cell swelling; high potassium ion concentration; cultured
astrocytes; glutamate release; bumetanide; intracellular chloride; excitatory amino acid
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INTRODUCTION |
NA+-k+-cl
cotransporter (NKCC) isoform 1 (NKCC1) has been shown to
play a role in cell volume regulation and K+ uptake in
astrocytes (10, 12, 33). NKCC1 activity in cultured rat
cortical astrocytes is significantly stimulated by high extracellular K+ concentration ([K+]o)
(32). Inhibition of NKCC1 activity by the potent
cotransporter inhibitor bumetanide decreases the
high-[K+]o-mediated uptake of
Cl and blocks astrocyte swelling (Ref. 33;
this issue). Moreover, administration of bumetanide in brain cortex
significantly reduces edema and infarct volume after focal
ischemia (41). Thus NKCC1 could contribute to an
overload of intracellular Na+, K+, and
Cl and cell swelling in conditions such as cerebral
ischemia, in which [K+]o is elevated.
Astrocytes release organic osmolytes such as excitatory amino acid
(EAA) under high-[K+]o conditions to regulate
cell volume (13, 18). One possible mechanism for the EAA
release under high-[K+]o conditions is via
volume-sensitive organic anion channels (VSOACs; Refs. 1,
27). High-[K+]o-induced
3H-labeled D-aspartate (Asp) release from
cultured astrocytes is inhibited by the anion channel inhibitors
L-644711 and dideoxyforskolin (27). We have reported (Ref.
33; this issue) that blocking of NKCC1 activity by
bumetanide significantly reduces
high-[K+]o-induced 14C-labeled
D-Asp release. These results imply that NKCC1-mediated swelling may contribute to the
high-[K+]o-triggered glutamate release.
The role of NKCC1 in high-[K+]o-triggered
swelling and glutamate release described above has been studied by
pharmacological techniques. Although bumetanide is a potent inhibitor
of the NKCCs, it also inhibits other ion transport proteins and
GABAA channels (26, 34). In this study, we
used astrocytes isolated from the NKCC1-deficient mice established by
Flagella et al.(6). We report here that
high-[K+]o-induced swelling was absent in
NKCC1/ astrocytes. A significant decrease in
intracellular Cl and Na+ and release of
glutamate was observed in NKCC1/ astrocytes. The
results suggest that NKCC1 is important in regulation of astrocyte
volume and intracellular Cl and Na+.
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MATERIALS AND METHODS |
Materials.
Bumetanide, digitonin, Triton X-100, monensin, and gramicidin were
purchased from Sigma (St. Louis, MO). Eagle's modified essential
medium (EMEM) and Hanks' balanced salt solution (HBSS) were from
Mediatech Cellgro (Herndon, VA). Fetal bovine serum was obtained from
Hyclone Laboratories (Logan, UT). Collagen type I was from
Collaborative Biomedical Products (Bedford, MA). 86RbCl was
purchased from NEN Life Science Products (Boston, MA). D-[14C]Asp was from American Radiolabeled
Chemicals (St. Louis, MO). Chloride-36 was purchased from Amersham
Pharmacia Biotech (Piscataway, NJ). Sodium-binding benzofuran
isophthalate (SBFI)-AM was purchased from Molecular Probes (Eugene,
OR). Pluronic acid was purchased from BASF (Ludwigshafen, Germany).
Animals and genotype analysis.
NKCC1 homozygous mutant and wild-type mice were obtained by breeding
gene-targeted NKCC1 heterozygous mutant mice, and genotypes were
determined by a polymerase chain reaction (PCR) of DNA from tail
biopsies as described previously (6).
Primary culture of mouse cortical astrocytes.
Dissociated cortical astrocyte cultures were established based on a
method used in our previous study of rat cortical astrocyte cultures
(32). Cerebral cortices were removed from 1-day-old NKCC1+/+ or NKCC1/ mice. The cortices were
incubated in a solution of 0.25 mg trypsin /ml HBSS for 25 min at
37°C. The tissue was then mechanically triturated and filtered
through nylon meshes. The dissociated cells were rinsed and resuspended
in EMEM containing 10% fetal bovine serum. Viable cells (1 × 104 cells /well) were plated in 24-well plates coated with
collagen type 1. Cultures were maintained in a 5% CO2
atmosphere at 37°C. The cultures were subsequently refed every 3 days
throughout the study. To obtain morphologically differentiated
astrocytes, confluent cultures (days 12-15 in culture)
were treated with EMEM containing 0.25 mM dibutyryl cAMP (DBcAMP) for 7 days to induce differentiation. DBcAMP has been widely used to mimic
neuronal influences on astrocyte differentiation (9, 35).
Experiments were routinely performed (see Figs. 4-10) on cultures
treated with DBcAMP for 7 days.
Gel electrophoresis and Western blotting.
Cortical astrocytes growing on culture dishes were washed with ice-cold
phosphate-buffered saline (PBS; pH 7.4) that contained 2 mM EDTA and
protease inhibitors. Cells were scraped from dishes and lysed by
sonication at 4°C (32). To obtain cellular lysates, cellular debris was removed by centrifugation and protein content of
the cellular lysate was determined (29). Samples and
prestained molecular mass markers (Bio-Rad) were denatured in SDS
reducing buffer (1:2 vol/vol; Bio-Rad) and heated at 37°C for 15 min
before gel electrophoresis. The samples were then electrophoretically separated on 6% SDS gels, and the resolved proteins were
electrophoretically transferred to a polyvinylidene difluoride membrane
(32). The blots were incubated in 7.5% nonfat dry milk in
Tris-buffered saline and then incubated with a primary antibody. After
rinsing, the blots were incubated with horseradish
peroxidase-conjugated secondary IgG. Bound antibody was visualized with
an enhanced chemiluminescence assay (ECL; Amersham). Anti-NKCC
monoclonal antibody against the human colonic T84 epithelial NKCC
(20) was used for detection of the cotransporter protein,
and the same blot was probed with anti-
actin antibody as a control.
A linear curve with 5-30 µg of protein has been established for
both anti-NKCC antibody and anti- actin antibody (32).
In addition, the linear range of the ECL exposure course on the film
was within 15-120 s (32). Therefore, 30 µg of
protein was loaded in all immunoblots of the present study. In
addition, the ECL exposure time was within 60 s.
Immunofluorescence staining.
