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Am J Physiol Cell Physiol 274: C1511-C1520, 1998;
0363-6143/98 $5.00
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Vol. 274, Issue 6, C1511-C1520, June 1998

Pharmacological characterization of swelling-induced D-[3H]aspartate release from primary astrocyte cultures

Eric M. Rutledge1, Michael Aschner1,2, and Harold K. Kimelberg1,3

Departments of 1 Pharmacology/Neuroscience and 3 Neurosurgery, Albany Medical College, Albany, New York 12208; and 2 Department of Pharmacology and Physiology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

During stroke or head trauma, extracellular K+ concentration increases, which can cause astrocytes to swell. In vitro, such swelling causes astrocytes to release excitatory amino acids, which may contribute to excitotoxicity in vivo. Several putative swelling-activated channels have been identified through which such anionic organic cellular osmolytes can be released. In the present study, we sought to identify the swelling-activated channel(s) responsible for D-[3H]aspartate release from primary cultured astrocytes exposed to either KCl or hypotonic medium. KCl-induced D-[3H]aspartate release was inhibited by the anion channel inhibitors 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), dideoxyforskolin, L-644711, ATP, ITP, 3'-azido-3'-deoxythymidine, DIDS, and tamoxifen but not by cAMP. The cell swelling caused by raised KCl was not inhibited by extracellular ATP or tamoxifen as measured by an electrical impedance method, which suggests that these anion channel inhibitors directly blocked the channel responsible for efflux. Extracellular nucleotides and DIDS, however, had no or only partial effects on D-[3H]aspartate release from cells swollen by hypotonic medium, but such release was inhibited by NPPB, dideoxyforskolin, and tamoxifen. Of the swelling-activated channels so far identified, our data suggest that a volume-sensitive outwardly rectifying channel is responsible for D-[3H]aspartate release from primary cultured astrocytes during raised extracellular K+ and possibly during hypotonic medium-induced release.

swelling-activated anion channels; extracellular ATP; tamoxifen; 5-nitro-2-(3-phenylpropylamino)benzoic acid; volume-sensing outwardly rectifying channel; excitatory amino acids

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

ALMOST ALL VERTEBRATE CELLS so far tested release free cellular inorganic and organic cations and anions, in this context termed osmolytes, when swollen by exposure to hypotonic media. This enables the cells to regulate their cell volumes back to normal, a process called regulatory volume decrease (RVD) (9, 15). The main inorganic osmolytes released during hypotonicity-induced swelling are K+ and Cl-, which probably contribute the most to RVD due to their high intracellular concentrations (9, 15). The main organic osmolytes are the amino acids and their derivatives. These include taurine, a major osmoregulator that is preferentially lost during cell swelling (33), and to a lesser extent glutamate, aspartate, gamma -aminobutyric acid, and alanine (21, 33, 37).

In various brain pathologies such as stroke and traumatic brain injury, there is marked overall brain and cellular swelling, categorized into vasogenic and cellular (cytotoxic) edema, respectively (25). Brain cellular edema is primarily due to a shift of osmolytes and brain water from the extracellular space into cells (20). Large changes in extracellular K+, reaching concentrations of up to 80 mM, have been measured with extracellular K+ electrodes in vivo in cerebral ischemia, (13, 43, 44). Such increases in extracellular K+ might be a key contributor to cellular edema during pathological conditions in the central nervous system, since raised extracellular K+ causes cell swelling due to Donnan diffusional forces (14), including swelling of astrocytes (19, 27, 48).

In many cultured cells, including primary cultured astrocytes, release of organic osmolytes is believed to be mediated by diffusion through swelling-activated anion channels rather than on co-transporters or exchangers (36, 37, 39, 40). Exposure of cells to hypotonic media usually generates outwardly rectifying current-voltage (I-V) curves in cells when the primary anions are inorganic or organic (2, 37). Anion channel proteins showing outwardly rectifying I-V curves are termed volume expansion-sensing outwardly rectifying (VSOR) channels or osmolyte anion channels (VSOAC) (46) and are assumed to play a role in cell volume regulation since inhibiting this current also inhibits RVD (31). These channels have been characterized pharmacologically and shown to be inhibited by DIDS, millimolar concentrations of extracellular nucleotides, tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), and dideoxyforskolin (31, 46).

