n-oleoylethanolamine and anandamide

Fatty acid amide hydrolase (FAAH) is responsible for hydrolysis of endocannabinoid, anandamide (AEA), and N-acyl ethanolamines such as palmitoylethanolamine (PEA) and N-oleoylethanolamide (OEA). Genetic deletion or pharmacological inactivation of FAAH shows site-specific elevation of AEA that plays a role in the modulation of pain and other neurodegenerative disorders. The review elaborates recent progress and current status of diverse structural classes of reversible and irreversible FAAH inhibitors. The discussion also addresses ligand-enzyme active site interactions and mechanism of enzyme inactivation, emerging approaches to novel FAAH inhibitors, and ongoing efforts to address gaps in therapeutic utility of FAAH inhibitors.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Cannabinoids; Endocannabinoids; Enzyme Inhibitors; Ethanolamines; Humans; Hydrolysis; Oleic Acids; Polyunsaturated Alkamides

Acylethanolamides are formed in the brain "on demand" from membrane phospholipids called N-acylated phosphatidylethanolamines. The acylethanolamides are signaling molecules of lipid nature, and this lipofilicity suggests an autocrine function. The acylethanolamides include palmitoylethanolamide (PEA), oleoylethanolamide (OEA), stearoylethanolamide (SEA), and several other quantitative minor species including anandamide (= arachidonoylethanolamide). PEA and OEA can activate several different receptors and inhibit some ion channels, e.g., PPARalpha, vanilloid receptor, K(+) channels (Kv4.3, Kv1.5), and OEA can activate GPR119 and inhibit ceramidases. Targets for SEA are less clear, but it has some cannabimimetic actions in rats in vivo. All acylethanolamides accumulate during neuronal injury, and injected OEA has neuroprotective effects, and PEA has anti-inflammatory effects as studied in the peripheral system. Several of the pharmacological effects seem to be mediated via activation of PPARalpha. Recently, injected OEA has been found to consolidate memories in rats. Inhibitors of the acylethanolamide-degrading enzyme FAAH can increase levels of all acylethanolamides including annandamide, and some of the pharmacological effects caused by these inhibitors may be explained by increased cerebral levels of OEA and PEA, e.g., suppression of nicotine-induced activation of dopamine neurons. Furthermore, through activation of PPARalpha, OEA and PEA may stimulate neurosteroid synthesis, thereby modulating several biological functions mediated by GABA(A) receptors. The existence of acylethanolamides in the mammalian brain has been known for decades, but it is first within the last few years that the putative biological functions of the three most abundant acylethanolamides species are starting to emerge.

Topics: Amides; Animals; Arachidonic Acids; Brain; Cytoprotection; Endocannabinoids; Ethanolamines; Humans; Nerve Degeneration; Neurons; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Receptors, Cannabinoid

Acylethanolamides (AEs) are a group of lipids occurring in both plants and animals. The best-studied AEs are the endocannabinoid anandamide (AEA), the anti-inflammatory compound palmitoylethanolamide (PEA), and the potent anorexigenic molecule oleoylethanolamide (OEA). AEs are biosynthesized in the gastrointestinal tract, and their levels may change in response to noxious stimuli, food deprivation or diet-induced obesity. The biological actions of AEs within the gut are not limited to the modulation of food intake and energy balance. For example, AEs exert potential beneficial effects in the regulation of intestinal motility, secretion, inflammation and cellular proliferation. Molecular targets of AEs, which have been identified in the gastrointestinal tract, include cannabinoid CB(1) and CB(2) receptors (activated by AEA), transient receptor potential vanilloid type 1 (TRPV1, activated by AEA and OEA), the nuclear receptor peroxisome proliferators-activated receptor-alpha (PPAR-alpha, activated by OEA and, to a less extent, by PEA), and the orphan G-coupled receptors GPR119 (activated by OEA) and GPR55 (activated by PEA and, with lower potency, by AEA and OEA). Modulation of AE levels in the gut may provide new pharmacological strategies not only for the treatment of feeding disorders but also for the prevention or cure of widespread intestinal diseases such as inflammatory bowel disease and colon cancer.

Topics: Amides; Analgesics; Animals; Anti-Inflammatory Agents; Antineoplastic Agents; Apoptosis; Arachidonic Acids; Caco-2 Cells; Dietary Fats; Eating; Endocannabinoids; Energy Metabolism; Ethanolamine; Ethanolamines; Feeding Behavior; Gastric Acid; Gastrointestinal Tract; Humans; Intestinal Absorption; Intra-Abdominal Fat; Oleic Acids; Palmitic Acids; Peroxisome Proliferator-Activated Receptors; Polyunsaturated Alkamides; TRPV Cation Channels

Anandamide and the other N-acylethanolamines, e.g. oleoylethanolamide (OEA), palmitoylethanolamide (PEA), and linoleoylethanolamide (LEA), may be formed by several enzymatic pathways from their precursors, which are the N-acylated ethanolamine phospholipids. The exact enzymatic pathways involved in their biosynthesis in specific tissues are not clarified. It has been suggested that endogenous anandamide could stimulate food intake by activation of cannabinoid receptors in the brain and/or in the intestinal tissue. On the other hand, endogenous OEA and PEA have been suggested to inhibit food intake by acting on receptors in the intestine. At present, there is no clear role for endogenous anandamide in controlling food intake via cannabinoid receptors, neither centrally nor in the gastrointestinal tract. However, OEA, PEA and perhaps also LEA may be involved in regulation of food intake by selective prolongation of feeding latency and post-meal interval. These N-acylethanolamines seem to be formed locally in the intestine, where they can activate PPARalpha located in close proximity to their site of synthesis. The rapid onset of OEA response and its reliance on an intact vagus nerve suggests that activation of PPARalpha does not result in formation of a transcription-dependent signal but must rely on an unidentified non-genomic signal that translates to activation of vagal afferents. Whether GPR119, TRPV1 and/or intestinal ceramide levels also contribute to the anorectic and weight-reducing effect of exogenous OEA is less clear. Prolonged intake of dietary fat (45 energy%) may promote over-consumption of food by decreasing the endogenous levels of OEA, PEA and LEA in the intestine.

Topics: Animals; Arachidonic Acids; Dietary Fats; Eating; Endocannabinoids; Ethanolamines; Fats; Linoleic Acids; Oleic Acids; Polyunsaturated Alkamides; Receptors, Cannabinoid

The gastrointestinal tract plays a pivotal role in the regulation of food intake and energy balance. Signals from the gastrointestinal tract generally function to limit ingestion in the interest of efficient digestion. These signals may be released into the bloodstream or may activate afferent neurones that carry information to the brain and its cognitive centres, which regulates food intake. The rate at which nutrients become systemically available is also influenced by gastrointestinal motility: a delay in gastric emptying may evoke a satiety effect. Recent evidence suggests that the endocannabinoid anandamide and the related acylethanolamide oleoylethanolamide are produced in the intestine and might regulate feeding behaviour by engaging sensory afferent neurones that converge information to specific areas of the brain. The intestinal levels of these acylethanolamides are inversely correlated to feeding, as food deprivation increases intestinal levels of anandamide (which acts in the gut as a 'hunger signal'), while it decreases the levels of oleoylethanolamide (which acts in the gut as a 'satiety signal'). Additionally, these acylethanolamides, whose gastric levels change in response to diet-induced obesity, alter gastrointestinal motility, which might contribute to their effect on food intake and nutrient absorption.

Topics: Animals; Appetite Regulation; Arachidonic Acids; Cannabinoid Receptor Modulators; Eating; Endocannabinoids; Energy Metabolism; Feeding Behavior; Gastrointestinal Motility; Gastrointestinal Tract; Homeostasis; Humans; Models, Biological; Neurosecretory Systems; Obesity; Oleic Acids; Polyunsaturated Alkamides; Satiation; Satiety Response; Signal Transduction

The endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG), as well as the related acylethanolamide oleoylethanolamide (OEA), have been implicated in energy expenditure (EE) regulation and metabolic diseases. Muscle (fat-free mass) and fat (fat mass) are metabolically active compartments and main determinants of EE.. To assess whether human muscle, adipose, and plasma endocannabinoids correlate with EE.. Muscle, adipose, and plasma AEA, 2-AG, and OEA concentrations were measured via liquid chromatography-mass spectrometry. EE was assessed by indirect whole-room calorimetry.. Clinical trial.. Obese/overweight Native Americans of full (n = 35) and at least half (n = 21) Southwestern heritage.. Twenty-four-hour EE, sleeping EE (SLEEP), resting EE (REE), respiratory quotient (RQ), and macronutrient oxidation.. In full Natives, muscle AEA concentration correlated with SLEEP (r = -0.65, P = 0.004) and REE (r = -0.53, P = 0.02). Muscle 2-AG was associated with SLEEP (r = -0.75, P = 0.0003). Adipose OEA concentration correlated with RQ (r = -0.47, P = 0.04) and lipid oxidation (r = 0.51, P = 0.03). Plasma OEA concentration was associated with SLEEP (r = -0.52, P = 0.04). After adjustment for major determinants, these lipids explained nearly 20% of the additional variance of the respective measure. Similarly, in Native Americans of at least half Southwestern heritage, investigated lipids correlated with EE measures.. Endocannabinoids in metabolically relevant peripheral tissues explained a large part of EE variation and may be involved in regulating EE. Dysregulation of peripheral endocannabinoids may predispose people to metabolic diseases via an effect on EE and lipid oxidation.

Topics: Adipose Tissue; Adult; Arachidonic Acids; Calorimetry, Indirect; Chromatography, Liquid; Endocannabinoids; Energy Metabolism; Female; Glycerides; Humans; Indians, North American; Lipid Metabolism; Male; Mass Spectrometry; Muscle, Skeletal; Obesity; Oleic Acids; Oxidation-Reduction; Polyunsaturated Alkamides; Respiration; Rest; Sleep; Southwestern United States

Food palatability increases food intake and may lead to overeating. The mechanisms behind this observation are still largely unknown.. The aims of this study were the following: 1) to elucidate the plasma responses of endocannabinoids, N-acylethanolamines, and gastrointestinal peptides to a palatable (sweet), unpalatable (bitter), and sensory-acceptable (tasteless control) food, and 2) to verify whether some of these bioactive compounds can serve as plasma biomarkers of food liking in humans.. Three puddings providing 60 kcal (35% from proteins, 62% from carbohydrates, and 3% from fats) but with different taste were developed. Twenty healthy subjects (11 women and 9 men; mean age 28 y and BMI 22.7 kg/m(2)), selected because they liked the puddings in the order sweet > control > bitter, participated in a randomized crossover study based on a modified sham feeding (MSF) protocol. Blood samples at baseline and every 5 min up to 20 min after the MSF were analyzed for gastrointestinal peptides, endocannabinoids, and N-acylethanolamines. Thirty minutes after the MSF, energy intake at an ad libitum breakfast was measured.. After the MSF, no response was observed in 7 of 9 gastrointestinal peptides measured. The plasma ghrelin concentration at 20 min after the sweet and bitter puddings was 25% lower than after the control pudding (P = 0.04), and the pancreatic polypeptide response after the sweet pudding was 23% greater than after the bitter pudding (P = 0.02). The plasma response of 2-arachidonoylglycerol after the sweet pudding was 37% and 15% higher than after the bitter (P < 0.001) and control (P = 0.03) puddings, respectively. Trends for greater responses of anandamide (P = 0.06), linoleoylethanolamide (P = 0.07), palmitoylethanolamide (P = 0.06), and oleoylethanolamide (P = 0.09) were found after the sweet pudding than after the bitter pudding. No differences in subsequent energy intake were recorded.. The data demonstrated that food palatability influenced some plasma endocannabinoid and N-acylethanolamine concentrations during the cephalic phase response and indicated that 2-arachidonoylglycerol and pancreatic polypeptide can be used as biomarkers of food liking in humans.

Topics: Adult; Amides; Arachidonic Acids; Blood Glucose; Body Mass Index; Cross-Over Studies; Edetic Acid; Endocannabinoids; Energy Intake; Ethanolamines; Female; Food Preferences; Ghrelin; Glycerides; Humans; Linear Models; Linoleic Acids; Male; Oleic Acids; Palmitic Acids; Pancreatic Polypeptide; Polyunsaturated Alkamides; Taste; Young Adult

Cannabidiol is a component of marijuana that does not activate cannabinoid receptors, but moderately inhibits the degradation of the endocannabinoid anandamide. We previously reported that an elevation of anandamide levels in cerebrospinal fluid inversely correlated to psychotic symptoms. Furthermore, enhanced anandamide signaling let to a lower transition rate from initial prodromal states into frank psychosis as well as postponed transition. In our translational approach, we performed a double-blind, randomized clinical trial of cannabidiol vs amisulpride, a potent antipsychotic, in acute schizophrenia to evaluate the clinical relevance of our initial findings. Either treatment was safe and led to significant clinical improvement, but cannabidiol displayed a markedly superior side-effect profile. Moreover, cannabidiol treatment was accompanied by a significant increase in serum anandamide levels, which was significantly associated with clinical improvement. The results suggest that inhibition of anandamide deactivation may contribute to the antipsychotic effects of cannabidiol potentially representing a completely new mechanism in the treatment of schizophrenia.

Topics: Acute Disease; Adult; Amides; Amisulpride; Antipsychotic Agents; Arachidonic Acids; Cannabidiol; Double-Blind Method; Drug Therapy, Combination; Endocannabinoids; Ethanolamines; Female; Humans; Male; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Psychiatric Status Rating Scales; Schizophrenia; Schizophrenic Psychology; Signal Transduction; Sulpiride; Young Adult

Endogenous cannabinoids activate cannabinoid receptors in the brain and elicit mood-altering effects. Parallel effects (eg, anxiolysis, analgesia, sedation) may be elicited by osteopathic manipulative treatment (OMT), and previous research has shown that the endorphin system is not responsible for OMT's mood-altering effects. The authors investigate whether OMT generated cannabimimetic effects for 31 healthy subjects in a dual-blind, randomized controlled trial that measured changes in subjects' scores on the 67-item Drug Reaction Scale (DRS). Chemical ionization gas chromatography and mass spectrometry were also used to determine changes in serum levels of anandamide (AEA), 2-arachidonoylglycerol (2-AG), and oleylethanolamide (OEA). In subjects receiving OMT, posttreatment DRS scores increased significantly for the cannabimimetic descriptors good, high, hungry, light-headed, and stoned, with significant score decreases for the descriptors inhibited, sober, and uncomfortable. Mean posttreatment AEA levels (8.01 pmol/mL) increased 168% over pretreatment levels (2.99 pmol/mL), mean OEA levels decreased 27%, and no changes occurred in 2-AG levels in the group receiving OMT. Subjects in the sham manipulative treatment group recorded mixed DRS responses, with both increases and decreases in scores for cannabimimetic and noncannabimimetic descriptors and no changes in sera levels. When changes in serum AEA were correlated with changes in subjects' DRS scores, increased AEA correlated best with an increase for the descriptors cold and rational, and decreased sensations for the descriptors bad, paranoid, and warm. The authors propose that healing modalities popularly associated with changes in the endorphin system, such as OMT, may actually be mediated by the endocannabinoid system.

Topics: Adult; Arachidonic Acids; Cannabinoid Receptor Modulators; Endocannabinoids; Female; Glycerides; Health Status Indicators; Humans; Male; Manipulation, Osteopathic; Oleic Acids; Polyunsaturated Alkamides

Patients with craniopharyngioma (CP) frequently suffer from morbid obesity. Endocannabinoids (ECs) are involved in weight gain and rewarding behavior but have not been investigated in this context.. Cross-sectional single-center study.. Eighteen patients with CP and 16 age- and sex-matched controls were included. Differences in endocannabinoids (2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (AEA)) and endocannabinoid-like molecules (oleoyl ethanolamide (OEA), palmitoylethanolamide (PEA), and arachidonic acid (AA) were measured at baseline and following endurance exercise. We further explored ECs-dynamics in relation to markers of HPA-axis activity (ACTH, cortisol, copeptin) and hypothalamic damage.. Under resting conditions, independent of differences in BMI, 2-AG levels were more than twice as high in CP patients compared to controls. In contrast, 2-AG and OEA level increased in response to exercise in controls but not in CP patients, while AEA levels decreased in controls. As expected, exercise increased ACTH and copeptin levels in controls only. In a mixed model analysis across time and group, HPA measures did not provide additional information for explaining differences in 2-AG levels. However, AEA levels were negatively influenced by ACTH and copeptin levels, while OEA levels were negatively predicted by copeptin levels only. There were no significant differences in endocannabinoids depending on hypothalamic involvement.. Patients with CP show signs of a dysregulated endocannabinoid system under resting conditions as well as following exercise in comparison to healthy controls. Increased 2-AG levels under resting conditions and the missing response to physical activity could contribute to the metabolic phenotype of CP patients.

Topics: Adrenocorticotropic Hormone; Adult; Arachidonic Acid; Arachidonic Acids; Case-Control Studies; Craniopharyngioma; Cross-Sectional Studies; Endocannabinoids; Endurance Training; Exercise; Female; Glycerides; Glycopeptides; Humans; Hydrocortisone; Hypothalamo-Hypophyseal System; Hypothalamus; Male; Middle Aged; Oleic Acids; Pituitary Neoplasms; Polyunsaturated Alkamides; Young Adult

The endocannabinoid (eCB) system is involved in diverse aspects of human physiology and behavior but little is known about the impact of circadian rhythmicity on the system. The two most studied endocannabinoids, AEA (ananamide) and 2-AG (2-arachidonoylglycerol), can be measured in peripheral blood however the functional relevance of peripheral eCB levels is not clear. Having previously detailed the 24-h profile of serum 2-AG, here we report the 24-h serum profile of AEA to determine if these two endocannabinoids vary in parallel across the biological day including a nocturnal 8.5-h sleep period. Further, we assessed and compared the effect of a physiological challenge, in the form of sleep restriction to 4.5-h, on these two profiles.. In this randomized crossover study, we examined serum concentrations of AEA across a 24-h period in fourteen young adults. Congeners of AEA, the structural analogs oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) were simultaneously assayed. Prior to 24-h blood sampling, each participant was exposed to two nights of normal (8.5 h) or restricted sleep (4.5 h). The two sleep conditions were separated by at least one month. In both sleep conditions, during the period of blood sampling, each individual ate the same high-carbohydrate meal at 0900, 1400, and 1900.. Mean 24-h concentrations of AEA were 0.697 ± 0.11 pmol/ml. A reproducible biphasic 24-h profile of AEA was observed with a first peak occurring during early sleep (0200) and a second peak in the mid-afternoon (1500) while a nadir was detected in the mid-morning (1000). The 24-h profiles for both OEA and PEA followed a similar pattern to that observed for AEA. AEA, OEA, and PEA levels were not affected by sleep restriction at any time of day, contrasting with the elevation of early afternoon levels previously observed for 2-AG.. The 24-h rhythm of AEA is markedly different from that of 2-AG, being of lesser amplitude and biphasic, rather than monophasic. These observations suggest distinct regulatory pathways of the two eCB and indicate that time of day needs to be carefully controlled in studies attempting to delineate their relative roles. Moreover, unlike 2-AG, AEA is not altered by sleep restriction, suggesting that physiological perturbations may affect AEA and 2-AG differently. Similar 24-h profiles were observed for OEA and PEA following normal and restricted sleep, further corroborating the validity of the wave-shape and lack of response to sleep loss observed for the AEA profile. Therapeutic approaches involving agonism or antagonism of peripheral eCB signaling will likely need to be tailored according to time of day.

Topics: Adolescent; Adult; Amides; Arachidonic Acids; Circadian Rhythm; Cross-Over Studies; Endocannabinoids; Ethanolamines; Female; Glycerides; Humans; Male; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Sleep; Young Adult

No data are available on whether a diet deficient of the essential fatty acids is able to modulate tissue levels of endocannabinoids and congeners. Male rats fed for 12 weeks a diet deficient of essential fatty acids, palmitic and oleic acids (EFAD), replaced with saturated fatty acids (SAFA), showed lowered n-3 and n-6 PUFAs levels in plasma, liver and adipose tissue, with concomitant steep increase of oleic and mead acids, while in hypothalamus no changes in PUFA concentration were detected and only palmitoleic acid was found increased. We found a reduction of anandamide and palmitoylethanolamide in liver and brain, while oleoylethanolamide increased significantly in liver and adipose tissue, associated to a 50 % body weight decrease. Changes in N-acylethanolamide profile may contribute to body weight reduction distinctive of EFA deficiency.

Topics: Adipose Tissue; Amides; Animals; Arachidonic Acids; Body Weight; Brain Chemistry; Endocannabinoids; Ethanolamines; Fatty Acids; Fatty Acids, Essential; Fatty Acids, Omega-3; Fatty Acids, Omega-6; Liver; Male; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Rats

Physical exercise is increasingly being promoted by health care for chronic pain conditions with beneficial outcomes, such as pain and fatigue reduction, and increased quality of life. Nevertheless, knowledge about biochemical consequences of physical exercise in chronic pain is still relatively poor. The endocannabinoid system has been suggested to play a role for acute exercise-induced reward and pain inhibition. The aim of this study is to investigate the chronic outcomes of resistance exercise on levels of endocannabinoids and related lipids in fibromyalgia (FM).. This study examine the outcomes of a 15-wk person-centered resistance exercise program on plasma levels of the lipid mediators; anandamide, 2-arachidonoylglycerol (2-AG), oleoylethanolamide, palmitoylethanolamide, and stearoylethanolamide (SEA) sampled from 37 women with FM and 33 healthy controls. The associations between clinical scorings of pain, depression, anxiety, fatigue, and muscle strength with levels of these lipid mediators before and after the exercise program are also analyzed.. After the 15-wk exercise program, anandamide levels were significantly increased, and SEA levels significantly decreased in FM. Pain intensity and depression scorings decreased and muscle strength increased, and in a multivariate context, muscle strength was positively associated with 2-AG levels after the resistance exercise program in FM.. The increased anandamide and decreased SEA in women with FM after the 15-wk program might point to a chronic effect of resistance exercise. Pain and depression scorings decreased in the FM group after the program, but no associations between pain, depression, and lipid level changes were assured.

