ATP-binding cassette transporter A subfamily 8 is a sinusoidal efflux

Subscriber access provided by UNIVERSITY OF ADELAIDE LIBRARIES

Article

ATP-binding cassette transporter A subfamily 8 is a sinusoidal efflux transporter for cholesterol and taurocholate in mouse and human liver Kazunari Sasaki, Masanori Tachikawa, Yasuo Uchida, Satoshi Hirano, Fumito Kadowaki, Michitoshi Watanabe, Sumio Ohtsuki, and Tetsuya Terasaki Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00679 • Publication Date (Web): 04 Jan 2018 Downloaded from http://pubs.acs.org on January 5, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

ATP-binding cassette transporter A subfamily 8 is a sinusoidal efflux transporter for cholesterol and taurocholate in mouse and human liver

Kazunari Sasaki1, Masanori Tachikawa1*, Yasuo Uchida1, Satoshi Hirano1, Fumito Kadowaki1, Michitoshi Watanabe1, Sumio Ohtsuki2, Tetsuya Terasaki1

1

Membrane Transport and Drug Targeting Laboratory, Graduate School of Pharmaceutical

Sciences, Tohoku University, Sendai 980-8578, Japan 2

Department of Pharmaceutical Microbiology, Faculty of Life Sciences, Kumamoto

University, Kumamoto, Japan

Running title: ATP-binding cassette transporter A subfamily 8 in the liver

*Address all correspondence to Masanori Tachikawa, Ph.D. Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aramaki, Aoba, Sendai 980-8578,

Japan.

Voice:

+81-22-795-6832,

FAX:

[email protected]

1

ACS Paragon Plus Environment

+81-22-795-6886,

Email:

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract

The ATP-binding cassette (ABC) transporter A subfamily 8 (ABCA8) belongs to the ABCA6-like transporters subgroup, which is distinct from the ABCA1-like subgroup in the ABCA family. The expression and function of the short-size human ABCA8 lacking one of the two ATP-binding domains for ATP hydrolysis, which are regularly present in the other ABCA transporters, have been reported. However, the functional differences between the short-size human ABCA8 and full-size human ABCA8 which has the two ATP-binding domains, remain unknown. The purpose of the present study was to clarify the tissue expression profiles of ABCA6-like and ABCA1-like subgroup transporters and the functional characteristics of ABCA8 in mouse and human. The tissue distribution of mouse ABCA (mABCA) transporter protein and the changes in mABCA8 protein expression levels in a mouse model of obstructive cholestasis were elucidated by means of quantitative targeted absolute proteomics (QTAP). The transport characteristics were clarified in a HEK293 cell line overexpressing full-size ABCA8 protein. QTAP and immunohistochemical analyses revealed that mABCA transporters exhibited the distinct protein expression patterns in the tissues, and mABCA8b, its mouse orthologue, was abundant in the liver and predominantly distributed in sinusoidal membranes of the hepatocytes. Further, protein expression of mABCA8b was decreased in the mouse cholestasis liver. Changes of mABCA8b expression level in cholestasis were similar to those of mABCA1, a sinusoidal cholesterol efflux transporter. Uptake and efflux assays showed that ABCA8 mediates efflux of [3H]cholesterol and [3H]taurocholate, while showed no significant efflux activity for [3H]estrone sulfate, [3H]digoxin, [3H]vinblastine, [3H]para-aminohippuric acid, [3H]oleic acid, [14C]nicotine, or

2


Page 2 of 52

Page 3 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


[3H]methotrexate. [3H]Cholesterol efflux was increased by extracellularly applied taurocholate. These results suggest that mABCA8b/ABCA8 functions as a sinusoidal efflux transporter for at least cholesterol and taurocholate in mouse and human liver.

