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Kedia S, Awal NM, Seddon J, Marder E. Sulfonylurea Receptor Pharmacology Alters the Performance of Two Central Pattern Generating Circuits in Cancer borealis. FUNCTION 2024; 5:zqae043. [PMID: 39293809 PMCID: PMC11577616 DOI: 10.1093/function/zqae043] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2024] [Revised: 09/12/2024] [Accepted: 09/13/2024] [Indexed: 09/20/2024] Open
Abstract
Neuronal activity and energy supply must maintain a fine balance for neuronal fitness. Various channels of communication between the two could impact network output in different ways. Sulfonylurea receptors (SURs) are a modification of ATP-binding cassette proteins that confer ATP-dependent gating on their associated ion channels. They are widely expressed and link metabolic states directly to neuronal activity. The role they play varies in different circuits, both enabling bursting and inhibiting activity in pathological conditions. The crab, Cancer borealis, has central pattern generators (CPGs) that fire in rhythmic bursts nearly constantly and it is unknown how energy availability influences these networks. The pyloric network of the stomatogastric ganglion and the cardiac ganglion (CG) control rhythmic contractions of the foregut and heart, respectively. Known SUR agonists and antagonists produce opposite effects in the two CPGs. Pyloric rhythm activity completely stops in the presence of a SUR agonist, and activity increases in SUR blockers. This results from a decrease in the excitability of pyloric dilator neurons, which are a part of the pacemaker kernel. The neurons of the CG, paradoxically, increase firing within bursts in SUR agonists, and bursting slows in SUR antagonists. Analyses of the agonist-affected conductance properties present biophysical effects that do not trivially match those of mammalian SUR-dependent conductances. We suggest that SUR-associated conductances allow different neurons to respond to energy states in different ways through a common mechanism.
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Affiliation(s)
- Sonal Kedia
- Biology Department and Volen Center, Brandeis University, Waltham, MA 02454, USA
| | - Naziru M Awal
- Biology Department and Volen Center, Brandeis University, Waltham, MA 02454, USA
| | - Jackie Seddon
- Biology Department and Volen Center, Brandeis University, Waltham, MA 02454, USA
| | - Eve Marder
- Biology Department and Volen Center, Brandeis University, Waltham, MA 02454, USA
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2
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Brennan S, Chen S, Makwana S, Esposito S, McGuinness LR, Alnaimi AIM, Sims MW, Patel M, Aziz Q, Ojake L, Roberts JA, Sharma P, Lodwick D, Tinker A, Barrett-Jolley R, Dart C, Rainbow RD. Identification and characterisation of functional K ir6.1-containing ATP-sensitive potassium channels in the cardiac ventricular sarcolemmal membrane. Br J Pharmacol 2024; 181:3380-3400. [PMID: 38763521 DOI: 10.1111/bph.16390] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2023] [Revised: 02/21/2024] [Accepted: 03/18/2024] [Indexed: 05/21/2024] Open
Abstract
BACKGROUND AND PURPOSE The canonical Kir6.2/SUR2A ventricular KATP channel is highly ATP-sensitive and remains closed under normal physiological conditions. These channels activate only when prolonged metabolic compromise causes significant ATP depletion and then shortens the action potential to reduce contractile activity. Pharmacological activation of KATP channels is cardioprotective, but physiologically, it is difficult to understand how these channels protect the heart if they only open under extreme metabolic stress. The presence of a second KATP channel population could help explain this. Here, we characterise the biophysical and pharmacological behaviours of a constitutively active Kir6.1-containing KATP channel in ventricular cardiomyocytes. EXPERIMENTAL APPROACH Patch-clamp recordings from rat ventricular myocytes in combination with well-defined pharmacological modulators was used to characterise these newly identified K+ channels. Action potential recording, calcium (Fluo-4) fluorescence measurements and video edge detection of contractile function were used to assess functional consequences of channel modulation. KEY RESULTS Our data show a ventricular K+ conductance whose biophysical characteristics and response to pharmacological modulation were consistent with Kir6.1-containing channels. These Kir6.1-containing channels lack the ATP-sensitivity of the canonical channels and are constitutively active. CONCLUSION AND IMPLICATIONS We conclude there are two functionally distinct populations of ventricular KATP channels: constitutively active Kir6.1-containing channels that play an important role in fine-tuning the action potential and Kir6.2/SUR2A channels that activate with prolonged ischaemia to impart late-stage protection against catastrophic ATP depletion. Further research is required to determine whether Kir6.1 is an overlooked target in Comprehensive in vitro Proarrhythmia Assay (CiPA) cardiac safety screens.
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Affiliation(s)
- Sean Brennan
- Department of Cardiovascular and Metabolic Medicine and Liverpool Centre for Cardiovascular Science, University of Liverpool, Liverpool, UK
| | - Shen Chen
- Department of Cardiovascular Sciences, University of Leicester, Leicester, UK
| | - Samir Makwana
- Department of Cardiovascular Sciences, University of Leicester, Leicester, UK
| | - Simona Esposito
- Department of Cardiovascular and Metabolic Medicine and Liverpool Centre for Cardiovascular Science, University of Liverpool, Liverpool, UK
- Department of Cardiovascular Sciences, University of Leicester, Leicester, UK
| | - Lauren R McGuinness
- Department of Cardiovascular and Metabolic Medicine and Liverpool Centre for Cardiovascular Science, University of Liverpool, Liverpool, UK
| | - Abrar I M Alnaimi
- Department of Cardiovascular and Metabolic Medicine and Liverpool Centre for Cardiovascular Science, University of Liverpool, Liverpool, UK
- Department of Cardiac Technology, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia
| | - Mark W Sims
- Department of Cardiovascular Sciences, University of Leicester, Leicester, UK
| | - Manish Patel
- Department of Cardiovascular Sciences, University of Leicester, Leicester, UK
| | - Qadeer Aziz
- William Harvey Research Institute, Queen Mary University of London, London, UK
| | - Leona Ojake
- William Harvey Research Institute, Queen Mary University of London, London, UK
| | - James A Roberts
- Department of Cardiovascular and Metabolic Medicine and Liverpool Centre for Cardiovascular Science, University of Liverpool, Liverpool, UK
| | - Parveen Sharma
- Department of Cardiovascular and Metabolic Medicine and Liverpool Centre for Cardiovascular Science, University of Liverpool, Liverpool, UK
| | - David Lodwick
- Department of Cardiovascular Sciences, University of Leicester, Leicester, UK
| | - Andrew Tinker
- William Harvey Research Institute, Queen Mary University of London, London, UK
| | - Richard Barrett-Jolley
- Department of Musculoskeletal and Ageing Science, University of Liverpool, Liverpool, UK
| | - Caroline Dart
- Department of Biochemistry, Cell and Systems Biology, University of Liverpool, Liverpool, UK
| | - Richard D Rainbow
- Department of Cardiovascular and Metabolic Medicine and Liverpool Centre for Cardiovascular Science, University of Liverpool, Liverpool, UK
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3
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Tektemur A, Etem Önalan E, Kaya Tektemur N, Dayan Cinkara S, Kılınçlı Çetin A, Tekedereli İ, Kuloğlu T, Türk G. Carbamazepine-induced sperm disorders can be associated with the altered expressions of testicular KCNJ11/miR-let-7a and spermatozoal CFTR/miR-27a. Andrologia 2020; 53:e13954. [PMID: 33372325 DOI: 10.1111/and.13954] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2020] [Revised: 11/29/2020] [Accepted: 12/15/2020] [Indexed: 12/22/2022] Open
Abstract
Male infertility is a global health problem, and the underlying molecular mechanisms are not clearly known. Ion channels and microRNAs (miRNAs), known to function in many vital functions in cells, have been shown to play a significant role in male infertility through changes in their expressions. The study aimed to evaluate the alterations of testicular and/or spermatozoal potassium voltage-gated channel subfamily J member 11 (KCNJ11), Cystic fibrosis transmembrane conductance regulator (CFTR), miR-let-7a and miR-27a expressions in carbamazepine-related male infertility. Here, we showed that carbamazepine reduced sperm motility, increased abnormal sperm morphology, and impaired hormonal balance as well as increased relative testis weight and decreased relative seminal vesicle weight. On the other hand, downregulated KCNJ11 and upregulated miR-let-7a expressions were determined in testis (p < .05). Also, downregulated KCNJ11 and upregulated CFTR and miR-27a expressions were found in spermatozoa (p < .05). Interestingly, altered testicular KCNJ11 and miR-let-7a expressions were correlated with decreased sperm motility and elevated sperm tail defect. Besides, spermatozoal CFTR and miR-27a expressions positively correlated with sperm tail defects. The results indicated a significant relationship between ion channel and/or miRNA expression alterations and impaired sperm parameters due to carbamazepine usage.
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Affiliation(s)
- Ahmet Tektemur
- Department of Medical Biology, Faculty of Medicine, Firat University, Elazig, Turkey
| | - Ebru Etem Önalan
- Department of Medical Biology, Faculty of Medicine, Firat University, Elazig, Turkey
| | - Nalan Kaya Tektemur
- Department of Histology and Embryology, Faculty of Medicine, Firat University, Elazig, Turkey
| | - Serap Dayan Cinkara
- Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Firat University, Elazig, Turkey
| | - Ayten Kılınçlı Çetin
- Department of Medical Biology and Genetics, Faculty of Medicine, Inonu University, Malatya, Turkey
| | - İbrahim Tekedereli
- Department of Medical Biology and Genetics, Faculty of Medicine, Inonu University, Malatya, Turkey
| | - Tuncay Kuloğlu
- Department of Histology and Embryology, Faculty of Medicine, Firat University, Elazig, Turkey
| | - Gaffari Türk
- Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Firat University, Elazig, Turkey
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Law NC, Marinelli I, Bertram R, Corbin KL, Schildmeyer C, Nunemaker CS. Chronic stimulation induces adaptive potassium channel activity that restores calcium oscillations in pancreatic islets in vitro. Am J Physiol Endocrinol Metab 2020; 318:E554-E563. [PMID: 32069073 PMCID: PMC7191410 DOI: 10.1152/ajpendo.00482.2019] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Insulin pulsatility is important to hepatic response in regulating blood glucose. Growing evidence suggests that insulin-secreting pancreatic β-cells can adapt to chronic disruptions of pulsatility to rescue this physiologically important behavior. We determined the time scale for adaptation and examined potential ion channels underlying it. We induced the adaptation both by chronic application of the ATP-sensitive K+ [K(ATP)] channel blocker tolbutamide and by application of the depolarizing agent potassium chloride (KCl). Acute application of tolbutamide without pretreatment results in elevated Ca2+ as measured by fura-2AM and the loss of endogenous pulsatility. We show that after chronic exposure to tolbutamide (12-24 h), Ca2+ oscillations occur with subsequent acute tolbutamide application. The same experiment was conducted with potassium chloride (KCl) to directly depolarize the β-cells. Once again, following chronic exposure to the cell stimulator, the islets produced Ca2+ oscillations when subsequently exposed to tolbutamide. These experiments suggest that it is the chronic stimulation, and not tolbutamide desensitization, that is responsible for the adaptation that rescues oscillatory β-cell activity. This compensatory response also causes islet glucose sensitivity to shift rightward following chronic tolbutamide treatment. Mathematical modeling shows that a small increase in the number of K(ATP) channels in the membrane is one adaptation mechanism that is compatible with the data. To examine other compensatory mechanisms, pharmacological studies provide support that Kir2.1 and TEA-sensitive channels play some role. Overall, this investigation demonstrates β-cell adaptability to overstimulation, which is likely an important mechanism for maintaining glucose homeostasis in the face of chronic stimulation.
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Affiliation(s)
- Nathan C Law
- Department of Biomedical Sciences Heritage College of Osteopathic Medicine, Ohio University, Athens, Ohio
- Diabetes Institute, Heritage College of Osteopathic Medicine, Ohio University, Athens, Ohio
| | | | - Richard Bertram
- Department of Mathematics and Programs in Neuroscience and Molecular Biophysics, Florida State University, Tallahassee, Florida
| | - Kathryn L Corbin
- Department of Biomedical Sciences Heritage College of Osteopathic Medicine, Ohio University, Athens, Ohio
- Diabetes Institute, Heritage College of Osteopathic Medicine, Ohio University, Athens, Ohio
| | - Cara Schildmeyer
- Department of Biomedical Sciences Heritage College of Osteopathic Medicine, Ohio University, Athens, Ohio
- Diabetes Institute, Heritage College of Osteopathic Medicine, Ohio University, Athens, Ohio
| | - Craig S Nunemaker
- Department of Biomedical Sciences Heritage College of Osteopathic Medicine, Ohio University, Athens, Ohio
- Diabetes Institute, Heritage College of Osteopathic Medicine, Ohio University, Athens, Ohio
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Panou MM, Antoni M, Morgan EL, Loundras EA, Wasson CW, Welberry-Smith M, Mankouri J, Macdonald A. Glibenclamide inhibits BK polyomavirus infection in kidney cells through CFTR blockade. Antiviral Res 2020; 178:104778. [PMID: 32229236 PMCID: PMC7322401 DOI: 10.1016/j.antiviral.2020.104778] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2020] [Revised: 03/07/2020] [Accepted: 03/18/2020] [Indexed: 02/08/2023]
Abstract
BK polyomavirus (BKPyV) is a ubiquitous pathogen in the human population that is asymptomatic in healthy individuals, but can be life-threatening in those undergoing kidney transplant. To-date, no vaccines or anti-viral therapies are available to treat human BKPyV infections. New therapeutic strategies are urgently required. In this study, using a rational pharmacological screening regimen of known ion channel modulating compounds, we show that BKPyV requires cystic fibrosis transmembrane conductance regulator (CFTR) activity to infect primary renal proximal tubular epithelial cells. Disrupting CFTR function through treatment with the clinically available drug glibenclamide, the CFTR inhibitor CFTR172, or CFTR-silencing, all reduced BKPyV infection. Specifically, time of addition assays and the assessment of the exposure of VP2/VP3 minor capsid proteins indicated a role for CFTR during BKPyV transport to the endoplasmic reticulum, an essential step during the early stages of BKPyV infection. We thus establish CFTR as an important host-factor in the BKPyV life cycle and reveal CFTR modulators as potential anti-BKPyV therapies.
BK polyomavirus (BKPyV) is life-threatening in those undergoing kidney transplant. BKPyV requires CFTR to infect primary kidney cells. Disrupting CFTR function pharmacologically reduces BKPyV infection. CFTR is required during BKPyV transport to the endoplasmic reticulum.
