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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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Acidic pH inhibits rotenone-induced ROS generation by RBM in the presence of glutamate and malate. Basic incubation medium supplemented with 1 mm Pi was used; concentration of rotenone was 1 μm. Statistical analysis was as follows. Data sets taken at pH 6.0, 6.5, and 7.0 were significantly different from that at pH 7.5 and 8.0 (n = 5–8; **, p < 0.01; ***, p < 0.001, t test).
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fig8: Acidic pH inhibits rotenone-induced ROS generation by RBM in the presence of glutamate and malate. Basic incubation medium supplemented with 1 mm Pi was used; concentration of rotenone was 1 μm. Statistical analysis was as follows. Data sets taken at pH 6.0, 6.5, and 7.0 were significantly different from that at pH 7.5 and 8.0 (n = 5–8; **, p < 0.01; ***, p < 0.001, t test).

Mentions: Blocking complex I with an inhibitor rotenone completely depolarized mitochondria (Fig. 7B) and dramatically stimulated ROS generation (11, 29). Fig. 8 shows that upon application of rotenone, regardless of depolarization, the rate of ROS generation increased with the increase of medium pH. Thus, all presented data show the same pattern of ROS production increase in conjunction with the alkalization of medium.


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)

Acidic pH inhibits rotenone-induced ROS generation by RBM in the presence of glutamate and malate. Basic incubation medium supplemented with 1 mm Pi was used; concentration of rotenone was 1 μm. Statistical analysis was as follows. Data sets taken at pH 6.0, 6.5, and 7.0 were significantly different from that at pH 7.5 and 8.0 (n = 5–8; **, p < 0.01; ***, p < 0.001, t test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig8: Acidic pH inhibits rotenone-induced ROS generation by RBM in the presence of glutamate and malate. Basic incubation medium supplemented with 1 mm Pi was used; concentration of rotenone was 1 μm. Statistical analysis was as follows. Data sets taken at pH 6.0, 6.5, and 7.0 were significantly different from that at pH 7.5 and 8.0 (n = 5–8; **, p < 0.01; ***, p < 0.001, t test).
Mentions: Blocking complex I with an inhibitor rotenone completely depolarized mitochondria (Fig. 7B) and dramatically stimulated ROS generation (11, 29). Fig. 8 shows that upon application of rotenone, regardless of depolarization, the rate of ROS generation increased with the increase of medium pH. Thus, all presented data show the same pattern of ROS production increase in conjunction with the alkalization of medium.

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