Abstract
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Ischemic factor-induced increases in cerebral microvascular endothelial cell Na/H exchange activity and abundance: evidence for involvement of ERK1/2 MAP kinase
Abstract
Brain edema forms rapidly in the early hours of ischemic stroke by increased secretion of Na, Cl, and water into the brain across an intact blood-brain barrier (BBB), together with swelling of astrocytes as they take up the ions and water crossing the BBB. Our previous studies provide evidence that luminal BBB Na-K-Cl cotransport (NKCC) and Na/H exchange (NHE) participate in ischemia-induced edema formation. NKCC1 and two NHE isoforms, NHE1 and NHE2, reside predominantly at the luminal BBB membrane. NKCC and NHE activities of cerebral microvascular endothelial cells (CMEC) are rapidly stimulated by the ischemic factors hypoxia, aglycemia, and AVP, and inhibition of NKCC and NHE activities by bumetanide and HOE642, respectively, reduces brain Na uptake and edema in the rat middle cerebral artery occlusion model of stroke. The present study was conducted to further explore BBB NHE responses to ischemia. We examined whether ischemic factor-stimulated NHE activity is sustained over several hours, when the majority of edema forms during stroke. We also examined whether ischemic factors alter NHE1 and/or NHE2 protein abundance. Finally, we conducted initial studies of ERK1/2 MAP kinase involvement in BBB NHE and NKCC responses to ischemic factors. We found that hypoxia, aglycemia, and AVP increase CMEC NHE activity through 5 h and that NHE1, but not NHE2, abundance is increased by 1- to 5-h exposures to these factors. Furthermore, we found that these factors rapidly increase BBB ERK1/2 activity and that ERK1/2 inhibition reduces or abolishes ischemic factor stimulation of NKCC and NHE activities.
it is well established that cerebral edema is a major contributor to morbidity and mortality in ischemic stroke (6, 16), yet the mechanisms by which cells of the neurovascular unit participate in edema formation are not well understood. It has been shown that formation of brain edema during the early hours of an ischemic insult involves increased secretion of Na, Cl, and water into the brain across the intact blood-brain barrier (BBB) (1, 24, 34), together with perivascular astrocyte uptake of the ions and water transported across the BBB, causing cytotoxic edema (2, 13, 16). These processes precede vasogenic edema, which generally occurs several hours after the onset of ischemia and involves influx of ions, water, and other plasma constituents from blood into brain following significant increases in BBB paracellular permeability (10, 11, 16, 23, 34). Our work, together with that of other laboratories, has provided evidence that BBB Na-K-Cl cotransport (NKCC) and Na/H exchange (NHE) contribute to edema formation. NKCC and NHE are located predominantly in the luminal membrane of BBB endothelial cells in situ (18, 31), as predicted for a role in secretion of ions from blood into brain. In addition, hypoxia, aglycemia, and AVP, three prominent factors present during cerebral ischemia, rapidly stimulate NKCC and NHE activity in cultured cerebral microvascular endothelial cells (CMEC) (9, 14, 15, 18, 28). Using the rat permanent middle cerebral artery occlusion (MCAO) model of stroke and NMR diffusion-weighted imaging and Na chemical shift imaging methods, we also found evidence that inhibition of BBB NKCC and/or NHE activity by intravenous administration of bumetanide and/or HOE642, respectively, significantly reduces edema and brain Na uptake (27, 31). Bumetanide and HOE642 also reduce brain infarct volume and improve neurological outcome following permanent MCAO (27, 31).
Our previous studies demonstrated that CMEC NHE activity is increased following 30-min exposures to hypoxia, aglycemia, or AVP (18). However, if NHE activity contributes to edema formation throughout the early hours of stroke, one would predict that stimulation of the BBB exchanger by one or more of the ischemic factors is sustained over the first several hours, when robust edema formation is occurring. Thus one goal of the present study was to evaluate the effects of our ischemic factors of interest on CMEC NHE activity following exposures of up to 5 h. Of the nine known isoforms of NHE, we have shown in immunoelectron microscopy studies that NHE1 and NHE2 are present in BBB endothelial cells, predominantly in the luminal membrane, in normoxic or ischemic brain (18, 27). However, we do not know which of these isoforms is responsible for the ischemic factor-induced increase in BBB NHE activity. As an initial approach to evaluate the responses of BBB NHE1 and NHE2 to ischemic factors, we also examined the abundance of these two isoforms in CMEC following 1- to 5-h exposures to hypoxia, aglycemia, and AVP.
The mechanisms by which ischemic factors stimulate BBB NKCC and NHE activity are not well understood. In recent studies of CMEC NKCC responses to ischemic factors, we demonstrated that AMP-activated protein kinase (AMPK) and the p38 and JNK MAP kinases are stimulated by hypoxia, aglycemia, and AVP in the cells and that pharmacological inhibition of AMPK, p38, or JNK reduces stimulation of NKCC activity by these ischemic factors (37, 38). However, other studies suggest that ERK1/2 MAP kinase (ERK) may play a role in ischemic factor activation of CMEC NKCC and/or NHE (32). ERK is activated in mouse and rat brain by permanent or reversible MCAO (33, 39) and in human brain by acute ischemia (35). Also, ischemia-induced activation of ERK has been shown to increase NHE activity in rat myocardium (25) and astrocytes and neurons (17, 20). Thus we also conducted an initial investigation to determine whether ERK participates in ischemic factor stimulation of CMEC NHE and NKCC activity.
We report that hypoxia, aglycemia, and AVP cause significant stimulation of CMEC NHE activity through at least 5 h. These factors also significantly increase NHE1, but not NHE2, protein abundance following 1- to 5-h exposures. In addition, we show that all three ischemic factors increase CMEC ERK activity and that the ERK inhibitor FR180204 reduces or abolishes ischemic factor stimulation of CMEC NHE, as well as NKCC, activity. Finally, we demonstrate the presence of ERK in BBB endothelial cells in situ.
MATERIALS AND METHODS
CMEC culture.
