An innovative method for rapid characterisation of phospholipase C activity: SB242,084 competitively antagonises 5-HT_2C receptor-mediated [³H]phosphatidylinositol depletion

Abstract.

6-chloro-5-methyl-1-[6-(2-methylpyridin-3-yloxy) pyridin-3-ylcarbamoyl] indoline (SB242,084) is a novel, selective 5-HT_2C receptor antagonist, but its actions at these sites have been little characterised at the cellular level. We employed a rapid and innovative approach to investigate its functional activity at phospholipase C (PLC)-coupled human 5-HT_2C receptors expressed in CHO cells. PLC activity was determined as a decrease in the [³H]phosphatidylinositol ([³H]PI) content of cell membranes. Serotonin (5-HT) stimulated [³H]PI depletion (pEC₅₀=8.74), and SB242,084, like mesulergine, completely reversed this action of 5-HT (pK _B=9.25 and 9.01, respectively). Further, in Schild analysis, SB242,084 behaved as a high affinity competitive antagonist, inducing a parallel, rightward displacement of the 5-HT stimulation isotherm without loss of maximum efficacy. The pA ₂ of 9.50 was similar to its binding affinity (pK _i=9.38). SB242,084 also displayed antagonist properties when PLC activity was examined by conventional determination of [³H]inositol phosphate generation. Employing this parameter, the potency of SB242,084 (pK _B=9.21) and that of mesulergine (pK _B=9.06) closely resembled those determined by [³H]PI depletion. In conclusion, determination of [³H]PI depletion constitutes a useful and novel technique to characterise agonist and antagonist properties of ligands at PLC-coupled receptors.