Alpha-adrenergic receptor stimulated phosphatidylinositol hydrolysis in the rat brain

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Alpha-adrenergic receptor stimulated phosphatidylinositol hydrolysis in the rat brain
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Raulli, Robert Emil, 1957-
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Receptors, Adrenergic, alpha   ( mesh )
Phosphatidylinositols   ( mesh )
Pharmacology and Therapeutics thesis Ph.D   ( mesh )
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Thesis:
Thesis (Ph.D.)--University of Florida, 1987.
Bibliography:
Bibliography: leaves 87-97.
Statement of Responsibility:
by Robert Emil Raulli.
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Typescript.
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Vita.

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ALPHA-ADRENERGIC RECEPTOR STIMULATED PHOSPHATIDYLINOSITOL
HYDROLYSIS IN THE RAT BRAIN



By




ROBERT EMIL RAULLI


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















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


ALPHA-ADRENERGIC STIMULATION OF
PHOSPHOINOSITIDE HYDROLYSIS
IN THE RAT BRAIN


By

Robert Emil Raulli

December 1987


Chairman: Fulton T. Crews
Major Department: Pharmacology and Therapeutics


Alphal-adrenergic stimulation in rat brain is known to stimulate

phosphoinositide (PI) hydrolysis. The PI response to norepinephrine

was found to vary regionally within the brain. Relative efficacies of

various phenylethylamine compounds remained consistent across

different brain regions suggesting phenylethylamines stimulate PI

hydrolysis via the same type of alphal receptor. The alpha2 agonist,

oxymetazoline, was unexpectedly found to stimulate PI hydrolysis.

Relative efficacies across various brain regions were not consistent

suggesting an alternative mechanism of PI stimulation.

Both phenylethylamines and imidazolines were found to compete for

the alphal antagonist [3H]-prazosin. Both classes of compounds

exhibit two sites of interaction with membrane [3H]-prazosin binding

sites when the assay is performed in TRIS buffer. Substituting a








modified Krebs buffer for TRIS buffer shifts the phenylethylamine

competition to a one-site interaction, while the imidazoline

interaction remained two-site. All phenylethylamines tested stimulate

PI hydrolysis in a dose-dependent manner. The imidazolines were not

as potent or efficacious in stimulating PI hydrolysis, nor did they

exhibit dose-effect relationships. The correlation coefficients

between Kd values (from membrane competition for [3H]-prazosin) and

ED50 values (from PI hydrolysis) were 0.96 and 0.33 for

phenylethylamines and imidazolines respectively.

The alphal antagonist prazosin was able to inhibit the PI

hydrolysis response to phenylethylamines, but not imidazolines. This

and the above data suggest that imidazolines are not acting via the

classical alphal receptor. Imidazolines, in fact, demonstrate

dose-dependent inhibition of norepinephrine stimulated PI hydrolysis,

thus acting as alphal antagonists.

To determine the regulation of norepinephrine-stimulated PI

hydrolysis rats were treated with reserpine acutely or chronically (5

mg/kg/day for 4 days or 0.25 mg/kg/day for 14 days). Reserpine, a

catecholamine depletor, increases the density of adrenergic receptors

in the CNS. Neither reserpine regimen had an effect on the ED50 value

or magnitude of norepinephrine-stimulated PI hydrolysis. Nor was a

change seen in the rate or amount of [3H]-inositol incorporation.

Thus, it appears that reserpine, at doses known to increase

beta-adrenergic receptor responsiveness, does not increase the

norepinephrine-stimulated PI hydrolysis response in rat brain.


















This dissertation is dedicated to my parents, Ernest and Jean, the

best parents in the world.














ACKNOWLEDGEMENTS


I would like to thank my advisor Fulton T. Crews for helping me

through this dissertation and for his generosity. I would also like

to thank my committee members Drs. Steve Childers, Mohan Raizada, and

Ralph Dawson. Special thanks are offered to committee members Steve

Baker for being an all-around good guy and Dr. Neims for spending time

with the students, teaching the things we couldn't learn in class.

Thanks are given also to the always helpful staff of secretaries.

Thanks are given to Connie, Ann, Denise, Jim, Rueben, Shelly, Kroll,

Roger, Rita, Shari, Kelly, Carolyn, Norb, Cathy, and many others for

their friendship, support, and encouragement. The author would also

like to acknowledge Cathy Morse for her help in preparing the graphics

and Bill Grimmett for typing this manuscript.















PREFACE


This dissertation is composed of an introduction, four chapters

written in standard manuscript style, and a general conclusions

section. I apologize for any redundancies that may exist.















TABLE OF CONTENTS


ACKNOWLEDGEMENTS ......................................... iii

PREFACE ................................................... iv

ABSTRACT .................................................. vii


INTRODUCTION ..........................

Synthesis of Phosphatidylinositol and
Polyphosphatidylinositol ..............
Mobilization of Intracellular Calcium .
The Role of Protein Kinase C ..........
Combined Effects of Calcium and
Protein Kinase C ......................
Summary of the Phosphatidylinositide
Cascade System ........................

BRAIN REGIONAL DISTRIBUTION
OF ALPHA ADRENERGIC-
STIMULATED PHOSPHOINOSITIDE
HYDROLYSIS ............................

Introduction ..........................
Methods ...............................
Results ...............................
Discussion ............................

DIFFERENCES IN IMIDAZOLINE AND
PHENYLETHYLAMINE ALPHA-ADRENERGIC
AGONISTS: COMPARISON OF BINDING
AFFINITY AND PHOSPHOINOSITIDE
RESPONSE ..............................

Introduction ..........................
Methods ...............................
Results ...............................
Discussion ............................


CHAPTER 1


CHAPTER 2


CHAPTER 3









REGULATION OF RAT BRAIN ALPHA1
RECEPTORS ....................


Introduction ...
Methods ........
Results ........
Discussion .....


EFFECTS OF nBM LESIONS ON
MUSCARINIC-STIMULATION OF
PHOSPHOINOSITIDE HYDROLYSIS ...........


Preface ........
Introduction ...
Methods ........
Results ........
Discussion .....


CONCLUSIONS ...........................


BIBLIOGRAPHY ..............................................

BIOGRAPHICAL SKETCH .......................................


CHAPTER 4


CHAPTER 5


CHAPTER 6














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


ALPHA-ADRENERGIC STIMULATION OF
PHOSPHOINOSITIDE HYDROLYSIS
IN THE RAT BRAIN


By

Robert Emil Raulli

December 1987

Chairman: Fulton T. Crews
Major Department: Pharmacology and Therapeutics


Alphal-adrenergic stimulation in rat brain is known to stimulate

phosphoinositide (PI) hydrolysis. The PI response to norepinephrine

was found to vary regionally within the brain. Relative efficacies of

various phenylethylamine compounds remained consistent across

different brain regions suggesting phenylethylamines stimulate PI

hydrolysis via the same type of alphal receptor. The alpha2 agonist,

oxymetazoline, was unexpectedly found to stimulate PI hydrolysis.

Relative efficacies across various brain regions were not consistent

suggesting an alternative mechanism of PI stimulation.

Both phenylethylamines and imidazolines were found to compete for

the alphal antagonist [3H]-prazosin. Both classes of compounds

exhibit two sites of interaction with membrane [3H]-prazosin binding









sites when the assay is performed in TRIS buffer. Substituting a

modified Krebs buffer for TRIS buffer shifts the phenylethylamine

competition to a one-site interaction, while the imidazoline

interaction remained two-site. All phenylethylamines tested stimulate

PI hydrolysis in a dose-dependent manner. The imidazolines were not

as potent or efficacious in stimulating PI hydrolysis, nor did they

exhibit dose-effect relationships. The correlation coefficients

between Kd values (from membrane competition for [3H]-prazosin) and

ED50 values (from PI hydrolysis) were 0.96 and 0.33 for

phenylethylamines and imidazolines respectively.

The alphal antagonist prazosin was able to inhibit the PI

hydrolysis response to phenylethylamines, but not imidazolines. This

and the above data suggest that imidazolines are not acting via the

classical alphal receptor. Imidazolines, in fact, demonstrate

dose-dependent inhibition of norepinephrine stimulated PI hydrolysis,

thus acting as alphal antagonists.

To determine the regulation of norepinephrine-stimulated PI

hydrolysis rats were treated with reserpine acutely or chronically (5

mg/kg/day for 4 days or 0.25 mg/kg/day for 14 days). Reserpine, a

catecholamine depletor, increases the density of adrenergic receptors

in the CNS. Neither reserpine regimen had an effect on the ED50 value

or magnitude of norepinephrine-stimulated PI hydrolysis. Nor was a

change seen in the rate or amount of [3H]-inositol incorporation.

Thus, it appears that reserpine, at doses known to increase

beta-adrenergic receptor responsiveness, does not increase the

norepinephrine-stimulated PI hydrolysis response in rat brain.


viii














CHAPTER 1
INTRODUCTION
The miracle of the human body is in many ways related to the

interaction of the numerous varied cell types and their ability to

maintain homeostasis. The division, growth, differentiation, and

specialized functions of cells and tissues are carefully regulated by

neurotransmitter, hormonal and humoral signals. Most cells are linked

to the appropriate signals through receptors on their cell surface.

Both the nervous system and immune system communicate with each other,

their cellular components and other tissues primarily through these

receptors on the exterior of the cell membrane. Although many of

these receptors are proteinaceous, they reside in a membrane which is

usually half lipid. This lipid plays a major part in the transduction

of the signal across the membrane to the interior of the cell. It

allows movement of the signal carrying receptors and proteins and

serves as a reservoir of messenger molecules which are released from

the membrane. Studies in recent years have clearly indicated that

there are two key transduction mechanisms involved in autonomic

receptors. The beta adrenergic receptors in the heart, lung, liver

and other tissues are linked to an enzyme on the inside of the plasma

membrane (adenylate cyclase) through a guanine nucleotide coupling

protein (Ns). The other two autonomic receptors, i.e. the alpha

adrenergic receptor and the muscarinic cholinergic receptor, are

linked to the hydrolysis of phosphoinositides, actual lipid components

1











of the membrane. Both of these signal transduction mechanisms are

dependent upon and/or regulated by membrane lipids. In addition, the

methylation of phospholipids is regulated by receptors and appears to

modulate and/or mediate certain cellular signals in various cells. A

fourth type of lipid involvement in signal transduction involves the

release of arachidonic acid, the precursor of prostaglandins,

leukotrienes and other bioactive metabolites which serve to transmit

and/or modulate cellular responses.

Tissues and organs are continuously regulated by signals from the

extracellular environment. The nervous, endocrine, and immune systems

constantly signal a variety of tissues and cells and simultaneously

process signals from those cells resulting in coordination of cellular

function. All cells that process extracellular signals need

mechanisms to transport that message into the cell. The transduction

system usually starts with membrane receptor recognition of the

hormone or neurotransmitter (cytosolic receptors for steroidal

hormones are notable exceptions). This recognition produces

conformational changes in the receptor. Molecular events involved in

receptor conformation changes trigger mechanisms by which the signal

can be passed from the cell surface through the lipid bilayer to the

cytosol. Systems using cyclic AMP as a second messenger are perhaps

the best characterized. In the transduction phase, receptor

occupation causes the GTP-dependent release of N protein components

that migrate through the membrane bilayer. These proteins bind to

adenylate cyclase on the inner membrane and stimulate (or inhibit) the

production of cAMP. The cAMP produced serves as the second messenger











to stimulate cAMP-dependent protein kinase (for review of this system

see Helmreich and Pfeuffer, 1985).

In this introduction, we will discuss the hydrolysis

of phosphatidylinositol as a transduction system since lipids are the

central component of this signal.


Synthesis of Phosphatidylinositol
and Polyphosphatidylinositol

Phosphatidylinositol (PI) is synthesized by the multi-step

pathway shown in Figure 1-1. Phosphatidic acid (PA) is transformed to

CDP-diacylglycerol by the enzyme CTP:diacyl-glycerophosphate

cytidyltransferase. CDP-diacylglycerol combines with inositol to form

PI and CMP. These processes take place in the endoplasmic reticulum.

PI can be further phosphorylated to phosphatidylinositol 4-phosphate

(Ptdlns 4P) and phosphatidylinositol 4,5-bisphosphate (Ptdlns 4,5P2)

by phosphatidylinositol kinases. Phosphomonoesterases are also

present that convert Ptdlns 4,5P2 to Ptdlns 4P and Ptdlns 4P back to

PI. Phosphorylation and dephosphorylation of the inositol head group

of PI are believed to take place in the plasma membrane (Hawthorne and

White, 1975). PI, Ptdlns 4P, and Ptdlns 4,5P2 exist in the membrane

in "steady-state" concentrations with PI serving as a reservoir for

polyphosphoinositides that are hydrolyzed during receptor activation.

Receptor occupation by an appropriate agonist leads to the rapid

activation of the enzyme phospholipase-C (PL-C) to yield the

hydrolysis products inositolpolyphosphate and diacylglycerol (DAG).






















Figure 1-1. The synthetic pathway of phosphatidylinositol.
Phosphatidylinositol is synthesized from phosphatidic acid in the
endoplasmic reticulum via the above multi-step pathway. In the
membrane phosphatidylinositol can be phosphorylated by
phosphatidylinositol kinase(s) to phosphatidylinositol 4-phosphate and
phosphatidylinositol 4,5-bisphosphate.




















