Endogenous Opioids Modulate the Growth of the Biliary Tree in the Course of Cholestasis
2006; Elsevier BV; Volume: 130; Issue: 6 Linguagem: Inglês
10.1053/j.gastro.2006.02.021
ISSN1528-0012
AutoresMarco Marzioni, Gianfranco Alpini, S. Saccomanno, Samuele De Minicis, Shannon Glaser, Heather Francis, L. Trozzi, Juliet Venter, Fiorenza Orlando, Giammarco Fava, C. Candelaresi, Giampiero Macarri, A. Benedetti,
Tópico(s)Pediatric Hepatobiliary Diseases and Treatments
ResumoBackground & Aims There is poor knowledge on the factors that modulate the growth of cholangiocytes, the epithelial cell target of cholangiopathies, which are diseases leading to progressive loss of bile ducts and liver failure. Endogenous opioids are known to modulate cell growth. In the course of cholestasis, the opioidergic system is hyperactive, and in cholangiocytes a higher expression of opioid peptide messenger RNA has been described. This study aimed to verify if such events affect the cholangiocyte proliferative response to cholestasis. Methods The presence of the δ opioid receptor (OR), μOR, and κOR was evaluated. The effects on cholangiocyte proliferation of the in vitro and in vivo exposure to their selective agonists, together with the intracellular signals, were then studied. The effects of the OR antagonist naloxone on cell growth were also tested both in vivo and in vitro. Results Cholangiocytes express all 3 receptors studied. δOR activation strongly diminished the proliferative and functional response of cholangiocytes to cholestasis, whereas μOR resulted in a slight increase in cell growth. The δOR signal is mediated by the IP3/CamKIIα/PKCα pathway, which inhibits the cAMP/PKA/ERK1/2/AKT cascade. In contrast, μOR activation stimulates the cAMP/PKA/ERK1/2/AKT cascade but does not affect the IP3/CamKIIα/PKCα pathway. The blockage of endogenous opioid peptides by naloxone further enhanced cholangiocyte growth both in vivo and in vitro. Conclusions The increase in opioid peptide synthesis in the course of cholestasis aims to limit the excessive growth of the biliary tree in the course of cholestasis by the interaction with the δOR expressed by cholangiocytes. Background & Aims There is poor knowledge on the factors that modulate the growth of cholangiocytes, the epithelial cell target of cholangiopathies, which are diseases leading to progressive loss of bile ducts and liver failure. Endogenous opioids are known to modulate cell growth. In the course of cholestasis, the opioidergic system is hyperactive, and in cholangiocytes a higher expression of opioid peptide messenger RNA has been described. This study aimed to verify if such events affect the cholangiocyte proliferative response to cholestasis. Methods The presence of the δ opioid receptor (OR), μOR, and κOR was evaluated. The effects on cholangiocyte proliferation of the in vitro and in vivo exposure to their selective agonists, together with the intracellular signals, were then studied. The effects of the OR antagonist naloxone on cell growth were also tested both in vivo and in vitro. Results Cholangiocytes express all 3 receptors studied. δOR activation strongly diminished the proliferative and functional response of cholangiocytes to cholestasis, whereas μOR resulted in a slight increase in cell growth. The δOR signal is mediated by the IP3/CamKIIα/PKCα pathway, which inhibits the cAMP/PKA/ERK1/2/AKT cascade. In contrast, μOR activation stimulates the cAMP/PKA/ERK1/2/AKT cascade but does not affect the IP3/CamKIIα/PKCα pathway. The blockage of endogenous opioid peptides by naloxone further enhanced cholangiocyte growth both in vivo and in vitro. Conclusions The increase in opioid peptide synthesis in the course of cholestasis aims to limit the excessive growth of the biliary tree in the course of cholestasis by the interaction with the δOR expressed by cholangiocytes. Cholangiopathies are chronic cholestatic diseases that affect cholangiocytes, the epithelial cells lining the intrahepatic biliary tree.1Alpini 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, 2Lazaridis K.N. Strazzabosco M. LaRusso N.F. The cholangiopathies disorders of biliary epithelia.Gastroenterology. 