1Department of Neurological Surgery and 2Department of Physiology, School of Medicine, University of Wisconsin, Madison, Wisconsin 53792
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sun, Dandan and
Sangita G. Murali.
Na+-K+-2Cl cotransporter in
immature cortical neurons: a role in intracellular Cl
regulation. Na+-K+-2Cl
cotransporter has been suggested to contribute to active intracellular Cl
accumulation in neurons at both early developmental
and adult stages. In this report, we extensively characterized the
Na+-K+-2Cl
cotransporter in
primary culture of cortical neurons that were dissected from cerebral
cortex of rat fetus at embryonic day 17. The
Na+-K+-2Cl
cotransporter was
expressed abundantly in soma and dendritic processes of cortical
neurons evaluated by immunocytochemical staining. Western blot analysis
revealed that an ~145-kDa cotransporter protein was present in
cerebral cortex at the early postnatal (P0-P9) and adult stages. There
was a time-dependent upregulation of the cotransporter activity in
cortical neurons during the early postnatal development. A substantial
level of bumetanide-sensitive K+ influx was detected in
neurons cultured for 4-8 days in vitro (DIV 4-8). The cotransporter
activity was increased significantly at DIV 12 and maintained at a
steady level throughout DIV 12-14. Bumetanide-sensitive K+
influx was abolished completely in the absence of either extracellular Na+ or Cl
. Opening of
-aminobutyric acid
(GABA)-activated Cl
channel or depletion of intracellular
Cl
significantly stimulated the cotransporter activity.
Moreover, the cotransporter activity was elevated significantly by
activation of N-methyl-D-aspartate ionotropic
glutamate receptor via a Ca2+-dependent mechanism. These
results imply that the inwardly directed Na+-K+-2Cl
cotransporter is
important in active accumulation of intracellular Cl
and
may be responsible for GABA-mediated excitatory effect in immature
cortical neurons.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Na+-K+-2Cl
cotransporters are important in renal salt reabsorption and in salt
secretion by epithelia (Haas 1994
; O'Grady et al. 1987
). They are also essential in maintenance and
regulation of ion gradient and cell volume in both epithelial and
nonepithelial cells. In Cl
-absorptive and
Cl
-secretive epithelia,
Na+-K+-2Cl cotransporters serve as the major
Cl
entry pathway and function in net transepithelial
movement of salt in concert with Cl
and K+
channels and Na+, K+/ATPase (O'Grady et
al. 1992
). Despite studies on the
Na+-K+-2Cl cotransporter in many cell types,
its physiological function and regulation in neurons are not yet defined.
It has been suggested that inwardly directed
Na+-K+-2Cl cotransporter
contributes to an active intracellular Cl
accumulation in
neurons (Alvarez-Leefmans et al. 1988
; Ballanyi and Grafe 1985
; Hara et al. 1992
; Misgeld
et al. 1986
). Therefore, Na+-K+-2Cl
cotransporter in
neurons may play an important role in determination of postsynaptic
responses to presynaptic stimulation as well as inhibitory
neurotransmitter
-aminobutyric acid (GABA). GABA is the major
inhibitory transmitter in the adult mammalian CNS (Krnjevic 1976
). It inhibits neuronal firing by increasing an inwardly
directed Cl
conductance mediated via GABAA
receptors. However, a different picture is present in neonatal neurons,
where GABA depolarizes and excites neuronal membranes (Cherubini
et al. 1991
; Luhmann and Prince 1991
). Moreover,
GABAA-receptor-mediated depolarizing responses can be
observed in adult hippocampal neurons, dorsal root ganglia as well as
sympathetic ganglion neurons (Alvarez-Leefmans et al.
1988
; Ballanyi and Grafe 1985
; Misgeld et
al. 1986
). These neurons keep their intracellular
Cl
concentration ([Cl
]i) at
levels higher than predicted from a passive distribution and the
Cl
equilibrium potential (ECl) is more
positive than the resting potential (Em). The depolarizing
effect of GABA is believed to result from activation of
GABAA receptor, with the consequent efflux of
Cl
, which transiently drives Em toward
ECl.
