Hyposmotic activation of ICl,swell in rabbit nonpigmented ciliary epithelial cells involves increased ClC-3 trafficking to the plasma membrane

2004 ◽  
Vol 82 (6) ◽  
pp. 708-718 ◽  
Author(s):  
John P Vessey ◽  
Chanjuan Shi ◽  
Christine AB Jollimore ◽  
Kelly T Stevens ◽  
Miguel Coca-Prados ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Kinase Inhibitor ◽  
Regulatory Volume Decrease ◽  
Membrane Dynamics ◽  
Cell Swelling ◽  
Cl Channels ◽  
Phosphoinositide 3 Kinase ◽  
Nonpigmented Ciliary Epithelial ◽  
Volume Decrease

In mammalian nonpigmented ciliary epithelial (NPE) cells, hyposmotic stimulation leading to cell swelling activates an outwardly rectifying Cl– conductance (ICl,swell), which, in turn, results in regulatory volume decrease. The aim of this study was to determine whether increased trafficking of intracellular ClC-3 Cl channels to the plasma membrane could contribute to the ICl,swell following hyposmotic stimulation. Our results demonstrate that hyposmotic stimulation reversibly activates an outwardly rectifying Cl– current that is inhibited by phorbol-12-dibutyrate and niflumic acid. Transfection with ClC-3 antisense, but not sense, oligonucleotides reduced ClC-3 expression as well as ICl,swell. Intracellular dialysis with 2 different ClC-3 antibodies abolished activation of ICl,swell. Immunofluorescence microscopy showed that hyposmotic stimulation increased ClC-3 immunoreactivity at the plasma membrane. To determine whether this increased expression of ClC-3 at the plasma membrane could be due to increased vesicular trafficking, we examined membrane dynamics with the fluorescent membrane dye FM1-43. Hyposmotic stimulation rapidly increased the rate of exocytosis, which, along with ICl,swell, was inhibited by the phosphoinositide-3-kinase inhibitor wortmannin and the microtubule disrupting agent, nocodazole. These findings suggest that ClC-3 channels contribute to ICl,swell following hyposmotic stimulation through increased trafficking of channels to the plasma membrane.Key words: ClC-3, NPE, cell swelling, membrane trafficking, ciliary body epithelium.

scholarly journals Inhibition of autophagic proteolysis by inhibitors of phosphoinositide 3-kinase can interfere with the regulation of glycogen synthesis in isolated hepatocytes

2002 ◽  
Vol 368 (3) ◽  
pp. 827-833 ◽  
Author(s):  
Peter F. DUBBELHUIS ◽  
Daphne A. VAN SLUIJTERS ◽  
Edward F.C. BLOMMAART ◽  
Lori A. GUSTAFSON ◽  
George M. VAN WOERKOM ◽  
...  
Keyword(s):  
Amino Acids ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Proteinase Inhibitors ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Regulatory Volume Decrease ◽  
Isolated Hepatocytes ◽  
Cell Swelling ◽  
Phosphoinositide 3 Kinase

Amino acid-induced cell swelling stimulates conversion of glucose into glycogen in isolated hepatocytes. Activation of glycogen synthase (GS) phosphatase, caused by the fall in intracellular chloride accompanying regulatory volume decrease, and activation of phosphoinositide 3-kinase (PI 3-kinase), induced by cell swelling, have been proposed as underlying mechanisms. Because PI 3-kinase controls autophagic proteolysis, we examined the possibility that PI 3-kinase inhibitors interfere with glycogen production due to their anti-proteolytic action. The PI 3-kinase inhibitor wortmannin inhibited endogenous proteolysis, the production of glycogen from glucose and the activity of active (dephosphorylated) GS (GSa) in the absence of added amino acids. The stimulation by amino acids of glycogen production and of GSa was only slightly affected by wortmannin. These effects of wortmannin could be mimicked by proteinase inhibitors. A combination of leucine, phenylalanine and tyrosine, which we showed previously to stimulate PI 3-kinase-dependent phosphorylation of ribosomal protein S6, did not stimulate glycogen production from glucose. In contrast with wortmannin, LY294002, another PI 3-kinase inhibitor, strongly inhibited both glycogen synthesis and GSa activity, irrespective of the presence of amino acids. Inhibition of glycogen synthesis by LY294002 could be ascribed in part to increased glycogenolysis and glycolysis. It is concluded that, in hepatocytes, activation of PI 3-kinase may not be responsible for the stimulation of glycogen synthesis by amino acids; LY294002 inhibits glycogen synthesis and stimulates glycogen breakdown by a mechanism that is unrelated to its action as an inhibitor of PI 3-kinase.