Cultured cells grown on collagen type I-coated coverslips were rinsed
with PBS (pH 7.4) and fixed with 4% paraformaldehyde in PBS for 40 min
at room temperature. After rinsing, cells were incubated with blocking
solution (10% normal goat serum, 0.4% Triton X-100, and 1% bovine
serum albumin in PBS) for 1 h. Cells were then incubated with
anti-glial fibrillary acidic protein (GFAP) polyclonal (1:100) or
anti-NKCC monoclonal antibodies (1:100) in blocking solution overnight
at 4°C. Cells were rinsed with PBS and incubated with goat
anti-mouse FITC-conjugated or goat anti-rabbit Texas red-conjugated
antibodies (1:200) for 2 h. Cell images were captured by laser
scanning confocal microscope (Bio-Rad MRC 1000) as described previously
(32). Bio-Rad MRC-1024 Laser Sharp software (version 2.1T)
was used to control the microscope and its settings. An identical
setting was used to capture the negative control and experimental images.
Measurement of relative cell volume changes in single cell.
Relative cell volume changes were estimated with video-enhanced
differential interference contrast (DIC) microscopy, as described in
our previous study (Ref. 33, this issue). The
same method has been reported by others (40). Astrocytes
cultured on collagen-coated coverslips were placed in an open-bath
imaging chamber (Warner Instruments, Hamden, CT; bath volume 40 µl)
on the stage of a Nikon TE 300 inverted epifluorescence microscope.
Astrocytes were equilibrated with isotonic HEPES-buffered minimal
essential medium (MEM, 312 mosmol/kgH2O) for 15 min
(33). Astrocytes were exposed sequentially to
HEPES-MEM (5 min), 75 mM [K+]o HEPES-MEM (10 min), HEPES-MEM (10 min), HEPES-MEM + 10 µM bumetanide (20 min),
75 mM [K+]o HEPES-MEM + 10 µM
bumetanide (10 min), and HEPES-MEM (10 min). In 75 mM
[K+]o HEPES-MEM, 75 mM
[K+]o was obtained by replacing NaCl in
HEPES-MEM solutions with equimolar KCl. A single astrocyte was
visualized with a Nikon ×60 Plan Apo oil-immersion objective lens, and
cell images were recorded every minute as described previously
(33). The mean cross-sectional area (CSA) of the cell body
was calculated with MetaMorph image-processing software. The control
CSA values were obtained when cells were exposed to HEPES-MEM only.
Relative changes in CSA (CSAr) were calculated as
experimental CSA divided by control CSA. After each experiment,
relative cell volume changes in response to HEPES-MEM calibration
buffers were measured. Salt concentrations in the buffers were held
constant, and the osmolality (238, 277, and 312 mosmol/kgH2O) was adjusted by varying the buffer
concentration of sucrose.
Assay for NKCC1 activity.
NKCC1 activity was measured as bumetanide-sensitive K+
influx with 86Rb as a tracer for K+
(32). Cultured astrocytes were equilibrated for 10-30
min at 37°C with isotonic HEPES-MEM (312 mosmol/kgH2O).
Cells were preincubated for 10 min in HEPES-MEM containing either 0 or
10 µM bumetanide. For assay of cotransporter activity, cells were
exposed to 1 µCi/ml of 86Rb in HEPES-MEM for 3 min in the
presence or absence of 10 µM bumetanide. 86Rb influx was
stopped by rinsing cells with ice-cold 0.1 M MgCl2. Radioactivity of the cellular extract in 1% SDS was analyzed by liquid
scintillation counting (1900CA; Packard, Downers Grove, IL).
K+ influx rate was calculated and expressed as nanomoles of
K+ per milligram of protein per minute. It has been
established that the slope of 86Rb uptake over 10 min is
linear in astrocytes (32). Bumetanide-sensitive K+ influx was obtained by subtracting the K+
influx rate in the presence of bumetanide from the total K+
influx rate. Quadruplicate determinations were obtained in each experiment throughout the study, and protein content was measured in
each sample with a method described previously (29).
Statistical significance in the study was determined by ANOVA
(Bonferroni-Dunn) at a confidence level of 95% (P < 0.05).
Intracellular Cl content measurement.
Cells on 24-well plates were preincubated for 30 min in HEPES-MEM
containing 5.8 mM [K+]o and 36Cl
(0.4 µCi/ml). A steady-state level of intracellular 36Cl
was established and maintained during the 30-min preincubation (33). The cells were then incubated in 75 mM
[K+]o HEPES-MEM containing 36Cl
(0.4 µCi/ml) in the presence or absence of 10 µM bumetanide for 4 or 13 min. Thus extracellular Cl concentration
([Cl]o) of 145 mM in HEPES-MEM was
maintained in 75 mM [K+]o, and the specific
activity of 36Cl was constant in 5.8 mM
[K+]o and 75 mM
[K+]o HEPES-MEM. Intracellular
36Cl content measurement was terminated by three washes
with 1 ml of ice-cold washing buffer (in mM: 118 NaCl, 26 NaHCO3, 1.8 CaCl2, pH 7.40). Radioactivity of
the cellular extract in 1% SDS was analyzed by liquid scintillation
counting (Packard 1900CA). In each experiment, specific activities
(counts/µmol × min) of 36Cl were determined for
each assay condition and used to calculate intracellular
Cl content (µmol/mg protein).
Intracellular Na+ measurement.
Intracellular Na+ concentration
([Na+]i) was measured with the fluorescent
dye SBFI-AM as described previously (25). Cultured astrocytes grown on collagen-coated coverslips were loaded with 10 µM
SBFI-AM at room temperature in HEPES-MEM containing 0.1% pluronic acid
as described in our previous report (Ref. 33, this issue). The coverslips were placed in an open-bath
imaging chamber containing HEPES-MEM at ambient temperature. With a
Nikon TE 300 inverted epifluorescence microscope and a ×40 Super Fluor oil-immersion objective lens, astrocytes were excited every 10 s
at 345 and 385 nm, and the emission fluorescences at 510 nm were
recorded. Images were collected and analyzed with MetaFluor image-processing software (33). An area on the coverslip
without cells was defined as the background region and used for
subtraction of baseline fluorometric intensities at 345 and 385 nm.
Approximately 65% of the SBFI fluorescence signals (340- to 380-nm
ratios) measured in this study represented changes of Na+
in the cytoplasm of astrocytes (33).
To monitor changes of [Na+]i, the SBFI-loaded
cells were equilibrated with HEPES-MEM for 20 min. Ratios of 340- to
380-nm fluorescence were recorded, and the bath chamber buffer was
changed with 75 mM [K+]o HEPES-MEM (10 min)
followed by HEPES-MEM (10 min). Absolute [Na+]i was determined for each cell by
calibrating the SBFI fluorescence ratio with solutions containing 0, 10, 20, 40 or 80 mM extracellular Na+ concentration
([Na+]o) and monensin (10 µM) and
gramicidin (3 µM) to equilibrate [Na+]o and
[Na+]i. The resulting ratios from each cell
were fit with a three-parameter hyperbolic curve from which
[Na+]i was calculated (4).
D-[14C]Asp release measurement.
Aspartate release was measured as described previously (Ref.
