Marianne Smith

Nitric oxide mediates interferon-gamma-induced hyperpermeability in cultured human intestinal epithelial monolayers.

OBJECTIVE Incubation with interferon-gamma has been shown to increase the permeability of cultured monolayers of intestinal epithelial cells. We sought to determine whether this phenomenon is mediated, at least in part, by increased production of nitric oxide. DESIGN Prospective, controlled, laboratory study. Human intestinal epithelial (Caco-2BBe) cells were grown as monolayers on permeable supports mounted in bicameral chambers. Permeability was assessed by adding fluorescein sulfonic acid (molecular weight = 478 daltons) to the apical compartment and determining the apical-to-basolateral clearance of the probe over a 24-hr period of incubation. SETTING Basic science laboratory. MEASUREMENTS AND MAIN RESULTS The permeability of monolayers to fluorescein sulfonic acid was significantly increased after incubation in the presence of interferon-gamma (250 to 1000 U/mL). The effect of interferon-gamma on permeability was dependent on both the concentration of the cytokine and the duration of exposure to it. Concentrations of nitric oxide oxidation products, nitrite and nitrate, in incubation media were increased after exposure of cells to interferon-gamma. When intestinal epithelial (Caco-2BBe) monolayers were incubated with interferon-gamma in the presence of inhibitors of nitric oxide synthase (NG-monomethyl-L-arginine NG-nitro-L-arginine-methyl ester, or NG-nitro-L-arginine), both of the effects of the cytokine (i.e., increased epithelial permeability and increased production of nitrite/nitrate) were attenuated. CONCLUSIONS These results suggest that upregulation of nitric oxide biosynthesis plays a pivotal role in the increase in permeability of intestinal epithelial (Caco-2BBe) monolayers induced by interferon-gamma. Increased production of nitric oxide induced by proinflammatory cytokines, such as interferon-gamma, may be an important factor contributing to gut mucosal hyperpermeability in sepsis.

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.

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

BACKGROUND We 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. METHODS Caco-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. RESULTS When 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. CONCLUSIONS Moderate decreases in pH ameliorate the hyperpermeability induced by metabolic inhibition, possibly by diminishing ATP depletion and blunting increases in [Ca2+]i.