Department of Internal Medicine, Scott and White Hospital, Temple, Texas 76508; and Texas A&M University Health Science Center College of Medicine, Temple, Texas 76504
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ABSTRACT |
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We assessed the effect of gastrin on ductal secretion in normal and bile duct-ligated (BDL) rats. The effect of gastrin on ductal secretion was examined in the presence of proglumide, a specific antagonist for gastrin receptor (GR). We isolated pure cholangiocytes from normal and BDL rats and assessed gastrin effects on secretin receptor (SR) gene expression and intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels. We examined the presence of GR mRNA in cholangiocytes by reverse transcription polymerase chain reaction (RT-PCR). In normal or BDL rats, gastrin produced no changes in spontaneous bile secretion. Simultaneous infusion of gastrin inhibited secretin-induced choleresis and bicarbonate output in BDL rats. In the presence of proglumide gastrin did not inhibit secretin-induced choleresis in BDL rats. Gastrin decreased in cholangiocytes from BDL rats 1) SR gene expression and 2) secretin-induced cAMP levels. With the use of RT-PCR, GR mRNA was detected in cholangiocytes. Similar to what is shown for secretin and somatostatin, we propose that the opposing effects of secretin and gastrin on cholangiocyte secretory activity regulate ductal secretion in rats.
bile duct ligation; biliary epithelium; hormones; proglumide; adenosine 3',5'-cyclic monophosphate
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INTRODUCTION |
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INTRAHEPATIC BILE DUCT epithelial cells or cholangiocytes are simple epithelia that line the complex network of interconnecting conduits of different diameters that form the intrahepatic biliary tree in the liver (4). Bile formation occurs in two steps, first by osmotically driven secretion from hepatocytes across the canalicular membrane (38) and then followed by hormone-regulated modification of bile in the intrahepatic biliary tree by a series of absorptive and secretory processes (1, 3-5, 8-10, 19, 27, 53). For example, the gastrointestinal hormone secretin stimulates ductal bile secretion (1, 3, 4, 30, 31, 53) by interaction with specific G-coupled protein receptors located on cholangiocytes (7), which causes an increase in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels (6, 8, 27, 30, 31, 53). Also, other reports (14, 15) have shown that vasoactive intestinal peptide (VIP) and bombesin stimulate ductal bile secretion. On the other hand, Tietz et al. (53) have shown that the hormone somatostatin inhibits both secretin-stimulated intracellular cAMP synthesis and exocytosis in vitro and secretin-stimulated choleresis in vivo in support of the concept that multiple gastrointestinal hormones regulate in concert ductal secretory processes (1, 3, 5, 6, 8, 14, 15, 30, 31, 53). The inhibitory actions of somatostatin in the liver are mediated through interaction with a subtype of receptor (SSTR2) on cholangiocytes (53).
In addition, cholangiocytes have the capacity to markedly proliferate after the application of specific pathological perturbations, including bile duct ligation (BDL) (1, 3, 4) and 70% partial hepatectomy (31). Both maneuvers induce an increase in secretin receptor (SR) gene expression (7, 8, 31) and secretin-induced intracellular cAMP levels in vitro (7, 31, 53) and secretin-stimulated bile secretion in vivo (1, 3, 31, 53), consistent with the concept that cholangiocyte proliferation is associated with increased ductal secretion (1, 3, 4, 7, 8, 31, 53).
Gastrin is a gastrointestinal hormone synthesized by gastric antral G cells (40). Gastrin stimulates the growth of enterochromaffin-like cells of the rat stomach (22). Gastrin stimulates gastric acid secretion and growth of the acid producing mucosa (21, 57). In addition, gastrin increases blood flow to the mucosa, participates in muscle contraction, and promotes growth in a number of epithelia including the gastrointestinal mucosa and colonical epithelial cells (37, 39, 45, 60). On the other hand, other studies indicate that gastrin does not have trophic effects in the oxyntic mucosal D cells (56) and liver (13). All of these trophic effects of gastrin occur through an interaction with specific receptors (28, 36, 50, 59, 60). Gastrin belongs to the family of cholecystokinin (CCK-A and CCK-B) receptors (11). CCK-A receptors are primarily located on pancreas and gallbladder, whereas CCK-B/gastrin receptors (GR) are located on the brain, smooth muscle cells, and parietal cells (28).
