Biphasic response of cardiac NO synthase isoforms to ischemic preconditioning in conscious rabbits

In conscious rabbits, a sequence of six 4-min coronary occlusion/4-min repe rfusion cycles, which elicits late preconditioning (PC), caused rapid activ ation of calcium-dependent nitric oxide (NO) synthase (NOS) [cNOS; endothel ial NOS (eNOS) and/or neuronal NOS (nNOS)], whereas calcium-independent NOS [inducible NOS (iNOS)] activity remained unchanged. The enhanced cNOS acti vity was associated with increased myocardial levels of NO2 and/or NO3 (NOx ). Twenty-four hours after ischemic PC was induced, the opposite pattern wa s observed, i.e., there was a pronounced increase in cytosolic iNOS activit y but no change in cNOS activity. The initial burst of ischemia-induced cNO S activity was not affected by pretreatment with the antioxidant N-2-mercap topropionyl glycine (MPG), the protein kinase C (PKC) inhibitor chelerythri ne, or the tyrosine kinase inhibitor lavendustin A, indicating that it is i ndependent of the generation of oxidant species and the activation of PKC a nd tyrosine kinases. In contrast, the delayed upregulation of iNOS 24 h aft er PC was prevented by pretreatment with N-omega-nitro-L-arginine, MPG, or chelerythrine before the PC ischemia, indicating that it is triggered by a signaling mechanism that involves the generation of NO, the formation of ox idant species, and the activation of PKC. Taken together, these results dem onstrate that, in conscious animals, ischemic PC elicits a biphasic respons e in cardiac NOS activity, i.e., an immediate activation of cNOS (most like ly eNOS) followed 24 h later by a delayed upregulation of iNOS. To our know ledge, this is the first study to directly measure NOS activity after brief myocardial ischemia in vivo. In conjunction with previous functional studi es, the data support a distinctive role of NOS isoforms in late PC, with eN OS serving as the trigger on day 1 and iNOS as the mediator on day 2.