Cell Volume Regulation in Cultured Human Retinal Müller Cells Is Associated with Changes in Transmembrane Potential

Müller cells are mainly involved in controlling extracellular homeostasis in the retina, where intense neural activity alters ion concentrations and osmotic gradients, thus favoring cell swelling. This increase in cell volume is followed by a regulatory volume decrease response (RVD), which is known to be partially mediated by the activation of K+ and anion channels. However, the precise mechanisms underlying osmotic swelling and subsequent cell volume regulation in Müller cells have been evaluated by only a few studies. Although the activation of ion channels during the RVD response may alter transmembrane potential (Vm), no studies have actually addressed this issue in Müller cells. The aim of the present work is to evaluate RVD using a retinal Müller cell line (MIO-M1) under different extracellular ionic conditions, and to study a possible association between RVD and changes in Vm. Cell volume and Vm changes were evaluated using fluorescent probe techniques and a mathematical model. Results show that cell swelling and subsequent RVD were accompanied by Vm depolarization followed by repolarization. This response depended on the composition of extracellular media. Cells exposed to a hypoosmotic solution with reduced ionic strength underwent maximum RVD and had a larger repolarization. Both of these responses were reduced by K+ or Cl− channel blockers. In contrast, cells facing a hypoosmotic solution with the same ionic strength as the isoosmotic solution showed a lower RVD and a smaller repolarization and were not affected by blockers. Together, experimental and simulated data led us to propose that the efficiency of the RVD process in Müller glia depends not only on the activation of ion channels, but is also strongly modulated by concurrent changes in the membrane potential. The relationship between ionic fluxes, changes in ion permeabilities and ion concentrations –all leading to changes in Vm– define the success of RVD.


Introduction
Glial cells in the sensory retina (Müller cells) are mainly involved in controlling osmotic and ionic homeostasis [1,2]. During intense neuronal activity, retinal cells can be surrounded by a hypoosmotic environment, since light-evoked changes in the ionic composition of the extracellular fluid cause a decrease in osmolarity, thus favoring glial swelling [3]. In most cell types this increase in cell volume is followed by a regulatory volume decrease response (RVD) partially mediated by the activation of K + and anion channels [4,5,6]. However, only a few studies have evaluated the mechanisms underlying cell volume regulation in Müller cells [7,8]. It has been reported that Müller cells in situ show an effective control of cell volume, that prevents cell swelling, probably due to the presence of K + channels K ir 4.1. The expression of these channels is altered in different pathologies such as retinal ischemia, ocular inflammation and diabetes, as well as in organ cultures [9,10,11,12].
Changes in the extracellular ion composition of the retina during neural activity also cause changes in transmembrane potential (V m ) and in the chemical gradients of most of the ions that determine RVD. In addition, the activation of ion channels during RVD may also alter V m . However, to our knowledge, no studies have investigated the putative link between cell volume regulation and V m in these cells.
The channels involved in the RVD response have been studied in different cell types, usually by evaluating changes in cell volume with and without blockers. The identification and characterization of these channels is typically performed through excised or whole cell patch clamp studies [13,14,15]. Though these methods undeniably offer important and reliable information on conductance changes during cell swelling, they fail to do so during cell volume regulation, since they do not preserve cell membrane integrity nor intracellular medium composition. This could explain the reason why only a few reports have been able to evaluate the RVD response in a more physiological context [16,17,18].
The aim of the present work is to characterize, for the first time, the RVD response in a retinal Müller cell line (MIO-M1) under different extracellular ionic conditions and to evaluate a possible association between RVD and changes in V m . Cell volume and V m changes were measured using fluorescent probe techniques. We also developed a mathematical model that provides information on electrochemical ion gradients and solutes fluxes during the RVD response.
Our results show that cell swelling and subsequent RVD is accompanied by V m depolarization followed by repolarization. However, this RVD response depends closely on the composition of extracellular media. Although K + and Cl 2 channels do play an important role in the RVD response of these cells, their contribution is evident only if a significant driving force for KCl efflux is present.

