Functional expression of P-glycoprotein in apical membranes of human intestinal Caco-2 cells. Kinetics of vinblastine secretion and interaction with modulators.

Abstract The functional expression of P-glycoprotein has been studied in confluent epithelial layers of human Caco-2 cells, a polarized, highly differentiated cell line demonstrating an intestinal absorptive cell phenotype. Expression of P-glycoprotein was localized, by indirect immunofluorescence with monoclonal antibody MRK16, to the apical brush-border, approximately 20 microns above the base of the cells. Functional, high capacity expression of P-glycoprotein in Caco-2 cell layers was demonstrated by the saturable secretion of vinblastine, a typical substrate, from basolateral to apical surfaces: Km 18.99 +/- 5.55 microM, Vmax 1285.9 +/- 281.2 pmol.cm-2 h-1. The direct correlation of apical P-glycoprotein expression with vinblastine net secretory flux was demonstrated by the reduction of this flux after treatment with MRK16 antibodies. Vinblastine secretory flux was also reduced by treatment with verapamil (R- and S-isomers with equal affinity), nifedipine, taxotere, and 1,9-dideoxyforskolin. Kinetic analyses suggest that the inhibition of vinblastine secretory flux by verapamil and nifedipine was competitive, while that by dideoxyforskolin was non-competitive, in nature. The polarized expression and activity of P-glycoprotein in Caco-2 cells is direct evidence for its secretory detoxifying function in the intestine, subserving at least one role of the gastrointestinal epithelial barrier.