Autocrine/paracrine regulation of the growth of the biliary tree by the neuroendocrine hormone serotonin
2005; Elsevier BV; Volume: 128; Issue: 1 Linguagem: Inglês
10.1053/j.gastro.2004.10.002
ISSN1528-0012
AutoresMarco Marzioni, Shannon Glaser, Heather Francis, Luca Marucci, A. Benedetti, Domenico Alvaro, Silvia Taffetani, Yoshiyuki Ueno, Tania Roskams, Jo Lynne Phinizy, Juliet Venter, Giammarco Fava, Gene LeSage, Gianfranco Alpini,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoBackground & Aims: The biliary tree is the target of cholangiopathies that are chronic cholestatic liver diseases characterized by loss of proliferative response and enhanced apoptosis of cholangiocytes, the epithelial cells lining the biliary tree. The endogenous factors that regulate cholangiocyte proliferation are poorly understood. Therefore, we studied the role of the neuroendocrine hormone serotonin as a modulator of cholangiocyte proliferation. Methods: The presence of the serotonin 1A and 1B receptors on cholangiocytes was evaluated. We then tested whether the activation of such receptors by the administration of the selective agonists modifies cholangiocyte proliferation and functional activity both in vivo and in vitro. In addition, the intracellular signal mediating the serotonin receptor action in cholangiocytes was characterized. We studied the expression and secretion of serotonin by cholangiocytes and the effects of the neutralization of the secreted hormone on the growth of the biliary tree. Results: Cholangiocytes express the serotonin 1A and 1B receptors. Their activation markedly inhibits the growth and choleretic activity of the biliary tree in the bile duct-ligated rat, a model of chronic cholestasis. Such changes are mediated by enhanced d-myo-inositol 1,4,5-triphosphate/Ca2+/protein kinase C signaling and the consequent inhibition of the adenosine 3′,5′-cyclic monophosphate/protein kinase A/Src/extracellular signal-regulated kinase 1/2 cascade. Cholangiocytes secrete serotonin, the blockage of which enhances cholangiocyte proliferation in the course of cholestasis. Conclusions: We observed the existence of an autocrine loop based on serotonin that limits the growth of the biliary tree in the course of chronic cholestasis. Our novel findings might open new approaches for the management of cholangiopathies. Background & Aims: The biliary tree is the target of cholangiopathies that are chronic cholestatic liver diseases characterized by loss of proliferative response and enhanced apoptosis of cholangiocytes, the epithelial cells lining the biliary tree. The endogenous factors that regulate cholangiocyte proliferation are poorly understood. Therefore, we studied the role of the neuroendocrine hormone serotonin as a modulator of cholangiocyte proliferation. Methods: The presence of the serotonin 1A and 1B receptors on cholangiocytes was evaluated. We then tested whether the activation of such receptors by the administration of the selective agonists modifies cholangiocyte proliferation and functional activity both in vivo and in vitro. In addition, the intracellular signal mediating the serotonin receptor action in cholangiocytes was characterized. We studied the expression and secretion of serotonin by cholangiocytes and the effects of the neutralization of the secreted hormone on the growth of the biliary tree. Results: Cholangiocytes express the serotonin 1A and 1B receptors. Their activation markedly inhibits the growth and choleretic activity of the biliary tree in the bile duct-ligated rat, a model of chronic cholestasis. Such changes are mediated by enhanced d-myo-inositol 1,4,5-triphosphate/Ca2+/protein kinase C signaling and the consequent inhibition of the adenosine 3′,5′-cyclic monophosphate/protein kinase A/Src/extracellular