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Zhu Q, Li M, Lu W, Wang Y, Li X, Cheng J. Transcriptomic Modulation Reveals the Specific Cellular Response in Chinese Sea Bass ( Lateolabrax maculatus) Gills under Salinity Change and Alkalinity Stress. Int J Mol Sci 2023; 24:ijms24065877. [PMID: 36982950 PMCID: PMC10056482 DOI: 10.3390/ijms24065877] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2023] [Revised: 02/27/2023] [Accepted: 03/12/2023] [Indexed: 03/30/2023] Open
Abstract
Salinity and alkalinity are among the important factors affecting the distribution, survival, growth and physiology of aquatic animals. Chinese sea bass (Lateolabrax maculatus) is an important aquaculture fish species in China that can widely adapt to diverse salinities from freshwater (FW) to seawater (SW) but moderately adapt to highly alkaline water (AW). In this study, juvenile L. maculatus were exposed to salinity change (SW to FW) and alkalinity stress (FW to AW). Coordinated transcriptomic responses in L. maculatus gills were investigated and based on the weighted gene co-expression network analysis (WGCNA), 8 and 11 stress-responsive modules (SRMs) were identified for salinity change and alkalinity stress, respectively, which revealed a cascade of cellular responses to oxidative and osmotic stress in L. maculatus gills. Specifically, four upregulated SRMs were enriched with induced differentially expressed genes (DEGs) for alkalinity stress, mainly corresponding to the functions of "extracellular matrix" and "anatomical structure", indicating a strong cellular response to alkaline water. Both "antioxidative activity" and "immune response" functions were enriched in the downregulated alkaline SRMs, which comprised inhibited alkaline specific DEGs, revealing the severely disrupted immune and antioxidative functions under alkalinity stress. These alkaline-specific responses were not revealed in the salinity change groups with only moderately inhibited osmoregulation and induced antioxidative response in L. maculatus gills. Therefore, the results revealed the diverse and correlated regulation of the cellular process and stress response in saline-alkaline water, which may have arisen through the functional divergence and adaptive recruitment of the co-expression genes and will provide vital insights for the development of L. maculatus cultivation in alkaline water.
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Affiliation(s)
- Qing Zhu
- Key Laboratory of Marine Genetics and Breeding (Ocean University of China), Ministry of Education, 5 Yushan Road, Qingdao 266003, China
- Key Laboratory of Tropical Aquatic Germplasm of Hainan Province, Sanya Oceanographic Institution, Ocean University of China, Sanya 572024, China
| | - Moli Li
- Key Laboratory of Marine Genetics and Breeding (Ocean University of China), Ministry of Education, 5 Yushan Road, Qingdao 266003, China
| | - Wei Lu
- Key Laboratory of Marine Genetics and Breeding (Ocean University of China), Ministry of Education, 5 Yushan Road, Qingdao 266003, China
| | - Yapeng Wang
- Key Laboratory of Marine Genetics and Breeding (Ocean University of China), Ministry of Education, 5 Yushan Road, Qingdao 266003, China
| | - Xujian Li
- Key Laboratory of Marine Genetics and Breeding (Ocean University of China), Ministry of Education, 5 Yushan Road, Qingdao 266003, China
| | - Jie Cheng
- Key Laboratory of Marine Genetics and Breeding (Ocean University of China), Ministry of Education, 5 Yushan Road, Qingdao 266003, China
- Key Laboratory of Tropical Aquatic Germplasm of Hainan Province, Sanya Oceanographic Institution, Ocean University of China, Sanya 572024, China
- Laboratory for Marine Fisheries Science and Food Production Processes, National Laboratory for Marine Science and Technology (Qingdao), 1 Wenhai Road, Qingdao 266237, China
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Donowitz M, Sarker R, Lin R, McNamara G, Tse CM, Singh V. Identification of Intestinal NaCl Absorptive-Anion Secretory Cells: Potential Functional Significance. Front Physiol 2022; 13:892112. [PMID: 35928564 PMCID: PMC9343792 DOI: 10.3389/fphys.2022.892112] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2022] [Accepted: 05/18/2022] [Indexed: 11/13/2022] Open
Abstract
Use of human enteroids studied in the undifferentiated and differentiated state that mimic the intestinal crypt and villus, respectively, has allowed studies of multiple enterocyte populations, including a large population of enterocytes that are transitioning from the crypt to the villus. This population expresses NHE3, DRA, and CFTR, representing a combination of Na absorptive and anion secretory functions. In this cell population, these three transporters physically interact, which affects their baseline and regulated activities. A study of this cell population and differentiated Caco-2 cells transduced with NHE3 and endogenously expressing DRA and CFTR has allowed an understanding of previous studies in which cAMP seemed to stimulate and inhibit DRA at the same time. Understanding the contributions of these cells to overall intestinal transport function as part of the fasting and post-prandial state and their contribution to the pathophysiology of diarrheal diseases and some conditions with constipation will allow new approaches to drug development.
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Affiliation(s)
- Mark Donowitz
- Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, United States
- Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, MD, United States
- *Correspondence: Mark Donowitz,
| | - Rafiquel Sarker
- Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, United States
| | - Ruxian Lin
- Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, United States
| | - George McNamara
- Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, United States
| | - Chung Ming Tse
- Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, United States
| | - Varsha Singh
- Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, United States
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Raraigh KS, Paul KC, Goralski JL, Worthington EN, Faino AV, Sciortino S, Wang Y, Aksit MA, Ling H, Osorio DL, Onchiri FM, Patel SU, Merlo CA, Montemayor K, Gibson RL, West NE, Thakerar A, Bridges RJ, Sheppard DN, Sharma N, Cutting GR. CFTR bearing variant p.Phe312del exhibits function inconsistent with phenotype and negligible response to ivacaftor. JCI Insight 2022; 7:148841. [PMID: 35315358 PMCID: PMC8986068 DOI: 10.1172/jci.insight.148841] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2021] [Accepted: 02/09/2022] [Indexed: 11/17/2022] Open
Abstract
The chloride channel dysfunction caused by deleterious cystic fibrosis transmembrane conductance regulator (CFTR) variants generally correlates with severity of cystic fibrosis (CF). However, 3 adults bearing the common severe variant p.Phe508del (legacy: F508del) and a deletion variant in an ivacaftor binding region of CFTR (p.Phe312del; legacy: F312del) manifested only elevated sweat chloride concentration (sw[Cl-]; 87-105 mEq/L). A database review of 25 individuals with F312del and a CF-causing variant revealed elevated sw[Cl-] (75-123 mEq/L) and variable CF features. F312del occurs at a higher-than-expected frequency in the general population, confirming that individuals with F312del and a CF-causing variant do not consistently develop overt CF features. In primary nasal cells, CFTR bearing F312del and F508del generated substantial chloride transport (66.0% ± 4.5% of WT-CFTR) but did not respond to ivacaftor. Single-channel analysis demonstrated that F312del did not affect current flow through CFTR, minimally altered gating, and ablated the ivacaftor response. When expressed stably in CF bronchial epithelial (CFBE41o-) cells, F312del-CFTR demonstrated residual function (50.9% ± 3.3% WT-CFTR) and a subtle decrease in forskolin response compared with WT-CFTR. F312del provides an exception to the established correlation between CFTR chloride transport and CF phenotype and informs our molecular understanding of ivacaftor response.
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Affiliation(s)
| | | | - Jennifer L Goralski
- University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
| | - Erin N Worthington
- University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
| | - Anna V Faino
- Seattle Children's Research Institute, Seattle, Washington, USA
| | - Stanley Sciortino
- California Department of Public Health, Genetic Disease Screening Program, Richmond, California, USA
| | - Yiting Wang
- University of Bristol, Bristol, United Kingdom
| | | | - Hua Ling
- Johns Hopkins University, Baltimore, Maryland, USA
| | | | | | | | | | | | | | | | - Amita Thakerar
- Rosalind Franklin University of Medicine and Science, Center for Genetic Diseases, North Chicago, Illinois, USA
| | - Robert J Bridges
- Rosalind Franklin University of Medicine and Science, Center for Genetic Diseases, North Chicago, Illinois, USA
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Wong R, Gu K, Ko Y, Patel P. Congenital absence of the vas deferens: Cystic fibrosis transmembrane regulatory gene mutations. Best Pract Res Clin Endocrinol Metab 2020; 34:101476. [PMID: 33353780 DOI: 10.1016/j.beem.2020.101476] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 10/22/2022]
Abstract
Congenital absence of the vas deferens (CAVD) is a rare genetic condition first discovered in the mid-18th century related to mutations in the cystic fibrosis transmembrane regulatory genes. The condition is typically found during work-up of male infertility, and the majority of cases can be diagnosed with complete history and physical examination and pertinent investigations. The condition can be separated into three subcategories, and genetic advances have led to a much better understanding behind the disease, its pathogenesis, and options for treatment. In this review, we discuss the genetics, pathogenesis, embryology, and diagnosis of treatment of CAVD. Future work in this area likely will aim to better understand the epigenetic factors that influence the development of the condition in order to identify potential upstream therapeutic targets.
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Affiliation(s)
- Rachel Wong
- Section of Urology, Department of Surgery, University of Manitoba, Winnipeg, Canada.
| | - Kaien Gu
- Department of Medicine, University of Manitoba, Winnipeg, Canada.
| | - Yool Ko
- Faculty of Science, Western University, London, Canada.
| | - Premal Patel
- Section of Urology, Department of Surgery, University of Manitoba, Winnipeg, Canada.
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Ma Z, Yuan D, Cheng X, Tuo B, Liu X, Li T. Function of ion transporters in maintaining acid-base homeostasis of the mammary gland and the pathophysiological role in breast cancer. Am J Physiol Regul Integr Comp Physiol 2019; 318:R98-R111. [PMID: 31553634 DOI: 10.1152/ajpregu.00202.2019] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
The incidence of breast cancer is increasing year by year, and the pathogenesis is still unclear. Studies have shown that the high metabolism of solid tumors leads to an increase in hypoxia, glycolysis, production of lactic acid and carbonic acid, and extracellular acidification; a harsh microenvironment; and ultimately to tumor cell death. Approximately 50% of locally advanced breast cancers exhibit hypoxia and/or local hypoxia, and acid-base regulatory proteins play an important role in regulating milk secretion and maintaining mammary gland physiological function. Therefore, ion transporters have gradually become a hot topic in mammary gland and breast cancer research. This review focuses on the research progress of ion transporters in mammary glands and breast cancer. We hope to provide new targets for the treatment and prognosis of breast cancer.
