Stimulation of Na+-K+-2Clminus cotransporter in neuronal cells by excitatory neurotransmitter glutamate

Dandan Sun1,2 and Sangita G. Murali1

Departments of 1 Neurological Surgery and 2 Physiology, School of Medicine, University of Wisconsin, Madison, Wisconsin 53792

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Na+-K+-2Cl- cotransporters are important in renal salt reabsorption and in salt secretion by epithelia. They are also essential in maintenance and regulation of ion gradients and cell volume in both epithelial and nonepithelial cells. Expression of Na+-K+-2Cl- cotransporters in brain tissues is high; however, little is known about their function and regulation in neurons. In this study, we examined regulation of the Na+-K+-2Cl- cotransporter by the excitatory neurotransmitter glutamate. The cotransporter activity in human neuroblastoma SH-SY5Y cells was assessed by bumetanide-sensitive K+ influx, and protein expression was evaluated by Western blot analysis. Glutamate was found to induce a dose- and time-dependent stimulation of Na+-K+-2Cl- cotransporter activity in SH-SY5Y cells. Moreover, both the glutamate ionotropic receptor agonist N-methyl-D-aspartic acid (NMDA) and the metabotropic receptor agonist (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) significantly stimulated the cotransport activity in these cells. NMDA-mediated stimulation of the Na+-K+-2Cl- cotransporter was abolished by the selective NMDA-receptor antagonist (+)-MK-801 hydrogen maleate. trans-ACPD-mediated effect on the cotransporter was blocked by the metabotropic receptor antagonist (+)-alpha -methyl-(4-carboxyphenyl)glycine. The results demonstrate that Na+-K+-2Cl- cotransporters in neurons are regulated by activation of both ionotropic and metabotropic glutamate receptors.

ionotropic glutamate receptors; metabotropic glutamate receptors; bumetanide

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

SODIUM-POTASSIUM-CHLORIDE cotransporters represent a family of integral membrane proteins that transport Na+, K+, and Cl- into and out of cells. The Na+-K+-2Cl- cotransporters have been identified in a wide variety of eukaryotic cells, and the stoichiometry of transported ions in most instances has been reported to be 1Na+:1K+:2Cl- (8, 25). The Na+-K+-2Cl- cotransporters are characterized by their specific, reversible inhibition by the sulfamoylbenzoic acid "loop" diuretics (furosemide, bumetanide, and benzmetanide). Because the cotransporter carries an electroneutral ion movement, the driving force for net transport is solely dependent on the chemical gradients of the three transported ions. Under physiological conditions, the Na+-K+-2Cl- cotransporter in most cells moves these ions inward, driven by both favorable inward Na+ and Cl- chemical gradients (8, 25). Thus the cotransporter plays a crucial role in vectorial salt transport in epithelial cells and ion gradients as well as cell volume regulation in epithelial and nonepithelial cells (8, 13, 24, 25).

Currently, only two distinct isoforms of the Na+-K+-2Cl- cotransporter (NKCC) have been identified: NKCC1 (7.0- to 7.5-kb transcript), which has a wide range of tissue distributions (6, 38), and NKCC2 (4.6- to 5.2-kb transcript), which has only been found in vertebrate kidney (7, 12, 27). Northern blot analysis has demonstrated that the expression level of NKCC1 mRNA is high in brain (6, 38). In situ hybridization and immunocytochemical studies have shown that NKCC1 proteins are abundantly present on plasma membranes of neurons throughout the rat brain (28).

Despite studies on the Na+-K+-2Cl- cotransporters in many cell types, the physiological function and regulation of NKCC1 protein in neuronal cells are little known. In the present study, human neuroblastoma SH-SY5Y cells were used to examine regulation of Na+-K+-2Cl- cotransport activity in neurons by excitatory neurotransmitter glutamate. Glutamate is the major excitatory neurotransmitter in the central nervous system (5, 22). Glutamate exerts its effect by activation of a family of heterogeneous glutamate receptors that couple to second-messenger systems (1, 11, 37). SH-SY5Y cells have been shown to express many properties of mature noradrenergic neurons, including the occurrence of neurosecretory granules, extension of neurites, and the Ca2+-dependent release of norepinephrine after plasma membrane depolarization (19). It has also been demonstrated that SH-SY5Y cells express both ionotropic and metabotropic glutamate receptors (20, 21) as well as the Na+-dependent glutamate-aspartate transporter (26). We report here that glutamate stimulates Na+-K+-2Cl- cotransport activity in SH-SY5Y neuronal cells. Activation of both the ionotropic N-methyl-D-aspartic acid (NMDA) receptor and metabotropic glutamate receptors stimulates Na+-K+-2Cl- cotransporter in SH-SY5Y cells.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell culture. Cultured human neuroblastoma SH-SY5Y cells were maintained in DMEM, supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml). Cells were grown on 24-well plates in DMEM, and experiments were performed on confluent cells.

