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Progress in Biophysics and Molecular Biology 108 (2012) 93e97

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Progress in Biophysics and Molecular Biology

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Review

Roles of cell volume in molecular and cellular biology

Jean-Marc Dubois*, Béatrice Rouzaire-Dubois 1

CNRS, Institut de Neurobiologie Alfred Fessard-FRC2118, Gif sur Yvette F-91198, France

a r t i c l e i n f o

Article history:Available online 13 December 2011

Keywords:Macromolecular crowdingIon channelsAquaporinsCancer cells

* Corresponding author. Fax: þ33 169 82 41 41.E-mail address: jmdubois@nbcm.cnrs-gif.fr (J.-M.

1 Passed away.

0079-6107/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.pbiomolbio.2011.12.001

a b s t r a c t

Extracellular tonicity and volume regulation control a great number of molecular and cellular functionsincluding: cell proliferation, apoptosis, migration, hormone and neuromediator release, gene expression,ion channel and transporter activity and metabolism. The aim of this review is to describe these effectsand to determine if they are direct or are secondarily the result of the activity of second messengers.

� 2011 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 932. Proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 933. Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 944. Migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 955. Hormone and neuromediator release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 956. Gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 957. Activation of ion channel and transporters activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 968. Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 969. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

1. Introduction

The cell volume and its regulation play a role in a great numberof molecular and cellular functions (Fig. 1). Beyond the change inextracellular osmolarity, the cell volume is dependent on severalparameters including the activity of ion channels and transporters(Wehner et al., 2003). Among these parameters the acute andchronic fluxes of monovalent ions (thick line in Fig.1) are importantregulators of cell volume, which in turn control several molecularand cellular functions. From this point of view, the activity of Kþ

and Cl� channels are, in relationship with other ions and moleculesincluding ATP and Ca2þ, a key feature of cell biology. For this reason,

Dubois).

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we have developed a new hypothesis based on many observationsthat cell volume controlled by ion fluxes is responsible for severalmolecular and cellular functions through membrane tension,concentration of several messengers and macromolecular crowd-ing, i.e. the relative concentration of intracellular molecules(Rouzaire-Dubois et al., 2004, 2009). In this review we describe theroles of cell volume in molecular and cell biology and in each casewe try to propose a physico-chemical interpretation of these roles.

2. Proliferation

During the cell cycle, cells must double their volume so thatdaughter cells have the same size that themother cell. In the 1970’s,(Cone,1971) and (Cone and Cone,1976) proposed a “unified theory”suggesting that intracellular Naþ level was of major mitogenicimportance. They showed that, in differentiated neurons from thecentral nervous system of chick embryo spinal cord, DNA synthesis

Metabolism

Activation of ion andtransporter pathways

Na+,K+Channels

and Cl-

CellVolume

Proliferation

Apoptosis

Migration

Hormone andneuromediator release

Geneexpression

Fig. 1. Schema showing roles of cell volume in molecular and cellular biology.

0

0.2

0.4

0.6

0 0.5 1 1.5 2 2.5Cell Volume (pl)

Rat

e of

cel

l pro

lifer

atio

n

Fig. 2. According to our results on rat glioma cells (Rouzaire-Dubois et al., 2004) therate of cell proliferation as a function of cell volume can be described by a bell-shapedcurve resulting from the combination of two inverse Boltzmann equations. Oneinterpretation of such a curve is that in different cell volume ranges, macromolecularcrowding reversibly activates cyclins and cyclin dependent kinase inhibitors.

J.-M. Dubois, B. Rouzaire-Dubois / Progress in Biophysics and Molecular Biology 108 (2012) 93e9794

and mitosis were induced by depolarization with different agentsthat produce a rise in the intracellular sodium ion concentrationand a decrease in the potassium ion concentration. The pharma-cological agents used were oubaine, veratridine and gramicidin.These agents induced a 3e20-fold increase in DNA synthesis andmitotic activity. The authors concluded that RNA and DNA synthesiswere caused by a significant shift in the intracellular Naþ level or tothe Naþ to Kþ ratio. They interpreted their results as due to a celldepolarization but did not propose any explanation of how celldepolarization and/or a rise in intracellular Naþ concentrationincrease mitosis.

