Distinct High-Affinity Binding Sites for Benzomorphan Drugs

Proc. Natd Acad. Sci. USA Vol. 78, No. 7, pp. 4309-4313, July 1981 Biochemistry

Distinct high-affinity binding sites for benzomorphan drugs and enkephalin in a neuroblastoma-brain hybrid cell line (multiple opiate receptors/stereospecific binding/ethylketocyclazocine/N-allylnorcyclazocine/neurotumor cell lines) RONALD W. MCLAWHON*, ROBERT E. WEST, JR.J, RICHARD J. MILLERt, AND GLYN DAWSON*t *Joseph P. Kennedy, Jr., Mental Retardation Research Center and Departments of Biochemistry and Pediatrics, and tDepartment of Pharmacological and Physiological Sciences, Pritzker School of Medicine, University of Chicago, Chicago, Illinois 60637 Communicated by Albert Dorfman, April 20, 1981

ABSTRACT The high-affminty binding of benzomorphan drugs mouse neuroblastoma-rat glioma NG108-15 cells (4, 9, 10). In (ethylketocyclazocine and N-allylnorcyclazocine) and [DAIa2,DLeu ] addition, the coexistence of 8 and A receptors in rat brain ho- enkephalin was examined in a mouse neuroblastoma-Chinese mogenates has been recently demonstrated (11) by the use of hamster brain clonal hybrid cell line (NCB-20). Scatchard analysis phenoxybenzamine to inactivate unprotected binding sites for of saturation binding isotherms indicated the presence of a single either enkephalin or morphine irreversibly. Thus, the existence binding site for 3H-labeled [DAla2,DLeu5]enkephalin (ld = 3 nM) ofphysically distinct enkephalin (8) and morphine (,u) receptors and multiple binding sites for [3H]ethylketocyclazocine (Kd = 4 appears to be established. and 20 nM) and N-[3H]allylnorcyclazocine (Kd = 0.5 and 15 nM). In contrast, the concept of specific receptors that respond Both ethylketocyclazocine and N-allylnorcyclazocine competed (K; preferentially to benzomorphan and as = 10 and 30 nM, respectively) with [3H][DAla2,DLeu5]enkephalin drugs (K or subtypes), binding in NCB-20 cells but neither [DAla2,DLeu]enkephalin originally proposed by Martin and colleagues (2, 3) to explain nor morphine could completely inhibit the specific binding of the different physiological effects of ethylketocyclazocine, ke- [3H]ethylketocyclazocine (7 nM) or N-[3H]allylnorcyclazocine tocyclazocine, and N-allynorcyclazocine, has not gained wide- (3 nM). Furthermore, not all benzomorphan drugs (e.g., ethyl- spread acceptance. In fact, there has been little biochemical ketocyclazocine) were totally efficacious in displacing 3 nM N- evidence to date in support of such a concept and it has been [3H]allylnorcyclazocine bindingin the presence or absence ofhigh suggested that 8 and ,u opiate receptors may alone mediate the concentrations of [DAIa2,DLeu lenkephalin. The data presented actions of benzomorphan drugs (6-8, 12). suggest that benzomorphan drugs interact with three distinct high- Such a two-site model (8 and ,u) does not adequately account affinity binding sites: (i) a site that binds enkephalin and morphine for the diverse pharmacological properties of opiates and ben- in addition to ethylketocyclazocine and N-allylnorcyclazocine; (ii) zomorphans. In this paper we present biochemical evidence for a site that binds both ethylketocyclazocine and N-allylnorcycla- the interaction ofbenzomorphan drugs with high-affinity bind- zocine but not enkephalin and morphine; and (iii) a site that binds ing sites that are distinct from receptors for enkephalin and N-allylnorcyclazocine but not enkephalin, morphine, or ethylke- morphine. tocyclazocine. The first of these sites was comparable to the 8 opiate receptor expressed in NG108-15 and N4TG1 cell lines based on the potency series obtained for various opiates and benzo- MATERIALS AND METHODS morphan drugs in competition studies with [HIDAla2,DLeu5]- enkephalin. However, the specific high-affinity benzomorphan [3H]Ethylketocyclazocine (15 Ci/mmol; 1 Ci = 3.7 x 101' binding sites thus far are unique and may represent biochemical becquerels), N-[3H]allylnorcyclazocine (33 Ci/mmol), and correlates of K and a opiate receptors which have been proposed [3H][Dla2,DLeu5]enkephalin (27 Ci/mmol) were purchased to exist on the basis of physiological studies. from New England Nuclear. The followingdrugs were generous gifts: morphine sulfate, etorphine, and naloxone hydrochloride, Physiological studies (1-3) have suggested that opiates and re- from B. Wainer (University ofChicago); N-allylnorcyclazocine, lated narcotic drugs (benzomorphans) may have four distinct ethylketocyclazocine, and the benzomorphan stereoisomers sites of action in the central nervous system-the so-called 8, UM1071-R and UM 1071-S, J. H. Woods (University of Mich- l, K, and o opiate receptors. However, attempts to resolve this igan); cyclazocine and ketocyclazocine, R. S. Zukin (Albert Ein- growing complexity biochemically by differential binding stud- stein College of Medicine); and [DAla2,DLeu5]enkephalin, S. ies have been hindered by the heterogeneous nature of nerve Wilkinson (Burroughs Wellcome). Bremazocine was obtained tissue. Some success has been reported in differentiating sep- from Sandoz and WIN 44,441-3 was from Sterling Winthrop. arate classes ofopiate receptors on the basis ofeffects ofsodium, Neurotumor hybrid cell lines NCB-20 and NG108-15 were GTP, and divalent cations (4-8) but direct evidence ofdiscrete generous gifts from J. Minna (Veterans Administration Hospi- receptor subpopulations has primarily come from studies in tal, Washington, DC) and W. A. Klee (National Institutes of peripheral tissue preparations and clonal neurotumor cell lines Health, Bethesda, MD), respectively. Cells were either grown (9, 10) enriched in a single high-affinity opiate binding site. as monolayer cultures on 100-mm Falcon plastic dishes as de- It is now generally accepted that opiate receptors (A subtype) scribed (13) or adapted to growth on 670-cm2 borosilicate tissue that respond preferentially to morphine may be found in guinea culture roller bottles (Bellco Glass) in modified Eagle's medium pig ileum (1), whereas opiate receptors (8 subtype) that respond supplemented with 10% fetal calf serum and an atmosphere of preferentially to enkephalin are in high concentration in the 90% air/10% CO2. Harvested cells were allowed to swell in 50 mouse vas deferens (1) and in mouse neuroblastoma N4TG1 and mM Tris-HCl (pH 7.4) for 30 min prior to disruption with a Polytron PT-20 homogenizer, and crude membrane fractions The publication costs ofthis article were defrayed in part by page charge were recovered by centrifugation at 50,000 X g for 30 min. payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. t To whom reprint requests should be addressed. 4309 Downloaded by guest on October 1, 2021 4310 Biochemistry: McLawhon et al. Proc. Nad Acad. Sci. USA 78 (1981.) Binding assays were performed via a modification ofthe pro- [3H][DAla2,DLeu5]enkephalin in each cell line. The affinities cedure described by Chang et al. (10). Aliquots ofcrude mem- of these binding sites were similar in the two cell lines (K-d = brane preparations [0.2 ml; 0.1-0.5 mg ofprotein as determined 3 nM), but the total number of sites in membrane preparations by the Lowry (14) procedure] in 50 mM Tris HCl (pH 7.4) were of NCB-20 cells (Bm: = 400 fmol/mg of protein) was 4-fold incubated for 45 min at 250C with various concentrations ofra- higher than that found in NG108-15 cells (Bma = 90 fmol/mg diolabeled ligand and unlabeled drug (final incubation volume of protein). 