Biliary excretion in primary rat hepatocytes cultured in a collagen-sandwich configuration

Xingrong Liu1, Edward L. LeCluyse1, Kenneth R. Brouwer2, Liang-Sheng L. Gan2, John J. Lemasters3, Bruno Stieger4, Peter J. Meier4, and Kim L. R. Brouwer1

1 Division of Drug Delivery and Disposition, School of Pharmacy, and 3 Department of Cell Biology and Anatomy, School of Medicine, University of North Carolina, Chapel Hill 27599; 2 Division of Bioanalysis and Drug Metabolism, Glaxo Wellcome, Inc., Research Triangle Park, North Carolina 27709; and 4 Division of Clinical Pharmacology, Department of Medicine, University Hospital, CH-8091 Zurich, Switzerland


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The objective of the present investigation was to examine the functional reestablishment of polarity in freshly isolated hepatocytes cultured between 2 layers of gelled collagen (sandwich configuration). Immunoblot analysis demonstrated that the canalicular multispecific organic anion transport protein (multidrug resistance-associated protein, Mrp2) was partially maintained in day 5 hepatocytes cultured in a sandwich configuration. Fluorescein-labeled taurocholate and carboxydichlorofluorescein were excreted into and concentrated in the bile canalicular lumen of day 5 sandwich-cultured hepatocytes, resulting in formation of fluorescent networks in standard buffer (intact bile canaliculi). Confocal microscopy studies demonstrated that 1) carboxydichlorofluorescein that had concentrated in the canalicular lumen was released into the incubation buffer in the presence of Ca2+-free buffer (disrupted bile canaliculi), and 2) rhodamine-dextran, an extracellular space marker, was only able to diffuse into the canalicular lumen in the presence of Ca2+-free buffer. The cumulative uptake of [3H]taurocholate in day 5 sandwich-cultured hepatocytes was significantly higher in standard buffer compared with Ca2+-free buffer, due to accumulation of taurocholate in canalicular spaces. When [3H]taurocholate was preloaded in the day 5 sandwich-cultured hepatocytes, taurocholate efflux was greater in Ca2+-free compared with standard buffer. The biliary excretion index of taurocholate, equivalent to the percentage of retained taurocholate in the canalicular networks, increased from ~8% at day 0 to ~60% at day 5 in sandwich-cultured hepatocytes. In summary, hepatocytes cultured in a collagen-sandwich configuration for up to 5 days establish intact canalicular networks, maintain Mrp2, reestablish polarized excretion of organic anions and bile acids, and represent a useful in vitro model system to investigate the hepatobiliary disposition of substrates.

taurocholate; carboxydichlorofluorescein; canalicular multispecific organic anion transporter; confocal microscopy


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MANY ENDOGENOUS AND exogenous compounds undergo hepatic uptake and biliary excretion via carrier-mediated transporters. A variety of techniques, involving the intact liver in vivo, the isolated perfused liver, isolated hepatocyte suspensions, short-term cultured hepatocyte couplets, membrane vesicles, and isolated transport proteins, have been employed to study these transport processes (3, 23). Numerous studies have utilized freshly isolated hepatocytes to examine the processes of biliary excretion. Tarao et al. (28) demonstrated that cholestasis impairs the biliary excretion of bile acids by measuring the efflux of bile acids in hepatocyte suspensions. Similarly, Oude Elferink et al. (24) utilized hepatocyte suspensions to study ATP-dependent efflux of oxidized glutathione and dinitrophenyl-glutathione. Studenberg and Brouwer (26) demonstrated the effects of p-hydroxyphenobarbital glucuronide on the canalicular excretion of acetaminophen glucuronide in hepatocyte suspensions. Nevertheless, the interpretation of these experiments is hampered by the fact that substrate efflux from isolated hepatocytes may be mediated by sinusoidal as well as canalicular excretion mechanisms. Because of the difficulties in differentiating between sinusoidal efflux and canalicular excretion in isolated hepatocytes, most transport studies utilizing hepatocyte suspensions are limited to investigating hepatic uptake processes (23).

