Mechanisms and significance of cell volume regulation

Mechanisms and Significance of Cell Volume Regulation Contributions to Nephrology Vol. 152 Series Editor Claudio Ro...

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Mechanisms and Significance of Cell Volume Regulation

Contributions to Nephrology Vol. 152

Series Editor

Claudio Ronco

Vicenza

Mechanisms and Significance of Cell Volume Regulation Volume Editor

Florian Lang

Tübingen

37 figures, 7 in color, and 4 tables, 2006

Basel · Freiburg · Paris · London · New York · Bangalore · Bangkok · Singapore · Tokyo · Sydney

Contributions to Nephrology (Founded 1975 by Geoffrey M. Berlyne)

Prof. Dr. Florian Lang Physiologisches Institut der Universität Tübingen Gmelinstrasse 5 DE–72076 Tübingen (Germany)

Library of Congress Cataloging-in-Publication Data Mechanisms and significance of cell volume regulation / volume editor, Florian Lang. p. ; cm. – (Contributions to nephrology, ISSN 0302-5144 ; v. 152) Includes bibliographical references and indexes. ISBN-13: 978-3-8055-8174-5 (hard cover : alk. paper) ISBN-10: 3-8055-8174-2 (hard cover : alk. paper) 1. Osmoregulation. 2. Cellular control mechanisms. 3. Homeostasis. I. Lang, Florian. II. Series. [DNLM: 1. Cell Size. 2. Ion Channels–physiology. 3. Ion Transport –physiology. 4. Signal Transduction–physiology. W1 CO778UN v.152 2006 / QU 375 M486 2006] QP90.6.M39 2006 572⬘.3–dc22 2006023031

Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents® and Index Medicus. Disclaimer. The statements, options and data contained in this publication are solely those of the individual authors and contributors and not of the publisher and the editor(s). The appearance of advertisements in the book is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements. Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. © Copyright 2006 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) www.karger.com Printed in Switzerland on acid-free paper by Reinhardt Druck, Basel ISSN 0302–5144 ISBN-10: 3–8055–8174–2 ISBN-13: 978–3–8055–8174–5

Contents

1 Cell Volume Regulatory Mechanisms An Introduction Friedrich, B. (Tübingen); Matskevich, I. (Edinburg); Lang, F. (Tübingen) 9 Cell Volume-Sensitive Chloride Channels: Phenotypic Properties and Molecular Identity Okada, Y. (Okazaki) 25 Cell Volume-Regulated Cation Channels Wehner, F. (Dortmund) 54 Sensors and Signal Transduction Pathways in Vertebrate Cell Volume Regulation Hoffmann, E.K.; Pedersen, S.F. (Copenhagen) 105 Activation of Kinases upon Volume Changes: Role in Cellular Homeostasis Alexander, R.T.; Grinstein, S. (Toronto) 125 Tonicity-Dependent Regulation of Osmoprotective Genes in Mammalian Cells Ferraris, J.D.; Burg, M.B. (Bethesda, Md.) 142 Ion Channels and Cell Volume in Regulation of Cell Proliferation and Apoptotic Cell Death Lang, F.; Shumilina, E. (Tübingen); Ritter, M. (Salzburg); Gulbins, E. (Essen).; Vereninov, A. (St. Petersburg); Huber, S.M. (Tübingen)

V

161 Cell Volume Regulatory Ion Transport in the Regulation of Cell Migration Jakab, M.; Ritter, M. (Salzburg) 181 Cell Volume Regulation in the Renal Papilla Beck, F.-X.; Neuhofer, W. (Munich) 198 Osmosensing and Signaling in the Regulation of Liver Function Schliess, F.; Häussinger, D. (Düsseldorf) 210 Cell Volume and Peptide Hormone Secretion Štrbák, V. (Bratislava) 221 Volume Changes in Neurons: Hyperexcitability and Neuronal Death Pasantes-Morales, H.; Tuz, K. (Mexico) 241 Pathophysiology of Red Cell Volume Browning, J.A.; Ellory, J.C. (Oxford); Gibson, J.S. (Cambridge) 269 Author Index 270 Subject Index

Contents

VI

Lang F (ed): Mechanisms and Significance of Cell Volume Regulation. Contrib Nephrol. Basel, Karger, 2006, vol 152, pp 1–8

Cell Volume Regulatory Mechanisms An Introduction

B. Friedricha, I. Matskevichb, F. Langc Departments of aMedicine, and cPhysiology, University of Tübingen, Tübingen, Germany; bCollege of Science and Engineering, The University of Edinburgh, Edinburgh, UK

Abstract Prerequisites for cell survival include avoidance of excessive alterations of cell volume. Cells counterbalance the osmolarity due to cellular accumulation of organic substances by uneven distribution of inorganic ions. They extrude Na in exchange for K by the Na/K ATPase. The cell membrane is less permeable to Na than to K. The K exit generates a cell-negative potential difference across the cell membrane which drives the exit of anions such as Cl thus decreasing intracellular osmolarity. Upon cell swelling, cells release ions through activation of K channels and/or anion channels, KCl-cotransport, or parallel activation of K/H exchange and Cl/HCO3 exchange. Upon cell shrinkage, cells accumulate ions through activation of Na, K, 2Cl cotransport, Na/H exchange in parallel to Cl/HCO3 exchange, or Na channels. Na taken up is extruded by the Na/K ATPase in exchange for K. Shrunken cells further accumulate organic osmolytes. They generate sorbitol and glycerophosphorylcholine and monomeric amino acids by altered metabolism and take up myoinositol (inositol), betaine, taurine and amino acids by Na coupled transport. They release osmolytes during cell swelling. Copyright © 2006 S. Karger AG, Basel

To survive, cells have to maintain their volume within certain limits. Profound alterations of cell volume will interfere with the integrity of the cell membrane and of the cytoskeletal architecture. Moreover, cellular function depends on the hydration of cytosolic proteins. Proteins and protein-bound water occupy a large portion of the cellular space (macromolecular crowding) leaving only a small fraction of cellular volume for free water. Loss or gain of cellular water in the range of only a few percent of cell volume has thus profound effects on protein and cell function. Water can freely move across the cell membrane which is, with only few exceptions, highly permeable to water. Driving forces for movement of water

include hydrostatic and osmotic pressure gradients. The impact of hydrostatic pressure is small as mammalian cell membranes do not withstand significant hydrostatic pressure gradients. Thus, water movement across cell membranes is governed by osmotic gradients and in order to avoid swelling or shrinkage, a cell has to achieve osmotic equilibrium across the cell membrane. At intracellular osmolarities exceeding extracellular osmolarity, water enters following its osmotic gradient and the cell swells. At extracellular osmolarities exceeding intracellular osmolarity water exits and the cell shrinks. A wide array of factors modify intra- or extracellular osmolarity and thus challenge the osmotic equilibrium across the cell membrane and thus cell volume. Cells utilize a variety of mechanisms to maintain cell volume constancy, including altered transport across the cell membrane and metabolism. Those mechanisms are under the control of hormones and mediators. Their influence on cell volume participates in the regulation of cellular function. Following cell swelling, volume regulatory mechanisms concert to decrease intracellular osmolarity and cell volume thus accomplishing regulatory cell volume decrease (RVD), upon cell shrinkage mirror-like mechanisms achieve an increase of intracellular osmolarity and cell volume thus accomplishing regulatory cell volume increase (RVI). The most rapid and efficient means to accomplish cell volume regulation is ion transport across the cell membrane. Following cell swelling ions are released, upon cell shrinkage ions are taken up by the respective transport systems. However, the use of ions in cellular osmoregulation is limited, since high inorganic ion concentrations interfere with the stability of proteins. Beyond that, altered ion gradients across the cell membrane interfere with cell function. For instance, an increase of intracellular Na activity decreases the chemical gradient for Na across the cell membrane, thus reducing the driving force for Ca2 extrusion via the Na/Ca2 exchanger and increasing intracellular Ca2 activity. To avoid excessive alterations of intracellular ion concentration cells thus employ in addition organic osmolytes for osmoregulation. Furthermore, cells modify their metabolism thus generating or disposing osmotically active organic substances. A given cell uses only part of the cell volume regulatory mechanisms described in the following. The large repertoire of cell volume regulatory transporters and osmolytes allows a selection of those, which have the least untoward impact on cell function. The present book is a collection of reviews on various aspects of cell volume regulation, written by the respective internationally leading experts. It updates our knowledge compiled in a similar book published earlier [Lang, 1998]. This brief chapter is a short introduction into the phenomenon of cell volume regulation and the major cell volume regulatory mechanisms. The

Friedrich/Matskevich/Lang

2

following chapters provide a more detailed discussion of the various aspects of cell volume regulation. Moreover, the readers may wish to consult several recent reviews on cell volume regulatory mechanisms [Adragna et al., 2004; Al Habori, 2001; Barvitenko et al., 2005; Gamba, 2005; Jakab et al., 2002; Lambert, 2004; Okada et al., 2001; Pedersen et al., 2001; Sabirov and Okada, 2004; Stein, 2002; Uchida and Sasaki, 2005; Wehner et al., 2003], and its role in cell proliferation and cells death [Bortner, 2005; Bosman et al., 2005; DuBois and Rouzaire-Dubois, 2004; Gomez-Angelats et al., 2000; Lang et al., 2005; Schreiber, 2005] for further information.

Cell Volume Regulatory Ion Transport

Intra- and extracellular osmolarities are in large part generated by inorganic ions. The high ion plasma membrane transport capacity allows rapid alterations of the osmotic gradient across the cell membrane. Thus, ion transport is the most powerful means to regulate cell volume. Ions in the Maintenance of Cell Volume: To maintain their metabolism, cells have to accumulate organic substances, which create an osmotic gradient across the cell membrane. They compensate the excessive cellular accumulation of substrates by uneven distribution of inorganic ions (fig. 1): The cells extrude Na in exchange for K by the Na/K ATPase. The cell membrane is less permeable to Na than to K. Thus, K but not Na is allowed to follow its chemical gradient. The chemical K gradient drives K exit through K channels. The K exit generates a cell-negative potential difference across the cell membrane which then drives the exit of anions such as Cl. Thus, cells maintain a low intracellular Cl concentration depending on the magnitude of the cell negative potential difference across the cell membrane: At a cell membrane potential of –18 mV the intracellular Cl concentration is in equilibrium only half of the extracellular Cl concentration (110 mM), i.e. intracellular Cl is under those conditions 55 mM lower than extracellular Cl concentration. The relatively low intracellular Cl concentration thus allows the cellular accumulation of organic substances. The high extracellular Na concentration is counterbalanced by the high intracellular K concentration. Thus, the cations do not directly contribute to a decrease of cellular osmolarity. The uneven cation concentrations are, however, prerequisites for the maintenance of the cell membrane potential. Inhibition of the Na/K ATPase eventually leads to decrease of intracellular K concentration and increase of intracellular Na concentration, the decrease of intracellular K concentration is followed by depolarization, Cl entry and thus swelling (fig. 1).

Cell Volume Regulatory Mechanisms

3

Na 145 15 Na

A

B

150 K

K 5 PD

10 Cl

C

Cl 110 D

Na A

Na

B K

Cl

PD Cl D

K C

Fig. 1. Ion transport in the maintenance of cell volume (upper panel) and its derangement following energy depletion (lower panel): Upper panel: The Na/K ATPase extrudes Na and accumulates K (A). The cellular K accumulation establishes a chemical gradient for K, which drives K exit through K channels (B). The efflux of positively charged K generates a cell negative potential difference (Vm) across the cell membrane (C) which drives Cl out of the cells (D). Thus, intracellular Cl activity is lower than extracellular Cl activity. Lower panel: Energy depletion compromises the function of the Na/K ATPase (A), and thus leads to dissipation of Na and K gradients, the decreased chemical driving force for K efflux (B) depolarizes the cell membrane (C) which in turn favors entry of Cl (D) and subsequent cell swelling.

Ion Release following Cell Swelling: Following cell swelling, cells need to decrease their osmolarity by stimulating exit of ions. Most cells release ions by activation of K channels and/or anion channels (fig. 2). The exit of KCl requires the activity of both ion channel types, since neither K nor anions can leave the cells without the respective counterion. If one of the channels is already active at the volume regulatory setpoint, cell swelling needs to activate only the other channel. Swelling of some cells activates unspecific cation channels. Since the electrochemical gradient favors entry rather then exit of cations, these channels cannot directly serve cell volume regulation. Instead, the channels allow the entry of Ca2 which in turn activates Ca2 sensitive K channels and/or Cl channels. KCl may exit as well by activation of KCl-cotransport. Some cells release cellular KCl via parallel activation of K/H exchange and Cl/HCO3 exchange. The H and HCO3, thus taken up in exchange for KCl,


4

A

HCO 3

Cl

H

AR G

B

Na Sorbitol

PDE F

Cl K Na

C

GPC Na D Na Cl E Osm

Fig. 2. Mechanisms of RVI: Cell shrinkage leads to parallel activation of Cl/HCO3 exchanger (A) and Na/H exchanger (B), of Na, K, 2Cl cotransport (C); Na channels (D) and Na coupled accumulation of organic osmolytes such as inositol, taurine and betaine (E). Furthermore, cell shrinkage leads to cellular accumulation of GPC through inhibition of its degradation by phosphodiesterase (F) and of sorbitol by activation of aldosereducase (G).

react via H2CO3 to CO2, which can easily exit the cell and is thus not osmotically relevant. Ion Uptake upon Cell Shrinkage: Cell shrinkage may activate the Na, K, 2Cl cotransport allowing cellular accumulation of NaCl and KCl. Cell shrinkage may further trigger Na/H exchange in parallel to Cl/HCO3 exchange. This tandem mediates the uptake of NaCl, since the H and HCO3 lost in exchange for NaCl is replenished in the cell from CO2 via H2CO3 (fig. 3). Na entering via Na, K, 2Cl cotransport or Na/H exchange is extruded by the Na/K ATPase in exchange for K. Thus, the transporters eventually lead to uptake of KCl. Some cells activate Na channels following shrinkage. The resulting depolarization drives Cl into the cell so that the net effect is cellular accumulation of NaCl. Again, the Na taken up is extruded by the Na/K ATPase in exchange for K. Some cells inhibit K and/or Cl channels upon cell shrinkage and thus avoid cellular ion loss.

Organic Osmolytes

In mammalian cells the most important organic osmolytes are polyols such as sorbitol and myoinositol, methylamines such as betaine and glycerophosphorylcholine (GPC), as well as amino acids and the amino acid derivative taurine. In contrast to inorganic ions, organic osmolytes do not destabilize but rather stabilize proteins and thus counteract the destabilizing effects of inorganic ions, some organic ions (spermidine), urea, heat shock, desiccation and presumably


5

Cl

A

K

HCO 3

HCO 3

B

Cl

Cl

K K Ca2 D

Osm

E

C

Fig. 3. Mechanisms of RVD: Cell swelling leads to activation of KCl-cotransport (A), anion channels (B), K channels (C) and channels releasing osmolytes such as sorbitol, inositol, taurine, and betaine (E). Ca2 permeable cation channels (D) do not directly serve cell volume regulation but increase cytosolic Ca2 activity leading to stimulation of Ca2 sensitive K channels.

radiation. The destabilizing effects of urea, for instance, are best counteracted by betaine and GPC and to a lesser extent by myoinositol. Osmolyte Accumulation by Metabolism: In shrunken cells, sorbitol is generated from glucose under the catalytic action of aldose reductase. The transcription of the enzyme is stimulated by osmotic cell shrinkage. The expression of the protein takes many hours and the appropriate increase of sorbitol concentration requires hours to days. GPC is produced from phosphatidylcholine by a phospholipase A2 which is distinct from the arachidonyl selective enzyme. GPC is degraded by a phosphodiesterase to glycerol-phosphate and choline. Cell shrinkage leads to inhibition of the phosphodiesterase and thus leads to cellular accumulation of GPC. Intracellular osmolarity is further generated by autophagic proteolysis, i.e. the degradation of cellular proteins to amino acids. Conversely, cell shrinkage decreases the de novo protein synthesis, thus avoiding the incorporation of single amino acids into the osmotically less effective peptides and proteins. Osmolyte Accumulation by Transport: The organic osmolytes myoinositol (inositol), betaine and taurine are taken up by specific Na coupled transporters.


6

The carriers further favor the cellular accumulation of Cl by depolarization of the cell membrane and in part by transport of Cl. The transporters thus mediate uptake of NaCl in parallel to organic osmolytes. Cell shrinkage stimulates the transcription of the carriers. Since the expression of the transporters is slow, full adaptation requires hours to days. Moreover, the efficiency of the system depends on the availability of osmolytes in extracellular fluid. Similar to the organic osmolytes, some amino acids are accumulated by cell volume sensitive Na coupled transport. For instance, expression and activity of amino acid transport system A is upregulated by cell shrinkage. Osmolyte Release: Cell swelling stimulates the release of organic osmolytes such as GPC, sorbitol, inositol, betaine and taurine. It appears that several osmolyte release mechanisms operate in parallel. Some of the release mechanisms are thought to be anion channels.

References Adragna NC, Fulvio MD, Lauf PK: Regulation of K-Cl cotransport: from function to genes. J Membr Biol 2004;201:109–137. Al Habori M: Macromolecular crowding and its role as intracellular signalling of cell volume regulation. Int J Biochem Cell Biol 2001;33:844–864. Barvitenko NN, Adragna NC, Weber RE: Erythrocyte signal transduction pathways, their oxygenation dependence and functional significance. Cell Physiol Biochem 2005;15:1–18. Bortner CD: Apoptotic volume decrease and nitric oxide. Toxicology 2005;208:213–221. Bosman GJ, Willekens FL, Werre JM: Erythrocyte aging: a more than superficial resemblance to apoptosis? Cell Physiol Biochem 2005;16:1–8. DuBois JM, Rouzaire-Dubois B: The influence of cell volume changes on tumour cell proliferation. Eur Biophys J 2004;33:227–232. Gamba G: Molecular physiology and pathophysiology of electroneutral cation-chloride cotransporters. Physiol Rev 2005;85:423–493. Gomez-Angelats M, Bortner CD, Cidlowski JA: Cell volume regulation in immune cell apoptosis. Cell Tissue Res 2000;301:33–42. Jakab M, Furst J, Gschwentner M, Botta G, Garavaglia ML, Bazzini C, Rodighiero S, Meyer G, Eichmueller S, Woll E, Chwatal S, Ritter M, Paulmichl M: Mechanisms sensing and modulating signals arising from cell swelling. Cell Physiol Biochem 2002;12:235–258. Lambert IH: Regulation of the cellular content of the organic osmolyte taurine in mammalian cells. Neurochem Res 2004;29:27–63. Lang F (ed): Cell Volume Regulation. Contrib Nephrol. Basel, Karger, vol 123. Lang KS, Lang PA, Bauer C, Duranton C, Wieder T, Huber SM, Lang F: Mechanisms of suicidal erythrocyte death. Cell Physiol Biochem 2005;15:195–202. Okada Y, Maeno E, Shimizu T, Dezaki K, Wang J, Morishima S: Receptor-mediated control of regulatory volume decrease (RVD) and apoptotic volume decrease (AVD). J Physiol 2001;532:3–16. Pedersen SF, Hoffmann EK, Mills JW: The cytoskeleton and cell volume regulation. Comp Biochem Physiol A Mol Integr Physiol 2001;130:385–399. Sabirov RZ, Okada Y: ATP-conducting maxi-anion channel: a new player in stress-sensory transduction. Jpn J Physiol 2004;54:7–14.


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Schreiber R: Ca2 signaling, intracellular pH and cell volume in cell proliferation. J Membr Biol 2005;205:129–137. Stein WD: Cell volume homeostasis: ionic and nonionic mechanisms. The sodium pump in the emergence of animal cells. Int Rev Cytol 2002;215:231–258. Uchida S, Sasaki S: Function of chloride channels in the kidney. Annu Rev Physiol 2005;67:759–778. Wehner F, Olsen H, Tinel H, Kinne-Saffran E, Kinne RK: Cell volume regulation: osmolytes, osmolyte transport, and signal transduction. Rev Physiol Biochem Pharmacol 2003;148:1–80.

Prof. Dr. Florian Lang Physiologisches Institut der Universität Tübingen Gmelinstrasse 5 DE–72078 Tübingen (Germany) Tel. 49 7071 297 2194, Fax 49 7071 293 073, E-Mail [email protected]


8


Cell Volume-Sensitive Chloride Channels: Phenotypic Properties and Molecular Identity Yasunobu Okada Department of Cell Physiology, National Institute for Physiological Sciences, Okazaki, Japan

Abstract Cell volume regulation is essential for the survival of cells. After osmotic swelling, animal cells show a regulatory volume decrease by releasing intracellular K⫹, Cl⫺ and water. In most cell types, volume-regulatory Cl⫺ efflux is induced by activation of electroconductive anion pathways. Among these volume-activated Cl⫺ channels, the most important and specific is a volume-sensitive outwardly rectifying (VSOR) Cl⫺ channel. The phonotypical properties have been well described. Extracellular application of anionic forms of ATP and glibenclamide give rise to voltage-dependent open-channel block of this channel, the fact suggesting that its outer vestibule and pore are larger and smaller, respectively, than the sizes of ATP and glibenclamide. Consistent with this prediction, the pore radius of VSOR Cl⫺ channel (0.63 nm) which has been recently determined is slightly smaller than the radii of ATP and glibenclamide. The activities of VSOR Cl⫺ channels are implicated not only in regulatory volume decrease but also in many other physiological or pathophysiological cell events including cell death induction. Despite their ubiquitous expression and physiological/pathophysiological significance, there is still a paucity of the molecular information of the VSOR Cl⫺ channel. Copyright © 2006 S. Karger AG, Basel

Regulatory Volume Decrease and Clⴚ Channels

Life and the activities of cells are maintained by the turnover (intracellular metabolism and membrane transport) of osmotically active substances. Intracellular osmolality is determined by the balance of anabolic and catabolic reactions as well as of influx and efflux of osmotically active substances. Therefore, many cell activities, which are closely associated with changes in

Antiporter system

Symporter system K⫹ CI H2O

Normal cell volume

H⫹

⫺

K⫹ H2O CI⫺

Osmotic swelling

Increased cell volume

RVD

K⫹

H 2O

HCO⫺ 3

NVI

Necrotic cell death

CI⫺

Channel system

Fig. 1. Schematic illustration of ionic mechanisms for the RVD. Three different systems have been reported depending on the cell types. Persistent cell swelling, called NVI, due to impairment of the RVD mechanism results in necrotic cell death.

metabolic reactions and membrane transport, inevitably result in osmotic perturbation to cells. Since most animal cell membranes are highly permeable to water, cells are destined to cope with cell volume changes. To attain volume regulation, animal cells adopt a strategy employing the same physicochemical principle. To drive water flux, which finally accomplishes cell volume adjustment, cells produce an osmotic difference between the intra- and extracellular spaces by inducing osmolyte transport (especially for the early phase of volume regulation) and by shifting cell metabolism to catabolism- or anabolismdominant conditions (especially for the late phase of volume regulation). After osmotic cell swelling and shrinkage, many cell types can rapidly show the regulatory volume decrease (RVD) and increase (RVI), respectively. RVD and RVI are accomplished mainly by inducing KC1 efflux and NaCl influx, respectively, which lead to flux of osmotically obligated water. Transport pathways involved in RVD and RVI have been investigated in a wide variety of cell types [1–7]. Three different systems are known to be responsible for volume regulatory KC1 efflux, as schematically shown in figure 1. Most cell types employ the electroconductive channel system, whereas erythrocytes and gallbladder epithelial cells mainly use the electroneutral symporter or antiporter system. In the face of swelling emergency cells should extrude chloride by using available anion channels, including cAMP-activated, Ca2⫹-activated, large-conductance (Maxi) and basally active (background) Cl⫺ channels in addition to volume-sensitive Cl⫺ channels [3].

Okada

10

Table 1. Properties of volume-sensitive outwardly rectifying (VSOR) Cl⫺ channels in human epithelial Intestine 407 cells Physiological properties Volume sensitivity ATP dependence Mg2⫹ dependence Fatty acid sensitivity

Biophysical properties Single-channel Voltage dependence Anion selectivity Pharmacological properties Cl⫺ channel blockers PGP inhibitors Sulfonylureas

Volume expansion-sensitive [3, 24] (but not mechano-sensitive) Cytosolic-free ATP requiring [20] Extracellular ATP blocking [22] Cytosolic-free Mg2⫹-sensitive [20] Sensitive to cis unsaturated arachidonate or oleate [18] Insensitive to trans unsaturated elaidate [18] Insensitive to saturated palmitate [18] Intermediate unitary conductance [19, 22] Outwardly rectifying [18, 19, 22] Inactivating at larger positive potentials [18] Low-field anion selectivity (Eisenman I sequence) [18] Sensitive to SITS, DIDS, NPPB, DPC, niflumate, flufenamate, and furosemide [3, 18] Sensitive to verapamil, nifedipine, cyclosporin A, tamoxifen and dideoxyforskolin [21] Sensitive to extracellular glibenclamide [23]

Among these volume-regulatory Cl⫺ channels, the most important or specific is a volume-sensitive outwardly rectifying (VSOR) Cl⫺ channel [3, 8, 9]. This channel has been referred as swelling-activated Cl⫺ channel or ICl,swell, volumedependent Cl⫺ channel or ICl,vol, volume-regulated anion channel or VRAC, and volume-sensitive organic osmolyte and anion channel or VSOAC [3, 8, 9]. However, it must be noted that inwardly rectifying ClC-2 Cl⫺ channels are also activated by cell swelling [10, 11], and that maxi-anion channels are activated by cell swelling and conductive to an organic osmolyte ATP [12–16]. To distinguish from ClC-2 and maxi-anion channels, we call this channel the VSOR Cl⫺ channel [3]. Phenotypic Properties of Human Epithelial VSOR Cl⫺ Channels

In a human small intestinal epithelial cell line (Intestine 407) RVD was first shown to be attained by parallel activation of Ca2⫹-activated K⫹ channels and Ca2⫹-independent volume-sensitive Cl⫺ channels [17]. The physiological, biophysical and pharmacological properties of the channel in Intestine 407 cells have been studied in our laboratory [18–27]. Table 1 summarizes those properties.

Cell Volume-Sensitive Chloride Channels

11

No single channel event No single channel event

Single channel events (C) Cl⫺

Osmotic Swelling Cl⫺

Patch pipette No whole-cell current activation

Whole-cell current activation (B)

a 140 mV 105 mV 90

40 mV

40 mV 60

40 mV

30

⫺60 mV

160 mV 20 pA

0 2s ⫺30 ⫺60 ⫺105 mV

40 mV

40 mV

⫺90

b

1,000 pA 0.5 s

40 mV

⫺80 mV

c

Fig. 2. Whole-cell and on-cell recordings after osmotic swelling. a Schematic illustrations of patch-clamp configurations and swelling-induced unfolding of membrane invaginations that are supported by the cytoskeletal meshwork. Single-channel and whole-cell currents of volume-sensitive Cl⫺ channel could be recorded only after unfolding of membrane invaginations. b Whole-cell volume-sensitive Cl⫺ channel currents in an osmotically swollen Intestine 407 cell. Current responses to positive command pulses to ⫹105 mV (with ⫹15 mV increments) from a holding potential at –105 mV. c On-cell single volume-sensitive Cl⫺ channel currents in a swollen Intestine 407 cell. Positive and negative command pulses

Okada

12

This VSOR Cl⫺ channel was activated in association with cell swelling, and no basal activity was found under the conditions where the cell volume was normal. The swelling-associated activity does not necessarily mean that an increase in membrane stretch or tension is responsible for activation of the Cl⫺ channel, because cells are known to have membrane invaginations or foldings [3, 7]. Actually, the membrane capacitance of Intestine 407 cells indicates that the cell membrane has sufficient reserve to allow for up to a 5-fold volume increase [3]. Enormous activation of whole-cell currents could be consistently observed after osmotic swelling (fig. 2). Simultaneous measurements of cell size and whole-cell currents demonstrated that, over a certain range above the threshold, the Cl⫺ current density increases in proportion to outer surface area as estimated by cell contour morphology [3] or the square of cell diameter of the spherical cells [24]. This fact indicates that the Cl⫺ channel can somehow sense volume expansion (without membrane stretch) due to unfolding of membrane invaginations or folds. The same behavior was also found in the VSOR Cl⫺ current in human epidermoid KB cells [28]. The concept that the channel activation is induced by unfolding of the membrane folds is consistent with the following observation. The single Cl⫺ channel event could be recorded only when the patch pipette was attached to the cell which had been swollen (fig. 2), but never recorded when giga-sealed on-cell attachment of pipette had been performed before the cell was rendered swollen (fig. 2a). Even without discernible cell swelling, the VSOR Cl⫺ channel was shown to be activated by intracellular application of GTP␥S in some (but not all) cell types [29–32], by reduction of intracellular ionic strength [33–35], by application of H2O2 [36, 37] or by stimulation with apoptotic inducers [37]. Thus, it is likely that activation of the VSOR Cl⫺ channel involves some signal related to cell swelling and the other above maneuvers. The candidate signals may include Src family tyrosine kinases [38–40] and PI3K [39, 41]. The single-channel conductance and anion selectivity are closely related to the properties of the channel pore. Intestine 407 cells exhibit an intermediate unitary conductance (27 pS at –50 mV, 37 pS at ⫹50 mV [19, 22]) and lowfield anion selectivity (with Eisenman I sequence). The double-patch clamp (simultaneous whole-cell and on-cell patch clamp) [19] and the ensemble average current of inside-out records [22] provided clear evidence that these single-channel events are responsible for the whole-cell current. Outward

to ⫹140 or ⫹160 mV and –60 or –80 mV were applied from a holding potential at ⫹40 mV. The intracellular zero-current potential was maintained at around 0 mV by exposing to 120 mM K⫹ bath solution. Closing and reopening events are indicated by downward and upward arrowheads, respectively.


13

Cl⫺ Extracellular

ATP

ATP

Intracellular

ATP

a

Cl⫺ Blocked

Open

Closed

SU

Cl⫺ ⫺

SU

ATP

b

ATP

ATP

SU

Extracellular

Intracellular

Cl⫺

Fig. 3. Schematic illustrations of the volume-sensitive Cl⫺ channel in the open, closed and blocked states. a ATP dependence. Intracellular ATP binding is required for maintaining the channel open. Extracellular ATP blocks the channel upon application of positive voltages. b Glibenclamide sensitivity. From the extracellular side, the uncharged form of the SU inhibits the channel, and the anionic form (SU⫺) may block the channel at positive potentials.

rectification and inactivation at large positive potentials are distinct properties of the Cl⫺ channel. Depolarization-induced blocking by extracellular anionic ATP suggests that the VSOR Cl⫺ channel possesses its outer vestibule larger than and the pore smaller than the size of ATP (fig. 3a). Recent our non-electrolyte partition studies [27] revealed the cut-off radius of the VSOR channel pore of 0.63 nm which is close to or slightly smaller than the radius of ATP4⫺ (0.6–0.7 nm). The most physiologically important property of VSOR Cl⫺ channel is the dependence of the channel activity on cytosolic free ATP (fig. 3a). The presence (presumably binding to the channel component) of intracellular free ATP, but without its hydrolysis, is a prerequisite to the channel activity [3, 20]. Thus, the phenotypic properties of the volume-sensitive Cl⫺ channel in human epithelial Intestine 407 cells are outward rectification, ATP dependency, low-field anion selectivity and inactivation kinetics at large positive potentials. These properties are shared by other epithelial cells [37, 42–51] (see table 2: Group 1).

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Table 2. ATP-dependent mammalian VSOR Cl⫺ channels Eisenman Inactivation References sequence kinetics Group 1 Epithelial

Neuronal

Human Intestine 407 HeLa HSG Cervical cancer Bovine NPCE Rabbit distal convoluted tubule Rat pancreatic duct Mouse Ml-CCD mIMCD-K2 liver Human Rat Rat C6 Human dermal Mouse NIH/3T3 Mouse cerebellar

Group 2 Fibroblast Myocyte Lymphocyte Carotid body

Mouse BALB/C-3T3 Guinea pig atrial Mouse and human T Rat type I Rat RINmSF Mouse HIT Mouse islet

Endothelial Glioma Fibroblast

Group 3 Pancreatic

I I

⫹ ⫹ ⫹

I I I I I I I

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

[18] [37, 42, 43] [44] [45] [46] [47] [48] [49] [50] [51] [30, 58] [59] [60–62] [63] [64] [65]

I I I

⫺ ⫺ ⫺ ⫺

[66] [53, 54, 67] [68] [69]

III III III

⫾ ⫾ ⫺

[70] [70, 71] [71]

I

The VSOR Cl⫺ channel in Intestine 407 cells exhibits broad specificity to many types of conventional blockers for Cl⫺ channels (SITS, DIDS, NPPB, DPC) [18], a bisphenol phloretin [26], arachidonic acid [18] and blockers for multidrug resistance P-glycoprotein (PGP) [21]. The volume-sensitive Cl⫺ channel was shown to be sensitive to glibenclamide, which is a member of sulfonylurea (SU) family and a well-known blocker of ATP-sensitive K⫹ (KATP) channel [52], in Intestine 407 cells [23] as well as other cell types [49, 53, 54]. In Intestine 407 cells, SU was found to exert an inhibitory action on the volume-sensitive Cl⫺ channel only from the extracellular side, the fact being in contrast to the effects on CFTR Cl⫺ channels [55], depolarization-activated outwardly rectifying Cl⫺ channels [56] and KATP channels [57]. Our studies on the


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mechanism performed by varying pH in Intestine 407 cells indicated that the uncharged form of glibenclamide voltage-independently inhibits instantaneous currents at micromolar concentrations (presumably by binding to the channel sites outside the electric field; fig. 3b), whereas the anionic form voltagedependently facilitates depolarization-induced channel inactivation at submillimolar concentrations [23]. The latter effect is similar to the extracellular ATP effect, and blocking (plugging) of the pore by anionic glibenclamide (SU⫺) would simply explain the mechanism (fig. 3b), because the radius of glibenclamide (0.696 nm calculated by R.Z. Sabirov: private communication) is also slightly larger than that of the VSOR channel pore.

Volume-Sensitive, Outwardly Rectifying, ATP-Dependent Cl⫺ Channel

Volume-sensitive Cl⫺ currents have been observed in a large variety of animal cell types, as previously reviewed [3, 8, 9]. Among them, those exhibiting outward rectification were most frequently found. In spite of its physiological importance, however, the ATP dependence was not investigated in all these VSOR Cl⫺ channels. Table 2 summarizes mammalian cell types which have been shown to exhibit dependence on cytosolic ATP. These ATP-dependent VSOR Cl⫺ channels fall into three groups. The first group (Group 1) is the channel exhibiting Eisenman I sequence anion selectivity and depolarizationinduced inactivation kinetics. This type was found not only in epithelial cells [18, 37, 42–51] but also in other many cell types [30, 58–65]. The second group (Group 2) exhibits Eisenman I-type anion selectivity but not inactivation kinetics [53, 54, 66–69]. The third one (Group 3), which was only found in pancreatic ␤ cells [70, 71], shows intermediate-field anion selectivity with Eisenman’s type III sequence, although the validity of this anion selectivity in ␤ cells may remain to be clarified [3]. The Molecular Identity of VSOR Clⴚ Channel

Three major candidate proteins (PGP, pICln and ClC-3) have been proposed as the molecules of volume-sensitive Cl⫺ channels [3, 72]. The first hypothesis (called the transporter/channel bifunctional hypothesis [3]) assumed that PGP switches from a drug pump to the channel upon osmotic swelling [73, 74]. However, this attractive hypothesis had to be finally withdrawn in the face of a large number of lines of conflicting evidence, as summarized in our previous reviews [3, 75]. The strongest evidence against this hypothesis is the fact that

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abolition of PGP expression using antisense oligonucleotides failed to inhibit the channel activity [21]. The second hypothesis (called the anchor-insertion channel hypothesis [3]) assumed that a cytosolic protein, pICln, forms the channel after insertion to the plasma membrane upon osmotic swelling, thereby exposing the glycine-rich nucleotide-binding site to the extracellular solution and conferring sensitivity to extracellular ATP, cAMP and cGMP on the channel [76, 77]. However, the volume-sensitive Cl⫺ current was not inhibited by extracellular cAMP and cGMP in Intestine 407 cells [10]. Moreover, Nilius and coworkers [78] have recently shown that mutation of the putative nucleotide-binding site of pICln does not affect the cAMP sensitivity of the pICln-associated Cl⫺ current in Xenopus oocytes, and that the phenotypic properties of pICln-associated Cl⫺ current are distinct from those of volume-sensitive Cl⫺ channels [79]. Furthermore, when reconstituted in planar lipid bilayers, pICln was found to exhibit often cation channel activity [80, 81] or sometimes anion channel activity, the properties of which are largely different from those of VSOR Cl⫺ channels [81, 82]. The ClC-3 hypothesis was next proposed by Duan et al. [83]. They showed that the basal activity of outwardly rectifying Cl⫺ current in mouse NIH/3T3 cells became huge, when the cloned guinea pig cardiac ClC-3 cDNA was expressed, and that the current was further increased by osmotic swelling. Based on the observations that the N579K mutant of the guinea pig ClC-3 brought about changes in selectivity from I⫺ ⬎ Cl⫺ to Cl⫺ ⬎ I⫺ and in rectification from outward to inward direction, they concluded that the ClC-3 gene encodes the volume-sensitive Cl⫺ channel. In fact, as summarized in table 3, the properties of guinea pig ClC-3-associated Cl⫺ currents are largely similar to those of the VSOR Cl⫺ channel in Intestine 407 cells with respect to intermediate unitary conductance, outward rectification, inactivation kinetics, anion selectivity of I⫺ ⬎ Cl⫺, blocking by extracellular ATP, DIDS sensitivity and tamoxifen sensitivity. However, significant basal activity, much lower open probability and inhibition by a protein kinase C activator (PDBu) of the guinea pig ClC-3-associated Cl⫺ current are distinct from those of VSOR Cl⫺ current in Intestine 407 cells (table 3). Unfortunately, however, no information is available for the most important physiological property, the dependence on cytosolic ATP. Enormous basal activity and sensitivity to a protein kinase C activator (TPA) were also reported by Kawasaki et al. [84] for the Cl⫺ current induced by expression of rat kidney-derived ClC-3 in Xenopus oocytes. Kawasaki et al. [85] also found that the rat ClC-3 stably expressed in CHO cells exhibits Cl⫺ currents with additional differences (table 3), such as a much larger unitary conductance (100 or 140 pS) and inhibition by intracellular Ca2⫹ in the physiological concentration range. However, it should be stressed that their study in inside-out patches [85] was performed in the absence of cytosolic ATP.


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Table 3. Properties of human epithelial VSOR, guinea pig heart ClC-3-associated, and rat kidney ClC-3-associated Cl⫺ channels

Single-channel conductance Open probability Rectification Outward current kinetics Anion selectivity Basal activity Volume sensitivity C kinase sensitivity Cytosolic Ca2⫹ sensitivity Cytosolic ATP dependence Extracellular ATP effect Pharmacology

Human VSOR

Guinea pig ClC-3

Rat ClC-3

Intermediate ⬃0.97 at ⫺100 mV Outward Inactivating I ⬎ Br ⬎ C None Swelling-activated TPA, OAG-insensitive None ATP requirement Blocking DIDS-sensitive Tamoxifen-sensitive

Intermediate ⬃0.6 at resting potential Outward Inactivating I⬎C Enormous Swelling-activated PDBu-sensitive ? ? Blocking DIDS-sensitive Tamoxifen-sensitive

Maxi ? Outward Non-inactivating I ⬎ Br ⬎ Cl Enormous Swelling-activated TPA-sensitive Inhibition by Ca None ? DIDS-sensitive

At variance with the ClC-3 hypothesis, essentially identical whole-cell VSOR Cl⫺ currents were observed in hepatocytes and pancreatic acinar cells [86] and parotid salivary acinar cells [87] isolated from ClC-3 knockout (Clcn3⫺1⫺) mice. Since the ClC-3 was first considered as the candidate molecule of VSOR Cl⫺ channel [83], we have recently examined the VSOR Cl⫺ channel current in Clcn3⫺1⫺ cardiomyocytes at macroscopical [88] and microscopical [89] levels. Between ventricular myocytes isolated from ClC-3-difficient mice and their wild-type mice, biophysical and pharmacological properties of the whole-cell [88] and single-channel [89] currents of VSOR Cl⫺ channels were found to be indistinguishable. Thus, it is now clear that molecular expression of ClC-3 is not required for functional expression of VSOR Cl⫺ channels in various cells including cardiomyocytes. Further investigation is needed before the molecular identity of VSOR Cl⫺ channel is determined.

Physiological and Pathological Significance

The activities of VSOR Cl⫺ channels are implicated not only in RVD [3, 7] but also in many other physiological cell activities, which are associated with changes in cell volume or shape, including cell proliferation [90–92], cell differentiation [45, 93, 94], insulin secretion [95], and the active uptake of amino acids in hepatocytes [96]. Recently, our group [37, 97] has provided firm

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evidence for an essential role of the VSOR Cl⫺ channel in induction of apoptotic volume decrease which is a prerequisite to apoptosis [98, 99]. Pathological implications of the VSOR Cl⫺ channel have been pointed out with respect to electrical instability of the heart [100], cataract formation [101], and the pathogenesis of cancer [45]. Also, ischemia–reperfusion-induced apoptotic death in cardiomyocytes [102] and anticancer drug-induced apoptotic death in cancer cells [103] were recently found to involve augmented activity of the VSOR Cl⫺ channel and to be inhibited by a Cl⫺ channel blocker. Metabolic inhibition due to prolonged hypoxia or ischemia produces cell swelling, called necrotic volume increase (NVI) [99], which, if it persists, results in necrotic cell death. The activity of the ATP-dependent volume-sensitive Cl⫺ channel should be inhibited by intracellular ATP depletion and also by an increase in the cytosolic-free Mg2⫹ induced by an ATP decrease. Actually, KCN-induced ATP depletion was found to inhibit activation of VSOR Cl⫺ currents in human endothelial cells [58]. Impaired activity of the VSOR Cl⫺ channel results in dysfunction of RVD and thereby inducing persistent NVI. Under lactacidosis conditions, the VSOR Cl⫺ channel activity in glial cells is also inhibited by intracellular acidification and thus leading to persistent swelling, which is initiated by uptake of Na⫹ and lactate⫺, and eventually to necrosis [104, 105]. Thus, the VSOR Cl⫺ channel is involved in induction of apoptosis when augmented by apoptotic stimuli, whereas inhibition of this channel by cytosolic ATP depletion or lactacidosis is involved in induction of necrosis. Such dual roles of the VSOR Cl⫺ channel in cell death induction were reviewed elsewhere [105].

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81 Furst J, Bazzini C, Jakab M, Meyer G, Konig M, Gschwentner M, Ritter M, Schmarda A, Botta G, Benz R, Deetjen P, Paulmichl M: Functional reconstitution of ICln in lipid bilayers. Pflugers Arch 2000;440:100–115. 82 Garavaglia ML, Rodighiero S, Bertocchi C, Manfredi R, Furst J, Gschwentner M, Ritter M, Bazzini C, Botta G, Jakab M, Meyer G, Paulmichl M: ICln channels reconstituted in heart-lipid bilayer are selective to chloride. Pflugers Arch 2002;443:748–753. 83 Duan D, Winter C, Cowley S, Hume JR, Horowitz B: Molecular identification of a volumeregulated chloride channel. Nature 1997;390:417–421. 84 Kawasaki M, Uchida S, Monkawa T, Miyawaki A, Mikoshiba K, Marumo F, Sasaki S: Cloning and expression of a protein kinase C-regulated chloride channel abundantly expressed in rat brain neuronal cells. Neuron 1994;12:597–604. 85 Kawasaki M, Suzuki M, Uchida S, Sasaki S, Marumo F: Stable and functional expression of the C1C-3 chloride channel in somatic cell lines. Neuron 1995;14:1285–1291. 86 Stobrawa SM, Breiderhoff T, Takamori S, Engel D, Schweizer M, Zdebik AA, Bösl MR, Ruether K, Jahn H, Draguhn A, Jahn R, Jentsch TJ: Disruption of ClC-3, a chloride channel expressed on synaptic vesicles, leads to a loss of the hippocampus. Neuron 2001;29:185–196. 87 Arreola J, Begenisich T, Nehrke K, Nguyen HV, Park K, Richardson L, Yang B, Schutte BC, Lamb FS, Melvin JE: Secretion and cell volume regulation by salivary acinar cells from mice lacking expression of the Clcn3 Cl⫺ channel gene. J Physiol (Lond) 2002;545:207–216. 88 Gong W, Xu H, Shimizu T, Morishima S, Tanabe S, Tachibe T, Uchida S, Sasaki S, Okada Y: ClC3-independent, PKC-dependent activity of volume-sensitive Cl⫺ channel in mouse ventricular cardiomyocytes. Cell Physiol Biochem 2004;14:213–224. 89 Wang J, Xu H, Morishima S, Tanabe S, Jishage K, Uchida S, Sasaki S, Okada Y, Shimizu T: Single-channel properties of volume-sensitive Cl⫺ channel in ClC-3-deficient cardiomyocytes. Jpn J Physiol 2005;55:379–383. 90 Voets T, Szücs G, Droogmans G, Nilius B: Blockers of volume-activated Cl⫺ currents inhibit endothelial cell proliferation. Pflugers Arch 1995;431:132–134. 91 Manolopoulos VG, Droogmans G, Nilius B: Hypotonicity and thrombin activate taurine efflux in BC3H1 and C2C12 myoblasts that is down regulated during differentiation. Biochem Biophys Res Commun 1997;232:74–79. 92 Lang F, Szabo I, Lepple-Wienhues A, Ritter M, Waldegger S, Gulbins E: Cell volume in the regulation of metabolism, cell proliferation and apoptotic cell death; in Okada Y (ed): Cell Volume Regulation: The Molecular Mechanism and Volume-Sensing Machinery. Elsevier, Amsterdam, 1998. 93 Voets T, Wei L, De Smet P, Van Driessche W, Eggermont J, Droogmans G, Nilius B: Downregulation of volume-activated Cl⫺ currents during muscle differentiation. Am J Physiol 1997;272:C667–C674. 94 Eder C, Klee R, Heinemann U: Involvement of stretch-activated Cl⫺ channels in ramification of murine microglia. J Neurosci 1998;18:7127–7137. 95 Best L: Glucose and ␣-ketoisocaproate induce transient inward currents in rat pancreatic beta cells. Diabetologia 1997;40:l–6. 96 Lidofsky SD, Roman RM: Alanine uptake activates hepatocellular chloride channels. Am J Physiol 1997;273:G849–G853. 97 Okada Y, Shimizu T, Maeno E, Tanabe S, Wang X, Takahashi N: Volume-sensitive chloride channels involved in apoptotic volume decrease and cell death. J Membr Biol 2006;209:1–9. 98 Maeno E, Ishizaki Y, Kanaseki T, Hazama A, Okada Y: Normotonic cell shrinkage due to disordered volume regulation is an early prerequisite to apoptosis. Proc Natl Acad Sci USA 2000;97:9487–9492. 99 Okada Y, Maeno E, Shimizu T, Dezaki K, Wang J, Morishima S: Receptor-mediated control of regulatory volume decrease (RVD) and apoptotic volume decrease (AVD). J Physiol (Lond) 2001;532:3–16. 100 Vandenberg JI, Bett GCL, Powell T: Contribution of a swelling-activated chloride current to changes in the cardiac action potential. Am J Physiol 1997;273:C541–C547. 101 Zhang JJ, Jacob TJ, Valverde MA, Hardy SP, Mintenig GM, Sepulveda FV, Gill DR, Hyde SC, Trezise AE, Higgins CF: Tamoxifen blocks chloride channels: A possible mechanism for cataract formation. J Clin Invest 1994;94:1690–1697.


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102 Wang X, Takahashi N, Uramoto H, Okada Y: Chloride channel inhibition prevents ROS-dependent apoptosis induced by ischemia-reperfusion in mouse cardiomyocytes. Cell Physiol Biochem 2005;16:147–154. 103 Ise T, Shimizu T, Lee EL, Inoue H, Kohno K, Okada Y: Roles of volume-sensitive Cl⫺ channel in cisplatin-induced apoptosis in human epidermoid cancer cells. J Membr Biol 2005;205:139–145. 104 Nabekura T, Morishima S, Cover TL, Mori S, Kannan H, Komune S, Okada Y: Recovery from lactacidosis-induced glial cell swelling with the aid of exogenous anion channels. Glia 2003;41: 247–259. 105 Okada Y, Maeno, Shimizu T, Manabe K, Mori S, Nabekura T: Dual roles of plasmalemmal chloride channels in induction of cell death. Pflugers Arch 2004;448:287–295.

Yasunobu Okada Department of Cell Physiology, National Institute for Physiological Sciences Myodaiji-cho Okazaki 444–8585 (Japan) Tel. ⫹81 564 55 7731, Fax ⫹81 564 55 7735, E-Mail [email protected]

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Cell Volume-Regulated Cation Channels Frank Wehner Max-Planck-Institut für molekulare Physiologie, Dortmund, Deutschland

Abstract Considering the enormous turnover rates of ion channels when compared to carriers it is quite obvious that channel-mediated ion transport may serve as a rapid and efficient mechanism of cell volume regulation. Whenever studied in a quantitative fashion the hypertonic activation of non-selective cation channels is found to be the main mechanism of regulatory volume increase (RVI). Some channels are inhibited by amiloride (and may be related to the ENaC), others are blocked by Gd3⫹ and flufenamate (and possibly linked to the group of transient receptor potential (TRP) channels). Nevertheless, the actual architecture of hypertonicity-induced cation channels remains to be defined. In some preparations, hypertonic stress decreases K⫹ channel activity so reducing the continuous K⫹ leak out of the cell; this is equivalent to a net gain of cell osmolytes facilitating RVI. The hypotonic activation of K⫹ selective channels appears to be one of the most common principles of regulatory volume decrease (RVD) and, in most instances, the actual channels involved could be identified on the molecular level. These are BKCa (or maxi K⫹) channels, IKCa and SKCa channels (of intermediate and small conductance, respectively), the group of voltage-gated (Kv) channels including their ␤ (or Kv ancilliary) subunits, two-pore K2P channels, as well as inwardly rectifying K⫹ (Kir) channels (also contributing to KATP channels). In some cells, hypotonicity activates non-selective cation channels. This is surprising, at first sight, because of the inside negative membrane voltage and the sum of driving forces for Na⫹ and K⫹ diffusion across the cell membrane rather favouring net cation uptake. Some of these channels, however, exhibit a PK/PNa significantly higher than 1, whereas others are Ca⫹⫹ permeable linking hypotonic stress to the activation of Ca⫹⫹ dependent ion channels. In particular, the latter holds for the group of TRPs which are specialised in the perception of a variety of different stimuli including mechanical and (hypo-) osmotic stress. As a peculiarity, phospholemman (PLM, a 72 AA peptide also employed in ion transport regulation) appears to be activated under, both, hypertonic and hypotonic conditions, preferentially operating as a cation and anion channel, respectively. Copyright © 2006 S. Karger AG, Basel

Given the transport rates of ion channels that are some 4–5 orders of magnitude higher than those achieved by carriers, any modulation of ion channel

activity under anisotonic conditions may serve as a fast and efficient mechanism of cell volume regulation. With the steep Na⫹ (inward) and K⫹ (outward) gradients across most cell membranes, the activation of Na⫹ and K⫹ permeable channels is clearly the prime mechanism for the rapid gain and export of cations during regulatory volume increase (RVI) and regulatory volume decrease (RVD), respectively. In addition, an inhibition of K⫹ channels may facilitate RVI by reducing the overall K⫹ leak out of a cell. Of note, there is increasing evidence for a role of transient receptor potential (TRP) channels in RVD (and possibly RVI), functioning as sensors, effectors and part of the signal transduction machinery. As a peculiarity, Phospholemman (PLM) appears to contribute to both RVI and RVD perhaps just by modulating its cation over anion selectivity.

RVI, the Activation of Cation Channels

In principle, the activation of a cation channel that does not discriminate much between Na⫹ and K⫹ will lead to both Na⫹ influx as well as K⫹ efflux. Because of the inside negative trans-membrane voltage, however, a significant net uptake of cations will be accomplished by such a channel that (together with an accompanying import of anions) will increase the overall intracellular osmotic activity considerably. Any increase in the relative Na⫹ over K⫹ permeability (PNa/PK) will, of course, significantly increase the efficiency of this mechanism. Just based on their pharmacology, one may distinguish a group of hypertonicity-induced cation channels that is inhibited by the diuretic amiloride (but that is insensitive to the anti-inflammatory drug flufenamate) from one that is insensitive to amiloride (but efficiently blocked by Gd3⫹ and flufenamate). Of note, from two preparations so far, a mixed type of pharmacology has also been reported. In confluent monolayers of rat hepatocytes, hypertonic stress leads to the activation of an amiloride-sensitive cation channel [1] that exhibits a PNa/PK of 1.4 [2]. The apparent Ki for amiloride binding is close to 5 ␮M and the overall sensitivity profile of the channel is EIPA (ethyl-isopropyl-amiloride) ⬎ amiloride ⬎ benzamil [3]. The cation channel is clearly the main mechanism of RVI with a relative contribution of 4:1:1 when compared to Na⫹/H⫹ anti-port (NHE1) and Na⫹–K⫹–2Cl⫺ symport (NKCC1) [4, 5]. In patch-clamp experiments in the cellattached configuration, hypertonicity-induced single channel events with a unitary conductance of 6 pS could be recorded [6]. In concentrations up to 1 mM, the hepatocyte channel is insensitive to Gd3⫹ and flufenamate (Wehner, unpublished). With respect to cell signalling, the hypertonic activation of cation channels in rat hepatocytes employs PKC [7], PLC, G proteins and tyrosine kinases [8]. Also of note,

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in HTC rat hepatoma cells, tyrosine kinases were found to be key elements in the exocytotic transfer of non-selective cation channels to the plasma membrane [9]. In U937 macrophages, hypertonicity induces inward currents with a distinct selectivity for Na⫹ over K⫹ and with an apparent Ki for amiloride that is close to 1 ␮M. Apparently, this current is related to the activation of a 6 pS channel [10]. In human red cell ghosts, hypertonic conditions induce a cation conductance with a PNa/PK close to unity. The cation channel is clearly sensitive to amiloride with a decrease of currents to some 60% of control with 100 ␮M [11]. These results are in line with earlier flux measurements on lamprey erythrocytes from which the hypertonic activation of an amiloride-sensitive Na⫹ transport was reported that could not be attributed to Na⫹/H⫹ anti-port (or Na⫹/Na⫹ self-exchange) [12]. In the principle cells of rat cortical collecting duct, hypertonic stress elicits a distinct depolarisation of membrane voltage and an increase of cell Na⫹. The inhibition of these effects by amiloride and Gd3⫹ was clearly additive, strongly implying the parallel activation of amiloride-sensitive and insensitive channels [13]. Hypertonicity-induced amiloride-sensitive cation channels appear to be related to the ENaC (the epithelial Na⫹ channel). This may be surprising, at first sight, given the high PNa/PK (ⱖ20) and Ki values close to 100 nM for amiloride inhibition as they are typically reported for the ENaC [14]. Quite obviously, however, both ion selectivity and sensitivity to amiloride are readily susceptible to subunit composition and mutational variability of ENaCs [14–18]. In rat hepatocytes, ␣-, ␤- and ␥-ENaC could be detected on the mRNA as well as on the protein level [3] and anti-sense oligo-nucleotides directed against ␣-ENaC decreased hypertonicity-induced currents by some 60% [2]. Non-pigmented human ciliary epithelial cells do express ␣-ENaC and conductive Na⫹ entry significantly contributes to RVI. In the range of 1–10 ␮M this Na⫹ conductance is inhibited by amiloride and, even more efficiently, by benzamil, the most effective blocker of the ENaC [19, 20]. From Xenopus oocytes expressing ␣-, ␤-, ␥-rENaC, it was reported that hypertonic stress increased and hypotonic stress decreased amiloride-sensitive currents, respectively, and these effects coincided with a decrease of PNa/PK from 20 (under isotonic conditions) to values of 11 and 6 [21]. For the same preparation, however, others found a decrease of Na⫹ currents under hypertonic conditions and virtually no effect of hypotonic stimulation [22] so that further studies are necessary to precisely define the osmo-sensitivity of the ENaC in the oocyte model system. In the second group, hypertonicity-induced cation channels are clearly amiloride-insensitive in concentrations up to 100 ␮M, but typically they are inhibited by flufenamate (at 100 ␮M) and Gd3⫹ (in the range of 10 ␮M to 1 mM). These channels are expressed in a variety of systems including human nasal epithelial cells [23], the colonic cell-lines CaCo-2 and HT29 [24, 25] and mouse

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collecting duct M1 cells [26]. They were also reported from monkey BSC-1 kidney cells, the rat vascular smooth muscle cell-line A10, mouse Neuro-2a cells [25, 26] and from the human cervix carcinoma cell-line HeLa [27]. Amilorideinsensitive channels may either be equally permeable to Na⫹, K⫹, Cs⫹ and Li⫹ but impermeable to NMDG⫹ (N-methyl-D-glucamine) or they exhibit a permeability to Li⫹ and NMDG⫹ that amounts to some 50% when compared to that of Na⫹ (see [6] for a comprehensive review). There are also considerable differences in the permeability of these channels to Cl⫺ with PNa/PCl values in the broad range of 60 [28] to 1.7 [24]. In some instances, channel activation is completely Ca⫹⫹ independent, in some preparations it may require a permissive Ca⫹⫹ activity of 1 ␮M on the cytosolic side. Nevertheless, the Ca⫹⫹ permeability of cation channels in the second group appears to be virtually zero [25, 28, 29]. Unitary conductances are in the narrow range of 15–27 pS [25, 28]. Of note, a considerable number of hypertonicity-induced and amilorideinsensitive cation channels are inhibited by cytosolic ATP concentrations that are in the lower millimolar range [25]. This raises some concerns as for their actual role in the RVI process. For some systems, on the other hand, the paramount importance of the cation channel for RVI could be clearly shown, as for instance for HeLa cells where the contribution of the channel significantly exceeds that of Na⫹/H⫹ antiport (and with no detectable part of Na⫹–K⫹–2Cl⫺ symport to this process at all) [27]. Interestingly, given its sizeable inhibition by SKF-96365 the cation channel in this preparation may be related to a member of the family of TRP channels [27], one of the very few hints available so far as for the molecular architecture of hypertonicity-induced amiloride-insensitive cation channels. Interestingly, hypertonicity-induced cation channels with a combined pharmacology also exist and these may reflect a molecular link between the two groups reported so far. In primary cultures of human hepatocytes, hypertonic stress induces a non-selective cation conductance that is completely blocked by Gd3⫹ and flufenamate and partially inhibited by amiloride (applied at 100 ␮M each; see fig. 1) [30]. Of note, the rate of proliferation in the human carcinoma cell-line HepG2 (most likely expressing the identical channel [31]) exhibits precisely the same pharmacological profile as channel inhibition (Bondarava, ter Veld, Wehner, unpublished). This could indicate that the cation channels of RVI may actually function as mediators of proliferation, just as the K⫹ channels of RVD are employed in the opposite process, namely apoptosis. siRNA directed against ␣-hENaC inhibited proliferation by 40% which, in the light of the above hypothesis, would provide additional indirect evidence for a contribution of the ENaC to hypertonicity-induced cation channels (Bondarava, Endl, Wehner, unpublished). In primary hepatocytes, the cation channel is virtually impermeable to Cl⫺ but, in a quasi physiological range, its activity critically depends on extracellular Cl⫺ concentration [30]. This Cl⫺ sensitivity was interpreted in

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##

Relative conductance (% of maximum activation)

100 80

** ##

60 40

***

***

20 0

Control Amiloride Iso

Gd3⫹ Flufenamate

Hyper

Fig. 1. Pharmacology of hypertonicity-induced whole-cell currents (plus 40 mOsm in the bath, ‘hyper’), in primary human hepatocytes. Data are normalised to the maximal conductance obtained (as ‘control’) and each compound was tested at 100 ␮M (from [30] with courtesy]. ## Significantly different from the isotonic value with p ⬍ 0.01. **,***Significantly different from the value at maximal hypertonic stimulation (control) with p ⬍ 0.01 and p ⬍ 0.001, respectively.

terms of a safety mechanism so that, under conditions of an outwardly directed Cl⫺ gradient, the activation of the non-selective cation channel may be too dangerous since it may lead to an actual overall release of osmolytes rather than to a net cellular uptake. Of note, with a PCa/PNa of 0.7 the hepatocyte channel is also clearly permeable to Ca⫹⫹. This peculiarity may indicate of a molecular correlation of the channel to TRPs [30]. In whole-cell recordings on mouse Ehrlich-Lettre-ascites (ELA) tumour cells, hypertonic conditions activate a cation channel that is non-selective for Na⫹, Li⫹ and K⫹, but virtually impermeable to choline⫹ and NMDG⫹ [32]. The channel is sensitive to both Gd3⫹ and amiloride with an inhibition by 60 and 40%, respectively, at 1 ␮M. Interestingly, the relative blocking efficiency of amiloride and its congeners is benzamil ⬎ amiloride ⬎ EIPA, i.e. the pharmacological profile of the channel is very similar to that of the ENaC [14, 16]. This renders ELA cells a most promising system for the molecular characterisation of hypertonicityinduced cation channels. In cell-attached measurements, the activation of a 14 pS channel could be observed [32]. Taken together, whenever studied in a quantitative manner the hypertonic activation of cation channels appears to be the main mechanism of RVI. Given the significant role that various RVI transporters (including Na⫹/H⫹ anti-port, Na⫹–K⫹–2Cl⫺ symport as well as Cl⫺ channels [33–36]) appear to play in cell proliferation, no wonder then that hypertonicity-induced cation channels may function as a very efficient mediator of this process (see fig. 2).