0.24 ml.) One milliliter ofice-cold 50mM Tris HCl (pH 7.4) was Scatchard Analysis of [3H]EthylketocyclazocineBinding. A added to each sample prior to filtration through Whatman GF/ nonlinear Scatchard plot was obtained for the specific binding B glass microfiber filters undervacuum, the filters were washed of [3H]ethylketocyclazocine in NCB-20 cell homogenates (Fig. twice with 5.0 ml ofice-cold buffer, and the bound radioactivity 2A) and could be resolved into two linear components. There was determined by liquid scintillation counting. Nonspecific were 3-4 times as many apparent low-affinity binding sites (Kd binding was measured in the presence of a large excess (1-100 = 20 nM; Bma = 1000 fmol/mg of protein) as there were ap- AuM) of the respective unlabeled ligand. Data used for calcu- parent high-affinity binding sites (Kd = 4 nM; Bma = 300 fmol/ lating kd, Kj, concentration for 50% inhibition of effect (IC50), mg of protein). In contrast, only a single high-affinity binding and Bma values were means of triplicate determinations with site for [3H]ethylketocyclazocine (Kd = 5 nM; Bma = 70 fmol/ variation between individual determinations (based on 40% mg ofprotein) could be identified on the basis ofScatchard anal- counting efficiency) never exceeding 5%. ysis in NG108-15 hybrid cells (Fig. 2B). Scatchard Analysis of N-[3H]Allylnorcyclazocine Binding. RESULTS The saturable binding ofN-[3H]allylnorcyclazocine in 'NCB-20 cells was also characterized by a nonlinear Scatchard plot (Fig. Scatchard Analysis of [3H][DAla2,DLeu5]Enkephalin-Bind- 3A) and was consistent with the expression ofmultiple binding ing. Saturable, high-affinity binding of [3H][DAla2,DLeu5]en- sites for benzomorphan drugs. Two linear components could be kephalin was observed in membrane preparations of neuro- resolved, an apparent high-affinity binding site (Yd(-- 0.5 nM; tumor hybrid cell lines NCB-20 and NG108-15. Scatchard plots Bmax = 100 fmol/mg of protein) and an apparent low-affinity of saturation binding isotherms were linear (Fig. 1) and sug- binding site (Kd = 15 nM; Bm. = 1000 fmol/mg of protein). gested the presence of a single high-affinity binding site for In NG108-15 cell homogenates (Fig. 3B) N-[3H]allylnorcyclazocine was found to interact with only a single bind-

A .1.0 - A 0.8

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1.5 0.4

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0.2 0.4 0.6 0.8 [3H][DAla2, DLeu5]Enkephalin bound, 0.2 0.4 0.6 (fmol/mg protein) x 10' [3H]Ethylketocyclazocine bound, (fmol/mg protein) x 10-2 FIG. 1. Scatchard analysis of [3H1[DAla2,DLeu'lenkephalin binding in NCB-20 (A) andNG108-15 (B) hybrid cells. Membrane prepa- FIG. 2. Scatchard analysis of [3Hlethylketocyclazocine binding in rations were incubated with [CH1[DAla2,mLeu5]enkephalin in the con- NCB-20 (A) and NG108-15 (B) hybrid cells. Membrane preparations centration range 0.3-20 nM and specific binding was assayed. were incubated with [3H]ethylketocyclazocine in the concentration Nonspecific binding was measured in the presence of 100 AM range 0.5-100 nM and specific binding wasassayed. Nonspecific bind- [DAla ,DLeu5]enkephalin. ing was measured' in the presence of 100 pM ethylketocyclazocine. Downloaded by guest on October 1, 2021 Biochemistry: McLawhon et al. Proc. Natl. Acad. Sci. USA 78 (1981) 4311

Table 1. Potencies of enkephalin, opiates, and benzomorphans for inhibiting [3H][DAla',DLeu5]enkephalin binding in NG108-15 0.4 - and NCB-20 hybrid cells Ki, nM Competitor NG108-15 NCB-20 [DAla2,DLeu5]Enkephalin 1 1

0.2 Etorphine 4 0.2 Morphine 90 100 Naloxone 200 100 Cyclazocine 9 20 x N-Allylnorcyclazocine 40 30 Ketocyclazocine 40 50 Ethylketocyclazocine 10 10 4 8 12 Bremazocine 2 1 0 WIN 44,441-3 0.5 5 0.3 Apparent dissociation constants (Ki) were calculated according to -B the equation of Cheng and Prusoff (15), Ki = IC50/[1 + ([L]/Kd)] in which [LI is the concentration of radiolabeled ligand, Kd is the dissociation constant (3 nM) for the radiolabeled ligand, and IC50 values are the concentrations of competingdrugs required to inhibitthe displace- 0.2- able binding of radiolabeled ligand by 50%. The binding of 4 nM [3H][DAla2,DLeu5]enkephalin in membrane preparations was assayed in the presence or absence of increasing concentrations of competing drugs. Nonspecific binding was measured in the presence of 100 ,uM [DAla2,DLeu 0.1 ]enkephalin.