Topics: Amides; Anxiety; Arachidonic Acids; Depression; Endocannabinoids; Ethanolamines; Exercise Therapy; Fatigue; Female; Fibromyalgia; Glycerides; Humans; Oleic Acids; Pain Management; Palmitic Acids; Polyunsaturated Alkamides; Resistance Training; Stearic Acids

Endocannabinoids are suggested to play a role in energy balance regulation.. We aimed to investigate associations of endocannabinoid concentrations during the day with energy balance and adiposity and interactions with 2 diets differing in protein content in participants in the postobese phase with prediabetes.. Participants (n = 38) were individually fed in energy balance with a medium protein (MP: 15:55:30% of energy from protein:carbohydrate:fat) or high-protein diet (HP: 25:45:30% energy from P:C:F) for 48 hours in a respiration chamber.. Associations between energy balance, energy expenditure, respiratory quotient, and endocannabinoid concentrations during the day were assessed.. Plasma-concentrations of anandamide (AEA), oleoylethanolamide (OEA), palmitoyethanolamide (PEA), and pregnenolone (PREG) significantly decreased during the day. This decrease was inversely related to body mass index (AEA) or body fat (%) (PEA; OEA). The lowest RQ value, before lunch, was inversely associated with concentrations of AEA and PEA before lunch. Area under the curve (AUC) of concentrations of AEA, 2-AG, PEA, and OEA were positively related to body fat% (P < .05).The HP and MP groups showed no differences in concentrations of AEA, OEA, PEA, and PREG, but the AUC of 2-arachidonoylglycerol (2-AG) was significantly higher in the HP vs the MP group.. In energy balance, only the endocannabinoid 2-AG changed in relation to protein level of the diet, whereas the endocannabinoid AEA and endocannabinoid-related compounds OEA and PEA reflected the gradual energy intake matching energy expenditure during the day.

Topics: Adipose Tissue; Adult; Aged; Aldehydes; Arachidonic Acids; Body Mass Index; Endocannabinoids; Energy Metabolism; Female; Humans; Male; Meals; Middle Aged; Obesity; Oleic Acids; Polyunsaturated Alkamides; Pregnenolone

Topics: Adult; Aged; Amides; Arachidonic Acids; Blood Pressure; Body Mass Index; Cardiovascular Diseases; Cholesterol; Diet, High-Protein; Endocannabinoids; Ethanolamines; Glycerides; Humans; Lipoproteins, LDL; Middle Aged; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Weight Loss

To determine if plasma concentrations of the N-acylethanolamines (NAEs) N-arachidonoylethanolamine (AEA), N-oleoylethanolamide (OEA) and N-palmitoylethanolamide (PEA) increase in women at high risk for preterm birth (PTB) and whether these could be used to predict preterm delivery and if so, how they compare with current methods.. Prospective cohort study.. A large UK teaching hospital.. 217 pregnant women were recruited between 24 and 34 gestational weeks at 'high-risk' for PTB, recruited from a prematurity prevention clinic or antenatal wards.. Plasma AEA, OEA, and PEA concentrations were measured using ultra-high performance liquid chromatography-tandem mass spectrometry whilst FAAH enzyme activity was measured by fluorometric radiometric assay and CL by ultrasound scan. The clinical usefulness of these measurements were determined by ROC and multivariate analyses.. AEA and PEA concentrations were significantly higher in women who delivered prematurely. An AEA concentration >1.095 nM predicted PTB, the gestational age at delivery and the recruitment to delivery interval (RTDI). A PEA concentration >17.50 nM only predicted PTB; FAAH enzyme activity was not related to these changes. Multivariate analysis (all variables) generated an equation to accurately predict the RTDI.. A single plasma AEA or PEA measurement can predict PTB. A single AEA measurement predicts the gestational age of delivery and the remaining period of pregnancy with reasonable accuracy and better than existing conventional tests thus offering a better window for primary prevention of PTB.

Topics: Amides; Amidohydrolases; Arachidonic Acids; Cohort Studies; Endocannabinoids; Ethanolamines; Female; Gestational Age; Humans; Obstetric Labor, Premature; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Pregnancy; Premature Birth; Prospective Studies; Risk Assessment; United Kingdom

In agreement with the neurodevelopmental hypothesis of schizophrenia, prenatal exposure of rats to the antimitotic agent methylazoxymethanol acetate (MAM) at gestational day 17 produced long-lasting behavioral alterations such as social withdrawal and cognitive impairment in the social interaction test and in the novel object recognition test, respectively. At the molecular level, an increased cannabinoid receptor type-1 (CB1) mRNA and protein expression, which might be due to reduction in DNA methylation at the gene promoter in the prefrontal cortex (PFC), coincided with deficits in the social interaction test and in the novel object recognition test in MAM rats. Both the schizophrenia-like phenotype and altered transcriptional regulation of CB1 receptors were reversed by peripubertal treatment (from PND 19 to PND 39) with the non-psychotropic phytocannabinoid cannabidiol (30 mg/kg/day), or, in part, by treatment with the cannabinoid CB1 receptor antagonist/inverse agonist AM251 (0.5 mg/kg/day), but not with haloperidol (0.6 mg/kg/day). These results suggest that early treatment with cannabidiol may prevent both the appearance of schizophrenia-like deficits as well as CB1 alterations in the PFC at adulthood, supporting that peripubertal cannabidiol treatment might be protective against MAM insult.

Topics: Amides; Animals; Arachidonic Acids; Cannabidiol; Disease Models, Animal; Endocannabinoids; Ethanolamines; Female; Glycerides; Hippocampus; Interpersonal Relations; Male; Methylazoxymethanol Acetate; Motor Activity; Oleic Acids; Palmitic Acids; Piperidines; Polyunsaturated Alkamides; Prefrontal Cortex; Pregnancy; Prenatal Exposure Delayed Effects; Puberty; Pyrazoles; Rats; Receptor, Cannabinoid, CB1; Recognition, Psychology; RNA, Messenger; Schizophrenia

Previous findings demonstrate a homeostatic role for oleoylethanolamide (OEA) signaling in the ethanol-related neuroinflammation and behavior. However, extensive research is still required in order to unveil the effects of OEA on a number of neurobiological functions such as adult neurogenesis, cell survival and resident neuroimmunity that become notably altered by alcohol. Daily consumption of ethanol (10%) for 2 weeks (6.3 ± 1.1 g/kg/day during last 5 days) caused hypolocomotor activity in rats. This effect appears to rely on central signaling mechanisms given that alcohol increased the OEA levels, the gene expression of OEA-synthesizing enzyme Nape-pld and the number of PPARα-immunoreactive neurons in the striatum. Ethanol-related neurobiological alterations such as a reduction in the number of microglial cells expressing iNOS (a cytokine-inducible immune defense) and in adult neural stem/progenitor cell (NSPC) proliferation (phospho-H3 and BrdU) and maturation (BrdU/β3-tubulin), as well as an increase in damage cell activity (FosB) and apoptosis (cleaved caspase 3) were also observed in the rat striatum. Pharmacological administration of OEA (10 mg/kg) for 5 days during ethanol exposure exacerbated ethanol-induced hypolocomotion and cell apoptosis in the striatum. Interestingly, OEA abrogated the impaired effects of ethanol on PPARα-positive cell population and NSPC proliferation and maturation. OEA also decreased astrocyte-related vimentin immunoreactivity and increased microglial cell population (Iba-1, iNOS) in the striatum. These results suggest that OEA-PPARα signaling modulates glial activation, cell apoptosis and NSPC proliferation and maturation in response to striatal-specific neurobiological alterations induced by prolonged ethanol intake in rats.

Topics: Alanine Transaminase; Alcohol Drinking; Amidohydrolases; Animals; Apoptosis; Arachidonic Acids; Aspartate Aminotransferases; Calcium-Binding Proteins; Caspase 3; Cell Proliferation; Cell Survival; Endocannabinoids; Ethanol; Ethanolamines; gamma-Glutamyltransferase; Glial Fibrillary Acidic Protein; Hepatobiliary Elimination; Locomotion; Male; Microfilament Proteins; Microglia; Neostriatum; Neurons; Oleic Acids; Phospholipase D; Polyunsaturated Alkamides; PPAR alpha; Proto-Oncogene Proteins c-fos; Rats; Rats, Wistar; Signal Transduction

Endocannabinoid signaling via anandamide (AEA) is implicated in a variety of neuronal functions and considered a promising therapeutic target for numerous emotion-related disorders. The major AEA degrading enzyme is fatty acid amide hydrolase (FAAH). Genetic deletion and pharmacological inhibition of FAAH reduce anxiety and improve emotional responses and memory in rodents and humans. Complementarily, the mechanisms and impact of decreased AEA signaling remain to be delineated in detail. In the present study, using the Cre/loxP system combined with an adeno-associated virus (AAV)-mediated delivery system, FAAH was selectively overexpressed in hippocampal CA1-CA3 glutamatergic neurons of adult mice. This approach led to specific FAAH overexpression at the postsynaptic site of CA1-CA3 neurons, to increased FAAH enzymatic activity, and, in consequence, to decreased hippocampal levels of AEA and palmitoylethanolamide (PEA), but the levels of the second major endocannabinoid 2-arachidonoyl glycerol (2-AG) and of oleoylethanolamide (OEA) were unchanged. Electrophysiological recordings revealed an enhancement of both excitatory and inhibitory synaptic activity and of long-term potentiation (LTP). In contrast, excitatory and inhibitory long-term depression (LTD) and short-term synaptic plasticity, apparent as depolarization-induced suppression of excitation (DSE) and inhibition (DSI), remained unaltered. These changes in hippocampal synaptic activity were associated with an increase in anxiety-like behavior, and a deficit in object recognition memory and in extinction of aversive memory. This study indicates that AEA is not involved in hippocampal short-term plasticity, or eLTD and iLTD, but modulates glutamatergic transmission most likely via presynaptic sites, and that disturbances in this process impair learning and emotional responses.

Topics: Amides; Amidohydrolases; Animals; Arachidonic Acids; Emotions; Endocannabinoids; Ethanolamines; Glutamic Acid; Glycerides; Hippocampus; Learning; Long-Term Potentiation; Long-Term Synaptic Depression; Male; Memory; Mice; Neuronal Plasticity; Neurons; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Synaptic Transmission; Up-Regulation

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

Oleoylethanolamide (OEA) is a non-cannabinoid acylethanolamide with multiple physiological roles that has been proposed to have antidepressant-like activity in preclinical models. OEA shares biosynthetic pathways with anandamide (AEA) a transmitter involved in affective disorders and anxiety in humans. However, although the participation of OEA in depression has been proposed, both, the contribution of OEA to the depressive phenotype and the effect of antidepressant therapy on circulating levels of this and related non-cannabinoid acylethanolamides in humans are basically unknown. The main objective of this study is to compare the plasma concentrations of OEA and related acylethanolamides in a sample of primary care patients with depression (n = 69) with those of healthy non-depressed patients (n = 47). At the time of admission to the study, 22 patients were under selective serotonin reuptake inhibitor (SSRI) antidepressant treatment and 47 patients were not receiving any type of intervention. In addition, plasma concentrations of the endocannabinoid 2-AG and two related monoacylglycerols were monitored. Plasma OEA concentrations were found to be elevated in depressed patients and to correlate with somatic symptoms of depression. Plasma concentrations of both, AEA and 2-AG, were found to be elevated also in depressed patients. Further analysis demonstrated that the elevation observed in the plasma concentrations of both, OEA and 2-AG, was associated to SSRI antidepressant therapy at the time of recruitment. Further clinical research is needed to understand whether SSRI-induced elevations in OEA levels contribute to the response to SSRI in depressed patients as described in preclinical models.

Topics: Adult; Antidepressive Agents; Arachidonic Acids; Depression; Endocannabinoids; Ethanolamines; Female; Healthy Volunteers; Humans; Male; Middle Aged; Monoglycerides; Oleic Acids; Polyunsaturated Alkamides; Primary Health Care; Selective Serotonin Reuptake Inhibitors

The endocannabinoid (eCB) system is implicated in the pathophysiology of depression and is responsive to acute exercise in healthy adults.. We aimed to describe acute changes in serum eCB across a prescribed moderate (MOD) and a self-selected/preferred (PREF) intensity exercise session in women with major depressive disorder (MDD) and determine relationships between changes in eCB and mood states.. Women with MDD (n = 17) exercised in separate sessions for 20 min on a cycle ergometer at both MOD or PREF in a within-subjects design. Blood was drawn before and within 10 min after exercise. Serum concentrations of eCB (anandamide [AEA], 2-arachidonoylglycerol) and related lipids (palmitoylethanolamine, oleoylethanolamine, 2-oleoylglycerol) were quantified using stable isotope-dilution, liquid chromatography/mass spectrometry/mass spectrometry. The profile of mood states and state-trait anxiety inventory (state only) were completed before, 10 min and 30 min postexercise.. Significant elevations in AEA (P = 0.013) and oleoylethanolamine (P = 0.024) occurred for MOD (moderate effect sizes: Cohen's d = 0.58 and 0.41, respectively). Significant (P < 0.05) moderate negative associations existed between changes in AEA and mood states for MOD at 10 min (depression, confusion, fatigue, total mood disturbance [TMD] and state anxiety) and 30 min postexercise (confusion, TMD and state anxiety). Significant (P < 0.05) moderate negative associations existed between 2-arachidonoylglycerol and mood states at 10 min (depression and confusion) and 30 min postexercise (confusion and TMD). Changes in eCB or related lipids or eCB-mood relationships were not found for PREF.. Given the broad, moderate-strength relationships between improvements in mood states and eCB increases after MOD, it is plausible that the eCB system contributes to the mood-enhancing effects of prescribed acute exercise in MDD. Alternative mechanisms are likely involved in the positive mood state effects of preferred exercise.

Topics: Adult; Affect; Amides; Arachidonic Acids; Depressive Disorder, Major; Endocannabinoids; Ethanolamines; Exercise; Female; Glycerides; Humans; Middle Aged; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides

The endocannabinoid (eCB) system is a modulatory system that is both altered by stress and mediates the effects of acute stress, including contributing to restoration of homeostasis. Earlier studies suggest that circulating eCBs are dysregulated in adults with post-traumatic stress disorder (PTSD); however, it is not known whether circulating eCBs remain responsive to stress. The purpose of this study was to examine eCB and psychological responses to physical (exercise) and psychosocial (Trier Social Stress Test) stressors, using a randomized, counterbalanced procedure in adults with PTSD and healthy controls (N = 20, mean age = 24, SD = 7 yrs). Results from mixed-design, repeated measures ANOVAs revealed significant increases (p <  .05) in N-arachidonoylethanolamine (AEA) and oleoylethanolamide (OEA) following exercise and psychosocial stress in both groups. However, only the control group exhibited a significant increase (p < .05) in 2-arachidonoylglycerol (2-AG) following exercise and psychosocial stress exposure. These data extend our current understanding of circulating eCB responsiveness in PTSD, and provide preliminary evidence to suggest that the eCB system is hypoactive in PTSD following exposure to physical and psychosocial stressors.

Topics: Adult; Arachidonic Acids; Chronic Disease; Endocannabinoids; Exercise; Glycerides; Humans; Male; Oleic Acids; Polyunsaturated Alkamides; Stress Disorders, Post-Traumatic; Stress, Psychological; Young Adult

The determination of endocannabinoids and endocannabinoid-like substances in biological human samples is a vibrant field of research with great significance due to postulated relevance of these substances in diseases such as Alzheimer's disease, multiple sclerosis, cancer and cardiovascular diseases. For a possible use as biomarker in early prediction or diagnosis of a disease as well as examination of a successful treatment, the valid determination of the analytes in common accessible human samples, such as plasma or serum, is of great importance. A method for the determination of arachidonoyl ethanolamide, oleoyl ethanolamide, palmitoyl ethanolamide, 1-arachidonoyl glycerol and 2-arachidonoyl glycerol in human K3EDTA plasma using liquid-liquid-extraction in combination with liquid chromatography-tandem-mass spectrometry has been developed and validated for the quantification of the aforementioned analytes. Particular emphasis was placed on the chromatographic separation of the isomers 1-arachidonoyl glycerol and 2-arachidonoyl glycerol, arachidonoyl ethanolamide and O-arachidonoyl ethanolamine (virodhamine) as well as oleoyl ethanolamide and vaccenic acid ethanolamide. During the validation process, increasing concentrations of 1-arachidonoyl glycerol and 2-arachidonoyl glycerol while storing plasma samples were observed. In-depth investigation of pre-analytical sample handling revealed rising concentrations for both analytes in plasma and for arachidonoyl ethanolamide, oleoyl ethanolamide and palmitoyl ethanolamide in whole blood, dependent on the period and temperature of storage. Prevention of the increase in concentration was not possible, raising the question whether human K3EDTA plasma is suitable for the determination of endocannabinoids and endocannabinoid-like substances. Especially the common practice to calculate the concentration of 2-arachidonoyl glycerol as sum of 1-arachidonoyl glycerol and 2-arachidonoyl glycerol is highly questionable because the concentrations of both analytes increase unequally while storing the plasma samples in the fridge.

Topics: Amides; Anticoagulants; Arachidonic Acids; Chromatography, High Pressure Liquid; Edetic Acid; Endocannabinoids; Ethanolamines; Glycerides; Humans; Liquid-Liquid Extraction; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Specimen Handling; Tandem Mass Spectrometry

Toll-like receptor (TLR)3 is a key component of the innate immune response to viral infection. The present study firstly examined whether sex differences exist in TLR3-induced inflammatory, endocrine, and sickness responses. The data revealed that TLR3-induced expression of interferon- or NFkB-inducible genes (IFN-α/β, IP-10, or TNF-α), either peripherally (spleen) or centrally (hypothalamus), did not differ between male and female rats, with the exception of TLR3-induced IFN-α expression in the spleen of female, but not male, rats 8 hr post TLR3 activation. Furthermore, TLR3 activation increased plasma corticosterone levels, induced fever, and reduced locomotor activity and body weight - effects independent of sex. Thus, the acute-phase inflammatory, endocrine, and sickness responses to TLR3 activation exhibit minimal sex-related differences. A further aim of this study was to examine whether enhancing endocannabinoid tone - namely, 2-arachidonylglycerol (2-AG) or N-arachidonoylethanolamine (AEA), exhibited similar effects on TLR3-induced inflammatory responses in male versus female rats. Systemic administration of the monoacylglycerol lipase (MAGL) inhibitor MJN110 and subsequent increases in 2-AG levels did not alter the TLR3-induced increase in IP-10, IRF7, or TNF-α expression in the spleen or the hypothalamus of male or female rats. In contrast, the fatty acid amide hydrolase (FAAH) inhibitor URB597 increased levels of AEA and related N-acylethanolamines, an effect associated with the attenuation of TLR3-induced inflammatory responses in the hypothalamus, but not the spleen, of male and female rats. These data support a role for FAAH, but not MAGL, substrates in the modulation of TLR3-induced neuroinflammatory responses, effects independent of sex.

Topics: Amides; Amidohydrolases; Animals; Arachidonic Acids; Body Temperature; Carbamates; Chemokine CXCL10; Corticosterone; Endocannabinoids; Estradiol; Ethanolamines; Female; Glycerides; Immunologic Factors; Interferons; Male; Monoacylglycerol Lipases; NF-kappa B; Oleic Acids; Palmitic Acids; Poly I-C; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Sex Factors; Signal Transduction; Succinimides; Toll-Like Receptor 3

Adolescence is characterized by endocannabinoid (ECB)-dependent refinement of neural circuits underlying emotion, learning, and motivation. As a result, adolescent cannabinoid receptor stimulation (ACRS) with phytocannabinoids or synthetic agonists like "Spice" cause robust and persistent changes in both behavior and circuit architecture in rodents, including in reward-related regions like medial prefrontal cortex and nucleus accumbens (NAc).. Here, we examine persistent effects of ACRS with the cannabinoid receptor 1/2 specific agonist WIN55-212,2 (WIN; 1.2 mg/kg/day, postnatal day (PD) 30-43), on natural reward-seeking behaviors and ECB system function in adult male Long Evans rats (PD 60+).. WIN ACRS increased palatable food intake, and altered attribution of incentive salience to food cues in a sign-/goal-tracking paradigm. ACRS also blunted hunger-induced sucrose intake, and resulted in increased anandamide and oleoylethanolamide levels in NAc after acute food restriction not seen in controls. ACRS did not affect food neophobia or locomotor response to a novel environment, but did increase preference for exploring a novel environment.. These results demonstrate that ACRS causes long-term increases in natural reward-seeking behaviors and ECB system function that persist into adulthood, potentially increasing liability to excessive natural reward seeking later in life.

Topics: Animals; Arachidonic Acids; Behavior, Animal; Benzoxazines; Cannabinoids; Eating; Endocannabinoids; Male; Morpholines; Motivation; Naphthalenes; Nucleus Accumbens; Oleic Acids; Polyunsaturated Alkamides; Rats; Rats, Long-Evans; Rats, Sprague-Dawley; Reward

Enhancing endogenous cannabinoid (eCB) signaling has been considered as a potential strategy for the treatment of stress-related conditions. Fatty acid amide hydrolase (FAAH) represents the primary degradation enzyme of the eCB anandamide (AEA), oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). This study describes a potent reversible FAAH inhibitor, SSR411298. The drug acts as a selective inhibitor of FAAH, which potently increases hippocampal levels of AEA, OEA and PEA in mice. Despite elevating eCB levels, SSR411298 did not mimic the interoceptive state or produce the behavioral side-effects (memory deficit and motor impairment) evoked by direct-acting cannabinoids. When SSR411298 was tested in models of anxiety, it only exerted clear anxiolytic-like effects under highly aversive conditions following exposure to a traumatic event, such as in the mouse defense test battery and social defeat procedure. Results from experiments in models of depression showed that SSR411298 produced robust antidepressant-like activity in the rat forced-swimming test and in the mouse chronic mild stress model, restoring notably the development of inadequate coping responses to chronic stress. This preclinical profile positions SSR411298 as a promising drug candidate to treat diseases such as post-traumatic stress disorder, which involves the development of maladaptive behaviors.

Topics: Acute Disease; Amides; Amidohydrolases; Animals; Anti-Anxiety Agents; Anxiety Disorders; Arachidonic Acids; Cannabinoid Receptor Agonists; Carbamates; Chronic Disease; Dioxanes; Endocannabinoids; Enzyme Inhibitors; Ethanolamines; Female; Gene Expression; Male; Mice; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Rats, Sprague-Dawley; Receptors, Cannabinoid; Stress, Psychological

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

Inhibition and genetic deletion of fatty acid-binding proteins (FABPs) 5 and 7 have been shown to increase the levels of the endocannabinoid anandamide as well as the related N-acylethanolamine's palmitoylethanolamide and oleoylethanolamide. This study examined the role of these FABPs on forced-swim (FS) behavior and on sucrose consumption in two experiments: (experiment 1) using wild-type (WT) mice treated with the FABP inhibitor SBFI26 or vehicle and (experiment 2) using WT and FABP5/7 deficient mice. Results from experiment 1 showed that acute treatment with SBFI26 did not have any effect on sucrose intake or FS behavior in mice. In experiment 2, male and female FABP5/7 deficient mice showed significant increases in sucrose consumption (25 and 21%, respectively) compared with their WT counterparts. In addition, immobility time during the FS was decreased by 27% in both male and female FABP5/7 knockout mice compared with their WT counterparts. The fact that such differences were seen between the acute pharmacological approach and the genetic approach (gene deletion) of FABP needs to be further investigated. The function of FABPs and their specific effects on endocannabinoid anandamide, oleoylethanolamide, and palmitoylethanolamide may play an important role in the development of reward and mood behaviors and could provide opportunities for potential therapeutic targets.

