Departments of 1Neurosurgery and 2Physiology, University of Wisconsin Medical School, Madison, Wisconsin 53792; 3Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, Ohio 45267; and 4Department of Cardiovascular Sciences, University of Leicester, Leicester LE2 7LX, United Kingdom
Submitted 10 December 2003 ; accepted in final form 4 March 2004
ABSTRACT
The ubiquitously expressed Na+/H+ exchanger isoform 1 (NHE1) functions as a major intracellular pH (pHi) regulatory mechanism in many cell types, and in some tissues its activity may contribute to ischemic injury. In the present study, cortical astrocyte cultures from wild-type (NHE1+/+) and NHE1-deficient (NHE1/) mice were used to investigate the role of NHE1 in pHi recovery and ischemic injury in astrocytes. In the absence of HCO3, the mean resting pHi levels were 6.86 ± 0.03 in NHE1+/+ astrocytes and 6.53 ± 0.04 in NHE1/ astrocytes. Removal of extracellular Na+ or blocking of NHE1 activity by the potent NHE1 inhibitor HOE-642 significantly reduced the resting level of pHi in NHE1+/+ astrocytes. NHE1+/+ astrocytes exhibited a rapid pHi recovery (0.33 ± 0.08 pH unit/min) after NH4Cl prepulse acid load. The pHi recovery in NHE1+/+ astrocytes was reversibly inhibited by HOE-642 or removal of extracellular Na+. In NHE1/ astrocytes, the pHi recovery after acidification was impaired and not affected by either Na+-free conditions or HOE-642. Furthermore, 2 h of oxygen and glucose deprivation (OGD) led to an 80% increase in pHi recovery rate in NHE1+/+ astrocytes. OGD induced a 5-fold rise in intracellular [Na+] and 26% swelling in NHE1+/+ astrocytes. HOE-642 or genetic ablation of NHE1 significantly reduced the Na+ rise and swelling after OGD. These results suggest that NHE1 is the major pHi regulatory mechanism in cortical astrocytes and that ablation of NHE1 in astrocytes attenuates ischemia-induced disruption of ionic regulation and swelling.
intracellular pH; cortical astrocytes; sodium/calcium exchange; ischemia; intracellular sodium
Pharmacological approaches have been used to elucidate the role of NHEs in astrocyte pHi regulation. In HCO3-free buffer, the NHE blocker amiloride decreased the resting pHi by 0.100.42 pH units in rat cortical astrocytes (23, 31). Similar decreases in the resting pHi by amiloride were found in rat cerebellar and hippocampal astrocytes (7, 26, 28). Amiloride abolished pHi recovery in astrocytes after NH4Cl prepulse acid loads (4, 21, 28, 31). However, the relative role of NHE1 in pHi regulation in cortical astrocytes is not clear because of the poor NHE1 isoform selectivity of amiloride. Thus definitive studies are warranted to assess the role of NHE1 in pHi recovery in cortical astrocytes.
To date, a genetic ablation approach has revealed an attenuated pHi recovery in freshly isolated CA1 neurons from NHE1 gene defect slow-wave epilepsy mutant mice (9, 41). However, it is not known how genetic ablation of NHE1 affects pHi recovery in cortical astrocytes. In the current study, both the more potent NHE1 inhibitor HOE-642 and genetic ablation approaches were taken to elucidate the function of NHE1 in mouse cortical astrocytes.
In the past decade, the results of a large body of research suggest that stimulation of NHE1 activity contributes to cardiac pathology after ischemia (1, 16). Inhibition of NHE1 has been shown to attenuate the detrimental consequences of myocardial ischemia and reperfusion (1). The principal mechanism underlying the cardioprotective actions of NHE1 inhibition is the attenuation of the intracellular Na+ accumulation during ischemia. However, limited information is available about the effects of NHE inhibition on cerebral ischemic damage. Pretreatment of gerbils with the amiloride derivative ethylisopropylamiloride (EIPA), a NHE inhibitor, significantly reduced the extent of CA1 pyramidal neuron loss after global ischemia (27). The NHE inhibitor SM-20220 (N-aminoiminomethyl-1-methyl-1-indole-2-carboxamide methanesulfonate) attenuated the increase in brain Na+ and water contents after 2 h of transient focal ischemia and 4 h of reperfusion in rats (18). It is, however, unknown whether these protective effects are attributable to inhibition of NHE activity in neurons and/or astrocytes. In addition, because these inhibitors are not isoform specific, it is not clear how dominant a role NHE1 plays compared with the other isoforms in the CNS.
In this study, we investigated the role of NHE1 in pHi recovery and cell damage in cultured cortical astrocytes after oxygen and glucose deprivation (OGD). We found that ablation of NHE1 activity decreased resting level pHi and impaired pHi recovery. OGD stimulated NHE1 activity in NHE1+/+ astrocytes; however, this increase in activity was absent in NHE1/ astrocytes and was blocked by HOE-642 in NHE1+/+ astrocytes. Inhibition or genetic ablation of NHE1 reduced the OGD-mediated rise in intracellular Na+ concentration ([Na+]i) and swelling, suggesting that the loss of NHE1 activity reduces ischemic injury in cortical astrocytes.
