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

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 cytochromesb H ,cc 1 , andaa 3 during cellular respiration. The rate of O2 consumption (VO2 ) remained constant until [O2 ] fell below 15 μM, whereas the onset of reduction of cytochromesaa 3 , part of the terminal ETC enzyme cytochromec 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.