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Fels B, Fischer F, Herrnboeck L, Beckers D, Niedzielski L, Roche P, Straeter A, Alesutan I, Borutta JT, Jaisser F, Staub O, Voelkl J, Kusche-Vihrog K. SECS, drugs, and Rac1&Rho: regulation of EnNaC in vascular endothelial cells. Pflugers Arch 2025:10.1007/s00424-025-03093-5. [PMID: 40402207 DOI: 10.1007/s00424-025-03093-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2024] [Revised: 04/11/2025] [Accepted: 05/04/2025] [Indexed: 05/23/2025]
Abstract
The endothelial ENaC (EnNaC) is mainly responsible for maintaining the mechanical properties of the endothelial cell surface, the sensitivity to the shear forces of the streaming blood and thus for vascular function. The correlation between EnNaC surface expression, the dynamics of the actin cortex, the mechanical stiffness, and nitric oxide release indicates a close structure-function relationship. Mechanical flexibility of the endothelial surface has been associated with proper vascular function, while chronic stiffening leads to endothelial dysfunction and the so-called 'stiff endothelial cell syndrome' (SECS). With the help of atomic force microscopy (AFM)-based nanoindentation and immunofluorescence staining in vitro and ex vivo, we investigated the underlying cellular mechanisms and signalling pathways of EnNaC-dependent endothelial behaviour. We were able to show that the interaction between EnNaC and the cortical cytoskeleton is mediated by the small GTPases RhoA, Rac1, and the Arp2/3 complex. The functional inhibition of EnNaC by the drugs amiloride and benzamil led to membrane removal of the channel within minutes. Furthermore, we could observe an involvement of mineralocorticoid receptor, SGK1 and Nedd4-2 in regulation of endothelial cell stiffness. Our study contributes further insights on complex regulation of EnNaC and elucidates its interaction with the actin cytoskeleton, which could be central to its role as a key regulator of vascular function in health and disease.
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Affiliation(s)
- Benedikt Fels
- Institute of Physiology, University of Lübeck, Ratzeburger Str. 160, 23562, Lübeck, Germany
- DZHK (German Research Centre for Cardiovascular Research), Partner Site Hamburg/Luebeck/Kiel, Luebeck, Germany
| | - Felix Fischer
- Institute of Physiology II, University of Münster, Münster, Germany
| | | | - David Beckers
- Institute of Physiology II, University of Münster, Münster, Germany
| | - Leon Niedzielski
- Institute of Physiology II, University of Münster, Münster, Germany
| | - Paul Roche
- Institute of Physiology II, University of Münster, Münster, Germany
| | | | - Ioana Alesutan
- Institute for Physiology and Pathophysiology, Johannes Kepler University Linz, Linz, Austria
| | - Johanna-Theres Borutta
- Institute of Physiology, University of Lübeck, Ratzeburger Str. 160, 23562, Lübeck, Germany
| | - Frederic Jaisser
- INSERM, UMRS 1166, ICAN, Sorbonne Université, Paris, France
- Université de Lorraine, INSERM Centre d'Investigations Cliniques-Plurithématique 1433, UMR 1116, CHRU de Nancy, Nancy, France
| | - Olivier Staub
- Department of Biomedical Sciences, University of Lausanne, Lausanne, Switzerland
| | - Jakob Voelkl
- Institute for Physiology and Pathophysiology, Johannes Kepler University Linz, Linz, Austria
- Department of Nephrology and Medical Intensive Care, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt Universität zu Berlin, Berlin, Germany
- DZHK (German Centre for Cardiovascular Research), Partner Site Berlin, Berlin, Germany
| | - Kristina Kusche-Vihrog
- Institute of Physiology, University of Lübeck, Ratzeburger Str. 160, 23562, Lübeck, Germany.
- DZHK (German Research Centre for Cardiovascular Research), Partner Site Hamburg/Luebeck/Kiel, Luebeck, Germany.
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Yu L, Bala N, Nguyen VAL, Kessler L, LaDisa JF, Alli AA. Activity and function of the endothelial sodium channel is regulated by the effector domain of MARCKS-like protein 1 in mouse aortic endothelial cells. Am J Physiol Cell Physiol 2025; 328:C1101-C1108. [PMID: 39982423 DOI: 10.1152/ajpcell.00425.2024] [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: 06/21/2024] [Revised: 07/11/2024] [Accepted: 02/14/2025] [Indexed: 02/22/2025]
Abstract
Enhanced endothelial sodium channel (EnNaC) functioning causes an increase in vessel stiffness. Here, we investigated the regulation of EnNaC in mouse aortic endothelial cells (mAoECs) by the actin cytoskeleton and lipid raft association protein myristoylated alanine-rich C-kinase substrate-like protein 1 (MLP1). We hypothesized that mutation of specific amino acid residues within the effector domain of MLP1 or loss of association between MLP1 and the anionic phospholipid phosphate PIP2 would significantly alter membrane association and EnNaC activity in mAoECs. mAoECs transiently transfected with a mutant MLP1 construct (three serine residues in the effector domain replaced with aspartate residues) showed a significant decrease in EnNaC activity compared with cells transfected with wild-type MLP1. Compared with vehicle treatment, mAoECs treated with the PIP2 synthesis blocker wortmannin showed less colocalization of EnNaC and MLP1. In other experiments, Western blot and densitometric analysis showed a significant decrease in MLP1 and caveolin-1 protein expression in mAoECs treated with wortmannin compared with vehicle. Finally, wortmannin treatment decreased sphingomyelin content and increased membrane fluidity in mAoECs. Taken together, these results suggest that constitutive phosphorylation of MLP1 attenuates the function of EnNaC in aortic endothelial cells by a mechanism involving a decrease in association with MLP1 and EnNaC at the membrane, whereas deletion of PIP2 decreases MLP1 expression and overall membrane fluidity.NEW & NOTEWORTHY In this study, we investigated the functional role of myristoylated alanine-rich C-kinase substrate-like protein 1 (MLP1) phosphorylation in regulating endothelial sodium channel (EnNaC) activity using mouse aortic endothelial cells for the first time. The results from this study will help elucidate the molecular mechanism by which aortic stiffness is regulated by EnNaC.
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Affiliation(s)
- Ling Yu
- Division of Nephrology, Hypertension, and Renal Transplantation, Department of Medicine, University of Florida College of Medicine, Gainesville, Florida, United States
| | - Niharika Bala
- Division of Nephrology, Hypertension, and Renal Transplantation, Department of Medicine, University of Florida College of Medicine, Gainesville, Florida, United States
| | - Van-Anh L Nguyen
- Division of Nephrology, Hypertension, and Renal Transplantation, Department of Medicine, University of Florida College of Medicine, Gainesville, Florida, United States
| | - Leah Kessler
- Division of Nephrology, Hypertension, and Renal Transplantation, Department of Medicine, University of Florida College of Medicine, Gainesville, Florida, United States
| | - John F LaDisa
- Section of Cardiology, Department of Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
- The Herma Heart Institute, Children's Wisconsin, Milwaukee, Wisconsin, United States
- Department of Biomedical Engineering, Marquette University and the Medical College of Wisconsin, Milwaukee, Wisconsin, United States
- Division of Cardiovascular Medicine, Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
- Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
| | - Abdel A Alli
- Division of Nephrology, Hypertension, and Renal Transplantation, Department of Medicine, University of Florida College of Medicine, Gainesville, Florida, United States
- Department of Physiology and Aging, University of Florida College of Medicine, Gainesville, Florida, United States
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Yu L, Bala N, Nguyen VAL, Kessler L, LaDisa JF, Alli AA. Activity and function of the endothelial sodium channel is regulated by the effector domain of MARCKS like protein 1 in mouse aortic endothelial cells. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.06.25.600595. [PMID: 38979152 PMCID: PMC11230428 DOI: 10.1101/2024.06.25.600595] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/10/2024]
Abstract
The endothelial sodium channel (EnNaC) plays an important role in regulating vessel stiffness. Here, we investigated the regulation of EnNaC in mouse aortic endothelial cells (mAoEC) by the actin cytoskeleton and lipid raft association protein myristoylated alanine-rich C-kinase substrate like protein 1 (MLP1). We hypothesized that mutation of specific amino acid residues within the effector domain of MLP1 or loss of association between MLP1 and the anionic phospholipid phosphate PIP2 would significantly alter membrane association and EnNaC activity in mAoEC. mAoEC transiently transfected with a mutant MLP1 construct (three serine residues in the effector domain replaced with aspartate residues) showed a significant decrease in EnNaC activity compared to cells transfected with wildtype MLP1. Compared to vehicle treatment, mAoEC treated with the PIP2 synthesis blocker wortmannin showed less colocalization of EnNaC and MLP1. In other experiments, Western blot and densitometric analysis showed a significant decrease in MLP1 and caveloin-1 protein expression in mAoEC treated with wortmannin compared to vehicle. Finally, wortmannin treatment decreased sphingomyelin content and increased membrane fluidity in mAoEC. Taken together, our results suggest constitutive phosphorylation of MLP1 attenuates the function of EnNaC in aortic endothelial cells by a mechanism involving a decrease in association with MLP1 and EnNaC at the membrane, while deletion of PIP2 decreases MARCKS expression and overall membrane fluidity.
