Endothelin-1 inhibits secretin-stimulated ductal secretion by interacting with ETA receptors on large cholangiocytes

Alessandra Caligiuri1, Shannon Glaser1, Rebecca E. Rodgers1, Jo Lynne Phinizy1, Willie Robertson1, Emanuela Papa1, Massimo Pinzani2, and Gianfranco Alpini1,3

1 Department of Internal Medicine and Medical Physiology, Scott and White Hospital and Texas A&M University Health Science Center College of Medicine, and 3 Central Texas Veterans Health Care System, Temple, Texas 76504; and 2 Istituto di Medicina Interna, Universita' di Firenze, 50134 Firenze, Italy

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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- 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).

Cholangiocytes are the target cells in a number of animal models of ductal hyperplasia, including bile duct ligation (BDL), alpha -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).

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).

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animal Model

Male Fischer 344 rats (150-200 g) were purchased from Charles River (Wilmington, MA) and were maintained at 22°C with a 12:12-h light-dark cycle and fed ad libitum with standard rat chow. Before each experiment, the animals were anesthetized with pentobarbital sodium (50 mg/kg ip). Experiments were performed in normal rats and rats with ductal hyperplasia induced by BDL for 2 wk (5, 6, 19, 50). Experimental protocols were conducted in compliance with institution guidelines.

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 for gamma -glutamyltranspeptidase (gamma -GT) (40), a cholangiocyte-specific marker (5, 7, 26)] was separated into two distinct subpopulations of small and large cholangiocytes by counterflow elutriation using a Beckman J6-MI centrifuge equipped with a JE-5.0 rotor (Beckman Instruments, Fullerton, CA). The two subpopulations of small and large cholangiocytes were obtained at the flow rates of 25 and 55 ml/min, respectively, and further purified by immunoaffinity purification (26) using a monoclonal antibody to an antigen ubiquitously expressed on all intrahepatic cholangiocytes (26). Cell number and viability were determined by trypan blue exclusion. Cell purity was assessed by histochemistry for gamma -GT (40). Contamination by hepatocytes and mesenchymal cells was determined by glucose-6-phosphatase histochemistry (49) and vimentin immunohistochemistry (2), respectively. Mean diameters of small and large cholangiocytes purified from both normal and BDL rats were measured by computerized image analysis using a technique recently described by us in cholangiocytes (10) and similar to that described in other cells (47).

Large 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 [alpha -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).

The following RNA probes were used: a 316-bp riboprobe complementary to rat GAPDH mRNA was obtained from cDNA purchased from Ambion. Both ETA and ETB cDNAs were generated in our laboratory by RT-PCR with the GeneAmp RNA PCR kit (Perkin-Elmer, Branchburg, NJ) using poly(A)+ mRNA obtained from pure preparations of cholangiocytes. Primers for ETA mRNA were based on the rat ETA sequence (31) (sense: 5'-GCCCTTGGAGAGACCTTATCTACGTG-3'; antisense: 5'-GGGTACCATGACGAAGCCGATT-3'), with an expected fragment length of 273 bp. Primers for ETB mRNA were based on the rat ETB sequence (41) (sense: 5'-TCCTGCATTAATCCAATCGCTG-3'; antisense: 5'-CTGGAGCGGAAGTTGTCGTATC-3'), with an expected fragment length of 130 bp. Standard RT-PCR conditions were used with 5 ng of poly(A)+ mRNA (35 step cycles: 30 s at 94°C, 30 s at 52°C, and 45 s at 72°C). Poly(A)+ mRNA was extracted from cholangiocytes by the Micro-Fast Track II kit (Invitrogen, San Diego, CA) according to the instructions supplied by the vendor. After ligation into the EcoR I site of pCR vector (TA cloning kit, Invitrogen) to confirm their identity, the PCR fragments were sequenced using a Sequenase version 2.0 kit (United States Biochemical, Cleveland, OH).

