We conclude that adenosine released during the preconditioning occlusion stimulates cardiac A1 receptors, which leaves the heart protected against infarction even after the adenosine has been withdrawn.

Rabbits and rats are becoming popular models for in vitro as well as in situ studies of myocardial infarction. In the present analysis we evaluated the results of several of our completed investigations and tested whether blood-free perfusate, anesthesia, or risk zone size affects infarction in these species. In addition, the influence of the method used for determining infarct size (histology or histochemistry) was examined in rabbits. All hearts experienced 30 min of regional ischemia followed by either 2-3 h of reperfusion in animals in which infarct size was assessed by staining with triphenyltetrazolium chloride or 72 h in those in which histological methods were used to measure infarct size. Eighteen rabbit and seven rat hearts perfused with Krebs buffer, seventeen open-chest rabbits, eight rats anesthetized with pentobarbital, and ten conscious rabbits were studied. Risk zone size measured with fluorescent particles was plotted against infarct size. Infarct size was linearly correlated with risk zone size and did not differ among models for each species. In rat hearts the regression line passed through the origin so that zero infarction occurred with zero risk zone size. However, in the rabbit heart there was no apparent infarction for risk zone sizes < 0.3 cm3. Although the relationship between risk zone and infarction was found to be remarkably independent of the model chosen, the nonzero intercept for the rabbit heart can be an important, previously unrecognized source of experimental variability when infarct size is expressed as a percentage of the risk zone.

Ischemit preconditioning in the rabbit is initiated by adenosine Al-receptor stimulation, which activates protein kinase C (PKC). Additionally, a,-adrenergic agonists can similarly protect ischemic myocardium, but there has been confusion about the role adenosine receptors play in this protection. To characterize the interaction between adrenergic and adenosine receptors and to study the possible role of PKC in this protection, we used isolated rabbit hearts perfused with oxygenated Krebs' buffer. All hearts were subjected to 30 minutes of regional myocardial ischemia and 2 hours of reperfusion. Infarct size was determined by triphenyltetrazolium staining. Pharmacologic preconditioning in hearts with a 5-minute phenylephrine (PE) infusion 10 minutes before the prolonged regional ischemia resulted in significantly smaller infarcts (9.7±+ 1.3% of risk area) than in control hearts (31.0±+2.6%, P<.05). This protection could be effectively blocked by administration of the a-adrenergic blocker phenoxybenzamine. Methoxamine, an ala-selective agonist, failed to protect, whereas the alb-selective antagonist chloroethylclonidine aborted the protective effect of PE. Polymyxin B, an inhibitor of PKC, also blocked the protective effect of PE, implying that PKC has an important role in preconditioning.P reconditioning the heart with 5 minutes of ischemia followed by 10 minutes of reperfusion renders it very resistant to ischemia from a subsequent ischemic insult. We have recently proposed that ischemic preconditioning is the result of an upregulation of protein kinase C (PKC) within the cardiac myocytes, probably through translocation of PKC into the membranes, where it can be activated by its cofactors. In this theory, occupancy of adenosine A1-receptors during the first coronary occlusion somehow upregulates PKC such that a PKC-mediated protective mechanism can be invoked during a second coronary occlusion.1,2 After a second episode of ischemia, adenosine receptors will again be populated with subsequent reactivation of previously upregulated PKC. Therefore, preconditioning appears to eliminate the expected time lag between receptor stimulation and protein phosphorylation when PKC is in this upregulated state. That PKC must phosphorylate protein very early in ischemia in order to afford protection is central to the hypothesis. Received March 28, 1994; accepted June 15, 1994 The adenosine receptor blocker 8-(p-sulfophenyl)theophylline (SPT) given at the same time as the PE infusion did not affect the protection, implying that an a1-agonist could initiate protection independent of adenosine, presumably by direct coupling to PKC. However, the protective effect of PE could be blocked if SPT were administered during the 30-minute regional ischemia. This observation suggested that adenosine receptor occupancy is necessary during long ischemia to reactivate PKC and mediate the protection. However, the addition of a second PE infusion beginning 5 minutes before and continuing throughout the long ischemic period restored th...