1 Department of Internal
Medicine and Medical Physiology, We studied the expression of endothelin-1 (ET-1)
receptors (ETA and
ETB) and the effects of ET-1 on
cholangiocyte secretion. The effects of ET-1 on cholangiocyte secretion
were assessed in normal and bile duct-ligated (BDL) rats by measuring
1) basal and secretin-induced
choleresis in vivo, 2) secretin
receptor gene expression and cAMP levels in small and large
cholangiocytes, and 3) luminal
expansion in response to secretin in intrahepatic bile duct units
(IBDU). ETA and
ETB receptors were expressed by small and large cholangiocytes. ET-1 had no effect on basal bile flow
or bicarbonate secretion in normal or BDL rats but decreased secretin-induced bicarbonate-rich choleresis in BDL rats. ET-1 decreased secretin receptor gene expression and secretin-stimulated cAMP synthesis in large cholangiocytes and secretin-induced luminal expansion in IBDU from normal or BDL rats. The inhibitory effects of
ET-1 on secretin-induced cAMP synthesis and luminal duct expansion were
blocked by specific inhibitors of the
ETA (BQ-610) receptor. ET-1
inhibits secretin-induced ductal secretion by decreasing secretin
receptor and cAMP synthesis, two important determinants of ductal
secretion.
biliary epithelium; bile duct ligation; adenosine
3',5'-cyclic monophosphate; secretin receptor
THE INTRAHEPATIC BILIARY tree consists of a complex,
ramified network of interconnecting tubular conduits of different
diameters and function lined by intrahepatic bile duct epithelial cells or cholangiocytes (7, 8, 10). Bile is formed at the canalicular domain
of hepatocytes, and, before it reaches the duodenum, it is extensively
modified by cholangiocyte secretion and reabsorption (5, 6, 19, 30,
50). Ductal bile secretion is regulated by a number of gastrointestinal
hormones, including secretin, gastrin, bombesin, and somatostatin (3,
5-10, 12, 15, 19, 28, 30, 50) and neuropeptides (11). Secretin,
for example, interacts with specific receptors expressed in rat liver
only by cholangiocytes (9), which leads to an increase in intracellular levels of the second messenger cAMP,
Cl Cholangiocytes are the target cells in a number of animal models of
ductal hyperplasia, including bile duct ligation (BDL), Endothelins (ET-1, ET-2, ET-3) are a family of vasoactive peptides that
interact with at least two types of receptors,
ETA and
ETB, which are distributed in
numerous organs (16). ET-1, a potent vasoconstrictor of 21 amino acids,
has a multifunctional role in a variety of tissues and cells (45, 53,
54). In the liver, ET-1 induces cholestasis, which is associated with an increase in portal pressure when administered to the isolated perfused rat liver (IPRL) (14, 25). ET-1 activates glycogenolysis, phosphoinositide synthesis, and intracellular free
Ca2+ oscillations in purified
hepatocytes (18, 43). Furthermore, rat sinusoidal endothelial cells in
culture synthesize ET-1 (37). Endothelin receptors have been detected
in rat hepatic stellate cells (23). ET-1 has been detected in human
gallbladder epithelial cells in culture (22) and in bile ducts in human
cirrhotic liver sections (35). However, no data exist regarding the
effects of ET-1 on spontaneous and secretin-regulated cholangiocyte
secretory processes in different segments of the rat intrahepatic
biliary tree. Given the fact that the intrahepatic biliary tree is
functionally heterogeneous with regard to ductal secretory processes
(1, 3, 8, 10) and that ET-1 plays an important role in the regulation
of secretion in a number of organs (21, 29, 33), we evaluated
1) if ET-1 receptors
(ETA and
ETB) are expressed (at the gene,
protein, and functional levels) in small and large cholangiocytes and
2) if ET-1 regulates basal and
secretin-stimulated ductal secretory activity in normal and diseased
liver following BDL.
