Nerve growth factor modulates the proliferative capacity of the intrahepatic biliary epithelium in experimental cholestasis
2004; Elsevier BV; Volume: 127; Issue: 4 Linguagem: Inglês
10.1053/j.gastro.2004.06.023
ISSN1528-0012
AutoresAlessandro Gigliozzi, Gianfranco Alpini, Gianluca Svegliati‐Baroni, Luca Marucci, Veronica Drudi Metalli, Shannon Glaser, Heather Francis, Maria Grazia Mancino, Yoshiyuki Ueno, Barbara Barbaro, A. Benedetti, A.F. Attili, Domenico Alvaro,
Tópico(s)Neuropeptides and Animal Physiology
ResumoBackground & Aims: We evaluated the expression of neurotrophins in rat cholangiocytes and the role and mechanisms by which nerve growth factor (NGF) modulates cholangiocyte proliferation. Methods: The expression of neurotrophins and their receptors was investigated by immunohistochemistry in liver sections and reverse-transcription polymerase chain reaction and immunoblots in isolated cholangiocytes. In vitro, the effect of NGF on cholangiocyte proliferation and signal transduction was investigated by immunoblotting for proliferating cell nuclear antigen, phosphorylated AKT (p-AKT), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), phosphorylated c-jun-N-terminal kinase, and phosphorylated p38. In vivo, rats that had undergone bile duct ligation (BDL) were treated with an anti-NGF antibody to immunoneutralize NGF and bile duct mass, proliferation, apoptosis, and inflammation were investigated by immunohistochemistry. Results: NGF and its TrkA receptor were expressed by normal rat cholangiocytes and up-regulated following BDL. Cholangiocytes secrete NGF, and secretion is increased in proliferating BDL cholangiocytes. In vitro, NGF stimulated cholangiocyte proliferation, which was associated with enhanced p-AKT and p-ERK1/2 expression. NGF proliferation in vitro was partially blocked by the MEK inhibitor (UO126) and completely ablated by the phosphatidylinositol 3-kinase inhibitor (wortmannin). In vitro, NGF and estrogens have an additive effect on cholangiocyte proliferation by acting on phosphorylated TrkA and p-ERK1/2. In vivo, immunoneutralization of NGF decreased bile duct mass in BDL rats, which was associated with depressed proliferation and enhanced apoptosis and with increased portal inflammation. Conclusions: Cholangiocytes secrete NGF and express NGF receptors. NGF induces cholangiocyte proliferation by activating the ERK and, predominantly, the phosphatidylinositol 3-kinase pathway and exerts an additive effect in combination with estrogens on proliferation. Background & Aims: We evaluated the expression of neurotrophins in rat cholangiocytes and the role and mechanisms by which nerve growth factor (NGF) modulates cholangiocyte proliferation. Methods: The expression of neurotrophins and their receptors was investigated by immunohistochemistry in liver sections and reverse-transcription polymerase chain reaction and immunoblots in isolated cholangiocytes. In vitro, the effect of NGF on cholangiocyte proliferation and signal transduction was investigated by immunoblotting for proliferating cell nuclear antigen, phosphorylated AKT (p-AKT), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), phosphorylated c-jun-N-terminal kinase, and phosphorylated p38. In vivo, rats that had undergone bile duct ligation (BDL) were treated with an anti-NGF antibody to immunoneutralize NGF and bile duct mass, proliferation, apoptosis, and inflammation were investigated by immunohistochemistry. Results: NGF and its TrkA receptor were expressed by normal rat cholangiocytes and up-regulated following BDL. Cholangiocytes secrete NGF, and secretion is increased in proliferating BDL cholangiocytes. In vitro, NGF stimulated cholangiocyte proliferation, which was associated with enhanced p-AKT and p-ERK1/2 expression. NGF proliferation in vitro was partially blocked by the MEK inhibitor (UO126) and completely ablated by the phosphatidylinositol 3-kinase inhibitor (wortmannin). In vitro, NGF and estrogens have an additive effect on