Abstract 78: Activation of A3 Adenosine Receptors Protects the Ischemic Reperfused Myocardium Via Recruitment of PI3K-AKT- iNOS Cell Survival Pathway
Recent studies have emphasized the important role that nitric oxide (NO) plays in protecting against myocardial ischemia reperfusion injury. It has been suggested that an increase in iNOS and enhancement of NO bioavailability provides protection in experimental myocardial infarction models. Our previous research has shown that activation of A3 adenosine receptors (A3R) protect the myocardium from ischemia reperfusion injury via anti-apoptotic/necrotic mechanisms. In the present study, we aimed at assessing whether the phosphatidylinositol 3 kinase (PI3K) - Akt - iNOS cell survival pathway was involved in A3R mediated cardioprotection. Isolated perfused rat hearts were subjected to 35mins ischemia followed by 120 mins reperfusion. Hearts underwent triphenyl tetrazolium staining for infarct size assessment, or were frozen for Western blot analysis. 2-Cl-IBMECA (A3R agonist, 100nM) was administered throughout reperfusion in the presence and absence of Wortmannin (PI3K inhibitor, 100nM), N(G)-nitro-L-arginine methyl ester (L-NAME) (nitric oxide synthase inhibitor, 100μM) or Aminoguanidine (AG) (iNOS inhibitor, 100μM). 2-Cl-IBMECA (100nM) when administered during reperfusion significantly reduced infarct size when compared to control (22.9 ± 7% vs. 64.3 ± 6%, p<0.05). The protection afforded by 2-Cl-IBMECA was significantly abrogated by co-administration of Wortmannin, L-NAME or AG (67.5 ± 5%, 76.6 ± 4%, 63.2 ± 7%, respectively, p<0.01). Western blot analysis further demonstrated that A3R activation during reperfusion induced a significant increase in p-AKT(Ser473) and iNOS expression in the presence of 2-Cl-IBMECA compared to control hearts. 2-Cl-IBMECA dependent upregulation of AKT and iNOS was blocked by their respective inhibitors Wortmannin and L-NAME. This is the first study to show that 2-Cl-IBMECA protects the ischemic reperfused heart via recruitment of the PI3K-AKT-iNOS cell survival pathway.