Endoplasmic
Reticulum -
Phosph tidic Acid
--- ,C-o0 vA +CTP
C o~c-oAw V + ,
,.-J- Diacylglycerol O-P-O-C 0 / CTP: iacylglyceroDhosphate
KInase 6- Cytidyltransferase
SC----- --- HO..CO
CO HO OH O V
-Co- ,- OH HC -0
C- 0 A= =/ Inositol C-0 .
HO-C O Diacylglycerol O-P-O-C 0 CDP-Diacylglycerol

o-P-o-
.^^ itCYTIDINE
Phosphollpase C

Membrane C'OAAAAMA
HO 0- 0 T




o-H -,-oc
S* P-0- 0 Phosphatidylinositol / J '


SO- 4-Phosphate(( /
C-O R
0 ( O-P-O-C 0 / /
p/ Phosphatldyllnosltol 4.5-Bsphosphate










Until recently the molecular events involved in PL-C stimulation were

unknown. Evidence is rapidly accumulating which suggests that PL-C

activation may be triggered by a guanine nucleotide binding protein

(Npi). It has been found that guanine nucleotides and guanine

nucleotide analogs can induce the breakdown of inositol phospholipids

in permeabilized cells (Cockcroft and Gomperts, 1985) and in membranes

(Litosch et al., 1985; Gonzales and Crews, 1985b). The time course

and products produced mimic receptor agonist induced breakdown. From

these studies and others we can propose a simplified mechanism.

Receptor activation causes the physical uncoupling of a guanine

nucleotide binding protein from the receptor. The protein is then

free to migrate through the bilayer or, perhaps to the cytosol to

interact with PL-C. This interaction causes a stimulation of PL-C

which acts to hydrolyze PI.

The PL-C activated by receptor stimulation is believed to be a

polyphosphatidylinositol specific one (Abdel-Latif et al., 1977;

Berridge, 1983). Abdel-Latif et al. demonstrated a significant loss

of 32P from triphosphoinositide with the addition of ACh and eserine

to 32p labelled rabbit iris muscle. ACh also decreased the

incorporation of 32p into Ptdlns 4,5P2, presumably by stimulating

breakdown. This decrease in 32p incorporation was accompanied by a

large increase in PA and PI labelling with no increase in PA and PI

content in the tissue. Berridge and co-workers demonstrated an

agonist-dependent increase in inositol 1,4-bisphosphate (DPI) and

inositol 1,4,5-trisphosphate (TPI) in blowfly salivary gland, rat

parotid gland and rat brain cortex (Berridge et al., 1983).











Streb et al. (1985) report an agonist-induced increase in the levels

of TPI of 106% over controls while the levels of DPI were increased by

64% over controls. Time course studies indicate that agonist-induced

TPI formation precedes that of inositol phosphate in blowfly salivary

gland and GH3 pituitary cells (Berridge et al., 1984; Rebecchi and

Gershengorn, 1983; Martin, 1983; Drummond et al., 1984). Together,

these results suggest that receptor-activated PL-C exhibits a

substrate specificity for Ptdlns 4,5P2. Many studies of this type

show an increase in inositol 1-phosphate (IP) as well as the increase

in inositol 1,4-bisphosphate (DPI). This may be the result of

enzymatic cleavage of phosphate groups from TPI. Another

interpretation is that PL-C can hydrolyze all phosphatidylinositols

and the IP and DPI produced either a) have effects that are specific

and as yet unidentified, or b) have no effects but allow for the

stoichiometric production of DAG, which can go on to stimulate protein

kinase-C. In any case, the hydrolysis of phosphoinositides can

produce 2 second messengers (inositolpolyphosphates and DAG) which can

initiate cascades involving Ca++ mobilization and protein kinase-C

activation.


Mobilization of Intracellular Calcium

In the early 1950s, Hokin and Hokin (1953) observed that

cholinergic drugs could stimulate the incorporation of 32P into PI and

PA of pancreatic slices. Since that time many other hormonal and

neurotransmitters have been found to produce similar effects on PI

metabolism in a variety of tissues (Berridge and Irvine, 1984). In

1975, Michell made the observation that all tissues that respond to an











agonist with a mobilization of intracellular Ca++ also exhibit PI

breakdown. He hypothesized that PI breakdown is the precursor of Ca++

mobilization (Michell, 1975).

The role of Ca++ in this cascade was controversial. Although it

was agreed that PI hydrolysis and increased intracellular Ca++ were

correlated, no cause and effect relationship existed. There were two

possibilities: one was that PI hydrolysis was stimulated by a

receptor mediated rise in intracellular Ca++; the other was PI

hydrolysis was the precursor of Ca++ elevation. In support of the

second theory, the addition of Ca++ ionophore in the presence of high

extracellular Ca++ failed to stimulate the breakdown of PI in a

variety of tissues. However, platelets, polymorphonuclear leukocytes,

nervous tissue and others do demonstrate PI hydrolysis with ionophore

(Michell and Kirk, 1981). Evidence exists in the platelet that

ionophore-stimulated PI hydrolysis has a different mechanism than that

of thrombin-induced hydrolysis and, therefore, may not be a good model

for this particular study (Lapetina et al., 1981). These studies

indicate that although the PI response can be independent of Ca++ in

many tissues, some tissues exhibit a PI response that is secondary to

an increase in Ca++. Gonzales and Crews (1985b) have shown that

guanine nucleotides and Ca++ can increase inositide hydrolysis in

isolated membranes in an additive manner. Thus, it is possible that

both Ca++ activated and/or Ca++ independent hydrolysis of PI can occur

depending on the cell type.

An interesting early theory suggesting the rise in intracellular
Ca++ is due to PI hydrolysis was put forth by Salmon and Honeyman










(1980). They believed that the hydrolysis of PI to DAG to PA by an

enzymatic phosphatase was the key event in this cascade. PA was known

to be a Ca++ ionophore in artificial systems (Tyson et al., 1976).

Experiments were performed on isolated frog smooth muscles that

exhibited contraction when stimulated with carbachol. They found that

carbachol produced an increase in PA content of the cells. In

addition, they demonstrated that addition of 1 jiM PA could mimic the

contractions produced by 100 AM carbachol. Thus they hypothesized

that PA was acting as a Ca++ ionophore, raising the intracellular Ca++

concentrations and causing smooth muscle contraction. However, in a

time-course study of vasopressin-induced PI hydrolysis in isolated

hepatocytes, the formation of PA was slower than Ca++ mobilization.

Also, the concentration of vasopressin required to produce maximum
Ca++ mobilization was much less than that required to produce maximum

levels of PA (Thomas et al., 1983). It is possible that PA plays a

minor role in the mobilization of Ca++, but more recent studies

suggest that the most important factor in mobilization of

intracellular Ca++ is TPI.

As mentioned above, inositol 1,4,5-trisphosphate (TPI) formation

precedes that of other phosphoinositols (Berridge et al., 1984;

Rebecchi and Gershengorn, 1983; Martin, 1983; Drummond et al., 1984).

These studies lead to the hypothesis that TPI could act as a second

messenger to stimulate Ca++ release from intracellular pools (Berridge

et al., 1984; Rebecchi and Gershengorn, 1983). This hypothesis was

put to the test by Streb et al. (1983). Using permeabilized rat

pancreatic acinar cells which allow phosphoinositols to cross the











plasma membrane they found that Ins 1,4,5P could release intracellular

Ca++. The release was concentration dependent, rapid, and specific

for Ins 1,4,5P. Using the mitochondrial poisons antimycin A or

oligomycin they determined that the Ins 1,4,5P induced Ca++ release

was not sensitive to mitochondrial poisons, suggesting a

non-mitochondrial Ca++ storage site. This storage of Ca++ was

dependent on the presence of ATP (Biden et al., 1984), and cellular

subfractionation studies determined that the storage site was the

microsomal fraction i.e. endoplasmic reticulum (Prentki et al., 1984;

Streb et al., 1984).

Recently, Irvine et al. (1984) discovered that stimulation of rat

parotid gland with carbachol produced the polyphosphoinositol isomer

inositol 1,3,4-trisphosphate. Subsequent examination showed that

inositol 1,4,5-trisphosphate is rapidly phosphorylated by a

3-phosphokinase to inositol 1,3,4,5-tetrakisphosphate (IP4) (Batty et

al., 1985). IP4 is a substrate for 5-phosphomonoesterase which

produces inositol 1,3,4-trisphosphate. Using sea urchin eggs, Irvine

and Moor (1986) found that microinjection of inositol

1,4,5-trisphosphate could fully activate eggs. Microinjections of

inositol 2,4,5-trisphosphate, an analog capable of mobilizing

intracellular Ca++ but not as potent as the 1,4,5 isomer, determined

that the 2,4,5 isomer was incapable of fully activating eggs

regardless of concentration. Coinjection of the 2,4,5 isomer with IP4

produced activation of eggs. The explanation offered by Irvine is

that the 2,4,5 isomer can cause the mobilization of intracellular Ca++

stores, but this alone is not enough to fully activate eggs. Ins










1,4,5-trisphosphate, which can mobilize intracellular Ca++ and be

metabolized to IP4, can fully activate eggs. Thus, Irvine suggests

that IP4 activates an extra source of Ca++, presumably a Ca++ channel

that allows extracellular Ca++ into the cytosol to trigger complete

egg activation.


The Role of Protein Kinase C

Since its discovery in 1978 by Nishizuka and coworkers (Takai et

al., 1977), protein kinase C (PK-C) has been shown to be very

important in regulation of cellular function (Nishizuka, 1984a). It

is widely distributed phylogenetically and is found in high

concentrations in the mammalian brain and spleen (Kuo et al., 1980),

suggesting an important role in nervous and immune system function.

The activity of PK-C is dependent on Ca++ and acidic phospholipids,

particularly phosphatidylserine (PS). Other phospholipids can

substitute for PS in vitro, but only in the presence of abnormally

high Ca++ concentrations (Takai et al., 1979), suggesting that PS is

critical for in vivo activity. In vitro, the Kd for Ca++ is about 60

AM. In the presence of DAG the Kd for Ca++ drops to the low AM range

and the affinity for PS but not other membrane lipids is greatly

increased (Takai et al., 1979). The tumor-promoting phorbol esters

mimic the effects of DAG in vitro. The effects of phorbol esters in

vivo are more complex because the stimulation of PK-C is very

long-lived as compared to the short-lived stimulation resulting from

the very labile DAG. Despite this, there is now little doubt that

PK-C is the cellular target for the effects of phorbol esters (for a

review of the role of tumor promotion see Nishizuka, 1984b).










As mentioned above, the receptor-stimulated activation of

phospholipase C produces two hydrolysis products in the membrane. One

is the aforementioned polyphosphatidylinositols, highly polar

compounds that migrate to the cytosol where they act as biochemical

second messengers. The other product is the highly lipophilic

substance diacylglycerol. The lipophilic DAG remains in the lipid

bilayer where it forms a quaternary complex with PK-C, membrane PS,

and Ca++ (Nishizuka, 1984). This active quaternary complex rapidly

phosphorylates other cellular enzymes altering their activity. This

model is supported by studies that demonstrate a shift of PK-C

activity from the cytosol to the membrane when phorbol esters are

incubated with parietal yolk sac cells (Kraft and Anderson, 1983).

Gonadotropin releasing factor also induces a shift of PK-C activity to

the membrane when incubated with isolated pituitary cells, presumably

through the receptor-mediated production of DAG (Hirota et al., 1985).

It is interesting to note that with normal aging, the activity of PK-C

in the cytosol markedly decreases, while the activity associated with

the membrane stays the same (Calderini et al., 1987). Since the

membrane bound PK-C is likely to be active in vivo, it is not

surprising that activity in this fraction is preserved during aging.

Phosphorylation by PK-C has been shown to regulate a wide variety

of cellular substrates in vivo and in vitro including EGF receptors

(Davis and Czech, 1984), beta-adrenergic receptors (Kelleher et al.,

1984), cytoskeletal proteins (Werth et al., 1983), enzymes involved in

glucose metabolism (Ahmad et al., 1984), and many others. Like most

protein kinases, PK-C has a broad substrate specificity. It will be











difficult to sort out the specific substrates that are most important

physiologically. The type of studies most likely to solve this

mystery are those combining biochemical evidence of protein

phosphorylation by PK-C plus biochemical or physiological changes in

response to transmitters or phorbol esters. Despite our ignorance it

is quite obvious that PK-C is an important part of the signal

transduction system that begins with receptor stimulated hydrolysis of

PI.


Combined Effects of Calcium and Protein Kinase C

Receptor-stimulated activation of PL-C can produce a rise in

intracellular Ca++ and PK-C activity. Thus, two bifurcating "arms"

exist in the signal transduction cascade. To examine the

physiological effect of an agonist in any system, both arms of the

cascade must be considered. In an elegant series of studies in the

laboratory of Nishizuka, it was found that one could mimic the effects

of thrombin, collagen, and platelet activating factor (PAF) on

platelets by adding A23187, a Ca++ ionophore, and 1-oleoyl-2-

acetylglycerol (OAG), a DAG analog that penetrates the cell membrane

to activate PK-C (Kaibuchi et al., 1983; Sano et al., 1983). Addition

of both compounds stimulated the phosphorylation of a 20K and 40K

molecular weight protein. The degree of phosphorylation of the

proteins correlated with the release of serotonin, whose release is

also stimulated by thrombin, collagen, and PAF. The addition of OAG

only produces phosphorylation of the 40K protein while A23187 only

stimulates the phosphorylation of the 20K protein, now known to be

myosin light chain kinase, presumably by calmodulin-dependent protein










kinase. In each case, there was no release of serotonin. Serotonin

release requires both DAG-PK-C and calcium-activated phosphoprotein.

Thus, both pathways together can act synergistically. Synergism of

these pathways has also been demonstrated on lysosomal enzyme release

in neutrophils (White et al., 1984; Robinson et al., 1984), histamine

release from mast cells (Heiman and Crews, 1985), and many other

systems (Hirasawa and Nishizuka, 1985).

Recent evidence suggests that PK-C exerts a negative feedback on

intracellular Ca++ levels (Drummond, 1985). In GH3 pituitary tumor

cells thyrotropin-releasing hormone (TRH) will stimulate PI hydrolysis

to form TPI and DAG. At low TRH receptor occupancy, [Ca]i increases

rapidly and declines slowly. As receptor occupancy increases, the

Ca++ signal duration is decreased due to an inhibitory component.

