PHORBOL ESTER-STIMULATED HYDROLYSIS OF PHOSPHATIDYLCHOLINE AND PHOSPHATIDYLETHANOLAMINE BY PHOSPHOLIPASE-D IN HELA-CELLS - EVIDENCE THAT THE BASAL TURNOVER OF PHOSPHOGLYCERIDES DOES NOT INVOLVE PHOSPHOLIPASE-D

12-O-Tetradecanoylphorbol-13-acetate (TPA) stimulated the release of [H-3]ethanolamine from HeLa cells prelabeled with [H-3]ethanolamine within 2 min, and of [H-3]choline from cells prelabeled with [H-3]choline after a lag of 10-20 min. This result suggests that TPA activates phospholipase D. Propranolol alone or propranolol plus TPA stimulated phosphatidic acid (PA) labeling in cells prelabeled with [H-3]hexadecanol. In the presence of ethanol, TPA stimulated the accumulation of labeled phosphatidylethanol (PEth); no PEth was formed in the absence of TPA. TPA-dependent PEth accumulation was not observed in cells pretreated with TPA to down-regulate protein kinase C, whereas propranolol-induced accumulation of PA was unaffected by TPA pretreatment. Incubation of prelabeled cells with propranolol alone caused a rapid loss of label and phospholipid mass from both phosphatidylethanolamine and phosphatidylcholine (PC) together with an accumulation of PA and phosphatidylinositol plus phosphatidylserine. When [H-3]hexadecanol-prelabeled cells were pulse labeled with P-32 to label nucleotide pools, propranolol induced the accumulation of both H-3- and P-32-labeled PA. When cells were prelabeled with lyso-PC double labeled with H-3 and P-32, and incubated with propranolol, only H-3-labeled PA accumulated, indicating that the pathways involved in the basal turnover of PC resulted in the loss of P-32 from the lipid. These results suggest that the basal turnover of phosphatidylethanolamine and PC involves the sequential actions of phospholipase C, diglyceride kinase, and PA phosphohydrolase.