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1
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Reshetnyak VI, Maev IV. New insights into the pathogenesis of primary biliary cholangitis asymptomatic stage. World J Gastroenterol 2023; 29:5292-5304. [PMID: 37899787 PMCID: PMC10600802 DOI: 10.3748/wjg.v29.i37.5292] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/19/2023] [Revised: 08/10/2023] [Accepted: 09/14/2023] [Indexed: 09/25/2023] Open
Abstract
Primary biliary cholangitis (PBC) is a chronic cholestatic progressive liver disease and one of the most important progressive cholangiopathies in adults. Damage to cholangiocytes triggers the development of intrahepatic cholestasis, which progresses to cirrhosis in the terminal stage of the disease. Accumulating data indicate that damage to biliary epithelial cells [(BECs), cholangiocytes] is most likely associated with the intracellular accumulation of bile acids, which have potent detergent properties and damaging effects on cell membranes. The mechanisms underlying uncontrolled bile acid intake into BECs in PBC are associated with pH change in the bile duct lumen, which is controlled by the bicarbonate (HCO3-) buffer system "biliary HCO3- umbrella". The impaired production and entry of HCO3- from BECs into the bile duct lumen is due to epigenetic changes in expression of the X-linked microRNA 506. Based on the growing body of knowledge on the molecular mechanisms of cholangiocyte damage in patients with PBC, we propose a hypothesis explaining the pathogenesis of the first morphologic (ductulopenia), immunologic (antimitochondrial autoantibodies) and clinical (weakness, malaise, rapid fatigue) signs of the disease in the asymptomatic stage. This review focuses on the consideration of these mechanisms.
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Affiliation(s)
- Vasiliy Ivanovich Reshetnyak
- Department of Propaedeutics of Internal Diseases and Gastroenterology, A.I. Yevdokimov Moscow State University of Medicine and Dentistry, Moscow 127473, Russia
| | - Igor Veniaminovich Maev
- Department of Propaedeutics of Internal Diseases and Gastroenterology, A.I. Yevdokimov Moscow State University of Medicine and Dentistry, Moscow 127473, Russia
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2
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Zhang F, Xiao X, Li Y, Wu H, Deng X, Jiang Y, Zhang W, Wang J, Ma X, Zhao Y. Therapeutic Opportunities of GPBAR1 in Cholestatic Diseases. Front Pharmacol 2022; 12:805269. [PMID: 35095513 PMCID: PMC8793736 DOI: 10.3389/fphar.2021.805269] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2021] [Accepted: 12/23/2021] [Indexed: 12/12/2022] Open
Abstract
GPBAR1, a transmembrane G protein-coupled receptor for bile acids, is widely expressed in multiple tissues in humans and rodents. In recent years, GPBAR1 has been thought to play an important role in bile homeostasis, metabolism and inflammation. This review specifically focuses on the function of GPBAR1 in cholestatic liver disease and summarizes the various pathways through which GPBAR1 acts in cholestatic models. GPBAR1 mainly regulates cholestasis in a holistic system of liver-gallbladder-gut formation. In the state of cholestasis, the activation of GPBAR1 could regulate liver inflammation, induce cholangiocyte regeneration to maintain the integrity of the biliary tree, control the hydrophobicity of the bile acid pool and promote the secretion of bile HCO3−. All these functions of GPBAR1 might be clear ways to protect against cholestatic diseases and liver injury. However, the characteristic of GPBAR1-mediated proliferation increases the risk of proliferation of cholangiocarcinoma in malignant transformed cholangiocytes. This dichotomous function of GPBAR1 limits its use in cholestasis. During disease treatment, simultaneous activation of GPBAR1 and FXR receptors often results in improved outcomes, and this strategy may become a crucial direction in the development of bile acid-activated receptors in the future.
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Affiliation(s)
- Fangling Zhang
- State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Xiaolin Xiao
- Hospital of Chengdu University of Traditional Chinese Medicine, School of Clinical Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Yong Li
- State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Hefei Wu
- State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Xinyu Deng
- State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Yinxiao Jiang
- State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Wenwen Zhang
- State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Jian Wang
- State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Xiao Ma
- State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Yanling Zhao
- Department of Pharmacy, The Fifth Medical Center of PLA General Hospital, Beijing, China
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3
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Franca A, Filho ACML, Guerra MT, Weerachayaphorn J, dos Santos ML, Njei B, Robert M, Lima CX, Vidigal PVT, Banales JM, Ananthanarayanam M, Leite MF, Nathanson MH. Effects of Endotoxin on Type 3 Inositol 1,4,5-Trisphosphate Receptor in Human Cholangiocytes. Hepatology 2019; 69:817-830. [PMID: 30141207 PMCID: PMC6351171 DOI: 10.1002/hep.30228] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/11/2018] [Accepted: 08/13/2018] [Indexed: 12/16/2022]
Abstract
Clinical conditions that result in endotoxemia, such as sepsis and alcoholic hepatitis (AH), often are accompanied by cholestasis. Although hepatocellular changes in response to lipopolysaccharide (LPS) have been well characterized, less is known about whether and how cholangiocytes contribute to this form of cholestasis. We examined effects of endotoxin on expression and function of the type 3 inositol trisphosphate receptor (ITPR3), because this is the main intracellular Ca2+ release channel in cholangiocytes, and loss of it impairs ductular bicarbonate secretion. Bile duct cells expressed the LPS receptor, Toll-like receptor 4 (TLR4), which links to activation of nuclear factor-κB (NF-κB). Analysis of the human ITPR3 promoter revealed five putative response elements to NF-κB, and promoter activity was inhibited by p65/p50. Nested 0.5- and 1.0-kilobase (kb) deletion fragments of the ITPR3 promoter were inhibited by NF-κB subunits. Chromatin immunoprecipitation (ChIP) assay showed that NF-κB interacts with the ITPR3 promoter, with an associated increase in H3K9 methylation. LPS decreased ITPR3 mRNA and protein expression and also decreased sensitivity of bile duct cells to calcium agonist stimuli. This reduction was reversed by inhibition of TLR4. ITPR3 expression was decreased or absent in cholangiocytes from patients with cholestasis of sepsis and from those with severe AH. Conclusion: Stimulation of TLR4 by LPS activates NF-κB to down-regulate ITPR3 expression in human cholangiocytes. This may contribute to the cholestasis that can be observed in conditions such as sepsis or AH.
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Affiliation(s)
- Andressa Franca
- Federal University of Minas Gerais (UFMG), Belo Horizonte, MG
| | | | - Mateus T. Guerra
- Section of Digestive Disease, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT
| | - Jittima Weerachayaphorn
- Section of Digestive Disease, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT,Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand
| | | | - Basile Njei
- Section of Digestive Disease, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT
| | - Marie Robert
- Department of Pathology, Yale University School of Medicine, New Haven, CT
| | | | | | - Jesus M. Banales
- Department of Liver and Gastrointestinal Diseases, Biodonostia Research Institute, Donostia University Hospital, University of the Basque Country (UPV/EHU), San Sebastian, Spain
| | | | - M. Fatima Leite
- Federal University of Minas Gerais (UFMG), Belo Horizonte, MG
| | - Michael H. Nathanson
- Section of Digestive Disease, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT
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4
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Rodrigues MA, Gomes DA, Nathanson MH. Calcium Signaling in Cholangiocytes: Methods, Mechanisms, and Effects. Int J Mol Sci 2018; 19:ijms19123913. [PMID: 30563259 PMCID: PMC6321159 DOI: 10.3390/ijms19123913] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2018] [Revised: 11/13/2018] [Accepted: 11/20/2018] [Indexed: 02/06/2023] Open
Abstract
Calcium (Ca2+) is a versatile second messenger that regulates a number of cellular processes in virtually every type of cell. The inositol 1,4,5-trisphosphate receptor (ITPR) is the only intracellular Ca2+ release channel in cholangiocytes, and is therefore responsible for Ca2+-mediated processes in these cells. This review will discuss the machinery responsible for Ca2+ signals in these cells, as well as experimental models used to investigate cholangiocyte Ca2+ signaling. We will also discuss the role of Ca2+ in the normal and abnormal regulation of secretion and apoptosis in cholangiocytes, two of the best characterized processes mediated by Ca2+ in this cell type.
