Contribution of chloride channels to volume regulation of cortical astrocytes

Kimberly A. Parkerson and Harald Sontheimer

Department of Neurobiology, Civitan International Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The objective of this study was to determine the relative contribution of Cl- channels to volume regulation of cultured rat cortical astrocytes after hypotonic cell swelling. Using a Coulter counter, we showed that cortical astrocytes regulate their cell volume by ~60% within 45 min after hypotonic challenge. This volume regulation was supported when Cl- was replaced with Br-, NO<UP><SUB>3</SUB><SUP>−</SUP></UP>, methanesulfonate-, or acetate- but was inhibited when Cl- was replaced with isethionate- or gluconate-. Additionally, substitution of Cl- with I- completely blocked volume regulation. Volume regulation was unaffected by furosemide or bumetanide, blockers of KCl transport, but was inhibited by Cl- channel blockers, including 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and niflumic acid. Surprisingly, the combination of Cd2+ with NPPB, DIDS, or niflumic acid inhibited regulation to a greater extent than any of these drugs alone. Volume regulation did not differ among astrocytes cultured from different brain regions, as cerebellar and hippocampal astrocytes exhibited behavior identical to that of cortical astrocytes. These data suggest that Cl- flux through ion channels rather than transporters is essential for volume regulation of cultured astrocytes in response to hypotonic challenge.

glia; ion channels; Coulter counter


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MOST CELLS POSSESS MECHANISMS to control or regulate their volume (18), and volume regulation has been clearly demonstrated for cultured rat cortical astrocytes in response to hypotonic challenge (14, 25). However, it appears that the mechanisms of volume regulation are ineffective in astrocytes in the brain after neurological insult, as swelling of astrocytes is a major complicating factor of conditions including ischemia, trauma, hyponatremia, and hepatic encephalopathy (16). Astrocytic swelling can decrease the volume of the extracellular space and have dramatic consequences on diffusion of neurotransmitters and on excitability (13). Additionally, swelling can have deleterious consequences on normal astrocytic functions such as K+, pH, amino acid, and transmitter homeostasis (12). Therefore, an impetus exists to provide a detailed assessment of the specific mechanisms that control cell volume in cultured cortical astrocytes, as this may provide insight into the persistence of astrocytic swelling in the brain.

In astrocytes, as in many other cell types, a significant body of evidence supports the notion that at least two mechanisms contribute to volume regulation in response to hypotonic challenge, namely, the efflux of inorganic ions such as K+ or Cl- and the release of organic osmolytes such as taurine (for review see Ref. 32). Pasantes-Morales and colleagues (26) suggested that these two mechanisms contribute approximately equally to volume regulation of cortical astrocytes. Less clear, however, are the pathways for osmolyte and Cl- release. Both inorganic and organic ions may be released through ion channels, such as volume-sensitive organic osmolyte-anion channels (VSOAC) (9, 10). Alternatively, organic osmolytes may be released through these channels, whereas Cl- may be released through other channels or carriers such as the KCl transporters (for review see Ref. 5).

The contribution of channel-mediated vs. carrier-mediated efflux of Cl- from cells depends not only on the complement of channels and transporters present but also on the distribution of Cl- across the cell membrane and the equilibrium potential of Cl- relative to the resting potential of the cell. In cultured astrocytes, evidence clearly points to an active accumulation of Cl- and a Cl- equilibrium potential positive to that of the K+-dominated resting potential (for review see Ref. 33). Therefore, activation of Cl- channels alone could effectively result in a net efflux of Cl-. Indeed, cultured astrocytes exhibit a depolarization (15, 17) during volume regulation that is consistent with activation of channels in which the permeant species exhibits an equilibrium potential positive to the resting potential of the cell.

Similarly, at least two studies have suggested an active accumulation of Cl- in astrocytes in situ (21, 34) although a study by Ballanyi and colleagues (1) instead suggested a passive distribution of Cl- in glial cells in situ. In contrast to active accumulation, such a passive distribution of Cl- would result in an equilibrium potential of Cl- that is similar to that of the resting potential of the cell. Therefore, a net efflux of Cl- through channels would be limited by a requirement for channel activation combined with cell hyperpolarization. In this case, a carrier-mediated component such as KCl cotransport that is coupled to an outward driving force for K+ might be required for effective Cl- efflux.

