Citation
Regulation of inducible nitric oxide synthase in murine proximal tubule epithelial cells

Material Information

Title:
Regulation of inducible nitric oxide synthase in murine proximal tubule epithelial cells
Creator:
Amoah-Apraku, Bismarck
Publication Date:
Language:
English
Physical Description:
ix, 133 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Department of Pharmacology and Therapeutics thesis Ph.D ( mesh )
Dissertations, Academic -- College of Medicine -- Department of Pharmacology and Therapeutics -- UF ( mesh )
Epithelial Cells ( mesh )
Gene Expression Regulation ( mesh )
Hypoxanthines -- pharmacology ( mesh )
Interferon Type II -- pharmacology ( mesh )
Kidney Tubules, Proximal ( mesh )
Lipopolysaccharides -- pharmacology ( mesh )
Nitric-Oxide Synthase -- drug effects ( mesh )
Nitric-Oxide Synthase -- genetics ( mesh )
Nitric-Oxide Synthase -- metabolism ( mesh )
Pteridines -- pharmacology ( mesh )
Pyrrolidines -- pharmacology ( mesh )
Research ( mesh )
Thiocarbamates -- pharmacology ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1995.
Bibliography:
Bibliography: leaves 117-132.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Bismarck Amoah-Apraku.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
002306848 ( ALEPH )
50189880 ( OCLC )
ALR0166 ( NOTIS )

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Full Text
129
Schmidt, H.H.H.W., Warner, T.D., Ishii, K. Sheng, H. and
Murad, F. Insulin secretion from pancreatic (J cells caused by
L-arginine-derived nitrogen oxides. Science 255: 721-723,
1992.
Schreck, R., Meier, B., Mannel, D.N., Droge, W. and Baeuerle,
P.A. Dithiocarbamates as potent inhibitors of nuclear
factor-kappa B activation in intact cells. J. Exp. Med. 175:
1181-1194, 1992.
Schuman, E.M. and Madison, D.V. A requirement for
intercellular messenger nitric oxide in long term-
potentiation. Science 254: 1503-1506, 1991.
Sharma, V.S., Isaacson, R.A., John, M.E., Waterman, M.R. and
Chevion, M. Reactions of nitric oxide with heme proteins:
studies on metmyoglobin, opossum methemoglobin and
microperoxidase. Biochemistry 22: 3897-3902, 1983.
Shibuki, K. and Okada, D. Endogenous nitric oxide release
required for long-term synaptic depression in the cerebellum.
Nature 349: 326-328, 1991.
Shultz, P.J., and Raij, L. Endogenously synthesized nitric
oxide prevents endotoxin-induced glomerular thrombosis. J.
Clin. Invest. 90: 1718-1725, 1992.
Shultz, P.J. Schorer, A.E. and Raij L. Effects of
endothelium-derived relaxing factor and nitric oxide on rat
mesangial cells. Am. J. Physiol. 258: F162-F167, 1990.
Shultz, P.J. and Tolins, J.P. Adaptation to increased
dietary salt intake in the rat. Role of endogenous nitric
oxide. J. Clin. Invest. 91: 642-650, 1993.
Snyder, S.H. and Bredt, D.S. Nitric oxide as a neuronal
messenger. Trends Pharmacol. Sci. 12: 125-128, 1991.
Stein, B., Baldwin Jr., A.S., Ballard, D.W., Greene, W.C.,
Angel, P. and Herrlich, P. Cross-coupling of the NF-kappa B
p65 and Fos/Jun transcription factors produces potentiated
biological function. EMBO. J. 12: 3879-3891, 1993.
Stoos, B.A., Carretero, O.A. and Garvin, J.L. Endothelium-
derived nitric oxide inhibits sodium transport by affecting
apical membrane channels in cultured collecting ducts cells.
J. Am. Soc. Nephrol. 4: 1855-1860, 1994.
Stuehr, D.J., Cho, H., Kwon, N.S., Weise, M. and Nathan, C.F.
Purification and characterization of the cytokine-induced
macrophage nitric oxide synthase: an FAD- and FMN-containing
flavoprotein. Proc. Natl. Acad. Sci. USA 88: 7773-7777, 1991
[a] .


75
protein (figure 4-3). Conversely, the combination of LPS and
IFN resulted in marked increases in iNOS protein (figure 4-3)
and nitrite accumulation (figure 4-1).
Effects of Lipopolvsaccharide and Interferon-y on Nuclear
Factor-KB Activation
To determine whether either LPS or IFN can activate NF-
kB in renal epithelium, I extracted nuclear proteins from MCT
cells treated with each of these agents alone or in
combination, and analyzed these extracts by EMSA using a
radio-labeled NF-kB consensus oligonucleotide as a probe.
As illustrated in figure 4-4, treatment with LPS or IFN
alone caused rapid nuclear translocation of NF-kB that
appeared by 15 min and was noticed at all time points studied
up to 24 h (data not shown). However, LPS appeared to be the
more efficacious activator of this transcription factor.
Moreover, the addition of IFN did not potentiate the
activation of NF-kB caused by LPS. The effect of LPS and IFN
on the nuclear translocation of NF-kB does not appear to be
concentration- or time-dependent after the initial nuclear
appearance within the concentration range and time interval
studied (data not shown).
Effect of Pvrrolidinedithiocarbamate on Nuclear Factor-KB
Activation. Inducible Nitric Oxide Svnthase mRNA and Protein
To investigate the potential role of NF-kB in the
induction of iNOS expression by LPS and IFN, I studied the


72
Table 5-2 Al-27 NMR Peak Data for Compounds III through VII.
Ligand
Compound
5, ppm for solid
6, ppm for
solution
pic
III
94
65
-198
15
mpic
IV
167
68
-202
-317
pza
V
0
70
hyp
VI
7
68
74
80
dipic
VII
85
68
162
-236
The solutions were prepared by heating the solid in d6-DMSO at
40C to produce clear colorless solutions. The peak data are
summarized in Table 5-2. The spectra are shown in Figures 5-2
through 5-6.
During the synthesis of compound V, the solution
containing pza and the aluminum ion began to turn a pale pink
color as the pH was raised, however, the solid formed is
white. Solutions containing varying aluminum ion to ligand
ratios were prepared by dissolving the appropriate amounts of
pza and A1 (N03) 3* 9H20, followed by the slow addition of solid
NaHC03 to raise the pH to 5. The solutions were submitted for
Al-27 NMR spectral analysis under the same experimental


30
C in dim light for 10 minutes, followed by
spectrophotometric absorbance readings at 543 nm (Beckman DU
650). Nitrite concentrations were determined by comparisons
with a standard sodium nitrite curve with double distilled
water as blank and expressed as nanomoles per mg of protein.
Protein Assay
Proteins were determined by the method of Lowry (Lowry
et al., 1951).
Northern Analysis
Total RNA was isolated by acid-phenol extraction
(Chomsczyski and Saachi, 1987 ) The RNA samples were
separated on agarose gel containing 2.2 M formaldehyde,
transferred to Nytran nylon membrane (Schleicher and Schuell,
Keene NH) and cross-linked by exposure to UV light.
Membranes were hybridized with a random prime-labeled
([32p]clcTP) full-length murine macNOS-cDNA (Xie et al., 1992,
Dinermann et al., 1993) overnight at 55 C followed by
washing under low stringency conditions (2X SSPE, 0.2% SDS,
65 C) and a high stringency wash (0.1X SSPE, 65 C) and
exposed to an x-ray film. Ribosomal (18s) RNA was used as
gel loading control.
Preparation of Primary Cultures of Rat Proximal Tubules
Male Sprague-Dawley rats weighing 150-175 g were
sacrificed by decapitation and their kidneys removed under


CHAPTER FIVE
TRANSCRIPTIONAL REGULATION OF NITRIC OXIDE SYNTHASE BY
NUCLEAR FACTOR-KB IN RENAL EPITHELIUM
Introduction
Computer analyses have identified numerous possible DNA
regulatory sequences within the promoter region of the iNOS
gene that are homologous to consensus DNA elements known to
bind nuclear transcription factors. These elements are known
to be involved in transcriptional responses to LPS and a
variety of cytokines (Xie et al. 1993, Lowenstein et al. ,
1993). They include NF-kB, AP-1, interferon-y regulatory
factor element (IRF-E) and tumor necrosis factor response
elements (Xie et al., 1993, Lowenstein et al., 1993).
The plethora of immunokines acting either alone or in
various combinations required to express iNOS activity in
different cell types may be explained by the fact that these
immunokines activate different nuclear factors, or perhaps,
different dimers of the same nuclear factor, which then bind
to DNA and influence gene transcription. This, therefore may
confer the tissue specificity of iNOS induction and its
response to stimulation by different immunokines. It is
reasonable to assume that macrophages, which utilize NO for
their cytotoxic functions, should be stimulated by microbes,
microbial products or cytokines whereas in proximal tubule
93


123
activator Tax of HTLV-1 binds to the NF-kB precursor pl05.
Oncogene 7: 1737-1742, 1992.
Ignarro, L.J. Biosynthesis and metabolism of endothelium-
derived nitric
560, 1990.
oxide.
Ann.
Rev. Pharmacol.
Toxicol. 30: 535-
Ignarro, L. J. ,
Buga,
G.M
. Wood, K S ,
Byrns, R.E. and
Chaudhuri, G. Endothelial-derived relaxing factor produced
and released from artery and vein is nitric oxide. Proc.
Natl. Acad. Sci. USA 84: 9265-9269, 1987.
Jorens, P.G., Van Overveld, F.J., Vermeir, P.A., Bult, H. and
Herman, A.G. Synergism between interleukin-1-p and
interferon-y, an inducer of nitric oxide synthase, in rat
fibroblasts. Eur. J. Pharmacol. 224: 7-12, 1992.
Junguero, D.C., Scott-Burden, T., Schini, V.B. and Vanhoutte,
P.M. Inhibition of cytokine-induced nitric oxide production
by transforming growth factor Bl in human smooth muscle
cells. J. Physiol. Lond. 454: 451-465, 1992.
Kamijo, R., Harada, H., Matsuyama, T., Bosland, M.,
Gerecitano, J., Shapiro, D., Le, J., Koh, S.I., Kimura, T.,
Green, S.J., Mak, T.W., Taniguchi, T. and Vicek, J.
Requirement for transcription factor IRF-1 in NO synthase
induction in macrophages. Science 263: 1612-1615, 1994.
Karupiah, G., Xie, Q-w., Buller, R.M.L., Nathan, C., Duarte,
C. and MacMicking, J.D. Inhibition of viral replication by
inter feron-y-induced nitric oxide synthase. Science 261:
1445-1448, 1993.
Kaufman, S. New tetrahydrobiopterin-dependent systems. Annu.
Rev. Nutr. 13: 261-286, 1993.
Ketteler, M., Border, W.A. and Noble, Nancy. Cytokines and
L-arginine in renal injury and repair. Am. J. Physiol. 267:
F197-F207, 1994.
Kilbourn, R.G. and Bellone, I. Endothelial cell production
of nitrogen oxides in response to interferon in combination
with tumor necrosis factor, interleukin-1, or endotoxin. J.
Natl. Cancer Inst. 82: 772-776, 1990.
Koesling, D., Schultz, G. and Bhme, E. Sequence homologies
between guanylyl cyclases and structural analogies to other
signal transducing proteins. FEBS Lett. 280: 301-306,1991.
Kubes, P., Suzuki, M. and Granger, D.N. Nitric oxide: an
endogenous modulator of leukocyte adhesion. Proc. Natl. Acad.
Sci. USA 88: 4651-4655, 1991.


73
(b)
Figure 5-?
DMSO.
Al-27 NMR Spectra of III, a) Solid, b) in d


60
saturating amounts could not be added to ensure constantly
available dihydropterines.
Discussion
In these studies, I have demonstrated that GTPCH is
required for NO production by proximal tubule epithelium and
that, when this enzyme is inhibited exogenous
dihydropteridines can restore NO synthesis through their
conversion to BH4 via the salvage pathway. This contrasts
with macrophage iNOS, which is fully active at levels of BH4
present in the resting state (Kwon et al. 1989, Nathan,
1994). On the other hand, MCT cells resemble hepatocytes,
vascular smooth muscle, fibroblasts and endothelial cells in
their requirement for BH4 synthesis during cytokine-induced
NO synthesis (Di Silvio et al., 1993, Werner-Felmayer et al.,
1990, Gross et al., 1991, Gross and Levi, 1992). Nitric
oxide synthesis by human endothelial cNOS is also limited by
BH4 (Werner-Felmayer et al. 1993, Rosenkranz-Weiss et al. ,
1994). In several of these cells, an inducible GTPCH is
coinduced with iNOS (Di Silvio et al. 1993, Gross et al. ,
1993). The design of this study however, does not allow
differentiation between inducible and constitutive isoforms
of GTPCH.
While confirming a similar requirement for de novo BH4
synthesis by MCT iNOS as in by several other nonrenal cells
(Di Silvio et al., 1993, Werner-Felmayer et al., 1990, Gross
and Levi, 1992), this study also identifies a major role for


15
Preparation of Al(ox) CH,QH (II). To avoid the lengthy
recrystallization required with I, the synthetic scheme was
modified to exclude DMSO. Al (N03) 3* 9H20 (0.181 g, 0.5 mmole)
was dissolved in 150 mL of H20. Oxine (0.220 g, 1.5 mmole)
was added as a solid and slowly dissolved in the acidic
aqueous solution to yield a pale yellow solution. The pH of
the mixture was slowly raised by dropwise additions of NaOH
until a very, thick yellow precipitate formed at pH = 5.2.
The solid was collected by filtration and recrystallized from
95% ethanol. Diffraction quality crystals were obtained by
recrystallization of the solid from the solution.
Picolinic Acid (pic)
Ligand reactivity. Picolinic acid is very soluble in
water and ethanol, but insoluble in organic solvents such as
ether. With a pKa of 5.4, it functions as a moderately weak
acid.
HO
Figure 2-4. Picolinic acid (pic).
Preparation of Al/pic complex (III) Al (N03) 3-9H20 (1.25
g, 3.33 mmole) was dissolved in 15 mL H20. In a separate
beaker, pic (1.23 g, 10.0 mmole) was dissolved in 20 mL H20.


LIST OF TABLES
Table Title Page
3-1 Vapor Diffusion Experimental Conditions
and Results 30
3-2 Gel Diffusion Experimental Conditions
and Results 32
3-3 Base Variation Experimental Conditions
and Results 35
3-4 Experimental Conditions for Crystal
Growth in Various Solvents and Results.. 37
4-1 Crystallographic Data for I and II 49
4-2 Final Atomic Coordinates and Isotropic
Thermal Parameters for 1 51
4-3 Hydrogen Atom Coordinates for 1 52
4-4 Bond Lengths for 1 53
4-5 Bond Angles for 1 53
4-6 Anisotropic Temperature Parameters for
Non-Hydrogen Atoms for 1 55
4-7 Final Atomic Coordinates and Isotropic
Thermal Parameters for II 58
4-8 Hydrogen Atom Coordinates for II 59
4-9 Bond Lengths for II 60
4-10 Bond Angles for II 60
4-11 Anisotropic Thermal Parameters for II 62
5-1 Al-27 NMR Peak Data and Aluminum
Coordination 68
v


63
angle. For Al(ox)3, the trans bond angles range from 168.3
to 173.9 for an average deviation from ideality of 8.8.
Aluminum-27 NMR Spectroscopy
The Al-27 NMR spectrum for la is shown in Figure 4-6.
Two different aluminum environments are present as exhibited
by the two wide peaks on the Al-27 NMR. The first peak is
centered around 54 ppm with a height of 580. The second,
smaller peak is centered around 221 ppm with a height of 160.
An Al-27 NMR study including hexacoordinate aluminum complexes
with aminopolycarboxylic acids reported that chemical shifts
range from 36.5 to 41.2 ppm.57 The complex with one of the
acids investigated by Iyer et al., has been characterized by
< 1 t i i i i 1 i i
800 BOO 4 00 200 0 -200 -400 -600 -BOO
Figure 4-6. Al-27 NMR Spectrum of the Solid la.


30
Table 3-1. Vapor Diffusion Experimental Conditions and
Results.
Ligand
[ligand], M
[Al3+] M
Result
pic
0.010
0.010
no solid
0.020
0.010
crystalline
0.030
0.010
crystalline
0.045
0.015
crystalline
mpic
0.010
0.010
no solid
0.020
0.010
no solid
0.030
0.010
crystalline
0.045
0.015
crystalline
dipic
0.016
0.008
crystalline
pza
0.010
0.033
crystalline
beaker because of the increased surface tension created by the
irregular surface. Once a single crystal sank to the bottom,
it did not grow any larger because the crystal growth only
occurred at the interface. Because of these limitations,
vapor diffusion is not an effective crystal growth technique
for the compounds under investigation.
Gel Diffusion
Theory
Vapor diffusion is limited because the reaction is
dependent upon the diffusion in only one direction. In
addition, the rates of diffusion through air and aqueous
solutions are fairly rapid. Diffusion of water soluble
compounds through an aqueous gel matrix is much slower.


95
emphasis on the NF-kB and IFN-activated protein binding sites
in the upstream and and NF-kB binding site in the downstream
areas in the promoter region of the iNOS gene.
Materials and Methods
Chemicals and Biologic Products
All restriction enzymes and other molecular biology
enzymes, DNA Wizard Miniprep, CAT Enzyme assay kit, were
purchased from Promega (Madison, WI).
Cloning of the Upstream Promoter Region
Two primers selected after careful study of the promoter
region of murine mac-iNOS, one designed with Hind III site at
the 5'-flanking end (BA I: 5' AGA AGC TTG ACT TTG ATA TGC ATA
TGC TGA AAT CCA T) and the other with a Sal I site at the 3'-
flanking end on the complementary DNA strand (BA II: 5' GAG
TCG ACG AAC AAG ACC CAA GCG TGA GGA A), were used to amplify
the 1.7 kilobase pair (kbp) promoter region from mouse
genomic DNA using the PCR (see DNA Amplification below). The
PCR product was treated with the Klenow fragment of DNA pol
I, restriction-digested with Hind III and Sal I. The PCR
product was subsequently isolated by agarose gel
electrophoresis and DEAE-cellulose.
This PCR product was subcloned to the Hind III/Sal 1
site of the pCAT-Basic and subjected to DNA sequence analysis
(Sanger Dideoxy Chain Termination, Sequenase Kit, IBI).


117
R = 4.38, wR = 5.62 and w = 4.38 for 109 parameters. Atomic
scattering factors used were those of International Tables for
X-ray Crystallography, vol. IV.
Discussion
X-rav Diffraction Study
Crystallographic data for X is summarized in Table 8-1.
Atomic positions for X are given in Table 8-2. The molecule
and atomic numbering scheme are given in Figure 8-1. All bond
lengths and angles including the sodium ion coordination are
listed in Table 8-3. The anisotropic thermal parameters for
the non-hydrogen atoms are given in Table 8-4.
One of the carboxylic acid groups in the tartaric acid
was deprotonated. 0(5) is bonded to H(3) at a bond length of
1.000 to form the intact carboxylic acid group. Of the
oxygen atoms in the molecule, 0(5) is the only donor atom that
is not within 3.0 of a sodium atom. H(3) is hydrogen bonded
at a distance of 1.568 to 0(1) in an adjacent hydrogen
tartrate ion. The 0(5)-H(3),-,0(l)' hydrogen bond is 0.43
shorter than the sum of two oxygen van der Waal's radii, but
is classified as a weak hydrogen bond because the H(3) is not
centered.66
The sodium ion is surrounded by eight oxygen atoms with
bond lengths between 2.426 and 2.827 Figure 8-2 shows a
detail of the coordination around the sodium atom. The
deviations from the least squares planes analysis for the


135
molecules at bond lengths of 2.795(2), 2.300(2) and 2.501(2)
, respectively. Because there are two hydrogen atoms each on
08, 09 and 010 at bond lengths between 0.71(4) and 0.90(4) ,
which create H-O-H bond angles between 99(4) and 106(5), it
seems reasonable that 08, 09 and 010 are water molecules and
not hydroxide ions.
There is extensive hydrogen bonding in VIII. H7 is
formally assigned to 07, but it is actually almost equidistant
between 07 and 07' in an adjacent dipic. The two oxygen atoms
are 2.4 72 apart and form a bond angle of 172 around H7.
The 07-H7-07' bond length is just slightly longer than the
average value, 2.44 reported for very strong hydrogen bonds
in similar compounds. Also, there is a hydrogen bond between
HI' and the equatorial water oxygen 010 and subsequently
between HlOa and 07'a in an adjacent molecule. In addition,
H8a is hydrogen bonded to 07a.
(CJhN(C0-,H) ) (CrH.NH (CO-J ) (NH, + ) (N0,~)
Compound IX is a complex salt of two pic molecules,
ammonium and nitrate. Although it is lacking an aluminum ion,
the compound does provide extensive discussion of the
placement and bonding of the hydrogen atoms. One pic is
protonated, by H(0), in the normal acid conformation.
However, the carboxylic acid group on the other pic is
formally deprotonated and, instead the hydrogen atom, H(N) is
located 0.975 from the ring nitrogen.


102
The apparently minor induction of CAT activity seen in
control- and IFN-treated cells may be attributed to the
presence of contaminating endotoxin during preparation of the
plasmid DNAs, which probably synergized with IFN. These
plasmid DNAs were prepared by transfection into competent E
coli bacteria. It is therefore difficult to avoid endotoxin
contamination, which in this case, was determined to be in
the order of 25 pg/ml by Limulus assay.
Discussion
In these studies, I characterized the role of NF-KB and
IFN-activated protein binding sites in the iNOS promoter
region in LPS- and IFN-induced gene transcription and also
investigated the mechanism of synergism between LPS and IFN
in this induction. The whole promoter region, as in pAiNOS-
CAT, conferred inducibility by LPS as well as synergistic
responsiveness to cotreatment with IFN. This is in agreement
with what is observed in macrophages where LPS alone, or in
combination with IFN, induces CAT activity in macrophages
transiently transfected with vectors bearing the iNOS
promoter region (Xie et al., 1993). The construct pBiNOS-CAT
responded to treatment with LPS but no synergism was noticed
on addition of IFN. This construct contains only the
downstream NF-KB binding site known to respond to LPS
treatment in macrophages (Xie et al. 1993). The inability
to respond to the synergistic effect of IFN could be due to
the absence in this construct of the upstream region that


31
sterile conditions and quickly placed in ice-cold Hank's
medium. The proximal tubules were then isolated as
previously described (Haverty et al., 1988, Toutain, 1992).
Light-Microscopic Immunocvtochemistrv
Murine proximal tubule cell iNOS immunoreactivity was
detected in cultured cells by the immunoperoxidase method
using a polyclonal antibody raised in rabbit against murine
mac-NOS. The antibody was kindly provided by Dr. Carl
Nathan, Cornell University, NY. It was raised against a
synthetic peptide that was constructed based on the published
mac-NOS sequence (Bogdan et al., 1993, Xie et al., 1992) and
prepared by Dr. Jeffrey R. Weidner and Richard A. Mumford,
Merck, Sharp & Dohme Research Laboratory. The MCT cell
monolayers grown in multichamber slides were fixed in ice-
cold acetone and processed for 1ight-microscopic
immunocytochemistry using the avidin-biotin-horseradish
peroxidase complex (ABC) technique (Vectastain ABC kit,
Vector Laboratories, Burlingame, CA). Endogenous peroxidase
activity was eliminated by incubation with 3% H2O2 for 30 min.
After treatment with blocking serum, the cells were incubated
with the primary antibody against macrophage-type NOS (mac-
NOS) (diluted 1:500) for 60 min. Following incubation with
the primary antibody the cell monolayers were rinsed with PBS
and incubated with the biotinylated secondary antibody for 30
min. After rinsing, the cells were incubated for 30 min with
the Vectastain ABC reagent followed by incubation with the


20
stimulated response element (y-IRE) and interferon-a-
stimulated response element (ISRE) (Eilers et al., 1993,
Martin et al. 1994). Recently, consensus sequences for
binding of several transcription factors involved in
conferring responsiveness to IFN have been identified in the
upstream promoter region of the iNOS gene thereby raising the
possibility of complex and tissue-specific regulation of the
expression of this gene by IFN (Lew et al., 1991, Lowenstein
et al. 1993, Xie et al. 1993). These sequences, which
include NF-kB, IRF-1, y-IRE, ISRE, GAS and AP-1 binding sites,
are all potential targets for IFN- or LPS-stimulated
signaling factors (Lew et al., 1991, Lowenstein et al., 1993,
Xie et al. 1993) and some reports even suggest that LPS-
induced macrophage iNOS expression is dependent on activation
and nuclear translocation of NF-kB (Xie et al. 1994). In
this study, I therefore focused on the role of NF-kB in the
mechanism of synergistic induction of iNOS activity by IFN
and LPS in proximal tubule epithelial cells.
The central hypothesis of this study was that IFN
potentiates the effects of LPS on iNOS induction by
modulating the LPS-stimulated activation and nuclear
translocation of dimers of NF-kB. I proposed that this
synergism may be due to IFN synergistically potentiating the
activation and nuclear translocation of NF-kB induced by LPS,
or activating different subunits which then dimerize with
those induced by LPS to produce synergistic iNOS gene
transcription. I tested this hypothesis by examining the


67
L/I + DAHP
Figure 3-5. Effects of sepiapterin on nitrite production by
MCT cells (48 h of induction) during GTPCH inhibition with
DAHP (6 iriM), CON, Control, L/I, LPS plus IFN, (n=3).


125
Lukic, M.L., Stosic-Grujicic, S., Ostojic, N., Chan, W.L. and
Liew, F.Y. Inhibition of nitric oxide generation affects the
induction of diabetes by streptozocin (sic) in mice. Biochem.
Biophys. Res. Commun. 178: 913-920, 1991.
Markewitz, B.A., Michael, J.R. and Kohan, D.E. Cytokine-
induced expression of a nitric oxide synthase in rat renal
tubule cells. J. Clin. Invest. 91: 2138-2143, 1993.
Marietta, M.A., Tayeh, M.A. and Hevel, J.M. Unraveling the
biological significance of nitric oxide. Biofactors 2: 219-
225, 1990.
Marsden, P.A. and Ballermann, B.J. Tumor necrosis factor
alpha activates soluble guanylate cyclase in bovine
glomerular mesangial cells via an L-arginine-dependent
mechanism. J. Exp. Med. 172: 1843-1852, 1990.
Martin, E., Nathan, C. and Xie, Q-w. Role of interferon
regulatory factor 1 in induction of nitric oxide synthase. J.
Exp. Med. 180: 977-984, 1994.
Mellion, B.T., Ignarro, L.J., Ohlstein, E.H., Pontecarvo,
E.G., Hyman, A.L. and Kadowitz, P.J. Evidence for the
inhibitory role of guanosine 35'-monophosphate in ADP-
induced human platelet aggregation in the presence of nitric
oxide and related dilators. Blood 57: 946-955, 1981.
Minnard, E.A., Shou, J., Naama, H. Cech, A., Gallagher, H.
and Daly, J.M. Inhibition of nitric oxide synthesis is
detrimental during endotoxemia. Arch. Surg. 129: 142-148,
1994.
Mitchell, H.H., Shonle, H.A. and Grindley, H.S. The origin
of nitrate in the urine. J. Biol. Chem. 24: 461, 1916.
Moneada, G. Palmer, R.M.J., and Higgs, E.A. Nitric oxide:
physiology, pathophysiology and pharmacology. Pharmacol Rev.
43: 109-142, 1991.
Morrissey, J.J., McCracken, R. Kaneto, H., Vehaskari, M. ,
Montani, D. and Klahr, S. Location of an inducible nitric
oxide synthase mRNA in the normal kidney. Kidney Int. 45:
998-1005, 1994.
Mukaida, N. Mahe, Y. and Matsushima, K. Cooperative
interaction of nuclear factor-kappa B and cis-regulatory
enhancer binding protein-like factor binding elements in
activating the interleukin-8 gene by pro-inflammatory
cytokines. J. Biol. Chem. 265: 21128-21138, 1990.
Muller, J.M., Ziegler-Heitbrock, H.W. and Baeuerle, P.A.
Nuclear factor-kappa B, a mediator of lipopolysaccharide
effects. Immunobiology 187: 233-256, 1993.


7
Figure 1-3. 9-Amino-l,2,3,4-tetrahydroacridine (THA).
its complexation to ligands containing a potential donor
nitrogen has been investigated. These ligands, shown in
Figure 1-4, include oxine, 2-methyl-oxine, picolinic acid, 6-
methyl-picolinic acid, pyrazinoic acid, hypoxanthine and 2,6-
dipicolinic acid.
Oxine is structurally very similar to the suspected
metabolite of THA.24 The manner in which oxine reacts with the
aluminum ion could be important to the reason that THA is
effective in alleviating Alzheimer's symptoms. 2-Methyloxine
is different from oxine only by the presence of the methyl
group adjacent to the ring nitrogen. The steric interaction
that could occur due to the presence of the methyl group could
change the length and strength of the Al-N bond as well as
prevent the formation of the tris complex.


57
therapeutic targets in its synthetic pathway. Hence,
understanding the biochemical events involved in renal
epithelial NO production and elucidating the control points
in BH4 synthesis has the potential of providing selective
means of therapeutic control of cytokine- and endotoxin-
induced NO-mediated renal tissue damage. In this study, I
investigated the role of GTPCH in the regulation of NO
synthesis by proximal tubule epithelium.
Materials and Methods
Chemicals and Biologic Products
Dulbecco's modified Eagle's medium (DMEM), L-arginine-
free Roswell Park Memorial Institute (RPMI) 1640 and all
other cell culture materials were purchased from Fisher
Scientific (Orlando, FL). Interferon-y (IFN, rat
recombinant) was purchased from Gibco BRL (Gaithersburg, MD).
Lipopolysaccharide (LPS, E coli serotype 026:B6),
sulfanilamide, methotrexate, 2,4-diamino-6-hydroxypyrimidine
(DAHP), sodium nitrite and N-(1-naphthyl)ethylenediamine
hydrochloride were purchased from Sigma Chemical Co. (St.
Louis, MO). Sepiapterin was purchased from Dr. B. Schircks
(Jonas, Switzerland) and 5,6,7,8-tetrahydro-L-biopterin was
purchased from Research Biochemicals Inc. (Natick, MA).


2
Zawadzki, 1980). This endothelium-derived relaxing factor
(EDRF) as it was then called, and which had a half-life of
about 6 seconds, was later identified as NO (Palmer et al. ,
1987, Ignarro et al. 1987). Over the past few years NO-
producing enzymes have been discovered in virtually every
tissue in numerous species and NO has become established as a
diffusible messenger mediating cell-to-cell interactions
throughout the body including immune cel1-mediated
cytotoxicity, inhibition of platelet aggregation, smooth
muscle relaxation and neuronal signaling (Ignarro et al.,
1987, Koesling et al., 1991, Bohrne et al., 1978, Radomski et
al., 1987, Mellion et al., 1981).
Isoforms of Nitric Oxide Svnthase
The nitric oxide synthase (NOS) enzyme family, the group
of enzymes that catalyze the biologic synthesis of NO,
comprises at least three distinct isoforms that can be
grouped into the constitutively expressed, Ca2+-calmodulin
dependent NOS isoforms (cNOS) found typically in neurons
(bNOS or NOS Type I) and endothelial cells (eNOS or NOS Type
III), and the immunologically inducible, Ca2+-independent NOS
found typically in macrophages (iNOS or NOS Type II)
(Forstermann et al. 1991, Stuehr and Griffith, 1992).
Calmodulin binding to cNOS subunits results in the transfer
of electrons from reduced nicotinic adenine dinucleotide
phosphate (NADPH) to a heme group at the active site within
each subunit of cNOS, initiating NO synthesis (Abu-Soud and


75
(a)
(b)
Figure 5-4. Al-27 NMR Spectra of V, a) Solid, b) in d6-DMSO.


27
Materials and Methods
Chemicals and Biologic Products
Dulbecco's modified Eagle's medium (DMEM), L-arginine-
free Roswell Park Memorial Institute (RPMI) 1640 and all
other cell culture materials were purchased from Fisher
Scientific (Orlando, FL). [IH]L-Arginine (specific activity:
77 Ci/mmol) was purchased from Amersham (Arlington Heights,
IL). AG50WX-8 Dowex (100-200 mesh) was purchased from Bio-Rad
(Melville, NY) Interferon-y (IFN, rat recombinant) was
purchased from Gibco BRL (Gaithersburg, MD) Radioactive
mac-iNOS-cDNA probes were prepared using a Stratagene (La
Jolla, CA) Prime-IT II random labeling kit. Other biologic
products were from Promega Co. (Madison, WI) .
Lipopolysaccharide (LPS, E coli serotype 026:B6),
dexamethasone, actinomycin D, cycloheximide, sulfanilamide,
sodium nitrite, N-(1-naphthyl)ethylenediamine hydrochloride
and N-nitro-L-arginine (NNA) were purchased from Sigma
Chemical Co. (St. Louis, MO).
Cell Cultures
Murine proximal convoluted tubule cells (MCT), were
passaged by trypsinization and cultured to confluence in
flat-bottom tissue culture dishes in Dulbeccos modified
Eagle's medium supplemented with 5% heat-inactivated fetal
bovine serum, 25 mM Hepes buffer, 10 mM nonessential amino
acids and 100 p.M sodium pyruvate. The cultures were


BIOGRAPHICAL SKETCH
Kim Browning was born in Orlando, Florida, on February
4, 1965. She graduated with honors from Maynard Evans High
School in 1983. In 1987, Kim graduated summa cum laude from
the University of Central Florida with a B.S. in chemistry and
began graduate studies at the University of Florida. Starting
in August 1992, she was an Instructor of Chemistry at the
Louisiana School for Math, Science and the Arts. Following
nomination by one of her students, Kim Browning was named to
Who's Who Among America's Teachers for 1994.
143


28
maintained at 37 C in a 5% CC>2/95% O2 environment during cell
culture and NOS induction. The MCT cell lines were kindly
provided by Dr. Eric Neilson (Univ. of Pennsylvania, PA) .
These cells were established from microdissected mouse
proximal tubules after stabilization by transfection with SV
40 virus and maintain many of the properties of
differentiated proximal tubule epithelial cells (Haverty et
al., 1988).
Determination of Nitric Oxide Synthase Activity
Nitric oxide synthase activity in intact MCT cells was
determined by measuring the conversion of [3H]L-arginine to
[3H]L-citrulline after separation of these amino acids by
anion exchange chromatography as previously described (Davda
et al. 1993). Briefly, control or experimentally treated
MCT cells were washed and then incubated in HEPES buffer
containing (in mM) HEPES 25 (pH 7.4, 37C) NaCl 140, KCl
5.4, CaCl2 1.8, MgCl2 1.0, glucose 5.0 and [3H]L-arginine 3
)j.Ci/ml for periods of time ranging from 0 to 120 min. The
reaction was terminated by washing the cells with ice-cold
Ca2+-free buffer containing 5 mM EDTA followed by addition of
1 ml 0.3 M HCIO4.
After neutralization of the tissue extract with 3.0 M
K2CO3, 50 M-l aliquots were taken for measurement of total
uptake of [3H]L-arginine and 500 |il aliquots applied to
columns containing DOWEX AG50WX8 (Na+ form) added as a 1:1
slurry in water. The columns were washed three times with 2


99
coenzyme A Controls contained CAT 0.1 U instead of cell
extract. The reaction was stopped with 0.5 ml ethyl acetate
which was also used to extract the chloramphenicol. The
upper organic layer was dried and taken up in 30 j.il of ethyl
acetate, 15 |il of which was spotted on silica gel thin layer
plates and ran with chloroformrmethanol (97:3 ascending).
After autoradiography of the separated butyrylated
chloramphenicol forms, spots were cut and counted. Data were
expressed as the amount of chloramphenicol butyrylated per
100 ig of extract. Protein was determined as described by
Bradford (Bradford, 1976). A p-galactosidase reporter
molecule was cotransfected into the cells and the p-
galactosidase assay, carried out according to manufacturer's
instructions, was used to normalize the results of the CAT
assay.
Results
The Upstream Promoter Region
Sequencing of the cloned promoter region confirmed it to
be almost identical to previously described iNOS promoter
sequence (Xie et al., 1993, Lowenstein et al. 1993), the
only differences being G for C in positions -617, -615, -613,
-611, -609, -607, -605, -603, -601, -599 and -21, A for G in
position -193, and A for C in position +24, all of them
outside regions described as putative transcription factor
binding sites (Xie et al., 1993).


102
The apparently minor induction of CAT activity seen in
control- and IFN-treated cells may be attributed to the
presence of contaminating endotoxin during preparation of the
plasmid DNAs, which probably synergized with IFN. These
plasmid DNAs were prepared by transfection into competent E
coli bacteria. It is therefore difficult to avoid endotoxin
contamination, which in this case, was determined to be in
the order of 25 pg/ml by Limulus assay.
Discussion
In these studies, I characterized the role of NF-kB and
IFN-activated protein binding sites in the iNOS promoter
region in LPS- and IFN-induced gene transcription and also
investigated the mechanism of synergism between LPS and IFN
in this induction. The whole promoter region, as in pAiNOS-
CAT, conferred inducibility by LPS as well as synergistic
responsiveness to cotreatment with IFN. This is in agreement
with what is observed in macrophages where LPS alone, or in
combination with IFN, induces CAT activity in macrophages
transiently transfected with vectors bearing the iNOS
promoter region (Xie et al., 1993). The construct pBiNOS-CAT
responded to treatment with LPS but no synergism was noticed
on addition of IFN. This construct contains only the
downstream NF-kB binding site known to respond to LPS
treatment in macrophages (Xie et al., 1993). The inability
to respond to the synergistic effect of IFN could be due to
the absence in this construct of the upstream region that


3
Stuehr, 1993, Wang et al., 1993). In contrast, calmodulin is
tightly bound to each subunit of iNOS even at very low
intracellular Ca2+ concentrations, an event that probably
occurs during synthesis, resulting in the permanent
activation of the enzyme (Cho et al., 1992). This makes the
addition of exogenous Ca2+ nonessential for iNOS activity.
The induction of iNOS by cytokines and endotoxin is
transcriptionally regulated and is inhibited by
glucocorticoids, interleukin (IL)-4, 8 and 10; transforming
growth factor-p(TGF-p) ; macrophage deactivating factor; and
protein synthesis inhibitors (Nussler and Billiar, 1993,
Radomski et al. 1990, Noonan and Noonan, 1977, Junguero et
al. 1992). It is probably only limited by substrate and
cofactor availability, protein turnover and product
inhibition. All NOS isoforms are homodimers of subunits
ranging from 130 to 160 kDa (Schmidt et al. 1991, Bredt et
al., 1991, Xie et al., 1992). The N-terminal portion of all
NOS isoforms contains binding motifs for NADPH, flavin-
mononucleotide (FMN) and flavin-adenine dinucleotide (FAD),
showing 50% amino acid identity with each other and 30-40%
overall sequence identity to NADPH-cytochrome P450 reductase
(Dinermann et al., 1993, Schmidt et al., 1993, Stuehr et al.,
1991 [a]). Additional binding sites have been postulated for
tetrahydrobiopterin (BH4), calmodulin and L-arginine (Schmidt
et al., 1993). All isoforms of NOS have consensus sites for
cAMP-dependent phosphorylation. Phosphorylation by protein
kinase C substantially diminishes cNOS activity but


CHAPTER 1
INTRODUCTION
Potential Aluminum Health and Environmental Issues
Traditionally considered to be a harmless element,
aluminum is widely used in the preparation of foodstuffs and
cosmetics. However, over the past couple of decades, aluminum
has been investigated with respect to a wide variety of
biological and environmental phenomena.
In the early 1980s, an extraordinarily high concentration
of aluminum was found, post mortem, as neurofibrillary tangles
and amyloid plaques in the brains of patients who had died of
Alzheimer's Disease.1 At first, aluminum was implicated as
the sole causative factor. To test this theory, several
studies were performed in various parts of the world to show
a causative relationship with respect to available aluminum
content in a drinking water and aluminum-containing products
such as antacids. The study on aluminum-containing products
reported no significant dose-response trend.2 The drinking
water studies showed a slight positive relationship for
aluminum content and a negative relationship for fluoride
content.1,3 However, in 1987, it was reported that the
precursor protein for the amyloid plaques was coded by a gene
1


128
reasonable sized peaks that would correspond to combinations
of aluminum, the ligand and hydroxide. However, there was a
small peak (0.11%) that would indicate the presence of the
tris complex. For IV, in addition to the ligand and its
decarboxylated fragment, there were three peaks probably due
to aluminum combined with one, two and three ligands. For V,
there were few large peaks; however, there is a small peak
(1.06%) that would indicate the tris complex. For VI, there
is no peak hear the mass for Al(hyp)3, but there is a small
peak (0.41%) that could be the tris complex after the loss of
part of one of its rings.
In each spectra, fragmentation has occurred to a
significant extent. The data for III, IV, and V indicate that
the tris complex was present; however, in all four spectra, no
conclusive results were found concerning the ratios of the
compounds in the sample mixtures.
Aluminum-27 NMR Spectroscopy
Aluminum-27 NMR would have proved a valuable technique if
the scale were more fully calibrated. The shift of the peak
is indicative of the geometry, symmetry and the type of atoms
surrounding the metal center. Since most of the research
reported for Al-27 NMR refers to exclusively oxygen atoms
bonded to the aluminum, it is not as useful as it could have
been. Most of the octahedral Al06 groups have shifts very
near 0 ppm, whereas the tetrahedral A104 groups are shifted to
near 80 ppm. However, the shifts reported for tris complexes


4
A complex, formed by a ligand substituting for water,
would be less stable and less likely to form if there were too
much steric strain. Coordination numbers of four, five and
six are most commonly reported for Al(III) .10 Too many ligands
around the metal center would be less stable due to crowding
caused by the relatively small metal ion radius, whereas too
few ligands would force Al(III) into a less favorable
geometry.
The formation of a five- or six-membered chelate ring
will provide stability to the complex due to the formation of
an unstrained ring structure. Organic rings of less than five
atoms are less stable because ring strain is created. If
chelation to a metal ion formed a small ring, the ligand would
be forced into a less stable configuration. The strain
occurring in the ligand is greater than if a larger ring were
formed. As a result, a less stable complex would be formed if
chelate rings are less than five-membered.
The donor atom bonded to the metal ion can affect the
stability of a complex. From equilibrium studies, a trend of
strength of complexation was determined as decreasing for the
ligands dicarboxylic acid, hydroxycarboxylic acid, carboxylic
acid and amino acid.16 Al(III), a relatively small ion with
a large positive charge, is a hard Lewis acid. Because oxygen
has a smaller size and larger negative charge, it has a
stronger attraction to the aluminum ion than nitrogen.10


42
and also modulate ion and acid transport across renal
epithelial cells membranes (Guzman et al., 1995, Tojo et al.,
1994 [a], Stoos et al. 1994). This suggests an important
role for NO in renal ion and acid-base homeostasis. Early
pathological findings in tubulointerstitial nephritis include
necrotic tubule epithelium with interstitial infiltrates of
lymphocytes and plasma cells which are cytokine-secreting
cells. Interferon-y is therefore likely to be an important
mediator of disorders involving T-cell responses such as
allograft rejection, autoimmune glomerulonephritis and
certain forms of tubulointerstitial nephritis, events in
which NO may play a mediating role (Langrehr et al., 1993
[a,b], Weinberg et al., 1994).
The discovery of the presence of iNOS in proximal
tubules has important implications due to the potential
pathophysiologic role of NO during immune-mediated and other
forms of renal injury. Migration of even small numbers of
inflammatory cells into the tubulointerstitium is likely to
lead to high local concentrations of cytokines such as IFN
and TNF-a which can then potentiate each other to induce iNOS
activity and stimulate the release of large amounts of NO by
proximal tubule cells. High concentrations of NO are
cytotoxic due to its conversion into toxic radicals like
peroxynitrites (Nussler and Billiar, 1993). Thus, proximal
tubule iNOS has the potential of playing a critical role in
the modulation of cellular injury during immune-mediated
responses.
Therefore, the existence of several different


130
Stuehr, D.J., Fasehun O.A., Kwon, N.S., Gross, S.S.,
Gonzalez, J.A., Levi, R. and Nathan, C.F. Inhibition of
macrophage and endothelial cell nitric oxide synthase by
diphenyleneiodonium and its analogues. FASEB J. 5: 98-103,
1991 [b].
Stuehr, D.J. and Griffith, O.W. Mammalian nitric oxide
synthases. Adv. Enzymol. 65: 287-346, 1992.
Stuehr, D.J., Gross, S.S., Sakuma, I., Levi, R. and Nathan,
C.F. Activated murine macrophages secrete a metabolite of
arginine with the bioactivity of endothelium-derived relaxing
factor and the chemical reactivity of nitric oxide. J. Exp.
Med. 169: 1011-1020, 1989.
Stuehr, D.J. and Marietta, M.A. Mammalian nitrate
biosynthesis: mouse macrophages produce nitrite and nitrate
in response to Escherichia coli lipopolysaccharide. Proc.
Natl. Acad. Sci. USA 82: 7738-7742, 1985.
Sun, S.C., Ganchi, P.A., Beraud, C., Ballard, D.W. and
Greene, W.C. Autoregulation of the NF-kappa B transactivator
Rel A (p65) by multiple cytoplasmic inhibitors containing
ankyrin motifs. Proc. Natl. Acad. Sci. USA. 91: 1346-1350,
1994.
Szab, C., Wu, C.C., Gross, S.S., Thiemermann, C. and Vane,
J.R. Interleukin-1 contributes to the induction of nitric
oxide synthase by endotoxin in vivo. Eur. J. Pharmacol. 250:
157-160, 1993.
Tayeh, M.A. and Marietta, M.A. Macrophage oxidation of L-
arginine to nitric oxide, nitrite and nitrate.
Tetrahydrobiopterin is required as a cofactor. J. Biol. Chem.
264: 19654-19658, 1989.
Terada, Y., Tomita, K., Nonoguchi, H. and Marumo, F.
Polymerase chain reaction localization of constitutive nitric
oxide synthase and soluble guanylate cyclase messenger RNAs
in microdissected rat nephron segments. J. Clin Invest. 90:
659-665, 1992.
Thanos, D. and Maniatis, T. The high mobility group protein
HMG I(Y) is required for NF-KB-dependent virus induction of
the human IFN-p gene. Cell 71: 777-789, 1992.
Tojo, A., Gross, S.S., Zhang, L., Tisher, C.C., Schmidt,
H.H.H.W., Wilcox, C.S. and Masden, K.M. Immunocytochemical
localization of distinct isoforms of nitric oxide synthase in
the juxtaglomerular apparatus of normal rat kidney. J. Am.
Soc. Nephrol. 4: 1438-1447, 1994 [a].


5 TRANSCRIPTIONAL REGULATION OF NITRIC OXIDE
SYNTHASE BY NUCLEAR FACTOR-KB IN RENAL EPITHELIUM.. 93
Introduction 93
Materials and Methods 95
Results 99
Discussion 102
6 SUMMARY AND CONCLUSIONS 113
LIST OF REFERENCES 117
BIOGRAPHICAL SKETCH 133
vi


35
Svneraistic Effect of Lipooolvsaccharide and Interferon-y
I next examined the interaction between LPS and IFN on
the induction of iNOS activity. Murine proximal tubule cells
were incubated with LPS at concentrations of 0.01, 0.1, 1.0,
and 10 |ig/ml either alone or in combination with 1.0 or 10
U/ml IFN, or IFN at concentrations of 1, 10, 50 and 100 U/ml
alone, or in combination with 0.1 (ig/ml LPS for 24 h.
Inducible NOS activity was determined following the
conversion of [3H]L-arginine to [3H]L-citrulline over 30 min.
Lipopolysaccharide caused only a small increase in iNOS
activity. At 10 ng/ml (the highest concentration studied),
LPS caused a 44% increase in iNOS activity compared to
control (figure 2-5B). However, the effect of LPS was
greatly potentiated by the addition of IFN, for example, 1
and 10 U/ml IFN potentiated the effect of 10 |ig/ml LPS on
iNOS activity, increasing this from 44% to 176% and 545% over
control, respectively. In contrast, IFN alone did not cause
any significant induction of iNOS activity compared to
control (figure 2-5A) However, combined with 0.1 |itg/ml LPS,
IFN caused a marked concentration-dependent increase in iNOS
activity (7.6-fold increase over control), which peaked at an
IFN concentration of 50 U/ml. Thus, whereas LPS and IFN by
themselves caused little or no induction of iNOS activity in
MCT cells, they synergized to produce a marked induction of
iNOS activity in these cells.


44
a
H
3
u
V
H
o
I 1 1 1 1 1
0 20 40 60 80 100
Time (min)
Figure 2-1. Time course of conversion of [3H]L-arginine to
[3H] L-citrulline in MCT cells treated with a combination of
LPS (0.1 |g/ml) and IFN (100 U/ml) for 24 h. N-nitro-L-
arginine (NNA, 300 |a,M) a competitive inhibitor of iNOS
completely abolished LPS/IFN-stimulated iNOS activity (^g: ng
of cell protein, n=6).


33
activity was determined by measuring the conversion of [3H]L-
arginine to [3H]L-citrulline over 30 min.
Induction Time Course of Inducible Nitric Oxide Synthase
Next, I examined the induction time course of iNOS
activity in MCT cells. Murine proximal tubule cells grown to
confluence in culture medium were incubated with 50 U/ml IFN
in combination with 0.1 p.g/ml LPS for 0, 4, 6, 8, 12 and 24 h
and iNOS activity measured as the conversion of [3H]L-
arginine to [3H]L-citrulline over 30 min.
There was a time-dependent induction of iNOS activity,
peaking to a 357% increase over control at 12 h (figure 2-2).
Thus, the induction of iNOS activity in MCT cells is time-
dependent. This lag period suggests the possibility that
protein synthesis or RNA transcription may be involved in the
induction of iNOS activity.
Effects of Lioopolvsaccharide and Interferon-y on Primary
Culture of Rat Proximal Tubule Cells
To establish that the expression of iNOS in MCT cells
was not an artifact of the transformation process in the MCT
cells, proximal tubules were isolated from rat kidneys as
described above and cultured to confluence. Nitrite
accumulation was then measured after 48 h incubation with IFN
(100 U/ml) or IFN plus LPS (0.1 (ig/ml) in the presence and
absence of NNA (300 jxM) .


A
B
Control IFN LPS LPS/IFN
pCiNOS-CAT
Figure 5-6. Effect of the removal of the downstream NF-kB binding site on the activation of
iNOS promoter by LPS and IFN. The MCT cells transfected with pCiNOS-CAT vector, which is
devoid of the downstream NF-KB binding site, were treated with LPS (1 n.g/ml) and IFN (100
U/ml) or their combination for 24 h followed by CAT assay of the cell lysates. There is the
loss of CAT inducibility by LPS, IFN or their combination; (n=3). A Autoradiograph, B
graphical presentation.
112


108
i Detail Showing Hydrogen Bonding in IX.
Figure 7-2


96
DNA Amplification
The reaction mixture was prepared with autoclaved
ultrafiltered water, 10X PCR buffer (Promega), MgC2 1.5 itiM,
dATP, dCTP, dGTP and dTTP 2 pM each (dNTPs), Primer BA I 2 (xM,
Primer BA II 2 p.M, Mouse genomic DNA 5 ng and 2.5 units of
Taq DNA polymerase in a total volume of 100 n.1. The mixture
was overlaid with 75 nl of mineral oil to reduce evaporation
or refluxing. The mixture was then subjected to repeated
cycles in a Perker-Elmer DNA Thermal Cycler at a template
melting temperature of 94 C (1 min), 53 C (2 min) and 72 C
(3 min) for a total of 25 cycles.
Purification of Amplified DNA
The PCR product was phenol:chloroform extracted and
precipitated overnight (or on dry ice for l1/2 h) from 0.3 M
NaOAc and ethanol, at -20 C. Recovery of DNA was monitored
by absorbance at 260 nm. Ten per cent of the total volume
was then ran on a 1% agarose gel in order to estimate the
molecular weight.
Subclonina of the DNA Fragment into : AT-Basic Vector and
Transfection of the Plasmids into HB101 Cells
The pCAT-Basic vector (figure 5-2B) was restriction
endonuclease digested with Hind III and Sal I as described
elsewhere. The PCR product was subcloned into the digested
vector, which was then transfected into HB101 cells.
Briefly, to a 100 jj. 1 of competent HB101 cells in a


24
multiple group comparisons as appropriate. The results are
presented as mean standard error of the indicated number of
experiments performed. Each blot shown is representative of
a series of experiments performed on at least three separate
occasions.


60
saturating amounts could not be added to ensure constantly
available dihydropterines.
Discussion
In these studies, I have demonstrated that GTPCH is
required for NO production by proximal tubule epithelium and
that, when this enzyme is inhibited exogenous
dihydropteridines can restore NO synthesis through their
conversion to BH4 via the salvage pathway. This contrasts
with macrophage iNOS, which is fully active at levels of BH4
present in the resting state (Kwon et al. 1989, Nathan,
1994). On the other hand, MCT cells resemble hepatocytes,
vascular smooth muscle, fibroblasts and endothelial cells in
their requirement for BH4 synthesis during cytokine-induced
NO synthesis (Di Silvio et al., 1993, Werner-Felmayer et al.,
1990, Gross et al., 1991, Gross and Levi, 1992). Nitric
oxide synthesis by human endothelial cNOS is also limited by
BH4 (Werner-Felmayer et al. 1993, Rosenkranz-Weiss et al. ,
1994). In several of these cells, an inducible GTPCH is
coinduced with iNOS (Di Silvio et al. 1993, Gross et al.,
1993). The design of this study however, does not allow
differentiation between inducible and constitutive isoforms
of GTPCH.
While confirming a similar requirement for de novo BH4
synthesis by MCT iNOS as in by several other nonrenal cells
(Di Silvio et al., 1993, Werner-Felmayer et al., 1990, Gross
and Levi, 1992), this study also identifies a major role for


Control
87
NF-kB Time course
15 min 30 min 60 min
z
2
z
u.
U-
(0
Q.
Q.
z
n
Ch
n
z
1
CO
Q.
co
Q.
_J
_l
U.
Ut
_l
UL
_i
U.
_l
-1
o.
E
g
+
a
E
o
o
c
o
Figure 4-4. Time course of the effects of IFN (100 U/ml) ,
LPS (1 jig/ml) and their combination on the nuclear
translocation of NF-kB in MCT cells. Control untreated cells
possess low basal nuclear levels of NF-kB; "+ comp" indicates
where the binding reaction was carried out in the presence of
100-fold excess unlabeled NF-kB oligonucleotide; "+ noncomp"
indicates where the reaction was carried out in the presence
of an irrelevant unlabeled oligonucleotide (AP-1). Both
competition and noncompetition controls were performed with
nuclear extracts from cells treated with a combination of LPS
and IFN for 60 min. Blot is representative of twelve
separate experiments.


9
(Werner-Felmayer et al., 1990); (vi) compounds that interfere
with NOS mRNA transcription by inhibiting critical nuclear
transcription factors; these include dithiocarbamates (e.g.,
pyrrolidinedithiocarbamate, PDTC), which are specific
inhibitors of nuclear factor-KB (NF-kB) (Schreck et al. ,
1992), and glucocorticoids, which act through mechanisms
still not fully understood, but probably involve repressing
transactivation by certain nuclear transcription factors that
are required for iNOS mRNA transcription; these compounds are
specific for iNOS.
Roles of Nitric Oxide in Mammalian Physiology
Molecular Targets
Once formed, NO can act as a paracrine substance or as
an intracellular messenger (Palmer et al., 1987, Ignarro et
al. 1987). At low concentrations (picomolar amounts), NO
solely stimulates soluble guanylyl cyclase (GC-S) by
nitrosation of its heme. This is currently thought to be the
main mechanism of action of NO on vascular smooth muscle
(Ignarro, 1990). When NO is formed in large quantities
(nanomolar amounts) as is seen in immunological iNOS
activation, in addition to sustained activation of guanylyl
cyclase, it also exerts cytostatic, cytotoxic and
cytoprotective actions in mammalian tissues as well as
antimicrobial activity towards certain pathogens (Saran et
al. 1990, Lipton et al. 1993, Karupiah et al., 1993,
Nussler, 1992). It can react with metalloproteins or thiol


ACKNOWLEDGEMENTS
I would like to thank the following individuals for
assisting me with my research and the preparation of my
dissertation. Analytical Services of the UF Chemistry
Department prepared the FAB Mass Spectra. Dr. Khalil A.
Abboud performed the x-ray analysis on the structure discussed
in chapter 6. John West produced the Al-27 NMR spectra for
solutions and solid samples. I would also like to acknowledge
the encouragement, patience and assistance readily available
from Gus and Ruth Palenik.
ii


86
139 kD-
Figure 4-3. Inducible NOS protein expression in MCT cells
before and after 12 h of treatment with LPS (1 ng/ml) alone
or in combination with IFN (100 U/ml). Blot is
representative of three separate experiments.


99
coenzyme A Controls contained CAT 0.1 U instead of cell
extract. The reaction was stopped with 0.5 ml ethyl acetate
which was also used to extract the chloramphenicol. The
upper organic layer was dried and taken up in 30 n1 of ethyl
acetate, 15 |j.l of which was spotted on silica gel thin layer
plates and ran with chloroform:methanol (97:3 ascending).
After autoradiography of the separated butyrylated
chloramphenicol forms, spots were cut and counted. Data were
expressed as the amount of chloramphenicol butyrylated per
100 jig of extract. Protein was determined as described by
Bradford (Bradford, 1976). A (3-galactosidase reporter
molecule was cotransfected into the cells and the p-
galactosidase assay, carried out according to manufacturer's
instructions, was used to normalize the results of the CAT
assay.
Results
The Upstream Promoter Region
Sequencing of the cloned promoter region confirmed it to
be almost identical to previously described iNOS promoter
sequence (Xie et al. 1993, Lowenstein et al. 1993), the
only differences being G for C in positions -617, -615, -613,
-611, -609, -607, -605, -603, -601, -599 and -21, A for G in
position -193, and A for C in position +24, all of them
outside regions described as putative transcription factor
binding sites (Xie et al., 1993).


43
tissue-specific regulatory signals for the induction of iNOS
may serve as a safeguard to prevent inappropriate or
uncontrolled production of NO. A better understanding of the
molecular mechanisms responsible for the tissue-specific
regulation of iNOS should allow us to design strategies to
prevent the NO-mediated tissue injury associated with various
pathological conditions.
In summary, I have demonstrated the presence of an iNOS
in renal proximal tubules that is markedly induced by a
combination of LPS and IFN, requires protein and mRNA
synthesis and its activity is inhibited by NNA, an analogue
of L-arginine. In contrast to its effects in murine
peritoneal macrophages, IFN by itself does not induce iNOS
expression in proximal tubule cells. It does, however,
markedly potentiate LPS-stimulated NO production by these
cells via mechanisms involving enhanced iNOS mRNA and protein
expression. Studies aimed at understanding the molecular
mechanisms that determine tissue-specific responses of iNOS
to various inducers are necessary for the development of more
rational and specific interventions to prevent NO-mediated
renal tissue injury.


122
Table 8-4. Anisotropic Thermal Parameters (2 x 103) for Non-
Hydrogen Atoms in X. Estimated Standard Deviations are given
in Parentheses.
Atom
u31
u22
u33
u23
u13
u12
Na
25(1)
23(1)
31(1)
5(1)
-2(1)
-2(1)
0(1)
13(1)
25(1)
38(2)
-6(1)
2(1)
1(1)
0(2)
18 (1)
23 (1)
38(2)
-2(1)
-5(1)
-3(1)
0(3)
19(1)
17(1)
35(2)
-6(1)
1(1)
2(1)
0(4)
24(1)
40(2)
16 (1)
3(1)
6(1)
-8(1)
0(5)
15(1)
36(1)
20(1)
2(1)
1(1)
-2 (1)
0(6)
25(1)
31(1)
27(1)
8(1)
-6(1)
-2(1)
0(7)
15(2)
28 (2)
40(2)
-7(1)
3 (2)
5(2)
C(l)
13 (2)
21(2)
17(2)
2(2)
0(2)
-1(2)
C (2)
13 (2)
18(2)
17(2)
-2 (2)
-1(2)
1(1)
C (3)
15(2)
21 (2)
13 (2)
0(2)
2 (1)
-2(2)
C (4)
18(2)
18(2)
19(2)
-5(2)
-1(1)
-1(2)
The anisotropic temperature factor exponent takes the form:
exp [-27r9h2a*2U11 + k2b*2U22 + l2c*2U33 + 2hka*b*U12 + 2hla*c*U13 +
2klb*c*U23) ]
Figure 8-2. Sodium Ion Coordination in X, Showing the Square
Antiprismatic, left, and Dodecahedral, right, Orientations.


A
B
Control
LPS/IFN
i-
.. h
o
<
.2 <
0)
(0
9
S 9
CD
w
CO (0
<
o
z
lL o
< .?
o
Q.
<
a
9 <
a a
Basal LPS/IFN
Figure 5-3. Demonstration of iNOS promoter activity by LPS and IFN in MCT cells. The MCT
cells transfected with pAiNOS-CAT vector or the promoterless pCAT-Basic vector were treated
with a combination of LPS (1 M.g/ml) and IFN (100 U/ml) for 24 h followed by CAT assay of
lysates. Treatment with LPS/IFN conferred inducibility of CAT activity to pAiNOS-CAT but
not to the promoterless pCAT-Basic. A Autoradiograph, B graphical presentation; (* p < 0.01
vs pAiNOS control, ** p < 0.01 vs pAiNOS [LPS/IFN], n=4).
109



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PAGE 144

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PAGE 145

81,9(56,7< 2) )/25,'$


C(21)
C(71)
C(51)
C(41)
0(11)
Figure 7-1. ORTEP of Compound IX.
0(2)
C(7)
C(3)
C(4)
C(5)


55
cases from cooperative interactions between iNOS inducers,
one of which may lack the ability to induce the synthesis of
cofactors required for full NOS activity. For instance,
whereas IFN stimulates marked NO production by murine
peritoneal macrophages, it is by itself ineffective in rat
aortic vascular smooth muscle (Deng et al., 1993, Hattori and
Gross, 1993, Gross and Levi, 1992). The latter appears to be
in part due to the inability of IFN to induce synthesis of
BH4 in this tissue (Hattori and Gross, 1993, Gross and Levi,
1992). Under these circumstances, the addition of LPS
results in induction of BH4 synthesis and leads to marked
concentration-dependent stimulation of NO production by IFN.
In murine macrophages, fibroblasts, lymphocytes and rat
pulmonary artery smooth muscle cells, IFN has been shown to
stimulate GTP-cyclohydrolase I induction (Werner et al.,
1989, Ziegler et al., 1990). This enzyme catalyzes the
cleavage of GTP to 7,8-dihydroneopterin triphosphate, the
rate-limiting step in BH4 synthesis. It is important to note
that NO formation from L-arginine is the only known BH4-
requiring reaction in which IFN plays an important role. It
can therefore be argued that cytokines like IFN probably
induce the formation of BH4 in order to provide a cofactor
for the generation of NO. In other circumstances, the
synergism between cytokines and endotoxin has been attributed
to less well-characterized interactions at the iNOS gene
transcription level (Geller et al., 1993 [b]).


59
Sepiapterin (5 mM) restored NO synthesis during GTPCH
blockade (figure 3-3), indicating that BH4 can be synthesized
via the salvage pathway in MCT cells. Sepiapterin alone
increased nitrite production in induced cells by 12.0 + 0.8%
(figure 3-3), suggesting that NO synthesis in MCT cells is
limited by their ability to synthesize BH4. Methotrexate
alone (10 mM) did not alter NO production in induced MCT
cells (figure 3-4), suggesting that iNOS does not normally
depend on the salvage pathway for BH4. However, methotrexate
did prevent the effect of sepiapterin during GTPCH blockade
(figure 3-4), confirming that sepiapterin is acting through
the salvage pathway.
Concentrations of sepiapterin between 1 and 8 mM
restored NO synthesis in a dose-dependent manner, whereas
concentrations higher than 10 mM had inhibitory effects
(figure 3-5). Thus, these results suggest that DAHP inhibits
GTPCH only partially and that, as reported for other cells
(Gross and Levi, 1992)), sepiapterin, in addition to being a
substrate for BH4 synthesis via the pterin salvage pathway,
is also a potent inhibitor of GTPCH in proximal tubules. At
incubation periods of 36 h and 48 h, the sepiapterin rescue
was less than expected (data not shown) This may be
because, at longer incubation periods, as the exogenously
added sepiapterin becomes depleted, the rescue then becomes
limited by the availability of dihydropterines. Due to the
inhibitory effect of high concentrations of sepiapterin,


CHAPTER 3
CRYSTAL GROWTH TECHNIQUES UTILIZED TO PRODUCE SINGLE
CRYSTALS SUITABLE FOR USE IN X-RAY DIFFRACTION STUDIES
Introduction
In order to characterize a compound by x-ray
crystallography, a single good crystal approximately 0.2 mm in
a dimension is required.61 When a suitable solvent is
available, crystals can be grown by slow evaporation of a
saturated solution of the compound. If a complex is not
soluble, a variety of crystal growth techniques can be
employed.61 The concept used in many methods involves
controlling the rate of reaction so that suitable crystals are
produced as the compound is formed. In this study, the rate
of reaction depends upon the availability of the aluminum ion,
the ligand and the base and the strength of the base. The
techniques discussed in this chapter include urea
decomposition, vapor and aqueous gel diffusion, variation of
the solvent system and the base strength.
Urea Decomposition
Theory
Urea decomposes into two molecules of ammonia and one
carbon dioxide when it reacts with water. The relatively slow
27


57
therapeutic targets in its synthetic pathway. Hence,
understanding the biochemical events involved in renal
epithelial NO production and elucidating the control points
in BH4 synthesis has the potential of providing selective
means of therapeutic control of cytokine- and endotoxin-
induced NO-mediated renal tissue damage. In this study, I
investigated the role of GTPCH in the regulation of NO
synthesis by proximal tubule epithelium.
Materials and Methods
Chemicals and Biologic Products
Dulbecco's modified Eagle's medium (DMEM), L-arginine-
free Roswell Park Memorial Institute (RPMI) 1640 and all
other cell culture materials were purchased from Fisher
Scientific (Orlando, FL) Interferon-y (IFN, rat
recombinant) was purchased from Gibco BRL (Gaithersburg, MD).
Lipopolysaccharide (LPS, E coli serotype 026:B6),
sulfanilamide, methotrexate, 2,4-diamino-6-hydroxypyrimidine
(DAHP) sodium nitrite and N-(1-naphthyl)ethylenediamine
hydrochloride were purchased from Sigma Chemical Co. (St.
Louis, MO). Sepiapterin was purchased from Dr. B. Schircks
(Jonas, Switzerland) and 5,6,7,8-tetrahydro-L-biopterin was
purchased from Research Biochemicals Inc. (Natick, MA).


CHAPTER SIX
SUMMARY AND CONCLUSIONS
The aims of this dissertation were to investigate the
regulation of inducible nitric oxide synthase in murine
proximal tubule epithelium, using the MCT cell line.
The major conclusions are: 1) murine proximal tubule
epithelial cells possess an immunokine-inducible isoform of
NOS; 2) expression of MCT iNOS activity requires protein
synthesis and mRNA transcription; 3) IFN potentiates the
induction of iNOS activity by LPS at the transcriptional
level but does not itself induce transcription or expression
of the enzyme; 4) inhibition of GTP cyclohydrolase I, the
rate-limiting enzyme in the de novo synthesis of BH4t
suppresses the maximum expression of iNOS activity in MCT
cells; this inhibition is reversed by concurrent activation
of the salvage pathway with sepiapterin, a substrate for BH4
formation via the salvage pathway; 5) LPS, IFN or their
combination cause a rapid activation and nuclear
translocation of NF-kB complexes in MCT cells, and that these
complexes are composed mainly of p65 with smaller amounts of
p50 but no c-Rel; 6) NF-KB activation is essential but not
sufficient for the induction of iNOS expression in MCT cells;
7) constructs bearing portions of the promoter region of iNOS
linked to a reporter gene, except for the upstream portion,
113


94
epithelial cells, exposure to excessive amounts of NO is
injurious and therefore a more stringent regulation of iNOS
activity may be required. Hence, iNOS activity is hardly
expressed by any of these immunokines acting alone in renal
epithelium. It is therefore important to characterize the
role of individual DNA regulatory elements in iNOS gene
expression in the different cell types.
Nuclear factor-KB is released from a cytoplasmic anchor
protein (IkB) upon immunokine stimulation, by phosphorylation
and proteolysis, allowing it to translocate into the nucleus
to bind its target DNA sequence and activate gene expression
(Muller et al. 1993, Henkel et al. 1993). The promoter
region of murine iNOS has been found to contain two putative
DNA binding sites for NF-kB (Xie et al. 1993). The
downstream one-third region contains one DNA binding site for
NF-kB which, in the mouse macrophage, has been found to be
responsive to LPS but unresponsive to the synergizing effect
of IFN (Xie et al. 1993). The upstream region which, in
mouse macrophage, was unresponsive to LPS but thought to be
critical for IFN responsiveness, contains the other putative
NF-kB binding site and also IRF-E, which is the IRF-1 binding
site needed for the synergistic effect of IFN (Xie et al.,
1993, Martin et al. 1994). In this study, I further
characterized the role of NF-kB in the transcription and
expression of the iNOS gene in MCT cells, and also
investigated the mechanism of the synergism between LPS and
IFN, using functional promoter analyses but with special


79
The failure of LPS to induce any iNOS activity in renal
epithelium could possibly be due to its inability to cause
the activation and nuclear translocation of c-Rel. However,
a combination of LPS and IFN that caused the expression of
iNOS activity also failed to induce the nuclear migration of
c-Rel. This could possibly be that whereas LPS alone may
require a p65/c-Rel dimer to activate iNOS gene expression in
macrophages, when combined with IFN in MCT cells, activation
of other processes by IFN leads to a cross-talk of pathways
resulting in the expression of iNOS activity independent of
c-Rel. These type of transcription factor interactions have
been reported between NF-kB and the Jun/Fos proteins (Stein
et al. 1993) as well as cis-regulatory enhancer binding
protein-like factor (Mukaida et al. 1990). Also, certain
proteins such as NF-IL-6, Tax and HMG I(Y) interact with NF-
kB and possibly function as necessary accessory proteins for
transactivation of the IL-6 (Leclair et al. 1992), HTLV-1
(Hirai et al. 1992) and IFN-p (Thanos and Maniatis, 1992)
genes, respectively. Other reports also suggest that, in
addition to c-Rel, activation of other proteins appears to be
necessary for iNOS expression in macrophages (Xie et al. ,
1994) Interferon-y may therefore be, among others, an
inducer of the activation and synthesis of these proteins.
This seems to be the case in macrophages where interferon
regulatory factor 1 (IRF-1) appears to be necessary for the
synergistic induction of iNOS by IFN and LPS (Martin et al.,
1994). Although this protein is yet to be described in renal


14
Preparation of Al (meox) CH,OH- H-,0 (I). DMSO (39 mL) was
placed in a 200 mL round bottom flask which was set in a 90C
oil bath to warm. Al2(S04)3*18 H20 (3.31 g, 10 mmole A1(H20 ) 63+)
was added and dissolved by stirring. 2-Methyloxine (5.02 g,
32 mmole) was subsequently added to yield a dark amber
solution. The solution was neutralized slowly by the addition
of 0.5 mL portions of diethylamine (7.5 mL, 72 mmol). The
resulting opaque mixture was stirred and heated for an
additional 10 minutes and then cooled to room temperature. A
dark amber solution (30 mL) was obtained by filtration through
a fine glass frit. After two months, large brown crystalline
clumps formed which were then redissolved in 15 mL methanol
and covered to slowly recrystallize. In two months, small
brown needles of diffraction quality were separated from the
solution.
Oxine (ox)
Ligand reactivity. 8-Hydroxyquinoline, commonly referred
to as oxine, has a similar solubility to meox. However, it is
more acidic, with a pKa of 9.0, and therefore, will be easier
to deprotonate the hydroxyl group.
HO
Figure 2-3. Oxine (ox).


36
Solvent System
Theory
When a crystal forms, occasionally solvent molecules are
incorporated to fill a space produced by the packing
arrangement. When a different solvent is used, the size and
shape of the new solvent may cause the molecules to pack in a
different manner. A possible result would be for the crystals
to grow differently. Crystallization in a series of solvents
is a common technique for the production of diffraction
quality crystals.61
Experimental
All syntheses were performed with a three-to-one ligand
to aluminum ion ratio. Unless otherwise noted, the
concentration of the ligand is equivalent to the concentration
of the base. The source of the aluminum ion was Al (N03) 3-9H20.
The experimental conditions and results are presented in Table
3-4 .
Results
Experiments performed in methyl or ethyl alcohol did not
produce a compound other than the sodium salt of the ligand
unless a substantial amount of water was present. However, in
the presence of water, pic and pza produce characteristic
unsuitable crystal forms. In DMSO, pic and pza both produced
a white solid that when heated to 100C to remove water, a
clear but brightly colored solution was formed. It is
possible that the solution's color was due to a decomposition


58
Table 4-7. Final Atomic Coordinates (xlO4) and Isotropic
Thermal Parameters (2xl03) for II. Estimated Standard
Deviations are given in Parentheses.
Atom
X
y
z
U
A1
0.4265 (3)
0.0274 (2)
0.7389(2)
38 (1)*
0(1)
0.2599 (5)
0.0105 (4)
0.7561 (3)
42(2)*
N (1)
0.4464(7)
0.0749(5)
0.8542 (4)
38 (3)*
C(ll)
0.5443 (10)
0.1074 (8)
0.9009(7)
54 (5) *
C (12)
0.5401(13)
0.1397(9)
0.9793 (8)
66(6)*
C (13)
0.4303(16)
0.1354 (9)
1.0089(7)
77(6) *
C (14)
0.3236(12)
0.1046(7)
0.9626(6)
51(5)*
C (15)
0.2008 (12)
0.0982 (9)
0.9863(7)
76(5)*
C (16)
0.1072(11)
0.0649(9)
0.9317(9)
71(6)*
C (17)
0.1220(9)
0.0336 (8)
0.8527(6)
50 (4) *
C (18)
0.2367(8)
0.0374(7)
0.8287(6)
39 (4) *
C (19)
0.3355 (8)
0.0739(6)
0.8841(5)
40 (4) *
0(2)
0.5956(5)
0.0229(5)
0.7371 (3)
33(2)*
N (2)
0.4551 (6)
-0.1228 (5)
0.7720 (4)
30 (3) *
C (21)
0.3771(9)
-0.1959(9)
0.7901 (5)
43(4)*
C (22)
0.4186(11)
-0.2922(9)
0.8134 (6)
55(5)*
C (23)
0.5385 (12)
-0.3132 (8)
0.8134(6)
57(5)*
C (24)
0.6254(9)
-0.2414(7)
0.7930 (5)
37(4)*
C (25)
0.7559(11)
-0.2558(7)
0.7925 (6)
52(5)*
C (26)
0.8301(10)
-0.1776(9)
0.7751 (6)
47 (4) *
C (27)
0.7783(9)
-0.0833 (8)
0.7574 (6)
44(4)*
C (28)
0.6537 (8)
-0.0632(7)
0.7555 (5)
31(4)*
C (29)
0.5780(7)
-0.1459(6)
0.7751 (4)
26 (3)*
0(3)
0.4114(5)
0.1581 (4)
0.7009 (3)
36(2)*
N (3)
0.3811(6)
-0.0107(5)
0.6223 (4)
31(3)*
C (31)
0.3649(9)
-0.0989 (7)
0.5836(6)
44 (4)*
C (32)
0.3391(10)
-0.1056(9)
0.5010(7)
59 (5)*
C (33)
0.3278 (9)
-0.0196(11)
0.4552 (6)
56 (5)*
C (34)
0.3404(8)
0.0749(9)
0.4916 (6)
44 (4)*
C (35)
0.3336 (10)
0.1669 (9)
0.4531(6)
59(5)*
C (36)
0.3520(9)
0.2581(8)
0.4940(7)
49 (5) *
C (37)
0.3796 (8)
0.2577(7)
0.5800(6)
45(4)*
C (38)
0.3880(8)
0.1655(7)
0.6218 (6)
36 (4)*
C (39)
0.3694(7)
0.0745(7)
0.5755(5)
30 (3)*
0(4)
0.9547 (8)
0.0461(7)
0.3444(7)
137 (5)*
C (4)
0.9648 (14)
0.1254 (13)
0.3919(11)
178(10)*
* Equivalent isotropic
U defined as
one third of the
trace of
the orthogonalized Ui;j tensor


89
PDTC
B
18s-
n
U4
LJ
PDTC
- +
139 kD-
Figure 4-6. Effect of PDTC (25 |a.M) on (A) iNOS mRNA
expression and (B) iNOS protein levels in MCT cells treated
with a combination of LPS (1 |ig/ml) and IFN (100 U/ml).
Cells were incubated with PDTC for 30 min before addition of
LPS and IFN and maintained during the entire period.
Inducible NOS mRNA and protein expression were measured at 6
h and 12 h, respectively. Blot is representative of three
separate experiments.


44
O 20 40 60 80 100
Time (min)
Figure 2-1. Time course of conversion of [3H]L-arginine to
[3H]L-citrulline in MCT cells treated with a combination of
LPS (0.1 ng/ml) and IFN (100 U/ml) for 24 h. N-nitro-L-
arginine (NNA, 300 |a.M) a competitive inhibitor of iNOS
completely abolished LPS/IFN-stimulated iNOS activity (^g: ng
of cell protein, n=6).


26
Apraku et al. 1993, Markewitz et al. 1993, Morrissey et
al., 1994). Nitric oxide has also been implicated in several
renal pathophysiologies such as acute interstitial nephritis,
glomerulonephritis and renal transplant rejection (Ketteler
et al. 1994, Langrehr et al. 1993 [a,b]). However, the
mechanisms that regulate NOS in the various renal cells have
not been extensively studied.
My preliminary studies suggest strong synergism between
LPS and IFN in stimulating iNOS activity in renal epithelial
cells although each, by itself, stimulates little or no iNOS
activity (Amoah-Apraku et al. 1993) This type of
synergism, which appears to vary among different tissues and
species, results in some cases from cooperative interactions
between iNOS inducers, one of which may lack the ability to
induce the synthesis of cofactors required for full NOS
activity. For instance, whereas IFN stimulates marked NO
production by murine peritoneal macrophages, it is by itself
ineffective in rat aortic vascular smooth muscle (Deng et
al. 1993, Hattori and Gross, 1993, Gross and Levi, 1992)
where it is limited by the inability to coinduce BH4
synthesis (Hattori and Gross, 1993, Gross and Levi, 1992).
This situation is remedied by the addition of LPS which
induces BH4 synthesis. In this study, I investigated the
presence of, as well as the characterization of iNOS in a
murine proximal tubule cell line, MCT cells, and in primary
cultures of rat proximal tubules.


58
Nitrite and Protein Assays
These assays were done as previously described in
Chapter Two.
Results
Effect of 2,4-Diamino-6-hvdroxvovrimidine on Murine Proximal
Tubule Cell Nitrite Production
To examine whether GTPCH, the rate-limiting enzyme in
the de novo synthesis of BH4 was required for NO synthesis, I
treated cells with a combination of LPS (0.1 ng/ml) and IFN
(100 U/ml) for 12 h in the presence or absence of the
specific GTPCH inhibitor DAHP (6 mM) followed by measurement
of nitrite accumulation in the culture medium.
Treatment with DAHP suppressed LPS/IFN-induced nitrite
accumulation by 53.1 + 3.4% (figure 3-2), indicating that NO
synthesis by MCT iNOS requires GTPCH activity.
Effects of Seoiaoterin and Methotrexate on Murine Proximal
Tubule Cell Nitrite Production
To investigate the contribution of the pterin salvage
pathway to the synthesis of BH4 for NO production, I used
sepiapterin, a dihydropteridine substrate for this pathway,
and methotrexate, an inhibitor of dihydrofolate reductase.
Experiments were designed to examine the effects of
dihydropteridine availability and dihydrofolate reductase
inhibition on NO synthesis in MCT cells treated as described
above.


UNIVERSITY OF FLORIDA
3 1262 08554 5233


121
results in L-arginine dependent mitochondrial iron-sulfur
enzymes in the macrophage effector cells. J. Immunol. 140:
2829-2838, 1988.
Eilers, A., Seegert, D. Schindler, C., Baccarini, M. and
Decker, T. The response of y-interferon activation factor is
under developmental control in cells of the macrophage
lineage. Mol. Cell Biol. 13: 3245-3254, 1993.
Frstermann, U., Schmidt, H.H., Pollock, J.S., Sheng, H. ,
Mitchell, J.A., Warner, T.D., Nakane, M. and Murad, F.
Isoforms of nitric oxide synthase: characterization and
purification from different cell types. Biochem Pharmacol
42: 1849-1857, 1991.
Franzoso, G. Bours, V. Park, S., Tomita-Yamaguchi, M. ,
Kelly, K. and Siebenlist, U. The candidate oncoprotein Bcl-3
is an antagonist of p50/NF-KB-mediated inhibition. Nature
359: 339-342, 1992.
Fried, M. and Crothers, D.M. Equilibrium and kinetics of lac
repressor-operator interactions by polyacrylamide gel
electrophoresis. Nucleic Acid Research 9: 6505-6525, 1981.
Furchgott, R.F. and Zawadzki, J.V. The obligatory role of
endothelial cells in the relaxation of arterial smooth muscle
by acetylcholine. Nature 288: 373-376, 1980.
Ganchi, P.A., Sun, S.C., Greene, W.C. and Ballard, D.W. A
novel NF-kappa B complex containing p65 homodimers:
implications for transcriptional control at the level of
subunit dimerization. Mol. Cell. Biol. 13: 7826-7835, 1993.
Garner, M.M. and Revzin A. A gel electrophoresis method for
quantifying the binding of proteins to specific DNA regions:
application to components of the Escherichia coli lactose
operon regulatory system. Nucleic Acid Research 9: 3047-3060,
1981.
Garthwaite, J. Glutamate, nitric oxide, and cell-cell
signaling in the nervous system. Trends Neurosci. 14: 60-67,
1991.
Geller, D.A., Di Silvio, M. Nussler, A.K., Wang, S.C.,
Shapiro, R.A., Simmons, R.L. and Billiar, T.R. Nitric oxide
synthase expression is induced in hepatocytes in vivo during
inflammation. J. Surg. Res. 55: 427-432, 1993 [a].
Geller, D.A., Nussler, A.K., Di Silvio, M., Lowenstein, C.J.,
Shapiro, R.A., Wang, S.C., Simmons, R.L. and Billiar, T.R.
Cytokines, endotoxin and glucocorticoids regulate the
expression of inducible nitric oxide synthase in hepatocytes.
Proc. Natl. Acad. Sci. USA 90: 522-526, 1993 [b] .


112
Table 7-7. Anisotropic Thermal Parameters (2xl03) for Non-
Hydrogen Atoms in IX. Estimated Standard Deviations are given
in Parentheses.
Atom
Un
u22
u33
u23
u13
u12
N (1)
37(2)
35(2)
29(1)
1(1)
9(1)
3(1)
C (2)
36(2)
32(2)
28 (2)
-2(1)
1(1)
2(2)
C (3)
50(3)
53(3)
31(2)
-2(2)
7(2)
11(2)
C (4)
57(3)
66(3)
47(3)
-2(3)
22 (2)
16(3)
C (5)
36(3)
64 (3)
52(3)
7(2)
9(2)
12 (2)
C (6)
41(3)
44 (3)
35(2)
8(2)
-2(2)
7(2)
C (7)
40(2)
37(2)
29 (2)
-5(2)
3(2)
2(2)
0(1)
41(1)
66(2)
35(1)
-2 (1)
9(1)
10(1)
0(2)
48(2)
67(2)
32 (1)
6(1)
2(1)
17(1)
N (11)
36(2)
46 (2)
29(1)
0(1)
5(1)
-4 (1)
C (21)
37(2)
34(2)
32(2)
1(1)
4(2)
-1(2)
C (31)
50(3)
60(3)
28 (2)
-5(2)
8 (2)
-12(2)
C (41)
63 (3)
84 (4)
38(3)
2(2)
24(2)
-16(3)
C (51)
39(3)
67(3)
47(3)
8(3)
10(2)
-12(3)
C (61)
40 (3)
53(3)
36(2)
-1(2)
2(2)
-10(2)
C (71)
42(3)
39(3)
30(2)
-2(2)
4(2)
-1(2)
0(11)
54 (2)
67(2)
35(1)
-2(1)
0(1)
-23 (2)
0(21)
60(2)
66 (2)
38 (1)
-5(1)
15(1)
-24(1)
N (2)
48(3)
57(3)
42(2)
3 (2)
10(2)
-5(2)
0 (IN)
112 (3)
104(3)
51(2)
8(2)
39(2)
-13(2)
0 (2N)
53(2)
117(4)
86(3)
39(3)
9(2)
-27(2)
0 (3N)
63(2)
129(3)
95(2)
20 (2)
-27(2)
-7(2)
N (3)
50(2)
68(3)
38(2)
4 (2)
19(2)
5(2)
The form
of the
thermal ellipsoid
is exp[-
-27T2 (h2a*2Ulx + k2b*2U2
+ 12c*2U33 + 2klb*c*U23 + 2hla*c*U13 + 2hka*b*U12) ]
Even though the bond lengths are significantly less than the
sum of the two radii, neither H(N) nor H(0) are centered but
instead are covalently bonded to a parent atom. Because of
the dissimilar bond lengths, these two interactions should be
classified as weak hydrogen bonds.66
The two other hydrogen bonds are also classified as weak.
The overall bond lengths for N(3)-H(1N)0(1)'' and N(3)-
H(4N) *0(2N) 3.038 and 2.895 respectively, are barely
less than the sum of the radii for an oxygen atom and a


30
C in dim light for 10 minutes, followed by
spectrophotometric absorbance readings at 543 nm (Beckman DU
650) Nitrite concentrations were determined by comparisons
with a standard sodium nitrite curve with double distilled
water as blank and expressed as nanomoles per mg of protein.
Protein Assay
Proteins were determined by the method of Lowry (Lowry
et al., 1951).
Northern Analysis
Total RNA was isolated by acid-phenol extraction
(Chomsczyski and Saachi, 1987). The RNA samples were
separated on agarose gel containing 2.2 M formaldehyde,
transferred to Nytran nylon membrane (Schleicher and Schuell,
Keene NH) and cross-linked by exposure to UV light.
Membranes were hybridized with a random prime-labeled
([32P]dCTP) full-length murine macNOS-cDNA (Xie et al., 1992,
Dinermann et al., 1993) overnight at 55 C followed by
washing under low stringency conditions (2X SSPE, 0.2% SDS,
65 C) and a high stringency wash (0.1X SSPE, 65 C) and
exposed to an x-ray film. Ribosomal (18s) RNA was used as
gel loading control.
Preparation of Primary Cultures of Rat Proximal Tubules
Male Sprague-Dawley rats weighing 150-175 g were
sacrificed by decapitation and their kidneys removed under


106
very important in the regulation of iNOS gene expression in
renal tubule epithelium. Interference with the activation or
activity of NF-kB may therefore be beneficial in suppressing
iNOS-mediated renal pathophysiologies such as acute
glomerulonephritis, acute tubular ischemia/reperfusion injury
and renal transplant rejection (Weinberg et al., 1994, Yu et
al. 1994, Langrehr et al. 1993 [a,b]). The inhibition of
NF-kB activation may provide a pharmacological basis for
interfering with these acute processes.