In vitro studies (16, 17) in the isolated perfused pig liver indicate that the liver plays an important role in the metabolism of circulating gastrin (i.e., gastrin-17 and gastrin-14, but not gastrin-34). However, no data exist regarding the role of gastrin in the regulation of ductal bile secretion in rat liver. Taking into consideration that a number of gastrointestinal hormones (1, 3, 5, 6, 8, 14, 15, 31, 53), including secretin and somatostatin (1, 3, 5, 6, 8, 31, 53), regulate in a cooperative fashion ductal bile secretion, we studied the effects of gastrin on 1) spontaneous and secretin-induced choleresis, 2) SR gene expression, and 3) both basal and secretin-stimulated cAMP synthesis. Finally, we examined for the presence of GR on cholangiocytes by reverse transcription polymerase chain reaction (RT-PCR). On the basis of these observations, we propose that gastrin inhibits in BDL rats secretin-stimulated ductal bile secretion in vivo by interaction with specific receptors on cholangiocytes through a decrease in SR gene expression and secretin-induced cAMP levels.
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MATERIALS AND METHODS |
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Animal model. Male Fischer 344 rats (150 to 200 g) were purchased from Charles River (Wilmington, MA). The animals were maintained in a temperature-controlled environment (20-22°C) with a 12:12-h light-dark cycle and had free access to standard rat chow and water before each experiment. The studies were conducted in normal rats and in rats with cholangiocyte proliferation induced by BDL for 14 days (1, 3). BDL was performed as previously described (1, 3, 7, 53). Before each experiment, animals were anesthetized with pentobarbital sodium (50 mg/kg ip). Study protocols were performed in compliance with the institution guidelines.
Materials. Reagents were purchased from Sigma Chemical (St. Louis, MO) unless otherwise indicated. Secretin was purchased from Peninsula Laboratories (Belmont, CA). Human gastrin I (i.e., gastrin-17) was purchased from (Calbiochem, La Jolla, CA). The mouse anti-cytokeratin 19 (CK-19) antibody was purchased from Amersham (Arlington Heights, IL). Radioimmunoassay (RIA) kits for the determination of intracellular cAMP levels in cholangiocytes from normal and BDL rats were purchased from Amersham.
In situ immunohistochemistry. The number of intrahepatic bile ducts present in frozen liver sections (6-µm thick) randomly obtained from normal and BDL rats was determined by immunohistochemistry for CK-19 (2, 31), a specific marker for cholangiocytes in rat liver (2, 4, 5). Only two to three intrahepatic bile ducts (stained for CK-19) were present within portal areas of frozen sections obtained from normal rat liver (results not shown). After BDL there was a marked increase in the number of intrahepatic bile ducts limited to portal areas (results not shown). These results are in agreement with a number of reports (1, 2, 53).
Isolation and phenotypic characterization of
cholangiocyte preparations from normal and BDL rat
livers. After standard collagenase perfusion, a mixed
nonparenchymal cell fraction (40-55% pure by -glutamyltranspeptidase) (7, 25) was obtained from intact portal
tracts of both normal and BDL rat liver and further purified by
immunoaffinity separation (5, 7, 25, 31, 53) using a monoclonal
antibody expressed by all intrahepatic cholangiocytes (25). By this
approach, virtually pure preparations of cholangiocytes were obtained
from both normal and BDL rat livers. Cell number and viability (
99%)
were assessed by trypan blue exclusion.
In vivo studies of biliary physiology.
Before being used for in vivo studies of biliary physiology, rats were
fed ad libitum. After anesthesia with pentobarbital sodium (50 mg/kg
ip), both normal and BDL rats were surgically prepared for bile
collection as previously described (1, 3, 31, 53). Briefly, the jugular
vein was cannulated with a PE-50 cannula (Clay-Adams, New York, NY) to
infuse either Krebs-Henseleit bicarbonate solution (KRH), gastrin,
gastrin plus secretin, or secretin alone dissolved in KRH (see below).
Blood was withdrawn every 10 min from one carotid artery to assess the
arterial hematocrit, which remained constant (41-45%) in all
animals 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 2.964 ml/h. Body temperature was
monitored with a rectal thermometer (Yellow Springs Instruments, Yellow
Springs, OH) and maintained at 37°C with a heating pad. When
steady-state bile flow was achieved (60-70 min from the beginning
of bile collection), we infused gastrin
(107 M) for 30 min, then
KRH for 60 min followed by simultaneous infusion of gastrin
(10
7 M) and secretin
(10
7 M) for 30 min, then
KRH for 60 min followed by infusion of secretin (10
7 M) for 30 min and a
final infusion of KRH for 60 min. In a separate set of experiments we
also determined in BDL rats the in vivo dose-dependent effects of
gastrin
(10
7-10
10
M) on both secretin-induced choleresis and bicarbonate output.
