glyceryl-2-arachidonate and Inflammation

It has been established that the endogenous cannabinoid (endocannabinoid) system plays key modulatory roles in a wide variety of pathological conditions. The endocannabinoid system comprises both cannabinoid receptors, their endogenous ligands including 2-arachidonoylglycerol (2-AG), N-arachidonylethanolamine (anandamide, AEA), and enzymes that regulate the synthesis and degradation of endogenous ligands which include diacylglycerol lipase alpha (DAGL-α), diacylglycerol lipase beta (DAGL-β), fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL), α/β hydrolase domain 6 (ABHD6). As the endocannabinoid system exerts considerable involvement in the regulation of homeostasis and disease, much effort has been made towards understanding endocannabinoid-related mechanisms of action at cellular, physiological, and pathological levels as well as harnessing the various components of the endocannabinoid system to produce novel therapeutics. However, drug discovery efforts within the cannabinoid field have been slower than anticipated to reach satisfactory clinical endpoints and raises an important question into the validity of developing novel ligands that therapeutically target the endocannabinoid system. To answer this, we will first examine evidence that supports the existence of an endocannabinoid system role within inflammatory diseases, neurodegeneration, pain, substance use disorders, mood disorders, as well as metabolic diseases. Next, this review will discuss recent clinical studies, within the last 5 years, of cannabinoid compounds in context to these diseases. We will also address some of the challenges and considerations within the cannabinoid field that may be important in the advancement of therapeutics into the clinic.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Agonists; Cannabinoids; Drug Discovery; Endocannabinoids; Glycerides; Humans; Inflammation; Metabolic Diseases; Mood Disorders; Nervous System Diseases; Pain; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Substance-Related Disorders

Endocannabinoids are key bioactive components of the endocannabinoid system, and the profound influence of endocannabinoids on the modulation of the immune system is being increasingly appreciated. The knowledge of endocannabinoid-immune cell crosstalk will pave the way to therapeutic implications of modulators of this pathway in autoimmune and chronic inflammatory disorders. Endocannabinoids seem to exert both anti-inflammatory and pro-inflammatory effects in specific contexts, based on specific receptor engagement and the downstream signalling pathways involved. In this review, we summarized the biosynthesis, signalling and degradation of two well-studied endocannabinoids-anandamide and 2-arachidonylglycerol in immune cells. Then, we discussed the effects of these two endocannabinoids on the functioning of major innate and adaptive immune cells, along with the choice of receptors employed in such interactions. Finally, we outline our current knowledge on the involvement of anandamide and 2-arachidonylglycerol in context of inflammation, allergies, autoimmunity and metabolic disorders.

Topics: Adaptive Immunity; Animals; Arachidonic Acids; Endocannabinoids; Glycerides; Humans; Immunity, Innate; Inflammation; Polyunsaturated Alkamides; Signal Transduction

Increased levels of endocannabinoids, 2-arachidonoylglycerol (2-AG) and arachidonoyl ethanolamide (AEA) have a pathophysiological role in the setting of cardiometabolic diseases. This systematic review was carried out to appraise the effect of omega-3 on cardiometabolic risk factors by highlighting the mediating effect of endocannabinoids. SCOPUS, PubMed, Embase, Google Scholar and ProQuest databases were searched until January 2020. All published English-language animal studies and clinical trials that evaluated the effects of omega-3 on cardiometabolic diseases with a focus on endocannabinoids were included. Of 1407 studies, 16 animal studies and three clinical trials were included for analysis. Eleven animal studies and two human studies showed a marked reduction in 2-AG and AEA levels following intake of omega-3 which correlated with decreased adiposity, weight gain and improved glucose homeostasis. Moreover, endocannabinoids were elevated in three studies that replaced omega-3 with omega-6. Omega-3 showed anti-inflammatory properties due to reduced levels of inflammatory cytokines, regulation of T-cells function and increased levels of eicosapentaenoyl ethanolamide, docosahexaenoyl ethanolamide and oxylipins; however, a limited number of studies examined a correlation between inflammatory cytokines and endocannabinoids following omega-3 administration. In conclusion, omega-3 modulates endocannabinoid tone, which subsequently attenuates inflammation and cardiometabolic risk factors. However, further randomized clinical trials are needed before any recommendations are made to target the ECS using omega-3 as an alternative therapy to drugs for cardiometabolic disease improvement.

Topics: Animals; Anti-Inflammatory Agents; Arachidonic Acids; Cardiovascular Diseases; Endocannabinoids; Fatty Acids, Omega-3; Glucose; Glycerides; Homeostasis; Humans; Inflammation; Oxylipins; Phospholipids; Polyunsaturated Alkamides; Risk Factors; Signal Transduction

2-Arachidonoyl-glycerol (2-AG) and arachidonyl-ethanolamide (AEA) are endocannabinoids that have been implicated in many physiologic disorders, including obesity, metabolic syndromes, hepatic diseases, pain, neurologic disorders, and inflammation. Their immunomodulatory effects are numerous and are not always mediated by cannabinoid receptors, reflecting the presence of an arachidonic acid (AA) molecule in their structure, the latter being the precursor of numerous bioactive lipids that are pro- or anti-inflammatory. 2-AG and AEA can thus serve as a source of AA but can also be metabolized by most eicosanoid biosynthetic enzymes, yielding additional lipids. In this regard, enhancing endocannabinoid levels by using endocannabinoid hydrolysis inhibitors is likely to augment the levels of these lipids that could regulate inflammatory cell functions. This review summarizes the metabolic pathways involved in the biosynthesis and metabolism of AEA and 2-AG, as well as the biologic effects of the 2-AG and AEA lipidomes in the regulation of inflammation.

Topics: Animals; Arachidonic Acids; Dendritic Cells; Endocannabinoids; Glycerides; Humans; Inflammation; Lipid Metabolism; Liver Diseases; Lymphocytes; Metabolic Syndrome; Neurodegenerative Diseases; Obesity; Pain; Phosphatidic Acids; Polyunsaturated Alkamides; Receptors, Cannabinoid

The endocannabinoid system is implicated in, and regulates, several physiological processes, ranging from food intake and energy balance to pain and inflammation. 2-Arachidonoylglycerol (2-AG) is a full agonist at the cannabinoid receptors which classically mediate its effects. The activity of this bioactive lipid is dependent on its endogenous levels, which are tightly controlled by several hydrolases, monoacylglycerol lipase and α/β-hydrolase domain 6 and 12. Moreover, 2-AG is also a substrate of cyclooxygenase-2, and this reaction leads to the formation of prostaglandin glycerol esters, the effects of which remain to be fully elucidated. In this review we discuss the multiple mechanisms by which 2-AG controls inflammation and the therapeutic potential of 2-AG metabolism inhibitors.

Topics: Animals; Anti-Inflammatory Agents; Arachidonic Acids; Cyclooxygenase 2; Drug Design; Endocannabinoids; Esters; Glycerides; Humans; Inflammation; Prostaglandins; Receptors, Cannabinoid

Crohn's disease and ulcerative colitis are two major forms of inflammatory bowel diseases (IBD), which are chronic inflammatory disorders of the gastrointestinal tract. These pathologies are currently under investigation to both unravel their etiology and find novel treatments. Anandamide and 2-arachidonoylglycerol are endogenous bioactive lipids that bind to and activate the cannabinoid receptors, and together with the enzymes responsible for their biosynthesis and degradation [fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL)] constitute the endocannabinoid system (ECS). The ECS is implicated in gut homeostasis, modulating gastrointestinal motility, visceral sensation, and inflammation, as well as being recently implicated in IBD pathogenesis. Numerous subsequent studies investigating the effects of cannabinoid agonists and endocannabinoid degradation inhibitors in rodent models of IBD have identified a potential therapeutic role for the ECS.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Cannabinoid Receptor Agonists; Disease Models, Animal; Endocannabinoids; Gastrointestinal Tract; Glycerides; Homeostasis; Humans; Inflammation; Inflammatory Bowel Diseases; Monoacylglycerol Lipases; Polyunsaturated Alkamides; Receptors, Cannabinoid

Among the several known fatty acid-derived chemical signals, the endogenous ligands of cannabinoid receptors type-1 and -2, two G-protein-coupled receptors involved in several aspects of mammalian physiology and pathology, are perhaps those the levels of which have proven to be most sensitive to the fatty acid composition of the diet. The two most studied such ligands, known as endocannabinoids, are N-arachidonoyl-ethanolamine and 2-archidonoylglycerol, and are found in tissues together with other N-acyl-ethanolamines and 2-acylglycerols, not all of which activate the cannabinoid receptors, although several of them do exhibit important pharmacological effects. In this review article, we describe literature data indicating that the tissue concentrations of the endocannabinoids and related signalling molecules, and hence the activity of the respective receptors, can be modulated by modifying the fatty acid composition of the diet, and particularly its content in long chain PUFAs or in long chain PUFA precursors. We also discuss the potential impact of these diet-induced changes of endocannabinoid tone on three of the major pathological conditions in which cannabinoid receptors have been involved, that is metabolic dysfunctions, inflammation and affective disorders.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Dietary Fats; Endocannabinoids; Energy Metabolism; Fatty Acids, Omega-3; Glycerides; Homeostasis; Humans; Inflammation; Metabolic Diseases; Mood Disorders; Polyunsaturated Alkamides; Signal Transduction; Stress, Physiological

The endogenous cannabinoid N-arachidonoyl ethanolamine (anandamide; AEA) produces most of its pharmacological effects by binding and activating CB(1) and CB(2) cannabinoid receptors within the CNS and periphery. However, the actions of AEA are short lived because of its rapid catabolism by fatty acid amide hydrolase (FAAH). Indeed, FAAH knockout mice as well as animals treated with FAAH inhibitors are severely impaired in their ability to hydrolyze AEA as well as a variety of noncannabinoid lipid signaling molecules and consequently possess greatly elevated levels of these endogenous ligands. In this mini review, we describe recent research that has investigated the functional consequences of inhibiting this enzyme in a wide range of animal models of inflammatory and neuropathic pain states. FAAH-compromised animals reliably display antinociceptive and anti-inflammatory phenotypes with a similar efficacy as direct-acting cannabinoid receptor agonists, such as Delta(9)-tetrahydrocannabinol (THC), the primary psychoactive constituent of Cannabis sativa. Importantly, FAAH blockade does not elicit any apparent psychomimetic effects associated with THC or produce reinforcing effects that are predictive of human drug abuse. The beneficial effects caused by FAAH blockade in these models are predominantly mediated through the activation of CB(1) and/or CB(2) receptors, though noncannabinoid mechanisms of actions can also play contributory or even primary roles. Collectively, the current body of scientific literature suggests that activating the endogenous cannabinoid system by targeting FAAH is a promising strategy to treat pain and inflammation but lacks untoward side effects typically associated with Cannabis sativa.

Topics: Amidohydrolases; Analgesics; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acids; Disease Models, Animal; Dronabinol; Drug Delivery Systems; Drug Evaluation, Preclinical; Endocannabinoids; Glycerides; Humans; Inflammation; Mice; Mice, Knockout; Pain; Peroxisome Proliferator-Activated Receptors; Polyunsaturated Alkamides; Rats; Receptors, Cannabinoid; Receptors, Opioid; TRPV Cation Channels

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Diglycerides; Endocannabinoids; Glycerides; Humans; Immune System; Inflammation; Ligands; Muscle Tonus; Muscle, Smooth, Vascular; Phosphatidylinositols; Protein Kinase C; Receptors, Cannabinoid; Signal Transduction

Marijuana has been used as a traditional medicine and a pleasure-inducing drug for thousands of years around the world, especially in Asia. Delta(9)-tetrahydrocannabinol, major psychoactive component of marijuana, has been shown to interact with specific cannabinoid receptors, thereby eliciting a variety of pharmacological responses in experimental animals and human. In 1990, the gene encoding a cannabinoid receptor (CB1) was cloned. This prompted the search for endogenous ligands. In 1992, N-arachidonoylethanolamine (anandamide) was isolated from pig brain as an endogenous ligand, and in 1995, 2-arachidonoylglycerol was isolated from rat brain and canine gut as another endogenous ligand. Both anandamide and 2-arachidonoylglycerol exhibit various cannabimimetic activities. The results of structure-activity relationship experiments, however, revealed that 2-arachidonoylglycerol, but not anandamide, is the intrinsic natural ligand for the cannabinoid receptor. 2-arachidonoylglycerol is a degradation product of inositol phospholipids that links the function of the cannabinoid receptors with the enhanced inositol phospholipid turnover in stimulated tissues and cells. The possible physiological roles of cannabinoid receptors and 2-arachidonoylglycerol in various mammalian tissues such as those of the nervous and inflammatory cells are demonstrated. Furthermore, the future development of therapeutic drugs coming from this endocannabinoid system are discussed.

