Alkylation of [3H]8-OH-DPAT binding sites in rat cerebral cortex and hippocampus

The binding of tritiated 8-hydroxy-2-(di-n-propyl-amino)tetralin, or [3H]8-OH-DPAT, to membranes from rat cerebral cortex and hippocampus could be inhibited by serotonin (5-HT) and buspirone, and by the 5-HT antagonists propranolol, NAN-190, pindolol, pindobind-5-HT(1A), WAY1OO135, spiperone and ritanserin. All competition curves, except for ritanserin, best fitted a two-site model. In vitro treatment of the membranes with N-ethylmaleimide (NEM), to alkylate sulfhydryl groups, caused dose-dependent decreases of binding; the inhibition curves were biphasic, and the effects irreversible. Reduction of disulfide bonds with L-dithiothreitol (L-DTT) also decreased binding, but in a monophasic way; these effects were fully reversible in cortex, but only partially reversible in hippocampus. In the latter region, but not in cerebral cortex, previous occupancy by [(3)H]8-OH-DPAT partially protected binding from 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 140 nM, of [(3)H]8-OH-DPAT. The saturation data were suggestive of a two-site receptor model incorporating a high-affinity site (K(H) of 0.3-0.5 nM) corresponding to the 5-HT(1A) 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 saturation curves from both control and EEDQ-treated rats were again best fitted to a two-site model. For EEDQ-treated animals, a drastic decrease of 5-HT(1A) receptor binding activity was noted; this loss was greater in hippocampus than in cerebral cortex. Since the decrease in 5-HT(1A) receptors was not associated with changes in low-affinity binding, the results suggest independent regulations of the two [(3)H]8-OH-DPAT binding proteins. Altogether, the present data further supports the notion that [(3)H]8-OH-DPAT, besides labelling 5-HT(1A) receptors, also binds to other structures in rat cerebral cortex and hippocampus.