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Lim JJ, Klaassen CD, Cui JY. Deciphering the cell type-specific and zonal distribution of drug-metabolizing enzymes, transporters, and transcription factors in livers of mice using single-cell transcriptomics. Drug Metab Dispos 2025; 53:100029. [PMID: 39919554 DOI: 10.1016/j.dmd.2024.100029] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2024] [Accepted: 11/08/2024] [Indexed: 02/09/2025] Open
Abstract
The liver contains multiple cell types, including resident cell types and immune cells. The liver is also categorized into 3 zones: periportal (zone 1), midzonal (zone 2), and centrilobular (zone 3). The goal of this study was to characterize the distribution of drug-processing genes (DPGs) in mouse liver using published single-cell and nuclei transcriptomic datasets, which were subjected to zonal deconvolution. Filtering, normalization, clustering, and differential expression analyses were performed using Seurat V5 in R. Hepatocytes were assigned to 3 zones based on known zonal markers and validated with published spatial transcriptomics data. Among the 195 DPGs profiled, most were expressed highest in hepatocytes (61.3%). Interestingly, certain DPGs were expressed most highly in nonparenchymal cells, such as in cholangiocytes (11.2%, eg, carboxylesterase [Ces] 2e, Ces2g), endothelial cells (7.2%, eg, aldo-keto reductase [Akr] 1c19, Akr1e1), Kupffer cells (5.3%, eg, Akr1a1, Akr1b10), stellate cells (5.1%, eg, retinoic acid receptor [Rar] α, Rarβ), myofibroblasts (2.9%, RAR-related orphan receptor [Rar] α), and a few were expressed in immune cell types. In hepatocytes, 72.4% of phase-I enzymes were enriched in zone 3. Phase-II conjugation enzymes such as UDP-glucuronosyltransferases (75%) were enriched in zone 3, whereas sulfotransferases (40%) were enriched in zone 1. Hepatic xenobiotic transporters were enriched in zone 3. The xenobiotic biotransformation-regulating transcription factors were enriched in zone 3 hepatocytes. The enrichment of DPGs in liver cell types, including non-parenchymal cells and zone 1 hepatocytes, may serve as an additional repertoire for xenobiotic biotransformation. SIGNIFICANCE STATEMENT: Our study is among the first to systematically characterize the baseline mRNA enrichment of important drug-processing genes in different cell types and zones in the liver. This finding will aid in further understanding the mechanisms of chemical-induced liver injury with improved resolution and precision.
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Affiliation(s)
- Joe Jongpyo Lim
- Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington; Environmental Health and Microbiome Research Center (EHMBRACE), Seattle, Washington
| | - Curtis Dean Klaassen
- Department of Pharmacology, Toxicology, and Therapeutics, School of Medicine, University of Kansas, Kansas City, Kanas.
| | - Julia Yue Cui
- Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington; Environmental Health and Microbiome Research Center (EHMBRACE), Seattle, Washington.
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Zhou Z, Qian J, Kini A, Riederer B, Römermann D, Gros G, Seidler U. Loss of luminal carbonic anhydrase XIV results in decreased biliary bicarbonate output, liver fibrosis, and cholangiocyte proliferation in mice. Pflugers Arch 2022; 474:529-539. [PMID: 35119514 PMCID: PMC8993780 DOI: 10.1007/s00424-021-02659-3] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2021] [Revised: 12/22/2021] [Accepted: 12/23/2021] [Indexed: 12/12/2022]
Abstract
Carbonic anhydrase XIV (Car14) is highly expressed in the hepatocyte, with predominance in the canalicular membrane and its active site in the extracellular milieu. The aim of this study is to determine the physiological relevance of Car14 for biliary fluid and acid/base output, as well as its role in the maintenance of hepatocellular and cholangiocyte integrity. The common bile duct of anesthetized car14-/- and car14+/+ mice was cannulated and hepatic HCO3- output was measured by microtitration and bile flow gravimetrically before and during stimulation with intravenously applied tauroursodeoxycholic acid (TUDCA). Morphological alterations and hepatic damage were assessed histologically and immunohistochemically in liver tissue from 3- to 52-week-old car14-/- and car14+/+ mice, and gene and/or protein expression was measured for pro-inflammatory cytokines, fibrosis, and cholangiocyte markers. Biliary basal and more so TUDCA-stimulated HCO3- output were significantly reduced in car14-/- mice of all age groups, whereas bile flow and hepatic and ductular morphology were normal at young age. Car14-/- mice developed fibrotic and proliferative changes in the small bile ducts at advanced age, which was accompanied by a reduction in bile flow, and an upregulation of hepatic cytokeratin 19 mRNA and protein expression. Membrane-bound Car14 is essential for biliary HCO3- output, and its loss results in gradual development of small bile duct disease and hepatic fibrosis. Bile flow is not compromised in young adulthood, suggesting that Car14-deficient mice may be a model to study the protective role of biliary canalicular HCO3- against luminal noxi to the cholangiocyte.
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Affiliation(s)
- Zhenzhen Zhou
- Department of Gastroenterology, Hannover Medical School, Hannover, Germany
- Department of Gastroenterology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Jiajie Qian
- Department of Gastroenterology, Hannover Medical School, Hannover, Germany
- Department of Gastrointestinal Surgery, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China
| | - Archana Kini
- Department of Gastroenterology, Hannover Medical School, Hannover, Germany
| | - Brigitte Riederer
- Department of Gastroenterology, Hannover Medical School, Hannover, Germany
| | - Dorothee Römermann
- Department of Gastroenterology, Hannover Medical School, Hannover, Germany
| | - Gerolf Gros
- Department of Molecular and Cell Physiology, Hannover Medical School, Hannover, Germany
| | - Ursula Seidler
- Department of Gastroenterology, Hannover Medical School, Hannover, Germany.
- Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Carl Neuberg Straße 1, 30625, Hannover, Germany.
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Huynh MT, Nguyen TT, Grison S, Lascols O, Fernandez E, Barbu V. Clinical characteristics and genetic profiles of young and adult patients with cholestatic liver disease. REVISTA ESPANOLA DE ENFERMEDADES DIGESTIVAS 2020; 111:775-788. [PMID: 31538484 DOI: 10.17235/reed.2019.6168/2019] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
BACKGROUND AND AIMS heterozygous ABCB4, ABCB11 and ATP8B1 sequence variants were previously reported to be associated with low phospholipid-associated cholelithiasis, intrahepatic cholestasis of pregnancy, benign recurrent intrahepatic cholestasis and biliary lithiasis. The present study aimed to identify the presence of sequence variations in genes responsible for Mendelian liver disorders in patients with cholestatic liver disease. METHODS targeted massive parallel sequencing of a panel of genes involved in bile acid homeostasis was performed in 105 young and adult patients with cholestatic liver disease in our laboratory for molecular diagnosis. The effects of novel variants were evaluated using bioinformatics prediction tools and the Protter and Phyre2 software programs were used to create 2D, 3D topology protein modeling. Genotype-phenotype correlation was established according to molecular analysis and clinical records. RESULTS twenty novel heterozygous ABCB4 sequence variations, one heterozygous ABCB4 large intragenic deletion and only one novel missense variant in ABCB11 and ATP8B1 were identified. Interestingly, heterozygous and homozygous SLC4A2 missense variants were detected in patients with low phospholipid-associated cholelithiasis. Two patients harbored heterozygous GPBAR1 variants. Common variants such as homozygous ABCB11 p.Val444Ala and heterozygous ABCG8 p.Asp19His were also identified in 12 cases. CONCLUSIONS forty-eight variants were identified in five genes including ABCB4, ABCB11, ATP8B1, SLC4A2 and GPBAR1, twenty-five of which were novel. This study expands the phenotypic and mutational spectrum in genes involved in bile acid homeostasis and highlights the genetic and phenotypic heterogeneity in patients with inherited liver disorders.