Topics: Analysis of Variance; Animals; Arachidonic Acids; Cyclobutanes; Dicarboxylic Acids; Endocannabinoids; Exploratory Behavior; Fatty Acid-Binding Protein 7; Fatty Acid-Binding Proteins; Female; Food Preferences; Freezing Reaction, Cataleptic; Gene Deletion; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neoplasm Proteins; Oleic Acids; Polyunsaturated Alkamides; Sex Factors; Sucrose; Swimming

Topics: Amides; Animals; Anorexia Nervosa; Arachidonic Acids; Dietary Fats; Dietary Supplements; Endocannabinoids; Ethanolamines; Feeding Behavior; Humans; Lipids; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Rats; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2

The endocannabinoid system plays a substantial role in analgesia.. To analyze N-arachidonoylethanolamine (AEA), N-oleoylethanolamine (OEA), linoleoyl ethanolamide (LEA), α-linoleoyl ethanolamine (α-LNEA), N-palmitoylethanolamine (PEA) and N-stearoyl ethanolamine (SEA) in two groups of patients having chronic pancreatic diseases.. Twenty-six patients with chronic pancreatitis, 26 patients with pancreatic ductal adenocarcinoma and 36 healthy subjects were studied. The visual analogic scale (VAS) was used for assessing pain immediately before the venipuncture to obtain blood in all subjects. Six endocannabinoids were measured in serum of the patients enrolled.. Only OEA, LEA and PEA serum levels were significantly higher in patients with pain as compared to those without. Using the cutoff values, the sensitivity and specificity of the various endocannabinoids in evaluating pain in patients with chronic pancreatitis and in those with pancreatic ductal adenocarcinoma were: 44.2% and 95.6% for AEA, 83.7% and 73.3% for LEA, 88.4% and 91.1% for LNEA, 81.4% and 82.2% for OEA, 81.4% and 88.9% for PEA, 86.0% and 88.9% for SEA, respectively.. Endocannabinoids are not useful in assessing pain in patients with chronic pancreatic diseases and they cannot replace a simple method such as VAS for assessing the pain and its intensity.

Topics: Abdominal Pain; Adolescent; Adult; Aged; Aged, 80 and over; Amides; Arachidonic Acids; Cancer Pain; Carcinoma, Pancreatic Ductal; Case-Control Studies; Endocannabinoids; Ethanolamines; Female; Humans; Linoleic Acids; Male; Middle Aged; Oleic Acids; Pain Measurement; Palmitic Acids; Pancreatic Neoplasms; Pancreatitis, Chronic; Polyunsaturated Alkamides; Predictive Value of Tests; ROC Curve; Stearic Acids; Young Adult

Cannabis-based drugs have been shown to be effective in inflammatory diseases. A number of endocannabinoids including N- arachidonoylethanolamide (anandamide, AEA) and 2-arachidonyl glycerol (2-AG) with activity at the cannabinoid receptors (CBR) CBR1 and CBR2, have been identified. Other structurally related endogenous fatty acid compounds such as oleoylethanolamide (OEA) and palmitoyl ethanolamide (PEA) have been identified in biological tissues. These compounds do not bind to CBR but might be involved in facilitating the actions of directly acting endocannabinoids and thus are commonly termed "entourage" compounds due to their ability to modulate the endocannabinoid system. The aim of this study was to evaluate the presence of endocannabinoids and entourage compounds in the synovial fluid of dogs with osteoarthritis subjected to arthrotomy of the knee joint. Cytokines and cytology were studied as well.. AEA, 2-AG, OEA and PEA were all present in the synovial fluid of arthritic knees and in the contralateral joints; in addition, a significant increase of OEA and 2AG levels were noted in SF from OA knees when compared to the contralateral joints.. The identification and quantification of endocannabinoids and entourage compounds levels in synovial fluids from dogs with OA of the knee is reported for the first time. Our data are instrumental for future studies involving a greater number of dogs. Cannabinoids represent an emerging and innovative pharmacological tool for the treatment of OA and further studies are warranted to evaluate the effectiveness of cannabinoids in veterinary medicine.

Topics: Animals; Arachidonic Acids; Dog Diseases; Dogs; Endocannabinoids; Ethanolamines; Female; Glycerides; Male; Oleic Acids; Osteoarthritis, Knee; Palmitic Acids; Pilot Projects; Polyunsaturated Alkamides; Synovial Fluid

Endocannabinoids are critical for rewarding behaviors such as eating, physical exercise, and social interaction. The role of endocannabinoids in mammalian sexual behavior has been suggested because of the influence of cannabinoid receptor agonists and antagonists on rodent sexual activity. However, the involvement of endocannabinoids in human sexual behavior has not been studied.. To investigate plasma endocannabinoid levels before and after masturbation in healthy male and female volunteers.. Plasma levels of the endocannabinoids 2-arachidonoylglycerol (2-AG), anandamide, the endocannabinoid-like lipids oleoyl ethanolamide and palmitoyl ethanolamide, arachidonic acid, and cortisol before and after masturbation to orgasm.. In study 1, endocannabinoid and cortisol levels were measured before and after masturbation to orgasm. In study 2, masturbation to orgasm was compared with a control condition using a single-blinded, randomized, 2-session crossover design.. In study 1, masturbation to orgasm significantly increased plasma levels of the endocannabinoid 2-AG, whereas anandamide, oleoyl ethanolamide, palmitoyl ethanolamide, arachidonic acid, and cortisol levels were not altered. In study 2, only masturbation to orgasm, not the control condition, led to a significant increase in 2-AG levels. Interestingly, we also found a significant increase of oleoyl ethanolamide after masturbation to orgasm in study 2.. Endocannabinoids might play an important role in the sexual response cycle, leading to possible implications for the understanding and treatment of sexual dysfunctions.. We found an increase of 2-AG through masturbation to orgasm in 2 studies including a single-blinded randomized design. The exact role of endocannabinoid release as part of the sexual response cycle and the biological significance of the finding should be studied further. Cannabis and other drug use and the attainment of orgasm were self-reported in the present study.. Our data indicate that the endocannabinoid 2-AG is involved in the human sexual response cycle and we hypothesize that 2-AG release plays a role in the rewarding consequences of sexual arousal and orgasm. Fuss J, Bindila L, Wiedemann K, et al. Masturbation to Orgasm Stimulates the Release of the Endocannabinoid 2-Arachidonoylglycerol in Humans. J Sex Med 2017;14:1372-1379.

Topics: Arachidonic Acids; Cannabinoid Receptor Agonists; Cannabinoid Receptor Modulators; Endocannabinoids; Female; Glycerides; Humans; Male; Masturbation; Oleic Acids; Orgasm; Polyunsaturated Alkamides

Exposure to chlorpyrifos (CPF) during the late preweanling period in rats inhibits the endocannabinoid metabolizing enzymes fatty acid hydrolase (FAAH) and monoacylglycerol lipase (MAGL), resulting in accumulation of their respective substrates anandamide (AEA) and 2-arachidonylglycerol (2-AG). This occurs at 1.0mg/kg, but at a lower dosage (0.5mg/kg) only FAAH and AEA are affected with no measurable inhibition of either cholinesterase (ChE) or MAGL. The endocannabinoid system plays a vital role in nervous system development and may be an important developmental target for CPF. The endocannabinoid system plays an important role in the regulation of anxiety and, at higher dosages, developmental exposure to CPF alters anxiety-like behavior. However, it is not clear whether exposure to low dosages of CPF that do not inhibit ChE will cause any persistent effects on anxiety-like behavior. To determine if this occurs, 10-day old rat pups were exposed daily for 7 days to either corn oil or 0.5, 0.75, or 1.0mg/kg CPF by oral gavage. At 12h following the last CPF administration, 1.0mg/kg resulted in significant inhibition of FAAH, MAGL, and ChE, whereas 0.5 and 0.75mg/kg resulted in significant inhibition of only FAAH. AEA levels were significantly elevated in all three treatment groups as were palmitoylethanolamide and oleoylethanolamide, which are also substrates for FAAH. 2-AG levels were significantly elevated by 0.75 and 1.0mg/kg but not 0.5mg/kg. On day 25, the latency to emerge from a dark container into a highly illuminated novel open field was measured as an indicator of anxiety. All three CPF treatment groups spent significantly less time in the dark container prior to emerging as compared to the control group, suggesting a decreased level of anxiety. This demonstrates that repeated preweanling exposure to dosages of CPF that do not inhibit brain ChE can induce a decline in the level of anxiety that is detectable during the early postweanling period.

Topics: Aging; Analysis of Variance; Animals; Animals, Newborn; Anxiety; Arachidonic Acids; Chlorpyrifos; Cholinesterase Inhibitors; Cholinesterases; Cohort Studies; Disease Models, Animal; Endocannabinoids; Female; Male; Oleic Acids; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Sex Factors

Acylethanolamides are a family of endogenous lipid mediators that are involved in physiological and behavioral processes associated with addiction. Recently, oleoylethanolamide (OEA) has been reported to reduce alcohol intake and relapse in rodents but the contribution of OEA and other acylethanolamides in alcohol addiction in humans is unknown. The present study is aimed to characterize the plasma acylethanolamides in alcohol dependence. Seventy-nine abstinent alcohol-dependent subjects (27 women) recruited from outpatient treatment programs and age-/sex-/body mass-matched healthy volunteers (28 women) were clinically assessed with the diagnostic interview PRISM according to the DSM-IV-TR after blood extraction for quantification of acylethanolamide concentrations in the plasma. Our results indicate that all acylethanolamides were significantly increased in alcohol-dependent patients compared with control subjects (p < 0.001). A logistic model based on these acylethanolamides was developed to distinguish alcohol-dependent patients from controls and included OEA, arachidonoylethanolamide (AEA) and docosatetraenoylethanolamide (DEA), providing a high discriminatory power according to area under the curve [AUC = 0.92 (95%CI: 0.87-0.96), p < 0.001]. Additionally, we found a significant effect of the duration of alcohol abstinence on the concentrations of OEA, AEA and DEA using a regression model (p < 0.05, p < 0.01 and p < 0.001, respectively), which was confirmed by a negative correlation (rho = -0.31, -0.40 and -0.44, respectively). However, acylethanolamides were not influenced by the addiction alcohol severity, duration of problematic alcohol use or diagnosis of psychiatric comorbidity. Our results support the preclinical studies and suggest that OEA, AEA and DEA are altered in alcohol-dependence during abstinence and that might act as potential markers for predicting length of alcohol abstinence.

Topics: Adult; Alcohol Abstinence; Alcoholism; Amides; Arachidonic Acids; Case-Control Studies; Dehydroepiandrosterone; Endocannabinoids; Ethanolamines; Female; Humans; Male; Middle Aged; Oleic Acids; Palmitic Acids; Polyethylene Glycols; Polyunsaturated Alkamides; Stearic Acids; Time Factors

Hedonic eating occurs independently from homeostatic needs prompting the ingestion of pleasurable foods that are typically rich in fat, sugar and/or salt content. In normal weight healthy subjects, we found that before hedonic eating, plasma levels of 2-arachidonoylglycerol (2-AG) were higher than before nonhedonic eating, and although they progressively decreased after food ingestion in both eating conditions, they were significantly higher in hedonic eating. Plasma levels of anandamide (AEA), oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), instead, progressively decreased in both eating conditions without significant differences. In this study, we investigated the responses of AEA, 2-AG, OEA and PEA to hedonic eating in obese individuals.. Peripheral levels of AEA, 2-AG, OEA and PEA were measured in 14 obese patients after eating favourite (hedonic eating) and non-favourite (nonhedonic eating) foods in conditions of no homeostatic needs.. Plasma levels of 2-AG increased after eating the favourite food, whereas they decreased after eating the non-favourite food, with the production of the endocannabinoid being significantly enhanced in hedonic eating. Plasma levels of AEA decreased progressively in nonhedonic eating, whereas they showed a decrease after the exposure to the favourite food followed by a return to baseline values after eating it. No significant differences emerged in plasma OEA and PEA responses to favourite and non-favourite food.. Present findings compared with those obtained in our previously studied normal weight healthy subjects suggest deranged responses of endocannabinoids to food-related reward in obesity.

Topics: Adult; Amides; Arachidonic Acids; Body Mass Index; Dietary Carbohydrates; Dietary Fats; Dietary Proteins; Endocannabinoids; Energy Intake; Ethanolamines; Feeding Behavior; Female; Glycerides; Humans; Male; Middle Aged; Nutritive Value; Obesity; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Satiation; Young Adult

The endocannabinoid system is dysregulated during obesity in tissues involved in the control of food intake and energy metabolism. We examined the effect of chronic exercise on the tissue levels of endocannabinoids (eCBs) and on the expression of genes coding for cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2) (Cnr1 and Cnr2, respectively) in the subcutaneous (SAT) and visceral adipose tissues and in the soleus and extensor digitorim longus (EDL) muscles, in rats fed with standard or high-fat diet. Twenty-eight male Wistar rats were placed on high-fat diet or standard diet (HFD and Ctl groups, respectively) during 12 weeks whereafter half of each group was submitted to an exercise training period of 12 weeks (HFD + training and Ctl + training). Tissue levels of eCBs were measured by LC-MS while expressions of genes coding for CB1 and CB2 receptors were investigated by qPCR. High-fat diet induced an increase in anandamide (AEA) levels in soleus and EDL (p < 0.02). In soleus of the HFD group, these changes were accompanied by elevated Cnr1 messenger RNA (mRNA) levels (p < 0.05). In EDL, exercise training allowed to reduce significantly this diet-induced AEA increase (p < 0.005). 2-Arachidonoylglycerol (2-AG) levels were decreased and increased by high-fat diet in SAT and EDL, respectively (p < 0.04), but not affected by exercise training. Unlike the HFD + training group, 2-AG levels in soleus were also decreased in the HFD group compared to Ctl (p < 0.04). The levels of eCBs and Cnr1 expression are altered in a tissue-specific manner following a high-fat diet, and chronic exercise reverses some of these alterations.

Topics: Amides; Animals; Arachidonic Acids; Body Composition; Diet, High-Fat; Endocannabinoids; Ethanolamines; Gene Expression Regulation; Glycerides; Hyperglycemia; Intra-Abdominal Fat; Male; Motor Activity; Muscle, Skeletal; Obesity; Oleic Acids; Organ Specificity; Palmitic Acids; Polyunsaturated Alkamides; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Subcutaneous Fat, Abdominal; TRPV Cation Channels; Weight Gain

Cannabinoids and modulators of the endocannabinoid system affect specific mechanisms that are critical to the establishment and development of endometriosis. The aim of this study was to measure the systemic levels of endocannabinoids and related mediators in women with and without endometriosis and to investigate whether such levels correlated with endometriosis-associated pain. Plasma and endometrial biopsies were obtained from women with a laparoscopic diagnosis of endometriosis (n = 27) and no endometrial pathology (n = 29). Plasma levels of endocannabinoids (N-arachidonoylethanolamine [AEA] and 2-arachidonoylglycerol [2-AG]) and related mediators (N-oleoylethanolamine [OEA] and N-palmitoylethanolamine [PEA]), messenger RNA expression of some of their receptors (cannabinoid receptor type 1 [CB1], CB2, transient receptor potential vanilloid type [TRPV1]), and the enzymes involved in the synthesis (N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D [NAPE-PLD]) and degradation (fatty acid amide hydrolase 1 [FAAH]) of AEA, OEA, and PEA were evaluated in endometrial stromal cells. The systemic levels of AEA, 2-AG, and OEA were elevated in endometriosis in the secretory phase compared to controls. The expression of CB1 was higher in secretory phase endometrial stromal cells of controls versus endometriosis. Similar expression levels of CB2, TRPV1, NAPE-PLD, and FAAH were detected in controls and endometriosis. Patients with moderate-to-severe dysmenorrhea and dyspareunia showed higher AEA and PEA levels than those with low-to-moderate pain symptoms, respectively. The association of increased circulating AEA and 2-AG with decreased local CB1 expression in endometriosis suggests a negative feedback loop regulation, which may impair the capability of these mediators to control pain. These preliminary data suggest that the pharmacological manipulation of the action or levels of these mediators may offer an alternative option for the management of endometriosis-associated pain.

Topics: Adult; Amides; Amidohydrolases; Arachidonic Acids; Endocannabinoids; Endometriosis; Ethanolamines; Female; Glycerides; Humans; Menstrual Cycle; Middle Aged; Oleic Acids; Palmitic Acids; Phospholipase D; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; RNA, Messenger; Stromal Cells; TRPV Cation Channels; Young Adult

The involvement of transient receptor vanilloid type-1 (TRPV1) channels in pain modulation by the brain remains understudied. The rostroventromedial medulla (RVM) plays a key role in conveying to the spinal cord pain modulatory influences triggered in higher brain centres, with co-existence of inhibitory (antinociceptive) and facilitatory (pronociceptive) effects. In spite of some reports of TRPV1 expression in the RVM, it remains unknown if endovanilloid signalling plays a direct role in local pain modulation. Here we used a model of diabetic neuropathy, the streptozotocin (STZ)-diabetic rat, to study the role of endovanilloid signalling in RVM-mediated pain modulation during chronic pain. Four weeks after diabetes induction, the levels of TRPV1 mRNA and fatty acid amide hydrolase (FAAH), a crucial enzyme for endovanilloid catabolism, in the RVM of STZ-diabetic rats were higher than control. The RVM of STZ-diabetic rats presented decreased levels of several TRPV1 endogenous ligands, namely anandamide (AEA), palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). Administration of capsaicin (a TRPV1 agonist) into the RVM decreased nociceptive behavioural responses in the inflammatory phase of the formalin test (phase 2). These findings suggest that diabetic neuropathy induces plastic changes of RVM endovanilloid signalling, indicating that TRPV1 may be a putative target for pain modulation in this chronic pain condition.

Topics: Amides; Amidohydrolases; Analgesics, Non-Narcotic; Animals; Arachidonic Acids; Capsaicin; Chronic Pain; Diabetes Mellitus, Experimental; Diabetic Neuropathies; Endocannabinoids; Ethanolamines; Formaldehyde; Male; Medulla Oblongata; Nociceptive Pain; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Rats, Wistar; RNA, Messenger; TRPV Cation Channels

Liver fatty acid-binding protein (FABP1, L-FABP) has high affinity for and enhances uptake of arachidonic acid (ARA, C20:4, n-6) which, when esterified to phospholipids, is the requisite precursor for synthesis of endocannabinoids (EC) such as arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG). The brain derives most of its ARA from plasma, taking up ARA and transporting it intracellularly via cytosolic fatty acid-binding proteins (FABPs 3,5, and 7) localized within the brain. In contrast, the much more prevalent cytosolic FABP1 is not detectable in the brain but is instead highly expressed in the liver. Therefore, the possibility that FABP1 outside the central nervous system may regulate brain AEA and 2-AG was examined in wild-type (WT) and FABP1 null (LKO) male mice. LKO increased brain levels of AA-containing EC (AEA, 2-AG), correlating with increased free and total ARA in brain and serum. LKO also increased brain levels of non-ARA that contain potentiating endocannabinoids (EC*) such as oleoyl ethanolamide (OEA), PEA, 2-OG, and 2-PG. Concomitantly, LKO decreased serum total ARA-containing EC, but not non-ARA endocannabinoids. LKO did not elicit these changes in the brain EC and EC* as a result of compensatory up-regulation of brain protein levels of enzymes in EC synthesis (NAPEPLD, DAGLα) or cytosolic EC chaperone proteins (FABPs 3, 5, 7, SCP-2, HSP70), or cannabinoid receptors (CB1, TRVP1). These data show for the first time that the non-CNS fatty acid-binding protein FABP1 markedly affected brain levels of both ARA-containing endocannabinoids (AEA, 2-AG) as well as their non-ARA potentiating endocannabinoids. Fatty acid-binding protein-1 (FABP-1) is not detectable in brain but instead is highly expressed in liver. The possibility that FABP1 outside the central nervous system may regulate brain endocannabinoids arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG) was examined in wild-type (WT) and FABP-1 null (LKO) male mice. LKO increased brain levels of arachidonic acid-containing endocannabinoids (AEA, 2-AG), correlating with increased free and total arachidonic acid in brain and serum. Read the Editorial Highlight for this article on page 371.

Topics: Animals; Arachidonic Acids; Brain; Endocannabinoids; Fatty Acid-Binding Proteins; Glycerides; Liver; Male; Mice, Inbred C57BL; Mice, Knockout; Oleic Acids; Polyunsaturated Alkamides

Anandamide (AEA) and N-oleoylethanolamine (OEA) are produced in the intestine and brain during fasting and satiety, respectively. Subsequently, AEA facilitates food intake via activation of cannabinoid type-1 receptors (CB1Rs) while OEA decreases food intake via activation of peroxisome proliferator-activated receptor-α (PPARα) and/or G-protein-coupled receptor 119 (GPR119). Neuronal activity in the gastrointestinal region of the autonomic insula (GI-Au-I) that rostrally adjoins the gustatory insula (Gu-I) increases during fasting, enhancing appetite while umami and sweet taste sensations in Gu-I enhances appetite in GI-Au-I, strongly suggesting the presence of a neural interaction between the Gu-I and GI-Au-I which changes depending on the concentrations of AEA and OEA. However, this possibility has never been investigated. In rat slice preparations, we demonstrate with voltage-sensitive dye imaging that activation of CB1Rs by AEA induces θ-rhythm oscillatory synchronization in the Gu-I which propagates into the GI-Au-I but stops at its caudal end, displaying an oscillatory coordination. The AEA-induced oscillation was abolished by a CB1R antagonist or OEA through activation of GPR119. Our results demonstrate that the neural coordination between the Gu-I and GI-Au-I is generated or suppressed by the opposing activities between CB1R and GPR119. This mechanism may be involved in the feeding behavior based on taste recognition.