Bovine CMEC were maintained in DMEM containing 5 mM d-glucose, 1 mM Na-pyruvate, 2 mM l-glutamine, 50 μg/ml gentamicin, 1 ng/ml basic fibroblast growth factor, 5% calf serum, and 5% horse serum in an atmosphere of 95% humidified air-5% CO2 at 37°C, as described previously (9, 18). Cells were grown to confluence on collagen- and attachment factor-coated (Cell Systems, Kirkland, WA) 6-well plates for Western blots, 96-well plates for NKCC assays, and 25-mm coverslips for NHE assays. Cells were refed fresh DMEM growth medium every 48 h until 2 days prior to the experiments, when medium was replaced with a 50:50 (vol/vol) mixture of DMEM containing 5% horse serum and 5% calf serum and astrocyte-conditioned medium containing 10% FBS (28, 30).
Treatment conditions.
The control treatment solution for our experiments was DMEM with 10 mM HEPES (HEPES-DMEM) containing (in mM) 5.6 d-glucose, 1.0 Na-pyruvate, 156 Na+, 119 Cl−, 5.3 K+, 1.8 Ca2+, 44.1 HCO3−, 0.91 H2PO4−, 0.81 Mg2+, and 0.81 SO4−. For ischemic factor treatments, CMEC were exposed to hypoxia, aglycemia, O2-glucose deprivation (OGD), or AVP, as described previously (9, 37, 38). For hypoxia experiments, CMEC monolayers grown on culture plates or coverslips were placed in a hypoxia chamber (Coy Laboratory Products, Grass Lake, MI) preset to 37°C humidified air with 5% CO2 and 19%, 7%, or 2% O2 to achieve a normoxic or moderate-to-severe hypoxic environment, respectively, as previously described (9, 38). O2 levels in the chamber and in treatment media were verified by a chamber O2 sensor/regulator and by a dissolved O2 sensor with a Checkmate II meter (Corning, Corning, NY). For aglycemia experiments, glucose and Na-pyruvate were omitted from HEPES-DMEM. In experiments testing the effects of AVP, HEPES-DMEM was added with 100 nM AVP. Aglycemic and AVP HEPES-DMEM solutions were equilibrated with 37°C humidified air with 5% CO2 and 19% O2. In OGD experiments, CMEC were treated with glucose- and pyruvate-free HEPES-DMEM equilibrated with 2% O2. Immediately before CMEC monolayers were exposed to treatment conditions, growth medium was removed from wells, and CMEC monolayers were rinsed once with the appropriate treatment medium equilibrated to the desired O2 level and then incubated in that medium for 5, 30, 60, 120, 180, or 300 min.
Western blot analysis.
Protein abundances for NHE1, NHE2, total ERK1/2, and phosphorylated (activated) ERK1/2 (p-ERK1/2) were assessed by Western blot analysis. Details of Western blot methods have been described by us previously (18, 37). Briefly, at the end of the treatment period, CMEC monolayers were rinsed with ice-cold PBS supplemented with protease inhibitors (Complete Protease Inhibitor Cocktail, Roche Diagnostic) and phosphatase inhibitors (100 nM NaF and 100 mM Na pyrophosphate). Cells were then lysed with PBS containing 5 mM EDTA, 20 mM HEPES, 150 mM NaCl, 50 mM Na2HPO4, 1% SDS, and protease and phosphatase inhibitors. Protein contents of the lysates were determined using the bicinchoninic acid method to ensure equal protein loadings for all gel lanes. CMEC lysates were denatured in SDS reducing buffer containing DTT (Invitrogen NuPage, Carlsbad, CA) and heated for 10 min at 70°C. Protein samples and prestained molecular weight markers (Bio-Rad, Carlsbad, CA) were loaded onto 7% or 12% Tris·HCl gels (PAGE Gold Precast, Lonza, Rockland, ME), separated by electrophoresis (Mini-Protean II, Bio-Rad, Hercules, CA), and transferred to polyvinylidene difluoride membranes using a Bio-Rad Trans-Blot apparatus. The blots were blocked with 7.5% BSA in PBS-0.1% Tween (PBST) for 1 h at room temperature and then incubated overnight at 4°C with appropriate primary antibodies (mouse monoclonal NHE1 and rabbit polyclonal NHE2 antibodies; Millipore, Billerica, MA) prepared at 1:2,000 dilutions with 7.5% milk-PBST. Rabbit polyclonal ERK and p-ERK (Thr202/Tyr204) antibodies (Cell Signaling Technology, Danvers, MA) were prepared at 1:1,000 dilutions in 7.5% BSA-PBST. Blots were rinsed three times with PBST and then incubated with appropriate secondary antibodies for 1 h at room temperature (horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG; Zymed Laboratories, San Francisco, CA) at 1:2,000 dilutions in 7.5% milk-PBST. Blots were rinsed three times with PBST, and protein was visualized using enhanced chemiluminescence (GE Healthcare, Buckinghamshire, UK) on an imaging machine (model LAS-4000, Fuji Film, Medford, UK). MultiGauge software (Science Lab 2005, FujiFilm) was used to quantitate band density. Densitometry values for abundances in hypoxia-, aglycemia-, AVP-, and OGD-treated cells were normalized to their respective normoxic controls, running as an internal standard on each gel. Lane positions for control and experimental lysates were randomized between experiments to avoid lane bias. For these Western blot assays, membranes were also stripped using 10 ml of stripping buffer (2% SDS, 65 mM Tris·HCl, and 100 mM β-mercaptoethanol, pH 6.8) and then reprobed for mouse monoclonal β-actin (Abcam, Cambridge, MA) at 1:5,000 dilutions.
NHE activity assay.