Thus far, four swelling-activated outwardly rectifying anion channels have been cloned and characterized, yet none have fulfilled all of the requirements to be the VSOR/VSOAC channel(s) (reviewed in Refs. 31, 46). These four channels are as follows. 1) ClC-2 and the recently cloned ClC-3 belong to a family of channels designated as ClC. The pharmacology differs within this family as well as differing from VSOR/VSOAC. ClC-2 is not inhibited by 100 µM DIDS, whereas ClC-3 is inhibited by DIDS and also by a variety of other proposed VSOR/VSOAC channel blockers such as extracellular ATP and tamoxifen. ClC-3 has an outwardly rectifying I-V curve, whereas ClC-2 has an inwardly rectifying I-V curve (6). 2) A swelling-activated channel termed the "maxi" anion channel has been identified in cultured cortical astrocytes, in neuroblastoma cells, and in the apical membrane of renal collecting duct cells (RCCT-28A cells). It shows multiple activation states with a large unitary conductance of 200-400 pS on hypotonicity-induced swelling. It is inhibited by DIDS, L-644711, NPPB, pertussis toxin, and inhibitors of protein kinase C (18, 41, 45). It shows a linear I-V curve. The maxi anion channel present in primary astrocyte cultures is also inhibited by L-644711, a derivative of the loop diuretic ethacrynic acid, which has previously been shown to be neuroprotective and which reduces K+-induced brain slice swelling (3). 3) P-glycoprotein, an ATP-dependent pump that appears to pump out exogenous substances from the central nervous system and is also potently inhibited by tamoxifen (24, 32), was thought to be identical to the VSOR channel, since it is inhibited by tamoxifen and dideoxyforskolin. However, recent data have challenged this idea (31). 4) Another cloned channel, termed ICln (34), has characteristics similar to those of the VSOR channel and is blocked by all the inhibitors mentioned but is also inhibited by millimolar concentrations of cAMP (31, 34).

In this study, we used the pharmacology just described to identify the channel(s) responsible for elevated KCl-induced release of D-[3H]aspartate from primary cortical astrocyte cultures.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. D-[3H]aspartate was obtained from Amersham (Arlington Heights, IL). All other chemicals were from Sigma Chemical (St. Louis, MO), except for NPPB and dideoxyforskolin, which were obtained from Research Biochemicals International (Natick, MA). L-644711 was a gift from Merck Pharmaceutical. Culture media and materials were obtained from GIBCO BRL (Grand Island, NY).

Cell culture. Primary astrocyte cultures were prepared from the cerebral cortex as previously described (8). In brief, the cerebral hemispheres of rat pups (Sprague-Dawley, 1 day postnatal) were removed, and the meninges were carefully dissected away. The cortexes were turned over from front to back, exposing the hippocampus, which was removed along with the meninges from the underside of the hemispheres. The tissue was extracted using three 10-min dissociations with Dispase II dissolved in Joklik S-MEM (Boehringer-Mannheim Biochemicals, neutral protease, Dispase grade II). The first extraction was discarded, and DNase (3 drops of 4 mg/ml DNase for 10 ml of S-MEM) was added for the second extraction. The dissociated cells were seeded and grown on poly-D-lysine-coated 18 × 18-mm coverslips (Bellco Biotechnology, Vineland, NJ). The cultures were used when the cells formed a confluent monolayer after ~3-4 wk. Immunocytochemistry showed that >95% of the cells stained positively for the astrocytic marker, glial fibrillary acidic protein.

Efflux measurements. Astrocytes grown on coverslips were incubated overnight in 2.5 ml of MEM containing 10% horse serum together with 4 µCi/ml D-[3H]aspartate (1 mCi/ml; sp act 86.4 mCi/mg aspartate). Radiolabeled D-aspartate was used as a nonmetabolizable marker for the behavior of intracellular glutamate and aspartate pools (7). The coverslips were inserted into a Lucite perfusion chamber with a cut-out depression in the bottom for the 18 × 18-mm glass coverslips. The chamber has a Teflon screw top and, when screwed down, leaves a height above the cells of ~100 µm (30).

