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Fig. 4. Tyrosine nitration inactivates PI 3-kinase/Akt-1 pro-survival pathway. (A) Immunoprecipitation with p85 subunit of PI 3-kinase and western blot analysis using anti-nitrotyrosine antibody showed that cells treated with high glucose (HG) or peroxynitrite (PN) had significantly more nitration on the regulatory p85 subunit compared with cells cultured in normal glucose (NG). (*P<0.05 compared to NG.) This effect was blocked by the specific peroxynitrite decomposition catalyst FeTPPS (2.5 µM) and the specific nitration inhibitor epicatechin (100 µM). (B) Immunoprecipitation with p110 subunit of PI 3-kinase and western blot analysis using p85 antibody showed that under high glucose (HG) or peroxynitrite (PN) treatments, the p85 subunit was hardly detected in the immunopreicipitate of the catalytic p110 subunit compared with cells cultured in normal glucose (NG). Cells were stimulated with VEGF (40 ng/ml) in the presence or absence of the peroxynitrite decomposition catalyst (FeTPPs). The association between p85 and p110 was restored by treatment with FeTPPs (2.5 µM). (C) High glucose (HG) and peroxynitrite (PN) decreased Akt-1 activity significantly compared to normal glucose (NG). Akt-1 kinase activity was restored by treatment of high glucose cultures with a specific peroxynitrite inhibitor (2.5 µM), NOS inhibitor (L-NAME, 0.5 mM) and superoxide dismutase (SOD, 100 U/ml). A western blot of phospho GSK-3, the substrate of Akt-1 kinase is shown at the top. (*P<0.05 compared to NG.) (D) Statistical analysis of caspase-3 activity showing significant increases in apoptosis in cells cultured in high glucose (HG) or treated with peroxynitrite (PN) compared to normal glucose (NG). These effects were blocked by the specific nitration inhibitor epicatechin (100 µM). Similar results were obtained in another three experiments. (*P<0.05 compared to NG.)
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