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Tamura T, Ashida R, Emori T, Itonoga M, Yamashita Y, Hatamaru K, Kawaji Y, Koutani H, Maekita T, Kitano M. Serum trypsin as an early predictor of post-endoscopic retrograde cholangiopancreatography pancreatitis. JOURNAL OF HEPATO-BILIARY-PANCREATIC SCIENCES 2024; 31:917-925. [PMID: 39183624 DOI: 10.1002/jhbp.12063] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 08/27/2024]
Abstract
BACKGROUND Serum amylase (AMY) levels measured 2-6 h after ERCP are a predictor of post-ERCP pancreatitis (PEP). Trypsin is one of the pancreatic enzymes elevated in the development of PEP. The study assessed whether serum trypsin (TRY) can predict early-stage PEP. METHODS This prospective study included patients who underwent ERCP from June 2022 to May 2023. TRY, AMY, serum pancreatic AMY (P-AMY), and serum lipase (LIP) levels were measured immediately after ERCP and 2 h later. The primary outcome was the diagnostic abilities of TRY levels measured immediately (0 h-TRY) and 2 h after (2 h-TRY) ERCP to predict PEP (compared with the other serum pancreatic enzymes). RESULTS Of 130 patients analyzed, 18 developed PEP. The sensitivity and specificity of 0 h-TRY were 83.3% and 69.6%, respectively, and those of 2 h-TRY were 88.9% and 72.3%, respectively. The area under the curve (AUC) for 0 h-TRY was significantly higher than that for 0 h-AMY (p = .006) and 0 h-P-AMY (p = .012), whereas the AUCs for 0 h-TRY and 0 h-LIP did not differ significantly (p = .563). The AUC for 2 h-TRY for predicting PEP was significantly higher than that for 2 h-AMY (p = .025), whereas there was no significant differences between the AUCs for 2 h-TRY and 2 h-P-AMY(p = .146), or between those for 2 h-TRY and 2 h-LIP (p = .792). The median increase ratio (expressed as a ratio relative to baseline) in TRY was highest among all of serum pancreatic enzymes tested immediately after ERCP (5.35, 1.72, 1.94, and 4.44 for TRY, AMY, P-AMY, and LIP, respectively). CONCLUSION Measuring TRY immediately after ERCP is useful for the early prediction of PEP.
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Affiliation(s)
- Takashi Tamura
- Second Department of Internal Medicine, Wakayama Medical University, Wakayama, Japan
| | - Reiko Ashida
- Second Department of Internal Medicine, Wakayama Medical University, Wakayama, Japan
| | - Tomoya Emori
- Department of Gastroenterology, Wakayama Rosai Hospital, Wakayama, Japan
| | - Masahiro Itonoga
- Second Department of Internal Medicine, Wakayama Medical University, Wakayama, Japan
| | - Yasunobu Yamashita
- Second Department of Internal Medicine, Wakayama Medical University, Wakayama, Japan
| | - Keiichi Hatamaru
- Second Department of Internal Medicine, Wakayama Medical University, Wakayama, Japan
| | - Yuki Kawaji
- Second Department of Internal Medicine, Wakayama Medical University, Wakayama, Japan
| | - Hiromu Koutani
- Second Department of Internal Medicine, Wakayama Medical University, Wakayama, Japan
| | - Takao Maekita
- Second Department of Internal Medicine, Wakayama Medical University, Wakayama, Japan
| | - Masayuki Kitano
- Second Department of Internal Medicine, Wakayama Medical University, Wakayama, Japan
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Song Z, Li S, Shang Z, Lv W, Cheng X, Meng X, Chen R, Zhang S, Zhang R. Integrating multi-omics data to analyze the potential pathogenic mechanism of CTSH gene involved in type 1 diabetes in the exocrine pancreas. Brief Funct Genomics 2024; 23:406-417. [PMID: 38050341 DOI: 10.1093/bfgp/elad052] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2023] [Revised: 10/19/2023] [Accepted: 11/09/2023] [Indexed: 12/06/2023] Open
Abstract
Type 1 diabetes (T1D) is an autoimmune disease caused by the destruction of insulin-producing pancreatic islet beta cells. Despite significant advancements, the precise pathogenesis of the disease remains unknown. This work integrated data from expression quantitative trait locus (eQTL) studies with Genome wide association study (GWAS) summary data of T1D and single-cell transcriptome data to investigate the potential pathogenic mechanisms of the CTSH gene involved in T1D in exocrine pancreas. Using the summary data-based Mendelian randomization (SMR) approach, we obtained four potential causative genes associated with T1D: BTN3A2, PGAP3, SMARCE1 and CTSH. To further investigate these genes'roles in T1D development, we validated them using a scRNA-seq dataset from pancreatic tissues of both T1D patients and healthy controls. The analysis showed a significantly high expression of the CTSH gene in T1D acinar cells, whereas the other three genes showed no significant changes in the scRNA-seq data. Moreover, single-cell WGCNA analysis revealed the strongest positive correlation between the module containing CTSH and T1D. In addition, we found cellular ligand-receptor interactions between the acinar cells and different cell types, especially ductal cells. Finally, based on functional enrichment analysis, we hypothesized that the CTSH gene in the exocrine pancreas enhances the antiviral response, leading to the overexpression of pro-inflammatory cytokines and the development of an inflammatory microenvironment. This process promotes β cells injury and ultimately the development of T1D. Our findings offer insights into the underlying pathogenic mechanisms of T1D.
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Affiliation(s)
- Zerun Song
- College of Bioinformatics Science and Technology, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin City, Heilongjiang Province, China
| | - Shuai Li
- College of Bioinformatics Science and Technology, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin City, Heilongjiang Province, China
| | - Zhenwei Shang
- College of Bioinformatics Science and Technology, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin City, Heilongjiang Province, China
| | - Wenhua Lv
- College of Bioinformatics Science and Technology, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin City, Heilongjiang Province, China
| | - Xiangshu Cheng
- College of Bioinformatics Science and Technology, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin City, Heilongjiang Province, China
| | - Xin Meng
- College of Bioinformatics Science and Technology, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin City, Heilongjiang Province, China
| | - Rui Chen
- College of Bioinformatics Science and Technology, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin City, Heilongjiang Province, China
| | - Shuhao Zhang
- College of Bioinformatics Science and Technology, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin City, Heilongjiang Province, China
| | - Ruijie Zhang
- College of Bioinformatics Science and Technology, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin City, Heilongjiang Province, China
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Tsomidis I, Voumvouraki A, Kouroumalis E. The Pathogenesis of Pancreatitis and the Role of Autophagy. GASTROENTEROLOGY INSIGHTS 2024; 15:303-341. [DOI: 10.3390/gastroent15020022] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/03/2025] Open
Abstract
The pathogenesis of acute and chronic pancreatitis has recently evolved as new findings demonstrate a complex mechanism operating through various pathways. In this review, the current evidence indicating that several mechanisms act in concert to induce and perpetuate pancreatitis were presented. As autophagy is now considered a fundamental mechanism in the pathophysiology of both acute and chronic pancreatitis, the fundamentals of the autophagy pathway were discussed to allow for a better understanding of the pathophysiological mechanisms of pancreatitis. The various aspects of pathogenesis, including trypsinogen activation, ER stress and mitochondrial dysfunction, the implications of inflammation, and macrophage involvement in innate immunity, as well as the significance of pancreatic stellate cells in the development of fibrosis, were also analyzed. Recent findings on exosomes and the miRNA regulatory role were also presented. Finally, the role of autophagy in the protection and aggravation of pancreatitis and possible therapeutic implications were reviewed.
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Affiliation(s)
- Ioannis Tsomidis
- Laboratory of Gastroenterology and Hepatology, University of Crete Medical School, 71500 Heraklion, Crete, Greece
| | - Argyro Voumvouraki
- 1st Department of Internal Medicine, AHEPA University Hospital, 54621 Thessaloniki, Greece
| | - Elias Kouroumalis
- Laboratory of Gastroenterology and Hepatology, University of Crete Medical School, 71500 Heraklion, Crete, Greece
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Peach CJ, Edgington-Mitchell LE, Bunnett NW, Schmidt BL. Protease-activated receptors in health and disease. Physiol Rev 2023; 103:717-785. [PMID: 35901239 PMCID: PMC9662810 DOI: 10.1152/physrev.00044.2021] [Citation(s) in RCA: 46] [Impact Index Per Article: 23.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2021] [Revised: 07/08/2022] [Accepted: 07/10/2022] [Indexed: 11/22/2022] Open
Abstract
Proteases are signaling molecules that specifically control cellular functions by cleaving protease-activated receptors (PARs). The four known PARs are members of the large family of G protein-coupled receptors. These transmembrane receptors control most physiological and pathological processes and are the target of a large proportion of therapeutic drugs. Signaling proteases include enzymes from the circulation; from immune, inflammatory epithelial, and cancer cells; as well as from commensal and pathogenic bacteria. Advances in our understanding of the structure and function of PARs provide insights into how diverse proteases activate these receptors to regulate physiological and pathological processes in most tissues and organ systems. The realization that proteases and PARs are key mediators of disease, coupled with advances in understanding the atomic level structure of PARs and their mechanisms of signaling in subcellular microdomains, has spurred the development of antagonists, some of which have advanced to the clinic. Herein we review the discovery, structure, and function of this receptor system, highlight the contribution of PARs to homeostatic control, and discuss the potential of PAR antagonists for the treatment of major diseases.
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Affiliation(s)
- Chloe J Peach
- Department of Molecular Pathobiology, College of Dentistry, New York University, New York, New York
- Department of Neuroscience and Physiology and Neuroscience Institute, Grossman School of Medicine, New York University, New York, New York
| | - Laura E Edgington-Mitchell
- Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, Australia
- Bluestone Center for Clinical Research, Department of Oral and Maxillofacial Surgery, New York University College of Dentistry, New York, New York
| | - Nigel W Bunnett
- Department of Molecular Pathobiology, College of Dentistry, New York University, New York, New York
- Department of Neuroscience and Physiology and Neuroscience Institute, Grossman School of Medicine, New York University, New York, New York
| | - Brian L Schmidt
- Department of Molecular Pathobiology, College of Dentistry, New York University, New York, New York
- Bluestone Center for Clinical Research, Department of Oral and Maxillofacial Surgery, New York University College of Dentistry, New York, New York
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Abstract
It has been 30 years since the first member of the protease-activated receptor (PAR) family was discovered. This was followed by the discovery of three other receptors, including PAR2. PAR2 is a G protein-coupled receptor activated by trypsin site-specific proteolysis. The process starts with serine proteases acting between arginine and serine, creating an N-terminus that functions as a tethered ligand that binds, after a conformational change, to the second extracellular loop of the receptor, leading to activation of G-proteins. The physiological and pathological functions of this ubiquitous receptor are still elusive. This review focuses on PAR2 activation and its distribution under physiological and pathological conditions, with a particular focus on the pancreas, a significant producer of trypsin, which is the prototype activator of the receptor. The role in acute or chronic pancreatitis, pancreatic cancer, and diabetes mellitus will be highlighted.
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Affiliation(s)
- Petr SUHAJ
- Department of Pathology and Molecular Medicine, Thomayer University Hospital, Prague, Czech Republic,Third Faculty of Medicine, Charles University, Prague, Czech Republic
| | - Tomas OLEJAR
- Department of Pathology and Molecular Medicine, Thomayer University Hospital, Prague, Czech Republic,Third Faculty of Medicine, Charles University, Prague, Czech Republic
| | - Radoslav MATEJ
- Department of Pathology and Molecular Medicine, Thomayer University Hospital, Prague, Czech Republic,Department of Pathology, University Hospital Kralovske Vinohrady, Prague, Czech Republic,Third Faculty of Medicine, Charles University, Prague, Czech Republic
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6
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Edwinson AL, Grover M. Measurement of novel intestinal secretory and barrier pathways and effects of proteases. Neurogastroenterol Motil 2019; 31:e13547. [PMID: 30843358 PMCID: PMC6407641 DOI: 10.1111/nmo.13547] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/28/2018] [Revised: 12/02/2018] [Accepted: 12/17/2018] [Indexed: 02/08/2023]
Abstract
The epithelial lining of the gastrointestinal (GI) tract in conjunction with the enteric nervous system (ENS) plays an important role in mediating solute absorption and secretion. A dysregulated ionic movement across the epithelium can result in GI diseases that manifest as either watery diarrhea or constipation. Hirschsprung disease is an example of an ENS disorder characterized by absence of enteric ganglia in distal gut resulting in obstructive phenotype. Receptor rearranged during transfection (RET) gene variants are the most commonly recognized genetic associations with Hirschsprung disease. In this issue of Neurogastroenterology and Motility, Russell et al demonstrate that RET mediates colonic ion transport through modulation of cholinergic nerves. They go on to show inhibition of RET can attenuate accelerated transit in a rat model. Normalizing secretory and absorptive defects has been an attractive therapeutic strategy. In addition to the intrinsic regulation of secretory processes, luminal mediators like bile acids, short-chain fatty acids, and proteases can affect both secretion and barrier function of the intestinal epithelium. Elevated levels of proteases have been identified in a wide range of GI diseases including irritable bowel syndrome. Proteases are known to cause visceral hypersensitivity and barrier disruption in vitro and in animal models. The goals of this review are to describe fundamental concepts related to intestinal epithelial secretion, the utility of Ussing chambers to measure ionic mechanisms and to discuss examples of novel signaling pathways; namely the RET signaling cascade in secretomotor neurons and effects of luminal proteases on barrier and ionic secretion.