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Hypertonic ⫹

Ca ⫹ ) (Na

Hypotonic RV

RV

I

K⫹

D K⫹

⫹

Ca ⫹ ) (Na

Apoptosis

Proliferation

Isotonic

Fig. 2. Isotonic cell shrinkage is one of the very early events in the induction of apoptosis, the programmed cell death. This shrinkage employs the same K⫹ channels that mediate the RVD of a cell under hypotonic stress. Accordingly, non-selective cation channels, which are the main mechanism of RVI under hypertonic conditions, may be part of the machinery of cell proliferation – as the process opposite to apoptosis.

RVI, the Inhibition of K⫹ Channels

In the majority of animal cells, the inside negative voltage reflects the high partial K⫹ conductance of the cell membrane in conjunction with the steep outwardly directed K⫹ gradient. Together, this generates a K⫹ current or, in other words, a continuous channel-mediated K⫹ leak out of the cell. In principle, the activation of Na⫹/K⫹-ATPase compensates for this K⫹ leak, however, because of the actual stoichiometry of 3Na⫹ over 2K⫹, the enzyme creates a significant further loss of cell osmolytes. Accordingly, any inhibition of K⫹ channels under hypertonic condition will have a dual effect on cell volume: It will reduce the K⫹ leak out of the cell per se and, as a consequence, this will then reduce the activity of Na⫹/K⫹-ATPase. Together this results in a net gain of cell osmolytes mediating or facilitating RVI. A shrinkage-induced decrease of cell membrane K⫹ conductance is reported from frog skin [37, 38], toad and rabbit urinary bladder [39, 40], rabbit proximal tubule [41, 42], the MDCK cell-line (derived from canine collecting duct) [43] and from human nasal epithelium [44]. In mouse liver, a reduction of K⫹ conductance appears to be the main mechanism of RVI [45, 46] and a transient inhibition of K⫹ channels is also supposed to contribute to the volume response of rat hepatocytes [1, 4]. Whole-cell recordings on isolated rat hippocampal neurons reveal a decrease of voltage-gated K⫹ currents under hypertonic

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stress [47]. In rabbit corneal epithelial cells, a K⫹ channel with a unitary conductance of 170 pS (in high K⫹ solutions) is identified and, apparently, a decrease in channel activity is responsible for the hypertonic reduction of whole-cell K⫹ conductance to some 40% of the control value [48]. In isolated rat colonic crypts, 50 and 100 mOsm of hypertonic stress depolarise membrane voltage by some 10 and 20 mV, respectively, coinciding with a decrease in whole-cell conductance to 70 and 50% [49]. On the molecular level, these effects appear to be related to a 16 pS K⫹ channel that is blocked by Ba⫹⫹ and charybdotoxin. Very likely, the hypertonic decrease in channel activity is triggered by a decrease of cellular Ca⫹⫹ [50]. RVD, the Activation of K⫹ Channels

The activation of K⫹ selective channels under hypotonic conditions has been reported from a variety of preparations [6]. Because of the steep outwardly directed K⫹ gradient, channel activation will lead to an immediate and sizeable release of K⫹ coinciding with a hyperpolarisation of membrane voltage. This hyperpolarisation will in turn facilitate conductive exit of Cl⫺ even if basal Cl⫺ channel activity is not changing. Likewise, conductive K⫹ release may just be elicited by the stimulation of Cl⫺ channels, Cl⫺ release and the resultant depolarisation of membrane voltage. The most effective mechanism of RVD will, of course, be the parallel activation of K⫹ and Cl⫺ channels. With the pronounced voltage-mediated coupling between both fluxes this may result in a quasi electroneutral mode of KCl export and osmotically obliged water. BKCa (or maxi K⫹) channels exhibit a linear current/voltage relation and a (big) unitary conductance that typically is in the range of 100–300 pS. They are activated by membrane depolarisation and micromolar concentrations of cell Ca⫹⫹ [51] and, very likely, these properties link BKCa channels to hypotonicityinduced and Ca⫹⫹ permeable cation channels as one of their triggering mechanisms. In most instances, maxi K⫹ channels are sensitive to Ba⫹⫹, quinine, tetraethylammonium (TEA) and (scorpion) charybdotoxin and, more selectively, they are blocked by (scorpion) iberiotoxin. BKCa channels are expressed in skeletal and smooth muscle, neurons and in various epithelia where they reside in the apical membrane [51]. Maxi K⫹ channels are designed as hetero-octamers with four pore-forming ␣-subunits (KCNMA1 or Slo, first cloned from Drosophila as dSlo [52]) and four auxiliary ␤-subunits [53–56]. ␤-Subunits have a proposed topology of spanning the membrane twice with both N and C-terminus inside the cell [51, 57]. Four isoforms of the ␤-subunit have been cloned to date (KCNMB1–4), which are mainly expressed in smooth muscle, endocrine cells, epithelia and the central nervous system. ␤-subunits are modulators of overall


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channel characteristics but apparently they may not be obligatory components of every BKCa channel [54, 57]. The ␣-subunit of BKCa channels is expressed in multiple splice variants, which generates a considerable functional diversity among different cell types; between species, however, it exhibits a high degree of homology [57, 58]. ␣-Subunits belong to the group of voltage gated K⫹ channels with six trans-membrane domains and with the typical GYGD sequence of the selectivity filter between TM 5 and 6 [59]; TM 4 includes a positively charged segment that probably is part of the voltage sensor [60, 61]. The ␣-subunits differ from other (voltage gated) K⫹ channels, however, with respect to one additional trans-membrane domain placing the N-terminus of the protein to the extracellular space and an enormous (800 amino acid) cytoplasmic C-terminal domain mediating Ca⫹⫹ sensitivity of the channel [57, 62]. Interestingly, the high unitary conductance of BKCa channels appears to be achieved by a set of surface charges facing the intra [60] and extracellular [63] space which concentrate K⫹ ions in the inner and outer vestibule of the pore. The hypotonic activation of maxi K⫹ channels is, first of all, reported from quite a number of different kidney cell preparations including Necturus and rabbit proximal tubule [64–66], the A3 cell line derived from rabbit thick ascending limb [67] as well as rat and rabbit collecting duct [68–72]. The effect was also observed in rat lacrimal gland cells [73], embryonic chick hepatocytes [74], guinea pig jejunal enterocytes [75], and in human colonic CaCo-2 [76] and G293 osteosarcoma cells [77]. Of note, the activity of many BKCa channels was found to be sensitive to membrane stretch as well. In some systems, this mechano-sensitivity is very likely to be indirect and due to a functional crosstalk to hypotonicity-induced and Ca⫹⫹ permeable cation channels [65, 67, 78]. In other tissues, however, BKCa channels appear to be directly sensitive to membrane stretch which may well contribute to their hypotonic mode of activation [72, 76, 77, 79]. Moreover, a (receptor-mediated) stimulation of maxi K⫹ channels by auto-/paracrine ATP release [59] as well as a remarkable complexity in BKCa channel tuning by protein kinases and phosphatases has been reported [51, 58, 59, 80], altogether offering a sophisticated repertoire for channel activation. Four genes are encoding for the family of intermediate to small conductance Ca⫹⫹ activated K⫹ channels, namely SK1 to SK4 (Kcnn1 to Kcnn4). The short cytosolic N-terminus of these channels is followed by six trans-membrane segments and a long intracellular C-terminal domain. Interestingly, calmodulin is constitutively attached to this domain via a specific (‘CaMBD’) motif close to the cell membrane that constitutes sensitivity of the channel to Ca⫹⫹ (with activating concentrations in the upper nanomolar range). Most likely, the members of this group function as homo-tetramers also including channel activation by calmodulin. The channel pore is probably related to a hydrophobic pocket

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between TM 5 and 6. SK channels are weakly inwardly rectifying (under symmetrical high K⫹ conditions) but their activity is not sensitive to membrane voltage [81–83]. They are specifically activated by the channel modulator 1-EBIO (1-ethyl-benzimmidazolinone) [82]. SK4 (KCNN4, IK1 or KCa4) is the only gene encoding for intermediate conductance Ca⫹⫹ activated (or IKCa) channels which typically exhibit a unitary conductance in the range of 20–80 pS. The IKCa channel was first identified in human tissues [84–86] and, shortly thereafter, the mouse and rat orthologues followed [87, 88]. IKCa channels are weakly inhibited by quinine but efficiently blocked by charybdotoxin and (more selectively) by clotrimazole. IKCa channels are insensitive to the bee venom toxin apamin, which clearly discriminates them from all isoforms of small conductance Ca⫹⫹ activated K⫹ channels (SKCa channels) [51, 84–86, 89]. IKCa channels are mainly detected in tissues rich in epithelia and endothelia from which a sizeable osmotic challenge of cells is to be expected [89]. In many systems, the activation of IKCa channels could be identified as the actual mechanism of hypotonic K⫹ release. In the human cell-line Intestine 407, for instance, hypotonic stress increases cell Ca⫹⫹ and activates a K⫹ selective channel with a unitary conductance between 30 and 18 pS (at –100 and ⫹100 mV, respectively, and with symmetrical high K⫹ solutions). The channel is inhibited by TEA, charybdotoxin and clotrimazol (but not by iberiotoxin and apamin) and molecular biology confirms the expression of IKCa (but not BKCa and SKCa) channels in these cells [90]. Interestingly, a hypotonicity-induced release of ATP and a stimulation of P2Y2 receptors appears to contribute to IKCa channel activation [91]. In human T lymphocytes, hypotonic stress activates a K⫹ conductance that is slightly inwardly rectifying and that is blocked by charybdotoxin as well as clotrimazole [92]. Transformed Madin-Darby canine kidney (MDCK-F) cells express a Ca⫹⫹ activated K⫹ channel that is inwardly rectifying with unitary conductances of 53 and 27 pS for inward and outward currents, respectively (under high K⫹ conditions). The channel is blocked by Ba⫹⫹, TEA and charybdotoxin, and it is activated by 1-EIBO. Also of note, it appears to be employed in, both, the RVD as well as the locomotion of MDCK-F cells [93–96]. In the A6 cell-line derived from Xenopus distal nephron, hypotonic stress leads to an increase of cell Ca⫹⫹ that parallels RVD and the activation of a TEA-sensitive K⫹ conductance [97, 98]. These effects appear to be related to a swelling induced K⫹ channel of 29 pS (at 0 mV in high K⫹ solutions) that is weakly inwardly rectifying and that is inhibited by quinine [99]. Extracellular ATP may contribute to the hypotonic activation of this channel since, in A6 cells, the compound elicits a TEA-sensitive K⫹ conductance [100]. The hypotonic activation of K⫹ channels that are in an intermediate conductance range has been reported for quite a number of additional preparations. In most instances, however, the actual contribution of IKCa channels


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to these experimental observations remains to be precisely defined (see [6] for review). SKCa channels (SK1 to SK3) exhibit a small conductance that (at 0 mV and under symmetrical high K⫹ conditions) amounts to some 4–18 pS [51, 82]. They are mainly expressed in the central nervous system but also found in other tissues including smooth muscle, endothelia, pancreas, liver and placenta [6]. In (electrically) excitable cells, SKCa channels generate a slow after-hyperpolarisation that limits the firing frequency of repetitive action potentials [51]. All SKCa channels are blocked by apamin but significant differences exist as for their relative sensitivities [51, 101]. The sensitivity to apamin is defined by a distinct group of amino acids in the deep pore of the channels and the same epitope also appears to determine the sensitivity of SKCa channels to D-tubocurarine, an additional quite selective blocker [51]. An actual role of SKCa channels in cell volume regulation is thus far only reported from human Mz-ChA-1 cholangiocarcinoma cells. In this system, hypotonic stress leads to a prominent increase of K⫹ conductance and this effect, as well as RVD, is partially inhibited by apamin. Ba⫹⫹ however, completely blocks RVD suggestive of additional K⫹ channels contributing to the process [9, 102, 103]. The ␣-subunits of voltage-gated K⫹ (Kv) channels typically consist of six trans-membrane domains (with the N and C-terminus in the cytoplasmic space), a region between TM 5 and 6 employed in K⫹ conduction and selectivity and a stretch of positively charged amino acids in TM 4 which is considered the key element for voltage sensing (see [104] for a comprehensive review). Kv channels assemble as a tetramer of ␣-subunits to form the aqueous ionconducting pore but the precise mechanism of channel gating is still a matter of debate [105]. The diversity of Kv channels originates from the large number of genes in this family, from alternative splicing and heteromeric assembly of ␣-subunits, post-translational modifications, as well as from numerous ancillary proteins or ␤-subunits. Currently, twelve subfamilies of ␣-subunits (Kv1–12) are recognised [104]. Kv channels were originally cloned from brain and cardiac muscle where they stabilise membrane voltage and modulate the repolarisation of action potentials [106, 107]. But they are also identified in a variety of other tissues including smooth muscle, kidney, pancreas, stomach, small intestine and colon, uterus, the auditory system, endothelial cells, T-lymphocytes, fibroblasts and spermatozoa [6, 104]. With very few exceptions, the unitary conductance of Kv channels is in the range of 5–25 pS. Kv channel blockers include charybdotoxin, 4-aminopyridine, correolide (a pentacyclic plant nortriterpenoid) as well as (scorpion) agitoxin and margatoxin [108, 109]. In many systems, Kv channels are found to contribute to RVD and/or to swelling induced K⫹ export. Mouse T-lymphocytes of the CTLL-2 clone, for instance, which are devoid of wild-type Kv channels do not exhibit any

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significant RVD response upon cell swelling. Transient transfection with Kv1.3 (Kcna3), however, almost completely restores volume regulation, cells regain voltage-dependent K⫹ currents and both effects are blunted by charybdotoxin [110]. In human T-lymphocytes, both hSK2 and Kv1.3 (KCNA3) contribute to K⫹ conductance and RVD, as was determined in a differential approach with margatoxin [92]. Transfection of mouse (Ltk⫺) fibroblasts with Kv1.5 (Kcna5) prevents isotonic cell swelling by dexamethasone, it elicits sizeable voltageactivated K⫹ currents and both effects are blocked by quinine [111]. From lightscattering measurements performed on mouse spermatocytes (and based on a pharmacological protocol), a potential contribution of Kv1.4, Kv1.5 as well as Kv4.1 (Kcnd1), Kv4.2 (Kcnd2) and Kv4.3 (Kcnd3) to RVD was postulated [112]. In mouse ventricular myocytes, hypotonic stress increases the fastinactivating K⫹ currents that are supposed to be mediated by Kv4.2 and Kv4.3 and expression of these channels in NIH/3T3 cells yields virtually identical results, also including the effects of channel phosphorylation and dephosphorylation [113]. Expression of Kv7.1 (Kv1.9, KCNQ1) and Kv7.4 (KCNQ4) in Xenopus oocytes yields voltage dependent K⫹ currents that precisely follow small changes in cell volume, with an increase and decrease under hypotonic and hypertonic conditions, respectively [114]. Of note, these effects critically depend on the actual water permeability of the oocyte membrane which had to be artificially elevated by coexpression of (AQP1) water channels. The auxiliary subunits KCNE1–4 are of no mayor significance for these effects and there is no detectable sensitivity of Kv7.2 (KCNQ2) and Kv7.3 (KCNQ3) to changes of oocyte volume [114]. Again in oocytes expressing AQP1, mouse Kv7.5 (Kcnq5) exhibits clear responses to volume changes as well and the osmosensitivity of this channel is even much higher than that of human Kv7.1 and Kv7.4. These Kv7.5 currents are also found to be potentiated by retigabine (a specific activator) and inhibited by linopiridine and XE-991 (specific blockers of KCNQ channels) [115]. Interestingly, N-terminal truncation of the human Kv7.1 channel and a treatment of oocytes with cytochalasin D abolishes volume sensitivity suggestive of a role of the actin cytoskeleton (and of defined motifs on the channel itself) in the regulatory machinery [114]. Also of interest concerning channel regulation is the serum- and glucocorticoid-inducible kinase SGK that is found to stimulate Kv1.3 and Kv1.9 currents. Most likely SGK does so by inactivating the ubiquitin-ligase Nedd4, thus (indirectly) increasing the density of Kv channels in the plasma membrane. Of note, by the same mechanism, SGK also appears to organise the abundance of the Kv ancillary protein KCNE1 (see [116] for review). There are five known members of the KCNE gene family of Kv ancillary or ␤-subunits. The founding member, MinK (historically, the ‘minimal K⫹ channel protein’, also named IsK) is coded for by the KCNE1 gene. MinK and related


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peptides (MiRPs) are integral membrane proteins with a single trans-membrane domain and the N-terminus in the extracellular space. In many instances, the stoichiometry and precise location of MiRPs within the channel complex is a matter of debate. ␤-Subunits may modulate the overall characteristics of Kv channels considerably and in many respects (see [104] for a comprehensive review). In Xenopus oocytes that were injected with MinK cRNA (and that were later found to endogenously express a KCNQ1 isoform [117]), a slowly developing voltage-activated K⫹ current becomes detectable. Under hypotonic conditions, this current is significantly increased and its activation is accelerated [118]. A contribution of MinK channels to the RVD process was also supposed for the vestibular dark cells of the gerbil inner ear [119, 120]. In mouse tracheal epithelium, RVD is insensitive to Ba⫹⫹ and apamin and only weakly inhibited by TEA. In contrast, clofilium (a rather selective blocker of the KCNQ1/KCNE1 complex) potently inhibits RVD and, in the KCNE1 (⫺/⫺) knockout mouse, there is no RVD detectable any more [121]. Expression of KCNQ1/KCNE1 in COS7-cells yields slowly activating (and non-inactivating) K⫹ currents as they are quite typical for a so-called delayed rectifier (IKs) channel [122, 123] (see also [124–129]). As one would also expect, KCNQ1 by itself is generating currents that are considerably smaller but that exhibit a much faster time-course of activation. Interestingly, hypotonic stress increases both KCNQ1/KCNE1 as well as KCNQ1 in COS7-cells. Of note, however, the actual degree of current stimulation is found to be very similar for both experimental conditions or, in other words, co-expression of KCNE1 with KCNQ1 does not appear to increase the relative sensitivity of volume-sensitive K⫹ currents [122]. K⫹ channels of the K2P family consist of subunits with four transmembrane helices and with the N and C-terminus inside the cell. Very typically, these subunits have two P domains in tandem (between TM 1,2 and 3,4) forming the K⫹ selective pore and, in every instance, a functional K2P channel is a dimer of these subunits. There is a widespread distribution of K2P channels in (electrically) excitable and non-excitable cells and, quite frequently, these channels make up the background K⫹ conductance of the cell membrane. In many cases, K2P channels are insensitive to most classical K⫹ channel blockers. Under symmetrical high K⫹ conditions, single channel conductance is in the broad range of 14–100 pS. Some K2P channels have a linear current-to-voltage relation, some exhibit slight inward or outward rectification. Of note, in K2P channels, the ‘typical’ GYG motif of the pore region is mutated to GFG or GLG (see [130–132]) for review). Interestingly, many K2P channels are directly sensitive to membrane stretch and there are three sub-families of K2P channels that appear to be relevant for hypotonicity-induced K⫹ release and RVD. These are TREK, for TWIK (Tandem of P domains in Weak Inward rectifier K⫹ channels)-RElated K⫹

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channels, TRAAK, for TWIK-Related, Arachidonic Acid-stimulated K⫹ channels, and TASK, for TWIK-related Acid-Sensing K⫹ channels [130, 131]. In whole-cell and inside-out patch-clamp measurements on COS cells expressing TREK-1 (KCNK2), membrane stretch induces K⫹ selective channels with a half-maximal activation at –23 mm Hg. TREK-1 is also activated by 10 ␮M arachidonic acid and its unitary conductance at ⫹50 mV is 48 pS [133]. There is virtually no voltage-dependence of channel activity. TREK-1 is insensitive to high concentrations of Ba⫹⫹, TEA and 4-aminopyridine but it is effectively blocked by quinine. Expression of the channel in Xenopus oocytes yields very similar effects and, in this system, TREK-1 is also activated by hypotonic cell swelling [133]. Cytosolic H⫹ appears to determine the set-point for the mechano-sensitivity of TREK-1 and a glutamate residue, E306, close to the C-terminal domain of the channel is a key element of this gating component [134]. In the same region, it is a specialised cluster of five positively charged residues that mediates the sensitivity of TREK-1 to many phospholipids, including PIP2 [135]. Interestingly, there is a reciprocal cross-talk between the actin cytoskeleton and TREK-1 (functionally including the H⫹ sensor E306 and the PKA phosphorylation site S333), so that the actin network tonically represses the mechano-sensitivity of the channel and TREK-1 expression triggers the formation of actin and ezrin-rich membrane protrutions [136]. TREK2 (KCNK10) shares many similarities with TREK-1 except that its expression is refined to a rather limited number of tissues like brain, spleen, testis and the intestine [137, 138]. When expressed in COS cells, TREK-2 forms channels of some 100 pS (in symmetrically high K⫹ solutions) that are activated by membrane stretch, low cell pH as well as by arachidonic acid and other unsaturated fatty acids [137, 138]. TRAAK (KCNK4) is the only member of the K2P channel family to be specifically expressed in the nervous system. In transfected COS cells, it forms a 45 pS channel that is activated by arachidonic acid, cytosolic acidification and membrane stretch, and the latter stimulus is synergised by a depolarisation of membrane voltage. TRAAK is blocked by Gd3⫹ and repressed by certain cytoskeletal elements, namely, the actin network and the microtubular system [139]. In whole-cell patch-clamp recordings on Ehrlich ascites tumour cells, TASK-2 (KCNK5) was identified as the mediator of hypotonicity-induced conductive K⫹ release and, when expressed in HEK293 cells, TASK-2 elicits a swelling-induced current that is very similar to the one in Ehrlich cells. This includes a stimulation at high extracellular pH values, the ion-selectivity profile and a specific block by clofilium, a class III antiarrhythmic drug. Moreover, expression of TASK-2 (but not TASK-1 and -3) could be shown in Ehrlich cells [140, 141]. A role of TASK related K⫹ channels in the RVD of Ehrlich cells was confirmed by others and, in their hands, the expression of TASK-1 could also be detected [142].


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Some 15 gene products have been identified to date that encode the subunits of inwardly rectifying K⫹ (Kir) channels. Every Kir subunit has only two trans-membrane helices bridged by a P-loop, and four of these subunits coassemble to form one functional Kir channel. This pattern is very similar to the structure of the bacterial orthologue KcsA, which actually was the first ion channel being crystallised [143]. Kir channels are classified into 5 subfamilies and among them, the Kir6.x isoforms make up the conductive elements of KATP channels that, very typically, are blocked by intracellular ATP (at millimolar concentrations) [144–146]. Four sulfonylurea receptors (SUR) of 17 TM helices each and with two ATP-binding cassettes combine with each Kir6.x tetramer to generate the fully operative KATP channel. Nevertheless, Kir6.x subunits by themselves contain the major ATP binding site that is located close to the C-terminal domain of the protein. The rectification of Kir channels is due to intracellular Mg⫹⫹ and polyamines which partially block outward currents but this effect is by far less pronounced with KATP channels than with any other member of this gene family. Most types of Kir channels are expressed in (electrically) excitable tissues but KATP channels are also found in the basal-lateral membrane of many epithelia. The single-channel conductance of KATP channels is in the range of 40–90 pS. Glibenclamide and tolbutamide block KATP channels and there is also a number of specific activators available including cromakalim, levcromakalim, pinacidil, nicorandil and diazoxide [144–147]. In cell-attached and inside-out patch-clamp recordings on isolated rat atrial myocytes, negative pressure induces a channel of 52 pS (in high K⫹ solutions) that is inhibited by ATP and tolbutamide. Moreover, the mechanic activation of the channel is potentiated by pinacidil. In the whole-cell configuration, hypotonic stress as well as pinacidil significantly increases membrane conductance and both currents exhibit a voltage dependence that is virtually identical [148]. In neonatal rat atrial appendage cardiomyocytes, KATP whole-cell currents can be activated by hypotonic stress or by the application of diazoxide. The excision of patches in an ATP free bath solutions leads to the immediate activation of KATP channels with a unitary conductance of 58 pS (under high K⫹ conditions). Channels are dose-dependently blocked by ATP with an apparent Ki of some 0.1 mM. Furthermore, RT-PCR techniques reveal the expression of Kir6.1, Kir6.2, SUR1A, SUR1B, SUR2A, and SUR2B in these cells [149]. In atrial cells from Kir6.2 KO mice, KATP currents are no longer detectable. Of note, the secretion of atrial natriuretic peptide (ANP) in response to (blood) volume expansion and atrial stretch was significantly increased in these animals. In addition, the duration of cardiac action potentials was shortened in WT but not in Kir6.2 KO mice. This suggests that KATP channels may rather serve as a negative feedback mechanism in the control of ANP release than being part of the secretory machinery [150]. It will be interesting to see if the hypotonic activation

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of atrial KATP channels just reflects their sensitivity to membrane stretch or if it represents an actual mechanism of cell volume regulation. Also of note in this respect is the role that KATP channels play in epithelial tissues where they are part of the ‘pump-leak’ machinery coupling (via ATP) the activity of Na⫹/K⫹ATPase to the basal-lateral electro-diffusive release of K⫹ [144]. In summary, the hypotonic activation of K⫹ channels is one of the most efficient mechanisms of cell volume regulation that has ever evolved. Interestingly, the K⫹ channels employed differ significantly with respect to their molecular architecture and biophysical properties which, on the other hand, underscores the general suitability of this principle of RVD. Also of note in this context is the isotonic volume decrease as it occurs as one of the very early events in the induction of apoptosis. Like RVD, this apoptotic volume decrease employs a variety of different K⫹ channels, as for instance BKCa, IKCa, Kv, K2P and Kir channels [151–155]. Once again, this proves the efficiency of conductive K⫹ release in defining cell volumes. Furthermore, it provides strong evidence for the actual functional coupling of cell volume regulation and the process of apoptosis.

RVD, the Activation of Cation Channels

In some cells, a hypotonic stimulation leads to the activation of non-selective cation channels. This is surprising, at first sight, since from the inside negative membrane voltage and the sum of driving forces for Na⫹ and K⫹ diffusion one would expect a net cation uptake rather than a cation release following this pathway. Of note, however, some of these channels are slightly more permeable for K⫹ than for Na⫹. In addition, certain channels exhibit a sizeable permeability to divalent cations and actually may participate in the Ca⫹⫹ signalling of RVD. Interestingly, some of the hypotonicity-induced and non-selective cation channels are directly sensitive to membrane stretch. In cell-attached patches on rat mesangial cells, hypotonic stress as well as negative pressure activates a cation channel with a PK/PNa of 4.7. The channel is impermeable to Ca⫹⫹ and Ba⫹⫹ and exhibits a unitary conductance of 76 pS (under symmetrical high K⫹ conditions). The mean open time of the channel increases with membrane depolarisation [156]. In the rat pheochromocytoma cell-line PC12, cell swelling and negative pressure induce a cation channel of 46 pS with high K⫹ and 27 pS with high Na⫹ in the pipette [157]. In whole-cell recordings on rabbit ventricular myocytes, hypotonic stress activates a cation conductance with a PK/PNa of 5.9 and with strong inward rectification. The cation conductance is blocked by micromolar concentrations of Gd3⫹; its Ca⫹⫹ permeability was not tested [158].


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In a second group, hypotonicity-induced channels do not discriminate much between monovalent cations and, interestingly, they exhibit a sizeable permeability to divalent cations as well. These channels are expressed in rat hepatocytes [159], in the cells of frog kidney proximal tubule [160], porcine cerebral capillaries [161, 162], rat atrium [163, 164] and guinea-pig gastric smooth muscle [165], as well as in mouse N1E115 neuroblastoma cells [166], Ehrlich ascites tumour cells [167] and frog [168–170] oocytes. In most instances, channels are blocked by micromolar concentrations of Gd3⫹ and unitary conductances are in the range of 15–40 pS. The founding member of the TRP super-family of gene products is the lightactivated cation channel of the transient receptor potential (Trp) in Drosophila [171]. Typically, TRP subunits are made up by six trans-membrane domains, a cytosolic N and C-terminus and a pore loop between TM 5 and 6, and these subunits are thought to co-assemble as homo or hetero-tetramers to form the functional cation selective channel (see [172, 173] for review). TRP channels are employed in a variety of sensational processes and in very many cases they appear to function as the actual mediators of stimulus-induced Ca⫹⫹ influx into a cell. In addition, they may contribute to the release of Ca⫹⫹ from intracellular stores. The subunits of TRP channels are divided into seven families: TRPC1–7 (‘C’ for ‘canonical’), TRPM1–8 (‘melastatin’), TRPV1–6 (‘vanilloid’), TRPP2,3,5 (‘polycystin’, with TRPP1,4,6–8 being non-conductive elements of these channels), TRPML1–3 (‘mucolipin’) and TRPA1 (‘ankyrin’). TRPN1 (‘NOMP’ for ‘no mechanoreceptor potential’) has so far only been identified in worm, fly and zebra fish [172, 173]. With the exception of TRPM4 and 5, all TRP channels are permeable to Ca⫹⫹ exhibiting PCa/PNa values of 0.3–10 or, in the case of TRPV5 and 6, of even higher than 100. TRPC1 has recently been identified as a functional component of the vertebrate mechano-sensitive and Ca⫹⫹ permeable channel that is directly gated by the tension in the lipid bilayer [174]. This is of note because, quite in contrast to bacterial systems, the activation of stretch-activated channels, in higher organisms, was supposed to employ additional auxiliary structures of a given cell for activation (see [175–177] for review). Nevertheless, the putative role of this channel in cell volume regulation remains to be elucidated (cf. to [178, 179]). TRPM3 is mainly found in the kidney and, in smaller amounts, in testis, brain and spinal chord. When expressed in HEK-293 cells, TRPM3 mediates Ca⫹⫹ uptake (triggered by intracellular Ca⫹⫹ store depletion) and a cation current that is significantly increased under hypotonic stress. These effects are blocked by Gd3⫹ and/or La3⫹ [180, 181]. In whole-cell and single-channel recordings on HeLa cells, a cation channel with a PCa/PNa(Cs) of 1.6 is observed that is activated by membrane stretch and by exposure to hypotonic media. The channel is inhibited by Mg⫹⫹ and blocked by Gd3⫹ and ruthenium red. Interestingly, channel

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activation is abolished by siRNA silencing of TRPM7 and the same maneuver blunts the RVD of HeLa cells. Moreover, in the HEK-293 system, membrane stretch as well as hypotonic stress induces Mg⫹⫹ sensitive currents, only after transfection of cells with TRPM7 cDNA [182]. In the nematode C. elegans, a number of ciliated neurons at the front edge are employed in the sensation of mechanical stimuli, osmolarity and various odorants. The prime mediator for the detection of these stimuli was identified as OSM-9, a Ca⫹⫹ permeable cation channel very closely related to the mammalian TRPV4 [183]. Four additional genes encoding a group of OSM-9/Capsaicin Receptor related (OCR1–4) proteins were then identified by homology screening and OSM-9 appears to always associate with at least one member of the OCR group to form the functional channel [184] (see also [185] for review). In Drosophila, two TRPV related proteins are expressed, namely IAV (‘inactive’, a homologue of OSM-9) and NAN (‘Nanchung’, more closely related to the OCR proteins). Both channels contribute to sound perception in the antennal chordotonal organ and when expressed in HEK and CHO-K1 cells, respectively, they mediate hypotonicity induced cation currents as well as an increase of cell Ca⫹⫹ [186, 187]. The mammalian orthologue TRPV4 (OTRPC4, VR-OAC, TRP12) is also activated by, both, hypotonicity and mechanical stress, although the latter effect does not appear to reflect an intrinsic property of the channel itself [172, 185]. Interestingly, TRPV4 appears to be expressed in particular in those tissues that exhibit a distinct swelling-induced Ca⫹⫹ influx, as for instance, kidney, liver, endothelial cells, heart and smooth muscle [185, 188–192] which may be interpreted in terms of its actual function as a volume sensor. It should be considered, however, that TRPV4 is also highly sensitive to diacylglycerol as well as to changes in temperature at values bracketing the physiological level [193]. Nevertheless, TRPV4 appears to be also employed in osmosensation on a systemic level since the channel may well be part of those structures of the hypothalamus controlling the secretion of anti-diuretic hormone [194, 195]. In murine aortic myocytes which express TRPV2, hypotonic as well as mechanical stress activates whole-cell cation currents and induces a sizeable increase of cell Ca⫹⫹. Both effects are inhibited by ruthenium red, an effective blocker of TRPV channels, and blunted by anti-sense oligo-nucleotides directed against TRPV2 [196]. In kidney, TRPP1 and TRPP2 (polycystic kidney disease [PKD]1 and PKD2) together contribute to the mechanosensation of fluid flow by stimulation of primary cilia and malfunctioning of these proteins contributes to the pathogenesis of PKD [197–199]. Taken together, TRPs are employed in the perception of a variety of different stimuli including mechanical and (hypo-) osmotic stress. Whereas, in many instances, their primary contribution to cell volume regulation, i.e. their function as transporters of osmolytes, remains to be elucidated, TRPs appear to


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operate as efficient integrators in cell signalling and, very likely, they do so in close proximity to the actual sensor of volume changes.

Phospholemman

Phospholemman (PLM, or FXYD1) is a small membrane protein of 15 kDa that was first purified, cloned and sequenced from dog heart [200]. The mature molecule consists of 72 amino acids with one single trans-membrane domain and with the C-terminus exposed to the cytoplasmic side. The name ‘phospholemman’ denotes the protein as a target for phosphorylation which, under most physiological conditions, is mediated by protein kinases A and C [200–203]. PLM belongs to a family of some seven proteins (in mammals) including the ␥-subunit of the Na⫹/K⫹-ATPase (FXYD2), mammary tumour marker 8 (MAT-8, or FXYD3) and corticosteroid hormone-induced factor (CHIF, or FXYD4) [203, 204]. These proteins share a signature of six conserved amino acids comprising the FXYD motif in the N-terminal region and two glycines and one serine in the transmembrane domain. Of note, FXYD proteins are widely distributed among mammals with the most prominent expression in tissues involved in fluid and solute transport [203, 204]. Accordingly, one of the major tasks of FXYD proteins appears to be the (tissue-specific) regulation of ion transport. PLM for instance, associates with various ␣-, ␤-isoenzymes of the Na⫹/K⫹-ATPase (see [203] for review), leading to a two-fold reduction in their affinity to cytosolic Na⫹ [205]. Furthermore, PLM could be clearly shown to modulate the activity of Na⫹/Ca⫹⫹ anti-port (NCX1) [206, 207]. When expressed in Xenopus oocytes, PLM induces anion currents that are activated by large hyperpolarisations of membrane voltage and, most likely, it does so by inducing endogenous oocyte channels [202, 208]. On the other hand, when reconstituted in lipid bilayers, PLM could be shown to form functional ion channels by itself which exhibit enormous unitary conductances in the range of 500–700 pS and rather slow gating kinetics [209]. It was also found that PLM exhibits two different modes of operation, namely a cation and an anion-permeable one, and that the protein appears to spontaneously switch between these modes [210, 211]. Interestingly, in solitary rat hepatocytes in primary culture, which exhibit a distinct RVI [212] and which do express PLM [213], hypertonic stress leads to the activation of a channel that very much resembles PLM with respect to its unitary conductance, its gating pattern and its non-selectivity for Na⫹ over K⫹. Furthermore, in Xenopus oocytes expressing rat hepatocyte PLM, hypertonic conditions induce a cation conductance and noise-analysis reveals the activation of a 670 pS channel [213].

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Over-expression of PLM in HEK293 cells significantly increases the membrane currents in response to hypotonic stress [211, 214] and it facilitates the release of Cl⫺ (I⫺) and taurine during RVD [214, 215]. Moreover, this osmolyte release could be markedly reduced be use of anti-PLM anti-sense oligo-nucleotides [216]. So apparently, PLM is activated under hypertonic as well as hypotonic conditions, preferentially operating as a cation and anion channel, respectively. One may speculate that, concerning membrane voltage and driving forces, this bimodal pattern of PLM operation would perfectly link electrogenic cation and anion transport to the actual demands of osmolyte uptake and release. The dual function of PLM as a cell volume-modulated ion channel and a regulator of membrane transport that is readily susceptible to differential upstream tuning by itself is suggestive of a more complex role of the protein in cell volume regulation than was appreciated so far.

Conclusion

In addition to the mere maintenance of homeostasis, the mechanisms of cell volume regulation contribute to a variety of most relevant physiological processes including the coordination of apical and basal-lateral transport in epithelia, the signalling on metabolic processes in the liver, the locomotion of cells, as well as the triggering of gene expression (see [6] for review). Moreover, many transporters of RVD and RVI are clearly employed in the processes of apoptosis and cell proliferation, respectively. Accordingly and taking the high efficiency of channel-mediated osmolyte transport into account, no wonder then that ion channels are of paramount importance for the physiology and patho-physiology of cells, in general.

Acknowledgement I wish to thank Bernd Nilius, Leuven, for stimulating discussions and for his comments on the manuscript.

References 1 2

Wehner F, Sauer H, Kinne RKH: Hypertonic stress increases the Na⫹ conductance of rat hepatocytes in primary culture. J Gen Physiol 1995;105:507–535. Böhmer C, Wehner F: The epithelial Na⫹ channel (ENaC) is related to the hypertonicity-induced Na⫹ conductance in rat hepatocytes. FEBS Lett 2001;494:125–128.


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193 Watanabe H, Vriens J, Suh SH, Benham CD, Droogmans G, Nilius B: Heat-evoked activation of TRPV4 channels in a HEK293 cell expression system and in native mouse aorta endothelial cells. J Biol Chem 2002;277:47044–47051. 194 Liedtke W, Friedman JM: Abnormal osmotic regulation in trpv4⫺/⫺ mice. Proc Natl Acad Sci USA 2003;100:13698–13703. 195 Mizuno A, Matsumoto N, Imai M, Suzuki M: Impaired osmotic sensation in mice lacking TRPV4. Am J Physiol 2003;285:C96–C101. 196 Muraki K, Iwata Y, Katanosaka Y, Ito T, Ohya S, Shigekawa M, Imaizumi Y: TRPV2 is a component of osmotically sensitive cation channels in murine aortic myocytes. Circ Res 2003;93: 829–838. 197 Nauli SM, Alenghat FJ, Luo Y, Williams E, Vassilev P, Li X, Elia AE, Lu W, Brown EM, Quinn SJ, Ingber DE, Zhou J: Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells. Nat Genet 2003;33:129–137. 198 Pazour GJ, San Agustin JT, Follit JA, Rosenbaum JL, Witman GB: Polycystin-2 localizes to kidney cilia and the ciliary level is elevated in orpk mice with polycystic kidney disease. Curr Biol 2002;12:R378–R380. 199 Yoder BK, Hou X, Guay-Woodford LM: The polycystic kidney disease proteins, polycystin-1, polycystin-2, polaris, and cystin, are co-localized in renal cilia. J Am Soc Nephrol 2002;13: 2508–2516. 200 Palmer CJ, Scott BT, Jones LR: Purification and complete sequence determination of the major plasma membrane substrate for cAMP-dependent protein kinase and protein kinase C in myocardium. J Biol Chem 1991;266:11126–11130. 201 Walaas SI, Czernik AJ, Olstad OK, Sletten K, Walaas O: Protein kinase C and cyclic AMP-dependent protein kinase phosphorylate phospholemman, an insulin and adrenaline-regulated membrane phosphoprotein, at specific sites in the carboxy terminal domain. Biochem J 1994;304:635–640. 202 Mounsey JP, Lu KP, Patel MK, Chen ZH, Horne LT, John JE, III, Means AR, Jones LR, Moorman JR: Modulation of Xenopus oocyte-expressed phospholemman-induced ion currents by co-expression of protein kinases. Biochim Biophys Acta 1999;1451:305–318. 203 Crambert G, Geering K: FXYD proteins: new tissue-specific regulators of the ubiquitous Na,KATPase. Sci STKE 2003;2003:RE1. 204 Sweadner KJ, Rael E: The FXYD gene family of small ion transport regulators or channels: cDNA sequence, protein signature sequence, and expression. Genomics 2000;68:41–56. 205 Crambert G, Füzesi L, Garty H, Karlish S, Geering K: Phospholemman (FXYD1) associates with Na,K-ATPase and regulates its transport properties. Proc Nat Acad Sci USA 2002;99: 11476–11481. 206 Zhang XQ, Qureshi A, Song J, Carl LL, Tian Q, Stahl RC, Carey DJ, Rothblum LI, Cheung JY: Phospholemman modulates Na⫹/Ca2⫹ exchange in adult rat cardiac myocytes. Am J Physiol 2003;284:H225–H233. 207 Ahlers BA, Zhang XQ, Moorman JR, Rothblum LI, Carl LL, Song J, Wang J, Geddis LM, Tucker AL, Mounsey JP, Cheung JY: Identification of an endogenous inhibitor of the cardiac Na⫹/Ca2⫹ exchanger, phospholemman. J Biol Chem 2005;280:19875–19882. 208 Moorman JR, Palmer CJ, John III JE, Durieux ME, Jones LR: Phospholemman expression induces a hyperpolarization-activated chloride current in Xenopus oocytes. J Biol Chem 1992;267:14551–14554. 209 Moorman JR, Ackerman SJ, Kowdley GC, Griffin MP, Mounsey JP, Chen ZH, Cala SE, O’Brian JJ, Szabo G, Jones LR: Unitary anion currents through phospholemman channel molecules. Nature 1995;377:737–740. 210 Kowdley GC, Ackerman SJ, Chen Z, Szabo G, Jones LR, Moorman JR: Anion, cation, and zwitterion selectivity of phospholemman channel molecules. Biophys J 1997;72:141–145. 211 Moorman JR, Jones LR: Phospholemman: a cardiac taurine channel involved in regulation of cell volume. Adv Exp Med Biol 1998;442:219–228. 212 Kirschner U, Tinel H, Rosin-Steiner S, Giffey A, Kinne RKH, Wehner F: Single rat hepatocytes in primary culture as a model system for the study of regulatory volume increase (RVI) in liver. Nova Acta Leopoldina 1998;306:299–303.

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Dr. Frank Wehner Max-Planck-Institut für molekulare Physiologie Otto-Hahn-Strasse 11 DE–44227 Dortmund (Germany) Tel. ⫹49 231 133 2225, Fax ⫹49 231 133 2699, E-Mail [email protected]


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Sensors and Signal Transduction Pathways in Vertebrate Cell Volume Regulation Else K. Hoffmann, Stine F. Pedersen Department of Biochemistry, Institute of Molecular Biology and Physiology, University of Copenhagen, Copenhagen, Denmark

Abstract The ability to control cell volume is fundamental for proper cell function. This review highlights recent advances in the understanding of the complex sequences of events by which acute cell volume perturbation alters the activity of osmolyte transport proteins in cells from vertebrate organisms. After cell swelling, the main effectors in the process of regulatory volume decrease are swelling-activated K⫹ and Cl⫺ channels, a taurine efflux pathway, and KCl cotransport. After cell shrinkage, the main effectors in the process of regulatory volume increase are Na⫹/H⫹ exchange, Na⫹, K⫹, 2Cl⫺ cotransport, and in some cells, shrinkageactivated Na⫹ channels. All of these proteins are regulated in a unique manner by cell volume perturbations. The molecular identity of most, although not all, of these transport pathways is now known. Among other important advances, this has lead to the identification of transporter binding partners such as protein kinases and phosphatases, cytoskeletal elements and lipids. Considerable progress has also been made recently in understanding the upstream elements in volume sensing and volume-sensitive signal transduction, and salient features of these systems will be discussed. In contrast to the simple pathway of osmosensing in yeast, cells from vertebrate organisms appear to exhibit multiple volume sensing systems, the specific mechanism(s) activated being cell type- and stimulus-dependent. Candidate sensors include integrins and growth factor receptors, while other early events include regulation of Rho family GTP binding proteins, Ste20-related protein kinases, and phospholipases, as well as cytoskeletal reorganization, Transient Receptor Potential channel-mediated Ca2⫹ influx, and generation of reactive oxygen species. Copyright © 2006 S. Karger AG, Basel

Even under steady state conditions, animal cells would swell and burst due to the Donnan effect of impermeable anions if net ion uptake was not compensated by the activity of the Na⫹, K⫹ ATPase [1–3]. In addition to this fundamental

volume-regulatory problem, a wide variety of cell types are challenged by volume perturbations resulting from changes in extra- or intracellular osmolarity under a variety of physiological or pathophysiological conditions (section ‘Physiological Relevance of Cell Volume Regulation’). The osmotic water permeabililty of animal cells is several orders of magnitude higher than that for Na⫹, K⫹, and Cl⫺. In many cells, this reflects in large part the presence of aquaporins (AQPs). The physiology and regulation of AQPs has been amply reviewed recently [4], and will not be dealt with here. However, it may be noted that some AQPs are volumeregulated, and appear to be relevant not only to osmotic water permeability but also to regulation of volume-sensitive ion transport [5, 6]. Depending on the time course of the anisosmotic challenge, several modes of volume regulation can be distinguished. Rapid changes in intra- or extracellular osmolarities elicit rapid changes in cell volume, followed by regulatory volume decrease (RVD) after cell swelling or regulatory volume increase (RVI) after cell shrinkage, respectively (for a more detailed description, see e.g. [7, 8]). When changes in osmolarity are slow and gradual, volume regulation can ‘keep up’, and the accompanying changes in cell volume may be negligible [9–11]. This phenomenon is known as isovolumetric volume regulation [12]. Finally, long-term exposure to anisosmotic conditions will, especially if the acute RVD or RVI is incomplete, elicit a wide array of adaptive changes, including, but not limited to, changes in the expression of volume-regulatory ion transporters. These have been extensively covered elsewhere [13–15], and will not be discussed here. The present contribution covers only the acute phase of cell volume regulation. Firstly, we discuss the individual osmolyte transport systems involved in acute RVD and RVI, respectively, and the volume-sensitive regulation mechanisms pertaining directly to the individual transporters. Secondly, we summarize and discuss central findings concerning the common upstream elements of volume sensing and volume-sensitive signal transduction events.

Physiological Relevance of Cell Volume Regulation

The physiological relevance of cell volume regulation is well recognized for free-living microorganisms [16] and has been particularly well described for yeast [17]. As evident from the above, many of the mechanisms involved in cell volume regulation are maintained in vertebrate organisms, in which they play important roles in a variety of physiological processes. Changes in extracellular osmolarity are experienced under physiological conditions, e.g. by intestinal epithelial cells, kidney epithelial cells, red blood cells traveling through the capillaries of these tissues [18], chondrocytes [19], as well as by brain cells [20].

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More commonly, cell volume perturbations occur as a consequence of changes in intracellular osmolyte concentration, and such changes are in fact an integral part of many physiological processes. This has been proposed, e.g. regulation of cell proliferation [21, 22], programmed cell death [23–26], cell migration [27, 28], cell metabolism [29] and epithelial secretion [14, 30–34]. Finally, cell volume perturbations are central to a number of clinically relevant pathophysiological conditions, including diabetes mellitus, dehydration states, cardiac and brain ischemia, and hypertonic resuccitation after brain edema [35–37].

Swelling-Activated Membrane Transport Systems

In the acute phase of RVD, the major membrane transport mechanisms in most cell types studied are: (i) swelling-activated K⫹ and Cl⫺ channels; (ii) a taurine efflux pathway, and (iii) KCl cotransport. The most salient features of the regulation of these transport pathways by cell volume are discussed below. More thorough descriptions of their structure, physiology and regulation may be found elsewhere [38–41]. Swelling-Activated K⫹ Channels (IK,vol) That K⫹ loss during RVD results from a swelling-activated conductive K⫹ efflux pathway was shown by unidirectional and net flux measurements in lymphocytes [42] and in Ehrlich ascites tumour (EAT) cells [43] and subsequently in many other cell types. A wide variety of K⫹ channels have been shown to be volume sensitive in various cell types, including: (i) Ca2⫹-activated K⫹ channels of small conductance (SK) [44, 45]; of intermediate conductance (IK) [44, 46–49] and large conductance (BK) [50, 51], although expression studies in oocytes indicated that the latter were only modestly volume sensitive [52]; (ii) Voltagedependent K⫹ channels including Kv1.3 or Kv1.5 [53, 54], Kv4.2/4.3 [55], as well as Kv7 channels (a.k.a. KCNQ) channels. Of these, KCNQ1, KCNQ4, and KCNQ5 channels are activated by cell swelling, whereas KCNQ2 and KCNQ3 are volume-insensitive [56–58]; (iii) Two-pore-domain K⫹ (K2P) channels, including the 2P-4TM, acid-sensitive potassium channel (TASK-2, aka KCNK5) [59–62], (TREK-1, a.k.a. KCNK2) and (TRAAK, a.k.a. KCNK4) [63–65]. Activation of IK,vol by Cell Swelling Membrane Stretch. Several K⫹ channels, including some K2P channels [63–67] and BK channels [68] appear to be activated both by cell swelling and directly by membrane stretch. At least in some cases, stretch-activated K⫹ channels can account for whole cell K⫹ currents in RVD [69]. However, this is not a universal mechanism of IK,vol activation, as in other cells, e.g. EAT cells,

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the swelling activated K⫹ channel cannot be activated directly by membrane stretch [47]. Cytoskeleton. The actin cytoskeleton has been implicated in the regulation of several swelling-activated K⫹ channels, including IK and SK, the swellinginduced activation of which was inhibited by cytochalasin treatment [44], KCNQ1 [56], and swelling-activated voltage-gated K⫹ channels in trigeminal ganglion neurons, the activation of which by cell swelling was stimulated by cytochalasin treatment [70]. The cortical actin crosslinking protein non-muscle filamin seemed to be required for swelling-induced K⫹ channel activation and RVD in melanoma cells [71]. A role for microtubules in swelling-induced K⫹ channel activation has also been suggested based on K⫹ flux measurements in rat colonic epithelium [72]. The Free, Intracellular Calcium Concentration ([Ca2⫹]i). In some cells, typically epithelial cells, Ca2⫹ influx is important for the RVD response [18, 73]. However, in other cell types, neither the RVD response [18, 74–76] nor swelling-induced activation of IK,vol [77] are dependent on a rise in [Ca2⫹]i. Consistent with this notion, the swelling-activation of Ca2⫹-activated K⫹ channels appears to be independent of a detectable increase in [Ca2⫹]i in several cell types studied [44, 52, 68], although an additional increase in [Ca2⫹]i will of course facilitate their activation. Autocrine signaling through ATP released to the extracellular medium contributes to RVD in many cell types [78, 79], at least in some cases via an increase in [Ca2⫹]i and activation of Ca2⫹-sensitive K⫹ channels [80]; for a discussion of the pathways of swelling-induced ATP-release, see [81]. However, this mechanism cannot ubiquitously account for activation of IK,vol. For instance, in EAT cells, addition of ATP elicits an increase in [Ca2⫹]i [82], yet does not activate IK,vol, but rather a Ca2⫹-activated, IK-like, charybdotoxin-sensitive channel [83]. Lipid Mediators. Arachidonic acid is released from membrane phospholipids during cell swelling in many cell types (see [84–87] and section ‘Phospholipase A2’ for the involvement of phospholipases in volume-sensing). Arachidonic acid at micromolar concentrations directly stimulates TRAAK [88], yet conversely arachidonic acid appears to inhibit TASK-2 [62]. Several of the eicosanoids resulting from arachidonic acid metabolism also play a role in activation of IK,vol (fig. 1). In EAT cells, leukotriene D4 (LTD4) activates IK,vol independent of an increase in [Ca2⫹]i [76, 77, 83], consistent with the role of LTD4 in RVD in these cells [89]. In human platelets, the 12-HPETE product, hepoxylin A activates IK,vol [90], and moreover, BK has been shown to be activated by 3,15 di-HETE and 3-HETE [91]. Finally, it may be noted that K2P


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Fig. 1. Comparison of the K⫹ current (IK) activated by cell swelling and LTD4. Panels (a) and (b) show the development of currents upon exposure of EAT cells to a hypotonic solution or to 5 nM LTD4 under isotonic conditions, respectively. High K indicates periods where extracellular Na⫹ was replaced by K⫹. Panels (c) and (d) show the current-voltage (IV) relation of IK,vol and of the IK activated by LTD4 (5 nM), respectively, measured in the standard or K⫹ rich extracellular solutions. IV relations for IK were obtained from the wholecell current corrected for any contribution from ICl as described in [62]. Results are from [62, 77]. Reproduced with permission from reference [405].

channels have been found to be inhibited by PtdIns(4,5)P2 [92], consistent with the decrease in PtdIns(4,5)P2 noted upon cell swelling [93]. Swelling-Activated Anion Current (ICl,vol) Due to the substantial exchange diffusion of anions across cell membranes particularly via the Cl⫺/HCO3⫺ exchanger [94], it was initially assumed that the basal cellular Cl⫺ conductance (gCl) was very high, and thus that an increase was not necessary for RVD (for a discussion, see [95]). However, studies in EAT cells later showed that gCl was much lower than gK [96], and increased in response to cell swelling along with gK [97, 98]. Subsequently, swelling-activated anion

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currents (ICl,vol), mediated by channels denoted volume-regulated anion channels (VRAC), or volume-sensitive, outwardly rectifying anion channels (VSOR), have been demonstrated in essentially all cell types studied [99, 100]. The biophysical and pharmacological characteristics of ICl,vol have been described in detail elsewhere [99, 100] and include: moderate outward rectification, a varying degree of depolarization-induced inactivation, an Eisenman I permeability sequence, and inhibition by classical Cl⫺ channel blockers such as DIDS and NPPB, and frequently also by tamoxifen. Although multiple candidates have been proposed, the molecular identity of VRAC is unknown (for a discussion, see e.g. [101]). The lack of inhibitors of high selectivity and affinity also hampers progress in the understanding of these channels [102]. Activation of ICl,vol by Cell Swelling Membrane Stretch and Composition. Activation of ICl,vol does not appear to involve membrane stretch [47, 100]. Decreases in membrane cholesterol content stimulate ICl,vol in several cell types [103–106] (fig. 2). Notably, cholesterol depletion has been shown to activate ICl,vol in non-swollen cells [106], suggesting that it modulates the volume-sensing pathway controlling ICl,vol activity. The modulation of ICl,vol by cholesterol is proposed not to involve a specific interaction of cholesterol with the channel, but rather a change in the physical properties of the membrane. This change appears to be at least in part related to changes in the F-actin cytoskeleton induced by cholesterol depletion [103, 107, 108]. ICl,vol stimulation by cholesterol depletion is also seen in caveolin-deficient Caco-2 cells and after sphingomyelinase treatment, and thus is not dependent on caveolae [105]. In fact, since cholesterol depletion is known to disrupt lipid rafts/caveolae, the stimulation of ICl,vol by cholesterol depletion is not easily reconcilable with the reported positive role for caveolae in ICl,vol regulation [109–111]. Cytoskeleton. Variable effects of F-actin disruption on ICl,vol have been reported in different cell types [93, 103, 112–115]. A number of studies have pointed to a stimulatory role of F-actin disruption on ICl,vol [112, 114, 116], and a role for F-actin dependent unfolding of membrane invaginations in ICl,vol activation by swelling has been suggested [100, 116]. In Ehrlich Lettre ascites (ELA) cells, isotonic ICl,vol activity was found to be stimulated, and swellingactivated activity to be partially inhibited by latrunculin B-induced F-actin disruption, suggesting that (different parts of the) actin cytoskeleton play a dual role, keeping ICl,vol silent under isotonic conditions, yet being involved in its shrinkage-induced stimulation [103]. On a similar note, it was recently proposed that activation of ICl,vol is dependent on, on the one hand a disruption of peripheral actin, and on the other, on signal transduction events requiring the integrity of perinuclear F-actin [55]. Also providing a link to the cytoskeleton,


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255 mOsm

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Fig. 2. Effect of cholesterol depletion on the swelling activated anion current (VRAC) in ELA cells and effect of hypotonic swelling on Rho activity in control cells and cholesterol depleted cells. a Cellular cholesterol content was decreased using empty M␤CD and anion current activation at ⫺55 and ⫹40 MV was measured after exposure to 225 mOsm using a fast ramp protocol. b ELA cells were exposed to Ringer solutions of the osmolarity indicated for 3 min. The GTP-bound Rho was collected using a GST-RBD pull down assay and the amount of precipitated GTP-Rho as well as total Rho was detected by SDS-PAGE and Western blotting using a monoclonal Rho antibody. Band intensity was quantified densitometrically. Rho-GTP band intensity values were normalized to the corresponding total band intensity values and are given relative to the value in the isotonic control. *, ** ⫽ Significant differences with p-values of ⬍0.05 and ⬍0.01, respectively. Redrawn from reference [103].

