urb-597 and glyceryl-2-arachidonate

Neuropathic pain refers to chronic pain that results from injury to the nervous system. The mechanisms involved in neuropathic pain are complex and involve both peripheral and central phenomena. Although numerous pharmacological agents are available for the treatment of neuropathic pain, definitive drug therapy has remained elusive. Recent drug discovery efforts have identified an original neurobiological approach to the pathophysiology of neuropathic pain. The development of innovative pharmacological strategies has led to the identification of new promising pharmacological targets, including glutamate antagonists, microglia inhibitors and, interestingly, endogenous ligands of cannabinoids and the transient receptor potential vanilloid type 1 (TRPV1). Endocannabinoids (ECs), endovanilloids and the enzymes that regulate their metabolism represent promising pharmacological targets for the development of a successful pain treatment. This review is an update of the relationship between cannabinoid receptors (CB1) and TRPV1 channels and their possible implications for neuropathic pain. The data are focused on endogenous spinal mechanisms of pain control by anandamide, and the current and emerging pharmacotherapeutic approaches that benefit from the pharmacological modulation of spinal EC and/or endovanilloid systems under chronic pain conditions will be discussed.

Topics: Amides; Amidohydrolases; Animals; Arachidonic Acid; Arachidonic Acids; Behavior; Benzamides; Carbamates; Endocannabinoids; Enzyme Inhibitors; Ethanolamines; Glycerides; Humans; Microglia; Neuralgia; Palmitic Acids; Peripheral Nerve Injuries; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Spinal Cord Injuries; TRPV Cation Channels

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

Neuropathic pain is often associated with depression. Enhancing endocannabinoids by fatty acid amide hydrolase (FAAH) inhibitors relieves neuropathic pain and stress-induced depressive-like behaviors in animal models. However, it is unclear whether FAAH inhibitor can relieve neuropathic pain-induced depression by or not by its antinociceptive effects.. Adult male Wistar rats with chronic constriction injury (CCI) to the sciatic nerve were treated with the systemic FAAH inhibitor URB597 (5.8 mg·kg·day, intraperitoneally) or peripherally acting FAAH inhibitor URB937 (1.6 mg·kg·d, intraperitoneally; n = 11-12). The treatment was applied from the 15th day after surgery and continued for 15 days. Mechanical withdrawal threshold was examined by Von Frey test before surgery and on the 28th day after CCI. Depressive-like behaviors were evaluated by forced swimming test (FST) and novelty-suppressed feeding (NSF) after 15-day treatment. The levels of anandamide and 2-arachidonoylglycerol in hippocampus were examined by liquid chromatography and mass spectrometry. Hippocampal neurogenesis including proliferation, differentiation, and survival of newborn cells was assessed by immunohistochemistry.. After CCI injury, the rats developed significantly nociceptive and depressive-like behaviors, indicated by persistent mechanical hypersensitivity in Von Frey test, significantly prolonged immobility time in FST (sham: 84.2 ± 13.4 seconds versus CCI: 137.9 ± 18.8 seconds; P < .001), and protracted latency to feed in NSF (sham: 133.4 ± 19.4 seconds versus CCI: 234.9 ± 33.5 seconds; P < .001). For the CCI rats receiving treatment, compared to vehicle placebo group, pain threshold was increased by both URB597 (3.1 ± 1.0 vs 11.2 ± 1.2 g; P < .001) and URB937 (3.1 ± 1.0 vs 12.1 ± 1.3 g; P < .001). Immobility time of FST was reduced by URB597 (135.8 ± 16.6 vs 85.3 ± 17.2 seconds; P < .001) but not by URB937 (135.8 ± 16.6 vs 129.6 ± 17.8 seconds; P = .78). Latency to feed in NSF was also reduced by URB597 (235.9 ± 30.5 vs 131.8 ± 19.8 seconds; P < .001) but not by URB937 (235.9 ± 30.5 vs 232.2 ± 33.2 seconds; P = .72). Meanwhile, CCI decreased the number of proliferating cells and reduced survival of new mature neurons in hippocampus. URB597 but not URB937 treatment improved these cellular deficits.. Inhibition of FAAH can improve depressive-like behaviors induced by neuropathic pain independent of its peripheral antinociceptive action. Enhanced neurogenesis in hippocampus might contribute to the antidepressive effects of URB597.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Behavior, Animal; Benzamides; Carbamates; Depression; Disease Models, Animal; Endocannabinoids; Enzyme Inhibitors; Feeding Behavior; Glycerides; Hippocampus; Locomotion; Male; Neuralgia; Neurogenesis; Pain Threshold; Polyunsaturated Alkamides; Rats, Wistar; Receptor, Cannabinoid, CB1; Signal Transduction; Swimming

