The dependence of brain mitochondria reactive oxygen species production on oxygen level is linear, except when inhibited by antimycin A

Abstract

Reactive oxygen species (ROS) are by-products of physiological mitochondrial metabolism that are involved in several cellular signaling pathways as well as tissue injury and pathophysiological processes, including brain ischemia/reperfusion injury. The mitochondrial respiratory chain is considered a major source of ROS; however, there is little agreement on how ROS release depends on oxygen concentration. The rate of H₂ O₂ release by intact brain mitochondria was measured with an Amplex UltraRed assay using a high-resolution respirometer (Oroboros) equipped with a fluorescent optical module and a system of controlled gas flow for varying the oxygen concentration. Three types of substrates were used: malate and pyruvate, succinate and glutamate, succinate alone or glycerol 3-phosphate. For the first time we determined that, with any substrate used in the absence of inhibitors, H₂ O₂ release by respiring brain mitochondria is linearly dependent on the oxygen concentration. We found that the highest rate of H₂ O₂ release occurs in conditions of reverse electron transfer when mitochondria oxidize succinate or glycerol 3-phosphate. H₂ O₂ production by complex III is significant only in the presence of antimycin A and, in this case, the oxygen dependence manifested mixed (linear and hyperbolic) kinetics. We also demonstrated that complex II in brain mitochondria could contribute to ROS generation even in the absence of its substrate succinate when the quinone pool is reduced by glycerol 3-phosphate. Our results underscore the critical importance of reverse electron transfer in the brain, where a significant amount of succinate can be accumulated during ischemia providing a backflow of electrons to complex I at the early stages of reperfusion. Our study also demonstrates that ROS generation in brain mitochondria is lower under hypoxic conditions than in normoxia. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Keywords: ROS generation; antimycin A; complex I; ischemia/reperfusion; mitochondria; reverse electron transfer.

Fig. 1.

Scheme of assessment of the effect of oxygen concentration on H₂O₂ release by intact mitochondria in different conditions. The reaction started with the addition of mitochondria (0.5 mg protein/ml) to the assay media containing 5 mM succinate and 1 mM glutamate (left panel) or 10 mM glycerol 3-phosphate and 1 μM antimycin A (right panel). Oxygen concentration as measured directly by the Oroboros respirometer was rapidly varied by continuously purging the headspace with argon (Ar) or air as detailed in the Materials and Methods section. (A, B) Representative registration traces of oxygen concentration (blue) and release of H₂O₂ measured with Amplex UltraRed (black). (C,D) The calculated rates of H₂O₂ release are shown as red traces. (E, F) H₂O₂ release rate vs. oxygen concentration were plotted and fitted with either a linear function (E, data from A) or a sum of both linear and hyperbolic functions (F, data from B). Corresponding equations are shown on the graphs.

Fig. 2.

Dependence of H₂O₂ release rate on the oxygen concentration in the absence of inhibitors. H₂O₂ release during non-phosphorylating (no ADP, open squares) and phosphorylating, state 3 (0.2 mM ADP added, open triangles) respiration was measured at different [O₂] as detailed in the Materials and Methods section. Mitochondria (0.1–0.5 mg protein/ml) were added to the assay media containing the following substrates: 2 mM malate and 5 mM pyruvate (A), 5 mM succinate and 1 mM glutamate (B), and 40 mM glycerol 3-phosphate (C). At least five different isolations of mitochondria were used and 5–6 traces for each experimental condition were averaged (n=5–6). Fit parameters of Eq 1 are shown on the graphs.

Fig. 3.

Dependence of H₂O₂ release rate on the oxygen concentration for mitochondria in the presence of respiratory chain inhibitors. Experimental conditions were the same as in Fig. 2. with the following substrates 2 mM malate and 5 mM pyruvate (A), 5 mM succinate and 1 mM glutamate (B) or 40 mM glycerol 3-phosphate (C). No ADP was present. The H₂O₂ release at different [O₂] in the presence of 1 μM rotenone (solid triangles) or 1 μM antimycin A (solid squares) was measured as described in the Materials and Methods. At least five different isolations of mitochondria were used and 5–9 traces for each experimental condition were averaged (n=5–9). Fit parameters of Eq 1 and Eq. 2 are shown on the graphs.

Fig. 4.

Respiration and H₂O₂ release by mitochondria oxidizing glycerol 3-phosphate. (A) Effect of glycerol 3-phosphate concentration on non-phosphorylating (no ADP added, white bars) and state 3 phosphorylating respiration (0.2 mM ADP, grey bars). (B) Effect of glycerol 3-phosphate concentration on non-phosphorylating H₂O₂ release (white bars), state 3 phosphorylating H₂O₂ release (grey bars), and antimycin A-inhibited H₂O₂ release (black). (C-E) Effect of complex II specific inhibitor atpenin A5 (1.5 nM, open triangles) on the dependence of H₂O₂ release rate on oxygen concentration in the presence of 1 μM rotenone (C), 1 μM rotenone and 25 nM uncoupler SF 6847 (D) or 1 μM rotenone and 2 μM complex III inhibitor myxothiazol (E). At least five different isolations of mitochondria were used and 5–6 traces for each experimental condition were averaged (n=5–6). Fit parameters of Eq. 1 are shown on the graphs. Concentration of glycerol 3-phosphate was 40 mM.