Proc. Natl. Acad. Sci. USA Vol. 77, No. 8, pp.4469-4473, August 1980 Biochemistry Possible role of distinct morphine and enkephalin receptors in mediating actions of benzomorphan drugs (putative K and o' agonists) (opiate receptors/physical dependence/neuroblastoma cells/ethylketocyclazocine/mixed opiate agonist-antagonist) KWEN-JEN CHANG, ELI HAZUM, AND PEDRO CUATRECASAS Department of Molecular Biology, The Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709 Communicated by George H. Hitchings, April 21, 1980

ABSTRACT The binding of many opiates and enkephalins 2), and 125I-labeled Sandoz's peptide, FK 33 824 (Tyr-D-Ala- to enkephalin (8) and morphine (es) receptors was compared Gly-N-Me-Phe-Met(O)ol) (3). Enkephalin receptors can be using three different binding assays: fi) 125I-labeled[DA-a, studied by using 3H-labeled natural enkephalins (2, 5) and D-Leu51enkephalin or I-4abeled[-Ala ,N-Me-Phe4,Met(Opol- enkephalin to brain membranes; (ih) [3Hkethylketocyclazocine '5I-labeled [D-Ala2,D-Leu5]enkephalin (1, 3). to brain membranes; and (iih) [3Hldiprenorphine and [3H]nal- Studies in chronic spinal dogs led Martin and colleagues to oxone to neuroblastoma cell and brain membranes, respectively. postulate the existence of three types of opiate receptors (pA, K, According to their relative binding potencies and the effects of and a) on the basis of the production of different pharmaco- Na+ and GTP on the binding to these two receptors, opiates and logical syndromes by morphine and benzomorphans (6, 7). enkephalins can be classified into seven classes: (i) morphine- Morphine, a ju agonist, supressed abstinence in morphine- type p agonists; (ii) enkephalin-type 8 agonists; (iii) mixed ago- dependent dogs, whereas benzomorphan derivatives (i.e., ke- nists-antagonists; (iv) putative K agonists; (v) putative or agonists; (vf) nalorphine-type antagonists; and (vit) opiate antagonists. tocyclazocine and ethylketocyclazocine), K agonists, neither Studies with [3Hethylketocyclazocine do not reveal specific x precipitated nor depressed abstinence despite exhibiting an- receptors distinct from those already described that bind mor- algesic effects. The morphine effects were completely anta- phine and enkephalins. The benzomorphan analogs ketocy- gonized by low doses of naloxone, whereas 30 times more of clazocine and ethylketocyclazocine (putative K agonists) and naloxone was required to block the effects of ketocyclazocine N-allylnormetazocine (putative a agonist) bind to morphine (p) and ethylketocyclazocine. N-Allylnormetazocine, a a agonist, and enkephalin (8) receptors with similarly high affinities. The potency of putative K agonists, measured by competition with caused behavioral excitation without analgesia (8) and it did binding of the 3H-labeled antagonist, is greatly reduced by the not depress abstinence in morphine- or cyclazocine-dependent presence of Na+ and GTP; the "Na+ and GTP ratios" are similar animals (8). to those of morphine and enkephalins. However, Na+ and GTP Although pharmacological studies suggest the possible exis- greatly decrease the potency of binding of putative or agonists tence of these three subtypes of opiate receptors, no biochemical to enkephalin receptors but only slightly decrease the binding evidence exists to support this hypothesis. Studies on struc- to morphine receptors. These data suggest that putative K ago- nists have agonistic activity toward both receptors, whereas ture-activity relationships of opiates and enkephalins provide putative a agonists behave as agonists for enkephalin receptors some evidence for the hypothesis that analgesia is mediated but have antagonist activity for morphine receptors. Mixed primarily by the morphine (or ,u) receptors (9, 10). The present agonist-antagonists also show smaller difference in affinity to studies demonstrate that putative