urb-597 and anandamide

Chronic pain is a persistent, complex condition that contributes to impaired mood, anxiety and emotional problems. Osteoarthritis (OA) is one of the major causes of chronic pain in adults and elderly people. A substantial body of evidence demonstrate that hippocampal neural circuits, especially monoamine dopamine and serotonin levels, contributes to negative affect and avoidance motivation experienced during pain. Current pharmacological strategies for OA patients are unsatisfying and the endocannabinoid system modulation might represent an alternative for the treatment of OA-related pain. In the present study, we used a rat model of osteoarthritis induced by intra-articular injection of sodium monoiodoacetate to assess, 28 days post-induction, the contribution of endocannabinoid system on the possible alteration in pain perception and affective behavior, in LTP and monoamine levels in the lateral entorhinal cortex-dentate gyrus pathway. The results show that OA-related chronic pain induces working memory impairment and depressive-like behavior appearance, diminishes LTP, decreases dopamine levels and increases serotonin levels in the rat dentate gyrus. URB597 administration (i.p., 1 mg/kg) reduces hyperalgesia and mechanical allodynia, improves recognition memory and depressive-live behavior, restores LTP and normalizes monoamine levels in the hippocampus. The effect was observed 60-120 min post-treatment and was blocked by AM251, which proves the action of URB597 via the CB

Topics: Amines; Animals; Chronic Pain; Dopamine; Endocannabinoids; Hippocampus; Hyperalgesia; Osteoarthritis; Rats; Serotonin

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

Arachidonoyl ethanolamide (anandamide, AEA) and 2-arachidonoylglycerol (2-AG) are the most studies endocannabinoids. AEA and 2-AG are degraded by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) enzymes, respectively. FAAH and MAGL enzymes are widely expressed in many tissues, including kidney. Recent works have depicted that AEA and 2-AG levels are associated with ischemia-reperfusion (IR) injury. In this study, we investigated the effects of MAGL inhibitor KML29 and FAAH inhibitor URB597 against kidney IR injury.. The kidneys of the rats underwent ischemia for 45 min and then reperfusion for 24 h. KML29 and URB597 were administered intraperitoneally with kidney IR to two different treatment groups.. IR application increased serum blood urea nitrogen (BUN), creatinine (Cre), interleukin-18 (IL-18), neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule-1 (KIM-1) levels, while these parameters were decreased following KML29 and URB597 administration. KML29 and URB597 administration also reduced the increased toll-like receptor-4 (TRL-4), phosphorylated-NF-κB, phosphorylated-IκB-α, tumor necrosis factor alpha (TNF-α), interleukin-1beta (IL-1β), interleukin-6 (IL-6), caspase-3 levels and histopathological damage in kidney tissue.. Our results reveal that MAGL inhibitor KML29 and FAAH inhibitor URB597 have a protective effect on kidney IR injury by preventing apoptosis and inflammation. Inhibition of MAGL and FAAH may be a new therapeutic strategy to prevent kidney IR injury.

Topics: Amidohydrolases; Animals; Endocannabinoids; Kidney; Monoacylglycerol Lipases; Monoglycerides; NF-kappa B; Rats; Reperfusion Injury; Toll-Like Receptor 4

Microglia, the resident immune cells of the central nervous system, play important roles in brain homeostasis as well as in neuroinflammation, neurodegeneration, neurovascular diseases, and traumatic brain injury. In this context, components of the endocannabinoid (eCB) system have been shown to shift microglia towards an anti-inflammatory activation state. Instead, much less is known about the functional role of the sphingosine kinase (SphK)/sphingosine-1-phosphate (S1P) system in microglia biology. In the present study, we addressed potential crosstalk of the eCB and the S1P systems in BV2 mouse microglia cells challenged with lipopolysaccharide (LPS). We show that URB597, the selective inhibitor of fatty acid amide hydrolase (FAAH)-the main degradative enzyme of the eCB anandamide-prevented LPS-induced production of tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β), and caused the accumulation of anandamide itself and eCB-like molecules such as oleic acid and

Topics: Animals; Anti-Inflammatory Agents; Endocannabinoids; Lipopolysaccharides; Mice; Microglia; Sphingosine; Tumor Necrosis Factor-alpha

The prototype endocannabinoid, anandamide activates both CB 1 and transient receptor potential vanilloid type 1 channels (TRPV1) receptor at different concentrations. At high concentrations, anandamide-mediated TRPV1 effects are opposite to its effects at low concentrations via CB 1 receptor. Thus, synaptic concentrations of anandamide govern the neuronal activity and consequently might affect the response of a drug. This study was undertaken to investigate the influence of high and low doses of anandamide on the anticonvulsant action of diazepam on the subcutaneous dose of pentylenetetrazole (PTZ) in Swiss mice weighing 20-25 g. Results revealed that intracerebroventricular administration of capsazepine (a TRPV1 antagonist: 1, 10, or 100 µg/mouse) and the low doses (10 µg/mouse) of anandamide, AM404 (anandamide transport inhibitor), or URB597 (fatty acid amide hydrolase inhibitor) augmented the anticonvulsant effect of diazepam. Conversely, higher dose of anandamide, AM404, URB597 (100 µg/mouse) as well as capsaicin (a TRPV1 agonist: 1, 10, or 100 µg/mouse) attenuated the protective effect of diazepam against PTZ-induced seizures. Thus, this study demonstrates that the effects of diazepam may be augmented by activating CB 1 receptors or dampened via TRPV1 receptors. The findings of the present study can be extrapolated to understand the use of TRPV1 blockers alone or in combination of benzodiazepines in the treatment of benzodiazepines-refractory status epilepticus, a condition associated with maladaptive trafficking of synaptic gamma-aminobutyric acid and glutamate receptors. However, potential clinical applications are needed to further support such preclinical studies.

Topics: Animals; Anticonvulsants; Capsaicin; Diazepam; Endocannabinoids; Mice; Pentylenetetrazole; Polyunsaturated Alkamides; Seizures; TRPV Cation Channels

Anandamide is an endocannabinoid that contributes to certain aspects of social behavior, like play and reward, by binding to cannabinoid receptor type 1 (CB1). Most interesting is the recent discovery that anandamide may be mobilized by oxytocin receptor activation under certain contexts, particularly in the nucleus accumbens.. Given the established role of oxytocin and the nucleus accumbens in the neurobiology of pair-bonding, we investigated whether systemic administration of brain-permeable modulators of the endocannabinoid system could alter preferential partner contact in both male and female prairie voles.. Specifically, we tested whether intraperitoneal administration of the neutral CB1 antagonist AM4113 (4.0-16.0 mg/kg) or the anandamide hydrolysis inhibitor URB597 (5.0-20.0 mg/kg) could prevent or facilitate partner preference formation, respectively. To further investigate the specificity of effects on partner preference, we repeated our URB597 dosing regimen on an additional group of females and tested their anxiety-related behavior in both an elevated-plus maze and a light/dark test.. AM4113 administration had no effect on partner preference. But while URB597 also had no effect on partner preference, low-dose females did increase absolute preferential contact with either the partner or the stranger; individual females spent significant contact time with either the partner or the stranger. None of our outcome measures in either anxiety test showed significant effects of treatment.. Our results reveal that experimentally increasing anandamide levels in female prairie voles can increase social contact with both a familiar and novel male via unknown mechanisms that are likely separate from anxiety reduction.

