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• W4235138617 abstract "Background & Aims: Hepatic production and release of endothelin 1 plays a central role in experimental hepatopulmonary syndrome after common bile duct ligation by stimulating pulmonary endothelial nitric oxide production. In thioacetamide-induced nonbiliary cirrhosis, hepatic endothelin 1 production and release do not occur, and hepatopulmonary syndrome does not develop. However, the source and regulation of hepatic endothelin 1 after common bile duct ligation are not fully characterized. We evaluated the sources of hepatic endothelin 1 production after common bile duct ligation in relation to thioacetamide cirrhosis and assessed whether transforming growth factor β1 regulates endothelin 1 production. Methods: Hepatopulmonary syndrome and hepatic and plasma endothelin 1 levels were evaluated after common bile duct ligation or thioacetamide administration. Cellular sources of endothelin 1 were assessed by immunohistochemistry and laser capture microdissection of cholangiocytes. Transforming growth factor β1 expression and signaling were assessed by using immunohistochemistry and Western blotting and by evaluating normal rat cholangiocytes. Results: Hepatic and plasma endothelin 1 levels increased and hepatopulmonary syndrome developed only after common bile duct ligation. Hepatic endothelin 1 and transforming growth factor β1 levels increased over a similar time frame, and cholangiocytes were a major source of each peptide. Transforming growth factor β1 signaling in cholangiocytes in vivo was evident by increased phosphorylation and nuclear localization of Smad2, and hepatic endothelin 1 levels correlated directly with liver transforming growth factor β1 and phosphorylated Smad2 levels. Transforming growth factor β1 also stimulated endothelin 1 promoter activity, expression, and production in normal rat cholangiocytes. Conclusions: Cholangiocytes are a major source of hepatic endothelin 1 production during the development of hepatopulmonary syndrome after common bile duct ligation, but not in thioacetamide-induced cirrhosis. Transforming growth factor β1 stimulates cholangiocyte endothelin 1 expression and production. Cholangiocyte-derived endothelin 1 may be an important endocrine mediator of experimental hepatopulmonary syndrome. Background & Aims: Hepatic production and release of endothelin 1 plays a central role in experimental hepatopulmonary syndrome after common bile duct ligation by stimulating pulmonary endothelial nitric oxide production. In thioacetamide-induced nonbiliary cirrhosis, hepatic endothelin 1 production and release do not occur, and hepatopulmonary syndrome does not develop. However, the source and regulation of hepatic endothelin 1 after common bile duct ligation are not fully characterized. We evaluated the sources of hepatic endothelin 1 production after common bile duct ligation in relation to thioacetamide cirrhosis and assessed whether transforming growth factor β1 regulates endothelin 1 production. Methods: Hepatopulmonary syndrome and hepatic and plasma endothelin 1 levels were evaluated after common bile duct ligation or thioacetamide administration. Cellular sources of endothelin 1 were assessed by immunohistochemistry and laser capture microdissection of cholangiocytes. Transforming growth factor β1 expression and signaling were assessed by using immunohistochemistry and Western blotting and by evaluating normal rat cholangiocytes. Results: Hepatic and plasma endothelin 1 levels increased and hepatopulmonary syndrome developed only after common bile duct ligation. Hepatic endothelin 1 and transforming growth factor β1 levels increased over a similar time frame, and cholangiocytes were a major source of