Vitamin E up-regulates arachidonic acid release and phospholipase A2 in megakaryocytes

The release of arachidonic acid is the rate limiting step in eicosanoid synthesis. In mammalian cells, the release of arachidonic acid is catalyzed by several enzymes. The 85 kDa cytosolic phospholipase A2 (cPLA2) is the key enzyme for the release reaction because of its specific acyl selectivity in phospholipid substrates. We have previously reported that vitamin E enrichment potentiates the arachidonic acid release as well as the spontaneous prostacyclin release in human endothelial cells. In contrast, similar enrichment of diets caused a dose-dependent suppression of platelet thromboxane synthesis. Therefore, the present study was undertaken to determine the effect of vitamin E on arachidonate release and phospholipaseA2 activity in a platelet precursor cell, the MEG-01 megakaryocyte cell line. When these cells were incubated with different concentrations of vitamin E, cellular incorporation was linear with the dosages of this vitamin. Determination of arachidonate release after labeling cells with [3H]-arachidonate showed that vitamin E enrichment caused a dose-dependent increase in ionophore A23187-induced [3H]-arachidonic acid release. Analysis of PLA2 activity showed that activity was detected in the cytosol and this activity was completely abolished by the addition of anti-cPLA2, antibody. Determination of cPLA2 activity demonstrated that vitamin E enrichment caused an increase in enzyme activity. Analysis of cPLA2 protein by Western blot revealed that vitamin E caused an increase in enzyme protein. These data showed that the potentiation of arachidonic acid release and cPLA2, activity by vitamin E was mediated by the enhanced expression of cPLA2 protein.