The effect of gastrin on both spontaneous and secretin-induced bile
flow and bicarbonate output was also examined in the presence of
proglumide
[N-(benzoyl)-L-glutamic
acid-1-di-n-propylamide] (75 mg · kg body
wt1 · h
1),
a specific antagonist for the GR (26, 59, 60). The dose of gastrin used
in the present studies was similar to that of other reports (26). When
spontaneous steady-state bile flow was achieved (60-70 min from
the beginning of bile collection), proglumide was infused until
steady-state bile flow was once more achieved. Then, in the presence of
proglumide, gastrin (10
7 M)
and secretin (10
7 M) were
infused together for 30 min, followed by a final infusion of KRH for 60 min. At the end of each experiment, the animal was euthanized with an
overdose of sedative (200 mg/kg ip) and the liver was removed and
weighed. During each experiment, bile was collected every 10 min in
preweighed tubes and immediately stored at
20°C before
determining bicarbonate concentration. Bile volume was determined by
weight, assuming a density of 1.0 g/ml. Bile flow was expressed as
microliters per minute per kilogram body weight. Bicarbonate
concentration (measured as total
CO2) in bile from control and
BDL rats was determined by a Natelson microgasometer apparatus
(Scientific Industries, Bohemia, NY).
Molecular localization of GRs on cholangiocytes from normal and BDL rats. To determine if the effects of gastrin on secretin-induced ductal bile secretion occur by interaction with specific receptors on cholangiocytes, we examined the genetic expression of GR mRNA by RT-PCR using cholangiocyte poly(A)+ mRNA obtained from normal and BDL rat liver. To examine for the presence of GR mRNA in cholangiocytes we used specific primers (sense 5'-TGTGCAACTTCCGCGTTCC-3' and antisense 5'-GCTGATGGTGGTATAGCTTAGCC-3', expected fragment length 444 bp) designed from the sequence encoding for the rat GR gene (28). Poly(A)+ mRNA from rat brain and yeast tRNA were the positive and negative controls, respectively, for the GR gene. The comparability of the total messenger RNA used was assessed by RT-PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the housekeeping gene (5, 7). Primers for GAPDH were based on the rat GAPDH sequence (sense 5'-GTGACTTCAACAGCAACTCCCATTC-3' and antisense 5'-GTTATGGGGTCTGGGATGGAATTGTG-3', expected fragment length 294 bp). The following RT-PCR conditions were used with 5 ng of poly(A)+ mRNA (35 step cycles: 30 s at 94°C, 30 s at 58°C, and 45 s at 72°C). To confirm the identity, the PCR fragments were sequenced using a Sequenase version 2.0 kit (United States Biochemical, Cleveland, OH). Poly(A)+ mRNA was extracted from isolated cholangiocytes (2 × 106) and tissues (100 mg) by the Micro-Fast Track II kit (Invitrogen, San Diego, CA) (7) according to the instructions supplied by the vendor. The relative intensity of the autoradiograms was determined by densitometry.
In vitro effect of gastrin on SR gene
expression. After isolation, pure preparations of
cholangiocytes (2.0 × 106) from both normal and BDL
rat liver were treated in the presence of 0.2% bovine serum albumin
(BSA) for 15 min at 37°C with gastrin (10
7 M), secretin
(10
7 M), gastrin plus
secretin (both at 10
7 M),
or 0.2% BSA (control) and subsequently analyzed for the genetic expression of the SR gene. We used rat heart and rat kidney RNA as
positive and negative controls, respectively, for the SR gene. The
equality of the RNA used was assessed by hybridization with the
housekeeping gene GAPDH (5, 7). Rat kidney and yeast tRNA were the
positive and negative controls, respectively, for the GAPDH gene. The
expression of selected messages was determined by lysate ribonuclease
protection assay (Direct Protect, Ambion, Austin, TX) (31) according to
the instructions of the vendor; each cell lysate sample contained 4.50 × 105 pure cholangiocytes
obtained from normal or BDL rat liver. This approach has been
previously used by us (31) to exactly quantitate SR gene expression
among cholangiocytes isolated from normal and partial hepatectomized
rat livers.
Antisense riboprobes were transcribed from linearized cDNA templates
with T7 or
SP6 RNA polymerase using
[-32P]UTP (800 Ci/mmol) (Amersham). The following
[32P]UTP labeled
single-stranded antisense riboprobes were used: a 316-bp riboprobe
encoding a complementary sequence for rat GAPDH mRNA was obtained from
cDNA purchased from Ambion and a 318-bp riboprobe encoding the message
for SR was transcribed from pGEM4Z-SR (a gift of Dr. N. LaRusso,
Rochester, MN) (7, 31).
