Ca2+-Dependent Cytoprotective Effects of Ursodeoxycholic and Tauroursodeoxycholic Acid on the Biliary Epithelium in a Rat Model of Cholestasis and Loss of Bile Ducts
2006; Elsevier BV; Volume: 168; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2006.050126
ISSN1525-2191
AutoresMarco Marzioni, Heather Francis, A. Benedetti, Yoshiyuki Ueno, Giammarco Fava, Juliet Venter, Ramona Reichenbach, Maria Grazia Mancino, Ryun Summers, Gianfranco Alpini, Shannon Glaser,
Tópico(s)Drug-Induced Hepatotoxicity and Protection
ResumoChronic cholestatic liver diseases are characterized by impaired balance between proliferation and death of cholangiocytes, as well as vanishing of bile ducts and liver failure. Ursodeoxycholic acid (UDCA) is a bile acid widely used for the therapy of cholangiopathies. However, little is known of the cytoprotective effects of UDCA on cholangiocytes. Therefore, UDCA and its taurine conjugate tauroursodeoxycholic acid (TUDCA) were administered in vivo to rats simultaneously subjected to bile duct ligation and vagotomy, a model that induces cholestasis and loss of bile ducts by apoptosis of cholangiocytes. Because these two bile acids act through Ca2+ signaling, animals were also treated with BAPTA/AM (an intracellular Ca2+ chelator) or Gö6976 (a Ca2+-dependent protein kinase C-α inhibitor). The administration of UDCA or TUDCA prevented the induction of apoptosis and the loss of proliferative and functional responses observed in the bile duct ligation-vagotomized rats. These effects were neutralized by the simultaneous administration of BAPTA/AM or Gö6976. UDCA and TUDCA enhanced intracellular Ca2+ and IP3 levels, together with increased phosphorylation of protein kinase C-α. Parallel changes were observed regarding the activation of the MAPK and PI3K pathways, changes that were abolished by addition of BAPTA/AM or Gö6976. These studies provide information that may improve the response of cholangiopathies to medical therapy. Chronic cholestatic liver diseases are characterized by impaired balance between proliferation and death of cholangiocytes, as well as vanishing of bile ducts and liver failure. Ursodeoxycholic acid (UDCA) is a bile acid widely used for the therapy of cholangiopathies. However, little is known of the cytoprotective effects of UDCA on cholangiocytes. Therefore, UDCA and its taurine conjugate tauroursodeoxycholic acid (TUDCA) were administered in vivo to rats simultaneously subjected to bile duct ligation and vagotomy, a model that induces cholestasis and loss of bile ducts by apoptosis of cholangiocytes. Because these two bile acids act through Ca2+ signaling, animals were also treated with BAPTA/AM (an intracellular Ca2+ chelator) or Gö6976 (a Ca2+-dependent protein kinase C-α inhibitor). The administration of UDCA or TUDCA prevented the induction of apoptosis and the loss of proliferative and functional responses observed in the bile duct ligation-vagotomized rats. These effects were neutralized by the simultaneous administration of BAPTA/AM or Gö6976. UDCA and TUDCA enhanced intracellular Ca2+ and IP3 levels, together with increased phosphorylation of protein kinase C-α. Parallel changes were observed regarding the activation of the MAPK and PI3K pathways, changes that were abolished by addition of BAPTA/AM or Gö6976. These studies provide information that may improve the response of cholangiopathies to medical therapy. Cholangiocytes are the epithelial cells that line the intrahepatic biliary tree.1Alpini G McGill JM LaRusso NF The pathobiology of biliary epithelia.Hepatology. 