MATERIALS AND METHODS
Materials
Eagle's modified essential medium (EMEM) and Hanks' balanced salt solution (HBSS) were obtained from Mediatech Cellgro (Herndon, VA). Fetal bovine serum (FBS) was obtained from Hyclone Laboratories (Logan, UT). Collagen type I was purchased from Collaborative Biomedical Products (Bedford, MA). The acetoxymethyl esters of 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF-AM) and sodium-binding benzofuran isophthalate (SBFI-AM) were obtained from Molecular Probes (Eugene, OR). Pluronic acid was purchased from BASF (Ludwigshafen, Germany). Nigericin, dibutyryl cAMP (DBcAMP), monensin, and gramicidin were purchased from Sigma (St. Louis, MO). HOE-642 was a kind gift from Aventis Pharma (Frankfurt, Germany).
Animals and Genotype Analysis
The NHE1 null mutant (NHE1/) mice used were generated as previously described (3). We obtained NHE1 homozygous mutant and wild-type (NHE1+/+) mice by breeding gene-targeted NHE1 heterozygous mutant mice. Tail biopsies were obtained from 1-day-old mice. Genotypes were determined by polymerase chain reaction (PCR) analysis of DNA from tail biopsies. For PCR analysis, three primers were included in the reaction mixture. Forward (5'-GTG ATC CCC ACC ATC TCA AG-3') and reverse (5'-CGA AGA CAA GGA TGT GTA GC-3') primers were used to amplify a 450-base pair product from the wild-type gene. For the mutant gene, a reverse primer (5'-GCA TGC TCC AGA CTG CCT TG-3') and the same forward primer were used to amplify a 330-base pair product. PCR amplification was performed as follows: 94°C for 3 min, 40 cycles of 94°C for 30 s and 60°C for 30 s, and 72°C for 30 s. PCR products were run and revealed on a 2% agarose gel.
Primary Culture of Mouse Cortical Astrocytes
Dissociated cortical astrocyte cultures were established as described previously (33). Cerebral cortices were removed from 1-day-old NHE1+/+ or NHE1/ mice. The cortices were incubated in a trypsin solution (0.25 mg/ml in HBSS) for 25 min at 37°C. The tissue was then mechanically triturated and filtered through nylon meshes. The dissociated cells were rinsed and resuspended in EMEM containing 10% FBS. Viable cells (1 x 104 cells/well) were plated in 24-well plates coated with collagen type 1 or on collagen-coated glass coverslips in 6-well plates. Cultures were maintained in a 5% CO2 atmosphere at 37°C and refed every 3 days throughout the study. To obtain morphologically differentiated astrocytes, we treated confluent cultures (10 days in culture, DIV10) with EMEM containing 0.25 mM DBcAMP to induce differentiation. DBcAMP has been widely used to mimic neuronal influences on astrocyte differentiation (14, 37). Experiments were routinely performed in DIV15DIV28 astrocytes. This culture age was chosen because immature astrocyte cultures are resistant to damage under ischemic conditions (15).
Gel Electrophoresis and Western Blotting
Cultured cells were washed with ice-cold phosphate-buffered saline (PBS, pH 7.4) that contained 2 mM EDTA and protease inhibitors (35). Cells were scraped from dishes and lysed by sonication at 4°C. To obtain cellular lysates, we removed cellular debris by centrifugation and determined protein content of the cellular lysate (36). Samples and prestained molecular mass markers (Bio-Rad, Hercules, CA) were denatured in SDS reducing buffer (1:2 by volume; Bio-Rad) and heated at 37°C for 15 min before gel electrophoresis. Thirty micrograms of lysate protein were loaded. The samples were then electrophoretically separated on 6% SDS-polyacrylamide gels, and the resolved proteins were electrophoretically transferred to a polyvinylidene difluoride membrane (35). The blots were incubated in 7.5% nonfat dry milk in Tris-buffered saline (TBS) and then incubated with a primary antibody. After being rinsed, the blots were incubated with horseradish peroxidase-conjugated secondary IgG. Bound antibody was visualized using the enhanced chemiluminescence assay (ECL; Amersham). Anti-NHE1 polyclonal antibody (G116) against rat NHE1 (17) was developed in the laboratory of L. L. Ng (University of Leicester) and used for detection of NHE1 protein in astrocytes. The same blot was probed with anti- actin antibody as a control.
After the ECL reaction, NHE1 protein bands on the film were scanned with a Hewlett-Packard ScanJet (4c/T) scanner. The relative molecular weight values for NHE1 protein band were obtained with UN-SCAN-It gel software (Silk Scientific, Orem, UT) on the basis of the assumption that the distance traveled by the protein band is inversely proportional to the log of the molecular weight of the protein band, using Bio-Rad prestained protein standards. A mean value was obtained from 7 experiments.
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 20 min at room temperature. After being rinsed, cells were incubated with blocking solution (10% normal goat serum, 0.4% Triton X-100, and 1% bovine serum albumin in PBS) for 1 h. Cells were then incubated with anti-glial fibrillary acidic protein (GFAP) monoclonal antibody (1:100) or anti-NHE1 rabbit polyclonal (G115) antibody (1:100) in blocking solution for 1 h. Cells were rinsed with PBS and incubated with goat anti-mouse Alexa 488- or anti-rabbit Alexa 595-conjugated antibodies (1:200) for 1 h. Cell images were captured by using a Nikon TE 300 inverted epifluorescence microscope (x20) with a Princeton Instruments MicroMax charge-coupled device (CCD) camera and MetaMorph image-processing software (Universal Imaging, Dowington, PA). Identical digital imaging acquisition parameters were used in both negative control and experimental images.