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Li Y, Martin TE, Hancock JM, Li R, Viswanathan S, Lydon JP, Zheng Y, Ye X. Visualization of preimplantation uterine fluid absorption in mice using Alexa Fluor™ 488 Hydrazide†. Biol Reprod 2023; 108:204-217. [PMID: 36308434 PMCID: PMC9930399 DOI: 10.1093/biolre/ioac198] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2022] [Revised: 10/06/2022] [Accepted: 10/24/2022] [Indexed: 11/12/2022] Open
Abstract
Uterine fluid plays important roles in supporting early pregnancy events and its timely absorption is critical for embryo implantation. In mice, its volume is maximum on day 0.5 post-coitum (D0.5) and approaches minimum upon embryo attachment ~D4.0. Its secretion and absorption in ovariectomized rodents were shown to be promoted by estrogen and progesterone (P4), respectively. The temporal mechanisms in preimplantation uterine fluid absorption remain to be elucidated. We have established an approach using intraluminally injected Alexa Fluor™ 488 Hydrazide (AH) in preimplantation control (RhoAf/f) and P4-deficient RhoAf/fPgrCre/+ mice. In control mice, bulk entry (seen as smeared cellular staining) via uterine luminal epithelium (LE) decreases from D0.5 to D3.5. In P4-deficient RhoAf/fPgrCre/+ mice, bulk entry on D0.5 and D3.5 is impaired. Exogenous P4 treatment on D1.5 and D2.5 increases bulk entry in D3.5 P4-deficient RhoAf/fPgrCre/+ LE, while progesterone receptor (PR) antagonist RU486 treatment on D1.5 and D2.5 diminishes bulk entry in D3.5 control LE. The abundance of autofluorescent apical fine dots, presumptively endocytic vesicles to reflect endocytosis, in the LE cells is generally increased from D0.5 to D3.5 but its regulation by exogenous P4 or RU486 is not obvious under our experimental setting. In the glandular epithelium (GE), bulk entry is rarely observed and green cellular dots do not show any consistent differences among all the investigated conditions. This study demonstrates the dominant role of LE but not GE, the temporal mechanisms of bulk entry and endocytosis in the LE, and the inhibitory effects of P4-deficiency and RU486 on bulk entry in the LE in preimplantation uterine fluid absorption.
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Affiliation(s)
- Yuehuan Li
- Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA
| | - Taylor Elijah Martin
- Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA
- Interdisciplinary Toxicology Program, University of Georgia, Athens, Georgia, USA
| | - Jonathan Matthew Hancock
- Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA
- Interdisciplinary Toxicology Program, University of Georgia, Athens, Georgia, USA
| | - Rong Li
- Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA
- Interdisciplinary Toxicology Program, University of Georgia, Athens, Georgia, USA
| | - Suvitha Viswanathan
- Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA
| | - John P Lydon
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA
| | - Yi Zheng
- Division of Experimental Hematology and Cancer Biology, Children’s Hospital Research Foundation, Cincinnati, Ohio, USA
| | - Xiaoqin Ye
- Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA
- Interdisciplinary Toxicology Program, University of Georgia, Athens, Georgia, USA
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Rapid shear stress-dependent ENaC membrane insertion is mediated by the endothelial glycocalyx and the mineralocorticoid receptor. Cell Mol Life Sci 2022; 79:235. [PMID: 35397686 PMCID: PMC8995297 DOI: 10.1007/s00018-022-04260-y] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2021] [Revised: 03/02/2022] [Accepted: 03/18/2022] [Indexed: 02/08/2023]
Abstract
The contribution of the shear stress-sensitive epithelial Na+ channel (ENaC) to the mechanical properties of the endothelial cell surface under (patho)physiological conditions is unclear. This issue was addressed in in vivo and in vitro models for endothelial dysfunction. Cultured human umbilical vein endothelial cells (HUVEC) were exposed to laminar (LSS) or non-laminar shear stress (NLSS). ENaC membrane insertion was quantified using Quantum-dot-based immunofluorescence staining and the mechanical properties of the cell surface were probed with the Atomic Force Microscope (AFM) in vitro and ex vivo in isolated aortae of C57BL/6 and ApoE/LDLR-/- mice. Flow- and acetylcholine-mediated vasodilation was measured in vivo using magnetic resonance imaging. Acute LSS led to a rapid mineralocorticoid receptor (MR)-dependent membrane insertion of ENaC and subsequent stiffening of the endothelial cortex caused by actin polymerization. Of note, NLSS stress further augmented the cortical stiffness of the cells. These effects strongly depend on the presence of the endothelial glycocalyx (eGC) and could be prevented by functional inhibition of ENaC and MR in vitro endothelial cells and ex vivo endothelial cells derived from C57BL/6, but not ApoE/LDLR-/- vessel. In vivo In C57BL/6 vessels, ENaC- and MR inhibition blunted flow- and acetylcholine-mediated vasodilation, while in the dysfunctional ApoE/LDLR-/- vessels, this effect was absent. In conclusion, under physiological conditions, endothelial ENaC, together with the glycocalyx, was identified as an important shear stress sensor and mediator of endothelium-dependent vasodilation. In contrast, in pathophysiological conditions, ENaC-mediated mechanotransduction and endothelium-dependent vasodilation were lost, contributing to sustained endothelial stiffening and dysfunction.
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Palma AG, Kotsias BA. The Effect of Dynasore Upon the Negative Interaction Between ENaC and CFTR Channels in Xenopus laevis Oocytes. J Membr Biol 2022; 255:61-69. [DOI: 10.1007/s00232-021-00212-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2021] [Accepted: 12/22/2021] [Indexed: 11/29/2022]
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Potent antitumor activity of a glutamyltransferase-derived peptide via an activation of oncosis pathway. Sci Rep 2021; 11:16507. [PMID: 34389740 PMCID: PMC8363616 DOI: 10.1038/s41598-021-93055-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2020] [Accepted: 03/08/2021] [Indexed: 12/09/2022] Open
Abstract
Hepatocellular carcinoma (HCC) still presents poor prognosis with high mortality rate, despite of the improvement in the management. The challenge for precision treatment was due to the fact that little targeted therapeutics are available for HCC. Recent studies show that metabolic and circulating peptides serve as endogenous switches for correcting aberrant cellular plasticity. Here we explored the antitumor activity of low molecular components in human umbilical serum and identified a high abundance peptide VI-13 by peptidome analysis, which was recognized as the part of glutamyltransferase signal peptide. We modified VI-13 by inserting four arginines and obtained an analog peptide VI-17 to improve its solubility. Our analyses showed that the peptide VI-17 induced rapid context-dependent cell death, and exhibited a higher sensitivity on hepatoma cells, which is attenuated by polyethylene glycol but not necrotic inhibitors such as z-VAD-fmk or necrostatin-1. Morphologically, VI-17 induced cell swelling, blebbing and membrane rupture with release of cellular ATP and LDH into extracellular media, which is hallmark of oncotic process. Mechanistically, VI-17 induced cell membrane pore formation, degradation of α-tubulin via influx of calcium ion. These results indicated that the novel peptide VI-17 induced oncosis in HCC cells, which could serve as a promising lead for development of therapeutic intervention of HCC.
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Morachevskaya EA, Sudarikova AV. Actin dynamics as critical ion channel regulator: ENaC and Piezo in focus. Am J Physiol Cell Physiol 2021; 320:C696-C702. [PMID: 33471624 DOI: 10.1152/ajpcell.00368.2020] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Ion channels in plasma membrane play a principal role in different physiological processes, including cell volume regulation, signal transduction, and modulation of membrane potential in living cells. Actin-based cytoskeleton, which exists in a dynamic balance between monomeric and polymeric forms (globular and fibrillar actin), can be directly or indirectly involved in various cellular responses including modulation of ion channel activity. In this mini-review, we present an overview of the role of submembranous actin dynamics in the regulation of ion channels in excitable and nonexcitable cells. Special attention is focused on the important data about the involvement of actin assembly/disassembly and some actin-binding proteins in the control of the epithelial Na+ channel (ENaC) and mechanosensitive Piezo channels whose integral activity has a potential impact on membrane transport and multiple coupled cellular reactions. Growing evidence suggests that actin elements of the cytoskeleton can represent a "converging point" of various signaling pathways modulating the activity of ion transport proteins in cell membranes.
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Chlorpromazine Induces Basolateral Aquaporin-2 Accumulation via F-Actin Depolymerization and Blockade of Endocytosis in Renal Epithelial Cells. Cells 2020; 9:cells9041057. [PMID: 32340337 PMCID: PMC7226349 DOI: 10.3390/cells9041057] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2020] [Revised: 04/13/2020] [Accepted: 04/19/2020] [Indexed: 12/11/2022] Open
Abstract
We previously showed that in polarized Madin-Darby canine kidney (MDCK) cells, aquaporin-2 (AQP2) is continuously targeted to the basolateral plasma membrane from which it is rapidly retrieved by clathrin-mediated endocytosis. It then undertakes microtubule-dependent transcytosis toward the apical plasma membrane. In this study, we found that treatment with chlorpromazine (CPZ, an inhibitor of clathrin-mediated endocytosis) results in AQP2 accumulation in the basolateral, but not the apical plasma membrane of epithelial cells. In MDCK cells, both AQP2 and clathrin were concentrated in the basolateral plasma membrane after CPZ treatment (100 µM for 15 min), and endocytosis was reduced. Then, using rhodamine phalloidin staining, we found that basolateral, but not apical, F-actin was selectively reduced by CPZ treatment. After incubation of rat kidney slices in situ with CPZ (200 µM for 15 min), basolateral AQP2 and clathrin were increased in principal cells, which simultaneously showed a significant decrease of basolateral compared to apical F-actin staining. These results indicate that clathrin-dependent transcytosis of AQP2 is an essential part of its trafficking pathway in renal epithelial cells and that this process can be inhibited by selectively depolymerizing the basolateral actin pool using CPZ.
