am-630 and glyceryl-2-arachidonate

The endocannabinoid 2-arachidonoylglycerol (2-AG) is an anti-nociceptive lipid, which is inactivated through cellular uptake and subsequent catabolism by monoacylglycerol lipase (MAGL). The present study aimed to explore the effects of inhibition of MAGL on intestinal permeability. We first tested it in differentiated CaCO2 cells after 21 days' culture. The rat model of water avoidance stress (WAS) was established, and rats were divided into four groups according to intervention. Rats received intraperitoneal injection (i.p.) of an MAGL inhibitor (JZL184) alone, JZL184 and a the cannabinoid receptor 1 (CB1) receptor antagonist (SR141716A), JZL184 and a cannabinoid receptor 2 (CB2) receptor antagonist (AM630) or vehicle alone (control). We analyzed the fluorescein isothiocyanate-dextran (FD4) permeability and 2-AG level. Expression of MAGL and tight-junction-associated proteins were detected by western blot. Compared with the control group, MAGL expression was higher and 2-AG levels lower among WAS rats. Intestinal permeability was increased following administration of JZL184 which occurred due to up-regulation of tight-junction-associated proteins Claudin-1, Claudin-2, Claudin-5 and Occludin. The effects of MAGL inhibition were mediated by CB1, indicating that MAGL may represent a novel target for the treatment of reduced intestinal permeability in the context of chronic stress.

Topics: Animals; Arachidonic Acids; Benzodioxoles; Caco-2 Cells; Claudin-1; Claudin-2; Claudin-5; Disease Models, Animal; Endocannabinoids; Glycerides; Humans; Indoles; Intestinal Mucosa; Intestines; Male; Monoacylglycerol Lipases; Occludin; Permeability; Piperidines; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Stress, Physiological

The hallmark of vaccines is their ability to prevent the spread of infectious pathogens and thereby serve as invaluable public health tool. Despite their medical relevance, there is a gap in our understanding of the physiological factors that mediate innate and adaptive immune response to vaccines. The endocannabinoid (eCB) system is a critical modulator of homeostasis in vertebrates. Our results indicate that macrophages and dendritic cells produce the endocannabinoid, 2-arachidonoyl-sn-glycerol (2-AG) upon antigen activation. We have also established that 2-AG levels are upregulated in the serum and in the lymph node of mice during vaccination. We hypothesized that the intrinsic release of eCBs from immune cells during activation by pathogenic antigens mitigate inflammation, but also suppress overall innate and adaptive immune response. Here we demonstrate, for the first time, that transient administration of the cannabinoid receptor 2 antagonist AM630 (10 mg/kg) or inverse agonist JTE907 (3 mg/kg) during immunization heightens the intensity and breadth of antigen-specific immune responses in young and aged mice through the upregulation of immunomodulatory genes in secondary lymphoid tissues.

Topics: Animals; Antibodies, Monoclonal; Arachidonic Acids; Cannabinoids; Dendritic Cells; Endocannabinoids; Female; Glycerides; Immunization; Immunomodulation; Immunophenotyping; Indoles; Macrophage Activation; Macrophages; Mice; Receptor, Cannabinoid, CB2

While activation of cannabinoid CB1 receptor (CB1R) regulates a variety of retinal neuronal functions by modulating ion channels in these cells, effect of activated cannabinoid receptors on Ca(2+) channels in retinal Müller cells is still largely unknown. In the present work we show that three subunits of T-type Ca(2+) channels, CaV3.1, CaV3.2 and CaV3.3, as well as one subunit of L-type Ca(2+) channels, CaV1.2, were expressed in rat Müller cells by immunofluorescent staining. Consistently, nimodipine- and mibefradil-sensitive Na(+) currents through L- and T-type Ca(2+) channels could be recorded electrophysiologically. The cannabinoid receptor agonist WIN55212-2 significantly suppressed Ca(2+) channel currents, mainly the T-type one, in acutely isolated rat Müller cells in a dose-dependent manner, with an IC50 of 3.98μM. The WIN55212-2 effect was not blocked by AM251/SR141716, specific CB1R antagonists. Similar suppression of the currents was observed when anandamide (AEA) and 2-arachidonoyl glycerol (2-AG), endogenous ligands of cannabinoid receptors, were applied. Moreover, even though CB2 receptors (CB2Rs) were expressed in rat Müller cells, the effects of WIN55212-2 and 2-AG on Ca(2+) channel currents were not blocked by AM630, a selective CB2R antagonist. However, the effect of AEA could be partially rescued by AM630. These results suggest that WIN55212-2 and 2-AG receptor-independently suppressed the Ca(2+) channel currents in Müller cells, while AEA suppressed the currents partially through CB2Rs. The existence of receptor-dependent and -independent mechanisms suggests that cannabinoids may modulate Müller cell functions through multiple pathways.