K+ influx determination. Na+-K+-2Cl- cotransport activity was measured as bumetanide-sensitive K+ influx, using 86Rb as a tracer for K+. Briefly, SH-SY5Y cells were equilibrated for 10-30 min at 37°C with an isotonic HEPES-buffered MEM (300 mosM). The concentrations of components in HEPES-MEM (mM) were 140 NaCl, 5.36 KCl, 0.81 MgSO4, 1.27 CaCl2, 0.44 KH2PO4, 0.33 Na2HPO4, 5.55 glucose, and 20 HEPES. Cells were then preincubated for 10 min in HEPES-MEM containing either 0 or 60-100 µM bumetanide. For assay of cotransport activity, cells were exposed to 1 µCi/ml of 86Rb in HEPES-MEM for 3 min, either in the presence or absence of 60-100 µM bumetanide. 86Rb influx was stopped by rinsing cells with ice-cold 0.1 M MgCl2. Radioactivity of cells extracted in 1% SDS was analyzed by liquid scintillation counting (Packard 1900CA, Downers Grove, IL). K+ influx rate was calculated as the slope of 86Rb uptake over time and expressed as nanomoles of K+ per milligram of protein per minute. Bumetanide-sensitive K+ influx was obtained by subtracting K+ influx rate in the presence of bumetanide from total K+ influx rate. Quadruplet determinations were obtained in each experiment, and protein content was measured in each sample using a method described by Smith et al. (32). Statistical significance in the study was determined by Student's t-test or ANOVA (P < 0.05).

Gel electrophoresis and Western blotting. SH-SY5Y cells growing on 100-mm tissue culture dishes were washed with ice-cold PBS (pH 7.4), which contained 2 mM EDTA and protease inhibitors, as described previously (33). Cells were scraped from dishes, suspended in PBS, and lysed by 30 s of sonication at 4°C with an ultrasonic processor (Sonics & Materials, Danbury, CT). To obtain cellular lysates, cellular debris was removed by a brief centrifugation at 420 g for 5 min. Protein content of the cellular lysate was determined by the Bradford method (4). Samples and prestained molecular mass markers (Bio-Rad) were denatured in SDS reducing buffer [9.2% SDS, 5% beta -mercaptoethanol, 50 mM Tris · HCl (pH 7.4), 35% sucrose, and 0.012% bromphenol blue] and heated at 37°C for 15 min before gel electrophoresis. The samples were then electrophoretically separated on 6% SDS gels (14), and the resolved proteins were electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane (0.45 µm, Millipore, Bedford, MA). The blots were incubated in 7.5% nonfat dry milk in Tris-buffered saline (TBS) for 2 h at room temperature and incubated overnight with a primary antibody. The blots were then rinsed five times with TBS and incubated with horseradish peroxidase-conjugated secondary IgG for 1 h. After five washes to remove unbound secondary antibody, bound antibody was visualized using the enhanced chemiluminescence assay (Amersham). T4 monoclonal antibody against the human colonic T84 epithelial Na+-K+-2Cl- cotransporter was used for detection of NKCC1 protein, as described by Lytle et al. (17). A monoclonal antibody against an NMDA-receptor isoform (NR-2B, 1 µg/ml) was used for analysis of NMDA-receptor expression. A rabbit polyclonal anti-rat metabotropic glutamate receptor (mGluR) type 1 IgG (0.75 µg/ml) was used to identify mGluR1 receptor expression in SH-SY5Y cells. Neuronal identity of SH-SY5Y cells was investigated by expression of neurofilament with a monoclonal antibody against neurofilament protein 200 (7.7 µg/ml).

For deglycosylation studies, cellular proteins were solubilized with 1% SDS and incubated in the presence of 1 unit deglycosidase F (Boehringer Mannheim Biomedicals, Indianapolis, IN) for 5 h at 37°C and separated by SDS-PAGE, as described above.