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Chambers L, Dorrance AM. Regulation of ion channels in the microcirculation by mineralocorticoid receptor activation. CURRENT TOPICS IN MEMBRANES 2020; 85:151-185. [PMID: 32402638 DOI: 10.1016/bs.ctm.2020.02.001] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
The mineralocorticoid receptor (MR) has classically been studied in the renal epithelium for its role in regulating sodium and water balance and, subsequently, blood pressure. However, the MR also plays a critical role in the microvasculature by regulating ion channel expression and function. Activation of the MR by its endogenous agonist aldosterone results in translocation of the MR into the nucleus, where it can act as a transcription factor. Although most of the actions of the aldosterone can be attributed to its genomic activity though MR activation, it can also act by nongenomic mechanisms. Activation of this ubiquitous receptor increases the expression of epithelial sodium channels (ENaC) in both the endothelium and smooth muscle cells of peripheral and cerebral vessels. MR activation also regulates activity of calcium channels, calcium-activated potassium channels, and various transient receptor potential (TRP) channels. Modification of these ion channels results in a myriad of negative consequences, including impaired endothelium-dependent vasodilation, alterations in generation of myogenic tone, and increased inflammation and oxidative stress. Taken together, these studies demonstrate the importance of studying the impact of the MR on ion channel function in the vasculature. While research in this area has made advances in recent years, there are still many large gaps in knowledge that need to be filled. Crucial future directions of study include defining the molecular mechanisms involved in this interaction, as well as elucidating the potential sex differences that may exist, as these areas of understanding are currently lacking.
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Affiliation(s)
- Laura Chambers
- Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI, United States
| | - Anne M Dorrance
- Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI, United States.
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Dee RA, Mangum KD, Bai X, Mack CP, Taylor JM. Druggable targets in the Rho pathway and their promise for therapeutic control of blood pressure. Pharmacol Ther 2019; 193:121-134. [PMID: 30189292 PMCID: PMC7235948 DOI: 10.1016/j.pharmthera.2018.09.001] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
The prevalence of high blood pressure (also known as hypertension) has steadily increased over the last few decades. Known as a silent killer, hypertension increases the risk for cardiovascular disease and can lead to stroke, heart attack, kidney failure and associated sequela. While numerous hypertensive therapies are currently available, it is estimated that only half of medicated patients exhibit blood pressure control. This signifies the need for a better understanding of the underlying cause of disease and for more effective therapies. While blood pressure homeostasis is very complex and involves the integrated control of multiple body systems, smooth muscle contractility and arterial resistance are important contributors. Strong evidence from pre-clinical animal models and genome-wide association studies indicate that smooth muscle contraction and BP homeostasis are governed by the small GTPase RhoA and its downstream target, Rho kinase. In this review, we summarize the signaling pathways and regulators that impart tight spatial-temporal control of RhoA activity in smooth muscle cells and discuss current therapeutic strategies to target these RhoA pathway components. We also discuss known allelic variations in the RhoA pathway and consider how these polymorphisms may affect genetic risk for hypertension and its clinical manifestations.
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Affiliation(s)
- Rachel A Dee
- Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Kevin D Mangum
- Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Xue Bai
- Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Christopher P Mack
- Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, USA; McAllister Heart Institute, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Joan M Taylor
- Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, USA; McAllister Heart Institute, University of North Carolina, Chapel Hill, NC 27599, USA.
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Shuyskiy LS, Levchenko VV, Negulyaev YA, Staruschenko AV, Ilatovskaya DV. Role of the Scaffold Protein MIM in the Actin-Dependent Regulation of Epithelial Sodium Channels (ENaC). Acta Naturae 2018; 10:97-103. [PMID: 30116621 PMCID: PMC6087825] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022] Open
Abstract
Epithelial Sodium Channels (ENaCs) are expressed in different organs and tissues, particularly in the cortical collecting duct (CCD) in the kidney, where they fine tune sodium reabsorption. Dynamic rearrangements of the cytoskeleton are one of the common mechanisms of ENaC activity regulation. In our previous studies, we showed that the actin-binding proteins cortactin and Arp2/3 complex are involved in the cytoskeleton-dependent regulation of ENaC and that their cooperative work decreases a channel's probability of remaining open; however, the specific mechanism of interaction between actin-binding proteins and ENaC is unclear. In this study, we propose a new component for the protein machinery involved in the regulation of ENaC, the missing-in-metastasis (MIM) protein. The MIM protein contains an IMD domain (for interaction with PIP2 -rich plasma membrane regions and Rac GTPases; this domain also possesses F-actin bundling activity), a PRD domain (for interaction with cortactin), and a WH2 domain (interaction with G-actin). The patch-clamp electrophysiological technique in whole-cell configuration was used to test the involvement of MIM in the actin-dependent regulation of ENaC. Co-transfection of ENaC subunits with the wild-type MIM protein (or its mutant forms) caused a significant reduction in ENaC-mediated integral ion currents. The analysis of the F-actin structure after the transfection of MIM plasmids showed the important role played by the domains PRD and WH2 of the MIM protein in cytoskeletal rearrangements. These results suggest that the MIM protein may be a part of the complex of actin-binding proteins which is responsible for the actin-dependent regulation of ENaC in the CCD.
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Affiliation(s)
- L. S. Shuyskiy
- Institute of Cytology of RAS, Tikhoretskij Ave. 4, St. Petersburg, 194064, Russia , Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA
| | - V. V. Levchenko
- Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA
| | - Y. A. Negulyaev
- Institute of Cytology of RAS, Tikhoretskij Ave. 4, St. Petersburg, 194064, Russia , Department of Medical Physics, Peter the Great St. Petersburg Polytechnic University, Politekhnicheskaya Str. 2, St. Petersburg, 195251, Russia
| | - A. V. Staruschenko
- Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA
| | - D. V. Ilatovskaya
- Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA , Medical University of South Carolina, Department of Medicine, Division of Nephrology, 96 Jonathan Lucas St, MSC 629 CSB 822, Charleston, SC 29425, USA
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13
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Herbert LM, Resta TC, Jernigan NL. RhoA increases ASIC1a plasma membrane localization and calcium influx in pulmonary arterial smooth muscle cells following chronic hypoxia. Am J Physiol Cell Physiol 2017; 314:C166-C176. [PMID: 29070491 DOI: 10.1152/ajpcell.00159.2017] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
Increases in pulmonary arterial smooth muscle cell (PASMC) intracellular Ca2+ levels and enhanced RhoA/Rho kinase-dependent Ca2+ sensitization are key determinants of PASMC contraction, migration, and proliferation accompanying the development of hypoxic pulmonary hypertension. We previously showed that acid-sensing ion channel 1a (ASIC1a)-mediated Ca2+ entry in PASMC is an important constituent of the active vasoconstriction, vascular remodeling, and right ventricular hypertrophy associated with hypoxic pulmonary hypertension. However, the enhanced ASIC1a-mediated store-operated Ca2+ entry in PASMC from pulmonary hypertensive animals is not dependent on an increase in ASIC1a protein expression, suggesting that chronic hypoxia (CH) stimulates ASIC1a function through other regulatory mechanism(s). RhoA is involved in ion channel trafficking, and levels of activated RhoA are increased following CH. Therefore, we hypothesize that activation of RhoA following CH increases ASIC1a-mediated Ca2+ entry by promoting ASIC1a plasma membrane localization. Consistent with our hypothesis, we found greater plasma membrane localization of ASIC1a following CH. Inhibition of RhoA decreased ASIC1a plasma membrane expression and largely diminished ASIC1a-mediated Ca2+ influx, whereas activation of RhoA had the opposite effect. A proximity ligation assay revealed that ASIC1a and RhoA colocalize in PASMC and that the activation state of RhoA modulates this interaction. Together, our findings show a novel interaction between RhoA and ASIC1a, such that activation of RhoA in PASMC, both pharmacologically and via CH, promotes ASIC1a plasma membrane localization and Ca2+ entry. In addition to enhanced RhoA-mediated Ca2+ sensitization following CH, RhoA can also activate a Ca2+ signal by facilitating ASIC1a plasma membrane localization and Ca2+ influx in pulmonary hypertension.
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Affiliation(s)
- Lindsay M Herbert
- Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center , Albuquerque, New Mexico
| | - Thomas C Resta
- Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center , Albuquerque, New Mexico
| | - Nikki L Jernigan
- Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center , Albuquerque, New Mexico
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14
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Ruhs S, Nolze A, Hübschmann R, Grossmann C. 30 YEARS OF THE MINERALOCORTICOID RECEPTOR: Nongenomic effects via the mineralocorticoid receptor. J Endocrinol 2017; 234:T107-T124. [PMID: 28348113 DOI: 10.1530/joe-16-0659] [Citation(s) in RCA: 51] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/21/2017] [Accepted: 03/27/2017] [Indexed: 12/12/2022]
Abstract
The mineralocorticoid receptor (MR) belongs to the steroid hormone receptor family and classically functions as a ligand-dependent transcription factor. It is involved in water-electrolyte homeostasis and blood pressure regulation but independent from these effects also furthers inflammation, fibrosis, hypertrophy and remodeling in cardiovascular tissues. Next to genomic effects, aldosterone elicits very rapid actions within minutes that do not require transcription or translation and that occur not only in classical MR epithelial target organs like kidney and colon but also in nonepithelial tissues like heart, vasculature and adipose tissue. Most of these effects can be mediated by classical MR and its crosstalk with different signaling cascades. Near the plasma membrane, the MR seems to be associated with caveolin and striatin as well as with receptor tyrosine kinases like EGFR, PDGFR and IGF1R and G protein-coupled receptors like AT1 and GPER1, which then mediate nongenomic aldosterone effects. GPER1 has also been named a putative novel MR. There is a close interaction and functional synergism between the genomic and the nongenomic signaling so that nongenomic signaling can lead to long-term effects and support genomic actions. Therefore, understanding nongenomic aldosterone/MR effects is of potential relevance for modulating genomic aldosterone effects and may provide additional targets for intervention.