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Affiliation(s)
- Adam L. Edwinson
- Division of Gastroenterology & Hepatology, Mayo Clinic, Rochester, MN, USA
| | - Madhusudan Grover
- Division of Gastroenterology & Hepatology, Mayo Clinic, Rochester, MN, USA,Correspondence: Madhusudan Grover, MD, Assistant Professor of Medicine and Physiology, Enteric NeuroScience Program, Division of Gastroenterology and Hepatology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA, Tel: 507-284-2478, Fax: 507-266-0350,
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Lin J, Xu Y, Wang Y, Huang S, Li J, Meti MD, Xu X, Hu Z, Liu J, He Z, Xu H. Dissection of binding of trypsin to its natural inhibitor Gensenoside-Rg1 using spectroscopic methods and molecular modeling. J Biomol Struct Dyn 2018; 37:4070-4079. [DOI: 10.1080/07391102.2018.1539411] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Affiliation(s)
- Jialiang Lin
- College of Life Sciences and Oceanography, Shenzhen Key Laboratory of Marine Bioresources and Ecology/Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen University, Shenzhen, China
| | - Yang Xu
- College of Life Sciences and Oceanography, Shenzhen Key Laboratory of Marine Bioresources and Ecology/Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen University, Shenzhen, China
- School of Science and Engineering, Chinese University of Hong Kong, Shenzhen, China
| | - Yuhan Wang
- College of Life Sciences and Oceanography, Shenzhen Key Laboratory of Marine Bioresources and Ecology/Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen University, Shenzhen, China
| | - Songyang Huang
- College of Life Sciences and Oceanography, Shenzhen Key Laboratory of Marine Bioresources and Ecology/Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen University, Shenzhen, China
| | - Junwei Li
- College of Life Sciences and Oceanography, Shenzhen Key Laboratory of Marine Bioresources and Ecology/Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen University, Shenzhen, China
| | - Manjunath D. Meti
- College of Life Sciences and Oceanography, Shenzhen Key Laboratory of Marine Bioresources and Ecology/Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen University, Shenzhen, China
| | - Xu Xu
- College of Life Sciences and Oceanography, Shenzhen Key Laboratory of Marine Bioresources and Ecology/Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen University, Shenzhen, China
| | - Zhangli Hu
- College of Life Sciences and Oceanography, Shenzhen Key Laboratory of Marine Bioresources and Ecology/Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen University, Shenzhen, China
| | - Johnson Liu
- School Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia
| | - Zhendan He
- School of Medicine, Shenzhen University, Shenzhen, China
| | - Hong Xu
- College of Life Sciences and Oceanography, Shenzhen Key Laboratory of Marine Bioresources and Ecology/Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen University, Shenzhen, China
- Key Laboratory of RF Circuits and Systems of Ministry of Education, Hangzhou Dianzi University, Hangzhou, China
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Kawaguchi S, Kikuyama M, Satoh T, Terada S. Use of Nasopancreatic Drainage for Severe Post-endoscopic Retrograde Cholangiopancreatography Pancreatitis: A Case Series. Intern Med 2018; 57:2657-2662. [PMID: 29709935 PMCID: PMC6191584 DOI: 10.2169/internalmedicine.0549-17] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Five patients complaining of severe pain due to severe post-endoscopic retrograde cholangiopancreatography pancreatitis (PEP) underwent nasopancreatic drainage (NPD) placement. Pain relief was achieved on the second, fourth, and fifth day in three, one, and one patients, respectively. Four patients underwent pancreatic juice culture; all were positive. Our results suggest that NPD can relieve severe PEP with severe pain. Bacteria-induced protease-activated receptor-2 activation may be associated with PEP.
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Affiliation(s)
- Shinya Kawaguchi
- Department of Gastroenterology, Shizuoka General Hospital, Japan
| | - Masataka Kikuyama
- Department of Gastroenterology, Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Japan
| | - Tatsunori Satoh
- Department of Gastroenterology, Shizuoka General Hospital, Japan
| | - Shuzo Terada
- Department of Gastroenterology, Shizuoka General Hospital, Japan
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9
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Das K, Prasad R, Singh A, Bhattacharya A, Roy A, Mallik S, Mukherjee A, Sen P. Protease-activated receptor 2 promotes actomyosin dependent transforming microvesicles generation from human breast cancer. Mol Carcinog 2018; 57:1707-1722. [DOI: 10.1002/mc.22891] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2018] [Revised: 08/01/2018] [Accepted: 08/15/2018] [Indexed: 01/16/2023]
Affiliation(s)
- Kaushik Das
- Department of Biological Chemistry; Indian Association for the Cultivation of Science; Kolkata India
| | - Ramesh Prasad
- Department of Biological Chemistry; Indian Association for the Cultivation of Science; Kolkata India
| | - Arpana Singh
- Department of Biological Chemistry; Indian Association for the Cultivation of Science; Kolkata India
| | - Anindita Bhattacharya
- Department of Biological Chemistry; Indian Association for the Cultivation of Science; Kolkata India
| | - Abhishek Roy
- Department of Biological Chemistry; Indian Association for the Cultivation of Science; Kolkata India
| | - Suman Mallik
- Department of Biological Chemistry; Indian Association for the Cultivation of Science; Kolkata India
| | - Ashis Mukherjee
- A Unit of Himadri Memorial Cancer Welfare Trust; Netaji Subhash Chandra Bose Cancer Research Institute; Kolkata India
| | - Prosenjit Sen
- Department of Biological Chemistry; Indian Association for the Cultivation of Science; Kolkata India
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10
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Jung SR, Seo JB, Deng Y, Asbury CL, Hille B, Koh DS. Contributions of protein kinases and β-arrestin to termination of protease-activated receptor 2 signaling. ACTA ACUST UNITED AC 2016; 147:255-71. [PMID: 26927499 PMCID: PMC4772372 DOI: 10.1085/jgp.201511477] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
Systematic imaging studies and modeling reveal new details of the regulation of the Gq-coupled GPCR, protease-activated receptor 2, by phosphorylation and β-arrestin. Activated Gq protein–coupled receptors (GqPCRs) can be desensitized by phosphorylation and β-arrestin binding. The kinetics and individual contributions of these two mechanisms to receptor desensitization have not been fully distinguished. Here, we describe the shut off of protease-activated receptor 2 (PAR2). PAR2 activates Gq and phospholipase C (PLC) to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol and inositol trisphosphate (IP3). We used fluorescent protein–tagged optical probes to monitor several consequences of PAR2 signaling, including PIP2 depletion and β-arrestin translocation in real time. During continuous activation of PAR2, PIP2 was depleted transiently and then restored within a few minutes, indicating fast receptor activation followed by desensitization. Knockdown of β-arrestin 1 and 2 using siRNA diminished the desensitization, slowing PIP2 restoration significantly and even adding a delayed secondary phase of further PIP2 depletion. These effects of β-arrestin knockdown on PIP2 recovery were prevented when serine/threonine phosphatases that dephosphorylate GPCRs were inhibited. Thus, PAR2 may continuously regain its activity via dephosphorylation when there is insufficient β-arrestin to trap phosphorylated receptors. Similarly, blockers of protein kinase C (PKC) and G protein–coupled receptor kinase potentiated the PIP2 depletion. In contrast, an activator of PKC inhibited receptor activation, presumably by augmenting phosphorylation of PAR2. Our interpretations were strengthened by modeling. Simulations supported the conclusions that phosphorylation of PAR2 by protein kinases initiates receptor desensitization and that recruited β-arrestin traps the phosphorylated state of the receptor, protecting it from phosphatases. Speculative thinking suggested a sequestration of phosphatidylinositol 4-phosphate 5 kinase (PIP5K) to the plasma membrane by β-arrestin to explain why knockdown of β-arrestin led to secondary depletion of PIP2. Indeed, artificial recruitment of PIP5K removed the secondary loss of PIP2 completely. Altogether, our experimental and theoretical approaches demonstrate roles and dynamics of the protein kinases, β-arrestin, and PIP5K in the desensitization of PAR2.
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Affiliation(s)
- Seung-Ryoung Jung
- Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195
| | - Jong Bae Seo
- Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195
| | - Yi Deng
- Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195
| | - Charles L Asbury
- Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195
| | - Bertil Hille
- Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195
| | - Duk-Su Koh
- Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195 Department of Physics, Pohang University of Science and Technology, Pohang, Kyungbuk, 790-784, Republic of Korea
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Pallagi P, Hegyi P, Rakonczay Z. The Physiology and Pathophysiology of Pancreatic Ductal Secretion: The Background for Clinicians. Pancreas 2015; 44:1211-1233. [PMID: 26465950 DOI: 10.1097/mpa.0000000000000421] [Citation(s) in RCA: 48] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The human exocrine pancreas consists of 2 main cell types: acinar and ductal cells. These exocrine cells interact closely to contribute to the secretion of pancreatic juice. The most important ion in terms of the pancreatic ductal secretion is HCO3. In fact, duct cells produce an alkaline fluid that may contain up to 140 mM NaHCO3, which is essential for normal digestion. This article provides an overview of the basics of pancreatic ductal physiology and pathophysiology. In the first part of the article, we discuss the ductal electrolyte and fluid transporters and their regulation. The central role of cystic fibrosis transmembrane conductance regulator (CFTR) is highlighted, which is much more than just a Cl channel. We also review the role of pancreatic ducts in severe debilitating diseases such as cystic fibrosis (caused by various genetic defects of cftr), pancreatitis, and diabetes mellitus. Stimulation of ductal secretion in cystic fibrosis and pancreatitis may have beneficial effects in their treatment.
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Affiliation(s)
- Petra Pallagi
- From the *First Department of Medicine, University of Szeged; and †Hungarian Academy of Sciences-University of Szeged Translational Gastroenterology Research Group, Szeged, Hungary
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Sedda S, Marafini I, Caruso R, Pallone F, Monteleone G. Proteinase activated-receptors-associated signaling in the control of gastric cancer. World J Gastroenterol 2014; 20:11977-11984. [PMID: 25232234 PMCID: PMC4161785 DOI: 10.3748/wjg.v20.i34.11977] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/04/2013] [Revised: 02/10/2014] [Accepted: 05/05/2014] [Indexed: 02/06/2023] Open
Abstract
Gastric cancer (GC) is the fourth most common cancer in the world and the second cause of cancer-related death. Gastric carcinogenesis is a multifactorial process, in which environmental and genetic factors interact to activate multiple intracellular signals thus leading to uncontrolled growth and survival of GC cells. One such a pathway is regulated by proteinase activated-receptors (PARs), seven transmembrane-spanning domain G protein-coupled receptors, which comprise four receptors (i.e., PAR-1, PAR-2, PAR-3, and PAR-4) activated by various proteases. Both PAR-1 and PAR-2 are over-expressed on GC cells and their activation triggers and/or amplifies intracellular pathways, which sustain gastric carcinogenesis. There is also evidence that expression of either PAR-1 or PAR-2 correlates with depth of wall invasion and metastatic dissemination and inversely with the overall survival of patients. Consistently, data emerging from experimental models of GC suggest that both these receptors can be important targets for therapeutic interventions in GC patients. In contrast, PAR-4 levels are down-regulated in GC and correlate inversely with the aggressiveness of GC, thus suggesting a negative role of this receptor in the control of GC. In this article we review the available data on the expression and role of PARs in GC and discuss whether manipulation of PAR-driven signals may be useful for interfering with GC cell behavior.