Topics: Animals; Arachidonic Acids; Benzoxazines; Calcium; Calcium Channels; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cells, Cultured; Dose-Response Relationship, Drug; Endocannabinoids; Ependymoglial Cells; Glycerides; Indoles; Male; Membrane Potentials; Morpholines; Naphthalenes; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant

Enhancement of the endocannabinoid (EC) system may reduce anticipatory nausea (AN).. The experiments evaluated the potential of the dual fatty acid amide hydrolase (FAAH)/monoacylglycerol lipase (MAGL) inhibitor, JZL195, on its own and combined with anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) to reduce contextually elicited gaping, a measure of AN in rats.. Following four context lithium chloride (LiCl) pairings, rats were injected with vehicle (VEH) or JZL195 (10 mg kg(-1), intraperitoneally) 105 min before an injection of VEH, 2-AG (1.25 mg kg(-1)), or AEA (5.0 mg kg(-1)). Fifteen minutes later, all rats were placed in the LiCl-paired context for 5 min and in a different context for a 15-min locomotor test. Whole brains were extracted for EC analysis. The potential of the CB1 antagonist, SR141716, to reverse the suppression of AN by both JZL195 and AEA and of the CB2 antagonist, AM630, to reverse the suppression of AN by JZL195 was then evaluated.. JZL195 suppressed gaping and elevated AEA, palmitoylethanolamine, and oleoylethanolamide. As the suppression of gaping was reversed by SR141716, but not by AM630, the effect was CB1 mediated. The suppressive effect of JZL195 on gaping, as well as elevation of AEA and 2-AG, was amplified by pretreatment with either AEA or 2-AG. On its own, AEA, but not 2-AG, also suppressed gaping-an effect that was also prevented by CB1 antagonism.. JZL195 reduces AN primarily by acting as a FAAH inhibitor, but MAGL inhibition is also indicated.

Topics: Amidohydrolases; Animals; Anticipation, Psychological; Arachidonic Acids; Brain; Cannabinoid Receptor Antagonists; Carbamates; Endocannabinoids; Enzyme Inhibitors; Glycerides; Indoles; Lithium Chloride; Male; Monoacylglycerol Lipases; Motor Activity; Nausea; Oleic Acids; Piperazines; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant

Cyclooxygenase (COX) possesses substrate affinity for the endocannabinoids (EC) anandamide (AEA) and 2-arachidonylglycerol (2-AG). We hypothesized that selective antagonism/activation of the cannabinoid receptors will increase COX activity and the availability of EC as substrates will lead to higher COX activity. Since the relationship between EC signaling of the endocannabinoid system (ECS) and the COX pathway in muscle has not been investigated, we examined agonist, antagonists and polyunsaturated fatty acid effects on ECS genes in myoblasts. At 50% confluency, C2C12 myoblasts were pretreated with 5 μM of the cannabinoid receptor (CB)2 inverse agonist AM630 for 2 h and one with both AM630 and 1 μM of the CB1 antagonist NESS0327. Cell cultures pretreated with AM630 were then administered with 25 μM of either arachidonic acid (20:4n6), eicosapentaenoate (EPA) (20:5n3), docosahexaenoate (DHA) (22:6n3), AEA or bovine serum albumin (vehicle control) for 24 h. Quantitative polymerase chain reaction analyses were performed looking at ECS and prostaglandin genes. Total COX activity and COX-1 protein were greater in the AM630+AEA-treated cells compared to all other cell cultures. The mRNA for the AEA synthesis enzyme N-acyl phosphatidylethanolamine phospholipase D and the 2-AG synthesis enzymes diacylglycerol lipase (DAGL)α and DAGLβ were higher in AM630+EPA-treated cells compared to the other groups. The mRNA levels of CB1 and CB2 were both highest in the AM630+EPA group. The mRNA for interleukin-6 and tumor necrosis factor-α was higher with AEA but lower with DHA and docosahexaenoyl ethanolamide (DHEA), supporting previous findings that the EC AEA supports activation of the COX system. These findings suggest that COX activity and protein levels are influenced by the ECS, specifically by the ligand AEA for CB1 and by inverse agonism of CB2.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Cannabinoid Receptor Antagonists; Endocannabinoids; Fatty Acids, Unsaturated; Gene Expression Regulation; Glycerides; Indoles; Inflammation; Interleukin-6; Lipoprotein Lipase; Mice; Myoblasts; Piperidines; Prostaglandin-Endoperoxide Synthases; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Tumor Necrosis Factor-alpha

