Membrane potential and delta pH dependency of reverse electron transport-associated hydrogen peroxide production in brain and heart mitochondria

Tímea Komlódi,Matilde Sassani,Attila Ambrus,László Tretter,Fanni F Geibl

doi:10.1007/s10863-018-9766-8

Abstract

Succinate-driven reverse electron transport (RET) is one of the main sources of mitochondrial reactive oxygen species (mtROS) in ischemia-reperfusion injury. RET is dependent on mitochondrial membrane potential (Δψm) and transmembrane pH difference (ΔpH), components of the proton motive force (pmf); a decrease in Δψm and/or ΔpH inhibits RET. In this study we aimed to determine which component of the pmf displays the more dominant effect on RET-provoked ROS generation in isolated guinea pig brain and heart mitochondria respiring on succinate or α-glycerophosphate (α-GP). Δψm was detected via safranin fluorescence and a TPP+ electrode, the rate of H2O2 formation was measured by Amplex UltraRed, the intramitochondrial pH (pHin) was assessed via BCECF fluorescence. Ionophores were used to dissect the effects of the two components of pmf. The K+/H+ exchanger, nigericin lowered pHin and ΔpH, followed by a compensatory increase in Δψm that led to an augmented H2O2 production. Valinomycin, a K+ ionophore, at low [K+] increased ΔpH and pHin, decreased Δψm, which resulted in a decline in H2O2 formation. It was concluded that Δψm is dominant over ∆pH in modulating the succinate- and α-GP-evoked RET. The elevation of extramitochondrial pH was accompanied by an enhanced H2O2 release and a decreased ∆pH. This phenomenon reveals that from the pH component not ∆pH, but rather absolute value of pH has higher impact on the rate of mtROS formation. Minor decrease of Δψm might be applied as a therapeutic strategy to attenuate RET-driven ROS generation in ischemia-reperfusion injury.

Highlights

There is a large body of experimental evidence demonstrating pathologically enhanced mitochondrial reactive oxygen species production in several diseases such as diabetes, neurodegenerative conditions including Alzheimer’s and Parkinson’s diseases, diabetes, and ischemia-reperfusion injury; for review see (Beal 1996; Giacco and Brownlee 2010; Chouchani et al 2014)
Succinate-driven mitochondrial reactive oxygen species (mtROS) production appears to have the highest rate in isolated murine mitochondria in the absence of ADP compared to NADH-linked substrates initiated mtROS (Korshunov et al 1997; Kwong and Sohal 1998; Votyakova and Reynolds 2001; Liu et al 2002; Zoccarato et al 2011); this is attributed primarily to reverse electron transport (RET) towards CI and partially to the forward electron transport (FET) towards CIII (Grivennikova and Vinogradov 2006; Treberg et al 2011; Zoccarato et al 2011; Quinlan et al 2013)
In contrast to Lambert and co-workers (Lambert and Brand 2004), we found that nigericin increased the rate of H2O2 generation by 52 ± 11% in succinate-respiring brain mitochondria (Fig. 1e)

Summary

Introduction

There is a large body of experimental evidence demonstrating pathologically enhanced mitochondrial reactive oxygen species (mtROS) production in several diseases such as diabetes, neurodegenerative conditions including Alzheimer’s and Parkinson’s diseases, diabetes, and ischemia-reperfusion injury; for review see (Beal 1996; Giacco and Brownlee 2010; Chouchani et al 2014). FADH2-linked substrates generate mtROS at a higher rate by supporting a reverse electron transport (RET) which occurs from Complex II (CII) or alphaglycerophosphate dehydrogenase (α-GPDH) to CI via the Qjunction (Treberg et al 2011). Succinate-driven mtROS production appears to have the highest rate in isolated murine mitochondria in the absence of ADP compared to NADH-linked substrates initiated mtROS (Korshunov et al 1997; Kwong and Sohal 1998; Votyakova and Reynolds 2001; Liu et al 2002; Zoccarato et al 2011); this is attributed primarily to RET towards CI and partially to the forward electron transport (FET) towards CIII (Grivennikova and Vinogradov 2006; Treberg et al 2011; Zoccarato et al 2011; Quinlan et al 2013). In the absence of ATP synthesis, FET secures the energy demands of RET

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