each peptide. Transforming growth factor β1 signaling in cholangiocytes in vivo was evident by increased phosphorylation and nuclear localization of Smad2, and hepatic endothelin 1 levels correlated directly with liver transforming growth factor β1 and phosphorylated Smad2 levels. Transforming growth factor β1 also stimulated endothelin 1 promoter activity, expression, and production in normal rat cholangiocytes. Conclusions: Cholangiocytes are a major source of hepatic endothelin 1 production during the development of hepatopulmonary syndrome after common bile duct ligation, but not in thioacetamide-induced cirrhosis. Transforming growth factor β1 stimulates cholangiocyte endothelin 1 expression and production. Cholangiocyte-derived endothelin 1 may be an important endocrine mediator of experimental hepatopulmonary syndrome. The hepatopulmonary syndrome (HPS) causes impaired oxygenation due to vasodilatation in the pulmonary microcirculation in patients with cirrhosis.1Fallon M. Abrams G. Pulmonary dysfunction in chronic liver disease.Hepatology. 2000; 32: 859-865Crossref PubMed Scopus (196) Google Scholar, 2Lange P.A. Stoller J.K. The hepatopulmonary syndrome.Ann Intern Med. 1995; 122: 521-529Crossref PubMed Scopus (338) Google Scholar HPS significantly increases mortality in affected patients, and no effective medical treatments are currently available.3Schenk P. Schoniger-Hekele M. Fuhrmann V. Madl C. Silberhumer G. Muller C. Prognostic significance of the hepatopulmonary syndrome in patients with cirrhosis.Gastroenterology. 2003; 125: 1042-1052Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar The pathogenesis of HPS remains an area of active investigation. Chronic common bile duct ligation (CBDL) in the rat is the only recognized model system for the study of HPS.4Fallon M.B. Abrams G.A. Luo B. Hou Z. Dai J. Ku D.D. The role of endothelial nitric oxide synthase in the pathogenesis of a rat model of hepatopulmonary syndrome.Gastroenterology. 1997; 113: 606-614Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar In this model, biliary cirrhosis is associated with increased pulmonary endothelial nitric oxide synthase (eNOS) levels, intrapulmonary vasodilatation, and gas exchange abnormalities analogous to human HPS.4Fallon M.B. Abrams G.A. Luo B. Hou Z. Dai J. Ku D.D. The role of endothelial nitric oxide synthase in the pathogenesis of a rat model of hepatopulmonary syndrome.Gastroenterology. 1997; 113: 606-614Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 5Carter E.P. Hartsfield C.L. Miyazono M. Jakkula M. Morris Jr, K.G. McMurtry I.F. Regulation of heme oxygenase-1 by nitric oxide during hepatopulmonary syndrome.Am J Physiol Lung Cell Mol Physiol. 2002; 283: L346-L353Crossref PubMed Scopus (107) Google Scholar Increased hepatic production and plasma levels of endothelin (ET)-1 accompanied by pulmonary microvascular endothelial ETB receptor overexpression occur at the onset of HPS after CBDL and stimulate eNOS-derived nitric oxide production in the microvascular endothelium.6Luo B. Abrams G.A. Fallon M.B. Endothelin-1 in the rat bile duct ligation model of hepatopulmonary syndrome correlation with pulmonary dysfunction.J Hepatol. 1998; 29: 571-578Abstract Full Text PDF PubMed Scopus (96) Google Scholar, 7Luo B. Liu L. Tang L. Zhang J. Stockard C. Grizzle W. Fallon M. Increased pulmonary vascular endothelin B receptor expression and responsiveness to endothelin-1 in cirrhotic and portal hypertensive rats a potential mechanism in experimental hepatopulmonary syndrome.J Hepatol. 