piperidines and glyceryl-2-arachidonate

In the past years, the relationship between the endocannabinoid system (ECS) and other hormonal and neuromodulatory systems has been intensively studied. G protein-coupled receptors (GPCRs) can stimulate endocannabinoid (eCB) production via activation of G(q/11) proteins and, in some cases, G(s) proteins. In this review, we summarize the pathways through which GPCR activation can trigger eCB release, as well as the best known examples of this process throughout the body tissues. Angiotensin II-induced activation of AT(1) receptors, similar to other G(q/11)-coupled receptors, can lead to the formation of 2-arachidonoylglycerol (2-AG), an important eCB. The importance of eCB formation in angiotensin II action is supported by the finding that the hypertensive effect of angiotensin II, injected directly into the hypothalamic paraventricular nucleus of anaesthetized rats, can be abolished by AM251, an inverse agonist of CB(1) cannabinoid receptors (CB(1)Rs). We conclude that activation of the ECS should be considered as a general consequence of the stimulation of G(q/11)-coupled receptors, and may mediate some of the physiological effects of GPCRs.

Topics: Angiotensin II; Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Endocannabinoids; Glycerides; GTP-Binding Protein alpha Subunits, Gq-G11; Humans; Paracrine Communication; Paraventricular Hypothalamic Nucleus; Piperidines; Pyrazoles; Rats; Receptor, Angiotensin, Type 1; Receptor, Cannabinoid, CB1

Endocannabinoids are endogenous compounds that bind to the same receptors as tetrahydrocannabinol, the active component in marijuana and hashish. They have been found to have many physiological and patho-physiological functions, including mood alteration, control of feeding and appetite, motor and co-ordination activities, analgesia, immune modulation and gut motility. In this review we aim to elucidate current knowledge as to their role in liver physiology and disease.. The major findings published to date concerning endocannabinoids and liver disease are described, and their implications with regard to understanding disease mechanisms, and the development of new treatments is considered.. Recently, endocannabinoids have been implicated in the hemodynamic alterations occurring in cirrhosis. These changes appear to be mediated via specific cannabinoid receptors (CB1) on splanchnic and hepatic vascular endothelium. Plasma levels of endocannabinoids also seem to be elevated in hepatitis, and are involved in apoptosis of hepatocytes by a membrane mechanism not related to a specific receptor. Other studies suggest a beneficial role for cannabinoids in reducing the inflammation of experimental hepatitis. In an animal model of acute hepatic failure, both endocannabinoids and the antagonist to the CB1 receptor have been found to have a beneficial effect on neurological and cognitive function.. Endocannabinoids appear to be involved in several aspects of acute and chronic liver disease, including vascular changes, modulation of inflammatory process and neurological function, Further research may provide new insights into the pathophysiology of liver disease, as well as a basis for novel treatment modalities.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Endocannabinoids; Glycerides; Humans; Liver Diseases; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant

The endocannabinoids, a family of endogenous lipids that activate cannabinoid receptors, are released from cells in a stimulus-dependent manner by cleavage of membrane lipid precursors. After release, the endocannabinoids are rapidly deactivated by uptake into cells and enzymatic hydrolysis. Endocannabinoid reuptake occurs via a carrier-mediated mechanism, which has not yet been molecularly characterized. Endocannabinoid reuptake has been demonstrated in discrete brain regions and in various tissues and cells throughout the body. Inhibitors of endocannabinoid reuptake include N-(4-hydroxyphenyl)-arachidonylamide (AM404), which blocks transport with IC50 (concentration necessary to produce half-maximal inhibition) values in the low micromolar range. AM404 does not directly activate cannabinoid receptors or display cannabimimetic activity in vivo. Nevertheless, AM404 increases circulating anandamide levels and inhibits motor activity, an effect that is prevented by the CB1 cannabinoid antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A). AM404 also reduces behavioral responses to dopamine agonists and normalizes motor activity in a rat model of attention deficit hyperactivity disorder. The endocannabinoids are hydrolyzed by an intracellular membrane-bound enzyme, termed anandamide amidohydrolase (AAH), which has been molecularly cloned. Several fatty acid sulfonyl fluorides inhibit AAH activity irreversibly with IC50 values in the low nanomolar range and protect anandamide from deactivation in vivo. alpha-Keto-oxazolopyridines inhibit AAH activity with high potency (IC50 values in the low picomolar range). A more thorough characterization of the roles of endocannabinoids in health and disease will be necessary to define the significance of endocannabinoid inactivation mechanisms as targets for therapeutic drugs.

Topics: Amidohydrolases; Arachidonic Acid; Arachidonic Acids; Biological Transport; Brain; Cannabinoid Receptor Modulators; Cannabinoids; Endocannabinoids; Glycerides; Piperidines; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Structure-Activity Relationship

Recent advances have dramatically increased our understanding of cannabinoid pharmacology: the psychoactive constituents of Cannabis sativa have been isolated, synthetic cannabinoids described and an endocannabinoid system identified, together with its component receptors, ligands and their biochemistry. Strong laboratory evidence now underwrites anecdotal claims of cannabinoid analgesia in inflammatory and neuropathic pain. Sites of analgesic action have been identified in brain, spinal cord and the periphery, with the latter two presenting attractive targets for divorcing the analgesic and psychotrophic effects of cannabinoids. Clinical trials are now required, but are hindered by a paucity of cannabinoids of suitable bioavailability and therapeutic ratio.

Topics: Amides; Amidohydrolases; Analgesics; Animals; Arachidonic Acids; Benzoxazines; Brain; Camphanes; Cannabinoid Receptor Modulators; Cannabinoids; Cell Membrane; Clinical Trials as Topic; Disease Models, Animal; Drug Design; Drug Interactions; Endocannabinoids; Enzyme Inhibitors; Ethanolamines; Glycerides; Humans; Injections, Spinal; Molecular Structure; Morpholines; Naphthalenes; Pain; Palmitates; Palmitic Acids; Piperidines; Plant Extracts; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Spinal Cord

Stress-related disorders are among the most prevalent psychiatric disorders, characterized by excess fear and enhanced avoidance of trauma triggers. Elucidating the mechanisms regulating temporally distinct aspects of innate and conditioned fear responses could facilitate novel therapeutic development for stress-related disorders. One potential target that has recently emerged is the endocannabinoid system, which has been reported to mediate the physiological response to stress and represents an important substrate underlying individual differences in stress susceptibility.. Here, we exposed male and female CD-1 mice to an innate predator stressor, 2MT (2-methyl-2-thiazoline), to investigate the ability of endocannabinoid signaling to modulate temporally distinct innate and conditioned fear behaviors.. We found that 2MT exposure increased amygdala 2-AG (2-arachidonoylglycerol) content and selectively increased excitability in central, but not basolateral, amygdala neurons. We also found that pharmacological 2-AG augmentation during stress exposure exacerbated both acute freezing responses and central amygdala hyperexcitability via cannabinoid receptor type 1- and type 2-dependent mechanisms. Finally, 2-AG augmentation during stress exposure reduced long-term contextual conditioned freezing, and 2-AG augmentation 24 hours after stress exposure reduced conditioned avoidance behavior.. Our findings demonstrate a bidirectional effect of 2-AG augmentation on innate and conditioned fear behavior, with enhancement of 2-AG levels during stress promoting innate fear responses but ultimately resulting in long-term conditioned fear reduction. These data could reconcile contradictory data on the role of 2-AG in the regulation of innate and conditioned fear-related behavioral responses.

Topics: Animals; Arachidonic Acids; Endocannabinoids; Female; Glycerides; Male; Mice; Odorants; Piperidines; Receptors, Cannabinoid

The endocannabinoid system modulates the stress coping strategies in the dorsolateral periaqueductal grey (dlPAG). The most relevant endocannabinoids, anandamide and 2-arachidonoylglycerol (2-AG) exert inhibitory control over defensive reactions mediated by the dlPAG. However, the protective role of anandamide is limited by its lack of effect in higher concentrations. Thus, the 2-AG emerges as a complementary target for developing new anxiolytic compounds. Nevertheless, the role of 2-AG on stress responsivity may vary according to the nature of the stimulus. In this study, we verified whether the dlPAG injection of 2-AG or inhibitors of its hydrolysis induce anxiolytic-like effects in male Wistar rats exposed to behavioral models in which physical stress (mild electric shock) is a critical component, namely the contextual fear conditioning test (CFC) and the Vogel conflict test (VCT). We also investigated the contribution of cannabinoid receptor type 1 (CB1) and type 2 (CB2) in such effects. The facilitation of 2-AG signaling in the dlPAG reduced contextual fear expression and exhibited an anxiolytic-like effect in the VCT in a mechanism dependent on activation of CB1 and CB2. However, the VCT required a higher dose than CFC. Further, the monoacylglycerol inhibitors, which inhibit the hydrolysis of 2-AG, were effective only in the CFC. In conclusion, we confirmed the anti-aversive properties of 2-AG in the dlPAG through CB1 and CB2 mechanisms. However, these effects could vary according to the type of stressor and the anxiety model employed.

Topics: Animals; Anti-Anxiety Agents; Arachidonic Acids; Endocannabinoids; Fear; Glycerides; Male; Periaqueductal Gray; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1

Hypoxia caused by ischemia induces acidosis and neuroexcitotoxicity, resulting in neuronal death in the central nervous system (CNS). Monoacylglycerol lipase (MAGL) is a modulator of 2-arachidonoylglycerol (2-AG), which is involved in retrograde inhibition of glutamate release in the endocannabinoid system. In the present study, we used positron emission tomography (PET) to monitor MAGL-positive neurons and neuroinflammation in the brains of ischemic rats. Additionally, we performed PET imaging to evaluate the neuroprotective effects of an MAGL inhibitor in an ischemic injury model.

Topics: Animals; Arachidonic Acids; Benzodioxoles; Brain; Brain Ischemia; Carbon Radioisotopes; Cell Hypoxia; Disease Models, Animal; Endocannabinoids; Glycerides; Infarction, Middle Cerebral Artery; Ischemic Stroke; Male; Minocycline; Monoacylglycerol Lipases; Neuroprotective Agents; Piperidines; Positron-Emission Tomography; Rats; Rats, Sprague-Dawley; Stroke; Tomography, X-Ray Computed

Alzheimer's disease (AD) is the most common cause of dementia, which affects more than 5 million individuals in the USA. Unfortunately, no effective therapies are currently available to prevent development of AD or to halt progression of the disease. It has been proposed that monoacylglycerol lipase (MAGL), the key enzyme degrading the endocannabinoid 2-arachidonoylglycerol (2-AG) in the brain, is a therapeutic target for AD based on the studies using the APP transgenic models of AD. While inhibition of 2-AG metabolism mitigates β-amyloid (Aβ) neuropathology, it is still not clear whether inactivation of MAGL alleviates tauopathies as accumulation and deposition of intracellular hyperphosphorylated tau protein are the neuropathological hallmark of AD. Here we show that JZL184, a potent MAGL inhibitor, significantly reduced proinflammatory cytokines, astrogliosis, phosphorylated GSK3β and tau, cleaved caspase-3, and phosphorylated NF-kB while it elevated PPARγ in P301S/PS19 mice, a tau mouse model of AD. Importantly, tau transgenic mice treated with JZL184 displayed improvements in spatial learning and memory retention. In addition, inactivation of MAGL ameliorates deteriorations in expression of synaptic proteins in P301S/PS19 mice. Our results provide further evidence that MAGL is a promising therapeutic target for AD.

Topics: Alzheimer Disease; Animals; Arachidonic Acids; Benzodioxoles; Cognition; Endocannabinoids; Female; Glycerides; Inflammation Mediators; Male; Maze Learning; Mice; Mice, Transgenic; Monoacylglycerol Lipases; Piperidines; tau Proteins; Tauopathies

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

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

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

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

Nicotine, the primary psychoactive component in tobacco, plays a major role in the initiation and maintenance of tobacco dependence and addiction, a leading cause of preventable death worldwide. An essential need thus exists for more effective pharmacotherapies for nicotine-use cessation. Previous reports suggest that pharmacological and genetic blockade of CB1 receptors attenuate nicotine reinforcement and reward; while exogenous agonists enhanced these abuse-related behaviors. In this study, we utilized complementary genetic and pharmacologic approaches to test the hypothesis that increasing the levels of the endocannabinoid 2-arachindonoylglycerol (2-AG), will enhance nicotine reward by stimulating neuronal CB1 receptors. Contrary to our hypothesis, we found that inhibition of monoacylglycerol lipase (MAGL), the primary catabolic enzyme of 2-AG, attenuates nicotine conditioned place preference (CPP) in mice, through a non-CB1 receptor-mediated mechanism. MAGL inhibition did not alter palatable food reward or Lithium Chloride (LiCl) aversion. In support of our findings, repeated MAGL inhibition did not induce a reduction in CB1 brain receptor levels or hinder function. To explore the potential mechanism of action, we investigated if MAGL inhibition affected other fatty acid levels in our CPP paradigm. Indeed, MAGL inhibition caused a concomitant decrease in arachidonic acid (AA) levels in various brain regions of interest, suggesting an AA cascade-dependent mechanism. This idea is supported by dose-dependent attenuation of nicotine preference by the selective COX-2 inhibitors valdecoxib and LM-4131. Collectively, these findings, along with our reported studies on nicotine withdrawal, suggest that inhibition of MAGL represents a promising new target for the development of pharmacotherapies to treat nicotine dependence.

Topics: Animals; Anti-Anxiety Agents; Arachidonic Acids; Benzodioxoles; Cannabinoid Receptor Agonists; Conditioning, Classical; Endocannabinoids; Enzyme Inhibitors; Glycerides; Male; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Knockout; Monoacylglycerol Lipases; Nicotine; Piperidines; Receptor, Cannabinoid, CB1; Reward; Tobacco Use Disorder

Modulation of the endocannabinoid system has been shown to alleviate neuropathic pain. The aim of this study was to evaluate if treatment with paclitaxel, a chemotherapeutic agent that induces neuropathic pain, affects endocannabinoid levels at a time when mice develop paclitaxel-induced mechanical allodynia. We also evaluated the peripheral antiallodynic activity of the endocannabinoid 2-arachidonoyl glycerol (2-AG) and an inhibitor of monoacylglycerol lipase (MAGL), an enzyme responsible for 2-AG hydrolysis.. Female BALB/c mice were treated intraperitoneally with paclitaxel to induce mechanical allodynia. Levels of the endocannabinoids, N-arachidonoylethanolamine (anandamide, AEA), 2-AG, and the N-acylethanolamines (NAEs), N-palmitoylethanolamide (PEA) and N-oleoylethanolamide (OEA), which are structurally-related to AEA, in the brain, spinal cord and paw skin were measured using LC-MS/MS. Protein expression of MAGL in the paw skin was measured using Wes™. The effects of subcutaneous (s.c.) injection of 2-AG and JZL184 (a MAGL inhibitor) into the right hind paw of mice with paclitaxel-induced mechanical allodynia were assessed using the dynamic plantar aesthesiometer. The effects of pretreatment, s.c., into the right hind paw, with cannabinoid type 1 (CB. The levels of 2-AG were reduced only in the paw skin of paclitaxel-treated mice, whilst the levels of AEA, PEA and OEA were not significantly altered. There was no change in the expression of MAGL in the paw skin. Administration of 2-AG and JZL184 produced antiallodynic effects against paclitaxel-induced mechanical allodynia in the injected right paw, but did not affect the uninjected left paw. The antiallodynic activity of 2-AG was antagonized by both AM251 and AM630.. These results indicate that during paclitaxel-induced mechanical allodynia there is a deficiency of 2-AG in the periphery, but not in the CNS. Increasing 2-AG in the paw by local administration of 2-AG or a MAGL inhibitor, alleviates mechanical allodynia in a CB

Topics: Analgesics; Animals; Arachidonic Acids; Benzodioxoles; Cannabinoid Receptor Agonists; Disease Models, Animal; Endocannabinoids; Enzyme Inhibitors; Female; Glycerides; Hyperalgesia; Mice, Inbred BALB C; Monoacylglycerol Lipases; Neuralgia; Paclitaxel; Piperidines; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Skin

Cannabinoids induce biphasic effects on memory depending on stress levels. We previously demonstrated that different stress intensities, experienced soon after encoding, impaired rat short-term recognition memory in a time-of-day-dependent manner, and that boosting endocannabinoid anandamide (AEA) levels restored memory performance. Here, we examined if two different stress intensities and time-of-day alter hippocampal endocannabinoid tone, and whether these changes modulate short-term memory.. Male Sprague-Dawley rats were subjected to an object recognition task and exposed, at two different times of the day (i.e., morning or afternoon), to low or high stress conditions, immediately after encoding. Memory retention was assessed 1 hr later. Hippocampal AEA and 2-arachidonoyl glycerol (2-AG) content and the activity of their primary degrading enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), were measured soon after testing.. Consistent with our previous findings, low stress impaired 1-hr memory performance only in the morning, whereas exposure to high stress impaired memory independently of testing time. Stress exposure decreased AEA levels independently of memory alterations. Interestingly, exposure to high stress decreased 2-AG content and, accordingly, increased MAGL activity, selectively in the afternoon. Thus, to further evaluate 2-AG's role in the modulation of short-term recognition memory, rats were given bilateral intra-hippocampal injections of the 2-AG hydrolysis inhibitor KML29 immediately after training, then subjected to low or high stress conditions and tested 1 hr later.. KML29 abolished the time-of-day-dependent impairing effects of stress on short-term memory, ameliorating short-term recognition memory performance.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzodioxoles; Emotions; Endocannabinoids; Glycerides; Hippocampus; Humans; Male; Memory, Short-Term; Monoacylglycerol Lipases; Piperidines; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Signal Transduction

Individuals with alcohol use disorder exhibit compulsive habitual behaviors that are thought to be, in part, a consequence of chronic and persistent use of alcohol. The endocannabinoid system plays a critical role in habit learning and in ethanol self-administration, but the role of this neuromodulatory system in the expression of habitual alcohol seeking is unknown. Here, we investigated the role of the endocannabinoid system in established alcohol habits using contingency degradation in male C57BL/6 mice. We found that administration of the novel diacyl glycerol lipase inhibitor DO34, which decreases the biosynthesis of the endocannabinoid 2-arachidonoyl glycerol (2-AG), reduced habitual responding for ethanol and ethanol approach behaviors. Moreover, administration of the endocannabinoid transport inhibitor AM404 or the cannabinoid receptor type 1 antagonist AM251 produced similar reductions in habitual responding for ethanol and ethanol approach behaviors. Notably, AM404 was also able to reduce ethanol seeking and consumption in mice that were insensitive to lithium chloride-induced devaluation of ethanol. Conversely, administration of JZL184, a monoacyl glycerol lipase inhibitor that increases levels of 2-AG, increased motivation to respond for ethanol on a progressive ratio schedule of reinforcement. These results demonstrate an important role for endocannabinoid signaling in the motivation to seek ethanol, in ethanol-motivated habits, and suggest that pharmacological manipulations of endocannabinoid signaling could be effective therapeutics for treating alcohol use disorder.

Topics: Alcohol Drinking; Animals; Arachidonic Acids; Benzodioxoles; Cannabinoid Receptor Antagonists; Central Nervous System Depressants; Drug-Seeking Behavior; Endocannabinoids; Ethanol; Glycerides; Habits; Lipoprotein Lipase; Lithium Chloride; Mice; Monoacylglycerol Lipases; Motivation; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1

Endocannabinoids (eCBs) anandamide (AEA) and 2-arachidonoylglycerol (2-AG) were shown to be involved in the basis of trauma-induced behavioral changes, particularly contextual conditioned fear, however, their ligand-specific effects and possible interactions are poorly understood. Here we assessed specific eCB effects and interactions on acquisition of contextual conditioned fear employing electric footshocks in a rat model. We selectively increased eCB levels by pharmacological blockade of the degrading enzymes of AEA by URB597 and 2-AG by JZL184 before traumatization either systemically or locally in relevant brain areas, the prelimbic cortex (PrL), ventral hippocampus (vHC) and basolateral amygdala (BLA). Following traumatization, a series of contextual reminders were conducted during which conditioned fear was assessed. While systemic URB597-treatment during traumatization only slightly enhanced the acquisition of contextual conditioned fear, administration of the compound in the PrL and vHC led to the acquisition of stable, lasting conditioned fear, resistant to extinction. These effects of URB597 were blocked by simultaneous administration of JZL184. Similar treatment effects did not occur in the BLA. Treatment effects were not secondary to alterations in locomotor activity or nociception. Our findings suggest that AEA and 2-AG functionally interact in the regulation of acquisition of contextual conditioned fear. AEA signaling in the PrL and vHC is a crucial promoter of fear acquisition while 2-AG potentially modulates this effect. The lack of eCB effects in the BLA suggests functional specificity of eCBs at distinct brain sites.

Topics: Animals; Arachidonic Acids; Benzamides; Benzodioxoles; Brain; Carbamates; Central Nervous System Agents; Conditioning, Psychological; Endocannabinoids; Fear; Glycerides; Male; Motor Activity; Nociception; Piperidines; Polyunsaturated Alkamides; Random Allocation; Rats, Wistar

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

The endocannabinoid system (ECS) comprises the cannabinoids anandamide and 2-arachidonoylglycerol and the cannabinoid receptors 1 and 2 (Cnr1 and Cnr2). The function of these receptors in relation to zebrafish larval behavior is poorly understood, even though the zebrafish larva has become a versatile animal model in biomedical research.. The objective of the present study is to characterize the function of Cnr1 and Cnr2 in relation to behavior in zebrafish.. Behavioral analysis of zebrafish larvae was performed using a visual motor response (VMR) test, which allows locomotor activity to be determined under basal conditions and upon a dark challenge.. Treatment with the non-specific Cnr agonists WIN55,212-2 and CP55,940 resulted in a decrease in locomotion. This was observed for both basal and challenge-induced locomotion, although the potency for these two effects was different, which suggests different mechanisms of action. In addition, WIN55,212-2 increased the reaction time of the startle response after the dark challenge. Using the Cnr1 antagonist AM251 and a cnr1. Taken together, these results show that Cnr1 activation by exogenous endocannabinoids modulates both basal and challenge-induced locomotor activity in zebrafish larvae and that these behavioral effects can be used as a readout to monitor the Cnr1 responsiveness in the zebrafish larva model system.

Topics: Animals; Arachidonic Acids; Cannabinoids; Dark Adaptation; Dose-Response Relationship, Drug; Endocannabinoids; Glycerides; Larva; Locomotion; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Zebrafish; Zebrafish Proteins

Monoacylglycerol lipase (MAGL) is the main enzyme implicated in the degradation of the most abundant endocannabinoid in the brain, 2-arachidonoylglycerol (2-AG), producing arachidonic acid (AA) and glycerol. MAGL pharmacological inhibition with JZL184 or genetic deletion results in an exacerbated 2-AG signaling and reduced synthesis of prostaglandins (PGs), due to the reduced AA precursor levels. We found that acute JZL184 administration, previously described to exert anti-inflammatory effects, and MAGL knockout (KO) mice display cerebellar, but not hippocampal, microglial reactivity, accompanied with increased expression of the mRNA levels of neuroinflammatory markers, such as cyclooxygenase-2 (COX-2). Notably, this neuroinflammatory phenotype correlated with relevant motor coordination impairment in the beam-walking and the footprint tests. Treatment with the COX-2 inhibitor NS398 during 5 days prevented the deficits in cerebellar function and the cerebellar microglia reactivity in MAGL KO, without affecting hippocampal reactivity. Altogether, this study reveals the brain region-specific response to MAGL inhibition, with an important role of COX-2 in the cerebellar deficits associated, which should be taken into account for the use of MAGL inhibitors as anti-inflammatory drugs.

Topics: Amidohydrolases; Animals; Arachidonic Acid; Arachidonic Acids; Benzodioxoles; Cerebellum; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Endocannabinoids; Glycerides; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Monoacylglycerol Lipases; Motor Activity; Neuroimmunomodulation; Nitrobenzenes; Piperidines; Signal Transduction; Sulfonamides

Topics: Actin Depolymerizing Factors; Actins; AMP-Activated Protein Kinases; Arachidonic Acids; Benzimidazoles; Blood Platelets; Calcium-Calmodulin-Dependent Protein Kinase Kinase; Cell Adhesion Molecules; Cycloheximide; Endocannabinoids; Glycerides; Humans; Microfilament Proteins; Myosin Light Chains; Naphthalimides; Phosphoproteins; Phosphorylation; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; Signal Transduction

Clinical reports suggest that rather than directly driving cocaine use, stress may create a biological context within which other triggers for drug use become more potent. We hypothesize that stress-induced increases in corticosterone "set the stage" for relapse by promoting endocannabinoid-induced attenuation of inhibitory transmission in the prelimbic cortex (PL).. We have established a rat model for these stage-setting effects of stress. In this model, neither a stressor (electric footshock) nor stress-level corticosterone treatment alone reinstates cocaine seeking following self-administration and extinction, but each treatment potentiates reinstatement in response to an otherwise subthreshold cocaine priming dose (2.5 mg/kg, intraperitoneal). The contributions of endocannabinoid signaling in the PL to the effects of stress-level corticosterone on PL neurotransmission and cocaine seeking were determined using intra-PL microinfusions. Endocannabinoid-dependent effects of corticosterone on inhibitory synaptic transmission in the rat PL were determined using whole-cell recordings in layer V pyramidal neurons.. Corticosterone application attenuated inhibitory synaptic transmission in the PL via cannabinoid receptor type 1 (CB. These findings suggest that stress-induced increases in corticosterone promote cocaine seeking by mobilizing 2-arachidonoylglycerol in the PL, resulting in CB

Topics: Animals; Arachidonic Acids; Cocaine; Cocaine-Related Disorders; Drug-Seeking Behavior; Endocannabinoids; Extinction, Psychological; Glucocorticoids; Glycerides; Male; Piperidines; Prefrontal Cortex; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Self Administration; Stress, Psychological

Exposure to a traumatic event may result in the development of post-traumatic stress disorder (PTSD). Endocannabinoids are crucial modulators of the stress response, interfere with excessive retrieval and facilitate the extinction of traumatic memories. Exposure therapy, combined with pharmacotherapy, represents a promising tool for PTSD treatment. We investigated whether pharmacological manipulations of the endocannabinoid system during extinction learning ameliorates the behavioral changes induced by trauma exposure. Rats were exposed to inescapable footshocks paired with social isolation, a risk factor for PTSD. One week after trauma, rats were subjected to three spaced extinction sessions, mimicking human exposure therapy. The anandamide hydrolysis inhibitor URB597, the 2-arachidonoylglycerol hydrolysis inhibitor JZL184 or the cannabinoid agonist WIN55,212-2 were administered before or after the extinction sessions. Rats were tested for extinction retention 16 or 36 days after trauma and 24-h later for social interaction. Extinction training alone reduced fear of the trauma-associated context but did not restore normal social interaction. Traumatized animals not exposed to extinction sessions exhibited reductions in hippocampal anandamide content with respect to home-cage controls. Noteworthy, all drugs exerted beneficial effects, but URB597 (0.1 mg/kg) induced the best improvements by enhancing extinction consolidation and restoring normal social behavior in traumatized rats through indirect activation of CB1 receptors. The ameliorating effects remained stable long after treatment and trauma exposure. Our findings suggest that drugs potentiating endocannabinoid neurotransmission may represent promising tools when combined to exposure-based psychotherapies in the treatment of PTSD.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Benzodioxoles; Benzoxazines; Cannabinoid Receptor Modulators; Carbamates; Disease Models, Animal; Electroshock; Endocannabinoids; Extinction, Psychological; Glycerides; Male; Morpholines; Naphthalenes; Piperidines; Psychotropic Drugs; Rats, Sprague-Dawley; Social Isolation; Stress Disorders, Post-Traumatic; Synaptic Transmission

Current therapeutics are not effective for orofacial neuropathic pain, and better options are needed. The present study used inferior orbital nerve (ION)-injured mice to investigate the effect of inhibiting monoacylglycerol lipase (MAGL), an enzyme that degrades the major endocannabinoid 2-arachydonoylgycerol (2-AG) in orofacial neuropathic pain. The head-withdrawal threshold to mechanical stimulation of the whisker pad was reduced on days 3, 5, and 7 after ION injury. Injection of JZL184, a selective inhibitor of MAGL, on day 7 after ION injury attenuated the reduction in head-withdrawal threshold at 2 h after administration. Moreover, the numbers of MAGL-immunoreactive neurons in the trigeminal subnucleus caudalis (Vc) and upper cervical spinal cord (C1-C2) were significantly greater in ION-injured mice than in sham-operated mice but were reduced after administration of JZL184. The increase in MAGL immunoreactivity suggests that increased 2-AG production is followed by rapid enzymatic degradation of 2-AG. JZL184 inhibited this degradation and thus increased 2-AG concentration in the brain, particularly in the Vc and C1-C2 regions, thus attenuating pain. Our findings suggest that inhibition of 2-AG degradation by MAGL inhibitors is a promising therapeutic option for treatment of orofacial neuropathic pain.

Topics: Animals; Arachidonic Acids; Behavior, Animal; Benzodioxoles; Endocannabinoids; Enzyme Inhibitors; Facial Pain; Glycerides; Male; Mice, Inbred C57BL; Monoacylglycerol Lipases; Neuralgia; Piperidines; Trigeminal Nerve Injuries

2-Arachidonoylglycerol (2-AG) and anandamide are two major endocannabinoids produced, released and eliminated by metabolic pathways. Anticonvulsive effect of 2-AG and CB1 receptor is well-established. Herein, we designed to investigate the anticonvulsive influence of key components of the 2-AG and anandamide metabolism. Tonic-clonic seizures were induced by an injection of Pentylenetetrazol (80 mg/kg, i.p.) in adult male Wistar rats. Delay and duration for the seizure stages were considered for analysis. Monoacylglycerol lipase blocker (JJKK048; 1 mg/kg) or alpha/beta hydroxylase domain 6 blocker (WWL70; 5 mg/kg) were administrated alone or with 2-AG to evaluate the anticonvulsive potential of these enzymes. To determine the CB1 receptor involvement, its blocker (MJ15; 3 mg/kg) was administrated associated with JJKK048 or WWL70. To assess anandamide anticonvulsive effect, anandamide membrane transporter blocker (LY21813240; 2.5 mg/kg) was used alone or associated with MJ15. Also, fatty acid amide hydrolase blocker (URB597; 1 mg/kg; to prevent intracellular anandamide hydrolysis) were used alone or with AMG21629 (transient receptor potential vanilloid; TRPV1 antagonist; 3 mg/kg). All compounds were dissolved in DMSO and injected i.p., before the Pentylenetetrazol. Both JJKK048 and WWL70 revealed anticonvulsive effect. Anticonvulsive effect of JJKK048 but not WWL70 was CB1 receptor dependent. LY2183240 showed CB1 receptor dependent anticonvulsive effect. However, URB597 revealed a TRPV1 dependent proconvulsive effect. It seems extracellular accumulation of 2-AG or anandamide has anticonvulsive effect through the CB1 receptor, while intracellular anandamide accumulation is proconvulsive through TRPV1.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Disease Models, Animal; Endocannabinoids; Glycerides; Male; Pentylenetetrazole; Piperidines; Polyunsaturated Alkamides; Rats, Wistar; Receptor, Cannabinoid, CB1; Seizures; TRPV Cation Channels

Activating the endocannabinoid system has become a major focus in the search for novel therapeutics for anxiety and deficits in fear extinction, two defining features of PTSD. We examined whether chronic treatment with the fatty acid amide hydrolase (FAAH) inhibitor URB597 (0.2, 0.3, 0.4 mg/kg, i.p.) or the CB1/2 receptor agonist WIN55,212-2 (0.25, 0.5 mg/kg, i.p.) injected for 3 weeks to rats exposed to the shock and reminders model of PTSD would attenuate post-stress symptoms and affect basolateral amygdala (BLA) and CA1 CB1 receptors. Exposure to shock and reminders enhanced acoustic startle response and impaired extinction. Rats exposed to shock and reminders and chronically treated with URB597 demonstrated normalized startle response and intact extinction kinetics. WIN55,212-2 only affected the startle response. The therapeutic effects of URB597 and WIN55,212-2 were found to be CB1 receptor dependent, as these effects were blocked when a low dose of the CB1 receptor antagonist AM251 (0.3 mg/kg, i.p. for 3 weeks) was co-administered. Moreover, URB597, but not WIN55,212-2, normalized the shock/reminders-induced upregulation in CB1 receptor levels in the BLA and CA1. One hour after the shock, N-arachidonoylethanolamine (AEA) was increased in the BLA and decreased in the CA1. Circulating 2-arachidonoylglycerol (2-AG) concentrations were decreased in shocked rats, with no significant effect in the BLA or CA1. FAAH activity was increased in the CA1 of shocked rats. Chronic cannabinoid treatment with URB597 can ameliorate PTSD-like symptoms suggesting FAAH inhibitors as a potentially effective therapeutic strategy for the treatment of disorders associated with inefficient fear coping.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Basolateral Nuclear Complex; Benzamides; Benzoxazines; CA1 Region, Hippocampal; Cannabinoid Receptor Antagonists; Carbamates; Dose-Response Relationship, Drug; Electric Stimulation; Endocannabinoids; Extinction, Psychological; Glycerides; Male; Morpholines; Naphthalenes; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Reflex, Startle; Stress Disorders, Post-Traumatic

The endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) is a known modulator of inflammation and ligand to both, pro-inflammatory cannabinoid receptor 1 (CB1) and anti-inflammatory CB2. While the role of both receptors in atherogenesis has been studied extensively, the significance of 2-AG for atherogenesis is less well characterized.. The impact of 2-AG on atherogenesis was studied in two treatment groups of ApoE-/- mice. One group received the monoacylglycerol lipase (MAGL)-inhibitor JZL184 [5 mg/kg i.p.], which impairs 2-AG degradation and thus causes elevated 2-AG levels, the other group received vehicle for four weeks. Simultaneously, both groups were fed a high-cholesterol diet. The atherosclerotic plaque burden was assessed in frozen sections through the aortic sinus following oil red O staining and infiltrating macrophages were detected by immunofluorescence targeting CD68. In vitro, the effect of 2-AG on B6MCL macrophage migration was assessed by Boyden chamber experiments. Transcription of adhesion molecules and chemokine receptors in macrophages was assessed by qPCR.. As expected, application of the MAGL-inhibitor JZL184 resulted in a significant increase in 2-AG levels in vascular tissue (98.2 ± 16.1 nmol/g vs. 27.3 ± 4.5 nmol/g; n = 14-16; p < 0.001). ApoE-/- mice with elevated 2-AG levels displayed a significantly increased plaque burden compared to vehicle treated controls (0.44 ± 0.03 vs. 0.31 ± 0.04; n = 14; p = 0.0117). This was accompanied by a significant increase in infiltrating macrophages within the atherosclerotic vessel wall (0.33 ± 0.02 vs. 0.27 ± 0.01; n = 13-14; p = 0.0076). While there was no alteration to the white blood counts of JZL184-treated animals, 2-AG enhanced macrophage migration in vitro by 1.8 ± 0.2 -fold (n = 4-6; p = 0.0393) compared to vehicle, which was completely abolished by co-administration of either CB1- or CB2-receptor-antagonists. qPCR analyses of 2-AG-stimulated macrophages showed an enhanced transcription of the chemokine CCL5 (1.59 ± 0.23 -fold; n = 5-6; p = 0.0589) and its corresponding receptors CCR1 (2.04 ± 0.46 -fold; n = 10-11; p = 0.0472) and CCR5 (2.45 ± 0.62 -fold; n = 5-6; p = 0.0554).. Taken together, elevated 2-AG levels appear to promote atherogenesis in vivo. Our data suggest that 2-AG promotes macrophage migration, possibly by the CCL5-CCR5/CCR1 axis, and thereby contributes to vascular inflammation. Thus, decreasing vascular 2-AG levels might represent a promising therapeutic strategy in patients suffering from atherosclerosis and coronary heart disease.

Topics: Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Apolipoproteins E; Arachidonic Acids; Atherosclerosis; Benzodioxoles; Cell Line; Cell Movement; Diet, High-Fat; Endocannabinoids; Glycerides; Macrophages; Male; Mice; Piperidines

The endocannabinoid system is a key modulator of memory consolidation for aversive experiences. We recently found that the fatty acid amide hydrolase (FAAH) inhibitor URB597, which increases anandamide levels by inhibiting its hydrolysis, facilitates memory consolidation through a concurrent activation of both cannabinoid receptor type 1 (CB1) and 2 (CB2). Here, we investigated the role played on memory consolidation by the other major endocannabinoid, 2-arachidonoylglycerol (2-AG). To this aim, we tested the effects of pharmacological inhibition of monoacylglycerol lipase (MAGL) through systemic administration of the MAGL inhibitor JZL184 to rats immediately after training of the inhibitory avoidance task. Pharmacological enhancement of 2-AG tone facilitated memory consolidation through activation of CB2 receptor signaling. Moreover, we found that increased 2-AG signaling prevented the activation of the mammalian target of rapamycin (mTOR) signaling pathway in the hippocampus through a CB2-dependent mechanism. Our results identify a fundamental role for 2-AG and CB2 receptors in the modulation of memory consolidation for aversive experiences.

Topics: Animals; Arachidonic Acids; Avoidance Learning; Benzodioxoles; Cannabinoid Receptor Modulators; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Glycerides; Hippocampus; Hydrolysis; Male; Memory Consolidation; Monoacylglycerol Lipases; Nootropic Agents; Piperidines; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Signal Transduction; TOR Serine-Threonine Kinases

Prominent motor deficits (e.g., chorea) that typify Huntington's disease (HD) arise following a prolonged prodromal stage characterized by psychiatric disturbances. Apathy, a disorder of motivation characterized by diminished goal-directed behavior, is one of the earliest and most common psychiatric symptoms in HD, but the underlying neurobiology is unclear and treatment options are limited. Alterations in the endocannabinoid (eCB) and dopamine systems represent prominent pathophysiological markers in HD that-similar to motivational deficits-present early and decline across disease progression. Whether changes in dopamine and eCB systems are associated with specific behavioral impairments in HD and whether these deficits are amenable to viable treatments is unknown. Here, we show that dopaminergic encoding of effortful drive progressively declines with age in an HD mouse model, and is restored by elevating tissue levels of the eCB 2-arachidonoylglycerol (2-AG) through targeted inhibition of its enzymatic degradation. This work supports aberrant dopaminergic encoding of reward as a neurobiological correlate of apathy in HD, and indicates that cannabinoid receptor-based therapies may benefit neuropsychiatric care for HD.

Topics: Aging; Animals; Apathy; Arachidonic Acids; Benzodioxoles; Conditioning, Operant; Disease Progression; Dopamine; Endocannabinoids; Glycerides; Huntington Disease; Male; Mice; Motivation; Piperidines; Pyrazoles; Reward

Topics: Analgesics; Animals; Antipruritics; Arachidonic Acids; Brain; Cell Line, Tumor; Cyclooxygenase Inhibitors; Endocannabinoids; Enzyme Inhibitors; Glycerides; Humans; Hydrolysis; Male; Mice; Mice, Inbred ICR; Monoacylglycerol Lipases; Pain; PC-3 Cells; Piperidines; Prostaglandins; Rats; Rats, Sprague-Dawley; Rodentia

Stress is a ubiquitous risk factor for the exacerbation and development of affective disorders including major depression and posttraumatic stress disorder. Understanding the neurobiological mechanisms conferring resilience to the adverse consequences of stress could have broad implications for the treatment and prevention of mood and anxiety disorders. We utilize laboratory mice and their innate inter-individual differences in stress-susceptibility to demonstrate a critical role for the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) in stress-resilience. Specifically, systemic 2-AG augmentation is associated with a stress-resilient phenotype and enhances resilience in previously susceptible mice, while systemic 2-AG depletion or CB1 receptor blockade increases susceptibility in previously resilient mice. Moreover, stress-resilience is associated with increased phasic 2-AG-mediated synaptic suppression at ventral hippocampal-amygdala glutamatergic synapses and amygdala-specific 2-AG depletion impairs successful adaptation to repeated stress. These data indicate amygdala 2-AG signalling mechanisms promote resilience to adverse effects of acute traumatic stress and facilitate adaptation to repeated stress exposure.

Topics: Amygdala; Animals; Anxiety; Arachidonic Acids; Behavior, Animal; Benzodioxoles; Disease Susceptibility; Dronabinol; Endocannabinoids; Excitatory Postsynaptic Potentials; Female; Glutamates; Glycerides; Hippocampus; Lipoprotein Lipase; Male; Mice, Inbred ICR; Mice, Knockout; Phenotype; Piperidines; Resilience, Psychological; Signal Transduction; Stress, Psychological; Synapses

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of the motor neuron system with limited therapeutic options. While an increasing number of ALS patients can be linked to a small number of autosomal-dominantly inherited cases, most cases are termed sporadic. Both forms are clinically and histopathologically indistinguishable, raising the prospect that they share key pathogenic steps, including potential therapeutic intervention points. The endocannabinoid system is emerging as a versatile, druggable therapeutic target in the CNS and its dysregulation is an early hallmark of neurodegeneration. Whether this is a defense mechanism or part of the pathogenesis remains to be determined. The neuroprotective and anti-inflammatory endocannabinoid 2-arachidonoylglycerol (2-AG), which is degraded by monoacylglycerol lipase (MAGL), accumulates in the spinal cords of transgenic models of ALS. We tested the hypothesis that this 2-AG increase is a protective response in the low-copy SOD1

Topics: Amyotrophic Lateral Sclerosis; Animals; Arachidonic Acids; Arginase; Benzodioxoles; Brain-Derived Neurotrophic Factor; Cytokines; Disease Models, Animal; Endocannabinoids; Female; Glycerides; Male; Mice; Mice, Transgenic; Molecular Targeted Therapy; Monoacylglycerol Lipases; Neuroglia; Neurons; Piperidines; Primary Cell Culture; Spinal Cord

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

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

Increasing the available repertoire of effective treatments for mood and anxiety disorders represents a critical unmet need. Pharmacological augmentation of endogenous cannabinoid (eCB) signaling has been suggested to represent a novel approach to the treatment of anxiety disorders; however, the functional interactions between two canonical eCB pathways mediated via anandamide (N-arachidonylethanolamine [AEA]) and 2-arachidonoylglycerol (2-AG) in the regulation of anxiety are not well understood.. We utilized pharmacological augmentation and depletion combined with behavioral and electrophysiological approaches to probe the role of 2-AG signaling in the modulation of stress-induced anxiety and the functional redundancy between AEA and 2-AG signaling in the modulation of anxiety-like behaviors in mice.. Selective 2-AG augmentation reduced anxiety in the light/dark box assay and prevented stress-induced increases in anxiety associated with limbic AEA deficiency. In contrast, acute 2-AG depletion increased anxiety-like behaviors, which was normalized by selective pharmacological augmentation of AEA signaling and via direct cannabinoid receptor 1 stimulation with Δ. Although AEA and 2-AG likely subserve distinct physiological roles, a pharmacological and functional redundancy between these canonical eCB signaling pathways exists in the modulation of anxiety-like behaviors. These data support development of eCB-based treatment approaches for mood and anxiety disorders and suggest a potentially wider therapeutic overlap between AEA and 2-AG augmentation approaches than was previously appreciated.