Topics: Alcohol Oxidoreductases; Animals; Appetite; Arachidonic Acids; Cerebral Cortex; Eating; Endocannabinoids; Ethanolamines; Fasting; Feeding Behavior; Female; Gene Expression; Intestines; Male; Neurons; Oleic Acids; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptors, G-Protein-Coupled; Satiation; Signal Transduction; Taste Perception; Theta Rhythm

The endocannabinoid system has been considered as a target for pharmacological intervention. Accordingly, inhibition of fatty acid amide hydrolase (FAAH), a degrading enzyme of the endocannabinoids N-arachidonoylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG) as well as of the endocannabinoid-like substances N-oleoylethanolamine (OEA) and N-palmitoylethanolamine (PEA), can cause augmented endogenous cannabinoid tone. Using liquid chromatography coupled with positive electrospray ionisation mass spectrometry, we herein describe a method to simultaneously quantify levels of AEA, OEA, PEA and 2-AG in cultured cells. The procedure was developed according to the FDA guidelines for bioanalytical methods validation. The limits of quantification (LOQs) were 0.05 pmol for AEA, 0.09 pmol for OEA, 0.10 pmol for PEA and 0.80 pmol for 2-AG when molecular ion monitoring was used. In H460 human lung carcinoma cells, basal levels of all four analytes ranged between 2 and 17 pmol mg(-1) protein with PEA showing the lowest and OEA the highest concentrations. Endocannabinoid levels observed in mesenchymal stem cells were of the same order of magnitude when compared to those in H460 human lung carcinoma cells.

Topics: Amides; Arachidonic Acids; Cell Line, Tumor; Chromatography, Liquid; Endocannabinoids; Ethanolamines; Glycerides; Humans; Limit of Detection; Lung Neoplasms; Mass Spectrometry; Mesenchymal Stem Cells; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Reproducibility of Results

A dysregulation of reward mechanisms was suggested in the pathophysiology of anorexia nervosa (AN), but the role of the endogenous mediators of reward has been poorly investigated. Endocannabinoids, including anandamide and 2-arachidonoylglycerol, and the endocannabinoid-related compounds oleoylethanolamide and palmitoylethanolamide modulate food-related and unrelated reward. Hedonic eating, which is the consumption of food just for pleasure and not homeostatic need, is a suitable paradigm to explore food-related reward.. We investigated responses of endocannabinoids and endocannabinoid-related compounds to hedonic eating in AN.. Peripheral concentrations of anandamide, 2-arachidonoylglycerol, oleoylethanolamide, and palmitoylethanolamide were measured in 7 underweight and 7 weight-restored AN patients after eating favorite and nonfavorite foods in the condition of no homeostatic needs, and these measurements were compared with those of previously studied healthy control subjects.. 1) In healthy controls, plasma 2-arachidonoylglycerol concentrations decreased after both types of meals but were significantly higher in hedonic eating; in underweight AN patients, 2-arachidonoylglycerol concentrations did not show specific time patterns after eating either favorite or nonfavorite foods, whereas in weight-restored patients, 2-arachidonoylglycerol concentrations showed similar increases with both types of meals. 2) Anandamide plasma concentrations exhibited no differences in their response patterns to hedonic eating in the groups. 3) Compared with 2-arachidonoylglycerol, palmitoylethanolamide concentrations exhibited an opposite response pattern to hedonic eating in healthy controls; this pattern was partially preserved in underweight AN patients but not in weight-restored ones. 4) Like palmitoylethanolamide, oleoylethanolamide plasma concentrations tended to be higher in nonhedonic eating than in hedonic eating in healthy controls; moreover, no difference between healthy subjects and AN patients was observed for food-intake-induced changes in oleoylethanolamide concentrations.. These data confirm that endocannabinoids and endocannabinoid-related compounds are involved in food-related reward and suggest a dysregulation of their physiology in AN. This trial was registered at ISRCTN.org as ISRCTN64683774.

Topics: Adolescent; Adult; Amides; Anorexia Nervosa; Arachidonic Acids; Case-Control Studies; Endocannabinoids; Energy Intake; Ethanolamines; Female; Glycerides; Healthy Volunteers; Humans; Male; Meals; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Retrospective Studies; Reward; Thinness; Young Adult

Endocannabinoids alter permeability at various epithelial barriers, and cannabinoid receptors and endocannabinoid levels are elevated by stroke, with potential neuroprotective effects. We therefore explored the role of endocannabinoids in modulating blood-brain barrier (BBB) permeability in normal conditions and in an ischaemia/reperfusion model.. Human brain microvascular endothelial cell and astrocyte co-cultures modelled the BBB. Ischaemia was modelled by oxygen-glucose deprivation (OGD) and permeability was measured by transepithelial electrical resistance. Endocannabinoids or endocannabinoid-like compounds were assessed for their ability to modulate baseline permeability or OGD-induced hyperpermeability. Target sites of action were investigated using receptor antagonists and subsequently identified with real-time PCR.. Anandamide (10 μM) and oleoylethanolamide (OEA, 10 μM) decreased BBB permeability (i.e. increased resistance). This was mediated by cannabinoid CB2 receptors, transient receptor potential vanilloid 1 (TRPV1) channels, calcitonin gene-regulated peptide (CGRP) receptor (anandamide only) and PPARα (OEA only). Application of OEA, palmitoylethanolamide (both PPARα mediated) or virodhamine (all 10 μM) decreased the OGD-induced increase in permeability during reperfusion. 2-Arachidonoyl glycerol, noladin ether and oleamide did not affect BBB permeability in normal or OGD conditions. N-arachidonoyl-dopamine increased permeability through a cytotoxic mechanism. PPARα and γ, CB1 receptors, TRPV1 channels and CGRP receptors were expressed in both cell types, but mRNA for CB2 receptors was only present in astrocytes.. The endocannabinoids may play an important modulatory role in normal BBB physiology, and also afford protection to the BBB during ischaemic stroke, through a number of target sites.

Topics: Arachidonic Acids; Astrocytes; Blood-Brain Barrier; Cells, Cultured; Coculture Techniques; Electric Impedance; Endocannabinoids; Endothelial Cells; Humans; Oleic Acids; Permeability; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB2; Reperfusion Injury; RNA, Messenger

Anandamide (AEA) is an endocannabinoid (EC) that modulates multiple functions in the CNS and that is released in areas of injury, exerting putative neuroprotective actions. In the present study, we have used intravital microscopy to analyze the role of the EC system in the glial response against an acute insult. Our data show that AEA modulates astroglial function in vivo by increasing connexin-43 hemichannel (HC) activity. Furthermore, the genetic inactivation of the AEA-degrading enzyme, fatty acid amide hydrolase (FAAH), also increased HC activity and enhanced the microglial response against an acute injury to the brain parenchyma, effects that were mediated by cannabinoid CB1 receptors. The contribution of ATP released through an astrocytic HC was critical for the microglial response, as this was prevented by the use of the HC blocker flufenamic acid and by apyrase. As could be expected, brain concentrations of AEA, palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) were elevated in FAAH-null mice, while 2-arachidonoylglycerol (2-AG) concentrations remained unaltered. In summary, these findings demonstrate that AEA modifies glial functions by promoting an enhanced pro-inflammatory glial response in the brain.

Topics: Adenosine Triphosphate; Amides; Amidohydrolases; Animals; Anti-Inflammatory Agents; Apyrase; Arachidonic Acids; Astrocytes; Brain; Brain Injuries; Connexin 43; Disease Models, Animal; Endocannabinoids; Ethanolamines; Flufenamic Acid; Glycerides; Lasers; Mice; Mice, Knockout; Mice, Transgenic; Microglia; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1

Regenerative activity in tissues of mesenchymal origin depends on the migratory potential of mesenchymal stem cells (MSCs). The present study focused on inhibitors of the enzyme fatty acid amide hydrolase (FAAH), which catalyzes the degradation of endocannabinoids (anandamide, 2-arachidonoylglycerol) and endocannabinoid-like substances (N-oleoylethanolamine, N-palmitoylethanolamine). Boyden chamber assays, the FAAH inhibitors, URB597 and arachidonoyl serotonin (AA-5HT), were found to increase the migration of human adipose-derived MSCs. LC-MS analyses revealed increased levels of all four aforementioned FAAH substrates in MSCs incubated with either FAAH inhibitor. Following addition to MSCs, all FAAH substrates mimicked the promigratory action of FAAH inhibitors. Promigratory effects of FAAH inhibitors and substrates were causally linked to activation of p42/44 MAPKs, as well as to cytosol-to-nucleus translocation of the transcription factor, PPARα. Whereas PPARα activation by FAAH inhibitors and substrates became reversed upon inhibition of p42/44 MAPK activation, a blockade of PPARα left p42/44 MAPK phosphorylation unaltered. Collectively, these data demonstrate FAAH inhibitors and substrates to cause p42/44 MAPK phosphorylation, which subsequently activates PPARα to confer increased migration of MSCs. This novel pathway may be involved in regenerative effects of endocannabinoids whose degradation could be a target of pharmacological intervention by FAAH inhibitors.

Topics: Adipose Tissue; Amides; Amidohydrolases; Arachidonic Acids; Benzamides; Carbamates; Cell Movement; Cells, Cultured; Endocannabinoids; Enzyme Inhibitors; Ethanolamines; Glycerides; Humans; Mesenchymal Stem Cells; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; PPAR alpha; Receptor, Cannabinoid, CB1; Serotonin

The dysregulation of the endocannabinoid system is associated with cardiometabolic complications of obesity. Allelic variants in coding genes for this system components may contribute to differences in the susceptibility to obesity and related health hazards. These data have mostly been shown in Caucasian populations and in severely obese individuals. We investigated a multiethnic Brazilian population to study the relationships among the polymorphism 385C>A in an endocannabinoid degrading enzyme gene (FAAH), endocannabinoid levels and markers of cardiometabolic risk. Fasting plasma levels of endocannabinoids and congeners (anandamide, 2-arachidonoylglycerol, N-oleoylethanolamide and N-palmitoylethanolamide) were measured by liquid chromatography-mass spectrometry in 200 apparently healthy individuals of both genders with body mass indices from 22.5 ± 1.8 to 35.9 ± 5.5 kg/m2 (mean ± 1 SD) and ages between 18 and 60 years. All were evaluated for anthropometric parameters, blood pressure, metabolic variables, homeostatic model assessment of insulin resistance (HOMA-IR), adiponectin, leptin, C-reactive protein, and genotyping. The endocannabinoid levels increased as a function of obesity and insulin resistance. The homozygous genotype AA was associated with higher levels of anandamide and lower levels of adiponectin versus wild homozygous CC and heterozygotes combined. The levels of anandamide were independent and positively associated with the genotype AA position 385 of FAAH, C-reactive protein levels and body mass index. Our findings provide evidence for an endocannabinoid-related phenotype that may be identified by the combination of circulating anandamide levels with genotyping of the FAAH 385C>A; this phenotype is not exclusive to mono-ethnoracial populations nor to individuals with severe obesity.

Topics: Adiponectin; Adult; Amides; Amidohydrolases; Anthropometry; Arachidonic Acids; Blood Pressure; Body Mass Index; Brazil; Endocannabinoids; Ethanolamines; Ethnicity; Female; Genotype; Glycerides; Homeostasis; Homozygote; Humans; Insulin Resistance; Male; Middle Aged; Obesity; Oleic Acids; Palmitic Acids; Phenotype; Polymorphism, Genetic; Polyunsaturated Alkamides; Prevalence; Risk Factors

To measure the circulating levels of endocannabinoids and related molecules at fasting, after acute hyperinsulinemia and after weight loss in insulin sensitive vs. insulin resistant obese postmenopausal women.. The sample consisted of 30 obese postmenopausal women (age: 58.9 ± 5.2 yrs; BMI: 32.9 ± 3.6 kg/m(2) ). Subjects underwent a 3-hour hyperinsulinaemic-euglycaemic clamp (HEC) (glucose disposal rate (M-value): 10.7 ± 3.3 mg min(-1) kg(-1) FFM) and 6-month weight loss intervention. Participants were classified as insulin sensitive obese (ISO) or insulin resistant obese (IRO) based on a predefined cutoff. Plasma levels of the endocannabinoids, anandamide (AEA), 2-arachidonoylglycerol (2-AG), and of the AEA-related compounds, palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), were measured by liquid chromatography-mass spectrometry.. IRO presented higher levels of 2-AG (P < 0.05) independently of the HEC and weight loss, whereas the HEC had an independent inhibitory effect on AEA, PEA, and OEA levels (P < 0.05) in both groups. Furthermore, there was an independent stimulatory effect of weight loss only on PEA levels in both groups (P < 0.05).. This study is the first to show that higher circulating levels of the endocannabinoid 2-AG are found in IRO compared to ISO postmenopausal women, and that weight loss is associated with an increase in PEA, a PPAR-α ligand.

Topics: Amides; Arachidonic Acids; Body Composition; Body Mass Index; Cholesterol, HDL; Cholesterol, LDL; Cohort Studies; Endocannabinoids; Ethanolamines; Female; Glucose Clamp Technique; Glycerides; Humans; Hyperinsulinism; Insulin; Insulin Resistance; Middle Aged; Obesity; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Postmenopause; Triglycerides; Weight Loss

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

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

Cannabinoids are effective antiemetics and the "endogenous cannabinoids" (endocannabinoids) are thought to modulate emesis in both humans and animal models. Endocannabinoids, their receptors and their metabolising enzymes are present in peripheral blood and a reduction in blood endocannabinoid concentration has been observed in individuals with excessive nausea and vomiting following parabolic flight manoeuvres. We tested the hypothesis that plasma endocannabinoid levels are similarly perturbed in women with hyperemesis gravidarum (HG), a condition where the aetiopathogenesis is still unknown, compared to normal pregnant controls.. Plasma N-arachidonoylethanolamine (anandamide), N-oleoylethanolamide and N-palmitoylethanolamide were quantified in women with HG (n = 15) and matched normal pregnant controls (n = 30) using UHPLC-ESI-MS/MS utilising an isotope dilution method and selective ion monitoring.. No significant differences in anandamide, oleoylethanolamide and palmitoylethanolamide levels were observed between the two groups. There were no significant correlations between these endocannabinoids and plasma haematocrit and serum urea or sodium concentrations.. These results would suggest that either the circulating endocannabinoids quantified may not be key modulating factors in HG or that the expected endocannabinoid system response to the stress induced by nausea and vomiting of early pregnancy remain unchanged in women with HG.

Topics: Adult; Amides; Arachidonic Acids; Case-Control Studies; Endocannabinoids; Ethanolamines; Female; Hematocrit; Humans; Hyperemesis Gravidarum; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Pregnancy; Sodium Chloride; Urea; Young Adult

Preclinical investigations have demonstrated that drugs of abuse alter the levels of lipid-based signalling molecules, including endocannabinoids (eCBs) and N-acylethanolamines (NAEs), in the rodent brain. In addition, several drugs targeting eCBs and/or NAEs are implicated in reward and/or seeking behaviours related to the stimulation of dopamine systems in the brain. In our study, the brain levels of eCBs (anandamide (AEA) and 2-arachidonoylglycerol (2-AG)) and NAEs (oleoylethanolamide (OEA) and palmitoylethanolamide (PEA)) were analyzed via an LC-MS/MS method in selected brain structures of rats during cocaine self-administration and after extinction training according to the "yoked" control procedure. Repeated (14days) cocaine (0.5mg/kg/infusion) self-administration and yoked drug delivery resulted in a significant decrease (ca. 52%) in AEA levels in the cerebellum, whereas levels of 2-AG increased in the frontal cortex, the hippocampus and the cerebellum and decreased in the hippocampus and the dorsal striatum. In addition, we detected increases (>150%) in the levels of OEA and PEA in the limbic areas in both cocaine treated groups, as well as an increase in the tissue levels of OEA in the dorsal striatum in only the yoked cocaine group and increases in the tissue levels of PEA in the dorsal striatum (both cocaine groups) and the nucleus accumbens (yoked cocaine group only). Compared to the yoked saline control group, extinction training (10days) resulted in a potent reduction in AEA levels in the frontal cortex, the hippocampus and the nucleus accumbens and in 2-AG levels in the hippocampus, the dorsal striatum and the cerebellum. The decreases in the limbic and subcortical areas were more apparent for rats that self-administered cocaine. Following extinction, there was a region-specific change in the levels of NAEs in rats previously injected with cocaine; a potent increase (ca. 100%) in the levels of OEA and PEA was detected in the prefrontal cortex and the hippocampus, whilst a drop was noted in the striatal areas versus yoked saline yoked animals. Our findings support the previous pharmacological evidence that the eCB system and NAEs are involved in reinforcement and extinction of positively reinforced behaviours and that these lipid-derived molecules may represent promising targets for the development of new treatments for drug addiction.

Topics: Amides; Animals; Arachidonic Acids; Brain; Cocaine; Conditioning, Operant; Endocannabinoids; Ethanolamines; Extinction, Psychological; Glycerides; Male; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Rats; Reinforcement, Psychology; Self Administration

The endocannabinoid (eCB) system has recently been implicated in both the pathogenesis of depression and the action of antidepressants. Here, we investigated the effect of acutely or chronically administering antidepressants [imipramine (IMI) (15 mg/kg), escitalopram (ESC) (10 mg/kg), and tianeptine (10 mg/kg)] on the levels of both eCBs [anandamide (AEA) and 2-arachidonoylglycerol (2-AG)] and N-acylethanolamines (NAEs) [palmitoylethanolamide (PEA) and oleoylethanolamide (OEA)] in various rat brain regions. We also examined the ability of the acute and chronic administration of N-acetylcysteine (NAC) (a mucolytic drug; 100 mg/kg) or URB597 (a fatty acid amide hydrolase inhibitor; 0.3 mg/kg), which have both elicited antidepressant activity in preclinical studies, to affect eCB and NAE levels. Next, we determined whether the observed effects are stable 10 days after the chronic administration of these drugs was halted. We report that the chronic administration of all investigated drugs increased AEA levels in the hippocampus and also increased both AEA and 2-AG levels in the dorsal striatum. NAE levels in limbic regions also increased after treatment with IMI (PEA/OEA), ESC (PEA), and NAC (PEA/OEA). Removing chronic ESC treatment for 10 days affected eCB and NAE levels in the frontal cortex, hippocampus, dorsal striatum, and cerebellum, while a similar tianeptine-free period enhanced accumbal NAE levels. All other drugs maintained their effects after the 10-day washout period. Therefore, the eCB system appears to play a significant role in the mechanism of action of clinically effective and potential antidepressants and may serve as a target for drug design and discovery.

Topics: Acetylcysteine; Amides; Amidohydrolases; Animals; Antidepressive Agents; Arachidonic Acids; Benzamides; Brain; Carbamates; Citalopram; Endocannabinoids; Enzyme Inhibitors; Ethanolamines; Expectorants; Glycerides; Imipramine; Male; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Rats, Wistar; Thiazepines

To determine whether changes in seminal plasma concentrations of the endogenous lipid signaling molecules palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) have significant effects on sperm quality.. Biochemical and physiological studies of human seminal plasma and spermatozoa.. Academic tertiary care medical center.. Ninety men attending an infertility clinic for semen analysis.. Palmitoylethanolamide and OEA extracted from seminal plasma were quantified by ultra high-performance liquid chromatography (HPLC)-tandem mass spectrometry. Patient sperm from semen with normal parameters were exposed in vitro to PEA or OEA to determine effects on sperm motility, viability, and mitochondrial activity.. The relationship between seminal plasma concentrations of PEA and OEA and sperm quality and the effect of these compounds on sperm motility, viability, and mitochondria activity in vitro.. Palmitoylethanolamide and OEA concentrations in seminal plasma were lower in men with asthenozoospermia and oligoasthenoteratozospermia compared with men with normal semen parameters. Palmitoylethanolamide and OEA rapidly and significantly improved sperm motility and maintained viability without affecting mitochondria activity in vitro.. Maintenance of normal PEA and OEA tone in human seminal plasma may be necessary for the preservation of normal sperm function and male fertility. Exocannabinoids found in Cannabis, such as delta-9-tetrahydrocannabinol and cannabidiol, could compete with these endocannabinoids upsetting their finely balanced, normal functioning and resulting in male reproductive failure.

Topics: Adult; Amides; Arachidonic Acids; Asthenozoospermia; Endocannabinoids; Ethanolamines; Humans; Male; Membrane Potential, Mitochondrial; Middle Aged; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Reproducibility of Results; Semen; Semen Analysis; Sensitivity and Specificity; Spermatozoa; Statistics as Topic; Young Adult

Obesity has emerged as a leading health threat but its biological basis remains insufficiently known, hampering the search for novel treatments. Here, we study oleoylethanolamide, a naturally occurring lipid that has been clearly implicated in weight regulation in animals. However, its role for weight regulation and obesity in humans is still unclear.. To investigate associations between plasma oleoylethanolamide levels and body mass index (BMI, calculated as weight in kilograms divided by height in meters squared) and functional magnetic resonance imaging response to food stimuli in obese patients and matched control participants.. Case-control study of 21 obese patients and 24 matched control participants. Obesity was defined as having a BMI of at least 30. The mean age of participants was 40.8 years and BMIs ranged from 18.2 to 47.5.. Interactions between plasma oleoylethanolamide levels and obesity on BMI and functional magnetic resonance imaging response to food stimuli.. Associations between oleoylethanolamide and BMI differed significantly depending on whether individuals were obese or not (P = .02). In obese individuals, oleoylethanolamide showed a trend toward a positive correlation with BMI (P = .06, ρ = 0.42), while this relationship was inverse for nonobese control participants (P = .07, ρ = -0.34). Similarly, we found significant interactions between oleoylethanolamide levels and obesity on food-related brain activation in cortical areas associated with reward processing and interoceptive signaling (P = .009). Specifically, nonobese individuals with higher oleoylethanolamide levels had higher insular brain activity (P < .001, ρ = 0.70); again, the relationship trended to be inverse for obese patients (P = .11, ρ = -0.36). These effects were not associated with plasma levels of leptin and anandamide, suggesting an independent role of oleoylethanolamide in hunger-associated interoceptive signaling. Analysis of food craving during the functional magnetic resonance imaging task suggested that the identified brain areas may be involved in suppressing food-liking reactions in nonobese individuals.. This study suggests that oleoylethanolamide-mediated signaling plays an important role for hedonic regulation of food craving and obesity in humans and thus may be a valuable target for developing novel antiobesity drugs.

