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

Abstract

NADPH oxidases are major sources of superoxide (O2∸) 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 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 O2∸ 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 O2∸ and H2O2 in intact RASMCs was analyzed by dihydroethidium and Amplex Red assay. Activity of NADPH oxidases was measured by NADPH-dependent O2∸ and H2O2 production using electron spin resonance (ESR) and 1-hydroxy-3-carboxy-pyrrolidine (CPH) in the membrane fraction in the absence of cytosolic superoxide dismutase. It was found that production of O2∸ by quiescent RASMC NADPH oxidases was five times less than H2O2 production. Stimulation of cells with AngII led to a 2-fold increase of O2∸ production by NADPH oxidases, with a small 15 to 30% increase in H2O2 formation. Depletion of Nox4 in RASMC led to diminished basal H2O2 production, but did not affect O2∸ or H2O2 production stimulated by AngII. In contrast, depletion of Nox1 in RASMC inhibited production of O2∸ and AngII-stimulated H2O2 in the membrane fraction and intact cells. Our data suggest that Nox4 produces mainly H2O2, while Nox1 generates mostly O2∸ that is later converted to H2O2. Therefore, Nox4 is responsible for basal H2O2 production, while O2∸ production in non-stimulated 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 O2∸ scavengers that do not affect H2O2.

Binding of EBP50 to Nox organizing subunit p47 phox is pivotal to cellular reactive species generation and altered vascular phenotype

“…To assess the role of scaffolding protein EBP50 in Nox1 activation, O 2 •-production was investigated in aortic VSMC from EBP50 KO vs. WT mice. It is well established that 100 nM AngII treatment significantly increases O 2 •-levels, and this increase is Nox1-derived (20)(21)(22)(23)(24)(25)(26). We corroborated these findings using concentration and time response experiments and by showing that siRNA against Nox1 abolished the signal (Fig.…”

NADPH oxidase-4 mediates protection against chronic load-induced stress in mouse hearts by enhancing angiogenesis

“…Second, several recent independent reports indicate that Nox4 generates predominantly H 2 O 2 (as further confirmed in the current study) whereas Nox2 primarily generates O 2 − (refs. [17][18][19][20]. These differences in regulation, activation mechanism, subcellular location, and ROS generation translate into isoform-specific actions in isolated cellular models (9,19,22,33).…”

“…[17][18][19][20]. These differences in regulation, activation mechanism, subcellular location, and ROS generation translate into isoform-specific actions in isolated cellular models (9,19,22,33). In the heart, previous studies in mouse models of defective Nox2 activation or its deletion showed that Nox2 is detrimental during remodeling, causing increased hypertrophy, apoptosis, and contractile dysfunction (10)(11)(12)(13)(14)(15).…”

“…Nox4 differs from Nox2 and other Nox enzymes in that it is regulated mainly by its expression level and does not require agonist stimulation or association with regulatory subunits for activation (16)(17)(18). Recent studies also suggest that it generates predominantly H 2 O 2 rather than O 2 − (17)(18)(19)(20). Previous work showed that Nox4 is expressed at a low level in the adult mammalian heart and that its abundance increases during pressure overload (11), but its pathophysiological functions in vivo are unknown.…”

Extracellular Superoxide Dismutase in Macrophages Augments Bacterial Killing by Promoting Phagocytosis

“…Identification of ROS production (superoxide, peroxynitrite, and other downstream single-electron reactive-oxygen intermediates) was accomplished by exposing the stimulated cells to either 50 mol/L cellpermeable CMH (1-hydroxy-3-methoxy-carbonyl-2,2,5,5-tetramethylpyrrolidine) or 50 mol/L cell-impermeable PPH (1-hydroxy-4-phosphono-oxy-2,2,6,6-tetramethylpiperidine) spin probes (Noxygen Science Transfer & Diagnostics GmbH, Elzach, Germany) and analyzed using a table-top EPR spectrometer (eScan; Bruker BioSpin, Billerica, MA). [31][32][33] Various amounts (in micromoles per liter) of CM radical were measured to generate a standard curve to quantify the amount of oxidants produced by wild-type and EC-SOD KO cells. EPR settings were as follows: field sweep, 50 G; microwave frequency, 9.78 GHz; microwave power 20 mW, modulation amplitude, 2 G; conversion time, 327 ms; time constant, 655 ms; and receiver gain, 1 ϫ 10 5 .…”