Vitamin E up-regulates arachidonic acid release and phospholipase A(2) in megakaryocytes

The release of arachidonic acid is the rate limiting step in eicosanoid syn thesis. In mammalian cells, the release of arachidonic acid is catalyzed by several enzymes. The 85 kDa cytosolic phospholipase A(2) (cPLA(2)) is the key enzyme for the release reaction because of its specific acyl selectivit y in phospholipid substrates. We have previously reported that vitamin E en richment potentiates the arachidonic acid release as well as the spontaneou s prostacyclin release in human endothelial cells. In contrast, similar enr ichment of diets caused a dose-dependent suppression of platelet thromboxan e synthesis. Therefore, the present study was undertaken to determine the e ffect of vitamin E on arachidonate release and phospholipase A(2), activity in a platelet precursor cell, the MEG-01 megakaryocyte cell line. When the se cells were incubated with different concentrations of vitamin E, cellula r incorporation was linear with the dosages of this vitamin. Determination of arachidonate release after labeling cells with [H-3]-arachidonate showed that vitamin E enrichment caused a dose-dependent increase in ionophore A2 3187-induced [H-3]-arachidonic acid release. Analysis of PLA(2) activity sh owed that activity was detected in the cytosol and this activity was comple tely abolished by the addition of anti-cPLA(2), antibody. Determination of cPLA(2) activity demonstrated that vitamin E enrichment caused an increase in enzyme activity. Analysis of cPLA(2) protein by Western blot revealed th at vitamin E caused an increase in enzyme protein. These data showed that t he potentiation of arachidonic acid release and cPLA(2), activity by vitami n E was mediated by the enhanced expression of cPLA(2) protein.