To measure efflux, the cells were perfused with HEPES-buffered solutions consisting of (in mM) 140 NaCl, 3.3 KCl, 0.4 MgSO4, 1.3 CaCl2, 1.2 KH2PO4, 10 D-(+)-glucose, and 25 HEPES (pH 7.4). KCl buffers (100 mM) were made by replacing Na+ with equimolar K+. The osmolarity of all buffers was measured by a freezing point osmometer (Advanced Instruments, Needham Heights, MA). Sucrose was added to adjust for differences in osmolarity between buffers to a value of 285-291 mosM. A 50:50 (vol/vol) ethanol-DMSO solvent was used to make a 50 mM stock solution of tamoxifen. DMSO was used to make a 100 mM stock solution of NPPB. When compounds were added in DMSO (or ethanol-DMSO), the same amount of solvent was added to media not containing inhibitors.

The Lucite chamber and a fraction collector were placed in an incubator set at 37°C, and the perfusate was collected in 1-min intervals. At the end of the experiment, the cells were lysed in 1 N NaOH, which released any D-[3H]aspartate left in the cells. The radioactivity was counted using a Packard Beckman LS 3801 liquid scintillation analyzer (Beckman Instruments, Irvine CA). Release for each time point was calculated by dividing the radioactivity released in a 1-min sample by the total radioactivity that would have been present at the beginning of that time point as calculated from the sum of the subsequent 1-min fractions plus the amount present in the cell digest at the end of the experiment. We term this "percent fractional release." The number of release experiments for each condition ranged from two to four, as indicated, using different coverslips from the same or different cultures. All values from the same experimental conditions were within 10% of each other. Percent inhibition was calculated using the total areas under the curve during the entire exposure to raised KCl or hypotonic medium.

Cell volume measurements. KCl-induced swelling was measured using an impedance method, as previously described (30). Briefly, a lock-in amplifier supplies a 5-V signal at 500 Hz through a 1-MOmega resistor in series with the chamber. Because the chamber resistance is relatively small, this gives an essentially constant current of 5 µA. The same Lucite chamber and 18 × 18-mm glass coverslips were used as in the perfusion experiments. Cell shrinkage or swelling produces a proportional increase or decrease in the volume of medium above the cells and therefore a decrease or increase, respectively, in the resistance of the chamber. Voltage across the chamber is thereby altered and detected by the amplifier system. Thus small changes in resistance that result from changes in astrocyte monolayer volume are measured.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of extracellular nucleotides and derivatives on KCl-induced D-[3H]aspartate release. A typical D-[3H]aspartate release response from primary cultured astrocytes exposed to 100 mM KCl alone is shown in the first response in Fig. 1A. There was usually, but not always, a small initial transient peak, followed by a slower, progressively increasing, second phase of release, which rapidly returned to baseline after a 3- to 4-min delay when the perfusate was returned to the normal 3.3 mM K+. We have previously shown that the initial transient peak represents reversal of the high-affinity glutamate uptake transporter and can be greatly enhanced by pretreatment with ouabain, whereas the larger, progressively increasing, second phase of release is due to KCl-induced cell swelling (27, 38). A similar control response to 100 mM KCl was obtained in every case before perfusing any inhibitor.


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  		Fig. 1.   Effects of 1 and 5 mM extracellular ATP on KCl-induced D-[3H]aspartate release. A: 2 consecutive 20-min 100 mM KCl exposures; 1st exposure was in absence of 1 mM ATP (control) and 2nd was in presence of 1 mM ATP. The 2 traces (open circle  and black-square) represent 2 experiments. Asterisk indicates a transient stimulation of release caused by 1 mM ATP in initial 4 min. Arrow, small initial transient peak. B: 3 consecutive 20-min 100 mM KCl exposures, with 2nd also containing 5 mM ATP. Inset: exposure to 5 mM ATP in normal isotonic (iso) 3.3 mM K+ medium. C: 40-min 100 mM KCl exposure in absence of 5 mM ATP (triangle ) and 2 separate 40-min 100 mM KCl exposures in presence of 5 mM ATP (open circle  and black-square). ATP always enhanced early release, but later release was suppressed by 5 mM ATP.

