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Am J Physiol Cell Physiol 294: C363-C371, 2008. First published October 24, 2007; doi:10.1152/ajpcell.00045.2007
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Shear stress and 17β-estradiol modulate cerebral microvascular endothelial Na-K-Cl cotransporter and Na/H exchanger protein levels

Elaine Chang,1 Martha E. O'Donnell,2 and Abdul I. Barakat1

Departments of 1Mechanical and Aeronautical Engineering and 2Physiology and Membrane Biology, University of California, Davis, California

Submitted 3 January 2007 ; accepted in final form 12 October 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Ion transporters of blood-brain barrier (BBB) endothelial cells play an important role in regulating the movement of ions between the blood and brain. During ischemic stroke, reduction in cerebral blood flow is accompanied by transport of Na and Cl from the blood into the brain, with consequent brain edema formation. We have shown previously that a BBB Na-K-Cl cotransporter (NKCC) participates in ischemia-induced brain Na and water uptake and that a BBB Na/H exchanger (NHE) may also participate. While the abrupt reduction of blood flow is a prominent component of ischemia, the effects of flow on BBB NKCC and NHE are not known. In the present study, we examined the effects of changes in shear stress on NKCC and NHE protein levels in cerebral microvascular endothelial cells (CMECs). We have shown previously that estradiol attenuates both ischemia-induced cerebral edema and CMEC NKCC activity. Thus, in the present study, we also examined the effects of estradiol on NKCC and NHE protein levels in CMECs. Exposing CMECs to steady shear stress (19 dyn/cm2) increased the abundance of both NKCC and NHE. Estradiol abolished the shear stress-induced increase in NHE but not NKCC. Abrupt reduction of shear stress did not alter NKCC or NHE abundance in the absence of estradiol, but it decreased NKCC abundance in estradiol-treated cells. Our results indicate that changes in shear stress modulate BBB NKCC and NHE protein levels. They also support the hypothesis that estradiol attenuates edema formation in ischemic stroke in part by reducing the abundance of BBB NKCC protein.

estrogen; ischemia; mechanotransduction; flow reduction; endothelium

DURING THE EARLY STAGES of cerebral ischemia, brain edema forms by a process involving increased secretion of Na and Cl across an intact blood-brain barrier (BBB) from the blood into the brain, with osmotically obliged water following (2, 23, 25). Our previous studies have provided evidence that a luminal BBB Na-K-Cl cotransporter (NKCC) participates in ischemia-induced cerebral edema formation. This includes the findings that the cotransporter is present in the luminal BBB membrane in situ and that inhibition of BBB NKCC activity by intravenous administration of the cotransport inhibitor bumetanide reduces edema formation, infarct, and brain Na uptake in the rat middle cerebral artery occlusion (MCAO) model of focal ischemia (32). In addition, we and others have shown that activity of NKCC in cultured cerebral microvascular endothelial cells (CMECs) is stimulated by hypoxia (8, 15), aglycemia (8), arginine vasopressin (AVP; 8, 14, 22), and endothelin (15, 41, 47), four prominent factors present during cerebral ischemia. There is also evidence suggesting a role for a BBB Na/H exchanger (NHE) in ischemia-induced cerebral edema formation and brain Na uptake. This includes the recent observations that CMEC NHE activity is stimulated by hypoxia, aglycemia, and AVP and that inhibition of the BBB NHE by intravenous administration of the specific NHE inhibitor cariporide reduces brain edema and Na uptake in the early hours of MCAO (17, 20, 46).

Estradiol has been shown to be neuroprotective in stroke (6, 12, 35, 45, 49). Clinical studies have documented that premenopausal women as well as postmenopausal women taking hormone replacement therapy (HRT) have less damage in stroke than men and postmenopausal women not taking HRT (12, 26, 51). In addition, we and others have shown previously that 17β-estradiol (E2) reduces brain infarct (6, 21, 35, 45, 49, 51) and edema (21) in male rats and female ovariectomized rats subjected to MCAO and that estradiol abolishes stimulation of CMEC NKCC activity by hypoxia and AVP (21). Estradiol also decreases NKCC protein abundance in CMECs (21). Collectively, these findings suggest that estradiol may play a role in attenuating ischemia-induced brain infarct and edema by reducing ischemia stimulation of BBB Na transporters that promote edema formation.

