Department of Physiology and Membrane Biology, University of California, Davis, California
Submitted 3 January 2005 ; accepted in final form 22 March 2005
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ABSTRACT |
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blood-brain barrier; stroke; cerebral ischemia; brain edema
If the BBB Na-K-Cl cotransporter does participate in edema formation during ischemia, then we predict that it should be stimulated by agents that are present during ischemia and associated with edema formation. One of the factors present during cerebral ischemia that may be responsible for triggering this increased BBB ion transport is the peptide arginine vasopressin (AVP). It has been shown previously that ischemia causes the central release of AVP from extrahypothalamic neural processes terminating on brain microvessels (10, 15, 24, 42), and AVP receptors have been demonstrated to be present on brain microvessels (22, 38). In addition, several studies have provided evidence that AVP plays a role in promoting brain edema. Brattleboro rats, genetically deficient in AVP, exhibit reduced edema formation in MCAO, whereas exogenous administration of AVP increases edema formation during ischemia in those animals (9). More recently, infarct volume in a rat focal embolic ischemic stroke model was shown to be reduced by an AVP receptor antagonist (41). Furthermore, we (34) have found previously that Na-K-Cl cotransporter activity of cultured bovine brain microvascular endothelial cells is stimulated by AVP.
The aim of the present study was to further evaluate the effects of AVP on the Na-K-Cl cotransporter of cerebral microvascular endothelial cells (CMECs). Here, we examined the sensitivity of the cotransporter to AVP as well as the receptor/signaling pathway employed by AVP in CMECs. We also evaluated the effect of AVP exposures ranging from minutes to hours to determine how the CMEC cotransporter responds to AVP with respect to both activity and cotransporter protein expression. We present evidence that CMEC Na-K-Cl cotransporter activity is stimulated by AVP in a V1 receptor-dependent manner that is also phospholipase C (PLC) and [Ca] dependent. In addition, we report here that while the AVP stimulation of CMEC cotransporter activity is rapid, occurring within minutes, it is also sustained in the continued presence of AVP. Overall, these findings add further support to the hypothesis that AVP-stimulated BBB cotransporter activity participates in edema formation during early hours stroke.
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MATERIALS AND METHODS |
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Bovine CMECs were maintained on collagen type I- and fibronectin-coated tissue culture flasks in Eagles minimal essential medium (EMEM) supplemented with 5% FBS and 2 ng/ml FGF (complete EMEM). When the cells were 80% confluent, the media were changed to a 50:50 mixture of complete EMEM plus C6 glial cell conditioned medium (C6CM), prepared as described previously (44). CMEC monolayers were maintained in EMEM-C6CM for 310 days before the cells were used for experiments. Human CMECs were cultured using the same method as for bovine cells with the exception that DMEM/F-12 medium was substituted for EMEM. In some experiments, CMECs were cultured in astrocyte conditioned medium (ACM), as described previously (44), rather than C6CM (see Figs. 6, A and B, and 7C). Our previous studies (34, 44) have shown that these two types of ACM are equally effective for studies of CMEC Na-K-Cl cotransport. Bovine CMECs were isolated as described previously (34) using bovine brains obtained from the University of California-Davis Meat Laboratory. Some of the bovine brain microvascular endothelial cells and all of the human brain microvascular cells were obtained from Cell Systems (Kirkland, WA). C6 glial cells were obtained from the American Type Culture Collection (Rockville, MD). Rat neonatal astrocytes were isolated as described previously (34, 44).
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This study was conducted in accordance with the Animal Use and Care Guidelines issued by the National Institutes of Health using a protocol approved by the Animal Use and Care Committee at the University of California, Davis. Bovine brains were removed within minutes of the animals death (University of California-Davis Meat Laboratory) and placed in ice-cold isolation buffer containing protease inhibitors (35, 44). Brain tissue was maintained at 4°C in buffered medium with protease inhibitors throughout the microvessel isolation procedure except for periods when the tissue was exposed to digestive enzymes at 37°C. After the meninges was removed, pieces of the cerebrum were gently dissected away from the cortical surface, treated with digestive enzymes, homogenized, and then separated from single cells and larger vessels by filtering through a series of different pore-sized meshes, based on previously described methods (43, 39a). Microvessels were used for K influx studies and Western blot analyses on the same day of isolation.
K Influx Assays
Assay of cultured cells. Na-K-Cl cotransport activity was measured as bumetanide-sensitive K influx using 86Rb as a tracer for K as previously described (33, 34). CMEC monolayers cultured on multiwell plates (24- or 96-well cluster plates) were pretreated with various conditions, as described in the figures. For these assays, CMEC monolayers were pretreated with agents to be tested (e.g., AVP) with or without 10 µM bumetanide in HEPES-buffered MEM and then assayed in identical media containing 86Rb. In addition to the various agents of interest, HEPES-buffered MEM pretreatment and assay media (both pH 7.4) contained (in mM) 144 Na, 147 Cl, 5.8 K, 1.2 Ca, 4.2 HCO3, 0.4 HPO4, 0.4 H2PO4, 0.4 Mg, 0.4 SO4, 5.6 glucose, and 20 HEPES. Pretreatment times varied with experiment and are specified in the figures. Assays were terminated by aspirating the wells and rapidly rinsing extracellular radioactivity from the monolayers using ice-cold 0.1 M MgCl2 and then extracting cells in 0.2% SDS for protein determination (Bradford method) and 86Rb quantitation (liquid scintillation analysis, Tri-Carb 2500 TR liquid scintillation counter). K influx was calculated as the slope of 86Rb uptake over time and expressed as micromoles of K per gram of protein per minute.
