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Departments of 1 Internal Medicine and 2 Medical Biochemistry and Genetics, 3 Division of Research and Education, 4 Medical Physiology, Scott and White Hospital and Texas A&M University System, Health Science Center, College of Medicine and 5 Central Texas Veterans Health Care System, Temple 76504; 6 Department of Gastroenterology, University of Ancona, Ancona, Italy; and 7 University of Texas, Houston, Texas 77030
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We sought to develop a cholangiocyte cell
culture system that has preservation of receptors, transporters, and
channels involved in secretin-induced secretion. Isolated bile duct
fragments, obtained by enzyme perfusion of normal rat liver, were
seeded on collagen and maintained in culture up to 18 wk. Cholangiocyte
purity was assessed by staining for 
-glutamyl transpeptidase
(-GT) and cytokeratin-19 (CK-19). We determined gene expression for
secretin receptor (SR), cystic fibrosis transmembrane conductance
regulator, Cl
/HCO
/HCO3
exchanger activity, secretin-stimulated Cl efflux, and
apical membrane-directed secretion in polarized cells grown on tissue
culture inserts. Cultured cholangiocytes were all -GT and CK-19
positive. The cells expressed SR and
Cl/HCO/HCO efflux were similar to freshly isolated
cholangiocytes. Forskolin (104 M) induced fluid
accumulation in the apical chamber of tissue culture inserts. In
conclusion, we have developed a novel cholangiocyte line that has
persistent HCO, and fluid
transport functions. This cell system should be useful to investigators
who study cholangiocyte secretion.
bicarbonate secretion; bile flow; intrahepatic bile ducts; adenosine 3',5'-cyclic monophosphate; secretin receptor
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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THE PRIMARY FUNCTION OF
CHOLANGIOCYTES lining the intrahepatic bile ducts is to secrete a
bicarbonate-rich bile in response to the hormone secretin (20,
39). Isolation of cholangiocytes or isolated intrahepatic bile
duct units (IBDU) fragments from rats or mice has been used
successfully by multiple investigators, but these techniques are
cumbersome, due to high expense and low yield of cells (3, 9, 18,
19, 24, 30, 34, 38). Furthermore, in vitro studies (1, 5,
13, 28) with freshly isolated cells or IBDU fragments are
limited by the lack of long-term (>24 h) viability of these cell
preparations. Cholangiocarcinoma cell lines have been also employed
successfully, but they suffer from the potential of undesired results
due to study of undifferentiated cells (29, 31, 33, 35).
Other investigators (1, 6, 7, 15) have successfully
isolated and cultured normal rat intrahepatic bile duct cells, but
secretory function characteristic of cholangiocytes (e.g.,
secretin-stimulated cAMP synthesis,
Cl/HCO/HCO
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Materials.
Reagents were purchased from Sigma (St. Louis, MO) unless otherwise
indicated. Porcine secretin was purchased from Peninsula (Belmont, CA).
The substrate for -glutamyltranspeptidase [-GT; N-(-L-glutamyl)-4-methoxy-2-naphthylamide]
was purchased form Polysciences (Warrington, PA).
