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The role of external and matrix pH in mitochondrial reactive oxygen species generation.

Selivanov VA, Zeak JA, Roca J, Cascante M, Trucco M, Votyakova TV - J. Biol. Chem. (2008)

Bottom Line: Matrix pH was manipulated by inorganic phosphate, nigericine, and low concentrations of uncoupler or valinomycin.In the absence of inorganic phosphate, when the matrix was the most alkaline, pH shift in the medium above 7 induced permeability transition accompanied by the decrease of ROS production.The phenomena revealed in this report are important for understanding mechanisms governing mitochondrial production of reactive oxygen species, in particular that related with uncoupling proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Associated Unit to Consejo Superior de Investigaciones Científicas, Institute of Biomedicine of the University of Barcelona, Barcelona, Spain.

ABSTRACT
Reactive oxygen species (ROS) generation in mitochondria as a side product of electron and proton transport through the inner membrane is important for normal cell operation as well as development of pathology. Matrix and cytosol alkalization stabilizes semiquinone radical, a potential superoxide producer, and we hypothesized that proton deficiency under the excess of electron donors enhances reactive oxygen species generation. We tested this hypothesis by measuring pH dependence of reactive oxygen species released by mitochondria. The experiments were performed in the media with pH varying from 6 to 8 in the presence of complex II substrate succinate or under more physiological conditions with complex I substrates glutamate and malate. Matrix pH was manipulated by inorganic phosphate, nigericine, and low concentrations of uncoupler or valinomycin. We found that high pH strongly increased the rate of free radical generation in all of the conditions studied, even when DeltapH=0 in the presence of nigericin. In the absence of inorganic phosphate, when the matrix was the most alkaline, pH shift in the medium above 7 induced permeability transition accompanied by the decrease of ROS production. ROS production increase induced by the alkalization of medium was observed with intact respiring mitochondria as well as in the presence of complex I inhibitor rotenone, which enhanced reactive oxygen species release. The phenomena revealed in this report are important for understanding mechanisms governing mitochondrial production of reactive oxygen species, in particular that related with uncoupling proteins.

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Ionophores valinomycin and FCCP have opposite effect on the rate of ROS release by brain mitochondria. A, recordings of membrane potential; B, recordings of ROS release. Basic medium without Pi (pH 6) was used. At the time points indicated by arrows, the following additions were made: mitochondria (Mt) (all traces), 20 pm valinomycin (traces 1A and 1B), and 2.5 nm FCCP (traces 2A and 2B). Concentration of K+ ions in the medium was 125 mm. To achieve a complete depolarization, the final addition of FCCP at 7 min was 200 nm.
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fig6: Ionophores valinomycin and FCCP have opposite effect on the rate of ROS release by brain mitochondria. A, recordings of membrane potential; B, recordings of ROS release. Basic medium without Pi (pH 6) was used. At the time points indicated by arrows, the following additions were made: mitochondria (Mt) (all traces), 20 pm valinomycin (traces 1A and 1B), and 2.5 nm FCCP (traces 2A and 2B). Concentration of K+ ions in the medium was 125 mm. To achieve a complete depolarization, the final addition of FCCP at 7 min was 200 nm.

Mentions: The difference in ROS production between the three conditions presented in Fig. 3 resulted, evidently, from the difference in corresponding matrix pH. In addition to the use of Pi and nigericin, application of the ionophores FCCP and valinomycin can be instrumental in manipulating matrix pH to study its role in mitochondrial ROS generation. Both of these ionophores can decrease membrane potential; however, protonophore FCCP acidifies the matrix by increasing proton leak, whereas valinomycin causes increase of matrix pH by carrying K+ into the matrix, decreasing ΔΨ and, thus, facilitating the proton ejection by the electron transport chain. To stress the qualitatively different effects of these ionophores, we applied a small concentration of FCCP that did not substantially change ΔΨ and a concentration of valinomycin that essentially depolarized mitochondria (Fig. 6A). As Fig. 6B shows, FCCP induced a dramatic decrease of ROS production, wheras valinomycin temporarily increased it.