Keywords: ABCA6-like subgroup transporters, ABCA1-like subgroup transporters, ABCA8, absolute protein expression level, tissue distribution, sinusoidal membrane, quantitative targeted absolute proteomics, cholestasis, liver, bile duct ligation, mass spectrometry

Abbreviations

ABC, ATP-binding cassette; BDL, bile duct ligation; DMEM, Dulbecco's modified Eagle's medium, LC-MS/MS, liquid chromatography-tandem mass spectrometry; OATP, organic anion transporting polypeptides; PAH, para-aminohippuric acid; QTAP, quantitative targeted absolute proteomics; RT-PCR, reverse transcription-polymerase chain reaction; SLC, solute carrier; SRM/MRM, selected reaction monitoring/multiple reaction monitoring

Abstract graphic

3



Introduction

The ATP-binding cassette transporter A (ABCA) subfamily 8 (ABCA8) belongs to the ABCA6-like transporters subgroup that is distinct from the ABCA1-like subgroup in the ABCA family1, 2. The ABCA transporters can be evolutionarily divided into two subgroups, i.e., ABCA6-like and ABCA1-like transporters. Accumulating evidence indicates that ABCA1-4, 7, and 12, belonging to the ABCA1-like subgroup, mediate efflux transport of bioactive lipids, including cholesterol and phospholipids, in various types of tissues and cells3-11. On the other hand, less is known about the functional roles of the ABCA6-like subgroup transporters, i.e., ABCA5-6, 8-10 in human and Abca5-6, 8a, 8b, and 9 in rodents, although several reports have described the tissue mRNA and protein expressions, as summarized in supplemental Table S1. Considering that the genes of ABCA6-like subgroup transporters form a cluster on human chromosome 17q24 and have 53-78% amino acid similarity2, it seems plausible that there are functional similarities among ABCA8 and the other ABCA6-like subgroup transporters. It has been reported that single nucleotide polymorphisms of ABCA6 affect blood lipid levels12 and also that gene expression of ABCA9 and A10 is cholesterol-responsive13, 14. In addition, high-level mRNA expressions of ABCA1,

ABCA6, ABCA8, and ABCA9 in human primary epithelial ovarian cancer tissues in vivo are statistically significantly associated with reduced survival in serous ovarian cancer patients15, whereas mRNA expressions of Abca5, Abca7, Abca8a, Abca8b, and Abca9 are down-regulated in mice with acute digoxin loading16. Thus, ABCA8 may transport certain lipids and drugs. Tsuruoka et al.17 used the human sequence of short-size ABCA8 (Gene accession no.

4


Page 4 of 52

Page 5 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


AB020629), which has only a single ATP-binding domains for ATP hydrolysis18, to show that the ABCA8 serves as an influx transporter of organic anions such as taurocholate, para-aminohippuric acid (PAH), estrone sulfate, and estradiol-β-glucuronide. On the other hand, other ABCA transporters have been identified as full-size transporters, which have two ATP-binding domains1. A variant of full-size ABCA8 that contains the two ATP-binding domains has been reported (Gene accession no. NM_001288985.1). Recent report has also demonstrated that the full-size ABCA8 mRNA with two ATP-binding domains can be amplified from a human liver cDNA library19, suggesting the full-size ABCA8 protein is predominantly expressed in human liver. However, it is not yet clear whether the two variants of ABCA8 have similar transport functions. A better understanding of the tissue-specific expression, changes in protein expression under pathological conditions such as cholestasis, and substrate spectrum of ABCA8 is needed to provide insight into its functional roles in the body, as well as to aid development of new drugs targeting lipid-related diseases. Knowledge on the changes in the protein expression levels under pathological conditions would give us a better idea of the transporter function. The cholestasis mouse model exhibits accumulation of cholesterol and taurocholate in liver and circulating blood20-22. Previous reports have demonstrated a decreased level of mouse ABCA1 (mABCA1) (a cholesterol efflux transporter)22, decreased levels of mouse organic anion transporting polypeptides (OATP) 1a123 and 1b224 (taurocholate influx transporters on the sinusoidal membranes), and increased levels of taurocholate efflux transporters (such as mABCC3)24. Thus, expression changes of transporters involved in transport of cholesterol and taurocholate appear to play key roles in the cholestasis model. Quantitative targeted absolute proteomics (QTAP)-based protein quantification has made it

