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Characterization of a stable cell line expressing human Na + /taurocholate cotransporting polypeptide for high throughput screening
Author(s) -
Roth Megan,
Alnouti Yazen,
Hagenbuch Bruno
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.796.12
Subject(s) - chemistry , cell culture , chinese hamster ovary cell , troglitazone , substrate (aquarium) , bile acid , carbenoxolone , caco 2 , biochemistry , receptor , cell , pharmacology , biology , intracellular , peroxisome proliferator activated receptor , ecology , genetics , gap junction
Human Na + /taurocholate cotransporting polypeptide (NTCP) is selectively expressed on the basolateral membrane of hepatocytes and mediates Na + ‐dependent uptake of conjugated bile acids. It has also been shown to transport non‐bile acid substrates, including some statins. To examine the substrate specificity of NTCP using high‐throughput screening, we established a stable Chinese Hamster Ovary cell line that transports taurocholate with K m and V max values of 18.4 μM and 6.9 nmoles/mg/min, respectively. To screen for interactions we determined uptake on 96‐well plates in the absence and presence of xenobiotics that affect hepatocytes. Potential substrates were tested directly using either radiolabels or LC‐MS/MS analysis. Numerous strong inhibitors were identified, including troglitazone and rosiglitazone (IC 50 s of 3.5 μM and 0.93 μM, respectively), perfluorodecanoic acid (IC 50 = 8 μM), fluvastatin (IC 50 = 40 μM) and carbenoxolone (IC 50 = 9 μM). Neither glitazone was a substrate of NTCP. Unexpectedly, pravastatin, which did not inhibit NTCP‐mediated transport, was a substrate for NTCP, indicating the potential presence of multiple binding sites. These results show that our cell line can be used for high‐throughput screening to identify xenobiotics that may interfere with enterohepatic circulation of bile acids, or are potential substrates of NTCP.