Properties of the phencyclidine (PCP) receptors

0278-5846m3 $0.00 + so Copyr@ht 0 1983 Per3amcm Press Ltd.

Rag. NewoPsychophmmacol b Biol. Psychiat. 1883, Vol. 7. pp. 451456 Printed in Great Britain. All ri&a reserved.

PROPERTIES OF THE PHENCYCLIDINE (Pm) RFmPTOBS

REM1 QUIRION1s2 and CANDACE B. PERT1 Iclinical Neuroscience Branch, NIMH NIH, Bethesda, MD, USA and ZDouglas Hospital Research Center Verdun, Quebec, Canada (Final form, June 1983) Abstract Quirion, R. and Candace B. Pert: Properties of the phencyclidine (PCP) receptors. Prog. Neuro-Psychopharmacol.and Biol. Psychiat. 1983;z (4-6):451-456. 1. 2. 3. 4.

5.

C3HlPhencyclidine (PCP, Angel Dust) receptors have been characterized using a rat brain binding section technique. C3H]PCP labels a single class of site in rat brain (KD = 46 nM; B E HIPCP binds to 10.5 fmol/slice). Ligand selectivity pattern strongly suggests t%t" -3 sites relevant for its pharmacologicalactions. Chronic PCP treat ent (10 mg/kg/day for 14 days) desreases the number of sites (B,,,) for [3H]PCP and ['5 Hlspiperone binding but not for [ Hldihydromorphine. These modifications could be related to the development of tolerance and dependence to PCP. Visualization of [3HlPCP binding sites shows high densities of receptors in cortical areas and hyppocampus. Lower densities are observed in caudate-putamen,nucleus accumbens, and amygdala. Negligible quantities of receptors are seen in brain stem and over white matter. The presence of specific C3HlPCP binding sites in rat brain suggests the possible existence of an endogenous ligand for this unique receptor.

Keywords: Angel Dust, autoradiography,endogenous ligand, phencyclidine (PCP), receptor binding Abbreviations: phencyclidine (PCP); gamna-aminobutyricacid (GABA) Introduction Phencyclidine (1-(l-phenylcyclohexyl)piperidine, PCP, Angel Dust) is currently a major drug of abuse in the United States (Petersen and Stillman 1978). PCP's pharmacologicalprofile is unlike any other psychoactive drugs and may provide an "acceptable"animal model of schizophrenia (Domino 1981). Up to a few years ago, very little was known about the mechanism of action of PCP. Various groups had suggested that PCP was interactingwith either muscarinic (Vincent et al 1980; Aronstam et al 1980), nicotinic (Albuquerqueet al 1980), dopaminergic (Doherty et al 1980; Johnson and Oeffinger 1981), serotoninergic (Martin et al 1979), GABAergic (Hsu et al 1980), and/or opiatergic (Vincent et al 1978) systems. Similar hypotheses were supported for morphine and congeners before the discovery of highly selective opiate receptors in rat brain (Pert and n der 1973, Simon et al 1973). In 1979 two independent groups reported the existence of Csij H PCP receptors in rat brain and suggested that they were the major site of action of PCP (Vincent et al 1979; Zukin and Zukin 1979). However, a few months later, another group reported that [3H]PCP specific binding was robably an artifact of the membrane filtration techni ue (Maayani and Weinstein 1980Y . To avoid these problems, we decided to investigate [31 H PCP binding using a brain autoradiographictechnique (Herkenham and Pert 1982) which allows biochemical characterizationas well as visualization of receptors 451