Topics: Animals; Arachidonic Acids; Drug Design; Endocannabinoids; Glycerides; Humans; Inflammation; Ligands; Neurotransmitter Agents; Polyunsaturated Alkamides; Receptors, Cannabinoid; Structure-Activity Relationship

Endocannabinoids are an emerging class of lipid mediators, which include amides and esters of long chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG) are the main endogenous agonists of cannabinoid receptors. Endotoxic shock is a potentially lethal failure of multiple organs that can be initiated by the inflammatory agent lipopolysaccharide (LPS), present in the outer membrane of gram-negative bacteria. LPS has been recently shown to stimulate the production of AEA in rat macrophages, and of 2-AG in rat platelets. The mechanism responsible for this effect has not been elucidated. On the other hand, mast cells are multifunctional bone marrow-derived cells found in mucosal and connective tissues and in the nervous system, where they play an essential role in inflammation. As yet, little is known about endogenous modulators and mechanisms of mast cell activation. Here, we review recent literature on the role of endocannabinoids in endotoxic shock and inflammation, and report our recent research on the effects of LPS on the production of AEA and 2-AG in human lymphocytes, and on AEA degradation by a specific AEA membrane transporter (AMT) and an AEA-degrading enzyme (fatty acid amide hydrolase, FAAH). We also report the ability of the HMC-1 human mast cells to degrade AEA through a nitric oxide-sensitive AMT and a FAAH. The role of endocannabinoids in HMC-1 degranulation is discussed as well. Taken together, it can be suggested that human lymphocytes and mast cells take part in regulating the peripheral endocannabinoid system, which can affect some activities of these cells.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Endocannabinoids; Fatty Acids, Unsaturated; Glycerides; Humans; Inflammation; Lymphocytes; Mast Cells; Polyunsaturated Alkamides; Shock, Septic

Evidence for the role of the cannabimimetic fatty acid derivatives (CFADs), i.e. anandamide (arachidonoylethanolamide, AEA), 2-arachidonoylglycerol (2-AG) and palmitoylethanolamide (PEA), in the control of inflammation and of the proliferation of tumor cells is reviewed here. The biosynthesis of AEA, PEA, or 2-AG can be induced by stimulation with either Ca(2+) ionophores, lipopolysaccharide, or platelet activating factor in macrophages, and by ionomycin or antigen challenge in rat basophilic leukemia (RBL-2H3) cells (a widely used model for mast cells). These cells also inactivate CFADs through re-uptake and/or hydrolysis and/or esterification processes. AEA and PEA modulate cytokine and/or arachidonate release from macrophages in vitro, regulate serotonin secretion from RBL-2H3 cells, and are analgesic in some animal models of inflammatory pain. However, the involvement of endogenous CFADs and cannabinoid CB(1) and CB(2) receptors in these effects is still controversial. In human breast and prostate cancer cells, AEA and 2-AG, but not PEA, potently inhibit prolactin and/or nerve growth factor (NGF)-induced cell proliferation. Vanillyl-derivatives of anandamide, such as olvanil and arvanil, exhibit even higher anti-proliferative activity. These effects are due to suppression of the levels of the 100 kDa prolactin receptor or of the high affinity NGF receptors (trk), are mediated by CB(1)-like cannabinoid receptors, and are enhanced by other CFADs. Inhibition of adenylyl cyclase and activation of mitogen-activated protein kinase underlie the anti-mitogenic actions of AEA. The possibility that CFADs act as local inhibitors of the proliferation of human breast cancer is discussed here.

Topics: Adjuvants, Immunologic; Amides; Animals; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Arachidonic Acids; Breast Neoplasms; Cannabinoids; Cell Division; Endocannabinoids; Ethanolamines; Glycerides; Humans; Inflammation; Male; Neoplasms; Palmitic Acids; Polyunsaturated Alkamides; Prostatic Neoplasms; Rats; Receptors, Growth Factor

The anti-inflammatory mechanisms of low-fat dairy product consumption are largely unknown. The objective of this study was to determine whether low-fat yogurt reduces biomarkers of chronic inflammation and endotoxin exposure in women. Premenopausal women (BMI 18·5-27 and 30-40 kg/m2) were randomised to consume 339 g of low-fat yogurt (yogurt non-obese (YN); yogurt obese (YO)) or 324 g of soya pudding (control non-obese; control obese (CO)) daily for 9 weeks (n 30/group). Fasting blood samples were analysed for IL-6, TNF-α/soluble TNF II (sTNF-RII), high-sensitivity C-reactive protein, 2-arachidonoyl glycerol, anandamide, monocyte gene expression, soluble CD14 (sCD14), lipopolysaccharide (LPS), LPS binding protein (LBP), IgM endotoxin-core antibody (IgM EndoCAb), and zonulin. BMI, waist circumference and blood pressure were also determined. After 9-week yogurt consumption, YO and YN had decreased TNF-α/sTNFR-RII. Yogurt consumption increased plasma IgM EndoCAb regardless of obesity status. sCD14 was not affected by diet, but LBP/sCD14 was lowered by yogurt consumption in both YN and YO. Yogurt intervention increased plasma 2-arachidonoylglycerol in YO but not YN. YO peripheral blood mononuclear cells expression of NF-κB inhibitor α and transforming growth factor β1 increased relative to CO at 9 weeks. Other biomarkers were unchanged by diet. CO and YO gained approximately 0·9 kg in body weight. YO had 3·6 % lower diastolic blood pressure at week 3. Low-fat yogurt for 9 weeks reduced biomarkers of chronic inflammation and endotoxin exposure in premenopausal women compared with a non-dairy control food. This trial was registered as NCT01686204.

Topics: Acute-Phase Proteins; Adult; Anthropometry; Arachidonic Acids; Biomarkers; C-Reactive Protein; Carrier Proteins; Chronic Disease; Cytokines; Diet; Dietary Fats; Endocannabinoids; Endotoxemia; Endotoxins; Female; Glycerides; Humans; Immunoglobulin M; Inflammation; Leukocytes, Mononuclear; Membrane Glycoproteins; Middle Aged; NF-kappa B; Obesity; Polyunsaturated Alkamides; Yogurt; Young Adult

The inflammatory sequence is the first phase of wound healing. Macrophages (MPhs) and mesenchymal stromal cells (MSCs) respond to an inflammatory microenvironment by adapting their functional activity, which polarizes them into the pro-inflammatory phenotypes M1 and MSC1. Prolongation of the inflammatory phase results in the formation of chronic wounds. The endocannabinoid system (ECS) possesses immunomodulatory properties that may impede this cellular phenotypic switch.. We investigated the immunosuppressive influence of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) on the M1 and MSC1 cytokine secretion. Lipopolysaccharides (LPS) were used as inflammagen to stimulate MPhs and MSCs. Both inflammatory phenotypes were co-exposed to AEA or 2-AG, the specific cannabinoid receptor CB2 agonist JWH-133 served as reference. The inflammatory responses were detected by CD80/163 immuno-labelling and by ELISA measures of secreted IL-6, IL-8, MIF, TNF-α, TGF-β, and VEGF.. M1 cells were found positive for CD80 expression and secreted less IL-6 and IL-8 than MSC1 cells, while both cell types produced similar amounts of MIF. TNF-α release was increased by M1, and growth factors were secreted by MSC1, only. Cannabinoid receptor ligands efficiently decreased the inflammatory response of M1, while their impact was less pronounced in MSC1.. The ECS down-regulated the inflammatory responses of MPhs and MSCs by decreasing the cytokine release upon LPS treatment, while CB2 appeared to be of particular importance. Hence, stimulating the ECS by manipulation of endo- or use of exogenous cannabinoids in vivo may constitute a potent therapeutic option against inflammatory disorders.

Topics: Arachidonic Acids; B7-1 Antigen; Cannabinoids; Cells, Cultured; Cytokines; Endocannabinoids; Glycerides; Humans; Immunosuppression Therapy; Inflammation; Lipopolysaccharides; Macrophages; Mesenchymal Stem Cells; Phenotype; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB2

Cannabinoids and the endocannabinoid system significantly contributes to the airway inflammation. Fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) are two main enzymes responsible for the metabolism of the endocannabinoids anandamide (AEA) and 2-arachydonoyl glycerol (2-AG), respectively. In the present study, we aimed to investigate the effects of local and systemic FAAH and MAGL inhibitor treatments in experimental airway inflammation and tracheal hyperreactivity in mice. Airway inflammation was induced by intranasal (i.n.) lipopolysaccharide (LPS) application (60 μl; 0,1 mg/ml in PBS) to mice and the control group received PBS. Systemic (intraperitoneal (i.p.)) or local (i.n.) FAAH inhibitor URB597 and MAGL inhibitor JZL184 treatments were administered 1h before LPS/PBS application. Fourty 8 h after LPS/PBS application, tracheas were removed to assess airway reactivity, and the lungs and bronchoalveolar lavage (BAL) fluids were isolated for histopathological evaluation, cytokine and endocannabinoid measurements. LPS application lead to an increase in 5-hydroxytryptamine (5-HT) contractions in isolated tracheal rings while carbachol contractions remained unchanged. The increased 5-HT contractions were prevented by both systemic and local URB597 and JZL184 treatments. Systemic treatment with URB597 and JZL184, and local treatment with JZL184 reduced peribronchial and paranchymal inflammation in the LPS group while i.n. application of URB597 worsened the inflammation in the lungs. Systemic URB597 treatment increased lung AEA level whereas it had no effect on 2-AG level. However, JZL184 treatment increased 2-AG level by either systemic or local application, and also elevated AEA level. Inflammation-induced increase in neutrophil numbers was only prevented by systemic URB597 treatment. However, both URB597 and JZL184 treatments abolished the increased TNF-α level either they are administered systemically or locally. These results indicate that FAAH and MAGL inhibition may have a protective effect in airway inflammation and airway hyperreactivity, and therefore their therapeutic potential for airway diseases should be further investigated.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Benzodioxoles; Carbamates; Cytokines; Endocannabinoids; Glycerides; Inflammation; Lipopolysaccharides; Lung; Male; Mice; Monoacylglycerol Lipases; Piperidines; Pneumonia; Polyunsaturated Alkamides; Respiratory Hypersensitivity

Kynurenine pathway metabolites and endocannabinoids both exert potent regulatory effects on the immune system, but the relationship between these molecules is unknown. The role of these immunobiological mediators in emotionality and personality traits is not previously characterized.. Interleukin-6 (IL-6), 2-arachidonoylglycerol (2-AG) and picolinic acid (PIC) were measured in the plasma of physically healthy individuals who had history of mood, anxiety, and personality disorders (n = 96) or who had no history of any psychiatric disorder (n = 56) by DSM-5 Criteria. Dimensional assessments of personality were performed using the Eysenck Personality Questionnaire (EPQ) and the Tridimensional Personality Questionnaire (TPQ).. Plasma IL-6 levels were significantly associated with plasma 2-AG levels and plasma PIC levels across all subjects. PIC levels were also negatively associated with 2-AG levels across all subjects, independent of IL-6 levels. In our analysis of the biological determinants of personality factors, we identified significant associations between IL-6 and novelty seeking assessment, and between PIC and neuroticism assessment.. These data provide evidence of a biological link between metabolites of the kynurenine pathway, the endocannabinoid system and IL-6 and suggest that these factors may influence personality traits.