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Affiliation(s)
- Stefanie Ruhs
- Julius Bernstein Institute of PhysiologyMartin Luther University Halle-Wittenberg, Halle, Germany
| | - Alexander Nolze
- Julius Bernstein Institute of PhysiologyMartin Luther University Halle-Wittenberg, Halle, Germany
| | - Ralf Hübschmann
- Julius Bernstein Institute of PhysiologyMartin Luther University Halle-Wittenberg, Halle, Germany
| | - Claudia Grossmann
- Julius Bernstein Institute of PhysiologyMartin Luther University Halle-Wittenberg, Halle, Germany
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15
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Montgomery DS, Yu L, Ghazi ZM, Thai TL, Al-Khalili O, Ma HP, Eaton DC, Alli AA. ENaC activity is regulated by calpain-2 proteolysis of MARCKS proteins. Am J Physiol Cell Physiol 2017; 313:C42-C53. [PMID: 28468944 PMCID: PMC5538800 DOI: 10.1152/ajpcell.00244.2016] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2016] [Revised: 04/13/2017] [Accepted: 04/15/2017] [Indexed: 12/27/2022]
Abstract
We previously demonstrated a role for the myristoylated alanine-rich C kinase substrate (MARCKS) to serve as an adaptor protein in the anionic phospholipid phosphate-dependent regulation of the epithelial sodium channel (ENaC). Both MARCKS and ENaC are regulated by proteolysis. Calpains are a family of ubiquitously expressed intracellular Ca2+-dependent cysteine proteases involved in signal transduction. Here we examine the role of calpain-2 in regulating MARCKS and ENaC in cultured renal epithelial cells and in the mouse kidney. Using recombinant fusion proteins, we show that MARCKS, but not the ENaC subunits, are a substrate of calpain-2 in the presence of Ca2+ Pharmacological inhibition of calpain-2 alters MARCKS protein expression in light-density sucrose gradient fractions from cell lysates of mouse cortical collecting duct cells. Calpain-dependent cleaved products of MARCKS are detectable in cultured renal cells. Ca2+ mobilization and calpain-2 inhibition decrease the association between ENaC and MARCKS. The inhibition of calpain-2 reduces ENaC activity as demonstrated by single-channel patch-clamp recordings and transepithelial current measurements. These results suggest that calpain-2 proteolysis of MARCKS promotes its interaction with lipids and ENaC at the plasma membrane to allow for the phosphatidylinositol 4,5-bisphosphate (PIP2)-dependent regulation of ENaC activity in the kidney.
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Affiliation(s)
- Darrice S Montgomery
- Department of Physiology and Functional Genomics and Department of Medicine Division of Nephrology, Hypertension, and Renal Transplantation, University of Florida College of Medicine, Gainesville, Florida
| | - Ling Yu
- College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, China; and
| | - Zinah M Ghazi
- Department of Physiology, Emory University School of Medicine, Atlanta, Georgia
| | - Tiffany L Thai
- Department of Physiology, Emory University School of Medicine, Atlanta, Georgia
| | - Otor Al-Khalili
- Department of Physiology, Emory University School of Medicine, Atlanta, Georgia
| | - He-Ping Ma
- Department of Physiology, Emory University School of Medicine, Atlanta, Georgia
| | - Douglas C Eaton
- Department of Physiology, Emory University School of Medicine, Atlanta, Georgia
| | - Abdel A Alli
- Department of Physiology and Functional Genomics and Department of Medicine Division of Nephrology, Hypertension, and Renal Transplantation, University of Florida College of Medicine, Gainesville, Florida;
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16
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Klemens CA, Edinger RS, Kightlinger L, Liu X, Butterworth MB. Ankyrin G Expression Regulates Apical Delivery of the Epithelial Sodium Channel (ENaC). J Biol Chem 2016; 292:375-385. [PMID: 27895120 DOI: 10.1074/jbc.m116.753616] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2016] [Revised: 11/11/2016] [Indexed: 11/06/2022] Open
Abstract
The epithelial sodium channel (ENaC) is the limiting entry point for Na+ reabsorption in the distal kidney nephron and is regulated by numerous hormones, including the mineralocorticoid hormone aldosterone. Previously we identified ankyrin G (AnkG), a cytoskeletal protein involved in vesicular transport, as a novel aldosterone-induced protein that can alter Na+ transport in mouse cortical collecting duct cells. However, the mechanisms underlying AnkG regulation of Na+ transport were unknown. Here we report that AnkG expression directly regulates Na+ transport by altering ENaC activity in the apical membrane. Increasing AnkG expression increased ENaC activity while depleting AnkG reduced ENaC-mediated Na+ transport. These changes were due to a change in ENaC directly rather than through alterations to the Na+ driving force created by Na+/K+-ATPase. Using a constitutively open mutant of ENaC, we demonstrate that the augmentation of Na+ transport is caused predominantly by increasing the number of ENaCs at the surface. To determine the mechanism of AnkG action on ENaC surface number, changes in rates of internalization, recycling, and membrane delivery were investigated. AnkG did not alter ENaC delivery to the membrane from biosynthetic pathways or removal by endocytosis. However, AnkG did alter ENaC insertion from constitutive recycling pathways. These findings provide a mechanism to account for the role of AnkG in the regulation of Na+ transport in the distal kidney nephron.
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Affiliation(s)
- Christine A Klemens
- From the Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
| | - Robert S Edinger
- From the Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
| | - Lindsay Kightlinger
- From the Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
| | - Xiaoning Liu
- From the Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
| | - Michael B Butterworth
- From the Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
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17
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Jella KK, Yu L, Yue Q, Friedman D, Duke BJ, Alli AA. Exosomal GAPDH from Proximal Tubule Cells Regulate ENaC Activity. PLoS One 2016; 11:e0165763. [PMID: 27802315 PMCID: PMC5089749 DOI: 10.1371/journal.pone.0165763] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2016] [Accepted: 10/17/2016] [Indexed: 12/19/2022] Open
Abstract
Exosomes are nanometer-scale, cell-derived vesicles that contain various molecules including nucleic acids, proteins, and lipids. These vesicles can release their cargo into adjacent or distant cells and mediate intercellular communication and cellular function. Here we examined the regulation of epithelial sodium channels in mpkCCD cells and distal tubule Xenopus 2F3 cells by exosomes isolated from proximal tubule LLC-PK1 cells. Cultured mpkCCD cells were stained with CTX coupled to a green fluorophore in order to label the cell membranes and freshly isolated exosomes from LLC-PK1 cells were labeled with the red lipophilic dye PKH26 in order to visualize uptake of exosomes into the cells. Single-channel patch clamp recordings showed the open probability of ENaC in Xenopus 2F3 cells and in freshly isolated split-open tubules decreased in response to exogenous application of exosomes derived from LLC-PK1 proximal tubule cells. Active GAPDH was identified within exosomes derived from proximal tubule LLC-PK1 cells. The effect on ENaC activity in Xenopus 2F3 cells was blunted after application of exosomes transfected with the GAPDH inhibitor heptelidic acid. Also, we show GAPDH and ENaC subunits associate in mpkCCD cells. These studies examine a potential role for exosomes in the regulation of ENaC activity and examine a possible mechanism for communication from proximal tubule cells to distal tubule and collecting duct cells.
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Affiliation(s)
- Kishore Kumar Jella
- Department of Radiation Oncology, Emory University School of Medicine, Atlanta, Georgia
| | - Ling Yu
- College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, Jiangsu Province, China
| | - Qiang Yue
- Department of Physiology, Emory University School of Medicine, Atlanta, Georgia
| | - Daniel Friedman
- Department of Physiology, Emory University School of Medicine, Atlanta, Georgia
| | - Billie J. Duke
- Department of Physiology, Emory University School of Medicine, Atlanta, Georgia
| | - Abdel A. Alli
- Department of Physiology and Functional Genomics and Department of Medicine Division of Nephrology, Hypertension, and Renal Transplantation, University of Florida College of Medicine, Gainesville, Florida, United States of America
- * E-mail:
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18
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The Epithelial Sodium Channel and the Processes of Wound Healing. BIOMED RESEARCH INTERNATIONAL 2016; 2016:5675047. [PMID: 27493961 PMCID: PMC4963570 DOI: 10.1155/2016/5675047] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/15/2016] [Accepted: 06/15/2016] [Indexed: 12/19/2022]
Abstract
The epithelial sodium channel (ENaC) mediates passive sodium transport across the apical membranes of sodium absorbing epithelia, like the distal nephron, the intestine, and the lung airways. Additionally, the channel has been involved in the transduction of mechanical stimuli, such as hydrostatic pressure, membrane stretch, and shear stress from fluid flow. Thus, in vascular endothelium, it participates in the control of the vascular tone via its activity both as a sodium channel and as a shear stress transducer. Rather recently, ENaC has been shown to participate in the processes of wound healing, a role that may also involve its activities as sodium transporter and as mechanotransducer. Its presence as the sole channel mediating sodium transport in many tissues and the diversity of its functions probably underlie the complexity of its regulation. This brief review describes some aspects of ENaC regulation, comments on evidence about ENaC participation in wound healing, and suggests possible regulatory mechanisms involved in this participation.
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19
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Bai X, Dee R, Mangum KD, Mack CP, Taylor JM. RhoA signaling and blood pressure: The consequence of failing to “Tone it Down”. World J Hypertens 2016; 6:18-35. [DOI: 10.5494/wjh.v6.i1.18] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/30/2015] [Revised: 11/24/2015] [Accepted: 01/22/2016] [Indexed: 02/06/2023] Open
Abstract
Uncontrolled high blood pressure is a major risk factor for heart attack, stroke, and kidney failure and contributes to an estimated 25% of deaths worldwide. Despite numerous treatment options, estimates project that reasonable blood pressure (BP) control is achieved in only about half of hypertensive patients. Improvements in the detection and management of hypertension will undoubtedly be accomplished through a better understanding of the complex etiology of this disease and a more comprehensive inventory of the genes and genetic variants that influence BP regulation. Recent studies (primarily in pre-clinical models) indicate that the small GTPase RhoA and its downstream target, Rho kinase, play an important role in regulating BP homeostasis. Herein, we summarize the underlying mechanisms and highlight signaling pathways and regulators that impart tight spatial-temporal control of RhoA activity. We also discuss known allelic variations in the RhoA pathway and consider how these polymorphisms may affect genetic risk for hypertension and its clinical manifestations. Finally, we summarize the current (albeit limited) clinical data on the efficacy of targeting the RhoA pathway in hypertensive patients.