33, this issue). Astrocytes grown on chamber
slides were incubated overnight in 1 ml of complete EMEM containing 2 µCi/ml of D-[14C]Asp (specific activity of
55 mCi/mmol). The perfusing rate of the perfusion chamber was 1.5 ml/min. This chamber allows a complete change of the perfusing buffer
within 2 min. The cells were perfused at a constant flow rate with
HEPES-MEM containing 5.8 or 75 mM [K+]o in
the presence or absence of 10 µM bumetanide. The buffers and
perfusion chamber were kept at 37°C. The perfusate was collected in
1-min intervals. At the end of the experiment, the cells were digested
in 1% SDS. The radioactivity of samples was measured by liquid
scintillation counting (Packard 1900CA). Calculation of fractional
release was based on the following formula: fractional release = Ct/[Sum(Ct:Cend) + Cremain], where Ct is
the cpm value in the effluent at time t,
Cend is the cpm value in the effluent at the end
of the experiment,
Sum(Ct:Cend) is the total
cpm value in the effluent from time t to the end
of the experiment (28), and Cremain
is the cpm value left in the cells at the end of the experiment.
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RESULTS |
Absence of NKCC1 protein expression in cultured cortical astrocytes
isolated from NKCC1/ mice.
Mating of heterozygous mice yielded live offspring of all three
genotypes in a Mendelian ratio of 1:2:1 (23% NKCC1+/+,
54% NKCC1+/, 23% NKCC1/). As shown in
Fig. 1A, a single DNA band
(~105 bp) was detected in a tail biopsy of the NKCC1+/+
mouse. In contrast, a larger DNA band (~156 bp) was found in the
NKCC1/ tail biopsy. In a heterozygous sample, both PCR
products were present.
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Fig. 1.
Genotyping and immunoblot
analysis for Na+-K+-Cl
cotransporter (NKCC) isoform 1 (NKCC1). A: DNA samples
isolated from tail biopsies were used as polymerase chain reaction
(PCR) templates. Three primers were included in the reaction mixture:
forward and reverse primers corresponding to an exon sequence of the
wild-type gene and a reverse primer complementary to sequence of the
mutant gene (6). B: cell differentiation was
induced by dibutyryl cAMP (DBcAMP). Cell lysates from cultured
astrocytes (NKCC1+/+ and NKCC1/) were
separated by 6% SDS-PAGE, transferred to a nitrocellulose membrane,
and probed with either an anti-NKCC monoclonal antibody or an anti-
actin antibody. The blots were visualized by enhanced
chemiluminescence. This is a representative experiment of
n = 3.
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To analyze expression of the NKCC1 protein in cultured
NKCC1/ cortical astrocytes, cellular lysates were
obtained from both cultured NKCC1+/+and
NKCC1/ astrocytes. Immunoblotting revealed that an
~152 ± 5.7 kDa protein was recognized by an anti-NKCC antibody
in undifferentiated (
DBcAMP) and differentiated (+DBcAMP)
NKCC1+/+ astrocytes (Fig. 1B). In contrast, no
protein band was detected by the anti-NKCC antibody in either
undifferentiated or differentiated NKCC1/ astrocytes.
The lack of NKCC1 bands was not due to an insufficient amount of
protein having been loaded, because, as shown on the same blot, a
similar amount of -actin protein (~58 ± 5.2 kDa) was
observed in samples from both NKCC1+/+ and
NKCC1/ astrocytes (Fig. 1B). These results
indicate that NKCC1 is absent in NKCC1/ astrocytes.
Further support is provided by the results of the immunofluorescence
study. As shown in Fig. 2, A
and D, both undifferentiated and differentiated
NKCC1+/+ astrocytes were positively stained for the glial
marker GFAP. Anti-NKCC antibody immunoreactive fluorescence signals
were observed in both undifferentiated and differentiated
NKCC1+/+ astrocytes (Fig. 2, B and
E). Colocalization of GFAP and the cotransporter is shown in
Fig. 2, C and F. This is consistent with our
previous findings in cultured rat cortical astrocytes (32). In NKCC1/ astrocytes, DBcAMP induced
differentiation in a similar fashion as in NKCC1+/+
astrocytes (Fig. 2, a and d). Expression of GFAP
was shown in both undifferentiated and differentiated
NKCC1/ astrocytes (Fig. 2, a and
d). However, no immunoreactive signals were detected in
NKCC1/ astrocytes with the anti-NKCC antibody (Fig. 2,
b and e). Only GFAP signals appeared in a
double-staining image (Fig. 2, c and f).
Together, these results demonstrate that the NKCC1 protein is not
expressed in undifferentiated and differentiated NKCC1/
astrocytes.
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Fig. 2.
Immunofluorescence staining for NKCC1 protein. A-F:
wild type (+/+). a-f: NKCC1 knock out (/).
A and a: non-DBcAMP-treated astrocytes,
anti-glial fibrillary acidic protein (GFAP) antibody staining.
B and b: non-DBcAMP-treated astrocytes, anti-NKCC
monoclonal antibody staining. C: double-staining images of
A and B. c: Double-staining images of
a and b. D and d:
DBcAMP-treated cells, anti-GFAP antibody staining. E and
e: DBcAMP-treated cells, anti-NKCC antibody staining.
F: double-staining images of D and E. f: Double-staining images of d and e.
Inset in f, a negative control in which a primary
antibody was omitted and the rest of the procedures were the same as in
A-E and a-e. Images were captured by a
laser scanning confocal microscope (Bio-Rad MRC 1000). Scale bar, 50 µm.
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Decrease in K+ influx in
NKCC1/ astrocytes under high
[K+]o.
Previous studies suggested that NKCC is important for K+
uptake in astrocytes and that cotransporter activity is significantly stimulated by high [K+]o (32,
37). In this study, we first examined whether the K+
influx rate was different in NKCC1+/+and
NKCC1/ astrocytes (undifferentiated) under 5.8 mM
[K+]o. As shown in Fig.
3A, at 5.8 mM
[K+]o the total K+ influx rate
was 111.8 ± 6.8 nmol/mg protein × min in
NKCC1+/+ astrocytes; however, it was decreased to 86.1 ± 4.8 nmol/mg protein × min in NKCC1/ astrocytes
(P > 0.05). 10 µM bumetanide did not significantly affect the K+ influx rate in either NKCC1+/+ or
NKCC1/ astrocytes under 5.8 mM
[K+]o. When NKCC1+/+ astrocytes
were exposed to 75 mM [K+]o, the total
K+ influx rate was increased to 348.7 ± 25.8 nmol/mg
protein × min (P < 0.05). Inhibition of
cotransporter activity by 10 µM bumetanide decreased the total
K+ influx rate by 20% (P < 0.05).
Interestingly, genetic ablation of NKCC1 caused a similar reduction in
the total K+ influx rate under 75 mM
[K+]o. Moreover, no additional reduction of
total K+ influx was observed when NKCC1/
astrocytes were treated with 10 µM bumetanide (Fig. 3A).