121
results in L-arginine dependent mitochondrial iron-sulfur
enzymes in the macrophage effector cells. J. Immunol. 140:
2829-2838, 1988.
Eilers, A., Seegert, D. Schindler, C., Baccarini, M. and
Decker, T. The response of y-interferon activation factor is
under developmental control in cells of the macrophage
lineage. Mol. Cell Biol. 13: 3245-3254, 1993.
Forstermann, U., Schmidt, H.H., Pollock, J.S., Sheng, H.,
Mitchell, J.A., Warner, T.D., Nakane, M. and Murad, F.
Isoforms of nitric oxide synthase: characterization and
purification from different cell types. Biochem Pharmacol
42: 1849-1857, 1991.
Franzoso, G. Bours, V. Park, S., Tomita-Yamaguchi, M. ,
Kelly, K. and Siebenlist, U. The candidate oncoprotein Bcl-3
is an antagonist of p50/NF-KB-mediated inhibition. Nature
359: 339-342, 1992.
Fried, M. and Crothers, D.M. Equilibrium and kinetics of lac
repressor-operator interactions by polyacrylamide gel
electrophoresis. Nucleic Acid Research 9: 6505-6525, 1981.
Furchgott, R.F. and Zawadzki, J.V. The obligatory role of
endothelial cells in the relaxation of arterial smooth muscle
by acetylcholine. Nature 288: 373-376, 1980.
Ganchi, P.A., Sun, S.C., Greene, W.C. and Ballard, D.W. A
novel NF-kappa B complex containing p65 homodimers:
implications for transcriptional control at the level of
subunit dimerization. Mol. Cell. Biol. 13: 7826-7835, 1993.
Garner, M.M. and Revzin A. A gel electrophoresis method for
quantifying the binding of proteins to specific DNA regions:
application to components of the Escherichia coli lactose
operon regulatory system. Nucleic Acid Research 9: 3047-3060,
1981.
Garthwaite, J. Glutamate, nitric oxide, and cell-cell
signaling in the nervous system. Trends Neurosci. 14: 60-67,
1991.
Geller, D.A., Di Silvio, M. Nussler, A.K., Wang, S.C.,
Shapiro, R.A., Simmons, R.L. and Billiar, T.R. Nitric oxide
synthase expression is induced in hepatocytes in vivo during
inflammation. J. Surg. Res. 55: 427-432, 1993 [a].
Geller, D.A., Nussler, A.K., Di Silvio, M., Lowenstein, C.J.,
Shapiro, R.A., Wang, S.C., Simmons, R.L. and Billiar, T.R.
Cytokines, endotoxin and glucocorticoids regulate the
expression of inducible nitric oxide synthase in hepatocytes.
Proc. Natl. Acad. Sci. USA 90: 522-526, 1993 [b].


137
intermediate of the two, the fit is closer for square
antiprismatic.
The only non-coordinated oxygen, 0(5), is located 1.000
from H(3), which is, in turn, hydrogen bonded at a distance
of 1.568 to 0(1) in an adjacent hydrogen tartrate ion.
Although it is significantly shorter than the sum of two van
der Waal's radii, this hydrogen bond is classified as weak
because the H(3) is not centered.
To check the absolute configuration of the compound,
least squares refinements for all non-hydrogen atom
coordinates were compared with those of the enantiomer. No
measurable difference was noted, probably because of the low
anomalous scattering factors for Mo Ka for atoms as small as
sodium. If radiation that has larger scattering factors had
been used, one enantiomer should have refined slightly better.
Summary
With the current focus on the health and environmental
issues related to aluminum, research in this field will most
certainly continue. However, because of the lack of
definitive characterization techniques, the progress in the
study of aluminum complexes with N,0 donor ligands will
proceed slowly.


49
0)
a
H
iH
iI
S
jj
H
o
I
d
=L
a
TJ
0 .01 0.1 1.0 10
LPS (Jig/ml)
Figure 2-5B. Synergistic interactions of LPS and IFN in MCT
cells: LPS treatment alone (0-10 (j.g/ml for 24 h; IFN)
caused minimal induction of iNOS activity. Simultaneous
addition of IFN (1 and 10 U/ml) markedly potentiated the
effect of LPS ((xg: ^g of cell protein, n=9) .


CHAPTER 8
ABSOLUTE CONFIGURATION OF SODIUM HYDROGEN (+)-TARTRATE
MONOHYDRATE
Introduction
In a study not covered in this work, the coordination and
crystal growth of aluminum with ligands, such as tartaric
acid, without a donor nitrogen was investigated.67 A solution
of the aluminum complex formed a glass upon evaporation. The
subject of this chapter, which does not contain aluminum, was
one of a few crystals grown from a mixed solvent system.
Structures of several bitartrate salts with alkali metals
and ammonium have been previously reported. However, the only
two to give absolute configurations are the isomorphous
potassium hydrogen ( + )-tartrate and ammonium hydrogen ( + ) -
tartrate. 68,69 As yet, there has been no structural data
published for the absolute configuration of a monohydrate of
a bitartrate salt.
Experimental
Synthesis
Materials. All materials and solvents were reagent grade
and used as supplied from manufacturer.
115


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19
medium frit. The white powder on the bottom of the beaker was
discarded. The solid collected by filtration was washed
thoroughly with water and dried in a desiccator for further
analysis.
Dipicolinic acid (dipic)
Ligand reactivity. Dipicolinic acid is slightly soluble
in hot water or ethanol and insoluble in all other solvents.
It is a moderately strong acid with a pKa of 2.2 for its first
dissociation.
HO OH
Figure 2-8. Dipicolinic acid (dipic).
Preparation of Al/dipic (VII). Dipic (4.00 g, 24.0
mmole) was added with stirring to 100 mL of H20 at room
temperature. NaHC03 (1.00 g, 11.9 mmole) was added as a solid
to partially neutralize the ligand. After the solution was
stirred for 20 minutes, the ligand dissolved to yield a clear,
colorless solution. A1 (N03) 3* 9H20 (2.99 g, 8.0 mmole) was
added as a solid to the solution. After ten minutes, the pH
measured 2.0 for the clear and colorless solution. Additional
NaHC03 (3.02 g, 36.0 mmole) was added slowly to the mixture.


116
Preparation of Na*C.H.g~-H.O (X) In 15 mL H20, ( + ) -
tartaric acid (1.475 g, 9.83 mmole) and Al (N03) 3- 9H20 (1.223 g,
3.26 mmole) was dissolved. NaHC03 (1.658 g, 19.7 mmole) was
added and bubbling ensued. When the solution was treated with
ethanol, a tacky white solid formed. The solution was
separated from the solid by filtration. From the filtrate,
large colorless hexagonal cylinders (X) (and characteristic
cubic NaN03 crystals) were obtained after evaporating
overnight. Elemental analysis: X: 24.64(10) % C, 3.55(3) % H,
0.21(9) % N, measured. C4H706Na: 27.60 % C, 4.05 % H, 0 % N,
calculated.
X-rav Crystallographic Analysis
A single crystal measuring approximately 0.8 x 0.8 x 0.6
mm was mounted on a glass fiber. All data were collected on
a Nicolet P3 Diffractometer, using graphite-monochromated Mo
Ka radiation. The unit cell dimensions were determined from
18 automatically centered reflections, 3.7<0<13.7; space
group by intensity statistics and satisfactory
structure solution and refinement; 1465 unique reflections,
1152 with J>3a(J), 0 s h s 9, -11 s k z 11, -13 s 1 s 13. Two
intensity standards measured every 98 reflections showed no
change during data collection. All non-hydrogen atoms were
located from an E-map. Four of the seven hydrogen atoms were
located from Fourier maps but no others from a difference
Fourier map. The program used for Fobs refinement was SHELXTL
Revision 5.1 (Sheldrick, 1985). Final refinement values were


tetrahydrobiopterin (BH4), an essential iNOS cofactor,
attenuated the increased LPS/IFN-stimulated nitrite
production. Sepiapterin, a substrate for the formation of
BH4 via the pterin salvage pathway, reversed this inhibition.
Electrophoretic mobility shift assays (EMSA) using specific
antibodies to p50, p65 and c-Rel subunits of nuclear factor-
kappa B (NF-kB) demonstrated that LPS and IFN treatment
induced the nuclear translocation of p50 and p65 but not c-
Rel. Cotreatment with Pyrrolidinedithiocarbamate (PDTC), a
specific NF-kB inhibitor, attenuated LPS- and IFN-stimulated
nuclear translocation of NF-kB as well as steady-state iNOS
mRNA levels, nitrite accumulation and protein synthesis.
Constructs pAiNOS-CAT and pBiNOS-CAT, prepared from the
promoter region of the iNOS gene, linked to a reporter gene
encoding chloramphenicol acetyltransferase and transiently
transfected into MCT cells, conferred inducibility of the
iNOS promoter by LPS but not by IFN. Construct pCiNOS-CAT,
which lacked the critical downstream NF-kB binding site, was
unresponsive to either LPS or IFN. Only construct pAiNOS-
CAT, which contained IFN response elements in addition to the
critical NF-kB binding site, conferred synergistic
inducibility by LPS and IFN, suggesting that this synergism
requires both NF-kB and IFN-induced proteins. In conclusion,
MCT iNOS is regulated at least at two levels:
transcriptionally, by transcription factors including NF-kB
and other IFN-induced proteins, and posttranscriptionally, by
BH4 availability.
IX


101
respectively. The hydroxyl groups coordinated in reported
structures are in deprotonated carboxylate groups instead of
acids.45'51 In the complexes where the acid group is intact,
the carbonyl, instead of the hydroxyl, oxygen is coordinated
to the metal ion.46,49 The other dipic has been formally
deprotonated, but it did not coordinate the metal ion through
the deprotonated hydroxy group. Instead, the carbonyl oxygen,
01'a, of the intact acid group is bonded to sodium at a
distance of 2.474(2) The coordination through a carbonyl
oxygen of an acid group has been reported for dipic.46'49 The
two axial and the remaining equatorial positions are occupied
by water molecules at bond lengths of 2.300(2), 2.501(2) and
2.795(2) respectively.
Hydrogen bonding
Although H7 is formally assigned to 07, it is actually
almost equidistant to 07' in the dipic coordinated to an
adjacent sodium ion. The symmetry operator for the 07'
hydrogen bonded to 07 is X, 1+Y, Z. 07 and the translated 07'
are 2.472 apart, forming a bond angle of 172 around H7. A
symmetrical hydrogen bond of significantly less than (>0.50 )
two van der Waal's radii constitutes a very strong hydrogen
bond.66 The 07-H7-07' bond length is just slightly longer than
the average value, 2.44 reported for dicarboxylates (RC02-
H-02CR),66 This interaction creates a network of very strong
hydrogen bonded chains throughout the crystal.


39
state mRNA levels by actinomycin D suggests a requirement for
mRNA transcription in iNOS induction in MCT cells.
Discussion
In these studies, I have described the characteristics
of a mouse renal proximal tubule inducible isoform of NOS
that shares many but not all the characteristics of the
macrophage iNOS. This proximal tubule iNOS is markedly
induced in a time- and concentration-dependent manner by
treatment with LPS and IFN, but is induced only modestly by
LPS alone. This induction requires protein and mRNA
synthesis and is inhibited by dexamethasone. Murine proximal
tubule cell iNOS activity is also blocked by NNA, a
competitive inhibitor of all NOS isoforms. In contrast to
its effects on mouse peritoneal macrophages (Bogdan et al.,
1993, Ding et al. 1988), IFN by itself did not induce the
expression of proximal tubule epithelial iNOS as illustrated
by the absence of iNOS activity, iNOS protein
immunoreact ivi ty and iNOS mRNA expression in MCT cells
treated with this cytokine. Nevertheless, IFN had a powerful
synergistic effect on LPS stimulation of iNOS expression in
these cells. This synergism, evidenced by the marked
enhancement of LPS-stimulated iNOS activity and iNOS protein
reactivity, is likely to occur at the level of transcription
since IFN also markedly augmented the stimulation of iNOS
mRNA expression induced by LPS. Although this effect could
be due to enhanced transcription or decreased degradation and


55
Table 4-6. Anisotropic Temperature Parameters (2 x 103) for
Non-Hydrogen Atoms for I. Estimated Standard Deviations are
given in Parentheses.
Atom
Un
u22
u33
u23
u13
u12
A1
41(1)
52(2)
47(1)
-5(1)
-2(1)
1(1)
0(1)
49 (4)
61(4)
61(3)
-5(3)
-8(3)
-1(3)
N (1)
39(4)
56 (4)
47(4)
-1(4)
7(3)
2(3)
C(ll)
45(5)
51(5)
55(5)
-4 (5)
6(4)
4(4)
C (12)
35(5)
57(6)
63(3)
-0(5)
9(5)
1(4)
C (13)
50(6)
51(6)
80 (7)
2 (5)
9(5)
-5(4)
C (14)
80 (8)
91(8)
54 (6)
22 (6)
8(6)
5(7)
C (15)
77(7)
74 (7)
64 (6)
15(6)
-17(6)
14(6)
C (16)
45(5)
73(6)
51(5)
7(5)
5(4)
9(5)
C (17)
61(7)
86 (8)
57(6)
-2(6)
-14(5)
-1(6)
C (18)
45 (6)
75(7)
54 (5)
-18(5)
-10 (4)
-1(5)
C (19)
34(5)
64(6)
53(5)
-7(4)
7(4)
-6(4)
C(10)
107(8)
59(6)
70(6)
10(5)
-9(6)
-16(6)
0(2)
41(3)
72(4)
41(3)
-15(3)
-6(3)
4 (3)
N (2)
47(4)
41(4)
44 (4)
3(3)
3(3)
-8(3)
C (21)
42 (5)
48 (5)
41(4)
4(4)
4 (4)
-8(4)
C (22)
57(6)
55(6)
51(5)
-5(5)
18 (5)
5(5)
C (23)
39(5)
78 (6)
47(5)
4 (5)
-8 (4)
-9(4)
C (24)
56(6)
84 (7)
49(5)
-1(5)
-15(5)
-12(5)
C (25)
47(5)
72(6)
61 (6)
-6(5)
-5(5)
7(5)
C (26)
41(5)
49 (5)
61(5)
4 (5)
7(5)
-2 (4)
C (27)
45(5)
50(5)
65(5)
-1(5)
21(5)
2(4)
C (28)
54(5)
42 (5)
55(5)
-15(4)
-3 (4)
6(4)
C (29)
58(6)
36(5)
45(4)
4(4)
6(4)
1(4)
C (20)
61(6)
58(6)
51(5)
-28 (4)
-4 (5)
9(5)
0(3)
49(3)
55(3)
51(3)
-3(3)
12 (3)
6(3)
N (3)
42(4)
55(5)
51(4)
-15(4)
-3(3)
5(3)
C (31)
34 (5)
72 (6)
46(5)
-6(5)
-2(4)
-6(4)
C (32)
44 (5)
69(7)
47(5)
9(5)
-6 (5)
-2(5)
C (33)
53(6)
86 (7)
74 (6)
22 (6)
6(5)
4(5)
C (34)
79(8)
100(9)
71(7)
19(7)
5(6)
-8(6)
C (35)
65(7)
148(11)
40(5)
6(6)
-8(5)
-30(7)
C (36)
39(5)
118(9)
37(5)
-20 (6)
-2(4)
-6(5)
C (37)
41(6)
133 (10)
46 (6)
-18(6)
13(5)
-15(6)
C (38)
71(7)
82 (7)
61(6)
-17(6)
-12(6)
12(6)
C (39)
48(5)
92 (7)
59 (6)
-21(5)
3 (4)
4(5)
C (30)
80 (7)
63 (7)
126(9)
-28(7)
-0(7)
27(6)
0(4)
113 (6)
94(5)
75(4)
-30(4)
11(4)
10(4)
C (4)
94 (9)
163(12)
79(8)
-5(8)
19(7)
-30(8)
0(5)
84 (5)
129(7)
136(7)
-8(5)
21(5)
6(5)
The form
of the
thermal ellipsoid
is exp[-
27T2 (h2a*2U31
+ l^b*2
U22 + 12c*2U33 + 2klb*c*U23 + 2hla*c*U13 + 2hka*b*U12) ]


07o
Figure 6-2.
ORTEP Representation of VIII.
vo


38
stimulate the appearance of iNOS immunoreactivity in MCT
cells most likely represents failure of this cytokine to
induce the synthesis of iNOS protein.
Effects of Lioopolvsaccharide and Interferon-v on Inducible
Nitric Oxide Svnthase mRNA
To investigate whether iNOS induction requires an
increase in steady-state mRNA levels, total RNA extraction
was done in cells incubated with LPS, IFN or LPS plus IFN in
the presence or absence of actinomycin D, cycloheximide and
dexamethasone for 4 h and a northern blot analysis done on
the extracts.
No iNOS mRNA was detected in Northern blots of controls
or IFN-treated cells (figure 2-9). However, LPS-treated
cells showed a significant increase in steady-state mRNA
levels first observed after 2 h and persisting for at least
12 h (data not shown). This was markedly potentiated by the
addition of IFN, blocked completely by actinomycin D and to
some extent by dexamethasone. Increases in steady-state iNOS
mRNA levels induced by LPS and LPS/IFN were also blocked by
coincubation with cycloheximide (data not shown). These
results suggest that there is a time-dependent increase in
steady-state iNOS mRNA levels with LPS treatment, and that
IFN, although ineffective by itself, acts in a synergistic
manner to enhance the increase in steady-state iNOS mRNA
levels induced by LPS. This synergism appears to require
protein synthesis. The inhibition of the increased steady-


66
Because the structure was confirmed by a second x-ray
analysis, the environment of the aluminum complex in solution
is known to be Al(ox)3 -- a distorted octahedral environment
with three nitrogen and three oxygen atoms. Possible
explanations for the intermediate peak at 73 ppm in the solid
not directly corresponding to the 54 ppm peak in the solution
include the presence of the solvent molecules and other
complex molecules in the crystal lattice, steric strains due
to packing or solvent effects. Since the location of each
nitrogen and oxygen atom around the aluminum atom has been
verified using x-ray diffraction techniques, the relatively
large value of 54 ppm should be added to the range of chemical
shifts reported for a distorted octahedral environment around
an aluminum ion.
Relatively few compounds with an AlN303 center have been
studied with both x-ray crystallography and Al-27 NMR
spectroscopy. Al(meox)3 and Al(ox)3 seem to be the least ideal
octahedral aluminum complexes with three nitrogen and three
oxygen atoms to be studied thus far.


80
epithelium, it is possible that it may also play a role in
the regulation of iNOS expression in this tissue.
Homodimers of p50 that reside constitutively in the
nucleus are believed to inhibit gene transcription by binding
to NF-kB sites. It requires the activation of the IxB-like
Bcl-3 proto-oncoprotein to dislodge these inhibitory dimers
in order to allow the incoming p50/p65 dimers to bind and
activate transcription (Franzoso et al. 1992). The basal
amounts of NF-kB found in control-treated MCT cells may be
homodimers of this type, thereby offering another explanation
for the failure of LPS to express iNOS activity in the cells.
Other reports also suggest that the plOO subunit of NF-KB2
inhibits p65-mediated transcription by preventing the
cytoplasmic dissociation of the p65-IxB complex during
immunokine activation (Sun et al. 1994). The function of
IFN therefore, may be the activation of processes leading to
the dislodging of the inhibitory effects of these subunits,
thus facilitating the binding of the p50/p65 dimers to their
nuclear DNA recognition sites.
The absence of iNOS activity after treatment with LPS,
and the total blockade of nitrite formation by PDTC despite
the fact that in both cases low levels of iNOS mRNA can be
detected, are intriguing. It is possible that very low
levels of iNOS activity may be present in these situations
and that these are below the detection capabilities of the
nitrite assay and immunoblotting. Alternatively, the iNOS
message or protein produced under these conditions may be


34
the presence of either a weak base or a low concentration of
a moderate base, the reaction should proceed slowly. In
addition, if the conjugate acid formed from the base is
volatile, the equilibrium will constantly shift towards the
products as the gaseous product evaporates. Furthermore,
because the chemical species present can alter crystal growth,
the production of twinned crystals and flower-like clusters
might not occur.
Experimental
Unless otherwise specified, the aluminum salt used was
Al (N03) 3* 9H20. All reactions were performed in aqueous systems
and the ligand to aluminum ion ratio was three-to-one. The
specific quantities and compounds used in the reaction are
shown in Table 3-3.
Results
No suitable crystals were formed except for the
KA1 (S04) 2* 12H20 reactant. However, the reactivity of the
ligands can be studied. The sulfate ion, with Kb of 8.3xl0"13,
is too weakly basic to react to produce an aluminum compound.
The acetate ion, with a Kb of 5.6xlO"10, was basic enough to
produce a crystalline product except for hypoxanthine, which
is a very weak acid. Although the reactions did proceed
slower than with the concentrated bicarbonate ion as the base,
single large crystals were not produced. The brown solution
produced in the reaction with hypoxanthine was probably due to
the ligand decomposing into a mixture of smaller amines.


12
1991, O'Dell et al. 1991. Bohrne et al. 1991, Schuman and
Madison, 1991, Nowak, 1992).
Nitric oxide also mediates glutamate neurotoxicity and
neuroprotection, a phenomenon dependent on its redox state
(Lipton et al. 1993). Reactions of NO with superoxide to
form peroxynitrite (0N00-) can lead to neurotoxicity and NMDA
receptor-mediated neuronal injury due to Ca2+-dependent
stimulation of NOS can be attributed, at least in part, to
this reaction (Lipton et al, 1993). In contrast, N0-
containing compounds in alternative redox states may prevent
neurotoxicity by mechanisms involving S-nitrosylation of
critical thiol groups at the NMDA receptor redox modulatory
site to down-regulate channel activity and excessive Ca2+
influx (Lipton et al., 1993, Dolye et al., 1981, Olson et
al., 1981, Saran et al. 1990).
Immunocytochemical techniques using anti-cNOS antibodies
have demonstrated the presence of NOS in intestinal myenteric
plexus and adrenal medullary ganglia (Snyder and Bredt,
1991). Non-adrenergic non-cholinergic (NANC) neurons in the
stomach and ileocolonic junction release an NO-like substance
when stimulated and other NO-responsive sites include the
penile corpus carvernosum, stomach, duodenum and esophagus
(Garthwaite, 1991), implying that NO may be playing an
important role as a neurotransmitter in these organs.


31
Because diffusion is slower, a concentration gradient is
produced over the distance from the interface.62 Also, the
semisolid gel is strong enough to support the crystals once
they form and flexible enough to not restrict growth as the
crystals enlarge. Aqueous gel diffusion is particularly
suited for these syntheses because the reactants are water
soluble and the products are insoluble. In addition, the
technique is regarded as a good method for the production of
large single crystals.62
Experimental
The agar gel used in the experiments was prepared by
dissolving the prescribed percentage of agar by weight in
water and heating to 98C. The solution was allowed to boil
at this temperature for 5 minutes and then cooled. Reagents
added to the agar were first dissolved in the minimum amount
of water and then completely mixed into the agar solution
before it hardened. The agar was poured into a large vial
with a screw top to ensure minimal solvent loss. To provide
a consistent reaction interface, any air bubbles were removed
using a disposable pipette before the agar set. Experiments
performed and results obtained are summarized in Table 3-2.
Results
A crystalline product was produced in most of the
experiments. The exceptions were when a white band was
produced in every case using hyp and twice with mpic. The
white band indicates that either very fine crystals formed or


47
X-rav Crystallographic Analysis
X-rav diffraction study of I. A needle shaped crystal
having the dimensions 0.22 x 0.16 x 0.14 mm was mounted on a
glass fiber for diffraction studies. All subsequent data were
collected on a Nicolet P3 Diffractometer, using filter-
monochromated Cu Ka radiation for I. The unit cell dimensions
were determined for 25 automatically centered reflections;
space group tetragonal P4/n by intensity statistics and
satisfactory structure solution and refinement; 2020 unique
reflections, 1945 with I>3ct(I); OshslO, OskslO, OslslO. Two
intensity standards, which were measured every 98 reflections,
showed no change during data collection. The program used for
Fobs refinement was SHELXTL Revision 5.1 (Sheldrick, 1985) All
non-hydrogen atoms were located from an E-map. Twenty of the
thirty hydrogen atoms were located using a difference Fourier
map. Final refinement values for 361 parameters were R =
8.67, wR = 10.67 and w = 9.07. Calculations and figures were
performed on an Eclipse Model 30 desktop computer. Atomic
scattering factors used in the SHELXTL program are the
analytical form given in International Tables for X-ray
Crystallography (1974).
X-rav diffraction study of II. Method and
instrumentation used to solve the X-ray structure of II were
the same as above with the following exceptions. A tabular
crystal having the dimensions 0.32 x 0.32 x 0.10 mm was
mounted. The data were collected using graphite-monochromated


103
bears the IRF-E, the binding site for IRF-1 demonstrated to
be critical for the synergistic role of IFN (Kami jo et al. ,
1994, Martin et al. 1994), as well as several other sites
for the binding of IFN-activated proteins such as y-IRE, GAS
and ISRE (Xie et al. 1993, Lowenstein et al. 1993). In
fact, in macrophages, site-directed mutagenesis that alter
the IRF-E site results in the loss of the synergistic effect
of IFN on LPS-stimulated iNOS activity (Martin et al., 1994).
Sequential deletion of the promoter region in constructs
transiently transfected into macrophages have shown that the
critical site for this IFN response may be located in the
region between -975 to -722 (figure 5-1) (Xie and Nathan,
1994) The response to the effect of LPS was similar in both
pAiNOS-CAT and pBiNOS-CAT. This suggests that the upstream
NF-kB binding site plays little or no role in the NF-kB-
mediated induction of gene transcription by LPS in MCT cells.
The inability of pCiNOS-CAT to elicit any response to
LPS, IFN or both may be due to the loss of the critical
downstream NF-KB binding site. The presence of the upstream
NF-kB binding site in addition to the IRF-E, GAS, ISRE and y-
IRE sites in this construct did not confer inducibility in
response to the immunokines, thus confirming that this NF-kB
binding site is irrelevant in NF-KB-mediated iNOS gene
transcription stimulated by LPS in MCT cells. Another reason
for the lack of response in this construct may be the loss of
the TATA box and the transcription initiation site, which
were deleted in preparing the construct. It must be stated


32
peroxidase substrate solution, diaminobenzidine. The cells
were then counterstained with hematoxylin, examined and
photographed under a light microscope (Zeiss Photomicroscope
II) .
Results
Measurement of Inducible Nitric Oxide Svnthase Activity:
f3H1L-Arqinine to f3h1L-Citrulline Conversion Time Course
To determine the presence of iNOS activity, MCT cells
were treated with a combination of LPS (0.1 (ig/ml) and IFN
(100 U/ml) for 24 h in the presence or absence of Nw-nitro-L-
arginine (NNA, 300 |iM) followed by measurement of the
conversion of [3H]L-arginine to [3H]L-citrulline over time.
These drug concentrations were selected based on previous
experiments on NOS activity in macrophages done in our
laboratory. Inducible NOS activity was measured after
incubation with [3H]L-arginine for 0, 10, 20, 30, 40, 50, 60
and 90 minutes. Control (untreated) and NNA-treated cells
were also incubated with [3H]L-arginine for 0, 10, 30, 60 and
90 min and iNOS activity measured.
Conversion of [3H]L-arginine to [3H]L-citrulline peaked
after 30 minutes (n=6) in LPS/IFN-induced cells (figure 2-1).
In control cells, there was no significant conversion whereas
NNA effectively blocked the conversion of [3H]L-arginine to
[3H]L-citrulline in LPS/IFN-induced cells. Thus, iNOS
activity was shown to be present in the MCT cell line after
induction with LPS and IFN. In subsequent experiments, iNOS


124
Kwon, N.S., Nathan, C.S. and Stuehr, D.J. Reduced biopterin
as a cofactor in the generation of nitrogen oxides by murine
macrophages. J. Biol. Chem. 264: 20496-20501, 1989.
Langrehr, J.M., Hoffman, R.A. Lancaster Jr., J.R. and
Simmons, R.L. Nitric oxide A new endogenous
immunomodulator. Transplantation 55: 1205-1212, 1993 [a],
Langrehr, J.M., White, D.A. Hoffman, R.A. and Simmons, R.L.
Macrophages produce nitric oxide at allograft sites. Ann.
Surg. 218: 159-166, 1993 [b].
Laychock, S.G., Mdica, M.E. and Cavanaugh, C.T. L-Arginine
stimulates cyclic guanosine 3',5'-monophosphate formation in
rat islets of Langerhans and RINm5F insulinoma cells:
evidence of L-arginine:nitric oxide synthase. Endocrinology
129: 3043-3052, 1991.
Leclair, K.P., Blanar, M.A. and Sharp, P.A. The p50 subunit
of NF-kB associates with the NF-IL6 transcription factor.
Proc. Natl. Acad. Sci. USA. 89: 8145-8149, 1992.
Levillain, O. Hus-Citharel, A., Morel, F. and Bankir, L.
Arginine synthesis in mouse and rabbit nephron: localization
and functional significance. Am. J. Physiol. 264 (Renal Fluid
Electrolyte Physiol. 33): F1038-F1045, 1993.
Lew, D.J., Decker, T., Strehlow, I. and Darnell, J.E.
Overlapping elements in the gualylate-binding protein gene
promoter mediate transcriptional induction by alpha and gamma
interferons. Mol. Cell Biol. 11: 182-191, 1991.
Lind, D.S., Copeland III, E.M. and Souba, W.W. Endotoxin
stimulates arginine transport in pulmonary artery endothelial
cells. Surgery 114: 199-205, 1993.
Lipton, S.A., Choi, Y., Pan, Z., Lei, H., Chen, H.V., Sucher,
N.J., Loscalzo, J., Singel, D.J. and Stamler, J.S. A redox-
based mechanism for the neuroprotective and neurodestructive
effects of nitric oxide and related nitroso-compounds. Nature
364: 626-632, 1993.
Lowenstein, C.J., Alley, E.W., Raval, P., Snowman, P.,
Snyder, S.H., Russel, S.W. and Murphy, W.J. Macrophage
nitric oxide synthase gene: two upstream regions mediate
induction by interferon-gamma and lipopolysaccharide. Proc.
Natl. Acad. Sci. USA 90: 9730-9734, 1993.
Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J.
Protein measurement with the Folin phenol reagent. J. Biol.
Chem. 193: 265-275, 1951.


CHAPTER TWO
CHARACTERIZATION OF INDUCIBLE NITRIC OXIDE SYNTHASE IN MURINE
PROXIMAL TUBULE EPITHELIUM
Introduction
Nitric oxide is an important mediator of vascular tone,
neurotransmission and immune responses (Moneada et al.,
1991). In addition, overproduction of NO has been implicated
in the pathogenesis of several cytotoxic and inflammatory
disorders. Nitric oxide is synthesized from L-arginine by
NOS in a reaction that produces stoichiometric amounts of L-
citrulline and requires NADPH, BH4 and flavin nucleotides as
cofactors. As described earlier, this enzyme family
comprises at least three distinct isoforms that can be
divided into two groups: the constitutively expressed, Ca2+-
calmodulin-dependent cNOS found typically in neurons (bNOS,
Type I) and endothelial cells (eNOS, Type III), and the
immunologically inducible, Ca2+-independent NOS found
typically in macrophages (iNOS, Type II) (Forstermann et al.,
1991, Stuehr and Griffith, 1992).
In the kidney, inducible and constitutive NOS protein or
mRNA has been detected in glomerular mesangial cells, macula
densa cells of the juxtaglomerular apparatus, proximal tubule
cells, the entire collecting duct as well as other epithelial
lining of the nephron (Marsden and Ballerman, 1990, Mundel et
25


LIST OF REFERENCES
Abu-Soud, H.M. and Stuehr, D.J. Nitric oxide synthases
reveal a role for calmodulin in controlling electron
transfer. Proc. Natl. Acad. Sci. USA 90: 10769-10772, 1993.
Alspaugh, J.A. and Granger, D.L. Inhibition of Cryptococcus
neoformans replication by nitrogen oxides supports the role
of these molecules as effectors of macrophage-mediated
cytostasis. Infect. Immun. 59: 2291-2296, 1991.
Amoah-Apraku, B., Chandler, L.J., Madsen, K.M., Tang, S.S.,
Ingelfinger, J.R. and Guzman, N.J. Regulation of inducible
nitric oxide synthase expression in murine proximal tubule
epithelial cells (Abstract). J. Am. Soc. Nephrol. 4:544,
1993.
Awolesi, M., Sessa, W. and Sumpio, B. Cyclic strain
upregulates nitric oxide synthase gene. FASEB 7: A755, 1993.
Baek, K.J., Thiel, B.A., Lucas, S. and Stuehr, D.J.
Macrophage nitric oxide synthase subunits: purification,
characterization and role of prosthetic groups and substrate
in regulating their association into dimeric enzymes. J.
Biol. Chem. 268: 21120-21129, 1993.
Baeuerle, P.A. and Baltimore, D. A 65-kD subunit of NF-kB is
required for inhibition of NF-kB by IkB. Genes Dev. 3: 1689-
1698, 1989.
Bellahsene, Z., Dhont, J.L. and Farriaux, J.P. Guanosine
triphosphate cyclohydrolase activity in rat tissues. Biochem.
J. 217: 59-65, 1984.
Billiar, T.R., Curran, R.D., Harbrecht, B.G., Stuehr, D.J.,
Demetris, A.J. and Simmons, R.L. Modulation of nitric oxide
synthesis in vivo: NG-monomethy1-L-arginine inhibits
endotoxin-induced nitrite/nitrate biosynthesis while
promoting hepatic damage. J. Leuk. Biol. 48: 568-569, 1990.
Blough, N.V. and Zafirou, O.C. Reaction of superoxide with
nitric oxide to form peroxinitrite in alkaline aqueous
solution. Inorg. Chem. 24: 3502-3504, 1985.
Bogdan, C., Vodovotz, Y., Paik, J. Xie, Q. and Nathan, C.
Traces of bacterial lipopolysaccharide suppress IFN-y-induced
nitric oxide synthase gene expression in primary mouse
macrophages. J. Immunol. 151: 301-309, 1993.
117


CHAPTER 4
SYNTHESIS, STRUCTURE AND AL-27 NMR STUDIES OF COMPLEXES
WITH OXINE AND 2-METHYLOXINE
Introduction
Oxine(ox) and 2-methyloxine(meox), shown in Figure 4-1,
contain the same N,0 donor group. Oxine has been extensively
Figure 4-1. Oxine, left; 2-Methyloxine, right.
studied for uses as a complexing agent for the quantitative
analysis of metal ions.64 The crystal structure of the
tris(oxinato)aluminum(III) with an occluded acetonylacetone
molecule was reported in a 1980 abstract as a distorted
octahedron with a meridional conformation.30 Two more recent
investigations confirm the structure of a tris complex between
the Al(III) ion and oxine.25,31 A drawing of the complex is
given in Figure 4-2.31 2-Methyloxine differs from oxine by the
substitution of a methyl group for the hydrogen alpha to the
nitrogen atom. The location of the methyl group could produce
41


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
a dissertation for the degree of Doctor of^^isPhy.
Fulton T. Crews
Professor of Pharmacology and
Therapeutics
as
This dissertation was submitted to tne Graduate Faculty
of the College of Medicine and to the Gra iuate School and was
accepted as partial fulfillment of the requirements for the
degree of Doctor of Philosophy.
Dean, Graduate School
August, 1995


104
however that the pCAT-Basic vector possesses its own TATA box
and transcription initiation site (Gorman et al. 1982).
Further studies to resolve this may best utilize constructs
where site-directed mutagenesis is used to alter only the
downstream NF-kB site, thus preserving the TATA box and the
transcriptional initiation site.
One intriguing finding of this study is the ability of
LPS alone to induce significant amount of CAT activity in
pAiNOS-CAT and pBiNOS-CAT transiently transfected into MCT
cells although LPS does not induce iNOS protein synthesis or
nitrite production in MCT cells (figure 4-1 & 4-3).
Lipopolysaccharide causes only moderate transcription of iNOS
mRNA in MCT cells. This may be because iNOS mRNA may possess
a number of bases at the 3'-flanking end not present in CAT
mRNA that makes it subject to increased degradation,
decreased stability or both, by proteins possibly coinduced
by LPS. The role of IFN may then be to activate pathways
that repress these proteins. Moreover, the promoter region
inserted into the construct may be too short and therefore
devoid of inhibitory binding sites present in the native
cells for proteins possibly coinduced by LPS. If such
proteins act as transcriptional or posttranscriptional
inhibitors of iNOS protein synthesis, then their absence from
the constructs may allow LPS to express CAT activity in
transfected cells but not iNOS in native cells. The
activities of such proteins may be those masked by pathways
activated by coincubation with IFN. It may well be part of


120
role of nitric oxide in interleukin-l-p -induced inhibition of
insulin secretion. J. Biol. Chem. 266: 21351-21354, 1991.
Corraliza, I.M., Campo, M.L., Fuentes, J.M., Campos-
Portuguez, S and Soler, G. Parallel induction of nitric
oxide and glucose-6-phosphate dehydrogenase in activated bone
marrow-derived macrophages. Biochem. Biophys. Res. Commun.
196: 342-347, 1993.
Davda, R.K., Chandler, L.J., Crews, F.T. and Guzman, N.J.
Ethanol enhances the endothelial nitric oxide synthase
response to agonists. Hypertention 21:939-943, 1993.
Davda, R.K., Chandler, L.J., Guzman, N.J. Protein kinase C
modulates receptor-independent endothelial nitric oxide
synthase activity. Eur. J. Pharmacol (Molecular
Pharmacology). 266: 237-244, 1994.
Davis, M.D., Kaufman, S. and Milstien, S. Distribution of
4a-hydroxytetrahydrobiopterin dehydratase in rat tissues.
Comparison with the aromatic amino acid hydroxylases. FEBS
Lett. 302: 73-76, 1992.
Deng, W. Thiel, B., Tannenbaum, C.S., Hamilton, T. A. and
Stuehr, D.J. Synergistic cooperation between T-cell
lymphokines for induction of the nitric oxide synthase gene
in murine peritoneal macrophages. J. Immunol. 151: 322-329,
1993 .
Dimmeler, S., Lottspeich, F. and Brue B. Nitric oxide
causes ADP-ribosylation and inhibition of glyceraldehyde-3-
phosphate dehydrogenase. J. Biol. Chem. 267: 1-4, 1992.
Dinermann, J.L., Lowenstein, C.J., Snyder, S.H. Molecular
mechanisms of nitric oxide regulation: potential relevance to
cardiovascular disease. Circulation Res. 73(2): 217-222,
1993.
Ding, A.H., Nathan, C.F. and Stuehr, D.J. Release of
reactive nitrogen intermediates from mouse peritoneal
macrophages. J. Immunol. 141: 2407-2412, 1988.
Di Silvio, M. Geller, D.A., Gross, S.S., Nussler, A.,
Freeswick, P., Simmons, R.L. and Billiar, R. Inducible
nitric oxide synthase activity in hepatocytes is dependent on
the coinduction of tetrahydrobiopterin synthesis. Adv. Exp.
Med. Biol. 338: 305-305, 1993.
Doyle, M.P. and Hoekstra, J.W. Oxidation of nitrogen oxides
bound by dioxygen in hemoproteins. J. Inorg. Biochem. 14:
351-358, 1981.
Drapier, J.C. and Hibbs, J.B. Differentiation of murine
macrophages to express non-specific toxicity for tumor cells


52
Figure 2-8. Immunoreactivity of iNOS protein in cultured MCT
cells. A: Control MCT cells; B: MCT cells treated with a
combination of IFN (100 U/ml) and LPS (1 ng/ml) for 24 h; Cs
MCT cells treated with IFN alone for 24 h. Inducible NOS was
detected by an immunoperoxidase method using a polyclonal
antibody raised in rabbit against mouse mac-NOS. Cells were
stained with hematoxylin. There is intense immunoreactivity
in cells treated with LPS/IFN compared to control or IFN-
treated cells. Picture is representative of three separate
experiments. Magnification x430.


118
Bogle, R.G., Baydoun, A.R., Pearson, J.D., Moneada, S. and
Mann, G.E. L-arginine transport is increased in macrophages
generating nitric oxide. Biochem. J. 284: 15-18, 1992.
Bhme, G.A., Bon, C., Stutzmann, J.M., Doble, A. and
Blanchard, J.C. Possible involvement of nitric oxide in long
term potentiation. Eur. J. Pharmacol. 199: 379-381, 1991.
Bhme, E., Graf, H. and Schultz, G. Effects of sodium
nitroprusside and other smooth muscle relaxants on cyclic
GMP formation on smooth muscle and platelets. Adv. Cycl.
Nucl. Res. 9: 131-143, 1978.
Boulanger, C. and Luscher, T.F. Release of endothelin from
the porcine aorta. Inhibition by endothelium-derived nitric
oxide. J. Clin. Invest. 85: 587-590, 1990.
Bours, V., Franzoso, G., Brown, K., Park, S., Azarenko, V. ,
Tomita-Yamaguchi, M., Kelly, K. and Siebenlist, U.
Lymphocyte activation and the family of NF-kB transcription
factor complexes. Current Topics Microbiol. Immunol. 182:
411-420, 1992.
Bradford, M.M. A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal. Biochem. 12: 248,
1976.
Bredt, D.S., Ferris, C.D., Snyder, S.H. Nitric oxide
synthase regulatory sites, phosphorylation by cyclic AMP-
dependent protein kinase, protein kinase C, and
calcium/calmodulin protein kinase: identification of flavin
and calmodulin binding sites. J. Biol Chem. 267: 10976-10981,
1992.
Bredt, D.S., Hwang, P.M., Glatt, C.E., Lowenstein, C., Reed,
R.R. and Snyder, S.H. Cloned and expressed nitric oxide
synthase structurally resembles cytochrome P-450. Nature 351:
741-748, 1991.
Bredt, D.S., Hwang, P.M. and Snyder, S.H. Localization of
nitric oxide synthase indicating a neural role for nitric
oxide. Nature 347: 768-770, 1990.
Bredt, D.S. and Snyder, S.H. Isolation of nitric oxide
synthetase, a calmodulin-requiring enzyme. Proc. Natl. Acad.
Sci. USA 87: 682-685, 1990.
Brezis, M. Heyman, S.N., Dinour, D., Epstein, F.H. and
Rosen, S. Role of nitric oxide in renal medullary
oxygenation. Studies in isolated and intact rat kidneys. J.
Clin. Invest. 88: 390-395, 1991.


CHAPTER ONE
INTRODUCTION
General
Mammalian cells have been known to produce oxides of
nitrogen since 1916 (Mitchell et al. 1916), an observation
verified and extended in the late 1970s and early 1980s when
it was shown that mammals produce nitrates and that this
production was enhanced by treatment with endotoxin (Stuehr
et al., 1985). To biologists in between these periods, these
compounds held little interest beyond their ability to cure
meat or aggravate pulmonary diseases even though compounds
that release nitric oxide (NO) have been used in clinical
medicine for over 150 years. This lack of interest in NO
could be because both biologists and clinicians were ignorant
of the endogenous mechanisms through which these compounds
exerted their clinical effects.
Biologic NO formation was independently discovered as a
physiologic regulator in blood vessels, macrophages and
neurons (Moneada et al. 1991). In a classic study,
Furchgott and Zawadzki demonstrated that in vessel segments
precontracted with norepinephrine, acetylcholine elicited
relaxation only if the endothelium was intact, suggesting
endothelial release of a short-lived substance that diffuses
to the smooth muscle to effect relaxation (Furchgott and
1


65
CQ
d)
4J
H
n
V
rt
0)
a
CQ
0)
40
LPS/IFN
-
+
+
+
+
DAHP
-
-
-
+
+
Sepiapterin
-
-
+
-
+
Figure 3-3. Effects of sepiapterin on nitrite production by
MCT cells in the absence or presence of DAHP, (mg: mg of cell
protein, n=9).


CHAPTER 9
CONCLUSION
Characterization of Aluminum Coordination Compounds
The coordination chemistry of aluminum with N,0 donor
ligands was investigated. Although a complex was formed with
each of the ligands in this study, only two were completely
characterized. Complexes I and II were sufficiently soluble
in ethanol or methanol to be recrystallized, while all others
were insoluble in every common solvent. It was determined
that the other compounds must not be the sodium salts of the
ligands, because the sodium salts were found to be very
soluble in water. The only methods that could be used to
characterize compounds III through VII were elemental
analysis, FABMS and Infra-red and Al-27 NMR spectroscopies.
Elemental Analysis
Since each ligand has a specific number of carbon and
nitrogen atoms, the presence of an organic solvent molecule or
an ammonium ion in a synthesized compound can easily be
confirmed. However, the amount of oxygen associated with
water can not be accurately determined because of the
uncertainty in the amount of hydrogen present. In addition,
since there is only a 2 g/mole difference between the mass of
125


53
Table 4-4. Bond Lengths () for I. Estimated
Deviations are given in Parentheses.
Al-O(l)
1.805(6)
Al-N(l)
2
Al-0(2)
1.858(5)
Al-N(2)
2
Al-0(3)
1.829(5)
Al-N(3)
2
0(1) -C (12)
1.347(10)
N(l)-C(ll)
1
N(l) -C (19)
1.347 (10)
C(ll)-C(12)
1
C(ll)-C(16)
1.430 (11)
C(12)-C(13)
1
C(13)-C(14)
1.412(13)
C(13)-H(13)
1
C(14)-C(15)
1.347(14)
C(14)-H(14)
1
C(15)-C(16)
1.407(13)
C(15)-H(15)
1
C(16)-C(17)
1.389 (13)
C(17)-C(18)
1
C(17)-H(17)
1.257(9)
C(18)-C(19)
1
C(18)-H(18)
1.200 (8)
C(19)-C(10)
1
C(10)-H(11)
0.983(9)
0(2) -C (22)
1
N(2) -C (21)
1.369(9)
N(2)-C(29)
1
C (21) -C (22)
1.418(11)
C(21)-C(26)
1
C(22)-C(23)
1.374(11)
C (23) -C (24)
1
C (24) -C (25)
1.354(12)
C(24)-H(24)
1
C(25)-C(26)
1.415(12)
C (25) -H (25)
1
C (26) -C (27)
1.435(11)
C (27) -C (28)
1
C (27) -H (27)
1.035(8)
C (28) -C (29)
1
C(28)-H(28)
1.055(7)
C (29) -C (20)
1
C(20)-H(21)
1.010 (8)
C(20)-H(22)
1
0(3) -C(32)
1.311(9)
N(3) -C (31)
1
N (3) -C (39)
1.332(11)
C (31) -C (32)
1
C (31) -C (36)
1.441(11)
C(32)-C(33)
1
C(33)-C(34)
1.435(14)
C(33)-H(33)
1
C (34) -C (35)
1.371 (16)
C(34)-H(34)
1
C(35)-C(36)
1.423(16)
C(35)-H(35)
1
C (36) -C (37)
1.436(15)
C (37) -C (38)
1
C (37) -H (37)
1.087(9)
C (38) -C (39)
1
C(38)-H(38)
1.239(10)
C (39) -C (30)
1
C (30) -H (31)
1.111(10)
C (30) -H (32)
1
0(4) -C (4)
1.432(15)
0(5) -H (5)
0
Table 4-5.
Bond Angles (
) for I. Estimated
Deviations are given in Parentheses.
0(1) -Al-N(1)
81.7(3)
C(22)-C(23)-C(24)
0(1) -Al-0(2)
92.0 (3)
C(23)-C(24)-C(25)
N(1)-Al-0(2)
173.1(3)
C(23)-C(24)-H(24)
0(1)-Al-N(2)
92.4 (2)
C(25)-C(24)-H(24)
N (1) -Al -N (2)
99.9 (2)
C(24)-C(25)-C(26)
0(2) -Al-N(2)
83.0 (2)
C(24)-C(25)-H(25)
0(1) -Al-0 (3)
173.9(3)
C(26)-C(25)-H(25)
N (1) -Al-0 (3)
92.5(2)
C (21) -C (26) -C (25)
Standard
.179(6)
.127(6)
.122 (6)
.372 (10)
.398(12)
.393 (12)
.146 (8)
.168(10)
.163(9)
.361 (14)
.431(11)
.504(12)
.351(10)
.362 (9)
.406(11)
.429 (12)
.167(8)
.102(8)
.356 (11)
.422 (11)
.488(11)
.181(8)
.361 (11)
.419 (12)
.407(13)
.186(9)
.186(11)
.190((9)
.310(15)
.452 (13)
.505 (13)
.154 (10)
.964(7)
Standard
118.6(7)
121.7(8)
116.9(7)
120.9 (8)
120.1(8)
128.9 (8)
110.6(7)
119.4(7)


130
Stuehr, D.J., Fasehun O.A., Kwon, N.S., Gross, S.S.,
Gonzalez, J.A., Levi, R. and Nathan, C.F. Inhibition of
macrophage and endothelial cell nitric oxide synthase by
diphenyleneiodonium and its analogues. FASEB J. 5: 98-103,
1991 [b].
Stuehr, D.J. and Griffith, O.W. Mammalian nitric oxide
synthases. Adv. Enzymol. 65: 287-346, 1992.
Stuehr, D.J., Gross, S.S., Sakuma, I., Levi, R. and Nathan,
C.F. Activated murine macrophages secrete a metabolite of
arginine with the bioactivity of endothelium-derived relaxing
factor and the chemical reactivity of nitric oxide. J. Exp.
Med. 169: 1011-1020, 1989.
Stuehr, D.J. and Marietta, M.A. Mammalian nitrate
biosynthesis: mouse macrophages produce nitrite and nitrate
in response to Escherichia coli lipopolysaccharide. Proc.
Natl. Acad. Sci. USA 82: 7738-7742, 1985.
Sun, S.C., Ganchi, P.A., Beraud, C., Ballard, D.W. and
Greene, W.C. Autoregulation of the NF-kappa B transactivator
Rel A (p65) by multiple cytoplasmic inhibitors containing
ankyrin motifs. Proc. Natl. Acad. Sci. USA. 91: 1346-1350,
1994.
Szab, C., Wu, C.C., Gross, S.S., Thiemermann, C. and Vane,
J.R. Interleukin-1 contributes to the induction of nitric
oxide synthase by endotoxin in vivo. Eur. J. Pharmacol. 250:
157-160, 1993.
Tayeh, M.A. and Marietta, M.A. Macrophage oxidation of L-
arginine to nitric oxide, nitrite and nitrate.
Tetrahydrobiopterin is required as a cofactor. J. Biol. Chem.
264: 19654-19658, 1989.
Terada, Y., Tomita, K., Nonoguchi, H. and Marumo, F.
Polymerase chain reaction localization of constitutive nitric
oxide synthase and soluble guanylate cyclase messenger RNAs
in microdissected rat nephron segments. J. Clin Invest. 90:
659-665, 1992.
Thanos, D. and Maniatis, T. The high mobility group protein
HMG I (Y) is required for NF-KB-dependent virus induction of
the human IFN-(3 gene. Cell 71: 777-789, 1992.
Tojo, A., Gross, S.S., Zhang, L., Tisher, C.C., Schmidt,
H.H.H.W., Wilcox, C.S. and Masden, K.M. Immunocytochemical
localization of distinct isoforms of nitric oxide synthase in
the juxtaglomerular apparatus of normal rat kidney. J. Am.
Soc. Nephrol. 4: 1438-1447, 1994 [a].


36
Effects of Exogenous Cofactors on Inducible Nitric Oxide
Synthase Activity Induced by Lipopolvsaccharide and
Interferon-v
Previous studies have suggested that the inability of
IFN to induce iNOS activity by itself in certain cells like
vascular smooth muscle may be due to lack of coinduction of
BH4 synthesis (Gross and Levi, 1992, Hattori and Gross,
1993). Thus, to determine whether the failure of IFN to
stimulate MCT iNOS activity in the absence of LPS was due to
its inability to induce synthesis of BH4 or lack of other
cofactors, I measured iNOS activity in crude cytosolic
extracts of MCT cells after the in vitro addition of
saturating concentrations of NADP (1 mM) FAD (4 |iM) and BH4
(10 jiM) .
Inter feron-y-treated MCT cells failed to show iNOS
activity even when this was measured in vitro after all the
required cofactors had been supplied (figure 2-6). Thus, the
apparent inability of IFN alone to stimulate MCT iNOS
activity does not appear to be primarily due to lack of BH4
synthesis or availability of other cofactors.
Effects of Lipooolvsaccharide and Interferon-y on Nitrite
Production
To correlate my findings with other methods of measuring
iNOS activity, I measured nitrites accumulated by MCT cells
after 48 h incubation with IFN alone (100 U/ml), LPS alone
(0.1 ng/ml) or IFN at concentrations of 1, 5, 10, 50 and 100
U/ml in combination with 0.1 ng/ml LPS.


48
Figure 2-5A. Synergistic interactions of LPS and IFN: IFN
treatment alone (0-100 U/ml for 24 h; LPS) failed to induce
iNOS activity in MCT cells. Simultaneous addition of LPS
(0.1 jig/ml; + LPS) resulted in a dose-dependent stimulation
of iNOS activity by IFN (|ig: |ig of cell protein, n=6) .


107
Hind III
-15 8 8 GACTTTGATATGCTGAAATCCATAAGCTGTGTGTGTGTGCAAGTGTGCA
-15 3 9 TGCGCATGTGTGCACATGAGTGTGCAGGTATATGTAGGAGCTAGAAGACAATCTCAGCTC
-1479 TTGTTTCCCAGGTTACCCAGCATCTCTCACCAGCCTGGAACCTGCCTAGTAGGCTAGGCT
-1419 GGCTGGCCAGCAAACCCTAGGCATATTCCTGTCTTTACCTCCCCAGAACTTATTGCAAAG
-13 5 9 GTGTGTCACCACACCCAGCATTTTATCATTGACCTATTGACTGGTGTGTGTGTGTGTGTG
-12 9 8 TGTGTTTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTAAATT
-1239 CCTTATCTCACCAACCCATGCCCAGCTTTTGAACTTAGGTCCTTGTACATGCAAGGCAAG
-117 9 CACTTTACCAACTGAGCCATCTCCCCAGCCCAATTACTTGATTTGTAATTCATTTATTCA
-1119 ATCAACAATTTATTTGTTCTCCCAACTATTGAGGCCACACACTTTTTGGGTGACTTAGTC
-10 5 9 TGTGTACCTCAGACAAGGGCAAAACACGAGGCTGAGCTGACTTTGGGGACCATGCGAAGA
NF-KB
-999 TGAGTGGACCCTGGCAGGATGTGCTAGGGGGATTTTCCCTCTCTCTGTTTGTTCCTTTTC
IRF-E
-939
-879
-819
-759
-699
-639
-579
-519
-459
-399
-339
-279
-219
-159
-99
-39
+ 22
CCCTAACACTGTCAATATTTCACTTTCATAATGGAAAATTCCATGCCATGTGTGAATGCT
TTATTGGAAGCATTGTAAGAAATTATAATTTATTCGTTTTTGTTTGTTTCTCAGAACAGG
GTTTTTCTGTGTAGTGTTCCTGGCTTATCCTAGAACTTACTCTGTAGACCAGGCTAGCCC
AAACTCAGGGATCAGCCTTTCTCTGTCTCCTGAATCCCGGGATTAAAGGCTTATGCCACC
ACACCCAGGTAGGACATTATAATCCTATATATAAGAAGTCACCCACACATACAAACACAC
ACACACCACACACACACACACAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA
GAGGGAGAGAGAGAGAGAGAGAGAGAATGCCACTGAGAAAAAAAATAAAAAGGCTTCACT
Spe,I
1
CAGCACAGCCCATCCACTATTCTGCCCAAGCTGACTTACTACTAGTGGGGAAATGCTGGT
Xba I
CAGACGGCATCTGTGCCCACAGCTTGCCTTCCATCCTTTCTAGAAAACCTCCTGATGAAT
GTGTCCTGGGCGTGTTGGAATATTGGCACCATCTAACCTCACTGAGAGAACAGACAGAAA
GCCAGAGAGCTCCGTGCCCAGAACAAAATCCCTCAGCAGCTGCAAGCCAGGGTATGTGGT
TTAGCTAAGAAAAGCCAGCCTCCCTCCCTAGTGAGTCCCAGTTTTGAAGTGACTACGTGC
TGCCTAGGGGCCACTGCCTTGGACGGACGACCAGGAAGAGATGGCCTTGCATGAGGATAC
ACCACAGAGTGATGTAATCAAGCACACAGACTAGGAGTGTCCATCATGAATGAGCTAACT
NF-KB
TGCACACCCAACTGGGGACTCTCCCTTTGGGAACAGTTATGCAAAATAGCTCTGCAGAGC
+1r^
CTGGAGGG'
cfrAT^AA'
TACGTGATGGCTGCTGCCAGGGTCACAACTTTACAGGGAGTTGAA
GAATGAGACTCTGGCCCCACGGGACACAGTGTCACTGGTTTGAAACTTCTCAGCCACCTT
Sal I
+ 82 GGTGAAGGGACTGAGCTGTTAGAGACACTTCTGAGGCTCCTCACGCTTGGGTCTTGTTC A'
$
Figure 5-1. The 1.7 kilobase pair 5'-flanking region of iNOS
cloned by PCR from mouse genomic DNA. Sequences are
designated that are homologous to the consensus sequences for
binding of NF-kB and IRF-1 (IRF-E). Also designated are the
restriction sites utilized in the preparation of the
constructs shown in figure 5-2A. The mRNA initiation site (+
1), TATA sequence (boxed).


79
Figure 5-7. Al-27 NMR Spectrum of an Aqueous
Containing 0.30 M Al3+ at pH 5.
Figure 5-8. Al-27 NMR Spectrum of an Aqueous
Containing 0.30 M Al3+ and 0.30 M pza at pH 5.
Solution
Solution


B
Control
IFN
LPS
LPS/IFN
pBiNOS-CAT
Figure 5-5. Effect of the removal of the upstream NF-kB binding site on the activation of
iNOS promoter by LPS and IFN. The MCT cells transfected with pBiNOS-CAT vector, which is
devoid of the upstream NF-kB binding site and the interferon response elements, were treated
with LPS (1 ng/ml) and IFN (100 U/ml) or their combination for 24 h followed by CAT assay of
the cell lysates. Only LPS conferred inducibility to pBiNOS-CAT, with the loss of the
synergistic effect
control, n=3)
of IFN. A Autoradiograph, B graphical presentation; (* p < 0.01 vs
111


25
Fast Atom Bombardment Mass Spectrometry
FABMS, a technique developed in the early 1980s, is used
to study the composition of a compound. The method has proven
useful for compounds of high molecular weight or low
volatility and for polar molecules.59 However, due to beam-
induced reduction or inefficient fragmentation, there is a
limit to its application.60 In FABMS, the sample is excited
by bombardment with high-energy atoms of either xenon or
argon, instead of the more traditional beam of high-energy
electrons.59 The charged molecules and fragments are then
separated by the use of a magnetic field. The data that can
be extracted include the molecular mass, or masses if the
sample is a mixture, and structural hints from the masses of
the fragments. Because of the low solubility of the samples,
the usefulness of this technique is limited.
X-rav Diffraction Techniques
Also known as x-ray crystallography, this method is used
to determine the location and type of each atom present in the
complex. The data required are collected by passing an x-ray
beam through a single crystal and measuring the intensity and
the angle of each diffracted beam relative to the incident
beam. The diffractions are caused by the electrons around
each atom scattering the x-ray beam. After preliminary
calculations, a test structure is proposed. The diffraction
pattern that would be produced by the test structure is


65
^ I I I' I I I I 'r"l l
800 600 400 200 0 -200 -400 -600 -800
Figure 4-7. Al-27 NMR Spectrum of the Solid Ila.
Figure 4-8. Al-27 NMR Spectrum of Al(ox)3 in C2H5OH Solution.


96
DNA Amplification
The reaction mixture was prepared with autoclaved
ultrafiltered water, 10X PCR buffer (Promega), MgCl2 1.5 mM,
dATP, dCTP, dGTP and dTTP 2 jiM each (dNTPs) Primer BA I 2 pM,
Primer BA II 2 piM, Mouse genomic DNA 5 ng and 2.5 units of
Taq DNA polymerase in a total volume of 100 |xl. The mixture
was overlaid with 75 pil of mineral oil to reduce evaporation
or refluxing. The mixture was then subjected to repeated
cycles in a Perker-Elmer DNA Thermal Cycler at a template
melting temperature of 94 C (1 min), 53 C (2 min) and 72 C
(3 min) for a total of 25 cycles.
Purification of Amplified DNA
The PCR product was phenol:chloroform extracted and
precipitated overnight (or on dry ice for IV2 h) from 0.3 M
NaOAc and ethanol, at -20 C. Recovery of DNA was monitored
by absorbance at 260 nm. Ten per cent of the total volume
was then ran on a 1% agarose gel in order to estimate the
molecular weight.
Subclonincr of the DNA Fragment into i \T-Basic: ctor and
Transfection of the Plasmids into HB101 Cells
The pCAT-Basic vector (figure 5-2B) was restriction
endonuclease digested with Hind III and Sal I as described
elsewhere. The PCR product was subcloned into the digested
vector, which was then transfected into HB101 cells.
Briefly, to a 100 p.1 of competent HB101 cells in a


all of them, I say thank you for their contributions to my
professional and personal development.
IV


Nitrites
(nmoles/mg)
84
Figure 4-1. Nitrite production in MCT cells treated with LPS
(1 ^g/ml) and IFN (100 U/ml) or their combination for 24 h,
(* pcO.OOl, mg: mg of cell protein, n=9).


93
monochromator utilizing Mo Ka radiation (X = 0.71073 ) The
cell parameters were refined for 50 reflections with 20.0s 26
s 22.0. 3305 reflections were collected using the w-scan
method. Four reflections were measured every 96 reflections
to monitor instrument and crystal stability (maximum
correction was 1 %). Absorption corrections were applied
based on measured crystal faces using SHELXTL plus (Sheldrick,
1990); absorption coefficient, /x = 0.16 mm'1 (min. and max.
transmission factors are 0.955 and 0.981, respectively).
The structure was solved by direct methods in SHELXTL
plus (Sheldrick, 1990) from which the locations of all of the
non-hydrogen atoms were obtained. During the initial
elemental assignment, an aluminum ion was entered as the
central metal atom. Fifteen hydrogen atoms were located from
a Difference Fourier map. The assignment of aluminum as the
central metal ion was changed to a sodium to obtain
electroneutrality. The structure was refined in SHELXTL plus
using full-matrix least squares. The non-hydrogen atoms were
treated anisotropically, whereas the hydrogen atoms were
refined with isotropic thermal parameters. H7 is disordered
between 07 and 07'. The disorder could not be resolved but
H7' was refined in a position approximately midway between 07
and 07'. 313 parameters were refined and E w (|F0| |Fc|)2
was minimized; w=l/(a | Fc|)2, a(F0) = 0.5kI~H{ [a (I) ]2 +
(0.02I)2}*, I (intensity) = (Ipeak Ibacicground) (scan rate), and
a (I) = (Ipealc + Ibackground)H (scan rate), k is the correction due to


131
Tojo, A., Guzman N.J., Garg, L.C., Tisher, C.C. and Masden,
K.M. Nitric oxide inhibits bafilomycin-sensitive H+-ATPase
activity in rat cortical collecting duct. Am. J. Physiol.
267: F509-F515, 1994 [b].
Toutain, H. Renal proximal tubule cell cultures for studying
drug-induced nephrotoxicity and modulation of phenotype
expression by medium components. Renal Failure 14(3): 371-
383, 1992.
Vincendeau, P. and Daulouede, S. Macrophage cytostatic
effect on Trypanosoma musculi involves an L-arginine-
dependent mechanism. J. Immunol. 146: 4338-4343, 1991.
Vincenti, M.P., Burrel, T.A. and Taffet, S.M. Regulation of
NF-kB activity in murine macrophages: effect of bacterial
lipopolysaccharide and phorbol ester. J. Cell Physiol. 150:
204-213, 1992.
Vodovotz, Y., Bogdan, D., Paik, J., Xie, Q-w. and Nathan, C.
Mechanisms of suppression of macrophage nitric oxide release
by transforming growth factor p. J. Exp. Med. 178: 605-613,
1993.
Wang, J., Stuehr, D.J., Ikeda-Saito, M. and Rousseau, D.L.
Heme coordination and structure of the catalytic site in
nitric oxide synthase. J. Biol. Chem. 268: 22255-22258, 1993.
Weinberg, J.B., Granger, D.L., Pisetsky, D.S., Seldin, M.F.,
Misukonis, M.A., Mason, S.N., Pippen, A.M., Ruiz, P., Wood,
E.R., and Gilkeson, G.S. The role of nitric oxide in the
pathogenesis of spontaneous murine autoimmune disease:
increased nitric oxide production and nitric oxide synthase
expression in MRL-lpr/lpr mice, and reduction of spontaneous
glomerulonephritis and arthritis by orally administered N-
monomethyl-L-arginine. J. Exp. Med. 179: 651-660, 1994.
Werner, E.R., Werner-Felmayer, G., Fuchs, D., Hausen, A.,
Reibnegger, G. and Wachter, H. Parallel induction of
tetrahydrobiopterin biosynthesis and indoleamine 2,3-
dioxygenase activity in human cells and cell lines by
interferon-y Biochem. J. 262: 861-866, 1989.
Werner, E.R., Werner-Felmayer, G. and Wachter, H.
Tetrahydrobiopterin and cytokines. Proc. Soc. Exp. Biol. Med.
203: 1-12, 1993.
Werner-Felmayer, G., Werner, E.R., Fuchs, D. Hausen, A.,
Reibnegger, G. Schmidt, K. Weiss, G. and Wachter, H.
Pteridine biosynthesis in human endothelial cells. Impact on
cycle oxide-mediated formation of cyclic GMP. J. Biol. Chem.
268: 1842-1846, 1993.


CHAPTER 5
ALUMINUM-27 NMR, INFRARED, MASS SPECTROMETRIC AND ELEMENTAL
ANALYSIS OF ALUMINUM COMPLEXES OF N,0 DONOR LIGANDS
Introduction
The techniques for the characterization of aluminum
complexes were discussed in Chapter 2. Because suitable
crystals were not produced for compounds III through VII, x-
ray crystallography could not be employed. Therefore, the
remaining methods discussed in Chapter 2 were used to
characterize the synthesized compounds.
Aluminum-27 NMR
Theory
A limited number of aluminum coordination compounds with
nitrogen as a donor atom have been analyzed by Al-27 NMR. The
Al-27 chemical shift is determined by the number and types of
atoms and the symmetry around the aluminum atom.54 The
chemical shifts as well as the donor atoms for some octahedral
environments in aluminum complexes are presented in Table 5-1.
All of the chemical shifts shown are relative to A1(H20) 63+.
Although shifts near 0 ppm and 80 ppm are generally
produced by octahedral and tetrahedral environments,
respectively, the peaks due to the hexavalent coordination
67


61
Table 4-10 -- continued.
0(1)-Al-0(3)
N(1)-Al-0(3)
0(2) -Al-0 (3)
N(2) -Al-0 (3)
0(1) -Al-N (3)
N(l) -Al-N (3)
0(2) -Al-N (3)
N(2) -Al-N(3)
0(3) -Al-N (3)
Al-0(1)-C(18)
Al-N(1)-C(ll)
Al-N(1)-C(19)
C(ll)-N(1)-C(19)
N(1)-C(ll)-C(12)
N(1)-C(ll)-H(11)
C(12)-C(ll)-H(11)
C(ll)-C(12)-C(13)
C(ll)-C(12)-H(12)
C (13) -C (12) -H (12)
C (12) -C (13) -C (14)
C (12) -C (13) -H (13)
C (14) -C (13) -H (13)
C(13)-C(14)-C(15)
C(13)-C(14)-C(19)
C(15)-C(14)-C(19)
C(14)-C(15)-C(16)
C(14)-C(15)-H(15)
C(16)-C(15)-H(15)
C(15)-C(16)-C(17)
C(15)-C(16)-H(16)
C(17)-C(16)-H(16)
C(16)-C(17)-C(18)
C(16)-C(17)-H(17)
C(18)-C(17)-H(17)
0(1) -C (18) -C (17)
0(1)-C(18)-C(19)
C(17)-C(18)-C(19)
N(1)-C(19)-C(14)
N(1)-C(19)-C(18)
C(14)-C(19)-C(18)
Al-0(2)-C(28)
Al-N(2)-C(21)
Al-N(2)-C(29)
C(21)-N(2)-C(29)
N(2)-C(21)-C(22)
N(2)-C(21)-H(21)
C(22)-C(21)-H(21)
C (21) -C (22) -C (23)
96.9(3)
92.0 (3)
93.9(3)
173.9(3)
90.1(3)
171.5(3)
95.3 (3)
91.8 (3)
84.1(3)
114.3(5)
130.9 (8)
111.2 (5)
117.9 (8)
123.0(11)
112.9 (11)
123.6(11)
118.5(11)
123.9(13)
117.3 (12)
121.5(11)
114.4 (17)
123.9(19)
127.0(11)
116.5 (12)
116.5(9)
118.0(11)
104.6(12)
135.8(15)
124.4 (12)
125.2(16)
110.3 (12)
119.2 (9)
118.9(11)
120.2(11)
123.8 (8)
118.7(8)
117.5(9)
122.6(8)
113.0 (8)
124.4 (9)
118.1(6)
132.2(6)
109.7(5)
118.2(7)
122.1(9)
117.9(10)
119.6(10)
118.2(11)
C (23) -C (24) -C (29)
C (25) -C (24) -C (29)
C(24)-C(25)-C(26)
C(24)-C(25)-H(25)
C(26)-C(25)-H(25)
C(25)-C(26)-C(27)
C(25)-C(26)-H(26)
C(27)-C(26)-H(26)
C(26)-C(27)-C(28)
C (26) -C (27) -H (27)
C(28)-C(27)-H(27)
0(2) -C (28) -C (27)
0(2) -C (28) -C (29)
C (27) -C (28) -C (29)
N (2) -C (29) -C (24)
N (2) -C (29) -C (28)
C(24)-C(29)-C(28)
Al-0(3)-C(38)
Al -N (3) -C (31)
Al-N(3)-C(39)
C (31) -N (3) -C (39)
N (3) -C (31) -C (32)
N (3) -C (31) -H (31)
C (32) -C (31) -H (31)
C (31) -C (32) -C (33)
C(31)-C(32)-H(32)
C(33)-C(32)-H(32)
C(32)-C(33)-C(34)
C(32)-C(33)-H(33)
C (34) -C (33) -H (33)
C(33)-C(34)-C(35)
C (33) -C (34) -C (39)
C(35)-C(34)-C(39)
C (34) -C (35) -C (36)
C(34)-C(35)-H(35)
C(36)-C(35)-C(35)
C (35) -C (36) -C (37)
C(35)-C(36)-H(36)
C (37) -C (36) -H (36)
C (36) -C (37) -C (38)
C (36) -C (37) -H (37)
C (38) -C (37) -H (37)
0(3) -C (38) -C (37)
0(3) -C (38) -C (39)
C (37) -C (38) -C (39)
N (3) -C (39) -C (34)
N (3) -C (39) -C (38)
C (34) -C (39) -C (38)
115.2 (9)
117.5(9)
120.9(9)
118.2(11)
120.9(13)
119.6(10)
128.4(11)
111.3(10)
123.4 (10)
121.7(11)
114.9(10)
127.7(9)
116.3(8)
116.1 (9)
123.3 (8)
114.1(7)
122.7(8)
122.7(8)
133.6 (6)
110.2(5)
116.1(7)
122.8 (9)
116.9(9)
120.2 (10)
102.2 (11)
122.5(11)
116.8(11)
120.1(10)
109.8(14)
130.0 (14)
127.1(10)
115.8(10)
117.1(10)
122.6 (10)
114.0(10)
123.1(11)
120.3(10)
129.7(10)
109.7(9)
119.9(9)
123.1(9)
116.5(9)
123.9(9)
118.6(8)
117.5(8)
124.9(8)
112.4(7)
122.7(9)


36
Effects of Exogenous Cofactors on Inducible Nitric Oxide
Synthase Activity Induced bv Lipopolvsacchari.de and
Interferon-y
Previous studies have suggested that the inability of
IFN to induce iNOS activity by itself in certain cells like
vascular smooth muscle may be due to lack of coinduction of
BH4 synthesis (Gross and Levi, 1992, Hattori and Gross,
1993). Thus, to determine whether the failure of IFN to
stimulate MCT iNOS activity in the absence of LPS was due to
its inability to induce synthesis of BH4 or lack of other
cofactors, I measured iNOS activity in crude cytosolic
extracts of MCT cells after the in vitro addition of
saturating concentrations of NADP (1 mM) FAD (4 |iM) and BH4
(10 jiM) .
Inter f eron-y-treated MCT cells failed to show iNOS
activity even when this was measured in vitro after all the
required cofactors had been supplied (figure 2-6). Thus, the
apparent inability of IFN alone to stimulate MCT iNOS
activity does not appear to be primarily due to lack of BH4
synthesis or availability of other cofactors.
Effects of Lipopolvsaccharide and Interferon-y on Nitrite
Production
To correlate my findings with other methods of measuring
iNOS activity, I measured nitrites accumulated by MCT cells
after 48 h incubation with IFN alone (100 U/ml), LPS alone
(0.1 ng/ml) or IFN at concentrations of 1, 5, 10, 50 and 100
U/ml in combination with 0.1 (ig/ml LPS.


69
has also been reported that the induction of macrophage iNOS
is dependent on the activation and nuclear translocation of
NF-kB (Xie et al., 1994).
Nuclear factor-KB proteins are a ubiquitous group of
transcription factors that exist mainly as inactive dimers in
the cytoplasm of cells bound to inhibitory proteins known as
inhibitors of kappa-B (IkB) (Muller et al. 1993). The most
important subunits of NF-kB are p50, p65 and c-Rel. Upon
cell activation, IkB dissociates from the NF-kB dimer, through
a process requiring both phosphorylation and proteolysis,
allowing this to migrate to the nucleus where it binds to its
DNA recognition site in the promoter region of various genes
to initiate transcription (Muller et al. 1993, Henkel et
al. 1993). The induction of macrophage iNOS by LPS is
dependent on the nuclear translocation of NF-kB heterodimers
containing mainly c-Rel and p65 subunits of NF-KB (Xie et
al., 1994). The c-Rel component of NF-kB had previously been
reported to have only modest transactivating activity (Muller
et al., 1993). Therefore, transactivation of the iNOS gene
constitutes an important novel role for this NF-kB protein in
macrophages.
Renal tubule epithelium has recently been found to
express iNOS but the regulation of this enzyme in the kidney
has not been extensively investigated (Amoah-Apraku et al.,
1993, Markewitz et al., 1993). In this study I examined the
role of NF-kB in the induction of renal epithelial iNOS and


124
Kwon, N.S., Nathan, C.S. and Stuehr, D.J. Reduced biopterin
as a cofactor in the generation of nitrogen oxides by murine
macrophages. J. Biol. Chem. 264: 20496-20501, 1989.
Langrehr, J.M., Hoffman, R. A. Lancaster Jr., J.R. and
Simmons, R.L. Nitric oxide A new endogenous
immunomodulator. Transplantation 55: 1205-1212, 1993 [a].
Langrehr, J.M., White, D.A. Hoffman, R.A. and Simmons, R.L.
Macrophages produce nitric oxide at allograft sites. Ann.
Surg. 218: 159-166, 1993 [b].
Laychock, S.G., Mdica, M.E. and Cavanaugh, C.T. L-Arginine
stimulates cyclic guanosine 3',5'-monophosphate formation in
rat islets of Langerhans and RINm5F insulinoma cells:
evidence of L-arginine:nitric oxide synthase. Endocrinology
129: 3043-3052, 1991.
Leclair, K.P., Blanar, M.A. and Sharp, P.A. The p50 subunit
of NF-kB associates with the NF-IL6 transcription factor.
Proc. Natl. Acad. Sci. USA. 89: 8145-8149, 1992.
Levillain, 0., Hus-Citharel, A., Morel, F. and Bankir, L.
Arginine synthesis in mouse and rabbit nephron: localization
and functional significance. Am. J. Physiol. 264 (Renal Fluid
Electrolyte Physiol. 33): F1038-F1045, 1993.
Lew, D.J., Decker, T., Strehlow, I. and Darnell, J.E.
Overlapping elements in the gualylate-binding protein gene
promoter mediate transcriptional induction by alpha and gamma
interferons. Mol. Cell Biol. 11: 182-191, 1991.
Lind, D.S., Copeland III, E.M. and Souba, W.W. Endotoxin
stimulates arginine transport in pulmonary artery endothelial
cells. Surgery 114: 199-205, 1993.
Lipton, S.A., Choi, Y., Pan, Z., Lei, H., Chen, H.V., Sucher,
N.J., Loscalzo, J., Singel, D.J. and Stamler, J.S. A redox-
based mechanism for the neuroprotective and neurodestructive
effects of nitric oxide and related nitroso-compounds. Nature
364: 626-632, 1993.
Lowenstein, C.J., Alley, E.W., Raval, P., Snowman, P.,
Snyder, S.H., Russel, S.W. and Murphy, W.J. Macrophage
nitric oxide synthase gene: two upstream regions mediate
induction by interferon-gamma and lipopolysaccharide. Proc.
Natl. Acad. Sci. USA 90: 9730-9734, 1993.
Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J.
Protein measurement with the Folin phenol reagent. J. Biol.
Chem. 193: 265-275, 1951.


97
polypropylene tube on ice, was added 5 ng plasmid DNA for 10
min and then heat-shocked for 45 seconds at exactly 42 C.
The tube was transferred to ice for 2 min and 900 |il SOC
medium (per 100 ml: bacto-tryptone 2 g, bacto-yeast extract
0.5 g, 1 M KC1 0.25 ml, 1 ml each of 1 M MgCl2, 1 M NaCl, 1 M
MgSC>4 and 2 M glucose) was added and incubated at 37 C with
shaking at 225 rpm for 60 min. Then, 100 |xl aliquots of the
transformation mix were plated onto LB plates (per liter:
bacto-yeast extract 5 g, bacto-tryptone 10 g, NaCl 5 g, agar
15 g, pH 7.5) with ampicillin (100 |ig/ml) and incubated at 37
C overnight. Filters were then lifted from the plates and
hybridized to 32p-iabeled PCR product. Colonies hybridizing
to the probe were picked up for miniprep amplification.
Preparation of Constructs of Inducible Nitric Oxide Synthase
A DNA fragment containing the promoter region of iNOS
from mouse genomic DNA was subcloned into the pCAT-Basic
vector (figure 5-2B) at the Hind III and Sal I to yield the
construct pAiNOS-CAT (figure 5-2A). The construct pBiNOS-CAT
was generated by the Spe I and the downstream Sal I fragment
and the pCiNOS-CAT, the Xba I and the upstream Hind III
fragment (figure 5-2A). These fragments were generated by
specific restriction endonuclease digestion with the
appropriate restriction enzymes. All the constructs were
sequenced to ensure that the appropriate deletions and
ligations have been obtained.


108
A
-1588
Hind
III
h
Xba I
Spe I
+ 141
JcatI PANOS-CAT
Sal I
-478
+ 141
CAT
pBiNOS-CAT
-1588
-421
CAT
pCiNOS-CAT
B
Figure 5-2. A: The CAT constructs. Dashed lines represent
deleted portions. B. pCAT-Basic Vector circle map and
sequence reference points.


20
stimulated response element (y-IRE) and interferon-a-
stimulated response element {ISRE) (Eilers et al., 1993,
Martin et al. 1994). Recently, consensus sequences for
binding of several transcription factors involved in
conferring responsiveness to IFN have been identified in the
upstream promoter region of the iNOS gene thereby raising the
possibility of complex and tissue-specific regulation of the
expression of this gene by IFN (Lew et al., 1991, Lowenstein
et al., 1993, Xie et al. 1993). These sequences, which
include NF-kB, IRF-1, y-IRE, ISRE, GAS and AP-1 binding sites,
are all potential targets for IFN- or LPS-stimulated
signaling factors (Lew et al., 1991, Lowenstein et al., 1993,
Xie et al., 1993) and some reports even suggest that LPS-
induced macrophage iNOS expression is dependent on activation
and nuclear translocation of NF-kB (Xie et al. 1994). In
this study, I therefore focused on the role of NF-kB in the
mechanism of synergistic induction of iNOS activity by IFN
and LPS in proximal tubule epithelial cells.
The central hypothesis of this study was that IFN
potentiates the effects of LPS on iNOS induction by
modulating the LPS-stimulated activation and nuclear
translocation of dimers of NF-kB. I proposed that this
synergism may be due to IFN synergistically potentiating the
activation and nuclear translocation of NF-kB induced by LPS,
or activating different subunits which then dimerize with
those induced by LPS to produce synergistic iNOS gene
transcription. I tested this hypothesis by examining the


86
139 kD-
Figure 4-3. Inducible NOS protein expression in MCT cells
before and after 12 h of treatment with LPS (1 |g/ml) alone
or in combination with IFN (100 U/ml). Blot is
representative of three separate experiments.


118
Table 8-1. Crystallographic Data for X
Complex
Na+ (C4H506) '* H20
Formula
C4H707Na
MW
190.08
Crystal system
Orthorhombic
Space group
P212121
a,
7.2393
b, A
8.6735
c,
10.5928
Vc, 3
665.12
Z
4
Dcalc* g/cm3
1.899
Radiation
Mo Ka
X,
0* 71069
HiMoKoi) cm'1
2.24
F(000)
388
Crystal dimensions
0.8 x 0.8 x 0.6 mm
Observed reflections
1152
Number of parameters
109
Final R
4.38
Rw
5.62


16
The solutions were combined to give a clear, colorless
solution. NaHC03 was added slowly as a solid until a thick,
white precipitate formed. The pH of the mixture measured 5.0.
The result was identical for a concentrated solution of NaOH.
The solid was collected by filtration through a glass frit,
washed thoroughly with water and dried for further analysis.
6-Methvlpicolinic acid (mpic)
Ligand reactivity. 6-Methylpicolinic acid has a
solubility similar to picolinic acid. With a pKa of 5.8, it
is only a slightly weaker acid the picolinic acid.
3
Figure 2-5. 6-Methylpicolinic acid (mpic).
Preparation of Al/mpic complex (IV) Al (N03) 3* 9H20 (1.24
g, 3.3 mmole) was dissolved in 15 mL H20. Mpic (1.37 g, 10.0
mmole) was dissolved in 20 mL H20 to produce a pale beige
solution. The solutions were combined to give a clear, almost
colorless solution. NaHC03 was slowly added as a solid until
a thick off-white precipitate formed. The pH of the resulting
solution was 6.0. The solid was collected by filtration
through a glass frit, washed thoroughly with H20 and dried for
further analysis.


CHAPTER THREE
GUANOSINE TRIPHOSPHATE CYCLOHYDROLASE I REGULATES NITRIC
OXIDE SYNTHESIS IN RENAL PROXIMAL TUBULES
Introduction
Tetrahydrobiopterin is an essential cofactor of NOS
because it is needed in the formation of the active dimeric
enzyme from iNOS monomers (Tayeh and Marietta, 1989, Baek et
al. 1993) and is tightly bound to the enzyme (Schmidt et
al., 1993). With the exception of macrophages, many cells
that possess iNOS have low BH4 in the resting state and NO
production is limited by their ability to synthesize this
cofactor (Di Silvio et al. 1993, Werner-Felmayer et al. ,
1990).
In proximal tubule epithelial cells, there is marked
synergism between LPS and IFN in stimulating iNOS activity.
However, unlike what is observed in macrophages, each of
these inducers, by itself, stimulates little or no iNOS
activity in these cells (Amoah-Apraku et al., 1993, Markewitz
et al., 1993). Synergism between cytokines and endotoxin in
their effects on the induction of iNOS expression has been
commonly observed in many other tissues (Blough and Zafirou,
1985, Deng et al. 1993, Pfeilschifter and Schwarzenbach,
1990, Szab et al. 1993). This synergism, that appears to
vary among different tissues and species, results in some
54


32
peroxidase substrate solution, diaminobenzidine. The cells
were then counterstained with hematoxylin, examined and
photographed under a light microscope (Zeiss Photomicroscope
II) .
Results
Measurement of Inducible Nitric Oxide Synthase Activity:
r3HlL-Arainine to 3H1L-Citrulline Conversion Time Course
To determine the presence of iNOS activity, MCT cells
were treated with a combination of LPS (0.1 |ag/ml) and IFN
(100 U/ml) for 24 h in the presence or absence of Nw-nitro-L-
arginine (NNA, 300 |i.M) followed by measurement of the
conversion of [3h]L-arginine to [3h]L-citrulline over time.
These drug concentrations were selected based on previous
experiments on NOS activity in macrophages done in our
laboratory. Inducible NOS activity was measured after
incubation with [3h]L-arginine for 0, 10, 20, 30, 40, 50, 60
and 90 minutes. Control (untreated) and NNA-treated cells
were also incubated with [3h]L-arginine for 0, 10, 30, 60 and
90 min and iNOS activity measured.
Conversion of [3h]L-arginine to [3h]L-citrulline peaked
after 30 minutes (n=6) in LPS/IFN-induced cells (figure 2-1).
In control cells, there was no significant conversion whereas
NNA effectively blocked the conversion of [3h]L-arginine to
[3h]L-citrulline in LPS/IFN-induced cells. Thus, iNOS
activity was shown to be present in the MCT cell line after
induction with LPS and IFN. In subsequent experiments, iNOS


8
NO synthesis through a feedback inhibition. As described
elsewhere, substrate and cofactor availability may also play
an important role in the regulation of NO production.
Pharmacologically, there are agents that promote or
inhibit NO release. Some agents mimic NOS by releasing NO.
These agents, termed NO donors, include organic nitrates and
sodium nitroprusside. They have been used clinically for
several years.
There are several classes of NOS inhibitors. These
include: (i) substrate analogues that are N^-subst ituted
analogues of arginine such as N^-mono-methyl-L-arginine (L-
NMMA) and N-nitro-L-arginine (NNA); these usually act as
competitive inhibitors of L-arginine when added along with
the necessary cofactors (Moneada et al., 1991); (ii) covalent
modifiers of NOS such as aromatic diphenylene iodoniums that
tend to compete with the nucleotide cofactors for binding to
NOS (Stuehr et al. 1991 [b]); (iii) carbon monoxide, which
inhibits NOS activity by binding to the heme moiety of the
enzyme (White and Marietta, 1992); (iv) compounds that bind
to calmodulin thereby impeding its binding to NOS, such as
calcineurin and trifluoroperazine; they are therefore
specific for cNOS which is dependent on Ca2+-calmodulin for
activity (Bredt and Snyder, 1990); (v) agents that inhibit
NOS activity by interfering with cofactor or substrate
availability such as substituted pyrimidines (e.g., 2,4-
diaminohydroxypyrimidine, DAHP); these inhibit GTP
cyclohydrolase I, the rate-limiting enzyme in BH4 synthesis


CHAPTER 6
A PENTAGONAL BIPYRAMIDAL SODIUM COMPOUND:
SYNTHESIS AND CRYSTAL STRUCTURE
Introduction
A naturally occurring ligand, 2,6-pyridinedicarboxylic
acid, shown in Figure 6-1, can act as an electron donor from
the nitrogen as well as the two carboxylate oxygens. Known by
the trivial name dipicolinic acid, this molecule has been
reported in a variety of compounds as a tridentate ligand.45'51
There are several modes of coordination of the carboxylate and
carboxylic acid groups. Dipic has been shown to coordinate
metal ions via the carbonyl oxygen in the carboxylic acid and
by the hydroxy group in the carboxylate ion. In addition, the
carboxylate oxygen atoms sometimes act as donor atoms to a
second metal ion, thereby linking together chains of
asymmetric units.47,48
HO OH
Figure 6-1. Dipicolinic acid.
90


14
The Renal System
In the kidney, inducible and constitutive NOS protein or
mRNA have been detected in mesangial cells, macula densa
cells of the juxtaglomerular apparatus, proximal tubule
cells, the entire collecting duct, as well as other
epithelial lining of the nephron (Marsden and Ballerman,
1990, Mundel et al., 1992, Terada et al., 1992, Tojo et al.,
1994 [b], Amoah-Apraku et al., 1993, Markewitz et al., 1993,
Morrissey et al., 1994).
Renal NO is involved in the regulation of renal vascular
resistance (Romero et al. 1992), medullary and glomerular
microcirculation and macula densa-mediated tubuloglomerular
feedback (Zatz and de Nucci, 1991, Brezis et al. 1991,
Wilcox et al. 1992). In addition, active induction of
nitric oxide inhibits proximal tubule Na+/K+-ATPase and ion
transport across renal tubule cell membranes (Guzman et al.,
1995, Stoos et al. 1994). Induced NO production also
inhibits H+-ATPase activity in rat cortical collecting ducts
(Tojo et al. 1994 [a]), suggesting that NO is probably
involved in the regulation of proton and bicarbonate
transport in the cortical collecting duct. Nitric oxide may
also mediate renal injury during allograft rejection, acute
tubular ischemia/reperfusion injury and autoimmune
glomerulonephritis (Langrehr et al. 1993 [b] Yu et al. ,
1994, Weinberg et al. 1994).