Cell Cultures
The MIO-M1 cell line (kindly provided by Dr. Astrid Limb, University College London, London, UK) is a spontaneously immortalized retinal Müller glial cell line, originated from human retina, that retained many characteristics of Müller cells [19]. Cells were grown as monolayers in the presence of Dulbecco's Modified Eagle Medium (DMEM)/glutamax supplemented with 10% fetal calf serum (FCS), containing 5 mg/ml streptomycin and 5 U/ml penicillin at 37uC in a humidified atmosphere containing 5% CO 2 . Cells were routinely subcultured every week, and those to be studied were grown on coverslips during 3-4 days before recording.

Solutions and Chemicals
Two different isoosmotic solutions were employed: 1-NaCl solution (ISO NaCl ) and 2-Mannitol solution (ISO Mannitol ), in which 50 mM NaCl were replaced with 100 mM Mannitol (Table 1). Hypoosmotic solutions were prepared from each isoosmotic solution by the removal of either NaCl (HYPO NaCl ) or Mannitol (HYPO Mannitol ), thus varying ion composition in the first case, or keeping it constant in the second (Table 1). Cells were set in an external isoosmotic solution for at least 10 minutes, and then a hypoosmotic shock was induced. All solutions were titrated to pH 7.40 using NaOH (Sigma-Aldrich), and osmolalities were routinely measured by a pressure vapor osmometer (Wescor).

Measurement of Cell Volume Changes and RVD Response
MIO-M1 cells grown on coverslips were mounted on a chamber and loaded with 6 mM BCECF-AM for 30 minutes at 20uC. The chamber was then placed on the stage of a Nikon TE-200 epifluorescence inverted microscope (Nikon Planfluor 40X oil immersion objective lens) as previously described [20]. Fluores- cence intensity was recorded by exciting BCECF at the isosbestic point (440 nm), where the fluorochrome is pH insensitive. Fluorescence data were acquired every 10 seconds using a charge coupled device camera (Hamamatsu C4742-95) connected to a computer with the Metafluor acquisition program (Universal Imaging Corporation, PA). The procedure used to estimate cell water volume was similar to the one previously described [20,21]. Changes in cell volume can be calculated as follows: where V is cell volume at time t; V 0 is cell volume at t = 0; F 0 represents the signal obtained from a small region of the cell (pinhole), when placed in equilibrium with an isoosmotic medium with an osmolality OsM 0 ; F t is the fluorescence from the same region at time t, when placed in equilibrium with a solution with an osmolality of OsM t and f b is the relative background. This parameter corresponds to the y intercept of a plot of F t /F 0 versus OsM 0 /OsM t , which represents relative fluorescence in the absence of osmolality changes. RVD after cell exposure to a hypoosmotic medium was calculated by the following equation: where (V/V 0 ) max is the maximal value of V/V 0 attained during hypoosmotic swelling (peak), and (V/V 0 ) t represents the value of V/V 0 observed at time t. RVD t thus denotes the magnitude of volume regulation at time t, with 100% RVD indicating complete volume regulation and 0% RVD indicating no volume regulation.

Measurement of Membrane Voltage Changes
Transmembrane potential was measured using DIBAC4 (3) , a slow response anionic dye which emission has previously been shown to be independent of cell volume changes [22]. The intracellular concentration of DIBAC4 (3) depends on V m following a Nernstian distribution [23,24]. Cells were loaded with 2.5 mM DIBAC4 (3) for 15 minutes at 20uC and placed on the stage of the same microscope described in the previous section. Excitation wavelength was 490 nm. Emitted light (above 520 nm) was recorded at 10 second intervals. Fluorescence intensity was monitored until it reached a stable value before starting the experiments. Fluorescence intensity changes after interventions were relativized to stationary values (F t /F 0 ) and data were corrected for background noise and drift. Calibration was made by adding 5 mM gramicidin and 2.5 mM DIBAC4 (3) to solutions containing different concentrations of NaCl replaced with N-Methyl-D-glucamine chloride (NMDGCl) (Figure 1). V m was calculated as: Data were obtained either from our own measurements in MIO-M1 cells or from literature, as follows: i-V cell and A c in isotonic conditions were estimated from confocal images as previously reported [43]; ii-P f was obtained from the measured cell volume changes during a hypoosmotic challenge using a modification of the Fick's law [20]; iii-V m corresponds to the value recorded using sharp electrode patch clamp technique by Limb et al [19]; iv-intracellular Na + , K + and Cl 2 concentration are the typical values for most cell types; vextracellular Na + , K + and Cl 2 concentrations are the same as in experimental solutions; vi-ion permeabilities were chosen in order to obtain the V m value of MIO-M1 cells; vii-the initial parameters presented for the Mannitol condition were obtained by simulating an extracellular solution change, from ISO NaCl to ISO Mannitol (see Materials and Methods section). doi:10.1371/journal.pone.0057268.t002 Intracellular concentrations of Na + and K + where assumed to be 18 mM and 132 mM, respectively. Three extracellular NaCl concentrations were tested, 126 mM, 70 mM and 0 mM, corresponding to membrane potentials of 23.32 mV, 217.33 mV and 283.47 mV, respectively. A 1% change in fluorescence corresponds to a V m variation of 2.2 mV, as calculated from the mean calibration curve (0.004560.002, X 6SD, n = 58).

