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Am J Physiol Gastrointest Liver Physiol 288: G327-G336, 2005. First published September 16, 2004; doi:10.1152/ajpgi.00227.2004
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LIVER AND BILIARY TRACT

Role of microtubules in estradiol-17{beta}-D-glucuronide-induced alteration of canalicular Mrp2 localization and activity

Aldo D. Mottino,1,2 Fernando A. Crocenzi,2 Enrique J. Sánchez Pozzi,2 Luis M. Veggi,2 Marcelo G. Roma,2 and Mary Vore1

1Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky; and 2Institute of Experimental Physiology, National University of Rosario, Rosario, Argentina

Submitted 18 May 2004 ; accepted in final form 8 September 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Estradiol-17{beta}-D-glucuronide (E2-17G) induces a marked but reversible inhibition of bile flow in the rat together with endocytic retrieval of multidrug resistance-associated protein 2 (Mrp2) from the canalicular membrane to intracellular structures. We analyzed the effect of pretreatment (100 min) with the microtubule inhibitor colchicine or lumicholchicine, its inactive isomer (1 µmol/kg iv), on changes in bile flow and localization and function of Mrp2 induced by E2-17G (15 µmol/kg iv). Bile flow and biliary excretion of bilirubin, an endogenous Mrp2 substrate, were measured throughout, whereas Mrp2 localization was examined at 20 and 120 min after E2-17G by confocal immunofluorescence microscopy and Western analysis. Colchicine pretreatment alone did not affect bile flow or Mrp2 localization and activity over the short time scale examined (3–4 h). Administration of E2-17G to colchicine-pretreated rats induced a marked decrease (85%) in bile flow and biliary excretion of bilirubin as well as internalization of Mrp2 at 20 min. These alterations were of a similar magnitude as in rats pretreated with lumicolchicine followed by E2-17G. Bile flow and Mrp2 localization and activity were restored to control levels within 120 min of E2-17G in animals pretreated with lumicolchicine. In contrast, in colchicine-pretreated rats followed by E2-17G, bile flow and Mrp2 activity remained significantly inhibited by 60%, and confocal and Western studies revealed sustained internalization of Mrp2 120 min after E2-17G. We conclude that recovery from E2-17G cholestasis, associated with exocytic insertion of Mrp2 in the canalicular membrane, but not its initial E2-17G-induced endocytosis, is a microtubule-dependent process.

bile secretion; endocytic compartment; colchicine; bilirubin

THE MULTIDRUG RESISTANCE-ASSOCIATED protein 2 (Mrp2; Abcc2) mediates the ATP-dependent transport of a wide range of amphiphilic anionic conjugates into bile (5, 35). Mrp2 plays an important role in elimination of potentially toxic endo- and xenobiotics, including bilirubin, hormones, drugs, and carcinogens, primarily as their glucuronide, glutathione (GSH), or sulfate conjugates. Mrp2 also mediates the active transport of oxidized GSH into bile and is believed to contribute to the active transport of reduced GSH at the canalicular level (22, 38). Because of the high concentrations of GSH in bile, and the role of such osmotically active solutes in the generation of bile flow, Mrp2 is considered critical to the generation of the bile acid-independent fraction of bile flow (1).

Experimental cholestasis in rats is associated with impaired Mrp2-mediated transport (4) and downregulation of Mrp2 expression (43). Changes in localization of Mrp2 from the canalicular membrane to a subapical membranous compartment of the hepatocyte is also thought to affect its function and has been linked to cholestasis. For example, in experimental obstructive cholestasis induced by chronic bile duct ligation, Mrp2 exhibits a substantial change from the normal clear pattern of staining outlining the canalicular membrane, toward a fuzzy and irregular staining pattern (36). Infusion of hyperosmolar buffer to the isolated perfused rat liver also induces a rapid retrieval of Mrp2 in pericanalicular vesicles in parallel with a decrease in bile flow (23). Treatment of rats with lipopolysaccharide (11), taurolithocholate (3), or phalloidin (41) also induces a rapid endocytic retrieval of Mrp2 from the canalicular membrane that correlates with decreased bile formation. More recently, we demonstrated that estradiol-17{beta}-D-glucuronide (E2-17G) induces endocytic retrieval of Mrp2 (33), thus accounting, at least in part, for decreased biliary secretion of GSH (34) and bile flow (29). In contrast, cAMP stimulates bile flow in the perfused rat liver (15) and sorting of Mrp2 from cytosol to the apical membrane in hepatocyte couplets (39, 40). Infusion of bile salts, such as taurocholate (19) and tauroursodeoxycholate (3), or perfusion with hypoosmotic buffer to induce hepatocyte swelling (23) also stimulate insertion of Mrp2 in the canalicular domain. The bile salt export pump (Bsep) is similarly regulated in a short-term fashion (9, 20). Taken together, the evidence indicates that transporter function and bile secretion are regulated, at least in part, by the dynamic endocytic retrieval and exocytic insertion of transporters between the canalicular membrane and an intracellular pool of vesicles.