Cisplatin has been used effectively to treat a variety of cancers but its use is limited by the development of painful peripheral neuropathy. Because the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) is anti-hyperalgesic in several preclinical models of chronic pain, the anti-hyperalgesic effect of JZL184, an inhibitor of 2-AG hydrolysis, was tested in a murine model of cisplatin-induced hyperalgesia. Systemic injection of cisplatin (1mg/kg) produced mechanical hyperalgesia when administered daily for 7 days. Daily peripheral administration of a low dose of JZL184 in conjunction with cisplatin blocked the expression of mechanical hyperalgesia. Acute injection of a cannabinoid (CB)-1 but not a CB2 receptor antagonist reversed the anti-hyperalgesic effect of JZL184 indicating that downstream activation of CB1 receptors suppressed the expression of mechanical hyperalgesia. Components of endocannabinoid signaling in plantar hind paw skin and lumbar dorsal root ganglia (DRGs) were altered by treatments with cisplatin and JZL184. Treatment with cisplatin alone reduced levels of 2-AG and AEA in skin and DRGs as well as CB2 receptor protein in skin. Combining treatment of JZL184 with cisplatin increased 2-AG in DRGs compared to cisplatin alone but had no effect on the amount of 2-AG in skin. Evidence that JZL184 decreased the uptake of [(3)H]AEA into primary cultures of DRGs at a concentration that also inhibited the enzyme fatty acid amide hydrolase, in conjunction with data that 2-AG mimicked the effect of JZL184 on [(3)H]AEA uptake support the conclusion that AEA most likely mediates the anti-hyperalgesic effect of JZL184 in this model.

Topics: Amides; Analgesics; Animals; Antineoplastic Agents; Arachidonic Acids; Benzodioxoles; Cells, Cultured; Cisplatin; Disease Models, Animal; Endocannabinoids; Ethanolamines; Ganglia, Spinal; Glycerides; Hyperalgesia; Indoles; Male; Mesencephalon; Mice; Mice, Inbred C3H; Monoacylglycerol Lipases; Morpholines; Neuralgia; Palmitic Acids; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Skin; Spinal Cord

Anandamide and 2-arachidonoylglycerol (2-AG) are the two main endocannabinoids, exerting their effects by activating type 1 (CB1r) and type 2 (CB2r) cannabinoid receptors. Anandamide inhibits anxiety-like responses through the activation of CB1r in certain brain regions, including the dorsolateral periaqueductal gray (dlPAG). 2-AG also attenuates anxiety-like responses, although the neuroanatomical sites for these effects remained unclear. Here, we tested the hypothesis that enhancing 2-AG signaling in the dlPAG would induce anxiolytic-like effects. The mechanisms involved were also investigated. Male Wistar rats received intra-dlPAG injections of 2-AG, URB602 (inhibitor of the 2-AG hydrolyzing enzyme, mono-acylglycerol lipase--MGL), AM251 (CB1r antagonist) and AM630 (CB2r antagonist). The behavior was analyzed in the elevated plus maze after the following treatments. Exp. 1: vehicle (veh) or 2-AG (5 pmol, 50 pmol, and 500 pmol). Exp. 2: veh or URB602 (30 pmol, 100 pmol or 300 pmol). Exp. 3: veh or AM251 (100 pmol) followed by veh or 2-AG (50 pmol). Exp. 4: veh or AM630 (1000 pmol) followed by veh or 2-AG. Exp. 5: veh or AM251 followed by veh or URB602 (100 pmol). Exp. 6: veh or AM630 followed by veh or URB602. 2-AG (50 pmol) and URB602 (100 pmol) significantly increased the exploration of the open arms of the apparatus, indicating an anxiolytic-like effect. These behavioral responses were prevented by CB1r (AM251) or CB2r (AM630) antagonists. Our results showed that the augmentation of 2-AG levels in the dlPAG induces anxiolytic-like effects. The mechanism seems to involve both CB1r and CB2r receptors.

Topics: Analysis of Variance; Animals; Anxiety; Arachidonic Acids; Biphenyl Compounds; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Disease Models, Animal; Dose-Response Relationship, Drug; Endocannabinoids; Glycerides; Indoles; Male; Maze Learning; Periaqueductal Gray; Piperidines; Pyrazoles; Rats; Rats, Wistar

