Citation
Characterization of brain type I receptors for adrenocorticosteroid hormones

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Title:
Characterization of brain type I receptors for adrenocorticosteroid hormones
Creator:
Emadian, Seyed Mohammad, 1958-
Publication Date:
Language:
English
Physical Description:
xiii, 185 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Cytosol ( jstor )
Gels ( jstor )
Kidneys ( jstor )
Ligands ( jstor )
Mineralocorticoids ( jstor )
Molybdates ( jstor )
Rats ( jstor )
Receptors ( jstor )
Sodium ( jstor )
Steroids ( jstor )
Corticotropin -- physiology ( mesh )
Dissertations, Academic -- Neuroscience -- UF ( mesh )
Neuroscience thesis Ph.D ( mesh )
Receptors, Cell Surface -- physiology ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1987.
Bibliography:
Bibliography: leaves 145-183.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Seyed Mohammad Emadian.

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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:
023161089 ( ALEPH )
17889880 ( OCLC )
AEL1997 ( NOTIS )
AA00006108_00001 ( sobekcm )

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Full Text













CHARACTERIZATION OF BRAIN TYPE I RECEPTORS
FOR ADRENOCORTICOSTEROID HORMONES












By

SEYED MOHAMMAD EMADIAN


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


1987




CHARACTERIZATION OF BRAIN TYPE I RECEPTORS
FOR ADRENOCORTICOSTEROID HORMONES
By
SEYED MOHAMMAD EMADIAN
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
1987


Dedicated to my parents and my wife without whose continuous
support this work would not have been possible


ACKNOWLEDGMENTS
The experiments in this dissertation were conducted under the
guidance of my mentor and chairman of my supervisory committee, Dr.
William G. Luttge. I am indebted to Dr. Luttge for being a superb
mentor, teacher, colleague, friend, and for his continuous support
throughout the course of my dissertation work. I further would like to
extend my gratitude to my other supervisory committee members, Drs. A.J.
Dunn, M.J. Fregly, M.B. Heaton and K.T. Shiverick, for their guidance
and critical evaluation of my dissertation. I am also grateful to Dr.
L.C. Garg for his participation in the discussion of this work and his
helpful and valuable contributions during our committee meetings. I
would like to thank Dr. C.L. Densmore and Ms. Y.-C. Chou for their
helpful comments and evaluation of the results of this work during our
laboratory meetings and Ms. Mary E. Rupp and Mrs. Cameron F. Bloom for
technical assistance. Last, but not least, I would like to acknowledge
the financial assistance provided to me by the Department of
Neuroscience and Center for Neurobiological Sciences during the course
of my graduate studies.
iii


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS iii
LIST OF TABLES vi
LIST OF FIGURES vii
ABBREVIATIONS ix
ABSTRACT xii
CHAPTERS
I REVIEW OF THE LITERATURE 1
Control of ALDO Secretion 4
Peripheral Site of Action and Effects of
Mineralocorticoids 5
Central Site of Action and Effects of
Mineralocorticoids 11
Molecular Mechanisms of Mineralocorticoid
Action 18
Mechanisms of Receptor Regulation 26
In Vitro Analysis of Type I Receptors
by Radioreceptor Assay 36
II EXPERIMENTAL PROCEDURES 43
Isotopes, Steroids and Other Chemicals 43
Experimental Animals 44
Buffers 44
Cytosol Preparation 45
Aging 45
Hydrophobic Interaction Chromatography 45
Steroid Binding Determination 46
Cytosolic Protein Concentration 47
III EFFECTS OF POLYHYDRIC AND MONOHYDRIC COMPOUNDS ON THE
STABILITY OF TYPE I RECEPTORS FOR ADRENOCORTICOSTEROID
HORMONES IN BRAIN CYTOSOL 48
Summary 48
Introduction 49
Materials and Methods 50
Results 50
Discussion 58


Proposed Mechanism of Action of Poly-
and Monohydric Compounds 58
Possible Mode of Action of Poly- and
Monohydric Compounds on Unoccupied
Type I Receptors in Crude Cytosol 64
IVA NOVEL EFFECT OF MOLYBDATE ON THE BINDING OF
[3H]ALDOSTERONE TO GEL FILTERED TYPE I
RECEPTORS IN BRAIN CYTOSOL 70
Summary 70
Introduction 71
Materials and Methods 72
Results 73
Discussion 88
VDIFFERENTIAL INACTIVATION OF TYPE II RECEPTORS FOR
ADRENAL STEROIDS IN WHOLE BRAIN CYTOSOL:
RECOVERY OF INTACT TYPE I RECEPTORS 93
Summary 93
Introduction 94
Materials and Methods 96
Results and Discussion 97
Effects of DTNB on Unoccupied Type I Receptors .... 97
Effects of DTNB on Occupied Receptors 101
Effects of DCC Pretreatment of Cytosol on
Ligand Binding Capacity of Unoccupied
Type I and Type II Receptors 106
VIIN VITRO TRANSFORMATION OF ALDOSTERONE-TYPE I
RECEPTOR COMPLEXES TO A DNA-BINDING STATE 122
Summary 122
Introduction 123
Materials and Methods 129
Results 130
Discussion 139
VIICONCLUDING REMARKS 142
REFERENCES 145
BIOGRAPHICAL SKETCH 184
v


LIST OF TABLES
Page
Table 2-1: Buffers 44
Table 4-1: Binding Parameters for Type I Receptors Obtained
from Crude and Gel Filtered Cytosol in the
Presence and Absence of 2 mM Molybdate 82
Table 5-1: Effects of 1 mM DTNB on Whole Brain Cytosolic
Unoccupied Type I Receptors 100
Table 5-2: Effects of Two Consecutive DCC Pretreatments
of Cytosol on the Residual DEX Specific
Binding Ill
Table 6-1: Comparison of the Binding of ALDO-Type I and
DEX-Type II Receptor Complexes to
DNA-cellulose Before and After Salt- and/or
Temperature-Induced Activation 132
Table 6-2: Effects of 5 mM Molybdate on the Binding of
ALDO-Type I Receptor Complexes to DNA-
Cellulose Before and After Salt- and
Temperature-Induced Activation 136
vi


LIST OF FIGURES
Page
Figure 3-1: Effects of Polyhydric Compounds on the Stability
of Unoccupied Type I Receptors in Whole Brain
Cytosol 52
Figure 3-2: Effects of 10% Glycerol on the Stability of
Unoccupied Type II Receptors in Whole brain
Cytosol 55
Figure 3-3: Effects of Monohydric Compounds on the Stability
of Unoccupied Type I Receptors in Whole Brain
Cytosol 57
Figure 3-4: Dose-Response Analysis of the Effects of Ethanol
on the Binding of ALDO to Type I Receptors in
Whole Brain Cytosol 60
Figure 3-5: Scatchard Analysis of the Effects of 10% Ethanol
on the Equilibrium Binding Parameters of ALDO
Binding to Type I Receptors in Whole Brain
Cytosol 62
Figure 3-6: Comparison of the Surface Hydrophobic Properties
of ALDO-Type I and DEX-Type II Receptor
Complexes on Pentyl-Agarose Columns 68
Figure 4-1: Effects of Gel Filtration, Molybdate and/or
Dithiothreitol on the Binding Capacity and
Stability of Unoccupied Type I Receptors From
Whole Brain Cytosol 75
Figure 4-2: Dose-Response Analysis of the Effects of Gel
Filtration and Molybdate on the Binding
Capacity and Stability of Unoccupied Type I
Receptors from Whole Brain Cytosol 78
Figure 4-3: Effects of Molybdate and/or Gel Filtration
on the Equilibrium Binding Parameters of ALDO
Binding to Unoccupied Type I Receptors from
Whole Brain Cytosol 81
v i i


Figure 4-4: Stability of Unoccupied Type I Receptors from
Whole Brain Cytosol Following Gel Filtration
in the Absence of Molybdate: Effects of
Molybdate Addition Following Gel Filtration
and Aging 85
Figure 4-5: Effects of Dilution and/or Molybdate on the
Binding Capacity of Unoccupied Type I
Receptors from Whole Brain Cytosol 87
Figure 5-1: Effects of 1 mM DTNB on Receptors Bound to
ALDO in Whole Brain Cytosol 103
Figure 5-2: Effects of DCC Pretreatment and/or Aging of
Unlabeled Whole Brain Cytosol on the Binding
of ALDO to Type I and DEX to Type II Receptors . 105
Figure 5-3: Effects of Pretreatment of Whole Brain Cytosol
with Various Concentrations of DCC and
Different Duration of DCC Pretreatment on
the Binding of DEX to Type II Receptors 109
Figure 5-4: Effects of DCC Pretreatment of Whole Brain
Cytosol on the Binding of ALDO and DEX to
Type I and Type II Receptors in the Presence
or Absence of a Steroid Competitor 114
Figure 5-5: Effects of 300 mM KC1 in the Presence and
Absence of 20 mM Molybdate on the Binding
of ALDO to Type I and DEX to Type II
Receptors 117
Figure 5-6: Effects of 300 mM KC1 on the Specific Binding
of ALDO and DEX Prior and Subsequent to DCC
Pretreatment 119
Figure 6-1: Comparison of the Surface Hydrophobic Properties
of Unactivated and Activated ALDO-Type I
Receptor Complexes on Pentyl-Agarose
Columns 138
viii


ABBREVIATIONS
All: angiotensin II
Ach: acetylcholine
ACTH: adrenocorticotropic hormone
ADP: adenosine diphosphate
AIPs: aldosterone-induced proteins
ALDO: aldosterone
ANF: atrial natriuretic factor
ASF: aldosterone-stimulating factor
ATPase: adenosine triphosphatase
B.,, : maximal binding
MAX
B : nonspecific binding
NS
B^: total binding
CBG: corticosteriod-binding globulin
CPM: counts per minute
CNS: central nervous system
CORT: corticosterone
CSF: cerebrospinal fluid
DCC: dextran-coated charcoal
DEAE-Cellulsoe: die thylaminoe thyl-Cellulose
DEX: dexamethasone
DNA: deoxyribonucleic acid
DNA-C: deoxyribonucleic acid-cellulose
DOC: deoxycorticosterone
IX


DOCA: deoxycorticosterone acelate
DPM: disintegration per minute
DTNB: 5,51-dithiobis (2-nitrobenzoic acid)
DTT: dithiothreitol
E: erythritol
EEDQ: N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinolone
EG: ethylene glycol
Et: ethanol
G: glycerol
HEPES: 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid
hn-RNA: heterogeneous nuclear-ribonucleic acid
: equilibrium dissociation constant
MMTS: methyl methanethiosulfonate
MMTV: mouse mammary tumor virus
mRNA: messenger ribonucleic acid
Mt: methanol
NAD+: nicotinamide-adenine dinucleotide
NADPH: nicotinamide-adenine dinucleotide phosphate
NEM: N-ethylmaleimide
NM: nuclear matrix
P: propanol
PMSF: phenylmethyl sulfonylfluoride
R: ribitol
RPM: revolutions per minute
S: sorbitol
SCO: subcommissural organ
SDS-PAGE: sodium dodocylsulfate-polyacrylamide gel electrophoresis
x


TA: triamcinolone acetonide
: melting temperature
X: xy 1 i to 1
xi


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
CHARACTERIZATION OF BRAIN TYPE I RECEPTORS
FOR ADRENOCORTICOSTEROID HORMONES
By
Seyed Mohammad Emad ian
May 1987
Chairman: William G. Luttge, Ph.D.
Major Department: Neuroscience
Preponderance of evidence suggests that the potent effects of
adrenocorticosteroid hormones on a number of physiological and
behavioral functions are initiated through stereospecific binding of
these steroids with soluble intracellular receptors, followed by the
interaction of the steroid-receptor complexes with the genome. Despite
the wealth of our knowledge regarding the properties of steroid hormone
receptors in general, practical difficulties such as lack of specific
available ligands, receptor instability under cell-free conditions, low
receptor concentration in whole tissue preparations, etc., have greatly
limited our knowledge of properties and regulation of Type I receptors
for adrenocorticosteroid hormones.
The work presented in this dissertation has overcome some of these
difficulties and describes a number of distinct in vitro properties
of these receptors: 1) Unlike Type II (glucocorticoid) receptors, the
extraction of unoccupied Type I receptors into an aqueous environment
was found to result in a reduction in the binding capacity of these
xii


receptors--possibly by inducing a conformation of the receptors with an
infolded hydrophobic steroid-binding pocket. A reduction in hydrophobic
interactions by including monohydric compounds in cytosol was shown to
result in an increase in the binding capacity of these receptors for the
ligand. 2) Although the stabilizing effects of molybdate on steroid
receptors is well established, this oxyanion was found to produce a
paradoxical effect on the binding capacity of unoccupied Type I
receptors in crude and gel filtered cytosol preparations: a reduction
in receptor binding capacity in crude, and an increase in receptor
binding capacity in gel filtered cytosol was observed in the presence of
molybdate. 3) Whereas unoccupied Type II receptors could be inactivated
by removal of endogenous sulfhydryl reducing agent from cytosol using
dextran-coated charcoal (DCC), Type I receptors appeared insensitive to
the effects of dithiothreitol (a potent sulfhydryl reducing reagent) and
DCC pretreatment of cytosol (a mild oxidizing condition). The
differences between these two receptors were used to inactivate
selectively Type II receptors. 4) Lastly, conditions that induced
transformation (activation) of glucocorticoid-Type II receptors to a
DNA-binding state (i.e., 300 mM KC1 and incubation at 22C) were found
to produce minimal activation of Type I receptors. Possible functional
and physiological significance of these findings is discussed.
xm


CHAPTER I
REVIEW OF THE LITERATURE
The actions of adrenocorticosteroid hormones on a wide variety of
physiological and behavioral functions in mammals are well characterized
(for review see Fregly and Luttge, 1982; Luttge, 1983; Rees and Gray,
1983; De Kloet 1984; Munck et al., 1984; Meyer, 1985; Funder, 1986;
De Kloet et al., 1986; McEwen et al., 1986b; Munck and Guyre, 1986). As
a result of extensive research, significant developments have occurred
in the past three decades with regard to the mechanism of action of
these hormones (for review see Reich and Scott, 1979; Fanestil and
Kipnowski, 1982; Lan et al., 1984; Rossier, et al., 1985; Marver, 1986;
Scheidereit et al., 1986; Slater et al., 1986). Although limited
experimental evidence suggests that the actions of adrenocorticosteroid
hormones may result from direct interactions of these steroids with
components of membrane (nongenomic response) (Chaplin et al., 1981;
Towle and Sze, 1983), the bulk of evidence suggests that steroid
hormones, in general, exert their effect via a stereospecific binding
with soluble intracellular protein receptors. The resulting
steroid-receptor complexes undergo certain physicochemical changes that
increase the overall affinity of the complexes for nuclei, chromatin and
DNA and other polyanions (for detail see Chapter VI). This
"transformation" process (also known as "activation") allows for a
direct interaction of the transformed complexes with nuclear acceptor
site(s) which in turn modulate the transcription of certain mRNAs and
hence cellular protein synthesis. It is important to note that steroid
1


2
hormone receptors are the only regulators of gene expression in the
eukaryotic cells thus far described. Indeed, recent studies have
characterized specific palindromic sequences within the DNA
molecule--hormone response elements--to which the transformed steroid
receptors bind in order to modulate the transcription of certain mRNAs
(e.g., Arnemann et al., 1987; Chambone et al., 1987; Drouin et al.,
1987; Karin et al., 1987; Miesfeld et al., 1987).
Long before isolation and purification of aldosterone (ALDO)
(Grundy et al., 1952; Simpson and Tait, 1952), the most potent naturally
occurring mineralocorticoid hormone, it was known that blood borne
"factors" controlled electrolyte balance within the body (Addison,
1855). Lucas (1926) provided the first evidence for the adrenal
cortical regulation of mineral metabolism by adrenalectomizing dogs.
However, despite these and many other preliminary observations, the
discovery of ALDO production by zona glomerulosa of the adrenal glands
came long after the discovery of the main corticosteroid hormones
produced by the zonae fasciculata and reticularis. The earlier
investigators attributed hyponatremia, hypochloremia, acidosis,
hyperkalemia, and other symptoms associated with Addison's disease to
the lack of deoxycorticosterone (DOC) secretion (Thorn et al., 1942).
Despite the potent mineralocorticoid activity of DOC, this theory was
discredited when extracts from adrenal glands were found to contain only
minute amounts of DOC. The discovery of ALDO came about some years
later as a result of improved quantitative bioassay and chromatographic
techniques (Grundy et al., 1952; Simpson and Tait, 1952; Simpson et al.,
1954; Simpson and Tait, 1955).


3
Because of the potent action of ALDO on mineral metabolism (for
detail see below), the high affinity binders for this steroid extracted
from the kidney were dubbed as "mineralocorticoid receptors." However,
since the endogenous glucocorticoid, corticosterone (CORT), was found to
have comparable affinty for these receptors (see below), a less
ambiguous term, Type I receptors, will be used in this dissertation to
refer to these receptors. This terminology will also be used to
distinguish these receptors from the lower affinity, higher capacity
ALDO binders, the Type II (glucocorticoid) receptors. Although the
principal target tissues for the action of ALDO in mammals have been
considered to be the kidney, intestines and salivary glands, because
electrolyte exchange occurs throughout the organism, other tissues may
also be considered as targets for ALDO. This claim is further supported
by the fact that Type I receptors have been identified in the pituitary
gland (Moguilewsky and Raynaud, 1980; De Nicola et al., 1981), parotid
gland (Funder et al., 1972), mammary gland (Quirk et al., 1983), lung
(Krozowski and Funder, 1981), bovine ciliary body (Starka et al., 1977),
- f
rabbit lens epithelium (Hampl et al., 1981), human ocular lens (Hampl,
et al., 1984; Starka et al., 1986), whole brain cytosol (Anderson and
Fanestil, 1976; Emadian et al., 1986), various brain regions
(Moguilewsky and Raynaud, 1980; De Nicola et al., 1981; Veldhuis et al.,
1982b; Coirini et al., 1983), spinal cord (Orti et al., 1986), human
mononuclear leukocytes (Armanini et al., 1986), rat epididymis (Hinton
and Keefer, 1985; Pearce et al., 1986a), "arterial walls" (Kornel, 1981;
Kornel et al., 1982a & b) and rat cochlear and vestibular organs (Rarey
and Luttge, 1987).


4
Control of ALDO Secretion
In mammals, several known physiological modulators of ALDO
secretion from the adrenal cortex include the renin-angiotensin II
system, potassium ion and adrenocorticotropic hormone (ACTH) An
extensive body of research has implicated the renin-angiotensin II
system as the principal regulator of ALDO secretion (e.g., Kaplan, 1965;
Davis, 1975; Fraser et al., 1979). Although ACTH stimulation of ALDO
steroidogenesis via the adenylate cyclase cascade mechanism is well
characterized, ACTH is apparently not involved in the maintenance of
ALDO secretion under basal conditions. Note, for example, that
suppression of ACTH secretion with dexamethasone (DEX), a synthetic
glucocorticoid, and other manipulations (e.g., hypophysectomy) does not
alter the circadian rhythm of ALDO secretion (Fraser et al., 1979).
Furthermore, infusion of ACTH leads to sustained increases in plasma
ALDO concentration, suggesting that ACTH may mediate only acute
increases in ALDO secretion physiologically (Kem et al., 1975).
A number of other factors including aldosterone-stimulating factor
(ASF, secreted by the anterior pituitary) (Carey and Sen, 1986); atrial
natriuretic factor (ANF, a family of peptides isolated from the atria of
rat, human and other species) (Chartier et al., 1984; Takagi et al.,
1986; Aguilera, 1987; Naruse et al., 1987; Sagnella et al., 1987);
acetylcholine, ACh (Kojima et al., 1986); dopamine (Carey and Drake,
1986; for review see Campbell et al., 1981); vasopressin (Woodcock et
al., 1986) and serotonin (Matsuoka et al., 1985) have been recently
implicated in the regulation of ALDO secretion. Large reductions in the
concentration of sodium in serum have also been shown to stimulate ALDO


5
secretion; however, the physiological role of this hyponatremia-induced
ALDO secretion awaits further exploration (Davis, 1975).
Peripheral Site of Action and Effects of Mineralocorticoids
As mentioned above, the main peripheral action of ALDO is to
maintain electrolyte balance in the organism. A secondary action is to
help maintain proper blood volume and pressure. These actions of ALDO
are achieved primarily through reabsorption of sodium and hence water
(due to osmotic activity of sodium) and excretion of potassium and
hydrogen ions by the kidney. In other words, an increase in the
circulating levels of ALDO results in a decrease in the ratio of sodium
to potassium in urine, hence an increase in plasma and extracellular
fluid volume. An ALDO-mediated reduction in the Na/K ratio is also
reported in saliva, sweat and feces.
The antinatriuretic and kaliuretic effects of ALDO on kidney are
well established. Due to a remarkable anatomical complexity and
specialization in this organ (for review see Jacobson, 1981), the exact
sites at which these effects are mediated remained controversial for
some time (Knox et al., 1980). Because ALDO, deoxycorticosterone
acetate (DOCA, an acetate derivative of DOC) and other steroids with
mineralocorticoid activity were found to influence electrolyte balance
without changing renal plasma flow and glomerular filtration rate
(Barger et al., 1958; Ganong and Mulrow, 1958), the distal tubule was
postulated to be the main locus of mineralocorticoid action (Hierholzer
et al., 1965; Wright and Giebisch, 1978). Studies with isolated,
perfused tubules from rabbit kidneys, however, provided evidence
suggesting that mineralocorticoids (administered acutely or chronically)
increase sodium reabsorption and potassium secretion in the cortical


6
collecting tubules and not the distal tubules (O'Neil and Helman, 1977;
Schwartz and Burg, 1978; Stokes et al., 1981). Furthermore,
mineralocorticoids were also found to increase discretely the
transtubular electrical potential (lumen negative) across the cortical
collecting tubules (Gross et al., 1975; Gross and Kokko, 1977), a
finding not confirmed in the study by Schwartz and Burg (1978).
Determination of pH profile along the rat papillary collecting duct by
micropuncture technique suggested the action of ALDO on distal papillary
collecting duct (Higashihara et al., 1984). In addition to the
possibility that ALDO may influence multiple segments of the nephron,
the uncertainty as to the site of action of mineralocorticoids in kidney
and other target tissues may also be due to species, tissue and/or
methodological differences between the aforementioned studies. For
example, in contrast to the results obtained from the rabbit nephron,
ALDO was found to normalize sodium transport in the rat distal tubule
(Hierholzer and Wiederholt, 1976; Horisberger and Diezi, 1983) without
changing the transtubular potential (Hierholzer and Wiederholt, 1976).
Moreover, ALDO action at receptor sites other that Type I receptors
cannot be ruled out. Note that the administration of DEX (a specific
synthetic glucocorticoid) to adrenalectomized rats has been shown
recently to increase Na+/H+ exchange activity in isolated renal
brush border membrane vesicles (Kinsella et al., 1985). Other
investigators, however, reported that glucocorticoids have kaliuretic
and not antinatriuretic effects (e.g., Bia et al., 1982; Campen et al.,
1983; for review see Field and Giebisch; 1985). The use of in situ
microperfusion of single superficial distal tubles in adrenalectomized
rats revealed no direct effect of DEX on potassium secretion by the


7
microperfused tubule; however, an overall increase in urinary flow and
sodium excretion was a common finding in these studies (Field et al.,
1984). In contrast, infusion of ALDO under identical conditions led to
a 90% stimulation of potassium secretion by the microperfused tubule and
a marked reduction in the kidney sodium excretion and urinary flow rate.
No apparent effect on kidney potassium excretion was observed under this
latter condition (Field et al., 1984). It is postulated that mineral
exchange represents the net result of direct and synergistic actions of
the steroids on renal tubules plus secondary effects mediated by changes
in urinary flow rate (Field et al., 1984). Parenthetically, recent
studies on the effects of corticosteroids on Na+ transport in distal
colon suggest a different cellular mechanism for ALDO and DEX (Na+
transport in response to ALDO, but not DEX, was found to be amiloride
sensitive) (Jorkasky et al., 1985). Furthermore, since basal plasma
levels of ALDO (5 ng/dl) in the presence of potassium loading
significantly increased transepithelial potential difference and the
area of the basolateral membrane in the late distal convolution (a
characteristic feature of "potassium adaptation" when
pharmacological doses of ALDO are used in potassium-loaded
adrenalectomized animals), Hirsch et al. (1984) postulated a
differential mechanism of ALDO action on different segments of nephron.
Note that neither the acute nor chronic administration of ALDO in the
absence of potassium loading has any effect on the late distal
convolution, whereas this latter treatment increased surface density of
the basolateral membrane in initial collecting tubule cells (Hirsch et
al., 1984).


8
Autoradiographic and binding assay studies focusing on the regional
distribution of Type I receptors in various segments of renal nephron
have yielded fruitful results regarding the potential site(s) of
mineralocorticoid action along the nephron. Radioreceptor assays of
cytosolic fractions obtained from rat kidney cortical tubules enriched
in proximal or distal segments revealed a higher concentration of
3
[ HjALDO binding in the latter (in the presence or absence of a
glucocorticoid competitor), although the precise location of Type I
receptors along the nephron was not identified (Scholer et al., 1979).
3 ]_6
In the rabbit nephron, high specific [HjALDO binding ( >10
mol/cm tubule length) was found in the branched, cortical and outer
medullary collecting tubules, whereas specific binding was negligible in
the proximal tubules, pars recta, medullary thick ascending limb,
cortical thick ascending limb and distal convoluted tubules ( <10 ^
mol/cm tubule length) (Doucet and Katz, 1981a, Farman et al., 1982a).
In contrast, using CORT as a ligand, receptor binding was demonstrated
throughout the rat nephron, albeit the greatest concentration was found
in the cortical collecting tubules (Lee et al., 1983). Autoradiographic
studies are consistent with radioreceptor assays, in that, specific ALDO
binding was observed along the distal and cortical collecting tubules
(Vandewalle et al., 1981; Farman et al., 1982b; Farman and Bonvalet,
1983), but not in the glomeruli (Farman et al., 1982c).
Recent research on the endocrine basis of experimental hypertension
suggest a role for a renally-independent (Berecek and Bohr, 1978;
Onoyama et al., 1979) intraarterial mechanism of mineralocorticoid
action (Kornel, 1981; Kornel et al., 1982a & b; for review see Kotchen
and Guthrie, 1980). Chronic administration of DOCA was found to


9
increase the turnover of arterial tissue potassium and chloride ions and
to increase cell membrane "leakiness" to Na+ (Friedman et al., 1975;
Jones and Hart, 1975; Friedman and Friedman, 1976). In agreement with
these results, ALDO was found to increase significantly sodium flux
along the porcine arterial wall (a tissue with morphological resemblance
to human arterial wall) (Llaurado et al., 1983). An increase in
peripheral resistance and vascular reactivity was also reported in the
DOCA-treated animals (Hansen and Bohr, 1975; Berecek and Bohr, 1977 &
1978). Treatment of normal subjects with the synthetic
mineralocorticoid, 9-Ct-fluorocortisol, for several weeks resulted in a
rise in blood pressure in those subjects, an effect which could be
reversed by administration of spirolactone (a mineralocorticoid
antagonist) (Nicholls et al., 1979).
Clinically, steroid-induced hypertension is usually associated with
three types of adrenal disorders. 1) Excess secretion of aldosterone
from an autonomous adrenocortical adenoma in primary hyperaldosteronism
(first described by Conn, 1955). This hypertension can be remedied
usually through surgical procedures and/or by administration of
antimineralocorticoids (e.g., spironolactones) (Lim et al., 1986). 2)
Cushing's syndrome, characterized by an excessive secretion of cortisol
(a glucocorticoid) and DOC, is accompanied by hypertension in about
80-90% of the patients (Gomez-Sanchez, 1982). 3) In patients with
11-3-dehydrogenase and 5ot-reductase ("apparent mineralocorticoid
excess," a juvenile disease characterized by low or undetectable levels
of renin and low plasma ALDO, accompanied by hypokalemia, Monder et al.,
1986) and 17a-hydroxylase deficiency (where there is an increase in
ACTH-induced ALDO production) there is a high incidence of hypertension.


10
The exact mechanism by which mineralocorticoids induce hypertension
awaits further elucidation. However, since mineralocorticoids can
influence electrolyte distribution within the arterial walls by
increasing sodium transport and since "specific" mineralocorticoid
receptors along the arterial, but not the venous, walls are known to
exist (Kornel, 1981; Kornel et al., 1982a & b; Moura and Worcel, 1984),
there is strong support for an intraarterial, mineralocorticoid-induced,
receptor-mediated basis for this form of experimental hypertension.
Studies investigating the effects of ALDO, CORT and DOCA on hypertension
in rats suggests that a common mediator of steroid-induced hypertension
may be an increase in extracellular fluid and plasma volume (Haack et
al., 1977; Ishii et al., 1985). Although aldosterone has been
implicated in the pathogenesis of essential hypertension in experimental
animal models (e.g., Nowaczynski et al., 1983), the role of
mineralocorticoids in essential hypertension is still a matter of
controversy (for review see Fraser et al., 1981; Gomez-Sanchez, 1982).
Recently, a central nervous system mediated mechanism of ALDO
induced-hypertension has been suggested (Gomez-Sanchez, 1986) (for
detail see below).
Some other peripheral effects of mineralocorticoid hormones include
a redistribution of potasium within the body in addition to the
kaliuretic effect discussed above (Young, 1979; Young and Jackson,
1982); a reduction in the concentration of zinc in plasma (Latman et
al., 1984); an increase in renal cortical and medullary citrate synthase
activity (Kinne and Kirsten, 1968) and synthesis (Law and Edelman,
1978b) a phenomenon independent of sodium transport (at least in
cultured cells) (Johnson and Green, 1981); an induction of renal


11
flavokinase enzymic activity (Trachewsky et al., 1985); an increase in
thymidine kinase synthesis in immature rat kidney (Bukhari et al.,
1985); an increase in guanylate cyclase activity in liver, kidney,
heart, lung, spleen and ilium (Vesely, 1980); an increase in isocitrate
dehydrogenase and glutamate-oxaloacetate transaminase activity in rat
kidney and toad urinary bladder (Kinne and Kirsten, 1968; Kirsten et
al., 1968; Kirsten et al., 1970; Kirsten and Kirsten, 1972); an
activation of intestinal mucosal carbonic anhydrase in adrenalectomized
rats (Scott and Sapirstein, 1975; Sapirstein and Scott, 1975; Voute and
Meier, 1978; Suzuki, 1981; Suzuki et al., 1983; Suzuki and Ozaki, 1984);
an increase in Na-K-ATPase activity along the nephron (Katz et al.,
1979; Garg et al., 1981; Le Hir et al., 1982; O'Neil and Dubinsky, 1984;
2+
for detail see below); an increase in intestinal Mg -HCO^ -
ATPase activity (Suzuki, 1981; Suzuki et al., 1983); a diminution of
bile flow and liver uptake and a decrease in biliary excretion of sodium
and water (Afifi et al., 1979; Afifi, 1981); an increase in red blood
cell sodium uptake (Rettori et al., 1969; Afifi et al., 1979; Stern et
al., 1983) and a non-genomically-induced increase in membrane
phospholipid polyunsaturated fatty acid content (Goodman, 1981).
Central Site of Action and Effects of Mineralocorticoids
Although specific macromolecular binding to ALDO in whole brain
cytosol was first described by Anderson and Fanestil (1976), prior to
and since this discovery investigators have implicated the involvement
of mineralocorticoids in the central nervous system (CNS) electrolyte
balance (Woodbury, 1958); neuronal electrical activities (Kraulis et
al., 1975); feedback mechanisms involving ALDO secretion (Dundore et
al., 1984; Birmingham et al., 1974; Grizzle and Dunlap, 1984);


12
inhibition of CORT-induced serotonin turnover in dorsal hippocampal and
3
raphe areas (by inhibiting [ H]CORT nuclear uptake) (De Kloet et
al., 1983); inhibition of brain growth (Devenport, 1979); influences on
glial-cell function (Beaumont, 1985) and inhibition of CORT-induced
exploratory behavior (Veldhuis et al., 1982a). In addition,
mineralocorticoids have been shown to augment passive avoidance
responses in adrenalectomized and adrenally intact "pre-stressed" rats,
an effect not observed with CORT administration (Weiss and Gray, 1973).
Although mineralocorticoid-induced hypertension by actions at renal
levels is well documented (see above), early reports implicated a role
for mineralocorticoid action on brain regulation of experimental
hypertension in rats (e.g., Finch et al., 1972; Zamir et al., 1979).
Recently, it was shown that minute doses of ALDO administered
intracerebroventricularly produce a significant rise in blood pressure
(Gomez-Sanchez, 1986). Infusion of ALDO in the presence of an equimolar
concentration of prorenone (a spironolactone with antimineralocorticoid
activity) antagonized the pressor response to ALDO. Studies of this
nature provide strong circumstantial evidence for hyper tensinogenic
effects of mineralocorticoids directly within the CNS; however, the
exact site(s) of action of mineralocorticoids on the CNS regulation of
hypertension remains to be elucidated.
Studies investigating the endocrine basis of salt appetite
implicate a central role for mineralocorticoid action (Fregly and
Waters, 1966; Wolf and Handal, 1966; Weisinger and Woods, 1971; McEwen
et al., 1986b). The classical work by Richter (1936) showed that
adrenalectomy was accompanied by sodium appetite in rats and that
treatment of adrenalec tomized rats with DOCA reduced sodium intake to


13
control levels (Richter, 1956). Similarly, a recent report by McEwen et
al. (1986b) showed that the administration of ALDO supressed salt intake
in adrenalectomized rats as measured in a two-bottle preference test
paradigm (findings that replicate the earlier report by Fregly and
Waters, 1966). The supression of salt appetite in response to ALDO
could be inhibited by co-administration of a molar excess of RU 28318 (a
specific synthetic antimineralocorticoid). It is noteworthy that this
effect of ALDO could not be mimicked by CORT, a proposed physiological
ligand for brain Type I receptors (for review see Funder, 1986) (for
more detail see below). In fact, only concentrations of CORT 500-fold
in excess of ALDO were able to inhibit the action of ALDO (McEwen et al.
1986b) .
In sheep deprived of sodium for 22 h, infusion of 500 mM Na+
via a lateral ventricular approach led to a marked reduction in salt
intake compared to animals that received 150 mM Na+ (Weisinger et
al., 1979). It is postulated that the modulation of Na+
concentration in cerebrospinal fluid (CSF) may translate into changes in
the concentration of Na+ in hypothalamic neurons subserving salt
appetite (Weisinger et al., 1979). Similarly, an earlier investigation
by Richter (1956) showed that low concentrations of sodium in plasma
stimulate salt appetite. The author (Ritcher, 1956) implicated (but did
not show) possible involment of hypothalamic neurons. Other CNS
structures have also been implicated in the regulation of salt intake;
for example, electrolytic lesions centered in the thalamic gustatory
subnucleus of the ventral posterior complex were shown to impair sodium
intake in adrenalectomized rats (Wolf and Dicara, 1974). It is
noteworthy that the impairment in salt appetite following such lesions


14
may be unrelated to the gustatory system since neocortical ablations
which resulted in extensive retrograde cell degeneration in this
diencephalic structure had no effect on salt appetite (Wolf et al.,
1970). Becuase other en passage fibers are damaged by the thalamic
lesion (e.g., ascending reticular pathways to the intralaminar nuclei,
ascending noradrenergic pathways to the hypothalamus and forebrain,
etc.), the involvement of other structures could not be ruled out.
Although a specific center(s) in the CNS through which
mineralocorticoids may mediate salt appetite and/or other functional
responses is (are) yet to be described, it is suggested that the actual
mechanism of salt appetite induction may be complex and involve a
synergistic association of a number of factors (Fregly and Rowland,
1985).
Since studies of the regional distribution of specific ALDO
receptors within the CNS have revealed distinct localization in certain
limbic structures such as the hippocampus, hypothalamus, indusium
griseum and amygdala, these brain regions have been implicated as
possible candidates for the central site of action of ALDO (Ermisch and
Ruhle, 1978; Moguilewsky and Raynaud, 1980; De Nicola et al., 1981;
Veldhuis et al., 1982b; Coirini et al., 1983; Birmingham et al., 1984;
Reul and De Kloet, 1985 & 1986). However, co-administration of "stress
levels" of CORT with ALDO was found to suppress preferentially ALDO
uptake by hippocampal formation, amygdala and septum, having little
effect on ALDO suppression of salt appetite or ALDO uptake in
circumventricular organs (McEwen et al., 1986b). Furthermore, bilateral
3
hippocampectomy, which depleted specific [ H]ALDO binding in the
residual structure by 80%, had no effect on the development of salt


15
appetite following adrenalectomy in these hippocampectomized rats (Kim,
1960; Magarinos et al., 1986). The increase in salt intake in
adrenalectomized-hippocampectomized animals was found to be suppressible
by administration of ALDO (Magarinos et al., 1986). These studies thus
suggest, at least in rats, that the hippocampus may not be the target
organ for the action of ALDO on salt appetite as elicited by
adrenalectomy.
McEwen et al. (1986b) have concluded that one or more of the
circumventricular organs, the dentate gyrus, Ammon's horn and/or
subiculum may be the most plausible candidates for the action of ALDO on
3
salt appetite since these structures show significant [ H]ALD0
uptake even in the presence of high circulating levels of CORT. This
notion is consistent with the theory that angiotensin II and ALDO act
synergistically to regulate salt intake (Epstein, 1982; Fluharty and
Epstein, 1983; Strieker, 1983; Sakai et al., 1986), and that some of
these circumventricular organs appear to contain angiotensin II
receptors that can be modulated by exposure to ALDO (Wilson et al.,
1986). Recently, it has been proposed that ALDO and angiotensin II
(All) may mediate thirst and salt appetite by influencing the activities
of septal neurons (Strieker, 1984). Lesions of the septal area in
hypovolemic rats were found to produce sensitization to the sodium
appetite-eliciting effects of All; however, this was only seen in the
presence of ALDO (Strieker, 1984). Although these studies reflect a
synergism between the two hormonal systems in inducing electrolyte and
water balance, it must be kept in mind that, in reality, water and
electrolyte homeostasis may involve complicated interactions of a number
of other systems (for review see Fregly and Rowland, 1985).


16
Furthermore, limited experimental evidence comparing adrenalectomized
mice, hamsters and rats suggests clear differences in the control of
salt appetite in these different species.
A developing area of research regarding mineralocortico id action on
the CNS is concerned with the role of a poorly understood CNS structure,
the subcommisural organ (SCO). The intraperitoneal or subcutaneous
administration of extracts from SCO was found to induce antinatriuresis
in dogs (Gilbert, 1963). Palkovits and Foldvari (1963) reported an
increased nuclear volume in the cells of the adrenal glomerulosa
indicating increased cellular activity following intraperitoneal
injection of SCO extracts. In contrast, lesions of the SCO decreased
nuclear volume as well as the width of the zona glomerulosa in rats
(Palkovits et al., 1965). An in vitro examination of the zona
glomerulosa cells from rats with SCO lesion revealed a marked reduction
in the rate of ALDO secretion (Palkovits et al., 1965). Furthermore,
mineralocorticoids reduced, and adrenalectomy or chronic dehydration
increased cellular activity in the SCO (Palkovits, 1968). Together,
these observations have led investigators to associate SCO with salt and
water homeostasis (for review see Ziegels, 1976).
Recently, Dundore et al. (1984) found that when ALDO was
administered in the "general vicinity" of the SCO (note that the
injections were performed via a cannula placed in the pineal recess
above the rostral two-thirds of the SCO), it produced a natriuresis (an
action opposite to those of blood-borne ALDO discussed above) and a
"site-specific" (related to cannula placement) reduction in the
cross-sectional area of the adrenal medulla without affecting cell
density. It is proposed, but not conclusively shown, that ALDO acts


17
upon SCO cells (and possibly other structures and cells in the vicinity
of SCO due to diffusion) to inhibit the trophic effects of SCO on
adrenal glomerulosa cells. This, in turn, would result in an increased
urinary Na/K ratio (Dundore et al., 1984). Although an attractive
hypothesis, this study fails to show a change in plasma concentration of
adrenocorticosteroids following intracerebral administration of ALDO.
Furthermore, intracerebral administration of ALDO was found to have no
significant effect on the cortical (steroid producing) cells of the
adrenal gland when examained histologically (Dundore et al., 1984). In
light of the above discrepancies and the fact that Crow (1967), who made
discrete lesions encompassing all or parts of the SCO observed no
changes in urine volume and concentration, it is apparent that the exact
role of the SCO in electrolyte metabolism awaits further experiments.
It is conceivable that research investigating 1) the direct effects of
SCO lesions on plasma levels of corticosteroids, 2) the localization
within the SCO of specific mineralocorticoid receptors by
autoradiographic and radioreceptor assay techniques and 3) the isolation
and purification of SCO "factor(s)" responsible for the aforementioned
effects is necessary to establish the physiological role of the SCO, and
its secretory factors in salt and water homeostasis.
Some other centrally mediated actions of mineralocorticoid hormones
include the regulation of food intake and maintenace of body weight,
(Devenport and Devenport, 1983; Devenport et al., 1983; Kenyon et al.,
1984); modulation of hypothalamic, thalamic and septal angiotensin II
receptor binding capacity (Wilson et al., 1986) and increase in cerebral
guanylate cyclase activity (Vesely, 1980).


