AJP - GI Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Gastrointest Liver Physiol 290: G640-G649, 2006. First published November 10, 2005; doi:10.1152/ajpgi.00354.2005
0193-1857/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/4/G640    most recent
00354.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (26)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rius, M.
Right arrow Articles by Keppler, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rius, M.
Right arrow Articles by Keppler, D.

LIVER AND BILIARY TRACT

Substrate specificity of human ABCC4 (MRP4)-mediated cotransport of bile acids and reduced glutathione

Maria Rius,1 Johanna Hummel-Eisenbeiss,1 Alan F. Hofmann,2 and Dietrich Keppler1

1Division of Tumor Biochemistry, German Cancer Research Center, Heidelberg, Germany; and 2Division of Gastroenterology, Department of Medicine, University of California-San Diego, La Jolla, California

Submitted 29 July 2005 ; accepted in final form 7 November 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The multidrug resistance protein ABCC4 (MRP4), a member of the ATP-binding cassette superfamily, mediates ATP-dependent unidirectional efflux of organic anions out of cells. Previous studies showed that human ABCC4 is localized to the sinusoidal membrane of hepatocytes and mediates, among other substrates, the cotransport of reduced glutathione (GSH) with bile acids. In the present study, using inside-out membrane vesicles, we demonstrated that human ABCC4 in the presence of physiological concentrations of GSH has a high affinity for the taurine and glycine conjugates of the common natural bile acids as well as the unconjugated bile acid cholate. Chenodeoxycholyltaurine and chenodeoxycholylglycine were the GSH cosubstrates with the highest affinities for ABCC4, with Km values of 3.6 and 5.9 µM, respectively. Ursodeoxycholyltaurine and ursodeoxycholylglycine were cotransported together with GSH by ABCC4 with Km values of 7.8 and 12.5 µM, respectively, but no transport of ursodeoxycholate and deoxycholate was observed. The simultaneous transport of labeled GSH and cholyltaurine or cholylglycine was demonstrated in double-labeled cotransport experiments with a bile acid-to-GSH ratio of ~1:22. Km values of the bile acids for ABCC4 were in a range similar to those reported for the canalicular bile salt export pump ABCB11. Under physiological conditions, the sinusoidal ABCC4 may compete with canalicular ABCB11 for bile acids and thereby play a key role in determining the hepatocyte concentration of bile acids. In cholestatic conditions, ABCC4 may become a key pathway for efflux of bile acids from hepatocytes into blood.

liver; multidrug resistance protein; sinusoidal transporter

BILE ACIDS ARE SYNTHESIZED from cholesterol in hepatocytes. After their biosynthesis, bile acids undergo enterohepatic circulation mediated by both hepatocyte and enterocyte transporters (18). In the hepatocyte, several members of the human ATP-binding cassette (ABC) superfamily have been identified as bile acid transporters, including the bile acid (salt) export pump ABCB11 (BSEP), which is the major canalicular bile salt transporter in hepatocytes (4, 24), and the multidrug resistance protein ABCC4 (MRP4), which functions as a bile acid efflux pump at the sinusoidal membrane of human hepatocytes (29). Although ABCC3 (MRP3) also is localized to the sinusoidal membrane (16), it has been reported to be a relatively poor transporter for bile acids (1, 37). In addition to the export pumps, several transporters in the sinusoidal membrane of hepatocytes mediate the uptake of bile acids (9), including the Na+-taurocholate cotransporting polypeptide NTCP (SLC10A1) and the sodium-independent organic anion transporting polypeptides OATP1B1 (OATP-C/OATP2) and OATP1B3 (OATP8) (18).

Hepatocytes are the major source of reduced glutathione (GSH) in plasma. As a physiological antioxidant, GSH is involved in many biochemical processes and serves as the major transport form of cysteine (5, 23). Therefore, GSH needs to be transported across the sinusoidal membrane of the hepatocyte to enable GSH delivery to other tissues (2). Three members of the human multidrug resistance protein family (ABCC/MRP) have been identified as GSH export pumps. The first is ABCC1 (MRP1; Refs. 20, 22), which is not expressed in hepatocytes in detectable amounts (14). The second is ABCC2 (MRP2), a low-affinity GSH transporter (25), which is localized to the canalicular membrane of hepatocytes (14). The third is ABCC4, which is localized to the sinusoidal membrane of hepatocytes and has been shown to function as a cotransporter for GSH and the conjugated bile acid cholyltaurine (C-tau, taurocholate) (29). In the coefflux by ABCC1 and ABCC4, GSH can be replaced by several GSH analogs, including S-methyl-glutathione and ophthalmate (22, 29).

ABCC4 has the lowest number of amino acids of the MRP family and transports a broad range of structurally diverse substances, including sulfated steroids as well as several drugs (17, 35). It is widely distributed in human epithelial cells, and in most of them, ABCC4 is localized to the basolateral membrane (19, 29, 30). An exception is the renal proximal tubular cell, in which ABCC4 has an apical localization (33). The ABCC4-mediated transport was potently inhibited by a widely used inhibitor of ATP-dependent ABCC transporters, the quinoline derivative MK571, as well as by known ABCC4 substrates, such as dehydroepiandrosterone 3-sulfate, methotrexate, and folate (29).

The localization of ABCC4 to the sinusoidal membrane of human hepatocytes, together with the identification of C-tau and GSH as cosubstrates for ABCC4, suggest important functions of this export pump in the physiology and pathophysiology of the liver (29). We previously proposed that ABCC4 mediates the cycling of bile acids across the sinusoidal membrane and along the sinusoid (29). The cycling of bile acids may act to regulate the intrahepatocyte concentration of bile acids, whereas the efflux of GSH serves as a source of GSH for other tissues. Bile acid efflux also could be of major importance in case of impaired bile acid transport into bile.

