CHARACTERIZATION OF SPECIFIC BINDING-SITES FOR [H-3] (D)-N-ALLYLNORMETAZOCINE IN RAT-BRAIN MEMBRANES

Abstract

Binding of [3H](d)-N-allylnormetazocine ([3H](d)-NANM) to rat brain membranes is stereospecific, reversible and saturable (Bmax [maximum bound concentration] = 260 fmol/mg of protein) and manifests moderately high affinity (Kd = 20 nM). The rank order of potency among opioidbenzomorphans and phencyclidine (PCP) [a drug of abuse] analogs for competition for [3H](d)-NANM-binding sites is as follows: (d)-NANM = PCP-3-OH > (d)-cyclazocine > N-ethylphenylcyclohexylamine > PCP > (l)-cyclazocine = dextrorphan > (d/l)-ethylketocyclazocine > (d/l)-bremazocine > (1)-NANM > 1-phenylcyclohexylamine > levorphanol. Other opioid ligands, relatively selective for each of the types of opioid binding sites other than .delta., such as morphine (.mu.), H-Tyr-D-Ala(Me)Phe-NH-CH2-OH (.mu.), D-Ala2-D-Leu5-enkephalin (.delta.), tifluadom (.kappa.), and U 50488 [trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzene acetamide] (.kappa.) as well as etorphine and naloxone were all unable to compete with [3H](d)-NANM for specific binding even at a concentration of 1 .mu.M. Regional distribution studies of [3H](d)-NANM-binding sites show high density in the hippocampus, thalamus, hypothalamus and amygdala and low density in cerebellum and nonfrontal neocortex membranes of the rat brain. These binding sites are very sensitive to protein-modifying enzymes and reagents such as trypsin and N-ethylmaleimide and to heat denaturation. These results provide direct biochemcial evidence for the existence of distinct (d)-NANM-binding sites in rat brain. Apparently PCP and several of its analogs and the dextrorotatory isomers of psychotomimetic benzomorphans may act at a common recognition site in rat CNS.