18
Molecular Mechanisms of Mineralocorticoid Action
Recent in vitro radioreceptor assay and ligand competition
techniques suggest that in cytosol lacking extravascular CBG, Type I
receptors have an equal affinity for ALDO and CORT (for review see
Funder, 1986). It is further suggested that some actions of CORT on the
CNS may be mediated through Type I receptors (e.g., De Kloet et al.,
1986; Magarinos et al., 1986). However, because in this dissertation
the emphasis is placed on the role of Type I receptors in electrolyte
homeostasis, this section will focus primarily on our current
understanding of the molecular mechanisms of action of ALDO and other
physiological mineralocorticoids.
Research on the mechanism of mineralocorticoid action has drawn
tremendous benefit from studies carried out using isolated nephrons, the
A6 cell line derived from the Xenopus kidney epithelial cells and
the sodium transporting amphibian epithelia (toad urinary bladder and
frog skin). These biological preparations can be maintained viable for
several hours at room temperature in incubation media of simple
composition. The simple morphological properties of these models render
them ideal in the study of the mechanism(s) of mineralocorticoid action;
however, it is noteworthy that although aldosterone is the naturally
occuring mineralocorticoid produced by the interrenal gland in anurans
(Carstensen et al., 1961; Crabbe, 1961), the "mineralocorticoid
response," at least in toad bladder appears to be mediated through
interactions with Type I as well as other glucocorticoid specific
receptors (Geering et al., 1985). Even though the authors dubbed these
other receptors as "type 2" glucocorticoid receptors, the possibility


19
they may be Type IB (for more detail see below) and thus distinct from
classical glucocorticoid (Type II) receptors should also be considered.
In the early 1960's, Williamson (1963) and Edelman and co-workers
(1963) independently proposed the classic two-step genomic mode of ALDO
action. The first step was the binding to specific soluble
intracellular receptors, following which, as a second step, the
steroid-receptor complexes would translocate into the nucleus where they
would interact with the cellular genomic machinery (for a discussion of
the nature of the nuclear acceptor site see Chapter VI). It is this
latter step that was proposed to result in the modulation of synthesis
of a number of structural and regulatory proteins. Several lines of
evidence appeared later in favor of this proposal. For instance, Sharp
and colleagues (1966b) were the first to report saturable ALDO binding
sites (receptors) in the toad bladder. Marver et al. (1974) later
reported that the mineralocorticoid antagonist, spirolactone (SC-26304),
when complexed with these receptors, had little or no affinity for
nuclear acceptor sites in vivo and in vitro. More recently, the
response to ALDO in the rat kidney was shown to depend clearly on the
ontogeny of Type I receptors in this target organ (Stephenson et al.,
1984a). Finally, it was possible to correlate the magnitude of
epithelial sodium transport with the binding of ALDO-receptor complexes
to chromatin and the increases in the synthesis of a specific class of
nonmethylated mRNAs and of proteins (Rossier et al., 1974). In this
regard, actinomycin-D (a DNA intercalator which binds specifically to
G--C base pairs and protrudes into the major grooves) was shown to block
ALDO-stimulated sodium transport by inhibiting new mRNA and protein
synthesis (Lahav et al., 1973; Rossier et al., 1974; Horisberger and


20
Diezi, 1984). Subsequent to these observations a number of studies
replicated the results described (for review see Edelman and Marver,
1980; Fanestil and Park, 1981; Rossier et al., 1985; Garty, 1986;
Marver, 1986) thus further confirming the original model proposed by
Edelman et al. (1963) and Williamson (1963).
It is now well established that mineralocorticoids, in general, are
capable of inducing the synthesis of a group of proteins in target
tissues generally referred to as aldosterone-induced proteins (AIPs).
In the rat kidney and toad urinary bladder, proteins with a variety of
molecular weights have been reported to exhibit increased incorporation
of radiolabeled precursor amino acids as a result of exposure to
aldosterone (Law and Edelman, 1978a & b; Geheb et al., 1981; Reich et
al., 1981; Yang et al; 1981; Blazer-Yost et al., 1982; Geering, et al.
1982; Geheb et al., 1983; Geheb et al., 1984). However, the mechanism
by which AIPs participate in the mineralocorticoid-induced physiological
and functional consequences such as sodium transport still remains a
matter of debate. In fact, only recently was it shown that some AIPs
can be immunoprecipitated by polyclonal antibodies specific for the a-
and B-subunits of Na-K-ATPase (Geering et al., 1982).
Current thinking on the role of AIPs, as related to the action of
mineralocorticoids, points to three distinct, but not necessarily
independent, theories: 1) the sodium pump theory, which argues for
increased sodium pump activity as a result of either activation of
pre-existing pumps or an increase in the synthesis of new pumps; 2) the
sodium permease theory, which holds that AIPs are involved in the
facilitation of passive sodium entry across the plasma membrane and 3)
the energy theory, which associates AIPs with the augmentation of


21
cellular energy, presumably by enhanced mitochondrial oxidative
phosphorylation. Although each of these theories has some experimental
support, thus far none has gained universal acceptance. It is important
to emphasize that the involvement of AIPs may not be limited to only one
of these mechanisms exclusively; rather the effects of
mineralocorticoids may reflect the contribution of AIPs to some
combination of these three general mechanisms. In this regard, Reich
and Scott (1979) proposed a unifying hypothesis that includes all three
of the above theories (for recent reviews see Rossier et al., 1985;
Garty, 1986; Marver, 1986).
Considerable controversy still surrounds the pump theory. In the
past decade, a large number of laboratories has investigated the
mineralocorticoid induction of Na-K-ATPase activity (e.g., Aperia et
al., 1981; Doucet and Katz, 1981b; Garg et al., 1981; Petty et al.,
1981; Geering et al., 1982; Osore and Gilbert, 1982; Cortas et al.,
1983; El Mernissi and Doucet, 1983; Stern et al., 1983; Aperia and
Larsson, 1984; El Mernissi and Doucet, 1984; O'Neil and Dubinsky, 1984;
Park and Edelman, 1984a & b; Geering et al., 1985; O'Neil and Hayhurst,
1985; Girardet et al., 1986; Johnson et al., 1986; Palmer and Speez,
1986). As a result of this intensive investigation, it is now well
established that following adrenalectomy there is a significant
reduction in Na-K-ATPase activity in nephrons; however, some
investigators find complete restoration of the enzymatic activity
following administration of ALDO (Petty et al., 1981, El Mernissi and
Doucet, 1983; El Mernissi and Doucet, 1984), whereas others find little
or no effect of ALDO on the restoration of enzyme activities in the
target tissues investigated (Doucet and Katz, 1981b).


22
In addition to this discrepancy in results, the studies cited above
also fail to prove that sodium pump activity plays a significant role in
the action of mineralocorticoids on transepithelial or nephron sodium
transport. Indeed, it is suggested that the observed increase in
Na-K-ATPase activity, at least in some target tissues, may be due to
interaction of mineralocorticoids with Type II receptors, since
long-term pharmacological doses of steroids are often required to
restore Na-K-ATPase activity to control levels. Recently, a number of
investigators (Rayson and Lowther, 1984; El Mernissi and Doucet, 1984;
Garg et al., 1985) reported that they were able to restore the
adrenalectomy-induced reduction in Na-K-ATPase activity in various
segments of rabbit nephron through DEX administration. Even though
similar results were reported by earlier investigators (Fisher et al.,
1975), the small doses of DEX used in the former reports (Rayson and
Lowther, 1984; El Menissi and Doucet, 1984; Garg et al., 1985), provide
stronger evidence that the stimulation of the Na-K-ATPase was probably
through Type II receptors. Note that although the radioreceptor assay
technique suggests that DEX binding to Type I receptors may be
negligible, the pharmacological doses of DEX administered in some of
these studies may allow sufficient interaction of this glucocorticoid
with these receptors to mediate mineralocorticoid action.
In contrast to this suggestion, El Mernissi and Doucet (1984) have
shown that ALDO administration increases the synthesis of new
Na-K-ATPase in the collecting tubules, whereas DEX stimulates the
activity of the pre-exisiting pumps in the thick ascending limb and
distal convoluted tubules. Thus, these results suggest that ALDO and
DEX may act through differential mechanisms at different portions of the


23
nephron. A differential action of ALDO in the rat proximal and distal
colon has also been described: ALDO response in distal, but not
proximal, colon could be inhibited by amiloride (an acylguanidine
diuretic that inhibits influx of Na+ through the apical membrane)
(Hirsch et al., 1985). In contrast, in vitro studies evaluating
short-circuit current in colon from adrenalectomized rats suggest that
although ALDO may be the physiological regulator of Na+ transport,
glucocorticoids may also mediate amiloride-sensitive sodium transport
(Will et al., 1985). Geering et al. (1985) maintain that, at least in
toad urinary bladder, ALDO binding to both Type I and Type II receptors
is required for full mineralocorticoid response.
It is also conceivable that DEX actions on Na-K-ATPase activity may
be mediated through a population of receptors distinct from Type I and
Type II receptors called Type IB. The presence of these receptors in a
number of tissues including the rat liver (Litwack and Rosenfield,
1975), colon (Bast et al., 1984) and kidney (Litwack and Rosenfield,
1975; Markovic et al., 1980; Mayer et al., 1983) has been confirmed (for
review see Mayer and Litwack, 1983). These receptors have also been
isolated from mouse lung cytosol (Goldman and Katsumata, 1986) and were
implicated to mediate the known anti-inf lamatory and teratogenic actions
of DEX and phenytoin (Katsumata et al., 1985). Some investigators,
however, have questioned the polymorphic nature of these receptors and
have argued that these atypical receptors may represent a proteolytic
fragment(s) generated from Type II receptors under in vitro
conditions (e.g., Sherman et al., 1983; Reichman et al., 1984). More
recently, Eisen et al., (1986) showed that kidney cytosol prepared in
the absence, but not the presence, of molybdate (for detail of possible


24
mechanism of action of this oxyanion see Chapter IV) showed specific
glucocorticoid binders characteristics of Type IB receptors (i.e., the
receptors did not react with a monoclonal antibody raised against Type
II receptors and they were not retained by DEAE-cellulose columns). In
contrast, Goldman and Katsumata (1986) have reported that Type IB
receptors could be detected in lung cytosol only when cytosol was
prepared in hypotonic buffers containing molybdate. Although the
questions as to whether Type IB receptors exist in vivo and whether
they have physiological significance remain to be established, it is
entirely possible that these receptors may be generated from Type II
receptors by specific proteases in target tissues that appear to contain
these receptors.
Experiments with adrenalectomized animals treated with amiloride
prior to injection of ALDO suggest that the increase in Na-K-ATPase
activity may be a secondary adaptation to sodium transport induced by
this steroid (Doucet and Katz, 1981b; Petty et al., 1981). Furthermore,
at least in toad urinary bladder, it appears that the stimulatory action
of ALDO on sodium transport is not mediated by the synthesis of new
(Geering et al., 1982, Park and Edelman, 1984b) or activation of the
pre-existing sodium pumps (Park and Edelman, 1984b).
The idea that AIPs may be a component of the plasma membrane (on
the luminal, urine, surface) of the cell, where they act as a sodium
permease to lower the resistance to Na+ entry into the cell, was
proposed originally by Crabbe and de Weer (1965) and Sharp et al.
(1966a) and subsequently gained experimental support through work in the
toad urinary bladder (Civan and Hoffman, 1971; Spooner and Edelman,
1975; Palmer et al., 1982; Park and Edelman, 1984a). All of these


25
studies, however, fail to demonstrate whether AIPs act as permeases
themselves, or whether they modulate the conformation or arrangement of
pre-existing permease molecules in the membrane such that they are more
accessible for Na+ entry. A recent study by Kipnowski et al. (1983)
found that ALDO fails to increase sodium transport in tissues where
transport was inhibited by N-ethoxycarbonyl-2-ethoxy-l,2-dihydro-
quinoline (EEDQ) prior to exposure to this steroid. In contrast to
results with cycloheximide (an inhibitor of protein synthesis), EEDQ did
alter ALDO stimulation of the osmotic water flow in response to
antidiuretic hormone. These data do not therefore support the notion
that AIPs are new sodium channels, but instead they are consistent with
the hormonal activation of pre-existing, nonfunctional channels.
The first evidence that mineralocorticoids may induce the synthesis
of a mitochondrial enzyme participating in tricarboxylic acid cycle was
obtained by Kirsten et al. (1968) who found that ALDO increased the
citrate synthase, isocitrate dehydrogenase, glutamate dehydrogenase,
glutamate-oxaloacetyl transaminase and malate dehydrogenase activity in
the toad bladder. The increase in the synthesis of mitochondrial
enzymes was postulated to reflect hormone-contro 1led mudulation of the
available ratio of ATP/ADP which, in turn, could alter both Na+
conductance and active transport. ALDO has also been shown to increase
the ratio of NADH/NAD+ in adrenalectomized rat kidneys (Kirsten and
Kirsten, 1972). Similarly, Law and Edelman (1978b) found an increase in
renal cortical and medullary citrate synthase activity three hours after
administration of ALDO to adrenalectomized rats. In this latter study
ALDO was found to enhance incorporation of radiolabeled methionine into
renal, but not hepatic, citrate synthase. The relevance of these


26
observations with reference to the energy theory of mineralocorticoid
action lies in the fact that actinomycin D and specific spironolactones
inhibit the ALDO-induced increase in mitochondrial enzymatic activities,
and that DEX has no effect on the hormonal induction (Law and Edelman,
1978b). Furthermore, this action of ALDO appears to be target specific:
ALDO had no detectable effect on the citrate synthase activity in the
renal papilla or the liver under the same conditions (Law and Edelman,
1978b).
In summary, although much has been done to expand our knowledge of
the molecular mechanisms of mineralocorticoid action, the exact
mechanism by which these hormones exert their effects still awaits
further exploration. It is, nevertheless, clear that the functional
consequences of mineralocorticoid action involves, at least in part, the
genetic machinery in target cells and an ultimate modulation of the
synthesis and/or activation of the pre-existing structural and
regulatory proteins.
Mechanisms of Receptor Regulation
There is considerable evidence to suggest that the in vivo
binding capacity of adrenocorticosteroid hormone receptors is subject to
change in response to a variety of intra- and extracellular signals
(McEwen, 1979; Muldoon, 1980; Svec, 1985b; De Kloet et al., 1986; McEwen
et al., 1986a). For example, hyperkalemic adrenalectomized rats were
shown to have an elevated renal cytosolic Type I receptor binding
capacity (Rafestin-Oblin et al., 1984). Similarly, adrenalectomy
(adrenocorticosteroid deprivation) alone was found to result in a
time-dependent increase in the maximal binding capacity of both Type I
and Type II receptors in all tissues investigated (Beato et al., 1974;


27
McEwen et al., 1974; Gregory et al., 1976; Ichii, 1981; Claire et al.,
1981; Muramatsu et al., 1983, Rafestin-Oblin et al., 1984; Turner, 1986;
Sarrieau, et al., 1986; Luttge and Rupp, unpublished). It is noteworthy
that adrenalectomy was found to induce a differential tissue-specific
up-regulation in Type I and Type II receptor binding capacity in mice.
In the three tissues investigated (i.e.; brain, liver and kidney),
adrenalectomy was found to induce the greatest up-regulation in the
brain cytosolic Type I receptors a 10- to 14-fold rise compared to
the adrenally intact control group as measured by saturation and
equilibrium binding analyses (Luttge and Rupp, unpublished).
Adrenalectomy also induced a similar tissue-specific up-regulation in
Type II receptors: The largest increase was found in the kidney
cytosol, followed by amygdala-entorhinal cortex, hippocampus, liver,
cerebral cortex, hypothalamus, pituitary and heart (Turner, 1986).
In Scatchard plots (Scatchard, 1949), an increase in the B, ,
MAX
as a mode of "up-regulation" of Type I and Type II receptors induced by
adrenalectomy is a common finding (Beato et al., 1974; McEwen et al.,
1974; Gregory et al., 1976; Claire et al., 1981; Ichii, 1981; Tornello
et al., 1982; Muramatsu et al., 1983; Rafestin-Oblin et al., 1984;
Sapolsky et al., 1984; Luttge and Rupp, unpublished). This increase in
the has been postulated to reflect an unmasking of receptors
previously occupied by endogenous ligands (Feldman, 1974; McEwen et al.,
1974; Giannopoulos, 1975), "activation" of an already available pool of
receptors to a ligand-binding state, an increase in the rate of receptor
synthesis (McEwen et al., 1974; Claire et al., 1981) and/or a reduction
in the rate of receptor degradation (Claire et al., 1981).


28
Treatment of adrenalectomized animals with CORT, ALDO and other
steroid agonists that show appreciable affinity for Type I and Type II
receptor binding sites results in a reduction in the ligand binding
capacity of these receptors (Grekin and Sider, 1981; Ichii, 1981;
Tornello et al., 1982; Muramatsu et al., 1983; Rafestin-Oblin et al.,
1984; Sarrieau et al., 1986; Luttge and Rupp, unpublished). Repeated
stress (a condition that induces the production of high circulating
levels of CORT) was also found to result in a significant reduction in
Type II receptor binding capacity in the rat hippocampus (Valeri et al.,
1978; Sapolsky et al., 1983; Sapolsky et al., 1984; Sapolsky, 1985;
Sapolsky et al., 1985b) and amygdala (Sapolsky et al., 1983; Sapolsky et
al., 1984). In addition to regulation of their own receptors, chronic
administration of stress level concentrations of corticosteroids was
found to have neurotoxic effects on hippocampal neurons (Sapolsky, 1985;
Sapolsky et al., 1985b). Similarly, in cultured cell lines (HeLa S3,
AtT-20 and GH^ cells), glucocorticoids were shown to "down-regulate"
their own receptors (Cidlowski and Cidlowski, 1981; Svec and Rudis,
1981; Raaka and Samuels, 1983; Svec, 1985a). This down-regulatory
effect of ligand on its own receptor has been reported for a number of
other receptors; e.g., progesterone receptors (Milgrom et al., 1973b;
Walters and Clark, 1979), thyroid hormone receptors (Raaka and Samuels,
1981), insulin receptors (Gavin et al., 1974; Huang and Cuatrecasas,
1975; Olefsky, 1976) and other peptide receptors (Catt et al., 1979;
Roth and Taylor, 1982).
In GH^ cells, incubation with triamcinolone acetonide (TA) was
found by McIntyre and Samuels (1985) to reduce the half-life of Type II
receptors by more than 50% (from 19.0 to 9.0 h) as determined through


29
the use of dense amino acid labeling of the receptors. Consistent with
this finding, in the presence of TA, the levels of Type II receptors in
these cells were half that measured in the control cells (i.e., about
130 vs. 260 fmole/100 ug DNA, respectively). The addition of TA to the
culture media had little effect on the rate of de novo receptor
synthesis. It was shown that TA promoted the activation of Type II
receptors (transformation to a state with increased affinity for DNA,
see Chapter VI), and that the activated form was degraded at a faster
rate (than the unactivated form) thus accounting for the reduction in
the receptor half-life (McIntyre and Samuels, 1985). This apparent
faster rate of degradation may represent the inability of the activated
receptors to rebind ligand, since recent work from our laboratory has
shown that following heat-induced transformation to a DNA-binding form
the activated receptors do not rebind ligand after dissociation (Chou
and Luttge, 1987). Recently, treatment of rat hepatoma culture cells
with DEX was shown to result in an initial increase (after 6 h),
followed by a reversible (after 72 h) 50% to 95% decrease in Type II
receptor mRNA (after 24 h) in these cells (Okret et al., 1986). In this
latter study, using an immunoprecipitation assay, Type II receptors were
shown to interact specifically with a Type II receptor cDNA clone, hence
providing a more direct evidence for the ligand-induced receptor
autoregulation (Okret et al., 1986).
The up- and down-regulation of the binding capacity of receptors
for adrenal steroids in the central nervous system raises the
possibility that receptor autoregulation, and hence modulation of brain
sensitivity to steroids, may ultimately be involved in such functional
consequences as the feed-back control mechanisms of adrenal steroid


30
secretion (Cake and Litwack, 1975; Sapolsky et al., 1985a; for review
see Sapolsky et al., 1986).
A second mode of receptor regulation, at least in
adrenocorticosteroid Type II and progesterone receptor systems, may
involve a rapid turning "on" and "off" of the unoccupied receptors in
response to different stimuli. This rapid and reversible loss of
steroid binding capacity has been shown to involve a phosphorylation-
dephosphorylation cycle of the receptors in vitro and possibly
in vivo (for review see Housley et al., 1984). In an earlier
report, Munck and co-workers (1972) proposed that Type II receptors in
thymocytes exist in two states; the steroid-binding form and those that
did not bind steroids. It was proposed that ATP was required to convert
the non-binding receptors to a state that bound steroid (Munck et al.,
1972). Consistent with this finding, receptor stability was reported to
be enhanced by including ATP and other nucleotides (a requirement for
the action of some phosphorylase enzymes) in cytosol preparations
(Nielsen et al., 1977b; Sando et al., 1979a & b). Our laboratory has
recently shown that exogenous ATP can also prevent the destabilizing
actions of as yet unknown microsomal components on Type II receptors
(Densmore and Luttge, 1985). The use of phosphatase inhibitors (e.g.,
fluoride and molybdate) in cytosol preparations has been shown to
improve considerably the stability of Type II receptors (Nielsen et al.,
1977b) Conversely, the addition of highly purified calf intestine
alkaline phosphatase to cytosol preparations markedly destabilized these
receptors (Nielsen et al., 1977a). A striking, positive correlation
between ATP concentration and Type II receptor binding capacity in a
human cell line (i.e., IM-9 cells) was also observed (Wheeler et al.,


31
1981). Lastly, affinity labeling of Type II receptors (from L-cells
32
grown in the presence of [ P]orthophosphate) with dexamethasone
21-mesylate has provided direct evidence suggesting that these receptors
may be phosphoproteins (Housley and Pratt, 1983). It is important to
note that although these data clearly favor the role of
phosphorylation-dephosphorylation as a possible mechanism of Type II
receptor regulation, there is as yet no experimental evidence to suggest
a similar mechanism for up- and down-regulation of Type I receptors.
A third mechanism for the potential regulation of Type II and
possibly Type I receptors (which may also be related to the
phosphorylation-dephosphorylation cycle (Housley et al., 1982, for
review see Housley et al., 1984)), is the reduction and oxidation of
sulfhydryl group(s) in the receptor macromolecule. It is well known
that thiol-disulfide exchanges can markedly affect the activity of a
number of protein macromolecules including androgen, estrogen,
progesterone, ecdysone and insulin receptors, lysozyme, immunoglobulins
and many digestive enzymes. In 1975, Rees and Bell claimed that they
were able to recover partially the loss in binding capacity of rat
thymic cytosol receptors by including sulfhydryl reducing reagents,
e.g.; dithiothreitol (DTT) and 2-mercaptoethanol, in their buffers. Two
years later, Granberg and Ballard (1977) reported that the addition of
2 mM DTT to rat lung cytosol increased the apparent binding of Type II
receptors 10-fold. This increase was proposed to be due to
transformation of an already-available pool of receptors to a
1igand-binding state. Similar effects were also seen in brain, uterus,
thymus and a number of other tissues whereas the presence or absence of
DTT was found to have little effect on Type II receptor binding capacity


32
in liver, kidney and heart cytosol. It was postulated that these latter
tissues contained endogenous, heat-stable, sulfhydryl reducing agents
which stabilize Type II receptor binding capacity. Interestingly, an
analysis of Type II receptors in fetal (Ballard et al., 1974) and adult
(Granberg and Ballard, 1977) rat lung revealed that exogenous DTT was
required only in the cytosol prepared from adults. Together these
observations suggest that variations in the production of sulfhydryl
reducing (and/or oxidizing) reagents may be responsible for differential
stabilization of Type II receptors in various tissues and in the same
tissue during different stages of development.
An endogenous protein molecule, capable of modulating steroid
binding capacity of the rat liver cytosolic unoccupied Type II
receptors, has recently been characterized by Pratt and co-workers
(Grippo et al., 1983 & 1985). This endogenous receptor-reducing agent
appears to be an NADPH-dependent thioredoxin. Extraction of this
stabilizing factor from liver cytosol by charcoal resulted in a
significant loss of unoccupied, but not occupied Type II receptor
binding capacity. Addition of DTT to the charcoal-ex trac ted cytosol
resulted in a complete recovery of Type II receptor binding capacity
(Grippo et al., 1983). Similar results have been obtained in our
laboratory. We found that charcoal extraction of steroid-free brain
cytosol prepared in HEPES [4(2-hydroxyethy1)-1-piperazineethanesulfonic
acid] buffer containing 10% glycerol and 2 mM DTT resulted in an
increase in the thermal instability and a decrease in the total binding
capacity of Type II receptors (Emadian et al., 1986). These effects
were reversible after the readdition of the exogenous DTT removed by the
charcoal treatment. In further investigating the effect of the presence


33
or absence of DTT, we found that the omission or removal of this
sulfhydryl reducing reagent produced a significant increase in the
3
apparent K, and a slight reduction in the of [ H]DEX
a MAX
binding to Type II receptors (Densmore et al., 1984b).
The effect of DTT on Type II receptor binding capacity appears to
be a tempera ture-independent phenomenon (Rees and Bell, 1975; Granberg
and Ballard, 1977; Grippo et al., 1983; Densmore et al., 1984; Emadian
et al., 1986), whereas the NADPH-dependent thioredoxin appears to act in
a temperature-dependent fashion (Grippo et al., 1983). These findings
suggest that the exogenous sulfhydryl reducing reagents may act
primarily on the receptor [or other component(s) of the system] to
maintain it in a reduced state -- the conformation necessary for steroid
binding. In contrast, the reducing effects of thioredoxin must be
mediated primarily through an enzymatic process (Grippo et al., 1983 &
1985). Thioredoxin reductase was found to be responsible for the
transfer of reducing equivalents from NADPH to thioredoxin. The reduced
form of the thioredoxin, in turn, is thought to maintain the receptors
in the steroid-binding state (Grippo et al., 1985). The transfer of the
reducing equivalent may be shown schematically as follows:
thioredoxin reductase
oxidized thioredoxin >- reduced thioredoxin
| NADPH
T
oxidized thioredoxin
+ ^ oxidized receptor
reduced receptor (incapable of binding ligand)
(ligand binding conformation)
The observation that occupied receptors from charcoal extracted
cytosol (i.e., in the absence of thioredoxin and/or DTT) appear to be
unaffected by charcoal treatment has raised the possiblity that the


34
sulfhydryl group(s) on the receptor may be located at or near the
steroid binding domain (Grippo et al., 1983). Alternatively, the
binding of ligand to Type II receptor could induce a conformational
change in the receptor molecule which renders more distant sulfhydryl
group(s) inaccessible for oxidation. With regards to this latter
hypothesis, a recent preliminary study from our laboratory suggests that
DTT may have no detectable effects on the sedimentation coefficient of
unoccupied Type II receptors (Densmore et al., 1984). It is important
to remember that the method of sucrose density gradient
ultracentrifugation may not be sensitive enough to reveal small
conformational changes in receptor.
Another possible regulatory role of the thiol-disulfide exchange
mechanism involves the interaction of steroid-receptor complexes with
the cellular genomic machinery. Preatreatment of DEX-receptor complexes
with sulfhydryl oxidizing reagents [e.g.; methyl methanethiosulfonate,
MMTS and 5,5'-dithiobis (2-nitobenzoic acid), DTNB] inhibits subsequent
binding of the activated receptor complexes to DNA-cellulose (an
in vitro index for receptor transformation) (Bodwell et al., 1984).
Interestingly, the inhibitory effect of these reagents can be reversed
by the addition of DTT. These results were interpreted to suggest that
sulfhydryl group(s), located at or near DNA-binding domain of the
activated steroid-receptor complex, may regulate the interaction of the
complex with the genome (Bodwell et al., 1984). It is noteworthy that
although MMTS and DTNB were shown to diminish DNA-cellulose binding of
the complexes, the authors (Bodwell et al., 1984) failed to refute
conclusively the possibility that these reagents inhibited DNA-binding
by acting as cross-linking reagents (i.e., cross-linking of the


35
activated receptors with other cytosolic components such as heat-shock
proteins, mRNAs, etc.) In fact, current data clearly favors the notion
that N-ethylmaleimide (NEM) and iodoacetamide (other sulfhydryl
modifying reagents) inhibit transformation of rat liver Type II
receptors (Kalimi and Love, 1980) probably through inhibition of subunit
dissociation (for detail see Chapter VI). Furthermore, it is possible
that the bulky thiomethyl and thionitrobenzoate complexes (formed by the
interaction of MMTS and DTNB with the receptors, respecively), rather
than the oxidation itself, inhibit the binding of the complexes to
DNA-cellulose. More recently, Type II receptors were shown to bind
nuclear matrix (a proteinaceous structural framework implicated in many
nuclear functions, for detail see Chapter VI) through intermolecular
disulfide bond formation (Kaufmann et al., 1986). Sodium
dodocylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the rat
hepatic Type II receptors extracted from nuclei isolated from animals
3
injected with [ H]TA in the absence of sulfhydryl-blocking (e.g.,
iodoacetamide) and cross-linking reagents yielded steroid-receptor
complexes that were disulfide-cross-linked to the nuclear matrix.
Conversly, isolation of the nuclei in presence of iodoacetamide,
revealed no receptor-nuclear matrix complex formation (Kaufmann et al.,
1986). These results provide a more direct evidence for the involvment
of sulfydryl groups in the binding of transformed steroid-receptor
complexes to nuclear components (for further discussion see Chapter VI).
Recently, structural analysis of the amino acid sequences of a number of
steroid receptors revealed the presence of a cysteine rich region in the
DNA-binding domain these receptors (e.g., Danielsen et al., 1987).


36
In contrast to our understanding of the factors regulating Type II
receptor binding activity, our knowledge concerning possible up- and
down-regulatory factors affecting Type I receptors remains scanty. As
discussed above, we have demonstrated several in vitro chemical
differences between the brain cytosolic Type I and Type II receptors
which may play an important role in the in vivo regulation of these
receptors. For example, although charcoal-extraction of steroid-free
brain cytosol in HEPES buffer lacking DTT causes a dramatic reduction in
Type II binding capacity (possibly by removal of endogenous
thioredoxin), this treatment has no effect on the binding capacity of
Type I receptors (Emadian et al., 1986; also see Chapter V). This
finding suggests that the labile thiol-disulfide exchange mechanism
described for Type II receptors may not be involved in the regulation of
Type I receptor binding capacity. In an effort to shed more light on
the role of possible factors and mechanisms involved in the in vitro
and/or in vivo regulation of Type I receptors, an investigation of
possible involvement of sulfhydryl groups in the ligand- and DNA-binding
capacity of these receptors was conducted in this dissertation (see
Chapters V and VI).
In Vitro Analysis of Type I Receptors by Radioreceptor Assay
A clear understanding of the physiological and behavioral
consequences of steroid hormone action requires an unambiguous
characterization of the receptors through which such actions presumably
are mediated. Our knowledge of the properties of Type I receptors as
well as the factors involved in the regulation of these receptors may
indeed lead to further discoveries regarding functional consequences of
these steroids. For example, the recognition of a specific


37
mineralocorticoid receptor led to the discovery of spironolactones
(mineralocorticoid antagonists), which have proven invaluable tools
clinically (as effective antihypertensive agents) and in basic sciences
(see above). Although numerous studies investigating the properties of
steroid hormone receptors have appeared in the literature within the
past two decades, the volume of research on the properties of Type I
receptors has been comparatively minimal (Liao et al., 1983). This is
partially due to the fact that in crude preparations ALDO and other
readily available synthetic mineralocorticoids bind to Type II receptors
with appreciable affinity (Rousseau et al., 1972; Funder et al., 1973a;
Emadian et al., 1986; Emadian and Luttge, unpublished; also see Chapter
V). This indiscriminatory binding of ALDO to Type II receptors can
produce confounding results thus hampering an independent analysis of
Type I receptors. Although the use of mathematical models has proven
effective in determination of binding parameters for Type I receptors
(e.g., Claire et al., 1978; Claire et al., 1985; Emadian et al., 1986),
failure to eliminate Type II-binding interferences has led a number of
investigators reach erroneous conclusions in their analysis of Type I
receptor properties (Grekin and Sider, 1980; Marver et al., 1972).
Recently, however, the introduction of very specific synthetic Type II
ligands (e.g., RU 26988, RU 28362 and RU 38486) has made the elimination
of Type II-binding contribution in crude cytosolic preparations
possible. When used in conjunction with radiolabeled ALDO, a molar
excess of the RU compound can saturate all Type II binding sites with
little or no cross-reactivity with Type I receptors, hence leaving these
latter sites intact for ALDO binding. This method has been utilized
successfully by this and a number of other laboratories (Moguilewsky and


38
Raynaud, 1980; Veldhuis et al., 1982b; Gomez-Sanchez and Gomez-Sanchez,
1983; Wrange and Yu, 1983; Emadian, et al., 1986).
In addition to these methods, Veldhuis et al. (1982b) reported that
they were able to eliminate non-linearities observed in the Scatchard
plots for ALDO binding by co-incubating hippocampal cytosol with
unlabeled CORT. Although the Scatchard plot of ALDO binding in the
presence of a 100-fold concentration of RU 26988 appears comparable to
that obtained in the presence of a 0.6-fold concentration of CORT
(Veldhuis et al., 1982b), in light of the fact that CORT has a higher
affinity for hippocampal Type I receptors (see below), a lower value of
equilibrium maximal binding in the presence of CORT is consistent with
the interaction of CORT with Type I receptors (for detail see below).
To eliminate the confounding effects of ALDO interaction with Type II
receptors, unless otherwise stated, the characterization of Type I
receptors in this dissertation was accomplished in the presence of a
500-fold molar excess of RU 26988, a concentration determined to
eliminate all Type II binding interferences (Emadian et al., 1986) (for
detail see Chapter II).
Another pertinent issue in the in vitro analysis of steroid
receptors in crude cytosolic preparations, in general, is possible
contamination and thus binding contribution by low affinity and high
capacity "non-specific binders" (of cytosolic, membrane or of plasma
origin). It is well established that ALDO binds with appreciable
affinity, especially at lower tempertures, to corticosteroid binding
globulin (CBG) (Sandberg et al., 1960; Daughaday et al., 1960; Meyer et
al., 1961; Davidson et al., 1962; Zager et al., 1976; Zipser et al.,
1980), and with a lower affinity to a^-acid glycoprotein and albumin


39
(Chen et al., 1961; Daughaday et al., 1960; Ganguly and Westphal, 1968).
Moreover, using competitive adsorption and gel filtration techniques,
Richardson and co-workers (1977) identified two other plasma
ALDO-binding proteins distinct from CBG, albumin and a^-acid
glycoprotein in human plasma. This has been confirmed by Katayama and
Yamaji (1982) who reported that this "ALDO binder" had an apparent
association constant for ALDO one order of magnitude higher than that
for CBG. Although there is no evidence that these plasma binders are
present in rodents, because 1) ligand binding assays are usually
performed at lower temperatures and 2) corticosteroid-binding globulin
(CBG) shows appreciable affinity for ALDO at lower tempertures, an
extensive in situ transcardiac perfusion of brains prior to cytosol
preparation will be performed. This will assure the elimination of
possible contribution of these binders in our in vitro
characterization of Type I receptors. Note that this perfusion was
deemed sufficient in eliminating these binders, since co-incubation of
3
whole brain cytosol with [ H]ALD0 and cortiuic acid (a synthetic
17a-acid derivitive of cortisol that specifically binds CBG, see Alexis
et al., 1983; Little, 1983; Sheppard and Funder, 1986) produced specific
Type I receptor binding similar in magnitude to that found in the
3
presence of [ H]ADL0 alone.
In addition to the above plasma binders, the presence of the
extravascular corticosterone binder (with identical physicochemical and
immunoreactive properties to the plasma CBG) has been identified in the
uterus (Milgrom and Baulieu, 1970; Guerigian et al., 1974; Al-Khouri and
Greenstein, 1980), liver (Koblinsky et al., 1972; Litwack et al., 1973;
Amaral et al., 1974; Suyemitsu and Terayama, 1975; Weiser et al., 1979),


40
kidney (Funder et al., 1973b; Feldman et al., 1973; Strum et al., 1975;
Weiser et al., 1979), pituitary and brain (Koch et al., 1976; De Kloet
and McEwen, 1976a & b; Al-Khouri and Greenstein, 1980; Gray and Luttge,
1982), and a number of other tissues (Mayer et al., 1975; Giannopoulos,
1976; Werthamer et al., 1973) (for review see Siiteri et al., 1982).
The order of steroid binding specificity for these CBG-like binders
determined by competition assays appears to be CORT > DOC >> ALDO >>
DEX. Although it has been shown conclusively that the synthesis of
CBG-like glucocorticoid binders (by convention, referred to as Type III
receptors) occurs in the liver (Perrot-Applanat and Milgrom, 1979;
Weiser et al., 1979), the possible functional roles and the origin of
these binders in other tissues have been the subject of considerable
controversy. Since a comprehensive review of the primary references
surrounding this issue is outside the scope of this dissertation, only
the recent findings relevant to the in vitro characterization of
Type I receptors will be reviewed.
More than a decade ago, it was established that in kidney there
were two distinct classes of corticosteroid binding proteins
representing the Type I and Type II receptors (Funder et al., 1973b).
It was further claimed that the hierarchy of steroid binding specificity
for Type I receptors were ALDO > DOC > CORT (Funder et al., 1973b).
Later, the presence of specific Type I receptors in a number of other
tissues including the CNS were reported (Anderson and Fanestil, 1976;
Moguilewsky and Raynaud, 1980; Lan et al., 1981). However, it appeared
that ALDO, DOC and CORT had equivalent affinity for these sites. The
results from these in vitro competition assays were taken as
evidence for possible in vivo occupancy of Type I receptors by


41
glucocorticoids in the rat kidney and brain (Lan et al., 1982; Veldhuis
et al., 1982b). In a separate study, Beaumont and Fanestil (1983)
reported that the steroid binding specificity Type I receptors in rat
brain could be altered by different experimental manipulations. These
investigators found that after elimination of Type II binding
interferences (in the presence of a molar excess of RU 26988) and plasma
CBG (and possibly Type III receptors), the order of steroid competition
for Type I sites (occupied with [ H]ALDO) was DOC > fludrocortisone >
CORT > ALDO > progesterone > DEX. However, the addition of small
quantities of dialyzed serum to the above preparations yielded a
competition hierarchy similar to that described for kidney, i.e., ALDO >
DOC > CORT. Several independent confirmations of the above observations
were reported for the rat kidney (Krozowski and Funder, 1983; Stephenson
et al., 1984b) and hippocampus (Krozowski and Funder, 1983; De Kloet et
al., 1984). In conclusion, it is suggested that CBG and CBG-like
binders, in addition to affecting the metabolic clearance rate of the
steriods to which they bind with high affinity (for review see Siiteri
et al., 1982), may act in vivo as sequestration sites or "sinks" for
corticosterone thus rendering the Type I sites available for ALDO
binding (Funder et al., 1973b; Krozowski and Funder, 1983; Stephenson et
al., 1984; Funder, 1986). It is further suggested that the hippocampal
Type I receptors are physiological CORT binders and may be involved in a
tonic (permissive) influence on brain function with septo-hippocampal
complex as a primary target (De Kloet et al., 1984; Reul and De Kloet,
1985; Funder, 1986; Magarinos et al., 1986; Reul and De Kloet, 1986).
Although the sequestration theory is an attractive hypothesis, further
experiments investigating the tissue availability of CORT (that is not


42
bound to CBG, other plasma binders and Type III receptors) and ALDO
under physiological conditions is required to establish this theory. In
this regard, preliminary studies suggest that the serum levels of ALDO
and CORT under "normal" conditions leads to nuclear uptake of both
hormones in the hippocampus as measured by radioimmunoassay (Yongue and
Roy, 1984). Recently, McEwen et al. (1986b) showed that, in
adrenalectomized rats, only "stress level" concentrations (a 500-fold
molar excess) of CORT was able to block the nuclear uptake of
3
[ HjALDO in limbic, but not circumventricular, areas. Further
functional relevence of these findings remain to be established.
A further caution regarding the results obtained from in vitro
competition assays also deserves mention: in the above experiments
cytosolic preparations were performed in the presence of molybdate and
DTT. Our own work has shown that although molybdate stabilizes both
Type I and Type II receptors, it causes a significant reduction in the
binding capacity of Type I receptors (Emadian et al., 1986; also see
Chapter IV). Furthermore, sulfhydryl reducing reagents such as DTT,
while having no effect on the binding of ALDO to Type I receptors,
result in an apparent increase in the equilibrium dissociation constant
of CORT from CBG (Westphal, 1983) and very likely from Type III
receptors. Therefore, under conditions that provide optimal stability
for all of these proteins, one may find different steroid-binding
hierarchy from those discussed above.


