Signal transduction of flumazenil-induced preconditioning in myocytes

The objective of this study was to examine the role of oxygen radicals, pro tein kinase C (PKC), and ATP-sensitive K+ (K-ATP) channels in mediating flu mazenil-produced preconditioning. Chick cardiomyocyte death was quantified using propidium iodide, and oxygen radical generation was assessed using 2' , 7'-dichlorofluorescin oxidation. Preconditioning was initiated with 10 mi n of ischemia followed by 10 min of reoxygenation. Alternatively, flumazeni l was infused for 10 min and removed 10 min before ischemia. Flumazenil (10 muM) and preconditioning increased oxygen radicals [1,693 +/- 101 (n = 3) and 1,567 +/- 98 (n = 3), respectively, vs. 345 +/- 53 (n = 3) in control] and reduced cell death similarly [22 +/- 3% (n = 5) and 18 +/- 2% (n = 6), respectively, vs. controls 49 +/- 5% (n = 8)]. Protection and increased oxy gen radicals by flumazenil were abolished by pretreatment with the antioxid ant thiol reductant 2-mercaptopropionyl glycine (800 muM; 52 +/- 10%, n = 6 ). Specific PKC inhibitors Go-6976 (0.1 muM) and chelerythrine (2 muM), giv en during ischemia and reoxygenation, blocked flumazenil-produced protectio n (47 +/-, n = 6). The PKC activator phorbol 12-myristate 13-acetate (0.2 m uM), given during ischemia and reoxygenation, reduced cell death similarly to that with flumazenil [17 +/- (n =6) and 22 +/- (n = 5)]. Finally, 5-hydr oxydecanoate (1 mM), a selective mitochondrial KATP channel antagonist give n during ischemia and reoxygenation, abolished the protection of flumazenil and phorbol 12-myristate 13-acetate. Thus flumazenil mimics preconditionin g to reduce cell death in cardiomyocytes. Oxygen radicals activate mitochon drial K-ATP channels via PKC during the process.