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Am J Physiol Cell Physiol 275: C416-C422, 1998;
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Vol. 275, Issue 2, C416-C422, August 1998

Intracellular ionic strength regulates the volume sensitivity of a swelling-activated anion channel

Carolyn L. Cannon1,2, Srisaila Basavappa1, and Kevin Strange1

1 Laboratory of Cellular and Molecular Physiology, Departments of Anesthesiology and Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232; and 2 Division of Respiratory Diseases, Children's Hospital, Boston, Massachusetts 02115

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell swelling activates an outwardly rectifying anion channel termed VSOAC (volume-sensitive organic osmolyte/anion channel). Regulation of VSOAC by intracellular electrolytes was characterized in Chinese hamster ovary cells by whole cell patch clamp. Elevation of intracellular CsCl concentration from 40 to 180 mM resulted in a concentration-dependent decrease in channel activation. Activation of VSOAC was insensitive to the salt gradient across the plasma membrane, the intracellular concentration of specific anions or cations, and the total intracellular concentration of cations, anions, or electrolytes. Comparison of cells dialyzed with either CsCl or Na2SO4 solutions demonstrated directly that VSOAC activation is modulated by intracellular ionic strength (µi). The relative cell volume at which VSOAC current activation was triggered, termed the channel volume set point, decreased with decreasing ionic strength. At µi = 0.04, VSOAC activation occurred spontaneously in shrunken cells. The rate of VSOAC activation was nearly 50-fold higher in cells with µi = 0.04 vs. those with µi = 0.18. We propose that µi modulates the volume sensor responsible for channel activation.

chloride channels; volume regulation; cell swelling; patch clamp; organic osmolytes

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

AN APPARENTLY UBIQUITOUS response to swelling in vertebrate cells is activation of an anion current termed ICl,swell. The general characteristics of this current include an Eisenman type I anion permeability sequence (I- > Br- > Cl- > F-), modest outward rectification, voltage-dependent inactivation at potentials above the Cl- equilibrium potential, inhibition by a wide variety of compounds including conventional anion transport inhibitors, block by extracellular nucleotides, and modulation of swelling-induced activation by intracellular ATP concentration (15, 21). A substantial body of evidence indicates that the channel responsible for ICl,swell is the major pathway for volume regulatory organic osmolyte loss (8, 21). Given this dual function, we have termed this channel VSOAC (volume-sensitive organic osmolyte/anion channel).

Patch-clamp studies of skate hepatocytes demonstrated that swelling-induced activation of VSOAC is inhibited in a concentration-dependent manner by increases in salt concentration of the patch pipette solution (6). We have recently shown a similar phenomenon in patch-clamped mammalian C6 glioma and Chinese hamster ovary (CHO) cells. Importantly, the inhibitory effect of elevated intracellular electrolytes can also be observed in intact C6 glioma cells (2). Elevation of cytoplasmic Na+, K+, and Cl- concentrations by activation of regulatory volume increase (RVI) transport pathways shifts the volume sensitivity of VSOAC such that larger degrees of cell swelling are required for activation. Interestingly, C6 cells still undergo regulatory volume decrease (RVD) even when the amount of cell swelling is insufficient to activate VSOAC and organic osmolyte efflux. This indicates that C6 cells possess RVD transport pathways that selectively mediate efflux of inorganic ions.

Our early studies on skate heptocytes (6) suggested that VSOAC activation was modulated by intracellular Cl- levels. However, we could not rule out the possibility that ionic strength and/or other physicochemical solution parameters were involved in channel regulation. The present investigations were therefore undertaken to define which of the parameters associated with changes in intracellular electrolyte concentration control channel activity. We demonstrate that cytoplasmic ionic strength controls both the volume set point and rate of swelling-induced activation of VSOAC.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell culture. CHO cells were grown in Ham's F-12 (GIBCO) with 10% fetal bovine serum and penicillin-streptomycin. After growth to 25-50% confluence, cells were acclimated to 400 mosmol/kgH2O Ham's F-12 (60 mM NaCl addition) for 24-48 h before experiments. Hypertonic growth medium was used so that cells could be patch clamped with pipette solutions containing high salt concentrations that were isotonic to the control extracellular bath. The effects of ionic strength described in this paper were also observed in cells grown in normotonic (i.e., 300 mosmol/kgH2O) medium (2).

