Abstract-The critical time for opening mitochondrial (mito) K ATP channels, putative end effectors of ischemic preconditioning (PC), was examined. In isolated rabbit hearts 29Ϯ3% of risk zone infarcted after 30 minutes of regional ischemia. Ischemic PC or 5-minute exposure to 10 mol/L diazoxide, a mito K ATP channel opener, reduced infarction to 3Ϯ1% and 8Ϯ1%, respectively. The mito K ATP channel closer 5-hydroxydecanoate (200 mol/L), bracketing either 5-minute PC ischemia or diazoxide infusion, blocked protection (24Ϯ3 and 28Ϯ6% infarction, respectively). However, 5-hydroxydecanoate starting 5 minutes before long ischemia did not affect protection. Glibenclamide (5 mol/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 (8Ϯ3%), implying memory as seen with other PC triggers. The protection by diazoxide was not blocked by 5 mol/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 mol/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 mol/L) or Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (7 mol/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. (Circ Res. 2000;87:460-466.)

Abstract-It has been assumed that all G i -coupled receptors trigger the protective action of preconditioning by means of an identical intracellular signaling pathway. To test this assumption, rabbit hearts were isolated and perfused with Krebs buffer. All hearts were subjected to a 30-minute coronary artery occlusion followed by 120 minutes of reperfusion. Risk area was measured with fluorescent particles and infarct size with triphenyltetrazolium chloride staining. Control hearts showed 29.1Ϯ2.8% infarction of the risk zone. A 5-minute infusion of acetylcholine (0.55 mmol/L) beginning 15 minutes before the 30-minute occlusion resulted in significant protection (9.2Ϯ2.7% infarction). This protection could be blocked by administration of 300 mol/L N-2-mercaptopropionyl glycine (MPG), a free radical scavenger, or by 200 mol/L 5-hydroxydecanoate (5-HD), a mitochondrial K ATP antagonist, for 15 minutes beginning 5 minutes before the acetylcholine infusion (35.2Ϯ3.9% and 27.8Ϯ2.4% infarction, respectively). Similar protection was observed with other known triggers, ie, bradykinin (0.4 mol/L), morphine (0.3 mol/L), and phenylephrine (0.1 mol/L), and in each case protection was completely abrogated by either MPG or 5-HD. In contrast, protection by adenosine or its analog N 6 -(2-phenylisopropyl) adenosine could not be blocked by either MPG or 5-HD. Therefore, whereas most of the tested agonists trigger protection by a pathway that requires opening of mitochondrial K ATP channels and production of free radicals, the protective action of adenosine is not dependent on either of these steps. Hence, it cannot be assumed that all G i -coupled receptors use the same signal transduction pathways to trigger preconditioning.

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Because protection against infarction afforded by ischaemic preconditioning, adenosine, or the A3 receptor agonist APNEA could not be blocked by DPCPX and because the potent A3 receptor antagonist BW A1433 blocked protection from ischaemic preconditioning, these data indicate that the protection of preconditioning is not exclusively mediated by the adenosine A1 receptor in rabbit heart and could involve the A3 receptor.