Elucidation of the intrahepatic pathways of canalicular transporter trafficking and their regulation may help to understand the cause of cholestasis at a molecular level and provide clues for novel therapies. The mechanism mediating targeting of canalicular transporters from intracellular sites to the plasma membrane induced by bile salts, cAMP, or cell swelling are only partially understood and may involve common signaling mediators (9). Whether microtubule integrity is essential for normal or increased sorting of canalicular transporters to the apical membrane in the hepatocyte is not clear. For example, taurocholate-stimulated recruitment of both Mrp2 and Bsep to the canalicular membrane is attenuated by colchicine, a microtubule inhibitor, in vivo (20), whereas colchicine blocks only the cAMP-induced recruitment of Mrp2 (31). Early studies by Haussinger et al. (13) reported involvement of microtubules in the swelling-induced stimulation of transcellular taurocholate transport and biliary excretion in the perfused rat liver; however, it is not known whether microtubules participate directly in canalicular insertion of Bsep. Data in isolated rat hepatocyte couplets point to the existence of a predominant microtubule-independent, but microfilament-dependent, trafficking of canalicular transporters during recovery of cell polarity (40). The nature of the opposite process, i.e., the endocytic internalization of canalicular transporters, is unknown. Rahner et al. (37) recently showed that bulk-phase and membrane-associated endocytic markers exposed to the canalicular membrane from the luminal side are readily internalized and directed to a subapical compartment in a clathrin-dependent manner. Whether a similar pathway accounts for canalicular transporter endocytosis is unknown. The role of microtubules in this process is far less known.

The decrease in bile flow induced by E2-17G in vivo is acute but reversible, since bile flow is completely restored within 120–180 min of its administration to rats (29). Endocytic internalization of Mrp2 induced by E2-17G is also reversible and parallels the changes in its transport activity (33). Mrp2-mediated transport of E2-17G across the canalicular membrane is also essential for E2-17G-induced cholestasis, since cholestasis is absent in TR– rats deficient in Mrp2 (16). Interestingly, infusion of taxol, a substrate of P-glycoprotein but not of Mrp2, antagonizes the cholestatic effect of E2-17G in the perfused rat liver (25). It is not known, however, whether taxol's protective effect is related to its ability to interact with microtubules. Cholestasis induced by lithocholic acid in vivo is partially attenuated by pretreatment of the animals with colchicine (2). Because taurolithocholic acid induces endocytic internalization of Mrp2 (3) and Bsep (10) in parallel with cholestasis, it seemed possible that colchicine's protective effects against lithocholic acid-induced cholestasis could be because of inhibition of microtubule-dependent retrieval of both transporters. In the current study, we explored the possibility that microtubules mediate trafficking of Mrp2 between the canalicular membrane and intracellular domains during the acute phase of cholestasis (20 min after E2-17G administration) and/or during reinsertion of Mrp2 in the canalicular membrane during the phase of recovery from cholestasis (120 min after E2-17G administration). Our results demonstrate that inhibition of microtubules with colchicine did not influence the magnitude of cholestasis or retrieval of Mrp2 induced by E2-17G. In contrast, integrity of microtubules was essential for the recovery of bile flow and the reinsertion of Mrp2 in the canalicular membrane during the phase of recovery from cholestasis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals. Leupeptin, aprotinin, phenylmethylsulfonyl fluoride (PMSF), pepstatin A, E2-17G, colchicine, lumicolchicine, methylbutane (isopentane), BSA, p-nitrophenol, UDP-glucuronic acid (ammonium salt), paraformaldehyde, and DMSO were from Sigma Chemical (St. Louis, MO). All other chemicals were of analytical grade purity, and used as supplied.

Animals and experimental protocols. Female Sprague-Dawley rats (180–210 g; Harlan Industries, Indianapolis, IN) were used throughout. The rats had free access to food and water and were maintained on a 12:12-h automatically timed light and dark cycle. All procedures involving animals were conducted in accordance with National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of Kentucky.

The rats were anesthetized with urethane (1,000 mg/kg ip) and thus maintained throughout. Body temperature was measured with a rectal probe and maintained at 37°C with a heating pad connected to a temperature regulator (model 73A; Yellow Springs Instruments, Yellow Springs, OH). The femoral vein and the common bile duct were cannulated with polyethylene tubing (PE-50 and PE-10, respectively). Saline was administered intravenously throughout the experiment to replenish body fluids. Colchicine or lumicolchicine (1.25 mM in DMSO-saline, 1:4; 1 µmol/kg) was administered via the femoral vein 30 min after bile duct cannulation (Fig. 1A). E2-17G (4.25 mM in DMSO-10% BSA in saline, 1:25; 15 µmol/kg) or solvent was administered 100 min later via the femoral vein. An E2-17G stock solution was prepared in DMSO (100 mM), followed by dilution in an appropriate volume of 10% BSA in saline to reach the desired final concentration. This solvent did not induce hemolysis, as demonstrated by the lack of increase in bilirubin excretion in bile in rats treated with lumicolchicine and solvent (Lumi + solv; Fig. 2A). Saline-propylene glycol-ethanol (10:4:1), used previously as the E2-17G solvent, was shown to increase bilirubin production (34), probably the result of ethanol-induced hemolysis.



