Departments of 1 Neurological Surgery, 2 Physiology, and 3 Surgery, School of Medicine, University of Wisconsin, Madison, Wisconsin 53792
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ABSTRACT |
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In this study, we examined the
Na+-K+-Cl cotransporter activity
and expression in rat cortical astrocyte differentiation. Astrocyte differentiation was induced by dibutyryl cAMP (DBcAMP, 0.25 mM) for
7 days, and cells changed from a polygonal to process-bearing morphology. Basal activity of the cotransporter was significantly increased in DBcAMP-treated astrocytes (P < 0.05).
Expression of an ~161-kDa cotransporter protein was increased by 91%
in the DBcAMP-treated astrocytes. Moreover, the specific
[3H]bumetanide binding was increased by 67% in the
DBcAMP-treated astrocytes. Inhibition of protein synthesis by
cyclohexamide (2-3 µg/ml) significantly attenuated the
DBcAMP-mediated upregulation of the cotransporter activity and
expression. The Na+-K+-Cl
cotransporter in astrocytes has been suggested to play a role in
K+ uptake. In 75 mM extracellular K+
concentration, the cotransporter-mediated K+ influx was
stimulated by 147% in nontreated cells and 79% in DBcAMP-treated
cells (P < 0.05). To study whether this high
K+-induced stimulation of the cotransporter is attributed
to membrane depolarization and Ca2+ influx, the role of the
L-type voltage-dependent Ca2+ channel was investigated. The
high-K+-mediated stimulation of the cotransporter activity
was abolished in the presence of either 0.5 or 1.0 µM of the L-type
channel blocker nifedipine or Ca2+-free HEPES buffer. A
rise in intracellular free Ca2+ in astrocytes was observed
in high K+. These results provide the first evidence that
the Na+-K+-Cl
cotransporter
protein expression can be regulated selectively when intracellular cAMP
is elevated. The study also demonstrates that the cotransporter in
astrocytes is stimulated by high K+ in a
Ca2+-dependent manner.
potassium uptake; bumetanide; L-type calcium channel; intracellular calcium; rat cortical astrocytes; dibutyryl-cAMP
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INTRODUCTION |
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UNDER PHYSIOLOGICAL
CONDITIONS, Na+-K+-Cl
cotransporter in most cells transports Na+, K+,
and Cl
into cells. This is driven by both favorable
inward Na+ and Cl
chemical gradients
(8, 28). Thus the cotransporter plays a crucial role in
vectorial salt transport in epithelial cells and cell volume regulation
in both epithelial and nonepithelial cells (8, 28).
Despite studies of the Na+-K+-Cl
cotransporters in many cell types, their function and regulation in
astrocytes have not been well understood. The
Na+-K+-Cl
cotransporter has been
shown to contribute to a baseline intracellular Na+
concentration ([Na+]i) in rat hippocampal
astrocytes under physiological conditions (32) and active
accumulation of Cl
in both mouse cortical astrocytes and
rat hippocampal astrocytes (1, 44). It has also been
proposed that inward transport of Na+ via the cotransporter
provides Na+ influx for
Na+-K+-ATPase function (43). The
involvement of the Na+-K+-Cl
cotransporter in astrocyte K+ uptake has been suggested by
several studies (17, 26, 45). Mouse astrocytes increased
their K+ content significantly when cells were exposed to
12 mM [K+]o (43). Furosemide, an
inhibitor of the Na+-K+-Cl
cotransporter, reduced the total net K+ uptake by 38%,
suggesting that stimulation of the
Na+-K+-Cl
cotransporter
contributes to K+ uptake (43).
Depending on the species and tissue types, the
Na+-K+-Cl cotransporter is
regulated by various hormonal factors and intracellular messengers,
such as cAMP, cGMP, and Ca2+/calmodulin (7, 10,
15), and by cell shrinkage. Generally, the
Na+-K+-Cl
cotransporter is
stimulated by cell shrinkage and is inhibited by cell swelling
(29). Interestingly, both hypertonic cell shrinkage and
hypotonic cell swelling have been shown to activate the cotransporter activity in rat astrocytes (18, 26, 41) and C6
glioma cells (2, 25). Cellular mechanisms underlying these
osmolarity-mediated effects on the cotransporter in astrocytes are not
yet clear. It appears that the swelling-activated
Na+-K+-Cl
cotransporter activity
in C6 cells is Ca2+/calmodulin dependent and
also is inhibited by the protein kinase inhibitors staurosporine and
polymyxin B (25). These studies imply that the
Na+-K+-Cl
cotransporter in
astroglial cells is regulated by complex intracellular signaling pathways.