signal-regulated kinase 1/2 cascade. Cholangiocytes secrete serotonin, the blockage of which enhances cholangiocyte proliferation in the course of cholestasis. Conclusions: We observed the existence of an autocrine loop based on serotonin that limits the growth of the biliary tree in the course of chronic cholestasis. Our novel findings might open new approaches for the management of cholangiopathies. The intrahepatic biliary tree is the target of several human diseases defined as cholangiopathies,1Alpini G. McGill J.M. LaRusso N.F. The pathobiology of biliary epithelia.Hepatology. 2002; 35: 1256-1268Crossref PubMed Scopus (126) Google Scholar characterized by chronic cholestasis leading to liver failure.1Alpini G. McGill J.M. LaRusso N.F. The pathobiology of biliary epithelia.Hepatology. 2002; 35: 1256-1268Crossref PubMed Scopus (126) Google Scholar, 2Desmet V.J. Vanishing bile duct disorders.Prog Liver Dis. 1992; 10: 89-121PubMed Google Scholar Recent studies have shown that such disorders are responsible for more than 20% of the liver transplantations among adults and for the 50% among pediatric patients in the United States.3Annual report of the U.S. Organ Procurement and Transplantation Network and the Scientific Registry for Transplant Recipients. 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Neuroendocrine features of reactive bile ductules in cholestatic liver disease.Am J Pathol. 1990; 137: 1019-1025PubMed Google ScholarSerotonin is a neuroendocrine hormone secreted by enterochromaffin cells throughout the entire gastrointestinal tract.18Suzuki A. Naruse S. Kitagawa M. Ishiguro H. Yoshikawa T. Ko S.B. Yamamoto A. Hamada H. Hayakawa T. 5-Hydroxytryptamine strongly inhibits fluid secretion in guinea pig pancreatic duct cells.J Clin Invest. 2001; 108: 749-756PubMed Google Scholar, 19Li Y. Hao Y. Zhu J. Owyang C. Serotonin released from intestinal enterochromaffin cells mediates luminal non-cholecystokinin-stimulated pancreatic secretion in rats.Gastroenterology. 2000; 118: 1197-1207Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar The local release of this hormone leads to marked changes in the functions of gastrointestinal epithelial cells. 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Therefore, in this investigation we posed the following questions: (1) Do cholangiocytes express functional serotonin 1A and 1B receptor subtypes? (2) What is the effect of the activation of these receptors on cholangiocyte proliferation and functional activity in the course of chronic cholestasis? (3) Which intracellular pathways are involved in the transduction of the serotoninergic intracellular signal? and (4) Is serotonin involved in the autocrine modulation of the cholangiocyte response to chronic cholestasis?Materials and methodsMaterialsReagents were purchased from Sigma Chemical (St. Louis, MO) unless otherwise indicated. The monoclonal mouse antibody against proliferating cellular nuclear antigen (PCNA) was obtained from DAKO (Kyoto, Japan). Intracellular cAMP and d-myo-inositol 1,4,5-triphosphate (IP3) levels were determined with radioimmunoassay (RIA) kits purchased from Amersham (Arlington Heights, IL). Serotonin receptor selective agonists and antagonists were purchased from Tocris (Ballwin, MO). Antibodies for immunoblotting and immunohistochemistry were purchased from Santa Cruz Biotechnologies Inc. (Santa Cruz, CA) unless otherwise indicated. The substrate for γ-glutamyltranspeptidase (γ-GT), N-(γ-l-glutamyl)-4-methoxy-2-naphthylamide, was purchased from Polysciences (Warrington, PA).Expression of the serotonin 1A and 1B receptor subtypes in cholangiocytesThe expression of serotonin receptors was evaluated by immunohistochemistry in formalin-fixed and paraffin-embedded liver sections. Sections were immersed