High glucose-induced effects on Na+-K+-2Cl- cotransport and Na+/H+ exchange of blood-brain barrier endothelial cells: involvement of SGK1, PKCßII, and SPAK/OSR1.

Hyperglycemia exacerbates edema formation and worsens neurological outcome in ischemic stroke. Edema formation in the early hours of stroke involves transport of ions and water across an intact blood-brain barrier (BBB), and swelling of astrocytes. We showed previously that high glucose (HG) exposures of 24 hours to 7 days increase abundance and activity of BBB Na+-K+-2Cl- cotransport (NKCC) and Na+/H+ exchange 1 (NHE1). Further, bumetanide and HOE-642 inhibition of these transporters significantly reduces edema and infarct following middle cerebral artery occlusion in hyperglycemic rats, suggesting that NKCC and NHE1 are effective therapeutic targets for reducing edema in hyperglycemic stroke. The mechanisms underlying hyperglycemia effects on BBB NKCC and NHE1 are not known. In the present study we investigated whether serum-glucocorticoid regulated kinase 1 (SGK1) and protein kinase C beta II (PKCßII) are involved in HG effects on BBB NKCC and NHE1. We found transient increases in phosphorylated SGK1 and PKCßII within the first hour of HG exposure, after 5-60 min for SGK1 and 5 min for PKCßII. However, no changes were observed in cerebral microvascular endothelial cell SGK1 or PKCßII abundance or phosphorylation (activity) after 24 or 48 h HG exposures. Further, we found that HG-induced increases in NKCC and NHE1 abundance were abolished by inhibition of SGK1 but not PKCßII, whereas the increases in NKCC and NHE activity were abolished by inhibition of either kinase. Finally, we found evidence that STE20/SPS1-related proline/alanine-rich kinase and oxidative stress-responsive kinase-1 (SPAK/OSR1) participate in the HG-induced effects on BBB NKCC.

Exacerbated brain edema in a rat streptozotocin model of hyperglycemic ischemic stroke: Evidence for involvement of blood-brain barrier Na-K-Cl cotransport and Na/H exchange.

Cerebral edema is exacerbated in diabetic ischemic stroke through poorly understood mechanisms. We showed previously that blood-brain barrier (BBB) Na-K-Cl cotransport (NKCC) and Na/H exchange (NHE) are major contributors to edema formation in normoglycemic ischemic stroke. Here, we investigated whether hyperglycemia-exacerbated edema involves changes in BBB NKCC and NHE expression and/or activity and whether inhibition of NKCC or NHE effectively reduces edema and injury in a type I diabetic model of hyperglycemic stroke. Cerebral microvascular endothelial cell (CMEC) NKCC and NHE abundances and activities were determined by Western blot, radioisotopic flux and microspectrofluorometric methods. Cerebral edema and Na in rats subjected to middle cerebral artery occlusion (MCAO) were assessed by nuclear magnetic resonance methods. Hyperglycemia exposures of 1-7d significantly increased CMEC NKCC and NHE abundance and activity. Subsequent exposure to ischemic factors caused more robust increases in NKCC and NHE activities than in normoglycemic CMEC. MCAO-induced edema and brain Na uptake were greater in hyperglycemic rats. Intravenous bumetanide and HOE-642 significantly attenuated edema, brain Na uptake and ischemic injury. Our findings provide evidence that BBB NKCC and NHE contribute to increased edema in hyperglycemic stroke, suggesting that these Na transporters are promising therapeutic targets for reducing damage in diabetic stroke.

Blood-brain barrier KCa3.1 channels: evidence for a role in brain Na uptake and edema in ischemic stroke.