Several studies support the notion that
Na+-K+-2Cl cotransporter may
function as an active Cl
transport system and is
responsible for the active accumulation of intracellular
Cl
in these cells. It has been demonstrated that the
steady-state intracellular Cl
activity levels in dorsal
root ganglion cells depend on the simultaneous presence of
extracellular Na+ and K+. The active
reaccumulation of Cl
after Cl
i
depletion is blocked by
Na+-K+-2Cl
cotransporter
inhibitor bumetanide (Alvarez-Leefmans et al. 1988
). In
addition, the depolarizing inhibitory postsynaptic potential of granule
cells is attenuated by furosemide, a less potent inhibitor of the
cotransporter (Misgeld et al. 1986
). These reports imply that the internal chloride activity of an individual neuron is regulated by inwardly directed
Na+-K+-2Cl
cotransporter system.
Currently, only two distinct isoforms of
Na+-K+-2Cl cotransporter (NKCC)
have been identified: NKCC1 (7.0- to 7.5-kb transcript) with a wide
range of tissue distributions (Delpire et al. 1994
; Xu et al. 1994
), and NKCC2 (4.6- to 5.2-kb transcript),
which has only been found in vertebrate kidney (Payne and
Forbush 1995
). Northern blot analysis has demonstrated that the
expression level of NKCC1 mRNA is abundant in brain (Delpire et
al. 1994
; Xu et al. 1994
). In situ hybridization
and immunocytochemical studies have shown that NKCC1 protein is
abundantly present on plasma membranes of neurons in rat neocortex as
well as CA1-CA3 of pyramidal cells in hippocampus (Payne et al.
1998
; Plotkin et al. 1997a
,b
).
In the present study, Na+-K+-2Cl
cotransporter was characterized extensively in primary culture of rat
cortical neurons. We demonstrated that
Na+-K+-2Cl
cotransporter was
expressed abundantly in cortical neurons at an early postnatal stage.
There was a time-dependent upregulation of the cotransporter activity
in cortical neurons during the early postnatal development. Function of
the cotransporter in cortical neurons was regulated by intracellular
Cl
and activation of glutamate ionotropic
N-methyl-D-aspartate (NMDA) receptor.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 here. Fetuses were removed at
embryonic day 17 from pregnant rats (Sprague Dawley). The cortices were dissected rapidly in Hanks balanced salt solution (HBSS) and minced with scissors. Cortical tissue was incubated in a trypsin solution (0.5 mg trypsin/ml in HBSS) for 25 min at 37°C. The tissue then was 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 mechanically trituration followed by
filtration through a strainer (70 µm). Cell viability was determined
by 0.01% eosin Y exclusion. 50-60% of dissociated cells were viable.
Viable cells (1 × 106 cells/well) were plated in
24-well plate coated with poly-D-lysine (10 µg/ml in 0.1 M boric acid). Cultures were maintained in a 5% CO2
atmosphere at 37°C. After 4 days, cultures were treated with
1-4 × 10
6 M cytosine-1-
-D arabinofuranoside (an
antimitotic 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 routinely were performed on 11-day-old cultures in vitro
(DIV) unless otherwise as indicated. The purity of cortical neuronal
culture was determined by immunocytochemical staining for
-tubulin,
a marker protein for neurons. Presence of astrocytes in culture was
determined by expression of glial fibrillary acidic protein. Seventy to
90% of cells in culture yielded by this preparation were neuronal cells.
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
; Sun et al. 1995
; Sun and O'Donnell
1996
). 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 (in 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 preincubated for 5 min in
HEPES-MEM containing either 0 or 100 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 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 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. To measure ouabain-sensitive
K+ influx, 86Rb influx rate was determined in
the presence of either 0 or 1 mM ouabain. Bumetanide and
ouabain-resistant K+ influx was measured in the presence of
both bumetanide (100 µM) and ouabain (1 mM). 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 (Benforroni/Dunn) at
a confidence level of 95% (P < 0.05).
Gel electrophoresis and Western blotting
Cortical neurons grown on culture dishes were washed with
ice-cold phosphate-buffered saline (PBS, pH 7.4), which 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). 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 (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 (1:2 by volume, Bio-Rad) and heated at
37°C for 15 min before gel electrophoresis. The samples and
prestained molecular mass markers (Bio-Rad) then were separated
electrophoretically on 6% SDS gels (Laemmli 1970
), and
the resolved proteins were transferred electrophoretically to a 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, then incubated overnight with a primary antibody. The
blots were rinsed with TBS and incubated with horseradish
peroxidase-conjugated secondary IgG for 1 h. 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)
. Monoclonal antibodies against NMDA
receptor NR-1 or isoform NR-2B were used for analysis of NMDA-receptor expression.