The G115 antibody was raised in rabbit against amino acids 627820 of the rat NHE1 sequence in the laboratory of L. L. Ng. The antibody was purified according to the same method described previously for the G116 antibody preparation (17).
Measurement of pHi
Solutions. The HEPES-buffered solution (pH 7.4) contained (in mM) 140 NaCl, 5.36 KCl, 0.81 MgSO4, 1.27 CaCl2, 0.44 KH2PO4, 0.33 Na2HPO4, 5.55 glucose, and 20 HEPES. Calibration solutions for pH contained (in mM) 130 KCl, 20 N-methyl-D-glucamine (NMDG), 0.81 MgSO4, 1.27 CaCl2, 5.55 glucose, and 20 HEPES plus 10 µM nigericin. In Na+-free solution, NaCl in the HEPES-buffered solution was replaced with an equimolar concentration of NMDG. In experiments in which CO2/HCO3-buffered solutions were used, HEPES was replaced with 26 mM NaHCO3 and equilibrated with 5% CO2. NH4+/NH3 solution was prepared by replacing 10 mM NaCl in either the HEPES-buffered solution or the CO2/HCO3-buffered solution with an equimolar concentration of NH4Cl.
pHi measurements. Cells grown on coverslips were incubated with 2.5 µM BCECF-AM in HEPES-buffered solution for 10 min at ambient temperature (see Figs. 25). For experiments in Fig. 6, cells were incubated for 2 h with 2.5 µM BCECF-AM under either normoxic control or OGD conditions (37°C). The coverslips were then washed in HEPES-buffered solution and placed in an open-bath imaging chamber (volume = 40 µl, model RC24; Warner Instruments, Hamden, CT) containing HEPES-buffered solution. The chamber was mounted on the stage of the TE 300 inverted epifluorescence microscope, and the astrocytes were visualized with a x40 Super Fluor oil-immersion objective lens (1.3 NA, 0.22 WD). The cells were excited every 1030 s at 440 and 490 nm, and the ratio of emission fluorescence at 535 nm was recorded. Images were collected by using a Princeton Instruments MicroMax CCD camera and analyzed with MetaFluor image-processing software (Universal Imaging). The ratio of fluorescence emissions (F490/F440) was calibrated by using the high-K+/nigericin technique (5). The background-corrected data were fit with a variant of the Michaelis-Menten equation (5). Typically, six cells were monitored in each coverslip. NHE1+/+ and NHE1/ astrocytes (75 cells from 4 different cultures) were exposed to the pH calibration solutions with pH 5.68.0. The calculated pK was not significantly different between NHE1+/+ and NHE1/ astrocytes (7.13 vs. 7.14).
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OGD Treatment
NHE1+/+ or NHE1/ cultures grown on coverslips in six-well plates were rinsed twice with an isotonic OGD solution (pH 7.4) containing (in mM) 0 glucose, 20 NaHCO3, 120 NaCl, 5.36 KCl, 0.33 Na2HPO4, 0.44 KH2PO4, 1.27 CaCl2, and 0.81 MgSO4. Cells were incubated in 1.0 ml of OGD solution in a hypoxic incubator at 37°C (Forma model 3130; Thermo Forma, Marietta, OH) equilibrated with 94% N2-1% O2-5% CO2. The oxygen level in the medium of cultured cells was monitored with an oxygen probe (model M1-730; Microelectrodes, Bedford, NH) and decreased to 23% after 60 min in the hypoxic incubator. The OGD incubation lasted 2 h. Normoxic control cells were incubated in 5% CO2 and atmospheric air in isotonic control buffer containing 5.5 mM glucose with the rest of the components in the buffer identical to the isotonic OGD solution.
Intracellular Na+ Measurement
Cultured astrocytes grown on coverslips were loaded with 10 µM SBFI-AM in HCO3-free HEPES-buffered solution containing 0.05% Pluronic acid for 45 min at room temperature (34). The coverslips were placed in the open-bath imaging chamber containing HCO3-free HEPES-buffered solution at ambient temperature. With the use of the Nikon TE 300 inverted epifluorescence microscope and a x40 Super Fluor oil-immersion objective lens, astrocytes were excited every 60 s at 345 and 385 nm, and the emission fluorescence at 510 nm was recorded. Images were collected and analyzed with the MetaFluor image-processing software as described previously (34). Absolute [Na+]i was determined in the cytoplasm of cells by calibrating the SBFI fluorescence ratio with solutions containing 0, 10, 20, 40, or 80 mM external Na+ ([Na+]o) plus monensin (10 µM) and gramicidin (3 µM), as described previously (33).
To minimize the dye loss and phototoxicity, we performed these experiments at ambient temperature. In one set of experiments, [Na+]i was measured and calibrated at 37°C (see Fig. 7B). In another set of experiments, [Na+]i was measured in CO2/HCO3-buffered solutions at 37°C (see Fig. 7C).
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NHE1+/+ or NHE1/ cultures grown on coverslips were placed in a closed-bath imaging chamber (volume = 36 µl, model RC20H; Warner Instruments). The chamber was mounted on a heated platform (model TC-344B; Warner Instruments) to allow regulation of buffer temperature (37°C). The chamber was perfused by gravity (0.25 ml/min) via buffer reservoirs containing either standard HEPES-buffered solution or OGD solution continuously bubbled with N2. Flow from the reservoirs was directed through a manifold, a flow valve, and an in-line oxygen monitor and then to the chamber. When flow was switched between the control reservoir and the OGD reservoir, the O2 level fell to 23% within 1.5 min, as monitored by an oxygen probe (model M1-730).