Topics: Adaptation, Ocular; Animals; Anti-Anxiety Agents; Anxiety; Arachidonic Acids; Benzodioxoles; Brain; Cannabinoid Receptor Agonists; Cyclohexanols; Disease Models, Animal; Dronabinol; Endocannabinoids; Excitatory Postsynaptic Potentials; Glycerides; Heterocyclic Compounds, 1-Ring; Locomotion; Male; Mice; Mice, Inbred ICR; Piperidines; Polyunsaturated Alkamides; Pyridines; Signal Transduction

In the era of combined antiretroviral therapy (cART), human immunodeficiency virus type 1 (HIV-1) is considered a chronic disease that specifically targets the brain and causes HIV-1-associated neurocognitive disorders (HAND). Endocannabinoids (eCBs) elicit neuroprotective and anti-inflammatory actions in several central nervous system (CNS) disease models, but their effects in HAND remain unknown. HIV-1 does not infect neurons, but produces viral toxins, such as transactivator of transcription (Tat), that disrupt neuronal calcium equilibrium and give rise to synaptodendritic injuries and cell death, the former being highly correlated with HAND. Consequently, we tested whether the eCBs N-arachidonoylethanolamine (anandamide/AEA) and 2-arachidonoyl-glycerol (2-AG) offer neuroprotective actions in a neuronal culture model. Specifically, we examined the neuroprotective actions of these eCBs on Tat excitotoxicity in primary cultures of prefrontal cortex neurons (PFC), and whether cannabinoid receptors mediate this neuroprotection. Tat-induced excitotoxicity was reflected by increased intracellular calcium levels, synaptodendritic damage, neuronal excitability, and neuronal death. Further, upregulation of cannabinoid 1 receptor (CB

Topics: Animals; Arachidonic Acids; Calcium; Cannabinoid Receptor Antagonists; Cell Survival; Cells, Cultured; Endocannabinoids; Glycerides; Mice; Mice, Inbred C57BL; Neurons; Neuroprotective Agents; Piperidines; Polyunsaturated Alkamides; Prefrontal Cortex; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; Synaptic Transmission; tat Gene Products, Human Immunodeficiency Virus

Tobacco withdrawal is associated with deficits in cognitive function, including attention, working memory, and episodic memory. Understanding the neurobiological mechanisms involved in these effects is crucial because cognitive deficits during nicotine withdrawal may predict relapse in humans.. We investigated in mice the role of CB. Memory impairment during nicotine withdrawal was blocked by the CB. These findings underline the interest of CB

Topics: Animals; Arachidonic Acids; Brain; Cannabinoid Receptor Antagonists; Endocannabinoids; GABAergic Neurons; Glycerides; Male; Memory; Mice; Mice, Inbred C57BL; Mice, Knockout; Neuronal Plasticity; Nicotine; Piperidines; Polyunsaturated Alkamides; Pyramidal Cells; Pyrazoles; Receptor, Cannabinoid, CB1; Receptors, GABA; Recognition, Psychology; Rimonabant; Substance Withdrawal Syndrome

Individuals who experience life-threatening psychological trauma are at risk of developing a series of chronic neuropsychiatric pathologies that include generalized anxiety, depression, and drug addiction. The endocannabinoid system has been implicated in the modulation of these responses by regulating the activity of the amygdala and the hypothalamic-pituitary-adrenal axis. However, the relevance of this signaling complex to the long-term consequences of traumatic events is unclear. Here we use an animal model of predatory stress-induced anxiety-like behavior to investigate the role of the endocannabinoid system in the development of persistent anxiety states. Our main finding is that rats exposed to the fox pheromone 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), a life-threatening stimulus for rodents, display a marked and selective increase in the mobilization of the endocannabinoid, 2-arachidonoyl-sn-glycerol (2-AG), in the amygdala. This effect lasts for at least 14 days after the stress has occurred. In addition, systemic or local pharmacological inhibition of monoacylglycerol lipase (MGL)-a lipid hydrolase that degrades 2-AG in presynaptic nerve terminals-elevates 2-AG levels and suppresses the anxiety-like behavior elicited by exposure to TMT. The results suggest that predator threat triggers long-term changes in 2-AG-mediated endocannabinoid signaling in the amygdala, and that pharmacological interventions targeting MGL might provide a therapeutic strategy for the treatment of chronic brain disorders initiated by trauma.

Topics: Amygdala; Animals; Anxiety; Arachidonic Acids; Benzodioxoles; Endocannabinoids; Enzyme Inhibitors; Glycerides; Male; Monoacylglycerol Lipases; Piperidines; Rats; Rats, Sprague-Dawley; Stress, Psychological; Thiazoles

Under some conditions, stress, rather than directly triggering cocaine seeking, potentiates reinstatement to other stimuli, including a subthreshold cocaine dose. The mechanisms responsible for stress-potentiated reinstatement are not well defined. Endocannabinoid signaling is increased by stress and regulates synaptic transmission in brain regions implicated in motivated behavior.. The objective of this study was to test the hypothesis that cannabinoid type 1 receptor (CB1R) signaling is required for stress-potentiated reinstatement of cocaine seeking in rats.. Following i.v. cocaine self-administration (2 h access/day) and extinction in male rats, footshock stress alone does not reinstate cocaine seeking but reinstatement is observed when footshock is followed by an injection of an otherwise subthreshold dose of cocaine (2.5 mg/kg, i.p.). CB1R involvement was tested by systemic administration of the CB1R antagonist AM251 (0, 1, or 3 mg/kg, i.p.) prior to testing for stress-potentiated reinstatement.. Stress-potentiated reinstatement was blocked by both 1 and 3 mg/kg AM251. By contrast, AM251 only attenuated food-reinforced lever pressing at the higher dose (i.e., 3 mg/kg) and did not affect locomotor activity at either dose tested. Neither high-dose cocaine-primed reinstatement (10 mg/kg, i.p.) nor footshock stress-triggered reinstatement following long-access cocaine self-administration (6 h access/day) was affected by AM251 pretreatment. Footshock stress increased concentrations of both endocannabinoids, N-arachidonylethanolamine and 2-arachidonoylglycerol, in regions of the prefrontal cortex.. These findings demonstrate that footshock stress increases prefrontal cortical endocannabinoids and stress-potentiated reinstatement is CB1R-dependent, suggesting that CB1R is a potential therapeutic target for relapse prevention, particularly in individuals whose cocaine use is stress-related.

Topics: Animals; Arachidonic Acids; Behavior, Addictive; Cocaine; Cocaine-Related Disorders; Endocannabinoids; Extinction, Psychological; Glycerides; Male; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Reinforcement, Psychology; Self Administration; Stress, Psychological

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

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

2-arachidonoylglycerol (2-AG) is an endogenous ligand of the cannabinoid CB1 receptor. This endocannabinoid and its hydrolyzing enzyme, monoacylglycerol lipase (MAGL), are present in encephalic regions related to psychiatric disorders, including the midbrain dorsolateral periaqueductal grey (dlPAG). The dlPAG is implicated in panic disorder and its stimulation results in defensive responses proposed as a model of panic attacks. The present work verified if facilitation of 2-AG signalling in the dlPAG counteracts panic-like responses induced by local chemical stimulation. Intra-dlPAG injection of 2-AG prevented panic-like response induced by the excitatory amino acid N-methyl-d-aspartate (NMDA). This effect was mimicked by the 2-AG hydrolysis inhibitor (MAGL preferring inhibitor) URB602. The anti-aversive effect of URB602 was reversed by the CB1 receptor antagonist, AM251. Additionally, a combination of sub-effective doses of 2-AG and URB602 also prevented NMDA-induced panic-like response. Finally, immunofluorescence assay showed a significant increase in c-Fos positive cells in the dlPAG after local administration of NMDA. This response was also prevented by URB602. These data support the hypothesis that 2-AG participates in anti-aversive mechanisms in the dlPAG and reinforce the proposal that facilitation of endocannabinoid signalling could be a putative target for developing additional treatments against panic and other anxiety-related disorders.

Topics: Animals; Arachidonic Acids; Biphenyl Compounds; Cannabinoid Receptor Antagonists; Disease Models, Animal; Dose-Response Relationship, Drug; Endocannabinoids; Fluorescent Antibody Technique; Glycerides; Male; N-Methylaspartate; Panic Disorder; Periaqueductal Gray; Piperidines; Pyrazoles; Rats, Wistar; Receptor, Cannabinoid, CB1

The cannabinoid 1 receptor (CB1) is an important regulator of energy metabolism. Reports of in vivo and in vitro studies give conflicting results regarding its role in insulin secretion, possibly due to circulatory factors, such as incretins. We hypothesized that this receptor may be a regulator of the entero-insular axis. We found that despite lower food consumption and lower body weight postprandial GLP-1 plasma concentrations were increased in CB1(-/-) mice compared to CB1(+/+) mice administered a standard diet or high fat/sugar diet. Upon exogenous GLP-1 treatment, CB1(-/-) mice had increased glucose-stimulated insulin secretion. In mouse insulinoma cells, cannabinoids reduced GLP-1R-mediated intracellular cAMP accumulation and subsequent insulin secretion. Importantly, such effects were also evident in human islets, and were prevented by pharmacologic blockade of CB1. Collectively, these findings suggest a novel mechanism in which endocannabinoids are negative modulators of incretin-mediated insulin secretion.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cell Line, Tumor; Cyclic AMP; Endocannabinoids; Genetic Predisposition to Disease; Glucagon-Like Peptide-1 Receptor; Glycerides; Humans; Insulin; Insulin Secretion; Islets of Langerhans; Male; Mice, Inbred C57BL; Mice, Knockout; Obesity; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1

Activation of cannabinoid CB1 receptors may offer new therapeutic strategies, but the efficiency of CB1 receptor agonists may be impaired by tolerance development upon prolonged administration. We compared the influence of repeated administration of Δ(9)-tetrahydrocannabinol (THC) 10 mg/kg on the motility and on basal and CB1 receptor-stimulated (35)S-GTPγS binding of adolescent and aged mice. Moreover, we determined the influence of JZL 184 (which inhibits the 2-arachidonoylglycerol, 2-AG, degrading enzyme monoacylglycerol lipase, MAGL) on (35)S-GTPγS binding and 2-AG levels of young adult mice. Mouse motility was tested in the open field. (35)S-GTPγS binding was studied in hippocampal membranes. THC and CP 55,940 were used as cannabinoid agonists in the behavioural and biochemical studies, respectively. 2-AG levels were quantified by liquid chromatography-multiple reaction monitoring. The THC (10 mg/kg)-induced hypomotility was stronger in untreated than in THC-pretreated adolescent mice but similar in both treatment groups of aged mice. Basal and stimulated (35)S-GTPγS binding was decreased in membranes from THC-pretreated adolescent but not affected in membranes from aged mice. Treatment of young adult mice with JZL 184 (4, 10 and 40 mg/kg) for 14 days did not affect basal binding. Stimulated binding tended to be decreased by 25 % only in mice treated with JZL 184 (40 mg/kg). Hippocampal 2-AG level was increased by JZL 184 at 40 and 10 but not affected at 4 mg/kg. In conclusion, CB1 receptor tolerance does not occur in aged mice pretreated with THC and in young adult mice treated with a low dose of the MAGL inhibitor JZL 184.

Topics: Age Factors; Animals; Arachidonic Acids; Behavior, Animal; Benzodioxoles; Cannabinoid Receptor Agonists; Dose-Response Relationship, Drug; Dronabinol; Drug Tolerance; Endocannabinoids; Enzyme Inhibitors; Genotype; Glycerides; Guanosine 5'-O-(3-Thiotriphosphate); Hippocampus; Male; Mice, Inbred C57BL; Mice, Knockout; Monoacylglycerol Lipases; Motor Activity; Phenotype; Piperidines; Receptor, Cannabinoid, CB1

Cannabinoids, such as Δ9-THC, act through an endogenous signaling system in the vertebrate eye that reduces IOP via CB1 receptors. Endogenous cannabinoid (eCB) ligand, 2-arachidonoyl glycerol (2-AG), likewise activates CB1 and is metabolized by monoacylglycerol lipase (MAGL). We investigated ocular 2-AG and its regulation by MAGL and the therapeutic potential of harnessing eCBs to lower IOP.. We tested the effect of topical application of 2-AG and MAGL blockers in normotensive mice and examined changes in eCB-related lipid species in the eyes and spinal cord of MAGL knockout (MAGL-/-) mice using high performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). We also examined the protein distribution of MAGL in the mouse anterior chamber.. 2-Arachidonoyl glycerol reliably lowered IOP in a CB1- and concentration-dependent manner. Monoacylglycerol lipase is expressed prominently in nonpigmented ciliary epithelium. The MAGL blocker KML29, but not JZL184, lowered IOP. The ability of CB1 to lower IOP is not desensitized in MAGL-/- mice. Ocular monoacylglycerols, including 2-AG, are elevated in MAGL-/- mice but, in contrast to the spinal cord, arachidonic acid and prostaglandins are not changed.. Our data confirm a central role for MAGL in metabolism of ocular 2-AG and related lipid species, and that endogenous 2-AG can be harnessed to reduce IOP. The MAGL blocker KML29 has promise as a therapeutic agent, while JZL184 may have difficulty crossing the cornea. These data, combined with the relative specificity of MAGL for ocular monoacylglycerols and the lack of desensitization in MAGL-/- mice, suggest that the development of an optimized MAGL blocker offers therapeutic potential for treatment of elevated IOP.

Topics: Administration, Topical; Animals; Anterior Chamber; Arachidonic Acids; Benzodioxoles; Ciliary Body; Cornea; Endocannabinoids; Glycerides; Immunohistochemistry; Intraocular Pressure; Mice; Mice, Inbred C57BL; Mice, Knockout; Monoacylglycerol Lipases; Monoglycerides; Piperidines; Rabbits; Tandem Mass Spectrometry

Incomplete overlap in the discriminative stimulus effects of Δ-tetrahydrocannabinol (THC) and the endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol has been reported in food-reinforced tasks. The aim of this study was to examine cannabinoid discriminative stimulus effects in a nonappetitive procedure. Adult male mice lacking the gene for AEA's major metabolic enzyme, fatty acid amide hydrolase (FAAH), and FAAH mice were trained to discriminate THC or AEA in a water T-maze, in which the response was swimming to an escape platform on the injection-appropriate side. JZL184, a monoacylglycerol lipase inhibitor, was also tested. FAAH mice showed faster acquisition than FAAH mice. THC and AEA fully substituted, with only minor cross-procedure potency variations. Incomplete substitution of JZL184 was observed in THC-trained FAAH mice in the water-maze task, as contrasted with full substitution in a food-reinforced nose-poke procedure. Stress-induced changes in AEA and/or 2-arachidonoylglycerol concentrations in the brain may have mediated this attenuation. JZL184 also partially substituted in AEA-trained FAAH mice in the water maze, suggesting incomplete overlap in the stimulus effects of AEA and JZL184. Through the use of a novel water-maze procedure, the present study supports the work of previous behavioral pharmacologists in showing the robustness of the discrimination paradigm.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzodioxoles; Brain; Discrimination, Psychological; Dronabinol; Endocannabinoids; Glycerides; Male; Maze Learning; Mice; Mice, Inbred C57BL; Mice, Knockout; Piperidines; Polyunsaturated Alkamides; Water

Endocannabinoids acting on cannabinoid-1 receptors (CB1Rs) are proposed to protect brain and spinal neurons from excitotoxic damage. The ability to recover from spinal cord injury (SCI), in which excitotoxicity is a major player, is usually investigated at late times after modulation of CB1Rs whose role in the early phases of SCI remains unclear. Using the rat spinal cord in vitro as a model for studying SCI initial pathophysiology, we investigated if agonists or antagonists of CB1Rs might affect SCI induced by the excitotoxic agent kainate (KA) within 24h from a transient (1h) application of this glutamate agonist. The CB1 agonist anandamide (AEA or pharmacological block of its degradation) did not limit excitotoxic depolarization of spinal networks: cyclic adenosine monophosphate (cAMP) assay demonstrated that CB1Rs remained functional 24h later and similarly expressed among dead or survived cells. Locomotor-like network activity recorded from ventral roots could not recover with such treatments and was associated with persistent depression of synaptic transmission. Motoneurons, that are particularly vulnerable to KA, were not protected by AEA. Application of 2-arachidonoylglycerol also did not attenuate the electrophysiological and histological damage. The intensification of damage by the CB1 antagonist AM251 suggested that endocannabinoids were operative after excitotoxic stimulation, yet insufficient to contrast it efficiently. The present data indicate that the early phases of excitotoxic SCI could not be arrested by pharmacologically exploiting the endocannabinoid system, consistent with the notion that AEA and its derivatives are more useful to treat late SCI phases.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Disease Models, Animal; Endocannabinoids; Glycerides; Kainic Acid; Locomotion; Motor Neurons; Neural Pathways; Neuroprotection; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats, Wistar; Receptor, Cannabinoid, CB1; Spinal Cord; Spinal Cord Injuries; Tissue Culture Techniques

Monoacylglycerol lipase (MAGL) is a serine hydrolase that acts as a principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG). In addition to terminating the signaling function of 2-AG, MAGL liberates arachidonic acid to be used as a primary source for neuroinflammatory prostaglandin synthesis in the brain. MAGL activity also contributes to cancer pathogenicity by producing precursors for tumor-promoting bioactive lipids. Pharmacological inhibitors of MAGL provide valuable tools for characterization of MAGL and 2-AG signaling pathways. They also hold great therapeutic potential to treat several pathophysiological conditions, such as pain, neurodegenerative disorders, and cancer. We have previously reported piperidine triazole urea, {4-[bis-(benzo[d][1,3]dioxol-5-yl)methyl]-piperidin-1-yl}(1H-1,2,4-triazol-1-yl)methanone (JJKK-048), to be an ultrapotent and highly selective inhibitor of MAGL in vitro. Here, we characterize in vivo effects of JJKK-048. Acute in vivo administration of JJKK-048 induced a massive increase in mouse brain 2-AG levels without affecting brain anandamide levels. JJKK-048 appeared to be extremely potent in vivo. Activity-based protein profiling revealed that JJKK-048 maintains good selectivity toward MAGL over other serine hydrolases. Our results are also the first to show that JJKK-048 promoted significant analgesia in a writhing test with a low dose that did not cause cannabimimetic side effects. At a high dose, JJKK-048 induced analgesia both in the writhing test and in the tail-immersion test, as well as hypomotility and hyperthermia, but not catalepsy.

Topics: Animals; Arachidonic Acids; Behavior, Animal; Benzodioxoles; Brain; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Glycerides; Hypothermia; Male; Mice; Monoacylglycerol Lipases; Nociception; Piperidines; Pyrazoles; Rimonabant

Stress and glucocorticoids stimulate the rapid mobilization of endocannabinoids in the basolateral amygdala (BLA). Cannabinoid receptors in the BLA contribute to anxiogenesis and fear-memory formation. We tested for rapid glucocorticoid-induced endocannabinoid regulation of synaptic inhibition in the rat BLA. Glucocorticoid application to amygdala slices elicited a rapid, nonreversible suppression of spontaneous, but not evoked, GABAergic synaptic currents in BLA principal neurons; the effect was also seen with a membrane-impermeant glucocorticoid, but not with intracellular glucocorticoid application, implicating a membrane-associated glucocorticoid receptor. The glucocorticoid suppression of GABA currents was not blocked by antagonists of nuclear corticosteroid receptors, or by inhibitors of gene transcription or protein synthesis, but was blocked by inhibiting postsynaptic G-protein activity, suggesting a postsynaptic nongenomic steroid signaling mechanism that stimulates the release of a retrograde messenger. The rapid glucocorticoid-induced suppression of inhibition was prevented by blocking CB1 receptors and 2-arachidonoylglycerol (2-AG) synthesis, and it was mimicked and occluded by CB1 receptor agonists, indicating it was mediated by the retrograde release of the endocannabinoid 2-AG. The rapid glucocorticoid effect in BLA neurons in vitro was occluded by prior in vivo acute stress-induced, or prior in vitro glucocorticoid-induced, release of endocannabinoid. Acute stress also caused an increase in anxiety-like behavior that was attenuated by blocking CB1 receptor activation and inhibiting 2-AG synthesis in the BLA. Together, these findings suggest that acute stress causes a long-lasting suppression of synaptic inhibition in BLA neurons via a membrane glucocorticoid receptor-induced release of 2-AG at GABA synapses, which contributes to stress-induced anxiogenesis.. We provide a cellular mechanism in the basolateral amygdala (BLA) for the rapid stress regulation of anxiogenesis in rats. We demonstrate a nongenomic glucocorticoid induction of long-lasting suppression of synaptic inhibition that is mediated by retrograde endocannabinoid release at GABA synapses. The rapid glucocorticoid-induced endocannabinoid suppression of synaptic inhibition is initiated by a membrane-associated glucocorticoid receptor in BLA principal neurons. We show that acute stress increases anxiety-like behavior via an endocannabinoid-dependent mechanism centered in the BLA. The stress-induced endocannabinoid modulation of synaptic transmission in the BLA contributes, therefore, to the stress regulation of anxiety, and may play a role in anxiety disorders of the amygdala.

Topics: Animals; Antiemetics; Anxiety; Arachidonic Acids; Basolateral Nuclear Complex; Benzoxazines; Calcium Channel Blockers; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Dexamethasone; Disease Models, Animal; Endocannabinoids; Enzyme Inhibitors; Glucocorticoids; Glycerides; Inhibitory Postsynaptic Potentials; Male; Morpholines; Naphthalenes; Piperidines; Pyrazoles; Rats; Rats, Wistar; Restraint, Physical; Rimonabant; Synaptic Transmission

The endocannabinoid 2-arachidonoylglycerol (2-AG) mediates retrograde synaptic depression including depolarization-induced suppression of excitation (DSE) and inhibition (DSI). 2-AG is degraded primarily by monoacylglycerol lipase (MAGL), which is expressed in neurons and astrocytes. Using knockout mice in which MAGL is deleted globally or selectively in neurons or astrocytes, we investigated the relative contribution of neuronal and astrocytic MAGL to the termination of DSE and DSI in Purkinje cells (PCs) in cerebellar slices. We report that neuronal MAGL plays a predominant role in terminating DSE at climbing fiber (CF) to PC synapses, while both neuronal and astrocytic MAGL significantly contributes to the termination of DSE at parallel fiber (PF) to PC synapses and DSI at putative Stellate cell to PC synapses. Thus, DSE and DSI at different synapses is not uniformly affected by global and cell type-specific knockout of MAGL. Additionally, MAGL global knockout, but not cell type-specific knockout, caused tonic activation and partial desensitization of the CB

Topics: Animals; Arachidonic Acids; Astrocytes; Benzodioxoles; Benzoxazines; Cerebellum; Endocannabinoids; Enzyme Inhibitors; Female; Glycerides; Male; Mice, Knockout; Monoacylglycerol Lipases; Morpholines; Naphthalenes; Neurons; Patch-Clamp Techniques; Piperidines; Purkinje Cells; Receptor, Cannabinoid, CB1; Synapses

The endocannabinoid ligand 2-arachidonoylglycerol (2-AG) is inactivated primarily by monoacylglycerol lipase (MAGL). We have shown recently that chronic treatments with MAGL inhibitor JZL184 produce antidepressant- and anxiolytic-like effects in a chronic unpredictable stress (CUS) model of depression in mice. However, the underlying mechanisms remain poorly understood. Adult hippocampal neurogenesis has been implicated in animal models of anxiety and depression and behavioral effects of antidepressants. We tested whether CUS and chronic JZL184 treatments affected adult neurogenesis and synaptic plasticity in the dentate gyrus (DG) of mouse hippocampus. We report that CUS induced depressive-like behaviors and decreased the number of bromodeoxyuridine-labeled neural progenitor cells and doublecortin-positive immature neurons in the DG, while chronic JZL184 treatments prevented these behavioral and cellular deficits. We also investigated the effects of CUS and chronic JZL184 on a form long-term potentiation (LTP) in the DG known to be neurogenesis-dependent. CUS impaired LTP induction, whereas chronic JZL184 treatments restored LTP in CUS-exposed mice. These results suggest that enhanced adult neurogenesis and long-term synaptic plasticity in the DG of the hippocampus might contribute to antidepressant- and anxiolytic-like behavioral effects of JZL184.

Topics: Animals; Anti-Anxiety Agents; Antidepressive Agents; Arachidonic Acids; Behavior, Animal; Benzodioxoles; Cannabinoid Receptor Agonists; Dentate Gyrus; Depression; Endocannabinoids; Glycerides; Hydrolysis; Male; Mice; Mice, Inbred C57BL; Monoacylglycerol Lipases; Neurogenesis; Neuronal Plasticity; Piperidines; Stress, Psychological

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

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

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

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

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

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

Distraction is used clinically to relieve and manage pain. It is hypothesized that pain demands attention and that exposure to another attention-demanding stimulus causes withdrawal of attention away from painful stimuli, thereby reducing perceived pain. We have recently developed a rat model that provides an opportunity to investigate the neurobiological mechanisms mediating distraction-induced analgesia, as these mechanisms are, at present, poorly understood. Given the well-described role of the endogenous cannabinoid (endocannabinoid; EC) system in the modulation of pain and attentional processing, the present study investigated its role in distraction-induced antinociception in rats.. Animals received the CB1 receptor antagonist/inverse agonist, rimonabant or vehicle intraperitoneally, 30 min prior to behavioural evaluation. Formalin-evoked nociceptive behaviour was measured in the presence or absence of a novel-object distractor. Liquid chromatography-tandem mass spectrometry was used to determine the levels of the endogenous cannabinoids anandamide and 2-arachidonoylglycerol (2-AG) in the ventral hippocampus (vHip).. Exposure to a novel object distractor significantly reduced formalin-evoked nociceptive behaviour. The novel object-induced reduction in nociceptive behaviour was attenuated by rimonabant. Novel object exposure was also associated with increased tissue levels of anandamide and 2-AG in the vHip.. These data suggest that the reduction in formalin-evoked nociceptive behaviour that occurs as a result of exposure to a novel object may be mediated by engagement of the EC system, in particular in the vHip. The results provide evidence that the EC system may be an important neural substrate subserving attentional modulation of pain.

Topics: Animals; Arachidonic Acids; Attention; Behavior, Animal; Cannabinoid Receptor Antagonists; Endocannabinoids; Exploratory Behavior; Fear; Glycerides; Hippocampus; Male; Nociception; Pain; Pain Measurement; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Receptor, Cannabinoid, CB1; Rimonabant

Endocannabinoids are strong modulators of emotionality and present a novel target for psychotropic drug development. Increasing evidence suggests that endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) affect behavior differentially. While the roles of anandamide have been investigated extensively, studies regarding the specific roles of 2-AG became possible only recently, and its involvement in social behaviors has not yet been studied.. We studied the impact of 2-AG signaling on aggression as a first attempt to characterize the role of this endocannabinoid in social behaviors.. 2-AG signaling was enhanced by the monoacylglycerol lipase inhibitor JZL184 (8, and 16 mg/kg) in mice later submitted to the resident/intruder paradigm.. JZL184 near completely abolished aggressiveness in residents and increased victimization (i.e., attacks by the opponent). Interestingly, the level of defensiveness remained unaltered, despite the large increase in bites received. The CB1 receptor blocker AM251 (0.5 mg/kg) did not influence the effects of JZL184. In intruders, JZL184 near completely suppressed bites and offensive behavior in a fashion similar to residents, but it also increased agitation and defensiveness during, and the corticosterone response to, aggressive encounters. Experiments involving the corticosterone synthesis inhibitor metyrapone (30 mg/kg) suggest that the suppression of biting and offensive behavior is directly influenced by JZL184, whereas increased agitation and defensiveness (seen in intruders only) are a secondary development of the stress-endocrine effects of JZL184.. 2-AG signaling emerges as a surprisingly strong negative modulator of aggressiveness, which warrants further studies into its general role in social behavior and the target receptors involved.

Topics: Aggression; Agonistic Behavior; Animals; Arachidonic Acids; Benzodioxoles; Cannabinoid Receptor Agonists; Corticosterone; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Glycerides; Male; Metyrapone; Mice; Monoacylglycerol Lipases; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Signal Transduction

Monoacylglycerol lipase (MAGL) represents a primary degradation enzyme of the endogenous cannabinoid (eCB), 2-arachidonoyglycerol (2-AG). This study reports a potent covalent MAGL inhibitor, SAR127303. The compound behaves as a selective and competitive inhibitor of mouse and human MAGL, which potently elevates hippocampal levels of 2-AG in mice. In vivo, SAR127303 produces antinociceptive effects in assays of inflammatory and visceral pain. In addition, the drug alters learning performance in several assays related to episodic, working and spatial memory. Moreover, long term potentiation (LTP) of CA1 synaptic transmission and acetylcholine release in the hippocampus, two hallmarks of memory function, are both decreased by SAR127303. Although inactive in acute seizure tests, repeated administration of SAR127303 delays the acquisition and decreases kindled seizures in mice, indicating that the drug slows down epileptogenesis, a finding deserving further investigation to evaluate the potential of MAGL inhibitors as antiepileptics. However, the observation that 2-AG hydrolysis blockade alters learning and memory performance, suggests that such drugs may have limited value as therapeutic agents.

Topics: Acetylcholine; Administration, Oral; Analgesics; Animals; Arachidonic Acids; Binding Sites; Brain; Cannabinoid Receptor Antagonists; Carbamates; Chromatography, High Pressure Liquid; Crystallography, X-Ray; Disease Models, Animal; Electric Stimulation; Endocannabinoids; Enzyme Inhibitors; Glycerides; Hippocampus; Humans; Hydrolysis; In Vitro Techniques; Learning; Long-Term Potentiation; Mass Spectrometry; Memory, Short-Term; Mice; Mice, Inbred C57BL; Mice, SCID; Monoacylglycerol Lipases; Pain; Piperidines; Protein Structure, Tertiary; Pyrazoles; Rimonabant; Seizures; Sulfonamides

Activation of G protein-coupled receptors (GPCRs) can induce vasoconstriction via calcium signal-mediated and Rho-dependent pathways. Earlier reports have shown that diacylglycerol produced during calcium signal generation can be converted to an endocannabinoid, 2-arachidonoylglycerol (2-AG). Our aim was to provide evidence that GPCR signaling-induced 2-AG production and activation of vascular type1 cannabinoid receptors (CB1R) is capable of reducing agonist-induced vasoconstriction and hypertension. Rat and mouse aortic rings were examined by myography. Vascular expression of CB1R was demonstrated with immunohistochemistry. Rat aortic vascular smooth muscle cells (VSMCs) were cultured for calcium measurements and 2-AG-determination. Inhibition or genetic loss of CB1Rs enhanced vasoconstriction induced by angiotensin II (AngII) or phenylephrine (Phe), but not by prostaglandin(PG)F2α. AngII-induced vasoconstriction was augmented by inhibition of diacylglycerol lipase (tetrahydrolipstatin) and was attenuated by inhibition of monoacylglycerol lipase (JZL184) suggesting a functionally relevant role for endogenously produced 2-AG. In Gαq/11-deficient mice vasoconstriction was absent to AngII or Phe, which activate Gq/11-coupled receptors, but was maintained in response to PGF2α. In VSMCs, AngII-stimulated 2-AG-formation was inhibited by tetrahydrolipstatin and potentiated by JZL184. CB1R inhibition increased the sustained phase of AngII-induced calcium signal. Pharmacological or genetic loss of CB1R function augmented AngII-induced blood pressure rise in mice. These data demonstrate that vasoconstrictor effect of GPCR agonists is attenuated via Gq/11-mediated vascular endocannabinoid formation. Agonist-induced endocannabinoid-mediated CB1R activation is a significant physiological modulator of vascular tone. Thus, the selective modulation of GPCR signaling-induced endocannabinoid release has a therapeutic potential in case of increased vascular tone and hypertension.

Topics: Angiotensin II; Animals; Aorta; Arachidonic Acids; Benzodioxoles; Calcium; Calcium Signaling; Dinoprost; Endocannabinoids; Gene Expression Regulation; Glycerides; GTP-Binding Protein alpha Subunits, Gq-G11; Hypertension; Lactones; Lipoprotein Lipase; Male; Mice; Mice, Knockout; Monoacylglycerol Lipases; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Orlistat; Phenylephrine; Piperidines; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Tissue Culture Techniques; Vasoconstriction

In the early stages, prostate cancer is androgen‑ dependent; therefore, medical castration has shown significant results during the initial stages of this pathology. Despite this early effect, advanced prostate cancer is resilient to such treatment. Recent evidence shows that derivatives of Cannabis sativa and its analogs may exert a protective effect against different types of oncologic pathologies. The purpose of the present study was to detect the presence of cannabinoid receptors (CB1 and CB2) on cancer cells with a prostatic origin and to evaluate the effect of the in vitro use of synthetic analogs. In order to do this, we used a commercial cell line and primary cultures derived from prostate cancer and benign prostatic hyperplasia. The presence of the CB1 and CB2 receptors was determined by immunohistochemistry where we showed a higher expression of these receptors in later stages of the disease (samples with a high Gleason score). Later, treatments were conducted using anandamide, 2-arachidonoyl glycerol and a synthetic analog of anandamide, methanandamide. Using the MTT assay, we proved that the treatments produced a cell growth inhibitory effect on all the different prostate cancer cultures. This effect was demonstrated to be dose-dependent. The use of a specific CB1 receptor blocker (SR141716) confirmed that this effect was produced primarily from the activation of the CB1 receptor. In order to understand the MTT assay results, we determined cell cycle distribution by flow cytometry, which showed no variation at the different cell cycle stages in all the cultures after treatment. Treatment with endocannabinoids resulted in an increase in the percentage of apoptotic cells as determined by Annexin V assays and caused an increase in the levels of activated caspase-3 and a reduction in the levels of Bcl-2 confirming that the reduction in cell viability noted in the MTT assay was caused by the activation of the apoptotic pathway. Finally, we observed that endocannabinoid treatment activated the Erk pathway and at the same time, produced a decrease in the activation levels of the Akt pathway. Based on these results, we suggest that endocannabinoids may be a beneficial option for the treatment of prostate cancer that has become nonresponsive to common therapies.

Topics: Adenocarcinoma; Apoptosis; Arachidonic Acids; Cell Cycle; Drug Screening Assays, Antitumor; Endocannabinoids; Glycerides; Humans; Male; MAP Kinase Signaling System; Neoplasm Proteins; Piperidines; Polyunsaturated Alkamides; Prostatic Hyperplasia; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; Signal Transduction; Tumor Cells, Cultured

As they age, Sprague-Dawley (SD) rats develop elevated systolic blood pressure associated with impaired baroreflex sensitivity (BRS) for control of heart rate. We previously demonstrated in young hypertensive (mRen2)27 rats that impaired BRS is restored by CB1 cannabinoid receptor blockade in the solitary tract nucleus (NTS), consistent with elevated content of the endocannabinoid 2-arachidonoylglycerol (2-AG) in dorsal medulla relative to normotensive SD rats. There is no effect of CB1 receptor blockade in young SD rats. We now report in older SD rats that dorsal medullary 2-AG levels are 2-fold higher at 70 versus 15 weeks of age (4.22 ± 0.61 vs. 1.93 ± 0.22 ng/mg tissue; P < 0.05). Furthermore, relative expression of CB1 receptor messenger RNA is significantly lower in aged rats, whereas CB2 receptor messenger RNA is significantly higher. In contrast to young adult SD rats, microinjection of the CB1 receptor antagonist SR141716A (36 pmole) into the NTS of older SD rats normalized BRS in animals exhibiting impaired baseline BRS (0.56 ± 0.06 baseline vs. 1.06 ± 0.05 ms/mm Hg after 60 minutes; P < 0.05). Therefore, this study provides evidence for alterations in the endocannabinoid system within the NTS of older SD rats that contribute to age-related impairment of BRS.

Topics: Age Factors; Aging; Animals; Arachidonic Acids; Baroreflex; Blood Pressure; Cannabinoid Receptor Antagonists; Endocannabinoids; Gene Expression Regulation; Glycerides; Heart Rate; Male; Mass Spectrometry; Microinjections; Piperidines; Pyrazoles; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Reverse Transcriptase Polymerase Chain Reaction; Rimonabant; RNA, Messenger; Solitary Nucleus

Cirrhosis is associated with blunted cardiovascular response to stimuli such as hemorrhage, but the mechanism remains unclear. We aimed to clarify the role of endocannabinoids in blunted hemorrhage response in cirrhotic rats.. Cirrhosis was induced by bile duct ligation (BDL). Hemodynamics were measured. Cannabinoid receptor-1 (CB1) antagonist, AM251, and macrophage inhibitor gadolinium chloride (GdCl3) were administered. Myocardial levels of anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) were measured and resident monocytes and macrophages quantified by immunohistochemistry. Isolated cardiomyocyte contractility was measured before and after incubation with monocytes from BDL and sham controls.. Hemorrhage significantly decreased arterial pressure and left ventricular dP/dT. After hemorrhage, these changes quickly reversed in controls, but were severely prolonged in BDL rats. Chronic AM251 treatment restored this impaired response. AEA and 2-AG levels were increased in BDL hearts and further increased after hemorrhage. Sham hearts showed virtually no monocytes or macrophages before or after hemorrhage, whereas BDL hearts had significantly more white blood cells which further increased after hemorrhage. GdCl3 treatment significantly reduced cardiac endocannabinoid levels both at baseline and after hemorrhage. This treatment also restored cardiovascular response to hemorrhage in BDL rats but did not affect sham controls. Monocytes isolated from BDL rats more potently inhibited cardiomyocyte contractility than sham control monocytes.. The cirrhotic heart showed increased monocyte recruitment and endocannabinoid levels. CB1 blockade or GdCl3 treatment restored blunted cardiovascular response to hemorrhage. Endocannabinoids released by monocytes blunt cardiac response to hemorrhage. Preventing monocyte recruitment or blocking endocannabinoid signaling may improve cardiovascular homeostasis in cirrhosis.

Topics: Animals; Arachidonic Acids; Blood Pressure; Cardiac Output; Endocannabinoids; Gadolinium; Glycerides; Hemorrhage; Liver Cirrhosis; Macrophages; Male; Monocytes; Myocardial Contraction; Myocardium; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Ventricular Function, Left

A number of studies have examined the ability of the endogenous cannabinoid anandamide to elicit Δ(9)-tetrahydrocannabinol (THC)-like subjective effects, as modeled through the THC discrimination paradigm. In the present study, we compared transgenic mice lacking fatty acid amide hydrolase (FAAH), the enzyme primarily responsible for anandamide catabolism, to wildtype counterparts in a THC discrimination procedure. THC (5.6 mg/kg) served as a discriminative stimulus in both genotypes, with similar THC dose-response curves between groups. Anandamide fully substituted for THC in FAAH knockout, but not wildtype, mice. Conversely, the metabolically stable anandamide analog O-1812 fully substituted in both groups, but was more potent in knockouts. The CB1 receptor antagonist rimonabant dose-dependently attenuated THC generalization in both groups and anandamide substitution in FAAH knockouts. Pharmacological inhibition of monoacylglycerol lipase (MAGL), the primary catabolic enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG), with JZL184 resulted in full substitution for THC in FAAH knockout mice and nearly full substitution in wildtypes. Quantification of brain endocannabinoid levels revealed expected elevations in anandamide in FAAH knockout mice compared to wildtypes and equipotent dose-dependent elevations in 2-AG following JZL184 administration. Dual inhibition of FAAH and MAGL with JZL195 resulted in roughly equipotent increases in THC-appropriate responding in both groups. While the notable similarity in THC's discriminative stimulus effects across genotype suggests that the increased baseline brain anandamide levels (as seen in FAAH knockout mice) do not alter THC's subjective effects, FAAH knockout mice are more sensitive to the THC-like effects of pharmacologically induced increases in anandamide and MAGL inhibition (e.g., JZL184).

Topics: Amidohydrolases; Analysis of Variance; Animals; Arachidonic Acids; Benzodioxoles; Brain; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Carbamates; Discrimination, Psychological; Dose-Response Relationship, Drug; Dronabinol; Endocannabinoids; Enzyme Inhibitors; Glycerides; Male; Mice; Mice, Knockout; Piperazines; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rimonabant

Chronic kidney disease, secondary to renal fibrogenesis, is a burden on public health. There is a need to explore new therapeutic pathways to reduce renal fibrogenesis. To study this, we used unilateral ureteral obstruction (UUO) in mice as an experimental model of renal fibrosis and microarray analysis to compare gene expression in fibrotic and normal kidneys. The cannabinoid receptor 1 (CB1) was among the most upregulated genes in mice, and the main endogenous CB1 ligand (2-arachidonoylglycerol) was significantly increased in the fibrotic kidney. Interestingly, CB1 expression was highly increased in kidney biopsies of patients with IgA nephropathy, diabetes, and acute interstitial nephritis. Both genetic and pharmacological knockout of CB1 induced a profound reduction in renal fibrosis during UUO. While CB2 is also involved in renal fibrogenesis, it did not potentiate the role of CB1. CB1 expression was significantly increased in myofibroblasts, the main effector cells in renal fibrogenesis, upon TGF-β1 stimulation. The decrease in renal fibrosis during CB1 blockade could be explained by a direct action on myofibroblasts. CB1 blockade reduced collagen expression in vitro. Rimonabant, a selective CB1 endocannabinoid receptor antagonist, modulated the macrophage infiltrate responsible for renal fibrosis in UUO through a decrease in monocyte chemoattractant protein-1 synthesis. Thus, CB1 has a major role in the activation of myofibroblasts and may be a new target for treating chronic kidney disease.

