1 Stroke Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda 20892; 2 Resuscitative Medicine Department, Naval Medical Research Center, Silver Spring 20910; 3 Section on Developmental Neurobiology, National Institute of Mental Health, National Institutes of Health, Bethesda 20892; and 4 Department of Neurosurgery, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287
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ABSTRACT |
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Previous studies have demonstrated that functional interaction between endothelin (ET)-1 and nitric oxide (NO) involves changes in Ca2+ mobilization and cytoskeleton in human brain microvascular endothelial cells. The focus of this investigation was to examine the possible existence of analogous interplay between these vasoactive substances and elucidate their signal transduction pathways in human brain capillary endothelial cells. The results indicate that ET-1-stimulated Ca2+ mobilization in these cells is dose-dependently inhibited by NOR-1 (an NO donor). This inhibition was prevented by ODQ (an inhibitor of guanylyl cyclase) or Rp-8-CPT-cGMPS (an inhibitor of protein kinase G). Treatment of endothelial cells with 8-bromo-cGMP reduced ET-1-induced Ca2+ mobilization in a manner similar to that observed with NOR-1 treatment. In addition, NOR-1 or cGMP reduced Ca2+ mobilization induced by mastoparan (an activator of G protein), inositol 1,4,5-trisphosphate, or thapsigargin (an inhibitor of Ca2+-ATPase). Interestingly, alterations in endothelial cytoskeleton (actin and vimentin) were associated with these effects. The data indicate for the first time that the cGMP-dependent protein kinase colocalizes with actin. These changes were accompanied by altered levels of phosphorylated vasodilator-stimulated phosphoprotein, which were elevated in endothelial cells incubated with NOR-1 and significantly reduced by ODQ or Rp-8-CPT-cGMPS. The findings indicate a potential mechanism by which the functional interrelationship between ET-1 and NO plays a role in regulating capillary tone, microcirculation, and blood-brain barrier function.
capillary endothelium; endothelin-1; nitric oxide; calcium mobilization; cytoskeleton
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INTRODUCTION |
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THE CENTRAL MICROCIRCULATORY BED (composed of small arteries, capillaries, and venules) is the main intermediary structure responsible for maintaining the cellular homeostasis in the brain. Its regulatory functions principally reside within the endothelium, which consists of a single layer of flat cells. In microvessels, the endothelium forms a continuous inner lining, whereas in the capillaries it provides a single cellular barrier between the blood and brain. These endothelial cells (EC) express a variety of biological activities, including the control of vascular tone and blood flow through the secretion of vasoactive substances. In addition, EC also regulate the passage of nutrients and cellular trafficking between the blood and underlining tissue [i.e., blood-brain barrier (BBB) permeability] (28). Among a number of vasoactive factors that play pivotal roles in regulating vascular reactivity are endothelin (ET)-1 and nitric oxide (NO), which are produced locally in adjunct cells or found in the circulation (1, 9, 22, 24, 32).
ET-1 is a member of the endothelin family, composed of two other isopeptides (ET-2 and ET-3), all of which contain 21 amino acids and 2 disulfide bonds. Each of these peptides is derived from separate genes and has different amino acid constituents (17, 42). Vascular EC only produce ET-1, but the secretion of this peptide (as well as ET-2 and ET-3) has also been demonstrated in other cells (20). The synthesis of ET-1 resembles that of other peptide hormones in which a precursor polypeptide is consequently cleaved to generate the active form.