Pasantes-Morales and colleagues (27, 30) provided additional evidence for KCl efflux through channels in cultured cerebellar astrocytes. In their studies, volume regulation of cerebellar astrocytes was inhibited by the K+ channel blocker quinidine and the Cl- channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) but was not affected by the KCl transport blocker bumetanide and only weakly inhibited by high concentrations of the KCl transport blocker furosemide. Kimelberg and Frangakis (14) similarly showed that volume regulation of cultured cortical astrocytes was not significantly affected by furosemide.

In this study, we examined volume regulation of astrocytes isolated from different brain regions, including cortex, hippocampus, and cerebellum. We found that all of these astrocytes, irrespective of brain region, showed a regulatory volume decrease in response to hypotonic challenge that depended on Cl- efflux through ion channels rather than transporters.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. Primary astrocyte cultures were established from Sprague-Dawley rats at postnatal days 0-2 by modification of the technique described by McCarthy and deVellis (22). The protocol was reviewed and approved by the Institutional Animal Care and Use Committee. The pups were placed on ice and decapitated. The brain tissue was dissected in ice-cold Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Grand Island, NY). The meninges and associated blood vessels were removed, and the cortex or region of interest was separated from surrounding regions. We then microscopically reexamined the tissue and removed any remaining blood vessels until the tissue was essentially vessel free. The tissue pieces were further minced, washed with DMEM, and incubated in enzyme solution (warmed to 37°C and bubbled with 100% O2 for 10 min before) for 20 min at 37°C. The enzyme solution consisted of DMEM supplemented with 20 U/ml papain, 1 mmol L-cysteine, 0.5 mmol EDTA, and 706 U/ml deoxyribonuclease (Worthington, Freehold, NJ). The cells were pelleted by centrifugation for 2 min. The supernatant was aspirated, and the remaining pellet was dissociated by trituration (10-15×) with a fire-polished Pasteur pipette into astrocyte medium supplemented with 10 U/ml penicillin, 10 µg/ml streptomycin, and 0.025 µg/ml amphotericin B (Life Technologies). Astrocyte medium consisted of DMEM and 7% heat-inactivated fetal calf serum (HyClone, Logan, UT). The cells were plated onto tissue culture-treated dishes (Fisher) and then kept at 37°C in a 95% O2-5% CO2 atmosphere. The medium was replaced every 3 days.

Cell volume measurements. Cell volume measurements were performed by electronic sizing with a Coulter counter Multisizer 3 (Beckman-Coulter, Miami, FL). Cells were washed in phosphate-buffered saline (PBS) and lifted by 3- to 5-min incubation with 0.05% trypsin and 0.53 mM EDTA (Life Technologies). Trypsin was inactivated by addition of an equal volume of astrocyte medium, and cells were pelleted by brief centrifugation. Cells were resuspended in bath solution of interest and passed through a 40-µm nylon cell strainer (Fisher). A single-cell suspension was verified by microscopic visualization of a sample aliquot. Cells were equilibrated for ~10 min before first reading. Cell volume measurements were obtained every minute, and each measurement was an average of 15,000 cells. Hypotonic challenge was applied after baseline readings.

Solutions for volume regulation experiments. The control NaCl bath solution for cell volume measurements was as follows (in mM): 140 NaCl, 2.5 KCl, 10.5 glucose, 25 HEPES, 1 CaCl2, and 1.2 MgCl2. Anion substitutions were made by replacement of 140 mM NaCl with the appropriate Na salt. Zero-calcium solution was made by omitting CaCl2 and by adding 500 µM EGTA. pH was adjusted to 7.4 with NaOH. Solution osmolarities were confirmed with a vapor pressure osmometer (Wescor 5500; Wescor, Logan, UT). The osmolarities of our isotonic solutions were 310 ± 5 mosM. Drugs were added directly to the bath solutions from stock solutions. Stock solutions of 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), niflumic acid, and bumetanide were dissolved at 1,000× final concentration in DMSO, CdCl2 was dissolved at 1,000× final concentration in double-distilled H2O, furosemide was dissolved at 500× final concentration in DMSO, and DIDS was dissolved at 250× final concentration in 0.1 M KHCO3. DMSO and KHCO3 did not affect volume regulation at their final concentrations. Solutions were made hyposmotic by addition of water.

