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Am J Physiol Cell Physiol 279: C1710-C1721, 2000;
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Vol. 279, Issue 6, C1710-C1721, December 2000

Regulation of Na+-K+-Clminus cotransporter in primary astrocytes by dibutyryl cAMP and high [K+]o

Gui Su1,2, Robert A. Haworth3, Robert J. Dempsey1, and Dandan Sun1,2

Departments of 1 Neurological Surgery, 2 Physiology, and 3 Surgery, School of Medicine, University of Wisconsin, Madison, Wisconsin 53792


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we examined the Na+-K+-Cl- cotransporter activity and expression in rat cortical astrocyte differentiation. Astrocyte differentiation was induced by dibutyryl cAMP (DBcAMP, 0.25 mM) for 7 days, and cells changed from a polygonal to process-bearing morphology. Basal activity of the cotransporter was significantly increased in DBcAMP-treated astrocytes (P < 0.05). Expression of an ~161-kDa cotransporter protein was increased by 91% in the DBcAMP-treated astrocytes. Moreover, the specific [3H]bumetanide binding was increased by 67% in the DBcAMP-treated astrocytes. Inhibition of protein synthesis by cyclohexamide (2-3 µg/ml) significantly attenuated the DBcAMP-mediated upregulation of the cotransporter activity and expression. The Na+-K+-Cl- cotransporter in astrocytes has been suggested to play a role in K+ uptake. In 75 mM extracellular K+ concentration, the cotransporter-mediated K+ influx was stimulated by 147% in nontreated cells and 79% in DBcAMP-treated cells (P < 0.05). To study whether this high K+-induced stimulation of the cotransporter is attributed to membrane depolarization and Ca2+ influx, the role of the L-type voltage-dependent Ca2+ channel was investigated. The high-K+-mediated stimulation of the cotransporter activity was abolished in the presence of either 0.5 or 1.0 µM of the L-type channel blocker nifedipine or Ca2+-free HEPES buffer. A rise in intracellular free Ca2+ in astrocytes was observed in high K+. These results provide the first evidence that the Na+-K+-Cl- cotransporter protein expression can be regulated selectively when intracellular cAMP is elevated. The study also demonstrates that the cotransporter in astrocytes is stimulated by high K+ in a Ca2+-dependent manner.

potassium uptake; bumetanide; L-type calcium channel; intracellular calcium; rat cortical astrocytes; dibutyryl-cAMP


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

UNDER PHYSIOLOGICAL CONDITIONS, Na+-K+-Cl- cotransporter in most cells transports Na+, K+, and Cl- into cells. This is driven by both favorable inward Na+ and Cl- chemical gradients (8, 28). Thus the cotransporter plays a crucial role in vectorial salt transport in epithelial cells and cell volume regulation in both epithelial and nonepithelial cells (8, 28). Despite studies of the Na+-K+-Cl- cotransporters in many cell types, their function and regulation in astrocytes have not been well understood. The Na+-K+-Cl- cotransporter has been shown to contribute to a baseline intracellular Na+ concentration ([Na+]i) in rat hippocampal astrocytes under physiological conditions (32) and active accumulation of Cl- in both mouse cortical astrocytes and rat hippocampal astrocytes (1, 44). It has also been proposed that inward transport of Na+ via the cotransporter provides Na+ influx for Na+-K+-ATPase function (43). The involvement of the Na+-K+-Cl- cotransporter in astrocyte K+ uptake has been suggested by several studies (17, 26, 45). Mouse astrocytes increased their K+ content significantly when cells were exposed to 12 mM [K+]o (43). Furosemide, an inhibitor of the Na+-K+-Cl- cotransporter, reduced the total net K+ uptake by 38%, suggesting that stimulation of the Na+-K+-Cl- cotransporter contributes to K+ uptake (43).

Depending on the species and tissue types, the Na+-K+-Cl- cotransporter is regulated by various hormonal factors and intracellular messengers, such as cAMP, cGMP, and Ca2+/calmodulin (7, 10, 15), and by cell shrinkage. Generally, the Na+-K+-Cl- cotransporter is stimulated by cell shrinkage and is inhibited by cell swelling (29). Interestingly, both hypertonic cell shrinkage and hypotonic cell swelling have been shown to activate the cotransporter activity in rat astrocytes (18, 26, 41) and C6 glioma cells (2, 25). Cellular mechanisms underlying these osmolarity-mediated effects on the cotransporter in astrocytes are not yet clear. It appears that the swelling-activated Na+-K+-Cl- cotransporter activity in C6 cells is Ca2+/calmodulin dependent and also is inhibited by the protein kinase inhibitors staurosporine and polymyxin B (25). These studies imply that the Na+-K+-Cl- cotransporter in astroglial cells is regulated by complex intracellular signaling pathways.

The present study was aimed at investigating the cellular mechanisms underlying cAMP- and high-[K+]o-mediated stimulation of the Na+-K+-Cl- cotransporter in primary astrocyte cultures. We report here that the cotransporter protein expression is upregulated in astrocytes treated with dibutyryl cAMP (DBcAMP). Moreover, the cotransporter activity in astrocytes in 75 mM [K+]o is significantly stimulated in a Ca2+-dependent manner.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Bumetanide and antineurofilament 200 monoclonal antibody were purchased from Sigma (St. Louis, MO). Eagle's modified essential medium (MEM) and Hanks' balanced salt solution were from Mediatech Cellgro (Herndon, VA). FBS was obtained from Hyclone Laboratories (Logan, UT). Collagen type I was from Collaborative Biomedical Products (Bedford, MA). 86RbCl was purchased from NEN Life Science Products (Boston, MA). [butyl-3H]bumetanide (15 Ci/mmol) was custom synthesized and HPLC purified by American Radiolabeled Chemicals (St. Louis, MO). Mouse anti-GLT-1 monoclonal IgG was from Chemicon International (Temecula, CA). Nifedipine and nimodepine were from Calbiochem (La Jolla, CA). Fura 2-AM was purchased from Molecular Probes (Eugene, OR).