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recent findings strongly point to a role of integrins in ICl,vol activation (section ‘Integrins and Growth Factor Receptors as Volume Sensors?’). [Ca2⫹]i. In most cells, activation of ICl,vol does not require an increase in [Ca ]i [40, 100, 117–119]. However, in a few cell types, Ca2⫹-dependence of ICl,vol has been proposed [120, 121], and moreover, similar to what was discussed above for IK,vol, in cell types in which RVD is associated with an increase in [Ca2⫹]i, a contribution from Ca2⫹-activated Cl⫺ channels is to be expected. 2⫹

Protein Phosphorylation and Dephosphorylation. Tyrosine kinase inhibitors attenuate, and tyrosine phosphatase inhibitors stimulate, ICl,vol in several cell types [122–125]. The regulation of ICl,vol by tyrosine phosphorylation– dephosphorylation events may however be complex and cell type-dependent. Thus, the Src family kinase p56lck was found to be involved in activation of ICl,vol in lymphocytes [122], whereas in human atrial myocytes, Src family kinases and the epidermal growth factor receptor (EGFR) tyrosine kinase were reported to have opposite effects on ICl,vol, with inhibition of Src kinases stimulating, and inhibition of EGRF kinase activity inhibiting ICl,vol, respectively [126]. Ser/thr protein phosphorylation events also appear to be important in control of ICl,vol. Thus, Rho kinase has also been shown to stimulate ICl,vol [127, 128], in congruence with the swelling-induced activation of Rho (section ‘Small GTP-Binding Proteins and Their Effectors’). Interestingly, myosin light chain kinase (MLCK) was found to stimulate ICl,vol in pulmonary artery endothelial cells [129], while a marked inhibitory effect of MLCK on ICl,vol was observed in NIH3T3 cells [128] (fig. 3). Reactive Oxygen Species. Reactive oxygen species (ROS), which are released in response to osmotic cell swelling in at least some cell types (section ‘Reactive Oxygen Species’), are emerging as important modulators of ICl,vol [130–133]. ROS-mediated ICl,vol stimulation could, however, be inhibited by osmotic cell shrinkage [133], indicating that ROS is not the volume signal per se, but a downstream modulator of channel function. Interestingly, EGF-mediated stimulation of ICl,vol was proposed to be ROS-mediated [133]. The mechanism of ROS-induced ICl,vol stimulation remains to be elucidated, although a role for phosphorylation-dependent events was tentatively suggested [132, 133]. Swelling-Activated Taurine Efflux Taurine (2-aminoethanesulphonic acid), a non-metabolized end product synthesized from cysteine, is a major organic osmolytes in many mammalian cells [39, 41, 134, 135]. The swelling-activated taurine leak pathway has been suggested to be identical to VRAC [136]. However, although the pore of VRAC


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Fig. 3. Involvement of Rho kinase and MLCK in regulation of ICl,vol. a Effect of Y27632 on mean current density in NIH3T3 cells. Cells were pre-incubated with (filled circles) or without (open circles) Y-27632 (10 ␮M) for 10 min, prior to exposure to a 30% hypotonic solution in the continued presence of Y-27632. Linear voltage ramps were applied every 10 s. The traces shown are representative of 6 (control) and 5 (Y-27632) experiments. The traces shown are paired experiments from the same day, and are representative of 38 (ctrl) and 6 (ML-7) independent experiments. b Effect of ML-7 on mean current density in wild type NIH3T3 cells. The cells were loaded with ML-7 (10 ␮M, filled circles) in the patch pipette for 3 min prior to initiating the experiments. Open circles show a representative control trace from the same day and passage. Linear voltage ramps were applied every 10 s, and currents measured at ⫹80 and ⫺60 mV. Current density was calculated from the current magnitude measured at ⫹80 and ⫺60 mV after full activation of the current by hypotonic exposure, divided by cell capacitance. Redrawn from reference [128].

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is large enough to permit amino acid efflux [101, 137], it is clear that in many cell types, taurine leaves the cell via a pathway different from VRAC [39, 138, 139]. Strongly supporting this notion, swelling-activated taurine release in the absence of Cl⫺ channel activity was directly demonstrated in Xenopus oocytes [140]. In skate erythrocytes, the taurine transport pathway has been shown to be the Cl⫺/HCO3⫺ exchanger (AE, band 3) [141, 142]. In mammalian cells, the molecular identity of the non-VRAC taurine efflux pathway is unknown. Regulation of the Taurine Efflux Pathway by Cell Swelling The regulation of swelling-activated taurine efflux has recently been reviewed in detail [39], and will only be considered briefly here. The main mechanisms proposed for regulation of this channel are: Tyrosine Phosphorylation and Dephosphorylation Events. Tyrosine kinase inhibitors attenuate, and tyrosine phosphatase inhibitors stimulate swellinginduced taurine efflux [39, 128, 141, 143–145]. Similar to what has been found for ICl,vol, Src family kinases appear to be important regulators of swellinginduced taurine efflux [141, 146]. Specifically, a role for p72syk and p56lyn was reported in skate red blood cells, apparently involving phosphorylation of the AE [141]. On the other hand, tyrosine phosphorylation alone could not account for swelling-activation of taurine efflux in the skate RBCs, which appeared to be dependent on vesicular insertion of AE proteins [147]. [Ca2⫹]i and Calmodulin. In some cells [148, 149], yet not in others [150], Ca influx is required for swelling-induced taurine efflux. Moreover, in some cells, Ca2⫹-mobilizing agonists potentiate swelling-induced taurine efflux [151]. Interestingly, calmodulin (CaM) appears to be required for swelling-activated taurine efflux in some cells [152], yet be inhibitory in others [39], possibly reflecting the involvement of different phospholipases (section ‘Phospholipase A2’). 2⫹

Lipid Mediators. A variety of lipid mediators derived from arachidonic acid have been found to play a role in swelling-induced activation of taurine efflux. Interestingly, the specific metabolite involved differs between cell types. LTD4 is involved in of the swelling-induced activation of taurine efflux in EAT cells [153], and 5-HETE stimulates swelling-induced taurine efflux in HeLa cells [154]. Arachidonic acid metabolites, yet not leukotrienes, were also implicated in regulation of osmolyte efflux in chondrocytes [39, 155]. Reactive Oxygen Species. ROS generated by cell swelling (section ‘Reactive Oxygen Species’) have been shown to potently stimulate swelling-induced taurine efflux in several cell types [143, 156]. The mechanism of ROS-mediated stimulation of this pathway is not fully elucidated, but ROS appeared to act


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downstream from phospholipase activation and the effect on taurine efflux was proposed to involve inhibition of a tyrosine phosphatase [39, 143]. Swelling-Activated KCl Cotransport Potassium-chloride cotransport has long been known to play a role in RVD in a wide range of cell types [38, 157–161]. The potassium-chloride cotransporters (KCCs) belong to the SLC12A family of cation-chloride cotransporters, and 4 isoforms, KCC1–4 (SLC12A4–7), have been cloned to date [38]. The KCCs are ⬃120 kDa protein with a proposed membrane topology of 12 transmembrane (TM) domains and a short N-terminal and long C-terminal cytoplasmic region [162]. The ubiquitous isoforms, KCC1 and KCC4, are both activated by cell swelling [163]. KCC2, which is neuronal-specific was found to be volumesensitive in some [164, 165], although not all [166] studies, and KCC3, which is expressed predominantly in kidney, heart, brain, and liver, also appears to be volume-sensitive [38, 167]. Activation of KCC by Cell Swelling Phosphorylation-Dephosphorylation Events. Substantial evidence supports a role for protein phosphorylation–dephosphorylation events in KCC regulation by swelling [161]. Early relaxation kinetic studies indicated that KCC activation during cell swelling is associated with net dephosphorylation, and, conversely, inactivation during cell shrinkage with net phosphorylation [168]. Consistent with this notion, a wide range of studies have demonstrated the inhibition of KCC by ser/thr phosphatase inhibitors, and the stimulation of KCC by ser/thr kinase inhibitors, respectively [161, 163, 169–173]. In most cases, protein phosphatase 1 (PP1) has been implicated as the regulatory phosphatase, however, recent evidence suggests that PP2A also plays a role [169]. The C-terminal domain of KCC1 contains consensus sites for phosphorylation by casein kinase and protein kinase C (PKC) [162]. KCC3, yet not KCC1 and KCC4, interact directly with the Ste20-related Proline-Alanine-rich-Kinase (SPAK) via the N-terminal domain [174]. SPAK has been proposed to phosphorylate and deactivate KCC2 in a manner dependent on a With-No-lysine (K) kinase (WNK4), a mechanism proposed to be central to the volume-sensitivity of KCC2 [164]. Conversely, overexpression of catalytically inactive SPAK resulted in isotonic KCC2 activation to a degree similar to that seen during cell swelling [175]. The roles of Ste20-related kinases and WNKs in volume sensing will be discussed below (section ‘Ser/thr Protein Kinases: Ste20 Related Kinases and With-No-Lysine (K) Kinases’). Finally, it may be noted that the MLCK inhibitor ML-7 stimulates KCC activity in red blood cells, albeit at a Ki unlikely to suggest the involvement of MLCK, and instead proposed to reflect modulation of a volume-sensitive KCC regulatory kinase other than MLCK [176].

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Shrinkage-Activated Membrane Transport Systems

In the acute phase of RVI, the major membrane transport mechanisms in most cell types studied are: (i) a Na⫹/H⫹ exchanger (NHE) operating in parallel with a Cl⫺/HCO3⫺ exchanger (AE), and (ii) a Na⫹, K⫹, 2Cl⫺ cotransporter (NKCC). In addition, shrinkage-activated Na⫹ channels play an important role in acute RVI in some cell types. A thorough description of the structure, physiology and regulation of these three classes of transporters by other stimuli than cell shrinkage is not the aim of the present contribution, and may be found elsewhere [36, 41, 177, 178]. The Na⫹/H⫹ Exchangers To date, nine mammalian isoforms of the NHE (SLC9A) family have been cloned and characterized. The NHEs are ⬃600–800 amino acid proteins with a proposed membrane topology of 12 TM domains and a long C-terminal cytoplasmic tail [177, 179]. NHE1–5 mediate Na⫹/H⫹ exchange across the plasma membrane (NHE3 and NHE5 are also found in recycling endosomes, reflecting their regulation by vesicular insertion/retrieval [180–182]), NHE6, NHE7, and NHE9 are thought to reside predominantly in intracellular organelles. Finally, the subcellular localization of NHE8 is controversial, with both plasma membrane and organellar localization reported [177, 183–185]. Of the plasma membranelocated isoforms, NHE1 is by far the most ubiquitous, being found in essentially every cell type studied. NHE1 consists of two major regions: an ⬃500 amino acid N-terminal domain consisting of 12 TM helices which is necessary and sufficient for ion translocation, and an ⬃300 amino acid cytosolic region with important roles in NHE1 regulation (for recent reviews, see e.g. [36, 177, 186]). NHE1 is robustly activated by osmotic shrinkage, and is likely the predominant isoform mediating RVI in the great majority of tissues [187, 188]. NHE2, NHE3, and NHE4 are predominantly expressed in epithelia of the kidney and gastrointestinal tract [177, 183]. The volume-sensitivity of NHE2 is somewhat controversial and perhaps cell-type dependent, with shrinkage-activation reported upon expression in AP1 cells [189], yet not in PS120 cells [190, 191]. NHE4 is potently shrinkageactivated and has been suggested to play a special role in volume regulation under conditions of very high osmolarity, as e.g. prevalent in the renal medulla [192]. In contrast to these isoforms, NHE3 is inhibited by shrinkage [189, 190, 193]. This is also the case for NHE5, which exhibits higher sequence homology to NHE3 than to the other NHE isoforms, and which is mainly found in the brain [194]. Regulation of NHE1 by Cell Shrinkage This section will focus on NHE1, since little is known about the mechanisms by which the other NHE isoforms are regulated by cell shrinkage. As will be


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evident, however, also the mechanism(s) of shrinkage-induced activation of NHE1 remain to be fully elucidated, although some progress has been made in recent years. It may be noted that shrinkage-activation of NHE1 appears to be dependent on cell shrinkage per se, rather than on an increase in intracellular ionic strength [195]. Shrinkage-activation of NHE1 does not appear to be associated with an increase in the number of exchangers in the plasma membrane, but rather with an increased affinity for intracellular H⫹ [196, 197]. Recent findings in dog red blood cells moreover suggested that shrinkage-activation of NHE1 involves a decreased inhibition of the exchanger by extracellular Na⫹ [198]. Protein-Protein and Protein-Lipid Interactions. NHE1 interacts directly, via its C-terminal cytoplasmic region, with a wide array of proteins. To date, the identified protein binding partners of NHE1 include calcineurin homologue protein [199], CaM [200, 201], carbonic anhydrase II, ezrin/radixin/moesin (ERM) proteins, PP1 [202], the Ste20-related protein kinase Nck-interacting kinase (NIK) [203] (not to be confused with Nf␬B-activating kinase, also abbreviated NIK), heat shock protein 70 [204], and when NHE1 is phosphorylated at Ser703, 14–3-3 protein interacts with this residue [205]. Below, we briefly discuss the evidence potentially implicating NHE1 binding partners in volume-sensitive regulation of NHE1. Calmodulin. Deletion of the high-affinity CaM binding site on NHE1 results in a constitutively active transporter, which is largely insensitive to cell shrinkage [200, 201], and CaM antagonists have been shown to inhibit shrinkage-induced NHE1 activation [206]. Shrinkage-activation of NHE1 does not require increases in [Ca2⫹]i, and is unaffected even when [Ca2⫹]i is strongly buffered [206], hence, CaM binding to NHE1 appears to be independent of [Ca2⫹]i. In CHO-K1 cells, a pathway involving hypertonicity-induced Jak2 activation and Jak2-dependent tyrosine phosphorylation of CaM was proposed to be involved in mediating the CaM–NHE1 interaction [207]. ERM Proteins and F-Actin. NHE1 interacts directly with ERM proteins, which thus link the exchanger to the actin cytoskeleton. This interaction plays a central role in NHE1-mediated control of cell morphology and motility [208, 209]. However, the fact that ERM proteins connect integral membrane proteins with the actin cytoskeleton may also makes them candidates for a volume-sensing pathway for NHE1 (see section ‘Cytoskeletal Rearrangement’). Cytochalasininduced disruption of F-actin generally [210, 211], although not ubiquitously [212, 213] has been found to have no effect on shrinkage-induced NHE1 activity. In contrast, the activity of both NHE3 (after acid-loading) [214], and NHE4 (after osmotic shrinkage) [210], is dependent on the integrity of F-actin.

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Nck-Interacting Kinase. Given the important role of Ste20-related kinases in osmosensing in yeast, and likely also in higher eukaryotes (see section ‘Ser/thr Protein Kinases: Ste20 Related Kinases and With-No-Lysine (K) Kinases’), it is notable that the Ste20-related kinase NIK binds directly to, and phosphorylates, NHE1 after stimulation by e.g. platelet derived growth factor (PDGF) [203]. The possible role of NIK in shrinkage-induced NHE1 activation has, to our knowledge, not yet been examined, and this is an important area for future research. Protein Phosphatase 1. The ser/thr protein phosphatase PP1 interacts directly with NHE1 [202]. PP1 appears to be the most important protein phosphatase involved in NHE1 dephosphorylation. Consistent with this, NHE1 is activated by ser/thr phosphatase inhibitors such as calyculin and okadaic acid [187, 215, 216] (fig. 4). Moreover, several ser/thr protein phosphatase, including PP1 and PP2A, have been found to be activated by cell swelling [217], and hence might be expected to be inhibited by cell shrinkage, consistent with increased NHE1 activation. PtdIns(4,5)P2 and Other Membrane Lipids. NHE1 interacts directly with the phospholipid phosphatidyl-inositol 4,5 bisphosphate (PtdIns(4,5)P2), and this interaction was shown to be required for full activity of the exchanger [218]. This is interesting in light of the fact that PtdIns(4,5)P2 levels have been found to be increased by cell shrinkage in both mammalian cell types [219, 220]; and plants [221], however, the possible involvement of PtdIns(4,5)P2 in volumedependent regulation of NHE1 has to our knowledge not been directly tested. After expression in CHO cells, yet not in AP1 cells, regulation of NHE1 by cell volume perturbations was sensitive to the lipid composition of the plasma membrane, such that cholesterol enrichment and other manipulations which thickened the membrane activated NHE1 [222]. Although the cell-type dependence of this phenomenon may argue against a general role for membrane lipids in the volume sensing pathway of NHE1, it is interesting to note that the swelling-activated Cl⫺ current, VRAC, is oppositely regulated by cholesterol such that cholesterol depletion activates the channel (section ‘Activation of ICl,vol by Cell Swelling’). It is not clear how NHE1 is regulated by the membrane lipid composition, but it may be relevant in this regard that the first extracellular loop of NHE1 has been assigned a role in volume-sensitivity by a mechanism tentatively proposed to involve a sensing of membrane curvature, strain, or via protein–lipid interactions [191]. Another possibility involves regulation dependent on the integrity of caveolae, to which NHE1 has been proposed to localize [223]. Protein Phosphorylation-Dephosphorylation Events. In contrast to activation by growth factors, which is at least in part dependent on direct NHE1 phosphorylation [224], removal of the majority of the protein kinase consensus sites


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Na⫹ content (mmol/kg dcs)

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Fig. 4. Regulation of NHE1 from Pleuronectes americanus red blood cells by protein phosphorylation and dephosphorylation. a Localization of the Pleuronectes americanus (pa)NHE1 in red blood cells from the winter flounder, P. americanus. b Activation of NHE1 by osmotic shrinkage and by the ser/thr protein phosphatase inhibitor calyculin A (CLA). Cellular Na⫹ content (mean ⫾ SEM) over time in P. americanus red blood cells. Cells were exposed to isotonic (control) or hypertonic (1.6 times isotonic osmolarity) saline in the absence or presence of calyculin A (100 nM), and cellular Na⫹ content was monitored over time using net flux measurements. c Hypotonic swelling inhibits the activation of NHE1 by calyculin A, indicating that this process is at least in part dependent on a volume-sensitive kinase. Experiments were carried out as in (b), except that where indicated (hypo), cells were exposed to hypotonic saline (0.5 times isotonic osmolarity). Data are mean ⫾ SEM at time 30 min after stimulation. d Phosphorylation of paNHE1 on serine residues is unaltered by osmotic shrinkage. Cells were exposed to either hypertonic (1.6 times isotonic osmolarity) saline or to isoproterenol, which stimulates paNHE1 via a pathway different from that activating the exchanger after osmotic shrinkage. Subsequently, paNHE1 was immunoprecipitated and subjected to SDS-PAGE gel electrophoresis and Western blotting using an antibody against phosphorylated serine. Data are normalized to the level of NHE1 in the samples [216]. * ⫽ Significant difference (p ⬍ 0.05) between CLA and hypo ⫹ CLA (c) or compared to the control value (d). Results are redrawn from reference [216, 372, 406].

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did not prevent NHE1 activation by cell shrinkage [196, 225]. In congruence with this, no detectable increase in the direct phosphorylation of NHE1 is noted upon shrinkage-induced activation [216, 226]; see [177, 216, 227] (fig. 4). Nonetheless, several lines of evidence support the involvement of protein phosphorylation events, albeit indirectly, in shrinkage-induced NHE1 activation. A number of ser/thr protein kinases, including PKC, p38 mitogen-activated protein kinases (MAPK), c-Jun N-terminal kinase 1 (JNK1) [187, 228], and the tyrosine kinase Jak2 [207] have been proposed to play a role, directly or indirectly, in shrinkageinduced NHE1 activation. Conversely, as noted above, ser/thr phosphatase inhibitors activate NHE1 and potentiate its shrinkage-induced activity [187, 215, 216]. Notably, activation of NHE1 by phosphatase-inhibitors is abolished when the cells are osmotically swollen, indicating that it is dependent on the activity of a shrinkage-activated kinase (fig. 4). Since the NHE1 binding proteins listed above are all phosphoproteins, it seems likely that the role of protein phosphorylation reflects a phosphorylation-dependence of the interaction between NHE1 and one or more of its binding partners, as suggested, e.g. for the Jak2-mediated tyrosine-phosphorylation-dependent NHE1-CaM interaction [207]. The Na⫹, K⫹, 2Cl⫺ Cotransporter The NKCCs belong to the cation-chloride cotransporter family, SLC12A. Two mammalian NKCC isoforms have been cloned: NKCC1 (SLC12A2), which is generally basolaterally targeted, and ubiquitously expressed, and NKCC2 (SLC12A1), which is apically targeted and kidney-specific [178]. NKCC1 is a ⬃1,200 amino acid protein with an apparent topology of 12 central TM domains and long cytosolic N- and C-termini. NKCC2 has a similar structure and exhibits about 60% homology to NKCC1 at the amino acid level [38, 178]. NKCC1 is potently activated by osmotic cell shrinkage [38, 178, 229–231]. However, given the fact that the driving force for transport of ions into the cell by NKCC1 is much less robust than that for inward Na⫹ transport by NHE1, the extent to which the activation of NKCC1 actually contributes to RVI is dependent on the mode of shrinkage, as excellently discussed by Russell [178]. The activity of NKCC2 is also stimulated by osmotic cell shrinkage, apparently in a manner involving phosphorylation of the same three threonine residues shown to be important for shrinkage-induced NKCC1 activation [232]. Regulation of NKCC1 by Cell Shrinkage Given its widespread expression, and since the great majority of studies were done on this isoform, only NKCC1 will be considered here. Protein-Protein Interactions. Although the number of identified binding partners is not as extensive as for NHE1, NKCC1 does exhibit regulated


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interactions with several proteins. These include PP1 [233], PP2A [234], the closely related Ste20 related kinases SPAK and Oxidative Stress Response kinase-1 (OSR1) [174, 235], and heat shock protein 90, although the latter probably only when NKCC1 is in its immature state [236]. NKCC1, SPAK, and p38 MAPK appear to form a complex, which has been proposed to function as a stress relay system modulating the activity of p38 MAPK [235]. Protein Phosphorylation and Dephosphorylation Events. In contrast to what was discussed above for NHE1, there is significant evidence for a major role for direct phosphorylation in shrinkage-activation of NKCC1 [178, 230, 231, 237]. A number of ser/thr protein kinases have been implicated in shrinkage-induced NKCC1 activation, albeit not necessarily such that the kinase in question directly phosphorylates NKCC1. These include MLCK [238, 239], JNK [240], PKC [229, 241] and extra cellular signal regulated kinase (ERK) [219]. Substantiating the notion that protein phosphorylation is central to NKCC1 function, inhibitors of PP1 and PP2A activate NKCC1 and potentiate and prolong its activation by osmotic shrinkage [230, 239, 242], and also increase NKCC1 phosphorylation [230]. MLCK, Myosin, and F-Actin. MLCK has been implicated in shrinkageinduced NKCC1 activation in multiple cell types, largely based on the inhibitory effect of the MLCK inhibitor ML-7 on NKCC1 activity [230, 238, 239]. Given that the MLCK target, myosin light chain (MLC) is phosphorylated upon cell shrinkage [230, 238], MLC phosphorylation was thought to play a central role in the process. However, in kidney epithelial cells, shrinkageinduced MLC phosphorylation yet not NKCC1 activation was Rho kinase dependent, whereas NKCC1 activation yet not peripheral MLC phosphorylation was blocked by ML-7 [243, 244]. These findings point to a lack of simple coupling of MLC phosphorylation and NKCC1 activation, although a basal level of myosin activity did appear to be required [243, 244]. Thus, at least in some cells, the effect of ML-7 on NKCC1 activity is independent of MLC phosphorylation, and in fact it is possible that high concentrations of ML-7 may affect a volume-sensitive kinase other than MLCK [176, 243]. On the other hand, in EAT cells, the Ki for inhibition of shrinkage-induced NKCC1 activity by ML-7 is in the nanomolar range, consistent with the involvement of MLCK and hence likely MLC [239]. The shrinkage-induced reinforcement of the cortical F-actin network and translocation of non-muscle myosin II to the cell periphery in these cells (fig. 5) is consistent with this notion (see also the section ‘Cytoskeletal Rearrangement’). Indeed, a number of studies have pointed to a major role for the actin-based cytoskeleton in regulation of NKCC1 [245–248], and there is evidence to suggest that an intact F-actin cytoskeleton is required both in the process of shrinkage-activation and in maintaining NKCC1 silent

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Hypertonic 1min

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Fig. 5. Effect of osmotic shrinkage and swelling on F-actin and myosin II organization in Ehrlich ascites tumor cells. EAT cells were incubated in isotonic medium or exposed for 1 min to hypertonic (600 mOsm) or hypotonic (225 mOsm) medium, and subsequently fixed, permeabilized, and labeled for F-actin (red) using rhodamine-phalloidin, and for non-muscle myosin II (green) using a monoclonal antibody raised against chicken gizzard myosin II. Images are 1 ␮m optical slices aquired by confocal laser scanning microscopy. Data are from reference [292].

under isotonic conditions [93, 245, 247–249]. In isolated membrane vesicles from EAT cells, which are devoid of myosin II and in which cortical F-actin is perturbed, NKCC1 exhibits a moderate constitutive activity [250], yet can no longer be shrinkage-activated, and is insensitive to ML-7 [249]. Since the transporter can still be stimulated by inhibition of PP1 and PP2A, it appears that the pathway of shrinkage-activation is specifically lost under these conditions, consistent with an important role of MLC and F-actin in the process [249]. This is an interesting example of how not only the activation of transport proteins, but also maintaining them in a quiescent state under non-stimulated conditions, may be dependent on the cytoskeleton. An apparently similar phenomenon appears to be operating for ICl,vol (section ‘Activation of ICl,vol by Cell Swelling’). WNKs and SPAK. Recent seminal findings point to a major role for a pathway involving With-No-lysine (K) Kinases (WNKs) and SPAK in the shrinkageinduced activation of NKCC1. The evidence implicating WNKs as relatively upstream elements in osmosensing will be discussed below (section ‘Ser/thr Protein Kinases: Ste20 Related Kinases and With-No-Lysine (K) Kinases’). Notably, both SPAK and OSR1 directly phosphorylate NKCC1 on the three specific threonine residues, which are also phosphorylated upon osmotic shrinkage [251]. A role for SPAK in activation of NKCC1 by osmotic shrinkage [164] and by low Cl⫺ conditions [252] has been demonstrated. As will be discussed in section ‘Ser/thr Protein Kinases: Ste20 Related Kinases and WithNo-Lysine (K) Kinases’, there is now significant evidence to suggest that shrinkage-activation of SPAK and OSR1 is mediated by WNKs. Specifically, it


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appears that a pathway consisting of WNK4 and SPAK, and requiring both protein–protein interaction between NKCC1 and SPAK, and direct SPAKmediated phosphorylation of NKCC1 is central to shrinkage-activation of NKCC1 [164, 175, 251, 253]. Hypertonicity-Induced Cation Channels Activation of cation channels upon cell shrinkage has been demonstrated in many cell types [41, 254]. There seems to be two types of hypertonicity-induced cation channels (HICCs); (i) amiloride insensitive non-selective cation channels of unknown molecular identity, with a conductance of 15–25 pS, which are inhibited by flufenamate as well as by Gd3⫹. Channels of this type have been described for a variety of preparations [255–258]. These channels are expected to be closed under most physiological conditions given their inhibition by cytosolic ATP in the milimolar range [256], hence, the extent to which they may contribute to RVI is uncertain, although such a contribution has been reported in HeLa cells [258]; (ii) amiloride-sensitive non-selective cation channels [41, 254, 259, 260]. These channels, at least some of which have been suggested to be related to epithelial Na⫹ channels (ENaC), are in general insensitive to Gd3⫹ and flufenamate [254]. Interestingly, the HICCs in ELA cells [261] and human hepatocytes [262] are both Gd3⫹ and amiloride-sensitive, and thus may represent a possible molecular link between these two groups of channels. Shrinkage-Induced Activation of HICCs The mechanism(s) of shrinkage-induced activation of HICCs are still relatively poorly understood. The involvement of G-proteins, phospholipase C (PLC), PKC, and tyrosine kinases has been suggested based on pharmacological studies in rat hepatocytes [263, 264]. A role for tyrosine-kinase mediated exocytotic insertion in shrinkage-induced HICC regulation has also been proposed [265]. Volume Sensing Pathways: Novel Players

Osmo- or volume sensors, while well described in yeast [17] have not been unequivocally identified in mammalian cells. A wide array of systems have, however, been shown to play important roles, and indeed, the co-existence of multiple mechanisms in any given cell type is likely [266]. Recent studies have pointed to a possible role of integrins in volume sensing [93, 267–270]. Other possible mechanisms include ligand-independent activation of growth factor- or cytokine receptors [268], activation of Ca2⫹-influx channels such as transient receptor potential (TRP) channels [271, 272], mechanical stress signals to plasma membrane/cytoskeletal components (e.g. caveolae, cortical F-actin,

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/ PCD

Fig. 6. A working model for integrin-mediated volume sensing in vertebrate cells. The figure summarizes some possible mechanisms linking osmotic cell shrinkage to cell volume regulation, cytoskeletal reorganization, and modulation of the balance between cell proliferation and death. See text for details.

ERM proteins), and intracellular systems, e.g. enzymes sensitive to macromolecular crowding, ionic strength, or [Cl⫺]i [266]. It is not the aim of the present review to comprehensively cover all the possible mechanisms initiating or modulating volume sensing. We will primarily focus on recent evidence implicating integrins and growth factor receptors as osmosensors in mammalian cells. Further downstream, the focus will be on the roles of small GTP binding (G-) proteins, cytoskeletal elements, With No lysine (K) kinases, Ste20-related kinases, and MAPKS in the transmission of the volume signal. A working model of how these systems may interact to provide a pathway from osmotic shrinkage to cell volume regulation, cytoskeletal reorganization, and modulation of cell proliferation and death is shown in figure 6. How May a Membrane Protein Sense Cell Volume Perturbations? A number of studies in recent years have increased the understanding of the fundamental mechanisms by which a membrane protein may sense cell volume stress [273–276]. The majority of such studies have been carried out for yeast or


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bacterial ion channels, however, many of the same fundamental principles should apply for other osmolyte transporters or TM sensors involved in volume-sensitive signaling. Studies of bacterial osmolyte transporters indicate that at the molecular level, the problem of how to sense cell volume perturbations has been solved differently by different transporter families. Mechanisms shown to play a role in various transporters include sensing of ionic strength or specifically of potassium, sensing of changes in membrane tension, and sensing of ionic-strength-mediated changes in protein–lipid interactions in the membrane bilayer [277]. There is also evidence specifically pointing to a role for the lipid bilayer in volume sensing [278]. With respect to a role for membrane tension in volume sensing, it may be noted that given the large ‘excess membrane surface’ found in most cell types in form of membrane invaginations, the cell membrane is likely to be unfolded rather than stretched after cell swelling, and volume-dependent changes in membrane tension or curvature may be of little importance in cell volume sensing [97, 273]. Integrins and Growth Factor Receptors as Volume Sensors? Integrins Integrins are a large family of heterodimeric membrane-spanning, cell adhesion proteins composed of ␣ and ␤ subunits, combining to form a wide variety of heterodimers [279]. Several studies have pointed to an important role for integrins both in the response to cell swelling [267, 270, 280, 281] and to shrinkage [93, 269, 282–285]. Integrin-mediated regulation of volume-sensitive ion transport appears to occur at the acute, post-translational level [280, 281] as well as at the transcriptional level, as hypertonicity-induced increase in renal expression of tonicity-responsive enhancer-binding protein and activation of osmolyte uptake were strongly attenuated in integrin ␣1-null mice [283]. There are many examples of regulation of ion transporters by integrins [286]. Of relevance to cell volume regulation, ␤1 integrins have been implicated in the regulation of a variety of K⫹ channels including Kv1.3 channels [287], Kv4.2 channels and Kv1.4 channels [288], and Ca2⫹-activated K⫹ channels [289]. Also ICl,vol has been shown to be integrin-regulated, and a role for Src and/or focal adhesion kinase (FAK) in the integrin-mediated signaling pathway leading to ICl,vol activation was suggested [130, 280, 281]. With respect to shrinkage-activated transporters, NHE1 is well-known to be activated by integrins [290] in a Rho-Rho-kinase mediated manner [291]. However, this pathway is unlikely to account for shrinkage-induced NHE1 activation, which is unaffected by inhibition of Rho kinase [292]. Growth Factor Receptors In Swiss 3T3 fibroblasts, hypotonic cell swelling has been found to activate the EGFR, leading to stimulation of PI3 kinase, and activation of taurine efflux

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[293]. Stimulation of the EGFR after hypotonic cell swelling has also been reported in other cellular systems [145, 293, 294]. On the other hand, hypertonic cell shrinkage has also been found to activate GFRs [268, 295, 296]. A mechanism involving ligand-independent receptor clustering as a consequence of shrinkage-induced membrane alteration or conformational changes in the receptor has been suggested [268]. In rat hepatocytes, shrinkage-induced EGFR activation was proposed to be downstream of ROS-mediated activation of the Src kinase family member Yes [295]. In apparent contrast, hypertonic shrinkage strongly reduced activation of Raf, Akt, ERK1, ERK2, and JNK downstream from EGFR activation [297]. Similarly, in NIH3T3 cells, ligand-dependent activation of the growth factor receptor, PDGFR␤, and the mitogenic MEK1/2ERK1/2 pathway were inhibited by osmotic shrinkage, whereas p38 MAPK and JNK activities were significantly up-regulated [298]. Limited evidence is available with respect to the signaling events linking integrins and GFRs to volumesensitive osmolyte transport. A wide array of event are initiated in response to integrin clustering/activation, including, but not limited to, cytoskeletal rearrangement, and recruitment/activation of Rho family small GTP-binding proteins, FAK, Src family tyrosine kinases, phosphatidyl-inositol 3 kinase, protein kinase B (PKB)/Akt, MLCK, and MAPKs [279, 299–301]. There is also evidence for direct interaction between Ste20-related protein kinases and integrins [302]. Many of these pathways are also activated downstream from GFR activation, and indeed, a substantial convergence of signaling pathways downstream from integrins and GFRs, at least in part involving cytoskeleton-dependent scaffolding within the focal adhesion complex has been suggested [303]. Another point of convergence is that integrin activation has been shown to elicit transactivation of GFRs [304]. For some of these pathways, there is substantial evidence for a role in regulation of volume-sensitive osmolyte transport, as will be described below (sections ‘Small GTP-Binding Proteins and Their Effectors’, ‘Cytoskeletal Rearrangement’, ‘Tyrosine Kinases: FAK and Src Family Kinases’, ‘Ser/thr Protein Kinases: Ste20 Related Kinases and With-NoLysine (K) Kinases’ and ‘Mitogen Activated Protein Kinases’). Other Upstream Mechanisms in Volume Sensing Transient Receptor Potential Channels TRP channels are a family of mostly non-selective cation channels, providing a major Ca2⫹ entry pathway in a wide variety of cell types [272, 305, 306]. The family is divided into 7 subfamilies, TRPC, TRPV, TRPM, TRPP, TRPML, TRPA, and TRPN, based on sequence homology [272, 305, 306]. The TRP subfamilies share a membrane topology of 6 predicted TM domains, cytosolic Nand C-termini, and a pore forming region betweenTM5 and TM6 [272, 305]. The N-terminal cytoplasmic domain of most of the TRP subfamilies contain an


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ankyrin repeat of variable length, which has been assigned a role in cytoskeletondependent mechanosensing via these channels [305]. The founding member of the TRPV family is the osmo-sensitive channel (OSM-9) in C. elegans, and TRPV4 was later identified as the mammalian homologue of this channel [307–309]. Substantial evidence has since pointed to a central role of TRPV4 in osmosensing, and another member of the TRPV family, TRPV2, has also been recognized as an osmosensitive channel [271, 310, 311]. Other TRP channels, e.g. TRPM3 [312] and TRPP2 [313] have also been suggested to be sensitive to hypotonic cell swelling, and several other TRPs are sensitive to fluid shear stress and/or membrane tension [272, 306, 311, 312]. Here, we will focus mainly on TRPV4, which is broadly distributed and for which the role in osmosensing is by far best understood. Several lines of evidence implicate TRPV4 as a major swelling-activated Ca2⫹ entry pathway: (i) when exogenously expressed, TRPV4 mediates swelling-activated Ca2⫹ influx [311, 316]; (ii) trpv4–/– mice exhibit a reduced ability to volume regulate [310, 311]; and (iii) TRPV4 expression rescues C. elegans osm-9 mutants from deficits in osmo- and mechanosensing [314]. Several, not necessarily all mutually exclusive, modes of activation of TRPV4 by hyposmotic stress have been put forward (for an excellent discussion, see [310]). Firstly, it may be noted that the channel appears to be activated by cell swelling per se, rather than by e.g. reduced ionic strength [311]. Direct membrane stretch did not appear to activate TRPV4 at least at room temperature, whereas at physiological temperature, TRPV4 was sensitive to fluid shear stress [311]. In apparent contrast, TRPV2 was found to be activated both by hypotonicity and by mechanical stretch [310, 311, 315]. However, as discussed by Liedtke, methodological problems in separating osmotic/fluid shear stress/mechanical stimuli makes the distinction between these activation modes somewhat challenging [310]. The N-terminal ankyrin repeat, which provide a putative link between the TRPs and the actin cytoskeleton, appeared to be required for TRPV2 function, and for the heat-dependent, yet not the swelling-dependent, activation of TRPV4 [305]. Phosphorylation of TRPV4 by Src family kinases, specifically Lyn, was first proposed to be involved in its activation by swelling [316]. Later studies provided evidence that the eicosanoid 5⬘, 6⬘-epoxyeicosatrienoic acid plays a central role in swelling-induced TRPV4 activation [271, 317], presumably downstream from the swelling-induced activation of PLA2. Finally, recent findings have implicated AQP5 in swelling-induced TRPV4 activation, possibly in a manner involving a direct interaction between the two proteins [6]. Phospholipase A2 The phospholipase A2 superfamily is divided into three major subfamilies, the Ca2⫹-dependent cytosolic PLA2 (cPLA2), the Ca2⫹-independent cytosolic PLA2 (iPLA2), and the secretory PLA2 (sPLA2) [318]. The involvement of

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phospholipase A2 activation and arachidonic acid release in swelling-induced activation of osmolyte transport has been reviewed elsewhere recently [39, 87], and will only be briefly summarized here. As noted above, osmotic cell swelling has been shown to be associated with an increase in PLA2-dependent arachidonic acid release in several cell types studied, including EAT cells [85], inner medullary collecting duct cells [86], and neuroblastoma cells [84]. In some cells, this release appears to be mediated by the Ca2⫹-dependent cytosolic PLA2 (cPLA2) [84, 319]. In EAT cells, immunocytochemical studies indicated that cPLA2alpha, but not cPLA2gamma, translocates to the nucleus upon cell swelling, and it was demonstrated that AA is predominantly released from the nuclear fraction [319]. The mechanism of swelling-induced activation and translocation of cPLA2 remains to be fully elucidated, since an increase in [Ca2⫹]i is generally required for cPLA2 activation, yet in many cell types including EAT cells, osmotic swelling is not assocated with a detectable increase in [Ca2⫹]i (for a discussion, see [87]). In other cell types, the swelling-induced release of arachidonic acid appears to involve iPLA2, and sPLA2, as suggested in NIH3T3 cells [143, 320]. Also for these phospholipases, the mechanism of swelling-induced activation remains to be elucidated (for a discussion, see [39]). A role for the actin cytoskeleton seems unlikely, as cytochalasins were without apparent effect on swelling-induced arachidonic acid release in NIH3T3 cells [87]. Notably, the activity of sPLA2 could be increased simply by swelling liposomes in which this phospholipase was reconstituted [321], indicating that changes in the organization of the lipid bilayer alone are sufficient to increase arachidonic acid release via this PLA2 [278]. Reactive Oxygen Species Recent studies have demonstrated that cell swelling is associated with the release of ROS [133, 143], and have, moreover, indicated an important role for ROS in the modulation of swelling-activated Cl⫺ and taurine efflux (see the sections Swelling-Activated Anion Current (ICl,vol) and Swelling-Activated Taurine Efflux). The mechanism(s) of swelling-induced ROS release is not fully elucidated, but appears to involve the NADPH oxidase [133, 143]. Upstream of the NADPH oxidase, a role for swelling-induced integrin activation is conceivable, by analogy with the pathway suggested for stretch-induced NADPH oxidase activation and ROS production [130]. Substantiating the notion that a given effector may be involved in both swelling- and shrinkage-dependent signaling cascades, there is also evidence that ROS are released in response to hypertonic stress [295, 321, 322]. Interestingly, the source of shrinkage-induced ROS production appears to be mitochondria rather than NADPH oxidase [322]. Swelling- and shrinkage-induced ROS release may occur within the same cell type, as in NIH3T3 cells, both swelling [143] and, to a lesser extent, shrinkage (Lambert IH, Friis M, unpublished) were associated with ROS release.


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Small GTP-Binding Proteins and Their Effectors A central role for small GTP-binding (G-) proteins of the Rho family in volume-sensitive signaling has been proposed in both yeast and higher eukaryotes [17, 323, 324]. The Rho family members, RhoA, Rac1 and Cdc42, are all activated by osmotic shrinkage in mammalian cells [244, 325–327]. Whereas the shrinkage-induced changes in the activity of Rho family G proteins have been shown to contribute importantly to shrinkage-induced cytoskeletal reorganization [244, 325, 326], their involvement in regulation of volume-sensitive osmolyte transport is less well understood. The major Rho effector Rho kinase does not appear to be involved in the shrinkage-induced activation of NHE1 or NKCC1 [244, 292, 324]. Rac appears to be important in the shrinkage-induced modulation of MAPK activity [24, 323]. Cdc42 has been found to be upstream of the shrinkage-induced activation of the Ste20-related kinase p21-activated kinase (PAK) [327] and FAK [328], as well as upstream of shrinkage-induced actin remodeling [325, 326], and may also, via PAK, be involved in the shrinkageinduced activation of p38 MAPK [329]. Conversely, osmotic swelling reduced Rho activity in ELA cells [103] (fig. 2), while Rho activity was unaffected by swelling in vascular enothelial cells [330]. Rho appears to play a permissive role in swelling-induced activation of ICl,vol [123, 127, 128, 330], and in congruence with this, the stimulating effect of cholesterol depletion on ICl,vol in ELA cells was associated with prevention of the swelling-induced reduction in Rho activity [103] (fig. 2). Overexpression of constitutively active RhoA potently stimulated RVD and swelling-induced K⫹ and taurine efflux from NIH3T3 cells (fig. 7). RhoA activity also sensitized ICl,vol in these cells to osmotic swelling [128]. Thus, at least in these cells, RhoA seems to be an upstream element modulating the activity of multiple swelling-activated osmolyte transporters. Finally, Rac and Cdc42 activation by hypotonicity has been reported in CHO cells [331]. Thus, the effect of cell volume perturbation on Rho family G proteins is at least in some cases cell type specific, and, moreover, a distinction must be made between permissive roles of these proteins and an causal role in the volume sensing pathways(s) per se (for a discussion, see [324]). The mechanisms by which changes in cell volume may modulate the activity of Rho family G proteins have recently been extensively discussed elsewhere [324] and will only be briefly summarized here. Given the known role of Rho family proteins downstream from integrin activation [279], a role for this pathway in their volume-sensitive regulation is conceivable. Both a decrease in cell volume per se, and an increase in intracellular ionic strength at constant volume is sufficient for shrinkage-induced Rho activation [244]. Rho protein activity is regulated by three classes of Rho protein regulators, the guanine nucleotide dissociation inhibitors, guanine nucleotide exchange factors, and GTPase activating proteins, and the volume-sensitivity of these proteins is as

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86 Rb⫹ efflux Maximal rate constant (min⫺1)

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Fig. 7. Effect of constitutively active Rho (RhoAV14) on RVD and swelling-induced K⫹and taurine efflux. a RVD rates were calculated from light scatter experiments as described in [128] as the slope of the relative cell volume traces in the initial 90 s after hypotonic exposure. Data are shown as means with SEM error bars, with the number of experiments indicated below each column. The RVD rates were significantly different from that of wild type cells in all RhoAV14 expressing clones. b The 86Rb⫹ efflux was used as a measure for K⫹ efflux and measured as indicated in [128]. At time 6 min, the extracellular osmolarity was reduced by the percentage shown. The figure shows the mean maximal efflux rate constant, obtained at time 6 min after hypotonic exposure, as a function on the decrease in extracellular osmolarity. c Wild type and RhoAV14C3 cells were preloaded with [3H]taurine for 2 h, followed by efflux measurements as described in [128]. At time 6 min, the extracellular osmolarity was reduced by 33%, from 300 to 200 mOsm l⫺1. Efflux rate constants were obtained as described in (b). The figure shows the mean maximal efflux rate constant obtained at time 6 min after hypotonic exposure, as a function on the decrease in extracellular osmolarity. * ⫽ Significantly different from the corresponding value in wild type cells. Redrawn from reference [128].


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yet unknown [324]. It may be noted that ERM proteins, which are rapidly activated by osmotic cell shrinkage [332, 333] can activate Rho by interaction with Rho-guanine nucleotide dissociation inhibitors [334]. Interestingly, ERM proteins may be an integrating element of a volume-dependent positive feedback loop, since they can also be activated downstream of Rho activation (see the section ‘Cytoskeletal Rearrangement’). Effectors immediately downstream from Rho proteins and of relevance for cell shrinkage include: (i) Rho kinase, which has been implicated in shrinkageinduced cytoskeletal reorganization [324] and integrin-mediated NHE1 activation; (ii) phosphatidylinositol-4-phosphate 5-kinase, the activity of which increases the cellular PtdIns(4,5)P2 level, a phenomenon seen after shrinkage in EAT cells [219] and other cells [220]; and (iii) the Ste20-related kinases. For the purpose of the present review, we will focus on the Ste20-related kinases, since the understanding of the role of these kinases in volume regulation has increased exponentially in recent years. For an excellent discussion of the involvement of other Rho effectors in signaling events following osmotic stress see [324]. Cytoskeletal Rearrangement Major rearrangements of the F-actin cytoskeleton following cell volume perturbations have been demonstrated in many cell types. Most commonly, the pattern is such that cell shrinkage is associated with an increase in net cellular F-actin content, particularly an increase in cortical F-actin, while the converse is true for cell swelling [93, 112, 335, 336]. Importantly, however, other patterns of F-actin rearrangement have also been demonstrated [123], likely reflecting the fundamental differences in F-actin organization and F-actin binding proteins between, for instance, adherent and non-adherent cell types. Notably, the peripheral and the perinuclear actin-associated cytoskeleton appear to respond differently to cell volume perturbations [337]. In addition to F-actin per se, a range of F-actin associated cytoskeletal proteins have been shown to be modulated by cell volume perturbations. These include the Arp2/3 complex and cortactin [325, 338] and non-muscle myosin II [292, 339], all of which have been shown to translocate to the cortical region in response to cell shrinkage (for myosin, see fig. 5), and ERM proteins, which are threonine-phosphorylated, consistent with activation, in shrunken cells [332, 333]. Cortactin is also tyrosine phosphorylated, in a manner dependent on the non-receptor tyrosine kinase Fer, an effect tentatively suggested to be downstream from PDGF-receptor clustering [338], however, this is not required for the shrinkage-induced cortactin translocation [325]. In EAT cells, the translocation of myosin II appeared to be at least in part dependent on Rho kinase and p38 MAPK [292]. The mechanism by which cortical ERM proteins are rapidly phosphorylated and activated upon cell shrinkage [332, 333] remains to be

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investigated, as does their possible causal role in volume sensing and/or volume-sensitive F-actin rearrangement. ERM protein activation is, however, known to be dependent on Rho kinase-mediated phosphorylation and association with PtdIns(4,5)P2 [334], consistent with the increased Rho activity [244] and increased cellular PtdIns(4,5)P2 levels [219, 220] in shrunken cells. Role of the Cytoskeleton in Regulation of Osmolyte Transport in RVD and RVI Studies in a wide range of cell types have pointed to the involvement of Factin in control of both RVD [336, 340–342] and RVI [93, 213, 336, 343]. However, it is emphasized that in other studies, disruption of F-actin had no apparent effect on either RVD [335, 344] or RVI [341, 344], suggesting that in congruence with the variable effects of volume-perturbations on F-actin, the causal involvement of the actin cytoskeleton in cell volume regulation is cell type dependent. Moreover, at least some of the discrepancy in the reported findings is likely to reflect methodological problems such as the concentration, isoform-, and cell-type specific effects of cytochalasins on F-actin [345], and the inherent unspecificity problems with inhibitor compounds. Future studies should address these questions using more targeted approaches. The role of the cytoskeleton in mechanosensing may be both global, as in the ‘tensegrity’ model [274] and local, involving organization of cortical microdomains and/or interaction with specific transporters [273]. Specifically, changes in F-actin organization may modulate volume-sensitive osmolyte transport at multiple levels, including (i) an upstream role in cell volume sensing; (ii) direct anchoring and/or regulation of transport proteins resident in the plasma membrane; (iii) vesicular insertion/removal of transporter proteins, and (iv) regulation of the signaling cascades controlling transporter function (for a review, see [93]). Here we will primarily focus on recent central findings regarding the possible upstream roles of F-actin in regulation of volume-sensitive signaling events. Examples of F-actin dependent regulation of specific osmolyte transporters are mentioned above in the sections covering individual transporters. Regulation of Swelling-Induced Calcium Influx. Cytochalasins inhibit swelling-induced Ca2⫹ influx in a variety of cell types [346–348]. In chick cardiomyocytes, the F-actin dependent Ca2⫹-current was a non-selective, Gd3⫹sensitive cation current [347], and in human syncytiotrophoblast a role for F-actin in swelling-induced activation of the polycystin-2 TRP channel was directly suggested [313]. Modulation of Cl⫺ Sensing. Interestingly, it was recently suggested that F-actin counteracts the modulation of Kv K⫹ channel function by [Cl⫺]i, presumably by


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stabilizing channel conformation and thus providing resistance to Cl⫺-dependent conformational changes [349]. A similar effect might underlie the observation that, as noted above (sections ‘Activation of IK,vol by Cell Swelling’ and ‘Activation of ICl,vol by Cell Swelling’) swelling-activation of both K⫹ and Cl⫺ channels is augmented by F-actin disruption. Regulation of Protein Kinases and Phospholipases. Many of the protein kinases of relevance to cell volume regulation associate with and are regulated by the F-actin cytoskeleton [93, 350]. Of note, SPAK has also been shown to translocate to the cytoskeletal fraction upon hypertonic challenge [351], and an interaction of SPAK with F-actin and MLC has been tentatively suggested to be involved in SPAK-mediated signaling after cell shrinkage [175]. While there is also evidence for a role for the cytoskeleton in regulation of PLA2 translocation and activity after other stimuli [352, 353], disruption of Factin had no effect on swelling-induced PLA2 activity in NIH3T3 cells [87]. Although the great majority of the evidence pointing to a role for the cytoskeleton in RVD and RVI has been directed at the actin-based cytoskeleton, a few studies point to the involvement other classes of cytoskeletal proteins. An apparent stabilization of microtubules following hypotonic swelling has been reported in hepatocytes [354], and a role for microtubuli in regulation of both swelling-induced [340, 355, 356] and shrinkage-induced [343, 357, 358] volumesensitive osmolyte transport has been reported. Finally, effects of cell volume perturbations on intermediate filaments [359] have also been reported, but to our knowledge, nothing is known about their potential involvement in volumesensitive signaling pathways. Tyrosine Kinases: FAK and Src Family Kinases FAK is an evolutionarily conserved non-receptor protein-tyrosine kinase localized within the focal adhesion [360]. Two fundamental roles of FAK in cellular signaling have been described: as a mediator of tyrosine phosphorylation of several substrates, and as a scaffold protein recruiting signaling molecules to the focal adhesion [361]. Hypertonic cell shrinkage has been found to stimulate FAK in a variety of cell types [328, 362, 363]. In Swiss 3T3 cells, osmotic shrinkage induced FAK phosphorylation both on Tyr577 in the kinase-activating loop and on the autophosphorylation site Tyr397, the latter dependent on the small G-protein Cdc42, yet independent of Rho, Rho kinase, phosphatidyl-inositol 3 kinase, p38 MAPK, and integrin/Src stimulation [328]. The mechanism of Cdc42-dependent FAK phosphorylation is unknown, but a role for aggregation and dimerization during cell shrinkage has been tentatively suggested, by analogy with previous findings on dimerization-induced FAK activation [328]. A role for the actin cytoskeleton, which is important for FAK activation by other stimuli [364], is also

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possible. No studies have to our knowledge demonstrated a role for FAK in RVI. Other functional effects of shrinkage-induced FAK activation have however been suggested, including a FAK-dependent strengthening of epithelial barrier function [365], and a protective effect against shrinkage-induced apoptosis [328]. Hypotonic cell swelling has also been shown to activate FAK in several cell types [366, 367], in a manner dependent on Rho family G proteins [366] or phosphatidyl-inositol 3 kinase [367]. A functional role of FAK in regulation of ICl,vol has been suggested in several cell types [123, 281]. On the other hand, FAK appeared to play an inhibitory role in regulation of ICl,vol in cardiomyocytes [368], whereas a lack of involvement of FAK in ICl,vol regulation was reported in endothelial cells [119]. A role for FAK in swelling-induced activation of Src and of ERK1/2 has also been proposed [367]. Similar to FAK, direct and indirect evidence indicates that multiple Src family kinases are activated both in response to cell swelling [122, 270, 369] and cell shrinkage [195, 295, 370]. The mechanism of Src activation by cell volume stress has yet to be fully elucidated, but a role for integrins upstream of cell swelling-mediated Src activation was recently demonstrated [270]. The evidence implicating Src kinases in volume regulation has been reviewed recently [371], and will not be discussed in detail here. Ser/Thr Protein Kinases: Ste20 Related Kinases and With-No-Lysine (K) Kinases The existence of a coordinated system of regulation of swelling- and shrinkage-activated osmolyte transporters by protein kinases and phosphatases has been proposed based on relaxation kinetic studies [168]. The finding that in general, ser/thr protein phosphatase inhibitors stimulate the shrinkage-activated transporters [215, 231, 239, 372], and inhibit the swelling-activated transporters [55, 161] is in congruence with this notion. These studies suggested the existence of a protein kinase inhibited by cell swelling, relieving an inhibitory effect of phosphorylation on the swelling-activated transporters, and conversely, activated by cell shrinkage resulting in the phosphorylation-dependent activation of the shrinkage-activated transporters. While such a scheme is undoubtedly to some extent much too simplistic, recent findings have in fact pointed to the existence of such a volume-sensitive kinase system with important roles in regulating both swelling- and shrinkage-activated transporters: The Ste20-related kinases include two subfamilies of ser/thr kinases: (i) the germinal centre kinases, which are mitogen-activated protein kinase kinase kinases kinases (MAP4Ks) [373, 374], and members of which are regulators of NHE1 and NKCC1 (sections ‘The Na⫹/H⫹ Exchangers’ and ‘The Na⫹, K⫹, 2Cl⫺Cotransporter’), and (ii) the PAKs, which are shrinkage-activated kinases with major roles in cytoskeletal organization [203, 324, 373, 375].


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The with-no-lysine (K) kinases are a family of four ser/thr protein kinases, WNK1–4. WNK1 and WNK4 were originally identified as mutated in the congenital hypertension and hyperkalemia disorder known as Gordon’s syndrome, or pseudohypoaldosteronism type II. It is now clear that the pathology of this syndrome at least in part reflects the important role of WNKs in regulation of paracellular Cl⫺ transport [376]. Recent evidence strongly suggests that this may at least in some cases be mediated via WNK-mediated regulation of the Ste20related kinases SPAK and/or OSR1. Thus, WNK1 and WNK4 bind, phosphorylate, and activate SPAK and OSR1 [164, 377, 378]. OSR1 has been shown to be activated by hypertonicity [379], and both OSR1 and SPAK are activated under low Cl⫺ conditions [253, 377]. Moreover, WNK1 was shown to mediate, via activation of SPAK and OSR1, phosphorylation of NKCC1 and of the kidneyspecific SLC12 family members, NKCC2 and the Na⫹, Cl⫺ cotransporter (NCC) [377]. Similarly, WNK4-dependent SPAK activation in turn lead to phosphorylation-dependent activation of NKCC1 and inhibition of KCC2, respectively [164]. Interestingly, WNK3 has also been shown to stimulate NKCC1 while inhibiting KCC1 and KCC2 in a phosphorylation-dependent manner [380, 381]. However, in the case of WNK3, SPAK and OSR1 do not seem to be required for this process, which has, rather, been proposed to involve an effect on cotransporter-directed phosphatase activity [380, 381]. WNKs modulate the activity not only of the SLC12 family, but of a wide range of ion transport proteins, including, interestingly, the swelling-activated TRP channel TRPV4 [382]. Importantly, other Ste20-related kinases than SPAK and OSR1 appear to be involved in regulation of volume-sensitive ion transporters. Thus, NIK binds and phosphorylates NHE1 and is central to its activation by PDGF [203], and germinal center kinase-3 (which may be a SPAK orthologue, see [374]) binds and regulates the volume-sensitive C. elegans Cl⫺ channel CLH-3b [383], for a review see [374]. The mechanism by which WNK activity is modulated by cell volume perturbations is unknown. WNK1 is activated by PKB [384], yet this may not be rapid enough to account for shrinkage-induced NKCC1 activation, and moreover, PKB signaling is most commonly [297, 385], although not always [386] found to be inhibited by osmotic shrinkage. A role for the actin-based cytoskeleton in volumesensitive regulation of SPAK has been tentatively suggested [175], and a similar scenario is possible for WNKs, however, this is at this point completely speculative. Moreover, it should be noted that both hypertonicity and, to a lesser extent, hypotonicity (by dilution of the medium) have been found to activate WNK1 [387]. Mitogen-Activated Protein Kinases That osmotic stress activates MAPKs was first described in S. cerevisiae [17] and later also found in mammalian cells [15]. MAPKs are activated by a

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Phospho p38 MAPK Phospho ERK1/2

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Protein phosphorylation Relative to isotonic value

* *

4

2

** *

* * 0

0

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Time of incubation (min)

Fig. 8. Shrinkage-induced p38 MAPK- and ERK1/2 phosphorylation in NIH3T3 cells. NIH3T3 cells were incubated in either isotonic (337 mOsmol l–1) or hypertonic (high NaCl) DMEM (687 mOsmol l–1), and cell lysates were subjected to SDS-PAGE and Western blot analysis at the indicated time intervals, using specific antibodies against total and phosphorylated ERK1/2 and p38 MAPK. The intensity of p38 MAPK or ERK1/2 phosphorylation was densitometrically quantified and is given relative to the isotonic control level. **, *** ⫽ Significantly different from the isotonic control condition, p-values ⬍0.01 and ⬍0.001, respectively. Data are redrawn from [24].

phosphorylation relay system, in which the MAPK kinases (MAP2Ks, a.k.a. MEKs or MKKs) MEK1/2, MKK3/6, and MKK4/7 dually phosphorylate and activate ERK, p38 MAPK, and JNK, respectively [388]. The MAP2Ks are again activated by MAP2K kinases (MAP3Ks), which are regulated by a wide array of signaling components including small G-proteins and Ste20-related protein kinases [388]. The three most well-described subfamilies of MAPKs found in mammalian cells, ERK, p38 MAPK, and JNK are all modulated by osmotic stress in a wide variety of cell types, although the specific pattern of MAPK modulation is cell type dependent [15, 187, 241, 389–392]. Most commonly, cell shrinkage will stimulate p38 MAPK [24, 187, 390, 391, 393] and JNK [298, 389, 390], whereas cell swelling will stimulate ERK [298, 394, 395]. In some cell types, the shrinkage-induced increase in p38 MAPK activity is accompanied by a transient decrease in ERK1/2 activity (fig. 8) which appears to be secondary to the shrinkage-induced increases in p38 MAPK activity and NHE1 activity [24, 389].


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The physiological consequences of MAPK activation by osmotic stress have been extensively described for the long-term effects of cell volume perturbations on cell proliferation, programmed cell death (PCD), and adaptive changes in osmolyte transporter expression, in which these kinases play a central role [15]. Broadly speaking, p38 MAPK and JNK tend to counteract cell proliferation and induce PCD, while the opposite is true for ERK [396, 397]. In agreement with this, cell swelling stimulates proliferation [21, 22], whereas cell shrinkage blocks cell cycle progression and induces PCD [23–25, 398, 399]. However, in many cell types, MAPKs are very rapidly modulated by osmotic volume perturbations, consistent with the time course of acute cell volume regulation. Indeed, roles for ERK, p38 MAPK, and JNK in regulation of shrinkage-induced osmolyte transport in the early phase of RVI have been proposed in several cell types [187, 228, 241, 391]. The upstream mechanisms by which cell volume modulate MAPK activity are only partially understood in higher eukaryotes. Elevation of extracellular osmolarity stimulates p38 MAPK and JNK both at increased and unaltered ionic strength [389]. Shrinkage-induced activation of p38 MAPK appears to involve Cdc42 and/or Rac [24, 323, 400], both of which are rapidly activated by osmotic shrinkage (section ‘Small GTP-Binding Proteins and Their Effectors’). A scaffold involving Rac, OSM (a proposed Ste50 homologue), MEKK3, and MKK3, possibly linking to F-actin as part of the osmosensing system, was found to be responsible for shrinkage-induced p38 MAPK activation in mammalian cells, by analogy with the HOG1 (the yeast p38 MAPK orthologue) signaling cascade in yeast [323]. The Ste20 kinases NIK and SPAK, regulators of the shrinkageactivated transporters NHE1 and NKCC1 (sections ‘The Na⫹/H⫹ Exchangers’ and ‘The Na⫹, K⫹, 2Cl⫺ Cotransporter’) have been found to be upstream activators of p38 MAPK and JNK, respectively [401, 402], and SPAK was proposed to modulate stress-induced p38 MAPK function [235]. Interestingly, NHE1 appears to play a major role in the shrinkage-induced inhibition of ERK, and a partial role in shrinkage-induced stimulation of JNK, in ELA cells [389], while in U937 cells, JNK activation by shrinkage was reported to be independent of NHE1 [403]. Finally, MAPK activity is also modulated by the activity of MAPK phosphatases, and there is evidence to suggest that cell volume-dependent regulation of MAPK phosphatases may be involved in regulation of MAPK activity [404]. Acknowledgements Work in the author’s laboratories is supported by the Danish Natural Sciences Research Council, the Danish Cancer Society, and the Danish Agency for Science, Technology and Innovation. Members of our laboratories are gratefully acknowledged for contributing unpublished data and insightful comments to this review.