5



Page 6 of 52

possible to determine the absolute protein expression levels of transporters in various tissues of human and mouse25-28. Whereas producing specific antibodies against ABCA6-like subgroup transporters is challenging due to the high amino acid sequence homology, QTAP is able in principle to distinguish even one amino acid difference in the target peptide to be quantified28. Therefore, the QTAP strategy should be suitable to elucidate the absolute protein expression profiles of ABCA6-like subgroup transporters, as well as the expression changes in disease models. The purpose of the present study was thus to clarify the absolute protein expression levels of mABCA1-like and mABCA6-like transporters in various tissues, as well as the changes in the protein expression levels in the obstructive cholestasis mouse model, by means of QTAP analyses, and also to investigate the localization of ABCA8 in human and mouse liver, and the substrate

spectrum

of

ABCA8

by

means

of

efflux

ABCA8-overexpressing HEK293 cells.

6


and

uptake

studies

in

Page 7 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Experimental Section

Animals Male ddY and C57BL/6 mice, 7 to 9 weeks old, were purchased from Japan SLC, Inc. (Shizuoka, Japan). The mice were housed in cages in a temperature, light, and humidity-controlled room for 4 weeks at maximum. The mouse model of obstructive cholestasis was prepared as described previously24. Briefly, under anesthesia with isoflurane, the abdominal cavity was opened and the common bile-duct was ligated with 4-0 surgical nylon, leaving the gall bladder intact. The abdominal muscle and wound were sutured with 4-0 surgical nylon and closed with instant adhesive. Sham surgeries were performed by the same method without the bile duct ligation (BDL) procedure. The liver was isolated at 48 h or 7 days after BDL. Tissues were snap-frozen in liquid nitrogen and stored at −80°C until analysis. All experiments were approved by the Institutional Animal Care and Use Committee in Tohoku University, and performed in accordance with Tohoku University guidelines.

Establishment of HEK293 cell line stably expressing ABCA8 (ABCA8/HEK293 cells) HEK293 cells stably expressing ABCA8 and control cells were generated by transfection with pCMV-myc-tag 3A (Invitrogen, Carlsbad, CA) containing the open reading frame of

ABCA8, or the empty vector (mock). ABCA8 cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR) from MTC Multiple Tissue cDNA Panel (Clontech, Palo Alto, CA). The amino acid sequence translated from the ABCA8 cDNA was identical to that reported in the NCBI database (Gene accession no. NM_001288985.1). The

7



transfection was performed using LipofectamineTM 2000 and Opti-MEM I medium (Invitrogen) according to the manufacturer’s protocol. Briefly, the cells were seeded at 90% confluence in a 10-cm-diameter dish containing antibiotic-free medium. The cells were incubated with the complex of the vector and Lipofectamine for 6 h. The medium was replaced with fresh medium without the antibiotics, and the transfected cells were passaged with medium containing G418 (Cayman, Ann Arbor, MI) to select vector-inserted cells. The cells were then routinely grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and 0.4 mg/mL G418 in a humidified incubator at 37°C and 5% CO2.

Preparation of plasma membrane/crude membrane fractions from mouse tissues and ABCA8/HEK293 cell line Brain, heart, ileum, jejunum, kidney, large intestine, liver, lung, skeletal muscle, pancreas, prostate, spleen, stomach, testicle, thymus of ddY mouse were each homogenized on ice in a Potter-Elvehjem homogenizer in 40 mL of hypotonic buffer [10 mM Tris–HCl, pH 7.4, 10 mM NaCl, 1.5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, and 1% v/v protease inhibitor cocktail (Sigma Chemical Co., St. Louis, MO)] per gram wet weight of tissues. Sequential centrifugations were performed to obtain the plasma membrane fractions of the brain, heart, ileum, jejunum, kidney, large intestine, liver, lung, skeletal muscle, pancreas, testicle, and thymus and the crude membrane fractions of the prostate, spleen and stomach as reported previously29. The plasma membrane fractions were used for protein quantification. In the cases of prostate, spleen, stomach, the crude membrane fractions were used for the absolute protein quantifications, because we could not obtain sufficient plasma membrane fraction (at least 50 µg is necessary for the methanol-chloroform precipitation method).