Topics: Animals; Anxiety; Arachidonic Acids; Arvicolinae; Behavior, Animal; Benzamides; Carbamates; Endocannabinoids; Exploratory Behavior; Female; Male; Maze Learning; Nucleus Accumbens; Pair Bond; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Receptors, Oxytocin; Social Behavior

Our study aimed to examine the effects of hypertension and the chronic administration of the fatty acid amide hydrolase (FAAH) inhibitor URB597 on vascular function and the endocannabinoid system in spontaneously hypertensive rats (SHR). Functional studies were performed on small mesenteric G3 arteries (sMA) and aortas isolated from SHR and normotensive Wistar Kyoto rats (WKY) treated with URB597 (1 mg/kg; twice daily for 14 days). In the aortas and sMA of SHR, endocannabinoid levels and cannabinoid CB

Topics: Acetylcholine; Amidohydrolases; Animals; Aorta; Arachidonic Acids; Benzamides; Carbamates; Endocannabinoids; Essential Hypertension; Hypertension; Male; Mesenteric Arteries; Nitroprusside; Polyunsaturated Alkamides; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptors, Cannabinoid; Vasoconstriction; Vasodilation

We studied the prolonged action of kainic acid on glutamatergic neurons in the dorsal hippocampus and the endocannabinoid-dependent protection against neurodegeneration. The pyramidal neurons of the CA3 field of the hippocampus, as well as granular and mossy cells of the dentate gyrus were examined. Light and electron microscopy revealed substantial damage to the components of the protein-synthesizing (rough endoplasmic reticulum, Golgi apparatus, and polyribosomes) and catabolic (lysosomes, autophagosomes, multivesicular structures, and lipofuscin formations) systems in all cells. Pyramidal and mossy neurons die mainly by the necrotic pathway. The death of granular cells occurred through both apoptosis and necrosis. The most vulnerable cells are mossy neurons located in the hilus. Activation of the endocannabinoid system induced by intracerebral injection of URB597, an inhibitor of degradation of endocannabinoid anandamide, protected the normal structure of the hippocampus and prevented neuronal damage and death induced by KA.

Topics: Animals; Arachidonic Acids; Autophagosomes; Benzamides; CA3 Region, Hippocampal; Carbamates; Dentate Gyrus; Endocannabinoids; Endoplasmic Reticulum; Excitatory Amino Acid Agonists; Golgi Apparatus; Kainic Acid; Lysosomes; Male; Microscopy, Electron; Necrosis; Nerve Degeneration; Polyunsaturated Alkamides; Pyramidal Cells; Rats; Rats, Wistar; Status Epilepticus

The ability of endocannabinoid (eCB) to change functional microglial phenotype can be explored as a possible target for therapeutic intervention. Since the inhibition of fatty acid amide hydrolase (FAAH), the main catabolic enzyme of anandamide (AEA), may provide beneficial effects in mice model of Alzheimer's disease (AD)-like pathology, we aimed at determining whether the FAAH inhibitor URB597 might target microglia polarization and alter the cytoskeleton reorganization induced by the amyloid-β peptide (Aβ). The morphological evaluation showed that Aβ treatment increased the surface area of BV-2 cells, which acquired a flat and polygonal morphology. URB597 treatment partially rescued the control phenotype of BV-2 cells when co-incubated with Aβ. Moreover, URB597 reduced both the increase of Rho protein activation in Aβ-treated BV-2 cells and the Aβ-induced migration of BV-2 cells, while an increase of Cdc42 protein activation was observed in all samples. URB597 also increased the number of BV-2 cells involved in phagocytosis. URB597 treatment induced the polarization of microglial cells towards an anti-inflammatory phenotype, as demonstrated by the decreased expression of iNOS and pro-inflammatory cytokines along with the parallel increase of Arg-1 and anti-inflammatory cytokines. Taken together, these data suggest that FAAH inhibition promotes cytoskeleton reorganization, regulates phagocytosis and cell migration processes, thus driving microglial polarization towards an anti-inflammatory phenotype.

Topics: Alzheimer Disease; Amidohydrolases; Amyloid beta-Peptides; Animals; Arachidonic Acids; Benzamides; Carbamates; Cell Line; Cell Movement; Cell Polarity; Cytokines; Cytoskeleton; Disease Models, Animal; Endocannabinoids; Mice; Microglia; Polyunsaturated Alkamides

Autism spectrum disorder (ASD) is a neurodevelopmental disorder, characterized by social interaction impairment, stereotypical/repetitive behaviors and emotional deregulation. The endocannabinoid (eCB) system plays a crucial role in modulating the behavioral traits that are typically core symptoms of ASD. The major molecular mechanisms underlying eCB-dependent long-term depression (eCB-LTD) are mediated by group 1 metabotropic glutamate receptor (mGluR)-induced removal of postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). Recently, modulation of anandamide (AEA), one of the main endocannabinoids in the brain, has been reported to alter social behaviors in genetic models of ASD. On this basis, we investigated the effects of treatment and the synaptic mechanism underlying AEA-mediated signaling in prenatal exposure to valproic acid (VPA) in rats. We found that the social deficits, repetitive behaviors and abnormal emotion-related behaviors in VPA-exposed offspring were improved after treatment with an inhibitor of AEA degrading enzyme, URB597. Using an integrative approach combing electrophysiological and cellular mechanisms, the results showed that the impaired eCB-LTD, abnormal mGluR-mediated LTD (mGluR-LTD) and decreased removal of AMPAR subunits GluA1 and GluA2 were reversed by URB597 in the prefrontal cortex (PFC) of VPA-exposed offspring. Taken together, these results provide the first evidence that rescue of the ASD-like phenotype by URB597 is mediated by enhancing the mechanism of removal of AMPAR subunits GluA1/2 underlying AEA signaling in the PFC in a VPA-induced model of ASD.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Autism Spectrum Disorder; Benzamides; Carbamates; Disease Models, Animal; Endocannabinoids; Enzyme Inhibitors; Female; Long-Term Synaptic Depression; Neuronal Plasticity; Polyunsaturated Alkamides; Prefrontal Cortex; Pregnancy; Prenatal Exposure Delayed Effects; Protein Transport; Rats; Receptors, AMPA; Receptors, Metabotropic Glutamate; Social Behavior; Valproic Acid

Topics: Amygdala; Animals; Arachidonic Acids; Arousal; Basolateral Nuclear Complex; Behavior, Animal; Benzamides; Carbamates; Depression; Disease Models, Animal; Endocannabinoids; Extinction, Psychological; Neuropeptide Y; Polyunsaturated Alkamides; Rats; Receptor, Cannabinoid, CB1; Receptors, Neuropeptide Y; Reflex, Startle; Social Behavior; Stress Disorders, Post-Traumatic

Fatty acid amide hydrolase (FAAH) is a membrane-bound homodimeric enzyme that in vivo controls content and biological activity of N-arachidonoylethanolamine (AEA) and other relevant bioactive lipids termed endocannabinoids. Parallel orientation of FAAH monomers likely allows both subunits to simultaneously recruit and cleave substrates. Here, we show full inhibition of human and rat FAAH by means of enzyme inhibitors used at a homodimer:inhibitor stoichiometric ratio of 1:1, implying that occupation of only one of the two active sites of FAAH is enough to fully block catalysis. Single W445Y substitution in rat FAAH displayed the same activity as the wild-type, but failed to show full inhibition at the homodimer:inhibitor 1:1 ratio. Instead, F432A mutant exhibited reduced specific activity but was fully inhibited at the homodimer:inhibitor 1:1 ratio. Kinetic analysis of AEA hydrolysis by rat FAAH and its F432A mutant demonstrated a Hill coefficient of ~1.6, that instead was ~1.0 in the W445Y mutant. Of note, also human FAAH catalysed an allosteric hydrolysis of AEA, showing a Hill coefficient of ~1.9. Taken together, this study demonstrates an unprecedented allosterism of FAAH, and represents a case of communication between two enzyme subunits seemingly controlled by a single amino acid (W445) at the dimer interface. In the light of extensive attempts and subsequent failures over the last decade to develop effective drugs for human therapy, these findings pave the way to the rationale design of new molecules that, by acting as positive or negative heterotropic effectors of FAAH, may control more efficiently its activity.