Osmotic Swelling and RVD Response Modeling
Cell volume and V m changes were simulated by using a mathematical model that implies the following assumptions: 1. Cells are non-polarized and cell membrane surface area available for solute and water transport (A c ) remains constant, regardless of cell volume changes. 2. The composition of extracellular solution remains constant, unless the solution is changed to induce an osmotic shock. Cell swelling occurs due to transmembrane water flux driven by osmotic gradients across the membrane. 3. Intracellular osmolarity is determined by the sum of Na + , K + , Cl 2 , and impermeant anions (X i ) concentrations. The value of the mean charge (z) of X i is assumed to be the one that fulfils the electroneutral condition (>21). 4. The membrane contains channels that allow the diffusive flux of ions (Na + , K + , and Cl 2 channels) and water (Aquaporins). To achieve a stationary condition, passive ion fluxes are assumed to be compensated by opposing active fluxes that do not balance those generated during RVD. Active fluxes were  assumed to be constant since several previous reports demonstrated that short-term volume regulation is not affected by them [25,26]. 5. RVD response is achieved by an increase in K + and/or Cl 2 permeabilities with a latency (t) that was arbitrarily determined to be 20 s after the initiation of swelling. Increases in ion permeabilities develop in a time-dependent exponential manner according to: Where P ion t is ion permeability at time t, P ion max is the maximum permeability achieved during RVD and P ion init is the initial permeability (Table 2). t init is the time at which permeability changes start and t p is a time constant (90 s) [27]. Mathematical model. Considering the assumptions indicated above, and given the initial values detailed in Table 2, the values for intracellular Na + mass (m Na ), K + mass (m K ), Cl 2 mass (m Cl ), osmotically active cell volume (V cell ) and V m were computed at each iteration step (0.1 seconds). Equilibrium potentials (Eq) were calculated by using the Nernst equation: Where R is the gas constant, T is the absolute temperature in Kelvin, F is the Faraday constant and [Ion] o is the external concentration of the ion in question. V m was determined by the Goldman-Hodgkin-Katz equation: Diffusive fluxes (J ion ) were given by [28,29]: , and a negative value of J ion indicates an outward flux. Furthermore, the time courses of m Na , m K and m Cl were given by: And from Fick's law of diffusion, the variation rate of Vcell is: Where Vw is the partial molar volume of water, Pf is the osmotic water permeability of the membrane and [X]o is the concentration of external impermeable solutes. Once external solutions were changed, cells were subjected to RVD activation after a latency (t) of 20 seconds (post solution change).

