ROS and NO trigger early preconditioning: relationship to mitochondrial K-ATP channel

Reactive oxygen species (ROS) and nitric oxide (NO) are implicated in induction of ischemic preconditioning. However, the relationship between these oxidant signals and opening of the mitochondrial ATP-dependent potassium (K-ATP) channel during early preconditioning is not fully understood. We observed preconditioning protection by hypoxia, exogenous H2O2, or PKC activator PMA in cardiomyocytes subjected to 1-h ischemia and 3-h reperfusion. Protection was abolished by K-ATP channel blocker 5-hydroxydecanoate (5-HD) in each case, indicating that these triggers must act upstream from the K-ATP channel. Inhibitors of NO synthase abolished protection in preconditioned cells, suggesting that NO is also required for protection. DAF-2 fluorescence (NO sensitive) increased during hypoxic triggering. This was amplified by pinacidil and inhibited by 5-HD, indicating that NO is generated subsequent to K-ATP channel activation. Exogenous NO during the triggering phase conferred protection blocked by 5-HD. Exogenous NO also restored protection abolished by 5-HD or N-omega-nitro-L-arginine methyl ester in preconditioned cells. Antioxidants given during pinacidil or NO triggering abolished protection, confirming that ROS are generated by K-ATP channel activation. Coadministration of H2O2 and NO restored PMA-induced protection in 5-HD-treated cells, indicating that ROS and NO are required downstream from the K-ATP channel. We conclude that ROS can trigger preconditioning by causing activation of the K-ATP channel, which then induces generation of ROS and NO that are both required for preconditioning protection.