Cyclosporin A Induced Internalization of the Bile Salt Export Pump in Isolated Rat Hepatocyte Couplets

Irene D. Román*,1, M. Dolores Fernández-Moreno*, Jesús A. Fueyo*, Marcelo G. Roma{dagger} and Roger Coleman{ddagger}

* Departamento de Bioquímica y Biología Molecular, Universidad de Alcalá, E-28871, Alcalá de Henares (Madrid), Spain; {dagger} Institute of Experimental Physiology, CONICET-University of Rosario, Argentina; and {ddagger} School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom

Received September 26, 2002; accepted November 14, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Isolated rat hepatocyte couplets were used to perform the comparative study of two widely used immunosuppressors, cyclosporin A (CsA) and tacrolimus (FK506) on hepatocanalicular function. We assessed canalicular function by counting the percentage of couplets that were able to accumulate the fluorescent cholephile, cholyl-lysyl-fluorescein (CLF), into the canalicular vacuole between the two cells, i.e., canalicular vacuole accumulation (CVA) of CLF. Compared to controls (DMSO-treated cells), CsA, in the approximate range of concentrations used therapeutically, caused inhibition of CVA of CLF, disorganization of the bile salt export pump (Bsep) localization at canalicular level resulting in its relocation into the cell, and disruption of the pericanalicular F-actin cytoskeleton. In contrast, FK506, at both approximately therapeutic and supratherapeutic concentrations, had no deleterious effect upon CVA of CLF, upon the localization of the bile salt transporter at the canalicular membrane, or on the organization of the pericanalicular F-actin cytoskeleton. These results point to transporter and cytoskeletal disorganization as contributors or determinants of CsA-induced cholestasis at canalicular level, whereas FK506 does not appear to produce these cholestasis-determining responses even at supratherapeutic concentrations.

Key Words: bile salt export pump (Bsep); cytoskeleton; F-actin; cyclosporin A; tacrolimus; hepatocyte.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bile formation is an osmotic process that critically depends on active secretion of osmotically active compounds from the liver into the bile canalicular lumen. Bile salts and glutathione are considered the major contributors to bile flow in rodents (Muller and Jansen, 1997Go, 1998Go). Excretion of a large variety of endogenous and exogenous compounds from hepatocytes into bile is an adenosine triphosphate (ATP)-dependent process, predominantly performed by members of the P-glycoprotein (Pgp) subfamily and the multidrug-resistence protein subfamily of the ATP-binding cassette protein superfamily (Muller and Jansen, 1997Go). At least four members of the Pgp subfamily are located at the canalicular membrane of rodent liver: mdr1a, mdr1b, mdr2, and Bsep (bile salt export pump, initially called sister gene of P-glycoprotein-spgp-). Gerloff et al. (1998)Go provided evidence that Bsep is exclusively present in the liver and most likely is the major canalicular bile salt export pump of mammalian liver. It is important to note that transport across the canalicular membrane is the rate-limiting step in overall hepatocellular bile salt excretion (Kullak-Ublick et al., 2000Go).

Cyclosporin A (CsA) is a powerful immunosuppressor drug widely used in transplantation procedures and in the treatment of several autoimmune diseases. However, its therapy is associated with numerous side effects, especially dose-related nephrotoxicity and hepatotoxicity (Kahan, 1989Go; Wiesner et al., 1990Go). The most common abnormalities related to hepatotoxicity are increases of serum bile salt levels, hyperbilirubinemia, and cholestasis (Kahan, 1989Go; Román et al., 1990Go). A relationship has been proposed between the cholestasis induced by CsA and the reduction of the bile salt-dependent fraction of bile flow due to an inhibition of uptake, synthesis, and/or ATP-dependent canalicular transport of bile salts in the liver (Ballantyne et al., 1989Go; Boelsterli et al., 1989Go; Ziegler and Frimmer, 1986Go), but the mechanism(s) underlying these effects remains largely unknown.

Tacrolimus (FK506), a macrolide immunosuppressant that possesses similar but more potent immunoppressant properties compared with CsA, is considered as an alternative primary immunosuppressant to CsA in hepatic transplantation (Peters et al., 1993Go). The effect of FK506 on bile flow is at the moment unclear. FK506 has been described as producing choleresis in dogs and rats (Deters et al., 2001Go; Reese et al., 1993Go) and cholestasis in rats at high doses (Sánchez-Campos et al., 1998Go). Little is known about its effect at the canalicular level. So far, the only information is that the trough levels of CsA or FK506 used in clinical trials in humans do not inhibit the mediator of multidrug resistance, the canalicular organic cation export pump (mdr1/P-glycoprotein) (Takeguchi et al., 1993Go).

