1Department of Neurological Surgery, 2Department of Physiology, and 3Department of Surgery, University of Wisconsin Medical School, Madison, Wisconsin 53792
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ABSTRACT |
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Schomberg, Stacey L.,
Gui Su,
Robert A. Haworth, and
Dandan Sun.
Stimulation of Na-K-2Cl Cotransporter in Neurons by Activation of
Non-NMDA Ionotropic Receptor and Group-I mGluRs.
J. Neurophysiol. 85: 2563-2575, 2001.
In a
previous study, we found that
Na+-K+-2Cl
cotransporter in immature cortical neurons was stimulated by activation
of the ionotropic N-methyl-D-aspartate (NMDA)
glutamate receptor in a Ca2+-dependent manner. In
this report, we investigated whether the Na+-K+-2Cl
cotransporter in immature cortical neurons is stimulated by non-NMDA glutamate receptor-mediated signaling pathways. Expression of the
Na+-K+-2Cl
cotransporter and metabotropic glutamate receptors (mGluR1 and 5) was
detected in cortical neurons via immunoblotting and immunofluorescence staining. Significant stimulation of cotransporter activity was observed in the presence of both
trans-(±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid
(trans-ACPD) (10 µM), a metabotropic glutamate
receptor (mGluR) agonist, and (RS)-3,5-dihydroxyphenylglycine
(DHPG) (20 µM), a selective group-I mGluR agonist. Both
trans-ACPD and DHPG-mediated effects on the cotransporter
were eradicated by
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-AM, a Ca2+ chelator. In addition,
DHPG-induced stimulation of the cotransporter activity was inhibited in
the presence of mGluRs antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) (1 mM) and also with selective mGluR1 antagonist
7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester
(CPCCOEt) (100 µM). A DHPG-induced rise in intracellular Ca2+ in cortical neurons was detected with
Fura-2. Moreover, DHPG-mediated stimulation of the cotransporter was
abolished by inhibition of Ca2+/CaM kinase II.
Interestingly, the cotransporter activity was increased by activation
of
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)
receptor. These results suggest that the
Na+-K+-2Cl
cotransporter in immature cortical neurons is stimulated by group-I mGluR- and AMPA-mediated signal transduction pathways. The effects are
dependent on a rise of intracellular
Ca2+.
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INTRODUCTION |
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Na+-K+-2Cl
cotransporter (NKCC) has been suggested to contribute to an active
intracellular Cl
accumulation in hippocampal
neurons, dorsal root ganglion (DRG), and sympathetic ganglion neurons
(Alvarez-Leefmans et al. 1988
; Ballanyi and
Grafe 1985
; Hara et al. 1992
; Misgeld et
al. 1986
). The steady-state intracellular
Cl
activity levels in DRG cells depend on the
simultaneous presence of extracellular Na+ and
K+ (Alvarez-Leefmans et al. 1988
).
After Cl
is blocked by
Na+-K+-2Cl
cotransporter inhibitor bumetanide (Alvarez-Leefmans et al.
1988
). A recent study by Sung et al. (2000)
reported that DRG neurons from NKCC1 knock-out mice have a more
negative EGABA than one in wild-type
neurons. The intracellular Cl
concentration in
NKCC1 null cells is significantly reduced, and an absence of
GABA-mediated depolarization was found in NKCC1 mutant DRG neurons
(Sung et al. 2000
). Therefore the
Na+-K+-2Cl
cotransporter in neurons may play an important role in determination of
postsynaptic responses to presynaptic stimulation, as well as the
inhibitory neurotransmitter GABA. GABA, the major inhibitory transmitter in the adult mammalian CNS (Krnjevic 1976
),
depolarizes and excites neuronal membranes in neonatal neurons
(Cherubini et al. 1991
; Luhmann and Prince
1991
). The depolarizing effect of GABA is believed to result
from activation of the GABAA receptor, along with
the consequent efflux of Cl
, which transiently
drives the resting potential toward the Cl
equilibrium potential (Cherubini et al. 1991
).
The depolarizing effect of GABA is of functional importance during
neuronal maturation and differentiation. Depolarizing action of GABA
subsequently activates N-methyl-D-aspartate
(NMDA) and voltage-gated Ca2+ channels that leads
to a rise in intracellular Ca2+ and activation of
a wide range of intracellular cascades (Ben-Ari et al.
1997). Thus GABA acts as an excitatory transmitter in early postnatal stage. Importantly, synergistic excitatory actions of GABAA and glutamergic NMDA receptor have been
found in the neonatal hippocampas (Ben-Ari et al. 1997
).
The interplay between GABA and glutamate-mediated excitatory actions is
required for the induction of synaptic plasticity during the
development (Belhage et al. 1998
; Ben-Ari et al.
1997
).
Despite an important role of the
Na+-K+-2Cl
cotransporter in the accumulation of intracellular
Cl
, little is known of how the cotransporter is
regulated in neurons. In a previous study, we found that the
cotransporter activity in immature cortical neurons is elevated by a
reduction of intracellular Cl
(Sun and
Murali 1999
). In addition, activation of an ionotropic glutamate NMDA receptor stimulates the cotransporter activity in rat
cortical neurons and human neuroblastoma SH-SY5Y cells (Sun and
Murali 1998
, 1999
). This stimulation is
specifically inhibited by NMDA receptor antagonists, MK801 and
ifenprodil (Sun and Murali 1998
, 1999
).