CHAPTER II
EXPERIMENTAL PROCEDURES
In order to avoid repetition, in this chapter a general scheme of
the materials and experimental procedures shared by the studies
described in Chapters III through VI will be presented. Materials and
methods that are unique to a given chapter will be described in that
particular chapter.
Isotopes, Steroids and Other Chemicals
3
The radiolabeled steroids; i.e., [6,7- H]Triamcinolone acetonide,
3
[ H]TA, 9a-fluoro-113, 16a,17,21-tetra-ol-pregna-1,4-diene-3,20-dione
3 3
(specific activity, SA 43.7 Ci/mmol); [6,7- H]dexamethasone, [ H]DEX,
9a-fluoro-16a-methylprednisolone (SA 37.3-44.1 Ci/mmol) and
[1,2,6,7-^H]aldosterone, [^HjALDO, 4-pregnen-113,21-diol-3,18,
20-trione (SA = 71.5-82.0 Ci/mmol) were purchased from New England
Nuclear. These radiolabeled steroids were checked for purity by
thin-layer silica gel chromatography using a chloroform-methanol (9:1,
volume:volume) solvent system. [^H]TA, [^H]DEX, [^H]ALDO,
Sephadex G-25 (bead size 20-80 u) and sodium molybdate were obtained
from Sigma. HEPES buffer [4(2-hydroxyethyl)-l-peperazineethanesulfonic
acid] and dithiothreitol (DTT) were obtained from Research Organic. RU
26988 [113173-dihydroxy-17a-(1-propionyl)-androsta-1,4,6-triene-3-one)]
and RU 26752 [3' (3-oxo-7a-propyl-173-hydroxy-4-androstene) propionic
acid lactone] were kindly supplied by Roussel-Uclaf (France). Prorenone
43


44
[ 3( 17$-hydroxy-6$, 7(3-methy lene-3-oxo-4-andros ten-170t-yl) propionic acid
y-lactone] was a gift from Searle Co. All other chemicals used in this
dissertation were reagent grade quality.
Experimental Animals
Adult female CD-I mice (Charles River Laboratories) were subjected
to a combined bilateral adrenalectomy and ovariectomy under barbiturate
anesthesia 3-6 days prior to use [a period shown to result in maximal
up-regulation of Type I receptors (Luttge and Rupp, unpublished
results)] in order to remove known sources of endogenous steroids. They
were then maintained on Purina Rodent Chow and 0.9% NaCl. On the day of
experiment, mice were anesthetized with ether and perfused slowly
through the heart with 15-20 ml ice-cold HEPES-buffered saline
(isotonic, pH 7.60).
Table 2-1
Buffers
20 mM 2 mM
BUFFER
HEPES
DTT
Na MoO.
2 4
A
+
-
-
D
+
+
-
M
+
-
2 mM
M20
+
-
20 mM
DM
+
+
2 mM
DM
20
+
+
20 mM
cytosolic concentration of the
component(s)
in Buffers D, M,
DM^q as well as
other cytosolic
components
(e *8 >
300 mM KC1; 10% mono- and polyhydric compounds) were obtained by a 9:1


45
dilution of cytosol with stock buffers containing a 10-fold
concentration of the desired component(s). Under such circumstances,
the control group was diluted accordingly with an equivalent volume of
Buffer A. Buffers were adjusted to pH 7.60 at 0-2C.
Cytosol Preparation
Brains were homogenized (2 x 10 strokes) at 1000 RPM in two volumes
of ice-cold Buffer A or Buffer A containing one or more of the desired
components (see specific experiments). A glass homogenizer with a
teflon pestle milled to a clearance of 0.125 mm on the radius was used
to minimize the rupture of brain cell nuclei. The crude homogenate was
centrifuged at 100K x g for 20 min and the resulting supernatant
recentrifuged at 100K x g for an additional 60 min to yield cytosol.
During this and all other procedures (except "aging" and column
chromatography) attempts were made to maintain the cytosol at 0C.
Aging
In order to examine thermal stability of unoccupied Type I and Type
II receptors, cytosol was incubated for various periods of time at 0
and/or 22C. Following such treatment, "aging" was quenched by
incubating the cytosol with steroid(s) for 24-48 h at 0C for specific
binding determination (see below) .
Hydrophobic Interaction Chromatography.
Pently-Agarose columns (0.7 x 21.5 cm) were used to compare the
surface hydrophobic properties of occupied Type I versus Type II
(Chapter III) as well as activated versus unactivated Type I receptors
(Chapter VI). In such experiments, cytosol in Buffer D^o was
incubated with radiolabeled ligand for Type I or Type II receptors for
48 h at 0C (see "Steroid Binding Determination"). Following incubation


46
with steroid(s), salt- and heat-induced activation (see Chapter VI),
bound-free separation on Sephadex G-25 (0.6 x 14.0 cm) columns were
performed as described previously (Luttge et al., 1984c). Subsequently,
0.8 ml [ H]steroid-bound macromolecular fraction eluted from the
G-25 column was loaded on pently-Agarose columns pre-equilibrated in
buffer D containing 50 mM molybdate and 600 mM KC1. Using this latter
buffer, 20-27 consecutive 0.8 ml fractions were collected directly into
scintillation vials for measurements of radioactivity (see below). In
some experiments, a second bound-free separation on G-25 columns were
performed on the "peak" fractions eluted from the pentyl agarose columns
to assure that the radioactivity eluted was indeed macromolecular-bound
in nature. All chromatographic procedures were performed at 4-6C.
Steroid Binding Determination
After appropriate treatment of cytosol, 0.24-4.0 ml aliquots were
added to pre-cooled glass tubes containing the desired steroid(s) (dried
under N^). The tubes were then allowed to incubate for 24-48 h in
ice. In single saturation dose studies, B determinations for Type
I receptors were performed in the presence of 10-20 nM [ H]ALDO and
500-fold molar excess of RU 26988. For Type II receptors, 20 nM
3 3
[ H]DEX or [ H]TA was used as the ligand. In some experiments,
3
[ HjDEX incubation was in the presence of a 500-fold molar excess of
RU 26752, prorenone or RU 26988 (see Chapter V). Parallel incubation
tubes containing an additional 200-fold molar excess of [^H]ALD0,
[^H]DEX or [^H]TA were used for the determination of B>T for
NS
Type I and Type II receptors, respectively. Equilibrium binding
parameters; i.e., K and BJV were determined using Scatchard's
a MAX
method (1949) as described previously (Emadian et al., 1986).


47
In all experiments, macromolecular-bound from free steroid was
separated at 4C using G-25 (0.6 x 14 cm) columns (for detail see Luttge
et al., 1984c). Bound fraction (0.8 ml) obtained from each column was
diluted with 5 ml Scinti-Verse flor (Fisher Scientific) in a
scitillation vial, vortexed vigorously and the radioactivity determined
in a Packard Model 2425 liquid Scintillation spectrometer. All counts
per minute (CPM) data were converted to disintegration per minute (DPM)
by an external standard channels ratio measurement. The counting
efficiency for tritium averaged about 35%.
Cytosolic Protein Concentration
The concentration of protein in cytosol preparations was determined
using the method described by Lowry et al. (1951). In crude
preparations, protein concentration ranged from 6.2 to 7.3 mg/ml
cytosol, whereas the values in gel filtered and 2-fold diluted
preparations (see Chapter IV) averaged about 3.3 and 3.6 mg/ml cytosol,
respectively.


CHAPTER III
EFFECTS OF POLYHYDRIC AND MONOHYDRIC COMPOUNDS ON THE STABILITY OF
TYPE I RECEPTORS FOR ADRENOCORTICOSTEROID HORMONES IN BRAIN CYTOSOL
Summary
We have shown previously that unoccupied Type I receptors for
adrenal steroids in brain cytosol lose their ability to bind
3
[ H]ALDO in a time- and temperature-dependent manner (Emadian
et al., 1986). Based on reports that sugars and polyvalent alcohols are
capable of stabilizing a variety of globular proteins, we made an
attempt in this study to stabilize Type I receptors by including
polyhydric compounds in our cytosol preparations. However, unlike our
expectations, the addition of 10% (weight:volume) ethylene glycol,
glycerol, erythritol, xylitol, ribitol or sorbitol to cytosol at 0C
failed to stabilize the receptors and in fact produced a slight
3
reduction in the binding of [ H]ALD0. The magnitude of this
reduction was greater when cytosol was "aged" for 2 h at 22C prior to
3
incubation with [ H]ALD0. In contrast to these results with
polyhydric compounds, the addition of 10% (weight:volume) ethanol to
3
cytosol maintained at 0C markedly increased the binding of [ H]ALD0
to Type I receptors. Under identical conditions, methanol slightly
increased, and propanol had no significant effect on the binding
capacity of these receptors. When cytosol was aged at 22C, however,
all of these monohydric compounds led to a marked loss in Type I
receptor binding capacity. An investigation of various doses of ethanol
at 0C on the subsequent binding of [ H]ALD0 to Type I receptors
48


49
yielded an inverse U-shaped curve with 10% ethanol producing the highest
level of specific binding, as reflected by an increase in maximal
binding in Scatchard plots, and 40% ethanol producing a complete loss in
Type I receptor binding capacity. In view of the above findings and
proposed mechanisms of action of mono- and polyhydric compounds, it is
suggested that Type I receptors in an aqueous cytosolic environment
undergo spontaneous conformational changes which promote the infolding
of hydrophobic steroid binding site(s) on the receptor surface, thus
leading to the observed reduction in Type I receptor binding capacity.
Introduc tion
Although receptors for steroid hormones appear to be relatively
stable under in vivo conditions (Raaka and Samuels, 1983), these
macromolecules are rendered labile under cell-free conditions. This
increased lability is particularly apparent when working with unoccupied
receptors (Puca et al., 1971; Pratt and Ishii, 1972; Bell and Munck,
1973; Rafestin-Oblin et al., 1977; Emadian et al., 1986), and it can be
a serious problem when experimental conditions necessitate that the
unoccupied receptors must remain stable for extended periods of time
(e.g., during the determination of their hydrodynamic and equilibrium
binding parameters). In previous work with unoccupied and occupied
Type I and Type II receptors for adrenal steroids in brain cytosol we
have systematically examined the stabilizing effectiveness of a number
of factors including temperature, ionic strength, chelators, molybdate,
dithiothreitol, dextran-coated charcoal adsorption, etc. (Gray, 1982;
Densmore et al., 1984a; Densmore et al., 1984b; Densmore and Luttge,
1985; Densmore et al., 1986a; Densmore et al., 1986b; Emadian and
Luttge, 1986; Emadian et al., 1986); however, even with our best buffer


50
formulations, we still observe a time- and temperature-dependent loss of
unoccupied Type I receptor binding capacity (Eraadian et al., 1986).
Work in other laboratories has shown that polyhydric compounds (e.g.,
sugars and polyvalent alcohols) can stabilize a wide range of globular
proteins, including a number of enzymes and receptors, against
time-dependent thermal degradation (Feil et al., 1972; Rafestin-Oblin
et al., 1977; Back et al., 1979; Ogle, 1980; Gekko and Timasheff, 1981a
& b; Ogle, 1983). In view of these successes, the experiments presented
here examine the effects of a number of mono- and polyhydric compounds
on the stability and binding capacity of unoccupied Type I receptors for
3
[ H]ALD0 in brain cytosol. The results from these studies suggest
that spontaneous conformational changes that occur as a consequence of
interaction between these receptors and their aqueous cytosolic
environment may ultimately lead to the infolding of hydrophobic steroid
binding site(s) on the surface of Type I receptors.
Materials and Methods
Chemicals. Reagent grade poly- and monohydric compounds were
purchased from Sigma.
Buffers. The final concentration of mono- and polyhydric compounds
in cytosol was obtained by adding concentrated stock buffers containing
each compound to the cytosol to yield the desired concentration of that
compound. The pH is all buffers was adjusted to 7.60 at 0-2C. All
other materials and methods were as described in Chapter II.
Results
Fig. 3-1 shows that a 10% (weight:volume) final concentration of
di- (ethylene glycol, EG), tri- (glycerol, G), tetra- (erythritol, E),
penta- (xylitol, X and ribitol, R) or hexavalent (sorbitol, S) alcohols


Figure 3-1. Effects of Polyhydric Compounds on the Stability of
Unoccupied Type I Receptors in Whole Brain Cytosol. Cytosol
prepared in Buffer A was either diluted with additional Buffer A (No
Poly-OH) or adjusted to 10% (weight:volume) ethylene glycol (EG),
glycerol (G), erythritol (E), Xylitol (X), ribitol (R) and sorbitol (S)
using a concentrated HEPES-buffered solution of each compound.
Subsequently, the cytosol was divided into two parts: one part was
3
incubated immediately with 10 nM [ H]ALD0 plus 5 uM RU 26988 and
either with (BV1) or without (B_) 2 uM [^HjALDO for 24 h 0C
ns r
(0-h open bars). The second part was aged either from 2 to 48 h at 0C
(2-, 24- and 48-h open bars) or for 2 h at 22C (closed bars) prior to
incubation with the steroids. At the end of the incubation period,
macromolecular-bound from free steroids were separated on Sephadex G-25
columns and specific binding was determined as described in Chapter II.
The number of independent replicate experiments is indicated near the
base of each bar. All data presented are based on the percent of the
non-aged, "No Poly-OH" control (*) group + SEM. 100% = 28 fmole/mg
protein.


52
No Poly-OH EG
R


53
to unlabeled brain cytosol produced a small, but significant (p < 0.05),
3
reduction in the binding of [ H]ALD0 to Type I receptors for adrenal
steroids. Aging unlabeled cytosol at 0C in the presence of any of
these polyhydric compounds did not attenuate, or exacerbate, the loss in
Type I receptor binding capacity seen in the control cytosol (No
Poly-OH). However, when cytosol was aged for 2 h at 22C, the reduction
3
in the subsequent binding of [ H]ALD0 to Type I receptors in the
presence of polyhydric compounds was nearly twice as great as in their
absence. Since a 10% concentration of glycerol has also been routinely
included by many investigators in buffers used for Type II receptors, we
3
examined the effects this compound on [ H]DEX binding. As shown in
Fig. 3-2, the addition of 10% glycerol to brain cytosol prior to
3
incubation with [ H]DEX fails to protect the time- and temperature-
dependent loss in Type II receptor binding capacity. It is noteworthy
that, in contrast to the results obtained with Type I receptors (Fig.
3-1), glycerol does not exacerbate the loss in binding capacity when
unoccupied Type II receptors are aged for 2 h at 22C.
Because polyhydric compounds failed to stabilize Type I (or
Type II) receptor binding capacity, we next investigated the effects of
3
monohydric alcohols on the binding of [ H]ALD0 to Type I receptors
(Fig. 3-3). In contrast to the data with polyhydric compounds, when a
10% (weight:volume) final concentration of methanol, and especially
ethanol, but not propanol, was added to cytosol immediately before the
steroids, we observed a significant increase in [ H]ALD0 binding to
Type I receptors. If cytosol was aged for 2 h at 0C prior to the
addition of steroids, ethanol still produced an increase in the
3
subsequent binding of [ H]ALD0 to Type I receptors, whereas methanol


Figure 3-2. Effects of 10% Glycerol on the Stability of Unoccupied
Type II Receptors in Whole Brain Cytosol. Cytosol prepared in
Buffer D was either diluted with additional Buffer D (No Poly-OH) or
adjusted to 10% (weight:volume) glycerol using a concentrated
HEPES-buffered solution of glycerol containing 2 mM DTT (G).
Subsequently, the cytosol was divided into two parts: one part was
3 ,
incubated immediately with 20 nM [ H]DEX and either with (B.,_)
NS
or without (B^) 4 uM [*H]DEX for 24 h at 0C (0-h open bars).
The second part was aged either from 2 to 48 h at 0C (2-, 24- and 48-h
open bars) or for 2 h at 22C (closed bars) prior to incubation with the
ligand. At the end of the incubation period, macromolecular-bound from
free steroid was separated on Sephadex G-25 columns and specific binding
was determined as described in Chapter II. The results are the mean
from 2 independent replicate experiments. All data presented are based
on the percent of the non-aged, "No Poly-OH" control (*) group.
100% = 300 fmole/mg protein.


55
No Poly- OH
G


Figure 3-3. Effects of Monohydrlc Compounds on the Stability of
Unoccupied Type I Receptors in Whole Brain Cytosoi. Cytosol
prepared in Buffer A was either diluted with additional Buffer A (No
Alcohol) or adjusted to 10% (weight:volume) methanol (Mt) ethanol (Et)
and propanol (P) using a concentrated HEPES-buffered solution of each
compound. Subsequently, the cytosol was divided into two parts: one
3
part incubated immediately with 10 nM [ H]ALD0 plus 5 uM RU 26988
and either with (BYT) or without (2 uM [^HlALDO for 24 h
Ni> T
at 0C (0-h open bars). The second part was aged for 2 h at 0 or 22C
(2-h open and closed bars, respectively) prior to incubation with the
steroids. At the end of the incubation period, macromolecular-bound
from free steroids were separated on Sephadex G-25 columns and specific
binding was determined as described in Chapter II. All data presented
are based on the percent of the non-aged, "No Alcohol" control (*) group
expressed as the mean + SEM from 6 independent replicate
experiments. 100% = 18 fmole/mg protein.


57
*
c
o
c
o
Ol
No Alcohol Mt
Et
P


58
had no effect and propanol produced a clear reduction in receptor
binding capacity. When cytosol preparations were aged for 2 h at 22C,
all three alcohols greatly increased the loss in Type I receptor binding
capacity.
An investigation of the effects of various concentrations of
3
ethanol on the binding of [ H]ALDO to cytosolic Type I receptors
revealed an inverse U-shaped curve with 10% ethanol producing the
greatest binding (120% of control) and 40% ethanol leading to a complete
inactivation of Type I receptors (1% of control) (Fig. 3-4).
Furthermore, a Scatchard (1949) analysis of the equilibrium binding data
revealed that the 10% concentration produced a 46% increase in the
3
K, and a 23% increase in the B of [ HlALDO binding to
d max
Type I receptors in brain cytosol (Fig. 3-5).
Discussion
Proposed Mechanism of Action of Poly- and Monohydric Compounds.
For proteins in an aqueous environment, the non-covalent weak
intramolecular interactions (i.e., electrostatic forces, hydrogen
bonding and Van der Waals forces) as well as the interactions between
protein molecules and their surrounding solvent (i.e., hydrophobic
interactions), act in concert to stabilize the functionally important
tertiary structure of proteins. In some proteins, additional stability
may result from cross-linking, metal complex formation and binding to
other ions and cofactors; however, it is often held that the hydrophobic
interactions, a consequence of net unfavorable interactions between
solvent (i.e., water molecules) and non-polar residues in the protein,
play the most important role in stabilizing proteins (e.g., Creighton,
1984; Fersht, 1985). Therefore, when proteins are extracted from the


Figure 3-4. Dose-Response Analysis of the Effects of Ethanol on the
Binding of ALDO to Type I Receptors in Whole Brain Cytosol. Cytosol
prepared in Buffer A was either diluted with additional Buffer A
(control, *) or adjusted to 1 to 40% (weight:volume) ethanol using a
concentrated HEPES-buffered solution of ethanol. Subsequently, all
3 ,
groups were incubated with 10 nM [ H]ALD0 plus 5 uM RU 26988 and
either with (BlT) or without (B) 2 uM [^HjALDO for 24 h at
0C. At the end of the incubation period, macromolecular-bound from
free steroids were separated on Sephadex G-25 columns and specific
binding was determined as described in Chapter II. All data presented
are based on the percent of 0% ethanol control (*) group expressed as
the mean + SEM from 3-12 independent replicate experiments.
100% = 23 fmole/mg protein.


Percent of Control*
60
Ethanol Concentration (% w:v)


Figure 3-5. Scatchard Analysis of the Effects of 10% Ethanol on the
Equilibrium Binding Parameters of ALDO Binding to Type I Receptors
in Whole Brain Cytosol. Cytosol prepared in Buffer A was either
diluted with additional Buffer A (open circles) or adjusted to 10%
(weight:volume) ethanol using a concentrated HEPES-buffered solution of
ethanol (closed circles). Subsequently, 0.25 ml aliquots from each group
3
was incubated with 0.5 to 40 nM [ H]ALD0 plus 500-fold molar excess
of RU 26988 and either with (B^) or without (B^) 200-fold molar
excess of [^H]ALD0 for 24 h at 0C. At the end of the incubation
period, macromolecular-bound from free steroids were separated on
Sephadex G-25 columns and specific binding was determined as described
in Chapter II. The Scatchard plot shown is representative from 4
independent replicate experiments. Binding parameters were calculated
using least square linear regression. Open circles: B =
max
21 fmole/mg protein, = 3.0 x 10 ^ M. Closed circles:
B =26 fmole/mg protein, K, = 4.4 x 10 ^ M.
max r d


62
0.05
OJO


63
intracellular environment into a medium that is more aqueous than
cytoplasm, it is not uncommon for them to undergo spontaneous
conformational changes leading to their functional inactivation. To
protect proteins against such in vitro denaturation, various
strategies have been utilized to reduce unfavorable solvent-solute
interactions .
In a series of studies by Gerlsma (1968 & 1970) and Gerlsma and
Stuur (1972 & 1974), polyhydric compounds were shown to increase the
melting temperature (T ) of chymotrypsinogen A, ribonuclease and
lysozyme and hence stabilize the native conformation of these proteins
at a higher temperature. In contrast, monohydric alcohols were found to
destabilize these proteins (i.e., lower the T ). On a molecule per
m
molecule basis, the increase in the produced by polyhydric
compounds was shown to be related directly to the number of hydroxyl
groups per molecule of the polyhydric compound, whereas the extent of
destabilization produced by monohydric compounds were shown to depend on
the length of their alkyl chain. On the basis of these results, Gerlsma
and Stuur (1972 & 1974) proposed that polyhydric compounds stabilize
proteins by decreasing the hydrogen bond rupturing capacity of the
solvent, whereas monohydric compounds augment such solvent-induced
hydrogen bond rupturing capacity. This proposed mechanism is somewhat
different from that set forth earlier by McDuffie et al. (1962), who
maintained that polyhydric compounds stabilize proteins by inducing the
tetrahedral conformation of water molecules at higher temperatures
which, in turn, increase the hydrophobic interactions. In support of
this hypothesis, Oakenfull & Fenwick (1979) showed that hydrophobic


64
interactions in aqueous-organic environments are maximized when the
three-dimensional hydrogen-bonded structure of water is most developed.
In a recent quantitative analysis by scanning calorimetric
technique, Back et al. (1979) have shown that sucrose and glycerol
strengthen pairwise hydrophobic interactions in aqueous solutions of
ovalbumin, lysozyme, a-chymotrypsinogen and conalbumin; however, both of
these polyhydric compounds were also shown to reduce the driving force
for transfer of a hydrophobic group from an aqueous to a nonpolar
environment. Other workers have argued that glycerol, the most common
polyhydric compound used to stabilize the activity and native structure
of enzymes and proteins (Feil et al., 1972; Rafestin-Oblin et al., 1977;
Ogle, 1980; Dias et al., 1981; Korge and Timpmann, 1983; Livesey et al.,
1983; Ogle, 1983; Ramaley and Vasantha, 1983; Fojo et al., 1985),
increases the hydrophobic interactions in chymotrypsinogen A,
a-chymotrypsin, y-lactoglobulin and ribonuclease A by being
preferentially excluded from the immediate domain of these proteins
(Gekko and Timasheff, 1981a & b; Timasheff et al., 1976). Collectively,
these results suggest that polyhydric compounds tend to strengthen the
pairwise hydrophobic interactions in proteins by promoting the
three-dimensional hydrogen-bonded structure of water, hence stabilizing
the native, more folded conformation of proteins.
Possible Mode of Action of Poly- and Monohydric Compounds on
Unoccupied Type I receptors in Crude Cytosol.
Recently we reported that, in contrast to unoccupied Type II
adrenal steroid receptors, unoccupied Type I adrenal steroid receptors
3
lose their binding capacity for [ H]ALD0 in crude brain cytosol in a
time- and temperature-dependent manner even in the presence of DTT,
molybdate and/or glycerol (Emadian et al., 1986). However, as discussed


65
above, since protein molecules are thought to exist in a dynamic state
in aqueous environments (Creighton, 1984), we now think that our earlier
findings with Type I receptors could be a reflection of the transient
fluctuations in the three-dimensional structure of the unoccupied
3
receptors in cytosol resulting in an exclusion of [ H]ALDO from its
specific hydrophobic binding site.
To arrive at this hypothesis, we first investigated the effects
polyhydric compounds with increasing ability to promote the tetrahedral
conformation of water molecules (i.e., di-, tri-, tetra-, penta-, and
hexavalent alcohols), hence an increased ability to enhance
3
intramolecular hydrophobic interactions, on the [ H]ALDO binding
capacity of unoccupied Type I receptors. As shown in Figure 3-1, all of
the polyhydric compounds tested failed to stabilize unoccupied Type I
receptors, in fact, all of them produced a slight reduction in the
3
specific binding of [ H]ALDO, especially when cytosol was aged for 2
h at 22C prior to binding determinations. These results suggest that
increases in the intramolecular hydrophobic interactions within the
unoccupied receptor, following the addition of polyhydric compounds to
aqueous cytosol preparations, may result in the preferential exclusion
of the steroid from its hydrophobic binding sites. If so, one would
expect that a reduction in intramolecular hydrophobic interactions
3
should increase the binding of [ HjALDO to Type I receptors. It is
noteworthy that, compared to Type I receptors, inclusion of 10% glycerol
in our buffers did not affect the binding of ligand to Type II receptors
(Figure 3-2). The differential effects of glycerol on Type I and Type
II receptors strongly suggested that there might be differences in the
overall surface hydrophobicity of these receptors. In fact, a


66
comparison of the surface hydrophobic properties of these receptors by
glass fiber filter assay (Luttge et al., 1984) and hydrophobic
interaction chromatography (Figure 3-6) revealed a greater overall
surface hydrophobicity for Type I receptors.
To test the hypothesis that a reduction in hydrophobic interactions
may increase the binding of ligand to Type I receptors, we next
investigated the effects of adding monohydric alcohols to brain cytosol.
As predicted, when compared to the non-aged, "no-alcohol" group, a 10%
final concentration of ethanol in cytosol increased the binding of
3
[ H]ALD0 to Type I receptors by about 20% (Figs. 3-3 and 3-4). This
effect was reflected in Scatchard plots by an increase in the B
J max
(Fig. 3-5). In spite of this increase, the ethanol-induced reductions
in intramolecular hydrophobic interactions also appear to reduce the
stability of unoccupied Type I receptors when they are aged at 0C, and
especially at 22C. This increase in the lability of the unoccupied
receptor could easily account for the shift seen in the Scatchard
plot (Fig. 3-5) (see Beck and Goren, 1983 for a theoretical explanation
of this phenomenon) and may be similar to the previously reported
temperature-dependent effects of ethanol on the stability of
ribonuclease and glutamate dehydrogenase (Shukuya and Schwert, 1960;
Brandts and Hunt, 1967).
In summary, the results described in this chapter provide indirect
3
support for the hypothesis that [ H]ALDO binding to Type I receptors
is dependent upon the exposure and stabilization of hydrophobic steroid
binding sites (and possibly other hydrophobic residues) on the surface
of the receptors. Since the comparatively stable in vivo
conformation of Type I receptors presumably allows for optimal ligand


Figure 3-6. Comparison of the Surface Hydrophobic Properties of
ALDO-Type I and DEX-Type II Receptor Complexes on Pentyl-Agarose
Columns. Cytosol prepared in buffer DM was incubated with either
3
10 nM [ H]ALD0 plus 5 uM RU 26988 (to label Type I receptors, open
3
circles) or 20 nM [ H]DEX (to label Type II receptors, closed
circles) at 0C for 48 h for determination of B The
T
determinations of B for Type I and Type II receptors were
IN u
performed in the presence of 2 uM [^H]ALD0 and 4 uM [^HjDEX,
respectively. Following separation of macromolecular-bound from free
steroids on Sephadex G-25 columns, hydrophobic interaction column
chromatography was performed as described in Chapter II. The data
presented are expressed as a percent of total B^ eluted from
pentyl-Agarose columns and are representative from 2 independent
replicate experiments.


Percent of Total Specific Binding
_ ro oj
o o o
o
00
T
T
T
T


69
binding, it seems likely that these receptors undergo a spontaneous
solvent-induced folding upon extraction into the more aqueous cytosolic
environment which results in a reduction in binding capacity and
stability. Although, the mechanism(s) and/or intracellular factors
producing the marked stability of Type I and other steroid receptors
in situ awaits further experiments, the present findings may provide
some clues as to the nature of their properties.


CHAPTER IV
A NOVEL EFFECT OF MOLYBDATE ON THE BINDING OF [ "h]ALDOSTERONE
TO GEL FILTERED TYPE I RECEPTORS IN BRAIN CYTOSOL
Summary
Recently, we reported that the addition of sodium molybdate to
crude cytosol at 0C results in a dose-dependent reduction in the
3
binding of [ H]ALDO to Type I receptors for adrenal steroids
(Emadian et al., 1986). In the experiments outlined here, we show that
Sephadex G-25 gel filtration of whole brain cytosol from
adrenalectomized and ovariectomized CD-I- female mice in the presence of
2 mM molybdate results in a 30-50% increase in the specific binding of
3 3
10 nM [ HjALDO to Type I receptors above the level of [ H]ALD0
binding seen in crude cytosol prepared in 20 mM HEPES buffer (control
group). All specific binding determinations were performed in the
presence of 5 uM RU 26988 a specific synthetic glucocorticoid known
3
to block [ H]ALD0 binding to Type II (glucocorticoid) receptors. In
Scatchard plots, this molybdate- and gel filtration-induced increase in
3
specific [ HjALDO binding was reflected as a 37% increase in maximal
binding (B ) with no change in the equilibrium dissociation
max n
constant (K^). In contrast, when gel filtration was performed in
the absence of molybdate, there was a marked reduction in the subsequent
3
specific binding of [ HjALDO to Type I receptors. In Scatchard
plots, this latter effect was reflected as a 62% reduction in the
B and a 2-fold increase in the K, when compared to the
max d
control group. The addition of 2 mM molybdate immediately following gel
70


71
3
filtration yielded specific [ HjALDO binding comparable to the
control group. Although the addition of 2 mM molybdate prevents the
loss in the binding capacity of unoccupied Type I receptors in crude
cytosol incubated at 22C, Type I receptors in gel filtered cytosol were
very unstable at 22C, and appeared insensitive to the protective
effects of 2, but not 10-100 mM, molybdate. Two mM dithiothreitol (DTT,
a potent sulfhydryl reducing reagent) was unable to prevent the gel
3
filtration-induced loss in [ H]ALD0 binding. Moreover, in gel
filtration experiments, the data obtained in the presence of 2 mM DTT
plus 2 mM molybdate did not differ from those obtained in the presence
of 2 mM molybdate alone. The effect of molybdate on Type I receptors
caused by its presence during gel filtration of cytosol is clearly
different from that seen with these receptors in crude cytosol
preparations, as well as those reported in the literature for other
steroid receptors. Possible mechanisms of action of molybdate on
unoccupied Type I receptors in crude and gel filtered cytosol are
discussed.
Introduc tion
In a series of reports from Pratt's laboratory it was shown that
the addition of sodium molybdate to crude cytosol preparations prevents
the heat-induced loss in the binding capacity of unoccupied
glucocorticoid (Type II) receptors (Nielsen et al., 1977b & c).
Subsequently, Toft and Nishigori (1979) showed that this transition
element oxyanion reversibly blocks the temperature-dependent
transformation (activation) of progesterone receptors to a DNA-binding
state. Later, numerous reports have shown similar stabilizing effects
of molybdate on other steroid receptors (Nishigori and Toft, 1980;


72
ShyamaLa and Leonard, 1980; MilLer et al., 1981) and proteins (Defay et
al., 1984; Nakada et al., 1985; Denison et al., 1986). In contrast to
these findings, recently we have reported that although molybdate
prevents the heat-induced inactivation of Type I receptors, at 0C, this
compound leads to a dose-dependent reduction in the subsequent binding
3
of [ H]ALD0 in crude whole brain cytosol preparations (Emadian et
al., 1986). In the experiments below, we will describe yet another
novel effect of molybdate on Type I receptors in Sephadex G-25 gel
filtered whole brain cytosol.
Materials and Methods
Chemicals. All chemicals used in the experiments in this chapter
were reagent grade quality.
Buffers. The cytosolic concentration of molybdate and DTT were
obtained by a 9:1 dilution of cytosol with stock buffers containing a
10-fold concentration of the desired component(s). Under such
circumstances, the control group was diluted accordingly with an
equivalent volume of Buffer A. The final pH for all buffers was
adjusted to 7.60 at 0-2C.
Sephadex G-25 Gel Filtration. Gel filtered receptor preparations
were obtained by loading 0.5 ml aliquots of unlabeled crude cytosol on
Sephadex G-25 columns (0.6 x 14.0 cm) pre-equilibrated in appropriate
buffer(s) at 4-5C. The sample on each column was allowed to penetrate
the gel and the column was then washed with 1.0 ml buffer.
Subsequently, the macromolecular fraction was collected using an
additional 1.0 ml homologous buffer.


73
Results
Gel filtration of cytosol prepared in Buffer A through Sephadex
G-25 columns (equilibrated and eluted with homologous buffer) led to a
3
70% reduction in the subsequent specific binding of [ H]ALD0 to Type
I receptors when compared to the control group (i.e., non-aged crude
cytosol prepared in Buffer A) (Fig. 4-1, A). There was no additional
loss in the binding capacity of Type I receptors when unlabeled crude or
gel filtered cytosol was aged for 2 h at 0C prior to incubation with
steroids; however, when the temperature during aging was raised to 22C,
we observed a 50% reduction in the subsequent Type I receptor binding to
3
[ H]ALD0 (Fig. 4-1, A; 2 h aging). With the exception of a slight
3
increase in [ H]ALD0 binding at 0C, similar results were obtained
when the above procedures were repeated in the presence of 2 mM DTT
(Fig. 4-1, D). This latter increase in B values was due to lower
u r
B^s values in crude and especially gel filtered cytosol preparations
containing 2 mM DTT. It should also be noted that unoccupied Type I
receptors in gel filtered cytosol containing DTT appeared to be more
susceptible to the 22C aging-induced loss in ligand binding.
In agreement with our earlier report (Emadian et al., 1986), at
0C, the addition of 2 mM molybdate to unlabeled crude cytosol produced
a slight reduction in the binding of [ H]ALD0 to Type I receptors
(Fig. 4-1, M). However, when the molybdate-containing cytosol was
subjected to Sephadex G-25 gel filtration, we observed a 50% increase in
3
the subsequent binding of [ H]ALDO to Type I receptors. Whereas
aging unlabeled crude or gel filtered cytosol in Buffer M at 0C for 2 h
had no significant effect on the measurable Type I receptor binding,
aging the unlabeled gel filtered, but not crude, cytosol at 22C for 2 h


Figure 4-1. Effects of Gel Filtration, Molybdate and/or
Dithiothreitol on the Binding Capacity and Stability of Unoccupied
Type I Receptors from Whole Brain Cytosol. Nine parts cytosol
prepared in Buffer A was diluted with either one part Buffer A (A) or
adjusted to 2 mM dithiothreitol (D), 2 mM molybdate (M) or 2 mM
dithiothreitol plus 2 mM molybdate (DM) using one part Buffer A
containing a 10-fold concentration of the respective component(s). Each
group either underwent the gel filtration procedure (for detail see
"Materials and Methods" section) at 4-5C (triangles) or was left
undisturbed at 4-5C (circles) during this process. Subsequently, a
portion of the crude or gel filtered cytosol from each group was either
incubated with 10 nM ["^H]ALD0 and 5 uM RU 26988 and minus (B^)
or plus (Bto) 2 uM [LH]ALD0 at 0C for 24 h for the
determination of specific binding or was aged at 0 (solid symbols) or
22C (open symbols) for 2 h prior to incubation with the steroids. Gel
filtration reduced the cytosolic protein concentration from 7.3 to
3.3 mg/ml. The data presented are from at least 5 independent replicate
experiments (except for the DM group) and are expressed as a percent of
control* (i.e., specific binding measured in non-aged crude cytosol
prepared in Buffer A) + standard error of the mean (SEM).
100% = 21 fmole/mg protein.


160
140
120
100
80
60
40
20
75
H D M DM
0 2
0 2 0 2 0 2
Duration of Aging (h)


76
led to a marked loss in the binding capacity of these receptors
(Fig. 4-1, M; 2 h aging). The data obtained in the presence of Buffer
DM did not differ appreciably from those described in the presence of
Buffer M with the exception, once again, of a slight exacerbation of the
3
heat-induced loss in the binding of [ H]ALDO to Type I receptors in
gel filtered cytosol following 2 h aging at 22C (Fig. 4-1, DM; 2 h
aging).
To determine the concentration of molybdate needed to produce the
3
highest increase in the binding of [ H]ALDO to Type I receptors
during gel filtration, we conducted a dose-response analysis of the
effects of this compound on Type I receptors during the gel filtration
procedure. Consistent with our previous observations (Emadian et al.,
1986) the addition of molybdate to HEPES buffer led to a dose-dependent
3
loss in [ H]ALDO specific binding to Type I receptors in crude
cytosol at 0C (Fig. 4-2). Aging the crude cytosol for 2 h at 0C
3
reduced the loss in [ H]ALDO binding seen with the two highest doses
of molybdate. In non-aged gel filtered cytosol, the maximal increase in
3
[ H]ALDO binding to Type I receptors occurred at 2 mM molybdate,
with 10-100 mM concentrations producing binding levels comparable to
those seen in the absence of molybdate (i.e., control group). It is
noteworthy that except for when molybdate was absent during gel
filtration, at all concentrations of this compound tested, the
measurable Type I receptor binding was higher in the non-aged gel
filtered cytosol than non-aged crude cytosol containing the same dose of
molybdate. Furthermore, at all concentrations of molybdate tested,
aging unlabeled gel filtered cytosol for 2 h at 0C, produced Type I
receptor binding comparable to the non-aged gel filtered preparations


Figure 4-2. Dose-Response Analysis of the Effects of Gel Filtration
and Molybdate on the Binding Capacity and Stability of Unoccupied
Type I Receptors from Whole Brain Cytosol. Nine parts cytosol
prepared in Buffer A was diluted with one part Buffer A containing 0,
20, 100, 200 or 1000 mM molybdate to bring the cytosol to a final
concentration of 0, 1, 2, 10, 20 or 100 mM molybdate, respectively.
Each group then either underwent the gel filtration procedure at 4-5C
(open symbols) or was left undisturbed at 4-5C during this process
(solid symbols). Subsequently, a portion of cytosol from each group was
3
either incubated with 10 nM [ H]ALD0 and 5 uM RU 26988 minus
(B ) or plus (B ) 2 uM [^H]ALD0 at 0C for 24 h for the
I NS
determination of specific binding (open and solid circles) or was aged
at 0 (open and solid squares) or 22C (open and solid triangles) for 2 h
prior to incubation with the steroids. Gel filtration reduced the
cytosolic protein concentration from 7.3 to 3.3 mg/ml. The data are
from at least 4 independent replicate experiments and are expressed as a
percent of control* (i.e., specific binding measured in non-aged crude
cytosol prepared in Buffer A) + standard error of the mean (SEM).
100% = 18 fmole/mg protein.


160
140
120
100
80
60
40'
20
78
0 2 10 20 DO
[Na ^ MoO^] mM


79
containing the same doses of molybdate. Consistent with the results in
Fig. 4-1, Type I receptors in unlabeled gel filtered cytosol containing
0 (i.e., Buffer A) or 2 mM molybdate (i.e., Buffer M) were found to be
extremely labile when aged for 2 h at 22C; however, increasing the
concentration of molybdate to 10-100 mM prevents this loss in binding
capacity. Moreover, as reported earlier (Emadian et al., 1986), when
crude, non-gel filtered cytosol preparations are aged for 2 h at 22C,
3
the highest level of [ H]ALD0 binding was observed in the presence
2 mM molybdate. Further increases in the concentration of molybdate
3
resulted in lower levels of [ H]ALD0 binding, comparable to those
measured in non-aged crude cytosol containing the same concentration of
molybdate.
To gain further insight about the nature of the effects of gel
filtration on the binding of Type I receptors in the presence and
absence of molybdate, Scatchard (1949) analyses of the equilibrium
binding data were performed (Fig. 4-3, Table 4-1). Gel filtration of
cytosol in Buffer M produced no effect on the but increased the
3
B of Type I receptors for [ HlALDO by 36% above the level
max J r r J
seen in crude cytosol prepared in Buffer A (control). Gel filtration of
cytosol in Buffer A produced a 62% reduction in the B and a
max
3
2-fold increase in the of [ HjALDO binding to Type I
receptors. The presence of 2 mM molybdate in crude cytosol produced a
slight increase in the but had no apparent effect on the
^max TyPe ^ recePtors when compared to the control group.
To see whether the gel filtration-induced loss in Type I receptor
binding capacity observed in Buffer A was reversible, we investigated
the effects of 2 mM molybdate added to cytosol at different time


Figure 4-3. Effects of Molybdate and/or Gel Filtration on the
Equilibrium Binding Parameters of ALDO Binding to Unoccupied Type I
Receptors from Whole Brain Cytosol. Nine parts cytosol prepared in
Buffer A was diluted with either one part Buffer A (solid circles and
triangles) or one part Buffer A containing 20 mM molybdate to bring the
cytosol to a final concentration of 2 mM molybdate (open circles and
triangles). Subsequently, each group either underwent the gel
filtration procedure (triangles) at 4-5C or was left undisturbed at
4-5C during this process (circles). All groups were then incubated at
0C with 0.1-40 nM [^H]ALD0 plus a 500-fold excess of RU 26988 and
either with (B^) or without (B^) a 200-fold excess of
[^H]ALDO at 0C for 24 h for the determination of specific binding.
3
The concentration of free [ H]ALD0 was determined in every tube by
subtracting B^ from the total radioactivity present. The values for
B and K, were obtained from the x-intercept and negative
max d t- b
inverse of the slope for each line, respectively, by least square linear
regression method. Gel filtration reduced the cytosolic protein
concentration from 7.3 to 3.3 mg/ml. The plots are representative of 2
independent replicate experiments.


