Phorbol diesters stimulate the accumulation of phosphatidate, phosphatidylethanol, and diacylglycerol in three cell types. Evidence for the indirect formation of phosphatidylcholine-derived diacylglycerol by a phospholipase D pathway and direct formation of diacylglycerol by a phospholipase C pathway

The effect of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), on phospholipid degradation was investigated in three cell lines of dissimilar origin, Madin-Darby canine kidney cells (MDCK), rat aorta smooth muscle cells (RASM), and bovine pulmonary artery endothelial cells (BPAE). In cells prelabeled with 3H]myristic acid, which is predominantly incorporated into phosphatidylcholine (PC), TPA treatment (80 nM) in the absence or presence of ethanol (2%) in the culture medium resulted in either the rapid generation of 3H]phosphatidate (PA) or the sustained accumulation of 3H]phosphatidylethanol (PEt), respectively. Increases in 3H]PA and 3H]PEt were paralleled by quantitative decreases in cellular 3H]PC radioactivity. TPA-induced 3H]PEt formation occurred in a similar fashion, irrespective of the presence of Ca2+ in the culture medium. The experiments demonstrate that TPA elicits PC degradation by phospholipase D (PLD) in cells of diverse origin. Data from further experiments revealed a complex relationship between TPA-induced 3H]PA and 3H]diacylglycerol (DG) generation in the three cell lines that was suggestive of dual pathways for the generation of 3H]DG. Experiments to discern the pathways for TPA-induced, PC-derived DG were conducted by comparing the variation of 3H]PA and 3H]DG formation in the absence and in the presence of increasing ethanol concentrations in the culture medium. With increasing amounts of ethanol, the formation of 3H]PA decreased at the expense of 3H]PEt formation, and depending upon the pathway operable, the amount of 3H]DG formed was either decreased, indicative of indirect formation of DG via PA phosphohydrolase, or not modified, indicative of DG formation by a direct phospholipase C (PLC) pathway. Increasing the concentration of ethanol in the medium blocked TPA-induced 3H]DG generation in MDCK cells in a concentration-dependent manner, while the formation of 3H]PEt increased at the expense of 3H]PA formation. In BPAE cells the presence of ethanol likewise reduced TPA-elicited formation of DG. Conversely, in two smooth muscle cell lines, RASM and A-10, ethanol was without influence on TPA-induced formation of 3H]DG, although 3H]PEt was generated at the expense of 3H]PA. In RASM cells prelabeled with 3H]choline, TPA induced the release to the medium of 3H]choline and 3H]phosphocholine, indicative of both PLD and PLC activation. These results show that TPA elicits DG formation from PC in MDCK cells predominantly by an indirect pathway, whereas in arterial smooth muscle cells DG is formed in part by the direct action of PLC.(ABSTRACT TRUNCATED AT 400 WORDS)