The present study was aimed at investigating the cellular mechanisms
underlying cAMP- and high-[K+]o-mediated
stimulation of the Na+-K+-Cl
cotransporter in primary astrocyte cultures. We report here that the
cotransporter protein expression is upregulated in astrocytes treated
with dibutyryl cAMP (DBcAMP). Moreover, the cotransporter activity in
astrocytes in 75 mM [K+]o is significantly
stimulated in a Ca2+-dependent manner.
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MATERIALS AND METHODS |
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Materials. Bumetanide and antineurofilament 200 monoclonal antibody were purchased from Sigma (St. Louis, MO). Eagle's modified essential medium (MEM) and Hanks' balanced salt solution were from Mediatech Cellgro (Herndon, VA). FBS 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). [butyl-3H]bumetanide (15 Ci/mmol) was custom synthesized and HPLC purified by American Radiolabeled Chemicals (St. Louis, MO). Mouse anti-GLT-1 monoclonal IgG was from Chemicon International (Temecula, CA). Nifedipine and nimodepine were from Calbiochem (La Jolla, CA). Fura 2-AM was purchased from Molecular Probes (Eugene, OR).
Primary culture of rat cortical astrocytes. Dissociated cortical astrocyte cultures were established based on a method described by Hertz et al. (14). Cerebral cortices were removed from 1-day-old rats (Sprague Dawley). The cortices were incubated in a trypsin solution (0.25 mg/ml of HBSS) 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 MEM containing 10% FBS. Viable cells (1 × 104 cells/well) were plated in 24-well plates coated with collagen type I. 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 MEM containing 0.25 mM DBcAMP for 7 days to induce differentiation. DBcAMP has been used widely to mimic neuronal influences on astrocyte differentiation (13, 40). Experiments were performed routinely on cultures treated with DBcAMP for 7 days. The identity of astrocytes in culture was determined by immunocytochemical staining for glial fibrillary acidic protein (GFAP) expression, a marker protein for astroglial cells. Staining for neurofilament 200 was used for identification of neurons. More than 95% of cells in culture yielded by this preparation were astrocytes.
Assay for
Na+-K+-Cl
cotransporter activity.
Na+-K+-Cl
cotransporter activity
was measured as bumetanide-sensitive K+ influx, using
86Rb as a tracer for K+ (38).
Cultured astrocytes were equilibrated for 10-30 min at 37°C with
an isotonic HEPES-buffered MEM (290 mosmol/l). The concentrations of
components in HEPES-MEM were (in mM) 140 NaCl, 5.36 KCl, 0.81 MgSO4, 1.27 CaCl2, 0.44 KH2PO4, 0.33 Na2HPO4,
4.4 NaHCO3, 5.55 glucose, and 20 HEPES. Cells were
preincubated for 10 min in HEPES-MEM containing either 0 or 10 µM
bumetanide. For assay of the cotransporter activity, cells were exposed
to 1 µCi/ml of 86Rb in HEPES-MEM for 3 min, either in the
presence or absence of 10 µM bumetanide. 86Rb influx was
stopped by rinsing cells with 0.1 M ice-cold 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 as the slope of
86Rb uptake over time and was expressed as nanomoles of
K+ per milligram of protein per minute.
Bumetanide-sensitive K+ influx was obtained by subtracting
the K+ influx rate in the presence of bumetanide from the
total K+ influx rate. In the
high-[K+]o study, 75 mM
[K+]o was obtained by replacing NaCl in
HEPES-MEM solutions with equimolar KCl. Quadruplicate determinations
were obtained in each experiment throughout the study, and protein
content was measured in each sample using a method described by Smith
et al. (35). Statistical significance in the study was
determined by ANOVA (Bonferroni/Dunn) at a confidence level of 95%
(P < 0.05).
Specific [3H]bumetanide binding assay. [3H]bumetanide binding activity was assessed in morphologically undifferentiated and differentiated astrocytes grown in 24-well plates. [butyl-3H]bumetanide (15 Ci/mmol) was synthesized and HPLC purified by American Radiolabeled Chemicals, according to the method described by Forbush and Palfrey (5). The binding medium consisted of 0.0-0.47 µM [3H]bumetanide in HEPES-MEM. Unlabeled bumetanide (100 µM) was used to determine nonspecific [3H]bumetanide binding. Cells were incubated in the binding medium for 20 min at 37°C. The reaction was stopped by 0.1 M ice-cold MgCl2. Radioactivity of the cellular extract in 1% SDS was measured by liquid scintillation counting. A fraction of sample was used for protein determination (35). In each experiment, quadruplicate determinations were obtained, and five experiments were performed in each study. The computer curve fitting of [3H]bumetanide binding data in astrocytes was obtained by a nonlinear least-squares fitting (Graphpad; GraphPad Software, San Diego, CA).
Gel electrophoresis and Western blotting.
