Estradiol-17{beta}-D-glucuronide induces endocytic internalization of Bsep in rats

Fernando A. Crocenzi,1 Aldo D. Mottino,1 Jingsong Cao,2 Luis M. Veggi,1 Enrique J. Sánchez Pozzi,1 Mary Vore,2 Roger Coleman,{dagger},3 and Marcelo G. Roma1

1Instituto de Fisiología Experimental, Universidad Nacional de Rosario, S2002LRL, Rosario, Argentina; 2Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky 40536-0305; and 3School of Biosciences, The University of Birmingham, Birmingham B15 2TT, United Kingdom

Submitted 2 December 2002 ; accepted in final form 11 April 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Endocytic internalization of the multidrug resistance-associated protein 2 (Mrp2) was previously suggested to be involved in estradiol-17{beta}-D-glucuronide (E217G)-induced cholestasis. Here we evaluated in the rat whether a similar phenomenon occurs with the bile salt export pump (Bsep) and the ability of DBcAMP to prevent it. E217G (15 µmol/kg iv) impaired bile salt (BS) output and induced Bsep internalization, as assessed by confocal microscopy and Western blotting. Neither cholestasis nor Bsep internalization occurred in TR- rats lacking Mrp2. DBcAMP (20 µmol/kg iv) partially prevented the decrease in bile flow and BS output and substantially prevented E217G-induced Bsep internalization. In hepatocyte couplets, E217G (50 µM) diminished canalicular accumulation of a fluorescent BS and decreased Bsep-associated fluorescence in the canalicular membrane; DBcAMP (10 µM) fully prevented both effects. In conclusion, our results suggest that changes in Bsep localization are involved in E217G-induced impairment of bile flow and BS transport and that DBcAMP prevents this effect by stimulating insertion of canalicular transporter-containing vesicles. Mrp2 is required for E217G to induce its harmful effect.

bile salt; cholestasis; dibutyryl-adenosine 3',5'-cyclic monophosphate; F-actin; TR- rats


BILE FORMATION IS AN OSMOTIC process driven by active secretion in the bile canaliculus of solutes across the hepatocyte apical membrane, followed by passive inflow of water (24). Bile salts (BS) are quantitatively the major constituents of bile and the main determinants of its volume. Active, concentrative transport of tauroand glycoconjugated BS through the canalicular membrane is principally mediated by the bile salt export pump (Bsep, Abcb11, originally called sister of P-glycoprotein), a member of the ATP-binding cassette family of membrane transporters (12). However, sulfated and glucuronidated BS are secreted by another member of this family, the multidrug resistance-associated protein 2 (Mrp2; Abcc2); Mrp2 also mediates the secretion of glutathione (32) and a number of glutathione, glucuronide, and sulfate conjugates into bile, thus playing a role in the generation of the bile acid-independent fraction of bile flow (BF; see Ref. 18).

Estradiol-17{beta}-D-glucuronide (E217G) is an endogenous estrogen metabolite that has been shown to induce an acute and completely reversible cholestasis in the rat. It impairs, in a dose-dependent manner, both the BS-dependent (26) and the BS-independent fractions of the BF (27). In a recent work, we have demonstrated that E217G, when administered in vivo to rats, induces a rapid endocytic internalization of Mrp2, which is associated with an impairment in the transport of the Mrp2 substrate, dinitrophenyl-S-glutathione (30). Pretreatment with DBcAMP, which stimulates the trafficking of intracellular vesicles containing membrane transporters in the canalicular pole (15, 34, 35), partially prevented both E217G-induced cholestasis and the alteration in localization and function of Mrp2 (30). Thus loss of functional Mrp2 from the canalicular membrane could contribute, at least in part, to E217G-induced inhibition of bile acid-independent BF.

The mechanisms by which E217G impairs BS secretion are poorly understood. It has been shown that Bsep traffics together with Mrp2 in the same microtubule-associated vesicles in rat liver (39). Mrp2 also undergoes endocytic internalization in cholestatic conditions, such as hyperosmotic perfusion (19), lipopolysaccharide treatment (9), taurolithocholate-induced cholestasis (3), and oxidative stress (38). Interestingly, Bsep undergoes internalization under the same conditions (6, 7, 37, 44), thus suggesting a close association between both canalicular transporters. Moreover, it was shown that integrity of Mrp2-mediated E217G transport across the canalicular membrane is essential for the inhibition of Bsep (14), which suggested an interference with Bsep-mediated transport of BS induced by an interaction between Bsep- and E217G-bound Mrp2.