The role of external and matrix pH in mitochondrial reactive oxygen species generation.

Selivanov VA, Zeak JA, Roca J, Cascante M, Trucco M, Votyakova TV - J. Biol. Chem. (2008)

Ionophores valinomycin and FCCP have opposite effect on the rate of ROS release by brain mitochondria. A, recordings of membrane potential; B, recordings of ROS release. Basic medium without Pi (pH 6) was used. At the time points indicated by arrows, the following additions were made: mitochondria (Mt) (all traces), 20 pm valinomycin (traces 1A and 1B), and 2.5 nm FCCP (traces 2A and 2B). Concentration of K+ ions in the medium was 125 mm. To achieve a complete depolarization, the final addition of FCCP at 7 min was 200 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2570889&req=5

fig6: Ionophores valinomycin and FCCP have opposite effect on the rate of ROS release by brain mitochondria. A, recordings of membrane potential; B, recordings of ROS release. Basic medium without Pi (pH 6) was used. At the time points indicated by arrows, the following additions were made: mitochondria (Mt) (all traces), 20 pm valinomycin (traces 1A and 1B), and 2.5 nm FCCP (traces 2A and 2B). Concentration of K+ ions in the medium was 125 mm. To achieve a complete depolarization, the final addition of FCCP at 7 min was 200 nm.
Mentions: The difference in ROS production between the three conditions presented in Fig. 3 resulted, evidently, from the difference in corresponding matrix pH. In addition to the use of Pi and nigericin, application of the ionophores FCCP and valinomycin can be instrumental in manipulating matrix pH to study its role in mitochondrial ROS generation. Both of these ionophores can decrease membrane potential; however, protonophore FCCP acidifies the matrix by increasing proton leak, whereas valinomycin causes increase of matrix pH by carrying K+ into the matrix, decreasing ΔΨ and, thus, facilitating the proton ejection by the electron transport chain. To stress the qualitatively different effects of these ionophores, we applied a small concentration of FCCP that did not substantially change ΔΨ and a concentration of valinomycin that essentially depolarized mitochondria (Fig. 6A). As Fig. 6B shows, FCCP induced a dramatic decrease of ROS production, wheras valinomycin temporarily increased it.

Bottom Line: Matrix pH was manipulated by inorganic phosphate, nigericine, and low concentrations of uncoupler or valinomycin.In the absence of inorganic phosphate, when the matrix was the most alkaline, pH shift in the medium above 7 induced permeability transition accompanied by the decrease of ROS production.The phenomena revealed in this report are important for understanding mechanisms governing mitochondrial production of reactive oxygen species, in particular that related with uncoupling proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Associated Unit to Consejo Superior de Investigaciones Científicas, Institute of Biomedicine of the University of Barcelona, Barcelona, Spain.

ABSTRACT
Reactive oxygen species (ROS) generation in mitochondria as a side product of electron and proton transport through the inner membrane is important for normal cell operation as well as development of pathology. Matrix and cytosol alkalization stabilizes semiquinone radical, a potential superoxide producer, and we hypothesized that proton deficiency under the excess of electron donors enhances reactive oxygen species generation. We tested this hypothesis by measuring pH dependence of reactive oxygen species released by mitochondria. The experiments were performed in the media with pH varying from 6 to 8 in the presence of complex II substrate succinate or under more physiological conditions with complex I substrates glutamate and malate. Matrix pH was manipulated by inorganic phosphate, nigericine, and low concentrations of uncoupler or valinomycin. We found that high pH strongly increased the rate of free radical generation in all of the conditions studied, even when DeltapH=0 in the presence of nigericin. In the absence of inorganic phosphate, when the matrix was the most alkaline, pH shift in the medium above 7 induced permeability transition accompanied by the decrease of ROS production. ROS production increase induced by the alkalization of medium was observed with intact respiring mitochondria as well as in the presence of complex I inhibitor rotenone, which enhanced reactive oxygen species release. The phenomena revealed in this report are important for understanding mechanisms governing mitochondrial production of reactive oxygen species, in particular that related with uncoupling proteins.

Show MeSH
Related in: MedlinePlus