An identical pattern of changes in the total K+ influx was
found in differentiated NKCC1+/+ and NKCC1/
astrocytes (data not shown). These data suggest that the cotransporter is important in K+ influx in astrocytes, particularly under
high-[K+]o conditions.
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Fig. 3.
High-extracellular K+ concentration
([K+]o)-mediated stimulation of NKCC1
activity. A: the effect of bumetanide and NKCC1 knock out on
the total K+ influx in both NKCC1+/+ and
NKCC1/ undifferentiated astrocytes. Undifferentiated
astrocytes were preincubated in HEPES-minimal essential medium (MEM)
containing 5.8 mM [K+]o for 10 min. Cells
were then exposed to either 5.8 or 75 mM
[K+]o HEPES-MEM for 10 min in the presence or
absence of 10 µM bumetanide (Bum). 86Rb influx was
assayed subsequently for 3 min in identical HEPES-MEM containing 1 µCi 86Rb. Data are means ± SE; n = 4-5. #P < 0.05 vs. 5.8 mM
[K+]o, * P < 0.05 vs.
NKCC1+/+ group under 75 mM [K+]o
(Bonferroni-Dunn). B: bumetanide-sensitive K+
influx in both NKCC1+/+ and NKCC1/
differentiated or undifferentiated astrocytes. Confluent cultured
astrocytes were treated with 0.25 mM DBcAMP to induce morphological
differentiation. Bumetanide-sensitive K+ influx was
obtained by subtracting the K+ influx rate in the presence
of bumetanide from the total K+ influx rate.
* P < 0.05 vs. 5.8 mM
[K+]o, #P < 0.05 vs.
NKCC1+/+ groups.
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The inhibitory effect of bumetanide on the total K+ influx
rate implies that cotransporter activity was increased in
NKCC1+/+ astrocytes under high
[K+]o. We next examined the
bumetanide-sensitive K+ influx rate in
NKCC1+/+and NKCC1/ astrocytes. Figure
3B shows that high [K+]o
stimulated the bumetanide-sensitive K+ influx in both
undifferentiated and differentiated NKCC1+/+ astrocytes. In
contrast, bumetanide-sensitive K+ influx in
NKCC1/ astrocytes was within experimental variation
under 5.8 mM [K+]o. It was not changed under
high-[K+]o conditions. This is in agreement
with the finding that bumetanide has no effect on the total
K+ influx rate in NKCC1/ astrocytes.
Decrease in 36Cl accumulation in
NKCC1/ astrocytes under high
[K+]o.
Pharmacological inhibition of NKCC suggested that the cotransporter
contributes to an accumulation of intracellular Cl in
astrocytes (15, 33). We investigated this further with NKCC1-deficient astrocytes. The basal levels of intracellular 36Cl were 0.48 ± 0.04 µmol/mg protein
when NKCC1+/+ astrocytes were exposed to 5.8 mM
[K+]o for 4 min (Fig.
4A). A similar level of
intracellular 36Cl was maintained after the cells were
incubated in 5.8 mM [K+]o for 13 min. The
intracellular 36Cl level was increased to 0.81 ± 0.06 µmol/mg protein when NKCC1+/+ astrocytes were exposed to
75 mM [K+]o for 4 min (P < 0.05) and maintained at 0.79 ± 0.05 µmol/mg protein after a
13-min incubation. Blocking of cotransporter activity by 10 µM
bumetanide significantly decreased the
high-[K+]o-induced 36Cl increase
in NKCC1+/+ astrocytes (P < 0.05; Fig.
4A). As shown in Fig. 4B, the basal 36Cl levels in NKCC1/ astrocytes were not
significantly different from those of NKCC1+/+ astrocytes
under 5.8 mM [K+]o (P > 0.05). However, when NKCC1/ astrocytes were exposed to
75 mM [K+]o for 4 min, no significant
increase in 36Cl accumulation was observed
(P > 0.05; Fig. 4B). After a 13-min incubation with 75 mM [K+]o, the
intracellular 36Cl level in the NKCC1/
astrocytes was slightly increased but still lower than in control NKCC1+/+ astrocytes and insensitive to bumetanide.
Moreover, 10 µM bumetanide did not significantly affect the
intracellular 36Cl levels in NKCC1/
astrocytes under either 5.8 or 75 mM [K+]o
(P > 0.05). Collectively, the results show that the
absence of NKCC1 in cortical astrocytes abolished the
high-[K+]o-mediated accumulation of
intracellular 36Cl.
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Fig. 4.
Time course of
high-[K+]o-induced Cl uptake
increase and an effect of bumetanide. The effect of bumetanide on
Cl uptake in NKCC1+/+ (A) and
NKCC1/ (B) astrocytes is shown. Astrocytes
were preincubated in 5.8 mM [K+]o HEPES-MEM
at 37°C for 10 min and then equilibrated in 5.8 mM
[K+]o HEPES-MEM with 36Cl (0.4 µCi/ml) for 30 min. Astrocytes were then treated with either 5.8 [K+]o or 75 mM
[K+]o HEPES-MEM containing 36Cl
(0.4 µCi/ml) in the presence or absence of 10 µM bumetanide for 4 or 13 min. Data are means ± SE; n = 5. * P < 0.05 vs. 5.8 mM
[K+]o, # P < 0.05 vs. 75 mM
[K+]o (Bonferroni-Dunn).
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Decrease in intracellular Na+ in
NKCC1/ astrocytes.
We previously observed that pharmacological inhibition of NKCC1 in rat
cortical astrocytes resulted in a decrease in basal levels of
[Na+]i by ~2 mM (33).
In the current study, the basal levels of [Na+]i were 19.0 ± 0.5 mM in
NKCC1+/+ astrocytes. In contrast,
[Na+]i was 16.9 ± 0.3 mM in
NKCC1/ astrocytes (P < 0.001; Fig.
5, A and B). In
response to high [K+]o, a compensatory
decrease in [Na+]i occurred in both
NKCC1+/+ and NKCC1/ astrocytes (Fig. 5,
A and B), which has been suggested to be attributable in part to activation of
Na+-K+-ATPase (19).
[Na+]i in NKCC1+/+
astrocytes recovered to basal levels after cells were returned to 5.8 mM [K+]o. However, in NKCC1/
astrocytes, [Na+]i remained significantly
lower than in NKCC1+/+ astrocytes. Thus the results suggest
that NKCC is important in maintaining intracellular Na+
under control conditions.
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Fig. 5.
Changes of intracellular Na+ concentration
([Na+]i) in NKCC1+/+ and
NKCC1/ astrocytes during high
[K+]o. A:
[Na+]i was determined in single cells
(NKCC1+/+ or NKCC1/) exposed to 5 min of
normal HEPES-MEM, followed by 10 min of 75 mM
[K+]o and 10 min of normal HEPES-MEM.
