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Extracellular tonicity and volume regulation control a great number of molecular and cellular functions including: 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 effects and to determine if they are direct or are secondarily the result of the activity of second messengers., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Cell volume controls many functions and is itself regulated. To study cell volume regulations, the mean volume of C6-BU-1 rat glioma cells was electronically measured under isotonic and anisotonic conditions. Two isotonic solutions were used containing either normal (solution 1) or low (solution 2) NaCl. Anisotonicity was induced by changing NaCl or sucrose concentrations in solutions 1 and 2, respectively. The cells behaved like perfect osmometers when the tonicity was increased. In contrast, just after hypotonic challenges, the cell volume was smaller than that predicted by a perfect osmometer. This deviation reveals a new mechanism, which we call the volume increase limitation (VIL). When hypotonicity was induced by decreasing NaCl, a classical slow regulatory volume decrease (RVD) was also observed in addition to VIL. The cells expressed aquaporin-1 sensitive to HgCl(2) and decreased by siRNA, which both reduced fast volume changes. In this study, we show that: (1) RVD is proportional to the change in external Cl(-) concentration and is inhibited by Cl(-) channel and K(+)-Cl(-) cotransporter blockers; (2) cell swelling due to the influx of H(2)O through aquaporins shows rectification with decreasing osmolarity and is sensitive to the internal Na(+) concentration; (3) VIL is linearly related with hypotonicity and is abolished in solutions 1 and 2 by the Na(+) ionophore monensin and in solution 1 by the Na(+)-K(+) ATPase inhibitor ouabain. These results suggest that VIL is triggered by the decrease in internal Na(+) caused by hyponatrema and cell swelling. In addition to RVD, VIL should protect cells during hyposmotic stress.

In the 1870's, Ludwig Boltzmann proposed a simple equation that was based on the notion of atoms and molecules and that defined the probability of finding a molecule in a given state. Several years later, the Boltzmann equation was developed and used to calculate the equilibrium potential of an ion species that is permeant through membrane channels and to describe conformational changes of biological molecules involved in different mechanisms including: open probability of ion channels, effect of molecular crowding on protein conformation, biochemical reactions and cell proliferation. The aim of this review is to trace the history of the developments of the Boltzmann equation that account for the behaviour of proteins involved in molecular biology and physiology.

Potassium channels, the most diverse superfamily of ion channels, have recently emerged as regulators of carcinogenesis, thus introducing possible new therapeutic strategies in the fight against cancer. In particular, the large conductance Ca(2+)-activated K(+) channels, often referred to as BK channels, are at the crossroads of several tumor-associated processes such as cell proliferation, survival, secretion and migration. Despite the high BK channel expression in osteosarcoma (OS), their function has not yet been investigated in this malignant bone pathology. Here, using stable RNA interference to reduce the expression of hSlo, the human pore-forming alpha-subunit of the BK channel, in human Cal72 OS cells, we show that BK channels play a functional role in carcinogenesis. Our results reveal for the first time that BK channels exhibit antitumoral properties in OS in vivo and affect the tumor microenvironment through the modulation of both chemokine expression and leukocyte infiltration., ((c) 2008 Wiley-Liss, Inc.)

Salicylhydroxamic acid (SHAM) inhibits the proliferation of cultured plant (Atriplex halimus) and murine neuroblastoma cells with IC50 of 90 and 250 microM, respectively. After 2 h of application, SHAM induces an acceleration of the neuroblastoma cell cycle from G1/S to G2 phases and, after 6 h, it induces an accumulation of the cells in S phase and a cell swelling. Up to 300 microM, SHAM is not cytotoxic and does not induce electrophysiological differentiation of neuroblastoma cells. When Drosophila females are grown in media containing 0.6-1.25 mM SHAM, the rate and number of laid eggs are increased. Furthermore, SHAM stimulates the different development stages from embryo to adult. A general interpretation of the effects of SHAM on cell proliferation and differentiation is proposed.