It remains unknown whether
Na+-K+-2Cl
cotransporter activity in cortical neurons is stimulated by non-NMDA ionotropic or metabotropic glutamate receptor (mGluR)-mediated signal
transduction pathways. mGluRs represent a large family of
G-protein-coupled receptors that activate multiple second-messenger systems. As a result, mGluRs affect many cellular processes that modulate voltage- and ligand-gated ion channels and contribute to
synaptic plasticity (Hollmann and Heinemann 1994
). This
report demonstrates that both non-NMDA ionotropic
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)
receptor- and group-I mGluR-mediated signal transduction pathways
stimulate the
Na+-K+-2Cl
cotransporter in immature cortical neurons. The effects of AMPA and
group-I mGluR on the cotransporter may result in re-accumulation of the
intracellular Cl
and thus reinforce the
depolarizing action of GABA in immature cortical neurons. In addition,
the cotransporter could also contribute to K+
re-accumulation after triggering of action potentials.
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METHODS |
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Primary culture of rat cortical neurons
Dissociated cortical neuron cultures were prepared using the
established method described by Hansson et al. (1984),
with a modification as described in the previous study (Sun and
Murali 1999
). Fetuses were removed at embryonic day
17 from pregnant rats (Sprague- Dawley). The cortices were rapidly
dissected in ice-cold Hanks balanced salt solution (HBSS). Cortical
tissue was incubated in a 10-ml trypsin solution (0.5 mg trypsin/ml in HBSS) for 25 min at 37°C. The tissue was then rinsed with HBSS and
resuspended in Eagle's modified essential medium (EMEM) supplemented with 10% fetal bovine serum and 10% horse serum. The dissociated cells were obtained by manual trituration followed by filtration through a strainer (70 µm). Viable cells (1 × 106 cells/well) were plated in 24-well plates
coated with poly-D-lysine. Cultures were maintained in a
5% CO2 atmosphere at 37°C. After 4 days,
cultures were treated with 4 × 10
6 M cytosine-1-
-D
arabinofuranoside (an anti-mitotic agent) to suppress the growth of
dividing astroglial cells. Seventy-two hours later, cultures were refed
with fresh EMEM supplemented with 10% fetal bovine serum and 10%
horse serum. Experiments were routinely performed on 11 day-old
cultures in vitro (DIV 11), unless otherwise as indicated. Seventy to
90% of the cells in culture yielded by this preparation were neuronal
cells, as described before (Sun and Murali 1999
).
K+ influx determination
Na+-K+-2Cl
cotransporter activity was measured as bumetanide-sensitive
K+ influx, using 86Rb as a
tracer for K+ (Sun and Murali
1998
, 1999
). Cultured neurons were equilibrated for 10-30 min at 37°C with an isotonic
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered minimal essential medium (MEM, 290 mOsm). 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 5 min in HEPES-MEM containing either 0 or 10 µM
bumetanide. A linear 86Rb influx curve
(r of 0.98) was obtained when it was assayed for 1, 2, 3, 4, and 7 min. 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 terminated by rinsing cells with
ice-cold 0.1 M MgCl2. Radioactivity of the cells,
which were 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 in nanomole 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 throughout the study, and protein
content was measured in each sample using a method described by
Smith et al. (1985)
. Statistical significance in the
study was determined by ANOVA (Bonferroni/Dunn) at a confidence level
of 95% (P < 0.05).
Measurement of changes of intracellular Ca2+
Cultured neurons grown on poly-D-lysine-coated
coverslips were loaded at room temperature for 1 h in HEPES-MEM
containing 5 µM fura-2 acetoxymethyl ester (AM). Subsequently, the
coverslips were rinsed with HEPES-MEM. The coverslip was put into a
cuvette at a 30° angle relative to excitation light path.
Fluorescence was measured using a spectrophotometer (Spex fluorescence
spectrophotometer CM111) at room temperature to minimize fura-2 dye
loss from cells. 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 previous studies (Haworth and
Redon 1998; Su et al. 2000
). The fluorescence
intensity with 1 mM Mn2+ was subtracted from the
value measured in the absence of Mn2+. The
360/380 ratio of the subtracted values was then calculated (Grynkiewicz et al. 1986
; Haworth and Redon
1998
). All solutions were perfused into 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.
Gel electrophoresis and western blotting
Cortical neurons grown on culture dishes were washed with
ice-cold phosphate-buffered saline (PBS, pH 7.4) that contained 2 mM
EDTA and protease inhibitors, as described previously (Sun et
al. 1995). Cells were scraped from dishes and suspended in PBS,
then lysed by 30 s of sonication at 4°C by an ultrasonic processor (Sonics and Materials, Danbury, CT). Cellular debris was
removed by a brief centrifugation at 420 g for 5 min to
obtain cellular lysates. Protein content of the cellular lysate was
determined (Smith et al. 1985
). Isolation of brain
cortex crude membrane from rats was done by homogenization and
centrifugation (Hillered and Ernster 1983
). Samples were
denatured in SDS reducing buffer (1:1 by volume) and heated at 37°C
for 15 min before gel electrophoresis. The samples and prestained
molecular mass markers (Bio-Rad) were then electrophoretically
separated on 6% SDS gels (Laemmli 1970
), and the
resolved proteins were electrophoretically transferred to a
polyvinylidene fluoride (PVDF) membrane (0.45 µm, Millipore Corporation, 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 incubated overnight with a primary antibody. The following day,
the blots were rinsed with TBS and incubated with horseradish peroxidase-conjugated secondary IgG for 1 h. The bound secondary antibody was visualized using the enhanced chemiluminescence assay (ECL, 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. (1995)
.