Statistics
Values are reported as mean 6 SEM, and n is the number of cells evaluated in each condition. Student's t Test for unpaired data was used according to the protocol; p,0.05 was considered a significant difference. Figure 6. Modeling of cells exposed to different extracellular media compositions. Time courses of V t /V 0 (A), V m /V m0 (B), J net (C) and V m , Eq Cl , Eq K (D) simulated in cells exposed to either HYPO NaCl or to HYPO Mannitol . At time = 0 extracellular osmolarity was reduced (DOsM = 100 mOsM) and after a delay of 20 s, P K and P Cl increased according to Equation 4. A negative value of J net indicates an outward flux. doi:10.1371/journal.pone.0057268.g006

Effect of Extracellular Media Composition on RVD Response in MIO-M1 Cells
We first characterized RVD response in MIO-M1 cells exposed to a hypoosmotic shock, generated, either varying or keeping constant ion composition (HYPO NaCl or HYPO Mannitol , respectively). Figure 2 shows the time course of relative cell volume changes (V/V 0 ) in response to these hypoosmotic gradients (DOsM = 100 mOsM). Though in both conditions cells respond to the hypoosmotic challenge with rapid swelling and thereafter exhibit RVD, kinetics were quite different in each case. In cells faced with HYPO NaCl , cell volume is restored more rapidly, as compared to HYPO Mannitol experiments. Indeed, the percentage of RVD at 10 minutes (% RVD 10 ) is significantly higher with the HYPO NaCl solution (Figure 2 insert). These results indicate that although MIO-M1 cells respond to cell swelling by triggering RVD, the magnitude of this response depends on extracellular media composition.
Since RVD is known to be attained by the activation of K + and Cl 2 conductances in most mammalian cell types [30], we further investigated RVD response in the presence of volume-sensitive Cl 2 channels blockers (NPPB) or K + channels blockers (Ba 2+ ), using cells exposed alternatively to either external media. Figure 3 illustrates that, in the presence of Ba 2+ , the time course of relative cell volume changes is not affected in MIO-M1 cells exposed to HYPO Mannitol (A) while it is significantly retarded in cells faced with HYPO NaCl (B). Thus, RVD is significantly decreased by Ba 2+ only when external NaCl concentration varies (HYPO NaCl ) (C). Similar results are observed in the presence of the volume-sensitive Cl 2 channels blocker NPPB ( Figure 4A-C). These results clearly indicate that K + and Cl 2 channels are involved in the RVD response of MIO-M1 cells; however, their participation is evident only under certain experimental conditions.

Effect of Extracellular Medium Composition on V m during a Hypoosmotic Challenge in MIO-M1 Cells
In this set of experiments, we evaluated V m after a hypoosmotic shock (DOsM = 100 mOsM) generated either varying or keeping a constant extracellular ion composition, and using the potentiometric dye DIBAC4 (3) . Figure 5A shows the time course of fluorescence changes (F t /F 0 ). The response in both conditions consists of an initial depolarization followed by a partial repolarization. The magnitude of this repolarization is assessed as the difference between peak maximum V m and V m 30 minutes after exposure to a hypoosmotic media (Vm max 2Vm min ). As seen in Figure 5D, repolarization is significantly larger in the HYPO NaCl condition than in the HYPO Mannitol condition.
We then evaluated whether Ba 2+ or NPPB treatments affect V m before and after the hypoosmotic shock. When Ba 2+ is added to cells exposed to isoosmotic solutions (Mannitol or NaCl), V m depolarizes (DV m = 5063 mV, n = 57), thus indicating that Eq K is negative in relation to V m . In contrast, the addition of NPPB induces hiperpolarization of V m (DV m = 25361.4 mV, n = 128), thus indicating that Eq Cl is positive in relation to V m . These Figure 7. Modeling of cell response to HYPO NaCl when P K is reduced. Time courses of V t /V 0 (A), V m /V m0 (B), J net (C) and V m , Eq Cl , Eq K (D) simulated in cells exposed to HYPO NaCl . Before the hypoosmotic shock, resting P K was reduced by half and remained constant throughout the entire simulation. At time = 0 extracellular osmolarity was reduced (DOsM = 100 mOsM) and after a delay of 20 s, P Cl 2but not P K 2 was increased, according to Equation 4. A negative value of J net indicates an outward flux. doi:10.1371/journal.pone.0057268.g007 changes in resting V m suggest that Ba +2 -sensitive K + channels and NPPB-sensitive Cl 2 channels contribute to resting potential. Interestingly, after the osmotic shock, the presence of Ba 2+ or NPPB does not affect the magnitude of repolarization in HYPO Mannitol , but does significantly reduce V m repolarization in the HYPO NaCl condition ( Figure 5B-D).
These results, together with those from the previous section, suggest that there may be an interplay between RVD and V m repolarization, and that both depend on extracellular media composition.

