title: Hepatic transport mechanisms of cholyl-L-lysyl-fluorescein.
abstract: 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(-/-), and Abcc3(-/-) mice. Human Na(+)-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 +/- 2.7, 3.3 +/- 2.0, and 3.7 +/- 1.0 microM, respectively. In Abcc2(-/-) 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(-/-) 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.
authors.affiliation: Liver Center, Academic Medical Center, Amsterdam, The Netherlands. d.r.dewaart@amc.nl.
authors.forename: Dirk R
authors.initials: DR
authors.lastname: de Waart
authors.forename: Stephanie
authors.initials: S
authors.lastname: Häusler
authors.forename: Maria L H
authors.initials: ML
authors.lastname: Vlaming
authors.forename: Cindy
authors.initials: C
authors.lastname: Kunne
authors.forename: Emanuel
authors.initials: E
authors.lastname: Hänggi
authors.forename: Hans-Jurgen
authors.initials: HJ
authors.lastname: Gruss
authors.forename: Ronald P J
authors.initials: RP
authors.lastname: Oude Elferink
authors.forename: Bruno
authors.initials: B
authors.lastname: Stieger
mesh_terms: D000818:Animals; D001646:Bile / Q000378:metabolism; D001692:Biological Transport; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002793:Cholic Acids / Q000493:pharmacokinetics*; D006224:Cricetinae; D003412:Cricetulus; D005452:Fluoresceins / Q000493:pharmacokinetics*; D005456:Fluorescent Dyes / Q000493:pharmacokinetics*; D006801:Humans; D007313:Insecta / Q000166:cytology / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism; D029363:Organic Anion Transporters, Sodium-Dependent / Q000378:metabolism; D029362:Organic Anion Transporters, Sodium-Independent / Q000235:genetics / Q000378:metabolism; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3; D027981:Symporters / Q000378:metabolism; D014162:Transfection
chemical_list: D002352:Carrier Proteins; D002793:Cholic Acids; D005452:Fluoresceins; D005456:Fluorescent Dyes; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D029363:Organic Anion Transporters, Sodium-Dependent; D029362:Organic Anion Transporters, Sodium-Independent; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3; D027981:Symporters; C000715087:ABCC2 protein, human; C421209:SLCO1B3 protein, human; C072313:cholyl-lysylfluorescein; C071908:sodium-bile acid cotransporter