Assay of microvessels. The microvessels were assayed for Na-K-Cl cotransport activity as bumetanide-sensitive K influx using the same protocol as for cultured monolayers with minor modifications. Specifically, microvessels in suspension were pretreated for 5 min in HEPES-buffered MEM containing 0 or 10 µM bumetanide with or without 100 nM AVP. The suspensions were then assayed for 5 min in the same media containing 86Rb. The assay was stopped by pipeting an aliquot of the microvessel suspension into ice-cold stop solution (0.1 M MgCl2) and immediately pelleting the microvessels with a microcentrifuge, aspirating the supernatant, and then counting radioactivity of the pellet. This method has been described previously (33).
Measurement of Intracellular [Ca]
Intracellular [Ca] ([Ca]i) of cultured CMECs was measured using the Ca-sensitive fluorescent dye fura-2, by a modification of previously described methods (12, 25). Briefly, CMEC monolayers cultured on collagen- and fibronectin-coated glass coverslips (22 x 9 mm) were preincubated for 60 min at 37°C in phenol red-free HEPES-buffered MEM containing 1 µM fura-2 AM. Slides were rinsed and then mounted in a temperature-regulated cuvette (maintained at 37°C) in a Hitachi F-2000 fluorescence spectrophotometer. [Ca]i was assessed as the ratio of fluorescence intensities (FI) measured at excitation wavelengths of 340 and 380 nm with the emission wavelength at 510 nm. Baseline FI ratios were measured first, and the agents of interest (AVP, AVP antagonist, or bradykinin) were then added to the cuvette. At the end of each experiment, 20 µM digitonin and then 3 mM EGTA were added to the cuvette to calibrate the system by determining the FI ratios at maximal [Ca] and zero [Ca], respectively. Cells on slides were evaluated for autofluorescence before being loaded with fura-2.
Gel Electrophoresis and Western Blot Analysis
Cell lysates of cultured CMECs and freshly isolated cerebral microvessels were prepared for Western blot studies as described previously (35, 44, 48). Cultured CMECs were scraped from culture dishes into an ice-cold solution of PBS with 2 mM EDTA (PBS-EDTA) containing protease inhibitors and then lysed by 30 s of sonication (XL 2020 Sonicator, Heat Systems) at 4°C in the protease inhibitor-containing PBS-EDTA solution. To prepare lysates of bovine cortical cerebral microvessels, freshly isolated microvessels were disrupted by sonication in PBS-EDTA-protease inhibitor solution. The lysates of cultured CMECs and cerebral microvessels were subsequently centrifuged at 7,600 g for 10 min (TL-100 Beckman ultracentrifuge), and the supernatant was retained. Each lysate preparation was analyzed in at least triplicate for protein content (Bradford method) to ensure equal loading of membrane protein into each gel lane. Lysate samples and prestained molecular weight markers (Bio-Rad) were denatured in SDS reducing buffer, heated to 100°C for 3 min, and then used immediately for gel electrophoresis. Protein samples were electrophoretically separated on 6% SDS gels (Bio-Rad Mini-PROTEAN II), and the resolved proteins were electrophoretically transferred to nitrocellulose membranes using a Bio-Rad Trans-Blot apparatus. The blots were incubated in 5% nonfat dry milk-Tris-buffered saline (TBS) for 2 h at room temperature. Subsequently, blots were incubated with T4 monoclonal antibody (which recognizes Na-K-Cl cotransporter protein) (26), rinsed five times with TBS, and then exposed to secondary antibody (horseradish peroxidase-conjugated goat anti-mouse IgG). After five washes to remove unbound secondary antibody, bound antibody was visualized using an enhanced chemiluminescence assay (Amersham). For every Western blot, each condition was run in at least duplicate lanes. Multiple separate experiments were conducted for each condition tested, and the order of loading various conditions among the gel lanes was randomized for each Western blot.