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20
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Alli AA, Bao HF, Liu BC, Yu L, Aldrugh S, Montgomery DS, Ma HP, Eaton DC. Calmodulin and CaMKII modulate ENaC activity by regulating the association of MARCKS and the cytoskeleton with the apical membrane. Am J Physiol Renal Physiol 2015; 309:F456-63. [PMID: 26136560 DOI: 10.1152/ajprenal.00631.2014] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2014] [Accepted: 06/24/2015] [Indexed: 11/22/2022] Open
Abstract
Phosphatidylinositol bisphosphate (PIP2) regulates epithelial sodium channel (ENaC) open probability. In turn, myristoylated alanine-rich C kinase substrate (MARCKS) protein or MARCKS-like protein 1 (MLP-1) at the plasma membrane regulates the delivery of PIP2 to ENaC. MARCKS and MLP-1 are regulated by changes in cytosolic calcium; increasing calcium promotes dissociation of MARCKS from the membrane, but the calcium-regulatory mechanisms are unclear. However, it is known that increased intracellular calcium can activate calmodulin and we show that inhibition of calmodulin with calmidazolium increases ENaC activity presumably by regulating MARCKS and MLP-1. Activated calmodulin can regulate MARCKS and MLP-1 in two ways. Calmodulin can bind to the effector domain of MARCKS or MLP-1, inactivating both proteins by causing their dissociation from the membrane. Mutations in MARCKS that prevent calmodulin association prevent dissociation of MARCKS from the membrane. Calmodulin also activates CaM kinase II (CaMKII). An inhibitor of CaMKII (KN93) increases ENaC activity, MARCKS association with ENaC, and promotes MARCKS movement to a membrane fraction. CaMKII phosphorylates filamin. Filamin is an essential component of the cytoskeleton and promotes association of ENaC, MARCKS, and MLP-1. Disruption of the cytoskeleton with cytochalasin E reduces ENaC activity. CaMKII phosphorylation of filamin disrupts the cytoskeleton and the association of MARCKS, MLP-1, and ENaC, thereby reducing ENaC open probability. Taken together, these findings suggest calmodulin and CaMKII modulate ENaC activity by destabilizing the association between the actin cytoskeleton, ENaC, and MARCKS, or MLP-1 at the apical membrane.
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Affiliation(s)
- Abdel A Alli
- Center for Cell and Molecular Signaling, Department of Physiology, Emory University School of Medicine; Atlanta, Georgia
| | - Hui-Fang Bao
- Center for Cell and Molecular Signaling, Department of Physiology, Emory University School of Medicine; Atlanta, Georgia
| | - Bing-Chen Liu
- Center for Cell and Molecular Signaling, Department of Physiology, Emory University School of Medicine; Atlanta, Georgia
| | - Ling Yu
- Center for Cell and Molecular Signaling, Department of Physiology, Emory University School of Medicine; Atlanta, Georgia
| | - Summer Aldrugh
- Center for Cell and Molecular Signaling, Department of Physiology, Emory University School of Medicine; Atlanta, Georgia
| | - Darrice S Montgomery
- Center for Cell and Molecular Signaling, Department of Physiology, Emory University School of Medicine; Atlanta, Georgia
| | - He-Ping Ma
- Center for Cell and Molecular Signaling, Department of Physiology, Emory University School of Medicine; Atlanta, Georgia
| | - Douglas C Eaton
- Center for Cell and Molecular Signaling, Department of Physiology, Emory University School of Medicine; Atlanta, Georgia
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21
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Amiloride-insensitive sodium channels are directly regulated by actin cytoskeleton dynamics in human lymphoma cells. Biochem Biophys Res Commun 2015; 461:54-8. [DOI: 10.1016/j.bbrc.2015.03.167] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2015] [Accepted: 03/28/2015] [Indexed: 01/03/2023]
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22
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Huveneers S, Daemen MJAP, Hordijk PL. Between Rho(k) and a hard place: the relation between vessel wall stiffness, endothelial contractility, and cardiovascular disease. Circ Res 2015; 116:895-908. [PMID: 25722443 DOI: 10.1161/circresaha.116.305720] [Citation(s) in RCA: 145] [Impact Index Per Article: 14.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Vascular stiffness is a mechanical property of the vessel wall that affects blood pressure, permeability, and inflammation. As a result, vascular stiffness is a key driver of (chronic) human disorders, including pulmonary arterial hypertension, kidney disease, and atherosclerosis. Responses of the endothelium to stiffening involve integration of mechanical cues from various sources, including the extracellular matrix, smooth muscle cells, and the forces that derive from shear stress of blood. This response in turn affects endothelial cell contractility, which is an important property that regulates endothelial stiffness, permeability, and leukocyte-vessel wall interactions. Moreover, endothelial stiffening reduces nitric oxide production, which promotes smooth muscle cell contraction and vasoconstriction. In fact, vessel wall stiffening, and microcirculatory endothelial dysfunction, precedes hypertension and thus underlies the development of vascular disease. Here, we review the cross talk among vessel wall stiffening, endothelial contractility, and vascular disease, which is controlled by Rho-driven actomyosin contractility and cellular mechanotransduction. In addition to discussing the various inputs and relevant molecular events in the endothelium, we address which actomyosin-regulated changes at cell adhesion complexes are genetically associated with human cardiovascular disease. Finally, we discuss recent findings that broaden therapeutic options for targeting this important mechanical signaling pathway in vascular pathogenesis.
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Affiliation(s)
- Stephan Huveneers
- From the Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Swammerdam Institute for Life Sciences (S.H., P.L.H.) and Department of Pathology (M.J.A.P.D.), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
| | - Mat J A P Daemen
- From the Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Swammerdam Institute for Life Sciences (S.H., P.L.H.) and Department of Pathology (M.J.A.P.D.), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
| | - Peter L Hordijk
- From the Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Swammerdam Institute for Life Sciences (S.H., P.L.H.) and Department of Pathology (M.J.A.P.D.), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
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23
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Tian J, Tep C, Benedick A, Saidi N, Ryu JC, Kim ML, Sadasivan S, Oberdick J, Smeyne R, Zhu MX, Yoon SO. p75 regulates Purkinje cell firing by modulating SK channel activity through Rac1. J Biol Chem 2014; 289:31458-72. [PMID: 25253694 PMCID: PMC4223344 DOI: 10.1074/jbc.m114.589937] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2014] [Revised: 09/11/2014] [Indexed: 12/22/2022] Open
Abstract
p75 is expressed among Purkinje cells in the adult cerebellum, but its function has remained obscure. Here we report that p75 is involved in maintaining the frequency and regularity of spontaneous firing of Purkinje cells. The overall spontaneous firing activity of Purkinje cells was increased in p75(-/-) mice during the phasic firing period due to a longer firing period and accompanying reduction in silence period than in the wild type. We attribute these effects to a reduction in small conductance Ca(2+)-activated potassium (SK) channel activity in Purkinje cells from p75(-/-) mice compared with the wild type littermates. The mechanism by which p75 regulates SK channel activity appears to involve its ability to activate Rac1. In organotypic cultures of cerebellar slices, brain-derived neurotrophic factor increased RacGTP levels by activating p75 but not TrkB. These results correlate with a reduction in RacGTP levels in synaptosome fractions from the p75(-/-) cerebellum, but not in that from the cortex of the same animals, compared with wild type littermates. More importantly, we demonstrate that Rac1 modulates SK channel activity and firing patterns of Purkinje cells. Along with the finding that spine density was reduced in p75(-/-) cerebellum, these data suggest that p75 plays a role in maintaining normalcy of Purkinje cell firing in the cerebellum in part by activating Rac1 in synaptic compartments and modulating SK channels.
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Affiliation(s)
- JinBin Tian
- the Department of Neuroscience, Ohio State University, Columbus, Ohio 43210, the Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, Texas 77030
| | - Chhavy Tep
- From the Department of Molecular and Cellular Biochemistry, the Biochemistry Program, and
| | - Alex Benedick
- From the Department of Molecular and Cellular Biochemistry
| | - Nabila Saidi
- From the Department of Molecular and Cellular Biochemistry
| | - Jae Cheon Ryu
- From the Department of Molecular and Cellular Biochemistry
| | - Mi Lyang Kim
- From the Department of Molecular and Cellular Biochemistry
| | - Shankar Sadasivan
- the Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, and
| | | | - Richard Smeyne
- the Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, and
| | - Michael X Zhu
- the Department of Neuroscience, Ohio State University, Columbus, Ohio 43210, the Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, Texas 77030
| | - Sung Ok Yoon
- From the Department of Molecular and Cellular Biochemistry,
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24
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Loirand G, Pacaud P. Involvement of Rho GTPases and their regulators in the pathogenesis of hypertension. Small GTPases 2014; 5:1-10. [PMID: 25496262 DOI: 10.4161/sgtp.28846] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Proper regulation of arterial blood pressure is essential to allow permanent adjustment of nutrient and oxygen supply to organs and tissues according to their need. This is achieved through highly coordinated regulation processes controlling vascular resistance through modulation of arterial smooth muscle contraction, cardiac output, and kidney function. Members of the Rho family of small GTPases, in particular RhoA and Rac1, have been identified as key signaling molecules playing important roles in several different steps of these regulatory processes. Here, we review the current state of knowledge regarding the involvement of Rho GTPase signaling in the control of blood pressure and the pathogenesis of hypertension. We describe how knockout models in mouse, genetic, and pharmacological studies in human have been useful to address this question.
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Key Words
- AT1 receptor, type 1 Ang II receptor
- Ang II, angiotensine II
- ENaCs, epithelial Na+ channels
- Et-1, endothelin-1
- GAPs, GTPase-activating proteins
- GEFs, exchange factors
- GTPase activating proteins
- GTPases
- MLC, 20 kDa-myosin light chain
- MLCK, MLC kinase
- MLCP, MLC phosphatase
- NA, noradrenaline
- NHE3, sodium-hydrogen exchanger isoform 3.
