Peroxynitrite (PN) effects on small intestinal enterocytes (IEC-6)

Abstract

Purpose: Our laboratory has shown that intestinal barrier failure following necrotizing enterocolitis or LPS peritonitis is associated with upregulation of inducible nitric oxide synthase, 3-nitrotyrosine residues (the PN footprints), and apoptosis in the ileum. The mechanism of PN-mediated gut barrier failure remains elusive. This study examines PN-mediated epithelial cell injury in vitro.Methods: We incubated IEC-6 cells with 12.5 μM PN or phosphate-buffered saline (PBS) or media for 60 minutes followed by a recovery period of 6, 12, 18, or 24 hours in culture media. Apoptosis was measured by fluorescent-activated cell sorting using the propidium iodide method and electron microscopy. We also tested the ability of pretreatment with 3-aminobenzamide (3AB) (1 mM), an inhibitor of poly-ADP ribose synthetase, to alter the response of IEC-6 cells to PN.Results: A 60-minute exposure to PN followed by an 18-hour recovery period resulted in significant apoptosis (24 ± 4%) in IEC-6 cells as compared with PBS (2.7 ± .5%); p < .015 (Fig. 1). Peroxynitrite induced apoptosis in IEC-6 cells in a time-dependent fashion. There was no significant difference between PN and PN + 3AB (24 ± 4 v 17 ± 4). Electron microscopy demonstrated marked injury.Conclusions: Peroxynitrite injures small intestinal epithelial cells. Thus, inhibition or scavengers of PN may prevent gut barrier failure after endotoxemia.