Simulation of Cell Volume and V m Changes under Different Extracellular Ion Composition
A mathematical model was designed to investigate why RVD and V m changes differ between cells exposed to a hypoosmotic challenge (DOsM = 100mOsM) by varying NaCl composition or by removing mannitol (and thus unchanging NaCl composition). The model considers two different conditions: one in which cells face the HYPO NaCl extracellular solution and another in which cells are exposed to the HYPO Mannitol extracellular solution ( Table 2). We calculated V cell changes, V m and E q as well as net ionic flux (J net ) under both conditions. Since RVD depends on the activation of K + and Cl 2 channels, a regulatory response was simulated by increasing both ion permeabilities at t = 20 s. Figure 6A shows that after the hypoosmotic challenge, cells exposed to HYPO NaCl initially swell and then partially restore their original volume reaching a new steady-state (%RVD 10 34%).
A similar response is observed in cells exposed to HYPO Mannitol , but their volume is re-established to a lesser extent (24%, Figure 6A). Simulated relative changes in V m show that the difference in %RVD 10 among both conditions is associated with a disparity in the magnitude of repolarization ( Figure 6B). As expected, under both conditions, Cl 2 exiting the cell is electrically coupled to K + efflux, thus leading to a quasi electroneutral KCl efflux followed by water. The reduced %RVD 10 in HYPO Mannitol , as compared to HYPO NaCl, is due to a reduced net osmolyte efflux ( Figure 6C). Net osmolyte efflux, and therefore RVD, ends just when V m reaches a new stationary value. The evolution of equilibrium potentials and V m during these simulations is illustrated in Figure 6D. Since in the HYPO NaCl condition hypoosmolarity is achieved by removing NaCl, immediately after the hypoosmotic shock, [Cl] o suddenly decreases, transiently increasing the equilibrium potential of this ion ( Figure 6D, t = 0, peak Eq Cl ). The subsequent swelling determines a depolarization associated with the dilution of intracellular K + . Since in this cell Cl 2 exhibits an equilibrium potential that is positive as compared to V m , when RVD is activated at 20 s, an additional increase in V m is produced due to the opening of Cl 2 channels. As intracellular Cl 2 concentration decreases, Eq Cl becomes more negative. In addition, K + permeability is also increased, a fact that tends to bring V m closer to Eq K . As a consequence, V m partially repolarizes during RVD ( Figure 6B and 6D).
As opposed to the HYPO NaCl condition, cells exposed to HYPO Mannitol keep extracellular NaCl composition constant; therefore, at the instant that extracellular solution is changed, Figure 8. Modeling of cell response to HYPO NaCl when P Cl is reduced. Time courses of V t /V 0 (A), V m /V m0 (B), J net (C) and V m , Eq Cl , Eq K (D) simulated in cells exposed to HYPO NaCl . Before the hypoosmotic shock, resting P Cl was reduced a tenfold and remained constant throughout the entire simulation. At time = 0 extracellular osmolarity was reduced (DOsM = 100 mOsM) and after a delay of 20 s, P K 2but not P Cl 2 was increased, according to Equation 4. A negative value of J net indicates an outward flux. doi:10.1371/journal.pone.0057268.g008 the concentrations of external and internal permeable ions remain constant. Thus, the substitution of the extracellular solution does not affect per se neither Eq Cl nor V m ( Figure 6D, t = 0 s, Eq Cl ). During cell swelling, the concentration of all intracellular species is reduced, which explains the increase of Eq K and the decrease of Eq Cl ( Figure 6D, t = 0-20 s). Mainly, it is the dilution of intracellular Cl 2 , together with the initial depolarization due to the opening of volume-activated Cl 2 channels, what reduces the electrochemical gradient for Cl 2 . As a consequence, V m repolarization and cell volume regulation are smaller than in the HYPO NaCl condition.