Intracellular cAMP levels. Both
spontaneous and hormone-induced intracellular cAMP levels were measured
in isolated cholangiocytes as previously described by us and others (5,
6, 27, 30, 31, 41, 53). After purification, pure preparations of
cholangiocytes (1.0 × 105)
were incubated for 1 h at 37°C to regenerate membrane proteins damaged by treatment with proteolytic enzymes (6, 27, 31, 48) and
subsequently stimulated in the presence of 0.2% BSA with gastrin
(107 M), secretin
(10
7 M), gastrin plus
secretin (both at 10
7 M),
or BSA for 5 min at 22°C, a temperature at which cAMP synthesis is
commonly measured (5, 8, 27, 30, 31, 41, 53). Before determination of
intracellular cAMP levels, a phosphodiesterase inhibitor, 0.5 mM
3-isobutylmethylxanthine, was added to all cholangiocyte preparations
used (5, 8, 27, 30, 31, 41, 53). After ethanol extraction, spontaneous
and agonist-induced cAMP formation was measured by RIA using commercial
kits (Amersham) according to the instructions supplied by the vendor.
Statistical 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 analysis of variance (ANOVA) if more than two groups were analyzed.
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RESULTS |
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In vivo studies of biliary physiology.
In normal rat liver, spontaneous bile flow (80.75 ± 6.69 µl · min1 · kg body
wt
1) and basal bicarbonate output (2.37 ± 0.15 µeq · min
1 · kg
body wt
1) were similar to
that of previous studies (1, 3, 30, 31, 53) (see Fig.
1, A and
B). As expected (1, 3, 30, 31, 53),
in normal rats secretin did not alter ductal bile secretion (75.72 ± 9.00 µl · min
1 · kg
body wt
1) or bicarbonate
output (2.41 ± 0.16 µeq · min
1 · kg body
wt
1) compared with their
corresponding basal values (80.75 ± 6.69 µl · min
1 · kg
body wt
1 and 2.37 ± 0.15 µeq · min
1 · kg
body wt
1, respectively, see
above) (Fig. 1, A and
B). In normal rats, intravenous
infusion of gastrin (10
7
M), alone or in combination with secretin
(10
7 M), did not induce
changes in bile secretion or bicarbonate output (see Fig. 1,
A and B).
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In agreement with a number of reports (1, 3, 30, 31, 53), after BDL
there was a significant increase in both spontaneous bile secretion
(99.88 ± 4.65 µl · min1 · kg
body wt
1,
P < 0.05) and bicarbonate output
(3.48 ± 0.20 µeq · min
1 · kg
body wt
1,
P < 0.05) compared with normal
control rats (80.75 ± 6.69 µl · min
1 · kg
body wt
1 and 2.37 ± 0.15 µeq · min
1 · kg
body wt
1, respectively)
(Fig. 1, A and
B). As expected (1, 3, 30, 31, 53),
in BDL rats secretin induced a massive increase in both bile secretion
(+79.37 ± 9.19 µl · min
1 · kg
body wt
1,
P < 0.05 vs. its corresponding basal
value) and biliary bicarbonate output (+7.24 ± 1.15 µeq · min
1 · kg
body wt
1 vs. its
corresponding basal value, P < 0.05)
(Fig. 1, A and
B). In BDL rats, gastrin alone
(10
7 M) did not cause
significant changes in bile secretion (106.75 ± 6.01 vs. 99.88 ± 4.65 µl · min
1 · kg
body wt
1 vs. its
corresponding basal value) or bicarbonate output (3.19 ± 0.30 vs. 3.48 ± 0.20 µeq · min
1 · kg
body wt
1 vs. its
corresponding basal value) (Fig. 1, A
and B). The dose-response curve
shows that gastrin, similar to what was observed at
10
7 M (Fig. 1,
A and
B), did not alter significantly bile
secretion or bicarbonate output when infused in vivo in BDL rats at the concentration of 10
10 M
(+9.81 ± 5.59 µl · min
1 · kg
body wt
1 and +0.069 ± 0.21 µeq · min
1 · kg
body wt
1, respectively, vs.
its corresponding basal value),
10
9 M (+1.97 ± 5.09 µl · min
1 · kg
body wt
1 and +0.33 ± 0.14 µeq · min
1 · kg
body wt
1, respectively, vs.
