JPET Fast Forward. Published on March 22, 2007 as DOI: 10.1124/jpet.106.117549 JPET FastThis article Forward. has not beenPublished copyedited on and Marchformatted. 22, The 2007 final version as DOI:10.1124/jpet.106.117549 may differ from this version.

JPET # 117549

Anandamide-mediated CB1/CB2 -independent NO production in rabbit aortic endothelial cells

LaTronya McCollum, Allyn C. Howlett and Somnath Mukhopadhyay1 Neuroscience of Drug Abuse Research Program Julius. L. Chambers Biomedical/Biotechnology Research Institute North Carolina Central University, Durham, NC 27707

Downloaded from

jpet.aspetjournals.org

at ASPET Journals on September 30, 2021

1

Copyright 2007 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on March 22, 2007 as DOI: 10.1124/jpet.106.117549 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 117549

Running title: Novel receptor mediated eNOS activation

1Corresponding author

Somnath Mukhopadhyay, Ph.D. Neuroscience of Drug Abuse Research Program J. L. Chambers Biomedical/Biotechnology Research Institute

North Carolina Central University Downloaded from 700 George Street, Durham, NC 27707 Ph # (919) 530-7762

FAX (919) 530-7760 jpet.aspetjournals.org [email protected]

Abbreviations used are: Abn-CBD, abnormal ; DAF-DA, 4-amino-5- methylamino-2',7'-difluorofluorescein diacetate; ECL, enhanced chemiluminescence; eNOS, at ASPET Journals on September 30, 2021 endothelial NO synthase; MAPK, mitogen-activated protein kinase; PCR, polymerase chain reaction; PKA, cyclic AMP-dependent protein kinase; PI3-kinase, phosphatidylinositol 3-kinase; PVDF, polyvinylidene difluoride; PTX, pertussis toxin; RAEC, rabbit aortic endothelial cells; RT-PCR, reverse transcription-polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate- polyacrylamide gel electrophoresis.

Abstract: 204 words Introduction: 491 words Discussion: 1391 words References: 40 Number of Figures : 7 Table: 0 Number of text pages (Abstract, Introduction, Methods, Result, Discussion, References, Fig. Legends: 18

2 JPET Fast Forward. Published on March 22, 2007 as DOI: 10.1124/jpet.106.117549 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 117549

ABSTRACT

We have previously shown that the endocannabinoid anandamide and its metabolically stable analog (R)-methanandamide produce vasorelaxation in rabbit aortic ring preparations in an endothelium-dependent manner that could not be mimicked by other CB1 receptor agonists (Mukhopadhyay et al., 2002). Here we show that (R)-methanandamide and stimulated NO production in rabbit aortic endothelial cells (RAEC) in a dose- dependent manner, but other CB1 and CB2 receptor agonists such as CP55940 and WIN55212 failed to do so. CB1 antagonists (also known as SR141716) or LY320135, and CB2 Downloaded from antagonist SR144528, failed to block (R)-methanandamide-mediated NO production in RAEC. However, anandamide receptor antagonist O-1918 blocked (R)-methanandamide-mediated NO

production in RAEC. Reverse transcriptase-polymerase chain reaction and Western blot analyses jpet.aspetjournals.org failed to detect the CB1 receptor in RAEC, making this a good model to study non-CB1 responses to anandamide. (R)-methanandamide produced eNOS phosphorylation via the activation of phosphoinositide 3-kinase-Akt signaling. Inhibition of Gi signaling with pertussis toxin, or phosphatidylinositol 3-kinase activity with LY294002, resulted in a decrease in (R)- at ASPET Journals on September 30, 2021 methanandamide-induced Akt phosphorylation and NO production. Results from this study suggest that in RAEC, (R)-methanandamide acts on a novel non-CB1 and non-CB2 anandamide receptor, and signals through Gi and PI3-kinase leading to Akt activation, eNOS phosphorylation and NO production.

3 JPET Fast Forward. Published on March 22, 2007 as DOI: 10.1124/jpet.106.117549 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 117549

To date, two cannabinoid receptors have been identified: CB1 cannabinoid receptors expressed predominantly in the brain (Howlett et al., 2004; Howlett et al., 2002) and also in peripheral tissues including the vascular endothelium (Kunos et al., 2002; Liu et al., 2000), and

CB2 cannabinoid receptors expressed in the cells of the immune and hematopoetic systems (Klein et al., 2003; Howlett et al., 2002), as well as in neurons (Van Sickle et al., 2005). Both

CB1 and CB2 cannabinoid receptors are coupled to Gi/Go proteins and inhibit adenylate cyclase (Howlett et al., 2004) and can also activate p42/44 mitogen activated protein kinase (MAPK) (Bouaboula et al., 1997; see Howlett 2004 review) and the phosphatidylinositol 3-kinase

(PI3K)/Akt pathway (Liu et al., 2000). Downloaded from

The endocannabinoid anandamide and its metabolically stable analog (R)-

methanandamide have been shown to produce hypotension and bradycardia in animal models jpet.aspetjournals.org (see (Kunos et al., 2000a; Kunos et al., 2000b; Kunos 2002) for review), and produce vasorelaxation in a number of vascular beds (Mukhopadhyay et al., 2002; Kunos 2002). Several diverse mechanisms have been suggested to explain these physiological responses, including 1) anandamide serving as an endothelial-derived hyperpolarizing factor (Randall et al., 1997); 2) at ASPET Journals on September 30, 2021 anandamide-stimulated CB1 cannabinoid receptors being sympathoinhibitory (Kunos 2002 for review); 3) anandamide-stimulated TRPVR vanilloid receptors promoting release of mediators (Di Marzo et al., 2002) or 4) anandamide altering the function of gap junctions (Chaytor et al., 1999). There is growing evidence over the last few years that anandamide-mediated vasodilation in the arterial (Mukhopadhyay et al., 2002), isolated mesenteric (see (Kunos et al., 2000a; Kunos et al., 2000b; Kunos 2002) for review) and some other vascular preparations (O'Sullivan et al.,

2004) is independent of CB1 or CB2 cannabinoid receptors, and produced via the activation of a putative non-CB1/CB2 anandamide receptor. Findings from our laboratory showed that in rabbit aortic ring preparations (Mukhopadhyay et al., 2002), anandamide or (R)-methanandamide, acting on a non-CB1/CB2 endothelial site, produced vasorelaxation. In that study, anandamide- or (R)-methanandamide-induced vasorelaxation was sensitive to pertussis toxin, and was blocked by nitric oxide (NO) synthase (NOS) inhibitors, suggesting that anandamide might act on an endothelial non-CB1/CB2 Gi-protein coupled receptor to activate endothelial NO synthase (eNOS) to produce NO.

4 JPET Fast Forward. Published on March 22, 2007 as DOI: 10.1124/jpet.106.117549 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 117549

In the present study, the endothelial cell response to the endocannabinoid anandamide has been investigated using cultured rabbit aortic endothelial cells (RAEC) as a model system. We have pharmacologically characterized the anandamide response leading to NO production, and investigated the molecular signaling mechanism of anandamide-mediated eNOS activation.

METHODS

Cell culture and reagents. RAEC primary culture was established by Dr. Mukhopadhyay in collaboration with Dr. Barry Chapnick (Saint Louis University School of Medicine). Cells were Downloaded from maintained at 37 °C under an atmosphere of 5% CO2 in phenol red-free endothelial basal media (EMB-A) (Clonetics Corp.) containing 2% fetal bovine serum and growth factors for endothelial

cells (Clonetics Bullet kit). Cells from passages three to five were used for experiments. jpet.aspetjournals.org

Measurement of NO. The production of NO was measured using 4-amino-5-methylamino- 2',7'- difluorofluorescein diacetate (DAF-DA) (Molecular Probes), which has been previously reported to be linearly related to NO production (Montagnani et al., 2001). Endothelial cells at ASPET Journals on September 30, 2021 were grown to 50% confluence on cover-slips in 24-well plates. Cells were incubated with serum-free EBM-A for 16 hr and then loaded with the NO-reactive dye DAF-DA (final concentration, 5 µM) for 40 min at 37 °C in the dark. DAF-DA is a cell-permeable compound that is converted to DAF-2 by intracellular esterases. DAF-2 forms a triazole derivative that emits light at 515 nm upon excitation at 489 nm in proportion to the amount of NO present inside the cells (Montagnani et al., 2001). After loading, cells were rinsed once with EBM-A, and treated with test compounds for 5 min in the dark at 37 °C. For antagonist treatment, cells were pretreated with antagonist for 30 min or as indicated prior to addition of agonist compounds. After drug treatment, cells were rinsed with Dulbecco’s phosphate-buffered saline (PBS: 140 mM NaCl, 0.9 mM CaCl2, 0.49 mM MgCl2, 2.6mM KCl, 8.0 mM KH2PO4, 0.14 mM Na2HPO4, pH 7.4), fixed with 2% glutaraldehyde at 4 oC, and washed twice with PBS. This method of measuring intracellular NO using aldehyde fixatives (2% glutaraldehyde) with DAF-2 DA has been extensively investigated (Sugimoto et al., 2000; Takumida et al., 2001) and found to be reliable for quantitation. The nuclei were stained with DAPI-dilactate (4’, 6-diamidino-2- phenylindole, dilactate; 0.3 µM). Slides were prepared using Prolong Mount and the

5 JPET Fast Forward. Published on March 22, 2007 as DOI: 10.1124/jpet.106.117549 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 117549 deconvolved images were visualized at 20X magnification using a Nikon 600 inverted epifluoroscence microscope and argon laser equipped with a Nikon DXM 1200 digital camera. Because the DAF-2 dye undergoes significant photobleaching, cells were maintained in the dark and were exposed to a light intensity of 20% for 3 s for each image captured. Background intensity was ascertained by treating the cells with the test compounds without DAF-DA, in which case, very little background intensity was observed. Fluorescence intensity was quantified using the Image Pro Plus 4.5 software, and all the images for DAF or DAPI were recorded at the same gain and exposure. The ratio of DAF to DAPI fluorescence intensity was used to normalize for the variation in cell number in a particular field. Each experiment was performed at least 5 Downloaded from times in duplicate.

RT-PCR.

Total RNA was isolated from confluent cultures of endothelial cells or tissues jpet.aspetjournals.org by using the Total RNA Isolation kit (Invitrogen). Total RNA (5 µg) was reverse-transcribed by random priming and incubation with 200 units of MMLV transcriptase at 37 °C for 1 hr. The resulting single-stranded cDNA (5 µl) was then subjected to 30 cycles of polymerase chain reaction (PCR) under the following conditions: denaturation at 95 °C for 5 min; amplification at ASPET Journals on September 30, 2021 cycles of 1 min at 94 °C, 1 min at 53 °C and 1 min at 72°C, with a 7 min extension at 72 °C during the last cycle. Each PCR mixture (100 µl) contained the cDNA template, 1 µM primers,

200 µM dNTPs, 1.5 mM MgCl2, 10 mM Tris-HCl (pH 9), 50 mM KCl, 0.1% Triton X-100 and

2.5 µM Taq polymerase. The primers used to amplify the human CB1 receptor gene (Gerard, 1991) corresponded to the following sequences in transmembrane segment II: 5’-CCTGGCGGTGGCAGACCTCC-3’ (sense) and transmembrane segment IV: 5’-GCAGCACGGCGATCACAATGG-3’(antisense). The expected size of the amplicons was

276 bp for the CB1 receptor. The PCR products were electrophoresed on a 2% agarose gel and visualized by ethidium bromide treatment.

Drug treatment, cell lysate preparation and Western blotting. RAEC were serum starved for 12 hrs and then treated with (R)-methanandamide or or abn-CBD (abnormal cannabidiol; (4-(3-

3,4-trans-p-menthadien-[1,8]-yl)olivetol) or CB1 and CB2 agonist CP55940 (cis-3R-[2-Hydroxy- 4-(1,1-dimethylheptyl)phenyl]-trans-4R-3(3-hydroxypropyl)-1R-cyclohexanol) or WIN55212-2 [(R)-(+)-[2,3-Dihydro-5-methyl-3-(4 morpholinyl methyl) pyrrolo[1,2,3-de]-1,4-benzoxazin-6-

6 JPET Fast Forward. Published on March 22, 2007 as DOI: 10.1124/jpet.106.117549 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 117549 yl]-1naphthalenylmethanone mesylate]as indicated. After experimental treatments, RAEC were washed twice with ice-cold PBS, pH 7.4, and scraped off the plate in lysis buffer (50 mM Tris- HCl, pH 7.4, 0.1 mM EDTA, 0.1 mM EGTA, 1% (v/v) Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 0.1% deoxycholic acid, 20 mM NaF, 1 mM sodium pyrophosphate, 1 mM sodium vanadate, 1 mM Pefabloc (serine protease inhibitor), 1.04 mM AEBSF [4-(3 aminoethylene benzenesulfonyl fluoride), 0.08 µM aprotinin, 20 µM leupeptin, 40 µM bestatin, 15 µM pepstatin A and 14 µM cysteine protease inhibitor E-64 (Protease Inhibitor Cocktail, Sigma Chemicals). Lysates were rotated for 1 hr at 4 °C and insoluble material was removed by centrifugation at

10,000 × g for 5 min at 4°C. Equal amounts of the denatured proteins were loaded on the wells Downloaded from and subjected to SDS-polyacrylamide gel (7.5%) electrophoresis (SDS-PAGE; Mini Protean III, Bio-Rad). Proteins were electrophoretically transferred to a polyvinylidene difluoride (PVDF)

membrane, blocked for 1 hr by incubation in Tris-buffered saline (10 mM Tris-HCl, pH jpet.aspetjournals.org 7.5, 100 mM NaCl) containing 5% (v/v) non-fat dry milk, followed by incubation (3 hr at room temperature) with primary antibodies (1:1000). Antibodies used were: mouse monoclonal anti- Akt antibodies; anti-phospho Ser473-Akt; anti-eNOS and anti-phospho Ser1177-eNOS

(Transduction Laboratories, Lexington, KY). The PVDF membranes were washed 3 times in at ASPET Journals on September 30, 2021 Tris-buffered saline containing 0.1% (v/v) Tween-20, before incubation for 1 hr with goat anti- mouse or goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:8000) (Jackson ImmunoResearch, West Grove, PA). Membranes were washed extensively with Tris- buffered saline-Tween-20 followed by water, and then developed using enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech).

Data analysis: Quantitative values of fluorescence intensities and band densities were normalized and analyzed using Graphpad Prism 4. Log concentration-response data were analyzed by non-linear regression analysis, and other data were compared to control using a Student’s t-test or one-way ANOVA.

RESULTS Effects of endocannabinoid and cannabinoid agonists on NO production in RAEC. The metabolically stable endocannabinoid analog (R)-methanandamide produced an increase in NO production in RAEC as detected by an increase in DAF-2 fluorescence intensity (Fig. 1A).

7 JPET Fast Forward. Published on March 22, 2007 as DOI: 10.1124/jpet.106.117549 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 117549

Quantitative analysis (Fig. 1B) of the fluorescence intensity upon treatment with (R)- methanandamide revealed a dose-dependent (1 nM-1 µM) increase in NO production in RAEC having an EC50 value (with 95% confidence intervals) of 9.4± 0.05 nM. We also tested the effect of abnormal cannabidiol (abn-CBD) on NO production in RAEC. Abn-CBD does not bind to the CB1 receptor (Mo et al., 2004), but has the ability to act as a relaxing factor in arterial preparations and has been implicated as a possible anandamide receptor specific agonist (Ho and Hiley, 2003; Offertaler et al., 2003). Like (R)-methanandamide, abn-CBD produced a significant increase in NO production compared to vehicle control in RAEC (Fig. 2A and 2B).

The concentrations of (R)-methanandamide and abn-CBD used in this study were based on their Downloaded from ability to produce NO-dependent vasorelaxation in aortic ring preparations as determined in previous studies (Mukhopadhyay et al., 2002; Ho and Hiley, 2003; Offertaler et al., 2003). We

then tested the effects of efficacious CB1 receptor agonists on NO production in RAEC. CB1 and jpet.aspetjournals.org

CB2 agonists CP55940 and WIN55212-2 failed to stimulate NO production in RAEC (Fig. 2C).

Both CP55940 and WIN55212-2 have been shown to be potent agonists of CB1 and CB2 receptors in cultured cells at less than1µM in different signal transduction assays that include inhibition of adenylyl cyclase, activation of mitogen activated protein kinase (MAPK), and at ASPET Journals on September 30, 2021 inhibition of Ca2+ channels (see Pertwee, 2006; Howlett et al., 2002 for review). Further, in our previous study with rabbit aortic ring preparations (Mukhopadhyay et al. 2002), CP55940 and WIN55212-2 failed to produce vasorelaxation at the concentration (1µM) used in this study. The failure of CB1 and CB2 agonists to stimulate NO production in RAEC suggests that (R)- methanandamide acts via a non-CB1/CB2 anandamide receptor in the RAEC to stimulate NO production. CP55940 or WIN55212-2 failed to inhibit NO-donor SIN-1 (3-Morpholinosydnonimine, HCl) and SNP (sodium nitroprusside)-mediated increases in the fluorescence intensity (data not shown), indicating that these compounds did not block the ability of the DAF assay to detect NO-mediated fluorescence in the cells.

To further examine the existence of a novel non-CB1/CB2 pharmacological target for anandamide in endothelial cells, CB1 antagonists rimonabant (SR141716; (N-piperidin-1-yl)-5- (4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide ) or LY320135

[6-methoxy-2-(4-methoxyphenyl)benzo[b]thien-3-yl][4-cyanophenyl]methanone) or CB2 antagonist SR144528 [N-[(1S)-Endo-1,3,3,-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-

8 JPET Fast Forward. Published on March 22, 2007 as DOI: 10.1124/jpet.106.117549 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 117549 methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide] were used to block (R)- methanandamide-stimulated NO production in RAEC. Both rimonabant and LY320135 at 1µM have been shown to be specific and potent antagonists for the CB1 receptor signal transduction in cultured cells when 1 µM of a potent agonist such as CP55940 was used (Pertwee, 2006; Howlett et al., 2002). Neither rimonabant (SR141716) nor LY320135 were able to block (R)- methanandamide-stimulated NO production in RAEC (Fig. 3A). Quantitation of fluorescence intensity showed that CB1 antagonists did not produce a significant decrease in (R)- methanandamide-stimulated fluorescence intensity for NO production (Fig. 3B). Similarly, the

CB2 antagonist SR144528 failed to block (R)-methanandamide-stimulated NO production in Downloaded from these cells (data not shown). On the otherhand, non-CB1/CB2 anandamide receptor antagonist O- 1918 [(−)-4-(3-3,4-trans-p-menthadien-(1,8)-yl)-orcinol)] significantly blocked anandamide-

mediated NO generation in RAEC (Fig. 3C). jpet.aspetjournals.org

In order to determine the presence of CB1 receptors in the RAEC, both mRNA and protein content of the cells was examined. As shown in Fig. 4A, reverse transcription- polymerase chain reaction (RT-PCR) of RNA preparations isolated from human umbilical vein at ASPET Journals on September 30, 2021 endothelial cells (HUVEC) (lane 3), and rat or rabbit brain (lanes 1 and 4), produced single discrete bands of the expected size (276 bp). However, no band was detectable using RAEC (lane 2). RNA without reverse transcription did not produce any amplicon (lane 5) indicating that there was no genomic DNA contamination. To determine the presence of CB1 receptor protein, Western blot analysis using anti-CB1 receptor antibody was performed with the HUVEC and RAEC cell membranes proteins. As shown in Fig. 4B, Western analysis of HUVEC membrane protein (17,000 X g pellet) produced a strong band at the expected apparent molecular weight for the CB1 receptor monomer (Lane 2). However, no bands were detected with RAEC cell membranes (lane 1). Collectively, results from RT-PCR and Western blot analysis indicate that RAEC cells do not express detectable amounts of CB1 receptor. This would allow the use of these cells to investigate endothelial cell responses to the endocannabinoid anandamide that are not mediated by the CB1 receptor. We have also tested for the presence of the CB2 receptor in these cells using Western blot techniques and failed to detect any CB2 receptor protein in RAEC (data not shown).

9 JPET Fast Forward. Published on March 22, 2007 as DOI: 10.1124/jpet.106.117549 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 117549

Signal transduction pathways for (R)-methanandamide-stimulated NO production. Results from previous studies have implicated the PI3-kinase-Akt pathway in eNOS activation and NO production. In order to characterize the role of the PI3-kinase pathway, we tested the effect of PI3-kinase inhibitor LY294002 on (R)-methanandamide-mediated NO production and Akt phosphorylation. As shown in Fig. 5A, LY294002 (1 µM) completely blocked (R)-methanandamide-stimulated NO production in RAEC. Quantitation of the fluorescence intensity showed a significant decrease (p < 0.05) in fluorescence intensity of (R)- Downloaded from methanandamide plus LY294002 compared to (R)-methanandamide alone (Fig. 5B). LY294002 failed to block NO-donor SIN-1 and SNP (sodium nitroprusside)-mediated increases in the

fluorescence intensity (data not shown) under similar conditions, suggesting that LY294002 did jpet.aspetjournals.org not block the ability of the DAF assay to detect NO-mediated fluorescence in the cells. Western blot analysis (Fig. 5C) showed that (R)-methanandamide stimulated Akt phosphorylation (lane 4) over basal phosphorylation (lane 1). Pretreatment with the PI3-kinase inhibitor LY294002 (lane

3), blocked (R)-methanandamide-mediated Akt phosphorylation almost completely. Unlike (R)- at ASPET Journals on September 30, 2021 methanandamide, the CB1 and CB2 agonist CP55940 failed to produce Akt phosphorylation (lane 2).

Pertussis toxin (PTX) acts to ADP-ribosylate Gi/o proteins, making the Gi unable to be stimulated by the receptor. To understand if Gi proteins are involved in the NO production, we tested the effect of PTX treatment on (R)-methanandamide-mediated NO production in RAEC. Overnight (16 hr) treatment with PTX (100 ng/ml) completely attenuated the (R)- methanandamide-stimulated NO production in RAEC (Fig. 6A). Quantitation of fluorescence intensity indicated that (R)-methanandamide-stimulated NO production in RAEC was completely abrogated by PTX pretreatment (Fig. 6B). The decrease in the ratio of DAF/DAPI fluorescence intensity following (R)-methanandamide plus PTX treatment, compared to (R)- methanandamide alone was nearly complete under these experimental conditions. We also tested the effect of PTX treatment on Akt phosphorylation in other endothelial cells (HUVEC) and found that (R)-methanandamide-stimulated Akt phosphorylation was completely inhibited by PTX pretreatment (unpublished observation). Collectively, these data indicate that (R)-

10 JPET Fast Forward. Published on March 22, 2007 as DOI: 10.1124/jpet.106.117549 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 117549 methanandamide signals via Gi proteins to PI3-kinase and Akt, which is one of the intrinsic pathways leading to eNOS activation and NO production in endothelial cells. It is known that eNOS can be regulated by phosphorylation mechanisms that include phosphorylation by Akt (Fulton et al., 2001). We tested the eNOS phosphorylation by (R)-methanandamide in RAEC. As shown in Fig. 7, (R)-methanandamide treatment (1 µM for 15 min) produced Ser1177 phosphorylation of eNOS in RAEC.

DISCUSSION

Downloaded from

The existence of a non-CB1/non-CB2 anandamide receptor has been suggested by our findings in rabbit aortic ring preparations (Mukhopadhyay et al., 2002), and findings by others in

rat mesenteric arteries [Offertaler et al., 2003; see (Kunos et al., 2000a; Kunos et al., 2000b; jpet.aspetjournals.org Kunos 2002) for review)) and other vascular preparations or in whole animals (see review (Kunos et al., 2000a; Kunos et al., 2000b; Kunos et al., 2002). A preliminary report that an orphan G-protein coupled receptor, GPR55, is capable of binding to cannabinoid ligands suggests that alternative receptors for anandamide may exist (Brown et al., 2005; Sjögren et al., at ASPET Journals on September 30, 2021 2005). Pharmacological characterization of the signal transduction response in cells that endogenously express the protein is the best argument for the existence of a putative receptor. Based upon the agonist and antagonist response profile, neither GPR55, nor the novel splice variant of the CB1 receptor, appear to be obvious candidates for the endothelial receptor described herein. The major caveat to dissecting the CB1 response apart from that of the anandamide receptor is 1) the lack of specific agonist ligands for the anandamide receptor; and

2) the expression of CB1 receptors in most of the vascular beds and endothelial cells [Liu et al., 2000; see (Kunos et al., 2000a; Kunos et al., 2000b; Kunos 2002) for review)]. Thus, the distinction of CB1 receptor responses from those of a novel anandamide receptor remains a challenge to investigators. In our earlier findings with rabbit aortic ring preparations, we described the existence of a novel non-CB1/non-CB2 endothelial anandamide receptor

(Mukhopadhyay et al., 2002). In that study, we described the failure of potent CB1 and CB2 agonists to mimic the anandamide-mediated vasorelaxation in rabbit aortic ring preparations. The current study used the cellular model of RAEC to study the pharmacology and mechanism of action of (R)-methanandamide. Here, using RT-PCR and Western blot analyses, we showed

11 JPET Fast Forward. Published on March 22, 2007 as DOI: 10.1124/jpet.106.117549 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 117549

that RAEC do not express CB1 receptor message or protein. This finding makes RAEC an excellent model to study the non-CB1/non-CB2 anandamide receptor response relative to various cellular responses, including NO production and angiogenesis (Perry et al., 2005).

The failure of potent CB1 and CB2 agonists CP55940 and WIN55212 to produce NO in RAEC supports our earlier findings in rabbit aortic ring preparations (Mukhopadhyay et al.,

2002), in which CB1and CB2 agonists WIN55212 and desacetyllevonantradol failed to produce vasorelaxation. Abn-CBD is a synthetic analog of the behaviorally inactive plant-derived cannabinoid, cannabidiol. Abn-CBD was first reported to be an inactive cannabinoid in Downloaded from neurobehavioral paradigms used to screen , but able to cause hypotension in dogs

(Adams et al., 1977). Abn-CBD does not bind to the rat brain CB1 even at µ

a very high concentrations up to 100 M, and is inactive in behavioral tetrad tests in mice at jpet.aspetjournals.org doses up to 60 mg/kg suggesting the non-psychoactive characteristic of the molecule. A detailed pharmacological study showed that abn-CBD (20 mg/kg i.v.) caused hypotension in both transgenic wild-type (+/+) and CB1 (-/-) C57BL/6 mice [see (Kunos et al., 2000a; Kunos et al.,

2000b; Kunos 2002) for review]. In that study, Abn-CBD produced endothelium-dependent at ASPET Journals on September 30, 2021 vasodilation in the buffer-perfused mesenteric vascular bed isolated from both transgenic CB1 (+/+) and (-/-) C57BL/6 mice. These findings promoted the candidacy of abn-CBD as an agonist for the novel vascular non-CB1 anandamide receptor. Our demonstration of the ability of abn- CBD and (R)-methanandamide to produce NO in RAEC supports the existence of an anandamide and abn-CBD-sensitive receptor in this cell. These findings corroborated those of Kunos and colleagues [see (Kunos et al., 2000a; Kunos et al., 2000b; Kunos 2002) for review] in mesenteric vascular bed preparations except that the abn-CBD-mediated vasodilatory response was not inhibited by L-NAME, suggesting that endothelial NO was not the only factor involved in the abn-CBD-mediated vasodilation.

Further support for the involvement of a non-CB1/non-CB2 anandamide receptor in (R)- methanandamide-mediated NO production in RAEC resulted from the experiments with antagonists. CB1 and CB2 receptor antagonists failed to block (R)-methanandamide-induced NO production while non-CB1/CB2 anandamide receptor antagonist O-1918 significantly blocked methanandamide-mediated NO production in RAEC. Similar to our findings, Offertaler and

12 JPET Fast Forward. Published on March 22, 2007 as DOI: 10.1124/jpet.106.117549 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 117549 colleagues (2003) also reported that O-1918 blocked abn-CBD mediated vasorelaxation in endothelium-intact mesenteric arteries and also blocked abn-CBD- mediated hypotensive effect. In the rabbit aortic ring preparations, rimonabant failed to block (R)-methanandamide-mediated endothelium-dependent vasorelaxation at 1 µM, and only partially (20%) blocked endothelium- independent vasorelaxation at a higher concentration of 20 µM (Mukhopadhyay et al., 2002).

This was not consistent with the inhibition of a CB1 receptor and we have discussed this in the previous study (Mukhopadhyay et al., 2002). In CB1 wild-type C57BL/6 mice, anandamide or the cannabinoid agonist HU210 mediated prolonged hypotension, which was blocked by pretreatment with rimonabant (3 mg/kg). These effects were completely absent in CB1 (-/-) Downloaded from C57BL/6 mice [see (Kunos et al., 2000a; Kunos et al., 2000b; Kunos 2002) for review]. In the buffer-perfused mesenteric vascular preparations from transgenic CB1 (-/-) mice, Abn-CBD

evoked long lasting vasodilation, which was significantly inhibited by rimonabant (1 to 5 µM). jpet.aspetjournals.org

In mesenteric preparations from CB1 (-/-) and CB2 (-/-) C57BL/6 mice, anandamide and Abn-

CBD caused vasodilation similar to that in preparations from CB1 (-/-) C57BL/6 mice [see (Kunos et al., 2000a; Kunos et al., 2000b; Kunos 2002) for review], indicating that anandamide and Abn-CBD act at a vascular site other than CB1 or CB2 receptors. at ASPET Journals on September 30, 2021

A non-CB1/non-CB2 site was reported to exist on glutamatergic terminals in the mouse hippocampus, where its activation by cannabinoids inhibited glutamatergic transmission (Hajos et al., 2001). The site in the hippocampus was susceptible to inhibition by rimonabant, but it differed from the endothelial cell response in that it could be activated by the synthetic cannabinoid WIN55212 (Hajos et al., 2001). Breivogel and colleagues reported an anandamide- and WIN55212-sensitive, non-CB1/non-CB2 site that could stimulate GTPγS binding to G- proteins in brain membranes from CB1 (-/-) C57BL/6 mice (Breivogel et al., 2001), and this target failed to be blocked with rimonabant. Another WIN55212-sensitive but rimonabant- insensitive, non-CB1/non-CB2 site identified in astrocytes led to inhibition of cAMP production (Sagan et al., 1999). Because the site we are describing in this paper, or described early in vascular preparations (Mukhopadhyay et al., 2002), is insensitive to WIN55212, it is very likely distinct from the non-CB1 sites described in the central nervous system above.

13 JPET Fast Forward. Published on March 22, 2007 as DOI: 10.1124/jpet.106.117549 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 117549

Endothelium-derived NO is produced by eNOS, which exhibits a complex regulatory mechanism. eNOS possesses oxidizing and reducing domains that are attached by a calmodulin binding site (for review, see (Fulton et al., 2001). Although eNOS was originally regarded as a strictly Ca2+/calmodulin-dependent enzyme, there is a growing body of evidence that eNOS is also activated in a Ca2+/CaM-independent manner when stimulated with agonists like adiponectin (Hattori et al., 2003) or insulin (Hartell et al., 2005), or by mechanical sheer-force (Boo 2006; Fleming et al., 1999). Further, several other positive regulators (heat shock protein 90, dynamin-2, signaling kinase Akt, MAPK) and negative regulators (caveolin, NO synthase interacting protein) have also been implicated in eNOS activity. Downloaded from

Phosphorylation of eNOS plays a vital role in the regulation of eNOS (see Shaul.2002 for

review). eNOS is primarily phosphorylated on Ser residues and to a lesser extent on Tyr or Thr jpet.aspetjournals.org residues. Multiple protein kinases including cyclic AMP-dependent protein kinase (PKA) (Boo, 2006; Michell et al., 2001), PI3K-Akt (Hartell et al., 2005), and MAPK (Anter et al., 2005) have been implicated in eNOS phosphorylation. Bernier and colleagues (Bernier et al., 2000) have 1177 documented that the vasodilator bradykinin activated eNOS by Ser phosphorylation via the at ASPET Journals on September 30, 2021 activation of MAPK. Our evidence that (R)-methanandamide produced an Akt-mediated phosphorylation at Ser1177 is in agreement with the results reported by Sessa and colleagues in bovine aortic endothelial cells stimulated with sphingosine-1-phosphate (Lin et al., 2003; Igarashi and Michel, 2001). PKC signaling acts in opposition by phosphorylating Thr495 residues and promoting phosphatase-mediated Ser1177 dephosphorylation (Li et al., 2004). Our data are consistent with eNOS activation via a PI3K-Akt pathway that involves a mechanism requiring Gi activation.

The findings reported in this study are important to our understanding of the endocannabinoid signaling system in endothelial cells. Anandamide is biosynthesized throughout the body, and it is beginning to be understood that CB1 receptors are also present in many cell types outside of the central nervous system. If we can characterize the pharmacology of alternative targets for anandamide’s action, we might be able to generate selective agonists directed at therapeutically beneficial responses to anandamide, such as vasodilation.

14 JPET Fast Forward. Published on March 22, 2007 as DOI: 10.1124/jpet.106.117549 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 117549

REFERENCES

Adams MD, Earnhardt JT, Martin BR, Harris LS, Dewey WL, and Razdan RK (1977) A cannabinoid with cardiovascular activity but no overt behavioral effects. Experientia 33:1204- 1205.

Anter E, Chen K, Shapira OM, Karas RH, and Keaney JF, Jr. (2005) p38 mitogen-activated protein kinase activates eNOS in endothelial cells by an estrogen receptor alpha-dependent Downloaded from pathway in response to black tea polyphenols. Circ.Res. 96:1072-1078.

Bernier SG, Haldar S, and Michel T (2000) Bradykinin-regulated interactions of the mitogen-

activated protein kinase pathway with the endothelial nitric-oxide synthase. J.Biol.Chem. jpet.aspetjournals.org 275:30707-30715.

Boo YC (2006) Shear stress stimulates phosphorylation of protein kinase A substrate proteins

Bouaboula M, Perrachon S, Milligan L, Canat X, Rinaldi-Carmona M, Portier M, Barth F, at ASPET Journals on September 30, 2021 Calandra B, Pecceu F, Lupker J, Maffrand JP, Le Fur G, and Casellas P (1997) A selective inverse agonist for central cannabinoid receptor inhibits mitogen-activated protein kinase activation stimulated by insulin or insulin-like growth factor 1. Evidence for a new model of receptor/ligand interactions. J.Biol.Chem. 272:22330-22339.

Breivogel CS, Griffin G, Di Marzo V, and Martin BR (2001) Evidence for a new G protein- coupled cannabinoid receptor in mouse brain. Mol.Pharmacol. 60:155-163.

Brown AJ, Ueno S, Suen K, Dowell SJ, and Wise A (2005) Molecular identification of GPR55 as a third G protein-coupled receptor responsive to cannabinoid ligands Symposium on the Cannabinoids, International Cannabinoid Research Society. p.16. Burlington, VT

Chaytor AT, Martin PE, Evans WH, Randall MD, and Griffith TM (1999) The endothelial component of cannabinoid-induced relaxation in rabbit mesenteric artery depends on gap junctional communication. J.Physiol 520:539-550.

15 JPET Fast Forward. Published on March 22, 2007 as DOI: 10.1124/jpet.106.117549 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 117549

Di Marzo V, De Petrocellis L, Fezza F, Ligresti A, and Bisogno T (2002) Anandamide receptors. Prostaglandins Leukot.Essent.Fatty Acids 66:377-391.

Fleming I, Bauersachs J, Schafer A, Scholz D, Aldershvile J, and Busse R (1999) Isometric contraction induces the Ca2+-independent activation of the endothelial nitric oxide synthase. Proc.Natl.Acad.Sci.U.S.A 96:1123-1128.

Fulton D, Gratton JP, and Sessa WC (2001) Post-translational control of endothelial nitric oxide synthase: why isn't calcium/calmodulin enough? J.Pharmacol.Exp.Ther. 299:818-824. Downloaded from Hajos N, Ledent C, and Freund TF (2001) Novel cannabinoid-sensitive receptor mediates inhibition of glutamatergic synaptic transmission in the hippocampus. Neuroscience 106:1-4.

Hartell NA, Archer HE, and Bailey CJ (2005) Insulin-stimulated endothelial nitric oxide release jpet.aspetjournals.org is calcium independent and mediated via protein kinase B. Biochem.Pharmacol. 69:781-790.

Hattori Y, Suzuki M, Hattori S, and Kasai K (2003) Globular adiponectin upregulates nitric

oxide production in vascular endothelial cells. Diabetologia 46:1543-1549. at ASPET Journals on September 30, 2021

Ho WS and Hiley CR (2003) Vasodilator actions of abnormal-cannabidiol in rat isolated small mesenteric artery. Br.J.Pharmacol. 138:1320-1332.

Howlett AC, Barth F, Bonner TI, Cabral G, Casellas P, Devane WA, Felder CC, Herkenham M, Mackie K, Martin BR, Mechoulam R, and Pertwee RG (2002) International Union of Pharmacology. XXVII. Classification of cannabinoid receptors. Pharmacol.Rev. 54:161-202.

Howlett AC, Breivogel CS, Childers SR, Deadwyler SA, Hampson RE, and Porrino LJ (2004) Cannabinoid physiology and pharmacology: 30 years of progress. Neuropharmacology 47 Suppl 1:345-358.

Igarashi J and Michel T (2001) Sphingosine 1-phosphate and isoform-specific activation of phosphoinositide 3-kinase beta. Evidence for divergence and convergence of receptor-regulated endothelial nitric-oxide synthase signaling pathways. J.Biol.Chem. 276:36281-36288.

16 JPET Fast Forward. Published on March 22, 2007 as DOI: 10.1124/jpet.106.117549 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 117549

Klein TW, Newton C, Larsen K, Lu L, Perkins I, Nong L, and Friedman H (2003) The cannabinoid system and immune modulation. J.Leukoc.Biol. 74:486-496.

Kunos G, Batkai S, Offertaler L, Mo F, Liu J, Karcher J, and Harvey-White J (2002) The quest for a vascular endothelial cannabinoid receptor. Chem.Phys.Lipids 121:45-56.

Kunos G, Jarai Z, Batkai S, Goparaju SK, Ishac EJ, Liu J, Wang L, and Wagner JA (2000a) Endocannabinoids as cardiovascular modulators. Chem.Phys.Lipids 108:159-168.

Kunos G, Jarai Z, Varga K, Liu J, Wang L, and Wagner JA (2000b) Cardiovascular effects of Downloaded from endocannabinoids-the plot thickens. Prostaglandins Other Lipid Mediat. 61:71-84.

Li Z, Carter JD, Dailey LA, and Huang YC (2004) Vanadyl sulfate inhibits NO production via threonine phosphorylation of eNOS. Environ.Health Perspect. 112:201-206. jpet.aspetjournals.org

Lin MI, Fulton D, Babbitt R, Fleming I, Busse R, Pritchard KA, Jr., and Sessa WC (2003) Phosphorylation of threonine 497 in endothelial nitric-oxide synthase coordinates the coupling of

L-arginine metabolism to efficient nitric oxide production. J.Biol.Chem. 278:44719-44726. at ASPET Journals on September 30, 2021

Liu J, Gao B, Mirshahi F, Sanyal AJ, Khanolkar AD, Makriyannis A, and Kunos G (2000) Functional CB1 cannabinoid receptors in human vascular endothelial cells. Biochem.J. 346:835- 840.

Michell BJ, Chen Z, Tiganis T, Stapleton D, Katsis F, Power DA, Sim AT, and Kemp BE (2001) Coordinated control of endothelial nitric-oxide synthase phosphorylation by protein kinase C and the cAMP-dependent protein kinase. J.Biol.Chem. 276:17625-17628.

Mo FM, Offertaler L, and Kunos G (2004) Atypical cannabinoid stimulates endothelial cell migration via a Gi/Go-coupled receptor distinct from CB1, CB2 or EDG-1. Eur.J.Pharmacol. 489:21-27.

Montagnani M, Chen H, Barr VA, and Quon MJ (2001) Insulin-stimulated activation of eNOS is independent of Ca2+ but requires phosphorylation by Akt at Ser(1179). J.Biol.Chem. 276:30392- 30398.

17 JPET Fast Forward. Published on March 22, 2007 as DOI: 10.1124/jpet.106.117549 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 117549

Mukhopadhyay S, Chapnick BM, and Howlett AC (2002) Anandamide-induced vasorelaxation in rabbit aortic rings has two components: G protein dependent and independent. Am.J.Physiol Heart Circ.Physiol 282:H2046-H2054.

O'Sullivan SE, Kendall DA, and Randall MD (2004) Heterogeneity in the mechanisms of vasorelaxation to anandamide in resistance and conduit rat mesenteric arteries. Br.J.Pharmacol. 142:435-442.

Offertaler L, Mo FM, Batkai S, Liu J, Begg M, Razdan RK, Martin BR, Bukoski RD, and Kunos

G (2003a) Selective ligands and cellular effectors of a G protein-coupled endothelial cannabinoid Downloaded from receptor. Mol.Pharmacol. 63:699-705.

Perry S, Johnson I, and Mukhopadhyay S (2005) Molecular Signaling Mechanism of jpet.aspetjournals.org Endogenous Cannabinoid Anandamide mediated Cell migaration and Angiogenesis. Abstracts, Proceedings of Am. Soc.Cell Biol.meeting, Sanfransisco, CA

Pertwee RG (2005) Pharmacological actions of cannabinoids Handb Exp Pharmacol.168:1-51. at ASPET Journals on September 30, 2021 Randall MD, McCulloch AI, and Kendall DA (1997) Comparative pharmacology of endothelium-derived hyperpolarizing factor and anandamide in rat isolated mesentery. Eur.J.Pharmacol. 333:191-197.

Sagan S, Venance L, Torrens Y, Cordier J, Glowinski J, and Giaume C (1999) Anandamide and WIN 55212-2 inhibit cyclic AMP formation through G-protein-coupled receptors distinct from CB1 cannabinoid receptors in cultured astrocytes. Eur.J.Neurosci. 11:691-699.

Shaul PW (2002) Regulation of endothelial nitric oxide synthase: location, location, location. Annu.Rev.Physiol 64:749-774.

Sjögren, S, Ryberg, E, Lindblom, A, Larsson, N, Åstrand, A, Hjorth, S, Andersson, A-K, Groblewski, T and Greasley, P (2005) A new receptor for cannabinoid ligands. Symposium on the Cannabinoids, International Cannabinoid Research Society. p.106. Burlington, VT

18 JPET Fast Forward. Published on March 22, 2007 as DOI: 10.1124/jpet.106.117549 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 117549

Sugimoto K, Fujii S, Takemasa T, Yamashita K. (2000) Detection of intracellular nitric oxide using a combination of aldehyde fixatives with 4,5-diaminofluorescein diacetate Histochem Cell Biol.113: 341-347.

Takumida M, Anniko M (2001) Detection of nitric oxide in the guinea pig inner ear, using a combination of aldehyde fixative and 4,5-diaminofluorescein diacetate. Acta Otolaryngol. 121: 460-464.

Downloaded from Van Sickle MD, Duncan M, Kingsley PJ, Mouihate A, Urbani P, Mackie K, Stella N, Makriyannis A, Piomelli D, Davison JS, Marnett LJ, Di M, V, Pittman QJ, Patel KD, and Sharkey KA (2005) Identification and functional characterization of brainstem cannabinoid CB2 jpet.aspetjournals.org receptors. Science 310:329-332. at ASPET Journals on September 30, 2021

19 JPET Fast Forward. Published on March 22, 2007 as DOI: 10.1124/jpet.106.117549 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 117549

FOOTNOTES

This work was supported by NIDA grants U24-DA12385 and K05-DA00182 (ACH), and American Heart Association Scientist Grant-in Aid 0060377Z (SM). A portion of this work was performed in partial fulfillment of the M.S. degree by LM, who was supported by NIGMS R25- GM66332 Bridge to the Ph.D. program.

Present address for LM and ACH

Dept. Physiology and Pharmacology Downloaded from Wake Forest University School of Medicine Winston-Salem, NC 27157

jpet.aspetjournals.org

at ASPET Journals on September 30, 2021

20 JPET Fast Forward. Published on March 22, 2007 as DOI: 10.1124/jpet.106.117549 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 117549

FIGURE LEGENDS

Fig. 1. Log concentration-response curve of (R)-methanandamide on NO production in RAEC. A. Cells were loaded with DAF-DA, treated with (R)-methanandamide at the indicated concentrations for 20 min, and processed for fluorescence imaging as described in Methods. B. Fluorescence intensity was quantitated as the ratio of [(DAF/DAPI) x100], and analyzed by GraphPad Prism 4.0. Data are presented as mean ± SEM of NO production from three separate experiments. The fluorescence intensity of the control cells (treated with vehicle) was 7.4 ± 0.6 Downloaded from as measured by the ratio of [(DAF/DAPI) x100].

Fig. 2. Effect of endocannabinoid and cannabinoid agonists on NO production in RAEC. Cells jpet.aspetjournals.org were loaded with DAF-DA, treated with the indicated agonist ligand at 1 µM for 20 min, and processed for fluorescence imaging as described in Methods. A. and C. are the representative images of the cells showing DAF or DAPI staining following agonist treatment as indicated. B.

Quantitation of the fluorescence intensity is the same as for Fig.1. Data are presented as the at ASPET Journals on September 30, 2021 mean ± SEM of DAF to DAPI ratios from three separate experiments.

Fig. 3. Effect of cannabinoid receptor antagonists on (R)-methanandamide-stimulated NO production in RAEC. A. DAF-DA loaded cells were treated with CB1 receptor antagonists rimonabant (1 µM) or LY320135 (1 µM) for 30 min at 37 °C prior to treatment with (R)- methanandamide (1µM) for an additional 20 min. Images of fluorescence in the RAEC are representative of four separate experiments having similar results. B. Quantitative analysis of the fluorescence intensity [(DAF/DAPI) x100], following (R)-methanandamide stimulation of NO production in the absence and presence of CB1 antagonists. No significant difference in the fluorescence intensity for (R)-methanandamide-stimulated NO production was observed following rimonabant and LY320135 treatment compared to (R)-methanandamide alone (p<0.05). Data are presented as mean ± SEM for four separate experiments and were analyzed by one-way ANOVA. C. Quantitative analysis of the fluorescence intensity [(DAF/DAPI) x100], following (R)-methanandamide stimulation of NO production in the absence and presence of non

CB1/non CB2 antagonist O-1918. The data were expressed as mean ± SEM from three separate

21 JPET Fast Forward. Published on March 22, 2007 as DOI: 10.1124/jpet.106.117549 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 117549 experiments for the treatments and were analyzed using an unpaired Student’s t-test (*p < 0.001; #p <0.05).

Fig. 4. Determination of CB1 receptors in endothelial cells and other tissues. A. RT-PCR was performed using total RNA from rat brain (lane 1), RAEC (lane 2), HUVEC (lane 3), rabbit brain (lane 4), RNA from rabbit brain without reverse transcription (lane 5) and rabbit liver (lane

6 using specific primers for the human CB1 receptor gene. Lane 8 contained molecular weight standard (not shown). B. Western blot detection of CB1 receptor in HUVEC and RAEC cell membranes. Western blot analysis was carried out with affinity-purified anti-CB1 receptor Downloaded from antibody as described in the text. Lanes 1 and 2 contained 20 µg RAEC and HUVEC P2 membrane proteins, respectively. This is a representative blot of three separate experiments

having similar results. jpet.aspetjournals.org

Fig. 5. Effect of PI 3-kinase inhibitor on (R)-methanandamide-stimulated NO production in RAEC. DAF-DA-loaded cells were treated with PI3-kinase inhibitor LY294002 (1 µM) for 30 minutes at 37 °C prior to treatment with (R)-methanandamide (1 µM) and fixation as previously at ASPET Journals on September 30, 2021 described in the Methods. A. Images of cells were from a representative experiment. B. In the presence of PI3-kinase inhibitor LY294002 there was no increase in fluorescence intensity upon (R)-methanandamide stimulation. The data were expressed as mean ± SEM from three separate experiments for the treatments as indicated (48.7 ± 3.99, 9.07 ± 2.07, 78.8 ± 3.4, and 14.8 ± 1.13) and were analyzed using an unpaired Student’s t-test (p < 0.0001). C. Western blot analysis of (R)-methanandamide stimulated Akt phosphorylation. Western blot analysis was carried out with anti-Akt and anti-phosphoAkt antibodies (1:1000). Equal amounts of protein (30 µg) were loaded: lane 1: control, lane 2: CP55940 (1 µM, for 15 min), lane 3: (R)-methanandamide (1 µM for 15 min) plus PI3-kinase inhibitor LY294002 (1 µM), lane 4: (R)-methanandamide (1 µM, for 15 min). This is a representative blot of three separate experiments having similar results.

Fig. 6. Effect of pertussis toxin treatment on (R)-methanandamide-mediated NO production in RAEC. Cells were pre-treated with PTX (100 ng/ml) for 16 hrs at 37 °C, followed by DAF-DA loading and treatment with (R)-methanandamide (1 µM). A. Images of fluorescent cells are representative of three separate experiments with similar results. B. The data were analyzed

22 JPET Fast Forward. Published on March 22, 2007 as DOI: 10.1124/jpet.106.117549 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 117549 using an unpaired student’s t-test with SEM (mean ± SEM: 67±3.78 and 2.5±1.47, n=3). (R)- methanandamide showed no significant increase in fluorescent intensity after treatment with PTX (p<0.0001).

Fig.7. (R)-methanandamide-mediated eNOS phosphorylation. Western blot analysis was carried out with anti-eNOSand anti-phospho (Ser1177) eNOS antibodies (1:1000). Equal amounts of protein (30 µg) were loaded: lane 1: control, lane 2: (R)-methanandamide (1 µM, for 15 min), This is a representative blot of four separate experiments having similar results. The band densities were quantitated and the ratio of [peNOS/eNOS x 100] band densities of control and Downloaded from methanandamide-treated samples were 34± 2.3 and 57± 4.4 respectively.

jpet.aspetjournals.org at ASPET Journals on September 30, 2021

23 Fig.1 A This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. JPET FastForward.PublishedonMarch22,2007asDOI:10.1124/jpet.106.117549

Methanandamide (nM): 10 100 1000

B

Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 30, 2021 30, September on Journals ASPET at Fig. 2 A This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. Control Methanandamide Abn-CBD JPET FastForward.PublishedonMarch22,2007asDOI:10.1124/jpet.106.117549 100 * *

B 75

50

25 (DAF/DAPI)x 100 Fluorescence Intensity 0 ControlControl methan-MA Abn-CBD Abn- . andamide CBD

C DAF DAF

DAPI DAPI

Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 30, 2021 30, September on Journals ASPET at Fig. 3 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion.

A JPET FastForward.PublishedonMarch22,2007asDOI:10.1124/jpet.106.117549

Methanandamide Methanandamide Methanandamide + Rimonabant + LY320135

B C 75 *

50

# 25 (DAF/DAPI)x 100 Fluorescence Intensity 0 Methanandamide + + + Methanandamide -- + + Rimonabant - + - O1918 - + - +

LY320135 --+

Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 30, 2021 30, September on Journals ASPET at Fig. 4 A This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. CB1R

(280 bp) JPET FastForward.PublishedonMarch22,2007asDOI:10.1124/jpet.106.117549

1 2 3 4 5 6

B

CB1R

RAEC HUVEC

Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 30, 2021 30, September on Journals ASPET at Fig.5 A DAF DAF DAPI This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. JPET FastForward.PublishedonMarch22,2007asDOI:10.1124/jpet.106.117549

Methanandamide + + + LY294002 - + + B C pAkt

Akt

MA - - + + CP55940 - + - -

LY294002 - - + -

Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 30, 2021 30, September on Journals ASPET at Fig. 6

A This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. JPET FastForward.PublishedonMarch22,2007asDOI:10.1124/jpet.106.117549

Methanandamide PTX + Methanandamide

80 B 70 60 50 40 30 20

(DAF/DAPI) X100 (DAF/DAPI) 10 Fluorescence Intensity Fluorescence 0 MA + +

PTX - +

Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 30, 2021 30, September on Journals ASPET at Fig. 7 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. JPET FastForward.PublishedonMarch22,2007asDOI:10.1124/jpet.106.117549

Phospho- eNOS(S1177)

eNOS

Control MA

Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 30, 2021 30, September on Journals ASPET at