Topics: Adult; Appetite Regulation; Arachidonic Acids; Body Mass Index; Case-Control Studies; Cerebral Cortex; Craving; Endocannabinoids; Female; Functional Neuroimaging; Humans; Leptin; Magnetic Resonance Imaging; Male; Obesity; Oleic Acids; Photic Stimulation; Polyunsaturated Alkamides; Young Adult

Neuropathic pain elevates spinal anandamide (AEA) levels in a way further increased when URB597, an inhibitor of AEA hydrolysis by fatty acid amide hydrolase (FAAH), is injected intrathecally. Spinal AEA reduces neuropathic pain by acting at both cannabinoid CB1 receptors and transient receptor potential vanilloid-1 (TRPV1) channels. Yet, intrathecal URB597 is only partially effective at counteracting neuropathic pain. We investigated the effect of high doses of intrathecal URB597 on allodynia and hyperalgesia in rats with chronic constriction injury (CCI) of the sciatic nerve. Among those tested, the 200 µg/rat dose of URB597 was the only one that elevated the levels of the FAAH non-endocannabinoid and anti-inflammatory substrates, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), and of the endocannabinoid FAAH substrate, 2-arachidonoylglycerol, and fully inhibited thermal and tactile nociception, although in a manner blocked almost uniquely by TRPV1 antagonism. Surprisingly, this dose of URB597 decreased spinal AEA levels. RT-qPCR and western blot analyses demonstrated altered spinal expression of lipoxygenases (LOX), and baicalein, an inhibitor of 12/15-LOX, significantly reduced URB597 analgesic effects, suggesting the occurrence of alternative pathways of AEA metabolism. Using immunofluorescence techniques, FAAH, 15-LOX and TRPV1 were found to co-localize in dorsal spinal horn neurons of CCI rats. Finally, 15-hydroxy-AEA, a 15-LOX derivative of AEA, potently and efficaciously activated the rat recombinant TRPV1 channel. We suggest that intrathecally injected URB597 at full analgesic efficacy unmasks a secondary route of AEA metabolism via 15-LOX with possible formation of 15-hydroxy-AEA, which, together with OEA and PEA, may contribute at producing TRPV1-mediated analgesia in CCI rats.

Topics: Amides; Amidohydrolases; Analgesia; Animals; Arachidonate 15-Lipoxygenase; Arachidonic Acids; Benzamides; Calcium Signaling; Carbamates; Diterpenes; Endocannabinoids; Ethanolamines; Flavanones; Glycerides; HEK293 Cells; Humans; Hyperalgesia; Injections, Spinal; Lipoxygenase Inhibitors; Male; Neuralgia; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Posterior Horn Cells; Rats; Rats, Wistar; Sciatic Nerve; Spinal Cord; TRPV Cation Channels

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

The endocannabinoid system (ECS) represents one of the major determinants of metabolic disorders. We investigated potential changes in the endogenous levels of anandamide (AEA), 2-arachidonoylglycerol (2-AG), N-oleoylethanolamine (OEA) and N-palmitoylethanolamine (PEA) in some peripheral organs and tissues of obese Zucker(fa/fa) and lean Zucker(fa/+) rats by qPCR, liquid chromatography mass spectrometry, western blot and enzymatic activity assays. At 10-12 weeks of age AEA levels were significantly lower in BAT, small intestine and heart and higher in soleus of Zucker(fa/fa) rats. In this tissue, also the expression of CB1 receptors was higher. By contrast in Zucker(fa/fa) rats, 2-AG levels were changed (and lower) solely in the small and large intestine. Finally, in Zucker(fa/fa), PEA levels were unchanged, whereas OEA was slightly lower in BAT, and higher in the large intestine. Interestingly, these differences were accompanied by differential alterations of the genes regulating ECS tone. In conclusion, the levels of endocannabinoids are altered during obesity in a way partly correlating with changes of the genes related to their metabolism and activity.

Topics: Amides; Animals; Arachidonic Acids; Blotting, Western; Chromatography, Liquid; Endocannabinoids; Ethanolamines; Glycerides; Male; Obesity; Oleic Acids; Palmitic Acids; Polymerase Chain Reaction; Polyunsaturated Alkamides; Rats; Rats, Zucker

Endocannabinoid (eCB) signaling has been identified as a modulator of adaptation to stress, and is integral to basal and stress-induced glucocorticoid regulation. Furthermore, interactions between eCBs and glucocorticoids have been shown to be necessary for the regulation of emotional memories, suggesting that eCB function may relate to the development of post-traumatic stress disorder (PTSD). To examine this, plasma eCBs were measured in a sample (n=46) drawn from a population-based cohort selected for physical proximity to the World Trade Center (WTC) at the time of the 9/11 attacks. Participants received a structured diagnostic interview and were grouped according to whether they met diagnostic criteria for PTSD (no PTSD, n=22; lifetime diagnosis of PTSD=24). eCB content (2-arachidonoylglycerol (2-AG) and anandamide (AEA)) and cortisol were measured from 8 a.m. plasma samples. Circulating 2-AG content was significantly reduced among individuals meeting diagnostic criteria for PTSD. The effect of reduced 2-AG content in PTSD remained significant after controlling for the stress of exposure to the WTC collapse, gender, depression and alcohol abuse. There were no significant group differences for AEA or cortisol levels; however, across the whole sample AEA levels positively correlated with circulating cortisol, and AEA levels exhibited a negative relationship with the degree of intrusive symptoms within the PTSD sample. This report shows that PTSD is associated with a reduction in circulating levels of the eCB 2-AG. Given the role of 2-AG in the regulation of the stress response, these data support the hypothesis that deficient eCB signaling may be a component of the glucocorticoid dysregulation associated with PTSD. The negative association between AEA levels and intrusive symptoms is consistent with animal data indicating that reductions in AEA promote retention of aversive emotional memories. Future work will aim to replicate these findings and extend their relevance to clinical pathophysiology, as well as to neuroendocrine and molecular markers of PTSD.

Topics: Aged; Alcoholism; Amides; Arachidonic Acids; Endocannabinoids; Ethanolamines; Ethnicity; Female; Glycerides; Humans; Hydrocortisone; Male; Middle Aged; Neuropsychological Tests; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Psychiatric Status Rating Scales; Sex Characteristics; Stress Disorders, Post-Traumatic; Terrorism

Cannabinoid-1 receptors (CB(1)) have an important role in nicotine reward and their function is disrupted by chronic nicotine exposure, suggesting nicotine-induced alterations in endocannabinoid (eCB) signaling. However, the effects of nicotine on brain eCB levels have not been rigorously evaluated. Volitional intake of nicotine produces physiological and behavioral effects distinct from forced drug administration, although the mechanisms underlying these effects are not known. This study compared the effects of volitional nicotine self-administration (SA) and forced nicotine exposure (yoked administration (YA)) on levels of eCBs and related neuroactive lipids in the ventral tegmental area (VTA) and other brain regions. Brain lipid levels were indexed both by in vivo microdialysis in the VTA and lipid extractions from brain tissues. Nicotine SA, but not YA, reduced baseline VTA dialysate oleoylethanolamide (OEA) levels relative to nicotine-naïve controls, and increased anandamide (AEA) release during nicotine intake. In contrast, all nicotine exposure paradigms increased VTA dialysate 2-arachidonoyl glycerol (2-AG) levels. Thus, nicotine differentially modulates brain lipid (2-AG, AEA, and OEA) signaling, and these modulations are influenced by the volitional nature of the drug exposure. Corresponding bulk tissue analysis failed to identify these lipid changes. Nicotine exposure had no effect on fatty acid amide hydrolase activity in the VTA, suggesting that changes in AEA and OEA signaling result from alterations in their nicotine-induced biosynthesis. Both CB(1) (by AEA and 2-AG) and non-CB(1) (by OEA) targets can alter the excitability and activity of the dopaminergic neurons in the VTA. Collectively, these findings implicate disrupted lipid signaling in the motivational effects of nicotine.

Topics: Animals; Arachidonic Acids; Choice Behavior; Endocannabinoids; Male; Nicotine; Oleic Acids; Polyunsaturated Alkamides; Rats; Rats, Wistar; Self Administration; Ventral Tegmental Area

Endocannabinoid levels are elevated in human and mouse atherosclerosis, but their causal role is not well understood. Therefore, we studied the involvement of fatty acid amide hydrolase (FAAH) deficiency, the major enzyme responsible for endocannabinoid anandamide degradation, in atherosclerotic plaque vulnerability.. We assessed atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) and ApoE(-/-)FAAH(-/-) mice. Before and after 5, 10, and 15 weeks on high-cholesterol diet, we analyzed weight, serum cholesterol, and endocannabinoid levels, and atherosclerotic lesions in thoracoabdominal aortas and aortic sinuses. Serum levels of FAAH substrates anandamide, palmitoylethanolamide (PEA), and oleoylethanolamide (OEA) were 1.4- to 2-fold higher in case of FAAH deficiency. ApoE(-/-)FAAH(-/-) mice had smaller plaques with significantly lower content of smooth muscle cells, increased matrix metalloproteinase-9 expression, and neutrophil content. Circulating and bone marrow neutrophil counts were comparable between both genotypes, whereas CXC ligand1 levels were locally elevated in aortas of FAAH-deficient mice. We observed enhanced recruitment of neutrophils, but not monocytes, to large arteries of ApoE(-/-) mice treated with FAAH inhibitor URB597. Spleens of ApoE(-/-)FAAH(-/-) mice had reduced CD4+FoxP3+regulatory T-cell content, and in vitro stimulation of splenocytes revealed significantly elevated interferon-γ and tumor necrosis factor-α production in case of FAAH deficiency.. Increased anandamide and related FAAH substrate levels are associated with the development of smaller atherosclerotic plaques with high neutrophil content, accompanied by an increased proinflammatory immune response.

Topics: Amides; Amidohydrolases; Animals; Aorta; Aortic Diseases; Apolipoproteins E; Arachidonic Acids; Atherosclerosis; Benzamides; Carbamates; Cells, Cultured; Chemokine CXCL1; Cholesterol; Disease Models, Animal; Endocannabinoids; Enzyme Inhibitors; Ethanolamines; Genotype; Inflammation Mediators; Interferon-gamma; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Smooth, Vascular; Neutrophil Infiltration; Neutrophils; Oleic Acids; Palmitic Acids; Phenotype; Plaque, Atherosclerotic; Polyunsaturated Alkamides; Spleen; T-Lymphocytes, Regulatory; Time Factors; Tumor Necrosis Factor-alpha

Studies from knockout mice suggest that perturbations in oviductal endocannabinoid levels, endocannabinoid receptors, or endocannabinoid degrading enzyme [fatty acid amide hydrolase (FAAH)] expression result in infertility secondary to physical trapping of embryos. Similar observations have been made in ectopic pregnant women together with a suggestion that the endocannabinoid receptor gene polymorphism 1359G/A (rs1049353) is associated with ectopic pregnancy. These observations led to the hypothesis that ectopic pregnancy is associated with a perturbation in levels of endocannabinoids and FAAH activity and that such changes are associated with impaired tubal function.. The objective of the study was to quantify the plasma levels of endocannabinoids (anandamide, oleoylethanolamide, and palmitoylethanolamide) and evaluate blood endocannabinoid metabolizing enzyme activities FAAH and N-acyl-phosphatidyl-ethanolamine phospholipase D (NAPE-PLD) in ectopic pregnancy and normal pregnant controls and relate that to β-human chorionic gonadotropin (β-hCG) levels. Additionally, we wanted to examine the effect of endocannabinoids on cilia beat frequency in Fallopian tube epithelial cells ex vivo.. Whole blood collected from ectopic and normal pregnancies was used for quantification of plasma endocannabinoid levels by ultra-HPLC-tandem mass spectrometry of FAAH and NAPE-PLD enzyme activities by radiometric assays, and β-hCG by immunoassay. Fallopian tube epithelial cells from healthy volunteers were treated with endocannabinoids and cilia beat frequency analyzed using a high-speed digital camera and CiliaFA software.. FAAH activity (P < .05) but not NAPE-PLD activity was significantly reduced in ectopic pregnancies. All 3 endocannabinoids levels were significantly higher (P < .05) in ectopic pregnancy. There was no correlation between endocannabinoids, enzyme activity, and β-hCG levels. Oleoylethanolamide (P < .05), but not methanandamide or palmitoylethanolamide, significantly decreased cilia beat frequency in Fallopian tube epithelial cells.. Elevated endocannabinoid levels and reduced FAAH activity are associated with ectopic pregnancy and may modulate tubal function, suggesting dysfunctional endocannabinoid action in ectopic implantation. Oleoylethanolamide may play a critical role in embryo-tubal transport.

Topics: Adult; Amides; Amidohydrolases; Arachidonic Acids; Cells, Cultured; Chorionic Gonadotropin, beta Subunit, Human; Cilia; Embryo Implantation; Endocannabinoids; Ethanolamines; Fallopian Tubes; Female; Humans; Luteal Phase; Oleic Acids; Palmitic Acids; Phospholipase D; Polyunsaturated Alkamides; Pregnancy; Pregnancy, Ectopic; Young Adult

The endocannabinoid system is known to have positive effects on depression partly through its actions on neurotrophins, such as Brain-Derived Neurotrophic Factor (BDNF). As BDNF is also considered the major candidate molecule for exercise-induced brain plasticity, we hypothesized that the endocannabinoid system represents a crucial signaling system mediating the beneficial antidepressant effects of exercise. Here we investigated, in 11 healthy trained male cyclists, the effects of an intense exercise (60 min at 55% followed by 30 min at 75% W(max)) on plasma levels of endocannabinoids (anandamide, AEA and 2-arachidonoylglycerol, 2-AG) and their possible link with serum BDNF. AEA levels increased during exercise and the 15 min recovery (P<0.001), whereas 2-AG concentrations remained stable. BDNF levels increased significantly during exercise and then decreased during the 15 min of recovery (P<0.01). Noteworthy, AEA and BDNF concentrations were positively correlated at the end of exercise and after the 15 min recovery (r>0.66, P<0.05), suggesting that AEA increment during exercise might be one of the factors involved in exercise-induced increase in peripheral BDNF levels and that AEA high levels during recovery might delay the return of BDNF to basal levels. AEA production during exercise might be triggered by cortisol since we found positive correlations between these two compounds and because corticosteroids are known to stimulate endocannabinoid biosynthesis. These findings provide evidence in humans that acute exercise represents a physiological stressor able to increase peripheral levels of AEA and that BDNF might be a mechanism by which AEA influences the neuroplastic and antidepressant effects of exercise.

Topics: Adult; Amides; Arachidonic Acids; beta-Endorphin; Bicycling; Brain-Derived Neurotrophic Factor; Cannabinoid Receptor Modulators; Chromatography, High Pressure Liquid; Depression; Endocannabinoids; Ethanolamines; Exercise; Glycerides; Hematocrit; Humans; Male; Mass Spectrometry; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; PPAR alpha; Reward; Young Adult

Hydrogen peroxide (H(2)O(2)) has been shown to participate in endothelium-derived hyperpolarising factor (EDHF)-mediated mechanisms. Vasorelaxation to the endocannabinoid-like N-oleoylethanolamine (OEA) and anandamide has been shown to be endothelium-dependent. Therefore, the principal aim was to investigate whether H(2)O(2) plays a role in vasorelaxation to endocannabinoids in rat mesenteric arteries. We have also investigated the effects of catalase on endothelium-dependent relaxations and vascular responses to H(2)O(2). First- (G1) and third- (G3) order branches of the superior mesenteric artery from male, Wistar rats were mounted in a wire myograph, contracted with methoxamine, and concentration-response curves to anandamide, OEA carbachol or H(2)O(2), were constructed. The influence of nitric oxide production and H(2)O(2) breakdown on these responses were then investigated using L-NAME (300 μM), and catalase (1000 Uml(-1)) respectively. In G1 mesenteric arteries, vasorelaxations to carbachol and H(2)O(2) were inhibited by L-NAME, but not by catalase. Responses to both anandamide and OEA were also unaffected by catalase. In G3 mesenteric arteries, endothelium-dependent relaxations to carbachol were modestly affected by L-NAME, unaffected by catalase alone, but their combination greatly inhibited vasorelaxation. Similarly, catalase inhibited vasorelaxation to anandamide and OEA, and combined treatment with L-NAME further reduced this response. In G1 mesenteric arteries, vasorelaxation to H(2)O(2) is predominantly mediated by nitric oxide. We conclude that in G3 arteries H(2)O(2) activity contributes towards EDHF-type responses and vasorelaxation to endocannabinoids, either directly or indirectly. Given the association between vascular pathophysiology and H(2)O(2), these findings may provide a mechanism whereby disease states may influence responses to endocannabinoid and related mediators.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Carbachol; Catalase; Endocannabinoids; Ethanolamines; Hydrogen Peroxide; In Vitro Techniques; Male; Mesenteric Arteries; NG-Nitroarginine Methyl Ester; Oleic Acids; Polyunsaturated Alkamides; Rats; Rats, Wistar; Vasodilation

The tissue-specific sources and regulated production of physiological signals that modulate food intake are incompletely understood. Previous work showed that L-Fabp(-/-) mice are protected against obesity and hepatic steatosis induced by a high-fat diet, findings at odds with an apparent obesity phenotype in a distinct line of aged L-Fabp(-/-) mice. Here we show that the lean phenotype in L-Fabp(-/-) mice is recapitulated in aged, chow-fed mice and correlates with alterations in hepatic, but not intestinal, fatty acid amide metabolism. L-Fabp(-/-) mice exhibited short-term changes in feeding behavior with decreased food intake, which was associated with reduced abundance of key signaling fatty acid ethanolamides, including oleoylethanolamide (OEA, an agonist of PPARα) and anandamide (AEA, an agonist of cannabinoid receptors), in the liver. These reductions were associated with increased expression and activity of hepatic fatty acid amide hydrolase-1, the enzyme that degrades both OEA and AEA. Moreover, L-Fabp(-/-) mice demonstrated attenuated responses to OEA administration, which was completely reversed with an enhanced response after administration of a nonhydrolyzable OEA analog. These findings demonstrate a role for L-Fabp in attenuating obesity and hepatic steatosis, and they suggest that hepatic fatty acid amide metabolism is altered in L-Fabp(-/-) mice.

Topics: Adiposity; Age Factors; Amidohydrolases; Animals; Arachidonic Acids; Body Weight; Chromosomes; Diet, Fat-Restricted; Endocannabinoids; Enzyme Activation; Fatty Acid-Binding Proteins; Fatty Liver; Feeding Behavior; Female; Lipid Metabolism; Liver; Mice; Mice, Inbred C57BL; Mice, Knockout; Obesity; Oleic Acids; Polyunsaturated Alkamides; PPAR gamma; Quantitative Trait Loci; Signal Transduction

Hedonic hunger refers to consumption of food just for pleasure and not to maintain energy homeostasis. In this condition, the subject eats also when not in a state of short-term energy depletion, and food is consumed uniquely because of its gustatory rewarding properties. The physiological mechanisms underlying this eating behavior are not deeply understood, but endogenous rewarding mediators like ghrelin and endocannabinoids are likely involved.. To explore the role of these substances in hedonic eating, we measured changes in their plasma levels in eight satiated healthy subjects after ad libitum consumption of highly palatable food as compared with the consumption of nonpalatable food in isoenergetic amounts with the same nutrient composition of the palatable food.. The consumption of food for pleasure was characterized by increased peripheral levels of both the peptide ghrelin and the endocannabinoid 2-arachidonoyl-glycerol. Levels of the other endocannabinoid anandamide and of anandamide-related mediators oleoylethanolamide and palmitoylethanolamide, instead, progressively decreased after the ingestion of both highly pleasurable and isoenergetic nonpleasurable food. A positive correlation was found between plasma 2-arachidonoyl glycerol and ghrelin during hedonic but not nonhedonic, eating.. The present preliminary findings suggest that when motivation to eat is generated by the availability of highly palatable food and not by food deprivation, a peripheral activation of two endogenous rewarding chemical signals is observed. Future research should confirm and extend our results to better understand the phenomenon of hedonic eating, which influences food intake and, ultimately, body mass.

Topics: Adult; Amides; Appetite; Arachidonic Acids; Blood Glucose; Cannabinoid Receptor Modulators; Endocannabinoids; Energy Intake; Ethanolamines; Feeding Behavior; Female; Ghrelin; Glycerides; Humans; Male; Oleic Acids; Palmitic Acids; Pilot Projects; Pleasure; Polyunsaturated Alkamides; Reference Values; Satiety Response; Young Adult

Elevating levels of endocannabinoids with inhibitors of fatty acid amide hydrolase (FAAH) is a major focus of pain research, purported to be a safer approach devoid of cannabinoid receptor-mediated side effects. Here, we have determined the effects of sustained pharmacological inhibition of FAAH on inflammatory pain behaviour and if pharmacological inhibition of FAAH was as effective as genetic deletion of FAAH on pain behaviour.. Effects of pre-treatment with a single dose, versus 4 day repeated dosing with the selective FAAH inhibitor, URB597 (i.p. 0.3 mg·kg⁻¹), on carrageenan-induced inflammatory pain behaviour and spinal pro-inflammatory gene induction were determined in rats. Effects of pain induction and of the drug treatments on levels of arachidonoyl ethanolamide (AEA), palmitoyl ethanolamide (PEA) and oleolyl ethanolamide (OEA) in the spinal cord were determined.. Single, but not repeated, URB597 treatment significantly attenuated the development of inflammatory hyperalgesia (P < 0.001, vs. vehicle-treated animals). Neither mode of URB597 treatment altered levels of AEA, PEA and OEA in the hind paw, or carrageenan-induced paw oedema. Single URB597 treatment produced larger increases in AEA, PEA and OEA in the spinal cord, compared with those after repeated administration. Single and repeated URB597 treatment decreased levels of immunoreactive N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) in the spinal cord and attenuated carrageenan-induced spinal pro-inflammatory gene induction.. Changes in the endocannabinoid system may contribute to the loss of analgesic effects following repeated administration of low dose URB597 in this model of inflammatory pain.

Topics: Amides; Amidohydrolases; Animals; Arachidonic Acids; Behavior, Animal; Benzamides; Carbamates; Disease Models, Animal; Drug Administration Schedule; Endocannabinoids; Ethanolamines; Inflammation; Male; Oleic Acids; Pain; Palmitic Acids; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Spinal Cord

Primary astrocytomas of grade 3 or 4 according to the classification system of the World Health Organization (high-grade astrocytomas or HGAs) are preponderant among adults and are almost invariably fatal despite the use of multimodal therapy. Here we show that the juvenile brain has an endogenous defense mechanism against HGAs. Neural precursor cells (NPCs) migrate to HGAs, reduce glioma expansion and prolong survival time by releasing endovanilloids that activate the vanilloid receptor (transient receptor potential vanilloid subfamily member-1 or TRPV1) on HGA cells. TRPV1 is highly expressed in tumor and weakly expressed in tumor-free brain. TRPV1 stimulation triggers tumor cell death through the branch of the endoplasmic reticulum stress pathway that is controlled by activating transcription factor-3 (ATF3). The antitumorigenic response of NPCs is lost with aging. NPC-mediated tumor suppression can be mimicked in the adult brain by systemic administration of the synthetic vanilloid arvanil, suggesting that TRPV1 agonists have potential as new HGA therapeutics.