BACKGROUND AND PURPOSE: KCa3.1, a calcium-activated potassium channel, regulates ion and fluid secretion in the lung and gastrointestinal tract. It is also expressed on vascular endothelium where it participates in blood pressure regulation. However, the expression and physiological role of KCa3.1 in blood-brain barrier (BBB) endothelium has not been investigated. BBB endothelial cells transport Na(+) and Cl(-) from the blood into the brain transcellularly through the co-operation of multiple cotransporters, exchangers, pumps, and channels. In the early stages of cerebral ischemia, when the BBB is intact, edema formation occurs by processes involving increased BBB transcellular Na(+) transport. This study evaluated whether KCa3.1 is expressed on and participates in BBB ion transport. METHODS: The expression of KCa3.1 on cultured cerebral microvascular endothelial cells, isolated microvessels, and brain sections was evaluated by Western blot and immunohistochemistry. Activity of KCa3.1 on cerebral microvascular endothelial cells was examined by K(+) flux assays and patch-clamp. Magnetic resonance spectroscopy and MRI were used to measure brain Na(+) uptake and edema formation in rats with focal ischemic stroke after TRAM-34 treatment. RESULTS: KCa3.1 current and channel protein were identified on bovine cerebral microvascular endothelial cells and freshly isolated rat microvessels. In situ KCa3.1 expression on BBB endothelium was confirmed in rat and human brain sections. TRAM-34 treatment significantly reduced Na(+) uptake, and cytotoxic edema in the ischemic brain. CONCLUSIONS: BBB endothelial cells exhibit KCa3.1 protein and activity and pharmacological blockade of KCa3.1 seems to provide an effective therapeutic approach for reducing cerebral edema formation in the first 3 hours of ischemic stroke.

Blood-brain barrier Na transporters in ischemic stroke.

Blood-brain barrier (BBB) endothelial cells form a barrier that is highly restrictive to passage of solutes between blood and brain. Many BBB transport mechanisms have been described that mediate transcellular movement of solutes across the barrier either into or out of the brain. One class of BBB transporters that is all too often overlooked is that of the ion transporters. The BBB has a rich array of ion transporters and channels that carry Na, K, Cl, HCO3, Ca, and other ions. Many of these are asymmetrically distributed between the luminal and abluminal membranes, giving BBB endothelial cells the ability to perform vectorial transport of ions across the barrier between blood and brain. In this manner, the BBB performs the important function of regulating the volume and composition of brain interstitial fluid. Through functional coupling of luminal and abluminal transporters and channels, the BBB carries Na, Cl, and other ions from blood into brain, producing up to 30% of brain interstitial fluid in healthy brain. During ischemic stroke cerebral edema forms by processes involving increased activity of BBB luminal Na transporters, resulting in "hypersecretion" of Na, Cl, and water into the brain interstitium. This review discusses the roles of luminal BBB Na transporters in edema formation in stroke, with an emphasis on Na-K-Cl cotransport and Na/H exchange. Evidence that these transporters provide effective therapeutic targets for reduction of edema in stroke is also discussed, as are recent findings regarding signaling pathways responsible for ischemia stimulation of the BBB Na transporters.

Ischemic factor-induced increases in cerebral microvascular endothelial cell Na/H exchange activity and abundance: evidence for involvement of ERK1/2 MAP kinase.

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.

Intravenous HOE-642 reduces brain edema and Na uptake in the rat permanent middle cerebral artery occlusion model of stroke: evidence for participation of the blood-brain barrier Na/H exchanger.

Cerebral edema forms in the early hours of ischemic stroke by processes involving increased transport of Na and Cl from blood into brain across an intact blood-brain barrier (BBB). Our previous studies provided evidence that the BBB Na-K-Cl cotransporter is stimulated by the ischemic factors hypoxia, aglycemia, and arginine vasopressin (AVP), and that inhibition of the cotransporter by intravenous bumetanide greatly reduces edema and infarct in rats subjected to permanent middle cerebral artery occlusion (pMCAO). More recently, we showed that BBB Na/H exchanger activity is also stimulated by hypoxia, aglycemia, and AVP. The present study was conducted to further investigate the possibility that a BBB Na/H exchanger also participates in edema formation during ischemic stroke. Sprague-Dawley rats were subjected to pMCAO and then brain edema and Na content assessed by magnetic resonance imaging diffusion-weighed imaging and magnetic resonance spectroscopy Na spectroscopy, respectively, for up to 210 minutes. We found that intravenous administration of the specific Na/H exchange inhibitor HOE-642 significantly decreased brain Na uptake and reduced cerebral edema, brain swelling, and infarct volume. These findings support the hypothesis that edema formation and brain Na uptake during the early hours of cerebral ischemia involve BBB Na/H exchanger activity as well as Na-K-Cl cotransporter activity.

