HETEROGENEITY OF OPIATE SUBJECTIVE BEHAVIORAL EFFECTS

 

Opiates, a large class of "morphine-like" drugs, exert a large range of pharmacological effects, in addition to analgesia. The heterogeneity of effects among opiates has been well known for many years. Most notable in this regard are the properties of the mixed agonist-antagonist opiates of the benzomorphan series. These compounds have been of special interest because of the desirability of developing a drug having morphine-like analgesic effects but lacking addictive potential. Cyclazocine, SKF-I0,047 (N-allylnorcyclazocine), and related drugs of the benzomorphan group differ from classical opiates in displaying psychotomimetic effects in humans and unique behavioral effects in animals (Haertzen, 1970; Holtzman, 1979). The syndromes associated with many of the benzomorphans depend dramatically on the species and vary qualitatively with the dose administered. At low dose many of these drugs produce effective analgesia in humans (Lasagna, 1954; Keats, 1964). In addition, they may clinically antagonize the analgesic and respiratory-depressant effects of classical opiates (Harris, 1964), precipitate a withdrawal syndrome in morphine-addicted patients, and prevent development of addiction to morphine (Martin, 1965). At the same time, these opiates may produce physical dependence when administered alone and a characteristi withdrawal syndrome differing from that of morphine upon drug cessation (Martin, 1965). At higher doses such benzomorphans produce a combination of sedation, "drunkenness," and psychosis differing from any morphine effect (Harris, 1964; Martin, 1967). The psychotomimetic effects include depersonalization, dysphoria, suspiciousness, and hallucinations (Haertzen, 1970).

 

In animal behavioral studies these drugs also produce both classical apiate (Killam, 1976; Steinert, 1973) and unique effects. The latter include lisruption of learned avoidance performances (Wray, 1972) and locomotor ,simulation not reversed by naloxone (Holtzman, 1973), constriction of pupils, lecreases in flexor reflex and skin twitch, sedation, ("K" effects) and/or anine delirium, tachycardia and tachypnea ("a" effects) (Gilbert, 1976; vIartin, 1976). On the basis of its properties in dogs, cyclazocine was noposed to be a p antagonist, K agonist, and a agonist (Martin, 1976); 3KF-10,047 was proposed to be a more selective j1 antagonist and a agonist with relatively few K properties (Gilbert, 1976).

 

Certain of the effects of SKF or cyclazocine in animals cannot be -eversed by the pure opiate antagonists naloxone or naltrexone (Holtzman, [969; Teal, 1980; Rosencrans, 1978). Other effects of these can be so -eversed, but only at significantly higher antagonist concentrations that are -equired to reverse morphine actions (Schaefer, 1978; Jasinski, 1968; Lord, [977). Thus, in humans and in animals the mixed agonist-antagonist opiates moduce a combination of morphine-like effects, anti-morphine effects, and .-pique sedative and psychotomimetic effects.

 

OPIATE RECEPTOR SUBCLASSES

 

The wide diversity of behavioral effects exhibited by these opiate analgesics raises the question as to whether these could be mediated by a tingle class of receptor sites. Heterogeneous opiate receptor populations were postulated by Martin and coworkers (1976) on the basis of neurophysiological and behavioral evidence. Striking differences in pharmacological responses to different types of narcotic analgesics and their inabilities to substitute for one mother in the suppression of withdrawal symptoms in addicted animals provided evidence for at least three receptor types in the dog CNS. These were termed (1) ,u receptors with which morphine-like drugs preferentially interact; (2) K receptors with which some benzomorphans such as ketooyclazocine interact; and (3) a receptors, for which the prototypic ligand is N-allylnorcyclazocine (SKF-10,047). Effects associated with p receptors included meiosis, bradycardia, hypothermia, analgesia and indifference. Effects characteristic of the K receptor included pupillary constriction, decreased flexor reflexes and sedation. The a syndrome involved mydriasis, tachycardia, and mania or "canine delirium," which Martin proposed to be the equivalent of psychotomimetic effects in man.