Inhibition of [3H]Ethylketocyclazocine and N-[3H]AI- lylnorcyclazocine Binding. The competitive effects of enkephalin, opiates, and benzomorphans against 7 nM [3H]ethylk- 0.2 0.4 0.6 0.8 etocyclazocine and 3 nM N-[3H]allylnorcyclazocine were studied N-[3H]Allylnorcyclazocine bound, in NCB-20 hybrid cells. At these concentrations, radiolabeled (fmol/mg protein) x 10-2 benzomorphan derivatives were directed equally toward apparent high- and low-affinity binding sites and it was therefore FIG. 3. Scatchard analysis ofN-[3H]allylnorcyclazocine binding in possible to investigate drug interactions at multiple sites si- NCB-20 (A) and NG108-15 (B) hybrid cells. Membrane preparations multaneously. The drug potency series against [3H]ethylketo- were incubated with N-[3Hlallylnorcyclazocine in the concentration cyclazocine and N-[3H]allylnorcylazocine was range 0.2-100 nM and specific binding was assayed. Nonspecific bind- binding (Table 2) ing was measured in the presence of 100 AM N-allylnorcyclazocine. markedly different from that seen with 4 nM [3H][DAla2, DLeu5]

Table 2. Potencies of enkephalin, and ing site having an affinity = 20 nM; = 90 of opiates, benzomorphans (Kd Bmi fmol/mg for protein) comparable to that of the apparent low-affinity site in inhibiting [3H]ethylketocyclazocine and N-[3Hlallylnor- NCB-20 cells. cyclazocine binding in NCB-20 hybrid cells Inhibition of[3H][DAla2,DLeu5]Enkephalin Binding. A wide IC50, nM range of opiate and benzomorphan drugs competed for high- [3H]Ethylketo- N-[3H]Allylnorcycl- affinity [3H][DAla2,DLeu5]enkephalin binding sites in NG108- Competitor cyclazocine azocine 15 and NCB-20 hybrid cells (Table 1). The relative potencies [DAla2,DLeU5]Enkephalin >105 of these drugs in displacing 4 nM (0.3) >105 (3) [3H][DAla2,DLeu5]enke- Etorphine >105 (0.3) >105 (0.4) phalin were essentially the same in the two cell lines Morphine >105 (40) >105 (80) ([DAla2,DLeu5]enkephalin = etorphine = bremazocine WIN Naloxone 105 105 44,441-3 > ethylketocyclazocine > cyclazocine - N-allylnor- Cyclazocine 100 100 cyclazocine > ketocyclazocine > morphine = naloxone), im- N-Allylnorcyclazocine 200 20 plying that the [3H][DAla2,DLeu5]enkephalin binding site in Ketocyclazocine 40 30 NCB-20 was identical to the previously characterized (4) op- Ethylketocyclazocine 20 25* iate receptor in NG108-15. In general, benzomorphans were Bremazocine 25 200 better competitors than morphine (a ,u agonist) but somewhat WIN 44,441-3 25 x 103 105 less potent than [DAla2,DLeu5]enkephalin (a 8 agonist)-how- The binding of 7 nM [3H]ethylketocyclazocine and 3 nM N-[3H]al- ever, both the putative K agonist bremazocine and the putative lylnorcyclazocine in membrane preparations was assayed in the pres- K antagonist WIN 44,441-3 had high affinities for [3H][DAla2, ence or absence of increasing concentrations of competing drugs. Non- DLeu5]enkephalin binding sites. The apparent KY obtained for specific binding was measured in the presence of 100 AM ethylketocy- ethylketocyclazocine and N-allynorcyclazocine (Table 1) were clazocine and 100 AMN-[3H]allylnorcyclazocine. Values inparenthesis in close agreement with Kd determined by Scatchard analysis represent IC15 values because radiolabeled ligand maximally dis- of saturation binding isotherms (Figs. 2 and 3), indicating that placed by competing drug was less than 30% of the radiolabeled ligand both specifically bound. [3H]ethylketocyclazocine and N-[3H]allylnorcyclazocine * This is an IC," value because radiolabeled ligand maximally dis- were binding to the same high-affinity site (8 receptor) placed by competing drug was less than 75% of the radiolabeled li- in NG108-15 cells as [3H][DAla2,DLeu5]enkephalin. gand specifically bound. Downloaded