Endocannabinoid signaling is terminated by enzymatic hydrolysis, a process that, for 2-Arachidonoylglycerol (2-AG), is mediated by monoacylglycerol lipase (MAGL). The piperidine carbamate, 4-nitrophenyl- 4-(dibenzo[d] [1,3]dioxol-5-yl (hydroxy) methyl) piperidine- 1-carboxylate (JZL184), is a drug that inhibits MAGL and presents high potency and selectivity. Thus, JZL184 increases the levels of 2-AG, an endocannabinoid that acts on the CB1 and CB2 cannabinoid receptors. Here, we investigated the effects of MAGL inhibition, with a single dose (16 mg/kg, intraperitoneally (i.p.)) of JZL184, in a murine model of lipopolysaccharide (LPS) -induced acute lung injury (ALI) 6, 24 and 48 hours after the inflammatory insult. Treatment with JZL184 decreased the leukocyte migration into the lungs as well as the vascular permeability measured through the bronchoalveolar lavage fluid (BAL) and histological analysis. JZL184 also reduced the cytokine and chemokine levels in the BAL and adhesion molecule expression in the blood and BAL. The CB1 and CB2 receptors were considered involved in the anti-inflammatory effects of JZL184 because the AM281 selective CB1 receptor antagonist (1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide) and the AM630 selective CB2 receptor antagonist ([6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)-methanone) blocked the anti-inflammatory effects previously described for JZL184. It was concluded that MAGL inhibition, and consequently the increase in 2-AG levels, produced anti-inflammatory effects in a murine model of LPS-induced ALI, a finding that was considered a consequence of the activation of the CB1 and CB2 receptors.

Topics: Acute Lung Injury; Animals; Arachidonic Acids; Benzodioxoles; Bronchoalveolar Lavage Fluid; Capillary Permeability; Cell Adhesion Molecules; Endocannabinoids; Glycerides; Indoles; Inflammation; Leukocytes; Male; Mice; Mice, Inbred C57BL; Monoacylglycerol Lipases; Piperidines; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2

Endocannabinoid system is reported to be activated during myocardial ischemia-reperfusion (IR) injury and protects against heart injury. We, therefore, observed changes in endocannabinoids levels during acute myocardial infarction (AMI) and myocardial IR injury and evaluated the role of cannabinoid-2 (CB2) receptor in infarct and IR heart injury. In contrast to 16 control patients with normal coronary artery angiogram, the endocannabinoid 2-arachidonoylglycerol level in the infarct-side coronary artery of 23 AMI patients increased significantly, with increased reactive oxygen species and tumor necrosis factor-α levels in both infarct-side coronary artery and radial artery. Then, 35 C57BL/6J mice were made into SHAM, AMI, or IR models. AMI and IR groups were treated with CB2-selective agonist HU308 ((+)-(1aH,3H,5aH)-4-[2,6-dimethoxy-4-(1,1-dimethylheptyl)phenyl]-6,6-dimethylbicyclo[3.1.1]hept-2-ene-2-carbinol), with or without CB2-selective antagonist AM630 [6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone through intraperitoneal injection. Compared with the SHAM, expressions of cannabinoid CB1/CB2 receptor proteins in AMI/IR animals were upregulated; production of 2-arachidonoylglycerol and anandamide and release of reactive oxygen species and tumor necrosis factor-α also increased. HU308 significantly decreased the infarct size and the levels of reactive oxygen species and tumor necrosis factor-α in AMI/IR animals. However, these effects were blocked by AM630. In conclusion, the endocannabinoid system was activated during AMI and IR, and CB2 receptor activation produces a protective role, thus offering a novel pharmaceutical target for treating these diseases.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Cannabinoids; Case-Control Studies; Coronary Angiography; Coronary Vessels; Disease Models, Animal; Endocannabinoids; Glycerides; Humans; Indoles; Injections, Intraperitoneal; Male; Mice; Mice, Inbred C57BL; Myocardial Infarction; Myocardial Reperfusion Injury; Radial Artery; Reactive Oxygen Species; Receptor, Cannabinoid, CB2; Tumor Necrosis Factor-alpha

Inflammatory bowel diseases (IBDs) are chronic inflammatory conditions for which new therapeutic approaches are needed. Genetic and pharmacological data point to a protective role of CB(1) and CB(2) cannabinoid receptor activation in IBD experimental models. Therefore, increasing the endogenous levels of 2-arachidonoylglycerol, the main full agonist of these receptors, should have beneficial effects on colitis. 2-Arachidonoylglycerol levels were raised in the trinitrobenzene sulfonic acid (TNBS)-induced colitis mouse model by inhibiting monoacylglycerol lipase (MAGL), the primary enzyme responsible for hydrolysis of 2-arachidonoylglycerol, using the selective inhibitor JZL184. MAGL inhibition in diseased mice increased 2-arachidonoylglycerol levels, leading to a reduction of macroscopic and histological colon alterations, as well as of colonic expression of proinflammatory cytokines. The restored integrity of the intestinal barrier function after MAGL inhibition resulted in reduced endotoxemia as well as reduced peripheral and brain inflammation. Coadministration of either CB(1) (SR141716A) or CB(2) (AM630) selective antagonists with JZL184 completely abolished the protective effect of MAGL inhibition on TNBS-induced colon alterations, thus demonstrating the involvement of both cannabinoid receptors. In conclusion, increasing 2-arachidonoylglycerol levels resulted in a dramatic reduction of colitis and of the related systemic and central inflammation. This could offer a novel pharmacological approach for the treatment of IBD based on the new protective role of 2-arachidonoylglycerol described here.