Patch-clamp recordings. CHO cells were grown in 35-mm culture dishes and dissociated by brief treatment with Ca2+- and Mg2+-free modified Hanks' solution. Dissociated cells were allowed to reattach to the poly-L-lysine-coated coverslip bottom of a bath chamber (model R-26G; Warner Instrument, Hamden, CT), which was mounted onto the stage of a Nikon Diaphot or TE 300 inverted microscope. Patch electrodes were pulled from 1.5-mm outer diameter borosilicate glass microhematocrit tubes (Fisher Scientific, St. Louis, MO) that had been silanized with dimethyldichlorosilane (Sigma Chemical, St. Louis, MO). Electrodes were not fire polished before use.

The standard bath solution contained (in mM) 140 NaCl, 5 MgSO4, 12 HEPES, 8 Tris, 5 glucose, 2 glutamine, and 100 sucrose (pH 7.4; osmolality = 400 mosmol/kgH2O). Cells were patch clamped with a pH 7.2 pipette solution that contained (in mM) 2 MgSO4, 20 HEPES, 6 CsOH, 1 EGTA, 0.5 GTP, and 2 ATP and variable concentrations of salt (see RESULTS). Osmolality was adjusted to 375-385 mosmol/kgH2O by addition of sucrose. The osmolality of all solutions was measured using a vapor pressure osmometer (Vapro model 5520; Wescor, Logan, UT). Electrodes had direct current resistances of 4-6 MOmega when filled with a pipette solution containing 140 mM CsCl. Cells were used only if the series resistance was no greater than ~140% of the pipette resistance.

An Axopatch 200A (Axon Instruments, Foster City, CA) patch-clamp amplifier was used to voltage clamp C6 cells following gigaseal formation and attainment of whole cell access. Command voltage generation, data digitization, and data analysis were carried out on an 80486, 100-MHz IBM-compatible computer (Optiplex 4100/MX, Dell Computer) using a DigiData 1200 AD/DA interface with pCLAMP software (Axon Instruments). Electrical connections to the amplifier were made using Ag-AgCl pellets and 3 M KCl-agar bridges.

Cell membrane potential was held at 0 mV. Whole cell currents were measured by varying membrane potential from -80 to +80 mV at 80 mV/s every 15 s. Swelling was induced by reducing bath osmolality to 325 mosmol/kgH2O via removal of sucrose. At the end of a patch-clamp recording, bath osmolality was returned to 400 mosmol/kgH2O and another cell was selected for study. Cells were exposed to hypotonicity approximately every 15-25 min and were exposed to at most four hypotonic shocks before they were removed and replaced with fresh cells. This number of repetitive hypotonic shocks had no effect on the rate or extent of current activation (data not shown).

Measurement of relative cell volume changes. Changes in the volume of patch-clamped cells were quantified by video-enhanced differential interference contrast microscopy (22). Cells were visualized using a Zeiss Neofluar ×63 (1.25 numerical aperture) oil-immersion objective lens and a Leitz ×32 (0.4 numerical aperture) condenser lens or a Nikon ×60 objective lens (0.7 numerical aperture) and a long-working-distance condenser lens (0.52 numerical aperture). Images were recorded using a super VHS video cassette recorder (model SVO-2000, Sony Electronics, San Jose, CA) and a Hamamatsu charge-coupled device camera (model C2400, Hamamatsu Photonics, Hamamatsu City, Japan).

The cross-sectional area (CSA) of single cells was quantified by digitizing recorded video images with an image processing computer board (MV-1000; MuTech, Woburn, MA) with 512 × 480 × 8 bit resolution and a 200-MHz Pentium computer (Dimension XPS M200s; Dell Computer, Austin, TX). Digitized images were displayed on the computer monitor, and cell borders were traced using a mouse and a computer-generated cursor. CSAs of the traced regions were determined by image analysis software (Optimas; Bioscan, Edmonds, WA). This image acquisition and analysis system allows detection of changes in CSA with an accuracy of ±2-3%. Cells dissociated from their growth substratum had a spherical morphology, and relative volume changes were therefore calculated as (experimental CSA/control CSA)3/2. Optical sectioning methods have demonstrated that this approach reliably tracks relative cell volume increases up to 250% of control values (unpublished observations). During swelling, a small percentage of cells exhibited bleb formation. These cells were excluded from the analysis of both volume changes and current activation.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of intracellular salt concentration on current activation. Figure 1 illustrates the effect of intracellular electrolyte concentration on cell swelling and VSOAC current activation. The predominant salt in the pipette solutions used in these experiments was CsCl. CsCl concentration was altered by isosmotic replacement with sucrose. Once the whole cell configuration had been attained and whole cell parameters adjusted, cells were dialyzed for 1.5-2 min with various pipette solutions before swelling was initiated. However, cells dialyzed with 40 mM CsCl exhibited spontaneous current activation in the absence of apparent swelling (discussed in more detail below). For these cells, therefore, bath osmolality was reduced as soon as current activation was observed (typically 15-30 s after obtaining the whole cell configuration).