scholarly journals The septin cytoskeleton facilitates membrane retraction during motility and blebbing

2012 ◽  
Vol 196 (1) ◽  
pp. 103-114 ◽  
Author(s):  
Julia K. Gilden ◽  
Sebastian Peck ◽  
Yi-Chun M. Chen ◽  
Matthew F. Krummel
Keyword(s):  
Plasma Membrane ◽  
Dendritic Spines ◽  
Membrane Trafficking ◽  
Shape Change ◽  
Critical Role ◽  
Regulatory Volume Decrease ◽  
Physiological Processes ◽  
Amoeboid Motility ◽  
Dynamic Shape ◽  
Volume Decrease

Increasing evidence supports a critical role for the septin cytoskeleton at the plasma membrane during physiological processes including motility, formation of dendritic spines or cilia, and phagocytosis. We sought to determine how septins regulate the plasma membrane, focusing on this cytoskeletal element’s role during effective amoeboid motility. Surprisingly, septins play a reactive rather than proactive role, as demonstrated during the response to increasing hydrostatic pressure and subsequent regulatory volume decrease. In these settings, septins were required for rapid cortical contraction, and SEPT6-GFP was recruited into filaments and circular patches during global cortical contraction and also specifically during actin filament depletion. Recruitment of septins was also evident during excessive blebbing initiated by blocking membrane trafficking with a dynamin inhibitor, providing further evidence that septins are recruited to facilitate retraction of membranes during dynamic shape change. This function of septins in assembling on an unstable cortex and retracting aberrantly protruding membranes explains the excessive blebbing and protrusion observed in septin-deficient T cells.

Cell volume regulation by the mouse zygote: mechanism of recovery from a volume increase

1997 ◽  
Vol 272 (6) ◽  
pp. C1854-C1861 ◽  
Author(s):  
D. G. Seguin ◽  
J. M. Baltz
Keyword(s):  
Regulatory Volume Decrease ◽  
Cell Volume Regulation ◽  
Cell Swelling ◽  
Channel Blockers ◽  
Volume Increase ◽  
K Channels ◽  
Cl Channel ◽  
Cl Channels ◽  
Mouse Zygotes ◽  
Volume Decrease

Mouse zygotes regulate their volumes after cell swelling. This regulatory volume decrease (RVD) is rapid and complete. RVD in zygotes was inhibited by K+ or Cl- channel blockers, indicating the participation of such channels in volume recovery. The channels are separate entities, as indicated by the ability of the cation ionophore gramicidin to restore RVD when K+ channels are blocked but not when Cl- channels are blocked. Intracellular Ca2+ concentration increased with cell swelling. Nevertheless, RVD occurred normally in zygotes loaded with the Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, which prevented Ca2+ from increasing above its normal resting concentration. Thus an increase in intracellular Ca2+ is not necessary for zygote RVD; consistent with this, inhibitors of Ca(2+)-activated K+ channels had little or no effect on RVD. RVD in zygotes was also completely inhibited by millimolar amounts of extracellular ATP. ATP has been shown to inhibit current passed by the volume-sensitive organic osmolyte-Cl- channel in other cells, and thus zygotes may have such a channel participating in RVD.

