THE BINDING OF K- AND a-OPIATES IN RAT BRAIN1

Detailed displacements of [3H]dihydromorphine by ketocyclazocine and SKF 10,047, [3H]ethylketocyclazocine by SKF 10,047, and C3H]SKF 10,047 by ketocyclazocine are all multiphasic, suggesting multiple binding sites. After treating brain tissue in vitro with naloxazone, all displacements lose the initial inhibition of 3H-ligand binding by low concentrations of unlabeled drugs. Together with Scatchard analysis of saturation experiments, these studies suggest a common site which binds EL-, K-, and u-opiates and enkephalins equally well and with highest affinity (Ko < 1 nM). The ability of unlabeled drugs to displace the low affinity binding of [3H]dihydromorphine (& = 3 IIM), [3H] ethylketocyclazocine (& = 4 nM), [3H]SKF 10,047 (& = 6 nM), and n-Ala2-n-Leu5-[3H]enkephalin (Ko = 5 nM) remaining after treating tissue with naloxazone demonstrates unique pharmacological profiles for each. These results suggest the existence of distinct binding sites for Kand u-opiates which differ from those sites which selectively bind morphine &cl) and enkephalin (6). Following the classification in viva of various groups of opiates according to their pharmacological effects (Martin et al., 1976), several attempts have been made to identify distinct receptors for the K and the u class of opiates, named after the prototypic drugs ketocyclazotine and SKF 10,047 (N-allylnormetazocine), respectively. Although several groups initially were unable to demonstrate these K and u sites (Hiller and Simon, 1979, 1980; Snyder and Goodman, 1980), a number of laboratories now have presented evidence strongly suggesting their presence, particularly of K sites (Kosterlitz and Patterson, 1980; McLawhon et al., 1981; Chang et al., 1980, 1981; Pfeiffer and Hertz, 1981; Wood et al., 1980). Earlier studies from our laboratory implied more than one [3H]ethylketocyclazocine site (Pasternak, 1980). Intensive investigations of high affinity [3H]ethylketocyclazocine binding suggested that it was the same as the high affinity [3H]dihydromorphine binding site and that this common high affinity site, the ~1 site (Wolozin and Pasternak, 1981; Zhang et al., 1981), mediated the analgesic effects of ketocyclazocine (Pasternak, 1980) in ad‘We wish to thank Drs. David Ahern and Jerome B. Posner for their assistance in these studies and Dr. Robert Willette for the generous gift of SKF 10,047. This work was supported in part by Grant PDT 169 from the American Cancer Society and Grant DA02615 from the National Institute of Drug Abuse. ‘Recipient of National Institute of Neurological and Communicative Disorders and Stroke Teacher Investigator Award 1 K07NS415. To whom correspondence should be addressed at Department of Neurology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. dition to those of morphine (Pasternak et al., 1980a, b), the enkephalins, and P-endorphin (Zhang and Pasternak, 1981; Pasternak, 1981). These studies focused on high affinity [3H]ethylketocyclazocine binding and did not address in great detail its lower affinity binding. Since earlier studies demonstrated that selective binding of morphine to p sites and enkephalins to S sites reflects differences in their lower affinity binding sites (Zhang and Pasternak, 1981; Zhang et al., 1981; Wolozin and Pasternak, 1981), we investigated the binding interactions of 3H-labeled ~1, K, u, and 6 ligands and their displacement by a number of unlabeled drugs in an effort to examine the possibility of Kand u-selective sites. Materials and Methods [3H]Dihydromorphine, [3H]ethylketocyclazocine, (+)[3H]SKF 10,047, D-Ala2-D-Leu5-[3H]enkephalin, and Formula 963 scintillation fluor were obtained from New England Nuclear Corp. (Boston, MA). D-Ala’-D-Leu5-Enkephalin was obtained from Peninsula Laboratories (San Carlos, CA), and (+)-SKF 10,047 was obtained from the National Institute of Drug Abuse. Male Sprague-Dawley rats were purchased from Charles River Breeding Laboratories (Wilmington, MA). Naloxazone was synthesized as previously reported (Pasternak and Hahn, 1980). Membranes were prepared and binding experiments were performed as previously described (Pasternak et al., 1975). In brief, washed membrane preparations were incubated with the 3H-ligand for 60 min at 25°C and samples (1 to 2 ml with 20 mg of tissue/ml) were filtered over Whatman GF/B filters and counted. Assays were