Short-term (3-8 h) cultured hepatocyte couplets have been employed to examine the biliary excretion of fluorescent compounds utilizing fluorescence microscopy. Graf and Boyer (10) demonstrated that polarized or vectorial transport function was restored and the canalicular lumen was sealed in hepatocyte couplets. However, the application of these techniques is limited because the substrate must contain a fluorescent chromophore. Historically, long-term cultures of primary hepatocytes (more than 24 h) have not been a suitable model for studying hepatobiliary transport due to the rapid deterioration of transport properties and other liver-specific functions and failure to maintain normal hepatocyte morphology (11, 17). For instance, Na+-dependent taurocholate uptake deteriorated within 3 days to 4% of the uptake exhibited by hepatocytes cultured for 3 h (21). Likewise, hepatocyte-derived cell lines often lack liver-specific transport functions (25). Many studies have been conducted to examine the influence of culture conditions on the expression of the liver-specific phenotype in hepatocyte cultures, including changes in medium composition, coculture with other epithelial cells, addition of chemical modulators, and alteration of the extracellular matrix environment (18). Primary rat hepatocytes cultured between two layers of gelled collagen form extensive bile canalicular networks and represent a successful approach to maintaining liver-specific functions including albumin secretion, cytochrome P-450 enzyme induction, and bile acid uptake (7, 17, 19, 22). Recently, it was established that hepatocytes cultured in a collagen-sandwich configuration for 5 days exhibited functional bile acid transport and partially maintained Na+-taurocholate cotransporting polypeptide (Ntcp) (22). In contrast, hepatocytes cultured under conventional conditions were unable to maintain Ntcp. More importantly, the hepatocytes cultured in a collagen-sandwich configuration formed extensive bile canalicular networks (17, 22). Talamini et al. (27) demonstrated that hepatocytes cultured in a sandwich configuration maintain functional polarity and form a sealed canalicular lumen. This model system allows for differentiation between sinusoidal and canalicular transport processes and thus may represent a useful tool for investigating the hepatobiliary disposition of substrates.

Several ATP-dependent primary active transport systems on the canalicular membrane have been characterized or postulated (23). The ATP-dependent canalicular multispecific organic anion transporter (multidrug resistance-associated protein, Mrp2) preferentially transports di- and multivalent organic anions other than bile acids, including glucuronide and glutathione conjugates (2, 13, 23). An ATP-dependent canalicular bile acid transporter that transports taurocholate in isolated rat canalicular liver plasma membrane vesicles has been described (1, 23). Sister of P-glycoprotein may represent the ATP-dependent taurocholate carrier on the canalicular membrane (8). In the present study, the maintenance of Mrp2 in sandwich-cultured hepatocytes was examined with immunoblot analysis. The functional activity of Mrp2 and the canalicular bile acid transporter were examined with the model substrates carboxydichlorofluorescein and taurocholate, respectively. The time course of reestablishment of vectorial transport was assessed quantitatively. Moreover, the utility of this in vitro model system to quantitate biliary excretion of substrates was examined.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals. Taurocholate and dexamethasone were purchased from Sigma Chemical (St. Louis, MO). [3H]taurocholate (3.4 Ci/mmol) and [14C]salicylic acid (salicylate) (120 mCi/mmol) were obtained from DuPont NEN (Boston, MA). Carboxydichlorofluorescein and rhodamine-dextran (10 kDa) were obtained from Molecular Probes (Eugene, OR). Fluorescein-labeled taurocholate was prepared by Dr. R. L. Bugianesi and kindly provided by Dr. C. P. Sparrow (Merck, Rahway, NJ). Collagenase (type I, class I) was obtained from Worthington Biochemical (Freehold, NJ). DMEM, fetal bovine serum, and insulin were purchased from GIBCO (Grand Island, NY). Rat tail collagen (type I) was obtained from Collaborative Biomedical Research (Bedford, MA). SDS-polyacrylaminde gel and nitrocellulose transfer membrane (0.45 µm) were purchased from Bio-Rad Laboratories (Hercules, CA). An anti-serum against the COOH terminus of Mrp2 was raised by immunizing rabbits with the peptide AGIENVNHTEL, which was coupled at the COOH terminus with an additional COOH to keyhole limpet hemocyanin with maleimidobensoyl-N-hydroxysuccinimide ester (Neosystem Laboratories, Strasbourg, France). Enhanced chemiluminescence (ECL) detection kit and Hyperfilm-ECL were purchased from Amersham Life Sciences (Buckinghamshire, UK). All other chemicals and reagents were of analytical grade and were readily available from commercial sources.

Animals. Male Wistar rats (250-280 g) from Charles River (Raleigh, NC) were used as liver donors. They were allowed free access to food (laboratory rodent diet 5001, PMI Feeds, St. Louis, MO) and water and were housed in a constant alternating 12-h light (6:00 AM to 6:00 PM) and dark cycle. All procedures were approved by the Institutional Animal Care and Use Committee at the University of North Carolina.

Preparation of culture dishes. Plastic culture dishes (60 mm) were precoated with rat tail collagen at least 1 day before the hepatocyte cultures were prepared. To obtain a simple (rigid) substratum, collagen solution (0.1 ml, 1.5 mg/ml) was added to each dish. Coated dishes were stored overnight in a sterile hood. Immediately before use, fresh medium was added to neutralize the collagen. To obtain a gelled collagen substratum, ice-cold neutralized collagen solution (0.1 ml, 1.5 mg/ml, pH 7.4) was spread onto each culture dish. Freshly coated dishes were placed at 37°C in a humidified incubator for ~1 h to allow the matrix material to gel, followed by addition of 3 ml DMEM to each dish and storage in a humidified incubator.

Isolation and culture of rat hepatocytes. Hepatocytes were isolated with a two-step perfusion method as reported previously (19). Hepatocyte suspensions were added to the precoated dishes at a density of 2 × 106 cells/60-mm dish. Approximately 1 h after the cells were plated, the medium was aspirated and 3 ml fresh DMEM was added. For hepatic transport studies, hepatocytes that had been seeded for 3-5 h without collagen overlay were defined as day 0 or short-term cultured hepatocytes.

