Bile salt–dependent efflux of cellular phospholipids... : Hepatology (original) (raw)

Original Articles

Bile salt–dependent efflux of cellular phospholipids mediated by ATP binding cassette protein B4

Morita, Shin-ya1; Kobayashi, Aya1; Takanezawa, Yasukazu2; Kioka, Noriyuki1; Handa, Tetsurou3; Arai, Hiroyuki2; Matsuo, Michinori1; Ueda, Kazumitsu1,*

1_Laboratory of Cellular Biochemistry, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan_

2_Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan_

3_Department of Biosurface Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan_

*Address reprint requests to: Laboratory of Cellular Biochemistry, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

Email: [email protected]

Received 3 October 2006; Accepted 11 January 2007

Published online in Wiley InterScience (www.interscience.wiley.com).

Grant sponsor: Japanese Ministry of Education, Culture, Sports, Science, and Technology; Grant Number: 15GS0301; Grant sponsor: Bio-oriented Technology Research Advancement Institution; Grant sponsor: Pharmaceutical and Medical Devices Agency, and Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists.

Potential conflict of interest: Nothing to report.

fax: (81) 75-753-6104

Abstract

Human ABCB4 (multidrug resistance [MDR]3 P-glycoprotein) is expressed in the canalicular membrane of the hepatocyte. ABCB4 has been shown to be required for phosphatidylcholine (PC) secretion into the bile and to translocate PC across the plasma membrane. To further investigate the function of ABCB4, we established a cell line stably expressing ABCB4 (human embryonic kidney [HEK]/ABCB4). The efflux of phospholipids from HEK/ABCB4 cells was remarkably increased by the addition of taurocholate. In addition, the cholesterol efflux from HEK/ABCB4 cells was also enhanced in the presence of taurocholate. Light scattering measurements suggested that the taurocholate monomer plays an important role in ABCB4-mediated lipid secretion. On the other hand, the efflux of phospholipids and cholesterol was not mediated by ABCB1 (MDR1) even in the presence of taurocholate. Taurocholate promoted the efflux of phospholipids and cholesterol from HEK/ABCB4 cells more efficiently than glycocholate and cholate. ABCB4-K435M and ABCB4-K1075M, Walker A lysine mutants, did not mediate the phospholipid and cholesterol efflux in the presence of taurocholate, suggesting that ATP hydrolysis is essential for the efflux. Verapamil completely inhibited the taurocholate-dependent efflux of phospholipids and cholesterol from HEK/ABCB4 cells. Mass spectrometry revealed that, in the presence of taurocholate, HEK/ABCB4 cells preferentially secreted PC compared to sphingomyelin. PC vesicles induced cholesterol diffusion from cell membrane, but did not accept cholesterol from ABCB4. Conclusion: ABCB4 mediates the efflux of phospholipids into the canalicular lumen in the presence of bile salts, and plays a crucial role in bile formation and lipid homeostasis. (HEPATOLOGY 2007.)

Abbreviations: ABC, ATP binding cassette; MDR3, multidrug resistance 3; PC, phosphatidylcholine; HEK, human embryonic kidney; NaTC, sodium taurocholate; NaGC, sodium glycocholate; NaC, sodium cholate; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; BSA, bovine serum albumin; LDH, lactate dehydrogenase; cmc, critical micelle concentration; WT, wild-type; SM, sphingomyelin; NBD, 7-nitro-2,1,3-benzoxadiazol group.