Topics: Adult; Anxiety Disorders; Arachidonic Acids; Cohort Studies; Endocannabinoids; Female; Glycerides; Humans; Inflammation; Interleukin-6; Kynurenine; Male; Middle Aged; Personality; Personality Disorders; Picolinic Acids; Receptors, Cannabinoid; Signal Transduction

Preclinical studies have demonstrated that the endocannabinoid system (ECS) plays an important role in the protection against intestinal inflammation and colorectal cancer (CRC); however, human data are scarce. We determined members of the ECS and related components of the 'endocannabinoidome' in patients with inflammatory bowel disease (IBD) and CRC, and compared them to control subjects. Anandamide (AEA) and oleoylethanolamide (OEA) were increased in plasma of ulcerative colitis (UC) and Crohn's disease (CD) patients while 2-arachidonoylglycerol (2-AG) was elevated in patients with CD, but not UC. 2-AG, but not AEA, PEA and OEA, was elevated in CRC patients. Lysophosphatidylinositol (LPI) 18:0 showed higher levels in patients with IBD than in control subjects whereas LPI 20:4 was elevated in both CRC and IBD. Gene expression in intestinal mucosal biopsies revealed different profiles in CD and UC. CD, but not UC patients, showed increased gene expression for the 2-AG synthesizing enzyme diacylglycerol lipase alpha. Transcripts of CNR1 and GPR119 were predominantly decreased in CD. Our data show altered plasma levels of endocannabinoids and endocannabinoid-like lipids in IBD and CRC and distinct transcript profiles in UC and CD. We also report alterations for less known components in intestinal inflammation, such as GPR119, OEA and LPI.

Topics: Adult; Aged; Aged, 80 and over; Arachidonic Acids; Colitis, Ulcerative; Colonic Neoplasms; Colorectal Neoplasms; Crohn Disease; Endocannabinoids; Female; Glycerides; Humans; Inflammation; Inflammatory Bowel Diseases; Male; Middle Aged; Oleic Acids; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Receptors, G-Protein-Coupled

Diacylglycerol lipase-β (DAGLβ) hydrolyzes arachidonic acid (AA)-esterified diacylglycerols to produce 2-arachidonoylglycerol (2-AG) and downstream prostanoids that mediate inflammatory responses of macrophages. Here, we utilized DAGL-tailored activity-based protein profiling and genetic disruption models to discover that DAGLβ regulates inflammatory lipid and protein signaling pathways in primary dendritic cells (DCs). DCs serve as an important link between innate and adaptive immune pathways by relaying innate signals and antigen to drive T cell clonal expansion and prime antigen-specific immunity. We discovered that disruption of DAGLβ in DCs lowers cellular 2-AG and AA that is accompanied by reductions in lipopolysaccharide (LPS) stimulated tumor necrosis factor α secretion. Cell-based vaccination studies revealed that DC maturation ex vivo and immunogenicity in vivo was surprisingly unaffected by DAGLβ inactivation. Collectively, we identify DAGLβ pathways as a means for attenuating DC inflammatory signaling while sparing critical adaptive immune functions and further expand the utility of targeting lipid pathways for immunomodulation.

Topics: Animals; Antigens; Arachidonic Acid; Arachidonic Acids; CD8-Positive T-Lymphocytes; Dendritic Cells; Diglycerides; Endocannabinoids; Female; Glycerides; Inflammation; Lipopolysaccharides; Lipoprotein Lipase; Macrophages; Male; Mice; Mice, Inbred C57BL; Signal Transduction; T-Lymphocytes; Tumor Necrosis Factor-alpha

Intestinal production of endocannabinoid and oleoylethanolamide (OEA) is impaired in high-fat diet/obese rodents, leading to reduced satiety. Such diets also alter the intestinal microbiome in association with enhanced intestinal permeability and inflammation; however, little is known of these effects in humans. This study aimed to 1) evaluate effects of lipid on plasma anandamide (AEA), 2-arachidonyl- sn-glycerol (2-AG), and OEA in humans; and 2) examine relationships to intestinal permeability, inflammation markers, and incretin hormone secretion. Twenty lean, 18 overweight, and 19 obese participants underwent intraduodenal Intralipid infusion (2 kcal/min) with collection of endoscopic duodenal biopsies and blood. Plasma AEA, 2-AG, and OEA (HPLC/tandem mass spectrometry), tumor necrosis factor-α (TNFα), glucagon-like peptide-1 (GLP-1), and glucose-dependent insulinotropic peptide (GIP) (multiplex), and duodenal expression of occludin, zona-occludin-1 (ZO-1), intestinal-alkaline-phosphatase (IAP), and Toll-like receptor 4 (TLR4) (by RT-PCR) were assessed. Fasting plasma AEA was increased in obese compared with lean and overweight patients ( P < 0.05), with no effect of BMI group or ID lipid infusion on plasma 2-AG or OEA. Duodenal expression of IAP and ZO-1 was reduced in obese compared with lean ( P < 0.05), and these levels related negatively to plasma AEA ( P < 0.05). The iAUC for AEA was positively related to iAUC GIP ( r = 0.384, P = 0.005). Obese individuals have increased plasma AEA and decreased duodenal expression of ZO-1 and IAP compared with lean and overweight subjects. The relationships between plasma AEA with duodenal ZO-1, IAP, and GIP suggest that altered endocannabinoid signaling may contribute to changes in intestinal permeability, inflammation, and incretin release in human obesity.

Topics: Adult; Alkaline Phosphatase; Arachidonic Acids; Dietary Fats; Duodenum; Endocannabinoids; Female; Gastric Inhibitory Polypeptide; Gene Expression; Glucagon-Like Peptide 1; Glycerides; GPI-Linked Proteins; Humans; Incretins; Inflammation; Male; Obesity; Occludin; Oleic Acids; Overweight; Permeability; Polyunsaturated Alkamides; Thinness; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha; Zonula Occludens-1 Protein

The innate immune response is mediated by primary immune modulators such as cytokines and chemokines that together with immune cells and resident glia orchestrate CNS immunity and inflammation. Growing evidence supports that the endocannabinoid 2-arachidonoylglycerol (2-AG) exerts protective actions in CNS injury models. Here, we used the acute phase of Theiler's virus induced demyelination disease (TMEV-IDD) as a model of acute neuroinflammation to investigate whether 2-AG modifies the brain innate immune responses to TMEV and CNS leukocyte trafficking. 2-AG or the inhibition of its hydrolysis diminished the reactivity and number of microglia at the TMEV injection site reducing their morphological complexity and modulating them towards an anti-inflammatory state via CB2 receptors. Indeed, 2-AG dampened the infiltration of immune cells into the CNS and inhibited their egress from the spleen, resulting in long-term beneficial effects at the chronic phase of the disease. Intriguingly, it is not a generalized action over leukocytes since 2-AG increased the presence and suppressive potency of myeloid derived suppressor cells (MDSCs) in the brain resulting in higher apoptotic CD4

Topics: Animals; Arachidonic Acids; Brain; Cardiovirus Infections; CD4-Positive T-Lymphocytes; Disease Models, Animal; Endocannabinoids; Female; Glycerides; Immunity, Innate; Inflammation; Mice; Microglia; Monoacylglycerol Lipases; Receptor, Cannabinoid, CB2; Theilovirus

Cyclooxygenase-2 (COX-2) catalyzes the formation of prostaglandins, which are involved in immune regulation, vascular function, and synaptic signaling. COX-2 also inactivates the endogenous cannabinoid (eCB) 2-arachidonoylglycerol (2-AG) via oxygenation of its arachidonic acid backbone to form a variety of prostaglandin glyceryl esters (PG-Gs). Although this oxygenation reaction is readily observed in vitro and in intact cells, detection of COX-2-derived 2-AG oxygenation products has not been previously reported in neuronal tissue. Here we show that 2-AG is metabolized in the brain of transgenic COX-2-overexpressing mice and mice treated with lipopolysaccharide to form multiple species of PG-Gs that are detectable only when monoacylglycerol lipase is concomitantly blocked. Formation of these PG-Gs is prevented by acute pharmacological inhibition of COX-2. These data provide evidence that neuronal COX-2 is capable of oxygenating 2-AG to form a variety PG-Gs in vivo and support further investigation of the physiological functions of PG-Gs.

Topics: Animals; Arachidonic Acids; Brain; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Endocannabinoids; Esters; Female; Glycerides; Inflammation; Lipopolysaccharides; Male; Mice, Inbred C57BL; Mice, Transgenic; Monoacylglycerol Lipases; Neurons; Oxidation-Reduction; Prostaglandins

Endocannabinoid-metabolizing enzymes are downregulated in response to lipopolysaccharide (LPS)-induced inflammation in mice, which may serve as a negative feedback mechanism to increase endocannabinoid levels and reduce inflammation. Increased plasma levels of the pro-inflammatory cytokine interleukin-6 (IL-6) and decreased fatty acid amide hydrolase (FAAH) activity in peripheral lymphocytes from individuals diagnosed with Huntington's disease (HD) suggests that a similar negative feedback system between inflammation and the endocannabinoid system operates in humans. We investigated whether CpG- (unmethylated bacterial DNA) and LPS-induced IL-6 levels in peripheral blood mononuclear cells (PBMCs) from non-HD and HD individuals modulated the activities of endocannabinoid hydrolases monoacylglycerol lipase (MAGL) and carboxylesterase (CES). Baseline plasma IL-6 levels and 2-arachidonoylglycerol (2-AG) hydrolytic activity in PBMC lysates were not different in HD and non-HD individuals. Inhibition of MAGL and CES1 activity in PBMCs using the inhibitors JZL184 and WWL113, respectively, demonstrated that MAGL was the dominant 2-AG hydrolytic enzyme in PBMCs, regardless of disease state. Correlative analyses of 2-AG hydrolytic activity versus enzyme abundance confirmed this conclusion. Flow cytometric analysis of PBMCs showed that MAGL and CES1 were primarily expressed in monocytes and to a lesser extent in lymphocytes. In conclusion, these data suggest that IL-6 did not influence 2-AG hydrolytic activity in human PBMCs; however, monocytic MAGL was shown to be the predominant 2-AG hydrolytic enzyme.

Topics: Arachidonic Acids; Biomarkers; Carboxylic Ester Hydrolases; Cytokines; Endocannabinoids; Gene Expression Regulation, Enzymologic; Glycerides; Humans; Hydrolysis; Inflammation; Inflammation Mediators; Leukocytes, Mononuclear; Lymphocytes

Topics: AMP-Activated Protein Kinases; Animals; Anti-Inflammatory Agents; Arachidonic Acids; Calcium-Calmodulin-Dependent Protein Kinase Kinase; Cell Differentiation; Diabetes Mellitus, Experimental; Embryonic Stem Cells; Endocannabinoids; Glucose; Glucose Transporter Type 4; Glycerides; Glycogen; Humans; Inflammation; Insulin Resistance; Male; Mice; Mice, Inbred C57BL; Myocytes, Cardiac; Rats; Rats, Inbred Lew; Receptor, Cannabinoid, CB1; RNA, Messenger; Signal Transduction; Tumor Necrosis Factor-alpha

The endocannabinoid system has previously been shown to play a role in the permeability and inflammatory response of the human gut. The goal of our study was to determine the effects of endogenous anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) on the permeability and inflammatory response of intestinal epithelium under normal, inflammatory, and hypoxic conditions. Human intestinal mucosa was modeled using Caco-2 cells. Human tissue was collected from planned colorectal resections. Accumulation of AEA and 2-AG was achieved by inhibiting their metabolizing enzymes URB597 (a fatty acid amide hydrolase inhibitor) and JZL184 (a monoacylglycerol lipase inhibitor). Inflammation and ischemia were simulated with TNF-α and IFN-γ and oxygen deprivation. Permeability changes were measured by transepithelial electrical resistance. The role of the CB

Topics: Amidohydrolases; Arachidonic Acids; Benzamides; Benzodioxoles; Caco-2 Cells; Carbamates; Colorectal Neoplasms; Cytokines; Electric Impedance; Endocannabinoids; Gene Expression Regulation; Glycerides; Humans; Inflammation; Intestinal Mucosa; Intestines; Monoacylglycerol Lipases; Oxygen Consumption; Permeability; Piperidines; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Tissue Culture Techniques