Materials. Bumetanide, L-glutamic acid, and anti-neurofilament 200 monoclonal antibody were purchased from Sigma (St. Louis, MO). DMEM was from GIBCO (Washington, DC). NMDA, AMPA-receptor agonist (±)-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), selective NMDA-receptor antagonist (+)-MK-801 hydrogen maleate, mGluR agonist (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD), and mGluR antagonist (+)-alpha -methyl-4-carboxyphenylglycine [(+)-MCPG] were purchased from Research Biochemicals International (Natick, MA). FBS was obtained from Hyclone Laboratories (Logan, UT). 86RbCl was purchased from NEN Life Science Products (Boston, MA). Rabbit anti-rat mGluR1 polyclonal IgG was from Upstate Biotechnology (Lake Placid, NY). Anti-NMDA-receptor (NR-2B) monoclonal antibody was purchased from Transduction Laboratories (Lexington, KY).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Dose- and time-dependent stimulation of Na+-K+-2Cl- cotransport activity by glutamate. To examine whether glutamate regulates Na+-K+-2Cl- cotransport activity in neurons, Na+-K+-2Cl- cotransporter activity in SH-SY5Y was assessed by bumetanide-sensitive K+ influx either in the presence or absence of glutamate. Data showed that glutamate induced a dose-dependent stimulation of Na+-K+-2Cl- cotransporter activity in SH-SY5Y (Fig. 1). The dose-response curve showed that 15 µM glutamate caused a significant stimulation of the cotransporter activity (P < 0.05). Glutamate still caused a stimulation of the cotransporter when the concentration in extracellular medium was increased to 75 µM (P < 0.05). The glutamate-mediated stimulation of Na+-K+-2Cl- cotransporter activity in SH-SY5Y was time dependent. 86Rb uptake in cells was assayed at 37°C in either the presence or absence of 15 µM glutamate for 0-15 min. Figure 2 demonstrated that bumetanide-sensitive K+ uptake was increased over time in the absence and presence of 15 µM glutamate. Moreover, in the absence as well as the presence of glutamate, bumetanide-sensitive K+ uptake rate seems to be faster in the period of 1-5 min than that between 5 and 15 min, which was reflected by the different slopes. The slope of the cotransport in glutamate-treated group was steeper than that of the control group, indicating that the cotransporter activity is stimulated by glutamate.


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Fig. 1.   Glutamate induces a dose-dependent stimulation of Na+-K+-2Cl- cotransporter activity in SH-SY5Y cells. Cultured cells were preincubated either in presence or absence of 60 µM bumetanide in HEPES-buffered medium for 4 min. In addition, cells were exposed to 0-75 µM glutamate for 10 min. To obtain bumetanide-sensitive 86Rb influx, 86Rb influx was subsequently assayed either in presence or absence of bumetanide for 3 min. Data are means ± SE; n = 3. Quadruplet determinations were obtained in each experiment. * P < 0.05 vs. control group by Student's t-test.


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Fig. 2.   Glutamate induces a time-dependent stimulation of Na+-K+-2Cl- cotransporter activity in SH-SY5Y cells. Cultured cells were preincubated in presence or absence of 60 µM bumetanide in HEPES-buffered medium for 4 min. 86Rb uptake in cells was assayed either in presence or absence of 15 µM glutamate for 0-15 min, as indicated on x-axis. In glutamate-treated groups, 15 µM glutamate and 86Rb were added to cells at same time. Data are means ± SE; n = 3. Quadruplet determinations were obtained in each experiment. * P < 0.05 vs. control group by Student's t-test.