Isolation of cholangiocytes for primary culture. IBDU fragments were obtained from male 344 Fisher rats (Charles River Laboratories, Wilmington, PA), as we previously reported (1). Rats were anesthetized with pentobarbital (50 mg/kg body wt) and the portal vein was cannulated and preperfused at 37°C with 250 ml of oxygenated buffer A [(in mM) 140 sodium chloride, 5.4 potassium chloride, 0.8 sodium phosphate, 25 HEPES, 0.5 EGTA tetrasodium salt]. The liver was removed, and perfusion was continued for 10 min at 37°C with 150 ml of buffer B (in mM: 140 sodium chloride, 5.4 potassium chloride, 0.8 sodium phosphate, 25 HEPES, 0.8 magnesium sulfate, 3 calcium chloride) containing 0.02% type 2 collagenase (Worthingham Biochemical, Freehold, NJ). These were allowed to settle on a petri dish and then, under phase contrast microscope (using a ×10 objective), IBDU fragments (70-100 µm in diameter) were removed by using a sterile pipette. IBDU fragments were suspended in rat tail collagen, which was allowed to solidify and was then incubated at 37°C with DMEM supplemented with (in µg/ml): 4 forskolin, 3.4 3,3',5-triiodo-L-thyromine, 0.4 dexamthasone, 5 gentamicin, and 50 trypsin inhibitor, plus 5% NuSerum IV, 5% FBS, 25 ng/ml EGF, 20 mM L-glutamine, 1% glyceryl monostearate, and 0.1 mM MEM nonessential amino acid solution. After 2 wk, the IBDU fragments, which had transformed into cystic structures were passaged. After dissolution of collagen, cystic bile duct fragments were washed in Ca2+-free HEPES-buffered saline, and were then washed with 1 ml of trypsin (0.25%) for 5 min followed by the addition of 10% FCS and 0.5 mg/ml soybean trypsin inhibitor in DMEM. After 10 min, dispersed cholangiocytes were allowed to settle, resuspended in growth medium, and plated onto collagen-coated, 60-mm plastic dishes, and maintained at 37°C in a humidified 5% CO2 incubator. Growth media were changed every 2-3 days. After 5-10 passages, the cholangiocytes began to proliferate to form a complete monolayer, and the contaminating fibroblasts disappeared. At that point, cells were seeded onto collagen-coated tissue culture inserts (1-cm diameter with 8-µm pore size; Transwell, Costar, Cambridge, MA) for morphological evaluation of polarity and physiological studies. Transepithelial resistance across the epithelial monolayer in the inserts was determined with an epithelial volt-ohm meter (World Precision Instruments, Sarasota, Florida).
Immunohistochemistry. Immunohistochemistry for cytokeratin-19 (CK-19) and vimentin was performed as previously described by us (21).
Transmission electron microscopy. Cell monolayers were fixed with 0.8% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 1 h at room temperature. Fixative was gently removed and the cells were rinsed in 0.1 M cacodylate buffer and incubated overnight in the same buffer containing 0.25 M sucrose. Fixation with 1% osmic acid in cacodylate buffer was carried out for 1 h and the cells were embedded in a mixture of Epon/Araldite after dehydration in alcohol and propylene oxide. Ultrathin sections were examined with an electron microscope (model 201; Philips, Eindoven, The Netherlands).
Molecular analysis.
Expression of selected genes [albumin, -GT, CK-19, SR, cystic
fibrosis transmembrane conductance regulator (CFTR), and
Cl/HCO
-32P]UTP (800 Ci/mmol; Amersham,
Arlington Heights, IL). The stability of gene expression was determined
by comparison of RNase protection assays at passages
9, 13, and 25.
-GT was transcribed from rat
pGEM4Z-GT (M-N Chobert, Crétel, France); a 350-bp riboprobe encoding for the message of the rat CK-19 gene was generated from pBlueScript CK-19 (a gift from A. Quaroni, Ithaca, NY); a probe 316-bases long, encoding sequences complementary to rat GAPDH mRNA, was
obtained from Ambion; a 318-bp riboprobe encoding the message for SR
was transcribed from pGEM4Z-SR (a gift of Dr. N. F. LaRusso, Mayo
Clinic, Rochester, MN); and a 348-bp riboprobe encoding the message for
Cl/HCO/HCOIntracellular cAMP levels.
Spontaneous and secretin-stimulated intracellular cAMP levels in
cultured cholangiocytes were determined as previously described (7). Cultured cholangiocytes were stimulated with 0.2%
BSA (basal) or secretin (107 M) in O.2% BSA for 5 min at
room temperature. After extraction with ethanol, cAMP levels were
determined by a commercially available kit (Amersham) according to the
instructions of the manufacturer. Intracellular cAMP levels were
expressed as fentomoles per 100,000 cells.
Secretory activity of cultured cholangiocytes.
