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(center dot-)) and hydrogen peroxide (H2O2) 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 hemecontaining 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(center dot-) and H2O2, 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(center dot-) and H2O2 in intact RASMCs was analyzed by dihydroethidium and Amplex Red assay. Activity of NADPH oxidases was Measured by NADPH-dependent O-2(center dot-) and H2O2 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(center dot-) by quiescent RASMC NADPH oxidases was five times less than H2O2 Production. Stimulation of cells with AngII led to a 2-fold increase of O-2(center dot-) production by NADPH oxidases, with a small 15 to 30% increase in H2O2 formation. Depletion of Nox4 in RASMCs led to diminished basal H2O2 Production, but did not affect O-2(center dot-) or H2O2 production stimulated by AngII. In contrast, depletion of Nox1 in RASMCs inhibited production of T and AngII-stimulated H2O2 in the membrane fraction and intact Our data Suggest that Nox4 produces mainly H2O2, while Nox1 generates mostly O-2(center dot-) that is later Cells. converted to H2O2, Therefore, Nox4 is responsible for basal H2O2 production, while O-2(center dot-) 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 H2O2 in vascular cells, and may partially account for the limited pharmacological effect of antioxidant treatments with O-2(center dot-) scavengers that do not affect H2O2. (c) 2008 Elsevier Inc. All rights reserved.