Stimulation of Na-K-2Cl Cotransporter in Neurons by Activation of Non-NMDA Ionotropic Receptor and Group-I mGluRs

Stacey L. Schomberg,1 Gui Su,1,2 Robert A. Haworth,3 and Dandan Sun1,2

 1Department of Neurological Surgery,  2Department of Physiology, and  3Department of Surgery, University of Wisconsin Medical School, Madison, Wisconsin 53792


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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+.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>i</SUB><SUP>−</SUP></UP> depletion, the active re-accumulation of 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 alpha -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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta -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-beta -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-mGluR1alpha and anti-mGluR5 polyclonal IgGs were from Chemicon International (Temecula, CA), and rabbit anti-GABAA receptor alpha 1 antibody was from Upstate Biotech (Lake Placid, NY).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 1 subunit. Using adult rat cortex as a positive control, a similar expression level of the alpha 1 subunit was detected in cortical tissue.



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 1. Expression of mGluR1alpha and mGluR5 in cultured cortical neurons. Cell lysates or crude membrane preparations of adult rat brain were separated by 6% SDS-block, transferred to a nitrocellulose membrane and probed with: (A) rabbit anti-mGluR1a polyclonal IgGs, (B) anti-mGluR5 polyclonal IgGs, (C) T4 anti-cotransporter monoclonal IgGs, or (D) anti-GABAA receptor alpha 1 subunit polyclonal IgGs. The Western blots were visualized by the enhanced chemiluminescence (ECL). These are representative blots of 3-4 experiments.

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-beta -tubulin antibody. As shown in Fig. 2A, cultured neurons were intensely stained with beta -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 beta -tubulin was detected in cortical neurons (yellow, Fig. 2C). A similar cellular colocalization pattern was found for mGluR5 and beta -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.



View larger version (131K):
[in this window]
[in a new window]
 
Fig. 2. Immunofluorescence staining. Cortical neurons were grown on poly-D-lysine-coated coverslips for 11 days. A: anti-tubulin (beta -subunit, type III) antibody staining image (1:100 dilution). B: anti-mGluR1a antibody staining image (1:100 dilution). C: double staining images of A and B. D: anti-tubulin (beta -subunit, type III) antibody staining image (1:100 dilution). E: anti-mGluR5 antibody staining image (1:100 dilution). F: double staining images of D and E. G: anti-Na+-K+-2Cl- cotransporter antibody staining image (T4, 1:150 dilution). H: anti-mGluR1a antibody staining image (1:150 dilution). I: double staining images of G and H. J: anti-Na+-K+-2Cl- cotransporter antibody staining image (T4, 1:150 dilution). K: anti-mGluR5 antibody staining image (1:150 dilution). L: double staining images of J and K. Small images in F and L were negative controls in which either FITC-conjugated goat anti-mouse IgG (1:200) or Texas-Red-conjugated goat anti-rabbit IgG (1:200) was used and the primary antibodies were omitted. The rest procedures were the same as in A-L. Images were captured by a laser-scanning confocal microscope (Bio-Rad MRC 1000). Scale bar: 17 µm in A-L.

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.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 3. Stimulation of Na+-K+-2Cl- cotransporter activity by trans-(±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) and (RS)-3,5-dihydroxyphenylglycine (DHPG). Na+-K+-2Cl- cotransporter activity was assessed by bumetanide-sensitive 86Rb influx. To obtain bumetanide-sensitive 86Rb influx, cells were preincubated for 10 min in conditions as described below, and influx was assayed either in the presence or absence of 10 µM bumetanide for 3 min at 37°C. A: cells were preincubated in HEPES-MEM containing 10 µM trans-ACPD, 10 µM trans-ACPD, and 10 µM bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA)-AM, or 10 µM BAPTA-AM for 10 min. B: cells were preincubated in HEPES-MEM containing either 20 µM DHPG or 20 µM DHPG plus 10 µM BAPTA-AM for 10 min. Inset: exposure of cells to 20 µM DHPG was for 1, 3, 4, 10, or 13 min. C: cells were preincubated in HEPES-MEM containing 100 µM 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt), 20 µM DHPG, 20 µM DHPG plus 1 mM (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) or 20 µM DHPG plus 100 µM CPCCOEt for 10 min. 86Rb influx was subsequently assayed in the presence of each for 3 min at 37°C. Data are means ± SE, n = 3-17. Quadruplet determinations were obtained in each experiment. * P < 0.05 vs. control, # P < 0.05 vs. trans-ACPD-treated (A) or # P < 0.05 vs. DHPG-treated (C) by ANOVA (Bonferroni/Dunn).

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+.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4. Change of intracellular Ca2+ in neurons by 20 µM DHPG. Intracellular Ca2+ measurement was performed as described in METHODS. Cells were loaded with 5 µM fura-2 AM at room temperature for 1 h. The 360/380 ratio was taken to indicate changes in intracellular Ca2+ levels. Changes in the 360/380 ratio were recorded either in HEPES-buffered MEM or presence of 20 µM DHPG plus 100 µM CPCCOEt (A) or 20 µM DHPG alone (B) in HEPES-buffered MEM at room temperature. Data are representative examples of 3-4 experiments. Summarized results were shown in C. Data are means ± SE, n = 3-4. * P < 0.05 vs. DHPG-induced 1st peak by Student's t-test (2-tailed).