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  		Fig. 1. Diagram of experimental protocols (A) and time-course changes in bile flow in response to colchicine and estradiol-17{beta}-D-glucuronide (E2-17G) (B). A: after a postsurgery stabilization period of 20 min, bile was collected to evaluate basal bile flow and biliary excretion of bilirubin. Colchicine (Col) or lumicolchicine (Lumi) (1 µmol/kg) and E2-17G (15 µmol/kg) or its solvent was administered iv at the time indicated. Gray areas represent the periods of bile collection and determination of biliary bilirubin excretion. Liver samples were taken for confocal immunofluorescence analysis of multidrug resistance-associated protein 2 (Mrp2), bile salt export pump (Bsep), and dipeptidyl-peptidase IV (DPPIV) and for Western studies of Mrp2 in liver membranes. B: bile flow was determined gravimetrically. Data represent means ± SE of 6–7 animals/group. E2-17G or solvent (Solv) was injected at the time indicated by the arrow, in which bile flow from the different groups was normalized to 100%. aCol + solv and Lumi + solv significantly different from Col + E2-17G and Lumi + E2-17G (P < 0.05). bCol + solv and Lumi + solv significantly different from Col + E2-17G and Lumi + E2-17G; Col + E2-17G significantly different from Lumi + E2-17G (P < 0.05). cCol + solv, Lumi + solv, and Lumi + E2-17G significantly different from Col + E2-17G (P < 0.01).

 


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  		Fig. 2. Effect of colchicine and E2-17G on the time course (A) and cumulative (B) biliary excretion of bilirubin. Total bilirubin concentration was determined in bile samples collected in 10-min intervals. Data represent means ± SE of 3–4 rats/group. A: arrow indicates the time at which E2-17G or solvent was injected, when biliary excretion of bilirubin was normalized to 100%. aCol + solv and Lumi + solv significantly different from Col + E2-17G and Lumi + E2-17G (P < 0.05). bCol + solv and Lumi + solv significantly different from Col + E2-17G and Lumi + E2-17G; Col + E2-17G significantly different from Lumi + E2-17G (P < 0.05). cCol + solv, Lumi + solv, and Lumi + E2-17G significantly different from Col + E2-17G (P < 0.05). dCol + solv, Lumi + solv, and Lumi + E2-17G significantly different from Col + E2-17G; Lumi + E2-17G significantly different from Col + solv and Lumi + solv (P < 0.05). B: cumulative excretion of bilirubin 120 min after E2-17G or solvent. eSignificantly different from Col + solv, Lumi + solv, and Lumi + E2-17G (P < 0.05).

 
Bile was collected at 10-min intervals (Fig. 1A), and bile flow was determined gravimetrically, assuming a density of 1 g/ml. To determine Mrp2 localization at the peak of cholestasis and after recovery from cholestasis, livers were perfused for 30 s with saline 20 or 120 min after E2-17G or solvent, and the major lobe was removed. A portion was gently frozen in liquid nitrogen and preserved at –80°C until used for membrane preparation for Western analysis or frozen in precooled isopentane for confocal immunofluorescence microscopy studies. The biliary excretion of total (unconjugated + conjugated) bilirubin was used as a measure of Mrp2 transport activity, since monoglucuronosyl and bisglucuronosyl bilirubin conjugates are prototypical Mrp2 substrates (17) and traces of unconjugated bilirubin present in bile most likely result from {beta}-glucuronidase-mediated hydrolysis of conjugates after their secretion in bile (28).

In additional experiments, we evaluated whether colchicine and E2-17G, administered individually or in combination, affect the integrity of hepatocyte microtubular network. For this purpose, 20 min after administration of E2-17G or its solvent, livers were perfused through the portal vein with PBS for 30 s and immediately fixed with PBS containing 4% (vol/vol) paraformaldehyde, as described previously (32). Immunofluorescent detection of {alpha}-tubulin was used as a marker of microtubular integrity.

Preparation of liver membranes and Western analysis. Membrane fractions enriched in plasma membranes or intracellular fractions enriched in microsomal membranes were prepared by differential centrifugation, as described previously (27, 33). Briefly, portions of the liver were homogenized in 0.3 M sucrose containing 0.1 mM PMSF, 25 µg/ml leupeptin, 5 µg/ml aprotinin, and 5 µg/ml pepstatin A (50 mg liver/ml buffer), an aliquot was reserved for Western analysis and assessment of enzyme markers, and the remaining homogenate was used for membrane preparation. Protein concentration was measured by the method of Lowry et al. (26), using BSA as a standard. The purity of plasma and microsomal membranes was confirmed using enzymatic assays for hepatic subcellular markers. 5'-Nucleotidase (plasma membrane), acid phosphatase (lysosomes), and aspartate aminotransferase (mitochondria) were assessed using commercial kits (Wiener Laboratories, Rosario, Argentina). Microsomal p-nitrophenol VDP-glucuronosyl transferase activity was determined as described previously (7). Data obtained are summarized in Table 1. Enrichment and purity of membranes were comparable to those reported previously (6, 7) and were not affected by any treatment or time period analyzed. Immunoblotting was performed with homogenates, mixed plasma membranes, and intracellular membranes as described (33) using a monoclonal antibody to human Mrp2 (M2 III-6; Alexis Biochemicals, Carlsbad, CA). Subsequent densitometry was performed using Gel-Pro Analyzer (Media Cybernetics, Silver Spring, MD) software.


