ALKYLATION OF [H-3] 8-OH-DPAT BINDING-SITES IN RAT CEREBRAL-CORTEX AND HIPPOCAMPUS

The binding of tritiated 8-hydroxy-2-(di-n-propyl-amino)tetralin, or [ H-3]8-OH-DPAT, to membranes from rat cerebral cortex and hippocampus c ould be inhibited by serotonin (5-HT) and buspirone, and by the 5-HT a ntagonists propranolol, NAN-190, pindolol, pindobind-5-HT1A, WAY100135 , spiperone and ritanserin. All competition curves, except for ritanse rin, best fitted a two-site model. In vitro treatment of the membranes with N-ethylmaleimide (NEM), to alkylate sulfhydryl groups, caused do se-dependent decreases of binding; the inhibition curves were biphasic , and the effects irreversible. Reduction of disulfide bonds with L-di thiothreitol (L-DTT) also decreased binding, but in a monophasic way; these effects were fully reversible in cortex, but only partially reve rsible in hippocampus. In the latter region, but not in cerebral corte x, previous occupancy by [H-3]8-OH-DPAT partially protected binding fr om the effects of both L-DTT and NEM, suggesting that the thiol groups in the receptor recognition site(s) of this brain region are readily accessible. The binding characteristics were examined with the aid of saturation curves, carried out with increasing concentrations, up to 1 40 nM, of [H-3]8-OH-DPAT. The saturation data were suggestive of a two -site receptor model incorporating a high-affinity site (KH of 0.3-0.5 nM) corresponding to the 5-HT1A receptor, and a low-affinity site (KL of ca 25 nM). After in vivo alkylations, carried out by treating rats with N-ethoxycarbonyl-2-ethoxy-1,2-dihydro-quinoline (EEDQ), the satu ration curves from both control and EEDQ-treated rats were again best fitted to a two-site model. For EEDQ-treated animals, a drastic decrea se of 5-HT1A receptor binding activity was noted; this loss was greate r in hippocampus than in cerebral cortex. Since the decrease in 5-HT1A receptors was not associated with changes in low-affinity binding, th e results suggest independent regulations of the two [H-3]8-OH-DPAT bi nding proteins. Altogether, the present data further supports the noti on that [H-3]8-OH-DPAT, besides labelling 5-HT1A receptors, also binds to other structures in rat cerebral cortex and hippocampus.