For decades, the connection between cell size and division has been the subject of controversy. While in yeast, cell size checkpoints coordinate cellular growth with cell-cycle progression, it has been recently shown that large and small Schwann cells proliferate at the same rate (Conlon and Raff, 2003, J Biol 2:7). From this point of view, it is important to know whether normal and tumoral cells are similar. During continuous culture of NG108-15 neuroblastoma x glioma cells, the rate of proliferation, cell size, and external pH changed in parallel. At constant pH, the cell size-proliferation relationship followed a bell-shaped curve, so that proliferation was optimal within a cell volume window. In contrast, external acidification decreased proliferation independently of cell size. Using electrophysiological techniques, we showed that changes in cell size were dependent on both the uptake of nutrients and the passive influx of ions. Furthermore, an increase in cell size was associated with an increase in total proteins/cell. Another way to influence cell growth and proliferation is to alter the activity of the PI-3 kinase and target of rapamycin (TOR) signaling pathway. In NG108-15 cells, pharmacological inhibition of these proteins with LY 294002 and rapamycin respectively decreased proliferation but did not modify cell size. In contrast, aphidicolin treated cells did not proliferate, but they continued to increase in size. Altogether these results indicate that the proliferation of NG108-15 cells is controlled by both cell size-dependent and independent mechanisms that include extracellular pH and PI-3 kinase activity., (2004 Wiley-Liss, Inc.)

While there is increasing evidence that Ca2+ plays an important role in regulating cell proliferation, the precise mechanisms have not been clearly elucidated so far. In order to gain insight into how Ca2+ controls cell division, the rate of proliferation, cell volume, viability and attachment to the culture support were measured in NG108-15 neuroblastoma cells in the presence of various extracellular Ca2+ concentrations ([Ca2+]o). Culture medium [Ca2+]o was decreased from 1.8 mmol/l to various values down to 1 micromol/l with EGTA. The rate of cell proliferation was almost independent of [Ca2+]o between 1.8 mmol/l and 45 micromol/l. It was decreased by about 50% at 12 umol/l Ca2+ and was almost zero in the presence of 1 micromol/l Ca2+. As we hawe shown previously (Rouzaire-Dubois and Dubois 1998) long-term hypertonicity increased the cell volume and decreased the rate of proliferation. The effects of hypertonicity and decrease in [Ca2+]o on cell proliferation were synergistic and can be described by cell size-dependent and independent mechanisms, respectively. Relative to control conditions (1.8 mmol/l Ca2+), decreases in [Ca2+]o to 12 and 1 micromol/l decreased the cell viability to 76 and 52% and the cell adhesion to dishes to 16 and 3%, respectively. Altogether, these results indicate that the effects of alteration in [Ca2+]o and cell size on neuroblastoma cell proliferation are independent and act on different signalling pathways controlling cell division.

Ion channels and cell volume control participate in a wide variety of cellular functions, including cell proliferation. According to the "pump-leak model" or the "double Donnan system", the cell volume is constant in physiological medium so long as the cell metabolism and the Na-K pump are not inhibited and the passive Na+ permeability is not dramatically increased. At short term, this model has been supported by a large number of experiments made on different cell types. However, at long term, it may be insufficient to describe the volume control because it does not take into account the fact that cells possess a large number of membrane transporters and interconnected volume regulatory mechanisms. In this review, we present recent results indicating that, in physiological conditions, ion channels may have important roles in cell volume control. Furthermore, we emphasize that cell proliferation and volume are phenomenologically correlated. On the basis of the macromolecular crowding theory, the possibility that the cell osmolyte and water content mediates this correlation is discussed.

Intracellular calcium ([Ca2+](i)), cell volume, membrane potential and currents were measured in neuroblastomaxglioma hybrid cells to gain insight into how [Ca2+](i) controls cell volume. [Ca2+](i) was increased by fluid shear stress, mechanical stimulation of the cells, the Ca2+ ionophore A23187, caffeine and thapsigargin. The increase in [Ca2+](i) induced by mechanical stimulation was decreased by about 50% by caffeine and abolished after incubation of the cells in a Ca2+-free solution. Mechanical stimulation by stirring the cell suspension induced cell shrinkage that was abolished by caffeine, but induced cell swelling in Ca2+-free solution. In the presence of caffeine, A23187 induced cell shrinkage whereas thapsigargin induced cell swelling. Both cell volume changes were inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid. The cells were hyperpolarized by fluid shear stress and A23187 and depolarized by caffeine, thapsigargin and intracellular EGTA. Under all these conditions, the membrane input resistance was decreased. Voltage-clamp experiments suggested that, in addition to an increased anionic current, fluid shear stress and A23187 increased a K+ current, whereas caffeine and intracellular Ca2+ chelation increased a non-selective cation current and thapsigargin increased both a K+ and a non-selective cation current. Taken together, these results suggest that, if cell volume is closely dependent on [Ca2+](i) and the activity of Cl- channels, its relative value is dependent on the ionic selectivity of co-activated channels and the membrane potential.