its corresponding basal value), or
10
8 M (+0.97 ± 3.10 µl · min
1 · kg
body wt
1 and +0.27 ± 0.33 µeq · min
1 · kg
body wt
1, respectively, vs.
its corresponding basal value). In contrast, simultaneous infusion of
gastrin and secretin (both at
10
7 M) markedly decreased
secretin-induced choleresis (133.26 ± 9.34 vs. 195.78 ± 22.88 µl · min
1 · kg
body wt
1,
P < 0.05) and biliary bicarbonate
output (5.41 ± 0.59 vs. 10.72 ± 1.11 µeq · min
1 · kg
body wt
1,
P < 0.05) in BDL rats (see Fig. 1,
A and
B). In a fashion similar to that
observed at the concentration of
10
7 M (Fig.
1A), gastrin also inhibited the
choleretic effect of secretin (i.e., +79.37 ± 9.19 µl · min
1 · kg
body wt
1; Fig.
2A) at
both 10
8 M (+22.81 ± 5.85 vs. +79.37 ± 9.19 µeq · min
1 · kg
body wt
1,
P < 0.05) and
10
9 M (+35.31 ± 10.95 vs. +79.37 ± 9.19 µl · min
1 · kg
body wt
1,
P < 0.05) (Fig.
2A). Similarly, gastrin markedly
decreased secretin-induced bicarbonate output (i.e., +7.24 ± 1.15 µeq · min
1 · kg
body wt
1; Fig.
2B) at both
10
8 M (+1.49 ± 0.45 vs.
+7.24 ± 1.15 µeq · min
1 · kg
body wt
1,
P < 0.05) and
10
9 M (+1.63 ± 0.71 vs.
+7.24 ± 1.15 µeq · min
1 · kg
body wt
1,
P < 0.05) (Fig.
2B).
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Consistent with the concept that specific GRs are present in
cholangiocytes, simultaneous infusion of gastrin
(107 M) and proglumide (75 mg · kg body
wt
1 · h
1)
did not inhibit secretin-induced choleresis (251.69 ± 15.38 vs.
149.88 ± 8.52, basal value, P < 0.05; Fig.
3B) or
secretin-stimulated bicarbonate output (14.21 ± 1.17 vs. 4.78 ± 0.07, basal value, P < 0.05; Fig.
3C) typical of BDL. As expected,
proglumide did not alter the effect of gastrin and secretin on bile
flow or bicarbonate excretion in normal rats (Fig. 3,
A and
C, respectively). In agreement with
previous reports (50), proglumide caused an increase in bile flow in
both normal (175.52 ± 12.16 vs. 85.77 ± 10.98 µl · min
1 · kg
body wt
1,
P < 0.05) and BDL (144.00 ± 6.02 vs. 109.00 ± 9.86 µl · min
1 · kg
body wt
1,
P < 0.05) rats (see Fig. 3,
A and
B). Similarly, proglumide increased
biliary bicarbonate excretion in both normal (5.74 ± 0.34 vs. 2.27 ± 0.21 µeq · min
1 · kg
body wt
1,
P < 0.05) and BDL (5.08 ± 0.36 vs. 3.24 ± 0.37 µeq · min
1 · kg
body wt
1,
P < 0.05) rats (Fig.
3C).
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Localization of GRs on cholangiocytes.
As shown in Fig. 4 by RT-PCR a 444-bp
product was detected in cholangiocytes from both normal and BDL rat
livers. Sequence analysis of the PCR fragment for GR mRNA showed 100%
homology to the published sequence for the GR gene (28). In a fashion
similar to that observed for other genes (e.g., SR and
SSTR2), the expression of GR
mRNA was greater (1.5-fold) in isolated cholangiocytes from BDL rats
compared with normal cholangiocytes (Fig.
4B). The expression of GAPDH mRNA
[the housekeeping gene (5, 7, 8)] was similar between normal and proliferating cholangiocytes in agreement with our previous
studies (5, 7, 8, 53).
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Effect of gastrin on SR gene
expression. To begin to investigate the mechanisms by
which gastrin, in concert with secretin, modulates ductal secretory
activity we studied, in cholangiocytes from both normal and BDL rats,
the effect of gastrin on the genetic expression of SR, an important
marker of ductal bile secretion (4, 5, 7, 31). The genetic expression
of GAPDH, the housekeeping gene used to assess the comparability of the
RNA used (5, 7, 8), was similar among basal and hormone-stimulated cholangiocytes (Fig.