Treatment of colitis has remained a major clinical challenge. The cannabinoid, 2-arachidonoyglycerol (2-AG), has shown beneficial effects in colitis, however, poor solubility or rapid hydrolysis may limit its efficiency. According to the high biocompatibility of carbon nanotubes (CNTs) and their ability for controlled drug delivery, we aimed to prepare multi-walled CNTs-2-AG (MWCNTs-2-AG) complex in order to improve the pharmacological profile of 2-AG and evaluate the therapeutic potential of this nanocomplex in a rat model of colitis.. Aminated MWCNTs-2-AG complex was prepared using acidified MWCNTs and then characterized by Fourier transform infrared spectroscopy and transmission electron microscopy. In vitro cytotoxicity of MWCNTs was evaluated. Colitis was induced by colonic instillation of trinitrobenzene sulfonic acid (TNBS) and the effects of 2-AG solution and various types of MWCNTs on the colonic tissue damage, inflammation, and oxidative stress were evaluated.. Aminated MWCNTs and MWCNTs-2-AG complex exhibited significantly lower cytotoxicity than acidified MWCNTs. Once daily intrarectal application of MWCNTs-2-AG complex (containing 2mg/kg of 2-AG) 2days before and 8days after the induction of colitis effectively reduced the macroscopic and microscopic injuries, malondialdehyde, tumour necrosis factor-α, and interlukin-1β concentrations, and myeloperoxidase activity. While, free 2-AG (2mg/kg), and acidified or aminated MWCNTs showed no beneficial effects.. Amino-functionalized MWCNTs appear as the suitable carriers for 2-AG which provide a sustained concentration for this cannabinoid leading to the promising therapeutic effects in the experimental colitis.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Agonists; Colitis; Disease Models, Animal; Drug Delivery Systems; Endocannabinoids; Glycerides; Inflammation; Male; Microscopy, Electron, Transmission; Nanotubes, Carbon; Oxidative Stress; Rats; Rats, Wistar; Spectroscopy, Fourier Transform Infrared; Trinitrobenzenesulfonic Acid

Although opioids are highly efficacious analgesics, their abuse potential and other untoward side effects diminish their therapeutic utility. The addition of non-opioid analgesics offers a promising strategy to reduce required antinociceptive opioid doses that concomitantly reduce opioid-related side effects. Inhibitors of the primary endocannabinoid catabolic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) show opioid-sparing effects in preclinical models of pain. As simultaneous inhibition of these enzymes elicits enhanced antinociceptive effects compared with single enzyme inhibition, the present study tested whether the dual FAAH-MAGL inhibitor SA-57 [4-[2-(4-chlorophenyl)ethyl]-1-piperidinecarboxylic acid 2-(methylamino)-2-oxoethyl ester] produces morphine-sparing antinociceptive effects, without major side effects associated with either drug class. SA-57 dose-dependently reversed mechanical allodynia in the constriction injury (CCI) of the sciatic nerve model of neuropathic pain and carrageenan inflammatory pain model. As previously reported, SA-57 was considerably more potent in elevating anandamide (AEA) than 2-arachidonyl glycerol (2-AG) in brain. Its anti-allodynic effects required cannabinoid (CB)

Topics: Acetamides; Analgesics; Analgesics, Opioid; Animals; Arachidonic Acid; Arachidonic Acids; Carbamates; Carrageenan; Dose-Response Relationship, Drug; Drug-Seeking Behavior; Endocannabinoids; Glycerides; Heroin; Hydrolysis; Hyperalgesia; Inflammation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Morphine; Neuralgia; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Sciatic Nerve; Self Administration

We investigated the effects of S-777469 (1-[[6-Ethyl-1-[4-fluorobenzyl]-5-methyl-2-oxo-1, 2-dihydropyridine-3-carbonyl]amino]-cyclohexanecarboxylic acid), a novel cannabinoid type 2 receptor (CB2) agonist, on 1-fluoro-2,4-dinitrobenzene (DNFB)-induced ear inflammation and mite antigen-induced dermatitis in mice. The oral administration of S-777469 significantly suppressed DNFB-induced ear swelling in a dose-dependent manner. In addition, S-777469 significantly alleviated mite antigen-induced atopic dermatitis-like skin lesions in NC/Nga mice. A histological analysis revealed that S-777469 significantly reduced the epidermal thickness and the number of mast cells infiltrating skin lesions. We demonstrated that S-777469 inhibited mite antigen-induced eosinophil accumulation in skin lesions and an endogenous CB2 ligand, 2-arachidonoylglycerol (2-AG)-induced eosinophil migration in vitro. Moreover, we confirmed that 2-AG levels significantly increased in skin lesions of mite antigen-induced dermatitis model. Together, these results suggest that S-777469 inhibits skin inflammation in mice by blocking the activities of 2-AG.

Topics: Animals; Arachidonic Acids; Cell Migration Assays, Leukocyte; Dermatitis, Atopic; Dinitrofluorobenzene; Dose-Response Relationship, Drug; Endocannabinoids; Glycerides; Inflammation; Male; Mice; Mite Infestations; Pyridones; Receptor, Cannabinoid, CB2; Skin

Monoglyceride lipase (MGL) is required for efficient hydrolysis of the endocannabinoid 2-arachidonoylglyerol (2-AG) in the brain generating arachidonic acid (AA) and glycerol. This metabolic function makes MGL an interesting target for the treatment of neuroinflammation, since 2-AG exhibits anti-inflammatory properties and AA is a precursor for pro-inflammatory prostaglandins. Astrocytes are an important source of AA and 2-AG, and highly express MGL. In the present study, we dissected the distinct contribution of MGL in astrocytes on brain 2-AG and AA metabolism by generating a mouse model with genetic deletion of MGL specifically in astrocytes (MKO(GFAP)). MKO(GFAP) mice exhibit moderately increased 2-AG and reduced AA levels in brain. Minor accumulation of 2-AG in the brain of MKO(GFAP) mice does not cause cannabinoid receptor desensitization as previously observed in mice globally lacking MGL. Importantly, MKO(GFAP) mice exhibit reduced brain prostaglandin E2 and pro-inflammatory cytokine levels upon peripheral lipopolysaccharide (LPS) administration. These observations indicate that MGL-mediated degradation of 2-AG in astrocytes provides AA for prostaglandin synthesis promoting LPS-induced neuroinflammation. The beneficial effect of astrocyte-specific MGL-deficiency is not fully abrogated by the inverse cannabinoid receptor 1 agonist SR141716 (Rimonabant) suggesting that the anti-inflammatory effects are rather caused by reduced prostaglandin synthesis than by activation of cannabinoid receptors. In conclusion, our data demonstrate that MGL in astrocytes is an important regulator of 2-AG levels, AA availability, and neuroinflammation.

Topics: Animals; Arachidonic Acids; Astrocytes; Behavior, Animal; Brain; Cytokines; Endocannabinoids; Female; Gene Deletion; Glial Fibrillary Acidic Protein; Glycerides; Inflammation; Lipopolysaccharides; Mice, Inbred C57BL; Mice, Knockout; Microglia; Monoacylglycerol Lipases; Nervous System; Organ Specificity; Receptor, Cannabinoid, CB1

Early-life inflammation has been shown to exert profound effects on brain development and behavior, including altered emotional behavior, stress responsivity and neurochemical/neuropeptide receptor expression and function. The current study extends this research by examining the impact of inflammation, triggered with the bacterial compound lipopolysaccharide (LPS) on postnatal day (P) 14, on social behavior during adolescence. We investigated the role that the endocannabinoid (eCB) system plays in sociability after early-life LPS. To test this, multiple cohorts of Sprague Dawley rats were injected with LPS on P14. In adolescence, rats were subjected to behavioral testing in a reciprocal social interaction paradigm as well as the open field. We quantified eCB levels in the amygdala of P14 and adolescent animals (anandamide and 2-arachidonoylglycerol) as well as adolescent amygdaloid cannabinoid receptor 1 (CB1) binding site density and the hydrolytic activity of the enzyme fatty acid amide hydrolase (FAAH), which metabolizes the eCB anandamide. Additionally, we examined the impact of FAAH inhibition on alterations in social behavior. Our results indicate that P14 LPS decreases adolescent social behavior (play and social non-play) in males and females at P40. This behavioral alteration is accompanied by decreased CB1 binding, increased anandamide levels and increased FAAH activity. Oral administration of the FAAH inhibitor PF-04457845 (1mg/kg) prior to the social interaction task normalizes LPS-induced alterations in social behavior, while not affecting social behavior in the control group. Infusion of 10ng PF-04457845 into the basolateral amygdala normalized social behavior in LPS injected females. These data suggest that alterations in eCB signaling following postnatal inflammation contribute to impairments in social behavior during adolescence and that inhibition of FAAH could be a novel target for disorders involving social deficits such as social anxiety disorders or autism.

Topics: Amidohydrolases; Amygdala; Animals; Arachidonic Acids; Behavior, Animal; Endocannabinoids; Female; Glycerides; Inflammation; Lipopolysaccharides; Male; Polyunsaturated Alkamides; Pyridazines; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Signal Transduction; Social Behavior; Urea

Traumatic brain injury (TBI) is an increasingly frequent and poorly understood condition lacking effective therapeutic strategies. Inflammation and oxidative stress (OS) are critical components of injury, and targeted interventions to reduce their contribution to injury should improve neurobehavioral recovery and outcomes. Recent evidence reveals potential protective, yet short-lived, effects of the endocannabinoids (ECs), 2-arachidonoyl glycerol (2-AG) and N-arachidonoyl-ethanolamine (AEA), on neuroinflammatory and OS processes after TBI. The aim of this study was to determine whether EC degradation inhibition after TBI would improve neurobehavioral recovery by reducing inflammatory and oxidative damage. Adult male Sprague-Dawley rats underwent a 5-mm left lateral craniotomy, and TBI was induced by lateral fluid percussion. TBI produced apnea (17±5 sec) and a delayed righting reflex (479±21 sec). Thirty minutes post-TBI, rats were randomized to receive intraperitoneal injections of vehicle (alcohol, emulphor, and saline; 1:1:18) or a selective inhibitor of 2-AG (JZL184, 16 mg/kg) or AEA (URB597, 0.3 mg/kg) degradation. At 24 h post-TBI, animals showed significant neurological and -behavioral impairment as well as disruption of blood-brain barrier (BBB) integrity. Improved neurological and -behavioral function was observed in JZL184-treated animals. BBB integrity was protected in both JZL184- and URB597-treated animals. No significant differences in ipsilateral cortex messenger RNA expression of interleukin (IL)-1β, IL-6, chemokine (C-C motif) ligand 2, tumor necrosis factor alpha, cyclooxygenase 2 (COX2), or nicotinamide adenine dinucleotide phosphate oxidase (NOX2) and protein expression of COX2 or NOX2 were observed across experimental groups. Astrocyte and microglia activation was significantly increased post-TBI, and treatment with JZL184 or URB597 blocked activation of both cell types. These findings suggest that EC degradation inhibition post-TBI exerts neuroprotective effects. Whether repeated dosing would achieve greater protection remains to be examined.

Topics: Animals; Arachidonic Acids; Benzamides; Benzodioxoles; Blood-Brain Barrier; Blotting, Western; Brain Injuries; Carbamates; Disease Models, Animal; Endocannabinoids; Glycerides; Immunohistochemistry; Inflammation; Male; Neuroprotective Agents; Piperidines; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Recovery of Function

N-Palmitoylethanolamine or palmitoylethanolamide (PEA) is an anti-inflammatory compound that was recently shown to exert peroxisome proliferator-activated receptor-α-dependent beneficial effects on colon inflammation. The actions of PEA are terminated following hydrolysis by 2 enzymes: fatty acid amide hydrolase (FAAH), and the less-studied N-acylethanolamine-hydrolyzing acid amidase (NAAA). This study aims to investigate the effects of inhibiting the enzymes responsible for PEA hydrolysis in colon inflammation in order to propose a potential therapeutic target for inflammatory bowel diseases (IBDs). Two murine models of IBD were used to assess the effects of NAAA inhibition, FAAH inhibition, and PEA on macroscopic signs of colon inflammation, macrophage/neutrophil infiltration, and the expression of proinflammatory mediators in the colon, as well as on the colitis-related systemic inflammation. NAAA inhibition increases PEA levels in the colon and reduces colon inflammation and systemic inflammation, similarly to PEA. FAAH inhibition, however, does not increase PEA levels in the colon and does not affect the macroscopic signs of colon inflammation or immune cell infiltration. This is the first report of an anti-inflammatory effect of a systemically administered NAAA inhibitor. Because NAAA is the enzyme responsible for the control of PEA levels in the colon, we put forth this enzyme as a potential therapeutic target in chronic inflammation in general and IBD in particular.