As an estimate of relative cell volume changes, relative cross-sectional areas (CSA) were monitored before and during OGD with the use of video-enhanced differential interference contrast (DIC) microscopy, as reported previously (33, 34). A single astrocyte was visualized by using a Nikon x60 Plan Fluor DIC objective lens, and an image was recorded. The mean CSA was calculated after the perimeter of the cell body was traced in triplicate with MetaMorph image-processing software as described in our previous study (34).
Statistics
In statistical analysis, n values refer to the number of coverslips in each experimental condition for Figs. 25 and 7. Significant differences were determined using ANOVA with the Bonferroni post hoc test for multiple comparisons. Data were considered significant if a 95% confidence level was reached.
RESULTS
NHE1 Expression in Cortical Astrocytes
The genotype of newborn mice was determined by PCR. As shown in Fig. 1A, a single DNA band (330 bp) was amplified from tail biopsies of NHE1/ mice. In contrast, a larger DNA band (
450 bp) was detected in NHE1+/+ tail biopsies. In a heterozygous sample, both PCR products were present. Immunoblotting was performed to analyze expression of the NHE1 protein in cortical astrocytes. An
96-kDa protein was recognized by an anti-NHE1 antibody in NHE1+/+ astrocytes (Fig. 1B). No protein band was detected by the anti-NHE1 antibody in NHE1/ astrocytes. A lack of NHE1 bands was not due to insufficient protein loading, because, as shown on the same blot, a similar amount of
-actin protein was observed in samples from both NHE1+/+ and NHE1/ astrocytes (Fig. 1B).
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Decrease in Resting pHi of NHE1/ Astrocytes
The pHi frequency distribution in NHE1+/+ astrocytes ranged from 6.5 to 7.2 (Fig. 2). The mean steady-state pHi in NHE1+/+ astrocytes was 6.86 ± 0.03 in the presence of a HEPES-buffered solution (pH 7.4, CO2/HCO3-free). This is in agreement with previous reports by others that astrocytes have a resting level pHi below neutral under HCO3-free conditions (4, 5, 21, 26, 28). However, the pHi in NHE1/ cells had a larger range (6.04 to 6.84) and was not normally distributed (Kolmogorov-Smirnov normality test, P < 0.05). The mean steady-state pHi in NHE1/ astrocytes was significantly lower (6.53 ± 0.04, P < 0.01). In the presence of CO2/HCO3, the mean steady-state pHi in NHE1/ astrocytes was increased relative to that observed in HEPES-buffered solution but remained significantly lower than in NHE1+/+ astrocytes (6.80 ± 0.02 vs. 7.00 ± 0.02, n = 6, 2 cultures, P < 0.001).
To further examine the role of NHE1 in the maintenance of resting pHi in astrocytes, we monitored pHi in NHE1+/+ and NHE1/ astrocytes in a Na+-free HEPES-buffered solution (Fig. 3A). In the absence of Na+, the pHi of NHE1+/+ astrocytes progressively decreased (Fig. 3A, a and b). The average decrease in pHi was 0.28 ± 0.05 pH unit (Fig. 3A, inset). When the NHE1+/+ astrocytes were returned to the HEPES-buffered solution, they rapidly recovered to a normal basal pHi (Fig. 3A, b and c). In response to Na+-free HEPES-buffered solution, the resting pHi of NHE1/ astrocytes further decreased by 0.13 ± 0.02 pH unit (Fig. 3A, inset). The possible mechanisms for this decrease are discussed later (see DISCUSSION).
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Impaired pHi Recovery in NHE1/ Astrocytes
We then studied how NHE1/ astrocytes regulate pHi after intracellular acidosis using the NH4+ prepulse technique. As shown in Fig. 4A, when either NHE1+/+ or NHE1/ astrocytes were exposed to 10 mM NH3/NH4+, pHi rapidly rose as NH3 diffused into the cell and combined with H+ to form NH4+ (Fig. 4A, a and b) and then declined slowly (Fig. 4A, b and c). When cells (either NHE1+/+ or NHE1/ astrocytes) were returned to the standard HCO3-free HEPES-buffered solution, pHi decreased sharply as NH4+ rapidly left cells as NH3, trapping H+ inside (Fig. 4A, c and d). After the acid load, pHi recovered within 2 min in NHE1+/+ astrocytes (Fig. 4A, d and e). However, in NHE1/ astrocytes, pHi recovery was delayed for 12 min and then slowly returned to near the basal pHi level over the following 10 min.