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Affiliation(s)
- Zhiyuan Ma
- Department of Thyroid and Breast Surgery, Affiliated Hospital of Zunyi Medical University, Zunyi, China
| | - Dumin Yuan
- Department of Gastroenterology, Affiliated Hospital of Zunyi Medical University, Zunyi, China.,Digestive Disease Institute of Guizhou Province, Zunyi, China
| | - Xiaoming Cheng
- Department of Thyroid and Breast Surgery, Affiliated Hospital of Zunyi Medical University, Zunyi, China
| | - Biguang Tuo
- Department of Gastroenterology, Affiliated Hospital of Zunyi Medical University, Zunyi, China.,Digestive Disease Institute of Guizhou Province, Zunyi, China
| | - Xuemei Liu
- Department of Gastroenterology, Affiliated Hospital of Zunyi Medical University, Zunyi, China.,Digestive Disease Institute of Guizhou Province, Zunyi, China
| | - Taolang Li
- Department of Thyroid and Breast Surgery, Affiliated Hospital of Zunyi Medical University, Zunyi, China
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SLC9A3 Affects Vas Deferens Development and Associates with Taiwanese Congenital Bilateral Absence of the Vas Deferens. BIOMED RESEARCH INTERNATIONAL 2019; 2019:3562719. [PMID: 30956978 PMCID: PMC6431446 DOI: 10.1155/2019/3562719] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/29/2018] [Revised: 01/06/2019] [Accepted: 02/03/2019] [Indexed: 12/21/2022]
Abstract
Background The pathophysiology of Taiwanese congenital bilateral absence of the vas deferens (CBAVD) is different from that in Caucasians. In particular, major cystic fibrosis transmembrane conductance regulator (CFTR) mutations and cystic fibrosis are absent in the former. Instead, deficiency in solute carrier family 9 sodium/hydrogen exchanger isoform 3 (SLC9A3) may play a role by generating obstructive azoospermia and degraded epithelial structure in the reproductive tract. Objectives The objective of the study was to test whether SLC9A3 variants cause Taiwanese CBAVD. Materials and Methods Six-month-old Slc9a3 -/-male mice were used to evaluate the effect of long-term SLC9A3 loss on the reproductive system. A case-control cohort of 29 men with CBAVD and 32 fertile men were genotyped for SLC9A3 variants. Results SLC9A3 was expressed and localized in the apical border of the epithelium of human vas deferens and glandular epithelium of the seminal vesicle. SLC9A3 deficiency specifically induces atrophy of vas deferens and unfolding of seminal vesicle mucosa in mice. Loss of SLC9A3 increased the incidence of CBAVD in humans from 3.1% to 37.9% (p < 0.001). Up to 75.9% of CBAVD patients carry at least one variant in either SLC9A3 or CFTR. Discussion Our findings build upon previous data associated with CBAVD pathogenesis. Here, we now report for the first time an association between CBAVD and loss of SLC9A3 and propose that specific defects in the reproductive duct due to SLC9A3 variants drive CBAVD development. Conclusion The data implicate loss of SLC9A3 as a basis of Taiwanese CBAVD and highlight SLC9A3 function in reproduction.
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7
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Massip-Copiz MM, Santa-Coloma TA. Extracellular pH and lung infections in cystic fibrosis. Eur J Cell Biol 2018; 97:402-410. [PMID: 29933921 DOI: 10.1016/j.ejcb.2018.06.001] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2018] [Revised: 06/01/2018] [Accepted: 06/12/2018] [Indexed: 12/11/2022] Open
Abstract
Cystic fibrosis (CF) is an autosomal recessive disease caused by CFTR mutations. It is characterized by high NaCl concentration in sweat and the production of a thick and sticky mucus, occluding secretory ducts, intestine and airways, accompanied by chronic inflammation and infections of the lungs. This causes a progressive and lethal decline in lung function. Therefore, finding the mechanisms driving the high susceptibility to lung infections has been a key issue. For decades the prevalent hypothesis was that a reduced airway surface liquid (ASL) volume and composition, and the consequent increased mucus concentration (dehydration), create an environment favoring infections. However, a few years ago, in a pig model of CF, the Na+/K+ concentrations and the ASL volume were found intact. Immediately a different hypothesis arose, postulating a reduced ASL pH as the cause for the increased susceptibility to infections, due to a diminished bicarbonate secretion through CFTR. Noteworthy, a recent report found normal ASL pH values in CF children and in cultured primary airway cells, challenging the ASL pH hypothesis. On the other hand, recent evidences revitalized the hypothesis of a reduced ASL secretion. Thus, the role of the ASL pH in the CF is still a controversial matter. In this review we discuss the basis that sustain the role of CFTR in modulating the extracellular pH, and the recent results sustaining the different points of view. Finding the mechanisms of CFTR signaling that determine the susceptibility to infections is crucial to understand the pathophysiology of CF and related lung diseases.
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Affiliation(s)
- María Macarena Massip-Copiz
- Laboratory of Cellular and Molecular Biology, Institute for Biomedical Research (BIOMED UCA-CONICET), The National Scientific and Technical Research Council (CONICET), and School of Medical Sciences, The Pontifical Catholic University of Argentina (UCA), Buenos Aires, Argentina
| | - Tomás Antonio Santa-Coloma
- Laboratory of Cellular and Molecular Biology, Institute for Biomedical Research (BIOMED UCA-CONICET), The National Scientific and Technical Research Council (CONICET), and School of Medical Sciences, The Pontifical Catholic University of Argentina (UCA), Buenos Aires, Argentina.
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Olesen CW, Vogensen J, Axholm I, Severin M, Schnipper J, Pedersen IS, von Stemann JH, Schrøder JM, Christensen DP, Pedersen SF. Trafficking, localization and degradation of the Na +,HCO 3- co-transporter NBCn1 in kidney and breast epithelial cells. Sci Rep 2018; 8:7435. [PMID: 29743600 PMCID: PMC5943355 DOI: 10.1038/s41598-018-25059-7] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2017] [Accepted: 04/13/2018] [Indexed: 01/28/2023] Open
Abstract
The Na+;HCO3− co-transporter NBCn1 (SLC4A7) is a major regulator of intracellular pH yet its trafficking and turnover are essentially unstudied. Here, we used MDCK-II and MCF-7 cells to investigate these processes in epithelial cells. GFP-NBCn1 membrane localization was abolished by truncation of the full NBCn1 C-terminal tail (C-tail) yet did not require the C-terminal PDZ-binding motif (ETSL). Glutathione-S-Transferase-pulldown of the C-tail followed by mass spectrometry analysis revealed putative interactions with multiple sorting-, degradation- and retention factors, including the scaffolding protein RACK1. Pulldown of FLAG-tagged deletion constructs mapped the RACK1 interaction to the proximal NBCn1 C-tail. Proximity Ligation Assay and co-immunoprecipitation confirmed that native NBCn1 interacts with RACK1 in a cellular context. Consistent with a functional role of this complex, RACK1 knockdown reduced NBCn1 membrane localization without affecting total NBCn1 expression. Notably, only non-confluent cells exhibited detectable NBCn1-RACK1 plasma membrane co-localization, suggesting that RACK1 regulates the trafficking of NBCn1 to the membrane. Whereas total NBCn1 degradation was slow, with a half-life of more than 24 h, one-third of surface NBCn1 was constitutively endocytosed from the basolateral membrane within 60 min. This suggests that a fraction of NBCn1 exhibits recycling between the basolateral membrane and intracellular compartment(s). Our findings have important implications for understanding NBCn1 regulation as well as its dysregulation in disease.
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Affiliation(s)
- Christina Wilkens Olesen
- Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen, Universitetsparken 13, DK-2100, Copenhagen Ø, Denmark
| | - Jens Vogensen
- Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen, Universitetsparken 13, DK-2100, Copenhagen Ø, Denmark
| | - Ida Axholm
- Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen, Universitetsparken 13, DK-2100, Copenhagen Ø, Denmark
| | - Marc Severin
- Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen, Universitetsparken 13, DK-2100, Copenhagen Ø, Denmark
| | - Julie Schnipper
- Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen, Universitetsparken 13, DK-2100, Copenhagen Ø, Denmark
| | - Isabella Skandorff Pedersen
- Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen, Universitetsparken 13, DK-2100, Copenhagen Ø, Denmark
| | - Jakob Hjorth von Stemann
- Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen, Universitetsparken 13, DK-2100, Copenhagen Ø, Denmark
| | - Jacob Morville Schrøder
- Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen, Universitetsparken 13, DK-2100, Copenhagen Ø, Denmark
| | - Dan Ploug Christensen
- Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen, Universitetsparken 13, DK-2100, Copenhagen Ø, Denmark.
| | - Stine Falsig Pedersen
- Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen, Universitetsparken 13, DK-2100, Copenhagen Ø, Denmark.
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Xie ZD, Guo YM, Ren MJ, Yang J, Wang SF, Xu TH, Chen LM, Liu Y. The Balance of [Formula: see text] Secretion vs. Reabsorption in the Endometrial Epithelium Regulates Uterine Fluid pH. Front Physiol 2018; 9:12. [PMID: 29422866 PMCID: PMC5788990 DOI: 10.3389/fphys.2018.00012] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2017] [Accepted: 01/05/2018] [Indexed: 12/26/2022] Open
Abstract
Uterine fluid contains a high concentration of HCO3- which plays an essential role in sperm capacitation and fertilization. In addition, the HCO3- concentration in uterine fluid changes periodically during the estrous cycle. It is well-known that the endometrial epithelium contains machineries involving the apical SLC26 family anion exchangers for secreting HCO3- into the uterine fluid. In the present study, we find for the first time that the electroneutral Na+/HCO3- cotransporter NBCn1 is expressed at the apical membrane of the endometrial epithelium. The protein abundance of the apical NBCn1 and that of the apical SLC26A4 and SLC26A6 are reciprocally regulated during the estrous cycle in the uterus. NBCn1 is most abundant at diestrus, whereas SLC26A4/A6 are most abundant at proestrus/estrus. In the ovariectomized mice, the expression of uterine NBCn1 is inhibited by β-estradiol, but stimulated by progesterone, whereas that of uterine SLC26A4/A6 is stimulated by β-estradiol. In vivo perfusion studies show that the endometrial epithelium is capable of both secreting and reabsorbing HCO3-. Moreover, the activity for HCO3- secretion by the endometrial epithelium is significantly higher at estrus than it is at diestrus. The opposite is true for HCO3- reabsorption. We conclude that the endometrial epithelium simultaneously contains the activity for HCO3- secretion involving the apical SLC26A4/A6 and the activity for HCO3- reabsorption involving the apical NBCn1, and that the acid-base homeostasis in the uterine fluid is regulated by the finely-tuned balance of the two activities.