The aim of the present study was to define the substrate specificity of ABCC4 toward common natural conjugated and unconjugated bile acids, as well as to quantify the stoichiometry of bile acid-GSH cotransport.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. [22,23-3H]deoxycholylglycine (DC-gly; 1.1 TBq/mmol), [22,23-3H]chenodeoxycholylglycine (CDC-gly; 0.4 TBq/mmol), [22,23-3H]-chenodeoxycholyltaurine (CDC-tau; 0.4 TBq/mmol), [22,23-3H]ursodeoxycholylglycine (UDC-gly; 0.4 TBq/mmol), [22,23-3H]ursodeoxycholyltaurine (UDC-tau; 0.4 TBq/mmol), [22,23-3H]cholate (0.9 TBq/mmol), [22,23-3H]ursodeoxycholate (UDCA; 0.4 TBq/mmol), and [22,23-3H]deoxycholate (1.1 TBq/mmol) were synthesized at the University of California, San Diego as described previously (32). [3H(G)]cholyltaurine (C-tau; 129.5 GBq/mmol), [24-14C]C-tau (1.7 GBq/mmol), and [glycine-2-3H]glutathione (GSH; 1.9 TBq/mmol) were obtained from PerkinElmer Life Science (Boston, MA). [Glycine-1-14C]cholylglycine (C-gly; 2.1 GBq/mmol) was obtained from American Radiolabeled Chemicals (St. Louis, MO). Unlabeled bile acids, GSH, acivicin, and dithiothreitol (DTT) were obtained from Sigma (St. Louis, MO).

Cell culture and cell lines. Chinese hamster lung fibroblasts V79 cells permanently expressing high levels of recombinant human ABCC4 (V79-ABCC4) were grown in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% (vol/vol) fetal bovine serum and 100 U/ml penicillin-streptomycin and kept at 37°C and 5% CO2 as described recently (29).

Preparation of membrane vesicles. Inside-out membrane vesicles from transfected V79 cells were prepared as described previously (13). In brief, the cells were lysed by incubation in hypotonic buffer (0.1 mM EDTA, 0.5 mM sodium phosphate, pH 7.0) for 1.5 h, followed by homogenization with a Potter-Elvehjem homogenizer. After centrifugation of the homogenate at 12,000 g (10 min at 4°C), the postnuclear supernatant was centrifuged at 100,000 g for 45 min at 4°C. The resulting pellet was suspended in incubation buffer (250 mM sucrose, 10 mM Tris·HCl, pH 7.4), homogenized with a tight-fitting Dounce (glass/glass) homogenizer, and layered over 38% sucrose in 5 mM HEPES/KOH, pH 7.4. After centrifugation at 280,000 g for 1.5 h at 4°C, the turbid layer at the interface was collected, washed by centrifugation in the incubation buffer (100,000 g), and passed 20 times through a 27-gauge needle for vesicle formation. Aliquots of the membrane vesicle suspension were frozen and stored in liquid nitrogen.

Vesicle transport studies. ATP-dependent transport of 3H-labeled bile acids into inside-out membrane vesicles was measured by rapid filtration through nitrocellulose filters essentially as described previously (13). Membrane vesicles (25 µg of protein) were incubated at 37°C in the presence of 4 mM ATP, 10 mM MgCl2, 10 mM creatine phosphate, 100 µg/ml creatine kinase, and labeled substrate in the presence or absence of 5 mM GSH in an incubation buffer containing 250 mM sucrose and 10 mM Tris·HCl at pH 7.4. DTT (1 mM) was added to the incubation buffer for measurements in the presence of GSH unless otherwise indicated. The final incubation volume was 55 µl, and the substrate concentrations are as indicated. Aliquots (15 or 20 µl) of the incubations were taken at the times indicated and filtered immediately through nitrocellulose filters (0.2-µm pore size; Millipore, Billerica, MA) presoaked in incubation buffer. Filters were rinsed with 8 ml of incubation buffer, dissolved in liquid scintillation fluid, and counted for radioactivity. In control experiments, ATP was replaced by an equal concentration of 5'-AMP. Rates of net ATP-dependent transport were calculated by subtracting values obtained in the presence of 5'-AMP as a blank from those in the presence of ATP.

ATP-dependent transport of [3H]GSH alone or together with 14C-labeled bile acid was measured by centrifugation of the vesicles through a gel matrix using NICK spin columns (13). This procedure was more efficient for the separation of extravesicular labeled substrates than for the filtration through nitrocellulose filters, because labeled substrates bound to the filters, causing high background radioactivity. Transport was performed as described above, except that assays were performed in a 55-µl final incubation volume containing 120 µg of vesicle protein. To minimize GSH catabolism by {gamma}-glutamyltransferase during transport, we preincubated membrane vesicles with 2 mM acivicin for 30 min at 37°C before measuring [3H]GSH transport (22) and added 5 mM DTT to the incubation buffer. NICK spin columns (0.2 g of Sephadex G-50 per 3.3 ml of incubation buffer; Amersham Bioscience, Uppsala, Sweden) were prepared by rinsing with incubation buffer and were centrifuged at 400 g for 3 min at 4°C immediately before use. Aliquots (10 µl) of the incubations were taken at the times indicated, diluted with ice-cold incubation buffer (final volume 100 µl), and immediately loaded onto the Sephadex G-50 columns. The columns were additionally rinsed with 100 µl of incubation buffer and centrifuged at 400 g for 3 min at 4°C. The effluents were collected, dissolved in liquid scintillation fluid, and counted for radioactivity. For the counting in the double-labeling experiments, a program was used that measures 14C and 3H radioactivity simultaneously. With the use of this program, the spillover of 14C into the 3H channel and vice versa was <5% and 0.01%, respectively. The transport rates were calculated as mentioned above.

