H2O2-induced O⨪2Production by a Non-phagocytic NAD(P)H Oxidase Causes Oxidant Injury

Abstract

Non-phagocytic NAD(P)H oxidases have been impliinduced stimulation of NAD(P)H oxidase activity in nonphagocytic cell types could represent a mechanism by which oxidant-mediated injury is amplified in vivo and identify these NAD(P)H oxidase enzymes as therapeutic targets for the amelioration of the biological effects of chronic oxidant stress. EXPERIMENTAL PROCEDURESCell Culture-SMC were prepared from 250 to 300-g male HarlanSprague Dawley rats as previously described (10, 11). Aortic adventitial fibroblasts were prepared from C57 BL/6 and from gp91 phox knock-out mice (Jackson Lab, Bar Harbor, ME) as described (12, 13). The cells were grown in minimum essential media supplemented with 10% fetal bovine serum and penicillin (100 IU/ml) and streptomycin (100 g/ml) in a humidified atmosphere containing 5% CO 2 at 37°C. Stocks were subcultured at subconfluence by trypsinization. All experiments were performed on cells between passages 8 and 20 grown to 70% to 95% confluence in 24-well plates, 35-or 60-mm dishes, or 4-well chamber slides.Superoxide Measurement by Enhanced Chemiluminescence-Lucigenin luminescence measurements were carried out using 5 M lucigenin, a concentration which is unlikely to induce redox cycling (14). Cells were grown to subconfluence in 35-mm dishes and growth arrested in serum-free medium for 24 h. After exposure to H 2 O 2 at the indicated concentrations and times, cells were washed twice with PBS, and 5 M lucigenin was added. Immediately afterward, NADPH (0.1 mM) was added, and after dark adaptation, luminescence was measured every 15 s for 5 min in a luminometer (Zylux Corp). Changes in luminescence were expressed as relative luminescent units per 10 5 cells per minute. No changes in luminescence were observed in the absence of NADPH, and addition of H 2 O 2 to lucigenin in PBS in the absence of cells did not increase chemiluminescence. Some experiments were also performed with coelenterazine (5 M), a structurally distinct luminescent probe which reportedly is not capable of redox cycling via autoxidation (15). Data were analyzed by ANOVA followed by Bonferroni t testing.Intracellular O 2 . Determination by Confocal Microscopy-SMC grown on chamber slides were treated as indicated under "Results" and in the figure legends, washed, and incubated with dihydroethidium (HE, 5

Mechanisms of H2O2-induced oxidative stress in endothelial cells

“…- (Fang et al 1998;Li et al 2000;Li et al 2001;Landmesser et al 2002;Li et al 2003). However, in this study, the presence or absence of fluorescence in individual cells was determined as a binary endpoint.…”

“…Recent studies have documented various effects of H 2 O 2 in the vascular milieu (Griendling et al 2000a;Li et al 2001;Landmesser et al 2002;Cai et al 2003b), including smooth muscle cell and endothelial cell migration, and activation of inflammatory signaling mechanisms such as NF-κB (Wolin et al 2002;Cai et al 2003b;Landmesser et al 2003;Davis et al 2004). H 2 O 2 has also been found to stimulate increased eNOS expression, eNOS activity, and nitric oxide production in endothelial cells (Drummond et al 2000;Thomas et al 2002;Cai et al 2003b).…”

Regulation of normal cell cycle progression by flavin-containing oxidases

“…Wild-type, gp91 phoxÀ/À (Li et al, 2001), p21 Waf1-/- (Deng et al, 1995) and p53 À/À MEFs (established in our laboratory) were derived from the C57BL/6 strain, and cultured as described (Azzam et al, 1996.…”

“…Similar to normal human fibroblasts (Figure 1b The DPI-induced G 1 checkpoint is mediated through NAD(P)H oxidase and not nitric oxide synthase Depending on cellular context and drug concentration, DPI inhibits several flavoprotein oxidoreductases, including NAD(P)H oxidase (Cross and Jones, 1986) and nitric oxide synthase (Stuehr et al, 1991). To examine the role of NAD(P)H oxidase in DPI modulation of G 1 to S transition, progression into S phase was investigated in DPI-treated wt and isogenic MEFs deficient in gp91 phox , the plasma membrane catalytic subunit of NAD(P)H oxidase in fibroblasts (Li et al, 2001). In contrast to wt cells (Figure 6a), isogenic gp91 phoxÀ/À MEFs were unaffected by DPI treatment: progression of cells pretreated with 0.05 or 0.15 mM DPI into S was similar to that of sham-treated cells (Figure 6d).…”

Effect of inhibitors of NADPH oxidase complex and mitochondrial ATP‐sensitive potassium channels on generation of dopaminergic neurons from neurospheres of mesencephalic precursors

“…ROS open up KATP channels (Zhang et al, 2002;Avshalumov and Rice, 2003), and mitochondrial ROS production can be stimulated by the opening of mitochondrial mitoKATP channels (Costa and Garlid, 2008;Kimura et al, 2005b). Recent studies have shown a ROSmediated relationship between the NADPH oxidase complex and the mitoKATP channels (Kimura et al, 2005a;Zhang et al, 2005;Sheh et al, 2007;Alberici et al, 2009) and that events leading to mitochondrial dysfunction or increased cytoplasmatic ROS have a significant upstream impact on increasing NADPH activity (Li et al, 2001;Wosniak et al, 2009). This suggests cross-talk signaling between both systems (reviewed by Daiber, 2010).…”