Given that our experimental results showed that the reduction of RVD by K + and Cl 2 channels blockers is only evident in cells exposed to HYPO NaCl , the following simulations were performed in this condition. Figure 7 shows the time course of V t /V 0 , Eq Cl , Eq K , V m and J net when K + permeability is decreased (reduced P K ), thus mimicking cells exposed to Ba 2+ , versus control conditions (control P K ). Experimental data indicate that the blockage of K + channels with Ba 2+ in MIO-M1 cells has two effects: 1) When added to an isoosmotic solution, V m depolarizes and 2) When added in the presence of a hypoosmotic shock, it significantly reduces RVD response. Simulations take into account these effects and therefore, even before the hypoosmotic shock, P K is reduced by half, remaining constant throughout the entire simulation time. Figure 7A shows that when cell P K is reduced, RVD response is lower as compared to control P K (%RVD 10 :13% vs. 34%, respectively). Even more, a reduced P K leads to a decrease in V m repolarization ( Figure 7B) together with a decrease in net osmolyte efflux ( Figure 7C). In addition, Figure 7D shows that Cl 2 electrochemical gradient is reduced while K + electrochemical gradient tends to increase. However, this rise in K + driving force does not contribute to RVD, since P K is reduced. Figure 8 shows the time course simulations of all the variables described above, when Cl 2 permeability is decreased (reduced P Cl ), thus imitating cells exposed to NPPB, versus control conditions (control P Cl ). Since treatment with NPPB leads to hyperpolarization and to a decrease in the RVD response, these effects are reproduced in our simulations. Before the hypoosmotic shock, P Cl is lowered tenfold and remains constant throughout the simulation. Simulations prove that when cells have a reduced P Cl , RVD response is completely absent, V m repolarization is significantly reduced and net osmolyte efflux is almost abolished ( Figure 8A-C). Figure 8D shows that, as a consequence of P Cl reduction, resting V m is hyperpolarized and the magnitude of the initial depolarization is diminished. Therefore K + electrochemical gradient is reduced throughout the entire simulation. On the contrary, Cl 2 electrochemical gradient is augmented; however, this increase in Cl 2 driving force cannot lead to successful RVD due to the low P Cl .

Discussion
In the present work we evaluated, for the first time, the RVD response in the immortalized retinal Müller cell line, MIO-M1, which maintains important functional characteristics of Müller cells [19]. However, since Müller cells function in vivo strongly depends on tissue structure and on the presence of other cell types [31], the extrapolation of our results to the in vivo condition may be limited. Nevertheless, taking in mind the appropriate considerations, cell culture is a simple and useful model to get insight into the complex machinery used by Müller cells to regulate their volume.
We showed that MIO-M1 cells respond to a hypoosmotic challenge with cell swelling and subsequent RVD 2which is at least in part mediated by K + and Cl 2 channels2 and that this process is associated to V m depolarization followed by repolarization. Our results demonstrate that RVD response depends on the composition of extracellular media. Cells exposed to a hypoosmotic solution with reduced ionic strength (HYPO NaCl ) underwent maximum RVD (,100%) and had a larger repolarization. Both of these responses were reduced by K + or Cl 2 channel blockers. On the other hand, cells facing a hypoosmotic solution with the same ionic strength (HYPO Mannitol ) as the isoosmotic solution showed a lower RVD (,75%) and a smaller repolarization and were not affected by blockers. Our mathematical model qualitatively described the observed changes in cell volume and V m . Simulations offered complementary information that explain how the opening of K + and Cl 2 channels, as well as changes in their electrochemical gradients, account for the differences in the RVD responses observed in cells exposed to HYPO NaCl versus HYPO Mannitol .
