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Difference between revisions of "Krumschnabel 2014 Methods Enzymol"

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|abstract=[[Image:O2k-Publications.jpg|80px|link=O2k-Publications: Topics|O2k-Publications: Topics]]
|abstract=[[Image:O2k-Publications.jpg|80px|link=O2k-Publications: Topics|O2k-Publications: Topics]]


Mitochondrial membrane potential (mtMP) is closely intertwined with oxidative phosphorylation (OXPHOS). The exact nature of the interactions of respiration (flux) and mtMP (force) under various physiological and pathological conditions remains unclear, partially due to methodological limitations. We introduce the combination of high-resolution respirometry and fluorometry with the OROBOROS Oxygraph-2k, using the widely applied mtMP indicator [[safranin]]. OXPHOS analysis with mouse brain homogenate revealed that safranin inhibits Complex I linked OXPHOS capacity at commonly applied concentrations and targets primarily the phosphorylation system, without effect on LEAK respiration. Complex II linked OXPHOS capacity was inhibited by <20% at 2 µM safranin sufficient for mtMP monitoring. mtMP was higher in the LEAK state without adenylates (''L''<sub>N</sub>) than at identical LEAK respiration after ADP stimulation and inhibition by oligomycin (''L''<sub>Omy</sub>). Maximum ET capacity was reached in uncoupler titrations before mtMP was fully collapsed, whereas respiration was inhibited at increasing uncoupler concentrations and further reduction of mtMP. Examining a pharmacologically induced state of Complex II dysfunction, mtMP was rather insensitive to 50% inhibition of OXPHOS, but responded strongly to addition of inhibitors when respiration was minimized by substrate depletion. The optimum uncoupler concentration supporting maximum ET capacity varied as a function of pharmacological intervention. Taken together, combined measurement of respiration and mtMP greatly enhances the diagnostic potential of OXPHOS analysis. Respirometric validation of inhibitory and uncoupling effects is mandatory for any fluorophore applied for probing mtMP, in any respiratory state, type of tissue and pathophysiological condition.
Mitochondrial membrane potential (mtMP) is closely intertwined with oxidative phosphorylation (OXPHOS). The exact nature of the interactions of respiration (flux) and mtMP (force) under various physiological and pathological conditions remains unclear, partially due to methodological limitations. We introduce the combination of high-resolution respirometry and fluorometry with the Oroboros Oxygraph-2k, using the widely applied mtMP indicator [[safranin]]. OXPHOS analysis with mouse brain homogenate revealed that safranin inhibits Complex I linked OXPHOS capacity at commonly applied concentrations and targets primarily the phosphorylation system, without effect on LEAK respiration. Complex II linked OXPHOS capacity was inhibited by <20% at 2 µM safranin sufficient for mtMP monitoring. mtMP was higher in the LEAK state without adenylates (''L''<sub>N</sub>) than at identical LEAK respiration after ADP stimulation and inhibition by oligomycin (''L''<sub>Omy</sub>). Maximum ET capacity was reached in uncoupler titrations before mtMP was fully collapsed, whereas respiration was inhibited at increasing uncoupler concentrations and further reduction of mtMP. Examining a pharmacologically induced state of Complex II dysfunction, mtMP was rather insensitive to 50% inhibition of OXPHOS, but responded strongly to addition of inhibitors when respiration was minimized by substrate depletion. The optimum uncoupler concentration supporting maximum ET capacity varied as a function of pharmacological intervention. Taken together, combined measurement of respiration and mtMP greatly enhances the diagnostic potential of OXPHOS analysis. Respirometric validation of inhibitory and uncoupling effects is mandatory for any fluorophore applied for probing mtMP, in any respiratory state, type of tissue and pathophysiological condition.
|keywords=High-resolution respirometry, Mitochondrial membrane potential, Safranin, Complex I, Complex II, OXPHOS analysis, electron transfer-pathway
|keywords=High-resolution respirometry, Mitochondrial membrane potential, Safranin, Complex I, Complex II, OXPHOS analysis, electron transfer-pathway
|mipnetlab=AT Innsbruck Oroboros, AT Innsbruck Gnaiger E, AT Innsbruck MitoCom
|mipnetlab=AT Innsbruck Oroboros, AT Innsbruck Gnaiger E, AT Innsbruck MitoCom

Revision as of 11:22, 23 January 2019

Publications in the MiPMap
O2k-Protocols contents

Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry. Methods Enzymol 542:163-81.