As mentioned in the introduction, 1-10 mM extracellular ATP inhibits the VSOR/VSOAC and the ICln channels in various cell types (31, 34, 46). The effect of 1 mM extracellular ATP on K+-induced swelling release is shown in the second response in Fig. 1A; 1 mM ATP caused the cells to release D-[3H]aspartate in a large transient phase, but the second, swelling-induced phase was unaffected. Figure 1B shows that at a higher concentration of ATP (5 mM) D-[3H]aspartate release also showed a rapid increase, but the release plateaued instead of progressively increasing. The initial stimulatory response was due to an effect of ATP itself, because the same concentration in isotonic medium also caused stimulation of D-[3H]aspartate release (as shown in Fig. 1B, inset).

Because the effects of ATP were complicated by the rapid initial stimulation of release, we reasoned that the effect of extracellular ATP would be more apparent over a longer period of exposure to 100 mM KCl, and therefore we doubled the exposure time to raised KCl. Figure 1C shows that 5 mM extracellular ATP again stimulated release (note larger y-axis scale in Fig. 1C compared with Fig. 1, A and B), but after 16 min of KCl exposure the control surpassed the inhibited 5 mM ATP + KCl release plateau and continued to increase for the remaining 24 min. The inhibition by 5 mM ATP of the second, swelling-induced phase of release is now much clearer.

Other nucleotide derivatives were tested for their effects on elevated K+-induced D-[3H]aspartate release, since some appear to be as good as or better than ATP at blocking the VSOR and ICln channels (31, 34). As shown in Fig. 2A, ITP inhibited the 100 mM K+-induced D-[3H]aspartate release, similar to ATP. A reproducible rebound of release occurred when cells were reperfused with medium of normal external K+ concentration after ITP exposure, but this was not investigated further. It may be a repolarization-driven efflux of D-[3H]aspartate through the still open channel. Another nucleotide derivative, 3'-azido-3'-deoxythymidine (AZT), was tested because, when the ICln channel gene was transfected into NIH/3T3 cells, micromolar concentrations of AZT inhibited the swelling-activated anion currents (10). AZT (1 mM) showed no effect on K+-induced release (data not shown) but ~55% inhibition occurred at 5 mM (Fig. 2B). This inhibitor differed from ATP and ITP, since the effect was only partially reversible, as shown in the third high-K+ exposure. Millimolar concentrations of cAMP also specifically blocked the ICln channel when expressed in Xenopus oocytes and NIH/3T3 cells (10, 34) but had no effect on VSOR channels (31). As shown in Fig. 2C, 5 mM cAMP had no effect on K+-induced release.


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  		Fig. 2.   Effect of several nucleotide derivatives on K+-stimulated release. A: 3 consecutive 100 mM KCl exposures; 2nd exposure was raised K+ exposure in presence of 5 mM ITP. B and C: effects of 5 mM 3'-azido-3'-deoxythymidine (AZT) and 5 mM cAMP, respectively, using same protocol as in A. Each panel shows 2 experiments (open circle  and black-square).

Effects of anion channel inhibitors on high K+-induced D-[3H]aspartate release. NPPB is a general anion channel inhibitor that inhibits all VSOR channels, ICln, and maxi channels. In Fig. 3A we show that 100 µM NPPB completely inhibited KCl-induced release. This effect was reversible, as shown in the third 100 mM KCl exposure in the absence of NPPB.


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  		Fig. 3.   Effect of several anion channel inhibitors on elevated K+-induced release of D-[3H]aspartate. A: 2 experiments (open circle  and black-square), showing 3 consecutive 20-min 100 mM KCl exposures, with 100 µM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) present in 2nd 100 mM KCl exposure. B: DIDS was perfused in 2nd KCl exposure; 1st KCl exposure was ~25 min in duration. C: same experiment as in A, but 100 µM dideoxyforskolin (DDF) was perfused in 2nd exposure to elevated K+. D: tamoxifen was added in 2nd exposure to elevated K+. Same percentage of DMSO or 50:50 (vol/vol) DMSO-ethanol was present throughout each experiment.