Cortical astrocytes growing on culture dishes were washed with ice-cold
PBS (pH 7.4) that contained 2 mM EDTA and protease inhibitors, as
described previously (36). Cells were scraped from dishes
and suspended in PBS and then were lysed by 30 s of sonication at
4°C by an ultrasonic processor (Sonics & Materials, Danbury, CT). To
obtain cellular lysates, cellular debris was removed by a
centrifugation at 420 g for 5 min. Protein content of the
cellular lysate was determined (35). Samples and
prestained molecular mass markers (Bio-Rad) were denatured in SDS
reducing buffer (1:2 by volume; Bio-Rad) and heated at 37°C for 15 min before gel electrophoresis. The samples were then
electrophoretically separated on 6% SDS gels (19), and
the resolved proteins were transferred electrophoretically to a
polyvinylidene difluoride membrane (0.45 µm; Millipore, Bedford, MA).
The blots were incubated in 7.5% nonfat dry milk in Tris-buffered
saline (TBS) for 2 h at room temperature and then were incubated
overnight with a primary antibody. The blots were then rinsed five
times with TBS and incubated with horseradish peroxidase-conjugated
secondary IgG for 1 h. After five washings, bound antibody was
visualized using the enhanced chemiluminescence (ECL) assay (Amersham).
T4 monoclonal antibody against the human colonic T84 epithelial
Na+-K+-Cl cotransporter
(21) was used for detection of the cotransporter protein.
To gain quantitative analysis of expression of
Na+-K+-Cl
cotransporter protein
and
-actin in astrocytes, 3-35 µg protein of the whole cell
lysate preparation were loaded on 6% SDS gels and probed with T4
antibody and anti-
-actin antibody as described above. The protein
bands on the film after the ECL reaction were scanned using a
Hewlett-Packard ScanJet (4c/T) scanner. The intensity of each protein
band was measured by UN-SCAN-It gel software (Silk Scientific, Orem,
UT). A linear curve was obtained within 5-30 µg protein for both
T4 antibody and anti-
-actin antibody (r = 0.98 and
0.96, respectively). In addition, to establish a linear curve for
exposure time of the ECL on the film, the blot with 5-30 µg
protein was exposed to a film for 10, 15, 20, 30, 40, 50, 60, 90, or
120 s. There was no
-actin signal observed in a 10-s exposure.
A linear curve of exposure time was found for both T4 antibody and
anti-
actin antibody within 15-120 s of exposure time
(r ~0.96). Therefore, either 15 or 30 µg of protein were loaded in all immunoblots of the present study. In addition, the
ECL exposure time was within 60 s. Monoclonal antibody against GLT-1 glutamate transporters was used for analysis of expression of the
glutamate transporter. For deglycosylation studies, cellular proteins
(50 µg) were solubilized with 1% SDS, incubated in the presence of 1 unit of N-glycosidase F (Boehringer Mannheim Biomedicals, Indianapolis, IN) for 4 h at 37°C, and separated by SDS-PAGE as described above.
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 being rinsed, cells were incubated with blocking solution (10% normal goat serum, 0.4% Triton X-100, and 1% BSA in PBS) for 1 h. Cells were then incubated with a primary antibody in blocking solution overnight at 4°C. Cells were rinsed with PBS and incubated with FITC-conjugated secondary antibodies for 2 h. The images of the cells were captured by a laser-scanning confocal microscope (MRC 1000; Bio-Rad) located in the University of Wisconsin-Madison W. M. Keck Neural Imaging Laboratory. The microscope scan head was mounted transversely to an inverted Nikon Diaphot 200. The laser was a 15 mM krypton-argon mixed-gas air-cooled laser, which emitted a strong line in exact alignment at 488 nm, and the 522DF32 filter block was used for FITC signals. The emitted green light signals were directed to the respective photomultiplier tubes. The 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 changes of intracellular Ca2+. Cultured astrocytes grown on collagen type I-coated coverslips were loaded at room temperature for 1 h in HEPES-MEM containing 5 µM fura 2-AM. Subsequently, the coverslips were rinsed with HEPES-MEM. The coverslip was put in a cuvette with a 30° angle in coordination with the excitation light path. Fluorescence was measured using a spectrophotometer (Spex fluorescence Spectrophotometer CM111) at room temperature. The excitation wavelength was alternated between 360 and 380 nm with 1-s integration time at each wavelength, and fluorescence intensity was monitored at an emission wavelength of 510 nm. Mn2+ (1 mM) was used at the end of each experiment to quench the cytosolic Ca2+-sensitive fluorescence, as described in a previous study (12). The fluorescence intensity with 1 mM Mn2+ was subtracted from the value measured in the absence of Mn2+. The 360- to 380-nm ratio (360/380 ratio) of the subtracted values was then calculated (6, 12). All solutions were perfused in the cuvette at a rate of 2.5 ml/min. The solution in the cuvette was exchanged with a time constant of 1.2 min.
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RESULTS |
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Expression of
Na+-K+-Cl
cotransporter in nontreated and DBcAMP-treated astrocytes.