cholangiocyte proliferation by acting on phosphorylated TrkA and p-ERK1/2. In vivo, immunoneutralization of NGF decreased bile duct mass in BDL rats, which was associated with depressed proliferation and enhanced apoptosis and with increased portal inflammation. Conclusions: Cholangiocytes secrete NGF and express NGF receptors. NGF induces cholangiocyte proliferation by activating the ERK and, predominantly, the phosphatidylinositol 3-kinase pathway and exerts an additive effect in combination with estrogens on proliferation. Cholangiocytes are the epithelial cells lining intrahepatic bile ducts that are characterized by marked proliferative capacities, which are evidenced under experimental conditions as well as during human cholangiopathies.1Alpini G. McGill J.M. LaRusso N.F. The pathobiology of biliary epithelia.Hepatology. 2002; 35: 1256-1268Crossref PubMed Scopus (124) Google Scholar, 2Alvaro D. Gigliozzi A. Attili A.F. Regulation and deregulation of cholangiocyte proliferation.J Hepatol. 2000; 33: 333-340Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 3Alvaro D. Biliary epithelium a new chapter in cell biology.Ital J Gastroenterol Hepatol. 1999; 31: 78-83PubMed Google Scholar At the experimental level, dietary manipulations, partial hepatectomy, or bile duct ligation (BDL) lead to marked proliferation of intrahepatic cholangiocytes.1Alpini G. McGill J.M. LaRusso N.F. The pathobiology of biliary epithelia.Hepatology. 2002; 35: 1256-1268Crossref PubMed Scopus (124) Google Scholar, 2Alvaro D. Gigliozzi A. Attili A.F. Regulation and deregulation of cholangiocyte proliferation.J Hepatol. 2000; 33: 333-340Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 3Alvaro D. Biliary epithelium a new chapter in cell biology.Ital J Gastroenterol Hepatol. 1999; 31: 78-83PubMed Google Scholar The BDL rat is the most widely used experimental model, in which a typical and selective cholangiocyte proliferation leads to a marked increase in intrahepatic bile duct mass.1Alpini G. McGill J.M. LaRusso N.F. The pathobiology of biliary epithelia.Hepatology. 2002; 35: 1256-1268Crossref PubMed Scopus (124) Google Scholar, 2Alvaro D. Gigliozzi A. Attili A.F. Regulation and deregulation of cholangiocyte proliferation.J Hepatol. 2000; 33: 333-340Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 3Alvaro D. Biliary epithelium a new chapter in cell biology.Ital J Gastroenterol Hepatol. 1999; 31: 78-83PubMed Google Scholar In the human pathology, cholangiocyte proliferation is a typical hallmark of all cholangiopathies, which condition, as a repair and compensatory mechanism, the evolution of the disease toward the terminal ductopenic stage.1Alpini G. McGill J.M. LaRusso N.F. The pathobiology of biliary epithelia.Hepatology. 2002; 35: 1256-1268Crossref PubMed Scopus (124) Google Scholar, 2Alvaro D. Gigliozzi A. Attili A.F. Regulation and deregulation of cholangiocyte proliferation.J Hepatol. 2000; 33: 333-340Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 3Alvaro D. Biliary epithelium a new chapter in cell biology.Ital J Gastroenterol Hepatol. 1999; 31: 78-83PubMed Google Scholar During proliferation, cholangiocytes display enhanced secretory activities and responsiveness to the secretory stimulus of various hormones and peptides, including secretin and acetylcholine.1Alpini G. McGill J.M. LaRusso N.F. The pathobiology of biliary epithelia.Hepatology. 2002; 35: 1256-1268Crossref PubMed Scopus (124) Google Scholar, 2Alvaro D. Gigliozzi A. Attili A.F. Regulation and deregulation of cholangiocyte proliferation.J Hepatol. 2000; 33: 333-340Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 3Alvaro D. Biliary epithelium a new chapter in cell biology.Ital J Gastroenterol Hepatol. 1999; 31: 78-83PubMed Google Scholar, 4Alvaro D. Alpini G. Jezequel A.M. Bassotti C. Francia C. Fraioli F. Romeo R. Marucci L. Le Sage G. Glaser S.S. 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, 5Le Sage G. Alvaro D. Benedetti A. Glaser S. Marucci L. Baiocchi L. Eisel W. Caligiuri A. Phinizy J.L. Rodgers R. 