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368 Walsh KB, Zhang J: Regulation of cardiac volume-sensitive chloride channel by focal adhesion kinase and Src kinase. Am J Physiol Heart Circ Physiol 2005;289:H2566–H2574. 369 Barfod ET, Moore AL, Melnick RF, Lidofsky SD: Src regulates distinct pathways for cell volume control through Vav and phospholipase Cgamma. J Biol Chem 2005;280:25548–25557. 370 Kapus A, Szaszi K, Sun J, Rizoli S, Rotstein OD: Cell shrinkage regulates Src kinases and induces tyrosine phosphorylation of cortactin, independent of the osmotic regulation of Na⫹/H⫹ exchangers. J Biol Chem 1999;274:8093–8102. 371 Cohen DM: SRC family kinases in cell volume regulation. Am J Physiol Cell Physiol 2005;288:C483–C493. 372 Pedersen SF, King SA, Rigor RR, Zhuang Z, Warren JM, Cala PM: Molecular cloning of NHE1 from winter flounder RBCs: activation by osmotic shrinkage, cAMP, and calyculin A. Am J Physiol Cell Physiol 2003;284:C1561–C1576. 373 Dan I, Watanabe NM, Kusumi A: The Ste20 group kinases as regulators of MAP kinase cascades. Trends Cell Biol 2001;11:220–230. 374 Strange K, Denton J, Nehrke K: Ste20-type kinases: evolutionarily conserved regulators of ion transport and cell volume. Physiology (Bethesda) 2006;21:61–68. 375 Bishop AL, Hall A: Rho GTPases and their effector proteins. Biochem J 2000;348(pt 2):241–255. 376 Kahle KT, Wilson FH, Lifton RP: Regulation of diverse ion transport pathways by WNK4 kinase: a novel molecular switch. Trends Endocrinol Metab 2005;16:98–103. 377 Moriguchi T, Urushiyama S, Hisamoto N, Iemura S, Uchida S, Natsume T, Matsumoto K, Shibuya H: WNK1 regulates phosphorylation of cation-chloride-coupled cotransporters via the STE20related kinases, SPAK and OSR1. J Biol Chem 2005;280:42685–42693. 378 Vitari AC, Deak M, Morrice NA, Alessi DR: The WNK1 and WNK4 protein kinases that are mutated in Gordon’s hypertension syndrome phosphorylate and activate SPAK and OSR1 protein kinases. Biochem J 2005;391:17–24. 379 Chen W, Yazicioglu M, Cobb MH: Characterization of OSR1, a member of the mammalian Ste20p/germinal center kinase subfamily. J Biol Chem 2004;279:11129–11136. 380 de Los HP, Kahle KT, Rinehart J, Bobadilla NA, Vazquez N, San Cristobal P, Mount DB, Lifton RP, Hebert SC, Gamba G: WNK3 bypasses the tonicity requirement for K-Cl cotransporter activation via a phosphatase-dependent pathway. Proc Natl Acad Sci USA 2006;103: 1976–1981. 381 Kahle KT, Rinehart J, de Los HP, Louvi A, Meade P, Vazquez N, Hebert SC, Gamba G, Gimenez I, Lifton RP: WNK3 modulates transport of Cl- in and out of cells: implications for control of cell volume and neuronal excitability. Proc Natl Acad Sci USA 2005;102:16783–16788. 382 Fu Y, Subramanya A, Rozansky D, Cohen DM: WNK kinases influence TRPV4 channel function and localization. Am J Physiol Renal Physiol 2006;290:F1305–F1314. 383 Denton J, Nehrke K, Yin X, Morrison R, Strange K: GCK-3, a newly identified Ste20 kinase, binds to and regulates the activity of a cell cycle-dependent ClC anion channel. J Gen Physiol 2005;125:113–125. 384 Vitari AC, Deak M, Collins BJ, Morrice N, Prescott AR, Phelan A, Humphreys S, Alessi DR: WNK1, the kinase mutated in an inherited high-blood-pressure syndrome, is a novel PKB (protein kinase B)/Akt substrate. Biochem J 2004;378:257–268. 385 Meier R, Thelen M, Hemmings BA: Inactivation and dephosphorylation of protein kinase Balpha (PKB␣) promoted by hyperosmotic stress. EMBO J 1998;17:7294–7303. 386 Terada Y, Inoshita S, Hanada S, Shimamura H, Kuwahara M, Ogawa W, Kasuga M, Sasaki S, Marumo F: Hyperosmolality activates Akt and regulates apoptosis in renal tubular cells. Kidney Int 2001;60:553–567. 387 Lenertz LY, Lee BH, Min X, Xu BE, Wedin K, Earnest S, Goldsmith EJ, Cobb MH: Properties of WNK1 and implications for other family members. J Biol Chem 2005;280:26653–26658. 388 Kyriakis JM, Avruch J: Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev 2001;81:807–869. 389 Darborg B, Rasmussen M, Nylandsted J, Hoffmann EK, Pedersen SF. The Na⫹/H⫹ exchanger, NHE1, differentially regulates mitogen-activated protein kinase subfamilies after osmotic shrinkage in Ehrlich Lettre Ascites cells. J Cell Physiol 2006; submitted.


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390 Gillis D, Shrode LD, Krump E, Howard CM, Rubie EA, Tibbles LA, Woodgett J, Grinstein S: Osmotic stimulation of the Na⫹/H⫹ exchanger NHE1:relationship to the activation of three MAPK pathways. J Membr Biol 2001;181:205–214. 391 Roger F, Martin PY, Rousselot M, Favre H, Feraille E: Cell shrinkage triggers the activation of mitogen-activated protein kinases by hypertonicity in the rat kidney medullary thick ascending limb of the Henle’s loop. Requirement of p38 kinase for the regulatory volume increase response. J Biol Chem 1999;274:34103–34110. 392 de Nadal E, Alepuz PM, Posas F: Dealing with osmostress through MAP kinase activation. EMBO Rep 2002;3:735–740. 393 Bode JG, Gatsios P, Ludwig S, Rapp UR, Haussinger D, Heinrich PC, Graeve L: The mitogenactivated protein (MAP) kinase p38 and its upstream activator MAP kinase kinase 6 are involved in the activation of signal transducer and activator of transcription by hyperosmolarity. J Biol Chem 1999;274:30222–30227. 394 Kim RD, Darling CE, Cerwenka H, Chari RS: Hypoosmotic stress activates p38, ERK 1 and 2, and SAPK/JNK in rat hepatocytes. J Surg Res 2000;90:58–66. 395 Sadoshima J, Qiu Z, Morgan JP, Izumo S: Tyrosine kinase activation is an immediate and essential step in hypotonic cell swelling-induced ERK activation and c-fos gene expression in cardiac myocytes. EMBO J 1996;15:5535–5546. 396 Chuang SM, Wang IC, Yang JL: Roles of JNK, p38 and ERK mitogen-activated protein kinases in the growth inhibition and apoptosis induced by cadmium. Carcinogenesis 2000;21:1423–1432. 397 Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME: Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 1995;270:1326–1331. 398 Dmitrieva NI, Michea LF, Rocha GM, Burg MB: Cell cycle delay and apoptosis in response to osmotic stress. Comp Biochem Physiol A Mol Integr Physiol 2001;130:411–420. 399 Nylandsted J, Jaattela M, Hoffmann EK, Pedersen SF: Heat shock protein 70 inhibits shrinkageinduced programmed cell death via mechanisms independent of effects on cell volume-regulatory membrane transport proteins. Pflugers Arch 2004;449:175–185. 400 Wesselborg S, Bauer MK, Vogt M, Schmitz ML, Schulze-Osthoff K: Activation of transcription factor NF-kappaB and p38 mitogen-activated protein kinase is mediated by distinct and separate stress effector pathways. J Biol Chem 1997;272:12422–12429. 401 Johnston AM, Naselli G, Gonez LJ, Martin RM, Harrison LC, DeAizpurua HJ: SPAK, a STE20/SPS1-related kinase that activates the p38 pathway. Oncogene 2000;19:4290–4297. 402 Su YC, Han J, Xu S, Cobb M, Skolnik EY: NIK is a new Ste20-related kinase that binds NCK and MEKK1 and activates the SAPK/JNK cascade via a conserved regulatory domain. EMBO J 1997;16:1279–1290. 403 Shrode LD, Rubie EA, Woodgett JR, Grinstein S: Cytosolic alkalinization increases stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) activity and p38 mitogen-activated protein kinase activity by a calcium-independent mechanism. J Biol Chem 1997;272:13653–13659. 404 Wu JJ, Bennett AM: Essential role for mitogen-activated protein (MAP) kinase phosphatase-1 in stress-responsive MAP kinase and cell survival signaling. J Biol Chem 2005;280:16461–16466. 405 Hoffmann EK, Hougaard C: Intracellular signalling involved in activation of the volume-sensitive K⫹ current in Ehrlich ascites tumour cells. Comp Biochem Physiol A Mol Integr Physiol 2001;130:355–366. 406 Pedersen SF: A novel NHE1 from red blood cells of the winter flounder: regulation by multiple signaling pathways; in Lauf P, Adragna N (eds): Cell Volume and Signaling. Adv Exp Med Biol. New York, NY, Springer Science ⫹ Business Media, 2005, vol 559, pp 89–98.

Stine F. Pedersen Department of Biochemistry, Institute of Molecular Biology and Physiology University of Copenhagen 13, Universitetsparken DK–2100 Copenhagen (Denmark) Tel. ⫹45 35321546, Fax ⫹45 35321567, E-Mail [email protected]

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Activation of Kinases upon Volume Changes: Role in Cellular Homeostasis R. Todd Alexander a,b, Sergio Grinsteinb,c a

Department of Pediatrics, bProgram in Cell Biology, Hospital for Sick Children, Department of Biochemistry, University of Toronto, Toronto, Canada

c

Abstract The cell is constantly encountering stimuli or conditions that can induce alterations to cell volume. Despite these challenges, cell volume remains relatively constant, by virtue of a series of complex and often redundant regulatory mechanisms. Acutely, an efflux of ions and organic osmolytes counteracts the tendency of cells to swell. Conversely when cells are exposed to perturbations that cause shrinkage, ions are rapidly imported. Chronic hypertonic stimulation increases the transcription of multiple genes whose products promote the intracellular accumulation of organic osmolytes. How the cell perceives these acute alterations in volume in order to signal the activation of specific channels, transporters and transcription factors has been the focus of a large body of research. In this article, we describe the central role that kinases play in mediating the cellular responses to alterations in cell volume. Although incompletely understood, these fundamental processes are key to cellular homeostasis and have obvious clinical implications. Copyright © 2006 S. Karger AG, Basel

Tight regulation of cellular volume is fundamental to life. Metabolic activity leads to the generation of intracellular osmoles, which tend to cause the cell to swell. In addition, alterations in the extracellular tonicity can either promote cell shrinkage or swelling, depending on the direction of the osmolarity gradient. Multiple mechanisms have evolved that enable cells to maintain their volume in response to these challenges to volume homeostasis (reviewed in [1, 2]). When cells are exposed to a perturbation that prompt an increase in cell volume, mechanisms exist that tend to minimize a potentially deleterious volume gain. The ability of cells to counteract imposed swelling is known as regulatory volume decrease (RVD) [3]. In general, RVD is accomplished through the net efflux from the cells of potassium and chloride ions, often accompanied by organic osmolytes. Transmembrane movement of these inorganic and organic

osmolytes occurs through specific channels and transporters that are activated in response to cell swelling. Conversely, mechanisms exist that enable cells to expand their volume in response to an imposed shrinkage. The processes that promote homeostatic volume gain are collectively termed regulatory volume increase (RVI). Often, shrinkage is acutely compensated by rapid intracellular accumulation of sodium and chloride, which in turn drives the passive entry of water into cells. This accumulation of inorganic ions is in general mediated by the activity of sodium–potassium–chloride cotransporters (NKCC), and/or the combined action of the sodium/hydrogen exchanger (NHE) and the bicarbonate/ chloride exchanger, also known as the anion exchanger (AE). When the acute compensatory mechanisms fail to fully restore normal cell volume following osmotically induced shrinkage, the resultant sustained increase in intracellular ionic strength can interfere with the normal function and even with the proper folding of many proteins. Thus, if exposed to increased extracellular osmolarity for long periods of time (hours to days), cells can regain their volume with modest changes in ionic strength by accumulating organic osmolytes. This is accomplished by activation of transcription factors like tonicity enhancer binding protein (TonEBP), that mediate the synthesis of proteins responsible for the intracellular synthesis or import of compensatory organic osmolytes [4]. The mode by which cells sense alterations in their volume is incompletely understood. In particular, the signaling pathways that lead to the activation of these channels and transporters involved in the restoration of physiological volume, in the face of destabilizing challenges, have been incompletely delineated. Nevertheless, some progress has been accomplished in recent years. The activation of various protein and lipid kinases in response to alterations in cell volume has been clearly documented. Further, some of these kinases have been shown to play a critical role in the activation of defined channels and transporters in response to cell volume. The objective of this chapter is to briefly summarize the existing knowledge of the involvement of kinases in signaling the maintenance of volume homeostasis. In the following sections, we will first outline the kinases known to be important for the activation of channels and transporters responsible for RVD. We will then outline the data supporting a role for altered kinase activity in triggering RVI. Lastly, we will explore the role of kinases in mediating the activation of TonEBP following prolonged exposure to extracellular hypertonicity.

Regulatory Volume Decrease

As mentioned briefly, rapid efflux of potassium and chloride ions is the primary process underlying RVD [3]. The channels responsible for the loss of

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Cl

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Fig. 1. Acute cell swelling mediated activation of kinases and their targets. CamK Calmodulin-dependent kinase, ERK extracellular signal-related kinase, KCl-cot potassium-chloride cotransporter, PKC protein kinase C, SGK serum and glucocorticoid-stimulated kinase.

potassium vary in different systems. Among the channels known to be involved in RVD are the large conductance potassium channels (BKCa), intermediate conductance channels activated by high levels of cytosolic calcium (IKCa), the tandem pore-domain acid-sensitive K (TASK) channels and voltage gated channels of the shaker family (KV). The nature of the channel(s) that mediate anion efflux during volume regulation remains the subject of controversy. A volume-responsive anion channel (VRAC) that is activated by cell swelling has been characterized electrophysiologically in a variety of cell types. However, the molecular identity of this channel is, as yet, undetermined. Of note, the loss of organic osmolytes can occur through channels, as well as transporters. In this context, it has been suggested that anion channels contribute to the efflux of organic osmolytes. The regulation of such volume-activated channels by protein kinases is addressed below (fig.1). K Channels In multiple tissue types and cell lines a clear involvement of protein kinase C (PKC) has been established in the modulation of volume-sensitive K efflux. Several lines of evidence support this contention. The pharmacological stimulation of PKC by diacylglycerol analogs mimics the effects of swelling on potassium efflux and potentiates RVD in human salivary ductal cells [5] and isolated perfused rat liver [6]. Conversely, in some systems pharmacological inhibition

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of PKC inhibits RVD, confirming a role for this enzyme in the activation of K efflux [6, 7]. Whether PKC alters the channels directly by phosphorylation during RVD is debatable. In hepatocytes, interference with cytoskeletal integrity induced by treatment with cytochalasin prevented PKC-mediated activation of the potassium current [7]. In this case, at least, it appears that an intact actin cytoskeleton is required for channel activation. In salivary ductal cells, alterations in intracellular calcium levels altered the volume sensitive potassium efflux, consistent with the involvement of conventional, calcium-sensitive PKC isoforms [5]. However, calcium could clearly affect potassium channels by several other means. Indeed, in many cell types calcium has been implicated in volume regulation [8–13], even in cases where PKC has not been invoked [14–16]. The effects of calcium may be direct, altering the gating of potassium channels without intervening phosphorylation events, or through calcium-sensitive kinases other than conventional PKC isoforms. Calcium and calmodulin-dependent kinases are prominent in this category. For example, activation of potassium channels in response to cell swelling is dependent on calcium/calmodulin stimulated kinase in villus epithelial cells, as documented by use of pharmacological inhibitors [17]. A similar pathway has also been implicated in RVD in mudpuppy red blood cells [18], which in addition utilize electroneutral exchangers for volume control. Despite the widespread involvement of calcium in potassium channel activation, requirement for the divalent cation is not universal, as some cell types display calcium-independent RVD [19–22]. Obviously, other regulatory cascades must be invoked in such cases. There is no compelling evidence that protein kinase A (PKA) triggers volume sensitive potassium efflux in response to cell swelling and pharmacological tests have failed to show any involvement [7, 23]. On the other hand, a role for tyrosine kinases has been suspected. Several potassium channels known to be involved in RVD are also affected by tyrosine kinases [24, 25]. The voltage gated channel hK1.5, a known mediator of RVD, associates with and can be phosphorylated by Src. Such phosphorylation inhibits potassium efflux through the channel [25], raising the possibility that dephosphorylation of constitutively phosphorylated sites may occur upon swelling. In other settings, tyrosine phosphorylation may potentiate RVD. The activity of the mouse calcium-dependent maxi-K channel is enhanced by phosphorylation catalyzed by the tyrosine kinases Pyk2 and Hck [26]. Similarly, the activity of BKCa channels is positively regulated by tyrosine phosphorylation, in this case, mediated by Src [27]. In this manner, modulation of tyrosine kinase activity could contribute to the development of RVD. However, definitive evidence linking the activation of tyrosine kinases by cellular volume to the modulation of potassium efflux is still lacking and in some instances, pharmacological data argue against this possibility. In one such instance, the

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application of genistein, a wide spectrum tyrosine kinase inhibitor, had no effect on RVD in villus epithelial cells [17]. Whilst mitogen-activated protein kinases (MAPK) appear to play a central role in the regulation of volume in response to cell shrinkage (reviewed in [28]), investigation of their role in RVD has been limited. In the perfused rat liver, hypoosmotic cell swelling activates ERK-2 and p38 MAPK, but not JNK-1. Pharmacological evidence suggests that K efflux may require the activation of p38 MAPK, but not that of ERK-2 [29]. Finally, cell swelling has also been reported to activate phosphatidylinositol 3-kinase and its products in turn stimulate protein kinase B (PKB/Akt). There is no evidence, however, that the volume sensitive K current promoted by swelling requires the activation of either of these kinases [29]. In summary, it is evident that the relationship between cell volume, potassium conductance and kinase activity is complex. Not only are multiple potassium channels sensitive to cell swelling and capable of initiating volume restoration, but an assortment of kinases appear to sense cell volume as well. The causal relationship of these events is not always clear, as it has often been established through the use of imperfect pharmacological agents. More specific manipulations, such as gene silencing or use of knockout animals, will be needed in the future to better delineate these interactions. Cl Channels The role of PKC in regulating the VRAC has been the subject of multiple studies, the results of which are seemingly contradictory. The best explanation for the observed discrepancies is that the effects of PKC on Cl efflux in response to cell swelling may differ depending on the cell type and/or animal species under investigation. The activity of VRAC in canine, chick and mouse cardiomyocytes [30–32], as well as in epithelial cells, is enhanced by the activation of PKC [33, 34]. In contrast, PKC activation inhibits VRAC currents in rabbit and guinea pig cardiomyocytes [35, 36], ciliary epithelial cells [37, 38], canine colonic and pulmonary arterial smooth muscle cells [39, 40], rat brain endothelial cells [41] and in a mouse inner medullary collecting duct cell line [42]. This distinct responsiveness may be attributable to differential involvement of ancillary proteins, since in some instances PKC inhibition of VRAC was shown to be mediated through P-glycoprotein [43, 44]. There is an abundance of evidence, both pharmacological and molecular in nature, to support a role for tyrosine kinases in the modulation of VRAC activity. Non-receptor tyrosine kinases including FAK and Src are activated by cell swelling [45–48]. In addition, Lck activity was demonstrated to be necessary for VRAC activation in human T lymphocytes, using genetically modified cells lacking the kinase or pharmacological inhibitors of its activity [49–51].


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Further, a receptor tyrosine kinase, EGFR, has been implicated in mediating VRAC in the case of rabbit cardiomyocytes [52]. In accordance with these findings, studies employing reasonably selective antagonists have demonstrated that an inhibition of tyrosine kinase activity impairs VRAC activation in astrocytes [53] and bovine endothelium [54]. However, as often occurs in this field, what appear to be diametrically opposing observations have also been reported: phosphotyrosine phosphatase inhibitors were reported to suppress VRAC in mouse fibrobasts [55] suggesting that phosphorylation is inhibitory to channel function. Consistent with tyrosine kinase-mediated inhibition of VRAC, targeting Src to caveolae in calf pulmonary endothelial cells, inhibited VRAC [56]. Two reports point to involvement of MAP kinases in the activation of VRAC, which may occur downstream of tyrosine kinase signaling [50, 53]. Other serine/ threonine kinases have also been implicated in the regulation of VRAC. The serum and glucocorticoid-stimulated kinase (SGK), which is known to be activated by cell swelling, was demonstrated to activate VRAC [57]. Another study supports a role for calmodulin in mediating VRAC in Ehrlich ascites tumor cells. In this system pimozide, a calmodulin blocker, inhibited the volumeinduced Cl current [58]. No direct evidence of phosphorylation was offered, but such data are consistent with activation of a calcium-calmodulin-dependent kinase (fig. 1). Experiments utilizing the Clostridium botulinum toxin C3 have implicated Rho signaling and an intact cytoskeleton in the activation of VRAC in intestinal epithelial cells [59]. In accordance with this concept, signaling through Rhoassociated kinase (ROK) and myosin light-chain (MLC)-kinase, which are key to the maintenance of cytoskeletal integrity, is also necessary for normal activation of VRAC [60, 61]. However, it is most likely that these effects are indirect and rather than pointing to the immediate mechanism of channel activation, reflect a need for an intact cytoskeleton to sense volume changes [62]. Kinases can also affect VRAC negatively. One study demonstrated that stimulation of PKA inhibited VRAC, while the converse effect was also observed, i.e. inhibition of PKA stimulated VRAC [63]. However, this was not observed in all cases: in cardiomyocytes PKA-induced phosphorylation failed to affect VRAC [64]. Lipid kinases may also contribute to chloride channel modulation during RVD. Pharmacological evidence utilizing wortmannin implicated PI3-kinase in the stimulation of VRAC [57, 65]. The variety of kinases invoked in VRAC regulation and the diversity of effects reported is rather bewildering. This may stem from bona fide differences among biological systems, but most likely reflects the use of different readouts and stimulatory conditions. These apparent discrepancies will be resolved only when systematic analyses of multiple kinases are performed in one or more

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systems where all variables are comparable and suitably controlled. Definitive establishment of the molecular identity of VRAC will be essential for a full understanding of the mode of action of protein and lipid kinases in volume regulation. K-Cl Cotransport In some cells RVD can be mediated, at least partly, by K and Cl symport (reviewed in [2]). There are four known isoforms of the symporter (KCC1–4). KCC2 is the only isoform not known to be activated by cell swelling [66]. Swelling-induced activation of K-Cl symport is related to the state of serine/threonine and/or tyrosine phosphorylation (reviewed in [67]), in an inverse manner. Indeed, swelling-induced K-Cl cotransport is stimulated by the non-specific kinase inhibitor staurosporine. Conversely, the phosphatase inhibitor calyculin A antagonizes symport activity, implicating serine/threonine kinases and phosphatases in swelling-mediated activation of K-Cl cotransport [68, 69]. Accordingly, more specific activation of PKC also resulted in inhibition of cotransport [70]. A similar relationship has been reported for tyrosine phosphorylation, pharmacological inhibition of protein tyrosine phosphatases antagonized swelling-induced K-Cl cotransport [68, 71]. Additionally, analysis of cell lines deficient in the tyrosine kinases Fgr and Hck revealed greatly activated K-Cl symport function, suggesting that Src-family tyrosine kinases antagonize the activation of K-Cl cotransport [72]. Thus, it appears that the volume-induced activation of K-Cl cotransport is caused by dephosphorylation of critical residues in the symporter and/or ancillary regulators, though exceptions to this notion have been reported, where receptor tyrosine kinase activation correlates with stimulation of transport [73, 74]. Organic Osmolyte Efflux Following cell swelling, some cells can effect a return to normal volume through the rapid efflux of organic osmolytes, notably taurine, aspartate, glycine and myo-inositol (reviewed in [2, 75]). Taurine is the major osmolyte involved in this process in most cells. The pathway utilized for taurine efflux is incompletely understood, but volume-sensitive chloride (anion) channels are thought to feature prominently. This dual-function, chloride and organic solute channel has been referred to as the volume-sensitive organic osmolyte-anion channel or VSOAC [76]. Like other volume-dependent systems, VSOAC is responsive to the activity of protein kinases, though the relationship is complex. The effect of PKC modulation on VSOAC contrasts between different systems, as was discussed for VRAC. In the rat supraoptic nucleus and in endothelial cells neither activation nor inhibition of PKC affected VSOAC [77, 78]. On the other hand, PKC


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activation was shown to either potentiate [30, 79] or inhibit [36, 39, 41] VSOAC. Similarly, PKA has been reported to potentiate [79, 80], inhibit [81] or have no affect [78, 82, 83] on VSOAC activity. Because VSOAC is not a welldefined, single molecular entity, these differences may be due to the involvement of different transporter/channel species in mediating the flux. Alternatively, the cellular context may modulate the behavior of a single molecular species in varying fashions. Pharmacological inhibitors have been used to implicate tyrosine kinase signaling in the regulation of VSOAC during RVD. In neurons the efflux of taurine is specifically inhibited by genistein, a tyrosine kinase inhibitor of ample spectrum, and potentiated by ortho-vanadate, a tyrosine phosphatase inhibitor [77, 83]. These findings, together with the large amount of data obtained analyzing Cl currents – previously shown to correlate with taurine efflux – support a role for tyrosine kinases in mediating RVD through the activation of VSOAC.

Regulatory Volume Increase

Exposure to a hypertonic extracellular milieu will cause rapid cell shrinkage. This is counteracted in most cells by stimulation of the influx of sodium and chloride. The accompanying osmotically-driven influx of water results in a compensatory cell swelling. This regulatory process has been termed RVI. During the course of RVI, sodium, the major extracellular cation, is frequently driven into the cells through an exchange for protons, a process mediated by the NHE. In other instances, sodium influx in response to hypertonicity is the result of cotransport with potassium and chloride. In all these instances net sodium uptake functions to restore intracellular volume in shrunken cells (for reviews see [1, 84, 85]) (fig. 2). At face value, the stoichiometric exchange of sodium for protons could be thought of as an osmotically ineffectual process, not one conducive to volume restoration. It should be kept in mind, however, that protons are buffered in the intracellular milieu. Replacement of the extruded protons by dissociation of cytosolic weak acids results in a net gain of sodium with negligible change in proton concentration, and thus net osmotic gain. Over time, pH tends to increase measurably despite the fact that the membrane is freely permeable to carbon dioxide. The elevated bicarbonate concentration in turn drives chloride into the cell via electroneutral AEs. This translates into a combined gain of sodium and chloride and extrusion of protons and bicarbonate. The two species translocated extracellularly can recombine to produce water and carbon dioxide, which can re-enter the cell. That NHE1 is the primary mechanism driving RVI in many cell types has been confirmed by deletion experiments. Indeed,

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HCO 3

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PKC

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2Cl K

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Fig. 2. Acute cell shrinkage mediated activation of kinases and their targets. AE Anion exchanger, Cam calmodulin, Jak2 janus kinase 2, MAPK mitogenactivated protein kinase, NHE1 sodium hydrogen exchanger isoform 1, NKCC sodium potassium chloride cotransporter, PKC protein kinase C.

mutant cells lacking NHE1 fail to regain volume after imposed shrinkage [86, 87]. A greatly increased intracellular tonicity will inhibit many normal intracellular processes. Therefore a more prolonged exposure to a hypertonic milieu will induce the import and synthesis of organic osmolytes, these include: betaine, taurine, inositol and sorbitol (reviewed in [28, 88]). The enhancement of solute uptake is accomplished, in part, through the activation of transcription factors like the tonicity-enhancer binding protein, TonEBP (fig. 3). Prolonged exposure to hypertonic medium leads to the nuclear localization of TonEBP and consequent transcription of BGT1 [89], SMIT [90, 91] and aldose reductase [92, 93]. Once transcribed these genes promote the intracellular import of betaine, inositol and taurine, and the conversion of D-glucose to sorbitol, respectively [4] (fig. 3). Na/H Exchange Cell shrinkage induced by exposure to a hypertonic medium stimulates the influx of sodium and chloride. The combined influx of the ion pair has been shown in multiple cell types to be mediated by NHE1 coupled functionally to bicarbonate/chloride exchange [94–99]. A primary role of NHE1 in RVI has been confirmed in fibroblastic cell lines deficient in this isoform. When exposed to a hyperosmolar extracellular environment such cells fail to display a significant compensatory swelling [100]. Pharmacological inhibition of NHE1 or the absence of extracellular sodium prevented the parallel stimulation of AE


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SMIT, AR BGT1 TonEBP D-Glucose

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Fig. 3. Chronic cell shrinkage mediated activation of kinases and their downstream affects. Chronic hyperosmolarity stimulates the nuclear localization of TonEBP through the activation of: protein kinase A (PKA), the tyrosine kinase Fyn and through a MAPK pathway involving p38, MAPK or extracellular signal-regulated kinase kinase kinase 3 (MEKK3), Rac GTPase, MAP kinase kinase 3 (MKK3) and osmosensing scaffold for MEKK3 (OSM). This results in the transcription and translation of the sodium/myo-inositol cotransporter (SMIT), aldose reductase (AR) and betaine/GABA transporter (BGT1) whose activities result in the intracellular accumulation of the organic osmoles: inositol, taurine, sorbitol and betaine. To get maximal expression, all three pathways must be activated.

in response to shrinkage, confirming that AE activation is secondary to the volume-induced activation of NHE [101]. The specific mechanism(s) responsible for NHE1 activation elicited by exposure to hypertonic extracellular medium and the consequent cell shrinkage are still incompletely understood. Although alteration in cell volume activates numerous kinases, the state of NHE1 phosphorylation is not significantly altered by cell shrinkage [86, 102–104]. This has been confirmed using both

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phosphopeptide mapping and by the persistence of cell shrinkage-induced activation of NHE1 in specific mutants lacking sites of phosphorylation [86, 105]. Several studies have investigated tyrosine kinase activation in response to cell shrinkage in relation to NHE activation [102, 103]. Although NHE1 is not directly phosphorylated in response to a decreased cell volume, inhibition of tyrosine phosphorylation has been shown to prevent shrinkage-induced activation of NHE1. The identity of the specific kinases responsible for this effect has only recently started to be elucidated. Garnovskaya et al. [106] demonstrated in CHO cells that cell shrinkage leads to the phosphorylation of Janus kinase 2 (Jak2), which in turn phosphorylates calmodulin. The phosphorylation of calmodulin parallels its physical interaction with, and the increased activity of, NHE1. Whether there is an upstream activator of Janus kinase in this system is unknown. Recent studies using whole-cell patch clamp and an oscillating pH electrode have suggested the existence of an associated protein necessary for the activation of NHE1 in response to decreased cell volume [107]. The identity of this molecule, and whether it is a kinase, is speculative at this time. Na/K/2Cl Symport There are two mediators of the electroneutral symport of Na/K and Cl (NKCC1 and NKCC2). They share many similarities to both Na/Cl symporters and K/Cl symporters. NKCC1 has been frequently implicated in the regulation of cell volume during RVI [1, 84, 108]. In contrast to NHE1 activation upon cell shrinkage, the activation of NKCC1 is clearly due to the direct phosphorylation of a serine/threonine residue of the cotransporter itself [109, 110]. The identity of the responsible kinase, however, is a matter of ongoing debate. PKC has been implicated in NKCC phosphorylation and its consequent activation upon cell shrinkage. In rat hepatocytes a specific PKC blocker prevented the activation, while a stimulator of PKC increased NKCC activity [111]. This is consistent with results in human tracheal epithelia showing that PKC and the MAP kinase ERK are involved in shrinkage-induced activation of NKCC [112]. Although MAPK-related kinases like JNK can phosphorylate NKCC in vitro [113], findings in muscle cells do not support a role for MAPKfamily enzymes in the activation of NKCC in response to cell shrinkage [114]. Hyperosmotic stress has been shown to result in the phosphorylation of the light chain of myosin [115–117]. This event coincides with the activation of NKCC upon cell shrinkage. Moreover ML-7, a MLC-kinase inhibitor prevents hypertonically-induced MLC phosphorylation and the activation of NKCC [115, 118, 119]. Rho, an upstream activator of ROK and MLC-kinase, is also activated by cell shrinkage [120]. However, inhibition of hyperosmolarity-induced MLC phosphorylation, through genetic means or by pharmacological inhibition of ROK or of Rho activation, fails to suppress NKCC activity [118, 120]. In contrast,


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cells lacking functional MLC are unable to activate NKCC upon shrinkage. This suggests that signaling through MLC is necessary for the activation of NKCC, but that increased phosphorylation of MLC is not essential [118]. TonEBP and the Late Response to Hypertonicity An acute increase in intracellular ionic strength by the mechanisms described above is followed by a slower accumulation of organic osmolytes. This compensatory response to an increased osmolarity on a chronic basis avoids a prolonged inhibition of macromolecular function induced by an increase in intracellular ionic strength. The acute intracellular accumulation of Na, K and Cl through RVI is followed by the induction of osmolyteaccumulating genes [121, 122]. This latter process is controlled by the binding of the TonEBP, also known as NFAT5 [92, 123], to a common genomic element named tonicity enhancer (TonE, also called osmotic response element or ORE) [124, 125]. This interaction, induced by cell shrinkage, results in the transcription of BGT1, SMIT, and AR and the consequent intracellular accumulation of betaine, sorbitol, taurine and inositol [4, 126]. Mice with a deletion in the TonEBP gene lack detectable BGT1, SMIT and AR expression in renal medullary cells and in their fibroblasts after exposure to a hyperosmolar environment [127]. The signals that cause intranuclear localization, binding and transcription of these genes during the late response to cell shrinkage are incompletely understood. However, a central role for the MAP kinases in the process has been established [28] and is detailed below. Although ERK and JNK are also activated by tonicity [128], p38 seems to play the central role in the activation of TonEBP by hypertonicity [28, 129, 130]. This was established through the use of pharmacological inhibitors of p38 MAP kinases in multiple cell lines. In the presence of such inhibitors hyperosmolarity failed to induce the transcription of SMIT, BGT1 and AR [131–133]. Further, expression of a mutant p38 MAPK inhibited TonEBP binding to TonE, as measured by reporter gene expression [134]. The upstream events leading to activation of p38 MAPK in response to hypertonicity have recently been elucidated. MEKK3 activates p38 in response to hypertonic stress [135]. In turn, MEKK3 itself is activated by hypertonicity through an association with a complex of proteins that include MKK3, Rac GTPase and a molecule called osmosensing scaffold for MEKK3 (OSM) [135]. The p38 MAPK pathway just described is the major, but not the exclusive mediator of TonEBP activation. In contrast to acute volume regulation where it has little effect, PKA contributes to the chronic volume regulatory response [136]. Both the pharmacological inhibition of PKA and dominant negative PKA inhibit the activation of TonEBP and the transcription of TonE reporter genes in response to cell shrinkage [136]. In contrast, pharmacological inhibition or transfection of

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dominant-negative PKC fails to alter gene expression mediated by TonEBP [137]. Yet, even the combined activation of p38 MAP Kinase and PKA is insufficient to produce a complete response to cell shrinkage. The activation of Fyn, a nonreceptor tyrosine kinase, and ATM is necessary to produce the maximal induction of TonEBP/TonE-mediated gene transcription in response to hypertonicity [134, 138–140]. Therefore, the process is complex and subject to regulation by various kinase pathways.

Concluding Remarks

When faced with an anisoosmotic environment cells have adopted a myriad of responses to maintain their volume. Acutely, the cells engage ion and other osmolyte-transporting systems and use pre-existing gradients to rapidly compensate for the alterations in volume and ionic strength that can have detrimental effects on cell metabolism and ultimately on survival. When facing sustained challenges, the cells also activate gene transcription and translation to promote solute transport and biosynthesis. In all cases, volume sensors and signals transducers are required and kinases have been invoked in the latter capacity. The evidence in some instances is compelling and the precise kinases involved have been identified. In most cases, however, the data accumulated to date are inconclusive, often confusing. In some instances, the reigning confusion may be attributed to the fact that the transporting entities have not been defined unambiguously and different molecular species may be involved in different cell types. In others, the cellular context may alter the manner in which a specified, well-defined transporter may respond to volume changes. This could result from variability in the volume sensor or signal transducing mechanisms. It is obvious that a more systematic approach to the study of volume regulation is required, minimizing the reliance on pharmacological inhibitors of imperfect selectivity. Examination of volume responses in cells where specific genes have been ablated or silenced using siRNA will improve our knowledge, while continuing identification of defined volume-responsive channels and transporters will ultimately complement the picture.

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119 Krarup T, Jakobsen LD, Jensen BS, Hoffmann EK: Na-K-2Cl cotransport in Ehrlich cells: regulation by protein phosphatases and kinases. Am J Physiol 1998;275(pt 1):C239–C250. 120 Di Ciano-Oliveira C, Sirokmany G, Szaszi K, et al: Hyperosmotic stress activates Rho: differential involvement in Rho kinase-dependent MLC phosphorylation and NKCC activation. Am J Physiol Cell Physiol 2003;285:C555–C566. 121 Neuhofer W, Bartels H, Fraek ML, Beck FX: Relationship between intracellular ionic strength and expression of tonicity-responsive genes in rat papillary collecting duct cells. J Physiol 2002; 543(pt 1):147–153. 122 Huang Z, Tunnacliffe A: Response of human cells to desiccation: comparison with hyperosmotic stress response. J Physiol 2004;558(pt 1):181–191. 123 Lopez-Rodriguez C, Aramburu J, Rakeman AS, Rao A: NFAT5, a constitutively nuclear NFAT protein that does not cooperate with Fos and Jun. Proc Natl Acad Sci USA 1999;96:7214–7219. 124 Rim JS, Atta MG, Dahl SC, Berry GT, Handler JS, Kwon HM: Transcription of the sodium/myoinositol cotransporter gene is regulated by multiple tonicity-responsive enhancers spread over 50 kilobase pairs in the 5-flanking region. J Biol Chem 1998;273:20615–20621. 125 Ferraris JD, Williams CK, Jung KY, Bedford JJ, Burg MB, Garcia-Perez A: ORE, a eukaryotic minimal essential osmotic response element. The aldose reductase gene in hyperosmotic stress. J Biol Chem 1996;271:18318–18321. 126 Ho SN: The role of NFAT5/TonEBP in establishing an optimal intracellular environment. Arch Biochem Biophys 2003;413:151–157. 127 Lopez-Rodriguez C, Antos CL, Shelton JM, et al: Loss of NFAT5 results in renal atrophy and lack of tonicity-responsive gene expression. Proc Natl Acad Sci USA 2004;101:2392–2397. 128 Berl T, Siriwardana G, Ao L, Butterfield LM, Heasley LE: Multiple mitogen-activated protein kinases are regulated by hyperosmolality in mouse IMCD cells. Am J Physiol 1997; 272(pt 2):F305–F311. 129 de Nadal E, Alepuz PM, Posas F: Dealing with osmostress through MAP kinase activation. EMBO Rep 2002;3:735–740. 130 Zarubin T, Han J: Activation and signaling of the p38 MAP kinase pathway. Cell Res 2005;15:11–18. 131 Denkert C, Warskulat U, Hensel F, Haussinger D: Osmolyte strategy in human monocytes and macrophages: involvement of p38MAPK in hyperosmotic induction of betaine and myoinositol transporters. Arch Biochem Biophys 1998;354:172–180. 132 Nadkarni V, Gabbay KH, Bohren KM, Sheikh-Hamad D: Osmotic response element enhancer activity. Regulation through p38 kinase and mitogen-activated extracellular signal-regulated kinase kinase. J Biol Chem 1999;274:20185–20190. 133 Sheikh-Hamad D, Di Mari J, Suki WN, Safirstein R, Watts BA 3rd, Rouse D: p38 kinase activity is essential for osmotic induction of mRNAs for HSP70 and transporter for organic solute betaine in Madin-Darby canine kidney cells. J Biol Chem 1998;273:1832–1837. 134 Ko BC, Lam AK, Kapus A, Fan L, Chung SK, Chung SS: Fyn and p38 signaling are both required for maximal hypertonic activation of the osmotic response element-binding protein/tonicityresponsive enhancer-binding protein (OREBP/TonEBP). J Biol Chem 2002;277:46085–46092. 135 Uhlik MT, Abell AN, Johnson NL, et al: Rac-MEKK3-MKK3 scaffolding for p38 MAPK activation during hyperosmotic shock. Nat Cell Biol 2003;5:1104–1110. 136 Ferraris JD, Persaud P, Williams CK, Chen Y, Burg MB: cAMP-independent role of PKA in tonicityinduced transactivation of tonicity-responsive enhancer/osmotic response element-binding protein. Proc Natl Acad Sci USA 2002;99:16800–16805. 137 Zhao H, Tian W, Cohen DM: Rottlerin inhibits tonicity-dependent expression and action of TonEBP in a PKCdelta-independent fashion. Am J Physiol Renal Physiol 2002;282: F710–F717. 138 Irarrazabal CE, Liu JC, Burg MB, Ferraris JD: ATM, a DNA damage-inducible kinase, contributes to activation by high NaCl of the transcription factor TonEBP/OREBP. Proc Natl Acad Sci USA 2004;101:8809–8814.


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139 Zhang Z, Ferraris JD, Irarrazabal CE, Dmitrieva NI, Park JH, Burg MB: Ataxia telangiectasiamutated, a DNA damage-inducible kinase, contributes to high NaCl-induced nuclear localization of transcription factor TonEBP/OREBP. Am J Physiol Renal Physiol 2005;289: F506–F511. 140 Nahm O, Woo SK, Handler JS, Kwon HM: Involvement of multiple kinase pathways in stimulation of gene transcription by hypertonicity. Am J Physiol Cell Physiol 2002;282:C49–C58.

Sergio Grinstein Cell Biology Program, The Hospital For Sick Children 555 University Avenue Toronto, ON M5G 1X8 (Canada) Tel. 1 416 813 5727, Fax 1 416 813 5028, E-Mail [email protected]

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Tonicity-Dependent Regulation of Osmoprotective Genes in Mammalian Cells Joan D. Ferraris, Maurice B. Burg Laboratory of Kidney and Electrolyte Metabolism, National Heart Lung Blood Institute, National Institutes of Health, DHHS, Bethesda, Md., USA

Abstract Cells in the renal medulla are normally exposed to levels of NaCl that are extremely high and that vary with concentration of the urine. Such high levels of NaCl cause cellular perturbations, including increased DNA double-strand breaks, increased oxidation of DNA and proteins, and cytoskeletal alterations. Despite these perturbations the cells are able to survive and function because of osmoprotective responses that include accumulation of compatible organic osmolytes and increased abundance of heat shock proteins and water channels. Many of the responses are initiated by increased gene transcription, directed by the transcription factor TonEBP/OREBP. Here, we review the sensors of hypertonicity, the signaling pathways to TonEBP/OREBP, and the ways in which it is activated to increase transcription. Multiple signals are involved, including some that arise directly from the cellular perturbations caused by hypertonicity. Although the combination of these signals is necessary for full osmotic activation of TonEBP/OREBP, no one of them, alone, is sufficient. We conclude that hypertonicity profoundly alters the state of cells, providing numerous interrelated inputs to the osmoregulatory network. Copyright © 2006 S. Karger AG, Basel

Hypertonicity, such as that produced by high NaCl, perturbs cells by causing osmotic efflux of water, resulting in cell shrinkage and elevation of the concentrations of all intracellular components including ions [1] and macromolecules [2]. Cells can adapt up to a certain hypertonicity, but higher levels can kill them by apoptosis [3, 4]. The osmolality of most mammalian extracellular fluids is maintained remarkably constant at approximately 290 mOsmol/kg by the urinary concentrating and diluting system. A notable exception is the renal medulla in which interstitial NaCl normally is continuously very high and varies with concentration of the urine.

We begin by discussing cellular perturbations caused by high NaCl in addition to those directly related to changes in cell volume, then discuss the osmoprotective mechanisms that allow the cells to survive and function.

Cellular Perturbations Caused by Hypertonicity

Increased DNA Breaks Acute elevation of NaCl increases the number of DNA double-strand breaks in cell culture [5], and the level remains high even after the cells adapt to the high NaCl and appear normal in other respects [6]. Further, cells in renal medullas, while exposed to the high level of NaCl that is normal there [7] contain many more DNA breaks than cells in the renal cortex, where NaCl is not elevated [6]. Lowering the level of NaCl leads to rapid repair of the excess DNA breaks both in cell culture and in vivo [6]. It is not entirely clear how high NaCl increases the number of DNA breaks. At least part of the explanation is that the hypertonicity inhibits repair of naturally occurring DNA breaks, so they accumulate [6]. However, generation of additional breaks has not been ruled out. Increased Reactive Oxygen Species High NaCl increases Reactive Oxygen Species (ROS) in cultures of renal cells [8–10] and ROS are high in renal medullas [11], where NaCl normally is elevated in vivo. ROS include hydrogen peroxide, hydroxyl radicals and superoxides [12]. Pathological elevation of ROS damages proteins, lipids, and DNA [12]. The hypertonicity-induced ROS oxidize proteins in cell culture and in vivo, and oxidize DNA in culture [8]. However, ROS also convey signals from cytokines, hormones and ions to downstream effectors, including transcription factors [12]. For example, signaling by ROS is involved in high NaCl-induced phosphorylation of Syk kinase [13] and superoxides and hydrogen peroxide regulate the activity of AP-1 and NF␬B transcription factors [14, 15]. Thus, ROS may damage cellular constituents, but they also have the potential to convey regulatory signals. Cytoskeletal Rearrangement Hypertonicity causes the F-actin cytoskeleton to reorganize, which is mediated by the Rho family GTPases, Rac and Cdc42 [16]. The F-actin binding protein, cortactin, translocates to the cortical cytoskeleton, which reinforces it. In addition, hypertonicity induces the phosphorylation and/or redistribution of myosin, mediated by activation of the Rho and Rho/ROK pathway [17]. One consequence of remodeling the actin cytoskeleton is increased mitochondrial ROS production, associated with integrin-mediated changes in the shape of the

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cells and activation of Rac and RhoA [18]. The GTPase mediated signals that initiate the hypertonicity-induced cytoskeletal rearrangement also act as signals for other aspects of osmoregulation, as will be seen.

Osmoprotective Genes

The initial, rapid response to hypertonicity-induced cell shrinkage is activation of transporters that bring inorganic ions into the cells, followed by osmotic influx of water. This restores cell volume but, in the process, elevates intracellular ionic strength. Since high levels of inorganic ions perturb the structure and function of cellular macromolecules [19], this is an undesirable state. Cells universally respond by accumulating compatible organic osmolytes, which, in effect, replace the excess inorganic ions, while at the same time maintaining cell volume. Compatible organic osmolytes do not perturb macromolecules nearly as much as do inorganic ions. The principal organic osmolytes accumulated in renal medullary cells are sorbitol, glycine betaine (betaine), myo-inositol (inositol), taurine and glycerophosphocholine [20]. Sorbitol accumulates through enzymatic conversion of glucose, catalyzed by aldose reductase (AR). Betaine, inositol and taurine are transported into the cell by the betaine/␥-aminobutyric acid transporter (BGT1), the sodium-inositol co-transporter, and the taurine transporter, respectively [20]. Synthesis of glycerophosphocholine from phosphatidylcholine increases, catalyzed by increased abundance of the phospholipase [21], neuropathy target esterase [unpublished data]. The protein abundance of each of these enzymes and transporters rises because of increased transcription of its gene, resulting in more of its mRNA [20]. The increased transcription results from activation of the transcription factor TonEBP/OREBP (tonicity-responsive enhancer-/osmotic response element binding protein) [22, 23]. TonEBP/OREBP also transactivates other genes involved in osmoprotection and urinary concentration, including those for heat shock protein 70 [24], vasopressin-activated urea transporters (UT-A) [25] and possibly aquaporin 2 [26]. Heat shock protein 70 is a protein chaperone that helps protect cells from high NaCl [27]. UT-A1 and UT-A2 increase renal interstitial urea which contributes to the cortico-medullary osmotic gradient necessary to concentrate urine. Aquaporin 2 increases water permeability of the renal collecting duct, which increases urine concentration in response to water restriction. Regulatory regions of genes that are transcriptional targets of TonEBP/OREBP contain one or more binding sites for TonEBP/OREBP, called osmotic response elements (OREs) [28–30] or tonicity-responsive enhancers (TonEs) [31–35].

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How TonEBP/OREBP is Activated by Hypertonicity

Increased mRNA and Protein Abundance Hypertonicity increases TonEBP/OREBP mRNA abundance in MadinDarby canine kidney (MDCK) cells [36], in HeLa cells [23] and in mouse inner medullary collecting duct (mIMCD3) cells [37]. In cell culture the increase of TonEBP/OREBP mRNA is transient with a time course that differs among cell lines. MDCK cell TonEBP/OREBP mRNA increases within 6 h, reaches a maximum of about threefold by 12 h and falls to 2-fold by 18 h [36]. Hela cell TonEBP/OREBP mRNA increases within 2 h, reaches a maximum at 6 h and decreases to basal level by 12 h [23]. mIMCD3 cell TonEBP/OREBP mRNA abundance peaks 2.4-fold after 4 h, then declines [37]. mRNA abundance is a balance of relative rates of synthesis and degradation. The hypertonicity-induced increase in TonEBP/OREBP mRNA is due to a transient (6 h) increase in its stability, mediated by elements within its 5⬘-UTR [37]. Stabilization by high NaCl is sufficient to explain the increase in mRNA without any change in transcription [37]. The stabilization of TonEBP/OREBP mRNA by hypertonicity was not been observed in MDCK cells, which led to an earlier conclusion that the mRNA rose because of increased transcription [36]. However, attempts to document an increase in transcription by nuclear run-on failed [23, 36]. The most likely explanation for the different results is that measurement of TonEBP/OREBP mRNA stability in the MDCK cells was started too late (not until after12 h of hypertonicity) to observe the short lived stabilization of the mRNA [37]. As a result of increased mRNA abundance, TonEBP/OREBP protein increases. In combination with tonicity-dependent nuclear translocalization, discussed below, the increased nuclear abundance of TonEBP/OREBP provides more TonEBP/OREBP protein to bind to OREs. In MDCK cells, the hypertonicity-induced increases in TonEBP/OREBP protein abundance and synthesis rate are proportional to the increase in mRNA abundance [36]. Similarly, in mIMCD3 cells, TonEBP/OREBP protein increases 3.0-fold, similar to the increase of mRNA [37]. Hypertonicity does not affect the rate of TonEBP/OREBP protein degradation [36]. Dimerization TonEBP/OREBP is constitutively dimerized, and the dimerization is necessary for several aspects of its hypertonicity-dependent activation. The N-terminus of TonEBP/OREBP contains two dimerization domains [38]. TonEBP/OREBP forms stable dimers (or higher order oligomers) in solution [39], independent of tonicity [38]. Dimerization of TonEBP/OREBP is required for its DNA binding [38]. The TonEBP/OREBP homodimer completely encircles its DNA target [40]. The constitutive dimerization of TonEBP/OREBP is also required for its ‘proper’ phosphorylation when NaCl is high [41] and for transactivation of its target genes [38].

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Phosphorylation Many transcription factors are regulated by single or multisite phosphorylation [42, 43], controlling nuclear localization [44], DNA binding [45] and transactivation [45–47]. TonEBP/OREBP becomes phosphorylated on serine and tyrosine residues within 30 min of increasing NaCl [48]. However, which amino acids are phosphorylated and their exact roles are the subject of ongoing research. Convincing indirect evidence points to certain amino acids, but even the phosphorylation of those particular amino acids has not been directly demonstrated. Defining the role of phosphorylation is complicated by the large number of potential phosphorylation sites in TonEBP/OREBP, namely 216 serines, 15 tyrosines, and 111 threonines. Despite these uncertainties, it seems likely that the phosphorylation of TonEBP/OREBP is involved in tonicitydependent regulation of its transcriptional activity, as detailed below. Nuclear Localization In order to bind to their cognate DNA elements, transcription factors must enter the nucleus. Proteins as large as TonEBP/OREBP (⬎50 kDa) are too big to diffuse freely through nuclear pores [44]. Rather, they are actively transported through the pores, associated with importins and exportins [44]. The relative rates of nuclear import and export determine the distribution between the cytoplasm and the nucleus. Binding to importins is mediated by nuclear localization sequences (NLS) in transcription factors. The TonEBP/OREBP NLS is located in its N-terminus [23, 39]. Binding to exportins is mediated by nuclear export sequences (NES) [44]. Preliminary reports have identified NESs, also in the N-terminus of TonEBP/OREBP [49]. The distribution of TonEBP/OREBP in the cell is tonicity-dependent. In MDCK cells, HeLa cells and Jurkat cells at ⬃300 mOsmol/kg, TonEBP/ OREBP is distributed between the cytoplasm and nucleus, whereas at 500 mOsmol/kg, most of the TonEBP/OREBP is in the nucleus [22, 23, 38], and at 135 mOsmol/kg in MDCK cells, almost all of the TonEBP/OREBP is in the cytoplasm [36]. Hypotonicity reduces transcriptional activity of TonEBP/OREBP [36, 50], which is at least partially explained by its absence from the nucleus. Recombinant TonEBP/OREBP containing only the N-terminal amino acids 1–547, localizes in the nucleus when NaCl is elevated. Thus, the C-terminal amino acids, 548–1531, are not required for the translocation [51]. The N-terminus of TonEBP/OREBP contains a series of amino acids homologous to a bipartite NLS [23, 52]. There are also two NES, NES-A and NES-B, in the N-terminus of TonEBP/OREBP [49]. NES-A is a protein domain that responds to the exportin, CRM1. Leptomycin B, which is an inhibitor of CRM1, blocks nucleocytoplasmic shuttling of TonEBP/OREBP at 300 mOsmol/kg [49]. Disruption of NES-A increases TonEBP/OREBP in the


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nucleus at 300 mOsmol/kg, but hypotonicity still causes nuclear export of this mutant indicating that NES-A is not responsible for nuclear export under hypotonic conditions [49]. On the other hand, NES-B is functional in nuclear export of TonEBP/OREBP under both normotonic and hypotonic conditions. Disruption of NES-B causes nuclear retention of TonEBP/OREBP regardless of tonicity [49]. A number of factors are known that affect the distribution of TonEBP/ OREBP between cytoplasm and nucleus. For example, ataxia telangiectasiamutated kinase (ATM), which is activated by high NaCl [53], contributes to nuclear translocation of TonEBP/OREBP [51]. In the absence of functional ATM, high NaCl-induced nuclear translocation of TonEBP/OREBP is reduced, as is nuclear translocation of recombinant 1–547 TonEBP/OREBP, and translocation is restored when the cells are reconstituted with active ATM [51]. Thus, ATM contributes to high NaCl-induced nuclear translocation of TonEBP/ OREBP through interaction at sites within amino acids 1–547. The effect of ATM could be direct or indirect, mediated by other factors in the signaling pathway. An additional factor is suggested by the observation that the 26S proteasome inhibitor, MG-132, blocks hypertonicity-induced nuclear localization of TonEBP/OREBP in MDCK cells [54]. This is reminiscent of the regulation of NF␬B [44]. Inactive NF␬B is retained in the cytoplasm by binding to I␬B. During activation of NF␬B, I␬B becomes phosphorylated, then degraded in the proteasome, releasing NF␬B to move into the nucleus. Although a similar system for regulation of TonEBP/OREBP is an attractive possibility, no TonEBP/ OREBP cytoplasmic binding protein analogous to I␬B has yet been identified. MG-132 also blocks high NaCl-induced nuclear localization of TonEBP/ OREBP in HepG2 cells, but not in COS-7 cells, indicating cell type dependence [51]. Finally, Cyclosporin A is also reported to inhibit hypertonicityinduced nuclear translocation of TonEBP/OREBP in MDCK cells [55], consistent with a role for calcineurin in nuclear localization. DNA Binding The DNA binding domain of TonEBP/OREBP is located in its N-terminus [23, 38]. Phosphorylation of other transcription factors controls or modifies their binding to DNA [43]. However, isolated TonEBP/OREBP binds to an ORE regardless of treatment with calf intestinal phosphatase, indicating that binding of TonEBP/OREBP to DNA does not require its own phosphorylation [48]. On the other hand, serine/threonine kinase activity apparently is involved in the signaling pathway that leads to binding of TonEBP/OREBP to DNA. TonEBP/OREBP binding to DNA is eliminated by treatment of cell extracts with calf intestinal phosphatase and by PP1, a serine/threonine phosphatase, but not by treatment with PTPase, a phosphotyrosine phosphatase [56].

