Sumbalova 2011 Abstract Bordeaux
|Sumbalova Z, Wiethuechter A, Fasching M, Gnaiger E (2011) Coupling control and substrate control of mitochondrial membrane potential and respiration in the mouse brain, and comparison with skeletal and cardiac muscle. Abstract MiP2011, Bordeaux.|
High-resolution respirometry was combined with an ion selective electrode system (TPP+; Oroboros O2k MultiSensor system; MiR06 at 37 °C) to measure respiration, JO2, and mt-membrane potential, ΔΨ, in three mouse brain preparations: isolated mitochondria, homogenate after 3 min centrifugation at 1300 g, and crude tissue homogenate.
• Keywords: High-resolution respirometry, OXPHOS, mitochondrial membrane potential, ROS production, brain mitochondria, O2k-Fluorimeter
Organism: Mouse Tissue;cell: Nervous system
Enzyme: Complex I, Complex II;succinate dehydrogenase Regulation: Substrate
HRR: Oxygraph-2k, TPP
Physiological substrate cocktails are required to reconstitute tricarboxylic acid cycle (TCA) function in mitochondrial (mt) preparations, to quantify maximum capacities of oxidative phosphorylation (OXPHOS, P) and of the Electron transfer-pathway (ET-pathway, E). Functional differences between mitochondria from different tissues and species are largely masked when restricting flux artificially by applying either Complex I (CI) or Complex II (CII) linked substrates which do not support convergent CI+II-linked electron input into the Q-junction . We applied and validated different protocols with substrate-uncoupler-inhibitor titrations (SUIT), monitoring simultaneously mt-membrane potential (TPP+) and respiration.
High-resolution respirometry was combined with an ion selective electrode system (TPP+; Oroboros O2k MultiSensor system; MiR06 at 37 °C) to measure respiration, JO2, and mt-membrane potential, ΔΨ, in three mouse brain preparations: isolated mitochondria, homogenate after 3 min centrifugation at 1300 g, and crude tissue homogenate. Coupling control and substrate control states  were established sequentially in SUIT protocols, and respiratory flux control patterns were compared between mouse brain and permeabilized fibres from skeletal muscle (gastrocnemius) and heart.
JO2 in the [[LEAK}} state (L; no ADP) with CI-linked substrates (pyruvate +malate +glutamate) represented only 0.05 and 0.07 of OXPHOS capacity (saturating [ADP]) in the brain and skeletal muscle (RCR= 22 and 14), but 0.29 in heart (RCR=3.4). OXPHOS capacity with CI-linked substrates constituted only 0.77 of physiological OXPHOS capacity (CI+II substrate cocktail, pyruvate +malate +glutamate +succinate) in brain, compared to a CI/CI+II flux ratio of 0.87 and 0.68 in skeletal muscle and heart. OXPHOS capacity was strongly limited by the phosphorylation system in the brain, as revealed by the increase of ADP-stimulated respiration by uncoupling, with a corresponding P/E flux control ratio of 0.77. In contrast, the P/E ratio was 0.94 (close to the maximum value of 1.0) in skeletal muscle, despite of the similar L/P coupling control ratio, and 0.92 in heart. Our results are in accordance with the investigation of Rossignol et al.  on tissue variation in the control of OXPHOS. While respiration is controlled mainly by the ET-pathway in the heart and skeletal muscle, the phosphorylation system (ANT, ATP synthase and phosphate carrier) exerts significant control over respiration in the brain.
In mt-preparations of brain, ΔΨ dropped by 20-25 mV as flux was increased by coupling control from the resting LEAK state to OXPHOS capacity, and dissipation of ΔΨ by uncoupling (FCCP) was accompanied by a further stimulation of flux in the non-coupled state E. Opposite to this coupling paradigm of an inverse ΔΨ/JO2 relationship, both ΔΨ and JO2 increased when flux was varied by substrate control. Under these conditions, ΔΨ increased with an increase of flux. The shift of ΔΨ was 4-6 mV both in the OXPHOS and LEAK state. The higher membrane potential supported by the CI+II substrate cocktail compared to CI-linked substrates requires re-investigations of ROS production and challenges the paradigm  that mitochondrial ROS plays a minor role during exercise and in active states of ATP turnover. Our results challenge the simplistic State 3/State 4 paradigm of mitochondrial respiratory coupling control and inverse regulation of ΔΨ. Substrate control is complementary to coupling control of mitochondrial respiration, as emphasized in the ingenious work of Chance and Williams .
Supported by FEMtech (NMVIT, Austria). Contribution to MitoCom Tyrol.
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