ly-320135 and anandamide

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 CB(1) cannabinoid receptor agonists (Am J Physiol 282: 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 CB(1) and CB(2) 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. CB(1) 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 CB(2) 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 CB(1) receptor in RAEC, making this a good model to study non-CB(1) responses to anandamide. (R)-Methanandamide produced endothelial nitric-oxide synthase (eNOS) phosphorylation via the activation of phosphoinositide 3-kinase-Akt signaling. Inhibition of G(i) 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-CB(1) and non-CB(2) anandamide receptor and signals through G(i) and phosphatidylinositol 3-kinase, leading to Akt activation, eNOS phosphorylation, and NO production.

Topics: Animals; Arachidonic Acids; Benzofurans; Benzoxazines; Camphanes; Cannabinoid Receptor Modulators; Cells, Cultured; Chromones; Cyclohexanols; Dose-Response Relationship, Drug; Endocannabinoids; Endothelial Cells; Enzyme Inhibitors; GTP-Binding Protein alpha Subunits, Gi-Go; Morpholines; Naphthalenes; Nitric Oxide; Pertussis Toxin; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Piperidines; Polyunsaturated Alkamides; Proto-Oncogene Proteins c-akt; Pyrazoles; Rabbits; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Resorcinols; Rimonabant; Signal Transduction

Noradrenaline and ATP are sympathetic co-transmitters. In the rat perfused mesenteric bed cannabinoids have been shown to modify the overall response to sympathetic nerve stimulation. This study has assessed whether cannabinoid receptor activation modulates differentially the noradrenergic and purinergic components of sympathetic vasoconstriction.. Rat mesenteric beds were perfused with physiological salt solution and the effects of cannabinoids on responses to nerve stimulation, or exogenous noradrenaline or alpha,beta-methylene ATP (alpha,beta-meATP; P2X receptor agonist) were determined after raising tone with U46619. The effects of cannabinoids on the noradrenaline and ATP components of sympathetic neurotransmission were assessed using the alpha 1-adrenoceptor antagonist, prazosin, or after P2X receptor desensitization with alpha,beta-meATP.. Anandamide, WIN 55,212-2 and CP55,940 attenuated sympathetic neurogenic vasoconstrictor responses. The inhibitory actions of anandamide and WIN 55,212-2 were blocked by LY320135, a CB1 receptor antagonist, but not by SR144528, a CB2 receptor antagonist. The inhibitory actions of CP55,940 were unaffected by LY320135 and SR144528. WIN 55,212-3, the inactive S(-) enantiomer of WIN 55,212-2, had no effect on sympathetic neurogenic responses. None of the cannabinoids affected contractile responses to exogenous noradrenaline or alpha,beta-meATP. Anandamide and WIN 55,212-2 inhibited both the noradrenaline and ATP components of the sympathetic neurogenic contractile responses, with effects on the ATP component being most marked.. These results indicate that prejunctional CB1-like receptors mediate the sympathoinhibitory action of anandamide and WIN 55,212-2, but not CP55,940, in the rat mesenteric bed. Cannabinoids inhibit both the noradrenergic and purinergic components of sympathetic neurotransmission.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Triphosphate; Adrenergic Fibers; Animals; Arachidonic Acids; Benzofurans; Benzoxazines; Camphanes; Cannabinoids; Cyclohexanols; Dose-Response Relationship, Drug; Electric Stimulation; Endocannabinoids; In Vitro Techniques; Male; Mesenteric Arteries; Morpholines; Muscle Tonus; Muscle, Smooth, Vascular; Naphthalenes; Norepinephrine; Perfusion; Polyunsaturated Alkamides; Prazosin; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Purinergic P2

The role of endocannabinoid signaling in the response of the brain to injury is tantalizing but not clear. In this study, transient middle cerebral artery occlusion (MCAo) was used to produce ischemia/reperfusion injury. Brain content of N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol were determined during MCAo. Whole brain AEA content was significantly increased after 30, 60 and 120 min MCAo compared with sham-operated brain. The increase in AEA was localized to the ischemic hemisphere after 30 min MCAo, but at 60 and 120 min, was also increased in the contralateral hemisphere. 2-Arachidonoylglycerol content was unaffected by MCAo. In a second set of studies, injury was assessed 24 h after 2 h MCAo. Rats administered a single dose (3 mg/kg) of the cannabinoid receptor type 1 (CB1) receptor antagonist SR141716 prior to MCAo exhibited a 50% reduction in infarct volume and a 40% improvement in neurological function compared with vehicle control. A second CB1 receptor antagonist, LY320135 (6 mg/kg), also significantly improved neurological function. The CB1 receptor agonist, WIN 55212-2 (0.1-1 mg/kg) did not affect either infarct volume or neurological score.

