National Institute on Drug Abuse

RESEARCH

MONOGRAPH SERIES

Sigma, PCP, and

NMDA Receptors

U.S. Department of Health and Human Services • Public Health Service • National Institutes of Health

133
Sigma, PCP, and NMDA

Receptors

Editors:

Errol B. De Souza, Ph.D.

Addiction Research Center

National Institute on Drug Abuse

Doris Clouet, Ph.D.

Division of Basic Research

National Institute on Drug Abuse

Edythe D. London, Ph.D.

Addiction Research Center

National Institute on Drug Abuse

Research Monograph 133

1993

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

Substance Abuse and Mental Health Services Administration

National Institute on Drug Abuse

5600 Fishers Lane

Rockville, MD 20857
ACKNOWLEDGMENT

This monograph is based on the papers and discussions from a technical

review on “Sigma, PCP, and NMDA Receptors Systems” held on September

27-28, 1989, in Baltimore, MD. The review meeting was sponsored by the

National Institute on Drug Abuse.

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Citation of the source is appreciated.

Opinions expressed in this volume are those of the authors and do not

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The U.S. Government does not endorse or favor any specific commercial

product or company. Trade, proprietary, or company names appearing in this

publication are used only because they are considered essential in the context

of the studies reported herein.

NIDA Research Monographs are indexed in the Index Medicus. They are

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lnformation Service, Chemical Abstracts, Current Contents, Psychological

Abstracts, and Psychopharmacology Abstracts.

National Institute on Drug Abuse

NIH Publication No. 93-3587

Printed 1993

i i
Contents

Phencyclidine Receptor Binding as a Probe of NMDA

Receptor Functioning: Implications for Drug Abuse

Research 1

Stephen R. Zukin and Daniel C. Javitt

Pharmacologic Regulation of the NMDA

Receptor-lonophore Complex 13

Kenneth M. Johnson, Lawrence D. Snell,

Aida I. Sacaan, and Susan M. Jones

Pharmacologic Characterizations of Receptors 41

Tsung-Ping Su

Studies of Receptors and Metabolic Responses to

Ligands in the Brain

E. D. London

Receptors and Signal Transduction: Negative

Modulation of Signaling Through Phosphoinositide-Linked

Receptor Systems

Wayne D. Bowen, Paul J. Tolentino,

Brian N. Kirschner, Paul Varghese,

Brian R. de Costa, and Kenner C. Rice

and Phencyclidine Receptors in the

Brain-Endocrine-Immune Axis

Seth A. Wolfe, Jr., and Errol B. De Souza

55

69

95

iii
A Role for Binding in the Antipsychotic Profile of BMY

14802? 125

Duncan P. Taylor, Michael S. Eison,

Sandra L. Moon, R. Francis

Schlemmer, Jr., Umesh A. Shukla,

Cam P. VanderMaelen, Frank D. Yocca,

Dennis J. Gallant, Susan H. Behling,

Christopher G. Boissard,

John P. Braselton, Houston H. Davis, Jr.,

Marlene N. Duquette, Raymond C. Lamy,

Judith M. Libera, Elaine Ryan, and

Robert N. Wright

Molecular Biology of PCP and NMDA Receptors 159

Leslie Kushner, Michael V.L. Bennett,

and R. Suzanne Zukin

Excitatory Amino Acid Neurotoxicity in the Developing

Brain 185

John W. McDonald and

Michael V. Johnston

Isolation and Characterization of an Endogenous Ligand

for the PCP and Receptors From Porcine, Rat, and

Human Tissue 207

Patricia C. Contreras, Nancy M. Gray,

Debora A. DiMaggio, Margaret E.

Bremer, and Julie A. Bussom

Summary and Future Directions 223

Edward F. Domino

iv
Phencyclidine Receptor Binding as a Probe of

NMDA Receptor Functioning: Implications for

Drug Abuse Research

Stephen R. Zukin and Daniel C. Javitt

INTRODUCTION

Phencyclidine (1 ,1-phenylcyclohexylpiperidine; PCP; “angel dust”), originally

developed as a general anesthetic in the late 1950s was found to produce loss

of consciousness and analgesia sufficient for the performance of surgical

procedures while not causing significant respiratory or cardiovascular

depression (Greifenstein et al. 1958; Meyer et al. 1959; Johnstone et al. 1958).

However, a significant proportion of patients subjected to PCP anesthesia

developed psychotic episodes typically lasting 12 to 72 hours but occasionally

as long as 7 to 10 days. These episodes were characterized by excitation,

unmanageability, paranoia, concreteness of thought (Greifenstein et al. 1958;

Meyer et al. 1959), and “maniacal episodes” (Johnstone et al. 1958).

Subanesthetic doses (0.1 mg/kg iv) were subsequently found to induce

psychotic episodes in normal volunteers and to rekindle presenting

symptomatology in recompensated schizophrenic subjects (Davies and Beech

1960; Luby et al. 1959, 1962). In addition, subanesthetic doses of PCP, but not

lisergic acid diethylamide (LSD), amobarbital, or amphetamine, could induce

abnormalities in tests of abstract reasoning, cognitive processing, attention,

motor function, and proprioception in normal volunteers that closely resembled

those seen in patients with chronic schizophrenia (Rosenbaum et al. 1959;

Cohen et al. 1962; Domino and Luby 1981).

Despite evoking behavioral effects apparently so dysphoric, PCP emerged as a

major drug of abuse during the 1970s (Petersen and Stillman 1978), and the

rate of PCP abuse increased again during the 1980s (Crider 1986). PCP

possesses powerful reinforcing and abuse-promoting effects (Balster 1986).

The key to developing interventions targeting the clinical and public-health

1
problems posed by PCP-like drugs will be found in elucidating the mechanisms

underlying their psychotomimetic and abuse-promoting properties.

PCP binds with high affinity to a specific brain PCP receptor (Zukin and Zukin

1979; Vincent et al. 1979; Reynolds et al. 1987). Several lines of evidence

indicate that the PCP receptor is a site within the ion channel gated by the

N-methyl-D-aspartate (NMDA)-type excitatory amino acid receptor. First, PCP

and NMDA receptors are co-localized in the central nervous system (Maragos

et al. 1988). Second, PCP receptor ligands have been shown to inhibit

NMDA-receptor-mediated conductances noncompetitively (Anis et al. 1983) in a

voltage- and use-dependent fashion (Honey et al. 1985; Huettner and Bean

1988). Finally, binding of PCP receptor ligands is enhanced by NMDA receptor

agonists, such as L-glutamate or NMDA, and is diminished by competitive

NMDA receptor antagonists, such as D(-)-2-amino-5-phosphonovaleric acid

(D(-)AP5) (Javitt et al. 1987; Fagg 1987; Loo et al. 1987). Such data suggest a

model in which NMDA and PCP receptors represent distinct sites associated

with a supramolecular NMDA receptor complex. The identification of

noncompetitive inhibition of NMDA receptor function as a possible mechanism

underlying the psychotomimetic effects of PCP suggests that elucidation of the

functioning of the NMDA receptor complex may reveal mechanisms relevant to

the pathogenesis and treatment of schizophrenia.

MECHANISMS OF PCP-NMDA RECEPTOR INTERACTION

To shed light on the mechanisms underlying NMDA receptor activation, binding

of the selective PCP receptor ligand [3H]MK-801 was determined in the

presence and absence of L-glutamate and either glycine or D-serine, agents

that had previously been shown to stimulate binding to PCP receptors

(Reynolds et al. 1987; Javitt and Zukin 1989a). L-glutamate mediates its actions

as an agonist at the NMDA recognition site, whereas both glycine and D-serine

mediate their actions at a non-strychnine-sensitive glycine recognition site

associated with the NMDA receptor complex. For these studies, rat forebrain

homogenates were subjected to extensive washing, freezing, and thawing to

reduce the high endogenous concentrations of L-glutamate and glycine present

in crude brain homogenate. Specific binding of [3H]MK-801 was determined at

12 to 16 time points between 5 minutes and 24 hours using a filtration

radioreceptor assay in the presence of a 5 mM Tris buffer system adjusted to

pH 7.4 and a low (30 μM) concentration of Mg2+ (Javitt and Zukin 1989b).

These studies produced three novel findings. First, analysis of association

curves using a computer-assisted, nonlinear-curve-fitting technique revealed

2
FIGURE 1. Association curves of 1 nM [3H]MK-801 under control conditions

(filled circles) or in the presence of 10 μM concentrations of

D(-)AP5 (open circles), glycine (open triangles), L-glutamate (filled

triangles), or L-glutamate plus glycine (open squares). Under

control conditions or in the presence of D(-)APS, binding was fit

best by single exponentials with apparent t1/2 values of 3.2 and 5.8

hours, respectively. In the presence of combined L-glutamate and

glycine, binding was best fit by a single exponential with an

apparent t1/2 of 12 minutes. In the presence of L-glutamate alone,

binding was best fit by dual exponentials with apparent t1/2 values

of 5.5 minutes and 1.6 hours for the fast (dashed line) and slow

(dotted line) components, respectively. (Reprinted from Javitt and

Zukin 1989b. Copyright 1989, American Society for Pharmacology

and Experimental Therapeutics.)

the presence of two distinct components of [3H]MK-801 binding: a fast

component with a t1/2 of approximately 5 minutes and a slow component with a

t1/2 of approximately 3 hours (figure 1).

3
This finding suggests that PCP-like agents do not gain access to their receptor

only via open channels, because channel-blocking drugs that interact

exclusively with open channels should manifest single exponential association

and dissociation (Starmer and Grant 1985). A model of PCP-NMDA receptor

interaction consistent with biexponential association of [3H]MK-801 would be

one in which PCP-like agents can gain access to their recognition site via two

distinct paths, each corresponding to one of the observed kinetic components of

binding. It has been suggested that channel-blocking drugs having pKa values

near physiological pH may associate with their binding sites via both fast,

hydrophilic and slow, hydrophobic paths (Starmer and Grant 1985). At pH 7.4,

MK-801 (pKa = 8.2 (Huettner and Bean 1988)) would exist in both deprotonated

and protonated forms. The former would be capable of association via both

hydrophilic and hydrophobic paths, while the latter would be capable of

association only via a hydrophilic path.

When channels were maximally activated in the presence of combined

L-glutamate and glycine, we found that more than 90 percent of [3H]MK-801

binding displayed fast kinetics of association, suggesting that the fast path

represents binding of [3H]MK-801 to its receptor following diffusion to the

binding site via a path corresponding to the open NMDA receptor channel. In

the absence of added L-glutamate or in the presence of D(-)AP5, more than

99 percent of [3H]MK-801 binding displayed slow kinetics of association,

suggesting that the slow path represents binding of [3H]MK-801 following

diffusion to the binding site via a path associated with closed NMDA receptor

channels. The latter path could involve diffusion of deprotonated [3H]MK-801

through the lipid bilayer, through hydrophobic domains of the receptor complex,

or through the (closed) NMDA-receptor gating mechanism. Biexponential

kinetics of association of [3H]MK-801 in the presence of L-glutamate alone

indicate that association via fast and slow paths can occur simultaneously, thus

supporting the concept that different underlying processes must be involved.

The ability of PCP-like agents to reach their binding site within closed NMDA

channels over the course of hours may be relevant to the course of PCP

intoxication because it suggests that PCP can reach its site of action even in

the absence of NMDA receptor activation.

A second finding of these studies (Javitt and Zukin 1989b) was that L-glutamate

significantly increased total steady-state [3H]MK-801 binding while D(-)AP5

significantly decreased total steady-state [3H]MK-801 binding (figure 2)

presumably by displacing endogenous agonists from the NMDA receptor. This

finding differs from those of previous studies, which had reported no change in

4
FIGURE 2. Specific binding (mean ± SEM) of 1 nM [3H]MK-801 to fast (open

bar) and slow (filled bar) components under control conditions or

in the presence of 10 μM concentrations of D(-)AP5 (AP5), glycine

(GLY), L-glutamate (L-GLU), or L-glutamate plus glycine. Total bar

height represents total steady-state binding of [3H]MK-807 under

conditions specified. Significant between-groups variation was

found for fast (p < .001), slow (p < .01), and total (p < .001)

steady-state binding. Values represent mean ± s.e.m. of 4 to 6

experiments. *p < .05 vs. control. ** p < .01 vs. control.

(Reprinted from Javitt and Zukin 1989b. Copyright 1989,

American Society for Pharmacology and Experimental

Therapeutics.)

equilibrium binding after the glutamate and glycine were added (Kloog et al.

1988; Bonhaus and McNamara 1988).

A third finding of our studies (Javitt and Zukin 1989b) was that the Hill

coefficient for stimulation of [3H]MK-801 binding by L-glutamate was

significantly greater than unity (figure 3). Although glycine shifted the

dose-response curve to the left, it did not alter the Hill coefficient significantly.

5
FIGURE 3. Stimulation of specific 1 nM [3H]MK-807 binding by L-glutamate in

the absence (filled circles) or presence (filled triangles) of 10 μM

glycine following 24-hour incubations. D(-)AP5 (50 μM) was added

under all conditions to decrease basal binding. Inset, Hill plots of

specific 1 nM [3H]MK-801 binding. Correlation coefficients (r) were

> .95 for both plots. In both the absence (filled circles) and

presence (filled triangles) of 10 μM glycine, Hill coefficients were

significant/y greater than unity (Reprinted from Javitt and Zukin

1989b. Copyright 1989, American Society for Pharmacology and

Experimental Therapeutics.)

This finding suggests that more than one molecule of agonist is required to

induce NMDA channel activation.

A model of NMDA receptor functioning that could account for these findings is

one involving the existence of two independent sets of agonist recognition sites

within each functional NMDA receptor complex. Occupation of both sets by

agonist would be required for channel activation and fast [3H]MK-801 binding.

Partial activation would not permit channel opening but would permit slow

diffusion of [3H]MK-801 to its binding site via a hydrophobic path. In the total

6
absence of agonist, however, the channel would remain closed and in a

conformation to which PCP receptor ligands could not bind. This model is

similar to those proposed to account for the functioning of nicotinic acetylcholine

(Hess et al. 1983) and GABAA (Aoshima et al. 1987) receptors.

If future studies confirm a functional similarity between NMDA and nicotinic

receptors, it may indicate that NMDA receptors are structurally homologous to

receptors of the Class I superfamily of ligand-gated channels, which includes

nicotinic, GABAA, and strychnine-sensitive glycine receptors as well (Barnard et

al. 1987).

ROLE OF NMDA RECEPTORS IN PSYCHOTOMIMETIC EFFECTS OF PCP

The ability of PCP-like agents to bind with high potency to a site within the

NMDA channel suggests that PCP-induced NMDA channel blockade may be

relevant to the clinical effects of PCP. The degree to which PCP receptors

mediate the psychotomimetic effects of PCP has been a subject of controversy

in the literature. Both haloperidol-sensitive sites (Su et al. 1988) and

monoamine reuptake sites (Smith et al. 1977; Garey and Heath 1976) sites

have been proposed as potential alternative receptors for mediation of the

psychotomimetic effects of PCP.

Several lines of evidence, however, support a unique role of the PCP receptor

in this regard. First, PCP receptors have been shown to mediate the

discriminative stimulus effects of PCP in rodents (Browne 1986). The rank order

of potency with which a large number of drugs from distinct chemical classes

can induce the PCP response in animals trained to discriminate PCP from

saline corresponds to their rank order of binding to PCP receptors and inducing

NMDA receptor blockade (Browne 1986). By contrast, agents that selectively

bind to the and/or dopamine reuptake sites neither induce PCP-like

discriminative stimulus effects nor antagonize the discriminative stimulus effects

induced by PCP receptor ligands (Browne 1986). These findings suggest that

PCP receptors mediate the interoceptive cues induced by PCP and PCP-like

agents.

Second, psychotomimetic effects similar to those induced by PCP can be

induced by ketamine, a related arylcyclohexylamine derivative (Siegel 1978).

The psychotomimetic effects of ketamine are induced by doses approximately

tenfold greater than PCP (Siegel 1978), which is consistent with ketamine’s

tenfold lower potency of binding to PCP receptors (Zukin and Zukin 1979;

7
Vincent et al. 1979). By contrast, ketamine is essentially inactive at both

dopamine reuptake (Vignon et al. 1988) and sites.

Finally, PCP-induced psychosis has been found to be associated with serum

concentrations of PCP as low as 20 nM, while serum concentrations greater

than 400 nM are associated with gross impairments in consciousness (Walberg

et al. 1986). PCP receptors have been shown to bind PCP with an affinity of

approximately 30 to 50 nM (Reynolds et al. 1987; Sircar et al. 1987; Wong et al.

1988) suggesting a highly significant degree of receptor occupancy by levels of

PCP present during low-dose PCP psychosis. By contrast, binding sites and

DA reuptake sites have been shown to bind PCP with affinities of approximately

600 nM (Wong et al. 1988) and 700 nM (Vignon et al. 1988) respectively. The

affinity of PCP for these sites is thus significantly lower than its affinity for PCP

receptors. Furthermore, the affinity of PCP for these sites suggests that they

would be affected only to a limited extent by concentrations of PCP that have

been demonstrated to cause robust psychotomimetic effects.

In summary, the unique behavioral effects of PCP and related drugs, which are

accompanied by a high degree of abuse potential, appear to be mediated at the

PCP receptor that is located within the NMDA-receptor-gated ion channel. Our

data from experiments measuring the effects of NMDA receptor activation on

binding of [3H]MK-801 to PCP receptors support a model of NMDA receptor

functioning in which two molecules of agonist are required for NMDA receptor

activation. This model is similar to models that have been proposed for the

nicotinic acetylcholine receptor, suggesting the possibility of functional and

structural homology between NMDA receptors and members of the Class I

superfamily of ligand-gated channels. The psychotomimetic effects of PCP are

observed at serum concentrations similar to the concentration at which PCP

binds to the NMDA-associated PCP receptor. It is hoped that future studies

may clarify mechanisms whereby agents influencing NMDA receptor

functioning may influence behaviors or lead to therapeutic approaches related

to drug abuse.

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11
ACKNOWLEDGMENTS

This work was supported in part by USPHS grants DA-03383 to SRZ and

MH-00631 to DCJ, grants from the Ritter Foundation and the David Berg Family

Fund for Research in Manic Depressive Illness to SRZ, and by the generous

support of the Department of Psychiatry of the Albert Einstein College of

Medicine, Herman M. van Pragg, M.D., Ph.D., chairman.

AUTHORS

Stephen R. Zukin, M.D.

Daniel C. Javitt, M.D.

Departments of Psychiatry and Neuroscience

Albert Einstein College of Medicine and Bronx Psychiatric Center

1300 Morris Park Avenue, F111

Bronx, NY 10461

12
Pharmacologic Regulation of the NMDA

Receptor-lonophore Complex

Kenneth M. Johnson, Lawrence D. Snell, Aida I. Sacaan, and

Susan M. Jones

INTRODUCTION

As Monaghan and colleagues (1989) pointed out in their recent review, the

effects of excitatory amino acids such as glutamate and aspartate are mediated

through at least five different receptor subtypes. Three are well known and have

been defined by antagonist pharmacology and the distinct depolarizing actions

of selective agonists (kainate, quisqualate, and N-methyl-D-aspartate [NMDA]).

The fourth appears to be an inhibitory autoreceptor, selectively activated by

AP4 (L-2-amino-4-phosphonobutyrate). AP4 has been demonstrated to inhibit

synaptic activation of dentate granule cells (Koerner and Cotman 1981)

olfactory cortex (Hearns et al. 1986), and hippocampal CA3 cells (Lanthorn et

al. 1984). The fifth receptor subtype appears to activate an oscillating C1-

current (Gundersen et al. 1984) that may be mediated by IP3 (Parker and Miledi

1987). This response is blocked by pertussis toxin (Sugiyama et al. 1987).

Glutamate and quisqualate-but not kainate, NMDA, or AP4—increase inositol

phosphate formation (Nicoletti et al. 1986; Schoepp and Johnson 1988;

Sladeczek et al. 1985) via a mechanism that is insensitive to the quisqualate

antagonists, joro spider toxin (Jackson and Usherwood 1988), or 6-cyano-2,

3-dihydroxy- 7-nitro-quinoxaline (CNQX) (Palmer et al 1988). This unusual

profile is amplified by the finding that the traditional quisqualate receptor

agonist, DL- amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA),

does not increase phosphatidyl inositol turnover: it is further enhanced by the

observation that quisqualate-induced inositol phosphate formation is blocked by

AP4 (Schoepp and Johnson 1988).

The NMDA receptor mediates ion flux through a channel permeable to Na+, K+,

and Ca++. Ion flux is voltage dependent and gated by Mg++ and phencyclidine

(PCP), which act at distinct sites within the channel itself. An additional role for

13
divalent cations has been illustrated by the voltage-independent inhibition of

NMDA by Zn++. Glycine also modulates channel activation by NMDA agonists

and may be required for the expression of receptor function. Recent work has

shown that polyamines act on two or more sites to regulate both glycine binding

and channel activation. In addition, both the NMDA and glycine receptor

recognition sites may exist in agonist- and antagonist-preferring conformations,

and the receptor state may be regulated by the relative concentrations of

glutamate, glycine, and polyamines. Thus, the actions of PCP in this complex

depend on the degree of activation of the ion channel, which in turn depends on

the relationship between at least five other receptors or binding sites. Therefore,

understanding the mechanism of action of PCP requires understanding the

various components of the complex and how they relate to one another. An

overview of the state of knowledge of this rapidly changing field is given below.

For information on other aspects of PCP pharmacology, see Johnson and

Jones (1990).

THE NMDA RECOGNITION SITE

[

3H]Glutamate has been widely used to specifically label each of the glutamate

receptors, including the NMDA subtype (Foster and Fagg 1984). More selective

NMDA agonists and antagonists have also been utilized to label the NMDA

receptor, but their use has been somewhat limited by their relatively poor

affinity. That is, the agonist [3H]NMDA (Foster and Fagg 1987) and the

antagonists [3H]D-AP5 (Olverman et al. 1988) [3H]DL-AP7 (Manallack and

Beart 1986; Ferkany and Coyle 1983) and [3H]3-[(±)-2-carboxypiperazine4-yl]propyl-1-phosphonate (CPP) (Harris et al. 1986; Murphy et al. 1987) have

equilibrium dissociation constants in the 200 to 2,000 nM range. However, a

recently synthesized piperidine analog of CPP, cis-4-phosphonomethyl-2-

piperidine carboxylic acid (CGS 19755), appears to be a selective and more

potent NMDA antagonist (Ki of 50 nM against [3H]CPP binding [Murphy et al.

1987]), and early indications are that [3H]CGS 19755 is the best radioligand

available at this time, at least for the antagonist-preferring state of the NMDA

receptor (see below).

Despite its lack of selectivity, L-[3H]glutamate is still the agonist of choice for

studies of the NMDA recognition site. Depending on the membrane preparation

(and perhaps the extent to which endogenous glutamate and aspartate are

removed), NMDA-displaceable L-[3H]glutamate binding varies between 25 and

90 percent of the total binding and has a Kc value between 50 and 200 nM

(Fagg and Baud 1988; Fagg and Matus 1984; Monahan and Michel 1987;

14
Monahan et al. 1988). Recently, the authors’ laboratory has described

high-affinity (36 nM KD), NMDA-displaceable L-[3H]glutamate binding that

constituted about 85 percent of the total binding in an easily prepared buffy coat

membrane preparation that can also be utilized for the study of [

3H]1 -[1-

(2-thienyl)cyclohexyl]piperidine (TCP) and [

3H]glycine binding (Jones et al. 1989).

Monaghan and colleagues (1988) have studied the autoradiographic distribution

of [3H]CPP and NMDA-sensitive [3H]glutamate binding sites and have found

their distribution to be heterogeneous. L[3H]gIutamate-specific binding was

relatively higher than that of [3H]CPP in the striatum and relatively lower in

portions of the thalamus and cortex. Glycine increased [3H]glutamate binding

but decreased [3H]CPP binding. These data led the authors to postulate that the

NMDA receptor exists in both agonist- and antagonist-preferring states and that

glycine may convert the antagonist-preferring state to the agonist-preferring

state.

In an attempt to verify this hypothesis, the effect of glycine and D-serine (0.3 to

100 μM) on [3H]CPP binding in cortical buffy coat membranes was determined.

No effect was observed (data not shown). Furthermore, the IC50 values for

inhibition of NMDA-sensitive L-[3H]glutamate binding to thalamic and striatal

membranes was determined for both NMDA and CPP. As can be seen in

table 1, 10 μM glycine had no effect on the apparent affinity of either agonist or

antagonist binding to membranes from the striatum (an area rich in

agonist-preferring sites) or the thalamus (an antagonist-preferring area). Thus, it

TABLE 1. Effect of 10 μM glycine on the inhibition of NMDA-specific

[

3H]glutamate binding by an agonist and antagonist in brain areas

relatively rich in agonist sites (striatum) and antagonist sites

(thalamus)

Addition

IC50 (μM)

Striatum Thalamus

NMDA CPP NMDA CPP

Control 1.45±0.17 1.47±0.20 1.85±1.20 1.90±0.26

10 μM glycine 1.80±0.46 1.20±0.49 2.10±0.45 1.39±0.61

NOTE: These data represent the mean±SE of two to four independent experiments carried out in

triplicate with six concentrations of displacing drug.

15
TABLE 2. Comparison of the affinity constants of NMDA agonists and

antagonists for [3H]glutamate and [3H]CPP binding sites in cortical

membranes

Ki (μM)

Agonist/Antagonist [

3H]glutamate [

3H]CPP Agonist ratio*

L-glutamate 0.025 0.081 3.24

L-homocysteate 0.214 0.582 2.72

NMDA 1.80 2.90 1.61

CGS 19755 0.195 0.048 0.246

CPP 1.50 0.117 0.078

* The higher number Indicates a greater relative affinity for the agonist-preferring NMDA receptor.

is unlikely that the data of Monaghan and coworkers (1988) can be explained

by an affinity shift in the NMDA recognition site.

That agonist- and antagonist-preferring states of the NMDA receptor exist was

first postulated by Fagg and Baud (1988). As shown in table 2, this postulate

has been verified by showing that agonists have a greater relative affinity for

sites labeled by [3H]glutamate, whereas antagonists have a higher relative

affinity for [3H]CPP-labeled sites.

THE PHENCYCLIDINE BINDING SITE

Lodge and colleagues first demonstrated that PCP and the dissociative

anesthetic ketamine blocked NMDA-induced depolarization of spinal neurons

while having no effect on either kainate- or quisqualate-induced depolarization

(Lodge et al. 1982; Anis et al. 1983). This finding was critical to understanding

the mechanism of action of PCP.

The mechanism by which PCP inhibits NMDA-mediated responses is

noncompetitive with respect to NMDA (Harrison and Simmonds 1985; Snell and

Johnson 1985; Jones et al. 1989). The block also exhibits voltage and use

dependence (Kemp et al. 1988; Honey et al. 1985; Snell et al. 1987; Mayer et

al. 1988; MacDonald et al. 1987; Woodruff et al. 1987). For example, using

whole-cell patch-clamp techniques in dissociated cultures of mouse

hippocampal neurons, Mayer and colleagues (1988) reported that

NMDA-activated channels have a mean open time of 5 to 6 ms. In contrast to

16
the rapid-onset blockade caused by the competitive antagonist AP5 (which had

no effect on mean open time), ketamine produced a slowly developing

reduction in mean channel open time (from about 6 ms to 3 ms). This effect

presumably reflects the high probability that open channels will be blocked by

ketamine before they would normally close. The recovery from ketamine

antagonism was also very slow and dependent on the presence of NMDA,

suggesting that ketamine can enter the open channel and be trapped unless

NMDA agonists are available to reopen the channel (Mayer et al. 1988;

MacDonald et al. 1987). This use dependence has also been noted for other

PCP-like ligands, including cyclazocine and MK-801 (Davies et al. 1988). Use

dependence was greatest for MK-801, followed by PCP and cyclazocine, and

then ketamine (which showed very little use dependence in a cortical wedge

preparation). The reason for these differences is unclear, but it may have to do

with the size and relative flexibility of the molecules; for example, MK-801 is a

rigid, planar, three-ring structure with a nitrogen bridge oriented perpendicular

to the central ring, whereas ketamine is a flexible, bicyclic structure.

A drug that exhibits substantial use dependence relative to its onset of action

could conceivably be utilized clinically as a pro-drug in that its antagonistic

effects would not be realized until endogenous NMDA agonists opened the

channel binding site. This possibility has been examined in vivo in rat spinal

cord neurons (Davies et al. 1988). However, in this paradigm the onset of block

by intravenous MK-801 was not altered by iontophoretic application of NMDA,

although the rate of recovery from MK-801 block was enhanced by NMDA

application. (The voltage dependence of the binding also partially accounts for

the accelerated rate of recovery from MK-801 block observed in cells held at

+30 mV [Huettner and Bean 1988].) This suggests that there may be enough

endogenous NMDA tone in vivo to allow MK-801 to act immediately as an

open-channel blocker or that MK-801 may gain access to its channel binding

site by rapidly diffusing through the membrane lipid bilayer.

Studies of the voltage dependence of the NMDA block by PCP and ketamine

have provided additional evidence for the channel localization of the PCP

binding site (Honey et al. 1985; Mayer et al. 1988; MacDonald et al. 1987;

Huettner and Bean 1988). In cultured hippocampal cells, blockade of NMDA

responses is much greater at hyperpolarized membrane potentials (-60 mV to

-70 mV) than at more depolarized potentials, and virtually no block is seen at

holding potentials greater than +30 mV. Equilibrium analysis of the ketamine

block at -60 mV revealed a Ki of 1.5 μM, whereas at +40 mV the Ki was

calculated at 49 μM (Mayer et al. 1988). Using these data, Mayer and

17
colleagues (1988) calculated that the fraction of the membrane electric field that

influences ketamine binding is close to 1.0. Although recently it has been

argued that the models used for this calculation may be inappropriate

(MacDonald and Nowak 1990), this suggests that the ketamine binding site is

deep within the channel, close to the cytoplasmic surface of the membrane.

Together, the voltage- and use-dependent open-channel block (and unblock)

may contribute to the pharmacologic uniqueness of the various drugs that act at

the PCP site. Although not well studied, it appears that voltage and use

dependence may be independent properties. For example, the ketamine block

is clearly voltage dependent and is similar to PCP in this regard (Honey et al.

1985) but shows relatively little use dependence (Davies et al. 1988). MK-801

blockade shows little voltage dependence during its onset, but the recovery of

blockade in the presence of NMDA does show marked voltage dependence

(Huettner and Bean 1988). MK-801 shows greater use dependence than PCP

and considerably more than ketamine (Davies et al. 1988). The relative

importance of these two properties is difficult to assess, but their significance

can be understood by realizing that these two properties tend to offset each

other in a functional sense. That is, inhibition of NMDA responses by PCP

depends on the presence of an NMDA agonist, but NMDA also depolarizes the

membrane, effectively decreasing the action of a given concentration of PCP.

Therefore, a compound that exhibits high use dependence relative to voltage

dependence might be a more selective and potent NMDA antagonist than one

that shows low use dependence but high voltage dependence.

The voltage-dependent properties of PCP blockade of the NMDA receptorionophore complex could also contribute to the uniqueness of PCP

pharmacology in a way that is interdependent with the selectivity of

endogenous excitatory amino acids for the several receptor subtypes. That is,

because glutamate can act at kainate, quisqualate, and NMDA receptors, its

ability to depolarize the cell membrane through quisqualate or kainate receptors

would theoretically partially negate the effects of PCP-like compounds acting at

NMDA receptors. However, aspartate, and particularly homocysteic acid, may

have more selectivity for NMDA receptors. Thus, the authors predict that PCP

would be a more potent antagonist of aspartate than of glutamate responses.

Such a finding would be important because these amino acids probably

mediate synaptic transmission in independent neuronal pathways, thus

affording an additional mechanism for PCP selectivity.

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The localization of the PCP binding site within the NMDA receptor-ionophore

complex has received considerable support from binding studies. Initially, it was

reported that the specific binding of [3H]TCP was reduced by progressively

washing brain membranes and that this reduction could be reversed by adding

glutamate back to the medium (Loo et al. 1986). Subsequent studies

demonstrated an excellent correlation between the potency of ligands in

activating NMDA receptors and in increasing [3H]TCP or [3H]MK-801 binding in

well-washed membranes (Foster and Wong 1987; Fagg 1987). This relationship

was further strengthened by reports that NMDA receptor antagonists inhibited

[

3H]TCP or [3H]MK-801 binding (Johnson et al. 1988a; Loo et al. 1987; Javitt et

al. 1987; Reynolds et al. 1987).

In addition, autoradiographic studies of [3H]TCP together with NMDAdisplaceable [3H]glutamate, [3H]CPP, or [3H]AP5 binding in brain slices have

shown a very high degree of co-localization of NMDA receptor sites and PCP

binding sites (Monaghan et al. 1988; Maragos et al. 1986, 1988; Jarvis et al.

1987). Binding of ligands to both sites was the highest in the stratum radiatum

and stratum oriens of hippocampal area CA1 , followed by the dentate gyrus,

entorhinal cortex, and layers I and II of the somatosensory and motor cortex

(Monaghan et al. 1988; Maragos et al. 1988). Intermediate levels were found in

the olfactory region, amygdala, thalamus, lateral septum, hippocampal area

CA3, and the caudate nucleus. Approximately 10 percent or less of the binding

found in area CA1 was found in the globus pallidus, medial septum, habenula,

hypothalamus, diagonal band, and cerebellum. The granule cell layer of the

cerebellum was the only area reported to be discordant in binding of the two

ligands (Maragos et al. 1988).

The mechanism by which NMDA agonists selectively stimulate ligand binding to

the receptor-ionophore complex has not been completely resolved. Initial

studies suggested that glutamate and NMDA either increased the affinity

(Foster and Fagg 1987; Loo et al. 1987; Javitt et al. 1987; Johnson et al. 1988 b)

or the apparent site density (Javitt et al. 1987) for [3H]TCP or [3H]MK-801

binding. Subsequently, it was determined that the time required for either ligand

to reach true equilibrium in highly washed membranes (nominally free of

glutamate) was 5 to 24 hours, depending on the ligand used and the

thoroughness with which the membranes were washed (Bonhaus and

McNamara 1988; Kloog et al. 1988a, 1988b; Johnson et al. 1989). Thus, earlier

studies using shorter incubation times were actually conducted at 50 to 70

percent of true equilibrium, making inferences from these experiments difficult.

19
More recent studies have suggested that glutamate and other NMDA agonists

increase both the rate of association and dissociation of [3H]TCP or [3H]MK-801

(Kloog et al. 1988a; Reynolds and Miller 1988) without altering the equilibrium

dissociation constant (Kloog et al. 1988a, 1988b). Although it has been stated

that the equilibrium Ko is similar to the kinetically determined Kc (Kloog et al.

1988a), closer examination of the kinetic data (Bonhaus and McNamara 1988;

Sacaan and Johnson 1990a) suggests that both association and dissociation

are multiphasic, making the KD (assuming classical bimolecular kinetics)

somewhat difficult to interpret. This is further complicated when considered in

the light of the effects that glycine has on channel kinetics (see below).

Nevertheless, the data thus far have been widely interpreted in a manner

consistent with open channel block by PCP-like ligands. That is, the increase in

the rates of binding and unbinding induced by NMDA agonists is due to an

increase in the fraction of time the channel binding site is accessible to [3H]TCP

or [3H]MK-801. This finding is supported by the observation that NMDA

antagonists such as AP5 decrease the rates of association and dissociation

(Bonhaus and McNamara 1988; Kloog et al. 1988a; Reynolds and Miller 1988).

Thus, there appears to be overwhelming evidence that NMDA agonists regulate

the binding of PCP-like ligands to the channel binding site by increasing

channel opening. However, whether NMDA agonists alone can induce a

conformational change in the channel that is associated with a conductance

increase or an affinity change in the PCP binding site or both is still a matter of

debate that may be settled only with the precise understanding of the role of

glycine.

The authors’ laboratory has consistently observed that glutamate and glycine

addition results in apparent increases in affinity of [3H]TCP binding (Johnson et

al. 1988b Jones et al. 1989). Although these data (under control conditions)

were collected before true equilibrium was attained, arguments have been

presented that suggest that this does not undermine the interpretation that

glutamate (in the presence of glycine) opens the NMDA-operated ion channel,

increases the accessibility of [3H]TCP to its binding sites, and alters the

conformation of the channel-associated PCP binding site (Johnson and Jones

1990).

One of the difficulties inherent in experiments trying to delineate the individual

actions of glutamate and glycine is their presence even in well-washed

membrane preparations. To overcome this problem the authors have conducted

equilibrium, saturation isotherms in the presence of a glycine antagonist

20
(7-chlorokynurenate) and an NMDA antagonist (CPP). These data are

summarized in table 3. In the presence of saturating concentrations of

7-chlorokynurenate (7CIKYN) and CPP, [3H]TCP binds with low affinity (Kc of

95 nM) to cortical membrane sites that have an apparent density of 1.2

pmol/mg protein (table 3). Addition of L-glutamate produces a concentrationdependent increase (about fourfold) in apparent site density but does not alter

the KD. The addition of 1 μM glycine produces no further increase in site density

but increases the binding affinity about fivefold. These data are very similar to

those recorded in the absence of antagonists and in the presence of maximally

effective concentrations of glutamate and glycine.

TABLE 3. Effect of glutamate and glycine on [3H]TCP binding at equilibrium

in the presence and absence of NMDA and glycine antagonists

Condition KD (nM) Bmax (pmol/mg)

100 μM CPP+3 μM 7CIKYN

CPP+7CIKYN+1 μM glutamate

CPP+7CIKYN+10 μM glutamate

CPP+7CIKYN+1 μM glutamate

+10 μM glycine

10 μM glutamate+10 μM glycine

95±56 1.2±0.3

110±3 2.0±0.3

124±19 4.3±0.4

21±2 4.2±0.2

11±0.5 4.2±0.1

NOTE: Experiments were conducted at 4°C over a 48-hour incubation period. Approximate KD,

values for glutamate and glycine in this preparation are 36 and 133 nM, respectively. Ki values for

CPP and 7-chlorokynurenate are 1.5 μM and 0.27 μM, respectively.

The authors interpret these data in the following manner. In the absence of

glutamate or glycine, [3H]TCP binds to approximately one-fourth of the available

channel sites. These channels are in a nonconducting, low-affinity state. If

these channels are continuously closed, then [3H]TCP must gain access

through the lipid bilayer (hydrophobic path). Alternatively, binding under these

conditions could represent binding to spontaneously active channels. In this

case, the hydrophobic path need not be postulated, as access would be

afforded through the open channel (hydrophilic path).

The presence of glutamate increases the apparent site density by increasing

the number of open channels and, hence, the overall accessibility to the

channel population. However, binding under these conditions is still to a

low-affinity site. The addition of glycine converts this population of sites from

low to high affinity. We postulate that this conversion results from a

21
glycine-induced conformational change in the channel as it goes from a

nonconducting to a conducting state. This change requires glutamate, as 10 μM

glycine has no significant effect on the KD in the presence of CPP alone (data

not shown). This concept is modeled in figure 1 and should be compared to that

derived by Zukin and Javitt elsewhere in this volume.

At this time, very little is known about the structure of the NMDA receptorionophore complex. Radiation inactivation analysis has indicated that the sites

labeled by [3H]TCP, [3H]glutamate (NMDA specific), and [3H]glycine have

FIGURE 1. Proposed model of how NMDA (N) and glycine (G) affect the

binding of TCP (T) to the NMDA-operated ion channel (IC). IC can

exist in a closed, nonconducting state (ICC). Binding of TCP to this

state is low because of the low probability (p=0.25) that the

channel will open in the absence of NMDA agonists. This

probability factor also includes the possibility that [3H]TCP may

gain access to its binding site by diffusion through a hydrophobic

path. NMDA shifts the equilibrium to a transition state, open with

respect to TCP (ICt-N), but still nonconducting. TCP binds to this

state with a relatively low affinity (KD=100 nM), but with a high

probability (p=1). Glycine does not produce a further increase in

accessibility (p), but converts the channel to an active, open,

conductive state (G-ICo

-N). This opening is associated with a

conformational change in the channel (and in the PCP binding

site) that is reflected by an increased binding affinity (KD=10 nM).

The postulated two binding sites each for glutamate and glycine

were deleted for simplicity.

22
molecular masses in the range of 115 kDa to 125 kDa (Honore et al. 1989).

Curiously, the site labeled by [3H]CPP appears to have a molecular mass of

209 kDa. This suggests that the recognition sites for NMDA and glycine are part

of the same protein that constitutes the channel binding site for PCP, but that

CPP may bind to a distinct but tightly coupled protein (Honore et al. 1989).

Receptor solubilization studies also suggest that the NMDA, glycine, and PCP

binding sites are tightly coupled, as the binding of [3H]TCP and[3H]MK-801 to a

solubilized protein is enhanced by NMDA agonists, glycine, and divalent cations

(Ambar et al. 1988; McKernan et al. 1989). Furthermore, injection of crude poly

(A) mRNA from rat brain into Xenopus oocytes results in the expression of a

cation channel opened by NMDA agonists (in the presence of glycine) and

blocked by PCP, MK-801, and Mg++ (Kleckner and Dingledine 1988; Kushner et

al. 1988; Fong et al. 1988). The NMDA complex is expressed by mRNA in the

range of 4.2 to 7.7 kb, suggesting that the complex consists of several

homologous subunits that might be encoded by mRNAs of different length,

presumably because of differences in the length of untranslated regions (Fong

et al. 1988). It seems likely that advances in knowledge of the molecular biology

of this complex will occur soon. It will be most interesting to see whether the

widely anticipated similarities with other receptor-operated ion channels

become a reality.