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Affiliation(s)
- Zhang-Dong Xie
- Key Laboratory of Molecular Biophysics of Ministry of Education, Department of Biophysics and Molecular Physiology, School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China
| | - Yi-Min Guo
- Key Laboratory of Molecular Biophysics of Ministry of Education, Department of Biophysics and Molecular Physiology, School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China
| | - Mei-Juan Ren
- Key Laboratory of Molecular Biophysics of Ministry of Education, Department of Biophysics and Molecular Physiology, School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China
| | - Jichun Yang
- Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China
| | - Shao-Fang Wang
- Wuhan National Laboratory for Optoelectronics, Britton Chance Center for Biomedical Photonics, Huazhong University of Science and Technology, Wuhan, China
| | - Tong-Hui Xu
- Wuhan National Laboratory for Optoelectronics, Britton Chance Center for Biomedical Photonics, Huazhong University of Science and Technology, Wuhan, China
| | - Li-Ming Chen
- Key Laboratory of Molecular Biophysics of Ministry of Education, Department of Biophysics and Molecular Physiology, School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China
| | - Ying Liu
- Key Laboratory of Molecular Biophysics of Ministry of Education, Department of Biophysics and Molecular Physiology, School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China
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Arias-Hidalgo M, Al-Samir S, Weber N, Geers-Knörr C, Gros G, Endeward V. CO 2 permeability and carbonic anhydrase activity of rat cardiomyocytes. Acta Physiol (Oxf) 2017; 221:115-128. [PMID: 28429509 DOI: 10.1111/apha.12887] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2017] [Revised: 04/11/2017] [Accepted: 04/14/2017] [Indexed: 11/30/2022]
Abstract
AIM To determine the CO2 permeability (PCO2 ) of plasma membranes of cardiomyocytes. These cells were chosen because heart possesses the highest rate of O2 consumption/CO2 production in the body. METHODS Cardiomyocytes were isolated from rat hearts using the Langendorff technique. Cardiomyocyte suspensions exhibited a vitality of 2-14% and were studied by the previously described mass spectrometric 18 O-exchange technique deriving PCO2 . We showed by mass spectrometry and by carbonic anhydrase (CA) staining that non-vital cardiomyocytes are free of CA and thus do not contribute to the mass spectrometric signal, which is determined exclusively by the fully functional vital cardiomyocytes. RESULTS Lysed cardiomyocytes yielded an intracellular CA activity for vital cells of 5070; that is, the rate of CO2 hydration inside the cell is accelerated 5071-fold. Using this number, analyses of the mass spectrometric recordings from cardiomyocyte suspensions yield a PCO2 of 0.10 cm s-1 (SD ± 0.06, n = 15) at 37 °C. CONCLUSION In comparison with the PCO2 of other cells, this value is quite high and about identical to that of the human red cell membrane. As no major protein CO2 channels such as aquaporins 1 and 4 are present in rat cardiac sarcolemma, the high PCO2 of this membrane is likely due to its low cholesterol content of about 0.2 (mol cholesterol)·(mol total membrane lipids)-1 . Previous work predicted a PCO2 of ≥0.1 cm s-1 from this level of cholesterol. We conclude that the low cholesterol establishes a PCO2 high enough to render the membrane resistance to CO2 diffusion almost negligible, even under conditions of maximal O2 consumption of the heart.
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Affiliation(s)
- M. Arias-Hidalgo
- Molekular- und Zellphysiologie and AG Vegetative Physiologie; Medizinische Hochschule Hannover; Hannover Germany
| | - S. Al-Samir
- Molekular- und Zellphysiologie and AG Vegetative Physiologie; Medizinische Hochschule Hannover; Hannover Germany
| | - N. Weber
- Molekular- und Zellphysiologie and AG Vegetative Physiologie; Medizinische Hochschule Hannover; Hannover Germany
| | - C. Geers-Knörr
- Molekular- und Zellphysiologie and AG Vegetative Physiologie; Medizinische Hochschule Hannover; Hannover Germany
| | - G. Gros
- Molekular- und Zellphysiologie and AG Vegetative Physiologie; Medizinische Hochschule Hannover; Hannover Germany
| | - V. Endeward
- Molekular- und Zellphysiologie and AG Vegetative Physiologie; Medizinische Hochschule Hannover; Hannover Germany
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11
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Bicarbonate in cystic fibrosis. J Cyst Fibros 2017; 16:653-662. [PMID: 28732801 DOI: 10.1016/j.jcf.2017.06.005] [Citation(s) in RCA: 57] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2017] [Revised: 06/22/2017] [Accepted: 06/22/2017] [Indexed: 11/20/2022]
Abstract
BACKGROUND Cystic fibrosis (CF, mucoviscidosis) is caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR), which is a chloride and bicarbonate channel necessary for fluid secretion and extracellular alkalization. For a long time, research concentrated on abnormal Cl- and Na+ transport, but neglected bicarbonate as a crucial factor in CF. METHODS The present short review reports early findings as well as recent insights into the role of CFTR for bicarbonate transport and its defects in CF. RESULTS The available data indicate impaired bicarbonate transport not only in pancreas, intestine, airways, and reproductive organs, but also in salivary glands, sweat duct and renal tubular epithelial cells. Defective bicarbonate transport is closely related to the impaired mucus properties and mucus blocking in secretory organs of CF patients, causing the life threatening lung disease. CONCLUSIONS Apart from the devastating lung disease, abrogated bicarbonate transport also leads to many other organ dysfunctions, which are outlined in the present review.
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12
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Yurkiv VA, Melikhov VI, Shubin VS. Effect of Ionophores on Activity of Na +,Cl -(HCO 3- )-ATPase. Bull Exp Biol Med 2016; 161:763-766. [PMID: 27783300 DOI: 10.1007/s10517-016-3504-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2015] [Indexed: 12/01/2022]
Abstract
We studied the effects of ionophores on activity of Na+,Cl-(HCO 3- )-ATPase. The most significant effect on the activity of this enzyme was produced by protonophore 2,4-dinitrophenol. The effect of this drug largely depended on the cation and anion composition and pH of the incubation medium and its pH. Activity of Na+,Cl-(HCO 3- )-ATPase increased at neutral and weakly alkaline pH and decreased at pH below 6.5-6.7. In control animals (without histamine injection) with very weak or absent Na+,Cl-(HCO 3- )-ATPase activity, the observed effect of the protonophore on ATPase activity was also virtually absent. The stimulatory effect of other ionophores (monensine, valinomycin, and A23187) was significantly weaker and depended on pH of the incubation medium, its cationic and anionic composition, and concentration of these ionophores.
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Affiliation(s)
- V A Yurkiv
- Research Institute of General Pathology and Pathophysiology, Russian Academy of Sciences, Moscow, Russia
| | - V I Melikhov
- Research Institute of General Pathology and Pathophysiology, Russian Academy of Sciences, Moscow, Russia
| | - V S Shubin
- Research Institute of General Pathology and Pathophysiology, Russian Academy of Sciences, Moscow, Russia.
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13
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Yurkiv VA, Melikhov VI, Shubin VS. Substrate Specificity of Na +,Cl -(HCO 3-)-ATPase. Bull Exp Biol Med 2016; 161:651-653. [PMID: 27709378 DOI: 10.1007/s10517-016-3477-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2015] [Indexed: 10/20/2022]
Abstract
We studied substrate specificity of Na+,Cl-(HCO3-)-ATPase. In most cases, replacement of ATP for other phosphate-containing substances resulted in not only pronounced suppression of phosphohydrolase reactions, but also dramatic changes of their responsiveness to the stimulating effect of monovalent ions. The data showed that Na+,Cl-(HCO3-)-ATPase is a highly specific enzyme for ATP.
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Affiliation(s)
- V A Yurkiv
- Research Institute of General Pathology and Pathological Physiology, Russian Academy of Sciences, Moscow, Russia
| | - V I Melikhov
- Research Institute of General Pathology and Pathological Physiology, Russian Academy of Sciences, Moscow, Russia
| | - V S Shubin
- Research Institute of General Pathology and Pathological Physiology, Russian Academy of Sciences, Moscow, Russia.
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14
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Abstract
INTRODUCTION Mutations in the cystic fibrosis transmembrane conductance regulator protein (CFTR) cause cystic fibrosis (CF), a disease with life threatening pulmonary and gastrointestinal manifestations. Recent breakthrough therapies restore function to select disease-causing CFTR mutations. Ivacaftor is a small molecule that increases the open channel probability of certain CFTR mutations, producing clear evidence of bioactivity and efficacy in pediatric CF patients. CFTR modulators represent a significant advancement in CF treatment. Extending these therapies to young CF patients is proposed to have the greatest long term impact, potentially preventing later disease. AREAS COVERED Here we summarize the research experience of CFTR modulators in pediatrics, focusing on ivacaftor and highlighting challenges in pediatric studies. As a result of these studies, ivacaftor has been approved in CF patients age 2 years and older who have one of ten CFTR mutations. EXPERT OPINION Conducting studies in young CF patients presents unique challenges, including small numbers of patients and difficulty selecting sensitive biomarkers and meaningful outcome measures. Adverse events may be more pronounced in children and deserve special attention. Ongoing efforts must focus on expanding and validating new biomarkers, innovative study design, and thorough monitoring of adverse events in children treated with CFTR modulators.
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Affiliation(s)
- Elizabeth L Kramer
- Division of Pulmonary Medicine, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave, Cincinnati, OH 45229
| | - John P Clancy
- Division of Pulmonary Medicine, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave, Cincinnati, OH 45229
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15
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Hou Y, Guan X, Yang Z, Li C. Emerging role of cystic fibrosis transmembrane conductance regulator - an epithelial chloride channel in gastrointestinal cancers. World J Gastrointest Oncol 2016; 8:282-288. [PMID: 26989463 PMCID: PMC4789613 DOI: 10.4251/wjgo.v8.i3.282] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/05/2015] [Revised: 10/21/2015] [Accepted: 12/18/2015] [Indexed: 02/05/2023] Open
Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR), a glycoprotein with 1480 amino acids, has been well established as a chloride channel mainly expressed in the epithelial cells of various tissues and organs such as lungs, sweat glands, gastrointestinal system, and reproductive organs. Although defective CFTR leads to cystic fibrosis, a common genetic disorder in the Caucasian population, there is accumulating evidence that suggests a novel role of CFTR in various cancers, especially in gastroenterological cancers, such as pancreatic cancer and colon cancer. In this review, we summarize the emerging findings that link CFTR with various cancers, with focus on the association between CFTR defects and gastrointestinal cancers as well as the underlying mechanisms. Further study of CFTR in cancer biology may help pave a new way for the diagnosis and treatment of gastrointestinal cancers.
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16
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Abstract
Cystic Fibrosis (CF) is a rare, multisystem disease leading to significant morbidity and mortality. CF is caused by defects in the cystic fibrosis transmembrane conductance regulator protein (CFTR), a chloride and bicarbonate transporter. Early diagnosis and access to therapies provides benefits in nutrition, pulmonary health, and cognitive ability. Several screening and diagnostic tests are available to support a diagnosis. We discuss the characteristics of screening and diagnostic tests for CF and guideline-based algorithms using these tools to establish a diagnosis. We discuss classification and management of common "diagnostic dilemmas," including the CFTR-related metabolic syndrome and other CFTR-associated diseases.
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Affiliation(s)
- John Brewington
- Division of Pulmonary Medicine, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, MLC 2021, 3333 Burnet Avenue, Cincinnati, OH 45229, USA
| | - J P Clancy
- Division of Pulmonary Medicine, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, MLC 2021, 3333 Burnet Avenue, Cincinnati, OH 45229, USA.