Immunocytochemical 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 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 mounted transversely 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 alignment at 488 nm, and the 522DF32 filter block was used for FITC signals. The transmitted green 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, antineurofilament 200, and antitubulin ( subunit)
monoclonal antibodies, and cytosine-1-
-D arabinofuranoside were
purchased from Sigma (St. Louis, MO). EMEM and HBSS were from Mediatech
Cellgro (Herndon, VA). NMDA and selective NMDA-receptor antagonist
ifenprodil tartrate were purchased from Research Biochemicals International (Natick, MA). Fetal bovine serum and horse serum were
obtained from Hyclone Laboratories (Logan, UT). 86RbCl was
purchased from NEN Life Science Products (Boston, MA). Mouse anti-NMDA
receptor (NR-1) monoclonal IgG was from Chemicon International
(Temecula, CA). Anti-NMDA receptor (NR-2B) monoclonal antibody was
purchased from Transduction Laboratories (Lexington, KY).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Localization of Na+-K+-2Cl
cotransporter in cortical neurons by immunocytochemical staining
To characterize localization of
Na+-K+-2Cl cotransporter in
primary cortical neurons, we performed immunocytochemical staining. Growth of cortical neurons at 11 days in culture is demonstrated in
Fig. 1A. It represents a
characteristic neuronal network composed of differentiated neurons. A
few large flat astrocytes were present, and they were 10-30% of the
total cell population in the culture. To confirm neuronal identity of
the cells, staining for
-tubulin was performed. Expression of
-tubulin in cortical neurons is shown in Fig. 1B. Soma,
dendrites and elongated axons of nerve cells possessed a high-density
of tubulin. Localization of the Na+-K+-2Cl
cotransporter protein
is shown in Fig. 1C. An abundant level of
Na+-K+-2Cl
cotransporter was
detected in both soma and dendritic processes of neurons with T4
anticotransporter monoclonal antibody. Figure 1E represents
a negative control study in which a primary antibody was omitted and
rest of the procedures were same as in Fig. 1, B and
C. This demonstrated that the images shown in Fig. 1,
B and C, were specific immunoreactive signals.
|
Determination of Na+-K+-2Cl
cotransporter activity in cortical neurons
Na+-K+-2Cl cotransporter
activity in cortical neurons was assessed by bumetanide-sensitive
K+ influx. Figure 2 shows
that bumetanide-sensitive K+ influx rate was 14.42 ± 1.65 nmol/mg protein/min (mean ± SE). Na+-K+-2Cl
cotransporter
comprised ~25% of the total unidirectional K+ influx. We
also examined two other K+ uptake pathways,
Na+, K+/ATPase and passive K+
influx. Na+, K+/ATPase activity was determined
by ouabain-sensitive K+ influx in cortical neurons. Passive
K+ influx was measured in the presence of both bumetanide
and ouabain. Data showed that ouabain-sensitive K+ influx
rate and bumetanide and ouabain-resistant K+ influx rate
were 21.49 ± 2.01 and 12.69 ± 1.76 nmol/mg protein/min, respectively (Fig. 2).
|
To further establish that bumetanide-sensitive K+ influx
represents the cotransporter activity in cortical neurons, we evaluated bumetanide-sensitive K+ influx in the absence of either
extracellular Na+ or Cl. Function of the
Na+-K+-2Cl
cotransporter requires
a simultaneous presence of the three transported ions. Thus removal of
either extracellular Na+ or Cl
would abolish
the cotransporter-mediated K+ influx. It is demonstrated in
Fig. 2 that bumetanide-sensitive K+ influx was abolished
completely when either extracellular Na+ was replaced with
an equimolar choline or Cl
was replaced with an equimolar
gluconate. These results further confirm that the bumetanide-sensitive
K+ influx indeed reflects the activity of the
Na+-K+-2Cl
cotransporter in
cortical neurons. Moreover, both total cellular K+ influx
and ouabain-sensitive K+ influx were inhibited
significantly in the absence of extracellular Na+. The
effect on the ouabain-sensitive K+ influx is possibly due
to a reduced intracellular Na+ in the absence of
extracellular Na+. In the absence of extracellular
Cl
, total cellular K+ influx was
significantly reduced (P < 0.05). In contrast, removal of extracellular Cl
did not have any statistically
significant effect on the ouabain-sensitive K+ influx rate.
