ATP-dependent transport of bile salts by rat multidrug resistance-associated protein 3 (Mrp3)

We have previously shown that cloned rat multidrug resistance-associated protein 3 (Mrp3) has the ability to transport organic anions such as 17 beta-estradiol 17-beta-D-glucuronide (E(2)17 beta G) and has a different substrate specificity from MRP1 and MRP2 in that glutathione conjugates are poor substrates for Mrp3 (Hirohashi, T,, Suzuki, H., and Sugiyama, Y, (1999) J, Biol. Chem. 274, 15181-15185). In the present study, the involvement of Mrp3 in the transport of endogenous bile salts was investigated using membrane vesicles from LLC-PK1 cells transfected with rat Mrp3 cDNA The ATP-dependent uptake of [H-3]taurocholate (TC), [C-14]glycocholate (GC), [H-3]taurochenodeoxycholate-3-sulfate (TCDC-S), and [H-3]taurolithocholate-3-sulfate (TLC-S) was markedly stimulated by Mrp3 transfection in LLC-PK1 cells. The extent of Mrp3-mediated transport of bile salts was in the order, TLC-S > TCDC-S > TC > GC. The K-m and V-max values for the uptake of TC and TLC-S were K-m = 15.9 +/- 4.9 mu M and V-max = 50.1 +/- 9.3 pmol/min/mg of protein and K-m = 3.06 +/- 0.57 mu M and V-max = 161.9 +/- 21.7 pmol/min/mg of protein, respectively. At 55 nM [H-3]E(2)17 beta G and 1.2 mu M [H-3]TC, the apparent K-m values for ATP were 1.36 and 0.66 mM, respectively, TC, GC, and TCDC-S inhibited the transport of [H-3]E(2)17 beta G and [H-3]TC to the same extent with an apparent IC50 of similar to 10 mu M. TLC-S inhibited the uptake of [H-3]E(2)17 beta G and [H-3]TC most potently (IC50 of similar to 1 mu M) among the bile salts examined, whereas cholate weakly inhibited the uptake (IC50 similar to 75 mu M). Although TC and GC are transported by bile salt export pump/sister of P-glycoprotein, but not by MRP2, and TCDC-S and TLC-S are transported by MRP2, but not by bile salt export pump/sister of P-glycoprotein, it was found that Mrp3 accepts all these bile salts as substrates, This information, together with the finding that MRP3 is extensively expressed on the basolateral membrane of human cholangiocytes, suggests that MRP3/Mrp3 plays a significant role in the cholehepatic circulation of bile salts.