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 Furthermore, proliferating cholangiocytes acquire phenotypical features of neuroendocrine epithelium, including (1) expression of neuroendocrine markers (chromogranin A, glycolipid A2-B4, S-100 protein, neural cell adhesion molecule) and acquisition of neuroendocrine granules6Desmet V. Roskams T. Van Eyken P. Ductular reaction in the liver.Pathol Res Pract. 1995; 191: 513-524Crossref PubMed Scopus (168) Google Scholar, 7Roskams T. Van den Oord J.J. De Vos R. Desmet V.J. Neuro-endocrine features or reactive bile ductules in cholestatic liver disease.Am J Pathol. 1990; 137: 1019-1025PubMed Google Scholar; (2) expression of the parathyroid hormone-related peptide, which is encoded by a growth factor-regulated early-response gene and is involved in the growth and differentiation of the cell8Roskams T. Campos R.V. Drucker D.J. Desmet V. Reactive human bile ductules express parathyroid hormone-related peptide.Histopathology. 1993; 23: 11-19Crossref PubMed Scopus (48) Google Scholar; (3) increased expression and response to endothelin9Caligiuri A. Glaser S. Rodgers R.E. Phinizy J.L. Robertson W. Papa E. Pinzani M. Alpini G. Endothelin-1 inhibits secretin-stimulated ductal secretion by interacting with ETA receptors on large cholangiocytes.Am J Physiol. 1998; 275: G835-G846PubMed Google Scholar; and (4) enhanced response to hormones/neuropeptides such as secretin, somatostatin, and acetylcholine.1Alpini G. McGill J.M. LaRusso N.F. The pathobiology of biliary epithelia.Hepatology. 2002; 35: 1256-1268Crossref PubMed Scopus (124) Google Scholar, 2Alvaro D. Gigliozzi A. Attili A.F. Regulation and deregulation of cholangiocyte proliferation.J Hepatol. 2000; 33: 333-340Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 3Alvaro D. Biliary epithelium a new chapter in cell biology.Ital J Gastroenterol Hepatol. 1999; 31: 78-83PubMed Google Scholar, 4Alvaro D. Alpini G. Jezequel A.M. Bassotti C. Francia C. Fraioli F. Romeo R. Marucci L. Le Sage G. Glaser S.S. 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, 5Le Sage G. Alvaro D. Benedetti A. Glaser S. Marucci L. Baiocchi L. Eisel W. Caligiuri A. Phinizy J.L. Rodgers R. 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, 10Tietz P. Alpini G. Pham L.D. LaRusso N.F. Somatostatin inhibits secretin-induced ductal choleresis in vivo and exocytosis by cholangiocytes.Am J Physiol. 1995; 269: G110-G118PubMed Google Scholar Nerve growth factor (NGF) is a member of the neurotrophin family, which regulates growth and differentiation of target tissues by acting on specific tyrosine kinase receptors of the Trk family (NTr).11Chao M.V. Neurotrophins and their receptors a convergence point for many signalling pathways.Nat Rev Neurosci. 2003; 4: 299-309Crossref PubMed Scopus (1671) Google Scholar, 12Skaper S.D. Nerve growth factor a neurokine orchestrating neuroimmune-endocrine functions.Mol Neurobiol. 2001; 24: 183-199Crossref PubMed Scopus (52) Google Scholar Different NTr have been identified, including TrkA, TrkB, TrkC, and the low-affinity p75 receptor.11Chao M.V. Neurotrophins and their receptors a convergence point for many signalling pathways.Nat Rev Neurosci. 2003; 4: 299-309Crossref PubMed Scopus (1671) Google Scholar, 12Skaper S.D. Nerve growth factor a neurokine orchestrating neuroimmune-endocrine functions.Mol Neurobiol. 2001; 24: 183-199Crossref PubMed Scopus (52) Google Scholar NGF and the related receptors have recently been identified in different non-nervous tissues where this neurotrophin plays a role in the regulation of proliferation, differentiation, remodeling, and inflammation.11Chao M.V. Neurotrophins and their receptors a convergence point for many signalling pathways.Nat Rev Neurosci. 2003; 4: 299-309Crossref PubMed Scopus (1671) Google Scholar, 12Skaper S.D. Nerve growth factor a neurokine orchestrating neuroimmune-endocrine functions.Mol Neurobiol. 