1 Department of Medical Physiology and Internal Medicine, Scott & White Hospital and Texas A&M University Health Science Center College of Medicine and Central Texas Veterans Health Care System, Temple, Texas 76504; and 2 Center for Basic Research in Digestive Diseases, Division of Gastroenterology and Hepatology, Departments of Internal Medicine and of Biochemistry and Molecular Biology, Mayo Medical School, Clinic, and Foundation, Rochester, Minnesota 55905
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ABSTRACT |
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We previously introduced the concept that intrahepatic bile duct epithelial cells, or cholangiocytes, are functionally heterogeneous. This concept is based on the observation that secretin receptor (SR) gene expression and secretin-induced cAMP synthesis are present in cholangiocytes derived from large (>15 µm in diameter) but not small (<15 µm in diameter) bile ducts. In work reported here, we tested the hypothesis that cholangiocytes are heterogeneous with regard to proliferative capacity. We assessed cholangiocyte proliferation in vivo by measurement of [3H]thymidine incorporation and in vitro by both [3H]thymidine incorporation and H3 histone gene expression in small ( fraction 1) and large ( fraction 2) cholangiocytes isolated from rats after bile duct ligation (BDL). In the two cholangiocyte subpopulations, we also studied basal somatostatin receptor (SSTR2) gene expression as well as the effects of somatostatin on 1) SR gene expression and secretin-induced cAMP synthesis and 2) [3H]thymidine incorporation and H3 histone gene expression. In normal rat liver, cholangiocytes, unlike hepatocytes, were mitotically dormant; after BDL, incorporation of [3H]thymidine markedly increased in cholangiocytes but not hepatocytes. When subpopulations of cholangiocytes were isolated after BDL, DNA synthesis assessed by both techniques was limited to large cholangiocytes, as was SSTR2 steady-state gene expression. In vitro, somatostatin inhibited SR gene expression and secretin-induced cAMP synthesis only in large cholangiocytes. Moreover, compared with no hormone, somatostatin inhibited DNA synthesis solely in large cholangiocytes. These results support the concept of the heterogeneity of cholangiocytes along the biliary tree, extend this concept to cholangiocyte proliferative activity, and imply that the proliferative compartment of cholangiocytes after BDL is located principally in the cholangiocytes lining large (>15 µm) bile ducts.
secretin; somatostatin; adenosine 3',5'-cyclic monophosphate; [3H]thymidine incorporation; H3 histone
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INTRODUCTION |
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INTRAHEPATIC BILE DUCT epithelial cells, or cholangiocytes, are simple epithelia that line the intrahepatic biliary tree, a complex three-dimensional network of tubular conduits of different diameters within the liver (8). A number of studies (2-5, 7-12, 14, 18, 19, 28, 35) have demonstrated that cholangiocytes modify bile of canalicular origin by a coordinated series of hormone-regulated secretory and absorptive processes. For example, secretin stimulates ductal bile secretion by interacting with specific receptors [present in the liver only on cholangiocytes (11)] through increases in the cAMP second messenger system (2, 4, 9, 10, 14, 18, 19, 35). In addition to secretin (2, 4, 9, 10, 14, 18, 19, 35), other gastrointestinal hormones, including gastrin and somatostatin (see below), have the capacity to regulate ductal absorptive and secretory activity (14, 27, 35).
Under basal conditions, cholangiocytes are thought to be mitotically
dormant (2, 5, 19). However, cholangiocytes proliferate markedly in
response to a number of perturbations including bile duct ligation
(BDL), -naphthylisothiocyanate feeding, and 70% hepatectomy (5-8, 19, 35). Not surprisingly, this proliferative response is accompanied by functional changes (5-8, 10, 19, 35).
For example, cholangiocyte proliferation after BDL or 70% hepatectomy
is coupled with an increase in secretin receptor (SR) gene expression
(10, 11, 19) and secretin-induced cAMP synthesis (11, 19, 35) and is
associated with a secretin-stimulated, bicarbonate-rich choleresis (5,
7, 14, 19, 35).