The aim of this work was to investigate whether E217G induces endocytic internalization of Bsep, as occurs with Mrp2 (30), and whether this phenomenon is associated with E217G-induced impairment of BS transport. Because DBcAMP was also found to increase the activity of bile acid transporters in isolated hepatocyte couplets (4), and the ATP-dependent transport of taurocholate, a Bsep substrate, in canalicular membrane vesicles (10), we also evaluated the capability of this transport modulator to prevent putative alterations in Bsep localization and function induced by E217G treatment. Finally, because integrity of Mrp2 activity is required for E217G-induced cholestasis (14), we ascertained whether changes in Bsep localization also occur in TR- rats, which lack Mrp2, during E217G treatment.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Chemicals

Leupeptin, aprotinin, PMSF, pepstatin A, methylbutane (isopentane), E217G, DBcAMP, 3{alpha}-hydroxysteroid dehydrogenase, Leibovitz-15 tissue culture medium, phalloidin-FITC, and DMSO were obtained from Sigma Chemical (St. Louis, MO). Cholyl-lysyl-fluorescein (CLF) was kindly provided by Dr. Charles O. Mills (Birmingham, UK). Collagenase type A from Clostridium histolyticum was from GIBCO (Paisley, UK). All other chemicals were of the highest analytic grade available from commercial sources.

Animals and Experimental Protocols

In in vivo experiments, female, Sprague-Dawley rats (180–210 g; Harlan Industries, Indianapolis, IN) were used. The rats had free access to food and water and were maintained on a 12:12-h automatically timed light-dark cycle. Adult female mutant TR- Wistar rats bred in the animal facility of the University of Kentucky, hereditarily deficient in Mrp2 and weighing 180–230 g, were also used for Bsep localization studies. 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 was 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. After 20 min of stabilization, bile was collected for 5 min (basal period). Next, E217G (4.25 mmol/l in saline-propylene glycol-ethanol, 10:4:1; 15 µmol/kg iv) was administered (26), and bile was collected for 5 min, 15 min after E217G administration.

To assess Bsep localization at the peak of cholestasis, the livers were perfused for 30 s with saline 20 min after E217G administration (26), and the major lobe was removed for Western blotting and confocal microscopy studies. Liver samples were frozen in liquid nitrogen, preserved at -80°C until used for membrane preparation, or frozen in precooled isopentane for immunofluorescence studies.

To evaluate the protective effect of DBcAMP on E217G-induced alterations in Bsep localization and activity, DB-cAMP (16.3 mmol/l in PBS, 20 µmol/kg iv; see Ref. 20) was administered 30 min before E217G. Bile was collected as described above. Liver samples for Western blotting and confocal microscopy studies were taken 20 min after E217G administration, as described above.

Assessment of BF and BS Output

BF was determined gravimetrically, assuming a bile density of 1 g/ml. BS concentration in bile was determined by the 3{alpha}-hydroxysteroid dehydrogenase procedure (41). BS output was calculated as the product between BF and BS concentration.

Preparation of Liver Membranes for Western Blot Analysis

Membrane fractions enriched in plasma membranes or intracellular microsomal membranes were prepared from liver by differential centrifugation. Portions of the liver were homogenized with a Teflon pestle (20 strokes at 3,000 rpm) in 0.3 mol/l sucrose containing 0.1 mmol/l PMSF, 25 µg/ml leupeptin, 5 µg/ml aprotinin, and 5 µg/ml pepstatin A (50 mg liver/ml buffer), and an aliquot was reserved for Western blot analysis. The remaining homogenate was used for the mixed membrane preparation, as described elsewhere (23). Purified canalicular membrane fractions were isolated according to the method of Meier et al. (25). Protein concentration was measured using BSA as a standard (22).