Primary culture of rat cortical astrocytes. Dissociated cortical astrocyte cultures were established based on a method described by Hertz et al. (14). Cerebral cortices were removed from 1-day-old rats (Sprague Dawley). The cortices were incubated in a trypsin solution (0.25 mg/ml of HBSS) for 25 min at 37°C. The tissue was then mechanically triturated and filtrated through nylon meshes (70 µm). The dissociated cells were rinsed and resuspended in MEM containing 10% FBS. Viable cells (1 × 104 cells/well) were plated in 24-well plates coated with collagen type I. Cultures were maintained in a 5% CO2 atmosphere at 37°C. The cultures were subsequently refed every 3 days throughout the study. To obtain morphologically differentiated astrocytes, confluent cultures (days 12-15 in culture) were then treated with MEM containing 0.25 mM DBcAMP for 7 days to induce differentiation. DBcAMP has been used widely to mimic neuronal influences on astrocyte differentiation (13, 40). Experiments were performed routinely on cultures treated with DBcAMP for 7 days. The identity of astrocytes in culture was determined by immunocytochemical staining for glial fibrillary acidic protein (GFAP) expression, a marker protein for astroglial cells. Staining for neurofilament 200 was used for identification of neurons. More than 95% of cells in culture yielded by this preparation were astrocytes.

Assay for Na+-K+-Cl- cotransporter activity. Na+-K+-Cl- cotransporter activity was measured as bumetanide-sensitive K+ influx, using 86Rb as a tracer for K+ (38). Cultured astrocytes were equilibrated for 10-30 min at 37°C with an isotonic HEPES-buffered MEM (290 mosmol/l). 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 10 min in HEPES-MEM containing either 0 or 10 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 10 µM bumetanide. 86Rb influx was stopped by rinsing cells with 0.1 M ice-cold MgCl2. Radioactivity of the cellular extract in 1% SDS was analyzed by liquid scintillation counting (1900CA; Packard, Downers Grove, IL). K+ influx rate was calculated as the slope of 86Rb uptake over time and was expressed as nanomoles of K+ per milligram of protein per minute. Bumetanide-sensitive K+ influx was obtained by subtracting the K+ influx rate in the presence of bumetanide from the total K+ influx rate. In the high-[K+]o study, 75 mM [K+]o was obtained by replacing NaCl in HEPES-MEM solutions with equimolar KCl. Quadruplicate 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. (35). Statistical significance in the study was determined by ANOVA (Bonferroni/Dunn) at a confidence level of 95% (P < 0.05).

Specific [3H]bumetanide binding assay. [3H]bumetanide binding activity was assessed in morphologically undifferentiated and differentiated astrocytes grown in 24-well plates. [butyl-3H]bumetanide (15 Ci/mmol) was synthesized and HPLC purified by American Radiolabeled Chemicals, according to the method described by Forbush and Palfrey (5). The binding medium consisted of 0.0-0.47 µM [3H]bumetanide in HEPES-MEM. Unlabeled bumetanide (100 µM) was used to determine nonspecific [3H]bumetanide binding. Cells were incubated in the binding medium for 20 min at 37°C. The reaction was stopped by 0.1 M ice-cold MgCl2. Radioactivity of the cellular extract in 1% SDS was measured by liquid scintillation counting. A fraction of sample was used for protein determination (35). In each experiment, quadruplicate determinations were obtained, and five experiments were performed in each study. The computer curve fitting of [3H]bumetanide binding data in astrocytes was obtained by a nonlinear least-squares fitting (Graphpad; GraphPad Software, San Diego, CA).

Gel electrophoresis and Western blotting. Cortical astrocytes growing on culture dishes were washed with ice-cold PBS (pH 7.4) that contained 2 mM EDTA and protease inhibitors, as described previously (36). Cells were scraped from dishes and suspended in PBS and then were lysed by 30 s of sonication at 4°C by an ultrasonic processor (Sonics & Materials, Danbury, CT). To obtain cellular lysates, cellular debris was removed by a centrifugation at 420 g for 5 min. Protein content of the cellular lysate was determined (35). Samples and prestained molecular mass markers (Bio-Rad) 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 were then electrophoretically separated on 6% SDS gels (19), and the resolved proteins were transferred electrophoretically to a polyvinylidene difluoride 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 then were 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 washings, bound antibody was visualized using the enhanced chemiluminescence (ECL) assay (Amersham). T4 monoclonal antibody against the human colonic T84 epithelial Na+-K+-Cl- cotransporter (21) was used for detection of the cotransporter protein. To gain quantitative analysis of expression of Na+-K+-Cl- cotransporter protein and beta -actin in astrocytes, 3-35 µg protein of the whole cell lysate preparation were loaded on 6% SDS gels and probed with T4 antibody and anti-beta -actin antibody as described above. The protein bands on the film after the ECL reaction were scanned using a Hewlett-Packard ScanJet (4c/T) scanner. The intensity of each protein band was measured by UN-SCAN-It gel software (Silk Scientific, Orem, UT). A linear curve was obtained within 5-30 µg protein for both T4 antibody and anti-beta -actin antibody (r = 0.98 and 0.96, respectively). In addition, to establish a linear curve for exposure time of the ECL on the film, the blot with 5-30 µg protein was exposed to a film for 10, 15, 20, 30, 40, 50, 60, 90, or 120 s. There was no beta -actin signal observed in a 10-s exposure. A linear curve of exposure time was found for both T4 antibody and anti-beta actin antibody within 15-120 s of exposure time (r ~0.96). Therefore, either 15 or 30 µg of protein were loaded in all immunoblots of the present study. In addition, the ECL exposure time was within 60 s. Monoclonal antibody against GLT-1 glutamate transporters was used for analysis of expression of the glutamate transporter. For deglycosylation studies, cellular proteins (50 µg) were solubilized with 1% SDS, incubated in the presence of 1 unit of N-glycosidase F (Boehringer Mannheim Biomedicals, Indianapolis, IN) for 4 h at 37°C, and separated by SDS-PAGE as described above.

Immunofluorescence staining. Cultured cells grown on collagen type I-coated coverslips were rinsed with PBS (pH 7.4) and fixed with 4% paraformaldehyde in PBS for 40 min at room temperature. After being rinsed, cells were incubated with blocking solution (10% normal goat serum, 0.4% Triton X-100, and 1% BSA in PBS) for 1 h. Cells were 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 2 h. The images of the cells were captured by a laser-scanning confocal microscope (MRC 1000; Bio-Rad) located in the University of Wisconsin-Madison W. M. Keck Neural Imaging Laboratory. The microscope scan head was mounted transversely to an inverted Nikon Diaphot 200. The laser was a 15 mM krypton-argon mixed-gas air-cooled laser, which emitted a strong line in exact alignment at 488 nm, and the 522DF32 filter block was used for FITC signals. The emitted green light signals were directed to the respective photomultiplier tubes. The Bio-Rad 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.

