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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
2-adrenergic receptor agonist UK 14,304 inhibits secretin-stimulated ductal secretion by downregulation of the cAMP system in bile duct-ligated rats1Central Texas Veterans Health Care System, 2Department of Medicine, and 3Systems Biology and Translational Medicine, 4Division of Research and Education, Scott & White Hospital and The Texas A&M University System Health Science Center, College of Medicine, Temple, Texas; 5University of Rome "La Sapienza," Polo Pontino, Latina; 6Department of Gastroenterology, Polytechnic University of Marche, Ancona, Italy; 7The University of Texas Houston Medical School, Houston, Texas; and 8Division of Gastroenterology, Tohoku University School of Medicine, Aobaku, Sendai, Japan
Submitted 22 January 2007 ; accepted in final form 16 July 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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PKA CFTR Cl–/HCO3– exchanger in cholangiocytes. We evaluated the expression of 
2A-, 2B-, and 2C-adrenergic receptors in cholangiocytes and the effects of the selective 2-adrenergic agonist UK 14,304, on basal and secretin-stimulated ductal secretion. In normal rats, we evaluated the effect of UK 14,304 on bile and bicarbonate secretion. In bile duct-ligated (BDL) rats, we evaluated the effect of UK 14,304 on basal and secretin-stimulated 1) bile and bicarbonate secretion; 2) duct secretion in intrahepatic bile duct units (IBDU) in the absence or presence of 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of the Na+/H+ exchanger isoform NHE3; and 3) cAMP levels, PKA activity, Cl– efflux, and Cl–/HCO3– exchanger activity in purified cholangiocytes. 2-Adrenergic receptors were expressed by all cholangiocytes in normal and BDL liver sections. UK 14,304 did not change bile and bicarbonate secretion of normal rats. In BDL rats, UK 14,304 inhibited secretin-stimulated 1) bile and bicarbonate secretion, 2) expansion of IBDU luminal spaces, and 3) cAMP levels, PKA activity, Cl– efflux, and Cl–/HCO3– exchanger activity in cholangiocytes. There was decreased lumen size after removal of secretin in IBDU pretreated with UK 14,304. In IBDU pretreated with EIPA, there was no significant decrease in luminal space after removal of secretin in either the absence or presence of UK 14,304. The inhibitory effect of UK 14,304 on ductal secretion is not mediated by the apical cholangiocyte NHE3. 2-Adrenergic receptors play a role in counterregulating enhanced ductal secretion associated with cholangiocyte proliferation in chronic cholestatic liver diseases. bicarbonate secretion; chloride efflux; gastrointestinal hormones; intrahepatic biliary epithelium; protein kinase A
Pathologically, cholangiocytes proliferate or are lost in chronic cholestatic liver diseases (i.e., cholangiopathies) (5, 9). The bile duct-ligated (BDL) rat model, which induces proliferation of cholangiocytes (4) with phenotypes similar to those of normal rats (62), allows us to evaluate changes in basal and secretin-stimulated bicarbonate-rich choleresis (4) following the in vivo administration of selected agonists or antagonists.
Several factors have been shown to change the choleretic effect of secretin. The gastrointestinal hormone gastrin inhibits secretin-stimulated choleresis via interaction with cholecystokinin-B/gastrin by Ca2+-dependent activation of protein kinase C isoforms (33, 35). Furthermore, somatostatin inhibits secretin-stimulated ductal secretion by interaction with somatostatin SSTR2 receptors by decreasing cAMP levels (3, 62). Endothelin-1 reduces secretin-stimulated ductal secretion in BDL rats by interacting with ETA receptors on cholangiocytes (21). The bile acids ursodeoxycholate and tauroursodeoxycholate inhibit secretin-stimulated ductal secretion in BDL rats by Ca2+-dependent activation of PKC- (1).
