Glucagon-Like Peptide-1 and Its Receptor Agonist Exendin-4 Modulate Cholangiocyte Adaptive Response to Cholestasis

Background & Aims: Cholangiopathies are characterized by progressive dysregulation of the balance between proliferation and death of cholangiocytes. In the course of cholestasis, cholangiocytes undergo a neuroendocrine transdifferentiation and their biology is regulated by neuroendocrine hormones. Glucagon-like peptide-1 (GLP-1), secreted by neuroendocrine cells, sustains β-cell survival in experimental diabetes and induces the neuroendocrine transdifferentiation of pancreatic ductal cells. GLP-1 receptor (GLP-1R) selective agonist exendin-4 is used in humans as a novel therapeutic tool for diabetes. The aim of this study was to define if GLP-1 modulates cholangiocyte biologic response to cholestasis. Methods: Expression of GLP-1R in cholangiocytes was determined. Effects on cholangiocyte proliferation of the in vitro and in vivo exposure to GLP-1 or exendin-4, together with the intracellular signals, were then studied. Synthesis of GLP-1 by cholangiocytes and the effects of GLP-1R blockage on their growth were also determined. Results: Cholangiocytes express the GLP-1 receptor, which is up-regulated in the course of cholestasis. GLP-1 and exendin-4 increase cholangiocyte growth both in vitro and in vivo. The GLP-1R signal is mediated by the phosphatidyl-inositol-3-kinase, cAMP/Protein Kinase A, and Ca2+-CamKIIα but not by the ERK1/2 and PKCα pathways. Proliferating cholangiocytes synthesize GLP-1: neutralization of its action by GLP-1R antagonist blunts cholangiocyte response to cholestasis. Conclusions: GLP-1 is required for the cholangiocyte adaptive response to cholestasis. Cholangiocytes are susceptible to the activation of GLP-1R and respond with increased proliferation and functional activity. Exendin-4 availability for employment in humans and these data may open novel perspectives for the medical treatment of cholangiopathies. Background & Aims: Cholangiopathies are characterized by progressive dysregulation of the balance between proliferation and death of cholangiocytes. In the course of cholestasis, cholangiocytes undergo a neuroendocrine transdifferentiation and their biology is regulated by neuroendocrine hormones. Glucagon-like peptide-1 (GLP-1), secreted by neuroendocrine cells, sustains β-cell survival in experimental diabetes and induces the neuroendocrine transdifferentiation of pancreatic ductal cells. GLP-1 receptor (GLP-1R) selective agonist exendin-4 is used in humans as a novel therapeutic tool for diabetes. The aim of this study was to define if GLP-1 modulates cholangiocyte biologic response to cholestasis. Methods: Expression of GLP-1R in cholangiocytes was determined. Effects on cholangiocyte proliferation of the in vitro and in vivo exposure to GLP-1 or exendin-4, together with the intracellular signals, were then studied. Synthesis of GLP-1 by cholangiocytes and the effects of GLP-1R blockage on their growth were also determined. Results: Cholangiocytes express the GLP-1 receptor, which is up-regulated in the course of cholestasis. GLP-1 and exendin-4 increase cholangiocyte growth both in vitro and in vivo. The GLP-1R signal is mediated by the phosphatidyl-inositol-3-kinase, cAMP/Protein Kinase A, and Ca2+-CamKIIα but not by the ERK1/2 and PKCα pathways. Proliferating cholangiocytes synthesize GLP-1: neutralization of its action by GLP-1R antagonist blunts cholangiocyte response to cholestasis. Conclusions: GLP-1 is required for