In 1984, De Coursey et al. observed that in humanT lymphocytesstimulated by phytohaemagglutinin, which is a potent mitogen forT lymphocytes, the pharmacological blockade of Ca2þ-activated andvoltage- dependent Kþ channels inhibited in a dose dependentmanner both 3H-Thymidine uptake and mitogenesis. While theseresults were clear the authors did not propose any interpretation ofhow Kþ channels control mitogenesis.

The fact that Kþ channel blockade inhibited mitogenesis andshould have induced a cell depolarization is in apparent contra-diction with the results of Cone and Cone (1976) who suggestedthat cell depolarization increased mitogenesis. However, it shouldbe noted that the experiments of Cone and Cone and De Courseyet al. weremade on different preparations namely: mature neuronson one hand and phytohaematogglutinin-stimulated T lympho-cytes on the other hand. The opposite effects of cell depolarizationcan be explained if the relationship between mitosis and somephysico-chemical parameters is a bell shape (see below). Later on,the Cones’ hypothesis has been abandoned but a great number ofreports showed that, in different cell types, the blockade of Kþ

channels was associated with an inhibition of mitogenesis and cellproliferation (reviewed by Dubois and Rouzaire-Dubois, 1993;Pardo, 2004; Wonderlin and Strobl, 1996).

Two hypotheses have been proposed to take account of the roleof Kþ channels in proliferation. The first one is that cell depolar-ization reduces the electrochemical gradient and the influx of Ca2þ

through non-voltage dependent Ca2þ channels. This has beendemonstrated under voltage-clamp conditions in human mela-noma cells (Nilius et al., 1993). Given that Ca2þ is necessary for celldivision (Munaron, 2002; Santella, 1998) this can explain thata decreased Ca2þ influx reduces cell mitosis. However, this inter-pretation does not take into account all the effects of Kþ channelson cell proliferation. First, tumour cells are almost insensitive toexternal Ca2þ concentration (Rouzaire-Dubois and Dubois, 2004).Second, Kþ channel blockade does not always significantly inducea cell depolarisation whereas it induces an inhibition of cell

proliferation (Rouzaire-Dubois and Dubois, 1991). Third, dependingon cell types, depolarization can induce either increase or inhibi-tion of mitosis.

More recently, we have proposed a new interpretation on therole on mitosis of ion channels and ion fluxes associated withosmotically water obliged fluxes (Rouzaire-Dubois and Dubois,1998; Rouzaire-Dubois et al., 2000). According to this hypothesis,the activity of proteins involved in cell cycle progression isdependent on their water environment, which is defined by themacromolecular crowding. On the basis of this theory, we showedon rat glioma cells that an increase in cell volume of small cellsincreased their rate of proliferation whereas an increase in cellvolume from its optimal value decreased their rate of proliferation.In other words, the rate of cell proliferation is a bell shape functionof the cell size (Fig. 2). According to Tzur et al. (2009), “the proba-bility of cell division varies independently with cell size and cellage”. However in the same article, the authors noted that “thelikelihood of division increases with cell size”. Moreover, the sameauthors emphasised that “beyond a critical size cell, the trend isreversed and growth rates decline with increased size”. Theseconclusions are in agreement with our observations (Rouzaire-Dubois et al., 2004) that the rate of cell proliferation is a bellshape function of the cell size.