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Affiliation(s)
| | - Truong-Tam Nguyen
- Service de Médecine Interne, Université de Médecine Pham Ngoc Thach, Viet Nam
| | - Sophie Grison
- Hôpitaux universitaires Est Parisien, hôpital Sain, Laboratoire commun de Biologie et Génétique Moléculaires, France
| | - Olivier Lascols
- Hôpitaux universitaires Est Parisien, hôpital Sain, Laboratoire commun de Biologie et Génétique Moléculaires, France
| | - Eric Fernandez
- Hôpitaux universitaires Est Parisien, hôpital Sain, Laboratoire commun de Biologie et Génétique Moléculaires, France
| | - Véronique Barbu
- Genetics Service, Laboratoire commun de Biologie et Génétique Moléculaires, Hôpitaux universitaires Est Parisien, hôpi, France
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Roma MG, Barosso IR, Miszczuk GS, Crocenzi FA, Pozzi EJS. Dynamic Localization of Hepatocellular Transporters: Role in Biliary Excretion and Impairment in Cholestasis. Curr Med Chem 2019; 26:1113-1154. [DOI: 10.2174/0929867325666171205153204] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2017] [Revised: 09/06/2017] [Accepted: 09/07/2017] [Indexed: 12/25/2022]
Abstract
Bile flow generation is driven by the vectorial transfer of osmotically active compounds from sinusoidal blood into a confined space, the bile canaliculus. Hence, localization of hepatocellular transporters relevant to bile formation is crucial for bile secretion. Hepatocellular transporters are localized either in the plasma membrane or in recycling endosomes, from where they can be relocated to the plasma membrane on demand, or endocytosed when the demand decreases. The balance between endocytic internalization/ exocytic targeting to/from this recycling compartment is therefore the main determinant of the hepatic capability to generate bile, and to dispose endo- and xenobiotics. Furthermore, the exacerbated endocytic internalization is a common pathomechanisms in both experimental and human cholestasis; this results in bile secretory failure and, eventually, posttranslational transporter downregulation by increased degradation. This review summarizes the proposed structural mechanisms accounting for this pathological condition (e.g., alteration of function, localization or expression of F-actin or F-actin/transporter cross-linking proteins, and switch to membrane microdomains where they can be readily endocytosed), and the mediators implicated (e.g., triggering of “cholestatic” signaling transduction pathways). Lastly, we discussed the efficacy to counteract the cholestatic failure induced by transporter internalization of a number of therapeutic experimental approaches based upon the use of compounds that trigger exocytic targetting of canalicular transporters (e.g., cAMP, tauroursodeoxycholate). This therapeutics may complement treatments aimed to transcriptionally improve transporter expression, by affording proper localization and membrane stability to the de novo synthesized transporters.
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Affiliation(s)
- Marcelo G. Roma
- Instituto de Fisiologia Experimental (IFISE) - Facultad de Ciencias Bioquimicas y Farmaceuticas (CONICET - U.N.R.), S2002LRL, Rosario, Argentina
| | - Ismael R. Barosso
- Instituto de Fisiologia Experimental (IFISE) - Facultad de Ciencias Bioquimicas y Farmaceuticas (CONICET - U.N.R.), S2002LRL, Rosario, Argentina
| | - Gisel S. Miszczuk
- Instituto de Fisiologia Experimental (IFISE) - Facultad de Ciencias Bioquimicas y Farmaceuticas (CONICET - U.N.R.), S2002LRL, Rosario, Argentina
| | - Fernando A. Crocenzi
- Instituto de Fisiologia Experimental (IFISE) - Facultad de Ciencias Bioquimicas y Farmaceuticas (CONICET - U.N.R.), S2002LRL, Rosario, Argentina
| | - Enrique J. Sánchez Pozzi
- Instituto de Fisiologia Experimental (IFISE) - Facultad de Ciencias Bioquimicas y Farmaceuticas (CONICET - U.N.R.), S2002LRL, Rosario, Argentina
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Brauner CJ, Shartau RB, Damsgaard C, Esbaugh AJ, Wilson RW, Grosell M. Acid-base physiology and CO2 homeostasis: Regulation and compensation in response to elevated environmental CO2. FISH PHYSIOLOGY 2019. [DOI: 10.1016/bs.fp.2019.08.003] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
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Parker MD. Mouse models of SLC4-linked disorders of HCO 3--transporter dysfunction. Am J Physiol Cell Physiol 2018; 314:C569-C588. [PMID: 29384695 DOI: 10.1152/ajpcell.00301.2017] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
The SLC4 family Cl-/[Formula: see text] cotransporters (NBCe1, NBCe2, NBCn1, and NBCn2) contribute to a variety of vital physiological processes including pH regulation and epithelial fluid secretion. Accordingly, their dysfunction can have devastating effects. Disorders such as epilepsy, hemolytic anemia, glaucoma, hearing loss, osteopetrosis, and renal tubular acidosis are all genetically linked to SLC4-family gene loci. This review summarizes how studies of Slc4-modified mice have enhanced our understanding of the etiology of SLC4-linked pathologies and the interpretation of genetic linkage studies. The review also surveys the novel disease signs exhibited by Slc4-modified mice which could either be considered to presage their description in humans, or to highlight interspecific differences. Finally, novel Slc4-modified mouse models are proposed, the study of which may further our understanding of the basis and treatment of SLC4-linked disorders of [Formula: see text]-transporter dysfunction.