B: mean [Na+]i during 5 min of
normal HEPES-MEM, the final 5 min of 75 mM
[K+]o, and the final 5 min after return to
normal HEPES-MEM. Data are means ± SE. NKCC1+/+
cells: n = 2, 6 coverslips, 29 cells;
NKCC1/ cells: n = 3, 6 coverslips, 34 cells. * P < 0.001 vs. NKCC1+/+
(Mann-Whitney rank sum test).
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Lack of
high-[K+]o-induced swelling
in NKCC1/ astrocytes.
A significant decrease in intracellular Cl and
K+ uptake in NKCC1/ astrocytes under high
[K+]o implies that ablation of NKCC1 may
prevent astrocyte swelling. Thus in the following experiments, relative
volume changes were examined in both NKCC1+/+ and
NKCC1/ astrocytes. As shown in Fig.
6A, when NKCC1+/+
astrocytes were exposed to 75 mM [K+]o,
CSAr reached a maximum value of 1.13 ± 0.02. Inhibition of NKCC1 activity by 10 µM bumetanide abolished the
high-[K+]o-induced swelling. Ten micromolar
bumetanide had no significant effect on the basal level of
CSAr (Fig. 6A). In contrast, no cell swelling
was observed when NKCC1/ astrocytes were exposed to
high [K+]o (Fig. 6B). Ten
micromolar bumetanide did not affect CSAr in NKCC1/ astrocytes under either control or
high-[K+]o conditions (Fig. 6B).
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Fig. 6.
High-[K+]o-induced changes in astrocyte
swelling in single cells. Mean relative cross-section area
(CSAr) in single astrocytes was determined in
NKCC1+/+ (A) and NKCC1/ (B)
cells exposed to 5 min of normal HEPES-MEM, followed by 10 min of 75 mM
[K+]o, 10 min of normal HEPES-MEM, 20 min of
normal HEPES-MEM + 10 µM bumetanide, 10 min 75 mM
[K+]o MEM + 10 µM bumetanide, and 10 min of normal HEPES-MEM. Results are plotted as the mean
CSAr during the final 5 min during normal HEPES-MEM or
normal HEPES-MEM + 10 µM bumetanide exposure and as maximum
CSAr during 75 mM [K+]o MEM or 75 mM [K+]o MEM + 10 µM bumetanide
exposure. Data are means ± SE for NKCC1+/+
(n = 3) and NKCC1/ (n = 3) cells. * P < 0.001 vs. 5.8 mM
[K+]o; #P < 0.001 vs. 75 mM
[K+]o (Mann-Whitney rank sum test).
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Pharmacological inhibition and genetic ablation of NKCC1 have similar
effects on astrocyte swelling. This indicates that NKCC1 is essential
in astrocyte volume regulation. Therefore, we further investigated how
NKCC1/ astrocytes respond to osmotic stress. First, we
evaluated regulatory volume increase (RVI) in both NKCC1+/+
and NKCC1/ astrocytes. As shown in Fig.
7A, when NKCC1+/+
astrocytes were exposed to hypertonic HEPES-MEM (367 mosmol/kgH2O), CSAr decreased quickly and was
0.92 ± 0.01 at maximum cell shrinkage. CSAr in
NKCC1+/+ recovered by 63.0 ± 4.4% at 20 min of
hypertonic exposure (Fig. 7, A and B,
inset). As NKCC1+/+ astrocytes were returned to
an isotonic HEPES-MEM (310 mosmol/kgH2O), CSAr
was further increased, followed by a secondary regulatory volume
decrease (RVD; Fig. 7A). As shown in Fig. 7B,
CSAr in NKCC1/ astrocytes decreased in
response to the hypertonic challenge. At maximum cell shrinkage,
CSAr was 0.93 ± 0.01 (Fig. 7B). Moreover, RVI was impaired in NKCC1/ astrocytes (Fig.
7B). Overall, there was only 4.3 ± 1.7% RVI in
NKCC1/ astrocytes at 20 min of hypertonic
exposure (Fig. 7B, inset). In
addition, after returning to isotonic conditions,
NKCC1/ astrocytes remained shrunken at a
CSAr of 0.96 ± 0.01, which was significantly lower
than basal levels (P < 0.01). These results indicate
that the NKCC1 is essential for RVI in mouse cortical astrocytes.
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Fig. 7.
Regulatory volume increase (RVI) in NKCC1+/+
and NKCC1/ astrocytes. Mean CSAr in single
astrocytes was determined in NKCC1+/+ (A) and
NKCC1/ (B) cells exposed to isotonic (310 mosmol/kgH2O) HEPES-MEM (5 min), followed by 20-min
hypertonic (367 mosmol/kgH2O) exposure and 10-min isotonic
exposure. Data are means ± SE; n = 6. Inset in B: % regulation in response to
hypertonic exposure in either NKCC1+/+ or
NKCC1/ single cells. See RESULTS for
calculation of % regulation. Data are means ± SE for
NKCC1+/+ (n = 2) and NKCC1/
(n = 3) cells. * P < 0.001 vs.
NKCC1+/+ (Mann-Whitney rank sum test).
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We next examined RVD in NKCC1+/+ and NKCC1/
astrocytes in response to a hypotonic challenge. When
NKCC1+/+ astrocytes were exposed to hypotonic HEPES-MEM
(247 mosmol/kgH2O), CSAr increased and reached
a maximum level of 1.11 ± 0.02 (Fig. 8A). Cell volume subsequently
decreased, and a RVD of 48.5 ± 5.0% developed after 20 min of
hypotonic exposure (Fig. 8, A and B, inset). In NKCC1/ astrocytes, cell swelling was
not significantly different from that of NKCC1+/+
astrocytes (CSAr = 1.08 ± 0.01 in
NKCC1/ vs. 1.11 ± 0.02 in NKCC1+/+;
P > 0.05). Interestingly, the rate of RVD in
NKCC1/ astrocytes was faster (CSAr = 0.002%/min in NKCC1+/+ vs. 0.009%/min in
NKCC1/ astrocytes; P < 0.05). After 20 min of hypotonic exposure, an RVD of 86.6 ± 8.3% was obtained,
which was significantly higher than the 48.5% in NKCC1+/+
astrocytes (Fig. 8B, inset). This suggests that
other ion extrusion systems may be upregulated in
NKCC1/ astrocytes.
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Fig. 8.