NHE activity of cultured CMEC was assessed as HOE642-sensitive, Na-dependent H flux using the well-established NH4 prepulse method and BCECF microspectrofluorometry, as described by others and us previously (3, 18, 22). By this method, H efflux from the cell that occurs during recovery from an acid load and is also Na-dependent and HOE642-sensitive is evaluated. Briefly, CMEC monolayers grown on collagen- and attachment factor-coated glass coverslips were pretreated in a hypoxia chamber (Coy Laboratory Products) for 1, 3, or 5 h at 37°C in a normoxic or hypoxic atmosphere in HEPES-buffered medium that contained (in mM) 144 Na, 147 Cl, 5.8 K, 1.2 Ca, 0.4 HPO4, 0.4 H2PO4, 0.4 Mg, 0.4 SO4, 5.6 glucose, and 20 HEPES (pH 7.45 and 290–300 mosM), as well as 0 or 100 nM AVP and 0 or 5.6 mM glucose. For hypoxia treatments, the HEPES-buffered media were preequilibrated with the desired O2 level in the hypoxia chamber before use. For all experimental conditions, 5 μM BCECF-AM (Molecular Probes, Eugene OR) was present during the last 30 min of the pretreatment period. For experiments assessing the effects of ERK1/2 inhibition, 20 μM FR180204 or vehicle was also present 30 min before and throughout the pretreatment and assay periods. After pretreatment, coverslips were placed in a closed-bath chamber assembly (with all pretreatment conditions maintained) and mounted on an epifluorescence microscope (Zeiss Axiovert). Cells were then superfused with the appropriate pretreatment HEPES buffer at a rate of 2 ml/min at 37°C for 5 min to wash out extracellular BCECF. Ratios of fluorescence intensities emitted at 535 nm after excitation at 490 and 440 nm (F490/F440) were collected every 10 s using a charge-coupled device camera and analyzed with OpenLab image-processing software. After baseline fluorescence intensities were measured, cells were superfused with 20 mM NH4Cl followed by Na-free (ChCl replacing NaCl) HEPES buffer solutions to acidify the cells. Subsequently, cells were superfused with 25 μM HOE642 in HEPES buffer. NHE activity was determined as the maximum rate of intracellular pH (pHi) recovery after superfusate was returned to Na-containing HEPES buffer solution without HOE642. NHE activity was expressed as H flux (mM H+/min), the product of ΔpHi/min and buffer capacity (β) at the corresponding pHi. For these experiments, β was calculated as β = (−18.134)(pH) + 136.31 on the basis of numerous measurements of β conducted under normoxic conditions over a number of years in this laboratory. In recent experiments conducted to determine whether hypoxia (5 h, 2% O2) altered the equation used to calculate β, we assessed values of β over the range of pHi from 6.3 to 7.1 from three separate coverslips each for normoxic and hypoxic conditions. We found that equations for β in normoxic and hypoxic CMEC were not significantly different (P = 0.655, by t-test), and the mean of those equations was β = (−18.2)(pH) + 131.4. Our finding that hypoxic exposures do not alter β is in agreement with a previous study of hypoxia and β in pulmonary endothelial cells (7).
At the end of each experiment, a one-point calibration was done by superfusion of CMEC with high-K+ buffer containing 10 μM nigericin and (in mM) 141 Cl, 135 K, 0.6 Ca, 1.02 Mg, 5.6 d-glucose, 20 HEPES, and 3.2 N-methyl-d-glucamine to determine F490/F440 at pH 7.0. In separate experiments, the high-K+/nigericin technique (4) was performed to construct a five-point calibration curve for F490/F440 vs. pHi for pHi 6.0, 6.5, 7.0, 7.5, and 8.0 (18). A serial NH4+ superfusion from 20 to 10, 5, 2.5, 1, 0.5, and then 0 mM NH4Cl in Na-free HEPES buffer was also done to calculate β using ΔNH4+/ΔpHi over a range of pHi pertinent to the BCECF studies. For each set of experiments, the mean prerecovery pHi values did not differ significantly among experimental conditions.
Exposure of CMEC to these ischemic factors did not alter baseline pHi values, i.e., those at the start of the experiment, prior to NH4 prepulse. Thus baseline pHi values for 1, 3, and 5 h of 2% O2 were 6.79 ± 0.03, 6.74 ± 0.03, and 6.80 ± 0.04, respectively, compared with 6.83 ± 0.02 for control. For aglycemia, baseline pHi values were 6.81 ± 0.04, 6.89 ± 0.05, and 6.84 ± 0.03 for 1, 3, and 5 h of aglycemia, respectively, compared with 6.93 ± 0.02 for control. For AVP, baseline pHi values were 6.89 ± 0.04, 6.96 ± 0.05, and 6.89 ± 0.02 for 1, 3, and 5 h of AVP, respectively, compared with 6.85 ± 0.03 for control. In addition, starting pHi values, i.e., those at the start of the pHi recovery H flux measurement, were not significantly different for 1-, 3-, and 5-h exposures to aglycemia and AVP compared with control (data not shown). For experiments evaluating 2% O2, the average starting pHi was slightly lower than control after 3- and 5-h (but not 1-h) exposures. Exclusion of the small number of hypoxia experiments with starting pHi values lower than control did not significantly alter the resulting mean values for NHE activity following exposure to 2% O2. CMEC exposed to the ERK inhibitor FR180204 also did not show significant differences between control and experimental conditions with respect to baseline pHi or starting pHi.
NKCC activity assay.
NKCC activity was calculated as ouabain-insensitive, bumetanide-sensitive K+ influx, with 86Rb used as a tracer for K+, as previously described by us (9, 28, 37, 38). Briefly, CMEC monolayers grown on multiwell plates were pretreated in a hypoxia chamber (Coy Laboratory Products) for 30–120 min at 37°C in a normoxic or hypoxic atmosphere in HEPES-buffered DMEM that also contained 0 or 100 nM AVP and 0 or 5.6 mM glucose. To test the effects of ERK1/2 inhibition, 20 μM FR180204 or vehicle was also present 30 min before pretreatment and throughout the pretreatment and assay periods. For assay of NKCC activity, ouabain (100 μM) with or without bumetanide (10 μM) was present during the last 5 min of pretreatment and during the assay. 86Rb (1 μCi/ml) was added to the media for 5 min, and the wells were aspirated and rinsed twice with ice-cold 0.1 M MgCl2 to terminate the assay. CMEC were lysed with 1% SDS and mixed in EcoLite liquid scintillation cocktail (MP Biomedicals, Solon, OH). 86Rb was detected using a liquid scintillation counter (Tri-Carb 2500TR), and protein contents of lysates were assessed using the bicinchoninic acid method.