by guest on October 1, 2021 4312 Biochemistry: McLawhon et al. Proc. Natl. Acad. Sci. USA 78 (1981) enkephalin (Table 1), suggesting the presence of binding sites distinct from 8 receptors. Of particular interest was the observation that at least 4000-fold higher concentrations of WIN 100 44,441-3 were required to inhibit the specific binding of .,- [3H]benzomorphans than the binding of[3H][DAla2,DLeu ]en- 0 kephalin. Furthermore, putative K agonists. (ethylketocyclazo- NQ cine, ketocyclazocine, and bremazocine) were more potent than c.) putative er agonists (N-allylnorcyclazocine and cyclazocine) in displacing [3H]ethylketocyclazocine binding in these cells. In c) contrast, bremazocine and cyclazocine were only weak com- 0 petitors of 3 nM N-[3H]allylnorcyclazocine whereas N-allylnor- a cyclazocine, ethylketocyclazocine, and ketocyclazocine were strong competitors. Only high concentrations of the. opiate antagonist naloxone could block the binding of either [3H]ethylk- 10 50 etocyclazocine and N-[3H]allylnorcyclazocine. Differences in the efficacies ofcertain drugs (Table 2) in displacing [3H]ethylketocyclazocine and N-[3H]allylnorcyclazocine binding provided additional evidence for the interaction of benzomorphan drugs with binding sites distinct from 8 re- 0bo ceptors in NCB-20 cells. The opiate agonists [DAla2,DLeul]enkephalin, etorphine, and morphine failed to displace more than *30% of either [3H]benzomorphan derivative in competition studies (Table 2). Good correlation of IC15 values obtained for these drugs with apparent KY in studies with [3H][DAla2, DLeu5]enkephalin (Table 1) suggested that the displaceable components of [3H]ethylketocyclazocine and N-[3H]al- -=c -10 -9 -8 -7 -6 -5 lylnorcyclazocine binding corresponded to interactions with log[competitorl enkephalin (8) receptors, whereas the nondisplaceable components represented binding to other sites. Although N-allyl- FIG. 4. Inhibition of the specific binding of N-[3H]allylnorcycla- norcyclazocine completely-inhibited the specific binding of zocine in NCB-20 hybrid. cells by the benzomorphan stereoisomers [3H]ethylketocyclazocine in NCB-20 cells (Table 2), ethylke- UM 1071-R (o) and UM 1071-S (o). The binding of 3 nM N- [3H]allylnorcyclazocine in membrane preparations was assayed in the tocyclazocine could only displace 75% of 3 nM N- presence or absence of increasing concentrations of competing drug. [3H]allylnorcyclazocine either in the presence or absence of 100 Nonspecific binding was measured in the presence of 100 uM N- AuM [DAla2,DLeu5]enkephalin. -This finding further implied allylnorcyclazocine. that one of the binding sites not shared by enkephalin, etorphine, or morphine also had little affinity for ethylketocyclazocine. ies in guinea pig ileum preparations (1, 19) have provided sup- The binding-ofibenzomorphan derivatives in NCB-20 cells port for the concept that benzomorphans, such as ethylketo- was stereospecific (Fig. 4) in that a 1000-fold difference in po- cyclazocine and bremazocine, may exert their pharmacological tency was observed for the stereoisomers UM 1071-R (IC50 = actions at sites other than those previously characterized as 10 nM) and UM1071-S (IC50 = 10,000 nM) in competition bind- morphine (,u) and enkephalin (8) receptors. *ing studies with 3 nM N-[3H]allylnorcyclazocine. This agrees In this investigation we have demonstrated that benzomor- with previous reports (16).that only the R enantiomer is active phan drugs interact stereospecifically with multiple binding in vivo. sites in a somatic cell hybrid NCB-20. One of the high-affinity binding sites appears to be identical to the enkephalin (8) re- DISCUSSION ceptor found in neurotumor cell.lines NG108-15 and N4TG1 Benzomorphans