Western Blot Studies

Immunoblotting and subsequent densitometry for Bsep were performed in homogenates, mixed and canalicular plasma membranes, and intracellular membranes, as previously described (5), using a rabbit antibody to mouse Bsep (Kamiya Biomedical, Seattle, WA). To check on the possibility of contamination of mixed plasma membranes with microsomes, we analyzed the content of microsomal UDP-glucuronosyltransferase in intracellular and plasma membrane preparations, using a polyclonal antibody developed against a phenol-conjugated UGT isoform (kindly provided by Dr. A. J. Dannenberg, Strang Cancer Prevention Center, New York, NY).

Confocal Microscopy and Image Analysis

Confocal microscopy was used to visualize internalization of Bsep from the canalicular domain and actin cytoskeleton organization. Briefly, liver samples were gently frozen in isopentane precooled in liquid nitrogen and stored at -80°C. Liver slices (5 µm) were prepared with a Zeiss Microm HM5000 microtome cryostat, air-dried for 2 h, and fixed for 10 min with 3% paraformaldehyde in PBS. For Bsep labeling, tissue sections were incubated overnight with the Bsep antibody (1:100). Sections were then washed five times with PBS and incubated with Cy2-conjugated donkey anti-rabbit IgG (1:100; Jackson ImmunoResearch Laboratory, West Grove, PA) for 1 h. After being washed three times with PBS and one time with distilled water, slices were air-dried, mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA), and analyzed in a confocal microscope (True Confocal Scanner Leica TCS SP II). For actin labeling, fixed tissue sections were incubated for 20 min with phalloidin-FITC (10 µg/ml in PBS), washed, mounted, and analyzed in a confocal microscope, as described for Bsep staining. For double labeling (Bsep + actin), tissue sections were exposed to phalloidin-FITC (10 µg/ml in PBS) after performing Bsep staining as described above. To ensure comparable staining and image capture performance for the different groups, liver slices were prepared the same day, mounted on the same glass slide in a single well, and subjected simultaneously to the staining procedure and confocal microscopy visualization.

Densitometric analysis of confocal pictures was performed as previously described (19), using the software Scion Image {beta}4.02 for Windows (Scion). Fluorescence intensity was measured over a line vertical to the canaliculus (8-µm length). For each section, data from 10 different canaliculi were collected and used for statistical comparison. Data were reproduced in three independent liver preparations. Each measurement was normalized to the sum of all intensities of the respective measurement. The variances from different conditions were compared by the Mann-Whitney test.

Studies in Isolated Rat Hepatocyte Couplets

Couplet isolation, enrichment, and culture. Isolated rat hepatocyte couplets (IRHC) were obtained from rat liver, according to the two-step collagenase perfusion procedure described by Wilton et al. (45), adapted from Gautam et al. (11). This initial preparation was further enriched in IRHC by centrifugal elutriation (47). The final preparation, containing 73 ± 5% of couplets with viability >95%, was plated in L-15 medium containing penicillin/streptomycin on 35-mm plastic culture dishes (2 ml/dish) at a density of 0.5 x 105 U/ml and incubated at 37°C for 4.5–5 h. This time was described to be sufficient for couplets to reach their maximal capability to transport and accumulate CLF in their canalicular vacuoles (45).

Assessment of IRHC canalicular function. BS secretory function was evaluated in IRHC by assessing the percentage of couplets (>50 per experiment) displaying in their canalicular vacuoles sufficient fluorescent BS analog, CLF, to make it visible under inverted fluorescent microscopy (45, 46). This parameter is recorded after 15 min of CLF exposure, when a maximum secretory stationary state has been reached (45). Because canalicular secretion rather than uptake is the rate-limiting step in the overall hepatocellular transport of BS (8), this parameter more likely reflects changes in the former, as the period of time allowed for CLF to accumulate apically (i.e., 15 min) is long enough to compensate for any change in the uptake process.

To perform this test, couplets were exposed to E217G (50 µM, in 2 µl DMSO, for 20 min; see Ref. 28). Next, E217G was removed by washing two times with L-15, and couplets were then exposed to CLF (2 µM) for 15 min. CLF was then removed, and canalicular vacuolar accumulation of CLF was assessed by using an inverted fluorescent microscope (Olympus IMT2-RFL; Olympus Optical, London, UK). A separate group of couplets was pretreated with DBcAMP (10 µM, 30 min) and further exposed to E217G (50 µM, 20 min).