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  		Table 1. Assessment of plasma and microsomal membrane purity

 
Confocal microscopy studies. To evaluate localization of Mrp2, confocal analysis was performed for Mrp2 and for the tight junctional-associated protein zonula occludens (ZO)-1, which was used to visualize the border of the bile canalicular structures, as described previously (23, 41). The tissue samples were cut in 5-µm sections with a Zeiss Microm HM5000 microtome cryostat and air-dried for 2 h before fixation with methanol (–20°C, 10 min). The staining and confocal microscopy were performed as described previously (33). Immunofluorescent intensity analysis of confocal images was performed as described previously (23), using the software Scion Image beta 4.02 for Windows (Scion). For each tissue section, four different images were captured, and data from four to six different canaliculi were collected per image and used for statistical analysis. Green and red immunofluorescence intensities were each measured over a line 8 µm in length and perpendicular to the canaliculus and normalized to the sum of all intensity under the respective curve. This makes the area under every curve equal and independent of the total amount of protein. The distribution of Mrp2 fluorescence (red channel), expressed as a percentage of the total, was then calculated for each canaliculi and compared statistically, using the Mann-Whitney test; any difference between groups thus reflects changes in localization along the 8-µm line. For the green channel (ZO-1 fluorescence), the distance between the two maximal intensities was determined as a good measure of the width of the respective canaliculus. The canalicular diameters, based on the distance of peak intensities under different experimental conditions, were compared by the two-sided Student's t-test. Analysis of confocal microscopy data was performed in a blinded manner. Data were replicated in at least three independent liver preparations. Thus a total of at least 48 canaliculi were analyzed per group.

Because of our findings on acute endocytic retrieval of Bsep after E2-17G administration (8), we further analyzed the effect of colchicine on E2-17G-induced internalization of Bsep relative to Mrp2. Double labeling was performed by incubating liver slices with the Mrp2 monoclonal antibody (1:100) and a rabbit polyclonal antibody to mouse Bsep (1:100; Kamiya Biomedical, Seattle, WA) overnight, followed by incubation with Cy2-conjugated donkey anti-rabbit and Cy3-conjugated donkey anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA), respectively, as described (8, 33). The effect of E2-17G and/or colchicine on localization of canalicular dipeptidyl-peptidase IV (DPPIV), relative to Mrp2, was also evaluated. The technical procedure was as described (33), except that liver slices were fixed with acetone (–20°C, 10 min) instead of methanol. Double labeling was performed by incubating the slices with a rabbit polyclonal antibody developed against rat Mrp2 (1:100, a generous gift from Dr. Peter Meier, Zurich) and a monoclonal antibody directed against rat DPPIV (1:100, a generous gift from Dr. Werner Reutter, Berlin) overnight, followed by incubation with Cy2-conjugated donkey anti-rabbit and Cy3-conjugated donkey anti-mouse secondary antibodies for 2 h.

To evaluate the integrity of the microtubular network, 20-µm sections of liver were fixed with paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and incubated overnight with monoclonal anti-{alpha}-tubulin (1:100; Sigma-Aldrich, St. Louis, MO), followed by incubation with Cy3-conjugated donkey anti-mouse secondary antibody for 2 h. Irrespective of whether E2-17G or its solvent was administered to the animals, treatment with colchicine induced a shift from reticulated to amorphous staining, with {alpha}-tubulin preferentially distributed in the periphery of the cells (images not shown). Monte et al. (32) noted a similar shift from reticulated to amorphous detection of {alpha}-tubulin in response to colchicine. These data confirm that colchicine disrupted the normal microtubular arrangement and that this effect was independent of E2-17G administration.

To ensure comparable staining and image capture performance for evaluation of Mrp2, Bsep, and DPPIV localization and microtubular network integrity among the different groups, liver slices were prepared the same day, mounted on the same glass slide, and subjected to the staining procedure and confocal microscopy analysis simultaneously.

We have previously demonstrated that the integrity of actin is preserved in response to treatment with E2-17G (8). In the current study, we evaluated the integrity of the structural protein {beta}-catenin and the tight junction protein occludin 20 min after administration of E2-17G. Immunofluorescence studies were performed as described above by using monoclonal anti-{beta}-catenin (1:100; Sigma-Aldrich) and anti-occludin (1:100; Zymed Laboratories, San Francisco, CA) antibodies.

Analytical methods. Total bilirubin (conjugated + unconjugated) was determined in bile immediately after bile samples were collected using a commercial kit (catalog no. 550-A; Sigma Chemical). Transport activity of Mrp2 was estimated as the biliary excretion rate of total bilirubin, calculated as the product of bile flow and the biliary concentration.

Statistical analysis. Data are presented as means ± SE. Statistical analysis was performed using one-way ANOVA, followed by the Bonferroni test, unless otherwise stated. Values of P < 0.05 were considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Influence of colchicine on E2-17G-induced cholestasis. Bile flow and biliary bilirubin excretion rates from the different experimental groups were normalized to 100% at the time of E2-17G or solvent administration to facilitate comparison among the groups. As shown in Fig. 1B, colchicine alone did not affect bile flow relative to lumicolchicine, its inactive isomer, throughout the experiment. E2-17G rapidly decreased bile flow to the same extent, 85–90%, in rats pretreated with either colchicine or lumicolchicine; this decrease was essentially the same as that shown after treatment with E2-17G alone (33). In contrast, the rate of recovery of bile flow clearly differed between rats pretreated with lumicolchicine (Lumi + E2-17G) and colchicine (Col + E2-17G). Although bile flow from Lumi + E2-17G animals had returned to control values within 120 min, bile flow from Col + E2-17G rats remained decreased by 60% at this time. The data indicate that colchicine neither prevented nor aggravated the magnitude of E2-17G cholestasis but rather interfered significantly with the spontaneous recovery of bile flow.

