Department of Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama
Submitted 13 October 2004 ; accepted in final form 14 January 2005
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ABSTRACT |
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voltage-gated chloride channel family; potassium-chloride cotransporters; peritumoral edema
Although the detailed mechanisms underlying RVD have been studied in only a few model systems, it is commonly understood that a volume decrease must involve the extrusion of organic and inorganic osmolytes followed by the obligatory movement of water (14, 28, 31, 34). Cl is the most abundant inorganic ion within the cell and, as such, is thought to be one of the major osmolytes involved in RVD. Previous studies showed that Cl efflux during RVD may occur through ion channels or transporters. In most cell types studied, swelling results in activation of Cl channels (2, 15, 32, 35, 39), and, particularly in mammalian cells, a swelling-activated Cl current (ICl,swell) has been shown to be ubiquitously expressed. ICl,swell is characterized as an outwardly rectifying current with anion selectivity sequence of I > Br > Cl > F > gluconate that is inhibited by a variety of nonspecific chloride channel inhibitors including both 5-nitro-2(3-phenylpropylamino)benzoic acid (NPPB) and DIDS (5, 26, 32, 39). The molecular identity of ICl,swell, however, remains unknown. Although their contribution to RVD is unclear, other swelling-activated Cl channels have been identified. For example, two members of the voltage-gated Cl channel family, ClC-2 and ClC-3, have been shown to activate in response to cell swelling (11, 17). ClC-2 generates currents that are inwardly rectifying, inhibited by NPPB and Cd2+, and have an anion selectivity sequence of Cl > Br > I (5, 26, 32, 42). Expression of ClC-3 generates outwardly rectifying, DIDS-sensitive currents (6, 26, 41). Although studies have shown inhibition of RVD by antisense oligonucleotides (13) and antibodies (43) generated against ClC-3, contradictory studies have shown intact volume regulation in transgenic animals with a genetic deletion of ClC-3 (38) or that expression of ClC-3 in HEK cells does not affect RVD (8). Other swelling-activated anion channels that may play a role in RVD include the yet to be identified maximal volume-sensitive anion channel current studied in astrocytes (39). Indeed, several pathways for Cl efflux may participate during RVD, because complete inhibition of this process in astrocytes, for example, requires a combination of Cl channel inhibitors (28).
The role of Cl transporters in RVD has been studied most extensively in red blood cells. In these cells, volume regulation occurs via Cl-dependent K+ flux, mediated by the activity of a K+-Cl cotransporter (KCC) (2022). The KCC family of proteins includes four members (KCC14), several of which have been shown to be activated by cell swelling (9, 24). Unlike Cl channels, which appear to be activated in almost all cell types in response to cell swelling, KCCs have been implicated mainly in volume regulation in red blood cells and a few types of epithelia, including, most recently, cervical cancer cells (36, 37).
Previous studies in astrocytes revealed that these nonmalignant cells undergo RVD aided by an increase in K+, Cl, and free amino acid efflux. Although Cl channel involvement in this process has been supported by inhibition of RVD (albeit to various degrees) by a variety of Cl channel inhibitors (14, 28, 31), the contribution of transporters to RVD in astrocytes has been inconclusive. A limited number of studies have explored volume regulation in a rat glioma cell line (C6) and have demonstrated RVD that was completely inhibited by Cl channel blockers (22, 25). Little is known regarding the properties of volume regulation in human glioma cells. Moreover, despite a wealth of previous research, the relative contribution of channels and transporters to RVD has not been clarified. In this study, we made an initial attempt to determine the mechanisms responsible for RVD in human glioma cells, with a specific emphasis on identifying the relative importance of Cl channels vs. cation-Cl cotransporters. Our data suggest, through various lines of experimental evidence, that 6070% of Cl efflux during RVD occurs through Cl channels, with up to 40% occurring through a transport-mediated process.
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MATERIALS AND METHODS |
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Cell volume measurements. Cell volumes were measured by electronic sizing with a Coulter Counter Multisizer 3 (Beckman-Coulter, Miami, FL) as previously described (28). The counter determines cell volume by measuring the voltage step that is created by the change in resistance that occurs when a cell displaces its volume in electrolyte solution as it passes through a small aperture. The aperture size used for these experiments was 100 µm.
To prepare the cells for volume measurements, cells were incubated for 3 min with 0.05% trypsin and 0.53 mM EDTA (Invitrogen, Carlsbad, CA). Trypsin was inactivated by the addition of an equal volume of glioma medium. After the cells were pelleted by brief centrifugation, the cells were resuspended in bath solution and passed through a 40-µm nylon cell strainer (Fisher). Cells were incubated in prewarmed or precooled bath solution for 10 min before the beginning of the first baseline measurement. The temperature of the cell suspension was maintained during the course of the experiment by pumping heated or cooled water from an external water bath through plastic tubing used to insulate the beaker and was monitored throughout the experiment. Cell volume measurements were obtained every minute, and each measurement was an average of 10,00020,000 cells. Five or six baseline volumes were recorded before the osmotic challenge was applied.
