Distinct roles of Nox1 and Nox4 in basal and angiotensin II-stimulated superoxide and hydrogen peroxide production.

NADPH oxidases are major sources of superoxide (O(2)(-)) and hydrogen peroxide (H(2)O(2)) in vascular cells. Production of these reactive oxygen species (ROS) is essential for cell proliferation and differentiation, while ROS overproduction has been implicated in hypertension and atherosclerosis. It is known that the heme-containing catalytic subunits Nox1 and Nox4 are responsible for oxygen reduction in vascular smooth muscle cells from large arteries. However, the exact mechanism of ROS production by NADPH oxidases is not completely understood. We hypothesized that Nox1 and Nox4 play distinct roles in basal and angiotensin II (AngII)-stimulated production of O(2)(-) and H(2)O(2). Nox1 and Nox4 expression in rat aortic smooth muscle cells (RASMCs) was selectively reduced by treatment with siNox4 or antisense Nox1 adenovirus. Production of O(2)(-) and H(2)O(2) in intact RASMCs was analyzed by dihydroethidium and Amplex Red assay. Activity of NADPH oxidases was measured by NADPH-dependent O(2)(-) and H(2)O(2) production using electron spin resonance (ESR) and 1-hydroxy-3-carboxypyrrolidine (CPH) in the membrane fraction in the absence of cytosolic superoxide dismutase. It was found that production of O(2)(-) by quiescent RASMC NADPH oxidases was five times less than H(2)O(2) production. Stimulation of cells with AngII led to a 2-fold increase of O(2)(-) production by NADPH oxidases, with a small 15 to 30% increase in H(2)O(2) formation. Depletion of Nox4 in RASMCs led to diminished basal H(2)O(2) production, but did not affect O(2)(-) or H(2)O(2) production stimulated by AngII. In contrast, depletion of Nox1 in RASMCs inhibited production of O(2)(-) and AngII-stimulated H(2)O(2) in the membrane fraction and intact cells. Our data suggest that Nox4 produces mainly H(2)O(2), while Nox1 generates mostly O(2)(-) that is later converted to H(2)O(2.) Therefore, Nox4 is responsible for basal H(2)O(2) production, while O(2)(-) production in nonstimulated and AngII-stimulated cells depends on Nox1. The difference in the products generated by Nox1 and Nox4 may help to explain the distinct roles of these NADPH oxidases in cell signaling. These findings also provide important insight into the origin of H(2)O(2) in vascular cells, and may partially account for the limited pharmacological effect of antioxidant treatments with O(2)(-) scavengers that do not affect H(2)O(2).