Measurement of changes of intracellular Ca2+. Cultured astrocytes grown on collagen type I-coated coverslips were loaded at room temperature for 1 h in HEPES-MEM containing 5 µM fura 2-AM. Subsequently, the coverslips were rinsed with HEPES-MEM. The coverslip was put in a cuvette with a 30° angle in coordination with the excitation light path. Fluorescence was measured using a spectrophotometer (Spex fluorescence Spectrophotometer CM111) at room temperature. 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 a previous study (12). The fluorescence intensity with 1 mM Mn2+ was subtracted from the value measured in the absence of Mn2+. The 360- to 380-nm ratio (360/380 ratio) of the subtracted values was then calculated (6, 12). All solutions were perfused in 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression of Na+-K+-Cl- cotransporter in nontreated and DBcAMP-treated astrocytes. Immunofluorescence staining was employed to characterize morphologically undifferentiated and differentiated astrocytes. In Fig. 1, A and B, cells were stained with monoclonal antibody against GFAP, a marker protein for astrocytes. Differentiated astrocytes induced by DBcAMP (0.25 mM, 7 days) changed from a polygonal (Fig. 1A) to process-bearing morphology (Fig. 1B), a morphology that is more characteristic of matured and reactive astrocytes in situ. Moreover, distribution of the intermediate filament GFAP extended into the processes (Fig. 1B). This is consistent with the finding that elevation of intracellular cAMP levels in cultured astrocytes by DBcAMP causes transformation of the flat- into the stellate-type astrocytes (13). Expression and distribution of the Na+-K+-Cl- cotransporter were examined in these astrocytes. The Na+-K+-Cl- cotransporter expression was recognized by T4 monoclonal antibody against the human colonic T84 epithelial Na+-K+-Cl- cotransporter (21). Immunoreactive signals with T4 monoclonal antibody were weak in the polygonal astrocytes (Fig. 1C). In DBcAMP-treated astrocytes, the intensity of the T4 antibody staining was enhanced throughout the cell, including the process (Fig. 1D). To gain quantitative analysis of T4 antibody immunoreactive signals in non-DBcAMP-treated vs. DBcAMP-treated cells, we acquired fluorescence intensity in each cell shown in Fig. 1, C and D, using a MetaMorph Imaging System software program (version 3.68; Universal Imaging, West Chester, PA). An average relative intensity in cell bodies was 73.07 ± 2.97 (n = 6) in non-DBcAMP-treated cultures (Fig. 1C). In contrast, in the DBcAMP-treated cultures, the average immunofluorescence intensity with T4 antibody in cell bodies was increased by ~92% (140.30 ± 7.03, n = 6, P < 0.05; Fig. 1D). However, protein distribution at cell bodies and processes may differ in untreated and treated astrocytes. In this experiment, we have only examined the immunoreactivity of T4 antibody in cell bodies. Therefore, this may only reflect a change of the cotransporter expression in the cell body region, and the total cellular cotransporter proteins may remain unchanged in untreated and treated astrocytes. Several additional approaches such as immunoblotting, specific [3H]bumetanide binding, and bumetanide-sensitive K+ influx assays were taken to further address the issue (see below). The purity of the primary astrocyte cultures in this preparation was demonstrated further by a negative staining for neurofilament 200 protein, a neuronal marker (Fig. 1E). More than 95% of cells yielded by this preparation were astrocytes, and the remaining cells were neurons or oligodendrocytes. Figure 1F represents a negative control study in which a primary antibody was omitted, and the rest of the procedures were the same as in Fig. 1, A-E. This demonstrates that the images shown in Fig. 1, A-E, are specific immunoreactive signals.


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  		Fig. 1.   Immunofluorescence staining for Na+-K+-Cl- cotransporter protein in primary astrocyte cultures. A: non-dibutyryl-cAMP (DBcAMP)-treated astrocytes, anti-glial fibrillary acidic protein (GFAP) staining. B: DBcAMP-treated cells (7 days), anti-GFAP staining. C: non-DBcAMP-treated astrocytes, T4 monoclonal antibody staining for the Na+-K+-Cl- cotransporter. D: DBcAMP-treated cells (7 days), T4 antibody staining. E: anti-neurofilament 200 staining. F: negative control in which a primary antibody was omitted, and the rest of the procedures were the same as in A-E. Images were captured by a laser-scanning confocal microscope (MRC 1000; Bio-Rad) Scale bar: 50 µm in A-F.

DBcAMP-induced morphological differentiation causes upregulation of astrocyte-specific protein expression, such as GFAP and glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) (13, 33, 40). We further investigated whether the Na+-K+-Cl- cotransporter expression differs in undifferentiated and differentiated astrocytes by immunoblotting. As shown in Fig. 2A, an ~161 ± 4.6-kDa cotransporter protein was recognized by T4 monoclonal antibody (n = 3). Expression of the cotransporter protein was significantly increased in differentiated astrocytes, and an ~91% increase was revealed by a densitometric analysis of the immunoblotting data (n = 3, P < 0.05). In a positive control study (Fig. 2B), induction of the glutamate transporter GLT-1 expression was clearly shown in differentiated astrocytes, a consistent finding with previous reports of others (33, 40). In contrast, no significant increase was seen in expression of beta -actin (20.0 ± 8.5%, P > 0.05). Glycosylation of the Na+-K+-Cl- cotransporter proteins has been found in many cell types (21). Deglycosylation of the cotransporter with N-glycosidase gave rise to a core protein band (138 ± 8 kDa, n = 5) in both non-DBcAMP-treated and DBcAMP-treated samples (Fig. 2C).


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  		Fig. 2.   Upregulation of Na+-K+-Cl- cotransporter (NKCC) expression in DBcAMP-treated astrocytes. Cell lysates were separated by 6% SDS-PAGE, transferred to a nitrocellulose membrane, and probed with either T4 monoclonal antibody or anti-beta actin antibody (A) or anti-glutamate transporter GLT-1 antibody (B). C: deglycosylation of protein samples with N-glucosidase was performed as described in MATERIALS AND METHODS, and the blots were probed with T4 antibody. The Western blots were visualized by enhanced chemiluminescence. Blots are representative of 3-5 experiments.

