Abstract

In the course of other experiments, we serendipitously observed that extracellular nicotinamide adenine dinucleotide (NAD⁺) ameliorated the development of epithelial hyperpermeability when monolayers of Caco-2 enterocyte-like cells were incubated with cytomix, a mixture containing interferon-γ, interleukin-1β, and tumor necrosis factor-α. We sought to characterize the effects of NAD⁺ on inflammation-induced epithelial barrier dysfunction using Caco-2 monolayers that were exposed to cytomix in the absence or presence of NAD⁺ or other purine-containing molecules. Paracellular barrier function measured as the apical-to-basolateral passage of fluorescein isothiocyanate-conjugated dextran (mol. wt. ∼4000) was preserved in a concentration-dependent manner when immunostimulated Caco-2 cells were exposed to extracellular NAD⁺. Incubation with NAD⁺ prevented cytomix-induced derangements in the expression and localization of the tight junction proteins occludin and zonula occludens-1 in Caco-2 cells. Treatment of cytomix-stimulated cells with NAD⁺ also blocked nuclear factor-κB (NF-κB) activation, inducible nitric-oxide synthase induction, and increased production of nitric oxide (NO·). Ileal mucosal permeability to fluorescein isothiocyanate-dextran mol. wt. ∼4000 was increased in mice 18 h after lipopolysaccharide (endotoxin) injection, but treatment of endotoxemic mice with NAD⁺ ameliorated the development of gut mucosal hyperpermeability. Thus, extracellular NAD⁺ seems to ameliorate inflammation-induced intestinal epithelial barrier dysfunction by inhibiting NF-κB activation and increased NO· production.