2004; 127: 1565-1577Google Scholar Such chronic cholestatic diseases represent a challenge for the clinician; because definitive medical treatments are not yet available, cholangiopathies often lead to liver failure. Indeed, 20% of liver transplants among adults and 50% of those among pediatric patients are due to these disorders.3Annual Report of the U. S. Organ Procurement and Transplantation Network and the Scientific Registry for Transplant Recipients: transplant data 1991–2000. Department of Health and Human Services, Health Resources and Services Administration, Office of Special Programs, Division of Transplantation, Rockville, MD2001Google Scholar Many of the problems that arise in the management of cholangiopathies can at least in part be ascribed to the poor knowledge about their pathophysiology. Cholangiopathies are indeed commonly characterized by an impaired proliferative response to duct injury. This, together with increased cell death, leads to vanishing of bile ducts.2Lazaridis K.N. Strazzabosco M. LaRusso N.F. The cholangiopathies disorders of biliary epithelia.Gastroenterology. 2004; 127: 1565-1577Google Scholar, 3Annual Report of the U. S. Organ Procurement and Transplantation Network and the Scientific Registry for Transplant Recipients: transplant data 1991–2000. Department of Health and Human Services, Health Resources and Services Administration, Office of Special Programs, Division of Transplantation, Rockville, MD2001Google Scholar However, very little is known about which factors are able to affect cholangiocyte growth, with particular regard to those endogenous molecules or systems that affect cholangiocyte proliferation either way.2Lazaridis K.N. Strazzabosco M. LaRusso N.F. The cholangiopathies disorders of biliary epithelia.Gastroenterology. 2004; 127: 1565-1577Google Scholar In recent years, several experimental models have been used to investigate those issues. In particular, it has been shown that if cholangiocytes do not proliferate in a normal state,2Lazaridis K.N. Strazzabosco M. LaRusso N.F. The cholangiopathies disorders of biliary epithelia.Gastroenterology. 2004; 127: 1565-1577Google Scholar marked growth of the biliary epithelium can be achieved, for example, by subjecting rodents to extrahepatic biliary obstruction by bile duct ligation (BDL).1Alpini 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, 2Lazaridis K.N. Strazzabosco M. LaRusso N.F. The cholangiopathies disorders of biliary epithelia.Gastroenterology. 2004; 127: 1565-1577Google Scholar, 4Glaser S. Benedetti A. Marucci L. Alvaro D. Baiocchi L. Kanno N. Caligiuri A. Phinizy J.L. Chowdury U. Papa E. LeSage G. Alpini G. Gastrin inhibits cholangiocyte growth in bile duct-ligated rats by interaction with cholecystokinin-B/Gastrin receptors via D-myo-inositol 1,4,5-triphosphate-, Ca(2+)-, and protein kinase C alpha-dependent mechanisms.Hepatology. 2000; 32: 17-25Google Scholar Such studies helped to clarify that cholangiocyte functional activity is closely coupled with cholangiocyte proliferation.1Alpini 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, 5Kanno N. LeSage G. Glaser S. Alpini G. Regulation of cholangiocyte bicarbonate secretion.Am J Physiol Gastrointest Liver Physiol. 2001; 281: G612-G625Google Scholar Nerves, neuropeptides, and neuroendocrine hormones play a significant role in the regulation of cholangiocyte biology.4Glaser S. Benedetti A. Marucci L. Alvaro D. Baiocchi L. Kanno N. Caligiuri A. Phinizy J.L. Chowdury U. Papa E. 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Autocrine/paracrine regulation of the growth of the biliary tree by the neuroendocrine hormone serotonin.Gastroenterology. 2005; 128: 121-137Google Scholar Interestingly, it has been shown that in the course of cholangiopathies, the biliary epithelium acquires neuroendocrine features that are not present in the normal liver12Roskams T. van den Oord J.J. De Vos R. Desmet V.J. Neuroendocrine features of reactive bile ductules in cholestatic liver disease.Am J Pathol. 1990; 137: 1019-1025Google Scholar; however, the link of this event to cholangiocyte biology is still obscure. We have recently shown that after BDL, cholangiocytes overexpress the neuroendocrine hormone serotonin, which is secreted in an autocrine-paracrine fashion to limit the growth of these cells associated to cholestasis.11Marzioni 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.D. Alpini G. Autocrine/paracrine regulation of the growth of the biliary tree by the neuroendocrine hormone serotonin.Gastroenterology. 2005; 128: 121-137Google Scholar There is increasing evidence that endogenous opioid peptides have receptor-mediated roles as growth regulators not only in neuronal but also in nonneuronal cells and tissues.13Glasel J.A. The effects of morphine on cell proliferation.Prog Drug Res. 2000; 55: 33-80Google Scholar Specifically, in the gastrointestinal tract, endogenous opioids have been shown to affect pancreatic,14Zagon I.S. Smith J.P. McLaughlin P.J. Human pancreatic cancer cell proliferation in tissue culture is tonically inhibited by opioid growth factor.Int J Oncol. 