 

More recently the evidence for distinct 3-endorphin, enkephalin, and possible dynorphin-mediated neuronal systems has lent support to the concept of multiple opiate receptors. In pharmacological and biochemical investigations, Kosterlitz and his co-workers (Lord, 1977) provided evidence for yet a fourth opiate receptor type. The 5 receptor was postulated to be the site at which the shorter enkephalin peptides preferentially interact. The depression of electrically-induced contractions in the guinea pig ileum and mouse vas deferens and the inhibition of radiolabelled opiate binding in brain were used as assays. The guinea pig ileum was shown to contain mainly p receptors, whereas the mouse vas deferens contained a mixture of p and the putative 5 receptor, with which the shorter enkephalin peptides preferentially interact. Thus, in the ileum, enkephalins are equal or slightly less potent than morphine whereas in the vas deferens, stable synthetic enkephalins are about 200 times more potent than is morphine. In addition, the guinea pig ileum would appear, on the basis of pharmacological evidence, to have many more K receptors than does the mouse vas deferens. Guinea pig brain appeared to parallel the mouse vas deferens most nearly in its receptor subclass distribution. By contrast, many neutoblastoma cell lines have been shown to bear only enkephalin or 5 receptors (Chang, 1978).

 

How mutually exclusive are the opiate receptor subtypes? On the basis of clinical and animal behavioral studies alone it is not possible to determine whether putative a ligands, for example, exert their effects through a unique population of "a" receptors alone or whether they cross-react with p and a receptors. In addition, such studies cannot determine whether there are distinct p, K, and a receptors, or whether the diverse effects of these opiates are mediated by binding in different manners to the same receptor.

 

Of particular interest is the finding that in pharmacological assays, (3-endorphin, unlike the shorter opioid peptides, interacts equally well with bath p and 5 receptors. This result suggests that met-enkephalin may be designed for a more specialized function than 0-endorphin. Recently, Snyder (1980) has suggested that met-enkephalin may be the endogenous ligand specifically targeted for mu receptors, whereas leuenkephalin is the endogenous delta ligand. We have prepared a radioiodinated sulfoxide-carbinol derivative of mets-enkephalin, FK 33-824 (Roemer, 1977), and have shown by competitive displacement analyses that this shorter opioid peptide also interacts equally well with the putative p and 5 receptors (Kream, 1979). On the basis of studies with enkephalin fragments, Chang and Cuatrecasas (1979) have suggested that it may be the hydrophobic group of the phenylalanine residue of enkephalin which is responsible for recognition by 5 receptors.

 

Among the narcotic opiates N-cyclopropylmethylnoretorphine and some mixed agonist-antagonist opiates appear to be equipotent in competing with p and 5 ligands and thus appear to interact equally well with both receptor types (Chang, 1979).

 

BIOCHEMICAL EVIDENCE FOR MU AND DELTA RECEPTORS

 

Substantiation for heterogenous opiate receptors comes from a variety )f pharmacological and biochemical approaches. Differences in pharmacological )rofiles of opiates and opioid peptides provide the first in vitro evidence for he existence of distinct opiate receptor subtypes (Lord, 1977; Waterfield, [977; Simantov, 1978; Lord, 1976; Kosterlitz, 1980). In particular, cornmrison of the results of binding studies in the brain with those of bioassays n preparations innervated by cholinergic or adrenergic nerves suggested that )piate receptor populations in both the central and peripheral systems are Heterogeneous (Lord, 1977; Lord, 1976). Opiate narcotic agonists and antagonists were found to be more potent inhibitors of the binding of labelled )piates than of labelled enkephalins, whereas the reverse was found to be rue of enkephalins and their analogs. Conclusions were based on the observation that the rank order of potency was different in different assay systems.

More recently, studies involving the competition of ligands for radioabelled opiate binding sites in specific brain regions (Chang, 1979; Simantov, 1978; Leslie, 1980) have provided further biochemical evidence for mu and lelta receptors, and indicate that these have somewhat different distributions :hroughout the brain. Thus the thalamus and hypothalamus were shown to be relatively enriched in mu receptors. In contrast, the frontal cortex and striatum appeared to have equivalent densities of these receptor types. 'These results have been confirmed by light microscopy autoradiography studies :Goodman, 1980) involving 125I-[D-Alaa, MePhe4, Met(0)5-ol] enkephalin as a

ligand and 125I-[D-Ala2, D-Leus] enkephalin as the 5 probe.