Topics: Amides; Amidohydrolases; Animals; Anti-Inflammatory Agents; Arachidonic Acids; Chromatography, High Pressure Liquid; Colitis; Colon; Cytokines; Disease Models, Animal; Endocannabinoids; Enzyme-Linked Immunosorbent Assay; Ethanolamines; Gene Expression Regulation; Glycerides; Inflammation; Inflammatory Bowel Diseases; Male; Mice; Mice, Inbred C57BL; Neutrophils; Palmitic Acids; Peroxidase; Piperidines; Pyridines; Taurine

Monoacylglycerol lipase (MAGL) is part of the endocannabinoid and the prostaglandin signaling system. MAGL degrades the endocannabinoid 2-arachidonoylglycerol (2-AG) into glycerol and arachidonic acid. MAGL-induced arachidonic acid is the primary source for prostaglandin synthesis in the brain. 2-AG mainly induces neuroprotective and anti-inflammatory effects, whereas prostaglandins are related to pro-inflammatory effects inducing neurotoxicity. Therefore, inhibition of MAGL represents a promising target for neurological diseases characterized by inflammation. However, as 2-AG is an agonist for the cannabinoid receptor 1 (CB1), inhibition of MAGL might be associated with unwanted cannabimimetic effects. Here, we show that oral administration of KML29, a highly selective inhibitor of MAGL, induced large and dose-dependent changes in 2-AG levels in vivo in brain and spinal cord of mice. Of note, MAGL inhibition by KML29 induced a decrease in prostaglandin levels in brain and most peripheral tissues but not in the spinal cord. MAGL expression was highest in fat, liver and brain, whereas the cytosolic phospholipase A2 (cPLA2), a further enzyme responsible for arachidonic acid production, was highly expressed in spinal cord, muscle and spleen. In addition, high doses (10 mg/kg) of KML29 induced some cannabimimetic effects in vivo in the tetrad test, including hypothermia, analgesia and hypomotility without induction of cataleptic behavior. In summary, inhibition of MAGL by KML29 represents a promising strategy for targeting the cannabinoid and prostaglandin system of the brain with only a moderate induction of cannabimimetic effects.

Topics: Adipose Tissue, Brown; Analgesics; Animals; Arachidonic Acid; Arachidonic Acids; Benzodioxoles; Brain Chemistry; Endocannabinoids; Enzyme Inhibitors; Female; Glycerides; Inflammation; Liver; Male; Mice; Monoacylglycerol Lipases; Motor Activity; Nociception; Piperidines; Prostaglandins; Quadriceps Muscle; Spinal Cord; Spleen

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

Monoacylglycerol lipase (MGL) is the main enzyme responsible for degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG). Selective inhibitors of MGL have antinociceptive effects upon acute administration and, therefore, hold promise as analgesics. To gain insight into the possible consequences of their prolonged administration, genetically modified mice with the knocked-out MGL gene were tested in several models of acute (phasic, tonic) and chronic (inflammatory, neuropathic) pain. MGL knockout mice showed normal acute phasic pain perception (pain thresholds) and no alleviation of pain perception in models of inflammatory and neuropathic pain. However, compared with wild-type controls, they showed significantly augmented nociceptive behavior in models of acute somatic and visceral tonic pain (formalin and acetic acid tests). The observed proalgesic changes in perception of tonic pain in MGL knockouts could have resulted from desensitization of cannabinoid receptors (known to occur after genetic inactivation of MGL). Supporting this notion, chronic pretreatment with the selective CB1 receptor antagonist AM 251 (employed to re-sensitize cannabinoid receptors in MGL knockouts) resulted in normalization of their tonic pain-related behaviors. Similar augmentation of tonic pain-related behaviors was replicated in C57BL/6N mice pretreated chronically with the selective MGL inhibitor JZL 184 (employed to pharmacologically desensitize CB1 receptors). These findings imply that prolonged use of MGL inhibitors, at doses causing close to complete inhibition of MGL enzymatic activity, not only have no beneficial analgesic effects, they may lead to exacerbation of some types of pain (particularly those with a tonic component).

Topics: Animals; Arachidonic Acids; Benzodioxoles; Cannabinoid Receptor Modulators; Endocannabinoids; Glycerides; Inflammation; Male; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Knockout; Neuralgia; Pain; Pain Threshold; Phosphotransferases (Alcohol Group Acceptor); Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1

Endocannabinoids are associated with multiple regulatory functions in several tissues. The main endocannabinoids, anandamide (AEA) and 2-arachidonylglycerol (2-AG), have been detected in the gingival crevicular fluid of periodontitis patients, but the association between periodontal disease or human periodontal ligament cells (hPdLCs) and endocannabinoids still remain unclear. The aim of the present study was to examine the effects of AEA and 2-AG on the proliferation/viability and cytokine/chemokine production of hPdLCs in the presence/absence of Porphyromonas gingivalis lipopolysaccharide (P. gingivalis LPS). The proliferation/viability of hPdLCs was measured using 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT)-assay. Interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) levels were examined at gene expression and protein level by real-time PCR and ELISA, respectively. AEA and 2-AG did not reveal any significant effects on proliferation/viability of hPdLCs in the absence of P. gingivalis LPS. However, hPdLCs viability was significantly increased by 10-20 µM AEA in the presence of P. gingivalis LPS (1 µg/ml). In the absence of P. gingivalis LPS, AEA and 2-AG did not exhibit any significant effect on the expression of IL-8 and MCP-1 expression in hPdLCs, whereas IL-6 expression was slightly enhanced by 10 µM 2-AG and not affected by AEA. In P.gingivalis LPS stimulated hPdLCs, 10 µM AEA down-regulated gene-expression and protein production of IL-6, IL-8, and MCP-1. In contrast, 10 µM 2-AG had an opposite effect and induced a significant up-regulation of gene and protein expression of IL-6 and IL-8 (P<0.05) as well as gene-expression of MCP-1 in P. gingivalis LPS stimulated hPdLCs. Our data suggest that AEA appears to have an anti-inflammatory and immune suppressive effect on hPdLCs' host response to P.gingivalis LPS, whereas 2-AG appears to promote detrimental inflammatory processes. In conclusion, AEA and 2-AG might play an important role in the modulation of periodontal inflammation.

Topics: Arachidonic Acids; Cell Proliferation; Cell Survival; Cells, Cultured; Cytokines; Endocannabinoids; Gene Expression Regulation; Glycerides; Humans; Inflammation; Inflammation Mediators; Lipopolysaccharides; Periodontal Ligament; Periodontitis; Polyunsaturated Alkamides

In regenerative medicine, platelet by-products containing factors physiologically involved in wound healing, have been successfully used in the form of platelet-rich plasma (PRP) for the topical therapy of various clinical conditions since it produces an improvement in tissue repair as well as analgesic effects. Measurement of endocannabinoids and related compounds in PRP revealed the presence of a significant amount of anandamide, 2-arachidonoylglycerol, palmitoylethanolamide, and oleoylethanolamide. Investigation of the activity of PRP on the keratinocyte cell line NCTC2544 in physiological and inflammatory conditions showed that, under inflammatory conditions, PRP induced in a statistically significant manner the production of these compounds by the cells suggesting that PRP might induce the production of these analgesic mediators particularly in the physiologically inflamed wounded tissue. Studies in a mouse model of acute inflammatory pain induced by formalin injection demonstrated a potent antinociceptive effect against both early and late nocifensive responses. This effect was observed following intrapaw injection of (1) total PRP; (2) lipids extracted from PRP; and (3) an endocannabinoid-enriched lipid fraction of PRP. In all conditions, antagonists of endocannabinoid CB1 and CB2 receptors, injected in the paw, abrogated the antinociceptive effects strongly suggesting for this preparation a peripheral mechanism of action. In conclusion, we showed that PRP and PRP lipid extract exert a potent antinociceptive activity linked, at least in part, to their endocannabinoids and related compound content, and to their capability of elevating the levels of these lipid mediators in cells.

Topics: Amides; Analgesics; Animals; Arachidonic Acids; Blotting, Western; Cell Line, Tumor; Endocannabinoids; Ethanolamines; Glycerides; Humans; Inflammation; Keratinocytes; Mice; Oleic Acids; Pain; Palmitic Acids; Platelet-Rich Plasma; Polyunsaturated Alkamides

Proinflammatory macrophages are key mediators in several pathologies; thus, controlling their activation is necessary. The endocannabinoid system is implicated in various inflammatory processes. Here we show that in macrophages, the newly characterized enzyme α/β-hydrolase domain 6 (ABHD6) controls 2-arachidonoylglycerol (2-AG) levels and thus its pharmacological effects. Furthermore, we characterize a unique pathway mediating the effects of 2-AG through its oxygenation by cyclooxygenase-2 to give rise to the anti-inflammatory prostaglandin D2-glycerol ester (PGD2-G). Pharmacological blockade of cyclooxygenase-2 or of prostaglandin D synthase prevented the effects of increasing 2-AG levels by ABHD6 inhibition in vitro, as well as the 2-AG-induced increase in PGD2-G levels. Together, our data demonstrate the physiological relevance of the interaction between the endocannabinoid and prostanoid systems. Moreover, we show that ABHD6 inhibition in vivo allows for fine-tuning of 2-AG levels in mice, therefore reducing lipopolysaccharide-induced inflammation, without the characteristic central side effects of strong increases in 2-AG levels obtained following monoacylglycerol lipase inhibition. In addition, administration of PGD2-G reduces lipopolysaccharide-induced inflammation in mice, thus confirming the biological relevance of this 2-AG metabolite. This points to ABHD6 as an interesting therapeutic target that should be relevant in treating inflammation-related conditions, and proposes PGD2-G as a bioactive lipid with potential anti-inflammatory properties in vivo.