Dideoxyforskolin, the inactive form of forskolin, was tested because recent data showed it to be a potent blocker of swelling-activated channels (16). DIDS, a relatively nonspecific anion transport inhibitor, also inhibits swelling-activated anion currents (46). Both of these inhibitors reduced KCl medium-induced D-[3H]aspartate release by >90% (Fig. 3, B and C).

Tamoxifen is another inhibitor of VSOR channels (46). It has previously been shown to inhibit both taurine and 125I efflux in HeLa cells with an IC50 of 1-2 µM (24). In Fig. 3D, we show that 15 µM tamoxifen inhibited KCl-induced release by 63 and 92% in the two experiments shown. As with DIDS, the tamoxifen effect is partially irreversible, so we do not show a third, control response.

Effect of temperature. Lowered temperature reduces glutamate release in vivo and is highly neuroprotective (4). If the release of D-[3H]aspartate in primary cultured astrocytes reflects to some degree the release of endogenous glutamate and aspartate in vivo from swollen astrocytes, then it is of interest to look at effects of reduced temperature on such release in vitro. We had previously shown that K+-induced, but not hypotonic medium-induced, D-[3H]aspartate release was inhibited by a reduction in temperature from 37 to 25°C (23). In the present study, ouabain was included to clearly differentiate the initial phase of D-[3H]aspartate release due to reversal of the excitatory amino acid (EAA) transporter and the second, swelling-induced phase. Figure 4 shows that when the temperature of the experiment was reduced from 37°C to room temperature (22-25°C) the second phase of release was inhibited, whereas the first phase was not.


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  		Fig. 4.   Effect of temperature on KCl-induced D-[3H]aspartate release. Ten minutes after reintroduction of isotonic medium after 1st 100 mM KCl exposure, incubator temperature was reduced to room temperature for 2nd exposure. After KCl exposure, incubator door was closed and, when 37°C was reached (as measured with a thermometer), raised KCl was reperfused (n = 3; means ± SE).

Effect of inhibitors and temperature on KCl-induced swelling. We used KCl medium to induce swelling because this is a likely way astrocytes swell in pathological states in vivo (see introduction). However, such swelling involves uptake of KCl followed by osmotically obligated water, and therefore anion channel blockers could inhibit such swelling by blocking Cl- influx. We therefore examined the effects of the anion transport inhibitors used, as well as reduced temperature, on KCl-induced swelling of astrocytes measured by the electrical impedance method (see Cell volume measurements). As shown in Fig. 5, the time course for KCl-induced swelling was similar to the second phase of D-[3H]aspartate release in that swelling also progressively increased for as long as the raised KCl medium was present. Figure 5B also shows that extracellular ATP at 10 mM had no effect on KCl-induced swelling. Tamoxifen (15 µM) had no effect over the initial 30 min of 100 mM KCl, but after 40 min the rate of swelling appeared to slow. DIDS (100 µM) initially stimulated elevated K+-induced swelling, but after 15 min swelling slowed (Fig. 5A). AZT (5 mM) dramatically slowed KCl-induced swelling (Fig. 5B). In the presence of 100 µM NPPB, treated cells initially shrank, but a small increase in cell volume followed (Fig. 5A). Remarkably, reducing the temperature from 37 to 22°C completely inhibited astrocytic K+-induced swelling (Fig. 5, A and B). Figure 5B also shows that 1 mM L-644711 almost completely inhibits cell swelling.


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  		Fig. 5.   Effect of anion channel blockers on KCl-induced swelling as measured by electrical impedance. KCl (100 mM) was introduced at time 0 and continued for 60 min, as indicated by bars. A voltage increase indicates an increased resistance reflecting a proportional decrease in the medium volume above cells caused by cell swelling (control, n = 5; ATP, n = 3; tamoxifen, n = 3; NPPB, n = 3; room temperature, n = 3). Effects of different inhibitors are shown at 37°C. Effect of room temperature (21-23°C) is also shown.