» PMID: 24862266 »O2k-brief

Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) Methods Enzymol

Abstract: O2k-Publications: Topics

Mitochondrial membrane potential (mtMP) is closely intertwined with oxidative phosphorylation (OXPHOS). The exact nature of the interactions of respiration (flux) and mtMP (force) under various physiological and pathological conditions remains unclear, partially due to methodological limitations. We introduce the combination of high-resolution respirometry and fluorometry with the Oroboros Oxygraph-2k, using the widely applied mtMP indicator safranin. OXPHOS analysis with mouse brain homogenate revealed that safranin inhibits Complex I linked OXPHOS capacity at commonly applied concentrations and targets primarily the phosphorylation system, without effect on LEAK respiration. Complex II linked OXPHOS capacity was inhibited by <20% at 2 µM safranin sufficient for mtMP monitoring. mtMP was higher in the LEAK state without adenylates (LN) than at identical LEAK respiration after ADP stimulation and inhibition by oligomycin (LOmy). Maximum ET capacity was reached in uncoupler titrations before mtMP was fully collapsed, whereas respiration was inhibited at increasing uncoupler concentrations and further reduction of mtMP. Examining a pharmacologically induced state of Complex II dysfunction, mtMP was rather insensitive to 50% inhibition of OXPHOS, but responded strongly to addition of inhibitors when respiration was minimized by substrate depletion. The optimum uncoupler concentration supporting maximum ET capacity varied as a function of pharmacological intervention. Taken together, combined measurement of respiration and mtMP greatly enhances the diagnostic potential of OXPHOS analysis. Respirometric validation of inhibitory and uncoupling effects is mandatory for any fluorophore applied for probing mtMP, in any respiratory state, type of tissue and pathophysiological condition. Keywords: High-resolution respirometry, Mitochondrial membrane potential, Safranin, Complex I, Complex II, OXPHOS analysis, electron transfer-pathway

O2k-Network Lab: AT Innsbruck Oroboros, AT Innsbruck Gnaiger E, AT Innsbruck MitoCom


Labels: MiParea: Respiration, Instruments;methods 

Stress:Mitochondrial disease  Organism: Mouse  Tissue;cell: Nervous system  Preparation: Homogenate 

Regulation: Uncoupler  Coupling state: LEAK, OXPHOS, ET  Pathway: N, S, ROX  HRR: Oxygraph-2k, O2k-Fluorometer, O2k-Protocol 

Safranin, O2k-Demo, O2k-MultiSensor, 1PGM;2D;3U-, SUIT-006 

SUIT protocols

  • O2k-Protocol: »MiPNet20.13 Safranin mt-membranepotential
  • Safranin used in the experiments for this publication was Safranin O from Sigma (# S2255, 25 g).
  • The fluorescence sensor settings were Amp Polarization voltage = 1000 , gain = 1000 (Figure 9.1) and Amp Polarization voltage = 500 , gain = 1000 (Figures 9.6 and 9.7)

Methods Enzymol

  • Galluzzi L, Kroemer G (ed) (2014) Conceptual background and bioenergetic/mitochondrial aspects of oncometabolism. Methods Enzymol 542:509 pp »Bioblast link«

O2k-Publications

» O2k-Publications: Instruments;methods
» O2k-Publications: O2k-Fluorometry

O2k-brief

» List of O2k-Publications presented as O2k-brief


Template:Keywords Force and membrane potential