Inducible nitric oxide synthase mediates delayed myocardial protection induced by activation of adenosine A(1) receptors - Evidence from gene-knockout mice

Background-The mechanism of delayed preconditioning induced by activation o f adenosine A(1) receptors (A(1)ARs) is not fully understood. We determined the role of inducible nitric oxide synthase (iNOS) in mediating adenosine- induced late cardioprotection using pharmacological inhibitors and iNOS gen e-knockout mice. Methods and Results-Adult male mice were treated with saline or an A,AR ago nist, 2-chloro-N-6-cyclopentyladenosine (CCPA). Twenty-four hours later, th e hearts were perfused in Langendorff mode and subjected to 30 minutes of g lobal ischemia followed by 30 minutes of reperfusion. 8-Cyclopentyl-1,3-dip ropylxanthine (DPCPX; 0.1 mg/kg IP) and S-methylisothiourea (SMT; 3 mg/kg I P) were used to block A(1)ARs and iNOS, respectively. Infarct size (IS) was measured by triphenyltetrazolium chloride staining, and iNOS expression wa s measured by Western blots. Myocardial IS was reduced from 24.0+/-3.2% in the saline group to 12.2+/-2.5% in CCPA-treated mice (P<0.05). The infarct- reducing effect of CCPA was abrogated by DPCPX (29.3 +/- 3.4%) and SMT (32. 3 +/- 2.6%) and was absent in mice with targeted ablation of iNOS (23.9+/-1 .6%). CCPA produced improvement in postischemic end-diastolic pressure, dev eloped pressure, and rate-pressure product, which was also blocked by DPCPX and SMT. Increased iNOS protein expression observed in CCPA-treated hearts was diminished by DPCPX. Conclusions-Selective activation of A(1)ARs produces delayed cardioprotecti on against ischemia/reperfusion injury in the mouse. Increased iNOS express ion concomitant with the lack of protective effect of A(1)AR activation in iNOS gene-knockout mice suggests a direct cause-and-effect relationship of iNOS in adenosine-induced late cardioprotection.