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
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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 MMeasurement 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 |
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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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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.
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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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