Ischemia-induced stimulation of cerebral microvascular endothelial cell Na-K-Cl cotransport involves p38 and JNK MAP kinases.

Previous studies have provided evidence that, in the early hours of ischemic stroke, a luminal membrane blood-brain barrier (BBB) Na-K-Cl cotransporter (NKCC) participates in ischemia-induced cerebral edema formation. Inhibition of BBB NKCC activity by intravenous bumetanide significantly reduces edema and infarct in the rat permanent middle cerebral artery occlusion model of ischemic stroke. We demonstrated previously that the BBB cotransporter is stimulated by hypoxia, aglycemia, and AVP, factors present during cerebral ischemia. However, the underlying mechanisms have not been known. Ischemic conditions have been shown to activate p38 and JNK MAP kinases (MAPKs) in brain, and the p38 and JNK inhibitors SB-239063 and SP-600125, respectively, have been found to reduce brain damage following middle cerebral artery occlusion and subarachnoid hemorrhage, respectively. The present study was conducted to determine whether one or both of these MAPKs participates in ischemic factor stimulation of BBB NKCC activity. Cultured cerebral microvascular endothelial cell NKCC activity was evaluated as bumetanide-sensitive (86)Rb influx. Activities of p38 and JNK were assessed by Western blot and immunofluorescence methods using antibodies that detect total vs. phosphorylated (activated) p38 or JNK. We report that p38 and JNK are present in cultured cerebral microvascular endothelial cells and in BBB endothelial cells in situ and that hypoxia (7% O(2) and 2% O(2)), aglycemia, AVP, and O(2)-glucose deprivation (5- to 120-min exposures) all rapidly activate p38 and JNK in the cells. We also provide evidence that SB-239063 and SP-600125 reduce or abolish ischemic factor stimulation of BBB NKCC activity. These findings support the hypothesis that ischemic factor stimulation of the BBB NKCC involves activation of p38 and JNK MAPKs.

The KCa3.1 blocker TRAM-34 reduces infarction and neurological deficit in a rat model of ischemia/reperfusion stroke.

Microglia and brain infiltrating macrophages significantly contribute to the secondary inflammatory damage in the wake of ischemic stroke. Here, we investigated whether inhibition of KCa3.1 (IKCa1/KCNN4), a calcium-activated K(+) channel that is involved in microglia and macrophage activation and expression of which increases on microglia in the infarcted area, has beneficial effects in a rat model of ischemic stroke. Using an HPLC/MS assay, we first confirmed that our small molecule KCa3.1 blocker TRAM-34 effectively penetrates into the brain and achieves micromolar plasma and brain concentrations after intraperitoneal injection. Then, we subjected male Wistar rats to 90 minutes of middle cerebral artery occlusion (MCAO) and administered either vehicle or TRAM-34 (10 or 40 mg/kg intraperitoneally twice daily) for 7 days starting 12 hours after reperfusion. Both compound doses reduced infarct area by ≈ 50% as determined by hematoxylin & eosin staining on day 7 and the higher dose also significantly improved neurological deficit. We further observed a significant reduction in ED1(+)-activated microglia and TUNEL-positive neurons as well as increases in NeuN(+) neurons in the infarcted hemisphere. Our findings suggest that KCa3.1 blockade constitutes an attractive approach for the treatment of ischemic stroke because it is still effective when initiated 12 hours after the insult.

Ischemia-induced stimulation of Na-K-Cl cotransport in cerebral microvascular endothelial cells involves AMP kinase.

