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Difference between revisions of "Uncoupler"

From Bioblast
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=== Noncoupled respiration ===
=== Noncoupled respiration ===
'''[[Noncoupled respiration]]''' is distinguished from general (pharmacological or mechanical) uncoupled respiration, to give a label to an effort to reach the ''fully uncoupled'' (non-coupled) state without inhibiting respiration. Non-coupled respiration, therefore, yields an estimate of [[ETS capacity]]. Experimentally uncoupled respiration may fail to yield an estimate of ETS capacity, due to inhibition of respiration above optimum uncoupler concentrations or insufficient stimulation by sub-optimal uncoupler concentrations. '''[[Optimum uncoupler concentration]]s''' for evaluation of (noncoupled) ETS capacity require inhibitor titrations <ref>Steinlechner-Maran R, Eberl T, Kunc M, Margreiter R, Gnaiger E (1996) Oxygen dependence of respiration in coupled and uncoupled endothelial cells. Am J Physiol Cell Physiol 271:C2053-61. [[Steinlechner-Maran_1996_Am J Physiol Cell Physiol |»PMID: 8997208]]«</ref>,<ref> Hütter E, Renner K, Pfister G, Stöckl P, Jansen-Dürr P, Gnaiger E (2004) Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts. Biochem J 380: 919-928. [[Huetter_2004_Biochem J |»Pubmed 15018610]]</ref>,<ref> Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial Dysfunction in Drug-Induced Toxicity (Dykens JA, Will Y, eds) John Wiley:327-52. [[Gnaiger_2008_POS |»Bioblast Access]]«</ref>
'''[[Noncoupled respiration]]''' is distinguished from general (pharmacological or mechanical) uncoupled respiration, to give a label to an effort to reach the ''fully uncoupled'' (non-coupled) state without inhibiting respiration. Non-coupled respiration, therefore, yields an estimate of [[ETS capacity]]. Experimentally uncoupled respiration may fail to yield an estimate of ETS capacity, due to inhibition of respiration above optimum uncoupler concentrations or insufficient stimulation by sub-optimal uncoupler concentrations. '''[[Optimum uncoupler concentration]]s''' for evaluation of (noncoupled) ETS capacity require inhibitor titrations <ref>Steinlechner-Maran R, Eberl T, Kunc M, Margreiter R, Gnaiger E (1996) Oxygen dependence of respiration in coupled and uncoupled endothelial cells. Am J Physiol Cell Physiol 271:C2053-61. [[Steinlechner-Maran_1996_Am J Physiol Cell Physiol |»PMID: 8997208]]«</ref>,<ref> Hütter E, Renner K, Pfister G, Stöckl P, Jansen-Dürr P, Gnaiger E (2004) Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts. Biochem J 380: 919-928. [[Huetter_2004_Biochem J |»PMID 15018610]]«</ref>,<ref> Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial Dysfunction in Drug-Induced Toxicity (Dykens JA, Will Y, eds) John Wiley:327-52. [[Gnaiger_2008_POS |»Bioblast Access]]«</ref>




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The optimum concentration of an uncoupler has to be determined for every biological system. It varies with incubation medium, sample concentratin, pharmacological treatment (with or without oligomycin), and pathophysiological state (e.g. induction of apoptosis). A single dose of uncoupler usually leads to an artefact in the estmation of maximum flux or electron transfer system capacity (for discussion, see [[Talk:Rogers_2011_PlosOne#Uncoupled_flux_does_not_reflect_electron_transfer_system_capacity|Artefacts by single dose uncoupling]]).
The optimum concentration of an uncoupler has to be determined for every biological system. It varies with incubation medium, sample concentratin, pharmacological treatment (with or without oligomycin), and pathophysiological state (e.g. induction of apoptosis). A single dose of uncoupler usually leads to an artefact in the estmation of maximum flux or electron transfer system capacity (for discussion, see [[Talk:Rogers_2011_PlosOne#Uncoupled_flux_does_not_reflect_electron_transfer_system_capacity|Artefacts by single dose uncoupling]]).