Topics: Acute Disease; Animals; Arachidonic Acids; Cells, Cultured; Chemokine CCL2; Collagen; Diabetes Mellitus; Disease Models, Animal; Endocannabinoids; Fibrosis; Gene Expression Profiling; Glomerulonephritis, IGA; Glycerides; Humans; Kidney; Ligands; Macrophages; Mice; Mice, Knockout; Myofibroblasts; Nephritis, Interstitial; Oligonucleotide Array Sequence Analysis; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; RNA, Messenger; Transforming Growth Factor beta1; Up-Regulation; Ureteral Obstruction

The contribution of two major endocannabinoids, 2-arachidonoylglycerol (2-AG) and anandamide (AEA), in the regulation of fear expression is still unknown.. We analyzed the role of different players of the endocannabinoid system on the expression of a strong auditory-cued fear memory in male mice by pharmacological means.. The cannabinoid receptor type 1 (CB1) antagonist SR141716 (3 mg/kg) caused an increase in conditioned freezing upon repeated tone presentation on three consecutive days. The cannabinoid receptor type 2 (CB2) antagonist AM630 (3 mg/kg), in contrast, had opposite effects during the first tone presentation, with no effects of the transient receptor potential vanilloid receptor type 1 (TRPV1) antagonist SB366791 (1 and 3 mg/kg). Administration of the CB2 agonist JWH133 (3 mg/kg) failed to affect the acute freezing response, whereas the CB1 agonist CP55,940 (50 μg/kg) augmented it. The endocannabinoid uptake inhibitor AM404 (3 mg/kg), but not VDM11 (3 mg/kg), reduced the acute freezing response. Its co-administration with SR141716 or SB366791 confirmed an involvement of CB1 and TRPV1. AEA degradation inhibition by URB597 (1 mg/kg) decreased, while 2-AG degradation inhibition by JZL184 (4 and 8 mg/kg) increased freezing response. As revealed in conditional CB1-deficient mutants, CB1 on cortical glutamatergic neurons alleviates whereas CB1 on GABAergic neurons slightly enhances fear expression. Moreover, 2-AG fear-promoting effects depended on CB1 signaling in GABAergic neurons, while an involvement of glutamatergic neurons remained inconclusive due to the high freezing shown by vehicle-treated Glu-CB1-KO.. Our findings suggest that increased AEA levels mediate acute fear relief, whereas increased 2-AG levels promote the expression of conditioned fear primarily via CB1 on GABAergic neurons.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Antagonists; Cannabinoids; Emotions; Endocannabinoids; Fear; GABAergic Neurons; Glycerides; Male; Mice; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant

We previously reported that intracerebroventricularly (i.c.v.) administered (±)-epibatidine (1, 5 or 10 nmol/animal), a nicotinic acetylcholine receptor agonist, dose-dependently induced secretion of noradrenaline and adrenaline (catecholamines) from the rat adrenal medulla by brain diacylglycerol lipase- (DGL), monoacylglycerol lipase- (MGL) and cyclooxygenase-mediated mechanisms. Diacylglycerol is hydrolyzed by DGL into 2-arachidonoylglycerol (2-AG), which is further hydrolyzed by MGL to arachidonic acid (AA), a cyclooxygenase substrate. These findings suggest that brain 2-AG-derived AA is involved in the (±)-epibatidine-induced response. This AA precursor 2-AG is also a major brain endocannabinoid, which inhibits synaptic transmission through presynaptic cannabinoid CB1 receptors. Released 2-AG into the synaptic cleft is rapidly inactivated by cellular uptake. Here, we examined a role of brain 2-AG as an endocannabinoid in the (±)-epibatidine-induced activation of central adrenomedullary outflow using anesthetized male Wistar rats. In central presence of AM251 (CB1 antagonist) (90 and 180 nmol/animal, i.c.v.), (±)-epibatidine elevated plasma catecholamines even at an ineffective dose (1 nmol/animal, i.c.v.). Central pretreatment with ACEA (CB1 agonist) (0.7 and 1.4 μmol/animal, i.c.v.), 2-AG ether (stable 2-AG analog for MGL) (0.5 and 1.0 μmol/animal, i.c.v.) or AM404 (endocannabinoid uptake inhibitor) (80 and 250 nmol/animal, i.c.v.) significantly reduced an effective dose of (±)-epibatidine- (5 nmol/animal, i.c.v.) induced elevation of plasma catecholamines, and AM251 (90 and 180 nmol/animal, i.c.v.) centrally abolished the reduction induced by 2-AG ether (1.0 μmol/animal, i.c.v.) or AM404 (250 nmol/animal, i.c.v.). Immunohistochemical studies demonstrated that (±)-epibatidine (10 nmol/animal, i.c.v.) activated DGLα-positive spinally projecting neurons in the hypothalamic paraventricular nucleus, a control center of central adrenomedullary system. These results suggest a possibility that a brain endocannabinoid, probably 2-AG, plays an inhibitory role in (±)-epibatidine-induced activation of central adrenomedullary outflow through brain CB1 receptors in the rat.

Topics: Adrenal Medulla; Animals; Arachidonic Acids; Bridged Bicyclo Compounds, Heterocyclic; Catecholamines; Dose-Response Relationship, Drug; Endocannabinoids; Glycerides; Immunohistochemistry; Male; Neurons; Neurotransmitter Agents; Nicotinic Agonists; Paraventricular Hypothalamic Nucleus; Piperidines; Pyrazoles; Pyridines; Rats, Wistar; Receptor, Cannabinoid, CB1

Central regulatory mechanisms for neurotransmitters of food intake vary among animals. Endocannabinoids have crucial role on central food intake regulation in mammals but its role has not been studied in layer-type chicken. Thus, in this study 6 experiments designed to evaluate effects of intracerebroventricular (ICV) administration of 2-AG (2-Arachidonoylglycerol, selective CB1 receptors agonist), SR141716A (selective CB1 receptors antagonist), JWH015 (selective CB2 receptors agonist), AM630 (selective CB2 receptors antagonist) on feeding behavior in 3 h food deprived neonatal layer-type chickens. In experiment 1, birds ICV injected with control solution and 2-AG (0.25, 0.5 and 1 μg). In experiment 2: control solution, SR141716A (6.25, 12.5 and 25 μg) were ICV injected to birds. In experiment 3 animals received: control solution, SR141716A (6.25 μg), 2-AG (1 μg) and co-injection of SR141716A+2-AG. In experiment 4, chickens received control solution and JWH015 (6.25, 12.5 and 25 μg). In experiment 5, control solution and AM630 (1.25, 2.5 and 5 μg) were injected. In experiment 6, the birds received control solution, AM630 (1.25 μg), JWH015 (25 μg) and co-administration of AM630+JWH015. Then, cumulative food intake was recorded until 120 min after injection. According to the results, 2-AG dose dependently increased cumulative food intake while SR141716A reduced appetite compared to control group (P < 0.05). Injection of 2-AG (1 μg) amplified food intake and its effect minimized by SR141716A (6.25 μg) (P < 0.05). Also, ICV injection of JWH015 (25 μg) dose dependently increased food intake and co-injection of JWH015+AM630 decreased JWH015-induced food intake (P < 0.05). These results suggest CB1 and CB2 receptors have an important role on ingestive behavior in FD3 neonatal layer-type chicken.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Agonists; Chickens; Eating; Endocannabinoids; Feeding Behavior; Glycerides; Infusions, Intraventricular; Piperidines; Pyrazoles; Rimonabant

To date, our understanding of the relative contribution and potential overlapping roles of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in the regulation of brain function and behavior is still limited. To address this issue, we investigated the effects of systemic administration of JZL195, that simultaneously increases AEA and 2-AG signaling by inhibiting their hydrolysis, in the regulation of socio-emotional behavior in adolescent and adult rats. JZL195, administered at the dose of 0.01mg/kg, increased social play behavior, that is the most characteristic social activity displayed by adolescent rats, and increased social interaction in adult animals. At both ages, these behavioral effects were antagonized by the CB1 cannabinoid receptor antagonist SR141716A and were associated with increased brain levels of 2-AG, but not AEA. Conversely, at the dose of 1mg/kg, JZL195 decreased general social exploration in adolescent rats without affecting social play behavior, and induced anxiogenic-like effects in the elevated plus-maze test both in adolescent and adult animals. These effects, mediated by activation of CB1 cannabinoid receptors, were paralleled by simultaneous increase in AEA and 2-AG levels in adolescent rats, and by an increase of only 2-AG levels in adult animals. These findings provide the first evidence for a role of 2-AG in social behavior, highlight the different contributions of AEA and 2-AG in the modulation of emotionality at different developmental ages and suggest that pharmacological inhibition of AEA and 2-AG hydrolysis is a useful approach to investigate the role of these endocannabinoids in neurobehavioral processes.

Topics: Aging; Animals; Anxiety; Arachidonic Acids; Brain; Cannabinoid Receptor Modulators; Carbamates; Endocannabinoids; Exploratory Behavior; Glycerides; Male; Piperazines; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats, Sprague-Dawley; Rimonabant; Social Behavior

Excessive exposure to infrasound, a kind of low-frequency but high-intensity sound noise generated by heavy transportations and machineries, can cause vibroacoustic disease which is a progressive and systemic disease, and finally results in the dysfunction of central nervous system. Our previous studies have demonstrated that glial cell-mediated inflammation may contribute to infrasound-induced neuronal impairment, but the underlying mechanisms are not fully understood. Here, we show that cannabinoid (CB) receptors may be involved in infrasound-induced neuronal injury. After exposure to infrasound at 16 Hz and 130 dB for 1-14 days, the expression of CB receptors in rat hippocampi was gradually but significantly decreased. Their expression levels reached the minimum after 7- to 14-day exposure during which the maximum number of apoptotic cells was observed in the CA1. 2-Arachidonoylglycerol (2-AG), an endogenous agonist for CB receptors, reduced the number of infrasound-triggered apoptotic cells, which, however, could be further increased by CB receptor antagonist AM251. In animal behavior performance test, 2-AG ameliorated the infrasound-impaired learning and memory abilities of rats, whereas AM251 aggravated the infrasound-impaired learning and memory abilities of rats. Furthermore, the levels of proinflammatory cytokines tumor necrosis factor alpha and interleukin-1β in the CA1 were upregulated after infrasound exposure, which were attenuated by 2-AG but further increased by AM251. Thus, our results provide the first evidence that CB receptors may be involved in infrasound-induced neuronal impairment possibly by affecting the release of proinflammatory cytokines.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Endocannabinoids; Glycerides; Hippocampus; Inflammation Mediators; Interleukin-1beta; Piperidines; Pyrazoles; Rats; Receptors, Cannabinoid; Ultrasonic Waves

Persistent CB1 cannabinoid receptor activity limits neurotransmitter release at various synapses throughout the brain. However, it is not fully understood how constitutively active CB1 receptors, tonic endocannabinoid signaling, and its regulation by multiple serine hydrolases contribute to the synapse-specific calibration of neurotransmitter release probability. To address this question at perisomatic and dendritic GABAergic synapses in the mouse hippocampus, we used a combination of paired whole-cell patch-clamp recording, liquid chromatography/tandem mass spectrometry, stochastic optical reconstruction microscopy super-resolution imaging, and immunogold electron microscopy. Unexpectedly, application of the CB1 antagonist and inverse agonist AM251 [N-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide], but not the neutral antagonist NESS0327 [8-chloro-1-(2,4-dichlorophenyl)-N-piperidin-1-yl-5,6-dihydro-4H-benzo[2,3]cyclohepta[2,4-b]pyrazole-3-carboxamine], significantly increased synaptic transmission between CB1-positive perisomatic interneurons and CA1 pyramidal neurons. JZL184 (4-nitrophenyl 4-[bis(1,3-benzodioxol-5-yl)(hydroxy)methyl]piperidine-1-carboxylate), a selective inhibitor of monoacylglycerol lipase (MGL), the presynaptic degrading enzyme of the endocannabinoid 2-arachidonoylglycerol (2-AG), elicited a robust increase in 2-AG levels and concomitantly decreased GABAergic transmission. In contrast, inhibition of fatty acid amide hydrolase (FAAH) by PF3845 (N-pyridin-3-yl-4-[[3-[5-(trifluoromethyl)pyridin-2-yl]oxyphenyl]methyl]piperidine-1-carboxamide) elevated endocannabinoid/endovanilloid anandamide levels but did not change GABAergic synaptic activity. However, FAAH inhibitors attenuated tonic 2-AG increase and also decreased its synaptic effects. This antagonistic interaction required the activation of the transient receptor potential vanilloid receptor TRPV1, which was concentrated on postsynaptic intracellular membrane cisternae at perisomatic GABAergic symmetrical synapses. Interestingly, neither AM251, JZL184, nor PF3845 affected CB1-positive dendritic interneuron synapses. Together, these findings are consistent with the possibility that constitutively active CB1 receptors substantially influence perisomatic GABA release probability and indicate that the synaptic effects of tonic 2-AG release are tightly controlled by presynaptic MGL activity and also by postsynaptic endovanilloid signaling and. Tonic cannabinoid signaling plays a critical role in the regulation of synaptic transmission. However, the mechanistic details of how persistent CB1 cannabinoid receptor activity inhibits neurotransmitter release have remained elusive. Therefore, electrophysiological recordings, lipid measurements, and super-resolution imaging were combined to elucidate those signaling molecules and mechanisms that underlie tonic cannabinoid signaling. The findings indicate that constitutive CB1 activity has pivotal function in the tonic control of hippocampal GABA release. Moreover, the endocannabinoid 2-arachidonoylglycerol (2-AG) is continuously generated postsynaptically, but its synaptic effect is regulated strictly by presynaptic monoacylglycerol lipase activity. Finally, anandamide signaling antagonizes tonic 2-AG signaling via activation of postsynaptic transient receptor potential vanilloid TRPV1 receptors. This unexpected mechanistic diversity may be necessary to fine-tune GABA release probability under various physiological and pathophysiological conditions.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Endocannabinoids; Enzyme Inhibitors; Female; gamma-Aminobutyric Acid; Glycerides; Hippocampus; Inhibitory Postsynaptic Potentials; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neurons; Piperidines; Pyrazoles; Pyridines; Receptor, Cannabinoid, CB1; Signal Transduction; Synapses; TRPV Cation Channels

Cannabinoid pharmacology has proven nettlesome with issues of promiscuity a common theme among both agonists and antagonists. One recourse is to develop allosteric ligands to modulate cannabinoid receptor signaling. Cannabinoids have come late to the allosteric table. The 'first-generation' negative and positive allosteric modulators (NAMs and PAMs) represent an important first effort. However, most studies have relied on synthetic agonists, often tested in over-expression systems rather than a defined neuronal model system that utilizes endogenously synthesized and released cannabinoids. We have systematically examined first-generation NAMs and a PAM on endocannabinoid modulation of synaptic transmission in cultured autaptic hippocampal neurons. These neurons exhibit CB1 and 2-arachidonoyl glycerol (2-AG)-mediated depolarization induced suppression of excitation (DSE) and therefore serve as a model to test CB1 modulators in a neuronal model of endogenous cannabinoid signaling. We find ORG27569, PSNCBAM-1, and PEPCAN12 attenuate DSE and do not directly inhibit CB1 receptors. Of these PSNCBAM-1 is the most efficacious while PEPCAN12 has the distinction of being an endogenous NAM. The reported NAMs pregnenolone and hemopressin as well as the reported PAM lipoxin A4 are without effect in this model of endocannabinoid signaling. In summary, three of the allosteric modulators evaluated function in a manner consistent with allosterism in a neuronal 2-AG-based model of endogenous cannabinoid signaling.

Topics: Allosteric Regulation; Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Cannabinoids; Endocannabinoids; Excitatory Postsynaptic Potentials; Glycerides; Hippocampus; Indoles; Mice; Neurons; Phenylurea Compounds; Piperidines; Pyridines; Receptor, Cannabinoid, CB1; Signal Transduction; Synaptic Transmission

Parkinson's disease (PD) is a common chronic neurodegenerative disorder, usually of idiopathic origin. Symptoms including tremor, bradykinesia, rigidity and postural instability are caused by the progressive loss of dopaminergic neurons in the nigrostriatal region of the brain. Symptomatic therapies are available but no treatment slows or prevents the loss of neurons. Neuroinflammation has been implicated in its pathogenesis. To this end, the present study utilises the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin to reproduce the pattern of cell death evident in PD patients. Herein, the role of a potential regulator of an immune response, the endocannabinoid system (ECS), is investigated. The most prevalent endocannabinoid, 2-arachidonoylglycerol (2-AG) (3 and 5mg/kg), was added exogenously and its enzymatic degradation inhibited to provide protection against MPTP-induced cell death. Furthermore, the addition of DFU (25mg/kg), a selective inhibitor of inflammatory mediator cyclooxygenase-2 (COX-2), potentiated these effects. Levels of 2-AG were shown to be upregulated in a time- and region-specific manner following MPTP administration, indicating that the ECS represents a natural defence mechanism against inflammation, potentiation of which could provide therapeutic benefits. The results expand the current understanding of the role that this signalling system has and its potential influence in PD.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Animals; Arachidonic Acids; Benzodioxoles; Brain; Cell Death; Cyclooxygenase 2; Disease Models, Animal; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Furans; Gait Disorders, Neurologic; Glycerides; Male; Mice; Mice, Inbred C57BL; Motor Activity; Neuroprotective Agents; Neurotoxins; Parkinson Disease; Piperidines; Time Factors; Tyrosine 3-Monooxygenase

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

The two most studied endocannabinoids are anandamide (AEA), principally catalyzed by fatty-acid amide hydrolase (FAAH), and 2-arachidonoyl glycerol (2-AG), mainly hydrolyzed by monoacylglycerol lipase (MGL). Inhibitors targeting these two enzymes have been described, including URB597 and URB602, respectively. Several recent studies examining the contribution of CB₁ and/or CB₂ receptors on the peripheral antinociceptive effects of AEA, 2-AG, URB597 and URB602 in neuropathic pain conditions using either pharmacological tools or transgenic mice separately have been reported, but the exact mechanism is still uncertain. Mechanical allodynia and thermal hyperalgesia were evaluated in 436 male C57BL/6, cnr1KO and cnr2KO mice in the presence or absence of cannabinoid CB₁ (AM251) or CB₂ (AM630) receptor antagonists in a mouse model of neuropathic pain. Peripheral subcutaneous injections of AEA, 2-AG, WIN55,212-2 (WIN; a CB₁/CB₂ synthetic agonist), URB597 and URB602 significantly decreased mechanical allodynia and thermal hyperalgesia. These effects were inhibited by both cannabinoid antagonists AM251 and AM630 for treatments with 2-AG, WIN and URB602 but only by AM251 for treatments with AEA and URB597 in C57BL/6 mice. Furthermore, the antinociceptive effects for AEA and URB597 were observed in cnr2KO mice but absent in cnr1KO mice, whereas the effects of 2-AG, WIN and URB602 were altered in both of these transgenic mice. Complementary genetic and pharmacological approaches revealed that the anti-hyperalgesic effects of 2-AG and URB602 required both CB₁ and CB₂ receptors, but only CB₂ receptors mediated its anti-allodynic actions. The antinociceptive properties of AEA and URB597 were mediated only by CB₁ receptors.

Topics: Animals; Arachidonic Acids; Behavior, Animal; Endocannabinoids; Glycerides; Hyperalgesia; Male; Mice; Mice, Knockout; Neuralgia; Pain Measurement; Pain Threshold; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2

High-content screening led to the identification of the N-isobutylamide guineensine from Piper nigrum as novel nanomolar inhibitor (EC50=290nM) of cellular uptake of the endocannabinoid anandamide (AEA). Noteworthy, guineensine did not inhibit endocannabinoid degrading enzymes fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL) nor interact with cannabinoid receptors or fatty acid binding protein 5 (FABP5), a major cytoplasmic AEA carrier. Activity-based protein profiling showed no inhibition of serine hydrolases. Guineensine also inhibited the cellular uptake of 2-arachidonoylglycerol (2-AG). Preliminary structure-activity relationships between natural guineensine analogs indicate the importance of the alkyl chain length interconnecting the pharmacophoric isobutylamide and benzodioxol moieties for AEA cellular uptake inhibition. Guineensine dose-dependently induced cannabimimetic effects in BALB/c mice shown by strong catalepsy, hypothermia, reduced locomotion and analgesia. The catalepsy and analgesia were blocked by the CB1 receptor antagonist rimonabant (SR141716A). Guineensine is a novel plant natural product which specifically inhibits endocannabinoid uptake in different cell lines independent of FAAH. Its scaffold may be useful to identify yet unknown targets involved in endocannabinoid transport.

Topics: Alkenes; Amidohydrolases; Analgesics; Animals; Arachidonic Acids; Biological Transport; Brain; Cannabinoid Receptor Antagonists; Catalepsy; Dose-Response Relationship, Drug; Endocannabinoids; Fatty Acid-Binding Proteins; Glycerides; Heterocyclic Compounds, 2-Ring; Humans; Hypothermia; Locomotion; Male; Mice; Mice, Inbred BALB C; Monoacylglycerol Lipases; Neoplasm Proteins; Piper; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Rimonabant; Serine Endopeptidases; Structure-Activity Relationship; U937 Cells

The endogenous cannabinoid (endocannabinoid) system plays a key role in the modulation of aversive and nociceptive behaviour. The components of the endocannabinoid system are expressed throughout the hippocampus, a brain region implicated in both conditioned fear and pain. In light of evidence that pain can impact on the expression of fear-related behaviour, and vice versa, we hypothesised that exogenous administration of the endocannabinoid 2-arachidonoyl glycerol (2-AG) into the ventral hippocampus (vHip) would differentially regulate fear responding in the absence vs. the presence of formalin-evoked nociceptive tone. Fear-conditioned rats showed significantly increased freezing and a reduction in formalin-evoked nociceptive behaviour upon re-exposure to a context previously paired with footshock. Bilateral microinjection of 2-AG into the vHip significantly reduced contextually induced freezing in non-formalin-treated rats, and reduced formalin-evoked nociceptive behaviour in non-fear-conditioned rats. In contrast, 2-AG microinjection had no effect on fear responding in formalin-treated rats, and no effect on nociceptive behaviour in fear-conditioned rats. The inhibitory effect of 2-AG on fear-related behaviour, but not pain-related behaviour, was blocked by co-administration of the cannabinoid receptor 1 (CB1) antagonist/inverse agonist rimonabant. Tissue levels of the endocannabinoids N-arachidonoylethanolamide (anandamide, AEA) and 2-AG were similar in the vHip of fear-conditioned rats receiving formalin injection and the vHip of fear-conditioned rats receiving saline injection. However, the levels of AEA and 2-AG were significantly lower in the contralateral ventrolateral periaqueductal grey of formalin-treated fear-conditioned rats than in that of their saline-treated counterparts. These data suggest that 2-AG-CB1 receptor signalling in the vHip has an anti-aversive effect, and that this effect is abolished in the presence of a persistent pain state.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Conditioning, Classical; Endocannabinoids; Fear; Freezing Reaction, Cataleptic; Glycerides; Hippocampus; Injections, Intraventricular; Mice; Nociception; Pain; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Receptor, Cannabinoid, CB1; Rimonabant

Insulin resistance is highly prevalent in patients with chronic hepatitis C (CHC) and to some extent accounts for fibrosis and reducing viral eradication. Activated cannabinoid 1 receptor (CB1R) signaling has been implicated in the development of phenotypes associated with insulin resistance and steatosis. We investigated the role of the endocannabinoid system in glucose metabolism disorders induced by hepatitis C virus (HCV) replication.. Human hepatic stellate cells (HSC; LX-2 cells) were co-cultured with Huh-7.5 cells or Huh-7.5 cells harboring HCV replicon (replicon cells). Endocannabinoid levels were then measured by liquid chromatography/mass spectrometry. The expression of CB1R and its downstream glucose metabolism genes in hepatocytes were determined by real-time PCR and Western blot. Glucose uptake by hepatocytes and glucose production were measured. Glucose metabolism tests and measurements of HCV RNA levels and nonstructural protein 5A (NS5A) levels were taken after treatment with CB1R agonist arachidonyl-2-chloroethanolamide (ACEA) or antagonist AM251.. Compared to the co-culture with Huh-7.5 cells, the level of 2-arachidonoylglycerol (2-AG) and the CB1R mRNA and protein levels increased in the co-culture of LX-2 cells with replicon cells. The activation of CB1R decreased AMP-activated protein kinase (AMPK) phosphorylation, inhibited cell surface expression of glucose transporter 2 (GLUT2), and suppressed cellular glucose uptake; furthermore, it increased cyclic AMP response element-binding protein H (CREBH), then up-regulated phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) genes and down-regulated the glucokinase (GK) gene, thus promoting glucose production. Interferon treatment restored the aforementioned changes. CB1R antagonist improved glucose metabolism disorders by an increase in glucose uptake and a decrease in glucose production, and inhibited HCV replication.. HCV replication may not only increase the 2-AG content, but may also up-regulate the expression of CB1R of hepatocytes, then change the expression profile of glucose metabolism-related genes, thereby causing glucose metabolism disorders of hepatocytes and promoting HCV replication. Treatment with CB1R antagonist improved glucose metabolism disorders and inhibited viral genome replication.

Topics: AMP-Activated Protein Kinases; Arachidonic Acids; Cell Line; Cell Survival; Coculture Techniques; Cyclic AMP Response Element-Binding Protein; Endocannabinoids; Genome, Viral; Glucose Metabolism Disorders; Glucose Transporter Type 2; Glucose-6-Phosphatase; Glycerides; Hepacivirus; Hepatic Stellate Cells; Hepatitis C, Chronic; Hepatocytes; Humans; Phosphorylation; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Real-Time Polymerase Chain Reaction; Receptor, Cannabinoid, CB1; RNA, Messenger; RNA, Viral; Signal Transduction; Up-Regulation; Virus Replication

Methamphetamine toxicity is associated with cell death and loss of dopamine neuron terminals in the striatum similar to what is found in some neurodegenerative diseases. Conversely, the endocannabinoid system (ECS) has been suggested to be neuroprotective in the brain, and new pharmacological tools have been developed to increase their endogenous tone. In this study, we evaluated whether ECS stimulation could reduce the neurotoxicity of high doses of methamphetamine on the dopamine system. We found that methamphetamine alters the levels of the major endocannabinoids, anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) in the striatum, suggesting that the ECS participates in the brain responses to methamphetamine. Δ(9)-tetrahydrocannabinol (THC), a cannabis-derived agonist of both CB1 and CB2 cannabinoid receptors, or inhibitors of the main enzymes responsible for the degradation of AEA and 2-AG (URB597 and JZL184, respectively), blunted the decrease in striatal protein levels of tyrosine hydroxylase induced by methamphetamine. In addition, antagonists of CB2, but not of CB1, blocked the preventive effects of URB597 and JZL184, suggesting that only the former receptor subtype is engaged in neuroprotection exerted by ECS stimulation. Finally, we found that methamphetamine increases striatal levels of the cytokine tumor necrosis factor alpha, an effect that was blocked by ECS stimulation. Altogether, our results indicate that stimulation of ECS prior to the administration of an overdose of methamphetamine considerably reduces the neurotoxicity of the drug through CB2 receptor activation and highlight a protective function for the ECS against the toxicity induced by drugs and other external insults to the brain. This article is part of the Special Issue entitled 'CNS Stimulants'.

Topics: Animals; Arachidonic Acids; Benzamides; Benzodioxoles; Cannabinoid Receptor Modulators; Carbamates; Central Nervous System Stimulants; Dronabinol; Endocannabinoids; Enzyme Inhibitors; Glycerides; Male; Methamphetamine; Mice, Inbred C57BL; Neostriatum; Neurotoxicity Syndromes; Piperidines; Polyunsaturated Alkamides; Random Allocation; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Tumor Necrosis Factor-alpha; Tyrosine 3-Monooxygenase

The cannabinoid CB1 receptors on the noradrenergic neurons in guinea pig hippocampal slices show an endogenous endocannabinoid tone. This conclusion is based on rimonabant, the facilitatory effect of which on noradrenaline release might be due to its inverse CB1 receptor agonism and/or the interruption of a tonic inhibition elicited by endocannabinoids. To examine the latter mechanism, a neutral antagonist would be suitable. Therefore, we studied whether O-2050 is a neutral CB1 receptor antagonist in the guinea pig hippocampus and whether it mimics the facilitatory effect of rimonabant. CB1 receptor affinity of O-2050 was quantified in cerebrocortical membranes, using (3)H-rimonabant binding. Its CB1 receptor potency and effect on (3)H-noradrenaline release were determined in superfused hippocampal slices. Its intrinsic activity at CB1 receptors was studied in hippocampal membranes, using (35)S-GTPγS binding. Endocannabinoid levels in hippocampus were determined by liquid chromatography-multiple reaction monitoring. O-2050 was about ten times less potent than rimonabant in its CB1 receptor affinity, potency and facilitatory effect on noradrenaline release. Although not affecting (35)S-GTPγS binding by itself, O-2050 shifted the concentration-response curve of a CB1 receptor agonist to the right but that of rimonabant to the left. Levels of anandamide and 2-arachidonoyl glycerol in guinea pig hippocampus closely resembled those in mouse hippocampus. In conclusion, our results with O-2050 confirm that the CB1 receptors on noradrenergic neurons of the guinea pig hippocampus show an endogenous tone. To differentiate between the two mechanisms leading to an endogenous tone, O-2050 is not superior to rimonabant since O-2050 may increase the inverse agonistic effect of endocannabinoids.

Topics: Animals; Arachidonic Acids; Benzoxazines; Cannabinoid Receptor Antagonists; Cerebral Cortex; Dronabinol; Drug Interactions; Endocannabinoids; Glycerides; Guinea Pigs; Hippocampus; In Vitro Techniques; Male; Morpholines; Naphthalenes; Norepinephrine; Piperidines; Polyunsaturated Alkamides; Pyrans; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant

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

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

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

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

Increased vascular sensitivity to angiotensin II (Ang II) is a marker of a hypertensive human pregnancy. Recent evidence of interactions between the renin-angiotensin system and the endocannabinoid system suggests that anandamide and 2-arachidonoylglycerol may modulate Ang II contraction. We hypothesized that these interactions may contribute to the enhanced vascular responses in hypertensive pregnancy. We studied Ang II contraction in isolated uterine artery (UA) at early gestation in a rat model that mimics many features of preeclampsia, the transgenic human angiotensinogen×human renin (TgA), and control Sprague-Dawley rats. We determined the role of the cannabinoid receptor 1 by blockade with SR171416A, and the contribution of anandamide and 2-arachidonoylglycerol degradation to Ang II contraction by inhibiting their hydrolyzing enzyme fatty acid amide hydrolase (with URB597) or monoacylglycerol lipase (with JZL184), respectively. TgA UA showed increased maximal contraction and sensitivity to Ang II that was inhibited by indomethacin. Fatty acid amide hydrolase blockade decreased Ang IIMAX in Sprague-Dawley UA, and decreased both Ang IIMAX and sensitivity in TgA UA. Monoacylglycerol lipase blockade had no effect on Sprague-Dawley UA and decreased Ang IIMAX and sensitivity in TgA UA. Blockade of the cannabinoid receptor 1 in TgA UA had no effect. Immunolocalization of fatty acid amide hydrolase and monoacylglycerol lipase showed a similar pattern between groups; fatty acid amide hydrolase predominantly localized in endothelium and monoacylglycerol lipase in smooth muscle cells. We demonstrated an increased Ang II contraction in TgA UA before initiation of the hypertensive phenotype. Anandamide and 2-arachidonoylglycerol reduced Ang II contraction in a cannabinoid receptor 1-independent manner. These renin-angiotensin system-endocannabinoid system interactions may contribute to the enhanced vascular reactivity in early stages of hypertensive pregnancy.

Topics: Amidohydrolases; Angiotensin II; Animals; Arachidonic Acids; Benzamides; Benzodioxoles; Blood Pressure; Carbamates; Disease Models, Animal; Endocannabinoids; Female; Glycerides; Humans; Hydrolysis; Hypertension, Pregnancy-Induced; Male; Monoglycerides; Piperidines; Polyunsaturated Alkamides; Pregnancy; Pregnancy, Animal; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Uterine Artery; Vasoconstriction

Changes in cannabinoid receptor expression and concentration of endocannabinoids have been described in Parkinson's disease; however, it remains unclear whether they contribute to, or result from, the disease process. To evaluate whether targeting the endocannabinoid system could provide potential benefits in the treatment of the disease, the effect of a monoacylglycerol lipase inhibitor that prevents degradation of 2-arachidonyl-glycerol was tested in mice treated chronically with probenecid and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTPp). Chronic administration of the compound, JZL184 (8 mg/kg), prevented MPTPp-induced motor impairment and preserved the nigrostriatal pathway. Furthermore, none of the hypokinetic effects associated with cannabinoid receptor agonism were observed. In the striatum and substantia nigra pars compacta, MPTPp animals treated with JZL184 exhibited astroglial and microglial phenotypic changes that were accompanied by increases in TGFβ messenger RNA expression and in glial cell-derived neurotrophic factor messenger RNA and protein levels. JZL184 induced an increase in β-catenin translocation to the nucleus, implicating the Wnt/catenin pathway. Together, these results demonstrate a potent neuroprotective effect of JZL184 on the nigrostriatal pathway of parkinsonian animals, likely involving restorative astroglia and microglia activation and the release of neuroprotective and antiinflammatory molecules.

Topics: Animals; Anti-Inflammatory Agents; Arachidonic Acids; Benzodioxoles; Corpus Striatum; Disease Models, Animal; Endocannabinoids; Glial Fibrillary Acidic Protein; Glycerides; Male; Mice, Inbred C57BL; Monoacylglycerol Lipases; Nerve Tissue Proteins; Neural Pathways; Neuroglia; Neuroprotective Agents; Parkinsonian Disorders; Piperidines; Probenecid; Substantia Nigra

It has been reported that direct activation of the cannabinoid CB1 receptor in epidermal growth factor (EGR)-stimulated PC-3 prostate cancer cells results in an anti-proliferative effect accompanied by a down-regulation of EGF receptors (EGFR). In the present study, we investigated whether similar effects are seen following inhibition of the endocannabinoid hydrolytic enzyme monoacylglycerol lipase (MGL).. CB1 receptor expression levels were found to differ greatly between two experimental series conducted using PC-3 cells. The monoacylglycerol lipase inhibitor JZL184 increased levels of 2-arachidonoylglycerol in the PC-3 cells without producing changes in the levels of anandamide and related N-acylethanolamines. In the first series of experiments, JZL184 produced a small mitogenic effect for cells that had not been treated with EGF, whereas an anti-proliferative effect was seen for EGF-treated cells. An anti-proliferative effect for the EGF-treated cells was also seen with the CB receptor agonist CP55,940. In the second batch of cells, there was an interaction between JZL184 and CB1 receptor expression densities in linear regression analyses with EGFR expression as the dependent variable.. Inhibition of MGL by JZL184 can affect EGFR expression. However, the use in our hands of PC-3 cells as a model to investigate the therapeutic potential of MGL inhibitors and related compounds is compromised by their variability of CB1 receptor expression.

Topics: Arachidonic Acids; Benzodioxoles; Cannabinoids; Cell Line, Tumor; Cell Proliferation; Cyclohexanols; Endocannabinoids; Enzyme Inhibitors; ErbB Receptors; Ethanolamines; Gene Expression Regulation, Neoplastic; Glycerides; Humans; Male; Monoacylglycerol Lipases; Piperidines; Polyunsaturated Alkamides; Prostate; Receptor, Cannabinoid, CB1; Signal Transduction

Endogenous cannabinoids (endocannabinoids) in the periaqueductal grey (PAG) play a vital role in mediating stress-induced analgesia. This analgesic effect of endocannabinoids is enhanced by pharmacological inhibition of their degradative enzymes. However, the specific effects of endocannabinoids and the inhibitors of their degradation are largely unknown within this pain-modulating region.. In vitro electrophysiological recordings were conducted from PAG neurons in rat midbrain slices. The effects of the major endocannabinoids and their degradation inhibitors on inhibitory GABAergic synaptic transmission were examined.. Exogenous application of the endocannabinoid, anandamide (AEA), but not 2-arachidonoylglycerol (2-AG), produced a reduction in inhibitory GABAergic transmission in PAG neurons. This AEA-induced suppression of inhibition was enhanced by the fatty acid amide hydrolase (FAAH) inhibitor, URB597, whereas a 2-AG-induced suppression of inhibition was unmasked by the monoacylglycerol lipase (MGL) inhibitor, JZL184. In addition, application of the CB1 receptor antagonist, AM251, facilitated the basal GABAergic transmission in the presence of URB597 and JZL184, which was further enhanced by the dual FAAH/MGL inhibitor, JZL195.. Our results indicate that AEA and 2-AG act via disinhibition within the PAG, a cellular action consistent with analgesia. These actions of AEA and 2-AG are tightly regulated by their respective degradative enzymes, FAAH and MGL. Furthermore, individual or combined inhibition of FAAH and/or MGL enhanced tonic disinhibition within the PAG. Therefore, the current findings support the therapeutic potential of FAAH and MGL inhibitors as a novel pharmacotherapy for pain.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Benzodioxoles; Carbamates; Endocannabinoids; Female; Glycerides; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Male; Monoacylglycerol Lipases; Neurons; Pain; Periaqueductal Gray; Piperidines; Polyunsaturated Alkamides; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Synaptic Transmission

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

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

The monoacylglycerol lipase (MAGL) inhibitor 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) produces antinociceptive and anti-inflammatory effects. However, repeated administration of high-dose JZL184 (40 mg/kg) causes dependence, antinociceptive tolerance, cross-tolerance to the pharmacological effects of cannabinoid receptor agonists, and cannabinoid receptor type 1 (CB1) downregulation and desensitization. This functional CB1 receptor tolerance poses a hurdle in the development of MAGL inhibitors for therapeutic use. Consequently, the present study tested whether repeated administration of low-dose JZL184 maintains its antinociceptive actions in the chronic constriction injury of the sciatic nerve neuropathic pain model and protective effects in a model of nonsteroidal anti-inflammatory drug-induced gastric hemorrhages. Mice given daily injections of high-dose JZL184 (≥16 mg/kg) for 6 days displayed decreased CB1 receptor density and function in the brain, as assessed in [(3)H]SR141716A binding and CP55,940 [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol]-stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate binding assays, respectively. In contrast, normal CB1 receptor expression and function were maintained following repeated administration of low-dose JZL184 (≤8 mg/kg). Likewise, the antinociceptive and gastroprotective effects of high-dose JZL184 underwent tolerance following repeated administration, but these effects were maintained following repeated low-dose JZL184 treatment. Consistent with these observations, repeated high-dose JZL184, but not repeated low-dose JZL184, elicited cross-tolerance to the common pharmacological effects of Δ(9)-tetrahydrocannabinol. This same pattern of effects was found in a rimonabant [(5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide)]-precipitated withdrawal model of cannabinoid dependence. Taken together, these results indicate that prolonged, partial MAGL inhibition maintains potentially beneficial antinociceptive and anti-inflammatory effects, without producing functional CB1 receptor tachyphylaxis/tolerance or cannabinoid dependence.

Topics: Analgesics; Animals; Anti-Inflammatory Agents, Non-Steroidal; Anti-Ulcer Agents; Arachidonic Acids; Benzodioxoles; Brain Chemistry; Cyclohexanols; Diclofenac; Dose-Response Relationship, Drug; Dronabinol; Drug Tolerance; Endocannabinoids; Glycerides; Guanosine 5'-O-(3-Thiotriphosphate); Male; Mice; Mice, Inbred C57BL; Monoacylglycerol Lipases; Pain Measurement; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; Stomach Ulcer; Substance Withdrawal Syndrome; Substance-Related Disorders

The basolateral amygdala (BLA) is a key substrate facilitating the expression of fear-conditioned analgesia (FCA). However, the neurochemical mechanisms in the BLA which mediate this potent suppression of pain responding during fear remain unknown. The present study investigated the role of cannabinoid1 (CB1) receptors and interactions with GABAergic (GABAA receptor) and glutamatergic (metabotropic glutamate receptor type 5; mGluR5) signalling in the BLA in formalin-evoked nociceptive behaviour and FCA in rats. Reexposure to a context previously paired with foot shock significantly reduced formalin-evoked nociceptive behaviour. Systemic or intra-BLA microinjection of the CB1 receptor antagonist/inverse agonist AM251 prevented this expression of FCA, while injection of AM251 into the central nucleus of the amygdala did not. The suppression of FCA by systemic AM251 administration was partially attenuated by intra-BLA administration of either the GABAA receptor antagonist bicuculline or the mGluR5 antagonist 2-methyl-6-(phenylethynyl) pyridine, (MPEP). Bilateral microinjection of MPEP, but not bicuculline, alone into the BLA enhanced formalin-evoked nociceptive behaviour. Postmortem analyses revealed that FCA was associated with a significant increase in tissue levels of anandamide in the BLA side contralateral to intraplantar formalin injection. In addition, fear-conditioned rats exhibited a robust formalin-induced increase in levels of 2-arachidonyl glycerol and N-palmitoylethanolamide in the ipsilateral and contralateral BLA, respectively. These data suggest that CB1 receptors in the BLA facilitate the expression of FCA, through a mechanism which is likely to involve the modulation of GABAergic and glutamatergic signalling.

Topics: Amygdala; Analgesia; Animals; Arachidonic Acids; Cannabinoid Receptor Agonists; Conditioning, Psychological; Dose-Response Relationship, Drug; Endocannabinoids; Ethanolamines; Excitatory Amino Acid Agents; Fear; Functional Laterality; GABA Agents; Glycerides; Injections, Intraventricular; Male; Pain; Pain Measurement; Piperidines; Pyrazoles; Rats; Receptor, Cannabinoid, CB1; Receptors, GABA; Receptors, Glutamate; Signal Transduction

The endocannabinoid 2-arachidonoylglycerol (2-AG) mediates activity-dependent depression of excitatory neurotransmission at central synapses, but the molecular regulation of 2-AG synthesis is not well understood. Here we identify a functional interaction between the 2-AG synthetic enzyme diacylglycerol lipase-α (DGLα) and calcium/calmodulin dependent protein kinase II (CaMKII). Activated CaMKII interacted with the C-terminal domain of DGLα, phosphorylated two serine residues and inhibited DGLα activity. Consistent with an inhibitory role for CaMKII in 2-AG synthesis, in vivo genetic inhibition of CaMKII increased striatal DGL activity and basal levels of 2-AG, and CaMKII inhibition augmented short-term retrograde endocannabinoid signaling at striatal glutamatergic synapses. Lastly, blockade of 2-AG breakdown using concentrations of JZL-184 that have no effect in wild-type mice produced a hypolocomotor response in mice with reduced CaMKII activity. These findings provide mechanistic insights into the molecular regulation of striatal endocannabinoid signaling with implications for physiological control of motor function.

