Arsenite stimulates plasma membrane NADPH oxidase in vascular endothelial cells.
Low-level arsenite treatment of porcine aortic endothelial cells (PAEC) stimulated 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 microM) stimulated a twofold increase in activity by 1 h, which was inhibited by the oxidase inhibitor 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 treated cells. In contrast, cytosolic Rac1 translocated to the membrane fractions of cells treated with arsenite or angiotensin II but not with tumor necrosis factor. Immunodepletion of p67(phox) blocked oxidase activity stimulated by all three compounds. However, depleting Rac1 inhibited responses only to arsenite and angiotensin II. These data demonstrate that stimulus-specific activation of NADPH oxidase in endothelial cells was the source of reactive oxygen in endothelial cells after noncytotoxic arsenite exposure.
Smith
K R
KR
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Klei
L R
LR
Barchowsky
A
A
D000818:Animals; D001011:Aorta / Q000166:cytology / Q000201:enzymology; D018053:Arsenites / Q000494:pharmacology*; D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D004789:Enzyme Activation; D019255:NADPH Oxidases / Q000378:metabolism*; D013552:Swine; D013997:Time Factors; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
D018053:Arsenites; D019255:NADPH Oxidases; D020830:rac1 GTP-Binding Protein; C015001:arsenite