Hyperpermeability of intestinal epithelial monolayers is induced by NO: Effect of low extracellular pH

Nitric oxide (NO .) increases the permeability of Caco-2BBe enterocytic monolayers. Many of the toxic effects of NO . are thought to be mediated by the peroxynitrite anion (ONOO-), which, under mildly acidic conditions, can rearrange to yield an intermediate with reactivity similar to toxic OH .. Accordingly, we assessed the permeability of Caco-2BBe cells grown on permeable supports for 24 h in media titrated to normal or acidic extracellular pH (pH(o)) with or without the NO . donors 3-morpholinosydnonimine (SIN-1) or sodium nitroprusside (SNP). Incubation with 2 mM SIN-1 at pH(o) 6.8 or 0.6 mM SNP at pH(o) 6.5 increased permeability (apical-to-basolateral flux of fluorescein sulfonic acid), whereas at pH(o) 7.4 permeability was unaffected by these concentrations of NO . donors. Accumulation of NO2-/NO3- in medium (index of NO . release) was not increased by incubation of cells with SIN-1 or SNP under mildly acidic conditions. Under acidic but not control conditions, incubation with SIN-1 caused disruption of perijunctional actin filaments as assessed by fluorescence microscopy. At pH(o) 6.8 and 6.5 (but not 7.4), SIN-1 significantly decreased intracellular levels of both ATP and glutathione. Incubation with 5 mM deferoxamine or 500 uM ascorbic acid (ONOO- scavengers) abrogated SIN-1-induced hyperpermeability. We conclude that mild acidosis augments NO .-induced intestinal epithelial permeability, possibly by promoting oxidant-mediated cytoskeletal damage and/or ATP depletion.