Received for publication May 5, 2005.
Revised August 2, 2005.
Accepted for publication August 10, 2005.

Functional assessment of multiple P-glycoprotein (P-gp) probe substrates: Influence of cell line and modulator concentration on P-gp activity

Abstract

Compounds known to modulate P-glycoprotein (P-gp) activity were evaluated in cell monolayers expressing P-gp for their effects on the secretory transport of P-gp substrates paclitaxel, vinblastine, and digoxin. Paclitaxel has been proposed to selectively interact with a binding site on P-gp that is distinct from the vinblastine and digoxin binding site. Using MDCK-MDR1, MDCK-WT, and Caco-2 cell monolayers, the basal-to-apical (BL-AP) apparent permeability (P_app) of [³H]paclitaxel, [³H]vinblastine, and [³H]digoxin in the presence of various concentrations of a series of structurally diverse P-gp substrates and modulators of P-gp function were determined. MDCK-WT cell monolayers demonstrated active secretory transport of all P-gp substrate probes, although the sensitivity to inhibition by verapamil was lower than that demonstrated in MDCK-MDR1 cell monolayers. When evaluated as competitive inhibitors, several known P-gp substrates had no effect or only a slight modulatory effect on the BL-AP P_app of all probe substrates in MDCK-MDR1 cells. The secretory transport of P-gp substrates in MDCK-WT cells was more sensitive to inhibition by known P-gp modulators compared to MDCK-MDR1 cells. Low concentrations of ketoconazole (1-3 µM) activated the BL-AP P_app of [³H]vinblastine and [³H]digoxin in MDCK-MDR1 cells, but not in MDCK-WT or Caco-2 cells. Determination of secretory transport in P-gp expressing cell monolayers such as MDCK-MDR1 and Caco-2 may be complicated by substrate cooperativity and allosteric binding, which may result in activation of P-gp. Additionally, expression of other efflux transporters in these cell lines introduces additional complexity in distinguishing which transporter is responsible for substrate recognition and transport.

Key words: ABC transporters, drug transport, membrane permeability, membrane transport, multi-drug resistance, p-glycoprotein, transporters

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