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 (10-7 M) in 0.2% BSA for 30 min at 37°C. The genetic expression of secretin receptor was subsequently determined by lysate RNase protection assay (Direct Protect, Ambion) according to the instructions from the manufacturer (see Molecular analyses of ETA and ETB in small and large cholangiocytes from normal and BDL rats). The comparability of the cell lysates was tested by hybridization with GAPDH, employed as the housekeeping gene (1-4, 19, 50). After exposure for 2 days, autoradiograms were quantified by scanning video densitometry using the ChemiImager 4000 low-light imaging system (Alpha Innotech).

After linearization of cDNA templates with the appropriate restriction endonuclease, antisense riboprobes were transcribed with T7 or SP6 RNA polymerase using [gamma -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 (10-7 M), ET-1 (10-10 to 10-7 M), or secretin (10-7 M) plus ET-1 (10-10 to 10-7 M) in the presence of 0.2% BSA. In separate sets of experiments, we also evaluated the effects of ET-1 (10-7 M) on basal and secretin-induced cAMP levels in the presence of BQ-610 (10-7 M), a specific inhibitor for the ETA receptor (24, 42). To ensure that BQ-610 does not differentially affect basal cAMP synthesis, cAMP levels of large cholangiocytes from normal or BDL rats were also determined in the absence of BQ-610. Finally, we evaluated the effects of ET-3 and S6c (both at 10-7 M) on basal and secretin-stimulated cAMP synthesis. Phosphodiesterase activity was inhibited by 0.5 mM IBMX. After ethanol extraction, spontaneous and agonist-induced intracellular cAMP levels were measured by RIA using commercial kits (Amersham) (2, 3, 8, 10, 19, 28, 30, 50).

Measurement 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 10-7 M), ET-1 (10 min at 10-7 M), or ET-1 plus secretin (10 min each, both at 10-7 M), in the absence or presence of the ETA inhibitor BQ-610. The methodology for measuring changes in bile duct volume in response to agonists has been described in great detail in previous studies by us and others (3, 34, 38).

In 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 (10-7 M) for 30 min, KRH until new steady state was reached, and then secretin plus ET-1 (both 10-7 M) for 30 min, followed by a final infusion of KRH. In separate sets of experiments, we also determined 1) the effect of ET-1 at different concentrations (10-10 to 10-7) and 2) the effect of ET-3 and S6c (both at 10-7 M) on both basal and secretin-induced choleresis. The dose of secretin (10-7 M) used in the present studies is the same as that of in vitro and in vivo studies from us and others (2, 3, 8, 10, 19, 28, 30, 50). In normal rats, blood secretin concentration ranges from 10-11 to 10-12 M (44, 52). The doses of ET-1 employed in the present studies (10-7 to 10-10 M, slightly above physiological doses) are, however, in the range of that used in other cell systems by a number of investigators (27, 32). The plasma concentrations of both ET-1 and ET-3 ranged from 10-11 to 10-12 M (20, 36). Throughout the experiment, bile was collected every 10 min and bile flow was determined by weight, assuming a density of 1.0 g/ml. Bile bicarbonate concentration (measured as CO2) was determined in bile by a Natelson microgasometer apparatus (Scientific Industries, Bohemia, NY).

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 ANOVA if more than two groups were analyzed.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Isolation, Morphological, and Phenotypic Characterizations of Small and Large Cholangiocytes or Large IBDU

We isolated virtually pure (by gamma -GT histochemistry) subpopulations of small and large cholangiocytes from both normal and BDL rat liver (results not shown). No hepatocytes or mesenchymal cells were detected in our cholangiocyte preparations (results not shown) (2). Cell viability was >99%. Pure preparations of large IBDU (by gamma -GT histochemistry) were obtained from normal and BDL rats (results not shown). The purity and morphological, phenotypic, and functional characteristics of isolated IBDU from normal or BDL rats have been described in great detail in previous studies (1, 3).

Molecular, 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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Fig. 1.   A: analysis of the genetic expression of endothelin-1 (ET-1) receptors (ETA and ETB) in small and large cholangiocytes purified from both normal and bile duct-ligated (BDL) rats by counterflow elutriation followed by immunoaffinity separation (see MATERIALS AND METHODS). In both small and large cholangiocytes, expression of the selected messages was determined by direct RNase protection assay using cell lysate samples each containing 4.5 × 105 pure cholangiocytes (see MATERIALS AND METHODS). Comparability of the RNA used was assessed by hybridization for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the housekeeping gene. Autoradiograms (n = 2) were quantified by densitometry (B). Note that the messages for ETA (273 bp) and ETB (130 bp) receptors were present in both small and large cholangiocytes purified from normal or BDL rats.