THE GLYCINE RECEPTOR

In studies of the effects of excitatory amino acids on cultured neurons, Johnson

and Ascher (1987) observed that glycine potentiated the effects of NMDA in a

strychnine-insensitive manner. The effects of glycine were mimicked by

D-serine and D-alanine and were completely blocked by NMDA antagonists,

suggesting that glycine was acting through a unique receptor to facilitate, or

perhaps to permit, the actions of NMDA. Patch-clamp analysis revealed that the

primary effect of glycine was to increase the frequency of channel opening

without changing the channel-open time or channel conductance (Johnson and

Ascher 1987) suggesting that glycine could regulate transitions to intermediate

states between binding NMDA agonists and ion channel gating (Mayer et al.

1989). Consistent with this suggestion, Javitt and Zukin (1989) have presented

a model based on near equilibrium binding of [3H]MK-801, which postulates that

glycine converts a glutamate-associated, closed conformation of the channel

(with a low affinity for [3H]MK-801) to a high-affinity, open conformation. This is

similar to the model derived above based on equilibrium analysis in the

presence of NMDA and glycine antagonists.

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Another prominent effect of glycine appears to be a reduction in the rapid

desensitization of NMDA receptors (Mayer et al. 1989). In other experiments in

which ambient glycine concentration was minimized, no NMDA response could

be obtained, suggesting an obligatory role for glycine (Kleckner and Dingledine

1988). However, Mayer and colleagues (1989) have pointed out that these

measurements were made under conditions insufficient to detect rapid changes

in receptor gating, and thus the apparent requirement for glycine may be due in

part to the ability of glycine to retard the rate of receptor desensitization.

The strychnine-insensitive glycine receptor has been studied using [3H]glycine

binding (Kishimoto et al. 1981) and autoradiography (Bristow et al. 1986). The

glycine receptor has a KD of about 100 to 250 nM for [3H]glycine. The relatively

high concentrations reported for cerebrospinal fluid (Connick and Stone 1988)

suggest that this receptor may be normally saturated. This hypothesis is

supported by the relatively low-affinity transport systems for glycine in forebrain

(Debler and Lajtha 1987) and by the lack of effect of exogenously applied

glycine on NMDA responses in cortical wedges (Fletcher and Lodge 1988;

Kemp et al. 1988). However, a recent study has found that iontophoretic

application of glycine selectively facilitated NMDA responses in neocortical

neurons (Thomson et al. 1989). Thus, the saturation of glycine receptors may

vary widely and may depend on the particular synapse. There may also be

pathological conditions in which the local glycine concentration is abnormal,

thereby adversely affecting normal synaptic transmission mediated by NMDA

receptors.

The structural requirements for activation of the glycine receptor have been

determined by measuring the effects of glycine and various analogs on [3H]TCP

binding under nonequilibrium conditions (Snell et al. 1988). The rank order

potency for simple amino acids is as follows:

glycine = D-serine > D-alanine > L-serine > L-alanine = L-valine >> D-valine

This rank order was found to correlate precisely with affinity for the site labeled

by [3H]glycine (Snell et al. 1988). Comparison with other analogs including

cysteine (Wong et al. 1987) phenylalanine, GABA, ß-alanine, glycine methyl

ester, glycinamide, and N-methylglycine revealed a strict structural specificity,

suggesting that the glycine receptor contains three points of attachment for

agonists: negative and positive ionic sites (corresponding to the amino

and carboxyl groups of glycine) and a hydrogen bond-donating site

(corresponding to the hydroxyl group of D-setine) (McBain et al. 1989;

Snell et al. 1988).

24
Recently, another cyclic glycine analog, 1-hydroxy-3-aminopyrolid-2-one

(HA-966) was reported to antagonize NMDA-induced depolarization in a

manner that was reversible by glycine (Fletcher and Lodge 1988). This

compound completely inhibits [3H]glycine binding with a Ki of about 5 μM and

has no effect on the NMDA recognition site at concentrations up to 100 μM

(L.D. Snell and K.M. Johnson, unpublished observations). HA-966 is

remarkably similar in structure to D-cycloserine, a moderately potent glycine

agonist (Johnson et al. 1988a), perhaps accounting for its inability to completely

inhibit NMDA responses. Alternatively, this finding may suggest that glycine is

not obligatory for NMDA function.

Kynurenic acid is a well-known, rather nonselective antagonist of NMDA,

kainate, and quisqualate receptors. The 7-chloro derivative of kynurenic acid is

a potent (Ki = 0.5 μM) and selective glycine antagonist (Kemp et al. 1988).

Kynurenic acid also inhibits [3H]glycine binding but is about 75 times less potent

and 70 times less selective (relative to the NMDA receptor) than

7-chlorokynurenic acid (Kemp et al. 1988). Comparisons with HA-966 suggest

that 7-chlorokynurenic acid may be an inverse agonist rather than a simple

antagonist (Foster and Kemp 1989). Recently, indole-2-carboxylic acid and its

5-chloro derivative have been reported to be selective but somewhat less

potent glycine antagonists (Huettner 1989).

INTERACTIONS BETWEEN NMDA AND GLYCINE RECEPTORS

NMDA agonists and antagonists have been shown to increase and decrease,

respectively, [

3H]glycine binding to a strychnine-insensitive site. Conversely,

glycine agonists tend to increase NMDA-sensitive [3H]glutamate binding, and

glycine antagonists decrease it. These effects are not due to pharmacologic

nonspecificity, as NMDA and glycine agonists reverse the effects of NMDA and

glycine antagonists on [3H]glycine and NMDA-sensitive [3H]glutamate binding,

respectively. The mechanisms responsible for this reciprocal interaction are

unclear, but some investigators found that changes in affinity accounted for the

effects of glycine (Fadda et al. 1988) and NMDA antagonists (Hood et al. 1990).

This finding suggests that the effects of NMDA and glycine agonists, as well as

antagonists, are potentiated via allosteric increases or decreases in the binding

affinity of the other receptor. This point has been shown in one study to have a

functional consequence in that the potency of glycine agonists for stimulation of

[

3H]TCP binding increases in the presence of glutamate (Hood et al. 1990).

25
As mentioned previously, there is evidence that the site labeled by [3H]CPP

may not be the same as that labeled by [3H]glutamate. Unpublished data from

this laboratory confirm the uniqueness of this site. Both glycine and D-serine

have no effect on [3H]CPP binding (L.D. Snell and K.M. Johnson, unpublished

observations) but decrease [3H]CGS 19755 binding (Kaplita and Ferkany 1990).

This decrease is in direct contrast to their stimulatory effect on NMDA-sensitive

[

3H]glutamate binding (Fadda et al. 1988; L.D. Snell and K.M. Johnson,

unpublished observations). Furthermore, the glycine antagonists

7-chlorokynurenate and HA-966 have differential effects on agonist and

antagonist binding. 7-Chlorokynurenate inhibits both NMDA-sensitive

[

3H]glutamate and [3H]CPP binding (L.D. Snell and K.M. Johnson, unpublished

observations), but HA-966 stimulates [3H]CPP binding (Compton et al. 1990;

L.D. Snell and K.M. Johnson, unpublished observations) and [3H]CGS 19755

binding (Kaplita and Ferkany 1990).

Further complexity is revealed by the observation that glycine inhibits [3H]CGS

19755 binding but not [3H]CPP binding (Kaplita and Ferkany 1990). Thus, at

this time it is exceedingly difficult to model the interactions between NMDA and

glycine receptors, particularly when the differential effects of glycine, HA-966,

and 7-chlorokynurenate on NMDA agonist and antagonist binding sites are

considered. It is also difficult to imagine the physiological significance of distinct

antagonist binding sites. Perhaps they are involved in mediating the effects of

endogenous antagonist molecules. The various interactions between glycine

and NMDA ligands are summarized in table 4.

ROLE OF POLYAMINES

Putrescine is derived from the decarboxylation of ornithine. This divalent

diamine is metabolized to the triamine spermidine, which in turn is converted to

spermine, a primary amine with the following structure: NH2-(CH2)3-NH-(CH2)4-

NH-(CH2)3NH2. Ransom and Stec (1988) reported that spermidine and

spermine, but not putrescine, increased [3H]MK-801 binding by a mechanism

that was enhanced by both glutamate and glycine and blocked by NMDA

antagonists. This report suggested that certain polyamines may modulate

NMDA receptor function through an intracellular mechanism. This finding

became more intriguing with the preliminary report that the effects of NMDA on

Ca2+ flux and neurotransmitter release were blocked by an irreversible inhibitor

of omithine decarboxylase (ODC) in a manner reversible by addition of

putrescine (Siddiqui et al. 1988). Furthermore, these investigators also reported

that NMDA produced a rapid, transient increase in ODC. Together, these data

26
TABLE 4. Summary of actions of NMDA and glycine ligands on binding at

several recognition sites

a

L.D. Snell and K.N. Johnson, unpublished observations

bCompton et al. 1989 CDanysz et al. 1989a

dDanysz et al. 1989b

eFadda et al. 1988

fKaplita and Ferkany 1990

gFoster and Kemp 1989 hHood et al. 1989a iHood et al. 1989b

jKessler et al. 1989 kMonaghan et al. 1988

lMonahan et al. 1988

mWhite et al. 1989

suggest that the actions of NMDA may be partially mediated by spermidine or

spermine or both.

As of this writing, the effect of NMDA on ODC activity and the effect of ODC

inhibition on NMDA responses have not yet been confirmed, but the effects of

spermine and spermidine on [3H]MK-801 binding have been confirmed

(Reynolds 1990). The authors’ laboratory has also confirmed this finding using

[

3H]TCP and has extended it to several synthetic polyamine analogs in an effort

to determine the structural requirements for this unique modulatory site (Sacaan

and Johnson 1990a). The number of nitrogen atoms in the polyamine backbone

have been found to play a major role in determining the efficacy of receptor

activation, and the number of methylene groups separating the nitrogens were

found to be a major determinant of affinity. Furthermore, it has been determined

that several diamine analogs, including putrescine, antagonize spermidineinduced [3H]TCP binding in a manner consistent with a noncompetitive

mechanism (Sacaan and Johnson 1990a). Because the metabolism of

27
putrescine to spermidine and spermine is reversible, these data imply that the

regulation of polyamine metabolism could be intimately involved in a complex

regulation of NMDA receptor function. It has been reported that SL 82,0715 and

ifenprodil may be antagonists at this polyamine site (Carter et al. 1988), but

Reynolds and Miller (1989) concluded that ifenprodil is a noncompetitive

antagonist at this site. Comparisons with the authors’ putrescine data suggest

that ifenprodil is about 100 times more potent and possibly more so, depending

on the concentration of glutamate and glycine (Reynolds and Miller 1989).

lfenprodil is of additional interest in this context because of its neuroprotective

effects in focal cerebral ischemia (Gotti et al. 1988).

Even more recently a spermidine analog, diethylenetriamine (Williams et al.

1990), and a putrescine analog, diguanidinobutane (Reynolds 1990) were

identified as putative competitive spermidine antagonists. This laboratory has

confirmed that arcaine (diguanidinobutane) competitively inhibits spermidineinduced [3H]TCP binding (Sacaan and Johnson 1990b). It was also observed

that arcaine competitively antagonizes Mg2+-induced [3H]TCP binding, thereby

suggesting that this previously poorly understood effect of Mg2+ was mediated

through the spermidine site, where it acts as a partial agonist (Sacaan and

Johnson 1990b). An obligatory role for polyamines was suggested by the

observation that arcaine completely inhibited both [3H]TCP binding and

NMDA-induced [3H]NE release from hippocampal slices (Sacaan and Johnson

1990c). The specificity of arcaine was demonstrated by the observations that

(1) it had no effect on either K+- or kainate-stimulated release, (2) it inhibited

NMDA-induced release noncompetitively, and (3) its inhibition of

NMDA-induced release was reversed by spermidine (Sacaan and Johnson

1990c). The reversal by spermidine was also interesting because it occurred

only in buffer containing 5 percent dimethyl sulfoxide (DMSO). It is postulated

that the spermidine site is intracellular and that DMSO increases the transport

or diffusion of spermidine across the neuronal membrane. Alternatively, the site

could be extracellular and the effect of DMSO could be due to its ability to

increase spermidine diffusion through the slice.

Further complexity is suggested by preliminary studies of polyamines on

[

3H]glycine binding (Johnson and Sacaan 1989). These experiments revealed

that spermine enhanced [3H]glycine binding by increasing the binding affinity

threefold. The structural requirements for activating the glycine receptor were

completely different from those for activating the NMDA ionophore, suggesting

the existence of at least two independent sites for polyamine modulation of the

NMDA receptor ionophore complex. It also was found that the spermine28
induced change in glycine receptor affinity was selective for agonists (i.e.,

spermine shifted the [3H]glycine displacement curve threefold to the left for

glycine and D-serine but had no effect on the displacement by the antagonists

7-chlorokynurenic acid and cycloleucine) (Sacaan and Johnson 1989). This

finding suggests that the glycine receptor may exist in agonist- and antagonistpreferring conformations. We propose that this effect of spermine may be

important physiologically in regulating the effects of an endogenous antagonist,

such as kynurenic acid.

CONCLUSIONS

As covered extensively in a recent review (Johnson and Jones 1990), the

mechanisms of action of PCP are extremely complex. Although it is not certain

exactly what role blockade of the NMDA receptor-ionophore plays in the

psychotomimetic and reinforcing effects of PCP, this mechanism clearly is

central to its anticonvulsant and neuroprotective effects in acute trauma, anoxia,

and hypoglycemia. Thus, if PCP (or related compounds) ultimately has any

impact in the therapy of chronic illnesses such as epilepsy, it will be necessary

to isolate pharmacologically the therapeutic effects from the psychoactive “side

effects.” It is unknown at this time if this is possible, but it is conceivable that, in

situations of acute trauma or stroke, the potential therapeutic benefit of

PCP-like drugs may outweigh these concerns.

Further research on PCP and similar drugs will be required to elucidate the

actions of these drugs and to understand the regulation of the NMDA

ionophore. To date, this complex has been shown to be regulated by several

mechanisms and by a diverse spectrum of effector molecules (figure 2). This

complexity affords an opportunity to subtly control receptor function, possibly

according to therapeutic needs. In addition to the voltage- and use-dependent

nature of the blockade by PCP-like drugs, the possibility that the makeup and

organization of NMDA receptor complexes vary regionally also provides an

opportunity for more selective modulation of receptor function. Thus, the

possibility of selectively targeting specific pathways for therapeutic intervention

is exciting and should be a fruitful area for future research.

29
FIGURE 2. Proposed model of the NMDA receptor ionophore complex.

Antagonists are shown on the right half of the complex or in the

channel itself; agonists are shown on the left half. There is no

known Zn2+ site or antagonist for the putative extracellular

spermine (SP) site. Occupation of the latter site by SP induces a

conformational change in the glycine (GLY) site that is seen as an

increased affinity for agonists, but not antagonists (Sacaan and

Johnson 7989). This site is not obligatory and has a distinct

pharmacology from the obligatory intracellular site activated by

Mg2+, Ca2+, Sr2+, and spermidine (SPMD). Activation of this site

results in an increased affinity for [3H]TCP or [3H]MK-801 binding

to the PCP site, as well as for Mg2+ and putrescine (PUT) binding

to their inhibitoty site that is thought to be in the channel.

Magnesium can also act as a partial agonist at the intracellular

SPD site, but at high receptor occupancy levels (by SPD)

magnesium acts as an antagonist at this site as well. At

concentrations above 1 mM, SPD acts at a distinct site as an

antagonist. The binding domains for SPD are depicted between

dashed lines to indicate the uncertainty of their locations. The

proposed transitional state induced by NMDA agonists in the

absence of glycine, as well as the allosteric interactions between

the NMDA and glycine recognition sites, are not included for ease

of presentation. APV= 2-amino-5-phosphonovaleric acid.

30
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NMDA receptor. Eur J Pharmacol 177:215-216, 1990.

Reynolds, I.J., and Miller, R.J. Multiple sites for the regulation of the

N-methyl-D-aspartate receptor. Mol Pharmacol 33:581-584, 1988.

Reynolds, I.J., and Miller, R.J. lfenprodil is a novel type of N-methyl-D-aspartate

receptor antagonist: Interaction with polyamines. Mol Pharmacol

36:758-765, 1989.

Reynolds, I.J.; Murphy, S.N.; and Miller, R.J. [3H]Labeled MK-801 binding to the

excitatory amino acid receptor complex from rat brain is enhanced by

glycine. Proc Natl Acad Sci U S A 84:7744-7748, 1987.

Sacaan, A.I., and Johnson, K.M. Spermine enhances binding to the glycine site

associated with the NMDA receptor channel complex. Mol Pharmacol

36:836-839, 1989.

Sacaan, A.I., and Johnson, K.M. Characterization of the stimulatory and

inhibitory effects of polyamines on [3H]TCP binding to the NMDA receptor

ionophore complex. Mol Pharmacol 37:572-577, 1990a.

Sacaan, A.I., and Johnson, K.M. Competitive inhibition of magnesium-induced

[

3H]TCP binding by arcaine: Evidence for a shared spermidine-magnesium

binding site. Mol Pharmacol 38:705-710, 1990b.

Sacaan, A.I., and Johnson, K.M. Spermidine reverses arcaine’s inhibition of

N-methyl-D-aspartate-induced hippocampal [3H]NE release. J Pharmacol

Exp Ther 255: 1060-1063, 1990c.

Schoepp, D.D., and Johnson, B.G. Excitatory amino acid agonist-antagonist

interactions at 2-amino-4-phosphonobutyric acid-sensitive quisqualate

receptors coupled to phosphoinositide hydrolysis in slices of rat

hippocampus. J Neurochem 50:1605-1613, 1988.

37
Siddiqui, F.; Iqbal, Z.; and Koenig, H. Polyamine dependence of NMDA

receptor-mediated Ca+2 fluxes and transmitter release from rat hippocampus.

Soc Neurosci Abstr 14:1048, 1988.

Sladeczek, F.; Pin, J.P.; Recasens, M.; Bockaert, J.; and Weiss, S. Glutamate

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317:717-719, 1985.

Snell, L.D., and Johnson, K.M. Antagonism of N-methyl-D-aspartate-induced

transmitter release in the rat striatum by phencyclidine-like drugs and its

relationship to turning behavior. J Pharmacol Exp Ther 235:50-57, 1985.

Snell, L.D.; Morter, R.S.; and Johnson, K.M. Glycine potentiates

N-methyl-D-aspartate-induced [3H]TCP binding to rat cortical membranes.

Neurosci Lett 83:313-317, 1987.

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activation of the glycine receptor that modulates the N-methyl-D-aspartate

operated ion channel. Eur J Pharmacol 156:105-110, 1988.

Sugiyama, H.; Ito, I.; and Hirono, C. A new type of glutamate receptor linked to

inositol phospholipid metabolism. Nature 325:531-533, 1987.

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NMDA-receptor mediated synaptic potentials in neocortical slices. Nature

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Pharmacological evidence for the existence of a polyamine recognition site.

Mol Pharmacol 36:575-581, 1990.

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1987.

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Functional consequences. Neuropharmacology 26:903-909, 1987.

ACKNOWLEDGMENT

The work described in this chapter was supported by National Institute on Drug

Abuse grant DA-02073.

38
AUTHORS

Kenneth M. Johnson, Ph.D.

Professor

Department of Pharmacology and Toxicology

University of Texas Medical Branch

Galveston, TX 77555-1031

Lawrence D. Snell, Ph.D

Instructor

Department of Pharmacology C236

Susan M. Jones, Ph.D.

Postdoctoral Fellow

Department of Physiology C240

University of Colorado Health Science Center

4200 East Ninth Avenue

Denver, CO 80262

Aida I. Sacaan, Ph.D.

Postdoctoral Fellow

CNS Research

Lilly Research Laboratories

Lilly Corporate Center

Indianapolis, IN 46285

39

Pharmacologic Characterizations

of Receptors

Tsung-Ping Su

Two binding sites, receptor and PCP receptor, have been proposed as

mediators of psychotomimetic effects exerted by certain benzomorphans and

phencyclidine (PCP) (Vincent et al. 1979; Zukin and Zukin 1979; Su 1981; Su

1982; Quirion et al. 1987). Exactly which receptor is responsible for the effects

is still unknown, however. Although a large number of reports have presented

pharmacological and behavioral results that indicated the activation of PCP

receptors (e.g., Shannon 1982; Brady et al. 1982; Johnson 1987), many studies

have also shown that receptors may mediate certain behavioral and

biochemical responses (Ceci et al. 1988; Steinfels et al. 1988; Bowen et al.

1988; Walker et al. 1988; lwamoto 1989a, 1989b; Woods, this volume).

Because no animal model can yet be said to definitively represent

psychotomimetic disturbances in humans, the results of those studies may be

interpreted as suggesting that both receptors may be related to

psychotomimesis or other psychological disturbances. It is noteworthy,

however, that at least seven potential antipsychotic drugs, which are efficacious

in preclinical tests, all possess high affinity for the receptor while exhibiting

minimal affinity for the PCP receptors (Largent et al. 1988). Our understanding

of the exact relationship between the two receptors and psychological

disturbances in humans can be clarified only when we obtain results from

clinical trials using drugs specific for each type of receptor.

In this chapter three areas concerning receptors will be addressed: (1)

endogenous ligands for receptors; (2) the structure-activity relationship of

a-selective compounds; and (3) the subcellular distribution of receptors.

ENDOGENOUS LIGANDS FOR RECEPTORS

The unique ligand specificity and distinctive central nervous system distribution

of receptors (Gundlach et al. 1986; Largent et al. 1986) have suggested that

41
endogenous ligand(s) for this receptor might exist. An early attempt to identify

endogenous ligands for the receptor employed a receptor binding assay and

yielded two putative endogenous ligands of about 10,000 and 4,000 daltons

(Su et al. 1986). Because many factors may interfere unpredictably with

receptor binding, we developed a bioassay system that, when coupled with

binding assays, would serve as a more selective tool for examining

endogenous ligands. This bioassay was a guinea pig vas deferens preparation.

An endogenous a ligand should be active in both the receptor binding assay

and the guinea pig vas deferens bioassay. The electrically induced twitches of

the guinea pig vas deferens were potentiated by drugs such as

d-SKF-10,047, d-pentazocine, and d-ketocyclazocine, whereas non-a drugs

such as morphine, DADLE, and I-ketocyclazocine were inactive (Vaupel and Su

1987). Further, the potentiation induced by drugs was blocked by putative

antagonists haloperidol and BW234U (Vaupel and Su 1987). Although this

bioassay system was not a very specific assay for drugs because the less

o-selective drugs such as PCP and l-SKF-10,047 were also active, the assay

did provide an adjunct tool for examining potential endogenous ligands.

Using the two assays, we identified from guinea pig brain extract an

endogenous ligand of about 485 daltons (Su and Vaupel 1988). The larger

molecular-weight substances found in the earlier study (i.e., 10,000 and

4,000 daltons from the receptor binding assay (Su et al. 1986) were inactive in

the guinea pig vas deferens assay (Su, unpublished observation). The

485-dalton material was obtained by fractionating a 700 to 200 molecular

weight mixture from a Sephadex G-50 column, the technique used in the earlier

study (Su et al. 1986). The mixture was then absorbed onto a Bio-Rex70 cation

exchanging resin (equilibrated in 0.05 M ammonium acetate), washed with a

linear gradient of 0.05 M to 1 M ammonium acetate, and then eluted with 1 N

acetic acid. The 1 N acetic-aid-eluted fraction was found to be active not only in

the receptor binding assay but also in the guinea pig vas deferens bioassay (Su

and Vaupel 1988). The active fraction from the Bio-Rex70 column was further

separated in a Sephadex G-15 column and the 485-dalton material obtained

(figure 1). Characterization of this material indicated that it is nonpeptidic and

may not contain nitrogen. Although this material may not be pure at present,

preliminary NMR data indicated that it may contain repetitive methylene group.

Final purification of this material, if successful, may provide an interesting clue

to our understanding of receptors.

42
FIGURE 1. Fractionation of an endogenous ligand in a Sephadex G-15

column. Samples applied to the column were obtained from the

guinea pig brain extract after a partial purification using

Sephadex G-50 and Bio-Rex70 columns. Twenty-five mg of the

lyophilized powder of the sample was applied to the column that

was preequilibrated in 0.1 M ammonium acetate buffer (pH 5.4).

The column (7.6 cm x 90 cm) was eluted with the same buffer and

fractions of 0.67 mL were collected. Each fraction was lyophilized

and tested in receptor binding assay using 2 nM of

[

3H]d-SKF-10,047. Percent inhibitions are shown on the y-axis.

Positions of molecular weight standards are indicated. V0

indicated void volume of the column.

The information collected initially suggested that the 485-dalton endogenous

ligand could bear some structural similarity to steroids. For example, the

material was not peptidic and presumably contained no nitrogen while

containing repetitive methylene groups. Further, the 485-dalton material was

active in an endocrine tissue, the guinea pig vas deferens (Su and Vaupel

1988). These properties suggested that some steroids could act at receptors

and be related to an endogenous ligand.

43
Our examination of a wide variety of steroids indicated that certain steroids

were indeed potent ligands (Su et al. 1988), with the gonadal steroids

progesterone and testosterone among the most potent (Su et al. 1988). The

affinity of progesterone for the receptor was about half that of the prototypic

drug d-SKF-10,047 (268 nM and 134 nM, respectively). Other steroids

possessing appreciable affinity for the receptor include deoxycorticosterone,

11ß-hydroxyprogesterone, pregnenolone sulfate, and corticosterone.

Steroids have been well known to bind at “steroid” receptors in peripheral as

well as neuronal tissues. The receptor, however, differs from traditionally

recognized “steroid” receptors. The “steroid” receptor is cytosolic and not

localized on cellular membranes, while receptors are membrane bound.

There are also other differences between these two receptors. For example,

potent, specific steroid ligands such as promegestone, RU 27987, estradiol,

and estriol, which bind at steroid receptors, were all inactive in the receptor

binding assay (Su et al. 1988). Further, a progesterone antagonist, RU 486,

which interacts specifically with the cytosolic progesterone receptor (Baulieu

1989) was inactive in receptor assays (Su, unpublished observation). The KD

values of the steroids binding to cytosolic steroid receptors were typically in the

range of about 0.1 to 10 nM (Gorski and Gannon 1976) while much higher KD

values were found for steroids binding to receptors (Su et al. 1988). Also,

steroids that were active at receptors in the brain were also active at o

receptors in the spleen cells (Su et al. 1988).

A question arises: What is the functional significance of the interaction of

steroids with receptors in the brain? or in the cells from the immune system?

All possible answers are purely speculative at this time. Because receptors

may mediate certain aspects of psychiatric disturbances, it is not unreasonable

to speculate that steroids may produce psychiatric disturbances by acting at

receptors.

Steroid abuse has recently aroused public awareness, especially abuse among

athletes. The adverse medical effects and major psychiatric disturbances

caused by steroid abuse are well documented (Annitto and Layman 1980;

Freinhar and Alvarez 1985; Wilson et al. 1974; Pope and Katz 1987, 1988). The

effects are even more dramatic because typical steroid abusers may routinely

use doses 10 to 100 times greater than those used clinically (Pope and Katz

1988). The possibility certainly exists that the receptor mediates

steroid-induced psychiatric disturbances, and, conversely, that some of those

behavioral effects may contribute to the abuse of steroids.

44
Steroids may also affect immune responses through interaction at receptors

in immune cells; receptors have been found in spleen cells and lymphocytes

(Su and Vaupel 1988; Su et al. 1988; Wolfe et al. 1988). The functional role of

receptors in these cells is the subject of speculation. These tissues contain

immune cells, so it is tempting to speculate that receptors in these cells may

be involved in cellular immune responses. If this speculation is true, then

steroids active at receptors should also be active in certain immunologic

events. Indeed, an interesting correlation was observed when the efficacies of

steroids in anti-inflammatory tests (Siiteri et al. 1977) were compared with their

affinities at receptors (Su et al. 1988). (A direct comparison of the potencies

could not be made because, in the anti-inflammatory tests, steroids were

delivered through silastic tubing and, thus, no determination of the local

concentration of the test steroids was made. However, the qualitative

correlation is striking.) Steroids that were active in the anti-inflammatory tests

were also active in receptor binding, including progesterone,

deoxycorticosterone, and corticosterone. Many steroids that were inactive in the

anti-inflammatory tests were also inactive in the receptor binding assay.

Those included estriol, estrone 3-hemisuccinate, 17ß-estradiol-17-

hemisuccinate, hydrocortisone, 17 hydroxyprogesterone, and pregnenolone

(Su et al. 1988). However, there were exceptions. For example, although

testosterone was one of the most potent in the receptor binding assay, it was

inactive in the anti-inflammatory test. These results suggest that receptors

may be involved in immune responses. Because receptors have also been

found in many endocrine tissues (Wolfe et al. 1989) it has been suggested that

receptors may provide a link between immune, endocrine, and central

nervous systems (Su et al. 1988). Therefore, ligands may have a wide

spectrum of therapeutic uses (Su et al. 1988).

STRUCTURE-ACTIVITY RELATIONSHIP OF SELECTIVE COMPOUNDS

Drugs that bind and are active at receptors encompass diverse classes of

pharmacological agents. The active compounds include neuroleptics such as

haloperiodol, chlopromazine, and fluphenazine (Su 1982; Tam and Cook 1984;

Largent et al. 1984; Martin et al. 1984; Weber et al. 1986; Contreras et al.

1987); benzomorphans such as d-pentazocine, l-pentazocine, d-cyclazocine,

and d-SKF-10,047 (Su 1982; Tam 1983; Largent et al. 1984; Martin et al. 1984;

Weber et al. 1986); antitussive agents such as dextromethorphan (Su 1982;

Largent et al. 1984); dissociative anesthetics such as phencyclidine (Su 1982;

Tam 1983; Largent et al. 1984; Martin et al. 1984); antihypertensives such as

propranolol (Su 1982; Tam 1983; Martin et al. 1984); antidepressants such as

45
imipramine (Su 1982; Largent et al. 1987) and sertraline (Schmidt et al. 1989);

a polymerization catalyzing agent, ditolyl-guanidine (Weber et al. 1986); a

compound structurally related to dopamine, (+)-3-PPP((+)-3-(3-hydroxyphenyl)-

N-(1-propyl)piperidine) (Largent et al. 1984); and steroids such as progesterone

(Su et al. 1988).

What do these compounds have in common, structurally, that leads to high

affinities at receptors? By synthesizing a large number of compounds and

comparing their activities in the receptor binding assay, Largent et al. (1987)

proposed that the basic pharmacophore for drugs to possess affinity at a

receptor is a phenylpiperidine with a lipophilic nitrogen substituent. Using

calculations based on energy and geometric fit of a series of o-selective drugs,

others reached the same conclusion, that the basic requirement for a

o-selective ligand is a substituted nitrogen and a phenyl hydrophilic center

separated by an intermediate chain (Manall`ack and Beart 1987; Manallack et al.

1988). It was also proposed in these reports that a receptor may interact with

a ligand through three hypothetical points on the receptor (Manallack and

Beart 1987; Manallack et al. 1988). An R1 and an R2 would each

perpendicularly project to the opposite side of the flat phenyl ring. An R3 site

that was relatively parallel to the R1-phenyl projection was proposed to receive

the vector provided by the lone pair electron on the nitrogen atom.

Similar results were obtained when several a-selective ligands such as

d-pentazocine, d-SKF-10,047, haloperidol, BW 234U, BMY 14,802,

remoxipride, and progesterone were examined based on molecular modeling

and electrostatic potential calculations (Gund et al. 1991). (Progesterone was

included in the study-although it does not contain nitrogen, because it is a

potent ligand.) The study suggested that the oxygen atom of the 20-carbonyl

group on progesterone must be the vector to the R3 hypothetical receptor point.

Further, the R1 and R2 receptor pocket can accommodate quite a variety of

lipophilic rings. The lipophilic ring does not have to be a phenyl ring, because

ring B of progesterone (a cyclohexyl ring) can substitute for a phenyl ring. Also,

the R1-R2 receptor pocket may tolerate lipophilic groups of quite different sizes,

because ring A of a steroid, immediately adjacent to ring B, does not destroy

progesterone activity at a receptor.

An important aspect of the results of the study by Gund et al. (1989) was

obtained from the calculation of the electrostatic potentials (ESP) on the van

der Waals surfaces of these o-selective ligands. ESP on progesterone

indicated a highly positively charged center around ring B of the molecule, i.e.,

46
the lipophilic area interacting with the R1-R2 hypothetical receptor points.

However, the ESP of other -selective ligands, when examined in their

protonated forms, had a positive center surrounding the nitrogen atoms. These

results represent a potential contradiction, as one would expect that the highly

positively charged centers of all these drugs would be located at the same end

of the molecules, where they share a common chemical-physical property to

interact with a common site on the receptor. The contradiction was resolved

when ESPs were examined on the unprotonated forms of the ligands. A

highly positively charged center on each drug was found to localize around the

lipophilic pocket-similar to the ESP on progesterone. These results indicated

that the unprotonated form of a drug is probably the molecular species that

interacts with receptors.

Ever since the demonstration of separate PCP and receptors, it has been

noted that phencyclidine, although more specific toward the PCP receptor, can

still interact with the receptor. The IC50 ratio of phencyclidine for the two

receptors is about 1/10 An interesting question can be asked: Is it

possible to modify the phencyclidine molecule to obtain compounds that are

more selective for the receptor? After an extra methylene group was inserted

into phencyclidine between the cyclohexyl and piperidine rings, the modified

compound became more selective than the parent compound for the receptor

(figure 2; Su and Parish, unpublished observation). The IC50 ratio of this new

compound for the two receptors is 17.8/1 The results suggest that by

increasing the length between the cyclohexyl ring and the amine moeity, one

may change PCP-selective compounds into -selective drugs. These results

indicate that the intermediate chain between the lipophilic ring and amine group

is probably essential for -selective compounds.

SUBCELLULAR DISTRIBUTION OF RECEPTORS

An examination of the subcellular distribution of receptor activity in rat brain

revealed that receptors were concentrated in the microsomal fraction (i.e., P3)

when compared with the crude synaptosomal fraction (i.e., P2) (McCann et al.

1989). Thus, receptors may not be localized in the synaptic area of plasma

membranes. Because microsomes contain many drug-metabolizing enzymes, it

was possible that the receptor may be an enzyme (McCann et al. 1989).

However, that speculation was made from data obtained from a relatively

“rough” fractionation of subcellular structures, and the authors also suggested

other possibilities such as localization on the nonsynaptic regions of plasma

membrane. The proof of cellular localization requires further fractionation and

47
FIGURE 2. Effect of inserting a methylene group between the arylcyclohexyl

ring and amine group of phencyclidine on the relative affinities for

and PCP receptors. The compound with a methylene insertion

was synthesized from phenylacetonitrile and 1,5-dihaloalkane

followed by an amine (Parish et al., in preparation). and PCP

receptor binding assays were performed using guinea pig brain

homogenates with 2 nM [3H]d-SKF-10,047 and 1 nM [3H]TCP as

the radioligands, respectively. IC50s represent the average from

three experiments, each assayed in quadruplicate.

comparison with known markers (McCann et al. 1989). In further studies, it was

found that the distribution patterns of receptors paralleled the patterns of a

general plasma membrane marker, 5’ nucleotidase, indicating the possibility

that receptors may reside in the nonsynaptic portion of the plasma membrane

(McCann and Su 1990). The distribution of receptors also differed from the

distribution of a marker for a major microsomal constituent-endoplasmic

reticulum (McCann and Su 1990). A better understanding of the exact

localization of the receptor may have to wait on examinations using

electronmicroscopic techniques coupled with immunocytochemistry.

48
Whether the receptor is an enzyme or not is by itself an interesting question.

It would be very revealing indeed (if the receptor turns out to be an enzyme)

to discover how an enzyme can play such interesting pharmacologic roles. So

far, however, no enzymatic activity has been demonstrated in receptor

preparations. In fact, in a report using liver homogenates, where there is an

abundance of drug-metabolizing enzymes, [

3H]d-SKF-1 0,047 was found

unchanged after incubation (Samovilova et al. 1988). This suggests that the

receptor may not be an enzyme. The biochemical nature of receptors

certainly deserves further investigation,

In conclusion, much remains to be learned about receptors. Our

understanding of this receptor is just beginning. Because of the potential

relation of receptor activity to psychiatric disturbances in humans, or other as

yet to be fully revealed actions, the studies reviewed in this chapter provide

significant information.

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Su, T.-P., and Vaupel, D.B. Further characterization of an endogenous ligand

(“SIGMAPHIN”) for sigma receptors in the brain. Soc Neurosci Abstr 14:545,

1988.

Su, T.-P.; Weissman, A.D.; and Yeh, S.Y. Endogenous ligands for sigma opioid

receptors in the brain (“SIGMAPHIN”): Evidence from binding assays. Life

Sci 38:2199-2210, 1986.

Tam, S.W. Naloxone-inaccessible receptor in rat central nervous system.

Proc Natl Acad Sci USA 80:6703-6707, 1983.

51
Tam, S.W., and Cook, L. Sigma opiates and certain antipsychotic drugs

mutually inhibit (+)-[3H]SKF 10,047 and [3H]haloperidol binding in guinea pig

brain membranes. Proc Natl Acad Sci USA 81:5618-5621, 1984.

Vaupel, B.D., and Su, T.-P. Guinea-pig vas deferens preparation may contain

both receptors and phencyclidine receptors. Eur J Pharmacol

139: 125-128, 1987.

Vincent, J.P.; Kartalovski, B.; Geneste, P.; Kamenka, J.M.; and Lazdinski, M.

Interaction of phencyclidine (“angel dust”) with a specific receptor in rat brain

membranes. Proc Natl Acad Sci USA 76:4678-4682, 1979.

Walker, J.M.; Matsumoto, R.R.; Bowen, W.D.; Gans, D.L.; Jones, K.D.; and

Walker, F.O. Evidence for a role of haloperiodol-sensitive -“opiate”

receptors in the motor effects of antipsychotic drugs. Neurology 38:961-965,

1988.

Weber, E.; Sonders, M.; Quarum, M.; McLean, S.; Pou, S.; and Keana, J.F.W.

1,3-Di(2[5-3Hjtolyl)guandine: A selective ligand that labels -type receptors

for psychotomimetic opiates and antipsychotic drugs. Proc Natl Acad Sci

USA 83:8784-8788, 1986.

Wilson, l.C.; Prange, A.J.; and Lara, P.P. Methyltestosterone and imipramine in

men: Conversion of depression to paranoid reaction. Am J Psychiatry

131:21-24, 1974.

Wolfe, S.A., Jr.; Culp, S.G.; and De Souza, E.B. Sigma receptors in endocrine

organs: Identification, characterization, and autoradiographic localization in

rat pituitary, adrenal, testis and ovary. Endocrinology 124:1160-1172, 1989.

Wolfe, S.A., Jr.; Kulsakdinun, C.; Battaglia, G.; Jaffe, J.H.; and De Souza, E.B.

Initial identification and characterization of sigma receptors in human

peripheral blood leukocytes. J Pharmacol Exp Ther 247:1114-1119, 1988.

Zukin, S.R., and Zukin, R.S. Specific [3H]phencyclidine binding in rat central

nervous system. Proc Natl Acad Sci USA 76:5372-5376, 1979.

ACKNOWLEDGMENT

The author appreciates the collaborations of Drs. Tamara M. Gund and Kenhiya

Shuklar on molecular modeling, Dr. Daniel W. Parish on structure-activity

relationship, and Dr. David J. McCann on subcellular fractionation studies.

52
AUTHOR

Tsung-Ping Su, Ph.D.

Chief, Neurochemistry Unit

Neuropharmacology Laboratory

Neuroscience Branch

Addiction Research Center

National Institute on Drug Abuse

P.O. Box 5180

Baltimore, MD 21224

53

Studies of Receptors and Metabolic

Responses to Ligands in the Brain

E. D. London

Binding sites were first characterized in rodent brain by using racemic tritiated

N-allylnormetazocine (NANM, SKF 10,047) (Su 1981, 1982), the prototypical

ligand (Martin et al. 1976). More recent studies of receptors have employed

tritiated d-NANM (Tam 1983; Martin et al. 1984; ltzhak et al. 1985; Mendelsohn

et al. 1985; Mickelson and Lahti 1985; Largent et al. 1986; Sircar et al. 1986),

(+)3-(3-hydroxyphenyI)-N-(1-propyl)-piperidine (PPP) (Largent et al. 1984),

di-o-tolylguanidine (DGT) (Weber et al. 1986), and haloperidol (HAL) (Tam and

Cook 1984; Downes et al. 1986; Contreras et al. 1987). In the studies

discussed below, our laboratory used [3H]HAL as a receptor ligand because

of its greater affinity for receptors than d-NANM, its negligible affinity for

phencyclidine (PCP) receptors, and because HAL has the potential for use in in

vivo human studies when labelled with the positron-emitting radionuclide F-l 8.

Although HAL binds to dopaminergic and -adrenergic receptors, we blocked

radioligand binding to these sites by adding spiperone to incubations. Assays

using various concentrations of spiperone to block the binding of 1 nM HAL

showed that 75 nM spiperone blocked 95 percent of the binding to one

population of sites without affecting binding to a lower affinity population in the

guinea pig brain; in samples from other subhuman species, 100 nM spiperone

was an optimal concentration for blocking binding to the higher affinity

population (Vu et al. 1990). In the human cerebellum, 50 nM spiperone

completely inhibited binding to one population of sites without appreciably

affecting another population of sites (Weissman et al. 1988b). The Ki values of

spiperone in competing for HAL binding to dopaminergic and -adrenergic

receptors are 0.25 nM (Bun et al. 1976) and about 4 nM (U'Prichard et al.