There is growing information regarding the effects of hepatic nerves and nerve receptor agonists on basal and secretin-stimulated ductal bile secretion (8, 31, 32, 44, 45). Interruption of the parasympathetic innervation (by total vagotomy) induces inhibition of secretin-stimulated ductal secretion of BDL rats (44). Adrenergic denervation by a single intraportal injection of 6-hydroxydopamine to BDL rats induces damage of cholangiocytes and decreases secretin-stimulated ductal secretion by decreasing cholangiocyte cAMP levels (31). The cholinergic agonist acetylcholine increases the stimulatory effects of secretin on cAMP levels and bicarbonate secretion by Ca2+/calcineurin-dependent modulation of adenylyl cyclase (8). The dopaminergic agonist quinelorane does not change basal cholangiocyte secretion but inhibits secretin-stimulated ductal secretion by increased phosphorylation of PKC-
and decreased PKA activity (32). The selective 1-adrenergic receptor agonist phenylephrine does not affect basal cholangiocyte secretory activity but increases secretin-stimulated ductal secretion by Ca2+-dependent activation of PKC- and PKC-
II (45). Neuropeptide Y (NPY)-positive nerves are present in extrahepatic bile ducts (18) and have been suggested to regulate bile flow by autocrine/paracrine mechanisms (26). Other studies have shown that NPY acts in the left dorsal vagal complex to stimulate bile acid-independent and bicarbonate-dependent bile secretion (possibly of ductal origin) by interaction with Y1 receptor subtypes (66). No information exists regarding the role of 2-adrenergic receptors in the regulation of basal and secretin-stimulated ductal secretion.
In this study, we evaluated whether 1) 2A-, 2B-, and 2C-adrenergic receptors are expressed by cholangiocytes in liver sections from normal and BDL rats; 2) the specific 2-adrenergic receptor agonist UK 14,304 [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine] (12, 41, 61) regulates basal and secretin-stimulated ductal secretion by changes in cAMP/PKA/CFTR/Cl–/HCO3– exchanger, which plays a key role in the regulation of cAMP-dependent cholangiocyte secretory activity (4, 6–8, 21, 23, 24, 32, 35, 40, 42, 48, 51, 54, 62); and 3) the effects of UK 14,304 on ductal secretion are modulated by the apical cholangiocyte NHE3 (54).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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2A-, 2B-, and 2C-adrenergic receptors were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti 2A-adrenergic receptor antibody (C-19; catalog no. sc-1478) is an affinity-purified goat polyclonal antibody raised against a peptide mapping at the COOH terminus of the 2A-adrenergic receptor of human origin. This antibody has been shown to specifically recognize 2A-adrenergic receptor in rats (36, 60). The anti 2B-adrenergic receptor antibody (C-19; catalog no. sc-1479) is an affinity-purified goat polyclonal antibody raised against a peptide mapping at the COOH terminus of the 2B-adrenergic receptor of human origin and has been shown to cross-react with rat and mouse (37). The 2C-adrenergic receptor (C-20; catalog no. sc-1480) is an affinity-purified goat polyclonal antibody raised against a peptide mapping at the COOH terminus of the 2C-adrenergic receptor of human origin and cross-reacts with rat and mouse (per data sheet from Santa Cruz Biotechnology). Porcine secretin (previously used in other studies to evaluate both in vivo and in vitro changes in cholangiocyte secretory activity) (1, 4, 42) was purchased from Peninsula Laboratories (Belmont, CA). The substrate for -glutamyltranspeptidase (-GT), N-(-L-glutamyl)-4-methoxy-2-naphthylamide, was purchased from Polysciences (Warrington, PA). RIA kits for the determination of intracellular cAMP levels were purchased from GE Healthcare (Arlington Heights, IL). Kits for the measurement of PKA activity were purchased from Promega (Madison, WI). The NHE inhibitor 5-(N-ethyl-N-isopropyl)amiloride (EIPA) (54) was purchased from RBI (Natick, MA). Animal Model Male Fischer 344 rats (150–175 g) were purchased from Charles River (Wilmington, MA). The animals were kept in a temperature-controlled environment (22°C) with a 12:12-h light-dark cycle. The studies were performed in normal and 1-wk BDL [for isolation of cholangiocytes and intrahepatic bile duct units (IBDU) (2, 3, 6, 33, 39, 42, 48)] or bile duct-incannulated [BDI; for bile collection (4)] rats. Whereas in BDL rats, extrahepatic bile duct obstruction is induced by double ligation of the common bile duct, BDI is achieved by insertion of a plastic cannula (occluded following insertion) into the common bile duct (4). In BDI rats, 7 days after the surgery, the previously installed bile duct cannula is freed from the abdominal ligatures, exteriorized, cut at its occluded end, and connected to a longer plastic tubing for bile flow and collection (4). The reason why we use BDL (rather than BDI) for cell and IBDU isolation is because of the higher mortality rate of the BDI surgery (due to the plastic cannula insertion) compared with BDL (Alpini G, unpublished observations). The in vitro studies, aimed to evaluate the intracellular mechanisms by which UK 14,304 regulates basal and secretin-stimulated ductal secretion, were performed in purified cholangiocytes (1, 3, 32, 35, 39, 42, 44–46, 48) and IBDU (2) from 1-wk BDL rats. Before each procedure, animals were anesthetized with pentobarbital sodium (50 mg/kg ip). Study protocols were performed according to the institutional guidelines and with Institutional Animal Care and Use Committee approval.