Topics: Aging; Amides; Amidohydrolases; Animals; Antineoplastic Agents; Apoptosis; Arachidonic Acids; Brain; Brain Neoplasms; Capsaicin; Cell Movement; Culture Media, Conditioned; Dopamine; Endocannabinoids; Ethanolamines; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, SCID; Neoplasm Proteins; Neural Stem Cells; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Real-Time Polymerase Chain Reaction; RNA, Small Interfering; TRPV Cation Channels; Tumor Cells, Cultured

An LC-MS/MS-ESI method has been validated for simultaneous estimation of the three endocannabinoids; N-arachidonoylethanolamine (AEA), N-oleoylethanolamine (OEA) and palmitoylethanolamide (PEA), in surrogate matrix using AEA-d (4) as an internal standard with highest sensitivity over the existing methods. Simple precipitation was used to extract analytes and these were subsequently analyzed on a monolithic column. Linear response function was established over the concentration range 12.3 to 1225 pg/ml for AEA (r > 0.994); 0.70 to 641 ng/ml for OEA (r > 0.999) and 0.54 to 321 ng/ml (r > 0.998) for PEA. The intra- and inter-day precision values met the acceptance to criteria as per US FDA guidelines. Analytes were found to be stable in the battery of stability studies. The method was applied to quantify endogenous levels of analytes in rat plasma.

Topics: Amides; Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Chromatography, Liquid; Endocannabinoids; Ethanolamines; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Rats; Reference Standards; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

The concentrations of the endocannabinoids 2-arachidonoylglycerol (2-AG) and N-arachidonylethanolamine (anandamide) were examined in rat brain cerebral cortex slices and surrounding medium. Basal concentrations of endocannabinoids were similar to those identified previously in rat brain, with anandamide content being much lower (19 pmol/g) than that of 2-AG (7300 pmol/g). In contrast, basal concentrations in the surrounding medium were proportionally much lower for 2-arachidonoylglycerol (16 pmol/mL) compared to anandamide (0.6 pmol/mL). Incubation of slices with glutamate receptor agonists, depolarizing concentrations of KCl, or ionomycin failed to alter tissue concentrations of endocannabinoids, while endocannabinoids in the medium were unaltered by elevated KCl. Cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester, an inhibitor of fatty acid amide hydrolase, significantly enhanced tissue concentrations of anandamide (and related N-acylethanolamines), without altering 2-AG, while evoking proportional elevations of anandamide in the medium. Removal of extracellular calcium ions failed to alter tissue concentrations of anandamide, but significantly reduced 2-AG in the tissue by 90% and levels in the medium to below the detection limit. Supplementation of the medium with 50 μM N-oleoylethanolamine only raised tissue concentrations of N-oleoylethanolamine in the presence of cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester and failed to alter either tissue or medium anandamide or 2-AG concentrations. These results highlight the ongoing turnover of endocannabinoids, and the importance of calcium ions in maintaining 2-AG concentrations in this tissue.

Topics: Amides; Amidohydrolases; Animals; Arachidonic Acids; Calcium; Calcium Signaling; Cannabinoid Receptor Modulators; Cerebral Cortex; Endocannabinoids; Ethanolamines; Glycerides; In Vitro Techniques; Inositol; Male; Monoacylglycerol Lipases; Oleic Acids; Palmitic Acids; Phospholipids; Polyunsaturated Alkamides; Potassium Chloride; Rats; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

Bioactive N-acylethanolamines include anandamide (an endocannabinoid), N-palmitoylethanolamine (an anti-inflammatory), and N-oleoylethanolamine (an anorexic). In the brain, these molecules are formed from N-acylphosphatidylethanolamines (NAPEs) by a specific phospholipase D, called NAPE-PLD, or through NAPE-PLD-independent multi-step pathways, as illustrated in the current study employing NAPE-PLD-deficient mice. Although N-acylethanolamine plasmalogen (1-alkenyl-2-acyl-glycero-3-phospho(N-acyl)ethanolamine, pNAPE) is presumably a major class of N-acylethanolamine phospholipids in the brain, its enzymatic conversion to N-acylethanolamines is poorly understood. In the present study, we focused on the formation of N-acylethanolamines from pNAPEs. While recombinant NAPE-PLD catalyzed direct release of N-palmitoylethanolamine from N-palmitoylethanolamine plasmalogen, the same reaction occurred in the brain homogenate of NAPE-PLD-deficient mice, suggesting that this reaction occurs through both the NAPE-PLD-dependent and -independent pathways. Liquid chromatography-mass spectrometry revealed a remarkable accumulation of 1-alkenyl-2-hydroxy-glycero-3-phospho(N-acyl)ethanolamines (lyso pNAPEs) in the brain of NAPE-PLD-deficient mice. We also found that brain homogenate formed N-palmitoylethanolamine, N-oleoylethanolamine, and anandamide from their corresponding lyso pNAPEs by a Mg(2+)-dependent "lysophospholipase D". Moreover, the brain levels of alkenyl-type lysophosphatidic acids, the other products from lyso pNAPEs by lysophospholipase D, also increased in NAPE-PLD-deficient mice. Glycerophosphodiesterase GDE1 can hydrolyze glycerophospho-N-acylethanolamines to N-acylethanolamines in the brain. In addition, we discovered that recombinant GDE1 has a weak activity to generate N-palmitoylethanolamine from its corresponding lyso pNAPE, suggesting that this enzyme is at least in part responsible for the lysophospholipase D activity. These results strongly suggest that brain tissue N-acylethanolamines, including anandamide, can be formed from N-acylated plasmalogen through an NAPE-PLD-independent pathway as well as by their direct release via NAPE-PLD.

Topics: Animals; Arachidonic Acids; Brain; Chlorocebus aethiops; COS Cells; Endocannabinoids; Ethanolamines; Male; Mice; Mice, Mutant Strains; Models, Biological; Oleic Acids; Phospholipase D; Plasmalogens; Polyunsaturated Alkamides; Signal Transduction

The antitumoral properties of endocannabinoids received a particular attention these last few years. Indeed, these endogenous molecules have been reported to exert cytostatic, apoptotic and antiangiogenic effects in different tumor cell lines and tumor xenografts. Therefore, we investigated the cytotoxicity of three N-acylethanolamines--N-arachidonoylethanolamine (anandamide, AEA), N-palmitoylethanolamine (PEA) and N-oleoylethanolamine (OEA)--which were all able to time- and dose-dependently reduce the viability of murine N1E-115 neuroblastoma cells. Moreover, several inhibitors of FAAH and NAAA, whose presence was confirmed by RT-PCR in the cell line, induced cell cytotoxicity and favored the decrease in cell viability caused by N-acylethanolamines. The most cytotoxic treatment was achieved by the co-incubation of AEA with the selective FAAH inhibitor URB597, which drastically reduced cell viability partly by inhibiting AEA hydrolysis and consequently increasing AEA levels. This combination of molecules synergistically decreased cell proliferation without inducing cell apoptosis or necrosis. We found that these effects are independent of cannabinoid, TRPV1, PPARα, PPARγ or GPR55 receptors activation but seem to occur through a lipid raft-dependent mechanism. These findings further highlight the interest of targeting the endocannabinoid system to treat cancer. More particularly, this emphasizes the great potential benefit of designing novel anti-cancerous therapies based on the association of endocannabinoids and inhibitors of their hydrolysis.

Topics: Amides; Animals; Antineoplastic Agents; Arachidonic Acids; Cannabinoid Receptor Modulators; Cell Line, Tumor; Cell Proliferation; Endocannabinoids; Ethanolamines; Metabolism; Mice; Neoplasms; Neuroblastoma; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides

Endocannabinoids are amides and esters of long chain fatty acids that can modulate ion channels through both receptor-dependent and receptor-independent effects. Nowadays, their effects on cardiac K(+) channels are unknown even when they can be synthesized within the heart. We have analyzed the direct effects of endocannabinoids, such as anandamide (AEA), 2-arachidonoylglycerol (2-AG), the endogenous lipid lysophosphatidylinositol, and cannabinoid analogues such as palmitoylethanolamide (PEA), and oleoylethanolamide, as well as the fatty acids from which they are endogenously synthesized, on human cardiac Kv4.3 channels, which generate the transient outward K(+) current (I(to1)). Currents were recorded in Chinese hamster ovary cells, which do not express cannabinoid receptors, by using the whole-cell patch-clamp. All these compounds inhibited I(Kv4.3) in a concentration-dependent manner, AEA and 2-AG being the most potent (IC(50) approximately 0.3-0.4 microM), while PEA was the least potent. The potency of block increased as the complexity and the number of C atoms in the fatty acyl chain increased. The effects were not mediated by modifications in the lipid order and microviscosity of the membrane and were independent of the presence of MiRP2 or DPP6 subunits in the channel complex. Indeed, effects produced by AEA were reproduced in human atrial I(to1) recorded in isolated myocytes. Moreover, AEA effects were exclusively apparent when it was applied to the external surface of the cell membrane. These results indicate that at low micromolar concentrations the endocannabinoids AEA and 2-AG directly block human cardiac Kv4.3 channels, which represent a novel molecular target for these compounds.

Topics: Amides; Animals; Arachidonic Acid; Arachidonic Acids; Cannabinoid Receptor Modulators; Cannabinoids; CHO Cells; Cricetinae; Cricetulus; Endocannabinoids; Ethanolamines; Fatty Acids; Glycerides; Heart; Humans; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Shal Potassium Channels

Endocannabinoids include anandamide (AEA) and 2-arachidonoylglycerol (2-AG). Endocannabinoid-related molecules like oleoyl-ethanolamine (OEA) and palmitoyl-ethanolamine (PEA) have also been identified. AEA contributes to the pathogenesis of cardiovascular alterations in experimental cirrhosis, but data on the endocannabinoid system in human cirrhosis are lacking. Thus, we aimed to assess whether circulating and hepatic endocannabinoids are upregulated in cirrhotic patients and whether their levels correlate with systemic haemodynamics and liver function.. The endocannabinoid levels were measured in peripheral and hepatic veins and liver tissue by isotope-dilution liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry. Systemic haemodynamics were assessed by the transthoracic electrical bioimpedance technique. Portal pressure was evaluated by hepatic venous pressure gradient.. Circulating AEA and, to a greater extent, PEA and OEA were significantly higher in cirrhotic patients than in controls. PEA and OEA were also increased in the cirrhotic liver tissue. AEA, OEA and PEA levels were significantly higher in peripheral than in the hepatic veins of cirrhotic patients, while the opposite occurred for 2-AG. Finally, circulating AEA, OEA and PEA correlated with parameters of liver function, such as serum bilirubin and international normalized ratio. No correlations were found with systemic haemodynamics.. The endocannabinoid system is upregulated in human cirrhosis. Peripheral AEA is increased in patients with a high model of end-stage liver disease score and may reflect the extent of liver dysfunction. In contrast, the 2-AG levels, the other major endocannabinoid, are not affected by cirrhosis. The upregulation of the endocannabinoid-related molecules, OEA and PEA, is even greater than that of AEA, prompting pharmacological studies on these compounds.

Topics: Adult; Amides; Arachidonic Acids; Bilirubin; Biomarkers; Cannabinoid Receptor Modulators; Case-Control Studies; Chromatography, Liquid; Electric Impedance; Endocannabinoids; Ethanolamines; Female; Glycerides; Hemodynamics; Humans; International Normalized Ratio; Italy; Liver; Liver Cirrhosis; Liver Function Tests; Male; Mass Spectrometry; Middle Aged; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Radioisotope Dilution Technique; Severity of Illness Index; Up-Regulation

Fatty acid amide hydrolase (FAAH) is the main degrading enzyme of the fatty acid ethanolamides anandamide (AEA) and oleoylethanolamide (OEA), which have opposite effects on food intake and energy balance. AEA, an endogenous ligand of CB(1) cannabinoid receptors, enhances food intake and energy storage, whereas OEA binds to peroxisome proliferator-activated receptors-alpha to reduce food intake and promoting lipolysis. To elucidate the role of FAAH in food intake and energy balance, we have evaluated different metabolic and behavioral responses related to feeding in FAAH-deficient (FAAH(-/-)) mice and their wild-type littermates.. Total daily food intake was similar in both genotypes, but high-fat food consumption was enhanced during the dark hours and decreased during the light hours in FAAH(-/-) mice. The reinforcing and motivational effects of food were also enhanced in FAAH(-/-) mice as revealed by operant behavioral paradigms. These behavioral responses were reversed by the administration of the selective CB(1) cannabinoid antagonist rimonabant. Furthermore, body weight, total amount of adipose tissue, plasma-free fatty acids and triglyceride content in plasma, liver, skeletal muscle and adipose tissue, were increased in FAAH(-/-) mice. Accordingly, leptin levels were increased and adiponectin levels decreased in these mutants, FAAH(-/-) mice also showed enhanced plasma insulin and blood glucose levels revealing an insulin resistance. As expected, both AEA and OEA levels were increased in hypothalamus, small intestine and liver of FAAH(-/-) mice.. These results indicate that the lack of FAAH predominantly promotes energy storage by food intake-independent mechanisms, through the enhancement of AEA levels rather than promoting the anorexic effects of OEA.

Topics: Adiposity; Amidohydrolases; Animals; Arachidonic Acids; Body Weight; Cannabinoid Receptor Modulators; Conditioning, Operant; Darkness; Dietary Fats; Eating; Endocannabinoids; Lipid Metabolism; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Motivation; Obesity; Oleic Acids; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rimonabant; Triglycerides

Topics: Amides; Arachidonic Acids; Biomarkers; Cannabinoid Receptor Modulators; Endocannabinoids; Ethanolamines; Glycerides; Hemodynamics; Humans; International Normalized Ratio; Liver; Liver Cirrhosis; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Severity of Illness Index; Up-Regulation

The endocannabinoids, anandamide and 2-AG, are produced by adipocytes, where they stimulate lipogenesis via cannabinoid CB1 receptors and are under the negative control of leptin and insulin. Endocannabinoid levels are elevated in the blood of obese individuals and nonobese type 2 diabetes patients. To date, no study has evaluated endocannabinoid levels in subcutaneous adipose tissue (SAT) of subjects with both obesity and type 2 diabetes (OBT2D), characterised by similar adiposity and whole body insulin resistance and lower plasma leptin levels as compared to non-diabetic obese subjects (OB).. The levels of anandamide and 2-AG, and of the anandamide-related PPARalpha ligands, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), in the SAT obtained by abdominal needle biopsy in 10 OBT2D, 11 OB, and 8 non-diabetic normal-weight (NW) subjects, were measured by liquid chromatography-mass spectrometry. All subjects underwent a hyperinsulinaemic euglycaemic clamp.. As compared to NW, anandamide, OEA and PEA levels in the SAT were 2-4.4-fold elevated (p < 0.05), and 2-AG levels 2.3-fold reduced (p < .05), in OBT2D but not in OB subjects. Anandamide, OEA and PEA correlated positively (p < .05) with SAT leptin mRNA and free fatty acid during hyperinsulinaemic clamp, and negatively with SAT LPL activity and plasma HDL-cholesterol, which were all specifically altered in OBT2D subjects.. The observed alterations emphasize, for the first time in humans, the potential different role and regulation of adipose tissue anandamide (and its congeners) and 2-AG in obesity and type 2 diabetes.

Topics: Adiposity; Adult; Amides; Arachidonic Acids; Cannabinoid Receptor Modulators; Diabetes Mellitus, Type 2; Endocannabinoids; Ethanolamines; Female; Humans; Lipids; Male; Middle Aged; Obesity; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Subcutaneous Fat

The endocannabinoid system promotes diverse effects on fat and glucose metabolism as well as on energy balance and sleep regulation. The role of N-acylethanolamides like oleoylethanolamide (OEA) and other endocannabinoids such as anandamide (AEA) and 2-arachidonyl-glycerol (2-AG) has not yet been investigated in patients with sleep apnea.. We measured circulating OEA, AEA and 2-AG in patients with sleep apnea (n = 20) and healthy control subjects (n = 57). Respiratory distress index (RDI) as measured by polysomnography was used as a quantitative index of sleep apnea.. In patients with sleep apnea OEA serum concentrations were significantly higher than in control subjects (8.4 pmol/ml (95% CI 6.9;9.9) vs. 4.0 (3.5;4.5); p<0.0001, adjusted for body mass index (BMI), fasting insulin, HDL and LDL cholesterol). In contrast, AEA (2.9 (95% CI 1.9;3.9) vs. 1.8 (1.4;2.1), p = 0.09) and 2-AG (20.0 (-14.5;54.5) vs. 32.8 (21.4;44.2), p = 0.56) were not significantly different between patients with sleep apnea and control subjects after adjustment. In the sleep apnea group, OEA serum concentrations were associated with RDI (r (2) = 0.28, p = 0.02) and BMI (r (2) = 0.32, p = 0.01). However, OEA was not associated with BMI in the control group (p = 0.10).. These results indicate that among the three analyzed fatty acid derivatives, OEA plays a specific role in patients with sleep apnea. Together with animal data, the 2-fold elevation of OEA serum concentrations could be interpreted as a neuroprotective mechanism against chronic oxidative stressors and a mechanism to promote wakefulness in patients with nocturnal sleep deprivation and daytime hypersomnolence.

Topics: Arachidonic Acids; Blood Glucose; Body Mass Index; Cannabinoid Receptor Modulators; Case-Control Studies; Endocannabinoids; Female; Glycerides; Humans; Insulin; Lipids; Male; Middle Aged; Oleic Acids; Polyunsaturated Alkamides; Sleep Apnea Syndromes

The endocannabinoid-like molecule N-oleoylethanolamine (OEA) is found in the small intestine and regulates food intake and promotes weight loss. The principal aim of the present study was to evaluate the vascular effects of OEA.. Perfused isolated mesenteric arterial beds were pre-contracted with methoxamine or high potassium buffers and concentration-response curves to OEA were constructed. Combinations of inhibitors to block nitric oxide production, sensory nerve activity, cyclooxygenase activity, potassium channels, chloride channels and gap junctions, and a cannabinoid CB(1) receptor antagonist, were used during these experiments. The effects of OEA on caffeine-induced contractions in calcium-free buffer were also assessed. Isolated thoracic aortic rings were used as a comparison.. OEA caused concentration-dependent vasorelaxation in rat isolated mesenteric arterial beds and thoracic aortic rings, with a greater maximal response in mesenteric vessels. This relaxation was sensitive to inhibition of sensory nerve activity and endothelial removal in both preparations. The cyclooxygenase inhibitor indomethacin reversed the effects of capsaicin pre-treatment in perfused mesenteric arterial beds and indomethacin alone enhanced vasorelaxation to OEA. The OEA-induced vasorelaxation was inhibited by a CB(1) receptor antagonist only in aortic rings. In mesenteric arteries, OEA suppressed caffeine-induced contractions in calcium-free buffer.. The vasorelaxant effects of OEA are partly dependent on sensory nerve activity and a functional endothelium in the vasculature. In addition, vasorelaxation to OEA is enhanced following cyclooxygenase inhibition. OEA may also interfere with the release of intracellular calcium in arterial preparations.

Topics: Animals; Aorta, Thoracic; Arachidonic Acids; Caffeine; Capsaicin; Chloride Channels; Endocannabinoids; Endothelium, Vascular; Ethanolamines; In Vitro Techniques; Indomethacin; Male; Mesenteric Arteries; Nitric Oxide; Oleic Acids; Piperidines; Polyunsaturated Alkamides; Potassium Channel Blockers; Prostaglandin-Endoperoxide Synthases; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Sensory Receptor Cells; Vasodilation

Endocannabinoids including N-acylethanolamides (NAEs) are a family of lipid-related signaling molecules implicated in many physiological and disease states which elicit their activities via the cannabinoid receptors. Anandamide (N-arachidonoylethanolamine, AEA) is the most characterized endocannabinoid and has been detected in many tissues and bio-fluids including human plasma and the central nervous system. The endocannabinoid-like NAEs, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) are described as entourage compounds because they illicit similar physiological effects to AEA but have little or no affinity for cannabinoid receptors. As entourage compounds, levels of these NAEs can greatly influence the efficacy of AEA yet there are few studies which measure these compounds in bio-fluids. Here we describe a rapid, highly sensitive, specific and highly reproducible ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the analysis of AEA, OEA, and PEA in human bio-fluids including plasma, serum, breast milk, and amniotic fluids. This validated method using deuterated (AEA-d(8), OEA-d(2), and PEA-d(4)) internal standards, represents an improvement over previous analyses in terms of run time (4 min), limit of detection (0.9 fmol on column for AEA and PEA and 4.4 fmol on column for OEA), precision (relative standard deviations of peak areas: 3.1% (AEA), 2.9% (OEA), and 5.4% (PEA) for 133 fmol on column) and accuracy (95.1-104.9%). The sensitivity and precision of the validated method described here suggests that this method is suitable for the analysis of AEA, OEA, and PEA in clinical samples and may be utilized for the investigation of bio-matrices containing limited amounts of NAEs.

Topics: Amides; Arachidonic Acids; Chromatography, Liquid; Endocannabinoids; Ethanolamines; Humans; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Tandem Mass Spectrometry

The endocannabinoids anandamide, palmitoylethanolamide and oleoylethanolamide have been detected in human seminal plasma and are bioactive lipids implicated in regulation of sperm motility, capacitation and acrosome reaction. Several methods exist for endocannabinoid quantification but none have been validated for measurement in human seminal plasma. We describe sensitive, robust, reproducible solid phase and isotope-dilution UHPLC-ESI-MS/MS methods for the extraction and quantification of anandamide, palmitoylethanolamide and oleoylethanolamide in human seminal plasma. Precision and accuracy were evaluated using pooled seminal plasma over a 4 day period. For all analytes, the inter- and intraday precision (CV%) was between 6.6-17.7% and 6.3-12.5%, respectively. Analyses were linear over the range 0.237-19nM for anandamide and oleoylethanolamide and 0.9-76nM for PEA. Limits of detection (signal-to-noise >3) were 50, 100 and 100fmol/mL and limits of quantification (signal-to-noise >10) were 100, 200 and 200fmol/mL, respectively for anandamide, palmitoylethanolamide and oleoylethanolamide. Anandamide and oleoylethanolamide were stable at -80°C for up to 4 weeks, but palmitoylethanolamide declined significantly. We assessed seminal plasma from 40 human donors with normozoospermia and found mean (inter-quartile range) concentrations of 0.21nM (0.09-0.27), 1.785nM (0.48-2.32) and 15.54nM (7.05-16.31) for anandamide, oleoylethanolamide and palmitoylethanolamide, respectively. Consequently, this UHPLC-ESI-MS/MS method represents a rapid, reliable and reproducible technique for the analysis of these endocannabinoids in fresh seminal plasma.