Topics: Animals; Arachidonic Acids; Benzodioxoles; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Corpus Striatum; Endocannabinoids; Gene Knockdown Techniques; Glycerides; HEK293 Cells; Humans; Lipoprotein Lipase; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Piperidines; Signal Transduction

The transient exposure of immature rodents to ethanol during postnatal day 7 (P7), which is comparable with the third trimester in human pregnancy, induces synaptic dysfunctions. However, the molecular mechanisms underlying these dysfunctions are still poorly understood. Although the endocannabinoid system has been shown to be an important modulator of ethanol sensitivity in adult mice, its potential role in synaptic dysfunctions in mice exposed to ethanol during early brain development is not examined. In this study, we investigated the potential role of endocannabinoids and the cannabinoid receptor type 1 (CB1R) in neonatal neurodegeneration and adult synaptic dysfunctions in mice exposed to ethanol at P7. Ethanol treatment at P7, which induces neurodegeneration, increased anandamide (AEA) but not 2-arachidonylglycerol biosynthesis and CB1R protein expression in the hippocampus and cortex, two brain areas that are important for memory formation and storage, respectively. N-Arachidonoyl phosphatidylethanolamine-phospholipase D (NAPE-PLD), glycerophosphodiesterase (GDE1), and CB1R protein expression were enhanced by transcriptional activation of the genes encoding NAPE-PLD, GDE1, and CB1R proteins, respectively. In addition, ethanol inhibited ERK1/2 and AKT phosphorylation. The blockade of CB1Rs before ethanol treatment at P7 relieved ERK1/2 but not AKT phosphorylation and prevented neurodegeneration. CB1R knock-out mice exhibited no ethanol-induced neurodegeneration and inhibition of ERK1/2 phosphorylation. The protective effects of CB1R blockade through pharmacological or genetic deletion resulted in normal adult synaptic plasticity and novel object recognition memory in mice exposed to ethanol at P7. The AEA/CB1R/pERK1/2 signaling pathway may be directly responsible for the synaptic and memory deficits associated with fetal alcohol spectrum disorders.

Topics: Animals; Animals, Newborn; Arachidonic Acids; Brain; Cannabinoid Receptor Antagonists; Endocannabinoids; Ethanol; Female; Gene Expression Regulation, Developmental; Glycerides; Male; Memory Disorders; Mice; Mice, Inbred C57BL; Mice, Knockout; Nerve Degeneration; Nerve Tissue Proteins; Neuronal Plasticity; Neuroprotective Agents; Phospholipase D; Phosphoric Diester Hydrolases; Phosphorylation; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; Signal Transduction; Synapses

Neural stem cells express cannabinoid CB1 and CB2 receptors and the enzymes for the biosynthesis and metabolism of endocannabinoids (eCBs). Here we have studied the role of neural stem cell-derived eCBs as autonomous regulatory factors during differentiation. First, we examined the effect of an indirect eCB precursor linoleic acid (LA), a major dietary omega-6 fatty acid, on the eCB system in neural stem/progenitor cells (NSPCs) cultured in DMEM/F12 supplemented with N2 (N2/DF) as monolayer cells. LA upregulated eCB system-related genes and 2-arachidonoylglycerol (2-AG), but not anandamide (AEA), levels. Glial fibrillary acidic protein (GFAP) was significantly higher under LA-enriched conditions, and this effect was inhibited by the cannabinoid receptor type-1 (CB1) antagonist AM251. Second, the levels of AEA and 2-AG, as well as of the mRNA of eCB system-related genes, were measured in NSPCs after γ-aminobutyric acid (GABA) treatment. GABA upregulated AEA levels significantly in LA-enriched cultures and increased the mRNA expression of the 2-AG-degrading enzyme monoacylglycerol lipase. These effects of GABA were reproduced under culture conditions using neurobasal media supplemented with B27, which is commonly used for neurosphere culture. GABA stimulated astroglial differentiation in this medium as indicated by increased GFAP levels. This effect was abolished by AM251, suggesting the involvement of AEA and CB1 in GABA-induced astrogliogenesis. This study highlights the importance of eCB biosynthesis and CB1 signalling in the autonomous regulation of NSPCs and the influence of the eCB system on astrogliogenesis induced by nutritional factors or neurotransmitters, such as LA and GABA.

Topics: Acetyltransferases; Analysis of Variance; Animals; Arachidonic Acids; Astrocytes; Cell Differentiation; Cells, Cultured; Endocannabinoids; gamma-Aminobutyric Acid; Glial Fibrillary Acidic Protein; Glycerides; Linoleic Acid; Mass Spectrometry; Mice; Neural Stem Cells; Piperidines; Polyunsaturated Alkamides; Pyrazoles; RNA, Messenger; Up-Regulation

Endocannabinoids and their attending cannabinoid type 1 (CB1) receptor have been implicated in animal models of post-traumatic stress disorder (PTSD). However, their specific role has not been studied in people with PTSD. Herein, we present an in vivo imaging study using positron emission tomography (PET) and the CB1-selective radioligand [(11)C]OMAR in individuals with PTSD, and healthy controls with lifetime histories of trauma (trauma-exposed controls (TC)) and those without such histories (healthy controls (HC)). Untreated individuals with PTSD (N=25) with non-combat trauma histories, and TC (N=12) and HC (N=23) participated in a magnetic resonance imaging scan and a resting PET scan with the CB1 receptor antagonist radiotracer [(11)C]OMAR, which measures the volume of distribution (VT) linearly related to CB1 receptor availability. Peripheral levels of anandamide, 2-arachidonoylglycerol, oleoylethanolamide, palmitoylethanolamide and cortisol were also assessed. In the PTSD group, relative to the HC and TC groups, we found elevated brain-wide [(11)C]OMAR VT values (F(2,53)=7.96, P=0.001; 19.5% and 14.5% higher, respectively), which were most pronounced in women (F(1,53)=5.52, P=0.023). Anandamide concentrations were reduced in the PTSD relative to the TC (53.1% lower) and HC (58.2% lower) groups. Cortisol levels were lower in the PTSD and TC groups relative to the HC group. Three biomarkers examined collectively--OMAR VT, anandamide and cortisol--correctly classified nearly 85% of PTSD cases. These results suggest that abnormal CB1 receptor-mediated anandamide signaling is implicated in the etiology of PTSD, and provide a promising neurobiological model to develop novel, evidence-based pharmacotherapies for this disorder.

Topics: Adult; Amides; Analysis of Variance; Arachidonic Acids; Brain; Endocannabinoids; Ethanolamines; Female; Glycerides; Humans; Hydrocortisone; Imidazoles; Logistic Models; Male; Palmitic Acids; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Radionuclide Imaging; Receptor, Cannabinoid, CB1; Stress Disorders, Post-Traumatic; Young Adult

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

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

The anti-obesity medication rimonabant, an antagonist of cannabinoid type-1 (CB(1)) receptor, was withdrawn from the market because of adverse psychiatric side effects, including a negative affective state. We investigated whether rimonabant precipitates a negative emotional state in rats withdrawn from palatable food cycling. The effects of systemic administration of rimonabant on anxiety-like behavior, food intake, body weight, and adrenocortical activation were assessed in female rats during withdrawal from chronic palatable diet cycling. The levels of the endocannabinoids, anandamide and 2-arachidonoylglycerol (2-AG), and the CB(1) receptor mRNA and the protein in the central nucleus of the amygdala (CeA) were also investigated. Finally, the effects of microinfusion of rimonabant in the CeA on anxiety-like behavior, and food intake were assessed. Systemic administration of rimonabant precipitated anxiety-like behavior and anorexia of the regular chow diet in rats withdrawn from palatable diet cycling, independently from the degree of adrenocortical activation. These behavioral observations were accompanied by increased 2-AG, CB(1) receptor mRNA, and protein levels selectively in the CeA. Finally, rimonabant, microinfused directly into the CeA, precipitated anxiety-like behavior and anorexia. Our data show that (i) the 2-AG-CB(1) receptor system within the CeA is recruited during abstinence from palatable diet cycling as a compensatory mechanism to dampen anxiety, and (ii) rimonabant precipitates a negative emotional state by blocking the beneficial heightened 2-AG-CB(1) receptor signaling in this brain area. These findings help elucidate the link between compulsive eating and anxiety, and it will be valuable to develop better pharmacological treatments for eating disorders and obesity.

Topics: Amygdala; Animals; Anorexia; Anti-Obesity Agents; Anxiety; Arachidonic Acids; Body Weight; Cannabinoid Receptor Antagonists; Corticosterone; Diet; Dietary Sucrose; Endocannabinoids; Female; Glycerides; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Rimonabant

The role of the endocannabinoid system in vomiting has been previously studied using several animal species. These investigations have clearly demonstrated an anti-emetic role for the eCB, anandamide, in these animal models; however, research concerning the role of 2-arhachidonoylglycerol (2AG) has been less clear. The aim of the present study was to assess the effects of exogenous 2AG administration in the house musk shrew, Suncus murinus. In Experiment 1, shrews were injected with vehicle or 2AG (1, 2, 5, 10 mg/kg) 15 min prior to behavioral testing in which the frequency of vomiting episodes was observed. In Experiment 2, shrews were pre-treated with 2AG (2, 5 mg/kg) prior to being administered the emetic drug, lithium chloride (LiCl). It was found that 2AG alone did not induce emesis, but interfered with vomiting in response to LiCl administration. The anti-emetic effects of 2AG in Suncus murinus do not appear to be mediated by CB1 receptors, as concomitant pretreatment with the CB1 receptor antagonist, SR141716, did not reverse the suppressive effects of 2AG. These results confirm that manipulations that increase levels of 2AG exert anti-emetic effects in the house musk shrew.

Topics: Animals; Antiemetics; Arachidonic Acids; Cannabinoid Receptor Agonists; Data Interpretation, Statistical; Dose-Response Relationship, Drug; Endocannabinoids; Female; Glycerides; Lithium Chloride; Male; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; Shrews; Vomiting

Endocannabinoids play essential roles in synaptic plasticity; thus, their dysfunction often causes impairments in memory or cognition. However, it is not well understood whether deficits in the endocannabinoid system account for the cognitive symptoms of schizophrenia. Here, we show that endocannabinoid-mediated synaptic regulation is impaired by the prolonged elevation of neuregulin-1, the abnormality of which is a hallmark in many patients with schizophrenia. When rat hippocampal slices were chronically treated with neuregulin-1, the degradation of 2-arachidonoylglycerol (2-AG), one of the major endocannabinoids, was enhanced due to the increased expression of its degradative enzyme, monoacylglycerol lipase. As a result, the time course of depolarization-induced 2-AG signaling was shortened, and the magnitude of 2-AG-dependent long-term depression of inhibitory synapses was reduced. Our study reveals that an alteration in the signaling of 2-AG contributes to hippocampal synaptic dysfunction in a hyper-neuregulin-1 condition and thus provides novel insights into potential schizophrenic therapeutics that target the endocannabinoid system.

Topics: Analysis of Variance; Animals; Animals, Newborn; Anti-Anxiety Agents; Arachidonic Acids; Benzodioxoles; Biophysics; Electric Stimulation; Endocannabinoids; Glycerides; Hippocampus; Inhibitory Postsynaptic Potentials; Long-Term Synaptic Depression; Methoxyhydroxyphenylglycol; Neural Inhibition; Neuregulin-1; Organ Culture Techniques; Patch-Clamp Techniques; Piperidines; Pyrimidines; Rats; Receptor, Cannabinoid, CB1; Synapses

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

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

Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate generates diacylglycerol, inositol 1,4,5-trisphosphate and protons, all of which can regulate TRPV1 activity via different mechanisms. Here we explored the possibility that the diacylglycerol metabolites 2-arachidonoylglycerol and 1-arachidonoylglycerol, and not metabolites of these monoacylglycerols, activate TRPV1 and contribute to this signaling cascade. 2-Arachidonoylglycerol and 1-arachidonoylglycerol activated native TRPV1 on vascular sensory nerve fibers and heterologously expressed TRPV1 in whole cells and inside-out membrane patches. The monoacylglycerol lipase inhibitors methylarachidonoyl-fluorophosphonate and JZL184 prevented the metabolism of deuterium-labeled 2-arachidonoylglycerol and deuterium-labeled 1-arachidonoylglycerol in arterial homogenates, and enhanced TRPV1-mediated vasodilator responses to both monoacylglycerols. In mesenteric arteries from TRPV1 knock-out mice, vasodilator responses to 2-arachidonoylglycerol were minor. Bradykinin and adenosine triphosphate, ligands of phospholipase C-coupled membrane receptors, increased the content of 2-arachidonoylglycerol in dorsal root ganglia. In HEK293 cells expressing the phospholipase C-coupled histamine H1 receptor, exposure to histamine stimulated the formation of 2-AG, and this effect was augmented in the presence of JZL184. These effects were prevented by the diacylglycerol lipase inhibitor tetrahydrolipstatin. Histamine induced large whole cell currents in HEK293 cells co-expressing TRPV1 and the histamine H1 receptor, and the TRPV1 antagonist capsazepine abolished these currents. JZL184 increased the histamine-induced currents and tetrahydrolipstatin prevented this effect. The calcineurin inhibitor ciclosporin and the endogenous "entourage" compound palmitoylethanolamide potentiated the vasodilator response to 2-arachidonoylglycerol, disclosing TRPV1 activation of this monoacylglycerol at nanomolar concentrations. Furthermore, intracerebroventricular injection of JZL184 produced TRPV1-dependent antinociception in the mouse formalin test. Our results show that intact 2-arachidonoylglycerol and 1-arachidonoylglycerol are endogenous TRPV1 activators, contributing to phospholipase C-dependent TRPV1 channel activation and TRPV1-mediated antinociceptive signaling in the brain.

Topics: Animals; Arachidonic Acids; Benzodioxoles; Endocannabinoids; Female; Ganglia, Spinal; Glycerides; HEK293 Cells; Humans; Male; Mice; Monoglycerides; Nociception; Piperidines; Rats; Sensory Receptor Cells; TRPV Cation Channels; Type C Phospholipases

JZL184 is a selective inhibitor of monoacylglycerol lipase (MAGL), the enzyme that preferentially catabolizes the endocannabinoid 2-arachidonoyl glycerol (2-AG). Here, we have studied the effects of JZL184 on inflammatory cytokines in the brain and plasma following an acute immune challenge and the underlying receptor and molecular mechanisms involved.. JZL184 and/or the CB₁ receptor antagonist, AM251 or the CB₂ receptor antagonist, AM630 were administered to rats 30 min before lipopolysaccharide (LPS). 2 h later cytokine expression and levels, MAGL activity, 2-AG, arachidonic acid and prostaglandin levels were measured in the frontal cortex, plasma and spleen.. JZL184 attenuated LPS-induced increases in IL-1β, IL-6, TNF-α and IL-10 but not the expression of the inhibitor of NFkB (IκBα) in rat frontal cortex. AM251 attenuated JZL184-induced decreases in frontal cortical IL-1β expression. Although arachidonic acid levels in the frontal cortex were reduced in JZL184-treated rats, MAGL activity, 2-AG, PGE₂ and PGD₂ were unchanged. In comparison, MAGL activity was inhibited and 2-AG levels enhanced in the spleen following JZL184. In plasma, LPS-induced increases in TNF-α and IL-10 levels were attenuated by JZL184, an effect partially blocked by AM251. In addition, AM630 blocked LPS-induced increases in plasma IL-1β in the presence, but not absence, of JZL184.. Inhibition of peripheral MAGL in rats by JZL184 suppressed LPS-induced circulating cytokines that in turn may modulate central cytokine expression. The data provide further evidence for the endocannabinoid system as a therapeutic target in treatment of central and peripheral inflammatory disorders.

Topics: Animals; Anti-Anxiety Agents; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acids; Benzodioxoles; Cannabinoid Receptor Antagonists; Cytokines; Encephalitis; Endocannabinoids; Enzyme Inhibitors; Frontal Lobe; Glycerides; Lipopolysaccharides; Male; Monoacylglycerol Lipases; Nerve Tissue Proteins; Peritonitis; Piperidines; Prostaglandins; Random Allocation; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Spleen

Cisplatin, a platinum-derived chemotherapeutic agent, produces mechanical and coldallodynia reminiscent of chemotherapy-induced neuropathy in humans. The endocannabinoid system represents a novel target for analgesic drug development. The endocannabinoid signaling system consists of endocannabinoids (e.g. anandamide (AEA) and 2-arachidonoylglycerol (2-AG)), cannabinoid receptors (e.g. CB(1) and CB(2)) and the enzymes controlling endocannabinoid synthesis and degradation. AEA is hydrolyzed by fatty-acid amide hydrolase (FAAH) whereas 2-AG is hydrolyzed primarily by monoacylglycerol lipase (MGL). We compared effects of brain permeant (URB597) and impermeant (URB937) inhibitors of FAAH with an irreversible inhibitor of MGL (JZL184) on cisplatin-evoked behavioral hypersensitivities. Endocannabinoid modulators were compared with agents used clinically to treat neuropathy (i.e. the opioid analgesic morphine, the anticonvulsant gabapentin and the tricyclic antidepressant amitriptyline). Cisplatin produced robust mechanical and cold allodynia but did not alter responsiveness to heat. After neuropathy was fully established, groups received acute intraperitoneal (i.p.) injections of vehicle, amitriptyline (30 mg/kg), gabapentin (100 mg/kg), morphine (6 mg/kg), URB597 (0.1 or 1 mg/kg), URB937 (0.1 or 1 mg/kg) or JZL184 (1, 3 or 8 mg/kg). Pharmacological specificity was assessed by coadministering each endocannabinoid modulator with either a CB(1) (AM251 3 mg/kg), CB(2) (AM630 3 mg/kg), TRPV1 (AMG9810 3 mg/kg) or TRPA1 (HC030031 8 mg/kg) antagonist. Effects of cisplatin on endocannabinoid levels and transcription of receptors (CB(1), CB(2), TRPV1, TRPA1) and enzymes (FAAH, MGL) linked to the endocannabinoid system were also assessed. URB597, URB937, JZL184 and morphine reversed cisplatin-evoked mechanical and cold allodynia to pre-cisplatin levels. By contrast, gabapentin only partially reversed the observed allodynia while amitriptyline, administered acutely, was ineffective. CB(1) or CB(2) antagonists completely blocked the anti-allodynic effects of both FAAH (URB597, URB937) and MGL (JZL184) inhibitors to mechanical and cold stimulation. By contrast, the TRPV1 antagonist AMG9810 blocked the anti-allodynic efficacy of both FAAH inhibitors, but not the MGL inhibitor. By contrast, the TRPA1 antagonist HC30031 did not attenuate anti-allodynic efficacy of any endocannabinoid modulator. When the levels of endocannabinoids were examined, cisplatin increased both anandami

Topics: Amidohydrolases; Analgesics; Animals; Antineoplastic Agents; Arachidonic Acids; Benzamides; Benzodioxoles; Cannabinoids; Carbamates; Cisplatin; Endocannabinoids; Enzyme Inhibitors; Ganglia, Spinal; Glycerides; Hyperalgesia; Lipid Metabolism; Male; Monoacylglycerol Lipases; Peripheral Nervous System Diseases; Piperidines; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; RNA, Messenger; Spinal Cord; TRPV Cation Channels

N-palmitoylethanolamine (PEA), an endogenous fatty acid ethanolamide, plays a key role in the regulation of the inflammatory response and pain through, among others, activation of nuclear peroxisome proliferator-activated receptors (PPAR-α). Endogenous cannabinoids play a protective role in several central nervous system (CNS) disorders, particularly those associated with neuronal hyperexcitability. We investigated the effects of PEA and the role of PPAR-α in absence epilepsy using the WAG/Rij rat model. PEA, anandamide (AEA), a PPAR-α antagonist (GW6471) and a synthetic CB1 receptor antagonist/inverse agonist (SR141716) were administered to WAG/Rij rats in order to evaluate the effects on epileptic spike-wave discharges (SWDs) on EEG recordings. We studied also the effects of PEA co-administration with SR141716 and GW6471 and compared these effects with those of AEA to evaluate PEA mechanism of action and focusing on CB1 receptors and PPAR-α. Both PEA and AEA administration significantly decreased SWDs parameters (absence seizures). In contrast, GW6471 was devoid of effects while SR141716 had pro-absence effects. The co-administration of SR141716 with PEA or AEA completely blocked the anti-absence effects of these compounds. GW6471 antagonized PEA's effects whereas it did not modify AEA's effects. Furthermore, we have also measured PEA, AEA and 2-AG (2-arachidonoylglycerol) brain levels identifying significant differences between epileptic and control rats such as decreased PEA levels in both thalamus and cortex that might contribute to absence epilepsy. Our data demonstrate that PEA has anti-absence properties in the WAG/Rij rat model and that such properties depend on PPAR-α and indirect activation of CB1 receptors. This article is part of the Special Issue entitled 'New Targets and Approaches to the Treatment of Epilepsy'.

Topics: Amides; Animals; Anticonvulsants; Arachidonic Acids; Calcium Channel Blockers; Cannabinoid Receptor Antagonists; Dose-Response Relationship, Drug; Electroencephalography; Endocannabinoids; Epilepsy, Absence; Ethanolamines; Glycerides; Injections, Intraventricular; Lipid Metabolism; Male; Oxazoles; Palmitic Acids; Piperidines; Polyunsaturated Alkamides; PPAR alpha; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Rimonabant; Tyrosine

2-Arachidonoylglycerol (2-AG) is recognized as a potent endocannabinoid, which reduces synaptic transmission through cannabinoid CB(1) receptors, and is hydrolyzed by monoacylglycerol lipase (MGL) to arachidonic acid (AA), a cyclooxygenase substrate. We already reported that centrally administered MGL and cyclooxygenase inhibitors each reduced the intracerebroventricularly (i.c.v.) administered bombesin-induced secretion of adrenal catecholamines, while a centrally administered CB(1)-antagonist potentiated the response, indirectly suggesting bidirectional roles of brain 2-AG (stimulatory and inhibitory roles) in the bombesin-induced response. In the present study, we separately examined these bidirectional roles using 2-AG and 2-AG ether (2-AG-E) (stable 2-AG analog for MGL) in rats. 2-AG (0.5 μmol/animal, i.c.v.), but not 2-AG-E (0.5 μmol/animal, i.c.v.), elevated basal plasma catecholamines with JZL184 (MGL inhibitor)- and indomethacin (cyclooxygenase inhibitor)-sensitive brain mechanisms. 2-AG-E (0.1 μmol/animal, i.c.v.) effectively reduced the bombesin (1 nmol/animal, i.c.v.)-induced elevation of plasma catecholamines with rimonabant (CB(1) antagonist)-sensitive brain mechanisms. Immunohistochemical studies demonstrated the bombesin-induced activation of diacylglycerol lipase α (2-AG-producing enzyme)-positive spinally projecting neurons in the hypothalamic paraventricular nucleus, a control center of central adrenomedullary outflow. These results directly indicate bidirectional roles of brain 2-AG, a stimulatory role as an AA precursor and an inhibitory role as an endocannabinoid, in the bombesin-induced central adrenomedullary outflow in rats.

Topics: Adrenal Medulla; Animals; Arachidonic Acids; Benzodioxoles; Bombesin; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Catecholamines; Cyclooxygenase Inhibitors; Drug Interactions; Endocannabinoids; Glycerides; Indomethacin; Injections, Intraventricular; Lipoprotein Lipase; Male; Monoacylglycerol Lipases; Neurotransmitter Agents; Paraventricular Hypothalamic Nucleus; Piperidines; Pyrazoles; Rats; Rimonabant

To evaluate the role of 2-arachidonoyl glycerol (2AG) in the regulation of nausea and vomiting using animal models of vomiting and of nausea-like behaviour (conditioned gaping).. Vomiting was assessed in shrews (Suncus murinus), pretreated with JZL184, a selective monoacylglycerol lipase (MAGL) inhibitor which elevates endogenous 2AG levels, 1 h before administering the emetogenic compound, LiCl. Regulation of nausea-like behaviour in rats by exogenous 2AG or its metabolite arachidonic acid (AA) was assessed, using the conditioned gaping model. The role of cannabinoid CB(1) receptors, CB(2) receptors and cyclooxygenase (COX) inhibition in suppression of vomiting or nausea-like behaviour was assessed.. JZL184 dose-dependently suppressed vomiting in shrews, an effect prevented by pretreatment with the CB(1) receptor inverse agonist/antagonist, AM251. In shrew brain tissue, JZL184 inhibited MAGL activity in vivo. In rats, 2AG suppressed LiCl-induced conditioned gaping but this effect was not prevented by AM251 or the CB(2) receptor antagonist, AM630. Instead, the COX inhibitor, indomethacin, prevented suppression of conditioned gaping by 2AG or AA. However, when rats were pretreated with a high dose of JZL184 (40 mg·kg(-1) ), suppression of gaping by 2AG was partially reversed by AM251. Suppression of conditioned gaping was not due to interference with learning because the same dose of 2AG did not modify the strength of conditioned freezing to a shock-paired tone.. Our results suggest that manipulations that elevate 2AG may have anti-emetic or anti-nausea potential.. This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

Topics: Animals; Arachidonic Acids; Behavior, Animal; Benzodioxoles; Brain; Endocannabinoids; Enzyme Inhibitors; Fear; Glycerides; Lithium Chloride; Male; Monoacylglycerol Lipases; Nausea; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Shrews; Vomiting

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

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

The cannabinoid receptor type 1 (CB1) is a G protein-coupled receptor that is activated in an autocrine fashion by the endocannabinoids (EC), N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG). The CB1 and its endogenous and synthetic agonists are emerging as therapeutic targets in several cancers due to their ability to suppress carcinoma cell invasion and migration. However, the mechanisms that the CB1 regulates cell motility are not well understood. In this study, we examined the molecular mechanisms that diminish cell migration upon the CB1 activation in prostate carcinoma cells. The CB1 activation with the agonist WIN55212 significantly diminishes the small GTPase RhoA activity but modestly increases the Rac1 and Cdc42 activity. The diminished RhoA activity is accompanied by the loss of actin/myosin microfilaments, cell spreading, and cell migration. Interestingly, the CB1 inactivation with the selective CB1 antagonist AM251 significantly increases RhoA activity, enhances microfilament formation and cell spreading, and promotes cell migration. This finding suggests that endogenously produced EC activate the CB1, resulting in chronic repression of RhoA activity and cell migration. Consistent with this possibility, RhoA activity is significantly diminished by the exogenous application of AEA but not by 2-AG in PC-3 cells (cells with very low AEA hydrolysis). Pretreatment of cells with a monoacylglycerol lipase inhibitor, JZL184, which blocks 2-AG hydrolysis, decreases the RhoA activity. These results indicate the unique CB1 signaling and support the model that EC, through their autocrine activation of CB1 and subsequent repression of RhoA activity, suppress migration in prostate carcinoma cells.

Topics: Actins; Arachidonic Acids; Benzoxazines; Biological Transport, Active; cdc42 GTP-Binding Protein; Cell Line, Tumor; Cell Membrane; Cell Movement; Endocannabinoids; Glycerides; Humans; Male; Morpholines; Myosins; Naphthalenes; Piperidines; Polyunsaturated Alkamides; Prostatic Neoplasms; Pyrazoles; rac1 GTP-Binding Protein; Receptor, Cannabinoid, CB1; rhoA GTP-Binding Protein

The endocannabinoid system is implicated in numerous physiopathological processes while more and more pieces of evidence wave the link between this complex machinery and cancer related phenomenon. In these lines, we confirmed the effects of 2-arachidonoylglycerol (2-AG), the main endocannabinoid, on neuroblastoma cells proliferation in vitro, and proved that some N-phenylmaleimide compounds that were previously shown as MAGL inhibitors can also inhibit type 2 topoisomerase. We also shed light on their antiproliferative effects on a neuroblastoma cell line. In order to establish a link between MAGL inhibition, topoisomerase inhibition and the effects on N1E-115 cells, we tested combinations of maleimides or known endocannabinoid metabolism inhibitors and 2-AG, the major MAGL substrate, on N1E-115 cells. However, none of the inhibitors tested, except the carbamate CAY10499, managed to increase 2-AG's effects. Even the MAGL reference inhibitor JZL184 failed to induce a stronger inhibition of proliferation.

Topics: Antineoplastic Agents; Arachidonic Acids; Benzodioxoles; Cannabinoid Receptor Modulators; Carbamates; Cell Proliferation; DNA Topoisomerases, Type II; Endocannabinoids; Etoposide; Glycerides; Humans; Maleimides; Monoacylglycerol Lipases; Neuroblastoma; Oxadiazoles; Piperidines; Topoisomerase II Inhibitors; Tumor Cells, Cultured

Different types of presynaptic inhibitory Gα(i/o) protein-coupled receptors usually do not act independently of each other but rather pre-activation of receptor X impairs the effect mediated via receptor Y. It is, however, unknown whether this interaction extends to the cannabinoid CB(1) receptor on cholinergic neurones and hence we studied whether its activation, pharmacological blockade, or genetic inactivation affects the function of other presynaptic inhibitory receptors. The electrically evoked acetylcholine or noradrenaline release was determined in superfused rodent tissues preincubated with (3)H-choline or (3)H-noradrenaline. The muscarinic M(2) receptor, Gα(i), and Gα(o) proteins were determined in hippocampal synaptosomes by Western blotting. Hippocampal anandamide and 2-arachidonoyl glycerol levels were determined by LC-MS/MS. The inhibitory effect of the muscarinic receptor agonist oxotremorine on acetylcholine release in hippocampal slices was increased by genetic CB(1) receptor ablation (mouse) and the CB(1) antagonist rimonabant (rat but not mouse) and decreased by a cannabinoid receptor agonist (mouse). In mouse tissues, CB(1) receptor ablation also increased the effect of a δ opioid receptor agonist on acetylcholine release in the hippocampus and the effect of oxotremorine on noradrenaline release in the vas deferens. CB(1) receptor ablation, to a very slight extent, increased Gα(o) protein levels without affecting either Gα(i) and M(2) receptor protein or the levels of anandamide and 2-arachidonoyl glycerol in the hippocampus. In conclusion, the CB(1) receptor shows an inhibitory interaction with the muscarinic and δ opioid receptor on cholinergic neurones in the rodent hippocampus and with the muscarinic receptor on noradrenergic neurones in the mouse vas deferens.

Topics: Analgesics, Opioid; Animals; Arachidonic Acid; Arachidonic Acids; Cerebral Cortex; Cholinergic Neurons; Endocannabinoids; Enkephalin, D-Penicillamine (2,5)-; Glycerides; GTP-Binding Protein alpha Subunits; Hippocampus; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscarinic Agonists; Oxotremorine; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptors, Muscarinic; Receptors, Opioid, delta; Rimonabant; Synaptosomes; Vas Deferens

The endocannabinoid system has recently emerged as a vital component of the stress response and is an appealing target for the treatment of mood and anxiety disorders. Additionally, corticolimbic endocannabinoid signaling is important for stress-induced regulation of emotional behavior. However, the mechanism by which this occurs remains elusive. Combining biochemical and behavioral analyses within the forced swim test, we examined whether stress-induced regulation of endocannabinoid signaling in the medial prefrontal cortex contributes to behavioral responses to stress, and whether these responses are dependent on serotonergic neurotransmission. Forced swim stress produced a rapid and pronounced reduction in medial prefrontal anandamide content, but had no effect on 2-arachidonoylglycerol content within this region. Local administration of the anandamide hydrolysis inhibitor URB597 (0.01μg) into the ventromedial region of the prefrontal cortex decreased passive coping responses and increased active behavioral strategies, a phenomenon which was blocked by local antagonism of the CB(1) receptor. Furthermore, local inhibition of anandamide hydrolysis within the medial PFC increased the firing rate of serotonergic neurons within the dorsal raphe, suggesting that prefrontal cortical endocannabinoid signaling may modulate stress coping behaviors through a regulation of serotonergic neurotransmission. Accordingly, serotonin depletion prevented the ability of inhibition of anandamide hydrolysis within the medial PFC to promote active stress coping responses. Collectively, these data argue that stress-induced changes in endocannabinoid signaling within the medial PFC modulate stress-coping behaviors through a regulation of serotonergic neurotransmission and provide a neuroanatomical framework by which we may understand the mechanisms subserving the antidepressant potential of the endocannabinoid system.

Topics: Action Potentials; Adaptation, Psychological; Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Antagonists; Carbamates; Endocannabinoids; Enzyme Inhibitors; Fenclonine; Glycerides; Male; Microinjections; Piperidines; Polyunsaturated Alkamides; Prefrontal Cortex; Pyrazoles; Raphe Nuclei; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Serotonergic Neurons; Signal Transduction; Stress, Psychological

There is extensive evidence that glucocorticoid hormones impair the retrieval of memory of emotionally arousing experiences. Although it is known that glucocorticoid effects on memory retrieval impairment depend on rapid interactions with arousal-induced noradrenergic activity, the exact mechanism underlying this presumably nongenomically mediated glucocorticoid action remains to be elucidated. Here, we show that the hippocampal endocannabinoid system, a rapidly activated retrograde messenger system, is involved in mediating glucocorticoid effects on retrieval of contextual fear memory. Systemic administration of corticosterone (0.3-3 mg/kg) to male Sprague-Dawley rats 1 h before retention testing impaired the retrieval of contextual fear memory without impairing the retrieval of auditory fear memory or directly affecting the expression of freezing behavior. Importantly, a blockade of hippocampal CB1 receptors with AM251 prevented the impairing effect of corticosterone on retrieval of contextual fear memory, whereas the same impairing dose of corticosterone increased hippocampal levels of the endocannabinoid 2-arachidonoylglycerol. We also found that antagonism of hippocampal β-adrenoceptor activity with local infusions of propranolol blocked the memory retrieval impairment induced by the CB receptor agonist WIN55,212-2. Thus, these findings strongly suggest that the endocannabinoid system plays an intermediary role in regulating rapid glucocorticoid effects on noradrenergic activity in impairing memory retrieval of emotionally arousing experiences.

Topics: Acoustic Stimulation; Adrenergic beta-Antagonists; Animals; Arachidonic Acids; Arousal; Benzoxazines; Cannabinoid Receptor Modulators; Conditioning, Classical; Corticosterone; Electroshock; Emotions; Endocannabinoids; Fear; Freezing Reaction, Cataleptic; Glucocorticoids; Glycerides; Hippocampus; Male; Memory; Morpholines; Naphthalenes; Piperidines; Propranolol; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptors, Glucocorticoid

We show that a fully functional endocannabinoid system is present in primary human melanocytes (normal human epidermal melanocyte cells), including anandamide (AEA), 2-arachidonoylglycerol, the respective target receptors (CB(1), CB(2), and TRPV1), and their metabolic enzymes. We also show that at higher concentrations AEA induces normal human epidermal melanocyte apoptosis (∼3-fold over controls at 5 μM) through a TRPV1-mediated pathway that increases DNA fragmentation and p53 expression. However, at lower concentrations, AEA and other CB(1)-binding endocannabinoids dose-dependently stimulate melanin synthesis and enhance tyrosinase gene expression and activity (∼3- and ∼2-fold over controls at 1 μM). This CB(1)-dependent activity was fully abolished by the selective CB(1) antagonist SR141716 or by RNA interference of the receptor. CB(1) signaling engaged p38 and p42/44 mitogen-activated protein kinases, which in turn activated the cyclic AMP response element-binding protein and the microphthalmia-associated transcription factor. Silencing of tyrosinase or microphthalmia-associated transcription factor further demonstrated the involvement of these proteins in AEA-induced melanogenesis. In addition, CB(1) activation did not engage the key regulator of skin pigmentation, cyclic AMP, showing a major difference compared with the regulation of melanogenesis by α-melanocyte-stimulating hormone through melanocortin 1 receptor.

Topics: alpha-MSH; Animals; Apoptosis; Arachidonic Acids; Blotting, Western; Cannabinoid Receptor Modulators; Cells, Cultured; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Dose-Response Relationship, Drug; Endocannabinoids; Gene Expression; Glycerides; HeLa Cells; Humans; Male; Melanins; Melanocytes; Mice; Microphthalmia-Associated Transcription Factor; Mitogen-Activated Protein Kinases; Models, Biological; Monophenol Monooxygenase; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Reverse Transcriptase Polymerase Chain Reaction; Rimonabant; RNA Interference

The endocannabinoid system (ECS) tightly controls emotional responses to acute aversive stimuli. Repeated stress alters ECS activity but the role played by the ECS in the emotional consequences of repeated stress has not been investigated in detail. This study used social defeat stress, together with pharmacology and genetics to examine the role of cannabinoid type-1 (CB(1)) receptors on repeated stress-induced emotional alterations. Seven daily social defeat sessions increased water (but not food) intake, sucrose preference, anxiety, cued fear expression, and adrenal weight in C57BL/6N mice. The first and the last social stress sessions triggered immediate brain region-dependent changes in the concentrations of the principal endocannabinoids anandamide and 2-arachidonoylglycerol. Pretreatment before each of the seven stress sessions with the CB(1) receptor antagonist rimonabant prolonged freezing responses of stressed mice during cued fear recall tests. Repeated social stress abolished the increased fear expression displayed by constitutive CB(1) receptor-deficient mice. The use of mutant mice lacking CB(1) receptors from cortical glutamatergic neurons or from GABAergic neurons indicated that it is the absence of the former CB(1) receptor population that is responsible for the fear responses in socially stressed CB(1) mutant mice. In addition, stress-induced hypolocomotor reactivity was amplified by the absence of CB(1) receptors from GABAergic neurons. Mutant mice lacking CB(1) receptors from serotonergic neurons displayed a higher anxiety but decreased cued fear expression than their wild-type controls. These mutant mice failed to show social stress-elicited increased sucrose preference. This study shows that (i) release of endocannabinoids during stress exposure impedes stress-elicited amplification of cued fear behavior, (ii) social stress opposes the increased fear expression and delayed between-session extinction because of the absence of CB(1) receptors from cortical glutamatergic neurons, and (iii) CB(1) receptors on central serotonergic neurons are involved in the sweet consumption response to repeated stress.

Topics: Adrenal Glands; Animals; Arachidonic Acids; Brain; Drinking; Eating; Emotions; Endocannabinoids; Food Preferences; Glycerides; Immobility Response, Tonic; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Motor Activity; Neurons; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; Stress, Psychological

The final motor output underlying behavior arises from an appropriate balance between excitation and inhibition within neural networks. Retrograde signaling by endocannabinoids adapts synaptic strengths and the global activity of neural networks. In the spinal cord, endocannabinoids are mobilized postsynaptically from network neurons and act retrogradely on presynaptic cannabinoid receptors to potentiate the locomotor frequency. However, it is still unclear whether mechanisms exist within the locomotor networks that determine the sign of the modulation by cannabinoid receptors to differentially regulate excitation and inhibition. In this study, using the lamprey spinal cord in vitro, we first report that 2-AG (2-arachidonyl glycerol) is mobilized by network neurons and underlies a form of modulation that is embedded within the locomotor networks. We then show that the polarity of the endocannabinoid modulation is gated by nitric oxide to enable simultaneously potentiation of excitation and depression of inhibition within the spinal locomotor networks. Our results suggest that endocannabinoid and nitric oxide systems interact to mediate inversion of the polarity of synaptic plasticity within the locomotor networks. Thus, endocannabinoid and nitric oxide shift in the excitation-inhibition balance to set the excitability of the spinal locomotor network.

Topics: Animals; Arachidonic Acids; Benzodioxoles; Cannabinoid Receptor Modulators; Endocannabinoids; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Female; Glycerides; Lactones; Lampreys; Locomotion; Male; N-Methylaspartate; Nerve Net; Neuronal Plasticity; Neurotransmitter Agents; Nitric Oxide; Orlistat; Patch-Clamp Techniques; Piperidines; Receptor, Cannabinoid, CB1; Spinal Cord; Synaptic Transmission

Stimulation of cannabinoid CB(1) receptors in nucleus accumbens shell has been shown to stimulate feeding and enhance positive 'liking' reactions to intraoral sucrose. This study examined the behavioural effects of noladin ether and 2-arachidonoylglycerol following infusion into accumbens shell, on chow intake and food preference in high-carbohydrate and high-fat preferring rats. Noladin ether, potently and dose-dependently stimulated chow intake as compared with 2-arachidonoylglycerol in free-feeding rats. In the diet preference paradigm, in which rats were given free access to both, high-carbohydrate (HC) and high-fat (HF) diets simultaneously, an intra-accumbens administration of noladin ether as well as 2-arachidonoylglycerol, preferentially enhanced fat consumption over carbohydrate in both HF- and HC-preferring rats. These effects were significantly attenuated by the CB(1) receptor antagonist, AM 251. These results suggesting that, the endocannabinoids through CB(1) receptors, affects appetite for specific dietary components. Both these agents exert a specific action on eating motivation and possibly promoting eating by enhancing the incentive value of food. Altogether these findings reinforce the idea that the endogenous cannabinoid system in the accumbens shell may be important to augment reward-driven feeding via modulation of CB(1) receptor signalling pathways.

Topics: Animals; Appetite; Arachidonic Acids; Cannabinoid Receptor Modulators; Diet; Diet, High-Fat; Dietary Carbohydrates; Eating; Endocannabinoids; Food Preferences; Glycerides; Hyperphagia; Male; Nucleus Accumbens; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Sucrose

Acute stress reduces pain sensitivity by engaging an endocannabinoid signaling circuit in the midbrain. The neural mechanisms governing this process and molecular identity of the endocannabinoid substance(s) involved are unknown. We combined behavior, pharmacology, immunohistochemistry, RNA interference, quantitative RT-PCR, enzyme assays, and lipidomic analyses of endocannabinoid content to uncover the role of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) in controlling pain sensitivity in vivo. Here, we show that footshock stress produces antinociception in rats by activating type 5 metabotropic glutamate receptors (mGlu(5)) in the dorsolateral periaqueductal gray (dlPAG) and mobilizing 2-AG. Stimulation of mGlu(5) in the dlPAG with DHPG [(S)-3,5-dihydroxyphenylglycine] triggered 2-AG formation and enhanced stress-dependent antinociception through a mechanism dependent upon both postsynaptic diacylglycerol lipase (DGL) activity, which releases 2-AG, and presynaptic CB(1) cannabinoid receptors. Pharmacological blockade of DGL activity in the dlPAG with RHC80267 [1,6-bis(cyclohexyloximinocarbonylamino)hexane] and (-)-tetrahydrolipstatin (THL), which inhibit activity of DGL-α and DGL-β isoforms, suppressed stress-induced antinociception. Inhibition of DGL activity in the dlPAG with THL selectively decreased accumulation of 2-AG without altering levels of anandamide. The putative 2-AG-synthesizing enzyme DGL-α colocalized with mGlu(5) at postsynaptic sites of the dlPAG, whereas CB(1) was confined to presynaptic terminals, consistent with a role for 2-AG as a retrograde signaling messenger. Finally, virally mediated silencing of DGL-α, but not DGL-β, transcription in the dlPAG mimicked effects of DGL inhibition in suppressing both endocannabinoid-mediated stress antinociception and 2-AG formation. The results indicate that activation of the postsynaptic mGlu(5)-DGL-α cascade triggers retrograde 2-AG signaling in vivo. This pathway is required for endocannabinoid-mediated stress-induced analgesia.