For determination of kinetic constants, transport rates were measured at five different substrate concentrations (2–50 µM bile acid). Km values were determined as the substrate concentration at half-maximal velocity of transport under these conditions with the use of double-reciprocal plots and direct curve fitting to the Michaelis-Menten equation.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
ATP-dependent transport of bile acids into membrane vesicles from transfected V79 cells. The ATP-dependent accumulation of 3H-labeled bile acid at the standard concentration of 5 µM in membrane vesicles from ABCC4- and vector-transfected cells was negligible in the absence of GSH compared with the presence of GSH for all tested bile acids (Fig. 1, B, D, and F, and Fig. 2B). The presence of 5 mM GSH caused an increase of the ATP-dependent 3H-labeled bile acid accumulation in membrane vesicles from ABCC4-transfected cells (Fig. 1, A, C, and E and Fig. 2A). Although membrane vesicles from vector-transfected cells also showed an ATP-dependent bile acid transport in the presence of GSH, transport mediated by the ABCC4-containing membrane vesicles was at least twofold higher, as shown in Table 1. No stimulation was observed in the presence of 1 mM DTT (not shown). Transport by the control membrane vesicles was consistent with the presence of endogenous hamster Abcc4 in the V79 cells. Although hamster Abcc4 in V79 cells could not be detected at the protein level by currently available antibodies, the presence of hamster Abcc4 mRNA in V79 cells was detected with RT-PCR using Abcc4-specific primers (not shown).


Figure 1
View larger version (34K):
[in this window]
[in a new window]
  		Fig. 1. Transport of bile acids into membrane vesicles from ABCC4-transfected (V79-ABCC4) and vector-transfected control (V79-Co) cells. Membrane vesicles (25 µg of protein) were incubated with 5 µM [3H]cholate (A and B), 5 µM [3H]deoxycholylglycine (DC-gly; C and D), or 5 µM [3H]chenodeoxycholylglycine (CDC-gly; E and F) in the absence (B, D, and F) or presence (A, C, and E) of 5 mM GSH. Vesicle-associated radioactivity was determined as described in MATERIALS AND METHODS. The rates of net ATP-dependent transport were calculated by subtracting transport in the presence of 4 mM 5'-AMP as a blank from transport in the presence of 4 mM ATP. Data represent mean (SD) values from a triplicate determination reproduced independently at least once.

 

Figure 2
View larger version (38K):
[in this window]
[in a new window]
  		Fig. 2. Transport and kinetic analysis of the glycine and taurine conjugates of ursodeoxycholate. Membrane vesicles (25 µg of protein) from V79-ABCC4 and V79-Co cells were incubated with 5 µM [3H]ursodeoxycholyltaurine (UDC-tau) in the absence (B) or presence (A) of 5 mM GSH. Vesicle-associated radioactivity was determined as described in MATERIALS AND METHODS. The rates of net ATP-dependent transport were calculated by subtracting transport in the presence of 4 mM 5'-AMP as a blank from transport in the presence of 4 mM ATP. Data represent mean (SD) values from at least 3 determinations. C: kinetic analysis of ABCC4-mediated [3H]UDC-tau ({blacktriangledown}) and [3H]ursodeoxycholylglycine (UDC-gly; {blacktriangleup}) transport in the presence of 5 mM GSH as described in MATERIALS AND METHODS. The Km values were calculated from double-reciprocal plots. Points represent mean (SD) values from 3 determinations. D: kinetic analysis of ABCC4-mediated [3H]cholylglycine C-gly ({square}) transport in the presence of 5 mM GSH and its inhibition by 20 µM of UDC-gly (bullet) and ursodeoxycholate (UDCA; {blacktriangleup}) determined as described in MATERIALS AND METHODS. The Km values were calculated from double-reciprocal plots. The Ki values for the competitive inhibition by UDC-gly and UDCA were 12.9 and 56.2 µM, respectively. Points represent mean (SD) values from a triplicate determination reproduced independently at least once.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Bile acid transport in the presence of 5 mM GSH in ABCC4-containing vesicles and in control vesicles

 
Vesicles from ABCC4-transfected V79 cells transported the following bile acids in a GSH-dependent manner as summarized in Table 1: for DC-gly, the rate was 31.6 pmol·mg protein–1·min–1 (Fig. 1C); for CDC-gly, 35.7 pmol·mg protein–1·min–1 (Fig. 1E); and for CDC-tau, 51.9 pmol·mg protein–1·min–1. For UDC-gly, the rate was 33.8 pmol·mg protein–1·min–1, and for UDC-tau, 36.8 pmol·mg protein–1·min–1 (Fig. 2A). For trihydroxy bile acids, the rate for C-gly was 19.4 pmol·mg protein–1·min–1, and that for C-tau was 47.1 pmol·mg protein–1·min–1. The unconjugated bile acid cholate was transported at a rate of 8.9 pmol·mg protein–1·min–1 (Fig. 1A). However, no transport could be detected for the unconjugated dihydroxy bile acids UDCA and deoxycholate in either ABCC4-containing vesicles or control vesicles under our experimental conditions. Transport rates of vesicles from vector-transfected V79 cells for the corresponding bile acids in the presence of GSH were much smaller, as summarized in Table 1.