are an enigmatic group of opioid drugs which (4, 9, 10), rat brain homogenates (1, 4, 11), and mouse vas def- produce a morphine-like depression ofthe nociceptive response erens (1). The other high-affinity sites expressed in NCB-20 yet fail to suppress withdrawal symptoms in morphine-depen- cells, which bind benzomorphan drugs preferentially, have not dent animals (2, 3, 16-19). Detailed studies of the differential been previously described. Both ethylketocyclazocine (a pu- response to opiates and benzomorphans in dogs led Martin and tative K agonist) and N-allylnorcyclazocine (a putative oagonist) coworkers (2, 3) to propose the existence of three discrete sub- bind to a site which is not occupied by enkephalin or the classical populations of opiate receptors-p, K, and o. The prototypical opiate agonists morphine and etorphine. Furthermore, N-al- A agonist morphine both suppressed abstinence and produced lylnorcyclazocine binds to a separate site having little affinity analgesia in morphine-dependent dogs, whereas ethylketocy- for enkephalin, morphine,.etorphine, or ethylketocyclazocine. clazocine and ketocyclazocine neither suppressed. nor precipi- Based on differences in the selectivity of the two specific ben- tated abstinence in morphine-dependent animals but did pro- zomorphan binding sites for ethylketocyclazocine, it may not duce naloxone-reversible analgesia (2, 3). It was therefore be unreasonable to suggest that these sites are equivalent to the concluded that ethylketocyclazocine has a site of action, des- K and o opiate receptor subtypes defined by Martin et al. (2). ignated as the K receptor, distinct from morphine (Au) receptors. Additional support for~the contention that specific receptors Another type of naloxone-reversible effect-behavioral excita- for benzomorphan drugs do exist comes from the observation tion without inducing analgesia or depressing abstinence in that [3H]ethylketocyclazocine binds to what has been tenta- morphine- and ketocyclazocine-dependent dogs-was ob- tively called a K site in guinea pig brain homogenates (20). The served (2) with. the benzomorphan derivative N-allylnorcycla- binding of [3H]ethylketocyclazocine was saturable, biphasic, zocine (SKF 10,047). To explain. this, the site mediating this and readily displaceable by etoThine. However, low concen- response was designated thear receptor. Behavioral studies in trations of morphine and [DAla ,DLeu'lenkephalin only dis- monkeys, rats, and mice (16-19) and electrophysiological stud- placed 35% of [3H]ethylketocyclazocine bound, implying that Downloaded by guest on October 1, 2021 Biochemistry: McLawhon et al. Proc. Nati Acad. Sci. USA 78 (1981) 4313 this ligand was interacting mainly with K sites (i.e., the 65% of We thank Mr. Daniel Cermak for his excellent technical assistance. total binding not displaced) and to a lesser extent with ,a and This work was supported in part by U.S. Public Health Service Grants 8 sites. Except that we do not find any evidence that etor- HD-06426, DA-02575, HD-09402, HD-04583, DA-02121, and GM- phine can displace [3H]ethylketocyclazocine or N-[3H]al- 07151. G. D. is a Joseph P. Kennedy, Jr., Scholar and the recipient of lylnorcyclazocine from specific benzomorphan binding sites in U.S. Public Health Service Career Development Award NS-00029. NCB-20 cells, our data are consistent with these findings (20). Our observations clearly indicate the presence of discrete 1. Lord, J. A. H., Waterfield, A. A., Hughes, J. & Kosterlitz, H. benzomorphan receptor subpopulations in nerve tissue. How- W. (1977) Nature (London) 267, 495-499. ever, a number ofrecent reports (6-8, have failed to 2. Martin, W. R., Eades, C. G., Thompson, J. A., Huppler, R. E. 12) provide & Gilbert, P. E. (1976)J. Pharmacol Exp. Ther. 197, 517-532. any corroborative evidence for the existence of K and a opiate 3. Gilbert, P. E. & Martin, W. R. (1976) J. Pharmacol Exp. Ther. receptors. Chang et al. (6) studied binding of "WI-labeled 198, 66-82. [DAla2,DLeu5]enkephalin and [3H]ethylketocyclazocine in rat 4. Chang, K.