1 Center for Basic Research in Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Medical School, Clinic, and Foundation, Rochester, Minnesota 55905; and 2 Medizinische Klinik m. S. Hepatologie, Gastroenterologie, Endokrinologie und Stoffwechsel, Universitätsklinikum Charité, Campus Virchow-Klinikum, Humboldt Universität Berlin, 13353 Berlin, Germany
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ABSTRACT |
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With an in vitro model using enclosed intrahepatic bile duct units (IBDUs) isolated from wild-type and somatostatin receptor (SSTR) subtype 2 knockout mice, we tested the effects of somatostatin, secretin, and a selective SSTR2 agonist (L-779976) on fluid movement across the bile duct epithelial cell layer. By RT-PCR, four of five known subtypes of SSTRs (SSTR1, SSTR2A/2B, SSTR3, and SSTR4, but not SSTR5) were detected in cholangiocytes in wild-type mice. In contrast, SSTR2A/2B were completely depleted in the SSTR2 knockout mice whereas SSTR1, SSTR3 and SSTR4 were expressed in these cholangiocytes. Somatostatin induced a decrease of luminal area of IBDUs isolated from wild-type mice, reflecting net fluid absorption; L-779976 also induced a comparable decrease of luminal area. No significant decrease of luminal area by either somatostatin or L-779976 was observed in IBDUs from SSTR2 knockout mice. Secretin, a choleretic hormone, induced a significant increase of luminal area of IBDUs of wild-type mice, reflecting net fluid secretion; somatostatin and L-779976 inhibited (P < 0.01) secretin-induced fluid secretion. The inhibitory effect of both somatostatin and L-779976 on secretin-induced IBDU secretion was absent in IBDUs of SSTR2 knockout mice. Somatostatin induced an increase of intracellular cGMP and inhibited secretin-stimulated cAMP synthesis in cholangiocytes; depletion of SSTR2 blocked these effects of somatostatin. These data suggest that somatostatin regulates ductal bile formation in mice not only by inhibition of ductal fluid secretion but also by stimulation of ductal fluid absorption via interacting with SSTR2 on cholangiocytes, a process involving the intracellular cAMP/cGMP second messengers.
bile flow; biliary secretion; cholestasis; secretin; receptors
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INTRODUCTION |
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BILE, MAINLY COMPOSED OF SOLUTES and water, is initially produced by hepatocytes and subsequently delivered to the intrahepatic bile ducts. The biliary tree not only delivers bile to the intestine but also participates in ductal bile formation by integrated absorptive and secretory processes that can contribute up to 40% of the daily output of bile depending on the species (4, 49). Recently, it has become clear that cholangiocytes selectively express a wide array of transporters, exchangers, channels, and receptors on their apical and basolateral plasma membrane domains that provide them with an enormous capacity to transfer water and solutes bidirectionally across the biliary epithelial barrier spontaneously or in response to hormones (26, 38). For example, both secretin and somatostatin receptors are expressed on the basolateral plasma membrane domain of cholangiocytes (9, 17, 31). Previous studies showed that secretin increases the intracellular levels of cAMP to stimulate exocytosis and thus increases ductal bile flow, resulting in choleresis; the molecular mechanisms likely involve membrane recycling of solute and ion transporters and water channels (17, 27). Although in vivo studies have demonstrated that somatostatin inhibits ductal bile flow, resulting in cholestasis (28, 31, 36, 50), the mechanism by which this hormone decreases ductal bile flow, especially its effects on cholangiocytes, is unclear.
Somatostatin is a cyclic tetradecapeptide with a wide spectrum of biological action mediated by five somatostatin receptor (SSTR) subtypes (SSTR1-SSTR5). These SSTRs are heterogeneously distributed in various tissues including the brain, gastrointestinal tract, pancreas, and pituitary gland (5, 18, 35). Somatostatin plays an important role in many physiological processes, such as neurotransmission, growth hormone release, cell proliferation, and inhibition of gastrointestinal motility and pancreatic enzyme secretion (6, 22, 42, 54). In the liver, somatostatin has been demonstrated to be cholestatic and to inhibit both basal and secretin-stimulated ductal bile secretion (24, 28, 31, 36, 50). However, there is only limited knowledge about SSTR expression on cholangiocytes and receptor subtypes that mediate the cholestatic effect of somatostatin. We previously demonstrated (50) that expression of SSTR2 increases markedly in cholangiocytes in rats after bile duct ligation, suggesting a role for SSTR2 in the regulation of ductal bile formation.