Increased blood-brain barrier (BBB) Na-K-Cl cotransporter activity appears to contribute to cerebral edema formation during ischemic stroke. We have shown previously that inhibition of BBB Na-K-Cl cotransporter activity reduces edema and infarct in the rat middle cerebral artery occlusion (MCAO) model of ischemic stroke. We have also shown that the BBB cotransporter is stimulated by the ischemic factors hypoxia, aglycemia, and arginine vasopressin (AVP), although the mechanisms responsible are not well understood. AMP-activated protein kinase (AMPK), a key mediator of cell responses to stress, can be activated by a variety of stresses, including ischemia, hypoxia, and aglycemia. Previous studies have shown that the AMPK inhibitor Compound C significantly reduces infarct in mouse MCAO. The present study was conducted to evaluate the possibility that AMPK participates in ischemic factor-induced stimulation of the BBB Na-K-Cl cotransporter. Cerebral microvascular endothelial cells (CMEC) were assessed for Na-K-Cl cotransporter activity as bumetanide-sensitive (86)Rb influx. AMPK activity was assessed by Western blot analysis and immunofluorescence methods using antibodies that detect total versus phosphorylated (activated) AMPK. We found that hypoxia (7% and 2% O(2)), aglycemia, AVP, and oxygen-glucose deprivation (5- to 120-min exposures) increase activation of AMPK. We also found that Compound C inhibition of AMPK reduces hypoxia-, aglycemia-, and AVP-induced stimulation of CMEC Na-K-Cl cotransporter activity. Confocal immunofluorescence of perfusion-fixed rat brain slices revealed the presence of AMPK, both total and phosphorylated kinase, in BBB in situ of both control and ischemic brain. These findings suggest that ischemic factor stimulation of the BBB Na-K-Cl cotransporter involves activation of AMPK.

Effects of estradiol on ischemic factor-induced astrocyte swelling and AQP4 protein abundance.

In the early hours of ischemic stroke, cerebral edema forms as Na, Cl, and water are secreted across the blood-brain barrier (BBB) and astrocytes swell. We have shown previously that ischemic factors, including hypoxia, aglycemia, and arginine vasopressin (AVP), stimulate BBB Na-K-Cl cotransporter (NKCC) and Na/H exchanger (NHE) activities and that inhibiting NKCC and/or NHE by intravenous bumetanide and/or HOE-642 reduces edema and infarct in a rat model of ischemic stroke. Estradiol also reduces edema and infarct in this model and abolishes ischemic factor stimulation of BBB NKCC and NHE. There is evidence that NKCC and NHE also participate in ischemia-induced swelling of astrocytes. However, little is known about estradiol effects on astrocyte cell volume. In this study, we evaluated the effects of AVP (100 nM), hypoxia (7.5% O(2)), aglycemia, hypoxia (2%)/aglycemia [oxygen glucose deprivation (OGD)], and estradiol (1-100 nM) on astrocyte cell volume using 3-O-methyl-d-[(3)H]glucose equilibration methods. We found that AVP, hypoxia, aglycemia, and OGD (30 min to 5 h) each significantly increased astrocyte cell volume, and that estradiol (30-180 min) abolished swelling induced by AVP or hypoxia, but not by aglycemia or OGD. Bumetanide and/or HOE-642 also abolished swelling induced by AVP but not aglycemia. Abundance of aquaporin-4, known to participate in ischemia-induced astrocyte swelling, was significantly reduced following 7-day but not 2- or 3-h estradiol exposures. Our findings suggest that hypoxia, aglycemia, and AVP each contribute to ischemia-induced astrocyte swelling, and that the edema-attenuating effects of estradiol include reduction of hypoxia- and AVP-induced astrocyte swelling and also reduction of aquaporin-4 abundance.

Engaging neuroscience to advance translational research in brain barrier biology.

The delivery of many potentially therapeutic and diagnostic compounds to specific areas of the brain is restricted by brain barriers, of which the most well known are the blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier. Recent studies have shown numerous additional roles of these barriers, including an involvement in neurodevelopment, in the control of cerebral blood flow, and--when barrier integrity is impaired--in the pathology of many common CNS disorders such as Alzheimer's disease, Parkinson's disease and stroke.

Cerebral metabolic alterations in rats with diabetic ketoacidosis: effects of treatment with insulin and intravenous fluids and effects of bumetanide.