This inhibition is mimicked by phorbol esters and bacterial PK-C.

Also the time course of DAG production in these cells agrees with the

onset of the inhibitory phase. These data suggest that DAG activation

of PK-C may feedback to turn off the increase in the intracellular

calcium level.

It is clear that there are tissue differences in the interaction

of PK-C stimulation with Ca++ mobilization. The differential effect

of PK-C on the Ca++ signal is probably dependent on the function of

that cell and the various mechanisms of regulation of that cell type.

In some cases the "arms" of this bifurcating cascade can antagonize

each other and strike a balance to regulate homeostasis; in other

cases they synergize to perform some cellular function.










Summary of the Phosphatidylinositide Cascade System

Having gone through the various steps of PI signal transduction

system, we can now construct a model (Figure 1-2). Receptor

occupation by an appropriate agonist causes an activation of PL-C,

possibly by a guanine nucleotide coupling protein that dissociates

from the receptor and binds to PL-C, activating the enzyme. PL-C will

hydrolyze Ptdlns 4,5P2 to form the products TPI and DAG. The water

soluble TPI migrates to the cytosol and somehow effects the release of

Ca++ from the endoplasmic reticulum. The lipid soluble DAG remains in

the membrane to form a quaternary complex with PS, Ca++, and PK-C,

resulting in an activation of the kinase. While the effects of TPI on

the intracellular Ca++ levels are fairly well characterized, the

effects of PK-C activation with respect to endogenous substrates and

biochemical responses are still unknown.

















a)
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CHAPTER 2
BRAIN REGIONAL DISTRIBUTION OF ALPHAI
ADRENERGIC-STIMULATED PHOSPHOINOSITIDE HYDROLYSIS


Introduction

The distribution of alphal receptors varies regionally within the

brain (Bremner and Greengrass, 1979; Young and Kuhar, 1980; Jones et

al., 1984). Although the results of these studies vary, probably due

to methodological differences, in general the cortex, hippocampus, and

olfactory bulb have a high density of alphal receptors while the brain

stem, hypothalamus, striatum, and cerebellum have moderate to low

densities of alphal receptors. Neuronal alphal receptors are coupled

to phosphoinositide (PI) hydrolysis as a second messenger (Brown et

al., 1984; Minneman and Johnson, 1984; Schoepp et al., 1984). Thus,

we have studied the regional distribution of alphal agonist-stimulated

PI hydrolysis for norepinephrine (NE) and other alphal agonists in the

rat brain.

Previous experiments with muscarinic receptors have suggested

that efficacy differences between agonists show larger variation than

potency (Gonzales and Crews, 1984). In fact, muscarinic agonists have

been separated into classes by binding properties and correlation to

agonist efficacy (Fisher et al., 1983). Furthermore, the efficacy of

muscarinic partial agonists was found to vary among brain regions.

Thus, we studied the maximal responses to full and partial alphal

agonists in several brain regions.

18










Classically, NE is a full alphal agonist. Phenylephrine (PHEN),

methoxamine (METH), and dihydroergotamine (DHE) are also characterized

as alphal agonists. 6-Fluoronorepinephrine (6-FLNE) is a

phenylethylamine derivative with a decreased beta-adrenergic component

(Auerbach et al., 1981). The imidazoline derivative oxymetazoline

(OXY), believed to be mostly alpha2-adrenergic, behaves as an alphal

agonist in rat vas deferens (Minneman et al., 1983); therefore, it was

included in this study.


Methods

Dissection of brain regions. Brains were dissected as follows.

The brain was rapidly removed and placed upright on a dissection

platform wetted with warm KRB buffer. The olfactory bulbs were

removed at the stalk, cut in half longitudinally, and placed on a

tissue chopper disk for slicing. The cortices were dissected using a

razor blade to cut slices 1 to 2 mm in thickness, avoiding underlying

white matter. After the cortical slices were removed, the remaining

cortex and corpus callosum were peeled back to reveal the intact

hippocampus. The septal hippocampal connections were cut and both

hippocampi were removed intact. The brain was then rotated so that

the inferior surface was up. A frontal section cut was made at the

optic chiasm. The striatum was dissected from the frontal piece by

trimming away the surrounding regions. The striatum was sliced with a

razor blade into slices 1 to 2 mm thick and then further sliced with a

tissue chopper. The hypothalamus was dissected by making frontal

section cuts at the optic chiasm and just caudal to the mammillary

bodies. Sagittal cuts were made approximately 2 mm lateral to the










central plane. A coronal cut was then made through the anterior

commissure to denote the upper surface of the hypothalamus. The brain

stem was sliced into sheets with a razor blade and further sliced with

a tissue chopper.

Determination of phosphoinositide hydrolysis. Phosphatidyl-

inositol hydrolysis was performed as described by Gonzales and Crews

(1984). Brain slices were carefully sliced and placed in warm,

oxygenated Krebs-Ringer bicarbonate buffer (KRB buffer). Slices were

minced with a McIlwain tissue chopper at 350 pm in perpendicular

directions. Slices were transferred to 50 ml conical flasks and

dispersed. The slices were washed four times with fresh oxygenated

KRB buffer. [3H]-inositol (myo-[2-3H]inositol, 16.3 Ci/mmole,

Amersham, UK) was added to a final concentration of 0.1 to 0.3 AM in a

volume approximately four times that of the settled slices and

incubated for 1 hour at 37C with enough agitation to prevent the

slices from settling. At the end of this incorporation period the

slices were washed with fresh, oxygenated KRB buffer, allowed to

settle and brought to a volume approximately 4 times that of the

packed slices. While gently swirling the slices 50 Al were

transferred to polypropylene tubes containing 190 Al of KRB buffer

with 10 mM LiCl substituted for NaCl. Reactions were started with the

addition of 10 pl of appropriate concentrations of agonist or buffer.

The tubes were gassed with 02:CO2 (95:5), capped tightly, and shaken

in an incubator at 37'C. The reaction was stopped with the addition

of 1 ml chloroform/methanol (1:2, v/v). An additional 0.35 ml of

distilled water and 0.35 ml of chloroform was added, and the tubes










were tightly capped, shaken, and centrifuged to separate the phases.

An 0.75 ml aliquot of the aqueous layer was removed, diluted to 3 ml

with distilled water, and a 1 ml aliquot of Dowex-1 (50% v/v) slurry

was added to each tube. The slurry containing bound inositol

phosphates was poured into a polypropylene column with a fritted disk.

Inositol phosphates were eluted directly into scintillation vials with

5 ml of 0.1 M formic acid/1.0 M ammonium format and 10 ml Liquiscint

added. An aliquot of the chloroform layer was placed into glass

scintillation vials, evaporated under an air stream, and 3 ml of OCS

scintillation fluid was added. The samples were counted in a Beckman

LS7500 scintillation counter at a 37% efficiency. The data were

expressed as total DPM of [3H]-inositol phosphates released/total DPM
[3H]-inositol incorporated x 100, otherwise called fractional release

x 100 (FR x 100).


Results
Stimulation of phosphoinositide hydrolysis by alpha-adrenergic

agents in various brain regions. Maximum responsiveness for NE was

determined by performing dose-response curves in the rat cerebral

cortex. Brain region studies were performed with NE concentrations 3

to 10-fold greater than what was needed for maximum response in

cerebral cortex. OXY reaches maximum at 1 mM in the rat cerebral

cortex; therefore, the data for OXY have been separated from the

phenylethylamine data.

NE-stimulated PI hydrolysis showed regional variation. The most

robust response to NE was found in the hippocampus, approximately

twice as responsive as the brain stem (weakest). The rank order for










regional responsiveness to NE is: hippocampus > olfactory bulb >

cortex > striatum > hypothalamus > brain stem (Figures 2-1 and 2-2).

Although OXY is fairly potent at stimulating contraction of vas

deferens (Minneman et al., 1983), little or no phosphoinositide

hydrolysis occurred at concentrations as high as 100 AM. Regional

differences were found at OXY concentrations of 1 mM. The cortex and

striatum exhibited the greatest response, approximately three times

greater than the brain stem. The rank order of responsiveness for 1

mM OXY is: cortex = striatum > hippocampus >> hypothalamus > brain

stem.

Examination of the relative efficacies of other phenylethylamine

compounds (Table 2-1) shows that the efficacy remains fairly

consistent across brain regions. METH is more effective than PHEN in

brain stem and olfactory bulb, while PHEN is more effective in

striatum. While these differences show a trend towards significance,

we conclude no great differences in efficacy occur from region to

region.

The relative efficacies for OXY are shown in Table 2-2.

Pronounced differences in regional relative efficacy exist for the

maximal OXY concentration. Clearly, the regional rank order of figure

efficacy for OXY contrasts with all the phenylethylamines studied.





























30- C] NE
0 PHEN
O] METH
0 25 Q 6FLNE

X
C 20 -





10 -





Cortex HlopocamouS Brain Stem Olfactory Bulb Striatum





Figure 2-1. Fractional release of various alpha-adrenergic agonists
in rat brain regions. Each bar represents 6 determinations from 2
separate experiments. Error bars indicate S.E.M. NE, norepinephrine;
PHEN, phenylephrine; METH, methoxamine; 6FLNE,
6-fluoronorepinephrine; DHE, dihydroergotamine.









24









30-
30E NE (100uM)
3 OXY (100uM)
0 25- 3 OXY (luM)

X
020


0 )



10-
ILL

5-



Cortex Hippo- Hypo- Striatum Brain Stem
campus thalamus




Figure 2-2. Comparison of the fractional release values from 100 uM
norepinephrine and concentrations of oxymetazoline (OXY) in various
rat brain regions. Each bar represents nine determinations from 3
separate experiments.
















TABLE 2-1

RELATIVE EFFICACIES OF PHENYLETHYLAMINE DERIVATIVES
IN VARIOUS BRAIN REGIONS


BRAIN OLFACTORY
DRUG CORTEX HIPPOCAMPUS STEM BULB STRIATUM


NE 1.00 1.00 1.00 1.00 1.00

PHEN 0.55 0.55 0.48 0.43 0.54

METH 0.53 0.57 0.60 0.52 0.43

6FLNE 0.74 0.68 0.68 ---- 0.72

DHE 0.09 0.12 0.12 0.14 ----


Fractional release values were determined in various brain regions
(6 determinations from 2 experiments). Relative efficacy values were
determined by taking the average fractional release for each drug and
dividing by the average fractional release value for NE in that brain
region. NE, norepinephrine; PHEN, phenylephrine; METH, methoxamine;
6FLNE, 6-flouronorepinephrine; DHE, dihydroergotamine.
















TABLE 2-2
RELATIVE EFFICACY OF OXYMETAZOLINE IN VARIOUS BRAIN REGIONS



BRAIN
OXY CORTEX HIPPOCAMPUS STEM HYPOTHALAMUS STRIATUM


100pM 0.110.05 0.090.04 0.260.12 0.110.04 0.140.03

ImM 0.570.07 0.390.04 0.180.07 0.270.05 0.760.05


Fractional release (FR) values (9 determinations from 3 experiments)
were determined and the average FR for 100 pM or ImM oxymetazoline
(OXY) in a brain region was divided by the average FR for NE in that
brain region. Data are expressed as relative efficacy S.E.M.










Discussion
Previous studies have shown that the number of alphal adrenergic

receptors varies regionally within the brain. Bremner and Greengrass

(1979), using [3H]-prazosin as their label, find the highest density

of alphal receptors in the frontal cortex > hypothalamic-pre-optic

area > striatum > cerebellum > basal hypothalamus. Studies using
[1251]-HEAT show the highest density of alphal receptors in the

frontal cortex > hippocampus > brain stem > olfactory bulb >

striatum > cerebellum (Jones et al., 1984). We have shown high levels

of responsiveness to NE in hippocampus, olfactory bulb, and cerebral

cortex, with a moderate response from striatum and hypothalamus.

Brain stem was the least responsive region tested. Cerebellum barely

exhibits a response to 100 uM NE (Gonzales and Crews, 1985a). These

data are in general agreement with radioligand studies performed on

rat brain homogenates and autoradiographic analysis of receptor

distribution.

All drugs tested are partial agonists compared to NE. The

phenylethylamine derivatives retain their relative efficacy

coefficients from region to region while OXY, surprisingly, varies

greatly. In addition, the PI response to OXY rose sharply from almost

zero to maximum with a ten-fold increase in concentration. Ruffolo et

al. (1977) found cross desensitization in the rat vas deferens between

different imidazolines but not between imidazolines and

phenylethylamines. Taken together, these data suggest that OXY may

have a different mechanism of PI hydrolysis than phenylethylamines.









28
Therefore, we decided to compare the interaction of a larger group of

imidazolines to phenylethylamines on alpha1 binding sites and

alpha-stimulated PI hydrolysis in cerebral cortex.














CHAPTER 3
DIFFERENCES IN IMIDAZOLINE AND PHENYLETHYLAMINE
ALPHA-ADRENERGIC AGONISTS: COMPARISON OF
BINDING AFFINITY AND PHOSPHOINOSITIDE RESPONSE


Introduction

The neurotransmitter NE can stimulate both alphal and alpha2

receptors in the brain alphal receptors are coupled to hydrolysis of

phosphoinositides (PI) (Kendall et al., 1985; Gonzales and Crews,

1985a) while neuronal alpha2 receptors are negatively coupled to

adenylate cyclase (Kitamura et al., 1985). In addition, these

receptor responses can be selectively inhibited by prazosin (aI) or

yohimbine (x2). In rat cerebral cortex, for example, prazosin is

three orders of magnitude more potent than yohimbine for inhibition of

NE-stimulated PI hydrolysis (Kendall et al., 1985).

Thus, in the brain, oc and o2 responses are dissimilar and

distinguishable with selective receptor antagonists.