1999; 14: 577-584Google Scholar colonic,15Zagon I.S. Hytrek S.D. McLaughlin P.J. Opioid growth factor tonically inhibits human colon cancer cell proliferation in tissue culture.Am J Physiol. 1996; 271: R511-R518Google Scholar and esophageal16Zagon I.S. Wu Y. McLaughlin P.J. 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Hepatic Met-enkephalin immunoreactivity is enhanced in primary biliary cirrhosis.Liver. 2002; 22: 107-113Google Scholar, 20Bergasa N.V. Rothman R.B. Vergalla J. Xu H. Swain M.G. Jones E.A. Central mu-opioid receptors are down-regulated in a rat model of cholestasis.J Hepatol. 1992; 15: 220-224Google Scholar, 21Bergasa N.V. Vergalla J. Swain M.G. Jones E.A. Hepatic concentrations of proenkephalin-derived opioids are increased in a rat model of cholestasis.Liver. 1996; 16: 298-302Google Scholar, 22Swain M.G. Rothman R.B. Xu H. Vergalla J. Bergasa N.V. Jones E.A. Endogenous opioids accumulate in plasma in a rat model of acute cholestasis.Gastroenterology. 1992; 103: 630-635Google Scholar Such discoveries led to an understanding of the fundamental role played by opioid peptides in the genesis of the pruritus of cholestasis.23Jones E.A. Bergasa N.V. The pruritus of cholestasis.Hepatology. 1999; 29: 1003-1006Google Scholar Interestingly, it has also been shown that in the course of experimental cholestasis, the messenger RNA for preproenkephalin, which codes for Met-enkephalin and Leu-enkephalin and Met-enkephalin–containing peptides, is expressed in the liver. In addition, Met-enkephalin immunoreactivity is expressed in the liver, particularly in cholangiocytes, of patients with primary biliary cirrhosis19Bergasa N.V. Liau S. Homel P. Ghali V. Hepatic Met-enkephalin immunoreactivity is enhanced in primary biliary cirrhosis.Liver. 2002; 22: 107-113Google Scholar (the most common of the cholangiopathies among adults)2Lazaridis K.N. Strazzabosco M. LaRusso N.F. The cholangiopathies disorders of biliary epithelia.Gastroenterology. 2004; 127: 1565-1577Google Scholar and of rats with cholestasis.24Bergasa N.V. Sabol S.L. Young III, W.S. Kleiner D.E. Jones E.A. Cholestasis is associated with preproenkephalin mRNA expression in the adult rat liver.Am J Physiol. 1995; 268: G346-G354Google Scholar Despite the abundant evidence strongly linking the opioidergic system to cholestasis, the meaning of that link is still obscure. Specifically, it is not known yet whether endogenous opioid peptides might affect cholangiocyte biology. Therefore, we developed a study that aimed to answer the following questions. (1) Do cholangiocytes express δOR, μOR, and κOR? (2) Does the activation of those receptors affect cholangiocyte proliferation and functional activity? (3) Which are the intracellular pathways that mediate the OR signal in cholangiocytes? (4) Do endogenous opioids participate in the neuroendocrine peptide loop that regulates cholangiocyte proliferation in the course of cholestasis? Reagents were purchased from Sigma Chemical Co (St Louis, MO) unless otherwise indicated. Intracellular adenosine 3′,5′-cyclic monophosphate (cAMP) and inositol 1,4,5-triphosphate (IP3) levels were determined with radioimmunoassay kits purchased from Amersham (Arlington Heights, IL). Antibodies for immunoblotting and immunohistochemistry were purchased from Santa Cruz Biotechnologies Inc (Santa Cruz, CA) unless otherwise indicated. The antibody anti–cytokeratin-19 was purchased from Novocastra (Milan, Italy). The expression of ORs was evaluated by immunohistochemistry in formalin-fixed and paraffin-embedded liver sections. To inactivate endogenous biotin, after immersion in hydrogen peroxide diluted in methanol (3%) to reduce endogenous peroxidase activity, sections were incubated with avidin and afterward with biotin. Liver sections were then incubated with or without the OR primary antibodies (1:20 dilution in 1× phosphate-buffered saline) overnight. After exposure to biotinylated secondary antibody, reactive sites were detected using peroxidase-conjugated streptavidin and diaminobenzidine (0.06%) in 1× phosphate-buffered saline containing 0.03% H2O2. Slides were counterstained with Mayer hematoxylin.25Bao L. Jin S.X. Zhang C. Wang L.H. Xu Z.Z. Zhang F.X. Wang L.C. Ning F.S. Cai H.J. Guan J.S. Xiao H.S. Xu Z.Q. He C. Hokfelt T. Zhou Z. Zhang X. Activation of delta opioid receptors induces receptor insertion and neuropeptide secretion.Neuron. 