Immunofluorescence detection of BBB endothelial ERK1/2 in situ.
This study was conducted in accordance with the animal use and care guidelines issued by the National Institutes of Health, and the protocol was approved by the Animal Use and Care Committee at the University of California, Davis. Normotensive male Sprague-Dawley rats (250–300 g; Charles River Laboratories, Wilmington, MA) were anesthetized by injection of pentobarbital sodium (1 mg/kg ip) and then either immediately perfusion-fixed or subjected to 60 min of MCAO and then perfusion-fixed. MCAO was performed by the intraluminal suture method, and brains were immediately perfusion-fixed at the end of the MCAO period using methods described previously (29, 31). Perfusion fixation was done via the ascending aorta through a right atrium incision, as described by us previously (18, 37, 38) with some modifications. Ice-cold saline (50 ml) followed by 4% paraformaldehyde (400 ml, 3 ml/min via peristaltic pump) was perfused to flush out blood. Brains were removed and further fixed in 4% paraformaldehyde for 24 h and then immersed in a series of sucrose solutions (10%, 20%, 30% sucrose, all in 0.1 M PBS) for 24 h each at 4°C. Brains were then embedded in optimal cutting temperature compound (Tissue-Tek OCT, Sakura Finetec, Torrance, CA), cooled to −80°C and cut on a cryostat (−20°C) into 20-μm thick sections that were directly mounted onto Fisherbrand Superfrost Plus microscope slides (Thermo Fisher Scientific, Waltham, MA). Sections were dried overnight at room temperature and stored at −20°C until use. For immunofluorescence staining, slide-mounted brain sections were washed with 0.1 M PBS (3 washes of 5 min each for all PBS wash steps), incubated for 20 min in 0.5% H2O2, and washed in PBS. For antigen retrieval, slides were incubated in 10 mM sodium citrate (in 0.1 M PBS and 0.05% Tween 20 at pH 6.0) for 30 min at 80°C and then in the same solution at room temperature for 30 min. Sections were washed again with PBS and then incubated in blocking solution (5% goat serum in 0.1 M PBS and 0.3% Triton X-100) for 2 h at room temperature. Slides were incubated with primary antibody (diluted in blocking solution) overnight at 4°C. To evaluate the in situ presence of ERK1/2, we used antibodies that specifically recognize ERK1/2 and p-ERK1/2 (both rabbit polyclonal at 1:200 dilution; Cell Signaling Technology). We also used antibodies to detect the astrocyte-specific marker glial fibrillary acidic protein (GFAP, guinea pig polyclonal, 1:500 dilution; Fitzgerald Industries International, Acton, MA) and SMI-71, a BBB endothelial cell-specific antibody (mouse monoclonal, 1:1,000 dilution; Covance, Emeryville, CA). After incubation with primary antibodies, slides were washed in PBS (3 times, 5 min each) and then incubated for 2 h at room temperature with secondary antibodies: goat anti-mouse for Alexa Fluor 647 (Life Technologies, Grand Island, NY), goat anti-guinea pig for Alexa Fluor 546, and goat anti-rabbit for Alexa Fluor 488. All secondary antibodies were diluted 1:1,000 with 5% goat serum in 0.1 M PBS. Slides were then washed with PBS, and coverslips were mounted using ProLong Gold antifade reagent (Life Technologies). Multichannel imaging was performed with a confocal microscope (model LSM 510, Carl Zeiss, Oberkochen, Germany) using a ×64 oil objective. Lasers with excitation wavelengths of 488 and 543 nm were used for Alexa Fluor 488 and 546, respectively. Emission wavelengths were set at 505- to 530-nm band pass for Alexa Fluor 488 and at 560-nm long pass for Alexa Fluor 546.
Materials.
Bovine CMEC and attachment factor were purchased from Cell Systems (Kirkland, WA); DMEM and l-glutamine from GIBCO-BRL (Grand Island, NY); gentamicin from AG Scientific (San Diego, CA); FBS and calf serum from Hyclone (Logan, UT); horse serum and AVP from Sigma (St. Louis, MO); bumetanide and ouabain from ICN Biomedicals (Costa Mesa, CA); 86Rb from Perkin-Elmer (Welles, MA); and FR180204 from Calbiochem (San Diego, CA). HOE642 was a gift from Sanofi-Aventis Pharmaceuticals (Bridgewater, NJ).
Statistics.
Values are means ± SE of at least five experiments. Statistical analyses were done using GraphPad Prism 4 software. Differences among treatment groups were determined by ANOVA or Student's t-test. P < 0.05 was considered to indicate significant difference.
RESULTS
Our previous studies showed that NHE1 and NHE2 are present in CMEC and that ischemic factors, including hypoxia, aglycemia, and AVP, stimulate NHE activity in the cells. This, together with the observation that NHE1 and NHE2 are present in BBB in situ, residing predominantly in the luminal BBB membrane, supports the hypothesis that NHE (NHE1 and/or NHE2) contributes to the increased secretion of Na and water into the brain during ischemic stroke, thereby participating in cerebral edema formation. We previously demonstrated that 30-min exposures to ischemic factors significantly increase CMEC NHE activity. If BBB NHE activity contributes to the robust cerebral edema formation that continues throughout the early hours of ischemic stroke, one would predict that stimulation of NHE by one or more ischemic factors is sustained over several hours. To address this, we evaluated the effects of 1-, 3-, and 5-h exposures to hypoxia, aglycemia, and AVP on CMEC NHE activity. Figure 1 shows that hypoxia (i.e., 2% O2) caused significant increases in NHE activity of the cells after 1-, 3-, and 5-h exposures. Thus NHE activity was increased by 34% and 71% after 1- and 5-h exposures, respectively. Aglycemia and AVP (100 nM) also significantly increased NHE activity after 1-, 3-, and 5-h exposures, with increases of 43% and 58% after 1- and 5-h aglycemia exposures, respectively, and by 62% and 35% after 1- and 5-h AVP exposures, respectively. These findings support the hypothesis that BBB NHE activity participates in cerebral edema formation throughout the early hours of ischemic stroke.