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Abstract
OBJECTIVE The objective of this study was to evaluate whether an uncontrolled activation of mast cells and macrophages through protease-activated receptor-2 (PAR-2) during acute pancreatitis could develop lung injury. METHODS Pancreatitis was induced in rats by intraductal infusion of sodium taurocholate. In a group of animals, PAR-2 antagonist or trypsin (TRP) inhibitor was intravenously administered before the pancreatitis induction. In additional groups, the animals were treated with PAR-2-activating peptide or pancreatic TRP. The myeloperoxidase (MPO) activity was measured to evaluate the progression of inflammation. RESULTS Plasma from the animals with pancreatitis and pancreatic TRP induced the secretion of mast cells and alveolar macrophages as well as increased the density of PAR-2 in the plasma membrane. The treatment of alveolar macrophages with TRP, tryptase, as well as PAR-1- and PAR-2-activating peptide led to an increase in calcium-triggered exocytosis. Similar results were obtained in acinar cells. The intravenous injection of PAR-2-activating peptide and TRP induced an increase in MPO activity in the lung. The intravenous injection of PAR-2 antagonist or TRP inhibitor before the pancreatitis induction could prevent the increase in MPO activity in the pancreas and the lung. CONCLUSIONS The TRP generated during acute pancreatitis could be involved in the progression of lung injury through the activation of PAR-2 in alveolar macrophages.
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Abstract
Abstract Many epithelial cells form polarized monolayers under in vivo and in vitro conditions. Typically, epithelial cells are cultured for differentiation on insert systems where cells are plated on a porous filter membrane. Although the cultured monolayers have been a standard system to study epithelial physiology, there are some limits: The epithelial cells growing inside the commercial inserts are not optimal to visualize directly through lenses on inverted microscopes. The cell images are optically distorted and background fluorescence is bright due to the filter membrane positioned between the cells and the lens. In addition, the cells are not easily accessible by electrodes due to the presence of tall side walls. Here, we present the design, fabrication, and practical applications of an improved system for analysis of polarized epithelial monolayers. This new system allows (1) direct imaging of cells without an interfering filter membrane, (2) electrophysiological measurements, and (3) detection of apical secretion with minimal dilution. Therefore, our culture method is optimized to study differentiated epithelial cells at the single-cell and subcellular levels, and can be extended to other cell types with minor modifications.
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Affiliation(s)
- Jong Bae Seo
- Department of Physiology and Biophysics, University of Washington, Seattle, Washington
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15
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Jung J, Lee MG. Role of calcium signaling in epithelial bicarbonate secretion. Cell Calcium 2014; 55:376-84. [PMID: 24598807 DOI: 10.1016/j.ceca.2014.02.002] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2014] [Revised: 02/03/2014] [Accepted: 02/04/2014] [Indexed: 12/24/2022]
Abstract
Transepithelial bicarbonate secretion plays a key role in the maintenance of fluid and protein secretion from epithelial cells and the protection of the epithelial cell surface from various pathogens. Epithelial bicarbonate secretion is mainly under the control of cAMP and calcium signaling. While the physiological roles and molecular mechanisms of cAMP-induced bicarbonate secretion are relatively well defined, those induced by calcium signaling remain poorly understood in most epithelia. The present review summarizes the current status of knowledge on the role of calcium signaling in epithelial bicarbonate secretion. Specifically, this review introduces how cytosolic calcium signaling can increase bicarbonate secretion by regulating membrane transport proteins and how it synergizes with cAMP-induced mechanisms in epithelial cells. In addition, tissue-specific variations in the pancreas, salivary glands, intestines, bile ducts, and airways are discussed. We hope that the present report will stimulate further research into this important topic. These studies will provide the basis for future medicines for a wide spectrum of epithelial disorders including cystic fibrosis, Sjögren's syndrome, and chronic pancreatitis.
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Affiliation(s)
- Jinsei Jung
- Department of Pharmacology and Brain Korea 21 Plus Project for Medical Sciences, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea; Department of Otorhinolaryngology, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea
| | - Min Goo Lee
- Department of Pharmacology and Brain Korea 21 Plus Project for Medical Sciences, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea.
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16
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Kim MH, Seo JB, Burnett LA, Hille B, Koh DS. Characterization of store-operated Ca2+ channels in pancreatic duct epithelia. Cell Calcium 2013; 54:266-75. [PMID: 23968882 PMCID: PMC3809127 DOI: 10.1016/j.ceca.2013.07.002] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2013] [Revised: 07/18/2013] [Accepted: 07/20/2013] [Indexed: 11/30/2022]
Abstract
Store-operated Ca2+ channels (SOCs) are activated by depletion of intracellular Ca2+ stores following agonist-mediated Ca2+ release. Previously we demonstrated that Ca2+ influx through SOCs elicits exocytosis efficiently in pancreatic duct epithelial cells (PDEC). Here we describe the biophysical, pharmacological, and molecular properties of the duct epithelial SOCs using Ca2+ imaging, whole-cell patch-clamp, and molecular biology. In PDEC, agonists of purinergic, muscarinic, and adrenergic receptors coupled to phospholipase C activated SOC-mediated Ca2+ influx as Ca2+ was released from intracellular stores. Direct measurement of [Ca2+] in the ER showed that SOCs greatly slowed depletion of the ER. Using IP3 or thapsigargin in the patch pipette elicited inwardly rectifying SOC currents. The currents increased ∼8-fold after removal of extracellular divalent cations, suggesting competitive permeation between mono- and divalent cations. The current was completely blocked by high doses of La3+ and 2-aminoethoxydiphenyl borate (2-APB) but only partially depressed by SKF-96365. In polarized PDEC, SOCs were localized specifically to the basolateral membrane. RT-PCR screening revealed the expression of both STIM and Orai proteins for the formation of SOCs in PDEC. By expression of fluorescent STIM1 and Orai1 proteins in PDEC, we confirmed that colocalization of the two proteins increases after store depletion. In conclusion, basolateral Ca2+ entry through SOCs fills internal Ca2+ stores depleted by external stimuli and will facilitate cellular processes dependent on cytoplasmic Ca2+ such as salt and mucin secretion from the exocrine pancreatic ducts.
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Affiliation(s)
- Mean-Hwan Kim
- Department of Physics, Pohang University of Science and Technology, Pohang, Republic of Korea
| | - Jong Bae Seo
- Department of Physiology & Biophysics, University of Washington, Seattle, Washington, USA
| | - Lindsey A. Burnett
- Department of Physiology & Biophysics, University of Washington, Seattle, Washington, USA
| | - Bertil Hille
- Department of Physiology & Biophysics, University of Washington, Seattle, Washington, USA
| | - Duk-Su Koh
- Department of Physics, Pohang University of Science and Technology, Pohang, Republic of Korea
- Department of Physiology & Biophysics, University of Washington, Seattle, Washington, USA
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17
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Effects of protease-activated receptors (PARs) on intracellular calcium dynamics of acinar cells in rat lacrimal glands. Histochem Cell Biol 2013; 140:463-76. [PMID: 23463389 DOI: 10.1007/s00418-013-1082-0] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/04/2013] [Indexed: 10/27/2022]
Abstract
Protease-activated receptors (PARs) represent a novel class of seven transmembrane domain G-protein coupled receptors, which are activated by proteolytic cleavage. PARs are present in a variety of cells and have been prominently implicated in the regulation of a number of vital functions. Here, lacrimal gland acinar cell responses to PAR activation were examined, with special reference to intracellular Ca(2+) concentration ([Ca(2+)]i) dynamics. In the present study, detection of acinar cell mRNA specific to known PAR subtypes was determined by reverse transcriptase polymerase chain reaction. Only PAR2 mRNA was detected in acinar cells of lacrimal glands. Both trypsin and a PAR2-activating peptide (PAR2-AP), SLIGRL-NH2, induced an increase in [Ca(2+)]i in acinar cells. The removal of extracellular Ca(2+) and the use of Ca(2+) channel blockers did not inhibit PAR2-AP-induced [Ca(2+)]i increases. Furthermore, U73122 and xestospongin C failed to inhibit PAR2-induced increases in [Ca(2+)]i. The origin of the calcium influx observed after activated PAR2-induced Ca(2+) release from intracellular Ca(2+) stores was also evaluated. The NO donor, GEA 3162, mimicked the effects of PAR2 in activating non-capacitative calcium entry (NCCE). However, both calyculin A (100 nM) and a low concentration of Gd(3+) (5 μM) did not completely block the PAR2-AP-induced increase in [Ca(2+)]i. These findings indicated that PAR2 activation resulted primarily in Ca(2+) mobilization from intracellular Ca(2+) stores and that PAR2-mediated [Ca(2+)]i changes were mainly independent of IP3. RT-PCR indicated that TRPC 1, 3 and 6, which play a role in CCE and NCCE, are expressed in acinar cells. We suggest that PAR2-AP differentially regulates both NCCE and CCE, predominantly NCCE. Finally, our results suggested that PAR2 may function as a key receptor in calcium-related cell homeostasis under pathophysiological conditions such as tissue injury or inflammation.
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18
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Michael ES, Kuliopulos A, Covic L, Steer ML, Perides G. Pharmacological inhibition of PAR2 with the pepducin P2pal-18S protects mice against acute experimental biliary pancreatitis. Am J Physiol Gastrointest Liver Physiol 2013; 304:G516-26. [PMID: 23275617 PMCID: PMC3602677 DOI: 10.1152/ajpgi.00296.2012] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Pancreatic acinar cells express proteinase-activated receptor-2 (PAR2) that is activated by trypsin-like serine proteases and has been shown to exert model-specific effects on the severity of experimental pancreatitis, i.e., PAR2(-/-) mice are protected from experimental acute biliary pancreatitis but develop more severe secretagogue-induced pancreatitis. P2pal-18S is a novel pepducin lipopeptide that targets and inhibits PAR2. In studies monitoring PAR2-stimulated intracellular Ca(2+) concentration changes, we show that P2pal-18S is a full PAR2 inhibitor in acinar cells. Our in vivo studies show that P2pal-18S significantly reduces the severity of experimental biliary pancreatitis induced by retrograde intraductal bile acid infusion, which mimics injury induced by endoscopic retrograde cholangiopancreatography (ERCP). This reduction in pancreatitis severity is observed when the pepducin is given before or 2 h after bile acid infusion but not when it is given 5 h after bile acid infusion. Conversely, P2pal-18S increases the severity of secretagogue-induced pancreatitis. In vitro studies indicate that P2pal-18S protects acinar cells against bile acid-induced injury/death, but it does not alter bile acid-induced intracellular zymogen activation. These studies are the first to report the effects of an effective PAR2 pharmacological inhibitor on pancreatic acinar cells and on the severity of experimental pancreatitis. They raise the possibility that a pepducin such as P2pal-18S might prove useful in the clinical management of patients at risk for developing severe biliary pancreatitis such as occurs following ERCP.