K and a agonists have high both receptors. These findings may have important implications affinity for both morphine (ti) and enkephalin (a) receptors. for understanding the differences in the pharmacological effects Although putative K agonists behave as agonists for both sub- of these drugs. types of the receptor, putative a agonists have agonist activity for enkephalin (6) receptors and mixed agonist-antagonist ac- Recently, two subtypes of opiate receptors, morphine (g) re- tivity for morphine (,i) receptors. No evidence for separate K ceptors and enkephalin (6) receptors, have been described receptors can be demonstrated. biochemically in brain membrane preparations from rats (1) and guinea pigs (2) by using radioisotope labeled opiates and MATERIALS AND METHODS enkephalins. Morphine receptors bind morphine preferentially, with an affinity about 10 times better than that for natural [3H]Naloxone (23 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels), and enkephalins. Enkephalin receptors, on the other hand, prefer [3H]ethylketocyclazocine (15 Ci/mmol) were purchased from natural enkephalins to morphine with a difference in affinity New England Nuclear. [3H]Diprenorphine (9 Ci/mmol) was of about 100-fold. The opiate antagonist naloxone binds to purchased from Amersham. 125I-Labeled[D-Ala2,D-Leu5]en- morphine receptors about 20 to 30 times better than to en- kephalin and 125I-[D-Ala2,N-Me-Phe4,Met(O)5ol]enkephalin kephalin receptors (1). Differences in the regional distribution were prepared as described (1, 3). The specific activity of of morphine and enkephalin receptors have also been described 125I-labeled peptides was 1-2 Ci/,jmol. Rat brain membranes in rat brain (3). Neuroblastoma cells (N4TG1 and NG108-15) were prepared by differential centrifugation in isotonic sucrose bear only enkephalin receptors (1, 4). solution as described (3). Neuroblastoma cell membranes were By using low concentrations of labeled opiates and enkeph- prepared from cultured monolayers of neuroblastoma cells alins of high specific radioactivity, it is possible to measure se- (N4TG1) grown in Dulbecco's modified Eagle's minimal es- lectively the interaction of ligands with either morphine or sential medium with 5% fetal bovine serum. The membranes enkephalin receptors. Morphine receptors can be identified and were suspended in 50 mM Tris-HCl (pH 7.7) for binding assays, characterized with 3H-labeled dihydromorphine, naloxone (1, which were performed (24°C, 60 min) essentially as described by using a filtration (GF/C) method (1, 3). Nonspecific binding The publication costs of this article were defrayed in part by page was determined in the presence of 1 ,uM of the respective un- charge payment. This article must therefore be hereby marked "ad- labeled ligand. vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: IC5o, concentration to produce 50% inhibition. 4469 Downloaded by guest on September 28, 2021 4470 Biochemistry: Chang et al. Proc. Nati. Acad. Sci. USA 77 (1980) RESULTS Potency of Opioids in Inhibiting the Binding of 125I4D- Ala2,D-Leu5]Enkephalin and 1251-D-Ala2,N-Me-Phe4,Met- (OpollEnkephalin to Rat Brain Membranes. By using these two 125I-labeled enkephalins as markers (1, 3) for binding to 0O enkephalin (3) and morphine (,i) receptors, respectively, the activities of putative K and ai ligands were compared (Table 1). .0 In contrast to morphine and enkephalins, ketocyclazocine, ethylketocyclazocine, and N-allylnormetazocine showed high affinity and less selectivity in competing with both labeled li- gands. Ethylketocyclazocine was 2 to 3 times more potent than ketocyclazocine, consistent with their known pharmacological potencies. Cyclazocine and other mixed agonist-antagonists, such as oxilorphan,. butorphanol, and pentazocine (3), also showed small differences in the binding to both receptor