Topics: Animals; Arachidonic Acids; Benzodioxoles; Colitis; Disease Models, Animal; Endocannabinoids; Endotoxemia; Enzyme Inhibitors; Glycerides; Humans; Indoles; Inflammation; Inflammation Mediators; Inflammatory Bowel Diseases; Male; Mice; Mice, Inbred C57BL; Monoacylglycerol Lipases; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; Trinitrobenzenesulfonic Acid

Endocannabinoids including anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are important lipid mediators for immunosuppressive effects and for appropriate homeostasis via their G-protein-coupled cannabinoid (CB) receptors in mammalian organs and tissues, and may be involved in wound healing in some organs. The physiological roles of endocannabinoids in periodontal healing remain unknown. We observed upregulation of the expression of CB1/CB2 receptors localized on fibroblasts and macrophage-like cells in granulation tissue during wound healing in a wound-healing model in rats, as well as an increase in AEA levels in gingival crevicular fluid after periodontal surgery in human patients with periodontitis. In-vitro, the proliferation of human gingival fibroblasts (HGFs) by AEA was significantly attenuated by AM251 and AM630, which are selective antagonists of CB1 and CB2, respectively. CP55940 (CB1/CB2 agonist) induced phosphorylation of the extracellular-regulated kinases (ERK) 1/2, p38 mitogen-activated protein kinase (p38MAPK), and Akt in HGFs. Wound closure by CP55940 in an in-vitro scratch assay was significantly suppressed by inhibitors of MAP kinase kinase (MEK), p38MAPK, and phosphoinositol 3-kinase (PI3-K). These findings suggest that endocannabinoid system may have an important role in periodontal healing.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Cell Proliferation; Endocannabinoids; Fibroblasts; Gingival Crevicular Fluid; Glycerides; Humans; Indoles; p38 Mitogen-Activated Protein Kinases; Periodontium; Phosphatidylinositol 3-Kinases; Phosphorylation; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Wound Healing

There are conflicting views in the literature as to whether cannabinoids have an impact on platelet activity and to what extent cannabinoid receptors are involved. This is an important issue to resolve because platelet effects of putative therapeutic cannabinoid inhibitors and stimulators will have an impact on their potential benefits and safety.. The data presented in this manuscript clearly show that the endocannabinoid 2-arrachidonyl glycerol can activate platelet activity, but that the effects are mediated through an aspirin-sensitive pathway that is not affected by cannabinoid receptor antagonists or FAAH inhibition, but is abolished by MAGL inhibition. The findings question the role of cannabinoid receptors in platelet function and suggest that platelet function is unlikely to be directly affected by cannabinoid receptor antagonists, at least in the acute phase.. Cannabinoid receptor-1 (CB(1)) antagonists suppress appetite and induce weight loss. Direct antagonism of CB(1) receptors on platelets might be an additional benefit for CB(1) antagonists, but the role of CB(1) receptors in platelets is controversial. We tested the hypothesis that the endocannabinoid, 2-arachidonyl glycerol (2-AG), induces platelet aggregation by a COX-mediated mechanism rather than through CB(1) receptor activation, in blood obtained from healthy volunteers and patients with coronary artery disease receiving low dose aspirin.. Aggregatory responses to the cannabinoids 2-AG and Delta(9)-THC were examined in blood sampled from healthy volunteers (n= 8) and patients (n= 12) with coronary artery disease receiving aspirin using whole blood aggregometry. The effects of CB(1) (AM251) and CB(2) (AM630) antagonists, as well as fatty acid amide hydrolase (FAAH) and monoacyl glycerol lipase (MAGL) inhibitors and aspirin on 2-AG-induced aggregation were also assessed.. AM251 (100 nm-30 microm) had no effect on platelet aggregation induced by either ADP (P= 0.90) or thrombin (P= 0.86). 2-AG, but not Delta(9)-THC, induced aggregation. 2-AG-induced aggregation was unaffected by AM251 and AM630 but was abolished by aspirin (P < 0.001) and by the MAGL inhibitor, URB602 (P < 0.001). Moreover, the aggregatory response to 2-AG was depressed (by >75%, P < 0.001) in blood from patients with coronary artery disease receiving aspirin compared with that from healthy volunteers.. 2-AG-mediated activation of platelets is via metabolism to arachidonic acid by MAGL, and not through direct action on CB(1) or CB(2) receptors, at least in the acute phase.