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Affiliation(s)
- Mark D Parker
- Department of Physiology and Biophysics, The State University of New York: The University at Buffalo , Buffalo, New York.,Department of Ophthalmology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo: The State University of New York , Buffalo, New York.,State University of New York Eye Institutes, University at Buffalo: The State University of New York , Buffalo, New York
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Heuer RM, Grosell M. Physiological impacts of elevated carbon dioxide and ocean acidification on fish. Am J Physiol Regul Integr Comp Physiol 2014; 307:R1061-84. [DOI: 10.1152/ajpregu.00064.2014] [Citation(s) in RCA: 258] [Impact Index Per Article: 23.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
Most fish studied to date efficiently compensate for a hypercapnic acid-base disturbance; however, many recent studies examining the effects of ocean acidification on fish have documented impacts at CO2 levels predicted to occur before the end of this century. Notable impacts on neurosensory and behavioral endpoints, otolith growth, mitochondrial function, and metabolic rate demonstrate an unexpected sensitivity to current-day and near-future CO2 levels. Most explanations for these effects seem to center on increases in Pco2 and HCO3− that occur in the body during pH compensation for acid-base balance; however, few studies have measured these parameters at environmentally relevant CO2 levels or directly related them to reported negative endpoints. This compensatory response is well documented, but noted variation in dynamic regulation of acid-base transport pathways across species, exposure levels, and exposure duration suggests that multiple strategies may be utilized to cope with hypercapnia. Understanding this regulation and changes in ion gradients in extracellular and intracellular compartments during CO2 exposure could provide a basis for predicting sensitivity and explaining interspecies variation. Based on analysis of the existing literature, the present review presents a clear message that ocean acidification may cause significant effects on fish across multiple physiological systems, suggesting that pH compensation does not necessarily confer tolerance as downstream consequences and tradeoffs occur. It remains difficult to assess if acclimation responses during abrupt CO2 exposures will translate to fitness impacts over longer timescales. Nonetheless, identifying mechanisms and processes that may be subject to selective pressure could be one of many important components of assessing adaptive capacity.
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Affiliation(s)
- Rachael M. Heuer
- Rosenstiel School of Marine and Atmospheric Science, University of Miami, Marine Biology and Fisheries, Miami, Florida
| | - Martin Grosell
- Rosenstiel School of Marine and Atmospheric Science, University of Miami, Marine Biology and Fisheries, Miami, Florida
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8
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Concepcion AR, Salas JT, Sarvide S, Sáez E, Ferrer A, López M, Portu A, Banales JM, Hervás-Stubbs S, Oude Elferink RPJ, Prieto J, Medina JF. Anion exchanger 2 is critical for CD8(+) T cells to maintain pHi homeostasis and modulate immune responses. Eur J Immunol 2014; 44:1341-51. [PMID: 24515893 DOI: 10.1002/eji.201344218] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2013] [Revised: 12/24/2013] [Accepted: 02/04/2014] [Indexed: 01/11/2023]
Abstract
Mitogenic stimulation of lymphocytes involves alkalinization of intracellular pH (pHi ). Subsequent pHi regulation may involve HCO3 (-) extrusion through Cl(-) /HCO3 (-) exchangers and/or Na(+) -HCO3 (-) co-transporters with acid-loading capability. Abnormalities in these mechanisms could result in immune dysfunctions, as suggested by the CD8(+) T-cell expansion encountered in mice lacking Ae2 (a widely expressed acid loader with electroneutral and Na(+) -independent Cl(-) /HCO3 (-) anion-exchange activity). Here we report that CD8(+) T cells but not CD4(+) T cells or other lymphocyte populations, are crucially dependent on Ae2 for pHi regulation. While total lymphocytes (including isolated CD4(+) T cells) exhibit Ae1 expression and Na(+) -HCO3 (-) co-transport with acidifying potential, CD8(+) T cells lack these acid-loading mechanisms. In Ae2-KO mice, CD4(+) but not CD8(+) T cells upregulate these potential Ae2 surrogates. As a consequence, Ae2-KO CD8(+) T cells exhibit alkalinized pHi , and dramatically increase their pHi upon CD3 stimulation. Moreover, stimulated Ae2-deficient CD8(+) T cells show enhanced intracellular production of IL-2 and membrane expression of its receptor IL-2Rα, together with increased cell proliferation and activation. These findings demonstrate that CD8(+) T cells are critically dependent on Ae2 for pHi homeostasis and tuning of cell proliferation and activation. Ae2 thus constitutes a novel target to modulate CD8(+) T-cell responses.
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Affiliation(s)
- Axel R Concepcion
- Center for Applied Medical Research (CIMA), Clinic and School of Medicine University of Navarra, Pamplona, Spain; CIBERehd, the "Carlos III" Institute of Health, Madrid, Spain
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Concepcion AR, Lopez M, Ardura-Fabregat A, Medina JF. Role of AE2 for pHi regulation in biliary epithelial cells. Front Physiol 2014; 4:413. [PMID: 24478713 PMCID: PMC3894451 DOI: 10.3389/fphys.2013.00413] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2013] [Accepted: 12/27/2013] [Indexed: 12/31/2022] Open
Abstract
The Cl−/HCO−3anion exchanger 2 (AE2) is known to be involved in intracellular pH (pHi) regulation and transepithelial acid-base transport. Early studies showed that AE2 gene expression is reduced in liver biopsies and blood mononuclear cells from patients with primary biliary cirrhosis (PBC), a disease characterized by chronic non-suppurative cholangitis associated with antimitochondrial antibodies (AMA) and other autoimmune phenomena. Microfluorimetric analysis of the Cl−/HCO−3 anion exchange (AE) in isolated cholangiocytes showed that the cAMP-stimulated AE activity is diminished in PBC compared to both healthy and diseased controls. More recently, it was found that miR-506 is upregulated in cholangiocytes of PBC patients and that AE2 may be a target of miR-506. Additional evidence for a pathogenic role of AE2 dysregulation in PBC was obtained with Ae2−/−a,b mice, which develop biochemical, histological, and immunologic alterations that resemble PBC (including development of serum AMA). Analysis of HCO−3 transport systems and pHi regulation in cholangiocytes from normal and Ae2−/−a,b mice confirmed that AE2 is the transporter responsible for the Cl−/HCO−3exchange in these cells. On the other hand, both Ae2+/+a,b and Ae2−/−a,b mouse cholangiocytes exhibited a Cl−-independent bicarbonate transport system, essentially a Na+-bicarbonate cotransport (NBC) system, which could contribute to pHi regulation in the absence of AE2.
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Affiliation(s)
- Axel R Concepcion
- Division of Gene Therapy and Hepatology, Center for Applied Medical Research (CIMA), School of Medicine, University of Navarra, and Ciberehd Pamplona, Spain
| | - María Lopez
- Division of Gene Therapy and Hepatology, Center for Applied Medical Research (CIMA), School of Medicine, University of Navarra, and Ciberehd Pamplona, Spain
| | - Alberto Ardura-Fabregat
- Division of Gene Therapy and Hepatology, Center for Applied Medical Research (CIMA), School of Medicine, University of Navarra, and Ciberehd Pamplona, Spain
| | - Juan F Medina
- Division of Gene Therapy and Hepatology, Center for Applied Medical Research (CIMA), School of Medicine, University of Navarra, and Ciberehd Pamplona, Spain
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Reshetnyak VI. Physiological and molecular biochemical mechanisms of bile formation. World J Gastroenterol 2013; 19:7341-7360. [PMID: 24259965 PMCID: PMC3831216 DOI: 10.3748/wjg.v19.i42.7341] [Citation(s) in RCA: 60] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/28/2013] [Revised: 09/27/2013] [Accepted: 09/29/2013] [Indexed: 02/06/2023] Open
Abstract
This review considers the physiological and molecular biochemical mechanisms of bile formation. The composition of bile and structure of a bile canaliculus, biosynthesis and conjugation of bile acids, bile phospholipids, formation of bile micellar structures, and enterohepatic circulation of bile acids are described. In general, the review focuses on the molecular physiology of the transporting systems of the hepatocyte sinusoidal and apical membranes. Knowledge of physiological and biochemical basis of bile formation has implications for understanding the mechanisms of development of pathological processes, associated with diseases of the liver and biliary tract.