Both NKCC and NHE have been found to be flow sensitive in large vessel endothelial cells. In aortic endothelium, flow-derived shear stress regulates NKCC protein levels (43, 44), reduces intracellular pH, and increases NHE activity (52, 53). However, nothing is known about how ion transporters of BBB endothelial cells are influenced by changes in shear stress and, in particular, how the abrupt reduction in cerebral blood flow that occurs during ischemic stroke may affect the BBB NKCC and NHE. The present studies were conducted to test the hypothesis that NKCC and NHE protein levels in CMECs are sensitive to changes in shear stress and that estradiol modulates this sensitivity. We show here that 1) steady shear stress causes a robust, sustained increase in CMEC NKCC protein levels and a modest, transient increase in NHE protein; 2) estradiol has no effect on the shear stress-induced increase in NKCC abundance but abolishes the shear stress-induced increase in NHE; and 3) abrupt reduction of shear stress causes a rapid decrease in NKCC abundance in estradiol-treated CMECs while having no effect on NKCC in the absence of estradiol and no effect on NHE abundance either in the presence or in the absence of estradiol.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
CMEC culture. Bovine CMECs (Cell Systems, Kirkland, WA) were cultured in DMEM containing 5.6 mM glucose and supplemented with 2 mM L-glutamine, 50 µg/ml gentamicin, 1 ng/ml basic fibroblast growth factor, 5% horse serum, and 5% calf serum. For flow experiments, cells were grown on 25 x 75-mm plastic slides (Permonox, Nalge Nunc; Rochester, NY) coated with attachment factor (Cell Systems). CMECs on slides were used for flow experiments as confluent monolayers 7–8 days after being plated. For estradiol treatments, CMECs were cultured in growth medium containing 1 nM E2 7–8 days before the flow experiments. CMECs were re-fed with E2-containing growth medium every 2 days. Media used in flow experiments testing the effects of E2 also contained 1 nM E2. Because serum contains estradiol, we evaluated the background estradiol level in our growth medium and found it to be 6.29 ± 0.46 pg/ml or 0.022 ± 0.0016 nM (n = 3), a value comparable to the level of estradiol that we have previously observed in ovariectomized rats (9.3 ± 1.9 pg/ml). Thus the background level is small relative to the 1 nM concentration used in the estradiol treatment in the experiments.

Flow experiments. CMECs on Permonox slides were subjected to a steady shear stress of 19 dyn/cm2 in a standard parallel-plate flow chamber as described previously (43). Briefly, the flow chamber was connected via tubing (Masterflex PharMed, Cole-Parmer Instruments; Vernon Hills, IL) to a recirculating flow loop. Cell culture medium was drawn into the loop from a feed reservoir by using a peristaltic flow pump (Cole-Parmer). The fluid subsequently passed through two buffer reservoirs to dampen pulsatility before entering the flow chamber. Flow exiting the chamber was recirculated back into the feed reservoir. Media in the flow loop were maintained at 37°C and 5% CO2-95% air. Cells not subjected to flow but otherwise handled similarly to those in the flow experiments served as controls. To investigate the effect of a sudden reduction in flow, cells were subjected to a steady shear stress of 19 dyn/cm2 for 24 h; the shear stress was then abruptly reduced to 4 dyn/cm2 for periods of 1, 2, or 3 h.

Western blotting. At the end of the flow exposure treatment period, slides were taken out of the flow chamber, were immediately rinsed twice with ice-cold PBS containing 5 mM EDTA (PBS-EDTA) plus protease inhibitors (Roche Diagnostic complete protease inhibitor cocktail tablet, containing chymotrypsin, thermolysin, papain, pronase, pancreatic extract, and trypsin) as described previously (8, 31, 50), and then were lysed in PBS-EDTA containing 1% SDS plus protease inhibitors. The protein concentration of each sample was determined by using the bicinchoninic acid method to ensure that there was equal loading of samples onto gel lanes. Lysates and prestained molecular markers (Bio-Rad, Hercules, CA) were denatured in SDS-reducing buffer containing dithiothreitol (Invitrogen NuPage, Carlsbad, CA), heated to 70°C for 10 min, and then loaded onto precast 7.5% Tris-glycine gels (PAGEr Gold Precast, Cambrex; Rockland, ME). Samples were electrophoretically separated (Bio-Rad Mini-Protean II) and transferred to polyvinylidene fluoride membranes (Immobilon P, Millipore; Billerica, MA) by using a Bio-Rad Trans-Blot apparatus. The membranes were blocked in PBS-milk (7.5% nonfat dry milk and 0.1% Tween 20 in PBS, pH 7.4) for 2–8 h at 4°C or 1 h at room temperature and were then incubated with either T4 monoclonal antibody (1:2,500 dilution in PBS-milk; University of Iowa Developmental Studies Hybridoma Bank, Iowa City, IA), which recognizes NKCC protein or 4E9 monoclonal antibody (1:3,000 dilution; Chemicon, Temecula, CA) which recognizes NHE isoform-1 protein (NHE1). The blots were rocked for 1 h at room temperature, washed 3 times with PBS-milk, and then incubated for 1 h with secondary antibody (horseradish peroxidase-conjugated goat anti-mouse IgG; Zymed, South San Francisco, CA). Bound secondary antibody was visualized by the enhanced chemiluminescence assay (Amersham Biosciences, Little Chalfont, UK) and a Fuji Film LAS-3000 Imaging System (Medford, UK). ImageQuant software (Molecular Dynamics, Sunnyvale, CA) was used to quantitate band intensity.