Upregulation of Na+-K+-2Cl
cotransporter activity in cortical neurons during the early postnatal
development
To investigate a role of the
Na+-K+-2Cl cotransporter in
active Cl
accumulation of neurons during the early
postnatal development, we evaluated the cotransporter activity in
cultures at 4, 6, 8, and 11-14 days in vitro (DIV 4-14). They
correspond to postnatal days 0-10 (P0-P10) because fetuses at
embryonic day 17 were used in the study and gestation period for rat is
21 days. As described in Fig. 3,
Na+-K+-2Cl
cotransporter activity
was 11.61 ± 1.19 nmol/mg protein/min at DIV 4 (P0). There was a
time-dependent upregulation of the cotransporter activity during the
first postnatal week. At DIV 11 (P7), bumetanide-sensitive K+ influx was increased to 16.27 ± 2.25 nmol/mg
protein/min, a 40% increase compared with the activity level at DIV 4 (P0). The cotransporter activity was further elevated to 36.29 ± 4.34 nmol/mg protein/min at DIV 13 (P9, P < 0.05) and
remained at the level of 31.23 ± 5.73 nmol/mg protein/min at DIV
14 (P10).
|
The level of Na+, K+/ATPase activity in cortical neurons was 20.20 ± 3.00 nmol/mg protein/min at DIV 4 (P0). The pump activity was developed further during the early postnatal stage. At DIV 11 (P7), the pump activity was increased to 35.23 ± 2.05 nmol/mg protein/min (P < 0.05), and this steady level was maintained through DIV 14 (P10). In contrast, passive K+ influx pathway, indicated by bumetanide and ouabain-resistant K+ uptake rate, was 7.24 ± 0.23 nmol/mg protein/min at DIV 4 (P0) and remained largely stable during the early postnatal development (P0-P10).
The total unidirectional K+ influx rate was significantly
up-regulated in neonatal neurons (DIV 12 and 13, P < 0.05). The change of the total K+ influx rate showed a
pattern that was similar to the one for bumetanide-and
ouabain-sensitive K+ influx. It implied that an increase in
the total cellular K+ uptake was largely due to an
upregulation of both the cotransporter and pump activities. Taken
together, the results demonstrate that a substantial level of the
Na+-K+-2Cl cotransporter activity
was detected in neonatal cortical neurons at the early postnatal
development. This active
Na+-K+-2Cl
cotransporter system
may be responsible for a higher intracellular Cl
level.
Development-dependent expression of
Na+-K+-2Cl cotransporter in
cortical neurons in the early postnatal stages
An increase in the cotransporter activity occurred in cortical
neurons during the early development (Fig. 3). To further understand the molecular mechanisms underlying this time-dependent change, we
tested whether it was due to an increase in expression of the cotransporter protein during the early postnatal development. Expression of Na+-K+-2Cl
cotransporter was detected with Western blot analysis (Fig.
4A) and quantified by
densitometric analysis (Fig. 4B). Figure 4A shows
that a substantial amount of the cotransporter protein was expressed in
neurons at DIV 4, and it was increased by ~17% at DIV 8 and 40% at
DIV 11 (n = 4, Fig. 4, A and B).
The cotransporter expression was increased further at DIV 13 and
remained high at DIV 14. The pattern of upregulation of the
cotransporter expression is in agreement with the time course of
bumetanide-sensitive K+ influx (Fig. 3). Upregulation
also occurred in
-actin protein expression, a "house-keeping"
protein (Fig. 4, A and B). However, the degree of
the changes in
-actin expression was less. In addition, a
time-dependent increase in expression of
subunit of
Na+/K+ ATPase also was found in neurons at DIV
12-14 (data not shown).
|
In light of these findings, we conducted a similar study to further investigate whether a development-dependent expression of the cotransporter protein occurs in vivo. Expression of the cotransporter protein in cerebral cortex was assessed in rat pups (Sprague Dawley) at postnatal day 0-9 (P0-P9). Western blot analysis shows that a substantial level of the cotransporter protein was found in cortex at P0, and it reached to a steady level at P2-P9 (Fig. 4C).
These results indicate that
Na+-K+-2Cl cotransporter system
in brain cortex is well developed at the early postnatal stage, and our
study suggests that it may play a role in Cl
homeostasis
in immature neurons.