Regulatory volume decrease (RVD) in NKCC1+/+
and NKCC1/ astrocytes. Mean CSAr in single
astrocytes was determined in NKCC1+/+ (A) or
NKCC1/ (B) cells exposed to isotonic (310 mosmol/kgH2O) HEPES-MEM (5 min), followed by 20-min
hypotonic (247 mosmol/kgH2O) exposure and 10-min isotonic
exposure. Data are means ± SE for NKCC1+/+
(n = 2) and NKCC1/ (n = 3) cells. Inset in B: % regulation in response
to hypotonic exposure in either NKCC1+/+ or
NKCC1/ single cells. See RESULTS for
calculation of % regulation. Data are means ± SE for
NKCC1+/+ (n = 2) and NKCC1/
(n = 3) cells. * P < 0.001 vs.
NKCC1+/+ (Mann-Whitney rank sum test).
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Decrease in
high-[K+]o-induced release
of D-[14C]Asp in NKCC1/
astrocytes.
We previously observed that NKCC1 activity in rat astrocytes
contributes to high-[K+]o-induced swelling
and glutamate release (33). If the
high-[K+]o-induced glutamate release is due
in part to swelling, we anticipated that preventing cell swelling by
inhibition of NKCC1 activity would reduce glutamate release. As shown
in Fig. 9A, in
NKCC1+/+ astrocytes there was a trace amount of
D-[14C]Asp release under 5.8 mM
[K+]o in the presence or absence of
bumetanide (data not shown in the latter case). A small increase in the
release of D-[14C]Asp occurred when cells
were exposed to 75 mM [K+]o (phase
I). The release was further developed with time and reached a peak
value after 20 min (phase II). On removal of the high-[K+]o medium, the release returned to
the resting level within 10 min. In the presence of 10 µM bumetanide,
the peak value of the D-[14C]Asp release
under 75 mM [K+]o was 0.65 ± 0.12%
(fractional release per minute; Fig. 9A). However, in the
absence of bumetanide, high [K+]o
induced substantially more D-[14C]Asp release
(P < 0.05; Fig. 9A). In summary, 10 µM
bumetanide has no effect on phase I of
D-[14C]Asp release; however, it blocked
~30% of the phase II release of
D-[14C]Asp under high
[K+]o (n = 12, P < 0.05; Fig. 9A and inset).
This is consistent with our finding in rat cortical astrocytes (Ref.
33, this issue).
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Fig. 9.
Effect of inhibition of NKCC1 on
high-[K+]o-mediated release of preloaded
14C-labeled D-Asp astrocytes. A: the effect of
bumetanide on the high-[K+]o-mediated
release of preloaded D-[14C]Asp.
NKCC1+/+ astrocytes loaded with
D-[14C]Asp were treated with 5.8 mM
[K+]o HEPES-MEM containing 10 µM Bum for 20 min followed by perfusion of 75 mM [K+]o + 10 µM Bum for 20 min. Cells were washed with 5.8 mM
[K+]o HEPES-MEM for 20 min and reexposed to
75 mM [K+]o HEPES-MEM for 20 min. Summarized
data are shown in inset. Data are mean ± SE % fractional release/min; n = 11-12.
* P < 0.05 vs. control in the absence of Bum
(Bonferroni-Dunn). B: a decrease in
high-[K+]o-mediated release of preloaded
D-[14C]Asp from NKCC1/
astrocytes. Cells were perfused with 5.8 mM
[K+]o HEPES-MEM for 15 min and then were
treated with 75 mM [K+]o for 20 min. Finally,
cells were reexposed to 5.8 mM [K+]o for 20 min. Inset, summarized data. Data are mean ± SE % fractional release/min; n = 11-12.
* P < 0.05 vs. NKCC1+/+ astrocytes
(Bonferroni-Dunn).
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The effect of bumetanide implies that NKCC1 may contribute to
D-[14C]Asp release under
high-[K+]o conditions. To investigate this
further, we examined D-[14C]Asp release in
NKCC1/ astrocytes. As shown in Fig. 9B,
similar to the results in NKCC1+/+ astrocytes, high
[K+]o triggered a small increase in
D-[14C]Asp release in NKCC1/
astrocytes (phase I). The peak release of
D-[14C]Asp in NKCC1/
astrocytes developed slowly and was smaller than in
NKCC1+/+ astrocytes (Fig. 9B). Compared with the
value in NKCC1+/+ astrocytes, the peak release was ~30%
less in NKCC1/ astrocytes (P < 0.05;
Fig. 9B and inset). This is in agreement with the
bumetanide-mediated effect in NKCC1+/+ astrocytes.
Together, the results suggest that NKCC1 contributes to the
high-[K+]o-induced glutamate release.
We hypothesized that high-[K+]o-induced
D-[14C]Asp release (phase II) is
mediated by volume-sensitive Cl channels because it has a
characteristic delayed onset rate and is partially blocked by
inhibition of NKCC1 activity. If this hypothesis is correct, blocking
of Cl channels should prevent
high-[K+]o-induced
D-[14C]Asp release. Therefore, we tested
whether blocking of Cl channels with 100 µM
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a
broad-spectrum Cl channel blocker, could reduce
D-[14C]Asp release. In NKCC1+/+
astrocytes, as shown in Fig.
10A, a trace level of
D-[14C]Asp release was found under control
conditions (0.16 ± 0.02%) and DIDS did not affect the basal
levels of release. In the absence of DIDS, the two-phase release of
D-[14C]Asp was the same as that described
above. In the presence of DIDS, there was no change in phase
I release; however, the peak release of phase II was
blocked by DIDS. In the DIDS-treated group, the peak release of
D-[14C]Asp during phase II was not
significantly different from basal levels (0.25 ± 0.04 vs.
0.16 ± 0.02% fractional release per minute, n = 6-12; Fig. 10A and inset). In
NKCC1/ astrocytes, DIDS did not significantly affect
the phase I release of D-[14C]Asp
(0.13 ± 0.01 vs. 0.17 ± 0.02% fractional release per
minute; P < 0.05). However, the remaining release
during phase II was abolished in the presence of DIDS (Fig.
10B and inset). Our data imply that phase
II of D-[14C]Asp release is largely
mediated by Cl channels.
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Fig. 10.
Effect of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)
on the high-[K+]o-mediated release of
preloaded D-[14C]Asp from cultured
NKCC1+/+ (A) and NKCC1/
(B) astrocytes. In the control experiments, cells were
perfused with 5.8 mM [K+]o for 15 min,
followed by 20 min perfusion with 75 mM
[K+]o, and were then reperfused with 5.8 mM
[K+]o for 20 min. In experiments to assess
the effects of DIDS, 100 µM DIDS was present during the entire
period. Cells were perfused with either 5.8 mM or 75 mM
[K+]o in the same order and for the same time
period as those in the control experiment. Insets,
summarized data. Data are means ± SE; n = 6-12. * P < 0.05 vs. 5.8 mM
[K+]o, #P < 0.05 vs. 75 mM
[K+]o (Bonferroni-Dunn).
