EGCG and adenosine receptor blocked by a nonspecific ADR antagonist 8-SPT but not by a nonspecific OPR antagonist naloxone, implying the involvement of ADR in EGCG-induced cardioprotection. We further tested the involvement of subtypes of ADR in the infarct limitation effect by EGCG with four different ADR subtypes (A1, A2A, A2B, and A3) antagonists. A1 and A2B ADR subtype antagonists attenuated the infarct-sparing effect by EGCG. Our data suggest, for the first time, that EGCG-induced cardioprotection may attenuate myocardial ischemia-reperfusion injury, at least in part, through ADR activation. Meanwhile, our present data clearly suggest that there is a functional coupling between EGCG and ADR in mediating EGCG-induced cardioprotection in the isolated rat heart, although the exact mechanism of this interaction between EGCG treatment and cardiac ADR is not known. One possible mechanism is that EGCG may further increase myocardial levels of adenosine. Adenosine is produced primarily through the metabolism of adenosine triphosphate (ATP) and its level increases during stressful situations when ATP utilization increases, such as in our myocardial ischemia reperfusion injury model. Therefore, it may be possible that EGCG further increase the release of adenosine. However, there is scant information about the adenosine level change by EGCG treat- ment in the heart. Navarro-Peran et al. [12] demonstrated that EGCG produces a significant increase in a specific ADR in colon cancer cells and adenosine can modulate different signaling pathways by binding to its specific receptors. In their study, A3 ADR antibody expression was significantly increased after EGCG treatment in cytosolic fractions of Caco-2 cells. Therefore, the change in adenosine level by EGCG treatment in the myocardial ischemia-reperfusion model should be determined in the future. Meanwhile, cardioprotection does not necessarily correlate with increased adenosine production. Thus, adenosine concentration is not crucial to the beneficial effects of ischemia- reperfusion of the injured rat heart [13]. In this regard, another possible mechanism might involve endogenous activation of ADR itself without increase in adenosine level. ADR is a GPCR and there are four subtypes, A1, A2A, A2B and A3. The activation of these ADR subtypes has been shown to be cardioprotective [14-16]. During the past two decades, numerous mechanisms of adenosine-mediated cardio- protection have been proposed. ADR activation has been suggested to reduce cell death through the mitochondrial KATP (mKATP) channel as well as protein kinase C and mitogen- activated protein kinase (MAPK) signaling [17,18]. Meanwhile, we previously reported the role of mKATP channel in EGCG- induced cardioprotection in isolated rat hearts [2]. The infarct limitation effect by EGCG was totally blocked by the non- selective KATP channel blocker glibenclamide and the selective mKATP channel blocker 5-hydroxydecanoate, implying that KATP 344 Vol. 63, No. 4, October 2012 channels, especially mKATP channels, play a crucial role in the cardioprotection by EGCG. Taken together, it is highly plausible that EGCG may provide cardioprotection via mKATP channel activation by ADR activation. However, the involvement of MAPK signaling in EGCG-induced cardioprotection is not clear. Previous studies in this laboratory have shown that that EGCG treatment did not increase the phosphorylation of ERK and Akt compared to untreated control hearts (data not shown). Therefore, it is not likely that ADR activation by EGCG involves MAPK downstream signaling. In the present study, we perfused EGCG targeting early reperfusion phase. Interestingly, it has been proposed that activation of both A1 and A3 ADR is cardioprotective when triggered ischemic period. For example, intracoronary injection of A1 ADR agonist N6-1-(phenyl-2R-isopropyl)adenosine reduced infarct size as effective as ischemic preconditioning in isolated rabbit heart [19]. In contrast, it has been demonstrated that the cardioprotection by activation of A2 ADR is beneficial during reperfusion [20,21]. Potent A2B ADR agonist NECA infused from 5 min reperfusion mimicked ischemic postcon- ditioning's effect on infarct size in in situ rabbit hearts [22]. In this study, EGCG-induced infarct limitation was blocked by A1 and A2B ADR. This suggests ADR may be involved in the EGCG treatment targeting both ischemia and reperfusion. In conclusion, the infarct reducing effect of EGCG appears to involve activation of ADR, especially A1 and A2B ADR, but not OPR in isolated rat heart. 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