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The cell volume, which controls numerous cellular functions, is theoretically linearly related with the inverse osmolarity. However, deviations from this law have often been observed. In order to clarify the origin of these deviations we electronically measured the mean cell volume of rat glioma cells under three different experimental conditions, namely: at different osmolarities and constant NaCl concentration; at different NaCl concentrations and constant osmolarity and at different osmolarities caused by changes in NaCl concentration. In each condition, the osmolarity was maintained constant or changed with NaCl or mannitol. We showed that the cell volume was dependent on both the extracellular osmolarity and the NaCl concentration. The relationship between cell volume, osmolarity and NaCl concentration could be described by a new equation that is the product of the Boyle-van't Hoff law and the Michaelis-Menten equation at a power of 4. Together, these results suggest that in hyponatriemia, the cell volume deviates from the Boyle-van't Hoff law because either the activity of aquaporin 1, expressed in glioma cells, is decreased or the reduced NaCl influx decreases the osmotically obliged influx of water.

The cell water content determines the cell volume, which in turn controls numerous cellular functions. The mean volume of rat glioma cells was electronically measured under isotonic and anisotonic conditions. Two types of isotonic solutions were used containing either high or low concentrations of NaCl, KCl or N-methylglucamineCl. In low salt solutions, osmolarity was maintained constant by the addition of sucrose or mannitol. Anisotonicity was induced by changing the concentration of electrolytes. As expected, the cell volume increased when the concentration of electrolytes was decreased from a high (165 mM) monovalent cation concentration. In contrast, the cell volume decreased when the concentration of electrolytes was decreased from a low (85 mM) monovalent cation concentration. Reciprocally and unexpectedly, the cell volume increased during a hyperosmotic challenge when the initial cation concentration was low, whereas it decreased when the initial cation concentration was high. These opposite volume changes observed during similar anisotonic challenges but starting from different electrolyte concentrations provide the first evidence that H2O is not only passively transported (downhill) through aquaporins but also follows ion fluxes (uphill).

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.)

The homeostasis of the central nervous system is highly controlled by glial cells and is dramatically altered in the case of glioma. In this respect, the complex connection between cell size and division is of particular importance and needs clarifying. In order to investigate this connection, cell number and volume were measured in C6 rat glioma cells under different experimental conditions, including continuous cell culture, Cl- channel blockade, and anisotonicity, and in the presence of an inhibitory conditioned medium collected from cell cultures or in a medium containing a low level of fetal calf serum. The rate of cell proliferation changed with cell volume in a bell-shaped manner, so that it is optimal within a cell volume window and appears to be controlled by low and high cell size checkpoints. The cell size-proliferation relationship can be defined by Boltzmann-like equations, which may reflect the effects of macromolecular crowding on proteins controlling the cell cycle progression. Altogether, these observations indicate that glioma cell proliferation is controlled predominantly but not exclusively by cell size-dependent mechanisms., (Copyright 2003 Wiley-Liss, Inc.)

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.

K+ and Cl- channels are involved in regulating the proliferation of a number of cell types. Two main hypotheses have been proposed to explain the mechanism by which these channels influence cell proliferation: regulation of membrane potential and regulation of cell volume. In order to test these hypotheses, we measured, under different experimental conditions, the volume, membrane potential and rate of proliferation of C6 glioma cells. Cells cultured in control medium for 1-4 days were compared with cells cultured for the same period of time in the presence of broad spectrum channel blockers: tetraethylammonium, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and Cs+, in hypertonic media (29% increased osmolarity with NaCl, KCl or sucrose), in hypotonic medium (23% decreased osmolarity with H2O) or in the presence of the specific channel blockers, i.e. mast cell degranulating peptide, charybdotoxin or chlorotoxin. In all of these conditions, we observed a close correspondence between the rate of proliferation and the mean cell volume. The proliferation decreased when volume increased. Moreover, whereas control cells were flattened, spindle-shaped, bipolar or multipolar, cells cultured in media supplemented with NPPB, KCl or CsCl were round with few processes. Of the agents tested, only KCl and Cs+ depolarized the cells. These results show that alterations of the rate of proliferation by K+ and Cl- channel blockers or anisotonia are closely related with changes in cell volume or form but are not correlated with changes in membrane potential.

