Pharmacological characterization of recombinant human 5-hydroxytryptamine1A receptors using a novel antagonist radioligand, [3H]WAY-100635.
The ligand binding characteristics of the recombinant human 5-HT1A receptor stably expressed in a Chinese Hamster Ovary (CHO) cell line are described using a selective agonist, [3H]8-OH-DPAT, and a novel antagonist radioligand, [3H]WAY-100635. The association of [3H]WAY-100635 was a time- and temperature-dependent process. Mn2+ > Ca2+ > Mg2+ reduced the specific [3H]WAY-100635 binding in a concentration-dependent manner, whereas Na+ and K+ were ineffective. Scatchard analyses revealed a homogeneous population of [3H]WAY-100635 recognition sites (Kd = 0.32 nM; Bmax = 162 fmol/mg of protein). Addition of divalent cations to the incubation medium produced a two-fold decrease in the binding affinity of [3H]WAY-100635 with no significant change in Bmax; GTP gamma S had no effect on Kd or Bmax parameters. [3H]WAY-100635 displayed a higher affinity (2-3 fold) for the 5-HT1A site when compared with [3H] 8-OH-DPAT binding under similar incubation conditions. Furthermore, [3H] 8-OH-DPAT labelled approximately 53-61% of total 5-HT1A sites recognised by [3H]WAY-100635. The competition binding profiles of [3H]WAY-100635 and [3H]8-OH-DPAT were highly correlated and consistent with the recognition of 5-HT1A receptors. Agonist competition curves with [3H]WAY-100635 were best-resolved into high- and low-affinity binding states, whereas partial agonist and antagonist curves were best-fit to one-site binding models. A significant correlation between the respective affinities of a range of agonists and antagonists at recombinant human and rodent hippocampal 5-HT1A binding sites (previously published) was also observed using [3H]WAY-100635 (r = 0.92; P < 0.0005) and [3H]8-OH-DPAT (r = 0.96; P < 0.0005). The availability of a novel, high-affinity antagonist radioligand, [3H]WAY-100635, will provide a useful tool for the further characterisation of 5-HT1A receptor pharmacology.
Khawaja
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Wyeth-Ayerst Research, Princeton, New Jersey 08543-8000, USA. khawajx@war.wyeth.com
Ennis
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Minchin
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D017371:8-Hydroxy-2-(di-n-propylamino)tetralin / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D006624:Hippocampus / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D010879:Piperazines / Q000378:metabolism*; D011725:Pyridines / Q000378:metabolism*; D011869:Radioligand Assay; D051381:Rats; D011985:Receptors, Serotonin / Q000235:genetics / Q000378:metabolism*; D044263:Receptors, Serotonin, 5-HT1; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012702:Serotonin Antagonists / Q000378:metabolism*; D017366:Serotonin Receptor Agonists / Q000378:metabolism; D014162:Transfection
D010879:Piperazines; D011725:Pyridines; D011985:Receptors, Serotonin; D044263:Receptors, Serotonin, 5-HT1; D011994:Recombinant Proteins; D012702:Serotonin Antagonists; D017366:Serotonin Receptor Agonists; C090413:N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide; D017371:8-Hydroxy-2-(di-n-propylamino)tetralin