Barnett R L, Ruffini L, Hart D, Mancuso P, Nord E P
Department of Medicine, School of Medicine, State University of New York at Stony Brook 11794.
Am J Physiol. 1994 Jan;266(1 Pt 2):F46-56. doi: 10.1152/ajprenal.1994.266.1.F46.
Previous studies from this laboratory have demonstrated that endothelin-1 (ET) stimulates phosphatidylinositol (PI) hydrolysis, activates dihydropyridine-insensitive Ca2+ channels, and promotes prostaglandin E2 (PGE2) accumulation in cultured rat renal medullary interstitial cells (RMIC). The mechanism whereby ET augments PGE2 production was explored in the current study. ET-evoked PGE2 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 PGE2 production, indicating that eicosanoid production was nonetheless a Ca(2+)-requiring process. Nanomolar concentrations of phorbol 12-myristate 13-acetate (PMA) alone did not stimulate PGE2 production, nor did PMA alter ET-stimulated PGE2 accumulation. Furthermore, downregulation of protein kinase C (PKC) by prolonged exposure of cells to PMA did not mitigate ET-mediated PGE2 production, demonstrating that PKC stimulation was not required for PGE2 production. ET stimulated PGE2 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 A2 (PLA2). Immunocytochemical studies including immunostaining, immunoblotting, and immunoprecipitation confirmed the presence of cytosolic PLA2 (cPLA2) in RMIC. In summary, ET stimulation of PGE2 production in RMIC is mediated via agonist activation of cPLA2 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.
该实验室先前的研究表明，内皮素-1（ET）可刺激磷脂酰肌醇（PI）水解，激活对二氢吡啶不敏感的Ca2+通道，并促进培养的大鼠肾髓质间质细胞（RMIC）中前列腺素E2（PGE2）的积累。本研究探讨了ET增加PGE2产生的机制。ET诱发的PGE2积累独立于细胞溶质游离Ca2+浓度（[Ca2+]i）的大幅增加，[Ca2+]i来源于细胞外或细胞内。用1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸螯合细胞内Ca2+可消除ET诱发的PGE2产生，表明类花生酸的产生仍然是一个需要Ca2+的过程。纳摩尔浓度的佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）单独不能刺激PGE2产生，PMA也不会改变ET刺激的PGE2积累。此外，通过将细胞长时间暴露于PMA来下调蛋白激酶C（PKC）并不会减轻ET介导的PGE2产生，这表明PGE2产生不需要PKC刺激。尽管纳摩尔浓度的PMA抑制了PI特异性磷脂酶C（PI-PLC），ET仍刺激了PGE2积累，这表明类花生酸的产生不是PI水解的下游事件。ET刺激花生四烯酸代谢产物释放，同时膜磷脂中的标记物丢失。磷脂酰乙醇胺是ET介导的磷脂酶A2（PLA2）激活的首选底物。包括免疫染色、免疫印迹和免疫沉淀在内的免疫细胞化学研究证实了RMIC中存在细胞溶质PLA2（cPLA2）。总之，ET刺激RMIC中PGE2产生是通过cPLA2的激动剂激活介导的，独立于PI-PLC的激活，提示与ET受体直接偶联。[Ca2+]i的组成水平而非[Ca2+]i的突然增加足以激活该受体-效应系统，对PKC无强制性要求。