Immunofluorescence staining was employed to characterize
morphologically undifferentiated and differentiated astrocytes. In Fig.
1, A and B, cells
were stained with monoclonal antibody against GFAP, a marker protein
for astrocytes. Differentiated astrocytes induced by DBcAMP (0.25 mM, 7 days) changed from a polygonal (Fig. 1A) to process-bearing
morphology (Fig. 1B), a morphology that is more
characteristic of matured and reactive astrocytes in situ. Moreover,
distribution of the intermediate filament GFAP extended into the
processes (Fig. 1B). This is consistent with the finding that elevation of intracellular cAMP levels in cultured astrocytes by
DBcAMP causes transformation of the flat- into the stellate-type astrocytes (13). Expression and distribution of the
Na+-K+-Cl
cotransporter were
examined in these astrocytes. The
Na+-K+-Cl
cotransporter
expression was recognized by T4 monoclonal antibody against the human
colonic T84 epithelial Na+-K+-Cl
cotransporter (21). Immunoreactive signals with T4
monoclonal antibody were weak in the polygonal astrocytes (Fig.
1C). In DBcAMP-treated astrocytes, the intensity of the T4
antibody staining was enhanced throughout the cell, including the
process (Fig. 1D). To gain quantitative analysis of T4
antibody immunoreactive signals in non-DBcAMP-treated vs.
DBcAMP-treated cells, we acquired fluorescence intensity in each cell
shown in Fig. 1, C and D, using a MetaMorph Imaging System software program (version 3.68; Universal Imaging, West
Chester, PA). An average relative intensity in cell bodies was
73.07 ± 2.97 (n = 6) in non-DBcAMP-treated
cultures (Fig. 1C). In contrast, in the
DBcAMP-treated cultures, the average immunofluorescence intensity with
T4 antibody in cell bodies was increased by ~92% (140.30 ± 7.03, n = 6, P < 0.05; Fig.
1D). However, protein distribution at cell bodies and
processes may differ in untreated and treated astrocytes. In this
experiment, we have only examined the immunoreactivity of T4 antibody
in cell bodies. Therefore, this may only reflect a change of the
cotransporter expression in the cell body region, and the total
cellular cotransporter proteins may remain unchanged in untreated and
treated astrocytes. Several additional approaches such as
immunoblotting, specific [3H]bumetanide binding, and
bumetanide-sensitive K+ influx assays were taken to further
address the issue (see below). The purity of the primary astrocyte
cultures in this preparation was demonstrated further by a negative
staining for neurofilament 200 protein, a neuronal marker (Fig.
1E). More than 95% of cells yielded by this preparation
were astrocytes, and the remaining cells were neurons or
oligodendrocytes. Figure 1F represents a negative control
study in which a primary antibody was omitted, and the rest of the
procedures were the same as in Fig. 1, A-E. This
demonstrates that the images shown in Fig. 1, A-E, are
specific immunoreactive signals.
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Increase of a specific [3H]bumetanide binding in
DBcAMP-treated astrocytes.
DBcAMP stimulates the Na+-K+-Cl
cotransporter protein expression in astrocytes, as discussed above. To
further characterize the upregulation of the cotransporter expression
by DBcAMP, we investigated whether the specific
[3H]bumetanide binding is increased in
DBcAMP-treated astrocytes. Specific [3H]bumetanide
binding to the cotransporter protein requires the simultaneous presence
of three transported ions and has been used as an index to reflect
functional cotransporters in many cell types (5, 9, 28).
[3H]bumetanide binding was performed in the absence
(total binding) and presence of 100 µM unlabeled bumetanide
(nonspecific binding). The total binding contains both a saturable
component and a linear component. The linear component represents
nonspecific [3H]bumetanide binding. The specific
[3H]bumetanide binding was determined by the difference
in the two components and is shown in Fig.
3. An increase in the specific [3H]bumetanide binding is evident in DBcAMP-treated
astrocytes (Fig. 3). The specific binding at 0.47 µM
[3H]bumetanide was 0.30 ± 0.07 pmol/mg protein in
nontreated astrocytes and increased to 0.58 ± 0.08 pmol/mg
protein in DBcAMP-treated astrocytes. The computer fit of the data in
Fig. 3 yields a value for maximal saturable binding of 0.49 ± 0.06 pmol/mg protein in nontreated astrocytes and of 0.82 ± 0.11 pmol/mg protein in DBcAMP-treated astrocytes. This reflects an ~67%
increase in the saturable specific binding (0.82/0.49). The
concentration of [3H]bumetanide required for half-maximal
saturable binding was 0.27 ± 0.07 µM in nontreated astrocytes
and 0.21 ± 0.07 µM in DBcAMP-treated astrocytes. These values
are consistent with the reported bumetanide affinity in many cell types
(28). These data imply that the amount of functional
cotransporter proteins is increased in DBcAMP-treated astrocytes by
67%, a quantitative agreement with upregulation of the cotransporter
expression revealed by immunoblot analysis. However, the bumetanide
affinity of the cotransporter remained unchanged in DBcAMP-treated
astrocytes.