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Affiliation(s)
- Margarita-Maria Panou
- School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, West Yorkshire, LS2 9JT, United Kingdom
| | - Michelle Antoni
- School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, West Yorkshire, LS2 9JT, United Kingdom
| | - Ethan L Morgan
- School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, West Yorkshire, LS2 9JT, United Kingdom
| | - Eleni-Anna Loundras
- School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, West Yorkshire, LS2 9JT, United Kingdom
| | - Christopher W Wasson
- School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, West Yorkshire, LS2 9JT, United Kingdom
| | | | - Jamel Mankouri
- School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, West Yorkshire, LS2 9JT, United Kingdom.
| | - Andrew Macdonald
- School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, West Yorkshire, LS2 9JT, United Kingdom.
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6
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Makrane H, Aziz M, Mekhfi H, Ziyyat A, Legssyer A, Melhaoui A, Berrabah M, Bnouham M, Alem C, Elombo FK, Gressier B, Desjeux JF, Eto B. Origanum majorana L. extract exhibit positive cooperative effects on the main mechanisms involved in acute infectious diarrhea. JOURNAL OF ETHNOPHARMACOLOGY 2019; 239:111503. [PMID: 30217790 DOI: 10.1016/j.jep.2018.09.005] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/30/2018] [Revised: 08/09/2018] [Accepted: 09/03/2018] [Indexed: 06/08/2023]
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE Origanum majorana L. (Lamiaceae) is commonly used in Moroccan folk medicine to treat infantile colic, abdominal discomfort and diarrhea. Liquid stools and abdominal discomfort observed in acute infectious diarrhea are the consequences of imbalance between intestinal water secretion and absorption in the lumen, and relaxation of smooth muscle surrounding the intestinal mucosa. AIM OF THE STUDY The objective of our study was to see if aqueous extract of Origanum majorana L. (AEOM) may exhibit an effect on those deleterious mechanisms. MATERIALS AND METHODS The effect of AEOM on electrogenic Cl- secretion and Na+ absorption, the two main mechanisms underlying water movement in the intestine, was assessed on intestinal pieces of mice intestine mounted, in vitro, in Ussing chambers. AEOM effect on muscle relaxation was measured on rat intestinal smooth muscle mounted in an isotonic transducer. RESULTS 1) AEOM placed on the serosal (i.e. blood) side of the piece of jejunum entirely inhibited in a concentration-dependent manner the Forskolin-induced electrogenic chloride secretion, with an IC50 = 654 ± 8 µg/mL. 2) AEOM placed on the mucosal (i.e. luminal) side stimulated in a concentration-dependent manner an electrogenic Na+ absorption, with an IC50 = 476.9 ± 1 µg/mL. 3) AEOM (1 mg/mL) inhibition of Forskolin-induced electrogenic secretion was almost entirely prevented by prior exposure to Ca++ channels or neurotransmitters inhibitors. 4) AEOM (1 mg/mL) proabsorptive effect was greater in the ileum and progressively declined in the jejunum, distal colon and proximal colon (minimal). 5) AEOM inhibited in a concentration-dependent manner smooth muscle Carbachol or KCl induced contraction, with an IC50 = 1.64 ± 0.2 mg/mL or 1.92 ± 0.8 mg/mL, respectively. CONCLUSION the present results indicate that aqueous extract of Origanum majorana L. exhibit positive cooperative effects on the main mechanisms that are involved in acute infectious diarrhea.
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Affiliation(s)
- Hanane Makrane
- Laboratory of Physiology, Genetic and Ethnopharmacology, Faculty of Sciences, Mohammed The First University, PB. 717, 60000 Oujda, Morocco
| | - Mohammed Aziz
- Laboratory of Physiology, Genetic and Ethnopharmacology, Faculty of Sciences, Mohammed The First University, PB. 717, 60000 Oujda, Morocco
| | - Hassane Mekhfi
- Laboratory of Physiology, Genetic and Ethnopharmacology, Faculty of Sciences, Mohammed The First University, PB. 717, 60000 Oujda, Morocco
| | - Abderrahim Ziyyat
- Laboratory of Physiology, Genetic and Ethnopharmacology, Faculty of Sciences, Mohammed The First University, PB. 717, 60000 Oujda, Morocco
| | - Abdelkhaleq Legssyer
- Laboratory of Physiology, Genetic and Ethnopharmacology, Faculty of Sciences, Mohammed The First University, PB. 717, 60000 Oujda, Morocco
| | - Ahmed Melhaoui
- Laboratory of Organic Chemistry, Macromolecular and Natural Products, Faculty of Sciences, Mohammed The First University, PB. 717, 60000 Oujda, Morocco
| | - Mohamed Berrabah
- Laboratory of Chemistry, Mineral and Analytical Solid, Department of Chemistry, Faculty of Sciences, Mohammed The First University, PB. 717, 60000 Oujda, Morocco
| | - Mohamed Bnouham
- Laboratory of Physiology, Genetic and Ethnopharmacology, Faculty of Sciences, Mohammed The First University, PB. 717, 60000 Oujda, Morocco
| | - Chakib Alem
- Laboratory of Biochemistry, Department of Biology, Faculty of Sciences & Techniques, Errachidia, Morocco
| | - Ferdinand Kouoh Elombo
- Laboratoires TBC, Faculty of Pharmaceutical and Biological Sciences, Lille, France; Laboratory of Pharmacology, Pharmacokinetics, and Clinical Pharmacy, Faculty of Pharmaceutical and Biological Sciences, Lille, France
| | - Bernard Gressier
- Laboratory of Pharmacology, Pharmacokinetics, and Clinical Pharmacy, Faculty of Pharmaceutical and Biological Sciences, Lille, France
| | | | - Bruno Eto
- Laboratoires TBC, Faculty of Pharmaceutical and Biological Sciences, Lille, France.
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Okada Y, Okada T, Sato-Numata K, Islam MR, Ando-Akatsuka Y, Numata T, Kubo M, Shimizu T, Kurbannazarova RS, Marunaka Y, Sabirov RZ. Cell Volume-Activated and Volume-Correlated Anion Channels in Mammalian Cells: Their Biophysical, Molecular, and Pharmacological Properties. Pharmacol Rev 2019; 71:49-88. [PMID: 30573636 DOI: 10.1124/pr.118.015917] [Citation(s) in RCA: 60] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
There are a number of mammalian anion channel types associated with cell volume changes. These channel types are classified into two groups: volume-activated anion channels (VAACs) and volume-correlated anion channels (VCACs). VAACs can be directly activated by cell swelling and include the volume-sensitive outwardly rectifying anion channel (VSOR), which is also called the volume-regulated anion channel; the maxi-anion channel (MAC or Maxi-Cl); and the voltage-gated anion channel, chloride channel (ClC)-2. VCACs can be facultatively implicated in, although not directly activated by, cell volume changes and include the cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, the Ca2+-activated Cl- channel (CaCC), and the acid-sensitive (or acid-stimulated) outwardly rectifying anion channel. This article describes the phenotypical properties and activation mechanisms of both groups of anion channels, including accumulating pieces of information on the basis of recent molecular understanding. To that end, this review also highlights the molecular identities of both anion channel groups; in addition to the molecular identities of ClC-2 and CFTR, those of CaCC, VSOR, and Maxi-Cl were recently identified by applying genome-wide approaches. In the last section of this review, the most up-to-date information on the pharmacological properties of both anion channel groups, especially their half-maximal inhibitory concentrations (IC50 values) and voltage-dependent blocking, is summarized particularly from the standpoint of pharmacological distinctions among them. Future physiologic and pharmacological studies are definitely warranted for therapeutic targeting of dysfunction of VAACs and VCACs.
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Affiliation(s)
- Yasunobu Okada
- Departments of Physiology and Systems Bioscience (Y.O.) and Molecular Cell Physiology (Y.M.), Kyoto Prefectural University of Medicine, Kyoto, Japan; Division of Cell Signaling, National Institute for Physiological Sciences, Okazaki, Japan (Y.O., T.O., M.R.I., M.K., R.Z.S.); Department of Physiology, School of Medicine, Fukuoka University, Fukuoka, Japan (K.S.-N., T.N.); Department of Cell Physiology, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Japan (Y.A.-A.); Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan (T.S.); Laboratory of Molecular Physiology, Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, Tashkent, Uzbekistan (R.S.K., R.Z.S.); and Research Institute for Clinical Physiology, Kyoto Industrial Health Association, Kyoto, Japan (Y.M.)
| | - Toshiaki Okada
- Departments of Physiology and Systems Bioscience (Y.O.) and Molecular Cell Physiology (Y.M.), Kyoto Prefectural University of Medicine, Kyoto, Japan; Division of Cell Signaling, National Institute for Physiological Sciences, Okazaki, Japan (Y.O., T.O., M.R.I., M.K., R.Z.S.); Department of Physiology, School of Medicine, Fukuoka University, Fukuoka, Japan (K.S.-N., T.N.); Department of Cell Physiology, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Japan (Y.A.-A.); Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan (T.S.); Laboratory of Molecular Physiology, Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, Tashkent, Uzbekistan (R.S.K., R.Z.S.); and Research Institute for Clinical Physiology, Kyoto Industrial Health Association, Kyoto, Japan (Y.M.)
| | - Kaori Sato-Numata
- Departments of Physiology and Systems Bioscience (Y.O.) and Molecular Cell Physiology (Y.M.), Kyoto Prefectural University of Medicine, Kyoto, Japan; Division of Cell Signaling, National Institute for Physiological Sciences, Okazaki, Japan (Y.O., T.O., M.R.I., M.K., R.Z.S.); Department of Physiology, School of Medicine, Fukuoka University, Fukuoka, Japan (K.S.-N., T.N.); Department of Cell Physiology, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Japan (Y.A.-A.); Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan (T.S.); Laboratory of Molecular Physiology, Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, Tashkent, Uzbekistan (R.S.K., R.Z.S.); and Research Institute for Clinical Physiology, Kyoto Industrial Health Association, Kyoto, Japan (Y.M.)
| | - Md Rafiqul Islam
- Departments of Physiology and Systems Bioscience (Y.O.) and Molecular Cell Physiology (Y.M.), Kyoto Prefectural University of Medicine, Kyoto, Japan; Division of Cell Signaling, National Institute for Physiological Sciences, Okazaki, Japan (Y.O., T.O., M.R.I., M.K., R.Z.S.); Department of Physiology, School of Medicine, Fukuoka University, Fukuoka, Japan (K.S.-N., T.N.); Department of Cell Physiology, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Japan (Y.A.-A.); Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan (T.S.); Laboratory of Molecular Physiology, Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, Tashkent, Uzbekistan (R.S.K., R.Z.S.); and Research Institute for Clinical Physiology, Kyoto Industrial Health Association, Kyoto, Japan (Y.M.)
| | - Yuhko Ando-Akatsuka
- Departments of Physiology and Systems Bioscience (Y.O.) and Molecular Cell Physiology (Y.M.), Kyoto Prefectural University of Medicine, Kyoto, Japan; Division of Cell Signaling, National Institute for Physiological Sciences, Okazaki, Japan (Y.O., T.O., M.R.I., M.K., R.Z.S.); Department of Physiology, School of Medicine, Fukuoka University, Fukuoka, Japan (K.S.-N., T.N.); Department of Cell Physiology, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Japan (Y.A.-A.); Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan (T.S.); Laboratory of Molecular Physiology, Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, Tashkent, Uzbekistan (R.S.K., R.Z.S.); and Research Institute for Clinical Physiology, Kyoto Industrial Health Association, Kyoto, Japan (Y.M.)
| | - Tomohiro Numata
- Departments of Physiology and Systems Bioscience (Y.O.) and Molecular Cell Physiology (Y.M.), Kyoto Prefectural University of Medicine, Kyoto, Japan; Division of Cell Signaling, National Institute for Physiological Sciences, Okazaki, Japan (Y.O., T.O., M.R.I., M.K., R.Z.S.); Department of Physiology, School of Medicine, Fukuoka University, Fukuoka, Japan (K.S.-N., T.N.); Department of Cell Physiology, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Japan (Y.A.-A.); Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan (T.S.); Laboratory of Molecular Physiology, Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, Tashkent, Uzbekistan (R.S.K., R.Z.S.); and Research Institute for Clinical Physiology, Kyoto Industrial Health Association, Kyoto, Japan (Y.M.)
| | - Machiko Kubo
- Departments of Physiology and Systems Bioscience (Y.O.) and Molecular Cell Physiology (Y.M.), Kyoto Prefectural University of Medicine, Kyoto, Japan; Division of Cell Signaling, National Institute for Physiological Sciences, Okazaki, Japan (Y.O., T.O., M.R.I., M.K., R.Z.S.); Department of Physiology, School of Medicine, Fukuoka University, Fukuoka, Japan (K.S.-N., T.N.); Department of Cell Physiology, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Japan (Y.A.-A.); Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan (T.S.); Laboratory of Molecular Physiology, Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, Tashkent, Uzbekistan (R.S.K., R.Z.S.); and Research Institute for Clinical Physiology, Kyoto Industrial Health Association, Kyoto, Japan (Y.M.)
| | - Takahiro Shimizu
- Departments of Physiology and Systems Bioscience (Y.O.) and Molecular Cell Physiology (Y.M.), Kyoto Prefectural University of Medicine, Kyoto, Japan; Division of Cell Signaling, National Institute for Physiological Sciences, Okazaki, Japan (Y.O., T.O., M.R.I., M.K., R.Z.S.); Department of Physiology, School of Medicine, Fukuoka University, Fukuoka, Japan (K.S.-N., T.N.); Department of Cell Physiology, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Japan (Y.A.-A.); Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan (T.S.); Laboratory of Molecular Physiology, Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, Tashkent, Uzbekistan (R.S.K., R.Z.S.); and Research Institute for Clinical Physiology, Kyoto Industrial Health Association, Kyoto, Japan (Y.M.)
| | - Ranohon S Kurbannazarova
- Departments of Physiology and Systems Bioscience (Y.O.) and Molecular Cell Physiology (Y.M.), Kyoto Prefectural University of Medicine, Kyoto, Japan; Division of Cell Signaling, National Institute for Physiological Sciences, Okazaki, Japan (Y.O., T.O., M.R.I., M.K., R.Z.S.); Department of Physiology, School of Medicine, Fukuoka University, Fukuoka, Japan (K.S.-N., T.N.); Department of Cell Physiology, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Japan (Y.A.-A.); Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan (T.S.); Laboratory of Molecular Physiology, Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, Tashkent, Uzbekistan (R.S.K., R.Z.S.); and Research Institute for Clinical Physiology, Kyoto Industrial Health Association, Kyoto, Japan (Y.M.)
| | - Yoshinori Marunaka
- Departments of Physiology and Systems Bioscience (Y.O.) and Molecular Cell Physiology (Y.M.), Kyoto Prefectural University of Medicine, Kyoto, Japan; Division of Cell Signaling, National Institute for Physiological Sciences, Okazaki, Japan (Y.O., T.O., M.R.I., M.K., R.Z.S.); Department of Physiology, School of Medicine, Fukuoka University, Fukuoka, Japan (K.S.-N., T.N.); Department of Cell Physiology, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Japan (Y.A.-A.); Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan (T.S.); Laboratory of Molecular Physiology, Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, Tashkent, Uzbekistan (R.S.K., R.Z.S.); and Research Institute for Clinical Physiology, Kyoto Industrial Health Association, Kyoto, Japan (Y.M.)
| | - Ravshan Z Sabirov
- Departments of Physiology and Systems Bioscience (Y.O.) and Molecular Cell Physiology (Y.M.), Kyoto Prefectural University of Medicine, Kyoto, Japan; Division of Cell Signaling, National Institute for Physiological Sciences, Okazaki, Japan (Y.O., T.O., M.R.I., M.K., R.Z.S.); Department of Physiology, School of Medicine, Fukuoka University, Fukuoka, Japan (K.S.-N., T.N.); Department of Cell Physiology, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Japan (Y.A.-A.); Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan (T.S.); Laboratory of Molecular Physiology, Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, Tashkent, Uzbekistan (R.S.K., R.Z.S.); and Research Institute for Clinical Physiology, Kyoto Industrial Health Association, Kyoto, Japan (Y.M.)