Increase of a specific [3H]bumetanide binding in DBcAMP-treated astrocytes. DBcAMP stimulates the Na+-K+-Cl- cotransporter protein expression in astrocytes, as discussed above. To further characterize the upregulation of the cotransporter expression by DBcAMP, we investigated whether the specific [3H]bumetanide binding is increased in DBcAMP-treated astrocytes. Specific [3H]bumetanide binding to the cotransporter protein requires the simultaneous presence of three transported ions and has been used as an index to reflect functional cotransporters in many cell types (5, 9, 28). [3H]bumetanide binding was performed in the absence (total binding) and presence of 100 µM unlabeled bumetanide (nonspecific binding). The total binding contains both a saturable component and a linear component. The linear component represents nonspecific [3H]bumetanide binding. The specific [3H]bumetanide binding was determined by the difference in the two components and is shown in Fig. 3. An increase in the specific [3H]bumetanide binding is evident in DBcAMP-treated astrocytes (Fig. 3). The specific binding at 0.47 µM [3H]bumetanide was 0.30 ± 0.07 pmol/mg protein in nontreated astrocytes and increased to 0.58 ± 0.08 pmol/mg protein in DBcAMP-treated astrocytes. The computer fit of the data in Fig. 3 yields a value for maximal saturable binding of 0.49 ± 0.06 pmol/mg protein in nontreated astrocytes and of 0.82 ± 0.11 pmol/mg protein in DBcAMP-treated astrocytes. This reflects an ~67% increase in the saturable specific binding (0.82/0.49). The concentration of [3H]bumetanide required for half-maximal saturable binding was 0.27 ± 0.07 µM in nontreated astrocytes and 0.21 ± 0.07 µM in DBcAMP-treated astrocytes. These values are consistent with the reported bumetanide affinity in many cell types (28). These data imply that the amount of functional cotransporter proteins is increased in DBcAMP-treated astrocytes by 67%, a quantitative agreement with upregulation of the cotransporter expression revealed by immunoblot analysis. However, the bumetanide affinity of the cotransporter remained unchanged in DBcAMP-treated astrocytes.


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  		Fig. 3.   Specific [3H]bumetanide binding to cultured rat astrocytes. [3H]bumetanide binding to astrocyte monolayers was assayed at different [3H]bumetanide concentrations as described in MATERIALS AND METHODS. In each experiment, quadruplicate determinations of binding were made either in the presence or absence of 100 µM unlabeled bumetanide (nonspecific and total binding, respectively). Specific [3H]bumetanide binding was defined as the difference between the total and nonspecific binding. Data are means ± SE from 7-8 experiments. The curves drawn were derived from a nonlinear least-squares curve fit (a single binding site model).

It appears that the maximal binding (Bmax) of 0.49 pmol [3H]bumetanide/mg protein in nontreated astrocytes was lower than reported values (~1.0 pmol/mg protein) in other cultured cell types (11, 24, 36). The low Bmax in nontreated astrocyes could be due to the Cl--mediated inhibition of the binding (7) or the Cl--mediated inhibitory effect on the cotransporter activity (20). In the present study, a HEPES-MEM with normal external Cl- concentration (147 mM) was used instead of a low Cl- buffer. To investigate whether a relatively low Bmax of the [3H]bumetanide observed in nontreated astrocytes is in part a result of the Cl--mediated inhibitory effect on the cotransporter activity, we have compared the basal activity of the cotransporter in normal extracellular Cl- concentration ([Cl-]o; 147 mM) and low [Cl-]o (15 mM). The basal levels of bumetanide-sensitive K+ influx were 25.70 ± 1.15 nmol · mg protein-1 · min-1 (n = 4) in the presence of normal [Cl-]o. In the presence of 15 mM [Cl-]o (equimolar methanesulfonate replaced Cl-), bumetanide-sensitive K+ influx was increased to 53.07 ± 2.17 nmol · mg protein-1 · min-1 (n = 4). This suggests that a relatively low Bmax of the [3H]bumetanide observed in nontreated astrocytes could in part be a result of the Cl--mediated inhibitory effect on the cotransporter activity.

Characterization of the Na+-K+-Cl- cotransporter activity in DBcAMP-treated astrocytes. It has been established that the Na+-K+-Cl- cotransporter is specifically and reversibly inhibited by bumetanide (IC50 ~0.2 µM) in many cells (28). Bumetanide has been shown to be more potent than furosemide in non-DBcAMP-treated rat astrocytes (41). In this study, we examined effects of bumetanide and furosemide on K+ influx in DBcAMP-treated astrocytes. As shown in Fig. 4, bumetanide (IC50 ~0.3 µM) is a more potent inhibitor than furosemide (IC50 ~10.1 µM) in DBcAMP-treated astrocytes, consistent with the observation in non-DBcAMP-treated astrocytes (41), C6 glial cells (2), and published data of other cell types (28). This is also in agreement with the specific [3H]bumetanide binding study demonstrated in Fig. 3.


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  		Fig. 4.   Effect of bumetanide and furosemide on Na+-K+-Cl- cotransporter activity in DBcAMP-treated astrocytes. Astrocytes treated with DBcAMP were preincubated in HEPES-MEM for 10 min at 37°C. 86Rb influx (1 µCi/ml) was assayed in the presence of different concentrations of inhibitors. Cotransporter activity (100%) was taken as the difference between the 86Rb influx in the absence and presence of either 100 µM bumetanide or 1 mM furosemide. In each experiment, quadruplicate determinations were performed. Curves are representative of 3 experiments. The curves drawn are computer fits of the data by a nonlinear least-squares program to a single binding site model.

In addition, the specificity of the bumetanide-mediated effect on the Na+-K+-Cl- cotransporter was established further by an ion substitution study. Function of the Na+-K+-Cl- cotransporter requires the simultaneous presence of the three transported ions. Thus removal of either extracellular Na+ or Cl- would abolish the cotransporter-mediated K+ influx. Moreover, the cotransporter activity was studied in high [K+]o conditions (see below). To further establish that bumetanide-sensitive K+ influx indeed reflects the cotransporter-mediated one, we evaluated bumetanide-sensitive K+ influx in the absence of either extracellular Na+ or Cl- in both 5.8 and 75 mM [K+]o. As shown in Fig. 5, in either 5.8 or 75 mM [K+]o, bumetanide-sensitive K+ influx in differentiated astrocytes was completely abolished when either extracellular Na+ was replaced with equimolar choline (P < 0.05) or Cl- was replaced with equimolar gluconate (P < 0.05). Gluconate is an impermeant substitute anion that can affect the ion activity of Ca2+ (16). To further establish a dependency of the Na+-K+-Cl- cotransporter on extracellular Cl-, equimolar methanesulfonate was used to substitute Cl-. Under control conditions, the bumetanide-sensitive K+ influx in DBcAMP-treated astrocytes was 43.20 ± 2.4 nmol · mg protein-1 · min-1 in the presence of 5.8 mM K+. In the presence of Cl-free conditions (equimolar methanesulfonate replaced Cl-), the bumetanide-sensitive K+ influx was reduced to 1.60 ± 0.87 nmol · mg protein-1 · min-1 (n = 3-5, P < 0.05). The latter value is consistent with the one obtained with gluconate substitution. These results further support the notion that the bumetanide-sensitive K+ influx indeed reflects the activity of the Na+-K+-Cl- cotransporter in differentiated astrocytes.