The optimum uncoupler (CCCP, FCCP, DNP) concentration for the noncoupled state varies over a large concentration range, depending on the medium ('binding' of uncoupler), type and concentration of sample.  This is true for various uncouplers, such as CCCP, FCCP and DNP. <ref> Steinlechner-Maran R, Eberl T, Kunc M, Margreiter R, Gnaiger E (1996) Oxygen dependence of respiration in coupled and uncoupled endothelial cells. Am J Physiol Cell Physiol 271:C2053-61. [[Steinlechner-Maran_1996_Am J Physiol Cell Physiol |»PMID: 8997208]]«</ref>  To evaluate the optimum concentration, a uncoupler titration has to be performed initially. For subsequent application series, we recommend a few titrations starting close to optimum concentration. <ref>Hütter E, Renner K, Pfister G, Stöckl P, Jansen-Dürr P, Gnaiger E (2004) Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts. Biochem J 380:919-28. [[Huetter_2004_Biochem J |»Pubmed 15018610]]«</ref>,<ref>Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopisies of human muscle. Methods Mol Biol 810:25-58. [[Pesta 2012 Methods Mol Biol |»Bioblast Access]]«</ref>  Optimum CCCP or FCCP concentrations range over an order of magnitude, from <0.5 to >4.0 µM.
The optimum uncoupler (CCCP, FCCP, DNP) concentration for the noncoupled state varies over a large concentration range, depending on the medium ('binding' of uncoupler), type and concentration of sample.  This is true for various uncouplers, such as CCCP, FCCP and DNP. <ref> Steinlechner-Maran R, Eberl T, Kunc M, Margreiter R, Gnaiger E (1996) Oxygen dependence of respiration in coupled and uncoupled endothelial cells. Am J Physiol Cell Physiol 271:C2053-61. [[Steinlechner-Maran_1996_Am J Physiol Cell Physiol |»PMID: 8997208]]«</ref>  To evaluate the optimum concentration, a uncoupler titration has to be performed initially. For subsequent application series, we recommend a few titrations starting close to optimum concentration. <ref>Hütter E, Renner K, Pfister G, Stöckl P, Jansen-Dürr P, Gnaiger E (2004) Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts. Biochem J 380:919-28. [[Huetter_2004_Biochem J |»PMID 15018610]]«</ref>,<ref>Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopisies of human muscle. Methods Mol Biol 810:25-58. [[Pesta 2012 Methods Mol Biol |»Bioblast Access]]«</ref>  Optimum CCCP or FCCP concentrations range over an order of magnitude, from <0.5 to >4.0 µM.


=== Uncoupler titration ===
=== Uncoupler titration ===

Revision as of 14:06, 17 January 2015


high-resolution terminology - matching measurements at high-resolution


Uncoupler

Description

An uncoupler is a protonophore (CCCP, FCCP, DNP) which cycles across the inner mt-membrane with transport of protons and dissipation of the electrochemical proton gradient. Mild uncoupling may be induced at low uncoupler concentrations, the noncoupled state of ETS capacity is obtained at optimum uncoupler concentration for maximum flux, whereas at higher concentrations an uncoupler-induced inhibition is observed. » MiPNet article

Abbreviation: U



MitoPedia topics: Uncoupler 

MitoPedia: Uncouplers

» MitoPedia: Uncouplers

Is respiration uncoupled - noncoupled - dyscoupled?

or loosely coupled?
Publications in the MiPMap
Gnaiger E (2014) Is respiration uncoupled - noncoupled - dyscoupled? Mitochondr Physiol Network 2014-04-18.


OROBOROS (2014) MiPNet

Abstract: Coupling of OXPHOS represents a complex concept. Uncoupler titrations provide an invaluable experimental tool.


O2k-Network Lab: AT Innsbruck Gnaiger E


Labels:




Regulation: Coupling efficiency;uncoupling  Coupling state: LEAK, ETS"ETS" is not in the list (LEAK, ROUTINE, OXPHOS, ET) of allowed values for the "Coupling states" property. 

HRR: Theory 


Uncoupled respiration

The uncoupled part of respiration in State P pumps protons to compensate for intrinsic uncoupling, which is a property of (a) the inner mt-membrane (proton leak), (b) the proton pumps (proton slip; decoupling), and (c) is regulated by molecular uncouplers (uncoupling protein, UCP1). Uncoupled and dyscoupled respiration are summarized as LEAK respiration. In contrast, noncoupled respiration is induced experimentally for evaluation of ETS capacity.[1],[2]

Uncoupled respiration - intrinsic

Uncoupling is used for intrinsic (physiological) uncoupling, appreciating the fact that we do not (never??) find mitochondria to be fully (mechanistically) coupled. In the ROUTINE (intact cells) and OXPHOS (mt-preparations) state of respiration, mitochondria are both, partially coupled and partially uncoupled. The uncoupled part of respiration in state P is larger than LEAK respiration evaluated in state L after inhibition of ATP synthase or adenine nucleotide translocase. This is due to the increase of mt-membrane potential in state L versus P, causing a corresponding increase of the proton leak driven by the higher proton motive force. As an approximation, however, the difference E-L yields an estimate of the physiological scope of uncoupling, or the pathological scope of dyscoupling.