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Parker MD, Boron WF. The divergence, actions, roles, and relatives of sodium-coupled bicarbonate transporters. Physiol Rev 2013; 93:803-959. [PMID: 23589833 PMCID: PMC3768104 DOI: 10.1152/physrev.00023.2012] [Citation(s) in RCA: 208] [Impact Index Per Article: 17.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
The mammalian Slc4 (Solute carrier 4) family of transporters is a functionally diverse group of 10 multi-spanning membrane proteins that includes three Cl-HCO3 exchangers (AE1-3), five Na(+)-coupled HCO3(-) transporters (NCBTs), and two other unusual members (AE4, BTR1). In this review, we mainly focus on the five mammalian NCBTs-NBCe1, NBCe2, NBCn1, NDCBE, and NBCn2. Each plays a specialized role in maintaining intracellular pH and, by contributing to the movement of HCO3(-) across epithelia, in maintaining whole-body pH and otherwise contributing to epithelial transport. Disruptions involving NCBT genes are linked to blindness, deafness, proximal renal tubular acidosis, mental retardation, and epilepsy. We also review AE1-3, AE4, and BTR1, addressing their relevance to the study of NCBTs. This review draws together recent advances in our understanding of the phylogenetic origins and physiological relevance of NCBTs and their progenitors. Underlying these advances is progress in such diverse disciplines as physiology, molecular biology, genetics, immunocytochemistry, proteomics, and structural biology. This review highlights the key similarities and differences between individual NCBTs and the genes that encode them and also clarifies the sometimes confusing NCBT nomenclature.
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Affiliation(s)
- Mark D Parker
- Dept. of Physiology and Biophysics, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106-4970, USA.
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12
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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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13
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Barosso IR, Zucchetti AE, Boaglio AC, Larocca MC, Taborda DR, Luquita MG, Roma MG, Crocenzi FA, Sánchez Pozzi EJ. Sequential activation of classic PKC and estrogen receptor α is involved in estradiol 17ß-D-glucuronide-induced cholestasis. PLoS One 2012; 7:e50711. [PMID: 23209816 PMCID: PMC3507741 DOI: 10.1371/journal.pone.0050711] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2012] [Accepted: 10/25/2012] [Indexed: 12/24/2022] Open
Abstract
Estradiol 17ß-d-glucuronide (E17G) induces acute cholestasis in rat with endocytic internalization of the canalicular transporters bile salt export pump (Abcb11) and multidrug resistance-associated protein 2 (Abcc2). Classical protein kinase C (cPKC) and PI3K pathways play complementary roles in E17G cholestasis. Since non-conjugated estradiol is capable of activating these pathways via estrogen receptor alpha (ERα), we assessed the participation of this receptor in the cholestatic manifestations of estradiol glucuronidated-metabolite E17G in perfused rat liver (PRL) and in isolated rat hepatocyte couplets (IRHC). In both models, E17G activated ERα. In PRL, E17G maximally decreased bile flow, and the excretions of dinitrophenyl-glutathione, and taurocholate (Abcc2 and Abcb11 substrates, respectively) by 60% approximately; preadministration of ICI 182,780 (ICI, ERα inhibitor) almost totally prevented these decreases. In IRHC, E17G decreased the canalicular vacuolar accumulation of cholyl-glycylamido-fluorescein (Abcb11 substrate) with an IC50 of 91±1 µM. ICI increased the IC50 to 184±1 µM, and similarly prevented the decrease in the canalicular vacuolar accumulation of the Abcc2 substrate, glutathione-methylfluorescein. ICI also completely prevented E17G-induced delocalization of Abcb11 and Abcc2 from the canalicular membrane, both in PRL and IRHC. The role of ERα in canalicular transporter internalization induced by E17G was confirmed in ERα-knocked-down hepatocytes cultured in collagen sandwich. In IRHC, the protection of ICI was additive to that produced by PI3K inhibitor wortmannin but not with that produced by cPKC inhibitor Gö6976, suggesting that ERα shared the signaling pathway of cPKC but not that of PI3K. Further analysis of ERα and cPKC activations induced by E17G, demonstrated that ICI did not affect cPKC activation whereas Gö6976 prevented that of ERα, indicating that cPKC activation precedes that of ERα. Conclusion: ERα is involved in the biliary secretory failure induced by E17G and its activation follows that of cPKC.
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Affiliation(s)
| | | | | | | | | | | | | | | | - Enrique J. Sánchez Pozzi
- Instituto de Fisiología Experimental (IFISE), Facultad de Ciencias Bioquímicas y Farmacéuticas (CONICET – U.N.R.), Rosario, Argentina
- * E-mail:
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Munoz-Garrido P, Fernandez-Barrena MG, Hijona E, Carracedo M, Marín JJG, Bujanda L, Banales JM. MicroRNAs in biliary diseases. World J Gastroenterol 2012; 18:6189-6196. [PMID: 23180938 PMCID: PMC3501766 DOI: 10.3748/wjg.v18.i43.6189] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/10/2012] [Revised: 07/05/2012] [Accepted: 08/14/2012] [Indexed: 02/06/2023] Open
Abstract
Cholangiopathies are a group of diseases primarily or secondarily affecting bile duct cells, and result in cholangiocyte proliferation, regression, and/or transformation. Their etiopathogenesis may be associated with a broad variety of causes of different nature, which includes genetic, neoplastic, immune-associated, infectious, vascular, and drug-induced alterations, or being idiopathic. miRNAs, small non-coding endogenous RNAs that post-transcriptionally regulate gene expression, have been associated with pathophysiological processes in different organs and cell types, and are postulated as potential targets for diagnosis and therapy. In the current manuscript, knowledge regarding the role of miRNAs in the development and/or progression of cholangiopathies has been reviewed and the most relevant findings in this promising field of hepatology have been highlighted.