2003; 38: 556-563Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar Chronic intravenous ET-1 infusion in animals that develop portal hypertension and increased pulmonary microvascular ETB levels without increased hepatic ET-1 production after partial portal vein ligation also increases pulmonary eNOS levels and triggers HPS.8Zhang M. Luo B. Chen S.J. Abrams G.A. Fallon M.B. Endothelin-1 stimulation of endothelial nitric oxide synthase in the pathogenesis of hepatopulmonary syndrome.Am J Physiol. 1999; 277: G944-G952PubMed Google Scholar Similarly, exogenous ET-1 directly increases eNOS expression and activity in an ETB receptor-dependent manner in pulmonary artery endothelial cells.9Liu L. Zhang M. Luo B. Abrams G.A. Fallon M.B. Biliary cyst fluid from common bile duct ligated rats stimulates eNOS in pulmonary artery endothelial cells a potential role in hepatopulmonary syndrome.Hepatology. 2001; 33: 722-727Crossref PubMed Scopus (23) Google Scholar In contrast, hepatic and plasma ET-1 levels do not increase and HPS does not develop in thioacetamide (TAA)-induced nonbiliary cirrhosis despite an increase in pulmonary microvascular ETB receptor expression.10Luo B. Liu L. Tang L. Zhang J. Ling Y. Fallon M.B. ET-1 and TNF-α in HPS analysis in prehepatic portal hypertension and biliary and nonbiliary cirrhosis in rats.Am J Physiol Gastrointest Liver Physiol. 2004; 286: G294-G303Crossref PubMed Scopus (93) Google Scholar Together, these observations indicate that hepatic ET-1 production and release is a critical event that drives pulmonary vascular eNOS-derived NO production during the onset of experimental HPS. The source and regulation of hepatic ET-1 production in experimental HPS have not been clearly defined. CBDL is unique among cirrhosis models in that marked progressive cholangiocyte proliferation occurs over time and could be an important source of ET-1 production. Both human and rodent cholangiocytes have been found to contain ET-1 in cirrhosis, although detailed analysis after CBDL has not been performed.6Luo B. Abrams G.A. Fallon M.B. Endothelin-1 in the rat bile duct ligation model of hepatopulmonary syndrome correlation with pulmonary dysfunction.J Hepatol. 1998; 29: 571-578Abstract Full Text PDF PubMed Scopus (96) Google Scholar, 11Koda W. Harada K. Tsuneyama K. Kono N. Sasaki M. Matsui O. Nakanuma Y. Evidence of the participation of peribiliary mast cells in regulation of the peribiliary vascular plexus along the intrahepatic biliary tree.Lab Invest. 2000; 7: 1007-1017Crossref Scopus (30) Google Scholar, 12Pinzani M. Milani S. DeFranco R. Grappone C. Caligiuri A. Gentilini A. Tosti-Guerra C. Maggi M. Failli P. Ruocco C. Gentilini P. Endothelin 1 is overexpressed in human cirrhotic liver and exerts multiple effects on activated hepatic stellate cells.Gastroenterology. 1996; 110: 534-548Abstract Full Text PDF PubMed Scopus (346) Google Scholar Proliferating biliary epithelium has also been recognized to contribute to the development of fibrosis after CBDL by producing a number of profibrogenic cytokines.13Saperstein L. Jirtle R. Farouk M. Thompson H.J. Chung K.S. Meyers W.C. Transforming growth factor-beta 1 and mannose 6-phosphate/insulin-like growth factor-II receptor expression during intrahepatic bile duct hyperplasia and biliary fibrosis in the rat.Hepatology. 1994; 19: 412-417Crossref PubMed Scopus (62) Google Scholar Among these, transforming growth factor (TGF)-β1 seems to be important, and TGF-β1 can modulate ET-1 production in several hepatic cell types14Eakes A.T. Olson M.S. Regulation of endothelin synthesis in hepatic endothelial cells.Am J Physiol. 1998; 274: G1068-G1076PubMed Google Scholar, 15Shao R. Shi Z. Gotwals P.J. Koteliansky V.E. George J. Rockey D.C. Cell and molecular regulation of endothelin-1 production during hepatic wound healing.Mol Biol Cell. 2003; 14: 2327-2341Crossref PubMed Scopus (47) Google Scholar and in renal ductular epithelium.16Schnermann J. Zhu X. Shu X. Yang T. Huang Y. Kretzler M. Briggs J. Regulation of endothelin production and secretion in cultured collecting duct cells by endogenous transforming growth factor-β.Endocrinology. 