In conclusion the precise mechanisms by which ion channelsregulate proliferation remain an enigma. The “Ca2þ theory” hasbeen the object of numerous reports (see Lang et al., 2005) but doesnot systematically explain the role of Ca2þ and depolarization incell proliferation. In contrast, the “volume theory” that we devel-oped has never been contradicted. In particular, Koegel et al. (2003)showed that the down-regulation of Ca2þ-activated Kþ channels inkeratinocytes decreased the rate of proliferation and increased thecell size. In view of all these observations, we conclude that the“volume theory” through macromolecular crowding is a betterinterpretation that the “Ca2þ theory” to explain the role of ionchannels and ion fluxes in cell proliferation.

3. Apoptosis

Programmed cell death or apoptosis is important in tissuemaintenance to compensate for cell proliferation by cell death.Apoptosis is characterised by DNA fragmentation, mitochondrialdamage and apoptotic volume decrease (AVD) that result inminimal inflammation to the surrounding tissue. In vitro, apoptosiscan be induced by staurosporine that induces a rapid AVD. This cell

J.-M. Dubois, B. Rouzaire-Dubois / Progress in Biophysics and Molecular Biology 108 (2012) 93e97 95

shrinkage is due to a Cl�, Kþ and osmotically obliged water effluxinducing a cytoplasmic condensation.

Recently, Ernest et al. (2008) showed that such a decrease in cellvolume could be prevented by inhibiting the efflux of Cl� throughchannels by the non-specific inhibitors NPPB, Cd2þ, DIDS and theKþ-Cl� inhibitor DIOA. All of these agents suppressed cell shrinkageand apoptosis and the authors concluded that cytoplasmiccondensation is necessary and sufficient to induce cell death.However, during prolonged cultures of rat glioma cells, we showedthat the cell volume decreased by about 50% whereas thepercentage of apoptotic cells remained inferior to 1% (Rouzaire-Dubois et al., 2004). Hence, we conclude that if cytoplasmiccondensation is necessary, it is not sufficient to induce apoptosis.Consequently, if the non-specific inhibitors of ion efflux reduce thecell size, they do not inhibit the intrinsic and extrinsic pathwaysthat induce DNA, mitochondrial damage and apoptosis.

4. Migration

Cells that present an aberrant migration, notably cancer cells,have the ability to modulate their volume and shape to movethrough the extracellular spaces. This property has been very wellstudied on gliomas that are the most deadly cancers. Recent studieson glioma cells in isotonic conditions showed that the changes incell volume and shape involved the secretion of Cl� and Kþ ionstriggered by an increase in intracellular Ca2þ (Wondergem et al.,2008) in association with water movements through aquaporin 1that is particularly expressed in glioma cells (McCoy andSontheimer, 2007; McFerrin and Sontheimer, 2006; Soroceanuet al., 1999). During migration in spatially restricted conditions,the cell shrink and become flat (Fig. 3) so that they invade thenarrow and tortuous spaces between normal cells and inducemetastasis.

Directional movement is a property common to all cell types.This movement is triggered by calcium influx through stretch-activated cation channels and by means of calcium inducedcalcium release (Wei et al., 2009, 2010). This local increase incalcium concentration triggers flickering of calcium microdomainsthat induce cytoskeleton dynamics such as actin remodelling, focaladhesion detachment and relocation, and cell migration (Fig. 3). Inorder to describe the migration of cancer cells, blockers of differentCl� and Kþ channels (chlorotoxine, tamoxifen and TEA) can be usedin several cancers and particularly in gliomas. However, thistherapy is inefficient because these drugs are toxic for normal cells.

In view of these conclusions, it should be noted that during G1phase in culture, the glioma cells are biphasic and flat. They diffuseand invade the Petri dish. In contrast, in G2M phase, the cells

Fig. 3. Schematic representation of cell shape change that occurs during invasion intospatially restricted pathways. N denotes the nuclear region. In isotonic conditions, thechange in Kþ and Cl�, accompanied with calcium flicker or triggered by Ca2þ influx andosmotically obliged water efflux, induces a change in cell shape and volume. The cellsbecome flat and can diffuse through narrow tortuous spaces between normal cells.(modified from Soroceanu et al., 1999 and Tzur et al., 2009).

become spherical and divide (personal observation). One way toimpede cell proliferation and invasion is to increase the cell volumewith different pharmacological agents (Rouzaire-Dubois et al.,2000). A remaining question is why cancer cells have the facultyto migrate out of their original tissue whereas normal cells do notdiffuse through the tortuous extracellular space? One possibleexplanation is that normal cells possess strong mechanisms ofregulatory volume increase (RVI) so that they remain larger thanthe extracellular restricted spaces.

