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
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ABSTRACT |
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
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INTRODUCTION |
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,
-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.
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MATERIALS AND METHODS |
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-M
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.
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RESULTS |
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 ( and ) 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 ( ) and 2 separate 40-min 100 mM KCl exposures in
presence of 5 mM ATP ( and ). ATP always enhanced early release,
but later release was suppressed by 5 mM ATP.
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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 ( and ).
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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 ( and ),
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.
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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).
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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.
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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 ( and
) are shown. A and
C: representative experiments are
shown.
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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 ( and ).
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 ( and ). 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.
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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
( and ) 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.
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DISCUSSION |
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
.

View larger version (25K):
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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.
 |
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