Topics: Animals; Anti-Inflammatory Agents; Arachidonic Acids; Biphenyl Compounds; Carbamates; Cell Line; Cells, Cultured; Cyclooxygenase 2; Endocannabinoids; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Esters; Female; Gene Expression; Glycerides; Glycerol; Inflammation; Interleukin-1beta; Intramolecular Oxidoreductases; Lipocalins; Lipopolysaccharides; Macrophage Activation; Macrophages; Male; Mice; Mice, Inbred C57BL; Monoacylglycerol Lipases; Prostaglandin D2; Reverse Transcriptase Polymerase Chain Reaction

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

Inflammatory pain presents a problem of clinical relevance and often elicits allodynia, a condition in which non-noxious stimuli are perceived as painful. One potential target to treat inflammatory pain is the endogenous cannabinoid (endocannabinoid) system, which is comprised of CB1 and CB2 cannabinoid receptors and several endogenous ligands, including anandamide (AEA). Blockade of the catabolic enzyme fatty acid amide hydrolase (FAAH) elevates AEA levels and elicits antinociceptive effects, without the psychomimetic side effects associated with Δ(9) -tetrahydrocannabinol (THC).. Allodynia was induced by intraplantar injection of LPS. Complementary genetic and pharmacological approaches were used to determine the strategy of blocking FAAH to reverse LPS-induced allodynia. Endocannabinoid levels were quantified using mass spectroscopy analyses.. FAAH (-/-) mice or wild-type mice treated with FAAH inhibitors (URB597, OL-135 and PF-3845) displayed an anti-allodynic phenotype. Furthermore, i.p. PF-3845 increased AEA levels in the brain and spinal cord. Additionally, intraplantar PF-3845 produced a partial reduction in allodynia. However, the anti-allodynic phenotype was absent in mice expressing FAAH exclusively in the nervous system under a neural specific enolase promoter, implicating the involvement of neuronal fatty acid amides (FAAs). The anti-allodynic effects of FAAH-compromised mice required activation of both CB1 and CB2 receptors, but other potential targets of FAA substrates (i.e. µ-opioid, TRPV1 and PPARα receptors) had no apparent role.. AEA is the primary FAAH substrate reducing LPS-induced tactile allodynia. Blockade of neuronal FAAH reverses allodynia through the activation of both cannabinoid receptors and represents a promising target to treat inflammatory pain.. This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Brain; Endocannabinoids; Enzyme Inhibitors; Female; Glycerides; Hyperalgesia; Inflammation; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Peripheral Nervous System; Piperidines; Polyunsaturated Alkamides; Pyridines; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Spinal Cord

Cannabinoid (CB) 2 is expressed on immune and inflammatory cells. Identification of 2-arachidonyl glycerol (2-AG) and anandamide as endogenous CB2 ligands has allowed investigations of the roles of CB2 and its endogenous ligand system in inflammatory cells. However, the roles of this receptor-ligand system in inflammatory and allergic immune responses in vivo have not been fully elucidated.. Two mouse allergy models, namely ear dermatitis induced by 2,4-dinitrofluorobenzene and allergic bronchitis induced by ovalbumin, were analyzed for 2-AG amounts in allergic tissues, with reference to allergic and inflammatory symptoms. To investigate the gene expression via CB2 in inflammatory cells, human promyelocytic HL-60 cells were stimulated by the CB2 ligand 2-AG ether and analyzed using a DNA microarray.. In the ear dermatitis model, the 2-AG amount increased upon serial 2,4-dinitrofluorobenzene challenges and was correlated with ear weight gain. The increased ear thickness in this allergy model was clearly suppressed in CB2 knockout mice, suggesting that the generated endogenous CB2 ligands induce ear thickness through aberrant inflammatory responses and remodeling mediated via CB2. In the allergic bronchitis model, the 2-AG level in bronchoalveolar lavage was increased and sustained during the elevation of inflammatory cell infiltration. The DNA microarray analysis of human HL-60 cells revealed that 2-AG ether induced expressions of not only inflammatory chemokines/cytokines but also of cell growth factors.. Our data strongly suggest that endogenous CB2 ligands upregulated upon disease progression in allergic models are involved in aberrant alterations of both inflammatory responses and tissue cell growth.

Topics: Animals; Arachidonic Acids; Bronchitis; Cannabinoids; Chemokines; Cytokines; Dermatitis, Allergic Contact; Disease Models, Animal; Endocannabinoids; Female; Gene Expression; Glycerides; Growth Substances; HL-60 Cells; Humans; Hypersensitivity; Inflammation; Ligands; Mice; Mice, Inbred BALB C; Mice, Knockout; Receptor, Cannabinoid, CB2

Muller cells play a prominent role in inflammatory conditions of the retina. They are part of the retinal innate immune response. The endocannabinoid system functions as an immune modulator in both the peripheral immune system as well as the central nervous system. We hypothesized that the neuroprotective ability of exogenous endocannabinoids in the retina is partially mediated through Muller glia. This study reports that exposure to endocannabinoids in activated but not resting primary human Muller glia inhibit production of several proinflammatory cytokines, while elevating anti-inflammatory mediators. Cytokine generation in activated Muller glia is regulated by endocannabinoids through the mitogen-activated protein kinase (MAPK) family at multiple signaling stages. Anandamide (AEA) acts to control MAPK phosphorylation through MKP-1. Both AEA and 2-arachidonoylglycerol (2-AG) inhibit the transcription factor NF-κB and increases the regulatory protein, IL1-R-associated kinase 1-binding protein 1. Endocannabinoids also increase expression of Tristetraprolin in activated Muller cells, which is implicated in affecting AU-rich proinflammatory cytokine mRNA. We demonstrate that exogenous application of AEA and 2-AG aid in retinal cell survival under inflammatory conditions by creating an anti-inflammatory milieu. Endocannabinoids or synthetic cannabinoid therapy may therefore orchestrate a molecular switch to bias the innate immune system suchthat the balance of pro- and anti-inflammatory cytokine generation creates a prosurvival milieu.

Topics: Adult; Aged; Aged, 80 and over; Arachidonic Acids; Cannabinoid Receptor Agonists; Cells, Cultured; Cytokines; Endocannabinoids; Glycerides; Humans; Immunity, Innate; Inflammation; Middle Aged; Mitogen-Activated Protein Kinases; Neuroglia; Phosphorylation; Polyunsaturated Alkamides; Retina; Tristetraprolin

The cannabinoid receptor-mediated analgesic effects of 2-arachidonoylglycerol (2-AG) are limited by monoacylglycerol lipase (MAGL). 4-nitrophenyl 4-[bis (1,3-benzodioxol-5-yl) (hydroxy) methyl] piperidine-1-carboxylate (JZL184) is a potent inhibitor of MAGL in the mouse, though potency is reportedly reduced in the rat. Here we have assessed the effects of spinal inhibition of MAGL with JZL184 on nociceptive processing in rats.. In vivo spinal electrophysiological assays in anaesthetized rats were used to determine the effects of spinal administration of JZL184 on spinal nociceptive processing in the presence and absence of hindpaw inflammation. Contributions of CB(1) receptors to these effects was assessed with AM251. Inhibition of 2-oleoylglycerol hydrolytic activity and alterations of 2-AG in the spinal cord after JZL 184 were also assessed.. Spinal JZL184 dose-dependently inhibited mechanically evoked responses of wide dynamic range (WDR) neurones in naïve anaesthetized rats, in part via the CB(1) receptor. A single spinal administration of JZL184 abolished inflammation-induced expansion of the receptive fields of spinal WDR neurones. However, neither spinal nor systemic JZL184 altered levels of 2-AG, or 2-oleoylglycerol hydrolytic activity in the spinal cord, although JZL184 displayed robust inhibition of MAGL when incubated with spinal cord tissue in vitro.. JZL184 exerted robust anti-nociceptive effects at the level of the spinal cord in vivo and inhibited rat spinal cord MAGL activity in vitro. The discordance between in vivo and in vitro assays suggests that localized sites of action of JZL184 produce these profound functional inhibitory effects.. This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.167.issue-8.

Topics: Amidohydrolases; Analgesics; Animals; Anti-Inflammatory Agents; Arachidonic Acids; Benzodioxoles; Carrageenan; Central Nervous System Sensitization; Drug Administration Routes; Endocannabinoids; Ethanolamines; Glycerides; Inflammation; Lipoprotein Lipase; Male; Mice; Mice, Inbred C57BL; Monoacylglycerol Lipases; Pain; Piperidines; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Species Specificity; Spinal Cord

The endocannabinoid 2-arachidonoylglycerol (2-AG) is biosynthesized by diacylglycerol lipases DAGLα and DAGLβ. Chemical probes to perturb DAGLs are needed to characterize endocannabinoid function in biological processes. Here we report a series of 1,2,3-triazole urea inhibitors, along with paired negative-control and activity-based probes, for the functional analysis of DAGLβ in living systems. Optimized inhibitors showed high selectivity for DAGLβ over other serine hydrolases, including DAGLα (∼60-fold selectivity), and the limited off-targets, such as ABHD6, were also inhibited by the negative-control probe. Using these agents and Daglb(-/-) mice, we show that DAGLβ inactivation lowers 2-AG, as well as arachidonic acid and eicosanoids, in mouse peritoneal macrophages in a manner that is distinct and complementary to disruption of cytosolic phospholipase-A2. We observed a corresponding reduction in lipopolysaccharide-induced tumor necrosis factor-α release. These findings indicate that DAGLβ is a key metabolic hub within a lipid network that regulates proinflammatory responses in macrophages.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Cell Line; Cytokines; Drug Discovery; Endocannabinoids; Glycerides; Inflammation; Lipid Metabolism; Lipoprotein Lipase; Macrophages, Peritoneal; Mice; Mice, Knockout; Neurons; Prostaglandins; Protein Isoforms; Proteome; Quantitative Structure-Activity Relationship; Signal Transduction; Triazoles; Tumor Necrosis Factor-alpha

The psychoactive component of the cannabis resin and flowers, delta9-tetrahydrocannabinol (THC), was first isolated in 1964, and at least 70 other structurally related 'phytocannabinoid' compounds have since been identified. The serendipitous identification of a G-protein-coupled cannabinoid receptor at which THC is active in the brain heralded an explosion in cannabinoid research. Elements of the endocannabinoid system (ECS) comprise the cannabinoid receptors, a family of nascent lipid ligands, the 'endocannabinoids' and the machinery for their biosynthesis and metabolism. The function of the ECS is thus defined by modulation of these receptors, in particular, by two of the best-described ligands, 2-arachidonoyl glycerol and anandamide (arachidonylethanolamide). Research on the ECS has recently aroused enormous interest not only for the physiological functions, but also for the promising therapeutic potentials of drugs interfering with the activity of cannabinoid receptors. Many of the former relate to stress-recovery systems and to the maintenance of homeostatic balance. Among other functions, the ECS is involved in neuroprotection, modulation of nociception, regulation of motor activity, neurogenesis, synaptic plasticity and the control of certain phases of memory processing. In addition, the ECS acts to modulate the immune and inflammatory responses and to maintain a positive energy balance. This theme issue aims to provide the reader with an overview of ECS pharmacology, followed by discussions on the pivotal role of this system in the modulation of neurogenesis in the developing and adult organism, memory processes and synaptic plasticity, as well as in pathological pain and brain ageing. The volume will conclude with discussions that address the proposed therapeutic applications of targeting the ECS for the treatment of neurodegeneration, pain and mental illness.

Topics: Arachidonic Acids; Brain; Cannabidiol; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Dronabinol; Electrical Synapses; Endocannabinoids; Glycerides; Humans; Inflammation; Neurodegenerative Diseases; Neurogenesis; Neuroprotective Agents; Nociceptors; Polyunsaturated Alkamides; Receptors, Cannabinoid; Synaptic Transmission

Acute exposure to bacterial lipopolysaccharide (LPS) is a potent inducer of immune response as well as hypophagia. Nevertheless, desensitization of responses to LPS occurs during long-term exposure to endotoxin. We induced endotoxin tolerance, injecting repeated (6LPS) LPS doses compared with single (1LPS) treatment. 1LPS, but not 6LPS group, showed decreased food intake and body weight, which was associated with an increased plasma leptin and higher mRNA expression of OB-Rb, MC4R, and SOCS3 in the hypothalamus. Hypophagia induced by 1LPS was associated with lower levels of 2-arachidonoylglycerol (2-AG), increased number of p-STAT3 neurons, and decreased AMP-activated protein kinase (AMPK) activity. Desensitization of hypophagia in the 6LPS group was related to high 2-AG, with no changes in p-STAT3 or increased p-AMPK. Leptin decreased food intake, body weight, 2-AG levels, and AMPK activity and enhanced p-STAT3 in control rats. However, leptin had no effects on 2-AG, p-STAT3, or p-AMPK in the 1LPS and 6LPS groups. Rats treated with HFD to induce leptin resistance showed neither hypophagia nor changes in p-STAT3 after 1LPS, suggesting that leptin and LPS recruit a common signaling pathway in the hypothalamus to modulate food intake reduction. Desensitization of hypophagia in response to repeated exposure to endotoxin is related to an inability of leptin to inhibit AMPK phosphorylation and 2-AG production and activate STAT3. SOCS3 is unlikely to underlie this resistance to leptin signaling in the endotoxin tolerance. The present model of prolonged inflammatory challenge may contribute to further investigations on mechanisms of leptin resistance.

