Problem with FCCP
- Correspondence to MIG-LIST, relating to lack of respiratory stimulation up to 1 µM FCCP, and a suggestion that titrations with DNP and to a lesser extent with CCCP are less problematic: http://sigs.nih.gov/mito/Pages/default.aspx
- In our hands, a careful uncoupler titration is the basis of any accurate assessment of the effect of uncouplers on mitochondrial respiration. Optimum concentrations for a specific uncoupler vary by more than an order of magnitude, depending on experimental conditions.
- Dinitrophenole (DNP) has as sharp an optimum concentration range as FCCC, but at 400-500 µM DNP compared to 2- 3 µM FCCP (in intact HUVEC in culture medium; PMID: 8997208)., Optimum concentrations cannot be extrapolated to other media, cell types or cell concentrations, mt-preparations, pathophysiological states or various pharmacological treatments. CCCP and FCCP have similar ranges of optimum concentration (CCCP>FCCP) for maximum electron transfer capacity (ETS), but CCCP is sigificantly less expensive:
- In respirometry with intact cells, uncoupler titrations are very informative in the absence versus presence of ATPase inhibitors (e.g. oligomycin, Omy). In several cases, uncoupler titrations after Omy inhibition do not even recover respiratory activity at pre-inhibition ROUTINE levels of respiration.
- -- Gnaiger Erich 06:03, 22 April 2014 (CEST)
- 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-C2061. »Bioblast link«
- Added note: as shown in Fig. 5 (see reference), the uncoupler concentration needs to be scaled relative to the optimum concentration for comparison of 'sharp' optimum concentration ranges of different uncouplers.
Which meaning of 'uncoupling'?
- I was getting increasingly worried about the fact that students have difficulties to understanding uncoupling or coupling in bioenergetics and mitochondrial physiology. In this context we should realize that conventional terminology is not particularly helpful. In fact, the meaning of 'uncoupling' (in the current literature and bioenergetics textbooks) must usually be deduced from the specific context, but this can be achieved only after the concept and context are clear. Are intact mitochondria partially uncoupled, or partially coupled?
- Exaggerating, we may be used to phrases such as:
- Uncoupling was studied in stressed cells by measurement of coupled respiration, evaluation of uncoupled respiration after inhibition with oligomycin, and FCCP titration to quantify uncoupled respiration.
- Perhaps this is intuitively more clear:
- I am even still worried about the different meanings of (1) intrinsic and (2) experimental uncoupling, but have not found a solution. The distinction between dyscoupling versus intrinsic (adaptive?) uncoupling is a fundamental challenge, not of terminology, but of understanding the physiological functions of intrinsic uncoupling.
- --Gnaiger Erich 09:01, 31 May 2011 (CEST)
Uncoupler titrations cause small jumps in oxygen concentration
- Q: When adding FCCP, the O2 concentration rises a bit. Does this mean that the cells are consuming or releasing oxygen?
- A: You add FCCP dissolved in ethanol. Ethanol has a high oxygen solubility, hence your titration is the cause of the increase in oxygen concentration.
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