Uncoupled respiration - experimental

Uncoupling is also used for directed experimental interventions to lower the degree of coupling, typically by application of established uncouplers (experimental use of a pharmacological intervention), less typical by freeze-thawing or mechanical crashing of mitochondrial membranes. Such experimental uncoupling can induce stimulation or inhibition of respiration.

Noncoupled respiration

Noncoupled respiration is distinguished from general (pharmacological or mechanical) uncoupled respiration, to give a label to an effort to reach the fully uncoupled (non-coupled) state without inhibiting respiration. Non-coupled respiration, therefore, yields an estimate of ETS capacity. Experimentally uncoupled respiration may fail to yield an estimate of ETS capacity, due to inhibition of respiration above optimum uncoupler concentrations or insufficient stimulation by sub-optimal uncoupler concentrations. Optimum uncoupler concentrations for evaluation of (noncoupled) ETS capacity require inhibitor titrations [3],[4],[5]


Dyscoupled respiration

Dyscoupled respiration is distinguished from intrinsically (physiologically) uncoupled and from extrinsic experimentally uncoupled respiration as an indication of extrinsic uncoupling (pathological, toxicological, pharmacological by agents that are not specifically applied to induce uncoupling, but are tested for their potential dyscoupling effect). Dyscoupling indicates a mitochondrial dysfunction. [6]


Continue the discussion


Experimental

Optimum uncoupler concentration

A titration of an uncoupler is necessary to achive the optimum concentration necessary for maximum stimulation of noncoupled respiration (ETS capacity) and to avoid inhibition of respiration by the too high uncoupler concentration. The underlying mechanism for the latter is not clear.

Uncouplers must be titrated carefully up to an optimum concentration for maximum stimulation of flux, since excess concentrations of uncoupler exert a strongly inhibitory effect.

See Steinlechner-Maran et al for a comparison of uncoupler titrations with FCCP and DNP from the ROUTINE state to the ETS state of cell respiration. [7] Uncoupler titrations after inhibition of respiration by oligomycin in the coupling control protocol with intact cells yield the sequence of ROUTINE respiration, LEAK respiration and ETS capacity, followed by inhibition to ROX. [8],[9] The highest accuracy of uncoupler titrations is achieved by titrations with the TIP2k at high concentrations of the stock solution. [10] Increasing the concentration in small steps, most accurately titrated by the TIP2k, is recommended (0.5 or 0.25 µM steps or even smaller).

The optimum concentration of an uncoupler has to be determined for every biological system. It varies with incubation medium, sample concentratin, pharmacological treatment (with or without oligomycin), and pathophysiological state (e.g. induction of apoptosis). A single dose of uncoupler usually leads to an artefact in the estmation of maximum flux or electron transfer system capacity (for discussion, see Artefacts by single dose uncoupling).

The optimum uncoupler (CCCP, FCCP, DNP) concentration for the noncoupled state varies over a large concentration range, depending on the medium ('binding' of uncoupler), type and concentration of sample. This is true for various uncouplers, such as CCCP, FCCP and DNP. [11] To evaluate the optimum concentration, a uncoupler titration has to be performed initially. For subsequent application series, we recommend a few titrations starting close to optimum concentration. [12],[13] Optimum CCCP or FCCP concentrations range over an order of magnitude, from <0.5 to >4.0 µM.

Uncoupler titration

In uncoupler titrations various uncouplers, such as CCCP, FCCP or DNP [14] are applied to uncouple mitochondrial electron transfer through Complexes I to IV from phosphorylation (Complex V or ATP synthase, ANT and phosphate transport), particularly with the aim to obtain the noncoupled state E with an optimum uncoupler concentration at maximum oxygen flux.


Uncoupling control ratio, UCR

Uncouplers may be used not only in isolated mitochondria or permeabilized tissue preparations, but also in intact cells. Uncouplers are permeable through the cell membrane, and intact cells contain energy substrates for mitochondrial respiration. The noncoupled (uncoupler-activated) state may be compared with ROUTINE respiration of the intact cells, in terms of the R/E or ROUTINE control ratio (compare: uncoupler control ratio, UCR). Or the non-coupled state may be the basis for evaluating LEAK respiration in the mitochondrial resting state induced by the addition of oligomycin (inhibitor of ATP synthase) or atractyloside (inhibitor of ANT), obtaing the L/E or LEAK control ratio (compare respiratory acceptor control ratio, RCR).

There are strong mathematical arguments to replace the conventional UCR and RCR by the corresponding flux control factors [15],[16] and flux control ratios.