1977) respectively. In contrast, spiperone shows an IC50 value between 10-7

and 10-6 M in inhibiting HAL binding to receptor (Tam and Cook 1984).

Therefore, the high-affinity, spiperone-sensitive binding of HAL apparently was

55
to dopaminergic or noradrenergic sites or both, and the lower affinity population

was the receptor.

Similarities in the binding properties (for reviews, see Zukin 1984; Quirion 1986;

Johnson 1987) and behavioral effects of NANM and PCP (Keats and Telford

1964; Snyder 1980; lwamoto and Martin 1981; Brady et al. 1982; Shannon

1982) led us to explore whether and PCP receptors showed similar

phylogenetic or development profiles. We studied and PCP receptor binding

in a wide variety of animal species, using [3H]HAL and [3H]TCP as the

respective ligands (Vu et al. 1990).

We performed saturation and single-concentration studies of both binding sites

on whole brains of various species, and the cerebellum [3H]HAL) and frontal

cortex ([3H]TCP) of the squirrel monkey. Kd values for sites varied about

five-fold, with the highest densities in the frog and guinea-pig brains. [3H]TCP

binding affinities at PCP receptors varied about four-fold, and maximum

densities of PCP receptors varied four- to five-fold. Binding at a single

concentration of ligand showed no orderly relationship between and PCP

receptor binding across the species tested (figure 1). However, the fact that

HAL and TCP binding were detected in all of the species tested indicates that

and PCP binding sites are phylogenetically old. The early appearance and

ubiquitous distribution of these sites suggest that they have a role in some

basic neurobiological function in the animal kingdom.

We observed marked similarities in the affinities of the various inhibitors for

receptors in guinea-pig and frog brains and PCP receptors in guinea-pig and

chicken brains, suggesting conservation of the pharmacological properties of

these receptors throughout vertebrate evolution. The results with receptors

showed relatively high affinity for d-NANM, d-pentazocine, I-butaclamol, and

HAL, which have been reported to be potent ligands. Pharmacological

characteristics of PCP receptor binding in the guinea-pig brain agreed with

published data on the guinea-pig and other rodent species.

Studies on the postnatal ontogeny of and PCP receptors yielded surprising

results (figure 2) (Majewska et al. 1989). Whereas the PCP receptor showed

development increase postnatally, there was no age-dependent change in the

receptor. The lack of postnatal development of receptors in the central

nervous system, as compared with postnatal changes in other classical

neurotransmitter receptors, and the fact that sites are much denser in

peripheral organs, such as the liver (Samovilova et al. 1989) and immune and

56
FIGURE 1. Phylogenetic distribution of [3H]HAL and [3H]TCP binding sites.

Assays of receptor binding were performed at a sing/e

concentration of [3H]HAL (1 nM) in the presence of 75 to 100 nM

spiperone. Data for sea anemone, planaria, earthworm, crayfish,

cricket, and hagfish are mean + SEM (bars) values from nine

samples. Data for other organisms are mean + SEM (bars) values

for at least three experiments, each done at least in duplicate.

PCP receptor binding was assayed with 2.5 nM [3H]TCP.

From Vu et al. (1990).

endocrine tissues (Wolfe et al. 1988, 1989), suggest a universal role for these

sites in cellular function.

57
FIGURE 2. Postnatal development of and PCP binding. Equilibrium binding

data of [3H]HAL and [3H]TCP were analyzed by the LIGAND

programs (Munson and Rodbard 1980). Data represent mean

values ± SEM from 3 to 6 individual experiments. * = statistically

different from previous age group, p < 0.05 by Bonferroni t

statistics. † = statistically different from 180-day-old rats; p < 0.05

by Dunnett’s test. From Majewska et al. (7989).

Despite such a possible universal role, several lines of evidence suggested that

receptors might be part of a system involved in psychotomimetic or

antipsychotic actions of drugs or endogenous substances in the brain. The

evidence included the following: (1) that HAL, a clinically useful antipsychotic

drug, has high affinity for receptors (Su 1982, Tam and Cook 1984; Largent et

al. 1988); (2) that several new, structurally unrelated potential antipsychotic

drug candidates have in common the ability to bind to receptors (Largent et

al. 1988); and (3) that canine delirium, produced by NANM, appeared to be

mediated through receptors (Vaupel 1983). We therefore studied receptors

in postmortem material from human brain, including autopsy material from

schizophrenics (Weissman et al. 1988a, 1988b).

58
Saturation and inhibition studies, performed in control cerebellar membranes,

revealed a single population of sites, with an average Kd of 1.4 nM, a Bmax of

about 350 fmol/mg protein, and pharmacological characteristics consistent with

the view that under our experimental conditions, [3H]HAL bound to a receptors

in the human brain. Inhibition studies (table 1) indicated that HAL was the most

potent drug tested, followed by d-pentazocine, di-o-tolylguanidine, and then

PPP. The d-isomers at NANM and pentazocine were more potent, as was the

l-isomer of butaclamol, as compared with the corresponding stereoisomers.

TCP and PCP were not potent, and apomorphine had negligible affinity.

An analysis of thirteen brain regions at a single ligand concentration showed the

highest level of binding in the cerebellar cortex, accumbens nucleus, and

cortical regions (see figure 3). There was little variation in the cortical

distribution of receptors. Pontine nuclei, thalamus, and spinal cord had the

TABLE 1. Inhibition of 1 nM [3H]haloperidol binding in human cerebellum

Drug

HAL

d-pentazocine

DTG

d-PPP

I-butaclaol

I-pentazocine

spiperone

d-NANM

d-butaclamol

TCP

PCP

I-NANM

apomorphine

Ki (nM)

1.8±1a

26±8

32 ± 9

72±8

263 ± 66

366±106

647 ± 244b

975 ± 381

2270 ± 240

3740 ± 290

5500 ± 430

7570 ± 229

>10,000

Relative potency

1.000

0.236

0.196

0.084

0.024

0.017

0.010

0.006

0.003

0.002

0.001

0.001

Negligible

NOTE: Ki values are means ± SEM from single experiments performed In triplicate on three brains,

using 13 to 15 concentrations of competing drug. Ki values were obtained with the LIGAND

programs (Munson and Rodbard 1980).

aKl

 was obtained using an estimate of nonspecific binding determined by adding d-NANM because

high concentrations of HAL reduced estimated nonspecific binding below that obtained with other a

ligands, suggesting that [3H]HAL In the presence of 5.0 nM spiperone bound specifically to non

sites with an affinity lower than for sites. For other inhibitor assays, nonspecific binding was

determined with an excess of unlabeled HAL.

bKi

 Is for the lower affinity (presumed a) spiperone-sensitive site. From Weissman et al. (1988b).

59
FIGURE 3. Regional distribution of specific binding (fmoles/mg protein) of

1 nM [3H]HAL to specific human brain regions in the presence of

50 nM spiperone. Receptor densities are coded as different

patterns. From Weissman et al. (1988b).

lowest levels of binding. Other areas, including limbic areas and components of

the extrapyramidal motor system, had intermediate levels.

Following the characterization of receptors in control brain, we assayed the

binding sites in postmortem brains from age-matched controls,

nonschizophrenic suicide victims, and schizophrenics. We observed

significantly lower densities of receptors in the temporal cortex in

schizophrenic brains (Weissman et al. 1988b). The possible explanations of the

decrement in receptors in the temporal cortex include an alteration in the level

60
of a putative endogenous ligand that may regulate the receptor. Alternatively,

schizophrenia may involve focal lesions of receptor-bearing neurons.

The demonstration of a specific alteration of receptors in schizophrenics

suggests that in vivo studies of schizophrenics using positron emission

tomography (PET) might be useful in the characterization and diagnosis of this

disorder. Therefore, we tested both radiolabeled HAL and d-NANM as potential

ligands for in vivo labelling of receptors in the brain (Weissman et al. 1987a).

We gave mice tracer quantities of [3H]HAL or [3H]d-NANM. In some mice which

received HAL, 0.5 mg/kg spiperone was given to block binding to

catecholaminergic sites. Other mice received 1 mg/kg unlabelled HAL to block

binding to catecholaminergic as well as sites, defining nonspecific binding.

Mice that were given injections of d-NANM received a pretreatment with

1 mg/kg TCP to block binding to PCP receptors, or 1 mg/kg each of HAL and

TCP to block binding to and PCP receptors (nonspecific binding). Both

radioligands showed high estimates of specific binding in the cerebellum,

cranial nerve nuclei of the medulla-pons, and midbrain, and low levels in the

hippocampus. The distribution of binding generally agreed with in vitro data

(figure 4).

FIGURE 4. Regional distribution in the mouse brain of in vivo binding of

[

3H]HAL in the presence of spiperone. From Weissman et al.

(1987a).

61
In addition to receptor mapping and quantitation, metabolic mapping with

deoxyglucose is a valuable approach to delineating the distribution of drug

action in the brain of an intact animal (McCulloch et al. 1982; London et al.

1986; London 1989). While receptor maps provide information about the

primary sites of action of drugs in the brain, deoxyglucose mapping provides

information about primary as well as secondary effects of drugs in the brain of

an intact animal. Radiolabeled deoxyglucose (2-deoxy-D-[1-14C]glucose (DG) is

used as a tracer for glucose metabolism (Sokoloff et al. 1977). Because

glucose is the primary substrate of the adult brain for energy metabolism, the

deoxyglucose method provides information about local brain function.

Recent interest in receptors has led to the introduction of several drugs that

bind to receptors and have been proposed as candidate antipsychotic agents

(Largent et al. 1988; Su et al. 1988). These drugs include BW 234U

(rimcazole), BMY 14802, and HR 375. We were interested in whether the

cerebral metabolic responses to these drugs relate to the distribution of

receptors and if the drugs produce a characteristic profile, which might

provide information on a mechanism for their potential antipsychotic action.

Therefore, we tested the acute effects of 30 mg/kg i.p. BW 234U and

BMY 14802 on local cerebral glucose utilization (LCGU) in rats by the DG

method (della Puppa and London 1989).

Both BW 234U and BMY 14802 produced effects on LCGU in epithalamic,

metathalamic, hypothalamic, and mesencephalic regions, as well as in cranial

nerve nuclei and the cerebellum (table 2). However, BMY 14802 increased

LCGU, whereas BW 234U reduced LCGU at the doses tested. It appears that

most of the changes in LCGU were driven by direct interactions with

receptors because the distribution of the effects on LCGU roughly paralleled the

distribution of receptors visualized autoradiographically using [3H]PPP as a

ligand (Largent et al. 1986). In this regard, affected regions such as the

paraventricular hypothalamic, medial mammillary and supramammillary nuclei;

cerebellum; granular layer of the dentate gyrus; locus ceruleus; and cranial

nerve nuclei contain high densities of receptors. However, no changes in

LCGU were observed in areas, such as the molecular layer of the dentate gyrus

and superficial layers of the cerebral cortex, which reportedly are devoid of

receptors.

We also tested the LCGU effects of d-NANM (della Puppa and London 1989;

Hill et al. 1989; della Puppa, Vu, Kimes, and London, unpublished findings),

which might be thought of as an agonist by virtue of its activity in behavioral

62
TABLE 2. Effects of ligands on glucose utilization in the rata

Brain regions

Vehicle

control

BMY 14802

(30 mg/kg)

Vehicle

control

BW 234U

(30 mg/kg)

Frontal cortex layers 1-3

Layer 4

Anterior cingulate cortex

Dentate gyrus granular

layer

Medial geniculate nucleus

Lateral habenula

Paraventricular nucleus

Medial mammillary

nucleus

Supramammillary nucleus

Inferior colliculus

Superior colliculus

Substantia nigra reticulata

Compacta

Locus ceruleus

Cochlear nucleus

Facial nucleus

Abducens nucleus

Lateral vestibular nucleus

Cerebellar vermis

83 ± 5.0

101 ± 4.9

103±3.1

71 ± 4.7

123±5.0

127±9.8

77 ± 3.5

122±8.3

82 ± 5.5

188±7.2

90 ± 4.6

62 ± 3.2

84 ± 3.8

68 ±4.8

100 ± 8.8

61 ± 3.7

61 ± 3.3

82 ± 5.2

69 ± 5.5

95 ± 2.4

102±3.5

112±2.7*

60 ± 3.1*

142 ± 5.3*

229 ± 2.5*

104±4.1*

149 ± 6.6*

97 ± 4.9*

196 ±5.2

100 ±4.2

80 ± 2.8*

105 ± 2.3*

90 ± 4.6*

138 ± 12.2*

83 ± 3.2*

66 ± 4.9

100 ± 3.4*

85 ± 1.9*

85 ± 8.3 96 ± 10.6

104±12.1 108 ± 12.1

111 ± 10.5 103 ± 7.7

56 ± 4.6

113±10.8

113±14.1

65 ± 9.6

52 ± 4.2

84 ± 3.5*

82 ± 8.4*

81 ±12.6

107±11.4

72 ± 7.8

146 ± 10.7

72 ± 5.3

54 ± 3.8

76 ± 7.7

60 ± 4.8

96 ± 6.9

50 ± 1.9

49 ± 2.1

78 ± 4.0

58 ± 5.9

84 ± 8.8

56 ± 9.7

97 ± 6.8*

52 ± 4.9*

44 ± 3.8

53 ± 5.6*

47 ± 5.7

94 ± 16.6

41 ±7.9

39 ± 4.4*

56 ± 6.1*

49 ± 6.5

a

LCGU values (μmol/100 g/min) are expressed as means ± SEM for n = 4 to 6 rats.

*Significant from control P < 0.05 according to one-way ANOVA.

From della Puppa and London (1969).

systems (Vaupel 1983; lwamoto and Martin 1981; Brady et al. 1982; Shannon

1982). The effect of d-NANM was dose-dependent. Although 0.5 mg/kg

d-NANM reduced LCGU in several brain regions, including the cerebellum and

cranial nerve nuclei (which are rich in receptors), higher doses were without

effect in these regions. In other regions, including the accumbens nucleus, the

globus pallidus, thalamic nuclei, the dentate gyrus, and the medial cortex,

d-NANM increased LCGU in a dose-dependent manner. These effects of

d-NANM resembled the acute effects of PCP on LCGU (Weissman et al.

19876). Therefore, it appears that low doses of d-NANM reduce LCGU by

interactions with receptors. The biphasic actions of d-NANM on LCGU is in

63
keeping with the relative affinities of the drug for and PCP receptors (Su

1982; Tam 1983; Largent et al. 1986).

The demonstration that cerebral metabolic effects of ligands are localized to

brain regions that show high densities of receptors is an important

demonstration that binding sites are coupled to functional activity.

Nonetheless, our metabolic studies did not assign agonist or antagonist

functions to the drugs tested on the basis of the direction of effects on LCGU. A

similar lack of separation of possible agonist from antagonist functions was

noted in the effect of various ligands, including HAL, to antagonize

carbachol-stimulated phosphoinositide turnover in vitro (Bowen et al. 1988). In

addition, whole-cell voltage-clamp studies in our laboratory demonstrated that

various ligands caused an apparent inward current, which was due to

blockade of a tonic outward K+ current (Bell et al. 1988). Although HAL blocked

the effect of d-pentazocine, consistent with an antagonist action of HAL, it was

also the most potent drug in blocking the outward K

+

 current. In fact, all

ligands tested produced the same effect. Therefore, studies in several systems

have failed to distinguish among agonist, reverse agonist, and antagonist

properties of ligands.

CONCLUSIONS

Receptor binding studies indicate that receptors are present, with conserved

pharmacologic properties, throughout vertebrate evolution. They differ from

classical neurotransmitter receptors in that they show no postnatal ontogeny in

the rat. Receptors are present in the human brain and are lost selectively

from the temporal cortex of schizophrenic brains. In vivo studies suggest that

receptors can be studied in the living human brain by PET imaging, possibly by

using HAL or other ligands labeled with positron-emitting radionuclides.

Metabolic studies support the view that receptors have functional significance,

for glucose utilization is affected by ligands in areas of brain that show high

densities of receptors. Therefore, metabolic mapping with DG provides a

functional index of -receptor activation. Both metabolic mapping studies and

other approaches have not yet provided us with a means of delineating agonist

or antagonist roles for various ligands.

64
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morphine-inaccessible sites in guinea-pig brain. Eur J Pharmacol 75:81-82,

1981.

Su, T.-P. Evidence for opioid receptor: Binding of [3H]SKF-10,047 to

etorphine-inaccessible sites in guinea-pig brain. J Pharmacol Exp Ther

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Su, T.-P.; Schell, S.E.; Ford-Rice, F.Y.; and London, E.D. Correlation of

inhibitory potencies of putative antagonists for receptors in brain and

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Tam, S.W. Naloxone-inaccessible receptor in rat central nervous system.

Proc Natl Acad Sci USA 80:6703-6707, 1983.

Tam, S.W., and Cook, L. a-Opiates and certain antipsychotic drugs mutually

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a radiolabeled agonist and antagonist at central nervous system

alpha-noradrenergic receptors. Mol Pharmacol 13:454-473, 1977.

Vaupel, D.B. Naltrexone fails to antagonize the effects of PCP and SKF

10,047 in the dog. Eur J Pharmacol 92:269-274, 1983.

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and distributions of and phencyclidine receptors in the animal kingdom. J

Neurochem 54:598-604, 1990.

Weber, E.; Sonders, M.; Quarum, M.; McLean, S.; Pou, S.; and Keana, J.F.W.

1,3-di(2[5-3H]tolyl)guanidine: A selective ligand that labels o-type receptors

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USA 83:8784-8788, 1986.

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3H]haloperidol and [3H]d-SKF 10,047 in mouse brain. Soc Neurosci Abstr

13:1130, 1987a.

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Weissman, A.D.; Su, T.-P.; Hedreen, J.C.; and London, E.D. receptors in

post-mortem human brains. J Pharmacol Exp Ther 247:2433, 1988b.

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AUTHOR

Edythe D. London, Ph.D.

Chief, Neuroimaging and Drug Action Section

Neuroscience Branch

Addiction Research Center

National Institute on Drug Abuse

P.O. Box 5180

Baltimore, MD 21224

68
Receptors and Signal Transduction:

Negative Modulation of Signaling Through

Phosphoinositide-Linked Receptor Systems

Wayne D. Bowen, Paul J. Tolentino, Brian N. Kirschner,

Paul Varghese, Brian R. de Costa, and Kenner C. Rice

INTRODUCTION

Receptors were first proposed by Martin and colleagues (1976) as a class of

opiate receptor. However, recent studies show that receptors are distinct from

any known drug or neurotransmitter receptor (Sonders et al. 1988). Several

drugs with central nervous system actions exhibit high to moderate affinity for

these sites. These drugs include dextrorotary opiates such as (+)-pentazocine

and dextromethorphan, antipsychotic dopamine antagonists such as haloperidol

and fluphenazine, and certain phencyclidine-related compounds.

Although the functions of these sites are still largely unknown, receptors are

likely to mediate at least some of the physiological or behavioral effects of these

compounds or both. Various studies have indicated that the binding site is a

functional entity. Ligands modulate electrically induced contractions of the

guinea pig ileum (Campbell et al. 1989) and contractions of other smoothmuscle tissue (Campbell et al. 1987; Vaupel and Su 1987). The rate of firing of

rubral neurons is slowed by selective ligands (Matsumoto and Walker 1988).

Microinjection of ligands into the rat substantia nigra or red nucleus produces

marked alterations in motor behavior and posture (Walker et al. 1988;

Matsumoto et al. 1990). This latter finding suggests a role of receptors in

neural regulation of movement and the possibility that these sites mediate some

of the adverse motor side effects of antipyschotic drugs. An involvement of

receptors in the motor disorder dystonia has also been suggested (Bowen et al.

1988a; Walker et al. 1988).

69
A step toward elucidating the role of receptors is to establish the transduction

mechanisms utilized or modulated by compounds. Several neurotransmitters

act as agonists at receptors that are coupled to phosphoinositide (PPI) turnover

(Berridge and Irvine 1984; Chuang 1989; Sladeczek 1987). The binding of

ligands to these receptors results in activation of phospholipase C, with the

resultant cleavage of phosphatidylinositol-4,5-bisphosphate (PIP2) and

production of diacylglycerol (DG) and inositol trisphosphate (IP3). IP3 causes

release of calcium from the endoplasmic reticulum. This effect results ultimately

in activation of calcium/calmodulin-dependent protein kinase. DG activates

protein kinase C. By activation of these kinases, a myriad of cellular responses

can be produced. Two transmitters known to signal through this pathway are

acetylcholine, via M1- (and possibly M3) muscarinic cholinergic receptors, and

norepinephrine, via -adrenergic receptors (Chuang 1989). We have

previously reported that receptors attenuated PPI turnover stimulated by the

cholinergic agonist carbachol (Bowen et al. 1988b). Here we further investigate

the relationship between receptors and PPI-linked receptor system.

MATERIALS AND METHODS

PPI Turnover

Synaptone urosomes were prepared and PPI turnover assayed as described

previously (Bowen et al. 1988b), using the same procedure originally described

by Gusovksy and Daly (1988) with minor modifications. Brains (minus

cerebellum) of 150 to 200 male Sprague-Dawley rats were homogenized in 10

volumes of oxygenated (95 percent O2/5 percent CO2) Krebs-Henseleit buffer,

pH 7.4 (118.5 mM NaCI, 4.7 mM KCI, 1.18 mM MgSO4, 2.5 mM CaCl2,

1.18 mM KH2PO4; 24.9 mM NaHCO3, 10.0 mM glucose) using five hand-driven

strokes in a Kontes Duall-24 glass-glass homogenizer. The homogenate was

centrifuged at 1,000 x g for 10 minutes and the pellet resuspended in

20 volumes Krebs-Henseleit supplemented with 50 mM Hepes, pH 7.4.

To a volume of the suspension equivalent to 320 μL/assay tube was added

[

3H]myo-inositol (23 Ci/mmol) to a final concentration of 0.8 to 1.0 μM (about 5

μCi per assay tube). Aliquots of 320 μL were removed and incubated for

60 minutes at 37°C to label the inositol lipid pool. Twenty microliters of 200 mM

LiCl were then added (10 mM final concentration) and incubation was allowed

to proceed for 10 more minutes before drugs were added. Test compound and

cholinergic or adrenergic agonist were then added. Test compound was always

added just before PPI agonist was added. Labeled synaptoneurosomes and

70
drugs were then incubated for 90 minutes in a final volume of 400 μL. All

incubations were carried out in capped 14.5-mm by 84-mm polypropylene tubes

under an atmosphere Of 95 percent O2/5 percent CO2.

After transferral to microfuge tubes and centrifugation for 5 minutes, the

synaptoneurosome pellet was washed once with 1 mL fresh buffer and then

extracted by resuspension in 1 mL 6 percent trichloroacetic acid. Lysed

synaptoneurosomes were removed by centrifugations, and the extract was

mixed with 1 mL of a slurry of BioRad anion exchange resin AG 1-X8, 100 to

200 mesh, formate form (1:1 , w:v resin:water). After incubation for 5 minutes at

25°C the slurry was placed into small polypropylene columns and the resin

washed four times with 1 mL of water to remove excess [3H]myo-inositol.

[

3H]lnositol-1-phosphate ([3H]IP1

) was then eluted directly into scintillation vials

by washing the column twice with 1 mL of 200-mM ammonium formate/100 mM

formic acid. After addition of 2 mL of Hydrofluor (National Diagnostics, Manville,

N.J.) and vigorous mixing, vials were counted 2 to 4 hours later to avoid

chemiluminescence. Previous studies have shown that measurement of [3H]IP1

gives sufficient indication of agonist-stimulated PPI turnover, for [3H]inositol

trisphosphate is rapidly degraded to [3H]IP1 under the conditions of this assay.

Receptor Binding

Muscarinic receptor binding was performed using either the lysed, frozen, crude

P2 membrane fraction (LFCP2) or synaptoneurosome fraction of male

Sprague-Dawley rat brain (minus cerebellum). LFCP2 was prepared as

described previously (Bowen et al. 19896). Synaptoneurosomes were prepared

as described above. [3H]Oxotremorine-M ([3H]Oxo-M, 87 Ci/mmol, 5 nM) was

incubated with about 500 μg membrane protein in 0.5 mL of Krebs-Henseleit/

Hepes, pH 7.4 for 60 minutes at 37°C. Nonspecific binding was determined in

the presence of 10 μM unlabeled oxotremorine-M. Assays were terminated by

dilution with 5 mL ice-cold phosphate-buffered saline (PBS), pH 7.4, and

vacuum filtration through glass-fiber filters. Filters were then washed twice with

ice-cold PBS.

Receptors were labeled using 3 nM [3H] (+)-3-(3-hydroxyphenyl)-N -

(1-propyl)piperidine ([3H] (+)-3-PPP, 98.9 Ci/mmol) and the crude P2 fraction

(LFCP2) of guinea pig brain. Incubations were carried out for 120 minutes in

0.5 mL 50 mM Tris-HCI, pH 8.0, at 25°C using 500 μg membrane protein.

Nonspecific binding was determined in the presence of 1 μM haloperidol.

Assays were terminated by dilution with 5 mL ice-cold 10 mM Tris, pH 8.0, and

71
vacuum filtration through glass-fiber filters. Filters were then washed twice with

5 mL buffer. Filters were soaked in 0.5 percent polyethyleneimine for at least 30

minutes at 25°C prior to use. Some assays were carried out, as specified,

with 5 nM [3H]1,3-di-stolylguanidine ([3H]DTG, 52.3 Ci/mmol) using rat brain

LFCP2 and Krebs-Henseleit/Hepes buffer. Protein concentration was

determined by the method of Lowry et al. (1951).

Materials

All scintillation counting was performed with a Packard model 4450 scintillation

spectrometer. [3H]Myo-inositol was obtained from American Radiochemicals,

Inc. (St. Louis, MO.). [3H] (+)3-PPP, [3H]DTG, and [3H]Oxo-M were obtained

from Dupont/New England Nuclear (Boston, Mass.). Carbachol, norepinephrine,

haloperidol, polyethyleneimine, Tris, and Hepes were obtained from Sigma

Chemicals (St. Louis, MO.). (+)-3-PPP and oxotremorine-M (Oxo-M) were

obtained from Research Biochemicals, Inc. (Natick, Mass.). DTG was from

Aldrich Chemical Co. (Milwaukee, Wis.). Levallorphan was obtained from the

National Institute on Drug Abuse (NIDA) (Rockville, Md.). (+)-Benzomorphans

and (+)-morphinans were synthesized in the laboratory of Dr. Kenner C. Rice.

BD614 and BD737 are, respectively, (±)-cis- and 1S, 2R(-)-cis-N-

[2-(3,4-dichlorophenyl)ethyl]- N-methyl-2-(l-pyrrolidinyl)cyclohexylamine.

Synthesis and characterization of these highly selective ligands is described

elsewhere (Rice et al., in press). The following abbreviations are used for

compounds: DEX (dextrallorphan), DTG (1,3-di-o+tolylguanidine), FLU

(fluphenazine), HAL (haloperidol), KCR 12-83.1 ((+)-N-cyclopropylmethylnordihydrocodeinone), LEV (levallorphan), MDEX (3-methoxydextrallorphan),

(+)-PENT ((+)-pentazocine), (+)-3-PPP ((+)-3-(3-hydroxyphenyl)-N-(1-

propyl)piperidine), and Red. HAL (reduced haloperidol).

RESULTS

Characterization of Effect and Determination of Specificity

The muscarinic cholinergic agonists carbachol and oxotremorine-M (Oxo-M)

and the -adrenergic agonist norepinephrine stimulated the basal rate of PPI

turnover in rat brain synaptoneurosomes in which the PPI pool had been

radiolabeled by preincubation with [3H]myo-inositol. At a concentration of

100 μM, carbachol and norepinephrine produced approximately a doubling of

the basal activity. Oxo-M at 10 μM produced 150 percent to 170 percent

stimulation of basal activity. The carbachol stimulation was completely blocked

72
by the competitive cholinergic antagonist atropine (100 μM). The

norepinephrine stimulation was completely blocked by the adrenergic

antagonist phenoxybenzamine (30 μM). These results are similar to those

observed with brain slices and confirm that synaptoneurosomes are a valid

system for the study of PPI turnover.

The effect of ligands on basal PPI metabolism is investigated in figure 1. HAL,

DTG, (+)-PENT, and (+)-3-PPP had a negligible effect on PPI turnover at

concentrations up to 100 μM. Above 100 μM, DTG and (+)-PENT produced a

slight inhibition of basal activity and reached only 20 percent inhibition at

1,000 μM. HAL, however, produced marked inhibition of basal activity at

concentrations above 100 μM. (+)-3-PPP produced no significant effect on

FIGURE 1. Effect of ligands on phosphoinositide turnover. Various

concentrations of the indicated ligands were incubated with

prelabeled rat brain synaptoneurosomes and production of [3H]IP1

measured as described in the methods section. Values are

expressed as percentage of basal activity and are the mean of

two to four experiments ± SEM. Each experiment was performed

in duplicate.

73
basal activity at concentrations up to 1,000 μM. (+)-Morphine, which lacks

affinity for receptors, was without effect on basal activity (not shown).

Figure 2 shows the effect of four representative ligands on the ability of two

cholinergic agonists, carbachol (panel A) and Oxo-M (panel B), to stimulate PPI

metabolism in rat brain synaptoneurosomes. (+)-PENT, HAL, DTG, and

dextrallorphan all produced concentration-dependent attenuation of the PPI

response. Dose curves were linear with least-squares-derived coefficients of

greater than 0.95. ED50 values were calculated from the least squares fits and

are shown in table 1 along with those of other ligands. The compounds are

from five different chemical classes, and all produced linear dose curves with

least squares coefficients of greater than 0.95. It is noteworthy that inhibition of

both carbachol- and Oxo-M-stimulated PPI turnover occurred at concentrations

of ligand below those required to produce significant effects on basal activity.

TABLE 1. Comparison of binding affinity and potency at inhibition of

cholinergic phosphoinositide turnover

Ligand

Ki (nM)

[

3H] (+)-3-PPP

ED50

Oxo-M

(μM)

Carbachol

BD 737 1.3± 0.3 3.1 ± 0.2 ND

BD 614 2.0± 0.4 4.7 ± 0.8 ND

(+)-Pentazocine 3.1 ± 0.3 7.2 ± 0.9 8.3

Haloperidol 3.8 ± 0.6 9.6 ± 0.8 35.4

Reduced haloperidol 5.1 ± 2.3 15.6 ± 0.6 ND

Dextrallorphan 16.1 ± 1.8 22.5 ± 1.0 27.0

DTG 20.5 ± 3.6 12.0 ± 2 25.0

Fluphenazine 26.3 ± 0.6 36.0 ± 1 ND

Methoxy-dextrallorphan 32.1 ± 8.9 15.4 ± 0.3 11.9

KCR 12-83.1 138.4 ± 15 147.0 ± 24 81.5

Levallorphan 7,540 ND 155.0

NOTE: Binding affinities of ligands were determined using the LFCP2 fraction of guinea pig brain

by competition of 0.05 to 10,000 nM test ligand with 3 nM [3H](+)-3-PPP. Assay conditions were as

described in the methods section. IC50 values were determined using the iterative curve-fitting

program CDATA (EMF Software, Baltimore, Md.), and Ki values were calculated using the

Cheng-Prusoff equation and a KD of 27.4 nM (Cheng and Prusoff 1973). ED50 values in the PPI

assays were determined from linear least squares fits of the dose curves for each compound.

Values for Oxo-M (10 μM) were determined from the individual least squares fits of data from two to

four experiments and are expressed as averages, μ SEM. Values for carbachol (100 μM) were

determined from a least squares fit of the averaged data from two or three experiments that varied

by less than 10 percent. All experiments were carried out in duplicate. ND = not determined.

74
FIGURE 2. Effect of representative ligands on the cholinergic

phosphoinositide response. Various concentrations of ligands

were incubated for 90 minutes with prelabeled

synaptoneurosomes in the presence of 100 μM carbachol

(panel A) or 10 μM Oxo-M (panel B). Values are expressed as the

percentage of the stimulation produced by cholinergic agonist in

the absence of a ligand and are the averages of two to four

experiments ± SEM. Each experiment was carried out in duplicate.
Binding affinities of various ligands to sites of guinea pig brain, determined by

competition with [3H](+)-3-PPP, are shown in table 1. Figure 3 shows the

correlation between binding affinities (Ki) and ED50 values at blocking

carbachol-stimulated (panel A) or Oxo-M-stimulated (panel B) PPI turnover in

rat brain synaptoneurosomes. Although the ED50 values for PPI inhibition fall in

the micromolar concentration range, whereas Ki values are in the nanomolar

range, the rank order of the compounds in the two assays is very similar. The

correlation coefficient for carbachol (7 compounds) was 0.81, while that for

Oxo-M (10 compounds) was 0.92. The high correlation between rank order of

affinities at sites and ED50, rank order suggests that attenuation of the

cholinergic PPI response is mediated by receptors.

Of particular note is the stereoselectivity between dextrallorphan and

levallorphan. Also, several (+)-opiates are structurally related to (+)-PENT and

dextrallorphan but lack affinity for receptors, exhibiting IC50 values versus

[

3H](+)-3-PPP of greater than 10,000 nM in guinea pig brain homogenates.

Compounds in this class are (+)-morphine, (+)-oxymorphone, (+)-naltrexone,

(+)-dihydrocodeinone, and (+)-nordihydrocodeinone (unpublished

observations). These compounds lacked ability to attenuate carbacholstimulated PPI turnover at concentrations up to 300 μM (Bowen et al. 1988a).

Sulpiride, like HAL, is an antipsychotic dopamine-D2 receptor antagonist.

However, unlike HAL, sulpiride lacks affinity for receptors and failed to

produce effects on carbachol-stimulated PPI metabolism at concentrations up

to 300 μM (Bowen et al. 1988a).

The only compound tested that gave anomalous results was (+)-3-PPP. As

shown in figure 4, this compound produced an effect that was markedly

different from that of the other ligands. (+)-3-PPP produced

concentration-dependent attenuation of the carbachol effect between 10 and

100 μM. However, at concentrations above 100 μM, no additional inhibition was

observed. A plateau was reached at about a 40 percent inhibition of the

carbachol stimulation. Little additional inhibition was observed at concentrations

as high as 1,000 μM. Other ligands, even those with lower binding affinity,

were able to produce complete inhibition of carbachol-stimulated PPI turnover.

The inability to produce a maximal effect suggests that (+)-3-PPP may have

partial agonist or antagonist properties in this system.

Figure 5 shows the effects of DTG and (+)-pentazocine, two highly selective

ligands (Weber et al. 1986; de Costa et al. 1989), on the ability of

norepinephrine to stimulate PPI hydrolysis. Like observations with cholinergic
FIGURE 3. Correlation of binding affinity to potency at inhibiting the

cholinergic PPI response. Data from table 7 were used to

construct correlation plots for binding Ki vs. inhibitory ED50

against carbachol- (panel A) or Oxo-M- (panel B) stimulated PPI

turnover. Correlation coefficients were r = 0.81 for carbachol and

r = 0.92 for Oxo-M.

77
agonists, the ability of norepinephrine to stimulate PPI metabolism was

attenuated in a concentration-dependent manner by these ligands. Consistent

with binding affinity, (+) PENT was more potent than DTG, with ED50 values

of 129 μM and 354 μM, respectively. However, compared with cholinergic

stimulation, adrenergic stimulation was less sensitive to inhibition by ligands.

The ED50 values for (+)-PENT and DTG were about 15 times higher for

norepinephrine stimulation than for carbachol stimulation. ED50 values for

(+)-PENT and DTG were, respectively, 18 and 30 times higher for

norepinephrine than for Oxo-M. (+)-Morphine and (+)-naltrexone, (+)-opiates

that lack affinity for receptors, failed to inhibit norepinephtine-stimulated PPI

turnover at concentrations up to 1,000 μM (not shown). These results suggest

that this effect on the adrenergic phosphositide response is also mediated by

receptors.

FIGURE 4. Dose curve for (+)-3-PPP inhibition of carbachol-stimulated PPI

metabolism. The indicated concentrations of (+)-3-PPP were

incubated with prelabeled synaptoneurosomes and 100 μM

carbachol. Values are expressed as the percentage of stimulation

observed with carbachol alone. Results are the average of two

experiments, ± SEM, each carried out in duplicate.

78
We investigated whether the inhibition by ligands of agonist-stimulated PPI

metabolism was competitive or noncompetitive by determining the effect of a

fixed concentration of ligand on the dose response of Oxo-M or

norepinephrine. These results are shown in figure 6. In panel A, the

concentration of Oxo-M was varied up to 1,000 μM in the presence and

absence of fixed concentrations of (+)-PENT. Oxo-M alone produced

dose-dependent stimulation of [3H]IP1 production; stimulation was greatest at

about 315 percent above basal. At 10 μM, (+)-PENT had little effect on the

concentration of Oxo-M required to produce half-maximal stimulation of PPI

turnover but reduced the maximal stimulation, even at large excess of agonist.

A significant rightward shift was observed with 100 μM (+)-PENT. However, the

largest effect was a decrease in maximal activity.

FIGURE 5. Effect of ligands on norepinephrine-stimulated PPI turnover.

Various concentrations of (+)-PENT or DTG were added to

prelabeled synaptoneurosomes prior to addition of 100 μM

norepinephrine as described in the methods section. Values are

expressed as a percentage of the stimulation of [3H]IP1 production

produced by norepinephrine alone. Results are the mean of two to

three experiments ± SEM. Each experiment was carried out in

duplicate.

79
FIGURE 6. Dose curves for oxotremorine-M- and norepinephrine-stimulated

PPI turnover in the absence and presence of (+)-PENT Various

concentrations of Oxo-M (panel A) or norepinephrine (panel B)

were incubated with prelabeled synaptoneurosomes in the

absence and presence of the indicated concentration of

(+)-PENT. Values are expressed as the percentage of stimulation

of [3H]IP1 production above basal activity and are the averages of

two to four experiments ± SEM. Each experiment was carried out

in duplicate.

80
Panel B shows results obtained with norepinephrine-stimulated PPI turnover

and (+)-PENT. With norepinephrine alone, maximal stimulation was 84 percent

above basal [3H]IP1 production, with half-maximal stimulation occurring at

approximately 60 μM norepinephrine. At high norepinephrine concentrations,

the amount of stimulation decreased, producing a bell-shaped curve. The

presence of 150 μM (+)-PENT had no effect on the concentration of

norepinephrine required to produce half-maximal stimulation but reduced the

maximal stimulation to 52 percent above basal. The bell-shaped nature of the

dose curve was still evident in the presence of (+)-PENT, and the inhibition

produced by 150 μM (+)-PENT was not overcome by up to 3,000 μM

norepinephrine. Thus, ligands appear to possess the ability to

noncompetitively inhibit the PPI response produced by muscarinic cholinergic

and -adrenergic agonists, reducing the maximal effect while not affecting the

ED50 of the agonists. That ligands produce noncompetitive inhibition of

cholinergic and adrenergic PPI turnover is consistent with mediation of the

effect through specific interaction with receptors.

Investigation of Mechanism

Because of the differential sensitivity of cholinergic and adrenergic PPI turnover

to inhibition and lack of effect at others points in the inositol lipid pathway

(Bowen et al. 1989b), we investigated the possible involvement of cholinergic

receptors in the mechanism of the response.

Using lysed, frozen crude P2 fraction of rat brain (LFCP2), we investigated the

ability of various ligands to compete for muscarinic binding sites labeled by

[

3H]Oxo-M. These results are shown in table 2. All of the active ligands were

found to exhibit weak affinity for muscarinic receptors as measured by inhibition

of [3H]Oxo-M binding. These affinities were greater than an order of magnitude

lower than affinities at receptors (table 1). Surprisingly, as shown in figure 7,

the potency to inhibit Oxo-M-stimulated PPI turnover correlated very well with

ability to inhibit [3H]Oxo-M binding (r = 0.92). This correlation was equal to the

correlation of rank order of potency with rank order in receptor affinity

(figure 3, panel B). These correlations suggested the possibility that the effect

could be mediated by either receptor activation or by a direct antagonist

action of ligands at the cholinergic receptor. Therefore, a series of

experiments was carried out to eliminate one or the other receptor as the

mediator of the effect.

81
FIGURE 7. Correlation of muscarinic binding affinity to potency a? inhibiting

oxotremorine-M-stimulated phosphoinositide turnover. The ability

of various ligands to displace [3H]Oxo-M was correlated with

potency at inhibiting Oxo-M-stimulated PPI turnover (table 7). The

correlation coefficient was r = 0.92.

The first experiments were aimed at determining whether the effect would be

evident under conditions where there is no occupation of muscarinic receptors

by ligand. The rationale is as follows. If synaptoneurosomes were pretreated

with (+)-PENT and then free ligands were removed, the receptor should

remain occupied, but any (+)-PENT bound to muscarinic receptors should

rapidly dissociate because of its low binding affinity. Upon subsequent

challenge with Oxo-M, stimulation would be inhibited if the effect is via sites

but not if (+)-PENT acts as a direct antagonist at cholinergic sites.

Intact synaptoneurosomes were labeled with [3H]myo-inositol and then

pretreated for 30 minutes with 50 μM (+)-PENT under the normal assay

conditions. The preparation was then centrifuged and the synaptoneurosome

pellet resuspended in the same medium except without (+)-PENT. The

preparation was then challenged with 10 μM Oxo-M to assess ability to

stimulate PPI turnover. Table 3 shows that, compared with synaptoneurosomes

pretreated with no ligand, the PPI response was inhibited in the (+)-PENT

pretreated samples. Stimulation was inhibited by 46 percent and 55 percent

during 15 and 90 minutes of incubation with Oxo-M, respectively.