Effects of extracellular nucleotides and derivatives on hypotonic medium-induced D-[3H]aspartate release. As shown in the previous section, some anion channel inhibitors blocked KCl-induced swelling. However, hypotonic medium-induced swelling, which involves the entry of water driven by osmotic gradients, should be unaffected by the anion channel blockers, and the pharmacology of the hypotonic medium-induced swelling-activated channel can be directly assessed and compared with KCl-induced swelling. The time course of hypotonic medium-induced D-[3H]aspartate release and swelling is very different from K+-induced swelling and release, as illustrated in Fig. 6 (see also Fig. 8 and Ref. 23). Hypotonic medium-induced D-[3H]aspartate release shows an immediate and rapid rate of D-[3H]aspartate release that peaks after 3-5 min and then declines. This decline in D-[3H]aspartate or taurine release in hypotonic medium mirrors cell volume regulation (RVD) and presumably reflects the progressive closure of channels as the cells restore normal volume (21, 33). Figure 6, A-D, shows that none of the nucleotides or nucleotide derivatives (10 mM ATP, 5 mM ITP, AZT, and cAMP) inhibited hypotonic medium-induced D-[3H]aspartate release. Indeed, 5 mM ITP and cAMP appeared to stimulate release.


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  		Fig. 6.   Effect of several nucleotides and their derivatives on hypotonic medium-induced release. In all experiments, inhibitors were present in 3rd hypotonic exposure and were compared with 2nd and 4th hypotonic exposures as controls. A, B, C, and D depict effects of ATP, ITP, AZT, and cAMP, respectively. Hypotonic medium (-100 mosM) was made by removing 50 mM NaCl. B and D: 2 separate experiments (open circle  and black-square) are shown. A and C: representative experiments are shown.

Effects of swelling-activated channel inhibitors on hypotonic medium-induced D-[3H]aspartate release. As shown in Fig. 7, A and B, the anion channel inhibitors NPPB and dideoxyforskolin (100 µM) blocked >90% of hypotonic medium-induced D-[3H]aspartate release, and this inhibition was reversible. DIDS (100 µM) only inhibited ~45% of D-[3H]aspartate release. L-644711 has previously been shown to inhibit K+- and hypotonic medium-induced D-[3H]aspartate release (23, 38). In Fig. 7D, >90 and 70% inhibition was seen at 1 and 0.3 mM L-644711, respectively.


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  		Fig. 7.   Effect of several anion channel inhibitors on hypotonicity-induced release. NPPB, DDF, DIDS, and L-644711 were added in 3rd hypotonic exposure. A: effect of 100 µM NPPB on 3rd hypotonicity-induced D-[3H]aspartate release; 2 separate experiments are shown (open circle  and black-square). B and C: same protocol as in A, except that 100 µM DDF and 100 µM DIDS were present in 3rd hypotonic exposure; 2 separate experiments are shown in each (open circle  and black-square). DMSO was present for entire NPPB and DDF experiments. D: effects of anion transport blocker L-644711 present in 3rd and 4th hypotonic exposures at 0.3 and 1 mM, respectively.

The effect of tamoxifen, a specific inhibitor of the VSOR/VSOAC channel, on D-[3H]aspartate hypotonic medium-induced release was more complicated. Tamoxifen (10 µM) inhibited 30% of D-[3H]aspartate release, with most of the release occurring within the first 4 min of hypotonic medium exposure (data not shown). On switching back to isotonic medium, there was a sharp peak of increased release that increased as the tamoxifen concentration increased, up to 20 µM. 51Cr release data suggest that this was due to cell lysis or loss (data not shown). However, a 10-min tamoxifen pretreatment not only inhibited >80% of D-[3H]aspartate release but also completely reduced 51Cr efflux, as shown in Fig. 8.