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).



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 5. High K+-induced stimulation of the cotransporter activity. A: change of intracellular Ca2+ in neurons by high extracellular K+ was measured. Cells were perfused with HEPES-MEM containing either 5.8 mM [K+]o or 75 mM [K+]o (as indicated in figure). [K+]o (75 mM) was obtained by replacing NaCl in HEPES-MEM buffer with equimolar KCl. Ca2+-free HEPES-MEM contained 0 mM CaCl2 and 100 µM EGTA. Data are representative examples of 4 experiments. Inset: a measurement was made at 37°C. B: to obtain bumetanide-sensitive 86Rb influx, cultured neurons were preincubated in HEPES-MEM containing either 5.8 or 75 mM [K+]o for 10 min. Na+-K+-2Cl- cotransporter activity was subsequently assessed by bumetanide-sensitive 86Rb influx for 3 min. For Ca2+-free conditions, cells were washed 3 times with Ca2+-free HEPES-MEM, then preincubated in Ca2+-free HEPES-MEM containing either 5.8 or 75 mM [K+]o for 10 min. Ca2+/CaM kinase II inhibitor KN62 (10 µM) was used. Data are means ± SE, n = 3. * P < 0.05 vs. control; # P < 0.05 vs. High K+-treated by ANOVA (Bonferroni/Dunn).

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.



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 6. Effect of alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) on the Na+-K+-2Cl- cotransporter activity and intracellular Ca2+. A: cells were preincubated in buffers containing either 0 or 25 µM AMPA or 25 µM AMPA plus 20 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) for 5-10 min. Na+-K+-2Cl- cotransporter activity was subsequently assessed by bumetanide-sensitive 86Rb influx for 3 min. Data are means ± SE, n = 4. Quadruplet determinations were obtained in each experiment. B: intracellular Ca2+ measurement was performed as described in the legend of Fig. 4. Changes in the 360/380 ratio were recorded either in HEPES-buffered MEM or the presence of 25 µM AMPA in HEPES-buffered MEM at room temperature. C: changes in the 360/380 ratio were recorded either in 25 µM AMPA or 20 µM CNQX plus 25 µM AMPA in HEPES-buffered MEM. Data are representative examples of 4-5 experiments in B and C. D: summarized results of B and C. Data are means ± SE, n = 4-5. * P < 0.05 vs. AMPA-induced 1st peak of Fig. 6B (Student's t-test, 2-tailed); # P < 0.05 vs. AMPA-induced 1st peak of Fig. 6C (Student's t-test, 2-tailed).

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.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 7. Effect of phosphorylation/dephosphorylation on the cotransporter activity. Cells were preincubated in HEPES-MEM as described in the Fig. 3 legend. Cells were incubated in HEPES-MEM containing either calyculin A (25 nM), DHPG (20 µM) alone, or 20 µM DHPG plus K252a (40 nM) for 5 min. 86Rb influx was assayed either in the presence or absence of 10 µM bumetanide for 3 min at 37°C. Data are means ± SE, n = 4-7. Quadruplet determinations were obtained in each experiment. * P < 0.05 vs. control; # P < 0.05 vs. DHPG-treated by ANOVA (Bonferroni/Dunn).

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.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 8. Inhibition of the Na+-K+-2Cl- cotransporter activity by KN62. Cells were preincubated in HEPES-MEM as described in the Fig. 3 legend. To examine the effect of the Ca2+/CaM kinase II inhibitor KN62, cells were incubated in HEPES-MEM containing 20 µM DHPG, 10 µM DHPG plus 10 µM KN62 or 20 µM DHPG plus 10 µM KN92 (a negative control of KN62) for 5 min. 86Rb influx was assayed either in the presence or absence of 10 µM bumetanide for 3 min at 37°C. Data are means ± SE, n = 5. Quadruplet determinations were obtained in each experiment. * P < 0.05 vs. control; # P > 0.05 vs. DHPG-treated by ANOVA (Bonferroni/Dunn).

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.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 9. Lack of an effect of PKC inhibitor RO-32-O432 on the DHPG-mediated stimulation of Na+-K+-2Cl- cotransporter. Cells were preincubated in HEPES-MEM. To examine the effect of the PKC inhibitor RO-32-O432, cells were incubated in HEPES-MEM containing 20 µM DHPG, 150 nM PMA, 3 µM RO-32-O432, 20 µM DHPG plus 3 µM RO-32-O432 or 150 nM PMA plus 3 µM RO-32-O432 for 5 min. 86Rb influx was assayed either in the presence or absence of 10 µM bumetanide for 3 min at 37°C. Data are means ± SE, n = 3-7. Quadruplet determinations were obtained in each experiment. * P < 0.05 vs. control; # P > 0.05 vs. control by ANOVA (Bonferroni/Dunn).

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    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.


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society