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under study. In this paper we briefly review evidence that strongly supports the existence of PCP receptors. Biochemical Characteristicsof C3HlPCP Binding. In the first serie of experiments, we investigated the effects of preincubation, ions, and temperature on CsHIPGP binding to rat brain sections. As shown in Table 1, pr incubation at O'C for 15 min in presence of a low concentration of NaCl doubled specific C3HIPCP binding to brain sections. Preincubationsat 25Y did not seem to be as efficient in increasing the signal-to-noiseratio. We believe that the increase in [3H]PCP binding after preincubation is probably due to the dissociation of a putative endogenous ligand from the binding sites. Following the preincubation we observed that the highest [3H]PCP specific binding is obtained with an incubation at 0°C for 45 min in 5.0 IrMTris HCl, pH 7.4 lus 50 ti sucrose. Incubationswith monovalent and/or divalent cations markedly decrease [sH]PCP binding (Table 1). Table 1 Effects of Preincubation and Various Ions on C3HlPCP Binding to Rat Olfactory Bulb Section Condition

% Optimal Binding

No preincubation Preincubation,5 min, 25'C Preincubation, 15 min, 25'C Preincubation,45 min, 25°C Preincubation, 5 min, O'C Preincubation, 15 min, 0“C Preincubation, 45 min, O'C Preincubation, 15 min O'C, 20 mM NaCla a + Incubation, 20 nElNaCl a + Incubation, 20 mM KC1 a + Incubation, 3 mM CaC12 a + Incubation, 3 mM MgC12

49 45 72 :: 84

aThis condition hereafter referred to as "a" Modified from Quirion et al 1981a Recently, Vignon et al ( 982) also reported in an extensive paper that many monovalent and divalent cations inhibit [4HIPCP binding in membrane preparations. Moreover, they cl arly demonstrated that the rapid membrane filtration binding Sechnique is applicable for [9H]PCP binding study when properly used. They also show that C HIPCP bi ding is sensitive to various proteases like trypsin, pronase, or papain, suggesting that [sHIPCP binding sites are of a protein nature. Finally, C3H]PCP binding was rapidly heat-inactivatedat temperatures over 5OY. We (Quirion et al 1981a) and others (Zukin et al 1983) obtained similar results in rat brain sections. All these results strongly show that under appropriate conditions it is possible to use binding assays (sections and/or membrane filtration) to characterize PCP receptors. Structure-ActivityRelationships. Various groups have reported the relative potency of vafious PCP analogs on EJHIPCP binding (Vincent et al 1979; Zukin and Zukin 1979; Quirion et al 1981a). We have seen that C3HlPCP binding is potently inhibited by analogs such as N-ethyl-phenyl-cyclohexylaminePCE) but not by others like ketamine (Table 2). Moreover, the relative potencies obtained in t3HlPCP binding assay correlated very well with those reported by Shannon (1981, 1982) and Holtzman (1980, 1982) in rat behavioral assays. More recently, Altura et al (1983) also reported that the relative potencies of PCP and congeners in inducing contrac ions of isolated dog cerebral arteries correlated very well with their activi ies in the C5HIPCP binding assay. Finally, using enantiomers of PCP, we have shown that [5HIPCP binding sites are stereoselective (Quirion et al 1981b). It strongly suggests that C3HlPCP binding sites are relevant to the pharmacologicaleffects induced by PCP and related compounds. Among the various other classes of drugs tested for their ability to compete for [3H]PCP