The rationale of the current study was therefore to test the effects of somatostatin on ductal bile formation in mice and to evaluate the role of SSTR2 in this process. We characterized the effects of somatostatin, secretin, and a selective agonist of SSTR2, L-779976, on net fluid secretion and absorption across the biliary epithelial barrier with an in vitro model using enclosed intrahepatic bile duct units (IBDUs) isolated from wild-type and SSTR2 knockout mice. SSTR gene expression in cholangiocytes, as well as effects of somatostatin and secretin on intracellular cAMP and cGMP synthesis, were measured in isolated, purified cholangiocytes. We found that somatostatin stimulates ductal fluid absorption and inhibits secretin-stimulated bile secretion in mice, resulting in decreased bile flow, a process involving regulation of intracellular cAMP/cGMP synthesis and mediated by interaction with SSTR2 on cholangiocytes.
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MATERIALS AND METHODS |
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Reagents. Somatostatin and secretin were purchased from Peninsula Laboratories (Belmont, CA). L-779976, a selective SSTR2 agonist (41), was provided by Dr. L. Koch (Merck Research Laboratories, Rahway, NJ), and the identification and characterization of this compound were described in detail in previous reports (41). Collagenase type II and trypsin were purchased from Worthington Biochemical (Lakewood, NJ). EGTA, DNase, collagenase type XI, and hyaluronidase were purchased from Sigma-Aldrich (St. Louis, MO). A monoclonal anti-mouse cytokeratin-19 antibody was from Amersham Life Science (Little Chalfont, UK), and a polyclonal antibody to SSTR2A/B was from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal rat anti-mouse epithelial cell adhesion molecule (EPCAM) antibody was generated from a G8.8 hybridoma cell line (Developmental Studies Hybridoma Bank, Iowa City, IA), and M-450 sheep anti-rat IgG Dynabeads were obtained from Dynal (Lake Success, NY). ELISA kits for the determination of intracellular cAMP and cGMP levels were purchased from Sigma-Aldrich.
Animals.
Mice deficient in SSTR2 [homozygous SSTR2 (/
) knockout] were
generated by gene targeting in mouse embryonic stem cells as previously
described (47, 55). Corresponding wild-type adult C57BL/129 mice (10-12 wk) were used as controls. Mice were
maintained under controlled conditions at 25°C with food and water
available ad libitum. All animal experimental protocols were approved
by the Animal Use and Care Committee of the Mayo Foundation.
Solutions. The composition of isotonic (290 mosmol/kgH2O) HEPES-buffered saline (HBS) was (in mM) 140 NaCl, 5.4 KCl, 0.8 Na2HPO4, 25 HEPES, 2.5 glucose, 2 CaCl2, and 0.8 MgSO4, pH 7.4. The composition of isotonic (290 mosmol/kgH2O) Krebs-Ringer bicarbonate (KRB) buffer was (in mM) 120 NaCl, 5.9 KCl, 1.2 Na2HPO4, 1 MgSO4, 1.25 CaCl2, 5 glucose, and 25 NaHCO3. The precise osmolality of solutions was determined with a freezing point osmometer (Osmette S; Precision System, Natick, MA).
Isolation of mouse cholangiocytes. Highly purified cholangiocytes were isolated from the livers of wild-type and SSTR2 knockout mice according to the method of Vierling and coworkers (13) with minor modifications. Briefly, mice were anesthetized with pentobarbital sodium (50 mg/kg ip). Livers were first perfused with HBS containing 0.02% EGTA through the portal vein to remove blood cells. Livers were then harvested, transferred to a temperature-controlled chamber at 37°C, and perfused for 10 min with HBS containing 0.05% collagenase type II (Worthington) and 2 mM CaCl2. Hepatocytes were removed by gentle, mechanical disruption of the Glisson's capsule followed by agitation for 10 min at 4°C. The biliary tree segments were then digested with a solution of 0.02% DNase, 0.032% collagenase type XI, and 0.048% hyaluronidase (Sigma) for 30 min at 37°C and passed three times through a 19-gauge needle. After cells were incubated with 0.08% trypsin (Worthington) for 15 min, passing three times through a 22-gauge needle and filtering through a 70-µm Falcon cell strainer, cholangiocytes were immunomagnetically isolated from other cell types with a rat anti-EPCAM antibody. Cholangiocytes opsonized with anti-EPCAM were bound to Dynabeads bearing anti-rat IgG and isolated in a magnetic field. The viability of freshly isolated cholangiocytes was >90% by Trypan blue exclusion, and the purity was >95% by positive immunostaining to cytokeratin-19, a specific protein marker for cholangiocytes.
