Phosphatidylcholine hydrolysis stimulated by phorbol myristate acetate is mediated principally by phospholipase D in endothelial cells

The mechanism of phosphatidylcholine (PC) degradation stimulated by phorbol myristate acetate (PMA) was investigated in bovine pulmonary artery endothelial cells prelabeled with methyl-3H]choline (3H]choline) or 9,10-3H]myristic acid (3H]myristic acid). Both labels were selectively incorporated into PC, and addition of PMA stimulated comparable losses of 3H from PC in cells prelabeled with 3H]choline or 3H]myristate. In cells prelabeled with 3H]choline, the loss of 3H from PC correlated with a rapid increase in intracellular free 3H]choline. The increase in intracellular 3H]choline stimulated by PMA was not preceded by an increase in any other 3H-labeled PC degradation product. PMA did not stimulate the formation of PC deacylation products in cells prelabeled with 3H]choline. In permeabilized cells prelabeled with 3H]choline, PMA stimulated the formation of 3H]choline but not 3H]phosphocholine. In intact cells prelabeled with 3H]myristate, the loss of 3H from PC induced by PMA correlated with the formation of 3H]phosphatidic acid (3H]PA) and 3H]diacylglycerol. In the presence of ethanol, PMA stimulated the formation of 3H]phosphatidylethanol (3H]PEt) at the expense of 3H]PA. The time-course of 3H]PEt formation was similar to the time-course of intracellular 3H]choline formation in cells stimulated with PMA. These data taken together support the notion that PC degradation in endothelial cells stimulated with PMA is mediated principally by phospholipase D. PC breakdown via phospholipase D was not observed in cells treated with phorbol esters incapable of interacting with protein kinase C. Activation of phospholipase D by phorbol esters was inhibited by long-term pretreatment of cells with PMA to down-regulate protein kinase C and by pretreatment of the cells with staurosporine. These data support the notion that activation of phospholipase D by phorbol esters is dependent upon protein kinase C.