Effects of 1–5 h of exposure to hypoxia, aglycemia, and AVP on cerebral microvascular endothelial cell (CMEC) Na/H exchange (NHE) activity. CMEC monolayers were exposed for 1, 3, or 5 h to 19% O2 (normoxic control) or 2% O2 in HEPES-DMEM (A), aglycemic normoxic media (glucose- and pyruvate-free HEPES-buffered DMEM at 19% O2; B), or normoxic HEPES-DMEM containing 100 nM AVP (C). Values are means ± SE; n = 13, 8, 6, and 6 for control, 1, 3, and 5 h, respectively, in A; 12, 7, 12, and 5 for control, 1, 3, and 5 h, respectively, in B; and 11, 6, 4, and 12 for control, 1, 3, and 5 h, respectively, in C. *P < 0.05 vs. control (by ANOVA with Dunnett's multiple-comparison post hoc test).
Our previous studies established the presence of NHE1 and NHE2 in bovine CMEC. To determine whether ischemic factor-induced elevation of CMEC NHE activity may be due in part to changes in NHE abundance in the cells, we used Western blot methods and antibodies that specifically recognize NHE1 or NHE2 to evaluate abundances of NHE1 and NHE2 in CMEC following 1-, 3-, and 5-h exposures of the cells to hypoxia, aglycemia, and AVP. As shown in Fig. 2, A and B, when CMEC were exposed to 2% O2, a more severe level of hypoxia as is found in the core of an ischemic focus, NHE1 abundance was significantly elevated following 1-, 3-, and 5-h exposures (52, 44, and 78% increases at 1, 3, and 5 h, respectively). Despite an apparent trend for increased NHE2 levels following exposure to 2% O2, no significant differences were observed (P = 0.14, 0.29, and 0.23 for 1-, 3-, and 5-h exposures, respectively). Figure 2, C and D, shows that NHE1 abundance was also increased following exposure to 7% O2, a moderate level of hypoxia as found in the penumbra of an ischemic focus. We found NHE1 abundance increases of 125%, 162%, and 140% following 1-, 3-, and 5-h exposures, respectively. However, only the 3-h exposure produced a statistically significant increase in NHE1 abundance. As shown in Fig. 3, A and B, exposure of CMEC to aglycemia significantly increased NHE1 abundance after 1 and 3 h (69 and 106% increases, respectively). A 5-h exposure also showed a trend for increased NHE1 abundance, albeit one that did not reach statistical significance. Aglycemia at any of the exposure times was without effect on NHE2 abundance. Finally, as shown in Fig. 3, C and D, when we evaluated the effects of AVP (100 nM) on CMEC NHE abundance, we again found that NHE1 was significantly increased after 1, 3, and 5 h of exposure (48, 62, and 77% increases, respectively), while NHE2 abundance was unchanged.
Effects of 1- to 5-h hypoxia exposures on NHE1 and NHE2 abundance in CMEC. CMEC monolayers were exposed to normoxic control conditions (19% O2) or 2% O2 (A and B) or 7% O2 (C and D) for 1, 3, or 5 h. Incubation medium in all cases was HEPES-buffered DMEM. Cell lysates were prepared and subjected to Western blot analysis using primary antibodies that recognize NHE1 protein (mouse monoclonal antibody) or NHE2 protein (rabbit polyclonal antibody). Top: representative Western blots. Bottom: abundance of NHE1 and NHE2. Values (means ± SE) are relative to internal normoxic control for each condition; n = 11, 10, 10, and 9 for control, 1, 3, and 5 h, respectively, in A; 8 for control, 1, 3, and 5 h in B; 7 for control and 5 for 1, 3, and 5 h in C; and 10, 6, 8, and 7 for control, 1, 3, and 5 h, respectively, in D. *P < 0.05 vs. control (by t-test).
Effects of 1- to 5-h aglycemia and AVP exposures on NHE1 and NHE2 abundance in CMEC. CMEC monolayers were exposed to control normoxic medium (HEPES-DMEM) or aglycemic normoxic medium (glucose- and pyruvate-free HEPES-DMEM at 19% O2; A and B) or AVP (100 nM in normoxic HEPES-DMEM; C and D) for 1, 3, or 5 h, and cell lysates were prepared and subjected to Western blot analysis for NHE1 and NHE2 as described in Fig. 2 legend. Top: representative Western blots. Bottom: abundance of NHE1 and NHE2. Values (means ± SE) are relative to internal normoxic control for each condition; n = 11, 8, 11, and 9 for control, 1, 3, and 5 h, respectively, in A; 12, 10, 12, and 12 for control, 1, 3, and 5 h, respectively, in B; 11 for control, 1, 3, and 5 h in C; and 12, 11, 10, and 10 for control, 1, 3, and 5 h, respectively, in D. *P < 0.05 vs. control (by t-test).
These findings support the possibility that changes in NHE1 abundance contribute to the increased NHE activity following ≥1-h exposures to ischemic factors (see discussion).