Materials
EMEM and DMEM/F-12 were purchased from GIBCO-BRL (Grand Island, NY). Hams F-10, penicillin-streptomycin, and glutamine were from Cellgro (Herndon, VA). FBS was obtained from HyClone (Logan, UT). Bumetanide was from ICN Biomedicals (Costa Mesa, CA), and 86Rb was purchased from DuPont-New England Nuclear (Boston, MA). T4 monoclonal antibody was obtained from the University of Iowa Developmental Studies Hybridoma Bank (Iowa City, IA). (Arg8)-vasopressin (AVP) and the V1 receptor agonists and antagonists were all purchased from Peninsula Laboratories (a division of Bachem; San Carlos, CA). These included the V1 antagonist [d(CH2) Tyr(Me)2Arg8]-vasopressin, also called Manning compound; the V1 antagonist Des-Gly9-[phenylacetyl1,D-Tyr(Et)2,Lys6,Arg8]-vasopressin, called here PhaaEt VP; the V2 antagonist [d(CH2)
D-Ile2,Ile4,Arg8,Ala-NH
]-vasopressin, called here D-Ile VP; the V1 agonist (Phe2,Orn8)-vasotocin, called here Orn VP; and the V2 agonist (deamino-Cys1,D-Arg8)-vasopressin (or desmopressin), called here DDAVP. PMA was from Sigma Chemical (St. Louis, MO). Fura-2 AM was purchased from Molecular Probes (Eugene, OR). BAPTA-AM, calphostin C, U-73122, and U-73343 were purchased from EMD Biosciences/Calbiochem (San Diego, CA).
Statistical Analysis
All values are presented as means ± SE. For each flux experiment, all conditions were tested in at least quadruplicate. Data shown were analyzed for significance using either ANOVA or Students t-test, as indicated in the figures. P values of <0.05 were considered to indicate significant differences. SAS Statview software (Cary, NC) was used for all data analyses.
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RESULTS |
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To investigate the hypothesis that ischemia-induced brain edema formation is mediated at least in part by AVP stimulation of the BBB Na-K-Cl cotransporter, we first examined the sensitivity of the CMEC cotransporter to stimulation by AVP. Figure 1 shows that treatment of cultured bovine CMECs with AVP caused a dose-dependent stimulation of Na-K-Cl cotransport activity, as assessed here as bumetanide-sensitive K influx. For these experiments, cells were pretreated with AVP for 5 min and assayed for a subsequent 5 min in the presence of AVP. The EC50 for this effect was 1 nM. To verify that AVP effects on the cotransporter are relevant to human BBB endothelial cells, we also tested the effects of AVP on cultured human CMECs. As shown in Fig. 2, we found that a 10-min exposure of cultured human CMEC monolayers to 100 nM AVP (5-min pretreatment plus 5-min assay) caused a 70% increase in cotransporter activity. This indicates that AVP stimulation of cotransporter activity is not limited to bovine CMECs but occurs in human cells as well. If AVP stimulation of Na-K-Cl cotransporter activity is relevant to BBB participation in edema formation during cerebral ischemia, then we should be able to demonstrate that AVP increases cotransport activity of intact brain microvessels as well as cultured CMECs. Thus we examined freshly isolated bovine cerebral microvessels for cotransporter activity. As shown in Fig. 2, we found that bumetanide-sensitive K influx is indeed present in intact brain microvessels and that it is stimulated by a 5-min exposure to 100 nM AVP in a manner similar to cultured CMECs. Thus, in the microvessels, AVP caused a rapid, robust stimulation of cotransporter activity just as it did in cultured CMECs. In the remainder of the experiments conducted for the present studies, we used cultured bovine or human CMECs.
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Vasopressin Stimulation of CMEC Na-K-Cl Cotransporter Activity Is Mediated Via V1 Vasopressin Receptors and Elevation of [Ca]i
AVP is well known to act via two different receptor types, V1 and V2 receptors, linked to activation of PLC and adenylate cyclase, respectively (37). To determine whether the action of AVP on the CMEC Na-K-Cl cotransporter is via V1 or V2 receptors, we evaluated the effects of selective V1 and V2 agonists and antagonists on bumetanide-sensitive K influx of our cultured CMECs. First, we evaluated the effects of the V1 agonist Orn VP and the V2 agonist DDAVP on bovine CMEC Na-K-Cl cotransporter activity. As depicted in Fig. 3, Orn VP (10 nM) caused an increase in cotransport activity of the same magnitude as seen with AVP (10 nM). However, the V2 agonist DDAVP (10 nM) was without effect on cotransporter activity. Orn VP and AVP in combination did not stimulate cotransporter activity above the level observed for either agent alone (data not shown). In these studies, we also evaluated the ability of V1 and V2 antagonists to inhibit AVP-stimulated CMEC cotransporter activity. Figure 4A shows that the V1 antagonist Manning compound (100 nM) abolished the stimulation of cotransporter activity observed with AVP (100 nM), as did the highly selective V1 antagonist PhaaEt VP (100 nM). The dose dependence of PhaaEt VP abolition of AVP-induced cotransporter stimulation is shown in Fig. 4B. Here, we found that increasing doses of PhaaEt VP reduced cotransporter activity in the presence of 10 nM AVP, with maximal inhibition occurring at PhaaEt VP concentrations of 50 nM and higher. In contrast, the V2 antagonist D-Ile VP had no effect on AVP-stimulated cotransporter activity. Neither PhaaEt VP nor D-Ile VP in the absence of AVP had an effect on the cotransporter (e.g., cotransporter activity under basal conditions was 18.46 ± 1.06 compared with 17.57 ± 1.01 µmol·g protein1·min1 for cells treated with 100 nM PhaaEt VP alone, n = 5).
