Mediating Actions Ofbenzomorphan Drugs (Putative K and O' Agonists)
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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