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Affiliation(s)
- E. S. Michael
- 1Department of Surgery, Tufts Medical Center, and Tufts University School of Medicine, Boston, Massachusetts; and
| | - A. Kuliopulos
- 2Molecular Oncology Research Institute, Tufts Medical Center, and Tufts University School of Medicine, Boston, Massachusetts
| | - L. Covic
- 2Molecular Oncology Research Institute, Tufts Medical Center, and Tufts University School of Medicine, Boston, Massachusetts
| | - M. L. Steer
- 1Department of Surgery, Tufts Medical Center, and Tufts University School of Medicine, Boston, Massachusetts; and
| | - G. Perides
- 1Department of Surgery, Tufts Medical Center, and Tufts University School of Medicine, Boston, Massachusetts; and
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19
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Lee MG, Ohana E, Park HW, Yang D, Muallem S. Molecular mechanism of pancreatic and salivary gland fluid and HCO3 secretion. Physiol Rev 2012; 92:39-74. [PMID: 22298651 DOI: 10.1152/physrev.00011.2011] [Citation(s) in RCA: 287] [Impact Index Per Article: 22.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Fluid and HCO(3)(-) secretion is a vital function of all epithelia and is required for the survival of the tissue. Aberrant fluid and HCO(3)(-) secretion is associated with many epithelial diseases, such as cystic fibrosis, pancreatitis, Sjögren's syndrome, and other epithelial inflammatory and autoimmune diseases. Significant progress has been made over the last 20 years in our understanding of epithelial fluid and HCO(3)(-) secretion, in particular by secretory glands. Fluid and HCO(3)(-) secretion by secretory glands is a two-step process. Acinar cells secrete isotonic fluid in which the major salt is NaCl. Subsequently, the duct modifies the volume and electrolyte composition of the fluid to absorb the Cl(-) and secrete HCO(3)(-). The relative volume secreted by acinar and duct cells and modification of electrolyte composition of the secreted fluids varies among secretory glands to meet their physiological functions. In the pancreas, acinar cells secrete a small amount of NaCl-rich fluid, while the duct absorbs the Cl(-) and secretes HCO(3)(-) and the bulk of the fluid in the pancreatic juice. Fluid secretion appears to be driven by active HCO(3)(-) secretion. In the salivary glands, acinar cells secrete the bulk of the fluid in the saliva that is driven by active Cl(-) secretion and contains high concentrations of Na(+) and Cl(-). The salivary glands duct absorbs both the Na(+) and Cl(-) and secretes K(+) and HCO(3)(-). In this review, we focus on the molecular mechanism of fluid and HCO(3)(-) secretion by the pancreas and salivary glands, to highlight the similarities of the fundamental mechanisms of acinar and duct cell functions, and to point out the differences to meet gland-specific secretions.
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Affiliation(s)
- Min Goo Lee
- Department of Pharmacology, Yonsei University College of Medicine, Seoul, Korea
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20
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Hoesli CA, Johnson JD, Piret JM. Purified human pancreatic duct cell culture conditions defined by serum-free high-content growth factor screening. PLoS One 2012; 7:e33999. [PMID: 22442738 PMCID: PMC3307781 DOI: 10.1371/journal.pone.0033999] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2011] [Accepted: 02/22/2012] [Indexed: 01/26/2023] Open
Abstract
The proliferation of pancreatic duct-like CK19+ cells has implications for multiple disease states including pancreatic cancer and diabetes mellitus. The in vitro study of this important cell type has been hampered by their limited expansion compared to fibroblast-like vimentin+ cells that overgrow primary cultures. We aimed to develop a screening platform for duct cell mitogens after depletion of the vimentin+ population. The CD90 cell surface marker was used to remove the vimentin+ cells from islet-depleted human pancreas cell cultures by magnetic-activated cell sorting. Cell sorting decreased CD90+ cell contamination of the cultures from 34±20% to 1.3±0.6%, yielding purified CK19+ cultures with epithelial morphology. A full-factorial experimental design was then applied to test the mitogenic effects of bFGF, EGF, HGF, KGF and VEGF. After 6 days in test conditions, the cells were labelled with BrdU, stained and analyzed by high-throughput imaging. This screening assay confirmed the expected mitogenic effects of bFGF, EGF, HGF and KGF on CK19+ cells and additionally revealed interactions between these factors and VEGF. A serum-free medium containing bFGF, EGF, HGF and KGF led to CK19+ cell expansion comparable to the addition of 10% serum. The methods developed in this work should advance pancreatic cancer and diabetes research by providing effective cell culture and high-throughput screening platforms to study purified primary pancreatic CK19+ cells.
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Affiliation(s)
- Corinne A Hoesli
- Michael Smith Laboratories and Department of Biological and Chemical Engineering, University of British Columbia, Vancouver, Canada.
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21
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ISHIGURO HIROSHI, YAMAMOTO AKIKO, NAKAKUKI MIYUKI, YI LANJUAN, ISHIGURO MARIKO, YAMAGUCHI MAKOTO, KONDO SHIHO, MOCHIMARU YUKA. Physiology and pathophysiology of bicarbonate secretion by pancreatic duct epithelium. NAGOYA JOURNAL OF MEDICAL SCIENCE 2012; 74:1-18. [PMID: 22515107 PMCID: PMC4831246] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/02/2022]
Abstract
HCO3- -rich fluid in the pancreatic juice (2-3 L/day) is secreted by epithelial cells lining the pancreatic duct tree, while digestive enzymes are secreted by acinar cells with a small amount of Cl- -rich fluid. Ductal HCO3- secretion is not only regulated by gastrointestinal hormones and cholinergic nerves but is also influenced by luminal factors: intraductal pressure, Ca2+ concentration, pathological activation of protease and bile reflux. The maximum HCO3- concentration of the juice under secretin stimulation reaches 140-150 mM. Thus pancreatic duct cells secrete HCO3- against a approximately 7-fold concentration gradient. HCO3- secretion critically depends on the activity of CFTR, a cAMP-dependent anion channel localized in the apical membrane of various epithelia. In the proximal part of pancreatic ducts close to acinar cells HCO3 secretion across the apical membrane is largely mediated by SLC26A6 CI- -HCO3- exchanger. In distal ducts where the luminal HCO3- concentration is already high, most of the HCO3- secretion is mediated by HCO3- conductance of CFTR. CFTR is the causative gene for cystic fibrosis. Loss of function due to severe mutations in both alleles causes typical cystic fibrosis characterized by dehydrated, thick, and viscous luminal fluid/mucus in the respiratory and gastrointestinal tract, pancreatic duct, and vas deferens. A compound heterozygote of mutations/polymorphisms (causing a mild dysfunction of CFTR) involves a risk of developing CFTR-related diseases such as chronic pancreatitis. In cystic fibrosis and certain cases of chronic pancreatitis, the pancreatic duct epithelium secretes a small amount of fluid with neutral-acidic pH, which causes an obstruction of the duct lumen by a protein plug or viscous mucus.
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Affiliation(s)
- HIROSHI ISHIGURO
- Laboratory of Human Nutrition, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - AKIKO YAMAMOTO
- Laboratory of Human Nutrition, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - MIYUKI NAKAKUKI
- Laboratory of Human Nutrition, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - LANJUAN YI
- Laboratory of Human Nutrition, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - MARIKO ISHIGURO
- Laboratory of Human Nutrition, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - MAKOTO YAMAGUCHI
- Laboratory of Human Nutrition, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - SHIHO KONDO
- Laboratory of Human Nutrition, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - YUKA MOCHIMARU
- Laboratory of Human Nutrition, Nagoya University Graduate School of Medicine, Nagoya, Japan
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22
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Pallagi P, Venglovecz V, Rakonczay Z, Borka K, Korompay A, Ózsvári B, Judák L, Sahin-Tóth M, Geisz A, Schnúr A, Maléth J, Takács T, Gray MA, Argent BE, Mayerle J, Lerch MM, Wittmann T, Hegyi P. Trypsin reduces pancreatic ductal bicarbonate secretion by inhibiting CFTR Cl⁻ channels and luminal anion exchangers. Gastroenterology 2011; 141:2228-2239.e6. [PMID: 21893120 PMCID: PMC3273991 DOI: 10.1053/j.gastro.2011.08.039] [Citation(s) in RCA: 63] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/26/2010] [Revised: 07/14/2011] [Accepted: 08/05/2011] [Indexed: 12/16/2022]
Abstract
BACKGROUND & AIMS The effects of trypsin on pancreatic ductal epithelial cells (PDECs) vary among species and depend on the localization of proteinase-activated receptor 2 (PAR-2). We compared PAR-2 localization in human and guinea-pig PDECs, and used isolated guinea pig ducts to study the effects of trypsin and a PAR-2 agonist on bicarbonate secretion. METHODS PAR-2 localization was analyzed by immunohistochemistry in guinea pig and human pancreatic tissue samples (from 15 patients with chronic pancreatitis and 15 without pancreatic disease). Functionally, guinea pig PDECs were studied by microperfusion of isolated ducts, measurements of intracellular pH and intracellular Ca(2+) concentration, and patch clamp analysis. The effect of pH on trypsinogen autoactivation was assessed using recombinant human cationic trypsinogen. RESULTS PAR-2 localized to the apical membrane of human and guinea pig PDECs. Trypsin increased intracellular Ca(2+) concentration and intracellular pH and inhibited secretion of bicarbonate by the luminal anion exchanger and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. Autoactivation of human cationic trypsinogen accelerated when the pH was reduced from 8.5 to 6.0. PAR-2 expression was strongly down-regulated, at transcriptional and protein levels, in the ducts of patients with chronic pancreatitis, consistent with increased activity of intraductal trypsin. Importantly, in PAR-2 knockout mice, the effects of trypsin were markedly reduced. CONCLUSIONS Trypsin reduces pancreatic ductal bicarbonate secretion via PAR-2-dependent inhibition of the apical anion exchanger and the CFTR Cl(-) channel. This could contribute to the development of chronic pancreatitis by decreasing luminal pH and promoting premature activation of trypsinogen in the pancreatic ducts.
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Affiliation(s)
- Petra Pallagi
- First Dept. of Medicine, University of Szeged, Szeged, Hungary
| | | | | | - Katalin Borka
- 2 Dept. of Pathology, Semmelweis University, Budapest, Hungary
| | - Anna Korompay
- 2 Dept. of Pathology, Semmelweis University, Budapest, Hungary
| | - Béla Ózsvári
- First Dept. of Medicine, University of Szeged, Szeged, Hungary
| | - Linda Judák
- First Dept. of Medicine, University of Szeged, Szeged, Hungary
| | - Miklós Sahin-Tóth
- Dept. of Molecular and Cell Biology, Henry M. Goldman School of Dental Medicine, Boston University, Boston, MA, USA
| | - Andrea Geisz
- First Dept. of Medicine, University of Szeged, Szeged, Hungary
- Dept. of Molecular and Cell Biology, Henry M. Goldman School of Dental Medicine, Boston University, Boston, MA, USA
| | - Andrea Schnúr
- First Dept. of Medicine, University of Szeged, Szeged, Hungary
- Dept. of Molecular and Cell Biology, Henry M. Goldman School of Dental Medicine, Boston University, Boston, MA, USA
| | - József Maléth
- First Dept. of Medicine, University of Szeged, Szeged, Hungary
| | - Tamás Takács
- First Dept. of Medicine, University of Szeged, Szeged, Hungary
| | - Mike A. Gray
- Institute for Cell & Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UK
| | - Barry E. Argent
- Institute for Cell & Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UK
| | - Julia Mayerle
- Department of Medicine A, Greifswald University Hospital, Greifswald, Germany
| | - Markus M. Lerch
- Department of Medicine A, Greifswald University Hospital, Greifswald, Germany
| | - Tibor Wittmann
- First Dept. of Medicine, University of Szeged, Szeged, Hungary
| | - Péter Hegyi
- First Dept. of Medicine, University of Szeged, Szeged, Hungary
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23
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Awla D, Abdulla A, Zhang S, Roller J, Menger MD, Regnér S, Thorlacius H. Lymphocyte function antigen-1 regulates neutrophil recruitment and tissue damage in acute pancreatitis. Br J Pharmacol 2011; 163:413-23. [PMID: 21244370 DOI: 10.1111/j.1476-5381.2011.01225.x] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
BACKGROUND AND PURPOSE Leucocyte infiltration is a rate-limiting step in the pathophysiology of acute pancreatitis (AP) although the adhesive mechanisms supporting leucocyte-endothelium interactions in the pancreas remain elusive. The aim of this study was to define the role of lymphocyte function antigen-1 (LFA-1) in regulating neutrophil-endothelium interactions and tissue damage in severe AP. EXPERIMENTAL APPROACH Pancreatitis was induced by retrograde infusion of sodium taurocholate into the pancreatic duct in mice. LFA-1 gene-targeted mice and an antibody directed against LFA-1 were used to define the role of LFA-1. KEY RESULTS Taurocholate challenge caused a clear-cut increase in serum amylase, neutrophil infiltration, CXCL2 (macrophage inflammatory protein-2) formation, trypsinogen activation and tissue damage in the pancreas. Inhibition of LFA-1 function markedly reduced taurocholate-induced amylase levels, accumulation of neutrophils, production of CXC chemokines and tissue damage in the pancreas. Notably, intravital microscopy revealed that inhibition of LFA-1 abolished taurocholate-induced leucocyte adhesion in postcapillary venules of the pancreas. In addition, pulmonary infiltration of neutrophils was attenuated by inhibition of LFA-1 in mice challenged with taurocholate. However, interference with LFA-1 had no effect on taurocholate-induced activation of trypsinogen in the pancreas. CONCLUSIONS AND IMPLICATIONS Our novel data suggest that LFA-1 plays a key role in regulating neutrophil recruitment, CXCL2 formation and tissue injury in the pancreas. Moreover, these results suggest that LFA-1-mediated inflammation is a downstream component of trypsinogen activation in the pathophysiology of AP. Thus, we conclude that targeting LFA-1 may be a useful approach to protect against pathological inflammation in the pancreas.