the cholangiocyte adaptive response to cholestasis. Cholangiocytes are susceptible to the activation of GLP-1R and respond with increased proliferation and functional activity. Exendin-4 availability for employment in humans and these data may open novel perspectives for the medical treatment of cholangiopathies. Cholangiocytes, the epithelial cells that line the intrahepatic biliary tree, are the target of cholangiopathies.1Lazaridis K.N. Strazzabosco M. LaRusso N.F. The cholangiopathies: disorders of biliary epithelia.Gastroenterology. 2004; 127: 1565-1577Google Scholar These diseases are a wide array of congenital or acquired disorders that share the feature of being chronic cholestatic conditions leading to liver failure. In particular, despite the different etiology, the progression of these diseases is determined by an abnormal cholangiocyte homeostasis, where cell death by apoptosis prevails over cholangiocyte proliferative response to liver injury.1Lazaridis K.N. Strazzabosco M. LaRusso N.F. The cholangiopathies: disorders of biliary epithelia.Gastroenterology. 2004; 127: 1565-1577Google Scholar Together, enhanced apoptosis and impaired proliferation lead to ductopenia, for example, loss of bile ducts, that is a unifying event in cholangiopathies.1Lazaridis K.N. Strazzabosco M. LaRusso N.F. The cholangiopathies: disorders of biliary epithelia.Gastroenterology. 2004; 127: 1565-1577Google Scholar Currently, there is no therapy effective in reestablishing the balance between cholangiocyte proliferation and death.1Lazaridis K.N. Strazzabosco M. LaRusso N.F. The cholangiopathies: disorders of biliary epithelia.Gastroenterology. 2004; 127: 1565-1577Google Scholar Thus, cholangiopathies represent a challenge for the clinician: 20% of liver transplants among adults and 50% of those among pediatric patients are due to these disorders.2Annual Report of the U.S. Organ Procurement and Transplantation Network and the Scientific Registry for Transplant Recipients. Department of Health and Human Services, Health Resources and Services Administration, Office of Special Programs, Division of Transplantation, Rockville, MD2001Google Scholar Unfortunately, the factors that regulate the balance between proliferation and death of cholangiocytes are still undefined; this certainly contributes to relent the development of effective therapies for cholangiopathies. In recent years it has been demonstrated that nerves, neuropeptides, and neuroendocrine hormones may play a significant role in this regard.3Alvaro D. Mancino M.G. Glaser S. Gaudio E. Marzioni M. Francis H. Alpini G. Proliferating cholangiocytes: a neuroendocrine compartment in the diseased liver.Gastroenterology. 2007; 132: 415-431Abstract Full Text Full Text PDF Scopus (234) Google Scholar In particular, we have recently demonstrated that in the course of cholestasis cholangiocytes synthesize some of these peptides (like serotonin, endogenous opioid peptides, or vascular endothelial growth factor),4Marzioni M. Glaser S. Francis H. Marucci L. Benedetti A. Alvaro D. Taffetani S. Ueno Y. Roskams T. Phinizy J.L. Venter J. Fava G. LeSage G. Alpini G. Autocrine/paracrine regulation of the growth of the biliary tree by the neuroendocrine hormone serotonin.Gastroenterology. 2005; 128: 121-137Abstract Full Text Full Text PDF Scopus (107) Google Scholar, 5Marzioni M. Alpini G. Saccomanno S. de Minicis S. Glaser S. Francis H. Trozzi L. Venter J. Orlando F. Fava G. Candelaresi C. Macarri G. Benedetti A. 