Pharmacological analysis of G-protein activation mediated by guinea-pig recombinant 5-HT_1B receptors in C6-glial cells: similarities with the human 5-HT_1B receptor

Abstract

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 [³⁵S]-GTPγS 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 [³H]-5-carboxamidotryptamine (5-CT) and [³H] - 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 [³H]-5-CT and [³H]-GR 125743 were similar. Ligand affinities were comparable to and correlated (r²: 0.74, P<0.001) with those determined at the recombinant h 5-HT_1B receptor.

3. [³⁵S]-GTPγS 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 (CP122638), rizatriptan and dihydroergotamine were between 0.76 and 0.85 compared to 5-HT. The potency of these agonists showed a positive correlation (r²: 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 [³⁵S]-GTPγS 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; pA₂ 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.

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