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  		Fig. 8.   Effect of tamoxifen on hypotonicity-induced D-[3H]aspartate release. Ten minutes before 3rd hypotonic exposure (hypo), 15 µM tamoxifen was perfused and both D-[3H]aspartate (triangle  and black-square) and 51Cr efflux (dotted and solid lines, respectively) were measured in each of two experiments. 51Cr efflux was used to determine whether release was due to cell lysis or loss. Same percentage of ethanol (0.06%) was present throughout an individual experiment.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Mechanisms of D-[3H]aspartate efflux caused by KCl-induced swelling. The inhibition of KCl-induced D-[3H]aspartate efflux and the lack of inhibition of KCl-induced swelling by ATP and tamoxifen are consistent with the VSOR/VSOAC channels being responsible for KCl-induced D-[3H]aspartate release in primary astrocyte cultures. The molecular identity of the VSOR channel is currently not known (see introduction), but in primary astrocyte cultures the ICln channel is not directly involved, because 5 mM cAMP had no effect on release (10, 31, 34). This may also explain why AZT, which inhibits the ICln channel expressed in NIH/3T3 cells with an IC50 of 20 µM, had no effect at 1 mM on KCl-induced D-[3H]aspartate release in primary astrocyte cultures. The inhibition seen at 5 mM AZT was probably due to inhibition of K+-induced swelling, as shown in Fig. 5B. The inhibition of KCl-induced release, but not the KCl-induced swelling, by extracellular nucleotides also suggests that the P-glycoprotein is not the channel, since no effect of extracellular ATP has been described for this intracellular ATP-dependent pump. It is possible that the ClC-3 channel or a variant thereof is involved, since, as previously shown, the pharmacology of this channel agrees with the data presented here (6, 31). The initial stimulation of D-[3H]aspartate release by ATP appears to be independent of KCl because stimulation by 5 mM ATP was also seen in isotonic medium. ATP might activate a nonspecific channel via activation of P2y receptors on cultured astrocytes, which would allow taurine (data not shown) and EAAs (see Fig. 1B, inset) to pass through. This release appears not to be mediated by a cell swelling process and was not further investigated. The enhanced initial phase of release in the third KCl exposure has previously been shown and could be due to phosphorylation of the glutamate transporter by Ca2+-activated second messengers (5).

Ten micromolar tamoxifen has been shown to inhibit 75% of the anion conductance in an M-1 cell line derived from mouse cortical collecting duct cells (28) and to inhibit ~90% of taurine, sorbitol, and thymidine efflux from HeLa cells when these cells were swollen by hypotonic media (12). In 3T3 fibroblasts, 1 mM ATP, 10 µM tamoxifen, and "knock down" expression of ICln protein by antisense oligonucleotides all blocked swelling-activated anion currents (11, 29). The effectiveness of tamoxifen in inhibiting the swelling-activated current varies widely from cell type to cell type, which is unexpected, since a VSOAC/VSOR-like channel is present in all cell types studied to date (29). It has also been suggested that tamoxifen inhibits release of osmolytes due to inhibition of calmodulin or a calmodulin-like binding site in the membrane (24). However, previous studies have shown that KCl- and hypotonicity-induced D-[3H]aspartate release is Ca2+ independent (30, 38).

One feature of KCl-induced swelling is the absence of apparent RVD. This is probably the result of K+ and Cl- contributing most to RVD during hypotonic exposure; in raised K+ media, net K+ efflux is reduced or eliminated, and uncharged amino acids are not in high enough concentration to counteract the swelling (47).

Pharmacology of hypotonic medium-induced release of D-[3H]aspartate. The rapidity and magnitude of hypotonic medium-induced swelling can probably activate a variety of swelling-activated anion channels. An example of this is nonpigmented epithelial retinal cells, which show three anion conductances when swollen by hypotonic media: a low conductance (6.7 pS), an intermediate conductance (18 pS) similar to the VSOR channel conductance, and a large conductance (100 pS). NPPB at a concentration of 100 µM blocked all three currents (49). However, except for the VSOR channel, it is still unclear which of these can also conduct amino acids. The data presented here show that tamoxifen pretreatment, dideoxyforskolin, and NPPB can all inhibit hypotonic medium-induced D-[3H]aspartate release and therefore suggest that the VSOAC channel is also involved during hypotonic medium-induced swelling. At 100 µM NPPB, a typical concentration used in other studies, complete inhibition of a swelling-activated anion conductance and organic osmolyte efflux was found (12). However, dideoxyforskolin and NPPB and the other lipophilic compounds may inhibit D-[3H]aspartate release via other mechanisms, such as reducing intracellular ATP concentrations, and not by directly blocking the channel (1). The mechanism by which 1 mM L-644711 (23, 38) blocks >90% of D-[3H]aspartate release remains unclear, since this concentration of drug has been shown to inhibit the maxi channel in primary cultured astrocytes (18), although a nonspecific effect of this drug on VSOAC channel activation cannot be ruled out and the relative amount of amino acid current through the maxi channel is unknown.