Such is not the case for vascular smooth muscle. A chemically

diverse variety of alpha adrenergic are known to contract vascular

smooth muscle. These contractions can be mediated by postsynaptic cI

and/or o2 receptors (Timmermans et al., 1979; Constantine et al.,

1980; Madjar, 1980). NE can induce contractions via both cl and o2

receptors. Unlike the thousand-fold difference seen in brain and rat

aorta, prazosin is only 20 times more potent than rauwolscine in cat

mesenteric artery (Skarby et al., 1983), ten times more potent in dog










splenic artery, and three times more potent in dog splenic vein

(Hieble and Woodward, 1984). Thus, the distinction between al and o2

mediated effects are less than clear.

Phenylethylamine derivatives of NE, such as phenylephrine and

methoxamine are selectively blocked by prazosin (van Meel et al.,

1981). The imidazoline cirazoline (CIR) is also selectively blocked

by prazosin (van Meel et al., 1981; Cavero et al., 1982). Other

imidazoline compounds, such as oxymetazolines (OXY), tramazoline

(TRAM), and naphazoline (NAPH), while believed to act at o2 receptors

(Timmermans and van Zwieten, 1982), may also contract via an alphal

component. For example, experiments on the alphal receptor-induced

contraction of rat vas deferens smooth muscle induced by NAPH, TRAM

and OXY indicate these compounds have an efficacy of 0.50, 0.68 and

0.72 compared to NE, respectively. In addition, 20 nM prazosin can

shift the dose-response curve for contraction 10-20 fold, suggesting

an alphal-receptor mediated contraction (Minneman et al., 1983).

We have compared classical phenylethylamine adrenergic drugs to

imidazolines in order to better understand the interaction of these

compounds with the alphal receptor. Our model system, rat cerebral

cortex, contains alphal receptors which are coupled to

phosphatidylinositol (PI) hydrolysis and selectively inhibited by

prazosin (Kendall et al., 1985; Gonzales and Crews, 1985a). We report

here that there are clear differences in the binding properties and

agonist activity of imidazoline alpha agonists when compared to

phenylethylamine alpha agonists.











Methods
Radioligand Binding. Rats were decapitated and their brains

placed into either ice-cold 50mM TRIS/HCl buffer containing 1mM EDTA

pH adjusted to 7.4 or modified Krebs-Ringer bicarbonate buffer (KRB

buffer) (minus glucose). Cerebral cortex was dissected away and

weighed. The cortex was minced in 20 volumes per wet weight of

ice-cold buffer and homogenized using a Tekmar Tissuemizer at setting

50 for 30 seconds. The homogenate was centrifuged at 42000 x g for 10

min at 4*C. The supernate was discarded and the pellet resuspended in

20 volumes of ice-cold buffer. The membranes were washed two

additional times and resuspended in 20 volumes of ice-cold buffer. An

aliquot was taken for protein determination. The assay was started

with the addition of approximately 0.3 mg of protein into test tubes

containing the buffer indicated, 10 nM [3H]prazosin

([7-methoxy-3H]prazosin, 83 Ci/mmole, Amersham, UK), and the indicated

concentration of competing ligand in a total volume of 0.5 ml.

Non-specific binding was determined in the presence of 10 gM

phentolamine. Non-specific binding was approximately 20% of total

binding. Incubations were performed at 25*C for 30 min and terminated

by rapid filtration onto Whatman GF/C filter paper. Filters were

washed three times with ice-cold buffer. Filters were placed in

scintillation vials and 10 ml of Liquiscint added. Vials were shaken

for 1 hour and counted in a Beckman LS7500 scintillation counter at 37

% efficiency.

Phosphatidylinositol Hydrolysis. See "Methods" section,

Chapter 2.











Data Analysis. Competition curves were analyzed using the

iterative, non-linear curve fitting program of Fletcher and Shrager as

described by McKinney and Coyle (1982). The criterion for

determination of one vs. two site fit was that previously used by

Fisher et al. (1983) and is as follows: when comparing the sum of

squared residuals for one vs. two site fit, if one was at least five

fold smaller than the other, the smaller sum of squared residuals was

considered the best fit. For cases where the sum of squared residuals

was less than five fold different, t-tests between actual occupancy

and predicted occupancy for one- and two-site fits were examined. If

the data were significantly different from one of the best fit models,

that model was eliminated and the other accepted. If both or neither

models were significantly different from the actual data, t-tests

between the predicted one and two site models were examined; if the

two models were not significantly different from each other the best

fit was taken to be one site.

ED50 and IC50 values were determined using probit analysis as

outlined by Goldstein (1964).

Protein Determination. Protein concentrations were determined

using the Coomassie blue dye method (Bradford, 1976).

Drugs. The source of drugs used is as follows: (-)-

norepinephrine bitartrate, (-)-epinephrine bitartrate, l-phenylephrine

HC1, oxymetazoline HC1, and naphazoline HCI (Sigma Chemical Co.,

St. Louis, MO), tramazoline HCl (Dr. Karl Thomae GMBH Biberach, FRG),

cirazoline HCL (L.E.R.S. Synthelabo Paris, France), prazosin HC1

(Pfizer Inc. Groton, CT), phentolamine mesylate (Ciba-Geigy Corp.










Summit, NJ), alphamethylnorepineprine HC1 (Sterling-Winthrop

Rensselaer, NY), methoxamine HC1 (Burroughs Wellcome Co. Research

Triangle Park, NC), guanfacine (gift from Dr. Micheal Katovich).

Results

Aqonist competition curves for [3Hl-prazosin binding in cerebral

cortical membranes. To determine the apparent affinity of the various

alpha agonists studied for alphal receptors, competition curves were

performed. To investigate the actions of sodium and other ions as

well as consistency in binding and response experiments, binding was

done in both TRIS buffer and KRB buffer. All agents tested inhibit

[3H]-prazosin binding (Figures 3-1 and 3-2). In TRIS buffer, all

agonists were found to best-fit two sites of interaction (Figure 3-1

and Table 3-1). Comparison of the high affinity Kd values indicate

that the imidazolines phentolamine and oxymetazoline had the highest

affinity, in the low nmol range, followed by EPI > METH > TRAM > NAPH

> CIR > NE with Kd values around 1 pM (Table 3-1). The least potent

agents were aMENE and phenylephrine. Low affinity sites show a marked

difference between the classes of compounds. Phenylethylamines have

low affinity sites with Kd values greater than 100 /iM, whereas the low

affinity sites for imidazolines have Kd values less than 30 pM. The

potency series for low affinity sites suggests that the imidazolines

have a higher affinity and therefore might be several times more

potent than phenylethylamines. Thus, the potency series for low

affinity sites is quite different from that of high affinity sites.

Furthermore, imidazolines interact with alphal receptors with an

affinity equal to or greater than that of NE.










To examine the effects of sodium and other ions competition

curves were also performed in KRB buffer. The imidazolines again have

higher affinities than NE. The imidazolines CIR (Figure 3-2) and TRAM

(data not shown) do not completely displace all the [3H]-prazosin

sites, leaving about 20% of the total label at saturating

concentrations of competitor. OXY and PHENTOL have the highest

affinities with Kd values in the low nM range; CIR, EPI, TRAM, PHEN,

and NE all have Kd values around 1 AM, whereas METH and ocMENE have the

lowest affinity with Kd values in the mid-AM range. Examination of

best-fit analysis shows that phenylethylamines exhibit a one-site

best-fit in KRB buffer while the imidazolines exhibit a two-site

interaction (Table 3-2). It is clear that imidazolines in general

have higher affinities for [3H]-prazosin sites in both TRIS buffer and

KRB buffer.

Aqonist-stimulated PI hydrolysis dose-response curves. The high

affinity for alphal receptors exhibited by imidazolines and their

known ability to contract vascular smooth muscle led us to test their

ability to stimulate phosphoinositide hydrolysis. To determine the

the potency and efficacy of various agonists, dose-response curves

were performed (Figures 3-3 and 3-4). The phenylethylamines, EPI, NE,

and oMENE are full agonists (Figure 3-3). Phenylephrine and

methoxamine (not shown) are partial agonists. The order of potency is

EPI > NE PHEN > oMENE > METH. Characteristic of alpha receptor

responses, stimulated PI hydrolysis is stereospecific as (+)-NE is

less potent and, surprisingly, less efficacious than (-)-NE (Table 3-

3). The alpha2 agonist clonidine (100 jM) did not stimulate PI



























60




44O


Figure 3-1. Competition curves for [3H]-prazosin binding by various
alpha-adrenergic compounds in TRIS buffer. Assays were performed on
rat cerebral cortex as described in "Methods." Values are expressed
as percent of maximum specific binding. For the sake of clarity only
the data points for oxymetazoline and cirazoline are presented. For
norepinephrine and oxymetazoline, data points represent 9
determinations from 3 separate experiments. Other points represent 6
determinations from 2 separate experiments.




























0
O60
E
E

2 40
OR-0


Log


(M)


Figure 3-2. Competition curves for [3H]-prazosin binding by various
alpha-adrenergic compounds in modified KRB buffer. Data are presented
as in Figure 3-1. Data points for NE represent 9 determinations from
3 separate experiments. All other data points represent 2 separate
experiments performed in triplicate.
















TABLE 3-1


ALPHA-ADRENERGIC COMPETITION FOR [3H]-PRAZOSIN
BINDING SITES IN TRIS BUFFER


Norepinephrine

Epinephrine

Phenylephrine

c Methylnore-
pinephrine

Methoxamine

Oxymetazoline

Cirazoline

Tramazoline

Naphazoline

Phentolamine


BEST
FIT

2 Site

2 Site

2 Site


Site

Site

Site

Site

Site

Site

Site


Kd (AM)
HIGH

1.44

0.44

11.75


5.20

0.87

0.024

1.34

1.11

1.20

0.01


Kd
LOW

298

271

540


249

112

2.87

21.43

17.31

27.55

3.29


Computer generated kinetic binding parameters obtained from
ligand competition for [IH]-prazosin binding in 50mM TRIS buffer, pH
7.4. Competition assays and data analysis were performed as described
in "Methods."
















TABLE 3-2

ALPHA-ADRENERGIC COMPETITION FOR [3H]-PRAZOSIN
BINDING SITES IN KREBS-RINGER BUFFER


Norepinephrine

Epinephrine

Phenylephrine

a Methylnore-
pinephrine

Methoxamine

Oxymetazoline

Cirazoline

Tramazoline

Naphazoline

Phentolamine


BEST
FIT

Site

Site

Site


Site

Site

Site

Site

Site

or 2

Site


N

3/3

2/2

2/2


2/2

3/3

2/2

2/2

2/2

2

2/2


Kd (1I
HIGH

3.20 0.22

0.84

3.15


4) K
LOI


58.20

36.9 t 13.0

0.0026

0.10

0.80

0.76/0.03

0.03


0.12

335.0

309.0

--/73.0

97.0


Computer generated kinetic binding parameters obtained from
ligand competition for [ H]-prazosin binding in modified KRB buffer
(minus glucose). Competition assays and data analysis were performed
as described in "Methods."


m

















28- A EPI
AX; A NE
c aE-MENE
24 A VPHEN

x20 /

Q)16-

c 12 -/
o


/ /!
4-


0 -6 -5 -4 -3
Log [Drug] (M)







Figure 3-3. Dose-response curves for phenylethylamine stimulation of
phosphoinositide hydrolysis in rat cerebral cortex. Data are
expressed as Fractional Release x 100 (see "Methods," Chapter 2).
Data points represent at least 9 determinations from at least 3
separate experiments. Error bars were omitted for clarity, but at no
point exceeded 2 fractional release units.




















20 /






0 *

LL 8





I I I
A....-y ------


0 -6 -5 -4 -3
Log [Drug] (M)











Figure 3-4. Dose-response curves for imidazoline stimulation of
phosphoinositide hydrolysis in rat cerebral cortex. Data are
expressed as described in Fig. 3-3. Data points represent at least 6
determinations from at least 2 separate experiments. The
dose-response curve for norepinephrine is presented for the sake of
comparison. The single point plus S.E.M. bar representing naphazoline
is the maximum response as determined from three dose-response curves.
















TABLE 3-3

ED50 AND EFFICACY VALUES FOR VARIOUS ALPHA AGONISIS


Drug Concentration ED50 (gM) Rel. Efficacy


EPI

NE

PHEN

xMENE

METH

(+)NE

CIR

OXY

TRAM

NAPH

DHE

6-FLNE

CLON

GUANF


100

100

100

100

100

1000

1000

1000

1000

1000

100

100

100

1000


1.41

3.29

5.06

19.08

37.57

110.76

250.00*

500.00*

600.00*

600.00*


0.10

0.54

0.98

2.66

3.62

29.00


1.00

1.00

0.66

0.95

0.64

0.50

0.76

0.46

0.42

0.15

0.09

0.81

0.00

0.80


ED0o values are determined as described in "Methods." Relative
efficacies are calculated from the maximal response to agonist divided
by the maximal response to norepinephrine.
* Due to the uncharacteristic shape of the imidazoline dose-response
curves, the normal probit method of ED50 determination was abandoned
and the ED50s were approximated by eye.











hydrolysis. The guanidine derivative guanfacine, which acts via the

alpha2 receptor, is efficacious but not potent with respect to PI

hydrolysis.

The dose-response curves for the imidazoline class of compounds

is shown in Figure 3-4. All imidazolines tested exhibit little or no

response until concentrations of about 100 uM are reached.

Imidazolines have a sharp dose-response curve, from essentially zero

to maximum in a ten-fold concentration range. The efficacies range

from 0.15 for NAPH to 0.76 for CIR (Table 3-3).

Correlation between Kd values and ED50 values for various alpha

agonists. Figure 3-5 shows the relationship between Kd values

obtained using KRB as the buffer and ED50 values for various agonists.

The correlation coefficient for the five phenylethylamines is 0.96.

