Departments of 1 Neurological Surgery and 2 Physiology, University of Wisconsin Medical School, Madison, Wisconsin 53792
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ABSTRACT |
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We hypothesized that high
extracellular K+ concentration
([K+]o)-mediated stimulation of
Na+-K+-Cl cotransporter isoform 1 (NKCC1) may result in a net gain of K+ and Cl
and thus lead to high-[K+]o-induced swelling
and glutamate release. In the current study, relative cell volume
changes were determined in astrocytes. Under 75 mM
[K+]o, astrocytes swelled by 20.2 ± 4.9%. This high-[K+]o-mediated swelling was
abolished by the NKCC1 inhibitor bumetanide (10 µM, 1.0 ± 3.1%; P < 0.05). Intracellular
36Cl
accumulation was increased from a
control value of 0.39 ± 0.06 to 0.68 ± 0.05 µmol/mg
protein in response to 75 mM [K+]o. This
increase was significantly reduced by bumetanide (P < 0.05). Basal intracellular Na+ concentration
([Na+]i) was reduced from 19.1 ± 0.8 to
16.8 ± 1.9 mM by bumetanide (P < 0.05).
[Na+]i decreased to 8.4 ± 1.0 mM under
75 mM [K+]o and was further reduced to
5.2 ± 1.7 mM by bumetanide. In addition, the recovery rate of
[Na+]i on return to 5.8 mM
[K+]o was decreased by 40% in the presence
of bumetanide (P < 0.05). Bumetanide inhibited
high-[K+]o-induced 14C-labeled
D-aspartate release by ~50% (P < 0.05).
These results suggest that NKCC1 contributes to
high-[K+]o-induced astrocyte swelling and
glutamate release.
cell swelling; high potassium ion concentration, cultured astrocytes; glutamate release; bumetanide; intracellular chloride
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INTRODUCTION |
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THE
NA+-k+-cl cotransporters
(NKCCs) are membrane proteins that mediate the coupled, electrically
neutral movement of Na+, K+, and
Cl
across the membrane of many cell types
(25). NKCC isoform 1 (NKCC1) is important for
accumulation of Cl
in neurons, astrocytes, and
oligodendrocytes (17, 25, 34). High intracellular
Cl
concentration ([Cl
]i)
makes possible the depolarizing action of GABA and glycine that opens
Cl
channels (1). The inhibition of
spontaneous epileptiform activity in rat hippocampal slices by
furosemide has been attributed to the blockade of K+ uptake
mediated by NKCC1 in hippocampal glial cells (13, 14). In
the recent study of Yan et al. (41), inhibition of NKCC1 by a more potent inhibitor, bumetanide, resulted in a significant reduction of edema and infarct volume in rat focal cerebral ischemia. In the current study, we investigated the role of NKCC1 in astrocyte swelling and glutamate release induced by elevated extracellular K+ concentration ([K+]o) to
further understand the contribution of glial NKCC1 in ischemic cerebral
damage. Both high [K+]o and glutamate release
are associated with ischemic cerebral damage (28).
High-[K+]o-induced astrocyte swelling has
been observed in both brain slices and cultured astrocytes. However,
the cellular mechanisms underlying
high-[K+]o-induced astrocyte swelling have
not been completely defined. NKCC has been implicated in
high-[K+]o-induced swelling in several cell
types, and high-[K+]o-induced swelling was
abolished either by the cotransporter inhibitor bumetanide or removal
of extracellular Cl or Na+ (39).
Furosemide blocked 70% of the
high-[K+]o-induced increase in intracellular
K+ content observed in cultured mouse cortical astrocytes
(36). In a recent study (32), we found that
the activity of NKCC1 in cultured rat cortical astrocytes was
significantly stimulated under 75 mM [K+]o.
This led us to hypothesize that this
high-[K+]o-induced stimulation of
cotransporter activity may cause Na+, K+, and
Cl
influx and result in swelling in astrocytes.
