Nitric oxide as a mediator of delayed pharmacological (A1 receptor triggered) preconditioning; is eNOS masquerading as iNOS?

Background: Nitric oxide (NO), synthesised from the inducible isoform of nitric oxide synthase (iNOS), is implicated in mediating second window of protection (SWOP)/delayed ischemic preconditioning. However the role of NO and iNOS in delayed pharmacological protection remains unclear and is the subject of this investigation. Methods: To test the hypothesis that iNOS is necessary for delayed pharmacological preconditioning, the adenosine A1 receptor agonist, 2-chloro N6 cyclopentyl adenosine (CCPA) (25 μg/kg i.v.) or saline was administered to wild type (WT) or iNOS gene knockout mice (KO). Twenty-four hours later, the hearts were isolated, Langendorff perfused and subjected to 35 min ischemia/30 min reperfusion prior to infarct size determination. Results: WT and KO control hearts had identical infarct sizes of 37±3% and 37±2%, respectively. CCPA significantly reduced infarct size in WT hearts to 22±2% and also, unexpectedly, in KO hearts (27±2%). This protection was abrogated with the non-specific NOS inhibitor, Nω nitro l-arginine methyl ester (l-NAME, 100 μM), and could be mimicked in naive hearts with the NO donor, donor S -nitroso N -acetyl dl penicillamine (SNAP, 1 μM). Delayed protection appeared to be mediated by NO synthesis in both WT and KO hearts. Additional studies using Western blot analysis demonstrated endothelial NOS (eNOS) upregulation and increased NO x release in both WT and KO hearts. Conclusions: This is the first study to demonstrate a role for eNOS in delayed A1 receptor triggered (pharmacological) preconditioning, potentially representing a new pharmacological target for protecting the ischemic heart.