Topics: Analgesia; Analysis of Variance; Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Cyclohexanones; Dose-Response Relationship, Drug; Electroconvulsive Therapy; Endocannabinoids; Excitatory Amino Acid Antagonists; Glycerides; Lipoprotein Lipase; Male; Methoxyhydroxyphenylglycol; Mice; Microscopy, Immunoelectron; Pain; Periaqueductal Gray; Piperidines; Protease Inhibitors; Pyrazoles; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Rimonabant; RNA, Messenger; RNA, Small Interfering; Synapses; Tandem Mass Spectrometry

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

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

The endocannabinoid 2-arachidonoyl glycerol (2-AG) acts as a retrograde messenger and modulates synaptic signaling e. g. in the hippocampus. 2-AG also exerts neuroprotective effects under pathological situations. To better understand the mechanism beyond physiological signaling we used Organotypic Entorhino-Hippocampal Slice Cultures (OHSC) and investigated the temporal regulation of 2-AG in different cell subsets during excitotoxic lesion and dendritic lesion of long range projections in the enthorhinal cortex (EC), dentate gyrus (DG) and the cornu ammonis region 1 (CA1).. 2-AG levels were elevated 24 h after excitotoxic lesion in CA1 and DG (but not EC) and 24 h after perforant pathway transection (PPT) in the DG only. After PPT diacylglycerol lipase alpha (DAGL) protein, the synthesizing enzyme of 2-AG was decreased when Dagl mRNA expression and 2-AG levels were enhanced. In contrast to DAGL, the 2-AG hydrolyzing enzyme monoacylglycerol lipase (MAGL) showed no alterations in total protein and mRNA expression after PPT in OHSC. MAGL immunoreaction underwent a redistribution after PPT and excitotoxic lesion since MAGL IR disappeared in astrocytes of lesioned OHSC. DAGL and MAGL immunoreactions were not detectable in microglia at all investigated time points. Thus, induction of the neuroprotective endocannabinoid 2-AG might be generally accomplished by down-regulation of MAGL in astrocytes after neuronal lesions.. Increase in 2-AG levels during secondary neuronal damage reflects a general neuroprotective mechanism since it occurred independently in both different lesion models. This intrinsic up-regulation of 2-AG is synergistically controlled by DAGL and MAGL in neurons and astrocytes and thus represents a protective system for neurons that is involved in dendritic reorganisation.

Topics: Animals; Arachidonic Acids; Astrocytes; Benzodioxoles; Cell Survival; Endocannabinoids; Female; Gene Knockdown Techniques; Glycerides; Hippocampus; Lipoprotein Lipase; Microglia; Monoacylglycerol Lipases; N-Methylaspartate; Neurons; Perforant Pathway; Piperidines; Protein Transport; Rats, Wistar; Transcription, Genetic; Up-Regulation

Peripheral and central endocannabinoids and cognate acylethanolamides (AEs) may play important but distinct roles in regulating energy balance.. We hypothesized that in humans central/peripheral endocannabinoids are differently associated with adiposity and energy expenditure and differ by race.. We examined associations of arachindonoylethanolamide, 2-arachidonoylglycerol, palmitoylethanolamide, and oleoylethanolamide (OEA) assayed in plasma and cerebrospinal fluid (CSF) with race, adiposity, and energy expenditure.. In this monitored clinical inpatient study, CSF was obtained by lumbar puncture in 27 individuals (12 Caucasian, 11 American Indian, and four African-American). Twenty-four hour and sleep energy expenditure were measured by indirect calorimetry in a respiratory chamber.. Samples were analyzed from a previous study originally designed to test a blood-brain barrier leptin transport deficit in human obesity.. CSF (but not peripheral) 2-arachidonoylglycerol was significantly increased in American Indians compared with Caucasians (18.48 ± 6.17 vs. 10.62 ± 4.58 pmol/ml, P < 0.01). In the whole group, peripheral AEs were positively but in CSF negatively associated with adiposity. However, in multivariate models adjusted for the other peripheral and CSF AEs, peripheral arachindonoylethanolamide was the only AE significantly associated with adiposity. Interestingly, CSF OEA concentrations were positively associated with adjusted 24 hour and sleep energy expenditure (r = 0.47, P < 0.05; r = 0.42, P < 0.05), but peripheral OEA was not.. These data indicate a central alteration of the endocannabinoid system in American Indians and furthermore show that AEs in both compartments play an important but distinct role in human energy balance regulation.

Topics: Absorptiometry, Photon; Adiposity; Amides; Anti-Obesity Agents; Arachidonic Acids; Blood Glucose; Cannabinoid Receptor Modulators; Endocannabinoids; Energy Metabolism; Ethanolamines; Ethnicity; Glycerides; Humans; Insulin; Leptin; Palmitic Acids; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant

BACKGROUND AND PURPOSE The endocannabinoid 2-arachidonoylglycerol (2-AG) is degraded primarily by monoacylglycerol lipase (MGL). We compared peripheral antinociceptive effects of JZL184, a novel irreversible MGL inhibitor, with the reversible MGL-preferring inhibitor URB602 and exogenous 2-AG in rats. EXPERIMENTAL APPROACH Nociception in the formalin test was assessed in groups receiving dorsal paw injections of vehicle, JZL184 (0.001-300 µg), URB602 (0.001-600 µg), 2-AG (ED(50)), 2-AG + JZL184 (at their ED(50)), 2-AG + URB602 (at their ED(50)), AM251 (80 µg), AM251 + JZL184 (10 µg), AM630 (25 µg) or AM630 + JZL184 (10 µg). Effects of MGL inhibitors on endocannabinoid accumulation and on activities of endocannabinoid-metabolizing enzymes were assessed. KEY RESULTS Intra-paw administration of JZL184, URB602 and 2-AG suppressed early and late phases of formalin pain. JZL184 and URB602 acted through a common mechanism. JZL184 (ED(50) Phase 1: 0.06 ± 0.028; Phase 2: 0.03 ± 0.011 µg) produced greater antinociception than URB602 (ED(50) Phase 1: 120 ± 51.3; Phase 2: 66 ± 23.9 µg) or 2-AG. Both MGL inhibitors produced additive antinociceptive effects when combined with 2-AG. Antinociceptive effects of JZL184, like those of URB602, were blocked by cannabinoid receptor 1 (CB(1)) and cannabinoid receptor 2 (CB(2)) antagonists. JZL184 suppressed MGL but not fatty-acid amide hydrolase or N-arachidonoyl-phosphatidylethanolamine phospholipase D activities ex vivo. URB602 increased hind paw 2-AG without altering anandamide levels. CONCLUSIONS AND IMPLICATIONS MGL inhibitors suppressed formalin-induced pain through peripheral CB(1) and CB(2) receptor mechanisms. MGL inhibition increased paw skin 2-AG accumulation to mediate these effects. MGL represents a target for the treatment of inflammatory pain.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzodioxoles; Biphenyl Compounds; Cannabinoid Receptor Modulators; Drug Interactions; Drug Therapy, Combination; Endocannabinoids; Glycerides; Male; Monoacylglycerol Lipases; Pain; Pain Measurement; Phospholipase D; Piperidines; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2

While endocannabinoid modulation of both GABAergic and glutamatergic synaptic transmission and plasticity has been extensively investigated, our understanding of the role of endocannabinoids in protecting neurons from harmful insults remains limited. 2-Arachidonoylglycerol (2-AG), the most abundant endogenous ligand and a full agonist for cannabinoid receptors, exhibits anti-inflammatory and neuroprotective effects via a CB1 receptor (CB1R)-mediated mechanism. However, it is still not clear whether 2-AG is also able to protect neurons from β-amyloid (Aβ)-induced neurodegeneration. Here, we demonstrate that exogenous application of 2-AG significantly protected hippocampal neurons in culture against Aβ-induced neurodegeneration and apoptosis. This neuroprotective effect was blocked by SR141716 (SR-1), a selective CB1R antagonist, but not by SR144528 (SR-2), a selective CB2R antagonist, or capsazepine (CAP), a selective transient receptor potential cation channels, subfamily V, member 1 (TRPV1) receptor antagonist. To determine whether endogenous 2-AG is capable of protecting neurons from Aβ insults, hippocampal neurons in culture were treated with URB602 or JZL184, selective inhibitors of monoacylglycerol lipase (MAGL), the enzyme hydrolyzing 2-AG. MAGL inhibition that elevates endogenous levels of 2-AG also significantly reduced Aβ-induced neurodegeneration and apoptosis. The 2-AG-produced neuroprotective effects appear to be mediated via CB1R-dependent suppression of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and nuclear factor-κB (NF-κB) phosphorylation and cyclooxygenase-2 (COX-2) expression. Our results suggest that elevation of endogenous 2-AG by inhibiting its hydrolysis has potential as a novel efficacious therapeutic approach for preventing, ameliorating or treating Alzheimer's disease.

Topics: Amyloid beta-Peptides; Animals; Apoptosis; Arachidonic Acids; Benzodioxoles; Biphenyl Compounds; Camphanes; Cannabinoid Receptor Antagonists; Cannabinoid Receptor Modulators; Capsaicin; Cell Culture Techniques; Drug Interactions; Endocannabinoids; Glycerides; Hippocampus; Monoacylglycerol Lipases; Nerve Degeneration; Peptide Fragments; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Rimonabant; Signal Transduction

We previously reported the involvement of brain diacylglycerol lipase and cyclooxygenase in intracerebroventricularly (i.c.v.) administered bombesin-induced secretion of noradrenaline and adrenaline from the adrenal medulla in rats. Diacylglycerol can be hydrolyzed by diacylglycerol lipase into 2-arachidonoylglycerol, which may be further hydrolyzed by monoacylglycerol lipase into arachidonic acid, a substrate of cyclooxygenase. 2-Arachidonoylglycerol is a major endocannabinoid, which can inhibit synaptic transmission by presynaptic cannabinoid CB(1) receptors. Released 2-arachidonoylglycerol is rapidly inactivated by uptake into cells and enzymatic hydrolysis. In the present study, we examined the involvement of brain 2-arachidonoylglycerol and its regulatory role in the bombesin-induced central activation of adrenomedullary outflow using anesthetized rats. The elevation of plasma noradrenaline and adrenaline induced by a sub-maximal dose of bombesin (1 nmol/animal, i.c.v.) was reduced by MAFP (monoacylglycerol lipase inhibitor) (0.28 and 0.7 μmol/animal, i.c.v.), JZL184 (selective monoacylglycerol lipase inhibitor) (0.7 and 1.4 μmol/animal, i.c.v.), ACEA (CB(1) receptor agonist) (0.7 and 1.4 μmol/animal, i.c.v.) and AM 404 (endocannabinoid uptake-inhibitor) (80 and 250 nmol/animal, i.c.v.), while AM 251 (CB(1) receptor antagonist) (90 and 180 nmol/animal, i.c.v.) potentiated the response induced by a small dose of bombesin (0.1 nmol/animal, i.c.v.). These results suggest a possibility that 2-arachidonoylglycerol is endogenously generated in the brain during bombesin-induced activation of central adrenomedullary outflow, thereby inhibiting the peptide-induced response by activation of brain CB(1) receptors in rats.

Topics: Adrenal Medulla; Animals; Arachidonic Acids; Benzodioxoles; Bombesin; Brain; Cannabinoid Receptor Modulators; Catecholamines; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Glycerides; Male; Monoacylglycerol Lipases; Piperidines; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1

Metastatic and primary bone cancers are usually accompanied by severe pain that is difficult to manage. In light of the adverse side effects of opioids, manipulation of the endocannabinoid system may provide an effective alternative for the treatment of cancer pain. The present study determined that a local, peripheral increase in the endocannabinoid 2-arachidonoyl glycerol (2-AG) reduced mechanical hyperalgesia evoked by the growth of a fibrosarcoma tumor in and around the calcaneous bone. Intraplantar (ipl) injection of 2-AG attenuated hyperalgesia (ED(50) of 8.2 μg) by activation of peripheral CB2 but not CB1 receptors and had an efficacy comparable to that of morphine. JZL184 (10 μg, ipl), an inhibitor of 2-AG degradation, increased the local level of 2-AG and mimicked the anti-hyperalgesic effect of 2-AG, also through a CB2 receptor-dependent mechanism. These effects were accompanied by an increase in CB2 receptor protein in plantar skin of the tumor-bearing paw as well as an increase in the level of 2-AG. In naïve mice, intraplantar administration of the CB2 receptor antagonist AM630 did not alter responses to mechanical stimuli demonstrating that peripheral CB2 receptor tone does not modulate mechanical sensitivity. These data extend our previous findings with anandamide in the same model and suggest that the peripheral endocannabinoid system is a promising target for the management of cancer pain.

Topics: Animals; Arachidonic Acids; Benzodioxoles; Bone Neoplasms; Calcaneus; Cannabinoid Receptor Antagonists; Dose-Response Relationship, Drug; Endocannabinoids; Fibrosarcoma; Ganglia, Spinal; Glycerides; Hyperalgesia; Male; Mice; Mice, Inbred C3H; Monoacylglycerol Lipases; Piperidines; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB2; Signal Transduction; Skin; Tibial Nerve

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

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

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

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

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

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

Two endogenous ligands for cannabinoid CB1 receptors, anandamide (N-arachidonoylethanolamine) and 2-arachidonoylglycerol (2-AG), have been identified and characterized. 2-AG is the most prevalent endogenous cannabinoid ligand in the brain, and electrophysiological studies suggest 2-AG, rather than anandamide, is the true natural ligand for cannabinoid receptors and the key endocannabinoid involved in retrograde signaling in the brain. Here, we evaluated intravenously administered 2-AG for reinforcing effects in nonhuman primates. Squirrel monkeys that previously self-administered anandamide or nicotine under a fixed-ratio schedule with a 60 s timeout after each injection had their self-administration behavior extinguished by vehicle substitution and were then given the opportunity to self-administer 2-AG. Intravenous 2-AG was a very effective reinforcer of drug-taking behavior, maintaining higher numbers of self-administered injections per session and higher rates of responding than vehicle across a wide range of doses. To assess involvement of CB1 receptors in the reinforcing effects of 2-AG, we pretreated monkeys with the cannabinoid CB(1) receptor inverse agonist/antagonist rimonabant [N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide]. Rimonabant produced persistent blockade of 2-AG self-administration without affecting responding maintained by food under similar conditions. Thus, 2-AG was actively self-administered by monkeys with or without a history of cannabinoid self-administration, and the reinforcing effects of 2-AG were mediated by CB1 receptors. Self-administration of 2-AG by squirrel monkeys provides a valuable procedure for studying abuse liability of medications that interfere with 2-AG signaling within the brain and for investigating mechanisms involved in the reinforcing effects of endocannabinoids.

Topics: Analysis of Variance; Animals; Arachidonic Acids; Behavior, Animal; Endocannabinoids; Extinction, Psychological; Glycerides; Infusions, Intravenous; Injections, Intravenous; Male; Motivation; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Reinforcement, Psychology; Rimonabant; Saimiri; Self Administration

Dysregulation in signaling of the endocannabinoid 2-arachidonoylglycerol (2-AG) is implicated in hyperresponsiveness to stress. We hypothesized that blockade of monoacylglycerol lipase (MGL), the primary enzyme responsible for 2-AG deactivation in vivo, would produce context-dependent anxiolytic effects in rats. Environmental aversiveness was manipulated by varying illumination of an elevated plus maze. Percentage open arm time and numbers of open and closed arm entries were measured in rats receiving a single intraperitoneal (i.p.) injection of either vehicle, the MGL inhibitor JZL184 (1-8mg/kg), the benzodiazepine diazepam (1mg/kg), the cannabinoid CB(1) receptor antagonist rimonabant (1mg/kg), or JZL184 (8mg/kg) coadministered with rimonabant (1mg/kg). JZL184 (8mg/kg) produced anxiolytic-like effects (i.e., increased percentage open arm time and number of open arm entries) under high, but not low, levels of environmental aversiveness. Diazepam produced anxiolytic effects in either context. Rimonabant blocked the anxiolytic-like effects of JZL184, consistent with mediation by CB(1). Anxiolytic effects of JZL184 were preserved following chronic (8mg/kg per day×6 days) administration. Chronic and acute JZL184 treatment similarly enhanced behavioral sensitivity to an exogenous cannabinoid (WIN55,212-2; 2.5mg/kg i.p.) 24 or 72h following the terminal injection, suggesting a pervasive effect of MGL inhibition on the endocannabinoid system. We attribute our results to alterations in emotion rather than locomotor activity as JZL184 did not alter the number of closed arm entries in the plus maze or produce motor ataxia in the bar test. Our results demonstrate that JZL184 has beneficial, context-dependent effects on anxiety in rats, presumably via inhibition of MGL-mediated hydrolysis of 2-AG. These data warrant further testing of MGL inhibitors to elucidate the functional role of 2-AG in controlling anxiety and stress responsiveness. Our data further implicate a role for 2-AG in the regulation of emotion and validate MGL as a therapeutic target.

Topics: Animals; Anti-Anxiety Agents; Anxiety; Arachidonic Acids; Benzodioxoles; Cannabinoid Receptor Modulators; Catalepsy; Diazepam; Endocannabinoids; Glycerides; Male; Monoacylglycerol Lipases; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Rimonabant; Signal Transduction

The endogenous cannabinoid 2-arachidonoylglycerol (2-AG) is described as a platelet agonist able to induce aggregation and to increase intracellular calcium. In the present report we have confirmed these data and demonstrated that the inhibitor of p38MAPK SB203580 and the inhibitor of cPLA(2) metabolism ETYA affect both these parameters. Thus, we aimed to define the role of p38MAPK/cytosolic phospholipase A(2) (cPLA(2)) pathway in 2-AG-induced human platelet activation. p38MAPK activation was assayed by phosphorylation. cPLA(2) activation was assayed by phosphorylation and as arachidonic acid release and thromboxane B(2) formation. It was shown that 2-AG in a dose- and time-dependent manner activates p38MAPK peaking at 10 µM after 1 min of incubation. The 2-AG effect on p38MAPK was not impaired by apyrase, indomethacin or RGDS peptide but it was significantly reduced by SR141716, specific inhibitor of type-1 cannabinoid receptor and unaffected by the specific inhibitor of type-2 cannabinoid receptor SR144528. Moreover, the incubation of platelets with 2-AG led to the phosphorylation of cPLA(2) and its activation. Platelet pretreatment with SB203580, inhibitor of p38MAPK, abolished both cPLA(2) phosphorylation and activation. In addition SR141716 strongly impaired cPLA(2) phosphorylation, arachidonic acid release and thromboxane B(2) formation, whereas SR144528 did not change these parameters. Finally platelet stimulation with 2-AG led to an increase in free oxygen radical species. In conclusion, data provide insight into the mechanisms involved in platelet activation by 2-AG, indicating that p38MAPK/cPLA(2) pathway could play a relevant role in this complicated process.

Topics: Apyrase; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Endocannabinoids; Glycerides; Humans; Imidazoles; In Vitro Techniques; Indomethacin; Oligopeptides; p38 Mitogen-Activated Protein Kinases; Phospholipases A2, Cytosolic; Phosphorylation; Piperidines; Pyrazoles; Pyridines; Reactive Oxygen Species; Rimonabant; Signal Transduction

We report a novel excitatory effect of cannabinoid agonists on action potential-independent GABAergic transmission in the rat dentate gyrus. Specifically, we find that both WIN55,212-2 and anandamide increase the frequency of miniature IPSCs (mIPSCs)recorded from hilar mossy cells without altering event amplitude, area, rise time, or decay. The effect of WIN55,212-2 on mIPSCs is insensitive to AM251 and preserved in CB1 −/− animals,indicating that it does not depend on activation of CB1 receptors. It is also insensitive to AM630 and unaffected by capsazepine suggesting that neither CB2 nor TRPV1 receptors are involved. Further, it is blocked by pre-incubation in suramin and by a selective protein kinase A inhibitor (H-89), and is mimicked (and occluded) by bath application of forskolin. Similar CB1 receptor-independent facilitation of exocytosis is not apparent when recording evoked IPSCs in the presence of AM251, suggesting that the exocytotic mechanism that produces WIN55,212-2 sensitive mIPSCs is distinct from that which produces CB1 sensitive and action potential-dependent release. Despite clear independence from action potentials, WIN55,212-2 mediated facilitation of mIPSCs requires calcium, and yet is insensitive to chelation of calcium in the postsynaptic cell. Finally, we demonstrate that both bath application of 2-arachidonoylglycerol(2-AG) and depolarization-induced release of endogenous cannabinoids have minimal effect on mIPSC frequency. Cumulatively, our results indicate that cannabinoid ligands can selectively facilitate action potential-independent exocytosis of GABA in the rat dentate gyrus, and further emphasize that this new cannabinoid sensitive signalling system is distinct from previously described CB1 receptor-dependent systems in numerous respects.

Topics: Action Potentials; Animals; Arachidonic Acids; Benzoxazines; Calcium; Cannabinoids; Endocannabinoids; Exocytosis; GABAergic Neurons; gamma-Aminobutyric Acid; Glycerides; Male; Morpholines; Mossy Fibers, Hippocampal; Naphthalenes; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Synaptic Transmission

Cannabinoid agonists are potential therapeutic agents because of their antinociceptive and anxiolytic-like effects, although an important caveat to their use is the possible adverse responses related to memory impairment. An alternative approach to circumvent this limitation consists of enhancing the concentration of the endocannabinoids anandamide and 2-arachidonoylglycerol.. Using low doses of the specific inhibitors of the endocannabinoid metabolizing enzymes fatty acid amide hydrolase, URB597, and monoacylglycerol lipase, JZL184, we analyzed their acute and chronic effects on memory consolidation, anxiolytic-like effects, and nociception in mice (n = 6-12 per experimental group).. We show that anandamide is a central component in the modulation of memory consolidation, whereas 2-arachidonoylglycerol is not involved in this process. Interestingly, both URB597 and JZL184 induce anxiolytic-like effects through different cannabinoid receptors. In addition, the results show that the antinociceptive and anxiolytic-like responses of both inhibitors, as well as their acute effects on memory consolidation, are maintained after chronic treatment.. These results dissociate the role of anandamide and 2-arachidonoylglycerol in memory consolidation and anxiety and reveal the interest of cannabinoid receptor 2 as a novel target for the treatment of anxiety-related disorders.

Topics: Amidohydrolases; Analgesics; Animals; Anti-Anxiety Agents; Arachidonic Acids; Benzamides; Benzodioxoles; Cannabinoid Receptor Antagonists; Carbamates; Drug Tolerance; Endocannabinoids; Glycerides; Hippocampus; Maze Learning; Mice; Mice, Inbred Strains; Mice, Knockout; Monoacylglycerol Lipases; Pain Measurement; Piperidines; Polyunsaturated Alkamides; Receptors, Cannabinoid; Recognition, Psychology; TOR Serine-Threonine Kinases

The mechanisms subserving the ability of glucocorticoid signaling within the medial prefrontal cortex (mPFC) to terminate stress-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis are not well understood. We report that antagonism of the cannabinoid CB(1) receptor locally within the mPFC prolonged corticosterone secretion following cessation of stress in rats. Mice lacking the CB(1) receptor exhibited a similar prolonged response to stress. Exposure of rats to stress produced an elevation in the endocannabinoid 2-arachidonoylglycerol within the mPFC that was reversed by pretreatment with the glucocorticoid receptor antagonist RU-486 (20 mg/kg). Electron microscopic and electrophysiological data demonstrated the presence of CB(1) receptors in inhibitory-type terminals impinging upon principal neurons within layer V of the prelimbic region of the mPFC. Bath application of corticosterone (100 nm) to prefrontal cortical slices suppressed GABA release onto principal neurons in layer V of the prelimbic region, when examined 1 h later, which was prevented by application of a CB(1) receptor antagonist. Collectively, these data demonstrate that the ability of stress-induced glucocorticoid signaling within mPFC to terminate HPA axis activity is mediated by a local recruitment of endocannabinoid signaling. Endocannabinoid activation of CB(1) receptors decreases GABA release within the mPFC, likely increasing the outflow of the principal neurons of the prelimbic region to contribute to termination of the stress response. These data support a model in which endocannabinoid signaling links glucocorticoid receptor engagement to activation of corticolimbic relays that inhibit corticosterone secretion.

Topics: Animals; Arachidonic Acids; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Corticosterone; Disease Models, Animal; Electric Stimulation; Endocannabinoids; Freezing Reaction, Cataleptic; gamma-Aminobutyric Acid; Glycerides; Hormone Antagonists; In Vitro Techniques; Long-Term Synaptic Depression; Male; Mice; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Knockout; Microscopy, Electron, Transmission; Mifepristone; Patch-Clamp Techniques; Piperidines; Prefrontal Cortex; Pyramidal Cells; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Signal Transduction; Stress, Psychological

Chronic stress is the primary environmental risk factor for the development and exacerbation of affective disorders, thus understanding the neuroadaptations that occur in response to stress is a critical step in the development of novel therapeutics for depressive and anxiety disorders. Brain endocannabinoid (eCB) signaling is known to modulate emotional behavior and stress responses, and levels of the eCB 2-arachidonoylglycerol (2-AG) are elevated in response to chronic homotypic stress exposure. However, the role of 2-AG in the synaptic and behavioral adaptations to chronic stress is poorly understood. Here, we show that stress-induced development of anxiety-like behavior is paralleled by a transient appearance of low-frequency stimulation-induced, 2-AG-mediated long-term depression at GABAergic synapses in the basolateral amygdala, a key region involved in motivation, affective regulation, and emotional learning. This enhancement of 2-AG signaling is mediated, in part, via downregulation of the primary 2-AG-degrading enzyme monoacylglycerol lipase (MAGL). Acute in vivo inhibition of MAGL had little effect on anxiety-related behaviors. However, chronic stress-induced anxiety-like behavior and emergence of long-term depression of GABAergic transmission was prevented by chronic MAGL inhibition, likely via an occlusive mechanism. These data indicate that chronic stress reversibly gates eCB synaptic plasticity at inhibitory synapses in the amygdala, and in vivo augmentation of 2-AG levels prevents both behavioral and synaptic adaptations to chronic stress.

Topics: Adaptation, Psychological; Amygdala; Animals; Anxiety Disorders; Arachidonic Acids; Benzodioxoles; Cannabinoid Receptor Modulators; Chronic Disease; Disease Models, Animal; Endocannabinoids; Glycerides; Male; Mice; Mice, Inbred ICR; Monoacylglycerol Lipases; Organ Culture Techniques; Piperidines; Stress, Psychological

Orexin A and B are hypothalamic peptides known to modulate arousal, feeding, and reward via OX1 and OX2 receptors. Orexins are also antinociceptive in the brain, but their mechanism(s) of action remain unclear. Here, we investigated the antinociceptive mechanism of orexin A in the rat ventrolateral periaqueductal gray (vlPAG), a midbrain region crucial for initiating descending pain inhibition. In vlPAG slices, orexin A (30-300 nm) depressed GABAergic evoked IPSCs. This effect was blocked by an OX1 [1-(2-methylbenzoxazol-6-yl)-3-[1,5]naphthyridin-4-yl urea (SB 334867)], but not OX2 [N-acyl 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride (compound 29)], antagonist. Orexin A increased the paired-pulse ratio of paired IPSCs and decreased the frequency, but not amplitude, of miniature IPSCs. Orexin A-induced IPSC depression was mimicked by (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone (WIN 55,212-2), a cannabinoid 1 (CB1) receptor agonist. 1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidyl)pyrazole-3-carboxamide (AM 251), a CB1 antagonist, reversed depressant effects by both agonists. Orexin A-induced IPSC depression was prevented by 1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122) and tetrahydrolipstatin, inhibitors of phospholipase C (PLC) and diacylglycerol lipase (DAGL), respectively, and enhanced by cyclohexyl[1,1'-biphenyl]-3-ylcarbamate (URB602), which inhibits enzymatic degradation of 2-arachidonoylglycerol (2-AG). Moderate DAGLα, but not DAGLβ, immunoreactivity was observed in the vlPAG. Orexin A produced an overall excitatory effect on evoked postsynaptic potentials and hence increased vlPAG neuronal activity. Intra-vlPAG microinjection of orexin A reduced hot-plate nociceptive responses in rats in a manner blocked by SB 334867 and AM 251. Therefore, orexin A may produce antinociception by activating postsynaptic OX1 receptors, stimulating synthesis of 2-AG, an endocannabinoid, through a Gq-protein-mediated PLC-DAGLα enzymatic cascade culminating in retrograde inhibition of GABA release (disinhibition) in the vlPAG.

Topics: Analysis of Variance; Animals; Animals, Newborn; Arachidonic Acids; Benzoxazines; Benzoxazoles; Biphenyl Compounds; Calcium Channel Blockers; Cannabinoid Receptor Modulators; Disease Models, Animal; Electric Stimulation; Endocannabinoids; Enzyme Inhibitors; Estrenes; gamma-Aminobutyric Acid; Glycerides; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Intracellular Signaling Peptides and Proteins; Lactones; Male; Morpholines; Naphthalenes; Naphthyridines; Neural Inhibition; Neural Pathways; Neuropeptides; Orexin Receptors; Orexins; Orlistat; Pain; Pain Measurement; Patch-Clamp Techniques; Periaqueductal Gray; Piperidines; Pyrazoles; Pyrrolidinones; Rats; Rats, Wistar; Receptors, G-Protein-Coupled; Receptors, Neuropeptide; Urea

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

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

The reason why neurons synthesize more than one endocannabinoid (eCB) and how this is involved in the regulation of synaptic plasticity in a single neuron is not known. We found that 2-arachidonoylglycerol (2-AG) and anandamide mediate different forms of plasticity in the extended amygdala of rats. Dendritic L-type Ca(2+) channels and the subsequent release of 2-AG acting on presynaptic CB1 receptors triggered retrograde short-term depression. Long-term depression was mediated by postsynaptic mGluR5-dependent release of anandamide acting on postsynaptic TRPV1 receptors. In contrast, 2-AG/CB1R-mediated retrograde signaling mediated both forms of plasticity in the striatum. These data illustrate how the eCB system can function as a polymodal signal integrator to allow the diversification of synaptic plasticity in a single neuron.

Topics: Animals; Arachidonic Acids; Biophysics; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Cannabinoid Receptor Modulators; Chromones; Cyclohexanones; Electric Stimulation; Endocannabinoids; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Glycerides; In Vitro Techniques; Long-Term Synaptic Depression; Neurons; Nimodipine; Patch-Clamp Techniques; Piperidines; Pyrazoles; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB2; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Septal Nuclei; Signal Transduction; Synapses; Time Factors; TRPV Cation Channels

The endocannabinoid system is a major regulator of food intake in many animal species. Studies conducted so far have mostly focused on mammals, and, therefore, in this study, the role of the endocannabinoid system in food intake in the sea bream Sparus aurata was investigated. The effect of different doses of the endocannabinoid anandamide (AEA), administered via water, was evaluated after different exposure times (30, 60 and 120 min) at both physiological and molecular levels. The results obtained indicate that fish exposed to AEA via water present approximately 1000-fold higher levels of AEA in both the brain and liver, which correlated with a significant increase in food intake and with the elevation of cannabinoid receptor 1 (CB(1)) and neuropeptide Y (NPY) mRNA levels in the brain. A peripheral effect of AEA was also observed, since a time-dependent increase in hepatic CB(1) mRNA and protein levels was detected. These effects were attenuated by the administration, again via water, of a selective cannabinoid CB(1) receptor antagonist (AM251). These findings indicate that the endocannabinoid AEA, at doses that stimulate food intake in fish, concomitantly stimulates the expression of the orexigenic peptide NPY as well that of its own receptor, thereby potentially enhancing its effect on food consumption. In agreement with a role of AEA in food intake in S. aurata, we found increased brain levels of both this and the other endocannabinoid, 2-arachidonoylglycerol (2-AG), following food deprivation.

Topics: Animals; Arachidonic Acids; Brain Chemistry; Cannabinoid Receptor Modulators; Eating; Endocannabinoids; Food Deprivation; Glycerides; Liver; Neuropeptide Y; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; RNA, Messenger; Sea Bream; Water

Both cyclooxygenase-1 and -2 are expressed in the spinal cord, and the spinal COX product prostaglandin E(2) (PGE(2)) contributes to the generation of central sensitization upon peripheral inflammation. Vice versa spinal COX inhibition is considered an important mechanism of antihyperalgesic pain treatment. Recently, however, COX-2 was shown to be also involved in the metabolism of endocannabinoids. Because endocannabinoids can have analgesic actions it is conceivable that inhibition of spinal COX produces analgesia not only by inhibition of PG synthesis but also by inhibition of endocannabinoid breakdown. In the present study, we recorded from spinal cord neurons with input from the inflamed knee joint and we measured the spinal release of PGE(2) and the endocannabinoid 2-arachidonoyl glycerol (2-AG) in vivo, using the same stimulation procedures. COX inhibitors were applied spinally. Selective COX-1, selective COX-2 and non-selective COX inhibitors attenuated the generation of spinal hyperexcitability when applied before and during development of inflammation but, when inflammation and spinal hyperexcitability were established, only selective COX-2 inhibitors reversed spinal hyperexcitability. During established inflammation all COX inhibitors reduced release of spinal PGE(2) almost equally but only the COX-2 inhibitor prevented breakdown of 2-AG. The reversal of spinal hyperexcitability by COX-2 inhibitors was prevented or partially reversed by AM-251, an antagonist at the cannabinoid-1 receptor. We conclude that inhibition of spinal COX-2 not only reduces PG production but also endocannabinoid breakdown and provide evidence that reversal of inflammation-evoked spinal hyperexcitability by COX-2 inhibitors is more related to endocannabinoidergic mechanisms than to inhibition of spinal PG synthesis.

Topics: Action Potentials; Animals; Arachidonic Acids; Arthritis, Experimental; Dinoprostone; Disease Models, Animal; Drug Administration Routes; Endocannabinoids; Enzyme Inhibitors; Glycerides; Indans; Injections, Spinal; Knee Joint; Male; Neurons; Pain Measurement; Physical Stimulation; Piperidines; Prostaglandin-Endoperoxide Synthases; Pyrazoles; Rats; Rats, Wistar; Spinal Cord; Statistics, Nonparametric

Bilateral olfactory bulbectomy (OBX) in rodents produces behavioral and neurochemical changes associated clinically with depression and schizophrenia. Most notably, OBX induces hyperlocomotion in response to the stress of exposure to a novel environment. We examined the role of the endocannabinoid system in regulating this locomotor response in OBX and sham-operated rats. In our study, OBX-induced hyperactivity was restricted to the first 3 min of the open field test, demonstrating the presence of novelty (0-3 min) and habituation (3-30 min) phases of the open field locomotor response. Levels of the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide were decreased in the ventral striatum, a brain region deafferented by OBX, whereas cannabinoid receptor densities were unaltered. In sham-operated rats, 2-AG levels in the ventral striatum were negatively correlated with distance traveled during the novelty phase. Thus, low levels of 2-AG are reflected in a hyperactive open field response. This correlation was not observed in OBX rats. Conversely, 2-AG levels in endocannabinoid-compromised OBX rats correlated with distance traveled during the habituation phase. In OBX rats, pharmacological blockade of cannabinoid CB(1) receptors with either AM251 (1 mg kg(-1) i.p.) or rimonabant (1 mg kg(-1) i.p.) increased distance traveled during the habituation phase. Thus, blockade of endocannabinoid signaling impairs habituation of the hyperlocomotor response in OBX, but not sham-operated, rats. By contrast, in sham-operated rats, effects of CB(1) antagonism were restricted to the novelty phase. These findings suggest that dysregulation in the endocannabinoid system, and 2-AG in particular, is implicated in the hyperactive locomotor response induced by OBX. Our studies suggest that drugs that enhance 2-AG signaling, such as 2-AG degradation inhibitors, might be useful in human brain disorders modeled by OBX.

Topics: Animals; Arachidonic Acids; Autoradiography; Cannabinoid Receptor Modulators; Chromatography, High Pressure Liquid; Cyclohexanols; Densitometry; Dopamine; Endocannabinoids; Environment; Glycerides; Limbic System; Lipid Metabolism; Male; Mass Spectrometry; Motor Activity; Olfactory Bulb; Piperidines; Pyrazoles; Radiopharmaceuticals; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Rimonabant; Signal Transduction

Although arachidonoyl ethanolamide (AEA or anandamide) is the first identified endocannabinoid, its roles in synaptic signaling and neuronal survival are still controversial. Here we report that AEA induced a dose-dependent elevation of the frequency of miniature excitatory postsynaptic currents (mEPSCs) in mouse hippocampal neurons in culture. This potentiation was not blocked by SR141716 or AM251, selective cannabinoid receptor antagonists, indicating that the AEA elevation of mEPSCs is not mediated via the CB1 receptor. Similarly, capsazepine and iodoresiniferatoxin, selective vanilloid receptor antagonists, and ryanodine also failed to inhibit the effect of AEA on mEPSCs. However, 2-APB and Xestospongin C, IP3 inhibitors, significantly attenuated AEA-induced increase in hippocampal excitatory synaptic transmission. Application of 3-deoxy-3-fluoro-d-myo-inositol 1,4,5-trisphosphate enhanced the frequency of mEPSCs and occluded the effect of AEA on mEPSCs. Our results suggest that AEA-produced stimulatory effect on excitatory glutamatergic synaptic transmission is likely mediated via an IP3 pathway.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Calcium Channel Blockers; Cells, Cultured; Electrophysiology; Endocannabinoids; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; Glycerides; Hippocampus; Inositol 1,4,5-Trisphosphate; Mice; Patch-Clamp Techniques; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; Ryanodine Receptor Calcium Release Channel; Signal Transduction; Synaptic Transmission; TRPV Cation Channels

Adenosine A(2A) receptor antagonists are psychomotor stimulants that also hold therapeutic promise for movement disorders. However, the molecular mechanisms underlying their stimulant properties are not well understood. Here, we show that the robust increase in locomotor activity induced by an A(2A) antagonist in vivo is greatly attenuated by antagonizing cannabinoid CB(1) receptor signaling or by administration to CB(1)(-/-) mice. To determine the locus of increased endocannabinoid signaling, we measured the amount of anandamide [AEA (N-arachidonoylethanolamine)] and 2-arachidonoylglycerol (2-AG) in brain tissue from striatum and cortex. We find that 2-AG is selectively increased in striatum after acute blockade of A(2A) receptors, which are highly expressed by striatal indirect-pathway medium spiny neurons (MSNs). Using targeted whole-cell recordings from direct- and indirect-pathway MSNs, we demonstrate that A(2A) receptor antagonists potentiate 2-AG release and induction of long-term depression at indirect-pathway MSNs, but not direct-pathway MSNs. Together, these data outline a molecular mechanism by which A(2A) antagonists reduce excitatory synaptic drive on the indirect pathway through CB(1) receptor signaling, thus leading to increased psychomotor activation.

Topics: Adenosine A2 Receptor Antagonists; Afferent Pathways; Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Central Nervous System Stimulants; Cerebral Cortex; Corpus Striatum; Endocannabinoids; Glutamic Acid; Glycerides; Long-Term Synaptic Depression; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Motor Activity; Neurons; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Pyrimidines; Receptor, Cannabinoid, CB1; Signal Transduction

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

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

When chronic alterations in neuronal activity occur, network gain is maintained by global homeostatic scaling of synaptic strength, but the stability of microcircuits can be controlled by unique adaptations that differ from the global changes. It is not understood how specificity of synaptic tuning is achieved. We found that, although a large population of inhibitory synapses was homeostatically scaled down after chronic inactivity, decreased endocannabinoid tone specifically strengthened a subset of GABAergic synapses that express cannabinoid receptors. In rat hippocampal slice cultures, a 3-5-d blockade of neuronal firing facilitated uptake and degradation of anandamide. The consequent reduction in basal stimulation of cannabinoid receptors augmented GABA release probability, fostering rapid depression of synaptic inhibition and on-demand disinhibition. This regulatory mechanism, mediated by activity-dependent changes in tonic endocannabinoid level, permits selective local tuning of inhibitory synapses in hippocampal networks.

Topics: Agatoxins; Animals; Arachidonic Acids; Benzamides; Benzoxazines; Biophysics; Calcium; Calcium Channel Blockers; Cannabinoid Receptor Modulators; Carbamates; Conotoxins; Dose-Response Relationship, Drug; Down-Regulation; Drug Interactions; Electric Stimulation; Endocannabinoids; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; gamma-Aminobutyric Acid; Glycerides; Hippocampus; Homeostasis; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Morpholines; Naphthalenes; Nerve Net; Neural Inhibition; Neurons; Patch-Clamp Techniques; Piperidines; Polyamines; Polyunsaturated Alkamides; Pyrazoles; Rats; Receptor, Cannabinoid, CB1; Rimonabant; Sodium Channel Blockers; Synapses; Tetrodotoxin

The present studies examined the effect of chronic neuropathic pain on cannabinoid receptor density and receptor-mediated G-protein activity within supraspinal brain areas involved in pain processing and modulation in mice. Chronic constriction injury (CCI) produced a significant decrease in WIN 55,212-2-stimulated [(35)S]GTPgammaS binding in membranes prepared from the rostral anterior cingulate cortex (rACC) of CCI mice when compared to sham-operated controls. Saturation binding with [(3)H]SR 141716A in membranes of the rACC showed no significant differences in binding between CCI and sham mice. Analysis of levels of the endocannabinoids anandamide (AEA) or 2-arachidonoylglycerol (2-AG) in the rACC following CCI showed no significant differences between CCI and sham mice. These data suggest that CCI produced desensitization of the cannabinoid 1 receptor in the rACC in the absence of an overall decrease in cannabinoid 1 receptor density or change in levels of AEA or 2-AG. These data are the first to show alterations in cannabinoid receptor function in the rostral anterior cingulate cortex in response to a model of neuropathic pain.