2001; 24: 183-199Crossref PubMed Scopus (52) Google Scholar However, no information exists regarding the role of neurotrophins in the modulation of cholangiocyte pathophysiology. The aims of this study were to evaluate (1) the expression of NGF, other neurotrophins, and the related receptors in cholangiocytes of normal and BDL rats and (2) the role and mechanisms of NGF in the regulation of cholangiocyte proliferation. Male 344 Fischer rats (225–250 g) were purchased from Charles River Italia (Calco, Italy) and fed ad libitum in a light- and temperature-controlled environment. The study protocols were in compliance with the institution’s guidelines. Reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated. BDL was performed as previously described.5Le Sage G. Alvaro D. Benedetti A. Glaser S. Marucci L. Baiocchi L. Eisel W. Caligiuri A. Phinizy J.L. Rodgers R. 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 Normal and 1-week BDL rats were used for immunohistochemistry in liver sections as well as for isolation of cholangiocytes. Cholangiocytes were isolated from normal or 1-week BDL rats by immunomagnetic separation4Alvaro D. Alpini G. Jezequel A.M. Bassotti C. Francia C. Fraioli F. Romeo R. Marucci L. Le Sage G. Glaser S.S. 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, 5Le Sage G. Alvaro D. Benedetti A. Glaser S. Marucci L. Baiocchi L. Eisel W. Caligiuri A. Phinizy J.L. Rodgers R. 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, 9Caligiuri A. Glaser S. Rodgers R.E. Phinizy J.L. Robertson W. Papa E. Pinzani M. Alpini G. Endothelin-1 inhibits secretin-stimulated ductal secretion by interacting with ETA receptors on large cholangiocytes.Am J Physiol. 1998; 275: G835-G846PubMed Google Scholar with a viability >90% (trypan blue exclusion). The purity of cholangiocyte preparations was assessed by (1) γ-glutamyltransferase-positive staining,13Rutemburg A.M. Kim H. Fishbein J.W. Hanker J.S. Wasserkrug H.L. Seligman A.M. Histochemical and ultrastructural demonstration of gamma-glutamyl transpeptidase activity.J Histochem Cytochem. 1969; 17: 517-526Crossref PubMed Scopus (1023) Google Scholar (2) glucose-6-phosphatase staining14Teutsch H.F. Improved method for the histochemical demonstration of glucose-6-phosphatase activity. A methodological study.Histochemistry. 1978; 57: 107-117Crossref PubMed Scopus (63) Google Scholar and reverse-transcription polymerase chain reaction (RT-PCR) for albumin (hepatocyte markers), (3) RT-PCR for fucose receptor (Kupffer cell marker), and (4) RT-PCR for von Willebrand factor (endothelial cell marker). All isolated cells were γ-glutamyltransferase positive, whereas glucose-6-phosphatase-positive cells were absent and RT-PCR for von Willebrand factor, fucose receptor, and albumin was negative in cell preparations from both normal and BDL livers, indicating absolute purity of cholangiocyte preparations. NGF was measured in the rat serum and in supernatant of isolated cholangiocytes from normal and 1-week BDL rats, suspended in 1× hepes buffer saline (HBS), by using a Sandwich ELISA Kit (Chemicon International, Inc. Temecula, CA) according to the manufacturer’s instructions. In the supernatant of isolated cholangiocytes from normal and 1-week BDL rats, we also measured K+ concentration as a marker of cell permeability by using QuikLYTE Integrated Multisensor Technology (Dade Behring, Inc., Deerfield, IL). To evaluate whether NGF secreted by proliferating cholangiocytes may induce cell proliferation, the supernatant of normal and BDL cholangiocytes (after 4 hours of incubation) was transferred into plates containing normal quiescent, freshly isolated cholangiocytes. After 4 hours of incubation in the presence or absence of an immunoneutralizing anti-NGF antibody (150 pg/mL of supernatant), the protein expression of proliferating cell nuclear antigen (PCNA) (Western blot) in cholangiocytes was measured as a marker of cell proliferation. Formalin-fixed, paraffin-embedded liver sections were stained with H&E for routine examination. To determine the degree of portal inflammation, inflammatory grading was blindly performed by an independent pathologist as previously described.15Ishak K. Baptista A. Bianchi L. Callea F. De Groote J. Gudat F. Denk H. Desmet V. Korb G. MacSween R.N. Histological grading and staging of chronic hepatitis.J Hepatol. 