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Endothelin stimulates phosphatidic acid formation in cultured rat mesangial cells: Role of a protein kinase C‐regulated phospholipase D
Author(s) -
Kester Mark,
Simonson Michael S.,
McDermott R. Guy,
Baldi Elisabetta,
Dunn Michael J.
Publication year - 1992
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041500319
Subject(s) - phosphatidylethanol , phosphatidic acid , phospholipase d , protein kinase c , diacylglycerol kinase , pld2 , phospholipase c , phospholipase , biochemistry , chemistry , biology , endocrinology , signal transduction , phospholipid , enzyme , membrane
We have previously reported that endothelin‐1 stimulates phospholipase C‐in‐duced hydrolysis of phosphatidylinositol‐4, 5‐bisphosphate, Other signal transduction pathways that hydrolyze alternative phospholipids through phospholipase D may also mediate endothelin‐stimulated cellular responses. We initially evaluated endothelin‐dependent generation of 32 P‐phosphatidic acid as an indirect indication of phospholipase D activity in rat mesangial cells. Endothelin (10 −7 M) induced an elevation of phosphatidic acid that was maximal at 15 min and persisted upward of 60 min. Pretreatment with the diacylglycerol‐kinase inhibitor, R59022, did not reduce formation of endothelin‐stimulated 32 P‐phosphatidic acid, demonstrating that the sequential actions of phospholipase C/diacylglycerol kinase do not contribute to endothelin‐stimulated phosphatidic acid formation. We next conclusively identified a role for phospholipase D in the generation of phosphatidic acid by assessing the formation of 3 H‐phosphatidylethanol from 3 H‐alkyl lyso glycerophosphocholine and exogenous ethanol. Endothelin stimulated 3 H‐alkyl phosphatidylethanol formation in the presence but not the absence of 0.5% ethanol. Also, endothelin induced a concomitant elevation of 3 H‐alkyl‐phosphatidic acid that was significantly reduced when the cells were exposed to exogenous ethanol, reflecting the formation of phosphatidylethanol. In addition, endothelin stimulated the release of 3 H‐choline and 3 H‐ethanolamine, demonstrating that additional phospholipids may serve as substrates for phospholipase D. Phorbol esters and synthetic diglycerides mimicked the effects of endothelin to stimulate phospholipase D and inhibitors of protein kinase C significantly reduced endothelin‐stimulated phospholipase D. In addition, endothelin did not stimulate phosphatidylethanol formation in protein kinase C down‐regulated cells. The calcium ionophore, ionomycin, did not stimulate phospholipase D and mesangial cells pretreated with BAPTA to chelate cytosolic calcium did not show a diminished endothelin‐stimulated phospholipase D. Thus these data demonstrate that mesangial cells possess a protein kinase C‐regulated phospholipase D activity that can be stimulated with endothelin.