Opening the mitochondrial
ATP-sensitive K(+) channel (
mitoK(ATP)) 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
mitoK(ATP)-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 K(
ATP) 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 K(
ATP) 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
mitoK(ATP)-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.