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(Circulation Research. 2000;87:323.)
© 2000 American Heart Association, Inc.

Cellular Biology

Human Brain Capillary Endothelium

2-Arachidonoglycerol (Endocannabinoid) Interacts With Endothelin-1

Ye Chen, Richard M. McCarron, Yukoh Ohara, Joliet Bembry, Nabil Azzam, Fred A. Lenz, Esther Shohami, Raphael Mechoulam, Maria Spatz

From the Naval Medical Research Center (R.M.M.C., Y.O.), Bethesda, Md; Department of Neurosurgery (F.A.L.), Johns Hopkins University School of Medicine, Baltimore, Md; Hebrew University (E.S., R.M.), Jerusalem, Israel; Stroke Branch (Y.C., J.B., N.A., M.S.), National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Md.

Correspondence to Maria Spatz, MD, National Institutes of Health, NINDS, Stroke Branch, 36 Convent Dr, MSC 4128, Bethesda, MD 20892-4128. E-mail spatzm{at}ninds.nih.gov

	Abstract

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Abstract—In brain, the regulatory mechanism of the endothelial reactivity to nitric oxide and endothelin-1 may involve Ca²⁺, cytoskeleton, and vasodilator-stimulated phosphoprotein changes mediated by the cGMP/cGMP kinase system.¹ Endothelium of human brain capillaries or microvessels is used to examine the interplay of endothelin-1 with the putative vasorelaxant 2-arachidonoyl glycerol, an endogenous cannabimimetic derivative of arachidonic acid. This study demonstrates that 2-arachidonoyl glycerol counteracts Ca²⁺ mobilization and cytoskeleton rearrangement induced by endothelin-1. This event is independent of nitric oxide, cyclooxygenase, and lipoxygenase and is mediated in part by cannabimimetic CB1 receptor, G protein, phosphoinositol signal transduction pathway, and Ca²⁺-activated K⁺ channels. The induced rearrangements of cellular cytoskeleton (actin or vimentin) are partly prevented by inhibition of protein kinase C or high levels of potassium chloride. The 2-arachidonoyl glycerol–induced phosphorylation of vasodilator-stimulated phosphoprotein is mediated by cAMP. These findings suggest that 2-arachidonoyl glycerol may contribute to the regulation of cerebral capillary and microvascular function.

Key Words: brain endothelial function • 2-arachidonoglycerol • endothelin-1 • endothelium • cannabinoids

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
An endogenous cannabinoid originally isolated from gut and brain, 2-arachidonoyl glycerol (2-AG), can be generated and released from blood and vascular cells.^{2 3 4} 2-AG is an intrinsic physiological ligand for the cannabinoid CB1 receptor³ ; these receptors are expressed in the brain and peripheral organs including vessels, platelets, and macrophages.^{4 5 6 7} 2-AG induces a rapid transient elevation in intracellular Ca²⁺ through activation of the CB1 receptor in NG108-15 cells⁸ ; this response is independent of cyclooxygenase and lipoxygenase.^{4 8} 2-AG also inhibits adenyl cyclase in mouse spleen cells and twitch responses in vas deferens.⁴ This novel bioactive lipid causes hypotension that may be attributed to its hyperpolarizing properties.⁹ It is suggested that induction of 2-AG release in endothelium occurs in parallel to nitric oxide (NO) and involves activation of cholinergic receptors.¹⁰ Previous studies in vivo and in vitro demonstrated that NO, the most effective endothelium-derived relaxing factor (EDRF) and endothelial-derived hyperpolarizing factor (EDHF),¹¹ may have a close functional relationship with the potent vasoconstrictor endothelin-1 (ET-1) in regulating the endothelium-dependent capillary and microvascular responses in the brain.¹