Kinetic analysis of ABCC4-mediated ATP-dependent bile acid transport in the presence of glutathione. To further characterize the affinity of ABCC4 for the different bile acids, we determined Km and Vmax values in ABCC4-containing vesicles for each bile acid in a concentration range from 2 to 50 µM and in the presence of 5 mM GSH (Table 2). CDC-tau was the bile acid with the lowest Km value of 3.6 µM, followed by CDC-gly with a Km value of 5.9 µM. DC-gly and C-tau showed Km values of 6.7 and 7.7 µM, respectively. The taurine and glycine conjugates of UDCA exhibited different Km values, with higher affinity of ABCC4 for UDC-tau (7.8 µM) than for UDC-gly (12.5 µM; Table 2 and Fig. 2C). The lowest affinity was observed for cholate (14.8 µM) and C-gly (25.8 µM; Table 2). Because membrane vesicles with similar transport rates for all bile acids, and thus similar Vmax values, were used, the transport efficiency could be calculated for each bile acid (Table 2). The ratio Vmax/Km obtained for the various bile acids resulted in the following ranking from high to low transport efficiency: CDC-tau > C-tau > DC-gly > UDC-tau > CDC-gly > UDC-gly > C-gly > cholate (Table 2).


View this table:
[in this window]
[in a new window]
  		Table 2. Kinetic constants for ABCC4-mediated bile acid cotransport in the presence of 5 mM GSH

 
Ursodeoxycholate conjugate-mediated inhibition and its kinetic characterization. ATP-dependent transport of the unconjugated bile acid UDCA into membrane vesicles from transfected cells was not detectable under our experimental conditions. However, ABCC4-mediated ATP-dependent transport of its taurine and glycine conjugates was observed in the presence of 5 mM GSH as noted above. Because UDCA is used widely in clinical practice to treat cholestatic liver disease, and because its dominant metabolites in humans are its glycine and taurine conjugates, we tested whether UDCA as well as UDC-gly and UDC-tau would inhibit C-gly transport in the presence of GSH (Fig. 2D). All three compounds, UDCA, UDC-gly, and UDC-tau, at a standard concentration of 20 µM caused a competitive inhibition of the C-gly-GSH cotransport with Ki values of 56.2 µM for UDCA (Fig. 2D), 12.9 µM for UDC-gly (Fig. 2D), and 10 µM for UDC-tau (not shown).

Double-labeling experiments for measuring simultaneous ATP-dependent transport of [3H]glutathione and 14C-labeled bile acids into vesicles from transfected V79 cells. To demonstrate the cotransport of GSH and bile acids, we measured the simultaneous ATP-dependent transport of 5 mM [3H]GSH with 5 µM [14C]C-tau or that of 5 mM [3H]GSH with different concentrations of [14C]C-gly (Fig. 3). High background of the GSH transport into vesicles from vector-transfected cells was obtained, which is probably due to other endogenous GSH efflux mechanisms present in all cells (23).


Figure 3
View larger version (21K):
[in this window]
[in a new window]
  		Fig. 3. Double-labeling experiments for measuring the simultaneous ATP-dependent cotransport of [3H]GSH and [14C]cholylglycine (C-gly) into vesicles from V79-ABCC4 and V79-Co cells. Membrane vesicles (120 µg of protein) were incubated at the same time with 5 mM [3H]GSH (A) and 5 µM [14C]C-gly (B). Vesicle-associated radioactivity was determined by centrifugation of the vesicles through a gel matrix as described in MATERIALS AND METHODS. The rates of net ATP-dependent transport were calculated by subtracting transport in the presence of 4 mM 5'-AMP as a blank from transport in the presence of 4 mM ATP. A and B represent the same transport measurement. Data represent mean (SD) values from a triplicate determination reproduced independently at least once.

 
The following transport rates were obtained in a 20-min time period for 5 mM GSH and 5 µM C-gly in double-labeling experiments: for GSH transport, 122.9 pmol·mg protein–1·min–1 in vesicles from ABCC4-transfected V79 cells and 41.4 pmol·mg protein–1·min–1 in vesicles from vector-transfected V79 cells (Fig. 3A); and for C-gly transport, 7.1 pmol·mg protein–1·min–1 in vesicles from ABCC4-transfected V79 cells and 3.4 pmol·mg protein–1·min–1 in vesicles from vector-transfected V79 cells (Fig. 3B). From these transport rates, ABCC4-mediated transport rates were subsequently obtained: 81.5 pmol·mg protein–1·min–1 for GSH and 3.7 pmol·mg protein–1·min–1 for C-gly, resulting in an approximate C-gly-to-GSH ratio of 1:22. When double-labeling experiments were performed at 5 mM GSH and 10 µM C-gly, the approximate C-gly-to-GSH ratio was maintained at 1:19 when the ABCC4-mediated transport was measured within the first 20 min: 157.6 pmol·mg protein–1·min–1 for GSH and 8.2 pmol·mg protein–1·min–1 for C-gly. At higher C-gly concentrations, the relative amounts of GSH and C-gly coefflux were not constant; however, much more GSH than C-gly was consistently cotransported.