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Affiliation(s)
- Michele Angela Rodrigues
- Section of Digestive Diseases, Department of Internal Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8019, USA.
| | - Dawidson Assis Gomes
- Section of Digestive Diseases, Department of Internal Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8019, USA.
- Department of Biochemistry and Immunology, Federal University of Minas Gerais. Av. Antônio Carlos, 6627, Belo Horizonte-MG 31270-901, Brazil.
| | - Michael Harris Nathanson
- Section of Digestive Diseases, Department of Internal Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8019, USA.
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5
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Oliva-Vilarnau N, Hankeova S, Vorrink SU, Mkrtchian S, Andersson ER, Lauschke VM. Calcium Signaling in Liver Injury and Regeneration. Front Med (Lausanne) 2018; 5:192. [PMID: 30023358 PMCID: PMC6039545 DOI: 10.3389/fmed.2018.00192] [Citation(s) in RCA: 56] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2018] [Accepted: 06/11/2018] [Indexed: 12/12/2022] Open
Abstract
The liver fulfills central roles in metabolic control and detoxification and, as such, is continuously exposed to a plethora of insults. Importantly, the liver has a unique ability to regenerate and can completely recoup from most acute, non-iterative insults. However, multiple conditions, including viral hepatitis, non-alcoholic fatty liver disease (NAFLD), long-term alcohol abuse and chronic use of certain medications, can cause persistent injury in which the regenerative capacity eventually becomes dysfunctional, resulting in hepatic scaring and cirrhosis. Calcium is a versatile secondary messenger that regulates multiple hepatic functions, including lipid and carbohydrate metabolism, as well as bile secretion and choleresis. Accordingly, dysregulation of calcium signaling is a hallmark of both acute and chronic liver diseases. In addition, recent research implicates calcium transients as essential components of liver regeneration. In this review, we provide a comprehensive overview of the role of calcium signaling in liver health and disease and discuss the importance of calcium in the orchestration of the ensuing regenerative response. Furthermore, we highlight similarities and differences in spatiotemporal calcium regulation between liver insults of different etiologies. Finally, we discuss intracellular calcium control as an emerging therapeutic target for liver injury and summarize recent clinical findings of calcium modulation for the treatment of ischemic-reperfusion injury, cholestasis and NAFLD.
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Affiliation(s)
- Nuria Oliva-Vilarnau
- Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
| | - Simona Hankeova
- Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden.,Faculty of Science, Institute of Experimental Biology, Masaryk University, Brno, Czechia
| | - Sabine U Vorrink
- Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
| | - Souren Mkrtchian
- Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
| | - Emma R Andersson
- Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden.,Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden
| | - Volker M Lauschke
- Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
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6
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Trampert DC, Nathanson MH. Regulation of bile secretion by calcium signaling in health and disease. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2018; 1865:1761-1770. [PMID: 29787781 DOI: 10.1016/j.bbamcr.2018.05.010] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/27/2018] [Revised: 05/12/2018] [Accepted: 05/16/2018] [Indexed: 12/15/2022]
Abstract
Calcium (Ca2+) signaling controls secretion in many types of cells and tissues. In the liver, Ca2+ regulates secretion in both hepatocytes, which are responsible for primary formation of bile, and cholangiocytes, which line the biliary tree and further condition the bile before it is secreted. Cholestatic liver diseases, which are characterized by impaired bile secretion, may result from impaired Ca2+ signaling mechanisms in either hepatocytes or cholangiocytes. This review will discuss the Ca2+ signaling machinery and mechanisms responsible for regulation of secretion in both hepatocytes and cholangiocytes, and the pathophysiological changes in Ca2+ signaling that can occur in each of these cell types to result in cholestasis.
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Affiliation(s)
- David C Trampert
- Section of Digestive Diseases, Department of Internal Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8019, USA
| | - Michael H Nathanson
- Section of Digestive Diseases, Department of Internal Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8019, USA.
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7
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Besnard A, Gautherot J, Julien B, Tebbi A, Garcin I, Doignon I, Péan N, Gonzales E, Cassio D, Grosse B, Liu B, Safya H, Cauchois F, Humbert L, Rainteau D, Tordjmann T. The P2X4 purinergic receptor impacts liver regeneration after partial hepatectomy in mice through the regulation of biliary homeostasis. Hepatology 2016; 64:941-53. [PMID: 27301647 DOI: 10.1002/hep.28675] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/20/2016] [Accepted: 05/25/2016] [Indexed: 02/06/2023]
Abstract
UNLABELLED Many regulatory pathways are involved in liver regeneration after partial hepatectomy (PH), to initiate growth, protect liver cells, and sustain remnant liver functions. Extracellular adenosine triphosphate rises in blood and bile after PH and contributes to liver regeneration, although purinergic receptors and mechanisms remain to be precisely explored. In this work we analyzed during regeneration after PH the involvement of P2X4 purinergic receptors, highly expressed in the liver. P2X4 receptor expression in the liver, liver histology, hepatocyte proliferation, plasma bile acid concentration, bile flow and composition, and lysosome distribution in hepatocytes were studied in wild-type and P2X4 knockout (KO) mice, before and after PH. P2X4 receptors were expressed in hepatocytes and Kupffer cells; in hepatocytes, P2X4 was concentrated in subcanalicular areas closely costained with lysosomal markers. After PH, delayed regeneration, hepatocyte necrosis, and cholestasis were observed in P2X4-KO mice. In P2X4-KO mice, post-PH biliary adaptation was impaired with a smaller increase in bile flow and HCO3 (-) biliary output, as well as altered biliary composition with reduced adenosine triphosphate and lysosomal enzyme release. In line with these data, lysosome distribution and biogenesis were altered in P2X4-KO compared with wild-type mice. CONCLUSION During liver regeneration after PH, P2X4 contributes to the complex control of biliary homeostasis through mechanisms involving pericanalicular lysosomes, with a resulting impact on hepatocyte protection and proliferation. (Hepatology 2016;64:941-953).