Purification of Cholangiocytes and IBDU
Cholangiocytes from 1-wk BDL rats were purified by immunoaffinity separation (3, 39, 44, 45, 48) using a monoclonal antibody (IgM; kindly provided by Dr. R. Faris, Brown University, Providence, RI) against an unidentified antigen expressed by all intrahepatic cholangiocytes (39). Cell viability (
97%) was determined by trypan blue exclusion. Purity of cholangiocytes was assessed using -GT histochemistry (59). Large polarized IBDU (>15 µm in diameter) were isolated from 1-wk BDL rats as described by our laboratory (2, 48). Hepatocytes from 1-wk BDL rats were isolated by standard collagenase perfusion (4).
Expression of 2A-, 2B-, and 2C-Adrenergic Receptors in Cholangiocytes
The expression of adrenergic receptors was evaluated using immunofluorescence for 2A-, 2B-, and 2C-adrenergic receptors and counterstained with CK-19 in consecutive liver sections (20 µm thick; 3 sections evaluated per each group of animals) from normal and 1 wk-BDL rats. Briefly, samples were fixed in 4% paraformaldehyde (in 1x phosphate-buffered saline, or PBS) for 10 min, followed by tissue permeabilization in PBST (1x PBS containing 0.2% Triton X-100). Nonspecific protein binding was blocked by 5% normal goat serum. Following incubation with primary antibodies against -2A-, 2B-, or 2C-adrenergic receptor (Santa Cruz Biotechnology), together with an anti-CK-19 antibody (raised in mouse, 1:50; Vision Biosystems, Norwell, MA), samples were rinsed with 1x PBS and subsequently incubated with Cy2-conjugated anti-mouse and Cy3-conjugated anti-goat antibodies, respectively (both diluted at 1:50; Jackson Immunochemicals, West Grove, PA). Negative controls were prepared in the presence of a blocking peptide for 2A-, 2B-, or 2C-adrenergic receptor (Santa Cruz Biotechnology). Samples were mounted with Antifade reagent containing 4,6-diamidino-2-phenylindole (Invitrogen, Carlsbad, CA) and imaged using an Olympus IX71 inverted confocal microscope (Tokyo, Japan).
Effect of UK 14,304 on Basal and Secretin-Stimulated Bile and Bicarbonate Concentration and Secretion
We evaluated the effects of the specific 2-adrenergic receptor agonist (12, 17, 41, 67) UK 14,304 (at doses of 1, 10, 25, 50, and 100 µM) (41) on basal and secretin-stimulated bile flow and bicarbonate concentration and secretion of 1 wk-BDI rats. In normal rats, we measured the effect of UK 14,304 (50 µM) (41) on basal and secretin-stimulated bile flow and bicarbonate concentration and secretion. The doses of UK 14,304 employed in the present studies are similar to those previously used in our laboratory (41) to evaluate the effect of this specific adrenergic receptor agonist on biliary growth. Following anesthesia with pentobarbital sodium, normal or 1 wk-BDI rats were surgically prepared for bile collection as described by our group (4). When steady-state bile flow was reached, normal or 1 wk-BDI rats were subsequently infused via a jugular vein with UK 14,304 (at the selected concentration for 30 min), Krebs-Ringer-Henseleit solution (KRH; 60 min), UK 14,304 (at the selected concentration) + secretin (100 nM) for 30 min, KRH for 60 min followed by secretin (100 nM) for 30 min, and by a final infusion of KRH for 30 min. In separate sets of experiments, to provide further evidence of the specificity of UK 14,304 action on ductal secretion, we evaluated the effect of UK 14,304 on secretin-stimulated bile and bicarbonate concentration and secretion in the presence of rauwolscine (0.1 µM; a specific 2-adrenergic receptor antagonist) (41). We also measured the effect of rauwolscine (0.1 mM) on basal and secretin-stimulated bile and bicarbonate concentration and secretion. Bile was collected every 10 min in preweighed tubes and immediately stored at –70°C before determination of bicarbonate concentration. Bicarbonate concentration (measured as total CO2) in the selected bile sample was determined using an ABL 520 blood gas system (Radiometer Medical, Copenhagen, Denmark).