Stimulation of the Na+-K+-2Cl
cotransporter by opening of GABA-activated Cl
channel
We hypothesize that
Na+-K+-2Cl cotransporter is an
essential mechanism in intracellular Cl
accumulation and
contributes to GABA-mediated depolarization in cortical neurons at the
early postnatal stages. If this hypothesis is valid, a decrease in
intracellular Cl
of cortical neurons by opening of
GABA-activated Cl
channel would accelerate the
cotransporter system in response to the loss of intracellular
Cl
. To test this possibility, we examined whether
bumetanide-sensitive K+ influx was stimulated by activation
of GABAA receptor. Cortical neurons were preincubated with
20-100 µM GABA in isotonic HEPES-buffered MEM at 37°C for 5 min.
Control cells were incubated with isotonic HEPES-buffered MEM at 37°C
for 5 min. 86Rb influx subsequently was assayed for 3 min.
Figure 5A shows that
bumetanide-sensitive K+ influx was stimulated significantly
by either 50 or 100 µM GABA (P < 0.05).
GABA-mediated effect (at 100 µM) on the total K+ influx
rate was statistically significant (P < 0.05). To
investigate that GABA-mediated effect was specifically via
GABAA receptor, we performed the following tests. First, we
tested if GABA-induced stimulation of the cotransporter was blocked by
a GABAA receptor antagonist (+)-bicuculline. As shown in
Fig. 5A, 10 µM (+)-bicuculline attenuated GABA-mediated
stimulation of the cotransporter activity. Bumetanide-sensitive
K+ influx was 39.33 ± 5.71 nmol/mg protein/min in the
presence of 100 µM GABA. It was reduced to 22.52 ± 4.11 nmol/mg
protein/min in the presence of both GABA and bicuculline (~43%
reduction). The latter value was not statistically significantly
different from a control of 15.87 ± 3.05 nmol/mg protein/min.
These results suggest GABA-mediated effect on the
Na+-K+-2Cl
cotransporter is
likely due to opening of GABA-activated Cl
channel.
|
To further strengthen this argument, we examined the effect of a
specific GABAA receptor agonist muscimol. Cells were
preincubated with 0-100 µM muscimol for 5 min. Bumetanide-sensitive
K+ influx was evaluated subsequently. As shown in Fig.
5B, bumetanide-sensitive K+ influx was
stimulated significantly by 30 µM muscimol (~93.4%, P < 0.05). It was increased further in the presence of
100 µM muscimol (105%, P < 0.05). In addition, the
total K+ influx rate also was increased by 30-100 µM
muscimol (P < 0.05). Bicuculline (10 µM) inhibited
muscimol-induced stimulation of the cotransporter activity from
59.89 ± 1.44 in the presence of 30 µM muscimol alone to
40.28 ± 6.69 nmol/mg protein/min in the presence of bicuculline
plus muscimol (P < 0.05). This result further supports
that opening of GABA-activated Cl channel stimulates the
Na+-K+-2Cl
cotransporter activity
probably signaled by loss of intracellular Cl
.
Stimulation of the Na+-K+-2Cl
cotransporter by depletion of intracellular Cl
If Na+-K+-2Cl cotransporter
is indeed important in intracellular Cl
regulation, the
cotransporter activity is anticipated to be sensitive to changes in
[Cl
]i, as described in Fig. 5. In this
study, we took another approach to test this hypothesis. A reduction in
[Cl
]i was achieved by preincubating cells
for 30 min in either 10, 20, or 30 mM
[Cl
]o. The different
[Cl
]o was obtained by substitution of
extracellular Cl
in HEPES-MEM buffer with equimolar
methanesulfonate (pH 7.4). Methanesulfonate was used for substitution
of Cl
because it has no effect on Ca2+ ion
activity (Kenyon and Gibbons 1977
). Bumetanide-sensitive K+ influx rate in cells then was examined for 2 min in
HEPES-MEM containing a normal level of 147 mM Cl
.
Reduction of intracellular Cl
by preincubation of cells
with 10-30 mM [Cl
]o significantly
stimulated the cotransporter activity in immature neurons by
140-190%, respectively (P < 0.05, Fig.
