Stimulation of
Na+-K+-2Cl
cotransporter in neuronal cells by excitatory neurotransmitter
glutamate
Dandan
Sun1,2 and
Sangita G.
Murali1
Departments of 1 Neurological
Surgery and 2 Physiology, School
of Medicine, University of Wisconsin, Madison, Wisconsin 53792
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ABSTRACT |
Na+-K+-2Cl
cotransporters are important in renal salt reabsorption and in salt
secretion by epithelia. They are also essential in maintenance and
regulation of ion gradients and cell volume in both epithelial and
nonepithelial cells. Expression of
Na+-K+-2Cl
cotransporters in brain tissues is high; however, little is known about
their function and regulation in neurons. In this study, we examined
regulation of the
Na+-K+-2Cl
cotransporter by the excitatory neurotransmitter glutamate. The cotransporter activity in human neuroblastoma SH-SY5Y cells was assessed by bumetanide-sensitive
K+ influx, and protein expression
was evaluated by Western blot analysis. Glutamate was found to induce a
dose- and time-dependent stimulation of
Na+-K+-2Cl
cotransporter activity in SH-SY5Y cells. Moreover, both the glutamate ionotropic receptor agonist
N-methyl-D-aspartic
acid (NMDA) and the metabotropic receptor agonist
(±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) significantly
stimulated the cotransport activity in these cells.
NMDA-mediated stimulation of the
Na+-K+-2Cl
cotransporter was abolished by the selective NMDA-receptor antagonist (+)-MK-801 hydrogen maleate.
trans-ACPD-mediated effect on the cotransporter was blocked by the metabotropic receptor antagonist (+)-
-methyl-(4-carboxyphenyl)glycine. The results demonstrate that
Na+-K+-2Cl
cotransporters in neurons are regulated by activation of both ionotropic and metabotropic glutamate receptors.
ionotropic glutamate receptors; metabotropic glutamate receptors; bumetanide
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INTRODUCTION |
SODIUM-POTASSIUM-CHLORIDE cotransporters represent a
family of integral membrane proteins that transport
Na+,
K+, and
Cl
into and out of cells.
The
Na+-K+-2Cl
cotransporters have been identified in a wide variety of eukaryotic cells, and the stoichiometry of transported ions in most instances has
been reported to be
1Na+:1K+:2Cl
(8, 25). The
Na+-K+-2Cl
cotransporters are characterized by their specific, reversible inhibition by the sulfamoylbenzoic acid "loop" diuretics
(furosemide, bumetanide, and benzmetanide). Because the
cotransporter carries an electroneutral ion movement, the driving force
for net transport is solely dependent on the chemical gradients of the
three transported ions. Under physiological conditions, the
Na+-K+-2Cl
cotransporter in most cells moves these ions inward, driven by both
favorable inward Na+ and
Cl
chemical gradients (8,
25). Thus the cotransporter plays a crucial role in vectorial salt
transport in epithelial cells and ion gradients as well as cell volume
regulation in epithelial and nonepithelial cells (8, 13, 24, 25).
Currently, only two distinct isoforms of the
Na+-K+-2Cl
cotransporter (NKCC) have been identified: NKCC1 (7.0- to 7.5-kb
transcript), which has a wide range of tissue distributions (6, 38),
and NKCC2 (4.6- to 5.2-kb transcript), which has only been found in vertebrate kidney (7, 12, 27). Northern blot analysis has demonstrated
that the expression level of NKCC1 mRNA is high in brain (6, 38). In
situ hybridization and immunocytochemical studies have shown that NKCC1
proteins are abundantly present on plasma membranes of neurons
throughout the rat brain (28).
Despite studies on the
Na+-K+-2Cl
cotransporters in many cell types, the physiological function and
regulation of NKCC1 protein in neuronal cells are little known. In the
present study, human neuroblastoma SH-SY5Y cells were used to examine
regulation of Na+-K+-2Cl
cotransport activity in neurons by excitatory neurotransmitter glutamate. Glutamate is the major excitatory neurotransmitter in the
central nervous system (5, 22). Glutamate exerts its effect by
activation of a family of heterogeneous glutamate receptors that couple
to second-messenger systems (1, 11, 37). SH-SY5Y cells have been shown
to express many properties of mature noradrenergic neurons, including
the occurrence of neurosecretory granules, extension of neurites, and
the Ca2+-dependent release of
norepinephrine after plasma membrane depolarization (19). It has also
been demonstrated that SH-SY5Y cells express both ionotropic and
metabotropic glutamate receptors (20, 21) as well as the
Na+-dependent glutamate-aspartate
transporter (26). We report here that glutamate stimulates
Na+-K+-2Cl
cotransport activity in SH-SY5Y neuronal cells. Activation of both the
ionotropic
N-methyl-D-aspartic
acid (NMDA) receptor and metabotropic glutamate receptors stimulates
Na+-K+-2Cl
cotransporter in SH-SY5Y cells.