1995; 22: 696-699Abstract Full Text PDF PubMed Scopus (4040) Google Scholar, 16Benedetti A. Di Sario A. Casini A. Ridolfi F. Bendia E. Pigini P. Tonnini C. D’Ambrosio L. Feliciangeli G. Macarri G. Svegliati-Baroni G. Inhibition of the Na(+)/H(+) exchanger reduces rat hepatic stellate cell activity and liver fibrosis an in vitro and in vivo study.Gastroenterology. 2001; 120: 545-556Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar For NGF and TrkA immunodetection, formalin-fixed, paraffin-embedded liver sections were obtained from normal or BDL rats. 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 anti-NGF or anti-TrkA (1:20 dilution in phosphate-buffered saline; Santa Cruz Biotechnology, Santa Cruz, CA) for 2 hours at room temperature, and the reactive sites were detected using a Dako LSAB kit (according to the manufacturer’s instructions) followed by incubation for 5 minutes with phosphate-buffered saline containing 0.06% diaminobenzidine and 0.01% H2O2 added just before use. To control the specificity of anti-NGF and anti-TrkA antibodies, the primary antibody was combined with a 5-fold excess of blocking peptide (Santa Cruz Biotechnology) and incubated overnight at 4°C (as recommended by the manufacturer). Total cellular RNA was extracted from cholangiocytes immunoisolated from normal or 1-week BDL rats by using the Micro-Fast Track II Kit (Invitrogen, San Diego, CA) according to the manufacturer’s instructions. Total RNA (1 μg) was used for first-strand complementary DNA synthesis by AMV reverse transcriptase (Roche Diagnostics, Mannheim, Germany). Degenerate oligonucleotide primers were synthesized based on the published sequence for the rat neurotrophins and related receptors as follows. NGF: 5′-CCAAGGACGCAGCTTTCTAT-3′ (forward), 5′-CTCCGGTGAGTCCTGTTGAA-3′ (reverse); brain-derived neurotrophic factor: 5′-ATTTGTCCGAGGTGGTAGTACTTCATC-3′ (forward), 5′-AGGAGGCTCCAAAGGCAC-TTGACT-3′ (reverse); neurotrophin 3: 5′-TTACAGGTGAACAAGGTGAT-3′ (forward), 5′-ACGAGTTTGTTGTT-TTCTGA-3′ (reverse); neurotrophin 5: 5′-CCCTGCGT-CAGTACTTCTTCGAGAC-3′ (forward), 5′-CTGGACGTCAGGCACGGCCTGTTC-3′ (reverse); TrkA: 5′-CTGAGGTCTCTGTCCAAGTC-3′ (forward), 5′-CCCAAAAG-GTGTTTCGTCCT-3′ (reverse); TrkB: 5′-GGCCAAGAATGA-ATATGGGAA-3′ (forward), 5′-TTGAGCTGG-CTGTTGGTGAT-3′ (reverse); TrkC: 5′-AGCTGCTCACTAACCTGCAGCATG-3′ (forward), 5′-GCTAAAGATCTCCCAAAGAA-TAAC-3′ (reverse); low-affinity NGF receptor: 5′-TGCTGCTGCTGC-TGATTCTA3′ (forward), 5′-GACCTTGGGATCCATCGAC-3′ (reverse); neurophilin 1: 5′-CGCCTGGTGAGCCCTGTGGTCTATT-3′ (forward), 5′-TGTTCTTGTCGCC-TTTCCCTTCTTC-3′ (reverse); albumin (hepatocytes): 5′TTGCCTTTTCCCAGTATCTCCA-3′ (forward), 5′ACACTCGTTTCTTTCGGGCT3′ (reverse); von Willebrand factor (endothelial cells): 5′GAGCGGTGCTCC-TTTGAGGA-3′ (forward), 5′-CACGTAGTCCTCGTGACAGC-3′ (reverse); fucose receptor (Kupffer cells): 5′GGAGGATGAAGGAGGCGGAACTG-3′ (forward), 5′GCCCCCAAGCAACTGCACC-3′ (reverse). Albumin, von Willebrand factor, and fucose receptor were used as markers of purity, whereas glyceraldehyde-3-phosphate dehydrogenase was used as housekeeping gene. Rat brain RNA was used as positive control in all cases with the exception of albumin, von Willebrand factor, and fucose receptor, where total rat liver was used as positive control. Standard RT-PCR conditions were used (35 step cycles: 60 seconds at 94°C, 45 seconds at 55°C, and 60 seconds at 72°C). PCR products were subcloned and sequenced. For Western blot analysis, cholangiocytes were solubilized in lysis buffer containing 0.125 mol/L Tris HCl (pH 6.8), 10% sodium dodecyl sulfate, 2 mmol/L phenylmethylsulfonyl fluoride, 2 mmol/L benzamidine, and 1% aprotinin at 4°C for 1 hour. After centrifugation at 