Using the high affinity or the low affinity constant from Table 3-2,

the imidazoline compounds do not approach the line of unity (r=0.33

and r=0.15 respectively). Correlation of the Kd values for high

affinity sites obtained using 50 mM TRIS buffer (pH 7.4) as the buffer

to ED50 values resulted in a correlation coefficient of 0.23 for the

phenylethylamines. Removal of the methoxamine values brings the

correlation coefficient to 0.71 for phenylethylamine high affinity

sites in TRIS buffer. Again, the imidazolines did not approach the

line of unity. These data clearly demonstrate the difference between

the two classes of compounds. Phenylethylamines exhibit a one to one

response characteristic of tight coupling between receptor occupation

and biochemical response. Imidazolines, although quite potent in

their ability to bind alphaI receptors, are weak in eliciting a
























CO METH/

I-
S/ -MENE
705- PHEN / -

EPI
6NE
06-


0)7-
-I

8-

I I I I I |
8 7 6 5 4 3
-Log Kd





Figure 3-5. Correlation between Kd values determined from competition
curves performed in KRB buffer to ED50 values for agonist stimulated
PI hydrolysis. All experiments were performed in rat cerebral
cortical membranes or tissue slices. The data are taken from Tables
3-1 through 3-3. The line represents the line of unity. CIR,
cirazoline; EPI, epinephrine; =MENE, alphamethylnorepinephrine; METH,
methoxamine; NAPH, naphazoline; NE, norepinephrine; OXY,
oxymetazoline; PHEN, phenylephrine; TRAM, tramazoline.











biochemical response. The apparent high affinity for alphal sites

shown by imidazolines contrasts with the weak potency for stimulation

of PI hydrolysis.

Antagonism of PI hydrolysis response by prazosin. To determine

whether or not these compounds were acting via alphal receptors, the

ability of prazosin to antagonize the response was tested (Figure 3-

6). As expected 1 pM prazosin was able to inhibit the PI response to

all phenylethylamines. The imidazoline-stimulated PI response was not

inhibited by 1 pM prazosin, nor was the guanfacine-induced PI response

inhibited. Similar results were obtained using 10 AM prazosin (data

not shown). These data suggest that imidazolines do not stimulate PI

hydrolysis via alphal receptors.

Ability of imidazolines to antagonize NE-stimulated PI

hydrolysis. The potent receptor binding properties of imidazolines

combined with their low efficacy suggest these compounds may exhibit

antagonist properties. To determine if the imidazolines can

antagonize NE-stimulated PI hydrolysis, dose-response curves for

oxymetazoline in the presence and absence of 100 AM NE were performed

(Figure 3-7). Increasing concentrations of OXY inhibit the

NE-stimulated PI hydrolysis. The stimulation by NE is almost

completely inhibited at 100 iM OXY. At this point, OXY begins to

stimulate PI hydrolysis and the NE + OXY curve exactly follows the OXY

curve. Similar results are obtained with CIR, TRAM and NAPH (data not

shown). These data indicate that imidazolines can antagonize

NE-induced stimulation of PI hydrolysis. It is interesting that the

response curve in the presence of 100 AM NE follows the imidazoline





























0
o
x 10







S20



10


A

O Control

S0 Prazosin (1uM)


EPI NE PHEN METH GUANF

B


/


Figure 3-6. Inhibition of agonist-stimulated PI hydrolysis by the
alphaI antagonist prazosin. Each bar represents nine determinations
from three separate experiments. Error bars represent the S.E.M.
Abbreviations are as in Figure 3-5, with the exception of GUANF,
guanfacine.


TRAM


NAPH

















30


25


20


15


10


0 -7


-6 -5 -4
Log [Oxymetazoline]


-3
(M)


Figure 3-7. Oxymetazoline and oxymetazoline plus norepinephrine
stimulation of PI hydrolysis. Data are expressed as FR X 100. Each
data point represents nine determinations from three separate
experiments. Error bars represent the S.E.M. NE, norepinephrine.


A Control
* +100 uM NE


A
\_





-&


I I I


l I
J | I |










biochemical response. The apparent high affinity for alphal sites

hypothesis that imidazolines are partial agonists and again suggests a

mechanism other than alphal-receptor stimulated PI hydrolysis.

Discussion

All adrenergic compounds tested compete with [3H]-prazosin for

alphaI receptors. Using 50 mM TRIS buffer, all tested compounds

exhibit two sites of interaction. Our data and that of others

(Glossmann and Hornung, 1980; Morrow and Creese, 1986) suggest two

affinity subtypes for alphal agonist binding. In addition, the

potency series for our data are in agreement with Hornung et al.

(1979). Clear differences arise between phenylethylamines and

imidazolines when the competition curves are performed in modified KRB

buffer. In this ionic millieu, imidazolines retain two sites of

interaction while phenylethylamines are converted to a one-site

interaction profile. This conversion of phenylethylamines to a one-

site interaction is in agreement with Glossman and Hornung (1980) who,

in rat brain membranes, calculates Hill coefficients in the presence

and absence of 150 mM NaCl. Sodium shifts the Hill coefficient for

NE, EPI, oMENE, and PHEN from less than one to one. This shift is

particularly dramatic for NE and ocMENE. The Hill coefficient for NAPH

was unaffected by Na+. Thus, it appears that sodium ion causes the

conversion of the phenylethylamine binding profile to one-site.

Dose-response curves for agonist stimulated PI hydrolysis show

glaring differences between the two classes of compounds.

Phenylethylamines exhibit classical dose-response relationships. The

calculated ED50 values correlate well with Kd values from competition











curves performed in KRB buffer. In contrast, imidazolines have sharp

peaks of activity, starting around 100 jiM and reaching maximum at 1

mM. The ED50 values for imidazolines do not correlate with any of the

high or low Kd values from competition curves performed in either TRIS

buffer or KRB buffer. The lack of correlation between ED50 values and

Kd values for imidazolines suggests a non-alphal receptor mediated

mechanism of PI hydrolysis. This is also supported by the fact that

the alphal receptor antagonist prazosin was unable to inhibit the

response due to imidazolines. Antagonists for various other receptor

systems, including rauwolscine, atropine, haloperidol and others, also

were unable to inhibit imidazoline-induced PI hydrolysis (data not

shown).

The finding that imidazolines can bind to alphal receptors and

stimulate PI hydrolysis, along with the fact that imidazolines

contract smooth muscle, would suggest imidazolines are alphal

agonists. Figure 3-6, however, demonstrates that imidazolines act as

antagonists at the alphal receptor. At 100 pM OXY the PI response to

NE is almost completely inhibited. This finding is supported by work

on the alphal receptor-mediated reduction of [3H]-acetylcholine

release from isolated rat atria (McDonough et al., 1986), where it was

determined that cirazoline and other imidazolines inhibit the NE-

induced reduction of [3H]-acetylcholine release. In addition, OXY and

CIR displace the dose-response curve for epinephrine-stimulated PI

hydrolysis in hepatocytes (Garcia-Sainz et al., 1985). Our data are

further supported by the fact that tolazoline and phentolamine, known

alphal antagonists, are imidazoline derivatives. Thus, imidazolines










are antagonists at alphal receptors that somehow stimulate PI

hydrolysis, leading to a cellular response.

Alphal receptor activation is associated with PI hydrolysis in

vascular smooth muscle cells (Villalobos-Molina et al., 1982; Garcia-

Sainz et al., 1985). Ambler et al. (1984) demonstrated a correlation

between PI hydrolysis and Ca++ mobilization in BC3H-1 muscle cells.

Chiu et al. (1987) obtained similar results in isolated rat aorta. It

is likely that the alphal receptor-stimulated PI hydrolysis provides

for the release of intracellular Ca++ stores that may act, directly or

in combination with other events, to stimulate smooth muscle

contraction. However, Chiu and co-workers (1986) find that in rat

aorta, NE, PHEN, and CIR stimulate both the influx of extracellular
Ca++ and release of intracellular Ca++ stores, while other agonists

stimulate the influx of extracellular Ca++ exclusively. Contractions

of the rat aorta by OXY, for instance, are completely dependent on

extracellular Ca++ (Godfraind et al., 1982). Also, although CIR can

stimulate contraction via intracellular and extracellular Ca++

pathways, pretreatment with phenoxybenzamine renders the pressor

response to CIR labile to Ca++ channel blockers (Ruffolo et al. 1984).

It has been postulated that there is a single alphal-adrenoceptor able

to activate two separate pathways of Ca++ mobilization or a single

alphal receptor that is exclusively coupled to either PL-C or

receptor-stimulated Ca++ channels (Chiu et al., 1986). Therefore, it

possible that contractions elicited by imidazolines are mediated

through an alphal receptor whose stimulation leads exclusively to

external Ca++ influx. NE stimulates both this external Ca++-coupled










receptor and a PL-C coupled receptor, while imidazolines block the PL-

C-coupled alphal receptor. Our data are consistent with the above

hypothesis; however, this hypothesis does not explain why imidazolines

stimulate PI hydrolysis.

Phospholipase-C is a Ca++-dependent enzyme and its activity is

affected by changes in intracellular Ca++ concentrations. The Ca++

ionophore A23187, presumably via Ca++ activation of PL-C, can

stimulate PI hydrolysis (Michell and Kirk, 1981; Gonzales et al.,

1986). In addition, veratrine, which opens Na+ channels, can

stimulate PI hydrolysis in rat brain, probably by activation of

voltage-dependent Ca++ channels (Maier and Rutledge, 1987).

Therefore, it is possible imidazoline receptor-activated Ca++ flux may

be responsible for the observed PI hydrolysis in these experiments.

In summary, the interaction of imidazolines and phenylethylamines

at alphal receptors, and stimulated PI hydrolysis shows striking

differences between the two classes of agonist. Also, imidazolines

can act as antagonists to NE-stimulated PI hydrolysis. Imidazoline-

stimulated PI hydrolysis is not blocked by a variety of receptor

antagonists including the alphal receptor antagonist prazosin. It is

possible that imidazoline-stimulated PI hydrolysis observed in our

experiments is the result of receptor-stimulated Ca++ influx.














CHAPTER 4
REGULATION OF RAT BRAIN ALPHA RECEPTORS


Introduction

Many neuronal and hormonal signal transduction systems are known

to be regulated by agonist stimulation (Fleming et al., 1973).

Changes in the normal levels of stimulation will induce changes within

the system to maintain homeostasis. Thus, an increase or decrease in

synaptic levels of neurotransmitter will generally cause a reciprocal

change in the responsiveness of the target tissue.

Reserpine, a catechol depletor, causes postjunctional

supersensitivity in the cat nictitating membrane (Trendelenburg,

1966), brain (Ungerstedt et al., 1975), and cardiovascular tissue

(Carrier, 1975). In addition, treatment with reserpine will increase

beta-adrenergic receptor density (Greenberg and Weiss, 1979) and

beta-adrenergic stimulation of adenylate cyclase activity (Dismukes

and Daly, 1974; Williams and Pirch, 1974). Alphal receptors coupled

to PI in the rat brain have not been studied to determine if they

follow classical super- and sub-sensitivity as established with smooth

muscle preparations and beta-adrenergic coupling to adenylate cyclase.

To determine if alphal receptor coupled PI hydrolysis exhibits

supersensitivity, we studied the PI response to NE in control and

reserpine treated rats after acute and chronic reserpine regimens.











Methods
Animals. Male Sprague-Dawley rats (200-300g) were housed in a

well ventilated room with food and water available ad libitum. Lights

were on between 8:00 a.m. and 4:00 p.m. Drugs were administered by

i.p. injection. Animals were killed by decapitation and their brains

rapidly removed and placed in ice cold 50 mM TRIS buffer adjusted to

pH 7.4 with HC1 for preparation of membranes, or 37*C KRB buffer for

preparation of brain slices.

Drugs. Reserpine was dissolved in propylene glycol:ethanol:

water (6:1:4) vehicle and sonicated 5 to 10 minutes. Reserpine was

administered by i.p. injection. Control rats received vehicle in

volumes identical to the treated group on a per kg basis.

Phosphoinositide hydrolysis. See "Methods," Chapter 2.

Radioligand binding. See "Methods," Chapter 3.

Adrenergic stimulation of adenylate cyclase in cerebral cortical

slices. Adrenergic stimulation of adenylate cyclase in cerebral

cortical slices was carried out by incubating the slices with

norepinephrine or isoproterenol as modified by Vetulani et al. (1976).

Cerebral cortices were dissected as described in Chapter 2. In

addition, the dissected cortices were further cut into slices 0.35 mm

thick in two perpendicular planes using a McIlwain tissue chopper.

Each control or drug-treated group consisted of cerebral cortical

slices obtained from three rats. Immediately following their

preparation, the slices were transferred to flasks containing 50 ml of

physiologic buffer, pH 7.4 (Kakiuchi and Rall, 1968) at 37C which had

been bubbled with 02/C02 (95:5). After the slices were gently










dispersed, they were washed four times with fresh physiologic buffer

at 37*C over a period of 10 minutes. The washed slices were then

dispersed in 50 ml fresh physiologic buffer and incubated for 45

minutes at 370C. The slices were continuously aggitated gently and

bubbled with a slow stream of 02/C02 during the incubation. After the

45 minute incubation, the slices were allowed to settle and the

supernatant buffer was removed and discarded. The slices were

resuspended in 50 ml of fresh physiologic buffer containing 5 mM

theophylline and were incubated an additional 15 minutes at 374C with

gentle aggitation and continuous bubbling of 02C02. Following the 15

minute incubation, the slices were again allowed to settle and the

supernatant buffer discarded. The slices were resuspended in 10 ml of

fresh physiologic buffer containing 5 mM theophylline at 37"C. One

milliliter aliquots of well-dispersed slices were then transferred to

separate tubes containing 2 ml of physiologic buffer with 5 mM

theophylline at 37C for incubation with buffer alone for

determination of basal cAMP production or with agonist. Incubation

with buffer or agonist was carried out in triplicate. To begin

stimulation, 0.33 ml of physiologic buffer or 10-fold concentrated

agonist was added. Preliminary experiments indicated a dose-dependent

increase in cAMP production stimulated by norepinephrine or

isoproterenol, with maximum stimulation occurring with 100 pM of

either agonist. Immediately after addition of buffer or agonist, the

tubes were rapidly gassed with a high pressure stream of 02/C02,

tightly capped, and gently aggitated for 10 minutes at 37*C. Care was

taken during this incubation to prevent the slices from settling on










the bottom of the tubes. Time course experiments done previously

indicated that maximum stimulation of cAMP production in cerebral

cortical slices by either norepinephrine or isoproterenol occurred

after 10 minutes of incubation. To stop the stimulation, the slices

were allowed to settle, and the supernatant buffer was aspirated and

discarded. Preliminary experiments found no detectable cAMP in the

supernatant buffer. Each tube containing slices then received 3 ml

boiling-hot 1 N HCI, and the slices were immediately homogenized with

a Tekmar Tissuemizer at setting 50 for approximately 30 seconds. The

homogenates were centrifuged at 27,000 x g for 30 minutes. The

pellets were dissolved in 1 ml 0.5 N NaOH for protein determination.