10
groups of other proteins such as ferritin which then acts as
a storage pool for NO (Reif and Simmons, 1990). The induced
(Type II) enzyme remains tonically activated and continuously
produces NO for the life of the active enzyme.
Nitric oxide reacts with O2 in both gaseous and aqueous
phases to form nitrogen dioxides (NO2) (Marietta et al.,
1990). Nitric oxide formed under reducing conditions as the
free molecule (NO')/ reacts with the superoxide anion (02_)
in aqueous solutions to yield peroxynitrite (ONOO-), which in
part mediates its cytotoxicity (Blough and Zafirou, 1985).
Under oxidizing redox conditions, NO is formed as the
nitrosonium ion (NO+) and reacts with thiol-containing
proteins to form S-nitrosothiols (RS-NO). In N-methyl-D-
aspartate type glutamate (NMDA) receptors, NO reacts with
cell surface thiols to form RS-NOs that afford protection
from receptor-mediated neurotoxicity by down-regulating
harmful receptor-mediated Ca2+ influx into the cells (Lipton
et al. 1993, Dolye et al. 1981, Olson et al. 1981, Saran
et al. 1990). Nitric oxide readily forms complexes with
transition metal ions and heme-containing proteins like
hemoglobin (Sharma et al. 1983). These reactions form the
basis for several NO assays. The inhibition by NO of iron-
containing mitochondrial enzymes like aconitase of the Krebs
cycle, succinate-ubiquinone oxidoreductase and NADPH-
ubiquinone oxidoreductase of the electron transport chain
occurs through the nitrosylation of their iron-sulfur (Fe-S)
complexes (Dimmeler et al., 1992, Stuehr et al., 1989,


5
R = H Al(mpp)3
=CH3 A1(dpp)3
= n-CHu Al (mhpp) 3
Figure 1-1. Representation of Aluminum Complexes with 3-
Hydroxy-4-pyridinone and its Derivatives. Nelson, Karpishin,
Rettig and Orvig, Inorganic Chemistry, 1988, 27, 1045-1051.
Aluminum Coordination Compounds
Complexes of Al(III) have been reported with many 0,0
donor ligands.17-23 For the derivatives of 3-hydroxy-4-
pyridinone, the complexes with the Al(III) ion, represented in
Figure 1-1, were synthesized by the addition of a base or
proton scavenger to a solution of ligand and Al(III).17 By a
similar technique, complexes were also formed with maltol and
its derivatives, isomaltol, oxalic acid, tropolone and 2,4-
pentanedione.18-22 Each of the compounds was characterized by
x-ray diffraction techniques as six coordinate tris chelates
with each ligand forming a five- or six-membered ring with the
central metal ion. The structure, shown in Figure 1-2, for
the complex of Al(III) with citric acid revealed a trinuclear
complex ion with each Al(III) in a distorted octahedral
environment.23


60
Table 4-9.
Deviations
Al-O(l)
Al-0(2)
Al-0(3)
N(1) -C(11)
C(ll)-C(12)
C(12)-C(13)
C(13)-C(14)
C(14)-C(15)
C(15)-C(16)
C(16)-C(17)
C(17)-C(18)
C(18)-C(19)
N(2) -C (21)
C(21)-C(22)
C(22)-C(23)
C(23)-C(24)
C(24)-C(25)
C(25)-C(26)
C (26) -C (27)
C (27) -C (28)
C (28) -C (29)
N(3)-C(31)
N(3) -C(31)
C(31)-C(32)
C(32)-C(33)
C (33) -C (34)
C(34)-C(35)
C (35) -C (36)
C (36) -C (37)
C (37) -C (38)
C(38)-C (39)
Table 4-10
Deviations
0(1)-Al-N(1
0(1) -Al-0 (2
N (1) -Al-0 (2
0(1) -Al-N (2
N(1) -Al-N(2
0(2) -Al-N(2
Bond Lengths (A) for II. Estimated Standard
are given in Parentheses.
1.884 (6)
Al-N(1)
2.026 (8)
1.842 (6)
Al-N(2)
2.077(8)
1.845(6)
Al-N(3)
2.026(7)
1.310 (13)
0(1)-C(18)
1.329 (12)
1.395 (18)
N(1)-C(19)
1.365 (12)
1.352 (22)
C(ll)-H(ll)
0.872 (12)
1.372 (19)
C(12)-H(12)
1.054 (13)
1.445(19)
C (13) -H (13)
0.761(12)
1.353(17)
C(14)-C(19)
1.407(14)
1.423(19)
C(15)-H(15)
0.793(11)
1.360 (14)
C(16)-H(16)
0.888(12)
1.413 (12)
C(17)-H(17)
0.926(10)
1.347(13)
0(2)-C(28)
1.320(11)
1.393 (16)
N(2)-C(29)
1.363(10)
1.332 (17)
C (21) -H (21)
1.028(10)
1.413 (16)
C(22)-H(22)
0.930 (12)
1.431(16)
C(23)-H(23)
0.670(10)
1.367 (16)
C (24) -C (29)
1.383(12)
1.387(15)
C(25)-H(25)
0.818(10)
1.375 (14)
C(26)-H(26)
0.983 (10)
1.434 (13)
C(27)-H(27)
0.839(11)
1.339 (12)
0(3) -C (38)
1.328(11)
1.339 (12)
N (3) -C (39)
1.372(11)
1.385(15)
C(31)-H(31)
0.940(10)
1.372 (18)
C(32)-H(32)
0.953(12)
1.392 (19)
C(33)-H(33)
0.910(11)
1.413 (17)
C(34)-C(39)
1.409(12)
1.357 (16)
C(35)-H(35)
1.059(10)
1.441 (15)
C(36)-H(36)
1.080(11)
1.406 (14)
C (37) -H (37)
1.094(10)
1.435 (13)
0(4) -C (4)
1.316(21)
Bond Angles () for II. Estimated Standard
are given in Parentheses.
)
82.8(3)
)
168.3 (3)
)
92.5(3)
)
87.6 (3)
)
92.7(3)
)
81.9 (3)
C(21)-C(22)-H
C(23)-C(22)-H
C(22)-C(23)-C
C(22)-C(23)-H
C (24) -C (23) -H
C(23)-C(24)-C
(22)
114 .
.7
(11)
(22)
127,
.0
(12)
(24)
122,
.9
(10)
(23)
112,
.3
(15)
(23)
120,
.2
(15)
(25)
127,
.2
(9)


CHAPTER FOUR
NUCLEAR FACTOR-kB REGULATES INDUCIBLE NITRIC OXIDE SYNTHASE
IN RENAL TUBULE EPITHELIUM
Introduction
Expression of iNOS is subject to strict and highly
tissue-specific transcriptional regulatory control (Nathan
and Xie, 1994 [a,b], Nussler et al. 1994 ). For example,
whereas LPS induces high levels of iNOS expression in
macrophages and microglia (Stuehr and Marietta, 1994, Xie et
al., 1993, Chandler et al. 1994), it induces only modest
levels of iNOS in hepatocytes, vascular smooth muscle and
renal epithelial cells and a combination of cytokines is
usually required for full expression of iNOS activity
(Nussler and Billiar, 1993, Szab et al., 1993, Amoah-Apraku
et al. 1993). Thus, the expression of iNOS appears to be
regulated differently in parenchymal cells compared to cells
such as macrophages and microglia whose main role is to
mediate immune responses.
Recent studies describe the presence in the murine
macrophage iNOS promoter of consensus sequences for the
binding of endotoxin and IFN-stimulated signaling proteins
including NF-kB and interferon-y regulatory factor (IRF-1)
(Xie et al. 1993), which may potentially constitute the
basis for the synergism observed between iNOS inducers. It
68


17
Pvrazinoic acid (pza)
Ligand reactivity. Pyrazinoic acid is slightly soluble
in hot water and ethanol and insoluble in organic solvents.
HO
Figure 2-6. Pyrazinoic acid (pza).
Preparation of Al/pza complex (V) Pza (3.00 g, 24.2
mmole) was added to 100 mL H20 but did not dissolve. In a
separate beaker, Al (N03) 3* 9H20 (3.02 g, 8.05 mmole) was
dissolved in 25 mL 95 % ethanol. The solutions were combined
and stirred to yield a 125 mL clear and colorless solution
with a white suspension. As NaHC03 (2.03 g, 24.2 mmole) was
slowly added as a solid, the suspension disappeared and the
mixture became a clear pale pink solution. When all the
NaHC03 had been added, the clear pink solution had a pH of
5.0. Additional NaHC03 was added until a solid white
precipitate formed. The pH of the opaque mixture measured
7.5. The solid was collected by filtration through a medium
glass frit and washed thoroughly with water and hot ethanol.
The solid was dried in a desiccator for further analysis.


66
DAHP + +
Sepiapterin + +
Methotrexate + +
Figure 3-4. Effects of methotrexate and sepiapterin on
nitrite production during GTPCH blockade with DAHP in MCT
cells. Cells were treated with LPS (0.1 (ig/ml) and IFN (100
U/ml) for 12 h in the presence of the indicated drugs.
Nitrite concentrations in culture medium from control cells
were less than 2 nmol/mg of protein per 12 h, (mg: mg of cell
protein, n=9).


78
these results indicate that the synergism between LPS and IFN
is not due to complementary effects of the activation of the
different NF-KB proteins by these agents.
Discussion
In these studies I have shown that LPS, IFN or their
combination cause rapid activation and nuclear translocation
of NF-kB complexes in renal tubule epithelium, consisting
mainly of p65 but with smaller amounts of p50 protein. This
is in agreement with what has been observed for most
functional heterodimers of NF-kB (Baeuerle and Baltimore,
1989, Ganchi et al., 1993) but in contrast to what is seen in
macrophages, where the p65/c-Rel heterodimer is reported to
play an important role in LPS-induced iNOS activity (Xie et
al., 1994). It is of interest to note that although LPS and
IFN each induced nuclear translocation of NF-KB complexes,
IFN failed to induce iNOS mRNA and LPS caused only a small
induction of iNOS mRNA transcription, not reflected in either
increase in iNOS protein synthesis or nitrite production.
These results indicate that nuclear translocation of NF-KB
alone is not sufficient for the induction of iNOS activity in
renal epithelium. Nevertheless, since cotreatment of LPS/IFN
with PDTC completely prevented the induction of iNOS
activity, it can be argued that NF-kB, though not sufficient
on its own, is necessary for iNOS expression in renal
epithelium.


26
Apraku et al. 1993, Markewitz et al. 1993, Morrissey et
al., 1994). Nitric oxide has also been implicated in several
renal pathophysiologies such as acute interstitial nephritis,
glomerulonephritis and renal transplant rejection (Ketteler
et al. 1994, Langrehr et al. 1993 [ a, b ] ) However, the
mechanisms that regulate NOS in the various renal cells have
not been extensively studied.
My preliminary studies suggest strong synergism between
LPS and IFN in stimulating iNOS activity in renal epithelial
cells although each, by itself, stimulates little or no iNOS
activity (Amoah-Apraku et al 1993) This type of
synergism, which appears to vary among different tissues and
species, results in some cases from cooperative interactions
between iNOS inducers, one of which may lack the ability to
induce the synthesis of cofactors required for full NOS
activity. For instance, whereas IFN stimulates marked NO
production by murine peritoneal macrophages, it is by itself
ineffective in rat aortic vascular smooth muscle (Deng et
al., 1993, Hattori and Gross, 1993, Gross and Levi, 1992)
where it is limited by the inability to coinduce BH4
synthesis (Hattori and Gross, 1993, Gross and Levi, 1992).
This situation is remedied by the addition of LPS which
induces BH4 synthesis. In this study, I investigated the
presence of, as well as the characterization of iNOS in a
murine proximal tubule cell line, MCT cells, and in primary
cultures of rat proximal tubules.


41
Gross, 1993). Since iNOS requires substrate concentrations
of BH4 for full enzymatic activity, the ability of a cytokine
to stimulate the synthesis of GTP cyclohydrolase I and that
of a given cell to carry out the synthesis of BH4 are both
critical in determining the magnitude of ensuing NO formation
(Di Silvio et al., 1993, Hattori and Gross, 1993). Thus, in
this study, the lack of stimulation of NO production by IFN
in intact MCT cells could have been due to the inability of
this cytokine to stimulate GTP cyclohydrolase I in a
situation analogous to what is seen in aortic vascular smooth
muscle. Under these circumstances, the addition of LPS could
have stimulated GTP cyclohydrolase I activity and BH4
synthesis thereby providing the missing cofactor. Therefore,
if the failure of IFN to stimulate iNOS activity was due to
lack of BH4, then providing an exogenous source of this
cofactor to cytosolic extracts of IFN-treated MCT cells would
be expected to fully restore iNOS activity. However, iNOS
activity was undetectable in IFN-treated MCT cell homogenates
despite the presence of saturating concentrations of
exogenously added BH4. Furthermore, the absence of iNOS mRNA
and iNOS protein immunoreactivity after IFN treatment is
consistent with the results of my functional studies and
indicates that, in contrast to what is seen in mouse
peritoneal macrophages, IFN does not induce MCT iNOS.
In the kidney, NO may mediate several physiological as
well as pathological functions. Recent reports suggest that
induced NOS decreases Na+/K+-ATPase and H+-ATPase activity


3
Stuehr, 1993, Wang et al., 1993). In contrast, calmodulin is
tightly bound to each subunit of iNOS even at very low
intracellular Ca2+ concentrations, an event that probably
occurs during synthesis, resulting in the permanent
activation of the enzyme (Cho et al., 1992). This makes the
addition of exogenous Ca2+ nonessential for iNOS activity.
The induction of iNOS by cytokines and endotoxin is
transcriptionally regulated and is inhibited by
glucocorticoids, interleukin (IL)-4, 8 and 10; transforming
growth factor-(3 (TGF-p) ; macrophage deactivating factor; and
protein synthesis inhibitors (Nussler and Billiar, 1993,
Radomski et al., 1990, Noonan and Noonan, 1977, Junguero et
al. 1992). It is probably only limited by substrate and
cofactor availability, protein turnover and product
inhibition. All NOS isoforms are homodimers of subunits
ranging from 130 to 160 kDa (Schmidt et al. 1991, Bredt et
al., 1991, Xie et al., 1992). The N-terminal portion of all
NOS isoforms contains binding motifs for NADPH, flavin-
mononucleotide (FMN) and flavin-adenine dinucleotide (FAD),
showing 50% amino acid identity with each other and 30-40%
overall sequence identity to NADPH-cytochrome P450 reductase
(Dinermann et al., 1993, Schmidt et al., 1993, Stuehr et al.,
1991 [a]). Additional binding sites have been postulated for
tetrahydrobiopterin (BH4), calmodulin and L-arginine (Schmidt
et al., 1993). All isoforms of NOS have consensus sites for
cAMP-dependent phosphorylation. Phosphorylation by protein
kinase C substantially diminishes cNOS activity but


95
emphasis on the NF-kB and IFN-activated protein binding sites
in the upstream and and NF-kB binding site in the downstream
areas in the promoter region of the iNOS gene.
Materials and Methods
Chemicals and Biologic Products
All restriction enzymes and other molecular biology
enzymes, DNA Wizard Miniprep, CAT Enzyme assay kit, were
purchased from Promega (Madison, WI).
Cloning of the Upstream Promoter Region
Two primers selected after careful study of the promoter
region of murine mac-iNOS, one designed with Hind III site at
the 5'-flanking end (BA I: 5 AGA AGC TTG ACT TTG ATA TGC ATA
TGC TGA AAT CCA T) and the other with a Sal 1 site at the 3'-
flanking end on the complementary DNA strand (BA II: 5' GAG
TCG ACG AAC AAG ACC CAA GCG TGA GGA A), were used to amplify
the 1.7 kilobase pair (kbp) promoter region from mouse
genomic DNA using the PCR (see DNA Amplification below). The
PCR product was treated with the Klenow fragment of DNA pol
I, restriction-digested with Hind III and Sal 1. The PCR
product was subsequently isolated by agarose gel
electrophoresis and DEAE-cellulose.
This PCR product was subcloned to the Hind III/Sal I
site of the pCAT-Basic and subjected to DNA sequence analysis
(Sanger Dideoxy Chain Termination, Sequenase Kit, IBI).


84
exist due to impurities if the synthesized compounds were the
tris complexes. Furthermore, the Al-27 NMR confirmed the
presence of more than one aluminum environment in all of the
samples except for V. As a result, the elemental percentages
would not match those of any particular compound.
Infrared Spectroscopy
The IR spectra for pic, mpic, pza and dipic and the
sodium salts were prepared on a Perkin-Elmer Fourier Transform
Infrared Spectrophotometer. IR spectra were also taken for
compounds III, IV, V and VII. The data for the carbonyl
stretching frequency in each compound is summarized in Table
5-5. Hypoxanthine was not included because it has no
carboxylic acid group.
Table 5-5. FTIR Carbonyl Peak Data, cm'1.
Ligand
Unreacted
ligand
Sodium
salt
Disodium
salt
Aluminum
compound
Compound
pic
1715
1584
-
1672
III
mpic
1677
1587
-
1683
IV
pza
1713
1612
-
1678
V
dipic
1693
1634
1731
1618
1610
1666
VII
Each aluminum compound has a carbonyl stretching
frequency between 1660 and 1690 cm"1, between characteristic
carboxylic acid and carboxylate ion regions.52 For III and V,
the single peak occurs between the peaks for the sodium salt


12
1991, O'Dell et al. 1991. Bohrne et al. 1991, Schuman and
Madison, 1991, Nowak, 1992).
Nitric oxide also mediates glutamate neurotoxicity and
neuroprotection, a phenomenon dependent on its redox state
(Lipton et al. 1993). Reactions of NO with superoxide to
form peroxynitrite (0N00-) can lead to neurotoxicity and NMDA
receptor-mediated neuronal injury due to Ca2 + dependent
stimulation of NOS can be attributed, at least in part, to
this reaction (Lipton et al, 1993) In contrast, N0-
containing compounds in alternative redox states may prevent
neurotoxicity by mechanisms involving S-nitrosylation of
critical thiol groups at the NMDA receptor redox modulatory
site to down-regulate channel activity and excessive Ca2 +
influx (Lipton et al. 1993, Dolye et al. 1981, Olson et
al., 1981, Saran et al. 1990).
Immunocytochemical techniques using anti-cNOS antibodies
have demonstrated the presence of NOS in intestinal myenteric
plexus and adrenal medullary ganglia (Snyder and Bredt,
1991). Non-adrenergic non-cholinergic (NANC) neurons in the
stomach and ileocolonic junction release an NO-like substance
when stimulated and other NO-responsive sites include the
penile corpus carvernosum, stomach, duodenum and esophagus
(Garthwaite, 1991), implying that NO may be playing an
important role as a neurotransmitter in these organs.


101
Promoter Activity of the 1.7 Kilobase Pair Fragment: and its
Truncated Plasmid Constructs
To investigate the role of NF-kB in the induction of
iNOS gene transcription by LPS and IFN, the constructs
described above were fused with the promoterless CAT reporter
gene, the pCAT-Basic reporter vector and transiently
transfected into MCT cells.
As reflected in the CAT activity shown in figure 5-3,
pAiNOS-CAT, the untruncated construct, conferred CAT
responsiveness by LPS (1 |xg/ml) in combination with IFN (100
U/ml) in contrast to the native vector. Treatment with LPS
alone (1 |ig/ml) resulted in a 3.9-fold increase in CAT
activity and cotreatment with IFN (100 U/ml) potentiated the
induction of CAT activity to 6.6-fold, which was blocked by
the addition of PDTC (25 nM) (figure 5-4). Interferon-y alone
(100 U/ml) did induce a small but significant CAT activity
(figure 5-4). Taken together, these findings show that the
effect of LPS and IFN on the promoter region is
transcriptional and that it requires NF-kB.
The construct pBiNOS-CAT, consisting of the downstream
portion linked to the CAT gene conferred inducibility by LPS
(1 |ig/ml) and a combination of LPS (1 ^g/ml) and IFN (100
U/ml) but not by IFN alone. However, in contrast to pAiNOS-
CAT, the synergistic effect of IFN was lost (figure 5-5).
The construct pCiNOS-CAT which lacked the downstream NF-kB
binding site was devoid of promoter activity in response to
treatment with LPS, IFN or their combination (figure 5-6).


23
pursued. (i) Cloning of the promoter region of mouse iNOS
from mouse genomic DNA. Two primers selected after careful
study of the promoter region of mac-iNOS, one designed with a
Hind III site at the 5'-flanking end and the other with a Sal
1 site at the 3'-flanking end on the complementary DNA
strand, were used to clone the 1.7-kilobase pair (kbp)
promoter region from mouse genomic DNA using the polymerase
chain reaction (PCR). (ii) Preparation of deletion
constructs of the 1.7 kbp and their insertion into a
chloramphenicol acetyltransferase (CAT) basic vector reporter
system (pCAT-Basic). Constructs were prepared to include or
exclude the consensus binding sequences for NF-kB and IFN-
activated proteins to enable me study the role of these
transcription factors on iNOS induction in MCT cells. (iii)
Transfection of the iNOS promoter-pCAT-Basic reporter
constructs into MCT cells and functional analysis of this
reporter as an indicator of iNOS activity. Experiments
included the transfection of pCAT constructs into MCT cells,
and subsequent measurement and comparison of CAT and iNOS
activities in the transfected cells after treatment with the
various inducers or their combinations.
Statistical Analysis
Where indicated, statistical analysis was performed
using Student's t-test for paired and unpaired data, or
analysis of variance (ANOVA) and subsequent Scheffe's F-test
(StatView II, Abacus Concepts, Inc. Berkeley, CA) for


67
140 -i
Sepiapterin ((IM)
L/I + DAHP
Figure 3-5. Effects of sepiapterin on nitrite production by
MCT cells (48 h of induction) during GTPCH inhibition with
DAHP (6 niM) CON, Control, L/I, LPS plus IFN, (n=3).


103
bears the IRF-E, the binding site for IRF-1 demonstrated to
be critical for the synergistic role of IFN (Kami jo et al. ,
1994, Martin et al. 1994), as well as several other sites
for the binding of IFN-activated proteins such as y-IRE, GAS
and ISRE (Xie et al. 1993, Lowenstein et al. 1993). In
fact, in macrophages, site-directed mutagenesis that alter
the IRF-E site results in the loss of the synergistic effect
of IFN on LPS-stimulated iNOS activity (Martin et al., 1994).
Sequential deletion of the promoter region in constructs
transiently transfected into macrophages have shown that the
critical site for this IFN response may be located in the
region between -975 to -722 (figure 5-1) (Xie and Nathan,
1994). The response to the effect of LPS was similar in both
pAiNOS-CAT and pBiNOS-CAT. This suggests that the upstream
NF-kB binding site plays little or no role in the NF-kB-
mediated induction of gene transcription by LPS in MCT cells.
The inability of pCiNOS-CAT to elicit any response to
LPS, IFN or both may be due to the loss of the critical
downstream NF-kB binding site. The presence of the upstream
NF-kB binding site in addition to the IRF-E, GAS, ISRE and y-
IRE sites in this construct did not confer inducibility in
response to the immunokines, thus confirming that this NF-KB
binding site is irrelevant in NF-KB-mediated iNOS gene
transcription stimulated by LPS in MCT cells. Another reason
for the lack of response in this construct may be the loss of
the TATA box and the transcription initiation site, which
were deleted in preparing the construct. It must be stated


21
possibly good crystals. The discussion of crystal growth
techniques is presented in detail in Chapter 3.
Methods of Characterization of Aluminum Complexes
There are several techniques that can be utilized to
characterize an aluminum complex that has been synthesized.
These methods include elemental analysis, Infra-red, Al-27 NMR
and ICP emission spectroscopies, Fast Atom Bombardment Mass
Spectrometry (FABMS) and x-ray diffraction techniques. The
first three can be performed upon the insoluble compounds III
through VII. ICP emission spectroscopy, FABMS and x-ray
diffraction techniques require that the samples are soluble.
Elemental Analysis
Elemental analysis can be used to determine the
percentage of all of the elements in a compound. CHN, which
can be performed on solid samples, only determines the
percentages of carbon, hydrogen and nitrogen. From these
percentages, the ratios of the number of carbon to hydrogen to
nitrogen atoms present in the compound can be calculated.
Since hydrogen has a very low atomic weight, the percentages
and ratios for this element have a large relative error.
Therefore, the ratio of carbon atoms to nitrogen atoms
provides the most valid numerical data. Since each ligand has
a specific number of carbon and nitrogen atoms, the presence
of an organic solvent molecule or an ammonium ion in a
synthesized compound can easily be confirmed.


56
Tetrahydrobiopterin synthesis occurs via two distinct
pathways (figure 3-1, Werner et al. 1993): (i) a de novo
synthetic pathway that utilizes GTP as a precursor and in
which GTP cyclohydrolase I (GTPCH) is the rate-limiting
enzyme. This is the main source of BH4 for iNOS in vascular
smooth muscle and hepatocytes (Stuehr and Griffith, 1992);
and (ii) a salvage pathway in which preexisting
dihydropteridines like dihydrobiopterin and sepiapterin are
converted to BH4 by dihydrofolate reductase. Constitutive
NOS requires only catalytic amounts of BH4 for full activity
whereas iNOS, on the other hand, requires substrate amounts.
There may therefore be a coinduction of BH4 synthesis with
iNOS induction in many cells.
Because only low levels of GTPCH activity are present in
the kidney and its role in providing BH4 for processes such
as renal aromatic amino acid hydroxylation has not been
demonstrated (Bellahsene et al., 1984, Rao and Kaufman, 1986,
Davis et al., 1992), the main function of renal GTPCH remains
to be defined. The presence of iNOS has recently been
reported in proximal tubule and collecting duct epithelium
(Markewitz et al., 1993, Amoah-Apraku et al., 1993). Because
NO may mediate renal injury during allograft rejection,
autoimmune glomerulonephritis (Langrehr et al. 1993 [a,b],
Weinberg et al., 1994), Na+/K+-ATPase and H+-ATPase activity
and therefore ion and acid transport across the renal tubule
epithelium (Guzman et al., 1995, Tojo et al,. 1994 [a], Stoos
et al., 1994), it will be important to identify potential


108
A
-1588
Hind III
h
Xba I
Spe I
+ 141
| CAT | pAlNOSCAT
Sal I
-478
+ 141
pBiNOS-CAT
-1588
-421
I CAT
pCiNOS-CAT
B
Figure 5-2. A: The CAT constructs. Dashed lines represent
deleted portions. B. pCAT-Basic Vector circle map and
sequence reference points.


2
Zawadzki, 1980). This endothelium-derived relaxing factor
(EDRF) as it was then called, and which had a half-life of
about 6 seconds, was later identified as NO (Palmer et al. ,
1987, Ignarro et al. 1987) Over the past few years NO-
producing enzymes have been discovered in virtually every
tissue in numerous species and NO has become established as a
diffusible messenger mediating cell-to-cell interactions
throughout the body including immune cel1-mediated
cytotoxicity, inhibition of platelet aggregation, smooth
muscle relaxation and neuronal signaling (Ignarro et al. ,
1987, Koesling et al., 1991, Bhme et al., 1978, Radomski et
al., 1987, Mellion et al., 1981).
Isoforms of Nitric Oxide Svnthase
The nitric oxide synthase (NOS) enzyme family, the group
of enzymes that catalyze the biologic synthesis of NO,
comprises at least three distinct isoforms that can be
grouped into the constitutively expressed, Ca2+-calmodulin
dependent NOS isoforms (cNOS) found typically in neurons
(bNOS or NOS Type I) and endothelial cells (eNOS or NOS Type
III), and the immunologically inducible, Ca2+-independent NOS
found typically in macrophages (iNOS or NOS Type II)
(Forstermann et al. 1991, Stuehr and Griffith, 1992).
Calmodulin binding to cNOS subunits results in the transfer
of electrons from reduced nicotinic adenine dinucleotide
phosphate (NADPH) to a heme group at the active site within
each subunit of cNOS, initiating NO synthesis (Abu-Soud and


74
Interferon-y (100 U/ml), which by itself was also ineffective
in stimulating nitrite production, potently synergized with
LPS to stimulate iNOS activity (figure 4-1).
Effects of Lipopolvsaccharide and Interferon-v on Inducible
Nitric Oxide Svnthase mRNA
To investigate the effects of LPS and IFN on steady-
state iNOS mRNA levels, total RNA extraction was done in
cells incubated with LPS, IFN, or LPS plus IFN. A northern
blot analysis was then done on the extracts.
Combined treatment with LPS and IFN was accompanied by
marked time-dependent increase in steady-state iNOS mRNA
levels that were initially detected after 2 h of treatment,
peaked at 6 h and decreased after 24 h (figure 4-2) .
Lipopolysaccharide treatment (1 |ig/ml for 6 h) caused only a
small increase in steady-state iNOS mRNA levels whereas none
was detected when the cells were treated with IFN alone
(figure 4-2) .
Effects of Lipopolvsaccharide and Interferon-v on Inducible
Nitric Oxide Svnthase Protein Levels
To determine the roles of LPS and IFN in inducing iNOS
protein synthesis, cells were treated with LPS (1 ng/ml)
alone or in combination with IFN (100 U/ml) and iNOS protein
was determined by Western analysis using a monoclonal
antibody against macrophage iNOS.
Despite the moderate amount of iNOS mRNA induced by LPS
(figure 4-2), it failed to induce detectable levels of iNOS


58
Nitrite and Protein Assays
These assays were done as previously described in
Chapter Two.
Results
Effect of 2.4-Diamino-6-hvdroxvpvrimidine on Murine Proximal
Tubule Cell Nitrite Production
To examine whether GTPCH, the rate-limiting enzyme in
the de novo synthesis of BH4 was required for NO synthesis, I
treated cells with a combination of LPS (0.1 ^g/ml) and IFN
(100 U/ml) for 12 h in the presence or absence of the
specific GTPCH inhibitor DAHP (6 mM) followed by measurement
of nitrite accumulation in the culture medium.
Treatment with DAHP suppressed LPS/IFN-induced nitrite
accumulation by 53.1 3.4% (figure 3-2), indicating that NO
synthesis by MCT iNOS requires GTPCH activity.
Effects of Seoiaoterin and Methotrexate on Murine Proximal
Tubule Cell Nitrite Production
To investigate the contribution of the pterin salvage
pathway to the synthesis of BH4 for NO production, I used
sepiapterin, a dihydropteridine substrate for this pathway,
and methotrexate, an inhibitor of dihydrofolate reductase.
Experiments were designed to examine the effects of
dihydropteridine availability and dihydrofolate reductase
inhibition on NO synthesis in MCT cells treated as described
above.


19
synergistically potentiating LPS-induced iNOS expression
probably at the level of mRNA transcription. This is
consistent with other studies that showed that single
cytokines stimulated only minor increases in mRNA levels in
rat cultured hepatocytes, but any double or triple
combinations of LPS, IL-1, IFN and tumor necrosis factor
(TNF-a) revealed synergistic increases in mRNA levels (Geller
et al. 1993 [b] ) The exact mechanism for this synergism,
however, is currently unknown.
The presence of specific DNA sequences in the promoter
regions of genes that can bind particular proteins confers on
a specific gene the ability to respond to various stimuli.
These DNA sequences play a critical role in producing tissue-
specific patterns of gene expression by binding transcription
factors that may be cytokine-activated but present only in
particular tissues. Hence the binding of these activated
transcription factors to the promoter region of DNA sequence
elements may result in the observed tissue-specific inducible
pattern of iNOS gene expression.
Lipopolysaccharide has been shown to induce the activity
of several of these transcription factors including NF-kB and
activator protein 1 (AP-1) binding proteins (Vincenti et al.,
1992, Eilers et al. 1993). Interferon-y-inducible factors
include y-interferon activated factor (GAF), which binds to
the y-activated site (GAS), interferon-y regulatory factor 1
(IRF-1), which binds to the interferon-y regulatory factor
element (IRF-E) and other factors that bind to interferon-y


17
rheumatoid arthritis, where NO may be responsible for the
pathological vasodilation and tissue damage.
The sensitivity to NO varies from cell to cell, being
cytotoxic in some but cytostatic in others. This may be
explained by the relative abundance and importance of Fe-S
containing enzymes in the various cells or their ability to
buffer the NO molecules with their thiol groups.
Other Cell Types
Hepatocytes have been shown to express iNOS during
inflammation (Geller et al., 1993 [a]) and both
cytoprotective and cytotoxic effects of NO have been
demonstrated in the liver (Billiar et al. 1990). This
seemingly ambiguous effect may be explained by the cytotoxic
and vasodilatory effects of NO. Hepatocyte glucose and urea
output are also inhibited by endogenously produced NO.
Whereas the former may be attributed to inhibition of key
enzymes in glycogenolysis and gluconeogenesis, the latter is
almost certainly due to the competitive use of the common
substrate L-arginine. Nitric oxide induction in hepatocytes
may contribute to host defense against infections like
malaria (Nussler et al. 1991). In both tumor cells and
fibroblasts, interferon-y (IFN) has been shown to inhibit
replication through NO generation. Recent reports suggest
that airway epithelial cells contain cNOS and that NO may
play an important role in diverse airway functions such as
maintaining vascular and airway smooth muscle tone, host


99
Table 6-6. Bond Angles () for all Atoms of Compound VIII
Estimated Standard Deviations given in Parentheses.
N-Na-01
64.28 (6)
N-Na-01'a
137.75 (7)
N-Na-09
86.75(7)
01-Na-07
126.54 (6)
01-Na-08
98.39(8)
Ol-Na-OlO
153.54 (6)
07-Na-08
82.41 (7)
O7-Na-O10
79.87 (6)
01'a-Na-09
91.12 (7)
08-Na-09
169.05(9)
09-Na-010
87.27 (8)
Na-N-C2
119.99(13)
Na-01-Cl
123.02 (14)
01-C1-C2
114.3 (2)
01-Cl-01a
124.5(2)
C1-C2-C3
120.4 (2)
C2-C3-C4
118.4(2)
C4-C5-C6
1.387(3)
C5-C6-C7
120.6(2)
07-C7-07a
124.8 (2)
07a-C7-C6
119.3(2)
Na-01'a-Cl'
129.10(14)
01'a-Cl'-C2'
123.0 (2)
N'-C2'-C3'
123.5 (2)
N'-C2'-Cl'
114.7 (2)
C3'-C4'-C5'
118.8 (2)
N'-C6'-C7'
118.3 (2)
N'-C6'-C5'
123.1(2)
07'-Cl'-C6'
116.4 (2)
Na-Ol-Hl
119. (2)
Na-07-H7
127. (2)
C2-C3-H3
116.5(15)
C3-C4-H4
122. (2)
C4-C5-H5
122.4 (14)
Cl'-01'-HI'
108.(2)
C4'-C3'-H3'
121.6(15)
C5'-C4'-H4'
119. (2)
C6'-C5'-H5'
118.3 (14)
Na-08-H8b
123 (3)
Na-09-H9a
115. (3)
H9a-09-H9b
106.(5)
Na-O10-H10b
107. (2)
H10a-O10-Hl'
113.(2)
N-Na-07
63.67 (6)
N-Na-08
103.38 (8)
N-Na-O10
141.59 (7)
Ol-Na-Ol'a
73.53 (6)
01-Na-09
89.69 (7)
07-Na-01'a
157.32 (6)
07-Na-09
98.73 (7)
01'a-Na-08
84.17(7)
Ol'a-Na-OlO
80.26 (6)
O8-Na-O10
82.20 (8)
C2-N-C6
117.8 (2)
Na-N-C6
120.31 (14)
Na-07-C7
119.87 (13)
01a-Cl-C2
121.2 (2)
N-C2-C3
123.1 (2)
N-C2-C1
116.5 (2)
C3-C4-C5
119.1(2)
N-C6-C7
116.8 (2)
N-C6-C5
122.6 (2)
07-C7-C6
115.9 (2)
C2'-N'-C6'
116.9(2)
01'-Cl'-C2'
113.1 (2)
01'-Cl'-01'a
123.8 (2)
Cl'-C2'-C3'
121.7 (2)
C2'-C3'-C4'
118.5 (2)
C4'-C5'-C6'
119.1 (2)
C5'-C6'-Cl'
118.7 (2)
01'-Cl'-07'a
125.5 (2)
07'a-C7'-C6'
118.1 (2)
Cl-Ol-Hl
115. (2)
C7-07-H7
113. (2)
C4-C3-H3
125.0 (15)
C5-C4-H4
119.(2)
C6-C5-H5
118.6(14)
C2'-C3'-H3'
119.9(15)
C3'-C4'-H4'
122. (2)
C4'-C5'-H5'
122.5(14)
Na-08-H8a
121. (2)
H8a-08-H8b
99. (4)
Na-09-H9b
106. (3)
Na-010-H10a
113. (2)
H10a-010-H10b
101. (3)
HlOb-OlO-HI'
132 (2)


65
40
CO 2*
0) ^
H
r. a)
LPS/IFN
DAHP
Sepiapterin
Figure 3-3. Effects of sepiapterin on nitrite production by
MCT cells in the absence or presence of DAHP, (mg: mg of cell
protein, n=9).


78
these results indicate that the synergism between LPS and IFN
is not due to complementary effects of the activation of the
different NF-kB proteins by these agents.
Discussion
In these studies I have shown that LPS, IFN or their
combination cause rapid activation and nuclear translocation
of NF-kB complexes in renal tubule epithelium, consisting
mainly of p65 but with smaller amounts of p50 protein. This
is in agreement with what has been observed for most
functional heterodimers of NF-kB (Baeuerle and Baltimore,
1989, Ganchi et al., 1993) but in contrast to what is seen in
macrophages, where the p65/c-Rel heterodimer is reported to
play an important role in LPS-induced iNOS activity (Xie et
al., 1994). It is of interest to note that although LPS and
IFN each induced nuclear translocation of NF-KB complexes,
IFN failed to induce iNOS mRNA and LPS caused only a small
induction of iNOS mRNA transcription, not reflected in either
increase in iNOS protein synthesis or nitrite production.
These results indicate that nuclear translocation of NF-KB
alone is not sufficient for the induction of iNOS activity in
renal epithelium. Nevertheless, since cotreatment of LPS/IFN
with PDTC completely prevented the induction of iNOS
activity, it can be argued that NF-kB, though not sufficient
on its own, is necessary for iNOS expression in renal
epithelium.


KEY TO ABBREVIATIONS
AP-1
ATPase
bh4
CAT
Activator Protein
Adenosine Triphosphatase
Tetrahydrobiopterin
Chloramphenicol Acetyl
Transferase
DAHP
DMEM
2,4-diaminohydroxypyrimidine
Dulbecco's Modified Eagle's
Medium
DMSO
EMSA
Dimethylsulfoxide
Electrophoretic Mobility
Shift Assay
FAD
GAS
GTP
GTPCH
IFN
I KB
IL
y- IRE
IRF-1
IRF-E
Flavin Adenine Dinucleotide
y-Activated Site
Guanosine 5'-Triphosphate
GTP Cyclohydrolase I
Interferon-y
Inhibitor of kappa B
Interleukin
y-Interferon Response Element
Interferon-y Response Factor
Interferon-y Response Factor
Element
ISRE
Interf eron-oc-Stimulated
LPS
MCT
NADH
Response Element
Lipopolysaccharide
Murine Proximal Tubule Cells
Nicotinamide Adenine
Dinucleotide
NADPH
Nicotinamide Adenine
Dinucleotide Phosphate
NF-KB
NNA
NO
NOS
cNOS
Nuclear Factor-kappa B
N^-Nitro-L-Arginine
Nitric Oxide
Nitric Oxide Synthase
Constitutive Nitric Oxide
Synthase
iNOS
Inducible Nitric Oxide
Synthase
PCR
PDTC
RPMI
Polymerase Chain Reaction
Pyrrolidinedithiocarbamate
Roswell Park Memorial
Institute
TNF-OC
Tumor Necrosis Factor
vil


43
tissue-specific regulatory signals for the induction of iNOS
may serve as a safeguard to prevent inappropriate or
uncontrolled production of NO. A better understanding of the
molecular mechanisms responsible for the tissue-specific
regulation of iNOS should allow us to design strategies to
prevent the NO-mediated tissue injury associated with various
pathological conditions.
In summary, I have demonstrated the presence of an iNOS
in renal proximal tubules that is markedly induced by a
combination of LPS and IFN, requires protein and mRNA
synthesis and its activity is inhibited by NNA, an analogue
of L-arginine. In contrast to its effects in murine
peritoneal macrophages, IFN by itself does not induce iNOS
expression in proximal tubule cells. It does, however,
markedly potentiate LPS-stimulated NO production by these
cells via mechanisms involving enhanced iNOS mRNA and protein
expression. Studies aimed at understanding the molecular
mechanisms that determine tissue-specific responses of iNOS
to various inducers are necessary for the development of more
rational and specific interventions to prevent NO-mediated
renal tissue injury.


76
effect of the NF-KB inhibitor PDTC on NF-kB activation, iNOS
protein and steady-state mRNA levels.
As shown in figure 4-5, treatment with PDTC (25 mM)
inhibited or attenuated the activation of NF-kB by LPS, IFN
or their combination. In addition, PDTC abolished the
synergism between LPS and IFN on iNOS steady-state mRNA
levels (figure 4-6A), blunted the expression of iNOS protein
(figure 4-6B), and completely prevented the formation of
nitrites induced by these agents (figure 4-7). Taken
together, these results suggest that NF-KB is necessary for
the induction of iNOS activity by LPS and IFN in renal
epithelium, but its activation alone does not explain the
synergism seen between these two inducers. Also, they
indicate that NF-kB activation is not sufficient for the
expression of this enzyme since neither IFN nor LPS alone is
able to induce iNOS activity despite activating NF-kB.
Effects of Nuclear Factor-KB Antibodies on Nuclear Factor-KB
Activation bv Lioopolvsaccharide and Interferon-y
To identify the components of NF-kB activated by LPS in
renal epithelium and compare them to those activated in
macrophages where LPS is known to be a potent inducer of
iNOS, I used IgG against the three major NF-kB subunits p50,
p65 (Rel-A) and c-Rel in an EMSA.
Consistent with previous reports (Xie et al. 1994),
LPS activated NF-kB complexes containing p50, p65 and c-Rel
in the mouse macrophage cell line RAW 264.7 (figure 4-8A).


88
The spectrum of VI, with the largest peak (m/z=137)
corresponding to L+l, does not provide much information on the
relative amounts of complexes present in the sample. The
remaining large peaks, m/z=165, m/z=177, and m/z=273, are
probably due to AlL+3, AlL(0H)-2 and 2HL+1, respectively. Two
of the smaller peaks, m/z=301 and m/z=313, are probably due to
AlL2+4 and A1L2(0H)-1. There is no peak present near m/z=432,
where A1L3 would be. The nearest peak, m/z=391, is smaller by
41 units. It seems reasonable that one ring is a complex
could have fragmented. The small peak at M-43 could be due to
the loss of part of one of the rings. By a similar
fragmentation, any AlL3 that could have been present may have
been responsible for this peak.
Conclusion
The structure of the compounds III through VII can not be
determined from the data provided by the three techniques
discussed in this chapter. Without more thorough calibration
of the Al-27 NMR scale, the available data merely confirms the
presence of the aluminum ion in more than one possible
environment in a single sample. In addition, without a pure
sample, elemental analysis provides little information beyond
the confirmation of the ligand's presence. Finally, the
carbonyl stretching frequency data confirms the coordination
of the aluminum ion to the ligand yet does not provide any
further information. Without the use of x-ray
Without the use


129
Schmidt, H.H.H.W., Warner, T.D., Ishii, K. Sheng, H. and
Murad, F. Insulin secretion from pancreatic P cells caused by
L-arginine-derived nitrogen oxides. Science 255: 721-723,
1992.
Schreck, R., Meier, B., Mannel, D.N., Droge, W. and Baeuerle,
P.A. Dithiocarbamates as potent inhibitors of nuclear
factor-kappa B activation in intact cells. J. Exp. Med. 175:
1181-1194, 1992.
Schuman, E.M. and Madison, D.V. A requirement for
intercellular messenger nitric oxide in long term-
potentiation. Science 254: 1503-1506, 1991.
Sharma, V.S., Isaacson, R.A., John, M.E., Waterman, M.R. and
Chevion, M. Reactions of nitric oxide with heme proteins:
studies on metmyoglobin, opossum methemoglobin and
microperoxidase. Biochemistry 22: 3897-3902, 1983.
Shibuki, K. and Okada, D. Endogenous nitric oxide release
required for long-term synaptic depression in the cerebellum.
Nature 349: 326-328, 1991.
Shultz, P.J., and Raij, L. Endogenously synthesized nitric
oxide prevents endotoxin-induced glomerular thrombosis. J.
Clin. Invest. 90: 1718-1725, 1992.
Shultz, P.J. Schorer, A.E. and Raij, L. Effects of
endothelium-derived relaxing factor and nitric oxide on rat
mesangial cells. Am. J. Physiol. 258: F162-F167, 1990.
Shultz, P.J. and Tolins, J.P. Adaptation to increased
dietary salt intake in the rat. Role of endogenous nitric
oxide. J. Clin. Invest. 91: 642-650, 1993.
Snyder, S.H. and Bredt, D.S. Nitric oxide as a neuronal
messenger. Trends Pharmacol. Sci. 12: 125-128, 1991.
Stein, B., Baldwin Jr., A.S., Ballard, D.W., Greene, W.C.,
Angel, P. and Herrlich, P. Cross-coupling of the NF-kappa B
p65 and Fos/Jun transcription factors produces potentiated
biological function. EMBO. J. 12: 3879-3891, 1993.
Stoos, B.A., Carretero, O.A. and Garvin, J.L. Endothelium-
derived nitric oxide inhibits sodium transport by affecting
apical membrane channels in cultured collecting ducts cells.
J. Am. Soc. Nephrol. 4: 1855-1860, 1994.
Stuehr, D.J., Cho, H., Kwon, N.S., Weise, M. and Nathan, C.F.
Purification and characterization of the cytokine-induced
macrophage nitric oxide synthase: an FAD- and FMN-containing
flavoprotein. Proc. Natl. Acad. Sci. USA 88: 7773-7777, 1991
[a] .


122
Golub, E.I., Kim, H. and Volsky, D.J. Transfection of DNA
into adherent cells by DEAE-dextran/DMSO method increases
drastically if cells are removed from surface and treated in
suspension. Nucleic Acids Res. 17: 4902, 1989.
Gorman, C.M., Moffat, L.F. and Howard, B.H. Recombinant
genomes which express chloramphenicol acetyltransferase in
mammalian cells. Mol. Cell Biol. 2: 1044, 1982.
Gross, S.S., Jaffe, E.A., Levi, R. and Kilbourn, R.G.
Cytokine-activated endothelial cells express an isotype of
nitric oxide synthase which is tetrahydrobiopterin-dependent,
calmodulin-independent and inhibited by arginine analogues
with a rank order of potency characteristic of activated
macrophages. Biochem. Biophys. Res. Commun. 178: 823-829,
1991.
Gross, S.S. and Levi, R. Tetrahydrobiopterin synthesis: an
absolute requirement for cytokine-induced nitric oxide
generation by vascular smooth muscle. J. Biol. Chem. 267:
25722-25729, 1992.
Gross, S.S., Levi, R., Madera, A., Park, K.H., Vane, J. and
Hattori, Y. Tetrahydrobiopterin is induced by LPS in
vascular smooth muscle and is rate-limiting for nitric oxide
production. Adv. Exp. Med. Biol. 338: 295-300, 1993.
Guzman, N.J., Fang, M.Z., Tang, S.S., Ingel finger, J. and
Garg, L.C. Autocrine inhibition of Na+/K+-ATPase by nitric
oxide in mouse proximal tubule epithelial cells. J. Clin.
Invest. 95: 2083-2088, 1995.
Hattori, Y. and Gross, S.S. GTP cyclohydrolase I mRNA is
induced by LPS in vascular smooth muscle: characterization,
sequence and relationship to nitric oxide synthase. Biochem.
Biophys. Res. Commun. 195: 435-441, 1993.
Haverty, T.P., Kelly, C.J., Hines, W.H., Amenta, P.S.,
Watanabe, M., Harper, R.A., Kefalides, N.A. and Neilson, E.G.
Characterization of a renal tubule epithelial cell line which
secretes the autologous target antigen of autoimmune
experimental interstitial nephritis. J. Cell Biol. 107: 1359-
1368, 1988.
Henkel, T., Machleit, T., Alkalay, I., Kronke, M. Ben-
Neriah, Y. and Baeuerle, P.A. Rapid proteolysis of I kappa
B-alpha is necessary for activation of transcription factor
NF-kappa B. Nature 365: 182-185, 1993.
Hirai, H., Fujisawa, J., Suzuki, K., Ueda, K., Muramutsu, M.,
Tsuboi, A., Arai, N. and Yoshida, M. Transcriptional


62
Anisotropic Thermal Parameters (2 x 103) for II.
Estimated Standard Deviations are given in Parentheses.
Atom
uxl
u22
U33
u23
u13
u12
Al
41(2)
41(2)
31(2)
-3 (2)
5(1)
-1(2)
0(1)
30(4)
59(5)
37(4)
0(3)
7(3)
2 (4)
N (1)
43 (5)
35(5)
34 (5)
-14 (4)
4(4)
-7(5)
C(ll)
62(9)
55(8)
48 (8)
11(6)
15(7)
-13(7)
C (12)
77(10)
59(9)
58(10)
-8(7)
-7(8)
-16 (8)
C (13)
127 (13)
56 (9)
41(8)
-24(7)
-16(9)
-6 (9)
C (14)
99(10)
27(7)
31(7)
2 (5)
27(7)
7(7)
C (15)
101(10)
70 (9)
65(8)
18(7)
45(8)
-2 (8)
C (16)
46 (8)
77(10)
100(12)
13 (9)
48 (8)
13(7)
C (17)
51(8)
49(7)
49 (8)
16(6)
4 (6)
10(6)
C (18)
30(7)
41(7)
51(8)
11(6)
22(5)
11(6)
C (19)
52 (7)
27(6)
43 (6)
-1(5)
15(5)
3(5)
0(2)
27(4)
29(4)
42 (4)
8(3)
8(3)
5(4)
N (2)
22 (5)
33(5)
33(5)
6 (4)
-3 (4)
2(4)
C (21)
51(8)
41(8)
36(7)
4 (6)
2(6)
-22(7)
C (22)
46(8)
64(9)
63 (8)
6(7)
33(7)
-2(7)
C (23)
87(10)
30(7)
62 (8)
-23(6)
35(7)
0(8)
C (24)
27 (7)
41(7)
43 (7)
4(6)
2(5)
6(6)
C (25)
71(9)
18 (7)
61(8)
4(6)
-11(7)
12(7)
C (26)
47(7)
46 (8)
48(7)
-4(7)
13(6)
12(7)
C (27)
42(8)
48 (8)
41(7)
7(6)
2(6)
7(6)
C (28)
25(6)
44 (7)
22(6)
10(5)
-2 (4)
10 (6)
C (29)
27(5)
32 (6)
17(5)
1(4)
1 (4)
-11(5)
0(3)
50(4)
29(4)
28(4)
-8(3)
1(3)
-8(4)
N(3)
41(5)
28 (5)
27(5)
3 (4)
8 (4)
10(4)
C (31)
54 (8)
45(8)
33(7)
-10(6)
13(6)
5(6)
C (32)
77(10)
70(10)
35(8)
-32(7)
22 (7)
-22 (8)
C (33)
34(7)
98(11)
39(7)
-24(8)
11(5)
-22(8)
C (34)
32 (7)
53(8)
44(7)
-11(7)
-7(5)
-2(6)
C (35)
81 (10)
44 (8)
49 (8)
16(7)
-4(7)
10(8)
C (36)
55(8)
38(8)
54 (8)
24(6)
9(6)
5(7)
C (37)
44(7)
35(7)
55 (8)
5(6)
2(6)
-14(6)
C (38)
26 (6)
50(8)
31(7)
-10(6)
-4 (5)
-1(6)
C (39)
21(5)
39(6)
31(6)
1(5)
2(4)
-5(5)
0(4)
84 (7)
93 (8)
229 (12)
-59(8)
8(7)
-6(6)
C (4)
120 (14)
61 (16)
270 (21)
-171(16)
85(14)
-71(13)
The form or the thermal ellipsoid is exp [-2ir2 (h2a*2U31 + k?b*2
U22 + 12c*2U33 + 2klb*c*U23 + 2hla*c*U13 + 2hka*b*U12)]


19
synergistically potentiating LPS-induced iNOS expression
probably at the level of mRNA transcription. This is
consistent with other studies that showed that single
cytokines stimulated only minor increases in mRNA levels in
rat cultured hepatocytes, but any double or triple
combinations of LPS, IL-1, IFN and tumor necrosis factor
(TNF-a) revealed synergistic increases in mRNA levels (Geller
et al. 1993 [b] ) The exact mechanism for this synergism,
however, is currently unknown.
The presence of specific DNA sequences in the promoter
regions of genes that can bind particular proteins confers on
a specific gene the ability to respond to various stimuli.
These DNA sequences play a critical role in producing tissue-
specific patterns of gene expression by binding transcription
factors that may be cytokine-activated but present only in
particular tissues. Hence the binding of these activated
transcription factors to the promoter region of DNA sequence
elements may result in the observed tissue-specific inducible
pattern of iNOS gene expression.
Lipopolysaccharide has been shown to induce the activity
of several of these transcription factors including NF-kB and
activator protein 1 (AP-1) binding proteins (Vincenti et al.,
1992, Eilers et al. 1993). Interferon-y-inducible factors
include y-interferon activated factor (GAF), which binds to
the y-activated site (GAS), interferon-y regulatory factor 1
(IRF-1), which binds to the interferon-y regulatory factor
element (IRF-E) and other factors that bind to interferon-y


85
LPS/IFN
Time (hr)
01 24 6 12 24 6 6
Figure 4-2. Inducible NOS mRNA expression in MCT cells
treated with LPS (1 ng/ml) and IFN (100 U/ml) for the
indicated periods of time or with each of these inducers
alone for 6 h. Blot is representative of three separate
experiments.


18
defense, pulmonary neurotransmission and ciliary motility
(Robbins et al., 1994 [a,b]).
It is likely that other beneficial and deleterious
effects of NO in a variety of cells will be discovered and
that modalities to selectively modulate iNOS expression or
local NO concentrations will be of immense therapeutic
benefit.
Rationale For The Thesis
The inducible isoform of NOS has been shown to be
subject to very strict and high tissue-specific regulatory
control (Nathan and Xie, 1994 [a,b], Nussler et al., 1994).
For example, whereas LPS induces high levels of iNOS
expression in macrophages and microglia (Stuehr and Marietta,
1985, Xie and Nathan, 1994), it induces only modest levels in
hepatocytes, vascular smooth muscle and renal epithelial
cells and a combination with cytokines is usually required
for full expression of iNOS activity (Nussler and Billiar,
1993, Amoah-Apraku et al., 1993). In preliminary studies, I
have established that mouse renal tubule epithelial cells
possess iNOS and that whereas LPS induces only very little
iNOS expression, coincubation with IFN results in a
synergistic expression of high levels of iNOS activity in
these cells (Amoah-Apraku et al., 1993).
I have also established that this was not due to the
lack of cofactor availability, suggesting that IFN may be
acting as a potent modulator of iNOS activity in MCT cells by


ALUMINUM COORDINATION COMPOUNDS
By
KIM E. BROWNING
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1995


70
characterized the NF-kB proteins activated by LPS and IFN in
this tissue.
Materials and Methods
Chemicals and Biologic Products
Dulbecco's modified Eagle's medium (DMEM), L-arginine-
free Roswell Park Memorial Institute (RPMI) 1640 and all
other cell culture materials were purchased from Fisher
Scientific (Orlando, FL) Interferon-y (IFN, rat
recombinant) was purchased from Gibco BRL (Gaithersburg, MD).
Radioactive mac-iNOS-cDNA probes were prepared using a
Stratagene (La Jolla, CA) Prime-IT II random labeling kit.
Other biologic products were from Promega Co. (Madison, WI).
Lipopolysaccharide (LPS, E coli serotype 026:B6),
sulfanilamide, pyrrolidinedithiocarmate (PDTC), sodium
nitrite, and N-(1-naphthyl)ethylenediamine hydrochloride were
purchased from Sigma Chemical Co. (St. Louis, MO).
Cell Cultures. Northern Blot Analysis, Nitrite and Protein
Assays
These procedures were carried out as previously
described in Chapter Two.
Preparation of Nuclear Extracts
Nuclear extracts were prepared by a modification of a
previously described procedure (Lew et al., 1991). Briefly,
cells cultured to confluence were treated with inducers at


Nitrite
(nmoles/mg)
51
250-,
200
150
100
50
Basal LPS IFN 1 5 10 50 100
0.1 (ig/ml 100 U/ml
LPS (0.1 jag/ml) + IFN (U/ml)
Figure 2-7. Concentration dependence of the effect of IFN
(0-100 U/ml for 48 h) on nitrite accumulation in the presence
of LPS (0.1 jog/ml) in MCT cells, (* p<0.05 vs control, mg: mg
of cell protein, n=3).


61
GTPCH in the renal parenchyma. Previous studies (Bellahsene
et al., 1984) have found GTP 8-formylhydrolase and neopterin
synthetic activities of GTPCH in rat kidneys, but the
physiologic significance of this remains unclear. In liver
and catecholamine-producing organs, GTPCH provides BH4 for
aromatic L-amino acid hydroxylation (Kaufman, 1993).
Phenylalanine hydroxylase activity has also been detected in
the kidney, but its function is unknown (Rao and Kaufman,
1986, Richardson et al. 1993). The renal hydroxylase
differs from the liver isoform in that it exists natively in
a very high state of activation of about 16-fold greater in
activity (Rao and Kaufman, 1986) However, it is not known
whether this activity is supported by BH4 synthesized de novo
or regenerated from dihydropteridines.
The regulation of NO synthesis by GTPCH and BH4
availability may be important therapeutically because it
provides a target for the blockade of the renal L-arginine:N0
system. Nitric oxide is a mediator of inflammation in
glomerulonephritis (Weinberg et al. 1994, Marsden and
Ballermann, 1990, Shultz et al. 1990) and kidney allograft
rejection (Langrehr et al. 1993 [a,b]) and of tubular
hypoxia/reoxygenation injury (Yu et al., 1994). On the other
hand, NO causes renal vasodilation, increases renal cortical
bloodflow (Shultz et al., 1993), protects against glomerular
thrombosis in endotoxemia and pregnancy (Shultz and Raij,
1992, Raij, 1994), and modulates T-cell proliferation
(Langrehr et al, 1993 [a]). It is therefore possible that


23
bond length for the carbonyl group would probably be between
that for the acid and the anion. Therefore, the carbonyl
stretching frequency should lie between those of the unreacted
ligand and its sodium salt.53
Aluminum-27 NMR Spectroscopy
Al-27 NMR is a technique used to probe into the
environment of aluminum in its compounds. A peak will be
present for each different aluminum atom. The shift of the
peak is indicative of the geometry, symmetry and the type of
atoms surrounding the metal center.
Most of the research reported for Al-27 NMR refers to
exclusively oxygen atoms bonded to the aluminum and the shifts
are referenced relative to A1(H20 ) 63+. Most of the octahedral
A106 groups have shifts very near 0 ppm, whereas the
tetrahedral A104 groups are shifted to near 80 ppm.54 However,
for an aluminophosphate, the shift of 41 ppm was attributed to
the tetrahedral A104 group in the compound.55 Also, the Al-27
NMR shifts reported for the distorted octahedral environments
in tris complexes of 0,0 bidentate ligands range from 36 to 41
ppm.17-18 These shifts are in the same range as reported for
some tetrahedral aluminum centers. Not all of the studies on
coordination compounds have been performed on exclusively
oxygen environments. The shift due to the octahedral
environment in fluoroaluminates was reported to range from
+1.4 to -13.2 ppm.56 In addition, an Al-27 NMR study including
hexacoordinate aluminum complexes formed with


9
Picolinic acid is a naturally occurring compound that has
been found bound to aluminum in human breast milk.44 Picolinic
acid is structurally similar to oxine, but without the second
ring, the flexibility of the ligand would be greater. If an
aluminum and picolinic acid complex could be formed and
characterized, a comparison of Al-0 and Al-N bond lengths
could be made with the oxine compounds. This analysis could
possibly determine if the nitrogen is coordinated to the
aluminum ion or held close to the ion simply because of its
location on the ring. 6-Methylpicolinic acid provides the
same potential comparisons for picolinic acid as 2-methyloxine
for oxine.
Pyrazinoic acid was chosen for its structural similarity
to picolinic acid. However, because of the second nitrogen
atom in the aromatic ring, the potential existed to have a
greater water solubility for an aluminum complex.
Hypoxanthine is formed in the animal body during the
breakdown of adenosine and nucleic acids. The two-ring
structure is similar to oxine; however, there are two notable
differences. First, there are three nitrogen atoms
substituted for ring carbons. Second, and probably more
important, is that one of the aromatic rings is a five
membered ring instead of six. As a result, the ring strain
should pull the nitrogen atom farther from the aluminum ion in
a coordination compound.


TABLE OF CONTENTS
page
ACKNOWLEDGEMENTS ii
LIST OF TABLES v
LIST OF FIGURES viii
ABSTRACT xi
CHAPTERS
1 INTRODUCTION 1
Potential Aluminum Health and
Environmental Issues 1
Coordination Chemistry of Aluminum.. 3
Aluminum Coordination Compounds 5
Aluminum Complexes with a Nitrogen
Donor Atom 6
2 SYNTHESIS AND CHARACTERIZATION OF
ALUMINUM COORDINATION COMPOUNDS 11
Aluminum as a Lewis Acid 11
Synthesis 12
Solubility of Aluminum Coordination
Compounds 20
Methods of Characterization of
Aluminum Complexes 21
3 CRYSTAL GROWTH TECHNIQUES UTILIZED TO
PRODUCE SINGLE CRYSTALS SUITABLE FOR
USE IN X-RAY DIFFRACTION STUDIES 27
Introduction 27
Urea Decomposition 27
Vapor Diffusion 28
Gel Diffusion 30
Base Strength 33
Solvent System 36
Aluminum Isopropoxide 38
Summary of Results 3 9
iii


CHAPTER FIVE
TRANSCRIPTIONAL REGULATION OF NITRIC OXIDE SYNTHASE BY
NUCLEAR FACTOR-KB IN RENAL EPITHELIUM
Introduction
Computer analyses have identified numerous possible DNA
regulatory sequences within the promoter region of the iNOS
gene that are homologous to consensus DNA elements known to
bind nuclear transcription factors. These elements are known
to be involved in transcriptional responses to LPS and a
variety of cytokines (Xie et al. 1993, Lowenstein et al.,
1993). They include NF-kB, AP-1, interferon-y regulatory
factor element (IRF-E) and tumor necrosis factor response
elements (Xie et al., 1993, Lowenstein et al., 1993).
The plethora of immunokines acting either alone or in
various combinations required to express iNOS activity in
different cell types may be explained by the fact that these
immunokines activate different nuclear factors, or perhaps,
different dimers of the same nuclear factor, which then bind
to DNA and influence gene transcription. This, therefore may
confer the tissue specificity of iNOS induction and its
response to stimulation by different immunokines. It is
reasonable to assume that macrophages, which utilize NO for
their cytotoxic functions, should be stimulated by microbes,
microbial products or cytokines whereas in proximal tubule
93


11
Drapier and Hibbs, 1988). These reactions may result in
enzyme inactivation, cytotoxicity or cell death. The
tumoricidal effect of NO may be mediated this way and
activated macrophages utilize this as a non-specific immune
defense mechanism against bacterial, protozoal and possibly
viral infections. Nitric oxide also induces ADP-ribosylation
and inhibition of the glycolytic enzyme glyceraldehyde-3-
phosphate dehydrogenase (Brue et al., 1990).
The Nervous System
Nitric oxide differs very much from conventional
neurotransmitters in that it is able to diffuse freely from
the point of synthesis to intracellular target sites in
neighboring cells independent of vesicular release, membrane
receptors or lipid cell boundaries. Though normal NO
neuronal actions have not been fully delineated, cerebellar
NOS has been cloned and antibodies to cNOS-related antigens
have been used to identify discreet populations in the
retina, striatum, hypothalamus, midbrain, posterior pituitary
and forebrain of the rat, suggesting a role for NO in
neuronal responses (Bredt et al., 1990, Bredt et al., 1991).
It has also been implicated specifically in some forms of
synaptic plasticity such as long-term synaptic potentiation
(LTP) and long-term synaptic depression (LTD), both of which
are considered elements of experience-driven synaptic network
remodeling underlying learning and memory (Shibuki and Okada,


53
9.5-
7.5-
0.24-
n
r
<
r*
r
o
*0
z
H
z
73
**3
O
Z
z
r
+
¡fr
es
H
r
cr

Z
+

M
X!
L
a
go
+
O
H
X
Figure 2-9. Effects of IFN (100 U/ml) and LPS (1 (ig/ml) on
iNOS steady-state mRNA levels after 4 h of treatment in MCT
cells. Interferon-Y enhances the effect of LPS but does not
by itself increase iNOS mRNA levels. Ribosomal (18s) RNA was
used as gel loading control which was comparable in all
lanes. Blot is representative of three experiments. Act:
actinomycin D (4 jrM) Dex: dexamethasone (20 jiM) .


Figure 5-3. Demonstration of iNOS promoter activity by LPS and IFN in MCT cells. The MCT
cells transfected with pAiNOS-CAT vector or the promoterless pCAT-Basic vector were treated
with a combination of LPS (1 ng/ml) and IFN (100 U/ml) for 24 h followed by CAT assay of
lysates. Treatment with LPS/IFN conferred inducibility of CAT activity to pAiNOS-CAT but
not to the promoterless pCAT-Basic. A Autoradiograph, B graphical presentation; (* p < 0.01
vs pAiNOS control, ** p < 0.01 vs pAiNOS [LPS/IFN], n=4).
109


81
highly unstable and unable to yield functionally active
enzyme due to the activities of other proteins possibly
coinduced by LPS. Interferon-y may therefore be activating
processes that repress these proteins and thereby decrease
iNOS mRNA degradation, stabilize iNOS mRNA and iNOS protein
or both, in renal epithelial cells. However, recent studies
in rat macrophages and hepatocytes do not support this
assertion (Nussler and Billiar, 1993). With regard to PDTC,
I cannot exclude the possibility that this compound may have
other as yet undetermined posttranscriptional and/or
posttranslational effects that prevent the expression of
iNOS.
The findings in these studies do not support my original
hypothesis with respect to the synergistic effects of LPS and
IFN in the activation of NF-kB. The signal observed with LPS
alone was about equal in magnitude to that observed for the
LPS and IFN combination. Unlike what has been previously
described in macrophages where IFN and IL-2 cooperatively
activate NF-kB to reach a threshold that may be required for
transcriptional activation of certain genes (Narumi et al. ,
1992), no such cooperative interaction was observed between
IFN and LPS in renal epithelium. However, it appears to
agree with what is seen in human U-937 histiocytic lymphoma
cells where IFN potentiates the cytotoxic effects of TNF, but
has no effect on the TNF-dependent NF-kB activation
(Chaturvedi et al. 1994) In addition, this study also
apparently excludes another pillar of my hypothesis, that is,


52
Table 4-3.
Hydrogen Atom
Coordinates
(xlO4) for I.
X
y
X
H (13)
0.8434
0.6558
-0.0747
H (14)
0.7812
0.6715
-0.2853
H (15)
0.7028
0.6083
-0.3354
H (17)
0.6493
0.4771
-0.3092
H (18)
0.6544
0.4089
-0.2414
H(101)
0.7131
0.3604
0.0660
H (24)
1.0569
0.4653
0.4023
H (25)
1.0875
0.4191
0.1925
H (27)
1.0614
0.3915
-0.0093
H (28)
0.9869
0.3763
-0.1527
H (201)
0.8500
0.4616
-0.1594
H (202)
0.8388
0.3809
-0.1610
H (3 3)
0.7949
0.3076
0.3074
H (34)
0.7034
0.2863
0.4238
H (35)
0.6609
0.3839
0.5353
H (37)
0.6266
0.4813
0.4740
H (38)
0.6482
0.6029
0.3477
H (301)
0.7000
0.6511
0.2198
H (302)
0.7772
0.6173
0.2270
H (5)
0.6325
0.7075
0.4869
aluminum(III)) which range from 2.054 to 2.110 .32 In
addition, they are even longer when compared to the Al-N bond
lengths of 2.028 to 2.073 in tris(oxinato)aluminum(III).31
The steric interaction of the methyl groups with the aromatic
rings is probably responsible for the lengthening of the Al-N
bonds in the tris(2-methyloxinato)aluminum(III) complex. The
longest Al-N bond length and shortest Al-0 bond length are to
ring 1. A potential cause may be that the molecule is
twisting outward to allow more space between the methyl group
on ring 2 and ring 1.


114
confer inducibility by LPS but not by IFN; synergistic
inducibility by both LPS and IFN was conferred only by the
entire promoter region.
This study has revealed several important differences
between renal epithelial iNOS and the enzyme in other tissues
and species. Synergism in some tissues appears to be the
result of cooperative interactions between iNOS inducers that
lead to stimulation of the synthesis of cofactors such as BH4
that are required for the expression of full activity. For
example, IFN markedly stimulates NO synthesis in murine
peritoneal macrophages (Deng et al., 1993) but is ineffective
in MCT cells. Also, LPS potentiates iNOS activity induced by
IFN in rat aortic vascular smooth muscle cells by inducing
BH4 synthesis (Gross and Levi, 1992, Hattori and Gross,
1993), but in MCT cells, even exogenous addition of these
cofactors to IFN- or LPS-treated cells has no effect.
Another important distinction is the inability of LPS to
cause the activation and nuclear translocation of the c-Rel
subunit of NF-kB in MCT cells even though in macrophages this
subunit is critical for the induction of iNOS activity (Xie
et al. 1993). The synergism between LPS and IFN in MCT
cells appears to be mainly at the level of transcription, as
is the case in hepatocytes (Geller et al. 1993), and
requires IFN-induced protein binding to the interferon-y
regulatory elements.
The role of NF-kB in the induction of iNOS in MCT cells
may be very complex.
Although IFN is able to cause the


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS iii
KEY TO ABBREVIATIONS vii
AB STRACT viii
CHAPTERS
1 INTRODUCTION 1
General 1
Isoforms of Nitric Oxide Synthase 2
Substrate and Cofactor Requirements 4
Synthesis of Nitric Oxide 6
Role of Nitric Oxide in Mammalian Physiology 9
Rationale for the Thesis 18
Specific Aims 21
Statistical Analysis 23
2 CHARACTERIZATION OF INDUCIBLE NITRIC OXIDE
SYNTHASE IN MURINE PROXIMAL TUBULE EPITHELIUM 25
Introduction 25
Materials and Methods 27
Results 32
Discussion 39
3 GUANOSINE TRIPHOSPHATE CYCLOHYDROLASE I
REGULATES NITRIC OXIDE SYNTHESIS IN RENAL
PROXIMAL TUBULES 54
Introduction 54
Materials and Methods 57
Results 58
Discussion 60
4 NUCLEAR FACTOR-KB REGULATES INDUCIBLE NITRIC OXIDE
SYNTHASE IN RENAL TUBULE EPITHELIUM 68
Introduction 68
Materials and Methods 70
Results 73
Discussion 78
v


all of them, I say thank you for their contributions to my
professional and personal development.
IV


57
Figure5_i. ORTEP of Al (ox) 3 Molecule Showing the Atomic
Numbering Scheme and Thermal Ellipsoids.


KEY TO ABBREVIATIONS
AP-1
ATPase
BH4
CAT
DAHP
DMEM
DMSO
EMSA
FAD
GAS
GTP
GTPCH
IFN
I KB
IL
y-IRE
IRF-1
IRF-E
ISRE
LPS
MCT
NADH
NADPH
NF-KB
NNA
NO
NOS
cNOS
iNOS
PCR
PDTC
RPMI
TNF-OC
Activator Protein
Adenosine Triphosphatase
Tetrahydrobiopterin
Chloramphenicol Acetyl
Transferase
2,4-diaminohydroxypyrimidine
Dulbecco's Modified Eagle's
Medium
Dimethylsulfoxide
Electrophoretic Mobility
Shift Assay
Flavin Adenine Dinucleotide
y-Activated Site
Guanosine 5'-Triphosphate
GTP Cyclohydrolase I
Interferon-y
Inhibitor of kappa B
Interleukin
y-Interferon Response Element
Interferon-y Response Factor
Interferon-y Response Factor
Element
Interferon-a-Stimulated
Response Element
Lipopolysaccharide
Murine Proximal Tubule Cells
Nicotinamide Adenine
Dinucleotide
Nicotinamide Adenine
Dinucleotide Phosphate
Nuclear Factor-kappa B
Nw-Nitro-L-Arginine
Nitric Oxide
Nitric Oxide Synthase
Constitutive Nitric Oxide
Synthase
Inducible Nitric Oxide
Synthase
Polymerase Chain Reaction
Pyrrolidinedithiocarbamate
Roswell Park Memorial
Institute
Tumor Necrosis Factor
vi 1


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of_^hq>losophy.
Fulton T. Crews
Professor of Pharmacology and
Therapeutics
This dissertation was submitted to tne Graduate Faculty
of the College of Medicine and to the Graduate School and was
accepted as partial fulfillment of the requirements for the
degree of Doctor of Philosophy.
Dean, Graduate School
August, 1995


5
synthetic pathway that utilizes GTP as a precursor, and a
salvage pathway with dihydrofolate reductase as the rate-
limiting enzyme that utilizes preexisting dihydropteridines
like dihydrobiopterin and sepiapterin. Constitutive NOS
requires only catalytic amounts of BH4 for full activity
whereas iNOS, on the other hand, requires substrate amounts.
There is thus a coinduction of BH4 synthesis with iNOS
induction (Werner-Felmayer et al., 1990).
Electrons donated by NADPH are essential for NO
synthesis (Abu-Soud and Stuehr, 1993, Wang et al., 1993). In
fact, some reports suggest that nitrite production and the
rate-1imiting enzyme in the NADPH-generating pentose
phosphate pathway, glucose-6-phosphate dehydrogenase (G-6-
PDH) are coinduced in macrophages (Corraliza et al. 1993).
This implies that metabolic pathways that compete for NADPH
could play an important role in determining the rate of
cellular NO production.
L-Arginine is the only physiologic nitrogen donor for
the production of NO by NOS. L-Arginine can be obtained by
uptake from extracellular fluid, by intracellular protein
degradation or by endogenous synthesis. Several reports
suggest that L-arginine transport increases in macrophages
stimulated to express iNOS and by endotoxin in endothelial
cells (Bogle et al. 1992, Closs et al. 1993, Lind et al.,
1993). Also, endotoxin-stimulated macrophages were more
active in both NO and L-arginine synthesis than in
unstimulated macrophages (Wu and Brosnan, 1992), suggesting


128
Richardson, S.B., Aspbury, R.A. and Fisher, M.J. The role of
reversible phosphorylation in the hormonal control of
phenylalanine hydroxylase in isolated rat proximal kidney
tubules. Biochem. J. 292: 419-424, 1993.
Robertson, F.M., Offner, P.J., Ciceri, D.P., Becker, W.K. and
Pruitt, B.J. Detrimental hemodynamic effects of nitric oxide
synthase inhibition in septic shock. Arch. Surg. 129: 149-
156, 1994.
Robbins, R.A., Barnes, P.J., Springall, D.R., Warren, J.B.,
Kwon, O.J., Buttery, L.D., Wilson, A.J., Geller, D. A. and
Polak, J.M. Expression of inducible nitric oxide synthase in
human lung epithelial cells. Biochem. Biophys. Res. Commun.
203: 209-218, 1994 [a].
Robbins, R.A., Springall, D.R., Warren, J.B., Kwon, O.J.,
Buttery, L.D., Wilson, A.J., Addock, I.M., Riveros-Moreno,
V. Moneada, S. and Polak, J.M. Inducible nitric oxide
synthase is increased in murine lung epithelial cells by
cytokine stimulation. Biochem. Biophys. Res. Commun. 198:
835-843, 1994 [b].
Romero, J.C., Lahera, V., Salom, M.G. and Biondi, M.L. Role
of endothelium-dependent relaxing factor nitric oxide on
renal function. J. Am. Soc. Nephrol. 2: 1371-1387, 1992.
Rosenkranz-Weiss, P., Sessa, W.C., Milstien, S., Kaufman, S.,
Watson, C.A. and Pober, J.S. Regulation of nitric oxide
synthesis by proinflammatory cytokines in human umbilical
vein endothelial cells: elevations in tetrahydrobiopterin
levels enhance endothelial nitric oxide synthase specific
activity. J. Clin. Invest. 93: 2236-2243, 1994.
Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular
Cloning: A Laboratory Manual. 2n<3 Edition, p. 31-57. Cold
Spring Harbor Press, Cold Spring Harbor, NY, 1989.
Saran, M., Michel, C. and Bors, W. Reaction of NO with 0^~.
Implications for the action of endothelium-derived relaxing
factor (EDRF). Free Radical Res. Commun. 10(4-5): 221-226,
1990.
Schmidt, H.H.H.W., Lohmann, S.M. and Walter, U. The nitric
oxide and cGMP signal transduction system: regulation and
mechanism of action. Biochem. et Biophysica. Acta. 1178: 153-
175, 1993.
Schmidt, H.H.H.W., Pollock, J.S., Nakane, M., Gorsky, L.D.,
Frstermann, U. and Murad, F. Purification of a soluble
isoform of guanylyl cyclase-activating factor synthase. Proc.
Natl. Acad. Sci. USA 88: 365-369, 1991.


91
The structure discussed in this chapter was initially
classified as an aluminum compound because of the experimental
conditions and the Al-27 NMR spectrum proving the existence of
an uncharacterized aluminum dipic complex. However, further
investigation, including structure refinement, supported the
presence of sodium as the central metal ion instead of
aluminum.
Experimental
Synthesis
Materials. All chemicals were used as supplied by the
manufacturer.
Preparation of Na (C.H.NO,) (C-.H.NO,) 3H.0 (VIII). With
stirring, 0.50 g agar was added to 100 mL H20. The
temperature of the mixture was slowly raised to and maintained
at 98C for 5 minutes. The 0.5 % agar solution was removed
from heat and allowed to cool. After the agar reached 50C,
1.68 g NaHC03 (20.0 mmole) was dissolved in the agar. Without
touching the sides, 15 mL of the basic agar medium was poured
into a vial (approximately 2 cm diameter) and allowed to cool
overnight. In 15 mL H20, both 0.1251 g Al (N03) 3-9H20 (0.33
mmole) and 0.1673 g dipicolinic acid (1.0 mmole) were
dissolved to provide a solution 0.022 M in Al3+ and 0.67 M in
ligand. The solution was transferred to the vial above the
solidified 0.20 M NaHC03 agar and tightly sealed to prevent
loss of solvent. After 24 days, clear colorless crystals had


22
of BH4 from guanosine triphosphate. I also investigated the
role of the salvage pathway in which dihydropteridines are
converted to BH4 by dihydrofolate reductase. Experiments
were designed to examine the effects of dihydropteridine
availability and dihydrofolate reductase inhibition using
sepiapterin, a dihydropteridine that is less toxic and is
taken up by cells easily compared to BH4, and methotrexate,
an inhibitor of dihydrofolate reductase.
Specific Aim 3
Identification and characterization of NF-kB as a
signaling element during the activation of MCT iNOS by IFN
and LPS. I investigated the time- and concentration-
dependence of the activation and nuclear translocation of NF-
kB by IFN and LPS using a radio-labeled oligonucleotide
containing a consensus binding site for NF-kB as a probe.
The presence of NF-kB in nuclear extracts was evaluated by
DNA Electrophoretic Mobility Shift Assay (EMSA) (Fried and
Crothers, 1981, Garner and Revzin, 1981) The presence or
absence of the various subunits of NF-kB was investigated in
supershift assays, using specific polyclonal IgG antibodies
against NF-kB subunits p50, p65 and c-Rel.
Specific Aim 4
Demonstration of the requirement for NF-kB and IFN-
activated proteins in iNOS induction using functional
promoter analysis. To achieve this, the following goals were


80
epithelium, it is possible that it may also play a role in
the regulation of iNOS expression in this tissue.
Homodimers of p50 that reside constitutively in the
nucleus are believed to inhibit gene transcription by binding
to NF-kB sites. It requires the activation of the IKB-Iike
Bcl-3 proto-oncoprotein to dislodge these inhibitory dimers
in order to allow the incoming p50/p65 dimers to bind and
activate transcription (Franzoso et al. 1992). The basal
amounts of NF-kB found in control-treated MCT cells may be
homodimers of this type, thereby offering another explanation
for the failure of LPS to express iNOS activity in the cells.
Other reports also suggest that the plOO subunit of NF-KB2
inhibits p65-mediated transcription by preventing the
cytoplasmic dissociation of the p65-lKB complex during
immunokine activation (Sun et al. 1994). The function of
IFN therefore, may be the activation of processes leading to
the dislodging of the inhibitory effects of these subunits,
thus facilitating the binding of the p50/p65 dimers to their
nuclear DNA recognition sites.
The absence of iNOS activity after treatment with LPS,
and the total blockade of nitrite formation by PDTC despite
the fact that in both cases low levels of iNOS mRNA can be
detected, are intriguing. It is possible that very low
levels of iNOS activity may be present in these situations
and that these are below the detection capabilities of the
nitrite assay and immunoblotting. Alternatively, the iNOS
message or protein produced under these conditions may be


88
IFN
LPS
PDTC
Figure 4-5. Effect of PDTC (25 ^M) on the nuclear
translocation of NF-kB in MCT cells treated with LPS (1
Hg/ml), IFN (100 U/ml), or their combination for 30 min. The
PDTC treatment was initiated 3 0 min before LPS and IFN were
added and maintained during the entire induction period.
Blot is representative of five separate experiments.


83
regulation of iNOS expression should allow us to identify
potential targets for future selective therapeutic
intervention.


75
protein (figure 4-3). Conversely, the combination of LPS and
IFN resulted in marked increases in iNOS protein (figure 4-3)
and nitrite accumulation (figure 4-1).
Effects of Lioopolvsaccharide and Interferon-y on Nuclear
Factor-KB Activation
To determine whether either LPS or IFN can activate NF-
kB in renal epithelium, I extracted nuclear proteins from MCT
cells treated with each of these agents alone or in
combination, and analyzed these extracts by EMSA using a
radio-labeled NF-kB consensus oligonucleotide as a probe.
As illustrated in figure 4-4, treatment with LPS or IFN
alone caused rapid nuclear translocation of NF-kB that
appeared by 15 min and was noticed at all time points studied
up to 24 h (data not shown). However, LPS appeared to be the
more efficacious activator of this transcription factor.
Moreover, the addition of IFN did not potentiate the
activation of NF-kB caused by LPS. The effect of LPS and IFN
on the nuclear translocation of NF-kB does not appear to be
concentration- or time-dependent after the initial nuclear
appearance within the concentration range and time interval
studied (data not shown).
Effect of Pyrrolidinedithiocarbamate on Nuclear Factor-KB
Activation. Inducible Nitric Oxide Synthase mRNA and Protein
To investigate the potential role of NF-kB in the
induction of iNOS expression by LPS and IFN, I studied the


70
characterized the NF-kB proteins activated by LPS and IFN in
this tissue.
Materials and Methods
Chemicals and Biologic Products
Dulbecco's modified Eagle's medium (DMEM), L-arginine-
free Roswell Park Memorial Institute (RPMI) 1640 and all
other cell culture materials were purchased from Fisher
Scientific (Orlando, FL) Interferon-y (IFN, rat
recombinant) was purchased from Gibco BRL (Gaithersburg, MD).
Radioactive mac-iNOS-cDNA probes were prepared using a
Stratagene (La Jolla, CA) Prime-IT II random labeling kit.
Other biologic products were from Promega Co. (Madison, WI).
Lipopolysaccharide (LPS, E coli serotype 026:B6),
sulfanilamide, pyrrolidinedithiocarmate (PDTC), sodium
nitrite, and N-(1-naphthyl)ethylenediamine hydrochloride were
purchased from Sigma Chemical Co. (St. Louis, MO).
Cell Cultures. Northern Blot Analysis, Nitrite and Protein
Assays
These procedures were carried out as previously
described in Chapter Two.
Preparation of Nuclear Extracts
Nuclear extracts were prepared by a modification of a
previously described procedure (Lew et al. 1991). Briefly,
cells cultured to confluence were treated with inducers at


94
epithelial cells, exposure to excessive amounts of NO is
injurious and therefore a more stringent regulation of iNOS
activity may be required. Hence, iNOS activity is hardly
expressed by any of these immunokines acting alone in renal
epithelium. It is therefore important to characterize the
role of individual DNA regulatory elements in iNOS gene
expression in the different cell types.
Nuclear factor-KB is released from a cytoplasmic anchor
protein (IkB) upon immunokine stimulation, by phosphorylation
and proteolysis, allowing it to translocate into the nucleus
to bind its target DNA sequence and activate gene expression
(Muller et al. 1993, Henkel et al. 1993). The promoter
region of murine iNOS has been found to contain two putative
DNA binding sites for NF-kB (Xie et al. 1993). The
downstream one-third region contains one DNA binding site for
NF-KB which, in the mouse macrophage, has been found to be
responsive to LPS but unresponsive to the synergizing effect
of IFN (Xie et al. 1993). The upstream region which, in
mouse macrophage, was unresponsive to LPS but thought to be
critical for IFN responsiveness, contains the other putative
NF-kB binding site and also IRF-E, which is the IRF-1 binding
site needed for the synergistic effect of IFN (Xie et al.,
1993, Martin et al. 1994). In this study, I further
characterized the role of NF-kB in the transcription and
expression of the iNOS gene in MCT cells, and also
investigated the mechanism of the synergism between LPS and
IFN, using functional promoter analyses but with special


14
The Renal System
In the kidney, inducible and constitutive NOS protein or
mRNA have been detected in mesangial cells, macula densa
cells of the juxtaglomerular apparatus, proximal tubule
cells, the entire collecting duct, as well as other
epithelial lining of the nephron (Marsden and Ballerman,
1990, Mundel et al., 1992, Terada et al., 1992, Tojo et al.,
1994 [b], Amoah-Apraku et al., 1993, Markewitz et al., 1993,
Morrissey et al., 1994).
Renal NO is involved in the regulation of renal vascular
resistance (Romero et al. 1992), medullary and glomerular
microcirculation and macula densa-mediated tubuloglomerular
feedback (Zatz and de Nucci, 1991, Brezis et al. 1991,
Wilcox et al. 1992). In addition, active induction of
nitric oxide inhibits proximal tubule Na+/K+-ATPase and ion
transport across renal tubule cell membranes (Guzman et al.,
1995, Stoos et al. 1994). Induced NO production also
inhibits H+-ATPase activity in rat cortical collecting ducts
(Tojo et al. 1994 [a]), suggesting that NO is probably
involved in the regulation of proton and bicarbonate
transport in the cortical collecting duct. Nitric oxide may
also mediate renal injury during allograft rejection, acute
tubular ischemia/reperfusion injury and autoimmune
glomerulonephritis (Langrehr et al. 1993 [b] Yu et al. ,
1994, Weinberg et al. 1994).


17
rheumatoid arthritis, where NO may be responsible for the
pathological vasodilation and tissue damage.
The sensitivity to NO varies from cell to cell, being
cytotoxic in some but cytostatic in others. This may be
explained by the relative abundance and importance of Fe-S
containing enzymes in the various cells or their ability to
buffer the NO molecules with their thiol groups.
Other Cell Types
Hepatocytes have been shown to express iNOS during
inflammation (Geller et al., 1993 [a]) and both
cytoprotective and cytotoxic effects of NO have been
demonstrated in the liver (Billiar et al. 1990). This
seemingly ambiguous effect may be explained by the cytotoxic
and vasodilatory effects of NO. Hepatocyte glucose and urea
output are also inhibited by endogenously produced NO.
Whereas the former may be attributed to inhibition of key
enzymes in glycogenolysis and gluconeogenesis, the latter is
almost certainly due to the competitive use of the common
substrate L-arginine. Nitric oxide induction in hepatocytes
may contribute to host defense against infections like
malaria (Nussler et al., 1991). In both tumor cells and
fibroblasts, interferon-y (IFN) has been shown to inhibit
replication through NO generation. Recent reports suggest
that airway epithelial cells contain cNOS and that NO may
play an important role in diverse airway functions such as
maintaining vascular and airway smooth muscle tone, host


13
Figure 2-1. Representation of a Tris Complex between Aluminum
and a Bidentate Ligand.
be dissolved in an appropriate solvent to be recrystallized.
The specific techniques used to synthesize the complexes
depend in part on the solubility and the acidity of the
particular ligand.
Materials
All materials and solvents used were reagent grade and
used as supplied by the manufacturer.
2-Methvloxine (meox)
Ligand reactivity. 8-Hydroxyquinaldine, represented here
as meox, is soluble in organic solvents such as ethanol and
dimethylsulfoxide (DMSO) but insoluble in water. With a pKa
of 10.1, meox is a rather weak acid that will require a
moderately strong base for deprotonation.
HO
Figure 2-2. 2-Methyloxine, meox.