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Affiliation(s)
- Aurore Besnard
- INSERM U1174, Université Paris Sud, Orsay, France.,Université Paris Sud, Orsay, France.,UPMC, Université Paris 06, Paris, France
| | - Julien Gautherot
- INSERM U1174, Université Paris Sud, Orsay, France.,Université Paris Sud, Orsay, France
| | - Boris Julien
- INSERM U1174, Université Paris Sud, Orsay, France.,Université Paris Sud, Orsay, France
| | - Ali Tebbi
- INSERM U1174, Université Paris Sud, Orsay, France.,Université Paris Sud, Orsay, France
| | - Isabelle Garcin
- INSERM U1174, Université Paris Sud, Orsay, France.,Université Paris Sud, Orsay, France
| | - Isabelle Doignon
- INSERM U1174, Université Paris Sud, Orsay, France.,Université Paris Sud, Orsay, France
| | - Noémie Péan
- INSERM U1174, Université Paris Sud, Orsay, France.,Université Paris Sud, Orsay, France
| | - Emmanuel Gonzales
- INSERM U1174, Université Paris Sud, Orsay, France.,Université Paris Sud, Orsay, France.,Hépatologie pédiatrique, Hôpital du Kremlin Bicêtre, Le Kremlin Bicêtre, France
| | - Doris Cassio
- INSERM U1174, Université Paris Sud, Orsay, France.,Université Paris Sud, Orsay, France
| | - Brigitte Grosse
- INSERM U1174, Université Paris Sud, Orsay, France.,Université Paris Sud, Orsay, France
| | - Bingkaï Liu
- INSERM U1174, Université Paris Sud, Orsay, France.,Université Paris Sud, Orsay, France
| | - Hanaa Safya
- INSERM U1174, Université Paris Sud, Orsay, France.,Université Paris Sud, Orsay, France
| | - Florent Cauchois
- INSERM U1174, Université Paris Sud, Orsay, France.,Université Paris Sud, Orsay, France
| | - Lydie Humbert
- UPMC, Université Paris 06, Paris, France.,ERL INSERM U 1057, Faculté de Médecine Pierre et Marie Curie, Paris, France
| | - Dominique Rainteau
- UPMC, Université Paris 06, Paris, France.,ERL INSERM U 1057, Faculté de Médecine Pierre et Marie Curie, Paris, France
| | - Thierry Tordjmann
- INSERM U1174, Université Paris Sud, Orsay, France.,Université Paris Sud, Orsay, France
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8
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Apical localization of inositol 1,4,5-trisphosphate receptors is independent of extended synaptotagmins in hepatocytes. PLoS One 2014; 9:e114043. [PMID: 25437447 PMCID: PMC4250053 DOI: 10.1371/journal.pone.0114043] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2014] [Accepted: 11/03/2014] [Indexed: 12/12/2022] Open
Abstract
Extended synaptotagmins (E-Syts) are a recently identified family of proteins that tether the endoplasmic reticulum (ER) to the plasma membrane (PM) in part by conferring regulation of cytosolic calcium (Ca2+) at these contact sites (Cell, 2013). However, the mechanism by which E-Syts link this tethering to Ca2+ signaling is unknown. Ca2+ waves in polarized epithelia are initiated by inositol 1,4,5-trisphosphate receptors (InsP3Rs), and these waves begin in the apical region because InsP3Rs are targeted to the ER adjacent to the apical membrane. In this study we investigated whether E-Syts are responsible for this targeting. Primary rat hepatocytes were used as a model system, because a single InsP3R isoform (InsP3R-II) is tethered to the peri-apical ER in these cells. Additionally, it has been established in hepatocytes that the apical localization of InsP3Rs is responsible for Ca2+ waves and secretion and is disrupted in disease states in which secretion is impaired. We found that rat hepatocytes express two of the three identified E-Syts (E-Syt1 and E-Syt2). Individual or simultaneous siRNA knockdown of these proteins did not alter InsP3R-II expression levels, apical localization or average InsP3R-II cluster size. Moreover, apical secretion of the organic anion 5-chloromethylfluorescein diacetate (CMFDA) was not changed in cells lacking E-Syts but was reduced in cells in which cytosolic Ca2+ was buffered. These data provide evidence that E-Syts do not participate in the targeting of InsP3Rs to the apical region. Identifying tethers that bring InsP3Rs to the apical region remains an important question, since mis-targeting of InsP3Rs leads to impaired secretory activity.
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9
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Amaya MJ, Nathanson MH. Calcium signaling and the secretory activity of bile duct epithelia. Cell Calcium 2014; 55:317-24. [PMID: 24612866 DOI: 10.1016/j.ceca.2014.02.003] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2013] [Revised: 02/03/2014] [Accepted: 02/04/2014] [Indexed: 12/20/2022]
Abstract
Cytosolic calcium (Cai(2+)) is a second messenger that is important for the regulation of secretion in many types of tissues. Bile duct epithelial cells, or cholangiocytes, are polarized epithelia that line the biliary tree in liver and are responsible for secretion of bicarbonate and other solutes into bile. Cai(2+) signaling plays an important role in the regulation of secretion by cholangiocytes, and this review discusses the machinery involved in the formation of Ca(2+) signals in cholangiocytes, along with the evidence that these signals regulate ductular secretion. Finally, this review discusses the evidence that impairments in cholangiocyte Ca(2+) signaling play a primary role in the pathogenesis of cholestatic disorders, in which hepatic bile secretion is impaired.
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Affiliation(s)
- Maria Jimena Amaya
- Section of Digestive Diseases, Department of Internal Medicine, Yale University, 333 Cedar Street, PO Box 208019, New Haven, CT 06520-8019, USA
| | - Michael H Nathanson
- Section of Digestive Diseases, Department of Internal Medicine, Yale University, 333 Cedar Street, PO Box 208019, New Haven, CT 06520-8019, USA.
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10
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Abstract
Intracellular free Ca(2+) ([Ca(2+)]i) is a highly versatile second messenger that regulates a wide range of functions in every type of cell and tissue. To achieve this versatility, the Ca(2+) signaling system operates in a variety of ways to regulate cellular processes that function over a wide dynamic range. This is particularly well exemplified for Ca(2+) signals in the liver, which modulate diverse and specialized functions such as bile secretion, glucose metabolism, cell proliferation, and apoptosis. These Ca(2+) signals are organized to control distinct cellular processes through tight spatial and temporal coordination of [Ca(2+)]i signals, both within and between cells. This article will review the machinery responsible for the formation of Ca(2+) signals in the liver, the types of subcellular, cellular, and intercellular signals that occur, the physiological role of Ca(2+) signaling in the liver, and the role of Ca(2+) signaling in liver disease.
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Affiliation(s)
- Maria Jimena Amaya
- Section of Digestive Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, USA
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11
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Abstract
Cholangiocytes are epithelial cells that line the intra- and extrahepatic ducts of the biliary tree. The main physiologic function of cholangiocytes is modification of hepatocyte-derived bile, an intricate process regulated by hormones, peptides, nucleotides, neurotransmitters, and other molecules through intracellular signaling pathways and cascades. The mechanisms and regulation of bile modification are reviewed herein.
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12
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Cruz LN, Guerra MT, Kruglov E, Mennone A, Garcia CRS, Chen J, Nathanson MH. Regulation of multidrug resistance-associated protein 2 by calcium signaling in mouse liver. Hepatology 2010; 52:327-37. [PMID: 20578149 PMCID: PMC3025771 DOI: 10.1002/hep.23625] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
UNLABELLED Multidrug resistance associated protein 2 (Mrp2) is a canalicular transporter responsible for organic anion secretion into bile. Mrp2 activity is regulated by insertion into the plasma membrane; however, the factors that control this are not understood. Calcium (Ca(2+)) signaling regulates exocytosis of vesicles in most cell types, and the type II inositol 1,4,5-triphosphate receptor (InsP(3)R2) regulates Ca(2+) release in the canalicular region of hepatocytes. However, the role of InsP(3)R2 and of Ca(2+) signals in canalicular insertion and function of Mrp2 is not known. The aim of this study was to determine the role of InsP(3)R2-mediated Ca(2+) signals in targeting Mrp2 to the canalicular membrane. Livers, isolated hepatocytes, and hepatocytes in collagen sandwich culture from wild-type (WT) and InsP(3)R2 knockout (KO) mice were used for western blots, confocal immunofluorescence, and time-lapse imaging of Ca(2+) signals and of secretion of a fluorescent organic anion. Plasma membrane insertion of green fluorescent protein (GFP)-Mrp2 expressed in HepG2 cells was monitored by total internal reflection microscopy. InsP(3)R2 was concentrated in the canalicular region of WT mice but absent in InsP(3)R2 KO livers, whereas expression and localization of InsP(3)R1 was preserved, and InsP(3)R3 was absent from both WT and KO livers. Ca(2+) signals induced by either adenosine triphosphate (ATP) or vasopressin were impaired in hepatocytes lacking InsP(3)R2. Canalicular secretion of the organic anion 5-chloromethylfluorescein diacetate (CMFDA) was reduced in KO hepatocytes, as well as in WT hepatocytes treated with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Moreover, the choleretic effect of tauroursodeoxycholic acid (TUDCA) was impaired in InsP(3)R2 KO mice. Finally, ATP increased GFP-Mrp2 fluorescence in the plasma membrane of HepG2 cells, and this also was reduced by BAPTA. CONCLUSION InsP(3)R2-mediated Ca(2+) signals enhance organic anion secretion into bile by targeting Mrp2 to the canalicular membrane.
