Limits...
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.

Show MeSH

Related in: MedlinePlus

Difference between the rates of ROS release calculated from the data presented in Fig. 3. Results of subtraction of ROS release rates are plotted versus corresponding pH of the medium. Circles, condition 1 to condition 3; squares, condition 2 to condition 3. Statistical analysis was as follows. Values of difference between condition 1 and condition 3 (circles) at pH 6.5 and 7 were significantly different from that at pH 6 (n = 3–10; **, p < 0.01, t test); values of difference between condition 2 and condition 3 (squares) at pH 7 were significantly different from that at pH 8 (n = 3–10; *, p < 0.05, t test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2570889&req=5

fig9: Difference between the rates of ROS release calculated from the data presented in Fig. 3. Results of subtraction of ROS release rates are plotted versus corresponding pH of the medium. Circles, condition 1 to condition 3; squares, condition 2 to condition 3. Statistical analysis was as follows. Values of difference between condition 1 and condition 3 (circles) at pH 6.5 and 7 were significantly different from that at pH 6 (n = 3–10; **, p < 0.01, t test); values of difference between condition 2 and condition 3 (squares) at pH 7 were significantly different from that at pH 8 (n = 3–10; *, p < 0.05, t test).

Mentions: The mentioned above comparison of the three conditions could give an approximate quantitative estimation of pH gradient and the role of matrix pH in ROS production. In the presence of nigericin, matrix pH is the same as external pH; therefore, subtracting the ROS production rate under condition 3 from that under two other conditions (as indicated in Fig. 9), we can deduce the role of matrix pH difference in ROS production. As Fig. 9 shows, at pH 6, the difference between ROS production rate under the condition when Pi and nigericin are absent (condition 1) and when they are present (condition 3) is 650 pmol/min/mg protein. The reason for this difference is different matrix pH, which is higher in the absence of Pi and nigericin (condition 1). The same difference in ROS production (650 pmol/min/mg prot) could be induced by a 0.5-unit pH shift from pH 6 to 6.5 in the presence of both Pi and nigericin (under condition 3) (Fig. 3A, circles). Therefore, if under condition 1 matrix pH defines this difference in ROS production, it must be not less than 6.5 when pH of the medium is 6.


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)

Difference between the rates of ROS release calculated from the data presented in Fig. 3. Results of subtraction of ROS release rates are plotted versus corresponding pH of the medium. Circles, condition 1 to condition 3; squares, condition 2 to condition 3. Statistical analysis was as follows. Values of difference between condition 1 and condition 3 (circles) at pH 6.5 and 7 were significantly different from that at pH 6 (n = 3–10; **, p < 0.01, t test); values of difference between condition 2 and condition 3 (squares) at pH 7 were significantly different from that at pH 8 (n = 3–10; *, p < 0.05, t test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig9: Difference between the rates of ROS release calculated from the data presented in Fig. 3. Results of subtraction of ROS release rates are plotted versus corresponding pH of the medium. Circles, condition 1 to condition 3; squares, condition 2 to condition 3. Statistical analysis was as follows. Values of difference between condition 1 and condition 3 (circles) at pH 6.5 and 7 were significantly different from that at pH 6 (n = 3–10; **, p < 0.01, t test); values of difference between condition 2 and condition 3 (squares) at pH 7 were significantly different from that at pH 8 (n = 3–10; *, p < 0.05, t test).
Mentions: The mentioned above comparison of the three conditions could give an approximate quantitative estimation of pH gradient and the role of matrix pH in ROS production. In the presence of nigericin, matrix pH is the same as external pH; therefore, subtracting the ROS production rate under condition 3 from that under two other conditions (as indicated in Fig. 9), we can deduce the role of matrix pH difference in ROS production. As Fig. 9 shows, at pH 6, the difference between ROS production rate under the condition when Pi and nigericin are absent (condition 1) and when they are present (condition 3) is 650 pmol/min/mg protein. The reason for this difference is different matrix pH, which is higher in the absence of Pi and nigericin (condition 1). The same difference in ROS production (650 pmol/min/mg prot) could be induced by a 0.5-unit pH shift from pH 6 to 6.5 in the presence of both Pi and nigericin (under condition 3) (Fig. 3A, circles). Therefore, if under condition 1 matrix pH defines this difference in ROS production, it must be not less than 6.5 when pH of the medium is 6.

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