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Characterization of the
Na+-K+-Cl
cotransporter activity in DBcAMP-treated astrocytes.
It has been established that the
Na+-K+-Cl
cotransporter is
specifically and reversibly inhibited by bumetanide (IC50
~0.2 µM) in many cells (28). Bumetanide has been shown
to be more potent than furosemide in non-DBcAMP-treated rat astrocytes
(41). In this study, we examined effects of bumetanide and
furosemide on K+ influx in DBcAMP-treated astrocytes. As
shown in Fig. 4, bumetanide (IC50 ~0.3 µM) is a more potent inhibitor than
furosemide (IC50 ~10.1 µM) in DBcAMP-treated
astrocytes, consistent with the observation in non-DBcAMP-treated
astrocytes (41), C6 glial cells
(2), and published data of other cell types
(28). This is also in agreement with the specific
[3H]bumetanide binding study demonstrated in Fig. 3.
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Effect of protein synthesis inhibition on
Na+-K+-Cl
cotransporter expression and activity.
It has been shown that an increase in intracellular cAMP level plays an
important role in the regulation of expression of several proteins in
astrocytes (13, 33, 40). Our results of immunofluorescence
staining, immunoblotting, and specific [3H]bumetanide
binding indicate that the cotransporter expression is elevated in
DBcAMP-treated astrocytes, and this might be due to an enhanced protein
synthesis. To further test this possibility, we examined the effect of
protein synthesis inhibition on the cotransporter expression. After
confluency, astrocytes were cultured either in normal MEM, MEM
containing 0.25 mM DBcAMP, or MEM containing 0.25 mM DBcAMP plus the
protein syntheses inhibitor cyclohexamide (2-3 µg/ml) for 7 days. Figure 6A
shows that expression of the cotransporter was
increased significantly in DBcAMP-treated cells. In contrast, the
DBcAMP-induced upregulation of the cotransporter expression was
inhibited in the presence of 2 µg/ml cyclohexamide. The effect of
cyclohexamide was summarized in Fig. 6B. Expression of the
cotransporter was increased by ~91% in DBcAMP-treated astrocytes. This stimulation of the protein expression was reduced significantly by
2 and 3 µg/ml cyclohexamide. In addition, there were no differences in the cotransporter expression levels in nontreated cells and cells
treated with DBcAMP plus 3 µg/ml cyclohexamide. These data further suggest that DBcAMP stimulates the cotransporter expression via
a protein synthesis mechanism.
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Ca2+-dependent stimulation of
Na+-K+-Cl
cotransporter by high
[K+]o in astrocytes.
The Na+-K+-Cl
cotransporter in astrocytes appears to play an important role in
K+ uptake (17, 26, 45). In non-DBcAMP-treated
astrocytes (18) and C6 glial cells
(39), the Na+-K+-Cl
cotransporter activity is substantially stimulated when
[K+]o reaches >50 mM. However, the mechanism
underlying this change has not been defined. We further extended this
investigation in differentiated astrocytes. Interestingly, 75 mM
[K+]o stimulates the cotransporter activity
in both nontreated and DBcAMP-treated astrocytes. In nontreated cells,
the cotransporter activity was elevated from a basal level of
17.37 ± 2.50 to 42.94 ± 7.77 nmol · mg
protein
1 · min
1 in the presence of
75 mM [K+]o (P < 0.05, Fig.
7A). The cotransporter
activity was stimulated further in DBcAMP-treated astrocytes, from a
control level of 38.64 ± 1.59 to 69.20 ± 4.81 nmol · mg protein
1 · min
1
in 75 mM [K+]o (P < 0.05).
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DISCUSSION |
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Na+-K+-Cl
cotransporter in astrocytes.
Na+-K+-Cl
cotransporter-mediated
K+ flux has been observed in primary cultures of rat
astrocytes (18, 26, 41) and C6 glioma cells
(2, 25, 39). In the present study, an ~161-kDa
Na+-K+-Cl
cotransporter protein
was recognized by T4 monoclonal antibody in both morphologically
undifferentiated and differentiated astrocytes. A core protein of 138 kDa is detected in these samples after deglycosylation. Moreover,
abundant T4 antibody immunoreactivity was found in primary cultures of
astrocytes. To date, only two distinct isoforms of Na+-K+-Cl
cotransporter (NKCC)
have been identified: NKCC1 (7.0- to 7.5-kb transcript) with a wide
range of tissue distributions (10, 27), and NKCC2 (4.6- to
5.2-kb transcript), which has only been found in vertebrate kidney
(10, 27). Several studies have demonstrated that rat brain
tissue expresses a high level of NKCC1 (30, 38). Although
T4 antibody reacts with both NKCC1 and NKCC2 isoforms of the
Na+-K+-Cl
cotransporters
(21), our immunoblotting and immunocytochemistry data
likely reflect expression of the NKCC1 isoform in primary cultures of astrocytes.