Each incubation tube contained from 6.0 to 10.0 mg of protein. The

supernatants were evaporated using a Savant rotary evaporator, and the

residues remaining were dissolved in 1 ml 25 mM TRIS-HCl buffer, pH

7.0 containing 5 mM theophylline and saved for assay of cAMP.

The cAMP content of the dissolved residue samples was determined

in triplicate using a protein binding assay modified from Brown et al.

(1971). 50 1l of sample was incubated with 60 pl of 25 mM TRIS-HCl

buffer, pH 7.0, 50 pl 0.8 nM [3H]-cAMP (Amersham; 41 Ci/mmole), and 40

pl of cAMP-dependent protein kinase (1.25 mg/ml; Sigma Corp.; binding

activity 0.09 pmol cAMP/mg) for 60 minutes in an ice-water bath.

Preliminary experiments indicated that the binding of [3H]-cAMP

reached equilibrium under these conditions. Total [3H]-cAMP binding

to the protein kinase was determined as above with the 50 1l of sample

replaced with 50 p1 of 25 mM TRIS-HCl buffer, pH 7.0. Nonspecific

binding of [3H]-cAMP was determined in the presence of 2.5 pM cAMP.










To terminate the incubation after 60 minutes, 70 pl of hydroxyapatite

(Sigma Corp.; diluted 1:1, v/v with 10 mM TRIS-HCI buffer, pH 7.0) was

added to each sample, the sample vortexed and allowed to stand at

least 6 minutes. The samples were then vacuum filtered over Whatman

GF/C filters and washed 3 times with 4 ml ice-cold 10 mM TRIS-HCl

buffer, pH 7.0. The filters were placed flat in vials and shaken for

at least 30 minutes in 1 ml 0.5 N HC1 to dissolve the hydroxyapatite.

Ten milliliters of Liquiscint scintillation fluid was added to each

vial and shaken to form a homogenous mixture. Radioactivity was

determined by counting on a Beckman Liquid Scintillation counter.

Specific [3H]-cAMP binding was calculated as the difference between

total and nonspecific binding. The quantity of cAMP in each sample

was determined by standard additions using the percent inhibition of

specific [3H]-cAMP binding by each sample. The standard curve was

constructed by the same protocol as above using known concentrations

of cAMP ranging from 2.5 nM to 500 nM. To confirm the specificity of

this assay for cAMP, several of the unknown samples as well as samples

with known cAMP content were incubated with 600 ig of 3',5'-cAMP

phosphodiesterase (Sigma Corp.; 0.02-0.05 U/mg) for 80 minutes at 37C

prior to assay. In all cases, preincubation with phosphodiesterase

prevented the inhibition of [3H]-cAMP binding to the protein kinase,

confirming the specificity of this assay for cAMP.
13Hl-inositol incorporation. Slices were made as described in

"Methods," Chapter 2. Slices were washed four times with warm,

oxygenated KRB buffer. Following the wash, the slices were allowed to

settle and the excess buffer removed leaving enough buffer to comprise









56
four volumes of settled slices. [3H]-inositol (16.3 Ci/mmol; Amersham

Corp.) was added to a final concentration of 0.1 to 0.3 AM. The tube

was gassed with 02/C02, capped, and incubated in a shaking water bath

at 37'C. Immediately after addition of radioactivity, and at

appropriate time points, a 200 Al aliquot of dispersed slices was

homogenized with a Tekmar Tissuemizer at setting 50 for 15 seconds.

Aliquots were taken for protein determination. A 500 gl aliquot of

the homogenate was placed in a Falcon 17 x 100 mm polypropylene tube.

Two milliliter of 2:1 methanol:chloroform was added. An additional

0.75 ml of distilled water and 0.75 ml chloroform was added. The

tubes were capped tightly and shaken vigorously for 5 minutes then

centrifuged for phase separation. The aqueous layer was aspirated and

a 200 gl aliquot of the chloroform layer was taken for determination

of radioactivity incorporated into membrane lipids. The chloroform

was evaporated under a stream of air and OCS scintillation fluid

(Amersham) was added. Radioactivity was determined using a Beckman

LS7500 liquid scintillation spectrophotometer. The data are expressed

as pmols [3H]-inositol incorporated/mg protein/minute.


Results
The effects of acute reserpine treatment on alphal-stimulated

phosphoinositide hydrolysis and beta-stimulated cyclic AMP production.

To determine the effects of reserpine treatment on alphal stimulated

PI hydrolysis, rats were treated (5mg/kg/day) for four days. This

regimen depletes brain catacholamines by 90% (Brodie et al., 1966).

NE dose-response curves following four days of treatment are shown in

Figure 4-1. The ED50 values for NE stimulation were 4.17 0.55 uM










for controls and 5.00 1.48 jM for reserpinized rats (n=3). It

appears that reserpine treatment does not alter the NE-stimulated PI

response.

As a positive control, isoproterenol-induced cyclic AMP

production was studied. Reserpine treatment had no effect on basal

cAMP production, but increased isoproterenol-stimulated cAMP

production from 15.14 1.30 pmol/mg protein to 29.49 1.42 pmol/mg

protein (P<0.001, n=3). Therefore, reserpine treatment (5mg/kg/day

for 4 days) was sufficient to alter beta-adrenergic stimulation of

adenylate cyclase but not the alphal-receptor stimulated PI

hydrolysis.

Effect of chronic reserpine treatment on alphal-stimulated PI

hydrolysis and beta-stimulated cyclic AMP production. Since the

alphal-stimulated PI response may require a longer course of treatment

before the results of NE depletion are evident, we gave reserpine for

14 days (0.25 mg/kg/day). Dose-response curves are shown in Figure 4-

2. The ED50 values were 3.26 and 2.71 pM for control and reserpine

treated rats, respectively (n=2). Clearly, no increase in alphal

receptor-stimulated PI hydrolysis is evident with a longer course of

reserpine treatment.

Chronic reserpine has no effect on basal cAMP production.

Chronic reserpine increased the isoproterenol-stimulated cAMP

production from 18.57 1.86 pmol/mg protein to 53.33 4.11 pmol/mg

protein (P<0.001, n=3). Similar to 4 days of reserpine treatment, 14

days of reserpine are also sufficient to alter beta-adrenergic















































U :. I I I I
0 0.1 0.3 1.0 3.0 10 30 100 300 1000
NE CONCENTRATION (uM; log scale)





Figure 4-1. Dose-response curves for NE-stimulated PI hydrolysis in
rats receiving acute reserpine treatment (5 mg/kg/day for 4 days) vs.
vehicle treated. PI hydrolysis was performed is described in
"Methods," Chapter 2 Data are expressed as [ H]-inositol released as
a percent of total [ H]-inositol incorporated. Each data point
represents the average of 9 determinations from 3 separate
experiments. Error bars represent S.E.M.


I I I I I

-4 CONTROL
*--- RESERPINE ---



I












.-*--























24 -0 CONTROL
S-11 RESERPINE ---" .

W 20--


S16--
0
-2- 12
I-6
0

8-U-

4



0 1.0 3.0 10 30 100 300
NE CONCENTRATION (uM; log scale)




Figure 4-2. Dose-response curves for NE-stimulated PI hydrolysis in
rats receiving chronic reserpine treatment (0.25 mg/kg/day for 14
days) ys. vehicle treated. PI hydrolysis was per ormed as described
in "Methods," Chapter 2 Data are expressed as [ H]-inositol released
as a percent of total [iH]-inositol incorporated. Each data point
represents the average of 6 determinations from 2 separate
experiments. Error bars represent S.E.M.


-4 ... |










stimulation of adenylate cyclase but not alphal-stimulated PI

hydrolysis.

Effect of chronic reserpine treatment on [3H1-inositol

incorporation. To determine if a decrease in [3H]-inositol

incorporation was masking the expected increase in the alphal

stimulated PI responsiveness in reserpinized rats, [3H]-inositol

incorporation was examined in rat cortical slices from rats treated

with reserpine (0.25 mg/kg/day for 14 days). There was an initial lag

phase with rates of 0.015 and 0.014 pmols/mg protein/min for control

and reserpine treated, respectively. The secondary rates were 0.0336

and 0.0319 pmols/mg protein/min for control vs. reserpine treated,

respectively (n=2). Thus, no change in inositol incorporation was

found.


Discussion
Although many adrenergic systems both in the brain and periphery

supersensitize in response to reserpine treatment, we find no increase

in alphal-adrenergic-stimulated PI hydrolysis with 4 or 14 days of

reserpine treatment. In contrast, Akhtar and Abdel-Latif (1986) found

that denervation of the sympathetic afferents to rabbit iris dilator

smooth muscle increased the alpha1-stimulated inositol trisphosphate

(TPI) accumulation and the muscle contraction in this tissue. In

addition, denervation has no effect on the density of alphal receptors

from rabbit iris (Page and Neufeld, 1978). These differences may be a

result of the model studied. In the cholinergic system, for example,

pharmacologic blockade of central cholinergic receptors produces an

increase in receptor density but physical lesions of central










cholinergic pathways do not. In addition, the same type of

experimental manipulation has different results when comparing central

vs. peripheral cholinergic systems (see "Discussion," Chapter 5).

Thus, the regulation of alphal responses may also be system specific.

Our findings are clearly different than classical descriptions of

drug-induced or denervation supersensitivity in the beta-adrenergic

system. Reduction of sympathetic stimulation will increase the

beta-adrenergic stimulation of adenylate cyclase. Treatment of rats

with reserpine will cause a 50 to 100 % increase in NE- and

isoproterenol-stimulated adenylate cyclase activity in rat brain (our

data, Dismukes and Daly, 1974; Williams and Pirch, 1974). Chemical

sympathectomy with 6-hydroxydopamine and chronic denervation also

cause an increase in adenylate cyclase activity (Sporn et al., 1976;

Weiss and Costa, 1967). Increased beta-adrenergic receptor density

was subsequently found to accompany the enhanced cAMP production

(Sporn et al., 1976; Weiss et al., 1979; Lefkowitz and Williams,

1978). Thus, reduction of sympathetic input results in

supersensitivity due to increased beta-adrenergic receptor-coupled

adenylate cyclase activity. The fact that we see no supersensitivity

is an unexpected finding. Perhaps rat brain alphal receptor-

stimulated PI hydrolysis simply does not supersensitize. It is also

possible that supersensitivity does occur, but at a mechanism beyond

receptor-stimulated PI hydrolysis. We know, for example, in smooth

muscle reserpine treatment results in a non-specific supersensitivity

to a variety of contractile agents (Nasseri et al., 1985). Changes in

resting membrane potential (Fleming and Westfall, 1975), changes in








62
Ca++ permeability or availability (Garrett and Carrier, 1971), and

enhanced cell-cell communication (Lee et al., 1975) have all been

proposed as possible mechanisms for this non-specific

supersensitivity. Thus, it may be that alphal receptor-stimulated PI

hydrolysis is not an appropriate response to study in this case.

In conclusion, both acute and chronic treatment with reserpine

caused an increase in beta-adrenergic receptor-stimulated adenylate

cyclase activity, but not alphal-adrenergic receptor-stimulated PI

hydrolysis.














CHAPTER 5
EFFECTS OF nBM LESIONS ON MUSCARINIC-STIMULATION
OF PHOSPHOINOSITIDE HYDROLYSIS


Preface
About the same time we were performing the regulation studies

with reserpinized rats, Dr. Gary Arendash from the University of South

Florida approached us about performing similar regulation studies in

rats lesioned at the nucleus basalis magnocellularis. We agreed to

perform the experiments and the results of those experiments are in

Chapter 4. Although this has little to do with the adrenergic system,

it does allow us to compare and contrast the regulation of

cholinergic-stimulated PI hydrolysis to the adrenergic system.


Introduction
Clinically Alzheimer's disease presents as a loss of cognitive

function (Coyle et al., 1983). This loss of cognitive function is

thought to be related to a loss of cholinergic innervation of the

cerebral cortex. Patients with Alzheimer's disease have a marked

decrease in the number of cholinergic neurons in the nucleus basalis

of Meynert (NBM), which projects to the cerebral cortex (Nagai et al.,

1983), and a significant loss of cortical choline acetyltransferase

(CAT) activity, a marker enzyme for cholinergic nerve terminals. Thus

a degeneration of the cholinergic projection from NBM to the cortex

occurs in Alzheimer's disease. We have investigated










muscarinic-cholinergic receptors in an animal model of Alzheimer's

disease. The nucleus basalis magnocellularis (nBM) in the rat

contains cholinergic neurons which project to the cerebral cortex of

the rat. Lesions of this nucleus result in decreased performance of

rats on memory related tasks and permanent decreases in cortical

cholinergic markers (Flicker et al., 1983; Kesner et al., 1986).