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17
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Brewington JJ, McPhail GL, Clancy JP. Lumacaftor alone and combined with ivacaftor: preclinical and clinical trial experience of F508del CFTR correction. Expert Rev Respir Med 2015; 10:5-17. [PMID: 26581802 DOI: 10.1586/17476348.2016.1122527] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator protein (CFTR), leading to significant morbidity and mortality. CFTR is a chloride and bicarbonate channel at the epithelial cell membrane. The most common CFTR mutation is F508del, resulting in minimal CFTR at the plasma membrane. Current disease management is supportive, whereas an ultimate goal is to develop therapies to restore CFTR activity. We summarize experience with lumacaftor, a small molecule that increases F508del-CFTR levels at the plasma membrane. Lumacaftor in combination with ivacaftor, a modulator of CFTR gating defects, improves clinical outcome measures in patients homozygous for the F508del mutation. Lumacaftor represents a significant advancement in the treatment of biochemical abnormalities in CF. Further development of CFTR modulators will improve upon current therapies, although it remains unclear whether this approach will provide therapies for all CFTR mutations.
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Affiliation(s)
- John J Brewington
- a Division of Pulmonary Medicine, Department of Pediatrics , Cincinnati Children's Hospital Medical Center and the University of Cincinnati , Cincinnati , OH , USA
| | - Gary L McPhail
- a Division of Pulmonary Medicine, Department of Pediatrics , Cincinnati Children's Hospital Medical Center and the University of Cincinnati , Cincinnati , OH , USA
| | - John P Clancy
- a Division of Pulmonary Medicine, Department of Pediatrics , Cincinnati Children's Hospital Medical Center and the University of Cincinnati , Cincinnati , OH , USA
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18
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Liu Y, Yang J, Chen LM. Structure and Function of SLC4 Family [Formula: see text] Transporters. Front Physiol 2015; 6:355. [PMID: 26648873 PMCID: PMC4664831 DOI: 10.3389/fphys.2015.00355] [Citation(s) in RCA: 46] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2015] [Accepted: 11/10/2015] [Indexed: 12/12/2022] Open
Abstract
The solute carrier SLC4 family consists of 10 members, nine of which are [Formula: see text] transporters, including three Na(+)-independent Cl(-)/[Formula: see text] exchangers AE1, AE2, and AE3, five Na(+)-coupled [Formula: see text] transporters NBCe1, NBCe2, NBCn1, NBCn2, and NDCBE, as well as "AE4" whose Na(+)-dependence remains controversial. The SLC4 [Formula: see text] transporters play critical roles in pH regulation and transepithelial movement of electrolytes with a broad range of demonstrated physiological relevances. Dysfunctions of these transporters are associated with a series of human diseases. During the past decades, tremendous amount of effort has been undertaken to investigate the topological organization of the SLC4 transporters in the plasma membrane. Based upon the proposed topology models, mutational and functional studies have identified important structural elements likely involved in the ion translocation by the SLC4 transporters. In the present article, we review the advances during the past decades in understanding the structure and function of the SLC4 transporters.
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Affiliation(s)
- Ying Liu
- Key Laboratory of Molecular Biophysics of Ministry of Education, Department of Biophysics and Molecular Physiology, School of Life Science and Technology, Huazhong University of Science and TechnologyWuhan, China
| | - Jichun Yang
- Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University Health Science CenterBeijing, China
| | - Li-Ming Chen
- Key Laboratory of Molecular Biophysics of Ministry of Education, Department of Biophysics and Molecular Physiology, School of Life Science and Technology, Huazhong University of Science and TechnologyWuhan, China
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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: 36] [Impact Index Per Article: 3.6] [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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20
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Namkoong E, Shin YH, Bae JS, Choi S, Kim M, Kim N, Hwang SM, Park K. Role of Sodium Bicarbonate Cotransporters in Intracellular pH Regulation and Their Regulatory Mechanisms in Human Submandibular Glands. PLoS One 2015; 10:e0138368. [PMID: 26375462 PMCID: PMC4573515 DOI: 10.1371/journal.pone.0138368] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2015] [Accepted: 08/28/2015] [Indexed: 01/16/2023] Open
Abstract
Sodium bicarbonate cotransporters (NBCs) are involved in the pH regulation of salivary glands. However, the roles and regulatory mechanisms among different NBC isotypes have not been rigorously evaluated. We investigated the roles of two different types of NBCs, electroneutral (NBCn1) and electrogenic NBC (NBCe1), with respect to pH regulation and regulatory mechanisms using human submandibular glands (hSMGs) and HSG cells. Intracellular pH (pHi) was measured and the pHi recovery rate from cell acidification induced by an NH4Cl pulse was recorded. Subcellular localization and protein phosphorylation were determined using immunohistochemistry and co-immunoprecipitation techniques. We determined that NBCn1 is expressed on the basolateral side of acinar cells and the apical side of duct cells, while NBCe1 is exclusively expressed on the apical membrane of duct cells. The pHi recovery rate in hSMG acinar cells, which only express NBCn1, was not affected by pre-incubation with 5 μM PP2, an Src tyrosine kinase inhibitor. However, in HSG cells, which express both NBCe1 and NBCn1, the pHi recovery rate was inhibited by PP2. The apparent difference in regulatory mechanisms for NBCn1 and NBCe1 was evaluated by artificial overexpression of NBCn1 or NBCe1 in HSG cells, which revealed that the pHi recovery rate was only inhibited by PP2 in cells overexpressing NBCe1. Furthermore, only NBCe1 was significantly phosphorylated and translocated by NH4Cl, which was inhibited by PP2. Our results suggest that both NBCn1 and NBCe1 play a role in pHi regulation in hSMG acinar cells, and also that Src kinase does not regulate the activity of NBCn1.
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Affiliation(s)
- Eun Namkoong
- Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul, 110-749, Korea
| | - Yong-Hwan Shin
- Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul, 110-749, Korea
| | - Jun-Seok Bae
- Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul, 110-749, Korea
| | - Seulki Choi
- Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul, 110-749, Korea
| | - Minkyoung Kim
- Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul, 110-749, Korea
| | - Nahyun Kim
- Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul, 110-749, Korea
| | - Sung-Min Hwang
- Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul, 110-749, Korea
| | - Kyungpyo Park
- Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul, 110-749, Korea
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21
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Abstract
Cation-coupled HCO3(-) transport was initially identified in the mid-1970s when pioneering studies showed that acid extrusion from cells is stimulated by CO2/HCO3(-) and associated with Na(+) and Cl(-) movement. The first Na(+)-coupled bicarbonate transporter (NCBT) was expression-cloned in the late 1990s. There are currently five mammalian NCBTs in the SLC4-family: the electrogenic Na,HCO3-cotransporters NBCe1 and NBCe2 (SLC4A4 and SLC4A5 gene products); the electroneutral Na,HCO3-cotransporter NBCn1 (SLC4A7 gene product); the Na(+)-driven Cl,HCO3-exchanger NDCBE (SLC4A8 gene product); and NBCn2/NCBE (SLC4A10 gene product), which has been characterized as an electroneutral Na,HCO3-cotransporter or a Na(+)-driven Cl,HCO3-exchanger. Despite the similarity in amino acid sequence and predicted structure among the NCBTs of the SLC4-family, they exhibit distinct differences in ion dependency, transport function, pharmacological properties, and interactions with other proteins. In epithelia, NCBTs are involved in transcellular movement of acid-base equivalents and intracellular pH control. In nonepithelial tissues, NCBTs contribute to intracellular pH regulation; and hence, they are crucial for diverse tissue functions including neuronal discharge, sensory neuron development, performance of the heart, and vascular tone regulation. The function and expression levels of the NCBTs are generally sensitive to intracellular and systemic pH. Animal models have revealed pathophysiological roles of the transporters in disease states including metabolic acidosis, hypertension, visual defects, and epileptic seizures. Studies are being conducted to understand the physiological consequences of genetic polymorphisms in the SLC4-members, which are associated with cancer, hypertension, and drug addiction. Here, we describe the current knowledge regarding the function, structure, and regulation of the mammalian cation-coupled HCO3(-) transporters of the SLC4-family.
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Affiliation(s)
- Christian Aalkjaer
- Department of Biomedicine, and the Water and Salt Research Center, Aarhus University, Aarhus, Denmark; Department of Physiology, Emory University School of Medicine, Atlanta, USA
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22
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Bernardino RL, Martins AD, Jesus TT, Sá R, Sousa M, Alves MG, Oliveira PF. Estrogenic regulation of bicarbonate transporters from SLC4 family in rat Sertoli cells. Mol Cell Biochem 2015; 408:47-54. [DOI: 10.1007/s11010-015-2481-4] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2015] [Accepted: 06/13/2015] [Indexed: 11/30/2022]
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23
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Kong SC, Giannuzzo A, Gianuzzo A, Novak I, Pedersen SF. Acid-base transport in pancreatic cancer: molecular mechanisms and clinical potential. Biochem Cell Biol 2014; 92:449-59. [PMID: 25372771 DOI: 10.1139/bcb-2014-0078] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
Solid tumors are characterized by a microenvironment that is highly acidic, while intracellular pH (pHi) is normal or even elevated. This is the result of elevated metabolic rates in the highly proliferative cancer cells, in conjunction with often greatly increased rates of net cellular acid extrusion. Studies in various cancers have suggested that while the acid extrusion mechanisms employed are generally the same as those in healthy cells, the specific transporters upregulated vary with the cancer type. The main such transporters include Na(+)/H(+) exchangers, various HCO3(-) transporters, H(+) pumps, and lactate-H(+) cotransporters. The mechanisms leading to their dysregulation in cancer are incompletely understood but include changes in transporter expression levels, trafficking and membrane localization, and posttranslational modifications. In turn, accumulating evidence has revealed that in addition to supporting their elevated metabolic rate, their increased acid efflux capacity endows the cancer cells with increased capacity for invasiveness, proliferation, and chemotherapy resistance. The pancreatic duct exhibits an enormous capacity for acid-base transport, rendering pHi dysregulation a potentially very important topic in pancreatic ductal adenocarcinoma (PDAC). PDAC - accounting for about 90% of all pancreatic cancers - has one of the highest cancer mortality rates known, and new diagnostic and treatment options are highly needed. However, very little is known about whether pH regulation is altered in PDAC and, if so, the possible role of this in cancer development. Here, we review current models for pancreatic acid-base transport and pH homeostasis and summarize current views on acid-base dysregulation in cancer, focusing where possible on the few studies to date in PDAC. Finally, we present new data-mining analyses of acid-base transporter expression changes in PDAC and discuss essential directions for future work.
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Affiliation(s)
- Su Chii Kong
- a Section for Cell and Developmental Biology, Department of Biology, Faculty of Science, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen, Denmark
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24
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Lee HJ, Kwon MH, Lee S, Hall RA, Yun CC, Choi I. Systematic family-wide analysis of sodium bicarbonate cotransporter NBCn1/SLC4A7 interactions with PDZ scaffold proteins. Physiol Rep 2014; 2:2/5/e12016. [PMID: 24844638 PMCID: PMC4098744 DOI: 10.14814/phy2.12016] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
NBCn1 (SLC4A7) plays a role in transepithelial HCO3 (-) movement and intracellular pH maintenance in many tissues. In this study, we searched PDZ proteins capable of binding to NBCn1. We screened a protein array membrane, on which 96 different class I PDZ protein peptides were blotted, with the C-terminal domain of NBCn1 fused to GST. Thirteen proteins were identified in these screens: MAGI-3, NHERF-1, NHERF-2, PSD-95, chapsyn-110, ERBIN, MALS-1, densin-180, syntrophins α1, β2, γ2, MUPP1, and PDZK1. After determining these binding partners, we analyzed the database of known and predicted protein interactions to obtain an NBCn1 interaction network. The network shows NBCn1 being physically and functionally associated with a variety of membrane and cytosolic proteins via the binding partners. We then focused on syntrophin γ2 to examine the molecular and functional interaction between NBCn1 and one of the identified binding partners in the Xenopus oocyte expression system. GST/NBCn1 pulled down syntrophin γ2 and conversely GST/syntrophin γ2 pulled down NBCn1. Moreover, syntrophin γ2 increased intracellular pH recovery, from acidification, mediated by NBCn1's Na/HCO3 cotransport. Syntrophin γ2 also increased an ionic conductance produced by NBCn1 channel-like activity. Thus, syntrophin γ2 regulates NBCn1 activity. In conclusion, this study demonstrates that NBCn1 binds to many PDZ proteins, which in turn may allow the transporter to associate with other physiologically important proteins.