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8
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Stefan SM, Wiese M. Small-molecule inhibitors of multidrug resistance-associated protein 1 and related processes: A historic approach and recent advances. Med Res Rev 2018; 39:176-264. [DOI: 10.1002/med.21510] [Citation(s) in RCA: 37] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2018] [Revised: 04/05/2018] [Accepted: 04/28/2018] [Indexed: 12/19/2022]
Affiliation(s)
- Sven Marcel Stefan
- Pharmaceutical Institute; Rheinische Friedrich-Wilhelms-University; Bonn Germany
| | - Michael Wiese
- Pharmaceutical Institute; Rheinische Friedrich-Wilhelms-University; Bonn Germany
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9
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Vázquez-Sánchez AY, Hinojosa LM, Parraguirre-Martínez S, González A, Morales F, Montalvo G, Vera E, Hernández-Gallegos E, Camacho J. Expression of K ATP channels in human cervical cancer: Potential tools for diagnosis and therapy. Oncol Lett 2018; 15:6302-6308. [PMID: 29849783 PMCID: PMC5962834 DOI: 10.3892/ol.2018.8165] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2017] [Accepted: 10/18/2017] [Indexed: 11/19/2022] Open
Abstract
Various ion channels, including ATP-sensitive potassium (KATP) channels, are expressed in cancer and have been suggested as potential tumor markers and therapeutic targets. KATP channels are composed of at least two types of subunit, an inwardly rectifying K+ channel (Kir6.x) and a sulfonylurea receptor (SUR). However, the association between KATP channels and cervical cancer remains elusive. The present study determined that the Kir6.2, SUR1 and SUR2 subunits are expressed in cervical cancer cell lines and/or human biopsies. The potential association of subunit expression with tumor differentiation and invasion was analyzed. The effect of the KATP channel blocker glibenclamide on the proliferation of cervical cancer cell lines was also studied. Five cervical cancer cell lines, two primary cultures of cervical cancer cells, one normal keratinocyte cell line and 74 human biopsies were used in the experiments. The mRNA and protein levels of the Kir6.2 subunit were assessed by reverse transcription-polymerase chain reaction and immunochemistry, respectively. Cell proliferation was evaluated by MTT assay. Kir6.2 subunit overexpression compared with control, was observed in some cervical cancer cell lines and cervical tumor tissues. Additionally, increased KATP channel expression was observed in high-grade, poorly differentiated and invasive human cervical cancer biopsies. Kir6.2 subunit expression was not observed in the majority of the non-cancerous cervical tissues. The effect of the KATP channel blocker glibenclamide on the proliferation of five different cervical cancer cell lines was studied, revealing that as Kir6.2 mRNA expression increased, the inhibitory effect of glibenclamide also increased. The results of the present study suggest, for the first time to the best of our knowledge, that the KATP channel subunits, Kir6.2 and SUR2, could potentially represent tools for diagnosing and treating cervical cancer.
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Affiliation(s)
- Alma Yolanda Vázquez-Sánchez
- Department of Pharmacology, Center for Research and Advanced Studies of The National Polytechnic Institute, Mexico City 07360, Mexico
| | - Luz María Hinojosa
- Service of Dysplasia, Gynecology and Obstetrics, 'Dr Manuel Gea González' Hospital General, Mexico City 14080, Mexico
| | - Sara Parraguirre-Martínez
- Division of Anatomical Pathology, 'Dr Manuel Gea González' Hospital General, Mexico City 14080, Mexico
| | - Aarón González
- Service of Colposcopy, Instituto Nacional de Cancerología, Mexico City 14080, Mexico
| | - Flavia Morales
- Medical Oncology, Instituto Nacional de Cancerología, Mexico City 14080, Mexico
| | - Gonzalo Montalvo
- Service of Gynecology, Instituto Nacional de Cancerología, Mexico City 14080, Mexico
| | - Eunice Vera
- Department of Pharmacology, Center for Research and Advanced Studies of The National Polytechnic Institute, Mexico City 07360, Mexico
| | - Elisabeth Hernández-Gallegos
- Department of Pharmacology, Center for Research and Advanced Studies of The National Polytechnic Institute, Mexico City 07360, Mexico
| | - Javier Camacho
- Department of Pharmacology, Center for Research and Advanced Studies of The National Polytechnic Institute, Mexico City 07360, Mexico
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10
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Krishnan V, Maddox JW, Rodriguez T, Gleason E. A role for the cystic fibrosis transmembrane conductance regulator in the nitric oxide-dependent release of Cl - from acidic organelles in amacrine cells. J Neurophysiol 2017; 118:2842-2852. [PMID: 28835528 DOI: 10.1152/jn.00511.2017] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2017] [Revised: 08/21/2017] [Accepted: 08/21/2017] [Indexed: 12/16/2022] Open
Abstract
γ-Amino butyric acid (GABA) and glycine typically mediate synaptic inhibition because their ligand-gated ion channels support the influx of Cl- However, the electrochemical gradient for Cl- across the postsynaptic plasma membrane determines the voltage response of the postsynaptic cell. Typically, low cytosolic Cl- levels support inhibition, whereas higher levels of cytosolic Cl- can suppress inhibition or promote depolarization. We previously reported that nitric oxide (NO) releases Cl- from acidic organelles and transiently elevates cytosolic Cl-, making the response to GABA and glycine excitatory. In this study, we test the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the NO-dependent efflux of organellar Cl- We first establish the mRNA and protein expression of CFTR in our model system, cultured chick retinal amacrine cells. Using whole cell voltage-clamp recordings of currents through GABA-gated Cl- channels, we examine the effects of pharmacological inhibition of CFTR on the NO-dependent release of internal Cl- To interfere with the expression of CFTR, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. We find that both pharmacological inhibition and CRISPR/Cas9-mediated knockdown of CFTR block the ability of NO to release Cl- from internal stores. These results demonstrate that CFTR is required for the NO-dependent efflux of Cl- from acidic organelles.NEW & NOTEWORTHY Although CFTR function has been studied extensively in the context of epithelia, relatively little is known about its function in neurons. We show that CFTR is involved in an NO-dependent release of Cl- from acidic organelles. This internal function of CFTR is particularly relevant to neuronal physiology because postsynaptic cytosolic Cl- levels determine the outcome of GABA- and glycinergic synaptic signaling. Thus the CFTR may play a role in regulating synaptic transmission.
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Affiliation(s)
- Vijai Krishnan
- Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana
| | - J Wesley Maddox
- Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana
| | - Tyler Rodriguez
- Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana
| | - Evanna Gleason
- Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana
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11
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Leonard CE, Hennessy S, Han X, Siscovick DS, Flory JH, Deo R. Pro- and Antiarrhythmic Actions of Sulfonylureas: Mechanistic and Clinical Evidence. Trends Endocrinol Metab 2017; 28:561-586. [PMID: 28545784 PMCID: PMC5522643 DOI: 10.1016/j.tem.2017.04.003] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/27/2017] [Revised: 04/18/2017] [Accepted: 04/19/2017] [Indexed: 12/19/2022]
Abstract
Sulfonylureas are the most commonly used second-line drug class for treating type 2 diabetes mellitus (T2DM). While the cardiovascular safety of sulfonylureas has been examined in several trials and nonrandomized studies, little is known of their specific effects on sudden cardiac arrest (SCA) and related serious arrhythmic outcomes. This knowledge gap is striking, because persons with DM are at increased risk of SCA. In this review, we explore the influence of sulfonylureas on the risk of serious arrhythmias, with specific foci on ischemic preconditioning, cardiac excitability, and serious hypoglycemia as putative mechanisms. Elucidating the relationship between individual sulfonylureas and serious arrhythmias is critical, especially as the diabetes epidemic intensifies and SCA incidence increases in persons with diabetes.
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Affiliation(s)
- Charles E Leonard
- Center for Pharmacoepidemiology Research and Training, Department of Biostatistics, Epidemiology, and Informatics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Center for Clinical Epidemiology and Biostatistics, Department of Biostatistics, Epidemiology, and Informatics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
| | - Sean Hennessy
- Center for Pharmacoepidemiology Research and Training, Department of Biostatistics, Epidemiology, and Informatics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Center for Clinical Epidemiology and Biostatistics, Department of Biostatistics, Epidemiology, and Informatics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Xu Han
- Center for Pharmacoepidemiology Research and Training, Department of Biostatistics, Epidemiology, and Informatics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Center for Clinical Epidemiology and Biostatistics, Department of Biostatistics, Epidemiology, and Informatics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - David S Siscovick
- The New York Academy of Medicine, New York, NY 10029, USA; Department of Epidemiology, School of Public Health, University of Washington, Seattle, WA 98195, USA
| | - James H Flory
- Center for Pharmacoepidemiology Research and Training, Department of Biostatistics, Epidemiology, and Informatics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Healthcare Policy and Research, Division of Comparative Effectiveness, Weill Cornell Medicine, Cornell University, New York, NY 10065, USA; Memorial Sloan Kettering Cancer Center, New York, NY 10022, USA
| | - Rajat Deo
- Center for Pharmacoepidemiology Research and Training, Department of Biostatistics, Epidemiology, and Informatics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Division of Cardiovascular Medicine, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
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12
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Abstract
Mitochondria are the "power house" of a cell continuously generating ATP to ensure its proper functioning. The constant production of ATP via oxidative phosphorylation demands a large electrochemical force that drives protons across the highly selective and low-permeable mitochondrial inner membrane. Besides the conventional role of generating ATP, mitochondria also play an active role in calcium signaling, generation of reactive oxygen species (ROS), stress responses, and regulation of cell-death pathways. Deficiencies in these functions result in several pathological disorders like aging, cancer, diabetes, neurodegenerative and cardiovascular diseases. A plethora of ion channels and transporters are present in the mitochondrial inner and outer membranes which work in concert to preserve the ionic equilibrium of a cell for the maintenance of cell integrity, in physiological as well as pathophysiological conditions. For, e.g., mitochondrial cation channels KATP and BKCa play a significant role in cardioprotection from ischemia-reperfusion injury. In addition to the cation channels, mitochondrial anion channels are equally essential, as they aid in maintaining electro-neutrality by regulating the cell volume and pH. This chapter focusses on the information on molecular identity, structure, function, and physiological relevance of mitochondrial chloride channels such as voltage dependent anion channels (VDACs), uncharacterized mitochondrial inner membrane anion channels (IMACs), chloride intracellular channels (CLIC) and the aspects of forthcoming chloride channels.
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Affiliation(s)
- Devasena Ponnalagu
- Department of Pharmacology and Physiology, Drexel University College of Medicine, 245 N. 15th Street, Room 8154, Mail Stop 488, Philadelphia, PA, 19102-1192, USA
| | - Harpreet Singh
- Department of Pharmacology and Physiology, Drexel University College of Medicine, 245 N. 15th Street, Room 8154, Mail Stop 488, Philadelphia, PA, 19102-1192, USA.
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13
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Koyanagi S, Kusunose N, Taniguchi M, Akamine T, Kanado Y, Ozono Y, Masuda T, Kohro Y, Matsunaga N, Tsuda M, Salter MW, Inoue K, Ohdo S. Glucocorticoid regulation of ATP release from spinal astrocytes underlies diurnal exacerbation of neuropathic mechanical allodynia. Nat Commun 2016; 7:13102. [PMID: 27739425 PMCID: PMC5067584 DOI: 10.1038/ncomms13102] [Citation(s) in RCA: 88] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2015] [Accepted: 09/02/2016] [Indexed: 12/26/2022] Open
Abstract
Diurnal variations in pain hypersensitivity are common in chronic pain disorders, but the underlying mechanisms are enigmatic. Here, we report that mechanical pain hypersensitivity in sciatic nerve-injured mice shows pronounced diurnal alterations, which critically depend on diurnal variations in glucocorticoids from the adrenal glands. Diurnal enhancement of pain hypersensitivity is mediated by glucocorticoid-induced enhancement of the extracellular release of ATP in the spinal cord, which stimulates purinergic receptors on microglia in the dorsal horn. We identify serum- and glucocorticoid-inducible kinase-1 (SGK-1) as the key molecule responsible for the glucocorticoid-enhanced release of ATP from astrocytes. SGK-1 protein levels in spinal astrocytes are increased in response to glucocorticoid stimuli and enhanced ATP release by opening the pannexin-1 hemichannels. Our findings reveal an unappreciated circadian machinery affecting pain hypersensitivity caused by peripheral nerve injury, thus opening up novel approaches to the management of chronic pain. Neuropathic pain hypersensitivity is known to undergo diurnal variations, although the underlying mechanisms are not clear. Using a sciatic nerve-injury mouse model, the authors find such diurnal changes are mediated by glucocorticoid induced enhancement of ATP release from astrocytes via pannexin-1 hemichannels.