82
TABLE 2. Affinity of various ligands for muscarinic receptors

Muscarinic IC50 (μM)

Ligand [

3H]Oxo-M

BD 737 5.9 ± 0.3

BD 614 4.8 ± 0.1

(+)-Pentazocine 5.6 ± 0.4

Haloperidol 7.5 ± 0.9

Reduced haloperidol 20±2

Dextrallorphan 37±2

DTG 12±2

Fluphenazine 58±5

Methoxy-dextrallorphan 31±6

KCR 12-83.1 78±14

NOTE: Concentrations of test iigand ranging from 0.1 to 500 μM were incubated with 5 nM

[

3H]Oxo-M and LFCP2 from rat brain as described in the methods section. IC50 values were

determined as in the caption to table 1. Values are averages of two to four experiments ± SEM.

Each experiment was carried out in duplicate.

TABLE 3. Effect of (+)-pentazocine pretreatment on stimulation of PPI

turnover by oxotremorine-M

Incubation time

Percent above basal activity with 10 μM Oxo-M

50 μM (+)-PENT

Control pretreated

15 minutes 69±6 37 ± 1

90 minutes 188±8 83±10

NOTE: Intact synaptoneurosomes were labeled with [

3H]myo-inositol and then pretreated for

30 minutes with 50 μM (+)-pentazocine under the normal assay conditions described in the

methods section. The preparation was then centrifuged and the synaptoneurosome pellet

resuspended in the same medium, except without (+)-PENT. The preparation was then challenged

for 15 or 90 minutes with 10 μM Oxo-M to assess ability to stimulate PPI turnover. Values are

expressed as percentage increases above basal activity. Results are averages of 2 experiments,

± SEM. Each experiment was carried out in duplicate.

In order to confirm that (+)-PENT did not occupy muscarinic receptors under the

conditions of this experiment, rat brain LFCP2 was again employed. LFCP2 was

incubated with 50 μM (+)-PENT. After centrifugation and resuspension in fresh

83
buffer, the degree of occupation of both muscarinic (5 nM [3H]Oxo-M) and

(5 nM [3H]DTG) was determined. Results are shown in table 4. Compared with

controls pretreated with no ligand, only 41 percent of the control [3H]DTG

binding remained after pretreatment, whereas all of the control [3H]Oxo-M

binding was recovered. Taken with the results described above, these data

demonstrate that (+)-PENT can inhibit Oxo-M-stimulated PPI turnover without

occupying the muscarinic receptor. The effect is therefore mediated solely by

occupation of receptors.

TABLE 4. Effect of (+)-pentazocine pretreatment on occupation of muscarinic

and receptors

Pretreatment condition

[

3H] Ligand bound (fmol/mg protein)

Muscarinic

5 nM [3H]Oxo-M 5 nM [3H]DTG

Control

50 μM (+)-PENT

47.1 ±8.5 58.9 ± 6.8

45.6 ± 5.6 23.9 ± 1.5

(96.8 ± 11.9 percent) (40.6 ± 2.5 percent)

NOTE: LFCP2 of rat brain was incubated with 50 μM (+)-PENT for 30 minutes at 37°C in

Krebs-Henselet/Hepes buffer. After centrifugation and resuspension In fresh buffer without

(+)-PENT, the degree of occupation of both muscarinic and receptors was determined. Muscarinic

and receptors were labeled with 5 nM [

3H]Oxo-M and [3H]DTG, respectively, using

Krebs-Henseleit/Hepes buffer as described In the methods section. Values for bound ligand

(fmol/mg protein) are the averages of three experiments, ± SEM. Each experiment was carried out

in triplicate. Numbers in parentheses are the percentages of recovery of control binding for the

respective [

3H] ligands.

Other data also serve to rule out direct binding to muscarinic receptors as the

mechanism for action. Scatchard analysis of [3H]Oxo-M binding to

synaptoneurosomes revealed labeling of an apparent single site with KD =

39 nM (see figure 8). The apparent Ki of (+)-PENT at this muscarinic binding

site is 5.6 μM (table 2). In the normal protocol for measuring the effect of

ligands on muscarinic PPI turnover, Oxo-M and (+)-PENT are present

simultaneously. Oxo-M was routinely used at a concentration of 10 μM (250

times higher than its observed KD), and (+)-PENT was effective at

concentrations approximating its muscarinic Ki (ED50 = 7.2 μM). Taking into

account the concentrations of (+)-PENT and Oxo-M present, their respective

affinities at the muscarinic binding site, and the expected levels of receptor

saturation under these conditions, it is highly unlikely that there would be

significant occupation of muscarinic receptors by effective levels of (+)-PENT.

84
FIGURE 8. Effect of (+)-PENT pretreatment on muscarinic receptors of

synaptoneurosomes. Intact synaptoneurosomes were pretreated

with 50 μM (+)-PENT under the identical conditions of the PPI

inhibition assay except that [3H]myo-inositol was replaced by

unlabeled myo-inositol. The tissue was then centrifuged and the

synaptoneurosome pellet was resuspended in fresh medium

containing no (+)-PENT Scatchard analysis of [3H]Oxo-M binding

was performed using increasing concentrations of radioligand and

the conditions described in the methods section. Results were

compared to synaptoneurosomes pretreated with no ligand.

Data were analyzed using the iterative curve fitting program

BDATA (EMF Software, Baltimore, Md.). Values are the averages

of five experiments, ± SEM. Each experiment was carried out in

duplicate. Control: KD = 39 ± 4 nM, Bmax = 151 ± 10 fmol/mg

protein; (+)-PENT-pretreated: KD = 32± 6 nM, Bmax = 78 ± 8

fmol/mg protein.

Oxo-M should effectively compete away (+)-PENT and preferentially occupy the

receptor. This supposition is even more true in view of the inability of even

1,000 μM Oxo-M to overcome the inhibition by 10 μM (+)-PENT (figure 6). Also,

when Oxo-M was allowed to prebind to the muscarinic receptor before addition

of (+)-PENT, inhibition of stimulation was still observed (not shown). Taken

85
together, these results strongly suggest that ligands exert their effects through

occupation of receptors and not by direct interaction with the muscarinic receptor.

To further investigate the possible involvement of muscarinic receptors in the

mechanism of the effect, we again used a pretreatment protocol coupled with

Scatchard analysis. Intact synaptoneurosomes were pretreated with 50 μM

(+)-PENT under the identical conditions of the usual assay except that

[

3H]myo-inositol was replaced by unlabeled myo-inositol. The tissue was

centrifuged and the synaptoneurosome pellet was resuspended in fresh

medium containing no (+)-PENT. Scatchard analysis of [3H]Oxo-M binding was

then performed. Results were compared with synaptoneurosomes pretreated

with no ligand and are shown in figure 8. In untreated synaptoneurosomes

[

3H]Oxo-M bound to an apparent single class of sites with KD = 39 ± 4 nM and

Bmax = 151 ± 10 fmol/mg protein. Pretreatment of synaptoneurosomes with

50 μM (+)-PENT resulted in a 50 percent reduction in the Bmax of [3H]Oxo-M

with no effect on the KD.

This effect cannot be due to the presence of residual (+)-PENT occupying the

muscarinic receptor, because the experiments described above with LFCP2

show that there is negligible muscarinic binding of (+)-PENT after pretreatment

under similar conditions. Furthermore, Scatchard analysis of [3H]Oxo-M binding

to LFCP2 in the simultaneous presence of 5.6 μM (+)-PENT showed that

(+)-PENT was a competitive inhibitor, increasing KD and having no effect on the

number of sites (not shown). Unlike the direct effect of (+)-PENT on [3H]Oxo-M

binding to LFCP2, the effect observed upon pretreatment of synaptoneurosomes

is solely a Bmax effect.

DISCUSSION AND SUMMARY

Functional roles of receptors are now beginning to be demonstrated

(Campbell et al. 1987; Vaupel and Su 1987; Bowen et al. 1988a, 1988b;

Matsumoto and Walker 1988; Walker et al. 1988; Campbell et al. 1989;

Matsumoto et al. 1990). The finding that sites attenuate the cholinergic and

adrenergic PPI response may suggest this as an underlying biochemical

mechanism by which ligands produce their effects.

Ligands from various structural classes inhibited the stimulation of PPI

turnover by cholinergic agonists in our study. Despite disparity between the

actual values for binding affinity and ED50 for PPI inhibition, there was

excellent correlation in the rank order of potencies of compounds in the two

86
assays. There is apparent specificity of this effect for the cholinergic PPI

response. Adrenergic signaling through receptors was less sensitive than

was cholinergic signaling through muscarinic (presumably M1) receptors.

Adams and Weber (1989) have reported inhibition of the carbachol-stimulated

PPI response but not the response to bradykinin in NCB-20 cells. In addition,

our preliminary data suggest that histamine stimulation is much more sensitive

to inhibition than is serotonin stimulation (not shown). It remains to be seen

what other PPI-linked systems can be modulated by receptors.

Mechanism of Action

The differential sensitivity of cholinergic and adrenergic stimulation, coupled

with lack of effect on either inositol phosphate hydrolysis or synthesis of inositol

lipids (Bowen et al. 1989b), led us to suspect the cholinergic receptor as the

target of action. One possibility was competitive or noncompetitive

antagonism by direct binding of ligands to cholinergic receptors. All the active

ligands did exhibit weak binding affinity to cholinergic receptors. Cholinergic

binding affinities were in the same concentration range as the ED50

concentration, and the correlation of inhibitory potency with cholinergic affinity

was the same as that with binding affinity (though it should be noted that poor

correlation was observed when [3H]quinuclidinyl benzilate was used as

cholinergic ligand instead of [3H]Oxo-M (Bowen et al. 1989b)).

However, several experiments ruled out direct antagonism of the cholinergic

response (i.e., a direct anticholinergic effect). The strongest evidence is that

(+)-PENT inhibition of Oxo-M-stimulated PPI turnover could be observed under

conditions where there was no occupation of [3H]Oxo-M binding sites. Also,

based on the high binding affinity of Oxo-M to muscarinic receptors and the

high concentration used to stimulate the response, there should be little

occupation of muscarinic receptors by ligands under the normal conditions of

the assay (both ligands present together). The noncompetitive nature of the

effect also argues against competitive antagonism at the receptor.

However, the cholinergic receptor does appear to be involved in the mechanism

of the effect. When intact synaptoneurosomes were pretreated with (+)-PENT,

a marked decrease in the number of [3H]Oxo-M binding sites was observed.

There was no change in the binding affinity. Thus, activation of receptors

causes a down-regulation of cholinergic receptors. This down-regulation

occurred in as little as 15 minutes. A mechanism involving decreased receptor

number is consistent with the observed o-induced decrease in maximal

87
response without change in the ED50 of the cholinergic agonist (figure 6). Also,

the decrease in Oxo-M-induced stimulation of PPI turnover as shown in table 3

was equivalent to the magnitude of the Bmax decrease shown in figure 8. These

observations are consistent with results of studies of the effect of cholinergic

receptor loss due to site-directed alkylation. In slices from cerebral cortex and

hippocampus, propylbenzilylcholine mustard was shown to cause a decrease in

carbachol-stimulated PPI metabolism that was equivalent to the loss of

receptors, without a change in the ED50 (Fisher and Snider 1987).

Figure 9 shows schematically the a-induced heterologous desensitization of the

cholinergic PPI response. Activation of receptors produces a signal,” which

causes a subsequent decrease in the number of cholinergic receptors and a

resultant desensitization of the cholinergic response. Preincubation with

(+)-PENT produced no effect on cholinergic binding when a preparation of

membrane fragments (LFCP2) was used instead of synaptoneurosomes (table

4). The difference between results observed with membrane fragments versus

synaptoneurosomes shows that an intact system is required in order to produce

the effect. This might suggest the involvement of ion gradients, soluble

enzymes or factors, intracellular compartments, or components sensitive to

freezing and thawing in the signal” that mediates the inhibitory effect. In

addition, because binding of some ligands is inhibited by guanine nucleotides

(Itzhak and Khouri 1988) it is possible that G-proteins are also involved in the

mechanism.

Though the effect does not appear to involve direct interaction of ligands

with the [3H]Oxo-M binding site, the high correlation of potency with the weak

muscarinic binding affinity cannot be ignored. This correlation is most likely a

consequence of the close relationship of rank order of affinity for compounds at

receptors and rank order at cholinergic receptors. For the 10 compounds

shown in tables 1 and 2, the correlation of Ki versus [3H]Oxo-M lC50 gave a

coefficient of r = 0.89 (graph not shown). Thus, although these compounds bind

to muscarinic receptors with much lower affinity than to sites, the rank order

of affinity is nearly the same. The explanation for this is not clear, and this

phenomenon deserves further investigation. However, it may suggest that the

topography of the muscarinic receptor resembles that of the receptor in some

way. Whether this effect has anything to do with the mechanism of action of

ligands remains to be demonstrated.

88
FIGURE 9. Schematic diagram of the effect of ligands on the cholinergic

PPI response.

Agonist vs. Antagonist Actions

Due to the current lack of functional assays for sites and lack of identified

endogenous ligands, it has been difficult to determine which compounds are

89
agonists and which compounds are antagonists at sites. An important

observation from this study is that all of the ligands tested produced effects in

the same direction. All produced inhibition of the PPI response. Neuroleptics

such as HAL and FLU produced the same effect as dextrorotary opiates such

as (+)-PENT and dextrallorphan. Similar observations were made in other

functional assays (Walker et al. 1988; Campbell et al. 1989; Matsumoto et al.

1990). Thus, there does not appear to be a clear agonist-antagonist relationship

between these classes of compounds.

Chronic HAL has been shown to cause down regulation of a receptors (Itzhak

and Alerhand 1989; Matsumoto et al. 1989), suggesting that HAL is an agonist

at sites. Because the other compounds behaved like HAL in the current study,

it is suggested that inhibition of the PPI response represents an agonist action

at receptors. Also, it is difficult to visualize how antagonism at sites would

cause inhibition of the PPI response, for one would then have to invoke a

requirement of agonism for efficient PPI signaling.

If other compounds are in fact agonists, then (+)-3-PPP could be

characterized as a partial agonist. It was the only ligand that failed to produce

complete inhibition of agonist-stimulated PPI turnover. (+)-3-PPP has been

shown to exhibit anomalous activity or lack of efficacy in other systems that

show biological functions of receptors (Campbell et al. 1989; Matsumoto et al.

1990). Further studies will be necessary to determine the agonist and

antagonist nature of compounds.

CONCLUSION

Attenuation of signaling via the PPI pathway is a potential mechanism by which

ligands could modulate the functions of neurotransmitters in vivo. Receptors

appear to be one of a growing number of receptors postulated to utilize a

variety of mechanisms to effect negative modulation of PPI turnover (Linden

and Delahunty 1989). Further studies will be aimed at the in vivo interactions of

neurotransmitters and ligands as well as further elucidation of the mechanism

of the effect. Furthermore, inhibition of the cholinergic PPI response provides

a convenient bioassay system for use in development and characterization of

novel agonists and antagonists.

90
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3H](+)-pentazocine, a highly potent and selective radioligand for sigma

receptors. FEBS Lett 251:53-58, 1989.

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Fisher, S.K., and Snider, R.M. Differential receptor occupancy requirements for

muscarinic cholinergic stimulation of inositol lipid hydrolysis in brain and in

neuroblastomas. Mol Pharmacol 32:81-90, 1987.

Gusovsky, F., and Daly, J.W. Formation of inositol phosphates in

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Sonders, M.S.; Keana, J.F.W.; and Weber, E. Phencyclidine and

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ACKNOWLEDGMENTS

This work was supported by National Institute of Neurological and

Communicative Disorders grant NS-26746 (WDB), NIDA grant DA-04988

(WDB), and funds from the Dystonia Medical Research Foundation.

AUTHORS

Wayne D. Bowen, Ph.D.

Paul J. Tolentino

Brian N. Kirschner

Paul Varghese

Section of Biochemistry

Division of Biology and Medicine

Box G-J263

Brown University

Providence, RI 02912

Brian R. de Costa, Ph.D.

Kenner C. Rice, Ph.D.

Laboratory of Medicinal Chemistry

National Institute of Diabetes and Digestive and Kidney Diseases

National Institutes of Health

Bethesda, MD 20892

93

and Phencyclidine Receptors in the

Brain-Endocrine-Immune Axis

Seth A. Wolfe, Jr., and Errol B. De Souza

INTRODUCTION

Recently there has been growing awareness of the existence of functional links

among the nervous, endocrine, and immune systems. Clinical studies have

associated affective disorders and psychological states such as bereavement,

depression, and anxiety with alterations in immune function in humans

(reviewed in Stein et al. 1985; Tecoma and Huey 1985). In animal studies,

immune modulation has been brought about by experimentally induced stress

(reviewed in Croiset et al. 1987; Stein et al. 1985) neural lesions (reviewed in

Rozman et al. 1985), and classical Pavlovian conditioning (Ader et al. 1983).

Candidates for mediators of these immunologic effects include a growing list of

neurotransmitters, neuroendocrine peptides, neurotropic factors, and their

receptors in lymphoid cells (reviewed in Blalock 1989). These substances may

be delivered via the bloodstream or secreted by nerves within target tissues.

Primary sensory afferents in lymphoid tissues have the potential to release

transmitter substances upon antidromic stimulation (Payan and Goetzl 1985)

and these tissues also contain sympathetic nerve fibers that establish

synaptic-like contacts with lymphocytes and macrophages (D.L. Felten 1987;

S.Y. Felten and Olschowka 1987).

There is also compelling evidence that the immune system modulates central

nervous system (CNS) and endocrine functions. Immune responses and

immune-cell products have been reported to produce changes in behavior,

hormone release, neurotransmitter turnover, and neuronal electrical activity

(reviewed in Besedovsky et al. 1986; Blalock 1989). Lymphokines and

monokines, such as interferon, interieukin-1 (IL-1), and thymosin, have

neurotropic effects (reviewed in Besedovsky et al. 1985) and immune-cell

products have been identified in the CNS (Breder et al. 1988). Leukocyte-cell

surface markers and specific membrane receptors for immune-cell products

95
have also been identified in the CNS and endocrine tissues (reviewed in

Blalock 1989). Finally, neuroendocrine peptides are produced by cells of the

immune system (reviewed in Blalock 1989). Thus, the nervous, endocrine, and

immune systems share common transmitter/communication molecules, are

physically and chemically interconnected, and they interact extensively.

Our interest, and the subject of this chapter, is receptors mediating the action of

phencyclidine drugs on the CNS-endocrine-immune axis. In humans and

mice, phencyclidine (PCP) and PCP analogs have been reported to depress a

variety of in vitro indices of immune function, such as [3H]thymidine and

[

3H]-2-deoxy-d-glucose uptake, mitogen-driven antibody production, and

lipopolysaccharide-driven IL-l production (Dornand et al. 1987; Khansari et al.

1984). Preliminary data suggest the presence of [3H]PCP binding sites on

human lymphocytes (Fudenberg et al. 1984), and PCP and Nallylnormetazocine

(SKF 10,047) have also been reported to alter neuroendocrine function. Both of

these compounds stimulate hypothalamic-pituitary-adrenocortical secretion

(Boggan et al. 1982; Pechnick et al. 1985) and suppress luteinizing hormone

(LH) (Pechnick et al. 1985) and prolactin (Pechnick et al. 1985; Saller et al.

1982) secretion in rats. d-[3H]SKF 10,047 binding sites have been

demonstrated in the anterior pituitary (Tam 1983) and PCP and SKF 10,047

also bind to cultured pituitary cells and alter LH release in vitro (Stojilkovic et al.

1987).

and PCP receptors in brain have been well characterized by others, most

notably those participating in this workshop. Because PCP and SKF 10,047

bind with high affinity to both PCP and receptors in brain (Largent et al. 1986;

Tam 1985) the identity of the receptor(s) mediating the immunologic and

endocrine effects of these compounds is unknown. Furthermore, it is unclear

whether the endocrine and immune effects of PCP and SKF 10,047 are

mediated in brain or through direct actions at the target endocrine organs or

lymphoid tissue. This chapter will focus on data from our recent studies on the

identification, characterization, and localization of these receptors in immune

and endocrine tissues. The characteristics of receptors in human peripheral

blood leukocytes (HPBLs) and rat pituitary, adrenal, testis, and ovary will be

compared with those of the well-characterized and PCP receptors in rat brain.

In these studies, these selective ligands were used in order to identify and

characterize and PCP receptors in peripheral tissues: [3H]haloperidol (Tam

and Cook 1984) 1,3-di(2-[5-3H]tolyl)guanidine ([3H]DTG) (Weber et al. 1986)

and d-3-(3-hydroxyphenyl)- N-(1-propyl)-2,3-[3H]piperidine ([3H]-3-PPP) (Largent

et al. 1986) to label receptors; and 3,4-[3H]-(N)-[1-(2-thienyl)cyclohexyl]-

96
piperidine ([3H]TCP) (Vignon et al. 1983) and d-[3H]5-methyl-10,11-dihydro5H-dibenzo-[a,d]+cyclohepten-5,10-imine ([3H]MK-801) (Bowery et al. 1988) to

label PCP receptors.

ABSENCE OF PCP RECEPTORS IN ENDOCRINE AND LYMPHOID TISSUES

Homogenate Binding Studies

In rat brain homogenates (minus the cerebellum), [3H]TCP binding was

saturable over the concentration range 2 to 100 nM and exhibited the expected

high affinity KD (mean ± SEM, n = 5) of 6.7 ± 0.9 nM, Bmax of 1233 ± 189

fmol/mg protein, and Hill coefficient value of 1.0 ± 0.1 (table 1). In contrast, in

the endocrine glands [3H]TCP binding was not saturable over this concentration

range, nor was high-affinity binding of [3H]TCP detected. Apparent KD values

ranged from 28 to 211 nM for pituitary, adrenal, testis, and ovary (table 1).

HPBLs were similar to rat endocrine tissues in this regard. In HPBLs, [3H]TCP

binding was not saturable within the concentration range of 2 to 80 nM, and

Scatchard analysis revealed a binding site with an approximate KD value (mean

± SEM, n = 3) of 62 ± 19 nM. This affinity of binding in HPBLs and rat endocrine

organs was not consistent with high-affinity PCP receptors and was kinetically

comparable to that seen in cerebellum (KD = 57 ± 10 nM, n = 3).

TABLE 1. [

3H]TCP binding in tissue homogenates

Tissue

Rat brain

Rat cerebellum

Rat pituitary

Rat adrenal

Rat testis

Rat ovary

Human PBLs

6.7 ± 0.9

57±10

110±31

211 ± 106

111 ±4

28±5

62±-19

Bmax

(fmol/mg-protein)

1,233 ± 189

1,399 ± 354

1,421 ± 17

2,606 ± 1 ,192

7,010 ± 155

978±158

1,818 ±293

NOTE: There was an absence of high-affinity PCP receptors in human PBL and rat endocrine

tissues. [3H]TCP saturation binding (2 to 80 nM for human PBL and 2 to 100 nM for other tissues)

was carried out in tissue homogenates as previously described. Nonspecific binding was defined in

parallel incubations containing 20 mM nonradioactive PCP. Data from Wolfe et al. 1988, 1989.

97
98
Autoradiography

In homogenate binding studies, a small number of high-affinity receptors would

not be detectable in the presence of a high density of lower affinity binding

sites, such as were seen in the above experiments. However, if high-affinity

sites were anatomically clustered, they might be revealed by in vitro receptor

autoradiography. Therefore, autoradiographic experiments were carried out in

the rat endocrine system using [3H]TCP to label PCP receptors. The most

notable feature of the resultant autoradiograms of pituitary (figure 1 D-F),

adrenal (figure 1G-I), testis (figure 1 J-L), and ovary (figure 1M-O) was the

absence of [3H]TCP-labeled PCP receptors under conditions that caused

appropriate, specific labeling of PCP receptors in the cerebral cortex and

Ammon’s horn-dentate gyrus regions of brain (figure 1A-C). Furthermore, the

pattern and intensity of nonspecific binding were identical to total binding,

indicating that no specific labeling of PCP receptors occurred in endocrine

tissues. Therefore, these autoradiographic studies confirmed the results of the

preceding homogenate binding experiments and demonstrated the absence of

high-affinity PCP receptors in pituitary, adrenal, testis, and ovary.

FIGURE 1. Autoradiographic localization of [3H]TCP-labeled PCP receptors in

rat brain and endocrine organs. Each horizontal row shows: left

(TOLLUIDINE BLUE) - brightfieldphotograph of toluidine

blue-stained section showing tissue histology; center (TOTAL) -

negative enlargement (similar appearance to darkfield

photograph) of [3H]Ultrofilm autoradiographic image of the same

section, showing the “total” distribution of silver grains (light areas)

indicating regions of [3H]TCP binding; and, right (NONSPECIFIC)

- autoradiogram of adjacent tissue section, under identical

incubation, exposure and photographic conditions, in which

specific [3H]TCP binding was blocked by the addition of 200 μM

PCP. In saggital sections of brain (B), the cerebral cortex (cc) and

Ammon’s horn - dentate gyrus (dg) regions had the highest

densities of [3H]TCP binding sites; this binding was blocked by

PCP (C), indicating the presence of specific PCP receptors in

these areas. In contrast, little or no [3H]TCP binding was seen in

pituitary (E), adrenal (H), testis (K) or ovary (N), and an identical

pattern was seen in the presence of excess PCP (F, I, L, and O,

respectively), indicating the absence of specific PCP receptors in

these sections. Bars = 4 mm. From Wolfe et al. 7989.

99
We sought to corroborate these findings using the novel PCP-receptor-selective

radioligand [3H]MK-801 (Bowery et al. 1988). [3H]MK-801 did produce a high

level of background labeling in endocrine tissues, but it was nonspecific,

because it was not blocked by the presence of 200 μM PCP (data not shown).

In all other respects, [3H]MK-801 autoradiograms were in agreement with those

produced by [3H]TCP. Specific binding of [3H]MK-801 was not seen in rat

pituitary, adrenal, testis, or ovary, although the conditions employed were

adequate to produce a pattern of specific labeling in the brain that was

comparable in intensity and distribution with the pattern that was obtained by

using [3H]TCP (data not shown).

Discussion

In these studies, we have used selective probes to label PCP receptors in

immunologic and endocrine tissues. In binding studies using washed

membranes from tissue homogenates, the PCP receptor that binds with high

affinity to [3H]TCP (Vignon et al. 1986) appeared to be either absent or present

at undetectably low levels in HPBLs and in rat pituitary, adrenal, testis, and

ovary. The nonsaturable binding of [3H]TCP that we observed in HPBLs, rat

cerebellum, and rat endocrine tissues had an affinity that was similar to the

lower affinity [3H]TCP binding site (KD value 50 to 80 nM) that has been

described in rat hindbrain and spinal cord (Vignon et al. 1986) and in

synaptosomal membranes of rat hippocampus (Haring et al. 1987). This lower

affinity [3H]TCP binding site remains to be characterized, but it is not thought to

be the traditional PCP receptor. The absence of PCP receptors in the endocrine

organs was confirmed by autoradiographic studies in slide-mounted sections

using the selective PCP receptor ligands [3H]TCP and [3H]MK-801.

PRESENCE OF RECEPTORS IN HUMAN PBL AND RAT ENDOCRINE

ORGANS

Homogenate Binding Studies

In homogenate binding studies, [3H]haloperidol was used to label receptors.

In these experiments [3H]haloperidol was prevented from binding to D2

dopamine and 5HT2 serotonin receptors by inclusion of excess nonradioactive

spiperone in all incubations. Under the conditions employed (Wolfe et al. 1988,

1989) [3H]haloperidol bound to membrane homogenates of HPBLs and rat

pituitary, adrenal, testis, and ovary in a specific, concentration-dependent

manner (representative experiment shown in figure 2). Specific binding was

100
FIGURE 2. Saturability and high affinity of [3H]haloperidol binding to human

PBL membranes. A. Representative saturation experiment

showing: total binding of [3H]haloperidol in the

presence of 25 nM spiperone; nonspecific binding in

the presence of 25 nM spiperone plus 30 mM I-butaclamol; and

specific (- - n - -) binding (the difference between total and

nonspecific) over a concentration range of 0.2 - 10 nM

[

3H]haloperidol. B. Scatchard plot of specific [3H]haloperidol

binding in the same experiment. Saturability of specific

[

3H]haloperidol binding is demonstrated in A, while the affinity of

binding (KD = 0.44 ± 0.10 nM), density of binding sites (Bmax = 752

± 737 fmol/mg protein), and fit of the points to a straight line (corr.

coef. = 0.99) is demonstrated in B. From Wolfe et al. 1988.

saturable, and Scatchard analysis revealed high-affinity binding, with similar KD

values of 0.4 nM in HPBLs, and 1.9 to 2.8 nM in endocrine tissues. These

[

3H]haloperidol binding sites were comparable to [3H]haloperidol-labeled sites in

rat cerebellum (KD = 0.5 to 1.02 nM), where a high density of receptors is

known to be present (Largent et al. 1986). In all of the tissues tested, the best fit

was obtained with a single-site model of [3H]haloperidol binding, and Hill

coefficients approached a value of unity (data not shown). The most striking

feature of these experiments was that the density of [3H]haloperidol binding

sites in HPBLs and rat endocrine organs was consistently greater than that

found in brain. HPBLs had a Bmax 1.8 times greater than that of rat cerebellum,

101
and the relative amounts for rat endocrine tissues were pituitary, 2.0; adrenal,

1.6; testis, 2.6; and ovary, 4.1 times greater than cerebellum (see figure 3).

The pharmacological profile of [3H]haloperidol binding was examined in

competition experiments by incubating rat cerebellar, HPBLs, and rat endocrine

tissue homogenates with [3H]haloperidol (in the presence of 25 nM spiperone)

plus increasing concentrations of drugs representing a wide range of potencies

for and PCP receptors. These results are summarized in table 2 and figures 4

and 5.

The pharmacology and stereospecificity of [3H]haloperidol-labeled sites in

human PBLs and rat pituitary, adrenal, testis, and ovary were consistent with

those previously reported using a variety of radioligands for receptors in brain

(Largent et al. 1984; Martin et al. 1984; Tam 1985; Tam and Cook 1984; Weber

FIGURE 3. Densities of [

3H]haloperidol-labeled receptors in endocrine and

lymphoid tissues. Saturation binding experiments were carried out

with [3H]haloperidol as previously described (0.2 -10 nM in human

peripheral blood leukocytes (HPBL), and 0.2 -15 nM in rat brain

and endocrine tissues). Spiperone (25 nM) was included in all

incubations, and nonspecific binding was defined in the presence

of 30 mM I-butaclamol. Data from Wolfe et al. 1988, 1989.

102
TABLE 2. Pharmacologic specificity of [3H]haloperidol-labeled sigma receptors

Drug Rat cerebellum Human PBL

Haloperidol 0.42 ± 0.05 (3) 0.30 ± 0.05 (3)

I-Butaclamol 21.8 ± 6.0 (3) 14.7 ± 5.9 (3)

Pentazocine 28.9 ± 16.8 (2) 19.7 ± 5.4 (2)

d-3-PPP 167 ± 64.6 (5) 283 ± 62.5 (5)

DTG 307 ± 97.0 (2) 1,386 ± 348 (2)

d-SKF 10,047 461 ±110(5) 1,666 ± 416 (5)

d-Butaclamol 1,851 ± 351 (3) 1,040 ± 251 (3)

I-SKF 10,047 3,850 ± 595 (5) 28,173 ± 5,260 (5)

d,lPCP 7,410 ± 6,986 (2) 19,628 ± 13,432 (2)

Levallorphan 9,624 ± 1,684 (4) 12,502 ± 4,984 (4)

TCP 10,41 1 ± 3,865 (3) 42,656 ± 11,406 (3)

MK-801 94,619 ± 23,489 (2) 212,968 ± 52,353 (2)

NOTE: The Ki values (nM, mean ± SEM) of drugs were determined at [3H]haloperidol (1 nM)

-labeled receptors in rat cerebellum and human PBL in the presence of 25 nM spiperone.

Nonspecific binding was defined in parallel incubations by the inclusion of 30 mM I-butaclamol.

From Wolfe et al. 1988.

et al. 1986). l-Butaclamol was 11 to 44 times as potent as d-butaclamol in

competing for [3H]haloperidol binding sites in rat endocrine tissues, and 70 to

80 times as potent in HPBL (representative experiment shown in figure 4).

Since d-butaclamol is selective for D2 dopamine receptors (Tam and Cook

1984), this pattern suggests that [3H]haloperidol is not binding to D2 dopamine

receptors under these experimental conditions. On the other hand, the

dstereoisomers of 3-PPP, pentazocine, and SKF 10,047 were more potent

than their /-forms in all of the tissues tested (figure 4 and table 2), which is

consistent with the stereospecificity of receptors in brain. The recently

described drug DTG and the PCP receptor ligands PCP, TCP, and MK-801

were also tested. DTG was 14 to 34 times as potent as PCP and TCP, and 154

to 308 times as potent as MK-801 (figure 4B and table 2) which confirms that

[

3H]haloperidol was labeling and not PCP receptors under the conditions

employed. These observations were further confirmed using [3H]DTG, which

bound HPBL in a specific, saturable manner and had kinetic properties (KD =

26.4 ± 3.6 nM) comparable with rat cerebellum (KD = 29.5 ± 3.5 nM) (data not

shown).

103
FIGURE 4. Pharmacologic profile of [3H]haloperido-labeled receptors in

HPBL. A. Competition by d- and I-stereoisomers of butaclamol

and SKF 10,047 with 1 nM [3H]haloperidol in the presence of

25 nM spiperone. I-Butaclamol is more potent than the

d-form and d-SKF 10,047 is more potent than

I-SKF 10,047 at this site. B. Competition by

haloperidol d-3-PPP (—x—), pentazocine (—+—-),

DTG levallorphan PCP and TCP

under identical conditions. From Wolfe et al. 1988.

104
FIGURE 5. Correlation of drug potencies of at [

3H]haloperidol-labeled

receptors in rat endocrine tissues with their respective potencies

in rat cerebellum. Numbers refer to the following compounds:

1, haloperidol; 2, d-pentazocine; 3, I-butaclamol; 4, I-pentazocine;

5, DTG; 6, d-3-PPP; 7, d-SKF 10,047; 8, I-3-PPP; 9, I-SKF

10,047; 10, d-butaclamol; 11, PCP; 12, TCP; 13, MK-801. From

Wolfe et al. 1989.

105
The rank orders of potency of the various drugs tested in HPBL and rat

endocrine tissues were similar to that of the rat cerebellum, and there was a

highly significant positive correlation (p < 0.001 in all tissues) of the Ki values of

the drugs in blood leukocytes and endocrine tissues with their Ki values in the

cerebellum. This relation is illustrated for the endocrine system in figure 5, in

which the Ki values of the drugs in the various tissues are plotted versus their Ki

values in the cerebellum. All such plots resulted in straight lines with slopes

closely approaching unity (0.99 for endocrine tissues and 0.87 for human PBLs)

and correlation coefficients (r values) of 0.91 for pituitary, 0.96 for HPBL, and

0.99 for rat adrenal, testis, and ovary.

Autoradiography

The drug [3H]-3-PPP gave a better signal-to-noise ratio than [3H]haloperidol in

binding to slide-mounted tissue sections and was therefore used instead of

[

3H]haloperidol to label receptors for in vitro receptor autoradiography.

Autoradiograms of rat pituitary, adrenal, testis, and ovary showed specific,

localized binding of [3H]-3-PPP (figure 6). In the pituitary, specific binding was

seen in all three lobes (figure 6D-F). Labeling was homogeneous within each

lobe, with highest densities of [3H]-3-PPP binding sites being present in the

anterior lobe and lower concentrations seen in the neural (posterior) and

intermediate lobes.

In the adrenal gland, [3H]-3-PPP binding was localized primarily in the outer

cortical regions (figure 6G-I). There was a slightly higher density of receptors

visualized in the capsule and the outer (zona glomerulosa and zona fasciculata)

than in the inner (zona reticularis) regions of the cortex. A low level of specific

binding was present in the adrenal medulla.

The testis displayed a high density of specific [3H]-3-PPP binding sites (figure

6J-L). Specific [3H]-3-PPP labeling in the seminiferous tubules was uniform and

comparable in intensity with the cerebellum of the brain (figure 6B). Interstitial

areas and the contents of the tubule lumens did not show visible radioligand

binding. The highest densities of receptors were found in the epididymis in the

two regions of the ductuli efferentes, and in the corpus and the initial segment

of the caput of the ductus epididymis. Lower intensity labeling was seen in the

rest of the caput, the proximal and distal cauda, and the luminal surfaces of the

pars epididymica and pars libera of the ductus deferens. The muscular layers of

the ductus deferens contained still-lower densities of receptors.

106
The ovary contained the greatest concentration of [3H]-3-PPP binding sites

found in the endocrine tissues examined, and the highest density of receptors

in the ovary was localized in the maturing follicles (figure 6M-O). A uniform,

lower density of receptors was seen throughout the remainder of the ovary

and in follicles not undergoing active development.

In addition to using [3H]-3-.PPP, we also carried out autoradiography using

[

3H]DTG to localize sigma receptors. [3H]DTG-generated autoradiograms were

in agreement with those presented above for [3H]-3-PPP. Specific labeling of

receptors was demonstrated in the anterior lobes of the pituitary, the adrenal

cortex, the noninterstitial areas of the testis, and ovarian follicles (data not

shown).

Effect of Hypophysectomy

In order to determine whether sigma receptors in endocrine organs require

trophic maintenance by pituitary hormones, hypophysectomized rats were also

examined in autoradiographic studies. Two to three weeks after

hypophysectomy, the adrenals, testes, and ovaries were visibly atrophied

relative to those of sham-operated animals. However, [3H]-3-PPP

autoradiography revealed that there was no apparent loss of receptors in the

adrenals or testes. In the adrenals, shrinkage of receptor-poor interstitial areas

caused a relative increase in receptor density of the spared tissues (figure 7A

and B). Consequently, the concentration of receptors in the adrenal cortex,

paucity of receptors in the medulla, and the gradation in density from outer

(zona glomerulosa and zona fasciculata) to inner (zona reticularis) regions of

the cortex was easier to visualize in the adrenals from the hypophysectomized

rats than it was in intact animals (figure 7B).

In the testis, the pattern of change was similar to that of the adrenal. As is

evident from the autoradiogram, labeling did not occur in interstitial areas of

testes from normal animals (figure 7C). While atrophy of interstitial areas and

shrinkage of the organ was evident, hypophysectomy had no effect on the

localization or density of receptors in the testis (figure 7D).

Because the estrus cycle ceased in hypophysectomized rats, no maturing

follicles were present in their ovaries. This resulted in loss of the high-density

[

3H]-3-PPP binding sites seen in the maturing follicles of sham-operated rats

(figure 7E and F). The remainder of the ovary, although reduced in size,

contained distribution and density of receptors comparable to that of ovaries

107
FIGURE 6. Autoradiographic localization of receptors in rat brain and

endocrine organs. Left column (TOLUlDlNE BLUE) - brightfield

photographs of toluidine blue-stained, slide-mounted sections

showing histology of brain (A), pituitary (D), adrenal (G), testis (J)

and ovary (M). Center column (TOTAL) - negative enlargements

of [3H]Ultrofilm autoradiograms showing binding of [3H]-3-PPP

(light areas) to the sections shown in A, D, G, J and M. Right

column (NONSPECIFIC) - autoradiograms of adjacent sections in

which specific [3H]-3-PPP binding was blocked by 10 mM

haloperidol. Specific binding was widely distributed in the brain (A,

B, C), and present in high densities in the cerebellum (cbl). In the

pituitary (D, E, F), the highest concentrations off [3H]-3-PPP

108
of sham-operated controls. Significant concentrations of [3H]-3-PPP binding

sites were present in quiescent follicles of normal and hypophysectomized

animals alike.

Discussion

The high-affinity, specific [3H]haloperidol and [3H]DTG binding that we

demonstrated in HPBLs and rat pituitary, adrenal, testis, and ovary exhibited a

pharmacology consistent with that of the receptor. The kinetic and

pharmacologic characteristics of [3H]haloperidoI-labeled binding sites in these

tissues were similar to those of receptors in rat cerebellum with respect to

their affinity (KD values), stereospecificity, and rank orders of drug potencies.

[

3H]Haloperidol binding was inhibited by nanomolar concentrations of the

receptor ligands l-butaclamol, d-pentazocine, DTG, d-3-PPP, and d-SKF

10,047. Competition binding studies, using drugs representing a wide range of

potencies for and PCP receptors, demonstrated similar pharmacologic

profiles of [3H]haloperidol binding in all of the tissues examined. In addition,

there were strong positive correlations between drug potencies at the

[

3H]haloperidol binding sites in lymphoid and endocrine tissues and their

respective potencies at [3H]haloperidol binding sites in rat cerebellum. The Ki

binding sites were found in the anterior lobes (a), with lower

densities present in the intermediate (i) and posterior (p, neural)

lobes. The adrenal gland (G, H, I) contained slightly higher

densities of binding sites in the outer (zona glomerulosa (g) and

zona fasciculata (f)) regions of the cortex than in the inner (zona

reticularis (r)) cortical areas. Binding sites were very sparse in the

adrenal medulla (m). The highest density of receptors in the

testis (J, K, L) was present in the epididymis in the ductuli

efferentes (de) and the initial segment (is) of the caput (ca).

Slightly lower concentrations were seen in the rest of the caput,

proximal cauda (pc) and distal cauda (dc), and luminal surfaces of

the pars epididymica (pe) and pars libera (pl) of the ductus

deferens. The seminiferous tubules (st) of the testis proper were

comparable in receptor density to the cerebellum of brain.