ET-1 induces the production of NO, which, in turn, reduces the secretion of ET-1. NO can also affect the binding of ET-1 to its receptor^{12 13 14} and alter the ET-1–stimulated Ca²⁺ mobilization and cytoskeleton arrangement in the endothelium derived from human cerebral capillaries (HBECs) and microvessels (HBMECs). This effect is mediated by the cGMP/cGMP kinase system and is associated with phosphorylation of vasodilator-stimulated phosphoprotein (VASP). The results of the present study indicate that endothelium possesses the intrinsic machinery required to balance the effects of NO and ET-1 and regulates cerebral capillary and microvascular function. This equilibrium may be disturbed in pathological conditions such as hypertension, arteriosclerosis, hemorrhagic shock, and diabetes mellitus.^{11 15 16} It is hypothesized that 2-AG may function as a vasorelaxant that can potentially interact with ET-1 to regulate endothelium-dependent vascular reactivity.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
2-AG was prepared by R. Mechoulam, Hebrew University. Masterpan (an activator of G protein) and ET-1 were from Peninsula Laboratories; BAPTA-AM, bisindolylmaleimide (selective inhibitor of protein kinase C [PKC]), charybdotoxin (blocker of several K⁺ channel subtypes), apamin (selective blocker of small-conductance Ca²⁺-activated K⁺ channels), and N^G-nitro-L-arginine methyl ester (L-NAME; NO synthase inhibitor) were from Calbiochem; fluo 3-AM and Texas Red-X phalloidin were from Molecular Probes; H7 (nonselective protein kinase inhibitors: PKA>PKC=PKG), H8 (nonselective protein kinase inhibitors: PKG>PKA>PKC), ouabain (inhibitor of Na⁺/K⁺ATPase), and quinine (inhibitor of Ca²⁺-dependent K⁺ channels) were from RBI; 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) (selective inhibitor of guanylyl cyclase) was from Alexis Biochemicals; and SR141716A, a selective CB1 receptor antagonist, was provided by SRI International. The monoclonal VASP antibody was from Transduction Laboratories; monoclonal vimentin antibody was from Dako Laboratories; and peroxidase-labeled antibody was from Amersham. Indomethacin acid (inhibitor of cyclooxygenase), nordihydroguaiaretic acid (inhibitor of lipoxygenase), BaCl (inhibitor of inwardly rectifying K⁺ channels), and all other chemicals were from Sigma Chemical.

Calcium and Inositol Phosphate Analyses
Endothelium HBECs and HBMECs were prepared and cultivated as previously described.¹⁷ The purity was >95% as determined by the presence of von Willebrand factor VIII and the absence of markers for astrocytes, microglia, and pericytes. Four cell lines (passages 6 to 13) were used in the present experiments.

The levels of Ca²⁺ in confluent cultures (24-well plates) were assessed with a 2.5-µmol/L fluo 3-AM fluorescent probe as previously described¹ using a CytoFluor II fluorescence multiwell plate reader (PerSeptive Biosystems) with a fluorescein filter pair (excitation 485±20 nm; emission 530±25 nm). Ca²⁺ concentration changes were expressed as fluorescence intensity using the formula: F(experimental)-F₀(initial)/F₀x100, which normalizes differences in dye loading and cell numbers. A modified technique was used to determine inositol 4,5 biphosphate (IP₂), and inositol 1,4,5-triphosphate (IP₃) formation was determined as previously described.¹⁸

Western Blotting
HBECs and HBMECs (35-mm dishes) were exposed to tested agents for the indicated times before lysis in 60 mmol/L Tris-HCl buffer containing 8 mol/L urea and 1% SDS (pH 6.8). Protein was determined using a Bio-Rad protein assay (Bio-Rad Laboratories). Cell proteins (5 µg) were electrophoresed (10% SDS-PAGE) and transferred onto 0.45-µm pore polyvinylidene difluoride membranes (Millipore). Blots were blocked with 10% goat serum in TNB-T buffer (150 mmol/L Tris, 50 mmol/L NaCl, and 0.05% Tween; pH 7.5) to reduce nonspecific staining and probed overnight with VASP antibody (1:3000) in TNB-T at 4°C and goat anti-mouse HRP-conjugated (1:6000). Protein bands were visualized with the enhanced chemiluminescence detection system (Amersham), and relative intensities were quantified by scanning densitometry (Eagle Eye II).

