THE SELECTIVE 5-HT1A ANTAGONIST RADIOLIGAND [H-3] WAY-100635 LABELS BOTH G-PROTEIN-COUPLED AND FREE 5-HT1A RECEPTORS IN RAT-BRAIN MEMBRANES

The tritiated derivative of the novel silent 5-HT1A receptor antagonis t WAY 100635 ethoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl) cycloh exane carboxamide] was tested as a potential radioligand of 5-HT1A rec eptors in the rat brain. Binding assays with membranes from various br ain regions showed that [H-3]WAY 100635 specifically bound to a homoge neous population of sites, with a K-d of 0.10 nM. The regional distrib ution of [H-3]WAY 100635 specific binding sites, as assessed in membra ne binding assays and by autoradiography of labelled brain sections, s uperimposed exactly over that of 5-HT1A receptors specifically labelle d by [H-3]8-hydroxy-2-(di-n-propylamino)tetralin ([H-3]8-OH-DPAT). Fur thermore, the positive correlation (r = 0.96) between the respective p K(i) values of a large series of ligands as inhibitors of the specific binding of [H-3]WAY 100635 and [H-3]8-OH-DPAT in hippocampal membrane s indicated that their pharmacological properties were similar. Nevert heless, marked differences also existed between [H-3]8-OH-DPAT and [H- 3]WAY 100635 specific binding, as the former was inhibited by 1-100 mu M GTP and GppNHp, whereas the latter was enhanced by these guanine nu cleotides. In contrast, Mn2+ (1-10 mM) increased the specific binding of [H-3]8-OH-DPAT, but inhibited that of [H-3]WAY 100635. Treatment of membranes with N-ethylmaleimide (1-5 mM) markedly reduced their capac ity to specifically bind [H-3]8-OH-DPAT, but slightly increased (at 1 mM) or did not affect (at 5 mM) their [H-3]WAY 100635 specific binding capacity. Finally, the B-max of [H-3]WAY 100635 specific binding site s was regularly 50-60% higher than that of [H-3]8-OH-DPAT in the same membrane preparations from various brain regions (hippocampus, septum, cerebral cortex). These data are compatible with the idea that wherea s [H-3]8-OH-DPAT only binds to G-protein-coupled 5-HT1A receptors, [H- 3]WAY 100635 is a high affinity ligand of both G-protein-coupled and f ree 5-HT1A receptor binding subunits in brain membranes.