Acidic conditions exacerbate interferon-gamma-induced intestinal epithelial hyperpermeability: Role of peroxynitrous acid

Objective: Nitric oxide (NO) derived from exogenous donors has been shown t o increase the permeability of cultured intestinal epithelial monolayers, a n effect that is augmented by mildly acidic conditions. Because interferon- gamma (IFN-gamma) also increases intestinal epithelial permeability, at lea st partly by an NO-dependent mechanism, we sought to determine whether IFN- gamma-induced hyperpermeability is increased under acidic conditions. Methods: Human intestinal epithelial (Caco-2(BBe)) cells were grown as mono layers on permeable supports in bicameral chambers. Permeability was assess ed by measuring transepithelial electrical resistance (TER) or the transepi thelial passage of fluorescein disulfonic acid. Inducible nitric oxide synt hase (iNOS) messenger RNA expression was determined by northern blot analys is. Concentrations of nitrite and nitrate (NO2-/NO3-), stable oxidation pro ducts of NO ., were determined using the Greiss reaction. Cellular adenosin e triphosphate (ATP) levels were determined using the luciferin/luciferase method. Measurements and Main Results: Incubation of Caco-2(BBe) monolayers with IN F-gamma (1000 units/mL) at an extracellular pH (pH(0)) of 7.4 increased per meability to fluorescein disulfonic acid and decreased TER, However, incuba tion of monolayers with IFN-gamma under mildly acidic conditions (i.e., pH( 0) 7.0-6.6) accelerated the decrease in TER and augmented the increase in p ermeability induced by the cytokine, IFN-gamma-induced iNOS messenger RNA e xpression and NO2-/NO3- accumulation in medium were unaffected by acidic co nditions. At pH, 7.4, incubation of Caco-2(BBe) monolayers with IFN-gamma ( 1000 units/mL) for 72 hrs had no effect on intracellular ATP content compar ed with monolayers simultaneously incubated under the same conditions but i n the absence of the cytokine, However, when the cells were incubated for 7 2 hrs with the same concentration of IFN-gamma under mildly acidic conditio ns (i.e., pH(0) 7.0 or 6,6), ATP levels were significantly decreased. At pH (0) 7.0, IFN-gamma-induced increases in permeability were ameliorated by ad dition of the following agent: 2-phenyl-4,4,5,5- tetramethylimidazoline-1-o xyl-3-oxide (a NO scavenger), N-G-monomethyl-L-arginine (a iNOS inhibitor), dimethyl sulfoxide (a hydroxyl radical scavenger), and ascorbate (a peroxy nitrous acid scavenger). Conclusion: Mild acidosis augments IFN-gamma-induced intestinal epithelial hyperpermeability and ATP depletion, possibly by fostering the formation of peroxynitrous acid and/or hydroxyl radical.