Immunocytochemistry
HBECs and HBMECs (coverslips) were fixed in 3.7% formaldehyde, permeabilized in 0.1% Triton X-100, blocked with 1% BSA, and stained for actin with Texas Red-X phalloidin as previously described.¹ Cells were subsequently incubated with VASP antibody (followed by FITC-goat anti-mouse IgG (1:200). Additional experiments used monoclonal vimentin antibody (1:200) for 1 hour followed by FITC-goat anti-mouse IgG (1:400) for 45 minutes. Coverslips were mounted in Vectashield (Vector Laboratories) and viewed by confocal microscopy (Carl Zeiss).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of 2-AG on ET-1–Induced Ca²⁺ Mobilization
ET-1 dose dependently (EC₅₀=20 nmol/L) stimulated intracellular Ca²⁺ mobilization, and all subsequent experiments used 20 nmol/L ET-1. 2-AG inhibited the ET-1–stimulated intracellular Ca²⁺ mobilization in a dose-dependent manner (Figure 1A). This response was not affected by 10 µmol/L L-NAME (Figure 1B) or 100 µmol/L L-NAME (not shown); 10 µmol/L indomethacin (Figure 1B) or 10 µmol/L nordihydroguaiaretic acid (not shown) were also ineffective. The stable 2-AG analog HU313 (arachidonoyl ester) reduced the ET-1–stimulated Ca²⁺ mobilization in a manner similar to 2-AG (not shown). Treatment with the selective CB1 receptor antagonist SR141716A dose dependently prevented the 2-AG reduction of ET-1–stimulated Ca²⁺ mobilization (Figure 1C).

View larger version (66K): [in this window] [in a new window]   Figure 1. Characterization of the effect of 2-AG on intracellular Ca2+ response of HBECs to ET-1. Confluent HBECs were exposed to 20 nmol/L ET-1 alone for 30 seconds or to 2-AG for 15 minutes in the presence or absence of various inhibitors. Data are mean±SEM of 3 to 14 experiments (as indicated for each panel) performed in quadruplicate and are expressed as a percentage of response observed with 20 nmol/L ET-1 alone; HBECs were derived from 4 cell lines. No significant differences in inhibition by 50 µmol/L 2-AG were observed in the various cultures; this concentration of 2-AG was used in all experiments unless indicated otherwise. A, Effect of increasing concentrations of 2-AG on ET-1–stimulated Ca2+ mobilization (n=14). B through F, Effect of the following factors on 50 µmol/L 2-AG–mediated inhibition of ET-1–stimulated Ca2+ mobilization: 10 µmol/L L-NAME for 40 minutes or 10 µmol/L indomethacin (Indo) for 30 minutes (n=4) (B); increasing concentrations of CB1 for 15 minutes (n=4) (C); 200 nmol/L BIS for 20 minutes (n=5) (D); 200 nmol/L quinine (QUI), 100 nmol/L apamin (APA), and 100 nmol/L charybdotoxin (CHB) for 15, 30, and 15 minutes, respectively (n=5) (E); and 15 and 50 mmol/L K+ (n=4) (F). * ,**Significant difference from ET-1 alone and from ET-1+50 µmol/L 2-AG, respectively; P<0.01 by ANOVA with Fisher’s protected least-significant difference. L-NAME or Indo alone (B) had no effect on ET-1–stimulated Ca2+ mobilization.

The addition of 2-AG alone gradually increased intracellular Ca²⁺ content, albeit to significantly lower levels (ie, 8% to 19%) than were observed with ET-1 alone. This effect was not observed in Ca²⁺-free medium. Treatment with SR141716A (1 µmol/L) inhibited (35%) the 2-AG–induced Ca²⁺ uptake; SR141716A alone had no effect on Ca²⁺ uptake.

The involvement of G proteins and certain second messengers in the 2-AG–mediated effects on ET-1–stimulated Ca²⁺ mobilization was next examined. Treatment with 2-AG reduced the Ca²⁺ mobilization stimulated by 5 µmol/L masterpan by 30%. In addition, 2-AG pretreatment reduced the ET-1–stimulated formation of IP₂ by 70% and IP₃ by 51%. The involvement of second messengers was also tested using the following inhibitors: 200 nmol/L bisindolylmaleimide (BIS); 30 µmol/L H7; 5 µmol/L H8; and 10 µmol/L ODQ. Only treatment with BIS hindered the 2-AG modulation of ET-1–stimulated Ca²⁺ mobilization (Figure 1D), indicating that this effect is partly mediated by PKC and not by cGMP or cAMP kinases.

HBECs were pretreated with agents implicated in the hyperpolarization of the cell membrane (ie, selective inhibitors of K⁺ channel or high [K⁺]) to further elucidate the possible pathways responsible for the 2-AG–induced effects. Quinine, apamin, or charybdotoxin partly blocked the 2-AG reduction of ET-1–stimulated intracellular Ca²⁺ mobilization (Figure 1E). Neither BaCl nor ouabain had any effect (not shown). Exposure of HBECs to 15 or 50 mmol/L K⁺ prevented the 2-AG–induced reduction of ET-1–stimulated intracellular Ca⁺ mobilization (Figure 1F).