79
The failure of LPS to induce any iNOS activity in renal
epithelium could possibly be due to its inability to cause
the activation and nuclear translocation of c-Rel. However,
a combination of LPS and IFN that caused the expression of
iNOS activity also failed to induce the nuclear migration of
c-Rel. This could possibly be that whereas LPS alone may
require a p65/c-Rel dimer to activate iNOS gene expression in
macrophages, when combined with IFN in MCT cells, activation
of other processes by IFN leads to a cross-talk of pathways
resulting in the expression of iNOS activity independent of
c-Rel. These type of transcription factor interactions have
been reported between NF-kB and the Jun/Fos proteins (Stein
et al. 1993) as well as cis-regulatory enhancer binding
protein-like factor (Mukaida et al. 1990). Also, certain
proteins such as NF-IL-6, Tax and HMG I(Y) interact with NF-
kB and possibly function as necessary accessory proteins for
transactivation of the IL-6 (Leclair et al. 1992), HTLV-1
(Hirai et al. 1992) and IFN-p (Thanos and Maniatis, 1992)
genes, respectively. Other reports also suggest that, in
addition to c-Rel, activation of other proteins appears to be
necessary for iNOS expression in macrophages (Xie et al. ,
1994). Interferon-y may therefore be, among others, an
inducer of the activation and synthesis of these proteins.
This seems to be the case in macrophages where interferon
regulatory factor 1 (IRF-1) appears to be necessary for the
synergistic induction of iNOS by IFN and LPS (Martin et al.,
1994). Although this protein is yet to be described in renal


59
Sepiapterin (5 mM) restored NO synthesis during GTPCH
blockade (figure 3-3), indicating that BH4 can be synthesized
via the salvage pathway in MCT cells. Sepiapterin alone
increased nitrite production in induced cells by 12.0 + 0.8%
(figure 3-3), suggesting that NO synthesis in MCT cells is
limited by their ability to synthesize BH4. Methotrexate
alone (10 mM) did not alter NO production in induced MCT
cells (figure 3-4), suggesting that iNOS does not normally
depend on the salvage pathway for BH4. However, methotrexate
did prevent the effect of sepiapterin during GTPCH blockade
(figure 3-4), confirming that sepiapterin is acting through
the salvage pathway.
Concentrations of sepiapterin between 1 and 8 mM
restored NO synthesis in a dose-dependent manner, whereas
concentrations higher than 10 mM had inhibitory effects
(figure 3-5). Thus, these results suggest that DAHP inhibits
GTPCH only partially and that, as reported for other cells
(Gross and Levi, 1992)), sepiapterin, in addition to being a
substrate for BH4 synthesis via the pterin salvage pathway,
is also a potent inhibitor of GTPCH in proximal tubules. At
incubation periods of 36 h and 48 h, the sepiapterin rescue
was less than expected (data not shown) This may be
because, at longer incubation periods, as the exogenously
added sepiapterin becomes depleted, the rescue then becomes
limited by the availability of dihydropterines. Due to the
inhibitory effect of high concentrations of sepiapterin,


106
Table 7-1. Crystallographic Data for IX
Compound
(C5H4N(C02H) ) (C5H4NH(C02) ) (NH4) (no3)
Formula
^12^14^07
MW
326.27
Crystal system
Monoclinic
Space group
P 2jc
a,
12.1985
b,
7.9230
c,
15.6065
0,
101.525
Vc, 3
1477.94
Z
4
Vcaio g/cm3
1.47
Radiation
Cu Koi
X,
1.54178
/x, cm'1
10.11
F(000)
679.86
Observed reflections
1573
Number of parameters
264
Final R
0.0580
Rw
0.0593
0.0593


50
Figure 2-6. Inducible NOS activity in crude homogenates of
MCT cells after 24 h of treatment with LPS (0.1 ng/ml), IFN
(100 U/ml) or a combination of both. In vitro iNOS activity
assay was performed in the presence of saturating
concentrations of NADPH, BH4, FAD and FMN. Even in the
presence of saturating concentrations of exogenously added
cofactors, IFN failed to induce iNOS activity (* p<0.05 vs
control, M-g: |og of cell protein, n=6) .


54
Table 4-5 -- Continued
0(2)-Al-0(3) 94
N(2)-Al-0(3) 86
0(1)-Al-N(3) 99
N (1) -Al-N (3) 91
0(2) -Al -N (3) 86
N (2) -Al-N (3) 164
0(3) -Al-N(3) 82
Al-0(1)-C(12) 118
Al-N(1)-C(11) 106
Al-N(1)-C(19) 135
C(ll)-N(l)-C(19) 118
N(l)-C(ll)-C(12) 115
N(l)-C(ll)-C(16) 123
C(12)-C(ll)-C(16) 120
0(1)-C(12)-C(ll) 117
0(1)-C(12)-C(13) 122
C(ll)-C(12)-C(13) 119
C(12)-C(13)-C(14) 118
C(12)-C(13)-H(13) 116
C(14)-C(13)-H(13) 124
C(13)-C(14)-C(15) 122
C(13)-C(14)-H(14) 126
C(15)-C(14)-H(14) 107
C(14)-C(15)-C(16) 120
C(14)-C(15)-H(15) 116
C(16)-C(15)-H(15) 121
C(ll)-C(16)-C(15) 118
C(ll)-C(16)-C(17) 116
C(15)-C(16)-C(17) 125
C(16)-C(17)-C(18) 121
C(16)-C(17)-H(17) 141
C(18)-C(17)-H(17) 93
C(17)-C(18)-C(19) 120
C(17)-C(18)-H(18) 101
C(19)-C(18)-H(18) 136
N(l)-C(19)-C(18) 120
N(l)-C(19)-C(10) 122
C(18)-C(19)-C(10) 117
C(19)-C(10)-H(101) 124
Al-0(2)-C(22) 116
Al-N(2)-C(21) 106
Al-N(2)-C(29) 134
C(21)-N(2)-C(29) 118
N(2)-C(21)-C(22) 116
N(2)-C(21)-C(26) 123
C(22)-C(21)-C(26) 119
0(2)-C(22)-C(21) 116
0(2)-C(22)-C(23) 122
C(21)-C(22)-C(23) 120
.0(2) C(21)-C(26)-C(27)
.8(2) C(25)-C(26)-C(27)
.1(3) C(26)-C(27)-C(28)
.6(2) C(26)-C(27)-H(27)
.7(2) C(28)-C(27)-H(27)
.8(3) C(27)-C(28)-C(29)
.8(2) C(27)-C(28)-H(28)
6(5) C(29)-C(28)-H(28)
3(5) N(2)-C(29)-C(28)
1(5) N(2)-C(29)-C(20)
5(6) C(28)-C(29)-C(20)
7(7) C(29)-C(20)-H(201)
3(7) C(29)-C(20)-H(202)
9(8) H(201)-C(20)-H(202)
7(7) Al-0 (3) -C (32)
8(7) Al-N (3) -C (31)
5(8) Al-N (3) -C (39)
6(8) C(31)-N(3)-C(39)
0(8) N(3)-C(31)-C(32)
1(8) N(3)-C(31)-C(36)
7(9) C(32)-C(31)-C(36)
6(9) 0(3)-C(32)-C(31)
9(8) 0(3)-C(32)-C(33)
2(9) C(31)-C(32)-C(33)
8(9) C(32)-C(33)-C(34)
3(8) C(32)-C(33)-H(33)
0(8) C(34)-C(33)-H(33)
3(8) C(33)-C(34)-C(35)
7(8) C(33)-C(34)-H(34)
0(8) C(35)-C(34)-H(34)
4(9) C(34)-C(35)-C(36)
9(7) C(34)-C(35)-H(35)
4(8) C(36)-C(35)-H(35)
5(7) C(31)-C(36)-C(35)
4(8) C(31)-C(36)-C(37)
3(7) C(35)-C(36)-C(37)
3(7) C(36)-C(37)-C(38)
2(7) C(36)-C(37)-H(37)
2(8) C(38)-C(37)-H(37)
1(5) C(37)-C(38)-C(39)
8(4) C(37)-C(38)-H(38)
7(5) C(39)-C(38)-H(38)
5(6) N(3)-C(39)-C(38)
9(7) N(3)-C(39)-C(30)
6(7) C(3 8)-C(39)-C(30)
5(7) C(39) C(30)-H(301)
8(7) C(39)-C(30)-H(302)
4(7) H(301)-C(30)-H(302)
8 (8)
116.8(7)
123.8 (7)
119.4(7)
119.6(7)
120.1(7)
121.3(7)
114.9 (7)
120.3 (7)
120.4 (7)
120.7(7)
118.9(7)
103.5(7)
118.0(6)
111.0(7)
116.6(5)
106.6 (5)
133.8 (6)
119.6(7)
115.5(7)
123.8(8)
120.7(8)
118.0(7)
122.2 (8)
119.7(7)
119.0(9)
116.5(7)
118.5(8)
121.4 (10)
108.5(9)
129.7(9)
121.0(9)
112.7(10)
119.0(10)
118.0(9)
113.3 (8)
128.7(8)
123.4 (8)
94.5(9)
142.1(11)
119.8(9)
137.2(9)
102.7(8)
120.1 (8)
121.6(8)
118.2(8)
119.3(9)
99.0(8)
111.5(8)


CHAPTER ONE
INTRODUCTION
General
Mammalian cells have been known to produce oxides of
nitrogen since 1916 (Mitchell et al. 1916), an observation
verified and extended in the late 1970s and early 1980s when
it was shown that mammals produce nitrates and that this
production was enhanced by treatment with endotoxin (Stuehr
et al., 1985). To biologists in between these periods, these
compounds held little interest beyond their ability to cure
meat or aggravate pulmonary diseases even though compounds
that release nitric oxide (NO) have been used in clinical
medicine for over 150 years. This lack of interest in NO
could be because both biologists and clinicians were ignorant
of the endogenous mechanisms through which these compounds
exerted their clinical effects.
Biologic NO formation was independently discovered as a
physiologic regulator in blood vessels, macrophages and
neurons (Moneada et al., 1991). In a classic study,
Furchgott and Zawadzki demonstrated that in vessel segments
precontracted with norepinephrine, acetylcholine elicited
relaxation only if the endothelium was intact, suggesting
endothelial release of a short-lived substance that diffuses
to the smooth muscle to effect relaxation (Furchgott and
1


12
creating a more stable complex is by the formation of one or
more five- or six-membered rings by complexation of two or
more functional groups belonging to the same donor molecule.
Most nitrogen donor atoms occur as the amine functional
group, whether aromatic or aliphatic. In amines, the nitrogen
atoms are more likely to become positively charged than
negatively. As a result, even though a nitrogen atom can
coordinate to an aluminum ion, the electrostatic attraction
between the metal ion and an oxygen donor tends to be stronger
and, therefore, forms more stable complexes.
Synthesis
The general technique for the synthesis of an aluminum
complex involves the combination of a soluble aluminum salt,
the ligand and a base. When the ligand is deprotonated, a
negatively charged donor atom is created. If the anionic
part of the ligand is a harder Lewis base than water, it is
then capable of displacing the neutral water molecule. If the
ligand can only carry one negative charge and this process is
repeated two more times, a neutral aluminum species is formed.
Such a non-charged species, especially with a shell of
aromatic carbon and hydrogen atoms, will generally have a
relatively low water solubility. Therefore, after a certain
quantity of the complex is formed, it will begin to
precipitate. The solid can then be collected by filtration,
rinsed and dried. Depending upon solubility, the complex may


16
The Immune System
That the cytotoxic effects of macrophages are due to
their ability to generate NO is well documented. These
include inhibition of mitochondrial respiration and DNA
synthesis mediated through NO-dependent inhibition of Fe-
containing enzymes (Dimmeler et al. 1992, Stuehr et al. ,
1989, Drapier and Hibbs, 1988). Macrophage NOS is the
inducible isoform, usually requiring cytokines or bacterial
products to stimulate its activity. In mammals, a
correlation between immunostimulation and elevated nitrate
and nitrite levels has been demonstrated (Stuehr et al.,
1985) and the L-arginine-NO pathway has been proposed to be
the primary nonspecific defense mechanism against
intracellular microorganisms and other pathogens such as
viruses, fungi, helminths and protozoa (Karupiah et al.,
1993, Alspaugh and Granger, 1991, Vincendeau and Daulouede,
1991, Nussler, 1992) .
Expression of iNOS is an inflammatory response to
infection or tissue injury that could be adaptive and
beneficial with respect to its antimicrobial action and
increased tissue perfusion, but it could also be detrimental,
such as occurs in autoimmune diseases or the marked
vasodilation seen in septic shock. Hence, the inhibition by
glucocorticoids of iNOS induction may explain, at least in
part, the therapeutic actions of these compounds in
conditions such as treatment of septic shock, asthma or


77
The combination of c-Rel and p65 IgGs resulted in an almost
total disappearance of the LPS-induced NF-KB complex in these
cells (data not shown) The formation of p65/c-Rel
heterodimers has been reported to be important in mediating
the transactivation of iNOS in macrophages (Xie et al. ,
1994) Treatment of MCT cells with LPS resulted in
activation of NF-kB complexes containing p65 and p50 proteins
(figure 4-8B). The activation of p65 appeared to be greater
in MCT cells than in RAW 264.7 macrophages. However,
contrary to the observation in macrophages, c-Rel activation
was not observed in renal epithelium. The addition of c-Rel
IgG to either p50 or p65 IgG failed to affect the respective
NF-kB complexes (data not shown). These results indicate
that the inability of LPS to induce iNOS activity in MCT
cells may be associated with the absence of c-Rel activation
by this agent, suggesting that c-Rel may be an important
determinant of tissue specificity of LPS-mediated iNOS
responses.
To determine whether the synergism between LPS and IFN
in iNOS induction could be related to interactions between
different NF-kB proteins activated selectively by each of
these agents, I investigated the effects of IFN alone and
combined LPS and IFN treatment on the activation of NF-kB
subunits in MCT cells. The profile of NF-kB was exactly the
same as that observed after LPS treatment alone, and is
characterized by predominant activation of p65, smaller
amounts of p50 and the absence of c-Rel (figure 4-9). Thus,


123
activator Tax of HTLV-1 binds to the NF-kB precursor pl05.
Oncogene 7: 1737-1742, 1992.
Ignarro, L.J. Biosynthesis and metabolism of endothelium-
derived nitric oxide. Ann. Rev. Pharmacol. Toxicol. 30: 535-
560, 1990.
Ignarro, L.J., Buga, G.M., Wood, K.S., Byrns, R.E. and
Chaudhuri, G. Endothelial-derived relaxing factor produced
and released from artery and vein is nitric oxide. Proc.
Natl. Acad. Sci. USA 84: 9265-9269, 1987.
Jorens, P.G., Van Overveld, F.J., Vermeir, P.A., Bult, H. and
Herman, A.G. Synergism between interleukin-l-p and
interferon-y, an inducer of nitric oxide synthase, in rat
fibroblasts. Eur. J. Pharmacol. 224: 7-12, 1992.
Junguero, D.C., Scott-Burden, T., Schini, V.B. and Vanhoutte,
P.M. Inhibition of cytokine-induced nitric oxide production
by transforming growth factor B1 in human smooth muscle
cells. J. Physiol. Lond. 454: 451-465, 1992.
Kamijo, R., Harada, H., Matsuyama, T., Bosland, M.,
Gerecitano, J., Shapiro, D., Le, J., Koh, S.I., Kimura, T.,
Green, S.J., Mak, T.W., Taniguchi, T. and Vicek, J.
Requirement for transcription factor IRF-1 in NO synthase
induction in macrophages. Science 263: 1612-1615, 1994.
Karupiah, G. Xie, Q-w., Buller, R.M.L., Nathan, C., Duarte,
C. and MacMicking, J.D. Inhibition of viral replication by
inter feron-y-induced nitric oxide synthase. Science 261:
1445-1448, 1993.
Kaufman, S. New tetrahydrobiopterin-dependent systems. Annu.
Rev. Nutr. 13: 261-286, 1993.
Ketteler, M. Border, W.A. and Noble, Nancy. Cytokines and
L-arginine in renal injury and repair. Am. J. Physiol. 267:
F197-F207, 1994.
Kilbourn, R.G. and Bellone, I. Endothelial cell production
of nitrogen oxides in response to interferon in combination
with tumor necrosis factor, interleukin-1, or endotoxin. J.
Natl. Cancer Inst. 82: 772-776, 1990.
Koesling, D. Schultz, G. and Bhme, E. Sequence homologies
between guanylyl cyclases and structural analogies to other
signal transducing proteins. FEBS Lett. 280: 301-306,1991.
Kubes, P., Suzuki, M. and Granger, D.N. Nitric oxide: an
endogenous modulator of leukocyte adhesion. Proc. Natl. Acad.
Sci. USA 88: 4651-4655, 1991.


139
(15) Motekaitis, R. J. ; Martell, A. E. Inorg. Chem. 1984, 23,
18-23 .
(16) Marklund, E.; hman, L.-O. Acta Chem. Scand. 1990, 44,
353-357.
(17) Nelson, W. O.; Karpishin, T. B.; Rettig, S. J.; Orvig,
C. Inorg. Chem. 1988, 27, 1045-1051.
(18) Finnegan, M. M.; Lutz, T. G.; Nelson, W. O.; Smith, A.;
Orvig, C. Inorg. Chem. 1987, 26, 2171-2176.
(19) Lutz, T. G.; Clevette, D. J.; Rettig, S. J.; Orvig, C.
Inorg. Chem. 1989, 28, 715-719.
(20) Taylor, D. Aust. J. Chem. 1978, 31, 1455-1462.
(21) Muetterties, E. L.; Guggenberger, L. J. J. Am. Chem.
SOC. 1972, 94, 8046-8054.
(22) McClelland, B. W. Acta Cryst. 1975, B31, 2496-2498.
(23) Feng, T. L.; Gurian, P. L.; Healy, M. D.; Barron, A. R.
Inorg. Chem. 1990, 29, 408-411.
(24) Sattin, A; Muhoberac, B. B.; Aprison, M. H.; Schauf, C.
L. Medical Hypotheses 1989, 29, 155-159.
(25) Schmidbaur, H; Lettenbauer, J.; Wilkinson, D. L.;
Mller, G. ; Kumberger, O. Z. Naturforsch, 1991, 46b,
901-911.
(26) Liu, S.; Rettig, S. J.; Orvig, C. Inorg. Chem., 1992,
31, 5400-5407.
(27) Hoveyda, H. R.; Karunaratne, V.; Rettig, S. J.; Orvig,
C. Inorg. Chem., 1992, 31, 5408-5416.
(28) Bossek, U.; Hanke, D.; Wieghardt, K.; Nuber, B.
Polyhedron, 1993, 12, 1-5.
(29) Liu, S.; Wong, E.; Karunaratne, V. ; Rettig, S. J.;
Orvig, C. Inorg. Chem., 1993, 32, 1756-1765.
(30) Simonsen, S. H.; Bechtel, D. W. American
Crystallographic Association, Ser. 2, 1980, 7, 23.
(31) Ul-Haque, M.; Horne, W.; Lyle, S. J. J. Cryst. Spect.
Res. 1991, 21, 411-417.
(32) Kushi, Y.; Fernando, Q. J. Am. Chem. Soc. 1970, 92,
91-95.


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
REGULATION OF INDUCIBLE NITRIC OXIDE SYNTHASE IN MURINE
PROXIMAL TUBULE EPITHELIAL CELLS
By
Bismarck Amoah-Apraku
August, 1995
Chairman: Nicolas J. Guzman, MD
Major Department: Pharmacology and Therapeutics
Nitric oxide (NO) is an important mediator of vascular
tone, neurotransmission and immune responses. The mechanisms
regulating inducible NO synthase (iNOS) expression in renal
tubule epithelium have not been well characterized. The
purpose of this study was to investigate the mechanism of
potentiation between interferon-y (IFN) and bacterial
lipopolysaccharide (LPS) in inducing iNOS activity in murine
proximal tubule epithelial (MCT) cells. Treatment with LPS
caused a small increase in iNOS activity whereas IFN had no
effect. Simultaneous treatment with LPS and IFN (LPS/IFN),
however, markedly increased iNOS activity. Treatment with
LPS/IFN in the presence of 2,4-diamino-6-hydroxypyrimidine
(DAHP), an inhibitor of the rate-limiting enzyme GTP-
cyclohydrolase I in the de novo synthesis of
vixi


127
Nussler, A.K., Liu, Z.Z., Di Silvio, M. Sweetland, M.A. ,
Geller, D.A., Lancaster, J.R., Billiar, T.R., Freeswick,
P.D., Lowenstein, C.L. and Simmons, R.L. Hepatocyte
inducible nitric oxide synthesis is influenced in vitro by
cell density. Am. J. Physiol. 267: C394-C401, 1994.
O'Dell, T.J., Hawkins, R.D., Kandel, E.R. and Arancio, 0.
Tests of the roles of two diffusible substances in long-term
potentiation: evidence for nitric oxide as a possible early
retrograde messenger. Proc. Natl. Acad. Sci. USA 88:11285-
11289, 1991.
Olson, J.S. Stopped-flow, rapid mixing measurements of
ligand binding to hemoglobin and red cells. Methods Enzymol.
76: 631-651, 1981.
Palmer, R.M.J., Ferrige, A.G. and Moneada, S. Nitric oxide
release accounts for the biological activity of endothelium-
derived relaxing factor. Nature 327: 524-526, 1987.
Petros, A., Lamb, G., Leone, A., Moneada, S., Bennett, D. and
Vallance, P. Effects of a nitric oxide synthase inhibitor in
humans with septic shock. Cardiovasc. Res 28: 34-39, 1994.
Pfeilschifter, J. and Schwarzenbach, H. Interleukin-1 and
tumor necrosis factor stimulate cGMP formation in rat
mesangial cells. FEBS Lett. 273: 185-187, 1990.
Radomski, M.W., Palmer, R.M.J. and Moneada, S. Endogenous
nitric oxide inhibits human platelet adhesion to vascular
endothelium. Lancet ii: 1057-1058, 1987.
Radomski, M.W., Palmer, R.M.J., and Moneada, S.
Glucocorticoids inhibit the expression of an inducible, but
not the constitutive nitric oxide synthase in vascular
endothelial cells. Proc. Natl. Acad. Sci. USA 87: 10043-
10047, 1990.
Raij L. Glomerular thrombosis in pregnancy: role of the L-
arginine-nitric oxide pathway. Kidney Int. 45: 775-781, 1994.
Rao, D.N. and Kaufman, S. Purification and state of
activation of rat kidney phenylalanine hydroxylase. J. Biol.
Chem. 261: 8866-8876, 1986.
Rees, D.D., Palmer, R.M.J. and Moneada, S. Role of
endothelium-derived nitric oxide in the regulation of blood
pressure. Proc. Natl. Acad. Sci. USA 86: 3375-3378, 1989.
Reif, D.W. and Simmons, R.D. Nitric oxide mediates iron
release from ferritin. Arch. Biochem. Biophys. 283: 537-541,
1990.


NO synthesis through a feedback inhibition. As described
elsewhere, substrate and cofactor availability may also play
an important role in the regulation of NO production.
Pharmacologically, there are agents that promote or
inhibit NO release. Some agents mimic NOS by releasing NO.
These agents, termed NO donors, include organic nitrates and
sodium nitroprusside. They have been used clinically for
several years.
There are several classes of NOS inhibitors. These
include: (i) substrate analogues that are N-substituted
analogues of arginine such as Nw-mono-methyl-L-arginine (L-
NMMA) and N-nitro-L-arginine (NNA); these usually act as
competitive inhibitors of L-arginine when added along with
the necessary cofactors (Moneada et al., 1991); (ii) covalent
modifiers of NOS such as aromatic diphenylene iodoniums that
tend to compete with the nucleotide cofactors for binding to
NOS (Stuehr et al. 1991 [b] ) ; (iii) carbon monoxide, which
inhibits NOS activity by binding to the heme moiety of the
enzyme (White and Marietta, 1992); (iv) compounds that bind
to calmodulin thereby impeding its binding to NOS, such as
calcineurin and trifluoroperazine; they are therefore
specific for cNOS which is dependent on Ca2+-calmodulin for
activity (Bredt and Snyder, 1990); (v) agents that inhibit
NOS activity by interfering with cofactor or substrate
availability such as substituted pyrimidines (e.g., 2,4-
diaminohydroxypyrimidine, DAHP) ; these inhibit GTP
cyclohydrolase I, the rate-limiting enzyme in BH4 synthesis


REGULATION OF INDUCIBLE NITRIC OXIDE SYNTHASE IN MURINE
PROXIMAL TUBULE EPITHELIAL CELLS
BY
BISMARCK AMOAH-APRAKU
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF .THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1995


40
be that x-ray quality crystals may never be grown for these
compounds.


69
HN
,CH2COOH
CH2COOH
(a)
HOOCCH2-N
CH2COOH
\:h2cooh
(b)
HOOCCH2
CH2CH20H
V
HOOCCH2
CH2COOH
/ NCH2CH2N NCH2CH2N '
HOOCCH2 CH2COOH H00CCH2// CH2COOH
(c) (d)
HOOCCH2
.CH2COOH
/
/
^N-CH-CH2-n(
H00CCH27 CH3 XCH2COOH
HOOCCH^
HOOCCH2
/CHaCOOH
xch2cooh
(e)
(f)
Structures of Ligands in Table 5-1.
Figure 5-1


104
however that the pCAT-Basic vector possesses its own TATA box
and transcription initiation site (Gorman et al., 1982).
Further studies to resolve this may best utilize constructs
where site-directed mutagenesis is used to alter only the
downstream NF-kB site, thus preserving the TATA box and the
transcriptional initiation site.
One intriguing finding of this study is the ability of
LPS alone to induce significant amount of CAT activity in
pAiNOS-CAT and pBiNOS-CAT transiently transfected into MCT
cells although LPS does not induce iNOS protein synthesis or
nitrite production in MCT cells (figure 4-1 & 4-3).
Lipopolysaccharide causes only moderate transcription of iNOS
mRNA in MCT cells. This may be because iNOS mRNA may possess
a number of bases at the 3'-flanking end not present in CAT
mRNA that makes it subject to increased degradation,
decreased stability or both, by proteins possibly coinduced
by LPS. The role of IFN may then be to activate pathways
that repress these proteins. Moreover, the promoter region
inserted into the construct may be too short and therefore
devoid of inhibitory binding sites present in the native
cells for proteins possibly coinduced by LPS. If such
proteins act as transcriptional or posttranscriptional
inhibitors of iNOS protein synthesis, then their absence from
the constructs may allow LPS to express CAT activity in
transfected cells but not iNOS in native cells. The
activities of such proteins may be those masked by pathways
activated by coincubation with IFN. It may well be part of


73
dishes in 20 mM Tris-HCl, pH 7.5, containing 0.1 mM PMSF, 2
mM EDTA, 0.5 mM EGTA, 10 mg/ml leupeptin and 1 mg/ml
aprotinin, centrifuged at 1000 rpm for 5 min and homogenized
on ice with 30 strokes in an Eppendorf tube with a disposable
homogenizer. Cellular proteins were solubilized in SDS,
separated by discontinuous polyacrylamide gels (5% stacking,
7.5% resolving gel, 40 mA for 5 h), and transferred to a 0.45
jim nitrocellulose membrane in 25 mM Tris, pH 8.3, 150 mM
glycine, and 20% vol/vol methanol for 15 h at 100 mA. The
nitrocellulose membrane was blocked with 10% nonfat dry milk
in PBS for 2 h and subsequently incubated with mouse anti-
mac-NOS monoclonal antibodies (1/250) for 4 h at 20 C .
After washing the membrane to remove excess antibody,
peroxidase-labeled anti-mouse secondary antibodies were
applied and the protein of interest identified by enhanced
chemiluminiscence (ECL, Amersham Corporation, Arlington
Heights, IL).
Results
Effects of L i popo lvsac char ide and Interferon-y on Nitrite
Accumulation in Murine Proximal Tubule Cells
To investigate the effects of LPS and IFN on nitrite
accumulation in MCT cells, I measured accumulated nitrite by
MCT cells after 24 h incubation with LPS (1 |ig/ml), IFN (100
U/ml) or in combination.
Incubation of MCT cells with LPS (1 ng/ml for 24 h)
stimulated only modest nitrite production (figure 4-1).


A
B
Control IFN LPS LPS/IFN
Figure 5-6. Effect of the removal of the downstream NF-kB binding site on the activation of
iNOS promoter by LPS and IFN. The MCT cells transfected with pCiNOS-CAT vector, which is
devoid of the downstream NF-kB binding site, were treated with LPS (1 |ig/ml) and IFN (100
U/ml) or their combination for 24 h followed by CAT assay of the cell lysates. There is the
loss of CAT inducibility by LPS, IFN or their combination; (n=3). A Autoradiograph, B
graphical presentation.
112


141
(49) Biagini-Cingi, M.; Chiesi-Villa, A.; Guastini, C.;
Nardelli, M. Gazz. Chim. Ital., 1972, 102, 1026-1033.
(50) Beveridge, K. A.; Bushnell, G. W. Can. J. Chem., 1979,
57, 2498-2503.
(51) Ou, C.-C.: Borowski, W. J.; Potenza, J. A.; Schugar, H.
J. Acta Cryst., 1977, B33, 3246-3248.
(52) Silverstein, R. M.; Bassler, G. C. ; Morrill, T. C.
"Spectrometric Identification of Organic Compounds" 4th
ed., John Wiley and Sons, New York, 1981.
(53) Cotton, F. A.; Wilkinson, G. "Advanced Inorganic
Chemistry: A Comprehensive Text"; Oxford University
Press, New York, 1985.
(54) Akitt, J. W. Prog, in NMR Spec., 1989, 21, 1-149.
(55) Wu, Y.; Chmelka, B. F.; Pines, A.; Davis, M. E.; Grobet,
P. J.; Jacobs, P. A. Nature, 1990, 346, 550-553.
(56) Dirken, P. J.; Jansen, J. B. H.; Schuiling, R. D. Amer.
Miner., 1992, 77, 718-724.
(57) Iyer, R. K. ; Karweer, S. B.; Jain, V. K. Magn. Reson.
in Chem., 1989, 27, 328-334.
(58) Polynova, T. N. ; Bel'Skaya, N. P.; Banus, D. T. G.;
Porai-Koishits, M. A.; Martynanko, L. I. J. Struct.
Chem., 1970,
11,
158 .
(59)
Rinehart,
K.
L. ,
Jr Science,
1982, 218, 254-260.
(60)
Reynolds,
D.
R. ;
Burn, J. L.
E.; Boggess, B.; Cook, K
D.; Woods, C. Inorg. Chem., 1993, 32, 5517-5521.
(61) Glusker, J. P.; Trueblood, K. N. "Crystal Structure
Analysis: A Primer" 2nd ed., Oxford University Press,
New York, 1985.
(62) Desiraju, G. R.; Curtin, D. Y.; Paul, I. C. J. Am.
Chem. Soc., 1977, 99, 6148.
(63) March. J. "Advanced Organic Chemistry: Reactions,
Mechanisms and Structure" 3rd ed., John Wiley and Sons,
New York, 1985.
(64) Hollinshead, R. G. W. "Oxine and its Derivatives" part
I; Butterworths, London, 1954.


101
Promoter Activity of the 1.7 Kilobase Pair Fragmei.t and its
Truncated Plasmid Constructs
To investigate the role of NF-kB in the induction of
iNOS gene transcription by LPS and IFN, the constructs
described above were fused with the promoterless CAT reporter
gene, the pCAT-Basic reporter vector and transiently
transfected into MCT cells.
As reflected in the CAT activity shown in figure 5-3,
pAiNOS-CAT, the untruncated construct, conferred CAT
responsiveness by LPS (1 |ig/ml) in combination with IFN (100
U/ml) in contrast to the native vector. Treatment with LPS
alone (1 |ig/ml) resulted in a 3.9-fold increase in CAT
activity and cotreatment with IFN (100 U/ml) potentiated the
induction of CAT activity to 6.6-fold, which was blocked by
the addition of PDTC (25 ^M) (figure 5-4). Interferon-y alone
(100 U/ml) did induce a small but significant CAT activity
(figure 5-4). Taken together, these findings show that the
effect of LPS and IFN on the promoter region is
transcriptional and that it requires NF-kB.
The construct pBiNOS-CAT, consisting of the downstream
portion linked to the CAT gene conferred inducibility by LPS
(1 |a.g/ml) and a combination of LPS (1 |ig/ml) and IFN (100
U/ml) but not by IFN alone. However, in contrast to pAiNOS-
CAT, the synergistic effect of IFN was lost (figure 5-5).
The construct pCiNOS-CAT which lacked the downstream NF-kB
binding site was devoid of promoter activity in response to
treatment with LPS, IFN or their combination (figure 5-6).


74
Interferon-y (100 U/ml), which by itself was also ineffective
in stimulating nitrite production, potently synergized with
LPS to stimulate iNOS activity (figure 4-1).
Effects of Lipopolvsaccharide and Interferon-y on Inducible
Nitric Qsifls...Synthage..mRNA
To investigate the effects of LPS and IFN on steady-
state iNOS mRNA levels, total RNA extraction was done in
cells incubated with LPS, IFN, or LPS plus IFN. A northern
blot analysis was then done on the extracts.
Combined treatment with LPS and IFN was accompanied by
marked time-dependent increase in steady-state iNOS mRNA
levels that were initially detected after 2 h of treatment,
peaked at 6 h and decreased after 24 h (figure 4-2).
Lipopolysaccharide treatment (1 ng/ml for 6 h) caused only a
small increase in steady-state iNOS mRNA levels whereas none
was detected when the cells were treated with IFN alone
(figure 4-2).
Effects of Lipopolvsaccharide and Interferon-y on Inducible
Nitric Oxide Svnthase Protein Levels
To determine the roles of LPS and IFN in inducing iNOS
protein synthesis, cells were treated with LPS (1 ng/ml)
alone or in combination with IFN (100 U/ml) and iNOS protein
was determined by Western analysis using a monoclonal
antibody against macrophage iNOS.
Despite the moderate amount of iNOS mRNA induced by LPS
(figure 4-2), it failed to induce detectable levels of iNOS


42
and also modulate ion and acid transport across renal
epithelial cells membranes (Guzman et al., 1995, Tojo et al.,
1994 [a], Stoos et al., 1994). This suggests an important
role for NO in renal ion and acid-base homeostasis. Early
pathological findings in tubulointerstitial nephritis include
necrotic tubule epithelium with interstitial infiltrates of
lymphocytes and plasma cells which are cytokine-secreting
cells. Interferon-y is therefore likely to be an important
mediator of disorders involving T-cell responses such as
allograft rejection, autoimmune glomerulonephritis and
certain forms of tubulointerstitial nephritis, events in
which NO may play a mediating role (Langrehr et al., 1993
[a,b], Weinberg et al., 1994).
The discovery of the presence of iNOS in proximal
tubules has important implications due to the potential
pathophysiologic role of NO during immune-mediated and other
forms of renal injury. Migration of even small numbers of
inflammatory cells into the tubulointerstitium is likely to
lead to high local concentrations of cytokines such as IFN
and TNF-a which can then potentiate each other to induce iNOS
activity and stimulate the release of large amounts of NO by
proximal tubule cells. High concentrations of NO are
cytotoxic due to its conversion into toxic radicals like
peroxynitrites (Nussler and Billiar, 1993). Thus, proximal
tubule iNOS has the potential of playing a critical role in
the modulation of cellular injury during immune-mediated
responses. Therefore, the existence of several different


This dissertation is dedicated to the blessed memory of my
dad who passed away while I was studying for this work, and
to all my loved ones, especially my beloved Margaret.


10
A naturally occurring ligand, 2,6-pyridinedicarboxylic
acid, has the potential to be an electron donor from the
nitrogen as well as the two carboxylate oxygens. This
molecule, also known by its common name dipicolinic acid, has
been reported in a variety of compounds as a tridentate
ligand.45'51 Because dipicolinic acid has the potential to
coordinate as a bidentate as well as a tridentate ligand,
there is more than one possible mode of coordination. If only
one of the carboxylate groups is deprotonated, then
dipicolinic acid should act as a bidentate ligand. In a
three-to-one ratio, the neutral tris complex could
theoretically be formed. Another type of complex could be
formed if both of the carboxylic acid groups on dipicolinic
acid were deprotonated. Were this to occur, dipicolinate
could act as a tridentate ligand by coordinating aluminum with
a nitrogen and both oxygen atoms.
The details of the investigation into the aluminum
complexes for these ligands are presented in the following
chapters. In addition, three compounds without aluminum were
inadvertently synthesized during this project. The structure
determinations for these compounds is also included.


6
OfUI
Figure 1-2. ORTEP of the Aluminum Citrate Complex, left, and
its Al304 Core, right. Feng, Gurian, Healy and Barron,
Inorganic Chemistry, 1990, 29, 408-411.
Aluminum Complexes with a Nitrogen Donor Atom
There are many naturally occurring ligands and biological
molecules containing nitrogen as a potential donor atom. For
example, the pharmaceutical 9-amino-1,2,3,4-tetrahydroacridine
(THA) pictured in Figure 1-3, has been used to alleviate
symptoms in Alzheimer's Disease patients. It has been
proposed that the aromatic ring is hydroxylated to yield a 0-
C-C-N donor group.24 Currently, the list of aluminum complexes
with a nitrogen as an electron donor is relatively short.25"43
From these studies, only seven coordination centers with N,0
donors have been confirmed by x-ray diffraction techniques.25-32
In an effort to further understand the behavior of Al(III),


ACKNOWLEDGMENTS
My first and utmost sincere thanks go to my committee
chairman and mentor, Dr. Nicolas J. Guzman, for his guidance,
support, infinite patience and invaluable contribution to my
education. Secondly, my thanks go to Dr. Jeffrey Harrison,
for his outstanding help, enthusiasm and encouragement with
the molecular biology aspects of this work. I also
respectfully thank the other members of my advisory
committee, Dr. Luiz Belardinelli, Dr. Fulton Crews and Dr.
Colin Sumners, for their thoughtful suggestions and support.
I am very grateful to Dr. Mao-Zhong Fang, Dr. Judson
Chandler, Dr. Rajesh Davda and Mina Salafranea for their
excellent technical assistance and companionship. I also
wish to thank members of the secretarial, fiscal and
administrative staff who ensured that I was duly registered
every semester and that the check for my subsistence was
constantly in the mail, and especially, Judy Adams, for her
help in preparing this manuscript. Many thanks go to my
fellow graduate students, who helped make my stay tolerable,
especially Scott Masten and Monica Sanghani.
Last, but not the least, I wish to thank Dr. Stephen P.
Baker for his encouragement, help and frequent advice and
proddings, as well as his confidence in me for nominating me
for several awards and competitions during my study tour. To
iii


4
phosphorylation by other kinases has no effect (Bredt et al.,
1992, Davda et al. 1994). Endothelial NOS has a
myristoylation site that could account for its association
with membranes thereby probably directing NO delivery to the
underlying vascular smooth muscle cells for the maintenance
of basal vascular tone, whereas other isoforms are largely
cytosolic (Dinermann et al., 1993).
The capacity to express iNOS exists in nearly every
mammalian tissue and iNOS has been expressed in such diverse
cells as macrophages, microglia, hepatocytes, vascular smooth
muscle, endothelial cells, pancreatic islet cells,
chondrocytes and kidney cells (Bredt et al., 1991, Chandler
et al., 1994, Charles et al., 1993, Forstermann et al., 1991,
Geller et al., 1993 [a,b], Lowenstein et al., 1993, Markewitz
et al. 1993). So far, almost all agents known to induce
iNOS are microbes, microbial products or inflammatory
cytokines, often exhibiting strong synergism between them and
variability between cell types in their responses to the
various inducing agents.
Substrate and Cofactor Requirements
All three isoforms of NOS are hemoproteins and are
therefore dependent on NADPH, FAD and FMN.
Tetrahydrobiopterin is also an essential cofactor of NOS and
is tightly bound to the enzyme (Schmidt et al., 1991, Schmidt
et al., 1993, Tayeh and Marietta, 1989). Tetrahydrobiopterin
synthesis occurs via two distinct pathways: a de novo


6-1 Dipicolinic acid 90
6-2 ORTEP Representation of VIII 96
7-1 ORTEP of Compound IX 107
7-2 Detail Showing Hydrogen Bonding in IX 108
8-1 ORTEP Drawing of X Showing the Atomic
Numbering Scheme 119
8-2 Sodium Ion Coordination in X, Showing
the Square Antiprismatic and
Dodecahedral Orientations 122
x


83
Table 5-4. Elemental Analysis Data for Compounds III through
VII and Pure Tris Complexes of the Ligands.
Compound
Data type
Carbon
Hydrogen
Nitrogen
III
% comp.
48.06
3.08
9.34
ratio
6.00
4.58
1.00
IV
% comp.
38.38
3.85
6.90
ratio
6.49
7.75
1.00
V
% comp.
26.12
4.30
11.85
ratio
5.15
10.18
2.00
VI
% comp.
32.74
3.03
30.90
ratio
4.94
5.49
4.00
VII
% comp.
33.84
3.57
5.40
ratio
7.31
9.25
1.00
A1(pic)3
% comp.
54.96
3.08
10.73
Al(mpic)3
% comp.
57.93
4.17
9.65
A1(pza)3
% comp.
45.46
2.29
21.21
Al(hyp)3
% comp.
41.67
2.08
38.89
Al(dipic)3
% comp.
48.01
2.30
8.00 !
the percentages that would be present if the samples were
unsolvated tris complexes of the ligand.
The data confirms the presence of the ligand in each of
the compounds because the molar ratios of carbon to nitrogen
are, for the most part, very close to those of the ligands.
However, because the amount of hydrogen is high, most likely
due to the presence of water molecules, the molar ratios
involving hydrogen do not correspond to those of the
deprotonated ligands. The percentages for the tris complexes
were included to illustrate the amount of error that would


27
Materials and Methods
Chemicals and Biologic Products
Dulbecco's modified Eagle's medium (DMEM), L-arginine-
free Roswell Park Memorial Institute (RPMI) 1640 and all
other cell culture materials were purchased from Fisher
Scientific (Orlando, FL). [3H]L-Arginine (specific activity:
77 Ci/mmol) was purchased from Amersham (Arlington Heights,
IL). AG50WX-8 Dowex (100-200 mesh) was purchased from Bio-Rad
(Melville, NY). Interferon-y (IFN, rat recombinant) was
purchased from Gibco BRL (Gaithersburg, MD) Radioactive
mac-iNOS-cDNA probes were prepared using a Stratagene (La
Jolla, CA) Prime-IT II random labeling kit. Other biologic
products were from Promega Co. (Madison, WI) .
Lipopolysaccharide (LPS, E coli serotype 026:B6),
dexamethasone, actinomycin D, cycloheximide, sulfanilamide,
sodium nitrite, N-(1-naphthyl)ethylenediamine hydrochloride
and N-nitro-L-arginine (NNA) were purchased from Sigma
Chemical Co. (St. Louis, MO).
Cell Cultures
Murine proximal convoluted tubule cells (MCT), were
passaged by trypsinization and cultured to confluence in
flat-bottom tissue culture dishes in Dulbecco's modified
Eagle's medium supplemented with 5% heat-inactivated fetal
bovine serum, 25 mM Hepes buffer, 10 mM nonessential amino
acids and 100 fiM sodium pyruvate. The cultures were


114
produced spaces suitable for the spatial requirements of
ammonium and nitrate ions.


63

GTP
GTP Cyclohydrolase I
(Blocked by DAHP)
Sepiapterin
Dihydroneopterin triphosphate
I
I
I
BH2
'bihydrofolate reductase
(Blocked by Methotrexate)
BH4
Figure 3-1. Tetrahydrobiopterin (BH4) synthetic pathways:
(I) de novo synthesis, and (II) dihydrofolate-dependent
salvage pathway (BH2, dihydrobiopterin).


8
(g)
Figure 1-4. Ligands with O-C-C-N Donor Group.
a) Oxine; b) 2-Methyloxine; c) Picolinic acid;
d) 6-Methylpicolinic acid; e) Pyrazinoic acid;
f) Hypoxanthine; g) Dipicolinic acid


49
Table 4-1. Crystallographic Data for I and II.
I II
Complex Al (meox) 3* CH3OH-H20 Al (ox) 3-CH3OH
Formula
AIC31H30N3O5
MW
551.55
Crystal system
Tetragonal
Space Group
P4/n
a,
22.1970
b, A
22.1970
c,
11.1804
0, 0
90.000
Vc, 3
5508.6
Z
8
Dcalc> g/cm3
1.33
Radiation
Cu Ka
X,
1.54178
H cm'1
8.95
F(000)
2095.61
Crystal
0.22 x 0.16 x
dimensions
20
0.0, 100.0
Observed
2020
reflections
Collected
1945
reflections
Number of
361
parameters
Final R
0.0867
Rw
0.1067
Al C28H22N304
491.48
Monoclinic
P21/n
10.8620
13.2381
16.8528
97.511
2402.5
4
1.36
Mo Ka
0.71069
4.49
951.81
0.14 0.32 x 0.32 x 0.10
1.0, 30.0
1421
1062
325
0.0918
0.0715


37
Lipopolysaccharide (0.1 |ig/ml) and IFN(100 U/ml) each
alone caused no significant accumulation of nitrite but there
was a concentration-dependent accumulation of nitrites in
cells treated with IFN in combination with LPS, peaking at
IFN concentration of 100 U/ml, a 30-fold increase over
control (figure 2-7). This pattern shows close correlation
with my measurements of iNOS activity obtained by assaying
[3H]L-citrulline. These results further confirm that iNOS
induction in MCT cells requires both LPS and IFN and is
concentration-dependent.
Effects of Interferon-y and Lipopolysaccharide on the
Expression of Inducible Nitric Oxide Synthase Protein
To investigate the effects of IFN on the expression of
iNOS protein in MCT cells, I performed immunocytochemical
studies using a rabbit polyclonal antibody against murine
mac-NOS. Cultured MCT cells were treated with LPS, IFN or a
combination of both and iNOS immunoreactivity was detected by
the immunoperoxidase method.
Inducible NOS immunoreactivity was absent in control
cells (figure 2-8A). Treatment with a combination of LPS and
IFN for 24 h resulted in intense staining for iNOS (2-8B)
whereas treatment with IFN alone did not elicit iNOS
immunoreactivity (2-8C). In addition, treatment with LPS
alone resulted only in modest staining for iNOS (data not
shown). These results are consistent with my functional iNOS
activity studies, and indicate that the inability of IFN to


110
Table 7-3. Hydrogen Atom Coordinates
Thermal Parameters (A2xl03) for IX.
Deviations are given in Parentheses.
(xlO4) and Isotropic
Estimated Standard
X
y
z
U
H (0)
0.5151
(34)
0.2867
(49)
1.0354
(23)
83
(13)
H (N)
0.2989
(31)
0.0187
(48)
0.7711
(23)
71
(12)
H (3)
0.3120
(26)
-0.0401
(41)
0.5199
(19)
48
(9)
H (4)
0.4919
(30)
-0.1649
(49)
0.5604
(21)
73
(11)
H (5)
0.5729
(27)
-0.1696
(42)
0.7086
(19)
44
(10)
H (6)
0.4657
(24)
-0.0765
(37)
0.8132
(17)
45
(9)
H (31)
0.3108
(23)
0.0578
(35)
1.0886
(16)
35
(8)
H (41)
0.1485
(28)
-0.0839
(44)
1.0807
(21)
66
(10)
H (51)
0.0395
(29)
-0.1688
(45)
0.9438
(20)
59
(10)
H (61)
0.1222
(27)
-0.1070
(41)
0.8227
(19)
47
(9)
H (IN)
0.0310
(28)
-0.0221
(44)
0.3510
(19)
65
(10)
H(2N)
0.1571
(35)
-0.0356
(53)
0.3391
(24)
85
(13)
H(3N)
0.1060
(28)
0.0656
(42)
0.4027
(21)
59
(10)
H(4N)
0.1065
(27)
0.1081
(44)
0.3224
(20)
57
(9)
Equivalent isotropic U defined as one third of the trace of
the orthogonalized Ui;j tensor
Table 7-4. Bond Lengths () for Non-Hydrogen Atoms in IX.
Estimated Standard Deviations are given in Parentheses.
N (1) -C (2)
1.350 (4)
C(2)-C(3)
1.370 (5)
C (3 ) -C (4)
1.387 (6)
C (4) -C (5)
1.369(6)
N(1)-C(6)
1.345 (5)
C(5)-C(6)
1.363 (7)
C(2)-C(7)
1.508 (5)
C (7) -0(1)
1.227 (5)
C (7) -0(2)
1.268 (4)
N(11)-C(21)
1.345 (4)
C (21) -C (31)
1.378(5)
C (31) -C (41)
1.380 (7)
C (41) -C (51)
1.388 (6)
C(51)-C(61)
1.372 (6)
N (11)-C(61)
1.338(5)
C(21)-C(71)
1.494 (5)
C (71) -0(11)
1.319 (4)
C (71) -0(21)
1.207 (5)
N (2) -0 (IN)
1.231(5)
N (2) -0 (2N)
1.219 (6)
N (2) -0 (3N)
1.223(5)


40
stabilization of iNOS mRNA, data from recent studies in rat
macrophages and hepatocytes seem to favor the former
mechanism (Nussler and Billiar, 1993). This is also
supported by the fact that the synergism at the level of mRNA
transcription requires protein synthesis, since
cycloheximide, a protein synthesis inhibitor, appears to
abolish the mRNA synthesis.
Synergistic interactions between IFN and other
immunomodulators in the induction of iNOS activity have also
been described in hepatocytes, lung fibroblasts, vascular
smooth muscle and mesangial cells (Geller et al. 1993 [b] ,
Gross and Levi, 1992, Jorens et al., 1992, Pfeilschifter and
Schwarzenbach, 1990). The molecular mechanisms involved in
the interactions among inducers in NO synthesis are not
completely understood and appear to vary among different
cells and cytokines. Interferon-y treatment does not
stimulate NO synthesis in rat hepatocytes despite inducing
iNOS mRNA transcription. Moreover, IFN has also been
reported to increase BH4 levels in murine macrophages,
fibroblasts, lymphocytes and rat pulmonary artery smooth
muscle by inducing the synthesis of GTP cyclohydrolase I, the
first and rate-limiting enzyme in the de novo pathway for the
synthesis of this cofactor (Di Silvio et al., 1993, Gross and
Levi, 1992, Hattori and Gross, 1993) Conversely, in rat
aortic vascular smooth muscle, IFN potentiates LPS-induced
synthesis of GTP cyclohydrolase I but does not itself
increase the levels of BH4 (Gross and Levi, 1992, Hattori and


50
Figure 4-4. ORTEP Drawing of the Al(meox)3 Molecule Showing
the Atomic Numbering Scheme and the Thermal Ellipsoids.


127
peaks for the sodium salt and the ligand. For mpic, the
stretching frequency is low, but the value for IV is
comparable to III and V. This would indicate that in the
aluminum compound, the ligand is coordinated to the metal ion
producing an environment different from that in either the
carboxylic acid and carboxylate ion. For VII, there are two
peaks present. The peak at 1666 cm'1 is in the region of the
other aluminum compounds and is, therefore, probably due to a
coordinated carboxylate group. The other peak could be due to
a differently reacting carboxylate group in one compound or
another compound with differently bonded carboxylate groups.
Despite the presence of more than one aluminum environment in
III, IV and V indicated by Al-27 NMR, there is only one
visible carbonyl stretching band. Either, the coordination is
so similar that the peaks are indistinguishable or the
aluminum is coordinated by a bond of the same strength in
every compound in the mixture.
Fast Atom Bombardment Mass Spectrometry
FABMS generally works well for large molecules by
providing structural evidence from fragmentation. It is due
to the occurrence of fragmentation that the ratios for the
mixtures in the samples could not be determined. For the
spectra of IV, V and VI, the m/z for the ligand is the largest
peak present. If the primary component of the solids were the
ligands, they would have been soluble. For III, the largest
peak was probably due to Al(pic) (H20)3+. There were also two


90
V
M
s
r wi
L v
£ E
LPS/IFN
Figure 4-7. Effect of PDTC (25 |iM) on nitrite production by
MCT cells treated with a combination of LPS (1 |xg/ml) and IFN
(100 U/ml) for 24 h. Cells were incubated with PDTC for 30
min before the addition of LPS and IFN, and during the entire
incubation period, (* p < 0.001 vs LPS/IFN, mg: mg of cell
protein, n=6).


126
Mundel, P., Bachmann, S., Bader, M., Fischer, A., Rummer, W.,
Mayer, B. and Kriz, W. Expression of nitric oxide synthase
in kidney macula densa cells. Kidney Int. 42: 1017-1019,
1992.
Nakaki, T., Nakayama, M. and Rato, R. Inhibition by nitric
oxide and nitric oxide-producing vasodilators of DNA
synthesis in vascular smooth muscle cells. Eur. J. Pharmacol.
1898: 347-353, 1990.
Narumi, S., Tebo, J.M., Finke, J.H. and Hamilton, T. A.
Interferon-y and interleukin-2 cooperatively activate NF-
kappa B in murine peritoneal macrophages. J. Immunol. 149:
529-534, 1992.
Nathan, C. Nitric oxide and biopterin: A study in
chiaroscuro. J. Clin. Invest. 93: 1875-1876, 1994.
Nathan, C. and Xie, Q-w. Nitric oxide synthases: roles,
tolls and control. Cell 78: 915-918, 1994 [a].
Nathan, C. and Xie, Q-w. Regulation of biosynthesis of
nitric oxide. J. Biol. Chem. 269: 13725-13728, 1994 [b] .
Nishida, R. Harrison, D.G., Navas, J.P., Fisher, A.A.,
Dockery, S.P., Uematsu, M., Nerem, R.M., Alexander, R.W. and
Murphy, T.J. Molecular cloning and characterization of the
constitutive bovine aortic endothelial cell nitric oxide
synthase. J. Clin. Invest. 90: 2092-2096, 1992.
Noonan, N.E. and Noonan, R.D. The effect of TLCR on
transcription and its role in modifying cell growth. J. Cell
Physiol. 92: 137-144, 1977.
Nowak, R. Corners of the mind: the cellular basis of memory
and learning. J. NIH Res. 4: 49-55, 1992.
Nussler, A.R. Nonparenchymal cell and hepatocyte interaction
in antimalarial activity: a central role for nitric oxide.
In: Hepatocvte and Ruoffer Cell Interactions, edited by T.R.
Billiar and R.D. Curran, Boca Raton, FL: CRC, 1992.
Nussler, A.R. and Billiar, T.R. Inflammation,
immunoregulation and inducible nitric oxide synthase. J.
Leukoc Biol. 54: 171-178, 1993.
Nussler, A.R., Drapier, J.C., Renia, L., Pied, S., Miltgen,
F., Gentilini, M. and Mazier, D. L-Arginine-dependent
destruction of intrahepatic malaria parasites in response to
tumor necrosis factor and/or interleukin-6 stimulation. Eur.
J. Immunol. 21: 227-230, 1991.


122
Golub, E.I., Kim, H. and Volsky, D.J. Transfection of DNA
into adherent cells by DEAE-dextran/DMSO method increases
drastically if cells are removed from surface and treated in
suspension. Nucleic Acids Res. 17: 4902, 1989.
Gorman, C.M., Moffat, L.F. and Howard, B.H. Recombinant
genomes which express chloramphenicol acetyltransferase in
mammalian cells. Mol. Cell Biol. 2: 1044, 1982.
Gross, S.S., Jaffe, E.A., Levi, R. and Kilbourn, R.G.
Cytokine-activated endothelial cells express an isotype of
nitric oxide synthase which is tetrahydrobiopterin-dependent,
calmodulin-independent and inhibited by arginine analogues
with a rank order of potency characteristic of activated
macrophages. Biochem. Biophys. Res. Commun. 178: 823-829,
1991.
Gross, S.S. and Levi, R. Tetrahydrobiopterin synthesis: an
absolute requirement for cytokine-induced nitric oxide
generation by vascular smooth muscle. J. Biol. Chem. 267:
25722-25729, 1992.
Gross, S.S., Levi, R., Madera, A., Park, K.H., Vane, J. and
Hattori, Y. Tetrahydrobiopterin is induced by LPS in
vascular smooth muscle and is rate-limiting for nitric oxide
production. Adv. Exp. Med. Biol. 338: 295-300, 1993.
Guzman, N.J., Fang, M.Z., Tang, S.S., Ingel finger, J. and
Garg, L.C. Autocrine inhibition of Na+/K+-ATPase by nitric
oxide in mouse proximal tubule epithelial cells. J. Clin.
Invest. 95: 2083-2088, 1995.
Hattori, Y. and Gross, S.S. GTP cyclohydrolase I mRNA is
induced by LPS in vascular smooth muscle: characterization,
sequence and relationship to nitric oxide synthase. Biochem.
Biophys. Res. Commun. 195: 435-441, 1993.
Haverty, T.P., Kelly, C.J., Hines, W.H., Amenta, P.S.,
Watanabe, M., Harper, R.A., Kefalides, N.A. and Neilson, E.G.
Characterization of a renal tubule epithelial cell line which
secretes the autologous target antigen of autoimmune
experimental interstitial nephritis. J. Cell Biol. 107: 1359-
1368, 1988.
Henkel, T., Machleit, T., Alkalay, I., Kronke, M., Ben-
Neriah, Y. and Baeuerle, P.A. Rapid proteolysis of I kappa
B-alpha is necessary for activation of transcription factor
NF-kappa B. Nature 365: 182-185, 1993.
Hirai, H., Fujisawa, J., Suzuki, K., Ueda, K., Muramutsu, M.,
Tsuboi, A., Arai, N. and Yoshida, M. Transcriptional


69
has also been reported that the induction of macrophage iNOS
is dependent on the activation and nuclear translocation of
NF-kB (Xie et al. 1994).
Nuclear factor-KB proteins are a ubiquitous group of
transcription factors that exist mainly as inactive dimers in
the cytoplasm of cells bound to inhibitory proteins known as
inhibitors of kappa-B (IkB) (Muller et al., 1993). The most
important subunits of NF-kB are p50, p65 and c-Rel. Upon
cell activation, IkB dissociates from the NF-kB dimer, through
a process requiring both phosphorylation and proteolysis,
allowing this to migrate to the nucleus where it binds to its
DNA recognition site in the promoter region of various genes
to initiate transcription (Muller et al. 1993, Henkel et
al. 1993) The induction of macrophage iNOS by LPS is
dependent on the nuclear translocation of NF-kB heterodimers
containing mainly c-Rel and p65 subunits of NF-kB (Xie et
al., 1994). The c-Rel component of NF-kB had previously been
reported to have only modest transactivating activity (Muller
et al., 1993). Therefore, transactivation of the iNOS gene
constitutes an important novel role for this NF-kB protein in
macrophages.
Renal tubule epithelium has recently been found to
express iNOS but the regulation of this enzyme in the kidney
has not been extensively investigated (Amoah-Apraku et al.,
1993, Markewitz et al., 1993). In this study I examined the
role of NF-kB in the induction of renal epithelial iNOS and


33
only Al (OH) 3 was produced. Although most of the products were
crystalline, only one experiment yielded single crystals of
the size and quality required for x-ray diffraction studies.
The structure for this compound is presented in Chapter 6.
All of the trials with picolinic acid gave similar
results. Large crystals were produced, however, all of these
were either twinned or double-twinned. Twinned crystals are
formed when two crystals grow together along a common face,
frequently producing a mirrored effect. Because the lattice
pattern in a twin may not be constant throughout the sample,
the x-rays would probably be diffracted differently than they
would in a single crystal of the same compound. As a result,
twinned crystals are not usually suitable for x-ray
diffraction studies.61 Pyrazinoic acid consistently produced
fairly large crystalline structures that were flower-like in
appearance. Examination with a microscope revealed that each
had the four striated branches arranged in a tetrahedral
orientation around a central point. Although they were large
enough, the crystalline products were not single crystals.
The other crystals formed in the experiments were too small to
be useful for x-ray crystallography.
Base Strength
Theory
The syntheses discussed utilized moderately strong bases
that are stronger than needed to deprotonate the ligands. In


80
Figure 5-9. Al-27 NMR Spectrum of an Aqueous
Containing 0.30 M Al3+ and 0.60 M pza at pH 5.
Figure 5-10. Al-27 NMR Spectrum of an Aqueous
Containing 0.30 M Al3+ and 0.90 M pza at pH 5.
Solution
Solution


CHAPTER 7
SYNTHESIS AND STRUCTURE OF A COMPLEX SALT CONTAINING
AMMONIUM NITRATE, PICOLINIC ACID AND
2-PYRIDINIUM CARBOXYLATE
Introduction
Picolinic acid, 2-pyridine carboxylic acid, can act as a
bidentate ligand by coordinating through the ring nitrogen and
a carboxylate oxygen. Also, because of the presence of the
accessible oxygen and nitrogen atoms, there is a potential for
a network of hydrogen bonding in the absence of a metal ion to
coordinate. The structure presented in this chapter, which
was synthesized prior to the work discussed in Chapter 1, is
another example of a compound, without aluminum, characterized
during this investigation.
Experimental
Synthesis
Materials. All materials and solvents were reagent grade
and used as supplied from the manufacturer.
Preparation of (C.H,N (CO.H) ) (CdLNH (CO-,) ) (NH. + ) (NO-, ) (IX) .
A 3-neck 300 mL round bottom flask was equipped with a
thermometer and condenser. Water (300 mL) A1 (N03) 3-9H20
(3.029 g, 8.07 mmole), urea (0.7830 g, 13.0 mmole) and pic
103


LO
1780
1995
.B&5
UNIVERSITY OF FLORIDA
1262 08554 5548


15
In the kidney, the proximal tubule is the major site of
L-arginine synthesis (Levillain et al. 1993). Also,
increased expression of cytokines occurs in many experimental
and human kidney diseases. Hence, the presence of constant
and abundant substrate coupled with the easy availability of
inducers make iNOS in the kidney a major factor in renal
pathophysiology. Thus, pathways regulating the renal L-
arginine:NO system such as the role of specific transcription
factors and cofactor availability, offer attractive
therapeutic targets.
The Endocrine System
The presence of NOS has been demonstrated in rat
pancreatic p islet cells (Laychock et al., 1991) and insulin
release in response to L-arginine is mediated by NO (Schmidt
et al. 1992). In mice, administration of streptozotocin
mimics diabetes mellitus, which is abolished by N-
substituted L-arginine analogues (Lukic et al 1991),
suggesting that this effect may be mediated by NO. It is now
clear that immune-mediated pancreatic islet cell destruction
is attributable to IL-l-induced NO formation (Corbett et al.,
1991). Also, the generation of NO in response to L-arginine
or tolbutamide appears to mediate insulin release in
pancreatic p cells (Laychock et al. 1991, Schmidt et al. ,
1992). Hence excessive production of NO or defective
responsiveness by NOS may play an important role in the
etiology of diabetes mellitus.


91
Figure 4-8. Characterization of NF-kB proteins activated by
LPS (1 fig/ml) in (A) RAW 264.7 macrophages and (B) MCT cells.
Nuclear extracts obtained after 30 min treatment with LPS
were analyzed by EMSA in the absence or presence of IgG
against p50, p65 and c-Rel. Marked activation of NF-kB is
observed after LPS treatment. In RAW 264.7 macrophages (A),
addition of p50 IgG results in "supershift" with the
appearance of an intense slower-migrating band consistent
with the presence of p50 protein. Addition of p65 IgG also
results in "supershift" with appearance of a less intense
band that migrates slower than the p50 complex. Addition of
c-Rel IgG results in marked reduction of the NF-kB complex
and "supershift" with appearance of a band that migrates
similar to p65. These findings are consistent with the
presence of a substantial amount of c-Rel protein in the
nuclei of LPS-treated RAW 264.7 macrophages. In MCT cells
(B) addition of p50 and p65 IgG results in "supershifts"
consistent with the presence of p50 and p65 proteins,
respectively. However, addition of c-Rel IgG fails to induce
"supershift" or substantially affect the NF-kB complex,
indicating the absence of significant amounts of c-Rel in the
nuclei of LPS-treated MCT cells. Blot is representative of
four separate experiments.


71
water molecules are present in the place of chelating ligands,
the smaller the chemical shift. Because some of the data
reported were for aqueous species without x-ray data, the
precise symmetry of several of the compounds can not be known.
However, it has been reported that the chemical shift for a
distorted octahedral compound will be larger than for one with
an ideal geometry.39 This could explain the smaller chemical
shift when more water molecules are present.
Although not included in this table, the results of the
Al-27 NMR spectra for Al(meox)3 (la) and Al(ox)3 (Ila) are
summarized below. In the solid la, the main peak appeared at
54 ppm, but there was also a smaller peak at 221 ppm. For the
solid Ila, there were three peaks present. The two larger
peaks, of almost equal height, occurred at 5 and 73 ppm. A
much smaller peak was present at -120 ppm. After Ila was
dissolved in ethanol and left to concentrate by evaporation
(lib), only one large peak was present at 54 ppm. It should
be noted that the crystals formed from this solution were
characterized by x-ray crystallography and the Al(ox)3
structure confirmed.
Experimental
Compounds III through VII were analyzed in the solid and
solution state using a NT-300 Al-27 NMR spectrophotometer with
spectrometer frequency 78.176839 MHz. The magic-angle
spinning frequency was between 1.1 and 2.4 kHz.


91
Figure 4-8. Characterization of NF-kB proteins activated by
LPS (1 ng/ml) in (A) RAW 264.7 macrophages and (B) MCT cells.
Nuclear extracts obtained after 30 min treatment with LPS
were analyzed by EMSA in the absence or presence of IgG
against p50, p65 and c-Rel. Marked activation of NF-kB is
observed after LPS treatment. In RAW 264.7 macrophages (A),
addition of p50 IgG results in "supershift" with the
appearance of an intense slower-migrating band consistent
with the presence of p50 protein. Addition of p65 IgG also
results in "supershift" with appearance of a less intense
band that migrates slower than the p50 complex. Addition of
c-Rel IgG results in marked reduction of the NF-kB complex
and "supershift" with appearance of a band that migrates
similar to p65. These findings are consistent with the
presence of a substantial amount of c-Rel protein in the
nuclei of LPS-treated RAW 264.7 macrophages. In MCT cells
(B) addition of p50 and p65 IgG results in "supershifts"
consistent with the presence of p50 and p65 proteins,
respectively. However, addition of c-Rel IgG fails to induce
"supershift" or substantially affect the NF-kB complex,
indicating the absence of significant amounts of c-Rel in the
nuclei of LPS-treated MCT cells. Blot is representative of
four separate experiments.


50
Figure 2-6. Inducible NOS activity in crude homogenates of
MCT cells after 24 h of treatment with LPS (0.1 ¡xg/ml), IFN
(100 U/ml) or a combination of both. In vitro iNOS activity
assay was performed in the presence of saturating
concentrations of NADPH, BH4, FAD and FMN. Even in the
presence of saturating concentrations of exogenously added
cofactors, IFN failed to induce iNOS activity (* p<0.05 vs
control, ng: (xg of cell protein, n=6) .


77
(a)
i i ~'~i ""i i r^ i
BOO >00 100 0 -100 -ZOO -900
(b)
Figure 5-6. Al-27 NMR Spectra of VII, a) Solid, b) in d6-
DMSO.


31
sterile conditions and quickly placed in ice-cold Hank's
medium. The proximal tubules were then isolated as
previously described (Haverty et al., 1988, Toutain, 1992).
Light-Microscopic Immunocvtochemistrv
Murine proximal tubule cell iNOS immunoreactivity was
detected in cultured cells by the immunoperoxidase method
using a polyclonal antibody raised in rabbit against murine
mac-NOS. The antibody was kindly provided by Dr. Carl
Nathan, Cornell University, NY. It was raised against a
synthetic peptide that was constructed based on the published
mac-NOS sequence (Bogdan et al., 1993, Xie et al., 1992) and
prepared by Dr. Jeffrey R. Weidner and Richard A. Mumford,
Merck, Sharp & Dohme Research Laboratory. The MCT cell
monolayers grown in multichamber slides were fixed in ice-
cold acetone and processed for 1ight-microscopic
immunocytochemistry using the avidin-biotin-horseradish
peroxidase complex (ABC) technique (Vectastain ABC kit,
Vector Laboratories, Burlingame, CA). Endogenous peroxidase
activity was eliminated by incubation with 3% H2O2 for 30 min.
After treatment with blocking serum, the cells were incubated
with the primary antibody against macrophage-type NOS (mac-
NOS) (diluted 1:500) for 60 min. Following incubation with
the primary antibody the cell monolayers were rinsed with PBS
and incubated with the biotinylated secondary antibody for 30
min. After rinsing, the cells were incubated for 30 min with
the Vectastain ABC reagent followed by incubation with the


This dissertation is dedicated to the blessed memory of my
dad who passed away while I was studying for this work, and
to all my loved ones, especially my beloved Margaret.


85
LPS/IFN
Time (hr)
01246 12 24 66
Figure 4-2. Inducible NOS mRNA expression in MCT cells
treated with LPS (1 ng/ml) and IFN (100 U/ml) for the
indicated periods of time or with each of these inducers
alone for 6 h. Blot is representative of three separate
experiments.


29
into the other reactants, usually confined to an aqueous
phase.
Experimental
Ammonia is a fairly volatile gas as well as a moderate
base. Because of its volatility, it can slowly be absorbed
into a solution of the aluminum ion and the ligand. An
ammonia chamber was created by sealing a large beaker
containing two small beakers or vials. One of the beakers
contains an aqueous ammonia solution and the other contains
the rest of the reactants. After sealing, the chamber becomes
saturated with ammonia vapor which diffuses into the acidic
aluminum ion and ligand solution. The solutions for the vapor
diffusion experiments were prepared by dissolving the ligand
and Al (N03) 3-9H20 in water as depicted in Table 3-1.
Results
After several days, a crystalline product began to form
in each experiment indicated. However, either the crystals
were too small or in inseparable clusters. The ammonia
reacted immediately upon contact with the surface of the
solution. Instead of single large crystals growing, clusters
formed and clung to the surface. The only single crystals
discovered were on the bottom of the beaker. Apparently, as
these single crystals grew, they became too heavy to be
supported by the surface tension of the solution and sank to
the bottom of the beaker. The clusters which continued to
grow on the surface probably did not sink to the bottom of the


13
The Cardiovascular System
Under normal physiologic conditions, basal vasodilator
tone is maintained by endothelial NO released in response to
shear stress on the vessel wall that causes entry of Ca2 +
across the plasma membrane and subsequent activation of cNOS
(Rees et al., 1989). It has also been shown that
inflammatory stimuli can induce iNOS-like activity in the
endothelium and vascular smooth muscle cells in amounts far
exceeding the agonist-triggered NO release from endothelial
and vascular smooth muscle cells (Kilbourn and Bellone, 1990,
Busse and Mulsch, 1990). Recent reports suggest that these
cytokines increase NOS activity by increasing BH4 synthesis
(Rozenkranz-Weiss et al., 1994). Thus, in addition to
maintaining basal vascular tone, NO of endothelial origin may
also play a role in the endotoxin-associated hypotension of
septic shock. Endothelial NO also inhibits platelet
aggregation, leukocyte adhesion, endothelin generation and
smooth muscle cell proliferation (Kubes et al., 1991,
Boulanger and Luscher, 1990, Nakaki et al., 1990). Defective
vasoregulation in diabetes, atherosclerosis and arterial
damage in normal aging may be due, in part, to scavenging of
NO by end products of advanced glycosylation and oxidized
lipoproteins (Bucala et al., 1991, Chin et al. 1992), and
defects in endothelial NO regulation could lead to vasospasm
and essential hypertension.


121
Table 8-3 -- Continued
(c)Torsion angles ()
0(1) -C(l) -C(2) -0(3)
0(1) -C(l) -C (2) -C (3)
0(2) -C(1) -C(2) -0(3)
0(2) -C(l) -C (2) -C (3)
0(3) -C (2) -C (3) -0(4)
0(3) -C (2) -C (3) -C (4)
C(l) -C (2) -C (3) -0(4)
C(l) -C (2) -C (3) -C (4)
0(4) -C (3) -C (4) -0(5)
0(4) -C (3) -C (4) -0(6)
C (2) -C (3) -C (4) -0(5)
C (2) -C (3) -C (4) -0(6)
for the bitartrate ion.
-170.3(3)
-48.4 (4)
10.7(5) **
132.6 (4)
67.4(4) **
-55.7 (4)
-53.7(4)
-176.8(3) **
-174.4 (3)
6.2(5) **
-51.3(4)
129.3 (4)
(d)Distances () and Symmetry Operators for Sodium Ion
Coordination.
Na-0(4)
Na-0(6)
Na-0(7)
Na-0 (22)
Na-0 (32)
Na-0 (l3)
Na-0 (43)
Na-0 (64)
2.616 (3)
2.648 (3)
2.416(4)
2.429 (3)
2.422(3)
2.827 (3)
2.566 (3)
2.622(3)
Symmetry operators: (2) 0.5-x, -y, -0.5+z; (3) 0.5-x, -1-y,
0.5+z; (4) 0.5+x, -0.5-y, -1-z
(e)Bonds () and Angles () around the Sodium Ion.
0...Na
. .0
0-0
O-Na-O
0(4) .
.0(6)
2.681(4)
61.2 (1)
0(6) .
0 (22)
3.322 (4)
81.6(1)
0(22) .
.0 (32)
2.616(4)
65.2(1)
0(4) .
.0 (32)
3.160(4)
77.6(1)
0(4) .
0 (22)
4.022 (4)
105.6 (1)
0(6) .
0 (32)
4.322 (4)
116.9(1)
0(7) .
0 (43)
2.912(5)
71.5(1)
0(43) .
0(13)
3.160(4)
71.5(1)
0(13) .
0 (64)
3.370(4)
76.3(1)
0(7) .
. 0 (64)
3.033(5)
73.9(1)
0(7) .
.0(13)
4.168(5)
105.0 (1)
0(43) .
.0 (64)
4.571 (4)
123.6(1)


136
A hydrogen-bonding network exists linking the pic
molecules to pic in other unit cells. H(N) is located 1.798
from the nitrogen atom in the second ring. H(0) is hydrogen
bonded to 0(2) in an adjacent pic molecule. Although, the
bond lengths for each of these hydrogen bonds are
significantly shorter that the sum of the two van der Waal's
radii, neither is considered strong hydrogen bond because of
the hydrogens are not centered. The two other hydrogen bonds,
N(3)-H(1N)-0(1) and N(3)-H(4N) O(2N) ', are also
classified as weak because the bond lengths are barely less
than the sum of the radii for oxygen and nitrogen. The
remaining hydrogen interactions do not qualify as even weak
hydrogen bonds.
Na+C.H.0.~- H.O
Compound X is sodium hydrogen (+)-tartrate monohydrate.
Although aluminum is not present in this compound, its
structure provides a discussion of octacoordinate geometries,
hydrogen bonding and absolute configuration. All of the
oxygens in the compound are coordinated to the sodium with the
notable exception of 0(5), the hydroxyl oxygen from the
protonated carboxylic acid group. The sodium ion is
surrounded by eight oxygen atoms, from four different tartrate
ions, with bond lengths between 2.426 and 2.827 .
Calculations of the deviations from the least squares planes
for square-antiprismatic and triangular dodecahedral
geometries indicate that, although the geometry is


tetrahydrobiopterin (BH4)# an essential iNOS cofactor,
attenuated the increased LPS/IFN-stimulated nitrite
production. Sepiapterin, a substrate for the formation of
BH4 via the pterin salvage pathway, reversed this inhibition.
Electrophoretic mobility shift assays (EMSA) using specific
antibodies to p50, p65 and c-Rel subunits of nuclear factor-
kappa B (NF-KB) demonstrated that LPS and IFN treatment
induced the nuclear translocation of p50 and p65 but not c-
Rel. Cotreatment with Pyrrolidinedithiocarbamate (PDTC), a
specific NF-kB inhibitor, attenuated LPS- and IFN-stimulated
nuclear translocation of NF-kB as well as steady-state iNOS
mRNA levels, nitrite accumulation and protein synthesis.
Constructs pAiNOS-CAT and pBiNOS-CAT, prepared from the
promoter region of the iNOS gene, linked to a reporter gene
encoding chloramphenicol acetyltransferase and transiently
transfected into MCT cells, conferred inducibility of the
iNOS promoter by LPS but not by IFN. Construct pCiNOS-CAT,
which lacked the critical downstream NF-KB binding site, was
unresponsive to either LPS or IFN. Only construct pAiNOS-
CAT, which contained IFN response elements in addition to the
critical NF-kB binding site, conferred synergistic
inducibility by LPS and IFN, suggesting that this synergism
requires both NF-kB and IFN-induced proteins. In conclusion,
MCT iNOS is regulated at least at two levels:
transcriptionally, by transcription factors including NF-kB
and other IFN-induced proteins, and posttranscriptionally, by
BH4 availability.
IX


132
the concentrated bicarbonate ion as the base, single large
crystals were not produced.
Solvent System
When methyl or ethyl alcohol was employed as the solvent,
experiments did not produce a compound other than the sodium
salt of the ligand unless a substantial amount of water was
present. However, in the presence of water, pic and pza
produce characteristic unsuitable crystal forms. In DMSO, pic
and pza both produced a white solid that when heated to 100C
to remove water, a clear but brightly colored solution was
formed. The same result was produced when a similar solution
of the sodium salt was heated. In addition, no solid remained
after the evaporation of the heated DMSO reaction mixture.
Aluminum Isopropoxide
When aluminum isopropoxide was employed, the solids
formed using aluminum isopropoxide were non-crystalline and
insoluble in any common solvent.
Crystal Growth Results
All of the trials with pic and pza gave consistent
results. For pic, large crystals were produced; however, all
of these were either twinned or double-twinned. Twinned
crystals, which are formed when two crystals grow together
along a common face, are not usually suitable for x-ray
diffraction studies. Pza consistently produced fairly large
crystalline structures that were flower-like in appearance.
Examination with a microscope revealed that each had the four


104
(4.009 g, 32.6 mmole) were added to the flask in order listed.
A stir bar was added and the mixture stirred until all solids
were dissolved. The reaction mixture was immersed in an oil
bath and maintained at a temperature of 99 to 102C for 24
hours. The clear colorless solution, which measured pH 6.5,
was removed by filtration from the white solid that had formed
during the reaction. After four months, small crystals (IX)
of x-ray quality had formed.
X-rav Crystallographic Analysis
A clear colorless crystal was mounted on a glass fiber
for diffraction studies. All subsequent data were collected
on a Nicolet P3 Diffractometer, using filter-monochromated Cu
Ka radiation. The unit cell dimensions were determined for 15
automatically centered reflections; space group V2x/c by
intensity statistics and satisfactory structure solution and
refinement; 2206 unique reflections, 1573 with I>3a(I);
0shsl4, 0sks9, -17slsl7. Two intensity standards measured
every 98 reflections showed no change during data collection.
The program used for Fobs refinement was SHELXTL Revision 5.1
(Sheldrick, 1985) Calculations and figures were performed on
a Model 30 Desktop Eclipse computer. Final refinement values
for 264 parameters were R = 5.80, R = 5.93 and w = 6.75.
Atomic scattering factors used were those of the International
Tables of X-ray Crystallography (1974) All non-hydrogen
atoms were located from an E-map. Hydrogen atoms were located
by a Difference Fourier map.


56
Tetrahydrobiopterin synthesis occurs via two distinct
pathways (figure 3-1, Werner et al. 1993): (i) a de novo
synthetic pathway that utilizes GTP as a precursor and in
which GTP cyclohydrolase I (GTPCH) is the rate-limiting
enzyme. This is the main source of BH4 for iNOS in vascular
smooth muscle and hepatocytes (Stuehr and Griffith, 1992);
and (ii) a salvage pathway in which preexisting
dihydropteridines like dihydrobiopterin and sepiapterin are
converted to BH4 by dihydrofolate reductase. Constitutive
NOS requires only catalytic amounts of BH4 for full activity
whereas iNOS, on the other hand, requires substrate amounts.
There may therefore be a coinduction of BH4 synthesis with
iNOS induction in many cells.
Because only low levels of GTPCH activity are present in
the kidney and its role in providing BH4 for processes such
as renal aromatic amino acid hydroxylation has not been
demonstrated (Bellahsene et al., 1984, Rao and Kaufman, 1986,
Davis et al., 1992), the main function of renal GTPCH remains
to be defined. The presence of iNOS has recently been
reported in proximal tubule and collecting duct epithelium
(Markewitz et al., 1993, Amoah-Apraku et al., 1993). Because
NO may mediate renal injury during allograft rejection,
autoimmune glomerulonephritis (Langrehr et al. 1993 [a,b],
Weinberg et al. 1994), Na+/K+-ATPase and H+-ATPase activity
and therefore ion and acid transport across the renal tubule
epithelium (Guzman et al., 1995, Tojo et al,. 1994 [a], Stoos
et al., 1994), it will be important to identify potential


86
Fast Atom Bombardment Mass Spectrometry
FABMS were prepared for III, IV, V and VI. Data from
these are given in Table 5-6. Most peaks smaller than ten
percent relative abundance have been omitted.
For III, the sample containing pic, the peak at m/z=204
is possibly due to A1L(H20)3+. There was no peak corresponding
to one ligand molecule, but a peak m/z=79 is probably due to
the ligand fragment left after the loss of carbon dioxide. In
addition, small peaks due to combinations of aluminum, the
ligand and hydroxide were present. The peaks for AlL(OH)2-2
(m/z = 181) AlL2 (OH) +3 (m/z = 291), and AlL3-3(OH) (m/z=342)
appear with m/z=291 being the weakest. Furthermore, a small
peak is present for A1L3+1. It seems likely that the free
ligand would more easily decarboxylate than a coordinated
ligand. Instead the coordinated ligand seems to lose a
hydroxyl group, leaving the coordination sphere intact.
Although the spectrum does support the presence of a tris
complex, the actual proportions of the complexes originally
present would be difficult to determine due to the extensive
fragmentation of the complexes and ligand molecules.
For sample IV, the largest peak corresponds to L+l, with
a decent size fragment due to the decarboxylation of the
ligand. Other prevalent peaks, m/z=166, m/z=299 and m/z=436
are most likely due to complexes such as AlL+3, A1L2, and
AlL3+l, respectively. Although there are many fragments
produced, with the ligand being the greatest in abundance,


73
dishes in 20 mM Tris-HCl, pH 7.5, containing 0.1 mM PMSF, 2
mM EDTA, 0.5 mM EGTA, 10 mg/ml leupeptin and 1 mg/ml
aprotinin, centrifuged at 1000 rpm for 5 min and homogenized
on ice with 30 strokes in an Eppendorf tube with a disposable
homogenizer. Cellular proteins were solubilized in SDS,
separated by discontinuous polyacrylamide gels (5% stacking,
7.5% resolving gel, 40 mA for 5 h), and transferred to a 0.45
|j.m nitrocellulose membrane in 25 mM Tris, pH 8.3, 150 mM
glycine, and 20% vol/vol methanol for 15 h at 100 mA. The
nitrocellulose membrane was blocked with 10% nonfat dry milk
in PBS for 2 h and subsequently incubated with mouse anti-
mac-NOS monoclonal antibodies (1/250) for 4 h at 20 C.
After washing the membrane to remove excess antibody,
peroxidase-labeled anti-mouse secondary antibodies were
applied and the protein of interest identified by enhanced
chemiluminiscence (ECL, Amersham Corporation, Arlington
Heights, IL).
Results
Effects of Li popo lvsac char ide and Interferon-v on Nitrite
Accumulation in Murine Proximal Tubule Cells
To investigate the effects of LPS and IFN on nitrite
accumulation in MCT cells, I measured accumulated nitrite by
MCT cells after 24 h incubation with LPS (1 ng/ml), IFN (100
U/ml) or in combination.
Incubation of MCT cells with LPS (1 |xg/ml for 24 h)
stimulated only modest nitrite production (figure 4-1) .