Secretory activity was measured by 1)
Cl/HCO efflux and influx,
and 3) accumulation of fluid secreted across the apical
membrane in cultured cholangiocytes obtained from passages 10-20. The Cl/HCO7 M) with 0.2% albumin for 5 min. In some studies, cultured cholangiocytes were pretreated
with 1 mM DIDS for 30 min before studies, which has previously been
shown to inhibit cholangiocyte Cl/HCO/HCO
permeability of apical membranes of confluent
cholangiocytes monolayers cultured on tissue culture inserts was
estimated by the measurement of intracellular
N-ethoxycarbonylmethyl-6-methoxyquinolinium bromide (MQAE)
fluorescence. MQAE, with its high Cl sensitivity, has
been used successfully to measure intracellular Cl
concentration ([Cl]i) in various cell types
(14, 25). Cells were loaded with 1 mM MQAE in a solution
containing (in mM) 101 Cl, 5 HEPES, 0.8 MgSO4, 1.0 NaH2PO4, 5.6 glucose, 1.8 Ca
acetate, 96 NaCl, 5.3 KCl, 50 mannitol, and 22 NaHCO3, plus
10% NCS at pH 7.4 for 2 h at 37°C. The cells were then washed
three times with the same solution (without MQAE) to remove MQAE and
the serum, and were then left for 10 min before measurements were
started. MQAE fluorescence intensity was measured by using excitation
and emission wavelengths of 360 and 460 nm, respectively.
Cl quenches MQAE in its excited state. Changes in MQAE
fluorescence intensity, therefore, inversely reflect changes in
[Cl]i. At the end of an experiment, the
monolayer was perfused with KSCN (120 mM) solution (buffered with 10 mM
HEPES-KOH, pH 7.2), which quenched MQAE fluorescence by >90%
(14, 25). For data analysis, fluorescence (F) at each time
interval was divided by the KSCN-quenched F value (F0). We
quantitatively compared the effects of secretin (107 M)
or BSA (control) on the rate of net apical Cl efflux and
influx. Relative rates of Cl influx and efflux were
computed from the time course of intracellular fluorescence and were
expressed as relative change in fluorescence by using the equation:
(
F/dt)/F0 · min1,
where F/dt is the initial rate of fluorescence change on
the addition or removal of Cl. Efflux or influx of
Cl across the apical membrane was assessed by the removal
and addition of Cl to the apical solutions, respectively.
The cell monolayer was first perfused with NaCl solution in both apical
and basolateral compartments. While MQAE fluorescence of the monolayer
was recorded, the apical Cl was replaced by equal molar
gluconate solution and 5 min later was then replaced by an NaCl
solution. In some studies (8), cultured cholangiocytes
were pretreated for 20 min with 10 µM 5-nitro-2-(3-phenylpropylamino)
benzoic acid (NPPB), which has been shown to block Cl
channels in cholangiocytes.
Fluid secretion was measured in cultured monolayers by two independent
techniques. In the first technique, the diameter of closed spaces
between cholangiocytes cultured in monolayer was observed under a phase
contrast microscope before and at 5-min intervals after the addition of
either forskolin 104 M in 0.2% albumin or 0.2% albumin
control. The change in volume of closed space was calculated as we
(1) previously described in IBDU units. Fluid excretion
was also determined in monolayers of cholangiocytes cultured on tissue
culture inserts as previously described for choroid plexus epithelial
cells (16). Once the cells had reached confluence, the
cholangiocytes on cell inserts were stimulated with either forskolin
(104 M) in 0.2% albumin or albumin control for 1-24
h. Increased weight was attributed to fluid secretion across the
cholangiocyte apical membrane into the chamber above the cells.
Secretion was quantified from the increase in weight of the insert and,
assuming a density of the fluid as 1 µl/mg, secreted fluid volume was determined.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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After 4 days, the IBDU fragments developed into cystic structures.