To begin to define the role of ET-1 on the modulation of basal and
secretin-stimulated ductal bile secretion, we investigated the effect
of ET-1 on 1) secretin receptor gene
expression and intracellular cAMP synthesis in purified cholangiocytes,
2) basal and secretin-induced lumen
expansion in large (>15 µm diameter) IBDU, and
3) spontaneous and secretin-induced
bile flow and biliary bicarbonate secretion in vivo. We studied the
expression of ET-1 receptors (ETA
and ETB) in purified
subpopulations of small and large cholangiocytes and subsequently
determined the receptor subtype responsible for these effects by using
a specific ETA receptor antagonist
(BQ-610) (24, 42) and ETB receptor
agonists [i.e., ET-3 and sarafotoxin 6c (S6c)] (14, 39,
55). We found that ET-1 receptors
(ETA and
ETB) are expressed in small and
large cholangiocytes from both normal and BDL rats and showed that ET-1 inhibited secretin-stimulated cAMP synthesis and secretin receptor gene
expression in large cholangiocytes and the secretin-induced ductal
luminal expansion in large, purified IBDU by selectively interacting
with ETA receptors. Similarly,
ET-1 decreased secretin-induced bicarbonate-rich choleresis and
bicarbonate secretion in BDL rats. The inhibitory effects of ET-1 on
cholangiocyte secretory processes were completely blocked by BQ-610, a
specific inhibitor of the ETA
receptor (24, 42).
Animal Model
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
channel opening and
activation of the
Cl
/bicarbonate exchanger
and secretin-induced bicarbonate-rich choleresis (1, 3, 5, 6, 10, 11,
17, 19, 28, 30, 50).
-naphthyl
isothiocyanate feeding, or 70% partial hepatectomy (5-7, 9, 10,
19, 30, 50). In BDL rats, the cholangiocyte proliferative response is
closely associated with an increase in secretin-stimulated choleresis
in vivo and with an augmented secretin receptor gene expression,
secretin-induced cAMP synthesis, and
Cl
/bicarbonate exchanger
activity in purified cholangiocytes (5, 6, 9, 19, 50). We have recently
shown in distinct subpopulations of cholangiocytes and intrahepatic
bile duct units (IBDU) that cholangiocytes are morphologically and
functionally heterogeneous along the length of the intrahepatic biliary
tree and that secretin-regulated transport of water and electrolytes
occurs in large (but not small) cholangiocytes and IBDU (3, 8, 10).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Materials
Reagents were purchased from Sigma Chemical (St. Louis, MO), unless otherwise indicated. Endothelins (ET-1 and ET-3) were purchased from Calbiochem (La Jolla, CA). BQ-610, a selective antagonist of the ETA receptor (24, 42), was purchased from Peptides International (Louisville, KY). The polyclonal antibodies (sheep antiserum) against the rat ETA and ETB receptors were purchased from Alexis (San Diego, CA). S6c, a specific agonist of the ETB receptor (39, 55), was purchased from American Peptide (Sunnyvale, CA). Dulbecco's PBS was obtained from Celox (Hopkins, MN). Secretin was purchased from Peninsula Laboratories (Belmont, CA). RIA kits for the determination of intracellular cAMP levels in pure preparations of small and large cholangiocytes were purchased from Amersham (Arlington Heights, IL).Isolation and Morphological and Phenotypic Characterization of Small and Large Cholangiocytes and Large IBDU From Normal and BDL Rats