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Additionally, treatment of cells with a p38 inhibitor or a MEK1 inhibitor, reduces TonEBP/OREBP DNA binding [57]. Increased Transactivational Activity In addition to having motifs that regulate nuclear localization and DNA binding, transcription factors have transactivation domains (TADs) that recruit and interact with a specific assembly of proteins (enhanceosome), as well as with the basal transcriptional complex that includes RNA polymerase. Transactivation of target genes by a transcription factor is regulated by post-translational modifications to the transcription factor, itself, and by its association with other proteins, including additional transcription factors [47, 58]. TonEBP/OREBP contains a TAD that is activated by exposure of cells to high NaCl [50]. The tonicity-regulated TAD is located within amino acids 1039–1249 [59]. Additional TADs are located within amino acids 1–76 and 1363–1476, but these are not sensitive to tonicity [59]. TonEBP/OREBP also has two modulation domains (amino acids 618–820 and 889–955) that potentiate the activity of the TADs but are not independently active. Only one of the modulation domains is responsive to tonicity (amino acids 618–820). All of these domains act synergistically to transactivate target genes in response to hypertonicity [59]. Phosphorylation of transcription factors regulates transactivation by recruiting coactivator proteins that contact the general transcriptional machinery [46] as well as by stabilizing homodimer–DNA complexes [45, 47]. High NaCl increases phosphorylation of recombinant TonEBP/OREBP-548–1531, which contains a functional, high NaCl-activated TAD [50]. Tyrosine and serine/threonine kinase inhibitors reduce the transactivational activity of TonEBP/OREBP at high NaCl [50]. This could be either due to a direct effect on the phosphorylation of TonEBP/OREBP, itself, or it could be indirect, mediated by a signaling partner or a transcription co-factor. In favor of an indirect effect is the failure to find any tonicity-dependent change in net phosphorylation of the TonEBP/OREBP TAD [59]. In favor of a direct effect is the inhibition caused by site-directed mutation of putative phosphorylation sites in TonEBP/OREBP [53], as discussed below. Regulation of TonEBP/OREBP Protein kinases, GTPases, ROS, scaffolding proteins, integrins, and phosphoinositides all contribute to regulation of TonEBP/OREBP, as follows. p38. p38 is a mitogen-activated protein kinase (MAPK), homologous to HOG1, the kinase that directs osmoprotective accumulation of glycerol in yeast [60, 61]. Hypertonicity activates p38 [62]. Inhibition of p38 by chemicals [63, 64] or by transfection of dominant negative constructs [64] reduces


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hypertonicity-induced activation of TonEBP/OREBP, including its transactivational activity, and also reduces mRNA abundance of its transcriptional targets, AR and BGT1 [64]. Thus, considerable evidence points to a role for p38 in osmoregulatory induction of TonEBP/OREBP. However, there are many conditions, not involving hypertonicity, that activate p38, but do not activate TonEBP/OREBP [65, 66]. This raises the question of why activation of p38 by other stimuli does not activate TonEBP/OREBP, whereas activation by hypertonicity does. In fact, the question is even more general, because, as will be discussed below, there are other perturbations and signals that also contribute to activation of TonEBP/OREBP during hypertonicity, but do not stimulate TonEBP/OREBP in the absence of hypertonicity. In the case of p38, a possible explanation is that a particular population of cellular p38 is specifically activated by an osmotically responsive assembly of proteins attached to the scaffolding protein, OSM [67]. As part of the hypertonicity-induced cytoskeletal rearrangement that is discussed above, OSM forms a complex that includes a GTPase (Rac), actin, MEKK3, MKK3, and p38. MEKK3 and MKK3/6 are MAPKKKs and MAPKKs, respectively, that are upstream activators of p38 [68]. Hypertonicity activates MEKK3 [67, 69] and MKK3/6 [70, 71]. Knock down of OSM or MEKK3 reduces the hypertonicity-induced the phosphorylation of p38 that is associated with its activation [67]. In these studies TonEBP/OREBP activity was not addressed, so linkage of TonEBP/OREBP activity to Rac and OSM remains conjectural. Nevertheless, over expression of recombinant active MEKK3 in MDCK cells increases both transcriptional activity of TonEBP/OREBP and mRNA abundance of its transcriptional target, BGT1 [72]. Also, knock down of native MEKK3 in HEK293 cells reduces BGT1 mRNA [72]. On the other hand, over expression of constitutively active MKK3 or MKK6 did not affect TonEBP/OREBP expression or function in other experiments [38], and dominant negative MKK3 did not affect TonEBP/OREBPdependent transcription [73], leaving some uncertainty as to the composition of the phospho-relay system by which hypertonicity activates TonEBP/OREBP via p38. Finally, it is important to note that inhibition of p38 only partially reduces the hypertonicity-induced increase of TonEBP/OREBP activity [64]. As will be seen, that is also true for other pathways that signal to TonEBP/OREBP. Evidently, full activation of TonEBP/OREBP requires a combination of signals, and no one signal, by itself, does the whole job. Fyn. Fyn is a Src-family tyrosine kinase that is activated by hypertonicityinduced cell shrinkage [74]. Chemical inhibition of Fyn, transfection of dominant negative Fyn or Fyn deficiency (Fyn⫺/⫺ cells) reduces high NaCl-induced TonEBP/OREBP transcriptional activity [64]. Thus, signaling through Fyn apparently contributes to the activation of TonEBP/OREBP. Fyn, like p38, targets the

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hypertonicity-dependent TonEBP/OREBP transactivation domain [64]. p38 and Fyn act independently. Their effects on TonEBP/OREBP are additive [64]. As described above, hypertonicity causes the F-actin binding protein, cortactin, to translocate to the cortical cytoskeleton [16]. In the process cortactin is tyrosinephosphorylated by the Src-family tyrosine kinase, FER, in a Fyn-dependent manner [75]. Nevertheless, although Fyn, itself, contributes to activation of TonEBP/OREBP, FER and cortactin phosphorylation apparently do not [64]. Protein Kinase A. Protein Kinase A (PKAc) is a serine/threonine kinase that is activated by cyclic AMP, accounting for many of the effects of that second messenger. High NaCl increases PKAc activity, but this activation does not involve cyclic AMP and, in fact, high NaCl does not increase cyclic AMP [76]. In HepG2 cells the PKAc inhibitor, H89, reduces high NaCl-dependent increases in TonEBP/OREBP transcriptional and transactivational activities, and mRNA abundance of its transcriptional targets, AR and BGT1 [76]. Further, transfection of constitutively active PKAc increases transcriptional and transactivational activities of TonEBP/OEBP, and dominant negative PKAc reduces TonEBP/OREBP transcriptional activity. Also, PKAc and TonEBP/OREBP reciprocally immunoprecipitate each other, indicative of physical association of the proteins. Thus, there is considerable evidence that activation of PKAc by hypertonicity contributes to activation of TonEBP/OREBP. The lack of dependence on cyclic AMP is curious, but not unprecedented. The contribution of PKAc to activation of another Rel family transcription factor, NF␬B, is also independent of cyclic AMP [77]. In the absence of stimuli, NF␬B resides in the cytoplasm in a ternary complex with PKAc and I␬B. I␬B interacts with the ATP-binding domain of the PKA catalytic subunit, keeping it inactive. Upon stimulation, I␬B becomes phosphorylated and degraded, releasing PKAc and NF␬B. The newly freed PKAc phosphorylates NF␬B, contributing to its transactivating activity. A similar model for the role of PKAc in activation of TonEBP/OREBP is attractive, but at this point evidence is lacking that PKAc directly phosphorylates TonEBP/OREBP or that a protein analogous to I␬B is involved [76]. Ataxia Telangiectasia-Mutated Kinase. ATM is a serine/threonine kinase that is activated by DNA double-strand breaks, notably those caused by ionizing radiation, and plays an important role in cell cycle arrest and DNA repair [78]. Upon stimulation it becomes autophosphorylated on serine 1981, which provides an indicator of its activity. Stimulation of ATM apparently depends on an accompanying perturbation of higher-order chromatin structure rather than the DNA breaks, themselves [78]. As discussed above, high NaCl causes DNA double-strand breaks [5, 6] and perturbs chromatin structure [79]. High NaCl activates ATM in HEK293 cells [53], presumably because of the DNA breaks.


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AT cells lack functional ATM. When AT cells are reconstituted with wild type ATM, TonEBP/OREBP transcriptional and transactivating activity increase, but not when the ATM that is transfected is mutated at serine 1981 to prevent its activation [53]. Also, ATM and TonEBP/OREBP reciprocally immunoprecipitate each other, indicative of physical association of the proteins. TonEBP/OREBP contains 3 serines within ATM consensus phosphorylation sites [53]. Mutation of the putative phosphorylation site at S1247 decreases transcriptional activity of TonEBP/OREBP when functional ATM is present, but not when it is absent [53]. Finally, as already discussed above, ATM activity contributes to high NaCl-induced nuclear translocation of TonEBP/OREBP [51]. Thus, there is considerable evidence that activation of ATM by hypertonicity contributes to activation of TonEBP/OREBP. Although ATM contributes to high NaCl-induced activation of TonEBP/ OREBP, induction of ATM by stimuli other than hypertonicity does not increase TonEBP/OREBP activity. Ionizing and ultraviolet radiation and high urea all activate ATM, but this does not lead to activation of TonEBP/OREBP [53]. Thus, activation of ATM increases TonEBP/OREBP activity only in the context of hypertonicity, underlying the importance of integration of the multiple responses to hypertonicity. Phosphoinositide 3-kinases. Phosphoinositide 3-kinases (PI3Ks) generate phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3), which mediates a wide variety of intracellular signaling pathways, that of insulin being a notable example [80]. There are three classes of PI3Ks. Class IA (PI3K-IA) enzymes consist of a p110 catalytic subunit complexed to a regulatory subunit, typically p85. Most studies of PI3K-IA concern its association with plasma membranes, where it is activated by tyrosine kinases. However, PI3K-IA is also located in the nucleus [81], where it interacts with other regulatory proteins [82]. High NaCl activates the PI3K-IA p110␣ catalytic subunit [83]. Dominant negative knock down of the PI3K-IA p85␣ regulatory subunit reduces high NaCl-induced transcriptional and transactivational activity of TonEBP/OREBP, and siRNA knock down of the PI3K-IA p110␣ catalytic subunit reduces TonEBP/OREBP driven transcription [83]. These effects of PI3K-IA are mediated by ATM [83]. Thus, knock down of PI3K-IA reduces high NaCl-induced activation of ATM and the effects of inhibiting PI3K-IA and ATM are equivalent and non additive. In contrast, activation of p38 is not mediated by PI3K-IA. We conclude that high NaCl activates PI3K-IA, which in turn contributes to full activation of TonEBP/OREBP via ATM. Reactive Oxygen Species. High NaCl increases intracellular ROS in cell culture [8–10], and ROS are elevated in the renal inner medulla [8, 11], where NaCl normally is elevated in vivo. The ROS derive from mitochondria [9, 11, 84]. If the

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high NaCl-induced ROS are lowered by antioxidants, transcriptional and transactivational activity of TonEBP/OREBP decrease [10], indicating that NaCl-induced ROS contribute to activation of TonEBP/OREBP. We speculate that p38 may mediate this effect of ROS. ROS are known to activate p38 [85] by inhibiting protein tyrosine phosphatases, and thus increasing the activating phosphorylation of p38 [86]. Hypertonicity inhibits activity of protein tyrosine phosphatases, which is prevented by antioxidants [85]. We conclude that high NaCl increases ROS, which contribute to activation of TonEBP/OREBP, possibly mediated by p38. Integrins. As discussed earlier, hypertonicity causes a cytoskeletal rearrangement that contributes to activation of TonEBP/OREBP. Integrins are extracellular matrix receptors that are linked to the cytoskeleton and can transduce signals that alter cell behavior. Hypertonicity increases mRNA and protein abundance of the cell adhesion molecule, ␤1-integrin, in MDCK cells [87]. TonEBP/OREBP protein abundance is reduced in primary cultures of inner medullary collecting duct cells from ␣1␤1-integrin null mice [88]. Also, when these mice are dehydrated, their inner medullas contain less sorbitol, betaine and inositol than do those of wild type mice, and have lower mRNA abundance of the sodium-inositol co-transporter and the sorbitol synthesizing enzyme, AR. Thus, ␣1␤1-integrin apparently contributes to hypertonicity-induced activation of TonEBP/OREBP. RNA Helicase A. RNA Helicase A (RHA) is a component of transcriptional complexes, involved in mediating target gene activation [89]. RHA binds to TonEBP/OREBP in vitro and is physically associated with TonEBP/OREBP in cells, as indicated by their mutual coimmunoprecipitation [90]. The binding of RHA to TonEBP/OREBP is independent of DNA binding and of TonEBP/ OREBP dimerization [90]. RHA interacts with the E’F’ loop in TonEBP/ OREBP [90], the loop that forms a secondary dimer interface involved in encirclement of DNA by TonEBP/OREBP [40]. Over expression of RHA reduces hypertonicity-induced increase of TonEBP/OREBP transcriptional activity [90], even when RHA is mutated so that it lacks catalytic activity. Hypertonicity reduces binding of RHA to TonEBP/OREBP. Thus, the interaction of RHA with TonEBP/OREBP apparently restricts TonEBP/OREBP activity, and this restriction is relaxed by hypertonicity.

Multiple Pathways Converge to Signal Full Hypertonicity-Induced Activation of TonEBP/OREBP

Increased ROS is necessary for full high NaCl-induced activation of TonEBP/OREBP, but elevation of ROS in the absence of hypertonicity does not


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activate TonEBP/OREBP [10]. Similarly, increased activity of ATM is necessary for full high NaCl-induced activation of TonEBP/OREBP, but activation of ATM in the absence of hypertonicity does not activate TonEBP/OREBP [53]. High urea, UV radiation [53], ionizing radiation and low NaCl [78] all activate ATM, yet none of these agents increases TonEBP/OREBP transcriptional activity in the absence of hypertonicity [53], and high urea [91] or low NaCl [50] actually decreases the activity. As detailed above, the kinases known to contribute to high NaCl-induced activation of TonEBP/OREBP include PKAc [76], p38 [63, 64], Fyn [64], ATM [53] and PI3K-IA [83]. Strikingly, interrupting any one of these pathways only partially reduces hypertonicity-induced increase in TonEBP/ OREBP activity. Thus, convergence of multiple signals is necessary for full hypertonicity-induced activation of TonEBP/OREBP. That raises the question of why the signaling system should be so complex. We speculate that the complexity is a safeguard against inappropriate activation of the osmoregulatory response. If organic osmolytes were accumulated in the absence of hypertonicity, cell volume would be increased or intracellular ionic strength would be decreased, both of which could perturb cell function. None of these signals is uniquely activated by hypertonicity. For example, ATM is activated by other causes of DNA damage, PI3K by insulin, etc. What is unique is not the individual signals themselves, but their combined response to hypertonicity. Furthermore, the signals are likely to be interrelated in complex networks. We already know that activation of PI3K is necessary for that of ATM [83]. We presume that there are similar relations between the other signaling molecules that remain to be discovered. What Is the Sensor of Hypertonicity? The immediate effect of hypertonicity is decreased cell volume, accompanied by stresses on the cytoskeleton and increased concentration of all intracellular components, including inorganic ions and macromolecules. There is evidence that each can signal an osmoregulatory response, including cell volume, per se [1, 92], macromolecular crowding [2], cytoskeleton [67, 93], and inorganic ions [1, 94]. Macromolecular crowding and ionic strength, in particular, have profound effects on biochemical processes, which could contribute to increased ROS [8–11], DNA breaks [5, 6], and altered activity of kinases and other enzymes that are involved in activating TonEBP/OREBP. No single mammalian osmosensor has yet been identified. It is possible that none exists, and that it is the complex array of changes that we just outlined that senses hypertonicity and triggers osmoregulatory responses. Beyond its immediate effects, cells adapt to hypertonicity, maintaining function and even proliferating, despite continuous presence of high NaCl. A prime example is the cells in the renal medulla, which normally are constantly

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exposed to extremely high concentrations of NaCl. The osmoregulatory responses, such as high levels of organic osmolytes, expression of heat shock proteins, and activity of TonEBP/OREBP, continue in adapted cells. Yet, the immediate effects of hypertonicity (cell shrinkage and increase in concentrations of intracellular components) have been alleviated by regulatory volume increase [93] and accumulation of organic osmolytes. That leaves the question of what the signal could be that maintains the osmoregulatory responses in adapted cells. A possibility is that the reversal of the immediate effects of hypertonicity is sufficient to keep the cells alive and functioning, but is not complete. Then, the remnants of cell shrinkage, macromolecular crowding, increased ionic strength, etc. might suffice to account for the continued DNA breaks [6] and oxidative stress [8] and to maintain osmoregulatory responses. Until now measurements in cell culture [94] and in vivo [95] have shown more or less complete recovery of cell volume and ionic strength in adapted cells. However, the precision of those assays might not have been sufficient to identify small, yet important, differences from cells not stressed by hypertonicity.

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Maurice B. Burg 9000 Rockville Pike 10 Center Dr., MSC 1603 Bethesda, MD 20892–1603 (USA) Tel. ⫹1 301 402 5714, Fax ⫹1 301 402 1443, E-Mail [email protected]


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Ion Channels and Cell Volume in Regulation of Cell Proliferation and Apoptotic Cell Death Florian Langa, Ekaterina Shumilinaa, Markus Ritter b, Erich Gulbinsc, Alexey Vereninovd, Stephan M. Hubera a

Department of Physiology, University of Tübingen, Tübingen, and cDepartment of Molecular Medicine, University of Essen, Essen, Germany; bInstitute of Physiology and Pathophysiology, Medical University Salzburg, Salzburg, Austria; dInstitute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia

Abstract Cell proliferation must be accompanied by increase of cell volume and apoptosis is typically paralleled by cell shrinkage. Moreover, profound osmotic cell shrinkage may trigger apoptosis. In isotonic environment cell volume changes require the respective alterations of transport across the cell membrane. Cell proliferation is typically paralleled by activation of K⫹ channels, which is required for the maintenance of the cell membrane potential, a critical determinant of Ca2⫹ entry through Ca2⫹ channels. The Ca2⫹ entry leads to oscillations of cytosolic Ca2⫹ activity which is followed by activation of Ca2⫹ dependent transcription factors and by depolymerization of the actin filament network. The latter disinhibits the Na⫹/H⫹ exchanger and Na⫹, K⫹, 2Cl⫺ cotransport thus leading to cell swelling. At some point transient activation of Cl⫺ channels is required leading to transient decrease of cell volume. Apoptosis is typically paralleled by sustained activation of Cl⫺ channels leading to Cl⫺, HCO3⫺ and osmolyte exit. The subsequent cell shrinkage and cytosolic acidification are not counter-regulated by activation of the Na⫹/H⫹ exchanger, which is inhibited and eventually degraded during apoptosis. At a later stage K⫹ exit through K⫹ channels decreases intracellular K⫹ concentration and facilitates cell shrinkage. Sustained or excessive increase of Ca⫹ triggers apoptotic cell death, typically paralleled by cell shrinkage due to activation of Ca2⫹ sensitive K⫹ channels. Cellular K⫹ loss and cell shrinkage are supportive but not required for the induction of apoptosis. On the other hand, several studies point to a critical role of K⫹-channel inhibition in the initiation of apoptosis. Thus, alterations of K⫹ channel and Ca2⫹ channel activities may participate in the triggering of both, cell proliferation and apoptosis. The impact of those channels depends on magnitude and temporal organization of channel activation and on the activity of further signaling mechanisms. Accordingly, the same ion channel blockers may interfere with both, cell proliferation and apoptosis depending on cell type, regulatory environment and condition of the cell. Copyright © 2006 S. Karger AG, Basel

The adjustment of cell numbers to ever changing functional requirements as well as the elimination and replacement of abundant or potentially harmful cells requires the finely tuned formation of new cells by cell proliferation and disposal of cells by apoptosis [Green and Reed, 1998]. Thus, cell proliferation and apoptosis are both well-controlled cellular mechanisms. Cell proliferation is stimulated by growth factors [Adams et al., 2004; Bikfalvi et al., 1998; Tallquist and Kazlauskas, 2004], apoptosis is triggered by either stimulation of respective receptors, such as CD95 [Fillon et al., 2002; Gulbins et al., 2000; Lang et al., 1998b, 1999], somatostatin receptor [Teijeiro et al., 2002] or TNF␣ receptor [Alexander et al., 2006; Lang et al., 2002a], by cell density [Long et al., 2003], by lack of growth factors [Sturm et al., 2004], thyroid hormones [Alisi et al., 2005], or adhesion [Davies 2003; Walsh et al., 2003], or by exposing cells to stressors such as oxidants [Rosette and Karin 1996; Wang et al., 2005], hypoxia [Cechowska-Pasko et al., 2006], radiation [Rosette and Karin, 1996], inhibition of glutamine synthetase [Rotoli et al., 2005], chemotherapeutics [Cariers et al., 2002; Wieder et al., 2001], energy depletion [Pozzi et al., 2002], choline deficiency [Albright et al., 2005; Yang et al., 2005], or osmotic shock [Bortner and Cidlowski, 1998, 1999; Lang et al., 1998a, 2000b; Maeno et al., 2000; Michea et al., 2000; Rosette and Karin, 1996]. Suicidal death is not limited to nucleated cells but may similarly affect erythrocytes, which may die through eryptosis [Lang et al., 2005a] erythrocyte senescence [Arese et al., 2005; Barvitenko et al., 2005; Bosman et al., 2005; Lang et al., 2005a], or neocytolysis [Rice and Alfrey, 2005]. Cell proliferation typically generates cells of similar size as the parent cells and thus requires at some point cell volume increase. Conversely, hallmarks of apoptosis include cell shrinkage [Lang et al., 1998a]. The alterations of cell volume are partially accomplished by transport across the cell membrane secondary to activation or inhibition of Cl⫺ channels, K⫹ channels, Ca2⫹ channels, Na⫹/H⫹ exchanger, Na⫹, K⫹, 2Cl⫺ cotransport and the Na⫹/K⫹ ATPase. The following brief review attempts to provide some insight into the role of ion transport across the cell membrane in the regulation of cell volume during cell proliferation and apoptosis. The paper will not be able to summarize the vast literature on cell volume, ion channels, cell proliferation and apoptosis. Rather, emphasis will be dedicated to cell proliferation in ras oncogene expressing fibroblasts, apoptosis of CD95 stimulated Jurkat lymphocytes and erythrocytes undergoing eryptosis.

Transport and Cell Volume in Cell Proliferation

As demonstrated in ras oncogene expressing cells [Lang et al., 2000c; Ritter and Woell, 1996] and illustrated in figure 1, mitogens such as bradykinin

Ion Channels and Cell Volume in Cell Proliferation and Apoptosis

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Mitogens Cl⫺

K⫹

PLC K⫹ IP3

Ca2⫹

Early phase Actin

Cell volume ICF

ECF

⫹

Na

⫹

⫺ Na⫹ K⫹ 2Cl

H

Later phase

Fig. 1. Transport and cell volume in the activation of cell proliferation by ras oncogene. The ras oncogene sensitizes the cells for the mitogen bradykinin, which stimulates the formation of inositol-(1,4,5) trisphosphate (InsP3). InsP3 triggers the release of Ca2⫹ from intracellular stores. The following activation of the Ca2⫹ release activated Ca2⫹ channel ICRAC results in Ca2⫹ entry. Ca2⫹ then activates Ca2⫹ sensitive K⫹ channels with subsequent K⫹ exit, hyperpolarization and Cl⫺ exit through Cl⫺ channels. The exit of KCl and the osmotically obliged water shrinks the cell. A transient cell shrinkage is required for the triggering of Ca2⫹- and cell membrane potential oscillations due to repetitive Ca2⫹ and K⫹ channel activation. The oscillations of Ca2⫹ depolymerize the cell actin filaments leading to disinhibition

Lang/Shumilina/Ritter/Gulbins/Vereninov/Huber

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trigger in ras oncogene expressing fibroblasts the formation of inositol-(1,4,5) trisphosphate (InsP3) with subsequent stimulation of intracellular Ca2⫹ release and activation of the Ca2⫹ release-activated Ca2⫹ channel ICRAC [Lang et al., 1991]. The Ca2⫹ entry is followed by activation of Ca2⫹-sensitive K⫹ channels leading to K⫹ exit and hyperpolarization, which drives Cl⫺ exit through Cl⫺ channels. The exit of KCl with the osmotically obliged water leads to cell shrinkage, which is apparently required for the initial triggering of oscillations of Ca2⫹ and cell membrane potential. The Ca2⫹ oscillations lead to depolymerization of the cell actin filaments [Dartsch et al., 1995; Lang et al., 1992; Lang et al., 2000c; Ritter et al., 1997] resulting in disinhibition of the Na⫹/H⫹ exchanger and/or the Na⫹, K⫹, 2Cl⫺ cotransporter (fig. 1). The following cellular accumulation of electrolytes and osmotically obliged water leads to increase of cell volume [Lang et al., 1998a]. The increase of cell volume is due to a shift of the cell volume regulatory set point towards larger cell volumes [Lang et al., 1992]. Activation of ICRAC, Ca2⫹ oscillations and depolymerization of the actin filament network all have been shown to be prerequisites of cell proliferation in ras oncogene expressing fibroblasts [Dartsch et al., 1995; Lang et al., 1992; Lang et al., 2000c; Ritter et al., 1997]. Activation of K⫹ channels is considered important for the stimulation of cell proliferation in a wide variety of further tissues [Braun et al., 2002; Patel and Lazdunski, 2004; Wang, 2004]. K⫹ channels are activated by growth factors [Enomoto et al., 1986; Faehling et al., 2001; Gamper et al., 2002; Lang et al., 1991; Liu et al., 2001; O’Lague et al., 1985; Sanders et al., 1996; Wiecha et al., 1998] and overactive in diverse tumour cells [DeCoursey et al., 1984; Mauro et al., 1997; Nilius and Wohlrab 1992; Pappas and Ritchie 1998; Pappone and Ortiz-Miranda 1993; Patel and Lazdunski 2004; Skryma et al., 1997; Strobl et al., 1995; Wang 2004; Zhou et al., 2003]. Accordingly, K⫹ channel inhibitors have been shown to disrupt cell proliferation [for review Wang, 2004]. K⫹ channel activation is considered of particular importance in the early G1 phase of the cell cycle [Wang et al., 1998; Wonderlin and Strobl, 1996]. The maintenance of cell membrane potential by K⫹ channels is a prerequisite for Ca2⫹ entry through ICRAC [Parekh and Penner, 1997; Tanneur et al., 2002]. Increases of cytosolic Ca2⫹ activity are required in the regulation of cell proliferation [Berridge et al., 1998, 2000, 2003; Parekh and Penner 1997; Santella et al., 1998; Santella 1998; Whitfield et al., 1995]. Along those lines

of the Na⫹/H⫹ exchanger and the Na⫹, K⫹, 2Cl⫺ cotransporter. The enhanced activity of these carriers eventually leads to accumulation of KCl with osmotically obliged water and thus to cell swelling.


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growth factors stimulate ICRAC [Qian and Weiss 1997]. Beyond their influence on the actin filament network the resulting Ca2⫹ oscillations regulate a wide variety of cellular functions relevant for cell proliferation [Berridge et al., 1998, 2000, 2003; Parekh and Penner, 1997]. In a wide variety of cells activation of anion channels has been observed during cell proliferation [Nilius and Droogmans 2001; Shen et al., 2000; Varela et al., 2004]. Along those lines anion channel blockers have been shown to interfere with cell proliferation [Jiang et al., 2004; Pappas and Ritchie, 1998; Phipps et al., 1996; Rouzaire-Dubois et al., 2000; Shen et al., 2000; Wondergem et al., 2001] and impaired cell proliferation has been observed in ClC3 deficient cells [Wang et al., 2002]. Parallel activation of K⫹ and Cl⫺ channels presumably mediates the transient cell shrinkage which is required for the triggering of cytosolic Ca2⫹ oscillations in ras oncogene expressing cells [Ritter et al., 1993].

Transport and Cell Volume in Apoptosis

Stimulation of the CD95 receptor in Jurkat lymphocytes, a typical model of receptor induced apoptosis (fig. 2), leads to activation of outwardly rectifying Cl⫺ channels (ORCC) [Szabo et al., 1998]. Similarly, TNF␣ or staurosporine induced apoptosis of various cell types is paralleled by Cl⫺ channel activation [Maeno et al., 2000; Okada et al., 2004]. The Cl⫺ channels activated by CD95 in Jurkat cells are similarly activated by osmotic cell swelling and serve regulatory cell volume decrease [Lepple-Wienhues et al., 1998]. Both cell swelling [Lepple-Wienhues et al., 1998] and stimulation of the CD95 receptor [Szabo et al., 1998] activate the Cl⫺ channels in Jurkat cells through the Src-like kinase Lck56. The kinase is activated by ceramide [Gulbins et al., 1997], which is released following stimulation of the CD95 receptor by sphingomyelinase-dependent hydrolysis of sphingomyelin. In lymphocytes from patients with cystic fibrosis the Cl⫺ channel ORCC is resistant to activation by protein kinase A but is activated by cell swelling and by exposure to active Lck56 [Lepple-Wienhues et al., 2001]. The activation of the Cl⫺ channels is required for full induction of apoptosis, which is blunted or even disrupted by the respective Cl- channel inhibitors. Specifically, Cl⫺ channel blockers have been shown to blunt the apoptosis of Jurkat cells following CD95 triggering [Szabo et al., 1998], the TNF␣- or staurosporine-induced apoptosis of various cell types [Maeno et al., 2000; Okada et al., 2004], apoptotic death of cortical neurons [Wei et al., 2004], the antimycin A-induced death of proximal renal tubules [Miller and Schnellmann, 1993], the GABA-induced enhancement of excitotoxic cell death of rat cerebral neurons [Erdo et al., 1991] and the ROS-induced cardiomyocyte apoptosis [Takahashi et al., 2005; Wang et al., 2005].


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CD95

Ca2⫹ Caspases Bcl2 BclX

SMase K⫹ Ceramide Lck56 Caspases Cl⫺ HCO⫺ 3 CO2 H⫹

Na⫹ Cell shrinkage Osm

Fig. 2. Transport and cell volume in the triggering of Jurkat cell apoptosis by CD95. CD95 stimulation is followed by activation of anion channels (ORCC), activation of osmolyte release channels, inhibition of Na⫹/H⫹-exchange, (early) inhibition of Kv1.3 K⫹channels and inhibition of ICRAC Ca2⫹ channels. After a delay of some 60 min the activation of the channels and the inhibition of the Na⫹/H⫹ exchanger lead to cell shrinkage.

Activation of Cl⫺ channels leads to cell shrinkage by triggering cellular loss of KCl. Some anion channels further allow the permeation of organic osmolytes such as taurine [Lang et al., 2003d], which are released by cells undergoing apoptosis [Lang et al., 1998b; Moran et al., 2000]. The loss of the organic osmolytes then contributes to cell shrinkage [Lang et al., 1998a].


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Moreover, organic osmolytes stabilize cellular proteins (for review see [Lang et al., 1998a] and their loss could indeed destabilize proteins). For instance, inhibition of inositol uptake has been shown to induce renal failure presumably due to apoptotic death of renal tubular cells [Kitamura et al., 1998]. Many Cl⫺ channels allow in addition the passage of HCO⫺ 3 and their activation thus leads to cytosolic acidification, a typical feature of cells entering into apoptosis [Lang et al., 2002a; Wenzel and Daniel, 2004]. Acidification may promote DNA fragmentation as DNase type II has its pH optimum in the acidic range (for review see [Shrode et al., 1997]). Along those lines, CD95-induced apoptosis is accelerated by inhibition of Na⫹/H⫹ exchange [Lang et al., 2000a]. CD95 triggering further leads to early inhibition of Kv1.3 K⫹ channels [Szabo et al., 1996, 1997, 2004], the cell volume regulatory K⫹ channel of those cells [Deutsch and Chen, 1993]. The channel protein is tyrosine phosphorylated upon CD95 receptor stimulation [Gulbins et al., 1997; Szabo et al., 1996] and its inhibition requires the src like tyrosine kinase Lck56 [Gulbins et al., 1997; Szabo et al., 1996]. Kv1.3 channel activity is governed by tyrosine phosphorylation [Holmes et al., 1996]. Similar to CD95 receptor triggering, the sphingomyelinase product ceramide inhibits Kv1.3 and induces apoptosis [Gulbins et al., 1997]. The early inhibition of Kv1.3 is followed by late activation of Kv1.3 [Storey et al., 2003]. The early inhibition of Kv1.3 channels prevents premature cell shrinkage which would otherwise interfere with the further signaling of apoptosis [Lang et al., 1998a]. The late activation of Kv1.3 channels during the execution phase of apoptosis is presumably required for cellular loss of KCl and subsequent apoptotic cell shrinkage [Storey et al., 2003]. Reports on the role of K⫹ channels in apoptosis of other cell types are conflicting. In some cells, inhibition of K⫹ channels favours [Bankers-Fulbright et al., 1998; Chin et al., 1997; Han et al., 2004; Miki et al., 1997; Pal et al., 2004; Patel and Lazdunski, 2004] and activation of K⫹ channels inhibits [Jakob and Krieglstein, 1997; Lauritzen et al., 1997] apoptosis. Along those lines mice carrying a mutated G-protein coupled inward rectifier K⫹ channel (Weaver mice) suffer from extensive neuronal cell death [Harrison and Roffler-Tarlov, 1998; Migheli et al., 1995, 1997; Murtomaki et al., 1995; Oo et al., 1996]. However, in other cell models apoptosis is stimulated by activation of K⫹ channels [Wei et al., 2004; Yu et al., 1997] and blunted by increase of extracellular K⫹ concentration [Colom et al., 1998; Lang et al., 2003d; Prehn et al., 1997] or inhibition of K⫹ channels [Gantner et al., 1995; Lang et al., 2003d]. Cellular loss of K⫹ appears to support apoptosis in a wide variety of cells [Beauvais et al., 1995; Benson et al., 1996; Bortner et al., 1997; Bortner and Cidlowski 1999, 2004; Gomez-Angelats et al., 2000; Hughes Jr et al., 1997; Hughes Jr and Cidlowski, 1999; Maeno et al., 2000; Montague et al., 1999; Perez et al., 2000; Yurinskaya et al., 2005a; Yurinskaya et al., 2005b].


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Inhibition of K⫹ channels is expected to depolarize the cell membrane, which in turn decreases Ca2⫹ entry through ICRAC [Parekh and Penner, 1997]. Moreover, CD95 triggering results in inhibition of ICRAC [Dangel et al., 2005; Lepple-Wienhues et al., 1999] (fig. 2). Inhibition of ICRAC presumably serves to abrogate activation and proliferation of lymphocytes and does not necessarily foster apoptotic cell death. In any case, increase of Ca2⫹ activity is not an early event in CD95 induced death of Jurkat lymphocytes. In arterial smooth muscle cells activation of L-type Ca2⫹ channels has even been reported to inhibit apoptosis [Tao et al., 2006]. In contrast to Ca2⫹ oscillations, sustained increases of cytosolic Ca2⫹ activity may trigger apoptosis [Berridge et al., 2000; Green and Reed 1998; Liu et al., 2005; Parekh and Penner 1997; Parekh and Putney Jr, 2005; Spassova et al., 2004]. The increase of Ca2⫹ could result from activation of Ca2⫹ channels or Ca2⫹ permeable cation channels. Cell volume-sensitive cation channels have been described to be expressed in diverse tissues, such as airway epithelial cells [Chan et al., 1992], mast cells [Cabado et al., 1994], macrophages [Gamper et al., 2000], vascular smooth muscle cells, colon carcinoma and neuroblastoma cells [Koch and Korbmacher, 1999], cortical collecting duct cells [Volk et al., 1995], and hepatocytes [Wehner et al., 1995; Wehner et al., 2000]. Cation channels could further be activated by Cl⫺ removal, as shown in salivary and lung epithelial cells [Dinudom et al., 1995; Marunaka et al., 1994; Tohda et al., 1994]. Whether or not those channels could participate in the stimulation of apoptosis during hyperosmotic or isotonic cell shrinkage remained, however, elusive.

Transport and Cell Volume in Eryptosis

Even though devoid of nuclei and mitochondria, key organelles in the apoptotic machinery of nucleated cells, erythrocytes may undergo suicidal death. Suicidal erythrocyte death or eryptosis is triggered by a sustained increase of cytosolic Ca2⫹ activity [Berg et al., 2001; Bratosin et al., 2001; Daugas et al., 2001; Lang et al., 2002b, 2003a]. Ca2⫹ may enter erythrocytes through Ca2⫹ permeable cation channels [Lang et al., 2003a]. The channels are activated by a variety of stimuli including osmotic shock [Huber et al., 2001], oxidative stress [Duranton et al., 2002], energy depletion [Lang et al., 2003a] and infection with the malaria pathogen Plasmodium falciparum [Brand et al., 2003; Duranton et al., 2003; Lang et al., 2004a]. Energy depletion presumably impairs the replenishment of GSH and thus weakens the antioxidative defence of the erythrocytes [Bilmen et al., 2001; Mavelli et al., 1984]. The cation channels are further activated by removal of intracellular and extracellular Cl⫺ [Duranton et al., 2002; Huber et al., 2001]. The cation channels are similar or


149

Osmotic shock, oxidative stress, energy depletion PGE2 NSC COX PLA

EPO

AA Ca2⫹

PAF

K⫹

SM Cl⫺

Ce

ram

ide

S

Phosphatidylserine

H2O

Fig. 3. Channel regulation in eryptosis. Several triggers including osmotic shock stimulate the release of PGE2 which activates Ca2⫹ permeable cation channels. Ca2⫹ then activates Ca2⫹ sensitive K⫹ channels, which lead to cell shrinkage due to loss of K⫹, hyperpolarization and Cl⫺ exit through Cl⫺ channels. Ca2⫹ in addition leads to scrambling of the cell membrane. Cell shrinkage further activates a phospholipase leading to formation of platelet activating factor, sphingomyelinase activation, ceramide formation and further stimulation of cell membrane scrambling.

identical to the Na⫹ and K⫹ permeability activated by incubation of human erythrocytes in low ionic strength [Bernhardt et al., 1991; Jones and Knauf, 1985; LaCelle and Rothsteto, 1966] or by depolarization [Bennekou, 1993; Christophersen and Bennekou, 1991; Kaestner et al., 1999]. The erythrocyte cation channels are activated by prostaglandin E2 (PGE2), which is released upon osmotic shock [Lang et al., 2005b] (fig. 3). Increased cytosolic Ca2⫹ concentrations trigger cell membrane scrambling [Zhou et al., 2002] and subsequent phosphatidylserine exposure at the erythrocyte surface [Lang et al., 2003a]. Phosphatidylserine exposure following osmotic shock is blunted by amiloride [Lang et al., 2003a] and ethylisopropylamiloride (EIPA) [Lang et al., 2003b], i.e. inhibitors of the cation channels [Lang et al., 2003a, b]. Accordingly, the Ca2⫹ permeable cation channels play a decisive role in apoptosis-like death of erythrocytes (eryptosis) [Brand et al., 2003; Lang et al., 2002b, 2003a]. Increased cytosolic Ca2⫹ activity further leads to stimulation of Ca2⫹sensitive K⫹ channels (Gardos channels) [Brugnara et al., 1993; Del Carlo et al.,


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2002; Dunn, 1998; Gardos, 1958; Grygorczyk and Schwarz, 1983; Leinders et al., 1992; Pellegrino and Pellegrini, 1998; Shindo et al., 2000]. The activation of the channels is followed by hyperpolarization of the cell membrane [Lang et al., 2003c], which drives Cl⫺ exit through Cl⫺ channels [Myssina et al., 2004]. The cellular loss of K⫹ and Cl⫺ and osmotically obliged water leads to cell shrinkage, which further supports the development of eryptosis [Lang et al., 2003c]. Along those lines, increase of extracellular K⫹ or pharmacological inhibition of the Gardos channels blunts not only erythrocyte shrinkage but as well eryptosis following exposure to the Ca2⫹ ionophore ionomycin [Lang et al., 2003c]. The cell shrinkage following Gardos channel activation stimulates the formation of platelet activating factor PAF, which in turn activates a sphingomyelinase and thus leads to formation of ceramide [Lang et al., 2005c]. Ceramide sensitizes the cell to the eryptotic effects of Ca2⫹ [Lang et al., 2004b, 2005c].

Conclusions

Alterations of cell volume and cell volume sensitive ion channels participate in the machinery leading to cell proliferation and apoptosis. They influence cytosolic pH and Ca⫹ concentrations. Cell proliferation is typically paralleled by early cell shrinkage requiring activation of Cl- and K⫹ channel activity, by cytosolic alkalinization due to activation of the Na⫹/H⫹ exchanger and by Ca2⫹ oscillations due to activation of Ca2⫹ channels. The Ca2⫹ oscillations are secondary to Ca2⫹ entry through Ca2⫹ release-activated channels ICRAC, which in turn require K⫹ channel-dependent maintenance of cell membrane polarization. The Ca2⫹ oscillations result in depolymerization of the actin filament network with subsequent disinhibition of Na⫹/H⫹ exchanger and/or Na⫹, K⫹, 2Cl⫺ resulting in cell swelling. Apoptosis is typically paralleled by cell shrinkage due to activation of K⫹ and/or Cl⫺ channels, organic osmolyte release and inhibition of the Na⫹/H⫹ exchanger. The activation of Cl⫺ channels and inhibition of Na⫹/H⫹ exchangers further result in cytosolic acidification. The Ca2⫹ channel ICRAC is inhibited during CD95 induced apoptosis. Apoptosis and eryptosis could be elicited by sustained Ca2⫹ entry through 2⫹ Ca permeable cation channels. Several channels play dual roles in both cell proliferation and apoptosis. Their effects depend on the temporal pattern and amplitude of channel activity as well as on the interplay with other channels, transporters and signaling pathways. Accordingly, oscillating K⫹ channel activity is typical for proliferating cells [Lang et al., 1991; Pandiella et al., 1989] whereas sustained K⫹ channel activation is typical for apoptosis [Lang et al., 2003c]. Oscillations of Ca2⫹


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channel activity with subsequent fluctuations of cytosolic Ca2⫹ concentration may depolymerize the cytoskeleton [Dartsch et al., 1995; Lang et al., 1992; Lang et al., 2000c; Ritter et al., 1997] or activate transcription factors [Whitfield et al., 1995] but are presumably too short lived for caspase activation [Whitfield et al., 1995] or triggering of cell membrane scrambling [Dekkers et al., 2002; Woon et al., 1999]. Beyond that the amplitude of channel activity may become decisive for the eventual outcome. For instance, the amplitude of TASK-3 K⫹ channel activity is one order of magnitude higher in apoptotic cells than in tumour cells [Patel and Lazdunski, 2004; Wang 2004]. Acknowledgments The authors acknowledge the meticulous preparation of the manuscript by Lejla Subasic. The work of the authors was supported by the Deutsche Forschungsgemeinschaft, Nr. La 315/4–3, La 315/6–1, Le 792/3–3, RUS 436, DFG Schwerpunkt Intrazelluläre Lebensformen La 315/11–2, and Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (Center for Interdisciplinary Clinical Research) 01 KS 9602 and the Russian Foundation for Basic Research (to A. Vereninow, projects no. 06-04-48060, 06-04-04000-DFG).

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Prof. Dr. F. Lang Physiologisches Institut der Universität Tübingen Gmelinstrasse 5 DE–72076 Tübingen (Germany) Tel. ⫹49 7071 29 72194, Fax ⫹49 7071 29 5618, E-Mail [email protected]


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Cell Volume Regulatory Ion Transport in the Regulation of Cell Migration M. Jakab, M. Ritter Institute for Physiology and Pathophysiology, Paracelsus Private Medical University, Salzburg, Austria

Abstract Cell migration is typically accomplished by the generation of protrusive mechanical forces and is achieved by repeated spatially and temporally coordinated cycles including the formation of a leading edge, the formation of new and disruption of older adhesions to the substratum, actomyosin based contractions and retraction of the trailing edge. Beside the well-described roles of the cytoskeleton and cell adhesions during these processes, a growing body of evidence indicates that the precise regulation of the cell volume is an indispensable prerequisite for coordinated cell migration. On the one hand during cell migration cell volume is continuously tormented by mechanical and morphological alterations, which pose changes to the intracellular hydrostatic pressure, metabolic changes and the formation or degradation of macromolecules like actin, which distort the osmotic equilibrium and the action of chemoattractants, hormones and transmitters, which frequently alter the electrical properties of a cell and thus cause cell swelling or shrinkage, respectively. On the other hand, a migrating cell actively has to govern cell volume regulatory ion transport mechanisms in order to create the appropriate micro- or even nanoenvironment in the intra- and/or extracellular space, which is necessary to guarantee the correct polarity and hence direction of movement of a migrating cell. This chapter will focus on the role of the cell volume regulatory ion transport mechanisms as they participate in the regulation of cell migration and special emphasis is given to their interplay with the cytoskeleton, their meaning for substrate adhesion and to the polarized fashion of their subcellular distribution. Copyright © 2006 S. Karger AG, Basel

Active migration of cells is a fundamental biological feature necessary for development and maintenance of life. Processes like fertilization, embryogenesis, tissue and organ modeling, wound healing, inflammation and immunity require the action of mobile cells. To achieve locomotion, cells have developed

highly specialized cellular mechanisms generating and coordinating propulsive forces [1–4]. Disturbance of these mechanisms can cause developmental malformation or life-threatening conditions like defective pathogen defence. Malignant transformation of cells is frequently accompanied by enhanced mobility of the tumor cells leading to formation of metastasis. Cell migration is not only restricted to professional motile cells like leukocytes, but can also be performed by various other cells like endothelia or epithelia. Outgrowth of neurite and activation of platelets are a functionally related processes [5–9]. Cells can either migrate randomly (chemokinesis), are attracted by chemical signals, called chemoattractants or chemokines, originating from other cells or from the pathogen itself (chemotaxis) or they are guided by substances bound to the substratum they are migrating on (haptotaxis) [10]. Stimulation of these cells is paralleled by metabolic changes as well as morphological alterations like cell shape change. Mechanical deformation occurs during diapedesis when cells have to squeeze in between other cells to pass epithelial or endothelial barriers [11, 12]. Considering the changes of cell shape and the forces generated within a moving cell, cell migration is expected to be accompanied by changes of cell volume. In order to fulfil their physiological role, migrating cells often have to invade regions of anisotonicity or sites of altered ionic composition, again challenging cell volume constancy. In addition, cells may change their volume in response to chemoattractants, hormones or transmitters altering their cellular metabolism or electrical properties [13]. Since the cell membrane cannot withstand substantial tension, excess distortion has to be counteracted by volume regulatory water fluxes across the cell membrane. Hence controlling its volume is certainly of critical importance for the migrating cell. Vice versa, disturbance of the cell volume homeostasis may impede coordinated migration as well as other cellular functions involving cell volume as an element of cellular signaling [14]. This chapter will focus on the role of the cell volume regulatory ion transport as it participates in the regulation of cell migration.

General Principles of Cell Locomotion

The directed locomotion of cells requires the generation of protrusive forces. The leading edge forms an actin-rich pseudopodial extension called lamellopodium, which guides the cell towards its target and which is regarded to act as the primary motor for cell protrusion. The cell movement is driven by repeated spatially and temporally coordinated cycles of leading edge protrusion, formation of new and disruption of older adhesions, actomyosin based contractions and retraction of the trailing edge. Different models have been proposed on

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how the cell generates the forces for the extension of the leading edge, all of them recognizing the cytoskeleton to play a major role [1, 2, 4, 6, 15–24]. In addition, cell migration requires accurate local changes of ion fluxes and transmembrane water movement accompanied by rapid changes of cell volume. According to the filament treadmilling model, actin monomers intercalate between the cell membrane and the forward-facing fast-growing ‘barbed’ ends of the actin filaments leading to actin polymerization and the assembling of a branched network of actin filaments which exert mechanical forces by pushing against the plasma membrane thus providing the driving force for the forward movement. While actin polymerization occurs preferentially at the leading edge, actin depolymerization occurs at free pointed actin ends. Dissociation of actin subunits from free rear-facing pointed ends allows for filament actin turnover by continuous addition of subunits to the barbed ends. The polymerization rate is regulated by the rate of monomer addition to the barbed ends and is limited by barbed end-capping proteins and depletion of the pool of available actin monomers [2, 6, 25–31]. Membrane protrusion may also occur in response to localized osmotic swelling caused by depolymerization of the submembraneous actin filament network in the lamellipodium. The focal breaking of the actin filaments and crosslinks – initiated by transmembrane signals at the site of maximal stimulation – causes rapid swelling due to osmotically driven water influx which protrudes the cell membrane at this point [1, 32]. In a recent study, it has been shown that in epithelial cells migration is mediated by two spatially colocalized but kinematically, kinetically, molecularly and functionally distinct actin networks. A narrow actin band assembles to a submembraneous actin network at the leading edge. This band is separated from the rest of the cytoskeleton and promotes the random protrusion and retraction of the leading edge. It is only weakly coupled to a colocalized second network, the lamella, which generates productive cell advance by actomyosin contraction that is transduced to substrate adhesion [33]. Accordingly, actomyosin based contraction may also promote cell migration [16, 17, 33, 34]. The ‘lipid flow’ model assumes that migrating cells undergo repeated cycles of endo- and exocytosis which could in addition drive forward movement of the cell [16, 35]. Dynamic plasma membrane internalization processes close to the leading edge have indeed been observed in migrating cells [36]. Clearly, migration requires well-tuned cycles of attachment and detachment of the cell with its substratum. While the newly formed leading edge has to be anchored to the surface, the rear of the cell has to release cell substratum contacts. The following recoil of the cell’s rear is expected to increase the hydrostatic pressure thus contributing to membrane protrusion at the leading edge [17, 37].


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Cell Volume Regulatory Ion Transport Mechanisms in Migrating Cells

As evident from the current models of locomotion, migrating cells are continuously subjected to changes in intracellular osmotic or hydrostatic pressure, which torment cell volume constancy and force the cell to counteract deviations from the actual set point volume. Rapid regulatory water fluxes are driven by ion transport across the cell membrane. Cell swelling triggers the extrusion of ions, thus accomplishing regulatory volume decrease (RVD), whereas cell shrinkage favors cellular uptake of ions to achieve regulatory volume increase (RVI). While these general principles are conserved among eukaryotic cells, each cell type may activate its individual set of ion transporters to perform cell volume regulation. The diverse mechanisms of RVI and RVD in different cells are summarized in a separate chapter.

Role of Cell Swelling in Cell Migration

An increase in neutrophil size variability is one of the most sensitive parameters indicating septic bacterial infections [38]. Neutrophils rapidly swell upon stimulation with fMLP, an effect that can be in part inhibited by selective blockers of the NHE1 isoform of the Na/H exchanger, or by replacement of extracellular Na, indicating, that this swelling is mediated by the Na/H exchanger. These blockers also effectively inhibit fMLP-stimulated migration of neutrophils with virtually no effect on the random migration. Obviously, the Na/H exchanger is not necessary for random migration, whereas stimulated migration requires full activity of the exchanger. Hypertonic activation of the Na/H exchanger does not stimulate cell migration per se or alter the random migration of the cells, but effectively suppresses fMLP-stimulated migration. Under these conditions the fMLP-induced cell volume increase is counteracted, indicating that cell swelling is a prerequisite for stimulated cell migration [39]. Similarly, migration of neutrophils is facilitated in hypoosmolar buffer and inhibition of neutrophil chemotaxis by Na/H exchange inhibitors can be overcome in hypoosmolar media [40]. Stimulation of neutrophils leads to massive formation of H resulting in intracellular acidosis [41]. The subsequent activation of NHE1 leads to recovery to the initial intracellular pH and secondary intracellular alkalinization. Upon inhibition of the Na/H exchanger the acidification is stronger and the secondary alkalinization is abolished, which highlights the crucial role of the exchanger in extruding cellular excess acid [39, 42]. The involvement of NHE1 in cell migration has also been demonstrated in human melanoma cells [43, 44], keratinocytes [45], human breast

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cancer cells [46, 47] and in spontaneously migrating transformed MDCK cells [48, 49]. In MDCK-F cells NHE1 is distributed to the leading edge of the cells and operates in parallel with the anion exchanger AE2 [44]. NHE-deficient MDCK-F cells migrate more slowly than normal MDCK-F cells. These cells express mainly the NBC1 isoform of Na/HCO 3 cotransporter which serves as the major acid extruder, mediator of propionate-induced cell volume increase, promotes migration during an acute intracellular acid load and increases migratory speed and displacement on a short time scale. NBC1 has, however, no effect on the long-term behavior of migrating MDCK-F cells [50]. In addition furosemide or bumetanide, blockers of the Na/K/2Cl cotransporter have been shown to impair cell migration in MDCK-F cells [51]. In neutrophils in which Na/H exchange activity is excluded, the calculated intracellular pH reached during respiratory burst is about 5 pH units below the value predicted from the rate of H generation. The excess H ions are extruded via H (equivalent) channels and a V-type H ATPase [52–55]. In addition neutrophils express an H/K ATPase which can be inhibited by the gastric proton pump blockers omeprazole and SCH28080. These inhibitors also suppress fMLPstimulated cell swelling as well as chemotaxis and haptotaxis [39]. Like neutrophils MDCK-F cells display omeprazole-sensitive migration, indicating that an H/K ATPase can at least partially substitute for NHE1 activity [44]. In PC12 cells nerve growth factor (NGF) stimulates the outgrowth of neurites, a process functionally related to cell migration. This differentiation process requires and is preceded by an increase in cell volume and is at least in part mediated by the Na/K/2Cl cotransporter [56]. As shown in figure 1, in PC12 cells the ability to perform RVD is strongly suppressed as an initial transient response to NGF. This down-regulation of the cell’s ability to counteract cell swelling precedes and obviously supports the NGF induced volume gain but is restored as soon as the new set point volume is reached prior to neurite outgrowth.

Role of Cell Shrinkage in Cell Migration

While cell swelling might be necessary to provide propulsive forces for the protrusion of the leading edge, cell shrinkage is expected to have an opposite effect on cell locomotion. Hypertonicity has been shown to hamper the formation of cell protrusions in and to reduce the elongation rate of growth cones in nerve cells [18]. However, the formation of filipodia is rather enhanced [57]. As mentioned above, cell migration is inhibited in hyperosmolar environment [39, 40, 58]. Hence cell shrinkage obviously suppresses cell migration. On the other hand, the cell volume increase during migration has to be counteracted by mechanisms of RVD to avoid excessive swelling. Alternatively, RVD might be necessary to


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Fig. 1. Cell volume regulation in rat pheochromocytoma (PC12) cells during stimulation with NGF. Upon stimulation PC12 increase their cell volume after ⬃12 h which reaches a new set point volume after ⬃36 h (a). This increase precedes the NGF stimulated outgrowth of neurites which becomes visible typically after a few days. Before and during the period of cell swelling the cells’ ability to down-regulate their volume after osmotic cell swelling (RVD) is drastically reduced, but is restored as soon as the new set point volume is reached (b). This immediate NGF-induced reduction of the RVD capacity obviously supports the cells to acquire a higher volume necessary for the following outgrowth of neurites.

restore the cell volume towards a resting value to allow for repeated cycles of migration associated cell swelling. In such a system oscillatory changes of cell volume are expected to occur during migration. Oscillatory activation of Ca2sensitive K channels have been observed in neutrophils [59] and spontaneously migrating MDCK-F cells. These channels have been identified as IK1, require Ca2 entry from the extracellular space and are activated by oscillatory elevations of the intracellular Ca2 concentration [51, 60, 61]. The same channels are important for migration of fibroblasts and melanoma cells [62], whereas voltagesensitive K channels (Kv1.3 and Kv3.1) are necessary for migration of lymphocytes [63] and embryonic nerve cells [64]. In MDCK-F cells a close correlation exists between inhibition of IK1 channel activity and inhibition of cell migration, which is abrogated upon complete inhibition of the K- or Ca2 channels. Measurements of the intermittent K fluxes revealed that about 20% of the total cellular K content is released during a cycle of K channel activation. The K ion exit has to be accompanied by parallel exit of anions to maintain electroneutrality, thus adding to loss of cellular osmotic activity and cell shrinkage. In a subsequent step K- and Cl ions are replenished by activation of the Na/K/2Cl cotransporter leading to cell volume increase. Hence, oscillations

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of the cell volume during migration of these cells might in fact occur [51, 65–68]. In p21Ha-ras transformed fibroblasts cell shrinkage induced by oscillatory K channel activation is a prerequisite for maintenance of the oscillations and thus serves as an element of a feedback mechanism [69]. Spontaneous oscillations of the cell membrane potential were also observed in macrophages. However, fMLPstimulated chemotaxis is not inhibited by K channel blockers in macrophages or neutrophils [59] or by high extracellular K concentration in newt eosinophils [70]. Stimulation of human neutrophils with various chemoattractants is accompanied by a ⬃50% reduction of cellular Cl content which is expected to be accompanied by cell shrinkage and could induce a catabolic state required to supply the energy for chemotaxis [14, 71, 72]. However, as outlined above, neutrophils rather swell in response to chemoattractants. It is tempting to speculate that Cl might leave the cell via Cl channels activated by chemoattractantmediated cell swelling. The finding, that inhibition of the Na/H exchanger [72] – known to suppress fMLP-induced cell swelling – also inhibits cellular Cl exit, is in favor of this idea. Recently, it has been shown that selective inhibition of swelling-activated Cl channels with tamoxifen strongly reduces neutrophil migration [73]. In monocytes Cl channel blockers inhibit chemotaxis, increase cell volume and decrease TNF-induced monocyte adhesion to endothelial cells. These results suggest that in monocytes Cl channels regulate transendothelial migration in a cell volume dependent manner [74]. In nasopharyngeal carcinoma (CNE-2Z) cells different properties of the swelling-activated Cl current have been described in migrating cells and non-migrating cells: migrating cells exhibit an altered anion permeability sequence, have a higher current density and a higher rate of RVD, both of which are stronger inhibited by specific blockers and the inhibition of migration is positively correlated with the blockage of RVD [75, 76]. In microglial cells the ramification process is dependent on transmembrane Cl transport [77]. In glioma cells inhibition of swelling-activated Cl channels inhibits both RVD and tumor cell migration. Interestingly in glioma cells Cl and K channels involved in RVD localize to lipid-raft domains on invadipodia. Thus, presumably by facilitating cell shape and cell volume changes, Cl channels in these cells may enable tumor invasiveness [78–80]. Upon activation platelets undergo a dramatic shape change and form lamellipodia analogous to migrating cells [5]. The major component mediating interaction of the platelets with the exctracellular matrix is the integrin 2b3 heterodimer [81]. It has recently been shown that this integrin complex specifically interacts with the multifunctional connector hub protein ICln [82] and plays an important role in integrin-mediated platelet activation [83]. ICln plays a crucial role in the evocation of ionic currents during RVD upon cell swelling, is transposed to the cell membrane upon cell swelling and interacts with cytoskeletal proteins like -actin, protein 4.1 or the non-muscle isoform of the alkali myosin light chain [84–86].