Measurement of IBDU Lumen Space
Polarized IBDU (>15 µm in diameter) (2, 48) and isolated hepatocyte couplets (49) from BDL rats were prepared as previously described by our laboratory (2, 48, 49) and cultured onto coverslips in minimum essential medium (GIBCO-BRL, Grand Island, NY) with fetal calf serum (10%) at 37°C for 24–48 h before the selected study. The coverslips were mounted on the stage of a Nikon TE2000-U inverted microscope with differential interference optics connected to a Cascade digital camera (Roper Scientific, Tucson, AZ), and images were acquired and processed using MetaMorph software version 6.0 (Universal Imaging, Downingtown, PA). The IBDU and hepatocyte couplets were superperfused with KRH gassed with 95% O2-5% CO2. The maximum diameter of the IBDU was measured and converted to volume as previously described by our group (2). The volume of the hepatocyte couplet canalicular space was determined by measuring the maximum diameter of the canalicular space and calculating the volume, assuming the space is spherical in shape (4/3
r3) (30). Data are expressed as percent change in volume from baseline. To evaluate whether NHE3 plays a role in UK 14,304 regulation of secretin-stimulated ductal secretion, IBDU were treated with 1) secretin (100 nM), a dose previously used by our group and others (35, 42, 48); 2) UK 14,304 (50 µM) (41) in the presence of secretin (100 nM); 3) EIPA (1 µM) in the presence of secretin (100 nM); and 4) EIPA (1 µM) in the presence of UK 14,304 (50 µM) + secretin (100 nM). IBDU, attached to coverslips, were pretreated for 30 min with UK 14,304 and/or EIPA before the study and treated throughout the experiment. We used the same dose of EIPA (1 µM) previously employed by Mennone et al. (54) to evaluate the role of NHE3 in fluid secretion and absorption in mouse and rat cholangiocytes.
Measurement of Intracellular cAMP Levels
We next evaluated in purified 1 wk-BDL cholangiocytes the effects of UK 14,304 (50 µM) on basal and secretin-stimulated cAMP levels, a key component of ductal secretory activity (2, 4, 7, 8, 32, 35, 42, 48, 62). Following purification, cholangiocytes from 1-wk BDL rats were incubated at 37°C for 1 h to regenerate membrane proteins (3, 32, 34, 35, 42, 45) damaged by enzyme treatment during the isolation procedure (42). Subsequently, cholangiocytes (1 x 105 cells) were stimulated at room temperature (2, 3, 6, 32, 35, 42, 44, 45, 48) with 1) 0.2% BSA (basal) or UK 14,304 (50 µM) for 5, 10, 15, and 30 min; 2) secretin (100 nM) with 0.2% BSA for 5 min (3, 32, 34, 35, 42, 45); or 3) UK 14,304 (50 µM, for 5 min) + secretin (100 nM for 5 min) (3, 32, 34, 35, 42, 45) with 0.2% BSA. To provide further evidence for the specificity of UK 14,304 action on ductal secretion, we also evaluated the effect of UK 14,304 (50 µM for 5 min at room temperature) on secretin-stimulated cAMP levels in the presence of preincubation (5 min) with rauwolscine (0.1 µM), an 2-adrenergic receptor antagonist (41). We also evaluated the effect of rauwolscine (0.1 µM) on basal or secretin-stimulated cAMP levels of BDL cholangiocytes. Following stimulation with the selected agonist/antagonist, 3-isobutylmethylxanthine, a phosphodiesterase inhibitor (56) that prevents cAMP degradation (3, 6, 8, 29, 32, 34, 35, 41, 42, 45, 46, 48, 62), was added to the cell suspension before ethanol extraction and RIA analysis (2, 3, 6, 32, 35, 42, 44, 45, 48) was performed in accordance with instructions from the vendor.
