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 [3H]ethanolamine from HeLa cells prelabeled with [3H]ethanolamine within 2 min, and of [3H]choline from cells prelabeled with [3H]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 [3H]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 [3H]hexadecanol-prelabeled cells were pulse labeled with 32P to label nucleotide pools, propranolol induced the accumulation of both 3H- and 32P-labeled PA. When cells were prelabeled with lyso-PC double labeled with 3H and 32P, and incubated with propranolol, only 3H-labeled PA accumulated, indicating that the pathways involved in the basal turnover of PC resulted in the loss of 32P 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.
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School of Biological Sciences, Flinders University, Bedford Park, South Australia.
Edwards
Y S
YS
Murray
A W
AW
D002794:Choline / Q000378:metabolism; D005233:Fatty Alcohols / Q000494:pharmacology; D006367:HeLa Cells / Q000201:enzymology*; D006801:Humans; D006868:Hydrolysis; D008244:Lysophosphatidylcholines / Q000378:metabolism; D010169:Palmitic Acids / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000378:metabolism*; D010714:Phosphatidylethanolamines / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D011433:Propranolol / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D005233:Fatty Alcohols; D008244:Lysophosphatidylcholines; D010169:Palmitic Acids; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; C005031:cetyl alcohol; D011433:Propranolol; D010739:Phospholipase D; D002794:Choline; D013755:Tetradecanoylphorbol Acetate