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Vasopressin V1 receptor activation can result not only in elevation of [Ca]i but also activation of PKC via PLC generation of diacylglycerol. In a previous study, we (34) reported that activation of PKC by PMA in bovine CMECs reduces Na-K-Cl cotransporter activity. In other studies, PMA has been reported to have either no effect or to stimulate rat and human CMEC Na-K-Cl cotransport activity (18, 43, 47). Thus, in the present studies, we more fully investigated the effects of PMA on CMEC cotransporter activity. As shown in Fig. 7A, PMA (10-min treatment) caused a dose-dependent inhibition of human CMEC Na-K-Cl cotransporter activity, with 10 nM PMA causing 55% inhibition and the maximal inhibition of 88% observed with 500 nM PMA (at 1 µM PMA, cotransporter activity in these cells was 0.74 ± 0.14 µmol·g protein1·min1). Exposure of cells to high doses of PMA (400 nM) for prolonged periods (>18 h) has been demonstrated to markedly decrease PKC activity through degradation of the protein after its increased recruitment to the plasma membrane (1, 13). Our previous studies of aortic endothelial cells have shown that treating the cells with 400 nM PMA for 48 h to downregulate PKC activity caused an increase in Na-K-Cl cotransporter activity both under basal conditions and in the presence of AVP (32). Figure 7B shows that the same appears to be true of the CMEC cotransporter. After 48-h pretreatment with 400 nM PMA, cotransporter activity of CMECs was significantly increased both under basal conditions and in the presence of 100 nM AVP. Results for human CMEC are shown in Fig. 7B. Similar results were obtained with bovine CMECs (data not shown). These findings suggest that PKC activity is inhibitory to the cotransporter in these cells, and thus the vasopressin V1 receptor-induced stimulation of the cotransporter most likely occurs via elevation of [Ca]i and not via activation of PKC. Another approach to evaluating the involvement of PKC in AVP effects is to use a PKC inhibitor. Thus we tested the effect of treating CMECs with the specific PKC inhibitor calphostin C. As shown in Fig. 7C, we found that a 5-min exposure of bovine CMECs to calphostin C (100 nM) significantly increased basal cotransporter activity with a magnitude not significantly different than that found with AVP (100 nM) alone. In these experiments, the effects of calphostin C and AVP were not additive, i.e., AVP in the presence of calphostin C did not increase cotransporter activity over that found in the presence of calphostin C alone. This observation will be considered further in the DISCUSSION. In any case, the findings shown in Fig. 7, AC, suggest that PKC activity is inhibitory to the CMEC cotransporter.
Effects of Sustained Vasopressin Exposure on Brain Microvascular Endothelial Cell Cotransporter Activity and Cotransporter Protein Abundance
Ischemia-induced edema forms in the presence of an intact BBB for up to 46 h, after which time the barrier begins to break down. While edema formation is fairly rapid, it appears to continue throughout this early period of ischemia. In this regard, it is of interest to determine whether AVP, which is present during ischemia, can elevate cotransporter activity for more than a few minutes and even throughout the early hours of ischemia. Thus, in the present study, we tested the effects of exposing CMEC to AVP over a time course of hours. Figure 8A shows that cotransporter activity of CMECs exposed to AVP (10 nM) for 4 h was significantly elevated over control levels (without AVP). Cotransport activity remained elevated even when CMECs were exposed to AVP for 18 and 36 h. The magnitude of stimulation by prolonged exposure to AVP was comparable with that observed with a 10-min AVP treatment (see Fig. 1), and there was no difference in the magnitude of cotransporter stimulation found among the 4-, 18-, or 36-h AVP treatments. To determine whether increased cotransporter activity occurring with 4 h or more of AVP exposure involves increased expression of the cotransporter protein, we evaluated cotransporter abundance in CMECs by Western blot analysis after 436 h of 10 nM AVP treatment (Fig. 8, B and C). We found that, although a 4-h AVP treatment caused a significant increase in cotransporter activity, it did not significantly increase the abundance of the cotransporter protein. In contrast, AVP treatments of 18 and 36 h increased cotransporter protein abundance 1.9- and 2.3-fold over control cotransporter abundance, respectively. Thus AVP appears to have two effects on the brain microvascular endothelial cells: a rapid stimulation of cotransporter activity and a slower onset elevation of cotransporter protein abundance in the cells.