[

3H]-3-PPP binding sites were not apparent in the interstitial areas

of the testis or epididymis. The ovary (M, N, 0) contained very

high concentrations of receptors in maturing follicles (mf), and

lower, but significant, densities in the resting follicles (rf). Bars = 4

mm. From Wolfe et al. 1989.

109
FIGURE 7. Effect of hypophysectomy on binding sites in endocrine organs.

Negative enlargements of autoradiograms of adrenals (B),

testis (D) and ovary (F) from hypophysectomized rats compared

to corresponding organs from sham-operated controls (A, C, and

E, respectively). Autoradiograms generated under identical

conditions as in Fig. 6. Bars = 4 mm. From Wolfe et al. 1989.

110
values that we obtained for competing drugs are similar to those reported by

others in rat (Largent et al. 1984, 1986) and in guinea pig brain (Tam and Cook

1984). Clearly, binding sites in lymphoid and endocrine organs have kinetic

and pharmacologic characteristics identical to those of the well-characterized

receptors present in brain.

Thus, the present studies suggest that the immunologic effects of PCP

(Dornand et al. 1987; Khansari et al. 1984) are mediated through actions on

rather than on PCP receptors. The immunosuppressive effects of PCP were

observed at relatively high concentrations (10 μM) of drug. This may relate in

part to the low affinity of PCP for receptors in PBLs (Ki = 20 μM; see table 2).

Although the relevance and characteristics of the low-affinity [3H]TCP binding

site in PBLs remains to be determined, actions of PCP at this binding site

resulting in immune modulation cannot be ruled out.

The mechanism by which PCP produces its immunosuppressant effects is

unknown, as the mechanism of action of the receptor has yet to be

elucidated. The work of ltzhak and Khouri (Itzhak and Khouri 1988; ltzhak 1989)

suggests that guanine nucleotides can modulate the binding affinity of the

receptor. Elsewhere in this volume, Bowen et al. present evidence that

receptors may be involved in negative modulation of phosphoinositide-linked

receptor signaling. Lymphocyte activation is well known to be modulated by

guanine nucleotide regulatory proteins and changes in phosphoinositide

turnover (reviewed in De Franco et al. 1987; lsakov et al. 1987). Another

putative mechanism of action may involve potassium channels. Potassium

channels are present on T lymphocytes and are activated during delivery of

“lethal hits” by cytotoxic T lymphocytes (Russell and Dobos 1983) and during

stimulation with mitogens such as phytohemagglutinin and succinyl

concanavalin A (DeCoursey et al. 1984; Matteson and Deutsch 1984). Because

PCP blocks potassium channels in the CNS (Albuquerque et al. 1981; Blaustein

and lckowicz 1983), and because blockade of these channels can suppress

lymphocyte proliferative responses (Decoursey et al. 1984) one can speculate

that receptors on HPBLs may play a role in regulating immune responses

through potassium channels.

The absence of PCP receptors and the localization of receptors to specific

areas within endocrine tissues suggest probable sites of action at which

agonists and antagonists may affect endocrine physiology. Using

membrane-homogenate binding techniques, other investigators have previously

reported d-[3H]SKF 10,047 binding sites localized primarily to the anterior lobe

111
of the pituitary (Tam 1983). In the present autoradiographic study, binding

sites were evident in all three lobes of the pituitary, but the highest densities

were found in the anterior lobe. The anterior lobe of the pituitary contains

several distinct cell types that secrete a variety of hormones. The homogeneous

distribution of binding sites within the anterior lobe probably reflects a

scattered distribution of the target cells and suggests a generalized action of

drugs on anterior pituitary function. Such action is evident from studies in the

literature reporting effects of receptor agonists including PCP and SKF 10,047

to stimulate adrenocorticotropic hormone (ACTH) secretion (Boggan et al.

1982; Pechnick et al. 1985) and to suppress LH (Pechnick et al. 1985) and

prolactin (Pechnick et al. 1985; Saller et al. 1982) secretion. Recently, Pechnick

et al. (1987) reported that the PCP-receptor-specific ligand MK-801 stimulated

the release of ACTH but did not affect the release of prolactin in the rat.

These data, in conjunction with reported effects of PCP and SKF 10,047 to

stimulate ACTH and to suppress prolactin secretion (see above), suggest that

the effects of these compounds on ACTH secretion may be mediated through

both PCP and receptors, while the effects on prolactin secretion appear to be

primarily mediated through actions on receptors. The absence of high-affinity

PCP receptors in the anterior pituitary suggests that these effects may be

mediated through high-affinity PCP receptors in the hypothalamus; moderate

densities of [3H]TCP-(figure 1B) and [3H]MK-801 -labeled PCP receptors were

present in the hypothalamus.

Both and PCP receptors appear to be involved in the ability of PCP and SKF

10,047 to suppress LH release. Evidence in support of receptors comes from

recent studies demonstrating direct actions of PCP and SKF 10,047 to inhibit

LH release in anterior pituitary cells (Stojilkovic et al. 1987). The in vivo effects

of PCP and SKF 10,047 on LH release may also be mediated through indirect

actions on hypothalamic gonadotropin-releasing hormone (GnRH) release.

Progesterone, at physiologically relevant concentrations, has been reported to

have a relatively high affinity for receptors and may represent an endogenous

ligand ford receptors (Su et al. 1988). If one assumes that progesterone acts

as an agonist at receptors, then binding sites in the pituitary and

hypothalamus may play important roles in the negative feedback effects of

progesterone to suppress GnRH and LH release. Recent studies have

established that PCP receptors in brain are coupled to glutamate receptors and

that PCP acts as an antagonist at this complex (Fagg 1987). Administration of

N-methyl-D-aspartic acid (NMDA), a glutamate receptor agonist, stimulates LH

secretion in rodents and monkeys (Gay and Plant 1987; Price et al. 1978;

112
Schainker and Cicero 1980) and this secretion can be blocked by

administration of the NMDA antagonist (d,l)-2-amino-5-phosphonopentanoic

acid (AP5) (Arslan et al. 1988; Gay and Plant 1987). It is conceivable that PCP

and SKF 10,047 antagonistic actions at the NMDA-PCP receptor complex in

hypothalamus may inhibit GnRH and LH release.

A variety of reports in the literature have documented potent effects of

haloperidol to alter anterior pituitary hormone secretion. Specifically, haloperidol

has been reported to stimulate prolactin, proopiomelanocortin-derived peptide,

and corticosterone secretion in rodents and in human subjects (Dickerman et al.

1974; Frantz and Sachar 1976; Höllt et al. 1982; Mueller et al. 1976). Although

haloperidol has a high affinity for D2 dopamine receptors, it has an equally high

affinity for binding sites. This suggests that some of the effects of haloperidol

on anterior pituitary hormone secretion may in fact be modulated through

actions at receptors. Consistent with this interpretation, agonists including

PCP and SKF 10,047 have been reported to suppress prolactin secretion

(Pechnick et al. 1985; Saller et al. 1982) whereas haloperidol (presumably

through antagonistic actions at or D2 dopamine receptors or both) stimulates

secretion (Dickerman et al. 1974). In contrast, the ability of haloperidol to

stimulate pituitary-adrenocortical secretion (Aimoto et al. 1980) does not appear

to be mediated through o-receptor antagonism, because haloperidol produces

the same effects as agonists, such as PCP and SKF 10,047 (Boggan et al.

1982; Pechnick et al. 1985).

In the adrenal glands, receptors were localized primarily in the adrenal cortex,

with significantly lower concentrations in the medulla. Because the densities of

binding sites were not decreased following hypophysectomy, these receptors

in the adrenal cortex are probably not present on cells that are dependent on

maintenance by trophic pituitary hormones. Therefore, binding sites in the

adrenal gland do not appear to be localized to glucocorticoid-producing cells.

Although o-receptor agonists including PCP and SKF 10,047 have been

reported to stimulate corticosterone secretion in mice and in rats (Boggan et al.

1982; Pechnick et al. 1985) these effects appear to be primarily mediated via

pituitary ACTH regulation, rather than through direct actions at the adrenal

gland, because they can be blocked by dexamethasone administration (Boggan

et al. 1982). Chronic PCP administration has been reported to increase adrenal

weight in rats (Mielke and Gallant 1982) presumably through sustained ACTH

secretion. The role of the binding sites in the adrenal gland in modulating

other adrenal functions remains to be elucidated.

113
There was a high density of binding sites in the testis. Binding sites were

uniformly distributed in the walls of the seminiferous tubules and were notably

absent in the interstitial areas and the contents of the tubule lumens. Like those

in the adrenal cortex, receptors in the testis were not altered after

hypophysectomy, suggesting that they were not dependent on trophic

maintenance from the pituitary gland. The paucity of receptors in interstitial

areas suggests that ligands may not have any direct action on the testis to

alter androgen secretion. The high densities of receptors in the epididymis

suggest that they may serve an important biologic function, possibly in transit of

sperm. The marked differences in the density of receptors within zones of the

epididymis appear to correspond to the tubule size and wall thickness in the

different regions of the ductuli efferentes, ductus epididymis, and ductus

deferens. On the other hand, the differences in the density of receptors in the

different areas of the epididymis may correspond to changes in cellular

composition of the duct epithelium in the different regions. At present, the

distinction between these two possibilities cannot be made and the role of

receptors in the epididymis remains to be determined. Fairly low densities of

binding sites were observed in the muscular areas of the ductus deferens.

Nevertheless, receptors in the vas deferens appear to be functional, as

evidenced by potentiation of the vas deferens muscle twitch after exposure to

agonists (Vaupel and Su 1987).

Of the various endocrine organs examined, the highest density of receptors

was present in the ovary. Within the ovary, the highest density of receptors

appeared to be localized to maturing follicles, which were lost in

hypophysectomized rats. It is of note that low to moderate concentrations of

binding sites were also present in quiescent follicles in both normal and

hypophysectomized animals, Although the role of receptors in the ovary is

unclear at present, the high density in this organ is consistent with a recent

report by Su et al. (1988) that progesterone has a relatively high affinity for

receptors.

Given the widespread distribution of receptors in the CNS, a significant

question is whether the binding sites are on elements of neural tissue within

endocrine organs or are, in fact, a component of the endocrine cells. Although

the resolution of the autoradiographic technique is not adequate to visualize

neuronal elements in the endocrine tissues, the distribution pattern of binding

sites is not consistent with the known location of neurons in the organs studied.

For example, receptors are present in high density in the anterior lobe of the

pituitary, with much lower concentrations present in the neurointermediate lobe.

114
On the other hand, the neuronal elements of the pituitary gland are primarily

localized in the intermediate and posterior lobes. Similarly, in the adrenals,

receptors are present in higher density in the cortex than in the medulla; the

medulla is derived from neural crest. The identity of the receptor-bearing cell(s)

within the testis and ovary is not known. However, the high density of binding

sites in maturing follicles, which decrease significantly after hypophysectomy,

suggests localization of these receptors to pituitary-hormone-regulated

endocrine cells. The case of PBLs is more clear-cut, because receptors were

characterized in single-cell suspensions of circulating leukocytes. These

suspensions contained no neural cells.

RELEVANCE OF RECEPTORS IN THE ENDOCRINE-IMMUNE AXIS

A striking feature of the pattern of receptor distribution that we have observed is

that PCP receptors are found only in brain, whereas receptors exist both in

brain and peripheral tissues. We are unaware of any exceptions to this rule.

This pattern is reminiscent of benzodiazepine receptors, which have central and

peripheral subtypes that are also differentially distributed between the CNS and

peripheral tissues (reviewed in Anholt et al. 1985a, 1985b; De Souza et al.

1985). However, unlike peripheral benzodiazepine receptors (Anholt et al.

1985b), receptors in the adrenal and testis are not dependent on trophic

maintenance by pituitary hormones.

Another striking feature of these results is the very high densities of receptors

in HPBLs and rat endocrine tissues. This observation suggests that

endogenous ligands may play an important physiological role in modulating

and integrating immune and endocrine functions. Because isolation and

characterization of endogenous PCP (Quirion et al. 1984) and (Contreras et

al. 1987; Su et al. 1986) ligands is only in preliminary stages at present (see

also chapter by Contreras et al. in this volume), it remains to be seen where

these endogenous substances are present in the body and by what means they

are delivered to target tissues. Furthermore, because lymphokines and

endocrine peptides can modulate CNS activity, alteration of the release of

hormones and lymphokines/cytokines could be an additional mechanism for the

central actions of agonists.

PCP induces a schizophrenia-like psychosis (Allen and Young 1978) and the

hypothesis has been put forward that in many cases schizophrenia may be

caused by genetic defects in PCP receptors, endogenous ligand structure,

regulation and expression of receptors and ligands, or alternatively to

115
autoimmune reactions against these receptors or ligands (Fudenberg et al.

1984). Because we now know that PCP acts at as well as at PCP receptors,

this hypothesis may be extended to include the system. If the hypothesis is

true, then some schizophrenics may also have endocrine and immunologic

abnormalities caused by the same disease process.

Lymphocytes of schizophrenic patients have been noted to have altered

morphology (Erban 1986; Fessel and Hirata-Hibi 1963; Kamp 1962); reduced

viability in culture (Erban 1965); and depressed functional activity as measured

by such in vitro assays as mitogen-driven proliferation, mixed lymphocyte

culture, generation of cytotoxic lymphocytes, and interferon production

(Liedeman and Prilipko 1978; Moises et al. 1985; Vartanian et al. 1978).

However, these phenomena may reflect both organic disease and drug effects,

because haloperidol is widely prescribed as a neuroleptic for these patients.

Haloperidol has extensive endocrine effects, which have been discussed.

Haloperidol’s effect on the immune system appears to be complex. Moises et

al. (1985) found that haloperidol in vitro had no effect on proliferation or

interferon production by normal human lymphocytes; Goldstein et al. (1980)

observed that proliferative responses of schizophrenic patients rebounded to

above-normal levels 4 weeks after withdrawal of neuroleptic therapy. In mice,

haloperidol suppressed in vivo antibody responses but not contact

hypersensitivity (Lovett et al. 1978). The receptor mechanisms involved in the

effects of haloperidol are unclear, because haloperidol binds to D2 dopamine

receptors and to adrenergic receptors (U'Prichard et al. 1977) as well as to

receptors; [3H]dopamine binding sites were recently detected in rat lymphocytes

(Ovadia and Abramsky 1987). Further study is therefore needed to determine

what immunologic and endocrine anomalies are an organic part of the

schizophrenic disease process and whether haloperidol’s effects on these

systems are modulated primarily by or dopamine receptors.

SUMMARY AND CONCLUSIONS

We have demonstrated the absence of PCP receptors and the presence of

receptors in PBLs and rat pituitary, adrenal, testis, and ovary that have kinetic

and pharmacologic characteristics comparable with the receptor found in the

CNS. The physiologic significance of the receptors in the various lymphoid

and endocrine tissues remains to be determined, but their presence in such

high densities suggests that endogenous ligands may play an important role

in regulating and integrating endocrine and immune responses. Thus, in

addition to their central actions, agonists including PCP and SKF 10,047 may

116
exert their immunosuppressive and endocrine effects directly through actions in

the pituitary and target organs. On the other hand, the effects of selective PCP

agonists on endocrine function appear to be mediated primarily through actions

in brain. Furthermore, the immunologic and endocrine effects of neuroleptics

such as haloperidol, which have previously been attributed primarily to actions

at D2 dopamine receptors, may also be mediated via receptors in brain,

lymphoid, and endocrine organs. Finally, the immune and neuroendocrine

systems may represent useful “windows to the brain” to assess the role of

and PCP receptors in the CNS.

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122
ACKNOWLEDGMENTS

We thank Charlene Jackson, R.N., and Dr. Hayden Braine of the Johns

Hopkins Hemopheresis Unit for kindly providing the human leukocytes used in

this study. We would also like to thank Steven Hurt of Dupont/New England

Nuclear for supplying us with [3H]DTG, and Dr. Edythe D. London of the NIDA

Addiction Research Center for a critical review of this manuscript.

AUTHORS

Seth A. Wolfe, Jr., Ph.D.

Laboratory of Neurobiology

Neuroscience Branch

Addiction Research Center

National Institute on Drug Abuse

P.O. Box 5180

Baltimore, MD 21224

Errol B. De Souza, Ph.D.

Chief, Laboratory of Neurobiology

Neuroscience Branch

Addiction Research Center

National Institute on Drug Abuse

P.O. Box 5180

Baltimore, MD 21224

123

A Role for Binding in the Antipsychotic

Profile of BMY 14802?

Duncan P. Taylor, Michael S. Eison, Sandra L. Moon,

R. Francis Schlemmer, Jr., Umesh A. Shukla,

Cam P. VanderMaelen, Frank D. Yocca, Dennis J. Gallant,

Susan H. Behling, Christopher G. Boissard,

John P. Braselton, Houston H. Davis, Jr.,

Marlene N. Duquette, Raymond C. Lamy, Judith M. Libera,

Elaine Ryan, and Robert N. Wright

INTRODUCTION

Although the dopamine D-2 antagonists (such as the phenothiazines,

thioxanthenes, and butyrophenones) have proven effective in managing of the

positive symptoms of schizophrenic disorders, the long-term outlook for patients is

not always hopeful. Only 25 percent of patients achieve full recovery with existing

drugs, while long-term care is required for another 25 percent of this population.

The relapse rate for those for whom drugs are effective averages 40 percent in the

first year after an acute illness. Should antipsychotic medication be discontinued at

any time in the first 4 years after an acute illness, the relapse rate rises to

65 percent (see Tantam and McGrath 1989 for a review). Moreover, side effects of

D-2 dopamine antagonist antipsychotic agents range from antihistaminic and

antimuscarinic properties, which are inconvenient and interfere with patient

compliance; to tardive dyskinesia, which is debilitating for patients and may put

physicians at risk for legal liabilities; to potentially life-threatening effects, such as

neuroleptic malignant syndrome, cardiotoxicity, and agranulocytosis.

As a consequence of these kinds of drawbacks of existing drugs, many

pharmaceutical companies have been engaging in a long-term search for more

effective, safer antipsychotic drugs. BMY 14802 is one compound to emerge

from this effort (figure 1; Yevich and Lobeck 1986) and it is our purpose to

profile this agent pharmacologically in order to explore the potential role of the

binding site in the treatment of schizophrenia.

125
Haloperidol

Clozapine

FIGURE 1. Chemical structures of current or potential antipsychotics.

126
PRECLINICAL PREDICTIONS OF ANTIPSYCHOTIC EFFICACY

BMY 14802 was originally identified as an antipsychotic candidate by its

activities in classical neuropharmacologic tests. Among these were the abilities

to block the conditioned avoidance response in rats, to inhibit apomorphineinduced stereotypy in rats, and to inhibit apomorphine-induced pole climbing in

mice (see table 1; Taylor et al. 1985; Bremer et al. 1989). In general the

potency of BMY 14802 in these tests was less than that of haloperidol but

equivalent to that of clozapine. In the somewhat more refined discriminated

(Sidman) avoidance test, BMY 14802 produced a profile similar to that seen

with clozapine in that avoidance was suppressed without a concomitant

inhibition of escape performance (see figure 2; Taylor et al. 1985). With the

more conventional antipsychotic agent chlorpromazine, a decrement in both of

these performance parameters is seen. In addition, BMY 14802 blocks the

induction of hyperlocomotion by the dopamine-mimetic agent amfonelic acid

(Matthews et al. 1986).

Further persuasive evidence supporting the potential for antipsychotic efficacy

comes from work on amphetamine-induced “psychotic” behavior in a primate

social colony (Schlemmer and Davis 1986; see table 2). Chronic

TABLE 1. Activity of selected agents in models that predict antipsychotic

efficacy, side effects, or other properties

Test BMY 14802 Clozapine Haloperidol

Inhibition of conditioned avoidance 26 27 2.8

response (ED,, mg/kg, p.o., in rat)

Inhibition of apomorphine-induced 33 49 0.5

stereotypy (ED,, mg/kg, p.o.,

in rat)

Inhibition of apomorphine-induced pole 60 10 1

climbing (MED, mg/kg, p.o.,

in mouse}

Induction of catalepsy (ED,,, IA(100)* IA(200)* 0.6

mg/kg, p.o., in rat)

Reversal of trifluoperazine-induced 17 >20* -

catalepsy (ED,, mg/kg, p.o., in rat)

NOTE: Data from Taylor et al. (1991). MED = minimally effective dose.

*IA = inactive at dose shown in parentheses.

127
FIGURE 2. Effects of current and potential antipsychotics on rats in a

discriminated avoidance task after dosing at 100 mg/kg, p.o. Solid

bars represent avoidance responding and open bars represent

escape responding, both as a percent of vehicle-treated controls.

Asterisks denote levels of responding that were significantly

different from controls at the p < 0.05 level. Data from Taylor et al.

(1991).

dextroamphetamine administration (1.6 mg/kg, n.g., b.i.d., 12 days) produces

intense scratching, inappropriate submissiveness, increased distancing,

decreased initiated social grooming, increased checking or vigilance, and

stereotyped behavior in stumptail macaques. Currently available antipsychotics

can block all of these manifestations, although the atypical agent clozapine

does not alter stereotyped behavior. When BMY 14802 is given concomitantly

(5 mg/kg, n.g., b.i.d., 12 days) with amphetamine, it attenuates or significantly

reverses all of these behaviors with the exception of stereotyped movements in

a profile resembling clozapine’s (see figure 3).

128
TABLE 2. Possible human behavioral correlates of signs exhibited by

macaques in social colonies following chronic amphetamine

intoxication

Behavioral sign

Increase in submissive gestures

Increase in scratching

Human correlate

Paranoia: perception of aggression or

hostility in a nonthreatening situation

Scratching in response to tactile

hallucinations (parasitosis)

Increase in distancing or spatial

isolation

Social withdrawal

Increase in checking or visual

scanning

Hypervigilance

Another persuasive line of evidence in predicting the potential antipsychotic

efficacy of BMY 14802 comes from the work of Wachtel and White (1987), who

showed that chronic administration of BMY 14802 at relatively low doses

(5 mg/kg, s.c., 28 days) depressed the number of neurons that fire in the A10

(ventral tegmental) region of the brain in a manner similar to the effect of all

clinically effective antipsychotic drugs. However, chronic administration of

BMY 14802 does not depress the number of neurons that fire in the A9

(substantia nigra) region, a profile similar to clozapine’s and one that is

dissimilar to the “neuroleptic” antipsychotics that produce movement disorders

as side effects (see next section).

PRECLINICAL PREDICTIONS OF MINIMAL SIDE EFFECT LIABILITY

Unlike the typical antipsychotics, BMY 14802 does not induce catalepsy in rats,

a test frequently employed to predict liability for the acute production of

extrapyramidal symptoms and the production of tardive dyskinesia after chronic

administration (Taylor et al. 1985; see table 1). Clozapine also fails to produce

catalepsy. In contrast to clozapine and all conventional antipsychotics,

BMY 14802 actually reverses catalepsy induced in rats by the neuroleptic

antipsychotics haloperidol and trifluoperazine (Taylor et al. 1985; Matthews

et al. 1986; Grimes et al. 1990, and submitted; see table 1). In addition,

BMY 14802 produces no movement abnormalities in macaque monkeys (see

table 3). This failure to produce abnormal movements cannot be attributed to

sedative effects of the drug, as haloperidol, which does produce abnormal

129
130
FIGURE 3. Effects of BMY 14802 on selected behaviors in social colonies of

macaque monkeys. Baseline (BL 1) measurements were taken by

a blinded observer during two l-hour observation periods.

Animals then received 7.6 mg/kg, n.g., (+)amphetamine, b.i.d. for

12 days (AMPH) and were rated for various behaviors each day.

After a 4-month washout (minimum), a second set of baseline

(BL2) observations was recorded. Following this, BMY 74802,

5 mg/kg, n.g., was administered b.i.d. for 72 days concomitantly

with amphetamine by previous regimen (74802 + AMPH).

A. Number of submissive gestures displayed by a nondominant

female. Data shown are mean values. For AMPH and BMY 74802

+ AMPH, data represent means of all observations on days 5

through 8. There was no increase in aggressive gestures received

by this animal (not shown). B. Incidence of scratching, distinct

from normal se/f-grooming, exhibited by a nondominant female.

Data shown are mean values throughout all observation periods.

C. Spatial isolation (>3 feet from other monkeys) by a

nondominant female. Data shown are mean values. For AMPH

and BMY 74802 + AMPH, data represent means of all

observations on days 5 through 8. D. Incidence of checking or

visual scanning by nondominant females. Data are means of total

incidence averaged over four animals. Data for AMPH and

BMY 74802 + AMPH represent means of all observations on days

5 through 8. E. Induction of stereotyped behaviors in nondominant

females. Data are means of total incidence averaged over four

animals. l significantly different from baseline, p < 0.05.

+

 significantly different from AMPH alone, p < 0.05.

movements, also increases time spent resting. It is significant that clinical trials

have revealed no signs of extrapyramidal symptoms at single doses of

BMY 14802 of 800 mg or multiple doses of 500 mg, q.i.d., for 21 days (Shukla

et al. 1990).

The ability of conventional D-2 dopamine antagonists to produce D-2 receptor

supersensitivity after chronic administration may be correlated with their ability

to provoke the disabling and sometimes irreversible syndrome of tardive

dyskinesia. Consistent with its atypical profile of effects, chronic administration

of BMY 14802 at behaviorally relevant doses produced no change in D-2

131
TABLE 3. Effects of agents on time spent in abnormal movements or resting

in stumptail macaque monkeys

Treatment

(dose, mg/kg, i.m.)

Saline

Haloperidol (0.1)

BMY 14802 (5)

Abnormal Checking

movements Resting Locomotion (number of

(sec) (sec) (sec) scans)

0 17±5 84±26 40 ± 1

68±22* 16±30* 54±28 24±2*

0 30±17 103±49 43 ± 1

NOTE: Data are mean ± SEM for observations of four animals. Abnormal movements included

bizarre posturing, tremor, rigidity, and slow movements.

*p < 0.01 vs saline (three-way partially crossed analysis of variance followed by least significant

differences method).

receptor binding while chronic administration of haloperidol did (see table 4;

Taylor et al. 1985, 1991).

Also, neuronal inactivation of A9 (substantia nigra) cells after chronic

administration of conventional D-2 dopamine antagonist drugs may contribute

to the production of tardive dyskinesia. Unlike typical agents such as

haloperidol, chlorpromazine, and metoclopramide (an antiemetic that produces

extrapyramidal side effects), which produce such inactivation, chronic treatment

of rats with BMY 14802 (2.5, 5, or 10 mg/kg/day, s.c., 28 days) produced no

change in the firing rate of A9 neurons, resulting in a pattern similar to that seen

with the atypical antipsychotic clozapine (Wachtel and White 1987; see above).

TABLE 4. Effect of chronic drug administration on D-2 dopamine receptor

binding in rat striata

Treatment (dose, mg/kg, p.o.) Bmax (fmole/mg protein)

Vehicle 228 ± 5 (5)

BMY 14802 (15) 233 ± 7 (4)

BMY 14802 (30) 212±7(5)

Haloperidol (3) 290 ± 9 (5)*

NOTE: Data are mean ± SEM for number of pooled preparations shown in parentheses. Each pool

contained striatal membranes from three rats, Bmex was determined by saturation analysis

employing [

3H]spiperone according to accepted methods (Taylor and Hyslop 1991).

*p < 0.05 vs vehicle (Student’s t-test, two-tailed).

132
ATYPICAL NEUROCHEMICAL PROFILE

Conventional D-2 dopamine antagonists such as haloperidol produce changes

in levels of dopamine and its metabolites in both mesolimbic and nigrostriatal

dopaminergic pathways (see figure 4; Taylor et al. 1991). The ability of

clozapine to selectively affect levels of dopamine and its metabolites in

mesolimbic brain regions has been hypothesized to underlie its atypical clinical

profile. BMY 14802 produces a clozapine-like profile in its regional effects on

levels of the dopamine metabolite dihydroxyphenylacetic acid (DOPAC; see

figure 4) in that effects are seen in a mesolimbic region, the nucleus

accumbens, at lower doses than those required to produce comparable effects

in the striatum. An additional distinction of BMY 14802 over haloperidol is the

ability of BMY 14802 to produce significant decreases in levels of dopamine in

both brain regions, whereas haloperidol lacks this effect (see figure 4). These

data demonstrate a preferential effect of BMY 14802 in mesolimbic brain

regions and support predictions of antipsychotic efficacy. The differential effects

of BMY 14802 relative to haloperidol on the mesolimbic and nigrostriatal

dopamine systems are consistent with the predictions and observations of

diminished side-effect liability noted above.

The atypical neurochemical profile of BMY 14802 is consistent with its in vitro

interactions with neurotransmitter receptors. Unlike conventional D-2 dopamine

antagonist antipsychotics such as haloperidol, BMY 14802 has very low affinity

for the D-2 receptor site (table 5; Taylor et al. 1985, 1991; Taylor and Dekleva

1988; Largent et al. 1988). Moreover, in vivo binding studies of serum

neuroleptic radioreceptor binding levels and ex vivo studies of [3H]spiperone

binding in the striata of rats receiving BMY 14802 suggest that biologically

relevant levels of metabolites of the drug, which may interact at D-2 dopamine

binding sites, are not generated (Taylor et al. 1985). These data are consistent

with the inability of chronic BMY 14802 administration to induce the proliferation

of D-2 dopamine receptor binding sites as noted above. Furthermore,

BMY 14802 does not bind to D-l dopamine receptors nor inhibit dopaminestimulated adenylate cyclase in the rat striatum (Yocca et al. 1986; Taylor et al.

1991). Finally, BMY 14802 displays low affinity for a variety of other receptor

sites: -adrenergic sites; benzodiazepine and aminobutyric acid

sites; glutamate (including N-methyl-D-aspartate, kainate, and quisqualate

types) and glycine (including both strychnine-sensitive and insensitive types)

sites; opiate sites, the dihydropyridine calcium channel antagonist site;

and 5-HT2 serotonergic sites (Taylor et al. 1985).

133
FIGURE 4. Effects of BMY 14802 and haloperidol on levels of DOPAC and

dopamine in selected brain regions. Tissues were dissected one

hour after drug administration, and DOPAC and dopamine levels

were determined by high-pressure liquid chromatography with

electrochemical detection. Regions investigated were striatum

(circles, solid lines) and nucleus accumbens (squares, broken

lines). Filled figures denote values that were significantly different

from vehicle-treated animals (p < 0.05, Student’s t-test,

two-tailed). All values for DOPAC produced by all doses of either

drug were significant/y different from vehicle. Adapted from Taylor

et al. (1991).

134
TABLE 5. Inhibition of in vitro radioligand binding by selected agents

[

3H] Ligand: spiperone SCH 23390 spiperone dizocilpine glycine

Site labeled: D-2 dopamine D-1 dopamine 5-HT2 serotonin phencyclidine glycine

Tissue used: striatum striatum cortex cortex cortex

Compound

BMY 14802 6,400 >100,000 1,700 >100,000 >100,000

Haloperidol 1.1 860 61 18,000 -

Clozapine 570 1,100 21 9,100 -

NOTE: Data are IC50 values in nM. Adapted from Taylor et al. (1965, 1991).

INTERACTIONS OF BMY 14802 AT THE BINDING SITE

Although the mechanism by which BMY 14802 achieves its various biologic

effects is unclear, a large body of evidence suggests that it may have an action

at the so-called binding site (see table 6; Taylor and Dekleva 1987; Taylor et

al. 1991). BMY 14802 exhibits high affinity in vitro for binding sites in guinea

pig brain (Taylor and Dekleva 1987) as well as rat brain (McCann et al. 1989;

Koe et al. 1989; Largent et al. 1988; Schmidt et al. 1989; Steinfels and Tam

1989). [

3H]BMY 14802 appears to label sites specifically in guinea pig brain

“sausage patties” (slices of frozen, minced tissues) with high affinity (KD =

73 nM, specific binding = 86 percent at 20 nM radioligand). The rank order of

potencies of various drugs to inhibit [3H]BMY 14802 binding is consistent with

the ranking of these drugs in inhibition of binding employing other

radioligands (see table 6; Taylor et al. 1991).

In addition, in vitro labeling of brain slices with [3H]BMY 14802 revealed a

distribution of binding with many similarities to the localization of

(+)-[3H]-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ((+)-[3H]3-PPP) labeling,

especially in cortical regions, hippocampus, cerebellar cortex, hypothalamus,

bed nucleus of the stria terminalis, periaqueductal grey, and superficial layers of

the superior colliculus (Taylor et al. 1991; Moon 1987; Gundlach et al. 1986).

Furthermore, BMY 14802 exhibits stereospecificity at the site, with the

R-dextrorotatory enantiomer being more potent than the S-levorotatory one

(Taylor and Dekleva 1987; Behling et al. 1990; Taylor et al. 1989, 1991).

135
TABLE 6. Inhibition of radioligand binding by selected agents

[

3H]Ligand:

Compound

In vivo assays, ED,

In vitro assays, IC50 (nM) (μmole/kg)

BMY

NANa (+)3-PPPb DTGC 14802 NAN (+)3-PPP DTG

Haloperidol 2.0 1.4

DTG 5.9 19

(+)3-PPP 28 24

R(+)BMY 14802 43 28

(±)BMY 14802 83 112

S(-)BMY 14802 420 310

(+)NAN 42 79

(-)NAN 600 2000

Rimcazole 1100 1000

2

22

91

32

-

140

620

3900

-

9.4 2 0.13 0.3

8.6 - 6 150

- 2 - 74

- - - -

33 9 13 59

- - - -

- 0.1 - 63

8600 6.1 - 310

- 19 24 -

NOTE: In vitro data are for binding to membranes from whole guinea pig brains (adapted from

Taylor and Dekleva 1987, 1988; Taylor et al. 1989, 1991). In vivo data are for binding to

membranes from mouse brains and are adapted from Ferris et al. (1988) and R.M. Ferris (personal

communication) for NAN, from Koe et al. (1989) for (+)3-PPP, and binding to rat brain membranes

for DTG, from Singh et al. (1990). The data for in vivo binding of DTG were derived assuming

inhibition occurred in a linear fashion according to the law of mass action for a single class of

noninteracting sites.

a N-allyl-N-normetazocine (SKF 10,047).

b

(+)-3-[3-hydroxyphenyl]- N-(l-propyl)piperidine.

C

1,3-di-o-tolylguanidine.

The inhibition of in vitro binding of (+)-[3H]-N-allylnormetazocine((+)-[3H]NAN,

(+)-SKF 10,047) and (+)-[3H]3-PPP by (+)BMY 14802 is competitive, although

its interaction at the site labeled by [3H]-1,3-di-o-tolylguanidine ([3H]DTG) is

more complex (see table 7). These data suggested that [3H]DTG labeled more

sites than (+)-[3H]3-PPP or (+)-[3H]NAN, but the membrane preparations used

in these studies were different. When a purified P2 membrane (Weber et al.

1986) preparation of guinea pig brain was studied in a pairwise fashion in

saturation analyses, we found that a significantly greater number of sites were

labeled by [3H]DTG than by (+)-[3H]3-PPP (Bmax values of 53 ± 12 and 20 ± 3

pmol/g wet weight, respectively, for three preparations; p < 0.05, Student’s

t-test for paired observations, two-tailed). These findings are consistent with

preliminary observations from a number of laboratories that suggest the

existence of multiple types and/or states of the site (Naper et al. 1989; Arnold

136
TABLE 7. Effect of R(+)BMY 14802 on in vitro radioligand equilibrium binding

parameters at sites in guinea pig brain membranes

Addition

Radioligand Parameter None R(+)BMY 14802

(+)NAN

(+)3-PPP

DTG

KD

43 110*

Bmax 17 20

KD

23 82*

Bmax 29 35

KD

51 120*

Bmax 40 28*

NOTE: For equilibrium analyses using (+)-[3H]NAN, the concentration of R(+)BMY 14802 was 50 nM

(Taylor and Dekleva 1987). For studies using (+)-[3H]3-PPP and [

3H]DTG, the concentration of

R(+)BMY 14802 was 30 nM.

*p < 0.05 vs no addition (Students t-test for paired observations, two-tailed).

TABLE 8. Characteristics of types of receptors

Receptor type

Property

Present in

Guinea pig CNS?

Rat CNS?

PC-12 cells?a

Labeled by

(+)-[3H]3-PPP?

[

3H]DTG?

(+)-[3H]benzomorphans?

Estimated molecular weights (kD)b

Yes Yes

No Yes

No Yes

Yes Yes

Yes Yes

Yes No

28 to 29 63,65

NOTE: This nomenclature is for discussion purposes only (taken from Reid et al. 1988).

aPharmacologic studies are discussed by Bowen et al. (1988).

bMolecular weights are from target size analyses presented by Arnold et al. (1988).

et al. 1988; Bowen and Hellewell 1988; Hellewell and Bowen 1988; Reid et al.

1988; Wu et al. 1990; see table 8).

137
Evidence from in vivo binding studies supports an interaction of BMY 14802

with sites. Ferris et al. (1988) used (+)-[3H]NAN to label these sites in mouse

brain, and the position of BMY 14802 in the rank order of potency after systemic

administration is consistent with its potency in in vitro binding studies (R.M.

Ferris, personal communication; see table 6). A similar result was obtained by

Koe et al. (1989) using (+)-[3H]3-PPP to label sites in mouse brain (see table 6).

Recently, Singh et al. (1990) were able to inhibit the in vivo binding of [3H]DTG

to sites in rat brain with BMY 14802 (see table 6).

Identifying a physiological function or behavior that is mediated by the binding

site has been problematic. However, several model systems have recently

emerged, and data from them are consistent with an interaction of BMY 14802

at sites. For instance, Bremer et al. (1989) noted that chronic administration

of haloperidol decreased the maximal number of binding sites for (+)-[3H]3-PPP

in rat brain while chronic administration of BMY 14802 increased this

equilibrium parameter. These opposite effects suggest that in some systems

one may be a “agonist” and the other an “antagonist” (see below). Recently,

BMY 14802 was shown to block the NMDA-evoked release of acetylcholine

from rat striatal slices in vitro, and this property was shared by the ligands

sabeluzole, haloperidol, (+)3-PPP, and DTG (Ryan et al. 1990).

Steinfels and Tam (1989) showed in physiological studies that the

dose-dependent inhibition of dopaminergic neurons in the substantia nigra

evoked by (+)-3-PPP could be completely reversed by BMY 14802. In addition,

pretreatment with BMY 14802 shifted the (+)-3-PPP dose-response curve to the

right, suggesting an agonist-antagonist relationship between the two

compounds. Monitoring extracellular recording in the CA3 region of dorsal

hippocampal pyramidal neurons in rats, Monnet and coworkers determined that

DTG and JO-1784, another ligand, could enhance the excitatory effect of

NMDA, and this enhancement was blocked by the ligands haloperidol,

BMY 14802, and rimcazole but not the D-2 dopamine antagonists spiperone or

sulpiride (Monnet et al. 1988, 1989; Debonnel et al. 1990). In the guinea pig

ileal longitudinal muscle/myenteric plexus BMY 14802, DTG and haloperidol

were found to inhibit serotonin- or electrically evoked muscle twitching, whereas

(+)-NAN and (+)cyclazocine potentiated such twitching (Campbell et al. 1989).

These authors suggest that the inhibitory ligands may act by inhibiting

acetylcholine release in this preparation. This explanation would be consistent

with the observations of Ryan et al. (1990; see above). In the perfused rat tail

artery Massamiri and Duckles (1990) observed that the order of potency for

138
FIGURE 5. Effect of drugs on (+)NAN-induced stereotyped circling in mice.

Saline or drug was administered 30 (BMY 14802) or 40 minutes

[(-)butaclamol] prior to testing. A 15 mg/kg, s.c., dose of (+)NAN

was administered 10 minutes prior to testing. Testing consisted of

a 10-minute observation period during which time circling activity

was assessed. Vehicle circling rates were 47±4 movements/10 min

for (-)butaclamol (n = 5 at all doses) and 29 ± 3 movements/10 min

for BMY 14802 (n = 8 at all doses). Further details are found in

Gallant (1988).

ligands, including BMY 14802, for the inhibition of contractile responses to

norepinephrine parallels their order of potency for the inhibition of in vitro binding.

Grimes et al. (1990) have noted In intact animals that the ligands rimcazole

and (+)-3-PPP as well as BMY 14802 reversed catalepsy induced by

haloperidol or trifluoperazine in rats, while DTG facilitated the effects of

139
TABLE 9. Is BMY 14802 a “antagonist”?

System

BMY 14802 blocks,

reverses, or its

Effect of effects are

BMY 14802 is opposite to

mimicked by: effects of:

Binding after chronic treatment

(Bremer et al. 1989)

Attenuation of dopaminergic cell firing

(Steinfels and Tam 1989)

NMDA-induced increase in CA3

pyramidal cell firing

(Monnet et al. 1988, 1989;

Debonnel et al. 1990)

Muscle twitching in guinea pig ileum

(Campbell et al. 1989)

Reversal of neuroleptic-induced

catalepsy (Grimes et al. 1990)

Blockade of stereotyped behavior

(lyengar et al. 1989)

Blockade of circling

- haloperidol

- (+)3-PPP

rimcazole

haloperidol

DTG

JO-1784

haloperidol

DTG

rimcazole

(+)3-PPP

-

(-)butaclamol

(+)NAN

(+)cyclazocine

DTG

(+)NAN

rimcazole

(+)NAN

subthreshold doses of the neuroleptics. In addition, we have recently observed

that BMY 14802 attenuated circling induced by (+)NAN in mice, an effect

mimicked by (-)butaclamol (see figure 5). Likewise, lyengar et al. (1989)

observed that BMY 14802 antagonized (+)NAN-evoked behaviors, such as

stereotypy and ataxia, in rats. Finally, lwamoto (1989) observed that

BMY 14802, as well as haloperidol and rimcazole, antagonized the so-called

syndrome,” particularly retropulsion and sideways circling, in rats. Data such as

these surely beg the question concerning the role of BMY 14802 as a potential

“antagonist.” A consideration of these data (collected in table 9) leads to the

realization that no other agent uniformly mimics the effects of BMY 14802,

although (+)NAN appears to consistently produce opposite effects. It would

appear to be premature at this time to term BMY 14802 a “antagonist,”

especially in view of the poorly characterized physiologic actions of ligands.