Media for overnight anion substitution were prepared identically to minimum essential medium (MEM; no. 51200, Life Technologies) except for the equimolar substitution of NaCl with the appropriate Na salt. The solutions consisted of (in mM) 116 Na salt, 5.4 KCl, 26 NaHCO3, 1 NaH2PO4, 0.8 MgSO4, 1.8 CaCl2, and 25.5 glucose with 10 mg/l phenol red, 1× MEM amino acids (Mediatech, Herndon, VA), and 1× MEM vitamin solution (Mediatech). During overnight incubation in these media, cells were kept at 37°C in a 95% O2-5% CO2 atmosphere. Volume regulation after overnight incubation in MEM-like solution with NaCl was similar to that of normal DMEM media (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 were normalized to baseline value. All data were plotted in Origin 6.0 (MicroCal, Northampton, MA) as means ± SE with the number of experiments performed (n). Significance (P) was determined by Student's t-test of relative volumes at 30 min after swelling. Percent regulation was determined as [1 - (relative volume at 30 min/peak relative volume)].


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Astrocytes exhibit volume regulation in response to hypotonic challenge. The first step in this study was to establish a consistent and reproducible cell swelling and regulation of cell volume in primary rat cortical astrocytes upon hypotonic challenge. Therefore, cells were suspended in a given volume of NaCl bath solution (see MATERIALS AND METHODS) and exposed to a 25%, 50%, or 70% reduction in osmolarity. Mean cell volume of 15,000 cells was monitored once every minute by electronic sizing with a Coulter counter (see MATERIALS AND METHODS). Figure 1A shows the mean normalized peak swelling response of astrocytes to these degrees of hypotonic challenge. On average, cells swelled to 122 ± 4%, 154 ± 12%, and 180 ± 10% of their original volume in response to a 25%, 50%, and 70% reduction in osmolarity, respectively. Addition of the same volume of isosmotic NaCl bath or sucrose solution produced no swelling (Fig. 1B). It should be noted that actual cell swelling was less than theoretically predicted for a given reduction in osmolarity, which could be attributed to nonosmotically active space within the cells. For astrocytes, Olson and Holtzman (24) approximated the nonsolvent volume of astrocytes to be 30%.


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Fig. 1.   Astrocytes exhibit volume regulation in response to hypotonic challenge. A: normalized mean peak cell volumes in response to 25%, 50%, and 70% reduction in osmolarity. B: normalized mean volume regulation in response to 50% hypotonic or isosmotic challenge for 45 min. , 50% Hypotonic regulation (control); , 50% hypotonic regulation with sucrose added to regain original osmolarity where indicated; , isosmotic NaCl or sucrose solution only.

In the presence of continued hypotonicity, cells began to decrease their volume as previously demonstrated by others (14, 25). Measurements were taken for 45 min, and Fig. 1B shows the mean volume regulation for 50% hypotonic challenge observed in nine separate cell preparations. Cell volume recovered to 125 ± 6% of the original volume after 15 min, 123 ± 6% after 30 min, and 121 ± 7% after 45 min. This corresponded to a regulation of cell volume by 52 ± 3% within 15 min, 56 ± 4% within 30 min, and 60 ± 4% within 45 min. Therefore, the majority of the regulatory response during this time period was completed within the first 15 min of hypotonic challenge. Addition of sucrose to restore original isosmotic conditions 10 min after swelling resulted in rapid recovery and undershoot of the original volume (Fig. 1B; n = 3). This undershoot was consistent with a loss of solutes from the cell during volume regulation in the 10 min after swelling (31).

Volume regulation of astrocytes requires anion flux. To determine the contribution of anion flux to volume regulation, we performed anion substitution experiments. We incubated cells overnight in media in which NaCl was replaced with equimolar Na salts of various monovalent anions (see MATERIALS AND METHODS). Overnight incubation allowed for maximal exchange of intracellular Cl- with the test anion. It has been shown that with only 20-min incubation in gluconate-- or NO<UP><SUB>3</SUB><SUP>−</SUP></UP>-containing media, the intracellular Cl- content decreases by 80% and 95%, respectively in cerebellar astrocytes (30). We did not measure the intracellular Cl- content after overnight substitution in cortical astrocytes. However, we did observe that the magnitude of volume regulation after overnight substitution differed from that after acute substitution for selected anions. An example of this is shown in Figs. 2B and 3A for isethionate- substitution. The enhanced inhibition of volume regulation after overnight substitution of isethionate- was consistent with successful intracellular substitution of Cl- with a less permeant anion. An exception to this was I- substitution, for which inhibition of regulation was observed after both acute and overnight exposure as explained below and in Acute I- substitution inhibits volume regulation of astrocytes.