RNA extraction and reverse transcription-polymerase chain reaction. Total cellular RNA was extracted from freshly isolated mouse cholangiocytes with Tri-Reagent (Sigma). Isolated cholangiocytes were lysed in 1 ml of Tri-Reagent per 5 × 106 cells with 2.5 µl of Glyco-Blue (Ambion, Austin, TX) added as a coprecipitant and incubated at room temperature for 5 min. After addition of 0.2 ml of 1-bromo-3-chloropropane per 1 ml of Tri-Reagent, samples were mixed vigorously, incubated for 15 min at room temperature, and centrifuged at 12,000 g for 15 min at 4°C. The aqueous phase was collected and transferred to a new tube; to this, 0.5 ml of isopropanol was added per 1 ml of Tri-Reagent used for the initial lysis. Samples were stored for 10 min at room temperature and centrifuged at 12, 000 g for 15 min at 4°C. After the supernatant was removed, the RNA pellet was washed with 1 ml of 75% ethanol and repelleted by centrifugation at 12,000 g for 15 min at 4°C. RNA was resuspended in RNA Secure Solution (Ambion), and the concentration and purity were determined by spectrophotometry.
Total RNA (5 µg) was reverse-transcribed to cDNA by using a Moloney murine leukemia virus reverse transcriptase kit (MMLV-RT kit; Life Technologies, Rockville, MD). RNA was first incubated with a random hexamer as primer at 65°C for 6 min and then reacted with a mixture in a total volume of 50 µl at 37°C for 50 min and 95°C for 5 min. The mixture contained first-strand reaction buffer, dithiothreitol, deoxynucleotide triphosphates, RNase inhibitor, and MMLV-RT. After reverse transcription (RT), cDNA was amplified with the polymerase chain reaction (PCR) with gene-specific primers designed to amplify a portion of the coding sequences of each of the five mouse SSTR genes (Table 1). The PCR reaction consisted of 1 cycle of 10-min denaturation at 94°C; 35 cycles of 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C; and a final extension step at 72°C for 10 min. Products of RT-PCR amplification were analyzed by 1.5% agarose gel electrophoresis, and the bands were visualized by ethidium bromide staining. Equal amounts of cDNA for each lane were analyzed, and all experiments were done in duplicate. Control PCR reactions were performed in parallel with template cDNA prepared from brain as positive control and using total RNA without RT as a genomic DNA contamination control. Sequencing was performed on all positive PCR products (Mayo Molecular Core Facility, Rochester, MN) to confirm the identity of amplified genes.
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Isolation of IBDUs from mouse liver. The technique used to isolate IBDUs from mice was developed by us and has been previously described in detail (12). Briefly, wild-type and SSTR2 knockout mice (10-12 wk) were anesthetized with pentobarbital sodium (50 mg/kg ip). The liver was perfused with ice-cold saline through the portal vein. Subsequently, 2-3 ml of liquid Trypan blue agar was injected into the portal vein. The liver was then removed and immersed in an ice-cold, preoxygenated HBS buffer. After the hepatic capsule and surface hepatocytes were removed, intrahepatic bile ducts were dissociated under a dissection microscope by using the Trypan blue agar-filled portal vein as reference. The dissociated bile duct was digested by shaking at 37°C for 10 min in an enzyme solution (containing RPMI 1640 supplemented with 0.032% collagenase XI, 0.016% DNase, and 0.03% hyaluronidase). Further microdissection was performed at higher magnification to remove residual hepatocytes, components of the portal veins and hepatic arteries, and excess connective tissues. The isolated intrahepatic bile ducts with luminal diameters ranging from 25 to 100 µm were cut into 0.5-mm segments and transferred to eight-well culture chambers that were coated with poly-L-lysine hydrobromide (Sigma). After being cultured 16-24 h in DMEM-F-12 medium plus 10% fetal bovine serum, penicillin (20,000 U/ml)-streptomycin (20,000 µg/ml), and gentamicin (20 µg/ml), the intrahepatic bile duct segments sealed spontaneously and formed enclosed IBDUs.