10,000g for 30 minutes at 4°C, the supernatant was recovered and protein concentration was determined with the Lowry method.17Lowry O.H. Rosebrough N.J. Farr A.L. Protein measurement with the folin phenol reagent.J Biol Chem. 1958; 193: 265-275Abstract Full Text PDF Google Scholar Cell extracts (100 μg) were diluted in 2× Laemmli sample buffer containing 0.3 mol/L 2-mercaptoethanol and resolved by 7.5% or 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Western blotting was performed as described4Alvaro D. Alpini G. Jezequel A.M. Bassotti C. Francia C. Fraioli F. Romeo R. Marucci L. Le Sage G. Glaser S.S. 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, 5Le Sage G. Alvaro D. Benedetti A. Glaser S. Marucci L. Baiocchi L. Eisel W. Caligiuri A. Phinizy J.L. Rodgers R. 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, 9Caligiuri A. Glaser S. Rodgers R.E. Phinizy J.L. Robertson W. Papa E. Pinzani M. Alpini G. Endothelin-1 inhibits secretin-stimulated ductal secretion by interacting with ETA receptors on large cholangiocytes.Am J Physiol. 1998; 275: G835-G846PubMed Google Scholar by using the following antibodies (obtained from Santa Cruz Biotechnology): (1) anti-PCNA mouse monoclonal antibodies (1:400 dilution), (2) anti-NGF rabbit polyclonal antibody (1:100 dilution), (3) anti-total TrkA (t-TrkA) rabbit polyclonal antibody (1:100 dilution), (4) anti-phosphorylated TrkA (p-TrkA) mouse monoclonal antibody (1:200 dilution), (5) anti-total extracellular signal-regulated kinase (ERK) 1/2 rabbit polyclonal antibody (1:1000 dilution), (6) anti-p-ERK1/2 (tyrosine-threonine diphosphorylated ERK 1/2 mouse monoclonal antibody, 1:1000 dilution), (7) anti-total AKT mouse monoclonal antibody (1:100 dilution), (8) anti-phosphorylated AKT (p-AKT) rabbit polyclonal antibody (1:250 dilution), (9) anti-total c-jun-N-terminal kinase (JNK) mouse monoclonal antibody (1:100 dilution), (10) anti-phosphorylated JNK mouse monoclonal antibody (dilution 1:100), (11) anti-total p38 mouse monoclonal antibody (dilution 1:200), and (12) anti-phosphorylated p38 mouse monoclonal antibody (1:200 dilution). For β-actin, we used a mouse monoclonal antibody obtained from Sigma Chemical Co. (1:10,000 dilution). As secondary antibodies, anti-mouse immunoglobulin G peroxidase conjugated (1:2000; Sigma Chemical Co.) or anti-rabbit immunoglobulin G peroxidase conjugated (1:10,000; Sigma Chemical Co.) were used. The intensity of the bands was determined by scanning video densitometry (Ultra Violet Products, Cambridge, England). Western blot analysis for t-TrkA and p-TrkA was also performed in cholangiocyte apical and basolateral plasma membranes prepared by isopyknic centrifugation on a 3-step sucrose gradient (38%, 34%, and 31% wt/wt) according to the technique previously described by Tietz et al.18Tietz P. Levine S. Holman R. Fretham C. LaRusso N.F. Characterization of apical and basolateral plasma membrane domains derived from cultured rat cholangiocytes.Anal Biochem. 1997; 254: 192-199Crossref PubMed Scopus (23) Google Scholar The purity of the membrane preparations was tested by measuring the activity of specific markers for the basolateral (i.e., Na+, K+-adenosine triphosphatase) and apical (i.e., alkaline phosphatase) domain of cholangiocyte membranes as described.18Tietz P. Levine S. Holman R. Fretham C. LaRusso N.F. Characterization of apical and basolateral plasma membrane domains derived from cultured rat cholangiocytes.Anal Biochem. 1997; 254: 192-199Crossref PubMed Scopus (23) Google Scholar Immediately after BDL, 200 μL of polyclonal neutralizing NGF antibody (400 μg/dose)19Gloster A. Diamond J. Sympathetic nerves in adult rats regenerate normally and restore pilomotor function during an anti-NGF treatment that prevents their collateral sprouting.J Comp Neurol. 1992; 326: 363-374Crossref PubMed Scopus (78) Google Scholar, 20Reinshagen M. Rohm H. Steinkamp M. Lieb K. Geerling I. von Herbay A. Flämig G. Eysselein V.E. Adler G. Protective role of neurotrophins in experimental inflammation of the rat gut.Gastroenterology. 