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pHi Recovery in Astrocytes Is Mediated by NHE1 Activity
The data in Fig. 4 indicate that initial pHi recovery after an acid load in astrocytes is exclusively mediated by NHE1 in the absence of HCO3. To further establish this point, we investigated the Na+ dependence of pHi recovery after an acid load in NHE1+/+ and NHE1/ astrocytes in the absence of HCO3 (Fig. 5, A and B). When NHE1+/+ or NHE1/ astrocytes were exposed to 10 mM NH3/NH4+, pHi increased (Fig. 5, A and B, a and b) and then declined gradually (Fig. 5, A and B, b and c). Both NHE1+/+ and NHE1/ astrocytes exhibited acidification after the NH4+ acid load (Fig. 5, A and B, c and d). In contrast to the results shown in Fig. 4, in the absence of extracellular Na+, NHE1+/+ astrocytes failed to regulate pHi (Fig. 5A, d and e) after acidosis. pHi in NHE1+/+ astrocytes decreased by 0.19 ± 0.04 pH unit/min at a pHi of 6.36 ± 0.012 (Fig. 5A, inset) during the first minute of recovery. NHE1/ astrocytes exhibited no pHi recovery in the absence of Na+ and no further decrease in pHi (0.02 ± 0.02 pH unit/min at a pHi of 6.09 ± 0.14) (Fig. 5A). NH3/NH4+ prepulse led to a significantly more acidification in NHE1/ astrocytes than in NHE1+/+ astrocytes in the absence of extracellular Na+ (P < 0.05). pHi recovered when NHE1+/+ cells were returned to the control conditions (Fig. 5A, e and f). However, there was only a slow recovery of pHi in NHE1/ astrocytes (Fig. 5A, e and f).
As shown in Fig. 5B, inhibition of NHE1 activity with the potent NHE1 blocker HOE-642 had a similar effect on pHi to that of removal of extracellular Na+. In the presence of 1 µM HOE-642, NH3/NH4+ triggered acid loading in NHE1+/+ and NHE1/ astrocytes (Fig. 5B, bd). However, after acidification, there was little increase in pHi in NHE1+/+ cells (Fig. 5B, d and e). The rate of pHi recovery in NHE1+/+ astrocytes in the presence of HOE-642 was 0.03 ± 0.1 pH units/min at a pHi of 6.35 ± 0.19 (Fig. 5B, inset). When HOE-642 was removed from the buffer, pHi rapidly returned to basal levels in NHE1+/+ astrocytes (Fig. 5B, e and f). In some NHE1/ astrocytes, pHi slowly increased despite NHE1 blockade (Fig. 5B, d and e). However, there was no change in the mean pHi recovery rate (0.00 ± 0.01 pH unit/min at a pHi of 6.12 ± 0.07) (Fig. 5B, inset). Returning NHE1/ astrocytes to the control solution had no effect on the rate of pHi recovery. The difference in the mean pHi during the initial pHi recovery between NHE1+/+ and NHE1/ astrocytes was not statistically significant.
pHi recovery was also measured in NHE1+/+ astrocytes from separate cultures in the presence of CO2/HCO3 (26 mM). In two experiments, the pHi recovery rates in NHE1+/+ astrocytes averaged 0.38 (1 coverslip, 6 cells) and 0.37 pH/min (1 coverslip, 6 cells), respectively. When NHE1 activity was blocked by HOE-642 (1 µM), the average pHi recovery rate was reduced to 0.11 ± 0.04 pH unit in NHE1+/+ astrocytes (n = 3, P < 0.001). These data suggest that NHE1 in astrocytes plays a dominant role in pHi recovery after acidosis, regardless of the presence of HCO3-dependent transport systems. In two experiments, the pHi recovery rates in NHE1/ astrocytes averaged 0.42 (1 coverslip, 20 cells) and 0.22 pH unit/min (1 coverslip, 20 cells), respectively. This implies that HCO3-dependent transport systems might be upregulated in NHE1/ astrocytes.
NHE1 Activity Was Elevated in Astrocytes After OGD
We hypothesize that NHE1 activity is stimulated in astrocytes and promotes the rapid extrusion of cytosolic H+ that is accumulated during OGD. First, we investigated whether pHi was reduced after 2 h of OGD. Figure 6A shows that at 2 min after 2 h of OGD in the presence of extracellular pH (pHo) of 7.4, pHi in NHE1+/+ astrocytes was significantly decreased from 7.09 ± 0.07 to 6.80 ± 0.02 (P < 0.05). Inhibition of NHE1 activity during OGD with HOE-642 (1 µM) further acidified NHE1+/+ astrocytes (6.66 ± 0.03 vs. 6.44 ± 0.01, P < 0.05). pHi was similarly reduced in NHE1/ astrocytes during OGD (6.65 ± 0.06 vs. 6.42 ± 0.13). These data suggest that OGD caused intracellular acidosis in astrocytes. This low pHi may subsequently stimulate NHE1 activity to extrude excessive H+ from astrocytes.
Under in vivo ischemic conditions, pHo decreases. Low pHo has been reported to inhibit NHE activity in astrocytes (22). Therefore, we investigated pHi changes in NHE1+/+ astrocytes after 2 h of OGD at pHo 6.6 and 2 min of reoxygenation at pHo 7.4. Figure 6A, inset, shows that pHi in NHE1+/+ astrocytes decreased to 6.67 ± 0.02 after 2 h of OGD at pHo 6.6, which is significantly less than pHi after 2 h of OGD at pHo 7.4 (6.80 ± 0.02, P < 0.05). Moreover, in NHE1+/+ astrocytes treated with 1 µM HOE-642, pHi decreased further to 6.48 ± 0.04 (P < 0.05). These findings imply that NHE1 in NHE1+/+ astrocytes is still active under OGD conditions at pHo 6.6.
