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. 2011 Feb 15;14(4):533-42.
doi: 10.1089/ars.2010.3213. Epub 2010 Oct 19.

NADPH oxidase 2 mediates intermittent hypoxia-induced mitochondrial complex I inhibition: relevance to blood pressure changes in rats

Shakil A Khan  1 , Jayasri Nanduri, Guoxiang Yuan, Brian Kinsman, Ganesh K Kumar, Joy Joseph, Balaraman Kalyanaraman, Nanduri R Prabhakar
Affiliations

NADPH oxidase 2 mediates intermittent hypoxia-induced mitochondrial complex I inhibition: relevance to blood pressure changes in rats

Shakil A Khan et al. Antioxid Redox Signal. .

Abstract

Previous studies identified NADPH oxidases (Nox) and mitochondrial electron transport chain at complex I as major cellular sources of reactive oxygen species (ROS) mediating systemic and cellular responses to intermittent hypoxia (IH). In the present study, we investigated potential interactions between Nox and the mitochondrial complex I and assessed the contribution of mitochondrial ROS in IH-evoked elevation in blood pressure. IH treatment led to stimulus-dependent activation of Nox and inhibition of complex I activity in rat pheochromocytoma (PC)12 cells. After re-oxygenation, Nox activity returned to baseline values within 3 h, whereas the complex I activity remained downregulated even after 24 h. IH-induced complex I inhibition was prevented by Nox inhibitors, Nox2 but not Nox 4 siRNA, in cell cultures and was absent in gp91(phox-/Y) (Nox2 knock-out; KO) mice. Using pharmacological inhibitors, we show that ROS generated by Nox activation mobilizes Ca(2+) flux from the cytosol to mitochondria, leading to S-glutathionylation of 75- and 50-kDa proteins of the complex I and inhibition of complex I activity, which results in elevated mitochondrial ROS. Systemic administration of mito-tempol prevented the sustained but not the acute elevations of blood pressure in IH-treated rats, suggesting that mitochondrial-derived ROS contribute to sustained elevation of blood pressure.