Immunoblot of SSTR2 in IBDUs. IBDUs isolated from both wild-type and SSTR2 knockout mice were cultured overnight and lysed with the M-PER mammalian protein extraction reagent (Pierce, Rockford, IL), and protein concentrations were determined with Bradford reagent according to the instructions of the supplier (Sigma-Aldrich). Twenty micrograms of lysate protein per lane was separated by SDS-polyacrylamide gel electrophoresis under reducing conditions and blotted onto nitrocellulose membranes. Membranes were sequentially incubated with the SSTR2 antibody (Santa Cruz Biotechnology) and then with 0.2 µg/ml of horseradish peroxidase-conjugated secondary antibody and revealed with enhanced chemiluminescence light substrate (ECL; Amersham).
Determination of ductal fluid absorption and secretion of IBDUs
in response to somatostatin, L-779976, and secretin.
Net ductal fluid absorption and secretion across biliary epithelia in
response to a cholestatic agent (somatostatin and L-779976) and a
choleretic agent (secretin) were determined with a quantitative image
analysis technique based on the changes of luminal area of enclosed
IBDUs, a methodology previously validated by us (39). Briefly, after 15-min incubation in an isotonic (290 mosmol/kgH2O) KRB buffer, enclosed and polarized IBDUs
isolated from wild-type and SSTR2 knockout mice were treated with
30-min exposure to 1) 107 M somatostatin,
2) 10
7 M L-779976, 3)
10
7 M secretin, 4) 10
7 M
secretin plus 10
7 M somatostatin, and 5)
10
7 M secretin plus 10
7 M L-779976 in an
isotonic (290 mosmol/kgH2O) KRB solution. Somatostatin, secretin, and L-779976 at a concentration of 10
7 M showed
most potent effects based on dose-response experiments and were
selected for the studies. Serial photographs of the same enclosed IBDU
before and after treatment were digitized, and the luminal area of
IBDUs was measured with an image analysis software program (Fig.
1). The results were expressed as percent
change in luminal area from basal values (without treatment).
Characterizations of isolated IBDUs from mice were previously described
by us (12). Those characterizations include retention of
in situ morphology with a lumen surrounded by a single layer of viable
epithelial cells with tight junctions between adjacent cells, an intact
basolateral structure, adequate response to hormones, and capabilities
for both absorptive and secretory activities. No obvious morphological changes, such as decrease of cholangiocyte viabilities by Trypan blue
staining or disruption of tight junctions or seals between adjacent
cells by electron microscopy, was observed in IBDUs after incubation
with somatostatin, secretin, or a combination (data not shown). To
further confirm that somatostatin and L-779976 do not affect the
function of tight junctions in cholangiocytes, an immortalized normal
mouse cholangiocyte cell line (a gift from Dr. Y. Ueno, Tohoku
University School of Medicine, Sendai, Japan) that expresses SSTR2 and
has been fully characterized previously (25) was grown on
12-well inserts to confluence to form polarized monolayers.
Transepithelial electrical resistance across the polarized cholangiocyte monolayers was assessed as described previously (7) before and after 30-min incubation with somatostatin
(10
7 M) or L-779976 (10
7 M) added to the
chambers. No significant change of transepithelial electrical
resistance was detected in the monolayers before and after 30-min
incubation with somatostatin (220.00 ± 7.21 and 222.00 ± 8.33
· cm2, respectively;
P > 0.05) or L-779976 (210.67 ± 10.48 and
206.00 ± 10.58
· cm2,
respectively; P > 0.05).
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Determination of cAMP and cGMP levels in cholangiocytes in
response to somatostatin and secretin.
Intracellular levels of cAMP and cGMP in response to somatostatin and
secretin in cholangiocytes isolated from both wild-type and SSTR2
knockout mice were measured by a modified ELISA method as previously
reported by us (50). Briefly, isolated cholangiocytes were
incubated at 37°C for 4 h to restore membrane proteins that might have been damaged by proteolytic enzyme digestion and
subsequently incubated for 30 min at 37°C in the presence of secretin
(107 M), somatostatin (10
7 M), or secretin
plus somatostatin (both at 10
7 M). Levels of cAMP and
cGMP in cholangiocytes were then detected with commercially available
ELISA kits (Sigma-Aldrich). The concentrations of cAMP and cGMP
detected were then calculated from standard curves and expressed in
femtomoles per 100,000 cells.