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Affiliation(s)
- Hye Jeong Lee
- Department of Pediatrics, Division of Hematology and Oncology, Vanderbilt University, Nashville, Tennessee, USA
| | - Min Hyung Kwon
- Department of Physiology, Emory University School of Medicine, Atlanta, Georgia, USA
| | - Soojung Lee
- Department of Physiology, Emory University School of Medicine, Atlanta, Georgia, USA
| | - Randy A Hall
- Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia, USA
| | - C Chris Yun
- Department of Medicine, Division of Digestive Disease, Emory University School of Medicine, Atlanta, Georgia, USA
| | - Inyeong Choi
- Department of Physiology, Emory University School of Medicine, Atlanta, Georgia, USA
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25
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Hong JH, Park S, Shcheynikov N, Muallem S. Mechanism and synergism in epithelial fluid and electrolyte secretion. Pflugers Arch 2013; 466:1487-99. [PMID: 24240699 DOI: 10.1007/s00424-013-1390-1] [Citation(s) in RCA: 50] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2013] [Revised: 10/16/2013] [Accepted: 10/17/2013] [Indexed: 01/04/2023]
Abstract
A central function of epithelia is the control of the volume and electrolyte composition of bodily fluids through vectorial transport of electrolytes and the obligatory H2O. In exocrine glands, fluid and electrolyte secretion is carried out by both acinar and duct cells, with the portion of fluid secreted by each cell type varying among glands. All acinar cells secrete isotonic, plasma-like fluid, while the duct determines the final electrolyte composition of the fluid by absorbing most of the Cl(-) and secreting HCO3 (-). The key transporters mediating acinar fluid and electrolyte secretion are the basolateral Na(+)/K(+) /2Cl(-) cotransporter, the luminal Ca(2+)-activated Cl(-) channel ANO1 and basolateral and luminal Ca(2+)-activated K(+) channels. Ductal fluid and HCO3 (-) secretion are mediated by the basolateral membrane Na(+)-HCO3 (-) cotransporter NBCe1-B and the luminal membrane Cl(-)/HCO3 (-) exchanger slc26a6 and the Cl(-) channel CFTR. The function of the transporters is regulated by multiple inputs, which in the duct include major regulation by the WNK/SPAK pathway that inhibit secretion and the IRBIT/PP1 pathway that antagonize the effects of the WNK/SPAK pathway to both stimulate and coordinate the secretion. The function of these regulatory pathways in secretory glands acinar cells is yet to be examined. An important concept in biology is synergism among signaling pathways to generate the final physiological response that ensures regulation with high fidelity and guards against cell toxicity. While synergism is observed in all epithelial functions, the molecular mechanism mediating the synergism is not known. Recent work reveals a central role for IRBIT as a third messenger that integrates and synergizes the function of the Ca(2+) and cAMP signaling pathways in activation of epithelial fluid and electrolyte secretion. These concepts are discussed in this review using secretion by the pancreatic and salivary gland ducts as model systems.
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Affiliation(s)
- Jeong Hee Hong
- Epithelial Signaling and Transport Section, Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institute of Health, Bethesda, MD, 20892, USA
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26
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Romero MF, Chen AP, Parker MD, Boron WF. The SLC4 family of bicarbonate (HCO₃⁻) transporters. Mol Aspects Med 2013; 34:159-82. [PMID: 23506864 DOI: 10.1016/j.mam.2012.10.008] [Citation(s) in RCA: 247] [Impact Index Per Article: 20.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2012] [Accepted: 08/28/2012] [Indexed: 01/13/2023]
Abstract
The SLC4 family consists of 10 genes (SLC4A1-5; SLC4A7-11). All encode integral membrane proteins with very similar hydropathy plots-consistent with 10-14 transmembrane segments. Nine SLC4 members encode proteins that transport HCO3(-) (or a related species, such as CO3(2-)) across the plasma membrane. Functionally, eight of these proteins fall into two major groups: three Cl-HCO3 exchangers (AE1-3) and five Na(+)-coupled HCO3(-) transporters (NBCe1, NBCe2, NBCn1, NBCn2, NDCBE). Two of the Na(+)-coupled transporters (NBCe1, NBCe2) are electrogenic; the other three Na(+)-coupled HCO3(-) transporters and all three AEs are electroneutral. In addition, two other SLC4 members (AE4, SLC4A9 and BTR1, SLC4A11) do not yet have a firmly established function. Most, though not all, SLC4 members are functionally inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). SLC4 proteins play important roles many modes of acid-base homeostasis: the carriage of CO2 by erythrocytes, the transport of H(+) or HCO3(-) by several epithelia, as well as the regulation of cell volume and intracellular pH.
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Affiliation(s)
- Michael F Romero
- Physiology & Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
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Parker MD, Boron WF. The divergence, actions, roles, and relatives of sodium-coupled bicarbonate transporters. Physiol Rev 2013; 93:803-959. [PMID: 23589833 PMCID: PMC3768104 DOI: 10.1152/physrev.00023.2012] [Citation(s) in RCA: 208] [Impact Index Per Article: 17.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
The mammalian Slc4 (Solute carrier 4) family of transporters is a functionally diverse group of 10 multi-spanning membrane proteins that includes three Cl-HCO3 exchangers (AE1-3), five Na(+)-coupled HCO3(-) transporters (NCBTs), and two other unusual members (AE4, BTR1). In this review, we mainly focus on the five mammalian NCBTs-NBCe1, NBCe2, NBCn1, NDCBE, and NBCn2. Each plays a specialized role in maintaining intracellular pH and, by contributing to the movement of HCO3(-) across epithelia, in maintaining whole-body pH and otherwise contributing to epithelial transport. Disruptions involving NCBT genes are linked to blindness, deafness, proximal renal tubular acidosis, mental retardation, and epilepsy. We also review AE1-3, AE4, and BTR1, addressing their relevance to the study of NCBTs. This review draws together recent advances in our understanding of the phylogenetic origins and physiological relevance of NCBTs and their progenitors. Underlying these advances is progress in such diverse disciplines as physiology, molecular biology, genetics, immunocytochemistry, proteomics, and structural biology. This review highlights the key similarities and differences between individual NCBTs and the genes that encode them and also clarifies the sometimes confusing NCBT nomenclature.
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Affiliation(s)
- Mark D Parker
- Dept. of Physiology and Biophysics, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106-4970, USA.
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Danielsen AA, Parker MD, Lee S, Boron WF, Aalkjaer C, Boedtkjer E. Splice cassette II of Na+,HCO3(-) cotransporter NBCn1 (slc4a7) interacts with calcineurin A: implications for transporter activity and intracellular pH control during rat artery contractions. J Biol Chem 2013; 288:8146-8155. [PMID: 23382378 PMCID: PMC3605633 DOI: 10.1074/jbc.m113.455386] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2013] [Indexed: 11/06/2022] Open
Abstract
Activation of Na(+),HCO3(-) cotransport in vascular smooth muscle cells (VSMCs) contributes to intracellular pH (pH(i)) control during artery contraction, but the signaling pathways involved have been unknown. We investigated whether physical and functional interactions between the Na(+),HCO3(-) cotransporter NBCn1 (slc4a7) and the Ca(2+)/calmodulin-activated serine/threonine phosphatase calcineurin exist and play a role for pHi control in VSMCs. Using a yeast two-hybrid screen, we found that splice cassette II from the N terminus of NBCn1 interacts with calcineurin Aβ. When cassette II was truncated or mutated to disrupt the putative calcineurin binding motif PTVVIH, the interaction was abolished. Native NBCn1 and calcineurin Aβ co-immunoprecipitated from A7r5 rat VSMCs. A peptide (acetyl-DDIPTVVIH-amide), which mimics the putative calcineurin binding motif, inhibited the co-immunoprecipitation whereas a mutated peptide (acetyl-DDIATAVAA-amide) did not. Na(+),HCO3(-) cotransport activity was investigated in VSMCs of mesenteric arteries after an NH4(+) prepulse. During depolarization with 50 mM extracellular K(+) to raise intracellular [Ca(2+)], Na(+),HCO3(-) cotransport activity was inhibited 20-30% by calcineurin inhibitors (FK506 and cyclosporine A). FK506 did not affect Na(+),HCO3(-) cotransport activity in VSMCs when cytosolic [Ca(2+)] was lowered by buffering, nor did it disrupt binding between NBCn1 and calcineurin Aβ. FK506 augmented the intracellular acidification of VSMCs during norepinephrine-induced artery contractions. No physical or functional interactions between calcineurin Aβ and the Na(+)/H(+) exchanger NHE1 were observed in VSMCs. In conclusion, we demonstrate a physical interaction between calcineurin Aβ and cassette II of NBCn1. Intracellular Ca(2+) activates Na(+),HCO3(-) cotransport activity in VSMCs in a calcineurin-dependent manner which is important for protection against intracellular acidification.
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Affiliation(s)
- Andreas A Danielsen
- Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark; Water and Salt Research Center, Aarhus University, DK-8000 Aarhus C, Denmark
| | - Mark D Parker
- Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106
| | - Soojung Lee
- Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark; Water and Salt Research Center, Aarhus University, DK-8000 Aarhus C, Denmark
| | - Walter F Boron
- Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106
| | - Christian Aalkjaer
- Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark; Water and Salt Research Center, Aarhus University, DK-8000 Aarhus C, Denmark
| | - Ebbe Boedtkjer
- Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark; Water and Salt Research Center, Aarhus University, DK-8000 Aarhus C, Denmark.
