Cellular hydration and intracellular ion concentrations can control gene expression (Higgins, Cairney, Stirling, Sutherland & Booth, 1987; Burg, Kwon & Kültz, 1996), and since the above cell volume changes developed within several hours, we hypothesized that they were due to alterations in the expression of genes encoding osmolyte transporters or channels. This hypothesis was confirmed by the observation that the protein translation inhibitor cycloheximide (20 µM) almost completely abolished long-term volume changes induced by hyper- and hypotonicity (Fig. 3).

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Figure 3. Effect of cycloheximide on anisotonicity induced long-term cell volume changes Relative volume of cells after 5 h incubation in hypertonic medium (40 % increase in osmolarity by the addition of NaCl) or 7·5 h incubation in hypotonic medium (24 % decrease in osmolarity by the addition of H2O) with or without 20 µM cycloheximide. Means + S.E.M. of 3 experiments. Volumes in anisotonic media with and without cycloheximide are expressed relative to volumes in isotonic media with and without cycloheximide, respectively.

Following these observations, we studied the effects on cell proliferation of alterations in the tonicity of the culture medium. Compared with control medium, hypotonic medium (addition of 30 % H₂O) decreased cell proliferation between day 1 and day 2 and then increased it by 32 ± 7 % (n = 4). In contrast, hypertonic medium (addition of 65 mM NaCl) induced a sustained 82 ± 3 % (n = 3) decrease in cell proliferation (Fig. 4). These alterations in cell proliferation may have been due to dilution of the culture medium (hypotonic condition) or to changes in the Na⁺ or Cl^- electrochemical gradients (hypo- and hypertonic conditions). Therefore, we performed control experiments in which the culture medium was diluted by 30 % with an isotonic solution of mannitol (control for the hypotonic condition) or was made hypertonic by the addition of 130 mM mannitol (control for the hypertonic condition with NaCl). Compared with control medium, the medium diluted with the mannitol solution induced a sustained 25 ± 2 % (n = 4) decrease in cell proliferation similar to that observed between day 0 and day 2 in the presence of hypotonic medium. When the culture medium was made hypertonic with mannitol (40 % increase in osmolarity), the cell volume was increased by 9 ± 2 % and the cell proliferation was decreased by 60 ± 2 % (n = 4).

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Figure 4. Effect of anisotonic media on cell proliferation A, cells were cultured in control () or in hypotonic (; 24 % decrease in tonicity by the addition of 30 % H2O) media. In isotonic and hypotonic media, the fetal calf serum concentration was 5 %. Means ± S.E.M. of 4 experiments. The numbers of cells at days 2 and 4 were significantly different in control and in hypotonic media (P < 0·05, Student's unpaired t test). B, cells were cultured in isotonic () or hypertonic (; 40 % increase in tonicity by the addition of 65 mM NaCl) media. Means ± S.E.M. of 3 experiments

Effects of Cl^- channel block on cell proliferation and volume

Blockers of volume-activated Cl^- channels have been shown to inhibit endothelial cell proliferation (Voets, Szücs, Droogmans & Nilius, 1995). Given that these channels play a key role in cell volume regulation (Nilius, Sehrer, De Smet, Van Driessche & Droogmans, 1995), one can ask whether, in isotonic conditions, the inhibition of cell proliferation induced by Cl^- channel blockers is due to or associated with cell swelling. In order to test this possibility, we studied the effects of NPPB on membrane current, cell proliferation and volume.

NPPB (50 µM) blocked an outwardly rectifying current which reversed at +1 ± 2 mV (n = 9) (Fig. 5A). This current was identified as a Cl^- current since, when the external NaCl concentration was reduced from 140 to 70 mM (the osmolarity being kept constant by the addition of 140 mM sucrose), its reversal potential was +14 ± 2 mV (n = 3) (Fig. 5B). The NPPB-sensitive current was Ca²⁺ independent since it was not affected by the addition of 1 mM EGTA to the pipette solution. Moreover, it has been shown by many groups that ATP is required for the sustained activation of Cl^- currents. We observed that the addition of 2 mM ATP to the pipette solution allowed recording of the NPPB-sensitive current for more prolonged periods (> 10 min) than without ATP (< 5 min). In addition to its effect on the Cl^- current (shown in Fig. 5 with CsCl in the pipette), 50 µM NPPB decreased the voltage-dependent K⁺ current (recorded with KCl in the pipette) by 20 % (not shown). This effect was independent of the voltage and the duration of depolarization.