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Affiliation(s)
- Darbaz Awla
- Department of Clinical Sciences, Malmö, Section of Surgery, Skåne University Hospital, Lund University, Malmö, Sweden
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24
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Rothmeier AS, Ruf W. Protease-activated receptor 2 signaling in inflammation. Semin Immunopathol 2011; 34:133-49. [PMID: 21971685 DOI: 10.1007/s00281-011-0289-1] [Citation(s) in RCA: 229] [Impact Index Per Article: 16.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2011] [Accepted: 09/05/2011] [Indexed: 12/17/2022]
Abstract
Protease-activated receptors (PARs) are G protein-coupled receptors that are activated by proteolytical cleavage of the amino-terminus and thereby act as sensors for extracellular proteases. While coagulation proteases activate PARs to regulate hemostasis, thrombosis, and cardiovascular function, PAR2 is also activated in extravascular locations by a broad array of serine proteases, including trypsin, tissue kallikreins, coagulation factors VIIa and Xa, mast cell tryptase, and transmembrane serine proteases. Administration of PAR2-specific agonistic and antagonistic peptides, as well as studies in PAR2 knockout mice, identified critical functions of PAR2 in development, inflammation, immunity, and angiogenesis. Here, we review these roles of PAR2 with an emphasis on the role of coagulation and other extracellular protease pathways that cleave PAR2 in epithelial, immune, and neuronal cells to regulate physiological and pathophysiological processes.
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Affiliation(s)
- Andrea S Rothmeier
- Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037, USA
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25
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Murrell M, Kamm R, Matsudaira P. Tension, free space, and cell damage in a microfluidic wound healing assay. PLoS One 2011; 6:e24283. [PMID: 21915305 PMCID: PMC3167843 DOI: 10.1371/journal.pone.0024283] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2011] [Accepted: 08/09/2011] [Indexed: 12/20/2022] Open
Abstract
We use a novel, microfluidics-based technique to deconstruct the classical wound healing scratch assay, decoupling the contribution of free space and cell damage on the migratory dynamics of an epithelial sheet. This method utilizes multiple laminar flows to selectively cleave cells enzymatically, and allows us to present a 'damage free' denudation. We therefore isolate the influence of free space on the onset of sheet migration. First, we observe denudation directly to measure the retraction in the cell sheet that occurs after cell-cell contact is broken, providing direct and quantitative evidence of strong tension within the sheet. We further probe the mechanical integrity of the sheet without denudation, instead using laminar flows to selectively inactivate actomyosin contractility. In both cases, retraction is observed over many cell diameters. We then extend this method and complement the enzymatic denudation with analogies to wounding, including gradients in signals associated with cell damage, such as reactive oxygen species, suspected to play a role in the induction of movement after wounding. These chemical factors are evaluated in combination with the enzymatic cleavage of cells, and are assessed for their influence on the collective migration of a non-abrasively denuded epithelial sheet. We conclude that free space alone is sufficient to induce movement, but this movement is predominantly limited to the leading edge, leaving cells further from the edge less able to move towards the wound. Surprisingly, when coupled with a gradient in ROS to simulate the chemical effects of abrasion however, motility was not restored, but further inhibited.
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Affiliation(s)
- Michael Murrell
- Department of Biological Engineering/Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
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26
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Kaneko D, Komatsu H, Ohama T, Sato K. Effects of trypsin on cytosolic calcium levels in the rat aortic endothelium. J Vet Med Sci 2011; 73:1001-5. [PMID: 21471694 DOI: 10.1292/jvms.11-0068] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
The effect of trypsin on vascular tone and the cytosolic calcium concentration ([Ca(2+)](i)) of endothelial and smooth muscle cells were examined in the rat aorta. A calcium indicator, fura-PE3, was used to measure [Ca(2+)](i) simultaneously with vascular tone. In the endothelium-intact rat aorta, carbachol and trypsin increased [Ca(2+)](i) in a dose-dependent manner. In the endothelium-denuded rat aorta, carbachol did not change [Ca(2+)](i), but trypsin slightly increased it. Addition of trypsin to the norepinephrine-stimulated rat aorta relaxed the muscle with an additional increase in [Ca(2+)](i). Under calcium-free conditions, trypsin induced a transient increase in [Ca(2+)](i). Trypsin-induced endothelium-dependent relaxation was inhibited by preincubation with l-NMMA, an endothelial NO synthase inhibitor, U-73122, a phospholipase C inhibitor, cyclopiazonic acid, a sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase blocker, and lanthanum, a nonselective Ca(2+) channel blocker. However, indomethacin, a nonselective cyclooxygenase inhibitor, and SKF-96365, a store-operated Ca(2+)-channel blocker, had no effect on the trypsin-induced relaxation. These results suggest that trypsin increases [Ca(2+)](i) in the endothelial cells through SKF-96365-insensitive Ca(2+) channels and regulates the release of NO, which results in relaxation of the rat aorta.
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Affiliation(s)
- Daijiro Kaneko
- Department of Veterinary Pharmacology, Yamaguchi University, 1677–1 Yoshida, Yamaguchi 753–8515, Japan
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27
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Abstract
There is an unacceptably high mortality in acute pancreatitis, which is due to the lack of specific treatments for the disease. A major reason stated to account for the inability to develop effective treatments is that there are multiple pathobiologic pathways activated in the acinar cell mediating pancreatitis making it difficult to choose molecular targets for therapeutic strategies. However, this reasoning limits opportunities for therapeutic development because it does include another important participant in pancreatitis - the pancreatic duct cells. The most recent advance in pancreatitis research is that depletion of both glycolytic and oxidative ATP synthesis is a common event in both acinar and ductal cells. Although ATP has a very short half-life in the blood and is hydrolysed to ADP, there is clear evidence that encapsulating ATP into liposomes can effectively drive ATP into the cells which can be effective in protecting them from necrosis. In this review, we will examine the effects of different insults associated with pancreatitis on both the acinar and ductal components of the exocrine pancreas pointing out the role of the ductal epithelial responses in both attenuating and increasing the severity of pancreatitis. In addition, we propose that exogenous ATP administration may restore ductal and acinar function providing therapeutic benefit.
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Affiliation(s)
- Péter Hegyi
- First Department of Medicine, University of Szeged, Szeged, Hungary.
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Orlichenko LS, Behari J, Yeh TH, Liu S, Stolz DB, Saluja AK, Singh VP. Transcriptional regulation of CXC-ELR chemokines KC and MIP-2 in mouse pancreatic acini. Am J Physiol Gastrointest Liver Physiol 2010; 299:G867-76. [PMID: 20671197 PMCID: PMC2957341 DOI: 10.1152/ajpgi.00177.2010] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Neutrophils and their chemoattractants, the CXC-ELR chemokines keratinocyte cytokine (KC) and macrophage inflammatory protein-2 (MIP-2), play a critical role in pancreatitis. While acute pancreatitis is initiated in acinar cells, it is unclear if these are a source of CXC-ELR chemokines. KC and MIP-2 have NF-κB, activator protein-1 (AP-1) sites in their promoter regions. However, previous studies have shown increased basal and reduced caerulein-induced AP-1 activation in harvested pancreatic tissue in vitro, which limits interpreting the caerulein-induced response. Moreover, recent studies suggest that NF-κB silencing in acinar cells alone may not be sufficient to reduce inflammation in acute pancreatitis. Thus the aim of this study was to determine whether acinar cells are a source of KC and MIP-2 and to understand their transcriptional regulation. Primary overnight-cultured murine pancreatic acini were used after confirming their ability to replicate physiological and pathological acinar cell responses. Upstream signaling resulting in KC, MIP-2 upregulation was studied along with activation of the transcription factors NF-κB and AP-1. Cultured acini replicated critical responses to physiological and pathological caerulein concentrations. KC and MIP-2 mRNA levels increased in response to supramaximal but not to physiological caerulein doses. This upregulation was calcium and protein kinase C (PKC), but not cAMP, dependent. NF-κB inhibition completely prevented upregulation of KC but not MIP-2. Complete suppression of MIP-2 upregulation required dual inhibition of NF-κB and AP-1. Acinar cells are a likely source of KC and MIP-2 upregulation during pancreatitis. This upregulation is dependent on calcium and PKC. MIP-2 upregulation requires both NF-κB and AP-1 in these cells. Thus dual inhibition of NF-κB and AP-1 may be a more successful strategy to reduce inflammation in pancreatitis than targeting NF-κB alone.
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Affiliation(s)
| | | | | | | | - Donna B. Stolz
- 2Cell Biology and Physiology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania; and
| | - Ashok K. Saluja
- 3Department of Surgery, University of Minnesota, Minneapolis, Minnesota
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29
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Long-term downregulation of protease-activated receptor-2 expression in distal colon in rats following bacillary dysentery. ACTA ACUST UNITED AC 2010; 163:49-56. [PMID: 20347884 DOI: 10.1016/j.regpep.2010.03.002] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2009] [Revised: 02/21/2010] [Accepted: 03/16/2010] [Indexed: 12/11/2022]
Abstract
The aim of this study was to determine changes of PAR-2 expression in distal colon and the sensitivity of colonic muscle to SLIGRL-NH2, the PAR-2-activating peptide (PAR-2-AP) following bacillary dysentery. Shigella flexneri was administrated intragastrically in healthy male rats to induce bacillary dysentery. The effect of SLIGRL-NH2 on the motility of colonic muscle strips were examined. The expression of PAR-2 was determined by immunohistochemistry and Western blotting. Intragastric administration of S.flexneri induced acute inflammation at the mucosa of the distal colon at 4-11 days, and the inflammation disappeared 18 days later. PAR-2-AP-induced TTX insensitive relaxation of the colonic muscle strips. This inhibitory effect on colonic circular muscle strips was reduced on days 11-35, but the carbachol-induced contraction did not change. PAR-2 was located at the colon smooth muscles cells and myenteric nerve plexus. The amount of PAR-2 expression in distal colon was down regulated on days 11-35. These data indicated that bacillary dysentery exerted a long-term downregulation on the expression of PAR-2 in distal colon. This might be the reason of the low sensitivity of the colon circular muscle strips to the PAR-2-AP-induced relaxation following intragastric administration of S.flexneri.
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30
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Jung SR, Hille B, Nguyen TD, Koh DS. Cyclic AMP potentiates Ca2+-dependent exocytosis in pancreatic duct epithelial cells. ACTA ACUST UNITED AC 2010; 135:527-43. [PMID: 20421376 PMCID: PMC2860593 DOI: 10.1085/jgp.200910355] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Exocytosis is evoked by intracellular signals, including Ca2+ and protein kinases. We determined how such signals interact to promote exocytosis in exocrine pancreatic duct epithelial cells (PDECs). Exocytosis, detected using carbon-fiber microamperometry, was stimulated by [Ca2+]i increases induced either through Ca2+ influx using ionomycin or by activation of P2Y2 or protease-activated receptor 2 receptors. In each case, the exocytosis was strongly potentiated when cyclic AMP (cAMP) was elevated either by activating adenylyl cyclase with forskolin or by activating the endogenous vasoactive intestinal peptide receptor. This potentiation was completely inhibited by H-89 and partially blocked by Rp-8-Br-cAMPS, inhibitors of protein kinase A. Optical monitoring of fluorescently labeled secretory granules showed slow migration toward the plasma membrane during Ca2+ elevations. Neither this Ca2+-dependent granule movement nor the number of granules found near the plasma membrane were detectably changed by raising cAMP, suggesting that cAMP potentiates Ca2+-dependent exocytosis at a later stage. A kinetic model was made of the exocytosis stimulated by UTP, trypsin, and Ca2+ ionophores with and without cAMP increase. In the model, without a cAMP rise, receptor activation stimulates exocytosis both by Ca2+ elevation and by the action of another messenger(s). With cAMP elevation the docking/priming step for secretory granules was accelerated, augmenting the releasable granule pool size, and the Ca2+ sensitivity of the final fusion step was increased, augmenting the rate of exocytosis. Presumably both cAMP actions require cAMP-dependent phosphorylation of target proteins. cAMP-dependent potentiation of Ca2+-induced exocytosis has physiological implications for mucin secretion and, possibly, for membrane protein insertion in the pancreatic duct. In addition, mechanisms underlying this potentiation of slow exocytosis may also exist in other cell systems.