Topics: Animals; Arachidonic Acids; Body Weight; Cyclic AMP-Dependent Protein Kinases; Diet; Dietary Fats; Eating; Endocannabinoids; Endotoxins; Gas Chromatography-Mass Spectrometry; Glycerides; Immunohistochemistry; Inflammation; Interleukin-10; Leptin; Lipopolysaccharides; Male; Phosphorylation; Rats; Rats, Wistar; Receptor, Melanocortin, Type 4; Receptors, Interleukin-10; Receptors, Leptin; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; STAT3 Transcription Factor; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins

The directional migration of neutrophils towards inflammatory mediators, such as chemokines and cannabinoids, occurs via the activation of seven transmembrane G protein coupled receptors (7TM/GPCRs) and is a highly organized process. A crucial role for controlling neutrophil migration has been ascribed to the cannabinoid CB(2) receptor (CB(2)R), but additional modulatory sites distinct from CB(2)R have recently been suggested to impact CB(2)R-mediated effector functions in neutrophils. Here, we provide evidence that the recently de-orphanized 7TM/GPCR GPR55 potently modulates CB(2)R-mediated responses. We show that GPR55 is expressed in human blood neutrophils and its activation augments the migratory response towards the CB(2)R agonist 2-arachidonoylglycerol (2-AG), while inhibiting neutrophil degranulation and reactive oxygen species (ROS) production. Using HEK293 and HL60 cell lines, along with primary neutrophils, we show that GPR55 and CB(2)R interfere with each other's signaling pathways at the level of small GTPases, such as Rac2 and Cdc42. This ultimately leads to cellular polarization and efficient migration as well as abrogation of degranulation and ROS formation in neutrophils. Therefore, GPR55 limits the tissue-injuring inflammatory responses mediated by CB(2)R, while it synergizes with CB(2)R in recruiting neutrophils to sites of inflammation.

Topics: Arachidonic Acids; Cannabinoid Receptor Modulators; cdc42 GTP-Binding Protein; Cell Degranulation; Cell Movement; Endocannabinoids; Glycerides; HEK293 Cells; HL-60 Cells; Humans; Inflammation; Neutrophil Activation; Neutrophils; rac GTP-Binding Proteins; RAC2 GTP-Binding Protein; Reactive Oxygen Species; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, G-Protein-Coupled; Signal Transduction

The inflammatory response plays an important role in the pathogenesis of many diseases in the central nervous system. Cannabinoids exhibit diverse pharmacological actions including anti-inflammatory activity. In this study, we tried to elucidate possible effects of cannabinoids on lipopolysaccharide (LPS)-induced expression of inflammatory cytokine mRNAs in rat cerebellar granule cells.. Inhibitory effects of cannabinoids on cytokine induction in cerebellar granule cells were determined by RT-PCR method.. In these cells, both mRNA and protein of cannabinoid receptor 1 (CB(1) ), but not CB(2) , were expressed. LPS (1 µg/ml) produced a marked increase in the induction of inflammatory cytokines, including interleukin-1β, interleukin-6 and tumour necrosis factor-α. CP55940, a synthetic cannabinoid analogue, concentration-dependently inhibited inflammatory cytokine expression induced by LPS. On the other hand, the endocannabinoids 2-arachidonoylglycerol and anandamide were not able to inhibit this inflammatory response. Notably, a CB(1) /CB(2) antagonist NESS0327 (3 µm) did not reverse the inhibition of cytokine mRNA expression induced by CP55940. GPR55, a putative novel cannabinoid receptor, mRNA was also expressed in cerebellar granule cells. Although it has been suggested that G(q) associates with GPR55, cannabinoids including CP55940 did not promote phosphoinositide hydrolysis and consequent elevation of intracellular Ca([2+]) concentration. Furthermore, a putative GPR55 antagonist, cannabidiol, also showed a similar inhibitory effect to that of CP55940.. These results suggest that the synthetic cannabinoid CP55940 negatively modulates cytokine mRNA expression in cerebellar granule cells by a CB and GPR55 receptor-independent mechanism.

Topics: Animals; Anti-Inflammatory Agents; Arachidonic Acids; Calcium; Cannabidiol; Cannabinoid Receptor Antagonists; Cannabinoid Receptor Modulators; Cannabinoids; Cerebellum; Cyclohexanols; Cytokines; Dose-Response Relationship, Drug; Endocannabinoids; Glycerides; Inflammation; Lipopolysaccharides; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptors, Cannabinoid; Receptors, G-Protein-Coupled; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Endocannabinoids have both anti-inflammatory and neuroprotective properties against harmful stimuli. We previously demonstrated that the endocannabinoid 2-arachidonoylglycerol (2-AG) protects hippocampal neurons by limiting the inflammatory response via a CB(1) receptor-dependent MAPK/NF-κB signalling pathway. The purpose of the present study was to determine whether PPARγ, an important nuclear receptor, mediates 2-AG-induced inhibition of NF-κB phosphorylation and COX-2 expression, and COX-2-enhanced miniature spontaneous excitatory postsynaptic currents (mEPSCs).. By using a whole-cell patch clamp electrophysiological recording technique and immunoblot analysis, we determined mEPSCs, expression of COX-2 and PPARγ, and phosphorylation of NF-kB in mouse hippocampal neurons in culture.. Exogenous and endogenous 2-AG-produced suppressions of NF-κB-p65 phosphorylation, COX-2 expression and excitatory synaptic transmission in response to pro-inflammatory interleukin-1β (IL-1β) and LPS were inhibited by GW9662, a selective PPARγ antagonist, in hippocampal neurons in culture. PPARγ agonists 15-deoxy-Δ(12,14) -prostaglandin J(2) (15d-PGJ(2)) and rosiglitazone mimicked the effects of 2-AG on NF-κB-p65 phosphorylation, COX-2 expression and mEPSCs, and these effects were eliminated by antagonism of PPARγ. Moreover, exogenous application of 2-AG or elevation of endogenous 2-AG by inhibiting its hydrolysis with URB602 or JZL184, selective inhibitors of monoacylglycerol lipase (MAGL), prevented the IL-1β- and LPS-induced reduction of PPARγ expression. The 2-AG restoration of the reduced PPARγ expression was blocked or attenuated by pharmacological or genetic inhibition of the CB(1) receptor.. Our results suggest that CB(1) receptor-dependent PPARγ expression is an important and novel signalling pathway in endocannabinoid 2-AG-produced resolution of neuroinflammation in response to pro-inflammatory insults.

Topics: Anilides; Animals; Arachidonic Acids; Benzodioxoles; Biphenyl Compounds; Cannabinoid Receptor Modulators; Cells, Cultured; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Endocannabinoids; Excitatory Postsynaptic Potentials; Glycerides; Hippocampus; Inflammation; Interleukin-1beta; Lipopolysaccharides; Mice; Monoacylglycerol Lipases; Neurons; NF-kappa B; Phosphorylation; Piperidines; PPAR gamma; Prostaglandin D2; Receptor, Cannabinoid, CB1; Rosiglitazone; Signal Transduction; Synaptic Transmission; Thiazolidinediones

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

Phospholipase A(2)(PLA(2)) enzymes are considered the primary source of arachidonic acid for cyclooxygenase (COX)-mediated biosynthesis of prostaglandins. Here, we show that a distinct pathway exists in brain, where monoacylglycerol lipase (MAGL) hydrolyzes the endocannabinoid 2-arachidonoylglycerol to generate a major arachidonate precursor pool for neuroinflammatory prostaglandins. MAGL-disrupted animals show neuroprotection in a parkinsonian mouse model. These animals are spared the hemorrhaging caused by COX inhibitors in the gut, where prostaglandins are instead regulated by cytosolic PLA(2). These findings identify MAGL as a distinct metabolic node that couples endocannabinoid to prostaglandin signaling networks in the nervous system and suggest that inhibition of this enzyme may be a new and potentially safer way to suppress the proinflammatory cascades that underlie neurodegenerative disorders.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Benzodioxoles; Brain; Cannabinoid Receptor Modulators; Cyclooxygenase 1; Cytokines; Eicosanoids; Endocannabinoids; Enzyme Inhibitors; Glycerides; Hydrolysis; Inflammation; Inflammation Mediators; Lipopolysaccharides; Liver; Lung; Metabolomics; Mice; Mice, Inbred C57BL; Monoacylglycerol Lipases; Neuroprotective Agents; Parkinsonian Disorders; Phospholipases A2; Piperidines; Prostaglandins; Signal Transduction

Topics: Animals; Arachidonic Acids; Brain; Cannabinoid Receptor Modulators; Endocannabinoids; Glycerides; Inflammation; Monoacylglycerol Lipases; Prostaglandins

Accumulating recent evidence suggests that cannabinoid-1 (CB(1)) receptor activation may promote inflammation and cell death and its pharmacological inhibition is associated with anti-inflammatory and tissue-protective effects in various preclinical disease models, as well as in humans.. In this study, using molecular biology and biochemistry methods, we have investigated the effects of genetic deletion or pharmacological inhibition of CB(1) receptors on inflammation, oxidative/nitrosative stress and cell death pathways associated with a clinically relevant model of nephropathy, induced by an important chemotherapeutic drug cisplatin.. Cisplatin significantly increased endocannabinoid anandamide content, activation of p38 and JNK mitogen-activated protein kinases (MAPKs), apoptotic and poly (ADP-ribose)polymerase-dependent cell death, enhanced inflammation (leucocyte infiltration, tumour necrosis factor-alpha and interleukin-1beta) and promoted oxidative/nitrosative stress [increased expressions of superoxide-generating enzymes (NOX2(gp91phox), NOX4), inducible nitric oxide synthase and tissue 4-hydroxynonenal and nitrotyrosine levels] in the kidneys of mice, accompanied by marked histopathological damage and impaired renal function (elevated creatinine and serum blood urea nitrogen) 3 days following its administration. Both genetic deletion and pharmacological inhibition of CB(1) receptors with AM281 or SR141716 markedly attenuated the cisplatin-induced renal dysfunction and interrelated oxidative/nitrosative stress, p38 and JNK MAPK activation, cell death and inflammatory response in the kidney.. The endocannabinoid system through CB(1) receptors promotes cisplatin-induced tissue injury by amplifying MAPK activation, cell death and interrelated inflammation and oxidative/nitrosative stress. These results also suggest that inhibition of CB(1) receptors may exert beneficial effects in renal (and most likely other) diseases associated with enhanced inflammation, oxidative/nitrosative stress and cell death.

Topics: Animals; Arachidonic Acids; Cell Death; Cisplatin; Disease Models, Animal; Endocannabinoids; Glycerides; Inflammation; Kidney; Male; Mice; Mice, Knockout; Morpholines; Nephritis; Oxidative Stress; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; Signal Transduction

Inflammation caused by activated macrophages and T lymphocytes may trigger plaque rapture in acute coronary syndrome (ACS). Anandamide and 2-arachidonylglycerol (2-AG) are macrophage-derived signal lipids and may be involved in the pathogenesis of ACS, but no clinical relevant data have been reported. In 43 acute myocardial infarction (AMI) patients (66 +/- 2 years), blood samples were obtained from the aortic root and the infarct-related coronary artery (IRA) using a PercuSurge system during primary percutaneous coronary intervention (PCI). In six patients with stable effort angina (SEA) (56 +/- 6 years), blood samples were obtained from the site of stenosis during elective PCI. In 25 of the 43 AMI patients, anandamide was detected in the serum. Serum anandamide level was 35 +/- 20 pmol/mL in the aorta and was significantly increased to 401 +/- 134 pmol/mL in the IRA (P < 0.01). 2-AG was undetectable in most of the patients. In patients with SEA, neither anandamide nor 2-AG was detected in the serum at the plaque site. In AMI patients with anandamide detected, left ventricular ejection fraction at 2 weeks after PCI was increased by 3.7 +/- 2.1% compared with that at the acute phase, while it was decreased by 3.0 +/- 1.8% in those without anandamide detected (P < 0.05). The serum anandamide level at the culprit lesion was elevated compared with the systemic level in a significant number of AMI patients, indicating the synthesis of anandamide at the IRA. Anandamide was suggested to be derived from ruptured plaque and may exert beneficial effects in humans.