When C-gly was replaced with C-tau, the cotransport with GSH was corroborated by the following transport rates: for GSH transport, 110.2 pmol·mg protein–1·min–1 in vesicles from ABCC4-transfected V79 cells and 9.7 pmol·mg protein–1·min–1 in vesicles from vector-transfected V79 cells; and for C-tau transport, 10.3 pmol·mg protein–1·min–1 in vesicles from ABCC4-transfected V79 cells and 5.5 pmol·mg protein–1·min–1 in vesicles from vector-transfected V79 cells (not shown). The resulting ABCC4-mediated C-tau-to-GSH ratio was 1:21. Under the same conditions, ATP-dependent transport of 5 mM [3H]GSH in the absence of bile acids into membrane vesicles from vector- and ABCC4-transfected V79 cells was detected with similar transport rates: 3.2 pmol·mg protein–1·min–1 for vector-transfected cells and 2.0 pmol·mg protein–1·min–1 for ABCC4-transfected cells (not shown).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we have examined in detail the bile acid specificity for GSH-bile acid cotransport by human ABCC4, an export pump localized to the sinusoidal membrane of human hepatocytes. Transport studies in inside-out membrane vesicles demonstrated ATP-dependent transport into ABCC4-containing membrane vesicles for the glycine- and taurine-conjugated bile acids predominating in human biliary bile acids, as well as for the conjugates of UDCA and the unconjugated bile acid cholate in the presence of physiological GSH concentrations (Figs. 1 and 2, Table 1). Whether other known ABCC4 substrates can influence GSH transport or whether their transport is modulated by GSH is currently unknown.

The Km values obtained for all bile acids in the presence of GSH were in the micromolar range and thus in the range of physiological concentrations (Table 2). The highest affinity was observed for CDC-tau with a Km value of 3.6 µM and the lowest affinity for C-gly with a Km value of 25.8 µM (Table 2). UDC-gly and UDC-tau, the amidated conjugates of UDCA, showed Km values of 7.8 and 12.5 µM (Fig. 2C), respectively, which are in the range of Km values obtained for most of the naturally occurring bile acids in humans (Table 2). CDC-tau followed by C-tau had the highest transport efficiency in ABCC4-containing membrane vesicles (Table 2). Transport affinities of all bile acids were comparable and almost identical to those obtained for the canalicular bile salt export pump ABCB11 (4, 24) with the difference that ABCC4-mediated bile acid transport is a GSH-dependent transport process. Thus the kinetic analysis reveals that ABCC4 is able, at least in principle, to compete with ABCB11 for bile acids in hepatocytes.

UDCA is widely used for the treatment of cholestatic liver diseases (26). In the hepatocyte, UDCA is rapidly conjugated with glycine and taurine. These conjugates may protect the liver against hepatotoxicity of endogenous bile acids by a variety of mechanisms (3). Although UDCA is present in human bile only in trace amounts, treatment with UDCA increases its concentration in plasma and bile, and UDCA becomes the predominant circulating bile acid (3). Although our data indicate that ABCC4 mediates ATP-dependent transport of UDC-tau and UDC-gly (Fig. 2, A and C), transport of labeled UDCA could not be demonstrated using membrane vesicles for reasons that need to be defined in future studies. However, UDCA was able to compete for the ABCC4-mediated ATP-dependent transport of C-gly (Fig. 2D), suggesting that UDCA also may be a substrate for the ABCC4-mediated GSH cotransport. Thus our results indicate that ABCC4 can contribute to the increase of UDCA conjugates, and possibly that of UDCA, in plasma during UDCA treatment of cholestatic liver diseases.

In an earlier study in our laboratory (29), measurements of ATP-dependent transport using 3H-labeled GSH in the presence of unlabeled C-tau suggested that ABCC4 cotransports GSH along with C-tau. Several other studies on ABCC1 (MRP1), which cannot be detected in human hepatocytes (14), have described the cotransport of GSH and several substances, including vincristine (20, 21). It has been proposed for ABCC1 that the protein contains a bipartite binding site for hydrophobic and anionic moieties (10). In our present transport system, ABCC4 may bind GSH as an anionic moiety, inducing a conformational change that allows the binding of bile acids to the other binding site, with this in turn leading to the transport of both cosubstrates. Direct evidence for cotransport has now been provided by double-labeling experiments, in which membrane vesicles were simultaneously incubated with both substrates, radiolabeled with different isotopes, i.e., in our study, [3H]GSH and [14C]C-tau or [14C]C-gly. These experiments demonstrated ABCC4-mediated simultaneous ATP-dependent transport of both compounds (Fig. 3, A and B). GSH cotransport was measured at a concentration of 5 mM, which is within the concentration range (1–10 mM) found in cells (2). This concentration was chosen on the basis of our earlier kinetic analysis (29), which indicated a Km value of 2.7 mM for GSH in the presence of 5 µM C-tau. Although glutathione S-transferases show dissociation constants between 10 and 200 µM for GSH (7), only a small proportion of hepatic GSH is used for glutathione S-transferase reactions (28), and the major proportion of hepatic GSH is effluxed across the basolateral membrane of hepatocytes (11).

For C-tau as well as for C-gly at concentrations between 5 and 10 µM and a constant concentration of 5 mM GSH, the bile acid-to-GSH ratio of ~1:22 indicates that GSH is cotransported along with bile acids at a far greater transport rate than the bile acids themselves. At higher C-gly concentrations, the bile acid-to-GSH ratio was no longer constant, but the amounts of GSH transported were invariably greater than the amounts of bile acid transported. Thus this transport process can be described as an obligatory coefflux of GSH and bile acids without a strict stoichiometry. Other cotransporters have been described with stoichiometries differing from 1:1, such as the human Na+-glucose cotransporter SGLT1, an apical membrane protein, which couples the transport of 2 Na+ ions and 1 glucose molecule with ~210 water molecules (34).