Pure subpopulations of small and large cholangiocytes were isolated from both normal and BDL rat liver as previously described (2, 8, 10). Briefly, after standard collagenase perfusion was performed, a mixed nonparenchymal cell fraction was obtained from undissociated liver tissue by digestion with a mixture of proteolytic enzymes as described by Ishii et al. (26). The cholangiocyte-enriched fraction [~50% pure by histochemistry forLarge IBDU from both normal and BDL rat liver were isolated and morphologically characterized as previously described by us (1, 3). After standard collagenase perfusion (3, 34, 38), the intrahepatic biliary tree was exposed by mechanical removal of parenchymal cells until only bile duct structures were present. The mixed bile duct fragments were placed in 50-mm petri dishes, visualized under phase-contrast optics (×10 objective) on a Nikon Diaphot inverted microscope (Tokyo, Japan), picked up by a 10-µl pipette, and subsequently transferred to the stage of a Nikon Diaphot microscope equipped with fluorescence and differential interference contrast optics. To separate large bile ducts from smaller bile ducts, we used a microscope-focused laser (3), an approach previously used in other cells (13). This approach was previously used by us for isolating small and large bile ducts from normal rat liver (3). Briefly, a nitrogen-pulsed dye laser (model LSl-377, LSD Industries, Cambridge, MA, coumarin 481 as a laser dye generating a 120-mJ/pulse, 20-Hz repetition rate energy output) was focused to the diffraction limit through the epilumination port of the fluorescence microscope containing a fluorescein filter cube (Omega Optical, Brattleboro, VT) to produce a very well-localized high-energy light beam (13). Typically, between one and five pulses were delivered to the junction of small and large ducts to cut and separate large ducts from the smaller bile ducts. Isolated large IBDU from normal or BDL rats were then placed in minimum essential medium (GIBCO BRL, Grand Island, NY) containing 10% FCS, allowed to settle on glass coverslips, previously coated with Cell-TAK, and incubated for 24 h at 37°C before effects of ET-1 on both basal and secretin-stimulated ductal lumen expansions were measured (see Measurement of Basal and Secretin-Induced Ductal Lumen Expansion in Large IBDU Purified From Both Normal and BDL Rats).
Molecular, Immunohistochemical, and Immunologic Analyses of ETA and ETB Receptors in Small and Large Cholangiocytes from Normal and BDL Rats
Molecular analyses of ETA and
ETB in small and large cholangiocytes from
normal and BDL rats.
The genetic expression of ETA,
ETB, and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [the
housekeeping gene (2, 4, 8-10, 19, 50)] in pure preparations
of small and large cholangiocytes purified from both normal and BDL
rats was determined by lysate RNase protection assay (Direct Protect,
Ambion, Austin, TX) according to the instructions of the manufacturer.
The RNase protection assay was performed with cell lysate samples each
containing 4.5 × 105
cholangiocytes. This technique has been previously used by us to
measure quantitative gene expression in purified cholangiocytes from
both normal and proliferating rat liver (2, 19, 30). This procedure has
also been used to determine steady-state levels of selected genes in
other cell systems (48). Briefly, an antisense 32P-labeled riboprobe was
transcribed from the selected linearized DNA template with T7 or SP6
RNA polymerase using
[-32P]UTP (800 Ci/mmol, Amersham). The full-length RNA transcript was purified by
excision from a 5% acrylamide-8 M urea denaturing gel with subsequent
elution for 3 h at 37°C into a solution of 0.5 M ammonium acetate,
1 mM EDTA, and 0.1% SDS. After hybridization at 45°C for 12 h, the
solution was treated with a mixture of RNase A-T1 (150-200 U/ml)
to digest unhybridized RNA chains and the protected hybrid was resolved
in a 5% acrylamide-8 M urea gel and detected by autoradiography at
70°C for 2 days. After exposure for 2 days, autoradiograms
were quantified by scanning video densitometry using the ChemiImager
4000 low-light imaging system (Alpha Innotech, San Leandro, CA).
