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.
Gozlan
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INSERM U288, Neurobiologie Cellulaire et Fonctionnelle, Faculté de Médecine Pitíe-Salpêtrière, Paris, France.
Thibault
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Laporte
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AM
Lima
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Hamon
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D017371:8-Hydroxy-2-(di-n-propylamino)tetralin / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D001667:Binding, Competitive / Q000187:drug effects; D001923:Brain Chemistry / Q000187:drug effects*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D008566:Membranes / Q000187:drug effects / Q000378:metabolism; D010879:Piperazines / Q000494:pharmacology*; D011725:Pyridines / Q000494:pharmacology*; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D011985:Receptors, Serotonin / Q000187:drug effects / Q000378:metabolism*; D012701:Serotonin / Q000378:metabolism; D012702:Serotonin Antagonists / Q000494:pharmacology*
D010879:Piperazines; D011725:Pyridines; D011985:Receptors, Serotonin; D012702:Serotonin Antagonists; D012701:Serotonin; C090413:N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide; D017371:8-Hydroxy-2-(di-n-propylamino)tetralin; D019204:GTP-Binding Proteins