cyclic-gmp and anandamide

In this study the effect of the endocannabinoid anandamide on platelet nitric oxide (NO)/cGMP pathway was investigated. Data report that anandamide in a dose-and time-dependent manner increased NO and cGMP levels and stimulated endothelial nitric oxide synthase (eNOS) activity. These parameters were significantly reduced by LY294002, selective inhibitor of PI3K and by MK2206, specific inhibitor of AKT. Moreover anandamide stimulated both eNOSser1177 and AKTser473 phosphorylation. Finally the anandamide effect on NO and cGMP levels, eNOS and AKT phosphorylation/activation were inhibited by SR141716, specific cannabinoid receptor 1 antagonist, supporting the involvement of anandamide binding to this receptor. Overall data of this report indicate that low concentrations of anandamide, through PI3K/AKT pathway activation, stimulates eNOS activity and increases NO levels in human platelets. In such way anandamide contributes to extend platelet survival.

Topics: Arachidonic Acids; Blood Platelets; Calcium; Citrulline; Cyclic GMP; Endocannabinoids; Enzyme Assays; Humans; Nitric Oxide; Nitric Oxide Synthase Type III; Phosphorylation; Platelet Aggregation; Polyunsaturated Alkamides; Proto-Oncogene Proteins c-akt; Signal Transduction

In our previous studies, CB(1) cannabinoid receptor agonists stimulated production of cyclic GMP and translocation of nitric oxide (NO)-sensitive guanylyl cyclase in neuronal cells (Jones et al., Neuropharmacology 54:23-30, 2008). The purpose of these studies was to elucidate the signal transduction of cannabinoid-mediated neuronal nitric oxide synthase (nNOS) activation in neuronal cells. Cannabinoid agonists CP55940 (2-[(1S,2R,5S)-5-hydroxy-2-(3-hydroxypropyl) cyclohexyl]-5-(2-methyloctan-2-yl)phenol), WIN55212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate), and the metabolically stable analog of anandamide, (R)-(+)-methanandamide stimulated NO production in N18TG2 cells over a 20-min period. Rimonabant (N-(piperidin-lyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-H-pyrazole-3-carboxamide), a CB(1) receptor antagonist, partially or completely curtailed cannabinoid-mediated NO production. Inhibition of NOS activity (N ( G )-nitro-L: -arginine) or signaling via Gi/o protein (pertussis toxin) significantly limited NO production by cannabinoid agonists. Ca(2+) mobilization was not detected in N18TG2 cells after cannabinoid treatment using Fluo-4 AM fluorescence. Cannabinoid-mediated NO production was attributed to nNOS activation since endothelial NOS and inducible NOS protein and mRNA were not detected in N18TG2 cells. Bands of 160 and 155 kDa were detected on Western blot analysis of cytosolic and membrane fractions of N18TG2 cells, using a nNOS antibody. Chronic treatment of N18TG2 cells with cannabinoid agonists downregulated nNOS protein and mRNA as detected using Western blot analysis and real-time polymerase chain reaction, respectively. Cannabinoid agonists stimulated NO production via signaling through CB(1) receptors, leading to activation of Gi/o protein and enhanced nNOS activity. The findings of these studies provide information related to cannabinoid-mediated NO signal transduction in neuronal cells, which has important implications in the ongoing elucidation of the endocannabinoid system in the nervous system.

Topics: Arachidonic Acids; Benzoxazines; Blotting, Western; Calcium; Cannabinoid Receptor Modulators; Cannabinoids; Cell Line; Cyclic GMP; Cyclohexanols; Endocannabinoids; Enzyme Activation; Enzyme Inhibitors; Guanylate Cyclase; Humans; Morpholines; Naphthalenes; Neurons; Nitric Oxide Synthase Type I; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction

In this study, we investigated whether the opioid system and the nitric oxide pathway were involved in the peripheral antinociception induced by a cannabinoid receptor agonist anandamide.. Hyperalgesia was induced by a subcutaneous injection of carrageenan (250 microg) into the plantar surface of the rat's hindpaw and measured by the paw pressure test 3h after injection. The weight in grams (g) required to elicit a nociceptive response, paw flexion, was determined as the nociceptive threshold.. Anandamide elicited a dose-dependent (50, 75, and 100 ng per paw) antinociceptive effect. The highest dose of anandamide did not produce antihyperalgesia in the contralateral paw, indicating a peripheral site of action. The CB(1) receptor antagonist AM251 (20, 40, 80 and 160mug per paw) antagonized peripheral antihyperalgesia induced by anandamide (100 ng), in a dose-dependent manner, suggesting CB(1) receptor activation. Anandamide-induced peripheral antihyperalgesia was reverted by blockers of the l-arginine/NO/cGMP pathway N(G)-nitro-l-arginine (NOARG; 24, 36 and 48 microg per paw) and 1H-[1,2,4] Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 25, 50 and 100 microg per paw), in a dose-dependent manner. Furthermore, opioid receptor antagonist naloxone (12.5, 25 and 50 microg per paw) antagonized the peripheral antihyperalgesia induced by anandamide.. This study provides evidence that the peripheral antinociceptive effect of the cannabinoid receptor agonist anandamide may result from l-arginine/NO/cGMP pathway activation and that the opioid system is also involved.

