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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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Inorganic phosphate (A) and nigericin (B) increase the value of membrane potential but decrease the rate of ROS release. The same relative scale of fluorescence was used for both dyes. A, membrane potential (traces 1–3) and ROS (traces 4–6). Basic incubation medium, pH 7, was used (see “Experimental Procedures”); 1 mm Pi was present (traces 1 and 4), or no Pi was present (traces 2, 3, 5, and 6). The additions of mitochondria (Mt) (all traces), 1 mm Pi (traces 3 and 6) and 200 nm FCCP (traces 1–3) were made at the time points indicated by arrows. B, basic incubation medium without Pi was used; pH was adjusted to 6.5. Trace 1, ROS; trace 2, membrane potential. At the time points indicated by arrows, the following additions were made: mitochondria (all traces), 100 nm nigericin (all traces), and 200 nm FCCP (trace 2).
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fig5: Inorganic phosphate (A) and nigericin (B) increase the value of membrane potential but decrease the rate of ROS release. The same relative scale of fluorescence was used for both dyes. A, membrane potential (traces 1–3) and ROS (traces 4–6). Basic incubation medium, pH 7, was used (see “Experimental Procedures”); 1 mm Pi was present (traces 1 and 4), or no Pi was present (traces 2, 3, 5, and 6). The additions of mitochondria (Mt) (all traces), 1 mm Pi (traces 3 and 6) and 200 nm FCCP (traces 1–3) were made at the time points indicated by arrows. B, basic incubation medium without Pi was used; pH was adjusted to 6.5. Trace 1, ROS; trace 2, membrane potential. At the time points indicated by arrows, the following additions were made: mitochondria (all traces), 100 nm nigericin (all traces), and 200 nm FCCP (trace 2).

Mentions: The pH dependences presented in Fig. 3 clearly show that pH itself, and not ΔpH, is an important determinant of ROS production. As can be seen, at pH ranging from 6 to 7, when mitochondria do not undergo permeability transition, ROS production under condition 1 is higher than under conditions 2 or 3, which could indicate an important role of matrix pH. The difference in ROS production can be seen when the conditions change in the course of the experiment, producing qualitatively the same outcome (i.e. the addition of Pi or nigericin always resulted in decrease of the ROS generation rate despite the increase in ΔΨ (Fig. 5, A and B) because of trading the ΔpH component of proton motive force to the electric one).


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)

Inorganic phosphate (A) and nigericin (B) increase the value of membrane potential but decrease the rate of ROS release. The same relative scale of fluorescence was used for both dyes. A, membrane potential (traces 1–3) and ROS (traces 4–6). Basic incubation medium, pH 7, was used (see “Experimental Procedures”); 1 mm Pi was present (traces 1 and 4), or no Pi was present (traces 2, 3, 5, and 6). The additions of mitochondria (Mt) (all traces), 1 mm Pi (traces 3 and 6) and 200 nm FCCP (traces 1–3) were made at the time points indicated by arrows. B, basic incubation medium without Pi was used; pH was adjusted to 6.5. Trace 1, ROS; trace 2, membrane potential. At the time points indicated by arrows, the following additions were made: mitochondria (all traces), 100 nm nigericin (all traces), and 200 nm FCCP (trace 2).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Inorganic phosphate (A) and nigericin (B) increase the value of membrane potential but decrease the rate of ROS release. The same relative scale of fluorescence was used for both dyes. A, membrane potential (traces 1–3) and ROS (traces 4–6). Basic incubation medium, pH 7, was used (see “Experimental Procedures”); 1 mm Pi was present (traces 1 and 4), or no Pi was present (traces 2, 3, 5, and 6). The additions of mitochondria (Mt) (all traces), 1 mm Pi (traces 3 and 6) and 200 nm FCCP (traces 1–3) were made at the time points indicated by arrows. B, basic incubation medium without Pi was used; pH was adjusted to 6.5. Trace 1, ROS; trace 2, membrane potential. At the time points indicated by arrows, the following additions were made: mitochondria (all traces), 100 nm nigericin (all traces), and 200 nm FCCP (trace 2).
Mentions: The pH dependences presented in Fig. 3 clearly show that pH itself, and not ΔpH, is an important determinant of ROS production. As can be seen, at pH ranging from 6 to 7, when mitochondria do not undergo permeability transition, ROS production under condition 1 is higher than under conditions 2 or 3, which could indicate an important role of matrix pH. The difference in ROS production can be seen when the conditions change in the course of the experiment, producing qualitatively the same outcome (i.e. the addition of Pi or nigericin always resulted in decrease of the ROS generation rate despite the increase in ΔΨ (Fig. 5, A and B) because of trading the ΔpH component of proton motive force to the electric one).

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