Immunohistochemistry for ETA and ETB in small and large cholangiocytes from normal and BDL rats. Immunohistochemistry for the ET-1 receptors (ETA and ETB) was performed on cytospin (100 g for 5 min) smears of small and large cholangiocytes purified from both normal and BDL rats. Before the staining procedure, the immunomagnetic beads [used for the purification of small and large cholangiocytes (8, 10)] were detached from the cells by enzymatic digestion as previously described by Ishii et al. (26). After purification, cytospin smears of small and large cholangiocytes were fixed in cold acetone for 10 min, air dried, and rinsed in 1× PBS, pH 7.2. The endogenous peroxidase activity was blocked with sodium azide-hydrogen peroxide; nonspecific binding was inhibited by adding 10% normal donkey serum. Cells were then incubated with the anti-ETA or ETB antibody (1:100 dilution) or sheep serum without the primary antibody (negative control) for 2 h at 22°C in a humidified chamber, washed with PBS, and incubated for 30 min at 22°C with biotin-conjugated donkey anti-sheep IgG. The immunoreactivity was detected with the indirect peroxidase procedure by using Elite Vectastain ABC kit (Vector Laboratories, Burlingame, CA). After the staining procedure, cells were lightly counterstained with hematoxylin and examined with a microscope (BX 40, Olympus Optical).
Western blot analyses of ETA and ETB receptors in small and large cholangiocytes from normal and BDL rats. Small and large cholangiocytes (5 × 106 cells) from normal or BDL rats were resuspended in RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, 2 mM EDTA, 10 mM sodium fluoride, 10 µg/ml leupeptin, 20 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride) and sonicated six times (30 s bursts). The cell lysates were immunoprecipitated with 4 µg of either sheep anti-ETA receptor or anti-ETB receptor antibody (Alexis). Proteins were resolved by SDS-12.5% PAGE and transferred onto a nitrocellulose filter (Bio-Rad, Hercules, CA). The filter was blocked using a 5% solution of nonfat dry milk in Tris-buffered saline (TBST, composed of 50 mM Tris, 150 mM NaCl, and 0.05% Tween 20). The filter was then incubated with rotation for 2 h at room temperature with either sheep anti-ETA receptor or anti-ETB receptor antibody (10 µg antibody/ml), washed five times with TBST, incubated with rotation for 1 h at room temperature with anti-goat IgG peroxidase conjugate (1:75,000 dilution), and washed five times with TBST. Proteins were visualized using chemiluminescence (Amersham Life Science, Little Chalfont, Buckinghamshire, UK). The intensity of the bands was determined by scanning video densitometry using the ChemiImager 4000 low-light imaging system (Alpha Innotech, San Leandro, CA).
Effect of ET-1 on Secretin Receptor Gene Expression
Small and large cholangiocytes purified from both normal and BDL rat liver were incubated with 0.2% albumin (BSA, control) or ET-1 (10After linearization of cDNA templates with the appropriate restriction
endonuclease, antisense riboprobes were transcribed with T7 or SP6 RNA
polymerase using
[-32P]UTP (800 Ci/mmol) (Amersham). The following riboprobes were used: a 318-bp
secretin receptor (SR) riboprobe was generated from linearized
pGEM4Z-SR (a gift of Dr. N. LaRusso, Mayo Clinic, Rochester, MN) and a
316-bp riboprobe complementary to rat GAPDH mRNA was transcribed from
cDNA purchased from Ambion.