In the present study we also evaluated the possibility that ischemic factor stimulation of CMEC NHE activity involves activation of ERK. We used Western blot methods and antibodies that specifically recognize activated ERK (p-ERK) and antibodies that recognize p-ERK or nonphosphorylated (total) ERK to test the effects of hypoxia, aglycemia, and AVP on activation of ERK in CMEC. Figure 4A shows prominent doublet bands (42 and 44 kDa) in Western blots for ERK and p-ERK, as predicted for this kinase. When cells were exposed to 7% or 2% O2 for 5, 30, 60, or 120 min, abundance of p-ERK bands increased, indicating activation of ERK. Quantitation of ERK and p-ERK abundances from multiple experiments revealed that p-ERK was significantly increased after 5- and 30-min exposures to 7% or 2% O2. No significant changes in total ERK abundance were observed for 7% or 2% O2 at any of the exposure times (quantitated data not shown). Exposure of CMEC to aglycemia or OGD also increased p-ERK abundance without a significant change in total ERK (Fig. 4B). p-ERK was significantly increased following 5-, 30-, and 60-min exposures to aglycemia and 5-, 30-, 60-, and 120-min exposures to OGD. When CMEC were exposed to AVP (100 nM), total ERK was again unchanged while p-ERK was increased. In this case, however, the increase in p-ERK was only observed after 5-min AVP exposure (Fig. 4C). These findings suggest that CMEC ERK activation by these three ischemic factors is rapid, with a two- to threefold increase in p-ERK by 5 min. However, the time course over which p-ERK remains elevated differs among the factors, with OGD and aglycemia having the most prolonged effect and AVP having the most short-lived effect.
Hypoxia, aglycemia, and AVP activation of ERK1/2 in CMEC. CMEC monolayers were exposed to normoxic control or hypoxia (19% and 2% O2, respectively, in HEPES-DMEM; A), aglycemic normoxic media or aglycemic hypoxic media (glucose- and pyruvate-free HEPES-buffered DMEM at 19% and 2% O2, respectively; B), or AVP (100 nM in normoxic HEPES-DMEM; C) for 5, 30, 60, or 120 min. Cell lysates were prepared and subjected to Western blot analysis using antibodies that recognize only phosphorylated (activated) ERK (p-ERK) or both p-ERK and nonphosphorylated (total) ERK protein. Top: representative Western blots. Doublet bands shown for both ERK and p-ERK are ~42 and 44 kDa. Bottom: abundance of p-ERK. Values (means ± SE) are relative to internal normoxic control for each condition; n = 6, 9, 8, 7, and 6 for 7% O2, 2% O2, aglycemia, O2-glucose deprivation (OGD), and AVP, respectively, at all time points. *P < 0.05 vs. control (by t-test).
If ERK participates in ischemic factor stimulation of NHE or NKCC activity, we should see that pharmacological inhibition of ERK reduces or abolishes the ability of ischemic factors to stimulate the Na transporters. As shown in Fig. 5, exposure of CMEC to 30 min of hypoxia (2% O2), aglycemia, or AVP (100 nM) stimulated NHE activity of the cells, as we have reported previously (18), and the highly selective ERK inhibitor FR180204 (30 μM) abolished stimulation of exchanger activity by each of the ischemic factors. While ERK1/2 inhibition reduced hypoxia, aglycemia, and AVP stimulation of CMEC NHE activity, it was without effect on NHE1 abundance increases after 5 h of exposure to the ischemic factors. Thus NHE1 abundance in cells exposed to 2% O2 for 5 h in the presence of FR180204 (30 μM) was 1.09 ± 0.30 fold of that observed in the absence of FR180204 (mean ± SE, n = 3 experiments). Similarly, for 5-h exposures to aglycemia and AVP, NHE1 abundance in the presence of the ERK inhibitor was 1.01 ± 0.034 and 0.98 ± 0.08 fold, respectively, of that in the absence of the inhibitor.
Effects of FR180204 on hypoxia-, aglycemia- and AVP-induced stimulation of CMEC NHE activity. CMEC monolayers were pretreated for 30 min with FR180204 (30 μM) or vehicle in normoxic HEPES-DMEM and exposed for 30 min to normoxic control (19% O2) or hypoxia (7% O2) in HEPES-DMEM (A), aglycemia (normoxic glucose- and pyruvate-free HEPES-DMEM; B), or AVP (100 nM in normoxic HEPES-DMEM; C) also containing FR180204 (30 μM) or vehicle, and CMEC NHE activity was assessed. Pretreatment conditions were maintained throughout the assay. Values are means ± SE; n = 5, 6, and 4 for control, hypoxia, and hypoxia + FR180204, respectively, in A and B and 4, 5, and 4 for control, hypoxia, and hypoxia + FR180204, respectively, in C. *P < 0.05 vs. vehicle (by ANOVA with Dunnett's multiple-comparison post hoc test).
Because our previous studies showed that hypoxia, aglycemia, and AVP also stimulate CMEC NKCC activity, in the present study we evaluated the effects of ERK inhibition of stimulation of NKCC activity by these ischemic factors. Figure 6 shows that when CMEC were exposed to hypoxia (7% O2) or aglycemia for 30 min, NKCC activity was significantly increased, as we have reported previously (9). Treatment of CMEC with FR180204 (30 μM) significantly reduced hypoxia-stimulated, and abolished aglycemia-stimulated, NKCC activity. These findings suggest that ERK plays a role in ischemic factor stimulation of BBB NHE and NKCC activities.
Effects of FR180204 on hypoxia- and aglycemia-induced stimulation of CMEC Na-K-Cl cotransporter (NKCC) activity. CMEC monolayers were pretreated for 30 min with FR180204 (30 μM) or vehicle and then exposed for 30 min to normoxic control (19% O2) or hypoxia (7% O2; A) or aglycemia (B) in media also containing FR180204 (30 μM) or vehicle. CMEC NKCC activity was assessed as ouabain-insensitive, bumetanide-sensitive K+ influx. Pretreatment conditions were maintained throughout the assay. Values are means ± SE for 6 and 5 separate experiments for A and B, respectively. #P < 0.05 vs. control (A and B); *P < 0.05 vs. 7% O2 in vehicle (A) or aglycemia in vehicle (B) (by ANOVA with Tukey's post hoc test).