This observed participation of K + and Cl 2 channels in the RVD response of MIO-M1 cells is in line with previous reports in glial cells [4,15,32,33]. It is interesting to note that in simulations, that only considered passive K + and Cl 2 fluxes as RVD mechanism, RVD 10 magnitude in HYPO NaCl was 34%, a value comparable to the experimental fraction of RVD 10 inhibited by the use of BaCl 2 and NPPB (,32% and ,25%, respectively). Then, the differences between absolute values of experimental and simulated RVD 10 (,70%), can be explained, at least in part, by the existence of other RVD mechanisms which are neither being inhibited by these drugs nor simulated. Indeed, organic osmolyte release during cell volume regulation has been widely described in glial cells [15,34]. Nevertheless, NPPB is also a well known blocker of this RVD mechanism and is likely affecting organic osmolyte release in our experiments. In fact, some previous evidence showed that in glial cells a residual fraction of organic osmolyte release was observed even after NPPB inhibition [35]. Moreover, in hyppocampal slices, where an exocytosis-mediated mechanism was proposed, a fraction of organic osmolyte release was not sensitive to NPPB [36]. Another possibility is that part of the RVD response could be associated to KCl efflux by KCC cotransporters although in other systems, RVD rates mediated by this transporter are lower than those measured in our study [15]. This, however, does not rule out their potential contribution to the RVD response in MIO-M1 cells. Finally, it cannot be discarded that the technique used in our study to assess RVD could be overestimating the real cell volume changes. Nevertheless, the high RVD rates observed in the MIO-M1 cells in our work are very close to previously reported data in glial cells using other techniques [4,15]. Although future studies are necessary to completely unmask RVD in MIO-M1 cells, it is likely that a combination of all the above described mechanisms explains the differences between experimental and simulated data.
Our data also demonstrated that, regardless of the activation of K + and Cl 2 channels, their contribution to the RVD response depends on the equilibrium potentials of these ions relative to the resting membrane potential of the cell. In fact, we found that the nature of the RVD response is affected by the steady-state condition previous to the osmotic shock. Our experiments indicated that, at steady-state, Eq K is more negative relative to V m while, as previously shown in glial cells [37,38], Eq Cl is less negative than the V m . MIO-M1 cells exposed to an isoosmotic solution in which NaCl was partially replaced with mannitol (ISO Mannitol ) had a slightly more negative V m than those exposed to a normal NaCl. When these cells are challenged with a hypoosmotic solution that keeps the same ionic strength, the intracellular compartment is diluted, thus reducing the chemical gradient of Cl 2 and K + , a fact that opposes KCl efflux, and thus RVD. On the other hand, when cells are exposed to HYPO NaCl , the intracellular compartment is also diluted, but since the external solution has a reduced Cl 2 concentration, the driving force for KCl efflux decreases to a lesser extent as compared to the HYPO Mannitol condition. Therefore, the magnitude of KCl efflux during cell volume regulation will depend not only of the activation of specific ion channels but also of the magnitude of the driving forces of these ions. This also explains why experiments with HYPO Mannitol , in which KCl efflux is diminished, the blockage of K + and Cl 2 channels does not affect RVD response.
In our experiments, regardless of the composition of the hypoosmotic solution, cell swelling causes V m depolarization. This depolarization could be at least partially explained by the dilution of intracellular K + , thus shifting Eq K and V m to less negative values and/or to the activation of channels corresponding to permeant species that exhibits an Eq positive to V m . We suggest that in MIO-M1 cells the opening of Cl 2 channels certainly contributes to cell depolarization during swelling, as previously described in other cells types [38,39]. In fact, the blockage of Cl 2 channels in the HYPO NaCl condition resulted in a markedly reduced swelling-induced depolarization. Nevertheless, it cannot be discarded that other ions with Eq that are more positive than V m could also account for this depolarization, as reported in others cell types [40,41,42].
Our results revealed that the gradual V m repolarization that coincides with RVD can be explained by the dissipation of Cl 2 and K + electrochemical gradients, which lead the cells to a new stationary-state. Clearly, the magnitude of this repolarization is lower in HYPO Mannitol , a condition in which these gradients are reduced as compared to HYPO NaCl . Even more, the blockage of K + and Cl 2 channels only in those conditions in which electrochemical gradients are considerable, like in HYPO NaCl , significantly reduces RVD and V m repolarization by preventing the dissipation of these gradients.
Altogether, experimental and theoretical observations allow us to propose that the efficiency of the RVD process in Müller glia depends not only on the activation of ion channels, but is also strongly modulated by concurrent changes in the membrane potential. Thus, the relationship between ion permeability changes and volume regulation is complex and increments in K + and Cl 2 ionic conductances do not necessarily induce osmolyte fluxes large enough to give rise to RVD.
A better understanding of the relationship between cell volume and V m in the central nervous system is of great interest, since neural activity itself as well as certain pathological conditions induce large alterations in extracellular fluid composition 2particularly in Na + , K + and Cl 2 concentrations2 which are associated to changes in these two interdependent baseline parameters, important for cellular function.