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MATERIALS AND METHODS |
Cell culture.
Cultured human neuroblastoma SH-SY5Y cells were maintained in DMEM,
supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin
(100 µg/ml). Cells were grown on 24-well plates in DMEM, and
experiments were performed on confluent cells.
K+ influx
determination.
Na+-K+-2Cl
cotransport activity was measured as bumetanide-sensitive
K+ influx, using
86Rb as a tracer for
K+. Briefly, SH-SY5Y cells were
equilibrated for 10-30 min at 37°C with an isotonic
HEPES-buffered MEM (300 mosM). The concentrations of components in
HEPES-MEM (mM) were 140 NaCl, 5.36 KCl, 0.81 MgSO4, 1.27 CaCl2, 0.44 KH2PO4,
0.33 Na2HPO4,
5.55 glucose, and 20 HEPES. Cells were then preincubated for 10 min in
HEPES-MEM containing either 0 or 60-100 µM bumetanide. For assay
of cotransport activity, cells were exposed to 1 µCi/ml of
86Rb in HEPES-MEM for 3 min,
either in the presence or absence of 60-100 µM bumetanide.
86Rb influx was stopped by rinsing
cells with ice-cold 0.1 M MgCl2. Radioactivity of cells extracted in 1% SDS was analyzed by liquid scintillation counting (Packard 1900CA, Downers Grove, IL).
K+ influx rate was calculated as
the slope of 86Rb uptake over time
and expressed as nanomoles of K+
per milligram of protein per minute. Bumetanide-sensitive
K+ influx was obtained by
subtracting K+ influx rate in the
presence of bumetanide from total
K+ influx rate. Quadruplet
determinations were obtained in each experiment, and protein content
was measured in each sample using a method described by Smith et al.
(32). Statistical significance in the study was determined by
Student's t-test or ANOVA
(P < 0.05).
Gel electrophoresis and Western blotting.
SH-SY5Y cells growing on 100-mm tissue culture dishes were washed with
ice-cold PBS (pH 7.4), which contained 2 mM EDTA and protease
inhibitors, as described previously (33). Cells were scraped from
dishes, suspended in PBS, and lysed by 30 s of sonication at 4°C
with an ultrasonic processor (Sonics & Materials, Danbury, CT). To
obtain cellular lysates, cellular debris was removed by a brief
centrifugation at 420 g for 5 min.
Protein content of the cellular lysate was determined by the Bradford
method (4). Samples and prestained molecular mass markers (Bio-Rad)
were denatured in SDS reducing buffer [9.2% SDS, 5%
-mercaptoethanol, 50 mM Tris · HCl (pH 7.4), 35%
sucrose, and 0.012% bromphenol blue] and heated at 37°C for
15 min before gel electrophoresis. The samples were then
electrophoretically separated on 6% SDS gels (14), and the resolved
proteins were electrophoretically transferred to a polyvinylidene
difluoride (PVDF) 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 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 washes to remove unbound secondary antibody, bound antibody
was visualized using the enhanced chemiluminescence assay (Amersham).
T4 monoclonal antibody against the human colonic T84 epithelial
Na+-K+-2Cl
cotransporter was used for detection of NKCC1 protein, as described by
Lytle et al. (17). A monoclonal antibody against an NMDA-receptor isoform (NR-2B, 1 µg/ml) was used for analysis of NMDA-receptor expression. A rabbit polyclonal anti-rat metabotropic glutamate receptor (mGluR) type 1 IgG (0.75 µg/ml) was used to identify mGluR1 receptor expression in
SH-SY5Y cells. Neuronal identity of SH-SY5Y cells was investigated by
expression of neurofilament with a monoclonal antibody against
neurofilament protein 200 (7.7 µg/ml).
For deglycosylation studies, cellular proteins were solubilized with
1% SDS and incubated in the presence of 1 unit deglycosidase F
(Boehringer Mannheim Biomedicals, Indianapolis, IN) for 5 h at 37°C
and separated by SDS-PAGE, as described above.