1996; 137: 5000-5008Crossref PubMed Scopus (29) Google Scholar TGF-β1 signaling involves binding to a heteromeric complex of 2 cell-surface receptors: type I and type II. Binding triggers phosphorylation of Smad2 and Smad3, followed by complex formation with Smad4 and translocation to the nucleus.17Dijke Pt, Hill C.S. New insights into TGF-β-Smad signalling.Trends Biochem Sci. 2004; 29: 265-273Abstract Full Text Full Text PDF PubMed Scopus (1032) Google Scholar Recently, a Smad-binding element has been identified in the ET-1 promoter that is critical for TGF-β1 induction of gene expression.18Rodriguez-Pascual F. Redondo-Horcajo M. Lamas S. Functional cooperation between Smad proteins and activator protein-1 regulates transforming growth factor-β-mediated induction of endothelin-1 expression.Circ Res. 2003; 92: 1288-1295Crossref PubMed Scopus (91) Google Scholar One hypothesis based on these observations is that proliferating biliary epithelial cells may be a major source of hepatic ET-1 production and that TGF-β1 might drive ET-1 expression in these cells after CBDL. The aim of this study was to define whether cholangiocytes are an important source of hepatic ET-1 production after CBDL and to investigate whether TGF-β1 may influence ET-1 expression in these cells and contribute to the development of HPS. To address this aim, we assessed the cellular localization and expression of hepatic ET-1, TGF-β1, and phospho-Smad2 (p-Smad2) after CBDL in comparison to TAA administration, particularly in biliary epithelial cells. We also directly evaluated whether TGF-β1 modulates ET-1 expression in normal rat cholangiocytes (NRCs). Our findings show that cholangiocytes are a major source of both ET-1 and TGF-β1 production after CBDL and that there is a temporal correlation in their expression in these cells. In addition, we found evidence of TGF-β1 activation in cholangiocytes on the basis of analysis of p-Smad2 levels and localization. Finally, we showed that TGF-β1 directly activates ET-1 expression in NRCs. Male Sprague–Dawley rats (200–250 g; Charles River, Wilmington, MA) were used in all experiments. CBDL was performed as previously described.19Easter D.W. Wade J.B. Boyer J.L. Structural integrity of hepatocyte tight junctions.J Cell Biol. 1983; 96: 745-749Crossref PubMed Scopus (67) Google Scholar, 20Chojkier M. Groszmann R.J. Measurement of portal-systemic shunting in the rat by using gamma-labeled microspheres.Am J Physiol. 1981; 240: G371-G375PubMed Google Scholar Normal control animals underwent mobilization of the common bile duct without ligation. Some rats were intraperitoneally injected with TAA 200 mg/kg body weight (Sigma-Aldrich, St Louis, MO) or saline 3 times each week for 2 or 8 weeks as previously described.10Luo B. Liu L. Tang L. Zhang J. Ling Y. Fallon M.B. ET-1 and TNF-α in HPS analysis in prehepatic portal hypertension and biliary and nonbiliary cirrhosis in rats.Am J Physiol Gastrointest Liver Physiol. 2004; 286: G294-G303Crossref PubMed Scopus (93) Google Scholar Five to 8 animals from each group (control; 1-, 2-, and 3-week CBDL; and 2- and 8-week TAA administration) were used. Plasma, tissue, and physiological measurements were similar in sham CBDL and saline-treated controls, and measurements were pooled for analysis. All animals had hepatic biochemical and histological analysis and measurements of portal venous pressure and spleen weight.4Fallon M.B. Abrams G.A. Luo B. Hou Z. Dai J. Ku D.D. The role of endothelial nitric oxide synthase in the pathogenesis of a rat model of hepatopulmonary syndrome.Gastroenterology. 