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Affiliation(s)
- Laura N. Cruz
- Section of Digestive Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT,Department of Parasitology, University of Saão Paulo, Saão Paulo, Brazil
| | - Mateus T. Guerra
- Section of Digestive Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT,Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, Brazil
| | - Emma Kruglov
- Section of Digestive Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT
| | - Albert Mennone
- Section of Digestive Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT
| | | | - Ju Chen
- Department of Medicine, University of California, San Diego, CA
| | - Michael H. Nathanson
- Section of Digestive Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT
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13
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Yu J, Sheung N, Soliman EM, Spirli C, Dranoff JA. Transcriptional regulation of IL-6 in bile duct epithelia by extracellular ATP. Am J Physiol Gastrointest Liver Physiol 2009; 296:G563-71. [PMID: 19136380 PMCID: PMC2660176 DOI: 10.1152/ajpgi.90502.2008] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The inflammatory cytokine IL-6 is essential for cell survival after liver injury. Bile duct epithelia (BDE) markedly upregulate IL-6 release after liver injury, but the mechanisms regulating this have not been defined. Purinergic signals induce multiple potent downstream effects in BDE, so the goals of this study were to determine whether extracellular ATP regulates BDE IL-6 transcription and to identify the molecular mechanisms regulating this process. Effects of extracellular nucleotides on IL-6 transcription in primary rat bile duct epithelia were assessed. The relative effects of cAMP and cytosolic calcium were determined by use of agonists and antagonists. The role of the cAMP response element (CRE) was determined by site-directed mutagenesis. We found that ATP potently upregulated IL-6 mRNA, and that the pharmacological profile for IL-6 upregulation was most consistent with the newly identified P2Y11 receptor. This occurred in a cAMP-dependent and calcium-dependent fashion. The effect of cAMP and calcium agonists on IL-6 promoter activity was synergistic, and mutation of the IL-6 CRE blocked upregulation by ATP. Taken together, these data show that extracellular ATP acts through a mechanism involving a rat P2Y receptor functionally related to the P2Y11 receptor, cAMP, and calcium signals and that the IL-6 promoter CRE to upregulate transcription of IL-6 in BDE. Since IL-6 has such critical importance in the liver, it is likely that this pathway is of great relevance to the understanding of hepatic response to injury.
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Affiliation(s)
- Jin Yu
- Yale University School of Medicine/Yale Liver Center, New Haven, Connecticut
| | - Nina Sheung
- Yale University School of Medicine/Yale Liver Center, New Haven, Connecticut
| | - Elwy M. Soliman
- Yale University School of Medicine/Yale Liver Center, New Haven, Connecticut
| | - Carlo Spirli
- Yale University School of Medicine/Yale Liver Center, New Haven, Connecticut
| | - Jonathan A. Dranoff
- Yale University School of Medicine/Yale Liver Center, New Haven, Connecticut
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Marzioni M, Fava G, Alvaro D, Alpini G, Benedetti A. Control of cholangiocyte adaptive responses by visceral hormones and neuropeptides. Clin Rev Allergy Immunol 2009; 36:13-22. [PMID: 18548352 PMCID: PMC2628969 DOI: 10.1007/s12016-008-8090-1] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Cholangiocytes, the epithelial cells lining the biliary tree, are the target cells in several liver diseases, termed cholangiopathies. Cholangiopathies are a challenge for clinicians and an enigma for scientists, as the pathogenetic mechanisms by which they develop, and the therapeutic tools for these diseases are still undefined. Several studies demonstrate that many visceral hormones, neuropeptides, and neurotransmitters modulate the adaptive changes of cholangiocytes to chronic cholestatic injury. The aim of this review is to present the recent findings that contributed to clarify the role of visceral hormones and neuropeptides in the regulation of the pathophysiology of cholestasis. These studies helped to shed light on some aspects of cholangiocyte pathophysiology, revealing novel perspectives for the clinical managements of cholangiopathies.
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Affiliation(s)
- Marco Marzioni
- Department of Gastroenterology, Università Politecnica delle Miarche, Nuovo Polo Didattico, III piano, Via Tronto 10, 60020, Ancona, Italy.
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Abstract
The formation of bile depends on the structural and functional integrity of the bile-secretory apparatus and its impairment, in different situations, results in the syndrome of cholestasis. The structural bases that permit bile secretion as well as various aspects related with its composition and flow rate in physiological conditions will first be reviewed. Canalicular bile is produced by polarized hepatocytes that hold transporters in their basolateral (sinusoidal) and apical (canalicular) plasma membrane. This review summarizes recent data on the molecular determinants of this primary bile formation. The major function of the biliary tree is modification of canalicular bile by secretory and reabsorptive processes in bile-duct epithelial cells (cholangiocytes) as bile passes through bile ducts. The mechanisms of fluid and solute transport in cholangiocytes will also be discussed. In contrast to hepatocytes where secretion is constant and poorly controlled, cholangiocyte secretion is regulated by hormones and nerves. A short section dedicated to these regulatory mechanisms of bile secretion has been included. The aim of this revision was to set the bases for other reviews in this series that will be devoted to specific issues related with biliary physiology and pathology.
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Abstract
The biliary tree is a complex network of conduits that begins with the canals of Hering and progressively merges into a system of interlobular, septal, and major ducts which then coalesce to form the extrahepatic bile ducts, which finally deliver bile to the gallbladder and to the intestine. The biliary epithelium shows a morphological heterogeneity that is strictly associated with a variety of functions performed at the different levels of the biliary tree. In addition to funneling bile into the intestine, cholangiocytes (the epithelial cells lining the bile ducts) are actively involved in bile production by performing both absorbitive and secretory functions. More recently, other important biological properties restricted to cholangiocytes lining the smaller bile ducts have been outlined, with regard to their plasticity (i.e., the ability to undergo limited phenotypic changes), reactivity (i.e., the ability to participate in the inflammatory reaction to liver damage), and ability to behave as liver progenitor cells. Functional interactions with other branching systems, such as nerve and vascular structures, are crucial in the modulation of the different cholangiocyte functions.
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Affiliation(s)
- Mario Strazzabosco
- Department of Internal Medicine, Yale University, New Haven 06504, Connecticut, USA.
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Marzioni M, Fava G, Benedetti A. Nervous and Neuroendocrine regulation of the pathophysiology of cholestasis and of biliary carcinogenesis. World J Gastroenterol 2006; 12:3471-3480. [PMID: 16773704 PMCID: PMC4087563 DOI: 10.3748/wjg.v12.i22.3471] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/13/2006] [Revised: 02/06/2006] [Accepted: 02/18/2006] [Indexed: 02/06/2023] Open
Abstract
Cholangiocytes, the epithelial cells lining the biliary ducts, are the target cells in several liver diseases. Cholangiopathies and cholangiocarcinoma generate interest in many scientists since the genesis. The developing mechanisms, and the therapeutic tools of these diseases are still undefined. Several studies demonstrate that many hormones, neuropeptides and neurotransmitters regulate malignant and non-malignant cholangiocyte pathophysiology in the course of chronic biliary diseases. The aim of this review is to present the findings of several studies published in the recent years that contributed to clarifying the role of nervous and neuroendocrine regulation of the pathophysiologic events associated with cholestasis and cholangiocarcinoma development. This manuscript is organized into two parts. The first part offers an overview of the innervation of the liver and the origin of neuroendocrine hormones, neurotransmitters and neuropeptides affecting cholangiocyte function and metabolism. The first section also reviews the effects played by several neuroendocrine hormones and nervous system on cholangiocyte growth, survival and functional activity in the course of cholestasis. In the second section, we summarize the results of some studies describing the role of nervous system and neuroendocrine hormones in the regulation of malignant cholangiocyte growth.