Assessment of Bsep and F-actin localization. IRHC were cultured on coverslips in petri dishes for 4–4.5 h and then exposed to E217G (50 µM, in 2 µl DMSO) or DMSO in controls for 20 min. Another group of couplets was pretreated with DBcAMP (10 µM, 30 min) and then exposed to E217G for a further 20-min period, in the presence of DBcAMP.

Couplet fixation and staining of Bsep were made based on the method previously described by Roma et al. (35) for Mrp2. Briefly, cells were incubated with rabbit anti-mouse Bsep (1:250) for 2 h, followed by incubation with FITC-labeled goat anti-rabbit IgG (1:100; Zymed, San Francisco, CA) for 40 min. Cells were then mounted and examined by fluorescence microscopy (Zeiss Axiovert 350TV microscope, equipped with plan-neofluar lenses).

Phalloidin-FITC labeling was employed to visualize F-actin, using a modification of the method described by Knutton et al. (17), as described by Wilton et al. (46). Cells were then mounted and examined by fluorescent microscopy, as described above for Bsep.

Monochrome images (20–30/group) were taken in 1-µm steps, captured on a CCD video camera (Hamamatsu Photonic System, Hamamatsu City, Japan). Out-of-focus flair was removed using a deconvolution program (Micro-Tome Mac; Vaytek, Fairfield, IL). Quantification of Bsep and F-actin fluorescence intensity in the canalicular and pericanalicular area, respectively, expressed as the percentage of total couplet fluorescent intensity, was carried out by using Open-lab digital imaging software (Improvision; Warwick Science Park, Coventry, UK). The canalicular space was identified on Bsep-labeled IRHC by superposing each fluorescent image with its respective phase-contrast image.

Statistical Analysis

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


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Effect of E217G on BF and BS Output in Control and DBcAMP-Pretreated Rats

Table 1 shows the effect of E217G, with or without DBcAMP pretreatment, on BF and BS output 20 min after E217G administration. At this time, a similar maximal decrease was reached in both parameters (approximately -83%, compared with the control group). DBcAMP, administered 30 min before E217G, showed a significant but partial ability to protect against E217G-induced diminution of BF and BS output. DBcAMP alone induced only a transient (~10 min) increase in BF immediately after DBcAMP injection, but no difference in this parameter was recorded by the time E217G (or E217G vehicle in controls) was administered, i.e., 30 min later (data not shown).


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Table 1. Impairment of BF and BS output by E217G and its prevention by DBcAMP pretreatment

 

Effect of E217G on Bsep and F-Actin Localization in Controls and DBcAMP-Pretreated Rats

Changes in Bsep localization in liver in response to E217G were analyzed in situ by confocal microscopy. Under control conditions (Fig. 1A), Bsep was mainly localized in the canalicular area, as previously reported (12, 37). During the acute phase of E217G-induced cholestasis, i.e., 20 min after its administration, Bsep was internalized from the canalicular space, redistributed in intracellular structures, and visualized as a scattered Bsep staining (Fig. 1B). Although DBcAMP did not modify per se the pattern of Bsep localization (Fig. 1C), pretreatment of rats with DBcAMP significantly prevented E217G-induced internalization of Bsep. This prevention was not complete, as some "fuzzy" staining of Bsep remained in some regions near the canaliculi (Fig. 1D). F-actin distribution in controls and in E217G-treated rats is shown in Fig. 1, E and F, respectively. F-actin cytoskeleton displayed a pericanalicular localization, and no difference was apparent between control and E217G-treated animals.



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Fig. 1. A-D: protective effect of DBcAMP on estradiol-17{beta}-D-glucuronide (E217G)-induced internalization of bile salt export pump (Bsep), as assessed by immunofluorescence analysis. Confocal microscopy analysis of Bsep was performed in rats treated with DBcAMP and E217G vehicles (control group; A), DBcAMP vehicle followed by E217G (E217G group; B), DBcAMP followed by E217G vehicle (DBcAMP group; C), or both DBcAMP and E217G (DBcAMP+E217G group; D). Specimens were collected 20 min later. In the control group, Bsep is mainly confined to the canalicular space. Treatment with E217G promoted disruption of the normal pattern of staining of Bsep and induced internalization of the transporter, represented by a fuzzy staining inside the cytoplasm of the cells. DBcAMP almost completely protected against Bsep retrieval during the acute phase of cholestasis (DBcAMP+E217G vs. E217G); some staining remained in the vicinity of the canalicular space (arrow). E and F: effect of E217G on actin cytoskeleton arrangement. No alteration in the normal pericanalicular localization of F actin (control group; E) was observed after E217G treatment (E217G group; F). Images are representative of at least 3 independent experiments/group.