To prepare sandwich-cultured hepatocytes, neutralized collagen solution (0.1 ml, 1.5 mg/ml, pH 7.4) was added to the monolayers 24 h after the cells were seeded. Cultures with collagen overlay were incubated for 45 min at 37°C in a humidified incubator to allow the collagen to gel before addition of DMEM. Medium was changed on a daily basis until the fifth day after the cells were seeded. These hepatocytes were referred to as day 5 or long-term cultured hepatocytes.

Immunoblot analysis. To prepare crude plasma membranes, hepatocyte cultures were rinsed once with 3 ml of ice-cold Hanks' balanced salt solution (standard buffer; in mM: 1.3 CaCl2, 0.8 MgSO4, 5.4 KCl, 0.4 KH2PO4, 0.3 Na2HPO4, 4.2 NaHCO3, 136.9 NaCl, and 5.6 D-glucose). Cells were collected by scraping into hypotonic lysis buffer (10 mM Tris · HCl, pH 7.4, 10 mM NaCl, 0.4 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) and incubated in an ice bath for 15 min. The swollen cells were disrupted with 30 strokes in a tightly fitting Dounce homogenizer. The nuclei were removed by centrifugation at 400 g for 10 min at 4°C. The pellet obtained by subsequent centrifugation at 30,000 g for 30 min at 4°C was used as the crude membrane fractions (9).

Proteins from the crude membrane fractions (50 µg) were subjected to 7.5% SDS-PAGE by the method of Laemmli (15). After proteins were transferred electrophoretically from SDS gels to nitrocellulose membranes, the blots were blocked with Tris-buffered saline containing 0.05% Tween 20 and 5% nonfat dry milk for 1 h at room temperature. Rabbit anti-serum raised against the COOH terminus of Mrp2 was used for the primary antibody in the immunoblot analysis. The specificity of this anti-serum has been confirmed previously in vesicles derived from Sf9 cells expressing Mrp2 and isolated canalicular plasma membrane vesicles (data not shown). The blots were probed with the polyclonal anti-Mrp2 rabbit serum at 1:4,000 dilution. Antibody binding was visualized with horseradish peroxidase-conjugated donkey anti-rabbit IgG serum at 1:2,000 dilution, followed by detection with Amersham ECL kit and exposure on Amersham Hyperfilm according to the manufacturer's instructions. The following molecular mass standards were used: 205-kDa myosin, 118-kDa beta -galactosidase, 85-kDa BSA, and 47-kDa ovalbumin (Bio-Rad Laboratories).

Fluorescence microscopy. For the carboxydichlorofluorescein biliary excretion studies, hepatocytes were incubated in standard buffer at 37°C for 10 min. Subsequently, each dish received 3 ml standard buffer containing 1 µg/ml of carboxydichlorofluorescein diacetate. The hepatocytes were incubated at 37°C for 5 min. Fluorescein-labeled taurocholate studies were performed utilizing a similar protocol. After the substrate was loaded, each dish was rinsed four times with 3 ml standard buffer to remove extracellular substrate before viewing with a Leitz Fluovert fluorescence microscope.

Laser scanning confocal microscopy. Fluorescence images were collected with a Bio-Rad (Cambridge, MA) MRC-600 laser scanning confocal microscope equipped with an argon-krypton multiline laser and mounted on a Nikon (Garden City, NY) diaphot inverted microscope. The objective lens was a ×60 numerical aperture 1.4 planapochromat. A pinhole setting of three to four was used to maximize optical sectioning, producing confocal optical sections of 3 µm thickness. Confocal machine settings (gain, black level, and neutral density filters) were set to maximize the dynamic range between background and the more intense canalicular fluorescence. Green fluorescence of fluorescein excited at 488 nm was collected through a 515-nm long-pass barrier filter (fluorescein channel). Red fluorescence of rhodamine-dextran excited at 568 nm was collected through a 585-nm long-pass barrier filter (rhodamine channel) (20). Sandwich-cultured hepatocytes (day 5) were rinsed twice with 3 ml standard buffer and preloaded with carboxydichlorofluorescein by addition of 10 µg of carboxydichlorofluorescein diacetate in 3 ml standard buffer and incubated at 37°C for 10 min. Thereafter, the monolayers were rinsed twice with 3 ml standard buffer to remove extracellular substrate. Rhodamine-dextran (2 mg/ml in standard buffer) was added to the day 5 sandwich-cultured hepatocytes. Images were collected immediately in the fluorescein channel and the rhodamine channel, respectively. Subsequently, the cultures were rinsed twice with 2 ml of Ca2+- and Mg2+-free Hanks' balanced salt solution containing 1 mM EGTA (Ca2+-free buffer), and the monolayers were maintained for 10 min in 2 mg/ml rhodamine-dextran solution prepared in Ca2+-free buffer before the images were collected again in the fluorescein and rhodamine channels, respectively.

