Nitric oxide mediates increased P-glycoprotein activity in interferon-γ-stimulated human intestinal cells

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Abstract
Patients with refractory inflammatory bowel disease (IBD) exhibit increased expression of intestinal P-glycoprotein (P-gp) as well as elevated luminal IFN-γ and nitric oxide (NO) levels. Using the in vitro Caco-2 cell culture model, we investigated whether these pathological mediators associated with the etiology of IBD affect functional activity of intestinal efflux systems. IFN-γ reduced cellular uptake of cyclosporin A (CysA) but not methotrexate (MTX) in a time- and concentration-dependent manner. Simultaneously, P-gp expression increased by approximately twofold. Coincubation with the inducible NO synthase inhibitor l- N6-(1-iminoethyl)lysine (l-NIL) dramatically reduced production of intracellular NO in response to IFN-γ stimulus. The presence of l-NIL also abrogated the cytokine-mediated increase in P-gp expression and function suggesting that NO is required for IFN-γ-mediated activation of this efflux system. Exposure of Caco-2 cells to the chemical NO donor S-nitroso- N-acetylpenicillamine (SNAP) produced a concentration-dependent decrease in intracellular CysA accumulation that was paralleled by an increase in P-gp expression. Both IFN-γ and SNAP enhanced DNA binding of NF-κB, whereas inclusion of l-NIL dramatically decreased this cytokine-induced effect on NF-κB binding. These results suggest that NO mediates IFN-γ-induced increase in expression and function of intestinal P-gp in the human Caco-2 cell culture model by altering DNA binding of NF-κB, which may enhance transcription of the ABCB1 gene encoding for this efflux system.
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