/ F (10 mg Protein
81


Table 4-1
Binding
Parameters
Cytoso1
for Type I Receptors Obtained from Crude and Gel
in the Presence and Absence of 2 mM Molybdate
Filtered
Na.MoO.
2 4
G-25
\
B C
max
- r
+
+
0.45
27 .4
0.95
-
-
0.45
20.1
0.97
+
-
0.60
20.3
0.96
-
+
0.82
7.6
0.89
a _
Experimental
conditions are
described under Fig.
4-3.
-15
10 mole/mg protein.


83
intervals following gel filtration. As shown in Fig. 4-4, adding 2 mM
molybdate to cytosol immediately following gel filtration in the
presence of Buffer A led to a significant recovery of Type I receptor
binding capacity; however, the magnitude of this recovery was only
3
sufficient to increase [ H]ALDO binding to the levels seen in the
control group and never to the levels observed in cytosol gel filtered
in the presence of 2 mM molybdate. The magnitude of this recovery was
also time-dependent, since only molybdate added 1 to 5 h following gel
filtration led to a significant recovery of the binding capacity of
Type I receptors. The addition of molybdate to unlabeled gel filtered
cytosol 24 h following gel filtration did not produce any increase in
the binding capacity of Type I receptors. The effects of 2 mM molybdate
on Type I receptor binding capacity in crude and gel filtered cytosol
were comparable to those described in Figs. 4-1 and 4-2.
A number of earlier reports have implicated the role of endogenous
small molecular weight "factors" in cytosol as possible modulators of
hormone binding to glucocorticoid receptors and subsequent activation
(transformation) and DNA binding of the steroid-receptor complexes
(Bailly et al., 1977; Ishohashi et al., 1980; Leach et al., 1982; Dahmer
et al., 1984). Since during Sephadex G-25 gel filtration many small
molecular weight components are removed from cytosol, we sought to
investigate the effects of dilution of cytosol (which in effect dilutes
the concentration of endogenous factor(s) possibly involved in the
3
inhibition of [ H]ALD0 binding to Type I receptors) on the binding
capacity of these receptors. As shown in Fig. 5-5, a 2-fold dilution of
crude cytosol prepared in Buffer A or incubated with 2 mM molybdate for
2 h prior to dilution, produced Type I receptor binding comparable to


Figure 4-4. Stability of Unoccupied Type I Receptors from Whole
Brain Cytosol Following Gel Filtration in the Absence of Molybdate:
Effects of Molybdate Addition Following Gel Filtration and Aging.
Nine parts cytosol prepared in Buffer A was diluted with either one part
Buffer A (-M) or Buffer A containing 20 mM molybdate to bring the
cytosol to a final concentration of 2 mM molybdate (+M, solid bars).
Each group then either underwent the gel filtration procedure at 4-5C
(+ G-25) or was left undisturbed at 4-5C during this process (- G-25).
Subsequently, the group that was gel filtered in the absence of
molybdate was divided into two parts. One part was brought to 2 mM
molybdate immediately or 1, 2, 5 and 24 h following gel filtration
(shaded bars). The second part was diluted accordingly with Buffer A
immediately or 1, 2, 5 and 24 h (hash marked bars) following gel
filtration. All other groups were diluted accordingly with appropriate
buffers. Following these steps, all groups incubated with 10 nM
3
[ H]ALD0 and 5 uM RU 26988 minus (B ) or plus (B ) 2 uM
1 Wo
[^H]ALD0 at 0C for 24 h for the determination of specific binding.
Gel filtration reduced the cytosolic protein concentration from 7.3 to
3.3 mg/ml. The data are from at least 3 independent experiments and are
expressed as a percent of control* (i.e., specific binding measured in
non-aged crude cytosol prepared in Buffer A) + standard error of the
mean (SEM). 100% = 26 fmole/mg protein.


Percent of Control
85


Figure 4-5. Effects of Dilution and/or Molybdate on the Binding
Capacity of Unoccupied Type I Receptors from Whole Brain Cytosol.
Nine parts cytosol prepared in Buffer A was incubated at 0C for 2 h
with either one part Buffer A (shaded bars) or Buffer A containing 20 mM
molybdate to bring the cytosol to a final concentration of 2 mM
molybdate (solid bars). One group either underwent the gel filtration
procedure at 4-5C in columns pre-equilibrated with (G-25, M) or without
(G-25) 2 mM molybdate. A second group was diluted by 2-fold (Dil.)
using either Buffer A or Buffer M, where appropriate. A third batch of
undiluted crude cytosol in Buffer A (the control*) or Buffer M (M) along
with the "Dil." groups remained at 4-5C during the gel filtration
process. Subsequently, all groups were incubated with 10 nM
[^H]ALD0 and 5 uM RU 26988 minus (B ) or plus (B ) 2 uM
1 No
[^H]ALD0 at 0C for 24 h for the determination of specific binding.
Dilution and gel filtration reduced the cytosolic protein concentration
from 7.3 to 3.6 and 3.3 mg/ml, respectively. The data are from 4
independent replicate experiments and are expressed as a percent of
control* + standard error of the mean (SEM). 100% = 25 fmole/mg
protein.


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UNIVERSITY OF FLORIDA
3 1262 08554 4012


CHARACTERIZATION OF BRAIN TYPE I RECEPTORS
FOR ADRENOCORTICOSTEROID HORMONES
By
SEYED MOHAMMAD EMADIAN
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

Dedicated to my parents and my wife without whose continuous
support this work would not have been possible

ACKNOWLEDGMENTS
The experiments in this dissertation were conducted under the
guidance of my mentor and chairman of my supervisory committee, Dr.
William G. Luttge. I am indebted to Dr. Luttge for being a superb
mentor, teacher, colleague, friend, and for his continuous support
throughout the course of my dissertation work. I further would like to
extend my gratitude to my other supervisory committee members, Drs. A.J.
Dunn, M.J. Fregly, M.B. Heaton and K.T. Shiverick, for their guidance
and critical evaluation of my dissertation. I am also grateful to Dr.
L.C. Garg for his participation in the discussion of this work and his
helpful and valuable contributions during our committee meetings. I
would like to thank Dr. C.L. Densmore and Ms. Y.-C. Chou for their
helpful comments and evaluation of the results of this work during our
laboratory meetings and Ms. Mary E. Rupp and Mrs. Cameron F. Bloom for
technical assistance. Last, but not least, I would like to acknowledge
the financial assistance provided to me by the Department of
Neuroscience and Center for Neurobiological Sciences during the course
of my graduate studies.
iii

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS iii
LIST OF TABLES vi
LIST OF FIGURES vii
ABBREVIATIONS ix
ABSTRACT xii
CHAPTERS
I REVIEW OF THE LITERATURE 1
Control of ALDO Secretion A
Peripheral Site of Action and Effects of
Mineralocorticoids 5
Central Site of Action and Effects of
Mineralocorticoids 11
Molecular Mechanisms of Mineralocorticoid
Action 18
Mechanisms of Receptor Regulation 26
In Vitro Analysis of Type I Receptors
by Radioreceptor Assay 36
II EXPERIMENTAL PROCEDURES 43
Isotopes, Steroids and Other Chemicals 43
Experimental Animals 44
Buffers 44
Cytosol Preparation 45
Aging 45
Hydrophobic Interaction Chromatography 45
Steroid Binding Determination 46
Cytosolic Protein Concentration 47
III EFFECTS OF POLYHYDRIC AND MONOHYDRIC COMPOUNDS ON THE
STABILITY OF TYPE I RECEPTORS FOR ADRENOCORTICOSTEROID
HORMONES IN BRAIN CYTOSOL 48
Summary 48
Introduction 49
Materials and Methods 50
Results 50
Discussion 58

Proposed Mechanism of Action of Poly-
and Monohydric Compounds 58
Possible Mode of Action of Poly- and
Monohydric Compounds on Unoccupied
Type I Receptors in Crude Cytosol 64
IVA NOVEL EFFECT OF MOLYBDATE ON THE BINDING OF
[3H]ALDOSTERONE TO GEL FILTERED TYPE I
RECEPTORS IN BRAIN CYTOSOL 70
Summary 70
Introduction 71
Materials and Methods 72
Results 73
Discussion 88
VDIFFERENTIAL INACTIVATION OF TYPE II RECEPTORS FOR
ADRENAL STEROIDS IN WHOLE BRAIN CYTOSOL:
RECOVERY OF INTACT TYPE I RECEPTORS 93
Summary 93
Introduction 94
Materials and Methods 96
Results and Discussion 97
Effects of DTNB on Unoccupied Type I Receptors .... 97
Effects of DTNB on Occupied Receptors 101
Effects of DCC Pretreatment of Cytosol on
Ligand Binding Capacity of Unoccupied
Type I and Type II Receptors 106
VIIN VITRO TRANSFORMATION OF ALDOSTERONE-TYPE I
RECEPTOR COMPLEXES TO A DNA-BINDING STATE 122
Summary 122
Introduction 123
Materials and Methods 129
Results 130
Discussion 139
VIICONCLUDING REMARKS 142
REFERENCES 145
BIOGRAPHICAL SKETCH 184
v

LIST OF TABLES
Page
Table 2-1: Buffers 44
Table 4-1: Binding Parameters for Type I Receptors Obtained
from Crude and Gel Filtered Cytosol in the
Presence and Absence of 2 mM Molybdate 82
Table 5-1: Effects of 1 mM DTNB on Whole Brain Cytosolic
Unoccupied Type I Receptors 100
Table 5-2: Effects of Two Consecutive DCC Pretreatments
of Cytosol on the Residual DEX Specific
Binding Ill
Table 6-1: Comparison of the Binding of ALDO-Type I and
DEX-Type II Receptor Complexes to
DNA-cellulose Before and After Salt- and/or
Temperature-Induced Activation 132
Table 6-2: Effects of 5 mM Molybdate on the Binding of
ALDO-Type I Receptor Complexes to DNA-
Cellulose Before and After Salt- and
Temperature-Induced Activation 136
vi

LIST OF FIGURES
Page
Figure 3-1: Effects of Polyhydric Compounds on the Stability
of Unoccupied Type I Receptors in Whole Brain
Cytosol 52
Figure 3-2: Effects of 10% Glycerol on the Stability of
Unoccupied Type II Receptors in Whole brain
Cytosol 55
Figure 3-3: Effects of Monohydric Compounds on the Stability
of Unoccupied Type I Receptors in Whole Brain
Cytosol 57
Figure 3-4: Dose-Response Analysis of the Effects of Ethanol
on the Binding of ALDO to Type I Receptors in
Whole Brain Cytosol 60
Figure 3-5: Scatchard Analysis of the Effects of 10% Ethanol
on the Equilibrium Binding Parameters of ALDO
Binding to Type I Receptors in Whole Brain
Cytosol 62
Figure 3-6: Comparison of the Surface Hydrophobic Properties
of ALDO-Type I and DEX-Type II Receptor
Complexes on Pentyl-Agarose Columns 68
Figure 4-1: Effects of Gel Filtration, Molybdate and/or
Dithiothreitol on the Binding Capacity and
Stability of Unoccupied Type I Receptors From
Whole Brain Cytosol 75
Figure 4-2: Dose-Response Analysis of the Effects of Gel
Filtration and Molybdate on the Binding
Capacity and Stability of Unoccupied Type I
Receptors from Whole Brain Cytosol 78
Figure 4-3: Effects of Molybdate and/or Gel Filtration
on the Equilibrium Binding Parameters of ALDO
Binding to Unoccupied Type I Receptors from
Whole Brain Cytosol 81
v i i

Figure 4-4: Stability of Unoccupied Type I Receptors from
Whole Brain Cytosol Following Gel Filtration
in the Absence of Molybdate: Effects of
Molybdate Addition Following Gel Filtration
and Aging 85
Figure 4-5: Effects of Dilution and/or Molybdate on the
Binding Capacity of Unoccupied Type I
Receptors from Whole Brain Cytosol 87
Figure 5-1: Effects of 1 mM DTNB on Receptors Bound to
ALDO in Whole Brain Cytosol 103
Figure 5-2: Effects of DCC Pretreatment and/or Aging of
Unlabeled Whole Brain Cytosol on the Binding
of ALDO to Type I and DEX to Type II Receptors . . 105
Figure 5-3: Effects of Pretreatment of Whole Brain Cytosol
with Various Concentrations of DCC and
Different Duration of DCC Pretreatment on
the Binding of DEX to Type II Receptors 109
Figure 5-4: Effects of DCC Pretreatment of Whole Brain
Cytosol on the Binding of ALDO and DEX to
Type I and Type II Receptors in the Presence
or Absence of a Steroid Competitor 114
Figure 5-5: Effects of 300 mM KC1 in the Presence and
Absence of 20 mM Molybdate on the Binding
of ALDO to Type I and DEX to Type II
Receptors 117
Figure 5-6: Effects of 300 mM KC1 on the Specific Binding
of ALDO and DEX Prior and Subsequent to DCC
Pretreatment 119
Figure 6-1: Comparison of the Surface Hydrophobic Properties
of Unactivated and Activated ALDO-Type I
Receptor Complexes on Pentyl-Agarose
Columns 138
viii

ABBREVIATIONS
All: angiotensin II
Ach: acetylcholine
ACTH: adrenocorticotropic hormone
ADP: adenosine diphosphate
AIPs: aldosterone-induced proteins
ALDO: aldosterone
ANF: atrial natriuretic factor
ASF: aldosterone-stimulating factor
ATPase: adenosine triphosphatase
B.,, : maximal binding
MAX
B : nonspecific binding
NS
B^: total binding
CBG: corticosteriod-binding globulin
CPM: counts per minute
CNS: central nervous system
CORT: corticosterone
CSF: cerebrospinal fluid
DCC: dextran-coated charcoal
DEAE-Cellulsoe: die thylaminoe thyl-Cellulose
DEX: dexamethasone
DNA: deoxyribonucleic acid
DNA-C: deoxyribonucleic acid-cellulose
DOC: deoxycorticosterone
IX

DOCA: deoxycorticosterone acelate
DPM: disintegration per minute
DTNB: 5,5'-dithiobis (2-nitrobenzoic acid)
DTT: dithiothreitol
E: erythritol
EEDQ: N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinolone
EG: ethylene glycol
Et: ethanol
G: glycerol
HEPES: 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid
hn-RNA: heterogeneous nuclear-ribonucleic acid
: equilibrium dissociation constant
MMTS: methyl methanethiosulfonate
MMTV: mouse mammary tumor virus
mRNA: messenger ribonucleic acid
Mt: methanol
NAD+: nicotinamide-adenine dinucleotide
NADPH: nicotinamide-adenine dinucleotide phosphate
NEM: N-ethylmaleimide
NM: nuclear matrix
P: propanol
PMSF: phenylmethyl sulfonylfluoride
R: ribitol
RPM: revolutions per minute
S: sorbitol
SCO: subcommissural organ
SDS-PAGE: sodium dodocylsulfate-polyacrylamide gel electrophoresis
x

TA: triamcinolone acetonide
: melting temperature
X: xy 1 i to 1
xi

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
CHARACTERIZATION OF BRAIN TYPE I RECEPTORS
FOR ADRENOCORTICOSTEROID HORMONES
By
Seyed Mohammad Emadian
May 1987
Chairman: William G. Luttge, Ph.D.
Major Department: Neuroscience
Preponderance of evidence suggests that the potent effects of
adrenocorticosteroid hormones on a number of physiological and
behavioral functions are initiated through stereospecific binding of
these steroids with soluble intracellular receptors, followed by the
interaction of the steroid-receptor complexes with the genome. Despite
the wealth of our knowledge regarding the properties of steroid hormone
receptors in general, practical difficulties such as lack of specific
available ligands, receptor instability under cell-free conditions, low
receptor concentration in whole tissue preparations, etc., have greatly
limited our knowledge of properties and regulation of Type I receptors
for adrenocorticosteroid hormones.
The work presented in this dissertation has overcome some of these
difficulties and describes a number of distinct in vitro properties
of these receptors: 1) Unlike Type II (glucocorticoid) receptors, the
extraction of unoccupied Type I receptors into an aqueous environment
was found to result in a reduction in the binding capacity of these
xii

receptors--possibly by inducing a conformation of the receptors with an
infolded hydrophobic steroid-binding pocket. A reduction in hydrophobic
interactions by including monohydric compounds in cytosol was shown to
result in an increase in the binding capacity of these receptors for the
ligand. 2) Although the stabilizing effects of molybdate on steroid
receptors is well established, this oxyanion was found to produce a
paradoxical effect on the binding capacity of unoccupied Type I
receptors in crude and gel filtered cytosol preparations: a reduction
in receptor binding capacity in crude, and an increase in receptor
binding capacity in gel filtered cytosol was observed in the presence of
molybdate. 3) Whereas unoccupied Type II receptors could be inactivated
by removal of endogenous sulfhydryl reducing agent from cytosol using
dextran-coated charcoal (DCC), Type I receptors appeared insensitive to
the effects of dithiothreitol (a potent sulfhydryl reducing reagent) and
DCC pretreatment of cytosol (a mild oxidizing condition). The
differences between these two receptors were used to inactivate
selectively Type II receptors. 4) Lastly, conditions that induced
transformation (activation) of glucocorticoid-Type II receptors to a
DNA-binding state (i.e., 300 mM KC1 and incubation at 22°C) were found
to produce minimal activation of Type I receptors. Possible functional
and physiological significance of these findings is discussed.
Xlll

CHAPTER I
REVIEW OF THE LITERATURE
The actions of adrenocorticosteroid hormones on a wide variety of
physiological and behavioral functions in mammals are well characterized
(for review see Fregly and Luttge, 1982; Luttge, 1983; Rees and Gray,
1983; De Kloet 1984; Munck et al., 1984; Meyer, 1985; Funder, 1986;
De Kloet et al., 1986; McEwen et al., 1986b; Munck and Guyre, 1986). As
a result of extensive research, significant developments have occurred
in the past three decades with regard to the mechanism of action of
these hormones (for review see Reich and Scott, 1979; Fanestil and
Kipnowski, 1982; Lan et al., 1984; Rossier, et al., 1985; Marver, 1986;
Scheidereit et al., 1986; Slater et al., 1986). Although limited
experimental evidence suggests that the actions of adrenocorticosteroid
hormones may result from direct interactions of these steroids with
components of membrane (nongenomic response) (Chaplin et al., 1981;
Towle and Sze, 1983), the bulk of evidence suggests that steroid
hormones, in general, exert their effect via a stereospecific binding
with soluble intracellular protein receptors. The resulting
steroid-receptor complexes undergo certain physicochemical changes that
increase the overall affinity of the complexes for nuclei, chromatin and
DNA and other polyanions (for detail see Chapter VI). This
"transformation" process (also known as "activation") allows for a
direct interaction of the transformed complexes with nuclear acceptor
site(s) which in turn modulate the transcription of certain mRNAs and
hence cellular protein synthesis. It is important to note that steroid
1

2
hormone receptors are the only regulators of gene expression in the
eukaryotic cells thus far described. Indeed, recent studies have
characterized specific palindromic sequences within the DNA
molecule--hormone response elements--to which the transformed steroid
receptors bind in order to modulate the transcription of certain mRNAs
(e.g., Arnemann et al., 1987; Chambone et al., 1987; Drouin et al.,
1987; Karin et al., 1987; Miesfeld et al., 1987).
Long before isolation and purification of aldosterone (ALDO)
(Grundy et al., 1952; Simpson and Tait, 1952), the most potent naturally
occurring mineralocorticoid hormone, it was known that blood borne
"factors" controlled electrolyte balance within the body (Addison,
1855). Lucas (1926) provided the first evidence for the adrenal
cortical regulation of mineral metabolism by adrenalectomizing dogs.
However, despite these and many other preliminary observations, the
discovery of ALDO production by zona glomerulosa of the adrenal glands
came long after the discovery of the main corticosteroid hormones
produced by the zonae fasciculata and reticularis. The earlier
investigators attributed hyponatremia, hypochloremia, acidosis,
hyperkalemia, and other symptoms associated with Addison's disease to
the lack of deoxycorticosterone (DOC) secretion (Thorn et al., 1942).
Despite the potent mineralocorticoid activity of DOC, this theory was
discredited when extracts from adrenal glands were found to contain only
minute amounts of DOC. The discovery of ALDO came about some years
later as a result of improved quantitative bioassay and chromatographic
techniques (Grundy et al., 1952; Simpson and Tait, 1952; Simpson et al.,
1954; Simpson and Tait, 1955).

3
Because of the potent action of ALDO on mineral metabolism (for
detail see below), the high affinity binders for this steroid extracted
from the kidney were dubbed as "mineralocorticoid receptors." However,
since the endogenous glucocorticoid, corticosterone (CORT), was found to
have comparable affinty for these receptors (see below), a less
ambiguous term, Type I receptors, will be used in this dissertation to
refer to these receptors. This terminology will also be used to
distinguish these receptors from the lower affinity, higher capacity
ALDO binders, the Type II (glucocorticoid) receptors. Although the
principal target tissues for the action of ALDO in mammals have been
considered to be the kidney, intestines and salivary glands, because
electrolyte exchange occurs throughout the organism, other tissues may
also be considered as targets for ALDO. This claim is further supported
by the fact that Type I receptors have been identified in the pituitary
gland (Moguilewsky and Raynaud, 1980; De Nicola et al., 1981), parotid
gland (Funder et al., 1972), mammary gland (Quirk et al., 1983), lung
(Krozowski and Funder, 1981), bovine ciliary body (Starka et al., 1977),
hf -
rabbit lens epithelium (Hampl et al., 1981), human ocular lens (Hampl,
et al., 1984; Starka et al., 1986), whole brain cytosol (Anderson and
Fanestil, 1976; Emadian et al., 1986), various brain regions
(Moguilewsky and Raynaud, 1980; De Nicola et al., 1981; Veldhuis et al.,
1982b; Coirini et al., 1983), spinal cord (Orti et al., 1986), human
mononuclear leukocytes (Armanini et al., 1986), rat epididymis (Hinton
and Keefer, 1985; Pearce et al., 1986a), "arterial walls" (Kornel, 1981;
Kornel et al., 1982a & b) and rat cochlear and vestibular organs (Rarey
and Luttge, 1987).

4
Control of ALDO Secretion
In mammals, several known physiological modulators of ALDO
secretion from the adrenal cortex include the renin-angiotensin II
system, potassium ion and adrenocorticotropic hormone (ACTH)• An
extensive body of research has implicated the renin-angiotensin II
system as the principal regulator of ALDO secretion (e.g., Kaplan, 1965;
Davis, 1975; Fraser et al., 1979). Although ACTH stimulation of ALDO
steroidogenesis via the adenylate cyclase cascade mechanism is well
characterized, ACTH is apparently not involved in the maintenance of
ALDO secretion under basal conditions. Note, for example, that
suppression of ACTH secretion with dexamethasone (DEX), a synthetic
glucocorticoid, and other manipulations (e.g., hypophysectomy) does not
alter the circadian rhythm of ALDO secretion (Fraser et al., 1979).
Furthermore, infusion of ACTH leads to sustained increases in plasma
ALDO concentration, suggesting that ACTH may mediate only acute
increases in ALDO secretion physiologically (Kem et al., 1975).
A number of other factors including aldosterone-stimulating factor
(ASF, secreted by the anterior pituitary) (Carey and Sen, 1986); atrial
natriuretic factor (ANF, a family of peptides isolated from the atria of
rat, human and other species) (Chartier et al., 1984; Takagi et al.,
1986; Aguilera, 1987; Naruse et al., 1987; Sagnella et al., 1987);
acetylcholine, ACh (Kojima et al., 1986); dopamine (Carey and Drake,
1986; for review see Campbell et al., 1981); vasopressin (Woodcock et
al., 1986) and serotonin (Matsuoka et al., 1985) have been recently
implicated in the regulation of ALDO secretion. Large reductions in the
concentration of sodium in serum have also been shown to stimulate ALDO

5
secretion; however, the physiological role of this hyponatremia-induced
ALDO secretion awaits further exploration (Davis, 1975).
Peripheral Site of Action and Effects of Mineralocorticoids
As mentioned above, the main peripheral action of ALDO is to
maintain electrolyte balance in the organism. A secondary action is to
help maintain proper blood volume and pressure. These actions of ALDO
are achieved primarily through reabsorption of sodium and hence water
(due to osmotic activity of sodium) and excretion of potassium and
hydrogen ions by the kidney. In other words, an increase in the
circulating levels of ALDO results in a decrease in the ratio of sodium
to potassium in urine, hence an increase in plasma and extracellular
fluid volume. An ALDO-mediated reduction in the Na/K ratio is also
reported in saliva, sweat and feces.
The antinatriuretic and kaliuretic effects of ALDO on kidney are
well established. Due to a remarkable anatomical complexity and
specialization in this organ (for review see Jacobson, 1981), the exact
sites at which these effects are mediated remained controversial for
some time (Knox et al., 1980). Because ALDO, deoxycorticosterone
acetate (DOCA, an acetate derivative of DOC) and other steroids with
mineralocorticoid activity were found to influence electrolyte balance
without changing renal plasma flow and glomerular filtration rate
(Barger et al., 1958; Ganong and Mulrow, 1958), the distal tubule was
postulated to be the main locus of mineralocorticoid action (Hierholzer
et al., 1965; Wright and Giebisch, 1978). Studies with isolated,
perfused tubules from rabbit kidneys, however, provided evidence
suggesting that mineralocorticoids (administered acutely or chronically)
increase sodium reabsorption and potassium secretion in the cortical

6
collecting tubules and not the distal tubules (O'Neil and Helman, 1977;
Schwartz and Burg, 1978; Stokes et al., 1981). Furthermore,
mineralocorticoids were also found to increase discretely the
transtubular electrical potential (lumen negative) across the cortical
collecting tubules (Gross et al., 1975; Gross and Kokko, 1977), a
finding not confirmed in the study by Schwartz and Burg (1978).
Determination of pH profile along the rat papillary collecting duct by
micropuncture technique suggested the action of ALDO on distal papillary
collecting duct (Higashihara et al., 1984). In addition to the
possibility that ALDO may influence multiple segments of the nephron,
the uncertainty as to the site of action of mineralocorticoids in kidney
and other target tissues may also be due to species, tissue and/or
methodological differences between the aforementioned studies. For
example, in contrast to the results obtained from the rabbit nephron,
ALDO was found to normalize sodium transport in the rat distal tubule
(Hierholzer and Wiederholt, 1976; Horisberger and Diezi, 1983) without
changing the transtubular potential (Hierholzer and Wiederholt, 1976).
Moreover, ALDO action at receptor sites other that Type I receptors
cannot be ruled out. Note that the administration of DEX (a specific
synthetic glucocorticoid) to adrenalectomized rats has been shown
recently to increase Na+/H+ exchange activity in isolated renal
brush border membrane vesicles (Kinsella et al., 1985). Other
investigators, however, reported that glucocorticoids have kaliuretic
and not antinatriuretic effects (e.g., Bia et al., 1982; Campen et al.,
1983; for review see Field and Giebisch; 1985). The use of in situ
microperfusion of single superficial distal tubles in adrenalectomized
rats revealed no direct effect of DEX on potassium secretion by the

7
microperfused tubule; however, an overall increase in urinary flow and
sodium excretion was a common finding in these studies (Field et al.,
1984). In contrast, infusion of ALDO under identical conditions led to
a 90% stimulation of potassium secretion by the microperfused tubule and
a marked reduction in the kidney sodium excretion and urinary flow rate.
No apparent effect on kidney potassium excretion was observed under this
latter condition (Field et al., 1984). It is postulated that mineral
exchange represents the net result of direct and synergistic actions of
the steroids on renal tubules plus secondary effects mediated by changes
in urinary flow rate (Field et al., 1984). Parenthetically, recent
studies on the effects of corticosteroids on Na+ transport in distal
colon suggest a different cellular mechanism for ALDO and DEX (Na+
transport in response to ALDO, but not DEX, was found to be amiloride
sensitive) (Jorkasky et al., 1985). Furthermore, since basal plasma
levels of ALDO (5 ng/dl) in the presence of potassium loading
significantly increased transepithelial potential difference and the
area of the basolateral membrane in the late distal convolution (a
characteristic feature of "potassium adaptation" when
pharmacological doses of ALDO are used in potassium-loaded
adrenalectomized animals), Hirsch et al. (1984) postulated a
differential mechanism of ALDO action on different segments of nephron.
Note that neither the acute nor chronic administration of ALDO in the
absence of potassium loading has any effect on the late distal
convolution, whereas this latter treatment increased surface density of
the basolateral membrane in initial collecting tubule cells (Hirsch et
al., 1984).

8
Autoradiographic and binding assay studies focusing on the regional
distribution of Type I receptors in various segments of renal nephron
have yielded fruitful results regarding the potential site(s) of
mineralocorticoid action along the nephron. Radioreceptor assays of
cytosolic fractions obtained from rat kidney cortical tubules enriched
in proximal or distal segments revealed a higher concentration of
3
[ HjALDO binding in the latter (in the presence or absence of a
glucocorticoid competitor), although the precise location of Type I
receptors along the nephron was not identified (Scholer et al., 1979).
3 — ]_6
In the rabbit nephron, high specific [ H]ALD0 binding ( >10
mol/cm tubule length) was found in the branched, cortical and outer
medullary collecting tubules, whereas specific binding was negligible in
the proximal tubules, pars recta, medullary thick ascending limb,
cortical thick ascending limb and distal convoluted tubules ( <10 ^
mol/cm tubule length) (Doucet and Katz, 1981a, Farman et al., 1982a).
In contrast, using CORT as a ligand, receptor binding was demonstrated
throughout the rat nephron, albeit the greatest concentration was found
in the cortical collecting tubules (Lee et al., 1983). Autoradiographic
studies are consistent with radioreceptor assays, in that, specific ALDO
binding was observed along the distal and cortical collecting tubules
(Vandewalle et al., 1981; Farman et al., 1982b; Farman and Bonvalet,
1983), but not in the glomeruli (Farman et al., 1982c).
Recent research on the endocrine basis of experimental hypertension
suggest a role for a renally-independent (Berecek and Bohr, 1978;
Onoyama et al., 1979) intraarterial mechanism of mineralocorticoid
action (Kornel, 1981; Kornel et al., 1982a & b; for review see Kotchen
and Guthrie, 1980). Chronic administration of DOCA was found to

9
increase the turnover of arterial tissue potassium and chloride ions and
to increase cell membrane "leakiness" to Na+ (Friedman et al., 1975;
Jones and Hart, 1975; Friedman and Friedman, 1976). In agreement with
these results, ALDO was found to increase significantly sodium flux
along the porcine arterial wall (a tissue with morphological resemblance
to human arterial wall) (Llaurado et al., 1983). An increase in
peripheral resistance and vascular reactivity was also reported in the
DOCA-treated animals (Hansen and Bohr, 1975; Berecek and Bohr, 1977 &
1978). Treatment of normal subjects with the synthetic
mineralocorticoid, 9-Ct-fluorocortisol, for several weeks resulted in a
rise in blood pressure in those subjects, an effect which could be
reversed by administration of spirolactone (a mineralocorticoid
antagonist) (Nicholls et al., 1979).
Clinically, steroid-induced hypertension is usually associated with
three types of adrenal disorders. 1) Excess secretion of aldosterone
from an autonomous adrenocortical adenoma in primary hyperaldosteronism
(first described by Conn, 1955). This hypertension can be remedied
usually through surgical procedures and/or by administration of
antimineralocorticoids (e.g., spironolactones) (Lim et al., 1986). 2)
Cushing's syndrome, characterized by an excessive secretion of cortisol
(a glucocorticoid) and DOC, is accompanied by hypertension in about
80-90% of the patients (Gomez-Sanchez, 1982). 3) In patients with
11-3-dehydrogenase and 5ot-reductase ("apparent mineralocorticoid
excess," a juvenile disease characterized by low or undetectable levels
of renin and low plasma ALDO, accompanied by hypokalemia, Monder et al.,
1986) and 17a-hydroxylase deficiency (where there is an increase in
ACTH-induced ALDO production) there is a high incidence of hypertension.

10
The exact mechanism by which mineralocorticoids induce hypertension
awaits further elucidation. However, since mineralocorticoids can
influence electrolyte distribution within the arterial walls by
increasing sodium transport and since "specific" mineralocorticoid
receptors along the arterial, but not the venous, walls are known to
exist (Kornel, 1981; Kornel et al., 1982a & b; Moura and Worcel, 1984),
there is strong support for an intraarterial, mineralocorticoid-induced,
receptor-mediated basis for this form of experimental hypertension.
Studies investigating the effects of ALDO, CORT and DOCA on hypertension
in rats suggests that a common mediator of steroid-induced hypertension
may be an increase in extracellular fluid and plasma volume (Haack et
al., 1977; Ishii et al., 1985). Although aldosterone has been
implicated in the pathogenesis of essential hypertension in experimental
animal models (e.g., Nowaczynski et al., 1983), the role of
mineralocorticoids in essential hypertension is still a matter of
controversy (for review see Fraser et al., 1981; Gomez-Sanchez, 1982).
Recently, a central nervous system mediated mechanism of ALDO
induced-hypertension has been suggested (Gomez-Sanchez, 1986) (for
detail see below).
Some other peripheral effects of mineralocorticoid hormones include
a redistribution of potasium within the body in addition to the
kaliuretic effect discussed above (Young, 1979; Young and Jackson,
1982); a reduction in the concentration of zinc in plasma (Latman et
al., 1984); an increase in renal cortical and medullary citrate synthase
activity (Kinne and Kirsten, 1968) and synthesis (Law and Edelman,
1978b) , a phenomenon independent of sodium transport (at least in
cultured cells) (Johnson and Green, 1981); an induction of renal

11
flavokinase enzymic activity (Trachewsky et al., 1985); an increase in
thymidine kinase synthesis in immature rat kidney (Bukhari et al.,
1985); an increase in guanylate cyclase activity in liver, kidney,
heart, lung, spleen and ilium (Vesely, 1980); an increase in isocitrate
dehydrogenase and glutamate-oxaloacetate transaminase activity in rat
kidney and toad urinary bladder (Kinne and Kirsten, 1968; Kirsten et
al., 1968; Kirsten et al., 1970; Kirsten and Kirsten, 1972); an
activation of intestinal mucosal carbonic anhydrase in adrenalectomized
rats (Scott and Sapirstein, 1975; Sapirstein and Scott, 1975; Voute and
Meier, 1978; Suzuki, 1981; Suzuki et al., 1983; Suzuki and Ozaki, 1984);
an increase in Na-K-ATPase activity along the nephron (Katz et al.,
1979; Garg et al., 1981; Le Hir et al., 1982; O'Neil and Dubinsky, 1984;
2+
for detail see below); an increase in intestinal Mg -HCO^ -
ATPase activity (Suzuki, 1981; Suzuki et al., 1983); a diminution of
bile flow and liver uptake and a decrease in biliary excretion of sodium
and water (Afifi et al., 1979; Afifi, 1981); an increase in red blood
cell sodium uptake (Rettori et al., 1969; Afifi et al., 1979; Stern et
al., 1983) and a non-genomically-induced increase in membrane
phospholipid polyunsaturated fatty acid content (Goodman, 1981).
Central Site of Action and Effects of Mineralocorticoids
Although specific macromolecular binding to ALDO in whole brain
cytosol was first described by Anderson and Fanestil (1976), prior to
and since this discovery investigators have implicated the involvement
of mineralocorticoids in the central nervous system (CNS) electrolyte
balance (Woodbury, 1958); neuronal electrical activities (Kraulis et
al., 1975); feedback mechanisms involving ALDO secretion (Dundore et
al., 1984; Birmingham et al., 1974; Grizzle and Dunlap, 1984);

12
inhibition of CORT-induced serotonin turnover in dorsal hippocampal and
3
raphe areas (by inhibiting [ H]CORT nuclear uptake) (De Kloet et
al., 1983); inhibition of brain growth (Devenport, 1979); influences on
glial-cell function (Beaumont, 1985) and inhibition of CORT-induced
exploratory behavior (Veldhuis et al., 1982a). In addition,
mineralocorticoids have been shown to augment passive avoidance
responses in adrenalectomized and adrenally intact "pre-stressed" rats,
an effect not observed with CORT administration (Weiss and Gray, 1973).
Although mineralocorticoid-induced hypertension by actions at renal
levels is well documented (see above), early reports implicated a role
for mineralocorticoid action on brain regulation of experimental
hypertension in rats (e.g., Finch et al., 1972; Zamir et al., 1979).
Recently, it was shown that minute doses of ALDO administered
intracerebroventricularly produce a significant rise in blood pressure
(Gomez-Sanchez, 1986). Infusion of ALDO in the presence of an equimolar
concentration of prorenone (a spironolactone with antimineralocorticoid
activity) antagonized the pressor response to ALDO. Studies of this
nature provide strong circumstantial evidence for hyper tensinogenic
effects of mineralocorticoids directly within the CNS; however, the
exact site(s) of action of mineralocorticoids on the CNS regulation of
hypertension remains to be elucidated.
Studies investigating the endocrine basis of salt appetite
implicate a central role for mineralocorticoid action (Fregly and
Waters, 1966; Wolf and Handal, 1966; Weisinger and Woods, 1971; McEwen
et al., 1986b). The classical work by Richter (1936) showed that
adrenalectomy was accompanied by sodium appetite in rats and that
treatment of adrenalec tomized rats with DOCA reduced sodium intake to

13
control levels (Richter, 1956). Similarly, a recent report by McEwen et
al. (1986b) showed that the administration of ALDO supressed salt intake
in adrenalectomized rats as measured in a two-bottle preference test
paradigm (findings that replicate the earlier report by Fregly and
Waters, 1966). The supression of salt appetite in response to ALDO
could be inhibited by co-administration of a molar excess of RU 28318 (a
specific synthetic antimineralocorticoid). It is noteworthy that this
effect of ALDO could not be mimicked by CORT, a proposed physiological
ligand for brain Type I receptors (for review see Funder, 1986) (for
more detail see below). In fact, only concentrations of CORT 500-fold
in excess of ALDO were able to inhibit the action of ALDO (McEwen et al.
1986b) .
In sheep deprived of sodium for 22 h, infusion of 500 mM Na+
via a lateral ventricular approach led to a marked reduction in salt
intake compared to animals that received 150 mM Na+ (Weisinger et
al., 1979). It is postulated that the modulation of Na+
concentration in cerebrospinal fluid (CSF) may translate into changes in
the concentration of Na+ in hypothalamic neurons subserving salt
appetite (Weisinger et al., 1979). Similarly, an earlier investigation
by Richter (1956) showed that low concentrations of sodium in plasma
stimulate salt appetite. The author (Ritcher, 1956) implicated (but did
not show) possible involment of hypothalamic neurons. Other CNS
structures have also been implicated in the regulation of salt intake;
for example, electrolytic lesions centered in the thalamic gustatory
subnucleus of the ventral posterior complex were shown to impair sodium
intake in adrenalectomized rats (Wolf and Dicara, 1974). It is
noteworthy that the impairment in salt appetite following such lesions

14
may be unrelated to the gustatory system since neocortical ablations
which resulted in extensive retrograde cell degeneration in this
diencephalic structure had no effect on salt appetite (Wolf et al.f
1970). Becuase other en passage fibers are damaged by the thalamic
lesion (e.g., ascending reticular pathways to the intralaminar nuclei,
ascending noradrenergic pathways to the hypothalamus and forebrain,
etc.), the involvement of other structures could not be ruled out.
Although a specific center(s) in the CNS through which
mineralocorticoids may mediate salt appetite and/or other functional
responses is (are) yet to be described, it is suggested that the actual
mechanism of salt appetite induction may be complex and involve a
synergistic association of a number of factors (Fregly and Rowland,
1985).
Since studies of the regional distribution of specific ALDO
receptors within the CNS have revealed distinct localization in certain
limbic structures such as the hippocampus, hypothalamus, indusium
griseum and amygdala, these brain regions have been implicated as
possible candidates for the central site of action of ALDO (Ermisch and
Ruhle, 1978; Moguilewsky and Raynaud, 1980; De Nicola et al., 1981;
Veldhuis et al., 1982b; Coirini et al., 1983; Birmingham et al., 1984;
Reul and De Kloet, 1985 & 1986). However, co-administration of "stress
levels" of CORT with ALDO was found to suppress preferentially ALDO
uptake by hippocampal formation, amygdala and septum, having little
effect on ALDO suppression of salt appetite or ALDO uptake in
circumventricular organs (McEwen et al., 1986b). Furthermore, bilateral
3
hippocampectomy, which depleted specific [ H]ALD0 binding in the
residual structure by 80%, had no effect on the development of salt

15
appetite following adrenalectomy in these hippocampectomized rats (Kim,
1960; Magarinos et al., 1986). The increase in salt intake in
adrenalectomized-hippocampectomized animals was found to be suppressible
by administration of ALDO (Magarinos et al., 1986). These studies thus
suggest, at least in rats, that the hippocampus may not be the target
organ for the action of ALDO on salt appetite as elicited by
adrenalectomy.
McEwen et al. (1986b) have concluded that one or more of the
circumventricular organs, the dentate gyrus, Ammon's horn and/or
subiculum may be the most plausible candidates for the action of ALDO on
3
salt appetite since these structures show significant [ H]ALD0
uptake even in the presence of high circulating levels of CORT. This
notion is consistent with the theory that angiotensin II and ALDO act
synergistically to regulate salt intake (Epstein, 1982; Fluharty and
Epstein, 1983; Strieker, 1983; Sakai et al., 1986), and that some of
these circumventricular organs appear to contain angiotensin II
receptors that can be modulated by exposure to ALDO (Wilson et al.,
1986). Recently, it has been proposed that ALDO and angiotensin II
(All) may mediate thirst and salt appetite by influencing the activities
of septal neurons (Strieker, 1984). Lesions of the septal area in
hypovolemic rats were found to produce sensitization to the sodium
appetite-eliciting effects of All; however, this was only seen in the
presence of ALDO (Strieker, 1984). Although these studies reflect a
synergism between the two hormonal systems in inducing electrolyte and
water balance, it must be kept in mind that, in reality, water and
electrolyte homeostasis may involve complicated interactions of a number
of other systems (for review see Fregly and Rowland, 1985).