Statistical analysis. All data are expressed as means ± SE and represent three independent experiments with five to seven IBDUs each, unless otherwise stated. The unpaired Student's t-test was used for statistical analysis. P < 0.05 was considered as statistically significant.
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RESULTS |
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Expression of SSTRs in isolated mouse cholangiocytes.
RT-PCR was carried out to identify transcripts encoding SSTRs in highly
purified mouse cholangiocytes. Reverse-transcribed cDNA from highly
purified mouse cholangiocytes was amplified by PCR with specific
primers for the five cloned murine SSTRs. As shown in Fig.
2A, PCR amplification of
reverse-transcribed mRNA prepared from highly purified cholangiocytes
immunoisolated from wild-type mice produced strong, sharp bands
corresponding to SSTR1, SSTR2A, SSTR2B, SSTR3, and SSTR4, whereas no
SSTR5 signal was detected. Two bands for SSTR2 have been identified
with primers that amplify SSTR2A and -2B. In the control experiments
using cDNA derived from mouse brain tissue as the template (Fig.
2B), all five SSTRs were amplified, consistent with previous
reports (8, 10, 53).
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Effects of somatostatin and L-779976 on ductal fluid absorption in
IBDUs isolated from wild-type and SSTR2 knockout mice.
In the absence of somatostatin, IBDUs isolated from wild-type mice
showed no change in the luminal area after incubation in an isotonic
(290 mosmol/kgH2O) buffer for 30 min, indicating the absence of net fluid movement across the epithelial barrier. After incubation with 107 M somatostatin in an isotonic KRB
buffer for 30 min, IBDUs isolated from wild-type mice displayed a
significant decrease (16.72 ± 1.85%; P < 0.01)
of luminal area (Fig. 3A),
reflecting net fluid absorption from the lumen. To identify whether
SSTR2 plays a role in somatostatin-mediated ductal fluid absorption, we
investigated the effects of a novel nonpeptidyl selective agonist
specific for SSTR2, L-779976, on bile fluid absorption across the
biliary epithelial barrier. Exposure of IBDUs isolated from wild-type mice with 10
7 M L-779976 for 30 min resulted in a
significant decrease (15.39 ± 1.83%; P < 0.01)
of IBDU luminal area (Fig. 3B), similar to the
somatostatin-stimulated decrease of luminal area in IBDUs. These data
suggest that somatostatin, as well as its SSTR2-selective agonist,
L-779976, directly induce net fluid absorption across the biliary
epithelial barrier by interacting with SSTR2.
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Effects of somatostatin and L-779976 on secretin-induced fluid
secretion in IBDUs isolated from wild-type and SSTR2 knockout mice.
To test the effect of somatostatin on choleretic hormone-stimulated
ductal bile secretion, we observed the effects of somatostatin and
L-779976 on secretin-induced ductal bile secretion in mice. Our
previous in vivo studies in the rat demonstrated that somatostatin inhibits secretin-stimulated ductal bile secretion
(50). In the absence of somatostatin or L-779976,
IBDUs isolated from wild-type mice showed a significant increase
(26.7 ± 3.56%) of the luminal area after incubation in an
isotonic (290 mosmol/kgH2O) buffer containing
107 M secretin for 30 min, indicating net fluid secretion
across the epithelial barrier. In the presence of somatostatin or
L-779976, a significant decrease (by 58.91% and 65.75%, respectively,
compared with secretin treatment alone) of secretin-induced ductal
fluid secretion was detected (Fig. 4),
suggesting that somatostatin, as well as its SSTR2-selective agonist,
L-779976, inhibits secretin-induced net fluid secretion across the
biliary epithelial barrier by interacting with SSTR2.
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Effects of somatostatin and secretin on intracellular levels
of cAMP and cGMP in cholangiocytes isolated from wild-type and SSTR2
knockout mice.
To explore the molecular mechanisms by which somatostatin
stimulates ductal bile absorption and inhibits secretin-induced ductal
bile secretion, we observed the effects of somatostatin on
intracellular levels of cAMP and cGMP in cholangiocytes in the presence
or absence of secretin. Cholangiocytes isolated from wild-type mice
showed a significant increase of intracellular cAMP level, but not
cGMP, after incubation with secretin for 30 min (Fig.