Ca(2+)-mobilizing hormones potentiate hypotonicity-induced activation of ionic conductances in Intestine 407 cells

1994 ◽  
Vol 267 (5) ◽  
pp. C1271-C1278 ◽  
Author(s):  
B. C. Tilly ◽  
M. J. Edixhoven ◽  
N. van den Berghe ◽  
A. G. Bot ◽  
H. R. de Jonge
Keyword(s):  
Tyrosine Phosphorylation ◽  
Regulatory Volume Decrease ◽  
Cell Types ◽  
Cell Swelling ◽  
Protein Tyrosine Phosphorylation ◽  
Acetoxymethyl Ester ◽  
Protein Tyrosine ◽  
Ionic Conductances ◽  
Cl Channels ◽  
Volume Decrease

Human Intestine 407 cells respond to hyposmotic stimulation by activating the conductive efflux of both Cl- and K+ (regulatory volume decrease) through pathways involving protein tyrosine phosphorylation (Tilly, B. C., N. van den Berghe, L. G. J. Tertoolen, M. J. Edixhoven, and H. R. de Jonge. J. Biol. Chem. 268: 19919-19922, 1993). Stimulation of the cells with hormones linked to the phospholipase C signaling cascade (e.g., bradykinin, histamine, or thrombin) or with the phosphotyrosine phosphatase inhibitor vanadate, potentiated the osmosensitive anion efflux by two- to threefold but did not affect anion efflux under isotonic conditions. No substantial increase in intracellular Ca2+ concentration ([Ca2+]i) was observed on mild hypotonicity-induced cell swelling. In addition, loading the cells with the intracellular Ca2+ chelator 1,2-bis(2-amino-phenoxy)ethane- N,N,N',N',-tetraacetic acid acetoxymethyl ester (BAPTA-AM) caused a partial reduction of the osmoshock-induced 125I- efflux but did not affect its potentiation by vanadate. In contrast, bradykinin transiently elevated [Ca2+]i, and its potentiation of the osmosensitive anion efflux was completely inhibited after BAPTA-AM loading. Both the Ca(2+)-mobilizing hormones as well as osmotic cell swelling rapidly triggered the phosphorylation of several proteins on tyrosine residues. However, the effects of the hormones, but not the effect of hypotonicity, on protein tyrosine phosphorylation was largely abolished in BAPTA-loaded cells. Taken together the results indicate a novel role for Ca(2+)-mobilizing hormones, although elevation of [Ca2+]i, in potentiating volume-sensitive ionic efflux even in cell types lacking the expression of Ca(2+)-activated Cl- channels in their plasma membrane.

scholarly journals HIV-1 requires Arf6-mediated membrane dynamics to efficiently enter and infect T lymphocytes

2011 ◽  
Vol 22 (8) ◽  
pp. 1148-1166 ◽  
Author(s):  
Laura García-Expósito ◽  
Jonathan Barroso-González ◽  
Isabel Puigdomènech ◽  
José-David Machado ◽  
Julià Blanco ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
T Lymphocytes ◽  
Viral Entry ◽  
Membrane Trafficking ◽  
Membrane Dynamics ◽  
Viral Fusion ◽  
Viral Attachment ◽  
Immunodeficiency Virus ◽  
Vesicular Stomatitis ◽  
Hiv 1

As the initial barrier to viral entry, the plasma membrane along with the membrane trafficking machinery and cytoskeleton are of fundamental importance in the viral cycle. However, little is known about the contribution of plasma membrane dynamics during early human immunodeficiency virus type 1 (HIV-1) infection. Considering that ADP ribosylation factor 6 (Arf6) regulates cellular invasion via several microorganisms by coordinating membrane trafficking, our aim was to study the function of Arf6-mediated membrane dynamics on HIV-1 entry and infection of T lymphocytes. We observed that an alteration of the Arf6–guanosine 5′-diphosphate/guanosine 5′-triphosphate (GTP/GDP) cycle, by GDP-bound or GTP-bound inactive mutants or by specific Arf6 silencing, inhibited HIV-1 envelope–induced membrane fusion, entry, and infection of T lymphocytes and permissive cells, regardless of viral tropism. Furthermore, cell-to-cell HIV-1 transmission of primary human CD4+T lymphocytes was inhibited by Arf6 knockdown. Total internal reflection fluorescence microscopy showed that Arf6 mutants provoked the accumulation of phosphatidylinositol-(4,5)-biphosphate–associated structures on the plasma membrane of permissive cells, without affecting CD4-viral attachment but impeding CD4-dependent HIV-1 entry. Arf6 silencing or its mutants did not affect fusion, entry, and infection of vesicular stomatitis virus G–pseudotyped viruses or ligand-induced CXCR4 or CCR5 endocytosis, both clathrin-dependent processes. Therefore we propose that efficient early HIV-1 infection of CD4+T lymphocytes requires Arf6-coordinated plasma membrane dynamics that promote viral fusion and entry.

