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

The critical time for opening mitochondrial (mito) K-ATP channels, putative end effecters of ischemic preconditioning (PC), was examined. In isolated rabbit hearts 29+/-3% of risk zone infarcted after 30 minutes of regional i schemia. Ischemic PC or 5-minute exposure to 10 mu mol/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 mu mol/L), bracketi ng either 5-minute PC ischemia or diazoxide infusion, blocked protection (2 4+/-3 and 28+/-6% infarction, respectively). However, 5-hydroxydecanoate st arting 5 minutes before long ischemia did not affect protection, Glibenclam ide (5 mu mol/L), another K-ATP channel closer, blocked the protection by P C only when administered early. These data suggest that K-ATP channel openi ng triggers protection but is not the final step. Five minutes of diazoxide followed by a 30-minute washout still reduced infarct size (8+/-3%), imply ing memory as seen with other PC triggers. The protection by diazoxide was not blocked by 5 mu mol/L chelerythrine, a protein kinase C antagonist, giv en either to bracket diazoxide infusion or just before the index ischemia. Bracketing preischemic exposure to diazoxide with 50 mu mol/L genistein, a tyrosine kinase antagonist, did not affect infarction, but genistein blocke d the protection by diazoxide when administered shortly before the index is chemia. 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 mu mol/L) or Mn(III)tetrakis(4-benzoi c acid) porphyrin chloride (7 mu mol/L), each of which is a free radical sc avenger, blocked protection, indicating that diazoxide triggers protection through free radicals, Therefore, mito K-ATP channels are not the end effec tors of protection, but rather their opening before ischemia generates free radicals that trigger entrance into a preconditioned state and activation of kinases.