Under physiological conditions in the hepatocyte, canalicular ABCB11 mediates the secretion of bile acids into bile (Fig. 4). In addition, ABCC4 in the sinusoidal membrane of hepatocytes should mediate active efflux of GSH and bile acids into the space of Disse and thence into sinusoidal blood (Fig. 4). Bile acids can then reenter into more portally situated hepatocytes by the Na+-taurocholate cotransporting polypeptide NTCP or the sodium-independent organic anion transporting polypepetides OATP1B1 and OATP1B3 (Fig. 4) (9). Such a reuptake of compounds from blood into the hepatocytes has been described in the isolated perfused rat liver (27). Pharmacokinetic analysis of this model indicated that the net sinusoidal efflux rate of dibromosulfophthalein is the result of sinusoidal efflux and partial reuptake of dibromosulfophthalein transported into the medium. In addition, Proost et al. (27) argued that biliary excretion and sinusoidal efflux of a compound may occur at the same time because of the involvement of different intracellular compartments. Thus the study by Proost et al. supports our proposed model of bile acid percolation along the sinusoid by ABCC4 when at the same time bile acids are excreted into bile by ABCB11. The requirement of GSH for the bile acid transport by ABCC4, but not by ABCB11, may be part of a regulatory mechanism to control intracellular concentrations of bile acids. In addition, hepatic GSH is predominantly released across the basolateral membrane of hepatocytes into the blood circulation as the major source of plasma GSH, cysteine, and cystine. Thus ABCC4 can be considered as a new pathway for the efflux of GSH into the systemic circulation, as pointed out in our earlier publication (29).


Figure 4
View larger version (26K):
[in this window]
[in a new window]
  		Fig. 4. Scheme of bile acid transport processes of the hepatocyte. Bile acids in hepatocytes are transported into bile by the bile salt export pump ABCB11 (BSEP). After enterohepatic circulation through the intestine, bile acids are taken up into the hepatocyte by the Na+-taurocholate cotransporting polypeptide NTCP (SLC10A1) and/or the organic anion transporters OATP1B1 (OATP-C/OATP2) and OATP1B3 (OATP8). ABCC4, which is localized to the sinusoidal membrane of hepatocytes (29), mediates high-affinity cotransport of GSH and bile acids into sinusoidal blood and thus competes for bile acid transport into bile via ABCB11. From sinusoidal blood, bile acids can then reenter into more portally located hepatocytes by the uptake transporters described.

 
Under cholestatic conditions, the concentration of substances, which are normally excreted into bile, may increase intracellularly and cause hepatotoxicity. One compensatory mechanism that prevents hepatotoxicity is the efflux of substances into blood across the sinusoidal membrane, as demonstrated for leukotriene metabolites formed in the hepatocytes of Mrp2-deficient mutant rats (8, 15) and presumably for bile acids that are excreted in urine in cholestatic liver disease.

The mechanisms that preserve hepatocytes from bile acid accumulation have been investigated in cholestatic animal models (6, 31). Mice lacking the farnesoid X receptor/bile acid receptor have a reduced expression of Abcb11 but an increased expression of Abcc4 mRNA as well as increased plasma concentrations and urinary excretion of bile acids (31). A similar adaptive response with increased Abcc4 protein expression has been observed in obstructive cholestasis in rats (6). However, species differences must be taken into account. A recent study in patients with progressive familial intrahepatic cholestasis, who have elevated serum concentrations of bile acids, showed a marked upregulation of ABCC4 protein and ABCC4 mRNA in BSEP-deficient patients suffering from mutations in the ABCB11 gene, as well as in ABCB4 (MDR3)-deficient patients with mutations in the ABCB4 (MDR3) gene (12). On the other hand, protein expression of ABCC3, which has been proposed to mediate bile acid transport (36), was unaltered in liver samples from these cholestatic patients (12). Comparison of the affinities of both transporters, ABCC3 and ABCC4, for bile acids indicates that ABCC4 is the better sinusoidal bile acid export pump compared with ABCC3. Thus ABCC4 appears to be a major player in buffering the intracellular concentration of bile acids in the hepatocyte, especially when canalicular secretion of bile acids is impaired. The molecular mechanisms by which ABCC4 is upregulated in cholestatic liver diseases are presently unknown.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by the Cooperation Program in Cancer Research of the Deutsches Krebsforschungszentrum (DKFZ) and Israeli's Ministry of Science, by collaboration between DKFZ and Pfizer Research Laboratories (Groton, CT), and by the Fonds der Chemischen Industrie, Frankfurt, Germany. Work at the University of California is supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-64891.