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  		Fig. 1.   Intracellular electrolyte levels alter swelling-induced activation of VSOAC (volume-sensitive organic osmolyte/anion channel) in a concentration-dependent manner. Cells were patch clamped with pipette solutions containing varying concentrations of CsCl, and swelling was induced at time 0 by reducing bath osmolality from 400 to 325 mosmol/kgH2O. Except when a 40 mM CsCl-containing pipette solution was used, cells were dialyzed for 1.5-2.0 min before swelling. With 40 mM CsCl, whole cell current activated spontaneously within 15-30 s after membrane rupture. In these cells, swelling was induced as soon as current activation was detected. Values are means ± SE (n = 3-17). Erev, reversal potential. Inset: cell volume changes were monitored during patch-clamp recordings with video-enhanced differential interference contrast microscopy. Images were recorded on videotape, and volume changes were quantified by computer image processing. Cells dialyzed with different salt solutions swell at the same rate and to a similar extent. Values are means ± SE (n = 3-16).

As shown in Fig. 1, reduction of CsCl levels enhanced current activation in a concentration-dependent manner. The initial rates of swelling in all four groups of cells were similar (Fig. 1, inset). Thus intracellular electrolyte concentration exerts its effect on the mechanism responsible for channel activation rather than altering the stimulus (i.e., cell swelling).

Intracellular ionic strength alters VSOAC activation. When intracellular CsCl concentration is altered, eight solution physicochemical parameters are changed simultaneously: 1) nonelectrolyte (i.e., sucrose) concentration, 2) transmembrane ion gradients, 3) Cl- concentration, 4) Cs+ concentration, 5) total anion concentration, 6) total cation concentration, 7) total electrolyte concentration (i.e., the sum of both cation and anion concentrations), and 8) ionic strength. Thus it is possible that any one or any combination of these parameters controls channel activation. We have shown previously that channel activation is not altered by the sucrose concentration of the patch pipette solution and therefore carried out experiments to assess the effects of electrolytes on swelling-induced current activation.

The effect of reductions in intracellular electrolyte levels on swelling-induced current activation may have been due to the imposition of a transmembrane electrolyte gradient. Two experiments were performed to test this possibility. First, cells were dialyzed with an 80 mM CsCl solution, and current activation was monitored in the presence of a bath solution containing 140 or 80 mM NaCl. As shown in Fig. 2A, swelling-induced current activation was the same in the presence and absence of a transmembrane electrolyte gradient. Cells were also dialyzed with 140 mM CsCl and then swollen in a hypotonic bathing medium containing either 140 or 40 mM NaCl. Reducing the bath electrolyte concentration did not enhance the rate of current activation.


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  		Fig. 2.   Transmembrane electrolyte gradients and intracellular anion or cation composition do not alter VSOAC activation. A: cells were dialyzed with pipette solutions (in) containing 80 or 140 mM CsCl. Current activation was monitored in the presence of hypotonic bath solutions (out) containing 40, 80, or 140 mM NaCl. Intracellular electrolyte concentration rather than the transmembrane electrolyte gradient alters current activation. Values are means ± SE (n = 4-18). B: cells were dialyzed with pipette solutions containing 5 mM CsCl and 75 mM NaCl, 75 mM sodium gluconate (NaGluc), or 75 mM NaBr. Results demonstrate that intracellular Cl- concentration and anion composition have no effect on swelling-induced current activation. Values are means ± SE (n = 8-18). C: cells were dialyzed with pipette solutions containing 5 mM CsCl and 75 mM NaCl, 75 mM KCl, or 75 mM LiCl. Swelling-induced current activation is not modified by intracellular cation composition. Values are means ± SE (n = 5-7). In all experiments, cells were dialyzed for 1.5-2.0 min and then swollen (time 0) by reducing bath osmolality to 325 mosmol/kgH2O. Rates of cell swelling were similar under all experimental conditions (data not shown).

