Regulation of the mitochondrial ATP-sensitive potassium channel in rat uterus cells by ROS

摘要

In previous study we demonstrated the presence of ATP-sensitive potassium current in the inner mitochondrial membrane, which was sensitive to diazoxide and glybenclamide, in mitochondria isolated from the rat uterus. This current was supposed to be operated by mitochondrial ATP-sensitive potassium channel (mitoKsubATP/sub). Regulation of the mitoKsubATP/sub in uterus cells is not studied well enough yet. It is well known that the reactive oxygen species (ROS) can play a dual role. They can damage cells in high concentrations, but they can also act as messengers in cellular signaling, mediating survival of cells under stress conditions. ROS are known to activate mitoKsubATP/sub during the oxidative stress in the brain and heart, conferring the protection of cells. The present study examined whether ROS mediate the mitoKsubATP/sub activation in myometrium cells. Oxidative stress was induced by rotenone. ROS generation was measured by 2′,7′-dichlorofluorescin diacetate. The massive induction of ROS production was demonstrated in the presence of rotenone. Hyperpolarization of the mitochondrial membrane was also detected with the use of the potential-sensitive dye DiOCsub6/sub (3,3′-dihexyloxacarbocyanine iodide). Diazoxide, a selective activator of mitoKsubATP/sub, depolarized mitochondrial membrane either under oxidative stress or under normal conditions, while mitoKsubATP/sub blocker glybenclamide effectively restored mitochondrial potential in rat myocytes. Estimated 1/2/sub value for diazoxide to mitoKsubATP/sub under normoxia was four times higher than under oxidative stress conditions: 5.01 ± 1.47?10-6 М and 1.24 ± 0.21?10-6 М respectively. The ROS scavenger N-acetylcysteine (NAC) successfully eliminates depolarization of mitochondrial membrane by diazoxide under oxidative stress. These results suggest that elimination of ROS by NAC prevents the activation of mitoKsubATP/sub under oxidative stress. Taking into account the higher affinity of diazoxide to mitoKsubATP/sub under stress conditions than under normoxia, we conclude that the oxidative stress conditions are more favourable than normoxia for the activation of mitoKsubATP/sub. Thus we hypothesize that the ROS regulate the activity of the mitoKsubATP/sub in myocytes.