Materials.
Bumetanide, L-glutamic acid, and
anti-neurofilament 200 monoclonal antibody were purchased from Sigma
(St. Louis, MO). DMEM was from GIBCO (Washington, DC). NMDA,
AMPA-receptor agonist (±)-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), selective NMDA-receptor antagonist (+)-MK-801 hydrogen maleate, mGluR agonist
(±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD), and mGluR
antagonist (+)-
-methyl-4-carboxyphenylglycine [(+)-MCPG]
were purchased from Research Biochemicals International (Natick, MA).
FBS was obtained from Hyclone Laboratories (Logan, UT).
86RbCl was purchased from NEN Life
Science Products (Boston, MA). Rabbit anti-rat
mGluR1 polyclonal IgG was from
Upstate Biotechnology (Lake Placid, NY). Anti-NMDA-receptor (NR-2B)
monoclonal antibody was purchased from Transduction Laboratories
(Lexington, KY).
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RESULTS |
Dose- and time-dependent stimulation of
Na+-K+-2Cl
cotransport activity by glutamate.
To examine whether glutamate regulates
Na+-K+-2Cl
cotransport activity in neurons,
Na+-K+-2Cl
cotransporter activity in SH-SY5Y was assessed by bumetanide-sensitive K+ influx either in the presence
or absence of glutamate. Data showed that glutamate induced a
dose-dependent stimulation of
Na+-K+-2Cl
cotransporter activity in SH-SY5Y (Fig. 1).
The dose-response curve showed that 15 µM glutamate caused a
significant stimulation of the cotransporter activity
(P < 0.05). Glutamate still caused a
stimulation of the cotransporter when the concentration in
extracellular medium was increased to 75 µM
(P < 0.05). The glutamate-mediated stimulation of
Na+-K+-2Cl
cotransporter activity in SH-SY5Y was time dependent.
86Rb uptake in cells was assayed
at 37°C in either the presence or absence of 15 µM glutamate for
0-15 min. Figure 2 demonstrated that
bumetanide-sensitive K+ uptake was
increased over time in the absence and presence of 15 µM glutamate.
Moreover, in the absence as well as the presence of glutamate,
bumetanide-sensitive K+ uptake
rate seems to be faster in the period of 1-5 min than that between
5 and 15 min, which was reflected by the different slopes. The slope of
the cotransport in glutamate-treated group was steeper than that of the
control group, indicating that the cotransporter activity is stimulated
by glutamate.

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Fig. 1.
Glutamate induces a dose-dependent stimulation of
Na+-K+-2Cl
cotransporter activity in SH-SY5Y cells. Cultured cells were
preincubated either in presence or absence of 60 µM bumetanide in
HEPES-buffered medium for 4 min. In addition, cells were exposed to
0-75 µM glutamate for 10 min. To obtain bumetanide-sensitive
86Rb influx,
86Rb influx was subsequently
assayed either in presence or absence of bumetanide for 3 min. Data are
means ± SE; n = 3. Quadruplet
determinations were obtained in each experiment.
* P < 0.05 vs. control group
by Student's t-test.
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Fig. 2.
Glutamate induces a time-dependent stimulation of
Na+-K+-2Cl
cotransporter activity in SH-SY5Y cells. Cultured cells were
preincubated in presence or absence of 60 µM bumetanide in
HEPES-buffered medium for 4 min.
86Rb uptake in cells was assayed
either in presence or absence of 15 µM glutamate for 0-15 min,
as indicated on x-axis. In
glutamate-treated groups, 15 µM glutamate and
86Rb were added to cells at same
time. Data are means ± SE; n = 3. Quadruplet determinations were obtained in each experiment.
* P < 0.05 vs.
control group by Student's t-test.
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Stimulation of
Na+-K+-2Cl
cotransporter by activation of both ionotropic NMDA receptor and mGluR.
To investigate the possible second-messenger signal pathways
responsible for glutamate-mediated stimulation of the cotransporter in
SH-SY5Y cells, it is necessary to determine which type of glutamate receptors are involved in the process. First, the effect of a potent
NMDA-receptor agonist, NMDA, was tested on activity of the
cotransporter. A dose-response study was conducted. SH-SY5Y cells in
24-well plates were incubated with different concentrations of NMDA in
isotonic HEPES-buffered MEM at 37°C for 10 min. Control SH-SY5Y
cells were incubated with isotonic HEPES-buffered MEM at 37°C for
10 min. Total K+ influx rate as
well as bumetanide-sensitive K+
influx rate were determined in both control and NMDA-treated cells.