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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Fig. 2.   Immunohistochemistry for ETA and ETB receptors in virtually pure (by gamma -glutamyltranspeptidase histochemistry) preparations of small and large cholangiocytes obtained from normal (A and C) or BDL (B and D) rats. Note that both small and large cholangiocytes purified from normal or BDL rats stained positively for ETA or ETB. Small and large cholangiocytes were obtained from both normal and BDL rats by counterflow elutriation (at the flow rates of 25 and 55 ml/min, respectively), followed by immunoaffinity purification using an antibody to an antigen expressed by all intrahepatic cholangiocytes (26). Before the staining procedure, immunomagnetic beads (used to purify cholangiocytes) were removed by enzyme digestion (see MATERIALS AND METHODS). Original magnification = ×250.

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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Fig. 3.   Western blot analysis of ET-1 receptors (ETA and ETB) in virtually pure subpopulations of small and large cholangiocytes purified from normal or BDL rat liver. The two subpopulations were obtained by counterflow elutriation followed by immunoaffinity purification (see MATERIALS AND METHODS). Note that a signal (migrating at 35 kDa) for the ETA and ETB receptors was expressed by both small and large cholangiocytes from normal or BDL rats. Quantitative expression of ETA or ETB receptors was determined by densitometry. Densitometric values are means of 2 experiments (results not shown).

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 (10-7 M), there was a significant decrease in secretin receptor gene expression in large cholangiocytes from both normal and BDL rat liver (Fig. 4). mRNA of GAPDH, the housekeeping gene (2, 3, 8, 10, 19, 50), was similarly expressed between the two subpopulations of small and large cholangiocytes purified from normal or BDL rats (see Fig. 4). Densitometric analyses of three experiments is shown in Fig. 4.


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Fig. 4.   Effect of ET-1 on the genetic expression of secretin receptor (SR) in small and large cholangiocytes purified from both normal and BDL rats. After purification, virtually pure preparations of small and large cholangiocytes were incubated with ET-1 (10-7 M) for 30 min at 37°C, and subsequently SR gene expression was determined by direct RNase protection assays (see MATERIALS AND METHODS). Comparability of the RNA used was assessed by hybridization for GAPDH, the housekeeping gene. Autoradiograms in A (n = 3) were quantified by densitometry (B) using the ChemiImager 4000 low-light imaging system. Densitometric values are means ± SE. * P < 0.05 vs. SR gene expression of large control cholangiocytes obtained from normal rats. Statistical analysis was performed with both ANOVA and unpaired t-test.

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 (10-7 M) induced a significant increase in intracellular cAMP levels in large (but not small) cholangiocytes purified from both normal (Fig. 5A) and BDL (Fig. 5B) rat liver. ET-1 (at 10-10 to 10-7 M) did not alter basal intracellular cAMP synthesis in small or large cholangiocytes from both normal and BDL rats (results not shown). In contrast, ET-1 inhibited secretin-stimulated cAMP synthesis in large cholangiocytes from both normal (Fig. 5A) and BDL (Fig. 5B) rats. In the presence of BQ-610, a specific antagonist of the ETA receptor (24, 42), ET-1 did not inhibit secretin-induced cAMP synthesis in large cholangiocytes from normal (Fig. 6A) or BDL (Fig. 6B) rats. BQ-610 did not alter basal cAMP levels in large cholangiocytes purified from normal or BDL rats (Fig. 6). No effect on basal or secretin-induced cAMP levels was seen when small or large cholangiocytes were treated with ET-3 or S6c (results not shown), specific agonists for the ETB receptor (14, 39, 55), which indicates that ET-1 modulates ductal secretory activity by interacting primarily, if not exclusively, with type A receptors.


