Submitted on June 3, 2004
Accepted on October 10, 2004
Nitric Oxide Mediates Increased P-glycoprotein Activity in Interferon-
-stimulated Human Intestinal Cells
Santosh G. Dixit1, Basilia Zingarelli2, Donna J. Buckley1, Arthur R. Buckley1, and Giovanni M. Pauletti1*
1 Division of Pharmaceutical Sciences, College of Pharmacy, University of Cincinnati, Cincinnati, OH, USA
2 Division of Critical Care, Children's Hospital Medical Center, Cincinnati, OH, USA
* To whom correspondence should be addressed. E-mail: gm.pauletti{at}uc.edu.
Patients with refractory inflammatory bowel disease (IBD) exhibit increased expression of
intestinal P-glycoprotein (P-gp) as well as elevated luminal interferon-
(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 ~2-fold. Coincubation with the inducible nitric oxide 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 nuclear factor 
B (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.
