Vasopressin and phorbol ester-stimulated phosphatidylcholine metabolism in mesangial cells

We have studied the effects of the vasoactive agents phorbol 12-myristate 13-acetate (PMA) and vasopressin (VP) on phosphatidylcholine metabolism in cultured rat glomerular mesangial cells. PMA and VP stimulate the incorporation of [3H]choline into phosphatidylcholine and the release of [3H]choline into the culture medium. VP, but not PMA, also increases the release of phosphorylcholine into the medium. This suggests that PMA specifically stimulates phospholipase D, whereas VP stimulates phospholipases C and D. Experiments were also conducted to look for production of phosphatidic acid and diacylglycerol, products of phospholipase D- and C-mediated breakdown of phosphatidylcholine. Treatment of cells prelabeled with [3H]myristic acid for 2.5 min with PMA or VP increases the content of [3H]myristic acid in diacylglycerol and phosphatidic acid. A dual labeling study ([3H]myristic acid and [14C]arachidonic acid) suggests that phosphatidylcholine is an important source of diacylglycerol in cells treated with VP and PMA. When PMA or VP are added to [3H]myristic acid-labeled cells in the presence of ethanol, increased labeling of phosphatidylethanol is seen as early as 2.5 min. Desensitization of protein kinase C by overnight treatment of cells with PMA blocked subsequent VP-stimulated formation of phosphatidylethanol and release of [3H]choline. When cells were simultaneously treated with VP and PMA, additive effects on phosphatidylethanol formation and [3H]choline release were observed.