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DISCUSSION |
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Our studies have revealed that AVP stimulates Na-K-Cl cotransporter activity of cultured bovine CMECs with an EC50 of 1 nM. This is consistent with previous estimates showing that the concentration of AVP in brain extracellular fluid due to central release of the peptide is in the 1 pg/ml range (109 M), varying from 1011 to 108 M depending on the physiological condition (24). In addition, the Kd for AVP-specific binding to cerebral microvessels has been reported to be between 1 and 3 nM (11, 23, 38). In the present studies, we have also demonstrated that nanomolar concentrations of AVP stimulate cotransporter activity of freshly isolated bovine cerebral microvessels, indicating that this AVP effect is not a culturing-induced phenomenon but has physiological relevance for the BBB. Furthermore, we found that AVP also stimulates cotransporter activity of cultured human CMECs, suggesting that the AVP effect on the cotransporter is relevant to the human BBB and not limited to bovine brain microvascular endothelial cells. It is noteworthy that in these studies we found cultured human CMECs and freshly isolated bovine microvessels to have lower basal cotransporter activity values compared with cultured bovine CMECs (
46 vs.
1720 µmol K·g protein1·min1). This is in keeping with the fact that previously reported values for basal Na-K-Cl cotransporter activity vary somewhat with species and cell culture preparation. In studies of cultured human microvascular endothelial cells, Spatz and coworkers (43) found basal activity the Na-K-Cl cotransporter to be in the range of 45 µmol·g protein1·min1, consistent with our findings for the human CMEC cotransporter. Reported values for rat CMEC basal cotransport activity vary from
7 to 11 µmol K·g protein1·min1 (17, 47). Basal cotransporter values for bovine CMEC range from
1012 to 1520 µmol µmol K·g protein1·min1 for cells without and with ACM, respectively (34). Despite these variations in basal Na-K-Cl cotransport activity, our present study shows that AVP is a potent and rapid stimulator of the cotransporter in both human and bovine brain microvascular endothelial cells. In the present studies, we also found that the peptide ANP decreases activity of the CMEC Na-K-Cl cotransporter and, furthermore, that it blocks stimulation of the cotransporter by AVP. Previous studies have shown that rats given intravenous ANP exhibit reduced edema formation during global cerebral ischemia (28) and also that intraventricular administration of ANP significantly reduces brain water and Na uptake in the rat MCAO model of cerebral edema (29). Our previous studies have shown that bumetanide inhibition of the BBB Na-K-Cl cotransporter in rats subjected to MCAO reduces cerebral edema formation. These findings, together with our present observation that the CMEC cotransporter is stimulated by AVP in a manner that can be inhibited by ANP, provide further support for the hypothesis that the BBB cotransporter is a central participant in ischemia-induced cerebral edema formation.
The present studies have also shown that nanomolar concentrations of the V1 vasopressin receptor agonist Orn VP stimulate CMEC cotransporter activity and that the AVP stimulation is abolished by nanomolar concentrations of the V1 vasopressin receptor antagonist PhaaEt VP. Neither the V2 vasopressin agonist DDAVP nor the V2 vasopressin antagonist had any effect on CMEC cotransporter activity. These findings are consistent with previous studies (36, 37) demonstrating that V1 receptors are present in brain microvessels. In addition, previous studies evaluating binding of AVP to various tissues have established that the Kd for V1 receptors is 13 nM and for V2 receptors is around 0.40.5 nM (2). Collectively, these findings suggest that AVP stimulates the CMEC cotransporter via a V1 receptor. This, together with the previous report (39) showing that AVP-induced brain edema is mediated by V1 receptors, lends further support to the hypothesis that AVP-stimulated BBB Na-K-Cl cotransporter activity contributes to cerebral edema formation.
The results of the present studies demonstrate that, characteristic of a V1 receptor-mediated event, AVP stimulation of the CMEC cotransporter is dependent on both PLC activation and elevation of [Ca]i. With respect to the latter, we have first shown that AVP induces a rapid increase in [Ca]i that is blocked by PhaaEt VP. Second, we have shown that AVP stimulation of CMEC cotransporter activity is lost if the cells are placed in Ca-free medium, if they are loaded with the Ca chelator BAPTA, or both. These findings indicate that the AVP effect occurs via a process that is dependent on elevation of [Ca]i and requires the presence of extracellular Ca. In this regard, CMECs appear to be similar to aortic endothelial cells in that AVP stimulation of Na-K-Cl cotransporter activity in those cells is also reduced by loading the cells with BAPTA and abolished in Ca-free medium (32). Determining the relative contributions of intracellular Ca stores and extracellular Ca to elevation of [Ca]i will require further investigation. However, it should be recognized that both PLC activation and intracellular store Ca release are known to be Ca-dependent phenomena. Thus it is possible that Ca influx from the extracellular space could indirectly, as well as directly, elevate [Ca]i. Both Ca influx and intracellular store Ca mobilization appear to contribute to V1 receptor-mediated [Ca]i elevation in hepatocytes (30) as well as astrocytes (50). Determining the signaling mechanisms whereby an AVP-induced elevation of [Ca]i may increase cotransporter activity is beyond the scope of the present study. However, in previous studies of the bovine aortic endothelial cell Na-K-Cl cotransporter, we (35) found that AVP both increases [Ca]i of the cells and increases phosphorylation of the cotransporter protein. We (45) have also shown previously that the phosphatase inhibitor calyculin A increases activity of the CMEC cotransporter and also increases phosphorylation of CMEC cotransporter protein. Thus it is likely that AVP stimulation of CMEC cotransporter activity involves an increase in phosphorylation of the cotransporter.