Immunofluorescence staining
Cultured cells grown on poly-D-lysine-coated coverslips were rinsed with PBS (pH 7.4) and fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. After rinsing, cells were incubated in blocking solution (10% normal goat serum, 0.4% Triton X-100 and 1% bovine serum albumin in PBS) for 1 h and then incubated with a primary antibody in blocking solution overnight at 4°C. Cells were rinsed with PBS and incubated with either Texas Red- or FITC-conjugated secondary antibodies for 1 h. The images of the cells were captured by the laser-scanning confocal microscope (BioRad MRC 1000) located in the University of Wisconsin-Madison W. M. Keck Neural Imaging Laboratory. The microscope scanhead was transversely connected to an inverted Nikon Diaphot 200. The laser was a 15-mM krypton/argon mixed gas air-cooled laser, which emits a strong line in exact co-alignment at 488 and 568 nm. The 522DF32 filter block was used for FITC and 605DF32 for rhodamine/Texas red signals. The transmitted green and red emitted light signals were directed to the respective photomultiplier tubes. The BioRad 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.
Materials
Bumetanide and cytosine-1--D arabinofuranoside were purchased
from Sigma (St. Louis, MO). EMEM and HBSS were from Mediatech Cellgro
(Herndon, VA). The mGluR agonist
trans-(±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid
(trans-ACPD) and the phosphatase inhibitor calyculin
A were purchased from Research Biochemicals International (Natick, MA). A CaM kinase II inhibitor KN62, its inactive control KN92, the general
kinase inhibitor K252a, and phorbol-12-myristate-13-acetate (PMA) were
from Calbiochem (San Diego, CA). Selective group-I mGluR agonist
(RS)-3,5-dihydroxyphenylglycine (DHPG), mGluR antagonist (RS)-1-aminoindan1,5-dicarboxylic acid (AIDA), selective group-I mGluR antagonist 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt), AMPA, and non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were from Tocris Cookson (Ballwin, MO). Fetal bovine and horse sera were obtained from Hyclone
Laboratories (Logan, UT). 86RbCl was purchased
from NEN Life Science Products (Boston, MA). Rabbit anti-mGluR1
and
anti-mGluR5 polyclonal IgGs were from Chemicon International (Temecula,
CA), and rabbit anti-GABAA receptor
1 antibody
was from Upstate Biotech (Lake Placid, NY).
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RESULTS |
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Expression of group-I mGluRs in cortical neurons
Group-I mGluRs (mGluR1 and mGluR5) are coupled to phospholipase C
(PLC) and increase the synthesis of inositol 1,4,5-triphosphate (IP3) (Hollmann and Heinemann
1994). Our immunoblotting analysis reveals that cortical
neurons cultured for 11 days in vitro expressed ~149 kDa mGluR1a and
~141 kDa mGluR5 (Fig.
1, A and
B). These results are in agreement with the reported
molecular weight mass of mGluR1 and mGluR5 (145-150 kDa)
(Casabona et al. 1997
). As a positive control, an
abundant expression of these two proteins was detected in crude
membrane preparation of adult rat brain. The same immunoblot was
reprobed with T4 anti-cotransporter antibody that recognizes both NKCC1
and NKCC2 isoforms, and Fig. 1C shows expression of the
Na+-K+-2Cl
cotransporter (NKCC1) in cortical neuron cultures. Since the Na+-K+-2Cl
cotransporter is important in regulation of intracellular
Cl
that could affect the GABA-mediated
responses in neurons, we have also investigated whether expression of
the GABAA receptor proteins could be detected in
the immature cortical neuron cultures. As shown in Fig. 1D,
a sharp protein band of ~52 kDa was found in cortical neuron cultures
using a specific antibody against the GABAA
receptor
1 subunit. Using adult rat cortex as a positive control, a
similar expression level of the
1 subunit was detected in cortical
tissue.
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To further reveal the cellular localization of the mGluRs and
cotransporter proteins, we stained cells with anti-mGluR1a or anti-mGluR5 antibodies as well as with anti--tubulin antibody. As
shown in Fig. 2A, cultured
neurons were intensely stained with
-tubulin antibody (type III, a
neuron-specific form). The same slide was double stained with
polyclonal anti-mGluR1a antibody. The immuno-reactive signals with
anti-mGluR1a antibody were found in cortical neurons (Fig.
2B). Colocalization of mGluR1a with
-tubulin was detected
in cortical neurons (yellow, Fig. 2C). A similar cellular
colocalization pattern was found for mGluR5 and
-tubulin (Fig. 2,
D-F). These data further confirmed expression of the mGluRs
in cultured cortical neurons. Interestingly, an abundant expression of
the
Na+-K+-2Cl
cotransporter (Fig. 2G) and mGluR1a (Fig. 2H) was
colocalized in cortical neurons (yellow, Fig. 2I). The
colocalization of the cotransporter and mGluR5 was found in cortical
neurons (Fig. 2, J-L). Insets in Fig. 2,
F and L, represent a negative control study in
which a primary antibody was omitted and either FITC-conjugated goat
anti-mouse IgG (F) or Texas Red-conjugated goat anti-rabbit IgG (L) was used. The rest of the procedures were the same
as in Fig. 2, A-L. This demonstrates that the images shown
in Fig. 2, A-L, are specific immunoreactive signals.
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Stimulation of Na+-K+-2Cl
cotransporter activity in cortical neurons via activation of group-I
mGluRs
The effect of activation of mGluRs on the cotransporter activity
was examined by exposing cells to trans-ACPD, an agonist of
group-I and group-II mGluRs. The
Na+-K+-2Cl
cotransporter activity was assessed by measuring bumetanide-sensitive K+ influx. Figure
3A shows that
bumetanide-sensitive K+ influx rate was 9.11 ± 0.99 nmol/mg protein/min (mean ± SE) under control conditions.
