Opening of mitochondrial K(ATP) channels triggers the preconditioned state by generating free radicals

Abstract

The critical time for opening mitochondrial (mito) K(ATP) channels, putative end effectors of ischemic preconditioning (PC), was examined. In isolated rabbit hearts 29±3% of risk zone infarcted after 30 minutes of regional ischemia. Ischemic PC or 5-minute exposure to 10 μmol/L diazoxide, a mito K(ATP) channel opener, reduced infarction to 3±1% and 8±1%, respectively. The mito K(ATP) channel closer 5-hydroxydecanoate (200 μmol/L), bracketing either 5-minute PC ischemia or diazoxide infusion, blocked protection (24±3 and 28±6% infarction, respectively). However, 5-hydroxydecanoate starting 5 minutes before long ischemia did not affect protection. Glibenclamide (5 μmol/L), another K(ATP) channel closer, blocked the protection by PC only when administered early. These data suggest that K(ATP) channel opening triggers protection but is not the final step. Five minutes of diazoxide followed by a 30-minute washout still reduced infarct size (8±3%), implying memory as seen with other PC triggers. The protection by diazoxide was not blocked by 5 μmol/L chelerythrine, a protein kinase C antagonist, given either to bracket diazoxide infusion or just before the index ischemia. Bracketing preischemic exposure to diazoxide with 50 μmol/L genistein, a tyrosine kinase antagonist, did not affect infarction, but genistein blocked the protection by diazoxide when administered shortly before the index ischemia. Thus, although it is not protein kinase C-dependent, the protection by diazoxide involves tyrosine kinase. Bracketing diazoxide perfusion with N-(2-mercaptopropionyl) glycine (300 μmol/L) or Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (7 μmol/L), each of which is a free radical scavenger, blocked protection, indicating that diazoxide triggers protection through free radicals. Therefore, mito K(ATP) channels are not the end effectors of protection, but rather their opening before ischemia generates free radicals that trigger entrance into a preconditioned state and activation of kinases.