1997; 113: 606-614Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 6Luo B. Abrams G.A. Fallon M.B. Endothelin-1 in the rat bile duct ligation model of hepatopulmonary syndrome correlation with pulmonary dysfunction.J Hepatol. 1998; 29: 571-578Abstract Full Text PDF PubMed Scopus (96) Google Scholar, 8Zhang M. Luo B. Chen S.J. Abrams G.A. Fallon M.B. Endothelin-1 stimulation of endothelial nitric oxide synthase in the pathogenesis of hepatopulmonary syndrome.Am J Physiol. 1999; 277: G944-G952PubMed Google Scholar, 21Fallon M.B. Abrams G.A. McGrath J.W. Hou Z. Luo B. Common bile duct ligation in the rat a model of intrapulmonary vasodilatation and hepatopulmonary syndrome.Am J Physiol. 1997; 272: G779-G784PubMed Google Scholar Blood and liver tissues were obtained from each animal. The study was approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham and conformed to National Institutes of Health guidelines on the use of laboratory animals. Liver samples were fixed in 10% neutral buffered formalin solution. Paraffin-embedded tissues were sectioned at 5 μm. Sections for histology were stained with the Masson trichrome stain.22Goldner J. A modification of Masson trichrome technique for routine laboratory purposes.Am J Pathol. 1938; 14: 237-243PubMed Google Scholar After preparation and blocking, sections were incubated with ET-1 (Peninsula, San Carlos, CA), cytokeratin 19 (CK19; Novocastra, Newcastle Laboratories, UK), a marker for bile epithelial cells, α-smooth muscle actin (α-SMA; Sigma-Aldrich), TGF-β1 (Santa Cruz, Santa Cruz, CA), or p-Smad2 (Ser465/467; Cell Signaling, Beverly, MA) antibodies, washed, and incubated with EnVision-labeled polymer (Dako, Carpinteria, CA). After diaminobenzidine (Biogenex, San Ramon, CA) development, sections were photographed by using an Axiophot microscope (Nikon, Melville, NY). Control sections were incubated with secondary antibody alone. Arterial blood was drawn from the femoral artery as previously described,21Fallon M.B. Abrams G.A. McGrath J.W. Hou Z. Luo B. Common bile duct ligation in the rat a model of intrapulmonary vasodilatation and hepatopulmonary syndrome.Am J Physiol. 1997; 272: G779-G784PubMed Google Scholar and blood gas analysis was performed on an ABL 520 radiometer (Radiometer America, Westlake, OH) in the clinical laboratory of the University of Alabama at Birmingham Hospital. The alveolar-arterial oxygen gradient was calculated as 150 − (Paco2/0.8) − Pao2. The pulmonary microcirculation was evaluated with an established technique. Cross-linked (2.5 × 106) colored polystyrene-divinylbenzene microspheres (size range, 5.5–10 μm; Interactive Medical Technologies, Irvine, CA) were injected through a femoral vein catheter after an aliquot of microspheres was removed to verify the numbers and sizes injected. A blood sample withdrawn from a femoral arterial catheter beginning at the time of femoral vein injection measured microspheres passing through the lung microcirculation. Numbers and sizes of microspheres were assessed by using a Leitz Laborlux microscope (Wetzlar, Germany) with a color video imaging and digital analysis system (Image Pro 5.0; Media Cybernetics, Silver Spring, MD) and counted directly. Intrapulmonary shunting was calculated as a percentage of intrapulmonary shunt fraction, as previously described.21Fallon M.B. Abrams G.A. McGrath J.W. Hou Z. Luo B. Common bile duct ligation in the rat a model of intrapulmonary vasodilatation and hepatopulmonary syndrome.Am J Physiol. 