Topics: Amides; Arachidonic Acids; Cannabinoid Receptor Modulators; Chromatography, High Pressure Liquid; Drug Stability; Endocannabinoids; Ethanolamines; Humans; Linear Models; Male; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Reproducibility of Results; Semen; Sensitivity and Specificity; Solid Phase Extraction; Tandem Mass Spectrometry

Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme within the amidase-signature family. It catalyzes the hydrolysis of several endogenous biologically active lipids, including anandamide (arachidonoyl ethanolamide), oleoyl ethanolamide, and palmitoyl ethanolamide. These endogenous FAAH substrates have been shown to be involved in a variety of physiological and pathological processes, including synaptic regulation, regulation of sleep and feeding, locomotor activity, pain and inflammation. Here we describe the biochemical and biological properties of a potent and selective FAAH inhibitor, 4-(3-phenyl-[1,2,4]thiadiazol-5-yl)-piperazine-1-carboxylic acid phenylamide (JNJ-1661010). The time-dependence of apparent IC(50) values at rat and human recombinant FAAH, dialysis and mass spectrometry data indicate that the acyl piperazinyl fragment of JNJ-1661010 forms a covalent bond with the enzyme. This bond is slowly hydrolyzed, with release of the piperazinyl fragment and recovery of enzyme activity. The lack of inhibition observed in a rat liver esterase assay suggests that JNJ-1661010 is not a general esterase inhibitor. JNJ-1661010 is >100-fold preferentially selective for FAAH-1 when compared to FAAH-2. JNJ-1661010 dose-dependently increases arachidonoyl ethanolamide, oleoyl ethanolamide, and palmitoyl ethanolamide in the rat brain. The compound attenuates tactile allodynia in the rat mild thermal injury model of acute tissue damage and in the rat spinal nerve ligation (Chung) model of neuropathic pain. JNJ-1661010 also diminishes thermal hyperalgesia in the inflammatory rat carrageenan paw model. These data suggest that FAAH inhibitors with modes of action similar to JNJ-1661010 may be useful clinically as broad-spectrum analgesics.

Topics: Amides; Amidohydrolases; Analgesics; Animals; Arachidonic Acids; Brain; Carrageenan; Disease Models, Animal; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Ethanolamines; Hot Temperature; Humans; Hydrolysis; Isoenzymes; Kinetics; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neuralgia; Oleic Acids; Pain; Pain Measurement; Pain Threshold; Palmitic Acids; Piperazines; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Reaction Time; Recombinant Proteins; Thiadiazoles

This study investigated the role of two fatty acid ethanolamides, the endogenous cannabinoid anandamide and its structural analog oleoylethanolamide in sleep deprivation of human volunteers. Serum and cerebrospinal fluid (CSF) samples were obtained from 20 healthy volunteers before and after a night of sleep deprivation with an interval of about 12 months. We found increased levels of oleoylethanolamide in CSF (P = 0.011) but not in serum (P = 0.068) after 24 h of sleep deprivation. Oleoylethanolamide is an endogenous lipid messenger that is released after neural injury and activates peroxisome proliferator-activated receptor-alpha (PPAR-alpha) with nanomolar potency. Exogenous PPAR-alpha agonists, such as hypolipidemic fibrates and oleoylethanolamide, exert both neuroprotective and neurotrophic effects. Thus, our results suggest that oleoylethanolamide release may represent an endogenous neuroprotective signal during sleep deprivation.

Topics: Adult; Arachidonic Acids; Cannabinoid Receptor Modulators; Chromatography, High Pressure Liquid; Endocannabinoids; Female; Humans; Lipid Metabolism; Male; Mass Spectrometry; Middle Aged; Neuroprotective Agents; Oleic Acids; Polyunsaturated Alkamides; PPAR alpha; Sleep Deprivation; Young Adult

Anandamide is a bioactive lipid binding to cannabinoid receptors. A homeostatic role for anandamide has been suggested in schizophrenia. We investigated its role in initial prodromal states of psychosis. We measured the levels of anandamide and its structural analog oleoylethanolamide in cerebrospinal fluid and serum of patients in the initial prodromal state (n=27) alongside healthy volunteers (n=81) using high-performance liquid chromatograph/mass spectrometry. Cerebrospinal anandamide levels in patients were significantly elevated. Patients with lower levels showed a higher risk for transiting to psychosis earlier. This anandamidergic up-regulation in the initial prodromal course may suggest a protective role of the endocannabinoid system in early schizophrenia.

Topics: Adult; Arachidonic Acids; Cannabinoid Receptor Modulators; Endocannabinoids; Female; Humans; Male; Mass Spectrometry; Oleic Acids; Polyunsaturated Alkamides; Psychotic Disorders; Young Adult

Central endocannabinoid signaling is known to be responsive to stressful stimuli; however, there is no research to date characterizing the effects of stress on peripheral endocannabinoid content. The current study examined serum content of the endocannabinoid ligands N-arachidonylethanolamide (anandamide; AEA) and 2-arachidonoylglycerol (2-AG), and the non-cannabinoid N-acyl ethanolamine (NAE) molecules palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) under basal conditions, immediately following the Trier Social Stress Test (TSST), and 30 min thereafter, in 15 medication-free women diagnosed with major depression, and 15 healthy matched controls. Basal serum concentrations of AEA and 2-AG, but not PEA or OEA, were significantly reduced in women with major depression relative to matched controls, indicating a deficit in peripheral endocannabinoid activity. Immediately following the TSST, serum 2-AG concentrations were increased compared to baseline; serum AEA concentration was unchanged at this time point. Serum concentrations of PEA and OEA were significantly lower than baseline 30 min following the cessation of the TSST. The magnitude of these responses did not differ between depressed and control subjects. These are the first data to demonstrate that the peripheral endocannabinoid/NAE system is responsive to exposure to stress.

Topics: Adult; Amides; Arachidonic Acids; Cannabinoid Receptor Modulators; Case-Control Studies; Depressive Disorder, Major; Endocannabinoids; Ethanolamines; Female; Glycerides; Humans; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Stress, Psychological

A simple and rapid analytical method is described for the simultaneous quantitative analysis of three different N-acylethanolamides in human biological samples: anandamide (AEA), oleoylethanolamide (OEA), and palmitoylethanolamide (PEA). The method is based on a new hybrid solid phase extraction-precipitation technology followed by ultra-performance liquid chromatography/mass spectrometry (UPLC/MS) analysis using d(4)-AEA as the internal standard. The method is linear up to 100ng/ml with a limit of quantitation of 50pg/ml for AEA and 100pg/ml for OEA and PEA. Good reproducibility, accuracy, and precision were demonstrated during the method validation. Application of this new methodology to the analysis of clinical study samples is presented.

Topics: Adolescent; Adult; Amides; Amidohydrolases; Arachidonic Acids; Calibration; Cannabinoids; Chromatography, High Pressure Liquid; Endocannabinoids; Enzyme Inhibitors; Ethanolamines; Female; Humans; Leukocytes, Mononuclear; Male; Middle Aged; Molecular Structure; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Reproducibility of Results; Semen; Solid Phase Microextraction; Tandem Mass Spectrometry; Young Adult

Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS. Therapies that affect the endocannabinoid (EC) system may have immunomodulatory, symptomatic and neuroprotective effects.. The aim of this study was to determine how levels of EC and related compounds are altered in MS.. Plasma and whole blood were collected from 24 MS patients (10 relapsing-remitting (RR); 8 secondary-progressive (SP); 6 primary-progressive (PP); 19 females; 25-66 years) and 17 controls (10 females; 22-62 years). Plasma EC and related compounds were quantified by liquid chromatography-tandem mass spectrometry. Fatty acid amide hydrolase (FAAH), cannabinoid receptors CB(1) and CB(2) mRNA were measured by quantitative reverse transcriptase-polymerase chain reaction.. Anandamide (AEA) and palmitoylethanolamide (PEA) were higher in RRMS compared to controls (p=0.001 and p=0.027). AEA, PEA and oleoylethanolamide were also increased in SPMS plasma (p=0.001, p=0.004, and p=0.005). PPMS patients had higher AEA plasma levels compared to controls (p=0.009). FAAH mRNA was decreased in SPMS (p=0.04) but not in RRMS or PPMS blood. CB(1) (p=0.012) and CB(2) mRNA (p=0.003) were increased in the PPMS.. The EC system is altered in MS. It may be dynamically modulated depending on the subtype of the disease, but further studies with larger subgroups are needed to confirm this.

Topics: Adult; Aged; Amides; Amidohydrolases; Arachidonic Acids; Brain; Brain Chemistry; Cannabinoid Receptor Modulators; Cannabinoids; Chromatography, Liquid; Cytoprotection; Disability Evaluation; Endocannabinoids; Ethanolamines; Female; Humans; Male; Mass Spectrometry; Middle Aged; Multiple Sclerosis; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Receptors, Cannabinoid; RNA, Messenger

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

The endocannabinoid N-arachidonoylethanolamide (anandamide) is co-synthesized with other N-acylethanolamides, namely N-palmitoylethanolamide (PEA) and N-oleoylethanolamide (OEA), which have been shown to potentiate anandamide responses (so-called 'entourage effects') in non-vascular tissues. It remains unclear whether such interactions occur in the circulation.. In rat isolated small mesenteric arteries, the effects of PEA and OEA on relaxation to anandamide and tissue contents of the N-acylethanolamides were examined under myographic conditions.. Anandamide-induced relaxation was potentiated by pretreatment with PEA (10 microM) or OEA (1 microM), or in combination. The potentiation by PEA and OEA was endothelium-independent and abolished by treatment with capsaicin (10 microM), which desensitizes the transient receptor potential vanilloid type 1 (TRPV1) receptor system, or by the TRPV1 receptor antagonist, N-(3-methoxyphenyl)-4-chlorocinnamide (SB366791) (2 microM). It was also observed at molar ratios of anandamide and PEA (or OEA) similar to those found in mesenteric arteries. PEA and inhibition of anandamide hydrolysis by 3'-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate (URB597) (1 microM) additively potentiated anandamide responses. On the other hand, PEA and OEA also induced vasorelaxation per se (rank order of potency: anandamide>OEA>PEA), but relaxation to the three N-acylethanolamides displayed different sensitivity to treatment with capsaicin, SB366791 and URB597. For example, relaxations to anandamide and OEA, but not PEA, were attenuated by both capsaicin and SB366791.. This study shows that PEA and OEA potentiate relaxant responses to anandamide through TRPV1 receptors in rat small mesenteric arteries. The congeners also induce vasorelaxation per se, suggesting a function for the N-acylethanolamides in vascular control.

Topics: Amides; Animals; Arachidonic Acids; Dose-Response Relationship, Drug; Drug Combinations; Endocannabinoids; Ethanolamines; Male; Mesenteric Artery, Superior; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Rats; Rats, Wistar; TRPV Cation Channels; Vasodilation

N-acylethanolamides (NAEs) are hydrophobic molecules synthesized in many tissues. An increase in the plasma levels of NAEs has been observed in human diseases. Previous studies have suggested that NAEs could exert a protective effect against oxidative stress. Aim of the study was to investigate whether NAEs (oleoylethanolamide, palmitoylethanolamide and anandamide), differing for acyl chain length and unsaturation, exert a protective role against plasma lipid peroxidation triggered by incubation with Cu2+2 or AAPH (2,2'-azobis(2-amidinopropane) dihydrochloride). Moreover, we investigated the effect of NAEs on the activity of HDL-associated paraoxonase (PON1), an enzyme involved in the antioxidant end anti-inflammatory role of human high density lipoproteins (HDL). The results demonstrated that the NAEs protect plasma lipids and PON1 activity against AAPH and/or copper-induced oxidation.

Topics: Adult; Amides; Amidines; Arachidonic Acids; Aryldialkylphosphatase; Endocannabinoids; Ethanolamines; Humans; Lipid Peroxidation; Middle Aged; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides

While it has long been recognized that Delta(9)-tetrahydrocannabinol (THC), the primary psychoactive constituent of cannabis, and other cannabinoid receptor agonists possess anti-inflammatory properties, their well known CNS effects have dampened enthusiasm for therapeutic development. On the other hand, genetic deletion of fatty acid amide hydrolase (FAAH), the enzyme responsible for degradation of fatty acid amides, including endogenous cannabinoid N-arachidonoyl ethanolamine (anandamide; AEA), N-palmitoyl ethanolamine (PEA), N-oleoyl ethanolamine (OEA), and oleamide, also elicits anti-edema, but does not produce any apparent cannabinoid effects. The purpose of the present study was to investigate whether exogenous administration of FAAs would augment the anti-inflammatory phenotype of FAAH (-/-) mice in the carrageenan model. Thus, we evaluated the effects of the FAAs AEA, PEA, OEA, and oleamide in wild-type and FAAH (-/-) mice. For comparison, we evaluated the anti-edema effects of THC, dexamethasone (DEX), a synthetic glucocorticoid, diclofenac (DIC), a nonselective cyclooxygenase (COX) inhibitor, in both genotypes. A final study determined if tolerance to the anti-edema effects of PEA occurs after repeated dosing. PEA, THC, DEX, DIC elicited significant decreases in carrageenan-induced paw edema in wild-type mice. In contrast OEA produced a less reliable anti-edema effect than these other drugs, and AEA and oleamide failed to produce any significant decreases in paw edema. Moreover, none of the agents evaluated augmented the anti-edema phenotype of FAAH (-/-) mice, suggesting that maximal anti-edema effects had already been established. PEA was the most effective FAA in preventing paw edema and its effects did not undergo tolerance. While the present findings do not support a role for AEA in preventing carrageenan-induced edema, PEA administration and FAAH blockade elicited anti-edema effects of an equivalent magnitude as produced by THC, DEX, and DIC in this assay.

Topics: Amidohydrolases; Analysis of Variance; Animals; Arachidonic Acids; Cannabinoids; Carrageenan; Dexamethasone; Disease Models, Animal; Dose-Response Relationship, Drug; Dronabinol; Drug Interactions; Edema; Endocannabinoids; Ethanolamines; Female; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Oleic Acids; Polyunsaturated Alkamides

The endocannabinoid system functions through two well characterized receptor systems, the CB1 and CB2 receptors. Work by a number of groups in recent years has provided evidence that the system is more complicated and additional receptor types should exist to explain ligand activity in a number of physiological processes.. Cells transfected with the human cDNA for GPR55 were tested for their ability to bind and to mediate GTPgammaS binding by cannabinoid ligands. Using an antibody and peptide blocking approach, the nature of the G-protein coupling was determined and further demonstrated by measuring activity of downstream signalling pathways.. We demonstrate that GPR55 binds to and is activated by the cannabinoid ligand CP55940. In addition endocannabinoids including anandamide and virodhamine activate GTPgammaS binding via GPR55 with nM potencies. Ligands such as cannabidiol and abnormal cannabidiol which exhibit no CB1 or CB2 activity and are believed to function at a novel cannabinoid receptor, also showed activity at GPR55. GPR55 couples to Galpha13 and can mediate activation of rhoA, cdc42 and rac1.. These data suggest that GPR55 is a novel cannabinoid receptor, and its ligand profile with respect to CB1 and CB2 described here will permit delineation of its physiological function(s).

Topics: Amino Acid Sequence; Animals; Arachidonic Acids; Binding Sites; Binding, Competitive; Cannabidiol; Cannabinoids; Cell Line; Cloning, Molecular; Cyclohexanols; Down-Regulation; Endocannabinoids; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Ligands; Mice; Molecular Sequence Data; Organ Specificity; Polymerase Chain Reaction; Polyunsaturated Alkamides; Rats; Receptors, Cannabinoid; Receptors, G-Protein-Coupled; RNA, Messenger; Signal Transduction; Structure-Activity Relationship

A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described for the simultaneous identification and quantification of eight endocannabinoid (EC) or related "entourage" compounds in rat brain tissue. Analytes were extracted and purified from rat brain tissue using an ethyl acetate/hexane solvent extraction, followed by a solid phase extraction (SPE) protocol. Chromatographic separation was achieved using a gradient elution, with a mobile phase of acetonitrile, formic acid, and ammonium acetate, at pH 3.6. A Thermo Hypersil C8 HyPurity Advance column (100x2.1 mm i.d., 3 microm) was used with a flow rate of 0.3 ml/min). Anandamide (AEA), 2-arachidonyl glycerol (2-AG), 2-arachidonylglyceryl ether (noladin ether), O-arachidonyl ethanolamide (virodhamine), 2-linoleoyl glycerol (2-LG), arachidonyl glycine, oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA) were quantified by positive ion tandem electrospray ionization mass spectrometry. Internal standards were deuterated AEA, deuterated 2-AG, and heptadecanoyl ethanolamide (HEA). Linearity was proven over the range of 25 fmol to 250 pmol, with a limit of detection of 25 fmol on column for all analytes except 2-AG, noladin ether, and 2-LG (250 fmol). This corresponded to a limit of quantification in biological tissue of 10 pmol/g for all analytes except 2-AG (100 pmol/g). Intra- and interday precision in biological tissue was routinely approximately 20% or lower, and accuracy was between 65% and 155%. This method was used to quantitatively profile regional differences in nine discrete rat brain regions for AEA, 2-AG, 2-LG, OEA, PEA, noladin ether, virodhamine, and arachidonyl glycine.

Topics: Animals; Arachidonic Acids; Brain Chemistry; Cannabinoid Receptor Modulators; Cannabinoids; Chromatography, Liquid; Endocannabinoids; Glycerides; Glycine; Male; Oleic Acids; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

We developed a new selective liquid chromatography-electrospray ionization-tandem mass spectrometry method for the identification and quantification of anandamide (AEA), an endogenous cannabinoid receptor ligand, and other bioactive fatty acid ethanolamides (FAEs) in biological samples. Detection limit (0.025 pmol for AEA and 0.1 pmol for palmitoylethanolamide (PEA) and oleoylethanolamide (OEA)) and quantification limit (0.2 pmol for AEA and 0.4 pmol for OEA and PEA) were in the high fmol to low pmol range for all analytes. Linear correlations (r(2)=0.99) were observed in the calibration curves for standard AEA over the range of 0.025-25 pmol and for standard PEA and OEA over the range of 0.1-500 pmol. This method provides a time-saving and sensitive alternative to existing methods for the analysis of FAEs in biological samples.

Topics: Amides; Arachidonic Acids; Chromatography, Liquid; Endocannabinoids; Ethanolamines; Female; Humans; Male; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

The endocannabinoid, arachidonoylethanolamide (AEA), and the peroxisome proliferator-activated receptor (PPAR)-alpha ligand, oleylethanolamide (OEA) produce opposite effects on lipogenesis. The regulation of OEA and its anti-inflammatory congener, palmitoylethanolamide (PEA), in adipocytes and pancreatic beta-cells has not been investigated. We report here the results of studies on acylethanolamide regulation in these cells during obesity and hyperglycaemia, and provide an overview of acylethanolamide role in metabolic control. We analysed by liquid chromatography-mass spectrometry OEA and PEA levels in: 1) mouse 3T3F442A adipocytes during insulin-induced differentiation, 2) rat insulinoma RIN m5F beta-cells kept in 'low' or 'high' glucose, 3) adipose tissue and pancreas of mice with high fat diet-induced obesity (DIO), and 4) in visceral fat or blood of obese or type 2 diabetes (T2D) patients. In adipocytes, OEA levels remain unchanged during differentiation, whereas those of PEA decrease significantly, and are under the negative control of both leptin and PPAR-gamma. PEA is significantly downregulated in subcutaneous adipose tissue of DIO mice. In RIN m5F insulinoma beta-cells, OEA and PEA levels are inhibited by 'very high' glucose, this effect being enhanced by insulin, whereas in cells kept for 24 h in 'high' glucose, they are stimulated by both glucose and insulin. Elevated OEA and PEA levels are found in the blood of T2D patients. Reduced PEA levels in hypertrophic adipocytes might play a role in obesity-related pro-inflammatory states. In beta-cells and human blood, OEA and PEA are down- or up-regulated under conditions of transient or chronic hyperglycaemia, respectively.

Topics: 3T3 Cells; Adipocytes; Adult; Aged; Amides; Animals; Arachidonic Acids; Diabetes Mellitus, Type 2; Endocannabinoids; Energy Metabolism; Ethanolamines; Female; Humans; Insulin-Secreting Cells; Leptin; Male; Mice; Mice, Inbred C57BL; Middle Aged; Models, Biological; Obesity; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; PPAR gamma; Review Literature as Topic; Structure-Activity Relationship

Although CB(1) receptor activation evokes neuroprotection in response to cannabinoids, some cannabinoids have been reported to be peroxisome proliferator activated receptor (PPAR) ligands, offering an alternative protective mechanism. We have, therefore, investigated the ability of a range of cannabinoids to activate PPAR alpha and for N-oleoylethanolamine (OEA), an endogenous cannabinoid-like compound (ECL), to evoke neuroprotection.. Assays of PPAR alpha occupancy and gene transactivation potential were conducted in cell-free and transfected HeLa cell preparations, respectively. In vivo estimates of PPAR alpha activation through fat mobilization and gene transcription were conducted in mice. Neuroprotection in vivo was investigated in wild-type and PPAR alpha gene-disrupted mice.. The ECLs OEA, anandamide, noladin ether and virodhamine were found to bind to the purified PPAR alpha ligand binding domain and to increase PPAR alpha-driven transcriptional activity. The high affinity synthetic CB(1/2) cannabinoid agonist WIN 55212-2 bound to PPAR alpha equipotently with the PPARalpha agonist fenofibrate, and stimulated PPARalpha-mediated gene transcription. The phytocannabinoid delta 9 tetrahydrocannabinol was without effect. OEA and WIN 55212-2 induced lipolysis in vivo, while OEA pre-treatment reduced infarct volume from middle cerebral artery occlusion in wild-type, but not in PPAR alpha-null mice. OEA treatment also led to increased expression of the NFkappa B-inhibitory protein, Ikappa B, in mouse cerebral cortex, while expression of the NFkappa B-regulated protein COX-2 was inhibited.. These data demonstrate the potential for a range of cannabinoid compounds, of diverse structures, to activate PPAR alpha and suggest that at least some of the neuroprotective properties of these agents could be mediated by nuclear receptor activation.

Topics: Animals; Arachidonic Acids; Benzoxazines; Cannabinoids; Cerebral Cortex; Cyclooxygenase 2; Dronabinol; Endocannabinoids; Fatty Acids, Unsaturated; Fenofibrate; HeLa Cells; Humans; I-kappa B Kinase; Ligands; Luciferases; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Morpholines; Naphthalenes; Neuroprotective Agents; Oleic Acids; Polyunsaturated Alkamides; PPAR alpha; Protein Binding; Recombinant Fusion Proteins; Transfection

The three amide oxyhomologues of the endolipids N-arachidonoylethanolamine (anandamide, AEA, 1a), N-oleoylethanolamine (OEA, 2a), and N-palmitoylethanolamine (PEA, 3a) have been prepared in a chemoselective way, capitalizing on the easy availability of O-[2-(triisopropylsilyoxy)ethyl]hydroxylamine (6) and the surprising complementary selectivity observed in the acylation of N-[2-(tert-butyldiphenylsilyloxy)ethyl]hydroxylamine (7) with the PPAA and the DCC/HOBT protocols. Reversal of the cannabinoid CB(1)/CB(2) receptor affinity ratio was observed for the first time in a derivative of anandamide (the O-alkyl-N-acyl hydroxylamine 1b), while the other oxyhomologues (1c and 1d) showed only marginal cannabimimetic activity. Compounds with unsaturated acyl chains generally retained vanilloid activity and showed an increased stability toward FAAH compared to their corresponding ethanolamides. Taken together, these observation show that oxyhomologation has a pronounced effect on both the pharmacodynamic and the pharmacokinetic properties of endogenous ethanolamides, suggesting a general relevance of this maneuver for the modification of amide pharmacophores.