Effect of UK 14,304 on PKA Activity PKA activity was measured in isolated 1 wk-BDL cholangiocytes (5 x 106) treated at 37°C with 1) 0.2% BSA (basal) for 30 min, 2) secretin (100 nM) for 30 min with 0.2% BSA, or 3) UK 14,304 (50 µM) for 10 min in the absence or presence of secretin (100 nM for 30 min) with 0.2% BSA. PKA activity was measured utilizing a nonradioactive Peptag cAMP-dependent protein kinase assay kit from Promega (Madison, WI) according to the instructions provided by the vendor and as described by our group (32, 48). Phosphorylated peptide bands were quantitated by scanning densitometry (ChemiImager 4000 low-light imaging system).
Cl– Efflux Studies Cl– efflux was assessed in purified cholangiocytes from 1-wk BDL rats as previously described (6, 28). Briefly, 3 x 106 cholangiocytes (attached to the immunomagnetic beads used for cell isolation) (3, 6, 8, 31–33, 35, 39, 42) were incubated for 2 h (1 h at 37°C and 1 h at room temperature) in a Cl–-rich buffer (140 mM NaCl, 4 mM KCl, 2 mM MgC12, l mM CaCl2, 1 mM KH2PO4, 10 mM glucose, and 10 mM HEPES, pH 7.4) containing 5 µCi/ml 36Cl– (>3 mCi/g; Amersham, Arlington Heights, IL). Cells were washed six times over 6 min with isotope-free buffer. Studies were initiated with the addition of 1 ml of isotope-free buffer. The buffer was changed every 60 s with a magnetic separator that binds the cells attached to the beads. After 180 s, the effect of agonists on 36Cl– efflux rates was assessed by adding 0.2% BSA (basal), secretin (100 nM + 0.2% BSA), or UK 14,304 (50 µM) in the absence or presence of secretin (100 nM). At the completion of the study, cells were lysed with NaOH (0.1 M) and radioactivity was determined by a liquid scintillation counter. 36Cl– efflux at each time point was measured as the rate of agonist-induced 36Cl– efflux relative to total remaining counts. Similar to our previous studies (6), basal or agonist-induced Cl– efflux data (expressed as 36Cl– efflux per minute) are shown at 300 s from the beginning of the experiments.
Evaluation of Cl–/HCO3– Exchanger Activity The Cl–/HCO3– exchanger activity was measured in isolated 1-wk BDL IBDU perfused with Krebs-Ringer-bicarbonate (53) by evaluating changes of intracellular pH (pHi) after abrupt Cl– removal/readmission from the perfusion medium (substitution with gluconate) (53). pHi was measured in single cholangiocytes of IBDU by using a microfluorimetric single-cell method with a Spex-AR-CM microsystem (Spex Industries, Edison, NJ) and BCECF-AM (Molecular Probes) as fluorescent probe (53). Two consecutive Cl– removal/readmission maneuvers were performed, the first used as the control and the second during exposure to secretin (100 nM), UK 14,304 (50 µM), or secretin + UK 14,304. The Cl–/HCO3– exchanger activity was determined by the rate of pHi increase after Cl– removal and the rate of pHi recovery after Cl– readmission (8, 53).
Statistical Analysis All data are means ± SE. Differences between groups were analyzed using Student's unpaired t-test when two groups were analyzed and ANOVA when more than two groups were analyzed.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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2A-, 2B-, and 2C-Adrenergic Receptors
We have performed immunofluorescence for 2A-, 2B-, and 2C-adrenergic receptors in liver sections from normal and 1-wk BDL rats and shown that all cholangiocytes express the three adrenergic receptor subtypes (Fig. 1, A–C). In addition, Fig. 1 shows each of the channels separately (red for the specific adrenergic receptor subtype and green for CK-19) as well as a merged image to clearly show cholangiocyte expression of adrenergic receptors. When immunofluorescence in liver sections was performed in the presence of the control peptide plus the primary antibody (competitive inhibition), no staining for 2A-, 2B-, and 2C-adrenergic receptors was observed in cholangiocytes (Fig. 1, A–C).