5). A significant increase of cGMP level,
but not cAMP, was detected in cholangiocytes of wild-type mice after a
30-min incubation with somatostatin (Fig. 5). In the presence of
somatostatin, a significant inhibition of secretin-induced cAMP
increase in cholangiocytes of wild-type mice was detected,
suggesting inhibition of secretin-induced cAMP synthesis by
somatostatin. However, no significant difference of intracellular cGMP
levels was detected between cells exposed to somatostatin alone and
cells treated with somatostatin plus secretin (Fig. 5).
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DISCUSSION |
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The major findings reported here relate to the molecular mechanisms by which somatostatin regulates ductal bile formation. Using an in vitro model of enclosed IBDUs isolated from both wild-type and SSTR2 knockout mice, we found that 1) four of the five known subtypes of SSTRs (SSTR1, SSTR2A/2B, SSTR3, and SSTR4) are expressed in wild-type cholangiocytes; 2) somatostatin and L-779976, a selective somatostatin analog specific to SSTR2, both directly stimulate ductal fluid absorption and inhibit secretin-induced ductal fluid secretion in mouse IBDUs; 3) deletion of SSTR2 leads to diminished increase of ductal fluid absorption and attenuated inhibition of secretin-stimulated fluid secretion induced by somatostatin or L-779976; and 4) somatostatin induces intracellular cGMP synthesis and inhibits secretin-stimulated cAMP synthesis in cholangiocytes, and deletion of SSTR2 completely blocks these effects of somatostatin. Our results provide the first functional evidence that somatostatin regulates murine ductal bile formation through both stimulation of bile fluid absorption and inhibition of fluid secretion via interacting with SSTR2 on cholangiocytes, a process that may involve the intracellular cAMP/cGMP second messengers.
Ductal bile formation is the net result of the bidirectional movement
of ion/water molecules across the biliary epithelial barrier, a process
regulated by hormones (4, 49). Cholangiocytes express a
wide array of transport, exchanger, channel, and receptor proteins on
their apical and basolateral plasma membrane domains. Those located in
or near the secretory pole (apical domain), such as the cystic fibrosis
transmembrane regulator (CFTR) Cl channel,
Cl
/HCO
/HCO
secretion. The increase of ion/solute
transport into the lumen generates an inward osmotic gradient and thus
drives water secretion into the lumen (3, 17, 27, 37, 39,
46). Consistent with previous in vivo studies (28, 31, 36,
50), we found a significant inhibitory effect on
secretin-stimulated ductal fluid secretion by somatostatin or its
agonist L-779976 in IBDUs isolated from wild-type mice. A significant
inhibition of secretin-stimulated cAMP synthesis in cholangiocytes by
somatostatin was detected, supporting the notion that somatostatin, by
blocking intracellular cAMP production (50), inhibits the
secretin-stimulated transport of AQP1 water channels and the activation
of the Cl
/HCO
Interestingly, when enclosed IBDUs were incubated with somatostatin or
the SSTR2-selective agonist L-779976 alone, a significant decrease of
luminal area was detected, suggesting that somatostatin can directly
induce bile fluid absorption, consistent with our previous studies
using enclosed IBDUs isolated from rat liver (7). A
decrease in bile flow by somatostatin has also been reported in in vivo
studies in the rat, dog, and human (18, 28, 31, 36). One
possible mechanism by which somatostatin may directly stimulate ductal
fluid absorption is to induce absorption of Na+ and
Cl from bile, thus generating an inward osmotic gradient
to drive water absorption. Intravenous administration of somatostatin
or its analogs induces a marked increase of both net Na+
and Cl
absorption in the intestine in the rat, rabbit,
and human (40, 43). Stimulation by somatostatin of
Na+-Cl
transport and consequent increases in
ion and water absorption have also been reported to occur in the
gallbladder (48). Absorption of other molecules via
transporters/exchangers expressed on the apical membrane of
cholangiocytes, such as glucose via SGLT1 and bile acids by ASBT, may
also be involved. Indeed, our most recent studies (29)
using a microperfused model of IBDUs isolated from rats showed that
cholangiocytes absorb glucose from the lumen, resulting in net water
absorption across the biliary epithelial barrier. Despite the fact that
the intrahepatic biliary tree is primarily thought to be a secretory
organ, our results provide new evidence that it can also absorb fluid
in response to hormones such as somatostatin.
cGMP has been reported as a second messenger for somatostatin in many
cell types (11, 30). In the present study, we found a
significant increase of intracellular cGMP level in cholangiocytes in
response to somatostatin, whereas somatostatin alone showed no effect
on the intracellular level of cAMP in cholangiocytes, suggesting that
cGMP may be the second messenger for somatostatin in cholangiocytes.
