Naoki Unno

Nitric oxide donor-induced hyperpermeability of cultured intestinal epithelial monolayers: role of superoxide radical, hydroxyl radical, and peroxynitrite.

Many of the cytopathic effects of nitric oxide (NO*) are mediated by peroxynitrite (PN), a product of the reaction between NO* and superoxide radical (O2*-). In the present study, we investigated the role of PN, O2*- and hydroxyl radical (OH*) as mediators of epithelial hyperpermeability induced by the NO* donor, S-nitroso-N-acetylpenicillamine (SNAP), and the PN generator, 3-morpholinosydnonimine (SIN-1). Caco-2BBe enterocytic monolayers were grown on permeable supports in bicameral chambers. Epithelial permeability, measured as the apical-to-basolateral flux of fluorescein disulfonic acid, increased after 24 h of incubation with 5.0 mM SNAP or SIN-1. Addition of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, an NO* scavenger, or Tiron, an O2*- scavenger, reduced the increase in permeability induced by both donor compounds. The SNAP-induced increase in permeability was prevented by allopurinol, an inhibitor of xanthine oxidase (a source of endogenous O2*-). Diethyldithiocarbamate, a superoxide dismutase inhibitor, and pyrogallol, an O2* generator, potentiated the increase in permeability induced by SNAP. Addition of the PN scavengers deferoxamine, urate, or glutathione, or the OH* scavenger mannitol, attenuated the increase in permeability induced by both SNAP and SIN-1. Both donor compounds decreased intracellular levels of glutathione and protein-bound sulfhydryl groups, suggesting the generation of a potent oxidant. These results support a role for PN, and possibly OH*, in the pathogenesis of NO* donor-induced intestinal epithelial hyperpermeability.

Acquired interferon gamma responsiveness during Caco-2 cell differentiation: effects on iNOS gene expression.

BACKGROUND Impairment of intestinal barrier function occurs under a variety of inflammatory conditions and is mediated at least in part by interferon γ (IFN-γ) induced nitric oxide (NO) production. Previous in vivo studies have shown that systemic lipopolysaccharide treatment caused an induction of the rat inducible nitric oxide synthase (iNOS) mRNA primarily in villus cells, rather than in undifferentiated crypt cells. AIMS To examine iNOS induction by IFN-γ in vitro as a function of enterocyte differentiation. METHODS Preconfluent and postconfluent Caco-2 cells were treated with IFN-γ in the presence or absence of various inhibitors. Northern analyses were performed to assess the magnitude of iNOS mRNA induction. IFN-γ receptor mRNA and protein levels were determined. RESULTS iNOS mRNA induction by IFN-γ occurred at two hours and was not blocked by cycloheximide, indicating that it is an immediate early response. iNOS induction and nitrite/nitrate increases were inhibited by dexamethasone and pyrrolidine dithiocarbamate, supporting an important role for the NF-κB transcription factor in this process. The stimulated iNOS induction was seen almost exclusively under conditions of cellular differentiation—that is, in postconfluent Caco-2 cells. This increased IFN-γ responsiveness seen in postconfluent Caco-2 cells correlated with an increased expression of IFN-γ receptor, whereas T84 and HT-29 cells did not show any significant alterations in either iNOS induction or IFN-γ receptor levels as a function of postconfluent growth. CONCLUSIONS With regard to iNOS mRNA induction, IFN-γ responsiveness is acquired during Caco-2 cell differentiation, perhaps related to an increase in the numbers of IFN-γ receptors.

Nitric oxide-induced hyperpermeability of human intestinal epithelial monolayers is augmented by inhibition of the amiloride-sensitive Na+-H+ antiport: Potential role of peroxynitrous acid

BACKGROUNDnNitric oxide (NO.) increases the permeability of cultured intestinal epithelial monolayers. NO. reacts with superoxide anion to form peroxynitrite anion, which can be protonated under mildly acidic conditions to form the potent and versatile oxidizing agent, peroxynitrous acid. We hypothesized that intracellular acidosis induced by the Na(+)-H+ antiport blocker, amiloride, would favor the formation of peroxynitrous acid and thereby augment hyperpermeability induced by the NO. donor, SIN-1.nnnMETHODSnCaco-2BBe human intestinal epithelial monolayers were grown on permeable supports in bicameral chambers. The permeability of monolayers was assessed by measuring the transepithelial flux of fluorescein disulfonic acid (FS).nnnRESULTSnIncubation of monolayers with SIN-1 increased permeability to FS. Adding amiloride augmented SIN-1-induced hyperpermeability. SIN-1 plus amiloride also decreased cellular adenosine triphosphate content and caused derangements of the actin-based cytoskeleton as demonstrated by fluorescence microscopy. Coincubation of monolayers with several free-radical or peroxynitrous acid scavengers (deferoxamine, mannitol, dimethyl sulfoxide, or ascorbate) ameliorated hyperpermeability induced by SIN-1 plus amiloride.nnnCONCLUSIONSnAmiloride augments NO.-induced intestinal epithelial permeability, apparently by promoting the development of intracellular acidosis and thereby favoring the formation of the peroxynitrous acid.

Acidic conditions ameliorate both adenosine triphosphate depletion and the development of hyperpermeability in cultured Caco-2BBe enterocytic monolayers subjected to metabolic inhibition

BACKGROUNDnWe recently reported that moderate degrees of adenosine triphosphate (ATP) depletion induced by chronic glycolytic inhibition or hypoxia increase the permeability of Caco-2BBe enterocytic monolayers. Interestingly, the development of lactic acidosis induced by anaerobic glycolysis ameliorates the development of hyperpermeability caused by chronic ATP depletion. We sought to further elucidate the mechanism(s) responsible for the apparent protection against epithelial hyperpermeability afforded by mild acidosis under conditions of metabolic inhibition.nnnMETHODSnCaco-2BBe monolayers growing on permeable supports in bicameral chambers were incubated with 2-deoxyglucose (2DOG) in a glucose-free (Glu-) environment to inhibit glycolysis. Permeability was determined by measuring the transepithelial flux of fluorescein sulfonic acid. Concentrations of intracellular calcium [Ca2+]i were determined fluorometrically by using fura-2.nnnRESULTSnWhen extracellular pH (pH0) was maintained at 7.4 or 5.5, incubation of monolayers for 24 hours with Glu-/2DOG increased permeability and depleted intracellular ATP levels. However, keeping pH0 at 7.0 to 6.0 ameliorated both the development of hyperpermeability and the depletion of ATP induced by Glu-/2DOG. These protective effects were observed under acidic conditions created either by addition to the medium of HCl or by incubation under an atmosphere containing 20% CO2. Incubation with Glu-/2DOG caused bulging of the apical membranes of cells (electron microscopy) and derangements in the perijunctional distribution of actin (fluorescence microscopy); however, these structural changes were ameliorated by mild acidosis. Acute chemical hypoxia at pH0 7.4 induced by Glu-/2DOG plus antimycin A decreased cellular ATP levels and elevated [Ca2+]i. Lowering pH0 to 6.8 ameliorated both the depletion of ATP and the increase in [Ca2+]i induced by Glu-/2DOG+antimycin A.nnnCONCLUSIONSnModerate decreases in pH ameliorate the hyperpermeability induced by metabolic inhibition, possibly by diminishing ATP depletion and blunting increases in [Ca2+]i.