- NO, nitric oxide
- NTS, nucleus tractus solitaries
- PDE5, type 5 phosphodiesterase
- PKG, cGMP-dependent protein kinase
- Rock, Rho-kinase
- SHR, spontaneously hypertensive rats
- SHRSP, stroke-prone SHR
- TxA2, thromboxane A2
- artery
- blood pressure
- cardiovascular
- eNOS, endothelial NO synthase
- exchange factors
- signal transduction
- small G proteins
- smooth muscle
- vasoconstriction
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25
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Pavlov TS, Levchenko V, Staruschenko A. Role of Rho GDP dissociation inhibitor α in control of epithelial sodium channel (ENaC)-mediated sodium reabsorption. J Biol Chem 2014; 289:28651-9. [PMID: 25164814 DOI: 10.1074/jbc.m114.558262] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
The epithelial sodium channel (ENaC) is expressed in the aldosterone-sensitive distal nephron where it performs sodium reabsorption from the lumen. We have recently shown that ENaC activity contributes to the development of salt-induced hypertension as a result of deficiency of EGF level. Previous studies revealed that Rho GDP-dissociation inhibitor α (RhoGDIα) is involved in the control of salt-sensitive hypertension and renal injury via Rac1, which is one of the small GTPases activating ENaC. Here we investigated the intracellular mechanism mediating the involvement of the RhoGDIα/Rac1 axis in the control of ENaC and the effect of EGF on ENaC in this pathway. We demonstrated that RhoGDIα is highly expressed in the cortical collecting ducts of mice and rats, and its expression is down-regulated in Dahl salt-sensitive rats fed a high salt diet. Knockdown of RhoGDIα in cultured cortical collecting duct principal cells increased ENaC subunits expression and ENaC-mediated sodium reabsorption. Furthermore, RhoGDIα deficiency causes enhanced response to EGF treatment. Patch clamp analysis reveals that RhoGDIα significantly decreases ENaC current density and prevents its up-regulation by RhoA and Rac1. Inhibition of Rho kinase with Y27632 had no effects on ENaC response to EGF either in control or RhoGDIα knocked down cells. However, EGF treatment increased levels of active Rac1, which was further enhanced in RhoGDIα-deficient cells. We conclude that changes in the RhoGDIα-dependent pathway have a permissive role in the Rac1-mediated enhancement of ENaC activity observed in salt-induced hypertension.
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Affiliation(s)
- Tengis S Pavlov
- From the Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
| | - Vladislav Levchenko
- From the Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
| | - Alexander Staruschenko
- From the Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
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26
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Zhou M, Fu J, Huang W, Shen L, Xiao L, Song Y, Liu Y. Increased cystic fibrosis transmembrane conductance regulators expression and decreased epithelial sodium channel alpha subunits expression in early abortion: findings from a mouse model and clinical cases of abortion. PLoS One 2014; 9:e99521. [PMID: 24914548 PMCID: PMC4051784 DOI: 10.1371/journal.pone.0099521] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2013] [Accepted: 05/15/2014] [Indexed: 11/23/2022] Open
Abstract
The status of the maternal endometrium is vital in regulating humoral homeostasis and for ensuring embryo implantation. Cystic fibrosis transmembrane conductance regulators (CFTR) and epithelial sodium channel alpha subunits (ENaC-α) play an important role in female reproduction by maintaining humoral and cell homeostasis. However, it is not clear whether the expression levels of CFTR and ENaC-α in the decidual component during early pregnancy are related with early miscarriage. CBA×DBA/2 mouse mating has been widely accepted as a classical model of early miscarriage. The abortion rate associated with this mating was 33.33% in our study. The decidua of abortion-prone CBA female mice (DBA/2 mated) had higher CFTR mRNA and protein expression and lower ENaC-α mRNA and protein expression, compared to normal pregnant CBA mice (BLAB/C mated). Furthermore, increased CFTR expression and decreased ENaC-α expression were observed in the uterine tissue from women with early miscarriage, as compared to those with successful pregnancy. In conclusion, increased CFTR expression and decreased ENaC-α expression in the decidua of early abortion may relate with failure of early pregnancy.
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Affiliation(s)
- Min Zhou
- Department of Obstetrics and Gynecology, West China Second University Hospital of Sichuan University, Chengdu, People's Republic of China
| | - Jing Fu
- Department of Obstetrics and Gynecology, West China Second University Hospital of Sichuan University, Chengdu, People's Republic of China
| | - Wei Huang
- Department of Obstetrics and Gynecology, West China Second University Hospital of Sichuan University, Chengdu, People's Republic of China
- * E-mail:
| | - Licong Shen
- Department of Obstetrics and Gynecology, West China Second University Hospital of Sichuan University, Chengdu, People's Republic of China
| | - Li Xiao
- Department of Obstetrics and Gynecology, West China Second University Hospital of Sichuan University, Chengdu, People's Republic of China
| | - Yong Song
- Department of Obstetrics and Gynecology, West China Second University Hospital of Sichuan University, Chengdu, People's Republic of China
| | - Ying Liu
- Sichuan University-The Chinese University of Hong Kong Joint Laboratory for Reproductive Medicine, West China Institute of Women and Children's Health, West China Second University Hospital, Sichuan University, Chengdu, People's Republic of China
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Reifenberger MS, Yu L, Bao HF, Duke BJ, Liu BC, Ma HP, Alli AA, Eaton DC, Alli AA. Cytochalasin E alters the cytoskeleton and decreases ENaC activity in Xenopus 2F3 cells. Am J Physiol Renal Physiol 2014; 307:F86-95. [PMID: 24829507 DOI: 10.1152/ajprenal.00251.2013] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Numerous reports have linked cytoskeleton-associated proteins with the regulation of epithelial Na(+) channel (ENaC) activity. The purpose of the present study was to determine the effect of actin cytoskeleton disruption by cytochalasin E on ENaC activity in Xenopus 2F3 cells. Here, we show that cytochalasin E treatment for 60 min can disrupt the integrity of the actin cytoskeleton in cultured Xenopus 2F3 cells. We show using single channel patch-clamp experiments and measurements of short-circuit current that ENaC activity, but not its density, is altered by cytochalasin E-induced disruption of the cytoskeleton. In nontreated cells, 8 of 33 patches (24%) had no measurable ENaC activity, whereas in cytochalasin E-treated cells, 17 of 32 patches (53%) had no activity. Analysis of those patches that did contain ENaC activity showed channel open probability significantly decreased from 0.081 ± 0.01 in nontreated cells to 0.043 ± 0.01 in cells treated with cytochalasin E. Transepithelial current from mpkCCD cells treated with cytochalasin E, cytochalasin D, or latrunculin B for 60 min was decreased compared with vehicle-treated cells. The subcellular expression of fodrin changed significantly, and several protein elements of the cytoskeleton decreased at least twofold after 60 min of cytochalasin E treatment. Cytochalasin E treatment disrupted the association between ENaC and myristoylated alanine-rich C-kinase substrate. The results presented here suggest disruption of the actin cytoskeleton by different compounds can attenuate ENaC activity through a mechanism involving changes in the subcellular expression of fodrin, several elements of the cytoskeleton, and destabilization of the ENaC-myristoylated alanine-rich C-kinase substrate complex.
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Affiliation(s)
- Matthew S Reifenberger
- Department of Physiology and the Center for Cell and Molecular Signaling, Emory University School of Medicine, Atlanta, Georgia
| | - Ling Yu
- Department of Physiology and the Center for Cell and Molecular Signaling, Emory University School of Medicine, Atlanta, Georgia
| | - Hui-Fang Bao
- Department of Physiology and the Center for Cell and Molecular Signaling, Emory University School of Medicine, Atlanta, Georgia
| | - Billie Jeanne Duke
- Department of Physiology and the Center for Cell and Molecular Signaling, Emory University School of Medicine, Atlanta, Georgia
| | - Bing-Chen Liu
- Department of Physiology and the Center for Cell and Molecular Signaling, Emory University School of Medicine, Atlanta, Georgia
| | - He-Ping Ma
- Department of Physiology and the Center for Cell and Molecular Signaling, Emory University School of Medicine, Atlanta, Georgia
| | - Ahmed A Alli
- Department of Physiology and the Center for Cell and Molecular Signaling, Emory University School of Medicine, Atlanta, Georgia
| | - Douglas C Eaton
- Department of Physiology and the Center for Cell and Molecular Signaling, Emory University School of Medicine, Atlanta, Georgia
| | - Abdel A Alli
- Department of Physiology and the Center for Cell and Molecular Signaling, Emory University School of Medicine, Atlanta, Georgia
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Hypotonic Regulation of Mouse Epithelial Sodium Channel in Xenopus laevis Oocytes. J Membr Biol 2013; 246:949-58. [DOI: 10.1007/s00232-013-9598-8] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2013] [Accepted: 09/24/2013] [Indexed: 10/26/2022]
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29
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Loirand G, Sauzeau V, Pacaud P. Small G Proteins in the Cardiovascular System: Physiological and Pathological Aspects. Physiol Rev 2013; 93:1659-720. [DOI: 10.1152/physrev.00021.2012] [Citation(s) in RCA: 79] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Small G proteins exist in eukaryotes from yeast to human and constitute the Ras superfamily comprising more than 100 members. This superfamily is structurally classified into five families: the Ras, Rho, Rab, Arf, and Ran families that control a wide variety of cell and biological functions through highly coordinated regulation processes. Increasing evidence has accumulated to identify small G proteins and their regulators as key players of the cardiovascular physiology that control a large panel of cardiac (heart rhythm, contraction, hypertrophy) and vascular functions (angiogenesis, vascular permeability, vasoconstriction). Indeed, basal Ras protein activity is required for homeostatic functions in physiological conditions, but sustained overactivation of Ras proteins or spatiotemporal dysregulation of Ras signaling pathways has pathological consequences in the cardiovascular system. The primary object of this review is to provide a comprehensive overview of the current progress in our understanding of the role of small G proteins and their regulators in cardiovascular physiology and pathologies.