Figure 3 showed that 10 µM of NMDA
significantly stimulated the cotransport activity in SH-SY5Y cells,
from a basal level of 6.34 ± 0.58 to 15.37 ± 2.42 nmol · mg
protein
1 · min
1
(mean ± SE, P < 0.05). It was
demonstrated in Table 1 that NMDA caused a
significant stimulation of
Na+-K+-2Cl
cotransporter activity in SH-SY5Y cells in a dose-dependent manner, with maximal stimulation at 20 µM and no stimulation at 100 µM. It
appears that the increase in the total
K+ influx in NMDA-stimulated cells
reflects the increase in the cotransporter activity as well as
bumetanide-insensitive K+ influx
pathway(s). To determine whether activation of mGluR is also involved
in glutamate-mediated stimulation of the cotransporter, the effect of a
mGluR agonist trans-ACPD on
cotransport activity was examined. Figure 3 demonstrated that 10 µM
trans-ACPD significantly stimulated
the cotransport activity in SH-SY5Y cells, from a basal level of 6.34 ± 0.58 to 9.93 ± 0.86 nmol · mg
protein
1 · min
1
(mean ± SE, P < 0.05).
trans-ACPD (20-100 µM) also
stimulated the
Na+-K+-2Cl
cotransporter substantially; however, that was not statistically significant (Table 1).

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Fig. 3.
Stimulation of
Na+-K+-2Cl
cotransporter by activation of both ionotropic
N-methyl-D-aspartic
acid (NMDA) receptor and metabotropic glutamate receptors (mGluR).
SH-SY5Y cells grown on 24-well plates were preincubated in 10 µM
(±)-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), 10 µM
glutamate, 10 µM NMDA, 10 µM
(±)-1-aminocyclopentane-trans-1,3-dicarboxylic
acid (trans-ACPD), or 10 µM NMDA + 10 µM trans-ACPD at 37°C for 10 min. Bumetanide-sensitive 86Rb
influx rates were determined in presence of 100 µM bumetanide.
86Rb influx was subsequently
assayed for 3 min. Data are means ± SE;
n = 4. Quadruplet determinations were
obtained in each experiment. * P < 0.05 vs. control group by Student's
t-test.
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To examine whether activation of AMPA ionotropic receptor is involved
in glutamate-mediated stimulation of the cotransporter activity, the
effect of AMPA receptor agonist AMPA on bumetanide-sensitive K+ influx was evaluated. It was
shown in Fig. 3 that activation of AMPA receptor had no effect on the
cotransporter activity.
To further investigate the relative contributions of NMDA and
trans-ACPD-mediated pathways, cells
were stimulated with NMDA and
trans-ACPD. As shown in Fig. 3, 10 µM NMDA plus 10 µM trans-ACPD did
not cause an additive effect on the cotransporter activity. This
implies that activation of these two receptors may lead to a final
common pathway which activates the cotransporter activity.
Effect of selective glutamate ionotropic receptor antagonist MK-801
on
Na+-K+-2Cl
cotransporter activity.
The results of Fig. 3 and Table 1 revealed that stimulation of the
cotransport activity by glutamate may be due to activation of both NMDA
receptors and mGluRs in SH-SY5Y cells. To further investigate this
hypothesis, we evaluated whether NMDA- or
trans-ACPD-induced stimulation of
Na+-K+-2Cl
cotransporter could be abolished by selective glutamate-receptor antagonists. We first studied whether NMDA-mediated stimulation of the
cotransporter could be blocked by the selective, noncompetitive NMDA-receptor antagonist MK-801. SH-SY5Y cells were exposed to an
isotonic HEPES-buffered MEM containing either 10 µM NMDA or 10 µM
NMDA plus 1 or 10 µM MK-801 at 37°C for 10 min. Figure
4 showed that neither 1 nor 10 µM MK-801
had a significant effect on a basal level of the cotransport activity.