Hypotonic medium-induced D-[3H]aspartate release is nucleotide insensitive, and 100 µM DIDS only inhibited ~45% of release, suggesting VSOAC is not involved (Figs. 6 and 8). However, a possible explanation for their ineffectiveness may be related to the type of medium used to cause swelling. Extracellular nucleotide inhibition is dependent on an inward electrochemical Cl- gradient that should increase ATP entry into the pore, thereby blocking the channel (17). Similarly, DIDS also appears to be voltage dependent, i.e., more inhibition occurs at positive cell membrane potentials (6, 26). Therefore, the larger cell membrane depolarization caused by exposing cultured astrocytes to 100 mM K+ allows extracellular ATP to block the swelling-activated VSOAC channel, in contrast to the smaller depolarization caused by hypotonic medium-induced swelling. Figure 9 summarizes the different swelling mechanisms and the respective swelling-activated channels responsible for D-[3H]aspartate release, based in part on the data presented in this paper. In hypotonic medium-induced swelling, there is rapid entry of water. There is also efflux of K+ via K+ channels and Cl- via the various anion channels, all of which contribute to RVD. K+-induced swelling is slow, and there is no apparent RVD because high KCl in the medium can enter the cell as easily as it can leave, so there is no net loss of the major osmolytes, K+ and Cl-.


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  		Fig. 9.   Model of influx and efflux mechanisms for swelling induced by hypotonic media (A) and by high-K+ media (B). A: hypotonic medium-induced swelling and subsequent release of various cellular components contributing to regulatory volume decrease. B: swelling and release due to exposure to raised external K+ medium. "Only" refers to KCl influx not being blocked (see text for further details).

Clinical relevance of K+-induced swelling of astrocytes. Astrocytes contribute substantially to the cellular swelling that occurs as a result of traumatic brain injury and ischemia (stroke) (20, 22). Inhibition of such swelling and glutamate release, either pharmacologically or by reduction of brain temperature, has been shown to be associated with improved neurological outcome and mortality (3, 4). From this and other studies, we may conclude that swollen astrocytes are possibly deleterious because of release of EAAs (21, 23, 38). The focus of the present study was to identify in primary astrocyte cultures the swelling-activated channel(s) responsible for such EAA release by using raised KCl to swell the cells, since elevated extracellular K+ is seen in both ischemia and traumatic brain injury (13, 43). Only further studies in vivo can show whether these processes actually occur in traumatic brain injury and stroke.

Currently, we have seen that in vivo an anion channel inhibitor partially blocks ischemia-induced release of glutamate and aspartate during ischemia in rat striatum as measured by microdialysis (42). NPPB (350 µM) and other channel inhibitors have been shown, using a cortical cup perfusion system, to reduce ischemia-induced glutamate, aspartate, and taurine release (35). Obtaining a better understanding of which type of swelling-activated channel(s) is responsible for EAA release during brain cellular edema will permit the design of more specific drugs to block these channels, which may, in turn, be therapeutically useful.

    ACKNOWLEDGEMENTS

We thank Dr. P. J. Feustel for helpful discussions of the manuscript and Dawn Conklin for technical assistance with the volume measurements.

    FOOTNOTES

This research was supported by National Institutes of Health Grants NS-35205 (to H. K. Kimelberg) and ES-07331 (to M. Aschner).

Address for reprint requests: H. K. Kimelberg, Div. of Neurosurgery, A-60, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208.

Received 21 October 1997; accepted in final form 25 February 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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