In quantifying NKCC and NHE protein levels, we opted not to use an internal loading control because of the larger question of whether or not an appropriate internal control exists within the context of shear studies. Shear stress has a profound effect on the expression and three-dimensional organization of the endothelial cell cytoskeleton. Therefore, the use of β-actin as a control is not without potential problems. GAPDH may pose fewer issues in this regard; however, whether GAPDH is a truly appropriate control for the conditions of these studies remains to be definitively established. Rather than relying on putative internal controls, we have opted to run all Western blot experiments, as well as all conditions within each experiment, in multiple replicates. We have shown in previous work (8) that this approach provides a high degree of confidence that loading errors contribute minimally to the results.

Materials. DMEM and L-glutamine were purchased from GIBCO-BRL (Grand Island, NY). Gentamicin was obtained from AG Scientific (San Diego, CA). E2 and horse serum were purchased from Sigma (St. Louis, MO). Calf serum was from HyClone (Logan, UT). T4 monoclonal antibody was obtained from the University of Iowa Developmental Studies Hybridoma Bank, and 4E9 antibody was obtained from Chemicon. Secondary antibodies were from Zymed. Unless otherwise stated, all other reagents were purchased from Invitrogen (Carlsbad, CA).

Statistical analysis. All experiments were repeated at least three times with at least two replicates for each experiment. Statistical analyses were performed by ANOVA followed by Fisher multiple-comparison or Dunnett posthoc tests using StatView software (SAS Institute, Cary, NC). Unless otherwise specified, data are presented as means ± SE, with n corresponding to the number of separate experiments. Differences in means were considered significant if P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
NKCC protein in cerebral CMECs is flow sensitive. As an initial approach to examine the sensitivity of CMECs to shear stress, we first evaluated the morphology of cells exposed to a steady shear stress of 19 dyn/cm2 in our flow chamber for 6, 12, or 24 h as described in MATERIALS AND METHODS. As shown in Fig. 1, CMECs exhibited morphological remodeling in response to shear stress compared with control cells maintained under static (i.e., no flow) conditions. As early as 6 h and continuing through at least 24 h of continuous shear stress, the cells progressively elongated, aligning in the direction of flow.


Figure 1
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  		Fig. 1. Effect of shear stress on cerebral microvascular endothelial cell (CMEC) shape. CMECs were exposed to either no flow (static control; A) or to a steady shear stress of 19 dyn/cm2 for 6, 12, or 24 h (B, C, and D, respectively) in a parallel-plate flow chamber. Note the progressive cell elongation occurring with exposure to 6, 12, and 24 h of flow.

 
To test our hypothesis that the BBB endothelial cell NKCC, which is stimulated by ischemic factors to participate in edema formation during stroke, is also influenced by changes in shear stress, we first evaluated the effect of shear stress on CMEC NKCC abundance. For this, CMECs (cultured under normal static conditions) were placed in a flow chamber and exposed to a steady shear stress of 19 dyn/cm2 for 6, 12, or 24 h, and NKCC abundance was subsequently determined by Western blot analysis. Figure 2 shows that exposing CMECs to 12 or 24 h of shear stress markedly increased the abundance of NKCC in the cells, with NKCC increasing approximately twofold after 12 h and nearly threefold after 24 h relative to control cells maintained under static conditions. No significant increase relative to the control was observed after 6 h of flow. Note that in the representative Western blots shown in Fig. 2A, single bands of ~170 kDa are observed in the static controls, while two bands at ~170 and ~135 kDa are seen for CMECs exposed to shear stress, consistent with the presence of both glycosylated and the less mature unglycosylated form of NKCC, respectively (33, 42). Because we have found previously that estradiol, which reduces edema and infarct in ischemic stroke, also reduces the abundance of NKCC in CMECs (21), we also evaluated the effect of estradiol on NKCC abundance in CMECs exposed to shear stress. For this, CMECs were pretreated with E2 (1 nM) for 7 days and were then subjected to 6, 12, or 24 h of shear stress, or to no flow (static control). As shown in Fig. 2, we found that the response of CMECs to shear stress with respect to NKCC abundance was not altered by estradiol, i.e., it was increased by flow whether or not the cells had been exposed to estradiol. However, in control cells maintained under static conditions (i.e., no flow), estradiol (1 nM, 7 days) significantly reduced NKCC abundance to 81.45 ± 0.05% of control without estradiol (n = 4 experiments; data not shown), in agreement with what we have reported previously (21). Note that the antibody used for our Western blots can detect both NKCC1 and NKCC2 isoforms of the Na-K-Cl cotransporter. However, it has been established that NKCC2 expression is restricted to kidney, and thus the NKCC isoform that we refer to in the present study is NKCC1.