A role for ERK in ischemia stimulation of BBB NHE and NKCC activity necessitates the presence of this kinase in the BBB endothelial cells in situ, not just in cultured CMEC. As an initial investigation of BBB ERK, we conducted confocal immunofluorescence studies using perfusion-fixed rat brain sections and antibodies that recognize ERK and p-ERK and also antibodies that specifically recognize GFAP, a marker of astrocytes, as well as SMI-71, a BBB-specific antibody. Figure 7A shows representative images of ERK or p-ERK (both green) and GFAP (red) in perfusion-fixed normoxic brain. Merged images show that ERK is present in BBB endothelial cells and astrocytes. p-ERK was also detected in BBB endothelial cells and astrocytes. Figure 7B shows representative images of ERK or p-ERK (both green) and SMI-71 (red). Merged images show again that ERK is present in the BBB endothelial cells and that activated ERK (p-ERK) is also detected in the BBB endothelial cells in situ. If ERK activation participates in stimulation of BBB NHE activity during ischemic stroke, we should also observe ERK and p-ERK in BBB of ischemic brain. We subjected rats to 60 min of permanent MCAO and then immediately perfusion-fixed the brains, prepared brain slices for confocal immunofluorescence studies, and evaluated the presence of ERK and p-ERK in the ischemic BBB endothelial cells in situ. Figure 7C shows representative images of ERK and p-ERK (green) and GFAP (red) staining. As in Fig. 7A, the merged images demonstrate the presence of ERK and p-ERK in the BBB endothelial cells (green) as well as astrocytes (orange). Figure 7D shows representative images of ERK and p-ERK (green) and SMI-71 (red) staining. The merged images show ERK and p-ERK in BBB endothelial cells (orange) surrounded by brain parenchyma (green). These findings are considered further in discussion.
Immunofluorescence detection of ERK and p-ERK in the blood-brain barrier (BBB) in situ. Perfusion-fixed normoxic (A and B) and ischemic (C and D) rat brains were cryosectioned (5 μm) and mounted on glass slides, and immunohistochemistry was performed with antibodies for ERK, p-ERK, and either the astrocyte marker glial fibrillary acidic protein (GFAP) or the BBB endothelial cell-specific antibody SMI-71. ERK and p-ERK (green) are detected in BBB endothelial cells and perivascular astrocytes. GFAP (red) appears in perivascular astrocytic end feet surrounding the BBB endothelial cells, and SMI-71 staining (red) appears in BBB endothelial cells. Merged images show ERK and p-ERK in BBB endothelial cells (green in A and C, orange in B and D), as well as astrocytes (orange in A and C). For A and B and for C and D, negative control images (Neg Control Merged) were generated using secondary antibodies only. Scale bars, 10 μm.
DISCUSSION
Previous studies from this group have provided evidence that BBB NHE activity is rapidly increased upon exposure to the ischemic factors hypoxia, aglycemia, and AVP. We have also shown that inhibition of BBB NHE activity by intravenous administration of HOE642 also reduces brain Na uptake and edema in the rat permanent MCAO model of stroke. Our hypothesis that BBB NHE is a substantial contributor to ischemia-induced edema formation during the early hours of stroke through increased secretion of Na, Cl, and water from blood into brain predicts that NHE activity will be elevated for at least several hours. However, the effects of ischemic factor exposure sustained over several hours, as occurs in stroke, on BBB NHE activity have not been known. Whether ischemic factors also alter abundance of NHE1 and/or NHE2 has not been previously examined. Finally, very little has been known about the signaling pathways responsible for ischemic factor stimulation of BBB NHE activity. In the present study we provide evidence that BBB NHE activity is increased by hypoxia, aglycemia, and AVP through ≥5 h and that NHE1, but not NHE2, abundance is also increased by these treatments. We also provide evidence that ERK participates in ischemic factor stimulation of BBB NHE activity.
The present study demonstrates for the first time that CMEC NHE activity is not only rapidly stimulated by hypoxia, aglycemia, and AVP but that it also remains elevated through at least several hours in the presence of these ischemic factors. This finding is consistent with the hypothesis that NHE contributes to edema formation throughout the early hours of ischemic stroke, when the majority of edema forms. Each of these ischemic factors is capable of independently maintaining elevated NHE activity. However, the response of CMEC NHE activity to combined exposures of these factors needs to be explored in future studies. Also, whether NHE activity remains elevated beyond 5 h, contributing to brain injury induced by more prolonged ischemia, either through further promoting cytotoxic edema or participating in development of vasogenic edema (26), remains to be determined. In a previous study we found that when CMEC are exposed to hypoxia, the cells do not exhibit significant swelling until ≥4 h of hypoxia, nor does the Na content of the cells increase (5), consistent with a role for NHE in BBB secretion of Na and water into the brain during the early stages of stroke. We also found that when CMEC do begin to swell, Na content increases with no change in K content of the cells and, furthermore, that swelling at 5 h is significantly reduced by treatment of the cells with HOE642 to inhibit NHE activity or bumetanide to inhibit NKCC activity, with the two inhibitors in combination abolishing any hypoxia-induced CMEC swelling (5). This suggests that NHE, as well as NKCC, contributes to BBB endothelial cell swelling as ischemia progresses. Swelling of the BBB endothelial cells is also predicted to contribute to ischemia-induced brain damage through further compression of brain tissue and consequent reduction of blood flow in neighboring regions. It is also possible, if not probable, that BBB endothelial cell swelling will contribute to increased BBB permeability through elevation of intracellular Na, leading to reversed Na/Ca exchange and consequent increases in cytosolic Ca levels (19), which have been linked to BBB breakdown (12). Additional studies are required to evaluate these possibilities.
In the present study we also demonstrate that hypoxia, aglycemia, and AVP increase abundance of CMEC NHE1 following exposures of up to 5 h, while no significant changes in NHE2 abundance are observed. This is consistent with the hypothesis that the ischemia-induced increase in CMEC NHE activity occurs at least in part by an increase in NHE1 abundance. However, further studies are needed to determine the relative contributions of NHE1 and NHE2, e.g., small interfering RNA experiments to evaluate the responses of CMEC NHE activity to ischemic factors after selective knockdown of NHE1 or NHE2. We have not yet observed other NHE isoforms in these cells (18). While the observation that increases in NHE1 abundance and NHE activity correlate fairly well is consistent with increased activity as a consequence of increased NHE protein, it is also likely that ischemia activation of signaling pathways contributes to increased NHE activity in the cells. Additional studies are needed to further examine the relationship between the increases in NHE1 abundance and NHE activity. For example, the degree to which activity of NHE1 protein residing in the plasma membrane is stimulated by ischemic factors independently of any increase in NHE1 protein abundance is not clear. Also, because the present study examined only total cell NHE abundance, future experiments should address whether abundance of NHE1 protein in the plasma membrane is altered during exposure to hypoxia, aglycemia, and/or AVP. For example, it is possible that increased NHE activity could arise in part from insertion of NHE1 proteins into the plasma membrane from intracellular membrane pools.
