title: How efficacious are 5-HT1B/D receptor ligands: an answer from GTP gamma S binding studies with stably transfected C6-glial cell lines.
abstract: The intrinsic activity of a series of 5-hydroxytryptamine (serotonin, 5-HT) receptor ligands was analysed at recombinant h5-HT1B and h5-HT1D receptor sites using a [35S]GTP gamma S binding assay and membrane preparations of stably transfected C6-glial cell lines. Compounds either stimulated or inhibited [35S]GTP gamma S binding to a membrane preparation containing either h5-HT1B or h5-HT1D receptors. The potencies observed for most of the compounds at the h5-HT1B receptor subtype correlated with their potencies measured by inhibition of stimulated cAMP formation on intact cells. Apparent agonist potencies in the [35S]GTP gamma S binding assay to C6-glial/h5-HT1D membranes were, with the exception of 2-[5-[3-(4-methylsulphonylamino)benzyl-1 2,4-oxadiazol-5-yl]-1H-indol-3-yl] ethanamine (L694247), 5- to 13-times lower than in the cAMP assay on intact cells. This suggests that receptor coupling in the h5-HT1D membrane preparation is less efficient than that in the intact cell. It further appeared that 6-times more h5-HT1D than h5-HT1B binding sites were required to attain a similar, maximal (73%), 5-HT-stimulated [35S]GTP gamma S binding response: Hence, the h5-HT1B receptor in C6-glial cell membranes could be more efficiently coupled, even though some compounds more readily displayed intrinsic activity at h5-HT1D receptor sites [e.g. dihydroergotamine and (2'-methyl-4'-(5-methyl[1,2,4]oxadiazol-3-yl)biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]amide (GR127935)]. Efficacy differences were apparent for most of the compounds (sumatriptan, zolmitriptan, rizatriptan, N-methyl-3-[pyrrolidin-2(R)-ylmethyl]-1H-indol-5-ylmethyl sulfonamide (CP122638), dihydroergotamine, naratriptan and GR127935) that stimulated [35S]GTP gamma S binding compared to the native agonist 5-HT. The observed maximal responses were different for the h5-HT1B and h5-HT1D receptor subtypes. Few compounds behaved as full agonists: L694247, zolmitriptan and sumatriptan did so at the h5-HT1B receptor and only L694247 at the h5-HT1D receptor. GR127935 (10 microM) exerted little effect on [35S]GTP gamma S binding via h5-HT1B receptors (10% stimulation), but potently (pA2: 9.11) antagonized h5-HT1B receptor-stimulated [35S]GTP gamma S binding. Ketanserin and methiothepin inhibited [35S]GTP gamma S binding (by 13-28%) in the absence of an agonist, but were potent and competitive antagonists in the presence of an agonist via h5-HT1B (methiothepin) and h5-HT1D (methiothepin and ketanserin) receptors. The results document the utility of using [35S]GTP gamma S binding studies to assess agonist efficacy, and to characterize 5-HT1B/D receptor ligands as apparently neutral antagonists and inverse agonists at the G-protein level.
authors.affiliation: Centre de Recherche Pierre Fabre, Laboratory of Cellular and Molecular Neurobiology, Castres, France. crpfl@starway.tm.fr
authors.forename: P J
authors.initials: PJ
authors.lastname: Pauwels
authors.forename: S
authors.initials: S
authors.lastname: Tardif
authors.forename: C
authors.initials: C
authors.lastname: Palmier
authors.forename: T
authors.initials: T
authors.lastname: Wurch
authors.forename: F C
authors.initials: FC
authors.lastname: Colpaert
mesh_terms: D002460:Cell Line; D000242:Cyclic AMP / Q000096:biosynthesis; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D008566:Membranes / Q000187:drug effects / Q000378:metabolism; D009457:Neuroglia / Q000187:drug effects / Q000378:metabolism*; D010957:Plasmids; D011869:Radioligand Assay; D011985:Receptors, Serotonin / Q000187:drug effects* / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012702:Serotonin Antagonists / Q000494:pharmacology; D017366:Serotonin Receptor Agonists / Q000494:pharmacology; D014162:Transfection
chemical_list: D008024:Ligands; D011985:Receptors, Serotonin; D011994:Recombinant Proteins; D012702:Serotonin Antagonists; D017366:Serotonin Receptor Agonists; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000242:Cyclic AMP; D019204:GTP-Binding Proteins