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

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

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 interaction between the endocannabinoid and ROS signaling systems has been demonstrated in different organs. Inhibitors of fatty acid amide hydrolase (FAAH), the key enzyme responsible for degradation of the endocannabinoid anandamide, are postulated to possess anti-hypertensive potential. Here, we compared the effects of hypertension and chronic FAAH inhibition by URB597 on the endocannabinoid system and redox balance in spontaneously hypertensive rats (SHR) and hypertensive deoxycorticosterone acetate (DOCA)-salt rats. Enhanced oxidative stress and lipid peroxidation were found in both hypertension models. Hypertension affected cardiac and plasma endocannabinoid systems in a model-dependent manner: anandamide and 2-arachidonoylglycerol levels decreased in SHR and increased in DOCA-salt. Cardiac CB

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Carbamates; Desoxycorticosterone Acetate; Endocannabinoids; Glycerides; Heart; Hypertension; Male; Myocardium; Oxidation-Reduction; Oxidative Stress; Polyunsaturated Alkamides; Rats, Inbred SHR; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2

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

Systemic activation of cannabinoid receptors often induces biphasic effects on emotional memory and anxiety depending on the levels of emotional arousal associated to the experimental context. The basolateral nucleus of the amygdala (BLA) represents a crucial structure for the ability of endocannabinoid (eCB) signaling to modulate emotional behaviour, and receives dense projections from brainstem arousal system nuclei. We examined whether changes in emotional arousal state would influence the ability of acute eCB manipulations within the BLA to modulate anxiety. Rats were tested in an elevated plus maze (EPM) under low or high arousal conditions. The low emotional arousal group was extensively handled and habituated to the experimental room and tested under red light condition, the high emotional arousal group was not handled or habituated and tested under high light condition. We examined amygdalar eCB anandamide (AEA) and 2-arachidonoylglycerol (2-AG) levels immediately after the EPM and the effects of intra-BLA administration of the AEA hydrolysis inhibitor URB597 or the 2-AG hydrolysis inhibitor KML29 on anxiety behaviour. The modulation of anxiety-like behaviour by eCBs in the BLA was strictly dependent on the environmental-associated emotional arousal. Pharmacologically-induced elevations of AEA or 2-AG in the BLA decreased anxiety under conditions of low emotional arousal. Conversely, when the level of emotional arousal increased, local eCB manipulation was ineffective in the modulation of the emotional arousal-induced anxiety response. These findings suggest that, depending on the emotional arousal state, eCB system is differentially activated to regulate the anxiety response in the amygdala and help to understand the state-dependency of many interventions on anxiety.

Topics: Animals; Anxiety; Arachidonic Acids; Arousal; Basolateral Nuclear Complex; Benzamides; Cannabinoid Receptor Agonists; Carbamates; Corticosterone; Emotions; Endocannabinoids; Epinephrine; Glycerides; Male; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Signal Transduction

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

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

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

Variations in environmental aversiveness influence emotional memory processes in rats. We have previously shown that cannabinoid effects on memory are dependent on the stress level at the time of training as well as on the aversiveness of the environmental context. Here, we investigated whether the hippocampal endocannabinoid system modulates memory retrieval depending on the training-associated arousal level. Male adult Sprague Dawley rats were trained on a water maze spatial task at two different water temperatures (19°C and 25°C) to elicit either higher or lower stress levels, respectively. Rats trained under the higher stress condition had better memory and higher corticosterone concentrations than rats trained at the lower stress condition. The cannabinoid receptor agonist WIN55212-2 (10-30 ng/side), the 2-arachidonoyl glycerol (2-AG) hydrolysis inhibitor JZL184 (0.1-1 μg/side), and the anandamide (AEA) hydrolysis inhibitor URB597 (10-30 ng/side) were administered bilaterally into the hippocampus 60 min before probe-trial retention testing. WIN55212-2 or JZL184, but not URB597, impaired probe-trial performances only of rats trained at the higher stressful condition. Furthermore, rats trained under higher stress levels displayed an increase in hippocampal 2-AG, but not AEA, levels at the time of retention testing and a decreased affinity of the main 2-AG-degrading enzyme for its substrate. The present findings indicate that the endocannabinoid 2-AG in the hippocampus plays a key role in the selective regulation of spatial memory retrieval of stressful experience, shedding light on the neurobiological mechanisms involved in the impact of stress effects on memory processing.. Endogenous cannabinoids play a central role in the modulation of memory for emotional events. Here we demonstrate that the endocannabinoid 2-arachidonoylglycerol in the hippocampus, a brain region crucially involved in the regulation of memory processes, selectively modulates spatial memory recall of stressful experiences. Thus, our findings provide evidence that the endocannabinoid 2-arachidonoylglycerol is a key player in mediating the impact of stress on memory retrieval. These findings can pave the way to new potential therapeutic intervention for the treatment of neuropsychiatric disorders, such as post-traumatic stress disorder, where a previous exposure to traumatic events could alter the response to traumatic memory recall leading to mental illness.