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Affiliation(s)
- Seung-Ryoung Jung
- Department of Physiology and Biophysics and 2 Department of Medicine, University of Washington, Seattle, Washington 98195, USA
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31
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Itkonen O. Human trypsinogens in the pancreas and in cancer. Scandinavian Journal of Clinical and Laboratory Investigation 2010; 70:136-43. [PMID: 20163205 DOI: 10.3109/00365511003615317] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
This study led to the development of monoclonal antibodies and time-resolved immunofluorometric methods recognizing human trypsinogen-1 and -2, respectively. Using these methods in normal sera the concentration of trypsinogen-1 was found to be higher than that of trypsinogen-2. However, in acute pancreatitis the concentration of serum trypsinogen-2 was 50-fold higher than in controls, whereas the difference in trypsinogen-1 concentration was only 15-fold. Serum samples from patients who had undergone pancreatoduodenectomy contained trypsinogen-2, while trypsinogen-1 was detected in only one of nine samples. Furthermore, in human ovarian cyst fluids tumor-associated trypsinogen-2 (TAT-2) is the predominant isoenzyme and in mucinous cyst fluids the levels of TAT-2 were associated with malignancy. These results suggest that (i) trypsinogen-2 could be used as a diagnostic marker for acute pancreatitis, (ii) its expression is not restricted to the pancreas, and (iii) TAT could be involved in ovarian tumor dissemination and breakage of tissue barriers. In ion exchange chromatography, isoelectric variants of the trypsinogen isoenzymes were seen. Mass spectrometric analysis of these revealed that pancreatic trypsinogens are sulfated at tyrosine 154 (Tyr154), whereas TAT-2 from a colon carcinoma cell line is not. Tyr154 is located within the primary substrate binding pocket of trypsin. Thus, Tyr154 sulfation is likely to influence substrate binding. The previously known differences in charge and substrate binding between pancreatic and tumor-associated trypsinogens are suggested to be caused by sulfation of Tyr154 in pancreatic trypsinogens.
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Affiliation(s)
- Outi Itkonen
- Hospital District of Helsinki and Uusimaa - HUSLAB and Department of Clinical Chemistry, University of Helsinki, Helsinki, Finland.
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32
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Horie A, Nagai K, Ohkura S, Ohama T, Komatsu H, Sato K. Proinflammatory cytokines suppress the expression level of protease-activated receptor-2 through the induction of iNOS in rat colon. J Vet Med Sci 2010; 71:1609-15. [PMID: 20046028 DOI: 10.1292/jvms.001609] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Protease-activated receptor (PAR)-2 plays important roles in intestinal inflammatory responses and also contributes to intestinal digestive motility. In the distal colon of a rat experimental colitis model, expression level of PAR-2 mRNA was decreased, and relaxation through PAR-2 activation was attenuated. This study shows the effects of proinflammatory cytokines on changes to PAR-2 in rat colonic smooth muscle using an organ culture method. Colonic inflammation was induced in rats by administering dextran sodium sulphate in drinking water. Organ culture of distal colonic smooth muscle layer of normal rat was performed for up to 3 days. In the experimental colitis rat, mRNA expression levels of proinflammatory cytokines such as IL-1beta and TNF-alpha increased with inflammation. After the incubation with IL-1beta and TNF-alpha for 3 days, trypsin (PAR-2 agonist)-induced relaxation was attenuated, simultaneous with suppression of PAR-2 mRNA expression. Conversely, in this preparation, mRNA expression levels of iNOS were significantly increased. When l-NMMA was added to the medium with IL-1beta and TNF-alpha, changes to PAR-2 by these cytokine recovered. Moreover, when samples were cultured with NOC-18 (slow-releasing NO donor) for 3 days, relaxation induced by trypsin and expression of PAR-2 mRNA were attenuated. These results suggest that suppression of PAR-2 expression under inflammatory conditions is at least partially induced by NO produced in the colonic muscularis externa by proinflammatory cytokines.
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Affiliation(s)
- Ai Horie
- Laboratory of Veterinary Pharmacology, Faculty of Agriculture, Yamaguchi University
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33
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Yang YL, Serrano MG, Sheoran AS, Manque PA, Buck GA, Widmer G. Over-expression and localization of a host protein on the membrane of Cryptosporidium parvum infected epithelial cells. Mol Biochem Parasitol 2009; 168:95-101. [PMID: 19631240 PMCID: PMC2752322 DOI: 10.1016/j.molbiopara.2009.07.004] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2009] [Revised: 06/26/2009] [Accepted: 07/13/2009] [Indexed: 12/30/2022]
Abstract
The genus Cryptosporidium includes several species of intestinal protozoan parasites which multiply in intestinal epithelial cells. The impact of this infection on the transcriptome of cultured host cells was investigated using DNA microarray hybridizations. The expression of 14 genes found to be consistently up- or down-regulated in infected cell monolayers was validated with RT PCR. Using immunofluorescence we examined the expression of Protease Activated Receptor-2, which is encoded by one of the up-regulated genes. In infected cells this receptor localized to the host cell membrane which covers the intracellular trophozoites and meronts. This observation indicates that the composition of the host cell membrane is affected by the developing trophozoite, a phenomenon which has not been described previously.
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Affiliation(s)
- Yi-Lin Yang
- Tufts Cummings School of Veterinary Medicine, Division of Infectious Diseases, 200 Westboro Road, North Grafton, MA 01536, USA
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34
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Abstract
PURPOSE OF REVIEW The pancreatic duct epithelium is remarkable for its capacity to secrete HCO(3)(-) ions at concentrations as high as 140 mmol/l. The properties of the key transporters involved in this process and the central role played by cystic fibrosis transmembrane conductance regulator (CFTR) are the main focus of this review. RECENT FINDINGS The Cl(-)/HCO(3)(-) exchanger at the apical membrane of pancreatic duct cells is now known to be SLC26A6. The 1: 2 stoichiometry and electrogenicity of this exchanger enable it to contribute to the secretion of HCO(3)(-) at high concentrations. The apical CFTR channels also appear to have sufficient HCO(3)(-) permeability to contribute directly to HCO(3)(-) secretion. There is a strong possibility that the Ca(2+)-activated Cl(-) channels at the apical membrane are members of the bestrophin family which, like CFTR, are also permeable to HCO(3)(-). More has been learned about the complex interactions between CFTR and other transporters within macromolecular complexes coordinated at the apical membrane by scaffolding proteins. Further details are also emerging of the protective paracrine roles of nucleotides, nucleosides, bile acids and trypsin in the regulation of ductal secretion. SUMMARY Most of the key transporters involved in Cl(-) and HCO(3)(-) secretion have now been identified and characterized. Current research focuses on the molecular interactions between these transporters and the ways in which they are regulated by extracellular signals.
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35
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Moraes TJ, Rafii B, Niessen F, Suzuki T, Martin R, Vachon E, Vogel W, Ruf W, O'Brodovich H, Downey GP. Protease-activated receptor (Par)1 alters bioelectric properties of distal lung epithelia without compromising barrier function. Exp Lung Res 2009; 35:136-54. [PMID: 19263282 DOI: 10.1080/01902140802490723] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Proteinases contribute to the pathogenesis of various lung diseases, partly through activating cell surface receptors by limited proteolytic cleavage. The authors provide evidence that in primary cultures of distal lung epithelia, basolateral protease-activated receptor 1 activation rapidly reduces transepithelial resistance but does not alter paracellular permeability to small uncharged solutes. Changes in transepithelial resistance were partially blocked by ion transport inhibitors and were completely blocked by placing cells in low chloride buffer. In vivo studies did not reveal enhanced lung permeability in response to pulmonary or intravenous administration of protease-activated receptor 1 activators. This information is relevant as strategies to inhibit protease-activated receptor 1 signaling are considered in order to preserve lung epithelial barrier function.
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Affiliation(s)
- Theo J Moraes
- Department of Medicine, The University of Toronto, Toronto, Ontario, Canada
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36
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Ji B, Gaiser S, Chen X, Ernst SA, Logsdon CD. Intracellular trypsin induces pancreatic acinar cell death but not NF-kappaB activation. J Biol Chem 2009; 284:17488-98. [PMID: 19383608 DOI: 10.1074/jbc.m109.005520] [Citation(s) in RCA: 60] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
Premature intracellular activation of the digestive enzyme trypsinogen is considered to be the initiating event in pancreatitis. However, the direct consequences of intracellular trypsin activity have not previously been examined. In the current study, a mutant trypsinogen (paired basic amino acid cleaving enzyme (PACE)-trypsinogen), which is activated intracellularly by the endogenous protease PACE, was developed. This new construct allowed for the first time direct examination of the effects of intracellular trypsin on pancreatic acinar cells. We found that PACE-trypsinogen was expressed in the secretory pathway and was activated within acinar cells. Expression of PACE-trypsinogen induced apoptosis of HEK293 cells and pancreatic acinar cells, as indicated by histology, DNA laddering, PARP cleavage, and caspase-3 activation. Cell death was blocked by the trypsin inhibitor Pefabloc but not by the pancaspase inhibitor benzyloxycarbonyl-VAD, indicating that caspase-independent pathways were also involved. However, intracellular trypsin had no significant effect on the activity of the proinflammatory transcription factor NF-kappaB. In contrast, extracellular trypsin caused cell damage and dramatically increased NF-kappaB activity. These data indicate that localization of active trypsin determines its effects on pancreatic acinar cells. This new model will greatly improve our understanding of the role of active trypsin in pancreatitis and its associated inflammatory response.
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Affiliation(s)
- Baoan Ji
- Department of Cancer Biology, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA
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37
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Namkung W, Yoon JS, Kim KH, Lee MG. PAR2 exerts local protection against acute pancreatitis via modulation of MAP kinase and MAP kinase phosphatase signaling. Am J Physiol Gastrointest Liver Physiol 2008; 295:G886-94. [PMID: 18755806 DOI: 10.1152/ajpgi.00053.2008] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
During acute pancreatitis, protease-activated receptor 2 (PAR2) can be activated by interstitially released trypsin. In the mild form of pancreatitis, PAR2 activation exerts local protection against intrapancreatic damage, whereas, in the severe form of pancreatitis, PAR2 activation mediates some systemic complications. This study aimed to identify the molecular mechanisms of PAR2-mediated protective effects against intrapancreatic damage. A mild form of acute pancreatitis was induced by an intraperitoneal injection of caerulein (40 microg/kg) in rats. Effects of PAR2 activation on intrapancreatic damage and on mitogen-activated protein (MAP) kinase signaling were assessed. Caerulein treatment activated extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK) within 15 min and maintained phosphorylation of ERK and JNK for 2 h in the rat pancreas. Although PAR2 activation by the pretreatment with PAR2-activating peptide (AP) itself increased ERK phosphorylation in rat pancreas, the same treatment remarkably decreased caerulein-induced activation of ERK and JNK principally by accelerating their dephosphorylation. Inhibition of ERK and JNK phosphorylation by the pretreatment with MAP/ERK kinase (MEK) or JNK inhibitors decreased caerulein-induced pancreatic damage that was similar to the effect induced by PAR2-AP. Notably, in caerulein-treated rats, PAR2-AP cotreatment highly increased the expression of a group of MAP kinase phosphatases (MKPs) that deactivate ERK and JNK. The above results imply that downregulation of MAP kinase signaling by MKP induction is a key mechanism involved in the protective effects of PAR2 activation on caerulein-induced intrapancreatic damage.