 

A direct and elegant approach has been that of cross-protection studies. Thus, it is expected that for the protection of a specific opiate receptor class against inactivation by alkylating agents, alkaloid opiates would protect alkaloid binding sites more effectively than would the enkephalins. Conversely, the enkephalin binding sites would be expected to be more effectively protected by enkephalins than by alkaloids. Using just such an approach, Robson and Kosterlitz (1979) showed that dihydromorphine (DHM) protects against inactivation of [3 H] DHM binding sites by phenoxybenzamine more effectively than does D-Ala2, D-Leus-enkephalin. Similarly, DADLE protects [3H] DADLE sites more effectively than does DHM. At the same time Simon and co-workers (Smith, 1980) showed that naltrexone and morphine are 20 and 8 times, respectively, more effective in protecting the binding of [3H] naltrexone than of [3H] enkephalin against inactivation by N-ethylmaleimide. DADLE and D-Ala2, Met5-enkephalinamide (DALA), however, were more effective (7 and 30 times, respectively) for the protection of [3H] DADLE binding. Together these studies have provided considerable substantiation for mu and delta receptors.

 

What is the molecular basis of these receptor differences? Possible molecular models include: (1) that p, 5, and other opiate receptors may be distinct polypeptide chains, the 3-dimensional structures of which are related but not identical; or (2) that these subclasses are the same polypeptide specie: in different lipid or membrane environments, in different conformational or aggregation states, or in the presence or absence of small effector-regulator molecules. Solubilization of the receptor species and its eventual purification and characterization should provide the size, subunit composition, and structural information to distinguish among these and other possibilities.

 

Although opiate receptor subclasses have been shown to differ with respect to ligand specificity, their interrelationships at the cellular level and possible functional distinctions have not been determined. Preliminary studies by ourselves (Zukin, 1980) and by others (Pert, 1980) suggest that p receptor binding may be significantly more sensitive to negative regulation by guanyl nucleotides than 5 receptor binding. Thus, p receptors could be functionally coupled to adenyl cyclase and 5 receptors not. Other approaches which would provide clarification of this issue include localization of opiate recepto: subtypes to pre- or postsynaptic membranes, in specific brain regions, and in peripheral tissue.

 

ARE THERE REALLY KAPPA AND SIGMA RECEPTORS?

 

Whereas a wide body of tantalizing data has provided evidence for the existence of distinct mu and delta receptors, attempts to establish the presence of kappa and sigma receptors have been more difficult. Thus, the first in vitro receptor binding studies involving [3H] ethylketocyclazocine (EKC, putative mu and kappa ligand) (Hiller, 1979; Pasternak, 1980; Chang, 1980; Kosterlitz, 1980) concluded that there were no distinct kappa receptors in brain. These investigations involved competitive displacement analyses and regional distribution studies of the [3H] EKC high affinity binding component only. These parameters were then compared with those previously reported for radiolabelled-p ligands. Other studies, notably those of Kosterlitz and Paterson (1980) and of Romer et al (1980), did lend support to the concept of kappa sites. The former showed in cross protection studies that EKC was far more effective than was morphine in preventing the inactivation of [3H] EKC binding sites by phenoxybenzamine. One possible explanation for these differing conclusions was that the Kosterlitz group worked with guinea pig brain, which they showed to have four times the density of kappa receptors than does rat brain. Romer and his co-workers (1980) studied the pharmacology and binding properties of bremazocine, a drug which they proposed to be a highly selective K agonist. In vivo, bremazocine produced both potent aid analgesia, and the K syndrome previously described by Martin for keto;yclazocine. No cross-tolerance to morphine was observed. In the guinea pig leum and mouse vas deferens assays, bremazocine inhibition of electricallynduced contractions was reversible by MR 2266 but not by naloxone. 3inding of [3H] bremazocine occurred to a single class of sites with a high affinity (KD = 0.55nM). In competition displacement analyses, MR 2266 and WIN 44,441-3 were shown to be much more potent than was morphine. Together these findings provide strong evidence that bremazocine interacts vith non-p opiate receptors, most likely K receptors.

 

Important evidence for K and a receptors has also been provided in two -ecent behavioral studies. Harris (1980) using the scheduled-controlled xhavior paradigm and Holtzman (1980) using the drug-discriminative stimulus assay have provided convincing evidence that p, K, and a drugs each nteract with distinct receptors.