Figure 2
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  		Fig. 2. Abundance of CMEC Na-K-Cl cotransporter (NKCC) protein after exposure to shear stress and 17β-estradiol (E2). A: representative Western blots of NKCC in CMECs subjected to 6, 12, or 24 h of either no flow (static control) or steady shear stress (19 dyn/cm2) following treatment with or without E2 (1 nM, 7 days). B: summary of NKCC abundance as determined by ImageQuant software analysis of Western blots. NKCC abundance values for CMECs exposed to 6, 12, or 24 h of shear stress are expressed relative to their corresponding static controls (6, 12, or 24 h of no flow). The vehicle control (0.01% ethanol) had no effect on NKCC expression in CMECs exposed to shear stress (data not shown). Values shown are means ± SE of 3, 3, and 4 experiments for cells exposed to 6, 12, and 24 h of shear stress, respectively, without E2 treatment and 4, 4, and 5 experiments for cells exposed 6, 12, and 24 h of shear stress, respectively, with E2 treatment. *Values significantly different from respective controls (P < 0.02). #Values significantly different from respective 6-h values (P < 0.004).

 
NHE protein in CMECs is flow sensitive. Because we have evidence that the BBB endothelial cell NHE also appears to be stimulated by ischemic factors such as hypoxia, aglycemia, AVP, and endothelin to participate in edema formation during stroke, we conducted studies to determine whether NHE is also influenced by changes in shear stress. Thus we exposed CMECs to a steady shear stress of 19 dyn/cm2 in our flow chamber for 6, 12, or 24 h. We subsequently determined NHE abundance by Western blot analysis. At least nine NHE isoform proteins have been identified to date, and the isoforms present in CMECs are currently under investigation. However, we have recently demonstrated that NHE1 and possibly NHE2 isoforms are present in CMECs. Because NHE1 is the ubiquitous form of NHE, we began in these studies by determining the abundance of NHE1. As shown in Fig. 3, we found that shear stress caused an increase of ~1.5-fold in NHE1 abundance after 12 h. Note that the band detected at ~110 kDa is consistent with NHE1. No significant change in abundance was found after 6 h of shear stress. Unlike the observed sustained increase in NKCC, we found that NHE1 abundance in CMECs exposed to shear stress returned to control static levels by 24 h. We also evaluated the effect of estradiol on NHE1 abundance in CMECs exposed to shear stress. CMECs were pretreated with E2 (1 nM, 7 days) and then subjected to 6, 12, or 24 h of shear stress, or to no flow (static control). As shown in Fig. 3, we found that estradiol abolished the transient increase in CMEC NHE1 abundance, i.e., NHE1 levels were not significantly different from control static levels after 6, 12, or 24 h of shear stress.


Figure 3
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  		Fig. 3. Abundance of CMEC Na/H exchanger (NHE) isoform-1 protein (NHE1) after exposure to shear stress and E2. A: representative Western blots of NHE1 in CMECs subjected to 6, 12, or 24 h of either no flow (static control) or steady shear stress (19 dyn/cm2) following treatment with or without E2 (1 nM, 7 days). B: summary of NHE1 abundance as determined by ImageQuant software analysis of Western blots. NHE1 abundance values for CMECs exposed to 6, 12, or 24 h of shear stress are expressed relative to their corresponding static controls (6, 12 or 24 h of no flow). The vehicle control (0.01% ethanol) had no effect on NHE1 expression (data not shown). Values shown are means ± SE of 3 experiments each for cells exposed to 6, 12, and 24 h of shear stress without E2 treatment and 3, 3, and 5 experiments for cells exposed 6, 12, and 24 h of shear stress, respectively, with E2 treatment. *Values significantly different from respective controls (P < 0.02). #Values significantly different from 12-h values in cells treated with E2 (P < 0.03).