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Fig. 5.   Effect of ET-1 on secretininduced cAMP levels of cholangiocytes purified from normal (A) or BDL (B) rats. After purification, small and large cholangiocytes were incubated at 37°C for 1 h and subsequently stimulated with 1) 0.2% BSA (control or basal), 2) 0.2% BSA plus secretin (10-7 M), or 3) 0.2% BSA plus ET-1 (10-10 to 10-7 M) plus secretin (10-7 M) (see MATERIALS AND METHODS). # Basal cAMP levels of large cholangiocytes from BDL rats differed from basal cAMP levels of normal large cholangiocytes, P < 0.05. * Intracellular cAMP synthesis of cholangiocytes stimulated with ET-1 plus secretin differed from secretin-stimulated cAMP levels, P < 0.05. ** P < 0.05 vs. basal cAMP levels. Data are means ± SE for 6 rats. Statistical analysis was performed with both ANOVA and unpaired t-test. ns, Not significant.


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Fig. 6.   In purified large cholangiocytes from normal (A) or BDL (B) rats, effect of ET-1 on secretin-induced cAMP synthesis was examined in the presence of the specific inhibitor of ETA (BQ-610) receptor (24, 42). After purification, small and large cholangiocytes from both normal and BDL rats were incubated at 37°C for 1 h, pretreated at 37°C for 10 min with BQ-610 (10-7 M), and subsequently stimulated with 1) 0.2% BSA (control), 2) ET-1 (10-7 M) in the presence of 0.2% BSA, 3) secretin (10-7 M) with 0.2% BSA, or 4) ET-1 (10-7 M) plus secretin (10-7 M) in the presence of 0.2% BSA (see MATERIALS AND METHODS). To ensure that BQ-610 does not alter differentially basal cAMP synthesis, cAMP levels of large cholangiocytes from normal or BDL rats were also determined in the absence of BQ-610. # Basal cAMP levels of large cholangiocytes from BDL rats differed from basal cAMP levels of normal large cholangiocytes, P < 0.05. * P < 0.05 vs. basal cAMP levels. Data are means ± SE for 6 rats. Statistical analysis was performed with both ANOVA and unpaired t-test.

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 (10-7 M), or ET-1 plus secretin (both at 10-7 M). An increase in bile duct lumen volume in large but not small IBDU from normal or BDL rats was observed after stimulation with secretin (10-7 M) (Fig. 7). In a fashion similar to that observed for cAMP synthesis, ET-1 alone did not alter IBDU lumen size (Fig. 7). In contrast, when administered in combination with secretin (both at 10-7 M), ET-1 inhibited secretin-induced lumen expansion in large IBDU from both normal and BDL rat liver (Fig. 7). The inhibitory effect of ET-1 on secretin-increased ductal lumen expansion was abolished by pretreating purified IBDU with BQ-610 (10-7 M) (Fig. 7).


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Fig. 7.   Effect of ET-1 on ductal secretion in large intrahepatic bile duct units (IBDU) from normal (A) or BDL (B) rats following the addition of secretin (10-7 M) or ET-1 plus secretin (both at 10-7 M). After purification, purified large IBDU were mounted in a perfusion chamber of a light microscope, and lumen size was measured before or 10 min after addition of secretin or ET-1 plus secretin. Note that ET-1 decreased the secretin-induced luminal expansion in large IBDU purified from both normal and BDL rats. Inhibitory effects of ET-1 on secretin-induced ductal lumen expansion were completely blocked by BQ-610, a specific inhibitor of the ETA receptor (24, 42). * P < 0.05 vs. basal value. #P < 0.05 vs. secretin-induced lumen expansion.

In Vivo Studies of Biliary Physiology

In normal rats, spontaneous bile secretion (73.96 ± 5.36 µl · min-1 · kg body wt-1) and biliary bicarbonate secretion (2.05 ± 0.11 µeq · min-1 · kg body wt-1) were similar to those reported in previous studies (5, 6, 19, 30, 50). Secretin had no stimulatory effects on bile flow or bicarbonate secretion in normal rats (results not shown). Intravenous infusion of ET-1 (10-10 to 10-7 M) did not alter basal bile flow or biliary bicarbonate secretion in normal rats (results not shown).

