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Sumbalova 2011 Abstract Kagoshima

From Bioblast
Sumbalova Z, Harrison DK, Gradl P, Fasching M, Gnaiger E (2011) Mitochondrial membrane potential, coupling control, H2O2 production, and the upper limit of mitochondrial performance. Abstract Kagoshima.

Link: Kagoshima, Japan

Sumbalova Z, Harrison DK, Gradl P, Fasching M, Gnaiger E (2011)

Event: Kagoshima

Electron gating through either Complex I (CI) or CII exerts an experimental limitation on OXPHOS capacity in mitochondrial preparations, artificially alters the production of reactive oxygen species (ROS), and restricts the driving force for generating the mitochondrial (mt) membrane potential. We applied physiological substrate cocktails to reconstitute tricarboxylic acid cycle function in mouse brain mitochondria to (i) support convergent CI+II-linked electron input into the Q-junction (Gnaiger 2009 Int J Biochem Cell Biol), (ii) quantify maximum capacities of oxidative phosphorylation (OXPHOS) and of the electron transfer system (ETS), and (iii) monitor simultaneously oxygen consumption (JO2) and mt-membrane potential (ΔΨ), and (iv) JO2 and hydrogen peroxide production (JH2O2). An inverse relationship between ΔΨ and JO2 and direct relation between ΔΨ and JH2O2 is well established when stimulating respiration by ADP and uncoupling. Applying CI- and/or CII-linked substrates, ΔΨ dropped by 20-25 mV as flux was increased by coupling control from the resting LEAK state to OXPHOS capacity (State 3), and JH2O2 decreased. Dissipation of ΔΨ by uncoupling (FCCP) was accompanied by a further stimulation of flux in the noncoupled ETS state (CI or CI+II substrates), comparable to human muscle mitochondria (Boushel_2007_Diabetologia; Pesta_2011_AJP). Opposite to this coupling paradigm of an inverse ΔΨ/JO2 relationship, both ΔΨ and JO2 increased significantly when the upper limit of OXPHOS capacity was obtained with convergent CI+II electron input (pyruvate +malate +glutamate +succinate). Despite the higher membrane potential supported by the CI+II substrate cocktail compared to CI-linked substrates, H2O2 production remained unchanged in the active OXPHOS state of respiration, but CI+II electron supply increased JH2O2 further in the passive LEAK state of respiration. The upper limit of respiratory capacity and the scope of ROS signalling, therefore, are significantly higher under conditions of physiological substrate supply compared with conventional minimal substrate combinations (Contribution to MitoCom Tyrol).

β€’ Keywords: High-resolution respirometry, OXPHOS, mitochondrial membrane potential, ROS production, brain mitochondria, O2k-Fluorimeter

β€’ O2k-Network Lab: AT Innsbruck Oroboros, AT Kolsass WGT, SK Bratislava Sumbalova Z

Product information

Labels: MiParea: Respiration, Instruments;methods 

Stress:Oxidative stress;RONS  Organism: Mouse  Tissue;cell: Nervous system 

Regulation: mt-Membrane potential 

HRR: Oxygraph-2k, O2k-Fluorometer, TPP