The endothelium not only secretes ET-1 but also contains two well-characterized receptors, namely ETA and ETB (17, 32). These receptors belong to the rhodopsin superfamily, and their action is mediated by a G protein. One of the effects of ET-1 binding to its receptor is the activation of a signal transduction pathway. More specifically, the stimulation of phospholipase C (PLC) leads to the formation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, resulting in the release of Ca2+. The rise in intracellular Ca2+ concentration ([Ca2+]i) is often associated with activation of the cellular contractive machinery (6, 29). It may also stimulate the NO production in the endothelium and other cells (2, 24). This compound is one of the smallest and most ubiquitous messenger molecules in mammals. NO is formed from L-arginine by the enzymatic action of nitric oxide synthase (NOS). There are three isoforms of this enzyme (endothelial, neuronal, and inducible NOS). The endothelium contains an acid-soluble isoform (endothelial NOS, eNOS) that is regulated by cofactors such as NADPH, tetrahydrobiopterin, and Ca2+/calmodulin. The vasorelaxant property of eNOS is attributed to the release of NO from endothelium. This event involves NO binding to the heme moiety in guanylyl cyclase, causing a conformational change that activates the enzyme and results in a rise of cGMP. In vivo and in vitro studies have examined the effects of ET-1 and NO on vascular tone and/or blood flow as well as BBB permeability (2, 12, 23, 36-38, 41). The results demonstrate a functional interplay between these two substances on the cellular secretory and/or receptor level. Recent investigations conducted on the endothelium derived from microvessels of human brain have shown that the close functional relationship between ET-1 and NO leads to rapid changes in [Ca2+]i and alterations in the cellular cytoskeleton. As mentioned above, the microcirculatory tree includes capillaries, which in general have been considered to be a passive segment of microcirculation even though they are the main constituents of the BBB. Therefore, the aim of this study was 1) to examine whether these cells possess the essential machinery and capacity to respond to ET-1 and/or NO and 2) to elucidate the signal transduction pathway accountable for the interaction.
This report clearly demonstrates that NO-mediated abrogation of ET-1-stimulated Ca2+ mobilization in human brain capillary endothelial cells (HBEC) involves the ETA receptor and postreceptor signal transduction pathway. It is also associated with cytoskeleton rearrangements and phosphorylation of vasodilator-stimulated phosphoprotein (VASP).
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MATERIALS AND METHODS |
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Materials
Endothelin-1 and mastoparan (Mas7; an activator of G protein) were obtained from Peninsula Laboratories (Belmont, CA). Fluo 3-AM and Texas red-X phalloidin were purchased from Molecular Probes (Eugene, OR). Anti-VASP was from Transduction Laboratories (Lexington, KY); monoclonal vimentin antibody was from Dako Laboratories (Carpinteria, CA). NOR-1 {(±)-(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexeneamide; an NO donor}, ODQ (1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one; a specific inhibitor of guanylyl cyclase), and Rp-8-CPT-cGMPS [Rp diastereomer of 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphorothioate, sodium salt; a selective inhibitor of protein kinase G] were purchased from Alexis Biochemicals (San Diego, CA). NG-nitro-L-arginine methyl ester (L-NAME; a competitive inhibitor of L-arginine), U-73122 (a selective inhibitor of PLC), and BQ123 and BQ788 (selective inhibitors of ETA and ETB receptors, respectively) were from Calbiochem (La Jolla, CA). Rabbit anti-cGMP-dependent protein kinase (anti-PKG) polyclonal antibody was a gift from Stressgen Biotechnologies (Victoria, BC, Canada). Thapsigargin, IP3, and 8-bromo-cGMP were obtained from Sigma Chemical (St. Louis, MO).Cell Culture
Human brain samples surgically removed for the treatment of idiopathic epilepsy were used to isolate capillary EC by mechanical dispersion, filtration, and dissociation as previously described (35). The purity of endothelium was >95% as determined by positive immunocytochemical staining for von Willebrand (factor VIII)-related antigen and incorporation of acetylated low-density lipoprotein. EC cultures used in experiments were obtained from at least six cell lines (passages 7-15).Measurements of Intracellular Ca2+ and Inositol Phosphates
For [Ca2+]i measurements, EC grown on 24-well plates were cultured to confluency and incubated with 2.5 µM fluo 3-AM fluorescent probe in complete saline buffer (137 mM NaCl, 5 mM KCl, 1 mM MgCl2, 25 mM sorbitol, 10 mM HEPES, and 3 mM CaCl2, pH 7.0) for 90 min at 37°C. After washing out unloaded probe, the background fluorescence was measured with a CytoFluor II fluorescence multiwell plate reader (PerSeptive Biosystems, Framingham, MA) using a fluorescein filter pair (excitation 485 ± 20 nm, emission 530 ± 25 nm). Tested substances were added and Ca2+ fluorescence was measured at indicated times. The [Ca2+]i changes were expressed as fluorescence intensity (F) by using the formula F =[F (experimental reading)Western Blot Analysis
EC grown on 35-mm dishes were washed three times with PBS and kept in serum-free medium (M199) overnight at 37°C. After exposure to tested agents, cells were lysed, and protein assays and Western blot analyses were determined as previously described (4).Immunocytochemistry
HBEC plated on coverslips were treated with various drugs as indicated, fixed in 3.7% formaldehyde (10 min), and permeabilized in 0.1% Triton X-100 (10 min) as previously described (4). Cells were stained with polyclonal anti-VASP (not shown) antibody or anti-PKG (1:100) antibody and either Texas red-phalloidin or monoclonal anti-vimentin and then viewed by Zeiss confocal microscope (Oberkochen, Germany) as previously described (3, 4). ![]() |
RESULTS |
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[Ca2+]i Mobilization
Response to ET-1.