One consequence of high-[K+]o-induced swelling is the stimulation of excitatory amino acid (EAA) release from astrocytes (18). The release of EAA under high-[K+]o conditions could be mediated by volume-sensitive organic anion channels (VSOACs; Refs. 2, 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 (26). We hypothesized that NKCC1 may play a role in a swelling-dependent release of EAA under high-[K+]o conditions.
We report here the effects of inhibition of NKCC1 activity on cell
swelling, intracellular Cl accumulation, changes of
intracellular Na+ concentration
([Na+]i), and release of
14C-labeled D-Asp in cultured cortical
astrocytes under high [K+]o.
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MATERIALS AND METHODS |
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Materials. Bumetanide, digitonin, Triton X-100, monensin, gramicidin, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) 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).
Primary culture of rat cortical astrocytes. Dissociated cortical astrocyte cultures were established as described previously (32). Cerebral cortices were removed from 1-day-old rats (Sprague-Dawley). The cortices were incubated in a trypsin solution for 25 min at 37°C. The tissue was then mechanically triturated and filtrated through nylon meshes (70 µm). 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 then 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 (11, 35). Experiments were routinely performed on cultures treated with DBcAMP for 7 days. More than 95% of cells in culture yielded by this preparation were astrocytes (32).
Measurement of relative cell volume changes in single cell.
Relative cell volume changes were determined in cultured single
astrocytes on coverslips with video-enhanced differential interference
contrast (DIC) microscopy (7, 10, 39). Astrocytes were
cultured on collagen-coated coverslips and placed in a home-made bath
chamber mounted on the stage of a Nikon TE 300 inverted epifluorescence microscope. The bath chamber was perfused continuously at room temperature at 1.0 ml/min, and the dead space between the perfusion pump and the bath chamber was 1.15 ml. Astrocytes were equilibrated with an isotonic HEPES-buffered minimal essential medium (MEM; 312 mosmol/kgH2O) for 15 min. The concentrations of
components in HEPES-MEM were (mM) 140 NaCl, 5.36 KCl, 0.81 MgSO4, 1.27 CaCl2, 0.44 KH2PO4, 0.33 Na2HPO4,
0.4 NaHCO3, 5.55 glucose, and 20 HEPES. Astrocytes were
perfused sequentially with HEPES-MEM (10 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 (1.4 NA, 0.21 WD). Cell images were recorded every
minute as 16-bit TIF files with a Princeton Instruments MicroMax
charge-coupled device (CCD) camera (model 1300 YHS; Roper Scientific,
Trenton, NJ). For each image, the cell body was traced three
separate times with a mouse and the mean cross-sectional area (CSA) of
the cell body was calculated with MetaMorph image-processing software
(Universal Imaging, Downingtown, PA). The control CSA values were
obtained when cells were exposed to HEPES-MEM only. Relative volume
changes were calculated as CSAr = experimental
CSA control CSA. On the same image, a peripheral astrocytic
process that was far distant from the cell body was selected and the
mean cross-sectional distance of the process (CSD) and relative CSD
(CSDr) were determined in a manner similar to the CSA
measurement. After each experiment, a calibration curve was
constructed by measuring relative cell volume changes in response to
HEPES-MEM calibration buffers in which salt concentrations 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 0-30 min in
HEPES-MEM containing 5.8 mM [K+]o and
36Cl (0.4 µCi/ml). 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
1-13 min. Thus 145 mM Cl
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. [Na+]i was measured with the fluorescent dye SBFI-AM as described by Rose and Ransom (23). Cultured astrocytes grown on coverslips were loaded with 10 µM SBFI-AM at room temperature for 90 min in HEPES-MEM containing 0.1% pluronic acid. The coverslips were placed in an open-bath imaging chamber (volume = 40 µl; series 20, Warner Instruments, Hamden, CT) containing HEPES-MEM at ambient temperature. The chamber was mounted on the stage of a Nikon TE 300 inverted epifluorescence microscope, and the astrocytes were visualized with a ×40 Super Fluor oil-immersion objective lens (1.3 NA, 0.22 WD). The cells were excited every 10 or 60 s at 345 and 385 nm, and the emission florescences at 510 nm were recorded. In some experiments, the data were processed with a nonparametric digital filter (Peakfit; SPSS, Chicago, IL) to improve the signal-to-noise ratio. Images were collected as 16-bit TIF files with a Princeton Instruments MicroMax CCD camera and analyzed with MetaFluor image-processing software. Cytoplasmic regions with minimum punctuate fluorescence staining were chosen for measurement of fluorescent intensity changes of SBFI. 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 and correction of autofluorescence of bumetanide. To determine the percentage of SBFI dye in cytoplasm, astrocytes were clamped at an extracellular Na+ concentration ([Na+]o) of 20 mM with a calibration solution (see below). A decrease in SBFI fluorescence at 340 nm was recorded after plasma membrane was permeabilized by 20 µM digitonin. A further release of the dye from organelles was induced subsequently by 1% Triton X-100.
