Gastroenterology

Gastroenterology

Volume 123, Issue 5, November 2002, Pages 1649-1658
Gastroenterology

Basicā€“Liver, Pancreas, and Biliary Tract
The human bile salt export pump: Characterization of substrate specificity and identification of inhibitors*,**,*

https://doi.org/10.1053/gast.2002.36591Get rights and content

Abstract

Background & Aims: The bile salt export pump (BSEP) is the major bile salt transporter in the liver canalicular membrane. Our aim was to determine the affinity of the human BSEP for bile salts and identify inhibitors. Methods: Human BSEP was expressed in insect cells. Adenosine triphosphatase (ATPase) assays were performed, and bile salt transport studies were undertaken. Results: The BSEP gene, ABCB11, was cloned and a recombinant baculovirus was generated. Infected insect cells expressed a 140-kilodalton protein that was absent in uninfected and in mock-infected cells. An ATPase assay showed BSEP to have a high basal ATPase activity. Transport assays were used to determine the Michaelis constant for taurocholate as 4.25 Ī¼mol/L, with a maximum velocity of 200 pmol Ā· mināˆ’1 Ā· mgāˆ’1 protein. Inhibition constant values for other bile salts were 11 Ī¼mol/L for glycocholate, 7 Ī¼mol/L for glycochenodeoxycholate, and 28 Ī¼mol/L for taurochenodeoxycholate. Cyclosporin A, rifampicin, and glibenclamide were proved to be competitive inhibitors of BSEP taurocholate transport, with inhibition constant values of 9.5 Ī¼mol/L, 31 Ī¼mol/L, and 27.5 Ī¼mol/L, respectively. Progesterone and tamoxifen did not inhibit BSEP. Conclusions: The human BSEP is a high-affinity bile salt transporter. The relative affinities for the major bile salts differ from those seen in rodents and reflect the different bile salt pools. BSEP is competitively inhibited by therapeutic drugs. This is a potentially significant mechanism for drug-induced cholestasis.

GASTROENTEROLOGY 2002;123:1649-1658

Section snippets

Generation of full-length ABCB11 complementary DNA

Total RNA from human liver was extracted by using RNAzol B (Biogenesis, Poole, England). First strand complementary DNA (cDNA) synthesis was performed by using C. therm. polymerase (Roche, Basal, Switzerland) in a 20 Ī¼L reaction containing 1 Ɨ reverse transcriptase buffer, 5 mmol/L MgCl2, 5 mmol/L dithiothreitol, 5% dimethyl sulfoxide, 1 Ī¼mol/L primer, 20 mmol/L deoxynucleoside triphosphates (AB gene, Epsom, England), 10 units RNAase inhibitor (Roche), and 6 units C. therm. polymerase. Products

Cloning of ABCB11 complementary DNA

The ABCB11 coding cDNA of 3.963 kilobases was amplified as 3 overlapping fragments (1, 2, and 3; see Figure 1). Each fragment was amplified with primers to introduce restriction endonuclease sites at the 5' and 3' ends to facilitate their cloning. The 3' end of ABCB11 was modified to encode 6 histidine residues at the carboxyl terminus of the recombinant protein. Unique restriction endonuclease sites in the coding sequence shared between overlapping fragments were used to reconstruct the

Discussion

Normal concentrations of bile salts in post sinusoidal blood are generally <10 Ī¼mol/L. The concentration is kept low by their removal from portal blood by a number of transporters located in the basolateral membrane of hepatocytes.24 The true intrahepatocyte concentration of free bile salts is not known. There are good reasons why concentrations should be kept lowā€”bile salts are highly cytotoxic detergents. This all points to the need for a high-affinity export system for bile salts in the

Acknowledgements

The authors thank Colin Dolphin for advice on insect cell membrane preparations, Charlotte Dyer and Andrew McKnight for their expert molecular biology guidance, David Dewin for critical reading of the manuscript, and Ann Mowat and family for their enthusiastic support.

References (41)

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*

Address requests for reprints to: Richard Thompson, M.D., Ph.D., Institute of Liver Studies, King's College Hospital, Denmark Hill, London SE5 9PJ, England. e-mail: [email protected]; fax: (44) 207-346-3700.

**

Supported by The Alex Mowat, Ph.D. Studentship Fund (to J.A.B.), The Wellcome Trust (to R.J.T.), The Children's Liver Disease Foundation (to S.S.S.), and The Medical Research Council (to K.J.L. and C.F.H.)

*

This article is dedicated to the memory of Professor Alex Mowat, Professor of Pediatric Hepatology at King's College Hospital, London, England (1990ā€“1995).

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