Rhodamine 123 requires carrier-mediated influx for its activity as a P-glycoprotein substrate in Caco-2 cells

Purpose. The purpose of this work was to elucidate transport pathways of the P-glycoprotein (P-gp) substrates rhodamine 123 (R123) and doxorubicin across Caco-2 cells. Methods. Experiments were designed to identify saturable and nonsaturable transport processes and transport barriers for R123 and doxorubicin transport across Caco-2 cells. Confocal laser scanning microscopy (CLSM) imaged R123 transport under normal conditions and in the presence of the P-gp inhibitor, GW918 ( used to abolish P-gp-mediated efflux activity). Results. R123 secretory P-app (P-app,P- BA) showed concentration dependence, whereas R123 absorptive P-app (P-app,P- AB) did not. Inhibition of P-gp efflux revealed that P-gp-mediated efflux had no effect on R123 or doxorubicin P-app,P- AB, but enhanced R123 and doxorubicin Papp, BA. In calcium-free medium, R123 P-app,P- AB increased 15-fold, indicating intercellular junctions are a barrier to R123 absorption. CLSM of R123 fluorescence during absorptive transport under normal conditions and in the presence of GW918 was identical, and was limited to paracellular space, confirming that P-gp is not a barrier to R123 absorption. CLSM revealed that R123 fluorescence during secretory transport under normal conditions and in the presence of GW918 was localized intracellularly and in paracellular space. R123 and doxorubicin uptake across Caco-2 cells basolateral membrane was saturable. Conclusions. R123 absorptive transport occurs primarily by paracellular route, whereas R123 secretory transport involves influx across BL membrane mediated solely by a saturable process followed by apically directed efflux via P-gp. Doxorubicin utilizes similar transport pathways to cross Caco-2 cells.