5A). As
shown in Fig. 5A, a 318-bp transcript for the SR gene was present in heart RNA (positive control) (5, 7, 8,
31). In agreement with our previous studies (5, 7, 8, 31), SR gene
expression was detected in normal cholangiocytes (Fig. 5,
A and
B). In normal cholangiocytes,
gastrin, secretin, or gastrin plus secretin (all at
107 M) had no effect on SR
gene expression (Fig. 5, A and
B). After BDL, there was an
approximately twofold increase in SR gene expression, a finding that
parallels our previous studies (7) (Fig. 5, A and
B). In cholangiocytes from BDL rats,
secretin (10
7 M) increases
(
70%) SR gene expression (Fig. 5,
A and
B) that parallels the increase in
ductal bile secretion observed during in vivo secretin infusion (Fig.
1A). In a fashion similar to that observed for ductal bile flow (Fig. 1), gastrin (in combination with
secretin) decreases (
50%) the genetic expression of SR in isolated
cholangiocytes from BDL rat livers (Fig. 5,
A and
B). Gastrin alone did not affect SR
gene expression (Fig. 5, A and B). Densitometric data are the means
of two experiments.
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Basal and hormone-regulated intracellular cAMP
levels. In normal cholangiocytes, basal intracellular
cAMP levels (18.71 ± 2.64 fmol/100,000 cells) were
similar to that reported in previous studies by us (31) and others (27)
(Fig. 6). Spontaneous intracellular cAMP
levels markedly increased in cholangiocytes from BDL rats compared
with normal cholangiocytes (28.98 ± 1.30 vs. 18.71 ± 2.64 fmol/100,000 cells, P < 0.05) (Fig.
6). As expected, secretin (107 M) markedly increased
intracellular cAMP levels in both normal (35.00 ± 3.93 fmol/100,000
cells, P < 0.05 vs. basal value) and hyperplastic (60.17 ± 3.36 fmol/100,000 cells,
P < 0.05 vs. basal value)
cholangiocytes (Fig. 6). Gastrin
(10
7 M) alone did not
affect basal intracellular cAMP levels in cholangiocytes from normal or
BDL rats (see Fig. 6), but markedly inhibited secretin-induced increases in cAMP levels in both normal (17.65 ± 0.43 vs. 35.00 ± 3.93 fmol/100,000 cells, P < 0.05) and proliferating cholangiocytes (19.40 ± 0.36 vs. 60.17 ± 3.36 fmol/100,000 cells, P < 0.05) (Fig. 6). The data closely parallel the inhibitory effect of
gastrin on bile secretion (Figs. 1 and 2) and SR gene expression
(Fig. 5).
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Similar to that observed with
107 M gastrin (Fig. 6),
dose-response analysis shows that gastrin did not affect intracellular cAMP synthesis in hyperplastic cholangiocytes at the concentration of
10
8 (23.40 ± 2.80 vs.
28.98 ± 1.30 fmol/100,000 cells, basal value), 10
9 (24.56 ± 5.35 vs.
28.98 ± 1.30 fmol/100,000 cells, basal value), or
10
10 M (16.72 ± 6.26 vs. 28.98 ± 1.30 fmol/100,000 cells, basal value). Parallel with
the changes in secretin-induced choleresis observed after gastrin
administration (Figs. 1 and 2), gastrin markedly decreased
secretin-induced cAMP synthesis in proliferating cholangiocytes at the
concentration of 10
8 (15.71 ± 3.25 vs. 60.17 ± 3.36 fmol/100,000 cells,
P < 0.05), 10
9 (23.98 ± 4.75 vs.
60.17 ± 3.36 fmol/100,000 cells, P < 0.05), and 10
10 M
(17.35 ± 0.85 vs. 60.17 ± 3.36 fmol/100,000 cells,
P < 0.05) (Fig.
7).
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DISCUSSION |
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These novel studies demonstrated the role of gastrin in the regulation
of secretin-stimulated ductal bile secretion in BDL rat liver. In
normal rats, in vivo infusion of gastrin at
107 M, alone or in
combination with secretin
(10
7 M), produced no
significant changes in bile secretion or biliary bicarbonate output. In
BDL rats, gastrin alone
(10
7-10
10
M) produced no significant changes in bile secretion or bicarbonate output. In contrast, simultaneous infusion of gastrin
(10
7-10
9
M) plus secretin (10
7 M)
markedly decreased both secretin-induced choleresis and biliary bicarbonate output in BDL rats. Gastrin, when infused simultaneously with proglumide [a specific inhibitor for the GR, (26, 59, 60)], did not inhibit secretin-induced choleresis or
secretin-stimulated bicarbonate output in BDL rats. In isolated
cholangiocytes, we have shown that gastrin decreased SR gene expression
and secretin-induced cAMP synthesis. Finally, we have detected GR mRNA
in cholangiocytes isolated from both normal and BDL rats by RT-PCR. On
the basis of these observations, we propose that gastrin inhibits
secretin-induced choleresis by interaction with specific receptors on
cholangiocytes by decreasing SR gene expression and secretin-induced
cAMP levels, two important regulatory determinants of ductal secretory
activity (5, 7, 8, 27, 31, 53).