In several series of experiments, we have previously demonstrated that cholangiocytes lining the intrahepatic biliary tree of normal rats are heterogeneous. These studies have employed both in vivo quantitative morphological techniques (3, 9) and in vitro functional assays using distinct subpopulations of cholangiocytes (9, 10) and intrahepatic bile duct fragments (3, 4) of different sizes. Indeed, we recently reported a technique for isolating subpopulations of cholangiocytes of different sizes from normal rat liver and showed (9) that both SR gene expression and secretin-induced increases in intracellular cAMP levels are differentially distributed among cholangiocyte subpopulations derived from different portions of the rat biliary tree (4, 9). Moreover, we demonstrated that the increased ductal secretory activity observed after BDL reflects transport processes restricted to selected cholangiocyte subpopulations (3, 10). Thus the morphological and functional heterogeneity of the epithelia lining the intrahepatic biliary tree has become a well-accepted concept (2-4, 9, 10).
Somatostatin, a cyclic tetradecapeptide, was first isolated from rat hypothalamus where it was shown to inhibit the secretion of growth hormone (37). Somatostatin is also inhibitory in a variety of other organs (25, 30); for example, it decreases secretory processes in the intestine (30) and pancreas (25) and inhibits growth of a variety of epithelial cells (32, 34, 36). In the liver, studies by us (35) and Ricci and Fevery (27) using in vivo models have shown that somatostatin is cholestatic and inhibits both basal and secretin-stimulated ductal bile secretion. Furthermore, we have shown that somatostatin inhibits both basal and secretin-induced exocytosis and secretin-stimulated increases in cAMP levels in isolated, highly purified cholangiocytes (35). Somatostatin is also antiproliferative in at least two circumstances: 1) it inhibits cholangiocyte proliferation in rats in response to BDL (36), and 2) we have recently shown that somatostatin prevents the growth of human cholangiocarcinoma cells in vitro and in vivo (32). Taken together, these observations strongly suggest that the cholangiocyte is the major target for somatostatin in the liver. All of these inhibitory actions of somatostatin occur through interaction with one of its receptor subtypes (i.e., SSTR2). The specific portion of the intrahepatic biliary tree where SSTR2 resides and where somatostatin exerts its effects on ductal secretory activity and on the proliferation of cholangiocytes is unknown. Indeed, no information currently exists as to whether or not cholangiocytes are heterogeneous with regard to their proliferative capacity (i.e., is there a specific proliferative compartment within the intrahepatic biliary tree).
To begin to explore these issues, we isolated from BDL rat liver two
subpopulations of cholangiocytes of different sizes, i.e.,
fraction 1 or small cholangiocytes [~8 µm in size
and originating from ducts <15 µm in diameter (3, 9, 10)] and
fraction 2 or large cholangiocytes [~14 µm in size
and originating from ducts >15 µm in diameter (3, 9, 10)],
and assessed proliferative capacity of the two subpopulations by
measurement of both
[3H]thymidine
incorporation and H3 histone gene
expression. In the two subpopulations, we also studied in vitro basal
SSTR2 gene expression and the effects of somatostatin
(107 M) on SR gene
expression, secretin-induced cAMP synthesis, and cholangiocyte
proliferation.