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Figure 5. NPPB blocks a Cl- current in isotonic conditions Currents were recorded during ramp potentials (100 mV s-1) applied from -60 to +60 mV. The pipette solution contained 140 mM CsCl to block K+ currents. NPPB was dissolved in DMSO which was added at the same concentration (0·25 %) to the control solution. A, current-voltage curves obtained in control (1) and after addition of 50 µM NPPB to the external solution (2). B, NPPB-sensitive current as a function of voltage with 140 mM NaCl (a) or 70 mM NaCl + 140 mM sucrose (b) in the external solution. Traces a and b were recorded from 2 different cells. In both cases, the liquid junction potential was corrected before seal formation.

The effects of NPPB (50 µM) on cell volume and proliferation are shown in Fig. 6. NPPB increased the mean cell volume by 12 % and decreased the rate of cell proliferation by 77 %. It must be noted that, in contrast to the effects of K⁺ channel blockers on cell volume, which developed within several hours, cell swelling induced by NPPB occurred within a few minutes.

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Figure 6. Effects of NPPB on cell volume and proliferation A, volume distributions of cells cultured with () or without () 50 µM NPPB. Means ± S.E.M. of 4 experiments. B, cell proliferation curves with () and without () 50 µM NPPB. Means ± S.E.M. of 4 experiments. In A and B, 0·25 % DMSO was added to the control medium.

Effects of antibiotics on cell proliferation and volume

In the present experimental conditions, the cells were cultured in the presence of penicillin (100 i.u. ml^-1) and streptomycin (100 µg ml^-1). Some antibiotics are known to inhibit cell proliferation (Sanders, Fiddes, Thompson, Philpott, Westgate & Kealey, 1996). Given that penicillin has been shown to decrease the Cl^- conductance in crustacean muscle (Hochner, Spira & Werman, 1976) and streptomycin has been shown to inhibit maxi-K channels in cochlear efferent nerve terminals (Takeuchi & Wangemann, 1993), we looked here for possible effects of antibiotics on membrane current, cell proliferation and volume. In order to see whether antibiotics alter K⁺ and/or Cl^- current, the membrane current was recorded alternately at -85 mV (the expected K⁺ equilibrium potential) and at 0 mV (the expected Cl^- equilibrium potential) before, during and after the application of antibiotics. Furthermore, the concentration of antibiotics was increased 5- or 10-fold compared with that of the culture medium in order to amplify their possible effects. Figure 7 shows that antibiotics decreased reversibly the current at 0 mV but did not alter the current at -85 mV. This effect, which was observed for cells cultured for 24 h in medium both with and without antibiotics, indicates that antibiotics block K⁺ channels and thus, as for classical K⁺ channel blockers, may alter cell proliferation and volume. To test this prediction and to avoid possible cytotoxic effects of the antibiotics, we did not increase the concentrations of the antibiotics but we studied the effects of their omission from the culture medium. Figure 8 shows that omission of antibiotics from the culture medium resulted in cell shrinkage and an increase in the rate of cell proliferation. In the absence of antibiotics, the mean cell volume was decreased by 10 ± 1 % and the rate of cell proliferation was increased by 32 ± 1 %.

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Figure 7. Effects of antibiotics on membrane currents The membrane current was recorded alternately at -85 and 0 mV (450 ms depolarizations to 0 mV applied at a frequency of 0·5 Hz from a holding potential of -85 mV). The antibiotics (1000 i.u. ml-1 penicillin and 1 mg ml-1 streptomycin) decreased the current at 0 mV () but had no effect on the current at -85 mV ().

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Figure 8. Effects of antibiotics on cell volume and proliferation A, volume distributions of cells cultured with () or without () antibiotics. Means ± S.E.M. of 3 experiments. B, cell proliferation curves with () and without () antibiotics. Means ± S.E.M. of 3 experiments.

Cell proliferation-volume relationship

All the preceding results indicate that any alteration in cell volume was associated with an inverse change in the rate of cell proliferation. In order to quantify the cell proliferation-volume relationship, the results were compiled and the relative rate of cell proliferation was plotted against the relative mean cell volume (Fig. 9). It can be seen from this relationship that the proliferation was fully inhibited when the cell volume was increased by about 25 %, and a 10 % decrease in cell volume was associated with a 30 % increase in the rate of proliferation.