Thus, chemical lesions of the nBM appear to mimic some of the

neurochemical and behavioral changes known to occur in Alzheimer's

disease.

Denervation induced receptor supersensitivity is a well known

phenomena at certain cholinergic synapses (Burt, 1978; Sachs et al.,

1979) and for several other neurotransmitter receptor systems (Fleming

et al., 1973). The purpose of this study was to determine the effects

of nBM lesions on muscarinic cholinergic receptor recognition sites

and on various aspects of phosphoinositide hydrolysis in rat cerebral

cortical slices. Muscarinic-cholinergic receptor stimulated

phosphoinositide hydrolysis has been well characterized as the second

messenger response to post-synaptic muscarinic receptors in rat

cerebral cortex (Fisher, et al., 1983; Gonzales and Crews, 1984). We

report here that more than three weeks after nBM lesions we find no

denervation supersensitivity in cerebral cortical muscarinic

receptors. The number of muscarinic-cholinergic receptor recognition

sites as determined by [3H]-QNB nor the apparent affinity of the sites

for agonists was altered. Furthermore, muscarinic stimulation of

phosphoinositide hydrolysis was not changed. These studies suggest

that no compensatory increase in muscarinic receptor density and/or










muscarinic responsiveness occurs during the loss of cerebral cortical

cholinergic innervation in Alzheimer's disease.


Methods
Animal preparation. Adult male Sprague-Dawley rats, weighing 265

to 335 g at surgery, were used in these studies. Animals were

maintained on a 14-h light, 10-h dark schedule and provided with

Purina rat chow and water ad libitum. NBM lesions were performed as

described previously (Arendash et al., 1972). Animals were

anesthetized with sodium pentobarbital (50 mg/kg) and placed in a

Trent Wells stereotaxic apparatus. Ibotenic acid was dissolved in

phosphate buffered saline (pH 7.4) at a concentration of 5 pg/pl. A

10 1l Hamilton syringe, equipped with a 30 gauge needle, was mounted

onto the probe drive of the stereotaxic apparatus and filled with

ibotenic acid solution. One microliter of this solution was infused

unilaterally into the right nBM at 2 dorsoventral sites (10 Ag

ibotenic acid total) utilizing the following coordinates: A6.9-7.3,

L2.6-2.7, V5.6-5.9 and V6.4-6.7 mm (Konig and Klippel, 1963). Each

infusion was done at the rate of 0.5 p1/minute and the needle was left

in place for 5 minutes following each infusion. The lesions typically

result in an approximately 80 to 90% destruction of the nBM (Arendash

et al., 1972). To verify lesions were comparable to those previously

characterized several rats were sacrificed, following lesions and

frontal cortices assayed for CAT activity according to modifications

(Lehman and Fibiger, 1978) of existing methodology (Fonnum, 1969). A

40% decrease in ipsilateral frontal cortex CAT activity was found when

cortical CAT activity from the lesioned hemisphere was compared to










activity in the contralateral unlesioned cortex (22.40 3.77 vs.

38.08 0.63 nMole/mg of protein/hr, n = 3). This nBM lesion-induced

cortical loss of CAT activity is consistent with a loss of the major

extrinsic cholinergic innervation to the neocortex from the nBM.

Phosphatidylinositol hydrolysis. See "Methods" section,

Chapter 2.

13HI-inositol incorporation. Slices were made as described in

"Methods," Chapter 2. Slices were washed four times with warm,

oxygenated KRB buffer. Following the wash, the slices were allowed to

settle and the excess buffer removed leaving enough buffer to comprise

four volumes of settled slices. [3H]-inositol (13.8 Ci/mmol; Amersham

Corp.) was added to a final concentration of 0.1 to 0.3 fM. The tube

was gassed with 02/C02, capped, and incubated in a shaking water bath

at 37*C. At appropriate time points, a 50 Al aliquot of packed slices

was removed and placed into 800 fl of ice-cold KRB buffer and

homogenized with a Tekmar Tissuemizer at setting 50 for 15 seconds. A

500 Al aliquot of the homogenate was placed in a Falcon 17 x 100 mm

polypropylene tube. Two ml of 1:2 chloroform/methanol (v/v) was

added. An additional 0.75 ml of distilled water and 0.75 ml

chloroform was added. The tubes were capped tightly and shaken

vigorously for 5 minutes then centrifuged for phase separation. The

aqueous layer was aspirated and a 200 Jl aliquot of the chloroform

layer was taken for determination of radioactivity incorporated into

membrane lipids. The chloroform was evaporated under a stream of air

and OCS scintillation fluid (Amersham) was added. Radioactivity was










determined using a Beckman LS7500 liquid scintillation

spectrophotometer.

13Hl-Quinuclidinyl Benzilate (QNB) Binding. Rats were
decapitated and their brains placed in ice-cold 50mM

tris(hydroxymethyl)aminomethane (TRIS buffer) containing 1mM EDTA, pH

adjusted to 7.4. Tissue from the lesioned and contralateral

non-lesioned fronto-parietal cerebral cortex was dissected away and

wet weights were determined. The tissues were then homogenized in 20

volumes of ice-cold 50mM TRIS/HCl buffer containing 1mM EDTA pH 7.4

using a Tekmar Tissuemizer at setting 50 for 30 seconds. The

homogenate was incubated on ice for 30 minutes. This treatment

removes endogenous neurotransmitter from the membranes (Potter et al.,

1984). The homogenate was then centrifuged for 10 minutes at 42000 x

g at 4'C. The pellet was resuspended in an ice-cold, modified KRB

buffer containing: 118 mM NaCl, 4.7 mM KC1, 0.75 mM CaC12, 1.18 mM

KH2PO4, 1.18 mM MgS04, 24.8 mM NaHCO3. The membranes were washed

twice more with modified KRB buffer and an aliquot was taken for

protein determination. Reactions were started with the addition of

120 pg of protein into modified KRB buffer containing 1 nM [3H]-QNB

(Amersham) and the appropriate concentration of carbachol in a total

volume of 2 ml. Non-specific binding was determined in the presence

of 1 AM atropine. Incubations were performed at 37*C for 60 minutes

and terminated by rapid filtration onto Whatman GF/B filter paper.

Filters were washed 3 times with ice-cold modified KRB buffer.

Filters were placed into scintillation vials and 10 ml of Liquiscint










added. Vials were shaken for 1 hour and radioactivity was measured in

a Beckman LS7500 scintillation counter.

Data Analysis. Competition curves were analyzed using an

iterative, nonlinear curve fitting program as described by McKinney

and Coyle (1982) (see "Data Analysis," Chapter 3). ED50 values were

determined using probit analysis as outlined by Goldstein (1964). The

data were analyzed by Student's t-test.


Results
Effect of nBM lesions on muscarinic-stimulated phosphoinositide

hydrolysis. To determine the effect of nBM lesions on cortical

muscarinic receptor responsiveness we measured carbachol-stimulated

hydrolysis of phosphatidylinositol. Dose-response curves for

carbachol stimulated phosphoinositide hydrolysis were determined in

control contralateral or nBM lesioned ipsilateral frontoparietal

cortical slices seven days post-lesion. The dose response curves to

carbachol in control and lesioned hemispheres were identical (Figure

5-1). To determine if changes might require longer periods of

denervation rats were studied approximately 3 weeks post-lesion. In

frontoparietal slices from animals 21 to 23 days post-lesion, again no

apparent change in the responsiveness of carbachol-stimulated PI

hydrolysis was seen (Figure 5-2). The ED50s were 95 MM t 20 AM (n =

3), for slices from control hemispheres and 129 MM 60 AM (n = 3) in

slices from lesioned hemispheres. Although calculated ED50 values

varied from experiment to experiment, control and lesioned curves were

paired within experiments and were consistently similar. Analysis of

variance between all carbachol stimulated groups indicates the ED50s










are not significantly different between control and lesioned

hemispheres at either one or three week time points (data not shown).

Maximal activity was reached at 1 to 3 mM carbachol and was similar

for both hemispheres studied.

To determine if nBM lesions alter the incorporation of

[3H]-inositol into slices from frontoparietal cerebral cortex, slices

were incubated up to one hour and the amounts of [3H]-inositol

incorporated into the phosphatidylinositol pool determined.

Incorporation was linear to at least 60 minutes and the rate was

similar in slices from control and lesioned hemispheres (Table 5-1).

These results indicate that ibotenate lesioning produces no change in

the ability of cortical slices to incorporate [3H]-inositol 22 days

post-lesion.

To investigate the possibility that lesioning can alter the rate

of carbachol-stimulated PI hydrolysis, we determined the time course

of carbachol-stimulated PI hydrolysis in unilaterally lesioned

frontoparietal cortex. In each hemisphere, cortical PI hydrolysis was

linear to 15 minutes, and reached plateau levels at approximately 40

minutes (Figure 5-3). At no time point was the hydrolysis in slices

from lesioned cortex significantly different from control values.

Since no effect on muscarinic receptor stimulated

phosphoinositide hydrolysis was found following unilateral lesions, we

compared the PI response to carbachol 25 to 28 days after bilateral

ibotenate lesions to that of sham- operated controls. Again, no

difference is seen between bilateral lesioned and sham control groups

(Table 5-2).



















So o %UurIMUL
V A LESION

I20- 'C1 --



--
0





E
a 5- -


o 1 10




CD -6 -5 -4 -3 -2

LOG [CARBACHOL] MOLAR









Figure 5-1. Dose-response curves for carbachol-stimulated PI
hydrolysis 7 days post nBM lesion. Slices were prepared from the
frontoparietal cortex ipsilateral to the nBM lesion. The
cQntralateral frontoparietal cortex served as control. The release of
[ H]-inositol phosphates after 60 minutes in the presence of agonist
was determined as described in "Methods," Chapter 2. The amount of
radioactivity found in the inositol phosphate fraction at time zero
was taken as the blank and subtracted from all values. Data points
are the means of nine determinations from three separate experiments.
Error bars indicate S.E.M. At no carbachol concentration are the two
groups significantly different as determined by Student's t test.








































0 -6 -5 -4 -3


LOG [CARBACHOLI MOLAR









Figure 5-2. Dose-response curves for carbachol-stimulated PI
hydrolysis 21 to 23 days post nBM lesion. Data were derived as
described in the legend to Figure 4-1. Shown are the means and S.E.M.
of at least six determinations from three separate experiments. At no
carbachol concentration are the two groups significantly different as
determined by Student's t-test.
















TABLE 5-1


INCORPORATION OF [3H]-INOSITOL
21-23 DAYS POST NBM


INTO BRAIN SLICES
LESION


Time (Min) Control Lesion


10 21.9 3.3 22.8 3.4

60 165.0 9.9 146.0 10.7

Rate 2.75 pmol/min 2.43 pmol/min



Slices from frontoparietal cortex ipsilateral to nBM lesion were
compared to frontoparietal cortical slices contralateral to oBM
lesion (control). Slices were incubated with 0.1 to 0.3 pM [ H]-
inositol for the indicated time and tested for their ability to
incorporate [ H]-ingsitol (see: "Methods," Chapter 5). Data are
expressed as pmol [ H]-inositol incorporated/50 ul packed slices.
Means SEM represent 4 separate determinations from two
experiments.












































TIME (MINUTES)







Figure 5-3. Time-course of carbachol-stimulated PI hydrolysis 21 to
23 days post nBM lesion. The release of [ H]-inositol phosphates at
various time points was determined as described in "Methods," Chapter
2. Data points represent the mean and S.E.M. of nine determinations
from three separate experiments. At no time point was the lesioned
data significantly different from controls (t-test).

















TABLE 5-2
CARBACHOL-STIMULATED PI HYDROLYSIS IN FRONTOPARIETAL
CORTICAL SLICES FROM BILATERALLY LESIONED
RATS VS. SHAM-OPERATED CONTROLS


Control Lesion

ED50 (AM) 26.6 1.0 25.3 (10.3,40.2)

Maximum 31.3 4.2 27.8 1.35
(10- M Carb)



ED50 values are expressed as iM. Maximum responses are expressed
as DPM [ H]-inositol phosphates released/total DPM [pH]-inositol
incorporated x 100. Control maximum values represent nine
determinations from three separate experiments. Control ED50
value is the average ED50 value the S.E.M. from three separate
experiments. Bilateral lesion maximum values represent six
determinations from two separate experiments. Lesion ED50 value
is the average ED50 from two experiments, actual ED50 values shown
in parentheses.










Effects of nBM lesions on muscarinic receptor density and binding

properties. To determine if there were changes in cortical muscarinic

binding sites 21 days post nBM lesions [3H]-QNB binding to rat

cortical membranes was determined. Scatchard analysis of [3H]-QNB

binding (Table 5-3) indicates no change in Bmax or affinity as

determined by Kd values of [3H]-QNB binding to frontoparietal cortical

membranes from lesioned and control hemispheres. Again, no difference

was found between the two groups. To investigate potential changes in

agonist binding, competition curves for [3H]-QNB sites by carbachol

were performed. Carbachol exhibited two-site displacement of [3H]-QNB

binding as indicated by the gradual slope of the curves in Figure 5-4

which was confirmed by computer modeling (Table 5-4). Membranes from

the lesioned hemispheres retained a two-site displacement of [3H]-QNB

by carbachol. The distribution of receptors was approximately 30%

high affinity and 70% low affinity for carbachol displacement. This

distribution was not altered by lesioning. Control Kd values for the

high and low affinity sites differed by approximately 100 fold in both

control and lesioned hemispheres (Table 5-4). None of the binding

parameters were found to be significantly different. Thus,

displacement of [3H]-QNB by carbachol is not altered by lesioning.