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Affiliation(s)
- Wan Namkung
- Dept. of Pharmacology, Yonsei Univ. College of Medicine, Seoul 120-752, Korea
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38
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Pancreatic duct secretion: experimental methods, ion transport mechanisms and regulation. J Physiol Biochem 2008; 64:243-57. [PMID: 19244938 DOI: 10.1007/bf03178846] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
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39
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Morla L, Crambert G, Mordasini D, Favre G, Doucet A, Imbert-Teboul M. Proteinase-activated receptor 2 stimulates Na,K-ATPase and sodium reabsorption in native kidney epithelium. J Biol Chem 2008; 283:28020-8. [PMID: 18678869 DOI: 10.1074/jbc.m804399200] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Proteinase-activated receptors 2 (PAR2) are expressed in kidney, but their function is mostly unknown. Since PAR2 control ion transport in several epithelia, we searched for an effect on sodium transport in the cortical thick ascending limb of Henle's loop, a nephron segment that avidly reabsorbs NaCl, and for its signaling. Activation of PAR2, by either trypsin or a specific agonist peptide, increased the maximal activity of Na,K-ATPase, its apparent affinity for sodium, the sodium permeability of the paracellular pathway, and the lumen-positive transepithelial voltage, featuring increased NaCl reabsorption. PAR2 activation induced calcium signaling and phosphorylation of ERK1,2. PAR2-induced stimulation of Na,K-ATPase Vmax was fully prevented by inhibition of phospholipase C, of changes in intracellular concentration of calcium, of classical protein kinases C, and of ERK1,2 phosphorylation. PAR2-induced increase in paracellular sodium permeability was mediated by the same signaling cascade. In contrast, increase in the apparent affinity of Na,K-ATPase for sodium, although dependent on phospholipase C, was independent of calcium signaling, was insensitive to inhibitors of classical protein kinases C and of ERK1,2 phosphorylation, but was fully prevented by the nonspecific protein kinase inhibitor staurosporine, as was the increase in transepithelial voltage. In conclusion, PAR2 increases sodium reabsorption in rat thick ascending limb of Henle's loop along both the transcellular and the paracellular pathway. PAR2 effects are mediated in part by a phospholipase C/protein kinase C/ERK1,2 cascade, which increases Na,K-ATPase maximal activity and the paracellular sodium permeability, and by a different phospholipase C-dependent, staurosporine-sensitive cascade that controls the sodium affinity of Na,K-ATPase.
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Affiliation(s)
- Luciana Morla
- Université Pierre et Marie Curie, Univ Paris 06, UMR 7134, 75005 Paris
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40
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Søreide K. Proteinase-activated receptor 2 (PAR-2) in gastrointestinal and pancreatic pathophysiology, inflammation and neoplasia. Scand J Gastroenterol 2008; 43:902-9. [PMID: 19086162 DOI: 10.1080/00365520801942141] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Of all the body systems, the gastrointestinal (GI) tract is the most exposed to proteinases. Proteolytic activity must thus be tightly regulated in the face of diverse environmental challenges, because unrestrained or excessive proteolysis leads to pathological GI conditions. The protease-activated receptor-2 (PAR-2) is expressed in numerous cell types within the GI tract, suggesting both multiple functions and numerous modes of receptor activation. Although best known as a pancreatic digestive enzyme, trypsin has also been found in other tissues and various cancers. Of interest, trypsin and PAR-2 act together in an autocrine loop that promotes proliferation, invasion and metastasis in neoplasia through various mechanisms. Trypsin and PAR-2 seem to act both directly and indirectly through activation of other proteinase cascades, including metalloproteinases. PAR-2 activation can participate in inflammatory reactions, be protective to mucosal surfaces, send or inhibit nociceptive messages, modify gut motility or secretory functions, and stimulate cell proliferation and motility. Several studies point to a role for the PARs in disease processes of the GI tract and pancreas ranging from inflammatory bowel disease, symptoms associated with irritable bowel syndrome, pain in pancreatitis, development of colon and other GI cancers, and even infectious colitis. Proteinases should not only be considered from the traditional view as digestive or degradative enzymes in the gut, but additionally as signalling molecules that actively participate in the spectrum of physiology and diseased states of the GI tract.
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Affiliation(s)
- Kjetil Søreide
- Department of Surgery, Stavanger University Hospital, Stavanger, Norway.
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41
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Laukkarinen JM, Weiss ER, van Acker GJD, Steer ML, Perides G. Protease-activated receptor-2 exerts contrasting model-specific effects on acute experimental pancreatitis. J Biol Chem 2008; 283:20703-12. [PMID: 18511423 DOI: 10.1074/jbc.m801779200] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Protease-activated receptor-2 (PAR2) is a 7-transmembrane G-protein-coupled tethered ligand receptor that is expressed by pancreatic acinar and ductal cells. It can be physiologically activated by trypsin. Previously reported studies (Namkung, W., Han, W., Luo, X., Muallem, S., Cho, K. H., Kim, K. H., and Lee, M. G. (2004) Gastroenterology 126, 1844-1859; Sharma, A., Tao, X., Gopal, A., Ligon, B., Andrade-Gordon, P., Steer, M. L., and Perides, G. (2005) Am. J. Physiol. 288, G388-G395) have shown that PAR2 activation exerts a protective effect on the experimental model of pancreatitis induced by supramaximal secretagogue (caerulein) stimulation. We now show that PAR2 exerts a worsening effect on a different model of experimental pancreatitis, i.e. one induced by retrograde pancreatic ductal infusion of bile salts. In vitro studies using freshly prepared pancreatic acini show that genetic deletion of PAR2 reduces bile salt-induced pathological calcium transients, acinar cell injury, and activation of c-Jun N-terminal kinase, whereas genetic deletion of PAR2 has the opposite or no effect on these pancreatitis-related events when they are elicited, in vitro, by caerulein stimulation. Studies employing a combination of trypsin inhibition and activation of PAR2 with the activating peptide SLIGRL show that all these differences indeed depend on the activation of PAR2. These studies are the first to report that a single perturbation can have model-specific and opposite effects on pancreatitis, and they underscore the importance of performing mechanistic pancreatitis studies using two dissimilar models of the disease to detect idiosyncratic, model-specific events. We suggest PAR2 activation exerts a worsening effect on the severity of clinical pancreatitis and that interventions interfering with PAR2 activation may be of benefit in the treatment of patients with severe pancreatitis.
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Affiliation(s)
- Johanna M Laukkarinen
- Department of Surgery, Tufts Medical Center, 750 Washington Street, Boston, MA 02111, USA
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42
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Sipe WEB, Brierley SM, Martin CM, Phillis BD, Cruz FB, Grady EF, Liedtke W, Cohen DM, Vanner S, Blackshaw LA, Bunnett NW. Transient receptor potential vanilloid 4 mediates protease activated receptor 2-induced sensitization of colonic afferent nerves and visceral hyperalgesia. Am J Physiol Gastrointest Liver Physiol 2008; 294:G1288-98. [PMID: 18325985 DOI: 10.1152/ajpgi.00002.2008] [Citation(s) in RCA: 107] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Protease-activated receptor (PAR(2)) is expressed by nociceptive neurons and activated during inflammation by proteases from mast cells, the intestinal lumen, and the circulation. Agonists of PAR(2) cause hyperexcitability of intestinal sensory neurons and hyperalgesia to distensive stimuli by unknown mechanisms. We evaluated the role of the transient receptor potential vanilloid 4 (TRPV4) in PAR(2)-induced mechanical hyperalgesia of the mouse colon. Colonic sensory neurons, identified by retrograde tracing, expressed immunoreactive TRPV4, PAR(2), and calcitonin gene-related peptide and are thus implicated in nociception. To assess nociception, visceromotor responses (VMR) to colorectal distension (CRD) were measured by electromyography of abdominal muscles. In TRPV4(+/+) mice, intraluminal PAR(2) activating peptide (PAR(2)-AP) exacerbated VMR to graded CRD from 6-24 h, indicative of mechanical hyperalgesia. PAR(2)-induced hyperalgesia was not observed in TRPV4(-/-) mice. PAR(2)-AP evoked discharge of action potentials from colonic afferent neurons in TRPV4(+/+) mice, but not from TRPV4(-/-) mice. The TRPV4 agonists 5',6'-epoxyeicosatrienoic acid and 4alpha-phorbol 12,13-didecanoate stimulated discharge of action potentials in colonic afferent fibers and enhanced current responses recorded from retrogradely labeled colonic dorsal root ganglia neurons, confirming expression of functional TRPV4. PAR(2)-AP enhanced these responses, indicating sensitization of TRPV4. Thus TRPV4 is expressed by primary spinal afferent neurons innervating the colon. Activation of PAR(2) increases currents in these neurons, evokes discharge of action potentials from colonic afferent fibers, and induces mechanical hyperalgesia. These responses require the presence of functional TRPV4. Therefore, TRPV4 is required for PAR(2)-induced mechanical hyperalgesia and excitation of colonic afferent neurons.
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Affiliation(s)
- Walter E B Sipe
- Department of Pediatrics, University of California San Francisco, San Francisco, CA 94143-0660, USA
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43
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Kim MH, Choi BH, Jung SR, Sernka TJ, Kim S, Kim KT, Hille B, Nguyen TD, Koh DS. Protease-activated receptor-2 increases exocytosis via multiple signal transduction pathways in pancreatic duct epithelial cells. J Biol Chem 2008; 283:18711-20. [PMID: 18448425 DOI: 10.1074/jbc.m801655200] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Protease-activated receptor-2 (PAR-2) is activated when trypsin cleaves its NH(2) terminus to expose a tethered ligand. We previously demonstrated that PAR-2 activates ion channels in pancreatic duct epithelial cells (PDEC). Using real-time optical fluorescent probes, cyan fluorescence protein-Epac1-yellow fluorescence protein for cAMP, PH(PLC-delta1)-enhanced green fluorescent protein for phosphatidylinositol 4,5-bisphosphate, and protein kinase Cgamma (PKCgamma)-C1-yellow fluorescence protein for diacylglycerol, we now define the signaling pathways mediating PAR-2 effect in dog PDEC. Although PAR-2 activation does not stimulate a cAMP increase, it induces phospholipase C to hydrolyze phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate and diacylglycerol. Intracellular Ca(2+) mobilization from inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores and a subsequent Ca(2+) influx through store-operated Ca(2+) channels cause a biphasic increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), measured with Indo-1 dye. Single-cell amperometry demonstrated that this increase in [Ca(2+)](i) in turn causes a biphasic increase in exocytosis. A protein kinase assay revealed that trypsin also activates PKC isozymes to stimulate additional exocytosis. Paralleling the increased exocytosis, mucin secretion from PDEC was also induced by trypsin or the PAR-2 activating peptide. Consistent with the serosal localization of PAR-2, 1 microm luminal trypsin did not induce exocytosis in polarized PDEC monolayers; on the other hand, 10 microm trypsin at 37 degrees C damaged the epithelial barrier sufficiently so that it could reach and activate the serosal PAR-2 to stimulate exocytosis. Thus, in PDEC, PAR-2 activation increases [Ca(2+)](i) and activates PKC to stimulate exocytosis and mucin secretion. These functions may mediate the reported protective role of PAR-2 in different models of pancreatitis.
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Affiliation(s)
- Mean-Hwan Kim
- Department of Physics, POSTECH, Pohang 790-784, Republic of Korea
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Weiss FU, Halangk W, Lerch MM. New advances in pancreatic cell physiology and pathophysiology. Best Pract Res Clin Gastroenterol 2008; 22:3-15. [PMID: 18206809 DOI: 10.1016/j.bpg.2007.10.017] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The mammalian pancreas originates from two developing buds on the dorsal and ventral side of the duodenum which fuse and convert into a single mixed gland, composed of exocrine and endocrine cells. In the adult organism, the exocrine pancreas consists of acinar and ductal cells which are organised in a lobular branched tissue architecture and secrete and transport digestive enzymes into the duodenum. Mature endocrine cells, which represent only 1-2% of the pancreatic organ volume, form aggregates of so called islets of Langerhans within the exocrine pancreatic tissue and control glucose homeostasis by secretion of glucagon, insulin and other hormones into the bloodstream. Pancreatitis is the most common and a potentially lethal disorder of the exocrine pancreas with limited therapeutic options. A major obstacle in the development of successful treatment strategies has, until today, been our limited knowledge of the disease pathophysiology. This review will summarise recent advances in our understanding of the physiological mechanisms involved in the early disease processes of the exocrine pancreas.