Electrophysiology.
Recordings of whole cell currents were made with an Axopatch 200A amplifier (Axon Instruments, Foster City, CA), following standard recording techniques (12). Patch pipettes were made with thin-walled borosilicate glass (TW150F-4; World Precision Instruments, Sarasota, FL) and an upright puller (PP-830; Narishige Instruments, Tokyo, Japan) and typically had resistances of 35 M. Current recordings were digitized online at 10 kHz and low-pass filtered at 2 kHz with a Digidata 1200 (Axon Instruments). pCLAMP 8.2 (Axon Instruments) was used to acquire and store data. Series resistance (Rs) was compensated to 80%, reducing voltage errors, and cells with a compensated Rs >10 M
were omitted. D54 cells were plated on glass coverslips and cultured for 3 days before experiments were performed. Standard bath solution was continuously exchanged at a rate of
1 ml/min.
Solutions. The control NaCl bath solutions contained the following (in mM): 130 NaCl, 5.0 KCl, 10.5 glucose, 32.5 HEPES, and 1 CaCl2. The pH of each solution was adjusted to 7.4 with NaOH, and the osmolarity of each solution was confirmed with a vapor pressure osmometer (Wescor 5500; Wescor, Logan, UT) to be 310 ± 10 mosM. Pipette solutions contained the following (in mM): 145 CsCl, 1 MgCl2, 10 EGTA, 10 HEPES sodium salt, pH adjusted to 7.3 with Tris-base. CaCl2 was added directly to pipette solution on the day of use at a concentration of 0.2 mM, resulting in a free Ca2+ concentration of 1.9 nM.
Drugs were added directly to bath solutions from stock solutions. Stock solutions of NPPB and tributyltin chloride (TBT) were dissolved at 1,000x final concentration in DMSO; bumetanide, R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid (DIOA), and DIDS were dissolved at 500x final concentration in DMSO; and CdCl2 was dissolved at 1,000x final concentration in double-distilled H2O (ddH2O). Control DMSO, at its final concentrations (0.1% and 0.2%), did not perturb cell volumes or affect volume regulation (data not shown).
Data analysis for volume regulation experiments.
Coulter Counter data were collected with Multisizer 3 software, and size listings were exported to Excel. Time points were rounded to whole minutes, and mean cell volumes (MCVs) were normalized to the average baseline value for a given experiment. All data were plotted in Origin 7.0 (MicroCal, Northampton, MA) as means ± SE with the number of experiments performed (n). Similar to previous studies (25), the volume-regulatory portion of the generated curve was fit with a first-order exponential decay function, generating a unique time constant () for each experiment. Post-RVD MCV was defined as the volume recorded at time = 3
± 1 (V3
), a time at which the volume is expected to be within 5% of its final value. Significance was determined by a Student's t-test with an
-value of P < 0.05.
Western blots. Cells in 100-mm culture dishes were rinsed with ice-cold PBS, scraped, and collected in 0.5 ml of 1:100 protease inhibitor cocktail (Sigma) in PBS. After a brief centrifugation, the pellet was resuspended in RIPA buffer with protease inhibitors. The sample was sonicated, gently mixed for 30 min, and centrifuged at 4°C. The proteins of the supernatant were separated by electrophoresis using a 7.5% SDS-polyacrylamide gel (Bio-Rad). Proteins were transferred onto polyvinylidene difluoride membranes (Millipore), probed with anti-ClC-2 and -5 antibodies from Alomone Labs (Jerusalem, Israel) and anti-ClC-1, -3, -4, -6, -7 and KCC1-4 antibodies from Alpha Diagnostics (San Antonio, TX), and visualized with horseradish peroxidase-conjugated anti-rabbit antibodies (Bio-Rad) and the ECL system (Amersham).