Topics: Animals; Arachidonic Acids; Arousal; Benzamides; Benzoxazines; Calcium Channel Blockers; Cannabinoid Receptor Agonists; Carbamates; Corticosterone; Dose-Response Relationship, Drug; Emotions; Endocannabinoids; Enzyme Inhibitors; Glycerides; Hippocampus; Male; Maze Learning; Mental Recall; Morpholines; Naphthalenes; Rats; Rats, Sprague-Dawley; Spatial Learning; Stress, Psychological; Temperature

In rodents, many exogenous and endogenous cannabinoids, such as anandamide (AEA) and 2-arachidonyl glycerol (2-AG), have been shown to play an important role in certain hippocampal memory processes. However, the mechanisms by which endogenous AEA regulate this processes are not well understood. Here the effects of AEA on long-term potentiation (LTP), hippocampal-dependent learning and memory tasks, pERK1/2, pCaMKIV, and pCREB signaling events in both cannabinoid receptor type 1 (CB1R) wild-type (WT) and knockout (KO) mice were assessed following administration of URB597, an inhibitor of the fatty acid amide hydrolase (FAAH). Acute administration of URB597 enhanced AEA levels without affecting the levels of 2-AG or CB1R in the hippocampus and neocortex as compared to vehicle. In hippocampal slices, URB597 impaired LTP in CB1R WT but not in KO littermates. URB597 impaired object recognition, spontaneous alternation and spatial memory in the Y-maze test in CB1R WT mice but not in KO mice. Furthermore, URB597 enhanced ERK phosphorylation in WT without affecting total ERK levels in WT or KO mice. URB597 impaired CaMKIV and CREB phosphorylation in WT but not in KO mice. CB1R KO mice have a lower pCaMKIV/CaMKIV ratio and higher pCREB/CREB ratio as compared to WT littermates. Our results indicate that pharmacologically elevated AEA impair LTP, learning and memory and inhibit CaMKIV and CREB phosphorylation, via the activation of CB1Rs. Collectively, these findings also suggest that pharmacological elevation of AEA beyond normal concentrations is also detrimental for the underlying physiological responses.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Calcium-Calmodulin-Dependent Protein Kinase Type 4; Carbamates; Cyclic AMP Response Element-Binding Protein; Endocannabinoids; Glycerides; Learning; Long-Term Potentiation; Male; MAP Kinase Signaling System; Maze Learning; Memory; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphorylation; Polyunsaturated Alkamides; Protein Processing, Post-Translational; Receptor, Cannabinoid, CB1; Spatial Memory

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

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

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

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

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

Previous studies have provided extensive evidence that administration of cannabinoid drugs after training modulates the consolidation of memory for an aversive experience. The present experiments investigated whether the memory consolidation is regulated by endogenously released cannabinoids. The experiments first examined whether the endocannabinoids anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) are released by aversive training. Inhibitory avoidance training with higher footshock intensity produced increased levels of AEA in the amygdala, hippocampus, and medial prefrontal cortex (mPFC) shortly after training in comparison with levels assessed in rats trained with lower footshock intensity or unshocked controls exposed only to the training apparatus. In contrast, 2-AG levels were not significantly elevated. The additional finding that posttraining infusions of the fatty acid amide hydrolase (FAAH) inhibitor URB597, which selectively increases AEA levels at active synapses, administered into the basolateral complex of the amygdala (BLA), hippocampus, or mPFC enhanced memory strongly suggests that the endogenously released AEA modulates memory consolidation. Moreover, in support of the view that this emotional training-associated increase in endocannabinoid neurotransmission, and its effects on memory enhancement, depends on the integrity of functional interactions between these different brain regions, we found that disruption of BLA activity blocked the training-induced increases in AEA levels as well as the memory enhancement produced by URB597 administered into the hippocampus or mPFC. Thus, the findings provide evidence that emotionally arousing training increases AEA levels within prefrontal-limbic circuits and strongly suggest that this cannabinoid activation regulates emotional arousal effects on memory consolidation.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Avoidance Learning; Benzamides; Carbamates; Emotions; Endocannabinoids; Glycerides; Limbic System; Memory; Polyunsaturated Alkamides; Prefrontal Cortex; Rats; Receptor, Cannabinoid, CB1

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

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

An additional class of endogenous lipid amides, N-arachidonoyl amino acids (Ara-AAs), is growing in significance in the field of endocannabinoids. The development, validation, and application of a sensitive and selective method to simultaneously monitor and quantify the level of Ara-AAs along with anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) in mouse brain has been established. The linearity of the method over the concentration ranges of 0.2-120 pg/μl for the standards of N-arachidonoyl amino acids, N-arachidonoyl alanine (NAAla), serine (NASer), γ-aminobutyric acid (NAGABA), and glycine (NAGly); 0.7-90 pg/μl for AEA-d(0)/d(8); and 7.5-950 pg/μl for 2-AG was determined with R(2) values of 0.99. Also the effects of the FAAH inhibitor URB 597 on the endogenous levels of these analytes were investigated. AEA and NASer brain levels exhibit a dose-dependent increase after systemic administration of URB 597, whereas NAGly and NAGABA were significantly decreased after treatment. NAAla and 2-AG were not altered after URB 597 treatment. The potential benefit of establishing this assay extends beyond the quantification of the Ara-AAs along with AEA and 2-AG in mouse brain, to reveal a variety of pharmacological effects and physiological roles of these analytes.