Our earlier studies on skate hepatocytes suggested that VSOAC activation was modified by intracellular Cl- levels. To test this possibility, cells were dialyzed with an 80 mM salt solution containing 5 mM CsCl and 75 mM NaCl, 75 mM NaBr, or 75 mM sodium gluconate. As shown in Fig. 2B, current activation was unaffected by replacement of Cl- with either Br- or gluconate. Replacement of Cl- with SO2-4 also had no effect on current activation (Fig. 3).


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  		Fig. 3.   Intracellular ionic strength (µ) regulates VSOAC activation. The µ of pipette solutions was held constant while total intracellular cation, anion, and electrolyte concentrations were varied simultaneously by replacement of 75 mM CsCl with 25 mM NaSO4 (µ = 0.08) or 135 mM CsCl with 45 mM NaSO4 (µ = 0.14). Swelling-induced current activation is enhanced by reducing intracellular µ from 0.14 to 0.08. In contrast, reductions of total cation, anion, and salt concentrations have no effect on current activation. Values are means ± SE (n = 6-10). In all experiments, cells were dialyzed for 1.5-2.0 min and then swollen (time 0) by reducing bath osmolality to 325 mosmol/kgH2O. Rates of cell swelling were similar under all experimental conditions (data not shown).

VSOAC activation may be modulated by the concentration of specific cations. This possibility was tested by dialyzing cells with 80 mM salt solutions containing 5 mM CsCl and 75 mM NaCl, 75 mM KCl, or 75 mM LiCl. Current activation was similar with all three cations (Fig. 2C).

The results discussed above demonstrate that VSOAC activation is modified by total anion concentration, total cation concentration, total electrolyte concentration, and/or ionic strength. It is not possible to experimentally isolate each of these variables, since a change in one also alters the other three. However, it is possible to hold ionic strength constant and simultaneously vary total anion, cation, and electrolyte concentration. Cells were therefore dialyzed with pipette solutions having an ionic strength of either 0.08 or 0.14. The 0.08 ionic strength solutions contained 80 mM CsCl or 5 mM CsCl plus 25 mM Na2SO4, whereas the 0.14 ionic strength solutions contained 140 mM CsCl or 5 mM CsCl plus 45 mM Na2SO4. As shown in Fig. 3, current activation occurred more slowly in the 0.14 ionic strength solutions. Importantly, current activation was similar in the pure Cl- and mixed Cl- and SO2-4 solutions with the same ionic strength despite the fact that total anion, cation, and electrolyte concentrations were considerably lower in the mixed solutions. These results demonstrate that VSOAC activation is modulated by intracellular ionic strength rather than other parameters that vary with changes in the electrolyte concentrations of the pipette solutions.

Ionic strength regulates VSOAC by altering volume set point and rate of activation. Analysis of the data in Fig. 1 revealed that decreases in intracellular ionic strength increased the rate of swelling-induced current activation (Fig. 4A). Reduction of ionic strength also decreased the channel volume set point, which is defined as the relative cell volume at which VSOAC activation is triggered (Fig. 4B).


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  		Fig. 4.   VSOAC volume set point and rate of activation are modulated by intracellular ionic strength. A: reducing intracellular µ from 0.18 to 0.04 increases rate of current activation by nearly 50-fold. Rates were obtained from data shown in Fig. 1 by performing linear regression analysis on the first 5-6 time points after current values began to rise above the baseline. B: reductions in intracellular ionic strength reduce VSOAC volume set point. Volume set point is defined as the relative cell volume at which VSOAC activation is triggered. Values illustrated by bullet  were obtained from data in Fig. 1 and Fig. 1, inset. Solid line is a linear regression through these points (y = 6.96x + 0.555). It was not possible to obtain a volume set point for cells dialyzed with 40 mM CsCl (µ = 0.04) using the experimental protocol defined in Fig. 1. Under these conditions, whole cell current activated without apparent cell swelling 15-30 s after membrane rupture. , Estimated volume set point obtained from the experiment shown in Fig. 5. This value falls on the line predicted by linear regression.