5. Hormone and neuromediator release

In various cell types including endocrine cells, neurons, leuco-cytes and exocrine pancreatic cells, cell swelling induced by anhyposmotic stress or by an isosmolar medium containing permeantmolecules such as ethanol or urea causes an immediate release ofpeptide hormones and enzymes (for review see Strbak and Greer,2000).

During cell swelling, the secretion of tyrosine releasinghormone, oxytocine, prolactine and insulin was not depressed byinhibition of stretch-activated channels, mercury-sensitive aqua-porins, protein kinase C, microtubules and microfilaments, NaeKATPase, Naþ and Kþ channels, Ca2þ influx and does not involvearachidonic acid metabolism, prostaglandins, and leucotrienes(Najvitova et al., 2003 and personal communication). These authorsconclude that hyposmotic medium does not trigger peptide releaseby a specific receptor-ligand binding but likely by its biophysicaleffect on secretory vesicles.

At the level of the nerve terminal, the effect of tonicity wasstudied by measuring the frequency of miniature end platepotentials (fMEPP) on innervating muscles. In this case, fMEPP isthe frequency of acetylcholine release (see review by Van der Klootand Molgo, 1994). Hypertonic solutions increase fMEPP whilehypotonic solutions decrease fMEPP. The effects of changing thetonicity in solutions are transitory evenwhen the extracellular Ca2þ

concentration is low. Black widow spider venom that increases theinflux of Naþ and Ca2þ (via the Naþ/Ca2þ exchanger) does notincrease spontaneous quantal release, which reflects the quantityof Ach molecules liberated during each MEPP. However, if thedivalent cation-free venom containing solutions is made hyper-tonic, fMEPP reversibly rises 20-fold (Misler and Hurlbut, 1979).

The effect of hypertonicity on fMEPP was also observed byexposure to hypertonic LiCl or KCl solutions and with permeablesolutions like glycerol, D- or L- glucose. Together, these observationssuggest that the cell volume and the intracellular Ca2þ concentra-tion,which is necessary for neuromediator release,modulate fMEPP.Consequently, hypertonicity and cell shrinkage increase intracel-lular Ca2þ and neuromediator release while hypotonicity and cellswelling decrease Ca2 and neuromediator release.

6. Gene expression

In mammalian kidney, the extracellular tonicity varies widelydue to the accumulation of NaCl. In order to avoid excessive cellshrinkage and apoptosis, the tonicity-responsive enhancer bindingprotein (TonEBP) regulates transcription of tonicity-responsivegenes (see review by Jeon et al., 2006). Under hypertonicity theosmotic gradient across the plasma membrane is maintainedconstant by the accumulation of compatible osmolytes such asmyo-inositol, betaine, sorbitol, taurine, and glycerophosphor-ylcholine (Garcia-Perez and Burg, 1991). In response to hyper-osmolarity TonEBP is translocated to the nucleus and stimulates theexpression of transporters including the sodium-myo-inositolcotransporter and the sodium-chloride-betaine cotransporter(Woo et al., 2000).