Efflux studies in sandwich-cultured hepatocytes. Hepatocytes cultured in a collagen-sandwich configuration were incubated in 3 ml of standard buffer at 37°C for 10 min. Each dish received 3 ml of standard buffer containing 1 µM [3H]taurocholate or 3.6 µM [14C]salicylate, followed by incubation at 37°C for 10 min. Subsequently, the incubation buffer was removed and cultures were washed four times with 3 ml of ice-cold standard buffer to quench the transport processes and remove extracellular substrate. Efflux was initiated by addition of 3 ml of standard buffer or Ca2+-free buffer to each dish. Aliquots of efflux buffer (0.1 ml) were removed at designated times and analyzed by liquid scintillation spectrometry.

Uptake studies in sandwich-cultured hepatocytes. Hepatocytes cultured in a collagen-sandwich configuration were incubated in 3 ml of standard buffer or Ca2+-free buffer at 37°C for 10 min. After the incubation buffer was removed, uptake was initiated by addition of 3 ml of standard buffer containing 1 µM [3H]taurocholate or 0.9 µM [14C]salicylate to each dish. After incubation for designated times, cumulative uptake was terminated by aspirating the incubation solution and rinsing four times with 3 ml of ice-cold standard buffer to remove extracellular substrate. Each rinse lasted 10 s. After washing, 2 ml of 1% Triton X-100 solution were added to culture dishes to lyse cells by shaking the dish on a shaker for 20 min at room temperature. An aliquot (1 ml) of lysate was analyzed by liquid scintillation spectrometry. All values for taurocholate uptake into cell monolayers were corrected for nonspecific binding to the collagen by subtracting taurocholate uptake determined in the appropriate control dishes in the absence of cells as described previously (22).

Protein assay. Bio-Rad DC protein assay kit (Bio-Rad Laboratories) was used to determine the protein concentration in the culture extracts using BSA as standard. Triton X-100 (1%) did not interfere with the assay.

Data analysis. Efflux or uptake data were normalized to the total solubilized protein content and are expressed as means ± SD from three to four separate preparations of hepatocytes. Differences between experimental groups were analyzed by multivariate analysis of variance. A P value of <0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Immunoblot analysis of Mrp2 in hepatocyte membranes. Immunoblot analysis of crude membrane fractions prepared from rat hepatocytes cultured on a gelled substratum or a rigid substratum (conventional condition) for 2-4 h (day 0), probed with antibody raised against the COOH-terminal peptide sequence of rat Mrp2, showed a band with a molecular mass of ~190 kDa (Fig. 1, lanes 1 and 3). Immunoblot analysis of crude membrane fractions isolated from hepatocytes cultured in a collagen-sandwich configuration or under conventional conditions (without the sandwich configuration) for 5 days also showed a band with molecular mass of ~190 kDa (Fig. 1, lanes 2 and 4). An increase in the molecular mass of Mrp2 by ~10 kDa was observed in the day 5 cultured hepatocytes.


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Fig. 1.   Immunoblot analysis of canalicular multispecific organic anion transport protein (Mrp2) in primary cultured rat hepatocytes. Lanes 1 and 2 were loaded with crude membranes isolated from hepatocytes cultured on a gelled substratum for 2-4 h (day 0) and in a sandwich configuration for 5 days, respectively. Lanes 3 and 4 were loaded with crude membranes from hepatocytes cultured on a rigid collagen substratum for 2-4 h and 5 days (conventional condition), respectively (50 µg protein in each lane). Mrp2 was detected at ~190 kDa.

Polarized excretion of carboxydichlorofluorescein in sandwich-cultured hepatocytes. The vectorial excretory activity of sandwich-cultured hepatocytes was examined with the fluorescent Mrp2 substrate, carboxydichlorofluorescein. Immediately after addition of carboxydichlorofluorescein diacetate to hepatocyte monolayers cultured for 4 h (day 0) or 5 days, strong fluorescence was observed in the cell interior. In day 0 hepatocytes, the carboxydichlorofluorescein remained localized predominantly in the cytoplasm of hepatocytes (Fig. 2, A and C). In day 5 sandwich-cultured hepatocytes, the strong intracellular fluorescence translocated rapidly into bile canalicular networks that surrounded each hepatocyte (Fig. 2D). In contrast, hepatocytes cultured under conventional conditions for 5 days failed to show these fluorescent networks (Fig. 2B).


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Fig. 2.   Fluorescence micrographs of hepatocytes cultured on a rigid collagen (A = day 0, B = day 5), on a gelled collagen (C = day 0), or in a sandwich configuration (D = day 5) after pretreatment with carboxydichlorofluorescein diacetate. In day 0 hepatocyte cultures, fluorescence was not concentrated between hepatocytes (A, C). In day 5 hepatocytes cultured in a collagen-sandwich configuration, fluorescence was exclusively localized within the bile canaliculi (D), demonstrating the functional activity of Mrp2 in the canalicular membrane. In day 5 hepatocytes cultured under conventional conditions, fluorescent networks were not observed (B). Bar = 60 µm.