However, both 1 and 10 µM MK-801 significantly abolished
NMDA-mediated stimulation of
Na+-K+-2Cl
cotransporter (Fig. 4). To verify that MK-801 exerts its effect specifically via blocking of the NMDA receptor, we also examined whether MK-801 had any effect on
trans-ACPD-mediated stimulation of the
cotransporter. SH-SY5Y cells were exposed to either 10 µM
trans-ACPD or 10 µM
trans-ACPD plus 1 or 10 µM MK-801 at
37°C for 10 min. As demonstrated in Fig. 4,
trans-ACPD-mediated stimulation of the
cotransport activity was not altered in the presence of either 1 or 10 µM MK-801. Thus MK-801 significantly inhibits the NMDA-mediated
effect by antagonizing NMDA receptor in SH-SY5Y cells. We then decided
to examine the effect of MK-801 on glutamate-induced stimulation of the
cotransporter. SH-SY5Y cells were incubated in the presence of either
10 µM glutamate or 10 µM glutamate plus 1 µM MK-801 for 10 min.
Bumetanide-sensitive K+ influx
rate in cells was 10.42 ± 0.59 nmol · mg
protein
1 · min
1
in the presence of glutamate alone but was reduced to 6.37 ± 1.52 nmol · mg
protein
1 · min
1
in the presence of both glutamate and MK-801; the latter was not
statistically significantly different from the value in the presence of
MK-801 alone. These results suggest that stimulation of the
Na+-K+-2Cl
cotransporter by glutamate is primarily mediated by activation of NMDA
glutamate receptors.

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Fig. 4.
Effect of noncompetitive NMDA-receptor antagonist MK-801 on NMDA- and
trans-ACPD-mediated stimulation of
Na+-K+-2Cl
cotransporter. SH-SY5Y cells were exposed to HEPES-buffered MEM
containing either 10 µM NMDA or 10 µM
trans-ACPD for 10 min. To test effect
of MK-801, cells were incubated in presence of either 1 or 10 µM
MK-801 for 10 min. To obtain bumetanide-sensitive
86Rb influx,
86Rb influx was subsequently
assayed either in presence or absence of 100 µM bumetanide for 3 min.
Data are means ± SE; n = 3-4.
Quadruplet determinations were obtained in each experiment.
* P < 0.05 vs. NMDA-treated
group by Student's t-test.
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mGluR-mediated stimulation of
Na+-K+-2Cl
cotransporter activity.
To further characterize metabotropic receptor-mediated stimulation of
Na+-K+-2Cl
cotransporter, a time-dependent stimulation of the cotransport system
was evaluated with 10 µM trans-ACPD.
SH-SY5Y cells were preincubated either in the presence or absence of 10 µM trans-ACPD in isotonic
HEPES-buffered MEM for 0-15 min. These experiments were performed
in either the presence or absence of 100 µM bumetanide, respectively.
K+ influx was assayed for 3 min in
those experiments. Figure
5A showed
that bumetanide-sensitive K+
influx rate was significantly elevated when cells were exposed to 10 µM trans-ACPD for 2 min (8.26 ± 0.62 nmol · mg
protein
1 · min
1
vs. a control level of 5.77 ± 1.26 nmol · mg
protein
1 · min
1,
P < 0.05). The
trans-ACPD-mediated stimulation of the
cotransporter was observed over 15 min of incubation period.

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Fig. 5.
Time-dependent stimulation of
Na+-K+-2Cl
cotransporter activity in SH-SY5Y by mGluR agonist
trans-ACPD and NMDA. SH-SY5Y cells
were preincubated in HEPES-buffered medium containing either 0 or 100 µM bumetanide for 10 min. To test effect of
trans-ACPD, cells were then exposed to
10 µM trans-ACPD at 37°C for
0-15 min (A). For NMDA
experiments, cells were incubated in presence of 20 µM NMDA at
37°C for 0-15 min (B).
Control cells were exposed to HEPES-buffered medium at 37°C for
0-15 min. To obtain bumetanide-sensitive (Bum-sen)
86Rb influx,
86Rb influx was assayed either in
presence or absence of 100 µM bumetanide for 3 min. Data are means ± SE; n = 3. * P < 0.05 vs. control group
by Student's t-test. Bum-res,
bumetanide resistant.
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To compare the temporal profile of the response to NMDA-mediated
pathway, a similar time-course study was conducted with 20 µM NMDA.
As shown in Fig. 5B, 20 µM NMDA
stimulated the cotransporter activity in a similar time-dependent
manner. Moreover, bumetanide-resistant K+ influx was also substantially
stimulated by NMDA.
mGluRs consist of three groups of heterogeneous receptor proteins (11,
37). trans-ACPD primarily activates
mGluR2 and mGluR3 receptors and also
activates mGluR1 and
mGluR5 receptors at higher
concentrations (10, 31). To further characterize trans-ACPD-mediated signal
transduction pathways, we examined whether the
trans-ACPD-mediated effect could be
blocked by a specific mGluR antagonist, (+)-MCPG. (+)-MCPG has been
reported to antagonize mGluR1
and mGluR2 but not
mGluR4 in transfected cells (10). Cells were incubated for 10 min in the presence of either 10 µM trans-ACPD or 10 µM
trans-ACPD plus 50 µM (+)-MCPG.