Topics: Allosteric Regulation; Allosteric Site; Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Biocatalysis; Carbamates; Catalytic Domain; Drug Design; Endocannabinoids; Enzyme Assays; Humans; Hydrolysis; Kinetics; Molecular Dynamics Simulation; Mutation; Polyunsaturated Alkamides; Protein Subunits; Rats

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

Hypertension is accompanied by oxidative stress, which can be modified by the functioning of the endocannabinoid system playing a prominent modulatory role in the brain. The present study tested whether chronic administration of the fatty acid amide hydrolase (FAAH) inhibitor [3-(3-carbamoylphenyl) phenyl]N-cyclohexylcarbamate (URB597) to rats with primary hypertension (SHR) can modify redox balance and consequently brain phospholipid metabolism. Experiments were conducted using SHRs and normotensive control Wistar-Kyoto rats treated by intraperitoneal injection with URB597 for 14 days. The biochemical parameters were assayed in the rats' brains. Inhibition of FAAH activity by URB597 resulted in an increase in anandamide and GPR55 receptor levels, as well as a decrease in CB

Topics: Animals; Arachidonic Acids; Benzamides; Brain; Carbamates; Disease Models, Animal; Endocannabinoids; Hypertension; Injections, Intraperitoneal; Male; Malondialdehyde; Oxidation-Reduction; Phospholipids; Polyunsaturated Alkamides; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, G-Protein-Coupled

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

The endocannabinoid system (ECS) regulates several physiological processes in the Central Nervous System, including the modulation of neuronal excitability via activation of cannabinoid receptors (CBr). Both glutaric acid (GA) and quinolinic acid (QUIN) are endogenous metabolites that, under pathological conditions, recruit common toxic mechanisms. A synergistic effect between them has already been demonstrated, supporting potential implications for glutaric acidemia type I (GA I). Here we investigated the possible involvement of a cannabinoid component in the toxic model exerted by QUIN + GA in rat cortical slices and primary neuronal cell cultures. The effects of the CB1 receptor agonist anandamide (AEA), and the fatty acid amide hydrolase inhibitor URB597, were tested on cell viability in cortical brain slices and primary neuronal cultures exposed to QUIN, GA, or QUIN + GA. As a pre-treatment to the QUIN + GA condition, AEA prevented the loss of cell viability in both preparations. URB597 only protected in a moderate manner the cultured neuronal cells against the QUIN + GA-induced damage. The use of the CB1 receptor reverse agonist AM251 in both biological preparations prevented partially the protective effects exerted by AEA, thus suggesting a partial role of CB1 receptors in this toxic model. AEA also prevented the cell damage and apoptotic death induced by the synergic model in cell cultures. Altogether, these findings demonstrate a modulatory role of the ECS on the synergic toxic actions exerted by QUIN + GA, thus providing key information for the understanding of the pathophysiological events occurring in GA I.

Topics: Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Agonists; Carbamates; Cell Survival; Cells, Cultured; Cerebral Cortex; Drug Synergism; Endocannabinoids; Female; Glutarates; Male; Neurons; Piperidines; Polyunsaturated Alkamides; Pregnancy; Pyrazoles; Quinolinic Acid; Rats; Rats, Inbred WF; Receptors, Cannabinoid

The dorsal hippocampus has a central role in modulating cardiovascular responses and behavioral adaptation to stress. The dorsal hippocampus also plays a key role in stress-associated mental disorders. The endocannabinoid system is widely expressed in the dorsal hippocampus and modulates defensive behaviors under stressful conditions. The endocannabinoid anandamide activates cannabinoid type 1 receptors and is metabolized by the fatty acid amide hydrolase enzyme.. We sought to verify whether cannabinoid type 1 receptors modulate stress-induced cardiovascular changes, and if pharmacological fatty acid amide hydrolase inhibition in the dorsal hippocampus would prevent the cardiovascular responses and the delayed anxiogenic-like behavior evoked by restraint stress in rats via cannabinoid type 1 receptors.. Independent groups received intra-dorsal-hippocampal injections of N-(piperidin-1yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-hpyrazole-3-carboxamide (AM251; cannabinoid type 1 receptor antagonist/inverse agonist, 10-300 pmol) and/or cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester (URB597; fatty acid amide hydrolase inhibitor, 10 pmol) before the restraint stress session. Cardiovascular response during restraint stress or later behavioral parameters were evaluated.. Acute restraint stress altered the cardiovascular response, characterized by increased heart rate and mean arterial pressure, as well as decreased tail cutaneous temperature. It also induced a delayed anxiogenic-like effect, evidenced by reduced open arm exploration in the elevated plus maze 24 h after stress. AM251 exacerbated the stress-induced cardiovascular responses after injection into the dorsal hippocampus. In contrast, local injection of URB597 prevented the cardiovascular response and the delayed (24 h) behavioral consequences of restraint stress, effects attenuated by pretreatment with AM251.. Our data corroborate previous results indicating that the hippocampal endocannabinoid system modulates the outcome of stress exposure and suggest that this could involve modulation of the cardiovascular response during stress exposure.

Topics: Amidohydrolases; Animals; Anxiety; Arachidonic Acids; Arterial Pressure; Behavior, Animal; Benzamides; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Carbamates; Disease Models, Animal; Endocannabinoids; Heart Rate; Hippocampus; Male; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Restraint, Physical; Skin Temperature; Stress, Psychological

The endocannabinoid system plays a key role in the control of emotional responses to environmental challenges. CB1 receptors are highly expressed within cortico-limbic brain areas, where they modulate stress effects on memory processes. Glucocorticoid and endocannabinoid release is influenced by circadian rhythm. Here, we investigated how different stress intensities immediately after encoding influence rat short-term memory in an object recognition task, whether the effects depend on circadian rhythm and if exogenous augmentation of anandamide levels could restore any observed impairment. Two separate cohorts of male adult Sprague-Dawley rats were tested at two different times of the day, morning (inactivity phase) or afternoon (before the onset of the activity phase) in an object recognition task. The anandamide hydrolysis inhibitor URB597 was intraperitoneally administered immediately after the training trial. Rats were thereafter subjected to a forced swim stress under low or high stress conditions and tested 1 h after training. Control rats underwent the same experimental procedure except for the forced swim stress (no stress). We further investigated whether URB597 administration might modulate corticosterone release in rats subjected to the different stress conditions, both in the morning or afternoon. The low stressor elevated plasma corticosterone levels and impaired 1 h recognition memory performance when animals were tested in the morning. Exposure to the higher stress condition elevated plasma corticosterone levels and impaired memory performance, independently of the testing time. These findings show that stress impairing effects on short-term recognition memory are dependent on the intensity of stress and circadian rhythm. URB597 (0.3 mg kg

