Na+-K+-2Clminus Cotransporter in Immature Cortical Neurons: A Role in Intracellular Clminus Regulation

Dandan Sun1,2 and Sangita G. Murali1

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 gamma -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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 gamma -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
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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 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-beta -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 beta -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 (beta  subunit) monoclonal antibodies, and cytosine-1-beta -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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -tubulin was performed. Expression of beta -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.



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Fig. 1. Expression of Na+-K+-2Cl- cotransporter proteins in cortical neurons. A: phase contrast image of cultured cortical neurons (11 days) obtained by Nikon TMS light microscope. Scale bar 50 µm. B: antitubulin (beta -subunit) antibody staining (1:50 dilution). Scale bar 25 µm. C: anti-Na+-K+-2Cl- cotransporter antibody staining (T4, 1:50 dilution). Scale bar 20 µm. D: negative control in which only the FITC-conjugated goat anti-mouse IgG (1:500) was used and the primary antibodies were omitted. The rest procedures were the same as in B and C. Scale bar 20 µm. Images in B-D were the confocal microscopic images (METHODS).

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).



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Fig. 2. Na+-K+-2Cl- cotransporter activity in cortical neurons. Na+-K+-2Cl- cotransporter activity was assessed by bumetanide-sensitive 86Rb influx. To obtain bumetanide-sensitive 86Rb influx, cells were preincubated in the presence of 100 µM bumetanide for 5 min. 86Rb influx was assayed either in the absence or presence of 100 µM bumetanide for 3 min at 37°C. Ouabain-sensitive K+ influx was determined in the presence of 1 mM ouabain for 3 min at 37°C. Passive K+ influx was determined in the presence of both bumetanide and ouabain for 3 min at 37°C. To determine the dependency of Na+-K+-2Cl- cotransporter on extracellular Na+ or Cl-, cortical neurons were preincubated in HEPES-buffered medium in which equimolar NaCl was replaced either with choline chloride or sodium gluconate for 10 min. 86Rb influx subsequently was assayed in Na+-free or Cl--free buffer containing either 100 µM bumetanide or 1 mM ouabain for 3 min. Data are means ± SE, n = 3. Quadruplet determinations were obtained in each experiment. * P < 0.05, vs. control by ANOVA (Benforroni/Dunn).

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).



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Fig. 3. Development-dependent upregulation of Na+-K+-2Cl- cotransporter activity in cortical neurons. Neurons were grown in 24-well plate for 4-14 days (DIV 4-14). Na+-K+-2Cl- cotransporter activity was assessed by bumetanide-sensitive 86Rb influx. To obtain bumetanide-sensitive 86Rb influx, 86Rb influx was assayed either in the presence or absence of 100 µM bumetanide for 3 min at 37°C. Ouabain-sensitive K+ influx was determined in the presence of 1 mM ouabain and passive K+ influx was determined in the presence of both bumetanide and ouabain for 3 min at 37°C. Data are means ± SE, n = 3. Quadruplet determinations were obtained in each experiment. * P < 0.05 vs. value at DIV 4 by ANOVA (Benforroni/Dunn).

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 beta -actin protein expression, a "house-keeping" protein (Fig. 4, A and B). However, the degree of the changes in beta -actin expression was less. In addition, a time-dependent increase in expression of alpha  subunit of Na+/K+ ATPase also was found in neurons at DIV 12-14 (data not shown).



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Fig. 4. Development-dependent expression of Na+-K+-2Cl- cotransporters in cortical neurons by Western blot analysis. Cellular lysate proteins or crude membrane proteins of cortex tissues were separated on 6% SDS gel and transferred to a PVDF membrane (METHODS). A: expression of the cotransporter protein in cortical neurons cultured for 4-14 days (DIV 4-14, 25 µg protein/lane) was determined by T4 monoclonal antibody. Expression of a "house-keeping" beta -actin protein also was shown in the same blot. B: densitometric analysis of Western blots for expression of the cotransporter and beta -actin protein in cortical neurons (DIV 4-14, n = 4). C: expression of the cotransporter protein in brain cortex of rats (postnatal day 0-9, 25 µg protein/lane). Blot was visualized by ECL. Data was a representative blot of 3-4 experiments.