Topics: Analgesics; Animals; Arachidonic Acids; Benzoxazines; Cannabinoid Receptor Modulators; Cell Membrane; Constriction; Disease Models, Animal; Endocannabinoids; Glycerides; Guanosine 5'-O-(3-Thiotriphosphate); Male; Mice; Mice, Inbred Strains; Models, Neurological; Morpholines; Naphthalenes; Pain; Piperidines; Polyunsaturated Alkamides; Prefrontal Cortex; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; Sulfur Radioisotopes; Tritium

Transcriptional silencing of the gene encoding the fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS). FMRP acts as a translational repressor at central synapses, and molecular and synaptic plasticity studies have shown that the absence of this protein alters metabotropic glutamate 5 receptors (mGlu5Rs)-mediated signaling. In the striatum of mice lacking FMRP, we found enhanced activity of diacylglycerol lipase (DAGL), the enzyme limiting 2-arachidonoylglicerol (2-AG) synthesis, associated with altered sensitivity of GABA synapses to the mobilization of this endocannabinoid by mGlu5R stimulation with DHPG. Mice lacking another repressor of synaptic protein synthesis, BC1 RNA, also showed potentiated mGlu5R-driven 2-AG responses, indicating that both FMRP and BC1 RNA act as physiological constraints of mGlu5R/endocannabinoid coupling at central synapses. The effects of FMRP ablation on DAGL activity and on DHPG-mediated inhibition of GABA synapses were enhanced by simultaneous genetic inactivation of FMRP and BC1 RNA. In double FMRP and BC1 RNA lacking mice, striatal levels of 2-AG were also enhanced compared with control animals and to single mutants. Our data indicate for the first time that mGlu5R-driven endocannabinoid signaling in the striatum is under the control of both FMRP and BC1 RNA. The abnormal mGlu5R/2-AG coupling found in FMRP-KO mice emphasizes the involvement of mGlu5Rs in the synaptic defects of FXS, and identifies the modulation of the endocannabinoid system as a novel target for the treatment of this severe neuropsychiatric disorder.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Corpus Striatum; Dronabinol; Endocannabinoids; Excitatory Amino Acid Antagonists; Fragile X Mental Retardation Protein; Gene Expression Regulation; Glycerides; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Lipoprotein Lipase; Membrane Potentials; Methoxyhydroxyphenylglycol; Mice; Mice, Knockout; Patch-Clamp Techniques; Piperidines; Protein Binding; Pyrazoles; Receptors, Kainic Acid; RNA, Small Cytoplasmic; Statistics, Nonparametric

We previously reported that intracerebroventricularly (i.c.v.) administered corticotropin-releasing factor (CRF) (0.5-3.0 nmol/animal) dose-dependently elevates plasma noradrenaline and adrenaline through brain phospholipase C-, diacylglycerol lipase- and prostanoids-mediated mechanisms in rats. Diacylglycerol produced by phospholipase C from phospholipids can be hydrolyzed by diacylglycerol lipase into 2-arachidonoylglycerol, which may be further hydrolyzed by monoacylglycerol lipase into arachidonic acid, a precursor of prostanoids. Recently, 2-arachidonoylglycerol has been recognized as a major brain endocannabinoid, which can modulate synaptic transmission through presynaptic cannabinoid CB(1) receptors. Released 2-arachidonoylglycerol is rapidly deactivated by uptake into cells and enzymatic hydrolysis. In the present study, therefore, we examined (1) the involvement of brain 2-arachidonoylglycerol, (2) the regulatory role of 2-arachidonoylglycerol as a brain endocannabinoid, and (3) the effect of exogenous cannabinoid receptor agonist, on the CRF-induced elevation of plasma noradrenaline and adrenaline using anesthetized rats. The elevation of both catecholamines induced by a submaximal dose of CRF (1.5 nmol/animal, i.c.v.) was reduced by i.c.v. administered MAFP (monoacylglycerol lipase inhibitor) (0.7 and 1.4 micromol/animal), AM 404 (endocannabinoid uptake-inhibitor) (80 and 250 nmol/animal) and ACEA (cannabinoid CB(1) receptor agonist) (0.7 and 1.4 micromol/animal), while AM 251 (cannabinoid CB(1) receptor antagonist) (90 and 180 nmol/animal, i.c.v.) potentiated the response induced by a small dose of CRF (0.5 nmol/animal, i.c.v.). These results suggest a possibility that 2-arachidonoylglycerol is endogenously generated in the brain during CRF-induced activation of central sympatho-adrenomedullary outflow, thereby inhibiting the peptide-induced response by activation of brain cannabinoid CB(1) receptors in anesthetized rats.

Topics: Adrenal Medulla; Anesthesia; Animals; Arachidonic Acids; Brain; Cannabinoid Receptor Agonists; Cannabinoid Receptor Modulators; Catecholamines; Corticotropin-Releasing Hormone; Dose-Response Relationship, Drug; Endocannabinoids; Glycerides; Male; Monoacylglycerol Lipases; Piperidines; Pyrazoles; Rats; Rats, Wistar; Sympathetic Nervous System

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

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

In vitro studies demonstrate that cannabinoid CB(1) receptors subserve activity-dependent suppression of inhibition in the neocortex. To examine this mechanism in vivo, we assessed the effects of local changes in CB(1) receptor activity on somatosensory cortex neuronal activation by whisker movement in rats.. Laser Doppler flowmetry and c-Fos immunohistochemistry were used to measure changes in local blood flow and neuronal activation, respectively. All drugs were applied directly to the cranium above the whisker barrel fields of the primary somatosensory cortex.. The CB(1) receptor agonist WIN55212-2 potentiated the hyperaemia induced by whisker movement and this potentiation was occluded by bicuculline. The CB(1) receptor antagonists, rimonabant and AM251, inhibited hyperaemic responses to whisker movement; indicating that activation of endogenous CB(1) receptors increased during whisker movement. Whisker movement-induced expression of c-Fos protein in neurons of the whisker barrel cortex was inhibited by rimonabant. Movement of the whiskers increased the 2-arachidonoylglycerol content in the contralateral, compared to the ipsilateral, sensory cortex.. These results support the hypothesis that endocannabinoid signalling is recruited during physiologically relevant activation of the sensory cortex. These data support the hypothesis that the primary effect of CB(1) receptor activation within the activated whisker barrel cortex is to inhibit GABA release, resulting in disinhibition of neuronal activation. These studies provide physiological data involving endocannabinoid signalling in activity-dependent regulation of neuronal activation and provide a mechanistic basis for the effects of cannabis use on sensory processing in humans.

Topics: Animals; Arachidonic Acids; Benzoxazines; Bicuculline; Cannabinoid Receptor Modulators; Endocannabinoids; Functional Laterality; Glycerides; Hyperemia; Male; Morpholines; Naphthalenes; Piperidines; Proto-Oncogene Proteins c-fos; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Rimonabant; Somatosensory Cortex; Vibrissae

Methamphetamine (METH) is a psychostimulant amphetamine that causes long-term dopaminergic neurotoxicity in mice. Hypodopaminergic states have been demonstrated to increase voluntary ethanol (EtOH) consumption and preference. In addition, the endocannabinoid system has been demonstrated to modulate EtOH drinking behaviour. Thus, we investigated EtOH consumption in METH-lesioned animals and the role of cannabinoid (CB) signalling in this EtOH drinking.. Mice were treated with a neurotoxic regimen of METH, and 7 days later exposed to increasing concentrations of drinking solutions of EtOH (3, 6, 10 and 20%). Seven days after neurotoxic METH, the following biochemical determinations were carried out in limbic forebrain: CB(1) receptor density and stimulated activity, 2-arachidonoyl glycerol (2-AG) and monoacylglycerol lipase (MAGL) activity, dopamine levels and dopamine transporter density.. EtOH consumption and preference were increased in METH-treated mice. Seven days after METH, a time at which both dopamine levels and density of dopamine transporters in limbic forebrain were decreased, CB(1) receptor density and activity were unaltered, but 2-AG levels were increased. At this same time-point, MAGL activity was reduced. The CB(1) receptor antagonist AM251 prevented the METH-induced increase in EtOH consumption and preference, while N-arachidonoyl maleimide, an inhibitor of MAGL, increased EtOH consumption and preference in both saline- and METH-treated mice.. An increase in endocannabinoid tone may be involved in the increased consumption of and preference for EtOH displayed by METH-lesioned mice as blockade of the CB(1) receptor decreased EtOH-seeking behaviours, whereas the MAGL inhibitor increased EtOH consumption.

Topics: Alcohol Drinking; Amidohydrolases; Animals; Arachidonic Acids; Central Nervous System Stimulants; Choice Behavior; Dopamine; Dopamine Plasma Membrane Transport Proteins; Endocannabinoids; Glycerides; Limbic System; Male; Methamphetamine; Mice; Mice, Inbred C57BL; Monoacylglycerol Lipases; Neurotoxicity Syndromes; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1

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

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

Obesity is characterised by altered gut microbiota, low-grade inflammation and increased endocannabinoid (eCB) system tone; however, a clear connection between gut microbiota and eCB signalling has yet to be confirmed. Here, we report that gut microbiota modulate the intestinal eCB system tone, which in turn regulates gut permeability and plasma lipopolysaccharide (LPS) levels. The impact of the increased plasma LPS levels and eCB system tone found in obesity on adipose tissue metabolism (e.g. differentiation and lipogenesis) remains unknown. By interfering with the eCB system using CB(1) agonist and antagonist in lean and obese mouse models, we found that the eCB system controls gut permeability and adipogenesis. We also show that LPS acts as a master switch to control adipose tissue metabolism both in vivo and ex vivo by blocking cannabinoid-driven adipogenesis. These data indicate that gut microbiota determine adipose tissue physiology through LPS-eCB system regulatory loops and may have critical functions in adipose tissue plasticity during obesity.

Topics: Adipogenesis; Adipose Tissue; Animals; Arachidonic Acids; Bacterial Translocation; Caco-2 Cells; Cannabinoid Receptor Modulators; Disease Models, Animal; Dronabinol; Endocannabinoids; Glycerides; Humans; Intestinal Mucosa; Intestines; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; Myeloid Differentiation Factor 88; Obesity; Permeability; Piperidines; Polyunsaturated Alkamides; Prebiotics; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; RNA, Messenger

2-Arachidonoylglycerol (2-AG) and anandamide are endocannabinoids that activate the cannabinoid receptors CB1 and CB2. Endocannabinoid signaling is terminated by enzymatic hydrolysis, a process that for anandamide is mediated by fatty acid amide hydrolase (FAAH), and for 2-AG is thought to involve monoacylglycerol lipase (MAGL). FAAH inhibitors produce a select subset of the behavioral effects observed with CB1 agonists, which suggests a functional segregation of endocannabinoid signaling pathways in vivo. Testing this hypothesis, however, requires specific tools to independently block anandamide and 2-AG metabolism. Here, we report a potent and selective inhibitor of MAGL called JZL184 that, upon administration to mice, raises brain 2-AG by eight-fold without altering anandamide. JZL184-treated mice exhibited a broad array of CB1-dependent behavioral effects, including analgesia, hypothermia and hypomotility. These data indicate that 2-AG endogenously modulates several behavioral processes classically associated with the pharmacology of cannabinoids and point to overlapping and unique functions for 2-AG and anandamide in vivo.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Behavior, Animal; Benzodioxoles; Cannabinoids; Dose-Response Relationship, Drug; Endocannabinoids; Glycerides; Hydrolysis; Male; Mice; Mice, Inbred C57BL; Monoacylglycerol Lipases; Piperidines; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2

Evidence from recent studies suggests that the endocannabinoid system participates in the regulation of lipid metabolism and body composition. We hypothesize that the system is activated by oxidized low-density lipoprotein (oxLDL) and regulates cellular cholesterol metabolism in macrophages.. Primary peritoneal macrophages isolated from Sprague-Dawley rats and RAW264.7 mice macrophages were cultured. A liquid chromatography/mass spectrometry (LC/MS) system was used to measure the endocannabinoid anandamide (AEA), 2-arachidonoylglycerol (2-AG), and cellular cholesterol levels in macrophages. The regulatory mechanisms of cellular cholesterol metabolism were also investigated by molecular biology methods. The results showed that the endocannabinoid system in macrophages was activated by oxLDL through elevation of the AEA and 2-AG levels and the up-regulation of the cannabinoid CB1 and CB2 receptor expression. Win55,212-2, a synthetic cannabinoid, promotes cellular cholesterol accumulation in macrophages, which was associated with an increase in the expression of CD36 and a decrease in the expression of ATP-binding cassette protein A1 (ABCA1) as mediated by an up-regulated peroxisome proliferator-activated receptor gamma (PPARgamma). AM251, a selective cannabinoid CB1 receptor antagonist, impaired the abilities of Win55,212-2-treated macrophages to accumulate cholesterol by down-regulating CD36 receptor expression and up-regulating ABCA1 expression.. We have demonstrated, for the first time, that the endocannabinoid system in macrophages is activated by oxLDL and that the activated endocannabinoid system promotes cellular cholesterol accumulation in macrophages. The results also indicate that selectively blocking the CB1 receptor can reduce oxLDL accumulation in macrophages, which might represent a promising therapeutic strategy for atherosclerosis.

Topics: Animals; Arachidonic Acids; ATP Binding Cassette Transporter 1; ATP-Binding Cassette Transporters; Benzoxazines; Cannabinoid Receptor Modulators; CD36 Antigens; Cell Survival; Cells, Cultured; Cholesterol; Dose-Response Relationship, Drug; Endocannabinoids; Glycerides; Lipoproteins, LDL; Macrophages, Peritoneal; Mice; Morpholines; Naphthalenes; Piperidines; Polyunsaturated Alkamides; PPAR gamma; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Signal Transduction

Endocannabinoids released by postsynaptic neurons inhibit neurotransmitter release from presynaptic axon terminals. One typical stimulus of endocannabinoid production is an increase of calcium concentration in postsynaptic neurons. The aim of the present study was to clarify whether depolarizing GABAergic synaptic input, by increasing calcium concentration in postsynaptic neurons, can trigger endocannabinoid production. Spontaneous GABAergic inhibitory postsynaptic currents (sIPSCs) were recorded in Purkinje cells in mouse cerebellar slices with patch-clamp pipettes containing 151 mM chloride (a usual recording mode). sIPSCs were depolarizing inward currents under this condition. Combined electrophysiological and fluorometric calcium imaging experiments indicated that sIPSCs frequently triggered calcium spikes. After the calcium spikes, a short-term suppression of sIPSCs occurred. This suppression was prevented by the CB(1) cannabinoid receptor antagonist rimonabant and the diacylglycerol lipase inhibitor orlistat, but not changed by URB597, an inhibitor of anandamide degradation. It is, therefore, likely that CB(1) receptors and 2-arachidonoylglycerol were involved. For testing the physiological significance of the above observation, we carried out experiments on brains of 3- to 5-day-old mice. The gramicidin-induced perforated patch-clamp mode was used for preserving the physiological intracellular chloride concentration of the neurons. Depolarizing GABAergic sIPSCs occurred under this condition, but at a very low rate. Rimonabant did not change the frequency of these sIPSCs, arguing against the persistence of an endocannabinoid tone. The results point to a new kind of trigger of endocannabinoid production: depolarizing GABAergic synaptic input can elicit endocannabinoid production in postsynaptic neurons by activating calcium channels. The produced endocannabinoid suppresses GABA release from presynaptic axon terminals.

Topics: Aniline Compounds; Animals; Animals, Newborn; Arachidonic Acids; Benzamides; Bicuculline; Biophysics; Calcium; Calcium Signaling; Cannabinoid Receptor Antagonists; Cannabinoid Receptor Modulators; Carbamates; Cerebellum; Electric Stimulation; Endocannabinoids; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Fluoresceins; GABA Agonists; GABA Antagonists; gamma-Aminobutyric Acid; Glycerides; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Lactones; Mice; Muscimol; Orlistat; Patch-Clamp Techniques; Piperidines; Purkinje Cells; Pyrazoles; Quinoxalines; Rimonabant; Signal Transduction; Synapses; Valine

Direct-acting cannabinoid receptor agonists are well known to reduce hyperalgesic responses and allodynia after nerve injury, although their psychoactive side effects have damped enthusiasm for their therapeutic development. Alternatively, inhibiting fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), the principal enzymes responsible for the degradation of the respective endogenous cannabinoids, anandamide (AEA) and 2-arachydonylglycerol (2-AG), reduce nociception in a variety of nociceptive assays, with no or minimal behavioral effects. In the present study we tested whether inhibition of these enzymes attenuates mechanical allodynia, and acetone-induced cold allodynia in mice subjected to chronic constriction injury of the sciatic nerve. Acute administration of the irreversible FAAH inhibitor, cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-yl ester (URB597), or the reversible FAAH inhibitor, 1-oxo-1-[5-(2-pyridyl)-2-yl]-7-phenylheptane (OL-135), decreased allodynia in both tests. This attenuation was completely blocked by pretreatment with either CB(1) or CB(2) receptor antagonists, but not by the TRPV1 receptor antagonist, capsazepine, or the opioid receptor antagonist, naltrexone. The novel MAGL inhibitor, 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) also attenuated mechanical and cold allodynia via a CB(1), but not a CB(2), receptor mechanism of action. Whereas URB597 did not elicit antiallodynic effects in FAAH(-/-) mice, the effects of JZL184 were FAAH-independent. Finally, URB597 increased brain and spinal cord AEA levels, whereas JZL184 increased 2-AG levels in these tissues, but no differences in either endo-cannabinoid were found between nerve-injured and control mice. These data indicate that inhibition of FAAH and MAGL reduces neuropathic pain through distinct receptor mechanisms of action and present viable targets for the development of analgesic therapeutics.

Topics: Amidohydrolases; Analgesics, Non-Narcotic; Animals; Arachidonic Acids; Benzamides; Benzodioxoles; Cannabinoid Receptor Modulators; Carbamates; Cold Temperature; Endocannabinoids; Enzyme Inhibitors; Glycerides; Hyperalgesia; Male; Mice; Mice, Inbred C57BL; Monoacylglycerol Lipases; Narcotic Antagonists; Pain; Pain Measurement; Peripheral Nervous System Diseases; Piperidines; Polyunsaturated Alkamides; Pyridines; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; TRPV Cation Channels

Involvement of the endocannabinoids in hyperphagia has been demonstrated, however, behavioral characterization of its role in food reinforcement is limited. The present study investigated whether 2-arachidonoyl glycerol, an endocannabinoid ligand, and rimonabant, a CB1 antagonist, change the reinforcing properties of food in gestationally undernourished rats (a putative model of obesity) vs controls. Albino dams were food deprived by 0 to 45% of their free-feeding weights up to day 18 of their gestational period. Their offspring were allowed to free-feed until postnatal day 75. Then, behavior of the offspring was placed under progressive ratio schedules of sucrose reinforcement. After baseline data were established, intraperitoneal injections of 2-AG (0.03-3.75 mg/kg), and rimonabant (SR141716, 0.3-3.0 mg/kg) were administered and compared across group. Results show gestationally undernourished (GU) rats as adults weighed less than controls at the time of testing and female offspring allowed to free-feed for over 35 weeks exhibited lower body weights than controls. Under baseline, GU rats had lower breakpoints than controls. 2-AG and rimonabant significantly increased and decreased, respectively, breakpoint and responses made per session, suggesting involvement of the cannabinoid system in food reinforcement. When comparing peak doses of 2-AG on breakpoint, gestationally undernourished rats exhibited lower peak doses than controls. These data suggest that under the gestation deprivation method employed, GU rats were thinner and had lower food reinforcer efficacy than controls, and may have heightened sensitivity to 2-AG.

Topics: Aging; Analysis of Variance; Animals; Arachidonic Acids; Body Weight; Cannabinoid Receptor Modulators; Conditioning, Operant; Dose-Response Relationship, Drug; Endocannabinoids; Feeding Behavior; Female; Fetal Nutrition Disorders; Glycerides; Male; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Reinforcement, Psychology; Rimonabant; Sucrose; Time Factors

Monoacylglycerol lipase (MAGL) is a principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG). We recently reported a piperidine carbamate, JZL184, that inhibits MAGL with high potency and selectivity. Here, we describe a comprehensive mechanistic characterization of JZL184. We provide evidence that JZL184 irreversibly inhibits MAGL via carbamoylation of the enzyme's serine nucleophile. Functional proteomic analysis of mice treated with JZL184 revealed that this inhibitor maintains good selectivity for MAGL across a wide range of central and peripheral tissues. Interestingly, MAGL blockade produced marked, tissue-specific differences in monoglyceride metabolism, with brain showing the most dramatic elevations in 2-AG and peripheral tissues often showing greater changes in other monoglycerides. Collectively, these studies indicate that MAGL exerts tissue-dependent control over endocannabinoid and monoglyceride metabolism and designate JZL184 as a selective tool to characterize the functions of MAGL in vivo.

Topics: Animals; Arachidonic Acids; Benzodioxoles; Brain; Cannabinoid Receptor Modulators; Endocannabinoids; Enzyme Inhibitors; Glycerides; Metabolic Networks and Pathways; Mice; Monoacylglycerol Lipases; Monoglycerides; Piperidines

Endocannabinoid (eCB) signaling mediates depolarization-induced suppression of excitation (DSE) and inhibition (DSI), two prominent forms of retrograde synaptic depression. N-Arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), two known eCBs, are degraded by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. Selective blockade of FAAH and MAGL is critical for determining the roles of the eCBs in DSE/DSI and understanding how their action is regulated. 4-Nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) is a recently developed, highly selective, and potent MAGL inhibitor that increases 2-AG but not AEA concentrations in mouse brain. Here, we report that JZL184 prolongs DSE in Purkinje neurons in cerebellar slices and DSI in CA1 pyramidal neurons in hippocampal slices. The effect of JZL184 on DSE/DSI is mimicked by the nonselective MAGL inhibitor methyl arachidonyl fluorophosphonate. In contrast, neither the selective FAAH inhibitor cyclohexylcarbamic acid 3'-carbomoylbiphenyl-3-yl ester (URB597) nor FAAH knockout has a significant effect on DSE/DSI. JZL184 produces greater enhancement of DSE/DSI in mouse neurons than that in rat neurons. The latter finding is consistent with biochemical studies showing that JZL184 is more potent in inhibiting mouse MAGL than rat MAGL. These results indicate that the degradation of 2-AG by MAGL is the rate-limiting step that determines the time course of DSE/DSI and that JZL184 is a useful tool for the study of 2-AG-mediated signaling.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Benzodioxoles; Cannabinoid Receptor Modulators; Carbamates; Electrophysiology; Endocannabinoids; Enzyme Inhibitors; Glycerides; Hydrolysis; Lipase; Mice; Mice, Inbred C57BL; Mice, Knockout; Patch-Clamp Techniques; Piperidines; Purkinje Cells; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Signal Transduction; Synapses

Depolarization-induced suppression of excitation (DSE) is a major form of cannabinoid-mediated short-term retrograde neuronal plasticity and is found in numerous brain regions. Autaptically cultured murine hippocampal neurons are an architecturally simple model for the study of cannabinoid signaling, including DSE. The transient nature of DSE--tens of seconds--is probably determined by the regulated hydrolysis of the endocannabinoid 2-arachidonoyl glycerol (2-AG). No less than five candidate enzymes have been considered to serve this role: fatty acid amide hydrolase (FAAH), cyclooxygenase-2 (COX-2), monoacylglycerol lipase (MGL), and alpha/beta-hydrolase domain (ABHD) 6 and 12. We previously found that FAAH and COX-2 do not have a role in determining the duration of autaptic DSE. In the current study, we found that two structurally distinct inhibitors of MGL [N-arachidonoyl maleimide and 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184)] prolong DSE in autaptic hippocampal neurons, whereas inhibition of ABHD6 by N-methyl-N-[[3-(4-pyridinyl)phenyl]methyl]-4'-(aminocarbonyl)[1,1'-biphenyl]-4-yl ester, carbamic acid (WWL70) had no effect. In addition, we developed antibodies against MGL and ABHD6 and determined their expression in autaptic cultures. MGL is chiefly expressed at presynaptic terminals, optimally positioned to break down 2-AG that has engaged presynaptic CB(1) receptors. ABHD6 is expressed in two distinct locations on autaptic islands, including a prominent localization in some dendrites. In summary, we provide strong pharmacological and anatomical evidence that MGL regulates DSE in autaptic hippocampal neurons and, taken together with other studies, emphasizes that endocannabinoid signaling is terminated in temporally diverse ways.

Topics: Animals; Arachidonic Acids; Benzodioxoles; Biphenyl Compounds; Cannabinoid Receptor Modulators; Cell Line; Cells, Cultured; Endocannabinoids; Excitatory Postsynaptic Potentials; Glycerides; Hippocampus; Humans; Mice; Monoacylglycerol Lipases; Neurons; Piperidines; Presynaptic Terminals; Pyrazoles; Rimonabant

Delta(9)-tetrahydrocannabinol (THC), the psychoactive component of marijuana, and other direct cannabinoid receptor (CB1) agonists produce a number of neurobehavioral effects in mammals that range from the beneficial (analgesia) to the untoward (abuse potential). Why, however, this full spectrum of activities is not observed upon pharmacological inhibition or genetic deletion of either fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), enzymes that regulate the two major endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG), respectively, has remained unclear. Here, we describe a selective and efficacious dual FAAH/MAGL inhibitor, JZL195, and show that this agent exhibits broad activity in the tetrad test for CB1 agonism, causing analgesia, hypomotilty, and catalepsy. Comparison of JZL195 to specific FAAH and MAGL inhibitors identified behavioral processes that were regulated by a single endocannabinoid pathway (e.g., hypomotility by the 2-AG/MAGL pathway) and, interestingly, those where disruption of both FAAH and MAGL produced additive effects that were reversed by a CB1 antagonist. Falling into this latter category was drug discrimination behavior, where dual FAAH/MAGL blockade, but not disruption of either FAAH or MAGL alone, produced THC-like responses that were reversed by a CB1 antagonist. These data indicate that AEA and 2-AG signaling pathways interact to regulate specific behavioral processes in vivo, including those relevant to drug abuse, thus providing a potential mechanistic basis for the distinct pharmacological profiles of direct CB1 agonists and inhibitors of individual endocannabinoid degradative enzymes.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Carbamates; Carboxylic Ester Hydrolases; Endocannabinoids; Glycerides; Mice; Molecular Structure; Monoacylglycerol Lipases; Motor Activity; Pain Measurement; Piperazines; Piperidines; Polyunsaturated Alkamides

The phospholipase A(2) (PLA(2))-prostanoid cascade is involved in cannabinoid receptor-mediated neuronal functions. We investigated the signaling mechanism for the release of arachidonic acid by cannabinoids, 2-arachidonoyl glycerol (2-AG) and HU210, in rat PC12 cells and in primary cultured cells from the mouse cerebellum. The effect of selective inhibitors for signaling pathways and/or enzymes (alpha type cytosolic PLA(2) (cPLA(2)alpha), G protein, Src kinases, phospholipase C, protein kinase C) was assessed. Methods included translocation of the chimeric protein GFP-cPLA(2)alpha, the activities of Src family kinases, Ca(2+)-dependent fluorescence and cyclic AMP accumulation. Treatment with 2-AG and HU210 at greater concentrations than 3 muM caused the release of arachidonic acid, and the response was inhibited by AM251 (an antagonist of cannabinoid CB(1) receptor) and by pyrrophenone (a selective inhibitor of cPLA(2)alpha) in PC12 cells. The cannabinoid treatment caused the intracellular translocation of cPLA(2)alpha and an increase in the intracellular Ca(2+) level. Treatment with HU210 caused tyrosine phosphorylation of Src and Fyn, and increased their kinase activities. Pretreatment with inhibitors of tyrosine kinases or phospholipase C abolished the cannabinoids-induced release of arachidonic acid and Ca(2+) response, and protein kinase C inhibitor reduced the release of arachidonic acid. 2-AG caused the release of arachidonic acid from cultured cells of the mouse cerebellum via similar mechanisms. These data reveal that cannabinoids activated cPLA(2)alpha in a Src-phospholipase C-protein kinase C-dependent manner probably via cannabinoid CB(1) receptor and/or CB(1)-like receptor in neuronal cells.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Cyclic AMP; Cytosol; Dronabinol; Endocannabinoids; Glycerides; Group IV Phospholipases A2; Mice; Mice, Inbred ICR; PC12 Cells; Phosphorylation; Piperidines; Protein Kinase C; Proto-Oncogene Proteins c-fyn; Pyrazoles; Rats; Receptor, Cannabinoid, CB1; Signal Transduction; src-Family Kinases; Type C Phospholipases

Mounting evidence suggests that the endocannabinoid system regulates energy metabolism through direct effects on peripheral tissues as well as central effects that regulate appetite. Here we examined the effect of cannabinoid receptor 1 (CB1) signaling on insulin action in fat cells. We examined effects of the natural CB1 agonist, 2-Arachidonoylglycerol (2-AG), and the synthetic CB1 antagonist, SR141716, on insulin action in cultured adipocytes. We used translocation of glucose transporter GLUT4 to plasma membrane (PM) as a measure of insulin action. 2-AG activation of the CB1 receptor promoted insulin sensitivity whereas antagonism by SR141716 reduced insulin sensitivity. Neither drug affected GLUT4 translocation in the absence of insulin or with high doses of insulin. Consistent with these results we found that insulin-stimulated phosphorylation of the protein kinase Akt was increased by 2-AG, attenuated by SR141716, and unaffected in the absence of insulin or by addition of high-dose insulin. These data provide a functional and molecular link between the CB1 receptor and insulin sensitivity, because insulin-stimulated phosphorylation of Akt is required for GLUT4 translocation to the PM. The sensitizing effects of 2-AG were abrogated by SR141716 and Pertussis toxin, indicating that the effects are mediated by CB1 receptor. Importantly, neither 2-AG nor SR141716 alone or in combination with maximal dose of insulin had effects on GLUT4 translocation and Akt phosphorylation. These data are consistent with a model in which the endocannabinoid system sets the sensitivity of the insulin response in adipocytes rather than directly regulating the redistribution of GLUT4 or Akt phosphorylation.

Topics: 3T3-L1 Cells; Adipocytes; Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Cell Membrane; Dose-Response Relationship, Drug; Endocannabinoids; Glucose Transporter Type 4; Glycerides; Insulin; Insulin Resistance; Mice; Pertussis Toxin; Piperidines; Proto-Oncogene Proteins c-akt; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Insulin; Rimonabant

This study was conducted to test the effects of 2-arachidonylglycerol (2-AG), an endocannabinoid, on aqueous humor outflow facility, to study the cellular mechanisms of 2-AG, and to investigate the possible existence and activity of monoacylgylcerol lipase (MGL), a 2-AG metabolic enzyme, in the trabecular meshwork (TM). The effects of 2-AG on aqueous humor outflow facility were measured using an anterior segment perfused organ culture model. The expression and activity of MGL in TM tissues were assessed using Western blot analysis and an enzyme activity assay respectively. 2-AG induced activation of p42/44 mitogen-activated protein (MAP) kinase was determined by Western blot analysis using an anti-phospho p42/44 MAP kinase antibody. AlexaFluor 488-labeled phalloidin staining was used to examine actin filament in cultured TM cells. Administration of 10nM of 2-AG caused a transient enhancement of aqueous humor outflow. In the presence of 100nM of LY2183240, an inhibitor of MGL, the effect of 10nM of 2-AG on outflow was prolonged by at least 4h. The 2-AG-induced enhancement of outflow was blocked by SR141716A, a CB1 antagonist, and SR144528, a CB2 antagonist. In Western blot studies, a 35kDa band representing MGL was detected on TM tissues with an anti-MGL antibody. The 2-AG enzymatic hydrolysis activity was detected in TM tissues and this activity was reduced by 70.1+/-5.3% with the addition of 100 nM of LY2183240. Treatment of trabecular meshwork cells with 10nM of 2-AG plus 100 nM LY2183240 for 5h evoked phosphorylation of p42/44 MAP kinase. The 2-AG-induced enhancement of p42/44 MAP kinase phosphorylation was blocked by pretreatment with SR141716A, SR144528, as well as PD98059, an inhibitor of the p42/44 MAP kinase pathway. In addition, the outflow-enhancing effect of 2-AG was blocked by pretreatment with PD98059. Furthermore, treatment with 2-AG plus LY2183240 caused rounding of TM cells and a reduction of actin stress fibers in TM cells. Pretreatment with SR141716A, SR144528, and PD98059 blocked these 2-AG-induced morphology and cytoskeleton changes in TM cells. In conclusion, the results from this study demonstrate that administration of 2-AG increases aqueous humor outflow facility and this effect of 2-AG is mediated through both the CB1 and CB2 cannabinoid receptors. In addition, this study reveals the existence and the activity of MGL, a 2-AG metabolizing enzyme, in the TM tissues. Furthermore, this study suggests that 2-AG-induced enhancement of o

Topics: Animals; Aqueous Humor; Arachidonic Acids; Calcium-Calmodulin-Dependent Protein Kinases; Camphanes; Cannabinoid Receptor Modulators; Cytoskeleton; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Flavonoids; Glycerides; Monoacylglycerol Lipases; Organ Culture Techniques; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; Signal Transduction; Sus scrofa; Trabecular Meshwork

Inhibition of the metabolism of the endocannabinoids, anandamide (AEA) and 2-arachidonyl glycerol (2-AG), by their primary metabolic enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively, has the potential to increase understanding of the physiological functions of the endocannabinoid system. To date, selective inhibitors of FAAH, but not MAGL, have been developed. The purpose of this study was to determine the selectivity and efficacy of N-arachidonyl maleimide (NAM), a putative MAGL inhibitor, for modulation of the effects of 2-AG. Our results showed that NAM unmasked 2-AG activity in a tetrad of in vivo tests sensitive to the effects of cannabinoids in mice. The efficacy of 2-AG (and AEA) to produce hypothermia was reduced compared with Delta(9)-tetrahydrocannabinol; however, 2-AG differed from AEA by its lower efficacy for catalepsy. All tetrad effects were partially CB(1) receptor-mediated because they were attenuated (but not eliminated) by SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-H-pyrazole-3-carboxamide HCl] and in CB(1)(-/-) mice. In vitro, NAM increased endogenous levels of 2-AG in the brain. Furthermore, NAM raised the potency of 2-AG, but not AEA, in agonist-stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate binding assay, a measure of G-protein activation. These results suggest that NAM is an MAGL inhibitor with in vivo and in vitro efficacy. NAM and other MAGL inhibitors are valuable tools to elucidate the biological functions of 2-AG and to examine the consequences of dysregulation of this endocannabinoid. In addition, NAM's unmasking of 2-AG effects that are only partially reversed by SR141716A offers support for the existence of non-CB(1), non-CB(2) cannabinoid receptors.

Topics: Animals; Arachidonic Acids; Drug Synergism; Endocannabinoids; Female; Glycerides; Guanosine 5'-O-(3-Thiotriphosphate); Maleimides; Mice; Mice, Inbred ICR; Monoacylglycerol Lipases; Motor Activity; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant

The endocannabinoid system is emerging as an integral component in central and peripheral regulation of feeding and energy balance. Our investigation analyzed behavioral roles for cannabinoid mechanisms of the pontine parabrachial nucleus (PBN) in modulating intake of presumably palatable foods containing fat and/or sugar. The PBN serves to gate neurotransmission associated with, but not limited to, the gustatory properties of food. Immunofluorescence and in vitro [(35)S]GTPgammaS autoradiography of rat tissue sections containing the PBN revealed the presence of cannabinoid receptors and their functional capability to couple to their G-proteins after incubation with the endocannabinoid 2-arachidonoyl glycerol (2-AG). The selective cannabinoid 1 receptor (CB(1)R) antagonist AM251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] prevented the response, demonstrating CB(1)R mediation of 2-AG-induced coupling. Microinfusions of 2-AG into the PBN in behaving rats robustly stimulated feeding of pellets high in content of fat and sucrose (HFS), pure sucrose, and pure fat (Crisco), during the first 30 min after infusion. In contrast, 2-AG failed to increase consumption of standard chow, even when the feeding regimen was manipulated to match baseline intakes of HFS. Orexigenic responses to 2-AG were attenuated by AM251, again indicating CB(1)R mediation of 2-AG actions. Furthermore, responses were regionally specific, because 2-AG failed to alter intake when infused into sites approximately 500 mum caudal to infusions that successfully stimulated feeding. Our data suggest that hedonically positive sensory properties of food enable endocannabinoids at PBN CB(1)Rs to initiate increases in eating, and, more generally, these pathways may serve a larger role in brain functions controlling behavioral responses for natural reward.

Topics: Analysis of Variance; Animals; Arachidonic Acids; Autoradiography; Behavior, Animal; Cannabinoid Receptor Modulators; Conditioning, Operant; Eating; Endocannabinoids; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Food Preferences; Glycerides; Guanosine 5'-O-(3-Thiotriphosphate); Male; Narcotic Antagonists; Peptides; Piperidines; Pons; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptors, Opioid, mu; Sulfur Isotopes; Time Factors; Xanthines

Anandamide and 2-arachidonoyl glycerol, referred to as endocannabinoids (eCBs), are the endogenous agonists for the cannabinoid receptor type 1 (CB1). Several pieces of evidence support a role for eCBs in the attenuation of anxiety-related behaviours, although the precise mechanism has remained uncertain. The fatty acid amid hydrolase (FAAH), an enzyme responsible for the degradation of eCBs, has emerged as a promising target for anxiety-related disorders, since FAAH inhibitors are able to increase the levels of anandamide and thereby induce anxiolytic-like effects in rodents. The present study adopted both genetic and pharmacological approaches and tested the hypothesis that FAAH-deficient (FAAH(-/-)) mice as well as C57BL/6N mice treated with an FAAH inhibitor (URB597) would express reduced anxiety-like responses. Furthermore, as it is known that anandamide can bind several other targets than CB1 receptors, we investigated whether FAAH inhibition reduces anxiety via CB1 receptors. FAAH(-/-) mice showed reduced anxiety both in the elevated plus maze and in the light-dark test. These genotype-related differences were prevented by the CB1 receptor antagonist rimonabant (3mg/kg). Moreover, URB597 (1mg/kg) induced an anxiolytic-like effect in C57BL/6N mice exposed to the elevated plus maze, which was prevented by rimonabant (3mg/kg). The present work provides genetic and pharmacological evidence supporting the inhibition of FAAH as an important mechanism for the alleviation of anxiety. In addition, it indicates an increased activation of CB1 receptors as a mechanism underlying the effects of FAAH inhibition in two models of anxiety.

Topics: Amidohydrolases; Analysis of Variance; Animals; Anxiety; Arachidonic Acids; Behavior, Animal; Benzamides; Carbamates; Disease Models, Animal; Endocannabinoids; Glycerides; Male; Maze Learning; Mice; Mice, Inbred C57BL; Mice, Knockout; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant

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

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

Cannabinoid signaling by CB1 receptors drives fibrogenesis and fat accumulation in liver. A report in this issue of Cell Metabolism (Jeong et al., 2008) now links hepatic stellate cells, a resident liver fibrogenic cell type, to the generation of steatosis through production of the endocannabinoid 2-arachidonoylglycerol (2-AG) after ethanol feeding, leading to paracrine stimulation of hepatocyte CB1 receptors.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Carnitine O-Palmitoyltransferase; Cells, Cultured; Diet, Fat-Restricted; Disease Models, Animal; Endocannabinoids; Ethanol; Fatty Acid Synthases; Fatty Acids; Fatty Liver, Alcoholic; Glycerides; Hepatocytes; Humans; Lipogenesis; Lipoprotein Lipase; Liver; Mice; Oxidation-Reduction; Paracrine Communication; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; Sterol Regulatory Element Binding Protein 1; Up-Regulation

Alcohol-induced fatty liver, a major cause of morbidity, has been attributed to enhanced hepatic lipogenesis and decreased fat clearance of unknown mechanism. Here we report that the steatosis induced in mice by a low-fat, liquid ethanol diet is attenuated by concurrent blockade of cannabinoid CB1 receptors. Global or hepatocyte-specific CB1 knockout mice are resistant to ethanol-induced steatosis and increases in lipogenic gene expression and have increased carnitine palmitoyltransferase 1 activity, which, unlike in controls, is not reduced by ethanol treatment. Ethanol feeding increases the hepatic expression of CB1 receptors and upregulates the endocannabinoid 2-arachidonoylglycerol (2-AG) and its biosynthetic enzyme diacylglycerol lipase beta selectively in hepatic stellate cells. In control but not CB1 receptor-deficient hepatocytes, coculture with stellate cells from ethanol-fed mice results in upregulation of CB1 receptors and lipogenic gene expression. We conclude that paracrine activation of hepatic CB1 receptors by stellate cell-derived 2-AG mediates ethanol-induced steatosis through increasing lipogenesis and decreasing fatty acid oxidation.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Carnitine O-Palmitoyltransferase; Cells, Cultured; Coculture Techniques; Diet, Fat-Restricted; Disease Models, Animal; Endocannabinoids; Ethanol; Fatty Acid Synthases; Fatty Acids; Fatty Liver, Alcoholic; Glycerides; Hepatocytes; Lipogenesis; Lipoprotein Lipase; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Oxidation-Reduction; Paracrine Communication; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; Sterol Regulatory Element Binding Protein 1; Up-Regulation

There is strong evidence that blocking CB1 receptors may reduce alcohol intake in alcohol-dependent individuals. However, there is still limited evidence that CB1 receptor antagonists may also be beneficial in the attenuation of alcohol withdrawal syndrome, even though alcohol withdrawal appears to be milder in CB1 receptor knockout mice. Here we have examined whether the CB1 receptor antagonist rimonabant (SR141716) can alleviate the behavioral symptoms and revert the neurochemical imbalance elicited by a 3-h interruption of chronic alcohol exposure (7.2% in the drinking water for 10 days) in male Wistar rats. Administration of rimonabant attenuated the strong anxiogenic traits of the animals that developed when regular alcohol intake was interrupted. This may reflect the correction of the GABA/glutamate imbalances developed by the animals that received rimonabant in various brain regions involved in emotional (e.g. prefrontal cortex) and motor (e.g. caudate-putamen and globus pallidus) responses. In addition, rimonabant also affected the dopamine deficits generated by alcohol abstinence in the amygdala and ventral-tegmental area, albeit to a lesser extent. However, this antagonist was unable to correct the impairment caused by alcohol abstinence in serotonin and neuropeptide Y. The endocannabinoid activity in the brain of alcohol-abstinent rats indicated that the behavioral and neurochemical improvements caused by rimonabant were not related to the attenuation of a possible increase in this activity generated by alcohol withdrawal. Conversely, the density of CB1 receptors was reduced in alcohol-abstinent animals (e.g. globus pallidus, substantia nigra), as were the levels of endocannabinoids and related N-acylethanolamines (e.g. amygdala, caudate-putamen). Thus, rimonabant possibly enhances an endogenous response generated by interrupting the regular use of alcohol. In summary, rimonabant might attenuate withdrawal symptoms associated with alcohol abstinence, an effect that was presumably due to the normalization of GABA and glutamate, and to a lesser extent, dopamine transmission in emotion- and motor-related areas.