Topics: Animals; Arachidonic Acids; Arousal; Benzamides; Carbamates; Circadian Rhythm; Corticosterone; Emotions; Endocannabinoids; Male; Memory, Short-Term; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Stress, Psychological

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

In rodent models, chronic exposure to cannabis' psychoactive ingredient, Δ9-tetrahydrocannabinol, during adolescence leads to abnormal behavior in adulthood. In female rats, this maladaptive behavior is characterized by endophenotypes for depressive-like and psychotic-like disorders as well as cognitive deficits. We recently reported that most depressive-like behaviors triggered by adolescent Δ9-tetrahydrocannabinol exposure can be rescued by manipulating endocannabinoid signaling in adulthood with the anandamide-inactivating enzyme FAAH inhibitor, URB597. However, the molecular mechanisms underlying URB597's antidepressant-like properties remain to be established.. Here we examined the impact of adult URB597 treatment on the cellular and functional neuroadaptations that occurred in the prefrontal cortex and dentate gyrus of the hippocampus upon Δ9-tetrahydrocannabinol during adolescence through biochemical, morphofunctional, and electrophysiological studies.. We found that the positive action of URB597 is associated with the rescue of Δ9-tetrahydrocannabinol-induced deficits in endocannabinoid-mediated signaling and synaptic plasticity in the prefrontal cortex and the recovery of functional neurogenesis in the dentate gyrus of the hippocampus. Moreover, the rescue property of URB597 on depressive-like behavior requires the activity of the CB1 cannabinoid receptor.. By providing novel insights into the cellular and molecular mechanisms of URB597 at defined cortical and hippocampal circuits, our results highlight that positive modulation of endocannabinoid-signaling could be a strategy for treating mood alterations secondary to adolescent cannabis use.

Topics: Adaptation, Psychological; Animals; Arachidonic Acids; Benzamides; Carbamates; Dentate Gyrus; Depression; Dronabinol; Endocannabinoids; Female; Marijuana Abuse; Neurogenesis; Neuronal Plasticity; Polyunsaturated Alkamides; Prefrontal Cortex; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Sexual Maturation; Signal Transduction; Synapses; Tissue Culture Techniques

Changes in endogenous cannabinoid homeostasis are associated with both ethanol-related neuroinflammation and memory decline. Extensive research is still required to unveil the role of endocannabinoid signaling activation on hippocampal microglial cells after ethanol exposure. Either microglial morphology, phenotype and recruitment may become notably altered after chronic alcohol-related neurodegeneration. Here, we evaluated the pharmacological effects of fatty-acid amide-hydrolase (FAAH) inhibitor URB597 (0.3 mg/kg), oleoylethanolamide (OEA, 10 mg/kg), arachidonoylethanolamide (AEA, 10 mg/kg), the CB1 receptor agonist ACEA (3 mg/kg) and the CB2 receptor agonist JWH133 (0.2 mg/kg) administered for 5 days in a rat model of subchronic (2 weeks) ethanol diet (11% v/v) exposure. URB597 turned to be the most effective treatment. URB597 increased microglial (IBA-1+) cell population, and changed morphometric features (cell area and perimeter, roughness, fractal dimension, lacunarity) associated with activated microglia in the hippocampus of ethanol-exposed rats. Regarding innate immune activity, URB597 specifically increased mRNA levels of toll-like receptor 4 (TLR4), glial fibrillary acidic protein (Gfap) and the chemokine stromal cell-derived factor 1 (SDF-1α/CXCL12), and elevated the cell population expressing the chemokine receptors CX3CR1, CCR2 and CCR4 in the ethanol-exposed rat hippocampus. Contrary to ethanol effect, URB597 reduced mRNA levels of Iba-1, Tnfα, IL-6 and the monocyte chemoattractant protein-1 (MCP-1/CCL2), as well as cell population expressing iNOS. URB597 effects on hippocampal immune system were accompanied by changes in short and long-term visual recognition memory. These results suggest that FAAH inhibition may modulates hippocampal microglial recruitment and activation that can be associated with improved hippocampal-dependent memory despite ethanol exposure.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Carbamates; Chemokines; Cytokines; Dentate Gyrus; Endocannabinoids; Enzyme Inhibitors; Ethanol; Gene Expression; Glial Fibrillary Acidic Protein; Hippocampus; Male; Memory; Memory, Long-Term; Memory, Short-Term; Microglia; Nitric Oxide Synthase Type II; Phenotype; Polyunsaturated Alkamides; Rats, Wistar; Receptors, Chemokine

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

Naturally occurring pyridone alkaloids as well as synthetic derivatives were previously shown to induce neurite outgrowth. However, the molecular basis for this biological effect remains poorly understood. In this work, we have prepared new pyridones, and tested the effect of thirteen 4-hydroxy-2-pyridone derivatives on the components of the endocannabinoid system. Investigation of binding affinities towards CB

Topics: Alkaloids; Amidohydrolases; Arachidonic Acids; Cyclooxygenase 2; Endocannabinoids; Humans; Inhibitory Concentration 50; Kinetics; Monoacylglycerol Lipases; Polyunsaturated Alkamides; Protein Binding; Pyridones; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; U937 Cells

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

Chronic pain and hyperalgesia, as well as pain resulting from episodes of vaso-occlusion, are characteristic features of sickle cell disease (SCD) and are difficult to treat. Since there is growing evidence that increasing local levels of endocannabinoids can decrease hyperalgesia, we examined the effects of URB597, a fatty acid amide hydrolase (FAAH) inhibitor, which blocks the hydrolysis of the endogenous cannabinoid anandamide, on hyperalgesia and sensitization of cutaneous nociceptors in a humanized mouse model of SCD. Using homozygous HbSS-BERK sickle mice, we determined the effects of URB597 on mechanical hyperalgesia and on sensitization of C-fiber nociceptors in vivo. Intraplantar administration of URB597 (10 μg in 10 μL) decreased the frequency of withdrawal responses evoked by a von Frey monofilament (3.9 mN bending force) applied to the plantar hind paw. This was blocked by the CB1 receptor antagonist AM281 but not by the CB2 receptor antagonist AM630. Also, URB597 decreased hyperalgesia in HbSS-BERK/CB2R sickle mice, further confirming the role of CB1 receptors in the effects produced by URB597. Electrophysiological recordings were made from primary afferent fibers of the tibial nerve in anesthetized mice. The proportion of Aδ- and C-fiber nociceptors that exhibited spontaneous activity and responses of C-fibers to mechanical and thermal stimuli were greater in HbSS-BERK sickle mice as compared to control HbAA-BERK mice. Spontaneous activity and evoked responses of nociceptors were decreased by URB597 via CB1 receptors. It is suggested that enhanced endocannabinoid activity in the periphery may be beneficial in alleviating chronic pain associated with SCD.