In the presence of 10 µM trans-ACPD, cotransporter activity was increased by approximately 2.6-fold (23.79 ± 3.30 nmol/mg protein/min, P < 0.05). Trans-ACPD
is an agonist of both group-I and group-II mGluRs, and it could
modulate the cotransporter either through stimulation of the
PLC/IP3-mediated pathways and/or protein kinase C
(PKC; group-I) or by down-regulation of cAMP (group-II)
(Hollmann and Heinemann 1994
). If the
trans-ACPD-mediated effect were via the group-I
mGluRs/IP3-mediated pathways, it would be dependent on changes of
intracellular Ca2+. To test this,
Ca2+ chelator
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid
(BAPTA)-AM was employed to prevent an increase of intracellular
Ca2+, and the effect of trans-ACPD was
then assessed. In the presence of the Ca2+
chelator BAPTA-AM (10 µM), the trans-ACPD-mediated
stimulation was abolished (Fig. 3A, P < 0.05). Unaccompanied, the Ca2+ chelator BAPTA-AM
substantially reduced the basal level of the cotransporter activity;
however, this effect was not statistically significant
(P > 0.05). The Ca2+ dependency
of the trans-ACPD-mediated effect suggests that it acts
primarily through the group-I mGluRs. This view was further supported
by a lack of effect of elevation of cAMP by forskolin on the
cotransporter activity. The levels of the bumetanide-sensitive K+ influx were 12.7 ± 1.34 nmol/mg
protein/min in control conditions and 12.10 ± 1.20 nmol/mg
protein/min in the presence of 50 µM forskolin (n = 5, P > 0.05). In the presence of both forskolin and
trans-ACPD, the cotransporter activity was 20.58 ± 1.28 nmol/mg protein/min, which was not statistically different from
one in the presence of trans-ACPD alone (23.78 ± 3.30 nmol/mg protein/min, Fig. 3A). This further suggests a role
of the group-I mGluRs.
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To further investigate the role of the group-I mGluRs on the
cotransporter, the effect of (RS)-3,5-DHPG, a selective group-I mGluRs
agonist, was tested. The bumetanide-sensitive K+
influx in cortical neurons was significantly increased in the presence
of 20 µM (RS)-3,5-DHPG, and this effect was abolished by the
Ca2+ chelator BAPTA-AM (Fig. 3B). This
finding was consistent with the results of the trans-ACPD
study. However, DHPG-induced stimulation of the cotransporter activity
is smaller than one triggered by trans-ACPD. The
mechanisms underlying the differences are not clear. As shown in Fig.
3B (inset), the cotransporter activity is
significantly stimulated after exposing cells to DHPG for 1 min. In
addition, a sustained stimulation of the cotransporter was observed
when neurons were exposed to DHPG for 3, 4, 10, or 13 min. To further
confirm that the DHPG-mediated effect is through activation of the
group-I mGluRs in cortical neurons, we evaluated whether the
DHPG-mediated stimulation could be inhibited by two antagonists, AIDA
and CPCCOEt, for the group-I mGluRs. Figure 3C demonstrates
that both AIDA and CPCCOEt significantly inhibited the DHPG-mediated
stimulation of the bumetanide-sensitive K+
influx. CPCCOEt alone had no significant effect on the basal levels of
the cotransporter activity (Fig. 3C, P > 0.05). These results strongly suggest that the
Na+-K+-2Cl
cotransporter in cortical neurons is stimulated by group-I mGluRs.
Changes of intracellular Ca2+ in cortical neurons
In light of these findings, we conducted the following experiments
to determine whether activation of group-I mGluRs causes an increase in
intracellular Ca2+ in cortical neurons.
Intracellular Ca2+ was monitored using a
Ca2+-sensitive dye fura-2, and an increase of
intracellular Ca2+ was reflected by an increase
of the 360/380 ratio. Intracellular Ca2+ remained
unchanged in the presence of control HEPES-MEM (Fig. 4, A and B). After
exposure of cells to 20 µM DHPG, the level of intracellular
Ca2+ began to rise within 2 min and reached a
peak level within 3 min. An oscillatory rise in
Ca2+ was often observed in the presence of DHPG.
The intracellular Ca2+ declined with time and
then remained at a plateau level for 4-9 min (Fig. 4B).
Removal of 20 µM DHPG resulted in a slow recovery of intracellular
Ca2+ to a basal level. The average time for the
sustained rise of the intracellular Ca2+ in the
presence of DHPG is 408.3 ± 38.9 s (n = 7).
It takes 205.4 ± 15.5 s (n = 10) for the
intracellular Ca2+ to return to the basal level
on removal of DHPG. Therefore there are a few minutes when
intracellular Ca2+ level remains elevated despite
removal of extracellular DHPG. Many biological events could occur
within this time period. To evaluate whether the DHPG-induced
intracellular Ca2+ rise is attributable to
group-I mGluRs-mediated pathways, we tested whether this
Ca2+ rise could be blocked by CPCCOEt, the
selective mGluR1 inhibitor. As shown in Fig. 4A, there were
no noticeable changes of the 360/380 ratio when cells were exposed to
both 20 µM DHPG and 100 µM CPCCOEt. To rule out a possibility that
the poor cellular response occurred when cells were challenged by the
second sequential stimulus in Fig. 4A, we compared the
changes of the 360/380 ratio in the first and second exposures of cells
to 20 µM DHPG in another set of experiments (Fig. 4B).
Figure 4B demonstrates that the second peak of the 360/380
ratio change was similar to the first one. We therefore believe that
the reduction of the Ca2+ rise in the second
stimulus in Fig. 4A is owing to inhibition of the group-I
mGluRs by CPCCOEt. Data analysis is summarized in Fig. 4C.
The average height of the first 360/380 ratio peak in the presence of
20 µM DHPG was 0.24 ± 0.04 (n = 4). In the presence of 20 µM DHPG and 100 µM CPCCOEt, the height of the second 360/380 ratio peak was 0.04 ± 0.02 (n = 4, P < 0.05, 2-tailed Student's t-test). In
contrast, in the absence of CPCCOEt, two sequential exposures of 20 µM DHPG gave rise to an average height of the 360/380 ratio changes
of 0.26 ± 0.06 (n = 3) and 0.20 ± 0.07 (n = 3), respectively. The differences between the two
responses were not statistically significant (P > 0.05, 2-tailed Student's t-test). These results further
support our hypothesis that the group-I mGluRs-induced stimulation of
the
Na+-K+-2Cl
cotransporter activity is due to an increase in intracellular Ca2+.