1997; 272: G779-G784PubMed Google Scholar NRCs (a kind gift of Dr Nicholas LaRusso, Mayo Clinic Rochester, Rochester, MN) were maintained and passaged in Dulbecco’s modified Eagle medium/F-12 medium (GIBCO, Grand Island, NY) containing 0.0l mL/mL nonessential amino acids, 0.01 mL/mL insulin-transferrin-selenium-S, 0.01 mL/mL lipid concentrate, 0.01 mL/mL vitamin solution, 20 μg/mL gentamicin, 393 ng/mL dexamethasone (Sigma-Aldrich), 30 μg/mL bovine pituitary extract, 25 ng/mL epidermal growth factor, 3.4 μg/mL triiodothyrodine (Sigma-Aldrich), 5% NuSerum IV (Becton Dickinson Labware, Lincoln Park, NJ), and 4.1 μg/mL forskolin (Sigma-Aldrich) on Collagen I Cellware dishes or plates (Becton Dickinson Labware). When cells reached 70% confluence, they were incubated in Dulbecco’s modified Eagle medium without any serum or supplements and stimulated with TGF-β1 (R&D Systems, Minneapolis, MN) at various concentrations (0.5, 2.5, 5.0, and 7.5 ng/mL) for various times (2, 4, 6, 8, and 14 hours). In a set of experiments, NRCs were treated with 10 μg/mL of pan-specific TGF-β polyclonal neutralizing antibody (R&D Systems) or nonspecific rabbit immunoglobulin G (Southern Biotechnology Associates, Birmingham, AL) for 20 minutes and then stimulated by TGF-β1 2.5 ng/mL for 6 hours for Northern blotting or for 24 hours for measurement of ET-1 in cell media and homogenate. ET-1 concentrations in liver, plasma, and the culture media and cell homogenates from NRCs were measured via radioimmunoassay (Phoenix Pharmaceuticals, Mountain View, CA). Liver tissues and NRCs were homogenized and prepared as previously described.6Luo B. Abrams G.A. Fallon M.B. Endothelin-1 in the rat bile duct ligation model of hepatopulmonary syndrome correlation with pulmonary dysfunction.J Hepatol. 1998; 29: 571-578Abstract Full Text PDF PubMed Scopus (96) Google Scholar Extraction of ET-1 from plasma and the media was accomplished by acidification and elution over Sep-Pak C18 columns (Waters, Milford, MA). Samples were subjected to radioimmunoassay with the use of a rabbit ET-1 antiserum. Recovery from the Sep-Pak C18 columns averaged 90%, and the sensitivity of the assay for ET-1 was 1.5–2.0 pg. Tissues were homogenized in radioimmunoprecipitation buffer in the presence of protease inhibitors, as previously described.4Fallon M.B. Abrams G.A. Luo B. Hou Z. Dai J. Ku D.D. The role of endothelial nitric oxide synthase in the pathogenesis of a rat model of hepatopulmonary syndrome.Gastroenterology. 1997; 113: 606-614Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 6Luo B. Abrams G.A. Fallon M.B. Endothelin-1 in the rat bile duct ligation model of hepatopulmonary syndrome correlation with pulmonary dysfunction.J Hepatol. 1998; 29: 571-578Abstract Full Text PDF PubMed Scopus (96) Google Scholar, 7Luo B. Liu L. Tang L. Zhang J. Stockard C. Grizzle W. Fallon M. Increased pulmonary vascular endothelin B receptor expression and responsiveness to endothelin-1 in cirrhotic and portal hypertensive rats a potential mechanism in experimental hepatopulmonary syndrome.J Hepatol. 2003; 38: 556-563Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar Equal concentrations of protein from liver were fractionated on Tris-HCl-ready gels (Bio-Rad Laboratories, Hercules, CA) and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ). Incubation with primary antibodies for CK19, α-SMA, TGF-β1, p-Smad2, or Smad2/3 (BD Biosciences, Palo Alto, CA) was followed by the addition of horseradish peroxidase-conjugated secondary antibodies and detection with enhanced chemiluminescence. Liver tissues were immediately frozen in OCT compound (Fisher, Suwanee, GA) and stored at −80°C until use. After rapid staining with H&E, 6-μm sections from frozen sections were microdissected under a PixCell II laser capture microscope with an infrared diode laser (Arcturus Engineering, Mountain View, CA), as previously described.23Emmert-Buck M.R. Bonner R.F. Smith P.D. Chuaqui R.F. Zhuang Z. Goldstein S.R. Weiss R.A. Liotta L.A. Laser capture microdissection.Science. 