Hepatocyte couplets constitute an in vitro technique for the study of bile canalicular function. Within 4 to 5 h of couplet isolation, the canalicular membrane reorganizes to establish a biliary pole directly between the two cells surrounded by tight junctions. The hepatocyte couplets can secrete biliary components ("primary bile") across their canalicular membranes into its vacuole (Graf and Boyer, 1990Go). Our previous studies using CsA in this experimental model revealed that CsA exerted a cholestatic effect, and that this might relate to an alteration in pericanalicular F-actin (Román and Coleman, 1994Go; Román et al., 1996Go).

The aim of the present work was to perform a comparative study of FK506 and CsA on canalicular function by studying their actions upon (1) canalicular secretion of a fluorescent bile salt, (2) subcellular localization of Bsep, the major canalicular bile salt export pump, and (3) the integrity of the pericanalicular F-actin cytoskeleton, which is necessary for normal localization of transporters at the canalicular membrane domain (Roma et al., 2000Go; Rost et al., 1999Go).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
Cholyl-lysyl-fluorescein was kindly provided by Dr. Charles O. Mills (Birmingham, UK) and its purity was confirmed according to the methods of Mills et al. (1991)Go; cyclosporin A (Sandoz Pharma AG, Switzerland) and tacrolimus (FK506, Fujisawa GmbH, Germany) were used. Collagenase (type A) from Clostridium histolyticum was purchased from Gibco (Paisley, UK). Bovine serum albumin (fraction V), Leibovitz-15 tissue culture medium (L-15), fluorescein isothiocyanate (FITC)-labeled-phalloidin, and other fine chemicals were obtained from Sigma Chemical Co. (Poole, Dorset, UK). FITC-labeled goat anti-rabbit immunoglobulin G was purchased from Zymed (San Francisco, CA). Dr. M. Muller, Wageningen, The Netherlands, kindly provided polyclonal antibody against Bsep (k12). All other chemicals were of reagent grade.

Animals.
Male Wistar rats (210–250 g body weight) from the Biomedical Services Unit of the University of Birmingham were used throughout. The animals were allowed free access to food (41B maintenance diet, Pillsbury, Birmingham, UK) and tap water ad libitum. Anesthesia was achieved with ketamine hydrochloride (Ketalar), 6 mg/100 g body weight (bw), and medetomidine (Domitor), 25 µg/100 g bw. Surgery was performed between 8:30 and 9:30 A.M. to minimize circadian variations. All experiments were conducted according to the criteria outlined in the Guiding Principles in the Use of Animals in Toxicology adopted by the Society of Toxicology in 1989.

Preparation of hepatocyte couplets.
Hepatocyte couplets were obtained from rat liver according to a two-step collagenase perfusion procedure (Wilton et al., 1993Go) adapted from Gautam et al. (1989)Go. This initial preparation contained 24% ± 4% (n = 25) of couplets with high viability (>90%), as assessed by the Trypan blue exclusion test. Couplets were further enriched (68 ± 6% of couplets with >95% viability) by centrifugal elutriation, as described elsewhere (Wilton et al., 1991Go).

Culture and treatment of hepatocyte couplets.
Hepatocytes were plated in L-15 containing 50 U/ml penicillin and 50-µg/ml streptomycin onto 35-mm plastic culture dishes (2 ml/dish) at a density of 0.5 x 105 units/ml, and incubated at 37°C for 4.5 h.

Then, cyclosporin A (100 nM or 1 µM, final concentrations) or FK506 (5 nM, 100 nM, 1 µM, or 3 µM, final concentrations) were added to the culture dishes as 10-µl aliquots dissolved in dimethyl-sulfoxide (DMSO).

DMSO has been used as the vehicle for CsA or FK506 in many other studies with hepatocytes (Kadmon et al., 1993Go; Takeguchi et al., 1993Go). DMSO itself did not affect the various experimental parameters in each experimental group in our studies (data not shown).