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We have demonstrated here that pharmacological inhibition of NKCC1
function or genetic ablation of the NKCC1 protein abolished high-[K+]o-induced swelling, but it only
blocked 30% of D-[14C]Asp release. This
suggests that, in addition to swelling, other signals under
high-[K+]o conditions may also stimulate
Cl channels. Exposing cells to high
[K+]o leads to an increase in intracellular
Ca2+ in rat and mouse astrocytes (21, 28, 32,
42). Preventing Ca2+ influx by exposing cells to
Ca2+-free HEPES-MEM inhibited the Ca2+ rise in
rat astrocytes (28, 32). However, astrocytes in situ may
use the mechanisms other than voltage-dependent Ca2+
channels to regulate intracellular Ca2+ signals (Ref.
2; see DISCUSSION). To test whether the
Cl channels could be activated via a
Ca2+-mediated pathway and subsequently lead to the release
of D-[14C]Asp, we evaluated whether removal
of extracellular Ca2+ could block
high-[K+]o-induced
D-[14C]Asp release. As shown in Table
1, 75 mM [K+]o
triggered a sevenfold increase in the peak release of
D-[14C]Asp during phase II release
in NKCC1+/+ astrocytes. In NKCC1/
astrocytes, only a fourfold increase was observed (P < 0.05). Interestingly, in Ca2+-free HEPES-MEM, 75 mM
[K+]o failed to induce
D-[14C]Asp release in either
NKCC1+/+ or NKCC1/ astrocytes (Table 1).
The results further support our hypothesis that Cl
channels could be stimulated in a Ca2+-dependent fashion
under high [K+]o and result in the glutamate
release. Cell swelling-mediated stimulation of the channels only
partially contributes to the high-[K+]o-mediated glutamate release.
| |
DISCUSSION |
Role for NKCC1 in regulation of intracellular
Na+, K+,
and Cl in astrocytes.
Previous pharmacological studies suggested that NKCC1 plays a role in
maintenance of intracellular Na+ and Cl
(25, 26). Inhibition of NKCC1 by bumetanide in rat
hippocampal (25) or cortical (33) astrocytes
reduces the basal levels of intracellular Na+ by ~2 mM.
Consistent with these studies, we observed a reduction of 2.1 mM in
basal [Na+]i in NKCC1-deficient cortical
astrocytes. The collective results further support the notion that the
steady state of Na+ influx via NKCC1 is important in
maintenance of resting [Na+]i in astrocytes.
We have also examined whether deletion of NKCC1 activity would affect
basal levels of K+ and Cl influx in
NKCC1/ astrocytes. Unlike
[Na+]i, our data indicate that the basal
levels of K+ influx and accumulation of intracellular
36Cl were not significantly different in
NKCC1/ and NKCC1+/+ cortical astrocytes.
This implies that a role for NKCC1 in maintenance of resting
intracellular levels of K+ and Cl in
astrocytes is negligible. It is possible that the intracellular K+ concentration is maintained by mechanisms such as
Na+-K+-ATPase and K+ channels,
whereas intracellular Cl could be regulated by other ion
transport pathways, including Cl/HCO
exchangers or Cl channels.
NKCC1 has been suggested to play a role in K+ uptake in
astrocytes under elevated [K+]o (10,
11, 37). Inhibition of NKCC1 in astrocytes by furosemide or low
[Cl]o significantly reduced K+
uptake (39) and spontaneous epileptiform activity in rat
hippocampal slices (12). We found previously
(32) that the bumetanide-sensitive K+ influx
rate was significantly increased in rat cortical astrocytes at 75 mM
[K+]o. In the current report, we demonstrate
that 1) the total K+ influx rate is increased in
NKCC1+/+ cortical astrocytes in the presence of 75 mM
[K+]o; 2) inhibition of NKCC1
activity by bumetanide decreases the total K+ influx rate
by 20%; and 3) ablation of NKCC1 in cortical astrocytes causes a similar reduction in total K+ influx rate.
Moreover, the bumetanide-mediated effect on the total K+
influx rate is absent in NKCC1/ cortical astrocytes.
Together, the results of our pharmacological and knockout studies
clearly suggest that NKCC1 is important in K+ uptake in
astrocytes under high-[K+]o conditions.
However, other K+ uptake pathways also play a major role
because 80% of the total K+ uptake rate in astrocytes was
not sensitive to bumetanide or genetic ablation of NKCC1. In addition,
we found that either inhibition of NKCC1 with bumetanide or genetic
ablation of NKCC1 blocks 33% of the
high-[K+]o-mediated Cl
accumulation in cortical astrocytes. NKCC1 appears to have a greater
effect on Cl uptake than on K+ uptake in
astrocytes, consistent with the 2:1 stoichiometry for Cl
and K+. These results support a role for NKCC1 in mediating
the net gain of both K+ and Cl in astrocytes
as [K+]o is elevated.
Role for NKCC1 in regulation of astrocyte volume.
NKCC1 is important in cell volume regulation in a variety of mammalian
cell types (24, 26). Inhibition of NKCC1 by bumetanide leads to cell shrinkage under isotonic conditions in cells such as
retinal pigment epithelia, ventricular myocytes, and vascular endothelial cells (26). However, bumetanide has no effect
on the basal volume of C6 glioma and vascular smooth muscle cells (26). In our study, neither inhibition of NKCC1 activity
by bumetanide nor genetic ablation of NKCC1 affected the baseline volume in astrocytes. One possible explanation for the lack of an
effect is that NKCC1 is not essential for maintenance of cell volume in
mouse cortical astrocytes. Other mechanisms, such as the coupled
activities of Na+/H+ and
Cl/HCO exchangers, which are expressed in astrocytes (3), may be able to maintain cell volume
under control conditions.
NKCC1 is one of the principal volume regulatory transporters in RVI
(24, 26). However, contradictory results about RVI have
been observed in rat astrocytes (24). No RVI was observed when cultured rat cortical astrocytes were exposed to a hypertonic medium made with mannitol or NaCl (17, 23). However, rat
astrocytes shrank and exhibited RVI in hypertonic buffer made with
sucrose (5). The basis of the lack of RVI in rat
astrocytes in these studies is unclear, although it could be
attributable to intracellular ion concentrations such as
Cl [see review by O'Neill (24)]. To our
knowledge, RVI in mouse cortical astrocytes has not been extensively
examined before. In the present study, NKCC1+/+ mouse
cortical astrocytes underwent RVI within 10 min after a hypertonic
shrinkage. Cell volume was completely recovered as NKCC1+/+
cortical astrocytes were returned to isotonic conditions. In contrast,
RVI was absent in NKCC1/ cortical astrocytes. In
addition, on returning cells to the isotonic solutions, cell volume of
NKCC1/ cortical astrocytes failed to recover to basal
levels. This is the first study to firmly establish that hypertonic RVI
occurs in mouse cortical astrocytes and that the absence of NKCC1
completely impairs the RVI function in mouse cortical astrocytes.