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Affiliation(s)
- Satoru Koyanagi
- Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.,Department of Glocal Healthcare, Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
| | - Naoki Kusunose
- Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
| | - Marie Taniguchi
- Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
| | - Takahiro Akamine
- Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
| | - Yuki Kanado
- Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
| | - Yui Ozono
- Department of Molecular and System Pharmacology, Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
| | - Takahiro Masuda
- Department of Life Innovation, Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
| | - Yuta Kohro
- Department of Life Innovation, Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
| | - Naoya Matsunaga
- Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.,Department of Glocal Healthcare, Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
| | - Makoto Tsuda
- Department of Life Innovation, Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
| | - Michael W Salter
- Program in Neuroscience and Mental Health, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.,Department of Physiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8.,University of Toronto Centre for the Study of Pain, Toronto, Ontario, Canada M5T 1P8
| | - Kazuhide Inoue
- Department of Molecular and System Pharmacology, Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
| | - Shigehiro Ohdo
- Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
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14
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Sabirov RZ, Merzlyak PG, Islam MR, Okada T, Okada Y. The properties, functions, and pathophysiology of maxi-anion channels. Pflugers Arch 2016; 468:405-20. [PMID: 26733413 DOI: 10.1007/s00424-015-1774-5] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2015] [Revised: 12/11/2015] [Accepted: 12/15/2015] [Indexed: 01/19/2023]
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15
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Raphemot R, Swale DR, Dadi PK, Jacobson DA, Cooper P, Wojtovich AP, Banerjee S, Nichols CG, Denton JS. Direct activation of β-cell KATP channels with a novel xanthine derivative. Mol Pharmacol 2014; 85:858-65. [PMID: 24646456 PMCID: PMC4014665 DOI: 10.1124/mol.114.091884] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2014] [Accepted: 03/19/2014] [Indexed: 11/22/2022] Open
Abstract
ATP-regulated potassium (KATP) channel complexes of inward rectifier potassium channel (Kir) 6.2 and sulfonylurea receptor (SUR) 1 critically regulate pancreatic islet β-cell membrane potential, calcium influx, and insulin secretion, and consequently, represent important drug targets for metabolic disorders of glucose homeostasis. The KATP channel opener diazoxide is used clinically to treat intractable hypoglycemia caused by excessive insulin secretion, but its use is limited by off-target effects due to lack of potency and selectivity. Some progress has been made in developing improved Kir6.2/SUR1 agonists from existing chemical scaffolds and compound screening, but there are surprisingly few distinct chemotypes that are specific for SUR1-containing KATP channels. Here we report the serendipitous discovery in a high-throughput screen of a novel activator of Kir6.2/SUR1: VU0071063 [7-(4-(tert-butyl)benzyl)-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione]. The xanthine derivative rapidly and dose-dependently activates Kir6.2/SUR1 with a half-effective concentration (EC50) of approximately 7 μM, is more efficacious than diazoxide at low micromolar concentrations, directly activates the channel in excised membrane patches, and is selective for SUR1- over SUR2A-containing Kir6.1 or Kir6.2 channels, as well as Kir2.1, Kir2.2, Kir2.3, Kir3.1/3.2, and voltage-gated potassium channel 2.1. Finally, we show that VU0071063 activates native Kir6.2/SUR1 channels, thereby inhibiting glucose-stimulated calcium entry in isolated mouse pancreatic β cells. VU0071063 represents a novel tool/compound for investigating β-cell physiology, KATP channel gating, and a new chemical scaffold for developing improved activators with medicinal chemistry.
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Affiliation(s)
- Rene Raphemot
- Departments of Anesthesiology (R.R., D.R.S., S.B., J.S.D.), Pharmacology (R.R., J.S.D.), and Molecular Physiology and Biophysics (P.K.D., D.A.J.) and Institutes of Chemical Biology (J.S.D.) and Global Health (J.S.D.), Vanderbilt University Medical Center, Nashville, Tennessee; Department of Medicine, University of Rochester Medical Center, Rochester, New York (A.P.W.); and Department of Cell Biology and Physiology (P.C., C.G.N.) and Center for the Investigation of Membrane Excitability Disorders (P.C., C.G.N.), Washington University School of Medicine in St. Louis, St. Louis, Missouri
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16
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Edlund A, Esguerra JLS, Wendt A, Flodström-Tullberg M, Eliasson L. CFTR and Anoctamin 1 (ANO1) contribute to cAMP amplified exocytosis and insulin secretion in human and murine pancreatic beta-cells. BMC Med 2014; 12:87. [PMID: 24885604 PMCID: PMC4035698 DOI: 10.1186/1741-7015-12-87] [Citation(s) in RCA: 108] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/09/2013] [Accepted: 04/10/2014] [Indexed: 01/07/2023] Open
Abstract
BACKGROUND Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene lead to the disease cystic fibrosis (CF). Although patients with CF often have disturbances in glucose metabolism including impaired insulin release, no previous studies have tested the hypothesis that CFTR has a biological function in pancreatic beta-cells. METHODS Experiments were performed on islets and single beta-cells from human donors and NMRI-mice. Detection of CFTR was investigated using PCR and confocal microscopy. Effects on insulin secretion were measured with radioimmunoassay (RIA). The patch-clamp technique was used to measure ion channel currents and calcium-dependent exocytosis (as changes in membrane capacitance) on single cells with high temporal resolution. Analysis of ultrastructure was done on transmission electron microscopy (TEM) images. RESULTS We detected the presence of CFTR and measured a small CFTR conductance in both human and mouse beta-cells. The augmentation of insulin secretion at 16.7 mM glucose by activation of CFTR by cAMP (forskolin (FSK) or GLP-1) was significantly inhibited when CFTR antagonists (GlyH-101 and/or CFTRinh-172) were added. Likewise, capacitance measurements demonstrated reduced cAMP-dependent exocytosis upon CFTR-inhibition, concomitant with a decreased number of docked insulin granules. Finally, our studies demonstrate that CFTR act upstream of the chloride channel Anoctamin 1 (ANO1; TMEM16A) in the regulation of cAMP- and glucose-stimulated insulin secretion. CONCLUSION Our work demonstrates a novel function for CFTR as a regulator of pancreatic beta-cell insulin secretion and exocytosis, and put forward a role for CFTR as regulator of ANO1 and downstream priming of insulin granules prior to fusion and release of insulin. The pronounced regulatory effect of CFTR on insulin secretion is consistent with impaired insulin secretion in patients with CF.
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Affiliation(s)
| | | | | | | | - Lena Eliasson
- Unit of Islet Cell Exocytosis, Lund University Diabetes Centre, Department Clinical Sciences in Malmö, Lund University, Clinical Research Centre, SUS Malmö, Jan Waldenströms gata 35, SE 205 02 Malmö, Sweden.
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Guinamard R, Hof T, Del Negro CA. The TRPM4 channel inhibitor 9-phenanthrol. Br J Pharmacol 2014; 171:1600-13. [PMID: 24433510 PMCID: PMC3966741 DOI: 10.1111/bph.12582] [Citation(s) in RCA: 73] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2013] [Revised: 12/17/2013] [Accepted: 01/08/2014] [Indexed: 01/06/2023] Open
Abstract
The phenanthrene-derivative 9-phenanthrol is a recently identified inhibitor of the transient receptor potential melastatin (TRPM) 4 channel, a Ca(2+) -activated non-selective cation channel whose mechanism of action remains to be determined. Subsequent studies performed on other ion channels confirm the specificity of the drug for TRPM4. In addition, 9-phenanthrol modulates a variety of physiological processes through TRPM4 current inhibition and thus exerts beneficial effects in several pathological conditions. 9-Phenanthrol modulates smooth muscle contraction in bladder and cerebral arteries, affects spontaneous activity in neurons and in the heart, and reduces lipopolysaccharide-induced cell death. Among promising potential applications, 9-phenanthrol exerts cardioprotective effects against ischaemia-reperfusion injuries and reduces ischaemic stroke injuries. In addition to reviewing the biophysical effects of 9-phenanthrol, here we present information about its appropriate use in physiological studies and possible clinical applications.
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Affiliation(s)
- R Guinamard
- EA 4650, Groupe Signalisation, Electrophysiologie et Imagerie des Lésions d'Ischémie-Reperfusion Myocardique, UCBN, Normandie UniversitéCaen, France
- Department of Applied Science, The College of William and MaryWilliamsburg, VA, USA
| | - T Hof
- EA 4650, Groupe Signalisation, Electrophysiologie et Imagerie des Lésions d'Ischémie-Reperfusion Myocardique, UCBN, Normandie UniversitéCaen, France
| | - C A Del Negro
- Department of Applied Science, The College of William and MaryWilliamsburg, VA, USA
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18
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Linsdell P. Cystic fibrosis transmembrane conductance regulator chloride channel blockers: Pharmacological, biophysical and physiological relevance. World J Biol Chem 2014; 5:26-39. [PMID: 24600512 PMCID: PMC3942540 DOI: 10.4331/wjbc.v5.i1.26] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/02/2013] [Revised: 11/15/2013] [Accepted: 12/11/2013] [Indexed: 02/05/2023] Open
Abstract
Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel causes cystic fibrosis, while inappropriate activity of this channel occurs in secretory diarrhea and polycystic kidney disease. Drugs that interact directly with CFTR are therefore of interest in the treatment of a number of disease states. This review focuses on one class of small molecules that interacts directly with CFTR, namely inhibitors that act by directly blocking chloride movement through the open channel pore. In theory such compounds could be of use in the treatment of diarrhea and polycystic kidney disease, however in practice all known substances acting by this mechanism to inhibit CFTR function lack either the potency or specificity for in vivo use. Nevertheless, this theoretical pharmacological usefulness set the scene for the development of more potent, specific CFTR inhibitors. Biophysically, open channel blockers have proven most useful as experimental probes of the structure and function of the CFTR chloride channel pore. Most importantly, the use of these blockers has been fundamental in developing a functional model of the pore that includes a wide inner vestibule that uses positively charged amino acid side chains to attract both permeant and blocking anions from the cell cytoplasm. CFTR channels are also subject to this kind of blocking action by endogenous anions present in the cell cytoplasm, and recently this blocking effect has been suggested to play a role in the physiological control of CFTR channel function, in particular as a novel mechanism linking CFTR function dynamically to the composition of epithelial cell secretions. It has also been suggested that future drugs could target this same pathway as a way of pharmacologically increasing CFTR activity in cystic fibrosis. Studying open channel blockers and their mechanisms of action has resulted in significant advances in our understanding of CFTR as a pharmacological target in disease states, of CFTR channel structure and function, and of how CFTR activity is controlled by its local environment.
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Madurga A, Mižíková I, Ruiz-Camp J, Vadász I, Herold S, Mayer K, Fehrenbach H, Seeger W, Morty RE. Systemic hydrogen sulfide administration partially restores normal alveolarization in an experimental animal model of bronchopulmonary dysplasia. Am J Physiol Lung Cell Mol Physiol 2014; 306:L684-97. [PMID: 24508731 DOI: 10.1152/ajplung.00361.2013] [Citation(s) in RCA: 48] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Arrested alveolarization is the pathological hallmark of bronchopulmonary dysplasia (BPD), a complication of premature birth. Here, the impact of systemic application of hydrogen sulfide (H2S) on postnatal alveolarization was assessed in a mouse BPD model. Exposure of newborn mice to 85% O2 for 10 days reduced the total lung alveoli number by 56% and increased alveolar septal wall thickness by 29%, as assessed by state-of-the-art stereological analysis. Systemic application of H2S via the slow-release H2S donor GYY4137 for 10 days resulted in pronounced improvement in lung alveolarization in pups breathing 85% O2, compared with vehicle-treated littermates. Although without impact on lung oxidative status, systemic H2S blunted leukocyte infiltration into alveolar air spaces provoked by hyperoxia, and restored normal lung interleukin 10 levels that were otherwise depressed by 85% O2. Treatment of primary mouse alveolar type II (ATII) cells with the rapid-release H2S donor NaHS had no impact on cell viability; however, NaHS promoted ATII cell migration. Although exposure of ATII cells to 85% O2 caused dramatic changes in mRNA expression, exposure to either GYY4137 or NaHS had no impact on ATII cell mRNA expression, as assessed by microarray, suggesting that the effects observed were independent of changes in gene expression. The impact of NaHS on ATII cell migration was attenuated by glibenclamide, implicating ion channels, and was accompanied by activation of Akt, hinting at two possible mechanisms of H2S action. These data support further investigation of H2S as a candidate interventional strategy to limit the arrested alveolarization associated with BPD.
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Affiliation(s)
- Alicia Madurga
- Dept. of Lung Development and Remodelling, Max Planck Institute for Heart and Lung Research, Parkstrasse 1, D-61231 Bad Nauheim, Germany.
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Patel BA. Mucosal adenosine triphosphate mediates serotonin release from ileal but not colonic guinea pig enterochromaffin cells. Neurogastroenterol Motil 2014; 26:237-46. [PMID: 24188286 DOI: 10.1111/nmo.12254] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/25/2013] [Accepted: 09/26/2013] [Indexed: 12/27/2022]
Abstract
BACKGROUND Mechanical stimulation of the mucosal epithelium results in increased serotonin (5-HT) release from enterochromaffin (EC) cells. Little is known about how this process varies in different regions of the intestinal tract; however, purines are felt to play a role. We studied the relationship between mechanical stimulation, adenosine triphosphate (ATP), and 5-HT release from ileal and colonic mucosal tissue. METHODS Amperometric recordings of ATP and 5-HT were carried out using an ATP biosensor and boron-doped diamond microelectrode. Levels of extracellular ATP and 5-HT were monitored using high performance liquid chromatography. KEY RESULTS Under basal conditions, 5-HT levels were significantly decreased in the ileum (p < 0.001) but not the colon in the presence of the P2 antagonist suramin (100 μM). Ecto-ATPase inhibitor ARL67156 (10 μM) elevated ATP levels in the ileum and colon (both p < 0.001), but only 5-HT levels in the ileum (p < 0.001). Exogenous ATP increased 5-HT release in the presence of tetrodotoxin in the ileum (p < 0.001), but had not effect in the colon. Mechanical stimulation increased levels of 5-HT in the ileum (p < 0.001) and colon (p < 0.01), but levels returned to baseline in the presence of suramin and MRS2179 in the ileum. The onset of 5-HT release was delayed following mechanical stimulation. The rise time of the ATP response was quicker than that of 5-HT during mechanical stimulation. CONCLUSIONS & INFERENCES During mechanical stimulation of the mucosal epithelium, ATP mediates 5-HT release from EC cells in the ileum, but not the colon. Mucosal 5-HT signaling following mechanical stimulation is varied in different regions of the intestinal tract.