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Figure 9. Cell proliferation-volume relationship The relative rate of cell proliferation and the relative mean cell volume were determined in the presence of TEA (1, 5 and 10 mM), 4-AP (0·2 and 2 mM), Cs+ (2·5, 5 and 10 mM) (), 50 µM NPPB (), in hypertonic medium (), in hypotonic medium () and without antibiotics (). In each case, cell volume and proliferation are expressed relative to control conditions where cells were cultured simultaneously in control culture medium. Means ± S.E.M. of 3-6 experiments in each condition. The curve was drawn from eqn (4) with Pmax = 1·32, V0·5 = 1·045 and k = 0·039.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

The results presented here support our previously proposed hypothesis (Rouzaire-Dubois & Dubois, 1991; Dubois & Rouzaire-Dubois, 1993) that mitogenesis inhibition induced by K⁺ channel blockers is associated with cell swelling. A similar observation has been reported recently by Xu et al. (1996) showing that 4-AP induced both G1 arrest and a volume increase in human myeloblastic ML-1 cells. We propose two possible explanations for these observations. The first and most attractive one is that cell swelling is the direct link between K⁺ channel block and inhibition of proliferation. The second interpretation is that either K⁺ channel activity independently regulates cell volume and proliferation or K⁺ channel blockers not only block K⁺ channels but also act on cell volume and/or DNA synthesis via other mechanisms. Such may be the case for 4-AP, which, independently of its action on K⁺ channels, is known to induce an increase in cytosolic pH and Ca²⁺ concentration (Guse, Roth & Emmrich, 1994; Pappas et al. 1994). These effects on pH_i and [Ca²⁺]_i may explain the fact that the cell volume increased faster with 4-AP than with TEA or Cs⁺ (Fig. 1B). Of the two explanations proposed above to account for the effects of K⁺ channel blockers on cell proliferation, the first one is strongly supported by our observations showing that alterations of cell volume by hypo- and hyperosmolarity were associated with changes in the rate of cell proliferation. One has to note that in hypotonic medium, the relative cell proliferation decreased between day 0 and day 2 and increased between day 2 and day 4 while the cell volume substantially decreased. The initial reduction in cell proliferation was probably due to the dilution of nutrients in the culture medium, since medium diluted with isotonic mannitol solution produced a sustained decrease in relative cell proliferation. It should also be noted that modifications of the osmolarity of the culture medium by adding either H₂O or NaCl cause alterations of the driving forces for a number of ion transporters (e.g. the Na⁺-H⁺ and the Cl^--HCO₃^- antiporters and the Na⁺-K⁺-Cl^- cotransporter) whose activities are thought to control cell proliferation (Horvat, Taheri & Salihagic, 1993; Panet, Markus & Atlan, 1994). We observed that hypertonicity induced with mannitol (i.e. at constant NaCl concentration) also increased cell volume and decreased cell proliferation. This suggests that the determining factor in the control of cell proliferation is the cell volume and not the activity of transporters per se, which, however, may secondarily alter cell volume. This last conclusion seems to be confirmed by the fact that a 40 % hypertonicity induced by the addition of 65 mM NaCl caused a more pronounced cell swelling (25 %) than a 40 % hypertonicity induced by the addition of 130 mM mannitol (9 %).

Apparently related changes in cell volume and rate of cell proliferation were also observed with the Cl^- channel blocker NPPB and after antibiotic omission. While antibiotics may have several unrelated effects on membrane permeabilities and cell metabolism, our results showing that they decreased the voltage-dependent K⁺ current may be sufficient to explain their action on the cell volume. We report here that, in addition to its effect on a membrane current identified as a Cl^- current, NPPB decreased the voltage-dependent K⁺ current. Consequently, the effect of NPPB on the cell volume may be mediated by a Cl^- and/or a K⁺ channel blockade. However, taking into account the fact that (1) NPPB was more efficient at blocking the Cl^- than the K⁺ current and (2) its effect on the cell volume was faster than that of classical K⁺ channel blockers, this suggests that NPPB-induced cell swelling is due mainly to Cl^- rather than K⁺ channel blockade. Following this conclusion, it would be interesting to test the effects on cell volume and proliferation of other Cl^- channel blockers. Quinine and tamoxifen have been shown to inhibit volume-activated Cl^- currents and proliferation of endothelial cells (Voets et al. 1995). However, quinine is also a potent blocker of K⁺ channels in several cell types (see Wonderlin & Strobl, 1996), and tamoxifen has been shown to inhibit, in the same concentration range, K⁺ current and proliferation of NG108-15 cells (Rouzaire-Dubois & Dubois, 1990).