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Huang J, Shan J, Kim D, Liao J, Evagelidis A, Alper SL, Hanrahan JW. Basolateral chloride loading by the anion exchanger type 2: role in fluid secretion by the human airway epithelial cell line Calu-3. J Physiol 2012; 590:5299-316. [PMID: 22802585 DOI: 10.1113/jphysiol.2012.236919] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Anion exchanger type 2 (AE2 or SLC4A2) is an electroneutral Cl(-)/HCO(3)(-) exchanger expressed at the basolateral membrane of many epithelia. It is thought to participate in fluid secretion by airway epithelia. However, the role of AE2 in fluid secretion remains uncertain, due to the lack of specific pharmacological inhibitors, and because it is electrically silent and therefore does not contribute directly to short-circuit current (I(sc)). We have studied the role of AE2 in Cl(-) and fluid secretion by the airway epithelial cell line Calu-3. After confirming expression of its mRNA and protein, a knock-down cell line called AE2-KD was generated by lentivirus-mediated RNA interference in which AE2 mRNA and protein levels were reduced 90%. Suppressing AE2 increased the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) by ∼70% without affecting the levels of NKCC1 (Na(+)-K(+)-2Cl(-) cotransporter) or NBCe1 (Na(+)-nHCO(3)(-) cotransporter). cAMP agonists stimulated fluid secretion by parental Calu-3 and scrambled shRNA cells >6.5-fold. In AE2-KD cells this response was reduced by ∼70%, and the secreted fluid exhibited elevated pH and [HCO(3)(-)] as compared with the control lines. Unstimulated equivalent short-circuit current (I(eq)) was elevated in AE2-KD cells, but the incremental response to forskolin was unaffected. The modest bumetanide-induced reductions in both I(eq) and fluid secretion were more pronounced in AE2-KD cells. Basolateral Cl(-)/HCO(3)(-) exchange measured by basolateral pH-stat in cells with permeabilized apical membranes was abolished in AE2-KD monolayers, and the intracellular alkalinization resulting from basolateral Cl(-) removal was reduced by ∼80% in AE2-KD cells. These results identify AE2 as a major pathway for basolateral Cl(-) loading during cAMP-stimulated secretion of Cl(-) and fluid by Calu-3 cells, and help explain the large bumetanide-insensitive component of fluid secretion reported previously in airway submucosal glands and some other epithelia.
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Affiliation(s)
- Junwei Huang
- Department of Physiology, McGill University, Montr´eal, QC, Canada
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16
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Baghdasaryan A, Claudel T, Gumhold J, Silbert D, Adorini L, Roda A, Vecchiotti S, Gonzalez FJ, Schoonjans K, Strazzabosco M, Fickert P, Trauner M. Dual farnesoid X receptor/TGR5 agonist INT-767 reduces liver injury in the Mdr2-/- (Abcb4-/-) mouse cholangiopathy model by promoting biliary HCO⁻₃ output. Hepatology 2011; 54:1303-12. [PMID: 22006858 PMCID: PMC3744065 DOI: 10.1002/hep.24537] [Citation(s) in RCA: 170] [Impact Index Per Article: 12.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
UNLABELLED Chronic cholangiopathies have limited therapeutic options and represent an important indication for liver transplantation. The nuclear farnesoid X receptor (FXR) and the membrane G protein-coupled receptor, TGR5, regulate bile acid (BA) homeostasis and inflammation. Therefore, we hypothesized that activation of FXR and/or TGR5 could ameliorate liver injury in Mdr2(-/-) (Abcb4(-/-)) mice, a model of chronic cholangiopathy. Hepatic inflammation, fibrosis, as well as bile secretion and key genes of BA homeostasis were addressed in Mdr2(-/-) mice fed either a chow diet or a diet supplemented with the FXR agonist, INT-747, the TGR5 agonist, INT-777, or the dual FXR/TGR5 agonist, INT-767 (0.03% w/w). Only the dual FXR/TGR5 agonist, INT-767, significantly improved serum liver enzymes, hepatic inflammation, and biliary fibrosis in Mdr2(-/-) mice, whereas INT-747 and INT-777 had no hepatoprotective effects. In line with this, INT-767 significantly induced bile flow and biliary HCO 3- output, as well as gene expression of carbonic anhydrase 14, an important enzyme able to enhance HCO 3- transport, in an Fxr-dependent manner. In addition, INT-767 dramatically reduced bile acid synthesis via the induction of ileal Fgf15 and hepatic Shp gene expression, thus resulting in significantly reduced biliary bile acid output in Mdr2(-/-) mice. CONCLUSION This study shows that FXR activation improves liver injury in a mouse model of chronic cholangiopathy by reduction of biliary BA output and promotion of HCO 3--rich bile secretion.
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Affiliation(s)
- Anna Baghdasaryan
- Laboratory of Experimental and Molecular Hepatology, Division of Gastroenterology and Hepatology, Department of Internal Medicine, Medical University of Graz, Graz, Austria
| | - Thierry Claudel
- Laboratory of Experimental and Molecular Hepatology, Division of Gastroenterology and Hepatology, Department of Internal Medicine, Medical University of Graz, Graz, Austria
- Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Medical University of Vienna, Vienna, Austria
| | - Judith Gumhold
- Laboratory of Experimental and Molecular Hepatology, Division of Gastroenterology and Hepatology, Department of Internal Medicine, Medical University of Graz, Graz, Austria
| | - Dagmar Silbert
- Laboratory of Experimental and Molecular Hepatology, Division of Gastroenterology and Hepatology, Department of Internal Medicine, Medical University of Graz, Graz, Austria
| | | | - Aldo Roda
- Laboratory of Bioanalytical and Analytical Chemistry, Department of Pharmaceutical Sciences, Alma Mater Studiorum, University of Bologna, Bologna, Italy
| | - Stefania Vecchiotti
- Laboratory of Bioanalytical and Analytical Chemistry, Department of Pharmaceutical Sciences, Alma Mater Studiorum, University of Bologna, Bologna, Italy
| | - Frank J. Gonzalez
- Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, MD
| | - Kristina Schoonjans
- Laboratory of Integrative and Systems Physiology, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
| | - Mario Strazzabosco
- Department of Internal Medicine, Section of Digestive Diseases, Yale University School of Medicine, New Haven, CT
- Department of Clinical Medicine and Prevention, University of Milan-Bicocca, Milan, Milan, Italy
| | - Peter Fickert
- Laboratory of Experimental and Molecular Hepatology, Division of Gastroenterology and Hepatology, Department of Internal Medicine, Medical University of Graz, Graz, Austria
| | - Michael Trauner
- Laboratory of Experimental and Molecular Hepatology, Division of Gastroenterology and Hepatology, Department of Internal Medicine, Medical University of Graz, Graz, Austria
- Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Medical University of Vienna, Vienna, Austria
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17
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Wendum D. [Liver disease associated with hereditary defects of hepatobiliary transporters]. Ann Pathol 2010; 30:426-31. [PMID: 21167428 DOI: 10.1016/j.annpat.2010.08.025] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2010] [Revised: 07/26/2010] [Accepted: 08/28/2010] [Indexed: 12/29/2022]
Abstract
The identification of biliary tranporters has enhanced our understanding of bile formation and some liver diseases. In this review, we first describe the main hepatobiliary transporters and their function. Then, some liver diseases related to mutations of biliary tranporters (FIC1/ATP8B1, BSEP/ABCB11, MDR3 /ABCB4 and MRP2/ABCC2) will be described with a focus on the pathological aspects. These diseases include progressive familial intrahepatic cholestasis (PFIC), benign recurrent intrahepatic cholestasis (BRIC), intrahepatic cholestasis of pregnancy, Dubin-Johnson's syndrome and low phospholipid associated cholelithiasis (LPAC).