DBcAMP-mediated upregulation of the
Na+-K+-Cl
cotransporter in astrocytes.
Neuronal influences have been suggested to play an important role in
astrocyte function. DBcAMP is widely used to mimic noradrenergic neuronal influences on astrocyte cultures (13). Maturation
of astrocyte cultures with DBcAMP is concomitant with induction and upregulation of several astrocyte proteins, including glial-specific protein GFAP and glial glutamate transporter GLT-1 (13, 33, 40). This DBcAMP-mediated effect could be mimicked by
coculturing astrocytes with neurons (33, 40). Induction of
the glutamate transporter GLT-1 in DBcAMP-treated cells was
demonstrated in our study as a positive control.
Ca2+-dependent stimulation of
Na+-K+-Cl
cotransporter activity under high
[K+]o.
It has been well documented that
Na+-K+-Cl
cotransporter activity
in many peripheral cell types is regulated by diverse second messenger
systems (8, 10). Inhibition of calmodulin activity significantly decreased the basal activity of the
Na+-K+-Cl
cotransporter in
C6 glioma cells (25). In contrast, blocking protein phosphatases with okadaic acid stimulated the cotransporter activity by approximately twofold in the absence of other stimuli (25). In this report, we found that reduction of
intracellular Ca2+ by incubation of astrocytes with
Ca2+-free HEPES buffer significantly decreased the basal
levels of the cotransporter activity. Taken together, these studies
suggest that a steady state of the cotransporter function in astrocytes is regulated by Ca2+-dependent mechanisms, and it could be
via protein phosphorylation.
Role of the
Na+-K+-Cl
cotransporter in K+ uptake in
astrocytes.
A high-[K+]o-mediated stimulation of the
cotransporter in astrocytes has been observed in other studies
(18, 43). In non-DBcAMP-treated rat cortical astrocytes
and C6 glial cells, the cotransporter activity is
stimulated significantly when [K+]o reaches
>50 mM (18, 39). In our study, the bumetanide-sensitive K+ influx in DBcAMP-treated astrocytes was stimulated by
79% in 75 mM [K+]o. Astrocytes in primary
cultures are known to accumulate K+ avidly
(43). Brain extracellular K+ increases in
response to numerous physiological and pathophysiological conditions in
the central nervous system (34). A few minutes of
anoxia/ischemia raises [K+]o to ~60 mM
(34). Therefore, elevation of the
Na+-K+-Cl
cotransporter activity
in astrocytes in high [K+]o could play an
important role in K+ uptake.
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ACKNOWLEDGEMENTS |
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We thank Dr. Mark Haas for providing the method and instruction on purification of [3H]bumetanide. We also thank Dr. Peter Lipton for helpful comments and suggestions.
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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 the American Heart Association (9630189N to D. Sun), National Institute of Neurological Disorders and Stroke Grant RO1 NS-38118 and NSF Career Grant IBN9981826 to D. Sun, and by a grant to the University of Wisconsin Medical School under the Howard Hughes Medical Institute Research Resources Program for Medical Schools. G. Su was supported by a Graduate School Research Grant from the University of Wisconsin-Madison and by a research fund from the Department of Neurological Surgery.
Address for reprint requests and other correspondence: D. Sun, Dept. of Neurological Surgery, School of Medicine, Univ. of Wisconsin, H4/332, Clinical Science 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.
Received 6 March 2000; accepted in final form 18 July 2000.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bevensee, MO,
Apkon M,
and
Boron WF.
Intracellular pH regulation in cultured astrocytes from rat hippocampus.
J Gen Physiol
110:
453-465,
1997
2.
Chassande, O,
Frelin C,
Farahifar D,
Jean T,
and
Lazdunski M.
The Na+/K+/Cl cotransport in C6 glioma cells. Properties and role in volume regulation.
Eur J Biochem
171:
425-433,
1988[Abstract].
3.
D'Andrea, L,
Lytle C,
Matthew JB,
Hofman P,
Forbush B, III,
and
Madara JL.
Na:K:2Cl cotransporter (NKCC) of intestinal epithelial cells.
J Biol Chem
271:
28969-28976,
1996
4.
Duffy, S,
and
MacVicar BA.
Potassium-dependent calcium influx in acutely isolated hippoampal astrocytes.
Neuroscience
61:
51-61,
1994[ISI][Medline].
5.
Forbush, B, III,
and
Palfrey C.
[3H]Bumetanide binding to membranes isolated from dog kidney outer medulla.