6
that iNOS and L-arginine biosynthetic activities were
coinduced. Thus, L-arginine transport and synthesis may be
rate-limiting for induced NO production. Hence, elucidation
of the biochemical events involved in NO production and
identification of the control points in cofactor and
substrate synthesis and transport have the potential for
providing selective targets for therapeutic control of
cytokine- and endotoxin-induced NO-mediated vascular shock
and tissue damage.
Synthesis of Nitric Oxide
Nitric oxide is synthesized from the terminal guanidino
nitrogen of L-arginine by NOS in a reaction that also
produces stoichiometric amounts of L-citrulline. This
reaction involves the transfer of five electrons from
molecular oxygen (O2) onto the substrate guanidino nitrogen
and requires NADPH, FMN, FAD and BH4 as cofactors. The
oxidation of L-arginine can be divided into two phases.
Phase I involves the hydroxylation of L-arginine to the
intermediate Nw-L-hydroxyarginine (OHArg), and consumes one
mole of NADPH. This reaction resembles a classical
cytochrome P450 hydroxylation and may involve the heme iron of
NOS. Phase II involves the oxidation of OHArg to L-
citrulline and NO, consumes another mole of NADPH as reducing
equivalent and clearly depends on BH4. In the absence of L-
arginine and BH4, the activation of molecular O2 by NOS
results in a divalent reduction of O2 to yield superoxide


116
synthesis are critical for the induction and full expression
of iNOS activity in MCT cells. Unlike what is observed in
macrophages, in mouse proximal tubule epithelium, LPS fails
to cause the nuclear translocation of the c-Rel subunit of
NF-kB. This subunit of may thus be an important determinant
of iNOS tissue specificity. Because cytokine-induced NO
production may cause tubular cell injury, the development of
inhibitors of proximal tubule BH4 synthesis, NF-kB and IRF-1
activation could provide important pharmacological tools for
future therapy of inflammatory renal diseases.
1


115
activation and nuclear translocation of NF-kB in MCT cells,
it does not cause the transcription of iNOS mRNA, translation
of iNOS protein or confer inducibility in pAiNOS-CAT. The
inhibition of iNOS activity in MCT cells by cycloheximide
demonstrates that protein synthesis is essential for iNOS
activation. Hence, it may be argued that the inability of
IFN to coinduce the synthesis of other vital proteins
necessary for the activation process may explain this
finding. On the other hand, LPS may be inducing the
synthesis of some of these proteins thereby explaining its
ability to confer inducibility in pAiNOS-CAT and pBiNOS-CAT.
However, with respect to iNOS, other processes activated by
IFN may still be vital.
The presence of iNOS in the MCT cell line offers a
simple but powerful tool for the investigation of the role of
NO in the kidney. This is in view of the fact that NO plays
a major role in several renal pathophysiological conditions
such as acute glomerulonephritis, kidney transplant rejection
and acute tubular ischemia-reperfusion injury (Weinberg et
al., 1994, Langrehr et al., 1993 [a,b], Yu et al., 1994).
In conclusion, mouse proximal tubule epithelial iNOS is
induced synergistically by LPS and IFN and it is regulated at
two levels: transcriptionally by transcription factors that
include NF-kB and possibly IRF-1, and posttranscriptionally
by BH4 availability. Whereas IFN does not induce iNOS
expression in proximal tubule cells, it markedly potentiates
the induction of iNOS by LPS. Also NF-kB activation and BH4


115
activation and nuclear translocation of NF-kB in MCT cells,
it does not cause the transcription of iNOS mRNA, translation
of iNOS protein or confer inducibility in pAiNOS-CAT. The
inhibition of iNOS activity in MCT cells by cycloheximide
demonstrates that protein synthesis is essential for iNOS
activation. Hence, it may be argued that the inability of
IFN to coinduce the synthesis of other vital proteins
necessary for the activation process may explain this
finding. On the other hand, LPS may be inducing the
synthesis of some of these proteins thereby explaining its
ability to confer inducibility in pAiNOS-CAT and pBiNOS-CAT.
However, with respect to iNOS, other processes activated by
IFN may still be vital.
The presence of iNOS in the MCT cell line offers a
simple but powerful tool for the investigation of the role of
NO in the kidney. This is in view of the fact that NO plays
a major role in several renal pathophysiological conditions
such as acute glomerulonephritis, kidney transplant rejection
and acute tubular ischemia-reperfusion injury (Weinberg et
al., 1994, Langrehr et al., 1993 [a,b], Yu et al., 1994).
In conclusion, mouse proximal tubule epithelial iNOS is
induced synergistically by LPS and IFN and it is regulated at
two levels: transcriptionally by transcription factors that
include NF-KB and possibly IRF-1, and posttranscriptionally
by BH4 availability. Whereas IFN does not induce iNOS
expression in proximal tubule cells, it markedly potentiates
the induction of iNOS by LPS. Also NF-kB activation and BH4


CHAPTER THREE
GUANOSINE TRIPHOSPHATE CYCLOHYDROLASE I REGULATES NITRIC
OXIDE SYNTHESIS IN RENAL PROXIMAL TUBULES
Introduction
Tetrahydrobiopterin is an essential cofactor of NOS
because it is needed in the formation of the active dimeric
enzyme from iNOS monomers (Tayeh and Marietta, 1989, Baek et
al. 1993) and is tightly bound to the enzyme (Schmidt et
al. 1993). With the exception of macrophages, many cells
that possess iNOS have low BH4 in the resting state and NO
production is limited by their ability to synthesize this
cofactor (Di Silvio et al. 1993, Werner-Felmayer et al. ,
1990) .
In proximal tubule epithelial cells, there is marked
synergism between LPS and IFN in stimulating iNOS activity.
However, unlike what is observed in macrophages, each of
these inducers, by itself, stimulates little or no iNOS
activity in these cells (Amoah-Apraku et al., 1993, Markewitz
et al., 1993). Synergism between cytokines and endotoxin in
their effects on the induction of iNOS expression has been
commonly observed in many other tissues (Blough and Zafirou,
1985, Deng et al. 1993, Pf eilschif ter and Schwarzenbach,
1990, Szab et al. 1993). This synergism, that appears to
vary among different tissues and species, results in some
54


LIST OF FIGURES
Figure Caption Page
1-1 Representation of Aluminum Complexes
with 3-Hydroxy-4-pyridinone and its
Derivatives 5
1-2 ORTEP of the Aluminum Citrate Complex
and its Al304 Core 6
1-3 9-Amino-1,2,3,4-tetrahydroacridine 7
1-4 Ligands with O-C-C-N Donor Group. Oxine;
2-Methyloxine; Picolinic acid;
6-Methylpicolinic acid; Pyrazinoic acid;
Hypoxanthine; Dipicolinic acid 8
2-1 Representation of a Tris Complex between
Aluminum and a Bidentate Ligand 13
2-2 2-Methyloxine 13
2-3 Oxine 14
2-4 Picolinic acid 15
2-5 6-Methylpicolinic acid 16
2-6 Pyrazinoic acid 17
2-7 Hypoxanthine 18
2-8 Dipicolinic acid 19
4-1 Oxine; Methyloxine 41
4-2 ORTEP Drawing of the Tris(oxinato)-
aluminum(III) without the Occluded
Acetonylacetone 42
4-3 PLUTO Line Drawing of the pi-Oxo-di (bis-
(2-methyloxinato)aluminum(III) Complex.. 43
viii


100
Table 6-7. Anisotropic Thermal Parameters (2xl03) for the Non-
Hydrogen Atoms of Compound VIII. Estimated Standard Deviations
given in Parentheses.
Atom
Uxi
u22
u33
u12
u13
u23
Na
58.7(6)
33.0(5)
26.5 (5)
-1.7(4)
-2.1(4)
-0.4(3)
N
36.2(10)
27.8(9)
26.4 (9)
-2.4 (8)
3.5(8)
-1.4(7)
01
84.8(14)
29.1(9)
26.8 (8)
-7.6(8)
-4.9(8)
-0.6(6)
Ola
90.4(15)
39.2(9)
34.9(9)
-18.9(9)
-5.0(9)
-12.6(7)
07
76.9(12)
23.6 (8)
29.9(8)
-5.1(8)
-4.4 (8)
-3.1(6)
07a
57.7(11)
24.6(8)
53.9(10)
-2.6(7)
-3.0(9)
4.7(7)
Cl
46.1(14)
35.5(12)
29.6(11)
-6.5(10)
-1.9(10)
-5.7(10)
C2
35.3 (12)
33.4(11)
27.2(11)
-3.1(9)
-4.2(9)
-3.3(9)
C3
44.3 (12)
44.0(14)
27.4(11)
-2.2(11)
-7.8(10)
-5.6(10)
C4
51. (2)
52.(2)
24.0(12)
-5.8(12)
-6.2(11)
5.0(10)
C5
47.4(15)
35.0(13)
34.6 (12)
-5.2(11)
-8.7(10)
8.1(10)
C6
33.0(12)
32.0(11)
29.7(11)
-1.9(9)
-5.3(9)
3.6(9)
C7
37.5(13)
25.2(11)
39.2(12)
-1.7(9)
-3.3 (10)
1.7(9)
N'
35.5(10)
22.6(8)
24.7(9)
0.2(7)
-2.1(7)
-2.2(7)
01'
93.2(15)
24.7 (8)
30.7(8)
-14.3(9)
-0.7(9)
-6.6(7)
01' a
76.2 (12)
26.9(8)
25.0 (8)
-6.2 (8)
-1.0(8)
-1.3(6)
07'
86.4 (14)
27.5 (8)
30.3 (9)
-11.3 (8)
-1.1(8)
-7.4(7)
01' a
94.0(15)
23.4 (9)
44.7(10)
-7.0(9)
12.6(10)
-2.7(7)
Cl'
44.1(13)
25.1(10)
24.4(11)
2.0(9)
-1.5(9)
-3.6(8)
C2'
39.9(13)
22.9(10)
25.4 (10)
0.6(9)
-1.8(9)
-4.3(8)
C3'
61. (2)
31.1(12)
24.4(11)
-4.4(11)
0.0(10)
-6.2(9)
C4'
66. (2)
40.1(13)
22.2 (11)
-6.9(12)
0.3(11)
1.2(10)
C5'
51.2(15)
28.2 (12)
30.6(11)
-4.1(10)
-3.4 (10)
2.2(9)
C6'
32.9(12)
23.8(10)
28.0 (11)
0.0(9)
-1.9(9)
-1.3(8)
01'
40.8 (13)
26.2 (11)
35.4 (12)
-3.3(9)
2.5(10)
-3.5(9)
08
64.3 (14)
41.0(11)
70.2(14)
4.8(11)
12.7(11)
6.8(10)
09
65.9 (13)
35.2 (10)
29.2(10)
-5.1(9)
-3.3(8)
0.6(7)
010
61.4(13)
27.1(9)
54.8(11)
-6.6(9)
3.6(9)
0.8(8)
The form of the thermal ellipsoid is exp [ -27r2 Ov'a*2^ + k2b*2U22
+ 12c*2U33 + 2hka*b*U12 + 2hla*c*U13 + 2klb*c*U23) ]
Sodium coordination
The sodium atom is in a pentagonal bipyramidal
environment created by two dipic ligands and three water
molecules. The dipic acting as a tridentate ligand has both
acid groups formally protonated. The hydroxyl oxygen atoms, 01
and 07, as well as ring nitrogen are coordinated to the central
atom at bond lengths of 2.482(2), 2.584(2) and 2.476(2) ,


41
Gross, 1993). Since iNOS requires substrate concentrations
of BH4 for full enzymatic activity, the ability of a cytokine
to stimulate the synthesis of GTP cyclohydrolase I and that
of a given cell to carry out the synthesis of BH4 are both
critical in determining the magnitude of ensuing NO formation
(Di Silvio et al., 1993, Hattori and Gross, 1993). Thus, in
this study, the lack of stimulation of NO production by IFN
in intact MCT cells could have been due to the inability of
this cytokine to stimulate GTP cyclohydrolase I in a
situation analogous to what is seen in aortic vascular smooth
muscle. Under these circumstances, the addition of LPS could
have stimulated GTP cyclohydrolase I activity and BH4
synthesis thereby providing the missing cofactor. Therefore,
if the failure of IFN to stimulate iNOS activity was due to
lack of BH4, then providing an exogenous source of this
cofactor to cytosolic extracts of IFN-treated MCT cells would
be expected to fully restore iNOS activity. However, iNOS
activity was undetectable in IFN-treated MCT cell homogenates
despite the presence of saturating concentrations of
exogenously added BH4. Furthermore, the absence of iNOS mRNA
and iNOS protein immunoreactivity after IFN treatment is
consistent with the results of my functional studies and
indicates that, in contrast to what is seen in mouse
peritoneal macrophages, IFN does not induce MCT iNOS.
In the kidney, NO may mediate several physiological as
well as pathological functions. Recent reports suggest that
induced NOS decreases Na+/K+-ATPase and H+-ATPase activity


105
Discussion
X-rav Diffraction Analysis
Crystallographic data for compound IX is summarized in
Table 7-1. The structure of the two organic molecules and the
hydrogen bonded network created between them is displayed in
Figures 7-1 and 7-2, respectively. The final atomic
coordinates are given in Table 7-2. Bond lengths and bond
angles are listed in Tables 7-3 and 7-4, respectively. The
anisotropic thermal parameters for the non-hydrogen atoms are
given in Table 7-5.
Hydrogen Bonding
The most notable feature of IX, despite the absence of an
aluminum ion, is the placement and bonding of the hydrogen
atoms. The carboxylic acid group from one pic is formally
deprotonated. The hydrogen atom, H(N), instead appears 0.975
from the ring nitrogen N(l) and thereby forms a zwitterion.
The other acid is protonated in the normal manner on the
carboxylate oxygen 0(11).
Each of these hydrogen atoms is bonded to an atom of
another pic. H(N) is located 1.798 from N(ll) of the second
ring. H(0) is hydrogen bonded to 0(2) in an adjacent pic
molecule (symmetry operator is 0.5+x, 0.5-y, 0.5+z). The
hydrogen bond created by N(1)-H(N)N(11) and 0(11)-
H(0)**0(2) are 0.327 and 0.417 shorter than the sum of two
van der Waal's radii (3.100 for N---N, 3.000 for 0*0).


81
complex experiencing the same effects as Al(ox)3 between solid
and solution phases. It should be noted that when Ila was
dissolved to form lib, the only peak present in the Al-27 NMR
spectrum was at 54 ppm. Also, there are no peaks near 70 for
any of the solid samples except for VI with hypoxanthine. It
does not seem likely for the ion [Al (DMSO) 6]3+ to be
responsible for these peaks since its chemical shift has been
reported as near 3 ppm.40 The peak at 7 ppm for the solid
sample VI could be a DMSO complex of aluminum.
The Al-27 NMR data confirms the presence of aluminum in
each sample but also suggests the existence of more than one
product for all of the samples except V. Furthermore, since
no coordination compounds have been assigned to the myriad of
chemical shifts present in the spectra, little information can
be gained about the environment of the aluminum ions in the
compounds that have been analyzed.
Compound V, with pyrazinoic acid, has only one peak in
both the solid and solution phases, but these occur at
distinctly different chemical shifts. The solution peak is at
70 ppm similar to the other solutions. The solid, however,
has one peak at 0 ppm, characteristic of Al(H20 ) 63+ or a
perfect octahedral environment. The series of solutions
containing aluminum ion and varying amounts of pza show that
in the absence of the ligand, there are no peaks between
approximately 2 and 60 ppm. The main peak in each spectra is
at 0 ppm, which is most probably due to a large amount of


13
The Cardiovascular System
Under normal physiologic conditions, basal vasodilator
tone is maintained by endothelial NO released in response to
shear stress on the vessel wall that causes entry of Ca2 +
across the plasma membrane and subsequent activation of cNOS
(Rees et al., 1989). It has also been shown that
inflammatory stimuli can induce iNOS-like activity in the
endothelium and vascular smooth muscle cells in amounts far
exceeding the agonist-triggered NO release from endothelial
and vascular smooth muscle cells (Kilbourn and Bellone, 1990,
Busse and Mulsch, 1990). Recent reports suggest that these
cytokines increase NOS activity by increasing BH4 synthesis
(Rozenkranz-Weiss et al., 1994). Thus, in addition to
maintaining basal vascular tone, NO of endothelial origin may
also play a role in the endotoxin-associated hypotension of
septic shock. Endothelial NO also inhibits platelet
aggregation, leukocyte adhesion, endothelin generation and
smooth muscle cell proliferation (Kubes et al., 1991,
Boulanger and Luscher, 1990, Nakaki et al., 1990). Defective
vasoregulation in diabetes, atherosclerosis and arterial
damage in normal aging may be due, in part, to scavenging of
NO by end products of advanced glycosylation and oxidized
lipoproteins (Bucala et al., 1991, Chin et al., 1992), and
defects in endothelial NO regulation could lead to vasospasm
and essential hypertension.


45
O 4 8 12 16 20 24 28
Time (h)
Figure 2-2. Time course of induction of iNOS activity in MCT
cells treated with a combination of LPS (0.1 (j.g/ml) and IFN
(100 U/ml). Inducible NOS activity is detectable at 4 h and
reaches maximum at 12 h. Data are mean S.E. (jug: |ig of cell
protein, n=6).


18
defense, pulmonary neurotransmission and ciliary motility
(Robbins et al., 1994 [a,b]).
It is likely that other beneficial and deleterious
effects of NO in a variety of cells will be discovered and
that modalities to selectively modulate iNOS expression or
local NO concentrations will be of immense therapeutic
benefit.
Rationale For The Thesis
The inducible isoform of NOS has been shown to be
subject to very strict and high tissue-specific regulatory
control (Nathan and Xie, 1994 [a,b], Nussler et al., 1994).
For example, whereas LPS induces high levels of iNOS
expression in macrophages and microglia (Stuehr and Marietta,
1985, Xie and Nathan, 1994), it induces only modest levels in
hepatocytes, vascular smooth muscle and renal epithelial
cells and a combination with cytokines is usually required
for full expression of iNOS activity (Nussler and Billiar,
1993, Amoah-Apraku et al., 1993). In preliminary studies, I
have established that mouse renal tubule epithelial cells
possess iNOS and that whereas LPS induces only very little
iNOS expression, coincubation with IFN results in a
synergistic expression of high levels of iNOS activity in
these cells (Amoah-Apraku et al., 1993).
I have also established that this was not due to the
lack of cofactor availability, suggesting that IFN may be
acting as a potent modulator of iNOS activity in MCT cells by


132
Werner-Felmayer, G., Werner, E.R., Fuchs, D., Hausen, A.,
Reibnegger, G. and Wachter, H. Tetrahydrobiopterin-dependent
formation of nitrite and nitrate in murine fibroblasts. J.
Exp. Med. 172: 1599-1607, 1990.
White, K.A. and Marietta, M.A. Nitric oxide synthase is a
cytochrome P-450 type hemoprotein. Biochemistry 31: 6627-
6631, 1992.
Wilcox, C.S., Schmidt, H.H.H.W., Murad, F., Gross, S.S.,
Taylor, G., Levi, R. and Welch, W.J. NO synthase in macula
densa regulates glomerular capillary pressure. Proc. Natl.
Acad. Sci. USA 89: 11993-11997, 1992.
Wu, G. and Brosnan, J.T. Macrophages can convert citrulline
into arginine. Biochem. J. 281: 45-48, 1992.
Xie, Q., Cho, H.J., Calaycay, J., Mumford, R.A., Swiderek,
K.M. Lee, T.T., Ding, A., Troso, T. and Nathan, C. Cloning
and characterization of inducible nitric oxide synthase from
mouse macrophage. Science 256: 225-228, 1992.
Xie, Q., Kashiwabara, Y. and Nathan, C. Role of
transcription factor NF-KB/Rel in the induction of nitric
oxide synthase. J. Biol. Chem. 269: 4705-4708, 1994.
Xie, Q. and Nathan, C. The high output nitric oxide pathway:
role and regulation. J. Leuk. Biol. 56: 576-582, 1994.
Xie, Q., Whisnant, R. and Nathan, C. Promoter of the mouse
gene encoding calcium-independent nitric oxide synthase
confers inducibility by interferon-y and bacterial
lipopolysaccharide. J Exp. Med. 177: 1779-1784, 1993.
Yu, L., Gengaro, P.E., Niederberger, M. Burke, T.J. and
Schrier, R.W. Nitrite oxide a mediator in rat tubular
hypoxia/reoxygenation injury. Proc. Natl. Acad. Sci. USA 91:
1691-1695, 1994.
Zatz, R. and de Nucci, G. Effects of acute nitric oxide
inhibition on rat glomerular microcirculation. Am. J.
Physiol. 261: F360-F363, 1991.
Ziegler, I., Schott, K., Lbbert, M.F., Schwulra, U. and
Bacher, A. Control of tetrahydrobiopterin synthesis in T-
lymphocytes by synergistic action of interferon-y and
interleukin-2. J. Biol. Chem. 265: 17026-17030, 1990.
Zweier, J.L., Kuppasamy, P. and Lutty, G.A. Measurement of
endothelial cell free radical generation: evidence for a
central mechanism of free radical injury in post ischemic
tissues. Proc. Natl. Acad. Sci. USA 85: 4046-4056 1988.


LIST OF REFERENCES
Abu-Soud, H.M. and Stuehr, D.J. Nitric oxide synthases
reveal a role for calmodulin in controlling electron
transfer. Proc. Natl. Acad. Sci. USA 90: 10769-10772, 1993.
Alspaugh, J.A. and Granger, D.L. Inhibition of Cryptococcus
neoformans replication by nitrogen oxides supports the role
of these molecules as effectors of macrophage-mediated
cytostasis. Infect. Iirnun. 59: 2291-2296, 1991.
Amoah-Apraku, B., Chandler, L.J., Madsen, K.M., Tang, S.S.,
Ingelfinger, J.R. and Guzman, N.J. Regulation of inducible
nitric oxide synthase expression in murine proximal tubule
epithelial cells (Abstract). J. Am. Soc. Nephrol. 4:544,
1993.
Awolesi, M. Sessa, W. and Sumpio, B. Cyclic strain
upregulates nitric oxide synthase gene. FASEB 1: A755, 1993.
Baek, K.J., Thiel, B.A., Lucas, S. and Stuehr, D.J.
Macrophage nitric oxide synthase subunits: purification,
characterization and role of prosthetic groups and substrate
in regulating their association into dimeric enzymes. J.
Biol. Chem. 268: 21120-21129, 1993.
Baeuerle, P.A. and Baltimore, D. A 65-kD subunit of NF-kB is
required for inhibition of NF-kB by IkB. Genes Dev. 3: 1689-
1698, 1989.
Bellahsene, Z., Dhont, J.L. and Farriaux, J.P. Guanosine
triphosphate cyclohydrolase activity in rat tissues. Biochem.
J. 217: 59-65, 1984.
Billiar, T.R., Curran, R.D., Harbrecht, B.G., Stuehr, D.J.,
Demetris, A.J. and Simmons, R.L. Modulation of nitric oxide
synthesis in vivo: NG-monomethy1-L-arginine inhibits
endotoxin-induced nitrite/nitrate biosynthesis while
promoting hepatic damage. J. Leuk. Biol. 48: 568-569, 1990.
Blough, N.V. and Zafirou, O.C. Reaction of superoxide with
nitric oxide to form peroxinitrite in alkaline aqueous
solution. Inorg. Chem.
24:
3502-3504,
1985.
Bogdan, C., Vodovotz,
Y. ,
Paik, J.,
Xie, Q. and Nathan, C.
Traces of bacterial lipopolysaccharide suppress IFN-y-induced
nitric oxide synthase gene expression in primary mouse
macrophages. J. Immunol. 151: 301-309, 1993.
117


10
groups of other proteins such as ferritin which then acts as
a storage pool for NO (Reif and Simmons, 1990). The induced
(Type II) enzyme remains tonically activated and continuously
produces NO for the life of the active enzyme.
Nitric oxide reacts with O2 in both gaseous and aqueous
phases to form nitrogen dioxides (NO2) (Marietta et al. ,
1990). Nitric oxide formed under reducing conditions as the
free molecule (NO-), reacts with the superoxide anion (O2-)
in aqueous solutions to yield peroxynitrite (ONOO-), which in
part mediates its cytotoxicity (Blough and Zafirou, 1985).
Under oxidizing redox conditions, NO is formed as the
nitrosonium ion (NO+) and reacts with thiol-containing
proteins to form S-nitrosothiols (RS-NO). In N-methyl-D-
aspartate type glutamate (NMDA) receptors, NO reacts with
cell surface thiols to form RS-NOs that afford protection
from receptor-mediated neurotoxicity by down-regulating
harmful receptor-mediated Ca2 + influx into the cells (Lipton
et al. 1993, Dolye et al. 1981, Olson et al. 1981, Saran
et al. 1990). Nitric oxide readily forms complexes with
transition metal ions and heme-containing proteins like
hemoglobin (Sharma et al., 1983). These reactions form the
basis for several NO assays. The inhibition by NO of iron-
containing mitochondrial enzymes like aconitase of the Krebs
cycle, succinate-ubiquinone oxidoreductase and NADPH-
ubiquinone oxidoreductase of the electron transport chain
occurs through the nitrosylation of their iron-sulfur (Fe-S)
complexes (Dimmeler et al., 1992, Stuehr et al. 1989,


A
B
LPS/IFN
+
Control IFN LPS LPS/IFN PDTC
pAiNOS-CAT
20 n
>
+3
u
<
<
o
LPS/IFN
Figure 5-4. Effects of LPS, IFN and PDTC on the activation of the iNOS promoter. The MCT
cells transfected with pAiNOS-CAT vector were treated with IFN (100 U/ml), LPS (1 |xg/ml) or
their combination in the absence or presence of PDTC (25 nM), followed by CAT assay of cell
lysates. Both LPS and LPS/IFN, but not IFN conferred CAT inducibility, which was blocked by
coincubation with PDTC. A Autoradiograph, B graphical presentation; (*, ** p < 0.05 and 0.01
respectively, vs control, *** p < 0.01 vs -PDTC, n=4)
110


48
Mo Ka radiation. The unit cell dimensions were determined for
17 automatically center reflections, 1.Os20s3O 0 ; space
group monoclinic P21/n; 1421 unique reflections, 1062 with
I>3a(I), 0shs8, OskslO, -13slsl3. Eighteen of the twenty-two
hydrogen atoms were located using a difference Fourier map.
Final refinement values for 325 parameters were R = 9.18, wR
= 7.15 and w = 7.13.
Discussion
X-ray diffraction analysis
Crystallographic data for each complex is summarized in
Table 4-1.
Al (meox) o CHnOH* H-,0 (I). An ORTEP drawing of the
Al(meox)3 molecule is given in Figure 4-4. Final atomic
coordinates for all non-hydrogen atoms and all hydrogen atoms
found, bond lengths and bond angles appear in Tables 4-2
through 4-5, respectively. The anisotropic thermal parameters
are listed in Table 4-6.
Despite the presence of the sterically hindering methyl
group adjacent to the nitrogen atom, the aluminum ion is
definitely coordinated to the nitrogen and oxygen atoms of
three 2-methyloxine ligands. The Al-N bond distances in I,
ranging from 2.122 to 2.179 correspond well with the
relatively long Al-N bond distance of 2.18 reported for
H3A1* 2N (CH3) 3.65 However, the Al-N bonds in I are significantly
longer than the Al-N bonds in i-oxo-di(bis(2-methyloxinato)


7
anions (O2 ) and hydrogen peroxide (H2O2) (Zweier et al. ,
1988).
Physiologically, synthesis of NO is regulated in various
ways. Firstly, the major stimulants of physiological
synthesis of NO are hemodynamic forces, notably shear stress
(Awolesi et al., 1993, Nishida et al., 1992). These and
several other agonists can activate NO synthesis within
seconds through pathways that are unaffected by
transcriptional or translational inhibition. Such agents
which include Ca2+ ionophores, excitatory amino acids,
acetylcholine and bradykinin, activate cNOS by eliciting
prompt elevation of intracellular Ca2+ (Ignarro et al., 1987,
Moneada et al., 1991) Secondly, agents termed inducers
stimulate synthesis of NOS in hours and require mRNA
transcription and protein synthesis (Geller et al. 1993
[a,b], Moneada et al. 1991) These include agents that
induce NO-mediated antitumor or antimicrobial activity such
as cytokines and bacterial lipopolysaccharide (LPS). These
agents typically activate iNOS and some of them interact
synergistically to enhance NO release. A third class of
agents includes those with the capacity to suppress NO
synthesis. A typical example of these agents is tissue
growth factor-p (TGF-P) which inhibits NO synthesis by
decreasing iNOS mRNA stability and translation, and by
increasing iNOS protein degradation (Vodovotz et al. 1993,
Junguero et al., 1992). Glucocorticoids may inhibit NO
synthesis through a similar mechanism and NO itself regulates


53
9.5-
7.5-
Figure 2-9. Effects of IFN (100 U/ml) and LPS (1 (ig/ral) on
iNOS steady-state mRNA levels after 4 h of treatment in MCT
cells. Interferon-y enhances the effect of LPS but does not
by itself increase iNOS mRNA levels. Ribosomal (18s) RNA was
used as gel loading control which was comparable in all
lanes. Blot is representative of three experiments. Act:
actinomycin D (4 jxM) Dex: dexamethasone (20 |1M) .


90
LPS/IFN
Figure 4-7. Effect of PDTC (25 |iM) on nitrite production by
MCT cells treated with a combination of LPS (1 |j.g/ml) and IFN
(100 U/ml) for 24 h. Cells were incubated with PDTC for 30
min before the addition of LPS and IFN, and during the entire
incubation period, (* p < 0.001 vs LPS/IFN, mg: mg of cell
protein, n=6).


28
reaction rate is increased by heat or a large concentration of
acid or base. Since urea is very water soluble, it is easily
dissolved in a dilute aqueous solution of an aluminum salt and
the ligand.
Experimental
Solutions with a three-to-one molar ratio of ligand to
aluminum ion were prepared by dissolving the 1.0 and 0.3
mmoles of the ligand and Al (N03) 3* 9H20, respectively, in 30 mL
of water. The ligands tested were pic, mpic, dipic, pza and
hyp. Upon gentle heating, 43 to 45C, ammonia, as well as
carbon dioxide bubbles, was gradually produced throughout the
solution and the compound was formed.
Results
For each ligand, the crystals that formed were neither
large nor of high quality. There was a very large incidence
of twinned crystals and inseparable clusters of crystalline
product, possibly as a result of the higher temperature.
Vapor Diffusion
Theory
One common technique to control the availability of the
reactants is through diffusion.61,62 By allowing the reactants
to only slowly combine, the reaction proceeds at a slower rate
than the formal concentrations would normally predict. As a
result larger crystals are generally produced. For vapor
phase diffusion, a gaseous reactant is slowly incorporated


LIST OF REFERENCES
(1) Liss, L., ed. "Aluminum Neurotoxicity"; Tathopox
Publishing, Inc., New York, 1980.
(2) Graves, A. B.; White, E.; Koepsell, T. D.; Reifler, B.
V.; van Belle, G; Larson, E. B. J. Clin. Epidemiol.
1990, 43, 35-44.
(3) Flaten, T. P. Proc. Sec. Intn. Symp. Geochem. Health
1987, London, Apr. 22-24, 1987.
(4) Goldgraber, D.; Lerman, M. I.; McBride, O. W. ;
Saffiotti, U.; Gajdusek, D. C. Science 1987, 235, 877-
880.
(5) Gitelman, H. J., ed. "Aluminum and Health: A Critical
Review"; Marcell Dekker, Inc., New York, 1989.
(6) Kaehny, W. D.; Hegg, A. P.; Alfrey, A. C. N. Engl. J.
Med. 1977, 296, 1389-1390.
(7) Sherrard, D. J. Sem. Nephrol. 1986, 6, No. 4, 5-11.
(8) Mayor, G. H.; Burnatowska-Hledin, M. Sem. Nephrol. 1986,
6, No. 4, 1-4.
(9)
Edwardson,
97.
J. A.; Candy, J. M.
Ann.
Med. 1989,
21
, 95-
(10)
Greenwood,
N. N.; Earnshaw,
A.
"Chemistry
of
the
Elements";
Pergamon Press, Ltd.
., Oxford, 1986.
(11)
Lewis, T.
E., ed. "Environmental Chemistry
and
Toxicology
of Aluminum"; Lewis
Publishers,
Inc.,
Chelsea, Michigan, 1989.
(12) Tam, S. C.; Williams, R. J. P. J. Inorg. Biochem. 1986,
26, 35-44.
(13) Driscoll, C. T. J. Royal Neth. Chem. Soc. 1987, 106,
402.
(14) Williams, R. J. P. J. Royal Neth. Chem. Soc. 1987, 106,
401.
138


114
confer inducibility by LPS but not by IFN; synergistic
inducibility by both LPS and IFN was conferred only by the
entire promoter region.
This study has revealed several important differences
between renal epithelial iNOS and the enzyme in other tissues
and species. Synergism in some tissues appears to be the
result of cooperative interactions between iNOS inducers that
lead to stimulation of the synthesis of cofactors such as BH4
that are required for the expression of full activity. For
example, IFN markedly stimulates NO synthesis in murine
peritoneal macrophages (Deng et al., 1993) but is ineffective
in MCT cells. Also, LPS potentiates iNOS activity induced by
IFN in rat aortic vascular smooth muscle cells by inducing
BH4 synthesis (Gross and Levi, 1992, Hattori and Gross,
1993), but in MCT cells, even exogenous addition of these
cofactors to IFN- or LPS-treated cells has no effect.
Another important distinction is the inability of LPS to
cause the activation and nuclear translocation of the c-Rel
subunit of NF-kB in MCT cells even though in macrophages this
subunit is critical for the induction of iNOS activity (Xie
et al. 1993). The synergism between LPS and IFN in MCT
cells appears to be mainly at the level of transcription, as
is the case in hepatocytes (Geller et al. 1993), and
requires IFN-induced protein binding to the interferon-y
regulatory elements.
The role of NF-kB in the induction of iNOS in MCT cells
may be very complex. Although IFN is able to cause the


129
of 0,0 bidentate ligands in distorted octahedral environments
range from 3 6 to 41 ppm. The data reported for hexacoordinate
aluminum complexes of N,0 donor atoms, which includes a study-
using aminopolycarboxylic acids and A1N303 compounds, list the
chemical shift range from 8.1 to 41.2 ppm. The spectra of
solids la and Ila revealed a mixture of aluminum environments.
For la, a large peak at 54 ppm and a smaller at 221 ppm. For
Ila, there are two peaks of equal height at 5 and 73 ppm. The
spectrum collected for lib provided a single peak at 54 ppm.
Because the structure was confirmed by a second x-ray
analysis, the environment of the aluminum complex in solution
is known to be Al(ox)3 -- a distorted octahedral environment
with three nitrogen and three oxygen atoms. A shift range of
54 to 73 ppm is probably due to the distorted meridional A103N3
center and should be added to the range of chemical shifts
reported for an octahedral environment around an aluminum ion.
Also, the difference could be due to the solvent molecule
present.
All of the solids except for V produced more than one
peak using Al-27 NMR. It is clear that each of these contains
a mixture of aluminum environments. However, in each
solution, there is a peak near 68 ppm which is rather close to
73 ppm. This could confirm the presence of a tris aluminum
complex or a common DMSO solvated aluminum species in
solution. Also, there are no peaks near 70 for any of the
solid samples except for VI with hypoxanthine. Since no


82
the possibility that the synergism between these two inducers
may be due to activation of different NF-kB subunits that
complement each other to induce iNOS gene transcription.
However, since this study was limited to the study of only
the three major subunits of NF-kB, the possibility of IFN
activating other less well-characterized subunits of NF-kB
cannot be completely ruled out. In spite of this, the
finding that LPS does not activate the nuclear translocation
of c-Rel in renal epithelium is very significant and may help
unravel tissue specificity in the regulation of iNOS
expression.
In summary, activation of NF-kB is necessary but not
sufficient for the induction of iNOS expression by LPS and
IFN in renal epithelium. The absence of c-Rel in the NF-kB
complexes translocated by these immunokines may, in part,
account for their inability to individually induce renal
epithelial iNOS. Therefore, c-Rel may be an important
determinant of the tissue specificity of iNOS expression.
This is consistent with the notion that the ability of the
various components of NF-kB to associate and form multiple
different dimers appear to be a major determinant of the
ability of these proteins to assign tissue-specific responses
to a given stimulus. Since overproduction of NO has been
associated with various pathological renal conditions
(Langrehr et al. 1993 [a,b], Weinberg et al. 1994, Yu et
al., 1994, Nussler et al., 1993), a better understanding of
the molecular mechanisms that determine the tissue-specific


CHAPTER SIX
SUMMARY AND CONCLUSIONS
The aims of this dissertation were to investigate the
regulation of inducible nitric oxide synthase in murine
proximal tubule epithelium, using the MCT cell line.
The major conclusions are: 1) murine proximal tubule
epithelial cells possess an immunokine-inducible isoform of
NOS; 2) expression of MCT iNOS activity requires protein
synthesis and mRNA transcription; 3) IFN potentiates the
induction of iNOS activity by LPS at the transcriptional
level but does not itself induce transcription or expression
of the enzyme; 4) inhibition of GTP cyclohydrolase I, the
rate-limiting enzyme in the de novo synthesis of BH4 f
suppresses the maximum expression of iNOS activity in MCT
cells; this inhibition is reversed by concurrent activation
of the salvage pathway with sepiapterin, a substrate for BH4
formation via the salvage pathway; 5) LPS, IFN or their
combination cause a rapid activation and nuclear
translocation of NF-kB complexes in MCT cells, and that these
complexes are composed mainly of p65 with smaller amounts of
p50 but no c-Rel; 6) NF-kB activation is essential but not
sufficient for the induction of iNOS expression in MCT cells;
7) constructs bearing portions of the promoter region of iNOS
linked to a reporter gene, except for the upstream portion,
113


29
ml of water and the eluant, whose sole radioactive component
was [3h]L-citrulline as determined by thin layer
chromatography, collected into vials. Ten ml of
scintillation cocktail was added to each vial and the
radioactivity quantified by liquid scintillation spectroscopy
(Beckman LS 6000SC). Inducible NOS activity was expressed as
[3h] L-citrulline DPMs per |ag of protein. For experiments in
which iNOS activity was measured in crude cell homogenates,
these were obtained from MCT cells grown to confluence in 100
mm dishes. The MCT cells were suspended in ice-cold 50 mM
Tris-HCl containing 0.1% 2-mercaptoethanol, 0.1 mM EGTA and
0.1 mM EDTA, and homogenized on ice by using a glass tissue
grinder with a Teflon pestle in the presence of 1 mM
phenylmethylsulfonyl fluoride (PMSF), 3 |iM leupeptin and 1 |jM
pepstatin A as previously described (Davda et al., 1993).
Aliquots of homogenates containing 100-200 p.g of protein were
added to the assay buffer devoid of Ca2 + and iNOS activity
was measured as described above in the presence of 1 mM
NADPH, 4 jiM flavin-adenine dinucleotide (FAD) 10 |iM BH4 and 3
[iCi/ml of [3h] L-arginine.
Nitrite Assay
Nitrites were measured by the addition of 200 n.1 of
freshly prepared Greiss reagent [0.75% sulfanilamide in 0.5 N
HC1 and 0.075% N-(1-naphthyl)ethylenediamine dihydrochloride
in double distilled water] to 200 |il of the culture medium
from treated and control cells, incubated with shaking at 25


44
shown in Figure 4-3, the aluminum ion in /-oxo-di (bis (2-
methyloxinato) -aluminum(III) ) is five coordinate with only two
2-methyloxine ligands each.32
The synthesis and characterization using x-ray
diffraction techniques of the tris complex Al(meox)3, as well
as A1(ox)3, are presented in this chapter. An ORTEP drawing
of each is included to display the distorted octahedral
environment around each aluminum ion. In addition, the
results of the Al-27 NMR analysis, including spectra, are
discussed for each complex.
Experimental
Synthesis
Materials. All materials and solvents were reagent grade
and used as supplied from manufacturer.
Preparation of Al (meox) CELOH-H-,0 (I). DMSO (39 mL) was
placed in a 200 mL round bottom flask which was set in a 90C
oil bath to warm. Al2(S04)3*18 H20 (3.31 g, 10 mmole A1(H20) 63+)
was added and dissolved by stirring. 2-Methyloxine (5.02 g,
32 mmole) was subsequently added to yield a dark amber
solution. The solution was neutralized slowly by the addition
of 0.5 mL portions of diethylamine (7.5 mL, 72 mmol). The
resulting opaque mixture was stirred and heated for an
additional 10 minutes and then cooled to room temperature. A
dark amber solution (30 mL) was obtained by filtration through
a fine glass frit. After two months, large brown crystalline


119
Figure 8-1. ORTEP Drawing of X Showing the Atomic Numbering
Scheme.


CHAPTER TWO
CHARACTERIZATION OF INDUCIBLE NITRIC OXIDE SYNTHASE IN MURINE
PROXIMAL TUBULE EPITHELIUM
Introduction
Nitric oxide is an important mediator of vascular tone,
neurotransmission and immune responses (Moneada et al. ,
1991). In addition, overproduction of NO has been implicated
in the pathogenesis of several cytotoxic and inflammatory
disorders. Nitric oxide is synthesized from L-arginine by
NOS in a reaction that produces stoichiometric amounts of L-
citrulline and requires NADPH, BH4 and flavin nucleotides as
cofactors. As described earlier, this enzyme family
comprises at least three distinct isoforms that can be
divided into two groups: the constitutively expressed, Ca2+-
calmodulin-dependent cNOS found typically in neurons (bNOS,
Type I) and endothelial cells (eNOS, Type III), and the
immunologically inducible, Ca2+-independent NOS found
typically in macrophages (iNOS, Type II) (Forstermann et al.,
1991, Stuehr and Griffith, 1992).
In the kidney, inducible and constitutive NOS protein or
mRNA has been detected in glomerular mesangial cells, macula
densa cells of the juxtaglomerular apparatus, proximal tubule
cells, the entire collecting duct as well as other epithelial
lining of the nephron (Marsden and Ballerman, 1990, Mundel et
25


REGULATION OF INDUCIBLE NITRIC OXIDE SYNTHASE IN MURINE
PROXIMAL TUBULE EPITHELIAL CELLS
BY
BISMARCK AMOAH-APRAKU
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1995


22
of BH4 from guanosine triphosphate. I also investigated the
role of the salvage pathway in which dihydropteridines are
converted to BH4 by dihydrofolate reductase. Experiments
were designed to examine the effects of dihydropteridine
availability and dihydrofolate reductase inhibition using
sepiapterin, a dihydropteridine that is less toxic and is
taken up by cells easily compared to BH4, and methotrexate,
an inhibitor of dihydrofolate reductase.
Specific Aim 3
Identification and characterization of NF-kB as a
signaling element during the activation of MCT iNOS by IFN
and LPS. I investigated the time- and concentration-
dependence of the activation and nuclear translocation of NF-
kB by IFN and LPS using a radio-labeled oligonucleotide
containing a consensus binding site for NF-kB as a probe.
The presence of NF-kB in nuclear extracts was evaluated by
DNA Electrophoretic Mobility Shift Assay (EMSA) (Fried and
Crothers, 1981, Garner and Revzin, 1981). The presence or
absence of the various subunits of NF-kB was investigated in
supershift assays, using specific polyclonal IgG antibodies
against NF-kB subunits p50, p65 and c-Rel.
Specific Aim 4
Demonstration of the requirement for NF-kB and IFN-
activated proteins in iNOS induction using functional
promoter analysis. To achieve this, the following goals were


100
Previous reports have identified the initiation site of
this promoter region by SI nuclease mapping and primer
extension (designated as +1 in figure 5-1) which is 30 bp
downstream of a TATA box (Xie et al. 1993, Lowenstein et
al. 1993). The TATA box is preceded by a number of
recognition sites for the binding of transcription factors
induced by immunokines and necessary for gene transcription
including NF-kB and IRF-1 (Xie et al., 1993).
The Plasmid Constructs
The pAiNOS-CAT plasmid (figure 5-A) construct represents
the insertion of the entire 1.7 kbp fragment into the vector;
pBiNOS-CAT, the Spe I and downstream Sal I fragment (-478 to
+141, figure 5-1) and pCiNOS-CAT, the Xba I and upstream Hind
III fragment (-1588 to -421, figure 5-1). The construct
pBiNOS-CAT contains one NF-kB consensus sequence. This part
of the promoter has been found to be responsive to LPS but
unresponsive to the synergizing effect of IFN in macrophages
(Xie et al., 1993). The construct pCiNOS-CAT contains
another NF-kB consensus sequence as well as the IRF-1 binding
site (IRF-E), GAS, ISRE and some y-IREs. In mouse
macrophages, this part of the promoter was unresponsive to
LPS but both regions were necessary for LPS-activated iNOS
expression since deletion of either region decreased the
activity of the reporter gene (Xie et al., 1993). Sequencing
of all the constructs confirmed that the appropriate
deletions and ligations have been obtained.


125
Lukic, M.L., Stosic-Grujicic, S., Ostojic, N. Chan, W.L. and
Liew, F.Y. Inhibition of nitric oxide generation affects the
induction of diabetes by streptozocin (sic) in mice. Biochem.
Biophys. Res. Conunun. 178: 913-920, 1991.
Markewitz, B.A., Michael, J.R. and Kohan, D.E. Cytokine-
induced expression of a nitric oxide synthase in rat renal
tubule cells. J. Clin. Invest. 91: 2138-2143, 1993.
Marietta, M.A., Tayeh, M.A. and Hevel, J.M. Unraveling the
biological significance of nitric oxide. Bio factors 2: 219-
225, 1990.
Marsden, P.A. and Ballermann, B.J. Tumor necrosis factor
alpha activates soluble guanylate cyclase in bovine
glomerular mesangial cells via an L-arginine-dependent
mechanism. J. Exp. Med. 172: 1843-1852, 1990.
Martin, E., Nathan, C. and Xie, Q-w. Role of interferon
regulatory factor 1 in induction of nitric oxide synthase. J.
Exp. Med. 180: 977-984, 1994.
Mellion, B.T., Ignarro, L.J., Ohlstein, E.H., Pontecarvo,
E.G., Hyman, A.L. and Kadowitz, P.J. Evidence for the
inhibitory role of guanosine 3',51-monophosphate in ADP-
induced human platelet aggregation in the presence of nitric
oxide and related dilators. Blood 57: 946-955, 1981.
Minnard, E.A., Shou, J., Naama, H. Cech, A., Gallagher, H.
and Daly, J.M. Inhibition of nitric oxide synthesis is
detrimental during endotoxemia. Arch. Surg. 129: 142-148,
1994.
Mitchell, H.H., Shonle, H.A. and Grindley, H.S. The origin
of nitrate in the urine. J. Biol. Chem. 24: 461, 1916.
Moneada, G., Palmer, R.M.J., and Higgs, E.A. Nitric oxide:
physiology, pathophysiology and pharmacology. Pharmacol Rev.
43: 109-142, 1991.
Morrissey, J.J., McCracken, R., Kaneto, H., Vehaskari, M.,
Montani, D. and Klahr, S. Location of an inducible nitric
oxide synthase mRNA in the normal kidney. Kidney Int. 45:
998-1005, 1994.
Mukaida, N. Mahe, Y. and Matsushima, K. Cooperative
interaction of nuclear factor-kappa B and cis-regulatory
enhancer binding protein-like factor binding elements in
activating the interleukin-8 gene by pro-inflammatory
cytokines. J. Biol. Chem. 265: 21128-21138, 1990.
Muller, J.M., Ziegler-Heitbrock, H.W. and Baeuerle, P.A.
Nuclear factor-kappa B, a mediator of lipopolysaccharide
effects. Immunobiology 187: 233-256, 1993.


134
bond angles for I and II have an average deviation of 9.4 and
8.8, respectively, from the ideal 180 angle. All of these
measurements prove that the Al(ox)3 is closer to the ideal
octahedral environment than Al(meox)3. The steric interaction
of the methyl groups with the aromatic rings is probably
responsible for the increased distortion from the octahedral
environment in the tris(2-methyloxinato)aluminum(III)
complex.
Na (C-,H1N01) (G,HcN0J 3H,0
For compound VIII, aluminum was initially assigned as the
central atom, although no seven coordinate aluminum complex
has been reported. However, during refinement, the number of
hydrogen atoms found indicated the loss of only one hydrogen
atom from the dipic and water molecules. A sodium ion was
then assigned as the metal ion to give a neutrally charged
compound.
The sodium ion is in a pentagonal bipyramidal environment
created by two dipic ligands and three water molecules. One
dipic, which has both acid groups deprotonated, acts as a
tridentate ligand. The bond lengths for the hydroxyl oxygen
atoms and the ring nitrogen 2.482 (2), 2.584(2) and 2.476(2) ,
respectively. The other dipic, which has been formally
deprotonated, is coordinated to the metal ion only through the
carbonyl oxygen of the acid group, at a distance of 2.474(2)
, rather than the deprotonated hydroxy group. The remaining
equatorial and the two axial positions are occupied by water


48
Figure 2-5A. Synergistic interactions of LPS and IFN: IFN
treatment alone (0-100 U/ml for 24 h; LPS) failed to induce
iNOS activity in MCT cells. Simultaneous addition of LPS
(0.1 (j.g/ml; + LPS) resulted in a dose-dependent stimulation
of iNOS activity by IFN (|xg: (j.g of cell protein, n=6) .


76
effect of the NF-kB inhibitor PDTC on NF-kB activation, iNOS
protein and steady-state mRNA levels.
As shown in figure 4-5, treatment with PDTC (25 mM)
inhibited or attenuated the activation of NF-kB by LPS, IFN
or their combination. In addition, PDTC abolished the
synergism between LPS and IFN on iNOS steady-state mRNA
levels (figure 4-6A), blunted the expression of iNOS protein
(figure 4-6B), and completely prevented the formation of
nitrites induced by these agents (figure 4-7). Taken
together, these results suggest that NF-kB is necessary for
the induction of iNOS activity by LPS and IFN in renal
epithelium, but its activation alone does not explain the
synergism seen between these two inducers. Also, they
indicate that NF-kB activation is not sufficient for the
expression of this enzyme since neither IFN nor LPS alone is
able to induce iNOS activity despite activating NF-kB.
Effects of Nuclear Factor-KB Antibodies on Nuclear Factor-KB
Activation bv Lipopolvsaccharide and Interferon-Y
To identify the components of NF-kB activated by LPS in
renal epithelium and compare them to those activated in
macrophages where LPS is known to be a potent inducer of
iNOS, I used IgG against the three major NF-kB subunits p50,
p65 (Rel-A) and c-Rel in an EMSA.
Consistent with previous reports (Xie et al., 1994),
LPS activated NF-kB complexes containing p50, p65 and c-Rel
in the mouse macrophage cell line RAW 264.7 (figure 4-8A).


113
nitrogen atom, 3.050 The remaining hydrogen interactions
have bond lengths greater than the sum of the two van der
Waal's radii and, therefore, do not qualify as even weak
hydrogen bonds.66
Although neither ammonia nor the ammonium ion were added
to the reaction mixture, NH/ was present in the crystal
analyzed. When urea is heated, especially in the presence of
acid, it decomposes by according to the following chemical
reaction. Under the slightly acidic conditions in the
H2NCONH2 + H20 2 NH3 + C02
filtrate, the ammonia was converted the its conjugate acid.
The four hydrogen atoms of the ammonium ion refined to
reasonable parameters. The four N-H bond lengths are 0.766,
0.769, 0.947 and 0.986 within normal limits. Also, the six
angles created by the hydrogen atoms around the nitrogen range
from 94.9 to 122.4.
Nature of the Products
The reaction mixture involved approximately a 4 : 3 : 1
ratio of ligand to base to aluminum ions. An excess of pic
was employed to ensure that the maximum amount of aluminum
could react. In consideration of the nature of the insoluble
white solid produced in later experiments, it seems reasonable
that the white solid formed in this synthesis contains
aluminum and pic. Since there was an excess of ligand used,
the unreacted pic was free to crystalize in a manner that


39
performed for pic and mpic, however, no solid was produced in
either experiment.
With no effort to exclude atmospheric water, preweighed
aluminum isopropoxide (2.6 g, 12 mmole) was stirred in 30 mL
of isopropyl alcohol. Approximately 25 mL of a viscous
solution was collected after the mixture was centrifuged to
remove any solid. Picolinic acid (1.3 g, 11 mmole) was
dissolved in 20 mL of isopropyl alcohol. The solutions were
combined and within 30 minutes, the mixture began to cloud.
After 24 hours, the solution was filtered, the solid washed
with isopropanol and tested for solubility. A similar
experiment was performed for pza with solid formation being
instantaneous.
Results
The solids formed using aluminum isopropoxide were non
crystalline. Furthermore, the products were not soluble in
any solvent and therefore could not be recrystallized.
Summary of Results
Although crystalline solids of the various complexes can
be readily formed using a variety of techniques, good single
crystals are very difficult to produce. Since the compounds
with pic and pza formed the same crystal forms under a variety
of conditions, it seems that packing of the molecules within
the crystal could be the cause. A probable conclusion would


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
REGULATION OF INDUCIBLE NITRIC OXIDE SYNTHASE IN MURINE
PROXIMAL TUBULE EPITHELIAL CELLS
By
Bismarck Amoah-Apraku
August, 1995
Chairman: Nicolas J. Guzman, MD
Major Department: Pharmacology and Therapeutics
Nitric oxide (NO) is an important mediator of vascular
tone, neurotransmission and immune responses. The mechanisms
regulating inducible NO synthase (iNOS) expression in renal
tubule epithelium have not been well characterized. The
purpose of this study was to investigate the mechanism of
potentiation between interferon-y (IFN) and bacterial
lipopolysaccharide (LPS) in inducing iNOS activity in murine
proximal tubule epithelial (MCT) cells. Treatment with LPS
caused a small increase in iNOS activity whereas IFN had no
effect. Simultaneous treatment with LPS and IFN (LPS/IFN),
however, markedly increased iNOS activity. Treatment with
LPS/IFN in the presence of 2,4-diamino-6-hydroxypyrimidine
(DAHP), an inhibitor of the rate-limiting enzyme GTP-
cyclohydrolase I in the de novo synthesis of


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS iii
KEY TO ABBREVIATIONS vii
ABSTRACT viii
CHAPTERS
1 INTRODUCTION 1
General 1
Isoforms of Nitric Oxide Synthase 2
Substrate and Cofactor Requirements 4
Synthesis of Nitric Oxide 6
Role of Nitric Oxide in Mammalian Physiology 9
Rationale for the Thesis 18
Specific Aims 21
Statistical Analysis 23
2 CHARACTERIZATION OF INDUCIBLE NITRIC OXIDE
SYNTHASE IN MURINE PROXIMAL TUBULE EPITHELIUM 25
Introduction 2 5
Materials and Methods 27
Results 32
Discussion 39
3 GUANOSINE TRIPHOSPHATE CYCLOHYDROLASE I
REGULATES NITRIC OXIDE SYNTHESIS IN RENAL
PROXIMAL TUBULES 54
Introduction 54
Materials and Methods 57
Results 58
Discussion 60
4 NUCLEAR FACTOR-KB REGULATES INDUCIBLE NITRIC OXIDE
SYNTHASE IN RENAL TUBULE EPITHELIUM 68
Introduction 68
Materials and Methods 70
Results 73
Discussion 78
v


(nmoles/mg)
51
Basal LPS IFN 1 5 10 50 100
0.1 (ig/ml 100 U/ml
LPS (0.1 (ig/ml) + IFN (U/ml)
Figure 2-7. Concentration dependence of the effect of IFN
(0-100 U/ml for 48 h) on nitrite accumulation in the presence
of LPS (0.1 (j.g/ml) in MCT cells, (* p<0.05 vs control, mg: mg
of cell protein, n=3).


CHAPTER 2
SYNTHESIS AND CHARACTERIZATION OF
ALUMINUM COORDINATION COMPOUNDS
Aluminum as a Lewis Acid
The only non-zero valence state available to aluminum is
a positive three. Since it can attain a positive charge, the
aluminum ion will readily accept electron pair donors and
function as a Lewis acid. The trivalent aluminum ion has a
rather small ionic radius of 0.50 .10 As a result, the
aluminum ion with a relatively high positive charge coupled
with a small ionic radius can be classified as a hard Lewis
acid.
In an aqueous solution of an aluminum salt, the metal ion
will be surrounded by six water molecules, each of which
donates an electron pair to the positive central ion. For a
ligand to replace the water molecule and coordinate the metal
ion, the complex formed by the exchange should be more stable.
One possibility is for the electron donor atom to be a small
atom, such as oxygen and nitrogen, and also have a stronger
negative charge. An example would be a deprotonated hydroxyl
group or carboxylate oxygen. With a negative charge, each of
these ions would function as harder Lewis bases and more
readily replace the water molecule. The other possibility for
11


85
and the ligand. This would indicate that in the aluminum
compound, the coordination of the ligand to the metal ion
produces a change in the double bond character that is
distinct from the perturbation caused by the presence of the
sodium near the carboxylate ion. For mpic, the ligand
stretching frequency is lower, probably due to the formation
of an acid dimer.52 However, the peak for IV is near those for
III and V, and, as such, has the ligand coordinated to the
aluminum ion. For VII, there are two peaks present. The peak
at 1666 cm"1 is in the region of the other aluminum compounds
and is, therefore, probably due to a coordinated carboxylate
group. The other peak is close to the one peak for the
disodium dipicolinate salt. The presence of two peaks could
indicate two differently reacting carboxylate groups in one
compound or two compounds with different carboxylate groups.
For the other aluminum compounds, it should be noted that,
despite the presence of more than one aluminum environment,
there is only one visible carbonyl stretching band. Either,
the ligand is coordinated by the same strength bond in every
compound in the mixture or the coordination is so similar that
there is not a distinguishable difference in the carbonyl
stretching frequency.


38
stimulate the appearance of iNOS immunoreactivity in MCT
cells most likely represents failure of this cytokine to
induce the synthesis of iNOS protein.
Effects of Lioopolysaccharide and Interferon-v on Inducible
Nitric Oxide Svnthase mRNA
To investigate whether iNOS induction requires an
increase in steady-state mRNA levels, total RNA extraction
was done in cells incubated with LPS, IFN or LPS plus IFN in
the presence or absence of actinomycin D, cycloheximide and
dexamethasone for 4 h and a northern blot analysis done on
the extracts.
No iNOS mRNA was detected in Northern blots of controls
or IFN-treated cells (figure 2-9). However, LPS-treated
cells showed a significant increase in steady-state mRNA
levels first observed after 2 h and persisting for at least
12 h (data not shown). This was markedly potentiated by the
addition of IFN, blocked completely by actinomycin D and to
some extent by dexamethasone. Increases in steady-state iNOS
mRNA levels induced by LPS and LPS/IFN were also blocked by
coincubation with cycloheximide (data not shown). These
results suggest that there is a time-dependent increase in
steady-state iNOS mRNA levels with LPS treatment, and that
IFN, although ineffective by itself, acts in a synergistic
manner to enhance the increase in steady-state iNOS mRNA
levels induced by LPS. This synergism appears to require
protein synthesis. The inhibition of the increased steady-


35
Svneraistic Effect of Lioopolvsaccharide and Interferon-y
I next examined the interaction between LPS and IFN on
the induction of iNOS activity. Murine proximal tubule cells
were incubated with LPS at concentrations of 0.01, 0.1, 1.0,
and 10 |ig/ml either alone or in combination with 1.0 or 10
U/ml IFN, or IFN at concentrations of 1, 10, 50 and 100 U/ml
alone, or in combination with 0.1 pg/ml LPS for 24 h.
Inducible NOS activity was determined following the
conversion of [3H]L-arginine to [3H]L-citrulline over 30 min.
Lipopolysaccharide caused only a small increase in iNOS
activity. At 10 ng/ml (the highest concentration studied),
LPS caused a 44% increase in iNOS activity compared to
control (figure 2-5B). However, the effect of LPS was
greatly potentiated by the addition of IFN, for example, 1
and 10 U/ml IFN potentiated the effect of 10 |j.g/ml LPS on
iNOS activity, increasing this from 44% to 176% and 545% over
control, respectively. In contrast, IFN alone did not cause
any significant induction of iNOS activity compared to
control (figure 2-5A) However, combined with 0.1 |Ltg/ml LPS,
IFN caused a marked concentration-dependent increase in iNOS
activity (7.6-fold increase over control), which peaked at an
IFN concentration of 50 U/ml. Thus, whereas LPS and IFN by
themselves caused little or no induction of iNOS activity in
MCT cells, they synergized to produce a marked induction of
iNOS activity in these cells.


98
Transfection of the Plasmid DNA into Murine Proxi:: al Tubule
Cells and Chloramphenicol Acetvl Transferase Assays
The MCT cells were transfected by a modification of the
DEAE-dextran method (Golub et al. 1989). Briefly, the cells
were seeded the day before transfection such that they were
about 60% confluent the next day. After cells were washed
twice with PBS, trypsinized and resuspended in RPMI with 10%
Nuserum, 10 pig of plasmid DNA was added per 2 X 106 cells per
1 ml of prewarmed RPMI ( (37 C, 10% Nuserum) containing
DEAE-dextran (100 pig/ml) and 50 mM Tris (pH 7.4) The
suspension was incubated at 37 C for 60 min followed by a 3
min shock with 10% DMSO at room temperature. The cells were
washed and distributed to 100-mm plates each with about 5 x
10 cells in 10 ml of complete medium, and incubated at 37 C
in 5% CO2. After 24 h, the medium was changed and inducers
added at the desired concentrations and incubated for another
24 h. The cells were then washed with ice-cold PBS,
resuspended in 0.25 mM Tris-HCl (pH 8), and subjected to
three cycles of freezing and thawing. The lysates were
centrifuged (11700 g for 10 min at 4 C) and the supernatant
heated at 60 C for 10 min to inactivate CAT inhibitors and
then centrifuged again as above. The supernatant was then
assayed for CAT activity by the thin layer chromatography
(TLC) method (Golub et al., 1989). Briefly, the assay
mixture contained (final vol 125 (1) 100 pig of cell extract
(in 50 pil) 69 pil of 0.25 mM Tris (pH 8), 3 |il of [14C]~
chloramphenicol (50 piCi/mmol) and 5 pi of 5 mg/ml n-butyryl


92
grown to the size suitable for x-ray diffraction studies.
Large crystals and the remaining solid was pasteured from the
agar, washed with water and dried for further analysis.
Elemental Analysis
The percentages of carbon, hydrogen and nitrogen in
compound VIII are listed in Table 6-1 with the calculated
values for the compounds Al (C7H3N04) (C7H4N04) 3H20 and
Na(C7H4N04) (C7H5N04) 3H20. Although the agreement is reasonable
Table 6-1. Elemental Analysis of VIII.
Na compound
VIII
Al compound
% c
40.99
40.92
40.79
% H
3.69
3.58
3.18
% N
6.83
6.69
6.79
for either set of data, the error for carbon and hydrogen is
one-half and one-fourth, respectively, for the sodium
compound. However, the percentage of nitrogen present is
slightly closer for the aluminum compound. Some of the error
in the percentages can be traced to the agar medium in which
VIII was grown. Although the crystals were pasteured out of
the agar and then washed, trace amounts of the cellulose gel
could affect the elemental analysis results.
X-rav Crystallographic Analysis
A clear colorless crystal measuring 0.34 x 0.26 x 0.13
mm was mounted on a glass fiber for diffraction studies. All
subsequent data were collected at room temperature on a
Siemans R3m/V diffractometer equipped with a graphite


131
Tojo, A., Guzman N.J., Garg, L.C., Tisher, C.C. and Masden,
K.M. Nitric oxide inhibits bafilomycin-sensitive H+-ATPase
activity in rat cortical collecting duct. Am. J. Physiol.
267: F509-F515, 1994 [b].
Toutain, H. Renal proximal tubule cell cultures for studying
drug-induced nephrotoxicity and modulation of phenotype
expression by medium components. Renal Failure 14(3): 371-
383, 1992.
Vincendeau, P. and Daulouede, S. Macrophage cytostatic
effect on Trypanosoma musculi involves an L-arginine-
dependent mechanism. J. Immunol. 146: 4338-4343, 1991.
Vincenti, M.P., Burrel, T.A. and Taffet, S.M. Regulation of
NF-kB activity in murine macrophages: effect of bacterial
lipopolysaccharide and phorbol ester. J. Cell Physiol. 150:
204-213, 1992.
Vodovotz, Y., Bogdan, D. Paik, J. Xie, Q-w. and Nathan, C.
Mechanisms of suppression of macrophage nitric oxide release
by transforming growth factor p. J. Exp. Med. 178: 605-613,
1993.
Wang, J., Stuehr, D.J., Ikeda-Saito, M. and Rousseau, D.L.
Heme coordination and structure of the catalytic site in
nitric oxide synthase. J. Biol. Chem. 268: 22255-22258, 1993.
Weinberg, J.B., Granger, D.L., Pisetsky, D.S., Seldin, M.F.,
Misukonis, M.A., Mason, S.N., Pippen, A.M., Ruiz, P. Wood,
E.R., and Gilkeson, G.S. The role of nitric oxide in the
pathogenesis of spontaneous murine autoimmune disease:
increased nitric oxide production and nitric oxide synthase
expression in MRL-lpr/lpr mice, and reduction of spontaneous
glomerulonephritis and arthritis by orally administered NG-
monomethyl-L-arginine. J. Exp. Med. 179: 651-660, 1994.
Werner, E.R., Werner-Felmayer, G. Fuchs, D., Hausen, A.,
Reibnegger, G. and Wachter, H. Parallel induction of
tetrahydrobiopterin biosynthesis and indoleamine 2,3-
dioxygenase activity in human cells and cell lines by
interferon-y Biochem. J. 262: 861-866, 1989.
Werner, E.R., Werner-Felmayer, G. and Wachter, H.
Tetrahydrobiopterin and cytokines. Proc. Soc. Exp. Biol. Med.
203: 1-12, 1993.
Werner-Felmayer, G., Werner, E.R., Fuchs, D., Hausen, A.,
Reibnegger, G. Schmidt, K., Weiss, G. and Wachter, H.
Pteridine biosynthesis in human endothelial cells. Impact on
cycle oxide-mediated formation of cyclic GMP. J. Biol. Chem.
268: 1842-1846, 1993.


38
product because the sodium salts of pic and pza produced the
same results when heated to 100C in DMSO. In addition, no
solid remained after the evaporation of the heated DMSO
reaction mixture.
Aluminum Isopropoxide
Theory
Used in the Meerwein-Ponndorf-Verley reduction, aluminum
isopropoxide, Al(OC3H7)3, could provide an alternative aluminum
reagent for a synthesis in a non-aqueous environment.63 In
theory, in the presence of a ligand with a hydroxyl group, one
of the isopropoxide groups could exchange with the ligand to
form free isopropyl alcohol. Removal of the isopropanol by
evaporation or distillation should drive the reaction towards
completion. This would produce a coordination compound
synthesized in the absence of water.
Experimental
In a glove bag flushed with nitrogen gas, aluminum
isopropoxide (0.80 g, 3.9 mmole) was transferred to a dried
weighing bottle. Pyrazinoic acid (0.52 g, 4.2 mmole) was
added as a solid. Isopropyl alcohol was dried over anhydrous
CaS04 and 20 mL was added to the solids. The bottle was
capped and transferred to a desiccator. After five days, a
white solid, which had formed on the bottom of the bottle, was
collected by filtration. Comparable experiments were


5-2 Al-27 NMR Peak Data for Compounds III
through VII 72
5-3 Al-27 NMR Data for Solutions Containing
0.30 M Al3+ and the Tabulated
Concentration of pza at pH 5 78
5-4 Elemental Analysis Data for Compounds
III through VII and Pure Tris Complexes
of the Ligands 83
5-5 FTIR Carbonyl Peak Data 84
5-6 FABMS Peak Data 87
6-1 Elemental Analysis of VIII 92
6-2 Crystallographic Data for Compound VIII... 95
6-3 Final Atomic Coordinates and Isotropic
Thermal Parameters for the Non-Hydrogen
Atoms of Compound VIII 97
6-4 Final Atomic Coordinates and Isotropic
Thermal Parameters for the Hydrogen
Atoms of Compound VIII 98
6-5 Bond Lengths of all Atoms of Compound
VIII 98
6-6 Bond Angles for all Atoms of Compound
VIII 99
6-7 Anisotropic Thermal Parameters for the
Non-Hydrogen Atoms of Compound VIII 100
7-1 Crystallographic Data for IX 106
7-2 Final Atomic Coordinates and Isotropic
Thermal Parameters for IX 109
7-3 Hydrogen Atom Coordinates and Isotropic
Thermal Parameters for IX 110
7-4 Bond Lengths for Non-Hydrogen Atoms in
IX 110
7-5 Bond Lengths involving Hydrogen Atoms in
IX Ill
7-6 Bond Angles in IX Ill
vi


71
specific concentrations and for specific incubation periods.
The cells were then harvested by scraping in ice-cold PBS and
centrifuged at 500 g for 10 min and homogenized in two packed
cell volumes (PCV) of hypotonic buffer (Tris-HCl 10 mM, pH
7.4, KCl 10 mM, MgCl2 3 mM, Dithiothreitol (DTT) 1 mM, PMSF
0.5 mM, leupeptin 2 nM) using a glass tissue grinder with a
Teflon pestle. The homogenates were centrifuged at 500 g for
10 min to pellet the nuclei. The nuclear pellet was
resuspended in 3 ml/109 cells of extraction buffer (Tris-HCl
10 mM, pH 7.4, KCl 0.3 M, MgCl2 3 mM, DTT 1 mM, PMSF 0.5 mM,
leupeptin 2 pM, glycerol 20% v/v) and kept on ice for 45 min,
with gentle rocking. The extract was then clarified by
centrifugation at 25000 g for 30 min. The clear supernatant
was then dialyzed against 50 volumes of dialysis buffer
(Tris-HCl 10 mM, pH 7.4, KCl 0.1 M, DTT 1 mM, PMSF 0.5 mM,
leupeptin 2 |iM, EDTA 0.2 mM, glycerol 20% v/v) for 1 h. The
dialyzed solution was centrifuged at 25000 g, aliquoted and
stored at -70 C for subsequent use.
Oligonucleotide Probes and DNA Gel Mobility Assay
The NF-kB consensus DNA binding site is formed by the
general decameric motif 5'-GGGRNNTYCC, where R is a purine, Y
a pyrimidine and N either of them (Muller et al., 1993, Bours
et al. 1992). I utilized a double-stranded consensus
oligonucleotide containing the sequence 5'-GGGACTTTCC and
radio-labeled by phosphorylation with [y-32P]ATP (3000


BIOGRAPHICAL SKETCH
The author was born and raised in Sunyani in the Brong
Ahafo Region of Ghana. He obtained his ordinary level
education at Tema Secondary School, graduating in 1978. He
had his sixth form education at the Presbyterian Boys' Sixth
Form Science College where he took his advanced level
examinations in 1980, majoring in physics, chemistry and
mathematics. In 1987, he graduated from the University of
Ghana Medical School with an MB ChB. He had a brief stint of
elective training in General Surgery at the Chase Farm
Hospital in Middlessex, London, in 1987. Between 1988 and
1989, he did six months of internship each in the Departments
of Obstetric & Gynecology and Internal Medicine at the Korle-
Bu Teaching Hospital in Accra, Ghana, after which he was
seconded as a medical officer to the Department of Pathology,
University of Ghana Medical School. He proceeded to the
University of Florida in 1991 where he started his graduate
education under the guidance of Dr. Nicolas J. Guzman in the
Department of Pharmacology and Therapeutics.
133


21
effects of IFN and LPS on the activation of NF-kB in MCT
cells, and by investigating the effects of eliminating or
including NF-kB and IFN-activated protein binding sites from
the promoter region of the iNOS gene on IFN- and LPS-induced
iNOS expression using a specific promoter-reporter system. I
restricted my studies to evaluating the role of NF-kB and its
main subunits p50, p65 and c-Rel, and also, that of NF-KB and
IFN-activated protein binding sites in IFN- and LPS-induced
iNOS activity in renal epithelial cells. The specific aims
of my study were as described below.
Specific Aims
Specific Aim 1
Characterization of iNOS in MCT cells. I investigated
the time- and concentration-dependence of IFN- and LPS-
stimulated iNOS induction as measured by the rate of [3h]L-
arginine to [3H]L-citrulline conversion, and the accumulation
of nitrites in the culture media of cells treated with the
above agents.
Specific Aim 2
Investigation of the cofactor requirements of LPS- and
IFN-induced iNOS expression in MCT cells. I investigated BH4
requirements, by utilizing 2,4-diamino-6-hydroxypyrimidine
(DAHP), an inhibitor of guanosine triphosphate cyclohydrolase
I (GTPCH), the rate-limiting enzyme in the de novo synthesis


116
synthesis are critical for the induction and full expression
of iNOS activity in MCT cells. Unlike what is observed in
macrophages, in mouse proximal tubule epithelium, LPS fails
to cause the nuclear translocation of the c-Rel subunit of
NF-kB. This subunit of may thus be an important determinant
of iNOS tissue specificity. Because cytokine-induced NO
production may cause tubular cell injury, the development of
inhibitors of proximal tubule BH4 synthesis, NF-kB and IRF-1
activation could provide important pharmacological tools for
future therapy of inflammatory renal diseases.
%


106
very important in the regulation of iNOS gene expression in
renal tubule epithelium. Interference with the activation or
activity of NF-kB may therefore be beneficial in suppressing
iNOS-mediated renal pathophysiologies such as acute
glomerulonephritis, acute tubular ischemia/reperfusion injury
and renal transplant rejection (Weinberg et al., 1994, Yu et
al., 1994, Langrehr et al., 1993 [a,b]). The inhibition of
NF-kB activation may provide a pharmacological basis for
interfering with these acute processes.


72
Ci/mmol) using T4 polynucleotide kinase (Promega, Madison,
WI) following standard protocols (Sambrook et al., 1989).
Electrophoretic mobility shift assays (EMSA) were
performed following previously published protocols with minor
modifications. The binding reaction was carried out by
adding 10 p.g nuclear extract to gelshift binding 5X buffer
[Tris-HCl 10 mM, pH 7.5, MgCl2 1 mM, EDTA 0.5 mM, DTT 0.5 mM,
NaCl 50 mM, glycerol 4% and poly(dl-dC)-poly(dl-dC) 50 mg/ml]
containing [y-32p]ATP-labeled NF-kB oligonucleotide probe in a
total volume of 10 jxl, incubated at room temperature for 20
min and the reaction terminated by addition of 1 nl gel
loading 10X buffer (Tris-HCl 250 mM, pH 7.5, bromophenol blue
0.2%, xylene cyanol 0.2%, glycerol 40%). The reactions were
then ran on a 4% nondenaturing acrylamide gel preran for 30
min before loading, for approximately 3 h at 100 V. The gel
was wrapped in plastic and exposed to x-ray film overnight.
Individual NF-kB subunits were identified by the presence of
supershift or the disappearance of the original NF-KB
complexes in EMSA after addition of specific polyclonal IgG
antibodies against p50, p65 and c-Rel (Santa Cruz
Biotechnology, Santa Cruz, CA) to the binding reaction.
Western Analysis
Expression of iNOS protein was determined by Western
analysis using a monoclonal antibody against macrophage iNOS
(Transduction Laboratories, Lexington, KY) and following
standard protocols. Briefly, MCT cells were lysed in culture


24
aminopolycarboxylic acids reported that the chemical shifts
range from 36.5 to 41.2 ppm.57 For one of the acids, EDTA, the
aluminum ion was determined by x-ray diffraction techniques to
be in a distorted octahedral environment in K[Al(EDTA)]2H20
coordinated to two nitrogen atoms and four oxygen atoms.58
However, there have been a limited number of studies reported
Al-27 NMR for the A1N303 centers produced by the formation of
a tris complex with a N,0 bidentate ligand. The shifts for
the four similar complexes range from 8.1 to 11.4 ppm.27 These
complexes contained an O-C-C-C-N group instead of the O-C-C-N
group present in each of the ligands in this study and had
coordination spheres fairly close to the ideal octahedron.
TCP Emission Spectroscopy
ICP emission spectroscopy depends upon the emission of
light at a specified wavelength after the aluminum ion has
been excited in the plasma. The concentration of aluminum can
be determined by comparing it to a calibration curve. If the
mass of the complex per volume is accurately known for the
test solution, then the molecular weight of the complex can be
determined. All of the emission lines for aluminum are
extremely weak and a rather high detection limit exists.
Therefore, not only is a solution required but it must be
relatively concentrated. This technique was not feasible to
employ due to the very low solubility of the synthesized
complexes.


4-4 ORTEP Drawing of the Al(meox)3 Molecule
Showing the Atomic Numbering Scheme and
the Thermal Ellipsoids 50
4-5 ORTEP of A1(ox)3 Molecule Showing the
Numbering Scheme and Thermal Ellipsoids. 57
4-6 Al-27 NMR Spectrum of the Solid la 63
4-7 Al-27 NMR Spectrum of the Solid Ila 65
4-8 Al-27 NMR Spectrum of Al(ox)3 in C2H50H
Solution 65
5-1 Ligands in Table 5-1. Iminodiacetic acid;
Nitrilotriacetic acid; N-(Hydroxyethyl)-
ethylenediaminetriacetic acid; Ethylene-
diaminetetraacetic acid; 1,2-Propylene
diaminetetraacetic acid; trans-1.2-
diaminocyclohexanetetraacetic acid;
Diethylenetriaminepentaacetic acid;
Pyromeconic acid; Maltol; 3-Hydroxy-2-
methyl-4-pyridinone; 3-Hydroxy-1,2-
dimethyl-4-pyridinone 69
5-2 Al-27 NMR Spectra of III, Solid, in
d6 DMSO 73
5-3 Al-27 NMR Spectra of IV, Solid, in d6-DMSO. 74
5-4 Al-27 NMR Spectra of V, Solid, in d6-DMSO.. 75
5-5 Al-27 NMR Spectra of VI, Solid, in d6-DMSO. 76
5-6 Al-27 NMR Spectra of VII, Solid, in
d6-DMSO 77
5-7 Al-27 NMR Spectrum of an Aqueous
Solution Containing 0.30 M Al3+ at pH 5.. 79
5-8 Al-27 NMR Spectrum of an Aqueous
Solution Containing 0.30 M Al3+ and
0.30 M pza at pH 5 79
5-9 Al-27 NMR Spectrum of an Aqueous
Solution Containing 0.30 M Al3+ and
0.60 M pza at pH 5 80
5-10 Al-27 NMR Spectrum of an Aqueous
Solution Containing 0.30 M Al3+ and
0.90 M pza at pH 5 80
IX


CHAPTER FOUR
NUCLEAR FACTOR-KB REGULATES INDUCIBLE NITRIC OXIDE SYNTHASE
IN RENAL TUBULE EPITHELIUM
Introduction
Expression of iNOS is subject to strict and highly
tissue-specific transcriptional regulatory control (Nathan
and Xie, 1994 [a,b], Nussler et al. 1994 ). For example,
whereas LPS induces high levels of iNOS expression in
macrophages and microglia (Stuehr and Marietta, 1994, Xie et
al., 1993, Chandler et al. 1994), it induces only modest
levels of iNOS in hepatocytes, vascular smooth muscle and
renal epithelial cells and a combination of cytokines is
usually required for full expression of iNOS activity
(Nussler and Billiar, 1993, Szab et al., 1993, Amoah-Apraku
et al. 1993). Thus, the expression of iNOS appears to be
regulated differently in parenchymal cells compared to cells
such as macrophages and microglia whose main role is to
mediate immune responses.
Recent studies describe the presence in the murine
macrophage iNOS promoter of consensus sequences for the
binding of endotoxin and IFN-stimulated signaling proteins
including NF-kB and interferon-7 regulatory factor (IRF-1)
(Xie et al. 1993), which may potentially constitute the
basis for the synergism observed between iNOS inducers. It
68


120
Table 8-2. Final Atomic Coordinates (xlO4) and Isotropic
Thermal Parameters (2xl03) for X. Estimated Standard
Deviations are given in Parentheses.
Atom
X
y
z
V
Na
0.0891(2)
-0.2074 (2)
-0.5731 (2)
0.0264(5)
0(1)
0.5928(3)
-0.2861(3)
-0.1605 (3)
0.0255(8)
0(2)
0.5694(4)
-0.0356 (3)
-0.1102 (3)
0.0263(9)
0(3)
0.2106(4)
-0.0054 (3)
-0.1288 (3)
0.0239(8)
0(4)
0.2807(4)
-0.1668 (4)
-0.3660(3)
0.0266(9)
0(5)
-0.0846(4)
-0.2826 (3)
-0.1586 (2)
0.0239(8)
0(6)
-0.0888(4)
-0.1869 (3)
-0.3552 (3)
0.0280(8)
C(l)
0.4874 (5)
-0.1563 (4)
-0.1356(4)
0.017(1)
C (2)
0.2762(5)
-0.1578 (4)
-0.1363 (4)
0.016(1)
C (3)
0.2026(5)
-0.2365 (4)
-0.2561(3)
0.017(1)
C (4)
-0.0054(5)
-0.2319 (4)
-0.2625(4)
0.019(1)
H (1)
0.246(7)
-0.209(7)
-0.054(5)
0.03(1)
H (2)
0.236(8)
-0.370(7)
-0.260 (5)
0.05(2)
H (3)
-0.221(7)
-0.297 (6)
-0.166(5)
0.05(2)
0(7)
0.0417(4)
-0.5245 (3)
-0.0093(3)
0.0257(9)
*Ueq='AZljUijai*aj*ai. .
Table 8-3. Bond Lengths () and Angles () for X. Estimated
Standard Deviations are given in Parentheses.
(a) Bond distances (A) for the bitartrate ion.
0(1)-C(l)
1.283(5)
C(1)-C(2)
1.527(5)
0(2)-C(l)
1.230(5)
C (2) -C (3)
1.540(6)
0(3) -C (2)
1.404 (5)
C (3) -C (4)
1.508(6)
0(4) -C (3)
1.428(5)
C (2) -H (1)
1.00(5)
0(5) -C (4)
1.314(5)
C (3) -H (2)
1.18(6)
0(6) -C (4)
1.218(5)
0(5) -H (3)
1.00(5)
(b) Bond angles () for the bitartrate ion.
0(1)
-C
(1)
-C (2)
114 ,
.6(3)
C (2)
-C
(3)
-C (4)
111,
.8
(3)
0(2)
-C
(1)
-C (2)
119,
.4(3)
0(4)
-C
(3)
-C(4)
110,
.3
(3)
0(1)
-C
(1)
-0(2)
125,
.9(3)
0(6)
-C
(4)
-C (3)
122,
.7
(3)
C(l)
-C
(2)
-C (3)
110,
.9(3)
0(5)
-C
(4)
-0(6)
124,
.5
(3)
0(3)
-C
(2)
-C(l)
109,
.2(3)
0(5)
-C
(4)
-C (3)
112.
.8
(3)
0(3)
-C
(2)
-C (3)
110,
.4 (3)
C (4)
-0
(5)
-H (3)
Ill
(3
)
0(4)
-C
(3)
-C (2)
110,
.3(3)


11
Drapier and Hibbs, 1988). These reactions may result in
enzyme inactivation, cytotoxicity or cell death. The
tumoricidal effect of NO may be mediated this way and
activated macrophages utilize this as a non-specific immune
defense mechanism against bacterial, protozoal and possibly
viral infections. Nitric oxide also induces ADP-ribosylation
and inhibition of the glycolytic enzyme glyceraldehyde-3-
phosphate dehydrogenase (Brue et al., 1990).
The Nervous System
Nitric oxide differs very much from conventional
neurotransmitters in that it is able to diffuse freely from
the point of synthesis to intracellular target sites in
neighboring cells independent of vesicular release, membrane
receptors or lipid cell boundaries. Though normal NO
neuronal actions have not been fully delineated, cerebellar
NOS has been cloned and antibodies to cNOS-related antigens
have been used to identify discreet populations in the
retina, striatum, hypothalamus, midbrain, posterior pituitary
and forebrain of the rat, suggesting a role for NO in
neuronal responses (Bredt et al., 1990, Bredt et al., 1991).
It has also been implicated specifically in some forms of
synaptic plasticity such as long-term synaptic potentiation
(LTP) and long-term synaptic depression (LTD), both of which
are considered elements of experience-driven synaptic network
remodeling underlying learning and memory (Shibuki and Okada,


15
In the kidney, the proximal tubule is the major site of
L-arginine synthesis (Levillain et al. 1993). Also,
increased expression of cytokines occurs in many experimental
and human kidney diseases. Hence, the presence of constant
and abundant substrate coupled with the easy availability of
inducers make iNOS in the kidney a major factor in renal
pathophysiology. Thus, pathways regulating the renal L-
arginine:NO system such as the role of specific transcription
factors and cofactor availability, offer attractive
therapeutic targets.
The Endocrine System
The presence of NOS has been demonstrated in rat
pancreatic p islet cells (Laychock et al., 1991) and insulin
release in response to L-arginine is mediated by NO (Schmidt
et al. 1992). In mice, administration of streptozotocin
mimics diabetes mellitus, which is abolished by Nw-
substituted L-arginine analogues (Lukic et al. 1991),
suggesting that this effect may be mediated by NO. It is now
clear that immune-mediated pancreatic islet cell destruction
is attributable to IL-l-induced NO formation (Corbett et al.,
1991). Also, the generation of NO in response to L-arginine
or tolbutamide appears to mediate insulin release in
pancreatic p cells (Laychock et al. 1991, Schmidt et al.,
1992). Hence excessive production of NO or defective
responsiveness by NOS may play an important role in the
etiology of diabetes mellitus.


82
unreacted A1(H20) 63+. However, in the spectra for solutions
with the ligand present, peaks near 7, 17 and 28 ppm are
present. These are possibly due to species formed from the
hydrated aluminum ion being involved in a series of pH
dependent equilibria. These equations seem to represent a
Al3+ + Hpza ** Al (pza)2+ + H+ (1)
Al(pza)2+ + Hpza ** Al(pza)2+ + H+ (2)
Al(pza)2+ + H20 ** Al (pza) 2 (OH) + H+ (3)
Al(pza)2+ + Hpza Al(pza)3(s) + H+ (4)
reasonable set of chemical reactions possible in an aqueous
solution with a ligand such as pza. Once the tris complex is
formed, it precipitates and, as a result, only three peaks
other than the hexaaquaaluminum(III) ion should be present.
The series is similar to that proposed for aluminum and
picolinic acid, a ligand of similar reactivity.34 Further
tests or x-ray studies to confirm the structure of the
complexes.
Elemental Analysis
Each of the solid samples were submitted for elemental
analysis. The results are summarized in Table 5-4 as
percentages of carbon, hydrogen and nitrogen present. Also
included are mole ratios, scaled to the number of nitrogen
atoms per ligand molecule, and, at the bottom of the table,


126
Mundel, P., Bachmann, S., Bader, M., Fischer, A., Rummer, W. ,
Mayer, B. and Kriz, W. Expression of nitric oxide synthase
in kidney macula densa cells. Kidney Int. 42: 1017-1019,
1992.
Nakaki, T., Nakayama, M. and Rato, R. Inhibition by nitric
oxide and nitric oxide-producing vasodilators of DNA
synthesis in vascular smooth muscle cells. Eur. J. Pharmacol.
1898: 347-353, 1990.
Narumi, S., Tebo, J.M., Finke, J.H. and Hamilton, T.A.
Interferon-y and interleukin-2 cooperatively activate NF-
kappa B in murine peritoneal macrophages. J. Immunol. 149:
529-534, 1992.
Nathan, C. Nitric oxide and biopterin: A study in
chiaroscuro. J. Clin. Invest. 93: 1875-1876, 1994.
Nathan, C. and Xie, Q-w. Nitric oxide synthases: roles,
tolls and control. Cell 78: 915-918, 1994 [a].
Nathan, C. and Xie, Q-w. Regulation of biosynthesis of
nitric oxide. J. Biol. Chem. 269: 13725-13728, 1994 [b] .
Nishida, R. Harrison, D.G., Navas, J.P., Fisher, A.A.,
Dockery, S.P., Uematsu, M., Nerem, R.M., Alexander, R.W. and
Murphy, T.J. Molecular cloning and characterization of the
constitutive bovine aortic endothelial cell nitric oxide
synthase. J. Clin. Invest. 90: 2092-2096, 1992.
Noonan, N.E. and Noonan, R.D. The effect of TLCR on
transcription and its role in modifying cell growth. J. Cell
Physiol. 92: 137-144, 1977.
Nowak, R. Corners of the mind: the cellular basis of memory
and learning. J. NIH Res. 4: 49-55, 1992.
Nussler, A.R. Nonparenchymal cell and hepatocyte interaction
in antimalarial activity: a central role for nitric oxide.
In: Heoatocvte and Ruoffer Cell Interactions, edited by T.R.
Billiar and R.D. Curran, Boca Raton, FL: CRC, 1992.
Nussler, A.R. and Billiar, T.R. Inflammation,
immunoregulation and inducible nitric oxide synthase. J.
Leukoc Biol. 54: 171-178, 1993.
Nussler, A.R., Drapier, J.C., Renia, L., Pied, S., Miltgen,
F., Gentilini, M. and Mazier, D. L-Arginine-dependent
destruction of intrahepatic malaria parasites in response to
tumor necrosis factor and/or interleukin-6 stimulation. Eur.
J. Immunol. 21: 221-230, 1991.


107
Hind III
-15 8 8 ^GACTTTGATATGCTGAAATCCATAAGCTGTGTGTGTGTGCAAGTGTGCA
-15 3 9 TGCGCATGTGTGCACATGAGTGTGCAGGTATATGTAGGAGCTAGAAGACAATCTCAGCTC
-14 7 9 TTGTTTCCCAGGTTACCCAGCATCTCTCACCAGCCTGGAACCTGCCTAGTAGGCTAGGCT
-1419 GGCTGGCCAGCAAACCCTAGGCATATTCCTGTCTTTACCTCCCCAGAACTTATTGCAAAG
-1359 GTGTGTCACCACACCCAGCATTTTATCATTGACCTATTGACTGGTGTGTGTGTGTGTGTG
-12 9 8 TGTGTTTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTAAATT
-1239 CCTTATCTCACCAACCCATGCCCAGCTTTTGAACTTAGGTCCTTGTACATGCAAGGCAAG
-1179 CACTTTACCAACTGAGCCATCTCCCCAGCCCAATTACTTGATTTGTAATTCATTTATTCA
-1119 ATCAACAATTTATTTGTTCTCCCAACTATTGAGGCCACACACTTTTTGGGTGACTTAGTC
-10 5 9 TGTGTACCTCAGACAAGGGCAAAACACGAGGCTGAGCTGACTTTGGGGACCATGCGAAGA
NF-KB
- 999 TGAGTGGACCCTGGCAGGATGTGCTAGGGGGATTTTCCCTCTCTCTGTTTGTTCCTTTTC
IRF-E
- 9 3 9 CCCTAACACTGTCAATATTTCACTTTCATAATGGAAAATTCCATGCCATGTGTGAATGCT
-879 TTATTGGAAGCATTGTAAGAAATTATAATTTATTCGTTTTTGTTTGTTTCTCAGAACAGG
-819 GTTTTTCTGTGTAGTGTTCCTGGCTTATCCTAGAACTTACTCTGTAGACCAGGCTAGCCC
-759 AAACTCAGGGATCAGCCTTTCTCTGTCTCCTGAATCCCGGGATTAAAGGCTTATGCCACC
- 69 9 ACACCCAGGTAGGACATTATAATCCTATATATAAGAAGTCACCC AC AC ATACAAACAC AC
- 6 3 9 ACACACCACACACACACACACAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA
-579 GAGGGAGAGAGAGAGAGAGAGAGAGAATGCCACTGAGAAAAAAAATAAAAAGGCTTCACT
Spe^I
-519 CAGCACAGCCCATCCACTATTCTGCCCAAGCTGACTTACTACTAGTGGGGAAATGCTGGT
Xba I
4 5 9 CAGACGGCATCTGTGCCCACAGCTTGCCTTCCATCCTTTCTAGAAAACCTCCTGATGAAT
-3 9 9 GTGTCCTGGGCGTGTTGGAATATTGGCACCATCTAACCTCACTGAGAGAACAGACAGAAA
- 3 3 9 GCCAGAGAGCTCCGTGCCCAGAACAAAATCCCTCAGCAGCTGCAAGCCAGGGTATGTGGT
- 2 7 9 TTAGCTAAGAAAAGCCAGCCTCCCTCCCTAGTGAGTCCCAGTTTTGAAGTGACTACGTGC
-219 TGCCTAGGGGCCACTGCCTTGGACGGACGACCAGGAAGAGATGGCCTTGCATGAGGATAC
-15 9 ACCACAGAGTGATGTAATCAAGCACACAGACTAGGAGTGTCCATCATGAATGAGCTAACT
NF-KB
-9 9 TGCACACCCAACTGGGGACTCTCCCTTTGGGAACAGTTATGCAAAATAGCTCTGCAGAGC
-39 CTGGAGGGi
gJtat^aa'
+1r*
TACGTGATGGCTGCTGCCAGGGTCACAACTTTACAGGGAGTTGAA
+22 GAATGAGACTCTGGCCCCACGGGACACAGTGTCACTGGTTTGAAACTTCTCAGCCACCTT
Sal I
+82 GGTGAAGGGACTGAGCTGTTAGAGACACTTCTGAGGCTCCTCACGCTTGGGTCTTGTTCA
$
Figure 5-1. The 1.7 kilobase pair 5'-flanking region of iNOS
cloned by PCR from mouse genomic DNA. Sequences are
designated that are homologous to the consensus sequences for
binding of NF-kB and IRF-1 (IRF-E). Also designated are the
restriction sites utilized in the preparation of the
constructs shown in figure 5-2A. The mRNA initiation site (+
1), TATA sequence (boxed).