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Convergence of IRBIT, phosphatidylinositol (4,5) bisphosphate, and WNK/SPAK kinases in regulation of the Na+-HCO3- cotransporters family. Proc Natl Acad Sci U S A 2013; 110:4105-10. [PMID: 23431199 DOI: 10.1073/pnas.1221410110] [Citation(s) in RCA: 59] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Fluid and HCO3(-) secretion is a vital function of secretory epithelia, involving basolateral HCO3(-) entry through the Na(+)-HCO3(-) cotransporter (NBC) NBCe1-B, and luminal HCO3(-) exit mediated by cystic fibrosis transmembrane conductance regulator (CFTR) and solute carrier family 26 (SLC26) Cl(-)/HCO3(-) exchangers. HCO3(-) secretion is highly regulated, with the WNK/SPAK kinase pathway setting the resting state and the IRBIT/PP1 pathway setting the stimulated state. However, we know little about the relationships between the WNK/SPAK and IRBIT/PP1 sites in the regulation of the transporters. The first 85 N-terminal amino acids of NBCe1-B function as an autoinhibitory domain. Here we have identified a positively charged module within NBCe1-B(37-65) that is conserved in NBCn1-A and all 20 members of the NBC superfamily except NBCe1-A. This module is required for the interaction and activation of NBCe1-B and NBCn1-A by IRBIT and their regulation by phosphatidylinositol 4,5-bisphosphate (PIP2). Activation of the transporters by IRBIT and PIP2 is nonadditive but complementary. Phosphorylation of Ser65 mediates regulation of NBCe1-B by SPAK, and phosphorylation of Thr49 is required for regulation by IRBIT and SPAK. Sequence searches using the NBCe1-B regulatory module as a template identified a homologous sequence in the CFTR R domain and Slc26a6 sulfat transporter and antisigma factor antagonist (STAS) domain. Accordingly, the R and STAS domains bind IRBIT, and the R domain is required for activation of CFTR by IRBIT. These findings reveal convergence of regulatory modalities in a conserved domain of the NBC that may be present in other HCO3(-) transporters and thus in the regulation of epithelial fluid and HCO3(-) secretion.
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Chan HC, Chen H, Ruan Y, Sun T. Physiology and Pathophysiology of the Epithelial Barrier of the Female Reproductive Tract. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2013; 763:193-217. [DOI: 10.1007/978-1-4614-4711-5_10] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
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The Exocrine Pancreas: The Acinar-Ductal Tango in Physiology and Pathophysiology. Rev Physiol Biochem Pharmacol 2013; 165:1-30. [DOI: 10.1007/112_2013_14] [Citation(s) in RCA: 75] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
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Lee S, Yang HS, Kim E, Ju EJ, Kwon MH, Dudley RK, Smith Y, Yun CC, Choi I. PSD-95 interacts with NBCn1 and enhances channel-like activity without affecting Na/HCO(3) cotransport. Cell Physiol Biochem 2012. [PMID: 23183381 DOI: 10.1159/000343332] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
BACKGROUND/AIMS The sodium/bicarbonate transporter NBCn1 plays an essential role in intracellular pH regulation and transepithelial HCO(3)(-) movement in the body. NBCn1 also has sodium channel-like activity uncoupled to Na/HCO(3) cotransport. We previously reported that NBCn1 interacts with the postsynaptic density protein PSD-95 in the brain. Here, we elucidated the structural determinant and functional consequence of NBCn1/PSD-95 interaction. METHODS RESULTS In rat hippocampal CA3 neurons, NBCn1 was localized to the postsynaptic membranes of both dendritic shafts and spines and occasionally to the presynaptic membranes. A GST/NBCn1 fusion protein containing the C-terminal 131 amino acids of NBCn1 pulled down PSD-95 from rat brain lysates, whereas GST/NBCn1-ΔETSL (deletion of the last four amino acids) and GST/NBCn2 (NCBE) lacking the same ETSL did not. NBCn1 and PSD-95 were coimmunoprecipitated in HEK 293 cells, and their interaction did not affect the efficacy of PSD-95 to bind to the NMDA receptor NR2A. PSD-95 has negligible effects on intracellular pH changes mediated by NBCn1 in HEK 293 cells and Xenopus oocytes. However, PSD-95 increased an ionic conductance produced by NBCn1 channel-like activity. This increase was abolished by NBCn1-ΔETSL or by the peptide containing the last 15 amino acids of NBCn1. CONCLUSION Our data suggest that PSD-95 interacts with NBCn1 and increases its channel-like activity while negligibly affecting Na/HCO(3) cotransport. The possibility that the channel-like activity occurs via an intermolecular cavity of multimeric NBCn1 proteins is discussed.
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Affiliation(s)
- Soojung Lee
- Department of Physiology, Emory University School of Medicine, Atlanta, GA 30322, USA
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Patterson K, Catalán MA, Melvin JE, Yule DI, Crampin EJ, Sneyd J. A quantitative analysis of electrolyte exchange in the salivary duct. Am J Physiol Gastrointest Liver Physiol 2012; 303:G1153-63. [PMID: 22899825 PMCID: PMC3517652 DOI: 10.1152/ajpgi.00364.2011] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
A healthy salivary gland secretes saliva in two stages. First, acinar cells generate primary saliva, a plasma-like, isotonic fluid high in Na(+) and Cl(-). In the second stage, the ducts exchange Na(+) and Cl(-) for K(+) and HCO(3)(-), producing a hypotonic final saliva with no apparent loss in volume. We have developed a tool that aims to understand how the ducts achieve this electrolyte exchange while maintaining the same volume. This tool is part of a larger multiscale model of the salivary gland and can be used at the duct or gland level to investigate the effects of genetic and chemical alterations. In this study, we construct a radially symmetric mathematical model of the mouse salivary gland duct, representing the lumen, the cell, and the interstitium. For a given flow and primary saliva composition, we predict the potential differences and the luminal and cytosolic concentrations along a duct. Our model accounts well for experimental data obtained in wild-type animals as well as knockouts and chemical inhibitors. Additionally, the luminal membrane potential of the duct cells is predicted to be very depolarized compared with acinar cells. We investigate the effects of an electrogenic vs. electroneutral anion exchanger in the luminal membrane on concentration and the potential difference across the luminal membrane as well as how impairing the cystic fibrosis transmembrane conductance regulator channel affects other ion transporting mechanisms. Our model suggests the electrogenicity of the anion exchanger has little effect in the submandibular duct.
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Affiliation(s)
- Kate Patterson
- Dept. of Mathematics, Univ. of Auckland, Auckland, New Zealand.
| | - Marcelo A. Catalán
- 2Secretory Mechanisms and Dysfunction Section, Division of Intramural Research, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland;
| | - James E. Melvin
- 2Secretory Mechanisms and Dysfunction Section, Division of Intramural Research, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland;
| | - David I. Yule
- 3Department of Pharmacology and Physiology and the Center for Oral Biology, University of Rochester Medical Center, Rochester, New York; and
| | - Edmund J. Crampin
- 4Auckland Bioengineering Institute and Department of Engineering Science, University of Auckland, Auckland, New Zealand
| | - James Sneyd
- 1Department of Mathematics, University of Auckland, Auckland, New Zealand;
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Park HW, Lee MG. Transepithelial bicarbonate secretion: lessons from the pancreas. Cold Spring Harb Perspect Med 2012; 2:2/10/a009571. [PMID: 23028131 DOI: 10.1101/cshperspect.a009571] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Many cystic fibrosis transmembrane conductance regulator (CFTR)-expressing epithelia secrete bicarbonate (HCO(3)(-))-containing fluids. Recent evidence suggests that defects in epithelial bicarbonate secretion are directly involved in the pathogenesis of cystic fibrosis, in particular by building up hyperviscous mucus in the ductal structures of the lung and pancreas. Pancreatic juice is one of the representative fluids that contain a very high concentration of bicarbonate among bodily fluids that are secreted from CFTR-expressing epithelia. We introduce up-to-date knowledge on the basic principles of transepithelial bicarbonate transport by showing the mechanisms involved in pancreatic bicarbonate secretion. The model of pancreatic bicarbonate secretion described herein may also apply to other exocrine epithelia. As a central regulator of bicarbonate transport at the apical membrane, CFTR plays an essential role in both direct and indirect bicarbonate secretion. The major role of CFTR in bicarbonate secretion would be variable depending on the tissue and cell type. For example, in epithelial cells that produce a low concentration of bicarbonate-containing fluid (up to 80 mm), either CFTR-dependent Cl(-)/HCO(3)(-) exchange or CFTR anion channel with low bicarbonate permeability would be sufficient to generate such fluid. However, in cells that secrete high-bicarbonate-containing fluids, a highly selective CFTR bicarbonate channel activity is required. Therefore, understanding the molecular mechanism of transepithelial bicarbonate transport and the role of CFTR in each specific epithelium will provide therapeutic strategies to recover from epithelial defects induced by hyposecretion of bicarbonate in cystic fibrosis.
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Affiliation(s)
- Hyun Woo Park
- Department of Pharmacology, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Korea
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35
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Boedtkjer E, Moreira JMA, Mele M, Vahl P, Wielenga VT, Christiansen PM, Jensen VED, Pedersen SF, Aalkjaer C. Contribution of Na+,HCO3(-)-cotransport to cellular pH control in human breast cancer: a role for the breast cancer susceptibility locus NBCn1 (SLC4A7). Int J Cancer 2012; 132:1288-99. [PMID: 22907202 DOI: 10.1002/ijc.27782] [Citation(s) in RCA: 96] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2012] [Accepted: 07/31/2012] [Indexed: 12/16/2022]
Abstract
Genome-wide association studies recently linked the locus for Na(+),HCO(3)(-)-cotransporter NBCn1 (SLC4A7) to breast cancer susceptibility, yet functional insights have been lacking. To determine whether NBCn1, by transporting HCO(3)(-) into cells, may dispose of acid produced during high metabolic activity, we studied the expression of NBCn1 and the functional impact of Na(+),HCO(3)(-)-cotransport in human breast cancer. We found that the plasmalemmal density of NBCn1 was 20-30% higher in primary breast carcinomas and metastases compared to matched normal breast tissue. The increase in NBCn1 density was similar in magnitude to that observed for Na(+)/H(+)-exchanger NHE1 (SLC9A1), a transporter previously implicated in cell migration, proliferation and malignancy. In primary breast carcinomas, the apparent molecular weight for NBCn1 was increased compared to normal tissue. Using pH-sensitive fluorophores, we showed that Na(+),HCO(3)(-)-cotransport is the predominant mechanism of acid extrusion and is inhibited 34 ± 9% by 200 μM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid in human primary breast carcinomas. At intracellular pH (pH(i) ) levels >6.6, CO(2)/HCO(3)(-)-dependent mechanisms accounted for >90% of total net acid extrusion. Na(+)/H(+)-exchange activity was prominent only at lower pH(i) -values. Furthermore, steady-state pH(i) was 0.35 ± 0.06 units lower in the absence than in the presence of CO(2)/HCO(3)(-). In conclusion, expression of NBCn1 is upregulated in human primary breast carcinomas and metastases compared to normal breast tissue. Na(+),HCO(3)(-)-cotransport is a major determinant of pH(i) in breast cancer and the modest DIDS-sensitivity is consistent with NBCn1 being predominantly responsible. Hence, our results suggest a major pathophysiological role for NBCn1 that may be clinically relevant.