 

Figure 2 depicts, in higher magnification, changes in Bsep localization after E217G treatment. Staining of actin was used to demarcate the canalicular space; Bsep and F actin are shown in red and green, respectively, whereas yellow staining indicates colocalization of both proteins. In control preparations, Bsep was mainly confined to the canalicular space (Fig. 2A), and few pericanalicular vesicles containing the transporters can be observed. Treatment with E217G induced disruption of the normal Bsep localization, which was visualized as an increased red staining outside the limits of the canaliculus, and an increased number of subapical Bsep-containing vesicular structures (Fig. 2B).



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Fig. 2. A and B: in vivo effect of E217G on Bsep immunofluorescent localization. F-actin staining, which normally displays pericanalicular localization, was used to demarcate the bile canaliculus. Yellow staining indicates colocalization of Bsep (red) and F actin (green). In solvent-treated rats, Bsep was mainly confined to the canaliculus (control; A), whereas E217G disrupted the normal canalicular localization of Bsep, visualized as an increment in the red staining outside the limits of the canaliculus (arrows) and in intracellular, vesicle-like structures (E217G; B; arrowheads). C and D: effect of E217G on Bsep immunofluorescent localization in TR- rats. Confocal microscopy analysis of Bsep was performed in TR- rats treated with E217G vehicle (control group; C) or E217G (E217G group; D). Specimens were collected 20 min later. Bsep (red) is mainly confined to the canalicular space, irrespective of whether E217G was administered or not.

 

Because the presence of Mrp2 in canalicular membranes is required for E217G to induce cholestasis (14), we studied whether E217G treatment changes Bsep localization in TR- rats, which are genetically deficient in Mrp2. Unlike the response in normal rats (Fig. 2B), E217G failed to induce any alteration in Bsep localization in TR- rats (Fig. 2D), such that 20 min after E217G administration, Bsep distribution was identical to that in TR- rats receiving only the vehicle. Consistent with previous work, E217G did not decrease BF in TR- rats relative to the control group (data not shown).

Densitometric analysis of Bsep fluorescence profiles in control and E217G-treated rats, with or without prior administration of DBcAMP (Fig. 3A), revealed a statistically significant broadening of the canalicular Bsep peak in the E217G group, and this was prevented by DBcAMP preadministration. Contrarily, a similar analysis for F-actin distribution revealed no changes after E217G administration to normal rats (Fig. 3B). E217G failed to induce any effect in Bsep fluorescence profiles in TR- rats (Fig. 3C); again, no change in F-actin distribution was recorded after E217G administration to TR- rats (data not shown).



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Fig. 3. A: densitometric analysis of fluorescence profiles of Bsep 20 min after E217G administration with ({square}) or without ({blacksquare}) preadministration of DBcAMP or in control conditions (without E217G administration) with ({circ}) or without ({bullet}) preadministration of DBcAMP. B: fluorescence distribution of F actin in control conditions ({bullet}) and 20 min after E217G administration ({circ}). C: fluorescence profiles of Bsep in TR- rats receiving E217G ({circ}) or its vehicle ({bullet}). Fluorescence distribution was recorded along a line (from -4to +4 µm) vertical to the canaliculi. Means ± SE of 10 measurements in each of the 3 individual experiments for each condition are shown. Statistical analysis of the fluorescence profiles of Bsep shown in A revealed that the E217G group was different from the remaining groups, whereas the E217G+DBcAMP group differed statistically from control, DBcAMP, and E217G groups (P < 0.05). No significant change in F-actin fluorescence profiles could be detected between control and E217G-treated rats (B) or in Bsep distribution profiles after administration of E217G to TR- rats (C). Statistical analysis was performed as described in MATERIALS AND METHODS.

