Cardioprotection during the final stage of the late phase of ischemic preconditioning is mediated by neuronal NO synthase in concert with cyclooxygenase-2

The infarct-sparing effect of the late phase of ischemic preconditioning (late PC) lasts for 72 hours. Upregulation of both cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS) has been shown to be essential to the protection in the initial stage of late PC (24 hours after PC); however, the mechanisms underlying the protection in the final stage of late PC (48 to 72 hours after PC) are unknown. Conscious rabbits were preconditioned with six cycles of 4-minute coronary occlusion/4-minute reperfusion. At 72 hours after PC, powerful protection against infarction was associated with increased myocardial levels of COX-2 mRNA, protein, and cardioprotective prostaglandins (PGI(2) and PGE(2)). The COX-2-selective inhibitor NS-398 completely blocked the protection. Surprisingly, iNOS expression was not increased at 72 hours; instead, upregulation of neuronal NO synthase (nNOS) was evident at both the mRNA (+266+/-20%, P<0.005) and the protein levels (+195 +/- 66%, P<0.005), which was accompanied by an increase in myocardial nitrite/nitrate (+20+/-4%, P<0.05). The nNOS-selective inhibitors N-propyl-L-arginine or S-ethyl N-[4-(trifluoromethyl) phenyl] isothiourea completely blocked the protection of late PC at 72 hours, whereas the iNOS-selective inhibitor S-methylisothiourea had no effect. In line with these findings, the disappearance of protection at 120 hours after PC was associated with the return of nNOS mRNA, protein, and activity to control levels. Although expression of COX-2 protein was still elevated at 120 hours, only a marginal increase in PGI(2) and PGE(2) levels was detected. In contrast to 72 hour after PC, nNOS was not upregulated at 24 hour after PC. We conclude that (1) the cardioprotection observed in the final stage of late PC ( 72 hour) is mediated by nNOS, not by iNOS, in concert with COX-2, and (2) nNOS-derived NO is required to drive COX-2 activity. These data identify, for the first time, a cardioprotective role of nNOS and demonstrate, surprisingly, that the mechanism of late PC differs at 72 hours ( nNOS) versus 24 hours (iNOS).