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  		Fig. 5.   Dependency of Na+-K+-Cl- cotransporter activity on extracellular Na+ and Cl-. Astrocytes treated with DBcAMP were preincubated in HEPES-MEM in which equimolar NaCl was replaced with either choline chloride or sodium gluconate for 10 min. 86Rb influx was assayed either in the presence or absence of 10 µM bumetanide for 3 min at 37°C. In the case of 75 mM extracellular K+ concentration ([K+]o), either choline chloride in HEPES-MEM was replaced with equimolar potassium chloride (Na+ free) or sodium gluconate was replaced with equimolar potassium gluconate (Cl- free). Data are means ± SE; n = 3. Quadruplicate determinations were obtained in each experiment *P < 0.05 vs. control group (5.8 mM [K+]o). #P < 0.05 vs. high [K+]o control group (in the presence of both Na+ and Cl-).

Effect of protein synthesis inhibition on Na+-K+-Cl- cotransporter expression and activity. It has been shown that an increase in intracellular cAMP level plays an important role in the regulation of expression of several proteins in astrocytes (13, 33, 40). Our results of immunofluorescence staining, immunoblotting, and specific [3H]bumetanide binding indicate that the cotransporter expression is elevated in DBcAMP-treated astrocytes, and this might be due to an enhanced protein synthesis. To further test this possibility, we examined the effect of protein synthesis inhibition on the cotransporter expression. After confluency, astrocytes were cultured either in normal MEM, MEM containing 0.25 mM DBcAMP, or MEM containing 0.25 mM DBcAMP plus the protein syntheses inhibitor cyclohexamide (2-3 µg/ml) for 7 days. Figure 6A shows that expression of the cotransporter was increased significantly in DBcAMP-treated cells. In contrast, the DBcAMP-induced upregulation of the cotransporter expression was inhibited in the presence of 2 µg/ml cyclohexamide. The effect of cyclohexamide was summarized in Fig. 6B. Expression of the cotransporter was increased by ~91% in DBcAMP-treated astrocytes. This stimulation of the protein expression was reduced significantly by 2 and 3 µg/ml cyclohexamide. In addition, there were no differences in the cotransporter expression levels in nontreated cells and cells treated with DBcAMP plus 3 µg/ml cyclohexamide. These data further suggest that DBcAMP stimulates the cotransporter expression via a protein synthesis mechanism.


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  		Fig. 6.   Effect of cyclohexamide (CHX) on cotransporter expression and activity. A: confluent astrocyte cultures were treated with either 0.25 mM DBcAMP alone or 0.25 mM DBcAMP plus 2 µg/ml cyclohexamide for 7 days. Proteins were separated by 6% SDS gel, transferred to a polyvinylidene difluoride membrane, and probed with T4 antibody, as described in Fig. 2. Data are representative of 3-4 experiments. B: densitometric analysis of immunoblots for expression of the cotransporter in astrocytes. Data are means ± SE; n = 3-4. *P < 0.05 vs. DBcAMP treated. C: non-DBcAMP-treated, DBcAMP-treated alone, or DBcAMP plus cyclohexamide (3 µg/ml)-treated astrocytes were preincubated in HEPES-MEM containing 5.8 mM [K+]o for 10 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. Quadruplicate determinations were obtained in each experiment. *P < 0.05 vs. control. #P < 0.05 vs. DBcAMP treated.

Next, we investigated whether the cotransporter activity was altered in DBcAMP-treated cells. An increase in the specific [3H]bumetanide binding in DBcAMP-treated astrocytes indicated such a possibility. Moreover, we evaluated whether cyclohexamide could abolish this effect. As shown in Fig. 6C, the basal level of the bumetanide-sensitive K+ influx rate was increased significantly in DBcAMP-treated astrocytes from 22.66 ± 4.88 to 42.11 ± 4.15 nmol · mg-1 · min-1 (P < 0.05), reflecting an 86% increase. Quantitatively, this is in an agreement with the increase of the cotransporter protein expression in DBcAMP-treated cells, as demonstrated in Figs. 2, 3, and 6B. Intriguingly, in the presence of both DBcAMP and cyclohexamide (3 µg/ml), DBcAMP-induced stimulation of the cotransporter activity was abolished (P < 0.05). The cotransporter activity under these conditions appears to be lower than in the control group; however, the differences were not statistically significant (P > 0.05).

Ca2+-dependent stimulation of Na+-K+-Cl- cotransporter by high [K+]o in astrocytes. The Na+-K+-Cl- cotransporter in astrocytes appears to play an important role in K+ uptake (17, 26, 45). In non-DBcAMP-treated astrocytes (18) and C6 glial cells (39), the Na+-K+-Cl- cotransporter activity is substantially stimulated when [K+]o reaches >50 mM. However, the mechanism underlying this change has not been defined. We further extended this investigation in differentiated astrocytes. Interestingly, 75 mM [K+]o stimulates the cotransporter activity in both nontreated and DBcAMP-treated astrocytes. In nontreated cells, the cotransporter activity was elevated from a basal level of 17.37 ± 2.50 to 42.94 ± 7.77 nmol · mg protein-1 · min-1 in the presence of 75 mM [K+]o (P < 0.05, Fig. 7A). The cotransporter activity was stimulated further in DBcAMP-treated astrocytes, from a control level of 38.64 ± 1.59 to 69.20 ± 4.81 nmol · mg protein-1 · min-1 in 75 mM [K+]o (P < 0.05).


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  		Fig. 7.   Ca2+-dependent stimulation of Na+-K+-Cl- cotransporter by high [K+]o. A: non-DBcAMP- and DBcAMP-treated astrocytes (MATERIALS AND METHODS) were preincubated in HEPES-MEM containing either 5.8 or 75 mM [K+]o for 10 min. [K+]o (75 mM) was obtained by replacing NaCl in HEPES-MEM buffer with equimolar KCl. Data are means ± SE; n = 5. *P < 0.05 vs. -DBcAMP controls (either 5.8 or 75 mM K+). #P < 0.05 vs. 5.8 mM [K+]o controls (either -DBcAMP or +DBcAMP). B: DBcAMP-treated astrocytes were washed 3 times with Ca2+-free HEPES-MEM and then were preincubated in Ca2+-free HEPES-MEM containing either 5.8 or 75 mM [K+]o for 10 min. Ca2+-free HEPES-MEM contained 0 mM CaCl2 and 100 µM EGTA. Data are means ± SE; n = 3. *P < 0.05 vs. HEPES-MEM with 5.8 mM [K+]o. #P < 0.05 vs. 75 mM [K+]o. C: DBcAMP-treated astrocytes were preincubated in HEPES-MEM containing either 0.5 or 10 µM nifedipine (Nif) with either 5.8 or 75 mM [K+]o for 10 min. Data are means ± SE; n = 8. *P < 0.05 vs. 5.8 mM [K+]o in HEPES. #P < 0.05 vs. 75 mM [K+]o. 86Rb influx was assayed either in the presence or absence of 10 µM bumetanide, and quadruplicate determinations were obtained in A-C.