118
Bogle, R.G., Baydoun, A.R., Pearson, J.D., Moneada, S. and
Mann, G.E. L-arginine transport is increased in macrophages
generating nitric oxide. Biochem. J. 284: 15-18, 1992.
Bhme, G.A., Bon, C., Stutzmann, J.M., Doble, A. and
Blanchard, J.C. Possible involvement of nitric oxide in long
term potentiation. Eur. J. Pharmacol. 199: 379-381, 1991.
Bhme, E., Graf, H. and Schultz, G. Effects of sodium
nitroprusside and other smooth muscle relaxants on cyclic
GMP formation on smooth muscle and platelets. Adv. Cycl.
Nucl. Res. 9: 131-143, 1978.
Boulanger, C. and Luscher, T.F. Release of endothelin from
the porcine aorta. Inhibition by endothelium-derived nitric
oxide. J. Clin. Invest. 85: 587-590, 1990.
Bours, V. Franzoso, G. Brown, K. Park, S., Azarenko, V. ,
Tomita-Yamaguchi, M., Kelly, K. and Siebenlist, U.
Lymphocyte activation and the family of NF-kB transcription
factor complexes. Current Topics Microbiol. Immunol. 182:
411-420, 1992.
Bradford, M.M. A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal. Biochem. 72: 248,
1976.
Bredt, D.S., Ferris, C.D., Snyder, S.H. Nitric oxide
synthase regulatory sites, phosphorylation by cyclic AMP-
dependent protein kinase, protein kinase C, and
calcium/calmodulin protein kinase: identification of flavin
and calmodulin binding sites. J. Biol Chem. 267: 10976-10981,
1992.
Bredt, D.S., Hwang, P.M., Glatt, C.E., Lowenstein, C., Reed,
R.R. and Snyder, S.H. Cloned and expressed nitric oxide
synthase structurally resembles cytochrome P-450. Nature 351:
741-748, 1991.
Bredt, D.S., Hwang, P.M. and Snyder, S.H. Localization of
nitric oxide synthase indicating a neural role for nitric
oxide. Nature 347: 768-770, 1990.
Bredt, D.S. and Snyder, S.H. Isolation of nitric oxide
synthetase, a calmodulin-requiring enzyme. Proc. Natl. Acad.
Sci. USA 87: 682-685, 1990.
Brezis, M. Heyman, S.N., Dinour, D., Epstein, F.H. and
Rosen, S. Role of nitric oxide in renal medullary
oxygenation. Studies in isolated and intact rat kidneys. J.
Clin. Invest. 88: 390-395, 1991.


29
ml of water and the eluant, whose sole radioactive component
was [3h]L-citrulline as
determined by
thin
layer
chromatography, collected
into
vials.
Ten
ml
of
scintillation cocktail was
added
to each
vial
and
the
radioactivity quantified by liquid scintillation spectroscopy
(Beckman LS 6000SC). Inducible NOS activity was expressed as
[3h]L-citrulline DPMs per jog of protein. For experiments in
which iNOS activity was measured in crude cell homogenates,
these were obtained from MCT cells grown to confluence in 100
mm dishes. The MCT cells were suspended in ice-cold 50 mM
Tris-HCl containing 0.1% 2-mercaptoethanol, 0.1 mM EGTA and
0.1 mM EDTA, and homogenized on ice by using a glass tissue
grinder with a Teflon pestle in the presence of 1 mM
phenylmethylsulfonyl fluoride (PMSF), 3 pM leupeptin and 1 ^M
pepstatin A as previously described (Davda et al. 1993).
Aliquots of homogenates containing 100-200 |ag of protein were
added to the assay buffer devoid of Ca2+ and iNOS activity
was measured as described above in the presence of 1 mM
NADPH, 4 |1M flavin-adenine dinucleotide (FAD) 10 |iM BH4 and 3
HCi/ml of [3h]L-arginine.
Nitrite Assay
Nitrites were measured by the addition of 200 nl of
freshly prepared Greiss reagent [0.75% sulfanilamide in 0.5 N
HC1 and 0.075% N-(1-naphthyl)ethylenediamine dihydrochloride
in double distilled water] to 200 |il of the culture medium
from treated and control cells, incubated with shaking at 25


50
BASAL IFN
LPS/IFN
LPS/IFN
+
NNA
Figure 2-3. Effects of LPS (0.1 ng/ml) and IFN (100 U/ml) on
rat primary culture proximal tubules. Interferon-y (100
U/ml) induces very little nitrite production. Addition of
LPS (0.1 |ig/ml) markedly potentiates the accumulation of
nitrites, which is inhibited by the addition of NNA (300 jiM) ,
(p < 0.05, mg: mg of cell protein, n=3).


3
Aluminum is the most abundant metallic element in the
earth's crust, but it is generally not available for uptake
into the biosphere. Under atmospheric conditions, elemental
aluminum can easily be oxidized to the trivalent aluminum ion,
that primarily exists as water insoluble mixed oxides and
silicates.10 When exposed to acidic conditions, such as in
acid rain, the oxides dissolve to yield a solution of
A1(H20) 63+ ions and aluminum ions coordinated to naturally
occurring ligands.11'12 As a mobile element, aluminum is
available for uptake by trees and aquatic organisms after it
has drained into lakes and rivers. As a result, increased
levels of aluminum have been implicated in the deforestation
and death of lakes commonly associated with acid rain.11,13
Because of the similar size and charges, aluminum could
compete with calcium, silicon and iron and interfere with
normal biological functions.3'14 In consideration of these
facts, an understanding of the reactivity and stability of
aluminum and its compounds warrants a thorough investigation.
Coordination Chemistry of Aluminum
In the presence of ligands other than water, the metal
ion can undergo exchange and loss of the water molecules.15
The factors controlling the coordination to a new ligand
include the formation of rings due to chelation, the geometry
around the metal center and the type of atom bonded to the
aluminum(III) ion.


43
steric strain and interfere with the nitrogen coordination to
a metal ion. The only complex involving aluminum(III) and 2-
methyloxine that has been characterized by x-ray
crystallography seems to reinforce the steric argument. As
C29
Figure 4-3. PLUTO Line Drawing of the /x-Oxo-di (bis
me thyloxinato) aluminum (I II) Complex. Kushi and Fernando,
Journal of the American Chemical Society, 1970, 92, 91-95.


83
regulation of iNOS expression should allow us
potential targets for future selective
intervention.
to identify
therapeutic


132
Werner-Felmayer, G., Werner, E.R., Fuchs, D., Hausen, A.,
Reibnegger, G. and Wachter, H. Tetrahydrobiopterin-dependent
formation of nitrite and nitrate in murine fibroblasts. J.
Exp. Med. 172: 1599-1607, 1990.
White, K.A. and Marietta, M.A. Nitric oxide synthase is a
cytochrome P-450 type hemoprotein. Biochemistry 31: 6627-
6631, 1992.
Wilcox, C.S., Schmidt, H.H.H.W., Murad, F., Gross, S.S.,
Taylor, G. Levi, R. and Welch, W.J. NO synthase in macula
densa regulates glomerular capillary pressure. Proc. Natl.
Acad. Sci. USA 89: 11993-11997, 1992.
Wu, G. and Brosnan, J.T. Macrophages can convert citrulline
into arginine. Biochem. J. 281: 45-48, 1992.
Xie, Q., Cho, H.J., Calaycay, J., Mumford, R.A., Swiderek,
K.M., Lee, T.T., Ding, A., Troso, T. and Nathan, C. Cloning
and characterization of inducible nitric oxide synthase from
mouse macrophage. Science 256: 225-228, 1992.
Xie, Q., Kashiwabara, Y. and Nathan, C. Role of
transcription factor NF-KB/Rel in the induction of nitric
oxide synthase. J. Biol. Chem. 269: 4705-4708, 1994.
Xie, Q. and Nathan, C. The high output nitric oxide pathway:
role and regulation. J. Leuk. Biol. 56: 576-582, 1994.
Xie, Q., Whisnant, R. and Nathan, C. Promoter of the mouse
gene encoding calcium-independent nitric oxide synthase
confers inducibility by interferon-y and bacterial
lipopolysaccharide. J Exp. Med. Ill: 1779-1784, 1993.
Yu, L., Gengaro, P.E., Niederberger, M. Burke, T.J. and
Schrier, R.W. Nitrite oxide a mediator in rat tubular
hypoxia/reoxygenation injury. Proc. Natl. Acad. Sci. USA 91:
1691-1695, 1994.
Zatz, R. and de Nucci, G. Effects of acute nitric oxide
inhibition on rat glomerular microcirculation. Am. J.
Physiol. 261: F360-F363, 1991.
Ziegler, I., Schott, K., Lbbert, M.F., Schwulra, U. and
Bacher, A. Control of tetrahydrobiopterin synthesis in T-
lymphocytes by synergistic action of interferon-y and
interleukin-2. J. Biol. Chem. 265: 17026-17030, 1990.
Zweier, J.L., Kuppasamy, P. and Lutty, G.A. Measurement of
endothelial cell free radical generation: evidence for a
central mechanism of free radical injury in post ischemic
tissues. Proc. Natl. Acad. Sci. USA 85: 4046-4056 1988.


89
A
PDTC
18s-
n
U
tuj
PDTC
- +
139 kD-
Figure 4-6. Effect of PDTC (25 |J.M) on (A) iNOS mRNA
expression and (B) iNOS protein levels in MCT cells treated
with a combination of LPS (1 jig/ml) and IFN (100 U/ml).
Cells were incubated with PDTC for 30 min before addition of
LPS and IFN and maintained during the entire period.
Inducible NOS mRNA and protein expression were measured at 6
h and 12 h, respectively. Blot is representative of three
separate experiments.


64
Figure 3-2. Effect of DAHP on nitrite production by MCT
cells; (L/I: LPS plus IFN). Data are mean + S.E. (* p < 0.05
vs L/I-treated, mg: mg of cell protein, n=9).


16
The Immune System
That the cytotoxic effects of macrophages are due to
their ability to generate NO is well documented. These
include inhibition of mitochondrial respiration and DNA
synthesis mediated through NO-dependent inhibition of Fe-
containing enzymes (Dimmeler et al. 1992, Stuehr et al. ,
1989, Drapier and Hibbs, 1988). Macrophage NOS is the
inducible isoform, usually requiring cytokines or bacterial
products to stimulate its activity. In mammals, a
correlation between immunostimulation and elevated nitrate
and nitrite levels has been demonstrated (Stuehr et al. ,
1985) and the L-arginine-NO pathway has been proposed to be
the primary nonspecific defense mechanism against
intracellular microorganisms and other pathogens such as
viruses, fungi, helminths and protozoa (Karupiah et al.,
1993, Alspaugh and Granger, 1991, Vincendeau and Daulouede,
1991, Nussler, 1992).
Expression of iNOS is an inflammatory response to
infection or tissue injury that could be adaptive and
beneficial with respect to its antimicrobial action and
increased tissue perfusion, but it could also be detrimental,
such as occurs in autoimmune diseases or the marked
vasodilation seen in septic shock. Hence, the inhibition by
glucocorticoids of iNOS induction may explain, at least in
part, the therapeutic actions of these compounds in
conditions such as treatment of septic shock, asthma or


89
crystallography, the structures of the aluminum coordination
compounds synthesized for this work are virtually impossible
to elucidate.


102
Two hydrogen atoms are located on 08, 09 and 010 at bond
lengths between 0.71(4) and 0.90(4) The H-O-H bond angles
created are 99(4), 106(5) and 101(3), respectively. Because
these bond angles and lengths are within normal values, it
seems reasonable that 08, 09 and 010 are water molecules and
not hydroxide ions. In addition, it should be noted that the
geometry around 010 is distorted tetrahedral if HI' and Na are
included. The bond angles around 010 range from 101(3) to
132(2) .
In addition, there are hydrogen bonds that involve the
water molecules. HI' is hydrogen bonded to 010 by a distance
of 1.743 to create an overall 01'-HI'010 bond length of
2.601 at an angle of 175. 07'a in an adjacent molecule
(symmetry operator x, 1+y, z) is hydrogen bonded to HlOa at a
distance of 1.788 to form an angle of 174 with 010. The
symmetry related molecule at x, 2+y, z has a hydrogen bond
from 07a to H8a in the original molecule. The 07a--*H8a
interaction has a 1.961 bond length and creates an overall
2.842 hydrogen bond with an angle of 167 for 08-**07a.
Although a crystal of the desired aluminum compound was
not synthesized, an intriguing seven coordinate complex sodium
salt has been characterized. An unusual mode of coordination
is documented by the bonding of the protonated hydroxyl oxygen
atom the central metal ion. Also, no crystal structures, for
either simple or complex salts, have been reported for dipic
with alkali metal ions.


77
The combination of c-Rel and p65 IgGs resulted in an almost
total disappearance of the LPS-induced NF-KB complex in these
cells (data not shown) The formation of p65/c-Rel
heterodimers has been reported to be important in mediating
the transactivation of iNOS in macrophages (Xie et al.,
1994). Treatment of MCT cells with LPS resulted in
activation of NF-kB complexes containing p65 and p50 proteins
(figure 4-8B). The activation of p65 appeared to be greater
in MCT cells than in RAW 264.7 macrophages. However,
contrary to the observation in macrophages, c-Rel activation
was not observed in renal epithelium. The addition of c-Rel
IgG to either p50 or p65 IgG failed to affect the respective
NF-kB complexes (data not shown). These results indicate
that the inability of LPS to induce iNOS activity in MCT
cells may be associated with the absence of c-Rel activation
by this agent, suggesting that c-Rel may be an important
determinant of tissue specificity of LPS-mediated iNOS
responses.
To determine whether the synergism between LPS and IFN
in iNOS induction could be related to interactions between
different NF-kB proteins activated selectively by each of
these agents, I investigated the effects of IFN alone and
combined LPS and IFN treatment on the activation of NF-kB
subunits in MCT cells. The profile of NF-kB was exactly the
same as that observed after LPS treatment alone, and is
characterized by predominant activation of p65, smaller
amounts of p50 and the absence of c-Rel (figure 4-9). Thus,


22
By determining the percent of carbon, hydrogen and
nitrogen present, the total percent of the other elements,
oxygen, aluminum and possibly sodium, can be calculated.
Since each ligand has a specific number of oxygen atoms, the
corresponding percentage of ligand oxygen can be calculated
and then subtracted to yield the percentage accounting for the
metal ion and the oxygen present as solvent molecules. The
oxygen associated with water can not be accurately determined
because of the uncertainty in the amount of hydrogen present.
In addition, because the substitution of a sodium and two
hydrogen atoms for an aluminum would only produce an error of
2 g/mole, is difficult to prove of disprove the presence of
aluminum, as opposed to sodium, based on elemental analysis
data.
Infra-red Spectroscopy
IR spectroscopy provides information about the strength,
and therefore the length, of a bond. The C=0 group in a
carboxylic acid has a very strong stretch in the region 1760
to 1700 cm'1.52 If the acid is deprotonated to form a
carboxylate anion, the carbonyl bond loses some of its double
bond character due to the delocalization of the electron pair
over both of the carboxylate oxygen atoms. As a result, the
carbonyl bond strength decreases as the bond length increases.
The stretching frequency of a longer bond is slower such that
the stretching band shifts to a range of 1650 to 1550 cm'1.52
If a complex is formed involving the carboxylate group, the


88
IFN
LPS
PDTC
+ +
+ +
+
Figure 4-5. Effect of PDTC (25 (0.M) on the nuclear
translocation of NF-kB in MCT cells treated with LPS (1
jig/ml), IFN (100 U/ml), or their combination for 30 min. The
PDTC treatment was initiated 3 0 min before LPS and IFN were
added and maintained during the entire induction period.
Blot is representative of five separate experiments.


46
for la. 69.36 % C, 5.04 % H, 8.09 % N for Al (meox) 3* H20.
Submitted for Al-27 NMR analysis.
Preparation of Al(ox), (Ila). Oxine (4.51 g, 31.1 mmole)
dissolved in 50 mL 100% C2H5OH to give dark amber solution.
Al (N03) 3* 9H20 (3.81 g, 10.2 mmole) was added to mixture with
stirring and a thick yellow precipitate formed. NaOH solution
was added until reaction cleared and then reformed a
precipitate. The bright yellow solid was collected by
filtration, washed thoroughly with H20 and C2H5OH, and dried.
Elemental analysis: 68.02 % C, 4.62 % H, 8.06 % N for Ila.
68.91 % C, 4.79 % H, 8.31 % N for Al (ox) 3 C2H50H. Submitted
for Al-27 NMR analysis.
Preparation of saturated Al (ox)CLHcOH solution (lib) .
Ila (0.5 g, 1 mmole) was added to 350 mL 100% C2HsOH and
stirred at room temperature for 1 hour. A bright yellow
solution was collected by filtration through a glass frit and
evaporated slowly to yield 50 mL of a dark orange solution.
Recrystallization of a small fraction yielded exclusively one
crystal form of fluorescent yellow crystals, confirmed by X-
ray crystallography to be Al (ox) 3-C2H5OH. The solution was
submitted for Al-27 NMR analysis.
Al-27 NMR spectral analysis. Al-27 NMR spectra were
collected on a NT-300 NMR spectrometer with spectrometer
frequency 78.176839 MHz. The samples were spun at the magic
angle at rates of 1.4 and 2.05 kHz for la and Ila,
respectively.


45
Time (h)
Figure 2-2. Time course of induction of iNOS activity in MCT
cells treated with a combination of LPS (0.1 |ag/ml) and IFN
(100 U/ml). Inducible NOS activity is detectable at 4 h and
reaches maximum at 12 h. Data are mean S.E. (|g: ng of cell
protein, n=6).


126
an aluminum and a sodium with two hydrogen atoms, it is
difficult to prove of disprove the presence of aluminum, as
opposed to sodium, based on CHN data.
The data confirms the presence of the ligand in each of
the compounds because the molar ratios of carbon to nitrogen
are, for the most part, very close to those of the ligands.
However, because the amount of hydrogen is high, most likely
due to the presence of water molecules, the molar ratios
involving hydrogen do not correspond to those of the
deprotonated ligands. No further conclusions can be drawn
from this technique.
Infra-red Spectroscopy
The information provided by Infra-red spectroscopy
includes the stretching frequency of the carbonyl group, and
therefore, also on its environmnet, length, strength and
double bond character. The C=0 group in a carboxylic acid has
stretch in the region 1760 to 1700 cm'1, while that for a salt
of the carboxylic acid shifts to between 1650 and 1550 cm'1,
because the sodium ion causes a perturbation. Theoretically,
in a complex involving the carboxylate group, the stretching
frequency for the carbonyl group would probably be different
from that of the acid and the sodium salt.
Each aluminum compound has a carbonyl stretching
frequency between 1660 and 1690 cm'1, distinctly different than
the characteristic carboxylic acid and carboxylate ion
regions.52 For III and V, the single peak occurs between the


142
(65) Heitsch, C. W. ; Nordman, C. E.; Parry, R. W. Inorg.
Chem., 1963, 2, 508-512.
(66) Emsley, J. Chem. Soc. Rev., 1980, 9, 91-124.
(67) Browning, K. R. Unpublished results.
(68) Buschman, J.; Luger, P. Acta Cryst. C, 1985, 41, 201-
203 .
(69) Spek, A. L. Acta Cryst. C, 1987, 43, 1633-1634.
(70) Hoard, J. L.; Silverton, J. V. Inorg. Chem., 1963, 2,
235-243.
(71) Lippard, S. J.; Russ, B. J. Inorg. Chem., 1968, 7, 1686-
1688 .
(71) Cromer, D. T.; Liberman, D. J. Chem. Phys., 1970, 53,
1891-1898.
(72) Dauben, C. H.; Templeton, D. H. Acta Cryst., 1955, 8,
841-842.


52
Figure 2-8. Immunoreactivity of iNOS protein in cultured MCT
cells. A: Control MCT cells; B: MCT cells treated with a
combination of IFN (100 U/ml) and LPS (1 |og/ml) for 24 h; C:
MCT cells treated with IFN alone for 24 h. Inducible NOS was
detected by an immunoperoxidase method using a polyclonal
antibody raised in rabbit against mouse mac-NOS. Cells were
stained with hematoxylin. There is intense immunoreactivity
in cells treated with LPS/IFN compared to control or IFN-
treated cells. Picture is representative of three separate
experiments. Magnification x430.


64
x-ray crystallography. The aluminum ion in K[Al(EDTA)] 2H20
has been shown to be in a distorted octahedral environment
coordinated to two nitrogen and four oxygen atoms.58 The Al-27
NMR shifts reported for tris complexes of 0,0 bidentate
ligands range from 36 to 41 ppm.17,18 The peak at 54 ppm is
probably due to the aluminum ion surrounded by three nitrogen
and three oxygen atoms in a rather distorted octahedral
environment. Two structures, with the A1N303 confirmed by x-
ray studies, had chemical shifts of 8.1 and 8.2 ppm.27 The
chemical shifts for I and II should be larger than these two
reported studies because environments around the aluminum in
I and II are much more distorted from the ideal octahedral
coordination27. The distorted octahedral environment could be
viewed as approaching a pentacoordinate geometry, which is
generally indicated with chemical shifts near 60 ppm. To
date, there has not been an aluminum complex reported with a
chemical shift near 220 ppm.
The Al-27 NMR spectrum of the solid Ila is shown in
Figure 4-7. There are three different aluminum environments
in the solid sample, as evidenced by the three peaks in the
Al-27 NMR spectrum. Two peaks of almost equal height, 587 and
601, are centered around 5 ppm and 73 ppm, respectively. A
third smaller peak appears centered at -120 ppm.
Solution lib was prepared to separate the mixture. The
Al-27 NMR spectrum of the solution, shown in Figure 4-8, has
only one rather broad peak = 5400 Hz) centered at 54 ppm.


Nitrites
(nmoles/mg)
84
Figure 4-1. Nitrite production in MCT cells treated with LPS
(1 |ig/ml) and IFN (100 U/ml) or their combination for 24 h,
(* p<0.001, mg: mg of cell protein, n=9).


94
decay and Lp effects, 0.02 is a factor used to down weight
intense reflections and to account for instrument instability.
The linear absorption coefficient was calculated from values
from the International Tables for X-ray Crystallography
(1974). Scattering factors for non-hydrogen atoms were taken
from Cromer & Mann (1968) with anomalous-dispersion
corrections from Cromer & Liberman (1970), while those of the
hydrogen atoms were from Stewart, Davidson & Simpson (1965) .
Discussion
X-rav Diffraction Analysis.
Crystallographic data for the compound are summarized in
Table 6-2. Compound VIII is shown as an ORTEP drawing in
Figure 6-2. Final atomic parameters for the non-hydrogen
atoms and the hydrogen atoms are given in Tables 6-3 and 6-4,
respectively. The bond lengths and bond angles for all atoms
appear in Tables 6-5 and 6-6. The anisotropic thermal for the
non-hydrogen atoms are listed in Table 6-7.
Initially, aluminum was assigned as the central atom,
although no seven coordinate aluminum complex has been
reported. However, during refinement, fifteen hydrogen atoms
were found, indicating the loss of only one hydrogen atom from
the dipic and water molecules. If aluminum were the metal
ion, two less hydrogen atoms should be present to give a
neutrally charged compound.


A
B
LPS/IFN
+
Control IFN LPS LPS/IFN PDTC
pAiNOS-CAT
Figure 5-4. Effects of LPS, IFN and PDTC on the activation of the iNOS promoter. The MCT
cells transfected with pAiNOS-CAT vector were treated with IFN (100 U/ml), LPS (1 |xg/ml) or
their combination in the absence or presence of PDTC (25 jiM) followed by CAT assay of cell
lysates. Both LPS and LPS/IFN, but not IFN conferred CAT inducibility, which was blocked by
coincubation with PDTC. A Autoradiograph, B graphical presentation; (*, ** p < 0.05 and 0.01
respectively, vs control, *** p < 0.01 vs -PDTC, n=4)
110


37
Table 3-4. Experimental Conditions for Crystal Growth in
Various Solvents and Results.
Solvent
Base
Ligand
Notes
CH3OH
0.10 M NaOH
pic
65C, no solid product
CH3OH
0.10 M NaHCOj
pic
50C, no solid product
C2H5OH
0.15 M NaOH
pic
60C, no solid product
c2h5oh
0.030 M NaHC03
pic
40C, no solid product
c2h5oh
0.033 M NaHC03
pic
50C, sodium picolinate
precipitated
75% C2H5OH
0.15 M NaHC03
pic
overnight, twinned
crystals at room temp
50% C2H5OH
0.15 M NaHCOj
pic
opaque solution within
30 minutes at room temp
25% C2H5OH
0.15 M NaHC03
pic
opaque solution within
5 minutes at room temp
c2h5oh
7 mM NH4C2H302
pic
room temp, no solid
hoch2ch2oh
0.15 M NaHC03
pic
45C, no solid product
DMSO
0.53 M NaHC03
pic
white solid formed;
when heated to 100C,
formed a clear peach
solution, no solid ppt
C2H5OH
0.045 M NaOH
pza
60C, no solid product
c2h5oh
0.034 M NaOH
pza
40C, no solid product
c2h5oh
5 mM NaHC03
pza
overnight at 50C,
flower-like clusters
DMSO
0.53 M NaHCOj
pza
white solid formed;
when heated to 100C,
formed a clear bright
yellow solution, no
solid remained
C2H5OH
0.093 M NaOH
4.7 mM
dipic
45C, sodium salt of
dipicolinic acid formed


130
chemical shifts for aluminum coordination compounds have been
reported for the variety of chemical shifts present in the
spectra, little information can be gained about the
environment of the aluminum ions in the compounds that have
been analyzed.
The spectra for compound V, with pyrazinoic acid, each
has only one peak for the solid and solution phases. The
solution peak is at 70 ppm similar to the other solutions.
However, the solid has a peak at 0 ppm, characteristic of
A1(H20 ) 63+.
X-rav Crystallography
X-ray crystallography is used to determine the location
and type of each atom present in the complex. The greatest
disadvantage to x-ray diffraction techniques is the need for
a single crystal approximately 0.2 mm in each dimension.
Since compounds III through VII could not be recrystallized,
a variety of crystal growth techniques, including urea
decomposition, vapor and aqueous gel diffusion, variation of
the solvent system and the base strength, were investigated.
Crystal Growth Techniques
Urea Decomposition
For urea decomposition, the crystals that formed with
each ligand were neither large nor of high quality. There was
a rather large incidence of twinned crystals and inseparable


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor?of Philosophy.
Nicolas sL^srzan, Chairman
Assistant Professor of Pharmacology
and Therapeutics
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Luiz^telardinelli
Professor of Pharmacology and
Therapeutics
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Jfrhy K. Harrison
A$s)is|ant Professor of Pharmacology
-'ana Therapeutics
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Colin Sumners
Professor of Physiology


92
Figure 4-9. Characterization of NF-kB proteins activated by
(A) IFN and (B) IFN/LPS in MCT cells. Nuclear extracts
obtained after 30 min of treatment with IFN (100 U/ml) or IFN
(100 U/ml) with LPS (1 ^ig/ml) were analyzed by EMSA in the
absence or presence of IgG against p50, p65 and c-Rel. The
results are similar to the profile obtained with LPS
treatment alone (see figure 4-8). Blot is representative of
four separate experiments.


71
specific concentrations and for specific incubation periods.
The cells were then harvested by scraping in ice-cold PBS and
centrifuged at 500 g for 10 min and homogenized in two packed
cell volumes (PCV) of hypotonic buffer (Tris-HCl 10 mM, pH
7.4, KCl 10 mM, MgCl2 3 mM, Dithiothreitol (DTT) 1 mM, PMSF
0.5 mM, leupeptin 2 |iM) using a glass tissue grinder with a
Teflon pestle. The homogenates were centrifuged at 500 g for
10 min to pellet the nuclei. The nuclear pellet was
resuspended in 3 ml/109 cells of extraction buffer (Tris-HCl
10 mM, pH 7.4, KCl 0.3 M, MgCl2 3 mM, DTT 1 mM, PMSF 0.5 mM,
leupeptin 2 |iM, glycerol 20% v/v) and kept on ice for 45 min,
with gentle rocking. The extract was then clarified by
centrifugation at 25000 g for 30 min. The clear supernatant
was then dialyzed against 50 volumes of dialysis buffer
(Tris-HCl 10 mM, pH 7.4, KCl 0.1 M, DTT 1 mM, PMSF 0.5 mM,
leupeptin 2 |iM, EDTA 0.2 mM, glycerol 20% v/v) for 1 h. The
dialyzed solution was centrifuged at 25000 g, aliquoted and
stored at -70 C for subsequent use.
Oligonucleotide Probes and DNA Gel Mobility Assay
The NF-kB consensus DNA binding site is formed by the
general decameric motif 5'-GGGRNNTYCC, where R is a purine, Y
a pyrimidine and N either of them (Muller et al., 1993, Bours
et al. 1992). I utilized a double-stranded consensus
oligonucleotide containing the sequence 5'-GGGACTTTCC and
radio-labeled by phosphorylation with [y-32P]ATP (3000


I
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor;of Philosophy.
Nicolas vL^Guzman, Chairman
Assistant Professor of Pharmacology
and Therapeutics
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree .of Doctor of Philosophy.
Luiz'-Belardinelli
Professor of Pharmacology and
Therapeutics
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy,
K. Harrison
ant Professor of Pharmacology
Therapeutics
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Cr
Colin Sumners
Professor of Physiology


37
Lipopolysaccharide (0.1 ^g/ml) and IFN(100 U/ml) each
alone caused no significant accumulation of nitrite but there
was a concentration-dependent accumulation of nitrites in
cells treated with IFN in combination with LPS, peaking at
IFN concentration of 100 U/ml, a 30-fold increase over
control (figure 2-7). This pattern shows close correlation
with my measurements of iNOS activity obtained by assaying
[3H]L-citrulline. These results further confirm that iNOS
induction in MCT cells requires both LPS and IFN and is
concentration-dependent.
Effects of Interferon-v and Lipopolysaccharide on the
Expression of Inducible Nitric Oxide Svnthase Protein
To investigate the effects of IFN on the expression of
iNOS protein in MCT cells, I performed immunocytochemical
studies using a rabbit polyclonal antibody against murine
mac-NOS. Cultured MCT cells were treated with LPS, IFN or a
combination of both and iNOS immunoreactivity was detected by
the immunoperoxidase method.
Inducible NOS immunoreactivity was absent in control
cells (figure 2-8A). Treatment with a combination of LPS and
IFN for 24 h resulted in intense staining for iNOS (2-8B)
whereas treatment with IFN alone did not elicit iNOS
immunoreactivity (2-8C). In addition, treatment with LPS
alone resulted only in modest staining for iNOS (data not
shown). These results are consistent with my functional iNOS
activity studies, and indicate that the inability of IFN to


47
Figure 2-4. Effects of protein synthesis inhibitor
cycloheximide (CH, 20 jxM), mRNA transcription inhibitor
actinomycin D (ACT, 4 ^M) and iNOS induction inhibitor
dexamethasone (DEXA, 20 p.M) on iNOS activity. The MCT cells
were treated with a combination of LPS (0.1 ng/ml) and IFN
(100 U/ml) for 12 h in the absence and presence of the
inhibitors. Protein and mRNA synthesis are required for full
iNOS activity (* p<0.05 vs control, ng: ng of cell protein,
n=6) .


105
the cross-talk reported between NF-kB and other
transcriptional factors like Jun/Fos and cis-regulatory
enhancer binding protein-like factor (Stein et al., 1993,
Mukaida et al. 1990). Another reason may be that CAT mRNA
transcription induced by LPS are more stable without any IFN-
induced influences. Studies utilizing longer inserts than
the one used in the present study or a northern blot analysis
of CAT mRNA may be needed to answer this question. In
macrophages and glial cells, LPS causes the induction of
substantial iNOS activity on its own (Stuehr et al., 1985,
Xie et al. 1993, Szab et al., 1993) It is reasonable for
cells primed to counter microbial invasion to be activated by
microbial products, but in the renal tubule epithelium,
excessive amounts of NO may be harmful. Hence a more
stringent control of iNOS activity is desirable. It must
also be noted that any small amount of cytokine contaminant
from the preparation of the DNAs could synergize with LPS to
produce this effect.
In summary, the downstream NF-kB binding site in the
iNOS promoter region is essential for NF-tcB-mediated LPS-
stimulated iNOS gene transcription in MCT cells. However,
the synergistic effect of IFN appears to be mediated through
a site located in the upstream portion of the promoter
region, possibly the IRF-1 binding site (IRF-E). The loss of
this region results in the loss of the synergistic role of
IFN in MCT cells, as is seen in pBiNOS-CAT. Nuclear factor-
kB is a ubiquitous transcription factor that appears to be


63

GTP
GTP Cyclohydrolase I
(Blocked by DAHP) y
Dihydroneopterin triphosphate
I
I
I
BH2
^Dihydrofolate reductase
(Blocked by Methotrexate)
BH4
Figure 3-1. Tetrahydrobiopterin (BH4) synthetic pathways:
(I) de novo synthesis, and (II) dihydrofolate-dependent
salvage pathway (BH2, dihydrobiopterin).


7-7 Anisotropic Thermal Parameters for
Non-Hydrogen Atoms in IX 112
8-1 Crystallographic Data for X 118
8-2 Final Atomic Coordinates and Isotropic
Thermal Parameters for X 120
8-3 Bond Lengths and Angles for X 120
8-4 Anisotropic Thermal Parameters for
Non-Hydrogen Atoms in X 122
8-5 Least Squares Planes Analysis of Square
Antiprismatic and Dodecahedral
Geometries for X 123
vii


128
Richardson, S.B., Aspbury, R.A. and Fisher, M.J. The role of
reversible phosphorylation in the hormonal control of
phenylalanine hydroxylase in isolated rat proximal kidney
tubules. Biochem. J. 292: 419-424, 1993.
Robertson, F.M., Offner, P.J., Ciceri, D.P., Becker, W.K. and
Pruitt, B.J. Detrimental hemodynamic effects of nitric oxide
synthase inhibition in septic shock. Arch. Surg. 129: 149-
156, 1994.
Robbins, R.A., Barnes, P.J., Springall, D.R., Warren, J.B.,
Kwon, O.J., Buttery, L.D., Wilson, A.J., Geller, D.A. and
Polak, J.M. Expression of inducible nitric oxide synthase in
human lung epithelial cells. Biochem. Biophys. Res. Commun.
203: 209-218, 1994 [a] .
Robbins, R.A., Springall, D.R., Warren, J.B., Kwon, O.J.,
Buttery, L.D., Wilson, A.J., Addock, I.M., Riveros-Moreno,
V. Moneada, S. and Polak, J.M. Inducible nitric oxide
synthase is increased in murine lung epithelial cells by
cytokine stimulation. Biochem. Biophys. Res. Commun. 198:
835-843, 1994 [b].
Romero, J.C., Lahera, V. Salom, M.G. and Biondi, M.L. Role
of endothelium-dependent relaxing factor nitric oxide on
renal function. J. Am. Soc. Nephrol. 2: 1371-1387, 1992.
Rosenkranz-Weiss, P., Sessa, W.C., Milstien, S., Kaufman, S.,
Watson, C. A. and Pober, J.S. Regulation of nitric oxide
synthesis by proinflammatory cytokines in human umbilical
vein endothelial cells: elevations in tetrahydrobiopterin
levels enhance endothelial nitric oxide synthase specific
activity. J. Clin. Invest. 93: 2236-2243, 1994.
Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular
Cloning: A Laboratory Manual. 2nc* Edition, p. 31-57. Cold
Spring Harbor Press, Cold Spring Harbor, NY, 1989.
Saran, M., Michel, C. and Bors, W. Reaction of NO with 0^~.
Implications for the action of endothelium-derived relaxing
factor (EDRF). Free Radical Res. Commun. 10(4-5): 221-226,
1990.
Schmidt, H.H.H.W., Lohmann, S.M. and Walter, U. The nitric
oxide and cGMP signal transduction system: regulation and
mechanism of action. Biochem. et Biophysica. Acta. 1178: 153-
175, 1993.
Schmidt, H.H.H.W., Pollock, J.S., Nakane, M., Gorsky, L.D.,
Frstermann, U. and Murad, F. Purification of a soluble
isoform of guanylyl cyclase-activating factor synthase. Proc.
Natl. Acad. Sci. USA 88: 365-369, 1991.


23
pursued. (i) Cloning of the promoter region of mouse iNOS
from mouse genomic DNA. Two primers selected after careful
study of the promoter region of mac-iNOS, one designed with a
Hind III site at the 5'-flanking end and the other with a Sal
I site at the 3'-flanking end on the complementary DNA
strand, were used to clone the 1.7-kilobase pair (kbp)
promoter region from mouse genomic DNA using the polymerase
chain reaction (PCR). (ii) Preparation of deletion
constructs of the 1.7 kbp and their insertion into a
chloramphenicol acetyltransferase (CAT) basic vector reporter
system (pCAT-Basic). Constructs were prepared to include or
exclude the consensus binding sequences for NF-kB and IFN-
activated proteins to enable me study the role of these
transcription factors on iNOS induction in MCT cells. (iii)
Transfection of the iNOS promoter-pCAT-Basic reporter
constructs into MCT cells and functional analysis of this
reporter as an indicator of iNOS activity. Experiments
included the transfection of pCAT constructs into MCT cells,
and subsequent measurement and comparison of CAT and iNOS
activities in the transfected cells after treatment with the
various inducers or their combinations.
Statistical Analysis
Where indicated, statistical analysis was performed
using Student's t-test for paired and unpaired data, or
analysis of variance (ANOVA) and subsequent Scheffe's F-test
(StatView II, Abacus Concepts, Inc. Berkeley, CA) for


34
Interferon-y alone produced a 46% increase in nitrite
accumulation (figure 2-3). This was markedly potentiated by
the addition of LPS (192%) and reversed by treatment with
NNA. This confirms that iNOS activity is present in proximal
tubule cells.
Effects of Cvcloheximide, Actinomvcin D and Dexame" iiasone on
Inducible Nitric Oxide Synthase Activity Induced by
Lipopolvsaccharide and Interferon-y
To test the hypothesis that LPS/IFN-induced iNOS
activity requires protein synthesis or mRNA transcription,
MCT cells grown to confluence were incubated with LPS (0.1
|ig/ml) in combination with IFN (100 U/ml) in culture medium
for 12 h, and in the absence or presence of cycloheximide (20
H.M) an inhibitor of protein synthesis, actinomycin D (4 |xM) ,
an inhibitor of mRNA transcription, or dexamethasone (20 (iM) ,
which inhibits iNOS induction in macrophages by an unknown
mechanism. Inducible NOS activity was determined as before.
Lipopolysaccharide in combination with IFN caused a 379%
increase over basal iNOS activity (figure 2-4). This induced
iNOS activity was attenuated by cycloheximide (90%, with
respect to LPS/IFN-treated cells), actinomycin D (93%) and
dexamethasone (65%). Thus cycloheximide, actinomycin D and
dexamethasone are able to inhibit the induction of iNOS
activity in MCT cells, demonstrating that this process
requires protein synthesis and mRNA transcription.


68
Table 5-1. Al-27 NMR Data and Aluminum Coordination.
Compound
6, ppm
N atoms
0 atoms
H20
Ref
Al (H20) 3 (IDA) a
18.2
1
2
3
57
Al (IDA) 2a
36.5
2
4
0
57
Al (H20) 2 (NTA) b
25.4
1
3
2
57
A1(H20) (HEDTA)c
32.8
2
3
1
57
Al(EDTA)d
41.2
2
4
0
57
Al(PDTA) e
40.7
2
4
0
57
Al(DCTA)f
40.5
2
4
0
57
Al (oz) 39
8.2
3
3
0
27
Al (BrOz) 3h
11.4
3
3
0
27
Al (moz) 3a
8.1
3
3
0
27
Al (aloz) 3j
9.0
3
3
0
27
Al (DTPA) 2'lt
37.5
1
5
0
57
Al (pa) 31
39
0
6
0
18
Al(ma) 3ra
40
0
6
0
18
Al (mpp) 3n
37
0
6
0
17
Al (dpp) 3
36
0
6
0
17
Notes: Ligands are shown in Figure 5-1 a) Iminodiacetic acid,
b)Nitrilotriacetic acid, c) N-(Hydroxyethyl)ethylenediamine-
triacetic acid, d) Ethylenediaminetetraacetic acid, e) 1,2-
Propylenediaminetetraacetic acid, f) trans-1,2-Diamino-
cyclohexanetetraacetic acid, g) 2-(2'-Hydroxyphenyl)-2-
oxazoline, h) 2-(5'-Bromo-2'-hydroxyphenyl)-2-oxazoline,
i) 2-(2'-Hydroxy-3'-methylphenyl)-2-oxazoline, j) 2-(2'-
Hydroxy-3'-allylphenyl)-2-oxazoline, k) Diethylenetriamine-
pentaacetic acid, 1) Pyromeconic acid, m) Maltol, n) 3-
Hydroxy-2-methyl-4-pyridinone, o) 3-Hydroxy-1,2-dimethyl-4-
pyridinone
compounds shown here lie about midway between the two. Also,
the compounds with one or more nitrogen atoms coordinated to
the aluminum are shifted farther than the similar complexes
that contain only oxygen donor atoms. In addition, the more


4 SYNTHESIS, STRUCTURE AND ALUMINUM-27 NMR
STUDIES OF COMPLEXES WITH OXINE AND
2 -METHYLOXINE 41
Introduction 41
Experimental 44
Discussion 48
5 ALUMINUM-27 NMR, INFRARED AND ELEMENTAL
ANALYSIS OF ALUMINUM COMPLEXES OF N,0
DONOR LIGANDS 67
Introduction 67
Aluminum-27 NMR 67
Elemental Analysis 82
Infrared Spectroscopy 84
Fast Atom Bombardment Mass
Spectrometry 86
Conclusion 88
6 A PENTAGONAL BIPYRAMIDAL SODIUM COMPOUND:
SYNTHESIS AND CRYSTAL STRUCTURE 90
Introduction 90
Experimental 91
Discussion 94
7 SYNTHESIS AND STRUCTURE OF A COMPLEX
COMPOUND WITH PICOLINIC ACID AND
AMMONIUM NITRATE 103
Introduction 103
Experimental 103
Discussion 105
8 SYNTHESIS AND ABSOLUTE CONFIGURATION OF
SODIUM ( + ) TARTRATE MONOHYDRATE 115
Introduction 115
Experimental 115
Discussion 117
9 CONCLUSION 125
Characterization of Aluminum
Coordination Compounds 125
Crystal Growth Techniques 130
X-ray Diffraction Studies 133
Summary 137
REFERENCES 138
BIOGRAPHICAL SKETCH 143
iv


40
stabilization of iNOS mRNA, data from recent studies in rat
macrophages and hepatocytes seem to favor the former
mechanism (Nussler and Billiar, 1993) This is also
supported by the fact that the synergism at the level of mRNA
transcription requires protein synthesis, since
cycloheximide, a protein synthesis inhibitor, appears to
abolish the mRNA synthesis.
Synergistic interactions between IFN and other
immunomodulators in the induction of iNOS activity have also
been described in hepatocytes, lung fibroblasts, vascular
smooth muscle and mesangial cells (Geller et al. 1993 [b],
Gross and Levi, 1992, Jorens et al. 1992, Pfeilschifter and
Schwarzenbach, 1990). The molecular mechanisms involved in
the interactions among inducers in NO synthesis are not
completely understood and appear to vary among different
cells and cytokines. Interferon-y treatment does not
stimulate NO synthesis in rat hepatocytes despite inducing
iNOS mRNA transcription. Moreover, IFN has also been
reported to increase BH4 levels in murine macrophages,
fibroblasts, lymphocytes and rat pulmonary artery smooth
muscle by inducing the synthesis of GTP cyclohydrolase I, the
first and rate-limiting enzyme in the de novo pathway for the
synthesis of this cofactor (Di Silvio et al., 1993, Gross and
Levi, 1992, Hattori and Gross, 1993). Conversely, in rat
aortic vascular smooth muscle, IFN potentiates LPS-induced
synthesis of GTP cyclohydrolase I but does not itself
increase the levels of BH4 (Gross and Levi, 1992, Hattori and


59
Table 4-8.
Hydrogen Atom
Coordinates (xlO4)
for II.
Atom
X
y
z
H (11)
0.6093
0.1143
0.8760
H (12)
0.6192
0.1599
1.0192
H (13)
0.4324
0.1558
1.0513
H (15)
0.2114
0.1052
1.0335
H (16)
0.0293
0.0557
0.9408
H (17)
0.0521
0.0261
0.8154
H (21)
0.2834
-0.1815
0.7796
H (22)
0.3567
-0.3365
0.8245
H (23)
0.5486
-0.3627
0.8086
H (25)
0.7843
-0.3126
0.8011
H (26)
0.9172
-0.1800
0.7650
H (27)
0.8209
-0.0347
0.7443
H (31)
0.3750
-0.1579
0.6148
H (32)
0.3176
-0.1677
0.4742
H (33)
0.3096
-0.0363
0.4026
H (35)
0.3219
0.1658
0.3898
H (36)
0.3406
0.3349
0.4728
H (37)
0.3823
0.3264
0.6163
The average intraligand O-Al-N bond angle for I is 82.5
which is slightly more acute than the average of 82.9 for II.
The interligand bond angles around aluminum range from 86.7
to 99.9 with an average of 92.8 for Al(meox)3. For Al(ox)3,
these bond angles, varying from 87.6 to 96.9, average to
92.5. The average of the bond angles for Al(ox)3 correspond
more closely to the 90 angles present in an ideal octahedron.
The comparison of the bond angles for the oxygen and
nitrogen atoms trans to each other corroborates that the
Al (ox)3 is closer to the ideal octahedral environment than
Al(meox)3. In I, the trans bond angles range from 164.8 to
173.9 for an average deviation 9.4 from an ideal 180 angle.


2
on chromosome 21.4 It is rather an interesting occurrence
that chromosome 21 should be implicated in Alzheimer's
Disease. Down's Syndrome patients have an extra copy of
chromosome 21. Furthermore, autopsies have shown that
virtually all Down's patients that survive into their thirties
or forties get Alzheimer's Disease.4 Upon further research,
the metal was also detected in victims of Lou Gehrig's
Disease, also known as Amyotrophic Lateral Sclerosis, and
other neurological disorders such as epilepsy.5
Ordinarily, ingested aluminum is excreted rapidly during
normal kidney function.6 Individuals with kidney failure,
however, are subject to aluminum accumulation in several
tissues. First, aluminum is incorporated onto the bone
surface by the osteoblasts in the place of calcium, thereby
weakening the structure.7 In addition, in healthy bone, the
osteoclasts remove the top layers so that the bone can
continually be rebuilt. After the aluminum has accumulated on
the bone surfaces, the osteoclasts cannot remove it.7 As a
result the damage to the bone cannot be repaired and aluminum
adsorption continues to cover the bone surfaces. Once the
bone surfaces are saturated, the aluminum then begins to
concentrate in the brain.7 Patients of dialysis dementia,
characterized by speech difficulties, seizures and personality
changes, have markedly elevated levels of aluminum in their
brain tissues in amyloid plaques similar to those in
Alzheimer's Disease patients.8,9


97
polypropylene tube on ice, was added 5 ng plasmid DNA for 10
min and then heat-shocked for 45 seconds at exactly 42 C.
The tube was transferred to ice for 2 min and 900 (il SOC
medium (per 100 ml: bacto-tryptone 2 g, bacto-yeast extract
0.5 g, 1 M KC1 0.25 ml, 1 ml each of 1 M MgCl2/ 1 M NaCl, 1 M
MgSC>4 and 2 M glucose) was added and incubated at 37 C with
shaking at 225 rpm for 60 min. Then, 100 (4.1 aliquots of the
transformation mix were plated onto LB plates (per liter:
bacto-yeast extract 5 g, bacto-tryptone 10 g, NaCl 5 g, agar
15 g, pH 7.5) with ampicillin (100 |4g/ml) and incubated at 37
C overnight. Filters were then lifted from the plates and
hybridized to 32p-iabeled PCR product. Colonies hybridizing
to the probe were picked up for miniprep amplification.
Preparation of Constructs of Inducible Nitric Oxide Svnthase
A DNA fragment containing the promoter region of iNOS
from mouse genomic DNA was subcloned into the pCAT-Basic
vector (figure 5-2B) at the Hind III and Sal 1 to yield the
construct pAiNOS-CAT (figure 5-2A). The construct pBiNOS-CAT
was generated by the Spe I and the downstream Sal I fragment
and the pCiNOS-CAT, the Xba I and the upstream Hind III
fragment (figure 5-2A). These fragments were generated by
specific restriction endonuclease digestion with the
appropriate restriction enzymes. All the constructs were
sequenced to ensure that the appropriate deletions and
ligations have been obtained.


92
Figure 4-9. Characterization of NF-kB proteins activated by
(A) IFN and (B) IFN/LPS in MCT cells. Nuclear extracts
obtained after 30 min of treatment with IFN (100 U/ml) or IFN
(100 U/ml) with LPS (1 (xg/ml) were analyzed by EMSA in the
absence or presence of IgG against p50, p65 and c-Rel. The
results are similar to the profile obtained with LPS
treatment alone (see figure 4-8). Blot is representative of
four separate experiments.


51
Table 4-2. Final Atomic Coordinates (xlO4) and Isotopic
Thermal Parameters (2xl03) for I. Estimated Standard
Deviations are given in Parentheses.
X
y
z
U
Al
0.8192
(1)
0.4832
(1)
0.1185
(2)
46
(1)
0(1)
0.8291
(2)
0.5569
(2)
0.0533
(5)
57
(2)
N (1)
0.7596
(3)
0.4725
(3)
-0.0348
(5)
48
(2)
C(ll)
0.7611
(4)
0.5262
(4)
-0.0956
(7)
50
(3)
C (12)
0.7996
(3)
0.5700
(4)
-0.0488
(8)
52
(3)
C (13)
0.8048
(4)
0.6254
(4)
-0.1064
(8)
60
(4)
C (14)
0.7716
(4)
0.6351
(5)
-0.2123
(8)
75
(4)
C (15)
0.7360
(4)
0.5926
(4)
-0.2612
(8)
72
(4)
C (16)
0.7295
(4)
0.5362
(4)
-0.2054
(7)
56
(3)
C (17)
0.6956
(4)
0.4883
(5)
-0.2484
(8)
68
(4)
C (18)
0.6939
(4)
0.4347
(4)
-0.1891
(8)
58
(3)
C (19)
0.7251
(3)
0.4276
(4)
-0.0781
(7)
50
(3)
C(10)
0.7220
(4)
0.3667
(4)
-0.0192
(8)
78
(4)
0(2)
0.8687
(2)
0.5023
(2)
0.2471
(4)
51
(2)
N (2)
0.9011
(3)
0.4510
(3)
0.0434
(5)
44
(2)
C (21)
0.9443
(3)
0.4564
(3)
0.1302
(6)
44
(3)
C (22)
0.9263
(4)
0.4830
(4)
0.2399
(7)
54
(3)
C (23)
0.9659
(3)
0.4875
(4)
0.3339
(7)
55
(3)
C (24)
1.0262
(4)
0.4667
(4)
0.3176
(7)
63
(4)
C (25)
1.0447
(4)
0.4420
(4)
0.2131
(8)
60
(3)
C (26)
1.0040
(4)
0.4367
(4)
0.1162
(7)
50
(3)
C (27)
1.0201
(4)
0.4124
(4)
0.0016
(7)
53
(3)
C (28)
0.9774
(3)
0.4079
(3)
-0.0848
(7)
50
(3)
C (29)
0.9172
(4)
0.4273
(3)
-0.1629
(7)
57
(3)
C (20)
0.8725
(4)
0.4221
(4)
-0.1629
(7)
57
(3)
0(3)
0.8059
(2)
0.4060
(2)
0.1692
(4)
51
(3)
N (3)
0.7455
(3)
0.5022
(3)
0.2332
(5)
49
(2)
C (31)
0.7314
(3)
0.4496
(4)
0.2893
(7)
51
(3)
C (32)
0.7656
(4)
0.3987
(4)
0.2537
(7)
53
(3)
C (33)
0.7542
(4)
0.3423
(4)
0.3063
(8)
71
(4)
C (34)
0.7092
(5)
0.3378
(5)
0.3980
(9)
83
(4)
0(35)
0.6764
(4)
0.3870
(6)
0.4337
(8)
85
(5)
C (36)
0.6866
(4)
0.4447
(5)
0.3822
(7)
64
(4)
C (37)
0.6561
(4)
0.5004
(5)
0.4062
(7)
73
(4)
C (38)
0.6687
(4)
0.5510
(5)
0.3514
(9)
71
(4)
C (39)
0.7148
(4)
0.5521
(4)
0.2593
(7)
66
(3)
C (30)
0.7277
(4)
0.6110
(4)
0.1977
(10)
90
(4)
0(4)
0.8433
(3)
0.5559
(3)
0.4583
(6)
94
(3)
C (4)
0.8249
(5)
0.5063
(6)
0.5319
(10)
112
(6)
0(5)
0.6755
(3)
0.7031
(3)
0.4787
(7)
117
(4)
Equivalent isotropic
U defined
as
one third
of the
¡ trace
the orthogonalized Ui;j tensor


82
the possibility that the synergism between these two inducers
may be due to activation of different NF-kB subunits that
complement each other to induce iNOS gene transcription.
However, since this study was limited to the study of only
the three major subunits of NF-kB, the possibility of IFN
activating other less well-characterized subunits of NF-kB
cannot be completely ruled out. In spite of this, the
finding that LPS does not activate the nuclear translocation
of c-Rel in renal epithelium is very significant and may help
unravel tissue specificity in the regulation of iNOS
expression.
In summary, activation of NF-kB is necessary but not
sufficient for the induction of iNOS expression by LPS and
IFN in renal epithelium. The absence of c-Rel in the NF-kB
complexes translocated by these immunokines may, in part,
account for their inability to individually induce renal
epithelial iNOS. Therefore, c-Rel may be an important
determinant of the tissue specificity of iNOS expression.
This is consistent with the notion that the ability of the
various components of NF-kB to associate and form multiple
different dimers appear to be a major determinant of the
ability of these proteins to assign tissue-specific responses
to a given stimulus. Since overproduction of NO has been
associated with various pathological renal conditions
(Langrehr et al. 1993 [a,b], Weinberg et al. 1994, Yu et
al., 1994, Nussler et al. 1993), a better understanding of
the molecular mechanisms that determine the tissue-specific


76
(a)
(b)
Figure 5-5. Al-27 NMR Spectra of VI, a) Solid, b) in d6-DMSO.


18
Hvpoxanthine (hyp)
Ligand reactivity. Hypoxanthine is not appreciably
soluble in any solvent; however, it will react with acid or
basic solutions to dissolve in water. It is a very weak acid
with a pKa of 12.
HO
Figure 2-7. Hypoxanthine (hyp).
Preparation of Al/hyr> complex (VI). Hyp (0.775 g, 5.69
mmole) was added to 80 mL of H20 with stirring and heated to
80C. This produced an opaque white solution in which the
ligand settled quickly as a fine intensely white powder when
not stirring. Al (N03) 3-9H20 (0.712 g, 1.90 mmole) was
dissolved in a separate 5 mL H20. The two solutions were
combined to yield a mixture of pH 4.0. The temperature was
maintained at 80C. NaHC03 (0.482 g, 5.73 mmole) was slowly
added as a solid. After addition of the base was complete,
the pH of the mixture was 7.5. A large amount of white solid
was still visible but only some settled to the bottom when
stirring was stopped. A fluffy cream white solid remained
suspended above the bottom of the beaker. The temperature was
lowered to 60C and the mixture continued to heat for one
hour. Without stirring, only a small amount of white powder
settled. The unstirred solution carefully filtered through a


24
multiple group comparisons as appropriate. The results are
presented as mean + standard error of the indicated number of
experiments performed. Each blot shown is representative of
a series of experiments performed on at least three separate
occasions.


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
ALUMINUM COORDINATION COMPOUNDS
By
Kim E. Browning
May 1995
Chairman: Gus J. Palenik, Ph. D.
Major Department: Chemistry
Long considered a harmless element, studies in recent
years have linked aluminum(III) with health and environmental
problems ranging from Alzheimer's Disease and Osteoporosis to
the deforestation and fish kills associated with acid rain.
The coordination chemistry of aluminum(III) must be further
understood to explain the mechanism by which aluminum(III) is
incorporated into the biosphere and what, if any, role it
might play in causing these disorders.
To date, the focus of aluminum(III) coordination
chemistry that has been conducted is with ligands containing
primarily oxygen donor atoms. Since many naturally occurring
compounds also contain nitrogen as a potential donor ligand,
the focus of this research has been to study the coordination
of aluminum(III) with naturally occurring compounds and
similar ligands that contain a potential N,0 donor moiety.
xi


5 TRANSCRIPTIONAL REGULATION OF NITRIC OXIDE
SYNTHASE BY NUCLEAR FACTOR-KB IN RENAL EPITHELIUM.. 93
Introduction 93
Materials and Methods 95
Results 99
Discussion 102
6 SUMMARY AND CONCLUSIONS 113
LIST OF REFERENCES 117
BIOGRAPHICAL SKETCH 133
vi


127
Nussler, A.K., Liu, Z.Z., Di Silvio, M. Sweetland, M.A. ,
Geller, D.A., Lancaster, J.R., Billiar, T.R., Freeswick,
P.D., Lowenstein, C.L. and Simmons, R.L. Hepatocyte
inducible nitric oxide synthesis is influenced in vitro by
cell density. Am. J. Physiol. 267: C394-C401, 1994.
O'Dell, T.J., Hawkins, R.D., Kandel, E.R. and Arancio, 0.
Tests of the roles of two diffusible substances in long-term
potentiation: evidence for nitric oxide as a possible early
retrograde messenger. Proc. Natl. Acad. Sci. USA 88:11285-
11289, 1991.
Olson, J.S. Stopped-flow, rapid mixing measurements of
ligand binding to hemoglobin and red cells. Methods Enzymol.
76: 631-651, 1981.
Palmer, R.M.J., Ferrige, A.G. and Moneada, S. Nitric oxide
release accounts for the biological activity of endothelium-
derived relaxing factor. Nature 327: 524-526, 1987.
Petros, A., Lamb, G., Leone, A., Moneada, S., Bennett, D. and
Vallance, P. Effects of a nitric oxide synthase inhibitor in
humans with septic shock. Cardiovasc. Res 28: 34-39, 1994.
Pfeilschifter, J. and Schwarzenbach, H. Interleukin-1 and
tumor necrosis factor stimulate cGMP formation in rat
mesangial cells. FEBS Lett. 273: 185-187, 1990.
Radomski, M.W., Palmer, R.M.J. and Moneada, S. Endogenous
nitric oxide inhibits human platelet adhesion to vascular
endothelium. Lancet ii: 1057-1058, 1987.
Radomski, M.W., Palmer, R.M.J., and Moneada, S.
Glucocorticoids inhibit the expression of an inducible, but
not the constitutive nitric oxide synthase in vascular
endothelial cells. Proc. Natl. Acad. Sci. USA 87: 10043-
10047, 1990.
Raij, L. Glomerular thrombosis in pregnancy: role of the L-
arginine-nitric oxide pathway. Kidney Int. 45: 775-781, 1994.
Rao, D.N. and Kaufman, S. Purification and state of
activation of rat kidney phenylalanine hydroxylase. J. Biol.
Chem. 261: 8866-8876, 1986.
Rees, D.D., Palmer, R.M.J. and Moneada, S. Role of
endothelium-derived nitric oxide in the regulation of blood
pressure. Proc. Natl. Acad. Sci. USA 86: 3375-3378, 1989.
Reif, D.W. and Simmons, R.D. Nitric oxide mediates iron
release from ferritin. Arch. Biochem. Biophys. 283: 537-541,
1990.


133
striated branches arranged in a tetrahedral orientation around
a central point.
X-rav Diffraction Studies
Despite many attempts with a variety of crystal growth
techniques, several compounds, I, II, and VIII through X, were
studied using x-ray crystallography.
Aluminum Complexes
Compound I, Al (meox) 3-CH3OH* H20, and compound II,
A1 (ox) 3* CH3OH, were the only aluminum-containing compounds
characterized by x-ray crystallography. In each complex, the
aluminum ion is in a distorted octahedral environment created
by three ligands. The three nitrogen and three oxygen atoms
are arranged in the meridional as opposed to the facial
conformation. Although II is quite similar to I, there are
notable differences in bond lengths and angles between the
aluminum, nitrogen and oxygen atoms. In Al(meox)3, the
average Al-0 bond lengths are shorter than for Al(ox)3. In
addition, the Al-N bond lengths for Al-N bonds are an average
of 0.100 longer for Al(meox)3. It seems reasonable that as
the Al-0 bond gets shorter, the ligand molecule pivots to also
provide a longer Al-N bond distance. The average intraligand
O-Al-N bond angle for I is 82.5 which is slightly more acute
than the average of 82.9 for II. The interligand bond angles
around aluminum average to 92.8 for Al(meox)3. For Al(ox)3,
these bond angles, have an average value of 92.5. The trans


66
LPS/IFN
+
+
+
+
DAHP
-
-
+
+
Sepiapterin
-
-
+
+
Methotrexate -
-
+
_
+
Figure 3-4. Effects of methotrexate and sepiapterin on
nitrite production during GTPCH blockade with DAHP in MCT
cells. Cells were treated with LPS (0.1 |ig/ml) and IFN (100
U/ml) for 12 h in the presence of the indicated drugs.
Nitrite concentrations in culture medium from control cells
were less than 2 nmol/mg of protein per 12 h, (mg: mg of cell
protein, n=9).


95
Table 6-2. Crystalloaraphic Data
for Compound VIII.
Compound
Na+ (C7H4N04') (C7H5N04) 3H20
Empirical formula
C14H9N208Na 3H20
Formula wt, g
410.27
Crystal system
Triclinic
Space group
P 1
a,
6.905(1)
Jb,
11.141 (1)
c,
11.209(1)
0, 0
85.64(1)
P.
82.28 (1)
?,
87.33(1)
vc, 3
851.4 (2)
z
2
Dcalc, g/cm3 (298K)
1.600
Radiation, X ()
Mo-Kc, 0.71073
/i, mm'1
0.16
F(000), electrons
424
Crystal dimensions (mm3)
0.34 x 0.26 x 0.13
2 0 range
20.0, 22.0
Observed reflections
3305
Number of parameters
313
Final R
4.26
4.99
4.99


of
123
Table 8-5. Least Squares Planes Analysis
Antiprismatic and Dodecahedral Geometries for X.
Least Squares Planes Atoms
Square Antiprism
(1) 013, 043, 064, 071
(2) 022, 032, 041, 061
Triangular Dodecahedron
(3) 022, 032, 043, 071
(4) 013, 041, 061, 064
Square
Square Antiprism
Dodecahedron
Deviations,
A, from
Least Squares
Planes
(1)
(2)
(3)
(4)
Na
-
_
0.1213
0.0240
013
-0.1585
-
-
0.2073
022
-
0.1014
0.3952
-
032
-
-0.1065
-0.2929
-
041
-
0.1043
-
0.4179
043
0.1860
-
-0.3538
-
061
-
-0.0993
-
-0.2759
064
0.1686
-
-
-0.3493
071
-0.1961
-
0.2515
-
Rmsd
0.1779
0.1029
0.3281
0.3224
Rmsd takes the
form [(EA2)/2]*
A
B
C
D
Least Squares Planes Equation Coefficients
-0.16 (54)
7.31 (38)
5.69 (20)
-6.19(13)
0.33 (39)
7.33(64)
5.64(16)
-3.301(81)
-1.2(1.2)
1.88(81)
10.18(39)
-6.46 (23)
1.39 (91)
8.5 (1.0)
-1.04(39)
-1.06(23)
Equations for Least Squares Planes take the form:
Ax + By + Cz = D


70
H00CCH2
CHzCOOH
NCH2CH2N; CH2COOH
/ \ /
H00CCH2 CH2CH2N,
(k)
CH2COOH
HO
(1)
HO
CHa
(m)
Figure 5-1 -- Continued


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor_jof_Philosophy.
Gus'J. Pajlenik, Chairman
Professor of Chemistry
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
'james M. Bonce11a
Associate Professor of Chemistry
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree ('&\
vid E. Richardson
Professor of Chemistry
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
\ll 3&
Koppaka V. Rao
Professor of Medicinal Chemistry
This dissertation was submitted to the Graduate Faculty
of the Department of Chemistry in the College of Liberal Arts
and Sciences and to the Graduate School and was accepted as
partial fulfillment of the requirements for the degree of
Doctor of Philosophy.
May 1995
Dean, Graduate School


32
Table 3-2. Gel Diffusion Experimental Conditions and
Results.
Bottom layer
Top layer
Ligand
Results
0.010 M Al3+
0.030 M solution
pic
twins
in 1% agar
of the sodium
mpic
white band
salt of the
dipic
small crystals
ligand
pza
fine crystals
hyp
white band
0.010 M sodium
Single
mpic
white band
salt of ligand
A1 (N03)3- 9H20
pza
flower-like
in 0.5% agar
crystal
hyp
white band
0.1 mM NaOH in
Aqueous solution
pic
twinned
1% agar and a
of 0.03 M Al3+
mpic
small crystals
thin layer of
and 0.10 M of
dipic
small crystals
i pure agar on
the ligand
pza
flower-like
top
hyp
white band
1.0 mM NaHC03
Aqueous solution
pic
small crystals
in 0.5% agar
of 0.006 M Al3+
mpic
small crystals
and 0.020 M of
dipic
good crystals*
the ligand
pza
flower-like
0.10 M pic and
0.03 M Al3+ in
1% agar
0.10 M solution
sodium acetate
pic
twinned and
double-twinned
crystals
0.10 M pic and
0.03 M Al3+ in
1% agar
0.10 M solution
sodium acetate
pic
twinned and
double-twinned
crystals
* See Chapter 8 for structure


6
that iNOS and L-arginine biosynthetic activities were
coinduced. Thus, L-arginine transport and synthesis may be
rate-limiting for induced NO production. Hence, elucidation
of the biochemical events involved in NO production and
identification of
the
control points
in cofactor
and
substrate
synthesis
and
transport
have
the potential
for
providing
selective
targets for
therapeutic control
of
cytokine- and endotoxin-induced NO-mediated vascular shock
and tissue damage.
Synthesis of Nitric Oxide
Nitric oxide is synthesized from the terminal guanidino
nitrogen of L-arginine by NOS in a reaction that also
produces stoichiometric amounts of L-citrulline. This
reaction involves the transfer of five electrons from
molecular oxygen (O2) onto the substrate guanidino nitrogen
and requires NADPH, FMN, FAD and BH4 as cofactors. The
oxidation of L-arginine can be divided into two phases.
Phase I involves the hydroxylation of L-arginine to the
intermediate NL-hydroxyarginine (OHArg), and consumes one
mole of NADPH. This reaction resembles a classical
cytochrome P450 hydroxylation and may involve the heme iron of
NOS. Phase II involves the oxidation of OHArg to L-
citrulline and NO, consumes another mole of NADPH as reducing
equivalent and clearly depends on BH4. In the absence of L-
arginine and BH4, the activation of molecular O2 by NOS
results in a divalent reduction of O2 to yield superoxide


ACKNOWLEDGMENTS
My first and utmost sincere thanks go to my committee
chairman and mentor, Dr. Nicolas J. Guzman, for his guidance,
support, infinite patience and invaluable contribution to my
education. Secondly, my thanks go to Dr. Jeffrey Harrison,
for his outstanding help, enthusiasm and encouragement with
the molecular biology aspects of this work. I also
respectfully thank the other members of my advisory
committee, Dr. Luiz Belardinelli, Dr. Fulton Crews and Dr.
Colin Sumners, for their thoughtful suggestions and support.
I am very grateful to Dr. Mao-Zhong Fang, Dr. Judson
Chandler, Dr. Rajesh Davda and Mina Salafranea for their
excellent technical assistance and companionship. I also
wish to thank members of the secretarial, fiscal and
administrative staff who ensured that I was duly registered
every semester and that the check for my subsistence was
constantly in the mail, and especially, Judy Adams, for her
help in preparing this manuscript. Many thanks go to my
fellow graduate students, who helped make my stay tolerable,
especially Scott Masten and Monica Sanghani.
Last, but not the least, I wish to thank Dr. Stephen P.
Baker for his encouragement, help and frequent advice and
proddings, as well as his confidence in me for nominating me
for several awards and competitions during my study tour. To
in


109
Table 7-2. Final Atomic Coordinates
Thermal Parameters (2xl03) for IX.
Deviations are given in Parentheses.
(xlO4) and Isotropic
Estimated Standard
x
U
N (1)
0.3376
(2)
-0.0187
(4)
0.7255
(2)
33
(1)
C (2)
0.2875
(3)
-0.0038
(4)
0.6405
(2)
33
(1)
C (3)
0.3433
(4)
-0.0584
(5)
0.5778
(3)
45
(2)
C (4)
0.4500
(4)
-0.1256
(7)
0.6024
(3)
55
(2)
C (5)
0.4987
(4)
-0.1375
(6)
0.6891
(3)
51
(2)
C (6)
0.4415
(4)
-0.0815
(6)
0.7503
(3)
42
(2)
C (7)
0.1709
(3)
0.0692
(5)
0.6225
(2)
36
(1)
0(1)
0.1249
(2)
0.0892
(3)
0.6848
(1)
47
(1)
0(2)
0.1316
(2)
0.1052
(3)
0.5431
(1)
50
(1)
N (11)
0.2504
(2)
0.0101
(4)
0.8798
(2)
37
(1)
C (21)
0.3060
(3)
0.0590
(4)
0.9592
(2)
35
(1)
C (31)
0.2699
(4)
0.0224
(6)
1.0353
(2)
46
(2)
C (41)
0.1719
(4)
-0.0674
(7)
1.0301
(3)
60
(2)
C (51)
0.1113
(4)
-0.1153
(6)
0.9488
(3)
51
(2)
C (61)
0.1553
(4)
-0.0758
(6)
0.8770
(3)
44
(2)
C (71)
0.4084
(4)
0.1621
(5)
0.9586
(2)
37
(2)
0(11)
0.4591
(2)
0.2102
(4)
1.0375
(2)
53
(1)
0(21)
0.4405
(2)
0.1991
(3)
0.8928
(1)
54
(1)
N (2)
0.8303
(3)
0.6893
(5)
0.8064
(2)
49
(1)
0 (IN)
0.8272
(3)
0.6306
(5)
0.8788
(2)
86
(1)
0 (2N)
0.9163
(3)
0.7521
(6)
0.7925
(2)
86
(2)
0 (3N)
0.7478
(3)
0.6798
(5)
0.7473
(2)
101
(2)
N(3)
0.1030
(3)
0.0370
(3)
0.3553
(2)
50
(1)
Equivalent isotropic U defined as one third of the trace of
the orthogonalized Ui;j tensor


7
anions (O2 ) and hydrogen peroxide (H2O2) (Zweier et al. ,
1988).
Physiologically, synthesis of NO is regulated in various
ways. Firstly, the major stimulants of physiological
synthesis of NO are hemodynamic forces, notably shear stress
(Awolesi et al., 1993, Nishida et al., 1992). These and
several other agonists can activate NO synthesis within
seconds through pathways that are unaffected by
transcriptional or translational inhibition. Such agents
which include Ca2+ ionophores, excitatory amino acids,
acetylcholine and bradykinin, activate cNOS by eliciting
prompt elevation of intracellular Ca2+ (Ignarro et al., 1987,
Moneada et al., 1991). Secondly, agents termed inducers
stimulate synthesis of NOS in hours and require mRNA
transcription and protein synthesis (Geller et al. 1993
[a,b], Moneada et al. 1991). These include agents that
induce NO-mediated antitumor or antimicrobial activity such
as cytokines and bacterial lipopolysaccharide (LPS). These
agents typically activate iNOS and some of them interact
synergistically to enhance NO release. A third class of
agents includes those with the capacity to suppress NO
synthesis. A typical example of these agents is tissue
growth factor-p (TGF-P) which inhibits NO synthesis by
decreasing iNOS mRNA stability and translation, and by
increasing iNOS protein degradation (Vodovotz et al. 1993,
Junguero et al., 1992). Glucocorticoids may inhibit NO
synthesis through a similar mechanism and NO itself regulates


46
4J
H
u
V
H
¡3
0)
g
CQ
(1)
NNA
Figure 2-3. Effects of LPS (0.1 ^g/ml) and IFN (100 U/ml) on
rat primary culture proximal tubules. Interferon-y (100
U/ml) induces very little nitrite production. Addition of
LPS (0.1 |xg/ml) markedly potentiates the accumulation of
nitrites, which is inhibited by the addition of NNA (300 pM),
(p < 0.05, mg: mg of cell protein, n=3).


131
clusters of crystalline product, possibly as a result of the
higher temperature.
Vapor Diffusion
For the diffusion of ammonia vapor, a crystalline product
began to form in each experiment after a few days. However,
either the crystals were too small or in inseparable clusters.
The ammonia reacted immediately upon contact with the surface
of the solution. The only single crystals discovered were on
the bottom of the beaker. Once a crystal became too heavy to
be supported by surface tension, it sank to the bottom and did
not grow any larger. The clusters which continued to grow on
the surface probably did not sink because of the increased
surface tension created by the irregular surface.
Gel Diffusion
For gel diffusion experiments, a crystalline product was
produced in most of the trials. The exceptions were when a
white band was produced in every case using hyp and twice with
mpic. The white band indicates that either very fine crystals
formed or only Al(OH)3 was produced. For the most part, he
crystals produced were too small, twinned or clusters.
However, one experiment, which yielded suitable single
crystals, led to the x-ray analysis of compound VIII.
Base Strength
For experiments varying the strength of the base, no
suitable crystals were formed except for the KAl (S04) 2* 12H20
reactant. Although the reactions did proceed slower than with


42
H2S H24
H15 HM
Figure 4-2. ORTEP Drawing of the Tris(oxinato)aluminum(III)
without the Occluded Acetonylacetone. Ul-Haque, Horne, and
Lyle, J. Cryst. Spec. Res., 1991, 21, 411-417.


34
Interferon-y alone produced a 46% increase in nitrite
accumulation (figure 2-3). This was markedly potentiated by
the addition of LPS (192%) and reversed by treatment with
NNA. This confirms that iNOS activity is present in proximal
tubule cells.
Effects of Cvcloheximide. Actinomvcin D and Dexamethasone on
Inducible Nitric Oxide Synthase Activity Induced by:
Lipopolvsaccharide and Interferon-y
To test the hypothesis that LPS/IFN-induced iNOS
activity requires protein synthesis or mRNA transcription,
MCT cells grown to confluence were incubated with LPS (0.1
jig/ml) in combination with IFN (100 U/ml) in culture medium
for 12 h, and in the absence or presence of cycloheximide (20
HM), an inhibitor of protein synthesis, actinomycin D (4 nM),
an inhibitor of mRNA transcription, or dexamethasone (20 |xM) ,
which inhibits iNOS induction in macrophages by an unknown
mechanism. Inducible NOS activity was determined as before.
Lipopolysaccharide in combination with IFN caused a 379%
increase over basal iNOS activity (figure 2-4). This induced
iNOS activity was attenuated by cycloheximide (90%, with
respect to LPS/IFN-treated cells), actinomycin D (93%) and
dexamethasone (65%). Thus cycloheximide, actinomycin D and
dexamethasone are able to inhibit the induction of iNOS
activity in MCT cells, demonstrating that this process
requires protein synthesis and mRNA transcription.


28
maintained at 37 C in a 5% CC>2/95% O2 environment during cell
culture and NOS induction. The MCT cell lines were kindly-
provided by Dr. Eric Neilson (Univ. of Pennsylvania, PA) .
These cells were established from microdissected mouse
proximal tubules after stabilization by transfection with SV
40 virus and maintain many of the properties of
differentiated proximal tubule epithelial cells (Haverty et
al., 1988).
Determination of Nitric Oxide Synthase Activity
Nitric oxide synthase activity in intact MCT cells was
determined by measuring the conversion of [3H] L-arginine to
[3H] L-citrulline after separation of these amino acids by
anion exchange chromatography as previously described (Davda
et al. 1993). Briefly, control or experimentally treated
MCT cells were washed and then incubated in HEPES buffer
containing (in mM) HEPES 25 (pH 7.4, 37C) NaCl 140, KCl
5.4, CaCl2 1.8, MgCl2 1.0, glucose 5.0 and [3H]L-arginine 3
(iCi/ml for periods of time ranging from 0 to 120 min. The
reaction was terminated by washing the cells with ice-cold
Ca2+-free buffer containing 5 mM EDTA followed by addition of
1 ml 0.3 M HCIO4.
After neutralization of the tissue extract with 3.0 M
K2CO3, 50 (J.1 aliquots were taken for measurement of total
uptake of [3H] L-arginine and 500 ja 1 aliquots applied to
columns containing DOWEX AG50WX8 (Na+ form) added as a 1:1
slurry in water. The columns were washed three times with 2


78
Table 5-3. Al-27 NMR Data for Solutions Containing 0.30 M Al3+
and the Tabulated Concentration of pza at pH 5.
conditions as all other samples. The data are summarized in
Table 5-3. The spectra are shown in Figures 5-7 through 5-10.
Results
Most of the solid and solution samples produced more than
one peak using Al-27 NMR. It is clear that each of these
contains a mixture of aluminum environments. However, in each
solution, there is a peak near 68 ppm which is rather close to
one of the large peaks, at 73 ppm, in the spectrum of the
solid Ila. This could confirm the presence of a tris aluminum


81
highly unstable and unable to yield functionally active
enzyme due to the activities of other proteins possibly
coinduced by LPS. Interferon-y may therefore be activating
processes that repress these proteins and thereby decrease
iNOS mRNA degradation, stabilize iNOS mRNA and iNOS protein
or both, in renal epithelial cells. However, recent studies
in rat macrophages and hepatocytes do not support this
assertion (Nussler and Billiar, 1993). With regard to PDTC,
I cannot exclude the possibility that this compound may have
other as yet undetermined posttranscriptional and/or
posttranslational effects that prevent the expression of
iNOS.
The findings in these studies do not support my original
hypothesis with respect to the synergistic effects of LPS and
IFN in the activation of NF-kB. The signal observed with LPS
alone was about equal in magnitude to that observed for the
LPS and IFN combination. Unlike what has been previously
described in macrophages where IFN and IL-2 cooperatively
activate NF-kB to reach a threshold that may be required for
transcriptional activation of certain genes (Narumi et al.,
1992), no such cooperative interaction was observed between
IFN and LPS in renal epithelium. However, it appears to
agree with what is seen in human U-937 histiocytic lymphoma
cells where IFN potentiates the cytotoxic effects of TNF, but
has no effect on the TNF-dependent NF-kB activation
(Chaturvedi et al. 1994). In addition, this study also
apparently excludes another pillar of my hypothesis, that is,


140
(33) DeSimone, J. M. ; Stangle, M. ; Riffle, J. S.; McGrath, J.
E. Makromol. Chem., Macromol. Symp., 1991, 42/43, 373-
385.
(34) Feng, T. L.; Tsangaris, J. M. ; Barron, A. R. Monatshefte
fur Chemie, 1990, 121, 113-118.
(35) Karweer, S. B.; Pillai, B. P.; Iyer, R. K. Magn. Res.
Chem. 1990, 28, 922-924.
(36) Jons, O.; Johansen, E. S. Inorg. Chim. Acta, 1988, 151,
129-132.
(37) Dzugan, S. T.; Goedken, V. L. Inorg. Chim. Acta, 1988,
154, 169-175.
(38) Addy, P.; Evans, D. F.; Sheppard, R. N. Inorg. Chim.
Acta, 1987, 127, L19-20.
(39) Gurian, P. L.; Cheatham, L. K.; Ziller, J. W.; Barron,
A. R. J. Chem. Soc. Dalton Trans., 1991, 1449-1456.
(40) Komatsu, N. ; Uokoi, M. ; Kubota, E. Bull. Chem. Soc.
Jpn., 1988, 61, 3746-3748.
(41) Hermnek, S.; Fusek, J.; Kfz, O.; Csensky, B.; erny,
Z. Z. Naturforsch, 1987, 42b, 539-545.
(42) Barron, A. R.; Lyons, D.; Wilkinson, G.; Motevalli, M.;
Howes, A.J.; Hursthouse, M. B. J. Chem. Soc. Dalton
Trans., 1986, 279-285.
(43) Shirk, A. E.; Shirk, J. S. Inorg. Chem., 1983, 22, 72-
77.
(44) Findlow, J. A.; Duffield, J. R. ; Evans, D. A.; Williams,
D. R. J. Royal Neth. Chem. Soc. 1987, 106, 403.
(45) Thich, J. C.; Ou, C. C.; Powers, D.; Vasiliou, B.;
Mastopaolo, D. ; Potenza, J. A. ; Schugar, H. J. J. Am.
Chem. Soc., 1976, 98, 1425-1433.
(46) Quaglieri, P. P.; Loiseleur, H.; Thomas, G. Acta Cryst.,
1972, B28, 2583-2590.
(47) Strahs, G.; Dickerson, R. E. Acta Cryst., 1968, B24,
571-578.
(48) Palmer, K. J.; Wong, R. Y.; Lewis, J. C. Acta Cryst.,
1972, B28, 223-228.


64
30 n
CQ
V
H
u
V
I
N
(0
a)
20
£ io
m
m
m
Basal
DAHP
L/I L/I+DAHP
Figure 3-2. Effect of DAHP on nitrite production by MCT
cells; (L/I: LPS plus IFN). Data are mean + S.E. (* p < 0.05
vs L/I-treated, mg: mg of cell protein, n=9).


47
Figure 2-4. Effects of protein synthesis inhibitor
cycloheximide (CH, 20 |iM), mRNA transcription inhibitor
actinomycin D (ACT, 4 ^M) and iNOS induction inhibitor
dexamethasone (DEXA, 20 jj.M) on iNOS activity. The MCT cells
were treated with a combination of LPS (0.1 |a.g/ml) and IFN
(100 U/ml) for 12 h in the absence and presence of the
inhibitors. Protein and mRNA synthesis are required for full
iNOS activity (* p<0.05 vs control, p.g: ^g of cell protein,
n=6) .


105
the cross-talk reported between NF-kB and other
transcriptional factors like Jun/Fos and cis-regulatory
enhancer binding protein-like factor (Stein et al. 1993,
Mukaida et al. 1990). Another reason may be that CAT mRNA
transcription induced by LPS are more stable without any IFN-
induced influences. Studies utilizing longer inserts than
the one used in the present study or a northern blot analysis
of CAT mRNA may be needed to answer this question. In
macrophages and glial cells, LPS causes the induction of
substantial iNOS activity on its own (Stuehr et al., 1985,
Xie et al., 1993, Szab et al., 1993). It is reasonable for
cells primed to counter microbial invasion to be activated by
microbial products, but in the renal tubule epithelium,
excessive amounts of NO may be harmful. Hence a more
stringent control of iNOS activity is desirable. It must
also be noted that any small amount of cytokine contaminant
from the preparation of the DNAs could synergize with LPS to
produce this effect.
In summary, the downstream NF-kB binding site in the
iNOS promoter region is essential for NF-KB-mediated LPS-
stimulated iNOS gene transcription in MCT cells. However,
the synergistic effect of IFN appears to be mediated through
a site located in the upstream portion of the promoter
region, possibly the IRF-1 binding site (IRF-E). The loss of
this region results in the loss of the synergistic role of
IFN in MCT cells, as is seen in pBiNOS-CAT. Nuclear factor-
kB is a ubiquitous transcription factor that appears to be


21
effects of IFN and LPS on the activation of NF-kB in MCT
cells, and by investigating the effects of eliminating or
including NF-KB and IFN-activated protein binding sites from
the promoter region of the iNOS gene on IFN- and LPS-induced
iNOS expression using a specific promoter-reporter system. I
restricted my studies to evaluating the role of NF-kB and its
main subunits p50, p65 and c-Rel, and also, that of NF-KB and
IFN-activated protein binding sites in IFN- and LPS-induced
iNOS activity in renal epithelial cells. The specific aims
of my study were as described below.
Specific Aims
Specific Aim 1
Characterization of iNOS in MCT cells. I investigated
the time- and concentration-dependence of IFN- and LPS-
stimulated iNOS induction as measured by the rate of [3h]L-
arginine to [3H]L-citrulline conversion, and the accumulation
of nitrites in the culture media of cells treated with the
above agents.
Specific Aim 2
Investigation of the cofactor requirements of LPS- and
IFN-induced iNOS expression in MCT cells. I investigated BH4
requirements, by utilizing 2,4-diamino-6-hydroxypyrimidine
(DAHP), an inhibitor of guanosine triphosphate cyclohydrolase
I (GTPCH), the rate-limiting enzyme in the de novo synthesis


UNIVERSITY OF FLORIDA
3 1262 08554 5233


BIOGRAPHICAL SKETCH
The author was born and raised in Sunyani in the Brong
Ahafo Region of Ghana. He obtained his ordinary level
education at Tema Secondary School, graduating in 1978. He
had his sixth form education at the Presbyterian Boys' Sixth
Form Science College where he took his advanced level
examinations in 1980, majoring in physics, chemistry and
mathematics. In 1987, he graduated from the University of
Ghana Medical School with an MB ChB. He had a brief stint of
elective training in General Surgery at the Chase Farm
Hospital in Middlessex, London, in 1987. Between 1988 and
1989, he did six months of internship each in the Departments
of Obstetric & Gynecology and Internal Medicine at the Korle-
Bu Teaching Hospital in Accra, Ghana, after which he was
seconded as a medical officer to the Department of Pathology,
University of Ghana Medical School. He proceeded to the
University of Florida in 1991 where he started his graduate
education under the guidance of Dr. Nicolas J. Guzman in the
Department of Pharmacology and Therapeutics.
133


98
Transfection of the Plasmid DNA into Murine Proximal Tubule
Cells and Chloramphenicol Acetvl Transferase Assays
The MCT cells were transfected by a modification of the
DEAE-dextran method (Golub et al. 1989). Briefly, the cells
were seeded the day before transfection such that they were
about 60% confluent the next day. After cells were washed
twice with PBS, trypsinized and resuspended in RPMI with 10%
Nuserum, 10 p.g of plasmid DNA was added per 2 X 106 cells per
1 ml of prewarmed RPMI ( (37 C, 10% Nuserum) containing
DEAE-dextran (100 ng/ml) and 50 mM Tris (pH 7.4). The
suspension was incubated at 37 C for 60 min followed by a 3
min shock with 10% DMSO at room temperature. The cells were
washed and distributed to 100-mm plates each with about 5 x
106 cells in 10 ml of complete medium, and incubated at 37 C
in 5% CO2. After 24 h, the medium was changed and inducers
added at the desired concentrations and incubated for another
24 h. The cells were then washed with ice-cold PBS,
resuspended in 0.25 mM Tris-HCl (pH 8), and subjected to
three cycles of freezing and thawing. The lysates were
centrifuged (11700 g for 10 min at 4 C) and the supernatant
heated at 60 C for 10 min to inactivate CAT inhibitors and
then centrifuged again as above. The supernatant was then
assayed for CAT activity by the thin layer chromatography
(TLC) method (Golub et al., 1989). Briefly, the assay
mixture contained (final vol 125 (il) 100 (ig of cell extract
(in 50 |xl), 69 p.1 of 0.25 mM Tris (pH 8), 3 p. 1 of [14C]-
chloramphenicol (50 nCi/mmol) and 5 (il of 5 mg/ml n-butyryl


62
the cytoprotective effects of proximal tubule NO could be
enhanced by increasing its synthesis with exogenous
dihydropteridines.
Nitric oxide synthase antagonists such as the L-arginine
analogues have been partially successful in limiting the
hypotension of endotoxic shock (Petros et al. 1994).
However, pulmonary hypertension, impaired cardiac output, and
decreased survival have been reported with the use of these
agents, possibly because of their lack of selectivity (Petros
et al., 1994, Minnard et al., 1994, Robertson et al., 1994).
On the basis of the different cell requirements for BH4
synthesis,
the inhibition
of
GTPCH
could
theoretically
provide an
alternative way
to
reduce
renal
NO production
while preserving normal macrophage and endothelial function.
In summary, full functional expression of iNOS in murine
proximal tubule epithelium requires GTPCH activity and the de
novo synthesis of BH4. In the absence of this, iNOS activity
is dependent on the availability of exogenous
dihydropteridines that are converted to BH4 via the salvage
pathway. The blockade of BH4 synthesis therefore constitutes
a promising therapeutic alternative for disorders in which
excessive NO production contributes to renal injury.