Topics: Adolescent; Adult; Aged; Arachidonic Acids; Aspirin; Blood Platelets; Cannabinoid Receptor Antagonists; Cannabinoid Receptor Modulators; Coronary Artery Disease; Endocannabinoids; Glycerides; Humans; Indoles; Male; Middle Aged; Piperidines; Platelet Activation; Platelet Aggregation; Platelet Aggregation Inhibitors; Pyrazoles; Young Adult

To date, it has been thought that cannabinoid receptors in CNS are primarily of the CB1R subtype, with CB2R expressed only in glia and peripheral tissues. However, evidence for the expression of CB2 type cannabinoid receptors at neuronal sites in the CNS is building through anatomical localization of receptors and mRNA in neurons and behavioural studies of central effects of CB2R agonists. In the medial entorhinal area of the rat, we found that blockade of CB1R did not occlude suppression of GABAergic inhibition by the non-specific endogenous cannabinoid 2-AG, suggesting that CB1R could not account fully for the effects of 2-AG. Suppression could be mimicked using the CB2R agonist JWH-133 and reversed by the CB2R inverse agonist AM-630, indicating the presence of functional CB2R. When we reversed the order of drug application AM-630 blocked the effects of the CB2R agonist JWH-133, but not the CB1R inverse agonist LY320135. JTE-907, a CB2R inverse agonist structurally unrelated to AM-630 elicited increased GABAergic neurotransmission at picomolar concentrations. Analysis of mIPSCs revealed that CB2R effects were restricted to action potential dependent, but not action potential independent GABA release. These data provide pharmacological evidence for functional CB2R at CNS synapses.

Topics: Animals; Arachidonic Acids; Benzofurans; Cannabinoid Receptor Modulators; Cannabinoids; Central Nervous System Agents; Dioxoles; Endocannabinoids; Entorhinal Cortex; gamma-Aminobutyric Acid; Glycerides; Hippocampus; In Vitro Techniques; Indoles; Male; Neural Inhibition; Neurons; Quinolones; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Synapses; Synaptic Transmission

Previously, we reported that intracerebroventricularly (i.c.v.) administered arginine-vasopressin evokes the secretion of noradrenaline and adrenaline from adrenal medulla through the brain phospholipase C- and diacylglycerol-mediated and cyclooxygenase-mediated mechanisms in rats. Diacylglycerol can be hydrolyzed by diacylglycerol lipase to 2-arachidonoyl-sn-glycerol, which may be further degradated by monoacylglycerol lipase to free arachidonic acid, a representative substrate of cyclooxygenase. Recently, 2-arachidonoyl-sn-glycerol has been recognized as a major endocannabinoid, which can modulate synaptic transmission in the brain. In the present experiment, therefore, we examined (1) a role of the brain 2-arachidonoyl-sn-glycerol as a precursor of arachidonic acid in the centrally administered vasopressin-induced elevation of plasma noradrenaline and adrenaline, and (2) a regulatory role of the brain 2-arachidonoyl-sn-glycerol as an endocannabinoid on the vasopressin-induced response, using urethane-anesthetized rats. The vasopressin (0.2 nmol/animal, i.c.v.)-induced elevation of plasma catecholamines was reduced by RHC-80267 (diacylglycerol lipase inhibitor) (1.3 and 2.6 micromol/animal, i.c.v.) and also reduced by MAFP (monoacylglycerol lipase inhibitor) (0.7 and 1.4 micromol/animal, i.c.v.). MAFP (1.4 micromol/animal, i.c.v.) also attenuated the 2-arachidonoyl-sn-glycerol (0.5 micromol/animal, i.c.v.)-induced elevation of plasma catecholamines. AM 251 (cannabinoid CB(1) receptor antagonist) (90 and 180 nmol/animal, i.c.v.) potentiated the vasopressin (0.2 nmol/animal, i.c.v.)-induced response, while AM 630 (cannabinoid CB(2) receptor antagonist) (198 and 793 nmol/animal, i.c.v.) was largely ineffective. In addition, WIN 55212-2 (cannabinoid CB receptor agonist) (188 and 470 nmol/animal, i.c.v.) dose-dependently reduced the vasopressin-induced response. These results suggest that the brain 2-arachidonoyl-sn-glycerol generated from diacylglycerol plays a role as a precursor of arachidonic acid in the centrally administered vasopressin-induced activation of the adrenomedullary outflow, and also negatively regulates the peptide-induced central response through the brain cannabinoid CB(1) receptors in rats.