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Fig. 2.   Volume regulation of astrocytes requires anion flux. A: comparison of volume regulation under control conditions and after overnight halide substitutions. B: comparison of volume regulation under control conditions and after overnight nonhalide substitutions. C: plot of %regulation for the halide and nonhalide substitutions after 30-min hypotonic challenge. Ace, acetate; Gluc, gluconate; Ise, isethionate; MeS, methanesulfonate.



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Fig. 3.   Acute I- substitution inhibits volume regulation of astrocytes. A: volume regulation after acute substitution of I- or isethionate- for Cl-. B: volume regulation in increasing concentrations of acute extracellular I-. C: plot of %regulation in increasing extracellular I- concentrations ([NaI]) compared with control after 30-min hypotonic challenge.

The morning after overnight substitution, cells were suspended in bath solution also containing the Na salt of the test anion in place of NaCl and volume regulation was measured in response to 50% hypotonic challenge. A series of halides (Br-, I-, NO<UP><SUB>3</SUB><SUP>−</SUP></UP>) and nonhalides (acetate-, gluconate-, isethionate-, methanesulfonate-) were examined for four cell preparations each, and the mean data are plotted in Fig. 2, A and B. As shown, Br-, NO<UP><SUB>3</SUB><SUP>−</SUP></UP>, methanesulfonate-, and acetate- were able to support volume regulation similarly to Cl-, whereas volume regulation was inhibited by I-, isethionate-, or gluconate-. The complete inhibition by I- and partial inhibition by either isethionate- or gluconate- suggested a strong dependence of volume regulation of cortical astrocytes on anion flux. We calculated the relative permeability sequence for the anion flux pathway involved in volume regulation of cortical astrocytes based on magnitudes of volume recovery after 30 min (Fig. 2C): Br- >=  Cl- >=  methanesulfonate- >=  NO<UP><SUB>3</SUB><SUP>−</SUP></UP> >=  acetate- > gluconate- >=  isethionate- > I-.

Acute I- substitution inhibits volume regulation of astrocytes. The Cl- > I- permeability sequence in volume regulation was surprising because many Cl- channels show a lyotropic permeability sequence in which I- is more permeable than Cl- (8). Therefore, this observation was further explored. Permeability ratios of I- to Cl- can be confounded by a blocking action of I- (8), and both intracellular and extracellular I- can reduce currents through some Cl- channels (6).

We tested whether I- could be showing an erroneously low permeability by exerting a blocking effect on the anion flux pathway involved in volume regulation of cortical astrocytes. Cells were acutely exposed to extracellular I- by suspension in bath solution containing 140 mM NaI (see MATERIALS AND METHODS). Interestingly, the acute I- substitution completely inhibited volume regulation of astrocytes in response to hypotonic challenge (Fig. 3A; n = 4). The effects of 1, 10, and 100 mM acute extracellular NaI substitution were also tested. The volume regulation curves are shown in Fig. 3B (n = 4 for each concentration), and the percent regulation after 30-min hypotonic challenge is compared with control in Fig. 3C. Increasing concentrations of I- resulted in increased inhibition of volume regulation of astrocytes and suggested that I- was indeed exerting a block on the anion flux pathway involved in volume regulation of astrocytes.

To show that inhibition of volume regulation by acute extracellular substitution was not a general effect of all anions with permeabilities less than Cl- (Fig. 2C), we also examined volume regulation in the presence of acute extracellular isethionate- substitution. In contrast to acute substitution of I-, acute substitution of isethionate- did not inhibit volume regulation (Fig. 3A; n = 3).

Cl- channel blockers, but not Cl- transport blockers, inhibit volume regulation of astrocytes. To examine the relative contribution of Cl- transporters vs. channels to volume regulation, the effects of several Cl- transport and channel blockers were investigated. First, drugs known to block the Na-K-2Cl and KCl families of transporters were tested. Either 200 µM furosemide or 50 µM bumetanide was applied to the suspension of astrocytes before swelling. Volume regulation was similar to control in the presence of either drug (Fig. 4A; n = 4 for each drug), suggesting that ion flux by these transporters is not necessary during the acute volume regulatory phase of cultured cortical astrocytes.