Effects of nBM lesions on phosphoinositide hydrolysis evoked by

other neurotransmitters. To determine if nBM lesions might alter















TABLE 5-3

SCATCHARD ANALYSIS OF [3H]-QNB BINDING IN MEMBRANES
PREPARED FROM RAT FRONTOPARIETAL CORTEX 21-23 DAYS POST NBM LESION


Control Lesion

Kd (pmol) 513 110 785 302

Bmax (pmol/mg protein) 3.67 0.42 3.08 0.98

r 0.984 0.991



Membranes were prepared as described in the [3H]-QNB binding
section of "Methods." In this c4se the membranes were incubated
with various concentrations of [VH]-QNB and assayed as described.
Means SEM of nine determinations from three separate experiments
are shown from kinetic parameters. Correlations are mean values
for three experiments.
















* CONTROL
A LESION


; I I 0 s i


S-6
-6


-2


Figure 5-4. Carbachol competition curves for specific [3H]-QNB
binding 21 to 23 days post nBM lesion. Membranes were prepared as
described ("Methods," Chapter 5) from frontoparietal cortex
ipsilateral to the nBM lesion. Membranes prepared from the
frontoparietal cortex contralateral to the nBM lesion served as
controls. Specific [3H]-QNB binding is expressed as per gram wet
weight of tissue. Shown are the means of nine determinations from
three separate experiments. The S.E.M. were deleted for the sake of
clarity, but at no point were they greater than 10 percent. No
significant difference between lesion and control groups exist (t-
test).


', I


.06 +


.051-


.04 +


.03 4-


.02 +


.01-


1 I


D


-5 -4 -3

LOG [CARBACHOL] MOLAR


It .


< I


1 1 '


. I
















TABLE 5-4


COMPUTE GENERATED KINETIC PARAMETERS OF CARBACHOL
FOR [IH]-QNB BINDING SITES IN RAT FRONTOPARIETAL
MEMBRANES 21-23 DAYS POST NBM LESION


Control


COMPETITION
CORTICAL


Lesion


Kd high (MM) 1.03 0.55 0.32 0.13

Kd low (AM) 74.95 29.80 57.02 25.55

% high 34.15 4.80 32.43 3.39

% low 65.85 4.80 67.57 3.39


Membranes were prepared as described in the legend to Figure 5-4.
Computer analysis is described in McKinney and Coyle (1982).
Shown are the carbachol dissociation constants (Kd) for high and
low affinity binding and the percentages of high and low affinity
binding. Each value is the mean S.E.M. of three separate
experiments. Competition curves were performed with a carbachol
concentration range from 1 MM to 10 mM, with each point done in
triplicate.










receptor-stimulated PI hydrolysis by other neurotransmitters the

response to other agents was investigated. In both 7 and 21 day

post-lesion studies using tissue from unilaterally lesioned rats, the

PI hydrolysis dose-response curves for norepinephrine (NE) were

identical (Table 5-5). In addition, PI hydrolysis in response to

maximal concentrations of the partial muscarinic agonist oxotremorine

(100 AM) and the excitatory amino acid glutamate (1 mM) were unchanged

by nBM lesions (Table 5-5). Thus, nBM lesions do not appear to alter

glutamate, alpha-adrenergic or cholinergic muscarinic receptor

stimulated PI hydrolysis.


Discussion
Denervation frequently will produce an increase in the

sensitivity of the effector organ (Fleming et al., 1973). The

increased sensitivity observed during decreased stimulation is a

compensatory homeostatic response to the loss of stimulation.

Denervation supersensitivity to acetylcholine has been found in both

skeletal muscle (Pestronk and Drachman, 1978) and superior cervical

ganglia (Dun et al., 1976). In nBM lesioned rats, we find no evidence

for a compensatory increase in muscarinic receptor sensitivity. The

number of muscarinic sites was not altered one or three weeks after

nBM lesions. This lack of change in receptor number was found with

either unilateral or bilateral lesions. These findings are in

agreement with similar studies in the cerebral cortex (McKinney and

Coyle, 1982), and hippocampus after septo-hippocampal lesions (Ben-

Barak and Dudai, 1980a; Yamayura and Snyder, 1974). In addition, it











TABLE 5-5

AGONIST-STIMULATED PI HYDROLYSIS IN BRAIN SLICES
FROM RATS 7 AND 21-23 DAYS AFTER NBM LESION


Control


7 Days Post-Lesion


Norepinephrine
ED50 (pM)


5.43


Maximum


Carbachol (10-3 M)

Oxotremorine (10-4 M)

Glutamate (10-3 M)

21 Days Post-Lesion

Norepinephrine
ED50 (pM)


Maximum


Carbachol (10-3 M)

Oxotremorine (10-4 M)

Glutamate (10-3 M)


24.10 1.27

22.04 1.25

7.62 1.30

12.39 0.74


2.40


23.62 1.29

30.00 2.50

8.92 0.28

12.98 0.49


25.30 0.93

21.50 1.07

7.41 1.00

11.84 0.73


5.05


26.52 0.79

24.00 4.50

10.44 0.67

13.30 0.62


Agonist-stimulated PI hydrolysis in brain slices from 7 and 21 to
23 days after nBM lesion. Frontoparietal slices ipsilateral to
nBM lesion were compared to the contralateral slices (control).
Slices were incubated with agonist for 60 minutes as described in
"Methods," Chapter 2. PI hydrolysis is expressed as DPM
[3H]-inositol phosphates released/total DPM [ H]-inositol
incorporated x 100. ED50 values were determined as outlined by
Goldstein (1964). Shown is the mean ED50 from two experiments.
ED50 values are expressed as pM. All values are means of six
determinations from two separate experiments except for carbachol
(9 determinations from 3 experiments.


Lesion


5.31










is consistent with the lack of change in receptor-stimulated PI

hydrolysis seen in our experiments. In contrast to these reports, one

study which measured muscarinic binding sites only in the dorsal

hippocampus found a 20% increase in muscarinic sites after septo-

hippocampal lesions (Westlind et al., 1981). Our finding in cerebral

cortex suggests that there is no denervation supersensitivity at

either the receptor level or the receptor coupling to PI hydrolysis.

Medial septum lesions increase the electrophysiological

responsiveness of hippocampal pyramidal cells to acetylcholine but not

carbachol (Bird and Aghajanian, 1975). The change in sensitivity to

acetylcholine was concluded to result from a loss of pre-synaptic

acetylcholinesterase following the lesion, with no change in receptor

responsiveness. Thus, this change in sensitivity is not via classical

denervation supersensitivity changes in receptor quantity or receptor

stimulated second messenger responses.

Although lesions of central cholinergic pathways do not appear to

result in denervation supersensitivity, chronic treatment with

antagonists will result in the expected increase in receptor density.

Chronic atropine (40 mg/kg for at least 5 days) produced an increase

in total receptors by 49%, due to a 100% increase in agonist high

affinity sites and 25% increase in agonist low affinity sites in the

rat cerebral cortex (McKinney and Coyle, 1982). Chronic atropine was

found to produce a dose-dependent increase in antagonist binding sites

in rat hippocampus (Westland et al., 1981). Treatment of neonatal

rats with scopolamine for 20 days resulted in a 20% increase in

hippocampal [3H]-QNB binding (Ben-Barak and Dudai, 1980b), whereas










septal lesions in rats 2 to 3 days after birth did not result in an

increased number of muscarinic sites in the hippocampus (Ben-Barak and

Dudai, 1980a). Inhibition of acetylcholinesterase by organophosphates

causes a decrease in muscarinic binding sites in the rat cortex (Costa

et al., 1986; McKinney and Coyle, 1982), and other rat brain regions

(Gazit et al., 1979), with an associated decrease in the

phosphoinositide hydrolysis response to carbachol in the cortex (Costa

et al., 1986). Thus, it appears that the central cholinergic system

can undergo sensitivity changes in response to pharmacological

manipulations.

Although cholinergic receptors increase following denervation in

the periphery (Dun et al., 1976; Pestronk and Drachman, 1978), or

pharmacologic blockade in the central nervous system, the overwhelming

evidence suggests that central lesions of cholinergic pathways do not

produce any increase in the number of receptors. It is possible that

in the central nervous system, a pre-synaptic factor is required for

post-synaptic receptor regulation. In the absence of the pre-synaptic

terminals, the factors) are unavailable and there is no post-synaptic

regulation in response to denervation. Regardless of the mechanism,

our data, and that of others suggest that central cholinergic lesions

have little or no effect on muscarinic receptor sensitivity (Ben-Barak

and Dudai, 1980a; McKinney and Coyle, 1982).

We have studied nBM lesions in the rat as a model for Alzheimer's

disease. Our finding that nBM lesions do not induce muscarinic

supersensitivity is clouded by the observation that in the rat,

lesioning the nBM results in an approximately 50% decrease in










frontoparietal cortical CAT activity (our data; Johnston et al.,

1981), whereas, in the brains of some Alzheimer's disease patients

larger decreases in cortical CAT activity have been reported (Coyle et

al., 1983). Our data indicating no denervation supersensitivity could

be due to this residual cholinergic innervation in the rat. However,

a variety of behavioral studies in rats indicate that nBM lesions

produce memory deficits in rats which mimic the symptomatology of

Alzheimer's disease (Flicker et al., 1983; Kesner et al., 1986).

Furthermore, the loss of CAT activity in patients with Alzheimer's

disease has been correlated with a loss of cognitive function (Perry

et al., 1978). An increase in muscarinic receptor sensitivity would

be expected to compensate for at least some loss of cholinergic

innervation. Taken together these data suggest that the lack of a

mechanism for cerebral cortical cholinergic supersensitivity in

response to loss of pre-synaptic input may underlie the correlation

between the loss of cholinergic innervation and the loss of cognitive

function in Alzheimer's disease.














CHAPTER 6
CONCLUSIONS


Norepinephrine and a variety of other adrenergic compounds

stimulate PI hydrolysis in the rat brain. Differences in

NE-stimulated PI hydrolysis were found when comparing various brain

regions. The relative efficacy for partial agonists remained

consistent from region to region with the exception of the imidazoline

derivative oxymetazoline. In addition, the PI hydrolysis response to

oxymetazoline rose sharply from almost zero at 100 jiM to maximum at 1

mM. This suggests that oxymetazoline may have a different mechanism

of stimulating PI hydrolysis than phenylethylamine derivatives.

Further comparison of imidazolines to phenylethylamines shows

that both classes of compounds compete for the tritiated alphal

antagonist prazosin in a biphasic manner in TRIS buffer. In addition,

imidazolines exhibit higher affinities for the [3H]-prazosin site than

phenylethylamines. When the competition assays were performed in KRB

buffer, the imidazolines retained two-sites of interaction as well as

their high affinity, while phenylethylamines were converted to

one-site of interaction. Thus, qualitative differences exist between

imidazolines and phenylethylamines with respect to alphal receptor

interactions.

The ability of phenylethylamines and imidazolines to stimulate PI

hydrolysis was examined. Phenylethylamines exhibit dose-respose










relationships with respect to PI hydrolysis, with norepinephrine,

epinephrine, and alphamethylnorepinephrine being full agonists.

Partial agonists include 6-flouronorepinephrine, phenylephrine, and

methoxamine. Imidazoline-stimulated PI hydrolysis, although

dose-related, does not exhibit classical dose-response

characteristics. This provides further evidence that imidazolines

stimulate PI hydrolysis via an alternative mechanism.

Comparison of Kd values from competition curves and ED50 values

obtained from agonist-induced PI hydrolysis demonstrate a strong

correlation between Kd values obtained in KRB buffer and ED50 values

for phenylethylamines but not imidazolines. Correlation coefficients

fall off sharply when the Kd values obtained using TRIS buffer are

compared to ED50 values. Thus, it is likely that the PI hydrolysis

observed for phenylethylamines is a result of interaction of these

compounds with the alphal receptor, whereas the imidazoline-induced PI

hydrolysis is not due to their interaction with the alphal receptor.

In addition, the data also suggest that physiologic buffers may be more

appropriate than TRIS buffer for studying alpha1 binding

characteristics.

The alphal receptor antagonist prazosin was able to inhibit the

PI response to all phenylethylamines tested. In contrast, prazosin

was unable to inhibit imidazoline-induced PI hydrolysis. In fact, a

wide variety of neurotransmitter receptor antagonists were unable to

inhibit imidazoline-induced PI hydrolysis. These results provide the

final proof that phenylethylamines evoke PI hydrolysis via interaction

with the alphal receptor, and imidazoline-stimulated PI hydrolysis is










unrelated to their interaction with the alphal receptor. In addition,

imidazolines exhibit a dose-dependent inhibition of NE-stimulated PI

hydrolysis and, therefore, can be classified as alphal receptor

antagonists. Furthermore, the inability of other antagonists to

inhibit imidazoline-induced PI hydrolysis suggests a previously

uncharacterized imidazoline receptor or a non-receptor mediated

mechanism to account for imidazoline-induced PI hydrolysis.

Investigations into the regulation of alphal receptor-stimulated

PI hydrolysis in the rat cerebral cortex reveals that both acute and

chronic treatments with reserpine do not cause an increase

NE-stimulated PI hydrolysis. In contrast, both acute and chronic

reserpine treatments produced an increase in beta-adrenergic

receptor-stimulated adenylate cyclase activity. Thus, it appears that

reduction of NE levels in the brain caused by reserpine treatment is

not sufficient to bring about an increase in alphal receptor

stimulated PI hydrolysis. Clearly, the regulation of alphal

receptor-stimulated PI hydrolysis differs from the regulation of the

beta-adrenergic system.

Reduction of the cholinergic input to the frontoparietal cerebral

cortex by lesioning the nucleus basalis magnocellularis does not

result in increased carbachol-stimulated PI hydrolysis. Nor does

lesioning produce any change in parameters that are likely to mask an

increased response such as [3H]-inositol incorporation, the rate of

carbachol-stimulated PI hydrolysis, and agonist binding to muscarinic

receptors. Like NE-stimulated PI hydrolysis, carbachol-stimulated PI

hydrolysis is not affected by loss of neurotransmitter input.














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