Assessment of canalicular activity.
Counting of couplets able to undergo canalicular vacuole accumulation (CVA) of cholyl-lysyl-fluorescein (CLF), expressed as a percentage of control couplets exhibiting this phenomenon, has been used to assess canalicular function; CLF is closely similar in both uptake kinetic parameters and secretory properties to those of its naturally occurring parent compound, cholylglycine, which is excreted into the bile canaliculus mainly by the bile salt export pump (Bsep) (Mills et al., 1997Go). After the 4.5-h culture period, DMSO or FK506 (5 nM, 100 nM, 1 µM, or 3 µM), were added to each plate in the presence of CLF (2 µM, final concentration) and incubated at 37°C for 15 min. Then, the cells were washed twice with 2 ml of L-15 and observed using an inverted fluorescence microscope (Olympus IMT2-RFL) equipped with a 100-W mercury light source and an incubator to maintain the cells at 37°C during observation. We chose a 15-min incubation period since a plateau in CLF secretion into the canalicular vacuoles is reached by this time (Fentem et al., 1990Go; Wilton et al., 1991Go).

Assessment of bile salt export pump (Bsep) localization.
Bsep was stained with the polyclonal antibody against Bsep (k12) (Vos et al., 1998Go). Bsep is the major canalicular bile salt export pump of mammalian liver (Gerloff et al., 1998Go).

After plating and treatment of couplets with DMSO, CsA (100 or 1 µM) or FK506 (100 nM, 1 µM, or 3 µM) for 15 min, cells were then incubated with NH4Cl (50 mM) in PBS, permeabilized with 0.2% Triton X-100 and 3% BSA in PBS, blocked with 3% goat serum and 3% BSA (1/1, v/v), stained with the polyclonal antibody against Bsep (k12) at a dilution of 1:250, and subsequently incubated with FITC-labeled goat anti-rabbit immunoglobulin G, according to Vos et al. (1998)Go. Coverslips were then inverted onto citifluor mounting solution to observe the stained cells.

The specimens were then examined under fluorescent light (excitation wavelength, 490 nm; emission wavelength, >525 nm) using a Zeiss microscope (Axiovert 350TV, Carl Zeiss Oberkochen Ltd., Welwyn Garden City, Herts, UK) equipped with Zeiss plan-neofluar lenses.

Assessment of phalloidin-FITC stained actin with confocal microscopy.
Phalloidin-FITC labeling of fixed cells allows visualization of the F-actin cytoskeleton (Watanabe and Phillips, 1986Go). After plating onto glass coverslips, hepatocyte couplets were incubated in L-15 at 37°C for 4.5 h. Cells were then treated with DMSO, CsA (1 µM), or FK506 (1 µM) for 15 min, fixed with 3% formalin in phosphate buffered saline, stored at 4°C until permeabilized with 0.1% Triton X-100 in PBS, and labeled with phalloidin-FITC (5 µg/ml in PBS) according to the method of Knutton et al. (1989)Go, as modified by Wilton et al. (1994)Go. Coverslips were then inverted onto citifluor mounting solution, to examine the stained cells under fluorescent light, as described above.

Image analysis.
Images were analyzed to quantify the total intensity of fluorescence within the couplet and, specifically, in the canalicular (for Bsep) and pericanalicular (for F-actin) regions. For this purpose, 20 to 30 monochrome images of couplets of every group, derived from three separate isolations, were taken in 1-µm steps, captured on a CCD video camera (Hamamatsu Photonic Systems Corp., Hamamatsu City, Japan) and out-of-focus flair removed using a deconvolution program (Micro-Tome Mac, Vaytek Ltd., Fairfield, IL). Total and pericanalicular or canalicular fluorescence was quantified by using Openlab Digital Imaging software (Improvision, Warwick Science Park, Coventry, UK).

Statistical analysis.
Results are expressed as mean ± SEM. Statistical analysis was performed using the Student’s t-test.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effect of CsA or FK506 on canalicular vacuole accumulation of CLF.
We had shown in a previous paper (Román et al., 1996Go) that concurrent incubation of CsA (5, 25, 50, 75, 100, or 500 nM, or 1 µM) and CLF for 15 min induces a concentration-dependent inhibition of CVA of CLF. This effect was confirmed in the present experiments (results not shown). However, when FK506 (5 or 100 nM, or 1 or 3 µM) was exposed to the cells for 15 min in the presence of CLF, canalicular vacuole accumulation of CLF did not change, relative to control values (i.e., cells treated with DMSO) (Table 1Go). Longer incubations with FK506 (30 min) were also without effect (data not shown). These results clearly reveal that, therapeutic/supratherapeutic concentrations of FK506 (Takeguchi et al., 1993Go) did not induce functional disruption of canalicular ability to accumulate bile salts, under conditions in which the disruptive effects of CsA were observed.