Resistance to osmotic lysis in BXD-31 mouse erythrocytes: association with upregulated K-Cl cotransport

1996 ◽  
Vol 270 (3) ◽  
pp. C866-C877 ◽  
Author(s):  
C. C. Armsby ◽  
A. K. Stuart-Tilley ◽  
S. L. Alper ◽  
C. Brugnara
Keyword(s):  
Normal Cell ◽  
Genetic Locus ◽  
Regulatory Volume Decrease ◽  
Osmotic Fragility ◽  
Cell Swelling ◽  
K Channel ◽  
Cell Dehydration ◽  
Osmotic Lysis ◽  
Volume Decrease ◽  
Characteristic Resistance

The decreased osmotic fragility and reduced K+ content of BXD-31 mouse erythrocytes arise from variation at a single genetic locus. We compared ion transport in erythrocytes from BXD-31 mice and the parental strain, DBA/2J. The strains had similar rates for Na-K pump, Na/H exchange, Na-K-2Cl cotransport, Ca2+ activated K+ channel, or AE1-mediated SO4 transport. In contrast, K-Cl cotransport was twice as active in BXD-31 as in DBA/2J cells. Cl- dependent K+ efflux from BXD-31 cells displayed steep activation by acid pH (with maximal transport occurring at pH 6.75), whereas DBA/2J erythrocytes displayed a far less dramatic response to pH. Both strains displayed regulatory volume decrease in response to cell swelling. However, a 62% greater loss of cell K+ via K-Cl cotransport was observed in the BXD-31 strain. Furthermore the decreased osmotic fragility of BXD-31 red blood cells was normalized by treatment with nystatin to achieve normal cell K+ and water content. Thus upregulated K-Cl cotransport induces cell dehydration and K+ deficit in BXD-31 erythrocytes and causes their characteristic resistance to osmotic lysis.

Physiological significance of hypotonicity-induced regulatory volume decrease: reduction in intracellular Cl− concentration acting as an intracellular signaling

2007 ◽  
Vol 292 (5) ◽  
pp. F1411-F1417 ◽  
Author(s):  
Hiroaki Miyazaki ◽  
Atsushi Shiozaki ◽  
Naomi Niisato ◽  
Yoshinori Marunaka
Keyword(s):  
Intracellular Signaling ◽  
Channel Blocker ◽  
Fluorescent Dyes ◽  
Regulatory Volume Decrease ◽  
Cell Swelling ◽  
Dependent Manner ◽  
Stimulatory Action ◽  
A6 Cells ◽  
Intracellular Signal ◽  
Volume Decrease

Regulatory volume decrease (RVD) occurs after hypotonicity-caused cell swelling. RVD is caused by activation of ion channels and transporters, which cause effluxes of K+, Cl−, and H2O, leading to cell shrinkage. Recently, we showed that hypotonicity stimulated transepithelial Na+ reabsorption via elevation of epithelial Na+ channel (α-ENaC) expression in renal epithelia A6 cells in an RVD-dependent manner and that reduction of intracellular Cl− concentration ([Cl−]i) stimulated the Na+ reabsorption. These suggest that RVD would reveal its stimulatory action on the Na+ reabsorption by reducing [Cl−]i. However, the reduction of [Cl−]i during RVD has not been definitely analyzed due to technical difficulties involved in halide-sensitive fluorescent dyes. In the present study, we developed a new method for the measurement of [Cl−]i change during RVD by using a high-resolution flow cytometer with a halide-specific fluorescent dye, N-(6-methoxyquinolyl) acetoethyl ester. The [Cl−]i in A6 cells in an isotonic medium was 43.6 ± 3.1 mM. After hypotonic shock (268 to 134 mosmol/kgH2O), a rapid increase of cell volume followed by RVD occurred. The RVD caused drastic diminution of [Cl−]i from 43.6 to 10.8 mM. Under an RVD-blocked condition with NPPB (Cl− channel blocker) or quinine (K+ channel blocker), we did not detect the reduction of [Cl−]i. Based on these observations, we conclude that one of the physiological significances of RVD is the reduction of [Cl−]i and that RVD shows its action via reduction of [Cl−]i acting as an intracellular signal regulating cellular physiological functions.