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To further strengthen our argument, we investigated whether elevation of intracellular Ca2+ in neurons via membrane depolarization could stimulate the cotransporter activity. Figure 5A shows that there was a sharp rise in intracellular Ca2+ when neurons were exposed to 75 mM K+. It reached a peak level in 1 min and declined to a sustained plateau phase after 5 min. This high K+-induced Ca2+ rise is primarily a result of Ca2+ influx from the extracellular compartment because the significant increase of Ca2+ was eradicated in the presence of 0 mM Ca2+/EGTA (Fig. 5A). In addition, this high K+-induced Ca2+ influx is reproducible. As shown in Fig. 5A, similar intracellular Ca2+ transient changes were repeatedly observed when a second stimulus was applied to the same monolayer of cells. Moreover, changes of intracellular Ca2+ were also observed when cortical neurons were exposed to high K+ at 37°C (inset of Fig. 5A). A contaminating Ca2+ level of 0.7 µM was measured in Ca2+-free HEPES-MEM without EGTA. Free Ca2+ ions in this Ca2+-free HEPES-MEM plus 100 µM EGTA was calculated to be 0.4 nM (MAXChelator program, Stanford University, Stanford, CA).
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We then examined whether the high K+-induced Ca2+ rise could stimulate the cotransporter activity in cortical neurons. Figure 5B demonstrates that the bumetanide-sensitive K+ influx was significantly increased by high K+ (P < 0.05) and that this stimulation was eliminated in the presence of 0 mM Ca2+/EGTA (P < 0.05). In contrast, the basal activity of the cotransporter in neurons was not significantly affected by 0 mM Ca2+/EGTA (P > 0.05). As shown in Fig. 5B, in the presence of Ca2+/CaM kinase II inhibitor KN62, the high K+-mediated stimulation of the cotransporter was significantly reduced (P < 0.05). Taken together, these data indicate that a rise in intracellular Ca2+ in the presence of high K+ or DHPG is a second messenger in regulation of the cotransporter in cortical neurons.
Effect of AMPA receptor activation on
Na+-K+-2Cl cotransporter activity
The opening of NMDA receptors was found to increase the
cotransporter activity in a Ca2+-dependent manner
(Sun and Murali 1999). In this study, we demonstrated that activation of group-I mGluRs stimulates the
Na+-K+-2Cl
cotransporter activity via a rise in intracellular
Ca2+. These findings prompted us to investigate
whether AMPA receptor-mediated membrane depolarization and/or increase
in Ca2+ permeability would have any effect on the
cotransporter activity. Cortical neurons were exposed to either control
HEPES-MEM or HEPES-MEM containing 25 µM AMPA for 5-10 min. As shown
in Fig. 6A,
Na+-K+-2Cl
cotransporter activity was 11.37 ± 1.10 nmol/mg protein/min under control conditions and increased to 17.27 ± 2.45 nmol/mg
protein/min in the presence of 25 µM AMPA (P < 0.05). This AMPA-induced stimulation can be blocked by non-NMDA
receptor antagonist CNQX (20 µM, Fig. 6A). We suspect that
this effect of AMPA on the cotransporter activity is owing to a rise of
intracellular Ca2+ evoked by activation of AMPA
receptor. Concomitantly, intracellular Ca2+
measurements revealed that a profound elevation of intracellular Ca2+ level occurred on exposing cortical neurons
to 25 µM AMPA (Fig. 6B). The rise of intracellular
Ca2+ remained at a sustained level in the
presence of 25 µM AMPA, and it declined slowly with time when AMPA
was removed from the perfusate. The intracellular
Ca2+ level did not return to the basal level as
it was observed in the cases of high K+ and DHPG
(Figs. 4A and 5A). A second exposure of cells to
AMPA also evoked a rise of intracellular Ca2+
(Fig. 6B). To confirm that AMPA-induced rise of
intracellular Ca2+ is mediated by activation of
the AMPA receptor, the non-NMDA receptor antagonist CNQX was tested in
the following set of experiments. Consistent with the previous
observations, the first exposure of cells to 25 µM AMPA triggered a
substantial rise of intracellular Ca2+ that
declined with time (Fig. 6C). Removal of AMPA did not result in a recovery of the intracellular Ca2+ level to
the basal level. In contrast, subsequent exposure of the cells to 20 µM CNQX completely abolished the Ca2+ rise in
response to the second AMPA stimulus, and no changes of the 360/380
ratio was found under these conditions (Fig. 6, C and
D). These results imply that activation of the AMPA receptor accounts for the rise of intracellular Ca2+ and
subsequent stimulation of the cotransporter activity in cortical neurons. Moreover, as summarized in Fig. 6D, the peak of
Ca2+ response to the second AMPA stimulus was
reduced by about 50% (0.30 ± 0.04, comparing to the 1st peak
value of 0.70 ± 0.07, P < 0.05). It is known
that AMPA receptor-mediated responses in neurons desensitize rapidly
(Hollmann and Heinemann 1994
; Otis et al.