 

Western blot analysis of Bsep content in plasma membrane and intracellular membranes was carried out to confirm the results obtained by confocal microscopy studies in normal rats. As shown in Fig. 4, Bsep detection was significantly increased in intracellular membranes in the E217G group during the acute phase of cholestasis, i.e., 20 min after estrogen administration (206.8 ± 26.8 arbitrary units vs. 103.7 ± 7.3, 95.9 ± 3.7, and 127.1 ± 11.1 arbitrary units for control, DBcAMP, and DBcAMP + E217G groups, respectively, P < 0.05). This increased detection of Bsep in intracellular membranes (~100% with respect to control group) is consistent with internalization of Bsep. Contrarily, the protein level in homogenates or mixed plasma membranes did not vary among groups (Fig. 4, densitometric data not shown); lack of change in plasma membrane-associated Mrp2 in E217G-treated rats remained even when mixed plasma membranes were further processed to obtain a purified canalicular membrane fraction (data not shown). A similar increment in intracellular membrane content without changes in purified canalicular membrane was reported to occur after retrieval of another canalicular ABC transporter, Mrp2, in phalloidin-induced cholestasis (36). The explanation for this experimental observation is not readily apparent, but it may reflect impossibility to separate efficiently submembranous structures containing Bsep, derived from canalicular membrane vesiculation, from the canalicular membrane itself by our ultracentrifugational approach. In line with this view, endocytosis of the canalicular membrane was shown to occur as a previous step for lysosomal degradation of apical membrane components (33), and submembranous, early endosomes were shown to have density similarities with the canalicular membrane (43). Whatever the reason, the increase in intracellular accumulation of Bsep in enriched microsomal membranes, instead, may reflect internalization in a deeper, late endosomal compartment, which has a higher density compared with the pericanalicular, endosomal compartment (43); this deep (late) endosomal compartment may contain endocytosed canalicular transporters that are ready to fuse with degradation compartments (31) and may only represent a small fraction of the total amount of transporters in the cells. Pretreatment with DBcAMP 30 min before E217G administration abolished the increase in Bsep levels in intracellular membranes induced by E217G. To validate the data obtained in microsomal membranes, we evaluated the effect of E217G on the content of UDP-glucuronosyltransferase, a model microsomal enzyme. No change in intracellular membrane content of UDP-glucuronosyltransferase was detected in the cholestatic group (data not shown), indicating that the simultaneous increase in Bsep content observed is more likely the result of Bsep internalization.



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Fig. 4. Western blot analysis showing E217G-induced internalization of Bsep and the protective effect of DBcAMP. Western blotting of Bsep was performed in liver membranes of controls, the E217G group, the DBcAMP group, and the DBcAMP+E217G group. Liver membrane fractions were obtained 20 min after E217G (or vehicle) administration. Each group contains samples from 3 independent experiments. Note that Bsep content in intracellular membranes was increased in E217G-treated rats, suggesting endocytic internalization of Bsep, and that this effect was prevented by DBcAMP (for quantitative data, see text). No change could be detected in Bsep levels in the plasma membrane or in the homogenate.

 

Effect of E217G on Bsep Function and Localization in IRHC

Under control conditions, 72 ± 2% of the couplets exhibited canalicular vacuolar accumulation of CLF. After incubation with E217G, the percentage of couplets accumulating CLF was reduced by 57% with respect to controls; this effect was completely prevented by DBcAMP pretreatment (Fig. 5). DBcAMP per se did not affect canalicular vacuolar accumulation of CLF.



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Fig. 5. Impairment by E217G of the canalicular vacuolar accumulation of cholyl-lysyl-fluorescein (CLF) in isolated rat hepatocyte couplets (IRHC) and its prevention by DBcAMP. Hepatocyte couplets were incubated with DBcAMP (10 µM, 30 min) and then were exposed to E217G (50 µM, 20 min). Canalicular accumulation of CLF was expressed as the percentage of total couplets displaying CLF in their canalicular vacuoles, referred to the control values. The no. of couplets analyzed was >50 in each experiment. Values are expressed as means ± SE of 4 independent cellular preparations. P < 0.05, significantly different from the control group (a) and significantly different from E217G group (b).

 

Immunofluorescent staining of Bsep was carried out to assess whether the impairment in canalicular vacuolar accumulation of CLF was associated with changes in the hepatocellular localization of the main canalicular BS transporter. In control couplets, Bsep was confined to the canalicular membrane or to vesicles located in the pericanalicular region (Fig. 6). E217G induced a marked relocation of Bsep in vesicles localized in the pericanalicular area and, more sparsely, over the remaining cell body (the respective phase-contrast images are shown as insets in each fluorescence image to help identify the location of the canalicular lumen). Consequently, the proportion of fluorescence intensity present in the canalicular membrane was diminished by 44% by E217G. DBcAMP pretreatment virtually normalized Bsep localization.