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Affiliation(s)
- Gervaise Loirand
- INSERM, UMR S1087; University of Nantes; and CHU Nantes, l'Institut du Thorax, Nantes, France
| | - Vincent Sauzeau
- INSERM, UMR S1087; University of Nantes; and CHU Nantes, l'Institut du Thorax, Nantes, France
| | - Pierre Pacaud
- INSERM, UMR S1087; University of Nantes; and CHU Nantes, l'Institut du Thorax, Nantes, France
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Haloui M, Tremblay J, Seda O, Koltsova SV, Maksimov GV, Orlov SN, Hamet P. Increased Renal Epithelial Na Channel Expression and Activity Correlate With Elevation of Blood Pressure in Spontaneously Hypertensive Rats. Hypertension 2013; 62:731-7. [PMID: 23959560 DOI: 10.1161/hypertensionaha.113.01295] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Affiliation(s)
- Mounsif Haloui
- From the Centre de Recherche, Centre Hospitalier de l’Université de Montréal (CRCHUM) – Technopôle Angus, QC, Canada (M.H., J.T., O.S., S.V.K., S.N.O.); and the Faculty of Biology, M.V. Lomonosov Moscow State University, Russia (G.V.M., S.N.O.)
| | - Johanne Tremblay
- From the Centre de Recherche, Centre Hospitalier de l’Université de Montréal (CRCHUM) – Technopôle Angus, QC, Canada (M.H., J.T., O.S., S.V.K., S.N.O.); and the Faculty of Biology, M.V. Lomonosov Moscow State University, Russia (G.V.M., S.N.O.)
| | - Ondrej Seda
- From the Centre de Recherche, Centre Hospitalier de l’Université de Montréal (CRCHUM) – Technopôle Angus, QC, Canada (M.H., J.T., O.S., S.V.K., S.N.O.); and the Faculty of Biology, M.V. Lomonosov Moscow State University, Russia (G.V.M., S.N.O.)
| | - Svetlana V. Koltsova
- From the Centre de Recherche, Centre Hospitalier de l’Université de Montréal (CRCHUM) – Technopôle Angus, QC, Canada (M.H., J.T., O.S., S.V.K., S.N.O.); and the Faculty of Biology, M.V. Lomonosov Moscow State University, Russia (G.V.M., S.N.O.)
| | - Georgy V. Maksimov
- From the Centre de Recherche, Centre Hospitalier de l’Université de Montréal (CRCHUM) – Technopôle Angus, QC, Canada (M.H., J.T., O.S., S.V.K., S.N.O.); and the Faculty of Biology, M.V. Lomonosov Moscow State University, Russia (G.V.M., S.N.O.)
| | - Sergei N. Orlov
- From the Centre de Recherche, Centre Hospitalier de l’Université de Montréal (CRCHUM) – Technopôle Angus, QC, Canada (M.H., J.T., O.S., S.V.K., S.N.O.); and the Faculty of Biology, M.V. Lomonosov Moscow State University, Russia (G.V.M., S.N.O.)
| | - Pavel Hamet
- From the Centre de Recherche, Centre Hospitalier de l’Université de Montréal (CRCHUM) – Technopôle Angus, QC, Canada (M.H., J.T., O.S., S.V.K., S.N.O.); and the Faculty of Biology, M.V. Lomonosov Moscow State University, Russia (G.V.M., S.N.O.)
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31
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Chubinskiy-Nadezhdin VI, Sudarikova AV, Nikolsky NN, Morachevskaya EA. Role of submembranous actin cytoskeleton in regulation of non-voltage-gated sodium channels. DOKL BIOCHEM BIOPHYS 2013; 450:126-9. [DOI: 10.1134/s1607672913030010] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2013] [Indexed: 11/23/2022]
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32
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Mondini A, Sassone F, Civello DA, Garavaglia ML, Bazzini C, Rodighiero S, Vezzoli V, Conti F, Torielli L, Capasso G, Paulmichl M, Meyer G. Hypertension-linked mutation of α-adducin increases CFTR surface expression and activity in HEK and cultured rat distal convoluted tubule cells. PLoS One 2012; 7:e52014. [PMID: 23284854 PMCID: PMC3528715 DOI: 10.1371/journal.pone.0052014] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2012] [Accepted: 11/08/2012] [Indexed: 11/18/2022] Open
Abstract
The CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) activity and localization are influenced by the cytoskeleton, in particular by actin and its polymerization state. In this study we investigated whether the expression of the hypertensive mutations of α-adducin (G460W-S586C in humans, F316Y in rats), an actin capping protein, led to a functional modification of CFTR activity and surface expression. The experiments were performed on HEK293 T cells cotransfected with CFTR and the human wild type (WT) or G460W mutated α-adducin. In whole-cell patch-clamp experiments, both the CFTR chloride current and the slope of current activation after forskolin addition were significantly higher in HEK cells overexpressing the G460W adducin. A higher plasma membrane density of active CFTR channels was confirmed by cell-attached patch-clamp experiments, both in HEK cells and in cultured primary DCT cells, isolated from MHS (Milan Hypertensive Strain, a Wistar rat (Rattus norvegicus) hypertensive model carrying the F316Y adducin mutation), compared to MNS (Milan Normotensive Strain) rats. Western blot experiments demonstrated an increase of the plasma membrane CFTR protein expression, with a modification of the channel glycosylation state, in the presence of the mutated adducin. A higher retention of CFTR protein in the plasma membrane was confirmed both by FRAP (Fluorescence Recovery After Photobleaching) and photoactivation experiments. The present data indicate that in HEK cells and in isolated DCT cells the presence of the G460W-S586C hypertensive variant of adducin increases CFTR channel activity, possibly by altering its membrane turnover and inducing a retention of the channel in the plasmamembrane. Since CFTR is known to modulate the activity of many others transport systems, the increased surface expression of the channel could have consequences on the whole network of transport in kidney cells.
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Affiliation(s)
- Anna Mondini
- Department of Life Sciences, Università degli Studi di Milano, Milano, Italy
| | - Francesca Sassone
- Department of Life Sciences, Università degli Studi di Milano, Milano, Italy
| | | | | | - Claudia Bazzini
- Department of Life Sciences, Università degli Studi di Milano, Milano, Italy
| | | | - Valeria Vezzoli
- Department of Life Sciences, Università degli Studi di Milano, Milano, Italy
| | - Fabio Conti
- Prassis Research Institute, Sigma Tau, Settimo M.se, Italy
| | - Lucia Torielli
- Prassis Research Institute, Sigma Tau, Settimo M.se, Italy
| | | | - Markus Paulmichl
- Institut of Pharmacology and Toxicology, Paracelsus Medical University, Salzburg, Austria
| | - Giuliano Meyer
- Department of Life Sciences, Università degli Studi di Milano, Milano, Italy
- * E-mail:
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33
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Ilatovskaya DV, Pavlov TS, Levchenko V, Staruschenko A. ROS production as a common mechanism of ENaC regulation by EGF, insulin, and IGF-1. Am J Physiol Cell Physiol 2012; 304:C102-11. [PMID: 23135700 DOI: 10.1152/ajpcell.00231.2012] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The epithelial Na(+) channel (ENaC) is a key transporter participating in the fine tuning of Na(+) reabsorption in the nephron. ENaC activity is acutely upregulated by epidermal growth factor (EGF), insulin, and insulin-like growth factor-1 (IGF-1). It was also proposed that reactive oxygen species (ROS) have a stimulatory effect on ENaC. Here we studied whether effects of EGF, insulin, and IGF-1 correlate with ROS production in the mouse cortical collecting duct (mpkCCD(c14)) cells. Western blotting confirmed the expression of the NADPH oxidase complex subunits in these cells. Treatment of mpkCCD(c14) cells with EGF, insulin, or IGF-1 evoked an increase in ROS production as measured by CM-H(2)DCF-DA fluorescence. ROS production caused by a xanthine-xanthine oxidase reaction also resulted in a significant elevation in short-circuit current through the mpkCCD(c14) monolayer. Transepithelial current measurements showed an acute increase of amiloride-sensitive current through the mpkCCD(c14) monolayer in response to EGF, insulin, or IGF-1. Pretreatment with the nonselective NADPH oxidase activity inhibitor apocynin blunted both ROS production and increase in ENaC-mediated current in response to these drugs. To further test whether NADPH oxidase subunits are involved in the effect of EGF, we used a stable M-1 cell line with a knockdown of Rac1, which is one of the key subunits of the NADPH oxidase complex, and measured amiloride-sensitive currents in response to EGF. In contrast to control cells, EGF had no effect in Rac1 knockdown cells. We hypothesize that EGF, insulin, and IGF-1 have a common stimulatory effect on ENaC mediated by ROS production.