 

Our approach has been to study the binding sites of [3H] cyclazocine putative u, K, and a ligand), [3H] EKC, and [3H] SKF-10,047 (putative mu and sigma ligand) by a combination of Scatchard, competitive displacement, tnd kinetic analyses (Zukin, 1981). In the first set of studies, we measured ;pecific [3H] cyclazocine binding, defined as total binding minus binding in he presence of 1 p.M non-radioactive cyclazocine, as a function of [31-1,1 .;yclazocine concentration. Specific binding was found to constitute approximately 92 percent of total binding at 1.0 nM [3f1] ligand and 67 percent of ;otal binding at 100 nM [3H] ligand. Binding data obtained using a centrifugation assay were shown to vary less than ± 5 percent from that obtained using the rapid filtration assay. Scatchard analyses revealed the [nteraction of [3H] cyclazocine with three distinct binding sites characterized by affinities of 0.2 nM, 10 nM and 70 nM (50 mM Tris-HC1 buffer, pH 7.4 it 4°C). In contrast, many radiolabelled classical opiates and opioid peptides xhibit biphasic binding, but do not exhibit binding to such a low affinity

 

The apparent KD of 0.2 nM for the high affinity cyclazocine site agreed closely with that reported for displacement by cyclazocine of [3H] naloxone binding to the opiate receptor. Addition of 15 nM naloxone to the incubation mixture resulted in elimination of [3H] cyclazocine binding to the tight sites with relatively little change in binding to the weak sites. When a series of opiates were tested for their abilities to displace specifically bound [3H] cyclazocine (1 nM), their rank order agreed with that for their displacement of [3H] naloxone. Together these findings indicated that the high affinity cyclazocine binding was occurring to the classical opiate receptor.

 

The only drugs other than cyclazocine-like opiates which proved active at displacing [3H] cyclazocine from the site of lowest affinit were PCP-like drugs. The relative potencies of a series of PCP analogs in displacement of [3H] cyclazocine (60 nM) in the presence of naloxone (60 nM) were similar to those for their abilities to displace [3H] PCP from its binding sites (Zukin, 1979; Zukin, 1981). The binding affinity of 70 nM observed for [3H] cyclazocine binding to the weak sites was consistent with the IC50 of 200 nM observed for displacement of [3H] PCP by cyclazocine. The total number of weak cyclazocine binding sites (820 fmol/mg protein) was similar to that of specific PCP sites (1100 fmol/mg protein) measured under the same conditions using polylysine-soaked filters. Addition of PCP (10 iiM) to the incubation mixture resulted in the disappearance of [3H] cyclazocine binding to the weak (70 nM) sites. Together, these findings suggested that the "low affinity" [3H] cyclazocine binding was occurring to the [3H] PCP binding site.

 

The high and low affinity [3H] cyclazocine sites exhibited differential sensitivities to sodium and also to the selective sulfhydryl reagent N-ethylmaleimide. In addition, all three sites exhibited greater than 50 percent loss of specific binding following incubation with trypsin (5 pg/m1) for 15 min at room temperature, and greater than 80 percent loss of specific binding following incubation at 60°C for 15 min in the absence of added reagents. Together, these findings indicated that all three sites have a proteinlike component.

 

In summary, competition analyses involving rank order determinations for a series of opiates and other drugs have indicated that the cyclazocine binding sites represent, in order of decreasing affinity: (1) the classical opiate receptor (the putative "ii" receptor); (2) a second as yet uncharacterized opiate binding site; and (3) the specific [3H] phencyclidine binding site.

 

In a second set of studies, we have studied the binding of [3f1] EKC and [3H]' SKF-10,047 to rat brain homogenates in order to provide further evidence for kappa and sigma receptors, and to attempt to understand the diverse actions of these drugs (Zukin, 1981). Scatchard analyses utilizing various competing drugs revealed the apparent interaction of [3H] EKC with two binding sites with affinities of 0.3 nM (Bm,, = 75 fmol/mg) and 22 nM (Bnu, = 60 fmol/mg). DHM or DADLE (20 nM) reduced [3H] EKC binding to high-affinity sites by approximately 50 percent but had no significant effec on binding to low-affinity sites. DHM (100 nM) plus DADLE (20 nM) reduced [3H] EKC binding to high-affinity sites by more than 90 percent while not affecting binding to weak sites. A series of opiates displaced binding of [3f1] EKC from high-affinity sites in rank order bremazocine > EKC > cyclazocine > DHM > DADLE while not affecting low-affinity [3H] EKC binding. Phencyclidine (PCP) selectively displaced [3H] EKC binding from low-affinity sites. The high- and low-affinity [3H] EKC sites exhibited differential sensitivities to sodium. For [3H] SKF-10,047 Scatchard analyses again revealed two distinct binding sites characterized by affinities of 4 nM (B„,„ = 160 fmol/mg) and 65 nM (Bf = 800 fmol/mg).