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RESULTS |
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Cl channel inhibitors limit RVD in human glioma cells. RVD has been shown in essentially all living cells to occur because of osmolyte release by different pathways (e.g., anion and cation channels, cotransporters, etc.). It was our objective to examine the underlying mechanisms in glioma cells. To this end, we chose to examine the robust RVD elicited by a 50% hypotonic challenge. As we were primarily interested in the role of Cl in RVD, we first sought to determine the relative contribution of Cl channels to RVD in these cells. We added the Cl channel inhibitors DIDS, NPPB, and Cd2+, which have been shown to inhibit different Cl channels (5, 32, 42), to the bath solution 10 min before measuring baseline volumes. To maintain a constant concentration, the drug was also added at the appropriate concentration to the ddH2O used to challenge the cells. Treatment with both NPPB (200 µM) and DIDS (200 µM), but not Cd2+ (250 µM), significantly inhibited RVD, as seen by comparing the mean responses in Fig. 2A and normalized post-RVD MCVs (Fig. 2B). Because Cd2+ may block different channels than DIDS or NPPB and was shown previously to synergistically inhibit volume regulation in combination with these drugs (28), we also studied the effect of Cd2+ in combination with NPPB. This combination showed a significantly greater inhibition than NPPB alone (Fig. 2, A and B), with almost complete inhibition of RVD. Similarly, whole cell recordings revealed that although NPPB was able to partially inhibit the hypotonically activated Cl current, significantly greater inhibition of this current could be achieved by applying the combination of NPPB and Cd2+ (Fig. 2C).
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Inhibition of Cl channels and transporters synergistically inhibits RVD. Although the hypotonically activated Cl current was greatly inhibited by the channel inhibitor combination of NPPB + Cd2+, the addition of DIOA was not able to provide further inhibition (Fig. 5, C and D), suggesting that the DIOA-sensitive component of this current was also sensitive to either NPPB or Cd2+. However, in the presence of the combination of the transport inhibitor DIOA and the Cl channel inhibitors NPPB and Cd2+, cells exposed to a 50% challenge exhibited a significantly greater inhibition of RVD than that observed with channel inhibitors or DIOA alone (Fig. 5, A and B), indicating that the inhibitory effect of DIOA cannot simply be explained by a nonspecific effect on Cl channels. Furthermore, although treatment with Cl channel or transport inhibitors alone did not affect the magnitude of the initial volume increase, this was not the case for cells treated with a combination of channel and transporter inhibitors, in which the initial volume increase was significantly greater than control cells. Indeed, under these conditions, cells behaved more like perfect osmometers. This finding suggests that the initial volume increase is limited not only by mechanical forces but also by quickly activated volume-regulatory mechanisms.
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DISCUSSION |
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This is indeed what we observed. Unlike astrocytes, which only show partial recovery of their cell volume (28, 31), glioma cells are able to regulate their volume back to baseline levels or even lower when exposed to an osmotic challenge. We also found that Cl channels account for 6070% of this process, whereas Cl transport via KCCs accounts for 3040%, primarily within the first 10 min of a challenge. The differential temperature sensitivity of channels vs. transporters allowed us to delineate their relative contributions. Finally, although we made no attempt to unequivocally identify the underlying proteins, we describe a "cast of characters" that includes ClC-2 and ClC-3 as Cl channels and KCC1 and KCC3b as Cl transporters.
Inhibition of RVD in human glioma cells by DIDS and NPPB is consistent with the involvement of several Cl channels in volume regulation. For example, ClC-2, ClC-3, and/or the protein responsible for ICl,swell among others (5, 29, 39) are all sensitive to these drugs. In our studies, however, the inhibition of RVD by either DIDS or NPPB was incomplete, but both drugs acted synergistically with Cd2+, suggesting that more than one population of Cl channels is involved. Although similar results have been obtained in astrocytes (28), these data stand in contrast to work published by Bond et al. (2) showing that treatment with 300 µM Cd2+ had no effect on volume regulation in T84 cells. Our inhibition studies suggest that in all likelihood at least two protein populations contribute to the Cl efflux during RVDa Cd2+-insensitive population and a Cd2+-sensitive population. One of these populations may be mediated by ICl,swell, which is inhibited by both NPPB and DIDS (5, 39) but insensitive to 300 µM Cd2+ (2), or by ClC-3, which is also sensitive to DIDS and has been shown to produce outwardly rectifying Cl current in D54-MG cells (29). The Cd2+-sensitive component may be mediated by ClC-2, which produces weakly rectifying currents in glioma cells (29) that are sensitive to NPPB and weakly inhibited by DIDS but, unlike ICl,swell and ClC-3, are highly sensitive to Cd2+. Although ClC-2 was previously dismissed as the protein responsible for ICl,swell because it is an inwardly rectifying current with an anion permeability sequence that differs from that reported for ICl,swell (32, 42), it may nonetheless have a role in RVD.
Although we have chosen to discuss the possibility of ClC-2 and ClC-3 involvement in RVD, we acknowledge that our data do not conclusively support the involvement of any one of these channels. Furthermore, we do not intend to make any conclusions about the involvement of ICl,swell in RVD or its identity. Clearly, the molecular identification of the proteins responsible for channel-mediated Cl efflux during RVD is an essential next step toward a better understanding this process in glioma cells.