Activation of the TASK-2 channel after cell swelling is dependent on tyrosine phosphorylation

2010 ◽  
Vol 299 (4) ◽  
pp. C844-C853 ◽  
Author(s):  
Signe Skyum Kirkegaard ◽  
Ian Henry Lambert ◽  
Steen Gammeltoft ◽  
Else Kay Hoffmann
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Tyrosine Phosphatase ◽  
Regulatory Volume Decrease ◽  
Janus Kinase ◽  
Cell Swelling ◽  
Hek 293 ◽  
Set Point ◽  
Phosphatase Inhibitor

The swelling-activated K+ currents ( IK,vol) in Ehrlich ascites tumor cells (EATC) has been reported to be through the two-pore domain (K2p), TWIK-related acid-sensitive K+ channel 2 (TASK-2). The regulatory volume decrease (RVD), following hypotonic exposure in EATC, is rate limited by IK,vol indicating that inhibition of RVD reflects inhibition of TASK-2. We find that in EATC the tyrosine kinase inhibitor genistein inhibits RVD by 90%, and that the tyrosine phosphatase inhibitor monoperoxo(picolinato)-oxo-vanadate(V) [mpV(pic)] shifted the volume set point for inactivation of the channel to a lower cell volume. Swelling-activated K+ efflux was impaired by genistein and the Src kinase family inhibitor 4-amino-5-(4-chloro-phenyl)-7-( t-butyl)pyrazolo[3,4- d]pyrimidine (PP2) and enhanced by the tyrosine phosphatase inhibitor mpV(pic). With the use of the TASK-2 inhibitor clofilium, it is demonstrated that mpV(pic) increased the volume-sensitive part of the K+ efflux 1.3 times. To exclude K+ efflux via a KCl cotransporter, cellular Cl− was substituted with NO3−. Also under these conditions K+ efflux was completely blocked by genistein. Thus tyrosine kinases seem to be involved in the activation of the volume-sensitive K+ channel, whereas tyrosine phosphatases appears to be involved in inactivation of the channel. Overexpressing TASK-2 in human embryonic kidney (HEK)-293 cells increased the RVD rate and reduced the volume set point. TASK-2 has tyrosine sites, and precipitation of TASK-2 together with Western blotting and antibodies against phosphotyrosines revealed a cell swelling-induced, time-dependent tyrosine phosphorylation of the channel. Even though we found an inhibiting effect of PP2 on RVD, neither Src nor the focal adhesion kinase (FAK) seem to be involved. Inhibitors of the epidermal growth factor receptor tyrosine kinases had no effect on RVD, whereas the Janus kinase (JAK) inhibitor cucurbitacin inhibited the RVD by 40%. It is suggested that the cytokine receptor-coupled JAK/STAT pathway is upstream of the swelling-induced phosphorylation and activation of TASK-2 in EATC.

Regulation of cell volume and intracellular pH in hyposmotically swollen rat osteosarcoma cells

1995 ◽  
Vol 73 (7-8) ◽  
pp. 535-544 ◽  
Author(s):  
C. Lo ◽  
J. Ferrier ◽  
H. C. Tenenbaum ◽  
C. A. G. McCulloch
Keyword(s):  
Cell Volume ◽  
Intracellular Ph ◽  
Binding Proteins ◽  
Kinase Inhibitor ◽  
Regulatory Volume Decrease ◽  
Cell Swelling ◽  
Osteosarcoma Cells ◽  
Extracellular Medium ◽  
Gtp Binding Proteins ◽  
Gtp Binding