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36
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Schrimpe-Rutledge AC, Fontès G, Gritsenko MA, Norbeck AD, Anderson DJ, Waters KM, Adkins JN, Smith RD, Poitout V, Metz TO. Discovery of novel glucose-regulated proteins in isolated human pancreatic islets using LC-MS/MS-based proteomics. J Proteome Res 2012; 11:3520-32. [PMID: 22578083 DOI: 10.1021/pr3002996] [Citation(s) in RCA: 50] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
The prevalence of diabetes mellitus is increasing dramatically throughout the world, and the disease has become a major public health issue. The most common form of the disease, type 2 diabetes, is characterized by insulin resistance and insufficient insulin production from the pancreatic beta-cell. Since glucose is the most potent regulator of beta-cell function under physiological conditions, identification of the insulin secretory defect underlying type 2 diabetes requires a better understanding of glucose regulation of human beta-cell function. To this aim, a bottom-up LC-MS/MS-based proteomics approach was used to profile pooled islets from multiple donors under basal (5 mM) or high (15 mM) glucose conditions. Our analysis discovered 256 differentially abundant proteins (∼p < 0.05) after 24 h of high glucose exposure from more than 4500 identified in total. Several novel glucose-regulated proteins were elevated under high glucose conditions, including regulators of mRNA splicing (pleiotropic regulator 1), processing (retinoblastoma binding protein 6), and function (nuclear RNA export factor 1), in addition to neuron navigator 1 and plasminogen activator inhibitor 1. Proteins whose abundances markedly decreased during incubation at 15 mM glucose included Bax inhibitor 1 and synaptotagmin-17. Up-regulation of dicer 1 and SLC27A2 and down-regulation of phospholipase Cβ4 were confirmed by Western blots. Many proteins found to be differentially abundant after high glucose stimulation are annotated as uncharacterized or hypothetical. These findings expand our knowledge of glucose regulation of the human islet proteome and suggest many hitherto unknown responses to glucose that require additional studies to explore novel functional roles.
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Masica DL, Sosnay PR, Cutting GR, Karchin R. Phenotype-optimized sequence ensembles substantially improve prediction of disease-causing mutation in cystic fibrosis. Hum Mutat 2012; 33:1267-74. [PMID: 22573477 DOI: 10.1002/humu.22110] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2011] [Accepted: 04/12/2012] [Indexed: 12/20/2022]
Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR) mutation is associated with a phenotypic spectrum that includes cystic fibrosis (CF). The disease liability of some common CFTR mutations is known, but rare mutations are seen in too few patients to categorize unequivocally, making genetic diagnosis difficult. Computational methods can predict the impact of mutation, but prediction specificity is often below that required for clinical utility. Here, we present a novel supervised learning approach for predicting CF from CFTR missense mutation. The algorithm begins by constructing custom multiple sequence alignments called phenotype-optimized sequence ensembles (POSEs). POSEs are constructed iteratively, by selecting sequences that optimize predictive performance on a training set of CFTR mutations of known clinical significance. Next, we predict CF disease liability from a different set of CFTR mutations (test-set mutations). This approach achieves improved prediction performance relative to popular methods recently assessed using the same test-set mutations. Of clinical significance, our method achieves 94% prediction specificity. Because databases such as HGMD and locus-specific mutation databases are growing rapidly, methods that automatically tailor their predictions for a specific phenotype may be of immediate utility. If the performance achieved here generalizes to other systems, the approach could be an excellent tool to help establish genetic diagnoses.
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Affiliation(s)
- David L Masica
- Department of Biomedical Engineering and Institute for Computational Medicine, Johns Hopkins University, Baltimore, Maryland, USA
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Liu Y, Wang DK, Chen LM. The physiology of bicarbonate transporters in mammalian reproduction. Biol Reprod 2012; 86:99. [PMID: 22262691 DOI: 10.1095/biolreprod.111.096826] [Citation(s) in RCA: 62] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
HCO(3)(-) plays critically important roles during virtually the entire process of reproduction in mammals, including spermatogenesis, sperm capacitation, fertilization, and development of early stage embryos. Therefore, the acid-base balance in the male and female reproductive tracts must be finely modulated. The fluid milieu in the epididymis is acidic, containing very low concentration of HCO(3)(-). In this acidic low HCO(3)(-) environment, mature sperm are rendered quiescent in the epididymis. In contrast, the luminal fluid in the female uterus and oviduct is alkaline, with very high concentration of HCO(3)(-) that is essential for sperm to fulfill fertilization. HCO(3)(-) transporter of solute carrier 4 (SLC4) and SLC26 families represent the major carriers for HCO(3)(-) transport across the plasma membrane. These transporters play critical roles in intracellular pH regulation and transepithelial HCO(3)(-) transport. The physiological roles of these transporters in mammalian reproduction are of fundamental interest to investigators. Here we review recent progress in understanding the expression of HCO(3)(-) transporters in reproductive tract tissues as well as the physiological roles of these transporters in mammalian reproduction.
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Affiliation(s)
- Ying Liu
- Department of Biological Sciences, Key Laboratory of Molecular Biophysics of Ministry of Education, Huazhong University of Science and Technology School of Life Science and Technology, Wuhan, China
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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: 283] [Impact Index Per Article: 21.8] [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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40
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Ford P, Rivarola V, Chara O, Blot-Chabaud M, Cluzeaud F, Farman N, Parisi M, Capurro C. Volume regulation in cortical collecting duct cells: role of AQP2. Biol Cell 2012; 97:687-97. [PMID: 15859948 DOI: 10.1042/bc20040116] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
BACKGROUND INFORMATION The renal CCD (cortical collecting duct) plays a role in final volume and concentration of urine by a process that is regulated by the antidiuretic hormone, [arginine]vasopressin. This hormone induces an increase in water permeability due to the translocation of AQP2 (aquaporin 2) from the intracellular vesicles to the apical membrane of principal cells. During the transition from antidiuresis to diuresis, CCD cells are exposed to changes in environmental osmolality, and cell-volume regulation may be especially important for the maintenance of intracellular homoeostasis. Despite its importance, cell-volume regulation in CCD cells has not been widely investigated. Moreover, no studies have been carried out till date to evaluate the putative role of AQPs during this process in renal cells. RESULTS In the present study, we have studied the regulatory cell-volume responses to hypo-osmotic or hyperosmotic challenges in two CCD cell lines: one not expressing AQPs and the other stably transfected with AQP2. We have used a fluorescent probe technique in which the acquisition of single-cell kinetic data can be simultaneously recorded with the intracellular pH. Experiments with hyperosmotic mannitol media demonstrated that, independent of AQP2 expression, CCD cells shrink but fail to show regulatory volume increase, at least under the studied conditions. In contrast, under hypo-osmotic shocks, regulatory volume decrease occurs and the activation of these mechanisms is more rapid in AQP2 transfected cells. This regulatory response takes place in parallel with intracellular acidification, which is faster in cells expressing AQP2. The acidification and the initial regulatory volume decrease response were inhibited by glibenclamide and BaCl2 only in AQP2 cells. CONCLUSIONS These results suggest that increases in the osmotic water permeability due to the expression of AQP2 are critical for a rapid activation of regulatory volume decrease mechanisms, which would be linked to cystic fibrosis transmembrane conductance regulator and to barium-sensitive potassium channels.
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Affiliation(s)
- Paula Ford
- Laboratorio de Biomembranas, Departamento de Fisiología y Biofísica, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, piso 7, 1121 Buenos Aires, Argentina
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Abstract
SLC4A gene family proteins include bicarbonate transporters that move HCO(3)(-) across the plasma membrane and regulate intracellular pH and transepithelial movement of acid-base equivalents. These transporters are Cl/HCO(3) exchangers, electrogenic Na/HCO(3) cotransporters, electroneutral Na/HCO(3) cotransporters, and Na(+)-driven Cl/HCO(3) exchanger. Studies of the bicarbonate transporters in vitro and in vivo have demonstrated their physiological importance for acid-base homeostasis at the cellular and systemic levels. Recent advances in structure/function analysis have also provided valuable information on domains or motifs critical for regulation, ion translocation, and protein topology. This chapter focuses on the molecular mechanisms of ion transport along with associated structural aspects from mutagenesis of particular residues and from chimeric constructs. Structure/function studies have helped to understand the mechanism by which ion substrates are moved via the transporters. This chapter also describes some insights into the structure of SLC4A1 (AE1) and SLC4A4 (NBCe1) transporters. Finally, as some SLC4A transporters exist in concert with other proteins in the cells, the structural features associated with protein-protein interactions are briefly discussed.
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Affiliation(s)
- Inyeong Choi
- Department of Physiology, Emory University, Atlanta, Georgia, USA.
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Abstract
IRBIT (IP(3)Rs binding protein released with IP(3)) is a protein originally identified by the Mikoshiba group as an inhibitor of IP(3) receptors function. Subsequently it was found to have multiple functions and regulate the activity of diverse proteins, including regulation of HCO(3)(-) transporters to coordinate epithelial HCO(3)(-) secretion and to determine localization of the Fip1 subunit of the CPSF complex to regulate mRNA processing. This review highlights the remarkably divers functions of IRBIT that are likely only a fraction of all the potential functions of this protein.
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Li C, Naren AP. CFTR chloride channel in the apical compartments: spatiotemporal coupling to its interacting partners. Integr Biol (Camb) 2010; 2:161-177. [PMID: 20473396 PMCID: PMC2989726 DOI: 10.1039/b924455g] [Citation(s) in RCA: 103] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel located primarily at the apical or luminal surfaces of epithelial cells in the airway, intestine, pancreas, kidney, sweat gland, as well as male reproductive tract, where it plays a crucial role in transepithelial fluid homeostasis. CFTR dysfunction can be detrimental and may result in life-threatening disorders. CFTR hypofunctioning because of genetic defects leads to cystic fibrosis, the most common lethal genetic disease in Caucasians, whereas CFTR hyperfunctioning resulting from various infections evokes secretory diarrhea, the leading cause of mortality in early childhood. Therefore, maintaining a dynamic balance between CFTR up-regulating processes and CFTR down-regulating processes is essential for maintaining fluid and body homeostasis. Accumulating evidence suggests that protein-protein interactions play a critical role in the fine-tuned regulation of CFTR function. A growing number of proteins have been reported to interact directly or indirectly with CFTR chloride channel, suggesting that CFTR might be coupled spatially and temporally to a wide variety of interacting partners including ion channels, receptors, transporters, scaffolding proteins, enzyme molecules, signaling molecules, and effectors. Most interactions occur primarily between the opposing terminal tails (amino or carboxyl) of CFTR protein and its binding partners, either directly or mediated through various PDZ scaffolding proteins. These dynamic interactions impact the channel function, as well as localization and processing of CFTR protein within cells. This article reviews the most recent progress and findings about the interactions between CFTR and its binding partners through PDZ scaffolding proteins, as well as the spatiotemporal regulation of CFTR-containing macromolecular signaling complexes in the apical compartments of polarized cells lining the secretory epithelia.