We then measured NHE1 activity in NHE1+/+ and NHE1/ astrocytes under both normoxic and OGD conditions. As shown in Fig. 6B, in normoxia-treated NHE1+/+ astrocytes, the rate of pHi recovery in response to the acid loading was 0.30 ± 0.01 pH unit/min at a pHi of 6.40 ± 0.11. After 2 h of OGD at pHo 7.4 and 15 min of reoxygenation, pHi recovery rate was increased to 0.53 ± 0.03 pH unit/min at a pHi of 6.42 ± 0.12 (P < 0.05). In contrast, pHi recovery rate after 2 h of OGD at pHo 7.4 and 15 min of reoxygenation was nearly absent in NHE1/ astrocytes or in HOE-642-treated NHE1+/+ astrocytes (0.03 ± 0.013 pH unit/min at a pHi of 6.13 ± 0.07 and 0.01 ± 0.01 pH unit/min at a pHi of 14 ± 0.05, respectively). These findings indicate that OGD treatment stimulates NHE1 activity and that NHE1 plays an important role in pHi recovery after OGD.
Figure 6C shows that 2 h of OGD at pHo 6.6 did not block the increase in pHi recovery rate in NHE1+/+ astrocytes (0.56 ± 0.05 pH unit/min at a pHi of 6.33 ± 0.0.14, P < 0.05). To further establish that NHE1 was responsible for the accelerated pHi recovery during reoxygenation, we then examined whether HOE-642 would block pHi recovery in NHE1+/+ astrocytes. Astrocytes underwent 2 h of OGD at pHo 6.6 in the presence of 1 µM HOE-642. pHi recovery was abolished by HOE-642, with a pHi recovery rate of 0.04 ± 0.01 pH unit/min at a pHi of 6.22 ± 0.11 (Fig. 6C). This indicates that NHE1 activity remains stimulated by low pHi, even under conditions that would be expected to cause low pHo-mediated inhibition.
An Increase in [Na+]i Was Significantly Attenuated in NHE1/ Astrocytes After OGD
NHE1+/+ and NHE1/ astrocytes were exposed to 2 h of OGD followed by 1 h of reoxygenation, and [Na+]i was determined at ambient temperature or 37°C. NHE1+/+ and NHE1/ astrocytes from sister cultures were incubated in normoxic control buffer in a normoxic incubator for 3 h. As shown in Fig. 7A, under control conditions, average [Na+]i measured at ambient temperature was 11.3 ± 2.3 mM in NHE1+/+ astrocytes and 10.9 ± 1.6 mM in NHE1/ astrocytes. After 2 h of OGD and 1 h of reoxygenation, [Na+]i increased to 61.2 ± 9.8 mM in NHE1+/+ astrocytes. However, in NHE1/ astrocytes, [Na+]i was 23.4 ± 5.0 mM, significantly lower than that in NHE1+/+ astrocytes (P < 0.001). Inhibition of NHE1 with 1 µM HOE-642 during OGD and reoxygenation also significantly attenuated the rise in [Na+]i (30.0 ± 8.3 mM; Fig. 7A) in NHE1+/+ astrocytes.
Figure 7B shows the resting [Na+]i levels measured at 37°C. Basal [Na+]i levels were 12.4 ± 0.1 mM in NHE1+/+ astrocytes and 12.1 ± 0.2 mM in NHE1/ astrocytes. After 2 h of OGD and 1 h of reoxygenation, [Na+]i levels increased to 40.9 ± 2.5 mM in NHE1+/+ astrocytes. In contrast, 2 h of OGD and 1 h of reoxygenation resulted in [Na+]i of only 20.9 ± 2.2 mM in NHE1+/+ astrocytes treated with 1 µM HOE-642 and [Na+]i of 22.0 ± 0.7 mM in NHE1/ astrocytes. Taken together, these data show that inhibition of NHE1 activity significantly attenuated intracellular Na+ accumulation after OGD.
The cells in Fig. 7, A and B, were exposed to 1 h of reoxygenation in the HCO3-free/ HEPES-buffered solution. Additional experiments were performed in the presence of HCO3 (26 mM) throughout 2 h of OGD and 1 h of reoxygenation. As shown in Fig. 7C, Na+ accumulation was significantly lowered in NHE1/ astrocytes or in NHE1+/+ astrocytes treated with HOE-642. Thus these results are in agreement with the Na+ accumulation data determined in the absence of HCO3 (Fig. 7, A and B). This finding implies that Na+-HCO3 cotransport systems contribute little to intracellular Na+ loading after OGD.
NHE1/ Astrocytes Exhibited Resistance to OGD-Mediated Swelling
Accumulation of intracellular Na+ leads to astrocyte swelling. CSA was measured as an estimate of cell swelling during OGD in NHE1+/+ and NHE1/ astrocytes. In control experiments, 100 min of perfusion with iso-HEPES did not cause any significant changes in CSA (data not shown). However, OGD induced a gradual increase in CSA over time in NHE1+/+ astrocytes. After 60 min, CSA increased by 26 ± 2% in NHE1+/+ astrocytes (Fig. 8, A and B). In contrast, treatment with 1 µM HOE-642 significantly attenuated the OGD-induced rise in CSA in NHE1+/+ astrocytes (9 ± 2%; Fig. 8B). In a similar manner, NHE1/ astrocytes exhibited only an 8 ± 2% increase in CSA after 60 min of OGD (Fig. 8, A and B).