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Neuroendocrine features of reactive bile ductules in cholestatic liver disease.Am J Pathol. 1990; 137: 1019-1025Google Scholar GLP-1 is secreted by enteroendocrine L cells and modulates the biology of a number of cells, by interacting with a specific G-protein coupled receptor (GLP-1R).8Brubaker P.L. Drucker D.J. Minireview: glucagon-like peptides regulate cell proliferation and apoptosis in the pancreas, gut, and central nervous system.Endocrinology. 2004; 145: 2653-2659Google Scholar, 9Drucker D.J. Biological actions and therapeutic potential of the glucagon-like peptides.Gastroenterology. 2002; 122: 531-544Abstract Full Text Full Text PDF Scopus (444) Google Scholar, 10Drucker D.J. Glucagon-like peptides: regulators of cell proliferation, differentiation, and apoptosis.Mol Endocrinol. 2003; 17: 161-171Google Scholar GLP-1 is known to modulate glucose homeostasis; specifically, it has been demonstrated that GLP-1 prevents pancreatic β-cell death by apoptosis and sustains their proliferation.8Brubaker P.L. Drucker D.J. Minireview: glucagon-like peptides regulate cell proliferation and apoptosis in the pancreas, gut, and central nervous system.Endocrinology. 2004; 145: 2653-2659Google Scholar, 9Drucker D.J. Biological actions and therapeutic potential of the glucagon-like peptides.Gastroenterology. 2002; 122: 531-544Abstract Full Text Full Text PDF Scopus (444) Google Scholar, 10Drucker D.J. Glucagon-like peptides: regulators of cell proliferation, differentiation, and apoptosis.Mol Endocrinol. 2003; 17: 161-171Google Scholar As a consequence, the GLP-1R selective agonist exendin-4 is now employed as a novel antidiabetic therapy.11Fineman M.S. Bicsak T.A. Shen L.Z. Taylor K. Gaines E. Varns A. Kim D. Baron A.D. Effect on glycemic control of exenatide (synthetic exendin-4) additive to existing metformin and/or sulfonylurea treatment in patients with type 2 diabetes.Diabetes Care. 2003; 26: 2370-2377Google Scholar Interestingly, among its many biologic properties, GLP-1 is able to induce the acquisition of a neuroendocrine phenotype by pancreatic ductal cells,10Drucker D.J. Glucagon-like peptides: regulators of cell proliferation, differentiation, and apoptosis.Mol Endocrinol. 2003; 17: 161-171Google Scholar, 12Bulotta A. Hui H. Anastasi E. Bertolotto C. Boros L.G. Di Mario U. Perfetti R. Cultured pancreatic ductal cells undergo cell cycle re-distribution and beta-cell-like differentiation in response to glucagon-like peptide-1.J Mol Endocrinol. 2002; 29: 347-360Google Scholar, 13Zhou J. Wang X. Pineyro M.A. Egan J.M. Glucagon-like peptide 1 and exendin-4 convert pancreatic AR42J cells into glucagon- and insulin-producing cells.Diabetes. 1999; 48: 2358-2366Google Scholar cells that share features in common with cholangiocytes, in terms of embryologic origin, morphology, functional activity, and response to injury.14Yang L. Li S. Hatch H. Ahrens K. Cornelius J.G. Petersen B.E. Peck A.B. In vitro trans-differentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells.Proc Natl Acad Sci U S A. 2002; 99: 8078-8083Google Scholar, 15Nagaya M. Kubota S. Isogai A. Tadokoro M. Akashi K. Ductular cell proliferation in islet cell neogenesis induced by incomplete ligation of the pancreatic duct in dogs.Surg Today. 