453

Phencyclidine receptors

binding sites, only a few have been shown to interact with it (Quirion et al 1981a; Zukin and Zukin 1979, 1982; Quirion and Pert 1982a,b). We observed that opiates classified as sigma agonists (e.g., cyclazocine, N-allyl-normetazocine)(Martin et al 1976) are potent displacers of [3H]PCP bindin in rat brain sections (Table 2). Similar results have been reported in behavioral tests 4Shannon 1982; Holtzman 1982; Brady et al 1982), electrophysiologicalstudies (Stringer et al 1983), and bioassays (Altura et al 1983). It strongly suggests that PCP and sigma "opiate" receptors are one entity with or without different subunits. We also observed that in the presence of amantadine, an anti iral drug which possess antiparkinsonianactivity (Grelak et al 1970), the affinity (Kd) of [rH]PCP for its receptors is significantly increased (Quirion and Pert 1982a). The number of sites (Bmax) does not appear to be changed by the presence of amantadine in the incubation buffer. Interestingly,rimantadine, an analogue of amantadine completely devoid of any central nervous system effects, is also totally inactive on the [3HlPCP binding site. It is tempting to speculate that some CNS effects of amantadine are related to an action of this drug on the PCP receptor complex. It is known that PCP and amantadine are able to stimulate the release of dopamine in various conditions (Doherty et al 1980; Johnson and Oeffinger 1981; Grelak et al 1970), and it may be that both compounds act on the same substrate to induce the release of dopamine in the brain. Another family of drugs which appears to interact with PCP receptors are calcium antagonists such as verapamil and its methoxy-derivativeD-600 (Table 2). We observed that verapamil and D-600 were able to displace C3HlPCP from its binding site in a dose-dependentmanner (Quirion and Pert 1982b). However, other calcium antagonists such as nifedipine and nitrendipine do not interact with [3H]PCP binding. This suggests that calcium antagonists interact with various substrates in the brain and that [3H]PCP receptors could be regulated by a Table 2 Relative Pot ncies of Various Drugs in Competition Experiments Against [5H]PCP Binding to Rat Olfactory Bulb Section Drug

Relative Potencyb

II&O,~ nM

--Phencyclidine (PCP) N-Ethyl-1-phenylcyclohexylamine (PCE) N-[l-(2-thienyl-cyclohexyllpiperidine (TCP) 1-(1-phenylcyclohexyl)pyrrolidine (PHP) N-Cl-(2-thienyl)cyclohexyllpyrrolidine (THP) Ketamine N-[l-(P-thienyl]cyclohez;yl]morpholine (TCM) 1-Piperidinocyclohexanecarbonitrile (PCC) Cyclazocine N-Allylnormetazocine(SKF 10,047) Metazocine Pentazocine Etorphine Ketocyclazocine Morphine Naloxone Oxotremorin Scopolamine Atropine Carbamylcholine Verapamil D-600 Nitrendipine Nifedipine

90 + 15 T 54 T 65 T

110 7

4:: 5.0 5.0

8.0

100 600 170 140

80

800 T 90 1700 + 130 > lb,000

10 5 < 0.001

300 3400 7000 > > > > 9000

T T 2: T 500 5ij,ooo 200,000 200,000 200,000 + 1600

30 0.03 0.01 < 0.002 0

16,000 > 1400 1700 > >

+ 2600 1~0,000 + 200 T 200 1~0,000 100,000

0.006 < 0.001

110 +

11,000 5

20

1850

80

x 0.01

0.008

: < 0.001 < 0.001

aIC50 is the concentrationthat displaces 50% of specifically-bound[3H]PCP. Mean + SEM of three determinations,each in triplicate. Modified from Quirion et al (1981a).

bother drugs that did not displace [3H]PCP at 0.1 mM: glycine, glutamic acid, dopamine, apormorphine, haloperidol, histamine, norepinephrine,propranolol, phentolamine, serotonin, methysergide, clonidine, diazepam, neurotensin, bombesin, somatostatin, substance P, pentagastrin, bradykinin, dynorphin, beta-endorphin,leu-enkephalin,and met-enkephalin.