ET-1 (0.1-100 nM) dose-dependently stimulated
[Ca2+]i mobilization in HBEC in the same
manner as that reported for human brain microvascular EC
(3). The maximal effect was observed at 100 nM with an
estimated EC50 = 20 nM (not shown). The response was completely inhibited by selective ETA receptor antagonists
(BQ123 or Ro-61-1790) but not by the selective ETB-receptor
antagonist (BQ788) (Table 1). The
selective inhibition of PLC (U-73112) also reduced the ET-1-stimulated
Ca2+ mobilization by 60.3 ± 8.5%. In contrast, 100 µM L-NAME (inhibitor of NOS) caused a twofold increase in
Ca2+ mobilization in the presence of ET-1.
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Modification of ET-1 effects.
To test the possibility of interplay between the endogenously or
exogenously derived NO and ET-1 on Ca2+ mobilization, we
pretreated the HBEC with L-arginine in the presence or
absence of L-NAME. As shown in Table 1,
L-arginine reduced the ET-1-stimulated Ca2+
mobilization. This response was prevented by L-NAME
treatment. The addition of NOR-1 (the exogenous NO donor) decreased the
ET-1-induced Ca2+ mobilization in a dose-dependent manner
(Fig. 1A). ODQ (the specific inhibitor of guanylyl cyclase) or Rp-8-CPT-cGMPS (a PKG
inhibitor) prevented the NOR-1-induced inhibition of ET-1-stimulated
Ca2+ mobilization (Fig. 1, B and C,
respectively). On the other hand, exposure of HBEC to 8-bromo-cGMP
dose-dependently decreased the ET-1-induced Ca2+
mobilization (Fig. 2A).
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Modification of ET-1 response distal to ETA receptor. To examine the possible interaction of NO with agents involved in the pathway of factors between the point of ET-1 stimulation (receptor binding) and Ca2+ mobilization, we exposed the HBEC to an activator of G protein (Mas7), IP3, or an inhibitor of Ca2+-ATPase (thapsigargin). As shown in Fig. 2B, NOR-1 inhibited Ca2+ mobilization induced by each of these substances in a manner similar to that observed with ET-1, albeit to a lesser extent. This effect was also mimicked by 8-bromo-cGMP pretreatment of HBEC (Fig. 2C). In addition, NOR-1 decreased the ET-1 stimulation of IP2 (30.0 ± 2.3%) and IP3 formation (32.0 ± 1.6%).
Cytoskeleton and VASP Response to ET-1 and/or NO
Because cGMP/cAMP systems play a role in the interaction between NO and ET-1 and their effects on Ca2+ mobilization, we first addressed the question of cGMP localization to assess whether this pathway may be involved in the cytoskeleton response to either NO or ET-1. Cells stained as depicted in Fig. 3 clearly demonstrate that PKG is present in these cells. PKG staining is manifested by green granules throughout the cytoplasm; their presence in close proximity to actin fibers is indicated by the readily visible yellow-green granules (Fig. 3, row 1, column 3). The more dense yellow-green granules are most predominantly observed in perinuclear locations in association with vimentin filaments (Fig. 3, row 2, column 3). The cells exposed to ET-1 show a marked increase in thickness and density of F-actin filaments (Fig. 3, row 3). Centrally stained green and yellow-green granules indicated areas of PKG, some colocalized with actin staining. The NOR-1 pretreated control cells (Fig. 3, row 4) showed a slight thinning of the actin fibers and PKG staining similar to that seen in untreated controls. The NOR-1-pretreated control cells that were subsequently exposed to ET-1 displayed a decrease in thickness and density of F-actin (Fig. 3, row 5) compared with cells treated with ET-1 alone. An increased colocalization of F-actin was apparent at the distal bundle of fibers (patches of yellow granules). Similar observations were seen with vimentin fibers (results not shown). Although not shown, the above results obtained with 100 µM NOR-1 and 50 nM ET-1 were also seen with cells treated with 50 µM NOR-1 and 20 nM ET-1.