6). In addition, the total K+
influx rate also was increased (P < 0.05). This
implies that the Na+-K+-2Cl
cotransporter is an essential Cl
entry pathway in
immature neurons. This study provided a second line of evidence that
Na+-K+-2Cl
cotransporter in
neurons is important in intracellular Cl
regulation.
|
Ca2+-dependent stimulation of
Na+-K+-2Cl cotransporter in
cortical neurons by NMDA receptor
Little is known about regulation of the
Na+-K+-2Cl cotransporter system
in neurons. We have observed that the cotransporter in neuroblastoma
SH-SY5Y cells was stimulated by excitatory neurotransmitter glutamate
(Sun and Murali 1998
). In this study, we extended our investigation in primary culture of cortical neurons. First, effect of
NMDA receptor activation on the cotransporter activity was studied.
Cortical neurons were preincubated with 40 µM NMDA in isotonic
HEPES-buffered MEM at 37°C for 5 min. 86Rb influx
subsequently was assayed for 3 min. The total K+ influx
rate was increased significantly from a basal level of 79.41 ± 4.55 to 102.24 ± 10.71 nmol/mg protein/min in the presence of 40 µM NMDA (P < 0.05). NMDA exerted a profound effect
on the cotransporter activity. The bumetanide-sensitive K+
influx in cortical neurons was significantly elevated by 40 µM NMDA,
from a basal level of 14.90 ± 1.72 to 33.42 ± 4.82 nmol/mg protein/min (P < 0.05, Fig.
7). This result is in an agreement with
our previous finding on the NMDA-mediated stimulation of the
cotransporter in neuroblastoma SH-SY5Y cells (Sun and Murali 1998
).
|
It has been reported that glutamate stimulates the pump activity
in cerebellar neurons (Marcida et al. 1996). We
speculated that Na+, K+/ATPase in cortical
neurons might be stimulated by activation of the NMDA receptor. To test
this possibility, we evaluated effect of NMDA on Na+,
K+/ATPase activity in cortical neurons. As shown in Fig. 7,
Na+, K+/ATPase in neurons was stimulated
significantly by 40 µM NMDA. The ouabain-sensitive K+
influx was increased from 48.94 ± 4.45 to 71.35 ± 9.51 nmol/mg protein/min (P < 0.05). In contrast,
activation of the NMDA receptor had no significant effect on passive
K+ influx pathway (P > 0.05, Fig. 7).
NMDA exerts its function in the CNS primarily through Ca2+ influx via NMDA receptors. Thus NMDA-mediated effect on the cotransporter could involve in an increase in intracellular Ca2+ via opening of the NMDA channels. This speculation was supported by the effect of NMDA receptor blocker ifenrodil and bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) in the following experiments. To investigate that NMDA-induced stimulation of the cotransporter activity was indeed due to activation of the NMDA receptor, we tested if NMDA receptor antagonist ifenprodil could block NMDA-mediated effect. Ifenprodil (15 µM) significantly attenuated NMDA-mediated stimulation of both bumetanide- and ouabain-sensitive K+ influx (Fig. 7). However, neither NMDA nor NMDA plus ifenprodil had any significant effect on the passive K+ influx. These data suggest that NMDA-mediated effect on ion transport systems was specifically due to activation of the NMDA receptor.
BAPTA-AM, a cell permeable Ca2+ chelator, was used to
further investigate the Ca2+-mediated process. If an
increase in intracellular Ca2+ was involved in
NMDA-mediated stimulation of the cotransporter activity, this effect
should be abolished by BAPTA-AM. In a control study, BAPTA-AM (50 µM)
reduced the basal activity of the cotransporter from 14.90 ± 1.72 to 9.19 ± 5.34 (nmol K+/mg protein/min). This
suggests that function of the cotransporter under physiological
conditions requires intracellular free Ca2+. A similar
finding was observed in glioma cells (G. Su, R. J. Dempsey, and D. Sun, unpublished observations). However, the basal level of
ouabain-sensitive K+ influx was unchanged in the presence
of BAPTA-AM (48.94 ± 4.45 nmol/mg protein/min vs. a control of
48.94 ± 4.45 nmol/mg protein/min). Neither was bumetanide and
ouabain-resistant K+ influx rate affected by BAPTA-AM (data
not shown). As shown in Fig. 8,
NMDA-mediated increase in bumetanide-sensitive K+ influx
was abolished completely by BAPTA-AM. Bumetanide-sensitive K+ influx rate was 33.42 ± 4.82 in the presence of
NMDA alone and reduced to 14.71 ± 3.43 in the presence of NMDA
plus BAPTA (P < 0.05). The latter was not
statistically significant from the control value (14.90 ± 1.72 nmol K+/mg protein/min). However, it was higher than one in
cells exposed to BAPTA-AM alone (9.19 ± 5.34 nmol
K+/mg protein/min). It implies that NMDA-mediated
regulation of the Na+-K+-2Cl
cotransporter also involved a process that is insensitive to intracellular-free Ca2+. Moreover, prevention of increase
in intracellular Ca2+ by BAPTA-AM significantly inhibited
NMDA-induced stimulation of Na+, K+/ATPase
activity. Neither NMDA nor NMDA plus BAPTA-AM had any effect on the
passive K+ influx (P > 0.05).