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Fig. 4.   Cl- channel blockers, but not Cl- transport blockers, inhibit volume regulation of astrocytes. A: volume regulation in the presence of bumetanide or furosemide. B: volume regulation in the presence of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), niflumic acid, or Cd2+. C: volume regulation in the presence of Cd2+ in combination with DIDS, NPPB, or niflumic acid.

Next, several drugs that are known to block Cl- channels were tested. Specifically, DIDS (200 µM), niflumic acid (200 µM), NPPB (123 µM), or Cd2+ (500 µM) was applied to the suspension of astrocytes 5 min before swelling and remained present throughout the experiment. Volume regulation of cortical astrocytes in the presence of each drug is shown in Fig. 4B (n = 4 for each drug). Both niflumic acid and NPPB significantly inhibited volume regulation, whereas DIDS slightly, but not significantly, inhibited and Cd2+ did not inhibit volume regulation.

Because Cd2+ has been shown to block Cl- currents that are distinct from those blocked by DIDS, niflumic acid, or NPPB (2, 3, 4, 7, 11), the effects of Cd2+ in combination with DIDS, niflumic acid, or NPPB were also tested. Surprisingly, each of the combinations was synergistic in inhibiting volume regulation, and the results are shown in Fig. 4C (n = 4 for each combination). The most likely explanation for this finding is the contribution of more than one type of Cl- channel to volume regulation of cortical astrocytes. Additionally, the differential effect of Cd2+ when combined with the other drugs suggests that the DIDS-, niflumic acid-, and NPPB-sensitive component may functionally compensate for the Cd2+-sensitive component when Cd2+ is applied alone.

Removal of extracellular Ca2+ does not mimic effects of cadmium. Cadmium is also a potent blocker of voltage-gated Ca2+ channels that are present in glial cells (20). Activation of Ca2+ channels causes a rise in intracellular Ca2+ that could act as a second messenger to influence volume regulation via downstream targets. These targets could include but are not limited to inhibition of Ca2+-activated Cl- channels. For example, inhibition of Ca2+-activated K+ channels could also influence volume regulation. Therefore, we wanted to test whether the inhibition of volume regulation by Cd2+ could be mediated through Ca2+ channels rather than direct inhibition of Cl- channels.

The following experiments examined whether a block of Ca2+ channels by Cd2+ could account for or contribute to the inhibition of volume regulation seen by combination of Cd2+ with DIDS, niflumic acid, or NPPB. We first compared volume regulation under control conditions and in the absence of Ca2+. By removing extracellular Ca2+ and adding 500 µM EGTA, we produced a functional block of Ca2+ channels. Removal of Ca2+ from the extracellular bath (n = 4) slightly, but not significantly, inhibited volume regulation of rat cortical astrocytes compared with control (Fig. 5A, but see Ref. 23). More importantly, we next compared volume regulation in the presence of DIDS alone, DIDS in combination with Cd2+, and DIDS in the absence of extracellular Ca2+. We expected that if the effects of Cd2+ were the result of Ca2+ channel block, volume regulation with DIDS in the absence of extracellular Ca2+ would be similar in magnitude to that of DIDS in the presence of Cd2+. However, removal of Ca2+ from the extracellular bath in the presence of DIDS (n = 4) resulted in volume regulation that was similar in magnitude to that of DIDS alone but different from that of DIDS in combination with Cd2+ (Fig. 5B). This highly suggested that Ca2+ channel block could not account for the inhibition of volume regulation by Cd2+.


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Fig. 5.   Removal of extracellular Ca2+ does not mimic the effects of Cd2+. A: comparison of volume regulation under control conditions and in 0 extracellular Ca2+. B: comparison of volume regulation in the presence of DIDS, DIDS + Cd2+, and DIDS in 0 extracellular Ca2+.

Different brain regions show similar volume regulation. The above data suggest a major role for Cl- channels in volume regulation of rat cortical astrocytes. We next tested whether this is a phenomenon that is specific for cortical astrocytes or whether similar Cl- channels are also involved in volume regulation of astrocytes from other brain regions. Volume regulation of cerebellar, hippocampal, and cortical astrocytes was compared. As shown in Fig. 6A (n = 4 for cerebellum, n = 4 for hippocampus, n = 9 for cortex), volume regulation was similar in astrocytes from all three brain regions. Also, volume regulation of each region was sensitive to I- and NPPB (n = 3 for cerebellum, Fig. 6B; hippocampus, not shown), suggesting that Cl- channels are important in volume regulation of astrocytes isolated from different regions of the brain.