8


Page 8 of 52

Page 9 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Plasma and crude membrane fractions were obtained from ABCA8/HEK293 cells and control HEK293 cells by means of sequential centrifugations as reported27. Protein concentrations were measured by the Lowry method using the DC protein assay reagent (Bio-Rad, Hercules, CA). The plasma and crude membrane fractions were stored at -80°C until analyzed.

Preparation of sinusoidal and canalicular plasma membrane fractions from mouse liver plasma membrane fraction Sinusoidal and canalicular plasma membrane fractions were prepared from mouse liver as reported previously30. Briefly, the mouse liver plasma membrane fraction obtained as described above was layered on top of a 31%, 34%, and 38% sucrose solution gradient, and centrifuged at 195,700 g for 3 h at 4°C. The turbid layers from the interface were sequentially collected, suspended in the suspension buffer, and centrifuged at 100,000 g for 40 min at 4°C. The canalicular plasma membrane fraction was obtained from the resultant pellets of turbid layers between the suspension buffer and 31% sucrose solution. The sinusoidal plasma membrane fraction was obtained from the resultant pellets of turbid layers between 34% and 38% sucrose solutions.

LC−MS/MS-based protein quantification analysis (QTAP) Simultaneous protein quantitation of target molecules was performed by using nanoLC-MS/MS with multiplexed selected/multiple reaction monitoring (SRM/MRM) as described previously29. Protein expression levels were determined as the absolute amounts of trypsin-generated specific target peptides whose sequences were selected based on in silico selection criteria25. The SRM/MRM transitions for quantification of each peptide were set as

9



Page 10 of 52

shown in supplemental Tables S2 and S3. The absolute expression amount of mABCA6 was calculated from the quantitative data obtained for a peptide generated from the common sequence of mABCA6, mABCA8a, and mABCA9 by subtracting the values obtained for peptides

specific

to

mABCA8a

and

mABCA9.

Preparation

of

trypsin-

and

lysylendopeptidase-treated samples, LC separation of peptides, and detection/quantification of the target peptides were carried out as we reported previously29, 31. The expression amount of each peptide was determined from one SRM transition or the average of up to four SRM transitions from one sample in one analysis. When the protein quantification values were determined for each sample from three or four SRM transitions in one analysis, the values were expressed as the mean±S.E.M. The variation of S.E.M. was used as a measure of the inter-analytical differences in the QTAP analyses. The protein expression levels in the livers of obstructive cholestasis and control mice were calculated from the protein quantification values of three or four independent mice and expressed as the mean±S.D. The variation of S.D. was used as a measure of the inter-individual differences in these studies. In cases where no signal peak was detected, the amount of peptide in the sample was defined as under the limit of quantification and the value (fmol/µg protein) was calculated as follows. Stfmol/ ISfmol = Peak AreaSt/ Peak AreaIS

Reverse transcription polymerase chain reaction (RT-PCR) analysis of ABCA8 cDNA synthesis from ABCA8/HEK293 cells and mock cells was performed as reported previously32. PCR was conducted with the specific primer sets through 1 cycle of 94 °C for 2 min, 30 cycles of 94 °C for 10 s, 60 °C for 5 s and 72 °C for 2 min, and 1 cycle of 72 °C for 5 min using PrimeSTAR Max polymerase (Takara, Shiga, Japan). The sequences of the specific

10


Page 11 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


primers were designed to amplify a specific region of the full-size ABCA8 variant (Gene accession no. NM_001288985.1): sense, 5’-GACAGAAAAGAAAGCTAACCTTTGG-3’; antisense, 5’-CAATTCCTAGGTCAGGATAGC-3’. Electrophoresis was performed with 1.5% agarose gel for 40 min at 135 V. The gel was stained with ethidium bromide.

Immunoblot analysis The crude membrane fractions obtained from ABCA8/HEK293 cells and mock cells were adjusted to the same protein concentration and the same amounts of sample buffer [10% 2-mercaptoethanol (Wako, Osaka, Japan), 4% sodium dodecyl sulfate (SDS) (Nacalai tesque, Kyoto, Japan), 125 mM Tris-HCl (pH 6.8),10% sucrose (Wako), 0.004% bromophenol blue] were used. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on 12% polyacrylamide gel.