2004; 34: 586-592Google Scholar, 16Chu J.Y. Yung W.H. Chow B.K. Secretin: a pleiotrophic hormone.Ann N Y Acad Sci. 2006; 1070: 27-50Google Scholar, 17Grapin-Botton A. Ductal cells of the pancreas.Int J Biochem Cell Biol. 2005; 37: 504-510Google Scholar Therefore, the aim of this study was to verify if GLP-1 is also able to modulate cholangiocyte pathobiology, also considering that GLP-1 serum levels are increased in the course of cholestasis.18Niwa T. Nimura Y. Niki I. Lack of effect of incretin hormones on insulin release from pancreatic islets in the bile duct-ligated rats.Am J Physiol Endocrinol Metab. 2001; 280: E59-E64Google Scholar Thus, we asked the following questions: (1) do cholangiocytes express the GLP-1R? (2) Does the activation of this receptor affect cholangiocyte proliferation and functional activity? (3) Which are the intracellular pathways that mediate the GLP-1R signal in cholangiocytes? (4) Is cholangiocyte proliferative response to cholestasis associated with the local release of GLP-1? Reagents were purchased from Sigma Chemical (St. Louis, MO) unless otherwise indicated. Intracellular cyclic adenosine 3′, 5′-monophosphate (cAMP) and D-myo-Inositol 1,4,5-triphosphate (IP3) levels were determined with RIA kits purchased from Amersham (Arlington Heights, IL). Antibodies for immunoblotting were purchased from Santa Cruz Biotechnologies Inc. (Santa Cruz, CA), unless differently indicated. The antibody anti-GLP1R was purchased by Alpha Diagnostic (San Antonio, TX); the antibody anti-Cytokeratin (CK)-19 was purchased from Novocastra (Milan, Italy). Exendin-4 and exendin-(9–39) were purchased from American Peptide Inc. (Sunnyvale, CA). Wortmannin was purchased from Sigma Chemical; PD98059, Rp-cAMPs, KN62, R0-32-0432, BAPTA/AM, and nitrendipine were purchased from Calbiochem (Milan, Italy). The expression of GLP-1R was evaluated by both reverse transcriptase-mediated polymerase chain reaction (RT-PCR) and immunoblots in cholangiocytes isolated both from normal rats and from rats subjected to 1-week bile duct ligation (BDL). Total RNA was extracted from freshly isolated cholangiocytes by TRIzol Reagent (Invitrogen, Carlsbad, CA). Two micrograms of total RNA were converted to cDNA with random primers using the M-MLV Reverse Transcriptase (Promega, Milan, Italy). PCR was performed by using the following primers: sense 5′ TGTACCTGAGCATAGGCTGG 3′, and antisense 5′ GCTCCCAGCTCTTCCGAAAC 3′ for GLP1-R.19Nakagawa A. Satake H. Nakabayashi H. Nishizawa M. Furuya K. Nakano S. Kigoshi T. Nakayama K. Uchida K. Receptor gene expression of glucagon-like peptide-1, but not glucose-dependent insulinotropic polypeptide, in rat nodose ganglion cells.Auton Neurosci. 2004; 110: 36-43Google Scholar Varying the number of PCR cycles did not change the relative differences between the samples, indicating that the PCR conditions were not within the plateau phase of amplification. The amplified products (453 bp) were subjected to electrophoresis on 1.6% agarose gel and stained with ethidium bromide. The intensity of the bands was determined by scanning video densitometry using the Chemi Doc imaging system (Bio Rad, Milan, Italy). Rat brain was used as positive control.19Nakagawa A. Satake H. Nakabayashi H. Nishizawa M. Furuya K. Nakano S. Kigoshi T. Nakayama K. Uchida K. Receptor gene expression of glucagon-like peptide-1, but not glucose-dependent insulinotropic polypeptide, in rat nodose ganglion cells.Auton Neurosci. 2004; 110: 36-43Google Scholar The expression of GLP-1R was also evaluated by immunoblots in whole cholangiocyte lysates. Rat brain and 0.2% bovine serum albumin (BSA) were used as positive and negative controls, respectively.19Nakagawa A. Satake H. Nakabayashi H. Nishizawa M. Furuya K. Nakano S. Kigoshi T. Nakayama K. Uchida K. Receptor gene expression of glucagon-like peptide-1, but not glucose-dependent insulinotropic polypeptide, in rat nodose ganglion cells.Auton Neurosci. 