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Dominant Role of NHE1 in pHi Regulation in Cortical Astrocytes
Resting pHi in NHE1/ astrocytes.
In this study, we found that the resting pHi in NHE1+/+ astrocytes was 6.86 and was decreased after removal of Na+ or application of the potent NHE1 inhibitor HOE-642. Genetic ablation of NHE1 in NHE1/ astrocytes resulted in a new "set point" for resting level pHi (6.53), which was significantly lower than in NHE1+/+ astrocytes in the absence of CO2/HCO3. In the presence of CO2/HCO3, despite an 0.24-pH unit increase, presumably by HCO3 transporters, the resting pHi in NHE1/ astrocytes remained significantly lower than in NHE1+/+ astrocytes. Thus both pharmacological inhibition and genetic ablation of NHE1 decrease the resting pHi in astrocytes. This suggests that NHE1 is active in maintaining the steady-state level of pHi in mouse cortical astrocytes. In contrast, NHE1 activity in many cell types is quiescent at resting pHi (8).
In NHE1/ astrocytes, removal of extracellular Na+ caused a small but significant decrease in pHi. This could be attributable to functions of other NHE isoforms, although this would require considerable upregulation of these isoforms in NHE1/ astrocytes. NHE1 is normally expressed in the CNS at a level 10 times that of the other isoforms (19). Alternatively, in the absence of extracellular HCO3, removal of external Na+ could acidify the cell by triggering reversal of the electrogenic Na+-HCO3 cotransporter (31).
Moreover, the resting pHi exhibited by NHE1/ astrocytes was significantly lower than that of NHE1+/+ astrocytes when NHE1 activity was inhibited by Na+-free buffer or the NHE1 inhibitor HOE-642. This implies that other pHi regulatory pathways are altered in NHE1/ astrocytes in addition to an absence of NHE1 activity. A steady-state pHi is affected by many factors, including cell growth and metabolism or mitochondrial function, in addition to membrane transport systems. NHE1-mediated H+ extrusion is only one of the factors in determination of the steady-state pHi in cells. To our knowledge, no studies have been done to compare rates of glucose consumption, lactate production, or other factors such as glycogen levels in NHE1+/+ to NHE1/ astrocytes during either normoxic or OGD conditions. Thus we do not know whether the proton production rate differs between NHE1+/+ and NHE1/ astrocytes. Future studies are needed to investigate whether acid loading systems are stimulated in NHE1/ astrocytes.
pHi recovery in NHE1 astrocytes after acid loading.
In response to NH4Cl-mediated acidosis, NHE1+/+ astrocytes restored pHi to the resting level within 12 min. This robust recovery was abolished when NHE1 activity was inhibited by either HOE-642 or removal of external Na+ in the absence of extracellular HCO3. The pHi recovery rate was decreased by 70% with HOE-642 in the presence of extracellular HCO3. This finding suggests that NHE1 is the dominant mechanism for alkalinizing astrocytes after acidosis.
pHi recovery in NHE1/ astrocytes after an acid load was delayed in the absence of extracellular HCO3 but not abolished. pHi recovery was quiescent in NHE1/ astrocytes during the first 2 min after an acid load, but the recovery rate gradually increased, and the original resting pHi level was reached within 10 min. The ability of the NHE1/ cells to slowly restore pHi may be due to upregulation of other NHE isoforms in NHE1/ astrocytes. This view is based on the following information: 1) the slow pHi recovery rate was Na+ dependent, because removal of extracellular Na+ abolished this regulation. This would argue against a role for the V-type ATPase that has been described in hippocampal astrocytes and does not require Na+ (26); 2) in HEPES buffer, HCO3-dependent transport activity (i.e., electrogenic Na+-HCO3 cotransporters) should be minimal, because the apparent Km for external HCO3 is 610 mM (13); and 3) a residual pHi recovery in NHE1/ astrocytes is resistant to 1 µM HOE-642. The apparent Ki for HOE-642 in transfected fibroblast cell lines is 0.05 µM for NHE1, 3 µM for NHE2, and 1 mM for NHE3 (30). Thus, in NHE1/ astrocytes, other NHEs are likely to be upregulated to compensate for the loss of NHE1 activity.
NHE1 activity is known to be stimulated in a dose-dependent manner below pHi 6.7 in astrocytes (21). In the current study, we found that pHi recovery was persistently impaired in NHE1/ astrocytes under pHi 6.10, compared with pHi 6.36 in NHE1+/+ astrocytes (Fig. 5). However, the sensitivity of BCECF, as determined by the ratio of the change in BCECF fluorescence with changes in pHi (FBCECF/
pH) decreases at low pHi. In a pilot study, we found that in NHE1+/+ astrocytes at pHi 6.15,
FBCECF/
pH was
25% of the maximum response we observed at pHi 7.25. In the current study, pHi in NHE1/ astrocytes decreased to 6.10 after NH4Cl pulse (Fig. 5). Thus the lack of pHi recovery in NHE1/ astrocytes could be due, in part, to an insensitivity of BCECF at low pHi.