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Affiliation(s)
- Frank Ulrich Weiss
- Department of Internal Medicine A, Ernst Moritz Arnd-University, Friedrich-Loeffler-STr. 23a, D17475 Greifswald, Germany
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45
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Proteinases and signalling: pathophysiological and therapeutic implications via PARs and more. Br J Pharmacol 2007; 153 Suppl 1:S263-82. [PMID: 18059329 DOI: 10.1038/sj.bjp.0707507] [Citation(s) in RCA: 215] [Impact Index Per Article: 11.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Proteinases like thrombin, trypsin and tissue kallikreins are now known to regulate cell signaling by cleaving and activating a novel family of G-protein-coupled proteinase-activated receptors (PARs 1-4) via exposure of a tethered receptor-triggering ligand. On their own, short synthetic PAR-selective PAR-activating peptides (PAR-APs) mimicking the tethered ligand sequences can activate PARs 1, 2 and 4 and cause physiological responses both in vitro and in vivo. Using the PAR-APs as sentinel probes in vivo, it has been found that PAR activation can affect the vascular, renal, respiratory, gastrointestinal, musculoskeletal and nervous systems (both central and peripheral nervous system) and can promote cancer metastasis and invasion. In general, responses triggered by PARs 1, 2 and 4 are in keeping with an innate immune inflammatory response, ranging from vasodilatation to intestinal inflammation, increased cytokine production and increased or decreased nociception. Further, PARs have been implicated in a number of disease states, including cancer and inflammation of the cardiovascular, respiratory, musculoskeletal, gastrointestinal and nervous systems. In addition to activating PARs, proteinases can cause hormone-like effects by other signalling mechanisms, like growth factor receptor activation, that may be as important as the activation of PARs. We, therefore, propose that the PARs themselves, their activating serine proteinases and their associated signalling pathways can be considered as attractive targets for therapeutic drug development. Thus, proteinases in general must now be considered as 'hormone-like' messengers that can signal either via PARs or other mechanisms.
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Saifeddine M, Seymour ML, Xiao YP, Compton SJ, Houle S, Ramachandran R, MacNaughton WK, Simonet S, Vayssettes-Courchay C, Verbeuren TJ, Hollenberg MD. Proteinase-activated receptor-2 activating peptides: distinct canine coronary artery receptor systems. Am J Physiol Heart Circ Physiol 2007; 293:H3279-89. [PMID: 17766477 DOI: 10.1152/ajpheart.00519.2007] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
In canine coronary artery preparations, the proteinase-activated receptor-2 (PAR2) activating peptides (PAR2-APs) SLIGRL-NH2and 2-furoyl-LIGRLO-NH2caused both an endothelium-dependent relaxation and an endothelium-independent contraction. Relaxation was caused at peptide concentrations 10-fold lower than those causing a contractile response. Although trans-cinnamoyl-LIGRLO-NH2, like other PAR2-APs, caused relaxation, it was inactive as a contractile agonist and instead antagonized the contractile response to SLIGRL-NH2. RT-PCR-based sequencing of canine PAR2revealed a cleavage/activation (indicated by underlines) sequence (SKGR/SLIGKTDSSLQITGKG) that is very similar to the human PAR2sequence (R/SLIGKV). As a synthetic peptide, the canine PAR-AP (SLIGKT-NH2) was a much less potent agonist than either SLIGRL-NH2or 2-furoyl-LIGRLO-NH2, either in the coronary contractile assay or in a Madin-Darby canine kidney (MDCK) cell PAR2calcium signaling assay. In the MDCK signaling assay, the order of potencies was as follows: 2-furoyl-LIGRLO-NH2≫ SLIGRL-NH2= trans-cinnamoyl-LIGRLO-NH2≫ SLIGKT-NH2, as expected for PAR2responses. In the coronary contractile assay, however, the order of potencies was very different: SLIGRL-NH2≫ 2-furoyl-LIGRLO-NH2≫ SLIGKT-NH2, trans-cinnamoyl-LIGRLO-NH2= antagonist. Because of 1) the distinct agonist (relaxant) and antagonist (contractile) activity of trans-cinnamoyl-LIGRLO-NH2in the canine coronary contractile assays, 2) the different concentration ranges over which the peptides caused either relaxation or contraction in the same coronary preparation, and 3) the markedly distinct structure-activity profiles for the PAR-APs in the coronary contractile assay, compared with those for PAR2-mediated MDCK cell calcium signaling, we suggest that the canine coronary tissue possesses a receptor system for the PAR-APs that is distinct from PAR2itself.
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MESH Headings
- Amino Acid Sequence
- Animals
- Calcium Signaling/drug effects
- Cell Line
- Coronary Vessels/drug effects
- Coronary Vessels/metabolism
- Dogs
- Dose-Response Relationship, Drug
- Endothelium, Vascular/drug effects
- Endothelium, Vascular/metabolism
- Epithelial Cells/drug effects
- Epithelial Cells/metabolism
- Indomethacin/pharmacology
- Molecular Sequence Data
- Oligopeptides/chemistry
- Oligopeptides/pharmacology
- RNA, Messenger/analysis
- Receptor, PAR-1/agonists
- Receptor, PAR-1/metabolism
- Receptor, PAR-2/agonists
- Receptor, PAR-2/genetics
- Receptor, PAR-2/metabolism
- Receptors, Neurokinin-1/metabolism
- Species Specificity
- Structure-Activity Relationship
- Vasoconstriction/drug effects
- Vasoconstrictor Agents/chemistry
- Vasoconstrictor Agents/pharmacology
- Vasodilation/drug effects
- Vasodilator Agents/chemistry
- Vasodilator Agents/pharmacology
- src-Family Kinases/metabolism
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Affiliation(s)
- Mahmoud Saifeddine
- Department of Pharmacology & Therapeutics, University of Calgary Faculty of Medicine, 3330 Hospital Drive N.W., Calgary, Alberta, Canada
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Kawabata A, Matsunami M, Sekiguchi F. Gastrointestinal roles for proteinase-activated receptors in health and disease. Br J Pharmacol 2007; 153 Suppl 1:S230-40. [PMID: 17994114 DOI: 10.1038/sj.bjp.0707491] [Citation(s) in RCA: 67] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
It has been almost a decade since the molecular cloning of all four members of the proteinase-activated receptor (PAR) family was completed. This unique family of G protein-coupled receptors (GPCRs) mediates specific cellular actions of various endogenous proteinases including thrombin, trypsin, tryptase, etc. and also certain exogenous enzymes. Increasing evidence has been clarifying the emerging roles played by PARs in health and disease. PARs, particularly PAR1 and PAR2, are distributed throughout the gastrointestinal (GI) tract, modulating various GI functions. One of the most important GI functions of PARs is regulation of exocrine secretion in the salivary glands, pancreas and GI mucosal epithelium. PARs also modulate motility of GI smooth muscle, involving multiple mechanisms. PAR2 appears to play dual roles in pancreatitis and related pain, being pro-inflammatory/pro-nociceptive and anti-inflammatory/anti-nociceptive. Similarly, dual roles for PAR1 and PAR2 have been demonstrated in mucosal inflammation/damage throughout the GI tract. There is also fundamental and clinical evidence for involvement of PAR2 in colonic pain. PARs are thus considered key molecules in regulation of GI functions and targets for development of drugs for treatment of various GI diseases.
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Affiliation(s)
- A Kawabata
- Division of Pharmacology and Pathophysiology, Kinki University School of Pharmacy, Higashi-Osaka, Japan.
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Lin KW, Park J, Crews AL, Li Y, Adler KB. Protease-activated receptor-2 (PAR-2) is a weak enhancer of mucin secretion by human bronchial epithelial cells in vitro. Int J Biochem Cell Biol 2007; 40:1379-88. [PMID: 18077203 PMCID: PMC2577811 DOI: 10.1016/j.biocel.2007.10.031] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2007] [Accepted: 10/29/2007] [Indexed: 11/21/2022]
Abstract
PAR-2, a member of a family of G-protein-coupled receptors, can be activated by serine proteases via proteolytic cleavage. PAR-2 expression is known to be upregulated in respiratory epithelium subsequent to inflammation in asthma and chronic obstructive pulmonary disease (COPD). Since these diseases also are characterized by excessive mucus production and secretion, we investigated whether PAR-2 could be linked to mucin hypersecretion by airway epithelium. Normal human bronchial epithelial (NHBE) cells in primary culture or the human bronchial epithelial cell lines, NCI-H292 and HBE-1, were used. NHBE, NCI-H292, and HBE-1 cells expressed prominent levels of PAR-2 protein. Short-term (30min) exposure of cells to the synthetic PAR-2 agonist peptide (SLIGKV-NH2) elicited a small but statistically significant increase in mucin secretion at high concentrations (100microM and 1000microM), compared to a control peptide with reversed amino acid sequence (VKGILS-NH2). Neither human lung tryptase nor bovine pancreatic trypsin, both PAR-2 agonists, affected NHBE cell mucin secretion when added over a range of concentrations. Knockdown of PAR-2 expression by siRNA blocked the stimulatory effect of the AP. The results suggest that, since PAR-2 activation only weakly increases mucin secretion by human airway epithelial cells in vitro, PAR-2 probably is not a significant contributor to mucin hypersecretion in inflamed airways.
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Affiliation(s)
- Ko-Wei Lin
- Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606, USA
| | - Joungjoa Park
- Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606, USA
| | - Anne L. Crews
- Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606, USA
| | | | - Kenneth B. Adler
- Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606, USA
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Jarry A, Dorso L, Gratio V, Forgue-Lafitte ME, Laburthe M, Laboisse CL, Darmoul D. PAR-2 activation increases human intestinal mucin secretion through EGFR transactivation. Biochem Biophys Res Commun 2007; 364:689-94. [PMID: 18028876 DOI: 10.1016/j.bbrc.2007.10.073] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2007] [Accepted: 10/12/2007] [Indexed: 12/26/2022]
Abstract
PAR-2 (protease-activated receptors-2) are G protein-coupled receptors whose action on mucin secretion by intestinal epithelial cells is still unknown. The aim of this study was to examine the effect of PAR-2 activation on mucin secretion in the human colonic goblet cell line HT29-Cl.16E and the intracellular pathways involved. We found that PAR-2 mRNA was constitutively expressed by HT29-Cl.16E cells as well as by isolated human normal colonocytes. The PAR-2-activating peptide SLIGKV-NH(2) elicited rapid mucin secretion in HT29-Cl.16E, which was partially inhibited by calcium chelator BAPTA. Inhibitors of MAPK activation (PD98059) and EGFR tyrosine kinase activity (AG1478) abrogated PAR-2-induced ERK1/2 and EGFR tyrosine phosphorylation, respectively, and subsequent mucin secretion. Finally, PAR-2-induced EGFR transactivation was involved upstream of ERK1/2 activation. Our results show that the activation of PAR-2 expressed by human intestinal epithelial cells enhances mucin secretion, a component of the intestinal innate defence, via a pathway involving EGFR transactivation.
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50
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Kawabata A, Matsunami M, Tsutsumi M, Ishiki T, Fukushima O, Sekiguchi F, Kawao N, Minami T, Kanke T, Saito N. Suppression of pancreatitis-related allodynia/hyperalgesia by proteinase-activated receptor-2 in mice. Br J Pharmacol 2007; 148:54-60. [PMID: 16520745 PMCID: PMC1617046 DOI: 10.1038/sj.bjp.0706708] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
1 Proteinase-activated receptor-2 (PAR2), a receptor activated by trypsin and tryptase, is abundantly expressed in the gastrointestinal tract including the C-fiber terminal, and might play a role in processing of visceral pain. In the present study, we examined and characterized the roles of PAR2 in pancreatitis-related abdominal hyperalgesia/allodynia in mice. 2 Caerulein, administered i.p. once, caused a small increase in abdominal sensitivity to stimulation with von Frey hairs, without causing pancreatitis, in PAR2-knockout (KO) mice, but not wild-type (WT) mice. 3 Caerulein, given hourly six times in total, caused more profound abdominal hyperalgesia/allodynia in PAR2-KO mice, as compared with WT mice, although no significant differences were detected in the severity of pancreatitis between the KO and WT animals. 4 The PAR2-activating peptide, 2-furoyl-LIGRL-NH(2), coadministered repeatedly with caerulein six times in total, abolished the caerulein-evoked abdominal hyperalgesia/allodynia in WT, but not PAR2-KO, mice. Repeated doses of 2-furoyl-LIGRL-NH(2) moderately attenuated the severity of caerulein-induced pancreatitis in WT animals. 5 Our data from experiments using PAR2-KO mice provide evidence that PAR2 functions to attenuate pancreatitis-related abdominal hyperalgesia/allodynia without affecting pancreatitis itself, although the PAR2AP applied exogenously is not only antinociceptive but also anti-inflammatory.
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Affiliation(s)
- Atsufumi Kawabata
- Division of Physiology and Pathophysiology, School of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-Osaka 577-8502, Japan.
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