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Fig. 6. Effect of E217G with or without DBcAMP pretreatment on Bsep localization in IRHC. Top: representative fluorescence microphotographs showing immunolocalization of Bsep in control and in E217G (50 µM, 20 min)-treated IRHC, in the presence or absence of DBcAMP (10 µM, 30 min). Phase-contrast images of every IRHC are shown as insets. Bottom: quantification of Bsep fluorescence intensity in the canalicular area, expressed as the percentage of total (canalicular membrane + cell body) fluorescent intensity. The canalicular space was identified by superposing each fluorescent image with its respective phase-contrast image. P < 0.05, significantly different from the control group (a) and significantly different from E217G group (b).

 

Effect of E217G on F-Actin Localization in IRHC

In control couplets, F actin was located in the pericanalicular area, surrounding the central canalicular vacuole. No difference in F-actin localization was apparent between control and E217G-treated couplets, as confirmed after fluorescence quantification by image analysis (proportion of fluorescence intensity in the pericanalicular area: 76 ± 2 vs. 73 ± 2% in control and E217G-treated IRHC, respectively, P > 0.05).


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
In the normal liver, Bsep is found mainly in the canalicular membrane (12, 37) and, in a lesser extent, in small vesicles located in the subcanalicular area (12). This study shows that, when administered either in vivo or to IRHC, E217G induced a marked disruption in the normal Bsep localization (Figs. 1, 2, and 6). This alteration was visualized 20 min after E217G treatment as an increased immunostaining of Bsep in the intracellular area, which is compatible with transporter-containing vesicles. The vesicular nature of this intracellular staining was even more evident in E217G-treated IRHC (Fig. 6). A similar pattern of endocytic internalization of Mrp2 by E217G was recently described in vivo (30), indicating that this effect is not specific for Bsep.

BS are the major constituents of bile, and their transport across the canalicular membrane, mainly mediated by Bsep, represents the rate-limiting step in the overall transport from sinusoidal blood to bile. It is likely, therefore, that an impairment in Bsep localization induced by E217G effectively contributes to the observed alterations on BS transport. Several lines of evidence from this and previous reports support this contention: 1) prevention of Bsep internalization by DBcAMP, a signaling molecule known to stimulate the trafficking of vesicles containing canalicular carriers to the apical pole (16, 34, 35), was associated with an improvement in the capability of the hepatocytes to secrete BS both in vivo (Table 1) and in IRHC (Fig. 5); 2) in TR-, Mrp2-deficient rats, where E217G was unable to inhibit BF, as previously reported (14), no Bsep internalization was observed; and 3) a consistent association between Bsep internalization and cholestasis resulting from decreased BS secretion has been described in other cholestatic conditions, such as those induced by hyperosmotic perfusion (37), endotoxemia (44), taurolithocholate administration (7), and oxidative stress (6).

The existence of functional alterations in Bsep other than those resulting from changes in Bsep localization cannot be ruled out and may well act in concert. Indeed, the existence of additional mechanism(s) of BS secretory failure is supported by our own results in DBcAMP-pretreated rats. In vivo, this signaling molecule almost completely prevented Bsep relocalization, as suggested by the presence of a control-like pattern of Bsep immunostaining (Fig. 1D) and normalization of Bsep content in intracellular membranes (Fig. 5). Despite this extensive normalization, BF and BS secretion failure were only partially prevented by DBcAMP (Table 1), suggesting that Bsep internalization is an important, but not the only, mechanism involved in the secretory failure induced by E217G. Based on studies of taurocholate transport in isolated membrane vesicles, Stieger et al. (40) suggested that E217G trans inhibits Bsep after being translocated into the canaliculus by Mrp2. However, the actual relevance of this in vitro finding in the situation in vivo is uncertain, as a further study by Huang et al. (14) showed that no decrease in BF occurs in isolated, perfused livers of TR- rats lacking Mrp2, even when the E217G concentrations achieved in bile were equivalent to those present in normal Wistar rats demonstrating cholestasis; this suggests that accumulation of E217G in the canalicular lumen alone is not sufficient to induce cholestasis. E217G also diminishes the canalicular plasma membrane fluidity by increasing its cholesterol-to-phospholipid ratio (1). Because membrane fluidity is known to modulate activity of the canalicular BS carrier system (29), the possibility also exists that E217G impairs Bsep activity by altering normal membrane composition.