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18
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Bronckers ALJJ, Lyaruu DM, Jansen IDC, Medina JF, Kellokumpu S, Hoeben KA, Gawenis LR, Oude-Elferink RPJ, Everts V. Localization and function of the anion exchanger Ae2 in developing teeth and orofacial bone in rodents. JOURNAL OF EXPERIMENTAL ZOOLOGY PART B-MOLECULAR AND DEVELOPMENTAL EVOLUTION 2009; 312B:375-87. [PMID: 19206174 DOI: 10.1002/jez.b.21267] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
To explore the functions of the anion exchanger 2 (Ae2) in the development of bones and teeth we examined the distribution of Ae2 in cells involved in the formation of teeth and surrounding bone in young hamsters, mice and rats. In all three species strongest immunostaining for Ae2 was obtained in basolateral membranes of maturation ameloblasts and in osteoclasts resorbing bone. In hamsters a weaker staining was also seen in the Golgi apparatus of secretory ameloblasts, young osteoblasts and osteocytes, odontoblasts and fibroblasts of the forming periodontal ligament. In adult Ae2(a,b) (-/-) mice, in which Ae2-targeted disruption precluded the expression of Ae2a, Ae2b1 and Ae2b2 isoforms, the immunostaining for Ae2 in ameloblasts and osteoclasts was totally abolished. The enamel formation was abnormal but teeth erupted, osteoclasts in jaw bone were functional and structure of dentin and bone was normal. In another mouse model, Ae2(-/-) mice in which the expression of all five Ae2 isoforms was disrupted, teeth failed to erupt and the alveolar bone proved poorly formed with giant but apparently functional osteoclasts. Our data indicate that basolaterally located Ae2a, Ae2b1 or Ae2b2 (or a combination of these) is present in maturation ameloblasts critical for the cells' normal functioning. Although isoforms of Ae2 were also present in basolateral membranes of osteoclasts, they proved to be not critical to osteoclast resorption of orofacial bone. Poorly formed bone and the failure of teeth to erupt seen in the Ae2(-/-) mice with gene disruption affecting all isoforms may result from secondary (systemic) changes that are different from Ae2(a,b) (-/-) mice.
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Affiliation(s)
- Antonius L J J Bronckers
- Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), MOVE Research Institute, University of Amsterdam and VU University Amsterdam, Amsterdam, The Netherlands.
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19
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Jansen IDC, Mardones P, Lecanda F, de Vries TJ, Recalde S, Hoeben KA, Schoenmaker T, Ravesloot JH, van Borren MMGJ, van Eijden TM, Bronckers ALJJ, Kellokumpu S, Medina JF, Everts V, Oude Elferink RPJ. Ae2(a,b)-deficient mice exhibit osteopetrosis of long bones but not of calvaria. FASEB J 2009; 23:3470-81. [PMID: 19564250 DOI: 10.1096/fj.08-122598] [Citation(s) in RCA: 52] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Extracellular acidification by osteoclasts is essential to bone resorption. During proton pumping, intracellular pH (pH(i)) is thought to be kept at a near-neutral level by chloride/bicarbonate exchange. Here we show that the Na(+)-independent chloride/bicarbonate anion exchanger 2 (Ae2) is relevant for this process in the osteoclasts from the long bones of Ae2(a,b)(-/-) mice (deficient in the main isoforms Ae2a, Ae2b(1), and Ae2b(2)). Although the long bones of these mice had normal numbers of multinucleated osteoclasts, these cells lacked a ruffled border and displayed impaired bone resorption activity, resulting in an osteopetrotic phenotype of long bones. Moreover, in vitro osteoclastogenesis assays using long-bone marrow cells from Ae2(a,b)(-/-) mice suggested a role for Ae2 in osteoclast formation, as fusion of preosteoclasts for the generation of active multinucleated osteoclasts was found to be slightly delayed. In contrast to the abnormalities observed in the long bones, the skull of Ae2(a,b)(-/-) mice showed no alterations, indicating that calvaria osteoclasts may display normal resorptive activity. Microfluorimetric analysis of osteoclasts from normal mice showed that, in addition to Ae2 activity, calvaria osteoclasts--but not long-bone osteoclasts--possess a sodium-dependent bicarbonate transporting activity. Possibly, this might compensate for the absence of Ae2 in calvaria osteoclasts of Ae2(a,b)(-/-) mice.
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Affiliation(s)
- Ineke D C Jansen
- Department of Periodontology, Academic Centre for Dentistry Amsterdam (ACTA), VU University Amsterdam, van der Boechorststraat 7, Amsterdam, Netherlands 1081 BT.
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20
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Alper SL. Molecular physiology and genetics of Na+-independent SLC4 anion exchangers. J Exp Biol 2009; 212:1672-83. [PMID: 19448077 PMCID: PMC2683012 DOI: 10.1242/jeb.029454] [Citation(s) in RCA: 170] [Impact Index Per Article: 10.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/12/2009] [Indexed: 01/12/2023]
Abstract
Plasmalemmal Cl(-)/HCO(3)(-) exchangers are encoded by the SLC4 and SLC26 gene superfamilies, and function to regulate intracellular pH, [Cl(-)] and cell volume. The Cl(-)/HCO(3)(-) exchangers of polarized epithelial cells also contribute to transepithelial secretion and reabsorption of acid-base equivalents and Cl(-). This review focuses on Na(+)-independent electroneutral Cl(-)/HCO(3)(-) exchangers of the SLC4 family. Human SLC4A1/AE1 mutations cause the familial erythroid disorders of spherocytic anemia, stomatocytic anemia and ovalocytosis. A largely discrete set of AE1 mutations causes familial distal renal tubular acidosis. The Slc4a2/Ae2(-/-) mouse dies before weaning with achlorhydria and osteopetrosis. A hypomorphic Ae2(-/-) mouse survives to exhibit male infertility with defective spermatogenesis and a syndrome resembling primary biliary cirrhosis. A human SLC4A3/AE3 polymorphism is associated with seizure disorder, and the Ae3(-/-) mouse has increased seizure susceptibility. The transport mechanism of mammalian SLC4/AE polypeptides is that of electroneutral Cl(-)/anion exchange, but trout erythroid Ae1 also mediates Cl(-) conductance. Erythroid Ae1 may mediate the DIDS-sensitive Cl(-) conductance of mammalian erythrocytes, and, with a single missense mutation, can mediate electrogenic SO(4)(2-)/Cl(-) exchange. AE1 trafficking in polarized cells is regulated by phosphorylation and by interaction with other proteins. AE2 exhibits isoform-specific patterns of acute inhibition by acidic intracellular pH and independently by acidic extracellular pH. In contrast, AE2 is activated by hypertonicity and, in a pH-independent manner, by ammonium and by hypertonicity. A growing body of structure-function and interaction data, together with emerging information about physiological function and structure, is advancing our understanding of SLC4 anion exchangers.