Effect of ET-1 on Intracellular cAMP Levels of Small and Large Cholangiocytes Purified From Both Normal and BDL Rats
We measured in small and large cholangiocytes from both normal and BDL rats the intracellular levels of cAMP, a functional assay for the secretin receptor (2, 3, 8, 10, 19, 28, 30, 50). Because antibodies against the secretin receptor are not commercially available, the cAMP assay allows us to closely correlate the molecular with the functional expression of the secretion receptor in purified cholangiocytes (8, 10, 19, 30, 50). After isolation, pure preparations of small and large cholangiocytes were incubated for 1 h at 37°C to restore surface proteins damaged by treatment with proteolytic enzymes (28, 30). Then, small and large cholangiocytes (1 × 105) were stimulated for 5 min at 22°C (8, 10, 19, 30, 50) with 0.2% BSA (control), secretin (10Measurement of Basal and Secretin-Induced Ductal Lumen Expansion in Large IBDU Purified From Both Normal and BDL Rats
Large IBDU were incubated for 24 h at 37°C in minimum essential medium containing 10% FCS to allow complete sealing of bile duct lumen (3, 34, 38). With the use of light microscopy to visualize the diameter of bile duct lumen, ductal fluid secretion was estimated from the changes in the volume of duct lumen (3, 34, 38) after stimulation at 37°C with BSA (control, 10 min), secretin (10 min at 10In Vivo Biliary Physiology
Normal and BDL rats were surgically prepared for bile collection as previously described (5, 6, 19, 30, 50). One jugular vein was incannulated with a PE-50 cannula (Clay-Adams, New York, NY) to infuse either Krebs-Henseleit bicarbonate solution (KRH), secretin, ET-1, or secretin plus ET-1 dissolved in KRH. Blood was withdrawn every 10 min from one carotid artery (by a PE-60 cannula) to assess the arterial hematocrit, which was constant (41-45%) in all rats during bile collection. The rate of fluid infusion was adjusted according to both the rate of bile flow and the value of the arterial hematocrit and ranged from 0.738 to 2.328 ml/h. Body temperature was monitored with a rectal thermometer (Yellow Springs Instrument, Yellow Springs, OH) and maintained at 37°C by using a heating pad. When steady-state spontaneous bile flow was achieved (60-70 min from the beginning of bile collection), we infused secretin (10Statistical Analysis
All data are expressed as means ± SE. The differences between groups were analyzed by Student's t-test when two groups were analyzed or ANOVA if more than two groups were analyzed. ![]() |
RESULTS |
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Isolation, Morphological, and Phenotypic Characterizations of Small and Large Cholangiocytes or Large IBDU
We isolated virtually pure (byMolecular, Immunohistochemical, and Immunologic Analyses of ETA and ETB Receptors in Small and Large Cholangiocytes
Molecular analysis. Molecular analysis of purified cholangiocytes shows that the transcripts for ETA (273 bp) and ETB (130 bp) receptors were present in both small and large cholangiocytes (Fig. 1A). Densitometric analysis of two experiments showed that no changes in ETA or ETB receptor gene expression were observed in small and large cholangiocytes purified from BDL rats compared with small and large cholangiocytes from normal rats (Fig. 1B). The quantitative expression of GAPDH (housekeeping gene) was similar in small and large cholangiocytes purified from normal or BDL rats (Fig. 1).
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Immunohistochemistry. At the protein level, parallel with the data shown in the molecular analysis of Fig. 1, immunohistochemistry of purified cholangiocytes (Fig. 2) shows positive staining for ETA or ETB receptors in both small and large cholangiocytes from normal or BDL rat liver.
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Western blot analysis. Immunoblotting analysis shows that a signal (migrating at 35 kDa) for the ETA and ETB receptors was expressed in both small and large cholangiocytes purified from normal or BDL rat liver (Fig. 3). Quantitative densitometric analysis of two experiments showed that no changes were observed in the protein expression of ETA or ETB receptors in small and large cholangiocytes purified from BDL rats compared with small and large cholangiocytes from normal rats (results not shown). The data closely parallel with the genetic expression of ETA or ETB receptors in small and large cholangiocytes purified from normal or BDL rats (see Fig. 1).