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Cytoskeleton, Intracellular Ca2ⴙ Homeostasis and Cell Volume

As outlined above, cell migration is tightly coupled to dynamic changes in actin architecture. Intracellular Ca2 acts as a key regulator of actin assembly [1, 87]. Many chemoattractive factors lead to an increase in intracellular Ca2 concentration by stimulation of Ca2 entry and/or release from intracellular stores [88]. In migrating neutrophils [89] eosinophils [70, 90], fibroblasts [91] and MDCK-F cells [92] localized increases in Ca2 occur at the rear of the cell, creating decreasing concentration gradients to the front of the cell. High Ca2 concentration at the rear of the cell promotes actin disassambly, while low Ca2 concentration at the leading edge favors actin polymerization. In order to guarantee coordinated cycles of cytoskeletal remodeling some migrating cells generate oscillations of intracellular Ca2 concentration like chemoattractant stimulated neutrophils [89] and spontaneously migrating transformed MDCK-F cells [65, 93, 94]. Oscillations of actin polymerization/depolymerization in response to chemoattractants occur in neutrophils [95]. Beside its function to maintain cell shape and to generate protrusive forces during migration, actin might influence cell volume regulation and migration by linkage to ion transporters. In Jurkat lymphoma cells, HL-60 cells and human neutrophils RVD proved to depend on both microtubules and microfilaments [96]. In neutrophils, fMLP-stimulated Ca2- and Na entry is augmented after disruption of actin filaments [97]. Grinstein et al. [98] have demonstrated that the NHE1 protein distributes along the border of lamellipodia and near the edge of cell processes and corresponds to sites of focal adhesions. This pattern of distribution may be involved in the generation of local osmotic gradients during migration. F-actin has been shown to interact with the NHE1 isoform of the Na/H exchanger. Upon disruption of the actin filaments by cytochalasin D the Na/H exchanger does not modify its activity but loses its focal accumulation with F-actin [99]. After cytochalasin D treatment, stimulation of fibroblasts with bradykinin causes rapid cell swelling due to Na/H exchange, whereas untreated cells respond to bradykinin with cell shrinkage. Similarly, in rabbit neutrophils the swelling effect of various chemoattractants was reversed to a shrinking effect following treatment of the cells with cytochalasin B [100]. In fibroblasts expressing the p21Ha-ras oncoprotein and in normal fibroblasts pretreated with lithium, oscillations of intracellular Ca2 trigger both the depolymerization of actin stress fiber network, activation of the Na/H exchanger and cell swelling. Obviously the cytoskeletal remodeling alters the cell volume regulatory properties and plays a permissive role for the Na/H exchanger to serve for cell volume increase [69, 101–103]. In MDCK cells transformed with the Moloney sarcoma virus (MSV) and in an invasive MSV-MDCK cell variant (MSV-MDCK-INV) expression of NHE1 is significantly increased in

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MSV-MDCK-INV cells relative to MSV-MDCK and MDCK cells. NHE1 is localized with -actin to the tips of MSV-MDCK-INV cell. Inhibition of NHE1 induces the disassembly of actin stress fibers and redistribution of the actin cytoskeleton in all cell types. The fact that NHE inhibition suppresses drug induced formation of actin stress fibers in MSV-MDCK cells indicates that NHE1 can regulate actin stress fiber assembly in transformed MSV-MDCK cells via its intracellular pH regulatory effect. Hence in these cells NHE1 is necessary to regulate the actin dynamics for adhesion and pseudopodial protrusion [48]. As mentioned above, migration of MDCK-F cells depends on the polarized activity of Ca2-sensitive K (IK1) channels. Both inhibition and activation of IK1 channels, either with specific drugs or by cell volume perturbations, reduce the rate of migration to the same extent as cytochalasin D. Inhibition of migration is accompanied either by actin depolymerization or polymerization with a strong correlation of the changes of migration and cytoskeletal remodeling. IK1 channel inhibition and cell swelling elicit a rise in intracellular Ca2, whereas IK1 channel activation has the opposite effect. IK channel-dependent perturbations of cell volume and anisotonicity elicit virtually identical effects on migration, actin filaments and intracellular Ca2. Hence cell volume is likely to be a link between IK channel activity, actin filaments and migration using intracellular Ca2 as an important coordinator [104]. Indeed inhibition of the plasma membrane Ca2 ATPase and the Na/Ca2 exchanger leads to an increase and inhibition of Ca2 channels to a decrease of the intracellular Ca2 concentration, respectively. All these treatments lead to a rapid impairment of cell migration, the most prominent effect being elicited by Na/Ca2 exchanger inhibition [105]. In CD14 mononuclear cells katacalcin, a polypeptide encoded by the calc-1 gene, elicits PKA/cAMP dependent migration at atomolar concentrations which is, however, accompanied by a lowering of the intracellular Ca2 concentration [106]. In CaCo-2 cells regulation of the Na/H exchanger by serum has been shown to involve an F-actin dependent mechanism [107]. In melanoma cells deficient in actin-binding protein, both cell volume regulation and cell migration are abrogated [108]. Cell swelling changes F-actin architecture in various cell types and F-actin has been shown to exert a modulatory effect on volume regulated Cl channels [13, 109]. In neutrophils the Cl/HCO 3 exchanger is linked to the cytoskeleton via ankyrin [110]. The Na /K /2Cl cotransporter is activated by disassembly of F-actin in intestinal cells and Ehrlich ascites tumor cells [111, 112]. In human hepatoblastoma (HepG2) cells, hypotonic stress stimulates cellular Ca2 influx via TRPV1 channels which is greater if cells are incubated with hepatocyte growth factor/scatter factor. This can, however, only be observed in cells with a migrating phenotype. The TRPV1-mediated increase of intracellular Ca2 may therefore give rise to the migratory phenotype and to cell


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locomotion [113]. A functional association between the osmosensitive TRPV4 channel and aquaporin 5 (AQP5) has recently been found in salivary gland cells. Both transporters are colocalized in the apical region of acini. Interestingly the hypotonicity-induced Ca2 entry via TRPV4, which is required for RVD, is dependent on intact AQP5 and hypotonicity increases the association and surface expression of AQP5 and TRPV4. Upon actin depolymerization these effects as well as RVD are reduced. Thus TRPV4 and AQP5 concertedly control RVD [114]. Recently, it has been described that AQP1 facilitates endothelial cell migration by a mechanism that may involve facilitated water transport across lamellipodia [32, 115].

Substrate Adhesion, Cell Spreading and Cell Volume

A highly regulated process in migration is the continuous assembly and disassembly of focal cell adhesions to the extracellular matrix (ECM). Focal adhesions are made up by an assembly of molecules including several kinases and adaptor molecules [116]. While adhesion formation occurs at the leading edge, disassembly occurs predominantly at the cell rear and at the base of protrusions. In most adhesion sites, integrin receptors mediate the binding to the ECM via their extracellular domains, and interact with the actin cytoskeleton via their cytoplasmic moieties. They anchor the cell to the ECM, allowing the contractile actomyosin system to pull the cell body and trailing edge forward. The intracellular domain of integrin-mediated adhesions contains a large number of proteins which mediate the mechanical linkage between the ECM and the cytoskeleton or participate in adhesion-mediated signaling [19, 37, 117–119]. Repeated transient increases in intracellular Ca2 supports detachment of integrins from their extracellular ligands. Neutrophils in which Ca2 transients are inhibited become stuck on ECM, but this can be restored by antibodies to 3 integrins or the v3 heterodimer, indicating that v3-like integrins are responsible for the Ca2-sensitive adhesion [120]. It has been shown that in different migrating cell types intracellular pH increases upon adhesion to the substratum by integrin mediated activation of the Na/H exchanger [121–125]. In human neutrophils, both cell spreading and activation of the Na/H exchanger are prevented by inhibition of actin filament assembly with cytochalasin D, suggesting that the cytoskeletal reorganization is required for activation of the Na/H exchanger [121]. Na/H exchanger NHE1 modulates initial steps in integrin signaling for the assembly of focal adhesions. In the absence of NHE1 activity, cell spreading is inhibited, the accumulation of integrins, paxillin, and vinculin at focal contacts as well as phosphorylation of p125FAK induced by integrin clustering are impaired. NHE1 seems to act downstream of RhoA to

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contribute a crucial signal to proximal events in integrin-induced cytoskeletal reorganization [126]. Furthermore a structural link between NHE1 and the actin binding proteins ezrin, radixin, and moesin (ERM) has been demonstrated. NHE1 and ERM proteins associate directly and colocalize in lamellipodia. Fibroblasts expressing NHE1 with mutations that disrupt ERM binding, but not ion translocation, show an impaired organization of focal adhesions and actin stress fibers, and attain an irregular cell shape [127, 128]. NHE1 also plays an important role in the regulation of the extracellular environment. Serum deprivation induces NHE1-dependent morphological and cytoskeletal changes in metastatic breast cancer (MDA-MB-435) cells via interaction with RhoA and Rac1, resulting in increased chemotaxis and invasion. Serum deprivation changes cell shape by reducing the amount of F-actin and inducing the formation of leading edge pseudopodia via Rac1- and RhoA-dependent action on NHE1 activity as a convergence point [46, 47]. In MDA-MB-231 breast tumor cells the hyaluronan receptor CD44, NHE1 and hyaluronidase-2 are closely associated in a complex and are enriched in plasma membrane microdomains. Stimulation of CD44 activates NHE1 which leads to changes in intracellular pH and acidification of the ECM. This in turn facilitates tumor cell invasion – a process that can be stopped by inhibition of NHE1 [129]. In human melanoma (MV3) cells migration depends on integrin 21. These cells reach a maximum motility at an extracellular pH of 7.0, but hardly migrate at higher or lower values, when NHE is inhibited, or when NHE activity is stimulated by cellular acid load. These procedures also cause changes in cell morphology and pH. The migration and morphology correlate with the strength of cell-matrix interactions, which is strongest at extracellular pH of 6.6 and weakens at alkaline conditions, upon NHE inhibition, or upon blockage of the integrin 21. This indicates that NHE activity affects migration of human melanoma cells by modulating the cell-matrix interactions. Migration is hampered when the interaction is too strong (acidic extracellular pH) or too weak (alkaline extracellular pH or NHE inhibition) [43]. The important role of NHE1 in response to cell stress has recently been reviewed [130]. Focal adhesion complexes might also be important in the regulation of swelling-activated anion currents. p125FAK, a protein tyrosine kinase regulating actin binding to focal adhesions, has been shown to be phosphorylated in response to cell swelling. Inhibition of the ras related GTPase p21rho, which is known to regulate actin cytoskeleton, inhibits both swelling-induced anion channel activation and pp125FAK phosphorylation, suggesting that actin is involved in the regulation of RVD [131]. p125FAK has been shown to contribute to the regulation of the swelling-activated Cl currents also in rat ventricular myocytes [132]. Beside actin, microtubules seem to be involved in volume regulation. RVD is inhibited in HL-60 cells, Jurkat cells and neutrophils


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upon treatment with colchicines and J774 macrophages respond to microtubule disassambly with anion channel activation [133].

Polarized Distribution of Cell Volume Regulatory Ion Transport Mechanisms

Considering the polarized cell shape of migrating cells and taking into account that locomotion requires both volume expansion at the leading edge and volume reduction at the trailing edge in a spatially and temporally coordinated manner, implies that the ion transporters regulating cell migration are also arranged in a polarized fashion. This has been demonstrated in an elegant series of studies in MDCK-F cells and other cells [49, 50, 68, 92, 134, 135]. In MDCK-F cells selective exposure of the lamellipodium to inhibitors of Na/H exchange slows migration of these cells, whereas exposure of the cell body to the inhibitor is without effect. Selective exposure of the cell body to K- or Ca2 channel inhibitors abrogates migration, whereas superfusion of the lamellipodium is without effect [68, 92]. By selective application of NHE1 blockers or inhibitors of the anion exchanger AE2 to the lamellipodium and by immunocytochemistry it has been shown that NHE1 and AE2 are concentrated at the front of MDCK-F cells and accumulate with actin [44]. As mentioned above, intracellular Ca2 concentration predominantly increases at the rear of some migrating cells [70, 89–92]. This might not only be necessary for spatial coordination of cytoskeletal rearrangement and actomyosin contraction, but also for polarized ion channel activation in the moving cell. Therefore, the mechanisms serving for RVD, i.e. K, Cl and Ca2 channels, are confined to the rear of the cell, whereas ion transporters mediating RVI, i.e. Na/H exchanger, Cl/HCO3 exchanger and Na/K/2Cl cotransport and presumably the H/K ATPase are sorted predominantly to the leading edge. Indeed MDCK-F preferentially swell at the lamellipodium and simultaneously shrink at the rear upon increasing the intracellular Ca2 concentration [136]. Consistent with this concept are the findings that exogenously expressed AQP1 or AQP4 enhances migration of epithelial cells, that AQP1 localizes to the leading edge of the cell membrane and that AQP1 expression produces more lamellipodia and a shorter mean residence time of these protrusions. This suggests that AQPs accelerate cell migration by facilitating the rapid turnover of membrane protrusions at the leading edge. As described above, actin cleavage and ion uptake at the tip of a lamellipodium could create local osmotic gradients that drive the influx of water across the cell membrane which in turn might then increase local hydrostatic pressure to cause cell membrane protrusion [32]. Figure 2 gives a tentative model of polarized distribution of ion transport mechanisms in a migrating cell.

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AQP

?

e

NCX

NKCC

AE

HkATPase

NHE

NBC

?

i

2CI

K

2

Na

Ca

H20

HCO 3

CI

Na

H

K

K Na

i PMCA e

?

K

CI

2

Ca

2

Ca

Fig. 2. Tentative model of the organization of cell volume regulatory ion transport mechanisms in a migrating human embryonic kidney (HEK-293T) cell. The cell was transfected with the farnesylated from of the yellow fluorescent protein which selectively stains the cell membrane. Ion transporters and aquaporins supporting cell swelling are sorted to the leading edge of the cell, which forms a fan like lamellipodium and directs the movement of the cell, whereas ion channels and transporters supporting volume reduction operate mainly at the trailing edge of the cell. NKCC Na/K/2Cl exchanger; NCX Na/Ca2 exchanger; AQP aquaporin; AE anion exchanger; NBC Na/HCO exchanger; 3 cotransporter; NHE Na /H PMCA plasma membrane Ca2 ATPase; i intracellular; e extracellular.


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Conclusions

Cell volume regulatory ion transport mechanisms are important elements in the regulation and control of cell migration. The ion and water fluxes across the cell membrane are not only triggered in response to challenges to cell volume constancy due to osmotic, contractile or mechanical forces created within a migrating cell, but also confer volume changes to the cell in a temporally and spatially coordinated manner, thus supporting the well tuned dynamics of migration. Several pathophysiological or even clinical disorders may disturb these mechanisms, thereby impeding coordinated migration. Likewise, defective cell volume regulation may contribute to enhanced or reduced mobility of cells, thus substantiating disorders like infections, chronic inflammatory processes, arteriosklerosis or formation of tumor metastasis. Cell volume regulatory mechanisms may be future targets for therapeutic intervention on a pharmacological, molecular or genetic level.

Acknowledgements This work has been supported by the Austrian Science Foundation (FWF) grant p14102-MED and the PMU grant to MR.

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Markus Ritter, MD Institute of Physiology and Pathophysiology Paracelsus Private Medical University Strubergasse 21 AT–5020 Salzburg (Austria) Tel. 43 0662 44 2002 1250, Fax: 43 0662 44 2002 1259, E-Mail [email protected]

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Cell Volume Regulation in the Renal Papilla Franz-X. Beck, Wolfgang Neuhofer Department of Physiology, University of Munich, Munich, Germany

Abstract Urine osmolality and interstitial solute concentration in the renal medulla are determined by the body’s hydration state. This implies that in the renal medulla extracellular solute concentrations, and hence the volume of medulla-resident cells, will vary drastically with the state of hydration. Medullary cells regulate their volume primarily by adjusting accumulation and release of low-molecular weight organic osmolytes appropriately. There is growing evidence that not only transcriptional processes, but also post-transcriptional mechanisms, such as targeting of transport molecules to the plasma membrane, contribute to the successful adaptation of medullary cells to extreme environmental conditions. Copyright © 2006 S. Karger AG, Basel

In contrast to the majority of cells in the mammalian organism, the cells of the renal medulla are exposed to wide fluctuations in extracellular osmolality during changes in the hydration status. These variations are primarily due to changes both in the activity of the renal medullary countercurrent system and in the concentrations of circulating antidiuretic hormone. Interstitial solute concentrations, which are high during water deprivation, may fall to values close to isosmolality following the ingestion of large amounts of fluid. The predominant solutes found in the interstitium of the medulla in antidiuresis are NaCl, a solute virtually excluded from the intracellular compartment, and urea, which penetrates most cell membranes readily. The low permeability of cell membranes to Na⫹ implies large variations in cell volume of medullary cells during the transition from antidiuresis to diuresis and vice versa. In the following, we describe strategies developed by medullary cells to meet these challenges.

Response to Falling Extracellular Osmolality

As described in greater detail below, renal medullary cells adapt osmotically to the high interstitial NaCl concentrations, prevailing in the inner medulla in antidiuresis [1], by accumulation of low-molecular weight organic substances, the so-called organic osmolytes. When, after a period of water shortage, i.e. a situation allowing medullary cells to fully adapt to their hypertonic environment, larger quantities of water are ingested, medullary tonicity falls rapidly [2, 3]. As a consequence, water enters the cells from a relative hypotonic environment leading to cell swelling and activation of pathways allowing the rapid efflux of organic osmolytes and hence reduction of cell volume [4–7]. Although volume-sensitive anion channels, permeable to a variety of inorganic anions and low-molecular weight organic osmolytes, have been implicated in osmolyte release [8, 9], neither the molecular identity of these channels nor the issue of whether medullary cells express more than one type of volume-sensitive anion channel, has been resolved to date. Hypotonicityevoked osmolyte efflux is inhibited by extracellular ATP, by intracellular ATP depletion and various anion channel blockers including niflumic acid, 5-nitro2-(3-phenylpropylamino)benzoate (NPPB), or 1,9-dideoxyforfolin [7]. In inner medullary collecting duct cells, efflux of taurine, betaine and sorbitol proceeds primarily across the basolateral membrane and depends on the transient increase in intracellular Ca⫹⫹ activity associated with falling extracellular tonicities [7, 10]. However, osmolyte efflux also across the apical membrane has been described for several epithelial cell lines derived from medulla-resident cells [11, 12]. For sorbitol, at least, release requires rearrangement of the submembrane actin network and relies on vesicle fusion with the plasma membrane [10]. Of interest, in a variety of cell types activation of the volume-sensitive anion channel is impeded by high intracellular ionic strength or, specifically, by high intracellular Cl⫺ concentrations [13–17]. While in non-medullary cells exposed to a hypotonic environment, efflux of K⫹ and Cl⫺, i.e. of inorganic electrolytes, is a key element in the regulatory volume decrease (RVD), this process is likely to play only a minor role in medullary cells subject to falling interstitial tonicities. This notion is based on the observation that the sum of Na⫹, Cl⫺ and K⫹ contents of papillary collecting duct cells decreases only moderately (from about 1,050 to 880 mmol/kg urea free dry weight) when the sum of interstitial Na⫹, Cl⫺ and K⫹ concentrations drops sharply (from about 1,050 in antidiuresis to some 310 mmol/l) after the administration of the loop diuretic furosemide [18].

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HCO3

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d gra a

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CH3 Cl O –O C CH N+ CH 2 3 CH3

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Na H Na Water loss

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Fig. 1. Following a rise in ambient tonicity, osmotically obliged water efflux causes cell shrinkage, which results in an increase in functionally impermeant intracellular electrolytes, i.e. the intracellular ionic strength. In the first step, cell volume recovers by regulatory volume increase, which however, entails a sustained elevation of the cellular ionic strength. To avoid the adverse effects of a persistently increased ionic strength, intracellular electrolytes are gradually replaced by metabolically neutral organic osmolytes.

Response to Rising Extracellular Osmolality

Stimulation of the renal concentrating mechanism after prolonged diuresis causes the extracellular NaCl concentration in the renal medulla to increase rapidly [3, 19] and osmotically obliged water efflux from medulla resident cells follows (fig. 1). This in turn leads to passive concentration of intracellular solutes, including intracellular inorganic electrolytes and hence to a rise in intracellular ionic strength [20]. Studies on isolated perfused papillary collecting ducts and medullary thick ascending limbs (mTALs) suggest that in this situation, recovery of cell volume is initiated by activation of Na⫹/H⫹ and Cl⫺/HCO3⫺ exchangers working in parallel, thus allowing the entry of NaCl and, in consequence, of water [21–25]. Hypertonicity-induced cell shrinkage is thus reversed by regulatory volume increase (RVI), but not the rise in intracellular ionic strength (fig. 1). The majority of Na ions entering papillary cells in this situation are probably exchanged against K⫹ via the Na⫹/K⫹-ATPase. This

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Fig. 2. Intracellular concentrations of organic osmolytes and inorganic electrolytes (Na⫹, K⫹ and Cl⫺) in papillary collecting duct cells of antidiuretic and diuretic rats and in proximal convoluted tubule cells of control rats. Data from reference [20, 127, 128.]

notion is based on the observation that cell Na⫹ concentration increases far less than cell K⫹ concentration when interstitial tonicity rises in the inner medulla [3, 20]. Apart from acute activation of the Na⫹/K⫹-ATPase by an increased intracellular Na⫹ concentration, the expression of this key transport molecule is enhanced in response to hypertonic stress [26–29]. Maintenance of a low intracellular Na⫹ concentration allows the continuous function of vitally important, Na⫹-dependent transporters, such as Na⫹/H⫹- and Na⫹/Ca⫹⫹-exchangers or Na⫹-dependent import of myo-inositol and betaine. It is thus not surprising that suppression of the hypertonicity-induced rise in the production of the Na⫹/K⫹ATPase ␥-subunit, a process controlled by Cl⫺ entry and activation of JNK2 and PI-3K, is associated with increased incidence of cell death in cultured medullary cells exposed to hypertonic stress [30]. Influx of Na⫹ and Cl⫺ and replacement of Na⫹ by K⫹ as described above assists the recovery of cell volume, but does not solve the problem of an elevated intracellular ionic strength caused by osmotic cell shrinkage. This problem is met by the gradual intracellular accumulation of organic osmolytes, primarily of the trimethylamines betaine and glycerophosphorylcholine, of the polyols myo-inositol and sorbitol and of free amino acids (fig. 1). A substantial portion of the inorganic monovalent ions is thus replaced by metabolically inert, i.e. ‘compatible’, organic osmolytes allowing the osmotic adaptation of medullary cells to high extracellular NaCl concentration at normal intracellular electrolyte concentrations (fig. 2) [31]. During the initial stages of osmolyte accumulation, enhanced expression of heat shock proteins (HSPs), specifically

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HSP70, may contribute to limiting the deterioration of macromolecule functions caused by elevated intracellular ionic strength [32, 33]. Betaine. In antidiuretic animals, betaine concentrations are much higher in the outer and inner medulla than in the cortex, consistent with its role as a compatible osmolyte [3, 34–37]. Accumulation of betaine by renal medullary cells is accomplished by the betaine ␥-amino-butyrate transporter (BGT1), a protein of 614 amino acids with sorting signals and basolateral retention motifs in the cytosolic C-terminal domain [38, 38–43], that combines the uphill transport of betaine with the downhill import of 3 Na⫹ and 1 or 2 Cl⫺ [40, 44]. A C-terminal PDZ-target motive interacts with LIN-7 in MDCK cells, thereby mediating retention of BGT-1 in the basolateral membrane [43]. It should be noted, however, that in cells derived from the thick ascending limb of the loop of Henle, tonicityresponsive betaine uptake is located mainly in the apical membrane [12]. Betaine is synthesized from choline via betaine aldehyde by choline dehydrogenase or the consecutive action of choline dehydrogenase and betaine aldehyde dehydrogenase [45]. Although medullary cells are capable of betaine synthesis [46], the main source of this osmolyte is probably the proximal tubule [47, 48]. Hence, during hypertonic stress, betaine production is activated mainly in the cortex [45]. There is evidence that under this condition betaine released from proximal tubule cells travels into the medulla via either the vasa recta or the loop of Henle and is then taken up by medullary cells either across the apical or basolateral membranes [49]. In this situation, accumulation of betaine by medullary cells is facilitated further by tonicity-induced transcriptional activation of the BGT1 gene [50–52]. Myo-Inositol. High intracellular concentrations of myo-inositol in response to elevated extracellular NaCl concentrations are achieved via the Na⫹-dependent myo-inositol transporter (SMIT), a member of the sodium/glucose transporter family encompassing 718 amino acids [53, 54]. This transporter presumably has 14 transmembrane domains and mediates the import of one myo-inositol molecule together with two Na⫹ [55, 56]. In cultured renal epithelial cells evidence for the location of SMIT in both apical and basolateral membranes has been obtained [12, 57–59]. Hence, uptake of myo-inositol may proceed across the apical and/or basolateral membrane, albeit mediated by different transport molecules. The basolateral membrane-resident transport molecule is probably the ‘classical’ SMIT (sometimes denoted ‘SMIT1’) with a Km for myo-inositol of about 50 ␮M [53, 55], while its apical counterpart, designated ‘SMIT2’, has a Km ⬎100 ␮M and 43% identity of its cDNA sequence with SMIT1 [59, 60]. The expression of both these transporters is enhanced by hypertonic stress [53, 59].


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In antidiuresis, myo-inositol concentrations are substantially higher in the inner and outer medulla than in the cortex and significantly reduced in the former kidney zones following the induction of diuresis [3, 5, 20, 35, 61, 62]. These findings are in accord with Northern blot analyses demonstrating corresponding intrarenal distribution and diuresis-induced response of SMIT mRNA abundance [52, 63, 64]. Sorbitol. In kidneys producing a concentrated urine, the tissue concentration of the polyol sorbitol increases steeply between the outer–inner medullary boundary and the tip of the papilla [3, 5, 6, 35, 65]. In the outer medulla and particularly in the cortex, sorbitol concentrations are low or even undetectable. After induction of diuresis, a rapid and drastic decrease of inner medullary sorbitol content is noted [37, 66]. Accumulation of sorbitol by renal medullary cells is achieved by aldose reductase-catalyzed, NADPH-dependent reduction of glucose to sorbitol. Degradation of sorbitol to fructose is catalyzed by sorbitol dehydrogenase in a NAD⫹-dependent oxidation reaction. Increased intracellular sorbitol concentrations during rising extracellular tonicities are achieved primarily by enhanced expression of aldose reductase [52, 64, 65, 67, 68]. Conversely, aldose reductase expression is reduced drastically during severe diuresis [52, 65, 67]. Changes in the expression and activity of sorbitol dehydrogenase appear to be less important for the regulation of intracellular sorbitol in response to altered tonicity [52, 67, 69]. After birth, when the concentrating ability of the mammalian kidney rises gradually, AR mRNA and AR immunoreactivity in the inner medulla also increase [70] and immature, inner mTALs are transformed into mature ascending thin limbs. Interestingly, this differentiation process is restricted to AR-positive mTAL cells, whereas AR-negative cells, not protected from tonicity-induced stress by elevated concentrations of organic osmolytes, are subject to apoptosis [70]. GPC. In antidiuresis, tissue GPC contents rise steeply along the corticopapillary axis [3, 5, 6, 35, 71]. This gradient is partially abolished following the induction of diuresis by either water-loading or administration of loop diuretics [3, 5, 20, 35, 66, 71] or in rats with hereditary hypothalamic diabetes insipidus (Brattleboro rats) [61]. In contrast to myo-inositol, betaine and sorbitol, which are concentrated by medullary cells either by enhanced uptake from the extracellular spaces (myoinositol, betaine) or increased intracellular production (sorbitol), GPC is accumulated in response to hypertonic stress primarily by curbed degradation. This view is based on observations showing that the degradation of GPC by GPC:choline phosphodiesterase is reduced or enhanced when extracellular solute concentrations rise or fall, respectively [72, 73]. On the other hand, the

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production of GPC, i.e. its release from phosphatidylcholine brought about by phospholipase A2, phospholipase A1 and lysophospholipase, is hardly altered by acute changes in extracellular solute concentrations [72, 74]. The regulation of intracellular GPC contents differs from that of myo-inositol, betaine or sorbitol in other respects: While intracellular accumulation of the latter three is determined primarily by the extracellular NaCl, but not urea, concentration, intracellular GPC concentrations increase not only in response to elevated extracellular NaCl, but also urea concentrations [73]. In agreement with these observations on MDCK cells is the close positive correlation between inner medullary GPC and urea concentrations [35, 36, 75]. Free Amino Acids. In addition to methylamines and polyols, free amino acids often are accumulated by animal cells exposed to a hypertonic environment. Accordingly, in cultured renal epithelial cells uptake of free amino acids via system A and the Na⫹- and Cl⫺-dependent taurine transporter (TauT) is activated by hypertonic stress [76–78]. After prolonged water deprivation, free amino acids, including taurine, contribute to the total pool of organic osmolytes also in the renal medulla [20, 36, 75]. TauT mRNA abundance increases in both the outer medulla and papilla following water restriction [79], but transcription of the TauT gene in vivo is probably not subject to extensive TonEBP-dependent regulation [80]. The uptake of free amino acids is thought to precede the relatively late onset of the accumulation of trimethylamines and polyols and to decline gradually as the intracellular concentrations of the latter rise [77, 81].

Regulation of Osmolyte Accumulation

Transcriptional Regulation Several genes involved in protection from osmotic stress share highly conserved cis-acting sequences within their 5⬘-flanking regions, termed tonicityresponsive enhancer, TonE (also called osmotic response element [ORE]). The corresponding transcriptional activator was cloned independently by several groups in the late 1990s and is known as tonicity responsive enhancer binding protein (TonEBP)/ORE binding protein (OREBP) or, based on its homology to nuclear factors of activated T-lymphocytes (NFAT), NFAT5 [82–84]. TonEBP is characterized by a Rel-like DNA binding domain, however unlike other members of the NFAT family, it is not regulated via calcineurin and does not form cooperative complexes with AP-1 (c-fos/c-jun) [83, 85]. Recently, it has been shown that TonEBP associates with RNA helicase A and that this inhibitory interaction is weakened by elevated ionic strength [86]. To date, consensus sequences for TonEBP have been identified in the genes for AR, BGT1, SMIT,


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␥-Subunit of Na/K-ATPase ↓

Osmolyte accumulating mechanisms ↓

Osmolyte efflux pathway(s) ↑

H2O

Intracellular ionic strength or/and [Cl⫺] ↓

Intracellular ionic strength or/and [Cl⫺] ↑

␥-Subunit of Na/K-ATPase ↑

Osmolyte accumulating mechanisms ↑

Osmolyte efflux pathway(s) ↓

H2O Diuresis

Antidiuresis

Fig. 3. Effect of changes in intracellular ionic strength or/and intracellular Cl concentration on processes relevant for cell volume regulation in the renal medulla.

TauT, and HSP70 [87–91]. These cis-acting sequences may occur at multiple sites in proximity to the transcriptional initiation site, but may also be spread over 50 kb upstream, as has been shown for SMIT [89]. The importance of TonEBP for the function and integrity of cells from the renal medulla has been demonstrated by targeted disruption of the TonEBP/ NFAT5 gene in mice [80]. These animals are characterized by hypoplastic kidneys, atrophy of the renal medulla, and an increased incidence of apoptosis in the inner medulla. Furthermore, medullary tubular epithelial cells show an increased nuclear/cytoplasmic ratio indicative of cell shrinkage [80]. These alterations are accompanied by reduced expression of AR, BGT1, and SMIT, suggesting an inability to maintain normal cell volume [80]. Although the importance of enhanced expression and action of genes involved in osmolyte accumulation for cells of the renal medulla has been demonstrated convincingly, the signaling events leading to transcriptional upregulation of these genes are much less clearly defined, particularly due to the complex regulation of TonEBP. Expression of TonEBP target genes correlates with the sum of the intracellular concentrations of monovalent ions, i.e. the cellular ionic strength [92–94]. Hence, intracellular ionic strength or intracellular Cl⫺ concentration may play a major role in orchestrating the various key processes allowing medullary cells to adapt to fluctuating extracellular tonicities: osmolyte accumulation and release and synthesis of the ␥-subunit of the Na⫹/K⫹-ATPase are modulated by changes in intracellular ionic strength or Cl⫺ concentration (fig. 3). Elevated transcriptional activity of TonEBP/NFAT5 requires proteasome activity, nuclear translocation and dimerization [85, 95]. In addition, TonEBP expression itself increases in response to hypertonicty and contributes to TonEBP activity [84]. Kinase Pathways. Numerous kinases, including MAPK, PKA, p38, Fyn, Syk, ataxia telangiectasia-mutated kinase (ATM), and DNA-PK have been implicated

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in TonEBP transactivation. Activation of all three major mammalian MAP kinase cascades (i.e. ERK, JNK, p38) has been demonstrated in response to hypertonicity [96–99]. Depletion of PKC by prolonged treatment with PMA abolishes tonicity-induced ERK activation [98], but fails to blunt the upregulation of BGT1 and SMIT [100], suggesting that ERK is not essential for TonEBP activation. In contrast, pharmacological inhibition of p38 dose-dependently attenuates tonicityinduced expression of BGT1 and HSP70 [101, 102]. This mechanism is reminiscent of the HOG-1 kinase pathway in yeast, a homolog of p38 in mammalian cells, which promotes accumulation of glycerol, which serves as an osmolyte in yeast cells. While a membrane-resident osmosensor that signals to HOG-1 has been identified in yeast, a corresponding mechanism that activates mammalian MAP kinases has not yet been found. Pharmacological inhibition of PKA and expression of a dominant negative PKA isoform reduce tonicity-induced expression of TonEBP target genes and reporter activity [103], suggesting that PKA signals to TonEBP. In addition, TonEBP contains two putative PKA phosphorylation sites and a physical interaction between PKA and TonEBP has been reported, however, this pathway appears to be independent of cAMP [103]. Furthermore, transfection with a constitutively active catalytic subunit of PKA increases TonEBP reporter activity under isotonic conditions, however not in cells exposed to osmotic stress [103]. Thus, in view of the latter two observations, the role of PKA in TonEBP transactivation is not yet clear. Recently, ATM kinase, a DNA damage-inducible kinase, has been implicated in signal transduction in response to osmotic stress [104]. Dimitrieva et al. have shown that medullary cells exposed to osmotic stress display numerous DNA double strand breaks, even when adapted to hypertonicity [105, 106]. This observation has been ascribed to an impaired DNA damage response, since proteins like Ku70, Ku80 and Mre11 exonuclease that would be expected to localize to the nucleus following DNA damage, reside in the cytosol rather than the nucleus by virtue of an increase in cellular ionic strength [107, 108]. Consequently, ATM, which is activated by autophosphorylation in response to DNA damage may contribute to TonEBP activation as evidenced by reduced TonEBP transcriptional activity by interference with ATM signaling [109]. The authors therefore suggested that DNA damage may represent a sensor for elevated cellular ionic strength. This mechanism, however would imply the presence of persistent DNA damage in renal medullary cells in situ, a finding that has not been documented consistently. Several other kinases including the non-receptor tyrosine kinases Fyn, Syk, and Pyk2, the serine/threonine kinase Pak2, and AKT have been implicated in osmosensing since these kinases are activated by osmotic stress or cell shrinkage [110–113]. Recently, however, the role of phosphorylation by tonicity-inducible protein kinases has been questioned because TonEBP does not


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appear to be a direct substrate [114]. It is thus possible that tonicity-inducible kinases affect TonEBP transactivation indirectly rather than by direct phosphorylation [114]. In conclusion, an array of kinase pathways converge on TonEBP transcriptional activation, but none of them appears to be sufficient in isolation. Cell-Matrix Interactions. Cell volume alterations may translate into mechanical forces which could be sensed through cell-matrix interactions. Integrins are heterodimeric transmembrane proteins consisting of a large ␣- and a small ␤-subunit that attach cells to extracellular matrix proteins and are linked to intracellular signaling mechanisms. ␤1-Integrin is induced by hypertonicity in MDCK cells within 2 h [115] and is up-regulated in the renal medulla of waterdeprived rats [116]. Furthermore, ␤1-integrin is associated physically with HBEGF and CD9 in mTAL cells in vivo [116], thereby forming an osmotically regulated membrane signaling complex that may transduce cell-matrix interactions into intracellular signals. This notion is supported by studies in mice lacking ␣1␤1-integrin [117]. These animals show decreased expression of AR and SMIT and diminished contents of organic osmolytes in the renal medulla following water deprivation, which coincides with increased incidence of apoptosis in the renal medulla [117]. These observations may result from impaired p38 signaling and subsequently reduced TonEBP levels as observed in ␣1-null mice. COX-2. COX-2 is expressed abundantly in the renal medulla and is thought to be a major determinant of medullary PGE2 production, particularly during antidiuresis. Recent evidence suggests that PGE2 not only modulates medullary electrolyte absorption and blood flow but also favors the adaptation of medullary cells to hyperosmolality. In mice lacking COX-2, or in rats with pharmacological inhibition of COX-2, dehydration causes apoptosis in renal medullary cells as a result of reduced expression of osmoprotective genes [118, 119]. Conversely, in MDCK cells the induction of osmoprotective genes in response to hypertonicity is more pronounced in the presence of PGE2. This effect is however not due to increased TonEBP transcriptional activity, abundance, or nuclear translocation. Reactive Oxygen Species. Elevated extracellular salt concentrations and angiotensin II promote superoxide production in the mTAL by virtue of stimulation of the Na⫹/K⫹-ATPase and NADPH oxidase, respectively [120, 121]. Zhou et al. [122] have demonstrated recently in 293 cells that hypertonicity increases the production of reactive oxygen species, including superoxide, which contribute to TonEBP transactivation and induction of BGT1. Conversely, nitric oxide, which combines with superoxide to form peroxynitrite, reduces TonEBP reporter activity in MDCK cells.

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Urea. Although present in large quantities in the renal medulla, urea enters most cells readily and therefore does not represent a major osmotic stress for medullary cells. Nevertheless, urea may affect the expression of genes involved in osmoadaptation by inhibiting tonicity-induced responses. Tian and Cohen have demonstrated that urea diminishes tonicity-induced TonEBP expression, reporter activity, and AR expression [123]. These effects are specific for urea, since another permeant solute, glycerol, is ineffective. In addition, urea-mediated repression of TonEBP expression and action was not due to inhibition of p38 or proteasomes [123]. Consistent data have been obtained by Lammers et al. [124] demonstrating that urea diminished up-regulation of BGT1 protein and transport activity following hypertonic stress. The physiological significance of these findings is however poorly understood. Post-Transcriptional Regulation Several lines of evidence suggest that post-translational modifications participate in the regulation of the amount of membrane-bound BGT1, and thereby transport activity. BGT1 contains several putative phosphorylation sites within the COOH-terminal region. Indeed, Massari et al. [125] have shown that phosphorylation of BGT1 by PKC, but not by PKA, causes clathrin-mediated internalization and intracellular accumulation of BGT1 in MDCK cells, which coincides with reduced transport activity. At least in the initial few hours following a rise in ambient tonicity, membrane targeting of BGT1, rather than increased abundance, appears to be the primary mechanism responsible for increased betaine uptake [126]. Transfected MDCK cells expressing a BGT1GFP chimeric protein exhibit cytoplasmic fluorescence under isotonic conditions. Following 6 h of hypertonic stress, however, GFP fluorescence localizes to the plasma membrane, while intracellular GFP fluorescence is diminished [126]. Interestingly, proteasome activity is not only required for nuclear translocation and activation of TonEBP, but also for membrane insertion and increased transport activity of BGT1 following osmotic stress [124, 126]. These observations suggest that additional, as yet unknown, TonEBP target genes promote membrane targeting of BGT1.

Concluding Remarks

The cells of the renal medulla are exposed to widely fluctuating interstitial solute concentrations that entail substantial variations in cell volume. In contrast to other cell types, renal medulla-resident cells achieve osmotic equilibrium with the interstitial compartment primarily by adjusting the intracellular amount of organic osmoeffectors (organic/compatible osmolytes) rather than


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inorganic electrolytes. Increasing evidence suggests that malfunction of these mechanisms may be important under pathophysiological conditions. Acknowledgments Work in the authors’ laboratory was supported by the Deutsche Forschungsgemeinschaft, the Deutsche Nierenstiftung and the Münchener Medizinische Wochenschrift.

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111 Miah SMS, Sada K, Tuazon PT, Ling J, Maeno K, Kyo S, Qu X, Tohyama Y, Traugh JA, Yamamura H: Activation of Syk protein tyrosine kinase in response to osmotic stress requires interaction with p21-activated protein kinase Pak2/␥PAK. Mol Cell Biol 2004;24:71–83. 112 Rizoli SB, Rotstein OD, Kapus A: Cell volume-dependent regulation of L-selectin shedding in neutrophils. A role for p38 mitogen-activated protein kinase. J Biol Chem 1999;274:22072–22080. 113 Terada Y, Inoshita S, Hanada S, Shimamura H, Kuwahara M, Ogawa W, Kasuga M, Sasaki S, Marumo F: Hyperosmolality activates Akt and regulates apoptosis in renal tubular cells. Kidney Int 2001;60:553–567. 114 Lee SD, Colla E, Sheen MR, Na KY, Kwon HM: Multiple domains of TonEBP cooperate to stimulate transcription in response to hypertonicity. J Biol Chem 2003;278:47571–47577. 115 Sheikh-Hamad D, Suki WN, Zhao W: Hypertonic induction of the cell adhesion molecule ␤1-integrin in MDCK cells. Am J Physiol 1997;273:C902–C908. 116 Sheikh-Hamad D, Yourker K, Truong LD, Nielsen S, Entman ML: Osmotically relevant membrane signaling complex: Association between HB-EGF, ␤1-integrin, and CD9 in mTAL. Am J Physiol 2000;279:C136–C146. 117 Moeckel GW, Zhang L, Chen X, Rossini M, Zent R, Pozzi A: Role of integrin ␣1␤1 in the regulation of renal medullary osmolyte concentration. Am J Physiol 2006;290:F223–F231. 118 Moeckel GW, Zhang L, Fogo AB, Hao C-M, Pozzi A, Breyer MD: COX2 activity promotes organic osmolyte accumulation and adaptation of renal medullary interstitial cells to hypertonic stress. J Biol Chem 2003;278:19352–19357. 119 Neuhofer W, Holzapfel K, Fraek ML, Ouyang N, Lutz J, Beck FX: Chronic COX-2 inhibition reduces medullary HSP70 expression and induces papillary apoptosis in dehydrated rats. Kidney Int 2004;65:431–441. 120 Mori T, Cowley AW Jr: Angiotensin II-NAD(P)H oxidase-stimulated superoxide modifies tubulovascular nitric oxide cross-talk in renal outer medulla. Hypertension 2003;42:588–593. 121 Mori T, Cowley AW Jr: Renal oxidative stress in medullary thick ascending limbs produced by elevated NaCl and glucose. Hypertension 2004;43:341–346. 122 Zhou X, Ferraris JD, Burg MB: Mitochondrial reactive oxygen species contribute to high NaClinduced activation of the transcription factor TonEBP/OREBP. Am J Physiol 2006;290:F1169–F1176. 123 Tian W, Cohen DM: Urea inhibits hypertonicity-inducible TonEBP expression and action. Am J Physiol 2001;280:F904–F912. 124 Lammers PE, Beck JA, Chu S, Kempson SA: Hypertonic upregulation of betaine transport in renal cells is blocked by proteasome inhibitor. Cell Biochem Funct 2005;23:315–324. 125 Massari S, Vanoni C, Longhi R, Rosa P, Pietrini G: Protein kinase C-mediated phosphorylation of the BGT1 epithelial ␥-aminobutyric acid transporter regulates its association with LIN7 PDZ protein. A post-translational mechanism regulating transporter surface density. J Biol Chem 2005;280:7388–7397. 126 Kempson SA, Beck JA, Lammers PE, Gens JS, Montrose MH: Membrane insertion of betaine/GABA transporter during hypertonic stress correlates with nuclear accumulation of TonEBP. Biochim Biophys Acta 2005;1712:71–80. 127 Beck FX, Dörge A, Rick R, Schramm M, Thurau K: The distribution of potassium, sodium and chloride across the apical membrane of renal tubular cells: effect of acute metabolic alkalosis. Pfluegers Arch 1988;411:259–267. 128 Pfaller W: Structure function correlation on rat kidney. Adv Anat Embryol Cell Biol 1982;70:1–106.

Franz-X. Beck, MD Department of Physiology, University of Munich Pettenkoferstrasse 12, DE–80336 Munich (Germany) Tel. ⫹49 89 2180 75534, Fax ⫹49 89 2180 75512 E-Mail [email protected]


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Osmosensing and Signaling in the Regulation of Liver Function Freimut Schliess, Dieter Häussinger Clinic for Gastroenterology, Hepatology and Infectiology, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany

Abstract Whereas the kidney has to cope with extremely high osmolarity during urinary concentration the liver is exposed to only moderate alterations in ambient osmolarity. More importantly, hormones, amino acids, and oxidative stress induce hepatocyte swelling or shrinkage within a narrow physiological range. It is meanwhile well-acknowledged that volume changes in different cell types trigger signal transduction events which contribute to the control and regulation of metabolism, transport and gene expression. For example, hepatocyte swelling induced by either hypoosmolarity, glutamine, ethanol, or insulin via activation of the p38-type mitogen-activated protein (MAP)-kinase mediates inhibition of autophagic proteolysis in perfused rat liver. On the other hand, dehydration of hepatocytes as triggered by hyperosmolarity produces insulin- and cytokine resistance and sensitizes cells to apoptotic stimuli. The volume-sensitivity of cell function relies on osmosensing structures which stimulate signal transduction in response to cell volume changes. This article focuses on recent developments regarding the understanding of osmosensing and signaling in the liver and its pathophysiological impact. Copyright © 2006 S. Karger AG, Basel

Cell Hydration and Cell Function

Moderate and well-tolerated cell volume changes are induced within minutes by changes of ambient osmolarity but also by hormones, amino acids, second messengers, and oxidative stress. Cell swelling due to a net uptake of inorganic and organic osmolytes is obligatory during the cell cycle, whereas cell shrinkage as a result of osmolyte release may play a role in apoptosis (apoptotic volume decrease [AVD]) [1–3]. Cell volume changes are registrated by osmosensing structures, thereby activating signals which contribute to control and regulation of metabolism and gene expression [4–7]. Anisoosmotically

exposed cells and tissues are a well-acknowledged experimental paradigm for the study of osmosensing and the osmosensitivity of signal transduction and metabolic pathways. Liver cell hydration-dependent pathways and functions have been reviewed extensively in the past [4, 7–13]. Changes in cell hydration within a narrow, physiological range markedly affect hepatic carbohydrate and protein metabolism as well as bile flow. A multitude of cellular signal transduction components, which are affected by anisoosmotic cell swelling or shrinkage has been characterized and it is meanwhile well-established that ‘osmosignaling’ integrates into the overall context of hormone- and nutrient-induced signal transduction. Hypoosmotic perfusion of rat liver rapidly activates mitogenactivated protein kinases of the p38- and the extracellular signal-activated protein kinase (Erk)-type, but not c-Jun-N-terminal kinases (JNK) [14, 15]. As shown recently, inhibition of autophagic proteolysis and regulatory volume decrease in response to hepatocyte swelling depends upon the p38 signal (fig. 1), whereas stimulation of bile acid secretion requires both, the Erk- and p38-dependent signaling [14, 15].

Osmosensing and Signaling in Hepatocytes

The investigation of osmosensing processes and structures considers among others macromolecular crowding, stretch-activated ion channels, the cytoskeleton, intracellular organelles and autocrine stimulation of signal transduction by release of mediators such as ATP [16–19]. Recent studies identified the integrin system as one major sensor of hepatocyte swelling [20–22]. Integrins are a family of extracellular matrix adhesion molecules, which play a part in ‘mechanotransduction’ and growth factor signaling [23–26]. In liver, the most important integrins are ␣1␤1, ␣5␤1 and ␣9␤1 [27–29]. In perfused rat liver the ␣5␤1 integrin is predominantly localized in the plasma membrane [22]. Hypoosmotic perfusion leads to appearance of the active conformation of the ␤1 subunit, which is largely absent under normoosmotic conditions [22]. The role of integrins as osmosensors is supported by the finding that integrin-inhibitory peptides exhibiting an RGD motif fully abolish hypoosmotic osmosignaling towards Src-type kinases, MAP-kinases and downstream metabolic events, including the stimulation of bile formation, proteolysis inhibition, and volume-regulatory K⫹-efflux [20, 21]. Inhibition of Src by PP-2 abolishes the hypoosmolarity-induced MAP-kinase activation, suggesting that the integrin/Src system localizes upstream of MAP-kinases [21]. In line with this not only PP-2, but also genistein inhibits the hypoosmotic Erk activation [30, 31]. It should be noted that RGD-peptides do not inhibit

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Hypoosmolarity Insulin

Glutamine

K

SIGNAL

2K

H

2K

Cell volume increase

Na

Na 3Na

PI 3-Kinase Na 2C K l a 3N

Integrins Src p38 MAPK Microtubules Proteolysis inhibition

Fig. 1. Swelling-dependent proteolysis inhibition critically depends on integrin-dependent cell volume sensing and signaling. Concentrative glutamine uptake leads to hepatocyte swelling, which is only in part counterregulated by a volume-regulatory K⫹ release. Insulin induces hepatocyte swelling by stimulating a PI 3-kinase-mediated net K⫹ uptake. Ambient hypoosmolarity triggers cell swelling due to osmotic water influx, which is in part counterregulated by a volumeregulatory loss of inorganic ions and organic osmolytes (not shown). Hepatocyte swelling induced by either glutamine, insulin or hypoosmolarity is registrated by the integrin system. In the case of hypoosmolarity and insulin it was shown that the ␤1 integrin subunit changes into the active conformation in a swelling-dependent manner. Integrin activation leads to a Src-mediated p38 MAPK activation which upstream (or at the side of) microtubule-dependent signals participates on inhibition of autophagic proteolysis. For references see text.

hypoosmotic hepatocyte swelling [21]. Thus, inhibition of osmosensing at the integrin level uncouples hepatocyte swelling from osmosignaling and its functional consequences. Whether integrins also sense hepatocyte shrinkage is currently unknown. However, hyperosmotic treatment of rat hepatocytes triggers a rapid generation of reactive oxygen species (ROS), followed by activation of the Src family kinase Yes, the epidermal growth factor receptor (EGFR) and JNK [32, 33]. This leads to an activation of the CD95 and sensitizes the hepatocyte towards CD95 ligand-induced apoptosis [32, 34]. The hyperosmotic ROS generation results from activation of NADPH oxidase isoforms, which were recently identified in rat hepatocytes [35]. Upstream events of hyperosmotic NADPH oxidase

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activation include a sphingomyelinase-catalyzed ceramide formation and ceramide-mediated activation of protein kinase C-␨, which phoshorylates p47phox, an essential subunit of NADPH oxidase [35]. The mechanism underlying the rapid sphingomyelinase activation in response to hyperosmotic hepatocyte shrinkage is currently under investigation.

Cell Hydration and Hepatic Insulin Signaling

Insulin induces hepatocyte swelling by osmotic water influx due to a PI 3kinase-dependent net uptake of K⫹ under participation of the Na⫹/K⫹/2Cl⫺ cotransporter NKCC1 [8, 36, 37]. Like hypoosmotic swelling [20, 21] cell swelling by insulin is registrated by the integrin system, leading to a Src-dependent activation of the p38 MAP-kinase and thereby inhibition of autophagic proteolysis [22]. Thus, integrin-dependent cell volume sensing and signaling integrates into the overall context of insulin signaling. Similar, sensing of glutamineinduced hepatocyte swelling by integrins feeds into Src-dependent p38 activation, which is critically required for autophagic proteolysis inhibition by this amino acid [21]. The resistance of suspended hepatocytes with regard to proteolysis inhibition by hypoosmolarity and insulin [38, 39] can be explained by the lack of integrin-dependent cell volume sensing due to the absence of integrin–matrix contacts [40]. Remarkably, integrins, Src, and p38 are not involved in mediation of swelling-independent proteolysis inhibition by phenylalanine [14, 21], excluding a non-specific interference of the used inhibitors with autophagic proteolysis. Different modes of integrin involvement in growth factor receptor signaling are well established [26]. Engagement of integrins may activate growth factor receptors [41] and/or increase efficacy of growth factor signaling by releasing costimulatory signals [42]. The studies reported here define a novel role of integrins in insulin signaling, namely sensing of cell swelling and initiation of osmosignaling cascades, which play an important part within the overall context of insulin signaling. Whereas insulin-induced cell swelling mediates insulin-dependent signals the dehydration of insulin target tissues contributes to insulin resistance. It is well known that endocrine and metabolic disturbances in severely diabetic patients are in part reversible already after adequate rehydration [43–45]. Vice versa experimental institution of systemic hyperosmolarity produces insulin resistance in healthy subjects [46]. A hyperosmotic insulin resistance was also observed in in vitro studies with isolated tissues and cell culture models [47–51]. At least some components of insulin signaling which are sensitive to hyperosmolarity are localized around the mammalian target of rapamycin


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(mTOR). For example, the hyperosmotic inhibition of insulin-induced glucose uptake, glycogen synthesis and lipogenesis in 3T3L1 adipocytes is related to a hyperosmotic inactivation of protein kinase B (PKB) [52] and a hyperosmotic inhibition of insulin-induced PKB activation [53]. Hyperosmotic suppression of PKB activation leads to the inability of insulin to activate the p70 ribosomal S6 protein kinase (p70S6-kinase) [53]. In monkey kidney CV1 cells hyperosmolarity decreases p70S6 kinase and phosphorylation of the eukaryotic initiation factor 4E (eIF4E) binding protein 4E-BP1 (4E-BP1) without affecting the PKB activity [54]. In rat H4IIE hepatoma cells hyperosmolarity interferes with the mTOR-dependent induction of the MAP-kinase phosphatase MKP-1 expression by insulin [55]. The hyperosmotic delay of MKP-1 induction was due to a suppression of the rapamycin-sensitive MKP-1 mRNA translation and was related to a sustained suppression of insulin-induced p70S6-kinase and 4EBP1 hyperphosphorylation [55]. In perfused rat liver hyperosmolarity prevents insulin-induced K⫹ uptake and cell swelling, thereby blunting the integrin-dependent p38 activation and the antiproteolytic insulin effect [36, 56]. Rapamycin does not prevent insulininduced K⫹ uptake, cell swelling and proteolysis inhibition [22]. For this reason, it seems unlikely that impairment of mTOR-dependent signaling accounts for hyperosmotic insulin resistance in this case. Recently, hyperosmolarity in H4IIE cells was shown to destabilize STAT3 by acceleration of its proteasomal degradation [57]. STAT3 depletion under dehydrating conditions is associated with impaired STAT3 Tyr705 phosphorylation in response to interleukin (IL)-22 [57]. Moreover, hyperosmolarity largely inhibits the activation by IL-6 or IL-22 of a reporter driven by a rat ␣2 macroglobulin promoter fragment containing the STAT3-binding tandem motiv [57]. Finally, hyperosmolarity potently antagonizes ␥-fibrinogen expression as induced by either IL-6 or IL-22 in HepG2 cells [57]. It is suggested that the acute phase response critically depends on the cellular hydration state. Whether STAT3 depletion plays a role in hyperosmotic insulin resistance remains to be proven. Interestingly, mice with liver-specific deficiency in STAT3 display insulin resistance associated with an increased hepatic expression of gluconeogenic genes [58].

Hepatocyte Hydration and Apoptosis

Cell shrinkage at the beginning of apoptosis (apoptotic volume decrease [AVD]) was considered as an early prerequisite for progression through the apoptotic program [59]. Signaling mechanisms upstream of AVD depend on cell type and stimulus under investigation and were already reviewed in more depth [12, 60]. As recently underlined [61], it is important to differentiate between


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CD95

Hyperosmotic dehydration

CD95

CD9 5

CD95 CD95

CD95

95

CD

CD

95

CD95 CD95

Glutamine Taurine Betaine L-JNKI SU6656 Protein kinase C-␨ pseudosubstrate

Fig. 2. Hyperosmotic trafficking of CD95 from intracellular compartments to the plasma membrane. Hyperosmotic sensitization of rat hepatocytes to CD95-induced apoptosis is related to the translocation of CD95 from intracellular pools to the plasma membrane. Hyperosmotic CD95 membrane trafficking is potently antagonized by glutamine, betaine and taurine, respectively. Also the JNK-inhibitory peptide L-JNKI, the Src-type kinase inhibitor SU6656 and the protein kinase C-␨ pseudosubstrate inhibitory peptide effectively antagonize hyperosmotic CD95 trafficking to the plasmamembrane. To what extent glutamine, taurine and betaine affect the hyperosmotic activation of JNKs and PKCs is currently not known. For references see text.

early shrinkage due to osmolyte release through specific transport proteins and a secondary cell volume reduction due to unspecific leakage of the cell membrane, plasma membrane fragmentation, and formation of apoptotic bodies. As illustrated recently [3], hyperosmotic shrinkage does not necessarily trigger apoptosis. For example, efficient RVI could protect cells from hyperosmotic apoptosis [62]. On the other hand, there is evidence that effective volume recovery is not always critical for protection from apoptosis. Other mechanisms which may protect from hyperosmotic apoptosis in some cells include activation of the PKB survival pathway [63], p53 activation [64], induction of the serum- and glucocorticoid-inducible kinase Sgk [65], expression of Hsp70 [66], cyclooxygenase-2 [67], or MKP-1 [68]. In cultured rat hepatocytes, moderate hyperosmolarity (405 mOsmol/l) activated the CD95 system [32, 34, 69]. Hyperosmolarity rapidly increased the production of ROS and trafficking of the CD95 from inside the hepatocyte to the plasma membrane [34] (fig. 2). Hyperosmotic CD95 trafficking to the plasma membrane involves a ROS-dependent tyrosine phosphorylation of the EGFR, association of CD95 with the tyrosine-phosphorylated EGFR, and phosphorylation of CD95 on Tyr232 and Tyr291 by the EGFR [32, 69]. Hyperosmotic CD95


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trafficking was antagonized by glutamine, taurine, and betaine [34] and by tyrosine nitration of the CD95 [70] (fig. 2). The latter interfers with CD95 tyrosine phosphorylation [70]. Also cAMP inhibited hyperosmotic CD95 activation in hepatocytes, which was explained by inhibition of EGFR phosphorylation and a protein kinase A (PKA)-dependent serine phosphorylation of CD95, which may act as an internalization signal [71]. Although the appearance of CD95 at the plasma membrane was associated with death inducing signaling complex (DISC) formation and activation of caspases 3 and 8, moderate hyperosmolarity was not sufficient to induce hepatocyte apoptosis [34], suggesting that apoptotic signals are counterbalanced by yet unknown survival signals. Like hyperosmolarity, CD95 ligand via generation of ROS in hepatocytes induced tyrosine phosphorylation of the EGFR, CD95/EGFR association, CD95 tyrosine phosphorylation, trafficking of the CD95 to the plasma membrane surface, and DISC formation, leading to the execution of apoptosis in this case [32, 69]. Similar to hyperosmotic CD95 membrane trafficking, CD95 ligand-induced membrane trafficking of CD95 and hepatocyte apoptosis was inhibited by CD95 tyrosine nitration [70]. Although ineffective to induce apoptosis by itself, hyperosmolarity sensitized the hepatocytes towards CD95 ligand-induced apoptosis [34], indicating a synergistic interplay between signals triggered by hyperosmotic shrinkage and CD95 ligand, respectively. A pro-apoptotic action of hyperosmolarity was also identified in hepatoma cells [68]. In H4IIE cells hyperosmolarity enabled the proteasome inhibitor MG-132 to induce poly-ADP-ribose-polymerase cleavage, which was sensitive to inhibition of p38 and JNK but not Erk. Further, caspase 3 activation by MG132 was increased by hyperosmolarity. Hyperosmolarity markedly augmented MG-132-inducd MKP-1 expression via increased induction of activator protein-1 (AP-1) DNA-binding activity and simultaneous stabilization of MKP-1 [68]. Increased MKP-1 expression by MG-132 under hyperosmotic conditions was associated with termination of MAP-kinase signaling [68]. MKP-1 was found to prevent apoptotic MAP-kinase signaling as induced, e.g. by TNF-␣ in rat mesangial cells [72] or by proteasome inhibitors in A1N4-myc human mammary epithelial and BT-474 breast carcinoma cells [73]. It was suggested that MKP-1 antagonizes proapoptotic signaling within a feed back-inhibitory loop which may be part of an adaptation mechanism allowing survival of H4IIE cells treated with MG-132 under hyperosmotic conditions [68]. As matters stand data support the view that AVD, if not even producing de novo death signals, at least can amplify apoptotic signals released by different apoptotic stimuli. Thus, AVD may further derange the balance between survival and death signals, thereby promoting execution of the apoptotic program. As reviewed recently [3], cell swelling triggers cell cycle progression. In line with this hypoosmotic swelling in multiple cell types was sufficient to activate


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the MAP-kinases Erk-1/Erk-2 and the PI 3-kinase [7, 11], which play a major role in mitogenic signaling. Indeed, hypoosmotic treatment of HepG2 cells potentiated proliferation of the cells, which depended on a PI 3-kinasemeditated AP1 activation [74]. A role of cell swelling for proliferation is also evident in liver regeneration. Following partial hepatectomy hepatocyte swelling due to substrate accumulation via system A was observed in vivo and inhibition of cell swelling antagonized liver regeneration [75]. Progression through the cell cycle depends on cell size [76] and it seems well conceivable that an acute cell size increase above a critical threshold by isoosmotic water retention represents an important trigger of proliferation. Accordingly, cell volume was increased in 3T3 cells by expression of oncogenic Ha-ras [77] and cell hyperhydration has been linked to tumor growth [78]. Cell swelling may not only play a role in cell cycle progression, but in addition can protect cells from apoptosis. Hypoosmotic swelling inhibits TGF␤-induced apoptosis in H4IIE rat hepatoma cells independently from the hypoosmotic NF-␬B activation observed in these cells [79]. A brief hypoosmotic treatment protected cardiomyocytes from doxorubicin-induced AVD and apoptosis, and this was observed even if doxorubicin was applicated 60 min after the hypoosmotic pulse [80]. It was shown that an elevation of intracellular cAMP accounts for this protection from doxorubicin [80]. In view of the above mentioned one is tempted to speculate that a hypoosmotic CD95 Ser phosphorylation downstream of PKA activation explains this effect.