Bumetanide-sensitive K+ influx was
then assessed in those cells. Figure 6
showed that the basal activity of the cotransporter was not changed by
50 µM (+)-MCPG. In contrast,
trans-ACPD-induced stimulation of
bumetanide-sensitive K+ influx was
significantly inhibited by 50 µM (+)-MCPG (from 21.46 ± 3.13 to 10.80 ± 2.03 nmol · mg
protein
1 · min
1,
P < 0.05). To verify that (+)-MCPG
exerts its effect specifically via blocking of metabotropic receptors,
we examined whether (+)-MCPG had any effect on the NMDA-mediated
pathway. As demonstrated in Fig. 6, NMDA-induced stimulation of the
cotransporter activity remained unchanged in the presence of (+)-MCPG.
This study further supports our hypothesis that stimulation of the
cotransporter by trans-ACPD is due to
activation of mGluR-mediated signal transduction pathways.

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Fig. 6.
Effect of a specific metabotropic receptor antagonist on
trans-ACPD-mediated stimulation of
Na+-K+-2Cl
cotransporter. SH-SY5Y cells were incubated at 37°C in
HEPES-buffered medium containing either 10 µM
trans-ACPD or 10 µM
trans-ACPD + 50 µM (+)-MCPG for 10 min. To test effect of (+)-MCPG on NMDA-mediated stimulation, cells
were exposed to either 10 µM NMDA or 10 µM NMDA + 50 µM (+)-MCPG
for 10 min. To obtain bumetanide-sensitive
86Rb influx,
86Rb influx was subsequently
assayed either in presence or absence of 100 µM bumetanide for 3 min
at 37°C. Data are means ± SE;
n = 3. Quadruplet determinations were
obtained in each experiment. * P < 0.05 vs. control group; #P < 0.05 vs. trans-ACPD-treated group by
ANOVA (Bonferroni-Dunn).
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Expression of
Na+-K+-2Cl
cotransporter protein and glutamate receptors in SH-SY5Y cells.
To further confirm neuronal identity of SH-SY5Y cells, expression of a
major component of neuronal intermediate filaments, neurofilament 200, was examined in SH-SY5Y cells by Western blot analysis. As shown in
Fig. 7A,
two protein bands were recognized by a monoclonal anti-neurofilament
200 antibody. These two major protein bands were 160- and 200-kDa
neurofilament proteins and represent different levels of
phosphorylation in these molecules during posttranslational
modification. A control experiment was performed by using a crude
membrane preparation of cerebral cortex from Sprague-Dawley rat and a
similar result was observed (Fig. 7A).

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Fig. 7.
Expression of
Na+-K+-2Cl
cotransporter and glutamate receptor proteins in SH-SY5Y cells by
Western blot analysis. Cellular lysate proteins were obtained from
SH-SY5Y cells by a sonication and brief centrifugation (see
MATERIALS AND METHODS). Proteins
were separated on 6% SDS gel and transferred to a PVDF membrane.
Expression of neurofilament 200 protein (both phosphorylated and
dephosphorylated) was determined by anti-neurofilament 200 protein
monoclonal antibody (SH-SY5Y, 15 µg protein; brain cortex, 10 µg
protein in A). To detect expression
of cotransporter, membrane was probed with T4 monoclonal antibody
(SH-SY5Y, 60 µg protein; brain cortex, 25 µg protein in
B). Expression of NMDA receptor in
SH-SY5Y cells was demonstrated by using anti-rat NR-2B antibody
(SH-SY5Y, 80 µg protein; brain cortex, 15 µg protein in
C). Expression of
mGluR1 proteins was detected by
using an anti-rabbit mGluR1
antibody (SH-SY5Y, 60 µg protein; brain cortex, 25 µg protein in
D). Deglycosylation of protein
samples with N-glycosidase in SH-SY5Y,
crude membrane preparation of cerebral cortex as well as C6 glioma
cells are shown in E. Blot was
visualized by enhanced chemiluminescence. Data are a representative
blot of 3 experiments.