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Affiliation(s)
- B A Patel
- School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, UK
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21
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Yin L, Vijaygopal P, MacGregor GG, Menon R, Ranganathan P, Prabhakaran S, Zhang L, Zhang M, Binder HJ, Okunieff P, Vidyasagar S. Glucose stimulates calcium-activated chloride secretion in small intestinal cells. Am J Physiol Cell Physiol 2014; 306:C687-96. [PMID: 24477233 DOI: 10.1152/ajpcell.00174.2013] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
The sodium-coupled glucose transporter-1 (SGLT1)-based oral rehydration solution (ORS) used in the management of acute diarrhea does not substantially reduce stool output, despite the fact that glucose stimulates the absorption of sodium and water. To explain this phenomenon, we investigated the possibility that glucose might also stimulate anion secretion. Transepithelial electrical measurements and isotope flux measurements in Ussing chambers were used to study the effect of glucose on active chloride and fluid secretion in mouse small intestinal cells and human Caco-2 cells. Confocal fluorescence laser microscopy and immunohistochemistry measured intracellular changes in calcium, sodium-glucose linked transporter, and calcium-activated chloride channel (anoctamin 1) expression. In addition to enhancing active sodium absorption, glucose increased intracellular calcium and stimulated electrogenic chloride secretion. Calcium imaging studies showed increased intracellular calcium when intestinal cells were exposed to glucose. Niflumic acid, but not glibenclamide, inhibited glucose-stimulated chloride secretion in mouse small intestines and in Caco-2 cells. Glucose-stimulated chloride secretion was not seen in ileal tissues incubated with the intracellular calcium chelater BAPTA-AM and the sodium-potassium-2 chloride cotransporter 1 (NKCC1) blocker bumetanide. These observations establish that glucose not only stimulates active Na absorption, a well-established phenomenon, but also induces a Ca-activated chloride secretion. This may explain the failure of glucose-based ORS to markedly reduce stool output in acute diarrhea. These results have immediate potential to improve the treatment outcomes for acute and/or chronic diarrheal diseases by replacing glucose with compounds that do not stimulate chloride secretion.
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Affiliation(s)
- Liangjie Yin
- Department of Radiation Oncology, University of Florida Shands Cancer Center, Cancer and Genetics Research Complex, Gainesville, Florida
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Simon F, Varela D, Cabello-Verrugio C. Oxidative stress-modulated TRPM ion channels in cell dysfunction and pathological conditions in humans. Cell Signal 2013; 25:1614-24. [PMID: 23602937 DOI: 10.1016/j.cellsig.2013.03.023] [Citation(s) in RCA: 60] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2012] [Revised: 03/25/2013] [Accepted: 03/28/2013] [Indexed: 10/27/2022]
Abstract
The transient receptor potential melastatin (TRPM) protein family is an extensive group of ion channels expressed in several types of mammalian cells. Many studies have shown that these channels are crucial for performing several physiological functions. Additionally, a large body of evidence indicates that these channels are also involved in numerous human diseases, known as channelopathies. A characteristic event frequently observed during pathological states is the raising in intracellular oxidative agents over reducing molecules, shifting the redox balance and inducing oxidative stress. In particular, three members of the TRPM subfamily, TRPM2, TRPM4 and TRPM7, share the remarkable feature that their activities are modulated by oxidative stress. Because of the increase in oxidative stress, these TRPM channels function aberrantly, promoting the onset and development of diseases. Increases, absences, or modifications in the function of these redox-modulated TRPM channels are associated with cell dysfunction and human pathologies. Therefore, the effect of oxidative stress on ion channels becomes an essential part of the pathogenic mechanism. Thus, oxidative stress-modulated ion channels are more susceptible to generating pathological states than oxidant-independent channels. This review examines the most relevant findings regarding the participation of the oxidative stress-modulated TRPM ion channels, TRPM2, TRPM4, and TRPM7, in human diseases. In addition, the potential roles of these channels as therapeutic tools and targets for drug design are discussed.
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Affiliation(s)
- Felipe Simon
- Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas and Facultad de Medicina, Universidad Andres Bello, Avenida Republica 239, 8370146, Santiago, Chile.
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Babes A, Fischer MJ, Filipovic M, Engel MA, Flonta ML, Reeh PW. The anti-diabetic drug glibenclamide is an agonist of the transient receptor potential Ankyrin 1 (TRPA1) ion channel. Eur J Pharmacol 2013; 704:15-22. [DOI: 10.1016/j.ejphar.2013.02.018] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2012] [Revised: 02/01/2013] [Accepted: 02/07/2013] [Indexed: 12/16/2022]
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Barr TP, Albrecht PJ, Hou Q, Mongin AA, Strichartz GR, Rice FL. Air-stimulated ATP release from keratinocytes occurs through connexin hemichannels. PLoS One 2013; 8:e56744. [PMID: 23457608 PMCID: PMC3574084 DOI: 10.1371/journal.pone.0056744] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2012] [Accepted: 01/14/2013] [Indexed: 01/17/2023] Open
Abstract
Cutaneous ATP release plays an important role in both epidermal stratification and chronic pain, but little is known about ATP release mechanisms in keratinocytes that comprise the epidermis. In this study, we analyzed ATP release from cultured human neonatal keratinocytes briefly exposed to air, a process previously demonstrated to trigger ATP release from these cells. We show that exposing keratinocytes to air by removing media for 15 seconds causes a robust, long-lasting ATP release. This air-stimulated ATP release was increased in calcium differentiated cultures which showed a corresponding increase in connexin 43 mRNA, a major component of keratinocyte hemichannels. The known connexin hemichannel inhibitors 1-octanol and carbenoxolone both significantly reduced air-stimulated ATP release, as did two drugs traditionally used as ABC transporter inhibitors (glibenclamide and verapamil). These same 4 inhibitors also prevented an increase in the uptake of a connexin permeable dye induced by air exposure, confirming that connexin hemichannels are open during air-stimulated ATP release. In contrast, activity of the MDR1 ABC transporter was reduced by air exposure and the drugs that inhibited air-stimulated ATP release had differential effects on this transporter. These results indicate that air exposure elicits non-vesicular release of ATP from keratinocytes through connexin hemichannels and that drugs used to target connexin hemichannels and ABC transporters may cross-inhibit. Connexins represent a novel, peripheral target for the treatment of chronic pain and dermatological disease.
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Affiliation(s)
- Travis P. Barr
- Pain Research Center, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham & Women's Hospital, Boston, Massachusetts, United States of America
| | - Phillip J. Albrecht
- Albany Medical College, Center for Neuroscience and Neuropharmacology, Albany, New York, United States of America
| | - Quanzhi Hou
- Albany Medical College, Center for Neuroscience and Neuropharmacology, Albany, New York, United States of America
| | - Alexander A. Mongin
- Albany Medical College, Center for Neuroscience and Neuropharmacology, Albany, New York, United States of America
| | - Gary R. Strichartz
- Pain Research Center, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham & Women's Hospital, Boston, Massachusetts, United States of America
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, United States of America
| | - Frank L. Rice
- Albany Medical College, Center for Neuroscience and Neuropharmacology, Albany, New York, United States of America
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Zertal-Zidani S, Busiah K, Edelman A, Polak M, Scharfmann R. Small-molecule inhibitors of the cystic fibrosis transmembrane conductance regulator increase pancreatic endocrine cell development in rat and mouse. Diabetologia 2013; 56. [PMID: 23178930 PMCID: PMC3536988 DOI: 10.1007/s00125-012-2778-8] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
AIMS/HYPOTHESIS The main objective of this work was to discover new drugs that can activate the differentiation of multipotent pancreatic progenitors into endocrine cells. METHODS In vitro experiments were performed using fetal pancreatic explants from rats and mice. In this assay, we examined the actions on pancreatic cell development of glibenclamide, a sulfonylurea derivative, and glycine hydrazide (GlyH-101), a small-molecule inhibitor of cystic fibrosis transmembrane conductance regulator (CFTR). We next tested the actions of GlyH-101 on in vivo pancreatic cell development. RESULTS Glibenclamide (10 nmol/l-100 μmol/l) did not alter the morphology or growth of the developing pancreas and exerted no deleterious effects on exocrine cell development in the pancreas. Unexpectedly, glibenclamide at its highest concentration promoted endocrine differentiation. This glibenclamide-induced promotion of the endocrine pathway could not be reproduced when other sulfonylureas were used, suggesting that glibenclamide had an off-target action. This high concentration of glibenclamide had previously been reported to inhibit CFTR. We found that the effects of glibenclamide on the developing pancreas could be mimicked both in vitro and in vivo by GlyH-101. CONCLUSIONS/INTERPRETATION Collectively, we demonstrate that two small-molecule inhibitors of the CFTR, glibenclamide and GlyH-101, increase the number of pancreatic endocrine cells by increasing the size of the pool of neurogenin 3-positive endocrine progenitors in the developing pancreas.
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Affiliation(s)
- S. Zertal-Zidani
- Inserm U845, Research Center Growth and Signalling, Faculté de Médecine, Université Paris Descartes, Sorbonne Paris Cité, Faculty Necker, 156 Rue de Vaugirard, 75015 Paris, France
| | - K. Busiah
- Inserm U845, Research Center Growth and Signalling, Faculté de Médecine, Université Paris Descartes, Sorbonne Paris Cité, Faculty Necker, 156 Rue de Vaugirard, 75015 Paris, France
| | - A. Edelman
- Inserm U845, Research Center Growth and Signalling, Faculté de Médecine, Université Paris Descartes, Sorbonne Paris Cité, Faculty Necker, 156 Rue de Vaugirard, 75015 Paris, France
| | - M. Polak
- Inserm U845, Research Center Growth and Signalling, Faculté de Médecine, Université Paris Descartes, Sorbonne Paris Cité, Faculty Necker, 156 Rue de Vaugirard, 75015 Paris, France
| | - R. Scharfmann
- Inserm U845, Research Center Growth and Signalling, Faculté de Médecine, Université Paris Descartes, Sorbonne Paris Cité, Faculty Necker, 156 Rue de Vaugirard, 75015 Paris, France
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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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Pouokam E, Bader S, Brück B, Schmidt B, Diener M. ATP-sensitive K(+) channels in rat colonic epithelium. Pflugers Arch 2012; 465:865-77. [PMID: 23262522 DOI: 10.1007/s00424-012-1207-7] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2012] [Revised: 12/10/2012] [Accepted: 12/10/2012] [Indexed: 11/30/2022]
Abstract
ATP-sensitive K(+) (KATP) channels couple the metabolic state of a cell to its electrical activity. They consist of a hetero-octameric complex with pore-forming Kir6.x (Kir6.1, Kir6.2) and regulatory sulfonylurea receptor (SUR) subunits. Functional data indicate that KATP channels contribute to epithelial K(+) currents at colonic epithelia. However, their molecular identity and their properties are largely unknown. Therefore, changes in short-circuit current (I sc) induced by the KATP channel opener pinacidil (5 10(-4) mol l(-1)) were measured in Ussing chambers under control conditions and in the presence of different blockers of KATP channels. The channel subunits expressed by the colonic epithelium were identified by immunohistochemistry and by RT-PCR. The K(+) channel opener, when administered at the serosal side, induced an increase in I sc consistent with the induction of transepithelial Cl(-) secretion after activation of basolateral K(+) channels, whereas mucosal administration of pinacidil resulted in a negative I sc. The increase in I sc evoked by serosal pinacidil was inhibited by serosal administration of glibenclamide (5 10(-4) mol l(-1)) and gliclazide (10(-6) mol l(-1)), but was resistant even against a high concentration (10(-2) mol l(-1)) of tolbutamide. In contrast, none of these inhibitors (administered at the mucosal side) reduced significantly the negative I sc induced by mucosal pinacidil. Instead, pinacidil inhibited Cl(-) currents across apical Cl(-) channels in basolaterally depolarized epithelia indicating that the negative I sc induced by mucosal pinacidil is due to a transient inhibition of Cl(-) secretion. In mRNA prepared from isolated colonic crypts, messenger RNA for both pore-forming subunits, Kir6.1 and Kir6.2, and two regulatory subunits (SUR1 and SUR2B) was found. Expression within the colonic epithelium was confirmed for these subunits by immunohistochemistry. In consequence, KATP channels are present in the basolateral membrane of the colonic epithelium; their exact subunit composition, however, has still to be revealed.
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Affiliation(s)
- Ervice Pouokam
- Institute for Veterinary Physiology and Biochemistry, Justus-Liebig-University, Frankfurter Str. 100, Giessen, Germany
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Liu X, O'Donnell N, Landstrom A, Skach WR, Dawson DC. Thermal instability of ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) channel function: protection by single suppressor mutations and inhibiting channel activity. Biochemistry 2012; 51:5113-24. [PMID: 22680785 DOI: 10.1021/bi300018e] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Deletion of Phe508 from cystic fibrosis transmembrane conductance regulator (CFTR) results in a temperature-sensitive folding defect that impairs protein maturation and chloride channel function. Both of these adverse effects, however, can be mitigated to varying extents by second-site suppressor mutations. To better understand the impact of second-site mutations on channel function, we compared the thermal sensitivity of CFTR channels in Xenopus oocytes. CFTR-mediated conductance of oocytes expressing wt or ΔF508 CFTR was stable at 22 °C and increased at 28 °C, a temperature permissive for ΔF508 CFTR expression in mammalian cells. At 37 °C, however, CFTR-mediated conductance was further enhanced, whereas that due to ΔF508 CFTR channels decreased rapidly toward background, a phenomenon referred to here as "thermal inactivation." Thermal inactivation of ΔF508 was mitigated by each of five suppressor mutations, I539T, R553M, G550E, R555K, and R1070W, but each exerted unique effects on the severity of, and recovery from, thermal inactivation. Another mutation, K1250A, known to increase open probability (P(o)) of ΔF508 CFTR channels, exacerbated thermal inactivation. Application of potentiators known to increase P(o) of ΔF508 CFTR channels at room temperature failed to protect channels from inactivation at 37 °C and one, PG-01, actually exacerbated thermal inactivation. Unstimulated ΔF508CFTR channels or those inhibited by CFTR(inh)-172 were partially protected from thermal inactivation, suggesting a possible inverse relationship between thermal stability and gating transitions. Thermal stability of channel function and temperature-sensitive maturation of the mutant protein appear to reflect related, but distinct facets of the ΔF508 CFTR conformational defect, both of which must be addressed by effective therapeutic modalities.
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Affiliation(s)
- Xuehong Liu
- Departments of Physiology & Pharmacology, Oregon Health & Science University, Portland, Oregon 97239, USA.