2002; 35: 1256-1268Crossref PubMed Scopus (124) Google Scholar Cholangiocytes are the target of chronic cholestatic liver diseases, also termed cholangiopathies, a group of disorders responsible for more than 20% of liver transplants among adults and 50% among pediatric patients in the United States.22001 Annual Report of the US Organ Procurement and Transplantation Network and the Scientific Registry for Transplant Recipients: Transplant Data 1991–2000. Rockville, Department of Health and Human Services, Health Resources and Services Administration, Office of Special Programs, Division of Transplantation, 2001Google Scholar Whatever the primary cause may be, cholangiopathies are almost invariably characterized by an abnormal balance between cholangiocyte proliferation and death, thus leading to chronic cholestasis, ductopenia, and liver failure.1Alpini G McGill JM LaRusso NF The pathobiology of biliary epithelia.Hepatology. 2002; 35: 1256-1268Crossref PubMed Scopus (124) Google Scholar, 3Desmet VJ Vanishing bile duct disorders.Prog Liver Dis. 1992; 10: 89-121PubMed Google Scholar The autonomic nervous system plays a crucial role in hepatic regeneration and metabolic regulation of liver cells.4Bioulac-Sage P Lafon ME Saric J Balabaud C Nerves and perisinusoidal cells in human liver.J Hepatol. 1990; 10: 105-112Abstract Full Text PDF PubMed Scopus (117) Google Scholar, 5Kato H Shimazu T Effect of autonomic denervation on DNA synthesis during liver regeneration after partial hepatectomy.Eur J Biochem. 1983; 134: 473-478Crossref PubMed Scopus (65) Google Scholar After liver transplantation, nerve fibers are resected. Despite the fact that an increasing body of knowledge exists on the impact of transplantation on liver allograft, the functional consequences of the loss of autonomic innervation have been neglected.4Bioulac-Sage P Lafon ME Saric J Balabaud C Nerves and perisinusoidal cells in human liver.J Hepatol. 1990; 10: 105-112Abstract Full Text PDF PubMed Scopus (117) Google Scholar, 5Kato H Shimazu T Effect of autonomic denervation on DNA synthesis during liver regeneration after partial hepatectomy.Eur J Biochem. 1983; 134: 473-478Crossref PubMed Scopus (65) Google Scholar, 6Sakamoto I Takahashi T Kakita A Hayashi I Majima M Yamashina S Experimental study on hepatic reinnervation after orthotopic liver transplantation in rats.J Hepatol. 2002; 37: 814-823Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar Little is known of the importance of liver innervation on the final outcome of transplantation, but it has been shown that adjuvant treatment with the bile acid ursodeoxycholic acid (UDCA) reduces allograft rejection.7Friman S Persson H Schersten T Svanvik J Karlberg I Adjuvant treatment with ursodeoxycholic acid reduces acute rejection after liver transplantation.Transpl Int. 1992; 5: S187-S189PubMed Google Scholar Cholangiocytes are normally mitotically dormant8LeSage G Glaser S Gubba S Robertson WE Phinizy JL Lasater J Rodgers R Alpini G Regrowth of the rat biliary tree after 70% partial hepatectomy is coupled to increased secretin-induced ductal bile secretion.Gastroenterology. 1996; 111: 1633-1644Abstract Full Text PDF PubMed Scopus (121) Google Scholar; however, their proliferation can be triggered in several experimental models, the most validated of which is the bile duct ligated (BDL) rat model.9Alpini G Glaser SS Ueno Y Pham L Podila PV Caligiuri A LeSage G LaRusso NF Heterogeneity of the proliferative capacity of rat cholangiocytes after bile duct ligation.Am J Physiol. 