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Affiliation(s)
- Marco Marzioni
- Department of Gastroenterology, Università Politecnica delle Marche, Nuovo Polo Didattico, III piano, Via Tronto 10, 60020 Ancona, Italy.
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18
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Minagawa N, Ehrlich BE, Nathanson MH. Calcium signaling in cholangiocytes. World J Gastroenterol 2006; 12:3466-70. [PMID: 16773703 PMCID: PMC4087562 DOI: 10.3748/wjg.v12.i22.3466] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/01/2006] [Revised: 01/14/2006] [Accepted: 01/24/2006] [Indexed: 02/06/2023] Open
Abstract
Cytosolic Ca2+ is an important second messenger in virtually every type of cell. Moreover, Ca2+ generally regulates multiple activities within individual cells. This article reviews the cellular machinery that is responsible for Ca2+ signaling in cholangiocytes. In addition, two Ca2+-mediated events in cholangiocytes are discussed: bicarbonate secretion and apoptosis. Finally, emerging evidence is reviewed that Ca2+ signaling is involved in the pathogenesis of diseases affecting the biliary tree and that Ca2+ signaling pathways can be manipulated to therapeutic advantage in the treatment of cholestatic disorders.
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Affiliation(s)
- Noritaka Minagawa
- Department of Medicine Pharmacology, Yale University School of Medicine, 1 Gilbert Street, Room TAC S241D, New Haven, CT 06519, USA
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Abstract
Primary canalicular bile undergoes a process of fluidization and alkalinization along the biliary tract that is influenced by several factors including hormones, innervation/neuropeptides, and biliary constituents. The excretion of bicarbonate at both the canaliculi and the bile ducts is an important contributor to the generation of the so-called bile-salt independent flow. Bicarbonate is secreted from hepatocytes and cholangiocytes through parallel mechanisms which involve chloride efflux through activation of Cl- channels, and further bicarbonate secretion via AE2/SLC4A2-mediated Cl-/HCO3- exchange. Glucagon and secretin are two relevant hormones which seem to act very similarly in their target cells (hepatocytes for the former and cholangiocytes for the latter). These hormones interact with their specific G protein-coupled receptors, causing increases in intracellular levels of cAMP and activation of cAMP-dependent Cl- and HCO3- secretory mechanisms. Both hepatocytes and cholangiocytes appear to have cAMP-responsive intracellular vesicles in which AE2/SLC4A2 colocalizes with cell specific Cl- channels (CFTR in cholangiocytes and not yet determined in hepatocytes) and aquaporins (AQP8 in hepatocytes and AQP1 in cholangiocytes). cAMP-induced coordinated trafficking of these vesicles to either canalicular or cholangiocyte lumenal membranes and further exocytosis results in increased osmotic forces and passive movement of water with net bicarbonate-rich hydrocholeresis.
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Affiliation(s)
- Jesús-M Banales
- Laboratory of Molecular Genetics, Division of Gene Therapy and Hepatology, University of Navarra School of Medicine, Clinica Universitaria and CIMA, Avda. Pio XII 55, E-31008 Pamplona, Spain
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20
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Glaser S, Alvaro D, Francis H, Ueno Y, Marucci L, Benedetti A, De Morrow S, Marzioni M, Mancino MG, Phinizy JL, Reichenbach R, Fava G, Summers R, Venter J, Alpini G. Adrenergic receptor agonists prevent bile duct injury induced by adrenergic denervation by increased cAMP levels and activation of Akt. Am J Physiol Gastrointest Liver Physiol 2006; 290:G813-G826. [PMID: 16339297 DOI: 10.1152/ajpgi.00306.2005] [Citation(s) in RCA: 52] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Loss of parasympathetic innervation after vagotomy impairs cholangiocyte proliferation, which is associated with depressed cAMP levels, impaired ductal secretion, and enhanced apoptosis. Agonists that elevate cAMP levels prevent cholangiocyte apoptosis and restore cholangiocyte proliferation and ductal secretion. No information exists regarding the role of adrenergic innervation in the regulation of cholangiocyte function. In the present studies, we investigated the role of adrenergic innervation on cholangiocyte proliferative and secretory responses to bile duct ligation (BDL). Adrenergic denervation by treatment with 6-hydroxydopamine (6-OHDA) during BDL decreased cholangiocyte proliferation and secretin-stimulated ductal secretion with concomitant increased apoptosis, which was associated with depressed cholangiocyte cAMP levels. Chronic administration of forskolin (an adenylyl cyclase activator) or beta(1)- and beta(2)-adrenergic receptor agonists (clenbuterol or dobutamine) prevented the decrease in cholangiocyte cAMP levels, maintained cholangiocyte secretory and proliferative activities, and decreased cholangiocyte apoptosis resulting from adrenergic denervation. This was associated with enhanced phosphorylation of Akt. The protective effects of clenbuterol, dobutamine, and forskolin on 6-OHDA-induced changes in cholangiocyte apoptosis and proliferation were partially blocked by chronic in vivo administration of wortmannin. In conclusion, we propose that adrenergic innervation plays a role in the regulation of biliary mass and cholangiocyte functions during BDL by modulating intracellular cAMP levels.
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Affiliation(s)
- Shannon Glaser
- Division of Research and Education, College of Medicine, Scott and White Hospital and The Texas A & M University System Health Science Center, Temple, 76504, USA
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21
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Marzioni M, Glaser S, Francis H, Marucci L, Benedetti A, Alvaro D, Taffetani S, Ueno Y, Roskams T, Phinizy JL, Venter J, Fava G, Lesage GD, Alpini G. Autocrine/paracrine regulation of the growth of the biliary tree by the neuroendocrine hormone serotonin. Gastroenterology 2005; 128:121-137. [PMID: 15633129 DOI: 10.1053/j.gastro.2004.10.002] [Citation(s) in RCA: 97] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
BACKGROUND & AIMS The biliary tree is the target of cholangiopathies that are chronic cholestatic liver diseases characterized by loss of proliferative response and enhanced apoptosis of cholangiocytes, the epithelial cells lining the biliary tree. The endogenous factors that regulate cholangiocyte proliferation are poorly understood. Therefore, we studied the role of the neuroendocrine hormone serotonin as a modulator of cholangiocyte proliferation. METHODS The presence of the serotonin 1A and 1B receptors on cholangiocytes was evaluated. We then tested whether the activation of such receptors by the administration of the selective agonists modifies cholangiocyte proliferation and functional activity both in vivo and in vitro. In addition, the intracellular signal mediating the serotonin receptor action in cholangiocytes was characterized. We studied the expression and secretion of serotonin by cholangiocytes and the effects of the neutralization of the secreted hormone on the growth of the biliary tree. RESULTS Cholangiocytes express the serotonin 1A and 1B receptors. Their activation markedly inhibits the growth and choleretic activity of the biliary tree in the bile duct-ligated rat, a model of chronic cholestasis. Such changes are mediated by enhanced d -myo-inositol 1,4,5-triphosphate/Ca 2+ /protein kinase C signaling and the consequent inhibition of the adenosine 3',5'-cyclic monophosphate/protein kinase A/Src/extracellular signal-regulated kinase 1/2 cascade. Cholangiocytes secrete serotonin, the blockage of which enhances cholangiocyte proliferation in the course of cholestasis. CONCLUSIONS We observed the existence of an autocrine loop based on serotonin that limits the growth of the biliary tree in the course of chronic cholestasis. Our novel findings might open new approaches for the management of cholangiopathies.