Influence of colchicine on E2-17G-induced alteration of Mrp2 activity. We analyzed the biliary excretion of total bilirubin throughout the experiment as a measure of Mrp2 transport activity (Fig. 2A). The biliary bilirubin excretion rate did not differ significantly between rats pretreated with colchicine and lumicolchicine. Although bilirubin excretion was slightly increased in solvent-treated rats treated with colchicine vs. lumicolchicine, these differences did not reach statistical significance at any time point. Thus neither colchicine nor its inactive isomer alone affected Mrp2 activity. In contrast, administration of E2-17G in the presence of either of these agents substantially changed the normal pattern of bilirubin excretion. In rats pretreated with lumicolchicine, E2-17G produced a significant decrease in bilirubin excretion during the acute phase of cholestasis, in agreement with decreased transport of the model exogenous Mrp2 substrate, dinitrophenyl-S-glutathione (33). This was followed by a rapid recovery and an "overshoot" in bilirubin excretion relative to that seen in Col + solv and Lumi + solv groups. This latter phenomenon likely resulted from excretion of bilirubin retained in the hepatocyte during the initial cholestasis. In support of this, the cumulative biliary excretion of bilirubin throughout the 120-min period was similar for lumicolchicine, colchicine, and Lumi + E2-17G groups (Fig. 2B). When E2-17G was administered to colchicine-pretreated rats, biliary bilirubin excretion was severely inhibited and had recovered only minimally by the end of the experiment. Changes in Mrp2 transport activity in this experimental group agreed well with changes in bile flow (Fig. 1B), suggesting a severe impairment in Mrp2 activity in addition to decreased bile flow. Cumulative bilirubin excretion in rats treated with Col + E2-17G (Fig. 2B) represented <30% of the other groups, indicating the inability of the livers to secrete the Mrp2 substrate, even under conditions of intrahepatic bilirubin accumulation.

Influence of colchicine on Mrp2 localization after E2-17G. Endocytic retrieval of Mrp2 from the canalicular membrane in intracellular compartments contributes, at least in part, to decreased bile flow and Mrp2 transport activity induced by different cholestatic agents (9, 14). Because microtubules have been implicated in the trafficking of transporters, we examined the effect of pretreatment with colchicine vs. lumicolchicine on the localization of Mrp2 in the canalicular membrane using two different approaches, confocal immunofluorescence microscopy and Western analysis. Figure 3 shows images of Mrp2 (red) and ZO-1 (green) immunofluorescent detection 20 and 120 min after E2-17G or solvent administration, during the time of maximal cholestasis and when bile flow was restored, respectively. Neither colchicine nor lumicolchicine alone affected the normal localization of Mrp2 in the canalicular membrane 20 min after treatment with solvent. In contrast, treatment with E2-17G significantly altered the normal pattern of staining, irrespective of pretreatment with colchicine or lumicolchicine. Within 20 min of E2-17G administration, Mrp2 was redistributed in intracellular structures, consistent with its endocytic retrieval from the canalicular membrane. This phenomenon coexisted with preserved localization of Mrp2 in some regions of the canalicular domain, indicating that Mrp2 internalization was only partial and not uniform. In animals pretreated with lumicolchicine, Mrp2 localization in the canalicular membrane was substantially restored to that in controls by 120 min after E2-17G administration, clearly indicating reversibility of Mrp2 internalization (Fig. 3, bottom left). In contrast, only partial restoration of Mrp2 localization in the canalicular membrane was observed in rats pretreated with colchicine. In this group, a significant red staining still remained associated with intracellular structures (Fig. 3, bottom right). As further controls for E2-17G-induced retrieval of Mrp2, we examined the localization of occludin and {beta}-catenin after E2-17G and found no detectable differences in their localization (data not shown).



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  		Fig. 3. Immunofluorescence assessment of Mrp2 localization in response to colchicine and E2-17G. Confocal immunofluorescence of Mrp2 (red) and zonula occludens (ZO)-1, a tight junction marker (green), and the merged images (yellow) during the acute phase of cholestasis (20 min) is shown for all experimental groups; similar data are shown for the time of recovery (120 min) for E2-17G-treated groups. White arrows indicate internalization of Mrp2, out of the limits of the canalicular domain, as indicated by double lines of ZO-1 staining. White arrowheads indicate Mrp2 retained inside the canalicular limits. Images are representative of at least 3 independent experiments/group. White bar: 10 µm.