Our initial investigation of signaling pathways that may mediate ischemic factor stimulation of CMEC NHE activity provides evidence for a role of ERK1/2 MAP kinase. Here, we found that all three ischemic factors cause significant increases in ERK activity. Moderate and severe hypoxia, as well as aglycemia and AVP, significantly increase p-ERK while having no effect on total ERK levels in the cells. While all three factors cause increases in ERK activity by 5 min, the subsequent time courses of ERK stimulation differ with ischemic factor. Aglycemia and OGD activation of ERK are sustained for ≥2 h, while hypoxia and AVP activation of ERK are transient, with ERK returning to control levels by 60 and 30 min, respectively. Our present studies further show that stimulation of CMEC NHE activity by 30-min exposures to hypoxia, aglycemia, or AVP is abolished by the specific ERK inhibitor FR180204, indicating that ischemic factor elevation of CMEC NHE activity is dependent on ERK activity. ERK has been reported to activate p90RSK to stimulate NHE activity in vascular smooth muscle cells (36) and also neurons (21). The events by which ERK stimulates CMEC NHE activity, as well as the relationship between ischemia-induced increases in NHE activity and NHE1 abundance, remain to be determined.
Our previous studies of ischemic factor effects on CMEC AMPK and the p38 and JNK MAP kinases (p38 and JNK) showed that the time courses of response varied with kinase and ischemic factor. Thus AMPK activation occurs by 5 min but is transient, disappearing after 5–30 min, depending on the factor (37). In contrast, p38 activation by ischemic factors is observed by 5 min and sustained through ≥120 min (38). JNK activation is rapid and transient in the presence of hypoxia, i.e., activated by 5 min but not sustained past 30 min, buts exhibits a slower onset and sustained activation with aglycemia and AVP (38). Thus, similar to AMPK and p38, ERK activation by all three factors is quite rapid, but the response to hypoxia and AVP is transient, as it is for AMPK, while the ERK response to aglycemia is sustained, as it is for p38. Whether continuous ERK activation is required for stimulation of CMEC NHE activity or a transient increase in ERK is sufficient remains to be determined, as does whether AMPK, p38, and/or JNK also participate in ischemia stimulation of CMEC NHE activity. Our present study also demonstrates that stimulation of CMEC NKCC activity by hypoxia and aglycemia is dependent on ERK. Our previous studies showing that ischemic factor-stimulated CMEC NKCC activity is also dependent on AMPK, p38, and ERK suggest that all four of these kinases also participate in ischemic factor effects on NHE as well as NKCC. Clarification of the relationships among these signaling pathways in ischemia-stimulated CMEC NHE and NKCC activities awaits further investigation.
A role for ERK in BBB endothelial cell NHE and NKCC responses to ischemic stroke is further supported by our observation that ERK is present in BBB endothelial cells in situ and that p-ERK is also detected in the cells. Previous immunohistochemistry and/or immunofluorescence studies of rodent brains for the presence of stress kinases, including ERK, have demonstrated ERK in neurons and astrocytes without addressing whether ERK is also present in BBB endothelial cells (8, 39, 40). Unless specific markers are used to positively identify BBB and perivascular astrocytes and to visually distinguish between the two, it is not possible to gain information about what kinases may be present and active in these closely apposed cells. A previous immunohistochemistry study examining ERK in human brain following stroke reported evidence for active ERK in penumbral microvessels (35). Again, however, markers were not used to distinguish BBB endothelial cells from neighboring cells. In the present study we used markers that revealed for the first time that ERK (total ERK and p-ERK) is present in BBB endothelial cells, as well as in the surrounding astrocyte end feet, in ischemic and normoxic brain. While this provides evidence further supporting a role for ERK in stimulation of BBB NHE activity during ischemic stroke, the confocal microscopy immunofluorescence methods used in this study do not readily provide a good quantitative assessment of ERK activation in ischemic brain tissue. Future experiments are needed to explore the time course and degree of ERK activation in BBB endothelial cells in vivo.
In summary, the present study provides evidence supporting the hypothesis that BBB NHE activity participates in cerebral edema formation throughout the early hours of ischemic stroke and that hypoxia, aglycemia, and AVP effects on NHE activity are mediated at least in part by ERK. We further show that while NHE1 and NHE2 are present in BBB endothelial cells, both CMEC and BBB in situ, only NHE1 abundance is increased upon exposure to hypoxia, aglycemia, and AVP. The relationship between ischemia-induced increases in NHE1 abundance and ERK-mediated increases in NHE activity of BBB endothelial cells requires further investigation.
GRANTS
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-039953 (M. E. O'Donnell), American Heart Association Western States Affiliate Predoctoral Fellowships 07PRE2060835; (T. I. Lam) and 10PRE3330016 (B. K. Wallace), and a University of California, Davis, Clinical Translational Science Center T32 Predoctoral Clinical Research Traineeship (N. Yuen). The investigation was conducted in part in a facility constructed with support from National Center for Research Resources Research Facilities Improvement Program Grant C06 RR-17348-01.
AUTHOR CONTRIBUTIONS
N.Y., T.I.L., B.K.W., and N.R.K. performed the experiments; N.Y., T.I.L., B.K.W., S.E.A., and M.E.O. analyzed the data; N.Y., T.I.L., B.K.W., S.E.A., and M.E.O. interpreted the results of the experiments; N.Y., T.I.L., B.K.W., and N.R.K. prepared the figures; N.Y. and T.I.L. drafted the manuscript; M.E.O. was responsible for conception and design of the research; M.E.O. edited and revised the manuscript; M.E.O. approved the final version of the manuscript.
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