We have previously found that adhesion fibroblasts exhibit lower apoptosis and higher protein nitration as compared with normal peritoneal fibroblasts. In this study, we sought to determine whether the decreased apoptosis observed in adhesion fibroblasts is caused by lower caspase-3 activity due to an increase in caspase-3 S-nitrosylation. For this study, we have utilized primary cultures of fibroblasts obtained from normal peritoneum and adhesion tissues of the same patient(s). Cells were treated with increasing concentrations of peroxynitrite and cell lysates were immunoprecipitated with anti-caspase-3 polyclonal antibody. The biotinylated proteins were detected using a nitrosylation detection kit. Caspase-3 activity and apoptosis were measured by colorimetric and TUNEL assays, respectively. Our results showed that caspase-3 S-nitrosylation is significantly higher in adhesion fibroblasts as compared with normal peritoneal fibroblasts. This increase in S-nitrosylation resulted in a 30% decrease in caspase-3 activity in adhesion fibroblasts. Peroxynitrite treatment resulted in a dose response increase in caspase-3 S-nitrosylation, leading to a decrease in caspase-3 activity and apoptosis in normal peritoneal fibroblasts. We conclude that S-nitrosylation of caspase-3 is the reason for its decreased activity and subsequent decrease in apoptosis of adhesion fibroblasts. The mechanism by which caspase-3 S-nitrosylation occurs is not fully understood. However, the role of hypoxia in the formation of peroxynitrite via superoxide production may suggest a possible mechanism.
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