In agreement with a number of reports (5, 6, 19, 30, 50), following BDL, both basal bile flow (127.86 ± 8.65 µl · min-1 · 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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Fig. 8.   Dose-dependent effect of ET-1 on secretin-induced choleresis (A) and biliary bicarbonate secretion (B) in BDL rats. When steady-state spontaneous bile flow was achieved (60-70 min from the beginning of bile collection), ET-1 (10-10 to 10-7 M) was simultaneously infused with secretin for 30 min, followed by a final infusion of Krebs-Henseleit bicarbonate solution for 60 min. * P < 0.05 vs. corresponding basal value. # P < 0.05 compared with secretin-induced increases in bile flow or biliary bicarbonate secretion. Data are mean ± SE for 6 rats. Statistical analysis was performed with both ANOVA and unpaired t-test. Note that scales on vertical axes are different.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 gamma -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 10-8 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.

    ACKNOWLEDGEMENTS

We thank Bryan Moss for his outstanding photographic and art work.

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Alpini, G., S. S. Glaser, W. Robertson, J. L. Phinizy, J. Lasater, R. Rodgers, and G. LeSage. Large but not small intrahepatic bile ducts (IBDU) from bile duct ligated (BDL) rats proliferate and are involved in secretin-induced ductal bile secretion (Abstract). Hepatology 24: A84, 1996.

2.   Alpini, G., S. Glaser, W. Robertson, J. L. Phinizy, R. Rodgers, A. Caligiuri, and G. LeSage. Bile acids stimulate proliferative and secretory events in large but not small cholangiocytes. Am. J. Physiol. 273 (Gastrointest. Liver Physiol. 36): G518-G529, 1997[Abstract/Free Full Text].

3.   Alpini, G., S. Glaser, W. Robertson, R. Rodgers, J. L. Phinizy, J. Lasater, and G. D. LeSage. Large but not small intrahepatic bile duct units are involved in secretin-regulated ductal bile secretion in normal rat liver. Am. J. Physiol. 272 (Gastrointest. Liver Physiol. 35): G1064-G1074, 1997[Abstract/Free Full Text].

4.   Alpini, G., S. Glaser, Y. Ueno, L. Pham, P. Podila, A. Caligiuri, G. LeSage, and N. F. LaRusso. Heterogeneity of the proliferative capacity of rat cholangiocytes following bile duct ligation. Am. J. Physiol. 274 (Gastrointest. Liver Physiol. 37): G767-G775, 1998[Abstract/Free Full Text].

5.   Alpini, G., R. Lenzi, L. Sarkozi, and N. Tavoloni. Biliary physiology in rats with bile ductular cell hyperplasia. Evidence for a secretory function of proliferated bile ductules. J. Clin. Invest. 81: 569-578, 1988[Medline].

6.   Alpini, G., R. Lenzi, W.-R. Zhai, P. A. Slott, M. H. Liu, L. Sarkozi, and N. Tavoloni. Bile secretory function of intrahepatic biliary epithelium in the rat. Am. J. Physiol. 257 (Gastrointest. Liver Physiol. 20): G124-G133, 1989[Abstract/Free Full Text].

7.   Alpini, G., J. O. Phillips, and N. F. LaRusso. The biology of the biliary epithelia. In: The Liver; Biology and Pathobiology (3rd ed.), edited by I. Arias, J. L. Boyer, N. Fausto, W. Jakoby, D. Schachter, and D. A. Shafritz. New York: Raven, 1994, p. 623-653.

8.   Alpini, G., S. K. Roberts, S. M. Kuntz, Y. Ueno, S. Gubba, P. Podila, G. LeSage, and N. F. LaRusso. Morphologic, molecular and functional heterogeneity of cholangiocytes from normal rat liver. Gastroenterology 110: 1637-1643, 1996.

9.   Alpini, G., C. Ulrich II, J. Phillips, L. Pham, L. Miller, and N. LaRusso. Upregulation of secretin receptor gene expression in rat cholangiocytes after bile duct ligation. Am. J. Physiol. 266 (Gastrointest. Liver Physiol. 29): G922-G928, 1994[Abstract/Free Full Text].

10.   Alpini, G., C. Ulrich, S. K. Roberts, J. O. Phillips, Y. Ueno, P. Podila, O. Colegio, G. LeSage, L. J. Miller, and N. F. LaRusso. Molecular and functional heterogeneity of cholangiocytes from rat liver after bile duct ligation. Am. J. Physiol. 272 (Gastrointest. Liver Physiol. 35): G289-G297, 1997[Abstract/Free Full Text].