Conclusion

It is meanwhile well-acknowledged that fluctuations of intracellular hydration release signals which are of (patho)physiological relevance. Recent investigations increased knowledge about cell volume-sensing mechanisms and their impact on signal transduction and subsequent alterations of metabolism and gene expression as induced by anisoosmolarity and under isoosmotic conditions by hormones and substrates, respectively. The understanding of how cell swelling integrates into the cell cycle machinery and how cell shrinkage interferes with insulin and cytokine signaling and sensitizes cells to apoptotic stimuli requires further scientific effort. Cell hydration may markedly affect the action of drugs. For example, cell hydration changes may switch the outcome of proteasome inhibitors from a non-toxic or even protective one to injury and apoptosis. From this it would be expected that the therapeutic efficacy and side effects of drugs critically depend on cell hydration. Although routinely monitoring of cell hydration in patients would provide valuable information in clinical medicine this is currently limited by methodological difficulties.


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Acknowledgements Our own studies reported herein were supported by Deutsche Forschungsgemeinschaft through Sonderforschungsbereich 575 ‘Experimental Hepatology’ (Düsseldorf).

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Prof. Dr. Dieter Häussinger Universitätsklinikum Düsseldorf, Klinik für Gastroenterologie Hepatologie und Infektiologie Moorenstrasse 5 DE–40225 Düsseldorf (Germany) Tel. ⫹49 211 811 7569, Fax ⫹49 211 811 8838, E-Mail [email protected]


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Cell Volume and Peptide Hormone Secretion Vladimír Štrbák Institute of Experimental Endocrinology, Centre of Excellence Acknowledged by European Commission, Slovak Academy of Sciences, Institute of Normal and Pathological Physiology, Slovak Medical University, Bratislava, Slovakia

Abstract In general way cell swelling evokes and shrinking inhibits exocytosis of proteins and peptides stored in secretory vesicles from various types of cells. Dynamics of this type of hormone secretion is indistinguishable from that induced by specific secretagogue. Peculiarities of swelling-induced secretion indicate an involvement of the unique signaling pathway. Hyposmotic stimulation of insulin secretion is independent from the extra- and intracellular Ca2⫹, does not involve other intracellular mediators of glucose stimulation, and could not be inhibited by noradrenaline. Swelling-induced peptide secretion is not essential for cell volume control. Hyposmotic stimulation is a useful research tool when natural or pharmacological secretagogue is unknown: Thyrotropin releasing hormone release from the heart slices, pancreatic islets and various brain structures was characterized by the stimulation by hypotonic medium. Swelling-induced exocytosis possesses limited selectivity; cells involved in water and salt regulation retain their specific response to osmotic stimuli; hypotonic medium evokes thyrotropin releasing hormone but not oxytocin (OT) release from hypothalamic paraventricular nucleus. Specific response (release after hyperosmotic stimulation) of intranuclear OT secretion in the paraventricular nucleus and the supraoptic nucleus could be obviated by GdCl3 and at these conditions OT release to swelling-inducing stimuli emerged. Swelling-induced hormone secretion can have pathophysiological implications. For example, a shift to anaerobic glycolysis and production of metabolites occurring in ischemia results in the increased intracellular osmolarity and cell swelling. Peptides and proteins released after swelling could play an important role in the pathophysiology of ischemia and be mediators of local or remote preconditioning when factors released at the place of ischemia have protective effect against ischemia-reperfusion injury. Moreover, the ischemic disruption of the osmotic receptors could result in a syndrome of inappropriate hormone secretion. Copyright © 2006 S. Karger AG, Basel

Cell volume regulation has received increasing attention as stimulus for a variety of intracellular phenomena [1–6] and is considered to be integrated into a signal transduction network regulating cell function [1–3]. Most of the cell reactions to volume changes are aiming at the renewal of initial volume status [4]. The defence against excess cell swelling is accomplished by a reduction of the intracellular osmolarity by release of organic- or inorganic osmolytes from the cell or by synthesis of osmotically less active macromolecules from their specific subunits. Swelling-induced peptide secretion represents important cellular reaction when material stored in secretory vesicles is expulsed as a secretory burst of peptide hormones or enzymes from various types of cells including endocrine, neurons, leukocytes or those of the exocrine pancreas (for review see [6]). Cell shrinking has most often suppressing effect of this type of secretion.

Cell Swelling-Induced Peptide Secretion and Cell Volume Regulation

Exocytosis of intravesicular material may help a cell to meet a relative extracellular hyposmotic challenge by expanding the plasmalemma through fusion with vesicular membrane. In contrast to electrolytes, export of proteins and peptides out of the cell does not change an osmotic balance between extraand intra-cellular space insofar as to have a significant effect on the cell volume regulation. Regulatory volume decrease (RVD) dependent on the transport of osmolytes outside of the swollen cell and swelling-induced peptide secretion could be affected in an opposite way by calcium; when medium Ca2⫹ is depleted or Ca2⫹ influx prevented by Ca2⫹ channel blockers, the amplitude of cell swelling is greater and the RVD slower [6, 7], in contrast, extracellular Ca2⫹ is not required and, in fact, negatively modulates osmotically-induced secretion in many cells [6–9]. The apparent negative modulation by Ca2⫹ influx of osmotically induced secretion in normal pituitary cells [7] may simply be due to an augmented signal intensity resulting from the greater magnitude and duration of cell swelling from an osmotic stimulus when Ca2⫹ influx is prevented than when it occurs. Another example of the opposite changes of the peptide secretion and RVD is blocking of Na⫹ and K⫹ channels. K⫹ efflux appears to play a critical role in the RVD after cell swelling, however, blocking Na⫹– K⫹-dependent ATPase with ouabain, blocking Na⫹ channels with tetrodotoxin or blocking K⫹ channels with TEA have no effect on hyposmolarity-induced hormone secretion in pituitary cells [6, 7]. It is therefore concluded that swelling-induced peptide secretion is not essential for cell volume control.

Swelling-Induced Secretion

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Peptides and Proteins Reported to Be Released by Cell Swelling

The release of almost all protein and peptide hormones tested in vitro was stimulated in a concentration-related manner by medium hyposmolarity, or isosmolar medium containing permeant molecules from various tissues, including pituitary (gonadotropins LH and FSH, adrenocorticotropin, melanocytestimulating hormone, thyrotropin, prolactin, beta endorphin [7–15]), pancreas (insulin [9, 16, 17], thyrotropin releasing hormone (TRH) [18], and glucagon [16]), hypothalamic neurons (gonadotropin-releasing hormone [19], TRH [10, 18, 20, 21]), cardiomyocytes (atriopeptin [22]) and juxtaglomerular kidney cells (renin [23]). Swelling can induce secretion in different parts of neurons – similar release of the TRH was evoked from the hypothalamic paraventricular nucleus (PVN) (mostly perikarya) and the median eminence (exclusively axon terminals) [20, 24]. Cell swelling induced exocytosis is not restricted to endocrine cells or hormones. Medium hyposmolarity also induces secretion of exocrine pancreatic enzymes [16] and myeloperoxidase from human polymorphonuclear leukocytes [25].

Specificity – Selectivity

The exocytosis of material stored in intracellular secretory vesicles induced by cell swelling is a broad phenomenon affecting many hormones and enzymes after exposure of the cells to relative hyposmolarity or treatment with permeant agents (for review see [6]). However, cell swelling-induced exocytosis possesses limited selectivity; cells specifically engaged in water and salt regulation retain their specific response to osmotic stimuli [10]. It is well established that direct stimulation of the hypothalamic PVN and supraoptic nuclei (SON) with hyperosmotic saline via microdialysis increases vasopressin and oxytocin (OT) levels in extracellular fluid [26–29] by a mechanism involving axons, dendrites and cell bodies [30]. Levels of these hormones also increase in plasma as a result of their release from axon terminals in the posterior pituitary. These neuropeptides are released within nuclei and into the blood in a Ca2⫹ dependent manner [26, 31, 32]. In our studies [10, 41], we have been using tissue explants of hypothalamic PVN and posterior pituitary for simultaneous TRH and OT examination. Under our experimental conditions, both neuropeptides were secreted into medium from the same tissue explants, though most likely from different neurons: parvocellular and magnocellular respectively. As expected [20, 21, 24, 33], TRH secretion was stimulated by cell swelling, while OT, neurohormone engaged in water and salt regulation, was not released after

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Fig. 1. Effect of 50 and 100 ␮M GdCl3 on cell swelling-induced (30% hyposmotic medium) TRH (upper panels) in PVN (a) and NH (b) and OT (lower panels, c, d) secretion from nucleus. Tissue explants were incubated for two subsequent 30-min periods in medium containing alternately basal (B) and hyposmotic (Hypo) medium. In the presence of GdCl3 also OT was released by hypotonicity from the PVN but not NH. Data are shown as mean ⫾ SE, n ⫽ 5 (*p ⬍ 0.05, ***p ⬍ 0.001). Adapted from reference [41]. NH ⫽ Neurohypophysis; OT ⫽ oxytocin; PVN ⫽ paraventricular nucleus.

cell swelling-inducing stimuli [10]. This was in good agreement with observations that ethanol inhibits the release of OT from the posterior pituitary induced by high K⫹ [34] or suckling [35] and that hypotonic stimuli hyperpolarize magnocellular neurosecretory cells via stretch-inactivated channels [36]. Excitatory effects of some peptides in these cells are also due to stimulation of stretchinactivated channels [37]. Further study (fig. 1) was performed to ascertain whether the swelling-induced OT secretion could be unmasked by the inhibition of the specific osmotic response using Ca2⫹ free medium and GdCl3, an


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inhibitor of stretch sensitive channels [38, 39] and a potent blocker of voltagesensitive Ca2⫹ channels [40]. We have shown [41] that an intranuclear OT secretion to hyposmotic stimulation within the PVN and the SON could be unmasked by the inhibiting specific response by 50 or 100 ␮M GdCl3. At these conditions secretory response to swelling-inducing stimuli emerged even in these structures. Apparently, swelling-induced exocytosis represents basic ancient mechanism of the reaction to hyposmolarity in most cells.

Mechanism of Signal Transduction

Secretion induced by extracellular hyposmolarity is due to the relative reduction of extracellular compared to intracellular osmolarity and not to dilution of any essential extracellular elements. If medium dilution is made with aqueous 5% mannitol [12] or by biologically inactive L-glucose [20] to maintain isosmolarity on both sides of the plasma membrane, no secretion is induced. If the medium is made hyperosmolar, secretion is not stimulated and is often suppressed [10, 21, 25, 42, 43]. However, on return of the cells to an isosmolar medium, there is a prompt dose-related burst of ‘off response’ secretion whose magnitude is proportional to that of the preceding hyperosmolarity [11]. This indicates that it is the relative difference in intracellular vs. extracellular osmolar concentration which causes exocytosis rather than the absolute osmolarity of either compartment. The dynamics of secretion induced by cell swelling closely resembles that induced by specific secretagogue. Perfusion of pituitary cells with 10 nM TRH (prolactin natural secretagogue), as well as cell swelling induced by a hypotonic solution (medium dilution with 30% H2O) or by depolarization (30 mM KCl), stimulates an immediate dose-related high-amplitude prolactin secretory burst, reaching a peak at 1–2 min followed by a decline to a low plateau within 5–10 min during a continuous exposure to the same stimulus. Repeated stimuli with 30-s long interstimulus intervals produce the same secretory response as continuous stimulation. For all three types of the stimuli, the secretory response to continuous exposure and refractory periods to repeated stimulation (⬍1 min) were essentially identical. An identical high-amplitude secretory burst was induced by exposure to TRH for times varying from 6 to 600 s. In contrast, for the conditions of 30% H2O and high KCl, the secretory amplitude was proportional to the exposure time between 6 and 60 s. While the TRH response was triggered by rapid specific receptor binding, a very short pulse would not have time to produce a sufficient transmembrane osmotic gradient or K⫹ difference. It is concluded that a hyposmotic medium does not trigger peptide release by the specific receptor-ligand binding [6, 44].

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Any changes in cellular physiology caused specifically by cell swelling would be initiated by distortion of the plasmalemma. Pressure-sensitive mechanoreceptors have been identified in a variety of cell types [45]. The trivalent lanthanide Gd3⫹ blocks mechanogated channels [38] and has diverse, often non-specific effects (reviewed by [40]). Gadolinium in the culture medium does not negatively modify ethanol-induced TRH release from isolated pancreatic islets, hypothalamic PVN or posterior pituitaries, or prolactin release from acutely dispersed anterior pituitary cells induced by hypotonicity [10]. These data indicate that signal transduction leading to exocytosis after cell swelling does not involve GdCl3 sensitive mechanogated channels [10]. Several studies showed that neither Na⫹ nor K⫹ channels play an essential role in hormone secretion induced by cell swelling [6, 10]. It was also demonstrated that signal transduction leading to exocytosis after cell swelling does not involve indomethacin and NDGA sensitive mediators including prostaglandins and leukotriens [6, 10]. Microtubules and microfilaments are important mechanical constituents of plasma membrane motion and migration of vesicles within the cell. However, colchicine or cytochalasin, which can respectively disrupt microtubule and microfilament function, have no effect on hyposmolarity-induced secretion [6, 7, 16], suggesting that these subcellular organelles are not involved. Role of Ca2⫹ The most striking and unusual feature of cell swelling-induced secretion is that it stimulates regulated secretion independent of both extracellular and intracellular Ca2⫹ concentration [6–9, 15–18] in contrast to most types of regulated secretion. Comparison of the Glucose- and Cell Swelling-Induced Insulin Secretion A comparison of the glucose- and swelling-induced insulin secretion showed that they exploit separate signal transduction pathways [9]. In contrast to glucose, hyposmotic stimulation is independent of both the extracellular and intracellular Ca2⫹, does not involve PKC activation, and could not be inhibited by noradrenaline indicating a novel signaling pathway for swelling-induced insulin secretion [9]. Stimulation by 20 mmol glucose or 30% hyposmolarity induced similar increase of insulin secretion and the hypotonic medium had an additive effect on the glucose-induced insulin secretion [9]. Resistance to Physiological Inhibitors Noradrenaline is physiological inhibitor of glucose-induced insulin secretion [46]. Alternative signaling pathways leading to insulin secretion independently from Ca2⫹ induced by black-widow spider neurotoxin ␣-latrotoxin or other types of Ca2⫹ independent stimulation of insulin secretion are inhibited by catecholamines [47–50]. In contrast, we have demonstrated that noradrenaline does


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not inhibit cell swelling-induced insulin secretion either in the presence or the absence of Ca2⫹ in the medium [9]. When noradrenaline was applied during combined stimulation by glucose and hypotonic medium, its inhibitory effect was partial, only the secretion induced by glucose but not that induced by the cell swelling was inhibited [9]. This indicates that different signaling pathways were used in parallel for glucose and hypotonicity during combined stimulation in Ca2⫹ containing medium and that swelling-induced insulin secretory pathway either bypasses steps affected by noradrenalin or acts at a more distal step of the cascade. TRH secretion from heart slices has attributes of regulated secretion – depending on the stimulus it could be either stimulated or inhibited [44]. Angiotensin II inhibits TRH secretion from heart tissue by a mechanism involving AT1 receptors [44]. Swelling-induced TRH secretion overrides inhibitory effect of angiotensin II [44]. Resistance to endogenous inhibitors may be common feature of the swellinginduced secretion. This fact could have serious pathophysiological implications. Biophysical Effect A failure to identify transduction pathway and its resistance to physiological inhibitors suggests that signaling of cell swelling-induced exocytosis bypasses conventional transduction steps and should be effective at the distal end of the cascade. It has been shown that secretory vesicle swelling is critical for exocytosis [51–53]. Stretching of vesicular and plasma membranes in the region of contact results in exposing areas of hydrophobic acyl chains leading to subsequent merging and fusion. Fusion rates are orders of magnitude higher if an osmotic gradient is applied [51]. The externalization of hormones or transmitters upon exocytosis of vesicles is augmented by secretion of water from the vesicle membrane through the widened fusion pore [53]. Considering these data, we hypothesized that cell swelling triggered exocytosis is a result of a direct biophysical effect of the osmotic gradient on secretory vesicles [54]. However, recently we discovered that INS-1E rat tumor beta cells [55] are unable to release insulin in response to cell swelling despite a good response to glucose challenge. It is hard to believe that secretory vesicles responding to glucose stimulation do not release insulin to volume changes if the vesicle swelling alone was the essential prerequisite of hypotonicity induced exocytosis.

Swelling as a Research Tool when Natural or Pharmacological Secretagogue Is Unknown

The cell swelling-induced protein/peptide hormone secretion as unspecific stimulator with only limited selectivity [10] appears to be a suitable tool to

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characterize peptide secretion in a novel location especially when natural secretagogue is unknown. Dynamics of the secretory response to cell swelling is similar to that induced by other specific and non-specific secretagogues. We have been using this approach to stimulate and characterize TRH secretion by heart tissue [44], endocrine pancreas [18, 21] and various brain structures [20, 21, 24, 33, 41, 43].

The Physiological Significance

The physiological significance of secretion induced by cell swelling remains to be clarified. Minor osmotic changes undoubtedly occur frequently as a result of local or general homeostatic or metabolic processes. More important changes take place at pathophysiological conditions. When tissue becomes ischemic there is a shift to anaerobic glycolysis and production of metabolites which can result in an increase of intracellular osmolarity by 70 and 130 mOsmol/l after 15 and 60 min, respectively, thus increasing transmembrane osmotic pressure differences and producing cell swelling [56]. Peptides and proteins released after swelling could play an important role in the pathophysiology of ischemia and could be mediators of local or remote preconditioning when factors released from the ischemic tissue have protective effect against ischemia-reperfusion injury [57]. Disruption of mechanosensitive gating in magnocellular neurosecretory cells could result in an inadequate secretory response (e.g. stimulation instead of inhibition and vice versa) of hormones engaged in water and salt metabolism regulation. This kind of phenomenon could play a role in the syndrome of inappropriate secretion of antidiuretic hormone [58] well known by clinicians and could be induced also experimentally by brain infarction [59].

Acknowledgement This work was supported by project nos. 23191/23 and 2/6158/26 of the Grant Agency VEGA.

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Vladimír Štrbák Institute of Experimental Endocrinology, Slovak Academy of Sciences Vlárska 3 SK–833 06 Bratislava (Slovakia) Tel. ⫹421 2 54774101, Fax ⫹421 2 54774809, E-Mail [email protected]

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Volume Changes in Neurons: Hyperexcitability and Neuronal Death Herminia Pasantes-Morales, Karina Tuz Department of Biophysics, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico

Abstract Hyponatremia propitiates and increases susceptibility to seizure episodes. In vitro, hyposmolarity induces hyperexcitability and epileptiform activity and increases the amplitude of excitatory postsynaptic potentials. Synaptic (increased glutamate vesicular release) and non-synaptic (swelling-induced extracellular space shrinkage and ephaptic interactions) might be responsible for the hyposmolarity effects on brain excitability. Neuronal volume constancy in hyponatremia is preserved by the isovolumetric regulation, relying importantly on organic osmolytes. Changes in cell volume are closely linked to neuronal death: swelling characterizes necrotic death as in acute ischemic episodes or brain trauma, whereas volume decrease is typical of apoptotic death. Swelling in necrotic death results from the intracellular Na increase followed by Cl and water influx. Na accumulation is due initially to the Na/K ATPase dysfunction and subsequently from the Na influx through the overactivated ionotropic glutamate receptors. A second wave of swelling generates by excitotoxic derived formation of reactive oxygen species, membrane lipoperoxidation and further ion overload. Excessive swelling contributes to membrane rupture and release of cell debris, propagating the damage to adjacent cells. Apoptotic death is characterized by cell volume decrease termed apoptotic volume decrease, which in neurons seems to occur by mechanisms remarkably similar to those operating in the hyposmotic swelling-activated volume regulatory decrease, i.e. channel-mediated efflux of K and Cl. A variety of K channels and the volume-regulated anion channel participate in apoptotic volume decrease. K has a protagonic role as an early element in neuronal apoptosis since a delayed rectifier K current IKDR is enhanced by apoptosis prior to the caspase activation, increased extracellular K and IKDR blockers attenuate apoptosis and intracellular K loss through ionophores induces apoptosis. Volume-regulated anion channel participates as well in the Cl efflux although its role and hierarchy in the apoptotic program are not well defined. Efflux of organic osmolytes, such as taurine participate as well in apoptotic volume decrease. Copyright © 2006 S. Karger AG, Basel

Swelling in hyposmotic or isosmotic conditions is closely linked to ion movements. Regulatory volume decrease (RVD) activated by hyposmotic swelling relies on the outflow of K, Cl and small organic osmolytes. Swelling in isosmotic conditions occurs by ion redistribution, modifying the ionic homeostasis in the cell. All these changes are of crucial importance in brain, because the extracellular/intracellular ionic equilibrium determines the resting potential and the discharge pattern of neurons, as excitatory and inhibitory synaptic events are driven by ionic gradients. Besides, some of the organic osmolytes released in connection with volume recovery play in the brain a prominent role as synaptic transmitters. Moreover, changes in cell volume, swelling or shrinkage, may be critical signals in directing the cell death type to necrosis or apoptosis.

Brain Cell Volume and Hyperexcitability

The influence of changes in cell volume on brain excitability was suggested by the seizure occurrence during acute hyponatremia, and the increased susceptibility to seizures in chronic hyponatremia or psychotic polydipsia. That the hyperexcitable condition was due to cell swelling and not to changes in the Na concentration in plasma, was demonstrated by seizure attenuation after infusion of hypertonic solutions or by water restriction. At the cellular level, also hyposmolarity induces hyperexcitability and increases evoked epileptiform activity as shown in CA3 neurons of hippocampal slices and in neocortical pyramidal neurons [Rosen and Andrew, 1990; Saly and Andrew, 1993]. Hyposmolarity also affects excitatory synaptic transmission, increasing the amplitude of excitatory postsynaptic potentials. These effects may result from events connected with cell swelling and volume recovery, occurring in both neurons and astrocytes. Cell swelling may increase brain excitability by one or both of these factors: (i) swelling-induced release of excitatory neurotransmitters, notably glutamate, (ii) reduction in the size of the extracellular space, propitiating ephaptic interactions and restraining the diffusion of neurotransmitters and depolarizing agents [Schwartzkroin et al., 1998]. The first possibility, i.e. a swelling-evoked release of excitatory neurotransmitters is documented in a variety of brain preparations [de la Paz et al., 2002; Franco et al., 2001; Kimelberg et al., 1990; Saransaari and Oja, 1999]. Interestingly, the glutamate efflux in hippocampal slices has features which deviate from the release of typical organic osmolytes represented by taurine. The efflux of taurine inactivates slowly, is sensitive to the Cl channel blockers NPPB and niflumic acid and is markedly reduced by tyrosine kinase blockers. Glutamate efflux, in contrast, is rapidly inactivated and is insensitive to all the agents which affected taurine, being only decreased by DIDS [Franco et al., 2001].

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Hyposmotic efflux of taurine and glutamate is also observed in rat cortical isolated nerve endings (synaptosomes) [Tuz et al., 2004] (fig. 1a, b). In the case of glutamate, the release results from a chain of events triggered by hyposmolarity and ultimately leading to an increase in synaptic vesicle discharge. The initial event is a Na-dependent depolarization, sensitive to La3, Gd3 and ruthenium red, mediated possibly by TRP channels. A subtype of this family of channels, the TRPV4, is almost exclusively present in the nervous tissue, it is osmotically- and mechano-sensitive and is blocked by Gd3, La3 and ruthenium red [Gunthorpe et al., 2002]. Depolarization is followed by a Na-dependent, La3 sensitive, PKC-modulated [Ca2]i rise, originated mostly from internal stores. The mitochondrial Ca2 pool released by activation of the mitochondrial Na/Ca2 exchanger, as result of the increase in cytosolic Na has an important contribution. [Ca2]i rise evoked by hyposmolarity has been reported in cerebellar granule neurons and in hippocampal pyramidal neurons, with contributions of extracellular Ca2 as well as of Ca2 released from internal sources [Borgdorff et al., 2000; Pasantes-Morales and Morales-Mulia, 2000]. Depolarization and [Ca2]i rise evoked by hyposmolarity leads to enhanced exocytosis in the nerve endings, which is Ca2-dependent and prevented by tetanus toxin (TeTX) (fig. 1). This vesicular release is the mechanism responsible for a fraction of the glutamate release from nerve endings, which accordingly, is La3- and Ca2-dependent, PKC-modulated and blocked by TeTX. Another fraction of this release occurs via the reversal operation of the carrier and is consequently suppressed by the transporter blockers [Tuz et al., 2004]. Hyposmolarity also increases the efflux of taurine and at a lesser extent, that of GABA (fig. 1b). Noteworthy, the hyposmotic release of taurine does not occur via the Ca2-dependent vesicular release but it has the features of the volumeactivated diffusion pathway, characteristic of the organic osmolyte outflow found in most cell types, i.e. reduced by Cl channel blockers and modulated by tyrosine kinase phosphorylation [Tuz et al., 2004]. The hyposmolarity-induced vesicular synaptic release of glutamate from nerve endings may explain in part, the increase in amplitude of spontaneous or evoked excitatory postsynaptic potentials found in CA3 cells in the hippocampus and in neocortical pyramidal neurons. Since hyposmolarity has no effect on the neuronal intrinsic properties such as resting membrane potential, cell input resistance, action potential threshold and duration, its effect on the excitatory postsynaptic potentials must reflect a synaptic phenomenon [Baraban and Schwartzkroin, 1998; Rosen and Andrew, 1990; Saly and Andrew, 1993]. The effects evoked by hyposmolarity resulting ultimately in enhanced exocytosis predict a more general effect of this condition on neurotransmitter release, even when the transmitter is unrelated to an osmolyte function. This was confirmed by data showing a hyposmolarity-evoked norepinephrine

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Hyposmolarity Na-dependent, La3/Gd3 sensitive depolarization Na TRPV4

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Fig. 1. Hyposmolarity and neurotransmitter release. a Events sequence of the hyposmolarity-induced release of neurotransmitters from isolated nerve endings (synaptosomes). The initial event is a Na-dependent, La3/Gd3 sensitive depolarization, followed by [Ca2]i increase from external and internal sources and activation of a PKC-dependent exocytotic release of neurotransmitters. b Time course of neurotransmitter release evoked by hyposmolarity from isolated nerve endings. Synaptosomes were prepared and loaded with 3 H-glutamate (•), 3H-GABA (䊏), 3H-norepinephrine (䉱) or 3H-taurine (䉲), washed and superfused (1 ml/min) during 3 min with isosmotic medium to obtain a constant basal efflux. At the arrow, the medium was replaced by 20% hyposmotic medium, and superfusion continued for 7 min. Results (radioactivity released per min) are expressed as percentage of the total radioactivity incorporated. SE is represented as vertical bars when they exceed the size of symbols. c Effect of EGTA-AM and TeTX on hyposmotic neurotransmitter release from synaptosomes obtained and treated as in (b). Bars represent the radioactivity released (%) at the peak release fractions (5–9). Empty bars represent net release (hyposmotic minus isosmotic release) as 100%. Dashed bars correspond to percentage decrease by EGTA-AM or TeTX.


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release from isolated nerve endings, which is fully dependent on depolarization, [Ca2]i rise and exocytosis [Tuz and Pasantes-Morales, 2005] (fig. 1c). In comparison with the norepinephrine release evoked by depolarizing concentration of K, the hyposmotic norepinephrine efflux is more dependent on intracellular than on extracellular sources of [Ca2]i rise and is insensitive to blockers of the voltage-dependent L-type Ca2 channels (fig. 1). Altogether these results confirm that despite the differences in the mechanism to induce depolarization and the source of [Ca2]i increase, the synaptic events elicited by hyposmolarity result at the end, on a TeTX-sensitive mechanism not different from the vesicular exocytosis characteristic of the classical release of neurotransmitters. This opens the intriguing possibility of a modulatory effect of changes in cell volume in synaptic transmission. Swelling may affect neuronal excitability also by non-synaptic mechanisms. Swelling of neurons, but particularly of astrocytes, leads to narrowing of the extracellular space. Astrocyte swelling occurs either in hyponatremia or during clearance of the high extracellular K resulting from intense neuronal activity. As cells expand, there is a reduction in the size of the extracellular space, enhancing ephaptic interactions. This is likely in the origin of the non-synaptic mechanisms of the hypersynchronous behavior of cortical neurons typical of seizures and of the synchronization of epileptiform activity. The influence of cell swelling and the mirrored extracellular space decrease may be of particular importance in brain regions such as the hippocampus where the tight packing of the cell somata restricts the size of the extracellular space. In line with this notion, hyperosmotic solutions block the high K-induced epileptiform activity [Dudek et al., 1998] and furosemide suppresses the neuronal synchronized activity generated by episodes of electrically evoked afterdischarges by reducing astrocyte swelling and extracellular space shrinkage [Hochman et al., 1995].

Neuronal Protection in Hyponatremia

Exposure of cultured neurons, astrocytes or brain slices to hyposmotic solutions has been a current experimental device to simulate hyponatremia in vivo. The typical response characterized by rapid swelling followed by return towards the original volume, i.e. the RVD, observed when cells are exposed to Synaptosomes were 15 min preincubated with 50 M EGTA-AM in Ca2 free medium containing 0.1 mM EGTA 10 mM MgCl2, or during 90 min in isosmotic medium in the presence of 50 nM TeTX. Other experimental details in Tuz et al. [2004, 2005]. Glu Glutamate, NE norepinephrine, tau taurine.


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sudden and large osmolarity reductions, has been useful to magnify the process and facilitate the identification of signals and mechanisms responsible for this adaptive cell response. However, changes of this magnitude probably never occur in brain under physiological conditions nor even in pathological situations. When the external osmolarity is gradually reduced (2.2 mOsm/l) some cells do not swell and others swell less than after an abrupt decrease in osmolarity. This is what was named isovolumetric regulation by Lohr and Grantham [1986] who first showed this phenomenon in renal proximal tubule cells. Exposed to gradual changes in osmolarity, renal cells were able to maintain a constant volume within a broad range of osmolarities. The term ‘isovolumetric regulation’ reflects the active nature of this process, as the unchanged volume is not due to the absence of swelling, but to a continuous volume adjustment accomplished by the extrusion of intracellular osmolytes. This paradigm has been applied to other cell types and marked differences have been found with respect to the efficiency of the process. Interestingly, these differences appear to be related to the contribution of amino acids. Cerebellar granule neurons, as renal cells, respond to the gradual decrease in external osmolarity by a constancy in cell volume even if osmolarity reductions reached up to 50% [Tuz et al., 2001] (fig. 2a). Glioma C6 cells and cultured astrocytes exhibit some swelling, but significantly lower that if an osmotic stimulus of the same magnitude is suddenly imposed [Ordaz et al., 2004a; 2004b] (fig. 2a). Finally, trout erythrocytes respond with similar swelling to gradual or sudden exposure to hyposmotic solutions [Godart et al., 1999]. The osmolytes involved in volume corrective mechanisms during isovolumetric regulation are the same as in RVD, i.e. K, Cl and organic molecules. The activation threshold of the osmolyte fluxes appears related to the efficiency of the different cell types to counteract the changes in external osmolarity. A correlation is observed between the efflux threshold of taurine and glutamate and the extent of swelling during gradual osmolarity changes. In cerebellar granule neurons, which show the typical isovolumetric regulation, the efflux of taurine and glutamate activates very early after the hyposmolarity reduction, as early as 2% for taurine [Tuz et al., 2001]. In astrocytes and C6 cells the efflux is delayed up to 15 and 39% for taurine and glutamate, respectively [Ordaz et al., 2004a; 2004b], and in trout erythrocytes which do not exhibit isovolumetric regulation, the efflux of taurine occurs in low amounts and is very delayed [Godart et al., 1999]. The higher ability of neurons as compared to astrocytes to resist to changes in external osmolarity, which seems based primarily on the contribution of organic osmolytes, may represent a protective mechanism to spare neurons from the deleterious consequences of swelling. In line with this notion is the interesting observation by Nagelhus et al. [1993] in the cerebellum


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Fig. 2. a Cell volume constancy i.e. isovolumetric regulation, in cultured cerebellar granule neurons (•) exposed to small and gradual changes in osmolarity (1.8 mOsm/min). The same treatment in astrocytes (䊏) increases cell volume but not at the extent observed when the osmotic change is suddenly imposed (䊏*). For experimental details see Tuz et al. [2001] and Ordaz et al. [2004a, b]. b Redistribution of taurine-like immunoreactivity in rat cerebellar cortex following water loading. Details of the experiment are in Nagelhus et al. [1993]. In control, isosmotic conditions, taurine is found accumulated in Purkinje cells (large arrows). Upon water loading taurine is transferred to the adjacent glial elements (arrows) which contain very low taurine in isosmotic conditions. As consequence of this redistribution, astrocytes swell while neurons are spared. The original distribution is restored after hyposmolarity correction. Reproduced from Naghelhus et al. [1993], with permission.

of water loaded rats. In the isosmotic condition, the cerebellar Purkinje cells contain high concentrations of taurine, while the nearby astrocytes contain essentially no taurine. In response to the osmolarity reduction there is a remarkable change in the location of taurine: all taurine is lost from the Purkinje cells and is then accumulated in astrocytes (fig. 2b). As consequence of this redistribution, astrocyte swell and neurons are spared. This notable protective action of astrocytes suggests the importance that for neuronal function may have the


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maintenance of cell volume, not only in the soma but also in dendrites, axons and nerve endings. Special mechanisms developed for preserving intact the cytoarchitecture of neurons appear to involve importantly organic osmolytes.

Swelling and Necrotic Neuronal Death

Brain cell swelling occurs during pathological conditions such as ischemia, epilepsies, trauma and hepatic encephalopathy. Vasogenic as well as cellular edema are found coincidently in these pathologies. Cellular swelling occurs also in pathologies associated with hyponatremia. Hyposmotic swelling results from a decrease in the external osmolarity and the subsequent water flow tending to establish a new osmotic equilibrium. Isosmotic (cytotoxic) swelling is generated by redistribution of ions or molecules responding to phenomena inherent to the pathology, such as the energetic failure and dissipation of Na gradients during hypoxia/ischemia, the increase of extracellular K during ischemia and epilepsies, or the ammonium accumulation during hepatic encephalopathy. Hyposmotic swelling, even if drastic, rarely results in cell death, while cytotoxic swelling commonly ends in excitotoxicity and necrotic death. It is generally accepted that cytotoxic swelling occurs mainly in astrocytes whereas neurons are less affected. This is in line with the known essential role of astrocytes in protecting neurons from the disturbing effects of changes in the composition of the extracellular space, or from excessive concentration of potentially toxic molecules. Typical examples of this role of astrocytes are the K clearance from the extracellular space by spatial buffering or the glutamate uptake persisting in astrocytes longer than in neurons due to their ability of facing the energy failure by generating ATP via the glycolitic pathway [rev. in Pasantes-Morales and Franco, 2005]. When the pathological conditions are too severe or prolonged, the protective mechanisms in astrocytes are exceeded and neurons could then be affected in several ways, including swelling occurrence. The mechanisms of swelling-induced necrotic neuronal death will be discussed essentially for the ischemic condition. During trauma, vasogenic is the predominant type of edema, while cellular edema is consequent to the ischemic conditions. Swelling in hepatic encephalopathy affects essentially astrocytes, since ammonia detoxification, which is the primary swelling inductor, occurs only in astrocytes [Butterworth, 2002]. The chain of events responsible for cytotoxic swelling and necrotic cell death in ischemia is initiated by the energy failure and the consequent Na/K ATPase dysfunction. The resultant elevation of intracellular Na and extracellular K is followed by depolarization and glutamate release. High extracellular


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Fig. 3. Swelling inductors in ischemic necrotic neuronal cell death. The energy failure produced by ischemia provokes Na/K ATPase dysfunction, intracellular Na increase, and depolarization. This is followed by Ca2-dependent glutamate release, both from exocytosis, reversal of the transporter and activation of the volume-sensitive Cl channel. The overfunction of ionotropic glutamate receptors further increases intracellular Na followed again by Cl and water. The massive Ca2 influx entry promotes production of ROS, membrane lipid peroxidation and a new wave of swelling due to ion overload through the injured membranes. Excessive swelling contributes to membrane rupture and release of cell debris, propagating the damage to adjacent cells.

glutamate levels resulting from depolarization cannot be removed by the Nadriven glutamate transporters since the Na gradient is dissipated. The transporters may even operate in a reverse mode, further increasing extracellular glutamate, and overfunction of ionotropic glutamate receptors leads to massive Na influx. Then, Cl and water influx driven by the intracellular high concentration of Na generates a first wave of cytotoxic swelling. Further glutamate release via the swelling-activated glutamate efflux pathway also contributes to the increase and persistence of glutamate in the extracellular space (fig. 3). A second wave of swelling occurs as consequence of Ca2 influx through the ionotropic glutamate receptors. This [Ca2]i rise produces reactive oxygen species (ROS) through the activation of prooxidant mechanisms, such as phospholipases, xantine oxidase and nitric oxid synthase. The free fatty acid release and the generation of several free radical species affects membrane integrity and favors ion overload and a further wave of Na and Ca2 increase (fig. 3). There is almost general agreement on that elevation of [Ca2]i is the primary


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cause of excitotoxic neuronal death, mediated by proteases of the family of calpains and cathepsins. At this state, swelling contributes to necrotic neuronal death propagation propitiating plasma membrane rupture and cell lysis. The release of cellular content and necrotic debris to the extracellular space precipitates damage to cells in the vicinity, via the chain of events involving ROS, membrane lipid peroxidation and Na and Ca2 overload through the affected membranes. Lactacidosis generated in ischemia and trauma is another factor of swelling and neuronal death. Lactate is formed during the operation of the glycolitic pathway in astrocytes, which provides an alternate source of energy as far as there is residual glucose. Cytotoxic swelling by lactacidosis occurs preferentially in astrocytes but there is evidence of neuronal swelling in vivo as well in cultured neurons exposed to extracellular lactate levels as those present in ischemia [Alojado et al., 1996; Staub et al., 1993]. Swelling is a function of the severity of acidosis and duration of the exposure, although not all neurons appear to be equally susceptible [Cronberg et al., 2005]. How neurons respond to cytotoxic swelling has not been examined in detail. Neurons exhibit a complex morphology, with specialized functions in soma, dendrites, axon or nerve terminals. Then, swelling may not be homogeneous and its functional consequences will depend on the affected area. There is no clear evidence of active volume recovery in neurons after cytotoxic swelling, but a low efficiency or inability is predictable, in view of the nature of the swelling inductors above discussed. Cell volume regulation relies on the expulsion of intracellular osmolytes, which occurs in general, via diffusion pathways or channels through which the osmolytes move following the gradient direction. In ischemia and epilepsies, the excessive extracellular K concentration resulting from the Na/K ATPase dysfunction and neuronal overexcitability will limit the operation of the volume-activated K channels. The ATP drop impairs also the volume-sensitive Cl channel which characteristically requires nonhydrolytic ATP binding. Organic osmolytes also have this ATP requirement [Okada et al., 2006]. In addition, the volume-sensitive Cl channel seems impaired by lactacidosis [Mori et al., 2002]. Altogether, these conditions accentuate the difficulty of brain cells, including neurons, to accomplish the set of reactions necessary to restore the normal cell volume in conditions of ischemic cytotoxic swelling.

Cell Volume and Apoptosis in Neurons

Apoptosis is a highly regulated process of cell deletion directed to eliminate a definite group of cells at a precise time, for preserving the optimal


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operation of tissues and organs. In the developing brain, apoptosis is an essential process to accomplish the numerical match of neuronal populations and their targets, for organizing the regional cytoarchitecture, and to establish functional synapses and building of neural circuitry. In the adult brain, apoptosis has some contribution to neuronal loss in acute neuropathologies, in some neurodegenerative disorders and in brain aging. Apoptosis occurs according to a strictly ordered set of biochemical events, one of which is a characteristic reduction in cell volume, termed apoptotic volume decrease (AVD). When the temporary resolution of apoptosis was established, it was clear that AVD is an early event in the program, occurring before the surge of other characteristic traits such as caspase 3 cleavage, cytochrome c release and translocation, endonuclease activation and DNA fragmentation. Moreover, it is now considered that AVD may be part of the causal signals and not only consequence of apoptosis [Bortner and Cidlowski, 2002, 2004]. Noteworthy, AVD is not followed by any compensatory mechanism directed to counteract the cell volume loss. There is no evidence of regulatory volume increase, which is apparently inhibited or overridden by an as yet unknown signal [Bortner and Cidlowsly, 2004]. Although AVD occurs in isosmotic conditions, it relies on the same mechanisms of RVD, i.e. the active translocation of intracellular osmolytes and osmotically obligated water flow. K and Cl are the main intracellular osmotically active solutes and are natural candidates to accomplish AVD. Some organic molecules also participate. K Efflux and AVD in Neurons, K Channels Involved The importance of K efflux and AVD as part of the apoptotic signaling chain is now well established, and there is reasonable agreement on that K outflow occurs via K channels [Bortner and Cidlowski, 2004; Burg et al., 2006; Yu, 2003]. Studies in mice cultured cortical neurons undergoing apoptosis by treatment with staurosporin or ceramide have shown AVD, K loss and the selective and early enhancement of a voltage-dependent delayed rectifier current (IKDR) in the apoptotic neurons. Decreasing cell K by the ionophores valinomycin and beauvericin induced apoptosis and accordingly, high external K levels suppressed apoptosis. The antiapoptotic effect of increasing external K was Ca2- and caspase-independent [Yu et al., 1997, 1999]. IKDR blockade by tetraethyl ammonium or clofilium also attenuates apoptosis. These results suggest an early location of K efflux and AVD in the apoptotic signaling chain in mice cortical neurons. However, a study in rat cortical neurons, showed that staurosporine-induced apoptosis is not reduced by tetraethyl ammonium, clofilium or high external K but it is prevented by SITS [Small et al., 2002]. These differences may be due to the higher concentration of staurosporine used in this study, which may induce both apoptosis and necrosis.


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Cerebellar granule neurons require for survival a depolarizing environment provided by 25 mM K or NMDA receptor agonists. Cell death prevention in this case depends on the Ca2 influx through voltage-gated channels [Alavez et al., 2003]. In conditions of low K, cells die by apoptosis which occurs concomitant to the increase of an outwardly rectifying K current named standing outward K current (IKso) [Lauritzen et al., 2003]. Apoptosis is induced by staurosporine in neuronal progenitor cells generated from mice striatal stem cells [Hribar et al., 2004]. Similar to mice cultured cortical neurons, a delayed rectifier K current already expressed in the neuronal precursors, is enhanced by staurosporine since the first days of differentiation. IKDR enhancement precedes the activation of caspase 3 and increasing external K reduced IKDR and attenuates apoptosis. These results are similar to those found in cortical neurons but in the neuronal progenitor cells the amplitude of IKDR is reduced by caspase blockers. Apoptotic cell death concurs with necrotic cell death in some neurodegenerative disorders as well as in ischemia and trauma. Necrotic neuronal death predominates at the ischemic focus while apoptotic cell death prevails in the perifocal area. Apoptosis is triggered and sustained by multiple factors concurrent with the ischemic condition: acidosis, increased expression of death receptors or proapoptotic molecules, activation of MAP kinases and generation of nitric oxid and peroxynitrite. Activation of apoptosis directly by nitric oxide in mice cortical neurons led to IKDR enhancement, K outflow and cell K loss, all resistant to the general caspase blocker zVAD [Bossy-Wetzel, 2004]. The same set of reactions occurs in cortical neurons in a thiol-oxidant model of apoptosis [Aizenman et al., 2000]. The -amyloid peptide linked to Alzheimer disease, enhances an outward K current in cortical neurons in conjunction with apoptosis [Yu et al., 1998]. In cerebellar Purkinje cells, apoptosis associated to lurcher gene occurs after activation of a voltage-gated channel, suggesting the involvement of K in the apoptotic program in this neurodegenerative condition [Norman et al., 1995]. The molecular identity of the ADV-linked K channels is now extensively investigated and from the diversity of cell types examined, including neurons, an important point emerged: there is apparently not a specific channel, devoted exclusively to permeate K for reducing cell volume during apoptosis. This function seems accomplished by K channels present in the non-apoptotic cell, and performing a variety of tasks in the physiological condition. The type of apoptosis-activated K channel may be cell specific and could be different according to the apoptotic inductor. K channels linked to AVD and apoptosis include: Kv channels (isoforms Kv1.1, Kv1.3, Kv1.5, Kv2.1), K2P channels (TASK-1 and TASK-3), HERG and BK channels. The KATP channels are involved in the intrinsic mid-and late phase of apoptosis [Yu, 2003]. Kv channels


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are those linked to AVD in cortical neurons. These channels fit an important requirement for the K exit route i.e. large K conductance and slow inactivation during depolarization. The Kv2.1 isoform carries a significant fraction of the enhanced IKDR in apoptotic cortical neurons. This is concluded by the effect of two dominant negative mutant forms of Kv2.1 decreasing IKDR, and complementary, the apoptosis attenuation in neurons deficient in functional Kv2.1 [Pal et al., 2003]. In cultured cerebellar granule neurons, the standing outward K current (IKso) which increases with cell maturation in the culture is suggested as the apoptotic K exit route. IKso has biophysical, pharmacological and regulation properties characteristic of the TASK-1 and TASK-3 2Pdomain K channels (K2P) [Lauritzen et al., 2003]. The following evidence connects IKso and K2P channels to apoptosis: (i) young cerebellar neurons lacking IKso are resistant to low K-induced cell death, (ii) conditions or agents decreasing K2P prevent apoposis in mature neurons, (iii) apoptosis occurs in hippocampal neurons lacking IKso after viral-induced expression of TASK-1 or TASK-3 channels (iiii) inactivation of endogenous TASK channels by expression of the dominant-negative loss of function TASK mutants, protects neurons from the low-K induced apoptosis [Lauritzen et al., 2003]. In some conditions, glutamate induces apoptosis and, similar to other apoptogenic models, K efflux is linked to apoptotic death. Kv channels, increased K permeability through the activated receptors or activation of high conductance Ca2-activated K channels are all proposed routes for K exit [Isaacson and Murphy, 2001; Yu, 2003]. Most studies on AVD in neurons have addressed to the early phase of the apoptotic program and the K efflux driven by plasmalemmal K currents. However, mitochondrial K channels may also be involved in the mid- and late phases characterized by mitochondrial depolarization and cytochrome c release. KATP channels are those commonly associated with these phenomena. AVD and Cl Fluxes K efflux is a characteristic trait of apoptosis in neurons as in many other cell types, but in order to effectively contribute to water outflow and AVD, it has to occur in conjunction with Cl exit. Activation of Cl currents in CD95induced apoptosis was first shown by Lang and coworkers in Jurkat cells [Lang et al., 2005; Szabo et al., 1998]. This was confirmed thereafter in other cell types in apoptosis driven by staurosporine, FAS ligand, TNF, ceramide or doxorubicin [Maeno et al., 2000; Okada et al., 2006]. The apoptosis-linked anion currents have properties in all similar to those carried by the volume-sensitive anion channel which plays a prominent role in RVD, but noteworthy in apoptosis this activation occurs in isosmotic conditions or even in shrinking, not swelling cells. The mechanism(s) or signals for the volume-sensitive Cl channel


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activation are as yet unknown, neither in RVD nor in AVD. Cl channel blockers, DIDS, NPPB or phloretin prevent staurosporine-induced apoptosis in neural PC12 and in neuroblastoma NG108–15 cells [Maeno et al., 2000], and SITS is effective in hippocampal neurons undergoing apoptosis by ischemia/reperfusion [Inoue et al., 2005]. However, a study in cortical neurons shows that DIDS, NPPB or phloretin provide only limited protection against apoptosis induced by staurosporine, ceramide or serum deprivation, while K channel blockers supply complete protection [Wei et al., 2004]. This failure of Cl channel blockers has been attributed to the low concentration of the blockers used in the cortical neurons preparation [Okada et al., 2006]. In addition to the volume-sensitive Cl channel, the voltage-dependent anion channel (VDAC) is proposed to participate in AVD. VDAC is a large conductance anion channel (300–400 pS), located at the outer mitochondrial membrane. In staurosporine-induced apoptosis in the hippocampal cell line HT-22 and in the human neuroblastoma cell line SK-N-MC, VDAC was found functionally expressed in the cell membrane in 48% of apoptotic cells and its blockade by functional antibodies or by high concentrations of sucrose reduce the number of apoptotic cells [Elinder et al., 2005]. In physiological conditions, membranal VDAC may function as an NADH-reductase involved in transmembrane redox regulation. The Cl/HCO3 exchanger is also suggested as mechanism for Cl outflow in apoptosis, based on the strong effect of the typical anion exchanger blocker DIDS, in suppressing the apoptotic program. This has been challenged by experiments showing no change in the antiapoptotic effect of DIDS in the absence of bicarbonate, a condition reducing the exchanger operation [Okada et al., 2006]. Signals for Activation of Ion Fluxes in Apoptosis The signals in the apoptotic process activating Cl and K fluxes for AVD remain largely unknown. Since various types of K channel are involved, a common signal may not operate. The stress-reacting MAPK p38 is suggested as a signal [McLaughlin et al., 2001] under the upstream influence of another MAPK, the MAPKKK also called ASK1 (apoptosis signal-regulating kinase). Inactive ASK-1 or expression of a dominant-negative form of ASK-1 suppresses IKDR and prevents apoptosis in cortical neurons [Aras and Aizenman, 2005]. Tyrosine kinases may also participate in the AVD-linked K fluxes in cortical neurons, since the tyrosine kinase general blockers herbimycin or lavendustin reduce IKDR in these cells [Yu et al., 1999]. Tyrosine kinases may be directly or indirectly associated with p38. Another proposed mechanism for apoptotic IKDR upregulation is the membrane insertion of preformed, endogenous channels as shown for Kv2.1 channels in cortical neurons [Pal et al., 2006]. This translocation requires t-SNARE proteins, the same involved in the


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exocytotic mechanism of neurotransmitter release. Disruption of SNARE proteins by botulin neurotoxin suppresses the apoptotic enhanced IKDR [Pal et al., 2006]. Cl outflow seems to occurs via the same anion channel in RVD and AVD, in spite of the different, even opposite volume of cells in the two conditions, suggesting a change in the volume set point for the channel gating. The mechanism for this adjustment in the set point is unknown. Reactive oxygen species or tyrosine kinases such as the Src kinase p56Lck might be involved [Lambert, 2004; Leppe-Wienhues et al., 1998]. A role for the cytosolic concentration of ATP has also been considered [Okada et al., 2006]. Volume Decrease or Ionic Homeostasis: Which Is the Apoptotic Signal? Cell volume decrease has been the key morphological trait to distinguish between necrotic and apoptotic death, but it is only in the last decade that insertion of AVD in the signaling chain of the apoptotic process has been demonstrated. Clearly AVD results essentially from K and Cl outflow, but the question remains of whether the relevant point in terms of apoptotic program, is the cell volume reduction or the decrease in the intracellular ion concentrations. If K or Cl at physiological levels could have an inhibitory influence on factors or reactions of the apoptotic chain, the activation of an efflux pathway and the consequent decrease in cell levels would relieve this inhibition. AVD would then be just the consequence of the ion extrusion. This hypothesis is supported by two types of results: (i) preventing K loss by increasing extracellular K interrupts apoptosis even when the K exit route is fully active and (ii) decreasing cell K concentrations with no activation of the apoptogen-induced K currents is sufficient to set in motion the apoptotic machinery. Caspases and nucleases may be the sites of K influence as suggested by the inhibitory effect of physiological K concentrations on the activity of caspase 3. The K threshold concentration for activating the apoptotic reactions has not been precisely defined [Bortner and Cidlowski, 2004]. A possible influence of Cl levels on the apoptotic steps has not being examined in detail. It is also unclear if K and Cl have equal importance as a relevant signal, or if only one of them is actively expelled as part of the apoptotic signaling chain, and the other one is just passively carried to support the persistence and magnitude of the active ion outflow. That Cl may play this passive role is suggested by a study in staurosporine-induced apoptosis in cortical neurons showing that DIDS prevents AVD but not caspase-3 nor DNA fragmentation, whereas K channel blockers have a full inhibitory action on all the apoptotic events [Wei et al., 2004]. The temporal sequence of apoptosis-linked Cl and K fluxes has not been examined in detail. In RVD the order of K and Cl efflux activation is dependent on the cell type. Thus, Cl channels activate prior to K channels in


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most epithelial cells and the opposite is found in non-epithelial cells [PasantesMorales and Morales-Mulia, 2000]. A role for Na in AVD and apoptosis has recently raised attention. An early and transient increase in intracellular Na has been detected in apoptotic Jurkat cells [Bortner and Cidlowki, 2003] which if suppressed, prevents K loss, cell shrinkage and DNA degradation. Na influx is a hallmark of necrotic death and therefore, if Na entry is also crucial for apoptosis, the influx mechanisms and their activation and inactivation signals must be critical for deciding the ultimate direction of the cell death type. AVD and Taurine Efflux Organic osmolytes contribute to RVD and they could also participate in AVD. Taurine efflux concurrent with apoptosis has been shown in a variety of cell types including cerebellar granule neurons. The mechanism of the apoptotic taurine release is not well characterized. In cerebellar granule neurons as well as in Jurkat cells, the Cl channel blockers which are very efficient in preventing taurine efflux in RVD do not decrease but increase the AVD-linked taurine efflux [Lang et al., 2000; Morán et al., 2000]. Also, the tyrosine kinase influence observed in taurine efflux in RVD is not found in AVD. Raising external taurine up to 20 mM, a condition likely preventing taurine efflux or/and cell taurine loss, attenuates apoptosis in cortical neurons [Huang et al., 2006]. Thus, the arguments raised about the key role of K cell loss and not AVD, are also valid for taurine. A consistent observation is that in cells treated with high external taurine, the apoptotic step corresponding to the assembly of apoptosomes and the further activation of caspase 9 is prevented [Takatani et al., 2004], suggesting an inactivating role of physiological concentrations of taurine in certain apoptotic signals. In line with this interpretation is the apoptotic death of photoreceptors in taurine transporter knockout mice [Heller-Stilb et al., 2002].

Final Comments

Excitability, hyperexcitability, hypersynchrony, survival and death, all have a link with one of the most ancient and preserved traits of the cell biology: the cell volume control. Even in the highly specialized neuron, cell volume influences its life cycle, from rescue to death, from proliferation to extinction, from rest to excitation. More question than answers still characterize our knowledge of this essential biological function. Among the most intriguing are the crossing points between RVD and AVD, while regulatory volume increase becomes protagonist by its apparently programmed repression. The nature of a volume


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sensor finely tuned to detect in RVD, the imperative of activating osmolyte outflow routes for rescue, and in AVD, activating apparently similar routes for death. The set point adjustment allowing the activation of the same channels in swollen or shrunken cells, the similarities or dissimilarities between the signaling cascades in RVD and AVD, connecting the same sensors and the same effectors, the sequence, consequence and interdependence of K and Cl fluxes leading cells to normality or to death, are still unsolved questions in the seminal topic of neuronal death and survival in pathologies related to changes in cell volume.

Acknowledegement The work of the authors was supported by grants No.IN206403 from DGAPA UNAM and 46465 from CONACYT.

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Mori S, Morishima S, Takasaki M, Okada Y: Impaired activity of volume-sensitive anion channel during lactacidosis-induced swelling in neuronally differentiated NG108–15 cells. Brain Res 2002;957: 1–11. Nagelhus EA, Lehmann A, Ottersen OP: Neuronal-glial exchange of taurine during hypo-osmotic stress: a combined immunocytochemical and biochemical analysis in rat cerebellar cortex. Neuroscience 1993;54:615–631. Norman DJ, Feng L, Cheng SS, Gubbay J, Chan E, Heintz N: The lurcher gene induces apoptotic death in cerebellar purkinje cells. Development 1995;121:1183–1193. Okada Y, Shimizu T, Maeno E, Tanabe S, Wang X, Takahashi N: volume-sensitive chloride channels involved in apoptotic volume decrease and cell death. J Membr Biol 2006;209:1–9. Ordaz B, Tuz K, Ochoa LD, Lezama R, Peña-Segura C, Franco R: Osmolytes and mechanisms involved in regulatory volume decrease under conditions of sudden or gradual osmolarity decrease. Neurochem Res 2004a;29:65–72. Ordaz B, Vaca L, Franco R, Pasantes-Morales H: Volume changes and whole cell membrane currents activated during gradual osmolarity decrease in C6 glioma cells: contribution of two types of K channels. Am J Physiol 2004b;286:C1399–C1409. Pal S, Hartnett KA, Nerbonne JM, Levitan ES, Aiznman E: Mediation of neuronal apoptosis by Kv2.1encoded potassium channels. J Neurosci 2003;23:4798–4802. Pal SK, Takimoto K, Aizenman E, Levitan ES: Apoptotic surface delivery of K channels. Cell Death Differ 2006;13:661–667. Pasantes-Morales H, Franco R: Astrocyte cellular swelling: mechanisms and relevance to brain edema; in Aschner M, Costa LG (eds): The Role of Glia in Neurotoxicity. ed 2, CRC Press, 2005, pp 173–190. Pasantes-Morales H, Morales-Mulia S: Influence of calcium on regulatory volume decrease: role of potassium channels. Nephron 2000;86:414–427. Rosen AS, Andrew RD: Osmotic effects upon excitability in rat neocortical slices. Neuroscience 1990;38:579–590. Saly V, Andrew RD: CA3 neuron excitation and epileptiform discharge are sensitive to osmolality. J Neurophysiol 1993;69:2200–2208. Saransaari P, Oja SS: Mechanisms of D-aspartate release under ischemic conditions in mouse hippocampal slices. Neurochem Res 1999;24:1009–1016. Schwartzkroin PA, Baraban SC, Hochman DW: Osmolarity, ionic flux, and changes in brain excitability. Epilepsy Res 1998;32:275–285. Small DL, Tauskela J, Xia Z: Role for chloride but not potassium channels in apoptosis in primary rat cortical cultures. Neurosci Lett 2002;334:95–98. Staub F, Mackert B, Kempski O, Peters J, Baethmann A: Swelling and death of neuronal cells by lactic acid. J Neurol Sci 1993;19:79–84. Szabo I, Lepple-Wienhues A, Kaba KN, Zoratti M, Gulbins E, Lang F: Tyrosine kinase-dependent activation of a chloride channel in CD95-induced apoptosis in T lymphocytes. Proc Natl Acad Sci USA 1998;95:6169–6174. Takatani T, Takahashi K, Ouzumi Y, Shikata E, Yamamoto Y, Ito T, Matsuda T, Schaffer SW, Fujio Y, Azuma J: Taurine inhibits apoptosis by preventing formation of the Apaf-1/caspase-9 apoptosome. Am J Physiol Cell Physiol 2004;287:C949–C953. Tuz K, Ordaz B, Vaca L, Quesada O, Pasantes-Morales H: Isovolumetric regulation mechanisms in cultured cerebellar granule neurons. J Neurochem 2001;79:143–151. Tuz K, Pasantes-Morales H: Hyposmolarity evokes norepinephrine efflux from synaptosomes by a depolarization- and Ca2-dependent exocytoic mechanism. Eur J Neurosci 2005;22:1636–1642. Tuz K, Peña-Segura C, Franco R, Pasantes-Morales H: Depolarization, exocytosis and amino acid release evoked by hyposmolarity from cortical synaptosomes. Eur J Neurosci 2004;19:916–924. Wei L, Xiao AY, Jin C, Yang A, Lu ZY, Yu SP: Effects of chloride and potassium channel blockers on apoptotic cell shrinkage and apoptosis in cortical neurons. Pflugers Arch 2004;448:325–334. Yu SP: Regulation and critical role of potassium homeostasis in apoptosis. Prog Neurol 2003;70: 363–386.


