title: Respective contribution of mitochondrial superoxide and pH to mitochondria-targeted circularly permuted yellow fluorescent protein (mt-cpYFP) flash activity.
abstract: Superoxide flashes are transient bursts of superoxide production within the mitochondrial matrix that are detected using the superoxide-sensitive biosensor, mitochondria-targeted circularly permuted YFP (mt-cpYFP). However, due to the pH sensitivity of mt-cpYFP, flashes were suggested to reflect transient events of mitochondrial alkalinization. Here, we simultaneously monitored flashes with mt-cpYFP and mitochondrial pH with carboxy-SNARF-1. In intact cardiac myocytes and purified skeletal muscle mitochondria, robust mt-cpYFP flashes were accompanied by only a modest increase in SNARF-1 ratio (corresponding to a pH increase of <0.1), indicating that matrix alkalinization is minimal during an mt-cpYFP flash. Individual flashes were also accompanied by stepwise increases of MitoSOX signal and decreases of NADH autofluorescence, supporting the superoxide origin of mt-cpYFP flashes. Transient matrix alkalinization induced by NH4Cl only minimally influenced flash frequency and failed to alter flash amplitude. However, matrix acidification modulated superoxide flash frequency in a bimodal manner. Low concentrations of nigericin (< 100 nM) that resulted in a mild dissipation of the mitochondrial pH gradient increased flash frequency, whereas a maximal concentration of nigericin (5 μm) collapsed the pH gradient and abolished flash activity. These results indicate that mt-cpYFP flash events reflect a burst in electron transport chain-dependent superoxide production that is coincident with a modest increase in matrix pH. Furthermore, flash activity depends strongly on a combination of mitochondrial oxidation and pH gradient.
authors: Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York 14642, USA.
authors: Lan
authors: L
authors: Wei-LaPierre
authors: Guohua
authors: G
authors: Gong
authors: Brent J
authors: BJ
authors: Gerstner
authors: Sylvie
authors: S
authors: Ducreux
authors: David I
authors: DI
authors: Yule
authors: Sandrine
authors: S
authors: Pouvreau
authors: Xianhua
authors: X
authors: Wang
authors: Shey-Shing
authors: SS
authors: Sheu
authors: Heping
authors: H
authors: Cheng
authors: Robert T
authors: RT
authors: Dirksen
authors: Wang
authors: W
authors: Wang
mesh_terms: D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001578:Benzopyrans / Q000494:pharmacology; D002478:Cells, Cultured; D045222:Electron Transport Chain Complex Proteins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007476:Ionophores / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008929:Mitochondria, Heart / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism*; D009284:Naphthols / Q000494:pharmacology; D009550:Nigericin / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D018892:Proton-Motive Force / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012235:Rhodamines / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D001578:Benzopyrans; D045222:Electron Transport Chain Complex Proteins; D005456:Fluorescent Dyes; D007476:Ionophores; D008164:Luminescent Proteins; D009284:Naphthols; D012235:Rhodamines; C091666:seminaphthorhodaminefluoride; C054562:yellow fluorescent protein, Bacteria; D000643:Ammonium Chloride; D013481:Superoxides; D009550:Nigericin