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Affiliation(s)
- Marco Marzioni
- Department of Medical Physiology, Scott & White Hospital, and Texas A&M University Health System Science Center, 702 Southwest H.K. Dodgen Loop, Temple, TX 76504, USA
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22
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Dranoff JA, Ogawa M, Kruglov EA, Gaça MDA, Sévigny J, Robson SC, Wells RG. Expression of P2Y nucleotide receptors and ectonucleotidases in quiescent and activated rat hepatic stellate cells. Am J Physiol Gastrointest Liver Physiol 2004; 287:G417-24. [PMID: 14764443 PMCID: PMC5241161 DOI: 10.1152/ajpgi.00294.2003] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Extracellular nucleotides regulate a variety of cellular activities, including proliferation of fibrogenic cells outside of the liver. However, the expression of receptors for extracellular nucleotides in hepatic stellate cells (HSC) is unknown. Thus our aims were to investigate the expression of mediators of nucleotide signaling in HSC and to determine whether extracellular nucleotides regulate HSC function. Confocal video microscopy was used to observe nucleotide-induced changes in cytosolic Ca(2+) (Ca(i)(2+)) in live HSC. P2Y receptor subtype expression and ectonucleotidase expression in quiescent and activated HSC were determined using RT-PCR, Northern blot, immunoblot, and confocal immunofluorescence. Functional ectonucleotidase activity was assessed using a colorimetric method. Nucleotide-sensitive procollagen-1 mRNA expression in activated HSC was assessed using real-time RT-PCR. Extracellular ATP increased Ca(i)(2+) in HSC; this was inhibited by the P2 receptor inhibitor suramin. Quiescent HSC expressed the P2Y subtypes P2Y(2) and P2Y(4) and were activated by ATP and UTP, whereas activated HSC expressed the P2Y subtype P2Y(6) and were activated by UDP and ATP. Activated but not quiescent HSC expressed the ectonucleotidase nucleoside triphosphate diphosphohydrolase 2, extracellular UDP tripled procollagen-1 mRNA expression in activated HSC, and this was inhibited by the P2Y receptor inhibitor suramin. HSC express functional P2Y receptors and switch the expression of P2Y receptor subtypes on activation. Moreover, HSC differentially regulate nucleoside triphosphate diphosphohydrolase expression after activation. Because activation of P2Y receptors in activated HSC regulates procollagen-1 transcription, P2Y receptors may be an attractive target to prevent or treat liver fibrosis.
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Affiliation(s)
- Jonathan A Dranoff
- Yale Univ. School of Medicine, Section of Digestive Diseases, 333 Cedar St. LMP 1080, New Haven, CT 06520, USA.
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23
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Glaser S, Alvaro D, Roskams T, Phinizy JL, Stoica G, Francis H, Ueno Y, Barbaro B, Marzioni M, Mauldin J, Rashid S, Mancino MG, LeSage G, Alpini G. Dopaminergic inhibition of secretin-stimulated choleresis by increased PKC-gamma expression and decrease of PKA activity. Am J Physiol Gastrointest Liver Physiol 2003; 284:G683-94. [PMID: 12505882 DOI: 10.1152/ajpgi.00302.2002] [Citation(s) in RCA: 56] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
To determine the role and mechanisms of action by which dopaminergic innervation modulates ductal secretion in bile duct-ligated rats, we determined the expression of D1, D2, and D3 dopaminergic receptors in cholangiocytes. We evaluated whether D1, D2 (quinelorane), or D3 dopaminergic receptor agonists influence basal and secretin-stimulated choleresis and lumen expansion in intrahepatic bile duct units (IBDU) and cAMP levels in cholangiocytes in the absence or presence of BAPTA-AM, chelerythrine, 1-(5-isoquinolinylsulfonyl)-2-methyl piperazine (H7), or rottlerin. We evaluated whether 1) quinelorane effects on ductal secretion were associated with increased expression of Ca(2+)-dependent PKC isoforms and 2) increased expression of PKC causes inhibition of PKA activity. Quinelorane inhibited secretin-stimulated choleresis in vivo and IBDU lumen space, cAMP levels, and PKA activity in cholangiocytes. The inhibitory effects of quinelorane on secretin-stimulated ductal secretion and PKA activity were blocked by BAPTA-AM, chelerythrine, and H7. Quinelorane effects on ductal secretion were associated with activation of the Ca(2+)-dependent PKC-gamma but not other PKC isoforms. The dopaminergic nervous system counterregulates secretin-stimulated ductal secretion in experimental cholestasis.
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Affiliation(s)
- Shannon Glaser
- Department of Internal Medicine, Texas A&M University System Health Sciences Center, College of Medicine, Temple, Texas 76504, USA
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Park SC, Yibchok-Anun S, Cheng H, Young TF, Thacker EL, Minion FC, Ross RF, Hsu WH. Mycoplasma hyopneumoniae increases intracellular calcium release in porcine ciliated tracheal cells. Infect Immun 2002; 70:2502-6. [PMID: 11953388 PMCID: PMC127901 DOI: 10.1128/iai.70.5.2502-2506.2002] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
We investigated the effects of intact pathogenic Mycoplasma hyopneumoniae, nonpathogenic M. hyopneumoniae, and Mycoplasma flocculare on intracellular free Ca2+ concentrations ([Ca2+]i) in porcine ciliated tracheal epithelial cells. The ciliated epithelial cells had basal [Ca2+]i of 103 +/- 3 nM (n = 217 cells). The [Ca2+]i increased by 250 +/- 19 nM (n = 47 cells) from the basal level within 100 s of the addition of pathogenic M. hyopneumoniae strain 91-3 (300 microg/ml), and this increase lasted approximately 60 s. In contrast, nonpathogenic M. hyopneumoniae and M. flocculare at concentrations of 300 microg/ml failed to increase [Ca2+]i. In Ca2+-free medium, pathogenic M. hyopneumoniae still increased [Ca2+]i in tracheal cells. Pretreatment with thapsigargin (1 microM for 30 min), which depleted the Ca2+ store in the endoplasmic reticulum, abolished the effect of M. hyoneumoniae. Pretreatment with pertussis toxin (100 ng/ml for 3 h) or U-73122 (2 microM for 100 s), an inhibitor of phospholipase C, also abolished the effect of M. hyopneumoniae. The administration of mastoparan 7, an activator of pertussis toxin-sensitive proteins G(i) and G(o), increased [Ca2+]i in ciliated tracheal cells. These results suggest that pathogenic M. hyopneumoniae activates receptors that are coupled to G(i) or G(o), which in turn activates a phospholipase C pathway, thereby releasing Ca2+ from the endoplasmic reticulum. Thus, an increase in Ca2+ may serve as a signal for the pathogenesis of M. hyopneumoniae.
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Affiliation(s)
- Seung-Chun Park
- Department of Biomedical Sciences, Iowa State University, Ames, Iowa 50011, USA
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25
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Dranoff JA, Masyuk AI, Kruglov EA, LaRusso NF, Nathanson MH. Polarized expression and function of P2Y ATP receptors in rat bile duct epithelia. Am J Physiol Gastrointest Liver Physiol 2001; 281:G1059-67. [PMID: 11557527 DOI: 10.1152/ajpgi.2001.281.4.g1059] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Extracellular nucleotides may be important regulators of bile ductular secretion, because cholangiocytes express P2Y ATP receptors and nucleotides are found in bile. However, the expression, distribution, and function of specific P2Y receptor subtypes in cholangiocytes are unknown. Thus our aim was to determine the subtypes, distribution, and role in secretion of P2Y receptors expressed by cholangiocytes. The molecular subtypes of P2Y receptors were determined by RT-PCR. Functional studies measuring cytosolic Ca2+ (Ca) signals and bile ductular pH were performed in isolated, microperfused intrahepatic bile duct units (IBDUs). PCR products corresponding to P2Y1, P2Y2, P2Y4, P2Y6, and P2X4 receptor subtypes were identified. Luminal perfusion of ATP into IBDUs induced increases in Ca that were inhibited by apyrase and suramin. Luminal ATP, ADP, 2-methylthioadenosine 5'-triphosphate, UTP, and UDP each increased Ca. Basolateral addition of adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), but not ATP, to the perifusing bath increased Ca. IBDU perfusion with ATP-gamma-S induced net bile ductular alkalization. Cholangiocytes express multiple P2Y receptor subtypes that are expressed at the apical plasma membrane domain. P2Y receptors are also expressed on the basolateral domain, but their activation is attenuated by nucleotide hydrolysis. Activation of ductular P2Y receptors induces net ductular alkalization, suggesting that nucleotide signaling may be an important regulator of bile secretion by the liver.