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Effect of ET-1 on Secretin Receptor Gene Expression in Purified Cholangiocytes
Because secretin receptor is a specific marker of ductal secretory activity (1, 3, 9, 19, 30), we investigated if ET-1 modulates the expression of this gene. The message for secretin receptor was present in large (but not small) cholangiocytes (8-10), and the expression of the secretin receptor gene was significantly (P < 0.05) increased in large cholangiocytes purified from BDL rats compared with large cholangiocytes from normal rat liver (see Fig. 4). When purified cholangiocytes were treated with ET-1 (10
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Intracellular cAMP Levels
Because antibodies against the secretin receptor are not commercially available, the secretin-stimulated cAMP synthesis is commonly used to measure the functional expression of the secretin receptor in cholangiocytes (1-4, 8, 10, 11, 19, 28, 30, 38, 50). Basal intracellular cAMP levels in small and large cholangiocytes from both normal (Fig. 5A) and BDL (Fig. 5B) rat liver were similar to those reported in previous studies (2, 8, 10). Basal cAMP levels of large cholangiocytes purified from BDL rats were higher than cAMP levels of large cholangiocytes purified from normal rats (see Figs. 5 and 6). Secretin (10
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Effect of ET-1 on Basal and Secretin-Stimulated Ductal Lumen Expansion
To directly assess that the inhibitory effects of ET-1 on secretin-induced ductal bile secretion occurred through a direct action on cholangiocytes rather than by ET-1-induced hemodynamic changes in the liver (51), we isolated large IBDU from both normal and BDL rats and studied their secretory activity in response to 0.2% BSA (control), secretin (10
|
In Vivo Studies of Biliary Physiology
In normal rats, spontaneous bile secretion (73.96 ± 5.36 µl · minIn agreement with a number of reports (5, 6, 19, 30, 50), following
BDL, both basal bile flow (127.86 ± 8.65 µl · min1 · kg
body wt
1) and basal
bicarbonate secretion (3.90 ± 0.50 meq · min
1 · kg
body wt
1) were
significantly (P < 0.05) increased
compared with normal rats. As shown in Fig.
8, secretin increased both bile flow and bicarbonate secretion. Similar to what was observed in normal rats,
ET-1 alone (10
10 to
10
7 M) did not alter basal
bile flow and biliary bicarbonate secretion in BDL rats (results not
shown). In contrast, simultaneous infusion of ET-1 and secretin
(10
7 M) markedly reduced,
in a dose-dependent fashion, both secretin-induced choleresis and
secretin-stimulated bicarbonate secretion (Fig. 8). In a fashion
similar to that observed for cAMP synthesis, ET-3 and S6c did not
affect spontaneous or secretin-induced choleresis or biliary
bicarbonate secretion (results not shown).
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DISCUSSION |
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The major findings of this study relate to the expression of ET-1
receptors (ETA and
ETB) in small and large
cholangiocytes purified from normal and BDL rats and to the inhibitory
effect of ET-1 (but not ET-3) on secretin-stimulated ductal bile
secretion in both normal and BDL rats. We isolated two pure (by -GT
histochemistry) and distinct subpopulations of small and large
cholangiocytes from normal and BDL rats and have shown that the
ETA and
ETB receptors are expressed (at
both the message and protein levels) by both small and large
cholangiocytes. At the functional level, ET-1 did not alter in vivo
spontaneous bile flow or biliary bicarbonate secretion in normal or BDL
rats but inhibited the stimulatory effects of secretin on both bile
flow and biliary bicarbonate secretion in BDL rats. Parallel with the
in vivo effects, ET-1 did not change basal cAMP synthesis but
significantly decreased secretin receptor gene expression and
secretin-induced cAMP levels in large cholangiocytes, the only
cholangiocyte subpopulation that responds physiologically to secretin
in rat liver (1-3, 8, 10). Moreover, ET-1 did not affect IBDU duct
lumen volume but inhibited the secretin-induced luminal ductal
expansion in large IBDU purified from both normal and BDL rats.
Finally, the inhibitory effects of ET-1 on both secretin-induced cAMP
synthesis and luminal duct expansion in large cholangiocytes and large
IBDU, respectively, were completely blocked by specific inhibitors of ETA (BQ-610) receptors (24, 42).
Consistent with the concept that ET-1 inhibits secretin-stimulated
ductal secretion by specifically interacting with
ETA (but not
ETB) receptors, we found that
the specific ETB receptor agonists
[ET-3 and S6c (14, 39, 55)] did not alter basal and
secretin-stimulated cAMP synthesis or bile flow. Together, the data
indicate that, in a fashion similar to that observed with somatostatin
(4, 50) and gastrin (19), ET-1 inhibits secretin-induced ductal bile
secretion by directly interacting with
ETA receptors on cholangiocytes,
which leads to a decrease of the expression of the secretin receptor
and to a decrease in secretin-stimulated cAMP levels, two important
regulatory determinants of ductal bile secretion (1-3, 7-10,
12, 19, 30, 50).
