FIGURE 7.

Ethanol-generated peroxynitrite induces tyrosine nitration of GCK, correlated with GCK down-regulation. A, in ethanol-fed mice, immunoprecipitated GCK was probed with anti-nitrotyrosine antibody (left panels). Shown are representative images of iNOS immunohistochemistry (brown) performed on pancreas sections from ethanol-fed mice (arrow, right panels). B, in vitro nitration. Recombinant GST-GCK was treated with proxynitrite (1 mm) for 2 h at 37 °C. Nitrated GCK were detected with a mouse monoclonal anti-nitrotyrosine antibody. C, after treatment with ethanol, endogenous GCK was immunoprecipitated and then subjected to Western blot (left panel). The ratio of nitrated GCK/immunoprecipitated GCK amounts are shown (right panel). *, p < 0.01; **, p < 0.05. D, effects of ethanol-induced GCK nitration or S-nitrosylation on GCK protein expression. E, effects of ATF3 on peroxynitrite-induced GCK nitration and GCK down-regulation. All of the results were obtained from three independent experiments. IP, immunoprecipitation; IB, immunoblot; WB, Western blot; Ab, antibody.