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INFLAMMATION/IMMUNITY/MEDIATORS

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

¹Division of Pharmaceutical Sciences, College of Pharmacy, University of Cincinnati, Cincinnati; and ²Children's Hospital Medical Center, Division of Critical Care, Cincinnati, Ohio

Submitted 3 June 2004 ; accepted in final form 10 October 2004

Patients with refractory inflammatory bowel disease (IBD) exhibit increased expression of intestinal P-glycoprotein (P-gp) as well as elevated luminal IFN- 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- 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- 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--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- and SNAP enhanced DNA binding of NF-B, whereas inclusion of L-NIL dramatically decreased this cytokine-induced effect on NF-B binding. These results suggest that NO mediates IFN--induced increase in expression and function of intestinal P-gp in the human Caco-2 cell culture model by altering DNA binding of NF-B, which may enhance transcription of the ABCB1 gene encoding for this efflux system.

Caco-2 cells; cytokines; efflux systems; signal transduction pathways