The IC50 for Inhibition of Digoxin Transport Across Confluent Cell Monolayers of P-gp Expressing Cell Lines is Often a Function of Inhibitor Binding to Both P-gp and an Unidentified Basolateral Digoxin Uptake Transporter

Abstract

Previously, we reported that the P-gp substrate digoxin requires basolateral and apical uptake transporter(s) to achieve the observed efflux kinetics across MDCKII-MDR1 (Netherlands Cancer Institute) confluent cell monolayers. We extend this kinetic analysis to include other cell lines, including MDCKII-MDR1 (National Institute of Health) and Caco-2. Digoxin transport across confluent cell monolayers was measured in the absence and presence of several P-gp substrates and/or inhibitors. Our kinetic analysis showed that digoxin transport in these other cell lines typically requires a basolateral digoxin uptake transporter. The B>A transport inhibition curve of digoxin was a conflation of inhibitor binding to both P-gp and to this basolateral digoxin uptake transporter. If a new compound is an inhibitor of digoxin transport across any of these cell-lines, the IC50 could be due to binding to P-gp or to the basolateral digoxin uptake transporter, or both. Finally, our analysis allowed us to estimate the efflux active P-gp surface density on the cell lines, which is the fraction of P-gp in the plasma membrane which successfully effluxes substrate into the apical chamber, which depends upon microvilli morphology.