Talk:MiPNet17.06 IOC66

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IOC66 Workshop experiments

2012-03-15 First Demo Experiment

Oxygraph EF (in both chambers the same SUIT-Protocol was applied)
DatLab File 2012-03-15 EF-03
mouse heart homogenate prepared with PBI Shredder (position 1 for 10 seconds, position 2 for 5 seconds) and the homogenate was completely washed out with 5 ml MitoOx2
respiration medium: MitoOx2
SUIT-Protocol: S + D5 + H2O2 + H2O2 + FCCP
the homogenate was added as well as AmR and HRP
addition of S (without rotenone) for LEAK respiration (without ADP) - succinate is the substrate for succinate dehydroganse (CII) resulting in succinate oxidation and accumulation of oxaloacetat (a stronger inhibitor of succinate dehydrogenase than malonate) that can not permeate the mitochondrial inner membrane - inhibition of CII and inhibition of LEAK respiration to an undefined extent
reverse electron flow from CII to CI is known to stimulate production of ROS species under these experimental conditions - represented by the increase of the fluorescence signal
high H2O2 flux may induce oxidative stress - partial dyscoupling of OXPHOS, thus potentially increasing LEAK respiration
addition of ADP inhibited respiration approx. 77 to 92% - drop down of the flux and the fluorescence signal (reproducible in both chambers)
no stimulation because of the inhibtion of CII by the accumulation of oxaloacetat (without rotenone: succinate - fumarat - malat - oxaloacetat that accumulates and inhibits CII)?
addition of rotenone: S(Rot) supports electron flux exclusively through CII; inhibition of the NADH-linked dehydrogenases through inhibition of NADH oxidation preventing accumulation of oxaloacetate
addition of FCCP in the end: further inhibition of respiration and no increase of the oxygen flux, no decrease in peroxide production (with FCCP the oxygen flux should increase due to a higher oxygen consumption) - hint: mitochondria already in an uncoupled state?


→ in both chambers, the highest H2O2 / O2 flux ratio (L with succinate) was 0.0057 or 0.57 %, which was diminished to 0.0009 or 0.09 % after the paradoxical inhibition by ADP. With 0.5 µM and 1 µM FCCP the H2O2 / O2 flux ratio was 0.0011 (0.011 %) and 0.0013 (0.013 %).

2012-03-16 Second Demo Experiment

Oxygraph EF (in both chambers different SUIT-Protocols were applied)
DatLab File 2012-03-16 EF-02
mouse heart homogenate prepared with PBI Shredder (position 1 for 10 seconds, position 2 for 5 seconds) and the homogenate was completely washed out with 5 ml MitoOx2
respiration medium: MitoOx2
SUIT-Protocol (chamber E): S + AmR/HRP + D0.15 + D5 + Omy + F + G + H2O2 + c
addition of small amounts of ADP (0.15 mM) already inhibits respiration in the presence of succinat (without rotenone) - 32.3 % inhibition
additional ADP (5 mM) inhibits respiration again - 38.6 % inhibition (70.9 % in total)
addition of ADP leads to a decrease in peroxid production
oligomycin (omy; for the evaluation of leak state in the presence of ADP): inhibition of ATP synthase and oxyen flux should be reduced; in the biological experiment the oxygen flux increased after addition of omy - omy can also act as an uncoupler but as FCCP further inhibited respiration and there was no increase of the oxygen flux (look at 2012-03-15 First Demo Experiment), omy did not act as an uncoupler in this experiment and the increase in the oxygen flux did not relay to uncoupling!
after addition of omy the O2 flux increased as well as the peroxid production but only to a small extend (this increase is not that comparable to the rise in peroxid production after reoxygenation!)
a rise in the oxygen concentration (reoxygenation with pure oxygen) increased the flux after titration of omy - direct influence of oxygen concentration on peroxid production (strong induction of peroxid production after reoxygenation)
titration of FCCP diminished the oxygen flux (no stimulation, no further uncoupling)
addition of glutamate as CI-linked substrate slightly increased the oxygen flux - 27.41 %
prominent cyt c effect (49.3 %) - quality of preparation? duration of the protocol - late application of cyt c?


SUIT-Protocol (chamber F): AmR/HRP + S + G + D0.15 + D5 + Omy + F + H2O2 + c
addition of succinate and glutamate for CI/CII-Leak respiration without ADP
* peroxide production with succinate alone was lower compared to chamber E
* addition of glutamate did not cause big changes in oxygen flux and peroxide production
titration of 0.15 mM ADP stimulated respiration (55.6 %) whereas addition of 5 mM ADP inhibited respiration about 3.9 % - but this inhibition could be due to oxygen limitation (no steady state reached!)
* there is a dependence of oxygen with homogenate but not that strong as with fibres (decrease of oxygen flux with decrease of oxygen concentration)
reoxygenation with pure oxygen increased the oxygen flux - similar to oxygen flux with 0.15 mM ADP and steady state!
reoxygenation increased the peroxide production
oligomycin reduced the oxygen flux but led to an increase in peroxide production
FCCP titration: did not cause really any effects - there was no increase in respiration (short stimulation of respiration but then inhibition) and peroxide production declined
mitochondria already dyscoupled?
high cytochrome c effect (31.7 %)


→ In both chambers, the highest H2O2 / O2 flux ratio (L with succinate) was 0.0112 (1.12 %) and 0.007 (0.70 %). The addition of ADP diminished the H2O2 / O2 flux ratio to 0.0015 (0.15 %) in the chamber with succinate alone. In the chamber with succinate and glutamate the ratio remained 0.007 (0.70 %) and was diminished to 0.0005 (0.05 %) after titration of ADP. Oligomycin caused an increase in the ratio of both chambers as indicated by 0.0026 (0.26 %) and 0.0036 (0.36%). With 0.5 µM FCCP the ratio was reduced to 0.0028 (0.28 %) and 0.0011 (0.11 %).

Notes

  • mitochondria uncoupled/dyscoupled already from the beginning?
  • investigation on CI is necessary (NADH)
  • conversion of ADP to ATP?
  • stimulation of respiration with succinate and glutamate - elimination of oxaloacetate through transamination?
  • P/E ratio > 1: ETS is less than OXPHOS capacity - experimental artefact (inhibitors of ATP synthase suppress ETS capacity, particularly in stressed cells)
  • cyt c effect: damage of the outer mitochondrial membrane
when should the addition of cyt c occur to evaluate the effect?
do not add cyt c in a Leak state - leads to activation of respiration unrelated to injury of the outer mitochondrial membrane
  • add rotenone together with succinate - rotenone inhibits CI by inhibition of NADH oxidation
  • huge cytochrome c effect as there was not a big stimulation of CI-linked respiration and also the titration of succinate did not really stimulate respiration


References


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