AVP V1 receptor activation of PLC can, through generation of diacylglcerol, also activate PKC. In a previous study, we (34) showed that activation of PKC by 10 nM PMA reduced CMEC cotransporter activity. Our present studies provide evidence that PKC does not appear to mediate AVP stimulation of cotransporter activity, and, in fact, PKC is inhibitory to the cotransporter. First, PMA potently and dose dependently reduced CMEC cotransporter activity with an IC50 of 10 nM in these cells. Second, downregulation of PKC activity (by 48-h exposure to 400 nM PMA) caused an increase in basal cotransporter activity and did not significantly reduce AVP stimulation of the cotransporter. Furthermore, we found that the PKC inhibitor calphostin C also increased basal CMEC cotransporter activity. These findings are in contrast to previous reports showing that PMA stimulates cotransporter activity of rat brain capillary endothelial cells and human large microvessel endothelial cells (18, 43) and that it is without effect on the cotransporter of clonal rat brain microvascular endothelial cells (47). The reasons for these discrepancies are unclear. However, they may be due to differences in the cultured cell preparations and/or doses of PMA tested. The bovine and human CMECs used in our studies were cultured with ACM to promote the BBB phenotype, whereas it appears that the cells in these other studies were not. Also, PMA was used at a dose of 10 µM in the rat capillary endothelial cell study, much higher than the nanomolar doses found to be inhibitory to the CMEC cotransporter in the present study, the same low doses that effectively stimulate PKC activity (35). It should be noted that the stimulatory effects of AVP and calphostin C on the CMEC cotransporter were not additive in our studies. There are two interpretations of this finding. One is that AVP stimulation of the cotransporter is blocked by calphostin C. This would imply that, unlike the high-dose PMA-induced downregulation of PKC, calphostin C inhibition of PKC activity can prevent AVP stimulation of the cotransporter. However, it remains to be clarified whether this apparent calphostin C inhibition of AVP effects does indeed occur through specific inhibition of PKC or whether calphostin C at the dose used exerts nonspecific effects. The second interpretation is simply that AVP and calphostin C are not additive because the cotransporter was maximally stimulated by either agent alone in these experiments. In any case, our studies consistently show that, at least in bovine and human CMECs, activation of PKC does not lead to increased cotransporter activity. Further studies are needed to clarify the mechanism whereby reduced PKC activity increases basal cotransporter activity in CMECs.
The results of the present study have also shown that while the stimulatory effect of AVP on CMEC Na-K-Cl cotransporter activity is rapid, occurring within minutes, it is also sustained for hours in the continued presence of AVP. This suggests that AVP may induce a prolonged elevation of BBB cotransporter activity throughout the early hours of stroke when the greatest portion of edema formation occurs (27, 40). Our studies have further revealed that prolonged exposure to AVP can also upregulate CMEC cotransporter protein expression but that this does not occur until after more than 4 h of AVP exposure. This suggests that an AVP-induced increase in Na-K-Cl cotransporter abundance does not contribute to ischemia-induced edema formation. Clarifying the significance of AVP-increased cotransporter abundance at 18 and 36 h, well after the early stages of ischemia-induced edema formation, is beyond the scope of our present study and will require future investigation.
In summary, the results of the present study provide evidence that AVP stimulates BBB Na-K-Cl cotransporter activity via a V1 receptor-dependent mechanism that is also PLC and [Ca]i dependent. Together with a previous report (39) showing that AVP-induced brain edema is mediated by V1 receptors and our previous finding that inhibition of the BBB Na-K-Cl cotransporter reduces edema formation in rats subjected to MCAO, these findings support the hypothesis that during cerebral ischemia, V1 receptor-mediated stimulation of BBB cotransporter activity is a major contributor to cerebral edema formation.
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GRANTS |
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FOOTNOTES |
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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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REFERENCES |
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---|
2. Barberis C and Tribollet E. Vasopressin and oxytocin receptors in the central nervous system. Crit Rev Neurobiol 10: 119154, 1996.[ISI][Medline]
3. Betz AL. Sodium transport from blood to brain: inhibition by furosemide and amiloride. J Neurochem 41: 11581164, 1983.[ISI][Medline]
4. Betz AL. Sodium transport in capillaries isolated from rat brain. J Neurochem 41: 11501157, 1983.[ISI][Medline]
5. Betz AL, Keep RF, Beer ME, and Ren X. Blood-brain barrier permeability and brain concentration of sodium, potassium, and chloride during focal ischemia. J Cereb Blood Flow Metab 14: 2937, 1994.[ISI][Medline]
6. Bourke RS, Kimelberg HK, Nelson LR, Barron KD, Auen EL, Popp AJ, and Waldman JB. Biology of glial swelling in experimental brain edema. Adv Neurol 28: 99109, 1980.[Medline]
7. Bronner LL, Kanter DS, and Manson JE. Primary prevention of stroke. N Engl J Med 333: 13921400, 1995.
8. Cserr HF, DePasquale M, Patlak CS, and Pullen RGL. Convection of cerebral interstitial fluid and its role in brain volume regulation. Ann NY Acad Sci 481: 123134, 1989.