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Intravenous infusion of UK 14,304 (at 1, 10, 25, 50 and 100 µM) did not change basal bile flow and bicarbonate concentration and secretion of BDI rats (Fig. 2A and Table 1). Parallel to other studies (4, 35, 62), intravenous infusion of secretin (100 nM) to 1-wk BDI rats increased bile flow and bicarbonate concentration and secretion (Fig. 2B and Table 1). At the doses of 1, 10, and 25 µM, UK 14,304 did not block secretin-stimulated choleresis (Fig. 2B) (which was similar to that observed in BDI rats infused only with secretin, Table 1 and Fig. 2B). UK 14,304 (at 50 and 100 µM) significantly inhibited secretin-stimulated bile flow and bicarbonate concentration and secretion (Fig. 2B and Table 1). To further support the specificity of the effects of UK 14,304 on ductal secretion, UK 14,304 inhibition of secretin-stimulated bile and bicarbonate secretion was prevented by the specific 2-adrenergic receptor antagonist rauwolscine (Table 1). When infused alone, rauwolscine (0.1 µM) did not change basal bile flow and bicarbonate concentration and secretion of BDI rats (Table 1). Furthermore, rauwolscine alone did not affect secretin-stimulated bicarbonate rich choleresis, which was similar to that observed in BDI rats infused only with secretin (not shown).
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UK 14,304 alone had no effect on the lumen space of IBDU compared with its basal value (not shown). Pretreatment of IBDU with UK 14,304 inhibited secretin-stimulated IBDU lumen expansion (41.2 ± 9.2 vs. 14.7 ± 2.8%, P < 0.05, Fig. 3, A and B). This finding is consistent with UK 14,304 inhibiting secretin-stimulated ductal secretion. The decrease in IBDU lumen space after removal of secretin stimulation has been shown recently to be due to fluid absorption mediated by NHE3 expressed on the cholangiocyte apical membrane (54). Consistent with this concept, EIPA virtually abolished the decrease in IBDU lumen space after removal of secretin (Fig. 3C). Consistent with a lack of effect of UK 14,304 on NHE3-mediated ductal absorption in IBDU, the decrease in lumen size of IBDU after removal of secretin (from 35 to 50 min, Fig. 3B) was still evident after pretreatment with UK 14,304. In contrast, in IBDU pretreated with a NHE3 inhibitor, EIPA, (54) there was no significant decrease in IBDU luminal space after removal of secretin in either the absence (Fig. 3C) or presence of UK 14,304 (Fig. 3D). EIPA slightly decreases the peak of lumen space expansion during secretin exposure (Fig. 3, Cvs. A, time 25–30 min); however, it should be noted that this slight decrease may be due to the EIPA-induced inhibition of basolateral NHE type 1 one that is partially (30%) involved in secretin-stimulated bicarbonate excretion (38). Together, our data show that the inhibitory effect of UK 14,304 on ductal secretion is not mediated by modulation of cholangiocyte NHE3.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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2A-, 2B-, and 2C-adrenergic receptors are expressed by all cholangiocytes from normal and BDL rats. We have demonstrated that the 2-adrenergic receptor agonist UK 14,304 (41) did not change bile flow and bicarbonate concentration and secretion of normal rats in vivo but inhibited secretin-stimulated bicarbonate-rich choleresis of proliferating cholangiocytes in BDI rats. UK 14,304 inhibition of secretin-stimulated bile flow and bicarbonate concentration and secretion of BDI rats was prevented by the administration of the specific 2-adrenergic receptor antagonist rauwolscine (41). Pretreatment of polarized BDL IBDU with UK 14,304, inhibited secretin-stimulated IBDU lumen expansion; the inhibitory effect of UK 14,304 on ductal secretion was not mediated by cholangiocyte NHE3. UK 14,304 did not change basal cAMP levels but inhibited secretin-stimulated cAMP levels of BDL cholangiocytes. In support of the specificity of the effects of UK 14,304 on ductal secretion, UK 14,304 inhibition of secretin-stimulated cAMP levels (a key component of ductal secretion) (2, 4, 7, 8, 32, 35, 42, 48, 62) was prevented by rauwolscine, a specific 2-adrenergic receptor antagonist (41). Furthermore, UK 14,304 inhibited secretin-stimulated PKA activity, Cl– efflux, and Cl–/HCO3– exchanger activity in purified cholangiocytes from BDL rats. Together, these findings indicate that 2-adrenergic receptors counteract secretin-induced ductal bicarbonate secretion chronic cholestatic liver diseases (induced by BDL) by coordinated inhibition of adenylyl cyclase, which reduces the secretin-induced activation of Cl– efflux and Cl–/HCO3– exchanger activity.
