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Báo cáo khoa học: Characterization of depolarization and repolarization phases of mitochondrial membrane potential fluctuations induced by tetramethylrhodamine methyl ester photoactivation

Depolarization and repolarization phases (D and R phases, respectively) of mitochondrial potential fluctuations induced by photoactivation of the fluorescent probe tetramethylrhodamine methyl ester (TMRM) were ana-lyzed separately and investigated using specific inhibitors and substrates. The frequency of R phases was significantly inhibited by oligomycin and aurovertin (mitochondrial ATP synthase inhibitors), rotenone (mitochond-rial complex I inhibitor) and iodoacetic acid (inhibitor of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase) | ềFEBS Journal Characterization of depolarization and repolarization phases of mitochondrial membrane potential fluctuations induced by tetramethylrhodamine methyl ester photoactivation Angela M. Falchi Raffaella Isola Andrea Diana Martina Putzolu and Giacomo Diaz Department of Cytomorphology University of Cagliari Monserrato Italy Keywords fluorescent probes mitochondria photoactivation potential fluctuations tetramethylrhodamine methyl ester TMRM Correspondence G. Diaz Department of Cytomorphology University of Cagliari I-40492 Monserrato Italy Fax 39 70 6754003 Tel 39 70 6754081 E-mail gdiaz@unica.it Received 5 October 2004 revised 21 January 2005 accepted 26 January 2005 doi 10.1111 j.1742-4658.2005.04586.x Depolarization and repolarization phases D and R phases respectively of mitochondrial potential fluctuations induced by photoactivation of the fluorescent probe tetramethylrhodamine methyl ester TMRM were analyzed separately and investigated using specific inhibitors and substrates. The frequency of R phases was significantly inhibited by oligomycin and aurovertin mitochondrial ATP synthase inhibitors rotenone mitochondrial complex I inhibitor and iodoacetic acid inhibitor of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase . Succinic acid mitochondrial complex II substrate given in the permeable form of dimethyl ester abolished the rotenone-induced inhibition of R phases. Taken together these findings indicate that the activity of both respiratory chain and ATP synthase were required for the recovery of the mitochondrial potential. The frequency of D phases prevailed over that of R phases in all experimental conditions resulting in a progressive depolarization of mitochondria accompanied by NAD P H oxidation and Ca2 influx. D phases were not blocked by cyclosporin A inhibitor of the permeability transition pore or o-phenyl-EGTA a Ca2 chelator suggesting that the permeability transition pore was not involved in mitochondrial potential .