The proteins

were electrophoretically transferred

onto

polyvinylidine fluoride (PVDF) membranes and incubated with blocking buffer (25 mM Tris-HCl, pH 8.0, 125 mM NaCl, 0.1% Tween20 and 4% skim milk). The PVDF membrane was incubated with rabbit anti-ABCA8 antibody (1:500 dilution; Abcam, Cambridge, MA) or mouse anti-myc antibody (1:2000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) in the blocking buffer overnight at 4°C. The blot was then probed with horseradish peroxidase-conjugated goat anti–rabbit or anti-mouse IgG as secondary antibodies in the blocking buffer for 1 h at room temperature. The bands were visualized by enhanced chemiluminescence (ECL, Amersham, Arlington Heights, IL) with HyperfilmTM-MP (Amersham).

Immunofluorescence staining of ABCA8 in ABCA8/HEK293 cells

11



ABCA8/HEK293 cells and mock cells were seeded on poly-D-lysine-coated 8-well culture slides (BD Bioscience, San Jose, CA) at a density of 1.0×105 cells/well. The cells were fixed in 4% paraformaldehyde (Merck, Darmstadt, Germany) in 0.1 M phosphate buffer (PB) containing 0.1 M NaH2PO4 (Wako) and 0.1 M Na2HPO4 (Wako) for 20 min. NaH2PO4 and Na2HPO4 were used for pH equilibration in the PB buffer. Sections were incubated with a solution of MeOH/CH3COOH/water (90: 5: 5) at -20°C and blocked for 30 min with 10% normal goat serum, then incubated overnight with rabbit polyclonal ABCA8 antibodies (HPA044914, Sigma-Aldrich, St Louis, MO) as a primary antibody in phosphate-buffered saline containing 0.1% Triton X and 4% bovine serum albumin at 4°C, followed by Alexa568-labeled goat anti-rabbit IgG (Invitrogen) as a secondary antibody in the same buffer for 1 hr. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Fluorescence images were taken with Zeiss Axio Observer Z1 (Zeiss, Göttingen, Germany) equipped with the specific filters for Alexa 568 and DAPI using Zeiss ZEN software. Contrast and brightness were modified by Photoshop software.

Immunofluorescence staining of ABCA8 in paraffinized human liver sections Paraffinized human liver sections were purchased from Wako Pure Chemical Industries (Osaka, Japan). They were dewaxed with xylene and hydrated with concentration-graded ethanol and distilled water. Heat-induced epitope retrieval was performed by autoclaving the sections with a target retrieval solution (Dako, Kyoto, Japan) according the manufacturer’s protocol. Blocking was done with PBS containing 10% normal goat serum and 0.2% Triton X-100 for 30 min. The sections were incubated overnight with rabbit polyclonal ABCA8 antibodies (HPA044914, Sigma-Aldrich) singly or in combination with mouse monoclonal

12


Page 12 of 52

Page 13 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


F-actin antibodies (ab205, Abcam) in PBS containing 5 % bovine serum albumin (BSA) at 4°C, followed by incubation for 2 hr with Alexa568-labeled goat-anti-rabbit IgG (Invitrogen) singly or in combination with Alexa488-labeled goat-anti-mouse as a secondary antibody in the same buffer. Fluorescence images were taken with Zeiss Axio Observer Z1 (Zeiss, Göttingen, Germany) equipped with the specific filters for Alexa 488 and Alexa 568 using Zeiss ZEN software. Contrast and brightness were modified by Photoshop software.