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Affiliation(s)
- Chunying Li
- Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, 540 E. Canfield Avenue, 5312 Scott Hall, Detroit, Michigan 48201, USA
| | - Anjaparavanda P. Naren
- Department of Physiology, University of Tennessee Health Science Center, 420 Nash, 894 Union Avenue, Memphis, Tennessee 38163, USA
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Park HJ, Rajbhandari I, Yang HS, Lee S, Cucoranu D, Cooper DS, Klein JD, Sands JM, Choi I. Neuronal expression of sodium/bicarbonate cotransporter NBCn1 (SLC4A7) and its response to chronic metabolic acidosis. Am J Physiol Cell Physiol 2010; 298:C1018-28. [PMID: 20147654 DOI: 10.1152/ajpcell.00492.2009] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
The sodium-bicarbonate cotransporter NBCn1 (SLC4A7) is an acid-base transporter that normally moves Na(+) and HCO(3)(-) into the cell. This membrane protein is sensitive to cellular and systemic pH changes. We examined NBCn1 expression and localization in the brain and its response to chronic metabolic acidosis. Two new NBCn1 antibodies were generated by immunizing a rabbit and a guinea pig. The antibodies stained neurons in a variety of rat brain regions, including hippocampal pyramidal neurons, dentate gyrus granular neurons, posterior cortical neurons, and cerebellar Purkinje neurons. Choroid plexus epithelia were also stained. Double immunofluorescence labeling showed that NBCn1 and the postsynaptic density protein PSD-95 were found in the same hippocampal CA3 neurons and partially colocalized in dendrites. PSD-95 was pulled down from rat brain lysates with the GST/NBCn1 fusion protein and was also coimmunoprecipitated with NBCn1. Chronic metabolic acidosis was induced by feeding rats with normal chow or 0.4 M HCl-containing chow for 7 days. Real-time PCR and immunoblot showed upregulation of NBCn1 mRNA and protein in the hippocampus of acidotic rats. NBCn1 immunostaining was enhanced in CA3 neurons, posterior cortical neurons, and cerebellar granular cells. Intraperitoneal administration of N-methyl-d-aspartate caused neuronal death determined by caspase-3 activity, and this effect was more severe in acidotic rats. Administering N-methyl-d-aspartate also inhibited NBCn1 upregulation in acidotic rats. We conclude that NBCn1 in neurons is upregulated by chronic acid loads, and this upregulation is associated with glutamate excitotoxicity.
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Affiliation(s)
- Hae Jeong Park
- Dept. of Physiology, Emory Univ., Atlanta, GA 30322, USA
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Riihonen R, Nielsen S, Väänänen HK, Laitala-Leinonen T, Kwon TH. Degradation of hydroxyapatite in vivo and in vitro requires osteoclastic sodium-bicarbonate co-transporter NBCn1. Matrix Biol 2010; 29:287-94. [PMID: 20079835 DOI: 10.1016/j.matbio.2010.01.003] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2009] [Revised: 12/01/2009] [Accepted: 01/06/2010] [Indexed: 01/19/2023]
Abstract
Dissolution of the inorganic bone matrix releases not only calcium and phosphate ions, but also bicarbonate. Electroneutral sodium-bicarbonate co-transporter (NBCn1) is expressed in inactive osteoclasts, but its physiological role in bone resorption has remained unknown. We show here that NBCn1, encoded by the SLC4A7 gene, is directly involved in bone resorption. NBCn1 protein was specifically found at the bone-facing ruffled border areas, and metabolic acidosis increased NBCn1 expression in rats in vivo. In human hematopoietic stem cell cultures, NBCn1 mRNA expression was observed only after formation of resorbing osteoclasts. To further confirm the critical role of NBCn1 during bone resorption, human hematopoietic stem cells were transduced with SLC4A7 shRNA lentiviral particles. Downregulation of NBCn1 both on mRNA and protein level by lentiviral shRNAs significantly inhibited bone resorption and increased intracellular acidification in osteoclasts. The lentiviral particles did not impair osteoclast survival, or differentiation of the hematopoietic or mesenchymal precursor cells into osteoclasts or osteoblasts in vitro. Inhibition of NBCn1 activity may thus provide a new way to regulate osteoclast activity during pathological bone resorption.
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Affiliation(s)
- Riikka Riihonen
- Bone Biology Research Consortium, Department of Cell Biology and Anatomy, Institute of Biomedicine, University of Turku, FIN-20520 Turku, Finland
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Boron WF, Chen L, Parker MD. Modular structure of sodium-coupled bicarbonate transporters. ACTA ACUST UNITED AC 2009; 212:1697-706. [PMID: 19448079 DOI: 10.1242/jeb.028563] [Citation(s) in RCA: 104] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Mammalian genomes contain 10 SLC4 genes that, between them, encode three Cl-HCO(3) exchangers, five Na(+)-coupled HCO(3) transporters (NCBTs), one reported borate transporter, and what is reported to be a fourth Cl-HCO(3) exchanger. The NCBTs are expressed throughout the body and play important roles in maintaining intracellular and whole-body pH, as well as contributing to transepithelial transport processes. The importance of NCBTs is underscored by the genetic association of dysfunctional NCBT genes with blindness, deafness, epilepsy, hypertension and metal retardation. Key to understanding the action and regulation of NCBTs is an appreciation of the diversity of NCBT gene products. The transmembrane domains of human NCBT paralogs are 50-84% identical to each other at the amino acid level, and are capable of a diverse range of actions, including electrogenic Na/HCO(3) cotransport (i.e. NBCe1 and NBCe2) and electroneutral Na/HCO(3) cotransport (i.e. NBCn1 and NBCn2), as well as Na(+)-dependent Cl-HCO(3) exchange (i.e. NDCBE). Furthermore, by the use of alternative promoters and alternative-splicing events, individual SLC4 genes have the potential to generate multiple splice variants (as many as 16 in the case of NBCn1), each of which could have unique temporal and spatial patterns of distribution, unitary transporter activity (i.e. flux mediated by one molecule), array of protein-binding partners, and complement of regulatory stimuli. In the first section of this review, we summarize our present knowledge of the function and distribution of mammalian NCBTs and their multiple variants. In the second section of this review we consider the molecular consequences of NCBT variation.
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Affiliation(s)
- Walter F Boron
- Department of Physiology, Case Western Reserve University Medical School, 10900 Euclid Avenue, Cleveland, OH 44106, USA
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Ducts isolated from the pancreas of CFTR-null mice secrete fluid. Pflugers Arch 2009; 459:203-14. [PMID: 19655163 DOI: 10.1007/s00424-009-0704-9] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2009] [Accepted: 07/21/2009] [Indexed: 01/01/2023]
Abstract
The pancreatic pathology in cystic fibrosis (CF) is normally attributed to the failure of ductal fluid secretion resulting from the lack of functional CF transmembrane conductance regulator (CFTR). However, murine models of CF show little or no pancreatic pathology. To resolve this dichotomy we analysed the transport mechanisms involved in fluid and electrolyte secretion by pancreatic ducts isolated from CFTR-null mice. Experiments were performed on cultured interlobular duct segments isolated from the pancreas of the Cftr(tm1Cam) strain of CFTR-null mouse. Fluid secretion to the closed luminal space was measured by video microscopy. The secretory response of ducts isolated from CF mice to cAMP-elevating agonists forskolin and secretin was significantly reduced compared with wild type but not abolished. The Cl(-)- and HCO(3) (-) -dependent components of the ductal secretion were affected equally by the absence of CFTR. The secretory response to carbachol stimulation was unaltered in CF ducts. Loading the ductal cells with the Ca2+ chelator BAPTA completely abolished carbachol-evoked secretion, but did not affect forskolin-evoked secretion in CF or wild-type ducts. We conclude that pancreatic duct cells from CF mice can secrete a significant amount of water and electrolytes by a cAMP-stimulated mechanism that is independent of CFTR and cannot be ascribed to the activation of calcium-activated chloride channels.
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Spence J. Pathway prediction by bioinformatic analysis of the untranslated regions of the CFTR mRNA. Genomics 2009; 94:39-47. [PMID: 19306924 DOI: 10.1016/j.ygeno.2009.03.002] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2009] [Revised: 03/07/2009] [Accepted: 03/11/2009] [Indexed: 11/17/2022]
Abstract
Mining the information contained within the genetic code in untranslated regions has proven difficult because of the ambiguity of microRNA and protein binding sites. This manuscript describes a bioinformatic screen that identifies long sequences with partial identity to the untranslated regions of the cystic fibrosis transmembrane regulator. This screen uncovered a long, evolutionarily conserved motif common to the 3' UTRs of the CFTR and SEC24A transcripts, and shorter, statistically significant motifs unique to either 5' or 3' UTRs. In addition, of the 140 transcripts identified in the screen that encode proteins with known protein interactions, 130 are linked to CFTR through protein interactions. The screen identified genes that are known to be involved in lung fibrosis, the inflammatory response of cystic fibrosis and sensitivity to Pseudomonas aeruginosa infections. The bioinformatic analysis of untranslated regions should prove to be a powerful adjunct to other tools for predicting pathways and relevant interactions.
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Affiliation(s)
- Jean Spence
- Omnitron Biosciences, P.O. Box 601002, San Diego, CA 92160, USA.
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Abstract
The family of mammalian bicarbonate transport proteins are involved in a wide-range of physiological processes. The importance of bicarbonate transport follows from the biochemistry of HCO(3)(-) itself. Bicarbonate is the waste product of mitochondrial respiration. HCO(3)(-) undergoes pH-dependent conversion into CO(2) and in doing so converts from a membrane impermeant anion into a gas that can diffuse across membranes. The CO(2)-HCO(3)(-) equilibrium forms the most important pH buffering system of our bodies. Bicarbonate transport proteins facilitate the movement of membrane-impermeant HCO(3)(-) across membranes to accelerate disposal of waste CO(2), control cellular and whole-body pH, and to regulate fluid movement and acid/base secretion. Defects of bicarbonate transport proteins manifest in diseases of most organ systems. Fourteen gene products facilitate mammalian bicarbonate transport, whose physiology and pathophysiology is discussed in the present review.
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Abstract
Adenosine (Ado) regulates diverse cellular functions in the lung through its local production, release, metabolism, and subsequent stimulation of G-protein-coupled P1 purinergic receptors. The A(2B) adenosine receptor (A(2B)AR) is the predominant P1 purinergic receptor isoform expressed in surface airway epithelia, and Ado is an important regulator of airway surface liquid (ASL) volume through its activation of the cystic fibrosis transmembrane conductance regulator (CFTR). Through a delicate balance between sodium (Na(+)) absorption and chloride (Cl(-)) secretion, the ASL volume is optimized to promote ciliary activity and mucociliary clearance, effectively removing inhaled particulates. When CFTR is dysfunctional, the Ado/A(2B)AR regulatory system fails to optimize the ASL volume, leading to its depletion and interruption of mucociliary clearance. In cystic fibrosis (CF), loss of CFTR function and resultant mucus stasis leaves the lower airways susceptible to mucus obstruction, chronic bacterial infection, relentless inflammation, and eventually panbronchiectasis. Adenosine triphosphate (ATP) also regulates transepithelial Cl(-) conductance, but through a separate system that relies on stimulation of P2Y(2) purinergic receptors, mobilization of intracellular calcium, and activation of calcium-activated chloride channels (CaCCs). These pathways remain functional in CF, and may serve a protective role in the disease. In this chapter, we will review our current understanding of how Ado and related nucleotides regulate CFTR and Cl(-) conductance in the human airway, including the regulation of additional intracellular and extracellular signaling pathways that provide important links between ion transport and inflammation relevant to the disease.
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