Acidic conditions exacerbate interferon-gamma-induced intestinal epithelial hyperpermeability: role of peroxynitrous acid.
OBJECTIVE
Nitric oxide (NO*) derived from exogenous donors has been shown to increase the permeability of cultured intestinal epithelial monolayers, an effect that is augmented by mildly acidic conditions. Because interferon-gamma (IFN-gamma) also increases intestinal epithelial permeability, at least 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-2BBe) cells were grown as monolayers on permeable supports in bicameral chambers. Permeability was assessed by measuring transepithelial electrical resistance (TER) or the transepithelial passage of fluorescein disulfonic acid. Inducible nitric oxide synthase (iNOS) messenger RNA expression was determined by northern blot analysis. Concentrations of nitrite and nitrate (NO2-/NO3-), stable oxidation products of NO*, were determined using the Greiss reaction. Cellular adenosine triphosphate (ATP) levels were determined using the luciferin/luciferase method.


RESULTS
Incubation of Caco-2BBe monolayers with INF-gamma (1000 units/mL) at an extracellular pH (pHo) of 7.4 increased permeability to fluorescein disulfonic acid and decreased TER. However, incubation of monolayers with IFN-gamma under mildly acidic conditions (i.e., pHo 7.0-6.6) accelerated the decrease in TER and augmented the increase in permeability induced by the cytokine. IFN-gamma-induced iNOS messenger RNA expression and NO2-/NO3- accumulation in medium were unaffected by acidic conditions. At pHo 7.4, incubation of Caco-2BBe monolayers with IFN-gamma (1000 units/mL) for 72 hrs had no effect on intracellular ATP content compared with monolayers simultaneously incubated under the same conditions but in the absence of the cytokine. However, when the cells were incubated for 72 hrs with the same concentration of IFN-gamma under mildly acidic conditions (i.e., pHo 7.0 or 6.6), ATP levels were significantly decreased. At pHo 7.0, IFN-gamma-induced increases in permeability were ameliorated by addition of the following agents: 2-phenyl-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide (a NO* scavenger), N(G)-monomethyl-L-arginine (a iNOS inhibitor), dimethyl sulfoxide (a hydroxyl radical scavenger), and ascorbate (a peroxynitrous acid scavenger).


CONCLUSIONS
Mild acidosis augments IFN-gamma-induced intestinal epithelial hyperpermeability and ATP depletion, possibly by fostering the formation of peroxynitrous acid and/or hydroxyl radical.
Unno
N
N
Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
Hodin
R A
RA
Fink
M P
MP
D000255:Adenosine Triphosphate / Q000032:analysis / Q000502:physiology; D001205:Ascorbic Acid / Q000494:pharmacology; D018938:Caco-2 Cells; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology*; D003497:Cyclic N-Oxides / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical; D017097:Electric Impedance; D016166:Free Radical Scavengers / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology; D007371:Interferon-gamma / Q000187:drug effects / Q000502:physiology*; D007413:Intestinal Mucosa / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D009608:Nitrous Acid / Q000276:immunology*; D030421:Peroxynitrous Acid; D013997:Time Factors; D019323:omega-N-Methylarginine / Q000494:pharmacology
D003497:Cyclic N-Oxides; D016166:Free Radical Scavengers; D007093:Imidazoles; D030421:Peroxynitrous Acid; C079391:2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D007371:Interferon-gamma; D000255:Adenosine Triphosphate; D019001:Nitric Oxide Synthase; D001205:Ascorbic Acid; D009608:Nitrous Acid; D004121:Dimethyl Sulfoxide