Arsenite stimulates plasma membrane NADPH oxidase in vascular endothelial cells

Low-level arsenite treatment of porcine aortic endothelial cells (PAEC) sti mulated superoxide accumulation that was attenuated by inhibitors of NAD(P) H oxidase. To demonstrate whether arsenite stimulated NADPH oxidase, intact PAEC were treated with arsenite for up to 2 h and membrane fractions were prepared to measure NADPH oxidase activity. Arsenite (5 muM) stimulated a t wofold increase in activity by 1 h, which was inhibited by the oxidase inhi bitor diphenyleneiodonium chloride. Direct treatment of isolated membranes with arsenite had no effect. Analysis of NADPH oxidase components revealed that p67(phox) localized exclusively to membranes of both control and treat ed cells. In contrast, cytosolic Rac1 translocated to the membrane fraction s of cells treated with arsenite or angiotensin II but not with tumor necro sis factor. Immunodepletion of p67(phox) blocked oxidase activity stimulate d by all three compounds. However, depleting Rac1 inhibited responses only to arsenite and angiotensin II. These data demonstrate that stimulus-specif ic activation of NADPH oxidase in endothelial cells was the source of react ive oxygen in endothelial cells after noncytotoxic arsenite exposure.