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Deachapunya C, Poonyachoti S, Krishnamra N. Site-specific regulation of ion transport by prolactin in rat colon epithelium. Am J Physiol Gastrointest Liver Physiol 2012; 302:G1199-206. [PMID: 22403794 DOI: 10.1152/ajpgi.00143.2011] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The effect of prolactin (PRL) on ion transport across the rat colon epithelium was investigated using Ussing chamber technique. PRL (1 μg/ml) induced a sustained decrease in short-circuit current (I(sc)) in the distal colon with an EC(50) value of 100 ng/ml and increased I(sc) in the proximal colon with an EC(50) value of 49 ng/ml. In the distal colon, the PRL-induced decrease in I(sc) was not affected by Na(+) channel blocker amiloride or Cl(-) channel blockers, NPPB, DPC, or DIDS, added mucosally. However, the response was inhibited by mucosal application of K(+) channel blockers glibenclamide, quinidine, and chromanol 293B, whereas other K(+) channel blockers, Ba(2+), tetraethylammonium, clotrimazole, and apamin, failed to have effects. The PRL-induced decrease in I(sc) was also inhibited by Na(+)-K(+)-2Cl(-) transporter inhibitor bumetanide, Ba(2+), and chromanol 293B applied serosally. In the transverse and proximal colon, the PRL-induced increase in I(sc) was suppressed by DPC, glibenclamide, and bumetanide, but not by NPPB, DIDS, or amiloride. The PRL-induced changes in I(sc) in both distal and proximal colon were abolished by JAK2 inhibitor AG490, but not BAPTA-AM, the Ca(2+) chelating agent, or phosphatidylinositol 3-kinase inhibitor wortmannin. These results suggest a segment-specific effect of PRL in rat colon, by activation of K(+) secretion in the distal colon and activation of Cl(-) secretion in the transverse and proximal colon. Both PRL actions are mediated by JAK-STAT-dependent pathway, but not phosphatidylinositol 3-kinase pathway or Ca(2+) mobilization. These findings suggest a role of PRL in the regulation of electrolyte transport in mammalian colon.
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Affiliation(s)
- Chatsri Deachapunya
- Department of Physiology, Faculty of Medicine, Srinakharinwirot University, Sukhumvit 23, Wattana, Bangkok 10110, Thailand.
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Liu J, Zhang Z, Xu Y, Feng T, Jiang W, Li Z, Hong B, Xie Z, Si S. IMB2026791, a xanthone, stimulates cholesterol efflux by increasing the binding of apolipoprotein A-I to ATP-binding cassette transporter A1. Molecules 2012; 17:2833-54. [PMID: 22399138 PMCID: PMC6268880 DOI: 10.3390/molecules17032833] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2011] [Revised: 02/16/2012] [Accepted: 02/24/2012] [Indexed: 11/24/2022] Open
Abstract
It is known that the ATP-binding cassette transporter A1 (ABCA1) plays a major role in cholesterol homeostasis and high density lipoprotein (HDL) metabolism. Several laboratories have demonstrated that ABCA1 binding to lipid-poor apolipoprotein A-I (apoA-I) will mediate the assembly of nascent HDL and cellular cholesterol efflux, which suggests a possible receptor-ligand interaction between ABCA1 and apoA-I. In this study, a cell-based-ELISA-like high-throughput screening (HTS) method was developed to identify the synthetic and natural compounds that can regulate binding activity of ABCA1 to apoA-I. The cell-based-ELISA-like high-throughput screen was conducted in a 96-well format using Chinese hamster ovary (CHO) cells stably transfected with ABCA1 pIRE2-EGFP (Enhanced Green Fluorecence Protein) expression vector and the known ABCA1 inhibitor glibenclamide as the antagonist control. From 2,600 compounds, a xanthone compound (IMB 2026791) was selected using this HTS assay, and it was proved as an apoA-I binding agonist to ABCA1 by a flow cytometry assay and western blot analysis. The 3H cholesterol efflux assay of IMB2026791 treated ABCA1-CHO cells and PMA induced THP-1 macrophages (human acute monocytic leukemia cell) further confirmed the compound as an accelerator of cholesterol efflux in a dose-dependent manner with an EC50 of 25.23 μM.
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Affiliation(s)
- Jikai Liu
- China Institute of Medical Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tiantanxili #1, Beijing 100050, China; (J.L.); (Z.Z.); (Y.X.); (T.F.); (W.J.); (Z.L.); (B.H.)
| | - Zhongbing Zhang
- China Institute of Medical Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tiantanxili #1, Beijing 100050, China; (J.L.); (Z.Z.); (Y.X.); (T.F.); (W.J.); (Z.L.); (B.H.)
| | - Yanni Xu
- China Institute of Medical Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tiantanxili #1, Beijing 100050, China; (J.L.); (Z.Z.); (Y.X.); (T.F.); (W.J.); (Z.L.); (B.H.)
| | - Tingting Feng
- China Institute of Medical Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tiantanxili #1, Beijing 100050, China; (J.L.); (Z.Z.); (Y.X.); (T.F.); (W.J.); (Z.L.); (B.H.)
| | - Wei Jiang
- China Institute of Medical Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tiantanxili #1, Beijing 100050, China; (J.L.); (Z.Z.); (Y.X.); (T.F.); (W.J.); (Z.L.); (B.H.)
| | - Zhuorong Li
- China Institute of Medical Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tiantanxili #1, Beijing 100050, China; (J.L.); (Z.Z.); (Y.X.); (T.F.); (W.J.); (Z.L.); (B.H.)
| | - Bin Hong
- China Institute of Medical Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tiantanxili #1, Beijing 100050, China; (J.L.); (Z.Z.); (Y.X.); (T.F.); (W.J.); (Z.L.); (B.H.)
| | - Zijian Xie
- Department of Physiology and Pharmacology, College of Medicine, University of Toledo, Toledo, OH 43614, USA
- Authors to whom correspondence should be addressed; (Z.X.); (S.S.); Tel.: +1-419-383-4182 (Z.X.); Fax: +1-419-383-2871 (Z.X.); Tel./Fax: +86-10-6318-0604 (S.S.)
| | - Shuyi Si
- China Institute of Medical Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tiantanxili #1, Beijing 100050, China; (J.L.); (Z.Z.); (Y.X.); (T.F.); (W.J.); (Z.L.); (B.H.)
- Authors to whom correspondence should be addressed; (Z.X.); (S.S.); Tel.: +1-419-383-4182 (Z.X.); Fax: +1-419-383-2871 (Z.X.); Tel./Fax: +86-10-6318-0604 (S.S.)
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31
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Klein AS, Schaefer M, Korte T, Herrmann A, Tannert A. HaCaT keratinocytes exhibit a cholesterol and plasma membrane viscosity gradient during directed migration. Exp Cell Res 2012; 318:809-18. [PMID: 22366262 DOI: 10.1016/j.yexcr.2012.02.007] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2011] [Revised: 01/27/2012] [Accepted: 02/08/2012] [Indexed: 11/29/2022]
Abstract
Keratinocyte migration plays an important role in cutaneous wound healing by supporting the process of reepithelialisation. During directional migration cells develop a polarised shape with an asymmetric distribution of a variety of signalling molecules in their plasma membrane. Here, we investigated front-to-back differences of the physical properties of the plasma membrane of migrating keratinocyte-like HaCaT cells. Using FRAP and fluorescence lifetime analysis, both under TIR illumination, we demonstrate a reduced viscosity of the plasma membrane in the lamellipodia of migrating HaCaT cells compared with the cell rears. This asymmetry is most likely caused by a reduced cholesterol content of the lamellipodia as demonstrated by filipin staining. siRNA-mediated silencing of the cholesterol transporter ABCA1, which is known to redistribute cholesterol from rafts to non-raft regions, as well as pharmacological inhibition of this transporter with glibenclamide, strongly diminished the viscosity gradient of the plasma membrane. In addition, HaCaT cell migration was inhibited by glibenclamide treatment. These data suggest a preferential role of non-raft cholesterol in the establishment of the asymmetric plasma membrane viscosity.
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Affiliation(s)
- Anke S Klein
- Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Universität Leipzig, Germany
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32
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Cui G, Song B, Turki HW, McCarty NA. Differential contribution of TM6 and TM12 to the pore of CFTR identified by three sulfonylurea-based blockers. Pflugers Arch 2011; 463:405-18. [PMID: 22160394 DOI: 10.1007/s00424-011-1035-1] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2011] [Revised: 09/14/2011] [Accepted: 09/30/2011] [Indexed: 02/06/2023]
Abstract
Previous studies suggested that four transmembrane domains 5, 6, 11, 12 make the greatest contribution to forming the pore of the CFTR chloride channel. We used excised, inside-out patches from oocytes expressing CFTR with alanine-scanning mutagenesis in amino acids in TM6 and TM12 to probe CFTR pore structure with four blockers: glibenclamide (Glyb), glipizide (Glip), tolbutamide (Tolb), and Meglitinide. Glyb and Glip blocked wildtype (WT)-CFTR in a voltage-, time-, and concentration-dependent manner. At V (M) = -120 mV with symmetrical 150 mM Cl(-) solution, fractional block of WT-CFTR by 50 μM Glyb and 200 μM Glip was 0.64 ± 0.03 (n = 7) and 0.48 ± 0.02 (n = 7), respectively. The major effects on block by Glyb and Glip were found with mutations at F337, S341, I344, M348, and V350 of TM6. Under similar conditions, fractional block of WT-CFTR by 300 μM Tolb was 0.40 ± 0.04. Unlike Glyb, Glip, and Meglitinide, block by Tolb lacked time-dependence (n = 7). We then tested the effects of alanine mutations in TM12 on block by Glyb and Glip; the major effects were found at N1138, T1142, V1147, N1148, S1149, S1150, I1151, and D1152. From these experiments, we infer that amino acids F337, S341, I344, M348, and V350 of TM6 face the pore when the channel is in the open state, while the amino acids of TM12 make less important contributions to pore function. These data also suggest that the region between F337 and S341 forms the narrow part of the CFTR pore.
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Affiliation(s)
- Guiying Cui
- Division of Pulmonology, Allergy/Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Center for Cystic Fibrosis Research, Children's Healthcare of Atlanta, Emory University School of Medicine, 2015 Uppergate Drive, Atlanta, GA 30322, USA
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33
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Lack of Restoration in Vivo by K-Channel Modulators of Jejunal Fluid Absorption after Heat Stable Escherichia coli Enterotoxin (STa) Challenge. J Trop Med 2011; 2011:853686. [PMID: 21760812 PMCID: PMC3134271 DOI: 10.1155/2011/853686] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2010] [Revised: 02/25/2011] [Accepted: 04/07/2011] [Indexed: 11/24/2022] Open
Abstract
Enhanced potassium ion permeability at the enterocyte basolateral membrane is assumed to facilitate sustained chloride ion and fluid secretion into the intestinal lumen during episodes of secretory diarrhoeal disease. To examine this concept in vivo, two potassium ion channel blockers and a channel opener were coperfused with E. coli heat stable STa enterotoxin to determine whether such compounds improved or worsened the inhibited fluid absorption. In the STa (80 ng/mL) challenged jejunal loop, the fluid absorption rate of 28.6 ± 5.8 (14) μL/cm/hr was significantly below (P < .001) the normal rate of 98.8 ± 6.2 (17) μL/cm/hr. Intraluminal (300 uM) glibenclamide added to STa perfused loops failed to improve the inhibited fluid absorption rate, which was 7.4 ± 3.2 (6) μL/cm/hr on coperfusion with STa. Similarly, on coperfusion with 30 uM clotrimazole, the fluid absorption rate with STa present remained inhibited at 11.4 ± 7.0 (4) μL/cm/hr. On coperfusion with intraluminal 1 uM cromakalim, STa reduced fluid absorption significantly (P < .02) to 24.7 ± 8.0 (10) μL/cm/hr, no different from STa challenge in the absence of cromakalim. Infusion i.v. with these agents also failed to restore fluid absorption after STa challenge. These observations do not support the proposed potassium ion permeability event as a necessary corollary of enterotoxin-mediated secretion. This makes it unlikely that modulators of such permeability prevent enterocyte secretion in diarrhoeal disease.
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Terao Y, Ayaori M, Ogura M, Yakushiji E, Uto-Kondo H, Hisada T, Ozasa H, Takiguchi S, Nakaya K, Sasaki M, Komatsu T, Iizuka M, Horii S, Mochizuki S, Yoshimura M, Ikewaki K. Effect of sulfonylurea agents on reverse cholesterol transport in vitro and vivo. J Atheroscler Thromb 2011; 18:513-30. [PMID: 21636950 DOI: 10.5551/jat.7641] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
AIM Reverse cholesterol transport (RCT) is a critical mechanism for the anti-atherogenic property of HDL. The inhibitory effect of the sulfonylurea agent (SUA) glibenclamide on ATP binding-cassette transporter (ABC) A1 may decrease HDL function but it remains unclear whether it attenuates RCT in vivo. We therefore investigated how the SUAs glibenclamide and glimepiride affected the functionality of ABCA1/ABCG1 and scavenger receptor class B type I (SR-BI) expression in macrophages in vitro and overall RCT in vivo. METHODS RAW264.7, HEK293 and BHK-21 cells were used for in vitro studies. To investigate RCT in vivo, 3H-cholesterol-labeled and acetyl LDL-loaded RAW264.7 cells were injected into mice. RESULTS High dose (500µM) of glibenclamide inhibited ABCA1 function and apolipoprotein A-I (apoA-I)-mediated cholesterol efflux, and attenuated ABCA1 expression. Although glimepiride maintained apoA-I-mediated cholesterol efflux from RAW264.7 cells, like glibenclamide, it inhibited ABCA1-mediated cholesterol efflux from transfected HEK293 cells. Similarly, the SUAs inhibited SR-BI-mediated cholesterol efflux from transfected BHK-21 cells. High doses of SUAs increased ABCG1 expression in RAW264.7 cells, promoting HDL-mediated cholesterol efflux in an ABCG1-independent manner. Low doses (0.1-100 µM) of SUAs did not affect cholesterol efflux from macrophages despite dose-dependent increases in ABCA1/G1 expression. Furthermore, they did not change RCT or plasma lipid levels in mice. CONCLUSION High doses of SUAs inhibited the functionality of ABCA1/SR-BI, but not ABCG1. At lower doses, they had no unfavorable effects on cholesterol efflux or overall RCT in vivo. These results indicate that SUAs do not have adverse effects on atherosclerosis contrary to previous findings for glibenclamide.