2004; 110: 36-43Google Scholar To determine the effect of GLP-1R activation on cholangiocyte proliferation, pure normal and BDL rat cholangiocytes were incubated for 5 hours at 37°C5Marzioni M. Alpini G. Saccomanno S. de Minicis S. Glaser S. Francis H. Trozzi L. Venter J. Orlando F. Fava G. Candelaresi C. Macarri G. Benedetti A. Endogenous opioids modulate the growth of the biliary tree in the course of cholestasis.Gastroenterology. 2006; 130: 1831-1847Google Scholar with (1) 0.2% BSA (control), or (2) increasing concentrations of either GLP-1 (1–100 nM)20Redondo A. Trigo M.V. Acitores A. Valverde I. Villanueva-Penacarrillo M.L. Cell signalling of the GLP-1 action in rat liver.Mol Cell Endocrinol. 2003; 204: 43-50Google Scholar, 21Ding X. Saxena N.K. Lin S. Gupta N.A. Anania F.A. Exendin-4, a glucagon-like protein-1 (GLP-1) receptor agonist, reverses hepatic steatosis in ob/ob mice.Hepatology. 2006; 43: 173-181Google Scholar or exendin-4 (the GLP-1R selective agonist 1–100 nM, 1 μmol/L).21Ding X. Saxena N.K. Lin S. Gupta N.A. Anania F.A. Exendin-4, a glucagon-like protein-1 (GLP-1) receptor agonist, reverses hepatic steatosis in ob/ob mice.Hepatology. 2006; 43: 173-181Google Scholar, 22Hui H. Nourparvar A. Zhao X. Perfetti R. Glucagon-like peptide-1 inhibits apoptosis of insulin-secreting cells via a cyclic 5′-adenosine monophosphate-dependent protein kinase A- and a phosphatidylinositol 3-kinase-dependent pathway.Endocrinology. 2003; 144: 1444-1455Google Scholar, 23Thorens B. Porret A. Buhler L. Deng S.P. Morel P. Widmann C. Cloning and functional expression of the human islet GLP-1 receptor Demonstration that exendin-4 is an agonist and exendin-(9-39) an antagonist of the receptor.Diabetes. 1993; 42: 1678-1682Google Scholar To study whether the changes observed in cholangiocytes after GLP-1R activation are mediated by the cAMP/Protein Kinase A (PKA), mitogen-activated protein kinases (MAPK), and phosphatidyl-inositol-3-kinase (PI3K) pathways, the experiments were also performed by preincubating normal rat cholangiocytes for 30 minutes at 37°C with (3) either Rp-cAMPs (100 μmol/L, a cAMP-dependent PKA inhibitor),5Marzioni M. Alpini G. Saccomanno S. de Minicis S. Glaser S. Francis H. Trozzi L. Venter J. Orlando F. Fava G. Candelaresi C. Macarri G. Benedetti A. Endogenous opioids modulate the growth of the biliary tree in the course of cholestasis.Gastroenterology. 2006; 130: 1831-1847Google Scholar or PD98059 (50 μmol/L, an MEK inhibitor),5Marzioni M. Alpini G. Saccomanno S. de Minicis S. Glaser S. Francis H. Trozzi L. Venter J. Orlando F. Fava G. Candelaresi C. Macarri G. Benedetti A. Endogenous opioids modulate the growth of the biliary tree in the course of cholestasis.Gastroenterology. 2006; 130: 1831-1847Google Scholar or wortmannin (100 nM, a PI3K inhibitor),5Marzioni M. Alpini G. Saccomanno S. de Minicis S. Glaser S. Francis H. Trozzi L. Venter J. Orlando F. Fava G. Candelaresi C. Macarri G. Benedetti A. Endogenous opioids modulate the growth of the biliary tree in the course of cholestasis.Gastroenterology. 2006; 130: 1831-1847Google Scholar respectively, followed by the incubation with GLP-1 (10 nmol/L)20Redondo A. Trigo M.V. Acitores A. Valverde I. Villanueva-Penacarrillo M.L. Cell signalling of the GLP-1 action in rat liver.Mol Cell Endocrinol. 