2000; 119: 368-376Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar (Sigma Chemical Co.) was administered intraperitoneally every day for 7 days (n = 8). BDL controls (n = 8) received nonimmune serum with the same modalities. In BDL rats treated with anti-NGF antibody or nonimmune serum, we determined (1) bile duct mass after immunohistochemical staining for γ-glutamyltransferase as previously described,4Alvaro D. Alpini G. Jezequel A.M. Bassotti C. Francia C. Fraioli F. Romeo R. Marucci L. Le Sage G. Glaser S.S. 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, 5Le Sage G. Alvaro D. Benedetti A. Glaser S. Marucci L. Baiocchi L. Eisel W. Caligiuri A. Phinizy J.L. Rodgers R. 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, 9Caligiuri A. Glaser S. Rodgers R.E. Phinizy J.L. Robertson W. Papa E. Pinzani M. Alpini G. Endothelin-1 inhibits secretin-stimulated ductal secretion by interacting with ETA receptors on large cholangiocytes.Am J Physiol. 1998; 275: G835-G846PubMed Google Scholar (2) apoptosis in situ in the liver by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) analysis as described,4Alvaro D. Alpini G. Jezequel A.M. Bassotti C. Francia C. Fraioli F. Romeo R. Marucci L. Le Sage G. Glaser S.S. 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, 5Le Sage G. Alvaro D. Benedetti A. Glaser S. Marucci L. Baiocchi L. Eisel W. Caligiuri A. Phinizy J.L. Rodgers R. 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, 9Caligiuri A. Glaser S. Rodgers R.E. Phinizy J.L. Robertson W. Papa E. Pinzani M. Alpini G. Endothelin-1 inhibits secretin-stimulated ductal secretion by interacting with ETA receptors on large cholangiocytes.Am J Physiol. 1998; 275: G835-G846PubMed Google Scholar and (3) PCNA protein expression in isolated cholangiocytes by immunoblotting. In addition, inflammation score was determined according to Ishak et al.15Ishak K. Baptista A. Bianchi L. Callea F. De Groote J. Gudat F. Denk H. Desmet V. Korb G. MacSween R.N. Histological grading and staging of chronic hepatitis.J Hepatol. 1995; 22: 696-699Abstract Full Text PDF PubMed Scopus (4040) Google Scholar Data are presented as arithmetic mean ± SEM. Statistical analysis was conducted using the paired or unpaired Student t test as appropriate or analysis of variance when multiple comparisons were performed. The expression of neurotrophins and neurotrophin receptors was investigated by RT-PCR in pure preparations of cholangiocytes isolated from normal (n = 3 cell preparations) or 1-week BDL rats (n = 3) by using gene-specific primers (Figure 1). NGF and TrkA (the preferred NGF receptor) were expressed in cholangiocytes isolated from both normal and BDL rat livers. Cholangiocytes isolated from normal rats also express the neurotrophin 4/5, whereas its preferred receptor TrkB was not detectable. The neurotrophin receptor TrkC and the low-affinity NGF receptor p75 were also expressed in normal and BDL rat cholangiocytes. In contrast, neurotrophin 3 and the brain-derived neurotrophic factor were absent in both normal and BDL rat cholangiocytes. Because neurophilin 1, the receptor for semaphorins, is involved in the modulation of NGF effects in different cells and tissues,11Chao M.V. Neurotrophins and their receptors a convergence point for many signalling pathways.Nat Rev Neurosci. 2003; 4: 299-309Crossref PubMed Scopus (1671) Google Scholar, 12Skaper S.D. Nerve growth factor a neurokine orchestrating neuroimmune-endocrine functions.Mol Neurobiol. 2001; 24: 183-199Crossref PubMed Scopus (52) Google Scholar its expression in rat cholangiocytes was also evaluated; this showed that neurophilin-1 messenger RNA was expressed by both normal and BDL rat cholangiocytes. RT-PCR for albumin (hepatocyte marker), von Willebrand factor (endothelial cell marker), and fucose receptor (Kupffer cell marker) was negative in both normal and BDL cholangiocytes (Fi
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