 

VOrmorphine (100 nM) markedly reduced binding of [3H] SKF-l0,047 to mly the high-affinity sites. Rank orders for opiate displacement of 4 nM [3H] SKF-10,047 were bremazocine = cyclazocine = levorphanol > SKF-10,047 > DADLE > morphine > pentazocine. PCP selectively decreased low-affinity 3KF-10,047 binding. These data provide biochemical evidence for interaction EKC and SKF-l0,047 with the a receptor (which we have previously dentified with the PCP receptor) and the /.2 receptor. The high-affinity EKC )inding may represent a combination of ti and K receptor populations.

 

ARE THE SIGMA RECEPTOR AND [3H] PCP BINDING SITE ONE AND THE SAME?

 

Another analgesic and anesthetic drug with prominent psychotomimetic ;ide effects is phencyclidine (PCP, or "angel dust"). Early trials in humans ihowed PCP to produce excellent anesthesia and analgesia (Luisada, 1978; .7hen, 1959). Early investigators coined the term "dissociative anesthesia" to lescribe the PCP-induced state of altered consciousness in which higher brain Functions seemed disconnected from awareness of the environment (Corssen, 1965). A significant number of patients suffered psychotic reactions during emergence from anesthesia. Oral PCP in subanesthetic doses proved able to control severe pain but also led to psychotic reactions (Griefenstein, 1958).

 

The psychotomimetic effects of PCP, noted in its earliest clinical trials, ranged from mild excitement to severe manic excitement and hallucinations (Vincent, 1979). Subanesthetic doses (0.1 mg/kg i.v.) induced body-image changes, feelings of estrangement, disorganization of thought, drowsiness and apathy in all subjects; feelings of inebriation, negativism, and hypnogogic states in a majority, and motor stereotypy in a large minority. The PCP-thought disorder included blocking echolalia, looseness of associations, neologisms and severe impairment of the capacity for abstraction; these are among the classical components of the schizophrenic syndrome. In this respect, PCP differs markedly from LSD and other hallucinogens which do not mimic the primary signs and symptoms of schizophrenia. The relationship between the analgesic, anesthetic, and psychotomimetic effects remains undetermined.

 

The question arises as to whether the diverse actions of PCP are mediated through interaction at specific receptor sites and whether these sites are related to the sites of action of other psychotomimetic drugs such as the opiate mixed agonist-antagonists. Preliminary studies by our laboratory (Zukin, 1979; 1981) and by others (Vincent, 1979) indicate that [3H] PCP binds to specific sites in animal nervous tissue. Maayani and Weinstein (1980) had suggested that the filtration method might be unsuitable for detecting the pharmacologically relevant binding sites of [3H] PCP. More recently however, we (Zukin, 1981) and others (Vincent, 1980; Quirion, 1981; McQuinn, 1981) have shown this method applicable kinetically and in terms of specificity to the detection of PCP receptors. Moreover, Quirion et al (1981) have described essentially identical sites using both direct binding to slide-mounted brain sections and autoradiography.

 

Several pieces of evidence suggest that the sigma receptor and PCP binding site may be the same. Thus, of a large number of opiates tested, only cyclazocine-like opiates have been shown to displace [3H] PCP binding. Conversely, PCP and its derivatives can inhibit binding of [31-1] cyclazocine to its lowest affinity (KD = 70 nM) binding site. Behaviorally too the two classes of drugs can produce similar effects.