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To assess whether ET-1 and/or NO also has an effect on VASP, we
examined the localization and phosphorylation of this protein. Positive
VASP capping of the terminal segments of F-actin was seen in HBEC (not
shown) and was similar to that previously reported in human brain
microvascular EC (4). It was not possible to differentiate
the nonphosphorylated from phosphorylated VASP because a specific
antibody to phosphorylated VASP was unavailable. However, VASP
phosphorylation by NOR-1 could be visualized by Western blot analysis
(Fig. 4). The average level of increased
phosphorylation of VASP was two- to sixfold higher than that of the
control. The NOR-1-induced phosphorylation of VASP was prevented by
pretreatment of HBEC with either ODQ or Rp-8-CPT-cGMPS. ET-1 also
decreased the NOR-1 stimulation of VASP phosphorylation even though
treatment with ET-1 alone slightly increased it (not shown).
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DISCUSSION |
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In the last decade numerous reports have described the involvement of ET-1 and NO in a variety of biological processes (22, 24, 32, 36). Their role in maintaining the vascular tone has been controversial. Nevertheless, many studies have demonstrated that inhibition of NOS reduces resting cerebral blood flow without affecting cerebral glucose utilization and oxygen consumption (16). The cerebral microcirculation has also been shown to be affected by NO, in part through a cGMP-dependent mechanism (10). On the other hand, the participation of ET-1 in controlling the vascular tone has been attributed mainly to its effect on the production of NO (2, 12, 15, 19). Several lines of evidence demonstrate the existence of the reciprocal relationship between the secretion and release of ET-1 and NO in creating a balanced system implicated in the regulation of blood flow. It has also been shown that inhibition of NOS in animals and human volunteers induces systemic (pulmonary and renal) vasoconstriction, which was abrogated by treatment with an ETA receptor antagonist (12, 19, 25, 41). Hypoperfusion induced by cerebral ischemia alone or in the presence of NOS inhibitors was also blocked by treatment with an ETA receptor antagonist (38). ET-1 and NO have also been demonstrated to affect the BBB integrity (23, 34, 36). However, little information is available regarding their interactive effects on the properties of human capillary endothelium (the main constituent of BBB), including their involvement in autocrine and paracrine events.
The results of this study unequivocally demonstrate that NO can interfere with HBEC responses evoked by ET-1. They are manifested by NO-mediated reduction of ET-1-stimulated Ca2+ mobilization associated with alteration of cytoskeleton arrangement (actin and vimentin) as well as with phosphorylation of VASP. These cells are capable of converting L-arginine to citrulline and producing NO through activation of eNOS (24). The L-arginine (NO substrate)-induced decrease of ET-1-stimulated Ca2+ mobilization and the reversal of this effect by L-NAME (the inhibitor of NOS activity) are illustrative of the autoregulatory property of HBEC. The demonstrated similar response of ET-1-stimulated Ca2+ mobilization to NOR-1 (the NO donor) underscores the assertion that the HBEC response to ET-1 can be abrogated by either exogenous or endogenous NO. It is known that ET-1 and NO, which are produced by endothelium or exogenously derived from other sources, can counteract each other at the secretory or receptor levels (2, 33, 37, 41). The ability of NO to counteract the effect of ET-1-induced Ca2+ transient was reported in platelets, smooth muscles, polymorphonuclear leucocytes, Chinese hamster ovary (CHO) cells stably transfected with ETA receptor cDNA, and human brain microvascular EC. However, in some of these cells this effect was not dependent on cGMP signal transduction (3, 11, 13, 26). In the present study, the capacity of NOR-1 to modulate Ca2+ mobilization stimulated by Mas7, IP3, and thapsigargin clearly indicates that NO may inhibit Ca2+ mobilization induced by ET-1 by mechanisms other than those just involving the ET-1 receptor. The prevention of NOR-1 reduction of ET-1-stimulated Ca2+ mobilization by ODQ or Rp-8-CPT-cGMPS (respective inhibitors of guanylyl cyclase and PKG activity) indicates that this event is mediated by the cGMP/PKG pathway. The cGMP-induced decrease of Ca2+ mobilization induced by ET-1, Mas7, IP3, or thapsigargin corroborates this contention.