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TABLE 1 Effect of FK506 on Couplet Canalicular Vacuole Accumulation of Cholyl-Lysyl-Fluorescein
 
Bile salt export pump (Bsep) localization after CsA or FK506 treatment.
The total couplet fluorescence (TF) and the canalicular fluorescence (CF) were measured, and the CF/TF ratio, which represents the relative amount of Bsep in the bile canaliculus, was calculated. The CsA (100 nM or 1 µM) group showed a significant decrease in CF/TF ratio (p < 0.001) with respect to its control group (DMSO), indicating a Bsep disorganization/decrease at canalicular membrane level. This effect was already at maximum at 100 nM CsA. In contrast, the cells treated with FK506 (100 nM, or 1 or 3 µM) showed no significant difference from either control in the timescale of these experiments (Fig. 1Go, Table 2Go).



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FIG. 1. FITC-labeled Bsep localization in rat hepatocyte couplets treated with DMSO (control), CsA (1 µM), or FK506 (1 µM) for 15 min. White indicates the highest intensity of fluorescein measured.

 

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TABLE 2 Couplet Bile Salt Export Pump Localization after Treatment with FK506 or Cyclosporin A
 
Pericanalicular actin disposition after CsA or FK506 treatment.
As we had shown in a previous paper (Román and Coleman, 1994Go), CsA (1 µM) induced a significant decrease in CF/TF ratio (p < 0.01) with respect to its control group (DMSO), indicating F-actin disruption. In contrast, the group of cells treated with FK506 at the same concentration showed no significant difference with controls, suggesting that F-actin integrity is preserved in the presence of FK506, in the time scale of these experiments (Fig. 2Go, Table 3Go).



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FIG. 2. Phalloidin-FITC-labeled F-actin localization in rat hepatocyte couplets treated with DMSO (control), CsA (1 µM), or FK506 (1 µM) for 15 min. White indicates the highest intensity of fluorescein measured.

 

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TABLE 3 Effect of FK506 or Cyclosporin A Treatment on F-Actin Distribution in Couplets
 
Another manifestation of the disorganization and relocation of the actin-cytoskeleton is the appearance of plasma membrane "blebs;" bleb formation appears to be a prominent feature in many forms of liver injury (Gores et al., 1990Go) and has already been described in cholestatic hepatic injury (Vital et al., 1982Go). Blebs were seen in CsA-treated cells in our previous studies (Román et al., 1996Go) and confirmed in the present work (results not shown). Cells treated with FK506, however, did not show these blebs at any of the concentrations of FK506 used.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previously, we had shown that CsA induced a concentration-dependent inhibition of CVA of CLF (Román et al., 1996Go). We have performed the present study to determine whether tacrolimus, an even more potent immunosuppressant, also has a similar harmful effect upon hepatocanalicular function. The lowest FK506 concentrations we have used in this study, i.e., 5 and 100 nM, are included as the recommended "trough level" and "peak level" of the drug in serum for clinical use, respectively (Takeguchi et al., 1993Go). The highest FK506 concentrations used here, i.e., 1 and 3 µM, aim to compare it to those used for CsA at supratherapeutic concentrations. Since FK506 showed no effect on CVA of CLF at any concentration (Table 1Go), our results suggest that FK506 is far less, if at all, harmful for the hepatobiliary system; this finding is compatible with the results obtained by a comparative study, showing that CsA, but not FK506, decreased both bile flow and biliary secretion of bile salts, cholesterol, and glutathione in the whole rat (Deters et al., 2001Go). Furthermore, even when, at higher concentrations, FK506 was shown to induce cholestasis in another study (Sánchez-Campos et al., 1998Go), it was suggested that a compound involved in the generation of the bile salt-independent bile flow, presumably glutathione, was affected by the immunosuppressant.