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MATERIALS AND METHODS |
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Animal Model
We used male Fischer 344 rats (200-225 g) from Charles River (Wilmington, MA). The animals were kept in a temperature-controlled room (20-22°C) with a 12:12-h light-dark cycle and fed ad libitum with standard rat food. The present experiments were performed in normal rats and in rats with cholangiocyte hyperplasia induced by BDL for 28 days. BDL was performed as previously described (5-7). Before each experiment, animals were anesthetized with pentobarbital sodium (50 mg/kg ip). Study protocols were performed in compliance with institutional guidelines.Materials
Reagents were obtained from Sigma Chemical (St. Louis, MO) unless otherwise indicated. Both secretin and somatostatin were purchased from Peninsula Laboratories (Belmont, CA). The mouse anticytokeratin 19 (CK-19) antibody was purchased from Amersham (Arlington Heights, IL). Radioimmunoassay kits for the determination of intracellular cAMP levels in purified cholangiocytes were purchased from Amersham. Dulbecco's PBS was obtained from Celox (Hopkins, MN). The monoclonal antibody to vimentin, an IgG1 against the 57-kDa intermediate filament vimentin (38), was purchased from BioGenex (San Ramon, CA). BSA was purchased from Calbiochem-Novabiochem (La Jolla, CA).In Situ Assessment of Cholangiocyte Proliferation in both Normal and BDL Rat Liver
In frozen liver sections (~6 µm thick) obtained randomly from both normal and BDL rat livers, immunohistochemistry for CK-19 was performed as previously described by us (6, 19).Ninety minutes after an intraperitoneal injection of 1 µCi/g body wt [methyl-3H]thymidine (6.70 Ci/mmol, Du Pont-New England Nuclear Products, Boston, MA), in situ nuclear labeling was determined in paraffin-embedded sections (~6 µm thick) from both normal and BDL rat livers. Briefly, after coating in Kodak NTB-2 emulsion (Eastman Kodak, Rochester, NY), the paraffin-embedded sections were exposed for 2 days, processed for standard autoradiography, and stained with hematoxylin and eosin by standard procedures.
Isolation and Phenotypic Characterization of Hepatocytes and Cholangiocytes
Hepatocytes were obtained from both normal and BDL rats as previously described (5, 6). After isolation, the purity of hepatocytes was assessed by histochemistry for glucose-6-phosphatase (33), a marker for hepatocytes (2, 5, 6), andMeasurement of Proliferative Capacity of Hepatocytes and Pooled Cholangiocytes
DNA synthesis in hepatocytes and pooled cholangiocytes from both normal and BDL rat livers was assessed by measurement of both [3H]thymidine incorporation (19) and H3 histone gene expression [an index of DNA synthesis (2, 3)] using RNase protection assays (9-11, 35). Both approaches have been previously used by us (2, 3, 19) to measure DNA synthesis in normal and proliferating cholangiocytes. Briefly, 90 min after an intraperitoneal injection of 1 µCi/g body wt [methyl-3H]thymidine, pure preparations of hepatocytes and pooled cholangiocytes from normal and BDL rat livers (~1.0 × 106 cells) were first treated with 3 M KOH at 37°C for 30 min, then with a solution containing 15% trichloroacetic acid and 6 N HCl at 4°C for 12 h. After the solution was centrifuged at 10,000 g for 20 min at 4°C, DNA was extracted from the cholangiocyte cell pellet with HClO4 at 80°C for 15 min. After the solution was spinned, the supernatant was transferred, and the radioactivity incorporated into DNA was measured. Before they were counted, samples were kept at 4°C in the dark overnight to avoid chemiluminescence. The results were expressed as 1 × 106 cpm/106 cholangiocytes. Data are means ± SE ofThe quantitative expression of H3
histone was assessed by RNase protection assays with the RPA II kit
(Ambion, Austin, TX) using total cellular RNA (20 µg) obtained from
hepatocytes or pooled cholangiocytes (~5.0 × 106) according to the method of
Chomczynski and Sacchi (13). The amount of the total cellular RNA
assayed was determined by both ultraviolet detection of ethidium
bromide-stained blots and absorption at 260 nm. The quality of the
total RNA samples was assessed by the ratio of absorbance at 260 nm to
absorbance at 280 nm. The equality of the total RNA used was assessed
by hybridization with glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
a housekeeping gene. After exposure for 2 days, autoradiograms were
quantified by densitometry. Antisense riboprobes were transcribed from
linearized cDNA templates with either
T7 or SP6 RNA polymerase using
[-32P]UTP (800 Ci/mmol, Amersham). We used the following
32P-labeled single-stranded
antisense riboprobes: a 204-bp riboprobe encoding for the
H3 histone gene was obtained from
Dr. S. Gupta (Albert Einstein Hospital, Bronx, NY) and a 316-bp
riboprobe encoding a complementary sequence for rat GAPDH mRNA was
purchased from cDNA purchased from Ambion. We used the following
controls: rat spleen (positive) and yeast transfer RNA (negative) for
the H3 histone gene as well as rat
kidney (positive) and yeast transfer RNA (negative) for the GAPDH gene.
