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

Abstract

Many of the cytopathic effects of nitric oxide (NO ·) are mediated by peroxynitrite (PN), a product of the reaction between NO · and superoxide radical (O ·− 2). In the present study, we investigated the role of PN, O ·− 2 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-2 BBe 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 O ·− 2 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 O ·− 2). Diethyldithiocarbamate, a superoxide dismutase inhibitor, and pyrogallol, an O ·− 2 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.