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Affiliation(s)
- Daria V Ilatovskaya
- Medical College of Wisconsin, Dept. of Physiology, 8701 Watertown Plank Rd., Milwaukee, WI 53226, USA
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34
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Sudarikova AV, Vassilieva IO, Morachevskaya EA, Negulyaev YA. Molecular and functional identification of sodium channels in K562 cells. ACTA ACUST UNITED AC 2012. [DOI: 10.1134/s1990519x12050124] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
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35
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Chen X, Zhu H, Liu X, Lu H, Li Y, Wang J, Liu H, Zhang J, Ma Q, Zhang Y. Characterization of Two Mammalian Cortical Collecting Duct Cell Lines with Hopping Probe Ion Conductance Microscopy. J Membr Biol 2012; 246:7-11. [PMID: 22961044 DOI: 10.1007/s00232-012-9495-6] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2012] [Accepted: 07/31/2012] [Indexed: 12/11/2022]
Affiliation(s)
- Xuewei Chen
- Department of Occupational Hygiene, Institute of Health and Environmental Medicine, Tianjin 300050, China
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36
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Shklyar TF, Dinislamova OA, Safronov AP, Blyakhman FA. Effect of cytoskeletal elastic properties on the mechanoelectrical transduction in excitable cells. J Biomech 2012; 45:1444-9. [DOI: 10.1016/j.jbiomech.2012.02.017] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2011] [Revised: 01/14/2012] [Accepted: 02/16/2012] [Indexed: 11/25/2022]
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37
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Assef YA, Ozu M, Marino GI, Galizia L, Kotsias BA. ENaC channels in oocytes from Xenopus laevis and their regulation by xShroom1 protein. Cell Physiol Biochem 2011; 28:259-66. [PMID: 21865733 DOI: 10.1159/000331738] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 07/08/2011] [Indexed: 11/19/2022] Open
Abstract
Shroom is a family of related proteins linked to the actin cytoskeleton. xShroom1 is constitutively expressed in X. oocytes and is required for the expression of amiloride sensitive sodium channels (ENaC). Oocytes were injected with α, β, and γ mENaC and xShroom1 sense or antisense oligonucleotides. We used voltage clamp techniques to study the amiloride-sensitive Na(+) currents (INa((amil))). We observed a marked reduction in INa((amil)) in oocytes co-injected with xShroom1 antisense. Oocytes expressing a DEG mutant β-mENaC subunit (β-S518K) with an open probability of 1 had enhanced INa((amil)) although these currents were also reduced when co-injected with xShroom1 antisense. Addition of low concentration (20 ng/ml) of trypsin which activates the membrane-resident ENaC channels led to a slow increase in INa((amil)) in oocytes with xShroom1 sense but had no effect on the currents in oocytes coinjected with ENaC and xShroom1 antisense. The same results were obtained with higher concentrations of trypsin (2 μg/ml) exposed during 2.5 min. In addition, fluorescence positive staining of plasma membrane in the oocytes expressing α, β and γ mENaC and xShroom1 sense were observed but not in oocytes coinjected with ENaC and xShroom1 antisense oligonucleotides. On this basis, we suggest that xShroom1-dependent ENaC inhibition may be through the number of channels inserted in the membrane.
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Affiliation(s)
- Yanina A Assef
- Laboratorio de Canales Iónicos, Instituto de Investigaciones Médicas Alfredo Lanari, University of Buenos Aires, IDIM- CONICET, Buenos Aires, Argentina
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38
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Pavlov TS, Ilatovskaya DV, Levchenko V, Mattson DL, Roman RJ, Staruschenko A. Effects of cytochrome P-450 metabolites of arachidonic acid on the epithelial sodium channel (ENaC). Am J Physiol Renal Physiol 2011; 301:F672-81. [PMID: 21697242 DOI: 10.1152/ajprenal.00597.2010] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Sodium reabsorption via the epithelial Na(+) channel (ENaC) in the aldosterone-sensitive distal nephron plays a central role in the regulation of body fluid volume. Previous studies have indicated that arachidonic acid (AA) and its metabolite 11,12-EET but not other regioisomers of EETs inhibit ENaC activity in the collecting duct. The goal of this study was to investigate the endogenous metabolism of AA in cultured mpkCCD(c14) principal cells and the effects of these metabolites on ENaC activity. Liquid chromatography/mass spectrometry analysis of the mpkCCD(c14) cells indicated that these cells produce prostaglandins, 8,9-EET, 11,12-EET, 14,15-EET, 5-HETE, 12/8-HETE, and 15-HETE, but not 20-HETE. Single-channel patch-clamp experiments revealed that 8,9-EET, 14,15-EET, and 11,12-EET all decrease ENaC activity. Neither 5-, 12-, nor 15-HETE had any effect on ENaC activity. Diclofenac and ibuprofen, inhibitors of cyclooxygenase, decreased transepithelial Na(+) transport in the mpkCCD(c14) cells. Inhibition of cytochrome P-450 (CYP450) with MS-PPOH activated ENaC-mediated sodium transport when cells were pretreated with AA and diclofenac. Coexpression of CYP2C8, but not CYP4A10, with ENaC in Chinese hamster ovary cells significantly decreased ENaC activity in whole-cell experiments, whereas 11,12-EET mimicked this effect. Thus both endogenously formed EETs and their exogenous application decrease ENaC activity. Downregulation of ENaC activity by overexpression of CYP2C8 was PKA dependent and was prevented by myristoylated PKI treatment. Biotinylation experiments and single-channel analysis revealed that long-term treatment with 11,12-EET and overexpression of CYP2C8 decreased the number of channels in the membrane. In contrast, the acute inhibitory effects are mediated by a decrease in the open probability of the ENaC. We conclude that 11,12-EET, 8,9-EET, and 14,15-EET are endogenously formed eicosanoids that modulate ENaC activity in the collecting duct.
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Affiliation(s)
- Tengis S Pavlov
- Dept. of Physiology, Medical College of Wisconsin, Milwaukee, 53226, USA
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Ilatovskaya DV, Pavlov TS, Levchenko V, Negulyaev YA, Staruschenko A. Cortical actin binding protein cortactin mediates ENaC activity via Arp2/3 complex. FASEB J 2011; 25:2688-99. [PMID: 21536685 DOI: 10.1096/fj.10-167262] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Epithelial Na(+) channel (ENaC) activity is regulated, in part, by the cortical cytoskeleton. Here we demonstrate that cortactin is highly expressed in the kidney cortex and polarized epithelial cells, and is localized to the cortical collecting duct. Coexpression of cortactin with ENaC decreases ENaC activity, as measured in patch-clamp experiments. Biotinylation experiments and single-channel analysis reveal that cortactin decreases ENaC activity via affecting channel open probability (P(o)). Knockdown of cortactin in mpkCCD(c14) principal cells results in an increase in ENaC activity and sodium reabsorption. Coimmunoprecipitation analysis shows direct interactions between cortactin and all three ENaC subunits in cultured and native cells. To address the question of what mechanism underlies the action of cortactin on ENaC activity, we assayed the effects of various mutants of cortactin. The data show that only a cortactin mutant unable to bind Arp2/3 complex does not influence ENaC activity. Furthermore, inhibitor of the Arp2/3 complex CK-0944666 precludes the effect of cortactin. Depolymerization of the actin microfilaments and inhibition of the Arp2/3 complex does not result in the loss of association between ENaC and cortactin. Thus, these results indicate that cortactin is functionally important for ENaC activity and that Arp2/3 complex is involved in this mechanism.
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Affiliation(s)
- Daria V Ilatovskaya
- Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226, USA
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40
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Karpushev AV, Levchenko V, Ilatovskaya DV, Pavlov TS, Staruschenko A. Novel role of Rac1/WAVE signaling mechanism in regulation of the epithelial Na+ channel. Hypertension 2011; 57:996-1002. [PMID: 21464391 DOI: 10.1161/hypertensionaha.110.157784] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
The epithelial Na(+) channel (ENaC) is an essential channel responsible for Na(+) reabsorption in the aldosterone-sensitive distal nephron. Consequently, ENaC is a major effector impacting systemic blood volume and pressure. We have shown recently that Rac1 increases ENaC activity, whereas Cdc42 fails to change channel activity. Here we tested whether Rac1 signaling plays a physiological role in modulating ENaC in native tissue and polarized epithelial cells. We found that Rac1 inhibitor NSC23766 markedly decreased ENaC activity in freshly isolated collecting ducts. Knockdown of Rac1 in native principal cells decreased ENaC-mediated sodium reabsorption and the number of channels at the apical plasma membrane. Members of the Wiskott-Aldrich syndrome protein (WASP) family play a central role in the control of the actin cytoskeleton. N-WASP functions downstream of Cdc42, whereas WAVEs are effectors of Rac1 activity. N-WASP and all 3 isoforms of WAVE significantly increased ENaC activity when coexpressed in Chinese hamster ovary cells. However, wiskostatin, an inhibitor of N-WASP, had no effect on ENaC activity. Immunoblotting demonstrated the presence of WAVE1 and WAVE2 and absence of N-WASP and WAVE3 in mpkCCD(c14) and M-1 principal cells. Immunohistochemistry analysis also revealed localization of WAVE1 and WAVE2 but not N-WASP in the cortical collecting duct of Sprague-Dawley rat kidneys. Moreover, patch clamp analysis revealed that Rac1 and WAVE1/2 are parts of the same signaling pathway with respect to activation of ENaC. Thus, our findings suggest that Rac1 is essential for ENaC activity and regulates the channel via WAVE proteins.
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Affiliation(s)
- Alexey V Karpushev
- Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226, USA
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41
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Karpushev AV, Ilatovskaya DV, Staruschenko A. The actin cytoskeleton and small G protein RhoA are not involved in flow-dependent activation of ENaC. BMC Res Notes 2010; 3:210. [PMID: 20663206 PMCID: PMC2918634 DOI: 10.1186/1756-0500-3-210] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2010] [Accepted: 07/27/2010] [Indexed: 11/13/2022] Open
Abstract
Background Epithelial cells are exposed to a variety of mechanical stimuli. Epithelial Na+ channels (ENaC) mediate sodium transport across apical membranes of epithelial cells that line the distal nephron, airway and alveoli, and distal colon. Early investigations into stretch sensitivity of ENaC were controversial. However, recent studies are supportive of ENaC's mechanosensitivity. This work studied whether flow-dependent activation of ENaC is modulated by changes in the state of the actin cytoskeleton and whether small GTPase RhoA is involved in flow-mediated increase of ENaC activity. Findings Pretreatment with Cytochalasin D and Latrunculin B for 20 min and 1-2 hrs to disassemble F-actin had no effect on flow-mediated increase of amiloride-sensitive current. Overexpression of ENaC with constitutively active (G14V) or dominant negative (T19N) RhoA similarly had no effect on flow-dependent activation of ENaC activity. In addition, we did not observe changes when we inhibited Rho-kinase with Y27632. Conclusions Our results suggest that the flow-dependent activation of ENaC is not influenced by small GTPase RhoA and modifications in the actin cytoskeleton.
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Affiliation(s)
- Alexey V Karpushev
- Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd,, Milwaukee, WI 53226, USA.
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