 
Effect of flow reduction on NKCC and NHE1 protein. To evaluate the possibility that an abrupt reduction in cerebral blood flow, as occurs in ischemic stroke, has an effect on BBB NKCC and/or NHE, we evaluated NKCC1 and NHE1 protein abundance in CMECs following reduction of shear stress. The typical flow reduction in ischemic stroke is on the order of ~80% (13). Thus we tested the effect of an abrupt 80% flow reduction on NKCC1 and NHE1 protein levels in CMECs that had been preconditioned with physiological levels of shear stress. Because clinical studies have determined that restoration of blood flow within a period of ~3 h is essential for avoiding permanent cerebral damage (13), we examined the effect of flow reduction over periods of 1, 2, and 3 h. For these studies, CMECs were "presheared" at a steady shear stress of 19 dyn/cm2 for 24 h, then the flow rate abruptly decreased to 4 dyn/cm2 for 1, 2, or 3 h. Fig. 4 shows that in the absence of estradiol, the sudden reduction in flow was without significant effect on NKCC abundance. It should be noted that the data in Fig. 4 are normalized to the static controls and that the 0-h shear stress values reflect NKCC increased expression due to the 24 h preshearing period. In CMECs exposed to estradiol (1 nM, 7 days) and presheared for 24 h at 19 dyn/cm2, abrupt reduction of shear stress caused a significant decrease in NKCC abundance after 1 and 2 h, with subsequent return to the 0-h baseline by 3 h.


Figure 4
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  		Fig. 4. Effect of an abrupt reduction in shear stress on CMEC NKCC protein abundance. A: representative Western blots of NKCC in CMECs subjected to a steady shear stress of 19 dyn/cm2 for 24 h, followed by an abrupt reduction in shear stress to 4 dyn/cm2 for 1, 2, or 3 h. Corresponding static controls (no flow) are also shown. In these experiments, the effects of flow reduction were evaluated in CMECs both without (top) and with (bottom) E2 treatment (1 nM, 7 days). B: summary of NKCC abundance in CMECs following abrupt shear stress reduction (with or without 7-day E2 treatment). NKCC abundance values are expressed relative to corresponding static controls. Values shown are means ± SE of 3, 8, 6, and 4 experiments for 0, 1, 2, and 3 h of reduced flow, respectively, for cells without E2 treatment and 4, 5, 3, and 3 experiments for 0, 1, 2, and 3 h of reduced flow, respectively, for E2-treated cells. *Value for E2-treated cells significantly different from value for untreated cells (P < 0.05). #Value significantly different from value for 0 h reduced flow in E2-treated cells (P < 0.01).

 
We also evaluated the effects of an abrupt reduction in flow on CMEC NHE1 abundance. Figure 5 shows that NHE1 abundance was not significantly altered by abrupt reduction of shear stress (from 19 dyn/cm2 to 4 dyn/cm2) whether or not the CMECs were treated with estradiol. Note that the 0 h of reduced shear stress indicates NHE1 abundance at the end of the 24-h exposure to 19 dyn/cm2 shear stress, which is not significantly different from static control NHE1 abundance values.


Figure 5
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  		Fig. 5. Effect of an abrupt reduction in shear stress on CMEC NHE1 protein abundance. A: representative Western blots of NHE1 in CMECs subjected to a steady shear stress of 19 dyn/cm2 for 24 h, followed by an abrupt reduction in shear stress to 4 dyn/cm2 for 1, 2, or 3 h. Corresponding static controls (no flow) are also shown. In these experiments, the effects of flow reduction were evaluated in CMECs both without (top) and with (bottom) E2 treatment (1 nM, 7 days). B: summary of NHE1 abundance in CMECs following abrupt shear stress reduction (with or without 7-day E2 treatment). NHE1 abundance values are expressed relative to corresponding static controls. Values shown are means ± SE of 3, 4, 3, and 4 experiments for 0, 1, 2, and 3 h of reduced flow, respectively, for cells without E2 treatment and 5, 4, 3, and 3 experiments for 0, 1, 2, and 3 h of reduced flow, respectively, for E2-treated cells.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we evaluated the possibility that expression levels of BBB NKCC and NHE proteins, both of which appear to participate in ischemia-induced edema formation, are influenced by changes in shear stress, a prominent factor in cerebral ischemia. We also investigated whether estradiol, known to attenuate ischemia-induced cerebral edema and infarct, may also modulate NKCC and NHE protein levels in CMECs. We demonstrate here that NKCC and NHE protein expression is indeed sensitive to both shear stress and estradiol but that the responses to these two stimuli, either alone or in combination, differ between the two Na transporters.

