alpha 1-adrenergic agonists precondition rabbit ischemic myocardium independent of adenosine by direct activation of protein kinase C.

Abstract

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...

Direct evidence that protein kinase C plays an essential role in the development of late preconditioning against myocardial stunning in conscious rabbits and that epsilon is the isoform involved.

“…Downey's group was the first to propose the hypothesis that activation of PKC plays a key role in the development of early PC against infarction (18)(19)(20). The PKC hypothesis of early PC has been supported by numerous investigations, which have demonstrated that various PKC blockers (i.e., H-7, staurosporine, chelerythrine, polymixin B) attenuate the protective effects of ischemic PC whereas PKC activators (i.e., PMA and 1-oleoyl 2-acetyl glycerol) mimic this protective effect in rabbits (23,(27)(28)(29), rats (21,24,25,30,33), dogs (31), and isolated rabbit cardiomyocyte preparations (22,32) as well as in isolated human atrial trabeculae (26). However, in contrast to the above studies, others have found that PKC inhibitors (H-7, staurosporine, bisindolylmaleimide, polymixin B) fail to block the protective effect of ischemic PC in dogs (35) and pigs (36,37).…”

“…Several studies (5,(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33) indicate that protein kinase C (PKC), an ubiquitous intracellular mediator (34), may play an important role in the genesis of ischemic PC. However, no information is currently available as to whether PKC plays a role in late PC against myocardial stunning.…”

Response to myocardial ischemia/reperfusion injury involves Bnip3 and autophagy

“…Hearts from anesthetized male Sprague-Dawley rats (225-250 g) were rapidly excised and cannulated onto the Langendorff perfusion apparatus using a protocol adapted from Tsuchida et al 36 The hearts were perfused with or without 50 nM TAT protein and subjected to I/R as described. 17 The Creatine Kinase (CK) activity in the coronary effluent was measured using a diagnostic kit (Stanbio Laboratory) and infarct size was measured using triphenyl tetrazolium staining.…”

Bnip3 functions as a mitochondrial sensor of oxidative stress during myocardial ischemia and reperfusion

“…Rat hearts were perfused in the Langendorff mode using a protocol adapted from Tsuchida et al (30). Hearts were perfused with Krebs-Ringer buffer at a constant pressure of 60 mmHg.…”