The maintenance of cell volume involves transduction of a volume-sensing signal into effectors of volume-regulatory transporters. After exposure to anisotonic conditions, cells undergo compensatory volume changes that are mediated by active transport and passive movement of ions and solutes. Intracellular pH (pHi) homeostasis may be compromised during these processes. We have studied pHi and some of the signal transduction mechanisms involved in the regulatory volume decrease (RVD) that occurs after exposure to hypoosmolar conditions in rat osteosarcoma cells, ROS 17/2.8. Cells were loaded with BCECF; pHi and cell volume were estimated by dual excitation ratio fluorimetry. Swelling of cells in 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES) buffered hypotonic medium induced a rapid cell swelling followed by an incomplete RVD of ~30% in suspended (i.e., round) cells and ~60% in attached (i.e., spread) cells that was independent of subpassage number. RVD was inhibited by ouabain, valinomycin, and high external [K+], all of which should reduce the cell membrane electrochemical gradient for K+. Inhibition of RVD was induced also by decreasing intracellular [Ca2+] with B APTA–AM and by depletion of Cl−, indicating the role of calcium-regulated K+ and Cl− efflux during RVD. Depolymerization of actin filaments by cytochalasin D prolonged the RVD three-fold and nonspecific activation of GTP-binding proteins up-regulated RVD. In attached cells the hypoosmolar-induced swelling caused a large reduction in pHi (~0.7 units), which was sustained as long as cells were in hypoosmotic medium. The reduction of pHi induced by cell swelling was inhibited by Na+-free extracellular medium, ouabain, the tyrosine kinase inhibitor genistein, and to a lesser extent by Cl−-free medium. However, amiloride failed to inhibit the hypoosmolar-induced reduction of pHi. Collectively these data indicate that RVD of ROS 17/2.8 cells in HEPES-buffered medium is dependent on conductive efflux of K+ and Cl− that is regulated by cell shape, actin, and GTP-binding proteins. The sustained inhibition of pHi homeostasis induced by cell swelling may reflect the existence of cell volume sensing mechanisms that operate through tyrosine kinases to regulate pHi.Key words: cell volume, pH, osteoblast, G proteins, actin.

scholarly journals Phosphatidylinositol(4,5)bisphosphate: diverse functions at the plasma membrane

2020 ◽  
Vol 64 (3) ◽  
pp. 513-531 ◽  
Author(s):  
Matilda Katan ◽  
Shamshad Cockcroft
Keyword(s):  
Plasma Membrane ◽  
Ion Channel ◽  
Tyrosine Kinases ◽  
Cell Biology ◽  
Second Messengers ◽  
Membrane Dynamics ◽  
Actin Dynamics ◽  
Ion Channel Regulation ◽  
Channel Regulation ◽  
Phosphoinositide 3 Kinase

Abstract Phosphatidylinositol(4,5) bisphosphate (PI(4,5)P2) has become a major focus in biochemistry, cell biology and physiology owing to its diverse functions at the plasma membrane. As a result, the functions of PI(4,5)P2 can be explored in two separate and distinct roles – as a substrate for phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K) and as a primary messenger, each having unique properties. Thus PI(4,5)P2 makes contributions in both signal transduction and cellular processes including actin cytoskeleton dynamics, membrane dynamics and ion channel regulation. Signalling through plasma membrane G-protein coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and immune receptors all use PI(4,5)P2 as a substrate to make second messengers. Activation of PI3K generates PI(3,4,5)P3 (phosphatidylinositol(3,4,5)trisphosphate), a lipid that recruits a plethora of proteins with pleckstrin homology (PH) domains to the plasma membrane to regulate multiple aspects of cellular function. In contrast, PLC activation results in the hydrolysis of PI(4,5)P2 to generate the second messengers, diacylglycerol (DAG), an activator of protein kinase C and inositol(1,4,5)trisphosphate (IP3/I(1,4,5)P3) which facilitates an increase in intracellular Ca2+. Decreases in PI(4,5)P2 by PLC also impact on functions that are dependent on the intact lipid and therefore endocytosis, actin dynamics and ion channel regulation are subject to control. Spatial organisation of PI(4,5)P2 in nanodomains at the membrane allows for these multiple processes to occur concurrently.

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