[3H]Cholesterol efflux assay [3H]Cholesterol efflux assay using ABCA8/HEK293 cells was conducted as reported previously33. ABCA8/HEK293 cells and mock cells were incubated with [3H]cholesterol (Perkin Elmer) at a concentration of 40.8 nM (2 µCi/mL) in antibiotics-free DMEM containing 10% fetal bovine serum for 24 h, then washed with PBS, and incubated for 2 h in the medium containing 0.1% BSA to allow equilibration of the labelled lipids with lipids in the intracellular pools. The cells were washed with PBS and then incubated in serum-free medium containing 0.1% BSA in the presence or absence of lipid acceptors such as apolipoprotein AI (Sigma-Aldrich) and taurocholate (Sigma-Aldrich) for 24 h. ApoAI and taurocholate were chosen as potent lipid acceptors of ABCA8 because previous reports have demonstrated that ApoAI and taurocholate play crucial roles in ABCA1-mediated cholesterol efflux34 and also in ABCB4-mediated cholesterol and phospholipids efflux35. The medium was collected and the cells were lysed with 5 M NaOH. The lysate was neutralized with 5 M HCl. Radioactivity in the medium and cell lysates were measured by scintillation counting (Beckman LS6500, Beckman). The efflux amount of radioactively labelled lipid into the medium was calculated as a percentage of total radioactivity in the cell lysate and medium.

13



Efflux data were calculated from four independent wells and expressed as the mean±S.E.M. The variation of S.E.M was used as a measure of the inter-wells differences in these studies.

Uptake assay ABCA8/HEK293 cells were seeded on poly-D-lysine-coated 24-well plates (AGC Techno Glass, Chiba, Japan) at a density of 1.3×105 cells/well. The mock cells were seeded at a density of 1.0×105 cells/well on similar poly-D-lysine-coated 24-well plates, because ABCA8/HEK293 cells grow more slowly than the mock cells. The cells were incubated for 48 hrs. The uptake assay was performed according to the methods previously reported36. The culture medium was removed and the cells were washed three times with the extracellular fluid (ECF) buffer (122 mM NaCl, 3 mM KCl, 0.4 mM K2HPO4, 25 mM NaHCO3, 1.2 mM MgSO4, 1.4 mM CaCl2, 10 mM D-glucose, 10 mM HEPES, pH 7.4). Then, the cells were incubated in ECF buffer containing [3H]taurocholic acid (1.00 µM) (PerkinElmer, Waltham, MA), [3H]digoxin (20.8 nM) (PerkinElmer), [3H]estrone sulfate (PerkinElmer) (10.9 nM), [3H]PAH (PerkinElmer) (136 nM), [3H]oleic acid (16.6 nM) (PerkinElmer), [3H]vinblastine (35.1 nM) (American Radiolabeled Chemicals, St Louis, MO), [3H]methotrexate (27.9 nM) (Moravek Biochemicals, Brea, CA), or [14C]nicotin (8.98 µM) (American Radiolabeled Chemicals) at 37°C for 60 min, based on a previous report17 on uptake and efflux activities in short-size ABCA8-expressing Xenopus laevis oocytes for several organic anions, presumably at an equilibrium state. This means that if ABCA8 mediates active efflux transport of one substrate from inside to outside the cell, the accumulation of the substrate in ABCA8/HEK293 cells would be smaller than that in mock cells, and vice versa. The test compounds were selected from among known substrates of ABC transporters and solute

14


Page 14 of 52

Page 15 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


carrier (SLC) family transporters, including those reported previously as substrates of ABCA817, in order to clarify the similarities and differences of substrate specificity compared with other ABC and SLC transporters. The uptake solution was removed and the cells were rapidly washed three times with ice-cold ECF buffer, dissolved in 5 M NaOH overnight, and then neutralized with 5 M HCl. The radioactivity of aliquots was determined with a liquid scintillation counter (Beckman LS6500, Beckman, Fullerton, CA). The cellular protein content was determined by the Lowry method using the DC protein assay reagent (Bio-Rad). Uptake was normalized to the protein content in the cell lysate and expressed as the cell-to-medium ratio (µL/mg protein), which was calculated by dividing the cellular uptake amount (pmol/mg protein) by the extracellular compound concentration in the medium (pmol/µL). Uptake data were calculated from four independent wells and expressed as the mean±S.E.M. The variation of S.E.M was used as a measure of the inter-wells differences in these studies.

Statistical analysis An F-test was performed to assess the equality of variance between two groups. According to the result of the F-test, Student’s t-test (equal variance) or Welch’s test (unequal variance) was performed to determine the statistical significance of differences between two groups. A value of p

ATP-binding cassette transporter A subfamily 8 is a sinusoidal efflux

Recommend Documents