1996
). Thus this decrease in changes of intracellular
Ca2+ could reflect AMPA receptor desensitization.
|
Effect of phosphorylation/dephosphorylation on the cotransporter activity
It is believed that the
Na+-K+-2Cl
cotransporter is activated when the cotransporter is phosphorylated at
multiple serine (Ser) and threonine (Thr) residues (Haas and
Forbush 1998
; Lytle 1998
; Russell
2000
). To determine whether this scenario occurs in group-I mGluRs-mediated signal transduction cascade, we first evaluated whether
the basal level of cotransporter activity in cortical neurons was
altered when either phosphatases or kinases were inhibited. As shown in
Fig. 7, DHPG consistently induced a
statistically significant stimulation of the cotransporter activity as
described above. In contrast, inhibition of phosphatases (pp-1 and
pp-2A) by calyculin A (25 nM) significantly stimulated cotransporter activity by twofold (from 14.53 ± 0.96 to 29.94 ± 1.95 nmol/mg protein/min, P < 0.05). These results suggest
that the cotransporter in cortical neurons is indeed regulated by
protein phosphorylation. K252a, a general kinase inhibitor with a broad
spectrum (with a potency for CaM kinase > PKA > PKC), has
been reported to block a nerve growth factor-induced
stimulation of the
Na+-K+-Cl
transporter in pheochromocytoma PC-12 cells (Leung et al.
1994
). In cortical neurons, bumetanide-sensitive
K+ influx rate was 20.84 ± 1.37 nmol/mg
protein/min in the presence of 20 µM DHPG. However, with both DHPG
(20 µM) and K252a (40 nM), the cotransporter activity was reduced to
10.39 ± 1.66 nmol/mg protein/min, the latter was not
statistically different from the control value (14.53 ± 0.96 nmol/mg protein/min, P > 0.05, Fig. 7). These data
suggest that a kinase-mediated phosphorylation may be involved in
group-I mGluR-induced effect.
|
To further elucidate possible mechanisms that underlie the group-I mGluR-mediated effect on the cotransporter, a role of Ca2+/CaM kinase II was examined. Ca2+/CaM kinase II is an abundant Ser/Thr kinase in neurons. In this study, we used KN62, a cell-permeable selective inhibitor of CaM kinase II. As shown in Fig. 8, in the presence of both DHPG and KN62, DHPG-mediated stimulation of the cotransporter activity was abolished (P < 0.05). In contrast, the effect of DHPG was not significantly altered by an inactive Ca2+/CaM kinase II inhibitor KN92 (P > 0.05). These results further support the notion that activation of Ca2+/CaM kinase II is involved in the DHPG-induced stimulation of the cotransporter activity.
|
It is known that agents that generate IP3 normally activate PKC through the concomitant generation of diacylglycerol. Therefore PKC could play a role in DHPG-mediated signal transduction. To test this possibility, we measured the effect of PKC inhibitor RO-32-0432 on the DHPG-mediated stimulation of the cotransporter activity. In this set of experiments (Fig. 9), 20 µM DHPG caused a significant stimulation of the cotransporter activity (from a basal level of 11.37 ± 1.10 to 17.85 ± 2.38 nmol/mg protein/min, P < 0.05, n = 3-6). In the presence of both DHPG (20 µM) and a selective cell-permeable PKC inhibitor RO-32-0432 (3 µM), the cotransporter activity was 17.19 ± 2.18 nmol/mg protein/min, and these values were not significantly different from one in the presence of DHPG alone (P > 0.05, n = 3). As a positive control, a potent PKC agonist PMA (150 nM) was used to stimulate PKC in cortical neurons. It was shown in Fig. 9 that the cotransporter activity was significantly stimulated by PMA, and this stimulation was abolished by PKC inhibitor R0-32-0432. Moreover, R0-32-0432 had no significant effect on the basal levels of the cotransporter activity. This study suggests that stimulation of the cotransporter activity by DHPG does not involve activation of PKC.
|
Contribution of astrocytes to 86Rb influx and intracellular Ca2+ measurements
Our cortical neuron culture preparation contains ~70-90%
neurons and 10-30% nonneuronal cells (primarily astrocytes and a few
oligodendrocytes). Neuronal survival and differentiation require input
from astrocytes. Therefore it is impossible to maintain a pure cortical
neuron culture without astrocytes. The astrocytes' contribution to the
bumetanide-sensitive 86Rb influx and
intracellular Ca2+ measurements in cortical
neuronal cultures in this study is minimum and negligible. This
argument is based on the following information. 1)
Differences in changes of the intracellular Ca2+
under high K+: the changes of 360/380 ratio of
fura-2 in astrocyte cultures (95% purity) were 0.26 ± 0.08 (n = 7) under high K+ (Su
et al. 2000). In contrast, we have observed much bigger changes of intracellular Ca2+ in cortical neuron cultures
with a mean value of 1.84 ± 0.14 (n = 5). If
there were a 30% of astrocyte contamination to the Ca2+ changes in neuronal cultures, it would be
only ~4% of the total measured Ca2+ changes
(0.26 × 0.3/1.84), and this value is within the range of
experimental variations. Therefore the astrocytes' contribution in
intracellular Ca2+ measurements is negligible.
2) No statistically significant effect of DHPG on the
cotransporter activity in astrocytes. The basal levels of the
bumetanide-sensitive K+ influx were 22.7 ± 4.8 nmol K+/mg protein/min in cortical astrocyte
cultures (Su et al. 2000
), while 12.73 ± 1.70 nmol
K+/mg protein/min was detected in cortical neuron
cultures. If there were a 30% contamination of astrocyte in the
culture (in a worst-case scenario), the astrocyte
Na+-K+-2Cl
cotransport could contribute 6.81 nmol K+/mg
protein/min to the basal levels of the bumetanide-sensitive K+ influx. We have conducted additional
experiments to investigate whether activation of mGluRs in astrocyte
cultures could stimulate the cotransporter activity. Although mGluRs
are expressed in astrocytes (Gallo and Ghiani 2000
), no
significant effect of 20 µM DHPG was found on the cotransporter
activity in astrocyte cultures (33.96 ± 3.15 nmol
K+/mg protein/min vs. 23.84 ± 4.68 nmol
K+/mg protein/min in the control group,
P = 0.13, n = 5-7). Taken together,
the bumetanide-sensitive 86Rb influx and
intracellular Ca2+ measurements reported in this
study are signals primarily from neurons in the cortical neuronal cultures.