2003; 204: 43-50Google Scholar or 0.2% BSA as above described. Furthermore, to determine whether the Ca2+ signaling transducts the GLP-1R message, cells were also preincubated with (4) either BAPTA/AM (an intracellular Ca2+ chelator, 5 μmol/L),5Marzioni M. Alpini G. Saccomanno S. de Minicis S. Glaser S. Francis H. Trozzi L. Venter J. Orlando F. Fava G. Candelaresi C. Macarri G. Benedetti A. Endogenous opioids modulate the growth of the biliary tree in the course of cholestasis.Gastroenterology. 2006; 130: 1831-1847Google Scholar or KN62 (10 μmol/L, a CamKinaseII inhibitor),5Marzioni M. Alpini G. Saccomanno S. de Minicis S. Glaser S. Francis H. Trozzi L. Venter J. Orlando F. Fava G. Candelaresi C. Macarri G. Benedetti A. Endogenous opioids modulate the growth of the biliary tree in the course of cholestasis.Gastroenterology. 2006; 130: 1831-1847Google Scholar or Ro-32-0432 (0.5 μmol/L, a Ca2+-dependent PKC inhibitor),5Marzioni M. Alpini G. Saccomanno S. de Minicis S. Glaser S. Francis H. Trozzi L. Venter J. Orlando F. Fava G. Candelaresi C. Macarri G. Benedetti A. Endogenous opioids modulate the growth of the biliary tree in the course of cholestasis.Gastroenterology. 2006; 130: 1831-1847Google Scholar or nitrendipine (1 μmol/L, a L-type Ca2+ channel antagonist)24Guibert C. Marthan R. Savineau J.P. 5-HT induces an arachidonic acid-sensitive calcium influx in rat small intrapulmonary artery.Am J Physiol Lung Cell Mol Physiol. 2004; 286: L1228-L1236Google Scholar followed afterward by the incubation with GLP-1 (10 nmol/L)20Redondo A. Trigo M.V. Acitores A. Valverde I. Villanueva-Penacarrillo M.L. Cell signalling of the GLP-1 action in rat liver.Mol Cell Endocrinol. 2003; 204: 43-50Google Scholar or 0.2% BSA as described above. To provide evidence that GLP-1 may act on cholangiocytes in an autocrine/paracrine fashion, 1-week BDL rat cholangiocytes were incubated for 5 hours at 37°C with (1) 0.2% BSA or (2) exendin-(9-39) (100 nmol/L,22Hui H. Nourparvar A. Zhao X. Perfetti R. Glucagon-like peptide-1 inhibits apoptosis of insulin-secreting cells via a cyclic 5′-adenosine monophosphate-dependent protein kinase A- and a phosphatidylinositol 3-kinase-dependent pathway.Endocrinology. 2003; 144: 1444-1455Google Scholar a selective GLP-1R antagonist).23Thorens B. Porret A. Buhler L. Deng S.P. Morel P. Widmann C. Cloning and functional expression of the human islet GLP-1 receptor Demonstration that exendin-4 is an agonist and exendin-(9-39) an antagonist of the receptor.Diabetes. 1993; 42: 1678-1682Google Scholar At the end of the incubation time, chosen, as in previous studies4Marzioni M. Glaser S. Francis H. Marucci L. Benedetti A. Alvaro D. Taffetani S. Ueno Y. Roskams T. Phinizy J.L. Venter J. Fava G. LeSage G. Alpini G. Autocrine/paracrine regulation of the growth of the biliary tree by the neuroendocrine hormone serotonin.Gastroenterology. 2005; 128: 121-137Abstract Full Text Full Text PDF Scopus (107) Google Scholar, 5Marzioni M. Alpini G. Saccomanno S. de Minicis S. Glaser S. Francis H. Trozzi L. Venter J. Orlando F. Fava G. Candelaresi C. Macarri G. Benedetti A. Endogenous opioids modulate the growth of the biliary tree in the course of cholestasis.Gastroenterology. 