1996; 274: 998-1001Crossref PubMed Scopus (2090) Google Scholar Briefly, a specific polymer film mounted on optically transparent caps (CapSure TF-100; Arcturus) was placed on the section. Bile epithelial cells or hepatocytes were captured by focal melting of membranes by the laser beam under visual control. The caps with captured epithelial cells were used as a lid for 500-μL microcentrifuge tubes containing RNA extraction buffer (Arcturus). Total RNA from captured epithelial cells was extracted by using a Pico Pure RNA isolation kit (Arcturus) according to the manufacturer’s recommendations. After deoxyribonuclease treatment (Invitrogen, Carlsbad, CA), RNA was eluted and stored at −80°C until use. All total RNA samples were first tested for quality on an Agilent Bioanalyzer 2100B by using an RNA Pico Lab Chip Kit (Agilent Technologies, Palo Alto, CA) and subsequently were amplified with the RiboAmp OA RNA Amplification Kit (Arcturus). The quality of the amplified RNA was again evaluated on an RNA Pico Lab Chip Kit (Agilent Technologies). Approximately 2 μg of total RNA from bile cholangiocytes or hepatocytes was reverse-transcribed by using the StrataScript first-strand synthesis system (Stratagene, La Jolla, CA). Complementary DNA (cDNA) was amplified with ET-1 and 18S primers and SYBR Green polymerase chain reaction (PCR) master mix (Applied Biosystems, Foster City, CA) by PCR with an iCycler real-time PCR detection system (Bio-Rad) for 40 cycles. Relative RNA levels were calculated by using the iCycler software and a standard equation (Applied Biosystems). ET-1 primers were designed as follows: sense primer, 5′-TCCTGCTCCTCCTTGATG-3′; antisense primer, 5′-TTCCCTTGGTCTGTGGTC-3′ (PCR product, approximately 164 base pairs [bp]). TGF-β1 primers were designed as follows: sense primer, 5′-CTACTGCTTCAGCTCCACAGA-3′; antisense primer, 5′-ACCTTGGGCTTGCGACC-3′ (PCR product, approximately 279 bp). 18S primers were designed as follows: sense primer, 5′-GAAACGGCTACCACATCC-3′; antisense primer, 5′-CACCAGACTTGCCCTCCA-3′ (PCR product, approximately 168 bp). ET-1 and TGF-β1 values were normalized to 18S values in each sample. Total NRC RNA was prepared by lysis in TRIzol reagent (GIBCO), electrophoresed, and transferred to Nytran membranes (Schleicher & Schuell, Keene, NH) followed by hybridization in Quikhyb (Stratagene) with a 0.5-kilobase rat ET-1 cDNA (a gift of Dr Tom Quertermous, Vanderbilt University) or a 0.5-kilobase rat glyceraldehyde-3-phosphate dehydrogenase cDNA (American Type Culture Collection, Manassas, VA) labeled with the DECAprime II kit (Ambion, Austin, TX). Imaging and quantification of signals normalized to glyceraldehyde-3-phosphate dehydrogenase were performed with phosphorimaging. Five micrograms of total RNA from NRCs was reverse-transcribed and amplified by using the ProSTAR first-strand reverse-transcription PCR (RT-PCR) kit (Stratagene). Sequences for the primers were designed on the basis of the published sequences for ET-124Tchekneva E. Lawrence M. Meyrick B. Cell-specific differences in ET-1 system in adjacent layers of main pulmonary artery. A new source of ET-1.Am J Physiol Lung Cell Mol Physiol. 2000; 278: L813-L821PubMed Google Scholar and TGF-β type I and II receptors16Schnermann J. Zhu X. Shu X. Yang T. Huang Y. Kretzler M. Briggs J. Regulation of endothelin production and secretion in cultured collecting duct cells by endogenous transforming growth factor-β.Endocrinology. 