Topics: Amides; Amidohydrolases; Animals; Arachidonic Acids; Cell Line; Chlorocebus aethiops; Endocannabinoids; Ethanolamines; Humans; Hydrolysis; Hydroxylamines; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Rats; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Structure-Activity Relationship; TRPV Cation Channels

The diurnal variations of the endocannabinoid arachidonoylethanolamine (anandamide, ANA) as well as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) were detected and quantified in cerebrospinal fluid (CSF), pons, hippocampus, and hypothalamus in the rat over 24 h using HPLC/MS. In CSF, the 3 compounds presented an increase in their concentration during the lights-on period and a remarkable decrease in their values during the lights-off period. In the pons, ANA, PEA and OEA showed the maximum values during the dark phase. On the other hand, we found that in the hippocampus, ANA increased its concentration during the lights-off period and PEA showed the highest peak at the beginning of the same period. OEA concentration showed no diurnal variations in the hippocampus. Finally, in the hypothalamus, ANA rose during the lights-on period whereas PEA and OEA presented the highest concentration at the end of the lights-off period. We postulate that all compounds are likely to be accumulated in parenchyma during the lights-off period (when animal is awake) and then, released into the CSF in order to reach target regions in turn to modulate diverse behaviors, such as feeding and sleep.

Topics: Amides; Animals; Arachidonic Acids; Brain Chemistry; Chromatography, High Pressure Liquid; Circadian Rhythm; Endocannabinoids; Ethanolamines; Hippocampus; Hypothalamus; Male; Mass Spectrometry; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Pons; Rats; Rats, Sprague-Dawley

The anorectic lipid oleoylethanolamide and the orexigenic lipid anandamide both belong to the group of N-acylethanolamines that are generated by the enzyme N-acylphosphatidylethanolamine-hydrolyzing phospholipase D. The levels of the two bioactive lipids were investigated in rat intestines after 24 h of starvation as well as after 1 and 4 h of re-feeding. Total levels of precursor phospholipids and N-acylethanolamines were decreased upon food-deprivation whereas the level of the anandamide precursor molecule was significantly increased. The level of 2-arachidonoyl-glycerol was unchanged as was the activity of N-acyltransferase, N-acylphosphatidylethanolamine-hydrolyzing phospholipase D, and fatty acid amide hydrolase upon starvation and re-feeding. It is concluded that remodeling of the amide-linked fatty acids of N-acylphosphatidylethanolamine is responsible for the opposite effects on levels of anandamide and oleoylethanolamide in intestines of food-deprived rats and not an alternative biochemical route for anandamide synthesis. Furthermore, linoleoylethanolamide, which accounted for more than 50 mol% of the endogenous pool of N-acylethanolamines, was found not to have the same inhibitory effect on food intake, as did oleoylethanolamide following oral administration.

Topics: Animals; Arachidonic Acids; Eating; Endocannabinoids; Food Deprivation; Intestinal Mucosa; Male; Oleic Acids; Phospholipids; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley

N-arachidonylethanolamine (AEA) accumulates during brain injury and postmortem. Because fatty acid amide hydrolase (FAAH) regulates brain AEA content, the purpose of this study was to determine its role in the postmortal accumulation of AEA using FAAH null mice. As expected, AEA content in immediately frozen brain tissue was significantly greater in FAAH-deficient (FAAH-/-) than in wild-type mice. However, AEA content was significantly lower in brains from FAAH-/- mice at 5 and 24 h postmortem. Similarly, wild-type mice treated in vivo with a FAAH inhibitor (URB532) had significantly lower brain AEA content 24 h postmortem compared with controls. These data indicate that FAAH contributes significantly to the postmortal accumulation of AEA. In contrast, the accumulations of two other N-acylethanolamines, N-oleoylethanolamine (OEA) and N-palmitoylethanolamine (PEA), were not reduced at 24 h postmortem in either the FAAH-/- mice or mice treated with URB532. FAAH-/- mice accumulated significantly less ethanolamine at 24 h postmortem compared with wild-type mice, suggesting that FAAH activity plays a role in the accumulation of ethanolamine postmortem. These data demonstrate that FAAH activity differentially affects AEA and OEA/PEA contents postmortem and suggest that AEA formation specifically occurs via an ethanolamine-dependent route postmortem.

Topics: Amides; Amidohydrolases; Animals; Arachidonic Acids; Brain; Endocannabinoids; Ethanolamine; Ethanolamines; Female; Hydrolysis; Lipid Metabolism; Male; Mice; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Transgenic; Oleic Acids; Palmitic Acids; Phosphatidylethanolamines; Pisum sativum; Polyunsaturated Alkamides; Postmortem Changes; Time Factors

Fatty acid amide hydrolase (FAAH) is an intracellular serine enzyme that catalyzes the hydrolysis of bioactive fatty acid ethanolamides such as anandamide and oleoylethanolamide (OEA). Genetic deletion of the faah gene in mice elevates brain anandamide levels and amplifies the effects of this endogenous cannabinoid agonist. Here, we show that systemic administration of the selective FAAH inhibitor URB597 (cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester; 0.3 mg/kg i.p.) increases anandamide levels in the brain of rats and wild-type mice but has no such effect in FAAH-null mutants. Moreover, URB597 enhances the hypothermic actions of anandamide (5 mg/kg i.p.) in wild-type mice but not in FAAH-null mice. In contrast, the FAAH inhibitor does not affect anandamide or OEA levels in the rat duodenum at doses that completely inhibit FAAH activity. In addition, URB597 does not alter the hypophagic response elicited by OEA (5 and 10 mg/kg i.p.), which is mediated by activation of peroxisome proliferator-activated receptor type-alpha. Finally, exogenously administered OEA (5 mg/kg i.p.) was eliminated at comparable rates in wild-type and FAAH-/- mice. Our results indicate that URB597 increases brain anandamide levels and magnifies anandamide responses by inhibiting intracellular FAAH activity. The results also suggest that an enzyme distinct from FAAH catalyzes OEA hydrolysis in the duodenum, where this lipid substance acts as a local satiety factor.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Body Temperature; Brain; Calcium Channel Blockers; Carbamates; Chromatography, High Pressure Liquid; Drug Synergism; Duodenum; Endocannabinoids; Enzyme Inhibitors; Feeding Behavior; Male; Mass Spectrometry; Mice; Mice, Inbred C57BL; Oleic Acids; Polyunsaturated Alkamides; PPAR alpha; Rats; Rats, Wistar

A new series of anandamide and N-oleoylethanolamine analogs, most of which with aromatic moieties in the head group region, has been synthesized and evaluated as inhibitors of anandamide uptake. Some of them efficaciously inhibit the uptake process with K(i) values in the low micromolar range (2.4-21.2microM). Strict structural requisites are needed to observe a significant inhibition and in no case inhibition of fatty acid amidohydrolase overlaps with inhibition of anandamide uptake.

Topics: Arachidonic Acids; Cell Line; Endocannabinoids; Humans; Ion Channels; Kinetics; Membrane Transport Modulators; Membrane Transport Proteins; Oleic Acids; Polyunsaturated Alkamides; Radioligand Assay; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Static Electricity; Structure-Activity Relationship; TRPV Cation Channels

The fatty acid oleoylethanolamide (OEA) is a satiety factor that excites peripheral vagal sensory nerves, but the mechanism by which this occurs and the molecular targets of OEA are unclear. In this study the ability of OEA to modulate the capsaicin receptor (TRPV1) was explored. OEA alone did not activate TRPV1 expressed in Xenopus oocytes under control conditions, but produced a differential modulation of agonist-evoked responses. OEA enhanced proton-gated TRPV1 currents, inhibited anandamide-evoked currents and had no effect on capsaicin-evoked responses. Following stimulation of protein kinase C (PKC), OEA alone directly activated TRPV1 channel with an EC50 of approximately 2 microm at room temperature. This effect was due to direct phosphorylation of TRPV1 because no responses to OEA were observed with mutant channels lacking critical PKC phosphorylation sites, S502A/S800A. In sensory neurons, OEA-induced Ca2+ rises that were selective for capsaicin-sensitive cells, inhibited by the TRPV1 blocker, capsazepine, and occurred in a PKC-dependent manner. Further, after PKC stimulation, OEA activated TRPV1 channels in cell-free patches suggesting a direct mode of action. Thus, TRPV1 represents a potential target for OEA and may contribute to the excitatory action of OEA on sensory nerves.

Topics: Animals; Arachidonic Acids; Calcium Channel Blockers; Capsaicin; Endocannabinoids; Female; Ganglia, Spinal; Gene Expression; Membrane Potentials; Neurons, Afferent; Oleic Acid; Oleic Acids; Oocytes; Patch-Clamp Techniques; Polyunsaturated Alkamides; Protein Kinase C; Rats; Receptors, Drug; Satiety Response; Xenopus laevis

This report presents a demonstration of ceramidase activity in the nuclear membrane or envelope of mammalian livers. The products of ceramidase reaction were identified by means of TLC for released fatty acid and HPLC for sphingosine. The ceramidase activity was maximum over a broad neutral to alkaline region ranging from pH 7.0 to 8.8. This activity was inhibited by N-oleoylethanolamine known as a specific inhibitor for ceramidase and by anandamide to a similar extent. The enzymatic study suggests that the nuclear ceramidase has different properties from other ceramidase reported previously. As sphingomyelinase, one of enzymes involved in the sphingomyelin cycle, are known to be present in the nuclear membrane, it is now evident that at least two enzymes involved in the sphingomyelin cycle are present in the nuclear membrane.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Cations, Divalent; Ceramidases; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Endocannabinoids; Enzyme Inhibitors; Ethanolamines; Hydrogen-Ion Concentration; Hydrolysis; In Vitro Techniques; Liver; Nuclear Envelope; Oleic Acids; Polyunsaturated Alkamides; Rats; Sphingosine; Swine

Excitotoxic insults such as stroke may induce release of fatty acid ethanolamides (FAEs), contributing to the downstream events in the ischemic cascade. We therefore studied release of FAEs such as anandamide, palmitylethanolamide (PEA), and oleylethanolamide (OEA) in the brain of a patient suffering from malignant hemispheric infarction treated with hypothermia.. A patient with life-threatening hemispheric stroke was treated with moderate hypothermia (33 degrees C) that was maintained for 3 days, followed by a 3-day rewarming period. Microdialysis was applied to measure glutamate, lactate, and glycerol by using a microdialysis analyzer. FAEs were measured by microdialysis coupled with high-performance liquid chromatography/mass spectrometry. Release of neuroprotective fatty amides occurred within the first day after ischemia and reached high concentrations for all 3 substances in tissue surrounding the primary ischemic lesion: anandamide up to 42 pmol/mL, PEA up to 120 pmol/mL, and OEA up to 242 pmol/mL. There was a significant correlation with elevation of lactate as early marker for the hypoxic insult.. This is the first report demonstrating release of FAEs in vivo during human stroke and may suggest contribution of the FAE signaling system to the pathophysiological events after ischemia.

Topics: Aged; Amides; Arachidonic Acids; Brain; Brain Chemistry; Brain Ischemia; Endocannabinoids; Ethanolamines; Extracellular Space; Glutamic Acid; Glycerol; Hemiplegia; Humans; Hypothermia, Induced; Lactic Acid; Male; Microdialysis; Monitoring, Physiologic; Oleic Acid; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Sleep Stages; Stroke

Recent studies suggest that the endocannabinoid system modulates feeding. Despite the existence of central mechanisms for the regulation of food intake by endocannabinoids, evidence indicates that peripheral mechanisms may also exist. To test this hypothesis, we investigated (1) the effects of feeding on intestinal anandamide accumulation; (2) the effects of central (intracerebroventricular) and peripheral (intraperitoneal) administration of the endocannabinoid agonist anandamide, the synthetic cannabinoid agonist R-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrol[1,2,3-de]-1,4-benzoxazin-6-yl)(1-naphthalenyl) methanone monomethanesulfonate (WIN55,212-2), and the CB1-selective antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide (SR141716A) on food intake in rats; and (3) the effects of sensory deafferentation on the modulation of feeding by cannabinoids. Food deprivation produced a sevenfold increase in anandamide content in the small intestine but not in the brain or stomach. Refeeding normalized intestinal anandamide levels. Peripheral but not central administration of anandamide or WIN55,212-2 promoted hyperphagia in partially satiated rats. Similarly, peripheral but not central administration of SR141716A reduced food intake. Capsaicin deafferentation abolished the peripheral effects of both cannabinoid agonists and antagonists, suggesting that these agents modulate food intake by acting on CB1 receptors located on capsaicin-sensitive sensory terminals. Oleoylethanolamide, a noncannabinoid fatty ethanolamide that acts peripherally, prevented hyperphagia induced by the endogenous cannabinoid anandamide. Pretreatment with SR141716A enhanced the inhibition of feeding induced by intraperitoneal administration of oleoylethanolamide. The results reveal an unexpected role for peripheral CB1 receptors in the regulation of feeding.

Topics: Animals; Appetite Regulation; Arachidonic Acids; Behavior, Animal; Benzoxazines; Brain; Cannabinoid Receptor Modulators; Capsaicin; Denervation; Dose-Response Relationship, Drug; Drug Synergism; Eating; Endocannabinoids; Feeding Behavior; Gastric Mucosa; Hyperphagia; Injections, Intraperitoneal; Injections, Intraventricular; Intestine, Small; Male; Morpholines; Naphthalenes; Oleic Acid; Oleic Acids; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Satiety Response

Endocannabinoids are lipid mediators thought to modulate central and peripheral neural functions. We report here gas chromatography-electron impact mass spectrometry analysis of human brain, showing that lipid extracts contain anandamide and 2-arachidonoylglycerol (2-AG), the most active endocannabinoids known to date. Human brain also contained the endocannabinoid-like compounds N-oleoylethanolamine, N-palmitoylethanolamine and N-stearoylethanolamine. Anandamide and 2-AG (0.16 +/- 0.05 and 0.10 +/- 0.05 nmol/mg protein, respectively) represented 7.7% and 4.8% of total endocannabinoid-like compounds, respectively. N-Palmitoyethanolamine was the most abundant (50%), followed by N-oleoyl (23.6%) and N-stearoyl (13.9%) ethanolamines. A similar composition in endocannabinoid-like compounds was found in human neuroblastoma CHP100 and lymphoma U937 cells, and also in rat brain. Remarkably, human meningioma specimens showed an approximately six-fold smaller content of all N-acylethanolamines, but not of 2-AG, and a similar decrease was observed in a human glioblastoma. These ex vivo results fully support the purported roles of endocannabinoids in the nervous system.

Topics: Amides; Animals; Arachidonic Acids; Brain Chemistry; Brain Neoplasms; Cannabinoid Receptor Modulators; Cannabinoids; Endocannabinoids; Ethanolamines; Gas Chromatography-Mass Spectrometry; Glioblastoma; Glycerides; Humans; Lymphoma; Meningioma; Neuroblastoma; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Rats; Rats, Wistar; Reference Values; Stearic Acids; Tumor Cells, Cultured; U937 Cells

Oleylethanolamide (OEA) is a natural analogue of the endogenous cannabinoid anandamide. Like anandamide, OEA is produced in cells in a stimulus-dependent manner and is rapidly eliminated by enzymatic hydrolysis, suggesting a function in cellular signalling. However, OEA does not activate cannabinoid receptors and its biological functions are still unknown. Here we show that, in rats, food deprivation markedly reduces OEA biosynthesis in the small intestine. Administration of OEA causes a potent and persistent decrease in food intake and gain in body mass. This anorexic effect is behaviourally selective and is associated with the discrete activation of brain regions (the paraventricular hypothalamic nucleus and the nucleus of the solitary tract) involved in the control of satiety. OEA does not affect food intake when injected into the brain ventricles, and its anorexic actions are prevented when peripheral sensory fibres are removed by treatment with capsaicin. These results indicate that OEA is a lipid mediator involved in the peripheral regulation of feeding.

Topics: Animals; Appetite Depressants; Arachidonic Acids; Drug Tolerance; Eating; Endocannabinoids; Feeding Behavior; Intestine, Small; Oleic Acid; Oleic Acids; Polyunsaturated Alkamides; Rats; Rats, Wistar

Anandamide amidohydrolase (AAH) catalyzes the hydrolysis of arachidonylethanolamide (anandamide), an endogenous cannabinoid receptor ligand. To delineate the structural requirements of AAH substrates, rat brain microsomal AAH hydrolysis of a series of anandamide congeners was studied using two reverse-phase high-performance liquid chromatography (RP-HPLC) assays developed in our laboratory. Arachidonamide (1) was found to be the best substrate with an apparent Km of 2.34 mM and a Vmax of 2.89 nmol/min/mg of protein. Although anandamide (2) has a similar Km value, its Vmax is approximately one-half that of arachidonamide. N, N-Bis(2-hydroxyethyl)arachidonamide (3) was not hydrolyzed, suggesting specificity for unsubstituted or mono-N-substituted arachidonamides. Analogues with a methyl group at the 1'-position of the ethanolamido headgroup were also found to have greater resistance to enzymatic turnover and therefore increased metabolic stability. The enzyme exhibited high stereoselectivity as the rate of hydrolysis of (R)-alpha-methanandamide (2.4%) (anandamide = 100%) was about 10-fold lower than that of its (S)-enantiomer (23%). In contrast, (R)-beta-methanandamide was 6-times more susceptible (121%) than the (S)-beta-enantiomer (21%). Interestingly, an inverse correlation was shown between AAH stereoselectivity and the brain cannabinoid receptor affinity as the enantiomers with high receptor affinity displayed low susceptibility to hydrolysis by AAH. Metabolic stability is also imparted to analogues with a short hydrocarbon headgroup as well as to those possessing 2-monomethyl or 2,2-dimethyl substituents. 2-Arachidonylglycerol and racemic 1-arachidonylglycerol were shown to be excellent AAH substrates. To identify AAH inhibitors, hydrolysis of anandamide was also studied in the presence of a select group of cannabimimetics. Of these, (-)-Delta8-THC and SR141716A, a biarylpyrazole CB1 antagonist, were found to inhibit enzymatic activity. These newly defined enzyme recognition parameters should provide a foundation for the rational development of stable, therapeutically useful anandamide analogues with high receptor affinity.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Brain; Cannabinoids; Endocannabinoids; Hydrolysis; Kinetics; Ligands; Microsomes; Polyunsaturated Alkamides; Rats; Receptors, Cannabinoid; Receptors, Drug; Stereoisomerism; Substrate Specificity

We measured endogenous cannabinoid release in dorsal striatum of freely moving rats by microdialysis and gas chromatography/mass spectrometry. Neural activity stimulated the release of anandamide, but not of other endogenous cannabinoids such as 2-arachidonylglycerol. Moreover, anandamide release was increased eightfold over baseline after local administration of the D2-like (D2, D3, D4) dopamine receptor agonist quinpirole, a response that was prevented by the D2-like receptor antagonist raclopride. Administration of the D1-like (D1, D5) receptor agonist SKF38393 had no such effect. These results suggest that functional interactions between endocannabinoid and dopaminergic systems may contribute to striatal signaling. In agreement with this hypothesis, pretreatment with the cannabinoid antagonist SR141716A enhanced the stimulation of motor behavior elicited by systemic administration of quinpirole. The endocannabinoid system therefore may act as an inhibitory feedback mechanism countering dopamine-induced facilitation of motor activity.

Topics: 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; Amides; Animals; Arachidonic Acids; Calcium; Cannabinoid Receptor Modulators; Corpus Striatum; Dopamine; Dopamine Agonists; Dopamine Antagonists; Endocannabinoids; Ethanolamines; Gas Chromatography-Mass Spectrometry; Glycerides; Hyperkinesis; Male; Microdialysis; Motor Activity; Oleic Acids; Palmitic Acids; Piperidines; Polyunsaturated Alkamides; Potassium; Pyrazoles; Quinpirole; Raclopride; Rats; Rats, Wistar; Receptors, Cannabinoid; Receptors, Dopamine D2; Receptors, Drug; Rimonabant; Salicylamides; Signal Transduction; Single-Blind Method; Sodium; Tetrodotoxin

Several analogues of the endogenous cannabinoid receptor ligand arachidonylethanolamide (anandamide) were synthesized and evaluated in order to study (a) the structural requirements for high-affinity binding to the CB1 and CB2 cannabinoid receptors and (b) their hydrolytic stability toward anandamide amidase. The series reported here was aimed at exploring structure-activity relationships (SAR) primarily with regard to stereoelectronic requirements of ethanolamido headgroup for interaction with the cannabinoid receptor active site. Receptor affinities, reported as Ki values, were obtained by a standard receptor binding assay using [3H]CP-55,940 as the radioligand, while stability toward the amidase was evaluated by comparing the Ki of each analogue in the presence and absence of phenylmethanesulfonyl fluoride (PMSF), a serine protease blocker and inhibitor of anandamide amidase. Introduction of a methyl group in the 1'- and 2'-positions or substitution of the ethanolamido headgroup with a butylamido group gave analogues with vastly improved biochemical stability. This is accomplished in some cases with increased receptor affinity. Conversely, oxazolyl and methyloxazolyl headgroups led to low-affinity analogues. Substitution of the hydroxyl group with electronegative substituents such as fluoro, chloro, allyl, and propargyl groups significantly increased receptor affinity but did not influence the biochemical stability. The 2'-chloro analogue of anandamide was found to have the highest affinity for CB1. Additionally, reversing the positions of the carbonyl and NH in the amido group produces retro-anandamides possessing considerably higher metabolic stability. Replacement of the arachidonyl tail with oleyl or linoleyl results in analogues with low affinities for both receptors. All of the analogues in this study showed high selectivity for the CB1 receptor over the peripheral CB2 receptor. The most potent analogues were tested for their ability to stimulate the binding of [35S]GTPgammaS to G-proteins and were shown to be potent cannabimimetic agonists. The results are discussed in terms of pharmacophoric features affecting receptor affinity and enzymatic stability.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Brain; Cannabinoids; Endocannabinoids; Guanosine 5'-O-(3-Thiotriphosphate); In Vitro Techniques; Mice; Polyunsaturated Alkamides; Radioligand Assay; Rats; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Serine Proteinase Inhibitors; Spleen; Structure-Activity Relationship; Tosyl Compounds