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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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pH dependence of mitochondrial respiration in state 3 (triangles), state 4 (squares), and state 4 with added oligomycin (inverse triangles), with succinate (A) and glutamate and malate (B) as substrates. Basic incubation medium (see “Experimental Procedures”) supplemented with 1 mm Pi was used. Statistical analysis was as follows. A, rates of respiration in state 3 taken at pH 8 were significantly different from that at pH 6–7 (n = 3–4; *, p < 0.05, t test). B, rates of respiration in state 3 taken at pH 8 were significantly different from that at pH 6–7.5; rates at pH 7 and 7.5 were significantly different from each other (n = 6; *, p < 0.05; **, p < 0.001, t test).
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fig1: pH dependence of mitochondrial respiration in state 3 (triangles), state 4 (squares), and state 4 with added oligomycin (inverse triangles), with succinate (A) and glutamate and malate (B) as substrates. Basic incubation medium (see “Experimental Procedures”) supplemented with 1 mm Pi was used. Statistical analysis was as follows. A, rates of respiration in state 3 taken at pH 8 were significantly different from that at pH 6–7 (n = 3–4; *, p < 0.05, t test). B, rates of respiration in state 3 taken at pH 8 were significantly different from that at pH 6–7.5; rates at pH 7 and 7.5 were significantly different from each other (n = 6; *, p < 0.05; **, p < 0.001, t test).

Mentions: Rat brain mitochondria respiratory characteristics measured in condition 2 and pH variations from 6 to 8 with succinate or glutamate plus malate as substrates are presented in Fig. 1. These data show that our mitochondrial preparations are good in coupling between respiration and phosphorylation in physiological range of pH. At pH 7, the addition of oligomycin inhibits respiration, whereas the addition of ADP (state 3 (St 3)) essentially stimulates it, so that the ratio of respiration rates (state 3/state oligomycin (St 3/St Oligo)) was 5.6 ± 0.6 (n = 3) for succinate and 6.5 ± 0.3 (n = 6) for glutamate plus malate. At alkaline pH, ADP stimulated respiration to a smaller degree, possibly because the lack of protons in the medium limits the process.


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)

pH dependence of mitochondrial respiration in state 3 (triangles), state 4 (squares), and state 4 with added oligomycin (inverse triangles), with succinate (A) and glutamate and malate (B) as substrates. Basic incubation medium (see “Experimental Procedures”) supplemented with 1 mm Pi was used. Statistical analysis was as follows. A, rates of respiration in state 3 taken at pH 8 were significantly different from that at pH 6–7 (n = 3–4; *, p < 0.05, t test). B, rates of respiration in state 3 taken at pH 8 were significantly different from that at pH 6–7.5; rates at pH 7 and 7.5 were significantly different from each other (n = 6; *, p < 0.05; **, p < 0.001, t test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: pH dependence of mitochondrial respiration in state 3 (triangles), state 4 (squares), and state 4 with added oligomycin (inverse triangles), with succinate (A) and glutamate and malate (B) as substrates. Basic incubation medium (see “Experimental Procedures”) supplemented with 1 mm Pi was used. Statistical analysis was as follows. A, rates of respiration in state 3 taken at pH 8 were significantly different from that at pH 6–7 (n = 3–4; *, p < 0.05, t test). B, rates of respiration in state 3 taken at pH 8 were significantly different from that at pH 6–7.5; rates at pH 7 and 7.5 were significantly different from each other (n = 6; *, p < 0.05; **, p < 0.001, t test).
Mentions: Rat brain mitochondria respiratory characteristics measured in condition 2 and pH variations from 6 to 8 with succinate or glutamate plus malate as substrates are presented in Fig. 1. These data show that our mitochondrial preparations are good in coupling between respiration and phosphorylation in physiological range of pH. At pH 7, the addition of oligomycin inhibits respiration, whereas the addition of ADP (state 3 (St 3)) essentially stimulates it, so that the ratio of respiration rates (state 3/state oligomycin (St 3/St Oligo)) was 5.6 ± 0.6 (n = 3) for succinate and 6.5 ± 0.3 (n = 6) for glutamate plus malate. At alkaline pH, ADP stimulated respiration to a smaller degree, possibly because the lack of protons in the medium limits the process.

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