-. & Cuatrecasas, P. (1979)J. Biol Chem. 254, 2610- brain homogenates and found no evidence for the interaction 2618. of benzomorphan drugs with sites other than those previously 5. Pert, C. B. & Taylor, D. (1979) in Endogenous and Exogenous characterized as and Opiate Agonists and Antagonists, ed. Way, E. L. (Pergamon, enkephalin (8) morphine (,4) receptors. New York), pp. 87-90. Therefore, they suggested that the analgesic effects ofputative 6. Chang, K.-J., Hazum, E. & Cuatrecasas (1980) Proc. Natl Acad. K agonists (e.g., ethylketocyclazocine) may be mediated through Sci. USA 77, 4469-4473. ,u receptors and that the behavioral effects ofputative a agonists 7. Pasternak, G. (1980) Proc. Natl Acad. Sci. USA 77, 3691-3694. (e.g., N-allylnorcyclazocine) may be mediated through 8 recep- 8. Snyder, S. H. & Goodman, R. R. (1980)J. Neurochem. 35, 5-15. tors. In independent studies, Pasternak (7) and Hiller and Si- 9. Klee, W. A. & Nirenberg, M. (1974) Proc. Nati Acad. Sci. USA mon (12) both concluded that 71, 3474-3477. high-affinity [3H]ethylketocycla- 10. Chang, K.-J., Miller, R. J. & Cuatrecasas, P. (1978) Mol Phar- zocine binding sites in ratbrain homogenates resembled mor- macol 14, 961-970. phine receptors. Pasternak found that sodium ions, divalent 11. Robson, L. E. & Kosterlitz, H. W. (1979) Proc. R. Soc. London cations, and GTP affect [3H]ethylketocyclazocine binding in a 205, 425-432. manner analogous to that seen with [3Hlmorphine; Hiller and 12. Hiller, J. M. & Simon, E. J. (1980)J. Pharmacol Exp. Ther. 214, Simon noted similarities in the regional distributionof [3H]- 516-519. and [3H]naloxone and 13. Dawson, G., Matalon, R. & Dorfman, A. (1972) J. Biol Chem. ethylketocyclazocine binding. Snyder 247, 5944-5950. Goodman (8) also failed to identify unique receptors for ben- 14. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. zomorphan drugs; they proposed that high-affinity binding of (1951)1. Biol. Chem. 193, 265-275. [3H]ethylketocyclazocine and N-[3H]allylnorcyclazocine corre- 15. Cheng, Y. C. & Prusoff, W. H. (1973) Biochem. Pharmacol. 22, sponded to interactions with ,A sites and that low-affinity bind- 3099-3108. ing occurred at 8 sites. 16. Hein, D. W., Young, A. M., Herling, S. & Woods, J. H. (1981) We would agree that benzomorphan drugs may exert some J. Pharmacol Exp. Ther., in press. 17. Swain, H. H. & Seevers, M. H. (1976) Addendum to the Pro- oftheir effects through enkephalin (8) and morphine (,u) recep- ceedings ofthe 38th Annual Scientific Meeting ofthe Committee tors, but a two-site model (8 and ,) cannot explain all of the on Problems of Drug Dependence, Vol. 2, pp. 768-787. pharmacological properties associated with this unique group 18. Teal, J. J. & Holtzman, S. G. (1980) Eur.J. Pharmacol 68, 1-10. of narcotics. Specifically, a two-site model cannot explain why 19. Romer, D., Buscher, D., Hill, R. C., Maurer, R., Petcher, T. J., benzomorphans fail to suppress or precipitate abstinence in Welle, H. B. A., Bakel, H. C. C. K. & Akkerman, A. M. (1980) Life Sci. 27, 971-978. morphine-dependent animals (2, 3, 16-19). Benzomorphan 20. Kosterlitz, H. W. & Paterson, S. J. (1980) Proc. R. Soc. London drugs must have sites of action that are distinct from those of 210, 113-122. enkephalin and morphine. We have presented evidence for their existence in a neurotumor hybrid cell line. Downloaded by guest on October 1, 2021