Endothelin stimulates phosphatidic acid formation in cultured rat mesangial cells: Role of a protein kinase C-regulated phospholipase D
✍ Scribed by Mark Kester; Michael S. Simonson; R. Guy McDermott; Elisabetta Baldi; Michael J. Dunn
- Publisher
- John Wiley and Sons
- Year
- 1992
- Tongue
- English
- Weight
- 929 KB
- Volume
- 150
- Category
- Article
- ISSN
- 0021-9541
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✦ Synopsis
Abstract
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.