Effect of 2-AG and ET-1 on Cytoskeleton and VASP
2-AG affected the control and the ET-1–stimulated cluster of F-actin (Figures 2C and 2D) and vimentin (Figures 3C and 3D); these changes were manifested by the rarefaction of filaments. Pretreatment with either 200 nmol/L BIS (Figures 2E and 2F) or 15 mmol/L KCl (not shown) partially prevented the thinning of F-actin; similar effects were observed on vimentin filaments (not shown). Positive staining for the actin binding protein VASP was localized at the terminal segments of F-actin filaments (Figures 2A through 2F). On the basis of morphology, it is not possible to differentiate the nonphosphorylated and phosphorylated VASP (specific phosphorylated VASP antibodies are currently unavailable). However, VASP phosphorylation, as seen by Western blot analysis (Figure 4), clearly demonstrated that 2-AG stimulation is prevented with H8; ET-1 had no effect. Pretreatment with H7 and BIS also had no effect (not shown).

View larger version (152K): [in this window] [in a new window]   Figure 2. Cytoskeleton F-actin and VASP in HBECs. Confluent HBECs grown on glass coverslips and processed as described in Materials and Methods and Figure 1 legend were exposed to medium alone (A); ET-1 (20 nmol/L) (B); 2-AG (50 µmol/L) (C); 2-AG followed by ET-1 (D); BIS (200 nmol/L) followed by 2-AG (E); and BIS followed by 2-AG followed by ET-1 (F). All cells were stained with phalloidin (red) and VASP (yellow-green) and examined with confocal fluorescence microscope (magnification x63). A, Cells display slender longitudinal and crisscrossing F-actin filaments that form bundles at the margins of overlapping cells; VASP is seen at the focal areas (yellow-green). B, ET-1–treated cells show a marked thickness and prominence of F-actin filaments with terminal caps of VASP. C and D, Both the control (C) and the ET-1–exposed HBECs pretreated with 2-AG (D) display rarefaction of the F-actin filaments capped with VASP. In contrast, HBECs pretreated with BIS display increased F-actin filaments that are more delicate in control cells (E) than in cells treated with 2-AG and ET-1 (F). The localization of VASP is unchanged but overshadowed by the F-actin filaments.

View larger version (164K): [in this window] [in a new window]   Figure 3. Cytoskeleton vimentin in HBECs. Cells on glass coverslips were processed as described in Materials and Methods and exposed to conditions as described in Figures 2A through 2D. All cells were treated with antibody for vimentin and examined with Zeiss Axioplan fluorescence microscopy (magnification x63). A, Control HBECs show a web of delicate intermediate filaments. B, Cells display very dense meshed fibers. C, HBECs pretreated with 2-AG show rarefaction of the web of intermediate filaments compared with control (A). D, HBECs pretreated with 2-AG before ET-1 display a rarefied web of fiber compared with cells exposed to ET-1 alone (B).

View larger version (36K): [in this window] [in a new window]   Figure 4. Western blot characterization of the effect of 2-AG on phosphorylation of VASP. Samples from HBECs treated with indicated compounds were analyzed for VASP, as described in Materials and Methods. The shift from 46 to 50 kDa indicates the apparent phosphorylation of VASP and is expressed as a ratio of the respective densities. The levels of increased phosphorylation compared with control for this representative experiment were 0.5-fold (ET-1), 3.5-fold (2-AG), 3.9-fold (2-AG+ET-1), and 0.9-fold (2-AG+H8).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study indicates that 2-AG reduces the ET-1–stimulated Ca²⁺ mobilization in HBECs and HBMECs. The results also indicate that the 2-AG–mediated effects may involve the cytoskeleton rearrangement of actin and vimentin filaments. 2-AG is the second arachidonic acid derivate (after anandamide) and is enzymatically hydrolyzed to arachidonic acid. Although these substances structurally resemble eicosanoids, they differ in their biosynthetic pathway.¹⁹ Both the endogenously produced 2-AG and anandamide are ligands for cannabinoid receptors.^{3 4 10} Studies suggested that these compounds act as neuromodulators and vasomodulators; previous reports demonstrate that these substances may affect the cardiovascular system (hypotension and bradycardia).^{4 9 20} So far, the vasoactive role of 2-AG as a putative EDRF/EDHF has not been extensively studied. Reports indicate that anandamide and 2-AG (among other EDRFs/EDHFs) can be released in parallel with NO and, in this way, contribute to endothelium-dependent relaxation.⁹ This event may occur through activation (direct or indirect) of vascular smooth muscle K⁺ channels.^{11 21} There are many conflicting reports regarding the effectiveness of anandamide or 2-AG as EDRF/EDRH agents, their dependence on activation of CB1 receptors, and their mediation of various factors (NO, cyclooxygenase, lipoxygenase).^{3 4 6 10 22 23} In addition, there is little information regarding possible interaction between these potential vasodilators with vasoconstrictors that could contribute to the maintenance of vascular homeostasis. The observed SR141716A-induced reversal of 2-AG reduction of ET-1–stimulated Ca²⁺ mobilization and inhibition of 2-AG–induced Ca²⁺ uptake implicates CB1 receptor in these responses. The lack of Ca²⁺ response to SR141716A alone supports this conclusion. These observations strongly suggest that HBECs and HBMECs have functional CB1 receptors. Although CB1 receptors were demonstrated in pial vessels, smooth muscle cells derived from cerebral microvessels, and peripheral vascular endothelium,^{3 6 7 24} this is the first demonstration of their presence in human brain endothelium.

Investigations indicated that the signal transduction pathway for the 2-AG modulation of ET-1–stimulated Ca²⁺ mobilization was independent of NO synthase, cyclooxygenase, and lipoxygenase activity (ie, treatment with inhibitors L-NAME, indomethacin, and nordihydroguaiaretic acid was ineffective). The effect of indomethacin is of particular interest. 2-AG, like anandamide, is metabolized to arachidonic acid, and we have observed that the effect of anandamide on ET-1–stimulated Ca²⁺ mobilization is cyclooxygenase-dependent (unpublished data, 2000). The demonstrated G protein, inositol phosphate hydrolysis, and PKC mediation of 2-AG–induced effects on ET-1–stimulated Ca²⁺ mobilization indicate that 2-AG affects the pathway distal to the receptor. The involvement of G protein is supported by studies showing that CB1 receptors have structural features of a G protein–coupled receptor.⁵ The additional findings are consistent with the CB1-coupled signal transduction pathways as stipulated by others (see Ameri⁵ for review). The capacity of inhibitors of Ca²⁺-dependent K⁺ channels or K⁺ ions to modulate 2-AG–induced reduction of ET-1–stimulated Ca²⁺ mobilization supports the reported involvement of these channels in vasorelaxation induced by EDHF.^{11 21} Because the specificity of some of the inhibitors used is not ensured or well investigated, various inhibitors were used to more accurately characterize the pathways.

The present findings represent a demonstration, for the first time, of the effects of 2-AG on the rarefaction of cytoskeletal filaments and the phosphorylation of the actin binding protein VASP. Although the exact mechanisms responsible for these effects are unknown, it is intriguing that the substances that phosphorylate VASP also rarefied the actin and vimentin assembly. Interestingly, similar results were observed with NO.^{1 25} VASP has been associated with actin filaments and local adhesion²⁶ and was suggested to impede or promote actin assembly.²⁷ These activities may involve substances known to elevate either cGMP or cAMP (ie, prostaglandin E₂, NO donors, respectively) because they stimulate phosphorylation of VASP.^{26 28}

The data indicate that 2-AG, a cannabinoid agonist, counteracts ET-1–induced cerebral capillary and microvascular endothelial responses (ie, Ca²⁺ mobilization and cytoskeleton rearrangement). The inhibition of this response by a CB1 receptor antagonist indicates that functional cannabinoid CB1 receptors may be expressed by these cells. The results represent the first direct evidence of a functional interaction between 2-AG and ET-1 and provide a potential alternative pathway for abrogating ET-1–inducible, endothelium-dependent capillary, and/or microvascular effects in the brain.

	Acknowledgments

 
This work was partially supported by grants from the National Institute on Drug Abuse of the National Institutes of Health (DA-09789) and the Office of Naval Research (61153N.MR04120.001.1804).

Received June 22, 2000; revision received July 12, 2000; accepted July 13, 2000.

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