Peroxynitrite inhibits enterocyte proliferation and modulates Src kinase activity in vitro

Overproduction of nitric oxide (NO) or its toxic metabolite, peroxynitrite (ONOO^-), after endotoxemia promotes gut barrier failure, in part, by inducing enterocyte apoptosis. We hypothesized that ONOO^- may also inhibit enterocyte proliferation by disrupting the Src tyrosine kinase signaling pathway, thereby blunting repair of the damaged mucosa. We examined the effect of ONOO^- on enterocyte proliferation and Src kinase activity. Sprague-Dawley rats were challenged with LPS or saline, whereas intestinal epithelial cell line cells were treated with ONOO^- or decomposed ONOO^- in vitro. Enterocyte proliferation in vivo and in vitro was measured by 5-bromo-2′-deoxyuridine (BrdU) or [ ³H]thymidine incorporation. Src kinase activity in cell lysates was determined at various times. LPS challenge in vivo and ONOO^- treatment in vitro inhibited enterocyte proliferation. ONOO^- treatment blunted the activity of Src and its downstream target, focal adhesion kinase, in a time-dependent manner. ONOO^- blocked mitogen (FBS, EGF)-induced enterocyte proliferation and Src phosphorylation while increasing Src nitration. Thus ONOO^- may promote gut barrier failure not only by inducing enterocyte apoptosis but also by disrupting signaling pathways involved in enterocyte proliferation.