1998; 274: G767-G775PubMed Google Scholar, 10Alpini 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: 569-578Crossref PubMed Scopus (286) Google Scholar The growth of the biliary tree after extrahepatic biliary obstruction by BDL is associated with a marked enhancement of cholangiocyte functional activity, eg, secretin-induced choleresis and intracellular cAMP synthesis.11Glaser SS Rodgers RE Phinizy JL Robertson WE Lasater J Caligiuri A Tretjak Z LeSage GD Alpini G Gastrin inhibits secretin-induced ductal secretion by interaction with specific receptors on rat cholangiocytes.Am J Physiol. 1997; 273: G1061-G1070PubMed Google Scholar, 12Kanno N LeSage G Glaser S Alpini G Regulation of cholangiocyte bicarbonate secretion.Am J Physiol. 2001; 281: G612-G625Google Scholar, 13LeSage G Alvaro D Benedetti A Glaser S Marucci L Eisel W Caligiuri A Baiocchi L Rodgers R Phinizy JL Francis H Alpini G Cholinergic system modulates growth, apoptosis and secretion of cholangiocytes from bile duct ligated rats.Gastroenterology. 1999; 117: 191-199Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar Cholinergic innervation of the liver is required for these proliferative and functional responses. We have demonstrated that severing the vagus nerve, which does not affect cholangiocyte growth or survival in the normal rat, abolishes the growth of the biliary tree after BDL.13LeSage G Alvaro D Benedetti A Glaser S Marucci L Eisel W Caligiuri A Baiocchi L Rodgers R Phinizy JL Francis H Alpini G Cholinergic system modulates growth, apoptosis and secretion of cholangiocytes from bile duct ligated rats.Gastroenterology. 1999; 117: 191-199Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 14Alvaro D Alpini G Jezequel AM Bassotti C Francia C Fraioli F Romeo R Marucci L Le Sage G Glaser SS Benedetti A Role and mechanisms of action of acetylcholine in the regulation of rat cholangiocyte secretory functions.J Clin Invest. 1997; 100: 1349-1362Crossref PubMed Scopus (114) Google Scholar, 15Marzioni M LeSage G Glaser S Patel T Marienfeld C Ueno Y Francis H Alvaro D Phinizy JL Tadlock L Benedetti A Marucci L Baiocchi L Alpini G Taurocholate prevents the loss of intrahepatic bile ducts due to vagotomy in bile duct ligated rats.Am J Physiol. 2003; 284: G837-G852Google Scholar In addition, vagotomy in BDL rats triggers cholangiocyte apoptosis and abolishes their functional activity. Together, the loss of proliferation and enhanced apoptosis lead to a significant reduction in the number of bile ducts.13LeSage G Alvaro D Benedetti A Glaser S Marucci L Eisel W Caligiuri A Baiocchi L Rodgers R Phinizy JL Francis H Alpini G Cholinergic system modulates growth, apoptosis and secretion of cholangiocytes from bile duct ligated rats.Gastroenterology. 1999; 117: 191-199Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 15Marzioni M LeSage G Glaser S Patel T Marienfeld C Ueno Y Francis H Alvaro D Phinizy JL Tadlock L Benedetti A Marucci L Baiocchi L Alpini G Taurocholate prevents the loss of intrahepatic bile ducts due to vagotomy in bile duct ligated rats.Am J Physiol. 2003; 284: G837-G852Google Scholar Bile acids play a major role in the regulation of cholangiocyte biology. Bile acids enter cholangiocytes by a specific transporter localized at the apical pole of the cell, thus altering cholangiocyte functional activity, proliferation, and apoptosis.15Marzioni M LeSage G Glaser S Patel T Marienfeld C Ueno Y Francis H Alvaro D Phinizy JL Tadlock L Benedetti A Marucci L Baiocchi L Alpini G Taurocholate prevents the loss of intrahepatic bile ducts due to vagotomy in bile duct ligated rats.Am J Physiol. 