Hollis 2003 Biochim Biophys Acta

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Hollis VS, Palacios-Callender M, Springett RJ, Delpy DT, Moncada S (2003) Monitoring cytochrome redox changes in the mitochondria of intact cells using multi-wavelength visible light spectroscopy. Biochim Biophys Acta 1607:191-202.


Hollis VS, Palacios-Callender M, Springett RJ, Delpy DT, Moncada S (2003) Biochim Biophys Acta

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 microM, whereas the onset of reduction of cytochromes aa(3), part of the terminal ETC enzyme cytochrome c oxidase, occurred at approximately 50 microM 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 (O(2)(-)) production at different [O(2)] and found there was no increase in O(2)(-) 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 O(2)(-) generation was observed, accompanied by translocation to the nucleus of the transcription factor NF-kappa B. 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 O(2)(-), which initiates the transcriptional activation of NF-kappa B as an early signaling stress response.

Labels: MiParea: Respiration, Instruments;methods 

Stress:Oxidative stress;RONS  Organism: Human  Tissue;cell: Blood cells  Preparation: Intact cells 

Regulation: Cyt c, Flux control, Inhibitor, Oxygen kinetics, Redox state  Coupling state: ROUTINE