Endothelin-induced changes of second messengers in cultured human ciliary muscle cells

To characterize the pharmacology of endothelin-induced changes in phospholipase C (PLC) activity, intracellular calcium concentration ([Ca2+]i) and cyclic adenosine monophosphate (cAMP) in cultured human ciliary muscle (HCM) cells. Changes in PLC activity of HCM cells were determined by production of [3H] inositol phosphates. [Ca2+]i was determined by single-cell dynamic fluorescence ratio imaging. Radioimmunoassays were used to determine cAMP and prostaglandin E2 (PGE2) concentrations. Endothelin-1 (ET-1) stimulated PLC (mean EC50 = 335 pM) and activated calcium mobilization in HCM cells. These effects were mediated by the endothelin ETA receptor subtype because at ETB receptor-selective agonist, sarafotoxin S6c, was ineffective. Additionally, effects of ET-1 were inhibited by pretreatment with a selective ETA agonist, BQ610 (mean pKi = 9.96 for PLC). ET-1 also stimulated the production of PGE2 (mean EC50 = 12.0 nM) and cAMP (mean EC50 = 5.2 nM) by these cells. PGE2 appeared to mediate the stimulatory effect of ET-1 on adenylyl cyclase because blockade of ET-1-induced PGE2 production by 10 microM indomethacin also completely blocked the ET-1-activated cAMP production. ET-1 stimulated PLC and increased [Ca2+]i in HCM calls by the ETA receptor subtype. ET-1 also increased cAMP production, an effect likely mediated by the enhanced production of prostaglandins.