|
Expression of Na+-K+-2Cl
cotransporter and NMDA receptors in cortical neurons by Western blot
analysis
The study described above demonstrated that cortical neurons
express a functional Na+-K+-2Cl
cotransporter system. In this experiment, we further evaluated the
cotransporter system by Western blot analysis. A control experiment was
performed by using a crude membrane preparation of cerebral cortex from
adult Sprague Dawley rat. Figure
9A demonstrates that neurofilament 200 (NF-200), a major component of neuronal intermediate filaments as a marker protein for neurons, was present in cortical neurons and brain cortex. This further confirmed the characteristic of
the primary cortical neuron culture used in this study. As shown in
Fig. 9B, an abundant expression of an ~145-kDa
cotransporter protein was detected in cortical neurons; this was
consistent with our observation in immunocytochemical staining study. A
similar molecular mass of the cotransporter protein was observed in
adult brain cortex.
|
Expression of ionotropic NMDA receptor NR-1 and NR-2B proteins, known to form a functional NMDA-gated Ca2+ channel complex, was shown in Fig. 9, C and D. An ~110-kDa NR-1 protein and 180-kDa NR-2B were expressed in both cortical neuron and cerebral cortex of adult rat.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression of functional
Na+-K+-2Cl cotransporter in
cortical neurons
To our knowledge, this is the first extensive study to
characterize functionally
Na+-K+-2Cl cotransporter in
primary culture of cortical neurons. In this report, we demonstrated
that cortical neurons express a functional Na+-K+-2Cl
cotransporter system
at the early postnatal life. Bumetanide-sensitive K+ influx
comprises approximately one-third of the total cellular unidirectional
K+ uptake. The
Na+-K+-2Cl
-cotransporter-mediated
K+ influx depends on the simultaneous presence of
extracellular Na+ and Cl
. Removal of either
extracellular Na+ or Cl
significantly
inhibits the Na+-K+-2Cl
cotransporter activity. It is necessary to point out that the bumetanide-sensitive K+ influx value was negative in the
absence of extracellular Cl
. This implies that
intracellular K+ accumulation somehow was increased by
bumetanide compared with one in Cl
-free medium without
bumetanide. One possible explanation is that removal of extracellular
Cl
probably activates outwardly directed
K+-Cl
cotransporter, and this K+
efflux is blocked by bumetanide. This process would be reflected by a
negative bumetanide-sensitive K+ influx value under
Cl
-free conditions.
Presence of the Na+-K+-2Cl
cotransporter in neurons was evaluated by Western blot analysis and
immunocytochemical staining. Immunocytochemical staining revealed a
strong immunoreactivity in soma and dendrites of cortical neurons with
T4 anti-cotransporter antibody. Interestingly, an ~145-kDa
cotransporter protein was observed in both cultured neurons and adult
rat cortex. The similar molecular mass of the cotransporter found in
neonatal neurons as well as adult cortex implies that a mature form of
the Na+-K+-2Cl
cotransporter was
developed in neurons at the early postnatal stages.