1996; 137: 5000-5008Crossref PubMed Scopus (29) Google Scholar as follows: ET-1 (PCR product, approximately 489 bp)—sense primer, 5′-TTGTGGCTTTCCAAGGAGCTCCAG-3′; antisense primer, 5′-TTGCTGATGGCCTCCAACCTTC-3′; TGF-β I receptor (PCR product, approximately 450 bp)—sense primer, 5′-GCACCATCTTCAAAAACAGG-3′; antisense primer, 5′-TCTTCACAGCAACTTCTTCT-3′; TGF-β II receptor (PCR product, approximately 385 bp)—sense primer, 5′-TCGGAATACACCACCAG-3′; antisense primer, 5′-AAGATCTTGACAGCCACGGT-3′; and α-actin (PCR product, approximately 400 bp)—sense primer, 5′-GACATGACAGACTACCTCAT-3′; antisense primer, 5′-AGACAGCACTGTGTTGGCAT-3′. PCR amplification reactions were performed in a PerkinElmer 2400 PCR machine (Foster City, CA), and RT-PCR products were electrophoresed on an agarose gel to show the amplified bands. Initial PCR amplification products from each set of primers were sequenced using an ABI PRISM Model 377 automated sequencer (Applied Biosystems, Foster City, CA) in a sequencing facility (University of Alabama at Birmingham, Birmingham, AL) to ensure accuracy. A plasmid (HPPET-LUC) containing −2459 to +165 bp of the human ET-1 promoter was provided by Dr Catherine Aversa (Bristol-Myers Squibb Pharmaceutical, Princeton, NJ).25Inoue A. Yanagisawa M. Takuwa Y. Mitsui Y. Kobayashi M. Masaki T. The human preproendothelin-1 gene.J Biol Chem. 1989; 264: 14954-14959Abstract Full Text PDF PubMed Google Scholar Plasmid pET-649 was constructed by insertion of the SacI-BglII restriction fragment from HPPET-LUC (−649 to +165 bp) into the luciferase reporter vector pGL2-Basic (Promega, Madison, WI). The insert for plasmid pET-184 (−184 to +165 bp) was generated from HPPET-LUC by PCR by using the sense primer 5′-GCGGGCGTCTGCTTCTGAAGTT-3′ and the antisense primer 5′-ATCTCAAAGCGATCCTTCAGCC-3′. The insert was cloned into pCR2.1 (Invitrogen) and sequenced to verify promoter orientation and sequence accuracy. Subsequently, the SacI-XhoI fragment was subcloned into pGL2-Basic. Plasmid pET-949 contains the full putative Smad-binding element, whereas the pET-184 does not.18Rodriguez-Pascual F. Redondo-Horcajo M. Lamas S. Functional cooperation between Smad proteins and activator protein-1 regulates transforming growth factor-β-mediated induction of endothelin-1 expression.Circ Res. 2003; 92: 1288-1295Crossref PubMed Scopus (91) Google Scholar The plasmid pRL-tk (Promega) was cotransfected in each experiment as a control for transfection efficiency. Transient cotransfection of either pET-649 and pRL-tk or pET-184 and pRL-tk into cultured NRCs was performed by using the TransFast Transfection Reagent (Promega) according to the manufacturer’s protocol. Briefly, the transfection mixture containing DNA (target plasmid/control plasmid, 40:1) and the transfection reagent was added into cells. After incubation, transfected cells were then subjected to various treatments. Luciferase activity was measured in the cellular extracts by using a Dual-Luciferase Reporter Assay Kit (Promega). After treatment, the cell lysates were collected after the addition of cell culture lysis reagent (Promega). The relative light units were then determined in a luminometer (MEM Instrument Inc., Hamden, CT) for a total of 10 seconds after a 5-second delay. The density of autoradiographic signals was assessed with an Astra 1200s scanner (UMAX, Fremont, CA) and quantitated with Scion ImagePC software (Scion, Frederick, MD). Data were analyzed with the Student t test or analysis of variance with Bonferroni correction for multiple comparisons between groups. Simple regression analysis was used to evaluate correlations between liver and/or plasma ET-1 content and liver CK19, TGF-β1, and p-Smad2 levels. Measurements are expressed as means ± SE. Statistical signifi" @default.
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• W4235138617 date "2005-08-01" @default.
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• W4235138617 title "Cholangiocyte Endothelin 1 and Transforming Growth Factor β1 Production in Rat Experimental Hepatopulmonary Syndrome" @default.
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