1/UCR = ROUTINE respiration / Noncoupled respiration = R/E; ROUTINE control ratio
Compare: L/E; LEAK control ratio
Compare: P/E; OXPHOS control ratio


When using uncouplers in mitochondrial preparations (mt-preparations: isolated mitochondria and permeabilized tissue or cells), different applications are distinguished:

  1. External energy substrates have to be added to the preparation, since the endogenous substrates of the cytoplasm have been removed. A residual amount of internal mitochondrial substrates may be removed, if necessary, by an initial addition of a very small amount of ADP to the mitochondrial medium (e.g. MiR06) containing inorganic phosphate.
  2. In mt-preparations, an uncoupler may be added as a methodological test for plasma membrane permeabilization. If the inital addition of ADP does not exert a stimulatory effect, subsequent addition of uncoupler will increase respiratory flux if permeabilization has not been achieved.
  3. The classical respiratory control ratio (RCR=State 3/State 4) may be compared with an uncoupler-induced respiratory control ratio. Uncoupler titrations are initiated in a resting state, to induce an activated, noncoupled state. In the absence of adenylates (no ADP, ATP or AMP added), or in State 4 of isolated mitochondria (in the presence of ATP after phosphorylation of ADP), titration of uncoupler stimulates respiration. If OXPHOS has been initiated by the addition of a saturating concentration of ADP (which is different in isolated mitochondria versus permeabilized tissue or cell preparations), the experiment may be continued by addition of oligomycin or atractyloside, to return to a LEAK state, followed by uncoupler titration.
  4. Respiratory flux in the noncoupled state is compared with OXPHOS (saturating ADP in the coupled state), to evaluate metabolic flux control by the phosphorylation system over the electron transfer capacity. Importantly, flux control by the phosphorylation system depends on the combination of substrates and inhibitors applied to activate various segments of the electron transfer system, and varies in different states of cytochrome c release.


References

  1. Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int J Biochem Cell Biol 41:1837-45. »PMID: 19467914«
  2. Gnaiger E (2014) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 4th ed. Mitochondr Physiol Network 19.12. OROBOROS MiPNet Publications, Innsbruck:80 pp. »Open Access«
  3. Steinlechner-Maran R, Eberl T, Kunc M, Margreiter R, Gnaiger E (1996) Oxygen dependence of respiration in coupled and uncoupled endothelial cells. Am J Physiol Cell Physiol 271:C2053-61. »PMID: 8997208«
  4. Hütter E, Renner K, Pfister G, Stöckl P, Jansen-Dürr P, Gnaiger E (2004) Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts. Biochem J 380: 919-928. »PMID 15018610«
  5. Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial Dysfunction in Drug-Induced Toxicity (Dykens JA, Will Y, eds) John Wiley:327-52. »Bioblast Access«
  6. Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Mark W, Steurer W, Saks V, Usson Y, Margreiter R, Gnaiger E (2004) Mitochondrial defects and heterogeneous cytochrome c release after cardiac cold ischemia and reperfusion. Am J Physiol Heart Circ Physiol 286:H1633–41. »Open Access«
  7. Steinlechner-Maran R, Eberl T, Kunc M, Margreiter R, Gnaiger E (1996) Oxygen dependence of respiration in coupled and uncoupled endothelial cells. Am J Physiol Cell Physiol 271:C2053-61. »PMID: 8997208«
  8. Hütter E, Renner K, Pfister G, Stöckl P, Jansen-Dürr P, Gnaiger E (2004) Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts. Biochem J 380:919-28. »Open Access«
  9. Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial Dysfunction in Drug-Induced Toxicity (Dykens JA, Will Y, eds) John Wiley:327-52. »Bioblast Access«
  10. Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial Dysfunction in Drug-Induced Toxicity (Dykens JA, Will Y, eds) John Wiley:327-52. »Bioblast Access«
  11. Steinlechner-Maran R, Eberl T, Kunc M, Margreiter R, Gnaiger E (1996) Oxygen dependence of respiration in coupled and uncoupled endothelial cells. Am J Physiol Cell Physiol 271:C2053-61. »PMID: 8997208«
  12. Hütter E, Renner K, Pfister G, Stöckl P, Jansen-Dürr P, Gnaiger E (2004) Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts. Biochem J 380:919-28. »PMID 15018610«
  13. Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopisies of human muscle. Methods Mol Biol 810:25-58. »Bioblast Access«
  14. Fontana-Ayoub M, Fasching M, Gnaiger E (2014) Selected media and chemicals for respirometry with mitochondrial preparations. Mitochondr Physiol Network 03.02(17):1-9. »Open Access«
  15. Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int J Biochem Cell Biol 41:1837-45. »PMID: 19467914«
  16. Gnaiger E. Biochemical coupling efficiency: from 0 to <1. Mitochondr Physiol Network. »Biochemical coupling efficiency«