Measurement of Genetic Expression of SSTR2 in Cholangiocyte Subpopulations from BDL Rat Liver
In the two cholangiocyte subpopulations, we measured the transcript for SSTR2 mRNA by RNase protection assays with the RPA II kit (Ambion) using 20 µg total cellular RNA (9-11) (see above). Total RNA was extracted by the acid guanidium thiocyanate-phenol chloroform single-step extraction method (13). The equality of total cellular RNA assayed was determined by hybridization with a cDNA encoding for the GAPDH gene (9-11, 35). A 252-bp riboprobe encoding for a complementary sequence for the mouse SSTR2 mRNA was a gift from Dr. G. Bell (The University of Chicago, Chicago, IL); a 316-bp riboprobe encoding for a complementary sequence for rat GAPDH mRNA was transcribed from a cDNA purchased from Ambion. We used the following controls: rat brain (positive) and yeast transfer RNA (negative) for the SSTR2 gene as well as rat kidney (positive) and yeast transfer RNA (negative) for the GAPDH gene. Steady-state levels of selected messages were quantified by densitometry.Determination of Proliferative Capacity of Cholangiocyte Subpopulations
To determine if somatostatin has differential inhibitory effects on proliferative processes of cholangiocyte subpopulations originating from different portions of the intrahepatic biliary tree, we studied in vitro the effect of somatostatin on DNA synthesis of small and large cholangiocytes isolated from BDL rat livers. Ninety minutes after an intraperitoneal injection of 1 µCi/g body wt [methyl-3H]thymidine, fraction 1 and fraction 2 were isolated by immunoaffinity purification and subsequently treated with 0.2% BSA (control) or somatostatin (10After incubation with 0.2% BSA (control) or somatostatin
(107 M) in 0.2% BSA for 15 min at 37°C, proliferative capacity of fraction 1 and
fraction 2 was assessed by measuring
H3 histone gene expression by
RNase protection assay (9-11, 35) (see above). The comparability
of the total RNA used was assessed by hybridization with GAPDH, the
housekeeping gene (9-11, 35). We used rat spleen (positive) and
yeast transfer RNA (negative) for the
H3 histone gene as well as rat
kidney (positive) and yeast transfer RNA (negative) for the GAPDH gene.
Steady-state levels of selected messages were quantified by
densitometry.
In Vitro Effect of Somatostatin on Ductal Secretory Activity of Cholangiocyte Subpopulations from BDL Rat Liver
Measurement of SR gene expression.
After purification, both fraction 1 and fraction
2 (~5.0 × 106 cells)
from BDL rat liver were stimulated in vitro with 0.2% BSA (control) or
somatostatin (107 M) in
0.2% BSA for 15 min at 37°C. After stimulation, the quantitative genetic expression of the SR gene was assessed by RNase protection assays with the RPA II kit (Ambion) (see above) using total cellular RNA (20 µg) obtained from fraction 1 and fraction
2 (stimulated with BSA or somatostatin) according to the method of
Chomczynski and Sacchi (13). The comparability of the total cellular
RNA used in our molecular studies was determined by hybridization with
GAPDH, a housekeeping gene (9-11, 35). The 318-bp riboprobe (encoding for SR mRNA) was generated from our rat SR cDNA clone (11); a
316-bp riboprobe encoding a complementary sequence for rat GAPDH mRNA
was transcribed from cDNA purchased from Ambion. In our molecular
analysis, we used the following controls: rat heart (positive) and
kidney (negative) for the SR gene and rat kidney (positive) and yeast
transfer RNA (negative) for the GAPDH gene.