Stimulation of Na+-K+-2Cl- cotransporter by activation of both ionotropic NMDA receptor and mGluR. To investigate the possible second-messenger signal pathways responsible for glutamate-mediated stimulation of the cotransporter in SH-SY5Y cells, it is necessary to determine which type of glutamate receptors are involved in the process. First, the effect of a potent NMDA-receptor agonist, NMDA, was tested on activity of the cotransporter. A dose-response study was conducted. SH-SY5Y cells in 24-well plates were incubated with different concentrations of NMDA in isotonic HEPES-buffered MEM at 37°C for 10 min. Control SH-SY5Y cells were incubated with isotonic HEPES-buffered MEM at 37°C for 10 min. Total K+ influx rate as well as bumetanide-sensitive K+ influx rate were determined in both control and NMDA-treated cells. Figure 3 showed that 10 µM of NMDA significantly stimulated the cotransport activity in SH-SY5Y cells, from a basal level of 6.34 ± 0.58 to 15.37 ± 2.42 nmol · mg protein-1 · min-1 (mean ± SE, P < 0.05). It was demonstrated in Table 1 that NMDA caused a significant stimulation of Na+-K+-2Cl- cotransporter activity in SH-SY5Y cells in a dose-dependent manner, with maximal stimulation at 20 µM and no stimulation at 100 µM. It appears that the increase in the total K+ influx in NMDA-stimulated cells reflects the increase in the cotransporter activity as well as bumetanide-insensitive K+ influx pathway(s). To determine whether activation of mGluR is also involved in glutamate-mediated stimulation of the cotransporter, the effect of a mGluR agonist trans-ACPD on cotransport activity was examined. Figure 3 demonstrated that 10 µM trans-ACPD significantly stimulated the cotransport activity in SH-SY5Y cells, from a basal level of 6.34 ± 0.58 to 9.93 ± 0.86 nmol · mg protein-1 · min-1 (mean ± SE, P < 0.05). trans-ACPD (20-100 µM) also stimulated the Na+-K+-2Cl- cotransporter substantially; however, that was not statistically significant (Table 1).


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Fig. 3.   Stimulation of Na+-K+-2Cl- cotransporter by activation of both ionotropic N-methyl-D-aspartic acid (NMDA) receptor and metabotropic glutamate receptors (mGluR). SH-SY5Y cells grown on 24-well plates were preincubated in 10 µM (±)-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), 10 µM glutamate, 10 µM NMDA, 10 µM (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD), or 10 µM NMDA + 10 µM trans-ACPD at 37°C for 10 min. Bumetanide-sensitive 86Rb influx rates were determined in presence of 100 µM bumetanide. 86Rb influx was subsequently assayed for 3 min. Data are means ± SE; n = 4. Quadruplet determinations were obtained in each experiment. * P < 0.05 vs. control group by Student's t-test.

                              
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Table 1.   Dose-dependent effect of NMDA and trans-ACPD on Na+-K+-2Cl- cotransporter activity in SH-SY5Y cells

To examine whether activation of AMPA ionotropic receptor is involved in glutamate-mediated stimulation of the cotransporter activity, the effect of AMPA receptor agonist AMPA on bumetanide-sensitive K+ influx was evaluated. It was shown in Fig. 3 that activation of AMPA receptor had no effect on the cotransporter activity.

To further investigate the relative contributions of NMDA and trans-ACPD-mediated pathways, cells were stimulated with NMDA and trans-ACPD. As shown in Fig. 3, 10 µM NMDA plus 10 µM trans-ACPD did not cause an additive effect on the cotransporter activity. This implies that activation of these two receptors may lead to a final common pathway which activates the cotransporter activity.

Effect of selective glutamate ionotropic receptor antagonist MK-801 on Na+-K+-2Cl- cotransporter activity. The results of Fig. 3 and Table 1 revealed that stimulation of the cotransport activity by glutamate may be due to activation of both NMDA receptors and mGluRs in SH-SY5Y cells. To further investigate this hypothesis, we evaluated whether NMDA- or trans-ACPD-induced stimulation of Na+-K+-2Cl- cotransporter could be abolished by selective glutamate-receptor antagonists. We first studied whether NMDA-mediated stimulation of the cotransporter could be blocked by the selective, noncompetitive NMDA-receptor antagonist MK-801. SH-SY5Y cells were exposed to an isotonic HEPES-buffered MEM containing either 10 µM NMDA or 10 µM NMDA plus 1 or 10 µM MK-801 at 37°C for 10 min. Figure 4 showed that neither 1 nor 10 µM MK-801 had a significant effect on a basal level of the cotransport activity. However, both 1 and 10 µM MK-801 significantly abolished NMDA-mediated stimulation of Na+-K+-2Cl- cotransporter (Fig. 4). To verify that MK-801 exerts its effect specifically via blocking of the NMDA receptor, we also examined whether MK-801 had any effect on trans-ACPD-mediated stimulation of the cotransporter. SH-SY5Y cells were exposed to either 10 µM trans-ACPD or 10 µM trans-ACPD plus 1 or 10 µM MK-801 at 37°C for 10 min. As demonstrated in Fig. 4, trans-ACPD-mediated stimulation of the cotransport activity was not altered in the presence of either 1 or 10 µM MK-801. Thus MK-801 significantly inhibits the NMDA-mediated effect by antagonizing NMDA receptor in SH-SY5Y cells. We then decided to examine the effect of MK-801 on glutamate-induced stimulation of the cotransporter. SH-SY5Y cells were incubated in the presence of either 10 µM glutamate or 10 µM glutamate plus 1 µM MK-801 for 10 min. Bumetanide-sensitive K+ influx rate in cells was 10.42 ± 0.59 nmol · mg protein-1 · min-1 in the presence of glutamate alone but was reduced to 6.37 ± 1.52 nmol · mg protein-1 · min-1 in the presence of both glutamate and MK-801; the latter was not statistically significantly different from the value in the presence of MK-801 alone. These results suggest that stimulation of the Na+-K+-2Cl- cotransporter by glutamate is primarily mediated by activation of NMDA glutamate receptors.