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Affiliation(s)
- J A Dranoff
- Section of Digestive Diseases, Yale University School of Medicine, New Haven, Connecticut 06520-8019, USA.
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26
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Abstract
The objective of this review article is to discuss the role of secretin and its receptor in the regulation of the secretory activity of intrahepatic bile duct epithelial cells (i.e., cholangiocytes). After a brief overview of cholangiocyte functions, we provide an historical background for the role of secretin and its receptor in the regulation of ductal secretion. We review the newly developed experimental in vivo and in vitro tools, which lead to understanding of the mechanisms of secretin regulation of cholangiocyte functions. After a description of the intracellular mechanisms by which secretin stimulates ductal secretion, we discuss the heterogeneous responses of different-sized intrahepatic bile ducts to gastrointestinal hormones. Furthermore, we outline the role of a number of cooperative factors (e.g., nerves, alkaline phosphatase, gastrointestinal hormones, neuropeptides, and bile acids) in the regulation of secretin-stimulated ductal secretion. Finally, we discuss other factors that may also play an important role in the regulation of secretin-stimulated ductal secretion.
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Affiliation(s)
- N Kanno
- Department of Internal Medicine, Scott & White Hospital and Texas A&M University System Health Science Center, College of Medicine, TX 76504, USA
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27
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Breitwieser GE, Gama L. Calcium-sensing receptor activation induces intracellular calcium oscillations. Am J Physiol Cell Physiol 2001; 280:C1412-21. [PMID: 11350736 DOI: 10.1152/ajpcell.2001.280.6.c1412] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Parathyroid hormone secretion is exquisitely sensitive to small changes in serum Ca2+concentration, and these responses are transduced via the Ca2+-sensing receptor (CaR). We utilized heterologous expression in HEK-293 cells to determine the effects of small, physiologically relevant perturbations in extracellular Ca2+ on CaR signaling via phosphatidylinositol-phospholipase C, using changes in fura 2 fluorescence to quantify intracellular Ca2+. Chronic exposure of CaR-transfected cells to Ca2+ in the range from 0.5 to 3 mM modulated the resting intracellular Ca2+concentration and the subsequent cellular responses to acute extracellular Ca2+ perturbations but had no effect on thapsigargin-sensitive Ca2+ stores. Modest, physiologically relevant increases in extracellular Ca2+concentration (0.5 mM increments) caused sustained (30–40 min) low-frequency oscillations of intracellular Ca2+ (∼45 s peak to peak interval). Oscillations were eliminated by 1 μM thapsigargin but were insensitive to protein kinase inhibitors (staurosporine, KN-93, or bisindolylmaleimide I). Staurosporine did increase the fraction of cells oscillating at a given extracellular Ca2+ concentration. Serum Ca2+ concentrations thus chronically regulate cells expressing CaR, and small perturbations in extracellular Ca2+ alter both resting intracellular Ca2+ as well as Ca2+ dynamics.
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Affiliation(s)
- G E Breitwieser
- Department of Physiology, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205, USA.
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28
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Kwiatkowski AP, McGill JM. Alternative splice variant of gamma-calmodulin-dependent protein kinase II alters activation by calmodulin. Arch Biochem Biophys 2000; 378:377-83. [PMID: 10860555 DOI: 10.1006/abbi.2000.1846] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous, multifunctional enzyme family involved in the regulation of a variety of Ca(2+)-signaling pathways. These family members are expressed from four highly homologous genes (alpha, beta, gamma, and delta) with similar catalytic properties. Additional isoforms of each gene, created by alternative splicing of variable regions I-XI, are differentially expressed in various cell types. gammaB, gammaC, gammaD, gammaE, gammaF, gammaGs, and gammaH CaMKII isoforms are expressed in the biliary epithelium; however, little is known about their roles in these cells. We began our studies into the function of these variable regions by examining the effects of variable region I on kinase activation and calmodulin binding. Activities and calmodulin binding properties of gammaB and gammaGs, which differ only by the exclusion or inclusion of this region, were compared. The K(0.5) for calmodulin was 2.5-fold lower for gammaGs than gammaB. In contrast, gammaB bound calmodulin more tightly in a calmodulin overlay assay. Mutation of variable regions I's charged residue, gammaGs-R318E, resulted in an enzyme with intermediate activation properties but a calmodulin affinity similar to gammaB. Thus, variable region I appears to modulate calmodulin sensitivity, in part, through charge-charge interactions. This altered threshold of activation may modulate cellular responses to gradients of Ca(2+)/calmodulin in the biliary tract.
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Affiliation(s)
- A P Kwiatkowski
- Division of Gastroenterology and Hepatology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
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Salter KD, Fitz JG, Roman RM. Domain-specific purinergic signaling in polarized rat cholangiocytes. Am J Physiol Gastrointest Liver Physiol 2000; 278:G492-500. [PMID: 10712270 DOI: 10.1152/ajpgi.2000.278.3.g492] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
In cholangiocytes, adenine nucleotides function as autocrine/paracrine signals that modulate ductular ion transport by activation of purinergic receptors. The purpose of these studies was to identify cellular signals that modulate ATP release and nucleotide processing in polarized normal rat cholangiocytes. In Ussing chamber studies, selective exposure of the apical and basolateral membranes to ATP or adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) stimulated increases in short-circuit current. Apical purinergic receptor agonist preference was consistent with the P2Y(2) subtype. In contrast, basolateral ADP was more potent in stimulating transepithelial currents, consistent with the expression of different basolateral P2 receptor(s). Luminometric analysis revealed that both membranes exhibited constitutive ATP efflux. Hypotonic exposure enhanced ATP release in both compartments, whereas decreases in ATP efflux during hypertonicity were more prominent at the apical membrane. Increases in intracellular cAMP, cGMP, and Ca(2+) also increased ATP permeability, but selective effects on apical and basolateral ATP release differed. Finally, the kinetics of ATP degradation in apical and basolateral compartments were distinct. These findings suggest that there are domain-specific signaling pathways that contribute to purinergic responses in polarized cholangiocytes.
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Affiliation(s)
- K D Salter
- Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
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Ishiguro H, Naruse S, Kitagawa M, Hayakawa T, Case RM, Steward MC. Luminal ATP stimulates fluid and HCO3- secretion in guinea-pig pancreatic duct. J Physiol 1999; 519 Pt 2:551-8. [PMID: 10457070 PMCID: PMC2269526 DOI: 10.1111/j.1469-7793.1999.0551m.x] [Citation(s) in RCA: 60] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/1999] [Accepted: 05/27/1999] [Indexed: 11/30/2022] Open
Abstract
1. The location of purinoceptors in the pancreatic duct and their role in regulating ductal secretion have been investigated by applying ATP and UTP to basolateral and luminal surfaces of pancreatic ducts isolated from the guinea-pig pancreas. 2. Changes in intracellular Ca2+ concentration were measured by microfluorometry in microperfused interlobular duct segments. Fluid and HCO3- secretion were estimated by monitoring luminal pH and luminal volume in sealed duct segments microinjected with BCECF-dextran. 3. Both ATP and UTP (1 microM) caused biphasic increases in intracellular Ca2+ concentration in pancreatic duct cells when applied to either the basolateral or luminal membrane. 4. Luminal application of both ATP and UTP evoked fluid and HCO3- secretion. The maximum response to 1 microM ATP or UTP was about 75 % of that evoked by secretin. By contrast, basolateral application of ATP or UTP inhibited spontaneous secretion by 52 % and 73 %, respectively, and secretin-evoked secretion by 41 % and 38 %, respectively. 5. The data suggest that luminal nucleotides may act in an autocrine or paracrine fashion to enhance ductal secretion while basolateral nucleotides, perhaps released from nerve terminals, may have an inhibitory effect. The fact that both apical and basolateral purinoceptors elevate intracellular Ca2+, but that they have opposite effects on secretion, suggests that additional signalling pathways are involved.