140
ADDITIONAL PROPERTIES OF BMY 14802

As part of our ongoing effort to identify compounds active in models of cerebral

ischemia and stroke, we routinely tested experimental agents in clinical

development for their ability to prevent nitrogen anoxia-induced lethality in rats,

to antagonize N-methyl-D-aspartic acid-(NMDA) induced convulsions in mice,

and to attenuate hippocampal neuronal damage after bilateral carotid occlusion

in the gerbil.

BMY 14802 exhibited activity in these models. Specifically, when given 15

minutes prior to a 1-minute exposure to a 100 percent nitrogen atmosphere, a

treatment that normally kills all rats so exposed, a 50 mg/kg, i.p., dose of

BMY 14802 protected all animals receiving the drug (see figure 6). Other

compounds, (e.g., haloperidol, sabeluzole, and ifenprodil) that bind to the site

(Taylor et al. 1990; Koe et al. 1990; Karbon et al. 1990; Contreras et al. 1990)

were also active in this model. In addition, 20 mg/kg, s.c., BMY 14802

significantly increased the dose of NMDA required to produce convulsions in

mice (see figure 7, table 10). The ligand sabeluzole was also active in this

model, although haloperidol was not. Moreover, Moon et al. (1990) have shown

that 25 mg/kg, i.p., BMY 14802 significantly attenuated the extent of

hippocampal lesions in gerbils subjected to bilateral carotid occlusion/

reperfusion (see table 11). Haloperidol also was active in this model, but

ifenprodil and sabeluzole were not. However, when given subacutely for 3

additional days after surgery, a significant attenuation in damage was seen with

sabeluzole. Further evidence of neuroprotective properties for BMY 14802

derived from the observation that this agent attenuated the NMDA-evoked

release of acetylcholine from rat striatal slices in vitro in a noncompetitive

fashion (Ryan et al. 1990, see above). Finally, Rao et al. (1990) have noted that

BMY 14802 reversed harmaline-induced elevations in cerebellar cyclic GMP levels.

That ligands could modulate NMDA-mediated events was suggested by

Monnet et al. (1988, 1989) who demonstrated that BMY 14802 blocked

enhancement by DTG of the excitatory effect of NMDA on CA3 hippocampal

pyramidal neurons (Debonnel et al. 1990). Unfortunately, further review of the

neuroprotective properties of other ligands, such as dextromethorphan,

(+)NAN, (+)3-PPP, DTG and its analogs, and haloperidol, lies outside the scope

of this article. However, the “ enigma” that these agents may play a role in

other physiologic responses in addition to schizophrenia is under active

investigation by many laboratories in addition to those referenced here.

141
TABLE 10. Effect of drug treatment on convulsant threshold (CD50) of NMDA

in mice

Drug

Dizocilpine

BMY 14802

Haloperidol

Sabeluzole

Dose

(mg/kg, s.c.) CD50, controls CD50, drug animals

1 117(103 to 131) Complete protectiona

20 98 (88 to 109) 143(114 to 360)

0.5 121 (100 to 139) 133(117 to 150)

5 113 (94 to 128) 179b

NOTE: CD50 values are for NMDA (mg/kg, i.p.) with 95 percent fiducial limits given in parentheses.

Values represent data obtained from 24 to 46 animals per group (from Taylor et al. 1990; Moon et

al. 1990).

aAnimals receiving dizocilpine exhibited no thrashing convulsions, and no deaths occurred after

receiving NMDA. However, those animals did engage in ‘popcorn” convulsions immediately

following administration of dizocilpine.

TABLE 11. Effect of drug pretreatment on extent of lesions of the

hippocampus of gerbils following bilateral carotid occlusion and

reperfusion

Drug

Dose

(mg/kg, i.p.) Control score Drug score

Dizocilpine 1 1.86 ± 0.09 (7) 0.78 ± 0.32 (6)a

BMY 14802 10 1.86 ± 0.09 (7) 1.50 ± 0.22 (5)

25 1.86 ± 0.09 (7) 0.69 ± 0.25 (7)a

Haloperidol 0.1 3.55 ± 0.45 (7) 2.28 ± 0.32 (6)a

lfenprodil 25 1.66 ± 0.24 (8) 1.19 ± 0.33 (5)a

Sabeluzole 5 3.10 ± 0.47 (7) 2.56 ± 0.43 (6)a

5

b

1.85 ± 0.30 (9) 0.81 ± 0.24 (9)a

NOTE: Data are mean score ± SEM for the number of animals given in parentheses. Drugs were

given at the indicated doses 1 hour before and after surgery. Artery clamps, used to achieve

occlusion, were released after a 15-minute period, and reperfusion/recovery proceeded for 4

subsequent days. Slices through the hippocampal level were evaluated on a scale of integers

denoting no damage (0) to total damage (4). Scores from slices for each animal were averaged, and

the mean of these average scores is reported here. Statistical comparisons were conducted using

the Mann-Whitney U-test, two-tailed (from Moon et al. 1990).

a

p < 0.05 vs respective control score.

bDose also given q.d. for three additional days.

142
FIGURE 6. Effect of selected agents on nitrogen-anoxia-induced lethality.

Groups of rats (8 to 72) were administered a drug, i.p., 30 or

15 minutes (BMY 14802 only, as indicated) prior to exposure to a

100 percent nitrogen atmosphere for 1 minute. This exposure kills

100 percent of untreated animals. The percent of treated animals

surviving was evaluated at 2 hours post-anoxia (from Taylor et al.

1990; Moon et al. 7990).

CAVEATS

Before reaching the conclusion that the many interesting, even exciting, effects

of BMY 14802 are solely due to its action at a sites, it is important to note two

additional properties of the agent. These are its ability to act as a partial agonist

at 5-HT1A receptors and the potential for its metabolism to a molecule with D-2

antagonist properties.

Like another pyrimidinylpiperazine, the anxiolytic drug buspirone (Taylor 1988)

BMY 14802 binds to 5-HT1A receptors (tables 12 and 13). That BMY 14802 may

143
FIGURE 7. Effect of selected agents on NMDA-induced convulsions. Groups

of mice (8 to 42) were administered a drug, S.C., 15 minutes prior

to doses of NMDA and observed for 60 minutes for clonic or tonic

convulsions. The dose producing convulsions in 50 percent of

animals, the CD50, and its 95 percent fiducial limits were

calculated using a log-logit transform and linear regression. See

table 10 for doses of drugs (from Taylor et al. 1990; Moon et al.

1990).

act as a 5-HT1A agonist has been suggested by the work of VanderMaelen and

Braselton (1990), who showed that the compound can inhibit the firing of

serotonergic cells in the dorsal raphe nucleus with a potency ratio compared to

buspirone that was close to that for binding to the 5-HT1A receptor (see

table 12). In addition, like buspirone, BMY 14802 can produce signs of the

serotonin syndrome in reserpinized rats.

144
TABLE 12. Comparison of activities of BMY 14802 and buspirone in selected

biological tests

Test Buspirone BMY 14802 Ratio

Inhibition of [3H]DPAT binding to

5-HT1A sites in vitro, IC50 (nM)

Inhibition of serotonergic cell firing

in rat dorsal raphe nucleus,

ED, (mg/kg, i.v.)

Production of 5-HT syndrome in

reserpinized rat, agonist indexC

(dose, mg/kg, p.o.)

Inhibition of 5-MeODMT-induced 5-HT

syndrome in reserpinized rat,

antagonist indexd (dose, mg/kg, p.o.)

Discriminable stimulus in pigeon

(mg/kg, i.m.)e

Inhibition of intraspecies ag

mice, ED50 (μmol/kg, i.p.)f

ression in

Reversal of neuroleptic-induced

catalepsy:

Trifluoperazine, ED50 (mg/kg, p.o.)

Haloperidol, drug dose x scoreh

Inhibition of (+)-[3H]-3-PPP binding to

sites in vitro, IC50 (nM)

Inhibition of conditioned avoidance

responding, ED, (mg/kg, p.o.)

Inhibition of apomorphine-induced

stereotypy in rats, ED, (mgkg, p.o.)

26

0.011 a

320

0.19b

0.29 (10) 0.65 (50)

0.85 (10) 0.40 (50)

0.056

9

5.6

64

3.6g

17

0.87 16.9

61 110

48i26

28j26

12

17

100

7

4.7

19.4

1.8

0.5

0.93

a

From VanderMaelen et al. 1986.

b

From VanderMaelen and Braselton 1990.

cAgonist index: a measure from 0 to 1.0, where 0 is no forepaw treading and 1.0 is forepaw treading

equivalent to that produced by 5 mg/kg, s.c., 5-MeODMT.

dAntagonist index: a measure ranging from 0 to 1.0, where 1.0 is complete antagonism of the forepaw

treading produced by 5 mg/kg, s.c., 5-MeODMT, and 0 is no antagonism of the forepaw treading.

e

From Vanecek et al. 1989.

f

From McMillen et al. 1988a.

g

From New et al. 1986.

h

From McMillen et al. 1988b.

i

From Yevich et al. 1983.

145
TABLE 13. Inhibition of in vitro radioligand binding by BMY 14802 and

BMY 14786

Binding R(+)BMY (±)BMY S(-)BMY BMY

site 14802 14802 14802 14786

28 110 310 140

5-HT1A 210 320 340 >1000

610 460 570 29

D-2 >1000 6400 >1000 490

NOTE: Data are mean Ki or IC50 (nM). binding assays were performed using (+)-[3H]3-PPP and

membranes from whole guinea pig brain. 5-HT1A, and D-2 binding assays were performed using

membranes from rat brain hippocampus, cortex, and striatum, respectively (from Behling et al.

1990).

Based on its ability to partially block the serotonin syndrome induced by

5-methoxydimethyltryptamine (5MeODMT) in reserpinized rats, BMY 14802

appears to behave as a partial agonist at 5-HT1A receptors. Moreover, in

pigeons, BMY 14802 can serve as a discriminative stimulus training cue to

which buspirone will generalize (Vanecek et al. 1989). McMillen et al. (1988a,

b) have suggested that the ability of buspirone and BMY 14802 to inhibit

intraspecies aggression in mice and to reverse haloperidol-induced catalepsy in

rats is a result of the affinity of these agents for 5-HT1A receptors and probable

partial agonist action at those sites (see table 12). Moreover, buspirone and

BMY 14802 also reverse trifluoperazine-induced catalepsy. For many of these

actions, the ratio of effective doses of buspirone and BMY 14802 is similar to

the ratio of their affinities for the 5-HT1A receptor (see table 12).

While these results suggest that at least some actions of BMY 14802 are produced

by its partial agonism at 5-HT1A receptors, they do not obviate a role for action at

the “receptor” in the potential antipsychotic effect of the drug. For instance,

Grimes et al. (1990 and submitted) recently showed that ligands, such as

(+)3-PPP and rimcazole, which do not possess affinity for 5-HT1A receptors, can

also reverse catalepsy induced by both trifluoperazine and haloperidol. While

buspirone is slightly more potent than (±)BMY 14802 in its affinity for binding

sites in vitro, it is less active in the traditional animal tests that identified BMY 14802

as an antipsychotic candidate, the inhibition of the conditioned avoidance response

and apomorphine-induced stereotypy (see table 12). In this regard it is useful to

recall that in a single trial in schizophrenics the efficacy of the 5-HT1A partial agonist

buspirone was doubtful (Sathananthan et al. 1975).

146
FIGURE 8. Structures of BMY 14802 and its precursor, BMY 74786, and of

haloperidol and its metabolite, reduced haloperidol.

BMY 14802 was originally prepared by reduction of a ketone precursor,

BMY 14786 (figure 8). This reaction is analogous to the reduction of haloperidol

to the carbinol metabolite formed in humans (figure 8; Forsman and Larsson

1978). Since reduced haloperidol may be oxidized to haloperidol in humans

(Midha et al. 1987) it was of interest to us to characterize the pharmacologic

profile of BMY 14786 and to determine if significant levels are formed in vivo

after administration of BMY 14802 in order to evaluate if this compound might

contribute to the in vivo effects seen after administration of the parent

compound.

The receptor binding profile of BMY 14786 is compared to that for the resolved

enantiomers of BMY 14802 as well as the racemic mixture in table 13. Like

BMY 14802, BMY 14786 exhibits affinity for the binding site. This observation

is in parallel with the recent finding of Bowen et al. (1990) that both haloperidol

and reduced haloperidol exhibit affinity for binding sites. Unlike BMY 14802,

BMY 14786 has low affinity for 5-HT1A receptors but high affinity for

adrenergic receptors. Finally, BMY 14786 exhibits modest affinity for D-2

dopamine receptors.

The behavioral consequences of this different profile for BMY 14786 are shown

in table 14. As previously noted, the R dextrorotatory enantiomer of BMY 14802

is more potent than S(-)BMY 14802 in inhibition of the conditioned avoidance

147
response and reversal of trifluoperazine-induced catalepsy, whereas

S(-)BMY 14802 is more potent in inhibition of apomorphine-induced stereotypy

(Taylor et al. 1989). BMY 14786 is about as potent as S4(-)BMY 14802 in

inhibition of the conditioned avoidance response and much less potent than this

enantiomer in inhibition of apomorphine-induced stereotypy. At high doses

BMY 14786 produces catalepsy in rats. Because such a finding might be of

concern if BMY 14786 were formed in vivo, we undertook an investigation on

the formation of bioactive metabolites.

After acute administration of BMY 14862, a serum radioreceptor assay for D-2

dopamine binding activity as well as ex vivo binding studies of D-2 dopamine

binding levels in the striata of rats at behaviorally relevant doses and time

points failed to support the hypothesis that significant levels of such metabolites

were formed (Taylor et al. 1985). Moreover, chronic administration of

BMY 14802 failed to alter D-2 dopamine receptor binding (table 4).

TABLE 14. Effects of BMY 14802 and BMY 14786 in selected behavioral tests

Test

R(+)BMY (±)BMY S(-)BMY BMY

14802 14802 14802 14786

Inhibition of 22a26 (20 to 35) 39 (30 to 50) 36 (27 to 49)

conditioned

avoidance

responding

Inhibition of 44 (38 to 50) 33 (23 to 48) 25 (17 to 37) 59 (48 to 73)

apomorphineinduced

stereotypy

Induction of >50b

>100b

>50b

144a

catalepsy

Reversal of 11 (7 to 19) 17 (11 to 26) 38 (27 to 54) not tested

trifluoperazineinduced

catalepsy

NOTE: Data are ED50 values (mg/kg. p.o.), with 95 percent fiducial limits given in parentheses,

for tests in rats (from Behling et al. 1990).

aDistribution of data not amenable to calculation of fiducial limits

bNo catalepsy observed at these doses.

148
FIGURE 9. Effect of administration of BMY 14802 or BMY 14786 on levels of

either agent in rat plasma. Groups of 4 to 6 rats received doses of

drug orally. Plasma fractions were prepared from blood samples

taken from jugular vein cannulae (50 mg/kg doses) or by cardiac

puncture (10 mg/kg doses). BMY 74802 and BMY 14786 were

assayed by reverse phase HPLC with fluorescence detection.

Levels of BMY 14802 are depicted as open symbols, while levels

of BMY 14786 are depicted as filled symbols. Dosing occurred

with R(+)BMY 14802 (upright triangles), racemic BMY 14802

(squares), S(-)BMY 14802 (inverted triangles), or BMY 14786

(circles).

More recently we have directly assayed the plasma of rats for levels of

BMY 14786 and BMY 14802 after administration of either agent (figure 9).

These studies have revealed that levels of BMY 14802 and BMY 14786 found

after dosing with the resolved enantiomers or the racemate appear to be dose

proportional. In addition BMY 14802 was found in plasma after administration of

149
the following drugs (descending order): R(+)BMY 14802, (±)BMY 14802,

S(-)BMY 14802, and BMY 14786. Furthemore, BMY 14786 was found in

plasma after administration of the same drugs, but there was a reversal in the

order of these drugs with respect to how much BMY 14786 was detected.

These observations permitted us to conclude that BMY 14802 and BMY 14786

are reversibly metabolized in the rat.

Recent phase 1 safety, tolerance, and pharmacokinetic studies in humans

demonstrated that quantifiable plasma concentrations of BMY 14786 were

observed only after higher doses of (±)BMY 14802 (500 mg or more, in single

doses), and these levels were 5 percent to 8 percent of the corresponding

BMY 14802 concentrations (Shukla et al. 1990). Levels of BMY 14786 below

10 percent of those of BMY 14802 were not detected in radioreceptor assays

(Taylor and Behling, unpublished findings). In toto, we conclude that formation

of biologically relevant amounts of an active metabolite of BMY 14802 does not

occur and that the pharmacologic profile observed to date can only be attributed

only to the properties intrinsic to the parent compound.

SUMMARY

BMY 14802 was identified as a potential antipsychotic drug in traditional model

systems, and this identification was confirmed in modem behavioral and

electrophysiological systems. The drug appears to be atypical as an

antipsychotic in its lack of activity in models predictive of the potential to

produce extrapyramidal side effects and tardive dyskinesia. Indeed, this

suggestion is corroborated by clinical findings to date.

The atypical profile of BMY 14802 extends to its neurochemical actions and

appears to find its basis in regionally selective, indirect modulation of the

dopamine system. Furthermore, BMY 14802 exhibits interactions with binding

sites in vitro and in vivo, a notion supported by data from neurophysiological,

behavioral, and biochemical investigations. BMY 14802 also appears to be

neuroprotective in some model systems and may have utility in the treatment of

stroke (Boissard et al. 1991). BMY 14802 appears to interact with 5-HT1A

receptors, but this interaction does not seem to contribute significantly to the

potential antipsychotic actions of the drug. Moreover, the formation of active

metabolites of BMY 14802 does not appear to occur in animals or humans to an

extent of physiological or behavioral relevance. If clinically efficacious,

BMY 14802 may treat the symptoms of schizophrenia by a mechanism novel

150
for antipsychotic drugs: regionally selective, indirect modulation of dopaminergic

systems by specific interaction at sites.

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155
ACKNOWLEDGMENTS

Graphics and artwork were provided by J. Stockla and M. Haas.

AUTHORS

Duncan P. Taylor, Ph.D., Senior Research Fellow

Michael S. Eison, Ph.D., Director

Sandra L. Moon, Ph.D., Associate Director

Cam P. VanderMaelen, Ph.D., Research Fellow

Frank D. Yocca, Ph.D., Associate Director

Susan H. Behling, B.S., Associate Research Scientist

Christopher G. Boissard, B.S., Associate Research Scientist

John P. Braselton, B.S., Senior Research Scientist

Houston H. Davis, Jr., B.A., Assistant Research Scientist

Marlene N. Duquette, A.S., Laboratory Technologist

Raymond C. Lamy, B.S., Assistant Research Scientist

Judith M. Libera, B.S., Assistant Research Scientist

Elaine Ryan, Associate Research Scientist

Robert N. Wright, B.S., Associate Research Scientist

CNS Biology

Pharmaceutical Research Institute

Bristol-Myers Squibb Company

P.O. Box 5100

Wallingford, CT 06492-7660

R. Francis Schlemmer, Jr., Ph.D., Associate Professor

Department of Pharmacodynamics

College of Pharmacy

University of Illinois at Chicago

833 South Wood Street

Chicago, IL 60612

Umesh A. Shukla, Ph.D., Senior Research Scientist

Metabolism and Pharmacokinetics

Pharmaceutical Research Institute

Bristol-Myers Squibb Company

P.O. Box 4755

Syracuse, NY 13221-2000

156
Dennis J. Gallant, Ph.D.

Department of Pharmacology

College of Pharmacy

University of Connecticut

Storrs, CT 06268

157

Molecular Biology of PCP and NMDA

Receptors

Leslie Kushner, Michael V.L. Bennett, and R. Suzanne Zukin

L-Glutamate, the major excitatory neurotransmitter of the central nervous

system, activates at least five receptors (Foster and Fagg 1984; Cotman and

lversen 1987; Mayer and Westbrook 1987; Monaghan et al. 1989), which can

be distinguished by the selective agonists N-methyl-D-aspartate (NMDA),

kainate, -amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA),

2-amino-4-phosphonobutyrate (L-AP4), and trans-1-amino-cyclopentyl1,3-dicarboxylate (ACPD) (table 1). Although most of the excitatory amino acid

(EAA) receptors are coupled to ligand-gated cation channels, the

ACPD-selective receptor is apparently G-protein linked.

Of the channel-forming EAA receptors, the NMDA receptor is unique in that it is

permeable to Ca2+ and is blocked by physiologic concentrations of Mg2+ in a

voltage-dependent manner (MacDermott et al. 1986; Mayer et al. 1987). This

Ca2+-gating property of the NMDA receptor is thought to be responsible for its

participation in long-term potentiation (Lynch et al. 1983; Harris et al. 1984;

Wigström and Gustafson 1984; Collingridge and Bliss 1987) memory formation

(Smith 1987), excitotoxicity (Schwartz et al. 1983; Jansco et al. 1984; Choi

1985; Wieloch 1985; Rothman and Olney 1987), and ischemic neuronal

damage (Simon et al. 1984). In addition, the NMDA-activated channel has been

shown to contain the pharmacologic receptor for phencyclidine (PCP) and has

been implicated in endogenous psychosis.

Until recently, little was known about the molecular properties of the EAA

receptors. A putative kainate receptor cDNA has been cloned and sequenced

and shown to direct the translation of kainate-gated channels in Xenopus

oocytes (Hollmann et al. 1989). Based on its predicted amino acid sequence,

the kainate receptor subunit has a relative molecular mass (Mr) of 99,800. This

mass is much larger than the sizes predicted for subunits of other cloned

ligand-gated channel receptors (the largest reported is 67 kD (Boulter et al.

159
TABLE 1. Excitatory amino acid receptors

Competitive Channel Allosteric Allosteric Antagonist at

Receptor Agonist antagonist blocker inhibitor potentiator potentiation site

NMDA NMDA CPP Mg2+ Zn2+ glycine 7-chlorokynurenate

L-glutamate CGS-1975 PCP D-serine cydoleuclne

L-aspartate D-AP5 MK-801 HA-966

lbotenate D-AP7

Kainate domoate CNQX

kainate DNQX

L-glutamate

AMPA AMPA GDEE

quisqualate CNQX

L-glutamate DNQX

L-AP4 L-AP4)

qulsqualate

L-glutamate

ACPD trans-ACPD

qulsqualate

L-glutamate

NOTE: NMDA=N-methyl-D-aspartate; AMPA=a-amino-3-hydroxy-5-methyl-4-isoxazpionate;

L-AP4=L-2-amino-7-phosphonoheptanoate; ACPD=1-amino-cyclopentyl-1,3-dicarboxylate;

CPP=3-3(2-carboxypiperazine-4-yl)propyl-1-phosphonate; CGS-1975=cis-4-phosphonomethyl-2-

piperidine carboxylic acid; D-AP5=D-2-amino-5-phosphonovalerate; D-AP7=D-2-amino-7-

phosphonoheptanoate; CNQX=6-cyano-7-nitroquinoxaline-2,3dione; DNQX=6,7-

dinitroquinoxaline-2,3-dione; GDEE=glutamate diethyl ester; PCP=phencyclidine;

MK-801=dibenzocycloheptenelmine; HA-966=3-amino-1-hydroxypyrrolid-2-one.

1987)), although it agrees quite well with the estimated size of the largest

protein component of the NMDA receptor, as determined by photoaffinitylabeling experiments in brain (Haring et al. 1987) and NCB-20 cells (Haring et

al. 1990a). Like other ligand-gated channels, this putative kainate receptor is

predicted to have four membrane-spanning domains and has a large N-terminal

extracellular domain that contains several putative glycosylation sites, a

“cys-cys loop” with partial homology to other ligand-gated channels, and a

cytoplasmic C-terminal domain (Barnard et al. 1987; Greeningloh et al. 1987).

Although direct comparison of the putative kainate receptor cDNA sequence

with the sequences of the subunits of other ligand-gated channel receptors

revealed no significant homology (Hollmann et al. 1989), allowance for

conservative substitutions and moderate length deletions give an alignment

with considerable homology (E. Barnard, personal communication). Thus, it

probably belongs to the family of other ligand-gated channel receptors. The

160
findings for the mammalian brain kainate receptor may provide important clues

to the structural features of the NMDA receptor.

The kainate-selective EAA receptor can be distinguished pharmacologically

from NMDA and quisqualate receptors. The kainate receptor is insensitive to

selective antagonists of the NMDA receptor. Although it is activated by

quisqualate (Evans et al. 1987) it can be distinguished from quisqualateactivated receptor subtypes by its sensitivity to domoate and lack of sensitivity

to the quisqualate-receptor subtype-specific agonists (see below) or the

antagonist glutamate diethyl ester (GDEE) (Haldeman and McLennan 1972;

London and Coyle 1979; McLennan and Liu 1982; Slevin et al. 1983). The

kainate receptor is a ligand-gated cation channel that is permeable to Na+ and

K

+

. Autoradiography of radioligand binding sites (Monaghan et al. 1983;

Greenamyre et al. 1985; Monaghan et al. 1985) patch clamp (MacDonald et al.

1987; Cull-Candy et al. 1988; Huettner and Bean 1988), and oocyte expression

studies (Lerma et al. 1989b) have shown that this receptor is distinct from other

excitatory amino acid receptors.

Quisqualate activates three distinct receptors or transducing mechanisms,

which can be distinguished by specific ligands as well as by functional

properties. GDEE (Krogsgaard-Larsen et al. 1980) 6-cyano-7-nitroquinoxaline2,3-dione (CNQX), and 6,7-dinitroquinoxaline-2,3-dione (DNQX) (Honoré et al.

1988) inhibit the AMPA type of quisqualate receptor. (CNQX and DNQX also

block the kainate receptor but are more specific than GDEE in not blocking the

NMDA receptor (Blake et al. 1988; Drejer and Honor 1988; Honoré et al. 1988).)

This receptor is a ligand-gated cation channel that gates Na+ and K+. L-AP4

acts at a site distinct from those activated by NMDA, kainate, or AMPA (Davies

and Watkins 1982; Yamamoto et al. 1983; Lanthom et al. 1984). However,

complete characterization of this site has not been reported. A third receptor,

activated by quisqualate, is coupled to inositol phosphate metabolism and has

been referred to as a metabolotropic receptor (Nicoletti et al. 1986; Sugiyama

et al. 1987). The glutamate analog ACPD is a specific agonist for this receptor.

The NMDA receptor is unique among ion-channel-forming excitatory amino acid

receptors in that it gates Ca2+ as well as Na+ and K+ (MacDermott et al. 1986;

Mayer et al. 1987). It contains several pharmacologically distinct binding sites,

which may have regulatory functions. These functions include a binding site

within the channel for Mg2+, which blocks the channel in a voltage-dependent

manner (Mayer et al. 1984; Nowak et al. 1984; Ascher and Nowak 1988); an

inhibitory binding site for Zn2+ and other divalent cations, which shows no

161
voltage dependence (Peters et al. 1987; Westbrook and Mayer 1987; Christine

and Choi 1990); an inhibitory binding site for Zn2+, which blocks the channel in

a voltage-dependent manner (Christine and Choi 1990); a site at which glycine

and several other small amino acids bind and act as coagonists (i.e., this site

must be occupied for glutamate or NMDA to cause channel opening) (Johnson

and Ascher 1987; Kleckner and Dingledine 1988; Lerma et al. 1990); and at

least one site for polyamines, which potentiate the maximum response and

increase the affinity of ligands for the glycine binding site (Sacaan and Johnson

1989; McGurk et al. 1991).

Considerable evidence suggests that the PCP receptor, which mediates the

psychotomimetic effects of PCP derivatives, opioids, and the dioxalanes (see

Zukin and Zukin 1988), is a site within the NMDA receptor channel complex.

PCP receptor ligands block NMDA-induced currents in a voltage- and

use-dependent manner (Honey et al. 1985; MacDonald et al. 1987; Bennett et

al. 1988; Huettner and Bean 1988; Kushner et al. 1988a; Lerma et al. 1989a,

1991) and specifically antagonize NMDA-induced neurotransmitter release

(Snell and Johnson 1985,1986; Drejer and Honoré 1987; Jones et al. 1987).

The antagonism of NMDA receptors by PCP receptor ligands is noncompetitive

with respect to NMDA (Honey et al. 1985; Martin and Lodge 1986; Javitt et al.

1987; Lerma et al. 1991). The drug potencies for blocking activity correlate with

potencies in receptor binding (Anis et al. 1983; Berry et al. 1984; Vincent et al.

1979; Zukin and Zukin 1979; Sircar et al. 1987) and behavioral assays

(Holtzman 1980; Hampton et al. 1982; Hayes and Balster 1985). NMDA and

glycine enhance binding of radiolabeled PCP receptor ligands, whereas

competitive antagonists of the NMDA binding site inhibit binding to the PCP

receptor in rat brain membranes (Javitt et al. 1987; Reynolds et al. 1987;

Benavides et al. 1988; Johnson et al. 1988; Kloog et al. 1988) or to solubilized

rat brain PCP receptors (Ambar et al. 1988; Haring et al. 19906). NMDA

receptors (labeled by [

3H]3-(2-carboxypiperazin-4-yl)propyl-1-phosphate (CPP)

or [3H]glutamate) and PCP receptors (labeled by [3H]1-[1-(2-thienyl)cyclohexyl]-

piperidine (TCP)) are colocalized throughout rat forebrain and midbrain

(Maragos et al. 1986; Jarvis et al. 1987; Maragos et al. 1988). [3H]Azido-PCP

photoaffinity labels five distinct polypeptides in NCBQO cell membranes;

binding to two of the polypeptides is inhibited by D-amino-5-phosphonovaleric

acid (D-AP5), a competitive inhibitor of the NMDA site (Haring et al. 1990a).

NMDA and PCP receptors are always coexpressed in Xenopus oocytes into

which rat brain mRNA, rat hippocampus or frontal cortex mRNA, NCB-20 cell

mRNA, or size-fractionated mRNA is injected (see below). Thus, it is apparent

162
that the two receptors (NMDA and PCP) exist as part of the same

supramolecular complex, with the PCP binding site located within the channel.

The NMDA receptor has attracted considerable attention because of its

proposed roles in long-term potentiation (Lynch et al. 1976; Stringer et al. 1983;

Harris et al. 1984; Wigström and Gustafson 1984; Morris et al. 1986;

Collingridge and Bliss 1987) memory formation (Morris et al. 1986; Morris

1988; Lincoln et al. 1988) and developmental structuring (Singer et al. 1986;

Stryker and Harris 1986; Cline and Debski 1987; Rauschecker and Hahn 1987).

In addition, it is involved in excitotoxicity (Schwartz et al. 1983; Simon et al.

1984; Wieloch 1985; Olney et al. 1987) and epilepsy (Herron et al. 1985;

Meldrum 1985; Dingledine et al. 1986; Mody and Heinemann 1987) and may

play a role in Alzheimer’s disease (Greenamyre et al. 1985, 1987; Geddes et al.

1986; Maragos et al. 1987) Huntington’s disease (Schwartz et al. 1983;

Greenamyre et al. 1985; Young et al. 1988), and schizophrenia (Kim and

Kornhuber 1982; Contreras et al. 1987; Zukin and Javitt, this volume). The

functional significance of the NMDA receptor has prompted several laboratories

to investigate the molecular nature of this receptor. This chapter will review

recent studies that used molecular biological approaches to gain a greater

understanding of the NMDA receptor. In particular, use of the Xenopus laevis

oocyte expression system and clonal cell lines for the study of this receptor will

be described.

EXPRESSION OF NMDA RECEPTORS IN XENOPUS OOCYTES INTO

WHICH RAT BRAIN mRNA HAS BEEN INJECTED

The Xenopus oocyte is a self-contained expression system that correctly

translates exogenous mRNAs encoding membrane proteins and is capable of

eukaryotic posttranslational modifications, including processing of precursor

molecules (Ghysdael et al. 1977) phosphorylation (Gedamu et al. 1978)

glycosylation (Colman et al. 1981; Lund et al. 1986) and assembly and

insertion of subunits into the plasma membrane with the correct orientation

(Sumikawa et al. 1981; Parker et al. 1985). Moreover, the oocyte provides a

precisely controlled environment for the study of receptor structure and function.

In the absence of protein purification and sequencing information, the Xenopus

oocyte system can be used for expression cloning (Masu et al. 1987; Julius et

al. 1988; Hollmann et al. 1989).

Recently two groups reported the expression of NMDA-activated channels in

Xenopus oocytes following injection of exogenous mRNA isolated from adult rat

163
brain (Verdoorn et al. 1987; Kushner et al. 1988a), primary cell cultures derived

from fetal rat brain (Verdoom et al. 1987) and a clonal cell line of neuronal

origin (Lerma et al. 1989a). NMDA-activated channels were distinguished from

other excitatory amino acid receptors by their electrophysiologic and

pharmacologic characteristics (Kushner et al. 1988a; Verdoorn and Dingledine

1988; Lerma et al. 1989b). NMDA application (with glycine) evoked an inward

current after short latency that rapidly desensitized to a steady level (Kushner et

al. 1988a; Lerma et al. 1989b). Kainate produced a more slowly rising,

nondesensitizing current also of short latency. Quisqualate induced both a

small, early, inward current and a much larger current characterized by long

latency and large oscillations, indicative of second-messenger-mediated

activation of endogenous Ca2+-activated Cl- channels (Sugiyama et al. 1987;

Kushner et al. 1988a; Verdoom and Dingledine 1988). These findings indicate

that rat brain mRNA directs the expression of NMDA-, kainate-, and two kinds

of quisqualate-activated excitatory amino acid receptors in the Xenopus oocyte.

The pharmacologic characteristics of the NMDA receptor expressed in the

oocyte were similar to the characteristics of the neuronal receptor. At a holding

potential of -60 mV, the half-maximal response to NMDA (in the presence of

10 μM glycine) was elicited at 16 μM (figure 1A). Oocytes that were not

subjected to injections or received injections of water did not respond to NMDA.

Glycine markedly potentiated the effect of NMDA (Verdoorn et al. 1987;

Kushner et al. 1988a) with a half-maximal effective concentration (EC50) of

0.19 μM (figure 1 B). There was essentially no response to 50 μM NMDA alone,

and 100 μM glycine alone had negligible effect. Glycine (or other agonists at the

glycine site) appeared to be an absolute requirement for the activation of the

NMDA receptor in Xenopus oocytes (Kleckner and Dingledine 1988; Lerma et

al. 1990), and rapid desensitization could not account for the failure of NMDA

responses in the absence of glycine (Lerma et al. 1990). The NMDA-activated

current could be blocked by the selective NMDA antagonist D-AP5 (Verdoorn et

al. 1987; Kushner et al. 1988a) (concentration for half-inhibition, IC50=3.3 μM)

(figure 1C) but not by its inactive enantiomer L-AP5. D-AP5 acted as a

competitive inhibitor in that it shifted the NMDA dose-response curve rightward

(i.e., the potency was decreased) without changing the slope or maximum

response (i.e., Hill coefficient [n] and Imax are constant) (Lerma et al. 1991);

competitive inhibition was also demonstrated by Schild analysis (Verdoorn et al.

1988). Inhibition by Zn2+ was noncompetitive (i.e., Imax was decreased but EC50

of NMDA was unchanged) (Lerma et al. 1991). The NMDA responses were

blocked by Mg2+ in a voltage-dependent manner (Verdoorn et al. 1987; Kushner

et al. 1988a). When 0.1 μM Mg2+ was included in the perfusion medium, the

164
FIGURE 1. Dose-response relations for N-methyl-D-aspartate- (NMDA)

induced currents in Xenopus oocytes subjected to injections of rat

brain mRNA (Kushner et al. 1988a). Cells were voltage clamped

at a holding potential of -60 mV. Drugs were applied in the bath.

Error bars indicate SEMs from three oocytes; several applications

were made to each oocyte at each concentration.

(A) NMDA-induced current in the presence of 10 μM glycine.

Responses (sample records inset) are graphed as both peak (—)

and steady-state (- - -) values. (B) Glycine potentiation of the

response to 50 μM NMDA normalized with respect to the

maximum response at 100 μM glycine. (C) D-AP5 inhibition of the

response to 50 μM NMDA with 10 μM glycine. (D) Mg2+ inhibition

of the response to 50 μM NMDA with 10 μM glycine.

relationship between the NMDA-induced current and voltage exhibited a

negative slope between holding potentials of -80 and -30 mV after which the

current decreased linearly with voltage to a reversal potential of approximately

-10 mV. When Mg2+ was omitted from the medium, the NMDA-induced current

165
FIGURE 2. Use dependence of PCP block and recovery from block of

NMDA-induced currents in Xenopus oocytes subjected to

injections of rat brain mRNA (unpublished data, with J. Lerma).

(A) PCP was applied 30 seconds before NMDA, and the response

was partially blocked (the plateau more than the peak, consistent

with use-dependent block). A test application of NMDA at

20 seconds showed the degree of block; further applications

showed recovery. (B) After full recovery PCP was applied for

2 minutes before NMDA. The initial response was a/most identical

to that in A, indicating that block in the absence of NMDA

developed very slowly. The continuous trace in the lower record

shows that after 2 minutes recovery was only slightly greater than

after 20 seconds as in A and markedly less than after 2 minutes in

A, when there had been two prior NMDA applications.

166
was a nearly linear function of voltage. The IC50 for Mg2+ was 8.3 μM at a

holding potential of -60 mV (figure 1 D). Inhibition by Mg2+ was noncompetitive

in that Imax was decreased, but EC50 of NMDA was unchanged (Lerma et al.

1991).

These results show that the distinguishing features of neuronal NMDA channels

are reproduced by NMDA receptors expressed in Xenopus oocytes following

injection of rat brain mRNA. In this system, as in spinal, striatal, cortical, and

hippocampal neurons, NMDA receptors are activated by NMDA, potentiated by

glycine, and blocked competitively by the antagonist D-AP5 and noncompetitively by Mg2+, which blocks in a voltage-dependent manner, and by Zn2+

.

To determine whether the NMDA channel expressed in Xenopus oocytes is

functionally associated with the PCP receptor, PCP receptor ligands were

tested for their ability to modulate NMDA-activated currents (Kushner et al.

1988a; Lerma et al. 1991). Application of PCP blocked the NMDA-evoked

current in a use-dependent manner (figure 2). When PCP was applied together

with NMDA, the initial peak of inward current was either essentially unchanged

or slightly reduced from the response to NMDA alone. Although the rate of

onset of block at a given concentration of PCP was faster at higher NMDA

concentrations, the degree of block was unaffected by the NMDA concentration

(Lerma et al. 1991). PCP blocked very slowly in the absence of NMDA;

responses to NMDA after a 20-second and a 2-minute exposure to PCP were

nearly identical and only slightly smaller than when NMDA and PCP were

applied together (Lerma et al. 1991). Recovery from PCP block also occurred

very slowly in the absence of NMDA; responses after a 2-minute saline wash

were only slightly greater than after a 20-second wash (Lerma et al. 1991).

Thus, both PCP block of the NMDA-induced current and relief of block were use

dependent, indicating that the PCP receptor is a site within the channel of the

NMDA receptor, which permits entry or egress of PCP only if the channel is

open. This use dependence is consistent with the action of PCP receptor

ligands on neurons (MacDonald et al. 1987; Huettner and Sean 1988).

The PCP receptor ligands tested were able to block, in a use-dependent

manner, the NMDA-evoked response in a rank order of potency that closely

matched the rank order of potency in binding to rat brain membranes (Vincent

et al. 1979; Zukin and Zukin 1979; Sircar et al. 1987) in electrophysiological

studies of neural tissues (Anis et al. 1983; Berry et al. 1984; Honey et al. 1985)

and in behavioral studies (Holtzman 1980; Hampton et al. 1982; Hayes and

Bolster 1985). The PCP receptor was stereoselective in that NMDA responses

167
were reduced by the active ligand dexoxadrol but hardly affected by its inactive

stereoisomer levoxadrol at the same concentration (Kushner et al. 1988a).

In addition, both PCP and dibenzocyclohepteneimine (MK-801), an extremely

potent PCP receptor ligand, inhibited NMDA-evoked currents without effect on

quisqualate- or kainate-evoked currents (Kushner et al. 1988a; Lerma et al.

1989b). These data indicate that many of the pharmacologic properties of PCP

receptors are reproduced in the oocyte system. Most important, in the large

number of oocytes expressing NMDA receptors that were tested, including

oocytes that received injections of mRNA from specific brain regions, PCP

receptor ligands reduced or blocked NMDA-induced current, demonstrating

coexpression of these receptors. Moreover, in size fractions prepared from rat

brain mRNA, the NMDA receptors always cofractionated with PCP receptors.

These results demonstrate that in this system PCP receptor ligands are acting

at the receptor implicated in PCP’s behavioral effects and that PCP and NMDA

receptors are always coexpressed in Xenopus oocytes. This study provides

important direct evidence in support of the hypothesis that the PCP receptor is

a binding site within the channel of the NMDA receptor protein.