2003; 284: G837-G852Google Scholar, 16Alpini G Glaser S Robertson W Phinizy JL Rodgers RE Caligiuri A LeSage G Bile acids stimulate proliferative and secretory events in large but not small cholangiocytes.Am J Physiol. 1997; 273: G518-G529PubMed Google Scholar, 17Alpini G Glaser SS Ueno Y Rodgers R Phinizy JL Francis H Baiocchi L Holcomb LA Caligiuri A LeSage GD 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, 18Alpini G Ueno Y Glaser SS Marzioni M Phinizy JL Francis H LeSage G Bile acid feeding increased proliferative activity and apical bile acid transporter expression in both small and large rat cholangiocytes.Hepatology. 2001; 34: 868-886Crossref PubMed Scopus (103) Google Scholar In particular, we have shown that cholinergic nerves and bile acids cooperatively modulate cholangiocyte survival. In fact, taurocholic acid feeding protects from the above-described loss of bile ducts induced by vagotomy.15Marzioni M LeSage G Glaser S Patel T Marienfeld C Ueno Y Francis H Alvaro D Phinizy JL Tadlock L Benedetti A Marucci L Baiocchi L Alpini G Taurocholate prevents the loss of intrahepatic bile ducts due to vagotomy in bile duct ligated rats.Am J Physiol. 2003; 284: G837-G852Google Scholar UDCA is a hydrophilic bile acid that is widely used for the treatment of various chronic cholestatic diseases.19Paumgartner G Beuers U Ursodeoxycholic acid in cholestatic liver disease: mechanisms of action and therapeutic use revisited.Hepatology. 2002; 36: 525-531Crossref PubMed Scopus (523) Google Scholar Despite several mechanisms that have been postulated,19Paumgartner G Beuers U Ursodeoxycholic acid in cholestatic liver disease: mechanisms of action and therapeutic use revisited.Hepatology. 2002; 36: 525-531Crossref PubMed Scopus (523) Google Scholar the cytoprotective effects of UDCA on cholangiocytes remains poorly understood. We have previously shown that feeding of UDCA or its taurine conjugate tauroursodeoxycholic acid (TUDCA) inhibits the proliferative response induced by BDL in rats.20Alpini G Baiocchi L Glaser S Ueno Y Marzioni M Francis H Phinizy JL Angelico M LeSage G Ursodeoxycholate and tauroursodeoxycholate inhibit cholangiocyte growth and secretion of BDL rats through activation of PKC alpha.Hepatology. 2002; 35: 1041-1052Crossref PubMed Scopus (112) Google Scholar Furthermore, TUDCA reduces the growth of cholangiocellular neoplastic cell lines.21Alpini G Kanno N Phinizy JL Glaser S Francis H Taffetani S LeSage G Tauroursodeoxycholate inhibits human cholangiocarcinoma growth via Ca2+-, PKC-, and MAPK-dependent pathways.Am J Physiol. 2004; 286: G973-G982Google Scholar Such effects are mostly determined by the UDCA- and TUDCA-induced activation of Ca2+-dependent protein kinase C-α (PKC-α).20Alpini G Baiocchi L Glaser S Ueno Y Marzioni M Francis H Phinizy JL Angelico M LeSage G Ursodeoxycholate and tauroursodeoxycholate inhibit cholangiocyte growth and secretion of BDL rats through activation of PKC alpha.Hepatology. 2002; 35: 1041-1052Crossref PubMed Scopus (112) Google Scholar, 21Alpini G Kanno N Phinizy JL Glaser S Francis H Taffetani S LeSage G Tauroursodeoxycholate inhibits human cholangiocarcinoma growth via Ca2+-, PKC-, and MAPK-dependent pathways.Am J Physiol. 2004; 286: G973-G982Google Scholar However, the effects of UDCA on cholangiocyte survival in the course of cholestasis, associated with the loss of bile ducts, is unknown. To address this, we posed the following questions. 1) Do UDCA and TUDCA protect the biliary tree from damage induced by vagotomy in BDL rats? 2) Are the effects of UDCA and TUDCA associated with changes in Ca2+ signaling? 