Topics: Animals; Anxiety; Appetite; Arachidonic Acids; Autoradiography; Benzoxazines; Brain Chemistry; Cannabinoid Receptor Modulators; Central Nervous System Depressants; Chromatography, High Pressure Liquid; Emotions; Endocannabinoids; Enkephalins; Ethanol; Ethanolamines; Glycerides; In Situ Hybridization; Male; Morpholines; Motor Activity; Naphthalenes; Neuropeptide Y; Neurotransmitter Agents; Piperidines; Polyunsaturated Alkamides; Protein Precursors; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Rimonabant; RNA, Messenger; Stress, Psychological; Substance Withdrawal Syndrome

The perivascular sensory nerve (PvN) Ca(2+)-sensing receptor (CaR) is implicated in Ca(2+)-induced relaxation of isolated, phenylephrine (PE)-contracted mesenteric arteries, which involves the vascular endogenous cannabinoid system. We determined the effect of inhibition of diacylglycerol (DAG) lipase (DAGL), phospholipase A(2) (PLA(2)), and cytochrome P-450 (CYP) on Ca(2+)-induced relaxation of PE-contracted rat mesenteric arteries. Our findings indicate that Ca(2+)-induced vasorelaxation is not dependent on the endothelium. The DAGL inhibitor RHC 802675 (1 microM) and the CYP and PLA(2) inhibitors quinacrine (5 microM) (EC(50): RHC 802675 2.8 +/- 0.4 mM vs. control 1.4 +/- 0.3 mM; quinacrine 4.8 +/- 0.4 mM vs. control 2.0 +/- 0.3 mM; n = 5) and arachidonyltrifluoromethyl ketone (AACOCF(3), 1 microM) reduced Ca(2+)-induced relaxation of mesenteric arteries. Synthetic 2-arachidonoylglycerol (2-AG) and glycerated epoxyeicosatrienoic acids (GEETs) induced concentration-dependent relaxation of isolated arteries. 2-AG relaxations were blocked by iberiotoxin (IBTX) (EC(50): control 0.96 +/- 0.14 nM, IBTX 1.3 +/- 0.5 microM) and miconazole (48 +/- 3%), and 11,12-GEET responses were blocked by IBTX (EC(50): control 55 +/- 9 nM, IBTX 690 +/- 96 nM) and SR-141716A. The data suggest that activation of the CaR in the PvN network by Ca(2+) leads to synthesis and/or release of metabolites of the CYP epoxygenase pathway and metabolism of DAG to 2-AG and subsequently to GEETs. The findings indicate a role for 2-AG and its metabolites in Ca(2+)-induced relaxation of resistance arteries; therefore this receptor may be a potential target for the development of new vasodilator compounds for antihypertensive therapy.

Topics: 8,11,14-Eicosatrienoic Acid; Acetylcholine; Animals; Arachidonic Acids; Calcium; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Glycerides; Lipoprotein Lipase; Male; Mesenteric Arteries; Miconazole; Peptides; Phenylephrine; Phospholipase A2 Inhibitors; Phospholipases A2; Piperidines; Potassium Channel Blockers; Potassium Channels, Calcium-Activated; Pyrazoles; Quinacrine; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptors, Calcium-Sensing; Rimonabant; Signal Transduction; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

Cannabis-based medicines have a number of therapeutic indications, including anti-inflammatory and analgesic effects. The endocannabinoid receptor system, including the cannabinoid receptor 1 (CB1) and receptor 2 (CB2) and the endocannabinoids, are implicated in a wide range of physiological and pathophysiological processes. Pre-clinical and clinical studies have demonstrated that cannabis-based drugs have therapeutic potential in inflammatory diseases, including rheumatoid arthritis (RA) and multiple sclerosis. The aim of this study was to determine whether the key elements of the endocannabinoid signalling system, which produces immunosuppression and analgesia, are expressed in the synovia of patients with osteoarthritis (OA) or RA.. Thirty-two OA and 13 RA patients undergoing total knee arthroplasty were included in this study. Clinical staging was conducted from x-rays scored according to Kellgren-Lawrence and Larsen scales, and synovitis of synovial biopsies was graded. Endocannabinoid levels were quantified in synovial fluid by liquid chromatography-mass spectrometry. The expression of CB1 and CB2 protein and RNA in synovial biopsies was investigated. Functional activity of these receptors was determined with mitogen-activated protein kinase assays. To assess the impact of OA and RA on this receptor system, levels of endocannabinoids in the synovial fluid of patients and non-inflamed healthy volunteers were compared. The activity of fatty acid amide hydrolase (FAAH), the predominant catabolic endocannabinoid enzyme, was measured in synovium.. CB1 and CB2 protein and RNA were present in the synovia of OA and RA patients. Cannabinoid receptor stimulation of fibroblast-like cells from OA and RA patients produced a time-dependent phosphorylation of extracellular signal-regulated kinase (ERK)-1 and ERK-2 which was significantly blocked by the CB1 antagonist SR141716A. The endocannabinoids anandamide (AEA) and 2-arachidonyl glycerol (2-AG) were identified in the synovial fluid of OA and RA patients. However, neither AEA nor 2-AG was detected in synovial fluid from normal volunteers. FAAH was active in the synovia of OA and RA patients and was sensitive to inhibition by URB597 (3'-(aminocarbonyl) [1,1'-biphenyl]-3-yl)-cyclohexylcarbamate).. Our data predict that the cannabinoid receptor system present in the synovium may be an important therapeutic target for the treatment of pain and inflammation associated with OA and RA.

Topics: Aged; Amidohydrolases; Arachidonic Acids; Arthritis, Rheumatoid; Blotting, Western; Cells, Cultured; Chromatography, Liquid; Cytokines; Endocannabinoids; Female; Fibroblasts; Glycerides; Humans; Knee Joint; Male; Mass Spectrometry; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Osteoarthritis; Phosphorylation; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Reverse Transcriptase Polymerase Chain Reaction; Rimonabant; RNA, Messenger; Synovial Fluid; Synovial Membrane

Endocannabinoids released from the postsynaptic neuronal membrane can activate presynaptic CB1 receptors and inhibit neurotransmitter release. In hippocampal slices, depolarization of the CA1 pyramidal neurons elicits an endocannabinoid-mediated inhibition of gamma-aminobutyric acid release known as depolarization-induced suppression of inhibition (DSI). Using the highly reduced neuron/synaptic bouton preparation from the CA1 region of hippocampus, we have begun to examine endocannabinoid-dependent short-term depression (STD) of inhibitory synaptic transmission under well-controlled physiological and pharmacological conditions in an environment free of other cells. Application of the CB1 synthetic agonist WIN55212-2 and endogenous cannabinoids 2-AG and anandamide produced a decrease in spontaneous inhibitory postsynaptic current (sIPSC) frequency and amplitude, indicating the presence of CB1 receptors at synapses in this preparation. Endocannabinoid-dependent STD is different from DSI found in hippocampal slices and the neuron/bouton preparation from basolateral amygdala (BLA) since depolarization alone was not sufficient to induce suppression of sIPSCs. However, concurrent application of the metabotropic glutamate receptor (mGluR) agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) and postsynaptic depolarization resulted in a transient (30-50 s) decrease in sIPSC frequency and amplitude. Application of DHPG alone had no effect on sIPSCs. The depolarization/DHPG-induced STD was blocked by the CB1 antagonist SR141716A and the mGluR5 antagonist MPEP and was sensitive to intracellular calcium concentration. Comparing the present findings with earlier work in hippocampal slices and BLA, it appears that endocannabinoid release is less robust in isolated hippocampal neurons.

Topics: Animals; Animals, Newborn; Arachidonic Acids; Benzoxazines; Cannabinoid Receptor Modulators; Drug Interactions; Endocannabinoids; Excitatory Amino Acid Antagonists; gamma-Aminobutyric Acid; Glycerides; Hippocampus; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Methoxyhydroxyphenylglycol; Morpholines; Naphthalenes; Neurons; Patch-Clamp Techniques; Piperidines; Pyrazoles; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Rimonabant; Synapses; Synapsins

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

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

In neuronal signalling mediated by the endocannabinoid 2-arachidonoylglycerol, both synthetic and inactivating enzymes operate within close proximity to the G(i/o)-coupled pre-synaptic CB(1) receptors, thus allowing for rapid onset and transient duration of this lipid modulator. In rat brain, 2-arachidonoylglycerol is inactivated mainly via hydrolysis by serine hydrolase inhibitor-sensitive monoacylglycerol lipase activity. We show in this study that comprehensive pharmacological elimination of this activity in brain cryosections by methyl arachidonylfluorophosphonate or hexadecylsulphonyl fluoride results in endocannabinoid-mediated CB(1) receptor activity, which can be visualized by functional autoradiography. URB597, a specific inhibitor of anandamide hydrolysis proved ineffective. TLC indicated that the bioactivity resided in 2-arachidonoylglycerol-containing fraction and gas chromatography-mass spectroscopy detected elevated levels of monoacylglycerols, including 2-arachidonoylglycerol in this fraction. Although two diacylglycerol lipase inhibitors, tetrahydrolipstatin (THL) and RHC80267, blocked the bulk of 2-arachidonoylglycerol accumulation in methyl arachidonylfluorophosphonate-treated sections, only THL reversed the endocannabinoid-dependent CB(1) receptor activity. Further studies indicated that at the used concentrations, THL rather specifically antagonized the CB(1) receptor. These findings confirm that in brain sections there is preservation of enzymatic pathways regulating the production of endogenous receptor ligands. Furthermore, the presently described methodology may serve as an elegant and intuitive approach to identify novel membrane-derived lipid modulators operating in the CNS.

Topics: Animals; Arachidonic Acids; Autoradiography; Benzamides; Binding, Competitive; Brain; Carbamates; Cryoultramicrotomy; Drug Interactions; Endocannabinoids; Enzyme Inhibitors; Glycerides; Guanosine 5'-O-(3-Thiotriphosphate); Male; Organophosphonates; Piperidines; Pyrazoles; Radiography; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Sulfones

2-arachidonoylglycerol (2-AG) is an endogenous ligand for the cannabinoid receptors with a variety of potent biological activities. In this study, we first examined the effects of potassium-induced depolarization on the level of 2-AG in rat brain synaptosomes. We found that a significant amount of 2-AG was generated in the synaptosomes following depolarization. Notably, depolarization did not affect the levels of other molecular species of monoacylglycerols. Furthermore, the level of anandamide was very low and did not change markedly following depolarization. It thus appeared that the depolarization-induced accelerated generation is a unique feature of 2-AG. We obtained evidence that phospholipase C is involved in the generation of 2-AG in depolarized synaptosomes: U73122, a phospholipase C inhibitor, markedly reduced the depolarization-induced generation of 2-AG, and the level of diacylglycerol was rapidly elevated following depolarization. A significant amount of 2-AG was released from synaptosomes upon depolarization. Interestingly, treatment of the synaptosomes with SR141716A, a CB1 receptor antagonist, augmented the release of glutamate from depolarized synaptosomes. These results strongly suggest that the endogenous ligand for the cannabinoid receptors, i.e. 2-AG, generated through increased phospholipid metabolism upon depolarization, plays an important role in attenuating glutamate release from the synaptic terminals by acting on the CB1 receptor.

Topics: Animals; Arachidonic Acids; Brain; Calcimycin; Calcium Channel Blockers; Camphanes; Diglycerides; Endocannabinoids; Estrenes; Fatty Acids; Glycerides; Male; Membrane Potentials; Neuromuscular Depolarizing Agents; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Pyrrolidinones; Rats; Rats, Wistar; Receptors, Cannabinoid; Rimonabant; Synaptosomes

Evidence indicates that the endocannabinoid, 2-arachidonoylglycerol (2-AG), increases food intake when injected into the nucleus accumbens shell (NAcS), thereby potentially activating hypothalamic nuclei involved in food intake regulation. We aimed to evaluate potential orexigenic effects of the endocannabinoid anandamide and of AA5HT, a fatty acid amide hydrolase (FAAH) inhibitor, and OMDM-1, an inhibitor of anandamide uptake, injected in the NAcS, as well as the effect of these treatments on activation of hypothalamic nuclei.. Drugs were given into the NAcS of rats and food intake quantified during the next 4 h. In other groups, after the same treatments the brains were processed for c-Fos immunohistochemistry with focus on hypothalamic nuclei. Additional groups were used to quantify endocannabinoid levels in the nucleus accumbens and the hypothalamus after AA5HT and OMDM-1 intra-NAcS injections.. Our results indicate that the above treatments stimulate food intake during 4 h post-injection. They also increase c-Fos immunoreactivity in hypothalamic nuclei. The CB(1) antagonist, AM251, blocked these effects. Finally, we found elevated levels of 2-AG, but not anandamide, after intra-NAcS injections of AA5HT.. These data support the involvement of the endocannabinoid system in feeding behavior at the level of the NAcS and hypothalamus. In addition, this is the first experimental demonstration that the pharmacological inhibition of endocannabinoid inactivation in the NAcS stimulates food intake, suggesting that the endocannabinoid degrading proteins can be a target for treating eating disorders.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Arcuate Nucleus of Hypothalamus; Benzyl Compounds; Cannabinoid Receptor Modulators; Eating; Endocannabinoids; Glycerides; Hypothalamus; Immunohistochemistry; Male; Nucleus Accumbens; Piperidines; Polyunsaturated Alkamides; Proto-Oncogene Proteins c-fos; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Serotonin; Time Factors

Allergic contact dermatitis affects about 5% of men and 11% of women in industrialized countries and is one of the leading causes for occupational diseases. In an animal model for cutaneous contact hypersensitivity, we show that mice lacking both known cannabinoid receptors display exacerbated allergic inflammation. In contrast, fatty acid amide hydrolase-deficient mice, which have increased levels of the endocannabinoid anandamide, displayed reduced allergic responses in the skin. Cannabinoid receptor antagonists exacerbated allergic inflammation, whereas receptor agonists attenuated inflammation. These results demonstrate a protective role of the endocannabinoid system in contact allergy in the skin and suggest a target for therapeutic intervention.

Topics: Animals; Arachidonic Acids; Camphanes; Cannabinoid Receptor Modulators; Cannabinoids; Chemokines; Dermatitis, Allergic Contact; Dinitrofluorobenzene; Disease Models, Animal; Down-Regulation; Dronabinol; Endocannabinoids; Female; Glycerides; Mice; Mice, Inbred C57BL; Mice, Knockout; Oligonucleotide Array Sequence Analysis; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; Skin; Up-Regulation

Cerebrovascular smooth muscle cells express the CB1 cannabinoid receptor and CB1 agonists produce vasodilatation of the middle cerebral artery (MCA). The thromboxane A2 mimetic, U-46619, increased the content of the endocannabinoid, 2-arachidonoylglycerol (2-AG) in the MCA and 2-AG moderated the vasoconstriction produced by U46619 in this tissue. The purposes of this study were to examine the extent to which 2-AG is catabolized by cerebral arteries and to determine whether blockade of 2-AG inactivation potentiates its feedback inhibition of U-44619-mediated vasoconstriction.. The diameters of isolated, perfused MCA from male rats were measured using videomicroscopy.. Exogenous 2-AG produces a CB1 receptor-dependent and concentration-related increase in the diameter of MCA constricted with 5-HT. The E (max) for 2-AG dilation is increased 4-fold in the presence of the metabolic inhibitors 3-(decylthio)-1,1,1-trifluropropan-2-one (DETFP), URB754 and URB597. To examine the role of catabolism in the effects of endogenous 2-AG, vasoconstriction induced by U-46619 was studied. DETFP and URB754, but not the fatty acid amide hydrolase inhibitor, URB597, significantly increased the EC(50) for U-46619. These data support a physiological role for endocannabinoid feedback inhibition in the effects of U-46619 and indicate that endogenously produced 2-AG is also efficiently catabolized within the MCA.. MCA express mechanisms for the efficient inactivation of 2-AG, providing further support for an endocannabinoid feedback mechanism that opposes thromboxane-mediated vasoconstriction. These data suggest that potentiation of endogenously produced 2-AG could be a novel therapeutic approach to the treatment of thrombotic stroke.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Amidohydrolases; Aniline Compounds; Animals; Arachidonic Acid; Arachidonic Acids; Benzamides; Benzoxazines; Carbamates; Dose-Response Relationship, Drug; Drug Synergism; Endocannabinoids; Enzyme Inhibitors; Glycerides; In Vitro Techniques; Male; Middle Cerebral Artery; Morpholines; Naphthalenes; Nimodipine; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Rimonabant; Thromboxanes; Vasoconstriction; Vasoconstrictor Agents

In the present study, we examined the occurrence and potential regulation of endocannabinoid release by cannabinoid CB1 receptors in the rat brain. To this end, we developed a highly sensitive (limit of sensitivity 30-300 amol) new analytical method, combining online brain microdialysis with solid-phase extraction-liquid chromatography-tandem mass spectrometry, which allowed the detection in real time of trace amounts of endocannabinoids in the extracellular fluid. In the hypothalamus, anandamide and 2-arachidonoyl-glycerol release was stimulated following depolarization via local administration of K(+), with or without addition of Ca(2+), or glutamate application. Inhibition of fatty acid amide hydrolase by systemic administration of intraperitoneal (i.p.) URB597 (0.5 mg/kg) induced an increase of anandamide, but not 2-arachidonoyl-glycerol, outflow. The CB1 receptor antagonist rimonabant (10 mg/kg i.p.) increased, whereas the CB1 agonist WIN55,212-2 (2.5 mg/kg i.p.) decreased, anandamide release. Interestingly, the same treatments induced opposite changes in 2-arachidonoyl-glycerol release. At a dose of 3 mg/kg i.p., which by itself did not affect endocannabinoid release, rimonabant fully antagonized the effect of WIN55,212-2 (2.5 mg/kg i.p.). Taken together, these results suggest that CB1 receptors are able to control the local release of endocannabinoids in the hypothalamus via a feedback mechanism and strengthen the view that anandamide and 2-arachidonoyl-glycerol have distinct physiological roles.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Benzoxazines; Cannabinoid Receptor Modulators; Cannabinoids; Carbamates; Chromatography, Liquid; Endocannabinoids; Extracellular Fluid; Glycerides; Hypothalamus; Male; Microdialysis; Morpholines; Naphthalenes; Piperidines; Polyunsaturated Alkamides; Potassium; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Rimonabant; Tandem Mass Spectrometry

Endocannabinoids (eCBs) mediate short- and long-term depression of synaptic strength by retrograde transsynaptic signaling. Previous studies have suggested that an eCB mobilization or release step in the postsynaptic neuron is involved in this retrograde signaling. However, it is not known whether this release process occurs automatically upon eCB synthesis or whether it is regulated by other synaptic factors. To address this issue, we loaded postsynaptic striatal medium spiny neurons (MSNs) with the eCBs anandamide (AEA) or 2-arachidonoylglycerol and determined the conditions necessary for presynaptic inhibition. We found that presynaptic depression of glutamatergic excitatory postsynaptic currents (EPSCs) and GABAergic inhibitory postsynaptic currents (IPSCs) induced by postsynaptic eCB loading required a certain level of afferent activation that varied between the different synaptic types. Synaptic depression at excitatory synapses was temperature-dependent and blocked by the eCB membrane transport blockers, VDM11 and UCM707, but did not require activation of metabotropic glutamate receptors, l-calcium channels, nitric oxide, voltage-activated Na(+) channels, or intracellular calcium. Application of the CB(1)R antagonist, AM251, after depression was established, reversed the decrease in EPSC, but not in IPSC, amplitude. Direct activation of the CB(1) receptor by WIN 55,212-2 initiated synaptic depression that was independent of afferent stimulation. These findings indicate that retrograde eCB signaling requires a postsynaptic release step involving a transporter or carrier that is activated by afferent stimulation/synaptic activation.

Topics: Animals; Arachidonic Acids; Benzoxazines; Calcium Channel Blockers; Cannabinoid Receptor Modulators; Corpus Striatum; Endocannabinoids; Furans; gamma-Aminobutyric Acid; Glutamic Acid; Glycerides; Long-Term Synaptic Depression; Membrane Potentials; Morpholines; Naphthalenes; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Receptor, Cannabinoid, CB1; Synapses; Synaptic Transmission

Endocannabinoids function as neurotransmitters and neuromodulators in the central nervous system via specific receptors and apparently have a neuroprotective role. We assumed that the endocannabinoid system could be involved in the pathogenesis of hepatic encephalopathy (HE), a neuropsychiatric syndrome due to liver disease. We used a mouse model of a thioacetamide induced fulminant hepatic failure. We found that the levels of the endocannabinoid 2-arachidonoyl-glycerol (2-AG) were elevated in the brain. Treatment with either 2-AG or with the CB1 receptor antagonist, SR141716A, improved a neurological score, activity and cognitive function. Activation of the CB2 receptor by a selective agonist, HU308, also improved the neurological score. 2-AG activity could be blocked with the specific CB2 receptor antagonist SR144528A. The CB1 receptor agonist noladin ether was inactive. We conclude that the endocannabinoid system may play an important role in the pathogenesis of HE. Modulation of this system either by exogenous agonists specific for the CB2 receptors or possibly also by antagonists to the CB1 receptors may have therapeutic potential.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Cognition; Dose-Response Relationship, Drug; Endocannabinoids; Female; Glycerides; Hepatic Encephalopathy; Liver; Liver Failure, Acute; Maze Learning; Mice; Mice, Inbred Strains; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; Thioacetamide

Recent work in our laboratories has demonstrated that an opioid-independent form of stress-induced analgesia (SIA) is mediated by endogenous cannabinoids [Hohmann et al., 2005. Nature 435, 1108]. Non-opioid SIA, induced by a 3-min continuous foot shock, is characterized by the mobilization of two endocannabinoid lipids--2-arachidonoylglycerol (2-AG) and anandamide--in the midbrain periaqueductal gray (PAG). The present studies were conducted to examine the contributions of spinal endocannabinoids to nonopioid SIA. Time-dependent increases in levels of 2-AG, but not anandamide, were observed in lumbar spinal cord extracts derived from shocked relative to non-shocked rats. Notably, 2-AG accumulation was of smaller magnitude than that observed previously in the dorsal midbrain following foot shock. 2-AG is preferentially degraded by monoacylglycerol lipase (MGL), whereas anandamide is hydrolyzed primarily by fatty-acid amide hydrolase (FAAH). This metabolic segregation enabled us to manipulate endocannabinoid tone at the spinal level to further evaluate the roles of 2-AG and anandamide in nonopioid SIA. Intrathecal administration of the competitive CB1 antagonist SR141716A (rimonabant) failed to suppress nonopioid SIA, suggesting that supraspinal rather than spinal CB1 receptor activation plays a pivotal role in endocannabinoid-mediated SIA. By contrast, spinal inhibition of MGL using URB602, which selectively inhibits 2-AG hydrolysis in the PAG, enhanced SIA through a CB1-selective mechanism. Spinal inhibition of FAAH, with either URB597 or arachidonoyl serotonin (AA-5-HT), also enhanced SIA through a CB1-mediated mechanism, presumably by increasing accumulation of tonically released anandamide. Our results suggest that endocannabinoids in the spinal cord regulate, but do not mediate, nonopioid SIA.

Topics: Analgesia; Analysis of Variance; Animals; Arachidonic Acids; Behavior, Animal; Benzamides; Carbamates; Dose-Response Relationship, Drug; Drug Interactions; Endocannabinoids; Glycerides; Male; Mass Spectrometry; Pain Measurement; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Reaction Time; Rimonabant; Serotonin; Spinal Cord; Stress, Psychological; Time Factors

Diverticulosis is a common disease of not completely defined pathogenesis. Motor abnormalities of the intestinal wall have been frequently described but very little is known about their mechanisms. We investigated in vitro the neural response of colonic longitudinal muscle strips from patients undergoing surgery for complicated diverticular disease (diverticulitis).. The neural contractile response to electrical field stimulation of longitudinal muscle strips from the colon of patients undergoing surgery for colonic cancer or diverticulitis was challenged by different receptor agonists and antagonists.. Contractions of colonic strips from healthy controls and diverticulitis specimens were abolished by atropine. The beta adrenergic agonist (-) isoprenaline and the tachykinin NK1 receptor antagonist SR140333 had similar potency in reducing the electrical twitch response in controls and diseased tissues, while the cannabinoid receptor agonist (+)WIN 55,212-2 was 100 times more potent in inhibiting contractions in controls (IC50 42 nmol/l) than in diverticulitis strips. SR141716, a selective antagonist of the cannabinoid CB1 receptor, had no intrinsic activity in control preparations but potentiated the neural twitch in diseased tissues by up to 196% in a concentration dependent manner. SR141716 inhibited (+)WIN 55,212-2 induced relaxation in control strips but had no efficacy on (+)WIN 55,212-2 responses in strips from diverticular disease patients. Colonic levels of the endogenous ligand of cannabinoid and vanilloid TRPV1 receptors anandamide were more than twice those of control tissues (54 v 27 pmol/g tissue). The axonal conduction blocker tetrodotoxin had opposite effects in the two preparations, completely inhibiting the contractions of control strips but potentiating those in diverticular preparations, an effect selectively inhibited by SR140333.. Neural control of colon motility is profoundly altered in patients with diverticulitis. Their raised levels of anandamide, apparent desensitisation of the presynaptic neural cannabinoid CB1 receptor, and the SR141716 induced intrinsic response, suggest that endocannabinoids may be involved in the pathophysiology of complications of colonic diverticular disease.

Topics: Adrenergic beta-Agonists; Adrenergic beta-Antagonists; Aged; Arachidonic Acids; Benzoxazines; Cannabinoid Receptor Modulators; Case-Control Studies; Colon; Diverticulum; Endocannabinoids; Female; Glycerides; Humans; Imidazoles; Isoproterenol; Male; Middle Aged; Morpholines; Muscle Contraction; Muscle, Smooth; Naphthalenes; Neurokinin-1 Receptor Antagonists; Piperidines; Polyunsaturated Alkamides; Propanolamines; Pyrazoles; Quinuclidines; Rimonabant; Substance P; Tetrodotoxin

Endocannabinoids act as neuroprotective molecules promptly released in response to pathological stimuli. Hence, they may represent one component of protection and/or repair mechanisms mobilized by dopamine (DA) neurons under ischemia. Here, we show that the endocannabinoid 2-arachidonoyl-glycerol (2-AG) plays a key role in protecting DA neurons from ischemia-induced altered spontaneous activity both in vitro and in vivo. Accordingly, neuroprotection can be elicited through moderate cannabinoid receptor type-1 (CB1) activation. Conversely, blockade of endocannabinoid actions through CB1 receptor antagonism worsens the outcome of transient ischemia on DA neuronal activity. These findings indicate that 2-AG mediates neuroprotective actions by delaying damage and/or restoring function of DA cells through activation of presynaptic CB1 receptors. Lastly, they point to CB1 receptors as valuable targets in protection of DA neurons against ischemic injury and emphasize the need for a better understanding of endocannabinoid actions in the fine control of DA transmission.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzoxazines; Brain Ischemia; Cannabinoid Receptor Modulators; Dopamine; Electrophysiology; Endocannabinoids; Glycerides; In Vitro Techniques; Male; Mice; Mice, Knockout; Morpholines; Naphthalenes; Neurons; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Rimonabant; Signal Transduction; Ventral Tegmental Area

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

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

The levels of the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) are under the negative control of leptin in the rodent hypothalamus. As leptin and endocannabinoids play opposite roles in the control of reproduction, we have investigated whether the impaired fertility typical of leptin-defective ob/ob mice is due, in part, to enhanced uterine endocannabinoid levels. We found that levels of both anandamide and 2-AG in the uterus of ob/ob mice are significantly elevated with respect to wild-type littermates, due to reduced hydrolase activity in the case of anandamide, and to reduced monoacylglycerol lipase and enhanced diacylglycerol lipase activity in the case of 2-AG. Furthermore, the process mediating endocannabinoid cellular uptake was also impaired in ob/ob mice, whereas the levels of cannabinoid and anandamide receptors were not modified. Although ineffective in wild-type mice, treatment of ob/ob mice with leptin re-established endocannabinoid levels and enzyme activities back to the values observed in wild-type littermates. Finally, treatment of ob/ob females with the CB1 receptor antagonist SR141716A did not improve their fertility, and inhibition of endocannabinoid inactivation with the endocannabinoid uptake inhibitor OMDM-1 in wild-type females did not result in impaired fertility.

Topics: Animals; Arachidonic Acids; Benzyl Compounds; Cannabinoid Receptor Modulators; Endocannabinoids; Female; Fertility; Glycerides; Leptin; Lipoprotein Lipase; Mice; Mice, Knockout; Monoacylglycerol Lipases; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Receptors, Leptin; Rimonabant; Up-Regulation; Uterus

Endocannabinoids and cannabinoid CB1 receptors play a role in the control of movement by modulating GABA, glutamate, and other neurotransmitters throughout the basal ganglia. Roles for abnormalities in endocannabinoid signaling in Parkinson's disease (PD) and the major side effect of current treatments, levodopa-induced dyskinesia (LID), have been suggested by rodent studies. Here we show that signaling by endocannabinoids contributes to the pathophysiology of parkinsonism and LID in MPTP-lesioned, non-human primate models of Parkinson's disease. In MPTP-lesioned marmosets previously treated with levodopa to establish LID, attenuation of CB1 signaling by systemic administration of rimonabant (1 and 3 mg/kg) had anti-parkinsonian actions, equivalent to a 71% increase in motor activity at 3 mg/kg. Rimonabant did not elicit dyskinesia. Co-administration of levodopa (8 mg/kg) and rimonabant (1 and 3 mg/kg) resulted in significantly less dyskinesia than levodopa alone, without significantly affecting the anti-parkinsonian action of levodopa. These data suggest that enhanced endocannabinoid signaling may be involved in the pathophysiology of both parkinsonism and LID. To define potential mechanisms by which such a role might be mediated, we determined the levels of the endocannabinoids anandamide and 2-arachidonyl glycerol (2-AG) throughout the basal ganglia in normal and three groups of MPTP-lesioned cynomolgus monkeys (untreated; acutely treated with L-DOPA, non-dyskinetic; long-term treated, with levodopa-induced dyskinesia). In the untreated, MPTP-lesioned primate, parkinsonism was associated with increases in both 2-AG (+88%) and anandamide (+49%) in the striatum, and of 2-AG (+97%) in the substantia nigra, changes that are consistent with the previously suggested role for endocannabinoids in mechanisms attempting to compensate for loss of dopamine in untreated parkinsonism. Increased levels of anandamide (+34%) in the external globus pallidus of MPTP-lesioned animals were normalized by levodopa treatment and may contribute to the generation of parkinsonian symptoms. However, no clear alteration in endocannabinoid levels could be correlated with the expression of LID. These data highlight the potential roles played by endocannabinoids and CB1 in PD and LID and suggest the need for further research to pursue the multiple therapeutic opportunities for manipulating this system in movement disorders.

Topics: Animals; Arachidonic Acids; Callithrix; Cannabinoid Receptor Modulators; Dyskinesia, Drug-Induced; Endocannabinoids; Female; gamma-Aminobutyric Acid; Glycerides; Levodopa; Male; MPTP Poisoning; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant

In the globus pallidus, cannabinoid CB(1) receptors are localized pre-synaptically on GABAergic neurons. We assessed the influence of the endocannabinoids, anandamide, 2-arachidonoyl-glycerol (2-AG) and noladin ether, on the uptake of [(3)H]-GABA in pallidal slices from rat. Both 2-AG and noladin ether increased [(3)H]-GABA uptake (by 40.8 +/- 8.0% and 38.4 +/- 12.5%). The effect of 2-AG was blocked by the cannabinoid CB(1) receptor antagonist AM 251. In contrast, neither anandamide nor the agonist WIN 55,212-2 had an effect on [(3)H]-GABA uptake. Different roles might be played by different endocannabinoids, both physiologically and in basal ganglia disorders, such as Parkinson's disease.

Topics: Animals; Arachidonic Acids; Binding, Competitive; Cannabinoid Receptor Modulators; Endocannabinoids; gamma-Aminobutyric Acid; Globus Pallidus; Glycerides; In Vitro Techniques; Male; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Tritium

Exogenous cannabinoids have been shown to significantly alter neuroendocrine output, presaging the emergence of endogenous cannabinoids as important signalling molecules in the neuroendocrine control of homeostatic and reproductive functions, including the stress response, energy metabolism and gonadal regulation. We showed recently that magnocellular and parvocellular neuroendocrine cells of the hypothalamic paraventricular nucleus and supraoptic nucleus (SON) respond to glucocorticoids by releasing endocannabinoids as retrograde messengers to modulate the synaptic release of glutamate. Here we show directly for the first time that both of the main endocannabinoids, anandamide (AEA) and 2-arachidonoyl glycerol (2-AG), are released in an activity-dependent fashion from the soma/dendrites of SON magnocellular neurones and suppress synaptic glutamate release and postsynaptic spiking. Cannabinoid reuptake blockade increases activity-dependent endocannabinoid levels in the region of the SON, and results in the inhibition of synaptically driven spiking activity in magnocellular neurones. Together, these findings demonstrate an activity-dependent release of AEA and 2-AG that leads to the suppression of glutamate release and that is capable of shaping spiking activity in magnocellular neurones. This activity-dependent regulation of excitatory synaptic input by endocannabinoids may play a role in determining spiking patterns characteristic of magnocellular neurones under stimulated conditions.

Topics: Animals; Arachidonic Acids; Benzoxazines; Benzyl Compounds; Cannabinoid Receptor Modulators; Cannabinoids; Endocannabinoids; Excitatory Postsynaptic Potentials; Glutamic Acid; Glycerides; In Vitro Techniques; Male; Morpholines; Naphthalenes; Neurons; Piperidines; Polyunsaturated Alkamides; Presynaptic Terminals; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptors, Presynaptic; Supraoptic Nucleus; Synaptic Transmission

Central nervous system responses to cannabis are primarily mediated by CB(1) receptors, which couple preferentially to G(i/o) G proteins. Here, we used calcium photometry to monitor the effect of CB(1) activation on intracellular calcium concentration. Perfusion with 5 microM CB(1) aminoalkylindole agonist, WIN55,212-2 (WIN), increased intracellular calcium by several hundred nanomolar in human embryonic kidney 293 cells stably expressing CB(1) and in cultured hippocampal neurons. The increase was blocked by coincubation with the CB(1) antagonist, SR141716A, and was absent in nontransfected human embryonic kidney 293 cells. The calcium rise was WIN-specific, being essentially absent in cells treated with other classes of cannabinoid agonists, including Delta(9)-tetrahydrocannabinol, HU-210, CP55,940, 2-arachidonoylglycerol, methanandamide, and cannabidiol. The increase in calcium elicited by WIN was independent of G(i/o), because it was present in pertussis toxin-treated cells. Indeed, pertussis toxin pretreatment enhanced the potency and efficacy of WIN to increase intracellular calcium. The calcium increases appeared to be mediated by G(q) G proteins and phospholipase C, because they were markedly attenuated in cells expressing dominant-negative G(q) or treated with the phospholipase C inhibitors U73122 and ET-18-OCH(3) and were accompanied by an increase in inositol phosphates. The calcium increase was blocked by the sarco/endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin, the inositol trisphosphate receptor inhibitor xestospongin D, and the ryanodine receptor inhibitors dantrolene and 1,1'-diheptyl-4,4'-bipyridinium dibromide, but not by removal of extracellular calcium, showing that WIN releases calcium from intracellular stores. In summary, these results suggest that WIN stabilizes CB(1) receptors in a conformation that enables G(q) signaling, thus shifting the G protein specificity of the receptor.

Topics: Analgesics; Animals; Arachidonic Acids; Benzoxazines; Calcium; Cannabinoids; Cell Line; Cyclohexanols; Cytoplasm; DNA, Complementary; Dronabinol; Endocannabinoids; Endoplasmic Reticulum; Excitatory Amino Acid Antagonists; Fluorescent Dyes; Fura-2; Glycerides; GTP-Binding Protein alpha Subunits, Gq-G11; Hippocampus; Humans; Immunosuppressive Agents; Morpholines; Naphthalenes; Neurons; Pertussis Toxin; Piperidines; Protein Binding; Protein Conformation; Pyrazoles; Rats; Receptor, Cannabinoid, CB1; Rimonabant; Ryanodine; Time Factors; Type C Phospholipases

Whether chronic cannabinoid consumption produces a dependent state comparable to that occurring with other drugs (e.g. the appearance of withdrawal signs when consumption is interrupted), and whether chronic cannabinoid consumption increases the risk of consuming other drugs of greater addictive power, are probably the two questions relating to cannabinoid addiction that provoke the most controversy. The present study was designed to further explore these two questions in laboratory animals. Firstly, we examined the effects of an acute challenge with SR141716 (an antagonist for the cannabinoid CB(1) receptor) in Delta(9)-tetrahydrocannabinol (Delta(9)-THC)-tolerant rats. This antagonist has been reported to precipitate a cannabinoid withdrawal syndrome. Thus, the administration of SR141716 to Delta(9)-THC-tolerant rats reduced inactivity in the open-field test and enhanced responses as tremor, turning and retropulsion-these responses that were only slightly enhanced in control rats. The administration of SR141716 increased the plasma prolactin and the corticosterone concentration in controls, but these increases were much lesser in Delta(9)-THC-tolerant rats. In addition, CRF-mRNA levels in the paraventricular hypothalamic nucleus, while reduced in SR141716-treated controls, were significantly increased in Delta(9)-THC-tolerant rats. The analysis of endocannabinoids also revealed that the administration of SR141716, which was mostly inactive in control rats, was able to reverse the changes in anandamide or 2-arachidonoylglycerol concentrations found in Delta(9)-THC-tolerant rats, in the striatum, limbic forebrain, diencephalon, cerebellum and brainstem, but not in the midbrain and hippocampus. As a second objective, we evaluated whether Delta(9)-THC-tolerant rats were more vulnerable to morphine in a self-administration paradigm. The Delta(9)-THC-tolerant and control rats self-administered morphine to a similar extent, in concordance with the similar values of dopaminergic activity in limbic and motor regions. In summary, our data indicate that Delta(9)-THC-tolerant rats were not more vulnerable to the reinforcing properties of morphine. However, they responded to the blockade of CB(1) receptors by exhibiting slightly but possibly relevant differences in behavioral, endocrine and molecular parameters compared to the response in non-tolerant rats. This is indicative of the existence of a withdrawal syndrome in cannabinoid-tolerant rats that is mild compare

Topics: Animals; Arachidonic Acids; Behavior, Animal; Brain; Cannabinoid Receptor Antagonists; Cannabinoid Receptor Modulators; Corticosterone; Dronabinol; Drug Administration Schedule; Drug Tolerance; Endocannabinoids; Glycerides; Male; Paraventricular Hypothalamic Nucleus; Piperidines; Polyunsaturated Alkamides; Prolactin; Pyrazoles; Rats; Rats, Wistar; Receptors, Cannabinoid; Receptors, Corticotropin-Releasing Hormone; Rimonabant; RNA, Messenger; Substance Withdrawal Syndrome

The control of endocannabinoid actions on cortical neurons by a putative carrier-mediated uptake is still poorly understood at the level of synaptic transmission. We investigated the effect of an endocannabinoid, 2-arachidonoyl glycerol (2-AG), on inhibitory postsynaptic currents (IPSCs) in hippocampal slices under physiological conditions, and when uptake was altered by a selective blocker or lower temperature. Bath application of 2-AG (20 micro m) caused a 40% reduction in the amplitude of IPSCs evoked in the perisomatic region of CA1 pyramidal neurons at room temperature; this effect could be blocked by a specific CB(1) receptor antagonist, AM251. By contrast, a smaller (20%) but significant suppression of inhibitory transmission was found by 2-AG at 33-35 degrees C. This reduced blocking effect at physiological temperature could be brought back to 40% by coapplying the endocannabinoid uptake blocker, AM404 (10 or 20 micro m) with 2-AG. In parallel experiments, we measured [(3)H]2-AG uptake at different temperatures in primary cultures prepared from cortical neurons. These data confirmed a striking inhibition of [(3)H]2-AG uptake at room temperature compared with values observed at 37 degrees C. Uptake could be significantly modified by anandamide, 2-AG and AM404, suggesting a common transporter for the two endocannabinoids. These findings together demonstrate the presence of an effective endocannabinoid uptake in cortical neurons, which could considerably alter the spatial and temporal constraints of endocannabinoid signalling at physiological temperature, and which may critically change the interpretation of findings at room temperature.