There are two possible mechanisms for the cotransporter to be stimulated under high [K+]o: a kinetic effect and/or a regulatory effect. The high [K+]o-induced stimulation of the cotransporter activity shown in Fig. 7A suggests a kinetic effect. Studies of properties of the cotransporter in C6 glioma cells have shown that the K0.5 value for K+ activation is 15 mM (2), whereas a K0.5 value of 2.7 mM was reported in cultured astrocytes (41). The cotransporter-mediated K+ influx in C6 glioma cells became saturated when [K+]o was >50 mM (25). In our previous study with C6 glioma cells, we found that the bumetanide-sensitive K+ influx was saturated when [K+]o was between 30 and 50 mM (39). However, there was a further sharp increase of the cotransporter activity when [K+]o reached >50 mM in C6 glioma cells (39) or 20-50 mM in DBcAMP-treated astrocytes (data not shown) and in nontreated astrocytes (18). This further increase of the bumetanide-sensitive K+ influx therefore could not be explained solely by a kinetic effect. We thus believe that a signal transduction-mediated regulatory effect contributes in part to the increase of the cotransporter activity under high [K+]o.

Ca2+ is involved in regulation of several ion flux systems, including the Na+-K+-Cl- cotransporter (8, 28, 38). Depolarization of the cellular plasma membrane under high [K+]o would activate voltage-dependent Ca2+ channels, and this could cause an increase in the intracellular concentration of Ca2+. To investigate this speculation, we first tested if high [K+]o-induced stimulation of the cotransporter in astrocytes was dependent on extracellular Ca2+. All experiments were performed in DBcAMP-treated astrocytes, unless indicated otherwise. As shown in Fig. 7B, basal levels of the bumetanide-sensitive K+ influx rate were 36.19 ± 3.70 nmol · mg protein-1 · min-1 in a control HEPES-MEM containing 1.27 mM CaCl2. The bumetanide-sensitive K+ influx rate was reduced to 22.94 ± 3.97 nmol · mg protein-1 · min-1 in the presence of 0 mM CaCl2 plus 100 µM EGTA (P < 0.05). Free Ca2+ in HEPES-MEM under the above conditions was calculated to be 0.4 nM (MAXChelator program; Stanford University). The results imply that an optimal level of intracellular Ca2+ is required to maintain the cotransporter activity under control conditions for DBcAMP-treated astrocytes. To investigate whether removal of extracellular Ca2+ may block only the DBcAMP-stimulated cotransporter activity in 5.8 mM [K+]o, the effect of Ca2+-free HEPES conditions on nontreated astrocytes was examined. Removal of external Ca2+ decreased bumetanide-sensitive 86Rb influx in nontreated astrocytes (from 19.15 ± 2.57 to 11.16 ± 1.59 nmol · mg protein-1 · min-1, P < 0.05; data are results of 12 replicate measurements). These data further suggest that Ca2+-free HEPES conditions affect the basal levels of the cotransporter activity not only in DBcAMP-treated but also in non-DBcAMP-treated astrocytes. Moreover, stimulation of the cotransporter activity in high [K+]o was significantly attenuated in Ca2+-free HEPES-MEM. The cotransporter activity at 75 mM [K+]o was reduced from 62.34 ± 8.49 to 31.51 ± 4.22 nmol · mg protein-1 · min-1 (P < 0.05; Fig. 7B). The latter value was not significantly different from that in Ca2+-free HEPES-MEM under normal K+ (P > 0.05). Thus removal of extracellular Ca2+ abolished the stimulation of the cotransporter activity under high [K+]o. These results suggest that high-[K+]o-mediated stimulation of the cotransporter activity in astrocytes required extracellular Ca2+.

We then investigated if blocking dihydropyridine-sensitive L-type Ca2+ channels could inhibit the high-[K+]o-mediated effect on the cotransporter. This was achieved by exposing cells to either 0.5 or 1.0 µM nifedipine, an L-type voltage-sensitive Ca2+ channel blocker. As shown in Fig. 7C, neither concentration of nifedipine caused any significant effect on the basal levels of the Na+-K+-Cl- cotransporter activity. However, high-[K+]o-induced stimulation of the bumetanide-sensitive K+ influx was abolished in the presence of 0.5 µM nifedipine. The cotransporter activity in high [K+]o was decreased from 74.47 ± 8.58 to 34.71 ± 3.71 nmol · mg protein-1 · min-1 by 0.5 µM nifedipine (P < 0.05, Fig. 7C). The latter level is similar to that of 31.51 ± 4.22 nmol · mg protein-1 · min-1 in Ca2+-free HEPES-MEM (Fig. 7B). In addition, the cotransporter activity was inhibited significantly by either 1.0 µM nifedipine (Fig. 7C) or 1.0 µM nimodipine (data not shown). These data further support our hypothesis that high-[K+]o-mediated stimulation of the cotransporter involves a Ca2+-dependent process.

To further strengthen this argument, we looked for changes in intracellular Ca2+ in astrocytes in the presence of high [K+]o by using fura 2 fluorescence. Exposing fura 2-loaded cells to 75 mM [K+]o led to an increase of the 360/380 ratio, which indicates a rise of intracellular Ca2+ levels (6). As shown in Fig. 8A, 2 min after cells were exposed to high [K+]o, the intracellular Ca2+ rise reached a peak level and then declined to a plateau within 3 min. The plateau level was sometimes up to 50% of the peak value, as shown in Fig. 8A. The intracellular Ca2+ levels gradually recovered to the basal levels after returning cells to the HEPES-MEM containing 5.8 mM [K+]o. When cells were exposed to 10 µM nifedipine followed by high [K+]o, a rise of the intracellular Ca2+ occurred within 2 min. However, the peak of the intracellular Ca2+ concentration ([Ca2+]i) rise was reduced (Fig. 8A). In many experiments, this peak was completely abolished by nifedipine. As shown in Fig. 8B, the average height of the first peak was 0.26 ± 0.08 (n = 7). In the presence of both high [K+]o and nifedipine, the average peak height of the 360/380 ratio was reduced to -0.01 ± 0.05 (P < 0.05, 2-tailed Student's t-test). We further investigated whether the lack of the intracellular Ca2+ rise in the presence of nifedipine could be due to a lack of the cellular response to the second high [K+]o stimulus rather than blocking of the L-type channel-mediated Ca2+ influx. In a different set of experiments, we compared the Ca2+ rises during first and second exposures of cells to high [K+]o, both without nifedipine. We found that the change of the first peak of the 360/380 ratio was 0.13 ± 0.02 (n = 7). The second exposure of cells to high [K+]o gave rise to a change of the ratio of 0.10 ± 0.02 (n = 7). These data were not statistically significantly different (P > 0.05, 2-tailed Student's t-test). This suggests that the loss of the second peak in Fig. 8B was truly an effect of nifedipine, which in turn implies that Ca2+ influx was predominantly through the L-type Ca2+ channels under high [K+]o.


