Molecular Pharmacology, Vol 20, 246-254, Copyright © 1981 by the American Society for Pharmacology and Experimental Therapeutics

Demonstration of [³H]Cyclazocine Binding to Mutiple Opiate Receptor Sites

¹ Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, and Department of Psychiatry, Mount Sinai School of Medicine, New York, New York 10029

Interaction of the psychotomimetic opiate cyclazocine with multiple receptor sites has been demonstrated biochemically. Cyclazocine has been postulated to interact with µ, , and opiate receptors (Martin et al., J. Pharmacol. Exp. Ther. 197:517-532 (1976).) In an effort to understand the molecular mechanisms involved in the neuropharmacological actions of this and closely related opiates we have studied the binding of [³H]cyclazocine to rat brain homogenates. Specific binding, defined as total binding minus binding in the presence of 10 µM nonradioactive cyclazocine, constitutes approximately 92% of total binding at 1.0 nM ³H-labeled ligand and 67% of total binding at 100 nM ³H-labeled ligand. Scatchard analyses utilizing various competing drugs reveal the apparent interaction of this drug with three distinct binding sites characterized by affinities of 0.2 nM, 11 nM, and 70 nM (50 nM Tris-HCl buffer, pH 7.4 at 4°). In contrast, many radiolabeled classical opiates and opioid peptides have been reported to exhibit biphasic binding but do not exhibit binding to a site of such low affinity. The high- and low-affinity [³H]cyclazocine sites exhibit differential sensitivities to sodium and also to the selective sulfhydryl reagent N-ethylmaleimide. In addition, all three sites exhibit greater than 50% loss of specific binding following incubation with trypsin (5 µg/ml) for 15 min at room temperature, and greater than 80% loss of specific binding following incubation at 60° for 15 min in the absence of added reagents. Together, these findings indicate that all three sites have a protein-like component. Competition analyses involving rank order determinations for a series of opiates and other drugs indicate that the cyclazocine binding sites represent, in order of decreasing affinity, the classical opiate receptor (the putative "µ" receptor), a second as yet uncharacterized opiate binding site, and the specific [³H]phencylidine binding site. Specific [³H]phencyclidine binding can be displaced by cyclazocine (IC₅₀ = 350 nM) and by related benzomorphans, but not by classical opiates such as morphine or naloxone. We thus propose a common binding site in rat nervous tissue for phencyclidine and some of the benzomorphan opiates.

Note:
ACKNOWLEDGMENTS We thank Hoffmann-La Roche, Bristol Laboratories, and the National Institute on Drug Abuse for their respective gifts of levorphanol, ketamine, and phencyclidine analogues; Dr. Eric Simon for nalorphine and pentazocine; Dr. Arthur Jacobson for cyclazocine and phenazocine; Dr. Agu Pert for metazocine; Dr. Alan Gintzler for ethylketocyclazocine and ketocyclazocine; and Sterling-Winthrop, Inc., for cyclorphan, etazocine, and WIN derivatives. We also thank Ms. Gail D. Federoff and Ms. Melissa Fitz for excellent technical assistance, and Dr. Gavril Pasternak for technical advice and the use of his computer program for nonlinear regression analysis.

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