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Affiliation(s)
- Yoshio Terao
- Division of Anti-aging, Department of Internal Medicine, National Defense Medical College, Saitama, Japan
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Characterization of a critical role for CFTR chloride channels in cardioprotection against ischemia/reperfusion injury. Acta Pharmacol Sin 2011; 32:824-33. [PMID: 21642951 DOI: 10.1038/aps.2011.61] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
AIM To further characterize the functional role of cystic fibrosis transmembrane conductance regulator (CFTR) in early and late (second window) ischemic preconditioning (IPC)- and postconditioning (POC)-mediated cardioprotection against ischemia/reperfusion (I/R) injury. METHODS CFTR knockout (CFTR(-/-)) mice and age- and gender-matched wild-type (CFTR(+/+)) and heterozygous (CFTR(+/-)) mice were used. In in vivo studies, the animals were subjected to a 30-min coronary occlusion followed by a 40-min reperfusion. In ex vivo (isolate heart) studies, a 45-min global ischemia was applied. To evaluate apoptosis, the level of activated caspase 3 and TdT-mediated dUTP-X nick end labeling (TUNEL) were examined. RESULTS In the in vivo I/R models, early IPC significantly reduced the myocardial infarct size in wild-type (CFTR(+/+)) (from 40.4% ± 5.3% to 10.4% ± 2.0%, n=8, P<0.001) and heterozygous (CFTR(+/-)) littermates (from 39.4% ± 2.4% to 15.4% ± 5.1%, n=6, P<0.001) but failed to protect CFTR knockout (CFTR(-/-)) mice from I/R induced myocardial infarction (46.9% ± 6.2% vs 55.5% ± 7.8%, n=6, P>0.5). Similar results were observed in the in vivo late IPC experiments. Furthermore, in both in vivo and ex vivo I/R models, POC significantly reduced myocardial infarction in wild-type mice, but not in CFTR knockout mice. In ex vivo I/R models, targeted inactivation of CFTR gene abolished the protective effects of IPC against I/R-induced apoptosis. CONCLUSION These results provide compelling evidence for a critical role for CFTR Cl(-) channels in IPC- and POC-mediated cardioprotection against I/R-induced myocardial injury.
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Valero MS, Pereboom D, Barcelo-Batllory S, Brines L, Garay RP, Alda JO. Protein kinase A signalling is involved in the relaxant responses to the selective β-oestrogen receptor agonist diarylpropionitrile in rat aortic smooth muscle in vitro. J Pharm Pharmacol 2011; 63:222-9. [PMID: 21235586 DOI: 10.1111/j.2042-7158.2010.01203.x] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
OBJECTIVES The oestrogen receptor β (ERβ) selective agonist diarylpropionitrile (DPN) relaxes endothelium-denuded rat aorta, but the signalling mechanism is unknown. The aim of this study was to assess whether protein kinase A (PKA) signalling is involved in DPN action. METHODS cAMP was measured by radioimmunoassay, HSP20 phosphorylation by 2D gel electrophoresis with immunoblotting, and membrane potential and free cytosolic calcium by flow cytometry. KEY FINDINGS DPN increased cAMP content and hyperpolarised cell membranes over the same range of concentrations as it relaxed phenylephrine-precontracted aortic rings (10-300 µM). DPN-induced vasorelaxation was largely reduced by the PKA inhibitors Rp-8-Br-cAMPS (8-bromoadenosine-3', 5'-cyclic monophosphorothioate, Rp-isomer) and H-89 (N-(2-bromocynnamyl(amino)ethyl)-5-isoquinoline sulfonamide HCl) (-73%) and by the adenylate cyclase inhibitor MDL12330A (cis-N-(2-phenylcyclopentyl)-azacyclotridec-1-en-2-amine)) (-65.5%). Conversely, the PKG inhibitor Rp-8-Br-cGMP was inactive against DPN vasorelaxation. In aortic smooth muscle segments, DPN increased PKA-dependent HSP20 phosphorylation, an effect reversed by H-89. Relaxant responses to DPN were modestly antagonised (-23 to -48% reduction; n=12 per compound) by the potassium channel inhibitors iberiotoxin, PNU-37883A, 4-aminopyridine, or BaCl(2) . All four potassium channel inhibitors together reduced DPN relaxation by 86±9% (n=12) and fully blocked DPN hyperpolarisation. CONCLUSIONS ERβ-dependent relaxation of rat aortic smooth muscle evokes an adenylate cyclase/cAMP/PKA signalling pathway, likely activating the cystic fibrosis transmembrane conductance regulator chloride channel and at least four potassium channels.
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Affiliation(s)
- Marta S Valero
- Department of Physiology and Pharmacology, School of Medicine, Zaragoza, Spain
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37
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Hashimoto Y, Shuto T, Mizunoe S, Tomita A, Koga T, Sato T, Takeya M, Suico MA, Niibori A, Sugahara T, Shimasaki S, Sugiyama T, Scholte B, Kai H. CFTR-deficiency renders mice highly susceptible to cutaneous symptoms during mite infestation. J Transl Med 2011; 91:509-18. [PMID: 21135815 DOI: 10.1038/labinvest.2010.193] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022] Open
Abstract
Pruritus, also known as itch, is a sensation that causes a desire to scratch. Prolonged scratching exacerbates skin lesions in several skin diseases such as atopic dermatitis. Here, we identify the cystic fibrosis transmembrane conductance regulator (CFTR/Cftr), an integral membrane protein that mediates transepithelial chloride transport, as a determinant factor in mice for the susceptibility to several cutaneous symptoms during mite infestation. Mice that endogenously express dysfunctional Cftr (Cftr(ΔF508/ΔF508)) show significant increase of scratching behavior and skin fibrosis after mite exposure. These phenotypes were due to the increased expression of nerve growth factor (NGF) that augments the sensitization of peripheral nerve fibers. Moreover, protein gene product 9.5 (PGP9.5)-positive neurites were abundant in the epidermis of mite-infested Cftr(ΔF508/ΔF508) mice. Furthermore, mite-infested Cftr(+/+) mice orally administered with a chloride channel inhibitor glibenclamide had higher scratching count and increased level of NGF than vehicle-treated mice. Consistently, mite extract-exposed primary and transformed human keratinocytes, treated with CFTR inhibitor, had significantly higher level of NGF mRNA compared with vehicle-treated, mite extract-exposed cells. These results reveal that CFTR in keratinocytes plays a critical role for the regulation of peripheral nerve function and pruritus sensation, and suggest that Cftr(ΔF508/ΔF508) mice may serve as a novel mouse model that represents NGF-dependent generation of pruritus.
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Affiliation(s)
- Yasuaki Hashimoto
- Department of Molecular Medicine, Graduate School of Pharmaceutical Sciences, Global COE Cell Fate Regulation Research and Education Unit, Kumamoto University, Kumamoto, Japan
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Bessadok A, Garcia E, Jacquet H, Martin S, Garrigues A, Loiseau N, André F, Orlowski S, Vivaudou M. Recognition of sulfonylurea receptor (ABCC8/9) ligands by the multidrug resistance transporter P-glycoprotein (ABCB1): functional similarities based on common structural features between two multispecific ABC proteins. J Biol Chem 2010; 286:3552-69. [PMID: 21098040 DOI: 10.1074/jbc.m110.155200] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023] Open
Abstract
ATP-sensitive K(+) (K(ATP)) channels are the target of a number of pharmacological agents, blockers like hypoglycemic sulfonylureas and openers like the hypotensive cromakalim and diazoxide. These agents act on the channel regulatory subunit, the sulfonylurea receptor (SUR), which is an ABC protein with homologies to P-glycoprotein (P-gp). P-gp is a multidrug transporter expressed in tumor cells and in some healthy tissues. Because these two ABC proteins both exhibit multispecific recognition properties, we have tested whether SUR ligands could be substrates of P-gp. Interaction with P-gp was assayed by monitoring ATPase activity of P-gp-enriched vesicles. The blockers glibenclamide, tolbutamide, and meglitinide increased ATPase activity, with a rank order of potencies that correlated with their capacity to block K(ATP) channels. P-gp ATPase activity was also increased by the openers SR47063 (a cromakalim analog), P1075 (a pinacidil analog), and diazoxide. Thus, these molecules bind to P-gp (although with lower affinities than for SUR) and are possibly transported by P-gp. Competition experiments among these molecules as well as with typical P-gp substrates revealed a structural similarity between drug binding domains in the two proteins. To rationalize the observed data, we addressed the molecular features of these proteins and compared structural models, computerized by homology from the recently solved structures of murine P-gp and bacterial ABC transporters MsbA and Sav1866. Considering the various residues experimentally assigned to be involved in drug binding, we uncovered several hot spots, which organized spatially in two main binding domains, selective for SR47063 and for glibenclamide, in matching regions of both P-gp and SUR.
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Affiliation(s)
- Anis Bessadok
- Service de Bioénergétique, Biologie Structurale et Mécanismes, URA 2096 CNRS, iBiTec-S, Commissariat à l'Energie Atomique-Saclay, 91191 Gif-sur-Yvette Cedex, France
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Abstract
Cystic fibrosis related diabetes (CFRD) is the most common co-morbidity in persons with cystic fibrosis (CF). As the life expectancy of persons with CF continues to increase, the need to proactively diagnose and aggressively treat CFRD and its potential complications has become more apparent. CFRD negatively impacts lung function, growth and mortality, making its diagnosis and management crucial in a population already at high risk for early mortality. Compared to type 1 and type 2 diabetes, CFRD is a unique entity, requiring a thorough understanding of its unique pathophysiology to facilitate the creation and utilization of an effective medical treatment plan. The physiology of CFRD is complex, likely consisting of a combination of insulin deficiency, insulin resistance and a genetic predisposition towards the development of diabetes. However, the hallmark of CFRD is insulin deficiency, necessitating the use of exogenous insulin as the mainstay of therapy. Insulin administration, in combination with a multidisciplinary team of health professionals with expertise in the care of patients with CF and CFRD, is the cornerstone of the care for these patients. The goals of treatment of the CFRD population are to reverse protein catabolism, maintain a healthy weight, and reduce acute and chronic diabetes complications. Creating a partnership between the treatment team and the patient is the ideal way to accomplish these goals and is essential for successful diabetes care.
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Affiliation(s)
- T A Laguna
- Department of Pediatrics, University of Minnesota School of Medicine and Amplatz Children's Hospital, Minneapolis, MN, USA.
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Howard AD, Verghese PB, Arrese EL, Soulages JL. Characterization of apoA-I-dependent lipid efflux from adipocytes and role of ABCA1. Mol Cell Biochem 2010; 343:115-24. [PMID: 20535530 DOI: 10.1007/s11010-010-0505-7] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2010] [Accepted: 05/21/2010] [Indexed: 10/19/2022]
Abstract
Adipose tissue is a major reservoir of cholesterol and, as such, it may play a significant role in cholesterol homeostasis. The aims of this study were to obtain a quantitative characterization of apolipoprotein A-I (apoA-I)-dependent lipid efflux from adipocytes and examine the role of ATP-binding cassette transporter A1 (ABCA1) in this process. The rates of apoA-I-induced cholesterol and phospholipid efflux were determined and normalized by cellular protein or ABCA1 levels. In order to allow a comparative analysis, parallel experiments were also performed in macrophages. These studies showed that apoA-I induces cholesterol efflux from adipocytes at similar rates as from macrophages. Enhancement of the expression of ABCA1 increased the rates of cholesterol efflux from both adipocytes and macrophages. The results also suggested that a non-ABCA1-dependent mechanism could make significant contributions to the rate of apoA-I-dependent cholesterol efflux when the expression levels of ABCA1 are low. Furthermore, the study of the effect of inhibitors of lipid efflux showed that glyburide and brefeldin A, which affect ABCA1 function, exerted strong and similar inhibitory effects on lipid efflux from both adipocytes and macrophages, whereas BLT1, an SRB-I inhibitor, only exerted a moderate inhibition. Overall these studies suggest that ABCA1 plays a major role in apoA-I-dependent lipid efflux from adipocytes and showed high similarities between the abilities of adipocytes and macrophages to release cholesterol in an apoA-I-dependent fashion.
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Affiliation(s)
- Alisha D Howard
- 147 Noble Research Center, Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078, USA
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Li H, Sheppard DN. Therapeutic potential of cystic fibrosis transmembrane conductance regulator (CFTR) inhibitors in polycystic kidney disease. BioDrugs 2010; 23:203-16. [PMID: 19697963 DOI: 10.2165/11313570-000000000-00000] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
In the common genetic disorder autosomal dominant polycystic kidney disease (ADPKD), kidney function is disrupted by multiple fluid-filled epithelial cysts. Cyst growth in ADPKD involves fluid accumulation within the cyst lumen driven by cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transepithelial Cl- secretion. This suggests that inhibitors of the CFTR Cl- channel might retard cyst growth. This review considers how knowledge of CFTR structure and function and its role in transepithelial salt and water movements provides insight into the mechanism of action of CFTR inhibitors. Some small molecules, termed open-channel blockers, inhibit directly the CFTR Cl- channel by physically obstructing the CFTR pore and preventing Cl- flow. By contrast, other small molecules, termed allosteric inhibitors, bind to CFTR at a site remote from the channel pore and interfere with conformational changes that open the pore. The application of high-throughput screening to CFTR drug discovery has led to the identification of new inhibitors of the CFTR Cl- channel including the thiazolidinone CFTR(inh)-172 and the glycine hydrazide GlyH-101. The demonstration that CFTR inhibitors retard cyst expansion and kidney enlargement in mouse models of ADPKD provides proof of concept for the use of small-molecule CFTR inhibitors in the treatment of ADPKD.
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Affiliation(s)
- Hongyu Li
- Department of Physiology and Pharmacology, University of Bristol, School of Medical Sciences, Bristol, UK
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Nishimura N, Reien Y, Matsumoto A, Ogura T, Miyata Y, Suzuki K, Nakazato Y, Daida H, Nakaya H. Effects of nicorandil on the cAMP-dependent Cl- current in guinea-pig ventricular cells. J Pharmacol Sci 2010; 112:415-23. [PMID: 20308804 DOI: 10.1254/jphs.09237fp] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/19/2022] Open
Abstract
In guinea-pig cardiomyocytes, a cAMP-dependent Cl(-) current (I(Cl,cAMP)) flows through a cardiac isoform of the cystic fibrosis transmembrane conductance regulator (CFTR), which belongs to a family of the ATP-binding cassette (ABC) proteins. Although several K(+)-channel openers and sulfonylurea ATP-sensitive K(+) (K(ATP))-channel blockers reportedly inhibit I(Cl,cAMP), effects of nicorandil on the Cl(-) current have not been evaluated. This study was conducted to examine the effects of nicorandil on I(Cl,cAMP) in isolated guinea-pig ventricular cells using patch clamp techniques. Nicorandil in concentrations higher than 300 microM enhanced the I(Cl,cAMP) preactivated by 0.1 microM isoproterenol. The isoproterenol-induced I(Cl,cAMP) was inhibited by 100 microM glibenclamide, but not by 100 microM pinacidil. SNAP (S-nitroso-N-acetyl-D,L-penicillamine, 10 microM), a nitric oxide (NO) donor, similarly enhanced the isoproterenol-induced I(Cl,cAMP). However, SG-86, a denitrated metabolite possessing K(+ )channel-opening action, failed to enhance the Cl(-) current. When the I(Cl,cAMP) was activated by 3-isobutyl-1-methylxanthine (IBMX, 30 microM), either nicorandil or SNAP failed to enhance the isoproterenol-induced I(Cl,cAMP). Thus, nicorandil enhances I(Cl,cAMP) in guinea-pig cardiomyocytes through an increase in intracellular cGMP, although direct modulation of I(Cl,cAMP) by NO cannot be completely excluded.