Topics: Anemia, Sickle Cell; Animals; Arachidonic Acids; Benzamides; Carbamates; Disease Models, Animal; Endocannabinoids; Enzyme Inhibitors; Evoked Potentials; Hemoglobins; Humans; Hydrolysis; Hyperalgesia; Indoles; Male; Mice; Mice, Transgenic; Morpholines; Nerve Fibers, Unmyelinated; Nociceptors; Pain Threshold; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2

Our current knowledge of the implications of endocannabinoids in fear and anxiety is largely based on fear conditioning paradigms and approach-avoidance conflicts. Here we establish the ethobehavioral beetle mania task (BMT), which confronts mice with an erratically moving robo-beetle. With the help of this task we demonstrate decreased tolerance yet increased avoidance responses to an approaching beetle in high-anxiety behavior (HAB) and BALBc mice compared to C57BL/6N, CD1 and normal-anxiety behavior (NAB) mice. Also DBA/2N mice showed decreased passive and increased active behavior, but followed the robo-beetle more often than HAB and BALBc mice. Treatment with diazepam (1 mg/kg) increased tolerance without affecting avoidance behavior in HAB mice. Treatment with the MAGL inhibitor JZL184 (8 mg/kg) increased flight behavior, but did not affect tolerance. The FAAH inhibitor URB597 (0.3 mg/kg), however, reduced flight behavior and enhanced tolerance to the robo-beetle. The latter effects were blocked by co-treatment with the CB1 receptor antagonist SR141716A (3 mg/kg), which failed to affect the behavior by itself. Taken together, we validate the BMT as a novel test for studying endocannabinoids beyond traditional paradigms and for assessing active fear responses in mice. Furthermore, we demonstrate panicolytic consequences of pharmacological enhancement of anandamide, but not 2-AG signaling.

Topics: Amidohydrolases; Animals; Anti-Anxiety Agents; Anxiety; Arachidonic Acids; Avoidance Learning; Behavior, Animal; Benzamides; Benzodioxoles; Cannabinoid Receptor Agonists; Carbamates; Diazepam; Endocannabinoids; Fear; Male; Mice, Inbred C57BL; Monoacylglycerol Lipases; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant

In the present study, we investigated the effects of acute pharmacological manipulation of the endocannabinoid (EC) system on the valence of cognitive judgement bias of rats in the ambiguous-cue interpretation (ACI) paradigm. To accomplish this goal, after initial behavioural training, different groups of rats received single, systemic injections of the irreversible anandamide (AEA) hydrolysis inhibitor URB597, the cannabinoid receptor type 1 (CB1) inverse agonist AM251, the cannabinoid receptor type 2 (CB2) inverse agonist AM630, the combination of URB597 and AM251, and a combination of URB597 and AM630 and were subsequently tested with the ACI paradigm. We report that URB597 at a dose of 1 mg/kg significantly biased animals towards positive interpretation of the ambiguous cue and that this effect was abolished by pre-treatment with AM251 (1 mg/kg) or AM630 (1 mg/kg). The CB1 and CB2 inverse agonists administered alone (1 mg/kg) had no statistically significant effects on the interpretation of the ambiguous cue by rats. Our findings suggest involvement of the endocannabinoid system in the mediation of optimistic judgement bias.

Topics: Acoustic Stimulation; Analysis of Variance; Animals; Arachidonic Acids; Benzamides; Bias; Calcium Channel Blockers; Carbamates; Cognition; Conditioning, Psychological; Cues; Discrimination, Psychological; Dose-Response Relationship, Drug; Drug Interactions; Endocannabinoids; Judgment; Male; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats

Cannabis use has been associated with an increased risk to develop schizophrenia as well as symptom exacerbation in patients. In contrast, clinical studies have revealed an inverse relationship between the cerebrospinal fluid levels of the endocannabinoid anandamide and symptom severity, suggesting a therapeutic potential for endocannabinoid-enhancing drugs. Indeed, preclinical studies have shown that these drugs can reverse distinct behavioral deficits in a rodent model of schizophrenia. The mechanisms underlying the differences between exogenous and endogenous cannabinoid administration are currently unknown. Using the phencyclidine (PCP) rat model of schizophrenia, we compared the effects on neuronal activity of systematic administration of delta-9-tetrahydrocannabinol (THC) with the fatty acid amide hydrolase inhibitor URB597. Specifically, we found that the inhibitory response in the prefrontal cortex to THC administration was absent in PCP-treated rats. In contrast, an augmented response to endocannabinoid upregulation was observed in the prefrontal cortex of PCP-treated rats. Interestingly, differential effects were also observed at the neuronal population level, as endocannabinoid upregulation induced opposite effects on coordinated activity when compared with THC. Such information is important for understanding why marijuana and synthetic cannabinoid use may be contraindicated in schizophrenia patients while endocannabinoid enhancement may provide a novel therapeutic approach.

Topics: Animals; Arachidonic Acids; Benzamides; Carbamates; Disease Models, Animal; Dronabinol; Endocannabinoids; Hippocampus; Male; Phencyclidine; Polyunsaturated Alkamides; Prefrontal Cortex; Rats; Rats, Sprague-Dawley; Schizophrenia; Up-Regulation

Exposure to stress during early development can exert profound effects on the maturation of the neuroendocrine stress axis. The endocannabinoid (ECB) system has recently surfaced as a fundamental component of the neuroendocrine stress response; however, the effect of early-life stress on neonatal ECB signaling and the capacity to which ECB enhancement may modulate neonatal stress responses is relatively unknown. The present study assessed whether exposure to early-life stress in the form of limited access to nesting/bedding material (LB) from postnatal (PND) day 2 to 9 alters neuroendocrine activity and hypothalamic ECB content in neonatal rats challenged with a novel immobilization stressor. Furthermore, we examined whether inhibition of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of anandamide (AEA) affects neuroendocrine responses in PND10 pups as a function of rearing conditions. Neonatal rats showed a robust increase in corticosterone (CORT) and adrenocorticotropin hormone (ACTH) secretion in response to immobilization stress, which was significantly blunted in pups reared in LB conditions. Accordingly, LB pups exhibited reduced stress-induced Fos immunoreactivity in the paraventricular nucleus of the hypothalamus, with no significant differences in hypothalamic ECB content. Administration of the FAAH inhibitor URB597 (0.3 mg/kg, ip) 90 min prior to immobilization stress significantly dampened stress-induced CORT release, but only in pups reared in LB conditions. These results suggest that rearing in restricted bedding conditions dampens the neuroendocrine response to stress, while augmenting AEA mitigates stress-induced alterations in glucocorticoid secretion preferentially in pups subjected to early-life stress.

Topics: Adrenocorticotropic Hormone; Amidohydrolases; Animals; Animals, Newborn; Arachidonic Acids; Behavior, Animal; Benzamides; Carbamates; Corticosterone; Endocannabinoids; Hydrolysis; Hypothalamus; Male; Neurosecretory Systems; Paraventricular Hypothalamic Nucleus; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Restraint, Physical; Stress, Physiological; Stress, Psychological

N-(4-hydroxyphenyl)-arachidonamide (AM404) is an anandamide transport inhibitor shown to reduce rewarding and relapse-inducing effects of nicotine in several animal models of tobacco dependence. However, the reinforcing/rewarding effects of AM404 are not clear.. We investigated whether AM404 maintains self-administration behavior or reinstates extinguished drug seeking in squirrel monkeys.. In monkeys with a history of anandamide or cocaine self-administration, we substituted injections of AM404 (1-100 μg/kg/injection). Using a 10-response, fixed-ratio schedule, self-administration behavior was maintained by AM404. Dose-response curves had inverted U shapes, with peak response rates occurring at a dose of 10 μg/kg/injection. In anandamide-experienced monkeys, we also demonstrated self-administration of another anandamide transport inhibitor VDM11. In addition to supporting self-administration, priming injections of AM404 (0.03-0.3 mg/kg) reinstated drug-seeking behavior previously reinforced by cannabinoids (∆(9)-tetrahydrocannabinol (THC) or anandamide) or cocaine. Both AM404 self-administration behavior and reinstatement of drug seeking by AM404 were reduced by treatment with the cannabinoid CB1 receptor antagonist/inverse agonist rimonabant (0.3 mg/kg). Moreover, the reinforcing effects of AM404 were potentiated by the treatment with the fatty acid amide hydrolase (FAAH) inhibitor URB597 (0.3 mg/kg) suggesting a major role of anandamide in these effects. Finally, AM404 (0.3 mg/kg) potentiated the reinforcing effects of anandamide but not those of cocaine.. In non-human primates, AM404 effectively reinforced self-administration behavior and induced reinstatement of drug-seeking behavior in abstinent monkeys. These effects appeared to be mediated by cannabinoid CB1 receptors. Therefore, compounds that promote actions of endocannabinoids throughout the brain by inhibiting their membrane transport may have a potential for abuse.