in 0.01 mol/L citrate buffer (pH 6.0) and irradiated in a microwave oven for 15 minutes. Liver sections were then incubated with or without the polyclonal primary antibody (1:20 dilution in 1× phosphate-buffered saline) for 2 hours at room temperature, and the reactive sites were detected by using a DAKO LSAB kit (according to the manufacturer’s instructions) followed by incubation for 5 minutes with 1× phosphate-buffered saline containing 0.06% diaminobenzidine and 0.01% H2O2. Slides were counterstained with Harris hematoxylin.Immunofluorescence was performed in acetone-fixed fresh-frozen liver sections and incubated with either the serotonin 1A or 1B antibody (both diluted 1:20) at 4°C overnight. Thereafter, after several washings, the samples were incubated with Alexa488-labeled secondary antibody (diluted 1:100; Molecular Probes, Eugene, OR) for 2 hours at room temperature. Then, the samples were mounted with antifade (Molecular Probes). These samples were evaluated by using confocal laser microscopy (Bio-Rad, Hercules, CA) with adequate filters.The expression of the receptors was also evaluated by immunoblots both in whole cells and after subcellular fractionation to separate cholangiocyte basolateral and apical membranes. Basolateral and apical fractions were obtained as previously described.29Tietz P.S. Holman R.T. Miller L.J. LaRusso N.F. Isolation and characterization of rat cholangiocyte vesicles enriched in apical or basolateral plasma membrane domains.Biochemistry. 1995; 34: 15436-15443Crossref PubMed Scopus (63) Google Scholar, 30Glaser S. Alvaro D. Roskams T. Phinizy J.L. Stoica G. Francis H. Ueno Y. Barbaro B. Marzioni M. Mauldin J. Rashid S. Mancino M.G. LeSage G. Alpini G. Dopaminergic inhibition of secretin-stimulated choleresis by increased PKC-gamma expression and decrease of PKA activity.Am J Physiol Gastrointest Liver Physiol. 2003; 284: G683-G694PubMed Google ScholarExperimental designIn vivo studiesMale Fischer 344 rats (150–175 g), purchased from Charles River Laboratories (Wilmington, MA), were kept in a temperature-controlled environment (20°C–22°C) with a 12-hour light/dark cycle and with free access to drinking water and standard rat chow.To study the effect of serotonin receptor agonist administration on cholangiocyte proliferation and functional activity, our studies were performed in (1) normal rats and rats with BDL7Alpini G. Glaser S. Ueno Y. Phinizy J.L. Rodgers R. Francis H. Baiocchi L. Holcomb L. Caligiuri A. LeSage G. 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Aggression escalated by social instigation or by discontinuation of reinforcement (“frustration”) in mice: inhibition by anpirtoline: a 5HT1B receptor agonist.Neuropsychopharmacology. 2002; 27: 171-181Crossref PubMed Scopus (121) Google Scholar; 1 nmol · kg−1 · h−1), or 8-hydroxy-DPAT and anpirtoline together for 1 week. 8-Hydroxy-DPAT and anpirtoline are known to have a very high affinity for their receptors: endogenous ligands do not represent an obstacle for their binding to the receptors.32Currie P.J. Braver M. Mirza A. Sricharoon K. Sex differences in the reversal of fluoxetine-induced anorexia following raphe injections of 8-OH-DPAT.Psychopharmacology. 2004; 172: 359-364Crossref PubMed Scopus (25) Google Scholar To study the effect of the blockage of endogenous serotonin on cholangiocyte proliferation, normal or BDL rats were treated by IP injections with a serotonin-neutralizing antibody (12 μg/d; Biomeda, Foster City, CA) or nonimmune serum for 1 week.The animals were fasted overnight before each experiment.7Alpini G. Glaser S. Ueno Y. Phinizy J.L. Rodgers R. Francis H. Baiocchi L. Holcomb L. Caligiuri A. LeSage G. Bile acid feeding induces cholangiocyte proliferation and secretion evidence for bile acid-regulated ductal secretion.Gastroenterology. 