33
activity was determined by measuring the conversion of [3H]L-
arginine to [3H]L-citrulline over 30 min.
Induction Time Course of Inducible Nitric Oxide Synthase
Next, I examined the induction time course of iNOS
activity in MCT cells. Murine proximal tubule cells grown to
confluence in culture medium were incubated with 50 U/ml IFN
in combination with 0.1 ^ig/ml LPS for 0, 4, 6, 8, 12 and 24 h
and iNOS activity measured as the conversion of [3H]L-
arginine to [3H]L-citrulline over 30 min.
There was a time-dependent induction of iNOS activity,
peaking to a 357% increase over control at 12 h (figure 2-2).
Thus, the induction of iNOS activity in MCT cells is time-
dependent. This lag period suggests the possibility that
protein synthesis or RNA transcription may be involved in the
induction of iNOS activity.
Effects of Lioooolvsaccharide and Interferon-y on Primary
Culture of Rat Proximal Tubule Cells
To establish that the expression of iNOS in MCT cells
was not an artifact of the transformation process in the MCT
cells, proximal tubules were isolated from rat kidneys as
described above and cultured to confluence. Nitrite
accumulation was then measured after 48 h incubation with IFN
(100 U/ml) or IFN plus LPS (0.1 (i.g/ml) in the presence and
absence of NNA (300 |iM) .


61
GTPCH in the renal parenchyma. Previous studies (Bellahsene
et al., 1984) have found GTP 8-formylhydrolase and neopterin
synthetic activities of GTPCH in rat kidneys, but the
physiologic significance of this remains unclear. In liver
and catecholamine-producing organs, GTPCH provides BH4 for
aromatic L-amino acid hydroxylation (Kaufman, 1993).
Phenylalanine hydroxylase activity has also been detected in
the kidney, but its function is unknown (Rao and Kaufman,
1986, Richardson et al., 1993). The renal hydroxylase
differs from the liver isoform in that it exists natively in
a very high state of activation of about 16-fold greater in
activity (Rao and Kaufman, 1986). However, it is not known
whether this activity is supported by BH4 synthesized de novo
or regenerated from dihydropteridines.
The regulation of NO synthesis by GTPCH and BH4
availability may be important therapeutically because it
provides a target for the blockade of the renal L-arginine:N0
system. Nitric oxide is a mediator of inflammation in
glomerulonephritis (Weinberg et al. 1994, Marsden and
Ballermann, 1990, Shultz et al. 1990) and kidney allograft
rejection (Langrehr et al. 1993 [a,b]) and of tubular
hypoxia/reoxygenation injury (Yu et al., 1994). On the other
hand, NO causes renal vasodilation, increases renal cortical
bloodflow (Shultz et al., 1993), protects against glomerular
thrombosis in endotoxemia and pregnancy (Shultz and Raij,
1992, Raij, 1994), and modulates T-cell proliferation
(Langrehr et al, 1993 [a]). It is therefore possible that


35
Table 3- 3 Base Variation Experimental Conditions and Results.
Ligand
Base
Time
Results
0.40 M pic
0.27 M S042 from
KAl (S04)2
overnight
KAl (S04)2 12H20
crystals formed
0.10 M pic
0.10 M NaC2H302
10 min
fine crystals
0.10 M pic
0.077 M NaC2H302
20 min
fine crystals
0.10 M pic
0.070 M NaC2H302
30 min
small crystals
7.5 mM pic
7.5 mM NaC2H302
1 week
no solid product
50 mM pic
2 M NaC2H302 and
9 M HC2H302
no solid product
14 mM pic
14 mM NH4C2H302
overnight
twinned crystals
11 mM pic
0.44 M NH4C2H302
and .07 M HC2H302
in 50 % acetone
no solid product
20 mM pic
20 mM NaHC03
30 min
twinned crystals
14 mM pic
14 mM NaHC03
2 hours
twinned crystals
7.5 mM pic
7.5 mM NaHC03
overnight
powdery solid
0.30 M pic
0.30 M pyridine
2 min
powdery solid
0.15 M pic
0.018 M pyridine
overnight
crystalline
15 mM dipic
7.5 mM S042' from
Al2 (S04) 3* 18H20
no solid product
pH rose from 1.5
to 4.5 overnight
8.3 mM
dipic
8.3 mM NaC2H302
3 days
small crystals
15 mM pza
7.5 mM S042' from
A12(S04)2 18H20
no solid product
6.4 mM pza
7.7 mM NaC2H302
overnight
flower-like
clusters
11 mM hyp
0.44 M NH4C2H302
and .07 M HC2H302
in 50 % acetone
2 weeks
brown solution,
no solid product


62
the cytoprotective effects of proximal tubule NO could be
enhanced by increasing its synthesis with exogenous
dihydropteridines.
Nitric oxide synthase antagonists such as the L-arginine
analogues have been partially successful in limiting the
hypotension of endotoxic shock (Petros et al. 1994).
However, pulmonary hypertension, impaired cardiac output, and
decreased survival have been reported with the use of these
agents, possibly because of their lack of selectivity (Petros
et al., 1994, Minnard et al., 1994, Robertson et al., 1994).
On the basis of the different cell requirements for BH4
synthesis, the inhibition of GTPCH could theoretically
provide an alternative way to reduce renal NO production
while preserving normal macrophage and endothelial function.
In summary, full functional expression of iNOS in murine
proximal tubule epithelium requires GTPCH activity and the de
novo synthesis of BH4. In the absence of this, iNOS activity
is dependent on the availability of exogenous
dihydropteridines that are converted to BH4 via the salvage
pathway. The blockade of BH4 synthesis therefore constitutes
a promising therapeutic alternative for disorders in which
excessive NO production contributes to renal injury.


98
Table 6-4. Final Atomic Coordinates (xlO4) and Isotropic
Thermal Parameters (2) for the Hydrogen Atoms of Compound
VIII. Estimated Standard Deviations are given in Parentheses.
Atom
X
y
z
u
HI
0 ,
.258
(5)
0 .
.459
(3)
-0
.041
(3)
0.
.076
(10)
H7
0 ,
.240
(5)
1,
. Oil
(3)
0
.081
(3)
0.
.114
(13)
H3
0 ,
.292
(4)
0 .
.649
(2)
-0 ,
.409
(2)
0
.051
(7)
H4
0.
.334
(4)
0.
. 856
(2)
-0
.479
(2)
0
.045
(7)
H5
0.
.326
(3)
1.
. 004
(2)
-0 ,
.341
(2)
0
.042
(6)
HI'
0 .
.345
(5)
0.
.644
(3)
0 .
.325
(3)
0.
.076
(10)
H3'
0 ,
.190
(4)
0 .
.431
(2)
0 ,
.528
(2)
0
.049
(7)
H4'
0 .
.112
(4)
0.
.236
(2)
0.
.617
(3)
0
.055
(7)
H5'
0 ,
.108
(4)
0 .
.072
(2)
0 .
.495
(2)
0
.046
(7)
H8a
-0.
. 076
(6)
0 .
.793
(4)
0.
.147
(3)
0.
.10
(13)
H8b
-0 .
. 085
(7)
0 .
.676
(4)
0 .
.164
(4)
0
.12
(2)
H9a
0.
.689
(6)
0 .
.666
(3)
-0,
. 082
(4)
0
.11
(2)
H9b
0 .
.704
(6)
0 .
. 736
(4)
-0 .
.012
(4)
0
.11
(2)
HlOa
0.
.325
(4)
0.
, 842
(3)
0.
.271
(3)
0
.062
(9)
HlOb
0 .
.511
(5)
0 .
.814
(3)
0 .
.223
(3)
0.
,069
(10)
Table 6-5. Bond Lengths () of all Atoms of Compound VIII.
Estimated Standard Deviations are given in Parentheses.
Na-N
2.476 (2)
Na-01
2.482 (2)
Na-07
2.584 (2)
Na-Ol'a
2.474 (2)
Na-08
2.300 (2)
Na-09
2.501 (2)
Na-O10
2.495(2)
N-C2
1.338 (3)
N-C6
1.337 (3)
Ol-Cl
1.304 (3)
Ola-Cl
1.219 (3)
01 -Cl
1.274 (3)
07a-C7
1.238 (2)
C1-C2
1.500 (3)
C2-C3
1.392 (3)
C3-C4
1.372 (3)
C4-C5
1.382 (3)
C5-C6
1.387 (3)
C6-C7
1.510 (3)
N'-C2'
1.346 (3)
N'-C6'
1.339 (2)
01'-Cl'
1.317 (3)
Ol'a-Cl'
1.209 (2)
01'-Cl'
1.271 (3)
07'a-C7'
1.226 (2)
Cl'-C2'
1.495 (3)
C2'-C3'
1.383 (3)
C3'-C4'
1.385 (3)
C4'-C5'
1.373 (3)
C5'-C6'
1.390 (3)
C6'-C7'
1.514 (3)
Ol-Hl
0.94(3)
07-H7
1.24 (4)
C3-H3
0.95(3)
C4-H4
0.90 (3)
C5-H5
0.98(2)
01'-HI'
0.86 (3)
C3'-H3'
0.95(3)
C4'-H4'
0.93 (3)
C5'-H5'
1.01(2)
08-H8a
0.90(4)
08-H8b
0.81(5)
09-H9a
0.76(4)
09-H9a
0.71(4)
O10-H10a
0.90(3)
O10-H10b
0.86 (3)


119
Brue, B. Molina y Vedia, L. and Lapetina, E.G. Agonist-
induced ADP-ribosylation of a cytosolic protein in human
platelets. Proc. Natl. Acad. Sci. USA 87: 3304-3308, 1990.
Bucala, R., Tracey, K.J. and Cerami, A. Advanced
glycosylation endproducts quench nitric oxide and mediate
defective endothelium-dependent vasodilation in experimental
diabetes. J. Clin. Invest. 87: 432-438, 1991.
Busse, R. and Mulsch, A. Induction of nitric oxide synthase
by cytokines in vascular smooth muscle cells. FEBS Lett. 275:
87-90, 1990.
Chandler, L.J., Guzman, N.J., Sumners, C. and Crews, F.T.
Induction of nitric oxide synthase in brain glial cells:
possible interactions with ethanol in reactive neuronal
injury, in Alcohol and Glial Cells (vol 27). National
Institute on Alcohol Abuse and Alcoholism Research Monograph,
edited by F.E. Lancaster, Bethesda, National Institutes of
Health, pp 195-214, 1994.
Charles, I.G., Palmer, R.M.J., Hickery, M.S., Bayliss, M.T.,
Chubb, A.P., Hall, V.S., Moss, D.W. and Moneada, S. Cloning,
characterization and expression of a cDNA encoding an
inducible nitric oxide synthase from the human chondrocyte.
Proc. Natl. Acad. Sci. USA 90: 11419-11423, 1993.
Chaturvedi, M.M., Higuchi, M. and Aggarwal, B.B. Effect of
tumor necrosis factors, interferons, interleukins and growth
factors on the activation of NF-kappa B: evidence for lack of
correlation with cell proliferation. Lymphokine Cytokine Res.
13: 309-313, 1994.
Chin, J.H., Azhar, S. and Hoffman, B.B. Inactivation of
endothelium-derived relaxing factor by oxidized lipoproteins.
J. Clin. Invest. 89: 10-18, 1992.
Cho, H.J., Xie, Q., Calaycay, J., Mumford, R.A., Swiderek,
K.M., Lee, T.D. and Nathan, C. Calmodulin is a subunit of
nitric oxide synthase from macrophages. J. Exp. Med. 176:
599, 1992.
Chomsczyski, P. and Sacchi, N. Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform
extraction. Anal. Biochem. 162: 156-159, 1987.
Closs, E.I., Lyons, R. Mitchell, M. and Cunningham, J.M.
Expression of cationic amino acid transporters in NO-
producing macrophages (Abstract). Endothelium 1:16, 1993.
Corbett, J.A., Lanscaster Jr., J.R., Sweetland, M.A. and
McDaniel, M.L. Interleukin-l-p -induced formation of EPR-
detectable iron-nitrosyl complexes of Islets of Langerhans:


4
phosphorylation by other kinases has no effect (Bredt et al.,
1992, Davda et al., 1994). Endothelial NOS has a
myristoylation site that could account for its association
with membranes thereby probably directing NO delivery to the
underlying vascular smooth muscle cells for the maintenance
of basal vascular tone, whereas other isoforms are largely
cytosolic (Dinermann et al. 1993).
The capacity to express iNOS exists in nearly every
mammalian tissue and iNOS has been expressed in such diverse
cells as macrophages, microglia, hepatocytes, vascular smooth
muscle, endothelial cells, pancreatic islet cells,
chondrocytes and kidney cells (Bredt et al. 1991, Chandler
et al. 1994, Charles et al., 1993, Forstermann et al., 1991,
Geller et al., 1993 [a,b], Lowenstein et al., 1993, Markewitz
et al. 1993). So far, almost all agents known to induce
iNOS are microbes, microbial products or inflammatory
cytokines, often exhibiting strong synergism between them and
variability between cell types in their responses to the
various inducing agents.
Substrate and Cofactor Requirements
All three isoforms of NOS are hemoproteins and are
therefore dependent on NADPH, FAD and FMN.
Tetrahydrobiopterin is also an essential cofactor of NOS and
is tightly bound to the enzyme (Schmidt et al., 1991, Schmidt
et al., 1993, Tayeh and Marietta, 1989). Tetrahydrobiopterin
synthesis occurs via two distinct pathways: a de novo


A
Control IFN LPS LPS/IFN
B
30 -i
t
O20
-P
rH
>1
pBiNOS-CAT
Basal IFN LPS LPS/IFN
Figure 5-5. Effect of the removal of the upstream NF-kB binding site on the activation of
iNOS promoter by LPS and IFN. The MCT cells transfected with pBiNOS-CAT vector, which is
devoid of the upstream NF-KB binding site and the interferon response elements, were treated
with LPS (1 |jg/ml) and IFN (100 U/ml) or their combination for 24 h followed by CAT assay of
the cell lysates. Only LPS conferred inducibility to pBiNOS-CAT, with the loss of the
synergistic effect of IFN. A Autoradiograph, B graphical presentation; (* p < 0.01 vs
control, n=3)
111


39
state mRNA levels by actinomycin D suggests a requirement for
mRNA transcription in iNOS induction in MCT cells.
Discussion
In these studies, I have described the characteristics
of a mouse renal proximal tubule inducible isoform of NOS
that shares many but not all the characteristics of the
macrophage iNOS. This proximal tubule iNOS is markedly
induced in a time- and concentration-dependent manner by
treatment with LPS and IFN, but is induced only modestly by
LPS alone. This induction requires protein and mRNA
synthesis and is inhibited by dexamethasone. Murine proximal
tubule cell iNOS activity is also blocked by NNA, a
competitive inhibitor of all NOS isoforms. In contrast to
its effects on mouse peritoneal macrophages (Bogdan et al. ,
1993, Ding et al. 1988), IFN by itself did not induce the
expression of proximal tubule epithelial iNOS as illustrated
by the absence of iNOS activity, iNOS protein
immunoreact ivity and iNOS mRNA expression in MCT cells
treated with this cytokine. Nevertheless, IFN had a powerful
synergistic effect on LPS stimulation of iNOS expression in
these cells. This synergism, evidenced by the marked
enhancement of LPS-stimulated iNOS activity and iNOS protein
reactivity, is likely to occur at the level of transcription
since IFN also markedly augmented the stimulation of iNOS
mRNA expression induced by LPS. Although this effect could
be due to enhanced transcription or decreased degradation and


REGULATION OF INDUCIBLE NITRIC OXIDE SYNTHASE IN MURINE
PROXIMAL TUBULE EPITHELIAL CELLS
BY
BISMARCK AMOAH-APRAKU
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1995

This dissertation is dedicated to the blessed memory of my
dad who passed away while I was studying for this work, and
to all my loved ones, especially my beloved Margaret.

ACKNOWLEDGMENTS
My first and utmost sincere thanks go to my committee
chairman and mentor, Dr. Nicolas J. Guzman, for his guidance,
support, infinite patience and invaluable contribution to my
education. Secondly, my thanks go to Dr. Jeffrey Harrison,
for his outstanding help, enthusiasm and encouragement with
the molecular biology aspects of this work. I also
respectfully thank the other members of my advisory
committee, Dr. Luiz Belardinelli, Dr. Fulton Crews and Dr.
Colin Sumners, for their thoughtful suggestions and support.
I am very grateful to Dr. Mao-Zhong Fang, Dr. Judson
Chandler, Dr. Rajesh Davda and Mina Salafranea for their
excellent technical assistance and companionship. I also
wish to thank members of the secretarial, fiscal and
administrative staff who ensured that I was duly registered
every semester and that the check for my subsistence was
constantly in the mail, and especially, Judy Adams, for her
help in preparing this manuscript. Many thanks go to my
fellow graduate students, who helped make my stay tolerable,
especially Scott Masten and Monica Sanghani.
Last, but not the least, I wish to thank Dr. Stephen P.
Baker for his encouragement, help and frequent advice and
proddings, as well as his confidence in me for nominating me
for several awards and competitions during my study tour. To
m

all of them, I say thank you for their contributions to my
professional and personal development.
IV

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS iii
KEY TO ABBREVIATIONS vii
ABSTRACT viii
CHAPTERS
1 INTRODUCTION 1
General 1
Isoforms of Nitric Oxide Synthase 2
Substrate and Cofactor Requirements 4
Synthesis of Nitric Oxide 6
Role of Nitric Oxide in Mammalian Physiology 9
Rationale for the Thesis 18
Specific Aims 21
Statistical Analysis 23
2 CHARACTERIZATION OF INDUCIBLE NITRIC OXIDE
SYNTHASE IN MURINE PROXIMAL TUBULE EPITHELIUM 25
Introduction 2 5
Materials and Methods 27
Results 32
Discussion 39
3 GUANOSINE TRIPHOSPHATE CYCLOHYDROLASE I
REGULATES NITRIC OXIDE SYNTHESIS IN RENAL
PROXIMAL TUBULES 54
Introduction 54
Materials and Methods 57
Results 58
Discussion 60
4 NUCLEAR FACTOR-KB REGULATES INDUCIBLE NITRIC OXIDE
SYNTHASE IN RENAL TUBULE EPITHELIUM 68
Introduction 68
Materials and Methods 70
Results 73
Discussion 78
v

5 TRANSCRIPTIONAL REGULATION OF NITRIC OXIDE
SYNTHASE BY NUCLEAR FACTOR-KB IN RENAL EPITHELIUM.. 93
Introduction 93
Materials and Methods 95
Results 99
Discussion 102
6 SUMMARY AND CONCLUSIONS 113
LIST OF REFERENCES 117
BIOGRAPHICAL SKETCH 133
vi

KEY TO ABBREVIATIONS
AP-1
ATPase
bh4
CAT
Activator Protein
Adenosine Triphosphatase
Tetrahydrobiopterin
Chloramphenicol Acetyl
Transferase
DAHP
DMEM
2,4-diaminohydroxypyrimidine
Dulbecco's Modified Eagle's
Medium
DMSO
EMSA
Dimethylsulfoxide
Electrophoretic Mobility
Shift Assay
FAD
GAS
GTP
GTPCH
IFN
I KB
IL
y- IRE
IRF-1
IRF-E
Flavin Adenine Dinucleotide
y-Activated Site
Guanosine 5'-Triphosphate
GTP Cyclohydrolase I
Interferon-y
Inhibitor of kappa B
Interleukin
y-Interferon Response Element
Interferon-y Response Factor
Interferon-y Response Factor
Element
ISRE
Interf eron-oc-Stimulated
LPS
MCT
NADH
Response Element
Lipopolysaccharide
Murine Proximal Tubule Cells
Nicotinamide Adenine
Dinucleotide
NADPH
Nicotinamide Adenine
Dinucleotide Phosphate
NF-KB
NNA
NO
NOS
cNOS
Nuclear Factor-kappa B
NW-Nitro-L-Arginine
Nitric Oxide
Nitric Oxide Synthase
Constitutive Nitric Oxide
Synthase
iNOS
Inducible Nitric Oxide
Synthase
PCR
PDTC
RPMI
Polymerase Chain Reaction
Pyrrolidinedithiocarbamate
Roswell Park Memorial
Institute
TNF-OC
Tumor Necrosis Factor
vil

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
REGULATION OF INDUCIBLE NITRIC OXIDE SYNTHASE IN MURINE
PROXIMAL TUBULE EPITHELIAL CELLS
By
Bismarck Amoah-Apraku
August, 1995
Chairman: Nicolas J. Guzman, MD
Major Department: Pharmacology and Therapeutics
Nitric oxide (NO) is an important mediator of vascular
tone, neurotransmission and immune responses. The mechanisms
regulating inducible NO synthase (iNOS) expression in renal
tubule epithelium have not been well characterized. The
purpose of this study was to investigate the mechanism of
potentiation between interferon-y (IFN) and bacterial
lipopolysaccharide (LPS) in inducing iNOS activity in murine
proximal tubule epithelial (MCT) cells. Treatment with LPS
caused a small increase in iNOS activity whereas IFN had no
effect. Simultaneous treatment with LPS and IFN (LPS/IFN),
however, markedly increased iNOS activity. Treatment with
LPS/IFN in the presence of 2,4-diamino-6-hydroxypyrimidine
(DAHP), an inhibitor of the rate-limiting enzyme GTP-
cyclohydrolase I in the de novo synthesis of
V1XX

tetrahydrobiopterin (BH4)( an essential iNOS cofactor,
attenuated the increased LPS/IFN-stimulated nitrite
production. Sepiapterin, a substrate for the formation of
BH4 via the pterin salvage pathway, reversed this inhibition.
Electrophoretic mobility shift assays (EMSA) using specific
antibodies to p50, p65 and c-Rel subunits of nuclear factor-
kappa B (NF-KB) demonstrated that LPS and IFN treatment
induced the nuclear translocation of p50 and p65 but not c-
Rel. Cotreatment with Pyrrolidinedithiocarbamate (PDTC), a
specific NF-kB inhibitor, attenuated LPS- and IFN-stimulated
nuclear translocation of NF-kB as well as steady-state iNOS
mRNA levels, nitrite accumulation and protein synthesis.
Constructs pAiNOS-CAT and pBiNOS-CAT, prepared from the
promoter region of the iNOS gene, linked to a reporter gene
encoding chloramphenicol acetyltransferase and transiently
transfected into MCT cells, conferred inducibility of the
iNOS promoter by LPS but not by IFN. Construct pCiNOS-CAT,
which lacked the critical downstream NF-KB binding site, was
unresponsive to either LPS or IFN. Only construct pAiNOS-
CAT, which contained IFN response elements in addition to the
critical NF-kB binding site, conferred synergistic
inducibility by LPS and IFN, suggesting that this synergism
requires both NF-kB and IFN-induced proteins. In conclusion,
MCT iNOS is regulated at least at two levels:
transcriptionally, by transcription factors including NF-kB
and other IFN-induced proteins, and posttranscriptionally, by
BH4 availability.
IX

CHAPTER ONE
INTRODUCTION
General
Mammalian cells have been known to produce oxides of
nitrogen since 1916 (Mitchell et al. , 1916), an observation
verified and extended in the late 1970s and early 1980s when
it was shown that mammals produce nitrates and that this
production was enhanced by treatment with endotoxin (Stuehr
et al., 1985). To biologists in between these periods, these
compounds held little interest beyond their ability to cure
meat or aggravate pulmonary diseases even though compounds
that release nitric oxide (NO) have been used in clinical
medicine for over 150 years. This lack of interest in NO
could be because both biologists and clinicians were ignorant
of the endogenous mechanisms through which these compounds
exerted their clinical effects.
Biologic NO formation was independently discovered as a
physiologic regulator in blood vessels, macrophages and
neurons (Moneada et al. , 1991). In a classic study,
Furchgott and Zawadzki demonstrated that in vessel segments
precontracted with norepinephrine, acetylcholine elicited
relaxation only if the endothelium was intact, suggesting
endothelial release of a short-lived substance that diffuses
to the smooth muscle to effect relaxation (Furchgott and
1

2
Zawadzki, 1980). This endothelium-derived relaxing factor
(EDRF) , as it was then called, and which had a half-life of
about 6 seconds, was later identified as NO (Palmer et al. ,
1987, Ignarro et al. , 1987) . Over the past few years NO-
producing enzymes have been discovered in virtually every
tissue in numerous species and NO has become established as a
diffusible messenger mediating cell-to-cell interactions
throughout the body including immune cel1-mediated
cytotoxicity, inhibition of platelet aggregation, smooth
muscle relaxation and neuronal signaling (Ignarro et al. ,
1987, Koesling et al., 1991, Bóhme et al., 1978, Radomski et
al., 1987, Mellion et al., 1981).
Isoforms of Nitric Oxide Svnthase
The nitric oxide synthase (NOS) enzyme family, the group
of enzymes that catalyze the biologic synthesis of NO,
comprises at least three distinct isoforms that can be
grouped into the constitutively expressed, Ca2+-calmodulin
dependent NOS isoforms (cNOS) found typically in neurons
(bNOS or NOS Type I) and endothelial cells (eNOS or NOS Type
III), and the immunologically inducible, Ca2+-independent NOS
found typically in macrophages (iNOS or NOS Type II)
(Forstermann et al. , 1991, Stuehr and Griffith, 1992).
Calmodulin binding to cNOS subunits results in the transfer
of electrons from reduced nicotinic adenine dinucleotide
phosphate (NADPH) to a heme group at the active site within
each subunit of cNOS, initiating NO synthesis (Abu-Soud and

3
Stuehr, 1993, Wang et al., 1993). In contrast, calmodulin is
tightly bound to each subunit of iNOS even at very low
intracellular Ca2+ concentrations, an event that probably
occurs during synthesis, resulting in the permanent
activation of the enzyme (Cho et al., 1992). This makes the
addition of exogenous Ca2+ nonessential for iNOS activity.
The induction of iNOS by cytokines and endotoxin is
transcriptionally regulated and is inhibited by
glucocorticoids, interleukin (IL)-4, 8 and 10; transforming
growth factor-f)(TGF-p) ; macrophage deactivating factor; and
protein synthesis inhibitors (Nussler and Billiar, 1993,
Radomski et al. , 1990, Noonan and Noonan, 1977, Junguero et
al. , 1992). It is probably only limited by substrate and
cofactor availability, protein turnover and product
inhibition. All NOS isoforms are homodimers of subunits
ranging from 130 to 160 kDa (Schmidt et al. , 1991, Bredt et
al., 1991, Xie et al., 1992). The N-terminal portion of all
NOS isoforms contains binding motifs for NADPH, flavin-
mononucleotide (FMN) and flavin-adenine dinucleotide (FAD),
showing 50% amino acid identity with each other and 30-40%
overall sequence identity to NADPH-cytochrome P450 reductase
(Dinermann et al., 1993, Schmidt et al., 1993, Stuehr et al.,
1991 [a]). Additional binding sites have been postulated for
tetrahydrobiopterin (BH4), calmodulin and L-arginine (Schmidt
et al., 1993). All isoforms of NOS have consensus sites for
cAMP-dependent phosphorylation. Phosphorylation by protein
kinase C substantially diminishes cNOS activity but

4
phosphorylation by other kinases has no effect (Bredt et al.,
1992, Davda et al., 1994). Endothelial NOS has a
myristoylation site that could account for its association
with membranes thereby probably directing NO delivery to the
underlying vascular smooth muscle cells for the maintenance
of basal vascular tone, whereas other isoforms are largely
cytosolic (Dinermann et al. , 1993).
The capacity to express iNOS exists in nearly every
mammalian tissue and iNOS has been expressed in such diverse
cells as macrophages, microglia, hepatocytes, vascular smooth
muscle, endothelial cells, pancreatic islet cells,
chondrocytes and kidney cells (Bredt et al. , 1991, Chandler
et al. , 1994, Charles et al., 1993, Forstermann et al., 1991,
Geller et al., 1993 [a,b], Lowenstein et al., 1993, Markewitz
et al. , 1993). So far, almost all agents known to induce
iNOS are microbes, microbial products or inflammatory
cytokines, often exhibiting strong synergism between them and
variability between cell types in their responses to the
various inducing agents.
Substrate and Cofactor Requirements
All three isoforms of NOS are hemoproteins and are
therefore dependent on NADPH, FAD and FMN.
Tetrahydrobiopterin is also an essential cofactor of NOS and
is tightly bound to the enzyme (Schmidt et al., 1991, Schmidt
et al., 1993, Tayeh and Marietta, 1989). Tetrahydrobiopterin
synthesis occurs via two distinct pathways: a de novo

5
synthetic pathway that utilizes GTP as a precursor, and a
salvage pathway with dihydrofolate reductase as the rate-
limiting enzyme that utilizes preexisting dihydropteridines
like dihydrobiopterin and sepiapterin. Constitutive NOS
requires only catalytic amounts of BH4 for full activity
whereas iNOS, on the other hand, requires substrate amounts.
There is thus a coinduction of BH4 synthesis with iNOS
induction (Werner-Felmayer et al., 1990).
Electrons donated by NADPH are essential for NO
synthesis (Abu-Soud and Stuehr, 1993, Wang et al., 1993). In
fact, some reports suggest that nitrite production and the
rate-limiting enzyme in the NADPH-generating pentose
phosphate pathway, glucose-6-phosphate dehydrogenase (G-6-
PDH), are coinduced in macrophages (Corraliza et al., 1993).
This implies that metabolic pathways that compete for NADPH
could play an important role in determining the rate of
cellular NO production.
L-Arginine is the only physiologic nitrogen donor for
the production of NO by NOS. L-Arginine can be obtained by
uptake from extracellular fluid, by intracellular protein
degradation or by endogenous synthesis. Several reports
suggest that L-arginine transport increases in macrophages
stimulated to express iNOS and by endotoxin in endothelial
cells (Bogle et al. , 1992, Closs et al. , 1993, Lind et al. ,
1993). Also, endotoxin-stimulated macrophages were more
active in both NO and L-arginine synthesis than in
unstimulated macrophages (Wu and Brosnan, 1992), suggesting

6
that iNOS and L-arginine biosynthetic activities were
coinduced. Thus, L-arginine transport and synthesis may be
rate-limiting for induced NO production. Hence, elucidation
of the biochemical events involved in NO production and
identification of the control points in cofactor and
substrate synthesis and transport have the potential for
providing selective targets for therapeutic control of
cytokine- and endotoxin-induced NO-mediated vascular shock
and tissue damage.
Synthesis of Nitric Oxide
Nitric oxide is synthesized from the terminal guanidino
nitrogen of L-arginine by NOS in a reaction that also
produces stoichiometric amounts of L-citrulline. This
reaction involves the transfer of five electrons from
molecular oxygen (O2) onto the substrate guanidino nitrogen
and requires NADPH, FMN, FAD and BH4 as cofactors. The
oxidation of L-arginine can be divided into two phases.
Phase I involves the hydroxylation of L-arginine to the
intermediate N^-L-hydroxyarginine (OHArg), and consumes one
mole of NADPH. This reaction resembles a classical
cytochrome P450 hydroxylation and may involve the heme iron of
NOS. Phase II involves the oxidation of OHArg to L-
citrulline and NO, consumes another mole of NADPH as reducing
equivalent and clearly depends on BH4. In the absence of L-
arginine and BH4, the activation of molecular O2 by NOS
results in a divalent reduction of O2 to yield superoxide

7
anions (O2 ) and hydrogen peroxide (H2O2) (Zweier et al. ,
1988).
Physiologically, synthesis of NO is regulated in various
ways. Firstly, the major stimulants of physiological
synthesis of NO are hemodynamic forces, notably shear stress
(Awolesi et al., 1993, Nishida et al., 1992). These and
several other agonists can activate NO synthesis within
seconds through pathways that are unaffected by
transcriptional or translational inhibition. Such agents
which include Ca2+ ionophores, excitatory amino acids,
acetylcholine and bradykinin, activate cNOS by eliciting
prompt elevation of intracellular Ca2+ (Ignarro et al., 1987,
Moneada et al., 1991) . Secondly, agents termed inducers
stimulate synthesis of NOS in hours and require mRNA
transcription and protein synthesis (Geller et al. , 1993
[a,b], Moneada et al. , 1991) . These include agents that
induce NO-mediated antitumor or antimicrobial activity such
as cytokines and bacterial lipopolysaccharide (LPS). These
agents typically activate iNOS and some of them interact
synergistically to enhance NO release. A third class of
agents includes those with the capacity to suppress NO
synthesis. A typical example of these agents is tissue
growth factor-p (TGF-(3) which inhibits NO synthesis by
decreasing iNOS mRNA stability and translation, and by
increasing iNOS protein degradation (Vodovotz et al. , 1993,
Junguero et al., 1992). Glucocorticoids may inhibit NO
synthesis through a similar mechanism and NO itself regulates

NO synthesis through a feedback inhibition. As described
elsewhere, substrate and cofactor availability may also play
an important role in the regulation of NO production.
Pharmacologically, there are agents that promote or
inhibit NO release. Some agents mimic NOS by releasing NO.
These agents, termed NO donors, include organic nitrates and
sodium nitroprusside. They have been used clinically for
several years.
There are several classes of NOS inhibitors. These
include: (i) substrate analogues that are N®-subst ituted
analogues of arginine such as Nw-mono-methyl-L-arginine (L-
NMMA) and N®-nitro-L-arginine (NNA); these usually act as
competitive inhibitors of L-arginine when added along with
the necessary cofactors (Moneada et al., 1991); (ii) covalent
modifiers of NOS such as aromatic diphenylene iodoniums that
tend to compete with the nucleotide cofactors for binding to
NOS (Stuehr et al. , 1991 [b] ) ; (iii) carbon monoxide, which
inhibits NOS activity by binding to the heme moiety of the
enzyme (White and Marietta, 1992); (iv) compounds that bind
to calmodulin thereby impeding its binding to NOS, such as
calcineurin and trifluoroperazine; they are therefore
specific for cNOS which is dependent on Ca2+-calmodulin for
activity (Bredt and Snyder, 1990); (v) agents that inhibit
NOS activity by interfering with cofactor or substrate
availability such as substituted pyrimidines (e.g., 2,4-
diaminohydroxypyrimidine, DAHP) ; these inhibit GTP
cyclohydrolase I, the rate-limiting enzyme in BH4 synthesis

9
(Werner-Felmayer et al., 1990); (vi) compounds that interfere
with NOS mRNA transcription by inhibiting critical nuclear
transcription factors; these include dithiocarbamates (e.g.,
pyrrolidinedithiocarbamate, PDTC), which are specific
inhibitors of nuclear factor-KB (NF-kB) (Schreck et al.,
1992), and glucocorticoids, which act through mechanisms
still not fully understood, but probably involve repressing
transactivation by certain nuclear transcription factors that
are required for iNOS mRNA transcription; these compounds are
specific for iNOS.
Roles of Nitric Oxide in Mammalian Physiology
Molecular Targets
Once formed, NO can act as a paracrine substance or as
an intracellular messenger (Palmer et al. , 1987, Ignarro et
al. , 1987). At low concentrations (picomolar amounts), NO
solely stimulates soluble guanylyl cyclase (GC-S) by
nitrosation of its heme. This is currently thought to be the
main mechanism of action of NO on vascular smooth muscle
(Ignarro, 1990). When NO is formed in large quantities
(nanomolar amounts), as is seen in immunological iNOS
activation, in addition to sustained activation of guanylyl
cyclase, it also exerts cytostatic, cytotoxic and
cytoprotective actions in mammalian tissues as well as
antimicrobial activity towards certain pathogens (Saran et
al. , 1990, Lipton et al . , 1993, Karupiah et al. , 1993,
Nussler, 1992). It can react with metalloproteins or thiol

10
groups of other proteins such as ferritin which then acts as
a storage pool for NO (Reif and Simmons, 1990). The induced
(Type II) enzyme remains tonically activated and continuously
produces NO for the life of the active enzyme.
Nitric oxide reacts with O2 in both gaseous and aqueous
phases to form nitrogen dioxides (NO2) (Marietta et al. ,
1990). Nitric oxide formed under reducing conditions as the
free molecule (NO-), reacts with the superoxide anion (O2-)
in aqueous solutions to yield peroxynitrite (ONOO-), which in
part mediates its cytotoxicity (Blough and Zafirou, 1985).
Under oxidizing redox conditions, NO is formed as the
nitrosonium ion (NO+) and reacts with thiol-containing
proteins to form S-nitrosothiols (RS-NO). In N-methyl-D-
aspartate type glutamate (NMDA) receptors, NO reacts with
cell surface thiols to form RS-NOs that afford protection
from receptor-mediated neurotoxicity by down-regulating
harmful receptor-mediated Ca2 + influx into the cells (Lipton
et al. , 1993, Dolye et al. , 1981, Olson et al. , 1981, Saran
et al. , 1990). Nitric oxide readily forms complexes with
transition metal ions and heme-containing proteins like
hemoglobin (Sharma et al. , 1983). These reactions form the
basis for several NO assays. The inhibition by NO of iron-
containing mitochondrial enzymes like aconitase of the Krebs
cycle, succinate-ubiquinone oxidoreductase and NADPH-
ubiquinone oxidoreductase of the electron transport chain
occurs through the nitrosylation of their iron-sulfur (Fe-S)
complexes (Dimmeler et al., 1992, Stuehr et al. , 1989,

11
Drapier and Hibbs, 1988). These reactions may result in
enzyme inactivation, cytotoxicity or cell death. The
tumoricidal effect of NO may be mediated this way and
activated macrophages utilize this as a non-specific immune
defense mechanism against bacterial, protozoal and possibly
viral infections. Nitric oxide also induces ADP-ribosylation
and inhibition of the glycolytic enzyme glyceraldehyde-3-
phosphate dehydrogenase (Bruñe et al., 1990).
The Nervous System
Nitric oxide differs very much from conventional
neurotransmitters in that it is able to diffuse freely from
the point of synthesis to intracellular target sites in
neighboring cells independent of vesicular release, membrane
receptors or lipid cell boundaries. Though normal NO
neuronal actions have not been fully delineated, cerebellar
NOS has been cloned and antibodies to cNOS-related antigens
have been used to identify discreet populations in the
retina, striatum, hypothalamus, midbrain, posterior pituitary
and forebrain of the rat, suggesting a role for NO in
neuronal responses (Bredt et al., 1990, Bredt et al., 1991).
It has also been implicated specifically in some forms of
synaptic plasticity such as long-term synaptic potentiation
(LTP) and long-term synaptic depression (LTD), both of which
are considered elements of experience-driven synaptic network
remodeling underlying learning and memory (Shibuki and Okada,

12
1991, O'Dell et al. , 1991. Bohrne et al. , 1991, Schuman and
Madison, 1991, Nowak, 1992).
Nitric oxide also mediates glutamate neurotoxicity and
neuroprotection, a phenomenon dependent on its redox state
(Lipton et al. , 1993). Reactions of NO with superoxide to
form peroxynitrite (0N00-) can lead to neurotoxicity and NMDA
receptor-mediated neuronal injury due to Ca2 + - dependent
stimulation of NOS can be attributed, at least in part, to
this reaction (Lipton et al, 1993) . In contrast, N0-
containing compounds in alternative redox states may prevent
neurotoxicity by mechanisms involving S-nitrosylation of
critical thiol groups at the NMDA receptor redox modulatory
site to down-regulate channel activity and excessive Ca2+
influx (Lipton et al. , 1993, Dolye et al. , 1981, Olson et
al., 1981, Saran et al. 1990).
Immunocytochemical techniques using anti-cNOS antibodies
have demonstrated the presence of NOS in intestinal myenteric
plexus and adrenal medullary ganglia (Snyder and Bredt,
1991). Non-adrenergic non-cholinergic (NANC) neurons in the
stomach and ileocolonic junction release an NO-like substance
when stimulated and other NO-responsive sites include the
penile corpus carvernosum, stomach, duodenum and esophagus
(Garthwaite, 1991), implying that NO may be playing an
important role as a neurotransmitter in these organs.

13
The Cardiovascular System
Under normal physiologic conditions, basal vasodilator
tone is maintained by endothelial NO released in response to
shear stress on the vessel wall that causes entry of Ca2 +
across the plasma membrane and subsequent activation of cNOS
(Rees et al., 1989). It has also been shown that
inflammatory stimuli can induce iNOS-like activity in the
endothelium and vascular smooth muscle cells in amounts far
exceeding the agonist-triggered NO release from endothelial
and vascular smooth muscle cells (Kilbourn and Bellone, 1990,
Busse and Mulsch, 1990). Recent reports suggest that these
cytokines increase NOS activity by increasing BH4 synthesis
(Rozenkranz-Weiss et al., 1994). Thus, in addition to
maintaining basal vascular tone, NO of endothelial origin may
also play a role in the endotoxin-associated hypotension of
septic shock. Endothelial NO also inhibits platelet
aggregation, leukocyte adhesion, endothelin generation and
smooth muscle cell proliferation (Kubes et al., 1991,
Boulanger and Luscher, 1990, Nakaki et al., 1990). Defective
vasoregulation in diabetes, atherosclerosis and arterial
damage in normal aging may be due, in part, to scavenging of
NO by end products of advanced glycosylation and oxidized
lipoproteins (Bucala et al., 1991, Chin et al. , 1992), and
defects in endothelial NO regulation could lead to vasospasm
and essential hypertension.

14
The Renal System
In the kidney, inducible and constitutive NOS protein or
mRNA have been detected in mesangial cells, macula densa
cells of the juxtaglomerular apparatus, proximal tubule
cells, the entire collecting duct, as well as other
epithelial lining of the nephron (Marsden and Ballerman,
1990, Mundel et al., 1992, Terada et al., 1992, Tojo et al.,
1994 [b], Amoah-Apraku et al., 1993, Markewitz et al., 1993,
Morrissey et al., 1994).
Renal NO is involved in the regulation of renal vascular
resistance (Romero et al. , 1992), medullary and glomerular
microcirculation and macula densa-mediated tubuloglomerular
feedback (Zatz and de Nucci, 1991, Brezis et al. , 1991,
Wilcox et al. , 1992). In addition, active induction of
nitric oxide inhibits proximal tubule Na+/K+-ATPase and ion
transport across renal tubule cell membranes (Guzman et al.,
1995, Stoos et al., 1994). Induced NO production also
inhibits H+-ATPase activity in rat cortical collecting ducts
(Tojo et al. , 1994 [a]), suggesting that NO is probably
involved in the regulation of proton and bicarbonate
transport in the cortical collecting duct. Nitric oxide may
also mediate renal injury during allograft rejection, acute
tubular ischemia/reperfusion injury and autoimmune
glomerulonephritis (Langrehr et al. , 1993 [b] , Yu et al. ,
1994, Weinberg et al. , 1994).

15
In the kidney, the proximal tubule is the major site of
L-arginine synthesis (Levillain et al. , 1993). Also,
increased expression of cytokines occurs in many experimental
and human kidney diseases. Hence, the presence of constant
and abundant substrate coupled with the easy availability of
inducers make iNOS in the kidney a major factor in renal
pathophysiology. Thus, pathways regulating the renal L-
arginine:NO system such as the role of specific transcription
factors and cofactor availability, offer attractive
therapeutic targets.
The Endocrine System
The presence of NOS has been demonstrated in rat
pancreatic p islet cells (Laychock et al., 1991) and insulin
release in response to L-arginine is mediated by NO (Schmidt
et al. , 1992). In mice, administration of streptozotocin
mimics diabetes mellitus, which is abolished by N*°-
substituted L-arginine analogues (Lukic et al . , 1991),
suggesting that this effect may be mediated by NO. It is now
clear that immune-mediated pancreatic islet cell destruction
is attributable to IL-l-induced NO formation (Corbett et al.,
1991). Also, the generation of NO in response to L-arginine
or tolbutamide appears to mediate insulin release in
pancreatic p cells (Laychock et al. , 1991, Schmidt et al. ,
1992). Hence excessive production of NO or defective
responsiveness by NOS may play an important role in the
etiology of diabetes mellitus.

16
The Immune System
That the cytotoxic effects of macrophages are due to
their ability to generate NO is well documented. These
include inhibition of mitochondrial respiration and DNA
synthesis mediated through NO-dependent inhibition of Fe-
containing enzymes (Dimmeler et al. , 1992, Stuehr et al. ,
1989, Drapier and Hibbs, 1988). Macrophage NOS is the
inducible isoform, usually requiring cytokines or bacterial
products to stimulate its activity. In mammals, a
correlation between immunostimulation and elevated nitrate
and nitrite levels has been demonstrated (Stuehr et al. ,
1985) and the L-arginine-NO pathway has been proposed to be
the primary nonspecific defense mechanism against
intracellular microorganisms and other pathogens such as
viruses, fungi, helminths and protozoa (Karupiah et al.,
1993, Alspaugh and Granger, 1991, Vincendeau and Daulouede,
1991, Nussler, 1992).
Expression of iNOS is an inflammatory response to
infection or tissue injury that could be adaptive and
beneficial with respect to its antimicrobial action and
increased tissue perfusion, but it could also be detrimental,
such as occurs in autoimmune diseases or the marked
vasodilation seen in septic shock. Hence, the inhibition by
glucocorticoids of iNOS induction may explain, at least in
part, the therapeutic actions of these compounds in
conditions such as treatment of septic shock, asthma or

17
rheumatoid arthritis, where NO may be responsible for the
pathological vasodilation and tissue damage.
The sensitivity to NO varies from cell to cell, being
cytotoxic in some but cytostatic in others. This may be
explained by the relative abundance and importance of Fe-S
containing enzymes in the various cells or their ability to
buffer the NO molecules with their thiol groups.
Other Cell Types
Hepatocytes have been shown to express iNOS during
inflammation (Geller et al., 1993 [a]) and both
cytoprotective and cytotoxic effects of NO have been
demonstrated in the liver (Billiar et al. , 1990). This
seemingly ambiguous effect may be explained by the cytotoxic
and vasodilatory effects of NO. Hepatocyte glucose and urea
output are also inhibited by endogenously produced NO.
Whereas the former may be attributed to inhibition of key
enzymes in glycogenolysis and gluconeogenesis, the latter is
almost certainly due to the competitive use of the common
substrate L-arginine. Nitric oxide induction in hepatocytes
may contribute to host defense against infections like
malaria (Nussler et al. , 1991). In both tumor cells and
fibroblasts, interferon-y (IFN) has been shown to inhibit
replication through NO generation. Recent reports suggest
that airway epithelial cells contain cNOS and that NO may
play an important role in diverse airway functions such as
maintaining vascular and airway smooth muscle tone, host

18
defense, pulmonary neurotransmission and ciliary motility
(Robbins et al., 1994 [a,b]).
It is likely that other beneficial and deleterious
effects of NO in a variety of cells will be discovered and
that modalities to selectively modulate iNOS expression or
local NO concentrations will be of immense therapeutic
benefit.
Rationale For The Thesis
The inducible isoform of NOS has been shown to be
subject to very strict and high tissue-specific regulatory
control (Nathan and Xie, 1994 [a,b], Nussler et al., 1994).
For example, whereas LPS induces high levels of iNOS
expression in macrophages and microglia (Stuehr and Marietta,
1985, Xie and Nathan, 1994), it induces only modest levels in
hepatocytes, vascular smooth muscle and renal epithelial
cells and a combination with cytokines is usually required
for full expression of iNOS activity (Nussler and Billiar,
1993, Amoah-Apraku et al., 1993). In preliminary studies, I
have established that mouse renal tubule epithelial cells
possess iNOS and that whereas LPS induces only very little
iNOS expression, coincubation with IFN results in a
synergistic expression of high levels of iNOS activity in
these cells (Amoah-Apraku et al., 1993).
I have also established that this was not due to the
lack of cofactor availability, suggesting that IFN may be
acting as a potent modulator of iNOS activity in MCT cells by

19
synergistically potentiating LPS-induced iNOS expression
probably at the level of mRNA transcription. This is
consistent with other studies that showed that single
cytokines stimulated only minor increases in mRNA levels in
rat cultured hepatocytes, but any double or triple
combinations of LPS, IL-1, IFN and tumor necrosis factor
(TNF-a) revealed synergistic increases in mRNA levels (Geller
et al. , 1993 [b] ) . The exact mechanism for this synergism,
however, is currently unknown.
The presence of specific DNA sequences in the promoter
regions of genes that can bind particular proteins confers on
a specific gene the ability to respond to various stimuli.
These DNA sequences play a critical role in producing tissue-
specific patterns of gene expression by binding transcription
factors that may be cytokine-activated but present only in
particular tissues. Hence the binding of these activated
transcription factors to the promoter region of DNA sequence
elements may result in the observed tissue-specific inducible
pattern of iNOS gene expression.
Lipopolysaccharide has been shown to induce the activity
of several of these transcription factors including NF-kB and
activator protein 1 (AP-1) binding proteins (Vincenti et al.,
1992, Eilers et al. , 1993). Interferon-y-inducible factors
include y-interferon activated factor (GAF), which binds to
the y-activated site (GAS), interferon-y regulatory factor 1
(IRF-1), which binds to the interferon-y regulatory factor
element (IRF-E) and other factors that bind to interferon-y

20
stimulated response element (y-IRE) and interferon-a-
stimulated response element (ISRE) (Eilers et al., 1993,
Martin et al. , 1994). Recently, consensus sequences for
binding of several transcription factors involved in
conferring responsiveness to IFN have been identified in the
upstream promoter region of the iNOS gene thereby raising the
possibility of complex and tissue-specific regulation of the
expression of this gene by IFN (Lew et al., 1991, Lowenstein
et al. , 1993, Xie et al. , 1993). These sequences, which
include NF-kB, IRF-1, y-IRE, ISRE, GAS and AP-1 binding sites,
are all potential targets for IFN- or LPS-stimulated
signaling factors (Lew et al., 1991, Lowenstein et al., 1993,
Xie et al. , 1993) and some reports even suggest that LPS-
induced macrophage iNOS expression is dependent on activation
and nuclear translocation of NF-kB (Xie et al. , 1994). In
this study, I therefore focused on the role of NF-kB in the
mechanism of synergistic induction of iNOS activity by IFN
and LPS in proximal tubule epithelial cells.
The central hypothesis of this study was that IFN
potentiates the effects of LPS on iNOS induction by
modulating the LPS-stimulated activation and nuclear
translocation of dimers of NF-kB. I proposed that this
synergism may be due to IFN synergistically potentiating the
activation and nuclear translocation of NF-kB induced by LPS,
or activating different subunits which then dimerize with
those induced by LPS to produce synergistic iNOS gene
transcription. I tested this hypothesis by examining the

21
effects of IFN and LPS on the activation of NF-kB in MCT
cells, and by investigating the effects of eliminating or
including NF-KB and IFN-activated protein binding sites from
the promoter region of the iNOS gene on IFN- and LPS-induced
iNOS expression using a specific promoter-reporter system. I
restricted my studies to evaluating the role of NF-kB and its
main subunits p50, p65 and c-Rel, and also, that of NF-KB and
IFN-activated protein binding sites in IFN- and LPS-induced
iNOS activity in renal epithelial cells. The specific aims
of my study were as described below.
Specific Aims
Specific Aim 1
Characterization of iNOS in MCT cells. I investigated
the time- and concentration-dependence of IFN- and LPS-
stimulated iNOS induction as measured by the rate of [3h]L-
arginine to [3H]L-citrulline conversion, and the accumulation
of nitrites in the culture media of cells treated with the
above agents.
Specific Aim 2
Investigation of the cofactor requirements of LPS- and
IFN-induced iNOS expression in MCT cells. I investigated BH4
requirements, by utilizing 2,4-diamino-6-hydroxypyrimidine
(DAHP), an inhibitor of guanosine triphosphate cyclohydrolase
I (GTPCH), the rate-limiting enzyme in the de novo synthesis

22
of BH4 from guanosine triphosphate. I also investigated the
role of the salvage pathway in which dihydropteridines are
converted to BH4 by dihydrofolate reductase. Experiments
were designed to examine the effects of dihydropteridine
availability and dihydrofolate reductase inhibition using
sepiapterin, a dihydropteridine that is less toxic and is
taken up by cells easily compared to BH4, and methotrexate,
an inhibitor of dihydrofolate reductase.
Specific Aim 3
Identification and characterization of NF-kB as a
signaling element during the activation of MCT iNOS by IFN
and LPS. I investigated the time- and concentration-
dependence of the activation and nuclear translocation of NF-
kB by IFN and LPS using a radio-labeled oligonucleotide
containing a consensus binding site for NF-kB as a probe.
The presence of NF-kB in nuclear extracts was evaluated by
DNA Electrophoretic Mobility Shift Assay (EMSA) (Fried and
Crothers, 1981, Garner and Revzin, 1981). The presence or
absence of the various subunits of NF-kB was investigated in
supershift assays, using specific polyclonal IgG antibodies
against NF-kB subunits p50, p65 and c-Rel.
Specific Aim 4
Demonstration of the requirement for NF-kB and IFN-
activated proteins in iNOS induction using functional
promoter analysis. To achieve this, the following goals were

23
pursued. (i) Cloning of the promoter region of mouse iNOS
from mouse genomic DNA. Two primers selected after careful
study of the promoter region of mac-iNOS, one designed with a
Hind III site at the 5'-flanking end and the other with a Sal
I site at the 3'-flanking end on the complementary DNA
strand, were used to clone the 1.7-kilobase pair (kbp)
promoter region from mouse genomic DNA using the polymerase
chain reaction (PCR). (ii) Preparation of deletion
constructs of the 1.7 kbp and their insertion into a
chloramphenicol acetyltransferase (CAT) basic vector reporter
system (pCAT-Basic). Constructs were prepared to include or
exclude the consensus binding sequences for NF-kB and IFN-
activated proteins to enable me study the role of these
transcription factors on iNOS induction in MCT cells. (iii)
Transfection of the iNOS promoter-pCAT-Basic reporter
constructs into MCT cells and functional analysis of this
reporter as an indicator of iNOS activity. Experiments
included the transfection of pCAT constructs into MCT cells,
and subsequent measurement and comparison of CAT and iNOS
activities in the transfected cells after treatment with the
various inducers or their combinations.
Statistical Analysis
Where indicated, statistical analysis was performed
using Student's t-test for paired and unpaired data, or
analysis of variance (ANOVA) and subsequent Scheffe's F-test
(StatViewâ„¢ II, Abacus Concepts, Inc. Berkeley, CA) for

24
multiple group comparisons as appropriate. The results are
presented as mean + standard error of the indicated number of
experiments performed. Each blot shown is representative of
a series of experiments performed on at least three separate
occasions.

CHAPTER TWO
CHARACTERIZATION OF INDUCIBLE NITRIC OXIDE SYNTHASE IN MURINE
PROXIMAL TUBULE EPITHELIUM
Introduction
Nitric oxide is an important mediator of vascular tone,
neurotransmission and immune responses (Moneada et al. ,
1991). In addition, overproduction of NO has been implicated
in the pathogenesis of several cytotoxic and inflammatory
disorders. Nitric oxide is synthesized from L-arginine by
NOS in a reaction that produces stoichiometric amounts of L-
citrulline and requires NADPH, BH4 and flavin nucleotides as
cofactors. As described earlier, this enzyme family
comprises at least three distinct isoforms that can be
divided into two groups: the constitutively expressed, Ca2+-
calmodulin-dependent cNOS found typically in neurons (bNOS,
Type I) and endothelial cells (eNOS, Type III), and the
immunologically inducible, Ca2+-independent NOS found
typically in macrophages (iNOS, Type II) (Forstermann et al.,
1991, Stuehr and Griffith, 1992).
In the kidney, inducible and constitutive NOS protein or
mRNA has been detected in glomerular mesangial cells, macula
densa cells of the juxtaglomerular apparatus, proximal tubule
cells, the entire collecting duct as well as other epithelial
lining of the nephron (Marsden and Ballerman, 1990, Mundel et
25

26
Apraku et al. , 1993, Markewitz et al. , 1993, Morrissey et
al., 1994). Nitric oxide has also been implicated in several
renal pathophysiologies such as acute interstitial nephritis,
glomerulonephritis and renal transplant rejection (Ketteler
et al. , 1994, Langrehr et al. , 1993 [ a, b ] ) . However, the
mechanisms that regulate NOS in the various renal cells have
not been extensively studied.
My preliminary studies suggest strong synergism between
LPS and IFN in stimulating iNOS activity in renal epithelial
cells although each, by itself, stimulates little or no iNOS
activity (Amoah-Apraku et al . , 1993) . This type of
synergism, which appears to vary among different tissues and
species, results in some cases from cooperative interactions
between iNOS inducers, one of which may lack the ability to
induce the synthesis of cofactors required for full NOS
activity. For instance, whereas IFN stimulates marked NO
production by murine peritoneal macrophages, it is by itself
ineffective in rat aortic vascular smooth muscle (Deng et
al., 1993, Hattori and Gross, 1993, Gross and Levi, 1992)
where it is limited by the inability to coinduce BH4
synthesis (Hattori and Gross, 1993, Gross and Levi, 1992).
This situation is remedied by the addition of LPS which
induces BH4 synthesis. In this study, I investigated the
presence of, as well as the characterization of iNOS in a
murine proximal tubule cell line, MCT cells, and in primary
cultures of rat proximal tubules.

27
Materials and Methods
Chemicals and Biologic Products
Dulbecco's modified Eagle's medium (DMEM), L-arginine-
free Roswell Park Memorial Institute (RPMI) 1640 and all
other cell culture materials were purchased from Fisher
Scientific (Orlando, FL). [3H]L-Arginine (specific activity:
77 Ci/mmol) was purchased from Amersham (Arlington Heights,
IL). AG50WX-8 Dowex (100-200 mesh) was purchased from Bio-Rad
(Melville, NY). Interferon-y (IFN, rat recombinant) was
purchased from Gibco BRL (Gaithersburg, MD) . Radioactive
mac-iNOS-cDNA probes were prepared using a Stratagene (La
Jolla, CA) Prime-IT II random labeling kit. Other biologic
products were from Promega Co. (Madison, WI) .
Lipopolysaccharide (LPS, E coli serotype 026:B6),
dexamethasone, actinomycin D, cycloheximide, sulfanilamide,
sodium nitrite, N-(1-naphthyl)ethylenediamine hydrochloride
and N®-nitro-L-arginine (NNA) were purchased from Sigma
Chemical Co. (St. Louis, MO).
Cell Cultures
Murine proximal convoluted tubule cells (MCT), were
passaged by trypsinization and cultured to confluence in
flat-bottom tissue culture dishes in Dulbecco's modified
Eagle's medium supplemented with 5% heat-inactivated fetal
bovine serum, 25 mM Hepes buffer, 10 mM nonessential amino
acids and 100 fiM sodium pyruvate. The cultures were

28
maintained at 37 °C in a 5% CC>2/95% O2 environment during cell
culture and NOS induction. The MCT cell lines were kindly-
provided by Dr. Eric Neilson (Univ. of Pennsylvania, PA) .
These cells were established from microdissected mouse
proximal tubules after stabilization by transfection with SV
40 virus and maintain many of the properties of
differentiated proximal tubule epithelial cells (Haverty et
al., 1988).
Determination of Nitric Oxide Synthase Activity
Nitric oxide synthase activity in intact MCT cells was
determined by measuring the conversion of [3H] L-arginine to
[3H] L-citrulline after separation of these amino acids by
anion exchange chromatography as previously described (Davda
et al. , 1993). Briefly, control or experimentally treated
MCT cells were washed and then incubated in HEPES buffer
containing (in mM) HEPES 25 (pH 7.4, 37°C) , NaCl 140, KCl
5.4, CaCl2 1.8, MgCl2 1.0, glucose 5.0 and [3H]L-arginine 3
(iCi/ml for periods of time ranging from 0 to 120 min. The
reaction was terminated by washing the cells with ice-cold
Ca2+-free buffer containing 5 mM EDTA followed by addition of
1 ml 0.3 M HCIO4.
After neutralization of the tissue extract with 3.0 M
K2CO3, 50 |¿1 aliquots were taken for measurement of total
uptake of [3H] L-arginine and 500 jj. 1 aliquots applied to
columns containing DOWEX AG50WX8 (Na+ form) added as a 1:1
slurry in water. The columns were washed three times with 2

29
ml of water and the eluant, whose sole radioactive component
was [3h]L-citrulline as determined by thin layer
chromatography, collected into vials. Ten ml of
scintillation cocktail was added to each vial and the
radioactivity quantified by liquid scintillation spectroscopy
(Beckman LS 6000SC). Inducible NOS activity was expressed as
[3h] L-citrulline DPMs per ¡ag of protein. For experiments in
which iNOS activity was measured in crude cell homogenates,
these were obtained from MCT cells grown to confluence in 100
mm dishes. The MCT cells were suspended in ice-cold 50 mM
Tris-HCl containing 0.1% 2-mercaptoethanol, 0.1 mM EGTA and
0.1 mM EDTA, and homogenized on ice by using a glass tissue
grinder with a Teflon pestle in the presence of 1 mM
phenylmethylsulfonyl fluoride (PMSF), 3 |iM leupeptin and 1 |jM
pepstatin A as previously described (Davda et al., 1993).
Aliquots of homogenates containing 100-200 p.g of protein were
added to the assay buffer devoid of Ca2 + and iNOS activity
was measured as described above in the presence of 1 mM
NADPH, 4 jiM flavin-adenine dinucleotide (FAD) , 10 |iM BH4 and 3
[iCi/ml of [3h] L-arginine.
Nitrite Assay
Nitrites were measured by the addition of 200 n.1 of
freshly prepared Greiss reagent [0.75% sulfanilamide in 0.5 N
HC1 and 0.075% N-(1-naphthyl)ethylenediamine dihydrochloride
in double distilled water] to 200 |il of the culture medium
from treated and control cells, incubated with shaking at 25

30
°C in dim light for 10 minutes, followed by
spectrophotometric absorbance readings at 543 nm (Beckman DU
650). Nitrite concentrations were determined by comparisons
with a standard sodium nitrite curve with double distilled
water as blank and expressed as nanomoles per mg of protein.
Protein Assay
Proteins were determined by the method of Lowry (Lowry
et al., 1951).
Northern Analysis
Total RNA was isolated by acid-phenol extraction
(Chomsczyski and Saachi, 1987 ) . The RNA samples were
separated on agarose gel containing 2.2 M formaldehyde,
transferred to Nytran nylon membrane (Schleicher and Schuell,
Keene NH) and cross-linked by exposure to UV light.
Membranes were hybridized with a random prime-labeled
([32p]cJCTP) full-length murine macNOS-cDNA (Xie et al., 1992,
Dinermann et al., 1993) overnight at 55 °C followed by
washing under low stringency conditions (2X SSPE, 0.2% SDS,
65 °C) and a high stringency wash (0.1X SSPE, 65 °C) , and
exposed to an x-ray film. Ribosomal (18s) RNA was used as
gel loading control.
Preparation of Primary Cultures of Rat Proximal Tubules
Male Sprague-Dawley rats weighing 150-175 g were
sacrificed by decapitation and their kidneys removed under

31
sterile conditions and quickly placed in ice-cold Hank's
medium. The proximal tubules were then isolated as
previously described (Haverty et al., 1988, Toutain, 1992).
Light-Microscopic Imrnunocvtochemistrv
Murine proximal tubule cell iNOS immunoreactivity was
detected in cultured cells by the immunoperoxidase method
using a polyclonal antibody raised in rabbit against murine
mac-NOS. The antibody was kindly provided by Dr. Carl
Nathan, Cornell University, NY. It was raised against a
synthetic peptide that was constructed based on the published
mac-NOS sequence (Bogdan et al., 1993, Xie et al., 1992) and
prepared by Dr. Jeffrey R. Weidner and Richard A. Mumford,
Merck, Sharp & Dohme Research Laboratory. The MCT cell
monolayers grown in multichamber slides were fixed in ice-
cold acetone and processed for 1ight-microscopic
immunocytochemistry using the avidin-biotin-horseradish
peroxidase complex (ABC) technique (Vectastain ABC kit,
Vector Laboratories, Burlingame, CA). Endogenous peroxidase
activity was eliminated by incubation with 3% H2O2 for 30 min.
After treatment with blocking serum, the cells were incubated
with the primary antibody against macrophage-type NOS (mac-
NOS) (diluted 1:500) for 60 min. Following incubation with
the primary antibody the cell monolayers were rinsed with PBS
and incubated with the biotinylated secondary antibody for 30
min. After rinsing, the cells were incubated for 30 min with
the Vectastain ABC reagent followed by incubation with the

32
peroxidase substrate solution, diaminobenzidine. The cells
were then counterstained with hematoxylin, examined and
photographed under a light microscope (Zeiss Photomicroscope
II) .
Results
Measurement of Inducible Nitric Oxide Svnthase Activity:
f3H1L-Arqinine to f3h1L-Citrulline Conversion Time Course
To determine the presence of iNOS activity, MCT cells
were treated with a combination of LPS (0.1 (ig/ml) and IFN
(100 U/ml) for 24 h in the presence or absence of Nw-nitro-L-
arginine (NNA, 300 |iM) , followed by measurement of the
conversion of [3H]L-arginine to [3H]L-citrulline over time.
These drug concentrations were selected based on previous
experiments on NOS activity in macrophages done in our
laboratory. Inducible NOS activity was measured after
incubation with [3H]L-arginine for 0, 10, 20, 30, 40, 50, 60
and 90 minutes. Control (untreated) and NNA-treated cells
were also incubated with [3H]L-arginine for 0, 10, 30, 60 and
90 min and iNOS activity measured.
Conversion of [3H]L-arginine to [3H]L-citrulline peaked
after 30 minutes (n=6) in LPS/IFN-induced cells (figure 2-1).
In control cells, there was no significant conversion whereas
NNA effectively blocked the conversion of [ 3H] L-arginine to
[3H]L-citrulline in LPS/IFN-induced cells. Thus, iNOS
activity was shown to be present in the MCT cell line after
induction with LPS and IFN. In subsequent experiments, iNOS

33
activity was determined by measuring the conversion of [3H]L-
arginine to [3H]L-citrulline over 30 min.
Induction Time Course of Inducible Nitric Oxide Synthase
Next, I examined the induction time course of iNOS
activity in MCT cells. Murine proximal tubule cells grown to
confluence in culture medium were incubated with 50 U/ml IFN
in combination with 0.1 p-g/ml LPS for 0, 4, 6, 8, 12 and 24 h
and iNOS activity measured as the conversion of [3H]L-
arginine to [3H]L-citrulline over 30 min.
There was a time-dependent induction of iNOS activity,
peaking to a 357% increase over control at 12 h (figure 2-2).
Thus, the induction of iNOS activity in MCT cells is time-
dependent. This lag period suggests the possibility that
protein synthesis or RNA transcription may be involved in the
induction of iNOS activity.
Effects of Lioopolvsaccharide and Interferon-y on Primary
Culture of Rat Proximal Tubule Cells
To establish that the expression of iNOS in MCT cells
was not an artifact of the transformation process in the MCT
cells, proximal tubules were isolated from rat kidneys as
described above and cultured to confluence. Nitrite
accumulation was then measured after 48 h incubation with IFN
(100 U/ml) or IFN plus LPS (0.1 (ig/ml) in the presence and
absence of NNA (300 jjM) .

34
Interferon-y alone produced a 46% increase in nitrite
accumulation (figure 2-3). This was markedly potentiated by
the addition of LPS (192%) and reversed by treatment with
NNA. This confirms that iNOS activity is present in proximal
tubule cells.
Effects of Cvcloheximide, Actinomvcin D and Dexame" iiasone on
Inducible Nitric Oxide Synthase Activity Induced by
Lipopolvsaccharide and Interferon-y
To test the hypothesis that LPS/IFN-induced iNOS
activity requires protein synthesis or mRNA transcription,
MCT cells grown to confluence were incubated with LPS (0.1
Hg/ml) in combination with IFN (100 U/ml) in culture medium
for 12 h, and in the absence or presence of cycloheximide (20
H.M) , an inhibitor of protein synthesis, actinomycin D (4 |xM) ,
an inhibitor of mRNA transcription, or dexamethasone (20 (iM) ,
which inhibits iNOS induction in macrophages by an unknown
mechanism. Inducible NOS activity was determined as before.
Lipopolysaccharide in combination with IFN caused a 379%
increase over basal iNOS activity (figure 2-4). This induced
iNOS activity was attenuated by cycloheximide (90%, with
respect to LPS/IFN-treated cells), actinomycin D (93%) and
dexamethasone (65%). Thus cycloheximide, actinomycin D and
dexamethasone are able to inhibit the induction of iNOS
activity in MCT cells, demonstrating that this process
requires protein synthesis and mRNA transcription.

35
Svneraistic Effect of Lipooolvsaccharide and Interferon-y
I next examined the interaction between LPS and IFN on
the induction of iNOS activity. Murine proximal tubule cells
were incubated with LPS at concentrations of 0.01, 0.1, 1.0,
and 10 |ig/ml either alone or in combination with 1.0 or 10
U/ml IFN, or IFN at concentrations of 1, 10, 50 and 100 U/ml
alone, or in combination with 0.1 (ig/ml LPS for 24 h.
Inducible NOS activity was determined following the
conversion of [3H]L-arginine to [3H]L-citrulline over 30 min.
Lipopolysaccharide caused only a small increase in iNOS
activity. At 10 ng/ml (the highest concentration studied),
LPS caused a 44% increase in iNOS activity compared to
control (figure 2-5B). However, the effect of LPS was
greatly potentiated by the addition of IFN, for example, 1
and 10 U/ml IFN potentiated the effect of 10 |ig/ml LPS on
iNOS activity, increasing this from 44% to 176% and 545% over
control, respectively. In contrast, IFN alone did not cause
any significant induction of iNOS activity compared to
control (figure 2-5A) . However, combined with 0.1 |itg/ml LPS,
IFN caused a marked concentration-dependent increase in iNOS
activity (7.6-fold increase over control), which peaked at an
IFN concentration of 50 U/ml. Thus, whereas LPS and IFN by
themselves caused little or no induction of iNOS activity in
MCT cells, they synergized to produce a marked induction of
iNOS activity in these cells.

36
Effects of Exogenous Cofactors on Inducible Nitric Oxide
Synthase Activity Induced bv Lipopolvsacchari.de and
Interferon-y
Previous studies have suggested that the inability of
IFN to induce iNOS activity by itself in certain cells like
vascular smooth muscle may be due to lack of coinduction of
BH4 synthesis (Gross and Levi, 1992, Hattori and Gross,
1993). Thus, to determine whether the failure of IFN to
stimulate MCT iNOS activity in the absence of LPS was due to
its inability to induce synthesis of BH4 or lack of other
cofactors, I measured iNOS activity in crude cytosolic
extracts of MCT cells after the in vitro addition of
saturating concentrations of NADP (1 mM) , FAD (4 |iM) and BH4
(10 jiM) .
Inter f eron-y-treated MCT cells failed to show iNOS
activity even when this was measured in vitro after all the
required cofactors had been supplied (figure 2-6). Thus, the
apparent inability of IFN alone to stimulate MCT iNOS
activity does not appear to be primarily due to lack of BH4
synthesis or availability of other cofactors.
Effects of Lipopolvsaccharide and Interferon-y on Nitrite
Production
To correlate my findings with other methods of measuring
iNOS activity, I measured nitrites accumulated by MCT cells
after 48 h incubation with IFN alone (100 U/ml), LPS alone
(0.1 fig/ml) or IFN at concentrations of 1, 5, 10, 50 and 100
U/ml in combination with 0.1 fig/ml LPS.

37
Lipopolysaccharide (0.1 |ig/ml) and IFN(100 U/ml) each
alone caused no significant accumulation of nitrite but there
was a concentration-dependent accumulation of nitrites in
cells treated with IFN in combination with LPS, peaking at
IFN concentration of 100 U/ml, a 30-fold increase over
control (figure 2-7). This pattern shows close correlation
with my measurements of iNOS activity obtained by assaying
[3H]L-citrulline. These results further confirm that iNOS
induction in MCT cells requires both LPS and IFN and is
concentration-dependent.
Effects of Interferon-y and Lipopolysaccharide on the
Expression of Inducible Nitric Oxide Synthase Protein
To investigate the effects of IFN on the expression of
iNOS protein in MCT cells, I performed immunocytochemical
studies using a rabbit polyclonal antibody against murine
mac-NOS. Cultured MCT cells were treated with LPS, IFN or a
combination of both and iNOS immunoreactivity was detected by
the immunoperoxidase method.
Inducible NOS immunoreactivity was absent in control
cells (figure 2-8A). Treatment with a combination of LPS and
IFN for 24 h resulted in intense staining for iNOS (2-8B)
whereas treatment with IFN alone did not elicit iNOS
immunoreactivity (2-8C). In addition, treatment with LPS
alone resulted only in modest staining for iNOS (data not
shown). These results are consistent with my functional iNOS
activity studies, and indicate that the inability of IFN to

38
stimulate the appearance of iNOS immunoreactivity in MCT
cells most likely represents failure of this cytokine to
induce the synthesis of iNOS protein.
Effects of Lioopolvsaccharide and Interferon-v on Inducible
Nitric Oxide Svnthase mRNA
To investigate whether iNOS induction requires an
increase in steady-state mRNA levels, total RNA extraction
was done in cells incubated with LPS, IFN or LPS plus IFN in
the presence or absence of actinomycin D, cycloheximide and
dexamethasone for 4 h and a northern blot analysis done on
the extracts.
No iNOS mRNA was detected in Northern blots of controls
or IFN-treated cells (figure 2-9). However, LPS-treated
cells showed a significant increase in steady-state mRNA
levels first observed after 2 h and persisting for at least
12 h (data not shown). This was markedly potentiated by the
addition of IFN, blocked completely by actinomycin D and to
some extent by dexamethasone. Increases in steady-state iNOS
mRNA levels induced by LPS and LPS/IFN were also blocked by
coincubation with cycloheximide (data not shown). These
results suggest that there is a time-dependent increase in
steady-state iNOS mRNA levels with LPS treatment, and that
IFN, although ineffective by itself, acts in a synergistic
manner to enhance the increase in steady-state iNOS mRNA
levels induced by LPS. This synergism appears to require
protein synthesis. The inhibition of the increased steady-

39
state mRNA levels by actinomycin D suggests a requirement for
mRNA transcription in iNOS induction in MCT cells.
Discussion
In these studies, I have described the characteristics
of a mouse renal proximal tubule inducible isoform of NOS
that shares many but not all the characteristics of the
macrophage iNOS. This proximal tubule iNOS is markedly
induced in a time- and concentration-dependent manner by
treatment with LPS and IFN, but is induced only modestly by
LPS alone. This induction requires protein and mRNA
synthesis and is inhibited by dexamethasone. Murine proximal
tubule cell iNOS activity is also blocked by NNA, a
competitive inhibitor of all NOS isoforms. In contrast to
its effects on mouse peritoneal macrophages (Bogdan et al. ,
1993, Ding et al. , 1988), IFN by itself did not induce the
expression of proximal tubule epithelial iNOS as illustrated
by the absence of iNOS activity, iNOS protein
immunoreact ivity and iNOS mRNA expression in MCT cells
treated with this cytokine. Nevertheless, IFN had a powerful
synergistic effect on LPS stimulation of iNOS expression in
these cells. This synergism, evidenced by the marked
enhancement of LPS-stimulated iNOS activity and iNOS protein
reactivity, is likely to occur at the level of transcription
since IFN also markedly augmented the stimulation of iNOS
mRNA expression induced by LPS. Although this effect could
be due to enhanced transcription or decreased degradation and

40
stabilization of iNOS mRNA, data from recent studies in rat
macrophages and hepatocytes seem to favor the former
mechanism (Nussler and Billiar, 1993) . This is also
supported by the fact that the synergism at the level of mRNA
transcription requires protein synthesis, since
cycloheximide, a protein synthesis inhibitor, appears to
abolish the mRNA synthesis.
Synergistic interactions between IFN and other
immunomodulators in the induction of iNOS activity have also
been described in hepatocytes, lung fibroblasts, vascular
smooth muscle and mesangial cells (Geller et al. , 1993 [b],
Gross and Levi, 1992, Jorens et al. , 1992, Pfeilschifter and
Schwarzenbach, 1990). The molecular mechanisms involved in
the interactions among inducers in NO synthesis are not
completely understood and appear to vary among different
cells and cytokines. Interferon-y treatment does not
stimulate NO synthesis in rat hepatocytes despite inducing
iNOS mRNA transcription. Moreover, IFN has also been
reported to increase BH4 levels in murine macrophages,
fibroblasts, lymphocytes and rat pulmonary artery smooth
muscle by inducing the synthesis of GTP cyclohydrolase I, the
first and rate-limiting enzyme in the de novo pathway for the
synthesis of this cofactor (Di Silvio et al., 1993, Gross and
Levi, 1992, Hattori and Gross, 1993). Conversely, in rat
aortic vascular smooth muscle, IFN potentiates LPS-induced
synthesis of GTP cyclohydrolase I but does not itself
increase the levels of BH4 (Gross and Levi, 1992, Hattori and

41
Gross, 1993). Since iNOS requires substrate concentrations
of BH4 for full enzymatic activity, the ability of a cytokine
to stimulate the synthesis of GTP cyclohydrolase I and that
of a given cell to carry out the synthesis of BH4 are both
critical in determining the magnitude of ensuing NO formation
(Di Silvio et al., 1993, Hattori and Gross, 1993). Thus, in
this study, the lack of stimulation of NO production by IFN
in intact MCT cells could have been due to the inability of
this cytokine to stimulate GTP cyclohydrolase I in a
situation analogous to what is seen in aortic vascular smooth
muscle. Under these circumstances, the addition of LPS could
have stimulated GTP cyclohydrolase I activity and BH4
synthesis thereby providing the missing cofactor. Therefore,
if the failure of IFN to stimulate iNOS activity was due to
lack of BH4, then providing an exogenous source of this
cofactor to cytosolic extracts of IFN-treated MCT cells would
be expected to fully restore iNOS activity. However, iNOS
activity was undetectable in IFN-treated MCT cell homogenates
despite the presence of saturating concentrations of
exogenously added BH4. Furthermore, the absence of iNOS mRNA
and iNOS protein immunoreactivity after IFN treatment is
consistent with the results of my functional studies and
indicates that, in contrast to what is seen in mouse
peritoneal macrophages, IFN does not induce MCT iNOS.
In the kidney, NO may mediate several physiological as
well as pathological functions. Recent reports suggest that
induced NOS decreases Na+/K+-ATPase and H+-ATPase activity

42
and also modulate ion and acid transport across renal
epithelial cells membranes (Guzman et al., 1995, Tojo et al.,
1994 [a], Stoos et al. , 1994). This suggests an important
role for NO in renal ion and acid-base homeostasis. Early
pathological findings in tubulointerstitial nephritis include
necrotic tubule epithelium with interstitial infiltrates of
lymphocytes and plasma cells which are cytokine-secreting
cells. Interferon-y is therefore likely to be an important
mediator of disorders involving T-cell responses such as
allograft rejection, autoimmune glomerulonephritis and
certain forms of tubulointerstitial nephritis, events in
which NO may play a mediating role (Langrehr et al., 1993
[a,b], Weinberg et al., 1994).
The discovery of the presence of iNOS in proximal
tubules has important implications due to the potential
pathophysiologic role of NO during immune-mediated and other
forms of renal injury. Migration of even small numbers of
inflammatory cells into the tubulointerstitium is likely to
lead to high local concentrations of cytokines such as IFN
and TNF-a which can then potentiate each other to induce iNOS
activity and stimulate the release of large amounts of NO by
proximal tubule cells. High concentrations of NO are
cytotoxic due to its conversion into toxic radicals like
peroxynitrites (Nussler and Billiar, 1993). Thus, proximal
tubule iNOS has the potential of playing a critical role in
the modulation of cellular injury during immune-mediated
responses.
Therefore, the existence of several different

43
tissue-specific regulatory signals for the induction of iNOS
may serve as a safeguard to prevent inappropriate or
uncontrolled production of NO. A better understanding of the
molecular mechanisms responsible for the tissue-specific
regulation of iNOS should allow us to design strategies to
prevent the NO-mediated tissue injury associated with various
pathological conditions.
In summary, I have demonstrated the presence of an iNOS
in renal proximal tubules that is markedly induced by a
combination of LPS and IFN, requires protein and mRNA
synthesis and its activity is inhibited by NNA, an analogue
of L-arginine. In contrast to its effects in murine
peritoneal macrophages, IFN by itself does not induce iNOS
expression in proximal tubule cells. It does, however,
markedly potentiate LPS-stimulated NO production by these
cells via mechanisms involving enhanced iNOS mRNA and protein
expression. Studies aimed at understanding the molecular
mechanisms that determine tissue-specific responses of iNOS
to various inducers are necessary for the development of more
rational and specific interventions to prevent NO-mediated
renal tissue injury.

44
a
â– H
3
u
V
â– H
o
I 1 1 1 1 1
0 20 40 60 80 100
Time (min)
Figure 2-1. Time course of conversion of [3H]L-arginine to
[3H] L-citrulline in MCT cells treated with a combination of
LPS (0.1 |¿g/ml) and IFN (100 U/ml) for 24 h. N®-nitro-L-
arginine (NNA, 300 |a,M) , a competitive inhibitor of iNOS
completely abolished LPS/IFN-stimulated iNOS activity (^g: ng
of cell protein, n=6).

45
O 4 8 12 16 20 24 28
Time (h)
Figure 2-2. Time course of induction of iNOS activity in MCT
cells treated with a combination of LPS (0.1 (j.g/ml) and IFN
(100 U/ml). Inducible NOS activity is detectable at 4 h and
reaches maximum at 12 h. Data are mean ± S.E. (p.g: |ig of cell
protein, n=6).

46
4J
â– H
u
V
â– H
a
0)
a
CQ
(1)
NNA
Figure 2-3. Effects of LPS (0.1 ^g/ml) and IFN (100 U/ml) on
rat primary culture proximal tubules. Interferon-y (100
U/ml) induces very little nitrite production. Addition of
LPS (0.1 |ig/ml) markedly potentiates the accumulation of
nitrites, which is inhibited by the addition of NNA (300 |xM) ,
(p < 0.05, mg: mg of cell protein, n=3).

47
Figure 2-4. Effects of protein synthesis inhibitor
cycloheximide (CH, 20 |iM), mRNA transcription inhibitor
actinomycin D (ACT, 4 ^M) and iNOS induction inhibitor
dexamethasone (DEXA, 20 |j.M) on iNOS activity. The MCT cells
were treated with a combination of LPS (0.1 |a.g/ml) and IFN
(100 U/ml) for 12 h in the absence and presence of the
inhibitors. Protein and mRNA synthesis are required for full
iNOS activity (* p<0.05 vs control, p.g: ^g of cell protein,
n=6) .

48
Figure 2-5A. Synergistic interactions of LPS and IFN: IFN
treatment alone (0-100 U/ml for 24 h; - LPS) failed to induce
iNOS activity in MCT cells. Simultaneous addition of LPS
(0.1 (j.g/ml; + LPS) resulted in a dose-dependent stimulation
of iNOS activity by IFN (|xg: (a.g of cell protein, n=6) .

49
0)
tí
•H
tí
U
4J
â– H
u
I
tn
tí.
e
a
•o
0 .01 0.1 1.0 10
LPS (|Xg/ml)
Figure 2-5B. Synergistic interactions of LPS and IFN in MCT
cells: LPS treatment alone (0-10 (xg/ml for 24 h; - IFN)
caused minimal induction of iNOS activity. Simultaneous
addition of IFN (1 and 10 U/ml) markedly potentiated the
effect of LPS (|ig: jxg of cell protein, n=9).

50
Figure 2-6. Inducible NOS activity in crude homogenates of
MCT cells after 24 h of treatment with LPS (0.1 ng/ml), IFN
(100 U/ml) or a combination of both. In vitro iNOS activity
assay was performed in the presence of saturating
concentrations of NADPH, BH4, FAD and FMN. Even in the
presence of saturating concentrations of exogenously added
cofactors, IFN failed to induce iNOS activity (* p<0.05 vs
control, M-g: |og of cell protein, n=6) .

(nmoles/mg)
51
250
200 -J
150 -A
100 -A
50 -A
Basal LPS IFN 1
0.1 ng/ml 100 U/ml
10
50 100
LPS (0.1 |ig/ml) + IFN (U/ml)
Figure 2-7. Concentration dependence of the effect of IFN
(0-100 U/ml for 48 h) on nitrite accumulation in the presence
of LPS (0.1 (j.g/ml) in MCT cells, (* p<0.05 vs control, mg: mg
of cell protein, n=3).

52
Figure 2-8. Immunoreactivity of iNOS protein in cultured MCT
cells. A: Control MCT cells; B: MCT cells treated with a
combination of IFN (100 U/ml) and LPS (1 |og/ml) for 24 h; C:
MCT cells treated with IFN alone for 24 h. Inducible NOS was
detected by an immunoperoxidase method using a polyclonal
antibody raised in rabbit against mouse mac-NOS. Cells were
stained with hematoxylin. There is intense immunoreactivity
in cells treated with LPS/IFN compared to control or IFN-
treated cells. Picture is representative of three separate
experiments. Magnification x430.

53
9.5-
7.5-
Figure 2-9. Effects of IFN (100 U/ml) and LPS (1 (ig/ral) on
iNOS steady-state mRNA levels after 4 h of treatment in MCT
cells. Interferon-y enhances the effect of LPS but does not
by itself increase iNOS mRNA levels. Ribosomal (18s) RNA was
used as gel loading control which was comparable in all
lanes. Blot is representative of three experiments. Act:
actinomycin D (4 nM) , Dex: dexamethasone (20 (iM) .

CHAPTER THREE
GUANOSINE TRIPHOSPHATE CYCLOHYDROLASE I REGULATES NITRIC
OXIDE SYNTHESIS IN RENAL PROXIMAL TUBULES
Introduction
Tetrahydrobiopterin is an essential cofactor of NOS
because it is needed in the formation of the active dimeric
enzyme from iNOS monomers (Tayeh and Marietta, 1989, Baek et
al. , 1993) and is tightly bound to the enzyme (Schmidt et
al. , 1993). With the exception of macrophages, many cells
that possess iNOS have low BH4 in the resting state and NO
production is limited by their ability to synthesize this
cofactor (Di Silvio et al. , 1993, Werner-Felmayer et al. ,
1990) .
In proximal tubule epithelial cells, there is marked
synergism between LPS and IFN in stimulating iNOS activity.
However, unlike what is observed in macrophages, each of
these inducers, by itself, stimulates little or no iNOS
activity in these cells (Amoah-Apraku et al., 1993, Markewitz
et al., 1993). Synergism between cytokines and endotoxin in
their effects on the induction of iNOS expression has been
commonly observed in many other tissues (Blough and Zafirou,
1985, Deng et al. , 1993, Pfeilschifter and Schwarzenbach,
1990, Szabó et al. , 1993). This synergism, that appears to
vary among different tissues and species, results in some
54

55
cases from cooperative interactions between iNOS inducers,
one of which may lack the ability to induce the synthesis of
cofactors required for full NOS activity. For instance,
whereas IFN stimulates marked NO production by murine
peritoneal macrophages, it is by itself ineffective in rat
aortic vascular smooth muscle (Deng et al., 1993, Hattori and
Gross, 1993, Gross and Levi, 1992). The latter appears to be
in part due to the inability of IFN to induce synthesis of
BH4 in this tissue (Hattori and Gross, 1993, Gross and Levi,
1992). Under these circumstances, the addition of LPS
results in induction of BH4 synthesis and leads to marked
concentration-dependent stimulation of NO production by IFN.
In murine macrophages, fibroblasts, lymphocytes and rat
pulmonary artery smooth muscle cells, IFN has been shown to
stimulate GTP-cyclohydrolase I induction (Werner et al.,
1989, Ziegler et al., 1990). This enzyme catalyzes the
cleavage of GTP to 7,8-dihydroneopterin triphosphate, the
rate-limiting step in BH4 synthesis. It is important to note
that NO formation from L-arginine is the only known BH4-
requiring reaction in which IFN plays an important role. It
can therefore be argued that cytokines like IFN probably
induce the formation of BH4 in order to provide a cofactor
for the generation of NO. In other circumstances, the
synergism between cytokines and endotoxin has been attributed
to less well-characterized interactions at the iNOS gene
transcription level (Geller et al., 1993 [b]).

