Endogenous NO regulates superoxide production at low oxygen concentrations by modifying the redox state of cytochrome c oxidase

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National Academy of Sciences

RESUMO

We have investigated in whole cells whether, at low oxygen concentrations ([O2]), endogenous nitric oxide (NO) modulates the redox state of the mitochondrial electron transport chain (ETC), and whether such an action has any signaling consequences. Using a polarographic-and-spectroscopic-coupled system, we monitored redox changes in the ETC cytochromes bH, cc1, and aa3 during cellular respiration. The rate of O2 consumption (VO2) remained constant until [O2] fell below 15 μM, whereas the onset of reduction of cytochromes aa3, part of the terminal ETC enzyme cytochrome c oxidase, occurred at ≈50 μM O2. Incubation of the cells with an inhibitor of NO synthase lowered significantly (P < 0.05) the [O2] at which reduction of the cytochromes occurred. We also measured intracellular superoxide (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathbf{O_{2}^{-}}}\end{equation*}\end{document}) production at different [O2] and found there was no increase in \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathbf{O_{2}^{-}}}\end{equation*}\end{document} generation in control cells, or those treated with the NO synthase inhibitor, when incubated at 21% O2. However, after 30-min exposure of control cells to 3% O2, an increase in \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathbf{O_{2}^{-}}}\end{equation*}\end{document} generation was observed, accompanied by translocation to the nucleus of the transcription factor NF-κB. Both of these responses were diminished by NO synthase inhibition. Our results suggest that endogenous NO, by enhancing the reduction of ETC cytochromes, contributes to a mechanism by which cells maintain their VO2 at low [O2]. This, in turn, favors the release of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathbf{O_{2}^{-}}}\end{equation*}\end{document}, which initiates the transcriptional activation of NF-κB as an early signaling stress response.

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