3) Does in vivo blockage of Ca2+ signaling neutralize the protective effects of UDCA and TUDCA on the vagotomy-induced damage of the biliary tree in the BDL rat? Finally, because the cytoprotective effect of UDCA and TUDCA on hepatocytes is mediated by the mitogen-activated protein kinase (MAPK) and by phosphatidyl-inositol-3-kinase (PI3K),22Qiao L Yacoub A Studer E Gupta S Pei XY Grant S Hylemon PB Dent P Inhibition of the MAPK and PI3K pathways enhances UDCA-induced apoptosis in primary rodent hepatocytes.Hepatology. 2002; 35: 779-789Crossref PubMed Scopus (130) Google Scholar, 23Schoemaker MH Conde de la Rosa L Buist-Homan M Vrenken TE Havinga R Poelstra K Haisma HJ Jansen PL Moshage H Tauroursodeoxycholic acid protects rat hepatocytes from bile acid-induced apoptosis via activation of survival pathways.Hepatology. 2004; 39: 1563-1573Crossref PubMed Scopus (189) Google Scholar we asked the following additional question: 4) Do the UDCA/TUDCA-induced changes in Ca2+ signaling subsequently modify the MAPK and PI3K pathways? Reagents were purchased from Sigma Chemical Co. (St. Louis, MO), unless differently indicated. Rat chow, containing either 1% UDCA (275 μmol/day), 1% TUDCA (275 μmol/day), or control diet (AIN-76), was prepared by Dyets Inc., Bethlehem, PA. Control chow (AIN 76) has the same composition of rat chow containing UDCA or TUDCA but does not contain bile acids. A radioimmunoassay kit for the determination of intracellular cAMP levels was purchased from Amersham (Arlington Heights, IL). The d-myo-inositol 1,4,5, trisphosphate (IP3) [3H] kit, for the determination of intracellular IP3 levels, was purchased from Amersham. Antibodies for immunohistochemistry or immunoblotting were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA), unless otherwise reported. BAPTA/AM and Gö6976 were purchased from Calbiochem (La Jolla, CA). Male Fischer 344 rats (150 to 175 g), purchased from Charles River (Wilmington, MA), were used in the study. Animals were kept in a temperature-controlled environment (20 to 22°C) with a 12-hour light-dark cycle with free access to drinking water and standard rat chow. As shown in Table 1, the study was mostly performed in eight experimental groups (groups 2 to 9) composed of 10 rats each. On day 0 (D0) for each group, six animals were subjected to BDL and four to BDI (bile duct incannulation).10Alpini 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: 569-578Crossref PubMed Scopus (286) Google Scholar, 15Marzioni M LeSage G Glaser S Patel T Marienfeld C Ueno Y Francis H Alvaro D Phinizy JL Tadlock L Benedetti A Marucci L Baiocchi L Alpini G Taurocholate prevents the loss of intrahepatic bile ducts due to vagotomy in bile duct ligated rats.Am J Physiol. 2003; 284: G837-G852Google Scholar, 20Alpini G Baiocchi L Glaser S Ueno Y Marzioni M Francis H Phinizy JL Angelico M LeSage G Ursodeoxycholate and tauroursodeoxycholate inhibit cholangiocyte growth and secretion of BDL rats through activation of PKC alpha.Hepatology. 2002; 35: 1041-1052Crossref PubMed Scopus (112) Google Scholar The six BDL rats were used either to obtain liver sections (n = 3) or for cholangiocyte purification (n = 3).15Marzioni M LeSage G Glaser S Patel T Marienfeld C Ueno Y Francis H Alvaro D Phinizy JL Tadlock L Benedetti A Marucci L Baiocchi L Alpini G Taurocholate prevents the loss of intrahepatic bile ducts due to vagotomy in bile duct ligated rats.Am J Physiol. 