Vacuolar H(+) ATPase (V-ATPase) activity is essential for many cellular processes, including intracellular membrane traffic, protein processing and degradation, and receptor-mediated endocytosis. Proton transport by V-ATPases could also play a role during cell transformation, tumorigenesis, and cell metastasis, and V-ATPase c-subunit overexpression was reported to be correlated with invasiveness of pancreatic tumors (Ohta et al., 1996). In the present work, we found that mRNAs encoding V-ATPase subunits are not overexpressed in C6 tumoral glioma cells when compared with immortalized astrocytes DI TNC1 and astrocytes in primary cultures. Accordingly, V-ATPase subunit mRNA levels are similar in human gliomas (grade II or IV) and in peritumoral tissues. A significant proportion (25%) of V-ATPase is present in the plasma membrane of both the C6 and the DI TNC1 astrocytic cells in culture. A bafilomycin-sensitive hyperpolarizing pump current through the plasma membrane was detected and measured after ionic channel inhibition, which corresponds most probably to an electrogenic transport of protons. This suggests that the plasma membrane V-ATPase is active. It could contribute to cytoplasmic pH regulation in astrocytic cells., (Copyright 2002 Wiley-Liss, Inc.)

Recently, we showed that at constant extracellular osmolarity, the volume of NG108-15 cells was dependent on the external NaCl concentration and we assumed that the responsible mechanism was mediated by background channels (Rouzaire-Dubois et al. 1999). In order to confirm this view, the mean cell volume and the background current of NG108-15 cells were measured under different experimental conditions, after blockade of specific volume regulating mechanisms and ion channels. When the external NaCl concentration was decreased, the reversal potential of the background current was shifted toward negative values and the membrane conductance decreased. Opposite effects were observed when the NaCl concentration was increased. Substitution of external Na+ with various monovalent cations altered the mean cell volume by: Rb+, +17%; Cs+, +15%; K+, +10%; Li+, -6%; choline, -9%; N-methylglucamine, -25% . The reversal potential of the background current and the membrane conductance were altered by these Na+ substitutes in such a way that the cell volume increased linearly with the background current at -60 mV. Substitution of external Cl- with various monovalent anions altered the mean cell volume by: I-, +4%; Br-, 0%; NO-, -3%; F-, -5%; isethionate, -30%; gluconate, -50%. Cl- substitutes did not significantly alter the background current at -60 mV, except F- which increased it by 39%. These results suggest that 1. the cell volume is dependent on ion fluxes through background channels; 2. electrogenic cation fluxes are larger than anionic ones and the background current is proportional to the difference between these fluxes; 3. whereas external cations do not interfere with anion fluxes, external anions alter cation fluxes.

Equinatoxin-II (EqTx-II), a cytotoxic protein (mol.wt 20 kDa) isolated from the sea anemone Actinia equina, was found to consistently increase the three-dimensional projected area of differentiated neuroblastoma (NG108-15) cells provided Ca(2+) was present in the medium. No swelling was detected when external NaCl was replaced by sucrose, but replacement of NaCl by Na-isethionate did not prevent the swelling, as revealed by confocal laser scanning microscopy. In addition, microspectrofluorometric measurements in cells preloaded with the Ca(2+) indicator fura-2/AM revealed that EqTx-II (100 nM) markedly increased the fluorescence (F(340)/F(380)) ratio indicating a rise of intracellular Ca(2+) concentration ([Ca(2+)](i)). The elevation of [Ca(2+)](i) exhibited two components that seem to be related to the kinetics of EqTx-II-induced Ca(2+) entry since pretreatment of cells with Ca(2+)-ATPase inhibitors (thapsigargin), Ca(2+) channel blockers (nifedipine and Gd(3+)) or prolonged exposure to a high K(+) (75 mM) medium did not alter EqTx-II-induced Ca(2+) signals. As far as we know, this is the first demonstration that EqTx-II causes swelling of neuroblastoma cells and that this effect is correlated both with an increase of [Ca(2+)](i) and needs the presence of extracellular Na(+). It is suggested that EqTx-II has the ability to insert into the plasma membrane of neuroblastoma cells and to form pores altering the membrane permeability and the intracellular osmolality, inducing a marked influx of water into the cells.