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Fig. 4.   Effect of noncompetitive NMDA-receptor antagonist MK-801 on NMDA- and trans-ACPD-mediated stimulation of Na+-K+-2Cl- cotransporter. SH-SY5Y cells were exposed to HEPES-buffered MEM containing either 10 µM NMDA or 10 µM trans-ACPD for 10 min. To test effect of MK-801, cells were incubated in presence of either 1 or 10 µM MK-801 for 10 min. To obtain bumetanide-sensitive 86Rb influx, 86Rb influx was subsequently assayed either in presence or absence of 100 µM bumetanide for 3 min. Data are means ± SE; n = 3-4. Quadruplet determinations were obtained in each experiment. * P < 0.05 vs. NMDA-treated group by Student's t-test.

mGluR-mediated stimulation of Na+-K+-2Cl- cotransporter activity. To further characterize metabotropic receptor-mediated stimulation of Na+-K+-2Cl- cotransporter, a time-dependent stimulation of the cotransport system was evaluated with 10 µM trans-ACPD. SH-SY5Y cells were preincubated either in the presence or absence of 10 µM trans-ACPD in isotonic HEPES-buffered MEM for 0-15 min. These experiments were performed in either the presence or absence of 100 µM bumetanide, respectively. K+ influx was assayed for 3 min in those experiments. Figure 5A showed that bumetanide-sensitive K+ influx rate was significantly elevated when cells were exposed to 10 µM trans-ACPD for 2 min (8.26 ± 0.62 nmol · mg protein-1 · min-1 vs. a control level of 5.77 ± 1.26 nmol · mg protein-1 · min-1, P < 0.05). The trans-ACPD-mediated stimulation of the cotransporter was observed over 15 min of incubation period.


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Fig. 5.   Time-dependent stimulation of Na+-K+-2Cl- cotransporter activity in SH-SY5Y by mGluR agonist trans-ACPD and NMDA. SH-SY5Y cells were preincubated in HEPES-buffered medium containing either 0 or 100 µM bumetanide for 10 min. To test effect of trans-ACPD, cells were then exposed to 10 µM trans-ACPD at 37°C for 0-15 min (A). For NMDA experiments, cells were incubated in presence of 20 µM NMDA at 37°C for 0-15 min (B). Control cells were exposed to HEPES-buffered medium at 37°C for 0-15 min. To obtain bumetanide-sensitive (Bum-sen) 86Rb influx, 86Rb influx was assayed either in presence or absence of 100 µM bumetanide for 3 min. Data are means ± SE; n = 3. * P < 0.05 vs. control group by Student's t-test. Bum-res, bumetanide resistant.

