PharmacologyBiochemistry&Behavior, Vol. 25, pp. 51-57, 1986.©AnkhoInternational Inc. Printed in the U.S.A. 0091-3057/86$3.00 + .00 Acylating Phencyclidines Irreversibly Enhance Brain Calcium Antagonist Binding GORDON T. BOLGER, .1 MICHAEL F. RAFFERTY,t BEN AVI WEISSMAN,* KENNER C. RICEr AND PHIL SKOLNICK* *Laboratory of Bioorganic Chemistry, and tLaboratory of Chemistry National Institutes of Health, Bethesda, MD 20892 Received 31 December 1985 BOLGER, G. T., M. F. RAFFERTY, B. A. WEISSMAN, K. C. RICE AND P. SKOLNICK. Acylating phencyclidines irreversibly enhance brain calcium antagonist binding. PHARMACOL BIOCHEM BEHAV 25(1) 51-57, 1986.-- Phencyclidine was previously shown to allosterically increase the apparent affinity of the dihydropyridine ([3H]nitrendipine) calcium antagonist binding site in a lysed synaptosomal membrane preparation of rat forebrain. Treat- ment of a similar preparation of mouse forebrain with 4-isothiocyanato- l-(1-phenylcyclohexyl) piperidine (FOURPHIT), an acylating phencyclidine derivative, resulted in a concentration dependent (0.1-10/xM), irreversible, increase in the appar- ent affinity of [3H]nitrendipine in contrast to the effects of phencyclidine which were reversible. The FOURPHIT isomer, 1-[l-(3-isothiocyanatophenyl) cyclohexyl] piperidine (METAPHIT), (10/~M) also irreversibly increased the apparent affin- ity of [SH]nitrendipine, but was much less efficacious than FOURPHIT. Phencyclidine blocked the irreversible increase in the apparent affinity of [SH]nitrendipine produced by FOURPHIT. The interactions of multivalent cations and the calcium antagonist diltiazem with the [aH]nitrendipine binding site were altered following treatment of membranes with FOUR- PHIT. These studies suggest that FOURPHIT irreversibly interacts with the same sites as PCP, and thus may be a useful tool with which to further probe both the behavioral and biochemical interactions between phencyclidine and the di- hydropyridine calcium antagonist binding site. Nitrendipine Dihydropyridine Calcium antagonists Calcium channels Acylating phencyclidines DIHYDROPYRIDINE calcium antagonists (DHPCA) are potent inhibitors of calcium currents in peripheral tissues [14, 31, 32]. High affinity binding sites for DHPCA have been characterized in both peripheral tissues [1, 2, 15, 28, 30, 33] and brain [19, 25, 26, 28]. In contrast to peripheral tissues (where the DHPCA binding site functions as a calcium chan- nel regulator [34]), the role of these sites in the central nerv- ous system (CNS) is still unclear. Nonetheless, recent biochemical [16, 22, 35] and behavioral [3, 6, 12, 21, 28] evidence clearly suggests that DHPCA binding sites in brain may play an important neuromodulatory role in the CNS. We recently reported [4,5] that the psychotomimetic phencyclidine (PCP) [9,17] and several related derivatives increased the apparent affinity of [SH]nitrendipine in rat and mouse brain [4,5]. The enhancement of [SH]nitrendipi~t' binding by PCP was via an aUosteric mechanism and dis- played structural dependence, brain region specificity, a possible modulation by endogenous tissue factors, and was potently inhibited by Ca ~+. Neither the local anesthetic properties of PCP [4,8] nor an interaction of PCP with its high affinity binding site in brain [4,27] could account for this effect on [aH]nitrendipine binding. These findings suggest that the DHPCA binding site in the CNS might represent a pharmacologically relevant locus of action for PCP [4,5]. We now report that the acylating PCP derivatives 4-isothiocyanato-l-(1-phenylcyclohexyl) piperidine (FOUR- PHIT) and 1-[ 1-(3-isothiocyanatophenyl) cyclohexyl) piperidine (METAPHIT) (Fig. 1) (the isomer characterized as an irreversible inhibitor of the [aH]PCP binding site in rat brain [27]), can irreversibly increase the apparent alTmity of [3H]nitrendipine for DHPCA binding sites in a lysed synap- tosomal membrane preparation of mouse forebrain; FOUR- PHIT being more efficacious than METAPHIT. The phar- macologic prof'de of FOURPHIT's actions suggests that it is interacting with the same site(s) as PCP to affect [3H]nitrendipine binding. Furthermore, through the use of FOURPHIT, it was determined that PCP interacts with two sites to alter [aH]nitrendipine binding. METHOD Tissue Preparation Male NIH mice (ICR, Veterinary Resources Branch, Na- tional Institutes of Health, Bethesda, MD: 18-22 g) were ~Requests for reprints should be addressed to Dr. G. T. Bolger, Laboratory of Bioorganic Chemistry, Bldg. 4, Room 212, Bethesda, MD 20892. 51