In addition to RVI, NKCC1 is also involved in a secondary RVI when
cells are returned to isotonic conditions after hypotonic cell swelling
(post-RVD RVI; Ref. 8). NKCC1+/+
cortical astrocytes exhibited RVD and post-RVD RVI. Interestingly, in
NKCC1/ cortical astrocytes, post-RVD RVI occurred but
at significantly slower rates (Fig. 8B). This implies that
other cell volume regulatory ion transport mechanisms can compensate
for the loss of NKCC1 during this process. However, it is unclear why
these ion transport mechanisms are functional after isosmotic shrinkage
but not in hypertonic shrinkage. It could be that a loss of osmotically
active molecules, such as KCl, under isosmotic shrinkage removes a
factor(s) that otherwise inhibits these ion transport mechanisms
(24). Moreover, the RVD rate in NKCC1/
cortical astrocytes is significantly faster than that in
NKCC1+/+ cortical astrocytes. We speculate that some ion
efflux pathways, such as K+ and Cl channels
or K-Cl cotransporters, could be upregulated in NKCC1/
cortical astrocytes. Further investigation is needed to verify this speculation.
Role for NKCC1 in
high-[K+]o-induced
astrocyte swelling and glutamate release.
Several studies using pharmacological approaches suggested that NKCC1
plays a role in K+ uptake and astrocyte swelling in high
[K+]o (37). In rat cortical
astrocytes, we reported that inhibition of NKCC1 with bumetanide
abolished the high-[K+]o-induced swelling
(33). A similar observation was made in choroid
plexus cells (40). In the current report, we conclude that
activation of NKCC1 is responsible for
high-[K+]o-induced swelling in mouse cortical
astrocytes. Blocking of NKCC1 activity or ablation of NKCC1 abolishes
high-[K+]o-induced swelling. We believe that
NKCC1 leads to cell swelling via excessive influx of Na+,
K+, and Cl, with accompanying H2O
under high [K+]o. This view is supported by
the following findings. 1) NKCC1 activity is significantly
stimulated in NKCC1+/+ astrocytes. 2) Either
inhibition of NKCC1 with bumetanide or ablation of NKCC1 significantly
reduces intracellular Cl content under high
[K+]o. Na+ entering the cell via
NKCC1 is then subsequently pumped out of the cell via
Na+-K+-ATPase. This speculation is based on our
finding that inhibition of Na+-K+-ATPase with 1 mM ouabain prevents the reduction of intracellular Na+ in
rat astrocytes under high [K+]o (data not
shown). A voltage-dependent stimulation of
Na+-K+-ATPase has been established
(7).
Transport mechanisms underlying the reduction in
[Na+]i under
high-[K+]o conditions remain unknown.
Longuemare et al. (19) recently suggested that an increase
in Na+-K+-ATPase activity, which is stimulated
by high [K+]o (16, 38), may
contribute to reduced [Na+]i. However,
because [Na+]i is normally a rate-limiting
factor for Na+-K+-ATPase, it is likely that
changes in Na+ entry mechanisms also contribute to the
reduction in [Na+]i. For example, astrocytes
express a plasma membrane Na+ conductance that has been
suggested to provide an important leak pathway for the
Na+-K+-ATPase (30) and a decrease
in inwardly directed Na+ currents has been observed under
conditions of high [K+]o (36).
Thus the reduced set point for [Na+]i during
high [K+]o could be the result of a balance
between the decrease in the inwardly directed Na+ current
and stimulation of intrinsic Na+-K+-ATPase activity.
High [K+]o causes glutamate release from
astrocytes in response to high-[K+]o-induced
swelling (1, 18). It is generally believed that astrocytes
reduce cell volume by the efflux of chloride, glutamate, and other
anions through VSOACs (1, 18, 27). However, the channels
responsible for VSOAC currents in astrocytes remain to be identified
(31). In addition to the swelling stimulus,
Cl channels are regulated by a variety of signal
transduction pathways, such as those involving
Ca2+/calmodulin, cAMP, and protein kinase C (1,
14).
In the current study, high [K+]o triggers the
release of D-[14C]Asp from both
NKCC1+/+ and NKCC1/ astrocytes. The
stilbene derivative DIDS abolished the
high-[K+]o-induced release of
D-[14C]Asp from both NKCC1+/+ and
NKCC1/ astrocytes. This implies that the release of
D-[14C]Asp is largely through VSOACs. The
slow onset kinetics of the D-[14C]Asp release
in our study (33) further supports this view. Interestingly, elimination of the
high-[K+]o-mediated swelling by bumetanide or
ablation of NKCC1 only blocks ~30% of the
D-[14C]Asp release. This suggests that under
high-[K+]o conditions, the VSOACs can be
activated by other mechanisms in addition to swelling. VSOAC
activation in astrocytes is reportedly dependent on
calmodulin and intracellular Ca2+ (22). The
Ca2+ chelator
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM and the calmodulin antagonist trifluoperazine significantly suppress [K+]o-induced taurine
release in rat astrocytes (22). Intracellular Ca2+ rise has been observed in both rat and mouse
astrocytes under high-[K+]o conditions
(2, 21, 28, 32). In cultured astrocytes, the
high-[K+]o-mediated Ca2+ influx
was blocked by voltage-operated Ca2+ channel inhibitors
(21, 32, 42). However, the depolarization-induced intracellular Ca2+ concentration
([Ca2+]i) increases in astrocytes in situ are
attributed to metabotropic glutamate receptor-mediated Ca2+
release from intracellular Ca2+ stores (2). In
the current study, the release of D-[14C]Asp
from astrocytes in the absence of cell swelling is abolished by removal
of extracellular Ca2+. Therefore, it is possible that a
rise in [Ca2+]i activates VSOACs and leads to
glutamate release. However, Ca2+-free medium plus
50-100 µM EGTA only moderately inhibits the [K+]o-induced taurine release
(22) and has no effect on glutamate release in rat
astrocytes (28). This is different from our observations on the release of D-[14C]Asp in mouse
astrocytes. The cause of this discrepancy is unclear. It could be due
to species differences (rat vs. mouse) or different cell culture
conditions (differentiated vs. undifferentiated astrocytes).
In summary, with the use of both pharmacological and transgenic
knockout approaches, we report here that NKCC1 contributes to baseline
[Na+]i in mouse cortical astrocytes. Ablation
of NKCC1 leads to complete impairment of RVI in mouse cortical
astrocytes. High-[K+]o-induced swelling and
accumulation of intracellular Cl are absent in
NKCC1/ astrocytes. In addition, the release of
D-[14C]Asp is inhibited by 30% in
NKCC1
cotransporter-null mice exhibit absence of swelling and decrease in
EAA release
TOP
ABSTRACT
INTRODUCTION