Topics: Amidohydrolases; Amino Acids; Animals; Arachidonic Acids; Benzamides; Brain; Carbamates; Chromatography, High Pressure Liquid; Endocannabinoids; Enzyme Inhibitors; gamma-Aminobutyric Acid; Glycerides; Mice; Polyunsaturated Alkamides; Tandem Mass Spectrometry

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

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

Aberrant Notch signaling has recently emerged as a possible mechanism for the altered neurogenesis, cognitive impairment, and learning and memory deficits associated with Alzheimer disease (AD). Recently, targeting the endocannabinoid system in models of AD has emerged as a potential approach to slow the progression of the disease process. Although studies have identified neuroprotective roles for endocannabinoids, there is a paucity of information on modulation of the pro-survival Notch pathway by endocannabinoids. In this study the influence of the endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol, on the Notch-1 pathway and on its endogenous regulators were investigated in an in vitro model of AD. We report that AEA up-regulates Notch-1 signaling in cultured neurons. We also provide evidence that although Aβ(1-42) increases expression of the endogenous inhibitor of Notch-1, numb (Nb), this can be prevented by AEA and 2-arachidonoylglycerol. Interestingly, AEA up-regulated Nct expression, a component of γ-secretase, and this was found to play a crucial role in the enhanced Notch-1 signaling mediated by AEA. The stimulatory effects of AEA on Notch-1 signaling persisted in the presence of Aβ(1-42). AEA was found to induce a preferential processing of Notch-1 over amyloid precursor protein to generate Aβ(1-40). Aging, a natural process of neurodegeneration, was associated with a reduction in Notch-1 signaling in rat cortex and hippocampus, and this was restored with chronic treatment with URB 597. In summary, AEA has the proclivity to enhance Notch-1 signaling in an in vitro model of AD, which may have relevance for restoring neurogenesis and cognition in AD.

Topics: Aging; Alzheimer Disease; Amyloid beta-Peptides; Amyloid Precursor Protein Secretases; Animals; Arachidonic Acids; Benzamides; Carbamates; Cells, Cultured; Cerebral Cortex; Endocannabinoids; Gene Expression Regulation, Enzymologic; Glycerides; Hippocampus; Male; Membrane Glycoproteins; Neurons; Peptide Fragments; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptor, Notch1; Signal Transduction; Up-Regulation

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

Evidence shows that the endocannabinoid system modulates the addictive properties of nicotine. In the present study, we hypothesized that spontaneous withdrawal resulting from removal of chronically implanted transdermal nicotine patches is regulated by the endocannabinoid system. A 7-day nicotine dependence procedure (5.2 mg/rat/day) elicited occurrence of reliable nicotine abstinence symptoms in Wistar rats. Somatic and affective withdrawal signs were observed at 16 and 34 hours following removal of nicotine patches, respectively. Further behavioral manifestations including decrease in locomotor activity and increased weight gain also occurred during withdrawal. Expression of spontaneous nicotine withdrawal was accompanied by fluctuation in levels of the endocannabinoid anandamide (AEA) in several brain structures including the amygdala, the hippocampus, the hypothalamus and the prefrontal cortex. Conversely, levels of 2-arachidonoyl-sn-glycerol were not significantly altered. Pharmacological inhibition of fatty acid amide hydrolase (FAAH), the enzyme responsible for the intracellular degradation of AEA, by URB597 (0.1 and 0.3 mg/kg, i.p.), reduced withdrawal-induced anxiety as assessed by the elevated plus maze test and the shock-probe defensive burying paradigm, but did not prevent the occurrence of somatic signs. Together, the results indicate that pharmacological strategies aimed at enhancing endocannabinoid signaling may offer therapeutic advantages to treat the negative affective state produced by nicotine withdrawal, which is critical for the maintenance of tobacco use.

Topics: Acute Disease; Amidohydrolases; Animals; Anxiety; Arachidonic Acids; Benzamides; Brain; Cannabinoid Receptor Modulators; Carbamates; Cotinine; Endocannabinoids; Glycerides; Implants, Experimental; Locomotion; Male; Maze Learning; Nicotine; Polyunsaturated Alkamides; Rats; Rats, Wistar; Substance Withdrawal Syndrome; Tobacco Use Cessation Devices; Weight Gain