In agreement with Huang et al. (14), E217G failed to show any cholestatic effect when administered to TR- rats. Our finding that Bsep also conserved its normal canalicular localization (Fig. 2D) confirms and extends the concept that Mrp2 is essential for E217G-induced inhibition of Bsep-mediated transport in that it influences not only Bsep activity but also its localization. This suggests that Mrp2 is acting as, or is associated with, a cholestatic receptor able to trigger an as yet uncharacterized chain of events leading to internalization of canalicular transporters, including Mrp2 itself. The nature of the interactions between Mrp2 and Bsep that lead to the apparent simultaneous retrieval is not known. Although a direct functional association between Mrp2 and the activity of another canalicular transport system, the swelling-activated Cl- channels, has been reported recently (21), no such functional association has been established for Mrp2 and Bsep. Clearly, further work will be required to understand the nature of these functional links.

Neither the mechanism by which E217G induces Bsep internalization nor the mechanism by which DB-cAMP prevents this effect can be addressed directly from our results. However, several recent reports provide some insights. A microtubule-dependent, vesicular pathway involved in insertion of canalicular transporters from a subapical, membranous compartment in their membrane domain has been revealed by using microtubule poisons under different conditions of stimulated vesicle-based trafficking of canalicular transporters, such as swelling (13), DBcAMP administration (2, 10), or taurocholate administration (10). On the other hand, apical endocytosis of protein components, which likely accounts for internalization of transporters, was recently demonstrated in polarized hepatic cells (33). Therefore, it is conceivable that a balance exists between canalicular transporter insertion and internalization, that E217G disturbs this balance either by inhibiting insertion or by stimulating internalization, or both. The first possibility is likely, since estrogens were shown to impair hepatocellular polarity by reducing motility of canalicular transporters containing vesicles toward the apical pole (42). cAMP has been shown to stimulate sorting of ATP-binding cassette transporters into the canalicular membrane, via a vesicular, partially microtubule-dependent pathway both in vivo (10) and in IRHC (34, 35). It is therefore tempting to speculate that cAMP induces its protective effect by shifting the insertion-internalization balance toward insertion, thus counteracting the shift toward internalization induced by E217G. In contrast, changes in the actin cytoskeleton, which was reported to alter normal localization (36) and targeting (35) of canalicular transporters in their membrane domain, seem not to be a contributing factor, as we found no alteration in F-actin distribution after E217G administration either in vivo or in IRHC.

In summary, the present data indicate that a marked endocytic internalization of Bsep from the canalicular membrane into intracellular, vesicular-like structures, accompanied by an impairment of Bsep transport activity, occurs in E217G-induced cholestasis and that these processes depend on the presence of Mrp2 in the canalicular membrane. cAMP, a signaling molecule known to stimulate targeting of canalicular transporters, is instrumental in counteracting these effects. Further investigation is necessary to elucidate the mechanism by which E217G induces internalization of Bsep, the role of Mrp2 in this process, and the mechanisms involved in the protective effect of cAMP.


    DISCLOSURES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This work was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica, Ministerio de Salud de la Nación (Beca Carrillo-Oñativia; to M. G. Roma), Fundación Antorchas, Universidad Nacional de Rosario (to F. A. Crocenzi and A. D. Mottino), and by National Institute of General Medical Sciences Grant GM-55343 (to M. Vore).


    ACKNOWLEDGMENTS
 
This work was presented in a preliminary form at the 37th Annual Meeting of the European Association for the Study of the Liver, Madrid, Spain, in April 2002 and was published in abstract form [J Hepatol 36, Suppl 1: 6, 2002].


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. G. Roma, Instituto de Fisiología Experimental, Facultad de Ciencias Bioquímicas y Farmacéuticas, Suipacha 570, S2002LRL, Rosario, Argentina (E-mail: ifise1{at}citynet.net.ar).

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.

{dagger} Deceased 14 January 2003 Back


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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
 DISCLOSURES
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