Two weeks later, IBDU fragments were passed onto collagen cell culture
plates. After 5-10 passages, the cholangiocytes began to
proliferate to form a complete monolayer. At this point, the cells
appeared morphologically homogeneous under phase contrast microscopy
(Fig. 1) and absence of contaminating
fibroblasts was also observed. The purity of cholangiocytes was
assessed by immunohistochemistry for CK-19, a cholangiocyte-specific
marker (27). One hundred percent of the cultured cells
were CK-19-positive (Fig. 2), which is
consistent with a cholangiocyte origin. In addition, the cultured cholangiocyte failed to stain for vimentin (Fig. 2), a marker for
Kupffer cells (26).
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Cholangiocytes, when seeded on collagen-coated cell inserts,
proliferated to confluency over a 1-wk period. At confluence, the
transepithelial resistance was 634 ± 124 
· cm2. The ultrastructural
morphological evaluation of cultured cholangiocytes grown on membranes
show polarized epithelial cells with surface microvilli and nuclei
closely adjacent to the membrane (Fig.
3). Golgi and submembrane vesicles were
closely adjacent to the membrane containing microvilli (Fig. 2).
Morphological features are consistent with polarized epithelial
structure, with the cholangiocyte basolateral membrane adjacent to the
culture membrane and the apical membrane opposite the culture membrane.
The morphological features were very similar to cholangiocytes observed
in situ.
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SR gene expression and secretin-stimulated cAMP synthesis.
The characteristic feature of cholangiocytes compared with the
remainder of cells in the liver is the presence of SR, CFTR, Cl/HCO/HCO-GT and CK-19 (two cholangiocyte-specific
markers) and GAPDH (the housekeeping gene) but were negative for
albumin mRNA (a marker for hepatocytes) (Fig. 4). Basal cAMP levels of
cultured cholangiocytes were similar to that previously reported
(5) in freshly isolated cholangiocytes (Fig.
5). When stimulated with secretin
(107 M for 10 min), cAMP levels in cultured
cholangiocytes increased by almost fivefold (Fig. 5). This increase is
similar in magnitude to the secretin-stimulated cAMP synthesis we
(5) previously observed in normal pooled or large
cholangiocytes isolated from rat liver.
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Cl/HCO
/HCO/HCO7 M secretin for 10 min, the rate of alkalization in secretin-stimulated cholangiocytes is
significantly greater than unstimulated cholangiocytes (0.43 ± 0.08 vs. 0.18 ± 0.04 pH U/min, P < 0.05).
Similarly, the total magnitude of the increase in intracellular pH was
greater in secretin-stimulated cholangiocytes compared with controls
(0.44 ± 0.09 vs. 0.19 ± 0.05 pH units, P < 0.05). Pretreatment with 1 mM DIDS inhibited (P < 0.05) the rate of alkalization in unstimulated and secretin-stimulated
cholangiocytes (0.03 ± 0.08 and 0.05 ± 0.04 pH U/min,
respectively). Data show the presence of a functioning secretin-stimulated Cl/HCO
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Secretin-stimulated chloride channel activity.
Chloride channel activity appears to be required for the generation of
ductal secretion by cholangiocytes (32). Chloride influx
and efflux across the apical membrane in cholangiocytes monolayer in
cell culture inserts were assessed by measuring the rate of change of
MQAE fluorescence during the removal and addition of chloride to the
upper (apical) chamber. In cholangiocytes pretreated with secretin
(107 M) for 5 min (compared to cholangiocytes treated
with BSA control), there was an accelerated rate of increase of
F/F0 after the removal of chloride and an accelerated rate
of decreasing F/F0 after the restitution of chloride (Fig.