CMECs in healthy brain are continually exposed to shear stress, whereas during cerebral ischemia they experience an abrupt reduction of shear stress. Given the evidence that ischemia increases Na transport across the BBB via processes involving NKCC and NHE, it is possible that abrupt reduction of shear stress has an effect on the activity and/or abundance of these BBB Na transporters. Thus in these studies we evaluated the response of CMECs to changes in fluid mechanical shear stress. Our finding that CMECs change morphology, aligning with the direction of flow within hours of changing from no flow to shear stress, is one indication that BBB endothelial cells are indeed sensitive to flow. In this regard, the CMECs respond to shear stress in a manner similar to aortic endothelial cells (43).

Our studies also reveal that CMEC expression of NKCC protein is notably sensitive to shear stress, with approximately twofold and threefold increases in NKCC abundance observed after 12 and 24 h, respectively, of imposing a steady shear stress of 19 dyn/cm2 on the cells. This response is largely similar to what we have observed previously for aortic endothelial cells (43). Because CMECs in vivo are constantly exposed to shear stress, NKCC protein levels at the end of the 24-h flow period may provide a better approximation of normal physiological NKCC levels than the static culture (no-flow) baseline values. Indeed, we view the increased NKCC abundance induced by chronically subjecting the cells to physiological levels of shear stress as suggestive that the cotransporter is important for normal BBB phenotype.

We have also found that while the imposition of steady shear stress on CMECs increases NKCC protein, an abrupt reduction in flow (to 4 dyn/cm2) is without significant effect on NKCC abundance. Thus it does not appear that the flow reduction occurring in cerebral ischemia contributes to BBB-mediated edema formation through increased expression of NKCC protein. The significance of the shear stress-induced increase in NKCC protein expression that occurs when CMECs are switched from no-flow conditions to steady shear stress is as yet unclear. However, this observation is consistent with previous findings made in aortic endothelial cells that the cotransporter is one of several proteins upregulated by shear stress and may be an important component of the endothelial phenotype under physiological flow conditions (43, 44).

Estradiol also influences expression of NKCC protein in CMECs, although the response varies depending on whether the cells are under static conditions, steady shear stress, or reduced flow conditions. As we have observed in a previous study, exposing CMECs to estradiol (1 nM) for 7 days significantly reduces NKCC abundance in cells maintained under static conditions. However, this effect is overridden by the shear stress-induced increase in NKCC, i.e., NKCC abundance is increased in CMECs exposed to shear stress regardless of whether or not the cells are treated with estradiol. While estradiol is without effect in CMECs subjected to shear stress, it appears to cause reduction of NKCC abundance in CMECs exposed to an abrupt reduction in flow. Our previous studies have shown that treating CMECs with estradiol for 1 to 3 h under static conditions causes a 50–60% decrease in NKCC abundance (21). This is consistent with the possibility that estradiol acts to reduce NKCC abundance in CMECs and that the effect is lost in cells exposed to steady shear stress, at least during the first 24 h of shear. Our findings suggest that the edema-reducing effects of estradiol do not include a decrease in NKCC protein abundance in the presence of steady shear stress.

Additional studies are needed to further clarify the CMEC response to estradiol in the presence of shear stress, including an evaluation of CMEC NKCC activity under these conditions. In our previous study on aortic endothelial cells (43), we had demonstrated that both NKCC protein levels and activity, as assessed by Western blot analysis and bumetanide-sensitive K influx, respectively, were increased in response to shear stress, although the magnitude of the increase in activity did not exactly match the increase in protein expression. More specifically, we observed that NKCC protein expression was increased by 2.3-fold after either 6 or 24 h of shear stress and that cotransporter activity was increased by 1.5-fold and 1.8-fold after 6 and 24 h of shear stress, respectively. In another recent study (30), we have shown that estradiol-induced decreases in NKCC protein expression are accompanied with similar decreases in cotransporter activity. In light of these previous findings, we would predict that the changes in NKCC protein levels documented here would be accompanied by changes in cotransporter activity.

Examination of the CMEC response to shear stress with respect to NHE1 protein levels revealed that NHE1 abundance is also increased in CMECs within hours of exposure to steady shear stress (19 dyn/cm2). However, in this case, the increase in NHE1 protein is more modest and occurs only after 12 h exposure, with NHE1 abundance returning to static control levels by 24 h. The significance of this shear stress-induced transient increase in NHE1 protein levels in CMECs is as yet unclear, and future experiments will need to evaluate the effect of imposing shear stress on NHE1 activity of the cells. Like NKCC, CMEC NHE1 protein levels are not altered when the cells are subjected to an abrupt reduction of flow (4 dyn/cm2 following 24 h of 19 dyn/cm2). Further studies will be needed to clarify whether shear stress, either physiological levels of shear or abrupt reduction of shear, alter NHE1 activity in CMECs. It should be noted that our recent studies indicate the presence of the NHE2 isoform in CMECs, and thus it is possible that shear stress may modulate expression of NHE2 as well as NHE1.