As noted earlier, cells dialyzed with a 40 mM salt solution exhibited spontaneous current activation that occurred within 30 s after membrane rupture and was not associated with observable cell swelling. The solid line in Fig. 4B is a linear regression through the volume set points obtained from cells dialyzed with 180, 140, and 80 mM salt solutions. With the assumption that the relationship between volume set point and ionic strength is linear below an ionic strength of 0.08, the regression predicts that VSOAC will activate in cells shrunken to a relative volume of 0.83 when they are dialyzed with 40 mM salt solution. We tested this prediction by performing the following experiment. The osmolality of a pipette solution containing 40 mM CsCl was reduced from ~380 to 360 mosmol/kgH2O by removal of sucrose. When the plasma membrane was ruptured, the cells shrank spontaneously due to the outwardly directed osmotic gradient (Fig. 5). In many cells, the shrinkage was followed by a slow reswelling and current activation. However, in eight cells, we observed that current activation occurred over a period of 1-3 min during which no reswelling was observed (Fig. 5). The relative volume (mean ± SE) of these cells 1 min after current recordings were started was 0.84 ± 0.03. 


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  		Fig. 5.   VSOAC activates spontaneously in shrunken cells dialyzed with a low ionic strength solution. Cells were dialyzed with a 360 mosmol/kgH2O pipette solution containing 40 mM CsCl (µ = 0.04). Bath osmolality was 400 mosmol/kgH2O. When the whole cell configuration was obtained, cells shrank 15-20% due to the outwardly directed osmotic gradient. Anion current activated spontaneously in the shrunken cells without measurable cell swelling. Values plotted are means ± SE (n = 8). Mean relative cell volume 1 min after membrane rupture was 0.84, which represents a maximal estimate of VSOAC volume set point when intracellular µ = 0.04. This estimated volume set point is plotted on Fig. 4B () and is close to the value predicted by linear regression analysis.

It is possible that the spontaneously activating and swelling-induced currents are due to the activity of different channels. We therefore examined the biophysical and pharmacological characteristics of the two currents (Table 1). Both currents had similar rectification and voltage sensitivity and were inhibited in a voltage-dependent manner and to the same extent by 100 µM DIDS. The channels responsible for the currents had similar relative anion permeabilities. Reversal potentials (Erev) for both currents were approximately -16 mV. With the assumption that relative cation permeability (Pcation/PCl) was 0.03, which we and others have estimated previously for VSOAC, Erev should have been approximately -30 mV. With an Erev of -16 mV, the Pcation/PCl estimated from the Goldman-Hodgkin-Katz equation of the spontaneously activating and swelling-activated current in cells dialyzed with 40 mM CsCl is ~0.24. Interestingly, these results are consistent with our recent studies of VSOAC in skate hepatocytes in which we observed that reduction in intracellular ionic strength substantially increased the relative cation permeability of the channel (6).

                              
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  		Table 1.   Characteristics of Cl - currents activated spontaneously or by swelling in cells dialyzed with a pipette solution containing 40 mM CsCl

Taken together, the data in Table 1 indicate that both the spontaneously activating and swelling-induced currents are due to the activity of the same channel. We conclude, therefore, that reduction of intracellular ionic strength to 0.04 decreases VSOAC volume set point to a relative volume of <= 0.84. This value is remarkably close to that predicted from the linear regression analysis shown in Fig. 4B.

The above results could be interpreted as indicating that a decrease in intracellular ionic strength induced by hypotonic swelling is the signal that triggers activation of VSOAC. In the whole cell patch-clamp mode, bulk cytoplasmic ionic strength is unlikely to change during water influx and cell swelling due to constant dialysis from the patch pipette solution. However, it is possible that localized changes in ionic strength occur close to the cell membrane. To test whether swelling-induced reductions in ionic strength were necessary for channel activation, cell volume was increased in the absence of osmotically induced transmembrane water flow by pressure injection of fluid from the patch pipette into the cell. Data shown in Fig. 6 illustrate the results from such an experiment. The cell was patch clamped using a pipette solution containing 140 mM CsCl. At time 0, cell volume was increased abruptly ~65% by pressure injection. Current activation was observed within 10 s after the volume increase. In five separate cells, the mean ± SE pressure injection-induced volume increase and rate of current activation were 120 ± 21% and 16.8 ± 3.6 pA · pF-1 · min-1, respectively. These results demonstrate clearly that swelling-induced reductions in ionic strength are not necessary for channel activation. We conclude that intracellular ionic strength alters how the "volume sensor" responsible for activation of VSOAC detects cell volume. Decreases in ionic strength shift the volume set point to lower values.


