The selective 5-HT1A antagonist radioligand [3H]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 antagonist WAY 100635 [N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl) cyclohexane carboxamide] was tested as a potential radioligand of 5-HT1A receptors in the rat brain. Binding assays with membranes from various brain regions showed that [3H]WAY 100635 specifically bound to a homogeneous population of sites, with a Kd of 0.10 nM. The regional distribution of [3H]WAY 100635 specific binding sites, as assessed in membrane binding assays and by autoradiography of labelled brain sections, superimposed exactly over that of 5-HT1A receptors specifically labelled by [3H]8-hydroxy-2-(di-n-propylamino) tetralin ([3H]8-OH-DPAT). Furthermore, the positive correlation (r = 0.96) between the respective pKi values of a large series of ligands as inhibitors of the specific binding of [3H]WAY 100635 and [3H]8-OH-DPAT in hippocampal membranes indicated that their pharmacological properties were similar. Nevertheless, marked differences also existed between [3H]8-OH-DPAT and [3H]WAY 100635 specific binding, as the former was inhibited by 1-100 microM GTP and GppNHp, whereas the latter was enhanced by these guanine nucleotides. In contrast, Mn2+ (1-10 mM) increased the specific binding of [3H]8-OH-DPAT, but inhibited that of [3H]WAY 100635. Treatment of membranes with N-ethylmaleimide (1-5 mM) markedly reduced their capacity to specifically bind [3H]8-OH-DPAT, but slightly increased (at 1 mM) or did not affect (at 5 mM) their [3H]WAY 100635 specific binding capacity. Finally, the Bmax of [3H]WAY 100635 specific binding sites was regularly 50-60% higher than that of [3H]8-OH-DPAT in the same membrane preparations from various brain regions (hippocampus, septum, cerebral cortex). These data are compatible with the idea that whereas [3H]8-OH-DPAT only binds to G-protein-coupled 5-HT1A receptors, [3H]WAY 100635 is a high affinity ligand of both G-protein-coupled and free 5-HT1A receptor binding subunits in brain membranes.