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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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Spontaneous depolarization of mitochondria takes place in the medium without Pi (condition 1) at pH 8. A, membrane potential; B, ROS. Additions were as follows. 2 μm CsA, 200 μm ADP, and 2 μm oligomycin were present in the medium before the addition of mitochondria (curve 2).
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fig4: Spontaneous depolarization of mitochondria takes place in the medium without Pi (condition 1) at pH 8. A, membrane potential; B, ROS. Additions were as follows. 2 μm CsA, 200 μm ADP, and 2 μm oligomycin were present in the medium before the addition of mitochondria (curve 2).

Mentions: At more alkaline pH (7.5 and 8), mitochondria undergo spontaneous depolarization. As Fig. 4 demonstrates, the loss of membrane potential can be delayed by the presence in the medium of the agents known to inhibit permeability transition pore opening; cyclosporine A together with ADP and oligomycin. This fact indicates that the loss of membrane potential in this condition is due to permeability transition pore opening.


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)

Spontaneous depolarization of mitochondria takes place in the medium without Pi (condition 1) at pH 8. A, membrane potential; B, ROS. Additions were as follows. 2 μm CsA, 200 μm ADP, and 2 μm oligomycin were present in the medium before the addition of mitochondria (curve 2).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Spontaneous depolarization of mitochondria takes place in the medium without Pi (condition 1) at pH 8. A, membrane potential; B, ROS. Additions were as follows. 2 μm CsA, 200 μm ADP, and 2 μm oligomycin were present in the medium before the addition of mitochondria (curve 2).
Mentions: At more alkaline pH (7.5 and 8), mitochondria undergo spontaneous depolarization. As Fig. 4 demonstrates, the loss of membrane potential can be delayed by the presence in the medium of the agents known to inhibit permeability transition pore opening; cyclosporine A together with ADP and oligomycin. This fact indicates that the loss of membrane potential in this condition is due to permeability transition pore opening.

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