DEMONSTRATlON OF PCP RECEPTORS IN THE

NEUROBLASTOMA-BRAIN HYBRID CELL LINE NCB-20

Clonal cell lines are particularly useful for purification and gene-cloning studies

of neurotransmitter receptors in that they provide a homogeneous population of

cells that can be grown in large quantity and subjected to precise physicochemical manipulation. In the search for a clonal cell line that possesses NMDA

receptors, we examined the receptors of the neuroblastoma-brain hybrid cell

line NCB-20. NCB-20 cells had been reported to have opioid receptors and a

benzomorphan-specific binding site on their membranes (McLawhon et al.

1981; West et al. 1983). Benzomotphans bind to opioid receptors and

receptors as well as to PCP receptors. Largent et al. (1986) demonstrated the

presence of receptors on the NCB-20 cell membrane. An important

unanswered question was whether NCB-20 cell membranes also have the PCP

receptor site associated with NMDA-activated channels, as in neuronal tissue.

Thus, highly specific radioligands were used to measure the density of opioid,

and PCP receptors on NCB-20 cells in quantitative receptor binding assays

(Kushner et al. 1988b).

168
NCB-20 cells were shown to possess high-affinity opioid receptors, but not

opioid receptors, on their cell membrane (Kushner et al. 19886). In

addition, these cells possessed distinct sites for opioids and PCP derivatives.

One site was labeled by [3H](+)N-allylnormetazocine ([3H](+)SKF 10,047).

Scatchard analysis of [3H](+)SKF 10,047 binding to NCBQO cell membranes

revealed an apparent single class of sites with a binding affinity of 69 nM and

receptor density of 4,100 fmol/mg protein (figure 3A). The rank order of potency

of drugs at that site was

(+)3-(3-hydroxyphenyl)- N-(1-propyl)piperidine ((+)3-PPP) > haloperidol >

(+)SKF 10,047 > (±)ethylketocyclazocine (EKC) > (μ)bremazocine >

N-[1-(2-thienyl)cyclohexyl]piperidine (TCP) > dexoxadrol.

This site was similar in its ligand selectivity to the haloperidol-sensitive

receptor of rat brain, confirming the previous report of Largent et al. (1986).

The other site was labeled by the potent phencyclidine derivative [3H]TCP.

Saturation curves and Scatchard analysis of equilibrium binding of the PCP

receptor-specific ligand [3H]TCP to NCB-20 cell membranes revealed binding to

be monophasic, saturable, and of high affinity (KD=335 nM, Bmax=9,300 fmol/mg

protein) (figure 3B). This density was approximately five times the density found

in rat brain and is equivalent to about 60,000 sites per cell. The rank order of

potency of drugs at this site was

TCP > (+)3-PPP > PCP > dexoxadrol > haloperidol > cyclazocine >

levoxadrol > (+)SKF 10,047;

ligands were inactive. Stereoselectivity at this site was demonstrated in

that dexoxadrol was more potent than levoxadrol. The drug potency profiles

characterized by displacement of [3H](+)SKF 10,047 and [3H]TCP binding fit

very well to a two-site model. Collectively, these data suggest that NCB-20 cell

membranes have a PCP receptor-like site and a haloperidol-sensitive o-like site.

Further analysis of [3H]TCP binding to NCB-20 cell membranes suggested the

presence of two distinct PCP sites that can be distinguished by their affinities

for MK-801 (Hating et al. 1990a). This finding is consistent with the finding that

PCP receptors in hindbrain exhibit an approximately tenfold lower affinity for

PCP than do forebrain PCP receptors (Haring et al. 1987), suggesting the

presence of different PCP receptor subtypes in brain. The NCB-20 cell line is

derived from a neoplasm of mouse spinal cord (Dawson et al. 1983) and would

be expected to have receptors that resemble more closely those of spinal cord

and hindbrain.

169
FIGURE 3. Saturation plots and Scatchard plots (insets) of specific binding to

PCP and receptors of NCB-20 cell membranes (Kushner et al.

1988b). Membranes (0.6 mg of protein/ml) were incubated with

[

3H]TCP (0.01 to 1 μM) (A) or (+)-[3H]SKF 10,047 (1 to 300 nM)

(B) in 5 mM Tris buffer, pH 7.4 at 4 °C for 45 minutes in the

presence or absence of unlabeled ligand. The Scatchard plots

were fit by a straight line using linear regression analysis. These

data are from a representative experiment that was performed in

triplicate and replica ted a minimum of three times.

170
To investigate the possibility that the [3H]TCP binding site of NCB-20 cell

membranes is the PCP receptor site of the NMDA-gated channel, NCB-20 cells

were tested electrophysiologically for their responses to NMDA. The majority of

tested passages failed to respond to NMDA (see Kushner et al. 1988b).

However, high-affinity, stereoselective binding to PCP receptors was observed

in all cell passages tested, and mRNA isolated from NCB-20 cells lacking

functional NMDA channels did cause expression of functional NMDA channels

in oocytes (Lerma et al. 1989a). In addition, [3H]azido-PCP photoaffinity labeled

five distinct polypeptides in NCB-20 cell membranes, and such binding to two

specific polypeptides (Mr 68K and 90K) was inhibited by inclusion of D-AP5 or

MK-801 in the binding reaction (Haring et al. 1990a). These data suggest that

expression of functional NMDA receptors in NCB-20 cells may be defective or

subject to regulation. The NCB-20 cell line is the only cultured cell line that has

been shown to have PCP receptors, although evidently not functional

NMDA-activated channels.

EXPRESSION OF THE NMDA RECEPTOR IN XENOPUS OOCYTES INTO

WHICH mRNA FROM NCB-20 CELLS WAS INJECTED

To further characterize the excitatory amino acid receptors of NCB-20 cells, we

injected poly(A+)RNA prepared from these cells into Xenopus oocytes. After

several days, inward currents were evoked by applying glutamate and NMDA

but not by kainate, quisqualate, or aminobutyric acid (GABA) (Lerma et al.

1989a). At a holding potential of -60 mV, NMDA (with 10 μM glycine)

application evoked an inward current at short latency that partially desensitized,

a response identical to that obtained after injection of rat brain mRNA (Kushner

et al. 1988a). No responses were observed for kainate (500 μM), quisqualate

(10 μM), or GABA (100 μM), compounds that evoke pronounced currents in

oocytes into which rat brain mRNA had been injected, or for glycine (100 μM) in

the absence of NMDA (Lerma et al. 1989a) (figure 4). Glutamate (100 μM)

activated an early current that resembled the response to NMDA. Of four mRNA

preparations, all encoded NMDA receptors. These results indicate that the

NCB-20 cells contain the precursor mRNA necessary to encode NMDA

receptors.

Properties of NCB-20 cell NMDA receptors expressed were typical of neuronal

NMDA receptors. The relation between the steady level of inward current and

NMDA concentration indicated a single-component response with an apparent

affinity (KD) of 22 μM and Hill coefficient of 1.3. As in neurons, glycine markedly

potentiated the response to NMDA (EC50 about 0.2 μM with 50 μM NMDA). The

171
NMDA-activated current was blocked by the competitive antagonist D-AP5 (at

50 μM NMDA, IC50 2.5 μM). NMDA currents were also blocked by Mg2+ at

inside negative potentials and by Zn2+.

PCP and MK-801 blocked NMDA-activated currents in a use-dependent

manner. Application of PCP together with NMDA resulted in an initial peak of

inward current that was little different from the response to NMDA alone.

Application of PCP alone for up to several minutes had no effect on subsequent

NMDA-evoked responses. Recovery from PCP block occurred rapidly in the

presence of agonist but very slowly in the absence of agonist. Consistent with

the results for rat brain NMDA receptors expressed in oocytes, these data

indicate that the channel of the NCB-20 cell NMDA receptors expressed must

be open in order for PCP receptor ligands to block or exit from it. Thus, the PCP

receptor encoded by NCB-20 mRNA appears to be within the NMDA channel.

In summary, the NMDA receptor, but not the kainate or quisqualate receptor, is

expressed in the oocyte after injection of NCB-20 cell mRNA. The NMDA

receptors expressed possess the salient pharmacological features of the

FIGURE 4. Expression of neurotransmitter receptors in Xenopus oocytes

injected with NCB-20 cell mRNA (Lerma et al. 1989a). Records

from an oocyte clamped at -60 mV, and subjected to injection of

mRNA 6 days previously. Horizontal bars indicate duration of drug

application. The small, brief, inward currents were due to

endogenous Ca2+-activated chloride channels; these currents

disappeared in these oocytes after injection of EGTA.

172
neuronal NMDA receptor, including the apparent location of the PCP receptor at

a site within the channel.

The absence of expression of kainate and quisqualate receptors in the oocyte

suggests the absence of the encoding mRNA from NCB-20 cells. If NCB-20

cells do not have mRNA encoding these excitatory amino acid (EAA) receptors,

they would be a relatively pure source of mRNA encoding the NMDA type of

EAA receptor.

SUMMARY

The studies described demonstrate that rat brain mRNA directs the synthesis of

at least four types of functional EAA receptors in the Xenopus oocyte system,

whereas in this system NCB-20 cell mRNA directs the synthesis of only the

NMDA type of EAA receptor. The NMDA channel expressed in the oocyte,

using either rat brain mRNA or NCB-20 cell mRNA, exhibits the pharmacologic

properties of the neuronal receptor, including the functional association with the

PCP receptor located within the NMDA-gated channel. The demonstration that

mRNA isolated from NCB-20 cells lacking functional NMDA-activated channels,

but bearing PCP binding sites, can encode functional NMDA-activated channels

in the oocyte indicates some defect or regulating step in posttranslational

processing or insertion of the receptors into the plasma membrane in the cell of

origin. This is the only cell line known to (1) have PCP receptors that appear to

be associated with NMDA receptors and (2) provide a homogeneous,

self-replicating population of cells that can be manipulated genetically and by

changing the extracellular environment. Consequently, the NCB-20 cell line will

be useful for the study of the NMDA receptor and its expression.

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ACKNOWLEDGMENTS

The authors acknowledge the fine collaborative efforts of Drs. S.-G. Fan,

R. Hating, J. Lerma, D.C. Spray, and S.R. Zukin, whose work (as cited in the

text) contributed to this manuscript. M. Eufemio, R. Kokoska, and D. Olive

assisted in binding assays. L. Cipriani assisted in the preparation of oocytes for

injection.

AUTHORS

Leslie Kushner, Ph.D.

Department of Urology

Long Island Jewish Medical Center

The Long Island Campus of Albert Einstein College of Medicine

New Hyde Park, NY 11042

Michael V.L. Bennett, D.Phil.

Chairman

Department of Neuroscience

Albert Einstein College of Medicine

1300 Morris Park Avenue

Bronx, NY 10461

R. Suzanne Zukin, Ph.D.

Department of Neuroscience

Albert Einstein College of Medicine

1300 Morris Park Avenue

Bronx, NY 10461

183

Excitatory Amino Acid Neurotoxicity in the

Developing Brain

John W. McDonald and Michael V. Johnston

INTRODUCTION

Excitatory neurotransmission in the mammalian central nervous system is

mediated principally by glutamate and structurally related compounds.

Excitatory effects of glutamate are mediated via activation of at least three

subtypes of glutamate receptors named by their preferential agonists,

N-methyl-D-aspartate (NMDA), quisqualate, and kainate. In addition to the role

of excitatory amino acids (EAAs) in transynaptic information coding, recent

evidence suggests that EAAs are involved in a variety of other physiologic

actions, including regulation and maintenance of neuronal cytoarchitecture as

well as regulation of certain forms of learning and memory (long-term

potentiation [LTP]). EAAs also have several physiologic roles in development,

including the regulation of neuronal survival, dendritic and axonal structure,

synaptogenesis, and activity-dependent synaptic plasticity. However, excessive

activation of EAA receptors and subsequent overactivation of some of the same

molecular mechanisms that mediate the structural changes associated with

synaptic plasticity can lead to neuronal destruction and death. Our current

understanding of the molecular mechanisms that mediate the structural

changes associated with synaptic plasticity can lead to neuronal destruction

and death. Our current understanding of the molecular mechanisms that

contribute to synaptic plasticity and to excitotoxicity are discussed along with

work from our laboratory related to the pathophysiologic characteristics of

excitotoxicity during development. We also reviewed this area in a longer recent

article (McDonald and Johnston 1990) and several figures from that review are

reproduced here with permission.

185
PHYSIOLOGIC ROLES OF EXCITATORY AMINO ACIDS DURING

DEVELOPMENT

EAAs may serve several physiologic functions during development and in the

adult brain, such as promotion of neuronal survival, differentiation, and

regulation of activity-dependent synaptic plasticity (Ascher and Nowak 1987;

Tsumoto et al. 1987). EAAs may also act as neurotrophic factors during

development (Balazs et al. 1988; McDonald and Johnston 1990).

MECHANISMS OF EXCITATORY AMINO ACID NEUROTOXICITY

Excessive activation of the cellular mechanisms that participate in neuronal

signaling and neural plasticity can initiate a series of intracellular biochemical

events that cause neuronal damage (referred to as excitotoxicity).

Overstimulation of each of the EAA receptor subtypes can initiate a cascade of

events resulting in neuronal injury in the developing and adult brain. The

mechanism underlying EAA neurotoxicity has been divided into two component

processes based on in vitro studies (figure 1) (Choi 1987, 1988; Rothman and

Olney 1987). The first involves depolarization of neuronal membranes and an

influx of sodium, chloride, and water. The second phase is characterized by

excessive calcium influx, primarily via NMDA receptor/channels, and activation

of secondary toxic events (Choi et al. 1988). This sequence of events appears

to be a major pathway leading to neuronal injury resulting from a variety of

acute brain insults. Under normal conditions excessive activation of EAA

receptors is prevented by the presynaptic high-affinity glutamate uptake system

that reduces the glutamate concentration in the synaptic cleft. When cellular

energy is depleted, high-affinity uptake of EAA is compromised (Silverstein et

al. 1986a). Excessive neuronal activation exacerbates any imbalance between

cellular energy requirements and energy availability. These mechanisms cause

metabolic stress and energy depletion and may ultimately produce excitotoxicity

(Novelli et al. 1988).

Overactivation of EAA receptors, particularly NMDA receptors, has been

implicated in the pathogenesis of neuronal injury in a variety of acute neurologic

disorders (Choi 1988; Rothman and Olney 1987) (figure 2).

ONTOGENY OF EXCITATORY AMINO ACID RECEPTORS

Synaptic markers of EAA neurotransmitter pathways change markedly during

the early postnatal period (Monaghan et al. 1989). The regional distribution of

186
FIGURE 1. This schematic diagram summarizes some of the mechanisms

that may contribute to EAA neurotoxicity. In vitro experiments

suggest that EAA neurotoxicity may have two components. The

first component, mediated by excessive activation of non-NMDA

receptors, is characterized by influx of Na+ followed by passive

influx of Cl- and H2O that may produce osmotic neuronal swelling.

These are acute events occurring within hours of exposure to

EAA agonists. The second, more prominent, component is

produced by overactivation of NMDA receptors, which leads to a

rise in the intracellular concentration of Ca2+. A sustained rise in

intracellular Ca2+ may trigger a biochemical cascade of events

that leads to neuronal injury and death. Furthermore, activation of

a subset of non-NMDA receptors coupled to polyphosphoinositide

hydrolysis may also elevate intracellular Ca2+. EAAs released

from synaptic terminals would further propagate neuronal injury.

Adapted from Choi 1988.

187
FIGURE 2. This diagram illustrates the possible role of EAA neurotoxicity in

acute brain injury. EAA neurotoxicity may be a common

mechanism in neuronal injury resulting from sustained seizures,

hypoxia-ischemia, and physical brain trauma. All these conditions

tend to elevate the extracellular concentration of EAAs via

different mechanisms. As detailed in figure 1, excessive activation

of EAA receptors, due to elevated concentrations of EAAs in the

synaptic cleft, can result in neuronal injury and death. Two

recurrent pathways could make these mechanisms

self-propagating. Adapted from Choi 1988.

NMDA- and quisqualate-sensitive glutamate binding is illustrated in figure 3.

High binding densities are present within the hippocampus and the outer layers

of the cerebral cortex followed by intermediate binding densities in the basal

ganglia. Lower binding densities are present in the brain stem and cerebellum.

In contrast to the high density of NMDA- and quisqualate-type glutamate

receptors in the postnatal day (PND)-7 brain, the density of kainate receptors is

relatively low compared to values in adults. High-affinity [3H]-kainate binding in

rat brain is low during the first postnatal week and then rises rapidly to achieve

188
FIGURE 3. Comparison of the distribution of (A) NMDA and (B)

quisqualate-sensitive [3H]glutamate binding in horizontal brain

sections from PND 7 rats. NMDA receptors were labeled with 40

nM [3H]glutamate in 50 mM Tris-acetate (pH 7.2 at 4°C)

containing 2.5 μM quisqualate. Quisqualate receptors were

labeled with 40 μM [3H]glutamate in 50 mM Tris-HCI (pH 7.2 at

4°C) containing 2.5 mM CaCl2 and 100 μm NMDA.

adult levels by postnatal week 3. In the striatum, [3H]-kainate binding densities

rise tenfold from 2 percent to 6 percent of adult values at PND 4 and to

70 percent at PND 21 (Campochiaro and Coyle 1978). By PND 28, receptor

densities are 80 percent of adult levels. A similar developmental profile of

kainate binding is present in the hippocampus, where binding remains relatively

low between PND 3 and 12 and then rises rapidly (twofold) to PND 17, when

adult levels are achieved (Ben-Ari et al. 1984). In contrast, quisqualate-sensitive

glutamate binding increases approximately threefold from PND 1 to maximal

adult levels at PND 7 in the rat striatum. The transient expression of

quisqualate-sensitive glutamate binding sites has been described in the globus

pallidus with peak binding densities present by PND 7 and thereafter declining

over 2 weeks to the very low levels present in adults (Greenamyre et al. 1987).

189
Using quantitative receptor autoradiography, we have recently described the

postnatal development of three receptors that constitute the NMDA

receptor/channel complex in the rat hippocampus (McDonald et al. 1989e).

NMDA-sensitive [

3H-glutamate binding, strychnine-insensitive [3H]-glycine

binding, and [3H]-TCP binding were measured to examine the ontogeny of

NMDA recognition sites, glycine modulatory sites, and PCP receptors,

respectively (McDonald and Johnston 1990; Wong et al. 1988).

Each receptor component of the NMDA receptor/channel complex has a unique

developmental profile in the hippocampal formation (figure 4). Furthermore,

each receptor component is transiently overexpressed during development.

NMDA-sensitive [3H]-glutamate binding exceeds adult levels by 50 percent to

120 percent in all regions examined, with peak densities occurring between

PND 10 through 28. In stratum radiatum CA1, binding increased slowly from

49 percent to 61 percent of adult value between PND 1 and PND 7, after which

binding rapidly rose to 151 percent of adult values at PND 14, remained

elevated through PND 28, and then decreased to adult levels. The ontogenic

profile of NMDA recognition site binding was similar in other hippocampal

regions.

The developmental changes we found in patterns of binding to each of the

receptor components of the NMDA receptor/channel complex are consistent

with postnatal changes in synaptogenesis. However, the relative

overexpression of the NMDA recognition sites with respect to glycine and PCP

receptor between PND 7 and PND 21 suggest preferential regulation of the

expression of these receptor components during development. These findings

are consistent with the hypothesis that multiple genetic forms of NMDA receptor

complexes exist (Stone and Burton 1988).

The physiologic relevance of the overexpression of the NMDA recognition sites

compared with the other components of the NMDA receptor/complex is unclear.

However, these changes parallel the maximal rate of change in stratum

radiatum pyramidal dendrites (Loy 1980; Pokorny and Yamamoto 1981). The

development of NMDA sites in stratum molecular dentate gyrus (SMDG)

parallels SMDG synaptogenesis (Crain et al. 1973). Dendritic growth and

afferent lamination of the entorhinal terminal zone (stratum moleculare) of the

dentate gyrus increase more rapidly between PND 4 and PND 10 than at later

times (Loy et al. 1977).

190
FIGURE 4. Comparison of postnatal developmental profiles of

NMDA-sensitive [3H]glutamate binding to NMDA recognition sites,

strychnine-insensitive [3H]giycine binding to glycine modulatory

sites, and [3H]TCP binding to PCP receptors in stratum radiatum

of the CA1 hippocampal subfield. Each data point represents the

relative binding densities (mean ± S. E. M.) compared with binding

densities in corresponding areas in adult rats (adult = PND 90).

Four animals were used at each age (except PND 21, n = 3).

Vertical error bars represent S. E. M.

191
The ontogeny of NMDA receptor binding could be related to several

developmental changes in the physiology of EAA receptors. In area CA3,

superfusion of NMDA elicits recurrent synchronized burst activity; the

epileptogenic effect of NMDA increases from PND 1 to PND 10 (King et al.

1989). Also, the susceptibility to convulsant-induced epileptiform activity

increases from PNDs 4 through 6 to PNDs 14 through 16 (Swann and Brady

1984; Chapman and Meldrum 1989). Specific NMDA receptor antagonists block

the seizure activity (Brady and Swann 1986). Developmental changes also

occur in the chemosensitivity of CA1 perinatal neurons to NMDA (Hamon and

Heinemann 1988). Maximal responses to NMDA occur between PND 12 and

PND 30 in stratum radiatum and correspond closely with the period of

overproduction of NMDA receptors in this area. The ontogeny of area CA1

NMDA-sensitive [3H]-glutamate binding parallels the development of LTP, an

experimental model of learning and memory, in area CA1 (Harris and Teyler

1984; Duffy and Teyler 1978). Nevertheless, the ontogeny of the absolute

number of hippocampal NMDA receptors does not correlate well with the

ontogeny of NMDA-induced brain injury that transiently peaks on PND 7

(McDonald et al. 1988b).

ROLE OF EXCITATORY AMINO ACIDS IN HYPOXIC-ISCHEMIC BRAIN

INJURY

Overactivation of NMDA-type glutamate receptors contributes to the

pathophysiology of hypoxic-ischemic neuronal injury in the adult brain, as

shown in figure 5. This diagram demonstrates a proposed schematic

representing the interrelationships among mechanisms that mediate

hypoxic-ischemic neuronal injury in the developing brain (Johnston et al. 1988).

Intracellular calcium overload probably plays an important role in causing cell

death. Calcium-activated release of free fatty acids and free radicals following

lipid degradation are also important steps producing toxic injury. Calcium-activated

proteases may also contribute to neuronal dysfunction and destruction. These

events can create a cycle of seizures and brain edema that could worsen

damage.

Our laboratory has examined the role of EAA in the pathogenesis of

hypoxic-ischemic brain injury by using a perinatal model of hypoxia-ischemia. In

this model, focal cerebral hypoxia-ischemia is produced by ligating the right

common carotid artery of PND 7 rats and exposing them to moderate hypoxia

(8 percent oxygen balance nitrogen) for 3 hours (Johnston 1983; McDonald et

192
FIGURE 5. Schematic representation of events that contribute to altered

neurologic function and neuronal cell death in perinatal

hypoxic-ischemic brain injury.

al. 1987). This procedure reliably produces focal ischemia and hypoxia in the

cerebral forebrain ipsilateral to the ligation while the brainstem and opposite

hemisphere are exposed primarily to hypoxia. Unilateral hypoxia-ischemia in

this model causes permanent neuronal injury in the corpus striatum, overlying

cortex, the hippocampus, globus pallidus, and thalamus.

193
To examine whether enhanced release and inhibition of uptake of EAA

contribute to the pathogenesis of hypoxic-ischemic brain injury in the immature

brain, we administered the noncompetitive NMDA antagonist MK-801 at various

times before or after injury in the model (Wong et al. 1986). In initial

experiments 1 mg/kg of MK-801 was administered intraperitoneally (i.p.) after

carotid ligation and just before onset of hypoxia, along with another dose

1.25 hours into hypoxia. This treatment protocol reduced hypoxic-ischemic

brain injury by 85 percent relative to saline-injected controls as assessed by

comparison of cerebral hemisphere weights 5 days following hypoxia-ischemia

(McDonald et al. 1987). A single dose of MK-801 at the onset or after

1.25 hours of hypoxia was similarly effective, while single injections at 1.5 or

2.5 hours into hypoxia were less effective. These data suggest that there is a

critical time threshold for drug intervention. Glycine antagonists are also

effective in this model (Uckele et al. 1989).

DEVELOPMENTAL ALTERATIONS IN EXCITATORY AMINO ACID

NEUROTOXICITY

Our studies indicate that the susceptibility of the brain to EAA neurotoxicity

changes considerably during development, and different brain regions and

neuronal types exhibit their own developmental profile of susceptibility to

selective EAA receptor agonists. The 7-day-old rat brain is most susceptible to

NMDA-induced brain injury, whereas quisqualate is less toxic and kainate is

relatively nontoxic on an equimolar basis (figure 6). In contrast, the adult brain

is most susceptible to kainate-induced brain injury, followed by injury from

NMDA and then from quisqualate.

Kainate is a potent neurotoxin in the adult brain, but it is much less toxic in the

immature brain. Nevertheless, kainate does produce prolonged tonic-clonic

seizures in immature animals. The susceptibility of striatal neurons to kainate

neurotoxicity initially appears at PND 7 and gradually rises to adult levels by

PND 21 (Campochiaro and Coyle 1978). The sensitivity to kainate neurotoxicity

parallels the development of glutamatergic innervation into the striatum as

measured by synaptosomal uptake of [3H]-glutamate. Similar developmental

trends of kainate toxicity have been reported in the hippocampus (Wolf and

Keilhoff 1984).

The susceptibility of the developing brain to NMDA toxicity transiently peaks

near PND 7 in rats (figure 7). The severity of brain injury produced by direct

intrastriatal infusion of NMDA is approximately 60 times greater at PND 7

194
FIGURE 6. Comparison of the severity of excitotoxic brain injury produced by

the selective EAA receptor agonists kainate, NMDA, and

quisqualate. PND 7 rats received unilateral intrastriatal injections

(right hemisphere) of either kainate (25 nmol/0.5 μL), NMDA

(25 nmol/0.5 μL), or quisqualate (100 nmol/0.5 mL). Animals

were sacrificed on PND 12. AlI three agonists produced prolonged

tonic-clonic seizures. Injections of NMDA produced the greatest

degree of brain injury whereas quisqualate injections produced

less. In contrast, kainate injections produced few signs of

neuronal injury.

195
FIGURE 7. Ontogeny of NMDA-mediated brain injury. Comparison of

Nissl-stained coronal brain sections at the level of the corpus

striatum from PND 1, 7, 14, and 21 rats that received unilateral

intrastriatal injections of 25 nmol(0.5 μL injection volume) NMDA.

Animals were sacrificed 5 days later. The severity of brain injury is

maximal at PND 7 the lesion is characterized by confluent

neuronal necrosis involving the striatum and extending into the

thalamus, dorsal hippocampus, and overlying neocortex.

Compared with PND 7, the severity of NMDA-mediated brain

injury is substantially reduced at PND 1, 14, and 21.

(McDonald et al. 1988a). NMDA toxicity peaks at different development time

points in various brain regions. It is noteworthy that the susceptibility of the

developing brain to NMDA parallels its susceptibility to hypoxic-ischemic brain

injury (Ikonomidou et al. 1989).

196
Susceptibility of striatal and hippocampal neurons to quisqualate-mediated

brain injury during postnatal development does not change as markedly,

although toxicity appears to be somewhat enhanced in younger animals

(Hastings et al. 1985; Silverstein et al. 1986b). However, the quisqualate

agonist AMPA (which binds to a subtype of quisqualate receptors) is

approximately 10 to 15 times more potent on an equimolar basis than

quisqualate as a neurotoxin when injected intercerebrally in PND 7 rats (data

not shown).

The noncompetitive NMDA antagonist MK-801 is approximately tenfold more

potent as a neuroprotective against NMDA- and hypoxia-ischemia-mediated

neuronal injury in immature rodents than in adults (McDonald et al. 1987;

McDonald et al. 1989c; Foster et al. 1987; Gill et al. 1987). Virtually complete

neuroprotection can be achieved with NMDA antagonists in the perinatal brain,

in contrast to only partial protection in adults.

CHARACTERIZATION AND PHARMACOLOGY OF NMDA-INDUCED BRAIN

INJURY

We have developed a model of NMDA-induced brain injury to characterize the

mechanism of EAA neurotoxicity and the effectiveness of neuroprotective

compounds against NMDA neurotoxicity in this model may relate to the

pathogenesis of hypoxia-ischemia brain injury (McDonald et al. 1987, 1989c,

and 1990).

In this model, unilateral intrastriatal injection of 25 nmol NMDA on PND 7

typically produces tonic-clonic seizure activity and results in ipsilateral

hemispheric necrosis 5 days later (McDonald et al. 1989c, 1990). Due to the

enhanced susceptibility of the PND 7 brain to NMDA toxicity and also to the

rapid phase of brain growth, the severity of brain injury can be quantified by

comparison of the weights of the injected and contralateral cerebral

hemispheres (McDonald et al. 1988b, 1989c,f). This method appears to provide

a reliable index of neuronal injury (McDonald et al. 1989c,f). There is a linear

relationship between the amount of NMDA (up to 25 nmol) injected into the

striatum and the severity of resulting brain injury (R2 = 0.97) and measurements

of regional cross-sectional areas (R2 = 0.98). Because of the sensitivity and

rapidity of this model, detailed pharmacologic dose-response relations of

neuroprotective compounds against NMDA-induced brain injury can be

examined.

197
We have systematically examined the neuroprotective characteristics of four

classes of NMDA receptor antagonists, anticonvulsants, receptor ligands, as

well as additional compounds that theoretically could protect against

excitotoxicity (McDonald et al. 1988a, 1989a,c,e,f). Only competitive and

noncompetitive NMDA antagonists were completely protective. Injury was

partially reduced by the ligands +PPP and haloperidol (36 percent reduction).

Of five anticonvulsants, only carbamazepine afforded partial protection

(36 percent).

TEMPORAL CHARACTERISTICS OF NMDA-MEDIATED BRAIN INJURY

The temporal progression of NMDA-mediated brain injury was examined by

varying the MK-801 posttreatment interval after intrastriatal NMDA injection.

One mg/kg MK-801 was administered (i.p.) to PND 7 rats at seven time

intervals after intrastriatal injection of 25 nmol NMDA. The severity of resulting

brain injury was assessed 5 days later on PND 12 by comparison of cerebral

hemisphere weight disparities. Administration of MK-801 up to 8 hours after the

NMDA injection reduced the severity of brain injury compared to saline-treated

controls (p < 0.001, ANOVA). The MK-801 posttreatment time interval was

linearly related to the severity of resulting brain injury between l-hour and

10-hour posttreatment intervals (R2 = 0.99). For every hour treatment was

delayed (between 2 and 10 hours) the severity of brain injury increased by

approximately 6.5 percent. Extension of the posttreatment interval to greater

than 8 hours did not attenuate NMDA-mediated brain injury. The ability of

MK-801 to reduce NMDA-mediated brain injury when treatment is delayed

suggests that the biochemical events initiated by overactivation of NMDA

receptors are, in part, reversible for up to one third of a day after the initial insult.

MK-801 ENHANCES NMDA-MEDIATED BRAIN INJURY AND INCREASES

NMDA RECOGNITION SITES

Another observation that may be clinically important is that administration of

MK-801 24 hours prior to intrastriatal injection of NMDA paradoxically enhances

NMDA-induced brain injury (McDonald et al. 1988c). One mg/kg MK-801 was

administered interperitoneally 0.5, 2, 6, 12, and 24 hours prior to intrastriatal

injection of NMDA. Animals were sacrificed 5 days later on PND 12, and the

severity of brain injury was assessed by comparison of the weights of injected

and contralateral cerebral hemispheres.

198
In these studies, MK-801 protected against NMDA toxicity when administered

0.5, 2, 6, and 12 hours prior to intrastriatal injection of NMDA in comparison to

saline-treated NMDA-injected controls (p < 0.001 vs. saline-treated,

NMDA-injected controls, ANOVA). In contrast, administration of MK-801

24 hours prior to intrastriatal injection of NMDA paradoxically enhanced the

severity of NMDA-mediated brain injury (p < 0.05 vs. saline-treated,

NMDA-injected controls, ANOVA). The severity of brain injury was greater in

groups that received MK-801 24 hours earlier than in saline-treated controls

(regression analysis, p < 0.001). The mortality rate did not differ between salineand MK-801 -treated groups.

The enhanced toxicity of NMDA produced by 24-hour MK-801 pretreatment was

blocked by a second injection of MK-801 15 minutes after intrastriatal injection

of 25 nmol NMDA. This observation suggests that the enhanced toxicity

produced by MK-801 pretreatment is mediated by NMDA receptor channel

activation. The effects of MK-801 pretreatment were selective for enhancement

of NMDA-mediated brain injury, since quisqualate-mediated brain injury was not

increased by a similar MK-801 pretreatment.

Quantitative receptor autoradiography was used to determine if alterations in

EAA receptor binding characteristics are associated with enhanced toxicity of

NMDA produced by MK-801 pretreatment. NMDA-sensitive [3H]-glutamate

binding was increased by up to 30 percent in comparison with saline-treated

controls in the four brain regions examined: CA1 and CA3 hippocampal

subfields corpus striatum and cingulate cortex (McDonald et al. 1989b).

The mechanisms by which MK-801 treatment alters NMDA-sensitive

[

3H]-glutamate and [3H]-TCP binding to NMDA receptor channels and

quisqualate-sensitive [3H]-glutamate and [3H]-TCP binding are unclear

(McDonald et al. 1990; Young et al. 1988). The increase in NMDA-sensitive

treated glutamate binding produced by MK-801 may reflect a compensatory

response to transient channel blockade.

The heightened sensitivity to NMDA neurotoxicity found 24 hours after MK-801

administration could relate to a persistent elevation in NMDA-sensitive

[

3H]-glutamate receptors after the neuroprotective effect of MK-801 has

diminished.

The pharmacology of MK-801- and PCP-like compounds in vivo in the

developing brain appears to be complex. In addition to blocking NMDA199
FIGURE 8. Schematic diagram relating the level of EAA receptor activation

and the balance between normal and delayed or disrupted

development and excitotoxicity. Evidence presented in this review

suggests there is an optimum level of EAA activation required for

normal development. However, excessive receptor activation can

produce neuronal injury, while under activation may delay or

disrupt normal development. Acute brain injury (e.g., hypoxiaischemia, etc.) can directly disrupt the normal course of

development. following brain injury, neuronal adaptive regulatory

mechanisms are activated. These compensatory processes, while

promoting neuronal plasticity and repair, may paradoxically

enhance the susceptibility of the brain to excitotoxic injury. A

corollary to this phenomenon is that the developing brain would

be predicted to be most vulnerable to excitotoxic injury during

periods when the capacity for synaptic plasticity is greatest.

responses and NMDA-mediated brain injury, they also regulate NMDA

recognition site binding in a complex manner.

SUMMARY AND CONCLUSIONS

EAA neurotransmitters participate in a variety of physiologic processes during

central nervous system development. EAAs function as neurotransmitters but

also regulate development of neuronal cytoarchitecture and neuronal

200
connectivity. EAAs play a major role in several forms of activity-dependent

synaptic plasticity including learning and memory, and stabilization and

elimination of synaptic connections during development. Similar molecular

mechanisms may underlie plastic changes during development and neuronal

destruction by overactivation of EAA receptors. A critical level of EAA

neurotransmitter activity is required for normal development. However, there

appears to be a continuum of physiologic reactions to EAA receptor activation:

underactivation can retard or disrupt normal development, whereas

overactivation can lead to neuronal injury and destruction (figure 8).

The susceptibility of the brain to excitotoxicity is dramatically altered during

postnatal development. As a result of these changes, the contribution of NMDA

receptor activation in excitotoxic brain injury may be greater during early

periods of postnatal development, whereas non-NMDA receptors may make a

greater contribution to excitotoxic injury in the adult brain.

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204
AUTHORS

John W. McDonald

Department of Neurology

Michael V. Johnson

Department of Neurology and Pediatrics

Johns Hopkins University School of Medicine

and Kennedy Research Institute

707 North Broadway

Baltimore, MD 21205

205

Isolation and Characterization of an

Endogenous Ligand for the PCP and

Receptors From Porcine, Rat, and Human

Tissue

Patricia C. Contreras, Nancy M. Gray, Debora A. DiMaggio,

Margaret E. Bremer, and Julie A. Bussom

Phencyclidine (PCP) is a dissociative anesthetic that alters sensory perception,

impairs memory, and induces a psychosis resembling schizophrenia (Luby et

al. 1959). Two different receptors have been characterized that mediate some

of the effects of PCP. One is the PCP receptor, which is distinguished by its

interaction with N-methyl-D-aspartic (NMDA) receptors (Lodge and Anis 1982;

Loo et al. 1986) and the inability of ligands, such as haloperidol and

(-)butaclamol, to label this site. The second receptor is the receptor, which is

distinguished by the finding that many neuroleptics label this site (Tam and

Cook 1984). Also, the distribution of PCP and receptors in the central nervous

system (CNS) is different (Largent et al. 1984; Contreras et al. 1987a; Gundlach

et al. 1985).

Several studies have examined the roles of PCP and receptors in the normal

functioning of endocrine (Su et al. 1988), immune (Su et al. 1988; Wolfe et al.

1988), and central nervous systems (Maragos et al. 1987; Mouradian et al.

1988), but very few have studied the role or presence of endogenous ligands

for PCP or receptors. Lack of a difference in PCP or receptors between

normal and disease states does not eliminate any possible role of PCP or

system because there could instead be changes in synthesis, metabolism, or

release of endogenous ligands, which have been identified by several groups.

Endogenous PCP-like compounds have been identified by extracting material

from porcine (Quirion et al. 1984) or bovine brains (Zukin et al. 1987) that

inhibits binding to PCP receptors. The material isolated from porcine brains has

been shown to bind selectively to PCP receptors, to have a specific distribution,

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to induce PCP-like effects, and to be peptidergic in nature (Quirion et al. 1984;

DiMaggio et al. 1988).

Endogenous like ligands have also been identified. One possible ligand is

progesterone (Su et al. 1988), which interacts with receptors with moderate

affinity. Several peptidergic components isolated from brain have also been

found to inhibit binding to receptors (Su et al. 1986; Contreras et al. 1987b).

These peptidergic like compounds have been shown to be different from

endogenous PCP-like compounds (Contreras et al. 1987b) and to bind

selectively to receptors (Su et al. 1986; DiMaggio et al. 1988).

The purpose of this study was to further characterize endogenous ligands

isolated from porcine brain and to determine whether endogenous PCP-like

material is also present in human tissue.

METHODS

Animals and Drugs

Sprague-Dawley rats weighing 200 to 250 g (Sasco Farms) were used in all

experiments. Each animal was used only once.

Phencyclidine HCI (PCP) and NMDA were obtained from Sigma Chemical

Company (St. Louis, MO). (+)-N-allylnormetazocine (NANM) was obtained from

Research Biochemicals Inc. (Wayland, Mass.). 1,3-Di-o-tolyl-guanidine (DTG)

was obtained from Aldrich Chemical Company (Milwaukee, Wis.). [3H]-TCP

(1-(2-thienyl)-cyclohexylpiperidine), [3H]-(+) 3-PPP (3-(3-hydroxyphenyl)-N -

(1-propyl)piperidine), and [3H]-(+)-N-allylnormetazocine were obtained from

New England Nuclear (Boston, Mass.).

Isolation of Endogenous PCP- and -Like Activity

PCP- and a-like activity was purified from porcine tissue as described by

Contreras and colleagues (1987b) and DiMaggio and coworkers (1988).

PCP- and o-like activity was isolated from rat brains, samples of human cortex,

and human cerebrospinal fluid (CSF) by homogenizing the tissue or mixing the

CSF with a solution of trifluoroacetic acid (TFA), hydrochloric acid, formic acid,

and sodium chloride. The mixture was centrifuged at 5,000 x g for 15 minutes.

The supernatant was extracted with ether and the aqueous phase titrated to a

208
pH of 4.0 with sodium hydroxide. Particulate matter was removed by filtering the

solution through a Millex-HV, 0.45 μm, durapore filter. Next, the solution was

chromatographed by analytical reverse-phase high-pressure liquid

chromatography (HPLC) using a C-18 column. Salts in the solution were first

eluted by washing the column (Vydac C-18 or Waters μBondapak C-18) for

20 minutes with 0.1 percent TFA, and then a linear gradient of acetonitrile (0 to

60 percent in 0.1 percent TFA) was used to fractionate the sample. Aliquots

from the fractions, collected at l-minute intervals, were assayed in a PCP or

radioreceptor assay.

Radioreceptor Assays

The PCP and radioreceptor assays were carried out as described by

Contreras and colleagues (1988). Briefly, the aliquots from the column were

lyophilized to dryness and then tested for the ability to inhibit the binding of

2 nM of [3H]-TCP, 6 nM of [3H]-(+)3-PPP, or 6 nM of (+)NANM. The assay was

incubated at room temperature for 60 minutes, then filtered through glass filters

(Schleicher & Schuell, #32), which were presoaked for 1 hour in 0.05 percent

polyethylenimine. Nonspecific binding was determined using 30 μM of PCP,

(+)NANM, or DTG.

NMDA-Induced Dopamine Efflux

The method used to measure PCP-like antagonism of NMDA-induced

dopamine release was modified from a super-fusion method described by

Middlemiss and coworkers (1988). Male Sprague-Dawley rats (200 to 250 g)

were decapitated and the brains removed. The nucleus accumbens was

removed and sliced to a thickness of 350 μm with a Mcllwain tissue chopper.

