title: Characterization of the stimulus for reactive oxygen species generation in calcium-overloaded mitochondria.
abstract: We have used two different probes with distinct detection properties, dichlorodihydrofluorescein diacetate and Amplex Red/horseradish peroxidase, as well as different respiratory substrates and electron transport chain inhibitors, to characterize the reactive oxygen species (ROS) generation by the respiratory chain in calcium-overloaded mitochondria. Regardless of the respiratory substrate, calcium stimulated the mitochondrial generation of ROS, which were released at both the mitochondrial-matrix side and the extra-mitochondrial space, in a way insensitive to the mitochondrial permeability transition pores inhibitor cyclosporine A. In glutamate/malate-energized mitochondria, inhibition at complex I or complex III (ubiquinone cycle) similarly modulated ROS generation at either mitochondrial-matrix side or extra-mitochondrial space; this also occurred when the backflow of electrons to complex I in succinate-energized mitochondria was inhibited. On the other hand, in succinate-energized mitochondria the modulation of ROS generation at mitochondrial-matrix side or extra-mitochondrial space depends on the site of complex III which was inhibited. These results allow a straight comparison between the effects of different respiratory substrates and electron transport chain inhibitors on ROS generation at either mitochondrial-matrix side or extra-mitochondrial space in calcium-overloaded mitochondria.
authors.affiliation: Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
authors.forename: Fernando P
authors.initials: FP
authors.lastname: Rodrigues
authors.forename: Cezar R
authors.initials: CR
authors.lastname: Pestana
authors.forename: Guilherme A
authors.initials: GA
authors.lastname: Dos Santos
authors.forename: Gilberto L
authors.initials: GL
authors.lastname: Pardo-Andreu
authors.forename: Antonio C
authors.initials: AC
authors.lastname: Santos
authors.forename: Sérgio A
authors.initials: SA
authors.lastname: Uyemura
authors.forename: Luciane C
authors.initials: LC
authors.lastname: Alberici
authors.forename: Carlos
authors.initials: C
authors.lastname: Curti
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D016572:Cyclosporine / Q000494:pharmacology; D004579:Electron Transport; D042967:Electron Transport Complex I / Q000037:antagonists & inhibitors / Q000378:metabolism; D014450:Electron Transport Complex III / Q000037:antagonists & inhibitors / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D018698:Glutamic Acid / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism; D008297:Male; D053078:Membrane Potential, Mitochondrial; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000378:metabolism; D010078:Oxazines / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012402:Rotenone / Q000494:pharmacology; D019802:Succinic Acid / Q000494:pharmacology
chemical_list: C110400:2',7'-dichlorodihydrofluorescein diacetate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D010078:Oxazines; D017382:Reactive Oxygen Species; D012402:Rotenone; C470430:Amplex Red; D018698:Glutamic Acid; D016572:Cyclosporine; D019802:Succinic Acid; D006861:Hydrogen Peroxide; D042967:Electron Transport Complex I; D014450:Electron Transport Complex III; D002118:Calcium