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  		Fig. 6.   VSOAC activates in cells swollen by pressure injection of fluid. Cell was patch clamped with a pipette solution containing 140 mM CsCl. Cell volume was increased ~65% at time 0 by placing positive pressure on the patch pipette and forcing fluid into the cytoplasm. Data shown are from a single cell. Mean ± SE values for rate of current activation and cell volume increase obtained in 5 separate cells are reported in RESULTS.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Our previous studies have demonstrated that intracellular electrolyte concentration alters the volume sensitivity of VSOAC in both patch-clamped and intact cells (2, 6). However, it was uncertain from these studies whether anion, cation, or total electrolyte concentration and/or ionic strength were responsible for modulation of channel activity. We have demonstrated directly in the present investigation that ionic strength is responsible for changes in channel volume set point and rate of activation. In a recent series of studies on endothelial cells, Nilius et al. (14) reached a similar conclusion.

Parker et al. (19) demonstrated recently that the volume set point for the swelling-activated KCl cotransporter in dog red blood cells is reduced by increased intracellular salt concentration. In other words, as cytoplasmic salt levels rise, less swelling is required to trigger activation of the cotransporter. This effect is opposite to that observed for VSOAC activation; elevation of salt concentration (i.e., ionic strength) renders the channel less sensitive to swelling.

Motais et al. (13) concluded that intracellular ionic strength plays a key role in controlling volume regulatory amino acid and KCl efflux from trout red blood cells. These investigators manipulated cytoplasmic ionic strength by swelling cells in the presence of 145 mM urea, 50 mM NH4Cl, or mixtures of urea and NH4Cl. Swelling-induced amino acid loss was an inverse function of cytoplasmic ionic strength. At high ionic strength, there was little or no swelling-induced amino efflux and volume regulation was instead mediated by coupled K+ and Cl- loss, presumably via the KCl cotransporter. Similarly, volume regulation occurs normally in C6 glioma cells that have elevated cytoplasmic ionic strength despite the fact that there is little or no VSOAC activation (2). This implies the existence of transport pathways, such as the KCl cotransporter, that selectively mediate volume regulatory efflux of electrolytes.

The differential effect of intracellular salt concentration on volume regulatory electrolyte and organic osmolyte transport pathways may have important physiological implications (2, 21). When cell swelling occurs concomitantly with elevated cytoplasmic ionic strength, it is advantageous for cells to use electrolytes selectively for RVD via activation of an "electrolyte-selective" transport pathway such as the KCl cotransporter. The loss of organic osmolytes under such conditions would mediate RVD but would also further concentrate intracellular electrolytes as cells undergo volume regulatory water loss and concomitant shrinkage. Changes in intracellular ionic strength may therefore play an important role in coordinating the activities of various volume regulatory transport pathways. This postulated coordinated regulation could in turn contribute to the long-term maintenance of cytoplasmic ionic strength.

There are at least two physiologically relevant conditions under which coordinated regulation of volume regulatory electrolyte and organic osmolyte efflux pathways might be beneficial for cell function. First, when cells are exposed to hypertonicity, they shrink and undergo an RVI response mediated initially by salt uptake (4), which increases intracellular ionic strength. If cells with elevated cytoplasmic ionic strength swell and then volume regulate by losing organic osmolytes, ionic strength will remain elevated. The use of organic osmolytes for RVD by cells that experience repetitive periods of shrinking and swelling could conceivably cause electrolytes to rise to damaging levels. Intertidal organisms and cells in the renal medulla, for example, may be exposed to repetitive, short-term changes in extracellular osmolality during tidal shifts and during changes in urinary concentrating ability, respectively.

Coordinated regulation of electrolyte and organic osmolyte efflux pathways might also be beneficial during RVD following cell swelling brought about by net salt influx, which is referred to as "isotonic swelling." Swelling induced by intracellular salt accumulation occurs during abrupt shifts in transmembrane ion transport and can occur under normal and pathophysiological conditions. Loss of organic osmolytes following isotonic swelling induced by inorganic ion accumulation would cause cytoplasmic ionic strength to rise further. Therefore, a mechanism that allows cells to monitor electrolyte levels and control the relative amounts of organic osmolytes and inorganic ions lost during RVD would allow the simultaneous regulation of volume and intracellular ionic strength.