Confocal microscopy was used to demonstrate that the fluorescence in the canalicular networks in Fig. 2D was due to transport of substrate from the cytoplasm into the canalicular lumen, which was separated from the incubation medium by junctional complexes. Carboxydichlorofluorescein, which is actively transported into bile, and rhodamine-dextran, an extracellular space marker, were employed simultaneously in this study to examine canalicular excretion and the integrity of the canalicular lumen in day 5 sandwich-cultured hepatocytes. After incubation in carboxydichlorofluorescein diacetate solution for 10 min, the monolayer was viewed with a laser scanning confocal microscope. In a process of confocal sectioning from the top to the bottom of the monolayer, an image on each focal plane appeared sequentially. In the fluorescein channel, a dark field was observed first; then fluorescent networks appeared (Fig. 3A) and were followed by a dark field. In contrast, in the rhodamine channel, a bright field was observed first and then a dark area appeared (which matched the location of hepatocytes and canalicular spaces viewed under the phase-contrast microscope), which was surrounded by a bright domain (Fig. 3B); subsequently, a bright field was observed. These image planes delineated the extracellular space above the monolayer, the intracellular and bile canalicular spaces across the monolayer, and the extracellular space below the monolayer. The fluorescent images were stable for at least 10 min in standard buffer. In contrast, after incubation in Ca2+-free buffer for 5 min, the fluorescent networks in the fluorescein channel were no longer visible (Fig. 3C) but the fluorescent networks in the rhodamine channel were visible (Fig. 3D).


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Fig. 3.   Effect of Ca2+ modulation on integrity of canalicular networks in hepatocytes cultured in a sandwich configuration. Day 5 sandwich-cultured hepatocytes were preloaded with carboxydichlorofluorescein and maintained in rhodamine-dextran solution in standard buffer. Confocal images were collected with a Bio-Rad MRC-600 confocal microscope in the fluorescein (A) and rhodamine (B) channels. After the monolayer was rinsed with Ca2+-free buffer twice, it was maintained for 5 min in Ca2+-free rhodamine-dextran solution before collection of images in the fluorescein (C) and rhodamine (D) channels. In Ca2+-free buffer, carboxydichlorofluorescein and rhodamine-dextran were able to move into the incubation medium and bile canalicular space, respectively, based on favorable concentration gradients. Bar = 30 µm.

Polarized excretion of taurocholate in sandwich-cultured hepatocytes. The vectorial transport of bile acids was examined with fluorescein-labeled taurocholate, a fluorescent bile acid (Fig. 4). Preliminary studies demonstrated that the fluorescein-labeled taurocholate was stable under the experimental conditions. Hepatocytes cultured for 5 days in a sandwich configuration were incubated with fluorescein-labeled taurocholate for 10 min. Fluorescence was localized predominantly in the canalicular spaces (Fig. 5).


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Fig. 4.   Structure of fluorescein-labeled taurocholate.



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Fig. 5.   Phase-contrast (A) and fluorescence (B) images of hepatocytes cultured in a sandwich configuration for 5 days after pretreatment with 1 µg/ml fluorescein-labeled taurocholate. Fluorescence was localized within the bile canaliculi (arrow), demonstrating the functional activity of the bile acid transporter in the canalicular membrane. Bar = 50 µm.

The efflux and cumulative uptake of [3H]taurocholate in sandwich-cultured hepatocytes with intact and disrupted bile canaliculi were examined to further demonstrate vectorial bile acid transport in day 5 sandwich-cultured hepatocytes. [14C]salicylate was utilized as a marker for simple diffusion because the biliary excretion of salicylate is negligible in vivo (16). After hepatocyte monolayers were preloaded with [3H]taurocholate or [14C]salicylate, substrate efflux was examined by measuring the amount of substrate appearing in standard or Ca2+-free buffer (Fig. 6). In hepatocytes cultured for ~4 h, [3H]taurocholate efflux in standard buffer and Ca2+-free buffer was similar. However, in day 5 sandwich-cultured hepatocytes, [3H]taurocholate efflux in Ca2+-free buffer was significantly higher than that in standard buffer (P < 0.01). In contrast, [14C]salicylate efflux failed to show any significant differences in standard and Ca2+-free buffer (P > 0.05).


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Fig. 6.   Efflux of [3H]taurocholate (A, B) and [14C]salicylate (C, D) in standard buffer (closed symbols) and Ca2+-free buffer (open symbols) from hepatocyte monolayers cultured for ~4 h (A, C) and hepatocytes cultured in a sandwich configuration for 5 days (B, D). Hepatocytes were preincubated for 10 min in standard buffer containing 1 µM [3H]taurocholate or 3.6 µM [14C]salicylate before efflux studies were conducted.

