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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), level of membrane potential (B), and rates of respiration of mitochondria oxidizing succinate (C). Basic medium (see “Experimental Procedures”) was used in all experiments with different supplementations: condition 1, basic medium alone (squares); condition 2, basic medium with 1 mm Pi (triangles); condition 3, basic medium with 1 mm Pi and 100 nm nigericin (circles). Statistical analysis was as follows. A, ROS rates taken at all conditions and all pH values were significantly different from each other (n = 3–10, p < 0.05), with an exception for conditions 2 and 3 at pH 8 (one-way analysis of variance test). Within the same condition, ROS rates taken at each pH were significantly different from that at the adjacent pH value (n = 3–10, p < 0.05), with the exceptions of the rates taken at pH 7.5 and 8 in condition 3 (t test). B, the levels of membrane potential measured at pH 7.0–8.0 for condition 1 were significantly different from that in condition 2 or 3 (n = 3–8; *, p < 0.05, analysis of variance test) and also from the membrane potential at pH 6.0–6.5 for the same condition (n = 3; +, p < 0.05, t test). C, at pH 8.0, rates of respiration measured in conditions 2 and 3 were significantly different from that in condition 1 (n = 3–8; +, p < 0.05, analysis of variance test). In condition 1, rates at pH 8.0 were significantly different from that at all other pH values (n = 7–8; ***, p < 0.0001, t test); in condition 3, rates at pH 7.5 were significantly different from that at pH 6.0 and 6.5 (n = 3–4; #, p < 0.05, t test).
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fig3: Effect of medium pH on rate of ROS release (A), level of membrane potential (B), and rates of respiration of mitochondria oxidizing succinate (C). Basic medium (see “Experimental Procedures”) was used in all experiments with different supplementations: condition 1, basic medium alone (squares); condition 2, basic medium with 1 mm Pi (triangles); condition 3, basic medium with 1 mm Pi and 100 nm nigericin (circles). Statistical analysis was as follows. A, ROS rates taken at all conditions and all pH values were significantly different from each other (n = 3–10, p < 0.05), with an exception for conditions 2 and 3 at pH 8 (one-way analysis of variance test). Within the same condition, ROS rates taken at each pH were significantly different from that at the adjacent pH value (n = 3–10, p < 0.05), with the exceptions of the rates taken at pH 7.5 and 8 in condition 3 (t test). B, the levels of membrane potential measured at pH 7.0–8.0 for condition 1 were significantly different from that in condition 2 or 3 (n = 3–8; *, p < 0.05, analysis of variance test) and also from the membrane potential at pH 6.0–6.5 for the same condition (n = 3; +, p < 0.05, t test). C, at pH 8.0, rates of respiration measured in conditions 2 and 3 were significantly different from that in condition 1 (n = 3–8; +, p < 0.05, analysis of variance test). In condition 1, rates at pH 8.0 were significantly different from that at all other pH values (n = 7–8; ***, p < 0.0001, t test); in condition 3, rates at pH 7.5 were significantly different from that at pH 6.0 and 6.5 (n = 3–4; #, p < 0.05, t test).

Mentions: Fig. 3A (squares) shows that the rate of ROS production under condition 1 is 3 times increased when the pH of media changed from 6 to 7, whereas the levels of membrane potential decreased (Fig. 3B, squares), and the respiration rate slightly increased (Fig. 3C, squares). Usually, high ΔΨ is considered to be a factor promoting ROS production (factor iii in the Introduction). Here the situation is controversial; ROS production is increased when ΔΨ is decreased. Obviously, substrate and oxygen concentration (factors ii and i) remain the same for all conditions; therefore, medium pH is the only factor correlating with increase of ROS.


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), level of membrane potential (B), and rates of respiration of mitochondria oxidizing succinate (C). Basic medium (see “Experimental Procedures”) was used in all experiments with different supplementations: condition 1, basic medium alone (squares); condition 2, basic medium with 1 mm Pi (triangles); condition 3, basic medium with 1 mm Pi and 100 nm nigericin (circles). Statistical analysis was as follows. A, ROS rates taken at all conditions and all pH values were significantly different from each other (n = 3–10, p < 0.05), with an exception for conditions 2 and 3 at pH 8 (one-way analysis of variance test). Within the same condition, ROS rates taken at each pH were significantly different from that at the adjacent pH value (n = 3–10, p < 0.05), with the exceptions of the rates taken at pH 7.5 and 8 in condition 3 (t test). B, the levels of membrane potential measured at pH 7.0–8.0 for condition 1 were significantly different from that in condition 2 or 3 (n = 3–8; *, p < 0.05, analysis of variance test) and also from the membrane potential at pH 6.0–6.5 for the same condition (n = 3; +, p < 0.05, t test). C, at pH 8.0, rates of respiration measured in conditions 2 and 3 were significantly different from that in condition 1 (n = 3–8; +, p < 0.05, analysis of variance test). In condition 1, rates at pH 8.0 were significantly different from that at all other pH values (n = 7–8; ***, p < 0.0001, t test); in condition 3, rates at pH 7.5 were significantly different from that at pH 6.0 and 6.5 (n = 3–4; #, p < 0.05, t test).
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fig3: Effect of medium pH on rate of ROS release (A), level of membrane potential (B), and rates of respiration of mitochondria oxidizing succinate (C). Basic medium (see “Experimental Procedures”) was used in all experiments with different supplementations: condition 1, basic medium alone (squares); condition 2, basic medium with 1 mm Pi (triangles); condition 3, basic medium with 1 mm Pi and 100 nm nigericin (circles). Statistical analysis was as follows. A, ROS rates taken at all conditions and all pH values were significantly different from each other (n = 3–10, p < 0.05), with an exception for conditions 2 and 3 at pH 8 (one-way analysis of variance test). Within the same condition, ROS rates taken at each pH were significantly different from that at the adjacent pH value (n = 3–10, p < 0.05), with the exceptions of the rates taken at pH 7.5 and 8 in condition 3 (t test). B, the levels of membrane potential measured at pH 7.0–8.0 for condition 1 were significantly different from that in condition 2 or 3 (n = 3–8; *, p < 0.05, analysis of variance test) and also from the membrane potential at pH 6.0–6.5 for the same condition (n = 3; +, p < 0.05, t test). C, at pH 8.0, rates of respiration measured in conditions 2 and 3 were significantly different from that in condition 1 (n = 3–8; +, p < 0.05, analysis of variance test). In condition 1, rates at pH 8.0 were significantly different from that at all other pH values (n = 7–8; ***, p < 0.0001, t test); in condition 3, rates at pH 7.5 were significantly different from that at pH 6.0 and 6.5 (n = 3–4; #, p < 0.05, t test).
Mentions: Fig. 3A (squares) shows that the rate of ROS production under condition 1 is 3 times increased when the pH of media changed from 6 to 7, whereas the levels of membrane potential decreased (Fig. 3B, squares), and the respiration rate slightly increased (Fig. 3C, squares). Usually, high ΔΨ is considered to be a factor promoting ROS production (factor iii in the Introduction). Here the situation is controversial; ROS production is increased when ΔΨ is decreased. Obviously, substrate and oxygen concentration (factors ii and i) remain the same for all conditions; therefore, medium pH is the only factor correlating with increase of ROS.

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