sites. Inhibitor, M The antagonist naloxone, was more potent toward morphine receptors. Diprenorphine, a potent antagonist and a derivative of oripavine, showed equal and high affinity to both subtypes of the receptor. Inhibition of Binding of [3H]Ethylketocyclazocine to Brain Membranes. The competition curve of morphine and [D-Ala,D-Leu5]enkephalin against [3H]ethylketocyclazocine binding to brain membranes showed a clear biphasic pattern (Fig. 1). Inhibition was complete at submicromolar concen- trations. Modified Scatchard plots reveal that about 65% of the bound [3H]ethylketocyclazocine is directed to the morphine high-affinity sites and 35% to the morphine low-affinity sites. These relative proportions are reversed (35% and 65%) with [D-Ala2,D-Leu5]enkephalin suggesting that the high-affinity sites for morphine probably correspond to the low-affinity sites for enkephalin. The calculated apparent dissociated constants % inhibition (Ks) for these high- and low-affinity sites do not differ signifi- FIG. 1. (Upper) Competition by morphine (0), [D-Ala2,D- cantly from the values obtained independently by using other Leu5jenkephalin (0), and ethylketocyclazocine (M) for the binding labeled opiates and enkephalins. of [3H]ethylketocyclazocine (0.5 nM, 15 Ci/mmol) to rat brain The competition of unlabeled ethylketocyclazocine against membranes. The experiments were carried out in 2 ml of 50 mM Tris-HCl buffer (pH 7.7) for 1 hr at room temperature. Values are [3H]ethylketocyclazocine also reveals biphasic curves (Fig. 1A), means of duplicate incubations. (Lower) Modified Scatchard plots but the differences in the slopes of the Scatchard plots (Fig. 1B) (3). are much smaller than with morphine and enkephalin. This suggests a more similar affinity of ethylketocyclazocine for both kephalin (6) receptots of neuroblastoma cells and the morphine receptor subtypes. These data do not support the hypothesis that (,u) receptors of rat brain can be characterized separately (Table there are separate K receptors that do not bind morphine and 2). Enkephalins were about 10 times more potent in neuro- enkephalins with high affinity. blastoma cell membranes (6) than in brain membranes. Mor- Comparison of Potencies Against the Binding of [3H} phine was about 100 times more potent in rat brain membranes Diprenorphine to Neuroblastoma Cell Membranes and of (,u) than in the neuroblastoma cell membranes. Levorphanol [3H]Naloxone to Rat Brain Membranes. By using the opiate was 20 times more potent in rat brain membranes than in antagonists [3H]diprenorphine and [3H]naloxone, the en- neuroblastoma cell membranes. Mixed agonists-antagonists had a slightly higher affinity for rat brain membranes than neuro- Table 1. Potency of putative IA, 6, and K ligands competing with blastoma cell membranes, but the differences are small (3). binding of 1251-[D-Ala2, N-Me-Phe4, Met(O)5o1l- and Ketocyclazocine, ethylketocyclazocine, and N-allylnormeta- 125I-[D-Ala2, D-Leu5jenkephalin zocine (SKF 10047) also showed only slight differences in these IC50, nM two systems. Naloxone and nalorphine exhibited about 20 times Morphine Enkephalin greater affinity to morphine receptors in rat brain membranes. Drug receptors receptors Diprenorphine showed very high and equal activity for both to con- [D-Ala2,Leu5]Enkephalin (5) 4 ± 0.2 1.5 d 0.1 sites. All of these data are essentially similar and thus Morphine (,u) 0.4 ± 0.2 35 + 5 firmatory of the data obtained by using 125I-labeled enkephalin Levorphanol (i') 0.3 : 0.1 4 + 1 analogs (Table 1). Cyclazocine 0.3 I 0.1 1.2 i 0.3 Effects of Na+ and GTP on the Competition of Binding Ketocyclazocine (K) 2.2 + 0.4 7.5 4 1.5 of 3H-Labeled Antagonists. Na+ and GTP selectively decrease Ethylketocyclazocine (K) 1.0 + 0.2 3.2 + 1.0 the potency of agonists but not (or do so to a lesser extent) of N-Allylnormetazocine (a) 