Role of constitutive nitric oxide synthase and peroxynitrite production in a rat model of splanchnic artery occlusion shock

Peroxynitrite, a potent cytotoxic oxidant formed by the reaction of nitric oxide with superoxide anion, is an important mediator of reperfusion injury. In a rodent model of mesenteric ischemia and reperfusion injury we evaluated the contribution of the constitutive and/or inducible nitric oxide synthase (cNOS or iNOS) in the formation of peroxynitrite. Splanchnic artery occlusion (SAO) shock was induced in rats by clamping both the superior mesenteric artery and the celiac trunk for 45 min, followed by release of the clamps (reperfusion). A significant peroxynitrite production was found in the plasma of the splanchnic occlusion shocked rats at 60 minutes after reperfusion. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine, a specific "footprint" of peroxynitrite, in the necrotic ileum and the aorta of shocked rats. No change in plasma levels of nitrate/nitrite, tissue iNOS expression (by western blotting detection) or iNOS activity was found in the intestine at 60 minutes after reperfusion. On the contrary, activity of the cNOS was reduced (approximately 50%) in the reperfused ischemic intestinal tissue. Treatment with N-G-nitro-L-arginine methyl ester, a non selective inhibitor of nitric oxide synthase (given at 3 mg/ kg i.v., 5 min prior to reperfusion), significantly reduced plasma level of peroxynitrite and the immunohistochemical staining for nitrotyrosine in the ileum and aorta. Our results suggest that during splanchnic artery occlusion shock peroxynitrite formation is likely to be correlated with nitric oxide production from constitutive nitric oxide synthase activation rather than from the inducible isoform enzyme.