Assessment of intracellular cAMP levels.
In BDL rat liver, intracellular cAMP levels of fraction 1 and fraction 2 were measured by commercially available
radioimmunoassay kits (Amersham) as previously reported by us (2, 9,
10, 18, 19, 28, 35). Purified cholangiocytes were incubated at 37°C
for 1 h to restore membrane proteins that may have been damaged by
proteolytic enzyme digestion and subsequently incubated for 5 min at
22°C (2, 9, 10, 18, 19, 28, 35) in the presence of 1% BSA with
secretin (107 M) or
secretin plus somatostatin (both at
10
7 M) before assessing
intracellular cAMP levels. Control cholangiocytes were incubated with
0.2% BSA under the same experimental conditions. In both control and
hormone-treated cholangiocytes, phosphodiesterase activity was
inhibited by addition of 3-isobutyl-1-methylxanthine (0.5 mM).
Statistical Analysis
All data are expressed as means ± SE. The differences between groups were analyzed by Student's unpaired t-test when two groups were analyzed or ANOVA if more than two groups were analyzed. ![]() |
RESULTS |
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In Situ Immunohistochemistry and Autoradiography
To validate our model of cholangiocyte hyperplasia after BDL, we estimated cholangiocyte proliferative capacity by both in situ immunohistochemistry for CK-19 [a cholangiocyte-specific marker (6, 9, 10, 19)] and standard in vivo autoradiography. Immunohistochemical studies show that only two to three intrahepatic bile ducts (stained for CK-19) were present in a normal rat liver section (Fig. 1A) in agreement with other studies (6, 19). After BDL, a marked increase in the number of bile ducts (stained for CK-19) was seen within portal areas (Fig. 1B), findings consistent with our previous studies (6, 19). In both normal and BDL rat livers, the lobular hepatic architecture was normal, indicating that BDL does not induce proliferative or morphological changes in hepatic lobules. In normal rats (Fig. 1C), no cholangiocyte nuclei were labeled with [3H]thymidine, whereas silver grains were observed in hepatocytes, the only liver epithelia that replicate in the normal state. After BDL (Fig. 1, D and E), cholangiocytes lining bile ducts had labeled nuclei, whereas no change in labeling was seen in other liver cells. The data are consistent with the concept that cholangiocytes selectively and markedly proliferate after BDL (5-7, 14, 35).
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Purification and Characterization of Hepatocytes and Cholangiocytes
Hepatocytes from both normal and BDL rats wereMeasurement of [3H]thymidine Incorporation in Isolated Hepatocytes and Isolated Pooled Cholangiocytes
In hepatocytes isolated from normal rat liver, [3H]thymidine incorporation was 238.31 ± 21.96 cpm/106 cells (Fig. 2), consistent with the concept that in normal rat liver hepatocytes have the capacity to replicate (see also Fig. 1C). In pooled cholangiocytes isolated from normal rat liver, [3H]thymidine incorporation was virtually absent (0.70 ± 0.48 cpm/106 cells) (Fig. 2), consistent with the notion that cholangiocytes are mitotically dormant under basal conditions (2, 5, 6, 19). After BDL, [3H]thymidine incorporation into purified hepatocytes did not change significantly (156.65 ± 44.07 cpm/106 cells, P > 0.05 vs. normal hepatocytes) (Fig. 2) concordant with the notion that hepatocytes do not proliferate after BDL (5-7). In contrast, radioactivity incorporated into the DNA of pooled cholangiocytes isolated after BDL markedly increased (97.89 ± 6.50 cpm/106 cells, P < 0.05 vs. normal pooled cholangiocytes), supporting the concept that cholangiocytes are the major proliferative target of BDL in rat liver (see also Fig. 1, D and E) (5-7, 10, 35). Similarly, H3 histone gene expression was present in normal hepatocytes and was not affected by BDL (Fig. 2). The expression of H3 histone gene was very low in normal pooled cholangiocytes but markedly increased (~90-fold) in proliferating cholangiocytes from BDL rat liver (Fig. 2). The expression of the housekeeping gene GAPDH was similar between hepatocytes and pooled cholangiocytes obtained from BDL rat liver (Fig. 2).