 

Recent animal-behavioral studies show that cyclazocine-like opiates display PCP-like properties. Teal and Holtzman (1980) proposed cyclazocine to possess both "opioid" and "nonopioid" properties based on their findings that the discriminative stimulus properties of the drug were only partially naloxone-reversible. In a subsequent discriminative stimulus study using rats trained to cyclazocine, Teal and Holtzman (1980) found that of twelve test compounds, ketocyclazocine, SKF-10,047, ethylketocyclazocine, PCP, ketamine, pentazocine, and levallorphan generalized to cyclazocine (the last two most weakly); morphine, nalorphine, amphetamine, mescaline, and LSD were inactive. Discriminative effects of cyclazocine were only partially reversible by naloxone. In a discriminative stimulus paradigm utilizing rats trained to PCP, Holtzman (1981) has found that animals trained to PCP generalized to cyclazocine, SKF-10,047 and cylorthan, whereas they do not generalize to nalorphine.

 

Thus, it would appear that opiates, much like adrenergic, cholinergic, and dopaminergic ligands, initiate their broad spectrum of diverse pharmacological actions by interaction with multiple receptor sites. It is interesting to note, on the one hand, the apparent complete cross-reactivity of p and 8 receptors. The finding that p and 8 sites exhibit differentiational sensitivity to GTP (Zukin, 1980; Pert, 1980) and the suggestion that they represent a single receptor in either a cyclase-coupled or uncoupled state (Gentleman et al 1981) lend support to the concept that p and 8 receptors are functionally related in analogy to dopamine type 1 and type 2 receptors. In contrast, there is no such cross-reactivity between p and either K or a receptors. The failure of morphine and other "classical" opiates, including the narcotic antagonists naloxone and naltrexone, to bind substantially to K sites and at all to a sites is of particular interest. Thus, it is intriguing to speculate that the a receptors do not represent "opiate" receptors at all, but rather a unique class of sites at which a class of chemically diverse psychotomimetic drugs, including some of the opiates and PCP, produce their distinctive effects.

 

REFERENCES

 

Chang K-J, Cooper BR, Hazum E and Cuatrecasas P: Multiple opiate receptors: Different regional distribution in the brain and differential binding of opiates and opioid peptides. Mol Pharmacol 16:91-104, 1979

Chang K-J and Cuatrecasas P: Multiple opiate receptors; enkephalins and morphine bind to receptors of different specificity. J Biol Chem 254:2610-2618, 1979

Chang K-J, Hazum E and Cuatrecasas P: Possible role of distinct morphine and enkephaline receptors in mediating actions of benzomorphan drugs (putative K and a receptors). Proc Nat Acad Sci USA 77:4469-4473, 1980

Chang K-J, Miller RJ and Cuatrecasus P: Interaction of enkephalin with opiate receptors in intact cultured cells. Mo! Pharmacol 14:961-970, 1978

hen G, Ensor CR, Russell D and Bohner B: The pharmacology of l-(1-phenylcyclohexyl) piperidine. HCI. J Pharmacol Exp Ther 127:241-250, 1959

2orssen G and Domino EF: Dissociative anesthesia: Further pharmacologic studies and first clinical experience with phencyclidine derivative C1-581. Anesth Analg 45:29-40, 1965

Zvans JM, Hogg MI, Lunn JN and Rosen M: Degree and duration by naloxone of effects of morphine in conscious subjects. Br Med J 2:589-591, 1974

-J'ilbert PE and Martin WR: The effects of morphine- and nalorphine-like drugs in the nondependent, morphine dependent and cyclazocine dependent chronic spinal dog. J Pharmacol Exp Therap 198:66-82, 1976

3oodman R, Snyder SH, Kuhar MJ and Young WS III: Differentiation of delta and mu opiate receptor localizations by light microscopic autoradiography. Proc Nat Acad Sci USA 77:6239-6243, 1980

Illiefenstein FE, DeVault M, Yoshitake MD and Cajewski JE: A study of a Iarylcyclohexylamine for anesthesia. Anesth Analg 37:283-294, 1958

qaertzen CR: Subjective effects of narcotic antagonists cyclazocine and nalorphine on the addiction research center inventory (ARCI). Psychopharmacologia 18:366-377, 1970

Harris RA: Interactions between narcotic agonists, partial agonists and antagonists evaluated by schedule-controlled bheavior. J Pharmacol Exp Ther 213:497-503, 1980

Harris LS and Pierson AK: Some narcotic antagonists in the benzomorphan series. J Pharmacol Exp Ther 143:141-148, 1964

Hiller JM and Simon El: 3H-ethylketocyclazocine binding: Lack of evidence for a separate K receptor in rat CNS. Eur J Pharmacol 60:389-390, 1979