16
Furthermore, limited experimental evidence comparing adrenalectomized
mice, hamsters and rats suggests clear differences in the control of
salt appetite in these different species.
A developing area of research regarding mineralocortico id action on
the CNS is concerned with the role of a poorly understood CNS structure,
the subcommisural organ (SCO). The intraperitoneal or subcutaneous
administration of extracts from SCO was found to induce antinatriuresis
in dogs (Gilbert, 1963). Palkovits and Foldvari (1963) reported an
increased nuclear volume in the cells of the adrenal glomerulosa
indicating increased cellular activity following intraperitoneal
injection of SCO extracts. In contrast, lesions of the SCO decreased
nuclear volume as well as the width of the zona glomerulosa in rats
(Palkovits et al., 1965). An in vitro examination of the zona
glomerulosa cells from rats with SCO lesion revealed a marked reduction
in the rate of ALDO secretion (Palkovits et al., 1965). Furthermore,
mineralocorticoids reduced, and adrenalectomy or chronic dehydration
increased cellular activity in the SCO (Palkovits, 1968). Together,
these observations have led investigators to associate SCO with salt and
water homeostasis (for review see Ziegels, 1976).
Recently, Dundore et al. (1984) found that when ALDO was
administered in the "general vicinity" of the SCO (note that the
injections were performed via a cannula placed in the pineal recess
above the rostral two-thirds of the SCO), it produced a natriuresis (an
action opposite to those of blood-borne ALDO discussed above) and a
"site-specific" (related to cannula placement) reduction in the
cross-sectional area of the adrenal medulla without affecting cell
density. It is proposed, but not conclusively shown, that ALDO acts

17
upon SCO cells (and possibly other structures and cells in the vicinity
of SCO due to diffusion) to inhibit the trophic effects of SCO on
adrenal glomerulosa cells. This, in turn, would result in an increased
urinary Na/K ratio (Dundore et al., 1984). Although an attractive
hypothesis, this study fails to show a change in plasma concentration of
adrenocorticosteroids following intracerebral administration of ALDO.
Furthermore, intracerebral administration of ALDO was found to have no
significant effect on the cortical (steroid producing) cells of the
adrenal gland when examained histologically (Dundore et al., 1984). In
light of the above discrepancies and the fact that Crow (1967), who made
discrete lesions encompassing all or parts of the SCO observed no
changes in urine volume and concentration, it is apparent that the exact
role of the SCO in electrolyte metabolism awaits further experiments.
It is conceivable that research investigating 1) the direct effects of
SCO lesions on plasma levels of corticosteroids, 2) the localization
within the SCO of specific mineralocorticoid receptors by
autoradiographic and radioreceptor assay techniques and 3) the isolation
and purification of SCO "factor(s)" responsible for the aforementioned
effects is necessary to establish the physiological role of the SCO, and
its secretory factors in salt and water homeostasis.
Some other centrally mediated actions of mineralocorticoid hormones
include the regulation of food intake and maintenace of body weight,
(Devenport and Devenport, 1983; Devenport et al., 1983; Kenyon et al.,
1984); modulation of hypothalamic, thalamic and septal angiotensin II
receptor binding capacity (Wilson et al., 1986) and increase in cerebral
guanylate cyclase activity (Vesely, 1980).

18
Molecular Mechanisms of Mineralocorticoid Action
Recent in vitro radioreceptor assay and ligand competition
techniques suggest that in cytosol lacking extravascular CBG, Type I
receptors have an equal affinity for ALDO and CORT (for review see
Funder, 1986). It is further suggested that some actions of CORT on the
CNS may be mediated through Type I receptors (e.g., De Kloet et al.,
1986; Magarinos et al., 1986). However, because in this dissertation
the emphasis is placed on the role of Type I receptors in electrolyte
homeostasis, this section will focus primarily on our current
understanding of the molecular mechanisms of action of ALDO and other
physiological mineralocorticoids.
Research on the mechanism of mineralocorticoid action has drawn
tremendous benefit from studies carried out using isolated nephrons, the
A6 cell line derived from the Xenopus kidney epithelial cells and
the sodium transporting amphibian epithelia (toad urinary bladder and
frog skin). These biological preparations can be maintained viable for
several hours at room temperature in incubation media of simple
composition. The simple morphological properties of these models render
them ideal in the study of the mechanism(s) of mineralocorticoid action;
however, it is noteworthy that although aldosterone is the naturally
occuring mineralocorticoid produced by the interrenal gland in anurans
(Carstensen et al., 1961; Crabbe, 1961), the "mineralocorticoid
response," at least in toad bladder appears to be mediated through
interactions with Type I as well as other glucocorticoid specific
receptors (Geering et al., 1985). Even though the authors dubbed these
other receptors as "type 2" glucocorticoid receptors, the possibility

19
they may be Type IB (for more detail see below) and thus distinct from
classical glucocorticoid (Type II) receptors should also be considered.
In the early 1960's, Williamson (1963) and Edelman and co-workers
(1963) independently proposed the classic two-step genomic mode of ALDO
action. The first step was the binding to specific soluble
intracellular receptors, following which, as a second step, the
steroid-receptor complexes would translocate into the nucleus where they
would interact with the cellular genomic machinery (for a discussion of
the nature of the nuclear acceptor site see Chapter VI). It is this
latter step that was proposed to result in the modulation of synthesis
of a number of structural and regulatory proteins. Several lines of
evidence appeared later in favor of this proposal. For instance, Sharp
and colleagues (1966b) were the first to report saturable ALDO binding
sites (receptors) in the toad bladder. Marver et al. (1974) later
reported that the mineralocorticoid antagonist, spirolactone (SC-26304),
when complexed with these receptors, had little or no affinity for
nuclear acceptor sites in vivo and in vitro. More recently, the
response to ALDO in the rat kidney was shown to depend clearly on the
ontogeny of Type I receptors in this target organ (Stephenson et al.,
1984a). Finally, it was possible to correlate the magnitude of
epithelial sodium transport with the binding of ALDO-receptor complexes
to chromatin and the increases in the synthesis of a specific class of
nonmethylated mRNAs and of proteins (Rossier et al., 1974). In this
regard, actinomycin-D (a DNA intercalator which binds specifically to
G--C base pairs and protrudes into the major grooves) was shown to block
ALDO-stimulated sodium transport by inhibiting new mRNA and protein
synthesis (Lahav et al., 1973; Rossier et al., 1974; Horisberger and

20
Diezi, 1984). Subsequent to these observations a number of studies
replicated the results described (for review see Edelman and Marver,
1980; Fanestil and Park, 1981; Rossier et al., 1985; Garty, 1986;
Marver, 1986) thus further confirming the original model proposed by
Edelman et al. (1963) and Williamson (1963).
It is now well established that mineralocorticoids, in general, are
capable of inducing the synthesis of a group of proteins in target
tissues generally referred to as aldosterone-induced proteins (AIPs).
In the rat kidney and toad urinary bladder, proteins with a variety of
molecular weights have been reported to exhibit increased incorporation
of radiolabeled precursor amino acids as a result of exposure to
aldosterone (Law and Edelman, 1978a & b; Geheb et al., 1981; Reich et
al., 1981; Yang et al; 1981; Blazer-Yost et al., 1982; Geering, et al.
1982; Geheb et al., 1983; Geheb et al., 1984). However, the mechanism
by which AIPs participate in the mineralocorticoid-induced physiological
and functional consequences such as sodium transport still remains a
matter of debate. In fact, only recently was it shown that some AIPs
can be immunoprecipitated by polyclonal antibodies specific for the a-
and B-subunits of Na-K-ATPase (Geering et al., 1982).
Current thinking on the role of AIPs, as related to the action of
mineralocorticoids, points to three distinct, but not necessarily
independent, theories: 1) the sodium pump theory, which argues for
increased sodium pump activity as a result of either activation of
pre-existing pumps or an increase in the synthesis of new pumps; 2) the
sodium permease theory, which holds that AIPs are involved in the
facilitation of passive sodium entry across the plasma membrane and 3)
the energy theory, which associates AIPs with the augmentation of

21
cellular energy, presumably by enhanced mitochondrial oxidative
phosphorylation. Although each of these theories has some experimental
support, thus far none has gained universal acceptance. It is important
to emphasize that the involvement of AIPs may not be limited to only one
of these mechanisms exclusively; rather the effects of
mineralocorticoids may reflect the contribution of AIPs to some
combination of these three general mechanisms. In this regard, Reich
and Scott (1979) proposed a unifying hypothesis that includes all three
of the above theories (for recent reviews see Rossier et al., 1985;
Garty, 1986; Marver, 1986).
Considerable controversy still surrounds the pump theory. In the
past decade, a large number of laboratories has investigated the
mineralocorticoid induction of Na-K-ATPase activity (e.g., Aperia et
al., 1981; Doucet and Katz, 1981b; Garg et al., 1981; Petty et al.,
1981; Geering et al., 1982; Osore and Gilbert, 1982; Cortas et al.,
1983; El Mernissi and Doucet, 1983; Stern et al., 1983; Aperia and
Larsson, 1984; El Mernissi and Doucet, 1984; O'Neil and Dubinsky, 1984;
Park and Edelman, 1984a & b; Geering et al., 1985; O'Neil and Hayhurst,
1985; Girardet et al., 1986; Johnson et al., 1986; Palmer and Speez,
1986). As a result of this intensive investigation, it is now well
established that following adrenalectomy there is a significant
reduction in Na-K-ATPase activity in nephrons; however, some
investigators find complete restoration of the enzymatic activity
following administration of ALDO (Petty et al., 1981, El Mernissi and
Doucet, 1983; El Mernissi and Doucet, 1984), whereas others find little
or no effect of ALDO on the restoration of enzyme activities in the
target tissues investigated (Doucet and Katz, 1981b).

22
In addition to this discrepancy in results, the studies cited above
also fail to prove that sodium pump activity plays a significant role in
the action of mineralocorticoids on transepithelial or nephron sodium
transport. Indeed, it is suggested that the observed increase in
Na-K-ATPase activity, at least in some target tissues, may be due to
interaction of mineralocorticoids with Type II receptors, since
long-term pharmacological doses of steroids are often required to
restore Na-K-ATPase activity to control levels. Recently, a number of
investigators (Rayson and Lowther, 1984; El Mernissi and Doucet, 1984;
Garg et al., 1985) reported that they were able to restore the
adrenalectomy-induced reduction in Na-K-ATPase activity in various
segments of rabbit nephron through DEX administration. Even though
similar results were reported by earlier investigators (Fisher et al.,
1975), the small doses of DEX used in the former reports (Rayson and
Lowther, 1984; El Menissi and Doucet, 1984; Garg et al., 1985), provide
stronger evidence that the stimulation of the Na-K-ATPase was probably
through Type II receptors. Note that although the radioreceptor assay
technique suggests that DEX binding to Type I receptors may be
negligible, the pharmacological doses of DEX administered in some of
these studies may allow sufficient interaction of this glucocorticoid
with these receptors to mediate mineralocorticoid action.
In contrast to this suggestion, El Mernissi and Doucet (1984) have
shown that ALDO administration increases the synthesis of new
Na-K-ATPase in the collecting tubules, whereas DEX stimulates the
activity of the pre-exisiting pumps in the thick ascending limb and
distal convoluted tubules. Thus, these results suggest that ALDO and
DEX may act through differential mechanisms at different portions of the

23
nephron. A differential action of ALDO in the rat proximal and distal
colon has also been described: ALDO response in distal, but not
proximal, colon could be inhibited by amiloride (an acylguanidine
diuretic that inhibits influx of Na+ through the apical membrane)
(Hirsch et al., 1985). In contrast, in vitro studies evaluating
short-circuit current in colon from adrenalectomized rats suggest that
although ALDO may be the physiological regulator of Na+ transport,
glucocorticoids may also mediate amiloride-sensitive sodium transport
(Will et al., 1985). Geering et al. (1985) maintain that, at least in
toad urinary bladder, ALDO binding to both Type I and Type II receptors
is required for full mineralocorticoid response.
It is also conceivable that DEX actions on Na-K-ATPase activity may
be mediated through a population of receptors distinct from Type I and
Type II receptors called Type IB. The presence of these receptors in a
number of tissues including the rat liver (Litwack and Rosenfield,
1975), colon (Bastí et al., 1984) and kidney (Litwack and Rosenfield,
1975; Markovic et al., 1980; Mayer et al., 1983) has been confirmed (for
review see Mayer and Litwack, 1983). These receptors have also been
isolated from mouse lung cytosol (Goldman and Katsumata, 1986) and were
implicated to mediate the known anti-inf lamatory and teratogenic actions
of DEX and phenytoin (Katsumata et al., 1985). Some investigators,
however, have questioned the polymorphic nature of these receptors and
have argued that these atypical receptors may represent a proteolytic
fragment(s) generated from Type II receptors under in vitro
conditions (e.g., Sherman et al., 1983; Reichman et al., 1984). More
recently, Eisen et al., (1986) showed that kidney cytosol prepared in
the absence, but not the presence, of molybdate (for detail of possible

24
mechanism of action of this oxyanion see Chapter IV) showed specific
glucocorticoid binders characteristics of Type IB receptors (i.e., the
receptors did not react with a monoclonal antibody raised against Type
II receptors and they were not retained by DEAE-cellulose columns). In
contrast, Goldman and Katsumata (1986) have reported that Type IB
receptors could be detected in lung cytosol only when cytosol was
prepared in hypotonic buffers containing molybdate. Although the
questions as to whether Type IB receptors exist in vivo and whether
they have physiological significance remain to be established, it is
entirely possible that these receptors may be generated from Type II
receptors by specific proteases in target tissues that appear to contain
these receptors.
Experiments with adrenalectomized animals treated with amiloride
prior to injection of ALDO suggest that the increase in Na-K-ATPase
activity may be a secondary adaptation to sodium transport induced by
this steroid (Doucet and Katz, 1981b; Petty et al., 1981). Furthermore,
at least in toad urinary bladder, it appears that the stimulatory action
of ALDO on sodium transport is not mediated by the synthesis of new
(Geering et al., 1982, Park and Edelman, 1984b) or activation of the
pre-existing sodium pumps (Park and Edelman, 1984b).
The idea that AIPs may be a component of the plasma membrane (on
the luminal, urine, surface) of the cell, where they act as a sodium
permease to lower the resistance to Na+ entry into the cell, was
proposed originally by Crabbe and de Weer (1965) and Sharp et al.
(1966a) and subsequently gained experimental support through work in the
toad urinary bladder (Civan and Hoffman, 1971; Spooner and Edelman,
1975; Palmer et al., 1982; Park and Edelman, 1984a). All of these

25
studies, however, fail to demonstrate whether AIPs act as permeases
themselves, or whether they modulate the conformation or arrangement of
pre-existing permease molecules in the membrane such that they are more
accessible for Na+ entry. A recent study by Kipnowski et al. (1983)
found that ALDO fails to increase sodium transport in tissues where
transport was inhibited by N-ethoxycarbonyl-2-ethoxy-l,2-dihydro-
quinoline (EEDQ) prior to exposure to this steroid. In contrast to
results with cycloheximide (an inhibitor of protein synthesis), EEDQ did
alter ALDO stimulation of the osmotic water flow in response to
antidiuretic hormone. These data do not therefore support the notion
that AIPs are new sodium channels, but instead they are consistent with
the hormonal activation of pre-existing, nonfunctional channels.
The first evidence that mineralocorticoids may induce the synthesis
of a mitochondrial enzyme participating in tricarboxylic acid cycle was
obtained by Kirsten et al. (1968) who found that ALDO increased the
citrate synthase, isocitrate dehydrogenase, glutamate dehydrogenase,
glutamate-oxaloacetyl transaminase and malate dehydrogenase activity in
the toad bladder. The increase in the synthesis of mitochondrial
enzymes was postulated to reflect hormone-contro 1led mudulation of the
available ratio of ATP/ADP which, in turn, could alter both Na+
conductance and active transport. ALDO has also been shown to increase
the ratio of NADH/NAD+ in adrenalectomized rat kidneys (Kirsten and
Kirsten, 1972). Similarly, Law and Edelman (1978b) found an increase in
renal cortical and medullary citrate synthase activity three hours after
administration of ALDO to adrenalectomized rats. In this latter study
ALDO was found to enhance incorporation of radiolabeled methionine into
renal, but not hepatic, citrate synthase. The relevance of these

26
observations with reference to the energy theory of mineralocorticoid
action lies in the fact that actinomycin D and specific spironolactones
inhibit the ALDO-induced increase in mitochondrial enzymatic activities,
and that DEX has no effect on the hormonal induction (Law and Edelman,
1978b). Furthermore, this action of ALDO appears to be target specific:
ALDO had no detectable effect on the citrate synthase activity in the
renal papilla or the liver under the same conditions (Law and Edelman,
1978b).
In summary, although much has been done to expand our knowledge of
the molecular mechanisms of mineralocorticoid action, the exact
mechanism by which these hormones exert their effects still awaits
further exploration. It is, nevertheless, clear that the functional
consequences of mineralocorticoid action involves, at least in part, the
genetic machinery in target cells and an ultimate modulation of the
synthesis and/or activation of the pre-existing structural and
regulatory proteins.
Mechanisms of Receptor Regulation
There is considerable evidence to suggest that the in vivo
binding capacity of adrenocorticosteroid hormone receptors is subject to
change in response to a variety of intra- and extracellular signals
(McEwen, 1979; Muldoon, 1980; Svec, 1985b; De Kloet et al., 1986; McEwen
et al., 1986a). For example, hyperkalemic adrenalectomized rats were
shown to have an elevated renal cytosolic Type I receptor binding
capacity (Rafestin-Oblin et al., 1984). Similarly, adrenalectomy
(adrenocorticosteroid deprivation) alone was found to result in a
time-dependent increase in the maximal binding capacity of both Type I
and Type II receptors in all tissues investigated (Beato et al., 1974;

27
McEwen et al., 1974; Gregory et al., 1976; Ichii, 1981; Claire et al.,
1981; Muramatsu et al., 1983, Rafestin-Oblin et al., 1984; Turner, 1986;
Sarrieau, et al., 1986; Luttge and Rupp, unpublished). It is noteworthy
that adrenalectomy was found to induce a differential tissue-specific
up-regulation in Type I and Type II receptor binding capacity in mice.
In the three tissues investigated (i.e.; brain, liver and kidney),
adrenalectomy was found to induce the greatest up-regulation in the
brain cytosolic Type I receptors — a 10- to 14-fold rise compared to
the adrenally intact control group as measured by saturation and
equilibrium binding analyses (Luttge and Rupp, unpublished).
Adrenalectomy also induced a similar tissue-specific up-regulation in
Type II receptors: The largest increase was found in the kidney
cytosol, followed by amygdala-entorhinal cortex, hippocampus, liver,
cerebral cortex, hypothalamus, pituitary and heart (Turner, 1986).
In Scatchard plots (Scatchard, 1949), an increase in the B, ,
MAX
as a mode of "up-regulation" of Type I and Type II receptors induced by
adrenalectomy is a common finding (Beato et al., 1974; McEwen et al.,
1974; Gregory et al., 1976; Claire et al., 1981; Ichii, 1981; Tornello
et al., 1982; Muramatsu et al., 1983; Rafestin-Oblin et al., 1984;
Sapolsky et al., 1984; Luttge and Rupp, unpublished). This increase in
the has been postulated to reflect an unmasking of receptors
previously occupied by endogenous ligands (Feldman, 1974; McEwen et al.,
1974; Giannopoulos, 1975), "activation" of an already available pool of
receptors to a ligand-binding state, an increase in the rate of receptor
synthesis (McEwen et al., 1974; Claire et al., 1981) and/or a reduction
in the rate of receptor degradation (Claire et al., 1981).

28
Treatment of adrenalectomized animals with CORT, ALDO and other
steroid agonists that show appreciable affinity for Type I and Type II
receptor binding sites results in a reduction in the ligand binding
capacity of these receptors (Grekin and Sider, 1981; Ichii, 1981;
Tornello et al., 1982; Muramatsu et al., 1983; Rafestin-Oblin et al.,
1984; Sarrieau et al., 1986; Luttge and Rupp, unpublished). Repeated
stress (a condition that induces the production of high circulating
levels of CORT) was also found to result in a significant reduction in
Type II receptor binding capacity in the rat hippocampus (Valeri et al.,
1978; Sapolsky et al., 1983; Sapolsky et al., 1984; Sapolsky, 1985;
Sapolsky et al., 1985b) and amygdala (Sapolsky et al., 1983; Sapolsky et
al., 1984). In addition to regulation of their own receptors, chronic
administration of stress level concentrations of corticosteroids was
found to have neurotoxic effects on hippocampal neurons (Sapolsky, 1985;
Sapolsky et al., 1985b). Similarly, in cultured cell lines (HeLa S3,
AtT-20 and GH^ cells), glucocorticoids were shown to "down-regulate"
their own receptors (Cidlowski and Cidlowski, 1981; Svec and Rudis,
1981; Raaka and Samuels, 1983; Svec, 1985a). This down-regulatory
effect of ligand on its own receptor has been reported for a number of
other receptors; e.g., progesterone receptors (Milgrom et al., 1973b;
Walters and Clark, 1979), thyroid hormone receptors (Raaka and Samuels,
1981), insulin receptors (Gavin et al., 1974; Huang and Cuatrecasas,
1975; Olefsky, 1976) and other peptide receptors (Catt et al., 1979;
Roth and Taylor, 1982).
In GH^ cells, incubation with triamcinolone acetonide (TA) was
found by McIntyre and Samuels (1985) to reduce the half-life of Type II
receptors by more than 50% (from 19.0 to 9.0 h) as determined through

29
the use of dense amino acid labeling of the receptors. Consistent with
this finding, in the presence of TA, the levels of Type II receptors in
these cells were half that measured in the control cells (i.e., about
130 vs. 260 fmole/100 ug DNA, respectively). The addition of TA to the
culture media had little effect on the rate of de novo receptor
synthesis. It was shown that TA promoted the activation of Type II
receptors (transformation to a state with increased affinity for DNA,
see Chapter VI), and that the activated form was degraded at a faster
rate (than the unactivated form) thus accounting for the reduction in
the receptor half-life (McIntyre and Samuels, 1985). This apparent
faster rate of degradation may represent the inability of the activated
receptors to rebind ligand, since recent work from our laboratory has
shown that following heat-induced transformation to a DNA-binding form
the activated receptors do not rebind ligand after dissociation (Chou
and Luttge, 1987). Recently, treatment of rat hepatoma culture cells
with DEX was shown to result in an initial increase (after 6 h),
followed by a reversible (after 72 h) 50% to 95% decrease in Type II
receptor mRNA (after 24 h) in these cells (Okret et al., 1986). In this
latter study, using an immunoprecipitation assay, Type II receptors were
shown to interact specifically with a Type II receptor cDNA clone, hence
providing a more direct evidence for the ligand-induced receptor
autoregulation (Okret et al., 1986).
The up- and down-regulation of the binding capacity of receptors
for adrenal steroids in the central nervous system raises the
possibility that receptor autoregulation, and hence modulation of brain
sensitivity to steroids, may ultimately be involved in such functional
consequences as the feed-back control mechanisms of adrenal steroid

30
secretion (Cake and Litwack, 1975; Sapolsky et al., 1985a; for review
see Sapolsky et al., 1986).
A second mode of receptor regulation, at least in
adrenocorticosteroid Type II and progesterone receptor systems, may
involve a rapid turning "on" and "off" of the unoccupied receptors in
response to different stimuli. This rapid and reversible loss of
steroid binding capacity has been shown to involve a phosphorylation-
dephosphorylation cycle of the receptors in vitro and possibly
in vivo (for review see Housley et al., 1984). In an earlier
report, Munck and co-workers (1972) proposed that Type II receptors in
thymocytes exist in two states; the steroid-binding form and those that
did not bind steroids. It was proposed that ATP was required to convert
the non-binding receptors to a state that bound steroid (Munck et al.,
1972). Consistent with this finding, receptor stability was reported to
be enhanced by including ATP and other nucleotides (a requirement for
the action of some phosphorylase enzymes) in cytosol preparations
(Nielsen et al., 1977b; Sando et al., 1979a & b). Our laboratory has
recently shown that exogenous ATP can also prevent the destabilizing
actions of as yet unknown microsomal components on Type II receptors
(Densmore and Luttge, 1985). The use of phosphatase inhibitors (e.g.,
fluoride and molybdate) in cytosol preparations has been shown to
improve considerably the stability of Type II receptors (Nielsen et al.,
1977b) . Conversely, the addition of highly purified calf intestine
alkaline phosphatase to cytosol preparations markedly destabilized these
receptors (Nielsen et al., 1977a). A striking, positive correlation
between ATP concentration and Type II receptor binding capacity in a
human cell line (i.e., IM-9 cells) was also observed (Wheeler et al.,

31
1981). Lastly, affinity labeling of Type II receptors (from L-cells
32
grown in the presence of [ P]orthophosphate) with dexamethasone
21-mesylate has provided direct evidence suggesting that these receptors
may be phosphoproteins (Housley and Pratt, 1983). It is important to
note that although these data clearly favor the role of
phosphorylation-dephosphorylation as a possible mechanism of Type II
receptor regulation, there is as yet no experimental evidence to suggest
a similar mechanism for up- and down-regulation of Type I receptors.
A third mechanism for the potential regulation of Type II and
possibly Type I receptors (which may also be related to the
phosphorylation-dephosphorylation cycle (Housley et al., 1982, for
review see Housley et al., 1984)), is the reduction and oxidation of
sulfhydryl group(s) in the receptor macromolecule. It is well known
that thiol-disulfide exchanges can markedly affect the activity of a
number of protein macromolecules including androgen, estrogen,
progesterone, ecdysone and insulin receptors, lysozyme, immunoglobulins
and many digestive enzymes. In 1975, Rees and Bell claimed that they
were able to recover partially the loss in binding capacity of rat
thymic cytosol receptors by including sulfhydryl reducing reagents,
e.g.; dithiothreitol (DTT) and 2-mercaptoethanol, in their buffers. Two
years later, Granberg and Ballard (1977) reported that the addition of
2 mM DTT to rat lung cytosol increased the apparent binding of Type II
receptors 10-fold. This increase was proposed to be due to
transformation of an already-available pool of receptors to a
1igand-binding state. Similar effects were also seen in brain, uterus,
thymus and a number of other tissues whereas the presence or absence of
DTT was found to have little effect on Type II receptor binding capacity

32
in liver, kidney and heart cytosol. It was postulated that these latter
tissues contained endogenous, heat-stable, sulfhydryl reducing agents
which stabilize Type II receptor binding capacity. Interestingly, an
analysis of Type II receptors in fetal (Ballard et al., 1974) and adult
(Granberg and Ballard, 1977) rat lung revealed that exogenous DTT was
required only in the cytosol prepared from adults. Together these
observations suggest that variations in the production of sulfhydryl
reducing (and/or oxidizing) reagents may be responsible for differential
stabilization of Type II receptors in various tissues and in the same
tissue during different stages of development.
An endogenous protein molecule, capable of modulating steroid
binding capacity of the rat liver cytosolic unoccupied Type II
receptors, has recently been characterized by Pratt and co-workers
(Grippo et al., 1983 & 1985). This endogenous receptor-reducing agent
appears to be an NADPH-dependent thioredoxin. Extraction of this
stabilizing factor from liver cytosol by charcoal resulted in a
significant loss of unoccupied, but not occupied Type II receptor
binding capacity. Addition of DTT to the charcoal-ex trac ted cytosol
resulted in a complete recovery of Type II receptor binding capacity
(Grippo et al., 1983). Similar results have been obtained in our
laboratory. We found that charcoal extraction of steroid-free brain
cytosol prepared in HEPES [4(2-hydroxyethy1)-1-piperazineethanesulfonic
acid] buffer containing 10% glycerol and 2 mM DTT resulted in an
increase in the thermal instability and a decrease in the total binding
capacity of Type II receptors (Emadian et al., 1986). These effects
were reversible after the readdition of the exogenous DTT removed by the
charcoal treatment. In further investigating the effect of the presence

33
or absence of DTT, we found that the omission or removal of this
sulfhydryl reducing reagent produced a significant increase in the
3
apparent K, and a slight reduction in the of [ H]DEX
a MAX
binding to Type II receptors (Densmore et al., 1984b).
The effect of DTT on Type II receptor binding capacity appears to
be a tempera ture-independent phenomenon (Rees and Bell, 1975; Granberg
and Ballard, 1977; Grippo et al., 1983; Densmore et al., 1984; Emadian
et al., 1986), whereas the NADPH-dependent thioredoxin appears to act in
a temperature-dependent fashion (Grippo et al., 1983). These findings
suggest that the exogenous sulfhydryl reducing reagents may act
primarily on the receptor [or other component(s) of the system] to
maintain it in a reduced state -- the conformation necessary for steroid
binding. In contrast, the reducing effects of thioredoxin must be
mediated primarily through an enzymatic process (Grippo et al., 1983 &
1985). Thioredoxin reductase was found to be responsible for the
transfer of reducing equivalents from NADPH to thioredoxin. The reduced
form of the thioredoxin, in turn, is thought to maintain the receptors
in the steroid-binding state (Grippo et al., 1985). The transfer of the
reducing equivalent may be shown schematically as follows:
thioredoxin reductase
oxidized thioredoxin >- reduced thioredoxin
| NADPH
T
oxidized thioredoxin
+ ^ oxidized receptor
reduced receptor (incapable of binding ligand)
(ligand binding conformation)
The observation that occupied receptors from charcoal extracted
cytosol (i.e., in the absence of thioredoxin and/or DTT) appear to be
unaffected by charcoal treatment has raised the possiblity that the

34
sulfhydryl group(s) on the receptor may be located at or near the
steroid binding domain (Grippo et al., 1983). Alternatively, the
binding of ligand to Type II receptor could induce a conformational
change in the receptor molecule which renders more distant sulfhydryl
group(s) inaccessible for oxidation. With regards to this latter
hypothesis, a recent preliminary study from our laboratory suggests that
DTT may have no detectable effects on the sedimentation coefficient of
unoccupied Type II receptors (Densmore et al., 1984). It is important
to remember that the method of sucrose density gradient
ultracentrifugation may not be sensitive enough to reveal small
conformational changes in receptor.
Another possible regulatory role of the thiol-disulfide exchange
mechanism involves the interaction of steroid-receptor complexes with
the cellular genomic machinery. Preatreatment of DEX-receptor complexes
with sulfhydryl oxidizing reagents [e.g.; methyl methanethiosulfonate,
MMTS and 5,5'-dithiobis (2-nitobenzoic acid), DTNB] inhibits subsequent
binding of the activated receptor complexes to DNA-cellulose (an
in vitro index for receptor transformation) (Bodwell et al., 1984).
Interestingly, the inhibitory effect of these reagents can be reversed
by the addition of DTT. These results were interpreted to suggest that
sulfhydryl group(s), located at or near DNA-binding domain of the
activated steroid-receptor complex, may regulate the interaction of the
complex with the genome (Bodwell et al., 1984). It is noteworthy that
although MMTS and DTNB were shown to diminish DNA-cellulose binding of
the complexes, the authors (Bodwell et al., 1984) failed to refute
conclusively the possibility that these reagents inhibited DNA-binding
by acting as cross-linking reagents (i.e., cross-linking of the

35
activated receptors with other cytosolic components such as heat-shock
proteins, mRNAs, etc.)» In fact, current data clearly favors the notion
that N-ethylmaleimide (NEM) and iodoacetamide (other sulfhydryl
modifying reagents) inhibit transformation of rat liver Type II
receptors (Kalimi and Love, 1980) probably through inhibition of subunit
dissociation (for detail see Chapter VI). Furthermore, it is possible
that the bulky thiomethyl and thionitrobenzoate complexes (formed by the
interaction of MMTS and DTNB with the receptors, respecively), rather
than the oxidation itself, inhibit the binding of the complexes to
DNA-cellulose. More recently, Type II receptors were shown to bind
nuclear matrix (a proteinaceous structural framework implicated in many
nuclear functions, for detail see Chapter VI) through intermolecular
disulfide bond formation (Kaufmann et al., 1986). Sodium
dodocylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the rat
hepatic Type II receptors extracted from nuclei isolated from animals
3
injected with [ H]TA in the absence of sulfhydryl-blocking (e.g.,
iodoacetamide) and cross-linking reagents yielded steroid-receptor
complexes that were disulfide-cross-linked to the nuclear matrix.
Conversly, isolation of the nuclei in presence of iodoacetamide,
revealed no receptor-nuclear matrix complex formation (Kaufmann et al.,
1986). These results provide a more direct evidence for the involvment
of sulfydryl groups in the binding of transformed steroid-receptor
complexes to nuclear components (for further discussion see Chapter VI).
Recently, structural analysis of the amino acid sequences of a number of
steroid receptors revealed the presence of a cysteine rich region in the
DNA-binding domain these receptors (e.g., Danielsen et al., 1987).

36
In contrast to our understanding of the factors regulating Type II
receptor binding activity, our knowledge concerning possible up- and
down-regulatory factors affecting Type I receptors remains scanty. As
discussed above, we have demonstrated several in vitro chemical
differences between the brain cytosolic Type I and Type II receptors
which may play an important role in the in vivo regulation of these
receptors. For example, although charcoal-extraction of steroid-free
brain cytosol in HEPES buffer lacking DTT causes a dramatic reduction in
Type II binding capacity (possibly by removal of endogenous
thioredoxin), this treatment has no effect on the binding capacity of
Type I receptors (Emadian et al., 1986; also see Chapter V). This
finding suggests that the labile thiol-disulfide exchange mechanism
described for Type II receptors may not be involved in the regulation of
Type I receptor binding capacity. In an effort to shed more light on
the role of possible factors and mechanisms involved in the in vitro
and/or in vivo regulation of Type I receptors, an investigation of
possible involvement of sulfhydryl groups in the ligand- and DNA-binding
capacity of these receptors was conducted in this dissertation (see
Chapters V and VI).
In Vitro Analysis of Type I Receptors by Radioreceptor Assay
A clear understanding of the physiological and behavioral
consequences of steroid hormone action requires an unambiguous
characterization of the receptors through which such actions presumably
are mediated. Our knowledge of the properties of Type I receptors as
well as the factors involved in the regulation of these receptors may
indeed lead to further discoveries regarding functional consequences of
these steroids. For example, the recognition of a specific

37
mineralocorticoid receptor led to the discovery of spironolactones
(mineralocorticoid antagonists), which have proven invaluable tools
clinically (as effective antihypertensive agents) and in basic sciences
(see above). Although numerous studies investigating the properties of
steroid hormone receptors have appeared in the literature within the
past two decades, the volume of research on the properties of Type I
receptors has been comparatively minimal (Liao et al., 1983). This is
partially due to the fact that in crude preparations ALDO and other
readily available synthetic mineralocorticoids bind to Type II receptors
with appreciable affinity (Rousseau et al., 1972; Funder et al., 1973a;
Emadian et al., 1986; Emadian and Luttge, unpublished; also see Chapter
V). This indiscriminatory binding of ALDO to Type II receptors can
produce confounding results thus hampering an independent analysis of
Type I receptors. Although the use of mathematical models has proven
effective in determination of binding parameters for Type I receptors
(e.g., Claire et al., 1978; Claire et al., 1985; Emadian et al., 1986),
failure to eliminate Type II-binding interferences has led a number of
investigators reach erroneous conclusions in their analysis of Type I
receptor properties (Grekin and Sider, 1980; Marver et al., 1972).
Recently, however, the introduction of very specific synthetic Type II
ligands (e.g., RU 26988, RU 28362 and RU 38486) has made the elimination
of Type II-binding contribution in crude cytosolic preparations
possible. When used in conjunction with radiolabeled ALDO, a molar
excess of the RU compound can saturate all Type II binding sites with
little or no cross-reactivity with Type I receptors, hence leaving these
latter sites intact for ALDO binding. This method has been utilized
successfully by this and a number of other laboratories (Moguilewsky and

38
Raynaud, 1980; Veldhuis et al., 1982b; Gomez-Sanchez and Gomez-Sanchez,
1983; Wrange and Yu, 1983; Emadian, et al., 1986).
In addition to these methods, Veldhuis et al. (1982b) reported that
they were able to eliminate non-linearities observed in the Scatchard
plots for ALDO binding by co-incubating hippocampal cytosol with
unlabeled CORT. Although the Scatchard plot of ALDO binding in the
presence of a 100-fold concentration of RU 26988 appears comparable to
that obtained in the presence of a 0.6-fold concentration of CORT
(Veldhuis et al., 1982b), in light of the fact that CORT has a higher
affinity for hippocampal Type I receptors (see below), a lower value of
equilibrium maximal binding in the presence of CORT is consistent with
the interaction of CORT with Type I receptors (for detail see below).
To eliminate the confounding effects of ALDO interaction with Type II
receptors, unless otherwise stated, the characterization of Type I
receptors in this dissertation was accomplished in the presence of a
500-fold molar excess of RU 26988, a concentration determined to
eliminate all Type II binding interferences (Emadian et al., 1986) (for
detail see Chapter II).
Another pertinent issue in the in vitro analysis of steroid
receptors in crude cytosolic preparations, in general, is possible
contamination and thus binding contribution by low affinity and high
capacity "non-specific binders" (of cytosolic, membrane or of plasma
origin). It is well established that ALDO binds with appreciable
affinity, especially at lower tempertures, to corticosteroid binding
globulin (CBG) (Sandberg et al., 1960; Daughaday et al., 1960; Meyer et
al., 1961; Davidson et al., 1962; Zager et al., 1976; Zipser et al.,
1980), and with a lower affinity to a^-acid glycoprotein and albumin

39
(Chen et al., 1961; Daughaday et al., 1960; Ganguly and Westphal, 1968).
Moreover, using competitive adsorption and gel filtration techniques,
Richardson and co-workers (1977) identified two other plasma
ALDO-binding proteins distinct from CBG, albumin and a^-acid
glycoprotein in human plasma. This has been confirmed by Katayama and
Yamaji (1982) who reported that this "ALDO binder" had an apparent
association constant for ALDO one order of magnitude higher than that
for CBG. Although there is no evidence that these plasma binders are
present in rodents, because 1) ligand binding assays are usually
performed at lower temperatures and 2) corticosteroid-binding globulin
(CBG) shows appreciable affinity for ALDO at lower tempertures, an
extensive in situ transcardiac perfusion of brains prior to cytosol
preparation will be performed. This will assure the elimination of
possible contribution of these binders in our in vitro
characterization of Type I receptors. Note that this perfusion was
deemed sufficient in eliminating these binders, since co-incubation of
3
whole brain cytosol with [ H]ALD0 and cortiuic acid (a synthetic
17a-acid derivitive of cortisol that specifically binds CBG, see Alexis
et al., 1983; Little, 1983; Sheppard and Funder, 1986) produced specific
Type I receptor binding similar in magnitude to that found in the
3
presence of [ H]ADL0 alone.
In addition to the above plasma binders, the presence of the
extravascular corticosterone binder (with identical physicochemical and
immunoreactive properties to the plasma CBG) has been identified in the
uterus (Milgrom and Baulieu, 1970; Guerigian et al., 1974; Al-Khouri and
Greenstein, 1980), liver (Koblinsky et al., 1972; Litwack et al., 1973;
Amaral et al., 1974; Suyemitsu and Terayama, 1975; Weiser et al., 1979),

40
kidney (Funder et al., 1973b; Feldman et al., 1973; Strum et al., 1975;
Weiser et al., 1979), pituitary and brain (Koch et al., 1976; De Kloet
and McEwen, 1976a & b; Al-Khouri and Greenstein, 1980; Gray and Luttge,
1982), and a number of other tissues (Mayer et al., 1975; Giannopoulos,
1976; Werthamer et al., 1973) (for review see Siiteri et al., 1982).
The order of steroid binding specificity for these CBG-like binders
determined by competition assays appears to be CORT > DOC >> ALDO >>
DEX. Although it has been shown conclusively that the synthesis of
CBG-like glucocorticoid binders (by convention, referred to as Type III
receptors) occurs in the liver (Perrot-Applanat and Milgrom, 1979;
Weiser et al., 1979), the possible functional roles and the origin of
these binders in other tissues have been the subject of considerable
controversy. Since a comprehensive review of the primary references
surrounding this issue is outside the scope of this dissertation, only
the recent findings relevant to the in vitro characterization of
Type I receptors will be reviewed.
More than a decade ago, it was established that in kidney there
were two distinct classes of corticosteroid binding proteins
representing the Type I and Type II receptors (Funder et al., 1973b).
It was further claimed that the hierarchy of steroid binding specificity
for Type I receptors were ALDO > DOC > CORT (Funder et al., 1973b).
Later, the presence of specific Type I receptors in a number of other
tissues including the CNS were reported (Anderson and Fanestil, 1976;
Moguilewsky and Raynaud, 1980; Lan et al., 1981). However, it appeared
that ALDO, DOC and CORT had equivalent affinity for these sites. The
results from these in vitro competition assays were taken as
evidence for possible in vivo occupancy of Type I receptors by

41
glucocorticoids in the rat kidney and brain (Lan et al., 1982; Veldhuis
et al., 1982b). In a separate study, Beaumont and Fanestil (1983)
reported that the steroid binding specificity Type I receptors in rat
brain could be altered by different experimental manipulations. These
investigators found that after elimination of Type II binding
interferences (in the presence of a molar excess of RU 26988) and plasma
CBG (and possibly Type III receptors), the order of steroid competition
for Type I sites (occupied with [ H]ALDO) was DOC > fludrocortisone >
CORT > ALDO > progesterone > DEX. However, the addition of small
quantities of dialyzed serum to the above preparations yielded a
competition hierarchy similar to that described for kidney, i.e., ALDO >
DOC > CORT. Several independent confirmations of the above observations
were reported for the rat kidney (Krozowski and Funder, 1983; Stephenson
et al., 1984b) and hippocampus (Krozowski and Funder, 1983; De Kloet et
al., 1984). In conclusion, it is suggested that CBG and CBG-like
binders, in addition to affecting the metabolic clearance rate of the
steriods to which they bind with high affinity (for review see Siiteri
et al., 1982), may act in vivo as sequestration sites or "sinks" for
corticosterone thus rendering the Type I sites available for ALDO
binding (Funder et al., 1973b; Krozowski and Funder, 1983; Stephenson et
al., 1984; Funder, 1986). It is further suggested that the hippocampal
Type I receptors are physiological CORT binders and may be involved in a
tonic (permissive) influence on brain function with septo-hippocampal
complex as a primary target (De Kloet et al., 1984; Reul and De Kloet,
1985; Funder, 1986; Magarinos et al., 1986; Reul and De Kloet, 1986).
Although the sequestration theory is an attractive hypothesis, further
experiments investigating the tissue availability of CORT (that is not

42
bound to CBG, other plasma binders and Type III receptors) and ALDO
under physiological conditions is required to establish this theory. In
this regard, preliminary studies suggest that the serum levels of ALDO
and CORT under "normal" conditions leads to nuclear uptake of both
hormones in the hippocampus as measured by radioimmunoassay (Yongue and
Roy, 1984). Recently, McEwen et al. (1986b) showed that, in
adrenalectomized rats, only "stress level" concentrations (a 500-fold
molar excess) of CORT was able to block the nuclear uptake of
3
[ HjALDO in limbic, but not circumventricular, areas. Further
functional relevence of these findings remain to be established.
A further caution regarding the results obtained from in vitro
competition assays also deserves mention: in the above experiments
cytosolic preparations were performed in the presence of molybdate and
DTT. Our own work has shown that although molybdate stabilizes both
Type I and Type II receptors, it causes a significant reduction in the
binding capacity of Type I receptors (Emadian et al., 1986; also see
Chapter IV). Furthermore, sulfhydryl reducing reagents such as DTT,
while having no effect on the binding of ALDO to Type I receptors,
result in an apparent increase in the equilibrium dissociation constant
of CORT from CBG (Westphal, 1983) and very likely from Type III
receptors. Therefore, under conditions that provide optimal stability
for all of these proteins, one may find different steroid-binding
hierarchy from those discussed above.