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Figure 7B demonstrated that T4
monoclonal antibody recognized an ~159-kDa cotransporter protein in
SH-SY5Y, whereas an ~145-kDa cotransporter protein was found in crude
membrane preparation of rat cerebral cortex. The size difference in the
cotransporter proteins has been reported in other studies (17, 28, 33) and reflects different levels of glycosylation in these proteins. Deglycosylation of these two samples with
N-glycosidase gave rise to a single
protein band (~130 kDa) in both SH-SY5Y and crude membrane
preparation of rat cerebral cortex (Fig.
7E).
Results in Figs. 1-6 of this study and work by others (20, 21)
indicated that heterogeneous glutamate receptors are important in
signal transduction in SH-SY5Y cells. In this experiment, we further
investigated expression of ionotropic NMDA receptor and mGluRs in
SH-SY5Y cells by Western blot analysis. Figure
7C showed that a 180-kDa
NR-2B-receptor protein was expressed in the rat cerebral cortex.
However, NR-2B-receptor protein in SH-SY5Y migrated faster. The cause
for this discrepancy is not apparent. An ~142-kDa mGluR1 protein was expressed in
both SH-SY5Y cells and rat cerebral cortex (Fig.
7D).
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DISCUSSION |
In this study, human neuroblastoma SH-SY5Y cells were used as a
neuronal cell model. Many properties of mature noradrenergic neurons
have been shown in SH-SY5Y cells (19). SH-SY5Y cells express ionotropic
NMDA and AMPA receptors as well as mGluRs, determined by
receptor-mediated activity and Northern blot analysis (20, 21).
Especially in the present study, expression of the NMDA-receptor isoform NR-2B and mGluR1
protein in SH-SY5Y was demonstrated by Western blot analysis
(Fig. 7, C and
D). In addition, neuronal identity
of SH-SY5Y cells was further characterized by an abundant expression of
neurofilament protein 200, a specific cellular marker for neurons (Fig.
7A).
Western blot analysis revealed that a 159-kDa protein was recognized in
cellular lysate preparation of SH-SY5Y by T4 monoclonal antibody (Fig.
7B). The molecular mass of the
cotransporter protein identified here is consistent with the sizes of
NKCC1 proteins observed in other cell types (8, 17, 35).
Bumetanide-sensitive K+ influx
represents ~38% of total K+
influx in SH-SY5Y cells (Table 1, Fig.
5B). The results of the present
study clearly indicate that
Na+-K+-2Cl
cotransporter in neuronal cells is regulated by the major excitatory neurotransmitter glutamate. Glutamate was found to induce a dose- and
time-dependent stimulation of
Na+-K+-2Cl
cotransporter activity in SH-SY5Y cells (Figs. 1 and 2). The novel
finding of this work also includes that activation of both NMDA and
metabotropic receptors is involved in stimulation of the
Na+-K+-2Cl
cotransporter. Both the glutamate ionotropic receptor agonist NMDA and
metabotropic receptor agonist
trans-ACPD significantly stimulated
the cotransport activity in these cells (Fig. 3). No significant effect
on the cotransporter activity was observed by AMPA-receptor agonist
AMPA (Fig. 3). Moreover, NMDA-mediated stimulation of
Na+-K+-2Cl
cotransport was specifically abolished by the selective NMDA-receptor antagonist MK-801 (Fig. 4). The presence of both NMDA and
trans-ACPD has no additive effect on
the stimulation of cotransporter activity in SH-SY5Y cells (Fig. 3).
This implies that activation of these two receptors may lead to a final
common pathway that activates the
Na+-K+-2Cl
cotransporter.
The mGluR agonist trans-ACPD caused a
dose- and time-dependent stimulation of the cotransporter (Fig.
5A, Table 1). Furthermore, the
trans-ACPD-mediated effect, but not
the NMDA-mediated one, was specifically inhibited by the metabotropic
receptor antagonist (+)-MCPG (Fig. 6). Expression of
mGluR1 protein was detected in SH-SY5Y cells. These results suggested that mGluRs may play a role in
regulation of
Na+-K+-2Cl
cotransporter in neurons. However, it appears that glutamate mediates
its effect on the cotransporter primarily through the NMDA receptor in
SH-SY5Y cells, which was evident in the study with the NMDA-receptor
antagonist MK-801 (Fig. 4).
