We have characterized the substrate specificity and mechanism of transport of the human multidrug resistance-associated protein 3 (MRP3). A murine fibroblastlike cell line generated from the kidneys of mice that lack Mdr1a/b and Mrp1 was retrovirally transduced with MRP3 cDNA. Stable clones overproducing MRP3 were resistant to the epipodophyllotoxins etoposide and teniposide but not to vincristine, doxorubicin, and cisplatin, drugs suggested to be MRP3 substrates by others.

Many of the transporters involved in the transport of bile acids in the enterohepatic circulation have been characterized. The basolateral bile-acid transporter of ileocytes and cholangiocytes remains an exception. It has been suggested that rat multidrug resistance protein 3 (Mrp3) fulfills this function. Here we analyse bile-salt transport by human MRP3. Membrane vesicles from insect ( Spodoptera frugiperda ) cells expressing MRP3 show time-dependent uptake of glycocholate and taurocholate. Furthermore, sulphated bile salts were high-affinity competitive inhibitors of etoposide glucuronide transport by MRP3 (IC50 approximately 10 microM). Taurochenodeoxycholate, taurocholate and glycocholate inhibited transport at higher concentrations (IC50 approximately 100, 250 and 500 microM respectively). We used mouse fibroblast-like cell lines derived from mice with disrupted Mdr1a, Mdr1b and Mrp1 genes to generate transfectants that express the murine apical Na+-dependent bile-salt transporter (Asbt) and MRP3. Uptake of glycocholate by these cells is Na+-dependent, with a K(m) and V(max) of 29+/-7 microM and 660 +/- 63 pmol/min per mg of protein respectively and is inhibited by several organic-aniontransport inhibitors. Expression of MRP3 in these cells limits the accumulation of glycocholate and increases the efflux from cells preloaded with taurocholate or glycocholate. In conclusion, we find that MRP3 transports both taurocholate and glycocholate, albeit with low affinity, in contrast with the high-affinity transport by rat Mrp3. Our results suggest that MRP3 is unlikely to be the principal basolateral bile-acid transporter of ileocytes and cholangiocytes, but that it may have a role in the removal of bile acids from the liver in cholestasis.

Understanding of the interactions between P‐glycoprotein and multidrug resistance (MDR) reversing agents is important in designing more effective MDR modulators. We examined transcellular transport of several MDR modulators by using a drug‐sensitive epithelial cell line, LLC‐PK1 and its transformant cell line, LLC‐GA5‐COL300, which expresses human P‐glycoprotein on the apical surface. Basal‐to‐apical transports of azidopine and diltiazem across the LLC‐GA5‐COL300 monolayer were increased and apical‐to‐basal transports were decreased compared to those across the LLC‐PK1 monolayer, indicating that P‐glycoprotein transports azidopine and diltiazem. Movements of nitrendipine and staurosporine across the epithelial monolayer were not affected by P‐glycoprotein. These results suggests that some MDR modulators exert their inhibitory effect not only by blocking the initial binding of anticancer drugs but throughout the course of the transport process.

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