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calcium channel. Already it has been shown that calcium antagonists displaced [3H]PCP binding in tray ish muscle (Edelfrawi et al 1982). Moreover, West et al (1983) recently reported that [5Hlethylketocyclazocinebinds to a lower affinity site in NCB-20 Hybrid neurotumor cells. This site is sensitive to PCP, its derivatives, calcium antagonists like verapamil, and to an alpha adrenergic antagonist, WB-4101. It will be of interest to investigate the interaction between PCP and WB-4101 in the brain. Chronic PCP treatment. A chronic PCP treatment (10 mg/kg/day for 14 days) caused a significant decrease in the number (Bmax) of [3H]PCP and C3H]spi erone binding sites in rat brain (Quirion et al 1982c; Robertson 1982, 1983). For [sH]PCP we observed a 33% decrease in the number of sites and for C3Hlspiperonethe Bmax is 31% lower in treated than in c ntrol rats. No changes in the affinity (Kd) of the receptors for C3HlPCP and [sHlspiperone were observed. Our results show that a chronic PCP treatment induced a decrease in the number of C3HlPCP binding sites in rat brain. Much evidence suggests that there is an inverse relationship between the number of receptors and the degree of receptor occupancy by agonists. Phencyclidine receptors also appear to be regulated that way. This down-regulationof the number of PCP sites induced by chronic PCP treatment may explain the development of tolerance and dependence to this drug, as observed in various species (Balster and Woolverton 1980; Chait and Balster 1978; Murray 1978; Pinchasi et al 1977, 1978). The diminution in the number of C3Hlspiperone binding sites, an antagonist of the O-2 dopamine receptor after chronic PCP treatment, is interesting, especially in regard to the "schizophrenia-like"effects induced by PCP. Since a direct interaction of PCP on dopamine binding sites is unlikely, the decrease in D-2 binding sites in rat striatum might be related to the various effects of PCP on the dopaminergic system. Already it has been shwon that PCP can induce the release of dopamine (Doherty et al 1980), which in a chronic situation induces a down-regulationof the number of D-2 dopamine binding sites. Another explanation might be that PCP can block the uptake of dopamine (Doherty et al 1980), thus inducing a decrease in the number of binding sites because of the higher than normal concentration of dopamine in the synaptic cleft. In any case, the diminution of D-2 dopamine binding sites may contribute in some way to the development of tolerance and dependence to PCP. Autoradiographicdistribution of [3H]PCP binding sites in rat brain. C3HlPhencyclidine receptors are widely distributed in rat brain (Quirion et al 1981a). Labeling was most striking in cortical areas. The hippocampus and the dentate gyrus contained high levels of specific C3HlPCP binding; labeling predominated in layers superficial to the pyramidal and granule cell layers. In the neocortex, labeling was dense in the outer five layers. In the cerebellum it predominated in he molecular layer. The striatum and the nucleus accumbens contained moderate levels of [5H]PCP receptors. In the brainstem, central gray, substantia nigra, interpeduncularnucleus, and periaqueductal gray areas were richer in binding sites than adjacent structures. Most of the thalamic and hypothalamic areas con ained low densities HIPCP binding of [3H]PCP receptors. In the spinal cord, the ventral horn is richer in [5' sites than the dorsal horn. Overall, C3HlPCP receptor distribution seems appropriate for a drug with "schizophrenomimetic"properties (Snyder 1980). Finally, our [3H]PCP receptor distribution data correlates well with regional brain metabolism localization (using 2-deoxyglucose as a probe) observed during ketamine anesthesia (Hamner et al 1982). Conclusions In summary, we and various other groups have shown that C3HlPCP binding is saturable, stereoselective,affected by various ions, and possesses a li and selectivity which correlates 9HlPhencyclidinebinding is well with its behavioral and electrophysiologicaleffects. L: also regionally distributed in the brain and down-regulatedafter chronic PCP treatment. Thus, we strongly believe that [3H]PCP labels a site in the brain which is highly relevant to its pharmacologicalactions (Pert and Quirion 1983). Moreover, it suggests the existence of an endogenous ligand for this peculiar receptor. Already we have preliminary evidence that indicates the presence of a peptide in brain that could act as an endogenous ligand for the PCP receptor (Quirion et al 1983, O'Donohue et al 1983). The complete characterizationof this new psychoactive substance will certainly generate a great deal of interest in the neurosciences. Acknowledgment Remi Quirion is a fellow of the Medical Research Council of Canada.

Phencyclidine

receptors

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