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SSTR2 mRNA in Cholangiocyte Subpopulations From BDL Rat Liver
As shown in Fig. 3, the message for GAPDH, the housekeeping gene, was similarly expressed in fraction 1 and fraction 2 of cholangiocytes obtained from BDL rat liver. In contrast, we found no expression of the message for SSTR2 in fraction 1, but unequivocal expression of this message in fraction 2 (Fig. 3).
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Effect of Somatostatin on Proliferative Capacity of Cholangiocyte Subpopulations From BDL Rat Liver
Measurement of [3H]thymidine
incorporation.
After purification, DNA synthesis was active principally in
fraction 2 (186.05 ± 13.63 cpm/106 cells,
P < 0.05 vs.
[3H]thymidine uptake
of fraction 1) obtained from BDL rat livers (Fig.
4). Indeed, DNA synthesis was virtually
absent in fraction 1 (1.56 ± 0.95 cpm/106 cells) (Fig. 4). In the
presence of somatostatin
(107 M), DNA synthesis
significantly decreased only in fraction 2 (84.06 ± 33.94 cpm/106 cells,
P < 0.05 vs.
[3H]thymidine uptake
of untreated cholangiocytes, 186.05 ± 13.63 cpm/106 cells) (Fig. 4). The
inhibitory effect of somatostatin on DNA synthesis of fraction
2 (Fig. 4) closely parallels the molecular data on
SSTR2 mRNA distribution obtained
by the RNase protection assay showing that the transcript for
SSTR2 is expressed by
fraction 2 but not fraction 1 (see Fig. 3).
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State-steady levels of H3 histone mRNA. As shown in Fig. 5, H3 histone mRNA was principally expressed in fraction 2 after BDL, being present at only very low levels in fraction 1. The data closely parallel the studies on [3H]thymidine incorporation (Fig. 4). In a fashion similar to that shown for [3H]thymidine incorporation in purified cholangiocytes (Fig. 4), somatostatin caused an approximately sixfold decrease in H3 histone gene expression in fraction 2 as compared with BSA-treated (control) cholangiocytes (Fig. 5). Consistent with the concept that SSTR2 mRNA is present principally in fraction 2 in rat liver (see Fig. 3), somatostatin did not decrease the expression of H3 histone mRNA in fraction 1 (Fig. 5).
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Effect of Somatostatin on Secretin-Induced Ductal Secretory Activity of Cholangiocyte Subpopulations From BDL Rat Liver
SR gene expression.
Because of the close coupling between cholangiocyte proliferation and
increased ductal bile secretion (5, 7, 14, 19), we next determined if
somatostatin also differentially inhibits ductal secretory activity in
small ( fraction 1) and large ( fraction 2) cholangiocytes from BDL rat livers. In agreement with our
previous studies in BDL rat liver (11), SR mRNA was principally
expressed in fraction 2 (Fig.
6). Parallel with decreases in the
proliferative capacity of cholangiocytes, somatostatin induced an
~50% decrease in SR gene expression in fraction 2.
Consistent with the cellular distribution of
SSTR2 mRNA in cholangiocytes (Fig.
3), somatostatin did not alter the genetic expression of SR in
fraction 1.