Topics: Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Antagonists; Carbamates; Cocaine; Dose-Response Relationship, Drug; Dronabinol; Drug-Seeking Behavior; Endocannabinoids; Male; Nicotine; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Reinforcement, Psychology; Reward; Rimonabant; Saimiri; Self Administration

Several lines of recent evidence suggest that endocannabinoids affect behavior by influencing the general patterns of challenge responding.. Here, we investigated the brain mechanisms underlying this phenomenon in rats.. The anandamide hydrolysis inhibitor URB597 was condensed into the tip of stainless steel cannulae, which were chronically implanted slightly above the prelimbic cortex (PRL) or the basolateral amygdala (BLA), two important regions of coping and endocannabinoid action. Thereafter, we investigated behavioral responsiveness to ambient light level in the elevated plus-maze and conditioned fear tests.. URB597 concentration was ~30 μg/mg protein in target areas; local brain anandamide levels increased threefold, without significant changes in 2-arachidonoylglycerol. High levels of illumination halved the time spent by controls in the open arms of the plus-maze. No similar decrease was observed in rats with URB597 implants in the PRL. High light decreased conditioned fear by 30 % in controls, but not in rats with prelimbic URB597 implants. Unresponsiveness to environmental challenges was not attributable to the anxiolytic effects of anandamide enhancement, as implants induced paradoxical anxiogenic-like effects under low light, which could be explained by effects on stimulus responsiveness rather than by effects on anxiety. URB597 implants targeting the BLA did not affect stimulus responsiveness.. Our findings show that elevated prelimbic anandamide signaling leads to less environment-dependent (more autonomous) behavioral responses to challenges, which is an attribute of active coping styles. These findings are discussed in light of two emerging concepts of endocannabinoid roles, particularly "emotional homeostasis" and "active coping."

Topics: Adaptation, Psychological; Animals; Anti-Anxiety Agents; Anxiety; Arachidonic Acids; Basolateral Nuclear Complex; Benzamides; Carbamates; Endocannabinoids; Glycerides; Male; Polyunsaturated Alkamides; Prefrontal Cortex; Rats; Rats, Wistar; Signal Transduction

The primary psychoactive ingredient of marijuana, Δ(9)-tetrahydrocannabinol (Δ(9)-THC), has medicinal value but also produces unwanted deleterious effects on cognitive function, promoting the search for improved cannabinergic therapeutics. The present studies used a battery of touchscreen procedures in squirrel monkeys to compare the effects of different types of cannabinergic drugs on several measures of performance including learning (repeated acquisition), cognitive flexibility (discrimination reversal), short-term memory (delayed matching-to-sample), attention (psychomotor vigilance), and motivation (progressive ratio). Drugs studied included the cannabinoid agonist Δ(9)-THC, fatty acid amide hydrolase (FAAH) inhibitor cyclohexylcarbamic acid 3-carbamoylbiphenyl-3-yl ester (URB597), and endocannabinoid anandamide and its stable synthetic analog methanandamide [(R)-(+)-arachidonyl-1'-hydroxy-2'-propylamide]. The effects of Δ(9)-THC and anandamide after treatment with the cannabinoid receptor type 1 inverse agonist/antagonist rimonabant [5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1Hpyrazole-3-carboxamide] and the FAAH inhibitor URB597, respectively, also were examined. The results showed the following: 1) Δ(9)-THC produced dose-related impairments of discrimination-based cognitive behavior with potency that varied across tasks (discriminative capability < learning < flexibility < short-term memory); 2) anandamide alone and URB597 alone were without effect on all endpoints; 3) anandamide following URB597 pretreatment and methanandamide had negligible effects on discriminative capability, learning, and reversal, but following large doses affected delayed matching-to-sample performance in some subjects; 4) all drugs, except anandamide and URB597, disrupted attention; and 5) progressive ratio breakpoints were generally unaffected by all drugs tested, suggesting little to no effect on motivation. Taken together, these data indicate that metabolically stable forms of anandamide may have lesser adverse effects on cognitive functions than Δ(9)-THC, possibly offering a therapeutic advantage in clinical settings.

Topics: Animals; Arachidonic Acids; Attention; Behavior, Animal; Benzamides; Carbamates; Cognition; Discrimination Learning; Dose-Response Relationship, Drug; Dronabinol; Endocannabinoids; Learning; Male; Memory, Short-Term; Motivation; Piperidines; Polyunsaturated Alkamides; Psychomotor Performance; Pyrazoles; Reversal Learning; Rimonabant; Saimiri

The endocannabinoid system is composed of endocannabinoids (such as anandamide), their target receptors (CB1 and CB2 receptors, CB1Rs and CB2Rs), the enzymes that degrade them (fatty-acid-amide-hydrolase (FAAH) for anandamide), and an endocannabinoid transporter. FAAH inhibition has been recently identified as having a critical involvement in behaviors related to nicotine addiction and has been shown to reduce the effect of nicotine on the mesolimbic dopaminergic system via CB1R and peroxisome proliferator-activated receptor alpha (PPARα). Thus, inhibition of FAAH may represent a novel strategy for smoking cessation, but its mechanism of action on relapse to nicotine seeking is still unknown.. The study aims to explore the mechanism of action of the inhibitor of FAAH activity, URB597, on relapse to nicotine seeking by evaluating the effect of the CB1R, CB2R, and PPARα antagonists on the attenuating effect of URB597 on cue-induced reinstatement of nicotine seeking in rats.. URB597 reduced cue-induced reinstatement of nicotine seeking, an effect that was reversed by the CB1R antagonist rimonabant, but not by the CB2R or PPARα antagonists AM630 and MK886, respectively.. These results indicate that URB597 reduces cue-induced reinstatement in rats through a CB1 receptor-dependent mechanism, and not via CB2R or PPARα. Since FAAH inhibition represent a novel and promising strategy for tobacco smoking cessation, dissecting how it produces its action may lead to a better understanding of the neurobiological mechanisms underlying nicotine addiction.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Antagonists; Carbamates; Cues; Drug-Seeking Behavior; Endocannabinoids; Male; Nicotine; Piperidines; Polyunsaturated Alkamides; PPAR alpha; Pyrazoles; Rats; Rats, Long-Evans; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; Tobacco Use Disorder

