The fraction of basal OCR (or ROUTINE respiration, as it is referred to in ) inhibited by addition of the ATP synthase inhibitor oligomycin gives an estimate of the respiration rate necessary to sustain cellular ATP turnover under basal conditions. When using intact cells, specific respirometric protocols are usually employed to evaluate mitochondrial function and can provide valuable information such as that described below. When the plasma membrane is permeabilized or isolated mitochondria are studied, the respiratory control ratio can be assessed in a medium supplemented with respiratory substrates by measuring the increase in OCR after the addition of ADP. Oxygen consumption rate (OCR) measurements are one of the preferred methods for mitochondrial function or dysfunction evaluation in cultured cells (for reviews see, ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist. JSR and ER-S are recipients of CAPES fellowships and IA is recipient of a FAPESP (#12/51288-1) fellowship. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.ĭata Availability: All relevant data are within the paper and its Supporting Information files.įunding: This work was supported by grants from the São Paulo Research Foundation (FAPESP, #11/50400-0, #10/51906-1 and #13/07937-8) and the Brazilian National Council for Scientific and Technological Development (CNPq). Received: DecemAccepted: FebruPublished: March 7, 2016Ĭopyright: © 2016 Ruas et al. Pizzo, Duke University Medical Center, UNITED STATES The inhibitory effect of an ATP synthase blocker on potent protonophore-induced maximal OCR may be associated with impaired metabolism of mitochondrial respiratory substrates.Ĭitation: Ruas JS, Siqueira-Santos ES, Amigo I, Rodrigues-Silva E, Kowaltowski AJ, Castilho RF (2016) Underestimation of the Maximal Capacity of the Mitochondrial Electron Transport System in Oligomycin-Treated Cells. We conclude that unless a previously validated protocol is employed, maximal ETS capacity in intact cells should be estimated without oligomycin. In permeabilized cells or isolated brain mitochondria incubated with respiratory substrates, only a minor inhibitory effect of oligomycin on CCCP-induced maximal OCR was observed. Lower maximal OCR and SRC values were obtained with the weaker protonophore 2,4-dinitrophenol, and these parameters were not affected by the presence of oligomycin. We replaced CCCP by FCCP, another potent protonophore and similar results were observed. Further experiments indicated that oligomycin cannot be replaced by the adenine nucleotide translocase inhibitors bongkrekic acid or carboxyatractyloside because, although these compounds have effects in permeabilized cells, they do not inhibit oxidative phosphorylation in intact cells. Underestimation of SRC also occurred when oxidative phosphorylation was fully inhibited by the ATP synthase inhibitor citreoviridin. The inhibitory effect of oligomycin on SRC was more pronounced in T98G cells and was observed in both suspended and attached cells. In the presence of oligomycin, the spare respiratory capacity (SRC), i.e., the difference between the maximal and basal cellular OCR, was underestimated by 25 to 45%. The results demonstrate that the presence of oligomycin or its A-isomer leads to underestimation of maximal ETS capacity. In the present study, human glioma (T98G and U-87MG) and prostate cancer (PC-3) cells were titrated with different concentrations of the protonophore CCCP to induce maximal oxygen consumption rate (OCR) within respirometers in a conventional growth medium. The maximal capacity of the mitochondrial electron transport system (ETS) in intact cells is frequently estimated by promoting protonophore-induced maximal oxygen consumption preceded by inhibition of oxidative phosphorylation by oligomycin.