Topics: Adrenal Medulla; Animals; Arachidonic Acids; Arginine Vasopressin; Benzoxazines; Brain; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cannabinoid Receptor Modulators; Catecholamines; Cyclohexanones; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Epinephrine; Glycerides; Indoles; Injections, Intraventricular; Male; Morpholines; Naphthalenes; Norepinephrine; Organophosphonates; Piperidines; Pyrazoles; Rats; Rats, Wistar

2-arachidonoyl glycerol (2-AG) is an endogenous cannabinoid with central antinociceptive properties. Its degradation is catalysed by monoacylglycerol lipase (MGL) whose activity is inhibited by URB602, a new synthetic compound. The peripheral antinociceptive effects of 2-AG and URB602 in an inflammatory model of pain are not yet determined. We have evaluated these effects with and without the cannabinoid CB(1) (AM251) and CB(2) (AM630) receptor antagonists.. Inflammation was induced in rat hind paws by intraplantar injection of formalin. Nociception was assessed behaviourally over the next 60 min, in 19 experimental groups: (1) control; (2-6) 2-AG (0.01-100 microg); (7) AM251 (80 microg); (8) AM251+2-AG (10 microg); (9) AM630 (25 microg); (10) AM630+2-AG (10 microg); (11-16) URB602 (0.1-500 microg); (17) 2-AG+URB602 (ED(50)); (18) AM251+URB602 (ED(50)); (19) AM630+URB602 (ED(50)). Drugs were injected s.c. in the dorsal surface of the hind paw (50 microl), 15 min before formalin injection into the same paw.. 2-AG and URB602 produced dose-dependent antinociceptive effects for the late phases of the formalin test with ED(50) of 0.65+/-0.455 mug and 68+/-14.3 microg, respectively. Their combination at ED(50) doses produced an additive antinociceptive effect. These effects were inhibited by AM630 but not by AM251 for 2-AG and by the two cannabinoid antagonists for URB602.. Locally injected 2-AG and URB602 decreased pain behaviour in a dose-dependent manner in an inflammatory model of pain. The antinociceptive effect of 2-AG was mediated by the CB(2) receptor.

Topics: Analgesics, Non-Narcotic; Animals; Arachidonic Acids; Biphenyl Compounds; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Glycerides; Indoles; Male; Monoacylglycerol Lipases; Pain Measurement; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2

Endocannabinoids have been implicated in protective effects in the heart and brain, but the mechanism of possible infarct-size-reducing effects remains controversial. Using a model of delayed preconditioning (PC), rats received the nitric oxide (NO) donor nitroglycerin (0.15 mg/h/kg) for 24 hours via transdermal application. Two days later, rat isolated perfused hearts were subjected to global, no-flow ischemia (20 min), and reperfusion (120 min). Cannabinoid receptor antagonists were given before no-flow throughout the protocol. Endocannabinoids were detected by liquid chromatography and mass spectrometry. NO-induced PC reduced the left ventricular infarct size from 40.9 +/- 3.9% to 27.5 +/- 3.8% (P < 0.05). Treatment with the specific CB1 cannabinoid receptor antagonist AM-251 (0.3 microM) prevented the protective effect of PC on infarct size (40.2 +/- 4.7%, P > 0.05 vs. controls). On the contrary, the specific CB2 receptor antagonist AM-630 (0.3 microM) did not alter infarct size (31.6 +/- 6.3%, P > 0.05 vs. PC alone). Recovery of left ventricular developed pressure and coronary flow was incomplete in control and NO-pretreated hearts and not consistently altered by cannabinoid receptor antagonists. PC increased the heart tissue content of the endocannabinoid 2-arachidonylglycerol (2-AG) from 4.6 +/- 1.0 nmol/g in controls to 12.0 +/- 2.1 nmol/g (P < 0.05). Tissue levels of the endocannabinoid arachidonylethanolamide (anandamide) remained unchanged (19.8 +/- 3.9 pmol/g vs. 19.5 +/- 4.8 pmol/g). 2-AG (1 microM) or its metabolically stable derivative noladinether (0.1 microM), given 30 minutes before ischemia/reperfusion in unpreconditioned hearts, mimicked the cardioprotective effects of PC and reduced infarct size. We conclude that delayed PC through transdermal nitroglycerin application increases the production of the endocannabinoid 2-AG which elicits protective effects against myocardial infarction via CB1 cannabinoid receptors which represents one new mechanism of NO-mediated PC.

