Late Preconditioning Elicited by Activation of Adenosine A3 Receptor in Heart: Role of NF- κ B, iNOS and Mitochondrial KATP Channel

Activation of adenosine A3 receptor (A3AR) protects against ischemia/reperfusion injury in the heart. However, the downstream signaling mechanisms leading to its delayed anti-ischemic effects remain unclear. We hypothesized that A3AR stimulation protects the heart via activation of nuclear transcription factor κ B (NF- κ B) and synthesis of inducible nitric oxide synthase (iNOS). Mice were treated with selective A3AR agonist, N6-(3-iodobenzyl) adenosine-5-N-methyluronamide (IB-MECA). Twenty-four h later, hearts were perfused in Langendorff mode and subjected to 30 min of global ischemia and 30 min of reperfusion. IB-MECA caused post-ischemic reduction in necrosis and improvement in myocardial performance which was abolished by A3AR antagonist, MRS1191. Electrophoretic mobility shift assay demonstrated increased NF- κ B binding in nuclear extracts following A3AR stimulation, which was diminished by MRS1191 and NF- κ B inhibitor, pyrrolidinediethyldithiocarbamate (PDTC). The cardioprotection was abrogated by PDTC and targeted ablation of p50 subunit of NF-κ B in mice. The inhibition of iNOS with S-methylisothiourea and targeted disruption of the iNOS gene also abolished the protective effect of A3AR stimulation. Expression of iNOS mRNA and NO production were enhanced after 6 and 24 h respectively of IB-MECA treatment. MRS1191 and PDTC blocked IB-MECA induced NO production after A3AR stimulation. MitoKATP channel blocker, 5-hydroxydecanoate abolished the protective effect of A3AR. For the first time, we have provided direct evidence of an essential role of NF- κ B activation and iNOS in A3AR-induced late preconditioning. Selective activation of A3AR with IB-MECA can be used to trigger long-lasting ischemic protection in the heart.