J.-M. Dubois, B. Rouzaire-Dubois / Progress in Biophysics and Molecular Biology 108 (2012) 93e9796

While TonEBP is widely expressed in mammalian kidney, it isalso expressed in various cells and tissues including bacteria, yeast,brain, endothelia and macrophages where it stimulates the accu-mulation of compatible osmolytes when they are exposed tohypertonicity (Handler and Kwon, 2001; Woo et al., 2000). Wepostulate that hypertonicity induces cell shrinkage that stimulatesthe expression of TonEBP and in turn the intracellular accumulationof NaCl and osmolytes by a process of RVI according to the scheme:

Hypertonicity

Cell shrinkage

Expression of TonEBP

Influx of NaCl and compatible osmolytes

Regulatory volume increase

7. Activation of ion channel and transporters activity

For several decades, it has been known that animal cells regulateprecisely their volume (Tosteson and Hoffman, 1960). Followinganisotonic challenges are changes in ion transport, and the cellvolume increases or decreases. Generally, these volume changes arecompensated by regulatory volume decrease (RVD) or increase(RVI) mediated by transport of ions through volume-activatedtransporters or channels (reviewed by Sarkadi and Parker, 1991)and osmotically obliged water fluxes through aquaporins.

The volume-activated transporters include the Kþ-Cl� cotrans-porter, the Kþ/Hþ exchanger, the Ca2þ/Naþ exchanger, the Naþ/Hþ

exchanger and the Naþ-Kþ-2Cl� cotransporter. In addition to thesetransporters, cell swelling activates Kþ and Cl� channels. All ofthese transport pathways contribute to maintain the cell volumeconstant and to regulate the balance between proliferation andapoptosis. However, their mode of activation remains unclear. Theycan also be acting through chemical or physical messengers such asintracellular pH, magnesium, Ca2þ, membrane stretch, cytoskeletonstructure or concentration of regulatory enzymes. The deregulationof these mechanisms may induce cell pathologies includingmyopathies, oedema, anaemia and cancer.

While acute hypotonicity induces a fast regulatory volumedecrease, chronic hypertonicity caused by the addition of NaCl, KCl,CsCl or sucrose to the isotonic medium induces first a fast cellshrinkage and then a slow (24e48 h) unexpected cell swelling sothat the cell volume becomes larger than in isotonic medium(Rouzaire-Dubois et al., 2000). We conclude that chronic hyperto-nicity induces a slow cell swelling due to an osmolyte influxthrough ion channels or transporters likely via the activation ofTonEBP.

8. Metabolism

Anisosmolarity influences the cell volume and metabolic activ-ities (Dubyak, 2004; Wehner et al., 2003). In recent years, it hasbecome evident that the cell volume is an important factor indefining the regulation of cell metabolism. Astrocyte swelling wasshown to stimulate glycogen synthesis (Dombro et al., 2000). Thusfurther acting as a signal for proliferation and playing a role ingliosis. In glioma cells, osmolarity affects the intracellular nucleo-side triphosphate level, rate of fatty acids biosynthesis and cyto-plasmic pH. Hyposmotic challenges lead to a biphasic response inthe cytosolic Ca2þ concentration and calcium-stimulated activation

of MAP kinases Erk 1 and Erk 2 (Sinning et al., 1997). The specificlipid composition of the membrane environment modulates thefunction of ion transport proteins (Rouzaire-Dubois et al., 1991) andtheir relative concentration (molecular crowding) influences theiractivity.

9. Conclusion

Modulations of cell volume and shape are involved in severalcellular functions. They are due to changes in Kþ and Cl� fluxesoften triggered by either an increase or a decrease in intracellularCa2þ concentration. These properties are involved in severalpathologies including renal failure, anaemia, myasthenia andcancer where the balance between cell proliferation and cell deathis modified. What is more, cancer cells have the faculty to changetheir volume and shape so that they diffuse into the tortuousextracellular space and create metastasis. For this reason, it isimportant to develop therapies that are not toxic for normal cellsbut specifically decrease Kþ and Cl� fluxes. While cell pathologiesare not systematically due to either dysfunction or associated withcell volume deregulation, this theory should be experimented byfurther experiments allowing to confirm that it is of importance incell functioning and disorders.

Acknowledgements

We thank Dr. Seana O’Regan for helpful comments on themanuscript.

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