Topics: Animals; Arachidonic Acids; Blood Pressure; Cannabinoid Receptor Modulators; Coronary Vessels; Endocannabinoids; Glycerides; Heart; Heart Rate; Indoles; Ischemic Preconditioning, Myocardial; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Nitric Oxide; Nitric Oxide Donors; Nitroglycerin; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Regional Blood Flow

This study examined the ability of the endocannabinoids 2-arachidonoyl glycerol (2-AG) and noladin ether as well as the synthetic cannabinoid CP-55,940 [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol] to regulate three intracellular effectors via CB2 receptors in transfected Chinese hamster ovary cells. Although the three agonists regulate all effectors with equivalent efficacy, the rank order of potencies differs depending on which effector is evaluated. Noladin ether and CP-55,940 most potently inhibit adenylyl cyclase, requiring higher concentrations to stimulate the extracellular signal-regulated kinase subgroup of the mitogen-activated protein kinases (extracellular signal-regulated kinase-mitogen-activated protein kinase; ERK-MAPK) and Ca(2+)-transients. In contrast, 2-AG most potently activates ERK-MAPK, necessitating greater concentrations to inhibit adenylyl cyclase and even higher amounts to stimulate Ca(2+)-transients. Endocannabinoids also seem to be more "efficient" agonists at CB2 receptors relative to synthetic agonists. 2-AG and noladin ether require occupancy of less than one-half the number of receptors to produce comparable regulation of adenylyl cyclase and ERK-MAPK, relative to the synthetic cannabinoid CP-55,940. The CB2 antagonist 6-iodo-2-methyl-1-[2-(4-morpholinyl)-ethyl]-1H-indol-3-yl](4-methoxyphenyl)-methanone (AM630) reverses the actions of all agonists except Ca(2+)-transient stimulation by 2-AG. However, the effect of 2-AG on Ca(2+)-transients is attenuated by a second CB2 antagonist N-[(1S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-1-pyrazole-3-carboxamide (SR144528). This suggests that 2-AG stimulates Ca(2+)-transients by binding to sites on CB2 receptors distinct from those occupied by AM630 and the other cannabinoids examined. Agonists produce no effects in pertussis toxin-treated cells. In summary, cannabinoid agonists distinctly bind to CB2 receptors and display different rank order of potencies and fractional receptor occupancies for regulation of intracellular effectors. These data provide direct evidence for agonist-directed trafficking of response by endocannabinoids acting at CB2 receptors.

Topics: Adenylyl Cyclases; Animals; Arachidonic Acids; Binding, Competitive; Blotting, Western; Calcium Signaling; Cannabinoid Receptor Modulators; Cell Membrane; CHO Cells; Cricetinae; Cyclic AMP; Cyclohexanols; DNA, Complementary; Endocannabinoids; Enzyme-Linked Immunosorbent Assay; Glycerides; GTP-Binding Protein alpha Subunits, Gi-Go; Humans; Indoles; Mitogen-Activated Protein Kinases; Receptor, Cannabinoid, CB2; Transfection

Pharmacological effects of cannabinoid ligands are thought to be mediated through cannabinoid CB1 and CB2 receptor subtypes. Sequence analysis revealed that rat and human cannabinoid CB2 receptors are divergent and share 81% amino acid homology. Pharmacological analysis of the possible species differences between rat and human cannabinoid CB2 receptors was performed using radioligand binding and functional assays. Pronounced species selectivity at the rat cannabinoid CB2 receptor (50- to 140-fold) was observed with AM-1710 (3-(1,1-Dimethyl-heptyl)-1-hydroxy-9-methoxy-benzo[c]chromen-6-one) and AM-1714 (3-(1,1-Dimethyl-heptyl)-1-9-dihydroxy-benzo[c]chromen-6-one). In contrast, JWH-015 ((2-Methyl-1-propyl-1H-indol-3-yl)-napthalen-1-yl-methanone) was 3- to 10-fold selective at the human cannabinoid CB2 receptor. Endocannabinoid ligands were more human receptor selective. Cannabinoid CB2 receptor antagonist, AM-630 ((6-Iodo-2-methyl-1-(2-morpholin-4-yl-ethyl)-1H-indol-3-yl)-(4-methoxy-phenyl)-methanone) was more potent at the rat receptor in radioligand binding and functional assays than that of the human receptor. The findings of the pharmacological differences between the human and rat cannabinoid CB2 receptors in this study provide critical information for characterizing cannabinoid ligands in in vivo rodent models for drug discovery purpose.

Topics: Animals; Arachidonic Acids; Benzoxazines; Binding, Competitive; Calcium; Cell Line; Chromones; Colforsin; Cyclic AMP; Cyclohexanols; DNA, Complementary; Dose-Response Relationship, Drug; Endocannabinoids; Glycerides; Humans; Indoles; Morpholines; Naphthalenes; Polyunsaturated Alkamides; Radioligand Assay; Rats; Receptor, Cannabinoid, CB2; Species Specificity; Transfection; Tritium