The present study has examined the role of vitamin E, a natural lipid antioxidant, in the production of diacylglycerol (DAG) and phosphatidic acid (PA) in thrombin-stimulated human endothelial cells. Cells were labelled with [H-3]myristate and the incorporation and distribution of [H-3]myristate into cellular lipids was not affected by vitamin E. However, in response to thrombin stimulation, considerably more PA and less DAG were formed in cells enriched with vitamin E. The time-course of thrombin stimulation indicated that vitamin E attenuated the accumulation of sustained DAG levels with a concomitant increase in PA. Direct determination of DAG mass further confirmed that vitamin E suppresses the accumulation of DAG induced by thrombin. In the presence of ethanol, the formation of [H-3]phosphatidylethanol (PEt) in [H-3]myristate-labelled cells stimulated by thrombin was unaffected by vitamin E enrichment. DL-Propranolol, a PA phosphohydrolase inhibitor, caused an accumulation of PA, without affecting DAG formation in either vitamin E-treated and untreated cells. This indicated that the increase in PA and decrease in DAG in vitamin E-treated cells was not due to a stimulation of phospholipase D or an inhibition of PA phosphohydrolase. Determination of inositol phosphates formation in response to thrombin showed that the change of DAG levels elicited by vitamin E was independent of phospholipase C-induced hydrolysis of inositol phospholipids. In contrast, analysis of DAG kinase activity revealed that vitamin E enrichment enhanced the activity of the enzyme in both basal and thrombin-stimulated cells. Taken together, these data indicated that vitamin E caused an increased conversion of DAG to PA by activating DAG kinase activity without causing any change in the activities of phospholipase D, PA phosphohydrolase or phospholipase C.