0.7 + 0.1 3.3 + 1.2 antagonists in competing with the binding of 3H-labeled an- Naloxone 1.0 0.2 15 8 tagonists to opiate receptors (11, 13, 14). These effects can be Diprenorphine 0.20 + 0.02 0.18 : 0.02 expressed by the ratio of the concentration to produce 50% in absence of Na+ or GTP IC50 values (the concentration of competing ligand causing 50% inhibition (IC5o) the presence and inhibition ofspecific binding) were estimated from competition curves (Table 3). Both Na+ and GTP decreased the potency of en- produced with 1251-labeled peptides at 0.05 nM. Values are expressed kephalins and morphine in competing with the binding of as mean h SEM for three to six separate experiments. 3H-labeled antagonists to both receptor subtypes. The effects Downloaded by guest on September 28, 2021 Biochemistry: Chang et al. Proc. Natl. Acad. Sci. USA 77 (1980) 4471

Table 2. Apparent dissociation constant (Ki) of opioids for Table 3. Effects of Na+ and GTP on the potency of opioids binding to enkephalin (6) and morphine (,u) receptors using Enkephalin 3H-labeled antagonists receptors Morphine Apparent Ki, nM ([3H]diprenor- receptors Enkephalin Morphine phine) ([3H]naloxone) receptors receptors Receptor Na+ GTP Na+ GTP ([3H]diprenor- ([3Hlnal- affinity Drug ratio ratio ratio ratio Drug phine) oxone) ratio* [Met]Enkephalin 2.8 1.5 4.0 4.0 [Met]Enkephalin 0.4 5.7 0.07 [Leu]Enkephalin 3.3 3.3 6.7 7.3 [Leu]Enkephalin 0.5 8.5 0.06 [D-Ala2,Leu5] [D-Ala2, Leu5]Enkephalin 0.4 2.8 0.14 Enkephalin 3.2 2.2 6.6 12 [D-Ala2, D-Leu5J- [D-Ala2,D-Leu51 Enkephalin 0.4 3.4 0.12 Enkephalin 3.2 1.6 5.8 10 Morphine 36 0.3 120 Morphine 3.6 2.4 20 36 Levorphanol 4 0.2 20 Levorphanol 12.5 18 10 Dextrorphan 700 Dextrorphan 3.8 Pentazocine 67 5.7 11.8 Pentazocine 2.5 1.8 5.6 1.4 Butorphanol 1.6 0.6 2.7 Butorphanol 2.0 3.3 3.5 4.0 Oxilorphan 0.9 0.4 2.3 Oxilorphan 1.6 1.6 2.2 1.8 Cyclazocine 2.5 0.3 8.3 Cyclazocine 2.0 3.5 2.4 Ketocyclazocine 5 3.4 1.5 Ketocyclazocine 3.3 5 8 8.3 Ethylketocyclazocine 1.5 0.6 2.5 Ethylketocyclazocine 7.2 6 30 20 N-Allylnormetazocine 1.3 0.9 1.4 N-Allylnormetazocine 5 7 1.8 4.0 Nalorphine 16 0.9 18 Nalorphine 1.0 3.3 Naloxone 16 0.6 27 Naloxone 0.9 1.2 1.0 2.5 Diprenorphine 0.25 0.2 1.3 Diprenorphine 0.9 0.9 1.0 1.0 The binding was performed with 1 nM [3H]diprenorphine (to Na+ and GTP ratios were calculated from the ratio of the IC50 neuroblastoma cell membrane) and 0.4 nM [3Hlnaloxone (to brain values in the presence and absence of Na+ (100 mM) or GTP (0.1 membrane), respectively. Kis were calculated according to Cheng and mM), with 1 nM [3H]diprenorphine (neuroblastoma cell membranes) Perusoff (12). Kj = IC5o/[1 + (L/Kd)] in which L is the concentration and 0.4 nM [3H]naloxone (brain membranes), respectively. of labeled ligand, Kd is the dissociation constant of labeled ligands (0.2 and 0.5 nM for [3Jdiprenorphine and [3]naloxone, respectively), likely results from the overlap (30%) of binding of 125I-[D-Ala2, and ICso values are the concentrations that inhibit the binding of la- D-Leu5]enkephalin to morphine receptors (1). On the basis of beled ligand by 50%. the relative binding potency to morphine (A) and enkephalin * Receptor affinity ratio is the ratio ofthe apparent Kis for enkephalin (6) receptors, and the ability of Na+ and GTP to alter the po- receptors and morphine receptors. tency for competing with the binding of 3H-labeled antagonists, various opiates can be classified into seven classes, as summa- were much more profound for binding to morphine receptors rized in Table 4. than to enkephalin receptors; the greatest effects occurred with i. Morphine, levorphanol (,u agonists), and the Sandoz full agonists, some effects were found for mixed agonist-an- peptide (FK 33824) have much higher affinity to morphine tagonists, and no or only slight effects were observed for an- receptors and behave as agonists