A population of NG108-15 neuroblastoma cells resistant to doxorubicin (NG/DOXR) was established. The cells exhibited a multidrug resistance phenotype with cross-resistance to vinblastin and colchicine, overexpression of a 170 kDa membrane protein identified as P-glycoprotein and reversal of resistance by verapamil and quinine. Compared with NG108-15 cells, NG/DOXR cells showed an increase in Na+ current density and a decrease in cyclic-AMP-activated Cl- current density with no change in K+- and volume-sensitive Cl- current densities. As previously observed in NG108-15 cells, the vacuolar-type H+-ATPase inhibitors bafilomycin A1 and nitrate induced membrane depolarizations in NG/DOXR cells. The resting potentials of sensitive and resistant cells were not significantly different, but the depolarizations evoked by these agents were significantly larger in NG/DOXR than in NG108-15 cells. The resting membrane potential of NG/DOXR cells, but not that of NG108-15 cells, was depolarized by verapamil, and this effect was abolished by bafilomycin. The volume-sensitive Cl- currents of drug-sensitive and drug-resistant cells were inhibited by a decrease in intracellular pH from 7.3 to 6.8. Whereas bafilomycin prevents activation of Cl- currents in both drug-sensitive and drug-resistant cells, verapamil inhibited the Cl- current only in NG/DOXR cells. The results are discussed in terms of the roles of cytoplasmic pH and membrane potential in multidrug resistance.

The contribution of an electrogenic proton pump to the membrane potential of neuroblastoma x glioma hybrid NG 108-15 cells was determined with whole-cell voltage and current recordings and cell volume measurements with the preparation bathed in symmetrical 140 mM KCl solutions. The effects of the K+ channel blockers tetraethylammonium and 4-aminopyridine and of the H+-ATPase inhibitor bafilomycin A1 on the membrane potential and input resistance revealed that the membrane potential is generated by an outward H+ pump current of 5-15 pA in equilibrium with an inward passive current. This conclusion is supported by both current- and voltage-clamp results obtained when the preparation was bathed in a Na+-containing external solution after K+ channel blockade with Cs+ in the pipette. Additional support was obtained by measurement of the volume of cells incubated in solutions containing 140 mM KCl. Tetraethylammonium induced a bafilomycin-sensitive increase in inward K+ current and an increase in cell volume of 76% which we believe to be a consequence of the K+ influx. Finally, comparison of membrane potentials obtained in experiments using Na+-containing external, and K+-containing pipette solutions and after K+ channel blockade with Cs+ in the pipette also showed that, under normal physiological conditions, the resting membrane potential is essentially determined by an electrogenic H+ pump.

The aim of this work was to examine the effects of changes in external K+ concentration (Ko) around its physiological value, of various K+ channels blockers, including internal Cs+, of vacuolar H(+)-ATPase inhibitors and of the protonophore CCCP on the resting potential and the voltage-dependent K+ current of differentiated neuroblastoma x glioma hybrid NG108-15 cells using the whole-cell patch-clamp technique. The results are as follows: (i) under standard conditions (Ko = 5 mM) the membrane potential was -60 +/- 1 mV. It was unchanged when Ko was decreased to 1 mM and was depolarized by 4 +/- 1 mV when Ko was increased to 10 mM. (ii) Internal Cs+ depolarized the membrane by 21 +/- 3 mV. (iii) The internal application of the vacuolar H(+)-ATPase inhibitors N-ethylmaleimide (NEM), NO3- and bafilomycin A1 (BFA) depolarized the membrane by 15 +/- 2, 18 +/- 2 and 16 +/- 2 mV, respectively. (iv) When NEM or BFA were added to the internal medium containing Cs+, the membrane was depolarized by 45 +/- 1 and 42 +/- 2 mV, respectively. (v) The external application of CCCP induced a transient depolarization followed by a prolonged hyperpolarization. This hyperpolarization was absent in BFA-treated cells. The voltage-dependent K+ current was increased at negative voltages and decreased at positive voltages by NEM, BFA and CCCP. Taken together, these results suggest that under physiological conditions, the resting potential of NG108-15 neuroblastoma cells is maintained at negative values by both voltage-dependent K+ channels and an electrogenic vacuolar type H(+)-ATPase.