Interestingly, we have observed that both NMDA and
trans-ACPD stimulated a portion of
K+ influx resistant to bumetanide
(Table 1, Fig. 5B). The nature of
NMDA and trans-ACPD-mediated
stimulation of bumetanide-insensitive K+ influx is not known. It has
been reported that the glutamate-receptor agonist
L-AP-4 stimulates an inwardly
rectifying K+ channel in
astrocytes that can be inhibited by
BaCl2 (9). It is possible that
upregulated bumetanide-insensitive
K+ influx may represent an
inwardly rectifying K+ channel in
SH-SY5Y cells. On the other hand, this bumetanide-insensitive K+ influx may be through
Na+-K+-ATPase;
however, it took 5-15 min for glutamate to maximally stimulate
Na+-K+-ATPase
activity in cerebellar neurons (18). The bumetanide-insensitive K+ influx appears to be stimulated
faster in our study (Fig. 5B).
There is evidence suggesting that
Na+-K+-2Cl
cotransporter activity in many peripheral cell types is regulated by
diverse second messenger systems (8, 25, 34).
Na+-K+-2Cl
cotransporter in some epithelial cells is activated by cell shrinkage and agents that elevate cAMP levels (16, 38). In contrast, it is found
that
Na+-K+-2Cl
cotransporter activity of endothelial cells and flounder intestine epithelial cells are inhibited by elevation of cAMP and cGMP (23, 36).
Studies of the
Na+-K+-2Cl
cotransporter have also shown that stimulation of the cotransporter by
hormones is mediated by Ca2+ in
endothelial cells (13, 23) and salivary acinar cells (29). However,
little is known about regulation and function of the Na+-K+-2Cl
cotransporter in neurons. The only neuronal preparation that has been
studied in depth is the squid giant axon. Bumetanide-sensitive cotransport activity in the squid giant axon, with a reported stoichiometry of
2Na+:1K+:3Cl
,
requires ATP and is regulated by intracellular
Cl
concentratoin (3, 30).
This process appears to be involved in
phosphorylation-dephosphorylation of the
Na+-K+-Cl
cotransporter (2). In pheochromocytoma PC-12 cells,
Na+-K+-2Cl
cotransporter is stimulated by nerve growth factor but inhibited by
phorbol ester-mediated protein kinase C activation and agents that
raise intracellular cAMP (15).
Activation of NMDA receptors and mGluRs causes an increase in
intracellular Ca2+ concentration
([Ca2+]i)
in SH-SY5Y cells (20). mGluR-receptor agonists
L-AP-4 and glutamate induced a
furosemide-sensitive cell swelling that was accompanied by a transient
increase in
[Ca2+]i
in type 1 astroglial cells (9). In the present study, we found NMDA to
induce a significant stimulation of NKCC1 protein activity, which was
specifically inhibited by MK-801. Taken together, glutamate could
cause an increase in
[Ca2+]i
of SH-SY5Y cells via NMDA-gated
Ca2+ channels and consequently
stimulate
Na+-K+-2Cl
cotransporter. In the present study,
trans-ACPD was also found to
significantly elevate
Na+-K+-2Cl
cotransporter activity in a time- and dose-dependent manner (Fig. 5A, Table 1). Therefore,
mGluR-mediated changes in Ca2+
and/or cAMP in SH-SY5Y cells could conceivably stimulate
Na+-K+-2Cl
cotransporter activity. Alteration of
Ca2+-cAMP second messenger
pathways by glutamate can modulate activities of protein kinases and
phosphatase and consequently regulate the function of the cotransport
system. It was recently reported that glutamate stimulates
Na+-K+-ATPase
activity by upregulating
Ca2+-dependent phosphatase
calcineurin, which appears to maintain the
phosphorylation-dephosphorylation state of
Na+-K+-ATPase
together with protein kinase C (18). It will be interesting in future
studies to investigate whether the glutamate-mediated effect involves
changes in phosphorylation of
Na+-K+-2Cl
cotransporter in neuronal cells.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Peter Lipton for many helpful discussions and for
reading the manuscript. We also thank Gui Su for excellent technical
assistance in protein deglycosylation experiments. The SH-SY5Y cell
line was kindly provided by Dr. John A. Payne.
 |
FOOTNOTES |
This work was supported in part by Scientist Development Grant 9630189N
from the National Center Affiliate of the American Heart Association
(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.
Address for reprint requests: D. Sun, Dept. of Neurological Surgery,
School of Medicine, University of Wisconsin, F4/311, Clinical Science
Center, 600 Highland Ave., Madison, WI 53792.
Received 19 December 1997; accepted in final form 3 June 1998.
 |
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