for both receptors, based on tagonists. Both ketocyclazocine and ethylketocyclazocine had their high Na+ and GTP ratios. high Na+ and GTP ratios, suggesting that they are likely to be ii. Enkephalins prefer enkephalin (6) to morphine (,t) re- full agonists for both receptors. In contrast, N-allylnormeta- ceptors and behave as agonists for both receptors (i.e., high Na+ zocine, a a agonist, had agonist types of Na+ and GTP ratios for and GTP ratios). enkephalin receptors and mixed agonist-antagonist types of iii. Mixed agonist-antagonists have much lower Na+ and ratios for morphine receptors. This suggests that a a agonist may GTP ratios than do morphine and enkephalins. These com- possess agonist activity for enkephalin receptors and mixed pounds exhibit only small differences in their affinity for both agonist-antagonist activity for morphine receptors. Nalorphine receptors, as indicated by a much smaller "receptor-affinity differed from naloxone in exhibiting a mixed.agonist-antago- ratio" than that of morphine (Table 2). nist-type of Na+ ratio for morphine receptors and a pure an- iv. Putative K agonists, such as ketocyclazocine and ethyl- tagonist-type of Na+ ratio for enkephalin receptors. ketocyclazocine, bind strongly to both receptors. The differ- ences in their affinity to morphine and enkephalin receptors DISCUSSION are very small. The receptor-affinity ratio is about 2 (Table 2). Three independent assays-with 125I-labeled enkephalin an- Na+ and GTP greatly decrease their potency in competing with alogs (Table 1), [3Hlethylketocyclazocine (Fig. 1), and 3H- the binding of 3H-labeled antagonists to both types of receptors. labeled opiate antagonists (Table 2)-show the differential The Na+ and GTP ratios are similar to those of morphine and affinity of morphine and enkephalins for their respective enkephalins, suggesting that they are potent agonists of similar subtype of opiate receptors. The slight difference in the potency affinity for both receptors of enkephalins exhibited in the binding assays using 1'5I-[D- These data are consistent with results of in vivo studies of Ala2,D-Leu5]enkephalin compared to [3H]diprenorphine most analgesia (8) and of studies on isolated tissues such as the mouse Downloaded by guest on September 28, 2021 4472 Biochemistry: Chang et al. Proc. Natl. Acad. Sci. USA 77 (1980)

Table 4. Summary and classification of opioids according to Putative K agonists do not supress narcotic abstinence signs when agonist and antagonist properties and affinity for the two subtypes given to morphine-dependent animals (6-8). When adminis- of receptors tered chronically, the signs during withdrawal are different Classification* from those seen in morphine withdrawal. They are not self- Enkephalin Morphine Main administered by rhesus monkeys at rates comparable to those Drug receptors receptors responses for codeine (8). The binding data clearly suggest that they are potent 1i and 5 agonists. These putative K agonists have lower Morphine (,u) Ag/L Ag/H Analgesia affinity than morphine to morphine (,g) receptors but higher Enkephalin (5) Ag/H Ag/M Behavioral affinity than morphine to enkephalin (5) receptors. Their unique pharmacological profile may be a consequence of their Ethylketocyclazocine (K) Ag/H Ag/H Analgesia & relative agonist potency toward the two receptor systems. Ketocyclazocine (K) Ag/M Ag/M behavioral Studies on the structure-activity relationships of opiates and enkephalin analogs have provided evidence for the hypothesis N-Allylnormetazocine (ar) Ag/H Mix or Behavioral that analgesia is mediated by morphine (,M) receptors (10). Stein Ant/H and Belluzzi (17) have provided supportive evidence that en- kephalins may serve as neurotransmitters or modulators in Mixed agonist-antagonists: neuronal systems for the mediation of satisfaction and reward. Cyclazocine Mix/M