Elements of the endocannabinoid system are strongly expressed in the basal ganglia where they suffer profound rearrangements after dopamine depletion. Modulation of the levels of the endocannabinoid 2-arachidonoyl-glycerol by inhibiting monoacylglycerol lipase alters glial phenotypes and provides neuroprotection in a mouse model of Parkinson's disease. In this study, we assessed whether inhibiting fatty acid amide hydrolase could also provide beneficial effects on the time course of this disease. The fatty acid amide hydrolase inhibitor, URB597, was administered chronically to mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTPp) over 5weeks. URB597 (1mg/kg) prevented MPTPp induced motor impairment but it did not preserve the dopamine levels in the nigrostriatal pathway or regulate glial cell activation. The symptomatic relief of URB597 was confirmed in haloperidol-induced catalepsy assays, where its anti-cataleptic effects were both blocked by antagonists of the two cannabinoid receptors (CB1 and CB2), and abolished in animals deficient in these receptors. Other fatty acid amide hydrolase inhibitors, JNJ1661010 and TCF2, also had anti-cataleptic properties. Together, these results demonstrate an effect of fatty acid amide hydrolase inhibition on the motor symptoms of Parkinson's disease in two distinct experimental models that is mediated by cannabinoid receptors.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Agonists; Carbamates; Disease Models, Animal; Endocannabinoids; Male; Mice; Mice, Inbred C57BL; Parkinsonian Disorders; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2

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

Direct activation of the cannabinoid CB1 receptor in the dorsolateral periaqueductal gray (dlPAG) inhibits anxiety- and panic-related behaviours in experimental animals. It has remained unclear, however, whether the local endocannabinoid signalling is recruited as a protective mechanism against aversive stimuli.. The present study tested the hypothesis that the endocannabinoid system counteracts aversive responses in the dlPAG-stimulation model of panic attacks.. All drugs were infused into the dlPAG of rats. Local chemical stimulation with N-methyl-D-aspartate (NMDA, 1 nmol) was employed to induce panic-like behavioural and cardiovascular responses in freely moving and anaesthetized animals, respectively. The neuronal activity in the dlPAG was investigated by c-Fos immunohistochemistry.. The selective CB1 receptor agonist, ACEA (0.005-0.5 pmol), prevented the NMDA-induced panic-like escape responses. More interestingly, increasing the local levels of endogenous anandamide with a fatty acid amide hydrolase (FAAH) inhibitor, URB597 (0.3-3 nmol), prevented both the behavioural response and the increase in blood pressure induced by NMDA. The effect of URB597 (3 nmol) was reversed by the CB1 receptor antagonist, AM251 (0.1 nmol). Moreover, an otherwise ineffective and sub-threshold dose of NMDA (0.5 nmol) was able to induce a panic-like response if local CB1 receptors were previously blocked by AM251 (0.1 nmol). Finally, URB597 prevented the NMDA-induced neuronal activation of the dlPAG.. The endocannabinoid system in the dlPAG attenuates the behavioural, cellular and cardiovascular consequences of aversive stimuli. This process may be considered for the development of additional treatments against panic and other anxiety-related disorders.

Topics: Amidohydrolases; Animals; Anxiety; Arachidonic Acids; Behavior, Animal; Benzamides; Carbamates; Disease Models, Animal; Endocannabinoids; Enzyme Inhibitors; Male; N-Methylaspartate; Panic Disorder; Periaqueductal Gray; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1

Painful neuropathy frequently develops as a consequence of commonly used chemotherapy agents for cancer treatment and is often a dose-limiting side effect. Currently available analgesic treatments are often ineffective on pain induced by neurotoxicity. Although peripheral administration of cannabinoids, endocannabinoids, and inhibitors of endocannabinoid hydrolysis has been effective in reducing hyperalgesia in models of peripheral neuropathy, including chemotherapy-induced peripheral neuropathy (CIPN), few studies have examined cannabinoid effects on responses of nociceptors in vivo. In this study we determined whether inhibition of fatty acid amide hydrolase (FAAH), which slows the breakdown of the endocannabinoid anandamide (AEA), reduced sensitization of nociceptors produced by chemotherapy. Over the course of a week of daily treatments, mice treated with the platinum-based chemotherapy agent cisplatin developed robust mechanical allodynia that coincided with sensitization of cutaneous C-fiber nociceptors as indicated by the development of spontaneous activity and increased responses to mechanical stimulation. Administration of the FAAH inhibitor URB597 into the receptive field of sensitized C-fiber nociceptors decreased spontaneous activity, increased mechanical response thresholds, and decreased evoked responses to mechanical stimuli. Cotreatment with CB1 (AM281) or CB2 (AM630) receptor antagonists showed that the effect of URB597 was mediated primarily by CB1 receptors. These changes following URB597 were associated with an increase in the endocannabinoid anandamide in the skin. Our results suggest that enhanced signaling in the peripheral endocannabinoid system could be utilized to reduce nociceptor sensitization and pain associated with CIPN.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Antagonists; Carbamates; Cisplatin; Endocannabinoids; Hydrolysis; Indoles; Male; Mice; Morpholines; Nerve Fibers, Unmyelinated; Nociception; Nociceptors; Peripheral Nervous System Diseases; Polyunsaturated Alkamides; Pyrazoles; Skin

Limbic forebrain endocannabinoid (eCB) signaling is critically involved in stress integration by modulating neurotransmitters release. The purpose of this study was to examine, by brain microdialysis, the effects of fatty acid amide hydrolase (FAAH) inhibition on noradrenergic and γ-aminobutyric acid (GABA)-ergic neurotransmission in the prefrontal cortex (PFC) and basolateral amygdala (BLA) of rats subjected to a 20-min swim stress. Microdialysis started on stress- and drug-naïve rats that were treated with the FAAH inhibitor URB597 (0.1 or 0.3 mg/kg) 30 min before undergoing the stress procedure. Dialysate samples were collected every 20 min from the beginning of the experiment. Concentrations of noradrenaline (NA) and GABA were determined by HPLC coupled to electrochemical and fluorescence detection, respectively. We found that neither URB597 treatment nor 20 min of swim stress exposure per se altered NA and GABA extracellular levels in PFC or BLA. Interestingly, rats treated with 0.1 mg/kg of URB597 followed by 20 min of stress showed significantly higher NA and GABA levels in PFC and BLA. These effects were absent in rats treated with 0.3 mg/kg URB597, indicating a dose-specific effect. Moreover, we found that the pretreatment with the CB1 receptor antagonist rimonabant blocked the URB597 effects on NA and GABA release in PFC and BLA of animals subjected to forced swimming. The present study might provide an important first step toward understanding the mechanisms through which URB597 modulates stress-induced neuroendocrine secretion and behavioral coping strategies.