The only effect of estradiol on NHE1 protein in CMECs was observed in cells exposed to steady shear stress. Estradiol (1 nM, 7-day pretreatment) abolished the transient increase in NHE1 abundance occurring at 12 h after the start of shear stress. Estradiol did not alter NHE1 abundance under static conditions or during abrupt reduction of flow. This suggests that estradiol attenuation of edema formation in ischemia does not involve reduction of BBB endothelial cell NHE1 protein abundance. Whether estradiol inhibition of the shear stress-induced transient increase in NHE1 protein is involved in estradiol edema reducing effects will need to be addressed by future studies evaluating estradiol effects on NHE1 activity in CMECs.

The mechanisms by which shear stress regulates NKCC expression in CMECs remain to be determined. In bovine aortic endothelial cells, changes in NKCC protein abundance occurring in response to shear stress appear to be modulated by flow-sensitive K+ and Cl– channels (43). It remains to be established whether or not the same occurs in CMECs. Previous studies have also shown that under static conditions, NKCC activity of both aortic endothelial cells and CMECs is regulated by changes in cell volume (28, 29). Therefore, whether the shear stress effects on CMEC NKCC activity involve changes in CMEC volume merits investigation. In fact, the effects of flow-sensitive ion channels and those of changes in cell volume on CMEC NKCC may not be entirely decoupled as it has been suggested that flow-activated Cl– channels may be identical to volume-regulated Cl– channels (27). Flow-induced cytoskeletal reorganization may also play a role in regulating CMEC NKCC expression. Bovine aortic endothelial cells exposed to either oscillatory or turbulent flow, both of which fail to induce cytoskeletal remodeling (which occurs in steady flow), undergo no changes in NKCC expression (43, 44). Therefore, a possible link between cytoskeletal changes and alterations in NKCC protein levels merits investigation.

With respect to possible mechanisms underlying the shear stress-induced transient increase in CMEC NHE1 protein expression, previous studies have shown that a steady shear stress of 13.4 dyn/cm2 elicits a decrease in intracellular pH in bovine aortic endothelial cells (53). Thus it is possible that such a decrease in intracellular pH could be involved in stimulating the shear stress-induced transient increase in CMEC NHE1 expression. In light of data demonstrating NHE1 activation following cell shrinkage (7), the possibility that the shear stress-induced transient upregulation of NHE1 may also involve changes in CMEC volume should also be considered. More recently, NHE1 has been found to play a role in cytoskeletal reorganization (3, 4, 10, 40). Therefore, it is also possible that shear stress-induced cytoskeletal remodeling and altered NHE1 expression in CMECs may be linked.

Determining the signaling pathways by which estradiol alters NKCC and NHE1 expression in CMECs should be the subject of future investigations. While increases in NKCC and NHE1 that occur after many hours of shear stress in the presence of estradiol are most likely the result of increased protein synthesis, the estradiol-induced decrease in NKCC abundance observed under static conditions or during 1–2 h of reduced flow may be caused by increased degradation and/or reduced synthesis. Our previous studies have shown that the estrogen receptor antagonist ICI-182,780 abolishes the downregulation of NKCC protein induced by physiological levels of estradiol (1 nM) in CMECs within 3 h of exposure (21), which suggests that this occurs via an estrogen receptor-mediated pathway. Estradiol binding to membrane estrogen receptors in endothelial cells appears to activate several signaling pathways including Akt kinase and MAP kinase pathways (9, 11, 38, 39) as well as cyclic AMP/protein kinase A (16, 24, 48) and cyclic GMP/nitric oxide pathways (9, 11). Whether any or all of these pathways are involved in the estradiol effects observed under conditions of either no flow or abrupt flow reduction remains to be determined.

Both NKCC and NHE1 have been reported to play a role in regulating the cell cycle in various systems (34, 36). Exposing endothelial cells to shear stress suppresses DNA synthesis, and shear stress removal restores baseline cell proliferation levels (1, 5). Therefore, a common element in the parameters examined in the present study is a possible effect on CMEC cycle. This issue certainly merits future investigation. We did not study cell cycle parameters in the present work because the studies were intended as an initial investigation of the possibility that shear stress reduction and estradiol modulate NKCC and NHE abundance in cerebral endothelial cells. Future studies should address whether the decreased NKCC expression observed in response to a sudden reduction in flow in the presence but not in the absence of estradiol is attributable at least partially to estradiol actions on cell cycle parameters.