In addition to regulating organic osmolyte efflux, ionic strength may also regulate organic osmolyte accumulation. Cell shrinkage increases the transcription of genes coding for organic osmolyte transporters and enzymes involved in their synthesis (1, 3). The rise in ionic strength brought about by shrinkage-induced water loss and/or by activation of RVI electrolyte accumulation pathways has been proposed by Burg and co-workers (1, 3) to play a key role in regulating the expression of these genes. Thus changes in cytoplasmic ionic strength may coordinate the activities of organic osmolyte accumulation and loss pathways.

Parker and co-workers (18, 19) have proposed that there is a common volume sensor and signaling system that coordinates the activity of several different volume-sensitive transporters in dog red blood cells. This hypothesis is based on observations that the volume sensitivity of three different ion transport pathways is modulated in a coordinated fashion by a variety of different experimental manipulations (18). Parker and co-workers have also postulated that the volume signal is a change in intracellular macromolecular crowding (11, 17, 19) and have suggested that ionic strength alters the association of a putative regulatory protein with various transport pathways (19).

The mechanism by which ionic strength modulates VSOAC volume set point is unknown. Indeed, there is little understanding of how VSOAC senses cell volume and how the volume signal is transduced into channel activation (15, 21). It is important to emphasize here that channel activation is not triggered by a swelling-induced reduction in intracellular ionic strength. As shown in Fig. 6, VSOAC can be activated in the absence of osmotically driven water influx simply by placing positive pressure on the patch pipette and forcing fluid into the cytoplasm, thereby expanding cell volume. Clearly, under such conditions, there is no change in cytoplasmic ionic strength. Although swelling-induced reductions in ionic strength may modulate VSOAC, other events associated with cell swelling must be involved in channel activation.

Mechanosensitivity has been invoked as playing an important role in regulating volume-sensitive channels (5, 15) and the KCl cotransporter (7). As described by Hamill and McBride (5), two broad classes of mechanisms have been implicated in conferring mechanosensitivity. These mechanisms are referred to as the "tethered model" and the "bilayer model." The tethered model envisions direct interactions between cytoskeletal proteins and proteins that comprise the transport pathway or its regulatory machinery. Changes in cytoskeletal tension or cytoskeletal-protein interactions induced by cell swelling could activate transport pathways directly or indirectly via changes in the activity of signal transduction pathways. The bilayer model arose from studies with purified and recombinant proteins reconstituted into artificial lipid membranes (16, 23). Cell swelling may induce changes in bilayer tension that activate the transport pathway through tension-induced changes in protein conformation (5).

Our ability to activate VSOAC in shrunken cells (Figs. 4 and 5) argues against a role for swelling-induced changes in bilayer mechanical properties. However, changes in cytoskeletal mechanical properties could be involved. It has been suggested that VSOAC activation is modulated by F-actin. Levitan et al. (9) demonstrated that disruption of F-actin with cytochalasin B or stabilization with phalloidin increased and decreased, respectively, the rate of VSOAC activation when cells were swollen slowly in the presence of small osmotic gradients. At higher rates of swelling, these compounds had no effect on channel activity. Using a different experimental protocol, Zhang et al. (24) have shown that cytochalasin B and phalloidin both inhibit VSOAC activation (10, 24). Cell swelling, via unknown mechanisms, has been shown to disrupt F-actin (9, 10, 15). Interestingly, F-actin can also be disrupted by reductions in ionic strength (12, 20). If a critical level of actin disassembly is required to trigger VSOAC activation, then it is possible to imagine that, in the presence of low intracellular ionic strength, less swelling is needed to reach that critical level. Under conditions of very low ionic strength, sufficient F-actin disassembly may occur to trigger VSOAC activation without swelling or even to trigger it in shrunken cells (see Fig. 5). Extensive studies using electrophysiological, biophysical, and molecular approaches will be needed to test this hypothesis.

    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants NS-30591 and DK-51610. C. L. Cannon was supported in part by National Institutes of Health training grant T32-HL-0763-3 and Grant CANNON 98DO from the Cystic Fibrosis Foundation.

    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: K. Strange, Vanderbilt University Medical Center, Dept. of Anesthesiology, 504 Oxford House, 1313 21st Ave. South, Nashville, TN 37232-4125.

Received 2 March 1998; accepted in final form 6 May 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Cell Physiol 275(2):C416-C422
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