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-HT(1B)) receptor stably transfected in rat C6-glial cells was characterized by monitoring G-protein activation in a membrane preparation with agonist-stimulated [(35)S]-GTP gamma S binding. The intrinsic activity of 5-HT receptor ligands was compared with that determined previously at the human recombinant 5-HT(1B) (h 5-HT(1B)) receptor under similar experimental conditions. 2 Membrane preparations of C6-glial/gp 5-HT(1B) cells exhibited [(3)H]-5-carboxamidotryptamine (5-CT) and [(3)H]- N- [4-methoxy-3,4-methylpiperazin-1-yl) phenyl]-3-methyl-4-(4-pyridinyl)benzamide (GR 125743) 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 [(3)H]-5-CT and [(3)H]-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-HT(1B) receptor. 3 [(35)S]-GTP gamma S binding to membrane preparations of C6-glial/gp 5-HT(1B) cells was stimulated by the 5-HT receptor agonists that were being investigated. The maximal responses of naratriptan, zolmitriptan, sumatriptan, N-methyl-3-[pyrrolidin-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 recombinant h 5-HT(1B) receptor. 1-naphthylpiperazine, (+/-)-cyanopindolol and (2'-methyl-4'-(5-methyl[1,2,4] oxadiazole-3-yl)biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]amide (GR 127935) elicited an even smaller response (E(max): 0.32 to 0.63). 4 The ligands 1'-methyl-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] (SB224289), methiothepin and ritanserin displayed inhibition of basal [(35)S]-GTP gamma S binding at concentrations relevant to their binding affinity for the gp 5-HT(1B) receptor. Methiothepin and SB224289 behaved as competitive antagonists at gp 5-HT(1B) receptors; pA(2) values were 9.74 and 8.73, respectively when 5-HT was used as an agonist. These estimates accorded with the potencies measured in antagonism of zolmitriptan-mediated inhibition of forskolin-stimulated cyclic AMP formation. Ketanserin acted as a weak antagonist (pK(B): 5.87) at gp 5-HT(1B) receptors. 5 In conclusion, the recombinant gp 5-HT(1B) receptor shares important pharmacological similarities with the recombinant h 5-HT(1B) receptor. The finding that negative activity occurs at these receptors further suggests that SB224289, methiothepin and ritanserin are likely to be inverse agonists.