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.



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Fig. 5. Stimulation of Na+-K+-2Cl- cotransporter by opening of GABA-gated Cl- channels. A: cortical neurons were preincubated with 20-100 µM GABA or 100 µM GABA plus 10 µM (+)-bicuculline in isotonic HEPES-buffered MEM at 37°C for 5 min. B: cells were preincubated with either muscimol (30-100 µM) alone or 30 µM muscimol plus 10 µM bicuculline for 5 min. Na+-K+-2Cl- cotransporter activity subsequently was assessed by bumetanide-sensitive 86Rb influx for 3 min. Data are means ± SE, n = 3-4. Quadruplet determinations were obtained in each experiment. * P < 0.05 vs. control, # P < 0.05 vs. muscimol (30 µM) by ANOVA (Benforroni/Dunn).

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.



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Fig. 6. Stimulation of Na+-K+-2Cl- cotransporter activity by reduction of intracellular [Cl-]. A reduction in [Cl-]i was achieved by preincubating cells for 30 min in HEPES-MEM buffer containing 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). Bumetanide-sensitive K+ influx rate in cells then was examined for 2 min in HEPES-MEM containing a normal level of 147 mM Cl-. Data are means ± SE, n = 3. Quadruplet determinations were obtained in each experiment. * P < 0.05 vs. control by ANOVA (Benforroni/Dunn).

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).



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Fig. 7. Effect of N-methyl-D-aspartate (NMDA) receptor antagonist ifenprodil on NMDA-mediated stimulation of the Na+-K+-2Cl- cotransporter. Cortical neurons were preincubated with either 40 µM NMDA or 40 µM NMDA plus 15 µM ifenprodil for 5 min. Control cells were incubated with isotonic HEPES-buffered MEM for 5 min. Na+-K+-2Cl- cotransporter activity subsequently was assessed by bumetanide-sensitive 86Rb influx. Ouabain-sensitive K+ influx was determined in the presence of 1 mM ouabain and passive K+ influx was determined in the presence of both bumetanide and ouabain for 3 min at 37°C. Data are means ± SE, n = 5-7. Quadruplet determinations were obtained in each experiment. * P < 0.05 vs. control; # P > 0.05 vs. control by ANOVA (Benforroni/Dunn).

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).



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Fig. 8. Effect of bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid acetoxymethyl(BAPTA-AM) on NMDA-mediated stimulation of the Na+-K+-2Cl- cotransporter. Cells were preincubated in buffers containing either 40 µM NMDA or 40 µM NMDA plus 50 µM BAPTA-AM for 5 min. Control cells were incubated with isotonic HEPES-buffered MEM. Na+-K+-2Cl- cotransporter activity subsequently was assessed by bumetanide-sensitive 86Rb influx for 3 min. Ouabain-sensitive K+ influx was determined in the presence of 1 mM ouabain, and passive K+ influx was determined in the presence of both bumetanide and ouabain for 3 min at 37°C. Data are means ± SE, n = 5-7. Quadruplet determinations were obtained in each experiment. * P < 0.05 vs. control; # P > 0.05 vs. control by ANOVA (Benforroni/Dunn).

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.



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Fig. 9. Expression of Na+-K+-2Cl- cotransporter and NMDA receptors in cortical neurons by Western blot analysis. Cellular lysate proteins from cortical neurons and brain cortex proteins of adult rat were separated on 6% SDS gel and transferred to a PVDF membrane. A: expression of neurofilament 200 protein were determined by antineurofilament 200 protein monoclonal antibody. Doublet bands result from different degree of phosphorylation. B: expression of the cotransporter was detected with T4 anticotransporter monoclonal antibody. C: expression of NMDA receptor NR-1. D: expression of NMDA receptor NR-2B. Blot was visualized by ECL. Data were representative blots of 3 experiments.

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.


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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.


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