Protective Effects of NHE1 Inhibition in Astrocytes
Little is known about the role of NHEs in ischemic damage in astrocytes. In this study, we examined whether NHE1 is involved in astrocyte damage after OGD. We first demonstrated that OGD induces acidosis in cortical astrocytes. OGD-induced intracellular acidosis may lead to stimulation of NHE1 activity allosterically by cytosolic H+ (25). In addition, prolonged intracellular acidosis can elevate NHE1 activity by ERK- and p90RSK-mediated phosphorylation (13a). We found that, measured at the same pHi levels (pHi 6.4 vs. 6.42), NHE1 activity in OGD-treated NHE1+/+ astrocytes was increased by 80% compared with that in normoxic NHE1+/+ astrocytes (Fig. 6B). This finding suggests that OGD-mediated stimulation of NHE1 is sustained.
After 2 h of OGD and 1 h of reoxygenation, [Na+]i in NHE1+/+ astrocytes rose to 5.4 times normal. In contrast, in NHE1/ astrocytes subjected to OGD and reoxygenation, [Na+]i was only
2.1 times normal. A similar attenuation of the rise in [Na+]i after OGD and reperfusion was observed in NHE1+/+ astrocytes (2.7 times normal) when NHE1 activity was inhibited by HOE-642. The data suggest that NHE1 activity in astrocytes plays an important role in Na+ loading during reoxygenation. Furthermore, OGD-mediated swelling was significantly reduced when NHE1 activity was eliminated by genetic ablation or inhibited by HOE-642.
However, blockade of NHE1 activity by HOE-642 only attenuated the Na+ loading by 60%. The mechanisms responsible for the remaining Na+ influx in astrocytes are unknown. Several other Na+ transport proteins could play a role in this Na+ loading, such as the Na+ channels, Na+-HCO3 cotransporters, or the Na-K-2Cl cotransporter. Na-K-2Cl cotransporter activity is stimulated in neurons (2) and astrocytes (Lenart B, Kintner DB, Shull GE, and Sun D, unpublished data) under OGD conditions. In addition, inhibition of Na+-K+-ATPase activity may also contribute to the Na+ accumulation.
Our data provide strong evidence that inhibition of NHE1 is partially protective against OGD-mediated damage in astrocytes. The cellular mechanisms underlying the role of NHE1 in ischemic astrocyte damage are not well understood. We demonstrated that NHE1 activity is stimulated during OGD and early reoxygenation. NHE1 triggers an acute accumulation of intracellular Na+ and swelling after OGD. A rise in [Na+]i and a reduction in the transmembrane Na+ gradient can worsen the energy state and exacerbate cell damage during ischemia. This view is supported by the report that 10 µM HOE-642 prevented a significant reduction of PCr/Cr in C6 glioma cells after hypoxia (11). It has been suggested that astrocyte death is related to the extent of Na+ influx under conditions in which Na+-K+-ATPase activity is not maintained (38). Thus the Na+ overload mechanism is likely the primary means whereby NHE1 contributes to ischemic astrocyte damage.
In addition, a rise in [Na+]i could reduce Ca2+ extrusion and/or reverse Na+/Ca2+ exchange and lead to accumulation of intracellular Ca2+ (32). This hypothesis is supported by several previous studies. Activation of NHE activity in hippocampal neurons by acidosis triggered Na+ influx and subsequent elevation in [Ca2+]i (40). The rise in both Na+ and Ca2+ was blocked by the NHE inhibitor EIPA and the Na+/Ca2+ exchange blocker dimethylbenzamil (40). Thus these findings suggest that stimulation of NHE activity can lead to accumulation of intracellular Na+ and Ca2+. In addition, reversal of Na+/Ca2+ exchange activity and subsequent Ca2+ influx have been observed in depolarized and glucose-deprived cerebellar granule cells (10). Abundant expression of Na+/Ca2+ exchanger proteins NCX1 and NCX2 has been found in cultured hippocampal astrocytes (39). When the Na+/Ca2+ exchanger inhibitor SEA0400 was applied to cultured astrocytes subjected to paradoxical Ca2+ challenge-induced injury, there was a reduction in Ca2+ influx, production of reactive oxygen species, and cell damage (20). Taken together, the results of these studies suggest that an initial intracellular Na+ overload and subsequent stimulation of Ca2+ influx may contribute to ischemic astrocyte damage. However, further studies are needed to determine whether stimulation of NHE1 activity would alter Na+/Ca2+ exchange and promote Ca2+ influx in astrocytes after OGD.
In summary, we have shown that both pharmacological inhibition and genetic ablation of NHE1 reduced the steady-state levels of pHi in cultured cortical astrocyte. Inhibition of NHE1 activity impaired pHi recovery after intracellular acidosis. NHE1 activity was elevated after OGD at either neutral or low pHo. In addition, NHE1/ astrocytes exhibited significantly less damage after OGD. These findings suggest that stimulation of NHE1 activity in astrocytes may contribute to disruption of ion homeostasis and ischemic damage.
GRANT
This work was supported in part by National Institute of Neurological Disorders and Stroke Grant R01 NS-38118 and National Science Foundation CAREER Award IBN9981826 (to D. Sun) and National Heart, Lung, and Blood Institute Grant R01 HL-61974 (to G. E. Shull).
ACKNOWLEDGMENTS
We thank Li Lin and Andy Look for excellent technical assistance.
Address for reprint requests and other correspondence: D. Sun, Dept. of Neurological Surgery, Univ. of Wisconsin Medical School, H4/332 Clinical Sciences Center, 600 Highland Ave., Madison, WI 53792 (E-mail address: sun{at}neurosurg.wisc.edu).
FOOTNOTES
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.
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