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Affiliation(s)
- Seth L Alper
- Renal Division and Molecular and Vascular Medicine Unit, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
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21
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Roma MG, Crocenzi FA, Mottino AD. Dynamic localization of hepatocellular transporters in health and disease. World J Gastroenterol 2008; 14:6786-801. [PMID: 19058304 PMCID: PMC2773873 DOI: 10.3748/wjg.14.6786] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Vesicle-based trafficking of hepatocellular transporters involves delivery of the newly-synthesized carriers from the rough endoplasmic reticulum to either the plasma membrane domain or to an endosomal, submembrane compartment, followed by exocytic targeting to the plasma membrane. Once delivered to the plasma membrane, the transporters usually undergo recycling between the plasma membrane and the endosomal compartment, which usually serves as a reservoir of pre-existing transporters available on demand. The balance between exocytic targeting and endocytic internalization from/to this recycling compartment is therefore a chief determinant of the overall capability of the liver epithelium to secrete bile and to detoxify endo and xenobiotics. Hence, it is a highly regulated process. Impaired regulation of this balance may lead to abnormal localization of these transporters, which results in bile secretory failure due to endocytic internalization of key transporters involved in bile formation. This occurs in several experimental models of hepatocellular cholestasis, and in most human cholestatic liver diseases. This review describes the molecular bases involved in the biology of the dynamic localization of hepatocellular transporters and its regulation, with a focus on the involvement of signaling pathways in this process. Their alterations in different experimental models of cholestasis and in human cholestatic liver disease are reviewed. In addition, the causes explaining the pathological condition (e.g. disorganization of actin or actin-transporter linkers) and the mediators involved (e.g. activation of cholestatic signaling transduction pathways) are also discussed. Finally, several experimental therapeutic approaches based upon the administration of compounds known to stimulate exocytic insertion of canalicular transporters (e.g. cAMP, tauroursodeoxycholate) are described.
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22
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Banales JM, Masyuk TV, Bogert PS, Huang BQ, Gradilone SA, Lee SO, Stroope AJ, Masyuk AI, Medina JF, LaRusso NF. Hepatic cystogenesis is associated with abnormal expression and location of ion transporters and water channels in an animal model of autosomal recessive polycystic kidney disease. THE AMERICAN JOURNAL OF PATHOLOGY 2008; 173:1637-46. [PMID: 18988797 DOI: 10.2353/ajpath.2008.080125] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Polycystic kidney (PCK) rats are a spontaneous model of autosomal recessive polycystic kidney disease that exhibit cholangiocyte-derived liver cysts. We have previously reported that in normal cholangiocytes a subset of vesicles contain three proteins (ie, the water channel AQP1, the chloride channel CFTR, and the anion exchanger AE2) that account for ion-driven water transport. Thus, we hypothesized that altered expression and location of these functionally related proteins contribute to hepatic cystogenesis. We show here that under basal conditions and in response to secretin and hypotonicity, cysts from PCK rats expanded to a greater degree than cysts formed by normal bile ducts. Quantitative reverse transcriptase-polymerase chain reaction, immunoblot analysis, and confocal and immunoelectron microscopy all indicated increased expression of these three proteins in PCK cholangiocytes versus normal cholangiocytes. AQP1, CFTR, and AE2 were localized preferentially to the apical membrane in normal rats while overexpressed at the basolateral membrane in PCK rats. Exposure of the cholangiocyte basolateral membrane to CFTR inhibitors [5-nitro-2-(3-phenylpropylamino)-benzoic acid and CFTRinh172], or Cl(-)/HCO(3)(-) exchange inhibitors (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate and 4-acetamido-4'-isothiocyanato-2,2'-stilbenedisulfonic acid disodium salt hydrate) blocked secretin-stimulated fluid accumulation in PCK but not in normal cysts. Our data suggest that hepatic cystogenesis in autosomal recessive polycystic kidney disease may involve increased fluid accumulation because of overexpression and abnormal location of AQP1, CFTR, and AE2 in cystic cholangiocytes. Therapeutic interventions that block the activation of these proteins might inhibit cyst expansion in polycystic liver disease.
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Affiliation(s)
- Jesús M Banales
- Miles and Shirley Fiterman Center for Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA
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23
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Recalde S, Muruzábal F, Looije N, Kunne C, Burrell MA, Sáez E, Martínez-Ansó E, Salas JT, Mardones P, Prieto J, Medina JF, Elferink RPJO. Inefficient chronic activation of parietal cells in Ae2a,b(-/-) mice. THE AMERICAN JOURNAL OF PATHOLOGY 2006; 169:165-76. [PMID: 16816370 PMCID: PMC1698767 DOI: 10.2353/ajpath.2006.051096] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
In parietal cells, basolateral Ae2 Cl(-)/HCO(3)(-) exchanger (Slc4a2) appears to compensate for luminal H(+) pumping while providing Cl(-) for apical secretion. In mouse and rat, mRNA variants Ae2a, Ae2b1, Ae2b2, and Ae2c2 are all found in most tissues (although the latter at very low levels), whereas Ae2c1 is restricted to the stomach. We studied the acid secretory function of gastric mucosa in mice with targeted disruption of Ae2a, Ae2b1, and Ae2b2 (but not Ae2c) isoforms. In the oxyntic mucosa of Ae2(a,b)(-/-) mice, total Ae2 protein was nearly undetectable, indicating low gastric expression of the Ae2c isoforms. In Ae2(a,b)(-/-) mice basal acid secretion was normal, whereas carbachol/histamine-stimulated acid secretion was impaired by 70%. These animals showed increased serum gastrin levels and hyperplasia of G cells. Immunohistochemistry and electron microscopy revealed baseline activation of parietal cells with fusion of intracellular H(+)/K(+)-ATPase-containing vesicles with the apical membrane and degenerative changes (but not substantial apoptosis) in a subpopulation of these cells. Increased expression of proliferating cell nuclear antigen in the oxyntic glands suggested enhanced Ae2(a,b)(-/-) parietal cell turnover. These data reveal a critical role of Ae2a-Ae2b1-Ae2b2 isoforms in stimulated gastric acid secretion whereas residual Ae2c isoforms could account to a limited extent for basal acid secretion.
