Nitric oxide mediates increased P-glycoprotein activity in interferon-{gamma}-stimulated human intestinal cells.
Patients with refractory inflammatory bowel disease (IBD) exhibit increased expression of intestinal P-glycoprotein (P-gp) as well as elevated luminal IFN-gamma 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-gamma 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-N(6)-(1-iminoethyl)lysine (l-NIL) dramatically reduced production of intracellular NO in response to IFN-gamma 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-gamma-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-gamma and SNAP enhanced DNA binding of NF-kappaB, whereas inclusion of l-NIL dramatically decreased this cytokine-induced effect on NF-kappaB binding. These results suggest that NO mediates IFN-gamma-induced increase in expression and function of intestinal P-gp in the human Caco-2 cell culture model by altering DNA binding of NF-kappaB, which may enhance transcription of the ABCB1 gene encoding for this efflux system.
College of Pharmacy, University of Cincinnati, 3223 Eden Ave., Cincinnati, OH 45267-0004, USA.
Santosh G
SG
Dixit
Basilia
B
Zingarelli
Donna J
DJ
Buckley
Arthur R
AR
Buckley
Giovanni M
GM
Pauletti
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D018938:Caco-2 Cells; D004247:DNA / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D015151:Immunoblotting; D015212:Inflammatory Bowel Diseases / Q000473:pathology; D007371:Interferon-gamma / Q000494:pharmacology*; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000166:cytology / Q000187:drug effects; D016328:NF-kappa B / Q000096:biosynthesis; D009569:Nitric Oxide / Q000378:metabolism / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D016328:NF-kappa B; D020030:Nitric Oxide Donors; D009569:Nitric Oxide; D026423:S-Nitroso-N-Acetylpenicillamine; D007371:Interferon-gamma; D004247:DNA; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II