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Figures

FIG. 1.
FIG. 1.
Time course and reversibility of Nox and mitochondrial complex I activities during IH. (A) Nox activity was determined in membrane fractions of PC12 cells treated with increasing durations of IH. Reversibility of the response was determined by placing IH60-treated cells under normoxia for varying periods. (B) Complex I activity was determined in mitochondrial fractions of PC12 cells treated with different durations of IH and after re-oxygenation. Data are mean ± SEM from four independent experiments run in triplicate. **p < 0.01 and *p < 0.05 compared to normoxic controls, respectively. IH60, cells exposed to 60 cycles of intermittent hypoxia; N, normoxia; Nox, NADPH oxidase; n.s., not significant; PC12, pheochromocytoma 12.
FIG. 2.
FIG. 2.
Nox mediates IH-induced complex I inhibition. (A, B) Effects of two structurally distinct Nox inhibitors 500 μM apocynin or 300 μM AEBSF on Nox and complex I activities in IH60-treated PC12 cells. (C) Effect of silencing Nox2 and Nox4 RNAs (siRNAs) on IH60-evoked complex I inhibition. Top panel represents Western blots of Nox2 and Nox4 protein expressions in cells treated with normoxia (lane 1), IH60 (lane 2), IH+scr RNA (lane 3), IH60+Nox2 siRNA (lane 4), and IH60+Nox4 siRNA (lane 5). Bottom panel presents average data (mean ± SEM) of complex I activity. (D, E) Effect of 10 days of IH treatment (IH10D) on Nox and complex I activities in brainstem tissue samples from wild-type and gp91 phox-/Y knock-out mice (n = 5 mice in each group). Data are mean ± SEM from three to five independent experiments. **p < 0.01 compared to normoxic controls. AEBSF, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride; scr, scrambled; Wt, wild type.
FIG. 3.
FIG. 3.
Ca2+ is required for IH-evoked complex 1 inhibition. (A) Effect of Ca2+ chelator BAPTA-AM (10 μM BAPTA) and RR, an inhibitor Ca2+ uniporter (10 μM) on complex I activity in IH60-treated PC12 cells. (B) Effect of ionomycin (1.4 μM), a Ca2+ ionophore on complex I activity in normoxic PC 12 cells with and without RR. **p < 0.01 compared to controls; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; RR, ruthenium red.
FIG. 4.
FIG. 4.
Kinetic analysis of the effects of Ca2+ on complex I activity. (A) Increasing Ca2+ concentrations inhibited complex I activity in control PC 12 cells (o-o) but not in IH exposed cells (▪-▪) (inset: plot of the reciprocal of complex I activity (1/V) versus Ca2+ concentration. Intercept represents the concentration required to produce 50% inhibition of the enzyme, that is, Ki. (B, C) Mitochondrial fractions were assayed for complex 1 activity with increasing NADH concentrations (0–300 μM) in control (N) and IH60-treated cells in the absence (o-o) or presence (▪-▪) of 20 μM Ca2+. Inset represents the Eadie-Hofstee plot of rate (V) versus V/S (S is the NADH concentration). Vmax and Km were calculated from the intercept and slope, respectively. Each data point represents the mean ± SEM from three independent experiments.
FIG. 5.
FIG. 5.
IH increases S-glutathionylation of mitochondrial complex I subunits. (A) Representative immunoblots showing increased S-glutathionylation of 75- and 55-kDa subunits of the complex I in IH60 and ionomycin (Iono; 1.4 μM)-treated PC12 cells and blockade of the responses by RR (10 μM; panels 1 and 2). Blockade of IH60-induced S-glutathionylation of complex I subunits by Nox inhibitor apocynin (Apo; 500 μM) and antioxidant (MnTMPyP; 50 μM; panel 3) and by Nox2 siRNA (panel 4). S-glutathionylation of complex I subunits in brain stem cell lysates from wild-type (Wt) and gp91phox-/Y mice treated with either 10 days of normoxia or IH (panel 5). Note the absence of increased S-glutathionylation of complex I subunits in tissues lysates from IH-treated gp91phox-/Y mice. Blockade of IH60-induced S-glutathionylation of complex I subunits by N-acetyl-cysteine (2 mM NAC), a precursor of glutathione (panel 6). (B) Ratio of oxidized from of glutathione/GSH was determined in normoxic (N) and IH60-treated PC12 cells as described in Materials and Methods. (C) IH-induced inhibition of complex I activity is prevented in the presence of 2 and 5 mM GSH or (D) 2 μM dithiothreitol (DTT). Data represent the mean ± SEM from three to five independent experiments **p < 0.01 compared to normoxic controls (N). GSH, reduced form of glutathione; MnTMPyP, manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin; NAC, N-acetyl-cysteine.
FIG. 6.
FIG. 6.
GSH prevents IH-evoked complex I inhibition by restoring Vmax. Complex I activity was determined as a function of NADH concentration with (▪-▪) and without (o-o) addition of 5 mM GSH in control (N) (A) and IH60-treated PC12 cells (B). Insets represent Eadie-Hofstee plots of rate (V) versus V/S (S is the NADH concentration). Vmax and Km are calculated from the intercept and slope, respectively.
FIG. 7.
FIG. 7.
IH increases mitochondrial ROS. Mitochondrial aconitase activity was determined as index of ROS. (A) Aconitase activity in the mitochondrial fractions from PC12 cells treated with normoxia (N) or to increasing durations of IH as indicated (IH cycles). For reversibility studies, PC12 cells exposed to IH60 were placed in normoxia for indicated duration (h). (B) Effect of apocynin (500 μM), BAPTA (10 μm), RR (10 μm), and MnTMPyP (50 μM), NAC (500 μM) on mitochondrial aconitase activity in IH60-treated PC12 cells. (C) IH-induced inhibition of mitochondrial aconitase activity was absent in PC12 cells transfected with Nox2 siRNA but not with Nox4 siRNA or scr RNAs. Data are mean ± SEM from three to four independent experiments. **p < 0.01 compared to normoxia. ROS, reactive oxygen species.
FIG. 8.
FIG. 8.
Effects of systemic administration of mitochondrial ROS scavenger mito-tempol or apocynin, a Nox inhibitor on blood pressure, mitochondrial aconitase, and NOX activity in rats exposed to 10 days IH. Adult rats were exposed to 10 days of IH and were treated daily with either saline (IH) or mito-tempol (10 mg/kg/day; intraperitoneal) or apocynin (10 mg/kg/day; intraperitoneal). Control experiments were performed on rats exposed to normoxia (N). (A) Mean blood pressure values measured ∼1 and ∼15 h after terminating 10 days of IH with and without mito-tempol or apocynin treatment. (B, C) Treatments with mito-tempol or apocynin prevented IH-induced decrease in mitochondrial aconitase and complex 1 activities. (D) IH-evoked increase in Nox activity was unaffected by mito-tempol but prevented by apocynin treatment. Data presented are mean ± SEM from six rats in each group. **p < 0.01.
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