HOW EFFICACIOUS ARE 5-HT1B D RECEPTOR LIGANDS - AN ANSWER FROM GTP-GAMMA-S BINDING-STUDIES WITH STABLY TRANSFECTED C6-GLIAL CELL-LINES/

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 [S-35]GTP gamma S binding assay and membrane p reparations of stably transfected C6-glial cell lines. Compounds eithe r stimulated or inhibited [S-35]GTP gamma S binding to a membrane prep aration containing either h5-HT1B or h5-HT1D receptors. The potencies observed for most of the compounds at the h5-HT1B receptor subtype cor related with their potencies measured by inhibition of stimulated cAMP formation on intact cells. Apparent agonist potencies in the [S-35]GT P gamma S binding assay to C6-glial/h5-HT1D membranes were, with the e xception of lamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indol-3-yl] ethanam ine (L694247), 5- to 13-times lower than in the cAMP assay on intact c ells. This suggests that receptor coupling in the h5-HT1D membrane pre paration is less efficient than that in the intact cell. It further ap peared that 6-times more h5-HT1D than h5-HT1B binding sites were requi red to attain a similar, maximal (73%), 5-HT-stimulated [S-35]GTP gamm a S binding response. Hence, the h5-HT1B receptor in C6-glial cell mem branes could be more efficiently coupled, even though some compounds m ore readily displayed intrinsic activity at h5-HT1D receptor sites [e. g. dihydroergotamine and -methyl[1,2,4]oxadiazol-3-yl)biphenyl-4-carbo xylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]amide (GR127935 )]. Efficacy differences were apparent for most of the compounds (suma triptan, zolmitriptan, rizatriptan, idin-2(R)-ylmethyl]-1H-indol-5-ylm ethylsulfonamide (CP122638), dihydroergotamine, naratriptan and GR1279 35) that stimulated [S-35]GTP gamma S binding compared to the native a gonist 5-HT. The observed maximal responses were different for the h5- HT1B and h5-HT1D receptor subtypes. Few compounds behaved as full agon ists: L694247, zolmitriptan and sumatriptan did so at the h5-HT1B rece ptor and only L694247 at the h5-HT1D receptor. GR127935 (10 mu M) exer ted little effect on [S-35]GTP gamma S binding via h5-HT1B receptors ( 10% stimulation), but potently (pA(2): 9.11) antagonized h5-HT1B recep tor-stimulated [S-35]GTP gamma S binding. Ketanserin and methiothepin inhibited [S-35]GTP gamma S binding (by 13-28%) in the absence of an a gonist, but were potent and competitive antagonists in the presence of an agonist via h5-HT1B (methiothepin) and h5-HT1D (methiothepin and k etanserin) receptors. The results document the utility of using [S-35] GTP gamma S binding studies to assess agonist efficacy, and to charact erize 5-HT1B/D receptor ligands as apparently neutral antagonists and inverse agonists at the G-protein level. (C) 1997 Elsevier Science Ltd .