Topics: Aged; Angina Pectoris; Angioplasty, Balloon, Coronary; Aorta; Arachidonic Acids; Coronary Vessels; Endocannabinoids; Female; Glycerides; Humans; Inflammation; Male; Middle Aged; Myocardial Infarction; Polyunsaturated Alkamides; Rupture, Spontaneous

Dietary (n-3) long-chain PUFA [(n-3) LCPUFA] ameliorate several metabolic risk factors for cardiovascular diseases, although the mechanisms of these beneficial effects are not fully understood. In this study, we compared the effects of dietary (n-3) LCPUFA, in the form of either fish oil (FO) or krill oil (KO) balanced for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) content, with a control (C) diet containing no EPA and DHA and similar contents of oleic, linoleic, and alpha-linolenic acids, on ectopic fat and inflammation in Zucker rats, a model of obesity and related metabolic dysfunction. Diets were fed for 4 wk. Given the emerging evidence for an association between elevated endocannabinoid concentrations and metabolic syndrome, we also measured tissue endocannabinoid concentrations. In (n-3) LCPUFA-supplemented rats, liver triglycerides and the peritoneal macrophage response to an inflammatory stimulus were significantly lower than in rats fed the control diet, and heart triglycerides were lower, but only in KO-fed rats. These effects were associated with a lower concentration of the endocannabinoids, anandamide and 2-arachidonoylglycerol, in the visceral adipose tissue and of anandamide in the liver and heart, which, in turn, was associated with lower levels of arachidonic acid in membrane phospholipids, but not with higher activity of endocannabinoid-degrading enzymes. Our data suggest that the beneficial effects of a diet enriched with (n-3) LCPUFA are the result of changes in membrane fatty acid composition. The reduction of substrates for inflammatory molecules and endocannabinoids may account for the dampened inflammatory response and the physiological reequilibration of body fat deposition in obese rats.

Topics: Animals; Anti-Inflammatory Agents; Arachidonic Acid; Arachidonic Acids; Cannabinoid Receptor Modulators; Cell Membrane; Cells, Cultured; Choristoma; Dietary Fats; Disease Models, Animal; Endocannabinoids; Euphausiacea; Fatty Acids, Omega-3; Glycerides; Heart; Inflammation; Intra-Abdominal Fat; Liver; Macrophages; Male; Obesity; Polyunsaturated Alkamides; Rats; Rats, Zucker; Shellfish; Triglycerides; Tumor Necrosis Factor-alpha

Endocannabinoids (eCBs) play a role in the modulation of neuroinflammation, and experimental findings suggest that they may be directly involved in the pathogenesis of multiple sclerosis (MS). The objective of our study was to measure eCB levels in the cerebrospinal fluid (CSF) of patients with MS.. Arachidonoylethanolamine (anandamide, AEA), palmotylethanolamide (PEA), 2-arachidonoylglycerol (2-AG) and oleoylethanolamide (OEA) levels were measured in the CSF of 50 patients with MS and 20 control subjects by isotope dilution gas-chromatography/mass-spectrometry. Patients included 35 patients with MS in the relapsing-remitting (RR) form of the disease, 20 in a stable clinical phase and 15 during a relapse, and 15 patients with MS in the secondary progressive (SP) form.. Significantly reduced levels of all the tested eCBs were found in the CSF of patients with MS compared to control subjects, with lower values detected in the SP MS group. Higher levels of AEA and PEA, although below those of controls, were found in the CSF of RR MS patients during a relapse. Higher levels of AEA, 2-AG and OEA were found in patients with MRI gadolinium-enhancing (Gd+) lesions.. The present findings suggest the presence of an impaired eCB system in MS. Increased CSF levels of AEA during relapses or in RR patients with Gd+ lesions suggest its potential role in limiting the ongoing inflammatory process with potential neuroprotective implications. These findings provide further support for the development of drugs targeting eCBs as a potential pharmacological strategy to reduce the symptoms and slow disease progression in MS.

Topics: Adult; Arachidonic Acids; Brain; Cannabinoid Receptor Modulators; Disability Evaluation; Disease Progression; Endocannabinoids; Female; Gas Chromatography-Mass Spectrometry; Glycerides; Humans; Inflammation; Magnetic Resonance Imaging; Male; Multiple Sclerosis; Oleic Acids; Polyunsaturated Alkamides; Severity of Illness Index

Hepatic ischemia-reperfusion (I/R) injury continues to be a fatal complication that can follow liver surgery or transplantation. We have investigated the involvement of the endocannabinoid system in hepatic I/R injury using an in vivo mouse model. Here we report that I/R triggers several-fold increases in the hepatic levels of the endocannabinoids anandamide and 2-arachidonoylglycerol, which originate from hepatocytes, Kupffer, and endothelial cells. The I/R-induced increased tissue endocannabinoid levels positively correlate with the degree of hepatic damage and serum TNF-alpha, MIP-1alpha, and MIP-2 levels. Furthermore, a brief exposure of hepatocytes to various oxidants (H2O2 and peroxynitrite) or inflammatory stimuli (endotoxin and TNF-alpha) also increases endocannabinoid levels. Activation of CB2 cannabinoid receptors by JWH133 protects against I/R damage by decreasing inflammatory cell infiltration, tissue and serum TNF-alpha, MIP-1alpha and MIP-2 levels, tissue lipid peroxidation, and expression of adhesion molecule ICAM-1 in vivo. JWH133 also attenuates the TNF-alpha-induced ICAM-1 and VCAM-1 expression in human liver sinusoidal endothelial cells (HLSECs) and the adhesion of human neutrophils to HLSECs in vitro. Consistent with the protective role of CB2 receptor activation, CB2-/- mice develop increased I/R-induced tissue damage and proinflammatory phenotype. These findings suggest that oxidative/nitrosative stress and inflammatory stimuli may trigger endocannabinoid production, and indicate that targeting CB2 cannabinoid receptors may represent a novel protective strategy against I/R injury. We also demonstrate that CB2-/- mice have a normal hemodynamic profile.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Disease Models, Animal; Endocannabinoids; Glycerides; Humans; Inflammation; Liver; Liver Diseases; Mice; Mice, Knockout; Oxidative Stress; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB2; Reperfusion Injury; Up-Regulation

The effect of WIN55212-2, a cannabinoid receptor agonist, on acute inflammation of mouse ear was investigated. We found that topical application of WIN55212-2 suppressed ear swelling induced by 12-O-tetradecanoylphorbol 13-acetate or 2-arachidonoylglycerol. Similar inhibition was observed with CP55940, another cannabinoid receptor agonist, and HU-308, a cannabinoid CB(2) receptor-selective agonist. WIN55212-2 also suppressed the infiltration of leukocytes induced by 12-O-tetradecanoylphorbol 13-acetate. On the other hand, WIN55212-3, an inactive enantiomer of WIN55212-2, exerted only small effects on inflammation. Notably, SR144528, a cannabinoid CB(2) receptor antagonist, also suppressed inflammatory reactions in mouse ear. Thus, both the cannabinoid CB(2) receptor agonist and antagonist are capable of reducing inflammatory reactions. We then investigated the mechanism underlying WIN55212-2-induced suppression of inflammation using cultured cells. We found that the addition of WIN55212-2 together with 2-arachidonoylglycerol blocked 2-arachidonoylglycerol-induced migration of human promyelocytic leukemia HL-60 cells that had been differentiated into macrophage-like cells. The restoration of 2-arachidonoylglycerol-desensitized cells and WIN55212-2-desensitized cells from an anergic condition was examined next. We found that 2-arachidonoylglycerol-treated cells rapidly recovered the capacity to respond to 2-arachidonoylglycerol. On the other hand, the anergic condition toward 2-arachidonoylglycerol continued for a longer period after pretreatment with WIN55212-2. These results suggest that the anti-inflammatory activity of WIN55212-2 is attributable, at least in part, to interference with the actions of the endogenous ligand, 2-arachidonoylglycerol.

Topics: Administration, Cutaneous; Animals; Arachidonic Acids; Benzoxazines; Calcium; Cannabinoid Receptor Agonists; Cannabinoids; Cell Movement; Cyclohexanols; Dose-Response Relationship, Drug; Ear, External; Endocannabinoids; Glycerides; HL-60 Cells; Humans; Inflammation; Intracellular Fluid; Leukocytes; Ligands; Male; Mice; Mice, Inbred ICR; Morpholines; Naphthalenes; Peroxidase; Tetradecanoylphorbol Acetate

The possible involvement of 2-arachidonoylglycerol (2-AG), an endogenous ligand for the cannabinoid receptors (CB1 and CB2), in contact dermatitis in mouse ear was investigated. We found that the level of 2-AG was markedly elevated in the ear following a challenge with oxazolone in sensitized mice. Of note, the swelling following the challenge was suppressed by either the administration of SR144528, a CB2 receptor antagonist, immediately after sensitization, or the administration of SR144528 upon the challenge. The effect of AM251, a CB1 receptor antagonist, was marginal in either case. It seems apparent, therefore, that the CB2 receptor and its endogenous ligand 2-AG are closely involved in both the sensitization phase and the elicitation phase of oxazolone-induced contact dermatitis. In line with this, we found that Langerhans cells (MHC class II(+)) contain a substantial amount of CB2 receptor mRNA, whereas keratinocytes (MHC class II(-)) do not. We also obtained evidence that the expression of mRNAs for proinflammatory cytokines following a challenge with oxazolone was markedly suppressed by treatment with SR144528. We next examined whether the CB2 receptor and 2-AG participate in chronic contact dermatitis accompanied by the infiltration of tissues by eosinophils. The amount of 2-AG in mouse ear dramatically increased following repeated challenge with oxazolone. Importantly, treatment with SR144528 attenuated both the recruitment of eosinophils and ear swelling in chronic contact dermatitis induced by repeated challenge with oxazolone. These results strongly suggest that the CB2 receptor and 2-AG play important stimulative roles in the sensitization, elicitation, and exacerbation of allergic inflammation.

Topics: Animals; Arachidonic Acids; Dermatitis, Contact; Ear; Endocannabinoids; Glycerides; Inflammation; Keratinocytes; Langerhans Cells; Ligands; Male; Mice; Mice, Inbred ICR; Oxazolone; Receptor, Cannabinoid, CB2; RNA, Messenger

2-Arachidonoylglycerol is an endogenous ligand for the cannabinoid receptors. Two types of cannabinoid receptors have been identified to date. The CB1 receptor is abundantly expressed in the brain, and assumed to be involved in the attenuation of neurotransmission. On the other hand, the physiological roles of the CB2 receptor, mainly expressed in several types of inflammatory cells and immunocompetent cells, have not yet been fully elucidated. In this study, we investigated possible pathophysiological roles of the CB2 receptor and 2-arachidonoylglycerol in acute inflammation in mouse ear induced by the topical application of 12-O-tetradecanoylphorbol-13-acetate. We found that the amount of 2-arachidonoylglycerol was markedly augmented in inflamed mouse ear. In contrast, the amount of anandamide, another endogenous cannabinoid receptor ligand, did not change markedly. Importantly, 12-O-tetradecanoylphorbol-13-acetate-induced ear swelling was blocked by treatment with SR144528, a CB2 receptor antagonist, suggesting that the CB2 receptor is involved in the swelling. On the other hand, the application of AM251, a CB1 receptor antagonist, exerted only a weak suppressive effect. The application of SR144528 also reduced the 12-O-tetradecanoylphorbol-13-acetate-induced production of leukotriene B(4) and the infiltration of neutrophils in the mouse ear. Interestingly, the application of 2-arachidonoylglycerol to the mouse ear evoked swelling, which was abolished by treatment with SR144528. Nitric oxide was suggested to be involved in the ear swelling induced by 2-arachidonoylglycerol. These results suggest that the CB2 receptor and 2-arachidonoylglycerol play crucial stimulative roles during the course of inflammatory reactions.

Topics: Administration, Topical; Animals; Arachidonic Acids; Camphanes; Cannabinoid Receptor Modulators; Dose-Response Relationship, Drug; Ear; Endocannabinoids; Glycerides; Inflammation; Ligands; Male; Mice; Mice, Inbred ICR; Pyrazoles; Receptor, Cannabinoid, CB2; Tetradecanoylphorbol Acetate

Topics: Animals; Arachidonic Acids; Breast Neoplasms; Cannabinoids; Cell Division; Eicosanoids; Endocannabinoids; Female; Glycerides; Humans; Inflammation; Polyunsaturated Alkamides; Rats; Rats, Wistar; Tumor Cells, Cultured; Urinary Bladder