Regulation of Apoptosis in Intestinal Epithelial Cells † 554

Abstract

Necrotizing enterocolitis (NEC) affects 10% of premature neonates. Although several potentially contributing mediators have been identified, the exact mechanism of disease pathogenesis is unknown. Increased epithelial apoptosis have been proposed to contribute to the epithelial demage in NEC. The goal of our studies was to identify mediators of epithelial apoptosis and the mechanism(s) of their action. IEC-6 rat small intestinal epithelial cells were subjected to treatment with inflammatory mediators known to induce NEC (PAF; 0.1-10μM, LPS; 10μg/ml) or a chemical NO donor (DETA; 31.125μM-1mM), and/or inhibitors of nitric oxide synthase(NOS) (LNAME; 1mM, AMT; 20nM) for 24hrs. Apoptosis was assessed using fluorescence TUNEL staining and fluorescence digital imaging microscopy or by quantifying histone-bound DNA fragments with ELISA. All effects on the rate of apoptosis were normalized to control values and were expressed as (percent control±S.E.M.; n>3). Treatment with either PAF, LPS or DETA significantly enhanced apoptosis in IEC-6 cells in a time- and concentration-dependent manner. Maximal effects were observed with 10μM PAF (292±26%), 10μg/ml LPS(326±39%) or with 250μM DETA (498±64%). The induction of apoptosis by DETA was due to NO release, since freshly isolated red blood cells, i.e. NO scavengers, completely obliterated the apoptosis-inducing effect of DETA, up to 1mM DETA concentration. Complete inhibition of all NOS activity with 1mM LNAME potently induced apoptosis (269±53%). In the presence of 1mM LNAME, low concentrations of DETA, i.e., 30-70μM DETA, were protective against LNAME-induced apoptosis, while high concentrations of DETA, i.e., 100-300μM, stimulated apoptosis. These data indicate that inflammatory mediators that induce NEC in vivo, elicit apotposis in cultured intestinal epithelial cells and that NO plays a complex role in the regulation of apoptosis in these cells. High concentrations of NO are pro-apoptotic, while low concentrations of NO are necessary to protect IEC-6 cells from apoptosis. Evaluating the exact relationship between PAF, LPS and NO in the regulation of epithelial apoptosis will contribute to our understanding of the cellular and molecular mechanisms that underlie the development of NEC.