The effects of ET-1 and/or NO on the transcriptional levels of ET-1, ETA, ETB, and eNOS were examined by RT-PCR. These preliminary experiments indicated that treatment with 50 µM NOR-1 for 1 min and/or with 20 nM ET-1 for 30 s had no effect on ET-1, ETA, ETB, or eNOS mRNA expression (results not shown). This finding is not surprising because the short period of HBEC exposure to either of these agents is not sufficient for induction of mRNA expression of the respective genes. Nevertheless, this negative result emphasizes that the noted endothelial response to ET-1 and NO is independent of, and/or distal to, transcriptional changes in the above genes.
The presence of PKG has been reported in a variety of cells; in the smooth muscle it was shown to colocalize with intermittent vimentin filaments (21, 27). The results shown here demonstrate that PKG colocalizes not only with vimentin but also with the actin filaments. Although PKG was reported to be present in the endothelium, its colocalization with actin filaments was not described previously. The morphological coexistence of PKG with actin observed in particular at the terminal segments of actin fibers after the combined treatment (NOR-1 + ET-1) strongly suggests a functional interaction. The previous and present demonstration of endothelial VASP in the same region of actin as that of PKG is of special interest. VASP belongs to a family of proline-rich proteins. It is a cytoskeleton-associated protein that interacts with profilin, vinculin (a component of adherens junctions), and zyxin (14). It has been shown to be a significant substrate for cyclic nucleotide-dependent kinases (i.e., cAMP, cGMP) in platelets and cardiovascular cells (14, 30, 31). Studies on EC derived from human umbilical veins have indicated that phosphorylation of VASP by PKG results in the loss of VASP and zyxin from focal adhesions. These and other investigations suggested that VASP phosphorylation might function as a negative regulator of actin activities (i.e., vasorelaxation, permeability) (7). The results of this study also clearly demonstrate that the phosphorylation of VASP is mediated by cGMP/PKG, because ODQ or Rp-8-CPT-cGMPS prevented the conversion of nonphosphorylated protein (46 kDa) to phosphorylated VASP (50 kDa). The precise mechanism responsible for the noted ET-1- and NO-induced cytoskeletal rearrangement is unknown. Previously, we suggested that the ET-1-stimulated F-actin formation might be mediated by ETA receptor and Rho, a member of the small GTPases (3). This supposition was recently confirmed by demonstrating that the ET-1-stimulated stress fiber formation is mediated through Rho/Rock in CHO transfected with a mutant ETA receptor (18).
The results presented here indicate that endothelial Ca2+ mobilization and cytoskeletal filaments can be rapidly altered by ET-1, NO, and/or cGMP. Each of these agents along with other vasoactive substances (i.e., thrombin, bradykinin, Ca2+, and cAMP) has been reported to influence cellular permeability in addition to vasomotor activities (5, 8, 23, 34, 40). This report demonstrates for the first time that HBEC possess the intrinsic capability to regulate the rapid, dynamic responses associated with contraction, relaxation, and permeability of large and small vessels. It is hereby suggested that such properties and mechanisms may be utilized by the HBEC in their capacity to regulate BBB function in addition to partaking in the control of vascular tone and cerebral blood flow.
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ACKNOWLEDGEMENTS |
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This work was partially supported by Work Unit funding obtained from the Office of Naval Research (602233N.333.120.A0102). The opinions expressed in this paper are those of the authors and do not reflect the official policy of the Department of Navy, Department of Defense, of the U.S. Government.
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. M. McCarron, Naval Medical Research Center, Resuscitative Medicine Dept., 503 Robert Grant Ave., Silver Spring, MD 20910-7500 (E-mail: mccarronr{at}nmrc.navy.mil).
10.1152/ajpcell.00305.2002
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