Cholyl-L-lysyl-fluorescein (CLF) is a fluorescent bile salt derivative that is being developed as an agent for determining in vivo liver function. However, the mechanisms of uptake and excretion by hepatocytes have not been rigorously studied. We have directly assessed the transport capacity of various hepatobiliary transporters for CLF. Uptake experiments were performed in Chinese hamster ovary cells transfected with human NTCP, OATP1B1, OATP1B3, and OATP2B1. Conversely, excretory systems were tested with plasma membrane vesicles from Sf21 insect cells expressing human ABCB11, ABCC2, ABCC3, and ABCG2. In addition, plasma clearance and biliary excretion of CLF were examined in wild-type, Abcc2(Ϫ/Ϫ), and Abcc3(Ϫ/Ϫ) mice. Human Na ϩ -dependent taurocholic-cotransporting polypeptide (NTCP) and ATP-binding cassette B11 (ABCB11) were incapable of transporting CLF. In contrast, high-affinity transport of CLF was observed for organic anion-transporting polypeptide 1B3 (OATP1B3), ABCC2, and ABCC3 with K m values of 4.6 Ϯ 2.7, 3.3 Ϯ 2.0, and 3.7 Ϯ 1.0 M, respectively. In Abcc2(Ϫ/Ϫ) mice biliary excretion of CLF was strongly reduced compared with wild-type mice. This resulted in a much higher hepatic retention of CLF in Abcc2(Ϫ/Ϫ) versus wild-type mice: 64 versus 1% of the administered dose (2 h after administration). In mice intestinal uptake of CLF was negligible compared with that of taurocholate. Our conclusion is that human NTCP and ABCB11 are incapable of transporting CLF, whereas OATP1B3 and ABCC2/Abcc2 most likely mediate hepatic uptake and biliary excretion of CLF, respectively. CLF can be transported back into the blood by ABCC3. Enterohepatic circulation of CLF is minimal. This renders CLF suitable as an agent for assessing in vivo liver function.
Organic anion transporting polypeptide (OATP) superfamily member 2B1 (OATP2B1) mediates the uptake of steroid hormone precursors and selected drugs in the placenta, liver, mammary gland, brain, and intestine. This action is modulated by sulfhydryl reagents. Common to all OATPs is a large extracellular loop between transmembrane domains IX and X with 10 conserved cysteines. To elucidate the structure-function relationship of this cysteine rich ectodomain, a truncated OATP2B1 lacking 10 extracellular cysteines (OATP2B1 ⌬489 -557 ) and 10 OATP2B1 mutants containing individual Cys-to-Ala substitutions were generated and expressed in CHO-K1 cells. The immunolocalization, cell-surface expression, transport activity, and free cysteine labeling with N-biotinoylaminoethylmethanethiosulfonate of mutant proteins and wild-type OATP2B1 were compared. OATP2B1 ⌬489 -557 accumulated intracellularly. Nine Cys-to-Ala substitutions, C489A, C495A, C504A, C516A, C520A, C539A, C541A, C553A, and C557A, were misprocessed, appearing predominantly as core-glycosylated, 60-kDa proteins and as 180-kDa complexes. Only C493A was a fully glycosylated 75-kDa protein expressed at the cell surface. Thapsigargin partially corrected the misprocessing of mutants. Compared with OATP2B1, C493A and C557A transported estrone-3-sulfate and dehydroepiandrosterone sulfate less efficiently, whereas all other mutants were functionally impaired. MTSEA labeled free cysteines in all Cys-to-Ala mutants but not in OATP2B1, suggesting that all 10 extracellular cysteines are normally disulfide-bonded. Our findings show that the trafficking and function of OATP2B1 is vulnerable to changes in the cysteine residues of extracellular loop IX-X.
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