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(Referência obtida automaticamente do Web of Science, por meio da informação sobre o financiamento pela FAPESP e o número do processo correspondente, incluída na publicação pelos autores.)

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

Texto completo
Autor(es):
Ruas, Juliana S. [1] ; Siqueira-Santos, Edilene S. [1] ; Amigo, Ignacio [2] ; Rodrigues-Silva, Erika [1] ; Kowaltowski, Alicia J. [2] ; Castilho, Roger F. [1]
Número total de Autores: 6
Afiliação do(s) autor(es):
[1] Univ Estadual Campinas, UNICAMP, Dept Patol Clin, Fac Ciencias Med, Campinas, SP - Brazil
[2] Univ Sao Paulo, Inst Quim, Dept Bioquim, BR-01498 Sao Paulo, SP - Brazil
Número total de Afiliações: 2
Tipo de documento: Artigo Científico
Fonte: PLoS One; v. 11, n. 3 MAR 7 2016.
Citações Web of Science: 9
Resumo

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. (AU)

Processo FAPESP: 12/51288-1 - Papel da restrição calórica na excitotoxicidade cerebral
Beneficiário:Ignacio Amigo de La Huerga
Linha de fomento: Bolsas no Brasil - Pós-Doutorado
Processo FAPESP: 13/07937-8 - Redoxoma
Beneficiário:Ohara Augusto
Linha de fomento: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs
Processo FAPESP: 11/50400-0 - Metabolismo energético, estado redox e funcionalidade mitocondrial na morte celular e em desordens cardiometabólicas e neurodegenerativas
Beneficiário:Aníbal Eugênio Vercesi
Linha de fomento: Auxílio à Pesquisa - Temático
Processo FAPESP: 10/51906-1 - Bioenergética, transporte iônico, balanço redox e metabolismo de DNA em mitocôndrias
Beneficiário:Alicia Juliana Kowaltowski
Linha de fomento: Auxílio à Pesquisa - Temático