Hydrogen peroxide-induced phospholipase D activation in human vascular smooth muscle cells

Exposure of human aortic smooth muscle cells to hydrogen peroxide (H2O2) caused a concentration- and time-dependent activation of both phospholipase D (PLD) and mitogen-activated protein (MAP) kinases. The formation of phosphatidylbutanol (PBut), a specific product of PLD activation in the presence of butanol, by 0.5 mM H2O2 was detectable within 5 min and lasted for at least 60 min where nearly 2-fold increase was observed. The pretreatment of cells with a selective MAP kinase kinase (MEK) inhibitor, PD 098059 prevented the H2O2-induced PBut formation in a concentration-dependent manner. In contrast, activations of PLD and MAP kinases were not affected by pretreatment of cells with 5 mu M Ro 31-8220, a protein kinase C (PKC) inhibitor, and by PKC down-regulation. H2O2-stimulated PLD activation and MAP kinase phosphorylation were abolished by removal of extracellular Ca2+. The PLD activity determined under conditions for the assay of GTP gamma S-dependent PLD was stimulated by 3.5-fold with GTP gamma S, and by 4.2-fold with ADP-ribosylation factor-i/GTP gamma S. Although 1 mM oleic acid increased PLD activity by 2.5-fold, the activity of GTP gamma S-dependent PLD was much higher than that of oleate-dependent one in membranes from human aortic smooth muscle cells. These observations lead us to speculate that 1) H2O2 induces PLD and MAP kinase activation in extracellular Ca2+-dependent and PKC-independent manner, 2) the activation of MAP kinases and extracellular Ca2+ are involved in H2O2-induced PLD activation, 3) oleate-dependent PLD is stimulated by H2O2 in human aortic smooth muscle cells.