INDUCTION AND ACTIVITY OF NITRIC-OXIDE SYNTHASE IN CULTURED HUMAN INTESTINAL EPITHELIAL MONOLAYERS

We have examined the induction and activity of inducible nitric oxide (NO) synthase (iNOS) in monolayers of DLD-1 cells, an epithelial cell line derived from a human intestinal adenocarcinoma. Induction of iNOS transcription by a combination of the cytokines interferon-gamma and IL-1 beta was inhibited by genistein, pyrrolidine dithiocarbamate, or dexamethasone and unaffected by pretreatment with ethylene glycol-bis( beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, basic fibroblast gr owth factor (bFGF), epidermal growth factor (EGF), or the isoflavone d aidzein. iNOS activity and NO synthesis were inhibited by nitro-L-argi nine methyl ester, N-G-monomethyl-L-arginine, S-methyl-isothiourea sul fate, or aminoethyl-isothiourea, but not by dexamethasone. NO synthesi s was potently inhibited by N-alpha-p-tosyl-lysine chloromethyl ketone and hypoxia. In the absence of cytokines, no iNOS induction was obser ved with oxidant stress (H2O2), growth factors (bFGF, EGF), hypoxia, o r hypoxia reoxygenation. We conclude that in this model of the human i ntestinal epithelium 1) cytokine-mediated induction of iNOS is Ca2+ in dependent, weakly steroid sensitive, and may involve the activation of nuclear factor-kappa B and a tyrosine kinase, and 2) iNOS activity is Ca2+-independent and inhibited by hypoxia, N-G-substituted L-arginine analogues, and isothioureas.