11.   Alvaro, D., G. Alpini, A. M. Jezequel, C. Bassotti, C. Francis, F. Fraioli, R. Romeo, G. LeSage, S. Glaser, and A. Benedetti. Role and mechanisms of acetylcholine in the regulation of cholangiocyte secretory functions. J. Clin. Invest. 100: 1349-1362, 1997[Abstract/Free Full Text].

12.   Alvaro, D., W. K. C. Cho, A. Mennone, and J. L. Boyer. Effect of secretin on intracellular pH regulation in isolated rat bile duct epithelial cells. J. Clin. Invest. 92: 1314-1325, 1993[Medline].

13.   Avery, L., and H. R. Horvitz. A cell that dies during wild-type C. elegans development can function as a neuron in a ced-3 mutant. Cell 51: 1071-1078, 1987[Medline].

14.   Bluhm, R. E., M. G. Frazer, M. Vore, C. W. Pinson, and K. F. Badr. Endothelins 1 and 3: potent cholestatic agents secreted and excreted by the liver that interact with cyclosporine. Hepatology 18: 961-968, 1993[Medline].

15.   Cho, W. K., A. Mennone, S. A. Ryderg, and J. L. Boyer. Bombesin stimulates bicarbonate secretion from rat cholangiocytes: implications for neural regulation of bile secretion. Gastroenterology 113: 311-321, 1997[Medline].

16.   Firth, J. D., and P. J. Ratcliffe. Organ distribution of the three rat endothelin messenger RNAs and the effects of ischemia on renal gene expression. J. Clin. Invest. 90: 1023-1031, 1992[Medline].

17.   Fitz, J. G., S. Basavappa, J. McGill, O. Melhus, and J. A. Cohn. Regulation of membrane chloride currents in rat bile duct epithelial cells. J. Clin. Invest. 91: 319-328, 1993[Medline].

18.   Gandhi, C. R., R. H. Behal, S. A. Harvey, T. A. Nouchi, and M. S. Olson. Hepatic effects of endothelin. Receptor characterization and endothelin-induced signal transduction in hepatocytes. Biochem. J. 287: 897-904, 1992[Medline].

19.   Glaser, S., R. Rodgers, J. L. Phinizy, W. Robertson, J. Lasater, A. Caligiuri, Z. Tretjak, G. LeSage, and G. Alpini. Gastrin inhibits secretin-induced ductal secretion by interaction with specific receptors on rat cholangiocytes. Am. J. Physiol. 273 (Gastrointest. Liver Physiol. 36): G1061-G1070, 1997[Abstract/Free Full Text].

20.   Gonick, H. C., Y. Ding, S. C. Bondy, Z. Ni, and N. D. Vaziri. Lead-induced hypertension: interplay of nitric oxide and reactive oxygen species. Hypertension 30: 1487-1492, 1997[Abstract/Free Full Text].

21.   Hosokawa, M., H. Tsukada, S. Ueda, M. Sakai, M. Okuma, K. Oda, M. Takimoto, and T. Okada. Regulation of ion transport by endothelins in rat colonic mucosa: effects of an ETA antagonist (FR139317) and an ETB agonist (IRL1620). J. Pharmacol. Exp. Ther. 273: 1313-1322, 1995[Abstract].

22.   Housset, C., A. Carayon, B. Housset, C. Legendre, L. Hannoun, and R. Poupon. Endothelin-1 secretion by human gallbladder epithelial cells in primary culture. Lab. Invest. 69: 750-755, 1993[Medline].

23.   Housset, C. N., D. C. Rockey, and D. M. Bissell. Endothelin receptors in rat liver: lipocytes as a contractile target for endothelin 1. Proc. Natl. Acad. Sci. USA 90: 9266-9270, 1993[Abstract].

24.   Illing, B., M. Horn, H. Han, S. Hahn, P. Bureik, G. Ertl, and S. Neubauer. Protective effect of the specific endothelin-1 antagonist BQ610 on mechanical function and energy metabolism during ischemia/reperfusion injury in isolated perfused rat hearts. J. Cardiovasc. Pharmacol. 27: 487-494, 1996[Medline].