J Biol Chem
258:
11787-11792,
1983
6.
Grynkiewicz, G,
Poenie M,
and
Tsien RY.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J Biol Chem
260:
3440-3450,
1985[Abstract].
7.
Haas, M.
Properties and diversity of Na-K-Cl cotransporters.
Annu Rev Physiol
51:
443-457,
1989[ISI][Medline].
8.
Haas, M.
The Na-K-Cl cotransporters.
Am J Physiol Cell Physiol
267:
C869-C885,
1994
9.
Haas, M,
and
Forbush B, III.
[3H]bumetanide binding to duck red cells.
J Biol Chem
261:
8434-8441,
1986
10.
Haas, M,
and
Forbush B, III.
The Na-K-Cl cotransporters.
J Bioenerg Biomembr
40:
161-172,
1998.
11.
Haas, M,
Mcbrayer DG,
and
Yankanskas JR.
Dual mechanisms for Na-K-Cl cotransporter regulation in airway epithelial cells.
Am J Physiol Cell Physiol
264:
C189-C200,
1993
12.
Haworth, RA,
and
Redon D.
Calibration of intracellular Ca transients of isolated adult heart cells labeled with fura-2 by acetoxymethyl ester loading.
Cell Calcium
24:
263-273,
1998[ISI][Medline].
13.
Hertz, L.
Dibutyryl cyclic AMP treatment of astrocytes in primary cultures as a substitute for normal morphogenic and "functiogenic" transmitter signals.
In: Molecular Aspects of Development and Aging of the Nervous System, edited by Lauder JM,
Privat A,
Giacobini E,
Timiras P,
and Venadakis A.. New York: Plenum, 1990, p. 227-243.
14.
Hertz, L,
Juurlink BHJ,
and
Szuchet S.
Cell cultures. In: Handbook of Neurochemistry (vol. 2), edited by Lajtha A.. New York: Plenum, 1985, vol. 8, p. 603-661.
15.
Jessen, BS,
Jessen F,
and
Hoffmann EK.
Na,K,Cl cotransport and its regulation in Ehrlich ascites tumor cells: Ca/Calmodulin and protein kinase C dependent pathways.
J Membr Biol
131:
161-178,
1993[ISI][Medline].
16.
Kenyon, JL,
and
Gibbons WR.
Effect of low-chloride solutions on action potential of sheep cardiac purkinje fibers.
J Gen Physiol
70:
635-660,
1977
17.
Kimelberg, HK.
Glial enzymes and ion transport.
In: Neural Trauma, edited by Popp AJ.. New York: Raven, 1979, p. 137-153.
18.
Kimelberg, HK.
Anisotonic media and glutamate-induced ion transport and volume responses in primary astrocyte cultures.
J Physiol (Paris)
82:
294-303,
1987[Medline].
19.
Laemmli, UK.
Cleavage of structural proteins during the assembly of the need of the bacteriophage T4.
Nature
227:
680-685,
1970[ISI][Medline].
20.
Lytle, C,
and
Forbush B, III.
Regulatory phosphorylation of the secretory Na-K-Cl cotransporter: modulation by cytoplasmic Cl.
Am J Physiol Cell Physiol
270:
C437-C448,
1996
21.
Lytle, C,
Xu JC,
Biemesderfer D,
and
Forbush B, III.
Distribution and diversity of Na-K-Cl cotransport proteins: a study with monoclonal antibodies.
Am J Physiol Cell Physiol
269:
C1496-C1505,
1995
22.
MacVicar, BA,
Hochman D,
Delay MJ,
and
Weiss S.
Modulaton of intracellular Ca2+ in cultured astrocytes by influx through voltage-activated Ca2+ channels.
Glia
4:
448-455,
1991[ISI][Medline].
23.
Maglova, LM,
Crowe WE,
Smith PR,
Altamirano AA,
and
Russell JM.
Na-K-Cl cotransport in human fibroblasts is inhibited by cytomegalovirus infection.
Am J Physiol Cell Physiol
275:
C1330-C1341,
1998
24.
Matthews, JB,
Smith JA,
Tally KJ,
Awtrey CS,
Nguyen H,
Rich J,
and
Madara JL.
Na-K-2Cl cotransporter in intestinal epithelial cells.
J Biol Chem
269:
15703-15709,
1994
25.
Mongin, AA,
Aksentsev SL,
Orlov SN,
Kvacheva ZB,
Mezen NI,
Fedulov AS,
and
Konev SV.
Swelling-induced activation of Na+, K+,2Cl cotransport in C6 glioma cells: kinetic properties and intracellular signaling mechanisms.
Biochim Biophys Acta
1285:
229-236,
1996[ISI][Medline].
26.
Mongin, AA,
Aksentsev SL,
Orlov SN,
Slepko NG,
Kozlova MV,
Maximov GV,
and
Konev SV.