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DISCUSSION |
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Stimulation of Na+-K+-2Cl
cotransporter activity by group-I mGluRs
In our previous study, a development-dependent expression of the
cotransporter was detected in cortical neurons and an approximately 145-kDa cotransporter protein was revealed in cortical neurons (Sun and Murali 1999). We also found that cotransporter
activity was significantly stimulated by a decrease of intracellular
Cl
via either opening of GABA-activated
Cl
channels or depletion of the intracellular
Cl
(Sun and Murali 1999
).
Despite the important role of the cotransporter in regulation of
intracellular Cl
, little is known about the
cotransporter protein regulation in neurons. We reported that the
Na+-K+-2Cl
cotransporter activity in human neuroblastoma SH-SY5Y cells is stimulated by the excitatory neurotransmitter glutamate (Sun and Murali 1998
). Glutamate is the major excitatory
neurotransmitter in the CNS and exerts its effect by activation of a
family of heterogeneous glutamate receptors that couple to
second-messenger systems (Hollmann and Heinemann 1994
).
We found that activation of ionotropic NMDA receptor stimulates the
Na+-K+-2Cl
cotransporter in cortical neurons (Sun and Murali 1999
).
In the present study, we investigated whether mGluR-mediated signal
transduction pathways affect the cotransporter in cortical neurons. Our
results indicate that activation of the group-I mGluRs stimulates the cotransporter in these cells. This conclusion is based on the following
evidence. First, both mGluR 1a and mGluR 5 are expressed in the
cultured immature cortical neurons. Second, activation of the group-I
mGluRs by its specific agonist DHPG significantly stimulates the
cotransporter activity, and this DHPG-induced stimulation is
specifically inhibited by the group-I mGluRs antagonists AIDA and
CPCCOEt. Since CPCCOEt is selective for mGluR1 and poorly blocks
mGluR5, the data suggest that the DHPG-mediated effect is, at least in
part, via activation of mGluR1.
Our results demonstrate that the group-I mGluRs-mediated effect on the
cotransporter is attributable to a rise in intracellular Ca2+. This view is supported by the two major
findings: 1) both trans-ACPD and DHPG-mediated
stimulation of the cotransporter is eradicated by BAPTA-AM, and
2) an increase in intracellular Ca2+
by DHPG is detected in cortical neurons, and this change in
Ca2+ is abolished when the group-I mGluRs are
blocked by CPCCOEt. Activation of the mGluRs results in synthesis of
IP3, which in turn releases
Ca2+ from intracellular
Ca2+ stores via IP3
receptors (Schoepp et al. 1990). Recently, activation of
mGluR1s was found to affect voltage-dependent
Ca2+ channels in cultured cerebellar neurons
(Chavis et al. 1996
). In hippocampal stratum
oriens/alveus interneurons, mGluRs agonist 1S,3R-ACPD induces
oscillatory membrane depolarizations and rises in intracellular
Ca2+ (Woodhall et al. 1999
). The
oscillatory responses generated by ACPD in these cells require a
coupling of Ca2+ entry through voltage-gated
Ca2+ channels and Ca2+
release from intracellular stores (Woodhall et al.
1999
). In our present study, we repeatedly observed oscillatory
Ca2+ rises evoked by DHPG in cultured cortical
neurons. Whether similar mechanisms are responsible for the
Ca2+ oscillation in this study remains to be determined.
In this study, depolarization of the plasma membrane by high
K+ results in a profound increase in
intracellular Ca2+, which leads to a greater
degree (2.4-fold) of the stimulation of the cotransporter activity.
Removal of extracellular Ca2+ abolishes the high
K+-induced intracellular
Ca2+ rise as well as stimulation of the
cotransporter activity. The stimulation of the cotransporter by high
K+ is also inhibited by
Ca2+/CaM kinase inhibitor KN62. This suggests
that the high K+-mediated effects depend on
Ca2+ entry via membrane depolarization. However,
it is not yet known whether the stimulation of the cotransporter
activity under these conditions is a response to a rise of
intracellular Ca2+ or a secondary response to a
decreased intracellular Cl. The cotransporter
is known to be activated by a decrease in intracellular
Cl
in cortical neurons (Sun and Murali
1999
), squid giant axon (Breitwieser et al.
1990
; Russell 2000
), and epithelium
(Lytle and Forbush 1992
). For instance, it is possible
that changes of intracellular Cl
could occur
under high K+-induced depolarization and that in
turn may have an effect on the cotransporter activity. Whether such a
secondary mechanism leads to the stimulation of the cotransporter
activity in rat cortical neurons requires further studies, although
that elevation of extracellular
[K+]o to 60-90 mM did
not decrease and actually increased intracellular Cl
in mouse cortical neurons (White et
al. 1992
).
Role of protein kinase(s) in group-I mGluRs-mediated effects
Protein kinase and protein phosphatase governs activation and
inactivation of the cotransporter, respectively (Haas
1994; Haas and Forbush 1998
; Russell
2000
). Phosphorylation of the
Na+-K+-2Cl
cotransporter by Ser/Thr protein kinases has been reported to stimulate
the cotransporter activity in other cells (Altamirano et al.
1995
; Haas 1994
; Lytle 1998
;
Sun and O'Donnell 1996
). Inhibition of phosphatase
activity by okadaic acid has a significant stimulatory effect on the
ion transporter activity in the squid giant axon (Altamirano et
al. 1995
). In the present report, the cotransporter activity in
cortical neurons is enhanced by twofold when phosphatases are blocked
by calyculin A. This implies that kinase(s) and phosphatase(s) under
physiological conditions tightly control the cotransporter activity.