To compare the temporal profile of the response to NMDA-mediated pathway, a similar time-course study was conducted with 20 µM NMDA. As shown in Fig. 5B, 20 µM NMDA stimulated the cotransporter activity in a similar time-dependent manner. Moreover, bumetanide-resistant K+ influx was also substantially stimulated by NMDA.

mGluRs consist of three groups of heterogeneous receptor proteins (11, 37). trans-ACPD primarily activates mGluR2 and mGluR3 receptors and also activates mGluR1 and mGluR5 receptors at higher concentrations (10, 31). To further characterize trans-ACPD-mediated signal transduction pathways, we examined whether the trans-ACPD-mediated effect could be blocked by a specific mGluR antagonist, (+)-MCPG. (+)-MCPG has been reported to antagonize mGluR1alpha and mGluR2 but not mGluR4 in transfected cells (10). Cells were incubated for 10 min in the presence of either 10 µM trans-ACPD or 10 µM trans-ACPD plus 50 µM (+)-MCPG. Bumetanide-sensitive K+ influx was then assessed in those cells. Figure 6 showed that the basal activity of the cotransporter was not changed by 50 µM (+)-MCPG. In contrast, trans-ACPD-induced stimulation of bumetanide-sensitive K+ influx was significantly inhibited by 50 µM (+)-MCPG (from 21.46 ± 3.13 to 10.80 ± 2.03 nmol · mg protein-1 · min-1, P < 0.05). To verify that (+)-MCPG exerts its effect specifically via blocking of metabotropic receptors, we examined whether (+)-MCPG had any effect on the NMDA-mediated pathway. As demonstrated in Fig. 6, NMDA-induced stimulation of the cotransporter activity remained unchanged in the presence of (+)-MCPG. This study further supports our hypothesis that stimulation of the cotransporter by trans-ACPD is due to activation of mGluR-mediated signal transduction pathways.


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Fig. 6.   Effect of a specific metabotropic receptor antagonist on trans-ACPD-mediated stimulation of Na+-K+-2Cl- cotransporter. SH-SY5Y cells were incubated at 37°C in HEPES-buffered medium containing either 10 µM trans-ACPD or 10 µM trans-ACPD + 50 µM (+)-MCPG for 10 min. To test effect of (+)-MCPG on NMDA-mediated stimulation, cells were exposed to either 10 µM NMDA or 10 µM NMDA + 50 µM (+)-MCPG for 10 min. To obtain bumetanide-sensitive 86Rb influx, 86Rb influx was subsequently assayed either in presence or absence of 100 µM bumetanide for 3 min at 37°C. Data are means ± SE; n = 3. Quadruplet determinations were obtained in each experiment. * P < 0.05 vs. control group; #P < 0.05 vs. trans-ACPD-treated group by ANOVA (Bonferroni-Dunn).

Expression of Na+-K+-2Cl- cotransporter protein and glutamate receptors in SH-SY5Y cells. To further confirm neuronal identity of SH-SY5Y cells, expression of a major component of neuronal intermediate filaments, neurofilament 200, was examined in SH-SY5Y cells by Western blot analysis. As shown in Fig. 7A, two protein bands were recognized by a monoclonal anti-neurofilament 200 antibody. These two major protein bands were 160- and 200-kDa neurofilament proteins and represent different levels of phosphorylation in these molecules during posttranslational modification. A control experiment was performed by using a crude membrane preparation of cerebral cortex from Sprague-Dawley rat and a similar result was observed (Fig. 7A).


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Fig. 7.   Expression of Na+-K+-2Cl- cotransporter and glutamate receptor proteins in SH-SY5Y cells by Western blot analysis. Cellular lysate proteins were obtained from SH-SY5Y cells by a sonication and brief centrifugation (see MATERIALS AND METHODS). Proteins were separated on 6% SDS gel and transferred to a PVDF membrane. Expression of neurofilament 200 protein (both phosphorylated and dephosphorylated) was determined by anti-neurofilament 200 protein monoclonal antibody (SH-SY5Y, 15 µg protein; brain cortex, 10 µg protein in A). To detect expression of cotransporter, membrane was probed with T4 monoclonal antibody (SH-SY5Y, 60 µg protein; brain cortex, 25 µg protein in B). Expression of NMDA receptor in SH-SY5Y cells was demonstrated by using anti-rat NR-2B antibody (SH-SY5Y, 80 µg protein; brain cortex, 15 µg protein in C). Expression of mGluR1 proteins was detected by using an anti-rabbit mGluR1 antibody (SH-SY5Y, 60 µg protein; brain cortex, 25 µg protein in D). Deglycosylation of protein samples with N-glycosidase in SH-SY5Y, crude membrane preparation of cerebral cortex as well as C6 glioma cells are shown in E. Blot was visualized by enhanced chemiluminescence. Data are a representative blot of 3 experiments.