Mix/H Analgesia & Liebeskind and colleagues (18, 19) reported that injection of Oxilorphan Mix/H Mix/H behavioral enkephalin into the lateral ventricle at low doses induces be- Butorphanol Mix/H Mix/H havioral epileptic seizures without analgesia. Morphine injection Pentazocine Mix/L Mix/M in the same fashion but at doses greatly exceeding the analgesic dose can also induce seizures. Such seizures are blocked by prior Nalorphine Ant/L Mix/H Analgesia administration of naloxone at doses higher than those required to antagonize analgesia. Analgesia without electroencephalo- Naloxone Ant/L Ant/H graphic changes is observed after injection of a high dose of Diprenorphine Ant/H Ant/H [Met]enkephalin or a low dose of morphine into the caudal * Ag, Ant, and Mix mean agonist, antagonist, and mixed agonist- midbrain periaqueductal gray matter, and seizures and other antagonist activity, respectively. L, M, and H denote low, medium, electroencephalographic changes without analgesia are seen and high affinity, respectively. following injection into the dorsomedial thalamus. It has been suggested that seizures are mediated by enkephalin (5) receptors vas deferens and guinea pig ileum (15). In these systems, put- in the dorsomedial thalamus and that analgesia is mediated by ative K agonists behave as potent agonists without significant morphine (,g) receptors in the periaqueductal gray matter. The antagonist activity. present studies show that these benzomorphan drugs which are v. The putative or agonist N-allylnormetazocine shows high hallucinatory and dysphoric bind to enkephalin receptors with and similar affinity for both receptors but exhibits different Na+ much higher affinity than morphine. All of these data suggest and GTP ratios for each. It has Na+ and GTP ratios greater than a close relationship between behavioral responses and en- those of enkephalins and morphine for enkephalin receptors. kephalin (5) receptors. However, these ratios are much lower than those of morphine Because putative K and ar agonists can bind to bothg and 6 and enkephalins for morphine receptors, being in the range receptors with similar high affinities, and because no evidence observed for mixed agonist-antagonists. Thus, it appears to exists for distinct receptors for ethylketocyclazocine, we can possess agonist properties for enkephalin receptors and antag- postulate (Table 4) that at low doses the typical ,u agonists in- onist activity for morphine receptors. In animal studies, N- teract with morphine (,g) receptors to elicit analgesia whereas allylnormetazocine did not induce analgesia but caused be- 6 agonists induce behavioral responses through interaction with havioral excitation (8). enkephalin (6) receptors. Typical putative K agonists may vi. Nalorphine has a Na+ ratio of 1 for enkephalin receptors produce both analgesia and behavioral responses by interacting and of 3.3 for morphine receptors. It binds to morphine re- with both opiate receptors. The putative a agonist N-allyl- ceptors with 20 times greater affinity than it does to enkephalin normetazocine may induce behavioral responses through receptors. Nalorphine appears to be a pure antagonist for en- binding to enkephalin receptors.* kephalin receptors and a mixed agonist-antagonist for morphine Vaught and Takemori (20) have shown that [Leu]enkephalin receptors. can potentiate the effects of morphine at doses that do not vii. Naloxone and diprenorphine are pure antagonists for produce analgesia. Attenuation of morphine tolerance by both receptors. Naloxone binds to morphine receptors with an [Metlenkephalin has also been reported recently (21). Thus, the affinity about 20 times greater than that for enkephalin re- possible interactions between enkephalin (5) and morphine (,u) ceptors. Diprenorphine shows high affinity with little prefer- receptors in the central