2003; 284: G837-G852Google Scholar, 20Alpini G Baiocchi L Glaser S Ueno Y Marzioni M Francis H Phinizy JL Angelico M LeSage G Ursodeoxycholate and tauroursodeoxycholate inhibit cholangiocyte growth and secretion of BDL rats through activation of PKC alpha.Hepatology. 2002; 35: 1041-1052Crossref PubMed Scopus (112) Google Scholar The four BDI animals were used for bile collection.10Alpini 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: 569-578Crossref PubMed Scopus (286) Google Scholar, 15Marzioni M LeSage G Glaser S Patel T Marienfeld C Ueno Y Francis H Alvaro D Phinizy JL Tadlock L Benedetti A Marucci L Baiocchi L Alpini G Taurocholate prevents the loss of intrahepatic bile ducts due to vagotomy in bile duct ligated rats.Am J Physiol. 2003; 284: G837-G852Google Scholar, 20Alpini G Baiocchi L Glaser S Ueno Y Marzioni M Francis H Phinizy JL Angelico M LeSage G Ursodeoxycholate and tauroursodeoxycholate inhibit cholangiocyte growth and secretion of BDL rats through activation of PKC alpha.Hepatology. 2002; 35: 1041-1052Crossref PubMed Scopus (112) Google Scholar To induce loss of bile ducts, animals of groups 3 to 9 (Table 1) were subjected to vagotomy.13LeSage G Alvaro D Benedetti A Glaser S Marucci L Eisel W Caligiuri A Baiocchi L Rodgers R Phinizy JL Francis H Alpini G Cholinergic system modulates growth, apoptosis and secretion of cholangiocytes from bile duct ligated rats.Gastroenterology. 1999; 117: 191-199Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 15Marzioni M LeSage G Glaser S Patel T Marienfeld C Ueno Y Francis H Alvaro D Phinizy JL Tadlock L Benedetti A Marucci L Baiocchi L Alpini G Taurocholate prevents the loss of intrahepatic bile ducts due to vagotomy in bile duct ligated rats.Am J Physiol. 2003; 284: G837-G852Google Scholar, 24Powley TL Prechtl JC Fox EA Berthoud HR Anatomical considerations for surgery of the rat abdominal vagus: distribution, paraganglia and regeneration.J Auton Nerv Syst. 1983; 9: 79-97Abstract Full Text PDF PubMed Scopus (56) Google Scholar This additional surgical procedure was performed on D0, immediately after BDL or BDI.Table 1Animal ModelsGroupSurgery*All the surgical procedures were performed on day 0 (D0); BDL was performed to obtain liver sections and to isolate cholangiocytes; BDI for bile collection.No. of animalsTreatment†All the treatments were started immediately after surgery and lasted one week.1-NRNone6Control diet + control injectionsNone42-BDLBDL6Control diet + control injectionsBDI43-BDL + vagotomyBDL + vagotomy6Control diet + control injectionsBDI + vagotomy44-BDL + vagotomy + UDCABDL + vagotomy6UDCA feeding + control injectionsBDI + vagotomy45-BDL + vagotomy + UDCA + BAPTA/AMBDL + vagotomy6UDCA feeding + BAPTA/AM injectionsBDI + vagotomy46-BDL + vagotomy + UDCA + Gö6976BDL + vagotomy6UDCA feeding + Gö6976 injectionsBDI + vagotomy47-BDL + vagotomy + TUDCABDL + vagotomy6TUDCA feeding + control injectionsBDI + vagotomy48-BDL + vagotomy + TUDCA + BAPTA/AMBDL + vagotomy6TUDCA feeding + BAPTA/AM injectionsBDI + vagotomy49-BDL + vagotomy + TUDCA + Gö6976BDL + vagotomy6TUDCA feeding + Gö6976 injectionsBDI + vagotomy4* All the surgical procedures were performed on day 0 (D0); BDL was performed to obtain liver sections and to isolate cholangiocytes; BDI for bile collection.† All the treatments were started immediately after surgery and lasted one week. Open table in a new tab Groups 2 to 3 (Table 1) received a control diet. In contrast, to study whether UDCA or TUDCA protects bile ducts from the damage observed in the BDL+ vagotomy model, groups 4 to 6 and 7 to 9 (Table 1) were fed a diet enriched with 1% UDCA or 1% TUDCA, respectively.20Alpini G Baiocchi L Glaser S Ueno Y Marzioni M Francis H Phinizy JL Angelico M LeSage G Ursodeoxycholate and tauroursodeoxycholate inhibit cholangiocyte growth and secretion of BDL rats through activation of PKC alpha.Hepatology. 