CHAPTER II
EXPERIMENTAL PROCEDURES
In order to avoid repetition, in this chapter a general scheme of
the materials and experimental procedures shared by the studies
described in Chapters III through VI will be presented. Materials and
methods that are unique to a given chapter will be described in that
particular chapter.
Isotopes, Steroids and Other Chemicals
3
The radiolabeled steroids; i.e., [6,7- H]Triamcinolone acetonide,
3
[ H]TA, 9a-fluoro-113, 16a,17,21-tetra-ol-pregna-1,4-diene-3,20-dione
3 3
(specific activity, SA 43.7 Ci/mmol); [6,7- H]dexamethasone, [ H]DEX,
9a-fluoro-16a-methylprednisolone (SA 37.3-44.1 Ci/mmol) and
[1,2,6,7-^H]aldosterone, [^HjALDO, 4-pregnen-113,21-diol-3,18,
20-trione (SA = 71.5-82.0 Ci/mmol) were purchased from New England
Nuclear. These radiolabeled steroids were checked for purity by
thin-layer silica gel chromatography using a chloroform-methanol (9:1,
volume :volume) solvent system. [^H]TA, [^H]DEX, [^H]ALDO,
Sephadex G-25 (bead size 20-80 u) and sodium molybdate were obtained
from Sigma. HEPES buffer [4(2-hydroxyethyl)-l-peperazineethanesulfonic
acid] and dithiothreitol (DTT) were obtained from Research Organic. RU
26988 [113»173-dihydroxy-17a-(1-propionyl)-androsta-1,4,6-triene-3-one)]
and RU 26752 [3' (3-oxo-7a-propyl-173-hydroxy-4-androstene) propionic
acid lactone] were kindly supplied by Roussel-Uclaf (France). Prorenone
43

44
[ 3( 17$-hydroxy-6$, 7(3-methy lene-3-oxo-4-andros ten-170t-yl) propionic acid
y-lactone] was a gift from Searle Co. All other chemicals used in this
dissertation were reagent grade quality.
Experimental Animals
Adult female CD-I mice (Charles River Laboratories) were subjected
to a combined bilateral adrenalectomy and ovariectomy under barbiturate
anesthesia 3-6 days prior to use [a period shown to result in maximal
up-regulation of Type I receptors (Luttge and Rupp, unpublished
results)] in order to remove known sources of endogenous steroids. They
were then maintained on Purina Rodent Chow and 0.9% NaCl. On the day of
experiment, mice were anesthetized with ether and perfused slowly
through the heart with 15-20 ml ice-cold HEPES-buffered saline
(isotonic, pH 7.60).
Table 2-1
Buffers
20 mM 2 mM
BUFFER
HEPES
DTT
Na MoO.
2 4
A
+
-
-
D
+
+
-
M
+
-
2 mM
M20
+
-
20 mM
DM
+
+
2 mM
DM„„
20
+
+
20 mM
cytosolic concentration of the
component(s)
in Buffers D, M,
DM^q as well as
other cytosolic
components
(e *8 • >
300 mM KC1; 10% mono- and polyhydric compounds) were obtained by a 9:1

45
dilution of cytosol with stock buffers containing a 10-fold
concentration of the desired component(s). Under such circumstances,
the control group was diluted accordingly with an equivalent volume of
Buffer A. Buffers were adjusted to pH 7.60 at 0-2°C.
Cytosol Preparation
Brains were homogenized (2 x 10 strokes) at 1000 RPM in two volumes
of ice-cold Buffer A or Buffer A containing one or more of the desired
components (see specific experiments). A glass homogenizer with a
teflon pestle milled to a clearance of 0.125 mm on the radius was used
to minimize the rupture of brain cell nuclei. The crude homogenate was
centrifuged at 100K x g for 20 min and the resulting supernatant
recentrifuged at 100K x g for an additional 60 min to yield cytosol.
During this and all other procedures (except "aging" and column
chromatography) attempts were made to maintain the cytosol at 0°C.
Aging
In order to examine thermal stability of unoccupied Type I and Type
II receptors, cytosol was incubated for various periods of time at 0
and/or 22°C. Following such treatment, "aging" was quenched by
incubating the cytosol with steroid(s) for 24-48 h at 0°C for specific
binding determination (see below) .
Hydrophobic Interaction Chromatography.
Pently-Agarose columns (0.7 x 21.5 cm) were used to compare the
surface hydrophobic properties of occupied Type I versus Type II
(Chapter III) as well as activated versus unactivated Type I receptors
(Chapter VI). In such experiments, cytosol in Buffer DM^q was
incubated with radiolabeled ligand for Type I or Type II receptors for
48 h at 0°C (see "Steroid Binding Determination"). Following incubation

46
with steroid(s), salt- and heat-induced activation (see Chapter VI),
bound-free separation on Sephadex G-25 (0.6 x 14.0 cm) columns were
performed as described previously (Luttge et al., 1984c). Subsequently,
0.8 ml [ H]steroid-bound macromolecular fraction eluted from the
G-25 column was loaded on pently-Agarose columns pre-equilibrated in
buffer D containing 50 mM molybdate and 600 mM KC1. Using this latter
buffer, 20-27 consecutive 0.8 ml fractions were collected directly into
scintillation vials for measurements of radioactivity (see below). In
some experiments, a second bound-free separation on G-25 columns were
performed on the "peak" fractions eluted from the pentyl agarose columns
to assure that the radioactivity eluted was indeed macromolecular-bound
in nature. All chromatographic procedures were performed at 4-6°C.
Steroid Binding Determination
After appropriate treatment of cytosol, 0.24-4.0 ml aliquots were
added to pre-cooled glass tubes containing the desired steroid(s) (dried
under N^). The tubes were then allowed to incubate for 24-48 h in
ice. In single saturation dose studies, B determinations for Type
I receptors were performed in the presence of 10-20 nM [ H]ALDO and
500-fold molar excess of RU 26988. For Type II receptors, 20 nM
3 3
[ H]DEX or [ H]TA was used as the ligand. In some experiments,
3
[ HjDEX incubation was in the presence of a 500-fold molar excess of
RU 26752, prorenone or RU 26988 (see Chapter V). Parallel incubation
tubes containing an additional 200-fold molar excess of [^H]ALD0,
[^H]DEX or [^H]TA were used for the determination of B>T„ for
NS
Type I and Type II receptors, respectively. Equilibrium binding
parameters; i.e., K and B„JV were determined using Scatchard's
a MAX
method (1949) as described previously (Emadian et al., 1986).

47
In all experiments, macromolecular-bound from free steroid was
separated at 4°C using G-25 (0.6 x 14 cm) columns (for detail see Luttge
et al., 1984c). Bound fraction (0.8 ml) obtained from each column was
diluted with 5 ml Scinti-Verse flúor (Fisher Scientific) in a
scitillation vial, vortexed vigorously and the radioactivity determined
in a Packard Model 2425 liquid Scintillation spectrometer. All counts
per minute (CPM) data were converted to disintegration per minute (DPM)
by an external standard channels ratio measurement. The counting
efficiency for tritium averaged about 35%.
Cytosolic Protein Concentration
The concentration of protein in cytosol preparations was determined
using the method described by Lowry et al. (1951). In crude
preparations, protein concentration ranged from 6.2 to 7.3 mg/ml
cytosol, whereas the values in gel filtered and 2-fold diluted
preparations (see Chapter IV) averaged about 3.3 and 3.6 mg/ml cytosol,
respectively.

CHAPTER III
EFFECTS OF POLYHYDRIC AND MONOHYDRIC COMPOUNDS ON THE STABILITY OF
TYPE I RECEPTORS FOR ADRENOCORTICOSTEROID HORMONES IN BRAIN CYTOSOL
Summary
We have shown previously that unoccupied Type I receptors for
adrenal steroids in brain cytosol lose their ability to bind
3
[ H]ALDO in a time- and temperature-dependent manner (Emadian
et al., 1986). Based on reports that sugars and polyvalent alcohols are
capable of stabilizing a variety of globular proteins, we made an
attempt in this study to stabilize Type I receptors by including
polyhydric compounds in our cytosol preparations. However, unlike our
expectations, the addition of 10% (weight:volume) ethylene glycol,
glycerol, erythritol, xylitol, ribitol or sorbitol to cytosol at 0°C
failed to stabilize the receptors and in fact produced a slight
3
reduction in the binding of [ H]ALD0. The magnitude of this
reduction was greater when cytosol was "aged" for 2 h at 22°C prior to
3
incubation with [ H]ALD0. In contrast to these results with
polyhydric compounds, the addition of 10% (weight:volume) ethanol to
3
cytosol maintained at 0°C markedly increased the binding of [ H]ALD0
to Type I receptors. Under identical conditions, methanol slightly
increased, and propanol had no significant effect on the binding
capacity of these receptors. When cytosol was aged at 22°C, however,
all of these monohydric compounds led to a marked loss in Type I
receptor binding capacity. An investigation of various doses of ethanol
at 0°C on the subsequent binding of [ H]ALD0 to Type I receptors
48

49
yielded an inverse U-shaped curve with 10% ethanol producing the highest
level of specific binding, as reflected by an increase in maximal
binding in Scatchard plots, and 40% ethanol producing a complete loss in
Type I receptor binding capacity. In view of the above findings and
proposed mechanisms of action of mono- and polyhydric compounds, it is
suggested that Type I receptors in an aqueous cytosolic environment
undergo spontaneous conformational changes which promote the infolding
of hydrophobic steroid binding site(s) on the receptor surface, thus
leading to the observed reduction in Type I receptor binding capacity.
Introduc tion
Although receptors for steroid hormones appear to be relatively
stable under in vivo conditions (Raaka and Samuels, 1983), these
macromolecules are rendered labile under cell-free conditions. This
increased lability is particularly apparent when working with unoccupied
receptors (Puca et al., 1971; Pratt and Ishii, 1972; Bell and Munck,
1973; Rafestin-Oblin et al., 1977; Emadian et al., 1986), and it can be
a serious problem when experimental conditions necessitate that the
unoccupied receptors must remain stable for extended periods of time
(e.g., during the determination of their hydrodynamic and equilibrium
binding parameters). In previous work with unoccupied and occupied
Type I and Type II receptors for adrenal steroids in brain cytosol we
have systematically examined the stabilizing effectiveness of a number
of factors including temperature, ionic strength, chelators, molybdate,
dithiothreitol, dextran-coated charcoal adsorption, etc. (Gray, 1982;
Densmore et al., 1984a; Densmore et al., 1984b; Densmore and Luttge,
1985; Densmore et al., 1986a; Densmore et al., 1986b; Emadian and
Luttge, 1986; Emadian et al., 1986); however, even with our best buffer

50
formulations, we still observe a time- and temperature-dependent loss of
unoccupied Type I receptor binding capacity (Eraadian et al., 1986).
Work in other laboratories has shown that polyhydric compounds (e.g.,
sugars and polyvalent alcohols) can stabilize a wide range of globular
proteins, including a number of enzymes and receptors, against
time-dependent thermal degradation (Feil et al., 1972; Rafestin-Oblin
et al., 1977; Back et al., 1979; Ogle, 1980; Gekko and Timasheff, 1981a
& b; Ogle, 1983). In view of these successes, the experiments presented
here examine the effects of a number of mono- and polyhydric compounds
on the stability and binding capacity of unoccupied Type I receptors for
3
[ H]ALD0 in brain cytosol. The results from these studies suggest
that spontaneous conformational changes that occur as a consequence of
interaction between these receptors and their aqueous cytosolic
environment may ultimately lead to the infolding of hydrophobic steroid
binding site(s) on the surface of Type I receptors.
Materials and Methods
Chemicals. Reagent grade poly- and monohydric compounds were
purchased from Sigma.
Buffers. The final concentration of mono- and polyhydric compounds
in cytosol was obtained by adding concentrated stock buffers containing
each compound to the cytosol to yield the desired concentration of that
compound. The pH is all buffers was adjusted to 7.60 at 0-2°C. All
other materials and methods were as described in Chapter II.
Results
Fig. 3-1 shows that a 10% (weight:volume) final concentration of
di- (ethylene glycol, EG), tri- (glycerol, G), tetra- (erythritol, E),
penta- (xylitol, X and ribitol, R) or hexavalent (sorbitol, S) alcohols

Figure 3-1. Effects of Polyhydric Compounds on the Stability of
Unoccupied Type I Receptors in Whole Brain Cytosol. Cytosol
prepared in Buffer A was either diluted with additional Buffer A (No
Poly-OH) or adjusted to 10% (weight:volume) ethylene glycol (EG),
glycerol (G), erythritol (E), Xylitol (X), ribitol (R) and sorbitol (S)
using a concentrated HEPES-buffered solution of each compound.
Subsequently, the cytosol was divided into two parts: one part was
3
incubated immediately with 10 nM [ H]ALD0 plus 5 uM RU 26988 and
either with (BV1„) or without (B_) 2 uM [^HjALDO for 24 h 0°C
ns r
(0-h open bars). The second part was aged either from 2 to 48 h at 0°C
(2-, 24- and 48-h open bars) or for 2 h at 22°C (closed bars) prior to
incubation with the steroids. At the end of the incubation period,
macromolecular-bound from free steroids were separated on Sephadex G-25
columns and specific binding was determined as described in Chapter II.
The number of independent replicate experiments is indicated near the
base of each bar. All data presented are based on the percent of the
non-aged, "No Poly-OH" control (*) group + SEM. 100% = 28 fmole/mg
protein.

52
No Poly-OH EG
R

53
to unlabeled brain cytosol produced a small, but significant (p < 0.05),
3
reduction in the binding of [ H]ALD0 to Type I receptors for adrenal
steroids. Aging unlabeled cytosol at 0°C in the presence of any of
these polyhydric compounds did not attenuate, or exacerbate, the loss in
Type I receptor binding capacity seen in the control cytosol (No
Poly-OH). However, when cytosol was aged for 2 h at 22°C, the reduction
3
in the subsequent binding of [ H]ALD0 to Type I receptors in the
presence of polyhydric compounds was nearly twice as great as in their
absence. Since a 10% concentration of glycerol has also been routinely
included by many investigators in buffers used for Type II receptors, we
3
examined the effects this compound on [ H]DEX binding. As shown in
Fig. 3-2, the addition of 10% glycerol to brain cytosol prior to
3
incubation with [ H]DEX fails to protect the time- and temperature-
dependent loss in Type II receptor binding capacity. It is noteworthy
that, in contrast to the results obtained with Type I receptors (Fig.
3-1), glycerol does not exacerbate the loss in binding capacity when
unoccupied Type II receptors are aged for 2 h at 22°C.
Because polyhydric compounds failed to stabilize Type I (or
Type II) receptor binding capacity, we next investigated the effects of
3
monohydric alcohols on the binding of [ H]ALD0 to Type I receptors
(Fig. 3-3). In contrast to the data with polyhydric compounds, when a
10% (weight:volume) final concentration of methanol, and especially
ethanol, but not propanol, was added to cytosol immediately before the
steroids, we observed a significant increase in [ H]ALD0 binding to
Type I receptors. If cytosol was aged for 2 h at 0°C prior to the
addition of steroids, ethanol still produced an increase in the
3
subsequent binding of [ H]ALD0 to Type I receptors, whereas methanol

Figure 3-2. Effects of 10% Glycerol on the Stability of Unoccupied
Type II Receptors in Whole Brain Cytosol. Cytosol prepared in
Buffer D was either diluted with additional Buffer D (No Poly-OH) or
adjusted to 10% (weight:volume) glycerol using a concentrated
HEPES-buffered solution of glycerol containing 2 mM DTT (G).
Subsequently, the cytosol was divided into two parts: one part was
3 ,
incubated immediately with 20 nM [ H]DEX and either with (B.,_)
NS
or without (B^) 4 uM [*H]DEX for 24 h at 0°C (0-h open bars).
The second part was aged either from 2 to 48 h at 0°C (2-, 24- and 48-h
open bars) or for 2 h at 22°C (closed bars) prior to incubation with the
ligand. At the end of the incubation period, macromolecular-bound from
free steroid was separated on Sephadex G-25 columns and specific binding
was determined as described in Chapter II. The results are the mean
from 2 independent replicate experiments. All data presented are based
on the percent of the non-aged, "No Poly-OH" control (*) group.
100% = 300 fmole/mg protein.

55
No Poly- OH
G

Figure 3-3. Effects of Monohydrlc Compounds on the Stability of
Unoccupied Type I Receptors in Whole Brain Cytosoi. Cytosol
prepared in Buffer A was either diluted with additional Buffer A (No
Alcohol) or adjusted to 10% (weight:volume) methanol (Mt) , ethanol (Et)
and propanol (P) using a concentrated HEPES-buffered solution of each
compound. Subsequently, the cytosol was divided into two parts: one
3
part incubated immediately with 10 nM [ H]ALD0 plus 5 uM RU 26988
and either with (BYT„) or without (B) 2 uM [^HlALDO for 24 h
Ni> T
at 0°C (0-h open bars). The second part was aged for 2 h at 0 or 22°C
(2-h open and closed bars, respectively) prior to incubation with the
steroids. At the end of the incubation period, macromolecular-bound
from free steroids were separated on Sephadex G-25 columns and specific
binding was determined as described in Chapter II. All data presented
are based on the percent of the non-aged, "No Alcohol" control (*) group
expressed as the mean + SEM from 6 independent replicate
experiments. 100% = 18 fmole/mg protein.

57
*
c
o
c
o
Q)
ÃœL
No Alcohol Mt
Et
P

58
had no effect and propanol produced a clear reduction in receptor
binding capacity. When cytosol preparations were aged for 2 h at 22°C,
all three alcohols greatly increased the loss in Type I receptor binding
capacity.
An investigation of the effects of various concentrations of
3
ethanol on the binding of [ HjALDO to cytosolic Type I receptors
revealed an inverse U-shaped curve with 10% ethanol producing the
greatest binding (120% of control) and 40% ethanol leading to a complete
inactivation of Type I receptors (1% of control) (Fig. 3-4).
Furthermore, a Scatchard (1949) analysis of the equilibrium binding data
revealed that the 10% concentration produced a 46% increase in the
3
K, and a 23% increase in the B of [ HlALDO binding to
d max
Type I receptors in brain cytosol (Fig. 3-5).
Discussion
Proposed Mechanism of Action of Poly- and Monohydric Compounds.
For proteins in an aqueous environment, the non-covalent weak
intramolecular interactions (i.e., electrostatic forces, hydrogen
bonding and Van der Waals forces) , as well as the interactions between
protein molecules and their surrounding solvent (i.e., hydrophobic
interactions), act in concert to stabilize the functionally important
tertiary structure of proteins. In some proteins, additional stability
may result from cross-linking, metal complex formation and binding to
other ions and cofactors; however, it is often held that the hydrophobic
interactions, a consequence of net unfavorable interactions between
solvent (i.e., water molecules) and non-polar residues in the protein,
play the most important role in stabilizing proteins (e.g., Creighton,
1984; Fersht, 1985). Therefore, when proteins are extracted from the

Figure 3-4. Dose-Response Analysis of the Effects of Ethanol on the
Binding of ALDO to Type I Receptors in Whole Brain Cytosol. Cytosol
prepared in Buffer A was either diluted with additional Buffer A
(control, *) or adjusted to 1 to 40% (weight:volume) ethanol using a
concentrated HEPES-buffered solution of ethanol. Subsequently, all
3 ,
groups were incubated with 10 nM [ HjALDO plus 5 uM RU 26988 and
either with (B ) or without (B„) 2 uM [^H]ALD0 for 24 h at
NS T
0°C. At the end of the incubation period, macromolecular-bound from
free steroids were separated on Sephadex G-25 columns and specific
binding was determined as described in Chapter II. All data presented
are based on the percent of 0% ethanol control (*) group expressed as
the mean + SEM from 3-12 independent replicate experiments.
100% = 23 fmole/mg protein.

Percent of Control*
60
Ethanol Concentration (% w:v)

Figure 3-5. Scatchard Analysis of the Effects of 10% Ethanol on the
Equilibrium Binding Parameters of ALDO Binding to Type I Receptors
in Whole Brain Cytosol. Cytosol prepared in Buffer A was either
diluted with additional Buffer A (open circles) or adjusted to 10%
(weight:volume) ethanol using a concentrated HEPES-buffered solution of
ethanol (closed circles). Subsequently, 0.25 ml aliquots from each group
3
was incubated with 0.5 to 40 nM [ H]ALD0 plus 500-fold molar excess
of RU 26988 and either with (B^) or without (B^,) 200-fold molar
excess of [^H]ALD0 for 24 h at 0°C. At the end of the incubation
period, macromolecular-bound from free steroids were separated on
Sephadex G-25 columns and specific binding was determined as described
in Chapter II. The Scatchard plot shown is representative from 4
independent replicate experiments. Binding parameters were calculated
using least square linear regression. Open circles: B =
max
21 fmole/mg protein, = 3.0 x 10 ^ M. Closed circles:
B =26 fmole/mg protein, K, = 4.4 x 10 ^ M.
max ° r ’ d

62
0.05
OJO

63
intracellular environment into a medium that is more aqueous than
cytoplasm, it is not uncommon for them to undergo spontaneous
conformational changes leading to their functional inactivation. To
protect proteins against such in vitro denaturation, various
strategies have been utilized to reduce unfavorable solvent-solute
interactions .
In a series of studies by Gerlsma (1968 & 1970) and Gerlsma and
Stuur (1972 & 1974), polyhydric compounds were shown to increase the
melting temperature (T ) of chymotrypsinogen A, ribonuclease and
lysozyme and hence stabilize the native conformation of these proteins
at a higher temperature. In contrast, monohydric alcohols were found to
destabilize these proteins (i.e., lower the T ). On a molecule per
m
molecule basis, the increase in the produced by polyhydric
compounds was shown to be related directly to the number of hydroxyl
groups per molecule of the polyhydric compound, whereas the extent of
destabilization produced by monohydric compounds were shown to depend on
the length of their alkyl chain. On the basis of these results, Gerlsma
and Stuur (1972 & 1974) proposed that polyhydric compounds stabilize
proteins by decreasing the hydrogen bond rupturing capacity of the
solvent, whereas monohydric compounds augment such solvent-induced
hydrogen bond rupturing capacity. This proposed mechanism is somewhat
different from that set forth earlier by McDuffie et al. (1962), who
maintained that polyhydric compounds stabilize proteins by inducing the
tetrahedral conformation of water molecules at higher temperatures
which, in turn, increase the hydrophobic interactions. In support of
this hypothesis, Oakenfull & Fenwick (1979) showed that hydrophobic

64
interactions in aqueous-organic environments are maximized when the
three-dimensional hydrogen-bonded structure of water is most developed.
In a recent quantitative analysis by scanning calorimetric
technique, Back et al. (1979) have shown that sucrose and glycerol
strengthen pairwise hydrophobic interactions in aqueous solutions of
ovalbumin, lysozyme, a-chymotrypsinogen and conalbumin; however, both of
these polyhydric compounds were also shown to reduce the driving force
for transfer of a hydrophobic group from an aqueous to a nonpolar
environment. Other workers have argued that glycerol, the most common
polyhydric compound used to stabilize the activity and native structure
of enzymes and proteins (Feil et al., 1972; Rafestin-Oblin et al., 1977;
Ogle, 1980; Dias et al., 1981; Korge and Timpmann, 1983; Livesey et al.,
1983; Ogle, 1983; Ramaley and Vasantha, 1983; Fojo et al., 1985),
increases the hydrophobic interactions in chymotrypsinogen A,
a-chymotrypsin, y-lactoglobulin and ribonuclease A by being
preferentially excluded from the immediate domain of these proteins
(Gekko and Timasheff, 1981a & b; Timasheff et al., 1976). Collectively,
these results suggest that polyhydric compounds tend to strengthen the
pairwise hydrophobic interactions in proteins by promoting the
three-dimensional hydrogen-bonded structure of water, hence stabilizing
the native, more folded conformation of proteins.
Possible Mode of Action of Poly- and Monohydric Compounds on
Unoccupied Type I receptors in Crude Cytosol.
Recently we reported that, in contrast to unoccupied Type II
adrenal steroid receptors, unoccupied Type I adrenal steroid receptors
3
lose their binding capacity for [ H]ALD0 in crude brain cytosol in a
time- and temperature-dependent manner even in the presence of DTT,
molybdate and/or glycerol (Emadian et al., 1986). However, as discussed

65
above, since protein molecules are thought to exist in a dynamic state
in aqueous environments (Creighton, 1984), we now think that our earlier
findings with Type I receptors could be a reflection of the transient
fluctuations in the three-dimensional structure of the unoccupied
3
receptors in cytosol resulting in an exclusion of [ H]ALDO from its
specific hydrophobic binding site.
To arrive at this hypothesis, we first investigated the effects
polyhydric compounds with increasing ability to promote the tetrahedral
conformation of water molecules (i.e., di-, tri-, tetra-, penta-, and
hexavalent alcohols), hence an increased ability to enhance
3
intramolecular hydrophobic interactions, on the [ H]ALDO binding
capacity of unoccupied Type I receptors. As shown in Figure 3-1, all of
the polyhydric compounds tested failed to stabilize unoccupied Type I
receptors, in fact, all of them produced a slight reduction in the
3
specific binding of [ H]ALDO, especially when cytosol was aged for 2
h at 22°C prior to binding determinations. These results suggest that
increases in the intramolecular hydrophobic interactions within the
unoccupied receptor, following the addition of polyhydric compounds to
aqueous cytosol preparations, may result in the preferential exclusion
of the steroid from its hydrophobic binding sites. If so, one would
expect that a reduction in intramolecular hydrophobic interactions
3
should increase the binding of [ HjALDO to Type I receptors. It is
noteworthy that, compared to Type I receptors, inclusion of 10% glycerol
in our buffers did not affect the binding of ligand to Type II receptors
(Figure 3-2). The differential effects of glycerol on Type I and Type
II receptors strongly suggested that there might be differences in the
overall surface hydrophobicity of these receptors. In fact, a

66
comparison of the surface hydrophobic properties of these receptors by
glass fiber filter assay (Luttge et al., 1984) and hydrophobic
interaction chromatography (Figure 3-6) revealed a greater overall
surface hydrophobicity for Type I receptors.
To test the hypothesis that a reduction in hydrophobic interactions
may increase the binding of ligand to Type I receptors, we next
investigated the effects of adding monohydric alcohols to brain cytosol.
As predicted, when compared to the non-aged, "no-alcohol" group, a 10%
final concentration of ethanol in cytosol increased the binding of
3
[ H]ALD0 to Type I receptors by about 20% (Figs. 3-3 and 3-4). This
effect was reflected in Scatchard plots by an increase in the B
J max
(Fig. 3-5). In spite of this increase, the ethanol-induced reductions
in intramolecular hydrophobic interactions also appear to reduce the
stability of unoccupied Type I receptors when they are aged at 0°C, and
especially at 22°C. This increase in the lability of the unoccupied
receptor could easily account for the shift seen in the Scatchard
plot (Fig. 3-5) (see Beck and Goren, 1983 for a theoretical explanation
of this phenomenon) and may be similar to the previously reported
temperature-dependent effects of ethanol on the stability of
ribonuclease and glutamate dehydrogenase (Shukuya and Schwert, 1960;
Brandts and Hunt, 1967).
In summary, the results described in this chapter provide indirect
3
support for the hypothesis that [ H]ALDO binding to Type I receptors
is dependent upon the exposure and stabilization of hydrophobic steroid
binding sites (and possibly other hydrophobic residues) on the surface
of the receptors. Since the comparatively stable in vivo
conformation of Type I receptors presumably allows for optimal ligand

Figure 3-6. Comparison of the Surface Hydrophobic Properties of
ALDO-Type I and DEX-Type II Receptor Complexes on Pentyl-Agarose
Columns. Cytosol prepared in buffer DM was incubated with either
3
10 nM [ H]ALD0 plus 5 uM RU 26988 (to label Type I receptors, open
3
circles) or 20 nM [ H]DEX (to label Type II receptors, closed
circles) at 0°C for 48 h for determination of B . The
T
determinations of B for Type I and Type II receptors were
IN u
performed in the presence of 2 uM [*H]ALD0 and 4 uM [^HjDEX,
respectively. Following separation of macromolecular-bound from free
steroids on Sephadex G-25 columns, hydrophobic interaction column
chromatography was performed as described in Chapter II. The data
presented are expressed as a percent of total B^ eluted from
pentyl-Agarose columns and are representative from 2 independent
replicate experiments.

T
OJ
O
L
Percent of Total Specific Binding
_ ro
o o
l i I.
On
00
T
T
T
T

69
binding, it seems likely that these receptors undergo a spontaneous
solvent-induced folding upon extraction into the more aqueous cytosolic
environment which results in a reduction in binding capacity and
stability. Although, the mechanism(s) and/or intracellular factors
producing the marked stability of Type I and other steroid receptors
in situ awaits further experiments, the present findings may provide
some clues as to the nature of their properties.

CHAPTER IV
A NOVEL EFFECT OF MOLYBDATE ON THE BINDING OF [ "h]ALDOSTERONE
TO GEL FILTERED TYPE I RECEPTORS IN BRAIN CYTOSOL
Summary
Recently, we reported that the addition of sodium molybdate to
crude cytosol at 0°C results in a dose-dependent reduction in the
3
binding of [ H]ALDO to Type I receptors for adrenal steroids
(Emadian et al., 1986). In the experiments outlined here, we show that
Sephadex G-25 gel filtration of whole brain cytosol from
adrenalectomized and ovariectomized CD-I female mice in the presence of
2 mM molybdate results in a 30-50% increase in the specific binding of
3 3
10 nM [ HjALDO to Type I receptors above the level of [ H]ALD0
binding seen in crude cytosol prepared in 20 mM HEPES buffer (control
group). All specific binding determinations were performed in the
presence of 5 uM RU 26988 — a specific synthetic glucocorticoid known
3
to block [ H]ALD0 binding to Type II (glucocorticoid) receptors. In
Scatchard plots, this molybdate- and gel filtration-induced increase in
3
specific [ HjALDO binding was reflected as a 37% increase in maximal
binding (B ) with no change in the equilibrium dissociation
max n
constant (K^). In contrast, when gel filtration was performed in
the absence of molybdate, there was a marked reduction in the subsequent
3
specific binding of [ HjALDO to Type I receptors. In Scatchard
plots, this latter effect was reflected as a 62% reduction in the
B and a 2-fold increase in the K, when compared to the
max d
control group. The addition of 2 mM molybdate immediately following gel
70

71
3
filtration yielded specific [ HjALDO binding comparable to the
control group. Although the addition of 2 mM molybdate prevents the
loss in the binding capacity of unoccupied Type I receptors in crude
cytosol incubated at 22°C, Type I receptors in gel filtered cytosol were
very unstable at 22°C, and appeared insensitive to the protective
effects of 2, but not 10-100 mM, molybdate. Two mM dithiothreitol (DTT,
a potent sulfhydryl reducing reagent) was unable to prevent the gel
3
filtration-induced loss in [ H]ALD0 binding. Moreover, in gel
filtration experiments, the data obtained in the presence of 2 mM DTT
plus 2 mM molybdate did not differ from those obtained in the presence
of 2 mM molybdate alone. The effect of molybdate on Type I receptors
caused by its presence during gel filtration of cytosol is clearly
different from that seen with these receptors in crude cytosol
preparations, as well as those reported in the literature for other
steroid receptors. Possible mechanisms of action of molybdate on
unoccupied Type I receptors in crude and gel filtered cytosol are
discussed.
Introduc tion
In a series of reports from Pratt's laboratory it was shown that
the addition of sodium molybdate to crude cytosol preparations prevents
the heat-induced loss in the binding capacity of unoccupied
glucocorticoid (Type II) receptors (Nielsen et al., 1977b & c).
Subsequently, Toft and Nishigori (1979) showed that this transition
element oxyanion reversibly blocks the temperature-dependent
transformation (activation) of progesterone receptors to a DNA-binding
state. Later, numerous reports have shown similar stabilizing effects
of molybdate on other steroid receptors (Nishigori and Toft, 1980;

72
ShyamaLa and Leonard, 1980; MilLer et al., 1981) and proteins (Defay et
al., 1984; Nakada et al., 1985; Denison et al., 1986). In contrast to
these findings, recently we have reported that although molybdate
prevents the heat-induced inactivation of Type I receptors, at 0°C, this
compound leads to a dose-dependent reduction in the subsequent binding
3
of [ H]ALD0 in crude whole brain cytosol preparations (Emadian et
al., 1986). In the experiments below, we will describe yet another
novel effect of molybdate on Type I receptors in Sephadex G-25 gel
filtered whole brain cytosol.
Materials and Methods
Chemicals. All chemicals used in the experiments in this chapter
were reagent grade quality.
Buffers. The cytosolic concentration of molybdate and DTT were
obtained by a 9:1 dilution of cytosol with stock buffers containing a
10-fold concentration of the desired component(s). Under such
circumstances, the control group was diluted accordingly with an
equivalent volume of Buffer A. The final pH for all buffers was
adjusted to 7.60 at 0-2°C.
Sephadex G-25 Gel Filtration. Gel filtered receptor preparations
were obtained by loading 0.5 ml aliquots of unlabeled crude cytosol on
Sephadex G-25 columns (0.6 x 14.0 cm) pre-equilibrated in appropriate
buffer(s) at 4-5°C. The sample on each column was allowed to penetrate
the gel and the column was then washed with 1.0 ml buffer.
Subsequently, the macromolecular fraction was collected using an
additional 1.0 ml homologous buffer.

73
Results
Gel filtration of cytosol prepared in Buffer A through Sephadex
G-25 columns (equilibrated and eluted with homologous buffer) led to a
3
70% reduction in the subsequent specific binding of [ H]ALD0 to Type
I receptors when compared to the control group (i.e., non-aged crude
cytosol prepared in Buffer A) (Fig. 4-1, A). There was no additional
loss in the binding capacity of Type I receptors when unlabeled crude or
gel filtered cytosol was aged for 2 h at 0°C prior to incubation with
steroids; however, when the temperature during aging was raised to 22°C,
we observed a 50% reduction in the subsequent Type I receptor binding to
3
[ H]ALD0 (Fig. 4-1, A; 2 h aging). With the exception of a slight
3
increase in [ H]ALD0 binding at 0°C, similar results were obtained
when the above procedures were repeated in the presence of 2 mM DTT
(Fig. 4-1, D). This latter increase in B values was due to lower
u r
B va^-ues crude and especially gel filtered cytosol preparations
containing 2 mM DTT. It should also be noted that unoccupied Type I
receptors in gel filtered cytosol containing DTT appeared to be more
susceptible to the 22°C aging-induced loss in ligand binding.
In agreement with our earlier report (Emadian et al., 1986), at
0°C, the addition of 2 mM molybdate to unlabeled crude cytosol produced
a slight reduction in the binding of [ H]ALD0 to Type I receptors
(Fig. 4-1, M). However, when the molybdate-containing cytosol was
subjected to Sephadex G-25 gel filtration, we observed a 50% increase in
3
the subsequent binding of [ H]ALDO to Type I receptors. Whereas
aging unlabeled crude or gel filtered cytosol in Buffer M at 0°C for 2 h
had no significant effect on the measurable Type I receptor binding,
aging the unlabeled gel filtered, but not crude, cytosol at 22°C for 2 h

Figure 4-1. Effects of Gel Filtration, Molybdate and/or
Dithiothreitol on the Binding Capacity and Stability of Unoccupied
Type I Receptors from Whole Brain Cytosol. Nine parts cytosol
prepared in Buffer A was diluted with either one part Buffer A (A) or
adjusted to 2 mM dithiothreitol (D), 2 mM molybdate (M) or 2 mM
dithiothreitol plus 2 mM molybdate (DM) using one part Buffer A
containing a 10-fold concentration of the respective component(s). Each
group either underwent the gel filtration procedure (for detail see
"Materials and Methods" section) at 4-5°C (triangles) or was left
undisturbed at 4-5°C (circles) during this process. Subsequently, a
portion of the crude or gel filtered cytosol from each group was either
incubated with 10 nM ["^H]ALD0 and 5 uM RU 26988 and minus (B^)
or plus (B„to) 2 uM [LH]ALD0 at 0°C for 24 h for the
determination of specific binding or was aged at 0 (solid symbols) or
22°C (open symbols) for 2 h prior to incubation with the steroids. Gel
filtration reduced the cytosolic protein concentration from 7.3 to
3.3 mg/ml. The data presented are from at least 5 independent replicate
experiments (except for the DM group) and are expressed as a percent of
control* (i.e., specific binding measured in non-aged crude cytosol
prepared in Buffer A) + standard error of the mean (SEM).
100% = 21 fmole/mg protein.