2003; 37: 121-133Google Scholar, 26Melone M. Brecha N.C. Sternini C. Evans C. Conti F. Etorphine increases the number of mu-opioid receptor-positive cells in the cerebral cortex.Neuroscience. 2000; 100: 439-443Google Scholar, 27Zhang N. Rogers T.J. Caterina M. Oppenheim J.J. 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Proinflammatory chemokines, such as C-C chemokine ligand 3, desensitize mu-opioid receptors on dorsal root ganglia neurons.J Immunol. 2004; 173: 594-599Google Scholar Rat brain and 0.2% bovine serum albumin (BSA) were used as positive and negative controls, respectively. To evaluate the effects of OR activation on cholangiocyte proliferation, pure normal and (1 week) BDL rat cholangiocytes were subjected to long-term incubation (5 hours) at 37°C with 0.2% BSA (control), with either [d-pen2,5]-enkephalin (DPDPE; 0.1 μmol/L, a δOR selective agonist)14Zagon I.S. Smith J.P. McLaughlin P.J. Human pancreatic cancer cell proliferation in tissue culture is tonically inhibited by opioid growth factor.Int J Oncol. 1999; 14: 577-584Google Scholar or [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO; 0.1 μmol/L, a μOR selective agonist),14Zagon I.S. Smith J.P. McLaughlin P.J. 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Furthermore, to investigate whether ORs transduct the message through the activation of Ca2+ signaling, cells were also preincubated with either 2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA)/AM (5 μmol/L, an intracellular Ca2+ chelator),8LeSage G.D. Marucci L. Alvaro D. Glaser S. Benedetti A. Marzioni M. Patel T. Francis H. Phinizy J.L. Alpini G. Insulin inhibits secretin-induced ductal secretion by activation of PKC alpha and inhibition of PKA activity.Hepatology. 2002; 36: 641-651Google Scholar, 11Marzioni 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.D. Alpini G. Autocrine/paracrine regulation of the growth of the biliary tree by the neuroendocrine hormone serotonin.Gastroenterology. 2005; 128: 121-137Google Scholar KN62 (10 μmol/L, a calcium-calmodulin kinase II inhibitor),30Wright D.C. Hucker K.A. Holloszy J.O. Han D.H. 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To study the effects of OR agonist administration on cholangiocyte proliferation and, as a consequence, on their functional activity, our studies were performed in (1) normal rats and rats with BDL1Alpini 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 (for cholangiocyte purification or liver sections) or bile duct incannulation (BDI) (BDI for bile collection) for 1 week1Alpini 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 and (2) normal, BDL, BDI (that immediately after BDL or BDI) rats treated with intraperitoneal injections of either DPDPE (10 mg · kg−1 · day−1)37Chen C. Pollack G.M. 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Roskams T. Phinizy J.L. Venter J. Fava G. LeSage G.D. Alpini G. Autocrine/paracrine regulation of the growth of the biliary tree by the neuroendocrine hormone serotonin.Gastroenterology. 2005; 128: 121-137Google Scholar Before each procedure, animals were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally). Study protocols were performed in compliance with the institution guidelines. Purification40Ishii 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 of cholangiocytes from normal or 1-week BDL rats was performed using a monoclonal antibody (immunoglobulin M, kindly provided by Dr R. Faris, Brown University, Providence, RI) against an unidentified membrane antigen expressed by all rat intrahepatic cholangiocytes.40Ishii 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 Separation of small and large cholangiocyte subpopulations was obtained by counterflow elutriation as previously reported.41Alpini G. Ulrich C. Roberts S. Phillips J.O. Ueno Y. Podila P.V. Colegio O. LeSage G.D. Miller L.J. LaRusso N.F. Molecular and functional heterogeneity of cholangiocytes from rat liver after bile duct ligation.Am J Physiol. 1997; 272: G289-G297Google Scholar At the end of each procedure, purity of cholangiocytes was assessed by cytochemistry for γ-glutamyltransferase,4Glaser S. Benedetti A. Marucci L. Alvaro D. Baiocchi L. Kanno N. Caligiuri A. Phinizy J.L. Chowdury U. Papa E. LeSage G. Alpini G. Gastrin inhibits cholangiocyte growth in bile duct-ligated rats by interaction with cholecystokinin-B/Gastrin receptors via D-myo-inositol 1,4,5-triphosphate-, Ca(2+)-, and protein kinase C alpha-dependent mechanisms.Hepatology. 2000; 32: 17-25Google Scholar, 9LeSage G.D. 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
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