   ACKNOWLEDGMENTS
 
We thank Anne T. Nies for many helpful discussions and critical reading of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Rius, Div. of Tumor Biochemistry, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany (e-mail: m.rius{at}dkfz.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Akita H, Suzuki H, Hirohashi T, Takikawa H, and Sugiyama Y. Transport activity of human MRP3 expressed in Sf9 cells: comparative studies with rat MRP3. Pharm Res 19: 34–41, 2002.[CrossRef][ISI][Medline]
  2. Ballatori N, Hammond CL, Cunningham JB, Krance SM, and Marchan R. Molecular mechanisms of reduced glutathione transport: role of the MRP/CFTR/ABCC and OATP/SLC21A families of membrane proteins. Toxicol Appl Pharmacol 204: 238–255, 2005.[CrossRef][ISI][Medline]
  3. Beuers U, Boyer JL, and Paumgartner G. Ursodeoxycholic acid in cholestasis: potential mechanisms of action and therapeutic applications. Hepatology 28: 1449–1453, 1998.[CrossRef][ISI][Medline]
  4. Byrne JA, Strautnieks SS, Mieli-Vergani G, Higgins CF, Linton KJ, and Thompson RJ. The human bile salt export pump: characterization of substrate specificity and identification of inhibitors. Gastroenterology 123: 1649–1658, 2002.[CrossRef][ISI][Medline]
  5. DeLeve LD and Kaplowitz N. Importance and regulation of hepatic glutathione. Semin Liver Dis 10: 251–266, 1990.[ISI][Medline]
  6. Denk GU, Soroka CJ, Takeyama Y, Chen W, Schuetz JD, and Boyer JL. Multidrug resistance-associated protein 4 is up-regulated in liver but down-regulated in kidney in obstructive cholestasis in the rat. J Hepatol 40: 585–591, 2004.[CrossRef][ISI][Medline]
  7. Dirr H, Reinemer P, and Huber R. X-ray crystal structures of cytosolic glutathione S-transferases. Implications for protein architecture, substrate recognition and catalytic function. Eur J Biochem 220: 645–661, 1994.[ISI][Medline]
  8. Guhlmann A, Krauss K, Oberdorfer F, Siegel T, Scheuber PH, Müller J, Csuk-Glänzer B, Ziegler S, Ostertag H, and Keppler D. Noninvasive assessment of hepatobiliary and renal elimination of cysteinyl leukotrienes by positron emission tomography. Hepatology 21: 1568–1575, 1995.[CrossRef][ISI][Medline]
  9. Hagenbuch B and Meier PJ. The superfamily of organic anion transporting polypeptides. Biochim Biophys Acta 1609: 1–18, 2003.[Medline]
  10. Jedlitschky G and Keppler D. Transport of leukotriene C4 and structurally related conjugates. Vitam Horm 64: 153–184, 2002.[ISI][Medline]
  11. Kaplowitz N, Aw TY, and Ookhtens M. The regulation of hepatic glutathione. Annu Rev Pharmacol Toxicol 25: 715–744, 1985.[CrossRef][ISI][Medline]
  12. Keitel V, Burdelski M, Warskulat U, Kuhlkamp T, Keppler D, Haussinger D, and Kubitz R. Expression and localization of hepatobiliary transport proteins in progressive familial intrahepatic cholestasis. Hepatology 41: 1160–1172, 2005.[CrossRef][ISI][Medline]
  13. Keppler D, Jedlitschky G, and Leier I. Transport function and substrate specificity of multidrug resistance protein. Methods Enzymol 292: 607–616, 1998.[ISI][Medline]
  14. Keppler D, König J, and Nies AT. Conjugate export pumps of the multidrug resistance protein (MRP) family in liver. In: The Liver: Biology and Pathobiology, edited by Arias IM, Boyer JL, Chisari FV, Fausto N, Schachter D and Shafritz DA. New York: Lippincott Williams & Wilkins, 2001, p. 373–382.
  15. Keppler D, Müller M, Klünemann C, Guhlmann A, Krauss K, Müller J, Berger U, Leier I, and Mayatepek E. Transport and in vivo elimination of cysteinyl leukotrienes. Adv Enzyme Regul 32: 107–116, 1992.[CrossRef][ISI][Medline]
  16. König J, Rost D, Cui Y, and Keppler D. Characterization of the human multidrug resistance protein isoform MRP3 localized to the basolateral hepatocyte membrane. Hepatology 29: 1156–1163, 1999.[CrossRef][ISI][Medline]
  17. Kruh GD and Belinsky MG. The MRP family of drug efflux pumps. Oncogene 22: 7537–7552, 2003.[CrossRef][ISI][Medline]
  18. Kullak-Ublick GA, Stieger B, and Meier PJ. Enterohepatic bile salt transporters in normal physiology and liver disease. Gastroenterology 126: 322–342, 2004.[CrossRef][ISI][Medline]
  19. Lee K, Klein-Szanto AJ, and Kruh GD. Analysis of the MRP4 drug resistance profile in transfected NIH3T3 cells. J Natl Cancer Inst 92: 1934–1940, 2000.[Abstract/Free Full Text]
  20. Loe DW, Almquist KC, Deeley RG, and Cole SP. Multidrug resistance protein (MRP)-mediated transport of leukotriene C4 and chemotherapeutic agents in membrane vesicles. Demonstration of glutathione-dependent vincristine transport. J Biol Chem 271: 9675–9682, 1996.[Abstract/Free Full Text]
  21. Loe DW, Deeley RG, and Cole SP. Characterization of vincristine transport by the Mr 190,000 multidrug resistance protein (MRP): evidence for cotransport with reduced glutathione. Cancer Res 58: 5130–5136, 1998.[Abstract/Free Full Text]
  22. Loe DW, Deeley RG, and Cole SP. Verapamil stimulates glutathione transport by the 190-kDa multidrug resistance protein 1 (MRP1). J Pharmacol Exp Ther 293: 530–538, 2000.[Abstract/Free Full Text]
  23. Meister A and Anderson ME. Glutathione. Annu Rev Biochem 52: 711–760, 1983.[CrossRef][ISI][Medline]
  24. Noe J, Stieger B, and Meier PJ. Functional expression of the canalicular bile salt export pump of human liver. Gastroenterology 123: 1659–1666, 2002.[CrossRef][ISI][Medline]
  25. Paulusma CC, van Geer MA, Evers R, Heijn M, Ottenhoff R, Borst P, and Oude Elferink RP. Canalicular multispecific organic anion transporter/multidrug resistance protein 2 mediates low-affinity transport of reduced glutathione. Biochem J 338: 393–401, 1999.[CrossRef][Medline]
  26. Paumgartner G and Beuers U. Mechanisms of action and therapeutic efficacy of ursodeoxycholic acid in cholestatic liver disease. Clin Liver Dis 8: 67–81, 2004.[CrossRef][Medline]
  27. Proost JH, Nijssen HMJ, Strating CB, Meijer DKF, and Groothuis GMM. Pharmacokinetic modeling of the sinusoidal efflux of anionic ligands from the isolated perfused rat liver: the influence of albumin. J Pharmacokinet Biopharm 21: 375–394, 1993.[CrossRef][ISI][Medline]
  28. Rinaldi R, Eliasson E, Swedmark S, and Morgenstern R. Reactive intermediates and the dynamics of glutathione transferases. Drug Metab Dispos 30: 1053–1058, 2002.[Abstract/Free Full Text]
  29. Rius M, Nies AT, Hummel-Eisenbeiss J, Jedlitschky G, and Keppler D. Cotransport of reduced glutathione with bile salts by MRP4 (ABCC4) localized to the basolateral hepatocyte membrane. Hepatology 38: 374–384, 2003.[ISI][Medline]
  30. Rius M, Thon WF, Keppler D, and Nies AT. Prostanoid transport by multidrug resistance protein 4 (MRP4/ABCC4) localized in tissues of human urogenital tract. J Urol 174: 2409–2414, 2005.[CrossRef][ISI][Medline]
  31. Schuetz EG, Strom S, Yasuda K, Lecureur V, Assem M, Brimer C, Lamba J, Kim RB, Ramachandran V, Komoroski BJ, Venkataramanan R, Cai H, Sinal CJ, Gonzalez FJ, and Schuetz JD. Disrupted bile acid homeostasis reveals an unexpected interaction among nuclear hormone receptors, transporters, and cytochrome P450. J Biol Chem 276: 39411–39418, 2001.[Abstract/Free Full Text]
  32. Sorscher S, Lillienau J, Meinkoth JL, Steinbach JH, Schteingart CD, Feramisco J, and Hofmann AF. Conjugated bile acid uptake by Xenopus laevis oocytes induced by microinjection with ileal poly A+ mRNA. Biochem Biophys Res Commun 186: 1455–1462, 1992.[CrossRef][ISI][Medline]
  33. Van Aubel RA, Smeets PH, Peters JG, Bindels RJ, and Russel FG. The MRP4/ABCC4 gene encodes a novel apical organic anion transporter in human kidney proximal tubules: putative efflux pump for urinary cAMP and cGMP. J Am Soc Nephrol 13: 595–603, 2002.[Abstract/Free Full Text]
  34. Wright EM and Loo DD. Coupling between Na+, sugar, and water transport across the intestine. Ann NY Acad Sci 915: 54–66, 2000.[ISI][Medline]
  35. Zelcer N, Reid G, Wielinga PR, Kuil AJ, van der Heijden Schuetz JD, and Borst P. Steroid- and bile acid-conjugates are substrates of human MRP4 (ABCC4). Biochem J 371: 361–367, 2003.[CrossRef][ISI][Medline]
  36. Zelcer N, Saeki T, Bot I, Kuil A, and Borst P. Transport of bile acids in multidrug-resistance-protein 3-overexpressing cells co-transfected with the ileal Na+-dependent bile-acid transporter. Biochem J 369: 23–30, 2003.[CrossRef][ISI][Medline]
  37. Zeng H, Liu G, Rea PA, and Kruh GD. Transport of amphipathic anions by human multidrug resistance protein 3. Cancer Res 60: 4779–4784, 2000.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Toxicol SciHome page
J. M. Maher, L. M. Aleksunes, M. Z. Dieter, Y. Tanaka, J. M. Peters, J. E. Manautou, and C. D. Klaassen
Nrf2- and PPAR{alpha}-Mediated Regulation of Hepatic Mrp Transporters after Exposure to Perfluorooctanoic Acid and Perfluorodecanoic Acid
Toxicol. Sci., December 1, 2008; 106(2): 319 - 328.
[Abstract] [Full Text] [PDF]

