Distinct roles of Nox1 and Nox4 in basal and angiotensin II-stimulated superoxide and hydrogen peroxide production - PubMed (original) (raw)

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

Sergey I Dikalov et al. Free Radic Biol Med. 2008.

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-carboxypyrrolidine (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 RASMCs led to diminished basal H2O2 production, but did not affect O2*- or H2O2 production stimulated by AngII. In contrast, depletion of Nox1 in RASMCs 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 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 O2*- scavengers that do not affect H2O2.

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Figures

Figure 1

Figure 1

Quantitative measurements of O2∸ (A) and H2O2 (B) in the xanthine oxidase system containing 0.1 mM xanthine and 2 mU/ml xanthine oxidase. Superoxide formation was assayed as SOD-inhibitable formation of 3-carboxy-proxyl (CP), while H2O2 was detected by peroxidase-acetamidophenol-mediated co-oxidation of CPH to CP-nitroxide, which was inhibited by catalase (20 µg/ml).

Figure 2

Figure 2

Analysis of NADPH oxidase activity by measurement of O2∸ and H2O2 production in the membrane fraction. (A) Accumulation of 3-carboxyproxyl (CP) in the membrane fraction of RASMCs (0.1mg/ml) followed by an increase in the low-field component of EPR spectrum (insert). The rates of O2∸ and H2O2 production were 184 pmol/mg/min and 658 pmol/mg/min, correspondingly. The O2∸ independent H2O2 production was 474 pmol/mg/min; (B) Detection of O2∸ in the membrane preparation of RASMCs (0.2 mg/ml) using CMH or CPH. Addition of cytosol inhibited O2∸ detection similar to SOD-supplementation; (C) Measurement of O2∸ production by NADPH oxidase in homogenate and membrane fraction of RASMCs (0.1mg/ml) at various concentrations of CPH; (D) Detection of O2∸ in the homogenate of RASMCs (0.2mg/ml) using CMH, CPH and CPH + SOD-inhibitor DETC.

Figure 3

Figure 3

Stimulation of NADPH oxidase activity in quiescent (Q) or proliferating (Prolif) RASMCs treated with AngII (100 nM) for 4-hours. Activity of NADPH oxidases was analyzed by O2∸ (A) and H2O2 (B) production measured in the membrane fraction using ESR spectroscopy as described in the Methods section. Data are from six to eight separate experiments (*P<0.01 Ang II vs unstimulated, **P<0.05 Proliferating vs Quiescent).

Figure 4

Figure 4

Expression of Nox1 and Nox4 catalytic subunits in AngII-stimulated (100 nM, 4-hours) quiescent (Q) or proliferating (Prolif) RASMCs measured by real-time PCR (A, B) and Western Blot analysis (C, D). Real-time PCR data are from five separate experiments. Western Blot shows typical expression of Nox1 and Nox4 in quiescent (Q) or proliferating (Prolif) RASMC.

Figure 5

Figure 5

Expression of Nox4 in siRNA treated RASMCs. RASMCs were treated with scrambled (Scr) or Nox4 siRNA (sinox4) for five days and then stimulated with 100 nM AngII for 4-hours. Expression of Nox4 was measured by real-time PCR (A) and Western Blot analysis (B). Data are average from five separate experiments ± Standard Error. Western Blot shows typical Western blot of Nox4 expression in scrambled or Nox4 siRNA treated RASMCs.

Figure 6

Figure 6

Analysis of O2∸ and H2O2 in Nox4-depleted RASMCs. (A) Production of intracellular O2∸ was measured by DHE/HPLC following accumulation of 2-hydroxyethidium using HPLC as described in Materials and Methods. RASMCs treated with scrambled (Scr) or Nox4 siRNA (sinox4) were stimulated with 100 nM AngII for 4-hours; (B) Cellular H2O2 was measured using the fluorescent probe Amplex Red and normalized by cellular protein. RASMCs were stimulated with AngII and incubated with Amplex Red for 2-hours. Accumulation of the fluorescent signal was blocked by supplementation with catalase (20 µg/ml) (not shown). (C) ESR measurements of NADPH oxidase activity in membrane preparations of siRNA-treated RASMC by analysis of NADPH-dependent O2∸ production; (D) Measurements of NADPH oxidase activity in membrane preparations of siRNA-treated RASMC by analysis of NADPH-dependent H2O2 production using ESR spectroscopy as described in Methods. Data are from 4 to 6 separate experiments (*P<0.05 AngII vs non-stimulated).

Figure 7

Figure 7

Analysis of NADPH oxidase activity in Nox1-depleted RASMCs. (A) Production of intracellular O2∸ measured by accumulation of 2-hydroxyethidium using DHE-HPLC as described in Methods. RASMCs treated with vector or AdASNox1 were stimulated with 100 nM AngII for 4-hours; (B) Cellular H2O2 was measured using the fluorescent probe Amplex Red and normalized by cellular protein. RASMCs were stimulated with 100 nM AngII and incubated with Amplex Red for 4-hours. Accumulation of fluorescent signal was blocked by supplementation with catalase (20 µg/ml). (C) NADPH oxidase activity measured by O2∸ production in the membrane fraction of RASMCs. (D) (D) NADPH-dependent H2O2 production in membrane preparations of RASMCs using ESR spectroscopy as described in Methods. (*P<0.05 vs AngII; **P<0.05 vs Vec+AngII).

Figure 8

Figure 8

Potential molecular mechanisms of O2∸ and H2O2 production by Nox1 and Nox4. One-electron reduction of oxygen produces a complex of ferric heme and superoxide. The complex of ferric Nox1—O2∸ may rapidly dissociate releasing free O2∸ molecule. Meanwhile, the complex of ferric Nox4—O2∸ may be stable enough to transfer the second electron from the flavin to the heme and then to O2∸, producing H2O2.

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