Meszaros 2017 MiP2017
Mitochondria isolated from mouse brain, liver and kidney were incubated in MiR05 respiration medium at 37°C in the Oroboros O2k high-resolution respirometer. Various substrate combinations were tested in OXPHOS, LEAK and ET states to investigate the effect of pathway and coupling control on mitochondrial p50. During SUIT protocols, kinetic data for oxygen was obtained with aerobic-anaerobic transitions in selected respiratory steady states using high time-resolution (0.2 s) data recording . p50 values were the calculated after corrections for zero oxygen concentration, zero oxygen signal drift, instrumental background oxygen flux and exponential time constant of the polarographic oxygen sensor were performed with a software package developed for internal laboratory use. In the second part of the study, COX inhibition was tested with sequential additions of NaNO2 to produce nitric oxide (NO), while the NO concentration was measured in the O2k chamber amperometrically (WPI). In another set of experiments, mitochondrial p50 was estimated to detect partial inhibition of COX by an unknown substance.
In our experiments, p50 values were independent of volume-specific flux (JV,O2) in the whole range tested (30-450 pmol*s-1*mL-1). Depending on the coupling state and substrate combination, p50 was determined to be 0.006 kPa (N(GM)L) – 0.04 kPa (NS(GM)P). Upon inhibition of COX by NO or in diagnostic experiments, several fold increases in p50 have been found (up to 0.15 kPa, NS(GM)P), which was not reflected in the oxygen flux at kinetic oxygen saturation (JO2,max). Furthermore, in diagnostic experimental runs, increasing p50 values correlated well with decreasing COX activity, as measured upon addition of ascorbate and TMPD.
In conclusion, affinity of isolated mitochondria for oxygen decreases already at a slight inhibition (and decrease in excess capacity) of COX. Mitochondrial p50 measurement is a quick, highly accurate and sensitive way for early detection of reduced COX excess capacity. Furthermore, the method presented here provides valuable data on the possible in vivo applicability of experimental results, reflecting low tissue oxygen concentrations. Determination of mitochondria oxygen dependence has implications in a wide range of biomedical research topics, e.g. in work with biologically active gases (NO, hydrogen sulfide, carbon monoxide, methane) which directly or indirectly influence COX and in detection of COX impairment and inhibition.
Labels: MiParea: Respiration, mt-Medicine
Organism: Mouse Tissue;cell: Nervous system, Liver, Kidney Preparation: Isolated mitochondria
Regulation: Oxygen kinetics Coupling state: LEAK, OXPHOS, ET Pathway: N, NS HRR: Oxygraph-2k
- Meszaros AT(1,2), Haider M(3), Gnaiger E(1,4)
- Oroboros Instruments, Innsbruck, Austria
- Inst of Surgical Research, Univ Szeged, Hungary
- Steinhauser Haider Technology Consulting OG, Innsbruck, Austria
- D.Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria. - firstname.lastname@example.org
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