The neurotransmitter acetylcholine (Ach) controls both excitatory and inhibitory synaptic transmission in the striatum. Here, we investigated the involvement of the endocannabinoid system in Ach-mediated inhibition of striatal GABA transmission, and the potential role of transient receptor potential vanilloid 1 (TRPV1) channels in the control of Ach-endocannabinoid coupling. We found that inhibition of Ach degradation and direct pharmacological stimulation of muscarinic M1 receptors reduced striatal inhibitory postsynaptic currents (IPSCs) through the stimulation of 2-arachidonoylglicerol (2AG) synthesis and the activation of cannabinoid CB1 receptors. The effects of M1 receptor activation on IPSCs were occlusive with those of metabotropic glutamate receptor 5 stimulation, and were prevented in the presence of capsaicin, agonist of TRPV1 channels. Elevation of anandamide (AEA) tone with URB597, a blocker of fatty acid amide hydrolase, mimicked the effects of capsaicin, indicating that endogenous AEA acts as an endovanilloid substance in the control of M1-dependent 2AG-mediated synaptic effects in the striatum. Accordingly, both capsaicin and URB597 effects were absent in mice lacking TRPV1 channels. Pharmacological interventions targeting AEA metabolism and TRPV1 channels might be considered alternative therapeutic routes in disorders of striatal cholinergic or endocannabinoid neurotransmission.

Topics: Acetylcholine; Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Modulators; Capsaicin; Carbamates; Corpus Striatum; Endocannabinoids; Enzyme Inhibitors; gamma-Aminobutyric Acid; Glycerides; Inhibitory Postsynaptic Potentials; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscarinic Agonists; Neural Inhibition; Organ Culture Techniques; Polyunsaturated Alkamides; Receptor, Metabotropic Glutamate 5; Receptor, Muscarinic M1; Receptors, Metabotropic Glutamate; Sensory System Agents; Synaptic Transmission; TRPV Cation Channels

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

Tetrahymena thermophila is a model organism for molecular and cellular biology. Previous studies from our group showed that Tetrahymena contains major components of the endocannabinoid system, such as various endocannabinoids and FAAH. In mammalian cells the endocannabinoid 2-arachidonoylglycerol is inactivated mainly by MAGL. In this study we showed that 2-arachidonoylglycerol and 2-oleoylglycerol are hydrolyzed by the combined actions of MAGL and FAAH. MAGL-like activity was examined in the presence of FAAH specific inhibitors, URB597 or AM374 and showed optimum pH of 8-9, apparent K(M) of 14.1μM and V(max) of 5.8nmol/min×mg. The enzyme was present in membrane bound and cytosolic isoforms; molecular mass was determined at ∼45 and ∼40kDa. MAGL and FAAH could also inactivate endogenous signaling lipids, which might play an important role in Tetrahymena as suggested in mammals. Tetrahymena could be used as a model system for testing drugs targeting enzymes of the endocannabinoid system.

Topics: Amidohydrolases; Arachidonic Acids; Benzamides; Carbamates; Cell Membrane; Cytosol; Endocannabinoids; Enzyme Inhibitors; Glycerides; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Molecular Weight; Monoacylglycerol Lipases; Palmitates; Protozoan Proteins; Tetrahymena thermophila

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

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

The endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are metabolised by cells by hydrolysis to arachidonic acid followed by esterification into phospholipids. Here, we report that nitric oxide (NO) donors significantly increase the amount of tritium accumulated in the cell membranes of RBL2H3 rat basophilic cells, 3T3-L1 mouse fibroblast cells and b.End5 mouse brain endothelioma cells following incubation of the intact cells with AEA labelled in the arachidonate part of the molecule. Similar results were seen with 2-AG and with arachidonic acid, whilst the NO donors reduced the accumulation of tritium after incubation of RBL2H3 cells with AEA labelled in the ethanolamine part of the molecule. Pretreatment of intact cells with NO donors did not increase the activity of the enzyme mainly responsible for metabolism of AEA, fatty acid amide hydrolase (FAAH). Furthermore, inhibition of FAAH completely blocked the effect produced by NO donors in cells with a large FAAH component, suggesting that for AEA, the effects were downstream of the enzyme. These data raise the possibility that the cellular processing of endocannabinoids following its uptake can be regulated by nitric oxide.

Topics: Amides; Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Modulators; Carbamates; Cell Line; Cell Membrane; Cyclic N-Oxides; Endocannabinoids; Ethanolamines; Free Radical Scavengers; Glycerides; Imidazoles; Lipid Metabolism; Mice; Nitric Oxide; Nitric Oxide Donors; Palmitic Acids; Polyunsaturated Alkamides; Rats; Signal Transduction; Tritium

The endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (N-arachidonoylethanolamine, AEA) are produced by neurons and other cells, including platelets, in a stimulus-dependent manner and act as signaling molecules; they are then inactivated through transport into cells followed by enzymatic degradation. A number of studies showed that monoacylglycerol lipase (MAGL) plays an important role in the degradation of 2-AG. In this study we investigated the enzymatic degradation of 2-acylglycerols in rabbit platelets and we characterized the responsible enzyme(s). [(3)H]2-AG and [(3)H]2-oleoylglycerol (2-OG) were both metabolized to [(3)H]glycerol and the respective fatty acid in a time and protein concentration-dependent manner, apparently by the action of MAGL activity. In the presence of the specific fatty acid amide hydrolase (FAAH) inhibitors URB597 and AM374, though, 2-OG hydrolysis was inhibited up to 55% in a concentration-dependent manner (IC(50) = 129.8 nM and 20.9 nM respectively). These results indicate the involvement of both MAGL and FAAH on 2-acylglycerol hydrolysis. MAGL was further characterized in the presence of URB597 and it was found that 2-monoacylglycerols were hydrolyzed in a time, pH and protein concentration-dependent manner and hydrolysis followed Michaelis-Menten kinetics, with an apparent K(M) of 0.11 microM and V(max) of 1.32 nmol/min*mg protein. Subcellular fractionation of platelet homogenate showed that MAGL activity was present in both the cytosolic and membrane fractions. In conclusion, the endocannabinoid 2-AG, as well as other 2-acylglycerols, are substrates of both FAAH and MAGL; the latter was characterized for the first time in platelets. In human platelets, under the same experimental conditions, the hydrolysis of 2-acylglycerols was higher and MAGL activity showed a different sensitivity against the inhibitors mentioned above. Finally, immunoblot analysis revealed the presence of MAGL, both in rabbit and human platelets, with a molecular mass of approximately 33 kDa.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Blood Platelets; Carbamates; Endocannabinoids; Enzyme Inhibitors; Glycerides; Glycerol; Humans; Hydrogen-Ion Concentration; Hydrolysis; Immunoblotting; Monoacylglycerol Lipases; Palmitates; Rabbits; Subcellular Fractions; Tritium

Very little is known about the processes regulating the cellular uptake of the endogenous cannabinoid 2-arachidonoylglycerol (2-AG). In the present study, we investigated whether inhibition of 2-AG hydrolysis reduced its uptake, i.e. whether this compound behaves in a manner analogous to the related endocannabinoid anandamide. The selective fatty acid amide hydrolase inhibitor URB597 (3'-(aminocarbamoyl)[1,1'-biphenyl]-3-yl)-cyclohexylcarbamate) completely blocked the hydrolysis of anandamide and reduced its uptake by about half in RBL2H3 basophilic leukaemia cells. In contrast, in these cells, in PC3 and R3327AT-1 prostate cancer cells and in Neuro-2a neuroblastoma cells, the compound had more modest effects upon the hydrolysis of 2-AG and did not affect its cellular uptake at all, indicating that in these cells fatty acid amide hydrolase does not regulate the uptake of 2-AG. The serine hydrolase inhibitor methylarachidonoyl fluoronophosphonate behaved like URB597 with respect to anandamide uptake by RBL2H3 and Neuro-2a cells, and inhibited the hydrolysis of 2-AG with IC50 values of 0.014, 0.052, 0.41 and approximately 1 microM for RBL2H3, PC3, AT-1 and Neuro-2a cells, respectively. MAFP (1 microM) did not significantly reduce the uptake of 2-AG by RBL2H3, PC3 and AT-1 cells but did reduce the uptake of this endocannabinoid by Neuro-2a cells. Arachidonoyl trifluoromethyl ketone and URB602 ([1,1'-biphenyl]-3-yl-carbamic acid, cyclohexyl ester) reduced the uptake of 2-AG by both RBL2H3 and Neuro-2a cells, but at the high concentrations needed, the compound also blocked the retention of these ligands by wells. It is concluded that unlike the situation for anandamide, hydrolysis of 2-AG does not regulate its cellular uptake in RBL2H3, AT-1 and PC3 cells, but may gate the uptake in Neuro-2a cells.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Biphenyl Compounds; Cannabinoid Receptor Modulators; Carbamates; Cell Line, Tumor; Endocannabinoids; Glycerides; Humans; Hydrolysis; Polyunsaturated Alkamides

HIV-1 infection has significant effect on the immune system as well as on the nervous system. Breakdown of the blood-brain barrier (BBB) is frequently observed in patients with HIV-associated dementia (HAD) despite lack of productive infection of human brain microvascular endothelial cells (HBMEC). Cellular products and viral proteins secreted by HIV-1 infected cells, such as the HIV-1 Gp120 envelope glycoprotein, play important roles in BBB impairment and HIV-associated dementia development. HBMEC are a major component of the BBB. Using cocultures of HBMEC and human astrocytes as a model system for human BBB as well as in vivo model, we show for the first time that cannabinoid agonists inhibited HIV-1 Gp120-induced calcium influx mediated by substance P and significantly decreased the permeability of HBMEC as well as prevented tight junction protein down-regulation of ZO-1, claudin-5, and JAM-1 in HBMEC. Furthermore, cannabinoid agonists inhibited the transmigration of human monocytes across the BBB and blocked the BBB permeability in vivo. These results demonstrate that cannabinoid agonists are able to restore the integrity of HBMEC and the BBB following insults by HIV-1 Gp120. These studies may lead to better strategies for treatment modalities targeted to the BBB following HIV-1 infection of the brain based on cannabinoid pharmacotherapies.