Figure 7B demonstrated that T4 monoclonal antibody recognized an ~159-kDa cotransporter protein in SH-SY5Y, whereas an ~145-kDa cotransporter protein was found in crude membrane preparation of rat cerebral cortex. The size difference in the cotransporter proteins has been reported in other studies (17, 28, 33) and reflects different levels of glycosylation in these proteins. Deglycosylation of these two samples with N-glycosidase gave rise to a single protein band (~130 kDa) in both SH-SY5Y and crude membrane preparation of rat cerebral cortex (Fig. 7E).

Results in Figs. 1-6 of this study and work by others (20, 21) indicated that heterogeneous glutamate receptors are important in signal transduction in SH-SY5Y cells. In this experiment, we further investigated expression of ionotropic NMDA receptor and mGluRs in SH-SY5Y cells by Western blot analysis. Figure 7C showed that a 180-kDa NR-2B-receptor protein was expressed in the rat cerebral cortex. However, NR-2B-receptor protein in SH-SY5Y migrated faster. The cause for this discrepancy is not apparent. An ~142-kDa mGluR1 protein was expressed in both SH-SY5Y cells and rat cerebral cortex (Fig. 7D).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In this study, human neuroblastoma SH-SY5Y cells were used as a neuronal cell model. Many properties of mature noradrenergic neurons have been shown in SH-SY5Y cells (19). SH-SY5Y cells express ionotropic NMDA and AMPA receptors as well as mGluRs, determined by receptor-mediated activity and Northern blot analysis (20, 21). Especially in the present study, expression of the NMDA-receptor isoform NR-2B and mGluR1 protein in SH-SY5Y was demonstrated by Western blot analysis (Fig. 7, C and D). In addition, neuronal identity of SH-SY5Y cells was further characterized by an abundant expression of neurofilament protein 200, a specific cellular marker for neurons (Fig. 7A).

Western blot analysis revealed that a 159-kDa protein was recognized in cellular lysate preparation of SH-SY5Y by T4 monoclonal antibody (Fig. 7B). The molecular mass of the cotransporter protein identified here is consistent with the sizes of NKCC1 proteins observed in other cell types (8, 17, 35). Bumetanide-sensitive K+ influx represents ~38% of total K+ influx in SH-SY5Y cells (Table 1, Fig. 5B). The results of the present study clearly indicate that Na+-K+-2Cl- cotransporter in neuronal cells is regulated by the major excitatory neurotransmitter glutamate. Glutamate was found to induce a dose- and time-dependent stimulation of Na+-K+-2Cl- cotransporter activity in SH-SY5Y cells (Figs. 1 and 2). The novel finding of this work also includes that activation of both NMDA and metabotropic receptors is involved in stimulation of the Na+-K+-2Cl- cotransporter. Both the glutamate ionotropic receptor agonist NMDA and metabotropic receptor agonist trans-ACPD significantly stimulated the cotransport activity in these cells (Fig. 3). No significant effect on the cotransporter activity was observed by AMPA-receptor agonist AMPA (Fig. 3). Moreover, NMDA-mediated stimulation of Na+-K+-2Cl- cotransport was specifically abolished by the selective NMDA-receptor antagonist MK-801 (Fig. 4). The presence of both NMDA and trans-ACPD has no additive effect on the stimulation of cotransporter activity in SH-SY5Y cells (Fig. 3). This implies that activation of these two receptors may lead to a final common pathway that activates the Na+-K+-2Cl- cotransporter.

The mGluR agonist trans-ACPD caused a dose- and time-dependent stimulation of the cotransporter (Fig. 5A, Table 1). Furthermore, the trans-ACPD-mediated effect, but not the NMDA-mediated one, was specifically inhibited by the metabotropic receptor antagonist (+)-MCPG (Fig. 6). Expression of mGluR1 protein was detected in SH-SY5Y cells. These results suggested that mGluRs may play a role in regulation of Na+-K+-2Cl- cotransporter in neurons. However, it appears that glutamate mediates its effect on the cotransporter primarily through the NMDA receptor in SH-SY5Y cells, which was evident in the study with the NMDA-receptor antagonist MK-801 (Fig. 4).