Using immunofluorescence in liver sections, we evaluated the expression of adrenergic receptors in bile ducts, showing that 2A-, 2B-, and 2C-adrenergic receptors are expressed by all cholangiocytes from normal and BDL rats. In support of our findings, a number of studies have shown that nerves and nerve receptors are distributed in the biliary epithelium. For example, in the liver, adrenergic and cholinergic nerves are located around the hepatic artery, the portal vein, and the biliary epithelium (57, 63). - and -adrenoreceptors were demonstrated in the common bile duct of guinea pigs and rabbits (65). Furthermore, the presence of functional 3-adrenergic receptors has been demonstrated in the extrahepatic common bile duct of guinea pigs (25). M3 acetylcholine, 1-, 2-, 1-, and 2-adrenergic, serotoninergic, D2 dopaminergic, GABAergic, and NPY receptors have been shown to be expressed by cholangiocytes (8, 11, 18, 27, 32, 41, 44, 45, 50) and to regulate secretory, proliferative, and apoptotic biliary functions (8, 11, 27, 32, 41, 44, 45, 50).
We next evaluated the involvement of 2-adrenergic receptors in modulating secretin-induced choleresis, since these adrenergic receptors have been shown to modulate secretory activities in different cell types (14, 52). In support of this concept, studies in isolated perfused rat liver have suggested regulation of bile flow by adrenergic nerves (13). Furthermore, our group has shown that the 1-adrenergic receptor agonist phenylephrine increases secretin-stimulated ductal secretion by Ca2+-dependent activation of PKC- and PKC-II (45). Moreover, in support of the concept that activation of 2-adrenergic receptors regulates cholangiocyte functions, our group has shown in previous studies that UK 14,304 inhibits cholangiocarcinoma growth by interacting with 2-adrenergic receptors on cholangiocarcinoma cell lines (41). However, no studies exist regarding the role of 2-adrenergic receptors in the regulation of ductal secretion.
The present study was performed in the BDL rat model where the in vivo effects of agonists/antagonists can confidently be attributed to alterations in biliary functions, since cholangiocytes are the predominant endogenous liver cells that proliferate following BDL, as evaluated by both in vivo and in vitro [3H]thymidine incorporation studies (3). In addition, the hyperplastic BDL model is characterized by marked in vivo amplification of cholangiocyte secretory activities and responsiveness to secretin (4, 35, 40, 48, 62), secretory features that are absent or minimal in the normal rat liver (4, 35, 46, 62). In fact, when infused intravenously, secretin-stimulated choleresis is not observed in vivo in normal rats, where cholangiocytes represent only 3–5% of the total liver mass (4). In the BDL model, secretin-stimulated bicarbonate-rich choleresis depends on the expansion of intrahepatic bile duct mass (3, 4), which results in upregulation of secretin receptors (3, 6, 7) and cAMP PKA CFTR Cl–/HCO3– exchanger (6, 32, 34, 35, 48) in proliferating cholangiocytes with subsequent amplification of basal and secretin-regulated cholangiocyte secretory activity (4, 35, 40, 48, 62). UK 14,304 inhibited secretin-stimulated bile flow and bicarbonate concentration and secretion in BDI but not in normal rats, indicating that the inhibitory effect of the 2-adrenergic agonist is specific for secretin-stimulated choleresis and does not affect the basal secretory activities of hepatocytes and cholangiocytes. In support of the specificity of the effects of UK 14,304 on secretin-induced choleresis, UK 14,304 inhibition of secretin-stimulated bile flow and bicarbonate concentration and secretion was blocked by the intravenous infusion of rauwolscine. In further support of the specificity of the in vivo inhibitory effects of UK 14,304 on secretin-stimulated choleresis, our group has previously shown that 1) the 1-adrenergic receptor agonist phenylephrine does not alter basal ductal secretion but increases secretin-stimulated cholangiocyte secretion (45) and that 2) the 1-adrenergic receptor agonist dobutamine does not affect basal or secretin-stimulated ductal secretion (45).