Topics: AIDS Dementia Complex; Amidohydrolases; Anti-HIV Agents; Arachidonic Acids; Benzamides; Blood-Brain Barrier; Brain; Cannabinoid Receptor Modulators; Carbamates; Cell Line; Coculture Techniques; Endocannabinoids; Endothelium, Vascular; Glycerides; HIV Envelope Protein gp120; HIV-1; Humans; Microcirculation; Receptor, Cannabinoid, CB1

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

The endocannabinoids, N-arachidonoylethanolamide (anandamide) and 2-arachidonoylglycerol (2-AG) are rapidly degraded by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL). Whilst these lipid mediators are known to modulate vascular tone, the extent to which they are inactivated via local metabolism in the vasculature remains unclear.. In rat isolated small mesenteric arteries, the regulatory role of FAAH, MGL and cyclooxygenase (COX) in relaxant responses to anandamide and 2-AG was evaluated by using inhibitors of these enzymes. Relaxations to non-hydrolysable analogues of endocannabinoids and arachidonic acid were also examined.. Relaxation to anandamide but not 2-AG was potentiated by the selective FAAH inhibitor, URB597 (1 microM). In contrast, MAFP (10 microM; an inhibitor of FAAH and MGL) enhanced responses to both anandamide and 2-AG. Inhibition of COX-1 by indomethacin (10 microM) potentiated relaxations to 2-AG, whereas inhibition of COX-2 by nimesulide (10 microM) potentiated anandamide-induced relaxation. With the exception of MAFP, effects of FAAH and COX inhibitors were dependent on the endothelium. Relaxation to methanandamide and noladin ether, the non-hydrolysable analogues of anandamide and 2-AG respectively, were insensitive to the enzyme inhibitors.. This study shows that local activity of FAAH, MGL and COX, which is present largely in the endothelium, limits the vasodilator action of endocannabinoids in rat small mesenteric arteries. Despite the differential roles played by these enzymes on relaxation to anandamide versus 2-AG, our results suggest that inhibitors of these enzymes enhance the vascular impact of endocannabinoids.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Modulators; Carbamates; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Dose-Response Relationship, Drug; Endocannabinoids; Endothelium, Vascular; Enzyme Inhibitors; Glycerides; Hydrolases; In Vitro Techniques; Lectins; Lectins, C-Type; Male; Membrane Proteins; Mesenteric Artery, Superior; Organophosphonates; Polyunsaturated Alkamides; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Wistar; Receptors, Cell Surface; Vasodilation; Vasodilator Agents

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

The phytocannabinoid cannabidiol (CBD) possesses no psychotropic activity amid potentially beneficial therapeutic applications. We here characterized interactions between CBD (1 microM) and the endocannabinoid system in cultured rat hippocampal cells. The CBD-induced Ca2+ rise observed in neurons and glia was markedly reduced in the presence of the endogenous cannabinoid anandamide in neurons, with no alteration seen in glia. Neuronal CBD responses were even more reduced in the presence of the more abundant endocannabinoid 2-arachidonyl glycerol, this action was maintained in the presence of the CB1 receptor antagonist AM281 (100 nM). Neuronal CBD responses were also reduced by pre-exposure to glutamate, expected to increase endocannabinoid levels by increasing in [Ca2+]i. Application of AM281 at 1 microM elevated CBD-induced Ca2+ responses in both cell types, further confirming our finding that endocannabinoid-mediated signalling is negatively coupled to the action of CBD. However, upregulation of endogenous levels of endocannabinoids via inhibition of endocannabinoid hydrolysis (with URB597 and MAFP) could not be achieved under resting conditions. Because delta9-tetrahydrocannabinol did not mimic the endocannabinoid actions, and pertussis toxin treatment had no effect on CBD responses, we propose that the effects of AM281 were mediated via a constitutively active signalling pathway independent of CB1 signalling. Instead, signalling via G(q/11) and phospholipase C appears to be negatively coupled to CBD-induced Ca2+ responses, as the inhibitor U73122 enhanced CBD responses. Our data highlight the interaction between exogenous and endogenous cannabinoid signalling, and provide evidence for the presence of an additional pharmacological target, sensitive to endocannabinoids and to AM281.