25.   Isales, C. M., M. H. Nathanson, and R. Bruck. Endothelin-1 induces cholestasis which is mediated by an increase in portal pressure. Biochem. Biophys. Res. Commun. 191: 1244-1251, 1993[Medline].

26.   Ishii, M., B. Vroman, and N. F. LaRusso. Isolation and morphological characterization of bile duct epithelial cells from normal rat liver. Gastroenterology 97: 1236-1247, 1989[Medline].

27.   Iwasaki, S., T. Homma, Y. Matsuda, and V. Kon. Endothelin receptor subtype B mediates autoinduction of endothelin-1 in rat mesangial cells. J. Biol. Chem. 270: 6997-7003, 1995[Abstract/Free Full Text].

28.   Kato, A., G. J. Gores, and N. F. LaRusso. Secretin stimulates exocytosis in isolated bile duct epithelial cells by a cyclic AMP-mediated mechanism. J. Biol. Chem. 267: 15523-15529, 1992[Abstract/Free Full Text].

29.   Leite, M. F., E. Page, and S. K. Ambler. Regulation of ANP secretion by endothelin-1 in cultured atrial myocytes: desensitization and receptor subtype. Am. J. Physiol. 267 (Heart Circ. Physiol. 36): H2193-H2203, 1994[Abstract/Free Full Text].

30.   LeSage, G., S. Glaser, S. Gubba, W. E. Robertson, J. L. Phinizy, J. Lasater, R. Rodgers, and G. Alpini. Regrowth of the rat biliary tree after 70% partial hepatectomy is coupled to increased secretin-induced ductal bile secretion. Gastroenterology 111: 1633-1644, 1996[Medline].

31.   Lin, H. Y., E. H. Kaji, G. K. Winkel, H. E. Ives, and H. F. Lodish. Cloning and functional expression of a vascular smooth muscle endothelin 1 receptor. Proc. Natl. Acad. Sci. USA 88: 3185-3189, 1991[Abstract].

32.   Markewitz, B. A., J. R. Michael, and D. E. Kohan. Endothelin-1 inhibits the expression of inducible nitric oxide synthase. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L1078-L1083, 1997[Abstract/Free Full Text].

33.   McDonough, P. M., S. L. Stella, and C. C. Glembotski. Involvement of cytoplasmic calcium and protein kinases in the regulation of atrial natriuretic factor secretion by contraction rate and endothelin. J. Biol. Chem. 269: 9466-9472, 1994[Abstract/Free Full Text].

34.   Mennone, A., D. Alvaro, W. Cho, and J. L. Boyer. Isolation of small polarized bile duct units. Proc. Natl. Acad. Sci. USA 92: 6527-6531, 1995[Abstract].

35.   Pinzani, M., S. Milani, R. De Franco, C. Grappone, A. Caligiuri, C. Tosti-Guerra, M. Maggi, P. Failli, C. Ruocco, and P. Gentilini. Endothelin 1 is overexpressed in human cirrhotic liver and exerts multiple effects on activated hepatic stellate cells. Gastroenterology 110: 534-548, 1996[Medline].

36.  Pollock, D. M., B. J. Divish, and T. J. Opgenorth. Stimulation of endogenous endothelin release in the anesthetized rat. J. Cardiovasc. Pharmacol. 22 Suppl.: S295-S298, 1993.

37.   Rieder, H., G. Ramadori, and K. H. Meyer zum BuBuschenfelde. Sinusoidal endothelial liver cells in vitro release endothelin: augmentation by transforming growth factor beta  and Kupffer cell-conditioned media. Klin. Wochenschr. 69: 387-391, 1991[Medline].

38.   Roberts, S., S. Kuntz, G. Gores, and N. LaRusso. Regulation of bicarbonate-dependent ductular secretion assessed by lumenal micropuncture of isolated rodent intrahepatic bile ducts. Proc. Natl. Acad. Sci. USA 90: 9080-9084, 1993[Abstract].