The observation that K⁺ channel blockers do not significantly affect the membrane potential of neuroblastoma cells and astrocytes (Miyake & Kurihara, 1983; Rouzaire-Dubois & Dubois, 1991; Gérard, Rouzaire-Dubois & Dubois, 1994; Pappas et al. 1994), but do inhibit their proliferation (Rouzaire-Dubois & Dubois, 1991; Pappas et al. 1994; present results) is apparently in contradiction with the idea that K⁺ channel activity regulates the ion influx-efflux ratio and the cell volume. The lack of effect of K⁺ channel blockers on membrane potential implies that K⁺ channels are closed at rest (Miyake & Kurihara, 1983) and thus cannot control cell volume. In contrast, if K⁺ channel blockers alter the cell volume via their effects on K⁺ channels, K⁺ channels must be open at rest and their blockade should induce a membrane depolarization. In fact, the reality should be intermediate between these two propositions, i.e. the resting open probability of K⁺ channels should be very small but not zero (Rouzaire-Dubois & Dubois, 1991). We have investigated this possibility in NG108-15 cells and we came to the conclusion that the resting membrane potential is maintained at negative values by K⁺ channels and electrogenic vacuolar-type H⁺-ATPases which both generate outward currents, the sum of which is in equilibrium with an inward passive current through non-selective channels (Gérard et al. 1994; Rouzaire-Dubois & Dubois, 1997b).

Assuming that the H⁺ pump behaves as an electrical current source having a negligible internal conductance (Lauger, 1991), a resting K⁺ conductance of 72 pS was calculated from the mean input conductances under control conditions (417 pS) and after K⁺ channel blockade with internal Cs⁺ (345 pS) (Rouzaire-Dubois & Dubois, 1997b). With a K⁺ equilibrium potential of -85 mV and a mean resting potential of -60 mV (Rouzaire-Dubois & Dubois, 1997b), this gives a resting K⁺ current of 1·8 pA. Assuming that the non-selective current reverses at a membrane potential of 0 mV, its amplitude at -60 mV (calculated from the input conductance in the presence of internal Cs⁺) is -20·7 pA and the H⁺ pump current is 18·9 pA. In such a situation, a K⁺ channel block should induce a maximum depolarization of 5 mV (60 - (18·9 × 10³/345)) but, in turn, may result in a noticeable cell swelling. Assuming that the influx-efflux ratio of osmolytes other than K⁺ (and Cl^-) remains unchanged and any change in intracellular osmotic pressure will result in a water flux and a volume alteration, the time (t) needed for the cell volume to increase by V upon a K⁺ current reduction of I_K can be calculated from the equation (derived from eqn (7) of Dubois & Rouzaire-Dubois, 1993):

t = (V/I_K)C_oF, (3)

where F is Faraday's constant (96 500 C mol^-1) and C_o is the total external cation concentration (0·15 M). From eqn (3) and a mean cell volume of 3·5 pl, a full inhibition of the K⁺ current induces a 20 % increase in cell volume within 94 min. This time is much shorter than that observed with TEA or Cs⁺ (Fig. 1B ). One explanation for this discrepancy is that, with the concentrations of K⁺ channel blockers used, the K⁺ current is not fully inhibited (Rouzaire-Dubois & Dubois, 1991) and the small depolarization induced by K⁺ channel blockers increases the unblocked voltage-dependent K⁺ conductance. This should result in an effective reduction of the K⁺ current which is smaller than that expected if the membrane potential was unchanged, and cell swelling should be slower. Another explanation is that the initial cell swelling induced by K⁺ channel blockers produces an increase in Cl^- and/or amino acid efflux (Kimelberg, Goderie, Higman, Pang & Waniewski, 1990; Nilius et al. 1995) and a partial regulatory volume decrease. In fact, eqn (3) predicts that with a K⁺ current reduction, the cell volume increases linearly and infinitely with time. This is not the case, which implies that cell swelling is slowed and limited by a decrease of osmolyte influx-efflux ratio.

