VITAMIN-E SUPPRESSES DIACYLGLYCEROL (DAG) LEVEL IN THROMBIN-STIMULATED ENDOTHELIAL-CELLS THROUGH AN INCREASE OF DAG KINASE-ACTIVITY

The present study has examined the role of vitamin E, a natural lipid antioxidant, in the production of diacylglycerol (DAG) and phosphatidi c acid (PA) in thrombin-stimulated human endothelial cells. Cells were labelled with [H-3]myristate and the incorporation and distribution o f [H-3]myristate into cellular lipids was not affected by vitamin E. H owever, 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 accum ulation of sustained DAG levels with a concomitant increase in PA. Dir ect determination of DAG mass further confirmed that vitamin E suppres ses the accumulation of DAG induced by thrombin. In the presence of et hanol, the formation of [H-3]phosphatidylethanol (PEt) in [H-3]myrista te-labelled cells stimulated by thrombin was unaffected by vitamin E e nrichment. 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 stimul ation of phospholipase D or an inhibition of PA phosphohydrolase. Dete rmination of inositol phosphates formation in response to thrombin sho wed that the change of DAG levels elicited by vitamin E was independen t of phospholipase C-induced hydrolysis of inositol phospholipids. In contrast, analysis of DAG kinase activity revealed that vitamin E enri chment 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 a ctivity without causing any change in the activities of phospholipase D, PA phosphohydrolase or phospholipase C.