Topics: Animals; Arachidonic Acids; Benzamides; Calcium; Cannabidiol; Cannabinoid Receptor Modulators; Carbamates; Cells, Cultured; Dronabinol; Endocannabinoids; Estrenes; Glutamic Acid; Glycerides; Hippocampus; Humans; Morpholines; Pertussis Toxin; Phosphodiesterase Inhibitors; Polyunsaturated Alkamides; Pyrazoles; Pyrrolidinones; Rats; Receptor, Cannabinoid, CB1; Signal Transduction

The N-acylethanolamines (NAEs) and 2-arachidonoylglycerol (2-AG) are bioactive lipids that can modulate inflammatory responses and protect neurons against glutamatergic excitotoxicity. We have used a model of focal cerebral ischemia in young adult mice to investigate the relationship between focal cerebral ischemia and endogenous NAEs. Over the first 24 h after induction of permanent middle cerebral artery occlusion, we observed a time-dependent increase in all the investigated NAEs, except for anandamide. Moreover, we found an accumulation of 2-AG at 4 h that returned to basal level 12 h after induction of ischemia. Accumulation of NAEs did not depend on regulation of N-acylphosphatidylethanolamine-hydrolyzing phospholipase D or fatty acid amide hydrolase. Treatment with the fatty acid amide hydrolase inhibitor URB597 (cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester; 1 mg/kg; i.p.) 1.5 h before arterial occlusion decreased the infarct volume in our model system. Our results suggest that NAEs and 2-AG may be involved in regulation of neuroprotection during focal cerebral ischemia in mice.

Topics: Analysis of Variance; Animals; Arachidonic Acids; Benzamides; Brain; Brain Infarction; Brain Ischemia; Carbamates; Disease Models, Animal; Endocannabinoids; Enzyme Inhibitors; Ethanolamines; Glycerides; Male; Mice; RNA, Messenger; Time Factors

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

Endogenous ligands for cannabinoid receptors ("endocannabinoids") include the lipid transmitters anandamide and 2-arachidonoylglycerol (2-AG). Endocannabinoids modulate a diverse set of physiological processes and are tightly regulated by enzymatic biosynthesis and degradation. Termination of anandamide signaling by fatty acid amide hydrolase (FAAH) is well characterized, but less is known about the inactivation of 2-AG, which can be hydrolyzed by multiple enzymes in vitro, including FAAH and monoacylglycerol lipase (MAGL). Here, we have taken a functional proteomic approach to comprehensively map 2-AG hydrolases in the mouse brain. Our data reveal that approximately 85% of brain 2-AG hydrolase activity can be ascribed to MAGL, and that the remaining 15% is mostly catalyzed by two uncharacterized enzymes, ABHD6 and ABHD12. Interestingly, MAGL, ABHD6, and ABHD12 display distinct subcellular distributions, suggesting that they may control different pools of 2-AG in the nervous system.

Topics: Animals; Arachidonic Acids; Benzamides; Biotin; Brain; Carbamates; Catalysis; Chlorocebus aethiops; COS Cells; Endocannabinoids; Enzyme Inhibitors; Glycerides; Hydrolases; Lactones; Membrane Proteins; Mice; Mice, Inbred C57BL; Monoacylglycerol Lipases; Organophosphorus Compounds; Orlistat; Proteomics; Subcellular Fractions; Transfection

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

The chemotherapeutic agent cisplatin may produce emesis via release of several neurotransmitters such as serotonin (5-HT), substance P and/or dopamine as well as production of prostaglandins (PGs). Administration of synthetic 2-arachidonoylglycerol (2-AG) but not of anandamide, which are two putative endocannabinoids, causes vomiting via its downstream metabolites such as arachidonic acid (AA) and PGs in the least shrew (Cryptotis parva). We report here that cisplatin (0, 5, 10 and 20 mg/kg, i.p.) causes dose- and time-dependent increases in brain tissue levels of 2-AG but not anandamide in this vomiting species. Concomitantly, intestinal tissue levels of both endocannabinoids are relatively reduced. Selective inhibitors [arachidonoyl-serotonin (AA-5-HT) and URB597, 0-5 and 0-10 mg/kg, i.p.] of one of the major endocannabinoid metabolic enzymes, the intracellular fatty acid amide hydrolase (FAAH), do not significantly prevent vomiting produced by emetic doses of i.p.-administered 2-AG, cisplatin or the dopamine receptor agonist apomorphine. At large doses (10 and 20 mg/kg, respectively), both FAAH inhibitors caused emesis per se. Administration of one selective uptake inhibitor of endocannabinoids, OMDM1 (0-5 mg/kg, i.p.), also did not significantly prevent emesis by the direct and indirect emetic stimuli, and likewise caused emesis by itself at a high (10 mg/kg) dose. However, another selective uptake inhibitor, VDM11, did not produce significant emesis per se and prevented emesis caused by apomorphine. Both the corticosteroid dexamethasone, and the cyclooxygenase inhibitor indomethacin, reduced vomiting produced by cisplatin. These data: (a) provide the first evidence that cisplatin causes a selective increase in 2-AG levels in the brain, and (b) support the established notion that 2-AG may produce some of its effects, including emesis, via downstream metabolites produced independently of FAAH.

Topics: Analysis of Variance; Animals; Apomorphine; Arachidonic Acids; Benzamides; Benzyl Compounds; Brain; Carbamates; Cisplatin; Dopamine Agonists; Dose-Response Relationship, Drug; Drug Interactions; Endocannabinoids; Female; Glycerides; Intestinal Mucosa; Intestines; Male; Radiation-Sensitizing Agents; Serotonin; Shrews; Time Factors; Vomiting