Role of Na+-K+-2Cl
cotransporter in regulation of intracellular Cl
levels
In neonatal neurons, GABA depolarizes neuronal membranes. This
GABA-mediated depolarization is Cl dependent and due to a
modified Cl
gradient in neonatal neurons
(Cherubini et al. 1991
). In this study, a substantial
level of bumetanide-sensitive cotransporter activity was detected in
cortical neurons at DIV 4 (P0). Moreover, the
Na+-K+-2Cl
cotransporter activity
was developed further at the end of the first postnatal week. The
inwardly directed Na+-K+-2Cl
cotransporter thus may function as an active inwardly directed Cl
transport system in neonatal cells. This hypothesis
was supported by our finding that the cotransporter activity was
stimulated significantly by a decrease of intracellular
Cl
via either opening of GABA-activated Cl
channels or depletion of the intracellular Cl
. Moreover
it has been reported that expression of outwardly directed K+-Cl
cotransporter proteins was detected at
postnatal day 10 and increased steadily to adult levels at day 28 (Sharp et al. 1998
). Taken together, early development
of the Na+-K+-2Cl
cotransporter
and delayed appearance of K+-Cl
cotransporter
would result a high level of intracellular Cl
. Because
Na+, K+/ATPase activity also is found to be
upregulated during the early postnatal period, it would remove
intracellular Na+ ion which is transported by the
Na+-K+-2Cl
cotransporter.
Therefore the Na+ electrochemical gradient, a driving force
for the cotransporter, would be maintained to support function of the
Na+-K+-2Cl
cotransporter system.
Regulation of Na+-K+-2Cl
cotransporter in cortical neurons by activation of NMDA receptors
We reported in our previous study that
Na+-K+-2Cl cotransporter activity
in human neuroblastoma SH-SY5Y cells was regulated by 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 (Choi and
Rothman 1990
; Hollmann et al. 1994
;
Nicolls 1995
). We found that activation of both
ionotropic NMDA receptor and metabotropic glutamate receptors
stimulates Na+-K+-2Cl
cotransporter in neuroblastoma cells (Sun and Murali
1998
).
In the present study, using an established primary culture of
cortical neurons, we further investigated whether the NMDA-mediated effect involved a Ca2+-dependent mechanism. NMDA receptors
constitute cation channels gated by the excitatory neurotransmitter
L-glutamate and mediate signal transduction in central
synapses. Most lasting cellular effects of NMDA receptor activation are
mediated by Ca2+ entering through the channel. To establish
that the NMDA-mediated effect on the cotransporter is due to an
increase of intracellular Ca2+, we examined if either
blocking of the NMDA channel or chelating intracellular
Ca2+ could block NMDA-mediated stimulation. In fact,
NMDA-mediated stimulation of the cotransporter activity was inhibited
by NMDA receptor antagonist ifenprodil. In addition, BAPTA-AM
completely abolished the stimulation of the cotransporter by NMDA.
Possible mechanisms of NMDA-mediated effect could involve
phosphorylation of the cotransporter protein by a Ser/Thr protein
kinase in the CNS, such as Ca2+/Cam kinase II.
Ca2+/Cam kinase II is stimulated by the NMDA-receptor
activation (Hollmann and Heinemann 1994; Nicolls
1995
). 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
; Lytle 1998
; Sun et al.
1996
). However, a substantial level of bumetanide-sensitive
K+ influx was insensitive to chelating of intracellular
free Ca2+. Thus it appears that a
Ca2+-independent regulatory mechanism also is involved in
NMDA-mediated stimulation of the cotransporter.
In summary, we have demonstrated that functional
Na+-K+-2Cl cotransporter system
is developed in cortical neurons in the early postnatal stages.
Function of the Na+-K+-2Cl
cotransporter in neurons is regulated by activation of the NMDA receptors. This partially involves an increase in intracellular Ca2+. Inwardly directed
Na+-K+-2Cl
cotransporter in
neurons represents an essential mechanism in regulation of
intracellular Cl
activity. Decrease of intracellular
Cl
via GABA-activated Cl
channels or
incubation of cells in reduced [Cl
]o
significantly stimulated the activity of the cotransporter. Thus our
study supports the notion that
Na+-K+-2Cl
cotransporter plays an
important role in intracellular Cl
activity.
Na+-K+-2Cl
cotransporter may be
responsible for GABA-mediated excitatory effect in immature neurons.
![]() |
ACKNOWLEDGMENTS |
---|
This work was supported in part by Scientist Development Grant 9630189N to D. Sun from National Center Affiliate of American Heart Association, a grant to the University of Wisconsin Medical School under the Howard Hughes Medical Institute Research Resources Program for Medical Schools, and Graduate School Research Grant 990275 to D. Sun from University of Wisconsin-Madison.
![]() |
FOOTNOTES |
---|
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.
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 1 October 1998; accepted in final form 9 December 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|