OBJECTIVE: Cerebral edema is a life-threatening complication of diabetic ketoacidosis (DKA) in children. Recent data suggest that cerebral hypoperfusion and activation of cerebral ion transporters may be involved, but data describing cerebral metabolic alterations during DKA are lacking. RESEARCH DESIGN AND METHODS: We evaluated 50 juvenile rats with DKA and 21 normal control rats using proton and phosphorus magnetic resonance spectroscopy (MRS). MRS measured cerebral intracellular pH and ratios of metabolites including ATP/inorganic phosphate (Pi), phosphocreatine (PCr)/Pi, N-acetyl aspartate (NAA)/creatine (Cr), and lactate/Cr before and during DKA treatment. We determined the effects of treatment with insulin and intravenous saline with or without bumetanide, an inhibitor of Na-K-2Cl cotransport, using ANCOVA with a 2 x 2 factorial study design. RESULTS: Cerebral intracellular pH was decreased during DKA compared with control (mean +/- SE difference -0.13 +/- 0.03; P < 0.001), and lactate/Cr was elevated (0.09 +/- 0.02; P < 0.001). DKA rats had lower ATP/Pi and NAA/Cr (-0.32 +/- 0.10, P = 0.003, and -0.14 +/- 0.04, P < 0.001, respectively) compared with controls, but PCr/Pi was not significantly decreased. During 2-h treatment with insulin/saline, ATP/Pi, PCr/Pi, and NAA/Cr declined significantly despite an increase in intracellular pH. Bumetanide treatment increased ATP/Pi and PCr/Pi and ameliorated the declines in these values with insulin/saline treatment. CONCLUSIONS: These data demonstrate that cerebral metabolism is significantly compromised during DKA and that further deterioration occurs during early DKA treatment--consistent with possible effects of cerebral hypoperfusion and reperfusion injury. Treatment with bumetanide may help diminish the adverse effects of initial treatment with insulin/saline.

Cerebral microvascular endothelial cell Na/H exchange: evidence for the presence of NHE1 and NHE2 isoforms and regulation by arginine vasopressin.

Blood-brain barrier (BBB) Na transporters are essential for brain water and electrolyte homeostasis. However, they also contribute to edema formation during the early hours of ischemic stroke by increased transport of Na from blood into brain across an intact BBB. We previously showed that a luminal BBB Na-K-Cl cotransporter is stimulated by hypoxia, aglycemia, and AVP and that inhibition of the cotransporter by intravenous bumetanide significantly reduces edema and infarct in the rat middle cerebral artery occlusion (MCAO) model of stroke. More recently, we found evidence that intravenous cariporide (HOE-642), a highly potent Na/H exchange inhibitor, also reduces brain edema after MCAO. The present study was conducted to investigate which Na/H exchange protein isoforms are present in BBB endothelial cells and to evaluate the effects of ischemic factors on BBB Na/H exchange activity. Western blot analysis of bovine cerebral microvascular endothelial cells (CMEC) and immunoelectron microscopy of perfusion-fixed rat brain revealed that Na/H exchanger isoforms 1 and 2 (NHE1 and NHE2) are present in BBB endothelial cells. Using microspectrofluorometry and the pH-sensitive dye BCECF, we found that hypoxia (2% O(2), 30 min), aglycemia (30 min), and AVP (1-200 nM, 5 min) significantly increased CMEC Na/H exchange activity, assessed as Na-dependent, HOE-642-sensitive H(+) flux. We found that AVP stimulation of CMEC Na/H exchange activity is dependent on intracellular Ca concentration and is blocked by V(1), but not V(2), vasopressin receptor antagonists. Our findings support the hypothesis that a BBB Na/H exchanger, possibly NHE1 and/or NHE2, is stimulated during ischemia to participate in cerebral edema formation.

Cerebral blood flow and cerebral edema in rats with diabetic ketoacidosis.

OBJECTIVE: Cerebral edema (CE) is a potentially life-threatening complication of diabetic ketoacidosis (DKA) in children. Osmotic fluctuations during DKA treatment have been considered responsible, but recent data instead suggest that cerebral hypoperfusion may be involved and that activation of cerebral ion transporters may occur. Diminished cerebral blood flow (CBF) during DKA, however, has not been previously demonstrated. We investigated CBF and edema formation in a rat model of DKA and determined the effects of bumetanide, an inhibitor of Na-K-Cl cotransport. RESEARCH DESIGN AND METHODS: Juvenile rats with streptozotocin-induced DKA were treated with intravenous saline and insulin, similar to human treatment protocols. CBF was determined by magnetic resonance (MR) perfusion-weighted imaging before and during treatment, and CE was assessed by determining apparent diffusion coefficients (ADCs) using MR diffusion-weighted imaging. RESULTS: CBF was significantly reduced in DKA and was responsive to alterations in pCO(2). ADC values were reduced, consistent with cell swelling. The reduction in ADCs correlated with dehydration, as reflected in blood urea nitrogen concentrations. Bumetanide caused a rapid rise in ADCs of DKA rats without significantly changing CBF, while saline/insulin caused a rapid rise in CBF and a gradual rise in ADCs. DKA rats treated with bumetanide plus saline/insulin showed a trend toward more rapid rise in cortical ADCs and a larger rise in striatal CBF than those observed with saline/insulin alone. CONCLUSIONS: These data demonstrate that CE in DKA is accompanied by cerebral hypoperfusion before treatment and suggest that blocking Na-K-Cl cotransport may reduce cerebral cell swelling.