The intrahepatic biliary tree is the principal site for
hormone-regulated ductal bile secretion in normal and
pathophysiological states including BDL (1, 3-5, 7-10, 14,
15, 19, 27, 53). The gastrointestinal hormone secretin stimulates both
in vivo (1, 3, 30, 31, 53) and in vitro (5, 8, 9, 27, 31, 53) ductal
bile secretion by interacting with specific receptors located in rat
liver solely on cholangiocytes (7). The interaction of secretin with
its receptor leads to an increase in cAMP (5, 8, 30, 31, 53), the most
important determinant of ductal secretory activity (5, 8, 27, 30, 31,
53). The increase in intracellular cAMP levels causes opening of
Cl channels (10, 19) and
Cl
/
exchanger activity (5, 9), which leads to the secretin-stimulated
bicarbonate-rich choleresis (1, 3, 31, 53). In addition, VIP and
bombesin stimulate ductal secretory activity both in vivo and in
isolated intrahepatic bile duct fragments (14, 15). Also, Tietz et al.
(53) have shown that somatostatin inhibits both secretin-induced cAMP
and exocytosis in vitro and secretin-induced choleresis in vivo by
interacting with specific (i.e.,
SSTR2) receptors on
cholangiocytes. In contrast to humans, where ductal bile secretion is
40% of total bile secretion (38), ductal bile flow in normal rats
represents only 10% of total bile volume (3), SR gene expression is
very low (7) and secretin does not increase bile secretion (1, 3, 20, 31, 53). In these studies, we employed the BDL model in which SR gene
expression and intracellular cAMP levels are elevated in vitro (7, 8,
53) and in which secretin stimulates ductal bile secretion in vivo (1,
3, 53).
Gastrin, which stimulates gastric acid secretion and growth of the acid
producing mucosa (21, 57), also has trophic effects in a number of
organs including pancreas and intestine (18, 39, 45, 60). On the other
hand, other investigators have shown that gastrin does not have
stimulatory effects in the oxyntic mucosal D-cells (56) or in the liver
(13). All these effects of gastrin on these organs occur by interaction
with specific GRs (28, 36, 50, 59, 60). GRs have also been found in brain (58), colon cancer (44), and small cell cancer of the lung (47).
Some studies (16, 17, 35) have shown that the liver is able to
metabolize circulating gastrin (i.e., gastrin-17 and gastrin-14, but
not gastrin-34). However, no data exist regarding the role of gastrin
in the regulation of ductal secretion in rat liver. The present data
indicate a direct interaction of gastrin on cholangiocytes (presumably
through an interaction with specific receptors) for an explanation of
the inhibitory effect of gastrin, both in vitro and in vivo, on
secretin-stimulated ductal bile secretion. To acquire evidence for the
presence of specific receptors for gastrin on cholangiocytes, we
studied the dose-dependent effect of gastrin (from
107 to
10
10 M) on secretin-induced
choleresis and bicarbonate output in vivo (Fig. 2,
A and
B) and secretin-induced cAMP
synthesis in vitro (Fig. 7), which is a functional assay for the SR (5,
8, 20, 27, 31, 53). The presence of an inhibitory effect of gastrin on
both secretin-induced choleresis and bicarbonate output (Fig. 2,
A and
B) and secretin-stimulated
intracellular cAMP (Fig. 7) at a physiological dose [blood
gastrin concentration of
10
9-10
10
M in rats (29)] supports the concept of specific, physiologically relevant receptors for gastrin on cholangiocytes. Furthermore, we have
also shown that in the presence of proglumide, a specific GR
antagoonist (26, 50, 59, 60), gastrin does not inhibit either
secretin-induced choleresis (Fig.