1999; 116: 179-186Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar Before each procedure, animals were anesthetized with sodium pentobarbital (50 mg/kg, IP). Study protocols were performed in compliance with institutional guidelines.In vitro studiesTo show that the effects of the serotonin receptor agonists on cholangiocyte proliferation are due to a specific interaction with serotonin receptors on cholangiocytes, pure cholangiocytes from 1-week BDL rats were incubated at 37°C with (1) 0.2% bovine serum albumin (BSA; control) for 5 hours; (2) 0.2% BSA for 1 hour followed by 8-hydroxy-DPAT (a serotonin 1A receptor agonist; 10 μmol/L)22Li Y. Wu X.Y. Zhu J.X. Owyang C. Intestinal serotonin acts as paracrine substance to mediate pancreatic secretion stimulated by luminal factors.Am J Physiol Gastrointest Liver Physiol. 2001; 281: G916-G923PubMed Google Scholar, 23Azmitia E.C. Modern views on an ancient chemical serotonin effects on cell proliferation, maturation, and apoptosis.Brain Res Bull. 2001; 56: 413-424Crossref PubMed Scopus (386) Google Scholar, 25Aune T.M. Golden H.W. McGrath K.M. Inhibitors of serotonin synthesis and antagonists of serotonin 1A receptors inhibit T lymphocyte function in vitro and cell-mediated immunity in vivo.J Immunol. 1994; 153: 489-498PubMed Google Scholar, 33Middlemiss D.N. Fozard J.R. 8-Hydroxy-2-(di-n-propylamino)-tetralin discriminates between subtypes of the 5-HT1 recognition site.Eur J Pharmacol. 1983; 90: 151-153Crossref PubMed Scopus (826) Google Scholar for 4 hours with 0.2% BSA; (3) (S)-WAY 100135 dihydrochloride (a serotonin 1A receptor antagonist; 10 μmol/L)22Li Y. Wu X.Y. Zhu J.X. Owyang C. Intestinal serotonin acts as paracrine substance to mediate pancreatic secretion stimulated by luminal factors.Am J Physiol Gastrointest Liver Physiol. 2001; 281: G916-G923PubMed Google Scholar, 23Azmitia E.C. Modern views on an ancient chemical serotonin effects on cell proliferation, maturation, and apoptosis.Brain Res Bull. 2001; 56: 413-424Crossref PubMed Scopus (386) Google Scholar for 1 hour followed by 8-hydroxy-DPAT for 4 hours with 0.2% BSA; (4) 0.2% BSA for 1 hour followed by anpirtoline (a serotonin 1B receptor agonist; 10 μmol/L)22Li Y. Wu X.Y. Zhu J.X. Owyang C. Intestinal serotonin acts as paracrine substance to mediate pancreatic secretion stimulated by luminal factors.Am J Physiol Gastrointest Liver Physiol. 2001; 281: G916-G923PubMed Google Scholar, 31de Almeida R.M. Miczek K.A. Aggression escalated by social instigation or by discontinuation of reinforcement (“frustration”) in mice: inhibition by anpirtoline: a 5HT1B receptor agonist.Neuropsychopharmacology. 2002; 27: 171-181Crossref PubMed Scopus (121) Google Scholar for 4 hours with 0.2% BSA; (5) SB 216641 hydrochloride (a serotonin 1B receptor antagonist; 10 μmol/L)22Li Y. Wu X.Y. Zhu J.X. Owyang C. Intestinal serotonin acts as paracrine substance to mediate pancreatic secretion stimulated by luminal factors.Am J Physiol Gastrointest Liver Physiol. 2001; 281: G916-G923PubMed Google Scholar, 34Przegalinski E. Siwanowicz J. Nowak E. Papla I. Filip M. Role of 5-HT(1B) receptors in the sensitization to amphetamine in mice.Eur J Pharmacol. 2001; 422: 91-99Crossref PubMed Scopus (41) Google Scholar for 1 hour followed by anpirtoline for 4 hours with 0.2% BSA; or (6) 0.2% BSA for 1 hour followed by 8-hydroxy-DPAT and anpirtoline together for 4 hours with 0.2% BSA.To show that the effects of the serotonin 1A and 1B receptor agonists on cholangiocyte proliferation are mediated by the cAMP/protein kinase A (PKA)/Src pathway cells from 1-week BDL rats were also incubated with (1) dybutyryl-cAMP (cAMP agonist; 500 μmol/L)35Roma M.G. Milkiewicz P. Elias E. Coleman R. 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