Activation of the group-I mGluRs gives rise to a smaller degree of
stimulation of the cotransporter compared with the effect of calyculin
A. Thus we speculate that activation of the group-I mGluRs may involve
a stimulation of a Ser/Thr protein kinase rather than inhibition of
phosphatase 1A or 2A. This view is supported by the finding that the
DHPG-mediated effect is abolished in the presence of K252a, a broad
spectrum kinase inhibitor with a potency for
Ca2+/CaM kinase > PKA > PKC. In
addition, we found that exposing cortical neurons to forskolin has no
effect on the cotransporter activity. It has been reported that agents
that raise cAMP or stimulate PKC lead to an inhibition of the
cotransporter in PC12 cells (Leung et al. 1994
). We
found that DHPG-mediated stimulation of the cotransporter is not
blocked by PKC inhibitor RO-32-0432, but RO-32-0432 does block the
PMA-mediated stimulation. Taken together, the effect of K252a on the
DHPG-mediated stimulation of the cotransporter is likely via inhibition
of Ca2+/CaM kinase, rather than PKA or PKC. Our
finding further shows that inhibition of Ca2+/CaM
kinase II by KN62 eliminates the DHPG-mediated effect on the
cotransporter. In contrast, an inactive form of the CaM kinase inhibitor KN92 has no significant effect. These results strongly suggest that DHPG-mediated effect involves an activation of
Ca2+/CaM kinase II. A
Ca2+/CaM-dependent regulation of the
cotransporter is found in Ehrlich Ascites tumor cells during regulatory
volume increase (Jensen et al. 1993
). However,
further studies are required to determine whether the
Ca2+/CaM-dependent pathways directly or
indirectly involve a phosphorylation of the cotransporter protein in
rat cortical neurons.
Significance of the glutamate receptor-mediated effects on the
Na+-K+-2Cl cotransporter
The significance of the glutamate non-NMDA or metabotropic
receptor-mediated stimulation of the cotransporter activity in neuronal function is unknown. To our knowledge, this is the first report on this observation. A rapid stimulation of the cotransporter activity by 10 µM glutamate (1 min) has also been observed in SH-SY5Y
neuroblastoma cells (Sun and Murali 1998). Given the
rapid stimulation of the cotransporter activity mediated by glutamate or the glutamate receptor agonists described above, it is possible that
stimulation of the cotransporter is physiologically relevant. A
synergistic interaction between GABAA and NMDA
receptors has been reported in formation of synchronous
Ca2+ oscillations and the synergistic excitatory
actions of GABAA and NMDA receptors in neonatal
brain (Ben-Ari et al. 1997
). Stimulation of the
Na+-K+-2Cl
cotransporter by glutamate in immature cortical neurons could increase
intracellular Cl
that would reinforce the
depolarizing actions of GABA. Moreover, stimulation of the
cotransporter by activation of the glutamate receptors could contribute
to K+ re-accumulation in neurons after triggering
of action potentials. The inward transport of Na+
via the cotransporter could also provide Na+
influx for Na+-K+-ATPase
function (Walz 1992
). In addition, the
glutamate receptor-mediated effect on the cotransporter may be
important under pathophysiological conditions. In our recent study,
administration of the potent ion transporter inhibitor bumetanide in
rat brain significantly reduced infarct volume, neuron death, and brain
edema after focal cerebral ischemia (Yan et al. 2000
).
Sustained elevation of extracellular glutamate concentration occurs in
cerebral ischemia and glutamate-mediated neurotoxicity is important in
neuronal cell death during cerebral ischemia (Choi and Rothman
1990
). Therefore stimulation of the Na+-K+-2Cl
cotransporter via activation of the glutamate receptors could contribute to Na+, Cl
influx, and cell damage. This view is supported by a recent study of
Inglefield and Schwartz-Bloom (1998)
. They showed that
exposure of hippocampal slices to NMDA caused a rise of intracellular
Cl
and that this effect of NMDA was dependent
on the extracellular Na+ and
Cl
(Inglefield and Schwartz-Bloom
1998
). The nature of the Na+ and
Cl
entry pathways after NMDA exposure is not
yet understood. Our findings imply that this could be a result of
stimulation of the Na+-K+-2Cl
cotransporter. Taken together, we report here that the
Na+-K+-2Cl
cotransporter in cortical neurons is regulated by group-I mGluR- and
AMPA receptor-mediated signal transduction pathways. The
mGluRs-mediated stimulation of the cotransporter is dependent on a rise
of intracellular Ca2+ and activation of
Ca2+/CaM kinase II.
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ACKNOWLEDGMENTS |
---|
We thank K. Nash for technical assistance in intracellular Ca2+ measurement. We also thank Dr. Peter Lipton for helpful comments and suggestions.
This work was supported in part by a Scientist Development Grant from National Center Affiliate of American Heart Association (9630189N), National Institute of Neurological Disorders and Stroke Grant RO1NS-38118, a National Science Foundation CAREER award (IBN9981826) to D. Sun, and a grant to the University of Wisconsin Medical School under the Howard Hughes Medical Institute Research Resources Program for Medical Schools. S. L. Schomberg was a recipient of a 2000-2001 Wisconsin/Hilldale Undergraduate/Faculty Research Award.
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FOOTNOTES |
---|
Address for reprint requests: D. Sun, Dept. of Neurological Surgery, University of Wisconsin Medical School, H4/332, Clinical Sciences Center, 600 Highland Ave., Madison, WI 53792 (E-mail: sun{at}neurosurg.wisc.edu).
Received 12 December 2000; accepted in final form 20 February 2001.
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REFERENCES |
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