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Affiliation(s)
- Sergio Recalde
- Laboratory of Experimental Hepatology, Academic Medical Center Liver Center, Amsterdam, The Netherlands
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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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25
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Pushkin A, Kurtz I. SLC4 base (HCO3 -, CO3 2-) transporters: classification, function, structure, genetic diseases, and knockout models. Am J Physiol Renal Physiol 2006; 290:F580-99. [PMID: 16461757 DOI: 10.1152/ajprenal.00252.2005] [Citation(s) in RCA: 116] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
In prokaryotic and eukaryotic organisms, biochemical and physiological processes are sensitive to changes in H(+) activity. For these processes to function optimally, a variety of proteins have evolved that transport H(+)/base equivalents across cell and organelle membranes, thereby maintaining the pH of various intracellular and extracellular compartments within specific limits. The SLC4 family of base (HCO(3)(-), CO(3)(2(-))) transport proteins plays an essential role in mediating Na(+)- and/or Cl(-)-dependent base transport in various tissues and cell types in mammals. In addition to pH regulation, specific members of this family also contribute to vectorial transepithelial base transport in several organ systems including the kidney, pancreas, and eye. The importance of these transporters in mammalian cell biology is highlighted by the phenotypic abnormalities resulting from spontaneous SLC4 mutations in humans and targeted deletions in murine knockout models. This review focuses on recent advances in our understanding of the molecular organization and functional properties of SLC4 transporters and their role in disease.
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Affiliation(s)
- Alexander Pushkin
- Division of Nephrology, David Geffen School of Medicine at UCLA, University of California-Los Angeles, 10833 Le Conte Avenue, Rm. 7-155 Factor Bldg., Los Angeles, CA 90095, USA
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26
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Abstract
Plasmalemmal Cl- -HCO3- exchangers regulate intracellular pH and [Cl-] and cell volume. In polarized epithelial cells, they contribute also to transepithelial secretion and reabsorption of acid-base equivalents and of Cl-. Members of both the SLC4 and SLC26 mammalian gene families encode Na+-independent Cl- -HCO3- exchangers. Human SLC4A1/AE1 mutations cause either the erythroid disorders spherocytic haemolytic anaemia or ovalocytosis, or distal renal tubular acidosis. SLC4A2/AE2 knockout mice die at weaning. Human SLC4A3/AE3 polymorphisms have been associated with seizure disorder. Although mammalian SLC4/AE polypeptides mediate only electroneutral Cl- -anion exchange, trout erythroid AE1 also promotes osmolyte transport and increased anion conductance. Mouse AE1 is required for DIDS-sensitive erythroid Cl- conductance, but definitive evidence for mediation of Cl- conductance is lacking. However, a single missense mutation allows AE1 to mediate both electrogenic SO4(2-) -Cl- exchange or electroneutral, H+-independent SO4(2)- -SO4(2-) exchange. In the Xenopus oocyte, the AE1 C-terminal cytoplasmic tail residues reported to bind carbonic anhydrase II are dispensable for Cl- -Cl- exchange, but required for Cl- -HCO3- exchange. AE2 is acutely and independently inhibited by intracellular and extracellular H+, and this regulation requires integrity of the most highly conserved sequence of the AE2 N-terminal cytoplasmic domain. Individual missense mutations within this and adjacent regions identify additional residues which acid-shift pHo sensitivity. These regions together are modelled to form contiguous surface patches on the AE2 cytoplasmic domain. In contrast, the N-terminal variant AE2c polypeptide exhibits an alkaline-shifted pHo sensitivity, as do certain transmembrane domain His mutants. AE2-mediated anion exchange is also stimulated by ammonium and by hypertonicity by a mechanism sensitive to inhibition by chelation of intracellular Ca2+ and by calmidazolium. This growing body of structure-function data, together with increased structural information, will advance mechanistic understanding of SLC4 anion exchangers.
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MESH Headings
- Acidosis, Renal Tubular/genetics
- Acidosis, Renal Tubular/metabolism
- Amino Acid Sequence
- Animals
- Anion Exchange Protein 1, Erythrocyte/chemistry
- Anion Exchange Protein 1, Erythrocyte/genetics
- Anion Exchange Protein 1, Erythrocyte/metabolism
- Anion Transport Proteins/chemistry
- Anion Transport Proteins/genetics
- Anion Transport Proteins/metabolism
- Antiporters/chemistry
- Antiporters/genetics
- Antiporters/metabolism
- Carbonic Anhydrases/metabolism
- Chloride-Bicarbonate Antiporters/chemistry
- Chloride-Bicarbonate Antiporters/genetics
- Chloride-Bicarbonate Antiporters/metabolism
- Elliptocytosis, Hereditary/genetics
- Elliptocytosis, Hereditary/metabolism
- Humans
- Hydrogen-Ion Concentration
- Molecular Sequence Data
- Multigene Family
- Mutation
- Polymorphism, Genetic
- Protein Conformation
- SLC4A Proteins
- Spherocytosis, Hereditary/genetics
- Spherocytosis, Hereditary/metabolism
- Structure-Activity Relationship
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Affiliation(s)
- Seth L Alper
- Molecular and Vascular Medicine Unit, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.
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27
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Shmukler BE, Kurschat CE, Ackermann GE, Jiang L, Zhou Y, Barut B, Stuart-Tilley AK, Zhao J, Zon LI, Drummond IA, Vandorpe DH, Paw BH, Alper SL. Zebrafish slc4a2/ae2 anion exchanger: cDNA cloning, mapping, functional characterization, and localization. Am J Physiol Renal Physiol 2005; 289:F835-49. [PMID: 15914778 DOI: 10.1152/ajprenal.00122.2005] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
Although the zebrafish has been used increasingly for the study of pronephric kidney development, studies of renal ion transporters and channels of the zebrafish remain few. We report the cDNA cloning and characterization of the AE2 anion exchanger ortholog from zebrafish kidney, slc4a2/ae2. The ae2 gene in linkage group 2 encodes a polypeptide of 1,228 aa exhibiting 64% aa identity with mouse AE2a. The exon-intron boundaries of the zebrafish ae2 gene are nearly identical to those of the rodent and human genes. Whole-mount in situ hybridization detects ae2 mRNA in prospective midbrain as early as the five-somite stage, then later in the pronephric primordia and the forming pronephric duct, where it persists through 72 h postfertilization (hpf). Zebrafish Ae2 expressed in Xenopus laevis oocytes mediates Na(+)-independent, electroneutral (36)Cl(-)/Cl(-) exchange moderately sensitive to inhibition by DIDS, is inhibited by acidic intracellular pH and by acidic extracellular pH, but activated by (acidifying) ammonium and by hypertonicity. Zebrafish Ae2 also mediates Cl(-)/HCO(3)(-) exchange in X. laevis oocytes and accumulates in or near the plasma membrane in transfected HEK-293 cells. In 24-48 hpf zebrafish embryos, the predominant but not exclusive localization of Ae2 polypeptide is the apical membrane of pronephric duct epithelial cells. Thus Ae2 resembles its mammalian orthologs in function, mechanism, and acute regulation but differs in its preferentially apical expression in kidney. These results will inform tests of the role of Ae2 in zebrafish kidney development and function.
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Affiliation(s)
- Boris E Shmukler
- Molecular Medicine and Renal Units, Beth Israel Deaconess Med. Ctr. E/RW763, 330 Brookline Ave., Boston, MA 02215, USA
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