Our study has focused primarily on the role of Cl movement through channels and transporters during RVD in glioma cells. However, we have not ruled out the involvement of other osmolytes, including osmoregulatory amino acids such as taurine, glutamate, and aspartate. Previous studies have shown swelling-induced efflux of these osmolytes in astrocytes and have suggested that this release is important in volume regulation (18, 31). Because several Cl channel blockers have been shown to inhibit the efflux of these osmolytes, it has been suggested that they may move through Cl channels or may be tightly coupled to Cl movement (16, 18, 33). Interestingly, when our cells were treated with TBT to disrupt the Cl gradient (to prevent passive movement through channels), RVD was essentially inhibited. Similarly, other studies have shown that swelling induced by an isotonic high-KCl medium, a condition under which the Cl gradient is not favorable for efflux, does not result in complete volume regulation even though the release of other organic osmolytes occurs (16). These data suggest that Cl movement is indeed necessary for RVD.
Inhibition of RVD in glioma cells by the KCC inhibitor DIOA strongly suggests that KCCs also play a role in volume regulation of these cells. Although KCCs have not been shown to play a role in the RVD of astrocytes (28), they have been shown to play a role in several other cell types studied (2022, 40), including other malignant cells. For instance, in the rat glioma cell line C6, the protein phosphatase inhibitor okadaic acid has been shown to inhibit RVD. Although it may affect many proteins, one protein known to be indirectly inhibited through the use of okadaic acid is the KCC, suggesting that this protein may, indeed, be involved in the volume regulation of these cells (21, 22). Although this evidence is indirect, a recent study of cervical cancer cells has shown that these malignant cells express more KCCs than their nonmalignant counterparts (normal cervical epithelium) and that inhibition of KCCs by DIOA limits the volume regulatory response in these cells. Interestingly, this study also showed that inhibition of KCCs also limited cell growth and the invasion of the tumor cells into surrounding tissues (36, 37). Further studies must be done to see whether KCCs play a role in the growth and invasion of glioma cells as well.
Our assertion that KCCs are involved in volume regulation of glioma cells was initially based on the inhibitory effect of 40 µM DIOA, believed to be a relatively specific KCC inhibitor (10). However, previous studies showed that at higher concentrations, i.e., 100 µM, this drug can also partially inhibit Cl and K+ channels (1, 3, 4). More specifically, a study showed that 100 µM DIOA was able to reduce a swelling-activated Cl conductance in human osteoblasts (4). Indeed, our own studies confirm that DIOA, even at the lower concentration of 40 µM, was able to reduce the hypotonically activated Cl current in our cells. We are quite confident, however, that in our studies DIOA affected primarily, if not exclusively, KCCs. This conclusion is based on our inability to observe any inhibitory effect of DIOA when ion transport was blocked by lowering temperature to 15°C, whereas at this temperature Cl channel blockers remained effective. Moreover, when we inhibited Cl channels with a combination of NPPB and Cd2+, DIOA had no further effect on the hypotonically activated Cl current but was able to inhibit the residual volume regulation that we attribute to KCC transport. Together these data strongly suggest that the inhibitory effects of DIOA on RVD are due, primarily, to its inhibition of KCCs.
We used an additional approach to verify the relative contribution of channels vs. transporters to RVD that proved to be quite instructive. Specifically, lowering the temperature to 15°C eliminated any contribution from DIOA-sensitive Cl transport (i.e., KCC activity). Under these conditions, the residual volume recovery was entirely blocked by NPPB + Cd2+, suggesting that this approach had isolated the channel-mediated component. Although in our cells volume regulation at 15°C appeared to have been entirely mediated by channels, this approach must be validated on a case-by-case basis as channel function may also be affected by lower temperatures. Although it is true that in general channel-mediated diffusion is less sensitive to temperature changes than energy-dependent ion transport, the gating of some Cl channels was recently shown to have an unusually high temperature dependence (44).
Interestingly, at 15°C cells in the presence of NPPB + Cd2+ behaved like perfect osmometers, achieving a doubling in cell size when osmolarity was reduced by 50%. Similarly, inhibition of RVD in C6 glioma cells (22, 25), thymocytes (1), and human cervical cancer cells (37) by different mechanisms (inhibition of K+ channels, Cl channels, or KCCs) each resulted in an increase in the magnitude of the initial rapid swell. These data demonstrate that cell volume is exclusively an osmotic property with no mechanical limitations.
The exciting new observation in this study is that both Cl channels and transporters play significant roles in volume regulation in human glioma cells. Furthermore, the contribution of these two components can be dissected by taking advantage of their relative temperature dependence, offering us a new method for studying the mechanisms of volume regulation. Future studies will have to determine whether these same mechanisms are involved in other aspects of cell behavior in which volume changes may be important, such as cell proliferation, death, migration, and invasion.
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GRANTS |
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ACKNOWLEDGMENTS |
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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. Section 1734 solely to indicate this fact.
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