Home page
 Drug Metab. Dispos.Home page
L. M. Aleksunes, Y. Cui, and C. D. Klaassen
Prominent Expression of Xenobiotic Efflux Transporters in Mouse Extraembryonic Fetal Membranes Compared with Placenta
Drug Metab. Dispos., September 1, 2008; 36(9): 1960 - 1970.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol SciHome page
L. M. Aleksunes, S. N. Campion, M. J. Goedken, and J. E. Manautou
Acquired Resistance to Acetaminophen Hepatotoxicity is Associated with Induction of Multidrug Resistance-Associated Protein 4 (Mrp4) in Proliferating Hepatocytes
Toxicol. Sci., August 1, 2008; 104(2): 261 - 273.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
S. N. Campion, R. Johnson, L. M. Aleksunes, M. J. Goedken, N. van Rooijen, G. L. Scheffer, N. J. Cherrington, and J. E. Manautou
Hepatic Mrp4 induction following acetaminophen exposure is dependent on Kupffer cell function
Am J Physiol Gastrointest Liver Physiol, August 1, 2008; 295(2): G294 - G304.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
M. Rius, J. Hummel-Eisenbeiss, and D. Keppler
ATP-Dependent Transport of Leukotrienes B4 and C4 by the Multidrug Resistance Protein ABCC4 (MRP4)
J. Pharmacol. Exp. Ther., January 1, 2008; 324(1): 86 - 94.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
M. J. Zamek-Gliszczynski, K.-i. Nezasa, X. Tian, A. S. Bridges, K. Lee, M. G. Belinsky, G. D. Kruh, and K. L. R. Brouwer
Evaluation of the Role of Multidrug Resistance-Associated Protein (Mrp) 3 and Mrp4 in Hepatic Basolateral Excretion of Sulfate and Glucuronide Metabolites of Acetaminophen, 4-Methylumbelliferone, and Harmol in Abcc3-/- and Abcc4-/- Mice
J. Pharmacol. Exp. Ther., December 1, 2006; 319(3): 1485 - 1491.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/4/G640    most recent
00354.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (26)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rius, M.
Right arrow Articles by Keppler, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rius, M.
Right arrow Articles by Keppler, D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.