7). Data indicate that secretin increases
the rate of both chloride influx and efflux across the apical membrane
of cholangiocytes. In seven experiments, the rate of apical chloride
efflux was 0.08 ± 0.02 vs. 0.02 ± 0.01 fluorescence arbitrary units/min in secretin and BSA treated cholangiocytes, respectively (P < 0.05). Similarly, the rate of apical
chloride influx was 0.07 ± 0.02 vs. 0.03 ± 0.01 fluorescence arbitrary units/min in secretin and BSA-treated
cholangiocytes, respectively (P < 0.05). Pretreatment
with the chloride channel inhibitor NPPB (10 µM) ablated the
secretin-stimulated cholangiocyte apical membrane chloride efflux
(NPPD; 0.02 ± 0.01 vs. control, 0.08 ± 0.02 arbitrary fluorescence units/min, P < 0.05) and the
secretin-stimulated cholangiocyte apical membrane influx (NPPD;
0.02 ± 0.01 vs. control, 0.07 ± 0.02 arbitrary fluorescence
units/min, P < 0.05).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Cholangiocytes are an important component of the liver, because
they are major contributors to bile secretion (
40% of the total bile
flow in man and 10% in rats) (11). Cholangiocytes are the
primary targets for specific liver diseases, such as primary biliary
cirrhosis, primary sclerosing cholangitis, and rejection after liver
transplantation (4). Understanding of the function and
pathology of cholangiocytes has significantly increased in the last 15 yr, primarily due to the ability of investigators to isolate pure
populations of cholangiocytes from experimental animals (2, 6,
13, 36). Further achievements have been partially impeded due to
the difficulty and high expense to isolate cholangiocytes. Although
other investigators (40) have previously established pure
cultured cholangiocytes (referred to as normal rat
cholangiocytes), the previous culture cholangiocyte systems have not been characterized by responses to secretin-induced secretion as we did in these studies. The present study expands the previous studies on cholangiocyte culture systems by the development of a new
cultured system that expresses SR, CFTR, and secretin-stimulated cAMP
synthesis, Cl/HCO
The primary culture of cholangiocytes utilized bile duct fragments
isolated from liver by enzymatic digestion. It is likely that
initiation of proliferating cholangiocytes in cell culture systems
requires coculture with fibroblast (and/or other cells types) within
the portal tract, because we have not had success in primary cultures
of isolated cholangiocytes (data not shown). Similar to previous
studies (40), noncholangiocyte cell populations are
eliminated with successive passages. Cell purity was demonstrated, by
using positive staining for CK-19, a cholangiocyte-specific marker and
negative staining for vimentin and albumin. The latter would exclude
even a small contamination from hepatocytes. In our cultured
cholangiocyte cell system, SR, CFTR, and
Cl/HCO/HCO/HCO
Secretin stimulation of cultured cholangiocytes secretion was
demonstrated by multiple techniques and was found to be similar to that
previously observed in freshly isolated cholangiocytes (5,
7). Each technique, however, evaluated a unique portion of
cholangiocyte secretory function. It is currently believed that
multiple transporters are responsible for hormone-stimulated ductal
secretion (11, 17). The initial event of secretin
stimulation of cholangiocyte secretion is increased intracellular cAMP
synthesis (11, 17). SR expression, and increased
secretin-stimulated intracellular cAMP levels in our cholangiocyte
cultured system are quite similar to freshly isolated cholangiocytes
(2). Increased cAMP is thought to activate apical chloride
channels and Cl/HCO/HCO/HCO/HCO/HCO/HCO
In summary, this study shows that intrahepatic cholangiocytes derived
from large intrahepatic bile duct fragments isolated from normal rat
liver can be successfully adapted to in vitro growth. The studies, for
the first time, establish a cholangiocyte cell culture system that
maintains all key elements of ductal secretion (SR expression,
secretin-stimulated cAMP, chloride channel activity,
Cl/HCO
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ACKNOWLEDGEMENTS |
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This work was supported by a grant award from Scott and White Hospital and Texas A&M University (to G. Alpini and G. LeSage); by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-54208 (to G. LeSage) and DK-958411 (to G. Alpini); a Veterans Affairs Merit Award (to G. Alpini); and by Grant MURST MM06215421 (to the Department of Gastroenterology, University of Ancona).
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. LeSage, Professor of Medicine, University of Texas Houston Medical School, 6431 Fannin St., MSB 4.234, Houston, TX 77030 (E-mail: gene.lesage{at}uth.tmc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 22, 2003;10.1152/ajpgi.00260.2002
Received 2 July 2002; accepted in final form 13 January 2003.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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