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 under different experimental conditions. Absolute [Na+]i was determined for each cell by standardization of the SBFI fluorescence ratio with calibration solutions containing 0, 10, 20, or 30 mM [Na+]o plus monensin (10 µM) and gramicidin (3 µM) to equilibrate [Na+]i and [Na+]o.D-[14C]Asp release measurement. Aspartate release was measured as described by Rutledge and Kimelberg (27). Astrocytes grown on chamber slides (Fisher, Pittsburgh, PA) were incubated overnight in 1 ml of complete EMEM containing 2 µCi/ml of D-[14C]Asp (specific activity of 55 mCi/mmol). Radiolabeled D-[14C]Asp is used as a nonmetabolizable marker for the intracellular glutamate and aspartate pool (27). Both of these amino acids are transported by the same glutamate carrier proteins (5, 8). A perfusion chamber was formed by a special lid that contains influx and efflux tubings in the chamber. The perfusing rate 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 from time t to the end of the experiment (27), and Cremain is the cpm value left in the cells at the end of the experiment.
D-[14C]Asp uptake assay. The assay was performed according to a method described by Kimelberg et al. (18). Cells were refed with EMEM on the evening before the uptake assay. On the following day, cells were washed four times (1 ml each) with HEPES-MEM to remove growth medium. The cells were preincubated with HEPES-MEM containing 5.8 mM [K+]o in the presence or absence of 10 µM bumetanide for 20 min at 37°C. In the high-[K+]o study, the cells were preincubated with 75 mM [K+]o HEPES-MEM in the presence or absence of 10 µM bumetanide for 20 min at 37°C. The buffer was then rapidly removed, and 0.5 ml of the same medium containing D-[14C]Asp (0.2 µCi/0.5 ml in each well) + unlabeled D-Asp (100 µM) was added to each well. The cells were then incubated for 1, 2, or 3 min. The uptake assay was terminated by three washes with ice-cold 0.1 M MgCl2 (1 ml each). Radioactivity of cellular extract in 1% SDS was analyzed by liquid scintillation counting. Uptake rate was determined by analyzing the slope of the uptake over time.
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RESULTS |
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Inhibition of NKCC1 abolishes
high-[K+]o-induced
astrocyte swelling.
To validate DIC microscopy for the determination of CSAr
and CSDr in single cells, we measured the changes in
CSAr in cultured astrocytes perfused with isotonic and
hypotonic HEPES-MEM buffers (Fig.
1A). CSAr
responded linearly (r = 0.999) as the osmolality of the
perfusion buffer decreased. CSAr returned to basal levels when cells were incubated in the isotonic buffer (data not shown). This
suggests that changes of CSAr in astrocytes measured by DIC microscopy can be used as an estimate of cellular volume changes, as
reported by others (7, 10, 39).
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Sustained elevation of cotransporter activity in presence of
high-[K+]o-induced
astrocyte swelling.
Cell swelling inhibits NKCC1 activity in many cell types
(25). To investigate whether
high-[K+]o-induced cell swelling under 75 mM
[K+]o affects cotransporter activity,
cotransporter activity was measured when astrocytes were exposed to
either 5.8 or 75 mM [K+]o for 1-12 min.