In normal rats, ductal bile flow consists of only 10% of the total
bile volume (6). Furthermore, secretin receptor gene expression and
secretin-induced cAMP response are very low (8, 9, 28, 30) and secretin
does not increase bile flow or biliary bicarbonate secretion in normal
rats (5, 6, 19, 30, 50). In the present studies, we used the BDL model
in which secretin receptor gene expression and intracellular cAMP levels are markedly increased in purified cholangiocytes (1, 9, 19, 50)
and in which secretin markedly increases bile flow and biliary
bicarbonate secretion in vivo (5, 6, 19, 50). The secretory and
reabsorptive processes of the intrahepatic biliary epithelium are
tightly regulated in a coordinated fashion by a number of
gastrointestinal hormones and neuropeptides (1-12, 15, 17, 19, 28,
30, 34, 38, 50). The hormone secretin, for example, induces in vivo an
increase in bile flow and biliary bicarbonate secretion (5, 6, 19, 30,
50) by interaction with specific receptors on cholangiocytes (9)
through an increase in the synthesis of the second messenger system,
cAMP (3, 8, 10, 19, 28, 30, 50). In contrast, somatostatin (4, 50) and
gastrin (19) regulate ductal bile secretion by counterposing the in
vivo choleretic effects of secretin by decreasing both secretin
receptor gene expression and secretin-stimulated intracellular cAMP
synthesis. The cholinergic system plays an important role in the
regulation of cholangiocyte secretory processes (11). For example, ACh
increases the secretin-induced stimulation of cAMP synthesis and
Cl/bicarbonate exchanger
activity in both IBDU and purified cholangiocytes by directly
interacting with M3 receptor
subtypes on cholangiocytes (11). All these studies demonstrate that the
secretin receptor and secretin-induced cAMP response are key factors in
the modulation of ductal bile secretion (1-4, 7-12, 19, 28,
30, 50). Our data are consistent and extend the concept that the
secretory processes of the intrahepatic biliary epithelium are
subjected to a tight regulation with stimulatory effects exerted by
secretin (3, 5-8, 10-12, 19, 30, 50), bombesin (15), and ACh (11) and an opposing inhibition by ET-1, somatostatin (4, 50), and
gastrin (19). Moreover, the data further support the notion that the
secretin receptor and some of the biological events associated with its
expression (i.e., cAMP response) tightly regulate the secretory
processes of cholangiocytes in both normal and proliferating rat liver
(1-4, 7-12, 19, 28, 30, 50). Indeed, secretin receptor and
secretin-induced cAMP responses are upregulated with all forms of
cholangiocyte proliferation studied to date in which we observed a
marked increase in ductal secretory activity (2, 9, 10, 19, 30, 50).
ET-1 has been shown (14, 25) to reduce bile flow in IPRL, an effect that is mediated by an increase in portal pressure. In our studies, ET-1 did not alter basal bile flow, which is due presumably to the different model used to measure bile flow (i.e., bile fistula rats vs. IPRL) during ET-1 infusion. The absence of ET-1 on basal bile flow suggests that ablation of secretin-induced choleresis by ET-1 is not due to cholestatic changes secondary to altered blood flow. Furthermore, to ensure that the inhibitory effects of ET-1 on secretin-induced ductal bile secretion are due to a direct interaction with cholangiocytes rather than an in vivo indirect effect of ET-1 [e.g., vasoconstriction or increased plasma levels of vasoactive hormones affecting cell function (16, 25, 46)] on cholangiocyte secretion, we examined the effects of ET-1 on ductal bile secretion in pure subpopulations of small and large cholangiocytes and polarized large IBDU that allow us to directly measure cholangiocyte secretory processes (1-3, 7-11, 19, 28, 30, 34, 38, 50).