 
Figure 4A shows that both groups receiving E2-17G exhibited a decrease in the peak height of the Mrp2 fluorescence profile together with an increased density of red color in the periphery (~1.0–3.0 µm from the center of the canaliculus). Comparison of the variance in the distribution of Mrp2 in the different groups confirmed a statistically significant (P < 0.005) alteration of the red fluorescence profile 20 min after E2-17G treatment in Lumi + E2-17G and Col + E2-17G groups vs. Lumi + solv and Col + solv groups. The sustained internalization of Mrp2 at 120 min in livers from rats pretreated with colchicine was also confirmed by comparison of the Mrp2 distribution and indicated a statistically significant difference (P < 0.005) between Lumi + E2-17G and Col + E2-17G groups. This difference is apparent in Fig. 4B, since a higher density of red color is found in the periphery (~0.5–2 µm from the canaliculus center) with a corresponding decrease in peak height. The effect produced by colchicine at 120 min was clearly differentiated from that produced at 20 min, since Lumi + E2-17G and Col + E2-17G curves were almost identical at this early time (Fig. 4A). In addition, the apparent visual difference in redistribution of Mrp2 between Fig. 4, A and B, may result from time differences in localization assessment (20 vs. 120 min after E2-17G), since cytosolic Mrp2 is expected to be closer to the center of the canaliculus at 120 min because of the partial reversibility of endocytosis in the Col + E2-17G group. A similar analysis for ZO-1 distribution revealed a double peak that was not affected by any of the treatments (Fig. 4, A and B, insets).



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  		Fig. 4. Immunofluorescent intensity analysis of fluorescence profiles of Mrp2 in response to colchicine and E2-17G. Fluorescence distribution of the Mrp2 fluorescence peak was recorded along a line (from –4 to +4 µm) perpendicular to the canaliculi. Data represent means ± SE. At least 3 different sections/experimental group were analyzed. For each section, 4 different images were captured, and data from 4 to 6 different canaliculi were collected/image and used for statistical comparison. Statistical analysis of the Mrp2 fluorescence profile 20 min after administration of E2-17G to either colchicine- or lumicolchicine-pretreated rats indicated a significant change (P < 0.005) in comparison with groups receiving solvent (A). Similar analysis shown in B indicated a significantly different (P < 0.005) profile of Mrp2 fluorescence between Col + E2-17G and Lumi + E2-17G groups 120 min after E2-17G administration. The pattern of ZO-1 distribution was not affected by any of the treatments (see insets).

 
Western analysis further confirmed Mrp2 internalization at 20 min, since intracellular membranes from Col + E2-17G and Lumi + E2-17G livers showed a significant increase in Mrp2 content of ~67% over their respective controls (Fig. 5). The Mrp2 content in total liver homogenate and the plasma membrane fraction did not change, as reported for both Mrp2 and Bsep after E2-17G treatment (8, 33). The sustained internalization of Mrp2 at 120 min was further confirmed by comparison of the Mrp2 content in intracellular membranes between Col + E2-17G and Lumi + E2-17G groups, which showed that more Mrp2 (~70%) was found in intracellular membranes in the Col + E2-17G vs. the Lumi + E2-17G groups (Fig. 5). The lack of change in the content of Mrp2 in the plasma membrane fraction after E2-17G likely results from the inability to separate submembranous structures derived from canalicular membrane vesiculation from the canalicular membrane itself by differential centrifugation. Submembranous, early endosomes have been shown to have a similar density as the canalicular membrane (44). In contrast, because expression of Mrp2 in microsomes is normally only about one-tenth that in the canalicular membrane (33), any increase in response to internalization is more readily detected.



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  		Fig. 5. Western analysis of Mrp2 in liver membranes in response to colchicine and E2-17G. Representative analysis of 20, 2, and 20 µg protein from total homogenates, mixed plasma membranes, and intracellular membranes, respectively. Additional experiments in 2 animals/group (data not shown) were included for final statistical analysis of densitometry (n = 4). After E2-17G or solvent (20 min), densitometric analysis (means ± SE) revealed a significant increase (P < 0.05) in Mrp2 content in intracellular membranes in Lumi + E2-17G and Col + E2-17G groups (241 ± 19 and 259 ± 17 arbitrary units, respectively) with respect to Lumi + solv and Col + solv groups (153 ± 8 and 148 ± 16 arbitrary units, respectively), whereas Mrp2 protein was not affected by treatments in homogenates or plasma membranes. At 120 min after E2-17G, the Mrp2 content in intracellular membranes in Col + E2-17G (210 ± 5 arbitrary units) with respect to Lumi + E2-17G (125 ± 6 arbitrary units) was significantly increased (P < 0.05, Student's t-test). No changes were observed in protein level in homogenates or plasma membrane between these same groups (data not shown).

 
Taken together, the data indicate that colchicine interfered with the spontaneous recovery of Mrp2 localization in the canalicular membrane through a mechanism mediated by disruption of the microtubule network and provide a likely explanation for the very slow recovery in the biliary excretion of bilirubin after pretreatment with colchicine (Fig. 2A).

Effect of colchicine and/or E2-17G on localization of Bsep and DPPIV relative to Mrp2. Figure 6, top, shows that Bsep (green) and Mrp2 (red) were colocalized to the canaliculus in the Lumi + solv group, as indicated by the predominance of yellow color. Both Bsep and Mrp2 underwent a significant internalization in the Lumi + E2-17G group, 20 min after E2-17G. Mrp2 (red fluorescence) is clearly seen in the cytoplasm next to the canaliculus in these animals. Bsep internalization also occurred, although to a lesser extent than Mrp2, since green intracellular vesicles were less frequently observed (Fig. 6, top). Red and green intracellular vesicles coexisted with yellow intracellular vesicles, indicating that Bsep and Mrp2 do not necessarily colocalize after endocytic internalization. Colchicine pretreatment did not change the pattern of Bsep and Mrp2 staining in liver from animals receiving either E2-17G or solvent (images not shown), as demonstrated for Mrp2 in Fig. 3. Figure 6, middle, shows that Bsep and Mrp2 largely returned to the canalicular membrane 120 min after E2-17G in animals pretreated with lumicolchicine. In contrast, colchicine administration interfered with the relocalization of Bsep, as well as Mrp2, back into the canalicular domain.