nervous system cannot be neglected. ence for either receptors. The relative occupancy of the two identified receptors by Putative K and a receptors have been proposed (6, 7) to help agonists and antagonists may determine the specific pharma- explain the different pharmacological effects observed with cological profile of a given drug at a particular dose. Thus, the ethylketocyclazocine, ketocyclazocine, and N-allylnormeta- potential for eliciting a large and complex variety of pharma- zocine. However, our studies using [3H]ethylketocyclazocine cological effects exists by such interactions with these two re- do not reveal any suggestive evidence of the existence of specific ceptors (Table 4). Putative K agonists which are not self-ad- K receptors. To the contrary, this drug binds strongly to both ministered by monkeys, and the mixed agonist-antagonists such morphine (,u) and enkephalin (6) receptors. as cyclazocine, butorphanol, oxilorphan, and pentazocine, In normal dogs, opposite effects on sleep, body temperature, and heart and respiratory rates have been reported for putative * However, because studies with labeled N-allylnormetazocine (not ,g and K agonists (16). The putative K agonists have also been available) were not carried out, the unlikely possibility of the exis- shown to cause dysphoric and hallucinatory effects in man. tence of separate a receptors cannot be ruled out categorically. Downloaded by guest on September 28, 2021 Biochemistry: Chang et al. Proc. Natl. Acad. Sci. USA 77 (1980) 4473

which are known to possess a lower potential for inducing (1979) in Mechanism of Pain and Analgesic Compounds, eds. physical dependence and addiction, exhibit only small differ- Beers, R. F., Jr. & Bassett, E. G. (Raven, New York), pp. 429- 445. ences in their affinity for both receptors. This again emphasizes 9. Pert, C. B. & Snyder, S. H. (1973) Science 179, 1011-1014. the importance of the relative potency of a compound on these 10. Kosterlitz, J. A. H. & Waterfield, A. A. (1975) Annu. Rev. Phar- two receptor sites. macol. 15, 29-47. 11. Pert, C. B. & Snyder, S. H. (1974) Mol. Pharmacol. 10, 868- The technical assistance of Mr. Mark Collins is gratefully acknowl- 879. edged. We thank Ms. Lydia J. Hernaez for assistance in growing the 12. Cheng, Y. C. & Prusoff, W. H. (1973) Biochem. Pharmacol. 22, neuroblastoma cells. 3099-3108. 13. Childers, S. R. & Snyder, S. H. (1978) Life Sci. 23, 759-762. 1. Chang, K.-J. & Cuatrecasas, P. (1979) J. Biol. Chem. 254, 14. Blum, A. J. (1978) Proc. NatI. Acad. Sci. USA 75, 1713-1717. 2610-2618. 15. Hutchinson, M., Kosterlitz, H. W., Leslie, F. M., Waterfield, A. 2. Lord, J. A. H., Waterfield, A. A., Hughes, J. S. & Kosterlitz, H. A. & Terenius, L. (1975) Br. J. Pharmacol. 55, 541-546. W. (1977) Nature (London) 267,495-500. 16. Pickworth, W. B. & Sharpe, L. G. (1979) Neuropharmacology 3. Chang, K.-J., Cooper, B. R., Hazum, E. & Cuatrecasas, P. (1979) 18,617-622. Mol. Pharmacol. 16,91-104. 17. Stein, L. & Belluzzi, J. D. (1978) Adv. Biochem. Psychophar- 4. Chang, K.-J., Miller, R. J. & Cuatrecasas, P. (1978) Mol. Phar- macol. 18,299-311. macol. 14, 961-970. 18. Frenk, H., Urca, G. & Liebeskind, J. C. (1978) Brain Res. 147, 5. Childers, S. R., Snowman, A. M. & Snyder, S. H. (1979) Eur. J. 327-337. Pharmacol. 55, 11-18. 19. Frenk, H., McCarty, B. G. & Liebeskind, J. C. (1978) Science 200, 6. Martin, W. R., Eades, C. G., Thompson, J. A., Huppler, R. E. & 335-7. Gilbert, P. E. (1976) J. Pharmacol. Exp. Ther. 197,517-532. 20. Vaught, J. L. & Takemori, A. E., (1979) in J. Pharmacol. Exp. 7. Gilbert, P. E. & Martin, W. K. (1976) J. Pharmacol. Exp. Ther. Ther. 211,280-283. 198, 66-82. 21. Graf, L., Miglecz, E., Bajusy, S. & Szekely, J. I. (1979) Eur. J. 8. Woods, J. H., Smith, C. B., Medzihradsky, F. & Swain, H. H. Pharmacol. 58, 345-3. Downloaded by guest on September 28, 2021