Holtzman SG: Narcotic antagonists as stimulants of behavior in the rat: Specific and nonspecific effects. In: MC Braude et al (eds.), Narcotic Antagonists. New York: Raven Press, pp 371-382, 1979

Holtzman SG: Phencyclidine-like discriminative effects of opioids in the rat. J Pharmacol Exp Ther 1981 (in press)

Holtzman SG and Jewett RE: Stimulation of behavior in the rat by cyclazocine: Effects of naloxone. J Pharmacol Exp Ther 187:380-390, 1973

lasinslci DR, Martin WR and Sapira JD: Antagonism of the subjective effects, behavioral,

papillary and respiratory depressant effects of cyclazocine by naloxone. Clin Pharmacol Ther 9:215-222, 1968

Keats AS and Telford I: RF Gould (ea.), Narcotic Antagonists as Analgesics: Chemical

Aspects in Molecular Modifications in Drug Design. Washington, DC: American Chemical Society, 1964

Killam KF' Jr, Brocco MJ and Robinson CA: Evaluation of narcotic and narcotic antagonist interactions in primates. Ann NY Acad Sci 281:331-335, 1976

Kosterlitz HW, Lord JAH, Paterson SJ and Waterfield AA: Effect of changes in the

structure of enkephalins and of narcotic analgesic drugs on their interactions with

it- and 6-receptors. Br J Pharmacol 68:33-342, 1980

Kosterlitz HW and Paterson W: Characterization of opioid receptors in nervous tissue.

Proc R Soc Lond 210:113-122, 1980

Kream RM and Zukin RS: Binding characteristics of a potent enkephalin analog.

Biochem Biophys Res Commun 90:99-109, 1979

Lasagna L and Beecher HK: The analgesic effectiveness of nalorphine and nalorphine-

morphine combinations in man. J Pharmacol Exp Ther 112:356-363, 1954

Leslie FM, Chavkin C and Cox BM: Ligand specificity of opioid binding sites in brain

and peripheral tissues. In: EL Way (ed.), Endogenous and Exogenous Opiate

Agonists and Antagonists. New York: Pergamon Press, pp 109-112, 1980

Lord JAH, Waterfield AA, Hughes J and Kosterlitz HW: Endogeneous opioid peptides:

Multiple agonists and receptors. Nature 267:495-500, 1977

Lord JAH, Waterfield AA, Hughes J and Kosterlitz HW: HW Kosterlitz (ed.), Multiple.

Opiate Receptors in Opiates and Endogenous Opioid Peptides. Amsterdam:

North-Holland Publishing, pp 275-280, 1976

Luisada PV: The phencyclidine psychosis: Phenomenology and treatment. In: RC

Petersen and RC Stillman (eds.), Phencylidine Abuse: An Appraisal. NIDA

Research Monograph 21, pp 241-253, 1978

Maayani S and Weinstein H: "Specific Binding" of 3H-phencyclidine: Artifacts of the

rapid filtration method. Life Sci 26:2011-2016, 1980

Martin WR: Opioid antagonists. Pharmacol Review 19:463-521, 1967

Martin WR, Eades CG, Thompson JA, Huppler RE and Gilbert PE: The effects of morphine

and nalorphine-like drugs in the nondependent and morphine-dependent chronic

spinal dog. J Pharmacol Exp Therap 197:517-532, 1976

Martin WR, Fraser HI', Gorodetzky CW and Rosenberg DE: Studies in the dependence

producing potential of the narcotic antagonists 2-cyclopropylmethy1-2-dydroxy--

5, 9 dimethy1-6,7 benzomorphan (cyclazocine, Win 20, 740, ARC II-C-3). J

Pharmacol Exp Therap 150:426-436, 1965

Martin WR and Gorodetzky CW: Demonstration of tolerance to and physical dependence

on N-allylnormorphine (nalorphine). J Pharmacol Exp Therap 150:437-442, 1965

McQuinn RL, Cone Li, Shannon HE and Su T-P: Phencyclidine: I. Structure activity

relationships of the cycloalkyl ring of phencyclidine. J Med Chem 1981 (in press)

Pasternak GW: Multiple opiate receptors: 3H-ethylketocyclazocine receptor binding and

ketocyclazocine analgesia. Proc Nat Acad Sci USA 77:3691-3694, 1980.