2006; 130: 1831-1847Google Scholar in light of the fact that the changes in the proliferating cell nuclear antigen (PCNA) protein expression are very early events in the proliferative process,25Maga G. Hubscher U. Proliferating cell nuclear antigen (PCNA): a dancer with many partners.J Cell Sci. 2003; 116: 3051-3060Google Scholar cells were lysed to obtain proteins for immunoblotting, as described below. All the above-described experiments were also reproduced extending the time of incubation with GLP-1, exendin-4, and exendin-(9–39) up to 12 hours to assess the changes in the Bromo-DeoxyUrydine (BrDU) incorporation, as previously demonstrated.5Marzioni M. Alpini G. Saccomanno S. de Minicis S. Glaser S. Francis H. Trozzi L. Venter J. Orlando F. Fava G. Candelaresi C. Macarri G. Benedetti A. Endogenous opioids modulate the growth of the biliary tree in the course of cholestasis.Gastroenterology. 2006; 130: 1831-1847Google Scholar Male Fischer 344 rats (150–175 g), purchased from Charles River (Milan, Italy), were maintained in a temperature-controlled environmol/Lent (20–22°C) with a 12-hour light–dark cycle and with free access to drinking water and to standard rat chow. To study the effects of GLP-1R activation on cholangiocyte proliferation and functional activity, our studies were performed in normal rats treated with either (1) control injections (n = 9) or (2) exendin-4 (0.1 μg/kg body weight, twice daily, IP, n = 9).26Ozyazgan S. Kutluata N. Afsar S. Ozdas S.B. Akkan A.G. Effect of glucagon-like peptide-1(7–36) and exendin-4 on the vascular reactivity in streptozotocin/nicotinamide-induced diabetic rats.Pharmacology. 2005; 74: 119-126Google Scholar After 1 week, 3 animals per group were subjected to the placement of a cannula in the bile duct, for bile collection.27Alpini G. Glaser S. Alvaro D. Ueno Y. Marzioni M. Francis H. Baiocchi L. Stati T. Barbaro B. Phinizy J.L. Mauldin J. LeSage G. Bile acid depletion and repletion regulate cholangiocyte growth and secretion by a phosphatidylinositol 3-kinase-dependent pathway in rats.Gastroenterology. 2002; 123: 1226-1237Google Scholar The 6 remaining animals per group were sacrificed for cholangiocyte purification or liver sections. To verify if GLP-1 is required for the cholangiocyte proliferative and functional response to cholestasis, we performed our studies in animals that, immediately after BDL (for cholangiocyte purification or liver sections, n = 6 per each group of treatment) or bile duct incannulation for bile collection (n = 6 per each group of treatment),28Alpini G. Lenzi R. Sarkozi L. Tavoloni N. Biliary physiology in rats with bile ductular cell hyperplasia Evidence for a secretory function of proliferated bile ductules.J Clin Invest. 1988; 81: 569-578Google Scholar were treated for 1 week with either (1) control injections or (2) exendin-(9–39) (12 μg/kg body weight, daily IP).29Tseng C.C. Zhang X.Y. Wolfe M.M. Effect of GIP and GLP-1 antagonists on insulin release in the rat.Am J Physiol. 1999; 276: E1049-E1054Google Scholar The animals were fasted overnight before each experiment.4Marzioni M. Glaser S. Francis H. Marucci L. Benedetti A. Alvaro D. Taffetani S. Ueno Y. Roskams T. Phinizy J.L. Venter J. Fava G. LeSage G. Alpini G. Autocrine/paracrine regulation of the growth of the biliary tree by the neuroendocrine hormone serotonin.Gastroenterology. 2005; 128: 121-137Abstract Full Text Full Text PDF Scopus (107) Google Scholar Before each procedure, animals were anesthetized with sodium pentobarbital (50 mg/kg IP). Study protocols were performed in compliance with the institution guidelines. Purification of cholangiocytes from normal or 1-week BDL rats was performed using a monoclonal antibody (IgM, kindly provided by Dr R. Faris, Brown University, Providence, RI) against an unidentified membrane antigen expressed by all rat intrahepatic cholangiocytes.30Ishii M. Vroman B. LaRusso N.F. Isolation and morphological characterization of bile duct epithelial cells from normal rat liver.Gastroenterology. 