Topics: Analgesics; Animals; Arachidonic Acids; Cannabinoid Receptor Agonists; Cannabinoid Receptor Modulators; Cyclic GMP; Dose-Response Relationship, Drug; Endocannabinoids; Male; Nitric Oxide; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptors, Opioid; Signal Transduction

Glaucoma pathophysiology appears to involve vascular deficits, which may contribute to initiation and progression of the disease. Anandamide, the endogenous cannabinoid ligand, and WIN55212-2, a synthetic cannabinoid agonist, are able to evoke concentration-dependent relaxations in bovine ophthalmic artery rings, precontracted with 5-hydroxytryptamine (5-HT) (1 microM). Endothelium removal reduces cannabinoid agonist potency and efficacy. The selective cannabinoid 1 (CB1) receptor antagonists SR141716A (100 nM) and AM251 (100 nM) cause a shift to the right in the concentration-response curves to anandamide and WIN55212-2 in arterial rings both in the presence and in the absence of endothelium. In endothelium-intact arteries, the nitric oxide synthase inhibitor, N(G)-monomethyl-L-arginine (L-NMMA, 300 microM), completely blocked the anandamide- and WIN55212-2-relaxant responses; by contrast, the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP, 100 microM) induced an increase in vasorelaxant responses to cannabinoid agonists. Relaxations to anandamide and WIN55212-2 were inhibited by iberiotoxin (IbTX, 200 nM), a blocker of large conductance, Ca2+-activated K+ channel (BK(Ca)), and by 4-aminopyridine (4-AP; 1 mM), a blocker of delayed rectifier K+ channel, whereas the blockade of K(ATP) channels by glibenclamide (5 microM) and of small conductance Ca2+-activated K+ channels (SK(Ca)) by apamin (100 nM) did not produce any effects. These data suggest that anandamide and WIN55212-2 relax the bovine ophthalmic artery by involving CB1 the cannabinoid receptor-sensitive pathway. In endothelium-intact arteries, relaxation occurs through activation of nitric oxide synthase cyclic GMP and Ca2+-activated K+ channels. They also cause endothelium-independent relaxation by involving potassium channel opening.

Topics: Animals; Arachidonic Acids; Benzoxazines; Calcium Channel Blockers; Cattle; Cyclic GMP; Endocannabinoids; Morpholines; Naphthalenes; Nitric Oxide; Ophthalmic Artery; Polyunsaturated Alkamides; Potassium Channel Blockers; Potassium Channels; Receptor, Cannabinoid, CB1; Vasodilation

In the isolated rat mesenteric bed, the 1 min perfusion with 100 nm anandamide, a concentration that did not evoke vasorelaxation, elicited an acute release of 165.1 +/- 9.2 pmol nitric oxide (NO) that was paralleled by a 2-fold increase in cGMP tissue levels. The rise in NO released was mimicked by either (R)-(+)-methanandamide or the vanilloid receptor agonists resiniferatoxin and (E)-capsaicin but not by its inactive cis-isomer (Z)-capsaicin. The NO release elicited by either anandamide or capsaicin was reduced by the TRPV1 receptor antagonists 5'-iodoresiniferatoxin, SB 366791 and capsazepine as well as by the cannabinoid CB(1) receptor antagonists SR 141716A or AM251. The outflow of NO elicited by anandamide and capsaicin was also reduced by endothelium removal or NO synthase inhibition, suggesting the specific participation of endothelial TRPV1 receptors, rather than the novel endothelial TRPV4 receptors. Consistently, RT-PCR showed the expression of the mRNA coding for the rat TRPV1 receptor in the endothelial cell layer, in addition to its expression in sensory nerves. The participation of sensory nerves on the release of NO was precluded on the basis that neonatal denervation of the myenteric plexus sensory nerves did not modify the pattern of NO release induced by anandamide and capsaicin. We propose that low concentrations of anandamide, devoid of vasorelaxing effects, elicit an acute release of NO mediated predominantly by the activation of endothelial TRPV1 receptors whose physiological significance remains elusive.

Topics: Anilides; Animals; Arachidonic Acids; Cannabinoid Receptor Antagonists; Capsaicin; Cinnamates; Cyclic GMP; Diterpenes; Dose-Response Relationship, Drug; Endocannabinoids; Endothelium, Vascular; In Vitro Techniques; Male; Mesenteric Artery, Superior; Nitric Oxide; Nitroarginine; Perfusion; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Rimonabant; RNA, Messenger; TRPV Cation Channels; Vasodilation