As shown in Fig. 3, under control
conditions, cotransporter activity was 34.3 ± 5.1 nmol/mg
protein × min (n = 5), and it did not change over
the entire time course. In contrast, the activity of NKCC1 increased to
the maximum level of 143.6 ± 38.1 nmol/mg protein × min
(n = 5; P < 0.05) after 1 min of exposure to high [K+]o. It decreased
gradually and reduced to 61.1 ± 9.0 nmol/mg protein × min
(n = 5, P < 0.05) at 4 min.
Cotransporter activity remained elevated during the rest of the
exposure time (P < 0.05). The results imply that
cotransporter activity remained stimulated under high
[K+]o despite astrocyte swelling.
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High-[K+]o-mediated
increase in Cl uptake is abolished by blocking of NKCC1.
To further examine whether NKCC1 contributes to
high-[K+]o-induced cell swelling, we measured
changes of intracellular Cl
content. Cells were
preequilibrated in HEPES-MEM with 36Cl (0.4 µCi/ml) for
0-30 min. A steady-state level of intracellular 36Cl
was obtained by only 4-min incubation
and maintained during the 30 min-equilibration (data not shown). Thus,
in the rest of the study, a 30-min preincubation was performed. After a
30-min equilibration with 36Cl (0.4 µCi/ml), the time
course of intracellular Cl
content changes was measured
under control or high-[K+]o conditions.
Intracellular 36Cl
content was 0.49 ± 0.04 µmol/mg protein (n = 8) after exposure of cells
to 5.8 mM [K+]o for 1 min (Fig.
4B). After 13 min of
incubation in 5.8 mM [K+]o, the intracellular
36Cl content was maintained at 0.39 ± 0.06 µmol/mg
protein (P > 0.05). However, at 1 min of incubation of
cells with 75 mM [K+]o,
36Cl
content increased to 0.67 ± 0.05 µmol/mg protein (P < 0.05). It reached a peak value
of 0.78 ± 0.07 µmol/mg protein (P < 0.05, n = 8) after 4 min of incubation with 75 mM
[K+]o. A sustained elevation of intracellular
36Cl
content was detected during the 13-min
incubation period. In contrast, in the presence of 10 µM bumetanide,
the high-[K+]o-induced intracellular
36Cl rise was significantly inhibited. The values of
intracellular 36Cl
content were significantly
less than in non-bumetanide-treated cells at 1, 4, 6, 10, or 13 min
(P < 0.05; n = 5). A small
36Cl increase was observed at 10-13 min. The
nature of the bumetanide-insensitive Cl
influx is
unclear. It could reflect Cl
influx via Cl
channels or reversal of outward K-Cl cotransport (21).
These results support a view that NKCC1 contributes to Cl
accumulation under high-[K+]o conditions and
may lead to cell swelling.
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[Na+]i measurement.
We next investigated whether stimulation of NKCC1 activity under high
[K+]o could affect
[Na+]i. First, intracellular localization of
SBFI dye in astrocytes was examined. Addition of 20 µM digitonin to
permeabilize the plasma membrane resulted in a decrease of the 340-nm
SBFI fluorescence signal (Na+-insensitive fluorescence) by
64.3 ± 6.9% (n = 1, 4 coverslips, 32 cells; Fig.
5A). This reflects a loss of
SBFI from the cytoplasm. When the detergent Triton X-100 (1%) was
subsequently added to cells, the 340-nm SBFI fluorescence signal
decreased to near zero. This is presumably the result of release of the
remaining SBFI dye from intracellular organelles. Typically, we
selected a region of the cell cytoplasm that was as free of punctuate
SBFI fluorescence as possible. Thus the SBFI fluorescence signals (340- to 380-nm ratios) measured in this study largely represent changes of
Na+ in the cytoplasm of astrocytes. This pattern of
intracellular SBFI dye localization in rat cortical astrocytes has been
reported in rat hippocampal astrocytes (23).
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Inhibition of NKCC1 attenuates
high-[K+]o-mediated
preloaded D-[14C]Asp release.
