P-GLYCOPROTEIN (P-GP) MEDIATED EFFLUX IN CACO-2 CELL MONOLAYERS - THEINFLUENCE OF CULTURING CONDITIONS AND DRUG EXPOSURE ON P-GP EXPRESSION LEVELS

The influence of cell culture conditions and previous drug exposure on P-glycoprotein (P-gp) expression levels in Caco-2 cells was determine d. In this study, the expression of P-gp is demonstrated (i) visually by confocal laser scanning microscopy (CLSM), (ii) functionally by tra nsport studies with substrates of the efflux pump, and (iii) quantitat ively by flow cytometry (FCM) analysis using specific monoclonal antib odies (anti P-gp MRK 16 as an external antibody and P-GlycoCheck C219 as an internal antibody). Trypsinization of the cells after reaching c onfluence led to a decrease of P-gp expression levels, while trypsiniz ation before reaching confluence led to an increase after long-term cu ltivation. Culturing the cells on polycarbonate filters did not elicit a significant change of P-gp expression over time in culture, whereas in plastic flasks (polystyrene) a decrease was detected. Using CLSM a strong fluorescence on the apical side of Caco-2 cell monolayers was observed, as a result of incubation with MRK 16 as primary and IgG Cy5 as secondary antibody; Previous drug exposure of the cells showed tha t verapamil, celiprolol, and vinblastine induced the P-gp expression, while metkephamid. (MKA) decreased the P-gp expression level as compar ed to the control. Permeation studies consolidated the theory that P-g p is expressed in the Caco-2 cells examined. For talinolol and MKA, a higher transport from basolateral to apical side than from apical to b asolateral could be measured. Incubation of the cell monolayer with MR K 16 reduced the secretion process to the apical side, but did not inf luence [H-3]-mannitol flux. Caco-2 cells seem to be a suitable,cell li ne model for P-gp-mediated secretion studies. However, the variability of the P-gp expression requires careful control when this model is to be used in quantitative structure/secretion studies.