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Affiliation(s)
- H Ishiguro
- Internal Medicine II, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8560, Japan.
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Nathanson MH, Burgstahler AD, Mennone A, Dranoff JA, Rios-Velez L. Stimulation of bile duct epithelial secretion by glybenclamide in normal and cholestatic rat liver. J Clin Invest 1998; 101:2665-76. [PMID: 9637700 PMCID: PMC508857 DOI: 10.1172/jci2835] [Citation(s) in RCA: 23] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
Cholestasis is a cardinal complication of liver disease, but most treatments are merely supportive. Here we report that the sulfonylurea glybenclamide potently stimulates bile flow and bicarbonate excretion in the isolated perfused rat liver. Video-microscopic studies of isolated hepatocyte couplets and isolated bile duct segments show that this stimulatory effect occurs at the level of the bile duct epithelium, rather than through hepatocytes. Measurements of cAMP, cytosolic pH, and Ca2+ in isolated bile duct cells suggest that glybenclamide directly activates Na+-K+-2Cl- cotransport, rather than other transporters or conventional second-messenger systems that link to secretory pathways in these cells. Finally, studies in livers from rats with endotoxin- or estrogen-induced cholestasis show that glybenclamide retains its stimulatory effects on bile flow and bicarbonate excretion even under these conditions. These findings suggest that bile duct epithelia may represent an important new therapeutic target for treatment of cholestatic disorders.
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Affiliation(s)
- M H Nathanson
- Liver Study Unit and Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
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Alvaro 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; 100:1349-62. [PMID: 9294100 PMCID: PMC508313 DOI: 10.1172/jci119655] [Citation(s) in RCA: 110] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
UNLABELLED We investigated, in isolated bile duct units (IBDU) and cholangiocytes isolated from normal rat liver, the occurrence of acetylcholine (ACh) receptors, and the role and mechanisms of ACh in the regulation of the Cl-/HCO3- exchanger activity. The Cl-/HCO3- exchanger activity was evaluated measuring changes in intracellular pH induced by acute Cl- removal/readmission. M3 subtype ACh receptors were detected in IBDU and isolated cholangiocytes by immunofluorescence, immunoelectron microscopy, and reverse transcriptase PCR. M1 subtype ACh receptor mRNA was not detected by reverse transcriptase PCR and M2 subtype was negative by immunofluorescence. ACh (10 microM) showed no effect on the basal activity of the Cl-/HCO3- exchanger. When IBDU were exposed to ACh plus secretin, ACh significantly (P < 0.03) increased the maximal rate of alkalinization after Cl- removal and the maximal rate of recovery after Cl- readmission compared with secretin alone (50 nM), indicating that ACh potentiates the stimulatory effect of secretin on the Cl-/HCO3- exchanger activity. This effect of ACh was blocked by the M3 ACh receptor antagonist, 4-diphenyl-acetoxy-N-(2-chloroethyl)-piperidine (40 nM), by the intracellular Ca2+ chelator, 1,2-bis (2-Aminophenoxy)- ethane-N,N,N', N'-tetraacetic acid acetoxymethylester (50 microM), but not by the protein kinase C antagonist, staurosporine (0.1 microM). Intracellular cAMP levels, in isolated rat cholangiocytes, were unaffected by ACh alone, but were markedly higher after exposure to secretin plus ACh compared with secretin alone (P < 0.01). The ACh-induced potentiation of the secretin effect on both intracellular cAMP levels and the Cl-/HCO3- exchanger activity was individually abolished by two calcineurin inhibitors, FK-506 and cyclosporin A (100 nM). CONCLUSIONS M3 ACh receptors are markedly and diffusively represented in rat cholangiocytes. ACh did not influence the basal activity of the Cl-/HCO3- exchanger, but enhanced the stimulation by secretin of this anion exchanger by a Ca2+-dependent, protein kinase C-insensitive pathway that potentiates the secretin stimulation of adenylyl cyclase. Calcineurin most likely mediates the cross-talk between the calcium and adenylyl cyclase pathways. Since secretin targets cholangiocytes during parasympathetic predominance, coordinated regulation of Cl-/HCO3- exchanger by secretin (cAMP) and ACh (Ca2+) could play a major role in the regulation of ductal bicarbonate excretion in bile just when the bicarbonate requirement in the intestine is maximal.
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Affiliation(s)
- D Alvaro
- Division of Gastroenterology, Department of Clinical Medicine, University of Rome, "La Sapienza," Viale dell'Universita' 37, 00185 Rome, Italy.
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Kwiatkowski AP, McGill JM. Electrolyte transport in biliary epithelia. THE JOURNAL OF LABORATORY AND CLINICAL MEDICINE 1997; 130:8-13. [PMID: 9242361 DOI: 10.1016/s0022-2143(97)90053-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Affiliation(s)
- A P Kwiatkowski
- Department of Medicine, Indiana University School of Medicine, and The Richard L. Roudebush Veterans Affairs Medical Center, Indianapolis 46202, USA
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Schlosser SF, Burgstahler AD, Nathanson MH. Isolated rat hepatocytes can signal to other hepatocytes and bile duct cells by release of nucleotides. Proc Natl Acad Sci U S A 1996; 93:9948-53. [PMID: 8790437 PMCID: PMC38535 DOI: 10.1073/pnas.93.18.9948] [Citation(s) in RCA: 177] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023] Open
Abstract
Intercellular communication among certain cell types can occur via ATP secretion, which leads to stimulation of nucleotide receptors on target cells. In epithelial cells, however, intercellular communication is thought to occur instead via gap junctions. Here we examined whether one epithelial cell type, hepatocytes, can also communicate via nucleotide secretion. The effects on cytosolic Ca2+ ([Ca2+]i) of mechanical stimulation, including microinjection, were examined in isolated rat hepatocytes and in isolated bile duct units using confocal fluorescence video microscopy. Mechanical stimulation of a single hepatocyte evoked an increase in [Ca2+]i in the stimulated cell plus an unexpected [Ca2+]i rise in neighboring noncontacting hepatocytes. Perifusion with ATP before mechanical stimulation suppressed the [Ca2+]i increase, but pretreatment with phenylephrine did not. The P2 receptor antagonist suramin inhibited these intercellular [Ca2+]i signals. The ATP/ADPase apyrase reversibly inhibited the [Ca2+]i rise induced by mechanical stimulation, and did not block vasopressin-induced [Ca2+]i signals. Mechanical stimulation of hepatocytes also induced a [Ca2+]i increase in cocultured isolated bile duct units, and this [Ca2+]i increase was inhibited by apyrase as well. Finally, this form of [Ca2+]i signaling could be elicited in the presence of propidium iodide without nuclear labeling by that dye, indicating that this phenomenon does not depend on disruption of the stimulated cell. Thus, mechanical stimulation of isolated hepatocytes, including by microinjection, can evoke [Ca2+]i signals in the stimulated cell as well as in neighboring noncontacting hepatocytes and bile duct epithelia. This signaling is mediated by release of ATP or other nucleotides into the extracellular space. This is an important technical consideration given the widespread use of microinjection techniques for examining mechanisms of signal transduction. Moreover, the evidence provided suggests a novel paracrine signaling pathway for epithelia, which previously were thought to communicate exclusively via gap junctions.
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Affiliation(s)
- S F Schlosser
- Liver Study Unit, Yale University School of Medicine, New Haven, CT 06520, USA
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