FUNCTIONAL EXPRESSION OF P-GLYCOPROTEIN IN APICAL MEMBRANES OF HUMAN INTESTINAL CACO-2 CELLS - KINETICS OF VINBLASTINE SECRETION AND INTERACTION WITH MODULATORS

The functional expression of P-glycoprotein has been studied in conflu ent epithelial layers of human Caco-2 cells, a polarized, highly diffe rentiated cell line demonstrating an intestinal absorptive cell phenot ype. Expression of P-glycoprotein was localized, by indirect immunoflu orescence with monoclonal antibody MRK16, to the apical brush-border, approximately 20 mum above the base of the cells. Functional, high cap acity expression of P-glycoprotein in Caco-2 cell layers was demonstra ted by the saturable secretion of vinblastine, a typical substrate, fr om basolateral to apical surfaces: K(m) 18.99 +/- 5.55 mum, V(max) 128 5.9 +/- 281.2 pmol.cm-2 h-1. The direct correlation of apical P-glycop rotein 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 verapami l (R- and S-isomers with equal affinity), nifedipine, taxotere, and 1, 9-dideoxyforskolin. Kinetic analyses suggest that the inhibition of vi nblastine secretory flux by verapamil and nifedipine was competitive, while that by dideoxyforskolin was non-competitive, in nature. The pol arized expression and activity of P-glycoprotein in Caco-2 cells is di rect evidence for its secretory detoxifying function in the intestine, subserving at least one role of the gastrointestinal epithelial barri er.