MiP2005: Session 6
Mitochondrial Physiology Network 10.9: 79-80 (2005) - download pdf
- Note added after publication: Abstracts published with A Garedew as first author have to be critically re-evaluated and the corresponding data will not be published as presented in these abstracts (Erich Gnaiger).
Respiratory coupling control ratio and respiratory capacity in cultured fibroblasts and myeloid progenitor cells. Effects of experimental cell density.
Assegid Garedew,1,2 A Naimi,1 B Haffner,1 J Troppmair,1 E Gnaiger1
1Daniel-Swarovski Research Lab., Dept. General Transplant Surgery, Innsbruck Medical Univ.; 2OROBOROS INSTRUMENTS, Innsbruck, Austria.- firstname.lastname@example.org
The uncoupling control ratio (UCR) is a sensitive index for the integrity of mitochondrial function in cells. The UCR is the respiratory ratio of uncoupled to physiologically controlled states, when intact cells are incubated in culture medium (routine respiration) or in the absence of exogenous energy substrates (endogenous respiration). Even in healthy control cells, large differences of UCR values are reported, with high UCR typically ranging from 2.5 to 3 at experimental cell densities of 0.2 to 1×106 ml-1 [1-3]. In contrast, UCR values of 1.1 to 1.9 are reported by other groups using experimental cell densities >3×106 ml-1 [4,5]. Hypothetically, cell-cell signalling may exert a density-dependent effect on routine respiration in the physiologically controlled state, whereas removing respiratory control by uncoupling should eliminate such potential density-related effects.
We measured respiratory activities of intact NIH3T3 fibroblasts (adherent) and mouse pro-myeloid 32D (suspension) cells, each at two cell densities, using the OROBOROS Oxygraph-2k for high resolution respirometry. Cells were grown to a standardized density. Cells from each culture flask (N=12 for each cell type) were suspended at low and high experimental densities (0.2 and 2.0×106∙ml-1 for NIH3T3; 0.5 and 5.0×106∙ml-1 for 32D). After recording routine respiration of cells suspended in 2 ml culture medium, ATP-synthase was inhibited by oligomycin (2 µg∙ml-1). Increasing the oligomycin concentration had no effect on respiration. Subsequently, stepwise FCCP titration was performed up to the optimum FCCP concentration required for maximum stimulation of respiration. Optimum FCCP was 6.3 ± 1.4 and 7.8 ± 2.5 µM at low cell density, and decreased significantly at high density by a factor of 1.4 and 1.6 in NIH3T3 and 32D, respectively. Finally, respiration was inhibited by 0.5 µM rotenone and 2.5 µM antimycin A. Respiratory oxygen flux was corrected on-line for instrumental background (DatLab 4), which is a standard procedure in high-resolution respirometry  and is particularly important at low cell densities, to eliminate corresponding methodological artefacts. Data were analysed by a paired t-test and presented as means ± SD.
Routine respiration was 58.7 ± 5.4 and 22.1 ± 3.5 pmol∙s-1∙10-6 cells in fibroblasts and 32D, respectively, independent of cell density. Uncoupling by FCCP stimulated respiration of fibroblasts 3.2 ± 0.17 fold and 2.9 ± 0.22 fold above routine levels at high and low cell density. An even more pronounced effect of cell density was observed in 32D cells (UCR was 2.7 ± 0.4 and 2.0 ± 0.4 at high and low density). Uncoupled respiration, therefore, decreased significantly by 13 % and 26 % in fibroblasts and 32D at low density. In contrast, respiration inhibited by oligomycin increased significantly at low density in both cells types (Fig. 1). The respiratory control ratio (uncoupled to oligomycin-inhibited respiration, RCRu/o) decreased two-fold at low cell density, from 9.5 to 5.7 in NIH3T3 and from 10.0 to 5.0 in 32D cells. This significant change of the RCRu/o at constant routine respiration was in direct contrast to hypothetical expectations on the effects of cell density. These density-related effects were comparable in magnitude to changes in RCRu/o reported in the context of oxidative stress , senescence , or cell cycle arrest .
The divergent effect of experimental cell density on respiration in the two consecutively induced metabolic states rules out experimental artefacts related to the oxygen measuring system and to potential errors in cell counting. Further control experiments were performed on digitonin-permeabilized 32D cells, which were suspended at the two different densities in mitochondrial medium MiR05. The optimum digitonin concentration was adjusted to cell density, and maximum respiration was measured with succinate/rotenone and 3 mM ADP. Cytochrome c had no effect and FCCP stimulated respiration by only 10 % above state 3. As expected, oxygen flow (respiration per million cells) was identical at low and high cell density.
Respiration inhibited by rotenone+ antimycin A (CRA) was 9.7 ± 2.8 and 3.0 ± 0.7 pmol∙s-1∙10-6 cells in intact NIH3T3 and 32D measured at low cell densities at an oxygen concentration of 80 µM in culture medium. After permeabilization of cells with digitonin in mitochondrial respiration medium MiR05, respiration inhibited by rotenone+antimycin A was significantly lower than in intact cells (3.1 ± 1.5 and 1.1 ± 0.6 pmol∙s-1∙10-6 in the two cells types). The difference provides a minimum estimate of non-mitochondrial respiration in intact cells, amounting to 10 % of routine respiration in both cell types, which increases at high oxygen concentration . At high cell density, the rotenone+antimycin A inhibited respiration was significantly lower in NIH3T3 and 32D cells, measured at 80 µM oxygen in culture medium, possibly indicating a correspondingly decreased ROS production at high protein concentration. Subtraction of the non-mitochondrial component from total respiration at different cell densities did not remove the density effect in the oligomycin-inhibited or uncoupled states. Permeabilized cells had identical rotenone+antimycin A inhibited respiration at high and low cell density.
Our results point to the importance of application of comparable cell densities in respiratory studies. Though our data clearly show that cell density affects UCR, this does not explain low UCR values reported in the literature with the use of high experimental cell density.
1. 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. 271: C2053-C2061.
2. Stadlmann S, Rieger G, Amberger A, Kuznetsov AV, Margreiter R, Gnaiger E (2002) H2O2-mediated oxidative stress versus cold ischemia-reperfusion:mitochondrial respiratory defects in cultured human endothelial cells. Transplantation 74: 1800-1803.
3. 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.
4. Villani G, Attardi G (1997) In vivo control of respiration by cytochrome c oxidase in wild-type and mitochondrial DNA mutation-carrying human cells. Proc. Natl. Acad. Sci. USA 94: 1166-1171.
5. Villani G, Greco M, Papa S, Attardi G (1998) Low reserve of cytochrome c oxidase capacity in vivo in the respiratory chain of a variety of human cell types. J. Biol. Chem. 273: 31829-31836.
6. Gnaiger E (2003) Oxygen conformance of cellular respiration. A perspective of mitochondrial physiology. Adv. Exp. Med. Biol. 543: 39-55.