The tissue slices were preincubated for 15 minutes at 37°C in a modified Krebs

buffer (NaCI, 135 mM; KCI, 5 mM; NaHCO3, 25 mM; KH2PO4, 1.25 mM;

MgS04, 1 mM; glucose, 10 mM; ascorbate, 0.11 mM; pargyline, 1 nM), pH 7.4,

containing 25 nM of [3H]-dopamine. The tissue slices were rinsed twice with the

Krebs buffer before three to four slices were placed on a mesh support and

incubated sequentially in the modified Krebs buffer containing 1 μM of

GBR-12909 and 100 μM of sulpiride at pH 7.4 for 30 minutes at 37°C and then

incubated in the modified Krebs buffer minus MgSO4 for 5 minutes at 37°C.

The effect of NMDA on dopamine release was determined by incubating tissue

sequentially for 1 minute in vials containing 0.5 mL of Krebs buffer minus

MgSO4, in the presence or absence of PCP, pig, or rat extracts or in

209
combination with 100 μM of NMDA. The amount of radioactivity remaining in

the tissue slices and the amount released into the incubation medium was

determined by liquid scintillation spectrometry. The percent of radioactivity

release into the incubation medium was calculated as the amount of

radioactivity in the incubate relative to the total amount in the slices at that time.

The amount of radioactivity released in response to NMDA, which is added in

fraction 5, is expressed as the following fractional release:

fractional release = percent radioactivity in fraction 5

percent radioactivity in fraction 4

The amount of radioactivity in the incubation medium was used as an index of

dopamine release. The effect of PCP and tissue extracts on NMDA-stimulated

dopamine release was compared using a Dunnett’s test subsequent to a

one-way analysis of variance.

Synthesis of ß-Endopsychosin (1-12)

The peptide was prepared using an Applied Biosystems Peptide Synthesizer

and Boc-protected amino acids on an amine resin. Coupling was achieved

using dicyclohexylcarbodiimide activation, and deprotection of the intermediate

Boc-peptide was achieved using trifluoroacetic acid in methylene chloride. The

peptide was cleaved and deprotected using anhydrous hydrogen fluoride and

purified on a C-l 8 reversed-phase column using an acetonitrile:water gradient

containing 0.05 percent trifluoroacetic acid.

Behavioral Assay

Rats were placed individually in rat cages for at least 1 hour in advance of the

experiment. DTG or synthetic peptide was administered by i.c.v. injection. The

behavior of each rat was scored using PCP rating scales developed by

Sturgeon and coworkers (1979) as described by Contreras and colleagues

(1986). Briefly, the rating scale for stereotyped behavior is (1) sniffing,

grooming, rearing, and chewing behavior; (2) undirected head movements or

reciprocal forepaw treading; (3) some circling, running backward, or

head-weaving behavior; (4) continuous circling, weaving, or running backward;

and (5) dyskinetic extension and flexion of head and limbs. The rating scale for

ataxia is (1) awkward movement, may fall when rears; (2) stumbles; (3) falls; (4)

cannot move beyond a small area, may support its weight on stomach or

haunches; and (5) unable to move. A rating of 5 was considered a 100-percent

response. ED50 values were determined using a Finney assay.

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RESULTS

Endogenous PCP-Like Activity

In previous reports the purification of endogenous PCP-like activity was

described (DiMaggio et al. 1988). To determine the distribution of this PCP-like

activity (inhibition of the binding of [3H]-TCP), areas of porcine brain were

extracted and fractionated over a C-18 column. The greatest concentration of

PCP-like activity in the brain was isolated from cortex, with smaller amounts

found in thalamus and hypothalamus and even smaller amounts in

hippocampus and cerebellum (figure 1). No PCP-like activity was detected in

pituitary or brainstem of porcine brains.

Endogenous PCP-like activity has also been isolated from sources other than

porcine brains, such as from the gastrointestinal tract (figure 1). The amount of

PCP-like activity found in the stomach is about sevenfold greater than that

found in cortex. PCP-like activity isolated from pancreas and small intestine was

greater than that found in CNS but less than that found in stomach. PCP-like

activity has also been found in the rat brain. The distribution of PCP-like activity

paralleled the distribution found in the porcine brain (figure 1). As in porcine

brain, there was little or no PCP-like activity detected in cerebellum or pituitary.

Unlike porcine brains, a small amount of PCP-like activity was detected in rat

brainstem.

To assess whether the endogenous PCP-like activity, like PCP, also

antagonized NMDA, the effect of endogenous PCP-like material on dopamine

basal and NMDA-stimulated efflux was determined (figure 2). In this paradigm,

PCP had no significant effect on the basal release of dopamine but inhibited

NMDA-stimulated release of dopamine in a dose-dependent manner. Like PCP,

an aliquot of endogenous PCP-like activity isolated from porcine or rat brain that

inhibited about 75 percent of the binding of [3H]-TCP had no significant effect on

basal release of dopamine. Addition of NMDA produced a stimulation of

dopamine release that was antagonized by both PCP and the endogenous

PCP-like activity isolated from rat and porcine brain (figure 3). However, an

equivalent volume from an HPLC fraction that contained a similar amount of

solvent as the active fractions did not antagonize NMDA-stimulated dopamine

release.

To assess whether there was endogenous PCP-like activity in human tissue,

pieces of human cortex were extracted and fractionated using a C-18 column

211
and reverse phase-HPLC (figure 4). Four samples of human cortex were

extracted and tested in the same manner with similar results. Inhibition of

binding of [3H]-TCP by aliquots from fractions 1 through 20 probably reflects

nonspecific inhibition due to salt because changes in PCP-like activity

paralleled changes in conductivity. There was not enough material to purify the

PCP-like activity, but it was clear that aliquots containing PCP-like activity were

present in three groups of fractions, a pattern also seen during the purification

of endogenous PCP-like material from porcine brain (Quirion et al. 1984).

Endogenous PCP-like activity was also detected in human CSF (5 mL)

(figure 5). The inhibition of the binding of [3H]-TCP detected in fractions 1

through 20 was probably nonspecific inhibition of the binding of [3H]-TCP

largely due to salt. Like porcine and rat brain, the endogenous PCP-like

FIGURE 1. Distribution of PCP-like activity from porcine and rat tissue.

Inhibition of the binding of [3H]-TCP by fractions extracted from

porcine or rat tissue was expressed as the equivalent

concentration of PCP that inhibits the same amount of binding of

[

3H]-TCP per gram of tissue.

212
material also eluted in three groups of fractions. The total amount of PCP-like

activity in CSF (32 pmol equiv. PCP/mL) is about 10 to 15 times less than that

detected in human or rat cortex.

Endogenous Like Activity

The isolation and purification of endogenous like activity has already been

described (Contreras et al. 1987b). The distribution of this activity in porcine

brain is shown in figure 6. Unlike the distribution of endogenous PCP-like

material, the greatest amount of like activity (inhibition of the binding of

[

3H]-(+)NANM) was found in the pituitary, with smaller amounts detected in

caudate and hypothalamus and even smaller amounts in frontal cortex,

hippocampus, and thalamus. Similarly, endogenous o-like activity was detected

in brainstem but not in cerebellum. The amount of endogenous o-like activity

FIGURE 2. Inhibition of NMDA-induced dopamine efflux by PCP. The percent

release of [3H]-dopamine from slices of the nucleus accumbens

was determined at 1-minute intervals. Vehicle or PCP at a

concentration of 0.1 μM was added to the

incubation buffer in fractions 3 through 7. NMDA (100 μM) was

added in fraction 5 of all groups except the control group

213
(nmol/g) was about 1,000 times greater than that of PCP-like activity (pmol/g) in

areas of the brain that contained both PCP- and o-like compounds.

Like the endogenous PCP-like material, the structure of the o-like compound is

at least in part peptidergic (DiMaggio et al. 1988). A small peptide consisting of

12 amino acids was synthesized. This peptide, ß endopsychosin (1-12)

inhibited the binding of [3H]-(+)NANM but not that of [3H]-TCP (figure 7).

This peptide also possessed o-like bioactivity. Like DTG, the peptide induced

stereotyped behavior and ataxia in a dose-dependent manner (figure 8).

FIGURE 3. Antagonism of NMDA-stimulated dopamine release by

endogenous PCP-like activity. The fractional release of [3H]-DA

was determined in fraction 5. When slices of the nucleus

accumbens were incubated with PCP (1 μM) or endogenous

PCP-like activity isolated from porcine or rat brains,

NMDA-stimulated dopamine release was significantly attenuated

(P<0.05).

214
DISCUSSION

This study extends earlier findings that endogenous PCP- and o-like ligands are

present in the CNS. The endogenous PCP-like ligand had been found to

specifically bind to PCP receptors (DiMaggio et al. 1988) and to have a specific

distribution in porcine brains. Since no PCP-like activity was detected in

pituitary or cerebellum, it is unlikely that the endogenous PCP-like ligand is an

artifact of the extraction or purification procedure. Endogenous PCP-like activity

was not limited to porcine brains but was detected in human CSF and tissue

from rat and human CNS, and porcine gastrointestinal tract. Since many

neuroactive peptides were first isolated from the gastrointestinal tract, it was not

FIGURE 4. An example of the isolation of endogenous PCP-like activity from

human cortex. A comparison showing the gradient of acetonitrile

used to fractionate a sample of human cortex and PCP-like

activity.

215
surprising that endogenous PCP-like activity was isolated from pancreas,

stomach, or small intestine, but it was surprising that the activity was greater

than that found in the CNS.

The finding that PCP antagonizes NMDA-stimulated dopamine release was

used to further explore the biological activity of the endogenous PCP-like

ligand. Like PCP, the PCP-like activity isolated from rat or porcine brain

inhibited NMDA-stimulated dopamine release, which was not mimicked by

HPLC fractions that were inactive in PCP radioreceptor assays. This result is

consistent with earlier reports that the endogenous PCP-like ligand has

PCP-like bioactivity in two other paradigms, inhibition of spontaneous firing of

cortical and hippocampal neurons and contralateral turning after intranigral

injection (Quirion et al. 1984; DiMaggio et al. 1988).

An endogenous o-like ligand has also been identified in porcine brains. The

endogenous -like ligand interacts selectively with receptors (DiMaggio et al.

1988) and has a specific distribution in CNS. Unlike the endogenous PCP-like

FIGURE 5. Isolation of endogenous PCP-like activity from human CSF. The

figure shows a comparison of the acetonitrile gradient used to

fractionate human CSF and PCP-like activity in the HPLC

fractions.

216
ligand, the largest concentration of o-like activity was isolated from pituitary.

This finding is consistent with the hypothesis that receptors may be involved

in modulation of endocrine function (Su et al. 1988).

The result of the authors’ studies to isolate and purify the endogenous ligand

has been the synthesis of a small peptide, ß endopsychosin (1-12). Like DTG,

this peptide produced o-like behaviors, which supports studies showing that

FIGURE 6. Distribution of endogenous -like activity in porcine brain. like

activity is expressed as the equivalent amount of (+)NANM

required to produce the same degree of inhibition of the binding of

[

3H]-(+)NANM ([3H]-(+)SKF 10,047).

217
endogenous -like activity isolated from porcine brain represents an

endogenous ligand for receptors and is not an artifact.

In summary, an endogenous PCP-like ligand can be detected in several types

of tissue, including human cortex and CSF; its distribution is different from that

of o-like activity; and it has PCP-like activity. The endogenous like ligand also

has a unique distribution, and a synthetic peptide derived from these studies

has o-like bioactivity.

FIGURE 7. Inhibition of -binding and PCP binding by (+)NANM and

ß-endopsychosin (1-12). Inhibition of the binding of [3H]-(+)NANM

by (+)NANM (A) or ß-endopsychosin (1-12) was determined.

The inhibition of the binding of [3H]-TCP by ß-endopsychosin

(1-12) (O) was also determined.

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FIGURE 8. Stereotyped behavior and ataxia induced by DTG and

ß-endopsychosin 1-12. DTG or ß-endopsychosin (1-12)

was administered by i.c.v. injection, and the ratings determined at

the time of peak effect (5 minutes) were used to determine the

dose-response curve for (A) stereotyped behavior and (B) ataxia.

219
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ACKNOWLEDGMENTS

Drs. J. Wu, W. Bunney, S. Potkin, and C. Sandman provided the human tissue

used in this study. This work was supported in part by U.S. Public Health

Service grant MH 44188-01.

AUTHORS

Patricia C. Contreras, Ph.D.

Research Scientist

Nancy M. Gray, Ph.D.

Research Scientist

Debora A. DiMaggio, Ph.D.

221
Margaret E. Bremer, B.S.

Research Associate

Julie A. Bussom

Summer Intern

CNSDR

G. D. Searle

St. Louis, MO 63198

222
Summary and Future Directions

Edward F. Domino

The following generalizations can be made as a result of this technical review:

First, much more research should be done in this area. Second, many research

areas have been clarified, although some aspects are still quite confused. Third,

the receptor story remains, in many respects, an enigma because most drugs

and chemicals, including ligands, have several different biological activities.

Hence, most are “dirty” in that their multiple pharmacologic actions tend to

confound interpretation of research findings. Obviously, new chemicals are

needed that are very selective and specific in their actions. To a large extent,

many current ligands are not “pure” enough in their mechanisms and sites of

action. Fourth, the researchers who made presentations are to be congratulated

on their contributions. Some aspects of this scientific field are very frustrating

because of their complexity. Nature tends to reveal its secrets slowly, which

means that these researchers worked very hard, sometimes under very

frustrating circumstances, to extract nature’s secrets; but that is what science is

all about: The end is worth the effort. Fifth, use of a more precise terminology

would result in better communication. During this 2-day review, there was

difficulty with the use of the terms “receptor” and "binding site.” Some speakers

referred to the receptor,” while others hedged and talked about a binding

site.” Most speakers did not hedge when they referred to the “PCP receptor,”

yet it is clear that the high-affinity PCP binding site is part of the glutamic acid

NMDA receptor subtype.

Is “PCP receptor” the appropriate term or the “PCP site in the NMDA receptor

complex”? Historically, the work of many scientists led to the concept of

chemicals acting in a biological system on “receptive substances.” Hence, the

term “drug receptor” or simply “receptor” was used to define the specific site at

which a drug or chemical acts in a functional molecular component in an

organism. A second definition of receptor has evolved that endogenous

hormones, neurotransmitters, and autocoids act on certain macromolecules,

usually in cell membranes as the first chemical messengers to trigger a series

of second, third, etc., chemical messenger events intracellularly. The targets of

223
drug action can include various extracellular and intracellular enzymes,

transport proteins, lipoproteins, and endogenous first messenger receptors,

among others. To define a drug receptor, many criteria must be met, including:

1. Saturability

2. Specificity

3. Correlation of binding affinity to biologic response

4. Reversibility

5. Reconstitution in an in vitro system

6. Presence of endogenous ligand

Readers are encouraged to extend this list and communicate their criteria to the

author. Clearly, the PCP binding site meets most of the criteria of a PCP drug

receptor. Although some researchers have promoted the binding site to the

status of the receptor, others still hedge because some of the abovementioned criteria have not been met.

At the beginning of the technical review, Dr. Doris H. Clouet stated that this is a

hot area of research but raised the issue as to whether the meeting would be

more illuminating or more confounding. The author believes it has been very

illuminating, although there are still many areas of confusion; that is the

challenge for future research.

Dr. Stephen R. Zukin stated that PCP abuse is still a problem, especially in

large cities, and predicts that it will become an even bigger problem as the war

on drugs reduces the availability of cocaine and amphetamine. Why some

people ingest PCP, which produces a global psychotic reaction with productive,

deficit, and hebephrenic symptoms of schizophrenia, is a mystery that further

research must solve. He described the historical controversy that took place

10 years ago concerning the existence of a PCP receptor in the brain and its

present relationship to the NMDA receptor complex. His chart illustrating the

many actions of PCP in relation to plasma PCP concentrations is very

important. Only the action of PCP on the NMDA receptor complex and its

224
actions on the NE, DA, and 5-HT amine uptake systems, as well as possibly its

ligand properties, seem to be pertinent in vivo.

The issue of precisely defining a versus a PCP receptor action still remains:

Are the or PCP sites true receptors? As described above, a drug receptor

must be distinguished from an endogenous neurotransmitter, hormone, or

autocoid as the molecular site of action of the drug.

Dr. Zukin emphasized an important analogy between the glutamic acid NMDA

receptor complex and the nicotinic ACh receptor complex. Both are members of

a receptor channel superfamily. He discussed the evidence for a model of

receptor ligand interactions requiring two molecules of ligand for both the ACh

and NMDA receptor. Research is clearly needed to isolate, clone, and define

the molecular details of the NMDA receptor complex, as is currently the case for

the nicotinic ACh receptor.

Dr. Kenneth M. Johnson provided us with evidence of the complexity of the

NMDA receptor, particularly its glycine modulating center. Clearly, the

schematic drawings of the NMDA receptor complex as published, for example,

by Reynolds and Miller (1988a), Yoneda et al. (1988), and Costa (1989) are too

incomplete to explain the facts that Dr. Johnson’s research team and others

have generated. Dr. Johnson promised us his view of the NMDA receptor

“cartoon” soon. To get an overview of what must be included, the schematic

drawing in figure 1 may be helpful. It is derived from other published schemes

and/or publications (Monaghan et al. 1989; Reynolds and Miller 1986b) but

includes additional crucial sites. For example, possible site(s) must also be

included for the role of tricyclic antidepressants (Reynolds and Miller 1988c;

Sernagor et al. 1989) a nonpsychotropic cannabinoid HU-211 (Feigenbaum et

al. 1989), as well as polyamines on the glycine modulating center with opposite

actions for spermine and spermidine. In view of their important intracellular

synthesis from ornithine, Dr. Johnson has suggested an intracellular site(s) for

polyamines, although an extracellular site(s) also may exist.

The glycine allosteric center is very complex, in which 7-Cl kynurenic acid (7-Cl

KYN) is a selective glycine antagonist and HA-966 is a negative allosteric

modulator. Dr. Johnson presented evidence that glycine causes a remarkable

facilitation of the NMDA receptor channel and thus enhances TCP binding to

the PCP site, The effects of glycine are antagonized by the competitive NMDA

antagonist APV. The stimulant effects of Mg++ are also blocked by APV. The

complexities of these relationships need much more research, as does the

225
FIGURE 1. Diagram of an NMDA glutamatergic receptor. This schematic is

based on an outline of the molecular configuration of the nicotinic

cholinergic receptor. Various binding sites are shown, including

the active site for flutamic acid (glutamate) and allosteric sites of

polyamines (spermine and spermidine), the glycine modulator

center for glycine, HA-966, 7-Cl KYN, and in the ion channel for

Mg++, TCA, and PGP. The symbols are as follows: CPP:

3-(carboxypiperazin-4-yl)-propyl-1-phosphate; PCP:

phencyclidine; TCA: tricyclic antidepressants; HA-966:

3-amino-1-hydroxypyrrolidone-2; and 7-Cl KYN:

7-chlorokynurenate. It is not known as yet if the polyamine site is

extracellular or intracellular. It is drawn as extracellular in this

diagram.

modulatory role of Zn++, tricyclic antidepressants, and others. Wroblewski and

colleagues (1989) and Wroblewski and Danysz (1989) have reviewed the

known molecular mechanisms of the modulation of glutamic-acid receptors,

especially the NMDA receptor, and their functional implications. One

subcategory of NMDA receptors in the cerebellum (where high-affinity PCP

binding sites are normally minimal but low-affinity sites are maximal)

increases c-fos proto-oncogene expression (Szekely et al. 1989a, 1989b).

226
Whether high-affinity PCP binding sites are in low density or low-affinity PCP

sites are in high density in primary cultures of neonatal rat cerebellar

granule cells, as in adult cerebellum, needs to be studied further.

As knowledge of the precise role of PCP and receptors increases, it may be

possible in the future to devise some ways to antagonize the actions of PCP in

vivo. Since the high-affinity PCP binding site is within an ion channel, it seems

unlikely that compounds like metaphit that acylate the PCP site will ever be

useful in vivo (Domino et al. 1987). Clearly, these are important directions for

future research that are of great interest to the mission of NIDA.

In contrast to the solid base of facts now accumulated regarding the PCP

receptor as the binding site within the channel of the NMDA glutamic acid

receptor complex, our knowledge of the receptor or binding site is very spotty.

The original hypothesis of Martin and his colleagues (1976) of opioid

receptors is now well accepted. However, the dysphoric and psychotomimetic

effects of nalorphine, which led them to study (±) SKF 10,047 as the prototypic

opioid receptor agonist, have been subdivided into opioid agonist and PCP

agonist-like effects.

A recommendation was made by the Committee on Terminology (Quirion et al.

1988) to drop the word “opioid” when referring to the receptor. This action

was approved by the participants of the second Joint U.S.-French Seminar on

Sigma and Phencyclidine-Like Compounds as Molecular Probes in Biology

meeting held at the University of Michigan in summer 1987. Musacchio (1990)

has been especially critical of the confusion among investigators and in the

literature regarding the term receptor.” He has pointed out that the

psychotomimetic and dysphoric effects of opioids are produced by the (-) and

not the (+) isomers. The psychotomimetic effects of the (-) opioids are

antagonized by naloxone. Thus, Martin and colleagues’ (1976) idea of a

receptor has evolved to take on an entirely new meaning in that ligand

stereospecificity is completely reversed and now ascribed to the (+) isomers,

which are nonopioid. Compounds such as (+) SKF 10,047 have high- as well as

low-affinity binding sites; the high-affinity site is called a receptor” and the

low-affinity site a “PCP receptor.” The reverse is true of PCP. The site

resembles the high-affinity dextromethorphan binding site. Whether the

dysphoric and psychotomimetic effects of (-) opioids can be explained by

receptor interactions or a still unknown opioid receptor is a subject for further

research.

227
The term receptor” in 1991 refers to a binding site shared by a variety of

chemicals (haloperidol through progesterone), with an order of potency

opposite to their binding affinity to the PCP receptor. To date, it is not possible

to distinguish between agonists and antagonists. Furthermore, their biological

and behavioral mechanisms of action are relatively unknown. However, this

Technical Review has been very helpful in shedding some light on the

receptor story.

Dr. Tsung-Ping Su provided us with a historical and chemical perspective of the

receptor involving a hydrophobic pocket, a side chain, and a nitrogen or a

20-keto group that acts as a nitrogen. He showed us a complex overlay of

molecular models of drugs, involving both bond length as well as molecular

orbital cloud structures with different distributions of charges. His scholarly

analysis reminded me of the hypothetical central nervous system receptor of

Lloyd and Andrews (1986) as modified for PCP by Manallack and Beart (1987)

FIGURE 2. Primary (R1, R2, R3, and N) and secondary (lipophilic clefts and

hydrogen bonding of phenyl substituents) binding sites of

PCP-like ligands as defined by Manallack et al. (1988). The

distance between the center of the phenyl ring and the nitrogen is

4.18 A.

228
and Manallack and co-workers (1986). Figure 2 summarizes their views, which

need to be integrated and contrasted with those of Dr. Su and his colleagues

regarding the molecular requirements for a receptor ligand. Especially

important will be an analysis of the role of stereoisomers to further define the

and PCP drug receptor complexes. Dr. Su’s molecular analysis led to the

synthesis of a methylene derivative of PCP that reversed the usual PCP versus

spectrum of activity based on binding assays. Future research will require a

description of the pharmacology of this compound and especially challenge

medicinal chemists to make more selective rigid stereoisomers. Clearly, the

site resides in membranes, and ligands carry no charge. Their Kd

 almost

triples with an increase of two pH units.

What is the functional role of receptors, especially in relation to major organ

systems, including the brain? What is their status in human psychoses? Dr.

Edythe London’s presentation was clearly a step in the direction of answering

these questions, specifically the role of receptors in schizophrenia. Several

crucial points were made:

1. Regional brain glucose utilization is unique following administration of

ligands. Ligands in the brain produce functional changes in glucose

utilization.

2. It is not possible to distinguish between agonists and antagonists.

3. Temporal lobe receptors are reduced in autopsy material from the brains

of paranoid schizophrenic patients. Is this due to down-regulation following

treatment with those neuroleptics (e.g., haloperidol) that have effects?

Obviously we need more research. We need to pursue PET studies in

drug-free patients, etc.

4. There is no postnatal increase in binding in contrast to PCP binding sites

in the brain. This is contrary to all we know about neurotransmitter

receptors, so there is much future work to be done.

Dr. Wayne D. Bowen tackled the crucial problem of receptors and signal

transduction. He reminded us that ligands have (1) smooth muscle,

(2) electrophysiologic, (3) motor dystonic, and (4) PI turnover effects. The latter

are new and exciting developments. Dr. Bowen pointed out that M1 ACh

receptor stimulated PI turnover by carbachol is decreased by drugs in a

noncompetitive manner. He systematically reviewed their possible mechanisms

229
of action. Although there is good correlation between Ml and receptor-ligand

effects, ligands act at the site to decrease Bmax. Their linkage to the Ml

cholinergic receptor to decrease PI turnover raises interest and all kinds of

exciting possible experiments.

Dr. Errol B. De Souza stressed the relationship between the nervous,

endocrine, and immune systems. He pointed out that some first messengers of

the nervous system (neurotransmitters) are also first messengers (hormones) in

the endocrine system and first messengers (autocoids) in the immune system

and vice versa. Although there are primarily high-affinity PCP receptor sites in

the cerebrum and spinal cord, there are many low-affinity PCP binding sites in

diverse tissues, including cerebellum, spleen, and lymphocytes. Some of the

pharmacological effects of PCP may be mediated by actions on low-affinity

receptors. The sites seem to be very important in leucocytes. Much future

research is needed to identify the function of receptors in T and B

lymphocytes.

In addition to the role of receptors in the immune system, it appears that such

receptors are of importance in the endocrine system. Why are receptors

present in highest concentrations in the ovary? Why do ligands suppress

luteinizing hormone (LH) and prolactin (PRL) hormone release? Will they inhibit

ovulation or alter the menstrual cycle? Dr. De Souza reminded us of the

analogy between high- and low-affinity benzodiazepine receptors in which the

high-affinity sites are in high density in the brain and the low-affinity sites are in

high density in the periphery. It appears that receptor function will be of

importance in the endocrine and immune systems as additional future research

is undertaken.

Dr. Brian de Costa provided hope that new ligands are being synthesized that

have more selective actions. He pointed out that the diastereoisomers of

U50,488 tend to separate opioid and effects. He and his colleagues

synthesized a series of benzeneacetamines in an attempt to obtain a highly

selective ligand. BD-737 is an especially promising compound whose

pharmacology needs to be studied extensively. Especially important is the

development of pairs of compounds, one of which is highly stereospecific for

effects.

Ketamine and other PCP-like drugs produce analgesia through a minor opioid

(Smith et al. 1987) and a major nonopioid (France et al. 1989a; Maurset et al.

1988, 1989, 1990) mechanism. Dr. Woods reported that the μ opioid antagonist

230
quadazocine antagonized alfentanil (μ)-induced analgesia more than U50,488

-induced analgesia but had no significant effect on PCP-like drug-induced

analgesia. The latter drugs included MK-801, PCP, ketamine, (+)SKF 20,047,

and dextrorphan. Interestingly, the competitive NMDA antagonist CGS 19755

also induced analgesia and anesthesia in a dose of 100 mg/kg i.v. with a very

slow onset (30 minutes to 2 hours) and long duration (24 to 48 hours) of action.

France and colleagues (19896) have shown that much smaller doses of CGS

19755 attenuated the rate-decreasing effects of NMDA in monkeys, but did not

produce ketamine-like discriminative stimulus effects. These observations will

stimulate much more research and the development of better competitive

NMDA antagonists. Especially important is the fact that CGS 19755 does not

appear to be self-administered, although this observation may be due to its very

slow onset of action.

One of the disappointing observations of both Dr. Balster and Dr. Woods was

that selective ligands do not produce unique and specific behavioral effects.

This strong conclusion is important in terms of what future direction should be

taken. Although specific ligands produce a large variety of animal behaviors,

including piano playing, sideways walking or jumping, convulsions, etc., these

behavioral effects are elicited by other drugs.

According to Dr. Woods, BMY 14802 appears to act behaviorally through the

5-HT1A rather than or DA receptors. Dr. Duncan P. Taylor reviewed the

antipsychotic profile of BMY 14802 in relation to other antipsychotic agents. The

former agent clearly has a most complex pharmacologic profile, including

binding to the receptor. The pharmacological actions of BMY 14802 are so

diverse that it will be a challenge to researchers to find the mechanism of its

antipsychotic effect should it prove to be useful in human therapy.

Another complexity of the in vivo pharmacology of BMY 14802 is its conversion

in the liver to BMY 14786. Thus, the analogy is that of reduced haloperidol

conversion to haloperidol and vice versa. Dr. Taylor reported that Umesch and

colleagues have observed that (+)BMY 14802 is not converted to the ketone

but (-)BMY 14802 is. He emphasized that better antipsychotic drugs should be

selective and stereospecific for the sites on postsynaptic dopaminergic

neurons. He also stressed the enigma as fertile territory for research.

The group of researchers represented by Dr. Leslie Kushner presented some of

their exciting findings on the molecular biology of NMDA receptors and the PCP

binding site. Of special importance are the voltage and use dependence of PCP

231
ligands. By using the Xenopus oocyte injected with mRNA, they have been able

to express NMDA, kainate, and quisqualate receptors. The NMDA receptor

complex behaves as expected with glycine potentiation (Kleckner and

Dingledine 1988), voltage dependency of Mg++, etc. Unique cell lines with

receptors are useful sources of mRNA for oocyte expression. Furthermore,

unique NMDA receptor forms (such as in the cerebellum) can be expressed.

This area of research shows much promise for improving the understanding of

the molecular mechanisms involved.

Dr. Michael V. Johnston presented a remarkable overview of NMDA receptormediated neurotoxicity in the developing brain. He contrasted the important

differences between the neonate and the adult with regard to glutamate toxicity

and selective vulnerability. The neonate brain is much more sensitive to NMDA

than to kainic acid compared with the adult brain. Especially important is the

observation that, of all of the PCP-like ligands, MK-801 is the most potent in

brain protection, and that this effect is independent of hypothermia. PCP itself is

not as potent and not as effective as MK-801. An important observation is that

MK-801, given 24 hours prior to NMDA, causes enhanced neurotoxicity in

contrast to its brain-protection actions when given after NMDA administration. It

appears that NMDA receptors play a crucial role in neuronal plasticity of the

newborn.

Dr. Patricia C. Contreras gave a status report of her group’s efforts in isolating

endogenous and PCP-like peptides. There has been steady progress

(DiMaggio et al. 1986, 1988; Contreras et al. 1987) but the problems of

isolation are enormous. One can only compliment Dr. Contreras for her efforts

in spite of innumerable impediments. Especially interesting and consistent with

an endocrine role, endogenous ligand activity is highest in the pituitary. This

is related to Dr. De Souza’s observation that the ovary and testes have a very

high density of receptors.

CONCLUSION

There is much work to be done. This Technical Review has shed new light on

the stranger-than-fiction developments that PCP- and o-related research has

revealed. We are still a long way from solving the problem of PCP abuse, but

only through more research is there any hope of doing so. In the process, some

useful therapeutic agents may emerge.

232
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Excerpta Medica, 1988. pp. 47-66.

AUTHOR

Edward F. Domino, M.D.

Professor of Pharmacology

University of Michigan

Ann Arbor, Ml 48109-0626

235

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94 PHARMACOLOGY AND TOXICOLOGY OF AMPHETAMINE AND RELATED

DESIGNER DRUGS. Khursheed Asghar, Ph.D.; Errol De Souza, Ph.D., eds.

NTIS PB #90-148958/AS $39.00

NCADI #M94

95 PROBLEMS OF DRUG DEPENDENCE, 1989. PROCEEDINGS OF THE 51st

ANNUAL SCIENTIFIC MEETING. THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC., Louis S. Harris, Ph.D., ed.

NTIS PB #90-237660/AS $67.00

NCADI #M95

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96 DRUGS OF ABUSE: CHEMISTRY, PHARMACOLOGY, IMMUNOLOGY, AND

AIDS. Phuong Thi Kim Pham, Ph.D. and Kenner Rice, Ph.D., eds.

NTIS PB #90-237678/AS $31.00

NCADI #M96

97 NEUROBIOLOGY OF DRUG ABUSE: LEARNING AND MEMOF

Erinoff, ed.

NTIS PB #90-237686/AS $31.00

NCADI #M97

98 THE COLLECTION AND INTERPRETATION OF DATA FROM HIDDEN

POPULATIONS. Elizabeth Y. Lambert, M.S., ed.

NTIS PB #90-237694/AS $23.00

NCADI #M98

99 RESEARCH FINDINGS ON SMOKING OF ABUSED SUBSTANCES. C. Nora

Chiang, Ph.D. and Richard L. Hawks, Ph.D., eds.

NTIS PB #91-141119 $23.00

NCADI #M99

100 DRUGS IN THE WORKPLACE: RESEARCH AND EVALUATION DATA VOL

II. Steven W. Gust, Ph.D.; and J. Michael Walsh, Ph.D., eds.

GPO Stock #017-024-=01458-3 $8.00

NCADI #M100

101 RESIDUAL EFFECTS OF ABUSED DRUGS ON BEHAVIOR. John W.

Spencer, Ph.D. and John J. Boren, Ph.D., eds.

GPO Stock #017-024-01426-7 $6.00 NTIS PB #91-172858/AS $31.00

NCADI #M101

102 ANABOLIC STEROID ABUSE. Geraline C. Lin, Ph.D. and Lynda Erinoff,

Ph.D., eds.

GPO Stock #017-024-01425-7 $8.00 NTIS PB #91-172866/AS $31.00

NCADI #M102

103 DRUGS AND VIOLENCE; CAUSES, CORRELATES, AND CONSEQUENCES.

Mario De La Rosa, Ph.D., Elizabeth Y. Lambert, M.S., and Bernard Gropper, Ph.D.,

eds.

GPO Stock #017-024-0/427-3 $9.00 NTIS PB #91-172841/AS $31.00

NCADI #M103

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104 PSYCHOTHERAPY AND COUNSELING IN THE TREATMENT OF DRUG

ABUSE. Lisa Simon Onken, Ph.D. and Jack D. Blaine, M.D., eds.

GPO Stock #017-024-01429-0 $4.00 NTIS PB #91-172874/AS $23.00

NCADI #M104

106 IMPROVING DRUG ABUSE TREATMENT. Roy W. Pickens, Ph.D.; Carl G.

Leukefeld, D.S.W.; and Charles R. Schuster, Ph.D., eds.

GPO Stock #017-024-01439-7 $12.00

NTIS PB #92-105873 Paperback $50.00 Microfiche $19.00

NCADI #M106

107 DRUG ABUSE PREVENTION INTERVENTION RESEARCH:

METHODOLOGICAL ISSUES. Carl G. Leukefeld, D.S.W., and William J. Bukoski,

Ph.D., eds.

GPO Stock #017-024-01441-9 $9.00

NTIS PB #92-160985 Paperback $35.00 Microfiche $17.00

NCADI #M107

108 CARDIOVASCULAR TOXICITY OF COCAINE: UNDERLYING MECHANISMS.

Pushpa V. Thadani, Ph.D., ed.

GPO Stock #017-024-01446-0 $7.00

NTIS PB #92-106608 Paperback $35.00 Microfiche $17.00

NCADI #M108

109 LONGITUDINAL STUDIES OF HIV INFECTION IN INTRAVENOUS DRUG

USERS: METHODOLOGICAL ISSUES IN NATURAL HISTORY RESEARCH. Peter

Hartsock, Dr.P.H., and Sander G. Genser, M.D., M.P.H., eds.

GPO Stock #017-024-01445-1 $4.50

NTIS PB #92-106616 Paperback $26.00 Microfiche $12.50

NCADI #M109

111 MOLECULAR APPROACHES TO DRUG ABUSE RESEARCH: VOLUME I.

Theresa N.H. Lee, Ph.D., ed.

GPO Stock #017-024-01455-

NTIS PB #92-135743 Paperback $35.00 Microfiche $17.00

NCADI #M111

112 EMERGING TECHNOLOGIES AND NEW DIRECTIONS IN DRUG ABUSE

RESEARCH. Rao S. Rapaka, Ph.D.; Alexandros Makriyannis, Ph.D.; and Michael

J. Kuhar, Ph.D., eds.

GPO Stock #017-024-01455-9 $11.00

NTIS PB #92-155449 Paperback $44.00 Microfiche $17.50

NCADI #M112

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113 ECONOMIC COSTS, COST EFFECTIVENESS, FINANCING, AND

COMMUNITY-BASED DRUG TREATMENT. William S. Cartwright, Ph.D., and

James M. Kaple, Ph.D., eds.

NTIS PB #92-155795 Paperback $35.00 Microfiche $17.00

NCADI #M113

114 METHODOLOGICAL ISSUES IN CONTROLLED STUDIES ON EFFECTS OF

PRENATAL EXPOSURE TO DRUG ABUSE. M. Marlyne Kilbey, Ph.D., and

Khursheed Asghar, Ph.D., eds.

GPO Stock #017-024-01459-1 $12.00

NTIS PB #92-146216 Paperback $43.00 Microfiche $17.00

NCADI #M114

115 METHAMPHETAMINE ABUSE: EPIDEMIOLOGIC ISSUES AND

IMPLICATIONS. Marissa A. Miller, D.V.M., M.P.H., and Nicholas J. Kozel, M.S.,

eds.

GPO Stock #017-024-01460-5 $4.00

NCADI #M115

116 DRUG DISCRIMINATION: APPLICATIONS TO DRUG ABUSE RESEARCH.

R.A. Glennon, Ph.D., T.U.C. Jarbe, Ph.D., and J. Frankenheim, Ph.D., eds.

GPO Stock #017-024-01470-2 $13.00

NCADI #M116

117 METHODOLOGICAL ISSUES IN EPIDEMIOLOGY, PREVENTION, AND

TREATMENT RESEARCH ON DRUG-EXPOSED WOMEN AND THEIR

CHILDREN. M. M. Kilbey, Ph.D. and K. Asghar, Ph.D., eds.

GPO Stock #017-024-01472-9 $12.00

NCADI #M117

118 DRUG ABUSE TREATMENT IN PRISONS AND JAILS. C.G. Leukefeld, D.S.W.

and F. M. Tims, Ph.D., eds.

GPO Stock #017-024-01473-7 $16.00

NCADI #M 118

119 PROBLEMS OF DRUG DEPENDENCE 1991: 53rd ANNUAL SCIENTIFIC

MEETING. The Committee on Problems of Drug Dependence, Inc. Louis Harris,

Ph.D., ed.

GPO Stock #017-024-01474-5 $22.00

NCADI #M119

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120 BlOAVAlLABlLlTY OF DRUGS TO THE BRAIN AND THE BLOOD-BRAIN

BARRIER. Jerry Frankenheim, Ph.D., and Roger M. Brown, Ph.D., eds.

GPO Stock #017-024-01481-8 $10.00

NCADI #M120

121 BUPRENORPHINE: AN ALTERNATIVE TREATMENT FOR OPIOID

DEPENDENCE. Jack D. Blaine, Ph.D. ed.

GPO Stock #017-024-01482-6 $5.00

NCADI #M121

123 ACUTE COCAINE INTOXICATION: CURRENT METHODS OF TREATMENT.

Heinz Sorer, Ph.D., ed.

GPO# 017-024-01501-6 $6.50

NCADI #M123

124 NEUROBIOLOGICAL APPROACHES TO BRAIN-BEHAVIOR INTERACTION.

Roger M. Brown, Ph.D., and Joseph Fracella, Ph.D., eds.

GPO #017-024-01492-3 $9.00

NCADI #M124

125 ACTIVATION OF IMMEDIATE EARLY GENES OF ABUSE. Reinhard Grzanna,

Ph.D., and Roger M. Brown, Ph.D., eds.

GPO# 017-024-01503-2 $7.50

NCADI #M125

126 MOLECULAR APPROACHES TO DRUG ABUSE RESEARCH VOLUME II:

STRUCTURE, FUNCTION, AND EXPRESSION. Theresa N.H. Lee, Ph.D., eds.

NCADI #M126

127 PROGRESS AND ISSUES IN CASE MANAGEMENT. Rebecca Sager Ashery,

D.S.W., ed.

GPO #017-024-01490-7 $14.00

NCADI #M127

128 STATISTICAL ISSUES IN CLINICAL TRIALS FOR TREATMENT OF OPIATE

DEPENDENCE. Ram B. Jain, Ph.D., ed.

GPO #017-024-01491-5 $6.50

NCADI #M128

129 INHALANT ABUSE: A VOLATILE RESEARCH AGENDA Charles Wm. Sharp,

Ph.D., Fred Beauvais, Ph.D., and Rechard Spence, Ph.D., eds.

GPO #017-024-01496-6 $12.00

NCADI #M129

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130 DRUG ABUSE AMONG MINORITY YOUTH: ADVANCES IN RESEARCH AND

METHODOLOGY. Mario De La Rosa, Ph.D., Juan-Luis Recio Adrados, Ph.D., eds.

NCADI #M130

131 IMPACT OF PRESCRIPTION DRUG DIVERSION CONTROL SYSTEMS ON

MEDICAL PRACTICE AND PATIENT CARE. James R. Cooper, Ph.D., Dorynne J.

Czechowicz, M.D., Stephen P. Molinari, J.D., R.Ph., and Robert C. Peterson, Ph.D.,

eds.

NCADI #M131

132 PROBLEMS OF DRUG DEPENDENCE, 1992: PROCEEDINGS OF THE 54TH

ANNUAL SCIENTIFIC MEETING OF THE COLLEGE ON PROBLEMS OF DRUG

DEPENDENCE. Louis Harris, Ph.D., ed.

GPO# 017-024-01502-4 $23.00

NCADI #M132

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NIH Publication No 93-3587

Printed 1993