9. Dickinson LD and Betz AL. Attenuated development of ischemic brain edema in vasopressin-deficient rats. J Cereb Blood Flow Metab 12: 681690, 1992.[ISI][Medline]
10. Dóczi T. Volume regulation of the brain tissuea survey. Acta Neurochir (Wien) 121: 18, 1993.[CrossRef][ISI][Medline]
11. Ermisch A. Peptide receptors of the blood-brain barrier and substrate transport into the brain. Prog Brain Res 91: 155161, 1992.[ISI][Medline]
12. Grynkiewicz G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 34403450, 1985.[Abstract]
13. Hepler JR, Earp HS, and Harden TK. Long-term phorbol ester treatment down-regulates protein kinase C and sensitizes the phosphoinositide signaling pathway to hormone and growth factor stimulation. J Biol Chem 263: 76107619, 1988.
14. Iadecola C. Mechanisms of cerebral ischemic damage. In: Cerebral Ischemia: Molecular and Cellular Pathophysiology, edited by Walz W. Totowa, NJ: Humana, 1999, p. 334.
15. Jójárt I, Joó F, Siklós L, and László FA. Immunohistochemical evidence for innervation of brain microvessels by vasopressin-immunoreactive neurons in the rat. Neurosci Lett 51: 259264, 1984.[CrossRef][ISI][Medline]
16. Kato H, Kogure K, Sakamoto N, and Watanabe T. Greater disturbance of water and ion homeostasis in the periphery of experimental focal cerebral ischemia. Exp Neurol 96: 118126, 1987.[CrossRef][ISI][Medline]
17. Kawai N, McCarron RM, and Spatz M. Effect of hypoxia on Na+-K+-Cl cotransport in cultured brain capillary endothelial cells of the rat. J Neurochem 66: 25722579, 1996.[ISI][Medline]
18. Kawai N, McCarron RM, and Spatz M. Na+-K+-Cl cotransport system in brain capillary endothelial cells: response to endothelin and hypoxia. Neurochem Res 21: 12591266, 1996.[ISI][Medline]
19. Keep RF. Potassium transport at the blood-brain and blood-CSF barriers. In: Frontiers in Cerebral Vascular Biology: Transport and Its Regulation, edited by Drewes LR and Betz AL. New York: Plenum, 1993, p. 4354.
20. Kimelberg HK. Cell swelling in cerebral ischemia. In: Cerebral Ischemia: Molecular and Cellular Pathophysiology, edited by Walz W. Totowa, NJ: Humana, 1999, p. 4568.
21. Kimelberg HK. Current concepts of brain edema. Review of laboratory investigations. J Neurosurg 83: 10511059, 1995.[ISI][Medline]
22. Kretzschmar R and Ermisch A. Arginine-vasopressin binding to isolated hippocampal microvessels of rats with different endogenous concentrations of the neuropeptide. Exp Clin Endocrinol 94: 151156, 1989.[ISI][Medline]
23. Kretzschmar R, Landgraf R, Gjedde A, and Ermisch A. Vasopressin binds to microvessels from rat hippocampus. Brain Res 380: 325330, 1986.[CrossRef][ISI][Medline]
24. Landgraf R. Central release of vasopressin: stimuli, dynamics, consequences. Prog Brain Res 91: 2939, 1992.[ISI][Medline]
25. Laskey RC, Adams DJ, Johns A, Rubanyi GM, and Breeman CV. Membrane potential and Na-K-pump activity modulate resting and bradykinin-stimulated changes in cytosolic free calcium in cultured endothelial cells from bovine aorta. J Biol Chem 265: 26132619, 1990.
26. Lytle C, Xu JC, Biemesderfer D, and IIIBF. Distribution and diversity of Na-K-Cl cotransport proteins: a study with monoclonal antibodies. Am J Physiol Cell Physiol 269: C1496C1505, 1995.
27. Menzies SA, Betz AL, and Hoff JT. Contributions of ions and albumin to the formation and resolution of ischemic brain edema. J Neurosurg 78: 257266, 1993.[ISI][Medline]
28. Nakao N, Itakura T, Yokote H, Nakai K, and Komai N. Effect of atrial natriuretic peptide on ischaemic brain edema. Acta Neurochir Suppl (Wien) 51: 201203, 1990.[Medline]
29. Naruse S, Takei R, Horikawa Y, Tanaka C, Higuchi T, Ebisu T, Ueda S, Sugahara S, Kondo S, Kiyota T, and Hayashi H. Effects of atrial natriuretic peptide on brain oedema: the change of water, sodium, and potassium contents in the brain. Acta Neurochir Suppl (Wien) 51: 118121, 1990.[Medline]
30. Nathanson MH, Moyer MS, Burgstahler AD, OCarroll AM, Brownstein MJ, and Lolait SJ. Mechanisms of subcellular cytosolic Ca2+ signaling evoked by stimulation of the vasopressin V1a receptor. J Biol Chem 267: 2328223289, 1992.