Pert CB and Taylor D: EL Way (ed.), Type 1 and Type 2 Opiate Receptors in Endogenous

and Exogenous Opiate Agonists and Antagonists. New York: Pergamon Press,

pp 87-90, 1980

Quirion R, Hammer R, Herkenham M and Pert CB: A phencyclidine/sigma opiate

receptor: Its visualization by tritium-sensitive film. Proc Nat Acad Sci USA

1981 (in press)

Robson LE and Kosterlitz HW: Specific protection of the binding sites of D-ale-D-leus-

enkephalin (6-receptors) and dihydrornorphine (A-receptors). Proc R Soc Lond

205:425-432, 1979

Roemer D, Buescher HH, Hill RC, Pless J, Bauer W, Cardinaux F, Closse A, Hauser D

and Hyguenip R: A synthetic enzephalin analogue with prolonged parenteral and

oral analgesic activity. Nature 268:547-549, 1977

Romer D, Buscher H, Hill RC, Maurer R, Petcher TJ, Welle HBA, Bakel HCCK and

Akkerman AM: Bremazocine: A potent, long-acting opiate kappa agonist. Life

Sci 27:971-978, 1980

Rosencrans JD, Chance WT and Spencer RM: The discriminative stimulus properties of

cyclazocine: Generalization studies involving nalorphine, morphine and LSD. Res Commun Chem Pathol Pharmacol 20:221-237, 1978

Schaefer GJ and Holtzman SG: Discriminative effects of cyclazocine in the squirrel monkey. J Pharmacol Exp Ther 205:291-301, 1978

Simatov R, Childers SR and Snyder SH: The opiate receptor binding interaction of 311-methionine enkephalin: An opioid peptide. Eur J Pharmacol 47:319-331, 1978

Smith JR and Simon EJ: Selective protection of stereospecific enkephalin and opiate

binding against inactivation by N-ethylmaleimide; evidence for two classes of opiate receptors. Proc Nat Acad Sci USA 77:281-284, 1980

Steinert HR, Holtzman SG and Jewett RE: Some agonistic actions of the morphine antagonist levallorphan on behavior and brain monoamines in the rat. Psychopharmacologia 31:35-46, 1973

Teal JJ and Holtzman SG: Discriminative stimulus effects of cyclazocine in the rat. J Pharmacol Exp Ther 212:368-376, 1980

Teal JJ and Holtzman SG: Discriminative stimulus effects of prototype opiate receptor agonists in monkeys. Eur J Pharmacol 68:1-10, 1980

Vincent JP, Kartalovski B, Geneste P, Kamenka JM and Lazdunski M: Interaction of phencyclidine ("angel dust") with a specific receptor in rat brain membranes. Proc Nat Acad Sci USA 76:4578-4682, 1979

Vincent J-P, Vignon J, Kartalovski B and Lazdunski M: Binding of phencyclidine to rat brain membranes: Technical aspects. Eur J Pharmacol 68:73-77, 1980

Vincent J-P, Vignon J, Kartalovski B and Lazdunski M: Compared properties of central and peripheral binding sites for phencyclidine. Eur J Pharmacol 68:79-82, 1980

Waterfield AA, Smokcum RWJ, Hughes J, Kosterlitz HW and Henderson G: In vitro pharmacology of the opioid peptides, enkephalins and endorphins. Eur J Pharmacol 43:107-116, 1977

Wray SR: A correlative evaluation of cyclazocine, LSD and naloxone on continuous

discriminated avoidance in rats. Psychopharmacologia 26:29-43, 1972 Zukin RS and Gintzler AR: Guanyl nucleotide interactions with opiate receptors in

guinea pig brain and ileum. Brain Res 186:486-491, 1980

Zukin SR, Margolis A and Zukin RS: Biochemical Evidence for Kappa and Sigma Receptors. 1981 (in preparation)

Zukin RS and Zukin SR: Demonstration of 3H-cyclazocine binding to multiple opiate receptor sites. Mol Pharm 1981 (in press)

Zukin SR and Zukin RS: Identification and characterization of 3H-phencyclidine binding to specific brain receptor sites in PCP. In: E Domino (ed.), Historical and Current Perspectives. Ann Arbor: NPP Books, 1981

Zukin SR and Zukin RS: Specific [3H] pencyclidine binding in rat central nervous system. Proc Nat Acad Sci USA 76:5372-5376, 1979