In cumulative uptake studies, sandwich-cultured hepatocytes were preincubated in standard or Ca2+-free buffer for 10 min before the cumulative uptake was conducted (Fig. 7). At day 0 and day 5, [3H]taurocholate uptake at various incubation times was greater in standard buffer than in Ca2+-free buffer (P < 0.05). At day 0, cumulative taurocholate uptake at 10 min in standard buffer was ~10% greater than in Ca2+-free buffer. At day 5, the cumulative uptake in standard buffer was 120% greater than in Ca2+-free buffer. In contrast, no significant differences were observed in the cumulative uptake of [14C]salicylate by the hepatocytes preincubated in standard and Ca2+-free buffer.


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Fig. 7.   Cumulative uptake of [3H]taurocholate (1 µM) (A, B) and [14C]salicylate (0.9 µM) (C, D) in hepatocyte monolayers cultured for ~4 h (A, C) and hepatocytes cultured in a sandwich configuration for 5 days (B, D). Hepatocytes were preincubated for 10 min in standard buffer (closed symbols) or Ca2+-free buffer (open symbols) before cumulative uptake studies were conducted.

Time course of reestablishment of polarized excretory function in sandwich-cultured hepatocytes. To examine the time course of the reestablishment of polarized excretory function in sandwich-cultured hepatocytes, cumulative uptake of the bile acid [3H]taurocholate (1 µM) at 10 min in the hepatocytes preincubated in standard and Ca2+-free buffer was assessed at various times after seeding (Fig. 8A). After the isolated hepatocytes were plated on the gelled collagen substratum, the cumulative uptake of taurocholate in standard buffer was greater than in Ca2+-free buffer and was maintained at similar levels for the first 5 h. Cumulative taurocholate uptake in both standard buffer and Ca2+-free buffer declined over time in culture. By 48 h, the cumulative taurocholate uptake in standard buffer was ~60% of that at 5 h. Between 96 and 120 h, taurocholate cumulative uptake was ~25% of that at 5 h.


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Fig. 8.   Relationship between the polarized excretory function of sandwich-cultured hepatocytes and culture time. A: after cultured hepatocyte monolayers were preincubated in standard buffer (solid bars) or in Ca2+-free buffer (open bars) for 10 min, cumulative uptake of 1 µM [3H]taurocholate was measured in the monolayers at 10 min. B: biliary excretion index of taurocholate, calculated from data shown in A, increased with culture time.

Polarized excretion of substrates may be quantitatively assessed on the basis of the following formula
Biliary excretion index = <FR><NU>uptake<SUB>standard buffer</SUB> − uptake<SUB>Ca<SUP>2+</SUP>-free buffer</SUB></NU><DE>uptake<SUB>standard buffer</SUB></DE></FR> × 100%
where, uptakestandard buffer and uptakeCa2+-free buffer represent the cumulative uptake of substrate at 10 min in the cultured hepatocytes preincubated in standard buffer and Ca2+-free buffer, respectively.

The biliary excretion index of taurocholate in sandwich-cultured hepatocytes at various culture times was calculated (Fig. 8B). In short-term cultured hepatocytes, the biliary excretion index of taurocholate was ~8%. The biliary excretion index of taurocholate increased with culture time to ~60% at 72 h. Thereafter, the biliary excretion index of taurocholate appeared to reach a plateau.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hepatocytes cultured in a conventional configuration (on rigid collagen) dedifferentiate and rapidly lose hepatic transport activity and other liver-specific functions. However, hepatocytes cultured in a collagen-sandwich configuration retain some liver-specific functions and form extensive bile canalicular networks (7, 17-19). The bile canaliculi reestablished in culture have morphological and biochemical characteristics similar to those observed in vivo, including microvilli in the bile canaliculi and localization of specific enzymes in the apical membrane (17, 27). The present investigation represents the first attempt to examine the functional activity of canalicular transporters in long-term cultured hepatocytes.

Recent studies demonstrated that Ntcp was partially maintained in day 5 hepatocytes cultured in a sandwich configuration but not under conventional conditions (22). Unlike Ntcp, Mrp2 was expressed in day 5 hepatocytes cultured under conventional conditions and in a sandwich configuration. The apparent molecular mass of Mrp2 in rat liver was ~190 kDa due to glycosylation (5, 29). Interestingly, the molecular mass in day 5 cultured hepatocytes was ~10 kDa greater than that in day 0 hepatocytes. Trauner et al. (29) noted a similar increase in the molecular mass of Mrp2 in common bile duct-ligated rats and suggested that posttranslational processing of this protein may be altered during cholestasis.