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Affiliation(s)
- Nami Nishimura
- Department of Pharmacology, Chiba University Graduate School of Medicine, Japan
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Mouse cystic fibrosis transmembrane conductance regulator forms cAMP-PKA-regulated apical chloride channels in cortical collecting duct. Proc Natl Acad Sci U S A 2010; 107:6082-7. [PMID: 20231442 DOI: 10.1073/pnas.0902661107] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in many segments of the mammalian nephron, where it may interact with and modulate the activity of a variety of apical membrane proteins, including the renal outer medullary potassium (ROMK) K(+) channel. However, the expression of CFTR in apical cell membranes or its function as a Cl(-) channel in native renal epithelia has not been demonstrated. Here, we establish that CFTR forms protein kinase A (PKA)-activated Cl(-) channels in the apical membrane of principal cells from the cortical collecting duct obtained from mice. These Cl(-) channels were observed in cell-attached apical patches of principal cells after stimulation by forskolin/3-isobutyl-1-methylxanthine. Quiescent Cl(-) channels were present in patches excised from untreated tubules because they could be activated after exposure to Mg-ATP and the catalytic subunit of PKA. The single-channel conductance, kinetics, and anion selectivity of these Cl(-) channels were the same as those of recombinant mouse CFTR channels expressed in Xenopus laevis oocytes. The CFTR-specific closed-channel blocker CFTR(inh)-172 abolished apical Cl(-) channel activity in excised patches. Moreover, apical Cl(-) channel activity was completely absent in principal cells from transgenic mice expressing the DeltaF508 CFTR mutation but was present and unaltered in ROMK-null mice. We discuss the physiologic implications of open CFTR Cl(-) channels on salt handling by the collecting duct and on the functional CFTR-ROMK interactions in modulating the metabolic ATP-sensing of ROMK.
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Inoue H, Takahashi N, Okada Y, Konishi M. Volume-sensitive outwardly rectifying chloride channel in white adipocytes from normal and diabetic mice. Am J Physiol Cell Physiol 2010; 298:C900-9. [PMID: 20107039 DOI: 10.1152/ajpcell.00450.2009] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The volume-sensitive outwardly rectifying (VSOR) chloride channel is ubiquitously expressed and involved in cell volume regulation after osmotic swelling, called regulatory volume decrease (RVD), in various cell types. In adipocytes, the expression of the VSOR channel has not been explored to date. Here, by employing the whole-cell patch-clamp technique, we examined whether or not the VSOR channel is expressed in white adipocytes freshly isolated from epididymal fat pads of normal (C57BL/6 or KK) and diabetic (KKA(y)) mice. Whole cell voltage-clamp recordings revealed that Cl(-) currents were gradually activated upon cell swelling induced by application of a hypotonic solution, both in normal and diabetic adipocytes. Although both the mean cell size (or cell capacitance) and the current magnitude in KKA(y) adipocytes were larger than those in C57BL/6 cells, the current density was significantly lower in KKA(y) adipocytes (23.32 +/- 1.94 pA in C57BL/6 adipocytes vs. 13.04 +/- 2.41 pA in KKA(y) adipocytes at +100 mV). Similarly, the current density in diabetic KKA(y) adipocytes was lower than that in adipocytes from KK mice (a parental strain of KKA(y) mice), which do not present diabetes until an older age. The current was inhibited by Cl(-) channel blockers, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and glibenclamide, or hypertonic solution, and showed outward rectification and inactivation kinetics at large positive potentials. These electrophysiological and pharmacological properties are consistent with those of the VSOR channel in other cell types. Moreover, adipocytes showed RVD, which was inhibited by NPPB. In KKA(y) adipocytes, RVD was significantly slower (tau; 8.42 min in C57BL/6 adipocytes vs. 11.97 min in KKA(y) adipocytes) and incomplete during the recording period (25 min). It is concluded that the VSOR channel is functionally expressed and involved in volume regulation in white adipocytes. RVD is largely impaired in adipocytes from diabetic mice, presumably as a consequence of the lower density of the functional VSOR channel in the plasma membrane.
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Affiliation(s)
- Hana Inoue
- Dept. of Physiology, Tokyo Medical Univ., 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan.
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Kloch M, Milewski M, Nurowska E, Dworakowska B, Cutting GR, Dołowy K. The H-loop in the second nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator is required for efficient chloride channel closing. Cell Physiol Biochem 2010; 25:169-80. [PMID: 20110677 DOI: 10.1159/000276549] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/23/2009] [Indexed: 11/19/2022] Open
Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a cAMP-activated chloride channel. The recent model of CFTR gating predicts that the ATP binding to both nucleotide-binding domains (NBD1 and NBD2) of CFTR is required for the opening of the channel, while the ATP hydrolysis at NBD2 induces subsequent channel closing. In most ABC proteins, efficient hydrolysis of ATP requires the presence of the invariant histidine residue within the H-loop located in the C-terminal part of the NBD. However, the contribution of the corresponding region (H-loop) of NBD2 to the CFTR channel gating has not been examined so far. Here we report that the alanine substitution of the conserved dipeptide HR motif (HR-->AA) in the H-loop of NBD2 leads to prolonged open states of CFTR channel, indicating that the H-loop is required for efficient channel closing. On the other hand, the HR-->AA substitution lead to the substantial decrease of CFTR-mediated current density (pA/pF) in transfected HEK 293 cells, as recorded in the whole-cell patch-clamp analysis. These results suggest that the H-loop of NBD2, apart from being required for CFTR channel closing, may be involved in regulating CFTR trafficking to the cell surface.
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Affiliation(s)
- Monika Kloch
- Department of Biophysics, Warsaw University of Life Sciences SGGW, Warsaw, Poland
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Cehak A, Burmester M, Geburek F, Feige K, Breves G. Electrophysiological characterization of electrolyte and nutrient transport across the small intestine in horses. J Anim Physiol Anim Nutr (Berl) 2009; 93:287-94. [PMID: 19646103 DOI: 10.1111/j.1439-0396.2008.00882.x] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
The aim of this study was to characterize the transport mechanisms of electrolytes and nutrients across the jejunum of nine healthy horses electrophysiologically. The stripped mucosa was mounted in Ussing chambers and tissue conductances (G(t)) and short circuit currents (I(sc)) were continuously monitored. After blocking the sodium and potassium channels with amiloride, tetraethylammonium chloride (TEA) and barium, chloride secretion was stimulated by carbachol and forskolin. Subsequently, chloride channels were inhibited by 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, CFTR(inh)-172, N-(2-naphtalenyl)-(3.5-dibromo-2.4-dihydroxyphenyl)methylene glycine hydrazide (GlyH-101) and glibenclamide and their dose-response effect was investigated. The response to glucose, l-alanine and glycyl-l-glutamine was determined at two different mucosal pH values (pH 7.4 and 5.4 respectively). Mean basal I(sc) was -0.47 +/- 0.31 microEq/cm(2)h and mean G(t) was 22.17 +/- 1.78 mS/cm(2). Amiloride and TEA did not alter the baseline I(sc). Barium, carbachol and forskolin significantly increased I(sc). Irrespective of the dose, none of the chloride inhibitors changed I(sc). All nutrients induced a significant increase in I(sc) with the increase being significantly higher at pH 7.4 than at pH 5.4. In conclusion, there is evidence that chloride secretion in horses may be different from respective transport mechanisms in other species. The glucose absorption is suggestive of a sodium-dependent glucose cotransporter 1. However, a decrease in luminal pH did not stimulate current response to peptides as shown for other mammals.
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Affiliation(s)
- A Cehak
- Department of Physiology, School of Veterinary Medicine Hannover, Hannover, Germany.
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O'Riordan SMP, Robinson PD, Donaghue KC, Moran A. Management of cystic fibrosis-related diabetes in children and adolescents. Pediatr Diabetes 2009; 10 Suppl 12:43-50. [PMID: 19754617 DOI: 10.1111/j.1399-5448.2009.00587.x] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/29/2022] Open
Affiliation(s)
- Stephen M P O'Riordan
- The Developmental Endocrinology Research Group, The Institute of Child Health, University College London, London, UK.
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Kanoo S, Alex AB, Tiwari AK, Deshpande SB. B(2) kinin receptors mediate the Indian red scorpion venom-induced augmentation of visceral reflexes via the nitric oxide cyclic guanosine monophosphate pathway. Acta Physiol (Oxf) 2009; 196:365-73. [PMID: 19133874 DOI: 10.1111/j.1748-1716.2008.01953.x] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
AIM This study was performed to delineate the kinin (receptor)-dependent pathways in the Indian red scorpion (Mesobuthus tamulus; MBT) venom-induced pulmonary oedema as well as the augmentation of cardio-pulmonary reflexes evoked by phenyldiguanide (PDG). METHODS In urethane-anaesthetized adult rats, the effect of venom on the PDG reflex responses (blood pressure, heart rate and respiration rate) and the pulmonary water content was ascertained using various antagonists(des- Arg, B(1) receptor antagonist; Hoe 140, B(2) receptor antagonist; N(omega)-nitro-l-arginine methyl ester (l-NAME), nitric oxide (NO) synthase inhibitor; methylene blue, soluble guanylate cyclase inhibitor; and glibenclamide, K(+)(ATP) channel blocker). The effect of phosphodiesterase V inhibitor (sildenafil citrate) on the reflex response and the pulmonary water content was also examined and compared with venom-induced responses. RESULTS Intravenous injection of PDG (10 microg kg(-1)) evoked apnoea, bradycardia and hypotension lasting >60 s. Exposure to MBT venom (100 microg kg(-1)) for 30 min augmented the PDG reflex responses by two times and increased the pulmonary water content, significantly. Hoe 140 blocked the venom-induced responses (augmentation of PDG reflex and increased pulmonary water content) whereas des-Arg did not. l-NAME, methylene blue or glibenclamide also blocked the venom-induced responses. Furthermore, sildenafil citrate (that increases cGMP levels) produced augmentation of PDG reflex response and increased the pulmonary water content as seen with venom. CONCLUSION The results indicate that venom-induced responses involve B(2) kinin receptors via the NO-dependent guanylate cyclase-cGMP pathway involving K(+)(ATP) channels.
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Affiliation(s)
- S Kanoo
- Department of Physiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221 005, Uttar Pradesh, India
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Sonawane ND, Zhao D, Zegarra-Moran O, Galietta LJV, Verkman AS. Nanomolar CFTR inhibition by pore-occluding divalent polyethylene glycol-malonic acid hydrazides. ACTA ACUST UNITED AC 2008; 15:718-28. [PMID: 18635008 DOI: 10.1016/j.chembiol.2008.05.015] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2007] [Revised: 05/16/2008] [Accepted: 05/21/2008] [Indexed: 12/30/2022]
Abstract
Inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have potential application as antisecretory therapy in cholera. We synthesized mono- and divalent CFTR inhibitors consisting of a malonic acid hydrazide (MalH) coupled via a disulfonic stilbene linker to polyethylene glycols (PEGs; 0.2-100 kDa). IC50 values for CFTR inhibition were 10-15 microM for the monovalent MalH-PEGs, but substantially lower for divalent MalH-PEG-MalH compounds, decreasing from 1.5 to 0.3 microM with increasing PEG size and showing positive cooperativity. Whole-cell patch-clamp showed voltage-dependent CFTR block with inward rectification. Outside-out patch-clamp showed shortened single-channel openings, indicating CFTR pore block from the extracellular side. Luminally added MalH-PEG-MalH blocked by >90% cholera toxin-induced fluid secretion in mouse intestinal loops (IC50 approximately 10 pmol/loop), and greatly reduced mortality in a suckling mouse cholera model. These conjugates may provide safe, inexpensive antisecretory therapy.
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Affiliation(s)
- N D Sonawane
- Department of Medicine and Physiology, 1246 Health Sciences East Tower, University of California, San Francisco, CA 94143-0521, USA
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Li J, Gao Z, Kehoe V, Xing J, King N, Sinoway L. Interstitial adenosine triphosphate modulates muscle afferent nerve-mediated pressor reflex. Muscle Nerve 2008; 38:972-7. [PMID: 18570238 DOI: 10.1002/mus.21014] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Previous work has shown that muscle contraction elevates interstitial adenosine triphosphate concentration ([ATP]i), which is likely due to the release of ATP from active skeletal muscle. ATP activation of purinergic receptors P2X on thin muscle afferent fibers further enhances cardiovascular responses to contraction. Thus, the purposes of this study were: (1) to examine the mechanisms by which ATP is released from muscle in response to mechanical stimulation; and (2) to study the effects of interstitial ATP concentrations on modulating pressor response to muscle contraction. Static contraction of the triceps surae muscle was evoked by electrical stimulation (at 5 HZ and 2.5 times motor threshold) of the tibial nerve in 9 anesthetized cats. Muscle interstitial ATP samples were collected from microdialysis probes inserted into the muscles. Dialysate ATP concentrations were determined using the luciferin-luciferase assay. In a control experiment, contraction was induced after 0.5 ml of saline was injected into the arterial blood supply of the hindlimb muscles. This increased [ATP]i by 220% (P < 0.05 vs. baseline). After gadolinium (1 mM), a blocker of mechanically sensitive channels, was injected into the muscles, contraction increased [ATP]i by 112% (P < 0.05 vs. control). In contrast, glibenclamide (an inhibitor of the ATP-binding cassette protein), monensin, and brefeldin A, which interfere with vesicular formation (or trafficking) and inhibit exocytosis, did not significantly affect ATP release by muscle contraction. In addition, a regression analysis showed that [ATP]i was linearly related to the pressor response to muscle contraction. The data suggest that ATP release from skeletal muscle is mediated via involvement of mechanosensitive channels. These findings further support a physiological role for release of ATP in modulating cardiovascular responses during static muscle contraction.
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Affiliation(s)
- Jianhua Li
- Penn State Heart and Vascular Institute, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.
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