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1 Dept of Biology, Portland State University, Portland, Oregon, United States
* To whom correspondence should be addressed. E-mail: garlid{at}pdx.edu.
Opening the mitochondrial ATP-sensitive K+ channel (mitoKATP) increases levels of reactive oxygen species (ROS) in cardiomyocytes. This increase in ROS is necessary for cardioprotection against ischemia-reperfusion injury; however the mechanism of mitoKATP-dependent stimulation of ROS production is unknown. We examined ROS production in suspensions of isolated rat heart and liver mitochondria, using fluorescent probes that are sensitive to hydrogen peroxide. When mitochondria were treated with the KATP channel openers diazoxide or cromakalim, their ROS production increased by 40-50%, and this effect was blocked by 5-hydroxydecanoate. ROS production exhibited a biphasic dependence on valinomycin concentration, with peak production occurring at valinomycin concentrations that catalyze about the same K+ influx as KATP channel openers. ROS production decreased with higher concentrations of valinomycin and with all concentrations of a classical protonophoretic uncoupler. Our studies show that the increase in ROS is due specifically to K+ influx into the matrix and is mediated by the attendant matrix alkalinization. Myxothiazol stimulated mitoKATP-dependent ROS production whereas rotenone had no effect. This indicates that the superoxide originates in complex I (NADH:ubiquinone oxidoreductase) of the electron transport chain.
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