Interestingly, we have observed that both NMDA and trans-ACPD stimulated a portion of K+ influx resistant to bumetanide (Table 1, Fig. 5B). The nature of NMDA and trans-ACPD-mediated stimulation of bumetanide-insensitive K+ influx is not known. It has been reported that the glutamate-receptor agonist L-AP-4 stimulates an inwardly rectifying K+ channel in astrocytes that can be inhibited by BaCl2 (9). It is possible that upregulated bumetanide-insensitive K+ influx may represent an inwardly rectifying K+ channel in SH-SY5Y cells. On the other hand, this bumetanide-insensitive K+ influx may be through Na+-K+-ATPase; however, it took 5-15 min for glutamate to maximally stimulate Na+-K+-ATPase activity in cerebellar neurons (18). The bumetanide-insensitive K+ influx appears to be stimulated faster in our study (Fig. 5B).

There is evidence suggesting that Na+-K+-2Cl- cotransporter activity in many peripheral cell types is regulated by diverse second messenger systems (8, 25, 34). Na+-K+-2Cl- cotransporter in some epithelial cells is activated by cell shrinkage and agents that elevate cAMP levels (16, 38). In contrast, it is found that Na+-K+-2Cl- cotransporter activity of endothelial cells and flounder intestine epithelial cells are inhibited by elevation of cAMP and cGMP (23, 36). Studies of the Na+-K+-2Cl- cotransporter have also shown that stimulation of the cotransporter by hormones is mediated by Ca2+ in endothelial cells (13, 23) and salivary acinar cells (29). However, little is known about regulation and function of the Na+-K+-2Cl- cotransporter in neurons. The only neuronal preparation that has been studied in depth is the squid giant axon. Bumetanide-sensitive cotransport activity in the squid giant axon, with a reported stoichiometry of 2Na+:1K+:3Cl-, requires ATP and is regulated by intracellular Cl- concentratoin (3, 30). This process appears to be involved in phosphorylation-dephosphorylation of the Na+-K+-Cl- cotransporter (2). In pheochromocytoma PC-12 cells, Na+-K+-2Cl- cotransporter is stimulated by nerve growth factor but inhibited by phorbol ester-mediated protein kinase C activation and agents that raise intracellular cAMP (15).

Activation of NMDA receptors and mGluRs causes an increase in intracellular Ca2+ concentration ([Ca2+]i) in SH-SY5Y cells (20). mGluR-receptor agonists L-AP-4 and glutamate induced a furosemide-sensitive cell swelling that was accompanied by a transient increase in [Ca2+]i in type 1 astroglial cells (9). In the present study, we found NMDA to induce a significant stimulation of NKCC1 protein activity, which was specifically inhibited by MK-801. Taken together, glutamate could cause an increase in [Ca2+]i of SH-SY5Y cells via NMDA-gated Ca2+ channels and consequently stimulate Na+-K+-2Cl- cotransporter. In the present study, trans-ACPD was also found to significantly elevate Na+-K+-2Cl- cotransporter activity in a time- and dose-dependent manner (Fig. 5A, Table 1). Therefore, mGluR-mediated changes in Ca2+ and/or cAMP in SH-SY5Y cells could conceivably stimulate Na+-K+-2Cl- cotransporter activity. Alteration of Ca2+-cAMP second messenger pathways by glutamate can modulate activities of protein kinases and phosphatase and consequently regulate the function of the cotransport system. It was recently reported that glutamate stimulates Na+-K+-ATPase activity by upregulating Ca2+-dependent phosphatase calcineurin, which appears to maintain the phosphorylation-dephosphorylation state of Na+-K+-ATPase together with protein kinase C (18). It will be interesting in future studies to investigate whether the glutamate-mediated effect involves changes in phosphorylation of Na+-K+-2Cl- cotransporter in neuronal cells.

    ACKNOWLEDGEMENTS

We thank Dr. Peter Lipton for many helpful discussions and for reading the manuscript. We also thank Gui Su for excellent technical assistance in protein deglycosylation experiments. The SH-SY5Y cell line was kindly provided by Dr. John A. Payne.

    FOOTNOTES

This work was supported in part by Scientist Development Grant 9630189N from the National Center Affiliate of the American Heart Association (to D. Sun) and by a grant to the University of Wisconsin Medical School under the Howard Hughes Medical Institute Research Resources Program for Medical Schools.

Address for reprint requests: D. Sun, Dept. of Neurological Surgery, School of Medicine, University of Wisconsin, F4/311, Clinical Science Center, 600 Highland Ave., Madison, WI 53792.

Received 19 December 1997; accepted in final form 3 June 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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