Topics: Animals; Animals, Newborn; Arachidonic Acids; Biological Transport; Cannabinoid Receptor Modulators; Dose-Response Relationship, Drug; Drug Interactions; Endocannabinoids; Excitatory Postsynaptic Potentials; Glycerides; Hippocampus; In Vitro Techniques; Male; Neural Inhibition; Neurons; Patch-Clamp Techniques; Piperidines; Pyrazoles; Rats; Rats, Wistar; Temperature; Tritium

In hippocampal pyramidal cells, a rise in Ca(2+) releases endocannabinoids that activate the presynaptic cannabinoid receptor (CB1R) and transiently reduce GABAergic transmission-a process called depolarization-induced suppression of inhibition (DSI). The mechanism that limits the duration of endocannabinoid action in intact cells is unknown. Here we show that inhibition of cyclooxygenase-2 (COX-2), not fatty acid amide hydrolase (FAAH), prolongs DSI, suggesting that COX-2 limits endocannabinoid action.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Drug Synergism; Endocannabinoids; Enzyme Inhibitors; Glycerides; Hippocampus; In Vitro Techniques; Isoenzymes; Male; Meloxicam; Membrane Potentials; Neural Inhibition; Patch-Clamp Techniques; Piperidines; Polyunsaturated Alkamides; Prostaglandin-Endoperoxide Synthases; Pyramidal Cells; Pyrazoles; Rats; Rats, Sprague-Dawley; Sulfonamides; Thiazines; Thiazoles

The arachidonic acid derivative, 2-arachidonoyl-glycerol (2-AG), was initially isolated from gut and brain; it is also produced and released from blood and vascular cells. Many of the 2-AG-induced cellular responses (i.e., neuromodulation, cytoprotection and vasodilation) are mediated by cannabinoid receptors CB1 and CB2. The findings presented here demonstrate the expression of CB1, CB2 and TRPV1 receptors on cerebromicrovascular endothelial cells (HBEC). The expression of TRPV1, CB1 and CB2 receptor mRNA and proteins were demonstrated by RT-PCR and polyclonal antibodies, respectively. The endocannabinoid 2-AG, and other related compounds [anandamide (ANA), methanandamide (m-ANA), N-(4-hydroxyphenyl-arachidonyl-ethanolamide) (AM404) and capsaicin] dose-dependently stimulated Ca2+ influx in HBEC. The selective TRPV1 receptor antagonist (capsazepine), CB1 receptor antagonist (SR141716A) and CB2 receptor antagonist (SR144528) inhibited these responses. The effects of capsaicin, a specific agonist for TRPV1 receptors, were inhibited by capsazepine, but only weakly by CB1 or CB2 receptor antagonists. 2-AG also induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP); this response was mediated by VR1 receptors. These studies clearly indicate that 2-AG and other related compounds may function as agonists on VR1 receptors, as well as CB1 and CB2 receptors, and implicated these factors in various HBEC functions.

Topics: Arachidonic Acids; Blood-Brain Barrier; Brain; Camphanes; Cannabinoid Receptor Agonists; Cannabinoid Receptor Modulators; Capsaicin; Cell Adhesion Molecules; Cells, Cultured; Cerebrovascular Circulation; Dose-Response Relationship, Drug; Drug Interactions; Endocannabinoids; Endothelium, Vascular; Glycerides; Humans; Ion Channels; Microcirculation; Microfilament Proteins; Phosphoproteins; Phosphorylation; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; RNA, Messenger; TRPV Cation Channels

Central cannabinoid systems have been implicated in appetite control through the respective hyperphagic and anorectic actions of CB1 agonists and antagonists. The motivational changes underlying these actions remain to be determined, but may involve alterations to food palatability.. The mode of action of cannabinoids on ingestion was investigated by examining the effects of exogenous and endogenous agonists, and a selective CB1 receptor antagonist, on licking microstructure in rats ingesting a palatable sucrose solution.. Microstructural analyses of licking for a 10% sucrose solution was performed over a range of agonist and antagonist doses administered to non-deprived, male Lister hooded rats.. Delta(9)-tetrahydrocannabinol (0.5, 1 and 3 mg/kg) and anandamide (1 mg/kg and 3 mg/kg) significantly increased total number of licks. This was primarily due to an increase in bout duration rather than bout number. There was a non-significant increase in total licks following administration of 2-arachidonoyl glycerol (0.2, 1.0 and 2.0 mg/kg), whereas administration of the CB1 antagonist SR141716 (1 mg/kg and 3 mg/kg) significantly decreased total licks. All drugs, with the exception of anandamide, significantly decreased the intra-bout lick rate. An exponential function fitted to the cumulative lick rate curves for each drug revealed that all compounds altered the asymptote of this function without having any marked effects on the exponent.. These data are consistent with endocannabinoid involvement in the mediation of food palatability.

Topics: Animals; Appetite Stimulants; Arachidonic Acids; Behavior, Animal; Cannabinoid Receptor Modulators; Cannabinoids; Dose-Response Relationship, Drug; Drinking Behavior; Dronabinol; Endocannabinoids; Glycerides; Male; Neurotransmitter Agents; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Sucrose; Time Factors

Cannabinoids, whether plant-derived, synthetic or endogenous, have been shown to stimulate appetite in the adult organism. We have reported previously that cannabinoid receptors play a critical role during the early suckling period: The selective cannabinoid CB(1) receptor antagonist N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141617A) permanently prevented milk ingestion in a dose-dependent manner, when administered to (Sabra, albino) mouse pups, within 1 day of birth. As a consequence, these pups died within the first week of life. We now generalize this finding to a different strain of mice (C57BL/6). Further, we show that cannabinoid CB(1) receptor blockade (20 mg/kg SR141716A) must occur within 24 h after birth as injection of SR141716A into 2- or 5-day-old pups had a much smaller effect or no effect at all, respectively. Cannabinoid CB(1) receptor knockout mice did not ingest milk on the first day of life, similarly to SR141716A-treated normal pups, as measured by the appearance of "milkbands". However, the knockout pups started to display milkbands from day 2 of life. Survival rates of cannabinoid CB(1) receptor knockout mice were affected significantly, but to a lesser extent than normal pups, by the administration of SR141716A. Daily administration of the endocannabinoid 2-arachidonoyl glycerol, or the synthetic agonists (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55,212-2, 5 mg/kg) or (-)-cis-3-[2-Hydroxy4-(1,1-dimethylheptyl) phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55,940, 5 or 20 mg/kg) did not promote survival or weight gain in CB(1)(-/-) pups. Our data support previous evidence for a critical role of cannabinoid CB(1) receptors for the initiation of suckling. Further, the present observations support the existence of an unknown cannabinoid receptor, with partial control over milk ingestion in newborns. Our data also suggest that the CB(1)(-/-) neonates possess a compensatory mechanism which helps them overcome the lack of cannabinoid CB(1) receptors.

Topics: Animals; Animals, Newborn; Animals, Suckling; Arachidonic Acids; Behavior, Animal; Benzoxazines; Cannabinoid Receptor Modulators; Cyclohexanols; Dose-Response Relationship, Drug; Drug Administration Schedule; Eating; Endocannabinoids; Female; Glycerides; Injections, Subcutaneous; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Milk; Morpholines; Naphthalenes; Piperidines; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Survival Rate; Time Factors

The aim of the present study was to assess the contribution of endogenous cannabinoids in the protective effect of ischemic preconditioning on the endothelial function in coronary arteries of the rat. Isolated rat hearts were exposed to a 30-min low flow ischemia (1 ml/min) followed by 20-min reperfusion, after which the response to the endothelium-dependent vasodilator, serotonine (5-HT), was compared with that of the endothelium-independent vasodilator, sodium nitroprusside (SNP). In untreated hearts, ischemia-reperfusion diminished selectively 5-HT-induced vasodilatation, compared with time-matched sham hearts, the vasodilatation to SNP being unaffected. A 5-min zero-flow preconditioning ischemia in untreated hearts preserved the vasodilatation produced by 5-HT. Blockade of either CB(1)-receptors with SR141716A or CB(2)-receptors with SR144528 abolished the protective effect of preconditioning on the 5-HT vasodilatation. Perfusion with either palmitoylethanolamide or 2-arachidonoylglycerol 15 min before and throughout the ischemia mimicked preconditioning inasmuch as it protected the endothelium in a similar fashion. This protection was blocked by SR144528 in both cases, whereas SR141716A only blocked the effect of PEA. The presence of CB(1) and CB(2)-receptors in isolated rat hearts was confirmed by Western blots. In conclusion, the data suggest that endogenous cannabinoids contribute to the endothelial protective effect of ischemic preconditioning in rat coronary arteries.

Topics: Amides; Animals; Arachidonic Acids; Blotting, Western; Camphanes; Cannabinoid Receptor Modulators; Cannabinoids; Coronary Vessels; Endocannabinoids; Endothelium, Vascular; Ethanolamines; Fatty Acids, Unsaturated; Glycerides; Heart; Ischemic Preconditioning, Myocardial; Male; Myocardium; Nitroprusside; Palmitic Acids; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Serotonin; Vasodilation

1 The purpose of this study was to determine whether endocannabinoids can protect the heart against ischaemia and reperfusion. 2 Rat isolated hearts were exposed to low-flow ischaemia (0.5-0.6 ml min(-1)) and reperfusion. Functional recovery as well as CK and LDH overflow into the coronary effluent were monitored. Infarct size was determined at the end of the experiments. Phosphorylation levels of p38, ERK1/2, and JNK/SAPK kinases were measured by Western blots. 3 None of the untreated hearts recovered from ischaemia during the reperfusion period. Perfusion with either 300 nM palmitoylethanolamide (PEA) or 300 nM 2-arachidonoylglycerol (2-AG), but not anandamide (up to 1 micro M), 15 min before and throughout the ischaemic period, improved myocardial recovery and decreased the levels of coronary CK and LDH. PEA and 2-AG also reduced infarct size. 4 The CB(2)-receptor antagonist, SR144528, blocked completely the cardioprotective effect of both PEA and 2-AG, whereas the CB(1)-receptor antagonist, SR141716A, blocked partially the effect of 2-AG only. In contrast, both ACEA and JWH015, two selective agonists for CB(1)- and CB(2)- receptors, respectively, reduced infarct size at a concentration of 50 nM. 5 PEA enhanced the phosphorylation level of p38 MAP kinase during ischaemia. PEA perfusion doubled the baseline phosphorylation level of ERK1/2, and enhanced its increase upon reperfusion. The cardioprotective effect of PEA was completely blocked by the p38 MAP kinase inhibitor, SB203580, and significantly reduced by the ERK1/2 inhibitor, PD98059, and the PKC inhibitor, chelerythrine. 6 In conclusion, endocannabinoids exert a strong cardioprotective effect in a rat model of ischaemia-reperfusion that is mediated mainly through CB(2)-receptors, and involves p38, ERK1/2, as well as PKC activation.

Topics: Amides; Animals; Arachidonic Acids; Biomarkers; Blotting, Western; Camphanes; Cannabinoid Receptor Modulators; Endocannabinoids; Ethanolamines; Glycerides; Heart; Imidazoles; L-Lactate Dehydrogenase; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Myocardial Infarction; Myocardial Ischemia; Myocardial Reperfusion Injury; p38 Mitogen-Activated Protein Kinases; Palmitic Acids; Piperidines; Protein Kinase C; Pyrazoles; Pyridines; Rats; Rats, Sprague-Dawley; Rimonabant; Signal Transduction

Abnormally high spiking activity can damage neurons. Signaling systems to protect neurons from the consequences of abnormal discharge activity have been postulated. We generated conditional mutant mice that lack expression of the cannabinoid receptor type 1 in principal forebrain neurons but not in adjacent inhibitory interneurons. In mutant mice,the excitotoxin kainic acid (KA) induced excessive seizures in vivo. The threshold to KA-induced neuronal excitation in vitro was severely reduced in hippocampal pyramidal neurons of mutants. KA administration rapidly raised hippocampal levels of anandamide and induced protective mechanisms in wild-type principal hippocampal neurons. These protective mechanisms could not be triggered in mutant mice. The endogenous cannabinoid system thus provides on-demand protection against acute excitotoxicity in central nervous system neurons.

Topics: Animals; Arachidonic Acids; Brain; Brain-Derived Neurotrophic Factor; Cannabinoids; Endocannabinoids; Epilepsy; Excitatory Amino Acid Agonists; Excitatory Postsynaptic Potentials; Furans; gamma-Aminobutyric Acid; Gene Expression Regulation; Genes, Immediate-Early; Glutamic Acid; Glycerides; Hippocampus; In Vitro Techniques; Kainic Acid; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Mitogen-Activated Protein Kinases; Mutation; Neurons; Neuroprotective Agents; Piperidines; Polyunsaturated Alkamides; Prosencephalon; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Signal Transduction

The endogenous cannabinoids N-arachidonylethanolamide (AEA) and 2-arachidonylglycerol (2-AG) are known to decrease intraocular pressure (IOP). Recently, a novel putative endogenous cannabinoid, noladin ether, was isolated in porcine and rat brains. In the present study, both the degradation of endogenous cannabinoids in ocular tissues and the effect on IOP of 2-AG and noladin ether were compared.. The rates of enzymatic degradation for AEA, 2-AG, and noladin ether were determined in bovine cornea and iris-ciliary body homogenates. 2-AG and noladin ether were dissolved in either hydroxypropyl-beta-cyclodextrin (HP-beta-CD) or propylene glycol and administered unilaterally to the rabbit eye. IOPs were measured in the treated and untreated eyes. The CB1 receptor antagonist AM251 was administered topically 15 minutes before the cannabinoids to investigate whether CB1 receptors mediate the effect on IOP produced by 2-AG and noladin ether.. Noladin ether degraded more slowly than either 2-AG or AEA in the iris-ciliary body and cornea homogenates. The effect on IOP of 2-AG was biphasic (i.e., an initial increase in IOP followed by a reduction in the treated eye). Noladin ether decreased IOP immediately after topical administration, and no initial IOP increase was observed in the treated eye. The CB1 receptor antagonist AM251 (25 micro g) blocked the effect on IOP of noladin ether but did not affect the action of 2-AG.. Topical administration of the novel putative endogenous cannabinoid noladin ether decreased IOP in rabbits. This IOP reduction was most probably mediated through the CB1 receptor. The effect on IOP of noladin ether differed from those of the known endogenous cannabinoids AEA and 2-AG, probably because of its more stable chemical structure.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Cattle; Ciliary Body; Cornea; Endocannabinoids; Enzyme Stability; Female; Glycerides; Intraocular Pressure; Iris; Male; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rabbits; Receptors, Cannabinoid; Receptors, Drug

Cannabinoids, including the endogenous cannabinoid or endocannabinoid, anandamide, modulate several gastrointestinal functions. To date, the gastrointestinal effects of the second putative endocannabinoid 2-arachidonoylglycerol (2-AG) have not been studied. In the present study using a shrew (Cryptotis parva) emetic model, 2-AG (0.25-10 mg/kg, i.p.) potently and dose-dependently increased vomiting frequency (ED(50) = 1.13 mg/kg) and the number of animals vomiting (ED(50) = 0.48 mg/kg). In contrast, neither anandamide (2.5-20 mg/kg) nor methanandamide (5-10 mg/kg) induced a dose-dependent emetogenic response, but both could partially block the induced emetic effects. Delta(9)-Tetrahydrocannabinol and its synthetic analogs reduced 2-AG-induced vomiting with the rank order potency: CP 55,940 > WIN 55,212-2 > Delta(9)-tetrahydrocannabinol. The nonpsychoactive cannabinoid, cannabidiol, was inactive. Nonemetic doses of SR 141716A (1-5 mg/kg) also blocked 2-AG-induced vomiting. The 2-AG metabolite arachidonic acid also caused vomiting. Indomethacin, a cyclooxygenase inhibitor, blocked the emetogenic effects of both arachidonic acid and 2-AG. CP 55,940 also blocked the emetic effects of arachidonic acid. 2-AG (0.25-10 mg/kg) reduced spontaneous locomotor activity (ED(50) = 11 mg/kg) and rearing frequency (ED(50) = 4.3 mg/kg) in the shrew, whereas such doses of both anandamide and methanandamide had no effect on locomotor parameters. The present study indicates that: 1) 2-AG is an efficacious endogenous emetogenic cannabinoid involved in vomiting circuits, 2) the emetic action of 2-AG and the antiemetic effects of tested cannabinoids are mediated via CB(1) receptors, and 3) the emetic effects of 2-AG occur in lower doses relative to its locomotor suppressant actions.

Topics: Animals; Antiemetics; Arachidonic Acids; Cannabinoid Receptor Modulators; Cannabinoids; Cyclohexanols; Dose-Response Relationship, Drug; Dronabinol; Emetics; Endocannabinoids; Female; Glycerides; Humans; Male; Motor Activity; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Shrews

Spasticity is a complicating sign in multiple sclerosis that also develops in a model of chronic relapsing experimental autoimmune encephalomyelitis (CREAE) in mice. In areas associated with nerve damage, increased levels of the endocannabinoids, anandamide (arachidonoylethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG), and of the AEA congener, palmitoylethanolamide (PEA), were detected here, whereas comparable levels of these compounds were found in normal and non-spastic CREAE mice. While exogenously administered endocannabinoids and PEA ameliorate spasticity, selective inhibitors of endocannabinoid re-uptake and hydrolysis-probably through the enhancement of endogenous levels of AEA, and, possibly, 2-arachidonoyl glycerol-significantly ameliorated spasticity to an extent comparable with that observed previously with potent cannabinoid receptor agonists. These studies provide definitive evidence for the tonic control of spasticity by the endocannabinoid system and open new horizons to therapy of multiple sclerosis, and other neuromuscular diseases, based on agents modulating endocannabinoid levels and action, which exhibit little psychotropic activity.

Topics: Amides; Animals; Arachidonic Acids; Brain; Cannabinoid Receptor Modulators; Cannabinoids; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Endocannabinoids; Ethanolamines; Glycerides; Humans; Mice; Mice, Inbred Strains; Multiple Sclerosis; Palmitic Acids; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Spasm; Spinal Cord

The endocannabinoid 2-arachidonoylglycerol (2-Delta(4)Ach-Gro) activates human platelets in platelet-rich plasma at physiological concentrations. The activation was inhibited by selective antagonists of CB(1) and CB(2) cannabinoid receptors, but not by acetylsalicylic acid. Human platelets can metabolize 2-Delta(4)Ach-Gro by internalization through a high affinity transporter (K(m) = 300 +/- 30 nM, V(max) = 10 +/- 1 pmol.min(-1).mg protein(-1)), followed by hydrolysis by a fatty acid amide hydrolase (K(m) = 8 +/- 1 microM, V(max) = 400 +/- 50 pmol.min(-1).mg protein(-1)). The anandamide transport inhibitor AM404, and anandamide itself, were ineffective on 2-Delta(4)Ach-Gro uptake by platelets, whereas anandamide competitively inhibited 2-Delta(4)Ach-Gro hydrolysis (inhibition constant = 10 +/- 1 microM). Platelet activation by 2-Delta(4)Ach-Gro was paralleled by an increase of intracellular calcium and inositol-1,4,5-trisphosphate, and by a decrease of cyclic AMP. Moreover, treatment of preloaded platelet-rich plasma with 2-Delta(4)Ach-Gro induced an approximately threefold increase in [(3)H]2-Delta(4)Ach-Gro release, according to a CB receptor-dependent mechanism. On the other hand, ADP and collagen counteracted the activation of platelets by 2-Delta(4)Ach-Gro, whereas 5-hydroxytryptamine (serotonin) enhanced and extended its effects. Remarkably, ADP and collagen also reduced [(3)H]2-Delta(4)Ach-Gro release from 2-Delta(4)Ach-Gro-activated platelets, whereas 5-hydroxytryptamine further increased it. These findings suggest a so far unnoticed interplay between the peripheral endocannabinoid system and physiological platelet agonists.

Topics: Adenosine Diphosphate; Amidohydrolases; Arachidonic Acids; Aspirin; Biological Transport; Blood Platelets; Calcium Channel Blockers; Camphanes; Cannabinoid Receptor Modulators; Collagen; Cyclic AMP; Endocannabinoids; Glycerides; Humans; Hydrolysis; Inositol 1,4,5-Trisphosphate; Kinetics; Piperidines; Platelet Activation; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Serotonin; Time Factors

We investigated whether 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, is involved in acetylcholine- and calcium ionophore A23187-induced relaxations in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME) and indomethacin, which is considered to be mediated by endothelium-derived hyperpolarizing factor (EDHF). In rabbit mesenteric arterial rings pre-constricted with noradrenaline, 2-arachidonoylglycerol caused concentration-dependent relaxation. The 2-arachidonoylglycerol-induced relaxations were not affected by endothelium removal. N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-caroxamide (SR141716A) and 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morholinyl-1H-pyrazole-3-carboxamide (AM281), cannabinoid CB(1) receptor antagonists, significantly attenuated 2-arachidonoylglycerol-induced relaxation and the acetylcholine-induced relaxation only slightly, but not the calcium ionophore A23187-induced relaxation. On the other hand, charybdotoxin plus apamin, K(+) channel blockers, significantly attenuated acetylcholine and calcium ionohore A23187-induced relaxations but not 2-arachidonoylglycerol-induced relaxations. These results suggest that 2-arachidonoylglycerol can cause relaxations via cannabinoid CB(1) receptors, but is not involved in EDHF-mediated relaxations.

Topics: Animals; Arachidonic Acids; Biological Factors; Calcimycin; Calcium Channel Blockers; Endocannabinoids; Glycerides; Ionophores; Male; Neurotransmitter Agents; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rabbits; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Vasodilation

Marijuana affects brain function primarily by activating the G-protein-coupled cannabinoid receptor-1 (CB1), which is expressed throughout the brain at high levels. Two endogenous lipids, anandamide and 2-arachidonylglycerol (2-AG), have been identified as CB1 ligands. Depolarized hippocampal neurons rapidly release both anandamide and 2-AG in a Ca2+-dependent manner. In the hippocampus, CB1 is expressed mainly by GABA (gamma-aminobutyric acid)-mediated inhibitory interneurons, where CB1 clusters on the axon terminal. A synthetic CB1 agonist depresses GABA release from hippocampal slices. These findings indicate that the function of endogenous cannabinoids released by depolarized hippocampal neurons might be to downregulate GABA release. Here we show that the transient suppression of GABA-mediated transmission that follows depolarization of hippocampal pyramidal neurons is mediated by retrograde signalling through release of endogenous cannabinoids. Signalling by the endocannabinoid system thus represents a mechanism by which neurons can communicate backwards across synapses to modulate their inputs.

Topics: Animals; Arachidonic Acids; Benzoxazines; Cannabinoid Receptor Modulators; Cannabinoids; Endocannabinoids; gamma-Aminobutyric Acid; Glycerides; Hippocampus; In Vitro Techniques; Morpholines; Naphthalenes; Neural Inhibition; Piperidines; Pyramidal Cells; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Receptors, Drug; Receptors, Metabotropic Glutamate; Rimonabant; Signal Transduction; Synapses; Synaptic Transmission

Leptin is the primary signal through which the hypothalamus senses nutritional state and modulates food intake and energy balance. Leptin reduces food intake by upregulating anorexigenic (appetite-reducing) neuropeptides, such as alpha-melanocyte-stimulating hormone, and downregulating orexigenic (appetite-stimulating) factors, primarily neuropeptide Y. Genetic defects in anorexigenic signalling, such as mutations in the melanocortin-4 (ref. 5) or leptin receptors, cause obesity. However, alternative orexigenic pathways maintain food intake in mice deficient in neuropeptide Y. CB1 cannabinoid receptors and the endocannabinoids anandamide and 2-arachidonoyl glycerol are present in the hypothalamus, and marijuana and anandamide stimulate food intake. Here we show that following temporary food restriction, CB1 receptor knockout mice eat less than their wild-type littermates, and the CB1 antagonist SR141716A reduces food intake in wild-type but not knockout mice. Furthermore, defective leptin signalling is associated with elevated hypothalamic, but not cerebellar, levels of endocannabinoids in obese db/db and ob/ob mice and Zucker rats. Acute leptin treatment of normal rats and ob/ob mice reduces anandamide and 2-arachidonoyl glycerol in the hypothalamus. These findings indicate that endocannabinoids in the hypothalamus may tonically activate CB1 receptors to maintain food intake and form part of the neural circuitry regulated by leptin.

Topics: Animals; Appetite Regulation; Arachidonic Acids; Cannabinoid Receptor Modulators; Cannabinoids; Cerebellum; Eating; Endocannabinoids; Female; Food; Glycerides; Hypothalamus; Leptin; Lipoprotein Lipase; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Obese; Obesity; Phospholipase D; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Rats, Zucker; Receptors, Cannabinoid; Receptors, Drug; Receptors, Leptin; Rimonabant; Signal Transduction

Anandamide (10(-7) and 10(-6) M) as well as a synthetic cannabinoid HU210 (10(-8) to 10(-6) M) suppressed the norepinephrine release evoked by perivascular nerve stimulation (PNS) of the rat heart Langendorff's preparation. The effects of HU210 and the lower dose of anandamide were completely blocked by the cannabinoid CB1-receptor antagonist AM251, while that of anandamide at 10(-6) M was partly mediated by arachidonate-derived metabolites. 2-Arachidonoylglycerol (2-AG), at 10(-6) M in the presence of DFP and indomethacin, increased PNS-evoked norepinephrine release, which was completely blocked by AM251. The present results suggest that the two endocannabinoids may oppositely participate in the CB1-receptor-mediated modulation of sympathetic norepinephrine release.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Cannabinoids; Dronabinol; Endocannabinoids; Glycerides; Male; Myocardium; Neurotransmitter Agents; Norepinephrine; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptors, Cannabinoid; Receptors, Drug; Sympathetic Nervous System

The endocannabinoid anandamide [N-arachidonoylethanolamine (AEA)] is thought to function as an endogenous protective factor of the brain against acute neuronal damage. However, this has never been tested in an in vivo model of acute brain injury. Here, we show in a longitudinal pharmacological magnetic resonance imaging study that exogenously administered AEA dose-dependently reduced neuronal damage in neonatal rats injected intracerebrally with the Na(+)/K(+)-ATPase inhibitor ouabain. At 15 min after injury, AEA (10 mg/kg) administered 30 min before ouabain injection reduced the volume of cytotoxic edema by 43 +/- 15% in a manner insensitive to the cannabinoid CB(1) receptor antagonist SR141716A. At 7 d after ouabain treatment, 64 +/- 24% less neuronal damage was observed in AEA-treated (10 mg/kg) rats compared with control animals. Coadministration of SR141716A prevented the neuroprotective actions of AEA at this end point. In addition, (1) no increase in AEA and 2-arachidonoylglycerol levels was detected at 2, 8, or 24 hr after ouabain injection; (2) application of SR141716A alone did not increase the lesion volume at days 0 and 7; and (3) the AEA-uptake inhibitor, VDM11, did not affect the lesion volume. These data indicate that there was no endogenous endocannabinoid tone controlling the acute neuronal damage induced by ouabain. Although our data seem to question a possible role of the endogenous cannabinoid system in establishing a brain defense system in our model, AEA may be used as a structural template to develop neuroprotective agents.

Topics: Animals; Animals, Newborn; Arachidonic Acids; Blotting, Western; Brain; Brain Edema; Brain Injuries; Cannabinoid Receptor Modulators; Cannabinoids; Disease Models, Animal; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Glycerides; Longitudinal Studies; Magnetic Resonance Imaging; Microinjections; Neurons; Ouabain; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

Anandamide and 2-arachidonoylglycerol (2-AG), two endogenous ligands of the CB1 and CB2 cannabinoid receptor subtypes, inhibit the proliferation of PRL-responsive human breast cancer cells (HBCCs) through down-regulation of the long form of the PRL receptor (PRLr). Here we report that 1) anandamide and 2-AG inhibit the nerve growth factor (NGF)-induced proliferation of HBCCs through suppression of the levels of NGF Trk receptors; 2) inhibition of PRLr levels results in inhibition of the proliferation of other PRL-responsive cells, the prostate cancer DU-145 cell line; and 3) CB1-like cannabinoid receptors are expressed in HBCCs and DU-145 cells and mediate the inhibition of cell proliferation and Trk/PRLr expression. Beta-NGF-induced HBCC proliferation was potently inhibited (IC50 = 50-600 nM) by the synthetic cannabinoid HU-210, 2-AG, anandamide, and its metabolically stable analogs, but not by the anandamide congener, palmitoylethanolamide, or the selective agonist of CB2 cannabinoid receptors, BML-190. The effect of anandamide was blocked by the CB1 receptor antagonist, SR141716A, but not by the CB2 receptor antagonist, SR144528. Anandamide and HU-210 exerted a strong inhibition of the levels of NGF Trk receptors as detected by Western immunoblotting; this effect was reversed by SR141716A. When induced by exogenous PRL, the proliferation of prostate DU-145 cells was potently inhibited (IC50 = 100-300 nM) by anandamide, 2-AG, and HU-210. Anandamide also down-regulated the levels of PRLr in DU-145 cells. SR141716A attenuated these two effects of anandamide. HBCCs and DU-145 cells were shown to contain 1) transcripts for CB1 and, to a lesser extent, CB2 cannabinoid receptors, 2) specific binding sites for [3H]SR141716A that could be displaced by anandamide, and 3) a CB1 receptor-immunoreactive protein. These findings suggest that endogenous cannabinoids and CB1 receptor agonists are potential negative effectors of PRL- and NGF-induced biological responses, at least in some cancer cells.

Topics: Arachidonic Acids; Binding Sites; Blotting, Western; Breast Neoplasms; Cannabinoid Receptor Modulators; Cannabinoids; Cell Division; Endocannabinoids; Female; Glycerides; Humans; Male; Neoplasms, Hormone-Dependent; Nerve Growth Factors; Piperidines; Polyunsaturated Alkamides; Prostatic Neoplasms; Pyrazoles; Receptor Protein-Tyrosine Kinases; Receptors, Cannabinoid; Receptors, Drug; Receptors, Nerve Growth Factor; Receptors, Prolactin; Rimonabant; Tumor Cells, Cultured

We examined the effect of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, on the intracellular free Ca(2+) concentrations in HL-60 cells that express the cannabinoid CB2 receptor. We found that 2-arachidonoylglycerol induces a rapid transient increase in intracellular free Ca(2+) concentrations in HL-60 cells. The response was affected by neither cyclooxygenase inhibitors nor lipoxygenase inhibitors, suggesting that arachidonic acid metabolites are not involved. Consistent with this notion, free arachidonic acid was devoid of any agonistic activity. Importantly, the Ca(2+) transient induced by 2-arachidonoylglycerol was blocked by pretreatment of the cells with SR144528, a CB2 receptor-specific antagonist, but not with SR141716A, a CB1 receptor-specific antagonist, indicating the involvement of the CB2 receptor but not the CB1 receptor in this cellular response. G(i) or G(o) is also assumed to be involved, because pertussis toxin treatment of the cells abolished the response. We further examined the structure-activity relationship. We found that 2-arachidonoylglycerol is the most potent compound among a number of naturally occurring cannabimimetic molecules. Interestingly, anandamide and N-palmitoylethanolamine, other putative endogenous ligands, were found to be a weak partial agonist and an inactive ligand, respectively. These results strongly suggest that the CB2 receptor is originally a 2-arachidonoylglycerol receptor, and 2-arachidonoylglycerol is the intrinsic natural ligand for the CB2 receptor that is abundant in the immune system.

Topics: Amides; Arachidonic Acids; Calcium Signaling; Camphanes; Cannabinoids; Cyclohexanols; Cyclooxygenase Inhibitors; Drug Interactions; Endocannabinoids; Ethanolamines; Glycerides; HL-60 Cells; Humans; Ligands; Lipoxygenase Inhibitors; Molecular Mimicry; Palmitic Acids; Pertussis Toxin; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; RNA, Messenger; Structure-Activity Relationship; Virulence Factors, Bordetella

Cannabinoids, including the endogenous ligand anandamide, elicit pronounced hypotension and bradycardia through the activation of CB1 cannabinoid receptors. A second endogenous cannabinoid, 2-arachidonoyl glycerol (2-AG), has been proposed to be the natural ligand of CB1 receptors. In the present study, we examined the effects of 2-AG on mean arterial pressure and heart rate in anesthetized mice and assessed the role of CB1 receptors through the use of selective cannabinoid receptor antagonists and CB1 receptor knockout (CB1(-/-)) mice. In control ICR mice, intravenous injections of 2-AG or its isomer 1-AG elicit dose-dependent hypotension and moderate tachycardia that are unaffected by the CB1 receptor antagonist SR141716A. The same dose of SR141716A (6 nmol/g IV) completely blocks the hypotensive effect and attenuates the bradycardic effect of anandamide. 2-AG elicits a similar hypotensive effect, resistant to blockade by either SR141716A or the CB2 antagonist SR144528, in both CB1(-/-) mice and their homozygous (CB1(+/+)) control littermates. In ICR mice, arachidonic acid (AA, 15 nmol/g IV) elicits hypotension and tachycardia, and indomethacin (14 nmol/g IV) inhibits the hypotensive effect of both AA and 2-AG. Synthetic 2-AG incubated with mouse blood is rapidly (<2 minutes) and completely degraded with the parallel appearance of AA, whereas anandamide is stable under the same conditions. A metabolically stable ether analogue of 2-AG causes prolonged hypotension and bradycardia in ICR mice, and both effects are completely blocked by SR141716A, whereas the same dose of 2-AG-ether does not influence blood pressure and heart rate in CB1(-/-) mice. These findings are interpreted to indicate that exogenous 2-AG is rapidly degraded in mouse blood, probably by a lipase, which masks its ability to interact with CB1 receptors. Although the observed cardiovascular effects of 2-AG probably are produced by an arachidonate metabolite through a noncannabinoid mechanism, the CB1 receptor-mediated cardiovascular effects of a stable analogue of 2-AG leaves open the possibility that endogenous 2-AG may elicit cardiovascular effects through CB1 receptors.

Topics: Anesthesia; Animals; Arachidonic Acids; Blood Pressure; Camphanes; Cardiovascular Agents; Cardiovascular Diseases; Dose-Response Relationship, Drug; Endocannabinoids; Female; Glycerides; Heart Rate; Hypotension; Indomethacin; Ligands; Male; Mice; Mice, Inbred ICR; Mice, Knockout; Piperidines; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Tachycardia

The monoacylglycerol 2-arachidonylglycerol is an endogenous ligand of cannabinoid receptors. We examined whether 2-arachidonylglycerol can influence excessive neuronal activity by investigating stimulation-induced population spikes and epileptiform activity in rat hippocampal slices. For this purpose, the effects of 2-arachidonylglycerol were compared with those of the synthetic cannabinoid agonist WIN 55,212-2. At concentrations of 10-50 microM, 2-arachidonylglycerol attenuated the amplitude of the orthodromic population spike and the slope of the field excitatory postsynaptic potential (field EPSP). However, the effect of the synthetic cannabinoid WIN 55,212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolol[ 1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone; 0.1 microM and 1 microM) was significantly higher than that of the endogenous ligand. At a concentration of 1 microM, WIN 55,212-2 completely suppressed the field EPSP. However, none of the investigated compounds did affect the presynaptic fiber spike of the afferents. The CB1 receptor antagonist SR 141716 (N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorphenyl)-4-methyl-3- pyrazole-carboxamide) blocked the inhibition evoked by the cannabinoids. Both 2-arachidonylglycerol (30 microM) and WIN 55,212-2 (100 nM) shifted the input-output curve of the postsynaptic spike and the field EPSP to the right and increased the magnitude of paired-pulse facilitation, indicating a presynaptic mechanism of action. 2-Arachidonylglycerol and WIN 55,212-2 attenuated the frequency of spontaneously occurring epileptiform burst discharges in CA3 elicited by omission of Mg2+ and elevation of K+ to 8 mM. The antiepileptiform effect of these cannabinoids was blocked by SR 141716. In conclusion, 2-arachidonylglycerol seems to limit neuronal excitability via cannabinoid receptors of the CB1 type. By acting predominantly at a presynaptic site, it is capable of reducing excitatory neurotransmission, a mechanism which might be involved in the prevention of excessive excitability leading to epileptiform activity.

Topics: Analgesics; Animals; Arachidonic Acids; Benzoxazines; Cannabinoids; Electric Stimulation; Endocannabinoids; Excitatory Postsynaptic Potentials; Glycerides; Hippocampus; Male; Morpholines; Naphthalenes; Neurons; Neurotransmitter Agents; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptors, Drug; Rimonabant

In recent years, cannabinoid receptors and their endogenous ligands (endocannabinoids) have been identified within the brain. The high density of CB1 cannabinoid receptors within the basal ganglia suggests a potential role for endocannabinoids in the control of voluntary movement and in basal ganglia-related movement disorders such as Parkinson's disease. However, whether endocannabinoids play a role in regulating motor behavior in health and disease is unknown. Here we report the presence in two regions of the basal ganglia, the globus pallidus and substantia nigra, of the endocannabinoids 2-arachidonoylglycerol (2AG) and anandamide. The levels of the latter compound are approximately threefold higher than those previously reported in any other brain region. In the reserpine-treated rat, an animal model of Parkinson's disease, suppression of locomotion is accompanied by a sevenfold increase in the levels of the 2AG in the globus pallidus, but not in the other five brain regions analyzed. Stimulation of locomotion in the reserpine-treated rat by either of the two selective agonists of D2 and D1 dopamine receptors, quinpirole and R-(+/-)-3-allyl-6-chloro-7, 8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (Cl-APB), respectively, results in the reduction of both anandamide and 2AG levels in the globus pallidus. Finally, full restoration of locomotion in the reserpine-treated rat is obtained by coadministration of quinpirole and the selective antagonist of the cannabinoid CB1 receptor subtype, SR141716A. These findings indicate a link between endocannabinoid signaling in the globus pallidus and symptoms of Parkinson's disease in the reserpine-treated rat, and suggest that modulation of the endocannabinoid signaling system might prove useful in treating this or other basal ganglia-related movement disorders.

Topics: Animals; Arachidonic Acids; Benzazepines; Cannabinoid Receptor Modulators; Cannabinoids; Dopamine Agonists; Endocannabinoids; Globus Pallidus; Glycerides; Humans; Male; Motor Activity; Parkinsonian Disorders; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Quinpirole; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Receptors, Drug; Reserpine; Rimonabant; Substantia Nigra; Tissue Distribution

Cannabinoids, including the endogenous ligand anandamide (arachidonyl ethanolamide), elicit pronounced hypotension in rats via activation of peripherally located CB1 cannabinoid receptors, which have been also implicated in endotoxin (lipopolysaccharide [LPS])-induced hypotension. The present study was designed to test the role of vascular CB1 receptors in cannabinoid- and endotoxin-induced mesenteric vasodilation. In the isolated, buffer-perfused rat mesenteric arterial bed precontracted with phenylephrine, anandamide induced long-lasting (up to 60 minutes) dose-dependent vasodilation (ED50: 79+/-3 nmol; maximal relaxation: 77+/-2%), inhibited by 0.5 to 5.0 micromol/L of the selective CB1 receptor antagonist SR141716A. Low doses of the calcium ionophore ionomycin also caused mesenteric vasodilation inhibited by SR141716A. The metabolically stable analogue R-methanandamide elicited mesenteric vasodilation (ED50: 286+/-29 nmol), whereas the potent synthetic CB1 receptor agonists WIN 55212-2 and HU-210 caused no change in vascular tone or only a minor dilator effect not affected by SR141716A, respectively. The endogenous ligand 2-arachidonyl glycerol caused no change in vascular tone, whereas Delta9-tetrahydrocannabinol and arachidonic acid caused mesenteric vasoconstriction. After endothelial denudation, the dilator response to anandamide was slightly reduced and was no longer inhibited by SR141716A. In preparations from LPS-pretreated rats, SR141716A alone caused a significant and prolonged increase in perfusion pressure, whereas it had no such effect in control preparations perfused in vitro with or without LPS or after endothelial denudation in preparations from rats pretreated with LPS. We conclude that anandamide-induced mesenteric vasodilation is mediated by an endothelially located SR141716A-sensitive "anandamide receptor" distinct from CB1 cannabinoid receptors and that activation of such receptors by an endocannabinoid, possibly anandamide, contributes to LPS-induced mesenteric vasodilation in vivo.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcium Channel Blockers; Cannabinoid Receptor Modulators; Dronabinol; Endocannabinoids; Endothelium, Vascular; Glycerides; Ligands; Male; Mesenteric Arteries; Muscle, Smooth, Vascular; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptors, Drug; Rimonabant; Vasoconstriction; Vasodilation

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

We examined the relative importance of G (Gi) protein-coupled brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors in preimplantation embryo development using agonists and antagonists specific to CB1-R and CB2-R. The results establish that endogenous cannabinoid ligands, anandamide and sn-2 arachidonoylglycerol, arrest embryo development in vitro, and this effect is reversed by CB1-R antagonists SR141716A or AM 251, but not by SR144528, a CB2-R antagonist. A CB2-R selective agonist AM 663 failed to affect embryo development. These results suggest that cannabinoid effects on embryo development are mediated by CB1-R. We also observed that delta9-tetrahydrocannabinol ([-]THC) infused in the presence of cytochrome P450 inhibitors interfered with blastocyst implantation. This adverse effect was reversed by coinfusion of SR141716A. The less active stereoisomer (+)THC plus the inhibitors failed to affect implantation. Analysis of tissue levels demonstrated that uterine accumulation of (-)THC occurred when it was infused in the presence of the P450 inhibitors. These results demonstrate that the uterus and perhaps the embryo have the cytochrome P450 enzymes to metabolize (-)THC and neutralize its adverse effects on implantation. Collectively, the present study demonstrates that cannabinoid effects on embryo development and implantation are mediated by embryonic and/or uterine CB1-R, but not CB2-R.

Topics: Animals; Arachidonic Acids; Brain; Cannabinoids; Cytochrome P-450 Enzyme Inhibitors; Dronabinol; Embryo Implantation; Embryo, Mammalian; Embryonic and Fetal Development; Embryonic Development; Endocannabinoids; Enzyme Inhibitors; Female; Glycerides; Male; Mice; Piperidines; Polyunsaturated Alkamides; Pregnancy; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

Low concentrations of 2-arachidonoylglycerol were found to induce rapid, transient elevation of intracellular free Ca2+ in NG108-15 cells (EC50 was 150 nM). Free arachidonic acid, 2-palmitoylglycerol, 2-oleoylglycerol, 2-linoleoylglycerol and 2-docosahexaenoylglycerol were inactive. Anandamide acted as a partial agonist. Importantly, desensitization was observed upon sequential challenge with 2-arachidonoylglycerol. Furthermore, cross-desensitization was observed between 2-arachidonoylglycerol and WIN 55212-2, a cannabinoid receptor agonist. Pretreatment of the cells with SR141716A, a cannabinoid receptor antagonist, abolished the activities of both 2-arachidonoylglycerol and WIN 55212-2. These results strongly suggest that 2-arachidonoylglycerol and WIN 55212-2 bind to a common cannabinoid receptor to elicit cellular responses and that 2-arachidonoylglycerol has some physiological role in nervous tissues.

Topics: Arachidonic Acids; Benzoxazines; Calcium; Cannabinoids; Dose-Response Relationship, Drug; Drug Interactions; Endocannabinoids; Glioma; Glycerides; Hybrid Cells; Ligands; Morpholines; Naphthalenes; Neuroblastoma; Neurons; Piperidines; Platelet Activating Factor; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant