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 cannabinoid receptor agonists (Am J Physiol282: H2046–H2054, 2002). Here, we show that (R)-methanandamide and abnormal cannabidiol stimulated nitric oxide (NO) production in rabbit aortic endothelial cells (RAEC) in a dose-dependent manner but that other CB1 and CB2 receptor agonists, such as cis-3R-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4R-3(3-hydroxypropyl)-1R-cyclohexanol (CP55940) and (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo-[1,2,3-d,e]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone (WIN55212-2), failed to do so. CB1 antagonists rimonabant [also known as SR141716; N-piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] and 6-methoxy-2-(4-methoxyphenyl)benzo[b]-thien-3-yl][4-cyanophenyl]methanone (LY320135) and CB2 antagonist N-[(1S)-endo-1,3,3,-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528) failed to block (R)-methanandamide-mediated NO production in RAEC. However, anandamide receptor antagonist (–)-4-(3–3,4-trans-p-menthadien-(1,8)-yl)-orcinol (O-1918) blocked (R)-methanandamide-mediated NO production in RAEC. Reverse transcriptase-polymerase chain reaction and Western blot analyses failed to detect the CB1 receptor in RAEC, making this a good model to study non-CB1 responses to anandamide. (R)-Methanandamide produced endothelial nitric-oxide synthase (eNOS) phosphorylation via the activation of phosphoinositide 3-kinase-Akt signaling. Inhibition of Gi signaling with pertussis toxin, or phosphatidylinositol 3-kinase activity with 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), resulted in a decrease in (R)-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 phosphatidylinositol 3-kinase, leading to Akt activation, eNOS phosphorylation, and NO production.
Footnotes
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This work was supported by National Institute on Drug Abuse Grants U24-DA12385 and K05-DA00182 (to A.C.H.) and American Heart Association Scientist Grant-in-aid 0060377Z (to S.M.). A portion of this work was performed in partial fulfillment of the Masters of Science degree by L.M., who was supported by National Institute of General Medical Sciences Grant R25-GM66332 Bridge to the Ph.D. program.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.106.117549.
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ABBREVIATIONS: CB, cannabinoid; MAPK, mitogen-activated protein kinase; PI3-kinase, phosphatidylinositol 3-kinase; NO, nitric oxide; eNOS, endothelial nitric-oxide synthase; RAEC, rabbit aortic endothelial cell(s); DAF-DA, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate; PBS, phosphate-buffered saline; DAPI, 4,6-diamidino-2-phenylindole; PCR, polymerase chain reaction; bp, base pairs; Abn-CBD, abnormal cannabidiol; CP55940, cis-3R-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4R-3(3-hydroxypropyl)-1R-cyclohexanol; WIN55212-2, [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinyl methyl) pyrrolo[1,2,3-d,e]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate; rimonabant, SR141716, (N-piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; LY320135, [6-methoxy-2-(4-methoxyphenyl)benzo[b]thien-3-yl][4-cyanophenyl]methanone; SR144528, N-[(1S)-endo-1,3,3,-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide; O-1918, (–)-4-(3–3,4-trans-p-menthadien-(1,8)-yl)-orcinol; RT-PCR, reverse transcriptase-polymerase chain reaction; HUVEC, human umbilical vein endothelial cell(s); LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; PTX, pertussis toxin; HU-210, (–)-7-OH-Δ6-tetrahydrocannabinol-dimethylheptyl; DAF, 4-amino-dimethylamino-2′,7′-difluorofluorescein.
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↵1 Current affiliation: Department Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, North Carolina.
- Received November 22, 2006.
- Accepted March 21, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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