Komlodi 2017 MiP2017
To this aim, isolated mouse brain and heart mitochondria respiring with the combination of NADH-linked substrates glutamate and malate and complex II-linked substrate, succinate in a closed-chamber respirometer combined with fluorimetry (Oroboros O2k High-Resolution FluoRespirometer). O2 concentration was varied from ≈170 µM to 15-20 µM with injection of N2 in the gas phase both in the LEAK and OXPHOS states. H2O2 production was determined by the highly specific Amplex UltraRed assay in MiR05 and KCl-based media. The rate of H2O2 generation was corrected for chemical background, changes of O2 level over time and for the sensitivity of the detection system.
Here we reveal that upon decrease of the O2 concentration from ≈170 µM to ≈20 µM, the rate of H2O2 formation is reduced as compared with normoxic H2O2 production in the LEAK but not in the OXPHOS state. After re-oxygenation, the rate of H2O2 generation again increases. At the same time, reducing the O2 concentration to ≈ 20 µM did not compromise O2 consumption, which is in line with the high affinity of mitochondrial respiration for O2 (p50 ≈ 0.03-0.04 kPa in OXPHOS).
Taken together, when reducing the O2 concentration to the physiological intracellular range in vitro, the rate of H2O2 production declines in a metabolic states-dependent manner. In contrast, respiration was not affected by the O2 concentration down to ≈ 20 µM neither in brain nor in heart. Based on this, we can assume that the H2O2 production is more sensitive to the changes of O2 concentration than the O2 consumption.
Labels: MiParea: Respiration
Stress:Oxidative stress;RONS, Hypoxia Organism: Mouse Tissue;cell: Heart, Nervous system Preparation: Isolated mitochondria
Regulation: Oxygen kinetics Coupling state: LEAK, OXPHOS Pathway: N, NS HRR: Oxygraph-2k
Amplex UltraRed, MitoEAGLE
Affiliations and support
- Timea K(1), Sobotka O(3), Gnaiger E(1,2)
- Oroboros Instruments, Innsbruck, Austria
- D. Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria
- Charles Univ, Fac Medicine Hradec Kralove, Dept Physiology, Hradec Kralove, Czech Republic. - email@example.com
- Contribution to European Union Framework Programme Horizon 2020 COST Action CA15203 MitoEAGLE.
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