56
Tetrahydrobiopterin synthesis occurs via two distinct
pathways (figure 3-1, Werner et al. , 1993): (i) a de novo
synthetic pathway that utilizes GTP as a precursor and in
which GTP cyclohydrolase I (GTPCH) is the rate-limiting
enzyme. This is the main source of BH4 for iNOS in vascular
smooth muscle and hepatocytes (Stuehr and Griffith, 1992);
and (ii) a salvage pathway in which preexisting
dihydropteridines like dihydrobiopterin and sepiapterin are
converted to BH4 by dihydrofolate reductase. Constitutive
NOS requires only catalytic amounts of BH4 for full activity
whereas iNOS, on the other hand, requires substrate amounts.
There may therefore be a coinduction of BH4 synthesis with
iNOS induction in many cells.
Because only low levels of GTPCH activity are present in
the kidney and its role in providing BH4 for processes such
as renal aromatic amino acid hydroxylation has not been
demonstrated (Bellahsene et al., 1984, Rao and Kaufman, 1986,
Davis et al., 1992), the main function of renal GTPCH remains
to be defined. The presence of iNOS has recently been
reported in proximal tubule and collecting duct epithelium
(Markewitz et al., 1993, Amoah-Apraku et al., 1993). Because
NO may mediate renal injury during allograft rejection,
autoimmune glomerulonephritis (Langrehr et al. , 1993 [a,b],
Weinberg et al. , 1994), Na+/K+-ATPase and H+-ATPase activity
and therefore ion and acid transport across the renal tubule
epithelium (Guzman et al., 1995, Tojo et al,. 1994 [a], Stoos
et al., 1994), it will be important to identify potential

57
therapeutic targets in its synthetic pathway. Hence,
understanding the biochemical events involved in renal
epithelial NO production and elucidating the control points
in BH4 synthesis has the potential of providing selective
means of therapeutic control of cytokine- and endotoxin-
induced NO-mediated renal tissue damage. In this study, I
investigated the role of GTPCH in the regulation of NO
synthesis by proximal tubule epithelium.
Materials and Methods
Chemicals and Biologic Products
Dulbecco's modified Eagle's medium (DMEM), L-arginine-
free Roswell Park Memorial Institute (RPMI) 1640 and all
other cell culture materials were purchased from Fisher
Scientific (Orlando, FL) . Interferon-y (IFN, rat
recombinant) was purchased from Gibco BRL (Gaithersburg, MD).
Lipopolysaccharide (LPS, E coli serotype 026:B6),
sulfanilamide, methotrexate, 2,4-diamino-6-hydroxypyrimidine
(DAHP) , sodium nitrite and N-(1-naphthyl)ethylenediamine
hydrochloride were purchased from Sigma Chemical Co. (St.
Louis, MO). Sepiapterin was purchased from Dr. B. Schircks
(Jonas, Switzerland) and 5,6,7,8-tetrahydro-L-biopterin was
purchased from Research Biochemicals Inc. (Natick, MA).

58
Nitrite and Protein Assays
These assays were done as previously described in
Chapter Two.
Results
Effect of 2,4-Diamino-6-hvdroxvpvrimidine on Murine Proximal
Tubule Cell Nitrite Production
To examine whether GTPCH, the rate-limiting enzyme in
the de novo synthesis of BH4 was required for NO synthesis, I
treated cells with a combination of LPS (0.1 ng/ml) and IFN
(100 U/ml) for 12 h in the presence or absence of the
specific GTPCH inhibitor DAHP (6 mM) followed by measurement
of nitrite accumulation in the culture medium.
Treatment with DAHP suppressed LPS/IFN-induced nitrite
accumulation by 53.1 + 3.4% (figure 3-2), indicating that NO
synthesis by MCT iNOS requires GTPCH activity.
Effects of Seoiaoterin and Methotrexate on Murine Proximal
Tubule Cell Nitrite Production
To investigate the contribution of the pterin salvage
pathway to the synthesis of BH4 for NO production, I used
sepiapterin, a dihydropteridine substrate for this pathway,
and methotrexate, an inhibitor of dihydrofolate reductase.
Experiments were designed to examine the effects of
dihydropteridine availability and dihydrofolate reductase
inhibition on NO synthesis in MCT cells treated as described
above.

59
Sepiapterin (5 mM) restored NO synthesis during GTPCH
blockade (figure 3-3), indicating that BH4 can be synthesized
via the salvage pathway in MCT cells. Sepiapterin alone
increased nitrite production in induced cells by 12.0 + 0.8%
(figure 3-3), suggesting that NO synthesis in MCT cells is
limited by their ability to synthesize BH4. Methotrexate
alone (10 mM) did not alter NO production in induced MCT
cells (figure 3-4), suggesting that iNOS does not normally
depend on the salvage pathway for BH4. However, methotrexate
did prevent the effect of sepiapterin during GTPCH blockade
(figure 3-4), confirming that sepiapterin is acting through
the salvage pathway.
Concentrations of sepiapterin between 1 and 8 mM
restored NO synthesis in a dose-dependent manner, whereas
concentrations higher than 10 mM had inhibitory effects
(figure 3-5). Thus, these results suggest that DAHP inhibits
GTPCH only partially and that, as reported for other cells
(Gross and Levi, 1992)), sepiapterin, in addition to being a
substrate for BH4 synthesis via the pterin salvage pathway,
is also a potent inhibitor of GTPCH in proximal tubules. At
incubation periods of 36 h and 48 h, the sepiapterin rescue
was less than expected (data not shown) . This may be
because, at longer incubation periods, as the exogenously
added sepiapterin becomes depleted, the rescue then becomes
limited by the availability of dihydropterines. Due to the
inhibitory effect of high concentrations of sepiapterin,

60
saturating amounts could not be added to ensure constantly
available dihydropterines.
Discussion
In these studies, I have demonstrated that GTPCH is
required for NO production by proximal tubule epithelium and
that, when this enzyme is inhibited exogenous
dihydropteridines can restore NO synthesis through their
conversion to BH4 via the salvage pathway. This contrasts
with macrophage iNOS, which is fully active at levels of BH4
present in the resting state (Kwon et al. , 1989, Nathan,
1994). On the other hand, MCT cells resemble hepatocytes,
vascular smooth muscle, fibroblasts and endothelial cells in
their requirement for BH4 synthesis during cytokine-induced
NO synthesis (Di Silvio et al., 1993, Werner-Felmayer et al.,
1990, Gross et al., 1991, Gross and Levi, 1992). Nitric
oxide synthesis by human endothelial cNOS is also limited by
BH4 (Werner-Felmayer et al. , 1993, Rosenkranz-Weiss et al. ,
1994). In several of these cells, an inducible GTPCH is
coinduced with iNOS (Di Silvio et al. , 1993, Gross et al. ,
1993). The design of this study however, does not allow
differentiation between inducible and constitutive isoforms
of GTPCH.
While confirming a similar requirement for de novo BH4
synthesis by MCT iNOS as in by several other nonrenal cells
(Di Silvio et al., 1993, Werner-Felmayer et al., 1990, Gross
and Levi, 1992), this study also identifies a major role for

61
GTPCH in the renal parenchyma. Previous studies (Bellahsene
et al., 1984) have found GTP 8-formylhydrolase and neopterin
synthetic activities of GTPCH in rat kidneys, but the
physiologic significance of this remains unclear. In liver
and catecholamine-producing organs, GTPCH provides BH4 for
aromatic L-amino acid hydroxylation (Kaufman, 1993).
Phenylalanine hydroxylase activity has also been detected in
the kidney, but its function is unknown (Rao and Kaufman,
1986, Richardson et al. , 1993). The renal hydroxylase
differs from the liver isoform in that it exists natively in
a very high state of activation of about 16-fold greater in
activity (Rao and Kaufman, 1986) . However, it is not known
whether this activity is supported by BH4 synthesized de novo
or regenerated from dihydropteridines.
The regulation of NO synthesis by GTPCH and BH4
availability may be important therapeutically because it
provides a target for the blockade of the renal L-arginine:N0
system. Nitric oxide is a mediator of inflammation in
glomerulonephritis (Weinberg et al. , 1994, Marsden and
Ballermann, 1990, Shultz et al. , 1990) and kidney allograft
rejection (Langrehr et al. , 1993 [a,b]) and of tubular
hypoxia/reoxygenation injury (Yu et al., 1994). On the other
hand, NO causes renal vasodilation, increases renal cortical
bloodflow (Shultz et al., 1993), protects against glomerular
thrombosis in endotoxemia and pregnancy (Shultz and Raij,
1992, Raij, 1994), and modulates T-cell proliferation
(Langrehr et al, 1993 [a]). It is therefore possible that

62
the cytoprotective effects of proximal tubule NO could be
enhanced by increasing its synthesis with exogenous
dihydropteridines.
Nitric oxide synthase antagonists such as the L-arginine
analogues have been partially successful in limiting the
hypotension of endotoxic shock (Petros et al. , 1994).
However, pulmonary hypertension, impaired cardiac output, and
decreased survival have been reported with the use of these
agents, possibly because of their lack of selectivity (Petros
et al., 1994, Minnard et al., 1994, Robertson et al., 1994).
On the basis of the different cell requirements for BH4
synthesis,
the inhibition
of
GTPCH
could
theoretically
provide an
alternative way
to
reduce
renal
NO production
while preserving normal macrophage and endothelial function.
In summary, full functional expression of iNOS in murine
proximal tubule epithelium requires GTPCH activity and the de
novo synthesis of BH4. In the absence of this, iNOS activity
is dependent on the availability of exogenous
dihydropteridines that are converted to BH4 via the salvage
pathway. The blockade of BH4 synthesis therefore constitutes
a promising therapeutic alternative for disorders in which
excessive NO production contributes to renal injury.

63
©
GTP
GTP Cyclohydrolase I
(Blocked by DAHP)
Sepiapterin
Dihydroneopterin triphosphate
I
I
I
BH2
'bihydrofolate reductase
(Blocked by Methotrexate)
BH4
Figure 3-1. Tetrahydrobiopterin (BH4) synthetic pathways:
(I) de novo synthesis, and (II) dihydrofolate-dependent
salvage pathway (BH2, dihydrobiopterin).

64
Figure 3-2. Effect of DAHP on nitrite production by MCT
cells; (L/I: LPS plus IFN). Data are mean + S.E. (* p < 0.05
vs L/I-treated, mg: mg of cell protein, n=9).

65
CQ
d)
4J
â– H
n
V
â– rt
0)
a
CQ
0)
40
LPS/IFN
-
+
+
+
+
DAHP
-
-
-
+
+
Sepiapterin
-
-
+
-
+
Figure 3-3. Effects of sepiapterin on nitrite production by
MCT cells in the absence or presence of DAHP, (mg: mg of cell
protein, n=9).

66
DAHP + +
Sepiapterin - + +
Methotrexate + . +
Figure 3-4. Effects of methotrexate and sepiapterin on
nitrite production during GTPCH blockade with DAHP in MCT
cells. Cells were treated with LPS (0.1 |ig/ml) and IFN (100
U/ml) for 12 h in the presence of the indicated drugs.
Nitrite concentrations in culture medium from control cells
were less than 2 nmol/mg of protein per 12 h, (mg: mg of cell
protein, n=9).

67
140 -i
Figure 3-5. Effects of sepiapterin on nitrite production by
MCT cells (48 h of induction) during GTPCH inhibition with
DAHP (6 niM) , CON, Control, L/I, LPS plus IFN, (n=3).

CHAPTER FOUR
NUCLEAR FACTOR-kB REGULATES INDUCIBLE NITRIC OXIDE SYNTHASE
IN RENAL TUBULE EPITHELIUM
Introduction
Expression of iNOS is subject to strict and highly
tissue-specific transcriptional regulatory control (Nathan
and Xie, 1994 [a,b], Nussler et al. , 1994 ). For example,
whereas LPS induces high levels of iNOS expression in
macrophages and microglia (Stuehr and Marietta, 1994, Xie et
al., 1993, Chandler et al. , 1994), it induces only modest
levels of iNOS in hepatocytes, vascular smooth muscle and
renal epithelial cells and a combination of cytokines is
usually required for full expression of iNOS activity
(Nussler and Billiar, 1993, Szabó et al., 1993, Amoah-Apraku
et al. , 1993). Thus, the expression of iNOS appears to be
regulated differently in parenchymal cells compared to cells
such as macrophages and microglia whose main role is to
mediate immune responses.
Recent studies describe the presence in the murine
macrophage iNOS promoter of consensus sequences for the
binding of endotoxin and IFN-stimulated signaling proteins
including NF-kB and interferon-y regulatory factor (IRF-1)
(Xie et al. , 1993), which may potentially constitute the
basis for the synergism observed between iNOS inducers. It
68

69
has also been reported that the induction of macrophage iNOS
is dependent on the activation and nuclear translocation of
NF-kB (Xie et al. , 1994).
Nuclear factor-KB proteins are a ubiquitous group of
transcription factors that exist mainly as inactive dimers in
the cytoplasm of cells bound to inhibitory proteins known as
inhibitors of kappa-B (IkB) (Muller et al., 1993). The most
important subunits of NF-kB are p50, p65 and c-Rel. Upon
cell activation, IkB dissociates from the NF-kB dimer, through
a process requiring both phosphorylation and proteolysis,
allowing this to migrate to the nucleus where it binds to its
DNA recognition site in the promoter region of various genes
to initiate transcription (Muller et al. , 1993, Henkel et
al. , 1993) . The induction of macrophage iNOS by LPS is
dependent on the nuclear translocation of NF-kB heterodimers
containing mainly c-Rel and p65 subunits of NF-kB (Xie et
al., 1994). The c-Rel component of NF-kB had previously been
reported to have only modest transactivating activity (Muller
et al., 1993). Therefore, transactivation of the iNOS gene
constitutes an important novel role for this NF-kB protein in
macrophages.
Renal tubule epithelium has recently been found to
express iNOS but the regulation of this enzyme in the kidney
has not been extensively investigated (Amoah-Apraku et al.,
1993, Markewitz et al., 1993). In this study I examined the
role of NF-kB in the induction of renal epithelial iNOS and

70
characterized the NF-kB proteins activated by LPS and IFN in
this tissue.
Materials and Methods
Chemicals and Biologic Products
Dulbecco's modified Eagle's medium (DMEM), L-arginine-
free Roswell Park Memorial Institute (RPMI) 1640 and all
other cell culture materials were purchased from Fisher
Scientific (Orlando, FL) . Interferon-y (IFN, rat
recombinant) was purchased from Gibco BRL (Gaithersburg, MD).
Radioactive mac-iNOS-cDNA probes were prepared using a
Stratagene (La Jolla, CA) Prime-IT II random labeling kit.
Other biologic products were from Promega Co. (Madison, WI).
Lipopolysaccharide (LPS, E coli serotype 026:B6),
sulfanilamide, pyrrolidinedithiocarmate (PDTC), sodium
nitrite, and N-(1-naphthyl)ethylenediamine hydrochloride were
purchased from Sigma Chemical Co. (St. Louis, MO).
Cell Cultures. Northern Blot Analysis. Nitrite and Protein
Assays
These procedures were carried out as previously
described in Chapter Two.
Preparation of Nuclear Extracts
Nuclear extracts were prepared by a modification of a
previously described procedure (Lew et al., 1991). Briefly,
cells cultured to confluence were treated with inducers at

71
specific concentrations and for specific incubation periods.
The cells were then harvested by scraping in ice-cold PBS and
centrifuged at 500 g for 10 min and homogenized in two packed
cell volumes (PCV) of hypotonic buffer (Tris-HCl 10 mM, pH
7.4, KCl 10 mM, MgCl2 3 mM, Dithiothreitol (DTT) 1 mM, PMSF
0.5 mM, leupeptin 2 nM) using a glass tissue grinder with a
Teflon pestle. The homogenates were centrifuged at 500 g for
10 min to pellet the nuclei. The nuclear pellet was
resuspended in 3 ml/109 cells of extraction buffer (Tris-HCl
10 mM, pH 7.4, KCl 0.3 M, MgCl2 3 mM, DTT 1 mM, PMSF 0.5 mM,
leupeptin 2 pM, glycerol 20% v/v) and kept on ice for 45 min,
with gentle rocking. The extract was then clarified by
centrifugation at 25000 g for 30 min. The clear supernatant
was then dialyzed against 50 volumes of dialysis buffer
(Tris-HCl 10 mM, pH 7.4, KCl 0.1 M, DTT 1 mM, PMSF 0.5 mM,
leupeptin 2 pM, EDTA 0.2 mM, glycerol 20% v/v) for 1 h. The
dialyzed solution was centrifuged at 25000 g, aliquoted and
stored at -70 °C for subsequent use.
Oligonucleotide Probes and DNA Gel Mobility Assay
The NF-kB consensus DNA binding site is formed by the
general decameric motif 5'-GGGRNNTYCC, where R is a purine, Y
a pyrimidine and N either of them (Muller et al., 1993, Bours
et al. , 1992). I utilized a double-stranded consensus
oligonucleotide containing the sequence 5'-GGGACTTTCC and
radio-labeled by phosphorylation with [y-32p]ATP (3000

72
Ci/mmol) using T4 polynucleotide kinase (Promega, Madison,
WI) following standard protocols (Sambrook et al. , 1989).
Electrophoretic mobility shift assays (EMSA) were
performed following previously published protocols with minor
modifications. The binding reaction was carried out by
adding 10 |ig nuclear extract to gelshift binding 5X buffer
[Tris-HCl 10 mM, pH 7.5, MgCl2 1 mM, EDTA 0.5 mM, DTT 0.5 mM,
NaCl 50 mM, glycerol 4% and poly(dl-dC)-poly(dl-dC) 50 mg/ml]
containing [y-32p]ATP-labeled NF-kB oligonucleotide probe in a
total volume of 10 |il, incubated at room temperature for 20
min and the reaction terminated by addition of 1 |xl gel
loading 10X buffer (Tris-HCl 250 mM, pH 7.5, bromophenol blue
0.2%, xylene cyanol 0.2%, glycerol 40%). The reactions were
then ran on a 4% nondenaturing acrylamide gel preran for 30
min before loading, for approximately 3 h at 100 V. The gel
was wrapped in plastic and exposed to x-ray film overnight.
Individual NF-kB subunits were identified by the presence of
supershift or the disappearance of the original NF-kB
complexes in EMSA after addition of specific polyclonal IgG
antibodies against p50, p65 and c-Rel (Santa Cruz
Biotechnology, Santa Cruz, CA) to the binding reaction.
Western Analysis
Expression of iNOS protein was determined by Western
analysis using a monoclonal antibody against macrophage iNOS
(Transduction Laboratories, Lexington, KY) and following
standard protocols. Briefly, MCT cells were lysed in culture

73
dishes in 20 mM Tris-HCl, pH 7.5, containing 0.1 mM PMSF, 2
mM EDTA, 0.5 mM EGTA, 10 mg/ml leupeptin and 1 mg/ml
aprotinin, centrifuged at 1000 rpm for 5 min and homogenized
on ice with 30 strokes in an Eppendorf tube with a disposable
homogenizer. Cellular proteins were solubilized in SDS,
separated by discontinuous polyacrylamide gels (5% stacking,
7.5% resolving gel, 40 mA for 5 h), and transferred to a 0.45
jim nitrocellulose membrane in 25 mM Tris, pH 8.3, 150 mM
glycine, and 20% vol/vol methanol for 15 h at 100 mA. The
nitrocellulose membrane was blocked with 10% nonfat dry milk
in PBS for 2 h and subsequently incubated with mouse anti-
mac-NOS monoclonal antibodies (1/250) for 4 h at 20 °C .
After washing the membrane to remove excess antibody,
peroxidase-labeled anti-mouse secondary antibodies were
applied and the protein of interest identified by enhanced
chemiluminiscence (ECLâ„¢, Amersham Corporation, Arlington
Heights, IL).
Results
Effects of L i popo lvsac char ide and Interferon-y on Nitrite
Accumulation in Murine Proximal Tubule Cells
To investigate the effects of LPS and IFN on nitrite
accumulation in MCT cells, I measured accumulated nitrite by
MCT cells after 24 h incubation with LPS (1 |ig/ml), IFN (100
U/ml) or in combination.
Incubation of MCT cells with LPS (1 ng/ml for 24 h)
stimulated only modest nitrite production (figure 4-1).

74
Interferon-y (100 U/ml), which by itself was also ineffective
in stimulating nitrite production, potently synergized with
LPS to stimulate iNOS activity (figure 4-1).
Effects of Lipopolvsaccharide and Interferon-v on Inducible
Nitric Oxide Svnthase mRNA
To investigate the effects of LPS and IFN on steady-
state iNOS mRNA levels, total RNA extraction was done in
cells incubated with LPS, IFN, or LPS plus IFN. A northern
blot analysis was then done on the extracts.
Combined treatment with LPS and IFN was accompanied by
marked time-dependent increase in steady-state iNOS mRNA
levels that were initially detected after 2 h of treatment,
peaked at 6 h and decreased after 24 h (figure 4-2) .
Lipopolysaccharide treatment (1 |ig/ml for 6 h) caused only a
small increase in steady-state iNOS mRNA levels whereas none
was detected when the cells were treated with IFN alone
(figure 4-2) .
Effects of Lipopolvsaccharide and Interferon-v on Inducible
Nitric Oxide Svnthase Protein Levels
To determine the roles of LPS and IFN in inducing iNOS
protein synthesis, cells were treated with LPS (1 ng/ml)
alone or in combination with IFN (100 U/ml) and iNOS protein
was determined by Western analysis using a monoclonal
antibody against macrophage iNOS.
Despite the moderate amount of iNOS mRNA induced by LPS
(figure 4-2), it failed to induce detectable levels of iNOS

75
protein (figure 4-3). Conversely, the combination of LPS and
IFN resulted in marked increases in iNOS protein (figure 4-3)
and nitrite accumulation (figure 4-1).
Effects of Lioopolvsaccharide and Interferon-y on Nuclear
Factor-KB Activation
To determine whether either LPS or IFN can activate NF-
kB in renal epithelium, I extracted nuclear proteins from MCT
cells treated with each of these agents alone or in
combination, and analyzed these extracts by EMSA using a
radio-labeled NF-kB consensus oligonucleotide as a probe.
As illustrated in figure 4-4, treatment with LPS or IFN
alone caused rapid nuclear translocation of NF-kB that
appeared by 15 min and was noticed at all time points studied
up to 24 h (data not shown). However, LPS appeared to be the
more efficacious activator of this transcription factor.
Moreover, the addition of IFN did not potentiate the
activation of NF-kB caused by LPS. The effect of LPS and IFN
on the nuclear translocation of NF-kB does not appear to be
concentration- or time-dependent after the initial nuclear
appearance within the concentration range and time interval
studied (data not shown).
Effect of Pyrrolidinedithiocarbamate on Nuclear Factor-KB
Activation. Inducible Nitric Oxide Synthase mRNA and Protein
To investigate the potential role of NF-kB in the
induction of iNOS expression by LPS and IFN, I studied the

76
effect of the NF-kB inhibitor PDTC on NF-kB activation, iNOS
protein and steady-state mRNA levels.
As shown in figure 4-5, treatment with PDTC (25 mM)
inhibited or attenuated the activation of NF-kB by LPS, IFN
or their combination. In addition, PDTC abolished the
synergism between LPS and IFN on iNOS steady-state mRNA
levels (figure 4-6A), blunted the expression of iNOS protein
(figure 4-6B), and completely prevented the formation of
nitrites induced by these agents (figure 4-7). Taken
together, these results suggest that NF-KB is necessary for
the induction of iNOS activity by LPS and IFN in renal
epithelium, but its activation alone does not explain the
synergism seen between these two inducers. Also, they
indicate that NF-kB activation is not sufficient for the
expression of this enzyme since neither IFN nor LPS alone is
able to induce iNOS activity despite activating NF-kB.
Effects of Nuclear Factor-KB Antibodies on Nuclear Factor-KB
Activation bv Lioopolvsaccharide and Interferon-y
To identify the components of NF-kB activated by LPS in
renal epithelium and compare them to those activated in
macrophages where LPS is known to be a potent inducer of
iNOS, I used IgG against the three major NF-kB subunits p50,
p65 (Rel-A) and c-Rel in an EMSA.
Consistent with previous reports (Xie et al. , 1994),
LPS activated NF-kB complexes containing p50, p65 and c-Rel
in the mouse macrophage cell line RAW 264.7 (figure 4-8A).

77
The combination of c-Rel and p65 IgGs resulted in an almost
total disappearance of the LPS-induced NF-KB complex in these
cells (data not shown) . The formation of p65/c-Rel
heterodimers has been reported to be important in mediating
the transactivation of iNOS in macrophages (Xie et al. ,
1994) . Treatment of MCT cells with LPS resulted in
activation of NF-kB complexes containing p65 and p50 proteins
(figure 4-8B). The activation of p65 appeared to be greater
in MCT cells than in RAW 264.7 macrophages. However,
contrary to the observation in macrophages, c-Rel activation
was not observed in renal epithelium. The addition of c-Rel
IgG to either p50 or p65 IgG failed to affect the respective
NF-kB complexes (data not shown). These results indicate
that the inability of LPS to induce iNOS activity in MCT
cells may be associated with the absence of c-Rel activation
by this agent, suggesting that c-Rel may be an important
determinant of tissue specificity of LPS-mediated iNOS
responses.
To determine whether the synergism between LPS and IFN
in iNOS induction could be related to interactions between
different NF-kB proteins activated selectively by each of
these agents, I investigated the effects of IFN alone and
combined LPS and IFN treatment on the activation of NF-kB
subunits in MCT cells. The profile of NF-kB was exactly the
same as that observed after LPS treatment alone, and is
characterized by predominant activation of p65, smaller
amounts of p50 and the absence of c-Rel (figure 4-9). Thus,

78
these results indicate that the synergism between LPS and IFN
is not due to complementary effects of the activation of the
different NF-kB proteins by these agents.
Discussion
In these studies I have shown that LPS, IFN or their
combination cause rapid activation and nuclear translocation
of NF-kB complexes in renal tubule epithelium, consisting
mainly of p65 but with smaller amounts of p50 protein. This
is in agreement with what has been observed for most
functional heterodimers of NF-kB (Baeuerle and Baltimore,
1989, Ganchi et al., 1993) but in contrast to what is seen in
macrophages, where the p65/c-Rel heterodimer is reported to
play an important role in LPS-induced iNOS activity (Xie et
al., 1994). It is of interest to note that although LPS and
IFN each induced nuclear translocation of NF-KB complexes,
IFN failed to induce iNOS mRNA and LPS caused only a small
induction of iNOS mRNA transcription, not reflected in either
increase in iNOS protein synthesis or nitrite production.
These results indicate that nuclear translocation of NF-KB
alone is not sufficient for the induction of iNOS activity in
renal epithelium. Nevertheless, since cotreatment of LPS/IFN
with PDTC completely prevented the induction of iNOS
activity, it can be argued that NF-kB, though not sufficient
on its own, is necessary for iNOS expression in renal
epithelium.

79
The failure of LPS to induce any iNOS activity in renal
epithelium could possibly be due to its inability to cause
the activation and nuclear translocation of c-Rel. However,
a combination of LPS and IFN that caused the expression of
iNOS activity also failed to induce the nuclear migration of
c-Rel. This could possibly be that whereas LPS alone may
require a p65/c-Rel dimer to activate iNOS gene expression in
macrophages, when combined with IFN in MCT cells, activation
of other processes by IFN leads to a cross-talk of pathways
resulting in the expression of iNOS activity independent of
c-Rel. These type of transcription factor interactions have
been reported between NF-kB and the Jun/Fos proteins (Stein
et al. , 1993) as well as cis-regulatory enhancer binding
protein-like factor (Mukaida et al. , 1990). Also, certain
proteins such as NF-IL-6, Tax and HMG I(Y) interact with NF-
kB and possibly function as necessary accessory proteins for
transactivation of the IL-6 (Leclair et al. , 1992), HTLV-1
(Hirai et al. , 1992) and IFN-f) (Thanos and Maniatis, 1992)
genes, respectively. Other reports also suggest that, in
addition to c-Rel, activation of other proteins appears to be
necessary for iNOS expression in macrophages (Xie et al. ,
1994). Interferon-y may therefore be, among others, an
inducer of the activation and synthesis of these proteins.
This seems to be the case in macrophages where interferon
regulatory factor 1 (IRF-1) appears to be necessary for the
synergistic induction of iNOS by IFN and LPS (Martin et al.,
1994). Although this protein is yet to be described in renal

80
epithelium, it is possible that it may also play a role in
the regulation of iNOS expression in this tissue.
Homodimers of p50 that reside constitutively in the
nucleus are believed to inhibit gene transcription by binding
to NF-kB sites. It requires the activation of the IxB-like
Bcl-3 proto-oncoprotein to dislodge these inhibitory dimers
in order to allow the incoming p50/p65 dimers to bind and
activate transcription (Franzoso et al. , 1992). The basal
amounts of NF-kB found in control-treated MCT cells may be
homodimers of this type, thereby offering another explanation
for the failure of LPS to express iNOS activity in the cells.
Other reports also suggest that the plOO subunit of NF-KB2
inhibits p65-mediated transcription by preventing the
cytoplasmic dissociation of the p65-IxB complex during
immunokine activation (Sun et al. , 1994). The function of
IFN therefore, may be the activation of processes leading to
the dislodging of the inhibitory effects of these subunits,
thus facilitating the binding of the p50/p65 dimers to their
nuclear DNA recognition sites.
The absence of iNOS activity after treatment with LPS,
and the total blockade of nitrite formation by PDTC despite
the fact that in both cases low levels of iNOS mRNA can be
detected, are intriguing. It is possible that very low
levels of iNOS activity may be present in these situations
and that these are below the detection capabilities of the
nitrite assay and immunoblotting. Alternatively, the iNOS
message or protein produced under these conditions may be

81
highly unstable and unable to yield functionally active
enzyme due to the activities of other proteins possibly
coinduced by LPS. Interferon-y may therefore be activating
processes that repress these proteins and thereby decrease
iNOS mRNA degradation, stabilize iNOS mRNA and iNOS protein
or both, in renal epithelial cells. However, recent studies
in rat macrophages and hepatocytes do not support this
assertion (Nussler and Billiar, 1993). With regard to PDTC,
I cannot exclude the possibility that this compound may have
other as yet undetermined posttranscriptional and/or
posttranslational effects that prevent the expression of
iNOS.
The findings in these studies do not support my original
hypothesis with respect to the synergistic effects of LPS and
IFN in the activation of NF-kB. The signal observed with LPS
alone was about equal in magnitude to that observed for the
LPS and IFN combination. Unlike what has been previously
described in macrophages where IFN and IL-2 cooperatively
activate NF-kB to reach a threshold that may be required for
transcriptional activation of certain genes (Narumi et al. ,
1992), no such cooperative interaction was observed between
IFN and LPS in renal epithelium. However, it appears to
agree with what is seen in human U-937 histiocytic lymphoma
cells where IFN potentiates the cytotoxic effects of TNF, but
has no effect on the TNF-dependent NF-kB activation
(Chaturvedi et al. , 1994) . In addition, this study also
apparently excludes another pillar of my hypothesis, that is,

82
the possibility that the synergism between these two inducers
may be due to activation of different NF-kB subunits that
complement each other to induce iNOS gene transcription.
However, since this study was limited to the study of only
the three major subunits of NF-kB, the possibility of IFN
activating other less well-characterized subunits of NF-kB
cannot be completely ruled out. In spite of this, the
finding that LPS does not activate the nuclear translocation
of c-Rel in renal epithelium is very significant and may help
unravel tissue specificity in the regulation of iNOS
expression.
In summary, activation of NF-kB is necessary but not
sufficient for the induction of iNOS expression by LPS and
IFN in renal epithelium. The absence of c-Rel in the NF-kB
complexes translocated by these immunokines may, in part,
account for their inability to individually induce renal
epithelial iNOS. Therefore, c-Rel may be an important
determinant of the tissue specificity of iNOS expression.
This is consistent with the notion that the ability of the
various components of NF-kB to associate and form multiple
different dimers appear to be a major determinant of the
ability of these proteins to assign tissue-specific responses
to a given stimulus. Since overproduction of NO has been
associated with various pathological renal conditions
(Langrehr et al. , 1993 [a,b], Weinberg et al. , 1994, Yu et
al., 1994, Nussler et al. , 1993), a better understanding of
the molecular mechanisms that determine the tissue-specific

83
regulation of iNOS expression should allow us to identify
potential targets for future selective therapeutic
intervention.

Nitrites
(nmoles/mg)
84
Figure 4-1. Nitrite production in MCT cells treated with LPS
(1 |ig/ml) and IFN (100 U/ml) or their combination for 24 h,
(* pcO.OOl, mg: mg of cell protein, n=9).

85
LPS/IFN
Time (hr)
01 24 6 12 24 6 6
Figure 4-2. Inducible NOS mRNA expression in MCT cells
treated with LPS (1 ng/ml) and IFN (100 U/ml) for the
indicated periods of time or with each of these inducers
alone for 6 h. Blot is representative of three separate
experiments.

86
139 kD-
Figure 4-3. Inducible NOS protein expression in MCT cells
before and after 12 h of treatment with LPS (1 ng/ml) alone
or in combination with IFN (100 U/ml). Blot is
representative of three separate experiments.

Control
87
NF-kB Time course
15 min 30 min 60 min
a
Figure 4-4. Time course of the effects of IFN (100 U/ml),
LPS (1 |jg/ml) and their combination on the nuclear
translocation of NF-kB in MCT cells. Control untreated cells
possess low basal nuclear levels of NF-kB; "+ comp" indicates
where the binding reaction was carried out in the presence of
100-fold excess unlabeled NF-kB oligonucleotide; "+ noncomp"
indicates where the reaction was carried out in the presence
of an irrelevant unlabeled oligonucleotide (AP-1). Both
competition and noncompetition controls were performed with
nuclear extracts from cells treated with a combination of LPS
and IFN for 60 min. Blot is representative of twelve
separate experiments.

88
IFN
LPS
PDTC
+ +
+ +
+
Figure 4-5. Effect of PDTC (25 (0.M) on the nuclear
translocation of NF-kB in MCT cells treated with LPS (1
jig/ml), IFN (100 U/ml), or their combination for 30 min. The
PDTC treatment was initiated 3 0 min before LPS and IFN were
added and maintained during the entire induction period.
Blot is representative of five separate experiments.

89
A
PDTC
18s- ^ ^
harf iarf
Figure 4-6. Effect of PDTC (25 |J.M) on (A) iNOS mRNA
expression and (B) iNOS protein levels in MCT cells treated
with a combination of LPS (1 p.g/ml) and IFN (100 U/ml).
Cells were incubated with PDTC for 30 min before addition of
LPS and IFN and maintained during the entire period.
Inducible NOS mRNA and protein expression were measured at 6
h and 12 h, respectively. Blot is representative of three
separate experiments.

90
LPS/IFN
Figure 4-7. Effect of PDTC (25 |iM) on nitrite production by
MCT cells treated with a combination of LPS (1 |j.g/ml) and IFN
(100 U/ml) for 24 h. Cells were incubated with PDTC for 30
min before the addition of LPS and IFN, and during the entire
incubation period, (* p < 0.001 vs LPS/IFN, mg: mg of cell
protein, n=6).

91
Figure 4-8. Characterization of NF-kB proteins activated by
LPS (1 ng/ml) in (A) RAW 264.7 macrophages and (B) MCT cells.
Nuclear extracts obtained after 30 min treatment with LPS
were analyzed by EMSA in the absence or presence of IgG
against p50, p65 and c-Rel. Marked activation of NF-kB is
observed after LPS treatment. In RAW 264.7 macrophages (A),
addition of p50 IgG results in "supershift" with the
appearance of an intense slower-migrating band consistent
with the presence of p50 protein. Addition of p65 IgG also
results in "supershift" with appearance of a less intense
band that migrates slower than the p50 complex. Addition of
c-Rel IgG results in marked reduction of the NF-kB complex
and "supershift" with appearance of a band that migrates
similar to p65. These findings are consistent with the
presence of a substantial amount of c-Rel protein in the
nuclei of LPS-treated RAW 264.7 macrophages. In MCT cells
(B) , addition of p50 and p65 IgG results in "supershifts"
consistent with the presence of p50 and p65 proteins,
respectively. However, addition of c-Rel IgG fails to induce
"supershift" or substantially affect the NF-kB complex,
indicating the absence of significant amounts of c-Rel in the
nuclei of LPS-treated MCT cells. Blot is representative of
four separate experiments.

92
Figure 4-9. Characterization of NF-kB proteins activated by
(A) IFN and (B) IFN/LPS in MCT cells. Nuclear extracts
obtained after 30 min of treatment with IFN (100 U/ml) or IFN
(100 U/ml) with LPS (1 (xg/ml) were analyzed by EMSA in the
absence or presence of IgG against p50, p65 and c-Rel. The
results are similar to the profile obtained with LPS
treatment alone (see figure 4-8). Blot is representative of
four separate experiments.

CHAPTER FIVE
TRANSCRIPTIONAL REGULATION OF NITRIC OXIDE SYNTHASE BY
NUCLEAR FACTOR-KB IN RENAL EPITHELIUM
Introduction
Computer analyses have identified numerous possible DNA
regulatory sequences within the promoter region of the iNOS
gene that are homologous to consensus DNA elements known to
bind nuclear transcription factors. These elements are known
to be involved in transcriptional responses to LPS and a
variety of cytokines (Xie et al. , 1993, Lowenstein et al. ,
1993). They include NF-kB, AP-1, interferon-y regulatory
factor element (IRF-E) and tumor necrosis factor response
elements (Xie et al., 1993, Lowenstein et al., 1993).
The plethora of immunokines acting either alone or in
various combinations required to express iNOS activity in
different cell types may be explained by the fact that these
immunokines activate different nuclear factors, or perhaps,
different dimers of the same nuclear factor, which then bind
to DNA and influence gene transcription. This, therefore may
confer the tissue specificity of iNOS induction and its
response to stimulation by different immunokines. It is
reasonable to assume that macrophages, which utilize NO for
their cytotoxic functions, should be stimulated by microbes,
microbial products or cytokines whereas in proximal tubule
93

94
epithelial cells, exposure to excessive amounts of NO is
injurious and therefore a more stringent regulation of iNOS
activity may be required. Hence, iNOS activity is hardly
expressed by any of these immunokines acting alone in renal
epithelium. It is therefore important to characterize the
role of individual DNA regulatory elements in iNOS gene
expression in the different cell types.
Nuclear factor-KB is released from a cytoplasmic anchor
protein (IkB) upon immunokine stimulation, by phosphorylation
and proteolysis, allowing it to translocate into the nucleus
to bind its target DNA sequence and activate gene expression
(Muller et al. , 1993, Henkel et al. , 1993). The promoter
region of murine iNOS has been found to contain two putative
DNA binding sites for NF-kB (Xie et al. , 1993). The
downstream one-third region contains one DNA binding site for
NF-KB which, in the mouse macrophage, has been found to be
responsive to LPS but unresponsive to the synergizing effect
of IFN (Xie et al. , 1993). The upstream region which, in
mouse macrophage, was unresponsive to LPS but thought to be
critical for IFN responsiveness, contains the other putative
NF-kB binding site and also IRF-E, which is the IRF-1 binding
site needed for the synergistic effect of IFN (Xie et al.,
1993, Martin et al. , 1994). In this study, I further
characterized the role of NF-kB in the transcription and
expression of the iNOS gene in MCT cells, and also
investigated the mechanism of the synergism between LPS and
IFN, using functional promoter analyses but with special

95
emphasis on the NF-kB and IFN-activated protein binding sites
in the upstream and and NF-kB binding site in the downstream
areas in the promoter region of the iNOS gene.
Materials and Methods
Chemicals and Biologic Products
All restriction enzymes and other molecular biology
enzymes, DNA Wizard Miniprep, CAT Enzyme assay kit, were
purchased from Promega (Madison, WI).
Cloning of the Upstream Promoter Region
Two primers selected after careful study of the promoter
region of murine mac-iNOS, one designed with Hind III site at
the 5'-flanking end (BA I: 5' AGA AGC TTG ACT TTG ATA TGC ATA
TGC TGA AAT CCA T) and the other with a Sal 1 site at the 3'-
flanking end on the complementary DNA strand (BA II: 5' GAG
TCG ACG AAC AAG ACC CAA GCG TGA GGA A), were used to amplify
the 1.7 kilobase pair (kbp) promoter region from mouse
genomic DNA using the PCR (see DNA Amplification below). The
PCR product was treated with the Klenow fragment of DNA pol
I, restriction-digested with Hind III and Sal 1. The PCR
product was subsequently isolated by agarose gel
electrophoresis and DEAE-cellulose.
This PCR product was subcloned to the Hind III/Sal I
site of the pCAT-Basic and subjected to DNA sequence analysis
(Sanger Dideoxy Chain Termination, Sequenase Kit, IBI).

96
DNA Amplification
The reaction mixture was prepared with autoclaved
ultrafiltered water, 10X PCR buffer (Promega), MgCl2 1.5 mM,
dATP, dCTP, dGTP and dTTP 2 pM each (dNTPs), Primer BA I 2 |xM,
Primer BA II 2 nM, Mouse genomic DNA 5 ng and 2.5 units of
Taq DNA polymerase in a total volume of 100 n.1. The mixture
was overlaid with 75 nl of mineral oil to reduce evaporation
or refluxing. The mixture was then subjected to repeated
cycles in a Perker-Elmer DNA Thermal Cycler at a template
melting temperature of 94 °C (1 min), 53 °C (2 min) and 72 °C
(3 min) for a total of 25 cycles.
Purification of Amplified DNA
The PCR product was phenol:chloroform extracted and
precipitated overnight (or on dry ice for l1/2 h) from 0.3 M
NaOAc and ethanol, at -20 °C. Recovery of DNA was monitored
by absorbance at 260 nm. Ten per cent of the total volume
was then ran on a 1% agarose gel in order to estimate the
molecular weight.
Subclonina of the DNA Fragment into : AT-Basic Vector and
Transfection of the Plasmids into HB101 Cells
The pCAT-Basic vector (figure 5-2B) was restriction
endonuclease digested with Hind III and Sal I as described
elsewhere. The PCR product was subcloned into the digested
vector, which was then transfected into HB101 cells.
Briefly, to a 100 p.1 of competent HB101 cells in a

97
polypropylene tube on ice, was added 5 ng plasmid DNA for 10
min and then heat-shocked for 45 seconds at exactly 42 °C.
The tube was transferred to ice for 2 min and 900 |il SOC
medium (per 100 ml: bacto-tryptone 2 g, bacto-yeast extract
0.5 g, 1 M KC1 0.25 ml, 1 ml each of 1 M MgCl2, 1 M NaCl, 1 M
MgSC>4 and 2 M glucose) was added and incubated at 37 °C with
shaking at 225 rpm for 60 min. Then, 100 |xl aliquots of the
transformation mix were plated onto LB plates (per liter:
bacto-yeast extract 5 g, bacto-tryptone 10 g, NaCl 5 g, agar
15 g, pH 7.5) with ampicillin (100 |ig/ml) and incubated at 37
°C overnight. Filters were then lifted from the plates and
hybridized to 32p-iabeled PCR product. Colonies hybridizing
to the probe were picked up for miniprep amplification.
Preparation of Constructs of Inducible Nitric Oxide Synthase
A DNA fragment containing the promoter region of iNOS
from mouse genomic DNA was subcloned into the pCAT-Basic
vector (figure 5-2B) at the Hind III and Sal I to yield the
construct pAiNOS-CAT (figure 5-2A). The construct pBiNOS-CAT
was generated by the Spe I and the downstream Sal I fragment
and the pCiNOS-CAT, the Xba I and the upstream Hind III
fragment (figure 5-2A). These fragments were generated by
specific restriction endonuclease digestion with the
appropriate restriction enzymes. All the constructs were
sequenced to ensure that the appropriate deletions and
ligations have been obtained.

98
Transfection of the Plasmid DNA into Murine Proximal Tubule
Cells and Chloramphenicol Acetvl Transferase Assays
The MCT cells were transfected by a modification of the
DEAE-dextran method (Golub et al. , 1989). Briefly, the cells
were seeded the day before transfection such that they were
about 60% confluent the next day. After cells were washed
twice with PBS, trypsinized and resuspended in RPMI with 10%
Nuserum, 10 p.g of plasmid DNA was added per 2 X 106 cells per
1 ml of prewarmed RPMI ( (37 °C, 10% Nuserum) , containing
DEAE-dextran (100 ng/ml) and 50 mM Tris (pH 7.4). The
suspension was incubated at 37 °C for 60 min followed by a 3
min shock with 10% DMSO at room temperature. The cells were
washed and distributed to 100-mm plates each with about 5 x
106 cells in 10 ml of complete medium, and incubated at 37 °C
in 5% CO2. After 24 h, the medium was changed and inducers
added at the desired concentrations and incubated for another
24 h. The cells were then washed with ice-cold PBS,
resuspended in 0.25 mM Tris-HCl (pH 8), and subjected to
three cycles of freezing and thawing. The lysates were
centrifuged (11700 g for 10 min at 4 °C) and the supernatant
heated at 60 °C for 10 min to inactivate CAT inhibitors and
then centrifuged again as above. The supernatant was then
assayed for CAT activity by the thin layer chromatography
(TLC) method (Golub et al., 1989). Briefly, the assay
mixture contained (final vol 125 (il) 100 |ig of cell extract
(in 50 |il), 69 p.1 of 0.25 mM Tris (pH 8), 3 p. 1 of [14C]-
chloramphenicol (50 nCi/mmol) and 5 p.1 of 5 mg/ml n-butyryl

99
coenzyme A . Controls contained CAT 0.1 U instead of cell
extract. The reaction was stopped with 0.5 ml ethyl acetate
which was also used to extract the chloramphenicol. The
upper organic layer was dried and taken up in 30 pi of ethyl
acetate, 15 pi of which was spotted on silica gel thin layer
plates and ran with chloroform:methanol (97:3 ascending).
After autoradiography of the separated butyrylated
chloramphenicol forms, spots were cut and counted. Data were
expressed as the amount of chloramphenicol butyrylated per
100 pg of extract. Protein was determined as described by
Bradford (Bradford, 1976). A p-galactosidase reporter
molecule was cotransfected into the cells and the p-
galactosidase assay, carried out according to manufacturer's
instructions, was used to normalize the results of the CAT
assay.
Results
The Upstream Promoter Region
Sequencing of the cloned promoter region confirmed it to
be almost identical to previously described iNOS promoter
sequence (Xie et al. , 1993, Lowenstein et al. , 1993), the
only differences being G for C in positions -617, -615, -613,
-611, -609, -607, -605, -603, -601, -599 and -21, A for G in
position -193, and A for C in position +24, all of them
outside regions described as putative transcription factor
binding sites (Xie et al., 1993).

100
Previous reports have identified the initiation site of
this promoter region by SI nuclease mapping and primer
extension (designated as +1 in figure 5-1) which is 30 bp
downstream of a TATA box (Xie et al. , 1993, Lowenstein et
al. , 1993) . The TATA box is preceded by a number of
recognition sites for the binding of transcription factors
induced by immunokines and necessary for gene transcription
including NF-kB and IRF-1 (Xie et al., 1993).
The Plasmid Constructs
The pAiNOS-CAT plasmid (figure 5-A) construct represents
the insertion of the entire 1.7 kbp fragment into the vector;
pBiNOS-CAT, the Spe I and downstream Sal 1 fragment (-478 to
+141, figure 5-1) and pCiNOS-CAT, the Xba I and upstream Hind
III fragment (-1588 to -421, figure 5-1). The construct
pBiNOS-CAT contains one NF-kB consensus sequence. This part
of the promoter has been found to be responsive to LPS but
unresponsive to the synergizing effect of IFN in macrophages
(Xie et al. , 1993). The construct pCiNOS-CAT contains
another NF-kB consensus sequence as well as the IRF-1 binding
site (IRF-E), GAS, ISRE and some y_IREs- In mouse
macrophages, this part of the promoter was unresponsive to
LPS but both regions were necessary for LPS-activated iNOS
expression since deletion of either region decreased the
activity of the reporter gene (Xie et al., 1993). Sequencing
of all the constructs confirmed that the appropriate
deletions and ligations have been obtained.

101
Promoter Activity of the 1.7 Kilobase Pair Fragmei.t and its
Truncated Plasmid Constructs
To investigate the role of NF-kB in the induction of
iNOS gene transcription by LPS and IFN, the constructs
described above were fused with the promoterless CAT reporter
gene, the pCAT-Basic reporter vector and transiently
transfected into MCT cells.
As reflected in the CAT activity shown in figure 5-3,
pAiNOS-CAT, the untruncated construct, conferred CAT
responsiveness by LPS (1 |ig/ml) in combination with IFN (100
U/ml) in contrast to the native vector. Treatment with LPS
alone (1 (ig/ml) resulted in a 3.9-fold increase in CAT
activity and cotreatment with IFN (100 U/ml) potentiated the
induction of CAT activity to 6.6-fold, which was blocked by
the addition of PDTC (25 piM) (figure 5-4). Interferon-y alone
(100 U/ml) did induce a small but significant CAT activity
(figure 5-4). Taken together, these findings show that the
effect of LPS and IFN on the promoter region is
transcriptional and that it requires NF-kB.
The construct pBiNOS-CAT, consisting of the downstream
portion linked to the CAT gene conferred inducibility by LPS
(1 |a.g/ml) and a combination of LPS (1 |ig/ml) and IFN (100
U/ml) but not by IFN alone. However, in contrast to pAiNOS-
CAT, the synergistic effect of IFN was lost (figure 5-5).
The construct pCiNOS-CAT which lacked the downstream NF-kB
binding site was devoid of promoter activity in response to
treatment with LPS, IFN or their combination (figure 5-6).

102
The apparently minor induction of CAT activity seen in
control- and IFN-treated cells may be attributed to the
presence of contaminating endotoxin during preparation of the
plasmid DNAs, which probably synergized with IFN. These
plasmid DNAs were prepared by transfection into competent E
coli bacteria. It is therefore difficult to avoid endotoxin
contamination, which in this case, was determined to be in
the order of 25 pg/ml by Limulus assay.
Discussion
In these studies, I characterized the role of NF-kB and
IFN-activated protein binding sites in the iNOS promoter
region in LPS- and IFN-induced gene transcription and also
investigated the mechanism of synergism between LPS and IFN
in this induction. The whole promoter region, as in pAiNOS-
CAT, conferred inducibility by LPS as well as synergistic
responsiveness to cotreatment with IFN. This is in agreement
with what is observed in macrophages where LPS alone, or in
combination with IFN, induces CAT activity in macrophages
transiently transfected with vectors bearing the iNOS
promoter region (Xie et al., 1993). The construct pBiNOS-CAT
responded to treatment with LPS but no synergism was noticed
on addition of IFN. This construct contains only the
downstream NF-kB binding site known to respond to LPS
treatment in macrophages (Xie et al. , 1993). The inability
to respond to the synergistic effect of IFN could be due to
the absence in this construct of the upstream region that

103
bears the IRF-E, the binding site for IRF-1 demonstrated to
be critical for the synergistic role of IFN (Kami jo et al. ,
1994, Martin et al. , 1994), as well as several other sites
for the binding of IFN-activated proteins such as y-IRE, GAS
and ISRE (Xie et al. , 1993, Lowenstein et al. , 1993). In
fact, in macrophages, site-directed mutagenesis that alter
the IRF-E site results in the loss of the synergistic effect
of IFN on LPS-stimulated iNOS activity (Martin et al., 1994).
Sequential deletion of the promoter region in constructs
transiently transfected into macrophages have shown that the
critical site for this IFN response may be located in the
region between -975 to -722 (figure 5-1) (Xie and Nathan,
1994). The response to the effect of LPS was similar in both
pAiNOS-CAT and pBiNOS-CAT. This suggests that the upstream
NF-kB binding site plays little or no role in the NF-kB-
mediated induction of gene transcription by LPS in MCT cells.
The inability of pCiNOS-CAT to elicit any response to
LPS, IFN or both may be due to the loss of the critical
downstream NF-kB binding site. The presence of the upstream
NF-kB binding site in addition to the IRF-E, GAS, ISRE and y-
IRE sites in this construct did not confer inducibility in
response to the immunokines, thus confirming that this NF-KB
binding site is irrelevant in NF-KB-mediated iNOS gene
transcription stimulated by LPS in MCT cells. Another reason
for the lack of response in this construct may be the loss of
the TATA box and the transcription initiation site, which
were deleted in preparing the construct. It must be stated

104
however that the pCAT-Basic vector possesses its own TATA box
and transcription initiation site (Gorman et al., 1982).
Further studies to resolve this may best utilize constructs
where site-directed mutagenesis is used to alter only the
downstream NF-kB site, thus preserving the TATA box and the
transcriptional initiation site.
One intriguing finding of this study is the ability of
LPS alone to induce significant amount of CAT activity in
pAiNOS-CAT and pBiNOS-CAT transiently transfected into MCT
cells although LPS does not induce iNOS protein synthesis or
nitrite production in MCT cells (figure 4-1 & 4-3).
Lipopolysaccharide causes only moderate transcription of iNOS
mRNA in MCT cells. This may be because iNOS mRNA may possess
a number of bases at the 3'-flanking end not present in CAT
mRNA that makes it subject to increased degradation,
decreased stability or both, by proteins possibly coinduced
by LPS. The role of IFN may then be to activate pathways
that repress these proteins. Moreover, the promoter region
inserted into the construct may be too short and therefore
devoid of inhibitory binding sites present in the native
cells for proteins possibly coinduced by LPS. If such
proteins act as transcriptional or posttranscriptional
inhibitors of iNOS protein synthesis, then their absence from
the constructs may allow LPS to express CAT activity in
transfected cells but not iNOS in native cells. The
activities of such proteins may be those masked by pathways
activated by coincubation with IFN. It may well be part of

105
the cross-talk reported between NF-kB and other
transcriptional factors like Jun/Fos and cis-regulatory
enhancer binding protein-like factor (Stein et al. , 1993,
Mukaida et al. , 1990). Another reason may be that CAT mRNA
transcription induced by LPS are more stable without any IFN-
induced influences. Studies utilizing longer inserts than
the one used in the present study or a northern blot analysis
of CAT mRNA may be needed to answer this question. In
macrophages and glial cells, LPS causes the induction of
substantial iNOS activity on its own (Stuehr et al., 1985,
Xie et al., 1993, Szabó et al., 1993). It is reasonable for
cells primed to counter microbial invasion to be activated by
microbial products, but in the renal tubule epithelium,
excessive amounts of NO may be harmful. Hence a more
stringent control of iNOS activity is desirable. It must
also be noted that any small amount of cytokine contaminant
from the preparation of the DNAs could synergize with LPS to
produce this effect.
In summary, the downstream NF-kB binding site in the
iNOS promoter region is essential for NF-KB-mediated LPS-
stimulated iNOS gene transcription in MCT cells. However,
the synergistic effect of IFN appears to be mediated through
a site located in the upstream portion of the promoter
region, possibly the IRF-1 binding site (IRF-E). The loss of
this region results in the loss of the synergistic role of
IFN in MCT cells, as is seen in pBiNOS-CAT. Nuclear factor-
kB is a ubiquitous transcription factor that appears to be

106
very important in the regulation of iNOS gene expression in
renal tubule epithelium. Interference with the activation or
activity of NF-kB may therefore be beneficial in suppressing
iNOS-mediated renal pathophysiologies such as acute
glomerulonephritis, acute tubular ischemia/reperfusion injury
and renal transplant rejection (Weinberg et al., 1994, Yu et
al. , 1994, Langrehr et al. , 1993 [a, b] ) . The inhibition of
NF-kB activation may provide a pharmacological basis for
interfering with these acute processes.

107
Hind III
-15 8 8 GACTTTGATATGCTGAAATCCATAAGCTGTGTGTGTGTGCAAGTGTGCA
-15 3 9 TGCGCATGTGTGCACATGAGTGTGCAGGTATATGTAGGAGCTAGAAGACAATCTCAGCTC
-1479 TTGTTTCCCAGGTTACCCAGCATCTCTCACCAGCCTGGAACCTGCCTAGTAGGCTAGGCT
-1419 GGCTGGCCAGCAAACCCTAGGCATATTCCTGTCTTTACCTCCCCAGAACTTATTGCAAAG
-1359 GTGTGTCACCACACCCAGCATTTTATCATTGACCTATTGACTGGTGTGTGTGTGTGTGTG
-12 9 8 TGTGTTTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTAAATT
-1239 CCTTATCTCACCAACCCATGCCCAGCTTTTGAACTTAGGTCCTTGTACATGCAAGGCAAG
-117 9 CACTTTACCAACTGAGCCATCTCCCCAGCCCAATTACTTGATTTGTAATTCATTTATTCA
-1119 ATCAACAATTTATTTGTTCTCCCAACTATTGAGGCCACACACTTTTTGGGTGACTTAGTC
-10 5 9 TGTGTACCTCAGACAAGGGCAAAACACGAGGCTGAGCTGACTTTGGGGACCATGCGAAGA
NF-KB
-999 TGAGTGGACCCTGGCAGGATGTGCTAGGGGGATTTTCCCTCTCTCTGTTTGTTCCTTTTC
IRF-E
-939
-879
-819
-759
-699
-639
-579
-519
-459
-399
-339
-279
-219
-159
-99
-39
+ 22
CCCTAACACTGTCAATATTTCACTTTCATAATGGAAAATTCCATGCCATGTGTGAATGCT
TTATTGGAAGCATTGTAAGAAATTATAATTTATTCGTTTTTGTTTGTTTCTCAGAACAGG
GTTTTTCTGTGTAGTGTTCCTGGCTTATCCTAGAACTTACTCTGTAGACCAGGCTAGCCC
AAACTCAGGGATCAGCCTTTCTCTGTCTCCTGAATCCCGGGATTAAAGGCTTATGCCACC
ACACCCAGGTAGGACATTATAATCCTATATATAAGAAGTCACCCACACATACAAACACAC
ACACACCACACACACACACACAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA
GAGGGAGAGAGAGAGAGAGAGAGAGAATGCCACTGAGAAAAAAAATAAAAAGGCTTCACT
Spe,I
1
CAGCACAGCCCATCCACTATTCTGCCCAAGCTGACTTACTACTAGTGGGGAAATGCTGGT
Xba I
CAGACGGCATCTGTGCCCACAGCTTGCCTTCCATCCTTTCTAGAAAACCTCCTGATGAAT
GTGTCCTGGGCGTGTTGGAATATTGGCACCATCTAACCTCACTGAGAGAACAGACAGAAA
GCCAGAGAGCTCCGTGCCCAGAACAAAATCCCTCAGCAGCTGCAAGCCAGGGTATGTGGT
TTAGCTAAGAAAAGCCAGCCTCCCTCCCTAGTGAGTCCCAGTTTTGAAGTGACTACGTGC
TGCCTAGGGGCCACTGCCTTGGACGGACGACCAGGAAGAGATGGCCTTGCATGAGGATAC
ACCACAGAGTGATGTAATCAAGCACACAGACTAGGAGTGTCCATCATGAATGAGCTAACT
NF-KB
TGCACACCCAACTGGGGACTCTCCCTTTGGGAACAGTTATGCAAAATAGCTCTGCAGAGC
+1r^
CTGGAGGG'
4tat4aa'
TACGTGATGGCTGCTGCCAGGGTCACAACTTTACAGGGAGTTGAA
GAATGAGACTCTGGCCCCACGGGACACAGTGTCACTGGTTTGAAACTTCTCAGCCACCTT
Sal I
+ 82 GGTGAAGGGACTGAGCTGTTAGAGACACTTCTGAGGCTCCTCACGCTTGGGTCTTGTTC A'
$
Figure 5-1. The 1.7 kilobase pair 5'-flanking region of iNOS
cloned by PCR from mouse genomic DNA. Sequences are
designated that are homologous to the consensus sequences for
binding of NF-kB and IRF-1 (IRF-E). Also designated are the
restriction sites utilized in the preparation of the
constructs shown in figure 5-2A. The mRNA initiation site (+
1), TATA sequence (boxed).

108
A
-1588
Hind
III
h
Xba I
Spe I
+ 141
JqAtI PAíNOS-CAT
Sal I
-478
+ 141
CAT
pBiNOS-CAT
-1588
-421
CAT
pCiNOS-CAT
B
Figure 5-2. A: The CAT constructs. Dashed lines represent
deleted portions. B. pCAT-Basic Vector circle map and
sequence reference points.

A
B
Control
LPS/IFN
i-
o
<
.2 <
0)
(0
9
S 9
CD
w
CO (0
<
o
z
lL o
< .?
o
a.
<
a
9 <
a a
Basal LPS/IFN
Figure 5-3. Demonstration of iNOS promoter activity by LPS and IFN in MCT cells. The MCT
cells transfected with pAiNOS-CAT vector or the promoterless pCAT-Basic vector were treated
with a combination of LPS (1 M.g/ml) and IFN (100 U/ml) for 24 h followed by CAT assay of
lysates. Treatment with LPS/IFN conferred inducibility of CAT activity to pAiNOS-CAT but
not to the promoterless pCAT-Basic. A Autoradiograph, B graphical presentation; (* p < 0.01
vs pAiNOS control, ** p < 0.01 vs pAiNOS [LPS/IFN], n=4).
109

A
B
LPS/IFN
+
Control IFN LPS LPS/IFN PDTC
pAiNOS-CAT
Figure 5-4. Effects of LPS, IFN and PDTC on the activation of the iNOS promoter. The MCT
cells transfected with pAiNOS-CAT vector were treated with IFN (100 U/ml), LPS (1 |xg/ml) or
their combination in the absence or presence of PDTC (25 jiM) , followed by CAT assay of cell
lysates. Both LPS and LPS/IFN, but not IFN conferred CAT inducibility, which was blocked by
coincubation with PDTC. A Autoradiograph, B graphical presentation; (*, ** p < 0.05 and 0.01
respectively, vs control, *** p < 0.01 vs -PDTC, n=4)
110

B
Control
IFN
LPS
LPS/IFN
pBiNOS-CAT
>1
4J
•H
>
•H
4J
U
<
Eh
<
U
30 -i
C
•S20-I
â– u
nJ
i—i
4J
<1)
Ü
<
o\° 10 -I
0 -l-l
M
Basal IFN LPS LPS/IFN
Figure 5-5. Effect of the removal of the upstream NF-kB binding site on the activation of
iNOS promoter by LPS and IFN. The MCT cells transfected with pBiNOS-CAT vector, which is
devoid of the upstream NF-kB binding site and the interferon response elements, were treated
with LPS (1 ng/ml) and IFN (100 U/ml) or their combination for 24 h followed by CAT assay of
the cell lysates. Only LPS conferred inducibility to pBiNOS-CAT, with the loss of the
synergistic effect
control, n=3)
of IFN. A Autoradiograph, B graphical presentation; (* p < 0.01 vs
111

A
B
Control IFN LPS LPS/IFN
Figure 5-6. Effect of the removal of the downstream NF-kB binding site on the activation of
iNOS promoter by LPS and IFN. The MCT cells transfected with pCiNOS-CAT vector, which is
devoid of the downstream NF-KB binding site, were treated with LPS (1 (ig/ml) and IFN (100
U/ml) or their combination for 24 h followed by CAT assay of the cell lysates. There is the
loss of CAT inducibility by LPS, IFN or their combination; (n=3). A Autoradiograph, B
graphical presentation.
112

CHAPTER SIX
SUMMARY AND CONCLUSIONS
The aims of this dissertation were to investigate the
regulation of inducible nitric oxide synthase in murine
proximal tubule epithelium, using the MCT cell line.
The major conclusions are: 1) murine proximal tubule
epithelial cells possess an immunokine-inducible isoform of
NOS; 2) expression of MCT iNOS activity requires protein
synthesis and mRNA transcription; 3) IFN potentiates the
induction of iNOS activity by LPS at the transcriptional
level but does not itself induce transcription or expression
of the enzyme; 4) inhibition of GTP cyclohydrolase I, the
rate-limiting enzyme in the de novo synthesis of BH4 f
suppresses the maximum expression of iNOS activity in MCT
cells; this inhibition is reversed by concurrent activation
of the salvage pathway with sepiapterin, a substrate for BH4
formation via the salvage pathway; 5) LPS, IFN or their
combination cause a rapid activation and nuclear
translocation of NF-kB complexes in MCT cells, and that these
complexes are composed mainly of p65 with smaller amounts of
p50 but no c-Rel; 6) NF-kB activation is essential but not
sufficient for the induction of iNOS expression in MCT cells;
7) constructs bearing portions of the promoter region of iNOS
linked to a reporter gene, except for the upstream portion,
113

114
confer inducibility by LPS but not by IFN; synergistic
inducibility by both LPS and IFN was conferred only by the
entire promoter region.
This study has revealed several important differences
between renal epithelial iNOS and the enzyme in other tissues
and species. Synergism in some tissues appears to be the
result of cooperative interactions between iNOS inducers that
lead to stimulation of the synthesis of cofactors such as BH4
that are required for the expression of full activity. For
example, IFN markedly stimulates NO synthesis in murine
peritoneal macrophages (Deng et al., 1993) but is ineffective
in MCT cells. Also, LPS potentiates iNOS activity induced by
IFN in rat aortic vascular smooth muscle cells by inducing
BH4 synthesis (Gross and Levi, 1992, Hattori and Gross,
1993), but in MCT cells, even exogenous addition of these
cofactors to IFN- or LPS-treated cells has no effect.
Another important distinction is the inability of LPS to
cause the activation and nuclear translocation of the c-Rel
subunit of NF-kB in MCT cells even though in macrophages this
subunit is critical for the induction of iNOS activity (Xie
et al. , 1993). The synergism between LPS and IFN in MCT
cells appears to be mainly at the level of transcription, as
is the case in hepatocytes (Geller et al. , 1993), and
requires IFN-induced protein binding to the interferon-y
regulatory elements.
The role of NF-kB in the induction of iNOS in MCT cells
may be very complex.
Although IFN is able to cause the

115
activation and nuclear translocation of NF-kB in MCT cells,
it does not cause the transcription of iNOS mRNA, translation
of iNOS protein or confer inducibility in pAiNOS-CAT. The
inhibition of iNOS activity in MCT cells by cycloheximide
demonstrates that protein synthesis is essential for iNOS
activation. Hence, it may be argued that the inability of
IFN to coinduce the synthesis of other vital proteins
necessary for the activation process may explain this
finding. On the other hand, LPS may be inducing the
synthesis of some of these proteins thereby explaining its
ability to confer inducibility in pAiNOS-CAT and pBiNOS-CAT.
However, with respect to iNOS, other processes activated by
IFN may still be vital.
The presence of iNOS in the MCT cell line offers a
simple but powerful tool for the investigation of the role of
NO in the kidney. This is in view of the fact that NO plays
a major role in several renal pathophysiological conditions
such as acute glomerulonephritis, kidney transplant rejection
and acute tubular ischemia-reperfusion injury (Weinberg et
al., 1994, Langrehr et al., 1993 [a,b], Yu et al., 1994).
In conclusion, mouse proximal tubule epithelial iNOS is
induced synergistically by LPS and IFN and it is regulated at
two levels: transcriptionally by transcription factors that
include NF-KB and possibly IRF-1, and posttranscriptionally
by BH4 availability. Whereas IFN does not induce iNOS
expression in proximal tubule cells, it markedly potentiates
the induction of iNOS by LPS. Also NF-kB activation and BH4

116
synthesis are critical for the induction and full expression
of iNOS activity in MCT cells. Unlike what is observed in
macrophages, in mouse proximal tubule epithelium, LPS fails
to cause the nuclear translocation of the c-Rel subunit of
NF-kB. This subunit of may thus be an important determinant
of iNOS tissue specificity. Because cytokine-induced NO
production may cause tubular cell injury, the development of
inhibitors of proximal tubule BH4 synthesis, NF-kB and IRF-1
activation could provide important pharmacological tools for
future therapy of inflammatory renal diseases.
1

LIST OF REFERENCES
Abu-Soud, H.M. and Stuehr, D.J. Nitric oxide synthases
reveal a role for calmodulin in controlling electron
transfer. Proc. Natl. Acad. Sci. USA 90: 10769-10772, 1993.
Alspaugh, J.A. and Granger, D.L. Inhibition of Cryptococcus
neoformans replication by nitrogen oxides supports the role
of these molecules as effectors of macrophage-mediated
cytostasis. Infect. Immun. 59: 2291-2296, 1991.
Amoah-Apraku, B., Chandler, L.J., Madsen, K.M., Tang, S.S.,
Ingelfinger, J.R. and Guzman, N.J. Regulation of inducible
nitric oxide synthase expression in murine proximal tubule
epithelial cells (Abstract). J. Am. Soc. Nephrol. 4:544,
1993.
Awolesi, M., Sessa, W. and Sumpio, B. Cyclic strain
upregulates nitric oxide synthase gene. FASEB 7: A755, 1993.
Baek, K.J., Thiel, B.A., Lucas, S. and Stuehr, D.J.
Macrophage nitric oxide synthase subunits: purification,
characterization and role of prosthetic groups and substrate
in regulating their association into dimeric enzymes. J.
Biol. Chem. 268: 21120-21129, 1993.
Baeuerle, P.A. and Baltimore, D. A 65-kD subunit of NF-kB is
required for inhibition of NF-kB by IkB. Genes Dev. 3: 1689-
1698, 1989.
Bellahsene, Z., Dhont, J.L. and Farriaux, J.P. Guanosine
triphosphate cyclohydrolase activity in rat tissues. Biochem.
J. 217: 59-65, 1984.
Billiar, T.R., Curran, R.D., Harbrecht, B.G., Stuehr, D.J.,
Demetris, A.J. and Simmons, R.L. Modulation of nitric oxide
synthesis in vivo: NG-monomethy1-L-arginine inhibits
endotoxin-induced nitrite/nitrate biosynthesis while
promoting hepatic damage. J. Leuk. Biol. 48: 568-569, 1990.
Blough, N.V. and Zafirou, O.C. Reaction of superoxide with
nitric oxide to form peroxinitrite in alkaline aqueous
solution. Inorg. Chem. 24: 3502-3504, 1985.
Bogdan, C., Vodovotz, Y., Paik, J. , Xie, Q. and Nathan, C.
Traces of bacterial lipopolysaccharide suppress IFN-y-induced
nitric oxide synthase gene expression in primary mouse
macrophages. J. Immunol. 151: 301-309, 1993.
117

118
Bogle, R.G., Baydoun, A.R., Pearson, J.D., Moneada, S. and
Mann, G.E. L-arginine transport is increased in macrophages
generating nitric oxide. Biochem. J. 284: 15-18, 1992.
Bóhme, G.A., Bon, C., Stutzmann, J.M., Doble, A. and
Blanchard, J.C. Possible involvement of nitric oxide in long
term potentiation. Eur. J. Pharmacol. 199: 379-381, 1991.
Bóhme, E., Graf, H. and Schultz, G. Effects of sodium
nitroprusside and other smooth muscle relaxants on cyclic
GMP formation on smooth muscle and platelets. Adv. Cycl.
Nucl. Res. 9: 131-143, 1978.
Boulanger, C. and Luscher, T.F. Release of endothelin from
the porcine aorta. Inhibition by endothelium-derived nitric
oxide. J. Clin. Invest. 85: 587-590, 1990.
Bours, V. , Franzoso, G. , Brown, K. , Park, S., Azarenko, V. ,
Tomita-Yamaguchi, M., Kelly, K. and Siebenlist, U.
Lymphocyte activation and the family of NF-kB transcription
factor complexes. Current Topics Microbiol. Immunol. 182:
411-420, 1992.
Bradford, M.M. A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal. Biochem. 72: 248,
1976.
Bredt, D.S., Ferris, C.D., Snyder, S.H. Nitric oxide
synthase regulatory sites, phosphorylation by cyclic AMP-
dependent protein kinase, protein kinase C, and
calcium/calmodulin protein kinase: identification of flavin
and calmodulin binding sites. J. Biol Chem. 267: 10976-10981,
1992.
Bredt, D.S., Hwang, P.M., Glatt, C.E., Lowenstein, C., Reed,
R.R. and Snyder, S.H. Cloned and expressed nitric oxide
synthase structurally resembles cytochrome P-450. Nature 351:
741-748, 1991.
Bredt, D.S., Hwang, P.M. and Snyder, S.H. Localization of
nitric oxide synthase indicating a neural role for nitric
oxide. Nature 347: 768-770, 1990.
Bredt, D.S. and Snyder, S.H. Isolation of nitric oxide
synthetase, a calmodulin-requiring enzyme. Proc. Natl. Acad.
Sci. USA 87: 682-685, 1990.
Brezis, M. , Heyman, S.N., Dinour, D., Epstein, F.H. and
Rosen, S. Role of nitric oxide in renal medullary
oxygenation. Studies in isolated and intact rat kidneys. J.
Clin. Invest. 88: 390-395, 1991.

119
Bruñe, B. , Molina y Vedia, L. and Lapetina, E.G. Agonist-
induced ADP-ribosylation of a cytosolic protein in human
platelets. Proc. Natl. Acad. Sci. USA 87: 3304-3308, 1990.
Bucala, R., Tracey, K.J. and Cerami, A. Advanced
glycosylation endproducts quench nitric oxide and mediate
defective endothelium-dependent vasodilation in experimental
diabetes. J. Clin. Invest. 87: 432-438, 1991.
Busse, R. and Mulsch, A. Induction of nitric oxide synthase
by cytokines in vascular smooth muscle cells. FEBS Lett. 275:
87-90, 1990.
Chandler, L.J., Guzman, N.J., Sumners, C. and Crews, F.T.
Induction of nitric oxide synthase in brain glial cells:
possible interactions with ethanol in reactive neuronal
injury, in Alcohol and Glial Cells (vol 27). National
Institute on Alcohol Abuse and Alcoholism Research Monograph,
edited by F.E. Lancaster, Bethesda, National Institutes of
Health, pp 195-214, 1994.
Charles, I.G., Palmer, R.M.J., Hickery, M.S., Bayliss, M.T.,
Chubb, A.P., Hall, V.S., Moss, D.W. and Moneada, S. Cloning,
characterization and expression of a cDNA encoding an
inducible nitric oxide synthase from the human chondrocyte.
Proc. Natl. Acad. Sci. USA 90: 11419-11423, 1993.
Chaturvedi, M.M., Higuchi, M. and Aggarwal, B.B. Effect of
tumor necrosis factors, interferons, interleukins and growth
factors on the activation of NF-kappa B: evidence for lack of
correlation with cell proliferation. Lymphokine Cytokine Res.
13: 309-313, 1994.
Chin, J.H., Azhar, S. and Hoffman, B.B. Inactivation of
endothelium-derived relaxing factor by oxidized lipoproteins.
J. Clin. Invest. 89: 10-18, 1992.
Cho, H.J., Xie, Q., Calaycay, J., Mumford, R.A., Swiderek,
K.M., Lee, T.D. and Nathan, C. Calmodulin is a subunit of
nitric oxide synthase from macrophages. J. Exp. Med. 176:
599, 1992.
Chomsczyski, P. and Sacchi, N. Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform
extraction. Anal. Biochem. 162: 156-159, 1987.
Closs, E.I., Lyons, R. , Mitchell, M. and Cunningham, J.M.
Expression of cationic amino acid transporters in NO-
producing macrophages (Abstract). Endothelium 1:16, 1993.
Corbett, J.A., Lanscaster Jr., J.R., Sweetland, M.A. and
McDaniel, M.L. Interleukin-l-p -induced formation of EPR-
detectable iron-nitrosyl complexes of Islets of Langerhans:

120
role of nitric oxide in interleukin-l-p -induced inhibition of
insulin secretion. J. Biol. Chem. 266: 21351-21354, 1991.
Corraliza, I.M., Campo, M.L., Fuentes, J.M., Campos-
Portuguez, S and Soler, G. Parallel induction of nitric
oxide and glucose-6-phosphate dehydrogenase in activated bone
marrow-derived macrophages. Biochem. Biophys. Res. Commun.
196: 342-347, 1993.
Davda, R.K., Chandler, L.J., Crews, F.T. and Guzman, N.J.
Ethanol enhances the endothelial nitric oxide synthase
response to agonists. Hypertention 21:939-943, 1993.
Davda, R.K., Chandler, L.J., Guzman, N.J. Protein kinase C
modulates receptor-independent endothelial nitric oxide
synthase activity. Eur. J. Pharmacol . (Molecular
Pharmacology). 266: 237-244, 1994.
Davis, M.D., Kaufman, S. and Milstien, S. Distribution of
4a-hydroxytetrahydrobiopterin dehydratase in rat tissues.
Comparison with the aromatic amino acid hydroxylases. FEBS
Lett. 302: 73-76, 1992.
Deng, W. , Thiel, B., Tannenbaum, C.S., Hamilton, T. A. and
Stuehr, D.J. Synergistic cooperation between T-cell
lymphokines for induction of the nitric oxide synthase gene
in murine peritoneal macrophages. J. Immunol. 151: 322-329,
1993 .
Dimmeler, S., Lottspeich, F. and Bruñe B. Nitric oxide
causes ADP-ribosylation and inhibition of glyceraldehyde-3-
phosphate dehydrogenase. J. Biol. Chem. 267: 1-4, 1992.
Dinermann, J.L., Lowenstein, C.J., Snyder, S.H. Molecular
mechanisms of nitric oxide regulation: potential relevance to
cardiovascular disease. Circulation Res. 73(2): 217-222,
1993.
Ding, A.H., Nathan, C.F. and Stuehr, D.J. Release of
reactive nitrogen intermediates from mouse peritoneal
macrophages. J. Immunol. 141: 2407-2412, 1988.
Di Silvio, M. , Geller, D.A., Gross, S.S., Nussler, A.,
Freeswick, P., Simmons, R.L. and Billiar, R. Inducible
nitric oxide synthase activity in hepatocytes is dependent on
the coinduction of tetrahydrobiopterin synthesis. Adv. Exp.
Med. Biol. 338: 305-305, 1993.
Doyle, M.P. and Hoekstra, J.W. Oxidation of nitrogen oxides
bound by dioxygen in hemoproteins. J. Inorg. Biochem. 14:
351-358, 1981.
Drapier, J.C. and Hibbs, J.B. Differentiation of murine
macrophages to express non-specific toxicity for tumor cells

121
results in L-arginine dependent mitochondrial iron-sulfur
enzymes in the macrophage effector cells. J. Immunol. 140:
2829-2838, 1988.
Eilers, A., Seegert, D. , Schindler, C., Baccarini, M. and
Decker, T. The response of y-interferon activation factor is
under developmental control in cells of the macrophage
lineage. Mol. Cell Biol. 13: 3245-3254, 1993.
Fórstermann, U., Schmidt, H.H., Pollock, J.S., Sheng, H. ,
Mitchell, J.A., Warner, T.D., Nakane, M. and Murad, F.
Isoforms of nitric oxide synthase: characterization and
purification from different cell types. Biochem Pharmacol
42: 1849-1857, 1991.
Franzoso, G. , Bours, V. , Park, S., Tomita-Yamaguchi, M. ,
Kelly, K. and Siebenlist, U. The candidate oncoprotein Bcl-3
is an antagonist of p50/NF-KB-mediated inhibition. Nature
359: 339-342, 1992.
Fried, M. and Crothers, D.M. Equilibrium and kinetics of lac
repressor-operator interactions by polyacrylamide gel
electrophoresis. Nucleic Acid Research 9: 6505-6525, 1981.
Furchgott, R.F. and Zawadzki, J.V. The obligatory role of
endothelial cells in the relaxation of arterial smooth muscle
by acetylcholine. Nature 288: 373-376, 1980.
Ganchi, P.A., Sun, S.C., Greene, W.C. and Ballard, D.W. A
novel NF-kappa B complex containing p65 homodimers:
implications for transcriptional control at the level of
subunit dimerization. Mol. Cell. Biol. 13: 7826-7835, 1993.
Garner, M.M. and Revzin A. A gel electrophoresis method for
quantifying the binding of proteins to specific DNA regions:
application to components of the Escherichia coli lactose
operon regulatory system. Nucleic Acid Research 9: 3047-3060,
1981.
Garthwaite, J. Glutamate, nitric oxide, and cell-cell
signaling in the nervous system. Trends Neurosci. 14: 60-67,
1991.
Geller, D.A., Di Silvio, M. , Nussler, A.K., Wang, S.C.,
Shapiro, R.A., Simmons, R.L. and Billiar, T.R. Nitric oxide
synthase expression is induced in hepatocytes in vivo during
inflammation. J. Surg. Res. 55: 427-432, 1993 [a].
Geller, D.A., Nussler, A.K., Di Silvio, M., Lowenstein, C.J.,
Shapiro, R.A., Wang, S.C., Simmons, R.L. and Billiar, T.R.
Cytokines, endotoxin and glucocorticoids regulate the
expression of inducible nitric oxide synthase in hepatocytes.
Proc. Natl. Acad. Sci. USA 90: 522-526, 1993 [b] .

122
Golub, E.I., Kim, H. and Volsky, D.J. Transfection of DNA
into adherent cells by DEAE-dextran/DMSO method increases
drastically if cells are removed from surface and treated in
suspension. Nucleic Acids Res. 17: 4902, 1989.
Gorman, C.M., Moffat, L.F. and Howard, B.H. Recombinant
genomes which express chloramphenicol acetyltransferase in
mammalian cells. Mol. Cell Biol. 2: 1044, 1982.
Gross, S.S., Jaffe, E.A., Levi, R. and Kilbourn, R.G.
Cytokine-activated endothelial cells express an isotype of
nitric oxide synthase which is tetrahydrobiopterin-dependent,
calmodulin-independent and inhibited by arginine analogues
with a rank order of potency characteristic of activated
macrophages. Biochem. Biophys. Res. Commun. 178: 823-829,
1991.
Gross, S.S. and Levi, R. Tetrahydrobiopterin synthesis: an
absolute requirement for cytokine-induced nitric oxide
generation by vascular smooth muscle. J. Biol. Chem. 267:
25722-25729, 1992.
Gross, S.S., Levi, R., Madera, A., Park, K.H., Vane, J. and
Hattori, Y. Tetrahydrobiopterin is induced by LPS in
vascular smooth muscle and is rate-limiting for nitric oxide
production. Adv. Exp. Med. Biol. 338: 295-300, 1993.
Guzman, N.J., Fang, M.Z., Tang, S.S., Ingel finger, J. and
Garg, L.C. Autocrine inhibition of Na+/K+-ATPase by nitric
oxide in mouse proximal tubule epithelial cells. J. Clin.
Invest. 95: 2083-2088, 1995.
Hattori, Y. and Gross, S.S. GTP cyclohydrolase I mRNA is
induced by LPS in vascular smooth muscle: characterization,
sequence and relationship to nitric oxide synthase. Biochem.
Biophys. Res. Commun. 195: 435-441, 1993.
Haverty, T.P., Kelly, C.J., Hines, W.H., Amenta, P.S.,
Watanabe, M., Harper, R.A., Kefalides, N.A. and Neilson, E.G.
Characterization of a renal tubule epithelial cell line which
secretes the autologous target antigen of autoimmune
experimental interstitial nephritis. J. Cell Biol. 107: 1359-
1368, 1988.
Henkel, T., Machleit, T., Alkalay, I., Kronke, M., Ben-
Neriah, Y. and Baeuerle, P.A. Rapid proteolysis of I kappa
B-alpha is necessary for activation of transcription factor
NF-kappa B. Nature 365: 182-185, 1993.
Hirai, H., Fujisawa, J., Suzuki, K., Ueda, K., Muramutsu, M.,
Tsuboi, A., Arai, N. and Yoshida, M. Transcriptional

123
activator Tax of HTLV-1 binds to the NF-kB precursor pl05.
Oncogene 7: 1737-1742, 1992.
Ignarro, L.J. Biosynthesis and metabolism of endothelium-
derived nitric oxide. Ann. Rev. Pharmacol. Toxicol. 30: 535-
560, 1990.
Ignarro, L.J., Buga, G.M., Wood, K.S., Byrns, R.E. and
Chaudhuri, G. Endothelial-derived relaxing factor produced
and released from artery and vein is nitric oxide. Proc.
Natl. Acad. Sci. USA 84: 9265-9269, 1987.
Jorens, P.G., Van Overveld, F.J., Vermeir, P.A., Bult, H. and
Herman, A.G. Synergism between interleukin-l-p and
interferon-y, an inducer of nitric oxide synthase, in rat
fibroblasts. Eur. J. Pharmacol. 224: 7-12, 1992.
Junguero, D.C., Scott-Burden, T., Schini, V.B. and Vanhoutte,
P.M. Inhibition of cytokine-induced nitric oxide production
by transforming growth factor B1 in human smooth muscle
cells. J. Physiol. Lond. 454: 451-465, 1992.
Kamijo, R., Harada, H., Matsuyama, T., Bosland, M.,
Gerecitano, J., Shapiro, D., Le, J., Koh, S.I., Kimura, T.,
Green, S.J., Mak, T.W., Taniguchi, T. and Vicek, J.
Requirement for transcription factor IRF-1 in NO synthase
induction in macrophages. Science 263: 1612-1615, 1994.
Karupiah, G. , Xie, Q-w., Buller, R.M.L., Nathan, C., Duarte,
C. and MacMicking, J.D. Inhibition of viral replication by
inter feron-y-induced nitric oxide synthase. Science 261:
1445-1448, 1993.
Kaufman, S. New tetrahydrobiopterin-dependent systems. Annu.
Rev. Nutr. 13: 261-286, 1993.
Ketteler, M. , Border, W.A. and Noble, Nancy. Cytokines and
L-arginine in renal injury and repair. Am. J. Physiol. 267:
F197-F207, 1994.
Kilbourn, R.G. and Bellone, I. Endothelial cell production
of nitrogen oxides in response to interferon in combination
with tumor necrosis factor, interleukin-1, or endotoxin. J.
Natl. Cancer Inst. 82: 772-776, 1990.
Koesling, D. , Schultz, G. and Bóhme, E. Sequence homologies
between guanylyl cyclases and structural analogies to other
signal transducing proteins. FEBS Lett. 280: 301-306,1991.
Kubes, P., Suzuki, M. and Granger, D.N. Nitric oxide: an
endogenous modulator of leukocyte adhesion. Proc. Natl. Acad.
Sci. USA 88: 4651-4655, 1991.

124
Kwon, N.S., Nathan, C.S. and Stuehr, D.J. Reduced biopterin
as a cofactor in the generation of nitrogen oxides by murine
macrophages. J. Biol. Chem. 264: 20496-20501, 1989.
Langrehr, J.M., Hoffman, R.A. Lancaster Jr., J.R. and
Simmons, R.L. Nitric oxide - A new endogenous
immunomodulator. Transplantation 55: 1205-1212, 1993 [a],
Langrehr, J.M., White, D.A. Hoffman, R.A. and Simmons, R.L.
Macrophages produce nitric oxide at allograft sites. Ann.
Surg. 218: 159-166, 1993 [b].
Laychock, S.G., Módica, M.E. and Cavanaugh, C.T. L-Arginine
stimulates cyclic guanosine 3',5'-monophosphate formation in
rat islets of Langerhans and RINm5F insulinoma cells:
evidence of L-arginine:nitric oxide synthase. Endocrinology
129: 3043-3052, 1991.
Leclair, K.P., Blanar, M.A. and Sharp, P.A. The p50 subunit
of NF-kB associates with the NF-IL6 transcription factor.
Proc. Natl. Acad. Sci. USA. 89: 8145-8149, 1992.
Levillain, O. , Hus-Citharel, A., Morel, F. and Bankir, L.
Arginine synthesis in mouse and rabbit nephron: localization
and functional significance. Am. J. Physiol. 264 (Renal Fluid
Electrolyte Physiol. 33): F1038-F1045, 1993.
Lew, D.J., Decker, T., Strehlow, I. and Darnell, J.E.
Overlapping elements in the gualylate-binding protein gene
promoter mediate transcriptional induction by alpha and gamma
interferons. Mol. Cell Biol. 11: 182-191, 1991.
Lind, D.S., Copeland III, E.M. and Souba, W.W. Endotoxin
stimulates arginine transport in pulmonary artery endothelial
cells. Surgery 114: 199-205, 1993.
Lipton, S.A., Choi, Y., Pan, Z., Lei, H., Chen, H.V., Sucher,
N.J., Loscalzo, J., Singel, D.J. and Stamler, J.S. A redox-
based mechanism for the neuroprotective and neurodestructive
effects of nitric oxide and related nitroso-compounds. Nature
364: 626-632, 1993.
Lowenstein, C.J., Alley, E.W., Raval, P., Snowman, P.,
Snyder, S.H., Russel, S.W. and Murphy, W.J. Macrophage
nitric oxide synthase gene: two upstream regions mediate
induction by interferon-gamma and lipopolysaccharide. Proc.
Natl. Acad. Sci. USA 90: 9730-9734, 1993.
Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J.
Protein measurement with the Folin phenol reagent. J. Biol.
Chem. 193: 265-275, 1951.

125
Lukic, M.L., Stosic-Grujicic, S., Ostojic, N., Chan, W.L. and
Liew, F.Y. Inhibition of nitric oxide generation affects the
induction of diabetes by streptozocin (sic) in mice. Biochem.
Biophys. Res. Commun. 178: 913-920, 1991.
Markewitz, B.A., Michael, J.R. and Kohan, D.E. Cytokine-
induced expression of a nitric oxide synthase in rat renal
tubule cells. J. Clin. Invest. 91: 2138-2143, 1993.
Marietta, M.A., Tayeh, M.A. and Hevel, J.M. Unraveling the
biological significance of nitric oxide. Biofactors 2: 219-
225, 1990.
Marsden, P.A. and Ballermann, B.J. Tumor necrosis factor
alpha activates soluble guanylate cyclase in bovine
glomerular mesangial cells via an L-arginine-dependent
mechanism. J. Exp. Med. 172: 1843-1852, 1990.
Martin, E., Nathan, C. and Xie, Q-w. Role of interferon
regulatory factor 1 in induction of nitric oxide synthase. J.
Exp. Med. 180: 977-984, 1994.
Mellion, B.T., Ignarro, L.J., Ohlstein, E.H., Pontecarvo,
E.G., Hyman, A.L. and Kadowitz, P.J. Evidence for the
inhi