Protective Effect of S-Nitrosylated a1-Protease Inhibitor on Hepatic Ischemia-Reperfusion Injury

S-Nitrosylated compounds (nitrosothiols; RS-NOs) function as nitric oxide (NO) reservoirs and preserve the antioxidant activities of NO. We found remarkable cytoprotection by an Snitrosylated protease inhibitor from human plasma, S-nitrosoa1-protease inhibitor (S-NO-a1-PI) that possesses a completely nitrosylated SH group, in hepatic ischemia-reperfusion injuries in rats. Liver ischemia was induced in rats by occluding both the portal vein and hepatic artery for 30 min and was followed by reperfusion. S-NO-a1-PI and control compounds such as native a1-PI, an NO synthase (NOS) inhibitor, and standard RS-NOs were given via the portal vein just after reperfusion was initiated. Liver injury was evaluated by measuring the extracellular release of liver enzymes (aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase). Infiltration of neutrophils and induction of apoptosis and heme oxygenase-1 (HO-1) in the liver were also examined. Maximal liver injury occurred at 3 h after reperfusion and then decreased gradually. Not only did S-NO-a1-PI treatment (0.1 mmol; 5.3 mg/rat) greatly reduce elevation of liver enzymes in plasma, as well as neutrophil accumulation and apoptotic change in liver, it also improved the impaired hepatic blood flow as assessed by laser Doppler flowmetry and potentiated the induction of HO-1 in the liver. Although native a1-PI moderately reduced liver injury, low molecular weight RS-NOs such as S-nitrosoglutathione and S-nitroso-N-acetyl penicillamine produced no obvious protective effect. An NOS inhibitor exacerbated the hepatic ischemia-reperfusion injuries. These results suggest that S-NO-a1-PI exerts a potent cytoprotective effect on ischemia-reperfusion liver injury by maintaining tissue blood flow, inducing HO-1, and suppressing neutrophil-induced liver damage and apoptosis. Nitric oxide (NO) produced in biological systems mediates a diverse array of physiological functions (Ignarro et al., 1988; Furchgott and Vanhoutte, 1989; Moncada and Higgs, 1993). In the vascular system, NO regulates organ blood flow, inhibits platelet aggregation, and attenuates neutrophil adherence (Ignarro et al., 1988; Kubes et al., 1991; Moncada and Higgs, 1993). A cytoprotective effect of NO has been reported for ischemia-reperfusion injuries in various organs (Tsao et al., 1990; Konorev et al., 1995; Liu et al., 1998; Ohmori et al., 1998; Cottart et al., 1999). Impairment of endothelial NO production may contribute to the pathogenesis of ischemia-reperfusion injuries, because a decrease in NO release can trigger neutrophil adherence and exudate into the ischemic area, which exacerbates reperfusion injury. It was recently proposed that various redox isoforms of NO have critical roles in the diverse physiological and pathophysiological events induced by NO. For example, biological S-nitrosation occurs via one-electron oxidation of NO catalyzed by heavy metal ions, such as copper and iron, and particularly by ceruloplasmin, which is a major multicoppercontaining plasma protein in mammals (Inoue et al., 1999; Akaike, 2000). Sulfhydryl-containing molecules such as glutathione are particularly susceptible to nitrosation; a nucleophilic attack by NO results in S-nitrosothiol adducts (nitrosothiols; RS-NOs) (Stamler et al., 1992; Akaike, 2000). Nitrosothiols may function as endogenous NO reservoirs and preserve the antioxidant activities of NO (Ignarro et al., 1981; Stamler et al., 1992; Gaston et al., 1993; Rauhala et al.,