3A) or biliary bicarbonate output
(Fig. 3B) in BDL rats, consistent
with the concept that gastrin inhibition of secretin-induced ductal
bile secretion occurs by interaction with specific receptors on
cholangiocytes. Finally, further evidence for the presence of receptors
on cholangiocytes comes from our in vitro molecular analysis (Fig. 4)
showing that a 444-bp product, 100% homology to the published sequence
for the GR gene (28), was detected in both normal and hyperplastic cholangiocytes. Upregulation of the genetic expression of GR in BDL rat
liver may increase cholangiocyte sensitivity to gastrin, leading to
inhibition of secretin-stimulated ductal bile secretion by gastrin in
BDL rats. Our studies are the first report of the presence of GRs in
the liver with evidence of an inhibitory effect of gastrin on SR gene
expression and secretin-induced cAMP levels in vitro and
secretin-stimulated bicarbonate rich choleresis in vivo.
Secretin and gastrin receptors are different with respect to organ or cell type distributions, binding of agonists or antagonists, second messenger systems, different physiological effects, and structures (57). On the basis of these differences, SR belongs to a receptor super family containing SR, VIP, and parathyroid hormone receptors (23, 24), whereas GR is in the family of CCK (CCK-A and CCK-B) receptors (11). GR employs Ca2+-dependent pathways (55) and not cAMP as a second messenger system (33, 51). Consistent with its known signaling pathway (33, 51, 55), gastrin did not alter basal intracellular cAMP levels or basal bile flow in BDL rats (Figs. 1 and 6). The lack of effect of gastrin on both spontaneous bile secretion in vivo (Fig. 1) and basal cAMP levels in vitro (Fig. 6) contrasts with somatostatin, which inhibits both spontaneous ductal bile flow and basal cAMP levels in cholangiocytes from BDL rats (53).
We have considered a number of other potential effects of gastrin on ductal bile flow other than through interaction with GR on cholangiocytes. For example, gastrin may increase somatostatin levels or reduce endogenous secretin release and thereby inhibit ductal bile flow (51). Yet, in previous studies (46, 49) gastrin had no effect on the release of somatostatin or secretin from isolated cells or circulatory levels in vivo. We have considered that gastrin may interact with its closely related CCK-A receptor, but previous studies have shown that stimulation of CCK-A receptors with CCK leads to an increase in ductal bile secretion rather than the decrease (42) we observed in this study. Although it can be argued that proglumide, which we employed in these studies as a specific GR blocker, has some effects on CCK-A receptors (34), the more specific blocker L-365,260 could not be used because of its poor solubility and bioavailability in vivo (12, 35).
Our demonstration of secretin expression and physiological responses of
GR in cholangiocytes has both physiological and pathophysiological relevance. In a fashion similar to that shown in the stomach (43), the
opposing effects of secretin and gastrin also regulate ductal bile
secretion. This study and others (1, 3-10, 14, 15, 19, 20, 27, 30,
31, 53) support the concept that the intrahepatic biliary epithelium is
subjected to a very tight hormonal regulation, with stimulatory effects
exerted by secretin (1, 3, 5, 8, 31, 53), bombesin (14), and VIP (15) and an opposing inhibition by somatostatin (53) and gastrin (20).
Because preliminary data (20, 32) indicate that, similar to
somatostatin (6, 52, 54), gastrin also exerts inhibitory effects on
cholangiocyte proliferation, the regulation of secretory processes
could be tightly coupled with that of proliferation and thus explain
the reason for the involvement of many different hormones
and/or neuropeptides. The inhibitory effects of gastrin on both
secretin-induced cAMP synthesis and biliary bicarbonate excretion
suggest that gastrin may affect (in addition to secretin and its
receptor) the function of a number of hormone-regulated ion
transporters (e.g., cystic fibrosis transmembrane regulator and
Cl/
exchanger). Finally, the presence of GR on cholangiocytes opens up new
therapeutic windows for the diagnosis and treatment of
cholangiocarcinoma. GR could be used similarly to somatostatin receptor
(52) to image cholangiocarcinomas with radiolabeled ligands.
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ACKNOWLEDGEMENTS |
---|
The authors thank Brian Moss for outstanding photographic and art work.
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FOOTNOTES |
---|
This work was supported by a grant award to both G. Alpini and G. D. LeSage from Scott & White Hospital and Texas A&M University Health Science Center, College of Medicine.
Portions of this study have been presented at the American Gastroenterological Association and have been published in abstract form (Gastroenterology 110: 1197, 1996).
Address for reprint requests: G. Alpini, Internal Medicine, Texas A & M Univ. Health Science Center, College of Medicine, Bldg. 147, Olin E Teague Veterans Center, 1901 South 1st St., Temple, TX, 76504.
Received 21 May 1997; accepted in final form 24 July 1997.
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