MECHANISM OF ENDOTHELIN ACTIVATION OF PHOSPHOLIPASE A(2) IN RAT RENAL MEDULLARY INTERSTITIAL-CELLS

Previous studies from this laboratory have demonstrated that endothelin-1 (ET) stimulates phosphatidylinositol (PI) hydrolysis, activates dihydropyridine-insensitive Ca2+ channels, and promotes prostaglandin E(2) (PGE(2)) accumulation in cultured rat renal medullary interstitial cells (RMIC). The mechanism whereby ET augments PGE(2) production was explored in the current study. ET-evoked PGE(2) accumulation proceeded independent of large increments in cytosolic free Ca2+ concentration ([Ca2+]i), derived from either extracellular or intracellular sources. Chelation of intracellular Ca2+ with 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'tetraacetic acid eliminated ET-evoked PGE(2) production, indicating that eicosanoid production was nonetheless a Ca2+ requiring process. Nanomolar concentrations of phorbol 12-myristate 13-acetate (PMA) alone did not stimulate PGE(2) production, nor did PMA alter ET-stimulated PGE(2) accumulation. Furthermore, downregulation of protein kinase C (PKC) by prolonged exposure of cells to PMA did not mitigate ET-mediated PGE(2) production, demonstrating that PKC stimulation was not required for PGE(2) production. ET stimulated PGE(2) accumulation despite PI-specific phospholipase C (PI-PLC) inhibition by nanomolar concentrations of PMA, indicating that eicosanoid production was not a downstream event of PI hydrolysis. ET stimulated arachidonic acid metabolite release in parallel with a loss of label from membrane phospholipids. Phosphatidylethanolamine was the preferred substrate for ET-mediated activation of phospholipase A(2) (PLA(2)). Immunocytochemical studies including immunostaining, immunoblotting, and immunoprecipitation confirmed the presence of cytosolic PLA(2) (cPLA(2)) in RMIC. In summary, ET stimulation of PGE(2) production in RMIC is mediated via agonist activation of cPLA(2) independent of activation of PI-PLC, suggesting direct coupling to the ET receptor. Constitutive levels of [Ca2+](i) rather than abrupt increments in [Ca2+](i) are sufficient for activation of this receptor-effector system, with no obligatory requirement for PKC.