Altogether the present results suggest that the link between ionic channel activity and cell proliferation is the cell volume rather than the membrane potential or another unknown mechanism controlled by ion channels. However, it should be noted that this conclusion may only be apparent. In particular: (1) although hypertonia induces long-term neuroblastoma cell swelling, it has also been shown to depress the voltage-dependent K⁺ current in hippocampal neurones (Huang & Somjen, 1997); and (2) alterations of cell volume can influence the membrane potential by activating or inhibiting Cl^- channels (Nilius et al. 1995). Moreover, although cell volume seems to play a major role in the control of neuroblastoma cell proliferation, in other cell types, depolarizations induced by K⁺ channel blockers may influence DNA synthesis via various mechanisms, including a modulation of Ca²⁺ influx.

Two ways in which cell swelling and shrinkage could influence cell proliferation are: (1) membrane deformation may modify the organization of cytoskeletal components; and (2) cell volume changes may alter intracellular ionic composition and concentrations. As a consequence of one or both of these modifications, the expression or activity of cell cycle-regulating proteins would be affected. In the model we proposed for the regulation of mitogenesis by K⁺ channels (Dubois & Rouzaire-Dubois, 1993), we examined the possibility that cell volume regulates DNA synthesis under the control of the concentration of an intracellular solute. In order to account for the K⁺ current-proliferation relationship we obtained, we assumed a co-operative action of this solute. However, since we show here that the proliferation was fully inhibited when the volume was increased by only 25 %, this co-operativity should be very high. Three hypotheses can be invoked to take account of this conclusion, namely: (1) a small initial change in the intracellular concentration of a solute such as Ca²⁺ can in turn be auto-amplified by a process such as Ca²⁺-induced Ca²⁺ release; (2) the intracellular solute is compartmentalized and its concentration is steeply dependent on the cell volume; and (3) a weak change in the solute concentration, inducing a small perturbation at an initial stage of the mitogen-activated protein kinase cascades, may in turn result in an important modification at the DNA duplication level. The nature of this solute remains unknown. As suggested by several authors (see Nilius & Droogmans, 1994), it may be calcium. Other possibilities are sodium or organic electrolytes, the transporter genes of which are modulated by osmolarity (Burg et al. 1996).

In the framework of the hypothesis that cell volume influences proliferation via the organization of cytoskeletal components, we can assume that a cell cycle-regulating protein has two configurations, resting and activated, and that cell swelling increases the probability of this protein being in the resting state. With such an assumption, the cell proliferation-volume relationship may be described by an equation similar to the so-called 'Boltzmann equation' used to described the open probability of voltage-dependent channels as a function of membrane potential (Hille, 1992). On the basis of this assumption, the curve in Fig. 9 was calculated using:

P = P_max / 1 + exp[(V - V_0·5)/k], (4)

where P and P_max are relative and maximum relative rate of cell proliferation, respectively, V is the relative cell volume, V_0·5 is the relative cell volume at P_max/2, and k is a steepness factor.

The last question which deserves attention is what molecular messenger sensitive to cell volume promotes the progression through the restriction point in late G1 phase. In view of the complexity of molecular processes underlying mitogenesis, it seems premature to propose a protein messenger able to transduce cell volume changes into positive or negative signals for mitogenesis. Nevertheless, some recent observations may give indications as to the identification of this messenger. Several proteins of the mitogen-activated protein kinase cascades and transcription factors have been shown to be activated by hypo- or hyperosmotic stresses (Schliess, Schreiber & Hussinger, 1995; Berl, Siriwardana, Ao, Butterfield & Heasley, 1997; Szaszi, Buday & Kapus, 1997). Moreover, Xu et al. (1996) showed that the suppression of K⁺ channel activity by 4-AP was associated with dephosphorylation of the retinoblastoma protein, pRb, which is known to play a key role in the transition from G1 into S phase. Whatever the mechanism by which ion channel pathways mediate signals involved in cell proliferation, the present results suggest that K⁺ channels have a major role in the system involved in cell volume regulation, and, if they are targeted by therapeutic drugs, could be used to influence cell proliferation.

	REFERENCES

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Acknowledgements

We are grateful to R. Kado for many discussions of this work and for review of the manuscript. We thank A. Ghazi and F. Giraud for assistance in optical density and cellocrit measurements.

Corresponding author

B. Rouzaire-Dubois: Laboratoire de Neurobiologie cellulaire et moléculaire, CNRS, 91198 Gif-sur-Yvette Cedex, France.

Email: beatrice{at}wat.nbcm.cnrs-gif.fr

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