Opening of ATP-sensitive potassium channels causes generation of free radicals in vascular smooth muscle cells

Recent evidence suggests that opening of mitochondrial K-ATP channels in cardiac muscle triggers the preconditioning phenomenon through free radical production. The present study tested the effects of K-ATP channel openers in a vascular smooth muscle cell model using the fluorescent probe MitoTracker (MTR) Red(TM) for detection of reactive oxygen species (ROS). Rat aortic smooth muscle cells (A7r5) were incubated with 1 muM reduced MTR (non-fluorescent) and the MTR oxidation product (fluorescent) was quantified. Thirty-minute pretreatment with either diazoxide (200 muM) or pinacidil (100 muM), both potent mitochondrial K-ATP channel openers, increased fluorescent intensity (FI) to 149 and 162% of control (p < 0.05 for both), respectively, and the K-ATP channel inhibitor 5-hydroxydecanoate (5HD) blocked it. Valinomycin, a potassium-selective ionophore, raised FI to 156% of control (p < 0.05). However, 5HD did not affect the valinomycin-induced increase in FI. Inhibition of mitochondrial electron transport (myxothiazol) or uncoupling of oxidative phosphorylation (dinitrophenol) also blocked either valinomycin- or diazoxide-induced increase in FI, and free radical scavengers prevented any diazoxide-mediated increase in fluorescence. Finally the diazoxide-induced increase in fluorescence was not. blocked by the PKC inhibitor chelerythrine, but was by HMR 1883, a putative surface K-ATP channel blocker. Thus opening of K-ATP channels increases generation of ROS via the mitochondrial electron transport chain in vascular smooth muscle cells. Furthermore, a potassium-selective ionophore can mimic the effect of putative mitochondrial K-ATP channel openers. We conclude that potassium movement through K-ATP directly leads to ROS production by the mitochondria.