To investigate whether cotransporter-mediated cell swelling is involved
in glutamate release, we examined whether blocking cotransporter
activity could inhibit aspartate release. As shown in Fig.
8A, under control conditions a
trace level of release of preloaded
D-[14C]Asp was detected. This is consistent
with a previous report (27). After 6 min of exposure to
high [K+]o, an increase in
D-[14C]Asp release occurred
(n = 4). The release developed progressively (Fig.
8A) and reached 1.5% fractional release of
D-[14C]Asp after 22 min (n = 4) in high [K+]o. On removal of
high-[K+]o medium,
D-[14C]Asp release returned to a resting
level within 12 min (n = 4; Fig. 8A). Figure
8B shows that exposing cells to 10 µM bumetanide did not
significantly affect basal D-[14C]Asp release
in 5.8 mM [K+]o. Moreover, in the presence of
10 µM bumetanide and 75 mM [K+]o, the peak
value of D-[14C]Asp release was only about
one-third of that in the absence of bumetanide (Fig. 8B).
This inhibition of aspartate release could be reversed by removal of 10 µM bumetanide (Fig. 8B). Figure 8C shows that
the average peak value of the fractional release under high
[K+]o was 1.52 ± 0.17%
(n = 4). In contrast, the fractional release was
0.68 ± 0.13% (n = 4) in the presence of
bumetanide (P < 0.05).
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DISCUSSION |
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Role of NKCC1 in high-[K+]o-induced astrocyte swelling. Astrocytes are thought to have a primary role in the clearance of K+ from the extracellular space in physiological and pathological conditions (38). Recently, Yan et al. (40) reported that NKCC1 protein is expressed in astrocytes in rat cortex, cerebellum, and hippocampus. An abundant level of NKCC1 protein is also detected in perivascular astrocytes (40). NKCC1 has been suggested to play a role in K+ uptake in cortical (36) or hippocampal (14) astrocytes. The cotransporter activity in astrocytes is significantly stimulated in response to high [K+]o in a Ca2+-dependent manner (32). Stimulation of NKCC1 under high [K+]o may result in cell swelling via a net increase of intracellular KCl and accompanying water. Cell swelling in rat hippocampal slices was detected with changes in intrinsic optical signals, and inhibition of NKCC1 increases extracellular space and blocks synchronized burst discharges (12).
The current study directly establishes that NKCC1 is responsible for a high-[K+]o-mediated swelling in cultured rat cortical astrocytes. This conclusion is based on the following findings. 1) Astrocyte swelling (cell body and process) occurs within 3-4 min in response to high [K+]o and reaches a peak level by 8 min. 2) Cotransporter activity is significantly stimulated when astrocytes are exposed to high [K+]o, and stimulation of the cotransporter precedes the swelling. 3) A high-[K+]o-mediated ClRole of NKCC1 in
high-[K+]o-mediated
intracellular Cl accumulation.
Astrocytes have a higher [Cl
]i level than
predicted by passive distribution of the ion (9). NKCC1 is
important for an accumulation of intracellular Cl
in
neurons (34), so it is conceivable that NKCC1 may also
contribute to intracellular Cl
accumulation in
astrocytes. In the current study, inhibition of NKCC1 with 10 µM
bumetanide did not significantly affect basal levels of intracellular
36Cl. This implies that basal
[Cl
]i can be maintained by other
Cl
influx mechanisms. When astrocytes were exposed to
high [K+]o intracellular 36Cl
content was increased by ~70%, and this increase in 36Cl
level was significantly reduced by the cotransporter inhibitor bumetanide. An increase in Cl
influx, accompanied by
K+ uptake, has been found in cultured mouse cortical
astrocytes under high [K+]o (37,
38). An increase of Cl
permeability by activation
of voltage-dependent Cl
channels has been proposed as a
major mechanism for the Cl
influx (37). Our
current study suggests that the NKCC1-mediated Cl
influx
also contributes to intracellular Cl
accumulation, and
this may subsequently lead to astrocyte swelling under high
[K+]o.
