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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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Effect of medium pH on rate of ROS release (A) and level of membrane potential of mitochondria oxidizing complex I substrates glutamate and malate (B and C). Basic incubation medium was supplemented with 1 mm Pi (A and C) and 100 nm nigericin (A). For traces of membrane potential (B and C), pH was adjusted to the values indicated by each trace. The additions of mitochondria (Mt), 1 mm Pi, 1 μm rotenone, 100 nm nigericin, and 200 nm FCCP were made at the time points indicated by the arrows. Statistical analysis was as follows. All data sets for ROS rates were statistically different from each other (n = 4; *, p < 0.05, t test).
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fig7: Effect of medium pH on rate of ROS release (A) and level of membrane potential of mitochondria oxidizing complex I substrates glutamate and malate (B and C). Basic incubation medium was supplemented with 1 mm Pi (A and C) and 100 nm nigericin (A). For traces of membrane potential (B and C), pH was adjusted to the values indicated by each trace. The additions of mitochondria (Mt), 1 mm Pi, 1 μm rotenone, 100 nm nigericin, and 200 nm FCCP were made at the time points indicated by the arrows. Statistical analysis was as follows. All data sets for ROS rates were statistically different from each other (n = 4; *, p < 0.05, t test).

Mentions: In the experiments described above, succinate was applied as a substrate. Used in much higher concentrations than normally produced in Krebs cycle, it stimulates high ROS production rate, and this was convenient to study the process, although the ROS production rates were much higher than could be observed under physiological conditions. This high concentration of succinate could be observed, however, at pathologies like ischemia (27, 28). More physiological substrates, glutamate and malate, produce much smaller amounts of ROS (11, 29). However, as Fig. 7A illustrates, the rates of ROS release in this case also significantly increased with the increase of medium pH. These experiments were performed in the presence of both Pi and nigericin to ensure the conversion of the ΔpH component of proton motive force into the electric one (condition 3). Fig. 7, B and C, shows that, indeed, the addition of inorganic phosphate and nigericin did increase membrane potential the same way as it was observed with succinate as substrate.


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)

Effect of medium pH on rate of ROS release (A) and level of membrane potential of mitochondria oxidizing complex I substrates glutamate and malate (B and C). Basic incubation medium was supplemented with 1 mm Pi (A and C) and 100 nm nigericin (A). For traces of membrane potential (B and C), pH was adjusted to the values indicated by each trace. The additions of mitochondria (Mt), 1 mm Pi, 1 μm rotenone, 100 nm nigericin, and 200 nm FCCP were made at the time points indicated by the arrows. Statistical analysis was as follows. All data sets for ROS rates were statistically different from each other (n = 4; *, p < 0.05, t test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig7: Effect of medium pH on rate of ROS release (A) and level of membrane potential of mitochondria oxidizing complex I substrates glutamate and malate (B and C). Basic incubation medium was supplemented with 1 mm Pi (A and C) and 100 nm nigericin (A). For traces of membrane potential (B and C), pH was adjusted to the values indicated by each trace. The additions of mitochondria (Mt), 1 mm Pi, 1 μm rotenone, 100 nm nigericin, and 200 nm FCCP were made at the time points indicated by the arrows. Statistical analysis was as follows. All data sets for ROS rates were statistically different from each other (n = 4; *, p < 0.05, t test).
Mentions: In the experiments described above, succinate was applied as a substrate. Used in much higher concentrations than normally produced in Krebs cycle, it stimulates high ROS production rate, and this was convenient to study the process, although the ROS production rates were much higher than could be observed under physiological conditions. This high concentration of succinate could be observed, however, at pathologies like ischemia (27, 28). More physiological substrates, glutamate and malate, produce much smaller amounts of ROS (11, 29). However, as Fig. 7A illustrates, the rates of ROS release in this case also significantly increased with the increase of medium pH. These experiments were performed in the presence of both Pi and nigericin to ensure the conversion of the ΔpH component of proton motive force into the electric one (condition 3). Fig. 7, B and C, shows that, indeed, the addition of inorganic phosphate and nigericin did increase membrane potential the same way as it was observed with succinate as substrate.

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