160
140
120
100
80
60
40
20
75
H D M DM
0 2
0 2 0 2 0 2
Duration of Aging (h)

76
led to a marked loss in the binding capacity of these receptors
(Fig. 4-1, M; 2 h aging). The data obtained in the presence of Buffer
DM did not differ appreciably from those described in the presence of
Buffer M with the exception, once again, of a slight exacerbation of the
3
heat-induced loss in the binding of [ H]ALDO to Type I receptors in
gel filtered cytosol following 2 h aging at 22°C (Fig. 4-1, DM; 2 h
aging).
To determine the concentration of molybdate needed to produce the
3
highest increase in the binding of [ H]ALDO to Type I receptors
during gel filtration, we conducted a dose-response analysis of the
effects of this compound on Type I receptors during the gel filtration
procedure. Consistent with our previous observations (Emadian et al.,
1986) , the addition of molybdate to HEPES buffer led to a dose-dependent
3
loss in [ H]ALDO specific binding to Type I receptors in crude
cytosol at 0°C (Fig. 4-2). Aging the crude cytosol for 2 h at 0°C
3
reduced the loss in [ H]ALDO binding seen with the two highest doses
of molybdate. In non-aged gel filtered cytosol, the maximal increase in
3
[ H]ALDO binding to Type I receptors occurred at 2 mM molybdate,
with 10-100 mM concentrations producing binding levels comparable to
those seen in the absence of molybdate (i.e., control group). It is
noteworthy that except for when molybdate was absent during gel
filtration, at all concentrations of this compound tested, the
measurable Type I receptor binding was higher in the non-aged gel
filtered cytosol than non-aged crude cytosol containing the same dose of
molybdate. Furthermore, at all concentrations of molybdate tested,
aging unlabeled gel filtered cytosol for 2 h at 0°C, produced Type I
receptor binding comparable to the non-aged gel filtered preparations

Figure 4-2. Dose-Response Analysis of the Effects of Gel Filtration
and Molybdate on the Binding Capacity and Stability of Unoccupied
Type I Receptors from Whole Brain Cytosol. Nine parts cytosol
prepared in Buffer A was diluted with one part Buffer A containing 0,
20, 100, 200 or 1000 mM molybdate to bring the cytosol to a final
concentration of 0, 1, 2, 10, 20 or 100 mM molybdate, respectively.
Each group then either underwent the gel filtration procedure at 4-5°C
(open symbols) or was left undisturbed at 4-5°C during this process
(solid symbols). Subsequently, a portion of cytosol from each group was
3
either incubated with 10 nM [ H]ALD0 and 5 uM RU 26988 minus
(B ) or plus (B ) 2 uM [^H]ALD0 at 0°C for 24 h for the
I NS
determination of specific binding (open and solid circles) or was aged
at 0 (open and solid squares) or 22°C (open and solid triangles) for 2 h
prior to incubation with the steroids. Gel filtration reduced the
cytosolic protein concentration from 7.3 to 3.3 mg/ml. The data are
from at least 4 independent replicate experiments and are expressed as a
percent of control* (i.e., specific binding measured in non-aged crude
cytosol prepared in Buffer A) + standard error of the mean (SEM).
100% = 18 fmole/mg protein.

160
140
120
100
80
60
40'
20
78
0 2 10 20 DO
[Na ^ MoO^] mM

79
containing the same doses of molybdate. Consistent with the results in
Fig. 4-1, Type I receptors in unlabeled gel filtered cytosol containing
0 (i.e., Buffer A) or 2 mM molybdate (i.e., Buffer M) were found to be
extremely labile when aged for 2 h at 22°C; however, increasing the
concentration of molybdate to 10-100 mM prevents this loss in binding
capacity. Moreover, as reported earlier (Emadian et al., 1986), when
crude, non-gel filtered cytosol preparations are aged for 2 h at 22°C,
3
the highest level of [ H]ALD0 binding was observed in the presence
2 mM molybdate. Further increases in the concentration of molybdate
3
resulted in lower levels of [ H]ALD0 binding, comparable to those
measured in non-aged crude cytosol containing the same concentration of
molybdate.
To gain further insight about the nature of the effects of gel
filtration on the binding of Type I receptors in the presence and
absence of molybdate, Scatchard (1949) analyses of the equilibrium
binding data were performed (Fig. 4-3, Table 4-1). Gel filtration of
cytosol in Buffer M produced no effect on the , but increased the
3
B of Type I receptors for [ HlALDO by 36% above the level
max J r r J
seen in crude cytosol prepared in Buffer A (control). Gel filtration of
cytosol in Buffer A produced a 62% reduction in the B and a
max
3
2-fold increase in the of [ HjALDO binding to Type I
receptors. The presence of 2 mM molybdate in crude cytosol produced a
slight increase in the , but had no apparent effect on the
B TyPe ^ recePtors when compared to the control group.
To see whether the gel filtration-induced loss in Type I receptor
binding capacity observed in Buffer A was reversible, we investigated
the effects of 2 mM molybdate added to cytosol at different time

Figure 4-3. Effects of Molybdate and/or Gel Filtration on the
Equilibrium Binding Parameters of ALDO Binding to Unoccupied Type I
Receptors from Whole Brain Cytosol. Nine parts cytosol prepared in
Buffer A was diluted with either one part Buffer A (solid circles and
triangles) or one part Buffer A containing 20 mM molybdate to bring the
cytosol to a final concentration of 2 mM molybdate (open circles and
triangles). Subsequently, each group either underwent the gel
filtration procedure (triangles) at 4-5°C or was left undisturbed at
4-5°C during this process (circles). All groups were then incubated at
0°C with 0.1-40 nM [^H]ALD0 plus a 500-fold excess of RU 26988 and
either with (B^) or without (B^) a 200-fold excess of
[^H]ALDO at 0°C for 24 h for the determination of specific binding.
3
The concentration of free [ H]ALD0 was determined in every tube by
subtracting B^ from the total radioactivity present. The values for
B and K, were obtained from the x-intercept and negative
max d t- b
inverse of the slope for each line, respectively, by least square linear
regression method. Gel filtration reduced the cytosolic protein
concentration from 7.3 to 3.3 mg/ml. The plots are representative of 2
independent replicate experiments.

/ F (10 mg Protein
81

Table 4-1
Binding
Parameters
Cytoso1
for Type I Receptors Obtained from Crude and Gel
in the Presence and Absence of 2 mM Molybdate
Filtered
Na_MoO.
2 4
G-25
\
B C
max
- r
+
+
0.45
27 .4
0.95
-
-
0.45
20.1
0.97
+
-
0.60
20.3
0.96
-
+
0.82
7.6
0.89
a _
Experimental
conditions are
described under Fig.
4-3.
-15
10 mole/mg protein.

83
intervals following gel filtration. As shown in Fig. 4-4, adding 2 mM
molybdate to cytosol immediately following gel filtration in the
presence of Buffer A led to a significant recovery of Type I receptor
binding capacity; however, the magnitude of this recovery was only
3
sufficient to increase [ H]ALDO binding to the levels seen in the
control group and never to the levels observed in cytosol gel filtered
in the presence of 2 mM molybdate. The magnitude of this recovery was
also time-dependent, since only molybdate added 1 to 5 h following gel
filtration led to a significant recovery of the binding capacity of
Type I receptors. The addition of molybdate to unlabeled gel filtered
cytosol 24 h following gel filtration did not produce any increase in
the binding capacity of Type I receptors. The effects of 2 mM molybdate
on Type I receptor binding capacity in crude and gel filtered cytosol
were comparable to those described in Figs. 4-1 and 4-2.
A number of earlier reports have implicated the role of endogenous
small molecular weight "factors" in cytosol as possible modulators of
hormone binding to glucocorticoid receptors and subsequent activation
(transformation) and DNA binding of the steroid-receptor complexes
(Bailly et al., 1977; Ishohashi et al., 1980; Leach et al., 1982; Dahmer
et al., 1984). Since during Sephadex G-25 gel filtration many small
molecular weight components are removed from cytosol, we sought to
investigate the effects of dilution of cytosol (which in effect dilutes
the concentration of endogenous factor(s) possibly involved in the
3
inhibition of [ H]ALD0 binding to Type I receptors) on the binding
capacity of these receptors. As shown in Fig. 5-5, a 2-fold dilution of
crude cytosol prepared in Buffer A or incubated with 2 mM molybdate for
2 h prior to dilution, produced Type I receptor binding comparable to

Figure 4-4. Stability of Unoccupied Type I Receptors from Whole
Brain Cytosol Following Gel Filtration in the Absence of Molybdate:
Effects of Molybdate Addition Following Gel Filtration and Aging.
Nine parts cytosol prepared in Buffer A was diluted with either one part
Buffer A (-M) or Buffer A containing 20 mM molybdate to bring the
cytosol to a final concentration of 2 mM molybdate (+M, solid bars).
Each group then either underwent the gel filtration procedure at 4-5°C
(+ G-25) or was left undisturbed at 4-5°C during this process (- G-25).
Subsequently, the group that was gel filtered in the absence of
molybdate was divided into two parts. One part was brought to 2 mM
molybdate immediately or 1, 2, 5 and 24 h following gel filtration
(shaded bars). The second part was diluted accordingly with Buffer A
immediately or 1, 2, 5 and 24 h (hash marked bars) following gel
filtration. All other groups were diluted accordingly with appropriate
buffers. Following these steps, all groups incubated with 10 nM
3
[ H]ALD0 and 5 uM RU 26988 minus (B ) or plus (B ) 2 uM
1 No
[^H]ALD0 at 0°C for 24 h for the determination of specific binding.
Gel filtration reduced the cytosolic protein concentration from 7.3 to
3.3 mg/ml. The data are from at least 3 independent experiments and are
expressed as a percent of control* (i.e., specific binding measured in
non-aged crude cytosol prepared in Buffer A) + standard error of the
mean (SEM). 100% = 26 fmole/mg protein.

Percent of Control
85

Figure 4-5. Effects of Dilution and/or Molybdate on the Binding
Capacity of Unoccupied Type I Receptors from Whole Brain Cytosol.
Nine parts cytosol prepared in Buffer A was incubated at 0°C for 2 h
with either one part Buffer A (shaded bars) or Buffer A containing 20 mM
molybdate to bring the cytosol to a final concentration of 2 mM
molybdate (solid bars). One group either underwent the gel filtration
procedure at 4-5°C in columns pre-equilibrated with (G-25, M) or without
(G-25) 2 mM molybdate. A second group was diluted by 2-fold (Dil.)
using either Buffer A or Buffer M, where appropriate. A third batch of
undiluted crude cytosol in Buffer A (the control*) or Buffer M (M) along
with the "Dil." groups remained at 4-5°C during the gel filtration
process. Subsequently, all groups were incubated with 10 nM
[^H]ALD0 and 5 uM RU 26988 minus (B ) or plus (B ) 2 uM
1 No
[^H]ALD0 at 0°C for 24 h for the determination of specific binding.
Dilution and gel filtration reduced the cytosolic protein concentration
from 7.3 to 3.6 and 3.3 mg/ml, respectively. The data are from 4
independent replicate experiments and are expressed as a percent of
control* + standard error of the mean (SEM). 100% = 25 fmole/mg
protein.

Dil. G-25 G-25 G-25 G-25
Percent of Control*
po o o 6 o o
no
o
00
*^l
T
T

88
the levels seen in the control group (i.e., non-diluted crude cytosol
prepared in Buffer A). Gel filtration of cytosol prepared in Buffer A
and/or incubated with 2 mM molybdate for 2 h prior to gel filtration
through Sephadex G-25 columns pre-equilibrated in Buffer M produced
Type I receptor binding 10 and 15% higher than that seen in the control
group, respectively. In contrast, gel filtration of the latter groups
through columns pre-equilibrated in Buffer A led to a significant loss
in the binding capacity of Type I receptors in both of these
preparations.
Discussion
The view that molybdate inhibits heat-, salt- and/or ammonium
sulfate-induced inactivation of the ligand binding capacity of
unoccupied, and the transformation (activation) of occupied,
glucocorticoid Type II and other steroid receptors is well accepted
(e.g., Leach et al., 1979; Sando et al., 1979a & b; Nishigori and Toft,
1980; Shyamala and Leonard, 1980; McBlain and Shyamala, 1980; Noma et
al., 1980; Housley et al., 1982; Murakami et al., 1982; Blanchardie et
al., 1984; Luttge and Densmore, 1984; Luttge et al., 1984a, b & d; Weisz
et al., 1984; Miller-Diener et al., 1985; Wilson et al., 1986); however,
despite the widespread use of this, and other transition metal oxyanions
(e.g., vanadate and tungstate), the exact mechanism(s) by which these
actions are mediated still remains unclear. The original reports
describing the profound stabilizing effects of molybdate on time- and
temperature-dependent inactivation of Type II receptors were based on
molybdate's purported phosphatase-inhibitor activity (Nielsen et al.,
1977b & c; Richards and Swisloki, 1979). The success of these early
studies led to the hypothesis that molybdate prevents both unoccupied

89
receptor inactivation and occupied receptor transformation by inhibiting
enzyme-induced dephosphorylation of the receptor (Sando et al.,
1979a & b; Barnett et al., 1980; Housley et al., 1982). However, the
observations that molybdate does not prevent alkaline phosphatase-
induced inactivation of unoccupied Type II receptors (Leach et al.,
1983), that other phosphatase inhibitors, including fluoride and
glucose-1-phosphate, do not prevent heat-induced transformation of
occupied Type II receptors (Nielsen et al., 1977b) and that molybdate
can still exert its stabilizing and transformation inhibiting actions
even when working with highly-purified (and presumably
phosphatase-lacking) receptor preparations (Weisz et al., 1984;
Miller-Diener et al., 1985) clearly suggests that phosphatase inhibition
is not the principal mechanism of molybdate's actions on steroid hormone
receptors. These and other observations have formed the basis for a new
hypothesis that molybdate may stabilize Type II, and possibly other
steroid, receptors by direct interactions with the receptor
macromolecules. In this regard, the formation of complexes between
molybdate and sulfhydryl moiety(ies) (possibly near the steroid binding
site(s)) has been proposed (Housley et al., 1982; Blanchardie et al.,
1984; Dahmer et al., 1984; Potgieter et al., 1986; Wilson et al., 1986).
Recently, we reported that although molybdate was very effective in
crude cytosol in preventing the time-dependent, heat-induced loss in
Type I receptor binding capacity, this stabilization was accompanied by
a molybdate dose-dependent (and time-independent) loss in the
ligand-binding capacity of these receptors (Emadian et al., 1986).
Tungstate has been reported to produce a similar dose-dependent
reduction in the binding capacity of progesterone receptors; however,

90
this effect was reflected as an increase in the K, with no effect on
d
the B (Murakami et al., 1982), whereas in our work with Type I
max J r
receptors, the reduction in binding was reflected as a decrease in the
B with no effect on the K, (Emadian et al., 1986). It is
max d
3
noteworthy that the increase in [ H]ALD0 binding to Type I receptors
following gel filtration of molybdate-containing cytosol shown in this
study, was also reflected as an increase in B with no effect on
max
Kd (Fig. 4-3).
If we assume that certain populations and/or conformations of
Type I receptors may have more steroid binding sites per molecule than
other populations and/or conformations, then the data from our previous
(Emadian et al., 1986) as well as our present studies suggest that
molybdate may mediate its facilitatory and inhibitory actions on Type I
receptor binding capacity by stabilizing different populations and/or
conformations of the receptor which in turn increase or decrease the
total number of specific binding sites available under different assay
conditions. The lack of an effect on the during both of these
situations suggests that all of the binding sites have a similar
3
affinity for [ H]ALDO. Similarly, the lack of curvature in the
Scatchard plots during both of these situations suggests that there are
no cooperative (positive or negative) interactions between the
3
[ H]ALD0 specific binding sites. Assuming, therefore, that the
number of steroid binding sites per Type I receptor molecule is not
fixed, then the data from the Sephadex G-25 gel filtration experiments
suggests that endogenous small molecular weight factors present in crude
cytosol may also be able to modulate this number in much the same
fashion as seen with molybdate. The concentration of this factor(s) in

91
crude cytosol (as well as in cytoplasm) appears to be in excess of that
needed for maximal binding, since a 50% reduction in the concentration
of this factor(s), as well as the receptor and other macromolecules, as
would be produced during Sephadex G-25 gel filtration, was shown to have
3
no effect on the number of [ H]ALD0 specific binding sites
(Fig. 4-5).
The addition of 2 mM molybdate to cytosol prior to the removal of
this putative factor(s) (i.e., prior to gel filtration) produces a
3
subtle reduction in [ H]ALD0 binding to Type I receptors (Emadian et
al., 1986; Figs. 4-1 through 4-5). Note that this effect is clearly
different from the increases in the absolute levels of receptor binding
capacity seen when either molybdate-containing or molybdate-free cytosol
is gel filtered in the presence of 2 mM molybdate (Figs. 4-1 through
4-5). When 2 mM molybdate is added after gel filtration there is a
time-dependent loss in its ability to stabilize/increase Type I receptor
binding capacity even when the cytosol is maintained at 0°C (Fig. 4-4).
Furthermore, although the addition of 2 mM molybdate either prior to, or
during gel filtration, produces a 15-50% increase in binding capacity,
this unliganded receptor preparation is very unstable when incubated at
22°C (Figs. 4-1 and 4-2). To stabilize unoccupied Type I receptors
during 22°C aging of the gel filtered cytosol, molybdate concentrations
above 10 mM were found to be required (Fig. 4-2).
On the basis of these results, we speculate that the addition of
2 mM molybdate prior to, or during gel filtration of brain cytosol
induces a thermally-labile conformation of the Type I receptor which has
a greater number of binding sites per molecule than found for Type I
receptors in crude cytosol (molybdate-free or molybdate-containing).

92
The reduction in binding seen when higher than 2 mM doses of molybdate
are used with gel filtered cytosol, may reflect the stabilization of a
thermally-resistant receptor conformation with an intermediate number of
3
[ HjALDO binding sites. Experiments investigating the above
possibilities are currently under way in our laboratory.

CHAPTER V
DIFFERENTIAL INACTIVATION OF TYPE II RECEPTORS FOR ADRENAL STEROIDS
IN WHOLE BRAIN CYTOSOL: RECOVERY OF INTACT TYPE I RECEPTORS AND
DISCOVERY OF A NEW CLASS OF TYPE II RECEPTORS
Summary
Recently we reported that whereas Type II receptors for
adrenocorticosteroids in brain cytosol preparations required a
sulfhydryl reducing reagent for optimal ligand binding, Type I receptors
manifested maximal binding capacity in the absence of reducing reagents
(Emadian et al., 1986). Furthermore, removal of endogenous reducing
agents from steroid-free cytosol (prepared in the absence of a reducing
reagent) by dextran-coated charcoal (DCC) was found to further
exacerbate the ligand binding capacity of Type II, but not Type I,
receptors (Emadian et al., 1986). To extend the observations reported
previously from this laboratory (Densmore et al., 1984a), in the
experiments below, we also found unoccupied Type II, but not Type I,
receptors lose their ligand binding capacity in the presence of 300 mM
KC1, especially when incubated at 0°C. In this chapter, the differences
between Type I and Type II receptors were utilized to differentially
inactivate the latter, in order to purify and characterize the nuclear
acceptor site(s) specific for Type I receptors in future experiments.
Although unoccupied Type I receptors were insensitive to the
effects of the reducing reagent, dithiothreitol (DTT), inclusion of 1 mM
5,51-dithiobis (2-nitrobenzoic acid) (DTNB) completely inactivated the
ligand binding capacity of these receptors. This DTNB-induced loss in
ligand binding could, however, be reversed by adding a 10-fold excess
93

94
concentration of DTT to the DTNB-treated cytosol. In contrast, the
3
effects of DTNB on receptors prelabeled with [ H]ALDO in the
presence or absence of RU 26988 was found to be minimal. Consistent
with our earlier report (Eraadian et al., 1986), DCC pretreatment of
steroid-free cytosol led to a marked reduction (80 to 90%) in the
binding capacity of Type II, but not Type I, receptors. Although this
loss in ligand binding capacity could be reversed by adding 2 mM DTT to
the DCC-pre treated cytosol, the magnitude of the recovery was dependent
on the concentration and/or the duration of DCC pretreatment. The
higher the concentration of DCC and/or the longer the duration of the
pretreatment, the less the magnitude of the recovery. Aging the DCC
pretreated preparations at 0°C caused a progressive loss in the
subsequent ligand binding capacity of unoccupied Type II, but not Type
I, receptors. Conversely, a loss in the ligand binding capacity of
unoccupied Type II, but not Type I, receptors was observed when
untreated cytosol preparations were aged at 0°C in the absence of DTT.
Use of a molar excess concentration of a number of synthetic ligands for
Type I and Type II receptors (e.g., RU 26988, prorenone and RU 26752) in
3 3
conjunction with [ H]ALDO or [ H]DEX revealed that the remaining
3
10 to 20% [ H]DEX binding seen following DCC pretreatment of cytosol
was mostly to a class of Type II receptors with chemical properties
distinct from the classical Type II receptors. Possible physiological
and methodological significance of these findings is discussed.
Introduction
Within the past three decades, numerous reports on the properties
as well as functional and physiological relevance of steroid hormone
receptors have appeared in the literature. Although much has been done

95
to expand our knowledge regarding the effects of raineralocorticoid
hormones on electrolyte balance (see Chapter I), the volume of research
on the properties of the receptors through which these effects are
mediated has been minimal by comparison (Liao et al., 1983). This is
partially due to the fact that in crude cytosol preparations
3
[ H]ALDO and other readily available radiolabeled synthetic ligands
for Type I receptors bind to Type II receptors with appreciable affinity
(Rousseau et al., 1972; Funder et al., 1973b; Emadian et al., 1986;
Emadian and Luttge, unpublished). Recently, however, the synthesis of a
number of very specific ligands for Type II receptors (e.g., RU 26988,
RU 28362 and RU 38486) has made the elimination of the confounding
3
contribution of [ HjALDO-binding to Type II receptors in crude
preparations possible (Moguilewsky and Raynaud, 1980; Veldhuis et al.,
1982b; Gomez-Sanchez and Gomez-Sanchez, 1983; Wrange and Yu, 1983;
Moguilewsky and Philibert, 1984; Emadian et al., 1986). Although this
competition method for the elimination of Type II receptors may be
acceptable under some experimental conditions (e.g., saturation and
equilibrium binding analyses, hydrodynamic parameter characterization
studies, etc.), at times, the presence of viable Type II receptors may
prove undesirable in crude preparations (e.g., during purification
procedure using steroid affinity gels, characterization of specific
3
nuclear acceptor sites for activated [ H]ALDO-Type I receptor
complexes, development of specific monoclonal antibodies for Type I
receptors, etc .).
Recently, we have demonstrated several in vitro chemical
differences between Type I and Type II receptors in brain cytosol which
may prove important in the in vivo regulation of these receptors.

96
For example, we showed that although charcoal-extraction of steroid-free
cytosol in HEPES buffer lacking dithiothreitol (DTT) causes a dramatic
reduction in Type II binding capacity (possibly by removal of endogenous
thioredoxin and NADPH), this treatment was found to have no effect on
the binding capacity of Type I receptors in the same preparations
(Emadian et al., 1986). Furthermore, DTT, while required for the
expression of optimal ligand binding capacity of Type II receptors, was
found to have no effect on unoccupied Type I receptors in whole brain
cytosol. In the experiments described here, we took advantage of the
chemical differences between Type I and Type II receptors and attempted
to differentially inactivate the Type II receptors in crude whole brain
cytosolic preparations. As a result of this work, we report here on the
discovery and possible functional and physiological relevance of a new
class of glucocorticoid binders, distinct from the classical Type II
receptors.
Materials and Methods
Chemicals. 5,51-dithiobis (2-nitrobenzoic acid) (DTNB, shown to be
a very specific sulfhydryl oxidizing reagent, Brucklehurst, 1979),
Dextran T70 and activated charcoal (Norite A) were obtained from Sigma
Chemical Co. All other chemicals used in these experiments were reagent
grade quality.
Buffers. The cytosolic concentration of the component(s) in Buffers
D, M20 and DM2q were obtained by a 9:1 dilution of cytosol with
stock buffers containing a 10-fold concentration of the desired
component(s). Under such circumstances, the control group was diluted
accordingly with an equivalent volume of Buffer A. Similarly, the final
concentration of DTNB was obtained by adding one volume of 10 mM

97
buffered solution of this compound to 9 volumes of cytosol to yield 1 mM
concentration of this compound in cytosol. Note that to dissolve 10 mM
DTNB in our buffers we had to start with buffers in acidic pH ranges
(i.e., 4.5-5.5) and then readjust the pH to 7.60 prior to use. All
other buffers were also adjusted to final pH 7.60 at 0-2°C prior to use.
DCC Pretreatment of Cytosol. The dextran-coated charcoal (DCC)
mixture was prepared overnight by adding a 1:2 ratio of dextran:charcoal
to Buffer A. Just prior to use, the desired volume of DCC suspension
was centrifuged at 2,230 x g for 10 min, the supernatant discarded and
an equivalent volume of cytosol added to the pellet. The DCC was
resuspended by gentle vortexing and the mixture incubated at 0°C for the
length of time indicated for each specific experiment. During this
incubation, the mixture was vortexed gently every 5 min. At the end of
the incubation period, the DCC was pelleted by centrifugation at
2,230 x g for 10 min and the resulting supernatant (i.e., DCC-pretreated
cytosol) carefully aspirated for subsequent steps. Control groups
underwent identical procedural steps in the absence of DCC in parallel
with their DCC-pretreated counterparts.
Results and Discussion
Effects of DTNB on Unoccupied Type I Receptors.
As mentioned above, we have recently shown that in crude
steroid-free cytosol preparations, unlike Type II receptors, Type I
receptors do not require a sulfhydryl reducing reagent for optimal
ligand binding (Emadian et al., 1986). Furthermore, we and others have
shown that unoccupied Type II receptors can be completely and reversibly
inactivated by specific sulfhydryl oxidizing reagents such as DTNB
(Bodwell et al., 1984; Densmore, 1986; Emadian and Luttge, unpublished).

98
Recently, in their investigation of the role of sulfhydryl groups as a
potential regulatory mechanism for Type I receptor binding capacity,
Tashima et al. (1984) employed a number of sulfhydryl oxidizing reagents
3
to induce inactivation of the swine kidney cytosolic "[ H]ALDO
receptors." "Mild" sulfhydryl oxidizing reagents such as DTNB were
found to promote a rapid but DTT-reversible inactivation of ALDO binding
capacity. Although this study may be accurate in terms of the reported
data, the interpretation of the results must take into account the
3
authors' failure to eliminate [ H]ALDO binding to Type II receptors.
Therefore, what Tashima et al. (1984) refer to as "aldosterone
receptors," is probably a heterogeneous population of Type I and Type II
receptors. Note that kidney is a rich source of Type II receptors
(Wrange and Yu, 1983; Emadian and Luttge, unpublished; Luttge and Rupp,
unpublished), and that ALDO binds to Type II with appreciable affinity
(Rousseau et al., 1972; Funder et al., 1973b; Raynaud et al., 1980;
Emadian and Luttge, unpublished; Luttge and Rupp, unpublished).
Because of the direct relevance of the study by Tashima et al.
(1984) to our intentions of wanting to chemically eliminate Type II
receptors in crude preparations, we investigated the effects of DTNB on
Type I receptors in whole brain cytosol. Note that in our studies we
3
included a 500-fold molar excess of RU 26988 to eliminate [ H]ALD0
cross-binding to Type II receptors. It is apparent that if DTNB is
found to have no effect on Type I receptors, this reagent could be used
in buffer preprations to inactivate Type II receptors selectively and
completely.
In agreement with the report by Tashima et al. (1984), the addition
3
of 1 mM DTNB to unlabeled cytosol virtually eliminated all [ H]ALD0

Table 5-1. Nine parts cytosol prepared in Buffer A incubated with either one
part Buffer A or adjusted to 1 mM DTNB using one part Buffer A containing 10 mM
DTNB for 1 h at 0°C. Subsequently, the group that received Buffer A was diluted
with either sufficient Buffer A containing 100 mM DTT to yield a final 10 mM
concentration of DTT (-DTNB, +DTT) or additional Buffer A (Control; -DTNB, -DTT)•
The DTNB group was either brought to 10 mM concentration of DTT (+DTNB, +DTT) or
diluted with an equivalent volume of 1 mM DTNB (+DTNB, -DTT). Following such
3
treatments, all groups incubated for 24 h at 0°C with 10 nM [ HjALDO plus 5 uM
RU 26988 and either with (B ) or without (B ) 2 uM [^H]ALD0 for the
Wb 1
determination of specific binding (B = B^ - B^) as described in
Chapter II. The data presented are from 3 independent replicate experiments and
are expressed as a percent of specific binding measured in the control group +
standard error of the mean (SEM). 100% = 29 fmole/mg protein.

Table 5-1
Effects of 1 tnM DTNB on Whole Brain Cytosolic
Unoccupied Type I Receptors
CONDITION
PERCENT OF CONTROL B + SEM
o r *“
Control (-DTNB, -DTT) 100 + 0
-DTNB, +DTT 108 + 5
+DTNB, -DTT 2+1
+DTNB, +DTT 90+5
100

101
binding to Type I receptors (Table 5-1). As is the case with Type II
receptors, this inactivation was found to be completely reversible by
replenishing the DTNB-treated cytosol with a 10-fold excess
concentration of DTT. Consistent with our earlier report (Emadian et
al., 1986), the addition of DTT to crude cytosol preparations had no
3
significant effect on the binding of [ H]ALD0 to Type I receptors.
Effects of DTNB on Occupied Receptors.
Since DTNB and other sulfhydryl oxidizing reagents were found to
act primarily on unoccupied receptors, earlier studies have implicated
that these reagents inactivate Type II receptors by interacting with
sulfhydryl group(s) in the steroid binding domain of these receptors
(Kalimi and Love, 1980; Bodwell et al., 1984). On the basis of these
reports and the fact that the equilibrium dissociation constant (K^)
3
of [ H]ALD0 from Type II receptors is about two orders of magnitude
greater than that from Type I receptors (Emadian et al., 1986), we
attempted to eliminate Type II receptors with DTNB in crude cytosol
3
preparations prelabeled with [ H]ALD0. Our predictions were that
3
because of the low affinity of [ H]ALD0 for Type II receptors, at
3
any given time, the chances of [ H]ALD0 dissociation from and
subsequent inactivation of Type II receptors by DTNB would be greater
3
than [ H]ALD0 reassociation with these receptors. As shown in Fig.
5-1, prolonged incubation of prelabeled cytosol with 1 mM DTNB (i.e., up
to 7 h) led to a slight, but significant loss in [ H]ALD0 binding
(in the presence and absence of RU 26988). It is noteworthy that the
3
inactivating effects of DTNB on [ H]ALD0-bound receptors were not as
dramatic as those seen with unoccupied Type I (and Type II) receptors.

Figure 5-1. Effects of 1 mM DTNB on Receptors Bound to ALDO in
Whole Brain Cytosol. Cytosol prepared in Buffer A was incubated
with 10 nM [^H]ALD0 (triangles) or 10 nM [^HjALDO plus 5 uM
RU 26988 (circles) and either with (BlT„) or without (B„) 2 uM
No T
[^H]ALD0 for 48 h at 0°C. After this incubation period, each group
either received sufficient Buffer A containing 10 mM DTNB to yield a
final 1 mM cytosolic concentration of this compound (open trangles and
circles) or diluted with an equivanlent volume of Buffer A (solid
triangles and circles). Subsequently, at various time points, a
fraction from each group was loaded on sephadex G-25 columns for steroid
binding determination as described in Chapter II. the data presented
are the mean from 3 independent replicate experiments and are expressed
as a percent of control* (i.e., specific binding measured in
3
[ H]ALD0 + RU 26988, -DTNB, 1 h aging group) + standard error of
the mean (SEM). 100% = 25 fmole/mg protein.

Percent of Control*
103
Duration of Aging at 0°C (h)
7

Figure 5-2. Effects of DCC Pretreatment and/or Aging of Unlabeled
WhoLe Brain Cytosol on the Binding of ALDO to Type I and DEX to
Type II Receptors. Cytosol prepared in 20 mM HEPES was either
pretreated for 20 min at 0°C with DCC (0.625% dextran plus 1.25%
charcoal) as described under "Materials and Methods" (open symbols) or
underwent the procedure in the absence of DCC (solid symbols).
Subsequently, each group was aged for 0, 1, 3 or 12 h at 0°C prior to
incubation with steroid(s). Following this aging, specific Type I
3
receptor binding was determined in the presence of 10 nM [ H]ALD0
plus 5 uM RU 26988 and either with (B ) or without (B ) 2 uM
NS X
[^H]ALD0 (circles). Type II receptor binding was determined in the
3
presence of 20 nM [ H]DEX and either with (B ) or without
NS
(B^,) 4 uM [^H]DEX (triangles). The data presented are the mean
from 2 independent replicate experiments and are expressed as a percent
of control* (no DCC pretreatment, 0-h aging group). 100% = 23 fmole/mg
protein for Type I and 341 fmol/mg protein for Type II receptors,
respectively.

105

106
Furthermore, it is apparent that with this strategy we were unable to
achieve a selective Type II receptor inactivation in these preparations.
Effects of DCC Pretreatment of Cytosol on Ligand Binding Capacity of
Unoccupied Type I and Type II Receptors.
As mentioned above, we reported earlier that DCC pretreatment of
crude cytosol preparations inactivates the ligand binding capacity of
Type II, but not Type I, receptors (Emadian et al., 1986). Since this
reversible and selective inactivation appeared to be partial, in the
next series of experiments we attempted to completely inactivate the
3
remaining specific [ H]DEX binding using several modified DCC
pretreatment procedures.
Because Type II, but not Type I, receptors in the absence of DTT
lose their ligand binding capacity in a time-dependent manner (Emadian
et al., 1986; Densmore, 1986), we sought to investigate the effects of
3
aging on the remaining [ H]DEX binding in DCC pretreated cytosol
preparations. It is important to emphasize that our expectations were
that the DCC pretreatment procedure would further exacerbate this DTT-
and time-dependent Type II receptor inactivation by removing endogenous
sulfhydryl reducing agents from cytosol. Consistent with our previous
report (Emadian et al., 1986), DCC pretreatment of cytosol in Buffer A
(note that this buffer lacks 10% glycerol used in our earlier studies)
3
selectively reduced the [ H]DEX binding by about 80% (Fig. 5-2).
Although aging the crude cytosol at 0°C produced a significant loss in
3
specific [ H]DEX binding, aging the DCC pretreated preparations for
up to 12 h caused no further loss on the receptor binding capacity of
this steroid. In contrast, unoccupied Type I receptors in the crude
untreated, but not DCC-pretreated cytosol preparations were unaffected
by aging for up to 12 h at 0°C. Parenthetically, this latter finding is

107
in agreement with our earlier observations that unoccupied Type I
receptors in crude whole brain cytosol prepared in buffer A are fairly
stable for up to 48 h at 0°C (Emadian and Luttge, unpublished, see
Chapter III).
To achieve our original goal of complete and selective inactivation
of Type II receptors in cytosol preparations, we next sought to
investigate the effects of DCC concentration and duration of cytosol
3
pretreatment on the inactivation of the remaining [ H]DEX binders.
As shown in Fig. 5-3, neither the concentration, nor the duration of
treatment with a fixed concentration of DCC mixture produced further
3
inactivating effect on the remaining [ H]DEX binders. Moreover, two
consecutive 20-min pretreatments of cytosol with DCC (0.1325% Dextran
3
plus 0.625% charcoal) produced [ H]DEX binding comparable to the
values seen with one DCC treatment (i.e., 9 vs. 11%, respectively)
(Table 5-2). It is noteworthy that an increase in the concentration,
duration and the number of DCC treatments significantly reduced the
3
[ H]DEX binding that could be recovered by adding 2 mM DTT and 20 mM
molybdate to the DCC pretreated preparations.
3
Because, 1) the specific [ H]DEX binding remaining after DCC
3
pretreatment of cytosol is approximately 1/10 of total [ H]DEX
binding in crude cytosol prepared in Buffer DM (an optimal buffer for
Type II receptors to bind ligand), 2) the concentration of Type I
receptors in whole brain cytosol preparations on a fmole/mg protein
basis is approximately 1/10 of the Type II receptors in the same
preparations (Wrange and Yu, 1983; Emadian et al., 1986), 3) Type I
receptors are unaffected by the DCC pretreatment procedures and 4) there
3
is a small, but significant [ H]DEX cross-binding with Type I

Figure 5-3. Effects of Pretreatment of Whole Brain Cytosol with
Various Concentrations of DCC and Different Duration of DCC
Pretreatment on the Binding of DEX to Type II Receptors. Cytosol
prepared in 20 mM HEPES buffer was either (a) pretreated for 20 min at
0°C with 1/8, 1/4, 1/2, 1, 2 or 4-fold concentration of DCC used in Fig.
5-2 (i.e., 0.625% Dextran to 1.25% charcoal); (b) pretreated with
0.3125% dextran and 0.625% charcoal mixture from 10 to 120 rain at 0°C as
described under "Materials and Methods" (the 1/2 group) or (c) underwent
the procedure in the absence of DCC (the 0 group). Subsequently, 9
parts of each group was diluted either with one part 20 mM HEPES buffer
containing 20 mM DTT and 200 mM molybdate to yield a final concentration
of 2 mM DTT and 20 mM molybdate in cytosol (open and hash marked bars)
or diluted appropriately with one part 20 mM HEPES buffer (solid bars).
Following these steps, all groups were incubated for 24 h at 0°C with 20
3
nM [ H]DEX and either with (B ) or without (B ) 4 uM
IN o i.
[^H]DEX. The data presented are the mean from at least 2
independent experiments and are expressed as a percent of control* (open
bar). 100% = 427 fmol/mg protein.

Percent of Control
109
0
1/8 1/4
1/2
100-
DCC _
+
4-
Pretreatment
Duration (min)
20
20
*h + 4- 4* ■+• 4-
10, 20,40,60,90,120 20 20 20

Table 5-2. Cytosol prepared in 20 mM HEPES buffer was pretreated once (+DCC)
or twice (+2x DCC) consecutively for 20 min each time at 0°C with DCC (i.e.,
0.3125% Dextran to 0.625% charcoal) as described under "Materials and methods,"
underwent the procedure in the absence of DCC (-DCC). Subsequently, 9 parts of
each group was diluted either with one part 20 mM HEPES buffer containing 20 mM
DTT and 200 mM molybdate to yield the composition of Buffer DM^q (+DM) or
diluted appropriately with one part 20 mM HEPES buffer (-DM). Following these
3
steps, all groups were incubated for 24 h at 0°C with 20 nM [ H]DEX and either
with (B ) or without (B ) 4 uM [^H]DEX. The data presented are the
Wu 1
mean from at least 2 independent replicate experiments and are expressed as a
3
percent of control (specific [ H]DEX binding in -DCC, +DM group) +
standard error of the mean (SEM) when the mean is from at least 3 independent
replicate experiments. 100% = 427 fmole/mg protein.
or

Table 5-2
Effects of Two Consecutive DCC Pretreatments of Cytosol
on the Residual DEX Specific Binding
CONDITION
PERCENT OF CONTROL + SEM
SP —
Control (-DCC, +DM) 100 + 0
-DCC, -DM 70+2
+DCC, -DM 11
+DCC, +DM 97
+2x DCC, -DM 9+1
+2x DCC, +DM
87 + 1
111

112
receptors (Raynaud et al., 1980), we found it essential to eliminate the
3
possibility that the remaining [ H]DEX binding following DCC
pretreatment represent binding of this ligand to Type I receptors.
In the next experiment, we measured Type I and Type II receptor
binding in DCC pretreated preparations in the presence and absence of a
"specific" Type I and Type II receptor blocker; i.e., prorenone and RU
26752 (antiraineralocorticoid spironolactones) and RU 26988 (a specific
3
antiglucocorticoid), respectively, in conjunction with [ H]ALD0 and
3
[ H]DEX. Despite the reported specificity of RU 26752 and prorenone
for Type I receptors (e.g., Claire et al., 1979 ; Coirini et al., 1985),
the introduction of a 500-fold molar excess of prorenone RU 26752 (Fig.
5-4) in incubation media produced an 80% and 99% reduction (-DCC, -DM)
in the measurable [ H]DEX binding, respectively. Furthermore, even
in the presence of prorenone, there was a small but rather significant
3
residual [ H]DEX binding after pretreatment of cytosol with DCC
(+DCC, -DM). As shown in Fig. 5-4, the magnitude of [^H]DEX binding
prior to as well as following DCC pretreatment was the same when the
incubation media contained a 500-fold molar excess of RU 26988 and RU
26752. By comparison, in the absence of RU 26988, there was a 50%
3
increase in the measurable specific [ H]ALD0 binding (i.e.,
3
[ HjALDO binding in the absence of RU 26988 was about 17% of total
3
[ H]DEX binding, whereas that seen in the presence of this Type II
receptor blocker was about 9%) (Fig. 5-4). As a caution, it is
important to note that although these synthetic steroids may manifest
biological specificity for the action of an endogenous steroid, one
cannot conclude binding specificity for the receptors through which
the action(s) of these endogenous steroids are presumably mediated.