Topics: Animals; Arachidonic Acids; Benzofurans; Benzoxazines; Blood Pressure; Brain Chemistry; Brain Infarction; Chromatography, Liquid; Disease Models, Animal; Dose-Response Relationship, Drug; Endocannabinoids; Hemodynamics; Infarction, Middle Cerebral Artery; Ischemic Attack, Transient; Male; Mass Spectrometry; Morpholines; Naphthalenes; Neurologic Examination; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Reperfusion Injury; Rimonabant; Tetrazolium Salts; Time Factors

The effects of three structurally related cannabinoids on human and rat recombinant vanilloid VR1 receptors expressed in human embryonic kidney (HEK293) cells and at endogenous vanilloid receptors in the rat isolated mesenteric arterial bed were studied. In the recombinant cells, all three were full agonists, causing concentration-dependent increases in [Ca(2+)](i) (FLIPR), with a rank order of potency relative to the vanilloids capsaicin and olvanil, of olvanil> or =capsaicin>AM404 ((allZ)-N-(4-hydroxyphenyl)-5,8,11,14-eicosatetraenamide)>anandamide>methanandamide. These responses were inhibited by the vanilloid VR1 receptor antagonist, capsazepine. In the mesenteric arterial bed, vasorelaxation was evoked by these ligands with a similar order of potency. The AM404-induced vasorelaxation was virtually abolished by capsaicin pretreatment. AM404 inhibition of capsaicin-sensitive sensory neurotransmission was blocked by ruthenium red, but not by cannabinoid CB(1) and CB(2) receptor antagonists. AM404 had no effect on relaxations to calcitonin gene-related peptide. These data demonstrate that the vasorelaxant and sensory neuromodulator properties of AM404 in the rat isolated mesenteric arterial bed are mediated by vanilloid VR1 receptors.

Topics: Acetylcholine; Animals; Arachidonic Acids; Benzofurans; Calcitonin Gene-Related Peptide; Calcium; Calcium Channel Blockers; Camphanes; Cannabinoids; Capsaicin; Cell Line; Dose-Response Relationship, Drug; Endocannabinoids; Humans; In Vitro Techniques; Mesenteric Arteries; Neurons, Afferent; Polyunsaturated Alkamides; Pyrazoles; Rats; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Ruthenium; Synaptic Transmission; Vasodilation; Vasodilator Agents

1. We have shown that the endocannabinoid anandamide and its stable analogue methanandamide relax rings of rabbit superior mesenteric artery through endothelium-dependent and -independent mechanisms that are unaffected by blockade of NO synthase and cyclooxygenase. 2. The endothelium-dependent component of the responses was attenuated by the gap junction inhibitor 18alpha-glycyrrhetinic acid (18alpha-GA; 50 microM), and a synthetic connexin-mimetic peptide homologous to the extracellular Gap 27 sequence of connexin 43 (43Gap 27, SRPTEKTIFII; 300 microM). By contrast, the corresponding connexin 40 peptide (40Gap 27, SRPTEKNVFIV) was inactive. 3. The cannabinoid CB1 receptor antagonist SR141716A (10 microM) also attenuated endothelium-dependent relaxations but this inhibition was not observed with the CB1 receptor antagonist LY320135 (10 microM). Furthermore, SR141716A mimicked the effects of 43Gap 27 peptide in blocking Lucifer Yellow dye transfer between coupled COS-7 cells (a monkey fibroblast cell line), whereas LY320135 was without effect, thus suggesting that the action of SR141716A was directly attributable to effects on gap junctions. 4. The endothelium-dependent component of cannabinoid-induced relaxation was also attenuated by AM404 (10 microM), an inhibitor of the high-affinity anandamide transporter, which was without effect on dye transfer. 5. Taken together, the findings suggest that cannabinoids derived from arachidonic acid gain access to the endothelial cytosol via a transporter mechanism and subsequently stimulate relaxation by promoting diffusion of an to adjacent smooth muscle cells via gap junctions. 6. Relaxations of endothelium-denuded preparations to anandamide and methanandamide were unaffected by 43Gap 27 peptide, 18alpha-GA, SR141716A, AM404 and indomethacin and their genesis remains to be established.

Topics: Acetylcholine; Animals; Arachidonic Acids; Benzofurans; Cannabinoid Receptor Modulators; Cannabinoids; COS Cells; Endocannabinoids; Gap Junctions; Glycyrrhetinic Acid; Indomethacin; Isoquinolines; Male; Mesenteric Artery, Superior; Muscle Relaxation; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Phenylephrine; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rabbits; Receptors, Cannabinoid; Receptors, Drug; Rimonabant