Stroke is a complex and multicomponent disease that involves many other factors in addition to flow reduction. Such factors include hypoxia, aglycemia, vasopressin, endothelin, inflammation, and pH changes. In the present work, our goal was to determine whether flow reduction, by itself, has an effect on NKCC and NHE expression in BBB microvascular endothelial cells and whether this effect is modulated by estradiol. We focused on NKCC and NHE because of previous evidence that these two transporters participate in edema formation and subsequent infarct observed in ischemic stroke. It is naturally possible, if not probable, that the various factors present during ischemic stroke interact so that separating the effects of shear reduction from other effects may be difficult. Indeed, a recent study using an in vitro BBB model reported that while flow cessation and reperfusion had only a modest impact on cytokine release and BBB permeability, the inclusion of leukocytes into the model significantly increased both parameters (18). Therefore, once the effects of flow reduction on ion transporters are established, subsequent studies should investigate how these transporters respond to changes in shear when other ischemic factors are also present.

In our flow experiments, we used a parallel-plate flow chamber similar to those used in numerous previous studies. Cells in parallel-plate flow chambers are exposed to both pressure and shear forces. Although we cannot exclude the possibility that pressure forces contributed to the observed response, we believe this is unlikely because the pressure drop across the flow chamber is rather small. For fully developed, steady, laminar flow of an incompressible fluid between two parallel plates, the pressure drop can be approximated by {Delta}p~3µQL/2h3W, where {Delta}p is the pressure drop across the length of the flow chamber, µ is the dynamic viscosity of the fluid, Q is the volumetric flow rate, L is the length of the flow chamber, h is the interplate spacing, and W is the chamber width. For the flow chamber and flow conditions we used, the pressure drop is ~200 Pa (~0.002 atm). Assuming the pressure level at the chamber outlet is atmospheric, the maximum pressure in the chamber would be at the chamber inlet and would be equivalent to exposing the cells to a hydrostatic head of 2 cmH2O. A survey of the literature on the effects of hydrostatic pressure on endothelial cells reveals that most of the previous studies that have reported pressure-induced biological responses were conducted at pressure levels of 20–150 mmHg (~25–200 cmH2O), considerably larger than the levels present here. There are a few studies that have reported endothelial responses, including intracellular calcium mobilization and altered integrin expression, in response to pressure levels as low as 4–5 cmH2O (19, 37); however, even these low levels are two to three times the maximal levels estimated to be present in the current work. Until the effects of pressure on NKCC and NHE protein are directly measured, however, one cannot completely exclude the possibility that pressure may have contributed to the observed response.

In summary, our studies demonstrate that NKCC abundance in BBB endothelial cells is increased by steady shear stress but is not affected by an abrupt reduction in shear stress as occurs in cerebral ischemia. Thus it appears that ischemia-induced elevation of BBB cotransporter activity and cerebral edema formation does not involve a flow reduction-induced increase in BBB NKCC abundance. Our studies also demonstrate that estradiol reduces expression of NKCC protein in BBB endothelial cells in the absence of shear stress or under reduced flow conditions but not in the presence of physiological levels of steady shear stress. This is consistent with the hypothesis that estradiol reduces BBB NKCC activity and edema formation during ischemia at least in part by reducing expression of NKCC protein in the endothelial cells. Finally, while NHE1 protein expression is transiently increased by shear stress in a manner that is blocked by estradiol, it is not affected by reduced flow, which suggests that ischemia-induced elevation of BBB NHE activity and cerebral edema formation also does not involve a flow reduction-induced increase in BBB NHE1 abundance.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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This work was supported in part by a National Institutes of Health grant (NINDS NS039953) and was conducted in part in a facility constructed with support from Research Facilities Improvement Program Grant C06 RR17348-01 from the National Center for Research Resources, National Institutes of Health.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. I. Barakat, Dept. of Mechanical and Aeronautical Engineering, Univ. of California, Davis, One Shields Ave., Davis, CA 95616 (e-mail: abarakat{at}ucdavis.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.


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T. I. Lam, P. M. Wise, and M. E. O'Donnell
Cerebral microvascular endothelial cell Na/H exchange: evidence for the presence of NHE1 and NHE2 isoforms and regulation by arginine vasopressin
Am J Physiol Cell Physiol, August 1, 2009; 297(2): C278 - C289.
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