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Prof. Herminia Pasantes-Morales Instituto de Fisiología Celular, UNAM Apartado Postal 70–600 04510 Mexico D.F. (Mexico) Tel. 52 55 5622 5588, Fax 52 55 5622 5607, E-Mail [email protected]


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Pathophysiology of Red Cell Volume Joseph A. Browninga, J. Clive Ellorya, John S. Gibsonb a

Department of Physiology, Anatomy and Genetics, Oxford, Department of Veterinary Medicine, Cambridge, UK

b

Abstract In the current work, we review three situations where red cell volume changes are important. Red cell apoptosis (eryptosis) accounts for the removal of ageing and damaged erythrocytes from the circulation by macrophages. Amongst other cellular responses, eryptosis is associated with net cytosolic KCl loss and concomitant cell shrinkage. KCl efflux is mediated by activation of Ca2⫹-activated K⫹ (Gardos) channels, permitting downhill movement of K⫹ and electrically obliged Cl⫺ through, as yet, incompletely described pathways. Red cells from patients suffering from sickle cell disease demonstrate progressive dehydration. Osmolyte loss is accounted for by the activation of two separate pathways. KCl cotransport, normally quiescent in red cells from HbA individuals, is activated under deoxygenated conditions and mediates net KCl efflux. Furthermore, intracellular Ca2⫹ is elevated, probably as a result of Ca2⫹ influx through a deoxygenation induced non-selective cation pathway termed Psickle. This results in Gardos channel activation coupled indirectly with Cl⫺ loss. Finally, a number of red cell stomatocytoses have been described where alterations to erythrocyte volume are the result of increased membrane cation permeability, in particular to Na⫹ and K⫹. The emerging significance of non-selective cation pathways is common to each of these conditions, and, although differences exist between their properties, particularly with regard to activation and ion selectivity, it is conceivable that they represent activation of closely related pathways. The recent finding that many hereditary stomatocytoses are caused by mutations to band 3 (AE-1) raises the possibility that modifications to this transporter could account for altered cation fluxes under different conditions. Copyright © 2006 S. Karger AG, Basel

Introduction

Red Cells as a Paradigm: Membrane Transport and Volume Regulation As a simple, readily available nuclear cell devoid of intracellular organelles, the human red cell has frequently been adopted as a paradigm for studying ion transport and cell volume [1]. However, in contrast to most other cell types,

including nucleated red cells, mature human red cells do not demonstrate classical regulatory volume responses. If human red cells are swollen or shrunken osmotically, they do not necessarily regain their starting volume. Despite this, volume changes are important to red cell function and survival, and the presence of a variety of volume-sensitive transport functions makes cell volume a particularly relevant consideration in red cell physiology [2]. The classical ‘pump-leak’ hypothesis of Tosteson and Hoffman was developed in 1960 using the red cell as a model [3], and illustrated the balance between the Na⫹/K⫹ pump and dissipative Na⫹ and K⫹ leak fluxes. Since then, the original hypothesis has been refined by the identification of new membrane transport pathways, but the basic concept of the balance between dissipative cation fluxes and active Na⫹ and K⫹ transport in concert with the colloid osmotic pressure of intracellular macromolecules (including haemoglobin) remains the basis for controlling red cell volume. Lew and Bookchin have pioneered a computer model of red cell volume, which has accounted successfully for known volume changes in some physiological situations, whilst also producing some valuable and superficially counter-intuitive predictions in other cases [4–6]. The model has been applied to the pathologies of malaria, sickle cell disease (SCD) and stomatocytoses and forms a robust basis from which to assess net ion fluxes, cell volume and pH. Since we last reviewed the subject in 1998 [7], there have been significant advances, particularly in the context of patch clamp studies, which have aided our understanding of the ground state cation permeability of the membrane and the possible role of non-selective cation channels under certain conditions [8–11]. In the current paper, we briefly review factors which are known to modify erythrocyte volume and focus on three pathophysiological situations, namely eryptosis, SCD and hereditary stomatocytoses. Leak Pathways and Cell Integrity The maintenance of cell volume implies a balance between ion pumps and leak pathways: the ‘pump-leak’ hypothesis. Increased membrane permeability to cations can cause the cell to swell via increased Na⫹, or shrink via K⫹ loss. Surprisingly, the number and nature of ion permeability routes in the human red cell membrane remains a difficult and controversial topic. Complications arise when radioisotope methods are used to measure fluxes, due to electrically silent exchange fluxes making large contributions to measured rates, and the multiplicity of transporters using cotransport of Na⫹. For example, the transport of the anion NaCO3⫺ via band 3 will contribute up to 40% of sodium influx at plasma Na⫹ concentrations [12]. Furthermore, there are significant Na⫹ symporter and anti-porter fluxes via, for example, Na⫹/H⫹ exchange (NHE), Na⫹/K⫹/2Cl⫺ cotransport (NKCC) and the amino acid transporters ASC and

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gly. The situation for K⫹ is less complicated, but K⫹Cl⫺ cotransport (KCC) and NKCC, as well as non-selective cation channels (NSCC), Psickle and the Ca2⫹activated K⫹ (Gardos) channel will contribute to transmembrane transport of K⫹. Pragmatically, electrophysiological measurements represent the best route for identification of electrogenic, conductive leak pathways. This has proved difficult due to technical problems with patch clamping red cells. Recently, however, a number of laboratories have had more success with this method, and data on membrane conductance have become more extensive [8, 13]. Classically, the conductance of human erythrocyte membranes is considered low in relation to other cell types and is mostly accounted for by anion conductivity. Indeed, the resting membrane potential of erythrocytes, rather than being dominated by the Nernst potential for K⫹, as is the case for most other cell types, is largely determined by the transmembrane distribution of Cl⫺ [1]. Intracellular Cl⫺ in erythrocytes is high (approximately 80 mM) and the membrane potential has been measured at about ⫺10 mV [14–16]. Hunter quantified the electrogenic movement of Cl⫺ and calculated an anion conductance of approximately 10 ␮S ⭈ cm⫺2 at 37⬚C using valinomycin [17]. This equates to approximately 15 pS per cell. A similar value was obtained by calculating the contribution to red cell membrane potential of the electrogenic Na⫹/K⫹ pump, and estimating the pump current as one-third of the Na⫹-dependent efflux [18]. However, in contrast to these data human red cell membranes extracted from ghosts were found to have a total conductance approximately one order of magnitude higher [19]. More recent whole-cell electrophysiological measurements have yielded very different values. Desai et al. [13] reported that erythrocytes exhibit a whole cell conductance of approximately 100 pS per cell – this was the first such report of this approach being used on human erythrocytes. Different values, some 1–2 orders of magnitude greater, have been reported by Lang and coworkers [9] using similar methodology and Rodighiero et al. [11]. Recently, reports from Lang’s laboratory have been towards the lower end of this scale, around 250 pS per cell [8], which are in reasonably close agreement with our own recent findings (unpublished data). These discrepancies between red cell conductance values measured using different approaches have not yet been resolved. However, given the recent interest in applying whole-cell patch clamp methodology to human erythrocytes, some reconciliation of the disparate values is required. The number and characteristics of the transport pathways relevant to red blood cell volume are not completely resolved, but a summary of our current understanding is summarised in table 1. A complete review of these pathways is beyond the scope of the present work. However, in a number of pathological conditions, particularly SCD and the hereditary stomatocytoses, as well as

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Table 1. Major human erythrocyte membrane transport pathways involved in regulation/determination of cell volume

Browning/Ellory/Gibson 244

Pathway

Major physiological permeants

Inhibitors

Important characteristics

Pathophysiological significance

Key references

Anion conductance (band 3?)

Cl–, HCO3–, NaCO3–

DIDS, SITS, NS1652, NS3623

‘Background’ anion conductance of RBC membranes

Mediates net anion efflux accompanying K⫹ in e.g. dehydrating sickle cells

[17, 145, 146, 166]

Gardos channel (Ca2⫹-activated K⫹ channel; hIK1, hSK4)

K⫹, Rb⫹, NH4⫹

Charybdotoxin, clotrimazole, extracellular Ca2⫹

Ca2⫹ activated K⫹ pathway, single channel conductance approximately 20 pS

RBC dehydration in sickle RBCs; erythrocyte shrinkage suring eryptosis

[24, 25, 36]

Voltage dependent NSCC

Na⫹, K⫹, Rb⫹, NH4⫹, Ca2⫹, Mg2⫹, Ba2⫹

Ruthenium red and La3⫹

Activated Em ⬎ ⫹ 30 mV, hysteresislike gating, approximately 35 pS single channel conductance, activated by carbachol

Eryptosis activating NSCC

Ca2⫹ and monovalent cations

EIPA, amiloride, Gd3⫹, Cl–

Activated by Cl– removal, PGE2, oxidation, shrinkage

Ca2⫹ influx during eryptosis; acute renal failure

[8, 9, 23, 169]

Psickle (NSCC)

Cations – monovalent,

DIDS, dipyridamole,

Stochastic activation upon

Ca2⫹ influx and dehydration in

[51, 66, 122, 125, 149]

[10, 158, 167, 168]

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divalent, organic osmolytes?

?Gd3⫹, ?Zn2⫹

deoxygenation, activation following HbS polymerisation

SCD

P-type Ca2⫹ channel

Ca2⫹

P-type Ca2⫹ channel blockers

Activated by LPA, 25% of RBCs participate

Gardos activation and cell shrinkage?

[170]

Mechanosensitive Ca2⫹ channel

Ca2⫹

Stretch channel blockers (Gd3⫹, neomycin and amiloride)

Activated by caffeine, mechanosensitive?


[171]

L-type Ca2⫹ channel

Ca2⫹

Partially by heparin and ryanodine

Activated by vanadate


[172]

Aquaporin (AQP1)

Water, CO2

Mercurials (pCMBS)

Mediate CO2 and water fluxes

Cell volume changes

[173, 174]

Band 3 (AE-1)

Cl–, HCO3–

DIDS, SITS, dipyridamole, niflumic acid

Facilitates blood CO2 carriage

[175, 176]

NHE1

Na⫹, H⫹

amiloride, EIPA, HOE 694

Modulation of O2/CO2 carriage?; NaCl influx (in parallel with band 3)

[177]

KCC1,3

K⫹, Cl–

DIOA, H25, H74, intracellular Mg2⫹

Activated by cell swelling, increased hydrostatic pressure, high Po2, low Po2 (sickle cells only)

Cell shrinkage in SCD

[68, 69, 97, 127, 178]

Table 1. (continued) Browning/Ellory/Gibson

Pathway

Major physiological permeants

Inhibitors

Important characteristics

Pathophysiological significance

Key references

NKCC1

Na⫹, K⫹, Cl–

Bumetanide, frusemide

Activated by cell shrinkage, low Po2; low net flux

K⫹(Na⫹)/H⫹ exchange

Na⫹, K⫹, H⫹

Quinacrine

Ca2⫹ATPase (PMCA)

Ca2⫹

Vanadate

Maintenance of low resting cytosolic Ca2⫹

[180]

Na⫹, K⫹ ATPase

Na⫹, K⫹

Ouabain

Na⫹ extrusion and prevention of cell swelling

[181]

Modified band 3 (AE-1)

Monovalent cations

DIDS, SITS

Reduced anion transport activity, increased cation conductivity

[179]

[153]

Hereditary stomatocytoses

[152, 154, 155]

246

eryptosis, increases in ion permeability lead to changes in red cell volume, morphology and eventually cell destruction. They are discussed in detail in the following.

Eryptosis

The events leading to apoptosis (programmed cell death), the process responsible for the removal of excess, damaged and ageing nucleated cells, have been the subject of considerable attention. However, until recently, it was assumed that similar mechanisms were not relevant to erythrocytes since many of the key features of apoptosis (e.g., nuclear condensation, DNA fragmentation and mitochondrial depolarisation) involve structures which are absent from mature erythrocytes [20]. However, raised intracellular Ca2⫹ (induced, e.g., by Ca2⫹ ionophores) can induce osmolyte loss and, of most interest to the current review, erythrocyte shrinkage, membrane blebbing and phosphatidylserine exposure, each of which are considered apoptotic mechanisms and which ultimately result in phagocytosis of erythrocytes by macrophages [21, 22]. Thus, it appears that modified apoptotic mechanisms, termed ‘eryptosis’ by Lang et al. [23], do operate in erythrocytes and underlie the removal of defective cells from the circulation, thereby avoiding potentially harmful haemolysis and haemoglobin release. Non-Selective Cation Channel The pathways which underlie eryptosis have now been elucidated in some detail (summarised in fig. 1). The central event is activation of a membrane channel permeable to monovalent and divalent cations which results in elevation of cytosolic Ca2⫹ [8, 9]. This, in turn, promotes activation of the Gardos channel (below) which mediates K⫹ loss together with Cl⫺ via parallel anion pathways [24, 25]. Together, these events result in osmotic water loss and account for the cell shrinkage characteristic of eryptosis. In parallel, raised intracellular Ca2⫹ activates a membrane scramblase which increases the level of phosphatidylserine in the outer leaflet of the erythrocyte membrane [26, 27]. This promotes phagocytosis of cells by macrophages, which express receptors for phosphatidylserine, and clears them from the circulation [28, 29]. Eryptosis can be induced by oxidative stress, Cl⫺ removal, and cell shrinkage [23]. Each of these stimuli has been shown, by direct whole-cell electrophysiological measurement, to activate the NSCC in the erythrocyte membrane [8, 9]. This conductance, which shares certain features with the non-selective cation conductance activated in erythrocytes by incubation in low ionic strength media, can be inhibited by amiloride, its analogue EIPA, and Gd3⫹ [8, 9]. The activation of this conductive pathway is mediated through synthesis and release of prostaglandin E2 (PGE2) and can be inhibited by phospholipase A2 inhibitors

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Osmotic shock

COX PGE2 PLA

PGE2 AA

PAF

Ca2⫹

SM

Ca2⫹

S rafts

Phosphatidylserine exposure

Fig. 1. Summary of mechanisms accounting for eryptosis of red cells. AA ⫽ Arachidonic acid; COX ⫽ cyclooxygenase; NSC ⫽ non-selective cation channel; PAF ⫽ platelet activating factor; PGE2 ⫽ prostaglandin E2; PLA ⫽ phospholipase A2; S ⫽ scramblase; SM ⫽ sphingomyelinase. Redrawn from reference [23].

(e.g., quinacrine) and cycloxygenase inhibitors (e.g., acetylsalicylic acid) [30]. In addition to the increase in intracellular Ca2⫹, production of platelet activating factor (PAF) stimulates a sphingomyelinase and ceramide formation [31]. Ceramide sensitises the scramblase to Ca2⫹ and permits phosphatidylserine exposure in the absence of raised cytosolic Ca2⫹. Interestingly, Cl⫺ and urea prevent red cell phosphatidylserine exposure following osmotic shock [32]. Cl⫺ inhibits the cation conductance as described above and urea inhibits sphingomyelinase thereby preventing ceramide formation. This is important, since it prevents eryptotic mechanisms from being activated as erythrocytes traverse the renal medulla. The Gardos Channel Electrogenic K⫹ efflux during eryptosis, as well as during SCD, is accounted for by the Ca2⫹-activated K⫹ (Gardos) channel. A red cell Ca2⫹-activated K⫹ permeability was first described by Gardos almost 50 years ago in red cells depleted of ATP [24], hence the eponym. Many features of the Gardos channel are well known: activation by intracellular Ca2⫹ levels with an EC50 of a few hundred nanomolar, irreversible inactivation in the absence of extracellular K⫹, channel conductance of about 20 pS and an inverse relationship with temperature


248

(see [33, 34] for recent reviews). Cytosolic Ca2⫹ is raised during eryptosis and SCD. Given the high resting anion conductance of erythrocyte membranes, Ca2⫹-induced increases in K⫹ permeability result in KCl efflux and cell shrinkage. This places the Gardos channel in a central role for the determination of cell volume. Here we focus on two aspects of the channel: its molecular identity and its regulation by factors other than Ca2⫹. The Gardos channel is a K⫹ channel of intermediate conductance, ascribed IK, small conductance (SK) or K⫹ channel calcium-activated intermediate/ small conductance subfamily N (KCNN) [35]. Of the various isoforms of SK channels, human reticulocyte RNA libraries were found to show only SK4, using RT-PCR [36]. These libraries were tested by ‘␤-profiling’, i.e., they contained only the ␤2 subunit of the Na pump, rather than the ␤1 which is present in white cells, consistent with the absence of contamination from white cells or platelets. The product of SK4 is present in erythroid precursors and mature human red cell ghosts. Expressed SK4 has some of the defining properties of the Gardos channel [36]. Unlike the molecular identity, however, the copy number of Gardos channels per red cell remains controversial. Estimates range from single figures to around a hundred [37], but, notwithstanding, distribution appears homogeneous throughout the red cell population [38]. Modulators of Gardos channel activity include the proteins calpromotin and calmodulin. Calpromotin is a soluble cytoplasmic protein, very similar or identical to NKEF-B and TSA. It is required for reconstitution of Gardos channel activity in sonicated vesicles [39]. The role of calmodulin remains somewhat enigmatic. As SK4 lacks an obvious Ca2⫹ recognition site, however, this may be conferred by calmodulin. The association of calmodulin with the Gardos channel is thought to be extremely tight, complicating the interpretation of the effect of anti-calmodulin reagents. Trifluoperizine, e.g., inhibits in some situations [40], but not in others [41]. There is also some evidence for regulation by protein phosphorylation. Thus a phosphorylation-promoting mixture (cAMP, MgATP and theophylline) modulates Ca2⫹ sensitivity [42] via an endogenous PKA. PKC inhibitors (staurosporine, chelerythrine, calphostin C) reduce Gardos channel activity in normal cells [43, 44]. In addition, various cytokines, chemokines and endothelins affect activity. Of relevance to eryptosis, PGE2 stimulates [45], probably via increasing Ca2⫹ influx [30, 46]. This may be an autocrine effect through endogenous production and release of PGE2 from ‘stressed’ red cells. In mouse red cells, endothelin-1 can activate Gardos activity via PKC [47]. In HbS cells, various stimulatory factors (including PAF, endothelin-1, IL-10 and RANTES), increase K⫹ flux through the Gardos channel whilst lowering Ca2⫹ affinity [44]. As for KCC, oxidants also affect Gardos channel activity with stimulation observed in both normal and HbS cells [48, 49]. In addition to its importance to eryptosis, many

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of these factors will be altered in patients with SCD, with modulation of Gardos channel activity potentially significant for the pathophysiology of the disease.

Sickle Cell Disease

Volume Abnormalities in SCD Shrinkage of HbS cells results from their abnormally high cation permeability [50–53], with loss of KCl obliging water to follow osmotically. Loss of cell water elevates [HbS], markedly promoting polymerisation of Hb, via reduced lag time for polymer formation on deoxygenation [54], with estimates suggesting that the lag is inversely proportional to [HbS]15–30. It is not surprising that understanding the mechanisms for solute loss, and preventing cell dehydration, remain an immediate goal of therapy for SCD [55, 56]. Three main transport systems are involved: KCC; the Ca2⫹-activated K⫹ channel; and the deoxygenation-induced cation channel (termed Psickle). The relative contribution of these to dehydration remains unclear, and probably varies [57–61]. KCC activity is abnormally high in sickle cells, and shows altered regulation. It mediates obligatorily coupled K⫹ and Cl⫺ efflux. As described above, the Gardos channel is a conductive K⫹ pathway. It is activated as intracellular Ca2⫹ levels rise to a few hundred nanomolar. In normal red cells, [Ca2⫹]i is kept below the threshold level by a low passive Ca2⫹ permeability and a highly active Ca2⫹ pump (PMCA) [62, 63], but in HbS cells a number of mechanisms permit elevation of [Ca2⫹]i. To allow rapid conductive K⫹ efflux, an anion, which will be Cl⫺ in normal circumstances, must follow, via indirect electrical coupling. Finally, a unique feature of HbS cells induced by deoxygenation is an additional ionic pathway ascribed Psickle. Its prime importance is to cause increased entry of Ca2⫹, thereby promoting Gardos channel activation. It can also mediate solute loss in two other, probably minor, ways. Thus, although under certain conditions Na⫹ entry and K⫹ loss are balanced [64–66], under physiological conditions, Psickle mediates a modest net loss of cations. These various pathways will interact. Thus, e.g., isosmotic loss of KCl will acidify the cell, via activation of Cl⫺/HCO3⫺ exchange, and thereby further stimulate KCC [51], which is activated by a fall in pH. Inhibiting solute loss through these various processes is an important strategy for ameliorating the effects of SCD. KCl Cotransport and Sickle Cell Disease Altered Expression of KCC and Altered Activity in Sickle Cells KCC is one of a superfamily of cation-chloride cotransporters which carry out a number of important roles within the body, including volume regulation, epithelial transport and intracellular ion homeostasis (notably regulation of the Cl⫺ equilibrium potential, especially in neurons) [67]. In fact, functional KCC


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activity was first described in red cells some 25 years ago, as a Cl⫺-dependent K⫹ flux stimulated by swelling [68] or thiol oxidation [69]. Physiologically, red cell KCC activity participates in the volume reduction during reticulocyte maturation and its activity subsequently declines in older cells [70–72], through loss of transporter per se [73] and probably its regulatory apparatus. Sickle cell KCC has an unusually high activity, contributing to the excessive solute loss [71, 74]. In part, this is due to a relatively young red cell population, following the regenerative anaemia of SCD. In addition, there are a number of other abnormal features of KCC in sickle cells, all ultimately associated with altered Hb [75]. To date, four isoforms of KCC have been cloned [73, 76–81]. There is some tissue specificity, e.g. KCC2 is confined to neurons, and, functionally, the various KCC isoforms have some differences in ion affinity and selectivity. Splice variants also exist, and, again, these display different transport characteristics [82–84]. Erythroid KCC was originally identified as KCC1 [78], but later an additional component of KCC3 was detected [82]. KCC1, KCC3 and KCC4 have all been identified in human erythroid tissue, although KCC1 appears to predominate. The abundance of KCC1 polypeptide is substantially higher in HbS or HbSC compared to HbA cells [73]. Splice variants, especially of KCC1 and KCC3, also occur but the relevance of this to red cell pathophysiology is not yet clear. HbS cells show a similar expression pattern to normal red cells, except that one KCC1 splice variant (exon 1) is over-expressed. HbA cells, by contrast, have more of the exon 1b splice variant. The latter has an N-terminal truncation which has several changes in phosphorylation sites (it lacks 2 serine and 2 tyrosine residues, but gains an extra serine phosphorylation site), which may be important in altering KCC activity. Furthermore, dominant negative effects can occur with KCC1 artificially truncated at the N-terminus (but not C-terminus), at least in the xenopus oocyte expression system [84]. Whether similar effects occur naturally is unknown. Less is known about the transcriptional control regulating KCC expression, however, recently a 5⬘ upstream promoter has been cloned [85]. The coding sequence and binding proteins show several unusual features for a mammalian promoter [85]. The extent to which altered transcription or translation, or any interaction between the expressed KCC proteins participate in the abnormal properties of KCC in HbS cells, and whether these correlate with the severity of SCD, awaits elucidation. Regulation of KCC by Cell Parameters The main variables controlling KCC activity are probably cell volume, intracellular pH and O2 tension. Differences occur between red cells from SCD patients and normal individuals. HbS cells are more sensitive to acid stimulation than HbA cells, when KCC activity is normalised to the maximal volume-stimulated flux in each sample [86]. They also show an exaggerated volume regulatory

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response to both cell swelling and acid pH, shrinking to a higher mean cell haematocrit. That these changes could be reversed in part by treatment with dithiothreitol suggests a role for reversible sulphydryl oxidation. These properties would support the rapid and pronounced dehydration of HbS cells, termed ‘fast track dehydration’ [58, 87]. Haemoglobin per se is clearly important in the response of KCC to various stimuli. It was proposed some years ago that [Hb], working through macromolecular crowding, may represent the actual ‘volume’ sensor of red cells [88, 89]. This is supported by more recent findings [90] which showed that white ghosts prepared from HbA or HbS lose volume and pH sensitivity of KCC. Readdition of Hb restored volume dependence, but not the pH sensitivity. Again, differences were apparent between HbA and HbS. With HbA, KCC activity was reduced at all volumes, whilst with HbS, by contrast, activity under hypotonic conditions was actually increased by adding HbS. Similar findings in transgenic mice experiments have shown that expression of HbS or HbC, but not HbA, stimulates KCC activity [91]. How Hb acts is not known, but an obvious membrane interaction concerns its propensity to bind to the cdb3 terminus of band 3, and thereby displace other proteins notably glycolytic enzymes [92, 93]. HbS cells also behave differently from HbA cells in respect of regulation of KCC by O2 tension. Whilst KCC in HbA cells is inhibited by deoxygenation [94], that in HbS cells is not [95, 96]. This feature is important because it would cause KCC in HbS cells to respond to low pH or urea in relatively hypoxic regions of the circulation, like active muscle or the vasa recta. The unusual response to O2 probably underlies the ability of both cyclic and continuous deoxygenation to support dehydration of HbS cells via KCC activity [52, 60, 66]. The deoxygenation-induced component of KCC activity correlates with desaturation of HbS with O2 and also morphological sickling, both in control HbS cells and those treated with ‘left shift’ reagents to increase the O2 affinity of HbS [97]. Crosslinkers, which prevent HbS polymerisation, also affect KCC activity [98]. The mechanism of this altered response to O2 remains unclear. Changes in free intracellular [Mg2⫹] were originally suggested as a possibility [99], but this appears to have relatively minor effects in intact cells. In fact, it has been demonstrated unequivocally that clamping Mg2⫹ does not obviate the different response of HbA and HbS cells [100]. In intact red cells, however, KCC activity in fully deoxygenated sickle cells is about 10% higher than in fully oxygenated cells. When intracellular [Mg2⫹] is carefully controlled, KCC activity is unaffected by full oxygenation/deoxygenation [97, 100]. Deoxygenation, through HbS polymerisation, will reduce the concentration of soluble Hb in the cytoplasm. An intriguing possibility follows, that sequestration of HbS into polymers is perceived as a marked increase in volume. Polymerisation would also presumably alter the displacement of


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other proteins, on sites such as cdb3, and may thereby alter the response of KCC to O2 tension. Regulation by Phosphorylation It has been apparent since seminal work in the early 1990s that protein phosphorylation is involved in regulation of KCC activity [101, 102]. Both serine-threonine and tyrosine residues are involved [103–105] and since gene sequences for KCC have become available it has been possible to identify putative phosphorylation sites. Whilst other transporters, including some of the cation-chloride cotransporters, are regulated directly by phosphorylation, it still remains unclear whether KCC or some regulatory intermediate phosphoprotein is involved. With our increased awareness of regulation of KCC activity by phosphorylation, an obvious possibility is that enzyme activity is altered in HbS cells. Indeed, abnormal protein phosphorylation has been observed in sickle cells over several decades [106, 107]. Syk and Srk PTK inhibitors in HbS cells suggest both stimulation and inhibition via phosphotyrosine residues. Whilst, PP1 activity correlates with KCC activity in both normal and sickle red cells [72, 108, 109], cotransport activity is always higher in sickle cells, suggesting a mechanism independent of membrane bound PP1 activity. One possibility is a defective inhibitory kinase in HbS cells, allowing higher levels of KCC. In addition, PP2a activity is lower in sickle cells compared to normal ones [72], which may suggest that it is not involved, or that HbA cells are simply older. Tyrosine phosphorylation of band 3 is elevated in sickle cells [110], perhaps because of oxidative stress, which is higher in sickle cells, as this stimulates PTKs [111] and may inhibit PTPs [112]. Protein phosphorylation also changes markedly with O2 tension with an overall reduction in phosphorylation of membrane targets inhibited by okadaic acid [43]. This phosphatase inhibitor also inhibits KCC activity in deoxygenated sickle cells [96]. On the other hand, deoxygenation is associated with increased PTK activity (and phosphorylation of band 3) via Src, but not Lyn [113, 114]. The PTK inhibitor tyrphostin T47 inhibits deoxygenation-induced K⫹ loss, probably via an effect on KCC activity [113], whilst the Src inhibitor pp2 inhibits KCC in oxygenated HbS cells. DHP, a Syk inhibitor, also inhibits in deoxygenated cells. These findings suggest that Srk inhibits KCC (hence the relative stimulation of KCC in the knockout mice [115]), whilst deoxygenation relieves this inhibition by activating Syk. In addition, recent evidence that Cl⫺ is required for swelling-induced activation of KCC may indicate the presence of Cl⫺ dependent PP/PKs [116]. Notwithstanding, these experiments tend to look at fully oxygenated or deoxygenated conditions, and none take into account the exquisite relationship between KCC and PO2 (fig. 2).

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120 (i) 100 Activity of Psickle or KCC

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60

40

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20

KCC in HbAA KCC in HbSS Psickle in HbSS

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Fig. 2. O2 dependence of KCl cotransport and Psickle in human red cells. (i) KCl cotransport in red cells from normal (HbAA) individuals is progressively inhibited as the cells are deoxygenated. (ii) By contrast, that in red cells from sickle cell patients (HbSS) shows a deoxygenation-induced component so that, in fully deoxygenated HbS cells, activity is similar (or slightly elevated) compared to that in fully oxygenated cells. (iii) The deoxygenation-induced component of KCC activity follows a similar O2 dependence to activation of Psickle, suggesting a similar underlying cause. (KCC activity was defined as Cl⫺-dependent component of K⫹ influx; Psickle as the K⫹ influx measured in the absence of Cl⫺; ouabain, bumetanide and clotrimazole were present in all cases.)

In summary, whilst our understanding of regulation by protein phosphorylation has improved, and differences in phosphorylation patterns in HbS cells represent an obvious possible mechanism underlying abnormal KCC activity, details remain hazy. The Deoxygenation-Induced Pathway, Psickle Psickle is an unusual permeability pathway characteristic of sickle cells. It is not observed in normal red cells, at least under physiological conditions, but rather becomes apparent when HbS cells are deoxygenated. Amongst other ions, it is permeable to Ca2⫹ and its significance to SCD is largely through mediating deoxygenation increases in intracellular Ca2⫹ and subsequent Gardos channel activation and cell shrinkage. Increased solute permeability becomes apparent at about 40 mm Hg (fig. 2) and is associated with polymerisation of HbS [66, 95, 97]. A shift in O2 affinity of HbS, e.g., by treating cells with 12C79 (which increases


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the O2 affinity of Hb [117], alters the O2 dependence of sickling and Psickle activation in parallel [97]. Thus Psickle activity correlates with polymerisation of HbS and sickling [118–120]. Activation of Psickle appears to be stochastic, occurring in only a proportion of the cells during each episode of deoxygenation [121]. Notwithstanding, how HbS polymers causes the increased membrane permeability of HbS cells remains unknown, although mechanical stress at the membrane imposed by the polymers has been suggested [118]. The organic cations tetramethylammonium, tetraethylammonium and N-methylglucamine are excluded [122]. Ion movements are non-saturable, voltage-dependent and not obligatorily coupled [66, 122]. In addition to representing an ionic permeability, Psickle also appears to be permeable to some non-electrolytes. Although Psickle is apparently impermeable to mannitol and erythritol [123] in cytochalasin B-treated HbS cells, we have recently shown that both sucrose and taurine can enter deoxygenated sickle cells under certain conditions (unpublished data). Notwithstanding these observations, the permeability characteristics of Psickle remain surprisingly poorly defined. A major issue is heterogeneity of red cells [51]. To date, studies on Psickle have necessarily been carried out under conditions which average the behaviour of a large number of cells. As for many of their properties, however, HbS cells are markedly heterogeneous in the magnitude of the permeability increase elicited by Psickle, with a few cells achieving particularly elevated values [124]. Recently, we have addressed this issue by applying the patch clamp technique to single sickle cells. We have demonstrated a deoxygenation-induced increase in whole cell conductance, which shares a number of properties of Psickle: non-selectivity of K⫹ over Na⫹, partial inhibition by DIDS (although this response is variable; fig. 3) and relative impermeability to NMDG. The application of this technique should facilitate progress on understanding Psickle. Finally, the molecular identity of the Psickle pathway remains unknown. A role for band 3 has been postulated [121]. Thus, band 3 is present in the membrane at the greatest copy number, and its inhibitor, DIDS, partially inhibits Psickle [125]. In this context, it is interesting that mutations in band 3 have been reported recently to confer a novel cation conductance in red cells of patients with hereditary stomatocytosis. Alternatively, a failure of the integrity of the lipid bilayer due to mechanical stress, a simple ‘hole’, is a possibility but features of Psickle, such as partial inhibition by some compounds and definite selectivity, suggest that this may not be the case. Furthermore, it has previously been demonstrated that sheer stress can increase cation conductance of HbA erythrocytes [126]. It is, therefore, entirely conceivable that Psickle is simply a manifestation of an endogenous erythrocyte pathway which is potentiated in HbS cells.

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I (pA)

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100

50 ⫺100

⫺50

50

100 Em (mV)

⫺50 ⫺100

Fig. 3. Deoxygenation-induced whole cell currents in red cells from sickle cell patients. Currents were recorded by the patch clamp technique in deoxygenated HbS cells in whole-cell configuration in the absence (open circles) and following addition (closed circles) of DIDS (100 ␮M) at 21⬚C. These currents are the first recordings from sickle cells, are markedly larger than those previously recorded from HbA cells [13] and probably represent the electrical manifestation of Psickle. They share a number of its characteristics, including partial DIDS inhibition. IV curve shows a representative trace of 4 recordings.

Treating SCD by Prevention of Cell Shrinkage Volume decrease via loss of solutes from sickle cells represents a critical feature in the pathogenesis of SCD. It is not surprising, therefore, that the search for inhibitors has been extensive. Provision of effective inhibitors against the three major transport pathways involved in dehydration would be a major advance in treatment of SCD. Although, to date, none have emerged, recent progress has been made. With regards to the electroneutral pathway KCC, although novel reagents such as DIOA and H74 have been useful for defining transporter activity in vitro [127], no specific inhibitors have been found. The transporter is sensitive to intracellular Mg2⫹, however, and modest elevation produces considerable inhibition [99, 128, 129], presumably by altering the PP/PK balance regulating KCC activity. For example, it has been suggested that high Mg2⫹ can inhibit KCC via Src tyrosine phosphorylation of PP1a [130]. Deoxygenated HbS cells have elevated Mg2⫹ permeability, via Psickle [131], and increased activity of Na⫹/Mg2⫹ exchange [132], and a reduction in free intracellular Mg2⫹ in HbS cells has been confirmed recently by NMR studies [133]. The most promising strategy to modulate KCC activity in vivo therefore has been augmentation of dietary Mg2⫹ intake. This procedure increases red cell Mg2⫹ levels, reduces KCC activity and increases red cell hydration in both transgenic mice models of SCD [134] and SCD patients [135]. A longer term clinical trial using oral Mg2⫹


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pidolate in SCD patients has shown similar benefits [136], whilst in the shorter term, intravenous administration of Mg2⫹ reduces the length of stay in hospital following a sickle cell crisis [137]. Oral Mg2⫹ pidolate is now being tested in clinical trials in homozygous SCD and in HbS/HbC (SC) disease, either as a single agent or in combination with hydroxyurea [138]. Inhibition of KCl efflux can also be achieved either by targeting the Gardos channel or the anion conductance of the red cell. The latter remains somewhat enigmatic, but normal levels of anion conductance are required to accompany fast loss of K⫹ concomitant with Gardos channel activation. The most promising approach to inhibition of the Gardos channel inhibition has been via clotrimazole and its derivatives. Clotrimazole is an imidazole antimycotic acting on cytochrome P450-related electron transport, first described as an inhibitor of the Ca2⫹-activated K⫹ channels in normal red cells in the early 1990s [139]. Subsequently, various imidazole compounds were shown to inhibit the Gardos channel in HbS cells in vitro [140] and to reduce sickle cell dehydration in SAD mice [141] and SCD patients [142] in vivo, although liver toxicity is a problem. Derivatives lacking the imidazole ring remain active and its exclusion will lessen potential side-effects [143]. Latterly, a novel compound ICA-17043 has been developed against the Gardos channel and it, too, is effective in SAD mice red cells in vitro and in vivo [144]. Finally, a newer approach has been to inhibit the anion conductance. Several compounds have been designed (e.g., NS1652 and NS3623), and are effective against sickle cell dehydration in vitro [109] and also in SAD mice models in vivo [145, 146]. Lastly, Psickle is an obvious target but although effective inhibitors would be of major benefit, especially by preventing Ca2⫹ entry and Gardos channel activation, none have emerged. Psickle is insensitive to standard cation transport inhibitors (such as furosemide, bumetanide, phloretin, quinine and amiloride [58, 65, 125], and whilst it is partially inhibited by band 3 inhibitors, such as DIDS [123, 125, 147], and also by dipyrimadole [148], these are not useful in vivo. The Ca2⫹ channel blocker nifedipine inhibits Ca2⫹ uptake through Psickle (by 30–40%) [147] and it may be possible to design a specific blocker. In addition, whilst some divalent cations are permeable through Psickle, some also inhibit its conductance. Older studies comparing several divalent cations found that the most effective inhibitor was Mn2⫹, with no significant difference for Ca2⫹, Sr2⫹, Ba2⫹ and Mg2⫹ [149]. Heterogeneity is also important here with inhibition by divalent cations greater in reticulocytes compared to dense cells [58, 149]. Recently, we have demonstrated that both Zn2⫹ and especially Gd3⫹ inhibit whole cell cation currents in deoxygenated HbS cells (unpublished data). Gd3⫹ is well known as an inhibitor of stretch-activated channels. Given that HbS polymers distort the red cell membrane, it may be that Psickle is a manifestation of this type of channel. Inhibition by Zn2⫹ is particularly exciting. Clinical trials with

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Zn2⫹ supplementation have shown improvement of SCD patients [150]. A reduction in Psickle permeability provides a rational explanation. Hereditary Stomatocytoses

Cation Leak and Stomatocytoses In marked contrast to SCD, hereditary stomatocytoses (and the related condition, hereditary spherocytosis) are extremely uncommon, but nevertheless are instructive in appreciating the pathophysiology of red cell volume. It is manifested in a group of rare haemolytic disorders of varying severity which are associated with alterations in cation composition, a changed red cell morphology, and increased cell volume [151, 152]. A significant fraction of these cases are classified as overhydrated stomatocytoses where the cells take on the characteristic shape with a stoma (mouth) in blood films. There is a considerable variability in the aetiology of individual cases, but the general profile of these disorders seems to be a big increase in the leakiness of the cells to Na⫹ and K⫹, with some compensation by an increased Na⫹/K⫹ pump activity, and stabilisation at an elevated cell volume. In most cases the cells have a shorter lifetime than normal red cells, eventually succumbing to haemolysis due to increased Na⫹ entry [152]. Temperature Effects There are two specific characteristics which are defining in many cases of stomatocytosis. The first is a novel, and variable temperature dependence of the passive cation fluxes in these cells, whilst the second is the recent identification of mutant forms of band 3 with this disorder. Ionic balance and volume in red cells is not just an equilibrium between a simple dissipative electrodiffusive cation leak flux and the Na⫹/K⫹ pump which therefore defines cell volume. Experimentally, ouabain and bumetanide, together with Ca2⫹ chelation, have been used to inhibit respectively the Na⫹/K⫹ pump, NKCC1 and the Gardos channel, but there may be additional systems (K⫹/H⫹ exchange [153], KCC, Na⫹-linked transporters) which may be components of the residual ‘leak’ flux. This is apparent when cation fluxes in the presence of the inhibitors outlined above are measured as a function of temperature in normal red cells (fig. 4a, open circles). Although K⫹ transport decreases on cooling as expected, the slope is rather shallow, and it passes through a minimum at 8⬚C and then rises, being significantly higher at 0⬚C than at 8⬚C. Cells from stomatocytic patients show important variations in this temperature profile, with about eight different possibilities. In particular, one large group show an increased permeability to K⫹ and Na⫹ at 37⬚C compared to controls (fig. 4a,


258

K+ influx

1.0

0.5

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Ser731Pro His734Arg Arg760Gln

1

2

3

4

5

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N-terminus Leu687Pro

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Fig. 4. a Temperature dependence of K⫹ (86Rb⫹) influx into erythrocytes from patients with cryohydrocytosis (closed circles) and healthy controls (open circles). For detail refer to text. b The membrane topology of band 3 illustrating amino acid substitutions which have been shown to account for hereditary stomtocytoses. For detail refer to text.

closed circles), which is exaggerated on cooling, to the extent that the condition has been identified as pseudohyperkalaemia, i.e. enough K⫹ leaks from the cells on cold (4⬚C) storage to give a false signal of raised circulating plasma K⫹. Cold storage becomes a volume stress, with cell swelling via Na⫹ entry and haemolysis in the cold [6].

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Stomatocytoses as Diseases of Band 3 Recently, a substantial number of these patients have been identified as demonstrating a mutation in the band 3 anion transport protein, with single point changes between amino acids 687–760 (fig. 4b) [154, 155]. Oocyte expression experiments revealed that these band 3 mutations result in cation rather than anion transport via band 3, essentially converting this anion exchanger into a nonselective monovalent cation channel, and thus accounting for the increased K and Na leak in these cells. There remains the question of the altered temperature sensitivity of this transport. It is tempting to speculate that the mutant band 3 has to adopt a particular conformation to function as a cation channel, and that the transition to this form is temperature-sensitive, i.e. facilitated by cooling. Temperature effects on band 3 are complex, and the region of the molecule where the mutations occur is not well-studied functionally. Dimerisation/oligomerisation may also be important in this aberrant band 3 function. Nevertheless, it is clear that abnormal band 3 function explains many, but not all (some may involve the protein stomatin band 7) cases of overhydrated stomatocytoses.

Future Perspectives

One major common property of red cells which emerges from the present brief review is the presence of quiescent non-selective cation channels (NSCCs) in the normal human red cell membrane [156–158]. Calcium influx through such NSCCs plays a role in promoting shrinkage via activation of the Gardos channel [25]. Further effects of calcium entry include activation of a scramblase altering extracellular exposure of PS [27] and triggering of other autocrine and paracrine effects [23]. Although showing differences in some characteristics, including inhibitor profiles and permeability to non-electrolytes, such NSCCs are active in disease processes such as malaria infection [159], and the two pathologies (SCD, overhydrated stomatocytoses) described here, and it is tempting to speculate that a common mechanism, possibly involving modification of band-3, being involved [160]. One further factor common to the pathophysiology of these conditions is oxidative stress, originating from reactive oxygen species generated at high levels in malaria [161] and SCD, and in haemolytic disorders such as glucose-6-phosphate dehydrogenase deficiency [162, 163]. Certainly, treatment of red cells with peroxynitrite [164] or t-butylhydroperoxide induces a reversible cation permeability via NSCCs [8]. Furthermore, modest shear forces produce channel activity which is greatly enhanced by oxidative stress [126, 165]. At present, the lack of good inhibitors makes definition of NSCCs difficult, but it is to be hoped that in the future such compounds will be identified so


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that the relationship betweem NSCCs and band-3 can be explored further, with the possibility of a common mechanism emerging for regulating cell shrinkage, particularly in pathophysiological situations such as SCD. Acknowledgements The authors would like to thank Action Medical Research and The Wellcome Trust for their support, and Miss Hannah Robinson for her technical assistance with the preparation of this manuscript. JAB is currently a Foulkes Foundation Fellow and is grateful for their support.

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Bernhardt I, Ellory JC: Red Cell Membrane Transport in Health Disease. Berlin, Springer-Verlag, 2003. Brugnara C: Erythrocyte membrane transport physiology. Curr Opin Hematol 1997;4:122–127. Tosteson DC, Hoffman JF: Regulation of cell volume by active cation transport in high and low potassium sheep red cells. J Gen Physiol 1960;44:169–194. Lew VL, Bookchin RM: Volume, pH, and ion-content regulation in human red cells: analysis of transient behavior with an integrated model. J Membr Biol 1986;92:57–74. Staines HM, Ellory JC, Kirk K: Perturbation of the pump-leak balance for Na(⫹) and K(⫹) in malaria-infected erythrocytes. Am J Physiol Cell Physiol 2001;280:C1576–C1587. Jarvis HG, et al: Cold storage of ‘cryohydrocytosis’ red cells: the osmotic susceptibility of the cold-stored erythrocyte. Br J Haematol 2003;122:859–868. Ellory JC, Gibson JS, Stewart GW: Pathophysiology of abnormal cell volume in human red cells. Contrib Nephrol 1998;123:220–239. Duranton SM, Huber SM, Lang F: Oxidation induces a Cl-dependent cation conductance in human red blood cells. J Physiol 2002;539:847–855. Huber SM, Gamper N, Lang F: Chloride conductance and volume-regulatory non-selective cation conductance in human red blood cell ghosts. Pflugers Arch 2001;441:551–558. Kaestner L, Bollensdorff C, Bernhardt I: Non-selective voltage-activated cation channel in the human red blood cell membrane. Biochim Biophys Acta 1999;1417:9–15. Rodighiero S, De Simoni A, Formenti A: The voltage-dependent nonselective cation current in human red blood cells studied by means of whole-cell and nystatin-perforated patch-clamp techniques. Biochim Biophys Acta 2004;1660:164–170. Nazaret C, et al: Inhibition of the Cl⫺/NaCO⫺ 3 anion exchanger by xipamide in human red blood cells. Eur J Pharmacol 1987;144:353–362. Desai SA, Bezrukov SM, Zimmerberg J: A voltage-dependent channel involved in nutrient uptake by the red blood cells infected with the malaria parasite. Nature 2000;406:1001–1004. Zidek W, et al: Intracellular chloride in essential hypertension. Clin Sci (Lond) 1985;68:45–47. Wilkins RJ, Browning JA, Ellory JC: Cell volume regulation reduced to size; in Kozlowski R (ed): Chloride Channels. Oxford, Isis Medical Publishing, 1999, pp 19–33. Deutsch CJ, et al: Transmembrane electrical and pH gradients across human erythrocytes and human peripheral lymphocytes. J Cell Physiol 1979;99:79–93. Hunter MJ: Human erythrocyte anion permeabilities measured under conditions of net charge transfer. J Physiol 1977;268:35–69. Hoffman JF, Laris PC: Membrane electrical parameters of normal human red blood cells. Soc Gen Physiol Ser 1984;38:287–293. Talekar SV, Bakore PV, Talekar VL: Electrical conductance characteristics of some human erythrocyte membranes. Phys Med Biol 1976;21:989–992.

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Professor J. Clive Ellory Department of Physiology, Anatomy and Genetics Sherrington Building, Parks Road Oxford OX1 3PT (UK) Tel. ⫹44 1865 272436, Fax ⫹44 1865 272488, E-Mail [email protected]


268

Author Index

Alexander, R.T. 105 Beck, F.-X. 181 Browning, J.A. 241 Burg, M.B. 125

Häussinger, D. 198 Hoffmann, E.K. 54 Huber, S.M. 142

Ritter, M. 142, 161 Jakab, M. 161

Ellory, J.C. 241

Lang, F. 1, 142

Ferraris, J.D. 125 Friedrich, B. 1

Matskevich, I. 1

Gibson, J.S. 241 Grinstein, S. 105 Gulbins, E. 142

Pasantes-Morales, H. 221 Pedersen, S.F. 54

Schliess, F. 198 Shumilina, E. 142 S¤tra´ k, V. 210 Tuz, K. 221

Neuhofer, W. 181 Okada, Y. 9

Vereninov, A. 142 Wehner, F. 25

269

Subject Index

Actin cytoskeleton cell migration 168–170 chloride sensing regulation 81, 82 osmolyte transport regulation 81 phospholipase regulation 82 protein kinase regulation 82–86 rearrangement in cell volume perturbation 80, 81, 126, 127 sodium/potassium/2chloride cotransporter cell shrinkage regulation role 70, 71 swelling-activated calcium influx regulation 81 swelling-activated potassium channel regulation 57 volume-sensitive outwardly rectifying chloride channel regulation 59, 61 Apoptosis eryptosis, see Red blood cell hepatocyte apoptotic volume decrease 202–205 ion transport and cell volume 146–149 neuronal apoptotic volume decrease 230–236 triggers 143 Aquaporins cell migration role 172 TRPV4-AQP5 interactions 170 volume regulation 55 ATM medullary cell osmolyte accumulation regulation 189 TonEBP regulation 133, 134

Band 3 anion transport protein, stomatocytosis mutations 260 Betaine medullary cell extracellular osmolality increase response 185 osmolyte activity 5 release 7 transport 6, 7 BKCa, regulatory volume decrease and activation 31, 32, 56 Calcium influx apoptosis 149–152 neuron necrosis role in swelling 229, 230 regulatory volume decrease response 57 swelling-induced secretion studies 215 Calcium-sensitive potassium channels cell migration role 169 Gardos channel, eryptosis ion transport and cell volume regulation 248–250 Calmodulin sodium/proton exchanger cell shrinkage regulation role 66 swelling-activated taurine efflux regulation 63 CD95 hepatocyte apoptotic volume decrease role 203, 204 voltage-gated potassium channel regulation 148 Cell migration cell shrinkage role 165–167 cell swelling role 164, 165

270

cytoskeleton, intracellular calcium homeostasis, and cell volume 168 locomotion principles 162, 163 modes 162 polarized distribution of cell volume regulatory ion transport mechanisms 172 substrate adhesion, cell spreading, and cell volume 170–172 Cell proliferation, ion transport, and cell volume 143–146 Cell-swelling-induced secretion, see Swelling-induced secretion Chloride apoptotic transport 146–149 equilibrium 3 neuronal apoptotic volume decrease role 233–235 uptake following cell shrinkage 5, 10 ClC-3, volume-sensitive outwardly rectifying chloride channels 17, 18 Cyclooxygenase-2 (COX-2), medullary cell osmolyte accumulation regulation 190 Cytoskeleton, see Actin cytoskeleton DNA breakage, hypertonicity induction 126 Epidermal growth factor receptor (EGFR), volume sensing 74, 75, 110 Epithelial sodium channel (ENaC), hypertonicity-induced amiloride-sensitive cation channels 27 Eryptosis, see Red blood cell Erythrocyte, see Red blood cell Focal adhesion kinase (FAK) actin cytoskeleton regulation 82, 83 cell migration role 171 Fyn medullary cell osmolyte accumulation regulation 189 TonEBP regulation 132, 133 Gardos channel, see Calcium-sensitive potassium channels

Subject Index

Glutamate, neuronal hypoosmotic efflux 222, 223 Glycerophosphorylcholine (GPC) medullary cell extracellular osmolality increase response 186, 187 metabolism and accumulation 6 osmolyte activity 5 release 7 Hepatocyte apoptotic volume decrease 202–205 hydration and cell function 198 osmosensing and signaling insulin 201, 202 integrins 199, 200 mitogen-activated protein kinase 199 Hereditary stomatocytoses band 3 anion transport protein mutations 260 red blood cell cation leak 258 temperature effects on red blood cells 258, 259 Hypertonicity-induced cation channels shrinkage-induced activation 72 types 72 Hyponatremia, neuroprotection 225–228 Insulin glucose-versus swelling-induced insulin secretion 215, 216 hepatocyte osmosensing and signaling 201, 202 Integrins hepatocyte osmosensing and signaling 199, 200 medullary cell osmolyte accumulation regulation 190 substrate adhesion, cell spreading, and cell volume 170, 171 TonEBP regulation 135 volume sensing 74 Inwardly rectifying potassium (Kir) channels classification 38 pharmacology 38 regulatory volume decrease and activation 38

271

Kir channels, see Inwardly rectifying potassium channels K2P channels neuronal apoptotic volume decrease role 233 regulatory volume decrease and activation 36, 37 Kv channels, see Voltage-gated potassium channels Lactic acidosis, swelling and neuron necrosis role 230 Lipids regulation of swelling-activated potassium channels 57, 58 sodium/proton exchanger cell shrinkage regulation role 67 swelling-activated taurine efflux regulation 63 Maxi potassium channels, regulatory volume decrease and activation 31, 32 Medullary cells extracellular osmolality decrease response 182 increase response betaine 185 glycerophosphorylcholine 186, 187 ion flux 183–185 myoinositol 185, 186 sorbitol 186 taurine 187 osmolyte accumulation regulation cyclooxygenase-2 190 extracellular matrix interactions 190 post-transcriptional regulation 191 protein kinases 188–190 reactive oxygen species 190 transcriptional regulation 187, 188 urea 191 Mitogen-activated protein kinase (MAPK) actin cytoskeleton regulation 84–86 hepatocyte osmosensing and signaling 199 medullary cell osmolyte accumulation regulation 188, 189 neuronal apoptotic volume decrease role 234

Subject Index

regulatory volume decrease regulation 109, 110 TonEBP regulation by p38 116, 117, 131, 132 Myoinositol, medullary cell extracellular osmolality increase response 185, 186 Myosin light chain kinase (MLCK), sodium/potassium/2chloride cotransporter cell shrinkage regulation role 70, 115, 116 Nck-interacting kinase, sodium/proton exchanger cell shrinkage regulation role 67 Neuron apoptotic volume decrease 230–236 brain cell volume and hyperexcitability 222, 223, 225 hyponatremia and neuroprotection 225–228 swelling and necrosis 228–230 Non-selective cation channels, eryptosis ion transport and cell volume regulation 247, 248 OREBP, see TonEBP Paraventricular nucleus (PVN), swellinginduced secretion 212–214 Peptide secretion, see Swelling-induced secretion Phosphoinositide 3-kinases, TonEBP regulation 134 Phospholemman (PLM) activation in cell volume change 42, 43 function 42 structure 42 Phospholipase A2 (PLA2) cell volume regulation 77 isoforms 76 Potassium release following cell swelling 4, 26, 30 uptake following cell shrinkage 5, 10, 31 Potassium/chloride cotransporter (KCC) isoforms 64, 251 regulatory volume decrease regulation 111

272

sickle cell disease cell volume regulation 251, 252 expression alterations 250, 251 oxygen tension effects 252, 253 phosphorylative regulation 253, 254 structure 64 swelling activation regulation by phosphorylation 64 Protein kinase A (PKA) medullary cell osmolyte accumulation regulation 189 TonEBP regulation 133 Protein kinase C (PKC) regulatory volume decrease response 107–109 sodium/potassium/2chloride cotransporter as substrate 115 Protein phosphatase-1, sodium/proton exchanger cell shrinkage regulation role 67 Reactive oxygen species (ROS) cell volume changes and release 77 hypertonicity induction 126 medullary cell osmolyte accumulation regulation 190 swelling-activated taurine efflux regulation 63, 64 TonEBP regulation 134, 135 volume-sensitive outwardly rectifying chloride channel regulation 61 Red blood cell eryptosis ion transport and cell volume regulation Gardos channel 248–250 non-selective cation channel 247, 248 overview 149–151, 247 ion transport and cell volume regulation leak pathways and cell integrity 242–247 overview 241, 242 non-selective cation channels 260, 261 sickle cell disease, see Sickle cell disease stomatocytoses, see Hereditary stomatocytoses Regulatory volume decrease (RVD) apoptotic volume decrease, see Apoptosis

Subject Index

calcium flux in response 57 cation channel activation 26–29, 39–42 chloride channels, see Volume-sensitive outwardly rectifying chloride channels overview 3, 10, 105 peptide secretion 211 Regulatory volume increase (RVI) overview 3, 10, 106, 107, 112 potassium channel inhibition 30, 31 Rho, cell volume regulation 78, 80, 110 RNA helicase A, TonEBP regulation 135 Serum- and glucocorticoid-inducible kinase (SGK), voltage-gated potassium channel regulation 35 Sickle cell disease (SCD) cell shrinkage prevention in treatment 256–258 cell volume abnormalities 250 deoxygenation-induced pathway 254, 255 potassium/chloride cotransporter cell volume regulation 251, 252 expression alterations 250, 251 oxygen tension effects 252, 253 phosphorylative regulation 253, 254 SK channels genes 32 regulatory volume decrease and activation 33, 34, 56 SK4 features 33 structure 32, 33 Sodium release following cell swelling 4, 26 swelling and neuron necrosis role 229, 230 uptake following cell shrinkage 5, 10 Sodium/calcium exchanger, cell migration role 169 Sodium/potassium ATPase cell volume regulation 30 function 3 medullary cells 183, 184 Sodium/potassium/2chloride cotransporter cell proliferation response 145 isoforms 69

273

Sodium/potassium/2chloride (continued) NKCC1 regulation by cell shrinkage actin cytoskeleton 70, 71 myosin light chain kinase 7, 115, 116 phosphorylation 70, 115, 116 protein-protein interactions 69–72 SPAK 71, 72 Sodium/proton exchanger (NHE) cell migration role 164, 165, 168–171 cell proliferation response 145 isoforms 65 NHE1 regulation by cell shrinkage calmodulin 66 lipids 67 Nck-interacting kinase 67 overview 66 phosphorylation 67, 69 protein phosphatase-1 67 protein-protein interactions 66, 67 regulatory volume increase role 113–115 Sorbitol medullary cell extracellular osmolality increase response 186 metabolism and accumulation 6 osmolyte activity 5 release 7 transport 6, 7 SPAK, see Ste20-related kinases Src kinases, actin cytoskeleton regulation 83 Ste20-related kinases (SPAK) actin cytoskeleton regulation 83 sodium/potassium/2chloride cotransporter cell shrinkage regulation role 71, 72 Stomatocytoses, see Hereditary stomatocytoses Supraoptic nuclei (SON), swelling-induced secretion 212–214 Swelling-induced secretion hypothalamic stimulation 212–214 peptides and proteins 212 physiological significance 217 research applications 216, 217 signaling

Subject Index

biophysical effect 216 calcium influx 215 glucose-versus swelling-induced insulin secretion 215, 216 overview 214, 215 Taurine hypertonicity response 127 medullary cell extracellular osmolality increase response 187 neuronal apoptotic volume decrease role 236 neuronal hypoosmotic efflux 223 osmolyte activity 5 swelling-activated efflux overview 7, 61, 63 regulation calcium and calmodulin 63 lipids 63 reactive oxygen species 63, 64 tyrosine phosphorylation 63 transport 6, 7, 111 Thyrotropin-releasing hormone (TRH), see Swelling-induced secretion TonEBP hypertonicity activation dimerization 128 DNA binding 130, 131 late response 116, 117 nuclear localization 129, 130 pathway interactions 135–137 phosphorylation 129 transactivational activity 131 transcription induction 128 medullary cell osmolyte accumulation regulation 187, 188 osmoprotection 127 regulation ATM 133, 134 Fyn 132, 133 integrins 135 mitogen-activated protein kinase p38 116, 117, 131, 132 phosphoinositide 3-kinases 134 protein kinase A 133 reactive oxygen species 134, 135 RNA helicase A 135

274

Transient receptor potential (TRP) channels classification 40 function 41, 42 structure 40 subunits 40, 41 TRPV4-AQP5 interactions 170 volume sensing 75, 76 Urea, medullary cell osmolyte accumulation regulation 191 Voltage-dependent anion channel (VDAC), neuronal apoptotic volume decrease role 234 Voltage-gated potassium (Kv) channels CD95 regulation 148 cell migration role 166 distribution 34 KCNE1 subunit 35, 36 neuronal apoptotic volume decrease role 232, 233 regulation 35 regulatory volume decrease and activation 34, 35, 56 structure 34

Subject Index

Volume-sensitive outwardly rectifying (VSOR) chloride channels antagonists 15, 16 ATP dependence 14, 16 candidate proteins 16–18 electrophysiology 13, 14 functions 18, 19 neuronal apoptotic volume decrease role 233, 234 nomenclature 11 pathology 19 properties of human intestinal cell channels 11, 13–15 swelling activation cytoskeleton regulation 59, 61 phosphorylative regulation 61, 109–111 reactive oxygen species regulation 61 Water, movement across membranes 1, 2 With-no-lysine kinase (WNK) actin cytoskeleton regulation 84 sodium/potassium/2chloride cotransporter cell shrinkage regulation role 71, 72

275

Mechanisms and significance of cell volume regulation

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Mechanisms and significance of cell volume regulation

Mechanisms in Transcriptional Regulation

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Apoptosis, Cell Signaling, and Human Diseases: Molecular Mechanisms, Volume 2

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Regulation of Sertoli Cell and Germ Cell Differentiation (Advances in Anatomy, Embryology and Cell Biology)

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