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(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.

The Role of the Sodium-Taurocholate Cotransporting Polypeptide (NTCP) and of the Bile Salt Export Pump (BSEP) in Physiology and Pathophysiology of Bile Formation

“…Besides different bile salts, estrone-3-sulfate and bromosulfophthalein are good substrates. Ntcp is also able to transport drugs conjugated to bile salts and hence is a potential drug target [208,323] [58]. This latter finding highlights the importance of the side chain a determinant for Ntcp substrates [17].…”

Bile Acid Signaling Is Involved in the Neurological Decline in a Murine Model of Acute Liver Failure

“…CLF exhibits aspects of transporter-mediated transport like endogenous bile acids and shows similar brain penetrance to tritiated taurocholate in models of chronic liver damage in our hands (data not shown). 24,25 Once again, at the time when minor neurological symptoms were evident, mice were anesthetized and transcardially perfused with ice-cold saline to remove the blood from the brain and the amount of fluorescence in homogenates of the frontal cortex was assessed as previously described. 26 …”

Transporters involved in the hepatic uptake of 99mTc-mebrofenin and indocyanine green

“…Flp-In CHO cells stably expressing human NTCP were generated by ligating the coding sequence of NTCP into pcDNA5/FRT (Invitrogen, Carlsbad, CA) as described previously (19). Control cells were transfected with the empty pcDNA5/FRT vector (CHO-pcDNA5/FRT).…”