1989; 97: 1236-1247Crossref Scopus (189) Google Scholar At the end of each procedure, purity of cholangiocytes was assessed by cytochemistry for γ-GT,4Marzioni M. Glaser S. Francis H. Marucci L. Benedetti A. Alvaro D. Taffetani S. Ueno Y. Roskams T. Phinizy J.L. Venter J. Fava G. LeSage G. Alpini G. Autocrine/paracrine regulation of the growth of the biliary tree by the neuroendocrine hormone serotonin.Gastroenterology. 2005; 128: 121-137Abstract Full Text Full Text PDF Scopus (107) Google Scholar, 5Marzioni M. Alpini G. Saccomanno S. de Minicis S. Glaser S. Francis H. Trozzi L. Venter J. Orlando F. Fava G. Candelaresi C. Macarri G. Benedetti A. Endogenous opioids modulate the growth of the biliary tree in the course of cholestasis.Gastroenterology. 2006; 130: 1831-1847Google Scholar, 31LeSage G. Alvaro D. Glaser S. Francis H. Marucci L. Roskams T. Phinizy J.L. Marzioni M. Benedetti A. Taffetani S. Barbaro B. Fava G. Ueno Y. Alpini G. Alpha-1 adrenergic receptor agonists modulate ductal secretion of BDL rats via Ca(2+)- and PKC-dependent stimulation of cAMP.Hepatology. 2004; 40: 1116-1127Google Scholar a specific marker for cholangiocytes.28Alpini G. Lenzi R. Sarkozi L. Tavoloni N. Biliary physiology in rats with bile ductular cell hyperplasia Evidence for a secretory function of proliferated bile ductules.J Clin Invest. 1988; 81: 569-578Google Scholar Cell viability at the end of the purification procedure was determined by trypan blue exclusion and was found >97%. The changes in cholangiocyte proliferation were assayed by both immunoblots for the PCNA protein expression4Marzioni M. Glaser S. Francis H. Marucci L. Benedetti A. Alvaro D. Taffetani S. Ueno Y. Roskams T. Phinizy J.L. Venter J. Fava G. LeSage G. Alpini G. Autocrine/paracrine regulation of the growth of the biliary tree by the neuroendocrine hormone serotonin.Gastroenterology. 2005; 128: 121-137Abstract Full Text Full Text PDF Scopus (107) Google Scholar, 5Marzioni M. Alpini G. Saccomanno S. de Minicis S. Glaser S. Francis H. Trozzi L. Venter J. Orlando F. Fava G. Candelaresi C. Macarri G. Benedetti A. Endogenous opioids modulate the growth of the biliary tree in the course of cholestasis.Gastroenterology. 2006; 130: 1831-1847Google Scholar and by the measurement of the BrDU incorporation5Marzioni M. Alpini G. Saccomanno S. de Minicis S. Glaser S. Francis H. Trozzi L. Venter J. Orlando F. Fava G. Candelaresi C. Macarri G. Benedetti A. Endogenous opioids modulate the growth of the biliary tree in the course of cholestasis.Gastroenterology. 2006; 130: 1831-1847Google Scholar for the in vitro studies. Changes in the bile duct mass by the quantitative immunohistochemistry CK-19, a specific marker of cholangiocytes,4Marzioni M. Glaser S. Francis H. Marucci L. Benedetti A. Alvaro D. Taffetani S. Ueno Y. Roskams T. Phinizy J.L. Venter J. Fava G. LeSage G. Alpini G. Autocrine/paracrine regulation of the growth of the biliary tree by the neuroendocrine hormone serotonin.Gastroenterology. 2005; 128: 121-137Abstract Full Text Full Text PDF Scopus (107) Google Scholar, 5Marzioni M. Alpini G. 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Autocrine/paracrine regulation of the growth of the biliary tree by the neuroendocrine hormone serotonin.Gastroenterology. 2005; 128: 121-137Abstract Full Text Full Text PDF Scopus (107) Google Scholar comparing the amount of loaded proteins by immunoblots for β-actin.4Marzioni