31. ODonnell M, Tran L, Lam TI, Liu XB, and Anderson SE. Bumetanide inhibition of the blood-brain barrier Na-K-Cl cotransporter reduces edema formation in the rat middle cerebral artery occlusion model of stroke. J Cereb Blood Flow Metab 24: 10461056, 2004.[CrossRef][ISI][Medline]
32. ODonnell ME. Endothelial cell sodium-potassium-chloride cotransport. Evidence of regulation by Ca2+ and protein kinase C. J Biol Chem 266: 1155911566, 1991.
33. ODonnell ME. Role of Na-K-Cl cotransport in vascular endothelial cell volume regulation. Am J Physiol Cell Physiol 264: C1316C1326, 1993.
34. ODonnell ME, Martinez A, and Sun D. Cerebral microvascular endothelial cell Na-K-Cl cotransport: regulation by astrocyte-conditioned medium. Am J Physiol Cell Physiol 268: C747C754, 1995.
35. ODonnell ME, Martinez A, and Sun D. Endothelial Na-K-Cl cotransport regulation by tonicity and hormones: phosphorylation of cotransport protein. Am J Physiol Cell Physiol 269: C1513C1523, 1995.
36. Ostrowski NL, Lolait SJ, Bradley DJ, OCarroll AM, Browstein MJ, and Young WS. Distribution of V1a and V2 vasopressin receptor messenger ribonucleic acids in rat liver, kidney, pituitary and brain. Endocrinology 131: 533535, 1992.[Abstract]
37. Ostrowski NL, Lolait SJ, and Young WS. Cellular localization of vasopressin V1a receptor messenger ribonucleic acid in adult male rat brain, pineal, and brain vasculature. Endocrinology 135: 15111528, 1994.[Abstract]
38. Pearlmutter AF, Szkrybalo M, Kim Y, and Harik SI. Arginine vasopressin receptors in pig cerebral microvessels, cerebral cortex and hippocampus. Neurosci Lett 87: 121126, 1988.[CrossRef][ISI][Medline]
39. Rosenberg GA, Estrada E, and Kyner WT. Vasopressin-induced brain edema is mediated by the V1 receptor. Adv Neurol 52: 149154, 1990.[Medline]
39. Sánchez del Pino MM, Hawkins RA, and Peterson DR. Neutral amino acid transport by the blood-brain barrier. Membrane vesicle studies. J Biol Chem 267: 2595125957, 1992.
40. Schielke GP, Moises HC, and Betz AL. Blood to brain sodium transport and interstitial fluid potassium concentration during focal ischemia in the rat. J Cereb Blood Flow Metab 11: 466471, 1991.[ISI][Medline]
41. Shuaib A, Wang CX, Yang T, and Noor R. Effects of nonapeptide V1 vasopressin receptor antagonist SR-49059 on infarction volume and recovery of function in a focal embolic stroke model. Stroke 33: 30333037, 2002.
42. Sorensen PS, Gjerris A, and Hammer M. Cerebrospinal fluid vasopressin in neurological and psychiatric disorders. J Neurol Neurosurg Psychiatry 48: 5057, 1985.[Abstract]
43. Spatz M, Merkel KN, Bembry J, and McCarron RM. Functional properties of cultured endothelial cells derived from large microvessels of human brain. Am J Physiol Cell Physiol 272: C231C239, 1997.
44. Sun D, Lytle C, and ODonnell ME. Astroglial cell-induced expression of Na-K-Cl cotransporter in brain microvascular endothelial cells. Am J Physiol Cell Physiol 269: C1506C1512, 1995.
45. Sun D and ODonnell ME. Astroglial-mediated phosphorylation of Na-K-Cl cotransporter in brain microvessel endothelial cells. Am J Physiol Cell Physiol 271: C620C627, 1996.
47. Vigne P, Farre AL, and Frelin C. Na+-K+-Cl cotransporter of brain capillary endothelial cells. Properties and regulation by endothelins, hyperosmolar solutions, calyculin A, and interleukin-1. J Biol Chem 269: 1992519930, 1994.
48. Yerby TR, Vibat CRT, Sun D, Payne JA, and ODonnell ME. Molecular characterization of the Na-K-Cl cotransporter of bovine aortic endothelial cells. Am J Physiol Cell Physiol 273: C188C197, 1997.
49. Young W, Rappaport ZH, Chalif DJ, and Flamm ES. Regional brain sodium, potassium, and water changes in the rat middle cerebral artery occlusion model of ischemia. Stroke 18: 751759, 1987.[Abstract]
50. Zhao L and Brinton RD. Vasopressin-induced cytoplasmic and nuclear calcium signaling in cultured cortical astrocytes. Brain Res 943: 117131, 2002.[CrossRef][ISI][Medline]