We found that ETA and
ETB (at both the gene and protein
levels) were expressed by small and large cholangiocytes purified from
normal or BDL rats. Our studies demonstrate a direct interaction of
ET-1 with ETA (but not
ETB) receptors on cholangiocytes
to explain the inhibitory effect that ET-1 has on secretin-induced increases in ductal bile secretion. The inhibitory effect of ET-1 on
secretin-induced cAMP synthesis in purified cholangiocytes and
secretin-induced bicarbonate-rich choleresis supports the concept of
specific, physiologically active receptors for ET-1 on cholangiocytes.
Surprisingly, we found that in normal rats secretin-stimulated cAMP
synthesis in large cholangiocytes is inhibited by even low
concentrations of ET-1, whereas in cholangiocytes from BDL rats the
inhibitory effect of ET-1 on secretin-induced cAMP synthesis is only
observed at 108 M. Perhaps
after BDL there is loss of ET-1 control over secretin-induced cholangiocyte secretory function. For example, the loss of negative feedback on cholangiocyte secretion by ET-1 may lead to higher ductal
secretion in BDL rats. This would be a potential mechanism for
increased ductal secretion in BDL that is independent of increased secretin receptor expression. In support of the view that ET-1 interacts with cholangiocytes through the
ETA receptor, we have demonstrated
that, in the presence of BQ-610, a specific inhibitor of
ETA (BQ-610) receptor (24, 42),
ET-1 does not inhibit secretin-stimulated cAMP synthesis in large
cholangiocytes and secretin-induced lumen duct expansion
in large IBDU. Moreover, in different sets of experiments, we have
demonstrated the presence of endothelin receptors
(ETA and
ETB) at both the RNA and protein
levels in rat cholangiocytes. Finally, consistent with the concept that
ET-1 inhibition of secretin-induced cholangiocyte secretory processes
occurs by interaction with ETA (but not ETB) receptors on
cholangiocytes, we have shown that ET-3 and S6c, specific agonists of
the ETB receptor (14, 39, 55), did
not alter basal or secretin-induced cAMP synthesis and
secretin-stimulated bicarbonate-rich choleresis. Because we found that
cholangiocytes also contain ETB
receptors, we suggest that ET-3 and S6c [specific agonists of the
ETB receptor (14, 39, 55)]
do not affect secretin-stimulated ductal secretory activity because
they are not involved in the cAMP signaling pathway of cholangiocytes.
We believe that our findings have important pathophysiological implications, since little is known about the cellular and molecular regulatory mechanisms of ductal bile secretion. This may lead to a better understanding of the mechanisms of enhanced cholangiocyte secretory processes in a number of cholestatic liver diseases, including extrahepatic biliary obstruction, primary biliary cirrhosis, and primary sclerosing cholangitis. Because endothelin receptor antagonists may be therapeutically employed in the future to promote collagen matrix degradation in patients with chronic liver diseases (39), it is important that we understand the effect of ET-1 on biliary physiology. In summary, the results of the present study provide further insight into the understanding of the regulatory mechanisms of ductal bile secretion and the potential involvement of ET-1 in clinical cholestatic conditions characterized by increased bile duct proliferation and ET-1 overexpression.
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ACKNOWLEDGEMENTS |
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We thank Bryan Moss for his outstanding photographic and art work.
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FOOTNOTES |
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This work was supported by a grant award to G. Alpini from Scott and White Hospital and Texas A&M University Health Science Center College of Medicine and by a Veterans Affairs Merit Award Grant. The work was also supported in part by a grant award from Scott and White Hospital to A. Caligiuri and S. Glaser.
Present address of A. Caligiuri: Istituto di Medicina Interna, Universita' di Firenze, 50134 Firenze, Italy.
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: G. Alpini, Internal Medicine and Medical Physiology, Texas A&M Univ. Health Science Center, College of Medicine and Central Texas Veterans Health Care System, Bldg. 147, Olin E. Teague Veterans Center, 1901 South 1st St., Temple, TX 76504.
Received 12 January 1998; accepted in final form 1 July 1998.
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