2002; 35: 1041-1052Crossref PubMed Scopus (112) Google Scholar The bile acid feeding started on D0, immediately after surgery, and lasted 1 week. Groups 2 to 4 and 7 (Table 1) received control injections. To verify whether the effects of UDCA and TUDCA were mediated by the Ca2+-dependent PKC signaling, groups 5 and 8 (Table 1) were also treated with daily intraperitoneal injections of BAPTA/AM (an intracellular Ca2+ chelator, 6 mg/kg body weight/day),25Jang YJ Ryu HJ Choi YO Kim C Leem CH Park CS Improvement of insulin sensitivity by chelation of intracellular Ca(2+) in high-fat-fed rats.Metabolism. 2002; 51: 912-918Abstract Full Text PDF PubMed Scopus (20) Google Scholar whereas groups 6 and 9 were treated with daily intraperitoneal injections of Gö6976 (a Ca2+-dependent PKC-α inhibitor, 2.35 mg/kg body weight, by a 0.05 mmol/L stock solution in 0.1% dimethyl sulfoxide).26Biswas DK Dai SC Cruz A Weiser B Graner E Pardee AB The nuclear factor kappa B (NF-kappa B): a potential therapeutic target for estrogen receptor negative breast cancers.Proc Natl Acad Sci USA. 2001; 98: 10386-10391Crossref PubMed Scopus (153) Google Scholar Treatments with injections started on D0, immediately after surgery, and lasted 1 week. As baseline control, we used 10 normal rats (NR, group 1) that did not undergo any surgery or treatment for 1 week (Table 1). Four of these animals were subjected to BDI on day 7 (D7), immediately before starting bile collection. BDL was performed as previously described.27Steiner JW Carruthers JS Kalifat SR The ductular cell reaction of rat liver in extrahepatic cholestasis. I. Proliferated biliary epithelial cells.Exp Mol Pathol. 1962; 1: 162-185Crossref PubMed Scopus (55) Google Scholar Briefly, under pentobarbital anesthesia (50 mg/kg, i.p.), the common bile duct was exposed through a paramedian incision, ligated twice (silk suture, 0.5 cm) and finally severed between the ligations. BDI was performed as previously described by us.10Alpini 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: 569-578Crossref PubMed Scopus (286) Google Scholar Briefly, under pentobarbital anesthesia (50 mg/kg, i.p.), the common bile duct was cannulated through a small abdominal incision. After free flow of bile was established, the bile duct cannula (4 to 5 cm long) was sealed at its open end with a flame and secured to the abdominal wall with several ligatures. The abdomen was then sutured, and the animal allowed to recover and kept in a standard rat cage. Total vagotomy was performed as described previously.15Marzioni M LeSage G Glaser S Patel T Marienfeld C Ueno Y Francis H Alvaro D Phinizy JL Tadlock L Benedetti A Marucci L Baiocchi L Alpini G Taurocholate prevents the loss of intrahepatic bile ducts due to vagotomy in bile duct ligated rats.Am J Physiol. 2003; 284: G837-G852Google Scholar, 17Alpini G Glaser SS Ueno Y Rodgers R Phinizy JL Francis H Baiocchi L Holcomb LA Caligiuri A LeSage GD 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, 24Powley TL Prechtl JC Fox EA Berthoud HR Anatomical considerations for surgery of the rat abdominal vagus: distribution, paraganglia and regeneration.J Auton Nerv Syst. 1983; 9: 79-97Abstract Full Text PDF PubMed Scopus (56) Google Scholar Briefly, a midline incision was made just anterior to the nasopharyngeal opening and anterior to the thoracic outlet, and the right carotid