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Fig. 6.   Different brain regions show similar volume regulation. A: comparison of volume regulation in cortical, cerebellar, and hippocampal astrocytes. B: inhibition of volume regulation by I- and NPPB in cerebellar astrocytes.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study addressed the contribution of Cl- channels to volume regulation of rat cortical astrocytes, and our data suggested that under our experimental conditions, Cl- channel-mediated efflux of anions is essential for volume regulation. Ion replacement studies suggest that volume regulation is supported by some anions that have previously been shown to permeate Cl- channels but not by anions with little or no permeability. Moreover, from this data, we constructed a relative permeability sequence for anions through the pathway mediating volume regulation. Both isethionate- and gluconate- inhibited volume regulation, in line with their typical poor permeability through Cl- channels. This clearly implied a dependence of volume regulation on anion flux. These data also suggested that the pathway involved was not exclusively dependent on Cl-, as expected for a transporter-mediated pathway, but rather allowed a number of different anions to permeate, as expected for a channel-mediated pathway.

Furthermore, we evaluated the effects of Cl- channel and transporter blockers on volume regulation. Neither bumetanide nor furosemide, selective blockers of Cl- transporters, affected volume regulation, whereas typical Cl- channel blockers inhibited volume regulation. Therefore, we propose that volume regulation of cultured astrocytes is dependent on an efflux of anions through Cl- channels and that to induce volume change these channels likely cooperate with separate cation and water channels.

Previous studies have shown that cortical astrocytes also release significant amounts of their intracellular osmolyte pool in response to hypotonic challenge (26). Necessarily, this efflux of osmolytes along with water also opposes increases in cell volume and is likely cooperative with KCl efflux. On the basis of their work on C6 glioma cells, Jackson and colleagues (9, 10) proposed that in response to cell swelling, Cl- and osmolytes permeate a common pathway that they termed volume-sensitive organic osmolyte-anion channel (VSOAC). Similarly, Roy (28) showed that amino acids could diffuse through anion channels in U-138 glioma cells. Our experiments did not address whether the Cl- channels that contribute to volume regulation of astrocytes also flux osmolytes. However, overnight substitution of Cl- with methanesulfonate-, which is structurally related to taurine, supported volume regulation similarly to control (Fig. 2B). The fact that methanesulfonate- but not other large anions such as isethionate- and gluconate- supported volume regulation was a bit surprising. However, it is possible that the Cl- channels involved in volume regulation show a higher than expected permeability to taurine and its analogs. Therefore, the Cl- channels involved in volume regulation of cortical astrocytes may regulate cell volume through both KCl and osmolyte efflux.

The molecular identity of the Cl- channels responsible for volume regulation is not known. Our pharmacology experiments did suggest that more than one channel population might be involved: one that is inhibited by NPPB and partially by niflumic acid and DIDS and one that is inhibited by Cd2+. Therefore, the biophysical properties that we observed, such as anion permeabilities, may have been derived from a combination of more than one channel. This may explain why our permeabilities did not follow either the typical lyotropic sequence described for many anion channels including typical swelling-activated Cl- channels nor the alternative sequence described for the voltage-gated Cl- channel ClC-1, in which larger anions have reduced permeability (8).

Most interesting was that either overnight or acute substitution of I- resulted in complete inhibition of volume regulation. This may suggest that I- sensitivity is a property shared among the different channels involved. Such sensitivity has been described for several anion channels including ClC-1 and cystic fibrosis transmembrane conductance regulator (CFTR) (6, 19, 29). More experiments are needed to characterize and identify the channel(s) involved, and this will be addressed in future electrophysiological and molecular experiments.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Neurological Disorders and Stroke Grant RO1-NS-36692 to H. Sontheimer. K. A. Parkerson was supported by the National Institutes of Health Medical Scientist Training Program.


    FOOTNOTES

Address for reprint requests and other correspondence: H. Sontheimer, 1719 6th Ave. South, CIRC 545, Birmingham, AL 35294 (E-mail: sontheimer{at}uab.edu).

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.

First published February 26, 2003;10.1152/ajpcell.00603.2002

Received 26 December 2002; accepted in final form 17 February 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 284(6):C1460-C1467
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