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  		Fig. 8.   Changes of intracellular Ca2+ in astrocytes in the presence of high extracellular K+. A: intracellular Ca2+ measurement was performed as described in MATERIALS AND METHODS. Cells on coverslips were perfused with HEPES-MEM containing 5.8 mM [K+]o. Exposure of cells to 75 mM [K+]o was indicated by the continuous line in the trace. Nifedipine (10 µM) was tested for Ca2+ influx via L-type voltage-dependent Ca2+ channels. Changes in the 360/380 ratio were taken to indicate changes in intracellular Ca2+ concentration. High [K+]o (75 mM) was obtained by replacing NaCl in HEPES-MEM buffer with equimolar KCl. The trace is representative of 7 experiments. B: analysis of data in A. Results show the peak height of the 360/380 ratio under 75 mM [K+]o either in the absence or presence of nifedipine (10 µM). Data are means ± SE; n = 7. *P < 0.05 vs. 1st peak (Student's t-test, 2-tailed).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Na+-K+-Cl- cotransporter in astrocytes. Na+-K+-Cl- cotransporter-mediated K+ flux has been observed in primary cultures of rat astrocytes (18, 26, 41) and C6 glioma cells (2, 25, 39). In the present study, an ~161-kDa Na+-K+-Cl- cotransporter protein was recognized by T4 monoclonal antibody in both morphologically undifferentiated and differentiated astrocytes. A core protein of 138 kDa is detected in these samples after deglycosylation. Moreover, abundant T4 antibody immunoreactivity was found in primary cultures of astrocytes. To date, only two distinct isoforms of Na+-K+-Cl- cotransporter (NKCC) have been identified: NKCC1 (7.0- to 7.5-kb transcript) with a wide range of tissue distributions (10, 27), and NKCC2 (4.6- to 5.2-kb transcript), which has only been found in vertebrate kidney (10, 27). Several studies have demonstrated that rat brain tissue expresses a high level of NKCC1 (30, 38). Although T4 antibody reacts with both NKCC1 and NKCC2 isoforms of the Na+-K+-Cl- cotransporters (21), our immunoblotting and immunocytochemistry data likely reflect expression of the NKCC1 isoform in primary cultures of astrocytes.

The role of the Na+-K+-Cl- cotransporter in astrocytes under physiological conditions has not been well defined. It has been proposed that inward transport of Na+ via the Na+-K+-Cl- cotransporter provides Na+ influx for Na+-K+-ATPase function, the so-called "transmembrane Na+ cycle" (43). In addition, it has been demonstrated that the Na+-K+-Cl- cotransporter contributes to a baseline [Na+]i in astrocytes under physiological conditions: application of furosemide or bumetanide to hippocampal astrocytes resulted in a slow, reversible decrease in [Na+]i by ~2 mM (32). The Na+-K+-Cl- cotransporter has been shown to contribute to active accumulation of Cl- in both mouse cortical astrocytes and rat hippocampal astrocytes (1, 44). The average resting [Cl-]i values are 36 ± 4 mM in rat hippocampal astrocyte cultures (1) and 25-30 mM in cultured mouse cortical astrocytes (44). These values are well above the equilibrium values that are predicted from the Cl- electrochemical equilibrium potentials. Inhibition of the cotransporter reduced the resting [Cl-]i by ~56% in rat hippocampal astrocyte cultures with 1 µM bumetanide (1). Equilibrated intracellular Cl- content was decreased by ~57% with 2 mM furosemide (44). These reports support the notion that a steady inward transport of Cl- via the Na+-K+-Cl- cotransporter contributes to active Cl- accumulation in astrocytes.

DBcAMP-mediated upregulation of the Na+-K+-Cl- cotransporter in astrocytes. Neuronal influences have been suggested to play an important role in astrocyte function. DBcAMP is widely used to mimic noradrenergic neuronal influences on astrocyte cultures (13). Maturation of astrocyte cultures with DBcAMP is concomitant with induction and upregulation of several astrocyte proteins, including glial-specific protein GFAP and glial glutamate transporter GLT-1 (13, 33, 40). This DBcAMP-mediated effect could be mimicked by coculturing astrocytes with neurons (33, 40). Induction of the glutamate transporter GLT-1 in DBcAMP-treated cells was demonstrated in our study as a positive control.

Interestingly, expression of the Na+-K+-Cl- cotransporter was increased by ~91% in DBcAMP-treated cells, as assessed by immunoblotting analysis. This was supported further by a 67% increase in the specific [3H]bumetanide binding and an elevated bumetanide-sensitive K+ influx in DBcAMP-treated astrocytes. This suggests that the newly synthesized cotransporter proteins are localized at the cell surface of DBcAMP-treated astrocytes. This speculation was supported further by upregulation of the basal level of the cotransporter activity in DBcAMP-treated astrocytes. In contrast, no significant change in expression of beta -actin was observed in DBcAMP-treated cells. Moreover, the DBcAMP-mediated upregulation of the cotransporter expression was abolished in the presence of the protein synthesis inhibitor cyclohexamide. cAMP-dependent regulation of the cotransporter has been investigated in different tissues. In many cell types, acute elevation of intracellular cAMP leads to stimulation of the cotransporter activity, which could be via direct or indirect mechanisms (10). In intestinal epithelial cells, activation of the cotransporter activity elicited by cAMP is accompanied by only a slight increase in surface expression of the cotransporter protein (3). Thus, to our knowledge, this study is the first report on the cAMP-mediated significant effect on the cotransporter protein expression.

Little is known about regulation of the Na+-K+-Cl- cotransporter expression. The study of Randall et al. (31) revealed that a promoter region of the secretory Na+-K+-Cl- cotransporter (bumetanide-sensitive cotransporter-2 or NKCC1) possesses a weak TATA box and binding sites for different transcription factors such as muscle enhancer factor 2, octamer transcription factor 1-2A, nuclear factor-kappa B, surfactant protein 1, and activator protein 2. These characteristics of the promoter region were also found in the nucleotide sequence of the brain PCR samples (31). Cytokines IL-1beta and IL-6 have been shown to upregulate NKCC1 mRNA and protein expression in endothelial cells (37, 42). However, both function and protein expression of NKCC1 were significantly inhibited by cytomegalovirus infection in human fibroblasts (23). Although the molecular mechanisms underlying DBcAMP-mediated upregulation of the cotransporter protein in astrocytes will need to be investigated further, it is plausible that elevation of intracellular cAMP via DBcAMP treatment alters either transcription or translation processes of the NKCC1 gene expression in astrocytes.

