Characterization of phospholipase C activity at h5-HT_2C compared with h5-HT_2B receptors: influence of novel ligands upon membrane-bound levels of [³H]phosphatidylinositols

Abstract.

Employing a novel, rapid and sensitive method for evaluation of phospholipase C (PLC) activity, the present study characterized the actions of diverse agonists and antagonists at human (h)5-HT_2C receptors expressed in Chinese Hamster Ovary (CHO) cells. In addition, affinities and efficacies at these sites were compared with those obtained at h5-HT_2B receptors.

5-HT elicited a robust and rapid reduction in levels of the pre-labelled, membrane-bound substrate of PLC, [³H]phosphatidylinositols ([³H]PI). The time-course of [³H]PI depletion paralleled that of [³H]inositol phosphate ([³H]IP) accumulation, as determined by conventional anion exchange chromatography. Inactivation of h5-HT_2C receptors with the alkylating agent, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), revealed a large receptor reserve, with half-maximal PLC activation induced by a concentration of 5-HT occupying only 5% of sites. In analogy to 5-HT (E _max=100%), DOI, MK212 and mCPP, as well as the novel ligands, Ro600332, Ro600175 and BW723C86, showed "full" efficacy at h5-HT_2C sites. Their efficacies were similar at h5-HT_2B sites, with the exception of mCPP and MK212, which acted as partial agonists. Further, lisuride and Ro600869 behaved as partial agonists and antagonists at h5-HT_2C and h5-HT_2B receptors, respectively. As concerns functional selectivity (potency for induction of [³H]PI depletion), only Ro600175 preferentially activated h5-HT_2B sites. In contrast, Ro600332 preferentially activated h5-HT_2C receptors. Amongst antagonists, RS102221 and SB242084 displayed a marked preference for h5-HT_2C sites, whereas LY266097, S33526 and SB204741 behaved as selective antagonists at h5-HT_2B receptors. At both h5-HT_2C and h5-HT_2B receptors, antagonist potency (pK _b) and binding affinity (pK _i) were highly correlated.

In conclusion, this rapid and innovative method for determination of PLC activity permitted characterization of an extensive range of novel ligands at h5-HT_2C receptors. Although several antagonists clearly differentiated h5-HT_2C from h5-HT_2B receptors under these conditions, highly selective agonists remain to be identified.