39.   Rockey, D. C., and R. A. Weisiger. Endothelin induced contractility of stellate cells from normal and cirrhotic rat liver: implications for regulation of portal pressure and resistance. Hepatology 24: 233-240, 1996[Medline].

40.   Rutemberg, A. M., H. Kim, J. W. Fishbein, J. S. Hanker, H. L. Wasserkrug, and A. M. Seligman. Histochemical and ultrastructural demonstration of gamma -glutamyl transpeptidase activity. J. Histochem. Cytochem. 17: 517-526, 1969[Medline].

41.   Sakurai, T., M. Yanagisawa, Y. Takuwa, H. Miyazaki, S. Kimura, K. Goto, and T. Masaki. Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. Nature 348: 732-735, 1990[Medline].

42.   Seki, H., M. G. Elder, and M. H. Sullivan. Endothelin-1 regulates human decidual cells through both A- and B-type receptors. Mol. Cell. Endocrinol. 114: 111-116, 1995[Medline].

43.   Serradeil-Le Gal, C., C. Jouneaux, A. Sanchez-Bueno, D. Raufaste, B. Roche, A. M. Preaux, J. P. Maffrand, P. H. Cobbold, J. Hanoune, and S. Lotersztajn. Endothelin action in rat liver. Receptors, free Ca2+ oscillations, and activation of glycogenolysis. J. Clin. Invest. 87: 133-138, 1991[Medline].

44.   Shiratori, K., S. Watanabe, and T. Takeuchi. Role of endogenous secretin and cholecystokinin in intraduodenal oleic acid-induced inhibition of gastric acid secretion in rats. Dig. Dis. Sci. 37: 397-402, 1992[Medline].

45.   Simonson, M. S. Endothelins: multifunctional renal peptides. Physiol. Rev. 73: 375-411, 1993[Free Full Text].

46.   Simonson, M. S., and M. J. Dunn. Cellular signaling by peptides of the endothelin gene family. FASEB J. 4: 2989-3000, 1990[Abstract].

47.   Stefano, G., P. Cadet, and B. Scharrer. Stimulatory effects of opioid neuropeptides on locomotory activity and conformational changes in invertebrate and human immunocytes: evidence for a subtype of delta receptor. Proc. Natl. Acad. Sci. USA 86: 6307-6311, 1989[Abstract].

48.   Strauss, K. I., and D. M. Jacobowitz. Quantitative measurement of calretinin and beta -actin mRNA in rat brain micropunches without prior isolation of RNA. Mol. Brain Res. 20: 229-239, 1993[Medline].

49.   Teutsch, H. F. Improved method for the histochemical demonstration of glucose-6-phosphatase activity. A methodological study. Histochemistry 57: 107-117, 1978[Medline].

50.   Tietz, P., G. Alpini, L. D. Pham, and N. F. LaRusso. Somatostatin inhibits secretin-induced ductal choleresis in vivo and exocytosis by cholangiocytes. Am. J. Physiol. 269 (Gastrointest. Liver Physiol. 32): G110-G118, 1995[Abstract/Free Full Text].

51.   Unneberg, K., M. Mjaaland, E. Helseth, and A. Revhaug. Effects of endothelin-1 on hepatic blood flow. HPB Surg. 9: 153-159, 1996[Medline].

52.   Watanabe, S., T. Takeuchi, and W. Y. Chey. Mediation of trypsin inhibitor-induced pancreatic hypersecretion by secretin and cholecystokinin in rats. Gastroenterology 102: 621-628, 1992[Medline].

53.   Watanabe, T., N. Suzuki, N. Shimamoto, M. Fujino, and A. Imada. Endothelin in myocardial infarction. Nature 344: 114, 1990[Medline].

54.   Yanagisawa, M., H. Kurihara, S. Kimura, Y. Tomobe, M. Kobayashi, Y. Matsui, and Y. Yazaki. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332: 411-415, 1988[Medline].

55.   Zhang, J. X., M. Bauer, and M. G. Clemens. Vessel- and target cell-specific actions of endothelin-1 and endothelin-3 in rat liver. Am. J. Physiol. 269 (Gastrointest. Liver Physiol. 32): G269-G277, 1995[Abstract/Free Full Text].


Am J Physiol Gastroint Liver Physiol 275(4):G835-G846
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