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 op- iate 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]al- lylnorcyclazocine was found to interact with only a single bind- A .1.0 - A 0.8 0.5 0.4 0 0 x x aL) a1) 1 2 3 a)! a m08 5 10 15 Po0 0 1.5 0.4 1.0 0.2 0.5 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 bind- ing in NCB-20 (A) andNG108-15 (B) hybrid cells.