Identification of polypeptides of the phencyclidine receptor of rat hippocampus by photoaffinity labeling with [3H]azidophencyclidine

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Abstract
Polypeptide components of the phencyclidine (PCP) receptor present in rat hippocampus were identified with the photolabile derivative of phencyclidine [3H]azidophencyclidine ([3H]AZ-PCP). The labeled affinity probe was shown to reversibly bind to specific sites in the dark. The number of receptor sites bound is equal to those labeled by [3H]PCP, and their pharmacology and stereospecificity are identical with those of the PCP/.sigma.-opiate receptors. The dissociation constant of [3H]AZ-PCP from these receptor is 0.25 .+-. 0.08 .mu.M. Photolysis of hippocampus membranes preequilibrated with [3H]AZ-PCP, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed the existence of five major labeled which membranes were incubated with [3H]AZ-PCP in the presence of various PCP analogues and opiates, indicate that labeling of both the Mr 90 000 band and the Mr 33 000 band is sensitive to relatively low concentrations (10 .mu.M) of potent PCP/.sigma. receptor ligands, while similar concentrations of levoxadrol, naloxone, morphine, D-Ala-D-Leu-enkephalin, atropine, propranolol, and serotonin were all ineffective. Stereoselective inhibition of labeling of the Mr 90 000 band and of the Mr 33 000 band was also observed by the use of dexoxadrol and levoxadrol. The Mr 33 000 band was not as sensitive as the Mr 90 000 band to inhibition by the selective PCP receptor ligands N-[1-(2-thienyl)cyclohexyl]piperidine and PCP. Strong inhibition of labeling of the Mr 33 000 bands by less selective PCP receptor ligands such as N-[1-(3-hydroxyphenyl)cyclohexyl]piperidine and (.+-.)-N-allylnormetazocine was also observed. The labeling of the other three polypeptides (Mr 62000, 49 000, and 40 000) was only mildly affected by dexoxadrol and (.+-.)-N-allylnormetazocine, suggesting that this interaction does not have a classical PCP/.sigma. receptor pharmacology. Thus, these labeled bands could be constituents of a second PCP receptor.