Abstract
As one of the Na+-dependent transporters responsible for the hepatic uptake of ligands, sodium taurocholate (TC) co-transporting polypeptide (NTCP) has been cloned from rat liver and its substrate specificity has been clarified by examining the inhibition of TC uptake mediated by NTCP. The contribution of NTCP to the Na+-dependent uptake of ligands into rat hepatocytes, however, still needs to be clarified. To determine the contribution of NTCP, we examined the uptake of ligands into primary cultured hepatocytes (cultured for 4 h) and into COS-7 cells, transiently expressing NTCP, and normalized the uptake of ligands with TC as a reference compound. Western Blot analysis indicated that NTCP was glycosylated much less extensively in the transfected COS-7 cells, although the expression level was comparable for the cultured hepatocytes and transfectant. Kinetic parameters for the Na+-dependent uptake of TC were similar for the cultured hepatocytes and NTCP-transfected COS-7 cells (Km = 17.7 vs. 17.4 μM;Vmax = 1.63 vs. 1.45 nmol/min/mg protein). Glycocholic acid and cholic acid were taken up by NTCP-transfected COS-7 cells. The contribution of NTCP to the Na+-dependent uptake of glycocholic acid into rat hepatocytes was approximately 80%, whereas that of cholic acid was 40%. In addition, the analysis indicated that the contribution of NTCP to the Na+-dependent uptake of several ligands (ouabain, ibuprofen, glutathione-conjugate of bromosulfophthalein, glucuronide- and sulfate-conjugates of 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole) was negligible. Thus, this is a convenient method to determine the contribution of NTCP to the uptake of ligands into hepatocytes. It is also suggested that multiple transport mechanisms are responsible for the Na+-dependent uptake of organic anions into hepatocytes.
Footnotes
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Send reprint requests to: Yuichi Sugiyama, Ph.D., Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7–3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
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↵1 This work was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan, and the Core Research for Evolutional Sciences and Technology of Japan Sciences and Technology Corporation.
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We would like to thank Eizai Co., Ltd., for providing labeled E3040 glucuronide and sulfate and Dr. PJ Meier, for providing anti-rat NTCP serum.
- Abbreviations:
- NTCP
- sodium taurocholate co-transporting polypeptide
- OATP
- organic anion transporting polypeptide
- TC
- taurocholate, taurocholic acid
- GCA
- glycocholate, glycocholic acid
- CA
- cholate, cholic acid
- BSP
- bromosulfophthalein
- BSP-SG
- glutathione-conjugate of bromosulfophthalein
- E3040
- 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole
- SD
- Sprague-Dawley
- Km
- Michaelis constant
- Vmax
- maximum transport velocity
- CLuptake
- uptake clearance
- DMEM
- Dulbecco’s modified Eagle’s medium
- BSA
- bovine serum albumin
- SSC
- saline sodium citrate
- SDS
- sodium dodecyl sulfate
- mEH
- microsomal epoxide hydrolase
- TBS-T
- Tris-buffered saline containing 0.05% Tween 20
- Received December 19, 1997.
- Accepted April 20, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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