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

Abstract

We have investigated in whole cells whether, at low oxygen concentrations ([O 2 ]), 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 b H , cc 1 , and aa 3 during cellular respiration. The rate of O 2 consumption (VO 2 ) remained constant until [O 2 ] fell below 15 μM, whereas the onset of reduction of cytochromes aa 3 , part of the terminal ETC enzyme cytochrome c oxidase, occurred at ≈50 μM O 2 . Incubation of the cells with an inhibitor of NO synthase lowered significantly ( P < 0.05) the [O 2 ] 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 [O 2 ] 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% O 2 . However, after 30-min exposure of control cells to 3% O 2 , 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 VO 2 at low [O 2 ]. 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.