artery was located and isolated. The vagus nerve was then blunt dissected and ligated. The incision was closed with 3-O Vicryl suture (Ethicon, Somerville, NJ) in an interrupted vertical mattress pattern. As internal controls, BDL rats were subjected to vagotomy, fed a control diet, and treated daily with BAPTA/AM or Gö6976, diluted in 0.1% dimethyl sulfoxide, as described above. We have previously shown that chronic intraperitoneal injections of dimethyl sulfoxide does not alter cholangiocyte apoptosis, proliferation, and secretion of BDL rats.15Marzioni M LeSage G Glaser S Patel T Marienfeld C Ueno Y Francis H Alvaro D Phinizy JL Tadlock L Benedetti A Marucci L Baiocchi L Alpini G Taurocholate prevents the loss of intrahepatic bile ducts due to vagotomy in bile duct ligated rats.Am J Physiol. 2003; 284: G837-G852Google Scholar, 17Alpini G Glaser SS Ueno Y Rodgers R Phinizy JL Francis H Baiocchi L Holcomb LA Caligiuri A LeSage GD 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 In a previous study we have widely characterized the control groups of rats subjected to BDL and fed either a UDCA- or TUDCA-enriched diet.20Alpini G Baiocchi L Glaser S Ueno Y Marzioni M Francis H Phinizy JL Angelico M LeSage G Ursodeoxycholate and tauroursodeoxycholate inhibit cholangiocyte growth and secretion of BDL rats through activation of PKC alpha.Hepatology. 2002; 35: 1041-1052Crossref PubMed Scopus (112) Google Scholar Therefore, we did not use these groups of animals in the study. The animals were fasted overnight before each experiment.17Alpini G Glaser SS Ueno Y Rodgers R Phinizy JL Francis H Baiocchi L Holcomb LA Caligiuri A LeSage GD 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 i.p.). Study protocols were performed in compliance with institutional guidelines. Pure cholangiocytes were obtained from the selected group of animals using a monoclonal antibody that selectively binds to an unidentified antigen expressed by intrahepatic rat cholangiocytes,17Alpini G Glaser SS Ueno Y Rodgers R Phinizy JL Francis H Baiocchi L Holcomb LA Caligiuri A LeSage GD 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, 28Ishii M Vroman B LaRusso NF Isolation and morphological characterization of bile duct epithelial cells from normal rat liver.Gastroenterology. 1989; 97: 1236-1247Abstract PubMed Google Scholar as previously described.10Alpini 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: 569-578Crossref PubMed Scopus (286) Google Scholar, 17Alpini G Glaser SS Ueno Y Rodgers R Phinizy JL Francis H Baiocchi L Holcomb LA Caligiuri A LeSage GD 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, 18Alpini G Ueno Y Glaser SS Marzioni M Phinizy JL Francis H LeSage G Bile acid feeding increased proliferative activity and apical bile acid transporter expression in both small and large rat cholangiocytes.Hepatology. 2001; 34: 868-886Crossref PubMed Scopus (103) Google Scholar, 28Ishii M Vroman B LaRusso NF Isolation and morphological characterization of bile duct epithelial cells from normal rat liver.Gastroenterology. 1989; 97: 1236-1247Abstract PubMed Google Scholar Purity of cholangiocytes was evaluated by histochemistry for γ-GT,29Rutenburg AM Kim H Fishbein JW Hanker JS Wasserkrug HL Seligman AM Histochemical and ultrastructural demonstration of g-glutamyl transpeptidase activity.J Histochem Cytochem. 1969; 17: 517-526Crossref PubMed Scopus (1023) Google Scholar a specific marker for cholangiocytes.10Alpini G Lenzi R Sarkozi L Tavoloni N Biliary physiology in rats with bi
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