That the specific target of the 2-adrenergic agonist is the bile duct epithelium was directly demonstrated in vitro in isolated IBDU, where UK 14,304 inhibited secretin-induced expansion of the IBDU lumen. The IBDU model consists of polarized epithelial cells with intact morphological and functional structures (2, 15, 16, 48, 53, 58) and has major advantages over other tools (e.g., bile fistula rats) because it allows for the direct measurement of ductal secretory activity by changes in lumen size in response to agonists/antagonists. This latter experiment performed in IBDU allowed us to exclude the possibility that the inhibitory effect of UK 14,304 in vivo on ductal secretion was a consequence of altered hepatic blood flow due to the 2-adrenergic agonist (22, 64).
We next explored the intracellular mechanisms (cAMP PKA CFTR Cl–/HCO3– exchanger) involved in the inhibition of secretin-stimulated choleresis by UK 14,304 in BDL cholangiocytes, and we first demonstrated that this specific 2-adrenergic receptor agonist does not change basal cAMP synthesis but inhibits secretin-induced cAMP levels. A possible explanation of why UK 14,304 does not change basal intracellular cAMP levels in BDL cholangiocytes but increases cAMP levels in the cholangiocarcinoma cell line Mz-ChA-1 (decreasing their growth) (41) is presumably due to the dysregulation of the cAMP-dependent signaling pathway in cancer cells (10, 19, 20, 43). In support of this concept, the cAMP-dependent protein kinases in lung adenomas are functionally different from those of normal lung (20). In addition, dissociation in the response of the adenylate cyclase system to thyrotropin and prostaglandin E2 has been shown in human thyroid carcinoma tissue (10). Functional changes in the regulatory subunit of the type II cAMP-dependent protein kinase isozyme have been observed during normal and neoplastic lung development (19). Moreover, differential cAMP response to selected agonists has been shown in normal versus neoplastic mouse lung epithelial cells (43).
In addition to cAMP levels, the sequence of other intracellular events induced by secretin and sustaining secretin-stimulated choleresis (2, 4–8, 32, 35, 40, 42, 48, 53) are all blocked by UK 14,304. In fact, secretin-induced PKA activity, Cl– efflux, and Cl–/HCO3– activity were all blocked by UK 14,304 in BDL cholangiocytes. cAMP PKA CFTR Cl–/HCO3– exchanger has been shown to play a pivotal role in the regulation of secretin-stimulated ductal bile secretion (2, 4–8, 32, 35, 40, 42, 48, 53). In support of this concept, the increase in secretin-stimulated choleresis (e.g., in rats with cholangiocyte hyperplasia following BDL) (4) is due to upregulation of cAMP PKA CFTR Cl–/HCO3– exchanger (3, 4, 6, 48, 62), whereas in pathological conditions of cholangiocyte damage (e.g., following CCl4 administration) (47), the inhibition of secretin-stimulated ductal secretory activity depends on the downregulation of cAMP PKA CFTR Cl–/HCO3– exchanger. To evaluate whether the inhibitory effect of UK 14,304 on secretin-stimulated ductal secretion is due to increased fluid absorption mediated by NHE3 expressed on the cholangiocyte apical membrane (54), we measured the effect of UK 14,304 on IBDU expansion in the presence of the NHE3 inhibitor EIPA (54). Our data show that the inhibitory effect of UK 14,304 on ductal secretion is not mediated by the apical cholangiocyte NHE3, but rather by downregulation of cAMP PKA CFTR Cl–/HCO3– exchanger.
In conclusion, our study demonstrates that cholangiocytes express 2A-, 2B-, and 2C-adrenergic receptors, which counterregulate secretin-induced choleresis in proliferating cholangiocytes. Cholangiocyte growth, a typical hallmark of cholangiopathies (9), is associated with enhanced ductal secretion (3–5, 7, 40, 62), which tends to compensate for the impaired secretory activity of damaged bile ducts. Our studies have important clinical implications indicating that the 2-adrenergic system (by changes in cAMP PKA CFTR Cl–/HCO3– exchanger) is involved in the regulation of secretory activities of proliferating cholangiocytes and suggesting potential pharmacological strategies for the modulation of bile secretion in the course of human cholangiopathies. Recent studies have demonstrated that the adrenergic signal is involved in modulating additional features characterizing chronic liver disease and, specifically, hepatic fibrosis. In fact, hepatic stellate cell activation (through autocrine signaling) requires adrenergic stimuli, and in animal models the loss of adrenergic signaling reduces hepatic fibrosis (55). Therefore, modulation of adrenergic signal could represent a potential strategy for the management of chronic liver damage.
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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.
* H. Francis and G. LeSage contributed equally to this work. 
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