Underestimation of the Maximal Capacity of the Mitochondrial Electron Transport System in Oligomycin-Treated Cells

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. 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 results demonstrate that the presence of oligomycin or its A-isomer leads to underestimation of maximal ETS capacity. 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 inhibitory effect of oligomycin on SRC was more pronounced in T98G cells and was observed in both suspended and attached cells. Underestimation of SRC also occurred when oxidative phosphorylation was fully inhibited by the ATP synthase inhibitor citreoviridin. 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. We replaced CCCP by FCCP, another potent protonophore and similar results were 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. 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. We conclude that unless a previously validated protocol is employed, maximal ETS capacity in intact cells should be estimated without oligomycin. The inhibitory effect of an ATP synthase blocker on potent protonophore-induced maximal OCR may be associated with impaired metabolism of mitochondrial respiratory substrates.

完整细胞中线粒体电子传递系统（mitochondrial electron transport system, ETS）的最大容量，通常可通过先以寡霉素抑制氧化磷酸化，再经质子载体诱导细胞达到最大耗氧量的方式进行估算。本研究针对人类胶质瘤细胞系（T98G、U-87MG）与前列腺癌细胞系（PC-3），在常规培养基中利用呼吸仪，以梯度浓度的质子载体CCCP（carbonyl cyanide m-chlorophenyl hydrazone）诱导细胞达到最大耗氧速率（oxygen consumption rate, OCR）。
实验结果显示，寡霉素或其A异构体的存在会导致ETS最大容量的估算值偏低。当存在寡霉素时，储备呼吸能力（spare respiratory capacity, SRC，即最大OCR与基础细胞OCR的差值）的估算值会被低估25%~45%。寡霉素对SRC的抑制作用在T98G细胞中更为显著，且在悬浮细胞与贴壁细胞中均有观测到。当氧化磷酸化被ATP合酶抑制剂citreoviridin完全抑制时，同样会出现SRC被低估的情况。
进一步实验表明，寡霉素无法被腺苷酸转位酶抑制剂孟加拉酸（bongkrekic acid）或羧基苍术苷（carboxyatractyloside）替代：尽管此类化合物对透化细胞具有作用，但它们无法抑制完整细胞中的氧化磷酸化过程。我们将CCCP替换为另一强效质子载体FCCP后，得到了相似的实验结果。使用较弱的质子载体2,4-二硝基苯酚时，测得的最大OCR与SRC值更低，且寡霉素的存在对这两项参数无显著影响。在透化细胞或添加呼吸底物孵育的分离脑线粒体中，仅观察到寡霉素对CCCP诱导的最大OCR存在微弱的抑制作用。
综上，除非采用此前已验证的实验方案，否则完整细胞的ETS最大容量应在不添加寡霉素的条件下进行估算。ATP合酶阻滞剂对强效质子载体诱导的最大OCR产生的抑制作用，可能与线粒体呼吸底物的代谢受损有关。