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  		Fig. 6. Immunofluorescence assessment of colocalization of Mrp2 with Bsep or DPPIV in response to colchicine and E2-17G. Confocal microscopy visualization of Mrp2 (red) and Bsep (green), and the merged images (yellow), 20 or 120 min after administration of E2-17G (top and middle) and of Mrp2 (green) and DPPIV (red), and the merged images, 20 min after administration of E2-17G (bottom). White arrows in top and middle indicate intracellular Bsep, whereas the white arrowhead indicates intracellular vesicles containing both Bsep and Mrp2. White arrows in bottom indicate intracellular DPPIV, whereas white arrowheads indicate intracellular Mrp2. Images are representative of at least 3 independent experiments/group. White bar: 10 µm.

 
Additional confocal studies evaluated the possibility that E2-17G-induced endocytosis also affected other canalicular membrane proteins, such as DPPIV. Simultaneous immunodetection of DPPIV (red) and Mrp2 (green) demonstrates a normal pattern of staining (prevalence of yellow color in the canaliculus, Fig. 6, bottom) in Lumi + solv animals. This pattern was significantly disrupted in the Lumi + E2-17G group, as intracellular red and green fluorescent vesicles are clearly visible. Colchicine pretreatment affected neither the normal pattern of staining in solvent-treated rats nor modified the disruption in this pattern induced by E2-17G (images not shown). DPPIV normalized its canalicular localization 120 min after administration of E2-17G in the Lumi + E2-17G group, whereas, in the Col + E2-17G group at 120 min, significant intracellular retention was still observed (images not shown), as already described for Mrp2 and Bsep.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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Once synthesized in the rough endoplasmic reticulum, ATP-dependent canalicular transporters traffic via the Golgi complex to the apical membrane (20). Recycling of transporters from a preexisting pericanalicular "storage" compartment is thought to serve as a reservoir, providing transporters to the canalicular domain according to physiological demands, followed by their endocytic internalization, once the demand is satisfied (20). Excessive internalization of transporters, however, could lead to cholestasis. In the E2-17G model of cholestasis, endocytic retrieval of Mrp2 is spontaneously reversible, most likely the result of rapid reinsertion of Mrp2 back to the canalicular membrane (33). Thus E2-17G represents a useful model of cholestasis to explore mechanisms mediating the bidirectional trafficking of Mrp2. Microtubule-dependent vesicle transport is an integral component of many of the membrane-trafficking events involved in endocytosis, secretion, transcytosis, and membrane organization and maintenance (12). Because microtubules are implicated in targeting of canalicular transporters from intracellular domains to the canalicular membrane (19, 20), we specifically focused on the potential role of microtubules in E2-17G-induced endocytic retrieval and spontaneous exocytic insertion of Mrp2 in the canalicular membrane.

In the present studies, pretreatment with colchicine vs. lumicolchicine had no significant effect on the initial E2-17G-induced decrease in bile flow or biliary excretion of bilirubin conjugates. Although the extent of retrieval of Mrp2 from the canalicular membrane is difficult to quantitate, there were again no clear differences in this measure between lumicolchicine- and colchicine-pretreated rats after E2-17G. In contrast, the recovery from cholestasis was markedly delayed in rats pretreated with colchicine vs. lumicolchicine. The recovery of bile flow, bilirubin excretion, and reinsertion of Mrp2 in the canalicular membrane were all still markedly inhibited at 120 min after colchicine relative to lumicolchicine pretreatment. These data indicate that retrieval of Mrp2 is a microtubule-independent phenomenon, whereas the restoration of normal localization of Mrp2 is critically dependent on microtubule integrity. As demonstrated by confocal immunofluorescence analysis, the effects of E2-17G and their spontaneous recovery, as well as the influence of microtubule integrity on these processes, were not restricted to Mrp2 but also affected Bsep and DPPIV. Whether E2-17G-induced endocytic internalization is a general phenomenon affecting all membrane constituents in a particular region of the canalicular membrane is not known.

Interestingly, we did not find any substantial change in either bile flow or in the localization or activity of Mrp2 after pretreatment with colchicine and solvent. These data imply that the dynamics of Mrp2 turnover or recycling from the storage compartment is not sufficiently rapid or extensive for the arrest of vesicle-containing microtubule-mediated Mrp2 trafficking to be reflected in a detectable change in Mrp2 localization over the time scale of these experiments (220 min). Alternatively, pathways independent of microtubules may account for the targeting of Mrp2 to its canalicular normal localization. For example, the resupply of ABC transporters to the canalicular membrane in the hepatocyte couplet model after loss of normal transporter localization during isolation is fully independent of microtubules but dependent on actin cytoskeleton integrity (40). Although it is accepted that the normal pattern of Mrp2 distribution results from a dynamic balance between its endocytic internalization and exocytic targeting from/to the canalicular membrane, the present data indicate that acute internalization of Mrp2, occurring in response to E2-17G, is unlikely to be a consequence of interruption of normal trafficking of the transporter from intracellular reservoir sites to the canalicular membrane, but rather, the result of the rapid endocytic retrieval of Mrp2 from the membrane.