Role of NKCC1 in [Na+]i. NKCC1 has been suggested to provide Na+ for Na+-K+-ATPase function in the so-called "transmembrane Na+ cycle" (36). Rose and Ransom (23) reported that application of 50 µM bumetanide to cultured hippocampal astrocytes caused a slow and reversible decrease in [Na+]i by <2 mM (14% of baseline). In our study, treatment of cortical astrocytes with 10 µM bumetanide caused a decrease in [Na+]i by 2.3 mM (12% of baseline), suggesting a role of the cotransporter in maintenance of a resting [Na+]i in cultured rat cortical astrocytes.
Consistent with reports by others (19, 38), we observed that [Na+]i in astrocytes significantly decreases when cells are exposed to high [K+]o. The decrease in [Na+]i can be partially attributed to the experimental decrease in [Na+]o (140 to 75 mM) that accompanies the high-[K+]o treatment. In addition, a stimulation of Na+-K+-ATPase by elevated external K+ may also play a role (36). In the current study, the decrease of [Na+]i was prevented by blocking Na+-K+-ATPase with 1 mM ouabain (data not shown). Moreover, inhibition of NKCC1 by bumetanide led to a further loss of [Na+]i by 3.2 mM under high [K+]o. However, the net decrease in [Na+]i was not changed in control vs. bumetanide-treated cells (Fig. 6B), which suggests that the Na+ efflux via Na+-K+-ATPase was not affected by bumetanide treatment. This implies that it is not NKCC1, but other Na+ influx mechanisms such as Na+ channel, Na+/H+ exchanger, or Na+/HCORole of NKCC1 in
high-[K+]o-induced
D-[14C]Asp release.
In the current study, high-[K+]o-induced
D-[14C]Asp release was detected in cultured
rat cortical astrocytes. High [K+]o could
induce glutamate release from astrocytes via nonvesicular mechanisms,
either a reversal of glutamate transporter or VSOACs (2,
27). Under physiological conditions the largest factor influencing the extracellular/intracellular glutamate equilibrium potential and the direction of the glutamate transporter is the Na+ gradient (19). Thus, if there were no
compensation of intracellular Na+, an increased
[K+]o plus decreased
[Na+]o would reduce the Na+
gradient and extracellular/intracellular glutamate equilibrium potential. This would result in the release of glutamate via reversal of the transporter (4). However, in the current study, the transmembrane Na+ gradient increased from 8.7 ± 1.7 to 12.9 ± 5.2 in response to the decrease in
[Na+]o during 75 mM
[K+]o incubation. Therefore, as discussed by
Longuemare et al. (19), it is unlikely that the reversal
of the glutamate transporter is a primary cause of the increase in
D-[14C]Asp release under 75 mM
[K+]o conditions. However, a fast-developing
small release of D-[14C]Asp, before the
delayed peak release (shown in Fig. 8), has been suggested to be
mediated via a reversal of the glutamate transporters (26,
27). This small release was enhanced by an increase of
intracellular Na+ with 1 mM ouabain and inhibited by the
glutamate transporter inhibitor threo-hydroxy -aspartic acid
(26, 27). A low [Na+]o at 75 mM
[K+]o and the low
[Na+]o-induced depolarization in astrocytes
(22) may facilitate the reversal of the glutamate transporters.
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ACKNOWLEDGEMENTS |
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The authors thank Dr. James Franklin for the use of the Nikon epifluorescence microscope in his laboratory. We also thank Dr. Robert Haworth for helpful discussion and comments.
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FOOTNOTES |
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This work was supported in part by a Scientist Development Grant from the National Center Affiliate of American Heart Association (no. 9630189N), National Institute of Neurological Disorders and Stroke Grant R01-NS-38118, and National Science Foundation CAREER Award IBN9981826 to D. Sun.
Address for reprint requests and other correspondence: D. Sun, Dept. of Neurological Surgery, Univ. of Wisconsin Medical School, H4/332 Clinical Sciences Center, 600 Highland Ave., Madison, WI 53792 (E-mail: sun{at}neurosurg.wisc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpcell.00478.2001
Received 9 October 2001; accepted in final form 7 December 2001.
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