PHARMACOLOGICAL ANALYSIS OF G-PROTEIN ACTIVATION MEDIATED BY GUINEA-PIG RECOMBINANT 5-HT1B RECEPTORS IN C6-GLIAL CELLS - SIMILARITIES WITH THE HUMAN 5-HT1B RECEPTOR

1 The guinea-pig recombinant 5-hydroxytryptamine(1B), (gp 5-HT1B) rece ptor stably transfected in rat C6-glial cells was characterized by mon itoring G-protein activation in a membrane preparation with agonist-st imulated [S-35]-GTP gamma S binding. The intrinsic activity of 5-HT re ceptor ligands was compared with that determined previously at the hum an recombinant 5-HT1B (h 5-HT1B) receptor under similar experimental c onditions. 2 Membrane preparations of C6-glial/gp 5-HT1B cells exhibit ed [H-3]-5-carboxamidotryptamine (5-CT) and [H-3]- N- [4-methoxy-3,4-m ethylpiperazin-1-yl) phenyl]-3-methyl-4-(4-pyridinyl)benzamide (GR 125 743) binding sites with a pK(d) of 9.62 to 9.85 and a B-max,,, between 2.1 to 6.4 fmol mg(-1) protein. The binding affinities of a series of 5-HT receptor ligands determined with [H-3]-5-CT and [H-3]-GR 125743 were similar. Ligand affinities were comparable to and correlated (r(2 ): 0.74, P<0.001) with those determined at the recombinant h 5-HT1B re ceptor. 3 [S-35]-GTP gamma S binding to membrane preparations of C6-gl ial/gp 5-HT1B cells was stimulated by the 5-HT receptor agonists that were being investigated. The maximal responses of naratriptan, zolmitr iptan, sumatriptan, din-2(R)-ylmethyl]-1H-indol-5-ylmethylsulphonamide (CP 122638), rizatriptan and dihydroergotamine were between 0.76 and 0.85 compared to 5-HT. The potency of these agonists showed a positive correlation (r(2): 0.72, P=0.015) with their potency at the recombina nt h 5-HT1B receptor. 1-naphthylpiperazine, (+/-)-cyanopindolol and (2 '-methyl-4'-(5-methyl[1,2,4] oxadiazole-3-yl)biphenyl-4-carboxylic aci d [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]amide (GR 127935) elicit ed an even smaller response (E-max: 0.32 to 0.63). 4 The ligands l-5-( 2'-methyl-4'-(5-methyl-1,2,4-oxadiazole-3-yl) biphenyl-4-carbonyl)-2,3 ,6,7-tetrahydrospiro [furo[2,3-f]indole-3-spiro-4'-piperidine] (SB2242 89), methiothepin and ritanserin displayed inhibition of basal [S-35]- GTP gamma S binding at concentrations relevant to their binding affini ty for the gp 5-HT1B receptor. Methiothepin and SB224289 behaved as co mpetitive antagonists at gp 5-HT1B receptors; pA(2) values were 9.74 a nd 8.73, respectively when 5-HT was used as an agonist. These estimate s accorded with the potencies measured in antagonism of zolmitriptan-m ediated inhibition of forskolin-stimulated cyclic AMP formation. Ketan serin acted as a weak antagonist (pK(B): 5.87) at gp 5-HT1B receptors. 5 In conclusion, the recombinant gp 5-HT1B receptor shares important pharmacological similarities with the recombinant h 5-HT1B receptor. T he finding that negative activity occurs at these receptors further su ggests that SB224289, methiothepin and ritanserin are likely to be inv erse agonists.