Swelling-induced K+ influx in cultured primary astrocytes.
Brain Res
655:
110-114,
1994[ISI][Medline].
27.
Mount, DB,
Delpire E,
Gamba G,
Hall AE,
Poch E,
Hoover RS,
and
Hebert SC.
The electroneutral cation-chloride cotransporters.
J Exp Biol
201:
2091-2102,
1998
28.
O'Grady, SM,
Palfrey HC,
and
Field M.
Characteristics and functions of Na-K-Cl cotransport in epithelial tissues.
Am J Physiol Cell Physiol
253:
C177-C192,
1987
29.
O'Neill, CW.
Physiological significance of volume-regulatory transporters.
Am J Physiol Cell Physiol
276:
C995-C1011,
1999
30.
Plotkin, MD,
Kaplan MR,
Perterson L,
Gullans SR,
Hebert SC,
and
Delpire E.
Expression of the Na+-K+-Cl cotransporter BSC2 in the nervous system.
Am J Physiol Cell Physiol
272:
C173-C183,
1997
31.
Randall, J,
Thorne T,
and
Delpire E.
Partial cloning and characterization of Slc12a2: the gene encoding the secretory Na-K-2Cl cotransporter.
Am J Physiol Cell Physiol
273:
C1267-C1277,
1997
32.
Rose, CR,
and
Ransom BR.
Intracellular sodium homeostasis in rat hippocampal astrocytes.
J Physiol (Lond)
491:
291-305,
1996[Abstract].
33.
Schlag, BD,
Vondrasek JA,
Munir M,
Kalandadze A,
Zelenaia OA,
Rothstein JD,
and
Robinson MB.
Regulation of the glial Na+-dependent glutamate transporters by cyclic AMP analogs and neurons.
Mol Pharmacol
53:
355-369,
1998
34.
Siesjo, BK.
Pathophysiology and treatment of focal cerebral ischemia. I. Pathophysiology.
J Neurosurg
77:
169-184,
1992[ISI][Medline].
35.
Smith, PK,
Krohn RI,
Hermanson GT,
Mallia AK,
Gartner FH,
Provenzano MD,
Fujimoto EK,
Goeke NM,
Olson BJ,
and
Klenk DC.
Measurement of protein using bicinchoninic acid.
Anal Biochem
150:
76-85,
1985[ISI][Medline].
36.
Sun, D,
Lytle C,
and
O'Donnell ME.
Astroglial cell-induced increase of Na-K-Cl cotransport protein expression in brain microvessel endothelial cells.
Am J Physiol Cell Physiol
269:
C1506-C1512,
1995
37.
Sun, D,
Lytle C,
and
O'Donnell ME.
IL-6 secreted by astroglial cells regulates Na-K-Cl cotransport in brain microvessel endothelial cells.
Am J Physiol Cell Physiol
272:
C1829-C1835,
1997
38.
Sun, D,
and
Murali SG.
Na+-K+-2Cl cotransporter in immature cortical neurons: a role in intracelluler Cl
regulation.
J Neurophysiol
81:
1939-1948,
1999
39.
Sun, D,
and
Su G.
Up-regulation of Na+-K+-Cl cotransporter activity in glial cells under high [K+]o: a Ca2+-dependent process (Abstract).
Soc Neurosci Abstr
24:
774,
1998.
40.
Swanson, RA,
Liu J,
Miller JW,
Rothstein JD,
Farrell K,
Stein BA,
and
Longuemare MC.
Neuronal regulation of glutamate transporter subtype expression in astrocytes.
J Neurosci
17:
932-940,
1997
41.
Tas, PWL,
Massa PT,
Kress HG,
and
Koschel K.
Characterization of an Na/K/Cl cotransport in primary cultures of rat astrocytes.
Biochim Biophys Acta
903:
411-416,
1987[ISI][Medline].
42.
Topper, JN,
Wasserman SM,
Anderson KR,
Falb D,
and
Gimbrone MA, Jr.
Expression of bumetanide-sensitive Na-K-Cl cotransporter BSC2 is differentially regulated by fluid mechanical and inflammatory cytokine stimuli in vascular endothelium.
J Clin Invest
99:
2941-2949,
1997
43.
Walz, W.
Role of Na/K/Cl cotransport in astrocytes.
Can J Physiol Pharmacol
70:
S260-S262,
1992[ISI][Medline].
44.
Walz, W,
and
Hertz L.
Comparison between fluxes of potassium and of chloride in astrocytes in primary cultures.
Brain Res
277:
321-328,
1983[ISI][Medline].
45.
Walz, W,
and
Hertz L.
Intense furosemide-sensitive potassium accumulation in astrocytes in the presence of pathologically high extracellular potassium levels.
J Cereb Blood Flow Metab
4:
301-304,
1984[ISI][Medline].