Figure 5-4. Effects of DCC Pretreatment of Whole Brain Cytosol on
the Binding of ALDO and DEX to Type I and Type II Receptors in the
Presence or Absence of a Steroid Competitor. Cytosol prepared in
20 mM HEPES was either pretreated for 20 rain at 0°C with DCC (0.1325%
dextran to 0.625% charcoal) as described under "Materials and Methods"
(+DCC) or underwent the procedure in the absence of DCC (-DCC).
Subsequently, nine parts cytosol from each group was diluted with either
one part 20 mM HEPES buffer containing 20 mM DTT and 200 mM molybdate to
yield a final concentration of 2 mM DTT and 20 mM molybdate in cytosol
(+DM) or diluted appropriately with one part 20 mM HEPES buffer (-DM).
Following such treatments, each group was incubated with the following
steroid(s) for 24 h at 0°C prior to specific binding determination, a)
20 nM [3H]DEX, b) 20 nM [3H]DEX + 10 uM Prorenone, c) 20 nM
[3H]DEX + 10 uM RU 26752, d) 20 nM[3H]DEX + 10 uM RÜ 26988, e)
10 nM [3H]ALD0 and f) Open bars: 10 nM [3H]ALDO + 5 uM RU
3 3
26988. Non-specific binding for [ H]ALD0 and [ H]DEX was
determined in the presence of 2 uM [^H]ALD0 and 4 uM [^H]DEX,
respectively. The data presented are the mean from 3 independent
replicate experiments and are expressed as a percent of control*
3
([ HjDEX binding in -DCC, +DM group) + standard error of the
mean (SEM). 100% = 460 fmole/mg protein.

Percent of Control
114
DM + + 4- +

115
Since the inactivating effects of DCC on Type II receptors was
partial, and because 300 tnM KCI was found to produce a time-dependent
loss in the binding capacity of unoccupied Type II, but not Type I
receptors (Fig. 5-5; Densmore et al. 1984a), we next attempted to
3
inactivate the residual 10-20% [ H]DEX binding by incubating DCC
pretreated cytosol preparations in the presence of 300 mM KCI.
Parenthetically, it is interesting to note that although 20 mM molybdate
3
leads to significant reduction in the subsequent binding of [ H]ALD0
to Type I receptors (Fig. 5-5, KM; also see Fig. 4-2 and Emadian et al.,
1986), in the presence of 300 mM KCI, the magnitude of specific
3
[ H]ALDO binding is even higher than the level seen in the absence
of these buffer components (Fig. 5-5, A). It is entirely possible that
300 mM KCI in conjunction with 20 mM molybdate induce a conformation of
Type I receptors that is optimal for ligand binding. This binding
increase is also seen in the presence of 10% ethanol (Chapter III) and 2
mM molybdate in gel filtered cytosol preparations (Chapter IV).
As shown in Fig. 5-6, 300 mM KCI was found to be ineffective in
3
further eliminating the [ H]DEX binders resistant to the
inactivating effects of DCC. Note that DCC and/or 300 mM KCI
3
significantly reduced the binding of [ H]ALD0 in the absence, but
not presence of RU 26988. Therefore, it appears that a significant
3
proportion of [ H]ALD0 binding in untreated cytosol in the absence
of RU 26988 is to Type II receptors sensitive to the inactivating
effects of DCC and KCI (i.e., the classical Type II receptors).
A possible mechanism by which KCI inactivates unoccupied Type II
receptors may be through subunit dissociation of the multimeric form of
the receptors; i.e., by generating the 92 Kda steroid-binding subunit

Figure 5-5. Effects of 300 mM KC1 in the Presence and/or Absence of
20 mM Molybdate on the Binding of ALDO to Type I and DEX to Type II
Receptors. Nine parts cytosol prepared in Buffer A was diluted
either with one part 20 mM HEPES buffer containing 200 mM molybdate (M),
3 M KC1 (K), 200 mM molybdate plus 3 M KC1 (KM) or 20 mM HEPES buffer
(A) to yield the concentration of the desired component(s) in cytosol.
Subsequently, each group either incubated with steroid(s) for 24 h at
0°C or aged at 0°C from 2 to 6 h prior to incubation with steroid(s).
Circles: Type I receptor binding was determined in the presence of 10
nM [^H]ALD0 and 5 uM RU 26988 (B^,) . Triangles: Type II
3
receptor binding was determined using 20 nM [ H]DEX (B^).
Non-specific binding for Type I and Type II receptors was determined in
the presence of 2 uM [^H]ALD0 and 4 uM [^H]DEX, respectively.
The data presented are the mean from 4 independent replicate experiments
and are expressed as a percent of control* (receptor binding in non-aged
cytosol in Buffer A) + standard error of the mean (SEM). 100% for
Type I receptors = 23 fmol/mg protein and 100% for Type II receptors =
275 fmol/mg protein.

117

Figure 5-6. Effects of 300 mM KC1 on the Specific Binding of ALDO
and DEX Prior and Subsequent to DCC Pretreatment. Cytosol prepared
in 20 mM HEPES buffer was either treated with DCC (+DCC), or underwent
the procedure in the absence of DCC (-DCC) as described under "Materials
and Methods." Subsequently, nine volumes of DCC- or non-DCC-treated
cytosol was diluted with either one volume of 3 M KC1 (+KC1) or one
volume of 20 mM HEPES buffer (-KC1). Following such treatments, each
group was incubated with the following steroid(s) for 24 h at 0°C: a)
20 nM [^H]DEX (solid bars), b) 10 nM [^H]ALD0 (shaded bars) and
3
c) 20 nM [ H]ALD0 + 5 uM RU 26988 (open bars). Non-specific binding
3 3
for [ H]ALD0 and [ H]DEX was determined in the presence of 2 uM
[^H]ALD0 and 4 uM [^HjDEX, respectively. The data presented are
the mean from 5 independent replicate experiments and are expressed as a
percent of control* (i.e., specific [ H]DEX binding measured in -DCC
and -KC1 group) + standard error of the mean (SEM). 100% = 315
fmol/mg protein.

Percent of Control
119
DCC
KCI
- + +
+ - +
+ +
- + - +
+ +
- + - +

120
(the "activated" receptor) (for detail se Chapter VI). Note that the
results from this and other laboratories suggest that the activated
Type II receptors in crude cytosol preparations are unable to rebind
glucocorticoids following ligand dissociation (Chou and Luttge, 1987).
The idea that KC1 may inactivate Type II receptors through subunit
dissociation is further supported by the fact that molybdate, which
prevents the salt induced activation of occupied Type II receptors (see
Chapter VI), is also capable of blocking the inactivating effects of KC1
on unoccupied Type II receptors (Fig. 5-5, KM).
3
Collectively, these data suggests that the [ H]DEX binding (and
some of [ HjALDO binding in the absence of RU 26988) remaining after
DCC pretreatment of cytosol represents, at least in part, ligand binding
to a new class of Type II receptors with chemical properties distinct
from the classical Type II receptors. Further evidence for this claim
comes from a close examination of the data presented in Fig. 5-2.
3
Whereas the receptors from DCC pretreated cytosol that bound [ H]DEX
were unaffected by aging the unlabeled cytosol at 0°C, Type I receptors
3
(i.e., [ HjALDO binding in the presence of RU 26988) in the same
preparations appeared to lose their ligand binding capacity in a
time-dependent manner at 0°C.
In conclusion, aside from the potential methodological
implications, the data presented here provide a number of suggestions
regarding the in vivo regulation of Type I and Type II receptors.
For example, it is apparent that the factors that are removed from
cytosol by DCC pretreatment and which have been implicated in the
in vivo regulation of Type II receptors (e.g., thioredoxin and
NADPH) (Grippo et al., 1983 & 1985), may not be involved in the

121
regulation of Type I receptors or the new class of Type II receptors
discussed above. In other words, there appears to be at least two
3
separate populations of [ H]DEX binders in brain. One population of
these binders is readily susceptible to up- and down-regulation by
sulfhydryl oxidation-reduction cycle, while a second population is
immune from this regulatory mechanism and may provide a constitutive
level of viable receptors even under challenging conditions. As an
example of one possible expression of this relationship, recent
experiments from our laboratory investigating the 1igand-induced
in vivo regulation of Type I and Type II receptors in the brain,
kidney and liver suggest that treating mice even with pharmacological
doses of corticosterone fails to produce a complete down-regulation of
Type II receptors in these tissues (Luttge and Rupp, unpublished). It
3
is entirely possible that the population of [ H]DEX binders
remaining after corticosterone treatment is enriched in the new class of
Type II receptors shown here to be resistant to the DCC pretreatment,
absence of DTT, high ionic strength and aging at 0°C. Investigation of
the above possibility as well as further physicochemical
characterization of this new class of Type II receptors is currently
under way in our laboratory.

CHAPTER VI
IN VITRO TRANSFORMATION OF ALDOSTERONE-TYPE I
RECEPTOR COMPLEXES TO A DNA-BINDING STATE
Summary
In order to modulate DNA transcription, steroid-receptor complexes
are thought to undergo a "transformation" (activation) process that
increases the affinity of the complexes for specific regions of DNA
called hormone response elements. It has been shown that once activated
androgen-, estrogen-, progesterone- and glucocorticoid-receptor
complexes attain physicochemical properties that are distinct from those
of the unactivated steroid-receptor complexes. In this chapter, an
investigation of the properties of ALDO-Type I receptors prior and
subsequent to exposure of brain cytosol to 300 mM KC1 and/or 22°C for
20 min (conditions known to result in complete activation of
glucocorticoid-Type II and other steroid-receptor complexes) was
conducted. Exposure to 300 mM KC1 and 22°C for 20 min, led to a 3-fold
increase in the binding of ALDO-Type I receptors to DNA-cellulose
(DNA-C) (6 and 19% binding for unactivated and activated receptors,
respectively). In contrast, identical experimental conditions produced
a 14-fold increase in the binding of Type II-receptor complexes to DNA-C
(4 and 54% binding for unactivated and activated receptors,
respectively). Molybdate, a known inhibitor of activation of all
steroid-receptor complexes, also prevented this salt- and
temperature-induced increase in the binding of ALDO-Type I receptors to
DNA-C. Dithiothreitol was found to have no effect on the binding of
activated ALDO-Type I and DEX-Type II receptors to DNA-C; however, the
1 99

123
presence of 5 mM molybdate in buffers during DNA-C batch assay produced
the largest difference in the magnitude of DNA-C binding between
unactivated and activated ALDO-Type I receptor complexes (i.e., 6 versus
20% in the presence and 13 versus 14% in the absence of this oxyanion).
Although previous work from our laboratory showed that activation of
glucocorticoid-Type II receptor complexes is followed by a marked change
in surface hydrophobic properties of these receptors, salt and heat
produced only subtle changes in surface hydrophobicity of ALDO-Type I
receptor complexes. Taken together, these results suggest that
conditions leading to complete activation of Type II and other steroid
receptors will only produce partial activation of ALDO-Type I receptor
complexes.
Introduction
Although the exact in vivo mechanisms or "factors" that bring
about activation of steroid-receptor complexes remain to be elucidated,
a number of in vitro manipulations are known to stimulate
activation. For example, in vitro activation of glucocorticoid-
Type II receptors can be triggered by increasing cytosolic ionic
strength and/or temperature (Higgins et al., 1973; Kalimi et al., 1975;
Muller et al., 1983; Luttge and Densmore, 1984; Luttge et al., 1984a, b
& d), gel filtration (Cake et al., 1976; Bailly et al.; 1977 & 1978;
Distelhorst and Benutto, 1984), dilution (Higgins et al., 1973; Goidl et
al., 1976), increase in pH (Bailly et al., 1978) and by addition of a
variety of chemicals such as ATP (Moudgil and John, 1980) and heparin
(McBlain and Shyamala, 1980; Yang et al., 1982; Hubbard and Kalimi,
1983). The involvement of cysteine and serine proteases have also been
implicated in the activation process (Hubbard and Kalimi, 1985).

124
Regardless of the factors eliciting activation, a number of
physicochemical changes have been shown to characterize this
transformation. For example, it has been demonstrated that the
activation of glucocorticoid-Type II receptor complexes is accompanied
by a conformational change resulting in the exposure of additional
positive charges on the surface of the complex (Milgrom et al., 1973a;
Luttge et al., 1984a & d). This increase in surface positivity can be
demonstrated by a reduction in affinity of the activated complexes for
diethylaminoethyl-cellulose (DEAE-cellulose) columns or filters (Sakaue
and Thompson, 1977), and an increase in the affinity for nuclei, DNA,
and other polyanions (Alberts and Herrick, 1971; Kalimi et al., 1975;
Luttge et al., 1984a, c & d). Activation is also characterized by an
increase in the overall surface hydrophobicity of the steroid-receptor
complexes. This increase in hydrophobicity (which may facilitate
nuclear membrane penetration and/or interaction with specific hormone
response elements on the DNA molecule) has been measured by an increase
in the binding of the complexes to glass-fiber filters (GF/C) (Luttge et
al., 1984b & d), aqueous dextran-polyethylene glycol two-phase
partitioning (Andreasen, 1982; Luttge et al., 1984d) and hydrophobic
interaction chromatographic techniques (Bruchovsky et al., 1981;
Geschwendt and Kittstein, 1980; Densmore, 1986; Densmore et al., 1986b).
Moreover, activation of a number of steroid-receptor systems has been
shown to be accompanied by a reduction in the rate of steroid
dissociation (e.g., McBlain et al., 1981; Moguilewsky and Philibert,
1984; Chou and Luttge, 1987).
Earlier studies regarding such alterations in the hydrodynamic
properties of the steroid-receptor complexes following activation

125
appeared inconsistent. In many cases, these inconsistencies were
primarily due to a failure to control spontaneous activation and
receptor degradation during prolonged ultracentrifugation and open
column analytical gel exclusion chromatographic procedures. Note that
the in vitro activation of a number of steroid-receptor complexes
can slowly occur even at near zero temperatures (Luttge and Densmore,
1984). The utilization of molybdate to inhibit activation during
cytosol preparations, labeling and prolonged hydrodynamic parameter
determination has allowed the generation of more consistent results
(e.g., Dahmer et al., 1981; Luttge and Densmore, 1984; Luttge et al.,
1984a, b & d). For example, our laboratory has reported that activation
is accompanied by a reproducible reduction in the sedimentation
coefficient of glucocorticoid-Type II receptor complexes (form 9.2 S to
a 3.8 S) (Luttge and Densmore, 1984; Luttge et al., 1984a, b & d). This
reduction in sedimentation coefficient is also accompanied by a
reduction in the Stoke's radius (from 7.7 nm to 5.8 nm) and the relative
molecular mass (from 297 kDa to 92 kDa) of the glucocorticoid-receptor
complexes (Luttge et al., 1984a & d) .
Although activation of steroid-receptor complexes is thought to be
a prerequisite step for genomic response, the exact nature of the
nuclear "acceptor" sites through which the complexes modulate cellular
events remained an enigma for some time. Hormone-receptor complexes
were shown to interact and be associated with a variety of partially
purified nuclear components including DNA (Higgins et al., 1973;
Tymoczko et al., 1984), RNA (Feldman et al., 1981; Rossini and
Barbiroli, 1983; Tymoczko and Phillips, 1983; Economidis and Rosseau,
1985; Reker et al., 1985; Tymoczko and Lee, 1985; Webb et al., 1986;

126
Ali and Vedeckis, 1987) and histones (Rallos et al., 1981). In
addition, it is becoming increasingly apparent that a salt- and
nuclease-resistant nuclear substructure, known as the "nuclear matrix,"
may play an important role in the genomic expression of hormone-receptor
action (for review see Barrack and Coffey, 1982; Capeo et al., 1982).
The nuclear matrix (NM) was shown to be enriched with newly
replicated DNA and heterogeneous nuclear RNA (hn-RNA) (Berezney and
Buckholtz, 1981; Ciejek et al., 1982; for review see Nelson et al.,
1986). These properties of NM, as well as the observations that in
target tissues high-affinity steroid binding sites are associated with
NM (Barrack and Coffey, 1980 & 1982; Barrack, 1983; Spelsberg et al.,
1983) , were taken as evidence for NM as the site for steroid-receptor
complex modulation of mRNA transcription (Barrack and Coffey, 1982;
Buttyan et al., 1983; Simmen et al., 1984; Kirsh et al., 1986).
Interestingly, it appears that different steroid-receptor complexes bind
preferentially to the NM of their respective target tissues (Buttyan et
al., 1983). Partial extraction of the DNA associated with NM resulted
in a loss in the specificity of the NM-steroid-receptor complex
interactions. It is thus proposed that the DNA associated with the NM
of target tissues may, at least in part, confer this binding specificity
(Buttyan et al., 1983).
DNA-cellulose competition assays have also been used to show that
activated steroid-receptor complexes manifest stereospecific binding to
DNAs containing specific sequences. For example, purified chick oviduct
progesterone-receptor complex was found to bind preferentially to a 1.7
kilobase DNA fragment flanking the 5' end of the chicken ovalbumin gene
with A--T rich base pair sequences (Hughes et al., 1981;

127
Compton et al., 1982; MuLvihilL et al., 1982; Compton et al., 1983).
Activated glucocorticoid-Type II receptor complexes were shown to
interact with high affinity at the promotor region of the mouse mammary
tumor (MMTV) proviral DNA (Pfahl, 1982). Recently, purified steroid
hormone receptors were shown to bind specifically to palindromic
sequences within the DNA molecule in order to modulate the transcription
of mRNAs (e.g., Wrange et al., 1986; Ameman et al., 1987; Chambone et
al., 1987 ; Drouin et al., 1987 ; Karin et al., 1987 ; Meisfeld et al.,
1987). Together, these observations suggest that activated
steroid-receptor complexes may act in a manner comparable to other DNA
regulatory proteins such that by interaction with specific sites on the
DNA molecule they may modulate the activity of adjacent promotor
regions, and hence selectively modulate the transcription of specific
DNA transcripts.
Despite the wealth of our knowledge regarding the physicochemical
changes accompanying activation of Type II, progesterone, estrogen and
androgen receptors, the existing information on the activation of Type I
receptors is limited and in most instances difficult to interpret. In
earlier studies, a major difficulty associated with the interpretation
of the results is the confounding effects of ALDO binding to Type II
receptors (Marver et al., 1972; Kolpakov et al., 1974; Kusch et al.,
1978; Farraan et al., 1981; Palem-Vliers et al., 1982; Lehoux et al.,
1984). Despite the lack of direct evidence regarding the activation of
Type I receptors in the studies cited above, indirect evidence clearly
favored the interaction of ALDO-receptor complexes with DNA. For
example, in addition to inhibiting the physiological action of ALDO,
certain spironolactones were shown to produce a dose-dependent reduction

128
3
in In vitro nucLear uptake of [ H]ALDO (Marver et al., 1972) and
chromatin binding (Swaneck et al., 1970). Furthermore, various DNA
intercalators (e.g., actinomycin D, ethidium bromide, netropsin and
proflavin sulfate) were shown to inhibit the binding of
3
[ H]ALDO-receptor complexes to chromatin extracts, thus ultimately
inhibiting the synthesis of AIPs (Edelman et al., 1963; Edelman and
Marver, 1980). The degree of this inhibition appeared to be related to
specificity and concentration of the intercala tor used, hence
implicating DNA as a candidate for complex formation (Edelman and
3
Marver, 1980). Lastly, site-specific and saturable nuclear [ H]ALD0
binding in a number of target tissues including the brain (Ermisch and
Ruhle, 1978; Birmingham et al., 1984), kidney (Vandewalle et al., 1981;
Farman et al., 1981, 1982a & b; Farman and Bonvalet, 1983) and toad
urinary bladder (Farman et al., 1978) was detected by autoradiographic
and other biochemical techniques.
Recently, two independent studies on activation of rat kidney
3
[ H]ALDO-Type I receptors (note that ALDO binding to Type II
receptors was prevented by including a molar excess of a glucocorticoid
competitor in the incubation media) have expressed difficulties in
achieving full activation of these receptors (measured by
DEAE-cellulose, Sephacryl S-300 and DNA-C column chromatographic
techniques) (Eisen and Harmon, 1986; Schulman et al., 1986). The
results from the experiments described in this chapter also suggest that
conditions resulting in full activation of brain DEX-Type II receptors
produce only partial activation of ALDO-Type I receptor complexes.

129
Materials and Methods
Chemicals. Double stranded calf thymus DNA-cellulose (DNA-C)
(5.7 mg DNA/g DNA-C) and PMSF were purchased from Sigma. All other
chemicals used in this chapter were reagent grade quality.
Buffers. Buffer K contained 20 mM HEPES and 300 mM KC1, Buffer DK
was buffer K plus 2 mM DTT, Buffer KM was Buffer K plus 20 mM molybdate
and Buffer DMK contained all of the above constituents. Buffer M,.
contained 20 mM HEPES and 5 mM molybdate. In some experiments, the
cytosolic concentration of Buffers K, DK, KM and DKM were obtained by
diluting 9 parts cytosol with one part buffer containing a 10-fold
concentration of the desired buffer. Alternatively, cytosol
preparations were eluted from Sephadex G-25 columns pre-equilibrated
with the desired buffer to bring the preparations to the composition of
that buffer (see specific experiments). All buffers were adjusted to pH
7.60 just prior to use.
Activation. Cytosol was incubated with steroid(s) at 0°C for 24 to
48 h to label Type I or Type II receptors (for detail see Chapter II).
Prior to initiation of activation, free steroid(s) were removed by gel
exclusion chromatography on Sephadex G-25 columns (0.6 x 14.0 cm)
pre-equilibrated in Buffer K, DK, KM or DMK (see specific experiments)
as described in Chapter II. To activate the steroid-receptor complexes,
samples obtained in the void volume were placed for 20 min in a water
bath maintained at 22°C. Activation was quenched by returning the
samples to ice bath followed by a second Sephadex G-25 gel filtration to
bring the preparations to buffer formulation desired and to remove
steroids dissociated during activation process.

130
DNA-C Binding Assay. Double stranded calf thymus DNA-C was prepared
overnight in Buffer A or Buffer A containing other component(s) (see
specific experiments) at a final concentration of 10 mg/ml. Just prior
to use, 0.8 ml aliquots of the slurry were transferred to separate test
tubes, the DNA-C pelleted by centrifugation at 2,280 x g in a model RC-3
Sorvall centrifuge for 10 min and the supernatant was aspirated
carefully and discarded. Subsequently, 0.8 ml samples of cytosol were
added to the DNA-C pellet. The DNA-C was then resuspended by gentle
vortexing and incubated in ice bath for 60 min. The assay tubes were
oscillated at 150 RPM throughout this incubation period and vortexed
gently every 10 to 15 min. At the end of the incubation period,
steroid-receptor complex bound to DNA-C was collected by centrifugation
at 2,280 x g for 10 rain, the supernatant aspirated and the DNA-C pellet
washed twice with 1.0 ml buffer identical in composition to the original
cytosolic buffer. The DNA-C was then transferred quantitatively to
scintillation vials for determination of bound radioactivity as
described in Chapter II.
Results
As shown in Table 6-1, unactivated ALDO-Type I and DEX-Type II
receptor complexes show minimal affinity for DNA-C (i.e., 6 and 4% when
maintained at 0°C in Buffer DMK, respectively). Whereas heating
DEX-Type II receptors in Buffer K or DK for 20 min at 22°C produced a
14-fold increase in the binding of these complexes to DNA-C (i.e, 54%
binding to DNA-C), under identical conditions, DNA-C binding for
ALDO-Type I receptor complexes did not exceed a 3-fold rise (i.e., 19%
binding to DNA-C in Buffer DK). Thus far, several other manipulations
(e.g., increasing the temperature and/or duration of activation) failed

Table 6-1. Nine parts cytosol prepared in Buffer was diluted either
with one part Buffer M^q containing 20 mM DTT or Buffer M^q alone to yield
cytosol with composition of Buffer DM^q or M^q, respectively. Type I
3
receptors were then labeled with 10 nM [ H]ALD0 in the presence of 5 uM RU
26988 and Type II receptors were labeled with 10 nM [^H]DEX. B„„
determinations for Type I and Type II receptors were in the presence of 2 uM
[^H]ALD0 and [^H]DEX, respectively. Following a 24-h incubation at 0°C
with the steroids, 0.5 ml fractions were loaded on Sephadex G-25 columns
(pre-equilibrated with Buffer K, KM, DK or DMK) to remove free steroids (and
molybdate under Buffer K and DK conditions) and to bring the cytosol to the
composition of the desired Buffers (i.e.; K, KM, DK or DMK). Subsequently,
sufficient 100 mM phenylmethyl sulfonylfluoride (PMSF) dissolved in absolute
ethanol was added to cytosol to yield a final 1 mM concentration of this compound.
Activation was then performed as described under "Materials and Methods" and DNA-C
binding assays were performed in the presence of Buffer DM,, as described under
"Materials and Methods."

Table 6-1
Comparison of the Binding of ALDO-Type I and DEX-Type II
Receptor Complexes to DNA-Cellulose Before and After Salt-
and/or Temperature-Induced Activation
BUFFER
TEMPERATURE
o
o
n
22° C
Type I
Type
II
Type
I
Type
II
DMK
6 + 1
A +
1
7 +
2
9 +
1
DK
8 + 2
41 +
2
19 +
1
54 +
1
KM
8 + 2
7 +
1
8 ±
1
H +
0
K
7 + 1
35 +
2
16 +
1
54 +
3
132

133
to produce DNA-C binding of ALDO-Type I receptor complexes greater than
20% (data not shown). Although 300 mM KC1 in the absence of heat was
able to increase DNA-C binding of DEX-Type II receptor complexes
significantly (i.e., 41 and 35% in Buffer DK and K, respectively), the
magnitude of the DNA-C binding of ALDO-Type I receptor complexes
maintaied at 0°C ranged from 6 to 8% under all buffer conditions
studied. As with other steroid hormone receptor systems, inclusion of
20 mM molybdate (a group VI transition metal oxyanion) prevented the
increases in DNA-C binding of ALDO-Type I and DEX-Type II receptors
following activation.
Recently, structural analysis of the amino acid sequence of the
steroid-binding subunit of purified Type II receptors has shown this
subunit to contain a cysteine rich region in the DNA-binding domain
(e.g., Danielsen et al., 1987). Earlier biochemical studies suggested
that the binding of activated glucocorticoid-Type II receptor complexes
to DNA required reduction of sulfhydryl groups in the DNA-binding region
(Kalimi and Love, 1980; Bodwell et al., 1984). However, the study
presented in Table 6-1 shows that DNA-C binding of salt- and
heat-activated DEX-Type II receptor complexes is identical in the
presence (Buffer DK) or absence (Buffer K) of the sulfhydryl reducing
reagent, DTT. In contrast, when maintained at 0°C, the DNA-C binding of
DEX-Type II receptor complexes in Buffer DK was slightly higher than
that seen in Buffer K (41 versus 35%, respectively). The reason for
this difference is unclear at this stage.
Since earlier experiments with Type II receptors suggested that the
presence of high concentrations of molybdate can prevent the binding of
activated Type II receptors to DNA-C (Luttge, unpublished results), and

134
because 5 mM molybdate was used during DNA-C binding to prevent
spontaneous activation, the effects of this reagent on the binding of
3
activated and unactivated [ H]ALDO-Type I receptor complexes to
DNA-C was next investigated. As shown in Table 6-2, the presence of
5 mM molybdate (Buffer M^) during DNA-C binding assay produced a
larger difference in the magnitude of the binding of unactivated (0°C)
and activated (22°C) Type I receptors (6 versus 20% DNA-C binding,
respectively) than that seen in the absence of molybdate (Buffer A) (13
versus 14% for unactivated and activated receptors, respectively).
Similar results were also obtained with Type II receptors: the
magnitude of difference between DNA-C binding of unactivated and
activated Type II receptors was greatest in the presence of 5 mM
molybdate (Luttge, unpublished results).
In addition to the increase in affinity for DNA-C following
activation, a recent extensive analysis of the surface hydrophobic
properties of activated and unactivated Type II receptors by hydrophobic
interaction chromatography showed that the activation of these receptors
is accompanied by an increase in the overall surface hydrophobicity of
these receptors (Densmore, 1986). In contrast, an examination of the
elution profile of ALDO-Type I receptors prior and subsequent to
attempted salt- and heat-induced activation from pently-Agarose columns
revealed that there were only slight differences between the unactivated
and "activated" receptors (Fig. 6-1). Furthermore, there were no
significant changes in the binding of ALDO-Type I receptor complexes to
DEAE or GF/C filters prior and subsequent to activation (data not
shown).

Table 6-2. Cytosol prepared in Buffer was incubated with 10 nM
[^H]ALD0 and 5 uM RU 26988 and either with (B„„) or without (Bm) 2 uM
NS T
[^H]ALD0 for 24 h at 0°C. Following this incubation period, 0.5 ml fractions
were loaded on Sephadex G-25 columns (pre-equilibrated with Buffer K) to remove
free steroids and molybdate and to bring the cytosol to the composition of Buffer
K. Subsequently, sufficient 100 mM phenylmethyl sulfonylfluoride (PMSF) dissolved
in absolute ethanol was added to cytosol to yield a final 1 mM concentration of
this compound. Activation was performed as described under "Materials and
Methods." DNA-C binding assays were performed in the presence of Buffer A or
Buffer Mr as described under "Materials and Methods."
5

Table 6-2
Effects of 5 mM Molybdate on the Binding of ALDO-Type I
Receptor Complexes to DNA-Cellulose Before and After
Salt- and Tempera ture-Induced Activation
TEMPERATURE
BUFFER
o
o
o
22°C
A
13
+
2
14
+
2
M5
6
+
1
20
+
1
136

Figure 6-1. Comparison of the Surface Hydrophobic Properties of
Unactivated and Activated ALDO-Type I Receptor Complexes on
Pentyl-Agarose Columns. Cytosol prepared in Buffer DM^ was
incubated with 10 nM [^H]ALD0 plus 5 uM RU 26988 and either with
(B ) or without (B ) 2 uM [^H]ALD0 for 24 h at 0°C.
No T
Following this incubation period, 0.5 ml fractions were loaded on
Sephadex G-25 columns (pre-equil ibra ted with Buffer DM^ or Buffer
DK) to remove free steroids (and molybdate under Buffer DK conditions)
and to bring the cytosol to the composition of the desired buffers
(i.e., 0^20 or • Subsequently, sufficient 100 mM phenylmethyl
sulfonylfluoride (PMSF) dissolved in absolute ethanol was added to
cytosol to yield 1 mM concentration of this compound. Activation was
then performed as described under "Materials and Methods." Unactivated
groups underwent the same procedural steps except for the 22°C
incabation. Pentyl-Agarose Hydrophobic interaction chromatography was
performed as described in Chapter II. The data presented are expressed
as a percent of total B loaded on the columns and are from 3
o t
independent replicate experiments.

138
25 -
Fraction Number
25

139
Discussion
According to the two-step model of steroid hormone action (see
Chapter I), a necessary step for modulation of DNA transcription is
transformation (activation) of the receptors to a state with an
increased affinity for DNA. The most commonly employed strategy for
in vitro activation of steroid receptors is by increasing cytosolic
salt concentration and/or temperature. Although this method has been
employed successfully for Type II and other steroid receptors, the
experiments described above, as well as two other recent reports (Eisen
and Harmon, 1986; Schulman et al., 1986), suggest distinct differences
between activation requirements of Type I and Type II receptors. In
these studies, a complete activation of Type I receptors (measured by
DEAE-cellulose column chromatography) could not be achieved: salt- and
temperature-treatment of cytosol gave two distinct peaks of
radioactivity one of which eluted at identical salt concentration as
3
that of unactivated [ H]ALDO-Type I receptor complexes.
Although earlier biochemical studies reported that nuclear uptake
of ALDO-Type I receptors is a temperature-dependent process,
autoradiographic techniques provide evidence for nuclear translocation
to be tempera ture-independent (Bonvalet et al., 1984; Farman et al.,
1984). It is suggested that although ALDO-receptor complex formation
may occur in cytoplasm, the apparent temperature-dependency of nuclear
"translocation" may be an artifact of biochemical techniques (Bonvalet
et al., 1984; Farman et al., 1984). Recently, evidence from both
biochemical, immunocytochemical and morphological studies have
challenged the classical two-step model of steroid hormone action. The
notion that unoccupied, unactivated steroid receptors may be localized

140
in the nucleus and that cytoplasmic localization may be an artifact of
preparations has been implied for Type I, Type II and sex steroid
receptor systems (e.g, Martin and Sheridan, 1982; Welshons et al., 1984
& 1985; Jensen et al., 1986; Pearce et al., 1986; for review see
Walters, 1985; Gasc and Baulieu, 1986).
In light of these recent findings, and several other chemical
differences between Type I and Type II receptors (Emadian et al., 1986)
one may argue that Type I receptors do not undergo the same
transformation process in order to modulate DNA transcription. In other
words, it is entirely possible that unoccupied Type I receptors are
associated with NM in a nonspecific manner. Upon binding ALDO, the
receptors may then undergo some conformational changes that allow
exposure of specific DNA-binding domains on the 1igand-receptor complex,
subsequent specific DNA-binding and hence modulation of mRNA synthesis.
Alternatively, cytosol may contain some molybdate-like factors that act
on Type I receptors and prevents the activation of the receptors (note
that small molecular weight inhibitors of activation of Type II and
other steroid receptors has been described previously). This latter
possibility appears more likely since the results discussed in Chapter
IV suggest the presence of such factor that inhibits optimal ligand
binding capacity of Type I receptors.
It is also noteworthy that 300 mM KC1 shown to activate Type II
receptors (Table 6-1) was also found to inactivate unoccupied Type II
receptors (see Chapter V). Because: 1) KCl and other agents capable of
activating steroid hormone receptors do so by dissociating the monomeric
steroid-binding subunit (92 kDa subunit) from the multimeric unactivated
receptors (297 kDa oligomeric form) [i.e., from heat shock proteins

141
(90 kDa subunit), RNAs, and possibly other molecules]; 2) following
ligand dissociation, activated Type II receptors appear to be unable to
rebind ligand. If it is assumed that the inactivating mode of action of
KC1 of unoccupied Type II receptors is through subunit dissociation
(Chapter V); since KC1 alone does not activate occupied (Table 6-1) and
inactivate unoccupied (Chapter V) Type I receptors, one may conclude
that the difficulty in activating Type I receptors resides in the fact
that subunit dissociation cannot occur because of the presence of
molybdate-like factors in the cytosol (Chapter IV) (note that molybdate
inhibits activation of steroid receptors by preventing subunit
dissociation).
Future experiments will therefore focus on 1) procedures that may
lead to full activation of Type I receptors (e.g., gel filtration of
unlabeled cytosol in the absence of 2 mM molybdate followed by salt- and
heat-induced activation); 2) characterization of possible endogenous
inhibitors of activation of ALDO-Type I receptors; 3) tissue specificity
of such factors and 4) physicochemical characterization of fully
activated Type I receptors.

CONCLUDING REMARKS
Among the many original observations described in this
dissertation, the most intriguing are the differences between Type I and
Type II receptors. Note that these differences further extend the
observations from an earlier study by our laboratory in which a number
of chemical differences between Type I and Type II receptors were
described (Emadian et al., 1986). Historically, the
adrenocorticosteroid hormone receptors were classified in terms of their
preference for a naturally occurring ligand. Therefore, on the basis
their apparent affinity for ALDO and CORT, Type I and Type II receptors
were classified as mineralo- and glucocorticoid receptors, respectively.
The recent observations that CORT, in the absence of extravascular CBG,
has an equal or greater affinity than ALDO for Type I receptors, has
challenged the traditional classification of these receptors (for review
see Funder et al., 1986; De Kloet et al., 1986). It is postulated that
in CNS structures such as the hippocampus (where there is little or no
extravascular CBG), the endogeous ligand for Type I receptors under
basal conditions is CORT. Furthermore, under basal, but not "stress,"
conditions, Type II receptors are said to remain largely unoccupied
(because of a 10-fold lower affinity of CORT for Type II than Type I
receptors). Accordingly, it is proposed that Type I receptors mediate
"tonic" influences of CORT on brain, whereas Type II receptors mediate
the stress effects of CORT as well as CORT action on feedback regulatory
mechanisms involving the hypothalamo-pituitary-adrenal axis (De Kloet et
al., 1986). At variance with this simplistic theory, Dallman and
142

143
co-workers (Dallman, personal communication) have recently shown that
during the late afternoon hours, when circulating levels of CORT are at
the acme of their circadian fluctuation, CORT-Type II receptor
interactions do indeed appear to mediate the feedback suppression of
ACTH. However, during the morning, when circulating levels of CORT are
at their nadir, CORT-Type I receptor interactions appear to be the
principle mechanism by which glucocorticoid suppression of ACTH is
mediated.
While recent studies have provided indirect evidence in support of
the above theoretical relationships, a clear proof for the proposed role
of CORT- versus ALDO-Type I receptor complexes is still lacking. For
example, preliminary studies from our laboratory suggest that CORT
administration to adrenalectomized mice down-regulates both Type I and
Type II receptors in brain, whereas ALDO down-regulates only Type I
receptors (Luttge and Rupp, unpublished results). Although at this
point it is not clear whether this Type I receptor down-regulation is a
direct effect of CORT-binding to Type I receptors or an indirect effect
of CORT-binding to Type II receptors (possibly through the production of
a protein which negatively modulates the gene for Type I receptor mRNA).
It is also noteworthy that although ligand binding is thought to be
a prerequisite for initiation of the genomic actions of steroid
hormones, this is by no means a sufficient step. In light of our
demonstrations of the distinct chemical differences between Type I and
Type II receptors, as well as the ease with which these receptors
undergo the transíormation(s) necessary to recognize nuclear acceptor
site(s), one may postulate that ALDO-Type I versus CORT-Type I receptor
complexes may be differentially prevented from eliciting a genomic

144
response by one or more regulatory mechanisms. Furthermore, this
selective modulation of Type I receptor activation may be target
specific. For example, Orti et al. (1986) reported no difficulties with
activation of ALDO-Type I receptor complexes obtained from spinal cord.
Experiments investigating such target and ligand specificities for Type
I receptor activation, similarties and differences between their nuclear
acceptor site(s), as well as the possible physiological and functional
relevance of such differences are currently under way in our laboratory.

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BIOGRAPHICAL SKETCH
The author was born on April 22, 1958, in Sari, Iran—a city of
about 100,000 population located 18 miles south of the Caspian Sea. The
author received his elementary and high school education as well as
several years of private formal training in painting and graphic arts in
Sari. Following graduation from Payam High School (May 1976), after
careful consideration, his parents decided to send him to the United
States for higher education training. Because of the author's primary
interest in Medicine, he entered Phillips University in Enid, Oklahoma,
as a Pre-Med student in the Fall of 1977.
In addition to academic achievements (e.g.; Outstanding Biology
Student award, 1979; research grant in the amount of $6,000, 1980; etc.)
at Phillips University, the author advanced to the presidency of the
local chapter of The Blue Key National Honor Fraternity (1981-82). As
part of his formal educational training, the author attended Harvard
University during summer of 1980. He graduated cum laude from Phillips
University with a Bachelor of Arts in biology and chemistry in May of
1982. While a student at Phillips University, the author worked as a
urology and orthopedics technician at a local hospital.
After failing to gain admission for an M.D.-Ph.D. program, with the
advice of his undergraduate mentor, the author decided to obtain his
Ph.D. degree first and then apply to a medical school. Although he was
admitted to several graduate schools, due to his genuine interest in
neuroendocrinology, the author chose to accept the offer from the
Department of Neuroscience, University of Florida, where he has been a
184

185
Ph.D. candidate since August 1982. The author's honors and
accomplishments during the past 5 years include research ass istantship
(1982-85), research fellowship (1985-87), presentation at a number of
local and national meetings and several full-length articles of his work
in a number of scientific refereed journals.

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.
William G. Luttge,'
Professor of Neuroscience
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.
^ '
Adrian J. Dunn
Professor of Neuroscience
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.
43
L
Ai¿á
Melvin J. Ui'regly /
_ .O
(J
Professor of Physiology
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.
Marieta B. Heaton
Associate Professor of
Neuroscience
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.
fkXjLÍ'K J ~ lócÁy
Kathleen T. Shiverick
Associate Professor of
Pharmacology and
Therapeutics

This dissertation was submitted to the 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 Philisophy.
May 1987
Dean, College of Medicine
Dean, Grj
uate School

UNIVERSITY OF FLORIDA
3 1262 08554 4012



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