The functional activity of Mrp2 was evaluated by examining the localization of the Mrp2 model substrate carboxydichlorofluorescein (14). Carboxydichlorofluorescein diacetate, which exhibits only weak fluorescence, rapidly penetrates into the hepatocyte plasma membrane and is hydrolyzed readily in the cytoplasm by intracellular esterases to a highly fluorescent product, carboxydichlorofluorescein (12). Carboxydichlorofluorescein is a high-affinity substrate for Mrp2 (14) and is rapidly transported across the canalicular membrane into bile. In short-term cultured hepatocytes, significant fluorescence was retained intracellularly; in contrast, in day 5 sandwich-cultured hepatocytes, negligible fluorescence was observed in the cytoplasm (Fig. 2, C and D, respectively). These results suggest that the functional activity of Mrp2 is lower in short-term cultured hepatocytes compared with long-term cultured hepatocytes, probably due to endocytosis of the membrane protein after isolation and a decrease in Mrp2 on the plasma membrane (10). In short-term cultured hepatocytes, fluorescence is concentrated between some of the hepatocytes, suggesting that the residual bile canaliculi have sealed and the hepatocytes have maintained some functional activity of Mrp2. These observations are in agreement with studies in hepatocyte couplets demonstrating that sealed canalicular spaces form ~3-5 h after seeding (10). However, the fluorescent carboxydichlorofluorescein did not accumulate in the spaces between the majority of short-term cultured hepatocytes, suggesting that the residual bile canaliculi may not be sealed completely or that Mrp2 is not functionally active in most of the short-term cultured hepatocytes. In day 5, sandwich-cultured hepatocytes, the fluorescent marker accumulated throughout the canalicular spaces, demonstrating the functional activity of Mrp2 on the canalicular membrane and functional integrity of the bile canalicular networks. In contrast, hepatocytes cultured under conventional conditions for 5 days failed to show these fluorescent networks.

In short-term cultured hepatocyte couplets, the canalicular lumen is sealed by a tight junction complex (10). LeCluyse et al. (17) demonstrated that the canalicular space in hepatocytes cultured in a sandwich configuration for 6 days is sealed by tight junction-like structures. Recently, Talamini et al. (27) demonstrated the existence of a junctional protein, uvomorulin (E-cadherin), in the hepatocytes cultured in a sandwich configuration. In the present study, the functional integrity of the canalicular networks was examined with two fluorescent markers, carboxydichlorofluorescein and rhodamine-dextran. The fluorescence intensity of the networks due to carboxydichlorofluorescein decreased markedly in the first minute after exposure of the hepatocytes to Ca2+-free buffer; fluorescence was not visible at the end of 5 min (Fig. 3C). Moreover, rhodamine-dextran, an extracellular marker, was unable to penetrate into the bile canaliculi in standard buffer. However, in Ca2+-free buffer, rhodamine-dextran readily diffused into the canalicular lumen. These studies indicated that in sandwich-cultured hepatocytes, the junctional complexes are an impermeable barrier between the canalicular lumen and the incubation buffer. This barrier can be disrupted within a few minutes by depletion of Ca2+ in the incubation medium. These results are consistent with the observation that the barrier function of tight junctions can be disrupted within a few minutes by exposure of Madin-Darby canine kidney cells to Ca2+-free buffer (6).

Fluorescent microscopy studies with a fluorescent bile acid, fluorescein-labeled taurocholate, demonstrated that this bile acid was taken up by hepatocytes and excreted extensively into the canaliculi of hepatocytes cultured in a sandwich configuration for 5 days. This observation indicated that the polarized transport systems for bile acids were reestablished in the long-term cultured hepatocytes. Furthermore, two experimental approaches were undertaken to evaluate the biliary excretion of nonfluorescent substrates in the cultured hepatocytes. These two methods are based on the observations that, in standard buffer, the integrity of the bile canalicular networks remains intact; in Ca2+-free buffer, the integrity of the canalicular space is disrupted, causing leakage of the canalicular contents.

In the efflux method, substrate was first loaded into hepatocytes followed by measurement of substrate efflux in standard buffer or in Ca2+-free buffer. Theoretically, when a cholephilic compound is taken up into sandwich-cultured hepatocytes, it should be excreted into the canalicular space if the transporter facilitating biliary excretion of this substrate remains functionally active. The efflux rate of a cholephilic compound should be higher in Ca2+-free buffer compared with standard buffer due to disruption of the tight junctions and leakage of substrate from the canalicular space where it has accumulated during substrate loading. In contrast, the efflux rate of a noncholephilic compound should be similar regardless of the extracellular Ca2+ concentrations in the efflux buffer because negligible amounts of substrate have accumulated in the canalicular space. In the present investigation, the canalicular transport function of bile acids was examined with taurocholate, a cholephilic compound. Salicylate, a noncholephilic compound, was utilized as a negative control. At each time point in day 5 sandwich-cultured hepatocytes, the efflux of [3H]taurocholate was greater in the Ca2+-free buffer compared with standard buffer. In contrast, efflux of [14C]salicylate was not significantly different in Ca2+-free and standard buffer in both day 0 and day 5 cultured hepatocytes. As expected, the efflux of [3H]taurocholate and [14C]salicylate in short-term cultured hepatocytes in Ca2+-free and standard buffer was not significantly different because extensive sealed canalicular networks had not formed.