However, the experimental data on the response of cholangiocytes to
extracellular cGMP are quite controversial. Cholangiocytes or bile
ducts from rat appear not to respond to extracellular cGMP (45,
52). Another report suggests that cell-permeant cGMP analogs may
even stimulate Cl secretion in a human bile duct
epithelial cell line. Cellular responses to extracellular cGMP vary in
different species and under different experimental conditions
(14, 15, 32). cGMP has opposite effects in the mucosal and
serosal compartments of the rat jejunum, i.e., mucosal cGMP increases
fluid secretion and serosal cGMP enhances fluid absorption (14,
15). Additional studies are currently under way to fully test
the importance of intracellular cGMP in cholangiocytes in
somatostatin-induced ductal bile absorption.
Somatostatin induces biological cell responses by interacting with specific receptors (SSTRs) expressed on the target cell membrane. Recent studies have shown that multiple SSTRs (SSTR1-SSTR5) are heterogeneously distributed in various tissues. For example, high levels of SSTRs are expressed in the brain, gastrointestinal tract, and pancreas (5, 18, 35). In the present study, we found that four of five known transcripts encoding SSTRs (SSTR1-4 but not SSTR5) were present in highly purified cholangiocytes immunoisolated from wild-type mice. Both of the two isoforms of SSTR2 (SSTR2A/2B), were identified in cholangiocytes as reported in other tissues (8, 53). No increase of intracellular cGMP was detected in cholangiocytes isolated from SSTR2 knockout mice after incubation with somatostatin. Deletion of SSTR2 also resulted in a much lower decrease of luminal area in IBDUs isolated from SSTR2 knockout mice after treatment with somatostatin compared with that in wild-type mice. Inhibition of secretin-stimulated cAMP synthesis by somatostatin was not detected in cholangiocytes isolated from SSTR2 knockout mice. Moreover, the inhibitory effect of both somatostatin and L-779976 on secretin-induced IBDU secretion was completely absent in IBDUs from SSTR2 knockout mice. Those findings strongly support the notion that somatostatin directly stimulates fluid absorption and inhibits fluid secretion via interacting with SSTR subtype 2 on cholangiocytes. Because there is still a small inhibitory effect seen with somatostatin in IBDUs of SSTR2 knockout mice (Fig. 3A), we cannot completely exclude the possibility of involvement of other SSTRs expressed on cholangiocytes. Moreover, the biliary tree is characterized by significant structural and functional heterogeneity of cholangiocytes including their receptor expression, ion transporting capabilities, and proliferative capacity. Previous reports demonstrated that, in rats, SSTR2 is expressed in large but not small bile ducts (1, 2). Further studies should address the heterogeneity of SSTR expression along the biliary tree and the role of somatostatin on other effects in cholangiocytes, such as proliferation (51).
In conclusion, with highly purified mouse cholangiocytes and an in vitro model using enclosed IBDUs isolated from wild-type and SSTR2 knockout mice, we have demonstrated that cholangiocytes express multiple SSTRs and that somatostatin regulates ductal bile formation through both stimulation of fluid absorption and inhibition of fluid secretion by interacting with SSTR2 on cholangiocytes in mice, a process that may be mediated by the intracellular cAMP/cGMP second messengers. Future studies will be necessary to define the molecular mechanisms of somatostatin-stimulated ductal bile absorption, for example, which ions or solutes are involved in generating osmotic gradients for water absorption.
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ACKNOWLEDGEMENTS |
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The authors acknowledge K. Jansen for invaluable advice on growth and maintenance of hybridoma cultures and D. Hintz for secretarial assistance. The G8.8 hybridoma cell line, developed by Dr. Andrew Farr, was obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the National Institute of Child Health and Human Development and maintained by the Department of Biological Sciences, University of Iowa, Iowa City, IA 52242.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-24031 and DK-57993 (to N. F. LaRusso), Deutsche Forschungsgemeinschaft (Str558/2-1, to M. Z. Strowski), the American Liver Foundation (to A.-Y. Gong), and the Mayo Foundation.
Address for reprint requests and other correspondence: N. F. LaRusso, Center for Basic Research in Digestive Diseases, Mayo Clinic, 200 First St., SW, Rochester, MN 55905 (E-mail: larusso.nicholas{at}mayo.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpcell.00313.2002
Received 8 July 2002; accepted in final form 30 December 2002.
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