Due to bile salt transport across the canalicular membrane is the rate-limiting step in overall hepatocellular bile-salt excretion (Kullak-Ublick et al., 2000Go), and since Bsep is the major canalicular bile salt export pump of mammalian liver (Gerloff et al., 1998Go), we performed a comparative study on Bsep distribution in CsA- and FK506-treated couplets. The results show a clear difference in localization of Bsep at the canalicular membrane between the two compounds. FK506 produced no change, as compared with controls (DMSO), even at the highest concentrations used in this study. In contrast, when the hepatocytes were treated with CsA, the canalicular staining of Bsep became irregular and "fuzzy," reflecting a decrease in the number of Bsep molecules at the canalicular level due to a relocation of these into vesicular compartments located in the cell body (Fig. 1Go). This relocation is reflected in a significant (p < 0.001) 45% reduction in the ratio of canalicular to total fluorescence (Table 2Go). This result is complementary with the findings showing that CsA induces cis-inhibition of Bsep-mediated taurocholate transport in Sf9-cell vesicles (Stieger et al., 2000Go). CsA is a competitive inhibitor of Bsep (Kadmon et al., 1993Go; Noe et al., 2001Go), and the immunosuppressor was shown to inhibit, in addition, the bile salt-stimulated Bsep ATPase activity (Noe et al., 2001Go). Therefore, both partial retrieval of Bsep from the canalicular membrane and inhibition of Bsep translocation activity of the transporters that have remained in the membrane can well act in concert to induce the overall reduction of the biliary bile salt secretion. Bsep relocalization was not present, however, when couplets were treated with FK506.

Pericanalicular F-actin alteration appears to be a specific marker of hepatocellular cholestasis (Thibault et al., 1992Go) resulting in impaired contractility, proposed to be necessary for bile propulsion within the biliary tree (Watanabe et al., 1991Go). Image analysis showed that, in FK506- or DMSO-treated couplets, F-actin labeling (with phalloidin-FITC) was far higher in the pericanalicular cytoskeleton than in the remainder of the couplet (Fig. 2Go, Table 3Go). In contrast, treatment of the couplets with CsA (1 µM), as we have previously shown at lower concentrations (100 or 500 nM) (Román et al., 1996Go), induced a significant reduction in this pericanalicular fluorescence caused by disruption of the F-actin cytoskeleton and its relocation at the cell periphery (Fig. 2Go, Table 3Go).

F-actin cytoskeletal integrity is required for normal localization of transporters at the canalicular membrane domain (Roma et al., 2000Go; Rost et al., 1999Go). Roma et al. (2000)Go showed that disruption of the actin cytoskeleton by cytochalasin D completely blocked the apical insertion of the canalicular transporter, mrp2, which remained in large vesicle-like structures in the cell body. Coincidentally, Rost et al. (1999)Go showed that the F-actin cytoskeletal poison, phalloidin, leads to mrp2 redistribution from the canalicular membrane to the cell body. Therefore, F-actin cytosketal alteration caused by CsA may explain the disturbance in Bsep cell localization induced by the immunosuppressant. Rigidification of the canalicular membrane by CsA, due to the increase in the cholesterol/phospholipid ratio, has been recently reported (Yasumiba et al., 2001Go); this change may affect the functional capacity of bile-salt transporters, as was shown to occur for TC transport in canalicular membrane preparations (Mills et al., 1987Go). Interestingly, we have previously shown that the membrane fluidizing compound, S-adenosyl-L-methionine, prevents both canalicular bile salt secretory function and cytoskeleton disorganization in couplets (Román et al., 1996Go). Because of the close association between actin cytoskeleton and the hepatocyte canalicular plasma membrane, membrane rigidification, actin cytoskeleton disruption, and impaired sorting of transporters into the canalicular membrane could be causally related events. Indeed, actin is anchored to the canalicular membrane via a number of intermediate proteins with regulatory properties, on bile secretory proteins (Fouassier et al., 2001Go). Therefore, it is tempting to speculate that actin-cytoskeleton disorganization can lead to impaired secretory function by either or both impaired sorting (Roma et al., 2000Go; Rost et al., 1999Go) and impaired intrinsic function.

We conclude that CsA-induced cholestasis may be in part due to Bsep relocalization/decrease at the canalicular membrane level, probably as a consequence of F-actin disruption at this level. On the contrary, the non-cholestatic immunosuppressant agent, FK506, does not exert any negative effect on canalicular function or cytoskeleton integrity at either therapeutic or supratherapeutic concentrations, therefore retaining a normal Bsep canalicular distribution; though this seems to be valid for the timescale of our experiments, it does not preclude other longer-term effects, but these are not possible to observe in hepatocyte couplets, which is a short-term culture model.


    ACKNOWLEDGMENTS
 
Polyclonal antibody against Bsep was kindly provided by M. Müller, Wageningen, The Netherlands. The present work was supported by Travel Awards from the Consejo Social of the Universidad de Alcalá and Wellcome Trust.


    NOTES
 
1 To whom correspondence should be addressed. Fax: 00-34-918 854585. E-mail: lola.roman{at}uah.es. Back


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 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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