The second experimental approach was to measure cumulative uptake of substrate. Cultured hepatocytes were preincubated in standard buffer or Ca2+-free buffer for 10 min. Subsequently, cultures were incubated in standard buffer with substrate for designated times and cumulative uptake of substrate in the cultures was quantitated. During uptake studies, substrate excreted into the intact bile canalicular networks will be stored there, whereas substrate excreted across the canalicular membrane when the integrity of the canalicular spaces has been disrupted will diffuse back into the incubation medium. Therefore, if a substrate can be taken up by the hepatocytes and excreted into the canaliculi, its apparent cumulative uptake should be greater in the hepatocytes pretreated in standard buffer than in the hepatocytes pretreated in Ca2+-free buffer. If a substrate cannot be excreted into the canaliculi, the cumulative uptake of substrate should be identical between the two treatments. The cumulative uptake of [3H]taurocholate was significantly higher in standard buffer relative to Ca2+-free buffer in day 5 sandwich-cultured hepatocytes. As a negative control, [14C]salicylate cumulative uptake in Ca2+-free and standard buffer did not show significant differences in either short- or long-term cultured hepatocytes. Interestingly, the cumulative uptake of [3H]taurocholate was ~10% greater in standard buffer compared with that in Ca2+-free buffer in short-term cultured hepatocytes. This may be due to the existence of hepatocyte couplets containing sealed canalicular lumens (10).

The differences in substrate efflux or cumulative uptake in standard and Ca2+-free buffer represent the extent of biliary excretion of substrate, assuming that Ca2+ depletion does not alter the hepatobiliary transport of the substrate other than by disruption of the junctional complexes. This assumption appears to be valid because the uptake of bile acids, such as cholate, has been reported to be independent of extracellular Ca2+ concentrations in freshly prepared hepatocyte suspensions (25). Similar results were obtained with taurocholate (data not shown). Furthermore, [14C]salicylate was employed as a simple diffusion marker because it is metabolized extensively by the liver and eliminated as metabolites exclusively in urine (16). Salicylate failed to show any significant difference in all of the treatments, suggesting that modulation of Ca2+ concentrations does not alter the diffusional properties of the plasma membrane.

Assessment of biliary excretory function should consider the fact that substrate uptake declines over time in sandwich-cultured hepatocytes. Polarized excretory function may be quantitatively assessed utilizing the biliary excretion index. In sandwich-cultured hepatocytes, the cumulative uptake of substrate in standard buffer represents the amount of substrate localized in the cytoplasm and bile canalicular networks; the cumulative uptake of substrate in Ca2+-free buffer represents the amount of substrate localized intracellularly. Therefore, the biliary excretion index represents the percentage of retained substrate in the monolayer that is localized in bile canaliculi. Theoretically, a large biliary excretion index indicates extensive polarized excretion of substrate. The biliary excretion index increased with time in culture for up to 4-5 days, suggesting that the functional polarity of sandwich-cultured hepatocytes was gradually established.

Boyer and Soroka (4) demonstrated that ~6% of cholylglycylamido fluorescein, a fluorescent bile acid, was secreted into the bile canalicular lumen of short-term cultured hepatocyte couplets with the use of image analysis and a similar calculation. The biliary excretion index of taurocholate measured in short-term cultured hepatocytes in the present studies was ~8%, presumably due to the existence of hepatocyte couplets with polarized excretory function. The biliary excretion index of taurocholate in long-term cultured hepatocytes was more than sixfold greater than in short-term cultured hepatocytes, demonstrating the reestablishment of polarized excretory function. These results are consistent with the previous observation that cultured hepatocytes develop morphological and functional polarity in 4-5 days (27).

In addition to utilizing the biliary excretion index to evaluate the functional polarity of cultured hepatocytes, measurement of the biliary excretion index for a given substrate may provide a novel approach to predict the hepatobiliary disposition of that substrate in vivo. For example, if the biliary excretion index of a substrate is as high as taurocholate, this substrate may be excreted extensively into bile in vivo. However, if the biliary excretion index of a substrate is much less than taurocholate, this substrate may not be secreted as extensively into bile in vivo, or the hepatobiliary transport system(s) for this substrate may not be maintained in the sandwich-cultured hepatocytes. Furthermore, the biliary excretion index may also be useful in predicting possible substrate interactions relevant to hepatobiliary disposition.

In summary, primary cultures of rat hepatocytes maintained in a collagen-sandwich configuration for up to 5 days establish intact canalicular networks, maintain Mrp2, reestablish polarized excretory function, and appear to express a more normal phenotype compared with conventional cultures. The taurocholate biliary excretion index, a measure of the relative amount of substrate in the bile lumen, may be a useful indicator of polarized excretory function and the relative extent of biliary excretion of a substrate.


    ACKNOWLEDGEMENTS

We thank Ting Qian for technical assistance with the confocal microscopy studies.


    FOOTNOTES

This work was supported by National Institute of General Medical Sciences Grant GM-41935.

This work was presented in part at the 47th Annual Meeting of the American Association for the Study of Liver Disease, November 8-12, 1996, Chicago, IL.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: K. L. R. Brouwer, Division of Drug Delivery and Disposition, School of Pharmacy, CB 7360, Beard Hall, Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7360 (E-mail: kbrouwer{at}unc.edu).

Received 5 May 1998; accepted in final form 29 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastroint Liver Physiol 277(1):G12-G21
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