The mechanism by which E2-17G induces retrieval of Mrp2, Bsep, and DPPIV is not known. Disruption of actin filament organization or function could also alter the localization of canalicular export pumps. In line with this, Rost et al. (41) demonstrated that treatment of rats with phalloidin severely disrupted the normal localization of Mrp2, P-glycoprotein, and DPPIV. However, on the basis of confocal immunofluorescence microscopy, we found that the structure of actin in rat liver was preserved after E2-17G treatment (8). It is nevertheless possible that more subtle changes in microfilament structure or function, not detectable by confocal microscopy, occur in response to E2-17G. Because actin filaments directly or indirectly interact with structural and regulatory proteins in the plasma membrane, it is also possible that E2-17G-induced disruption of these interactions leads to transporter retrieval. Recently, Kikuchi et al. (18) found that mice lacking radixin, a protein that cross links actin filaments and plasma membrane proteins, develop conjugated hyperbilirubinemia associated with loss of Mrp2 from the canalicular membrane, suggesting that radixin is required for its normal canalicular localization. More recently, Kojima et al. (21) showed that the colocalization of radixin and Mrp2 observed in normal human liver was disrupted in patients with stage III primary biliary cirrhosis and that radixin staining almost disappeared in canalicular areas with an irregular Mrp2 staining. We therefore examined whether E2-17G-induced Mrp2 retrieval from the canalicular membrane involved disruption of a putative radixin-Mrp2 interaction and found some degree of dissociation between radixin and Mrp2, since both proteins partially relocalized to different intracellular structures after E2-17G (unpublished observations). These results agree well with a recent preliminary report that susceptibility to estrogen-induced cholestasis in female vs. male rats is associated with loss of radixin from the canalicular membrane (42). However, although disruption of radixin-Mrp2 could represent a potential mechanism triggering endocytic internalization of Mrp2, we cannot exclude the possibility that it is the result of such internalization. Kubitz et al. (24) have suggested protein kinase C (PKC) as a potential mediator regulating the normal "canalicular" localization of Mrp2 in Hep G2 cells; treatment of Hep G2 cells with the PKC activator phorbol 12-myristate,13-acetate rapidly decreased both the apical localization and transport activity of Mrp2. Interestingly, this relocalization of Mrp2 in Hep G2 cells was not inhibited by colchicine. Whatever mechanism is involved, the present data indicate that microtubules are not required for trafficking of Mrp2 from the canalicular membrane to the putative subapical compartment in the current model of E2-17G-induced cholestasis. These data also suggest that taxol's ability to protect against E2-17G cholestasis (25) is independent of its action to stabilize microtubules.

The reversibility of E2-17G-induced cholestasis is apparently the result of spontaneous reinsertion of canalicular transporters back in the apical membrane (33). In the current study, we further demonstrate that this phenomenon is largely dependent on microtubule integrity. This clearly indicates that both Mrp2 and Bsep follow distinct routes of trafficking during retrieval and reinsertion. Because cAMP is a well-known stimulator of microtubule-dependent sorting of canalicular transporters in the apical domain (20), it is now evident why pretreatment with cAMP is more effective in accelerating recovery than preventing E2-17G cholestasis (33). Results from analysis of localization of Mrp2 assessed 120 min after E2-17G were consistent with data on transport activity, indicating that pretreatment with colchicine markedly decreased the ability to restore transport of Mrp2 substrates. The delayed recovery of bile flow from colchicine-pretreated animals is likely the result of delayed reinsertion of both Mrp2 and Bsep (8, 34). We currently demonstrated that endocytic internalization of Bsep is reversible and that its reinsertion is also dependent on microtubule integrity. Thus it is possible that inhibition of recovery of bile acid-dependent bile flow by colchicine also contributed to the substantial delay observed in restoration of total bile flow. Finally, the current data suggest that therapeutic strategies based on stimulation of microtubule-dependent vesicular transport could be effective for treatment of cholestasis induced by drugs, such as monohydroxylated bile salts (3, 10) and antidepressants (30), which promote retrieval of canalicular transporters.

In summary, the current data demonstrated that the marked endocytic internalization of Mrp2 from the canalicular membrane occurring early after E2-17G administration is a microtubule-independent phenomenon. In contrast, integrity of microtubules is essential for the exocytic insertion of Mrp2 in the canalicular membrane, the recovery of bile flow, and the biliary excretion of Mrp2 substrates.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of General Medical Sciences Grant GM-55343 to M. Vore and by grants from Agencia Nacional de Promoción Científica y Tecnológica, Consejo Nacional de Investigaciones Científicas y Técnicas, and Fundación Antorchas, Argentina, to A. D. Mottino.


   ACKNOWLEDGMENTS
 
We express our gratitude to Drs. Mary E. Jennes, Mary Gail Engle, and Bruce Maley for kind help in performing confocal microscope studies. We also express our gratitude to Drs. Fabiana García and Flavia Carreras for invaluable technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Vore, Graduate Center for Toxicology, 306 Health Sciences Research Bldg., Univ. of Kentucky, Lexington, KY 40536-0305 (E-mail: maryv{at}uky.edu)

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.


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 DISCUSSION
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 REFERENCES
 

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