Hypoxia effects on cell volume and ion uptake of cerebral microvascular endothelial cells.

Increased transport of Na across an intact blood-brain barrier (BBB) contributes to cerebral edema formation in ischemic stroke. Our previous studies have shown that ischemic factors stimulate activity of a luminal BBB Na-K-Cl cotransporter, and we have hypothesized that during ischemia, the cotransporter together with the abluminal Na/K pump mediates increased transport of Na from blood into the brain. However, it is possible that elevated Na-K-Cl cotransporter activity could also cause cell swelling if it outpaces ion efflux pathways. The present study was conducted to evaluate the effects of hypoxia on intracellular volume of BBB cells. Cerebral microvascular endothelial cell (CMEC) monolayers were exposed to varying levels of hypoxia for 1 to 5 h in an O(2)-controlled glove box, and cell volume was assessed using 3-O-methyl-D-[(3)H]glucose and [(14)C]sucrose as markers of total and extracellular water space, respectively. Cells exposed to either 7.5%, 3%, or 1% O(2) showed gradual increases in volume (compared with 19% O(2) normoxic controls) that became significant after 3 or more hours. By ion chromatography methods, we also found that a 30-min exposure to 7.5% O(2) caused an increase in bumetanide-sensitive net Na uptake by the cells without increasing cell Na content. CMEC Na content was significantly increased, however, following 3 or more hours of exposure to 7.5% O(2). These findings are consistent with the hypothesis that during cerebral ischemia, the BBB Na-K-Cl cotransporter is stimulated to mediate transendothelial uptake of Na into the brain and that increased cotransporter activity also contributes to gradual swelling of the cells.

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

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/cm(2)) 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.

Estradiol reduces activity of the blood-brain barrier Na-K-Cl cotransporter and decreases edema formation in permanent middle cerebral artery occlusion.

Estrogen has been shown to protect against stroke-induced brain damage, yet the mechanism is unknown. During the early hours of stroke, cerebral edema forms as increased transport of Na and Cl from blood into brain occurs across an intact blood-brain barrier (BBB). We showed previously that a luminal BBB Na-K-Cl cotransporter is stimulated by hypoxia and arginine vasopressin (AVP), factors present during cerebral ischemia, and that inhibition of the cotransporter by intravenous bumetanide greatly reduces edema in rats subjected to permanent middle cerebral artery occlusion (MCAO). The present study was conducted to determine whether estrogen protects in stroke at least in part by reducing activity of the BBB cotransporter, thereby decreasing edema formation. Ovariectomized rats were subjected to 210 mins of permanent MCAO after 7-day or 30-min pretreatment with 17beta-estradiol and then brain swelling and 2,3,5-triphenyltetrazolium chloride staining were assessed as measures of brain edema and lesion volume, respectively. Diffusion-weighed imaging was used to monitor permanent MCAO-induced decreases in apparent diffusion coefficient (ADC) values, an index of changes in brain water distribution and mobility. Na-K-Cl cotransporter activity of cerebral microvascular endothelial cells (CMECs) was assessed as bumetanide-sensitive K influx and cotransporter abundance by Western blot analysis after estradiol treatment. Estradiol significantly decreased brain swelling and lesion volume and attenuated the decrease in ADC values during permanent MCAO. Estradiol also abolished CMEC cotransporter stimulation by chemical hypoxia or AVP and decreased cotransporter abundance. These findings support the hypothesis that estrogen attenuates stimulation of BBB Na-K-Cl cotransporter activity, reducing edema formation during stroke.