Ca2+-dependent stimulation of Na+-K+-Cl- cotransporter activity under high [K+]o. It has been well documented that Na+-K+-Cl- cotransporter activity in many peripheral cell types is regulated by diverse second messenger systems (8, 10). Inhibition of calmodulin activity significantly decreased the basal activity of the Na+-K+-Cl- cotransporter in C6 glioma cells (25). In contrast, blocking protein phosphatases with okadaic acid stimulated the cotransporter activity by approximately twofold in the absence of other stimuli (25). In this report, we found that reduction of intracellular Ca2+ by incubation of astrocytes with Ca2+-free HEPES buffer significantly decreased the basal levels of the cotransporter activity. Taken together, these studies suggest that a steady state of the cotransporter function in astrocytes is regulated by Ca2+-dependent mechanisms, and it could be via protein phosphorylation.

The increase of bumetanide-sensitive K+ influx by high [K+]o observed in this study is not a simple kinetic effect of extracellular K+ on the cotransporter. We found that the high-[K+]o-induced stimulation is completely abolished by either removal of extracellular Ca2+ or blocking of L-type voltage-dependent Ca2+ channels. If the high-[K+]o-mediated effect was a kinetic one, blockage of L-type voltage-dependent Ca2+ channels or removal of extracellular Ca2+ would affect the activity of the cotransporter under 5.8 and 75 mM [K+]o to a similar degree. Our results show that blocking of L-type voltage-dependent Ca2+ channels did not cause any significant effect on the basal levels of the Na+-K+-Cl- cotransporter activity. In contrast, it completely abolished the high-[K+]o-induced stimulation of the bumetanide-sensitive K+ influx. Moreover, the cotransporter activities were not statistically significantly different in the absence of extracellular Ca2+ in either 5.8 or 75 mM [K+]o. We therefore believe that the cotransporter activity is stimulated under high [K+]o in part via Ca2+-mediated signal transduction pathways.

It has been reported that voltage-dependent Ca2+ channels in astrocytes are activated under high-[K+]o conditions (4). Elevation of [K+]o to 50 mM caused an increase of [Ca2+]i to 150 nM-1 µM above resting levels in acutely isolated hippocampal astrocytes. Moreover, a high-[K+]o-evoked increase in [Ca2+]i was blocked by removal of external Ca2+ and was suppressed markedly by the Ca2+ channel blocker verapamil (4). We demonstrated that high [K+]o caused a rise of intracellular Ca2+ in rat astrocytes, and this increase in intracellular Ca2+ was decreased significantly by 10 µM nifedipine. These results imply that the stimulation of the cotransporter activity by high [K+]o is attributable to an increase in intracellular Ca2+. Conceivably, activation of Ca2+-mediated second messenger pathways could modulate activities of protein kinases and/or phosphatases and consequently regulate the function of the cotransporter.

We have repeatedly detected the 360/380 ratio increase under high [K+]o. However, the changes are smaller than that reported by MacVicar et al. (4, 22). The reason for the discrepancy is unclear but could be due to differences in species and experimental methods. In those studies, single cells from either acutely isolated rat hippocampal asytrocytes (4) or mouse cortical astrocyte primary cultures (22) were used. In this report, the Ca2+ measurement was performed on rat cortical astrocyte monolayers. The 360/380 fluorescence intensity ratio represents the average values of the entire cell population on the monolayer. Although the 360/380 ratio changes under high [K+]o reported here are small, they are reproducible and can be inhibited significantly by nifedipine.

Role of the Na+-K+-Cl- cotransporter in K+ uptake in astrocytes. A high-[K+]o-mediated stimulation of the cotransporter in astrocytes has been observed in other studies (18, 43). In non-DBcAMP-treated rat cortical astrocytes and C6 glial cells, the cotransporter activity is stimulated significantly when [K+]o reaches >50 mM (18, 39). In our study, the bumetanide-sensitive K+ influx in DBcAMP-treated astrocytes was stimulated by 79% in 75 mM [K+]o. Astrocytes in primary cultures are known to accumulate K+ avidly (43). Brain extracellular K+ increases in response to numerous physiological and pathophysiological conditions in the central nervous system (34). A few minutes of anoxia/ischemia raises [K+]o to ~60 mM (34). Therefore, elevation of the Na+-K+-Cl- cotransporter activity in astrocytes in high [K+]o could play an important role in K+ uptake.

In summary, we report here that an ~161-kDa cotransporter protein is expressed abundantly in both morphologically undifferentiated and differentiated astrocytes. The morphologically differentiated astrocytes induced by DBcAMP expressed a significantly higher basal activity of the cotransporter. This was attributable to an upregulation of the cotransporter protein expression. Moreover, 75 mM [K+]o significantly stimulated the cotransporter activity in astrocytes in a Ca2+-dependent manner. The high-[K+]o-mediated stimulation of the cotransporter activity was abolished in the presence of the L-type channel blocker nifedipine or Ca2+-free HEPES buffer. These results provide the first evidence that the Na+-K+-Cl- cotransporter protein expression can be regulated selectively when intracellular cAMP is elevated. The study also demonstrates that the cotransporter in astrocytes is stimulated by high [K+]o in a Ca2+-dependent manner.


    ACKNOWLEDGEMENTS

We thank Dr. Mark Haas for providing the method and instruction on purification of [3H]bumetanide. We also thank Dr. Peter Lipton for helpful comments and suggestions.


    FOOTNOTES

This work was supported in part by a Scientist Development Grant from the National Center Affiliate of the American Heart Association (9630189N to D. Sun), National Institute of Neurological Disorders and Stroke Grant RO1 NS-38118 and NSF Career Grant IBN9981826 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. G. Su was supported by a Graduate School Research Grant from the University of Wisconsin-Madison and by a research fund from the Department of Neurological Surgery.

Address for reprint requests and other correspondence: D. Sun, Dept. of Neurological Surgery, School of Medicine, Univ. of Wisconsin, H4/332, Clinical Science Center, 600 Highland Ave., Madison, WI 53792 (E-mail: sun{at}neurosurg.wisc.edu).

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 6 March 2000; accepted in final form 18 July 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Cell Physiol 279(6):C1710-C1721
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