Topics: Analysis of Variance; Animals; Arachidonic Acids; Basolateral Nuclear Complex; Benzamides; Carbamates; Chromatography, High Pressure Liquid; Disease Models, Animal; Endocannabinoids; Extracellular Fluid; gamma-Aminobutyric Acid; Male; Microdialysis; Norepinephrine; Polyunsaturated Alkamides; Prefrontal Cortex; Rats; Rats, Wistar; Stress, Psychological; Swimming

Panic attacks, a major feature of panic disorder, can be modelled in rats by exposing animals to stimuli that induce escape reactions, such as the elevated T-maze or the activation of the dorsolateral periaqueductal grey. Since the cannabinoid CB1 receptor modulates various types of aversive responses, this study tested the hypothesis that enhancement of endocannabinoid signalling in the dorsolateral periaqueductal grey inhibits panic-like reactions in rats. Local injection of the CB1 agonist, arachidonoyl 2-Chloroethylamide (0.005-0.5 pmol), attenuated the escape response from the open arm of the elevated T-maze, a panicolytic effect. The anandamide hydrolysis inhibitor, URB597 (0.3-3 nmol), did not induce consistent results. In the test of dorsolateral periaqueductal grey stimulation with d,l-homocysteic acid, arachidonoyl 2-Chloroethylamide, at the lowest dose, attenuated the escape reaction. The highest dose of URB597 also inhibited this response, contrary to the result obtained in the elevated T-maze. This effect was reversed by the CB1 antagonist, AM251 (100 pmol). The present results confirm the anti-aversive property of direct CB1 receptor activation in the dorsolateral periaqueductal grey. The effect of the anandamide hydrolysis inhibitor, however, could be detected only in a model employing direct stimulation of this structure. Altogether, these results suggest that anandamide signalling is recruited only under certain types of aversive stimuli.

Topics: Animals; Arachidonic Acids; Benzamides; Carbamates; Disease Models, Animal; Dose-Response Relationship, Drug; Endocannabinoids; Escape Reaction; Male; Maze Learning; Panic Disorder; Periaqueductal Gray; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Signal Transduction

Inducible or neuronal nitric oxide synthase gene deletion increases or decreases anxiety-like behavior in mice, respectively. Since nitric oxide and endocannabinoids interact to modulate defensive behavior, the former effect could involve a compensatory increase in basal brain nitric oxide synthase activity and/or changes in the endocannabinoid system. Thus, we investigated the expression and extinction of contextual fear conditioning of inducible nitric oxide knockout mice and possible involvement of endocannabinoids in these responses.. We evaluated the effects of a preferential neuronal nitric oxide synthase inhibitor, 7-nitroindazol, nitric oxide synthase activity, and mRNA changes of nitrergic and endocannabinoid systems components in the medial prefrontal cortex and hippocampus of wild-type and knockout mice. The effects of URB597, an inhibitor of the fatty acid amide hydrolase enzyme, which metabolizes the endocannabinoid anandamide, WIN55,212-2, a nonselective cannabinoid agonist, and AM281, a selective CB1 antagonist, on contextual fear conditioning were also evaluated.. Contextual fear conditioning expression was similar in wild-type and knockout mice, but the latter presented extinction deficits and increased basal nitric oxide synthase activity in the medial prefrontal cortex. 7-Nitroindazol decreased fear expression and facilitated extinction in wild-type and knockout mice. URB597 decreased fear expression in wild-type and facilitated extinction in knockout mice, whereas WIN55,212-2 and AM281 increased it in wild-type mice. Nonconditioned knockout mice showed changes in the mRNA expression of nitrergic and endocannabinoid system components in the medial prefrontal cortex and hippocampus that were modified by fear conditioning.. These data reinforce the involvement of the nitric oxide and endocannabinoids (anandamide) in stress-related disorders and point to a deregulation of the endocannabinoid system in situations where nitric oxide signaling is increased.

Topics: Animals; Arachidonic Acids; Benzamides; Benzoxazines; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Carbamates; Conditioning, Psychological; Endocannabinoids; Enzyme Inhibitors; Extinction, Psychological; Fear; Freezing Reaction, Cataleptic; Hippocampus; Indazoles; Male; Mice, Inbred C57BL; Mice, Knockout; Morpholines; Naphthalenes; Nitric Oxide; Nitric Oxide Synthase Type II; Polyunsaturated Alkamides; Prefrontal Cortex; Pyrazoles; Receptor, Cannabinoid, CB1; RNA, Messenger; Stress, Psychological

The pharmacological inhibition of anandamide (AEA) hydrolysis by fatty acid amide hydrolase (FAAH) attenuates pain in animal models of osteoarthritis (OA) but has failed in clinical trials. This may have occurred because AEA also activates transient receptor potential vanilloid type 1 (TRPV1), which contributes to pain development. Therefore, we investigated the effectiveness of the dual FAAH-TRPV1 blocker OMDM-198 in an MIA-model of osteoarthritic pain. We first investigated the MIA-induced model of OA by (1) characterizing the pain phenotype and degenerative changes within the joint using X-ray microtomography and (2) evaluating nerve injury and inflammation marker (ATF-3 and IL-6) expression in the lumbar dorsal root ganglia of osteoarthritic rats and differences in gene and protein expression of the cannabinoid CB1 receptors FAAH and TRPV1. Furthermore, we compared OMDM-198 with compounds acting exclusively on FAAH or TRPV1. Osteoarthritis was accompanied by the fragmentation of bone microstructure and destroyed cartilage. An increase of the mRNA levels of ATF3 and IL-6 and an upregulation of AEA receptors and FAAH in the dorsal root ganglia were observed. OMDM-198 showed antihyperalgesic effects in the OA model, which were comparable with those of a selective TRPV1 antagonist, SB-366,791, and a selective FAAH inhibitor, URB-597. The effect of OMDM-198 was attenuated by the CB1 receptor antagonist, AM-251, and by the nonpungent TRPV1 agonist, olvanil, suggesting its action as an "indirect" CB1 agonist and TRPV1 antagonist. These results suggest an innovative strategy for the treatment of OA, which may yield more satisfactory results than those obtained so far with selective FAAH inhibitors in human OA.

Topics: Activating Transcription Factor 3; Amidohydrolases; Anilides; Animals; Arachidonic Acids; Benzamides; Capsaicin; Carbamates; Cinnamates; Disease Models, Animal; Endocannabinoids; Ganglia, Spinal; Gene Expression; Hyperalgesia; Inflammation; Interleukin-6; Lumbar Vertebrae; Male; Osteoarthritis; Pain; Pain Management; Pain Measurement; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; TRPV Cation Channels

There is evidence to suggest that a dysregulation of endocannabinoid signaling may contribute to the etiology and pathophysiology of migraine. Thus, patients suffering from chronic migraine or medication overuse headache showed alterations in the activity of the arachidonoylethanolamide (AEA) degrading enzyme fatty acid amide hydrolase (FAAH) and a specific AEA membrane transporter, alongside with changes in AEA levels. The precise role of different endocannabinoid system components is, however, not clear. We have therefore investigated mice with a genetic deletion of the two main cannabinoid receptors CB1 and CB2, or the main endocannabinoid degrading enzymes, FAAH and monoacylglycerol lipase (MAGL), which degrades 2-arachidonoylglycerol (2-AG), in a nitroglycerine-induced animal model of migraine. We found that nitroglycerin-induced mechanical allodynia and neuronal activation of the trigeminal nucleus were completely abolished in FAAH-deficient mice. To validate these results, we used two structurally different FAAH inhibitors, URB597 and PF3945. Both inhibitors also dose-dependently blocked nitroglycerin-induced hyperalgesia and the activation of trigeminal neurons. The effects of the genetic deletion of pharmacological blockade of FAAH are mediated by CB1 receptors, because they were completely disrupted with the CB1 antagonist rimonabant. These results identify FAAH as a target for migraine pharmacotherapy.

Topics: Amidohydrolases; Analgesics; Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Antagonists; Carbamates; Disease Models, Animal; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Hyperalgesia; Male; Mice, Inbred C57BL; Mice, Knockout; Migraine Disorders; Monoacylglycerol Lipases; Nitroglycerin; Pain Measurement; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; Touch; Trigeminal Nuclei

Silencing the gene FMR1 in fragile X syndrome (FXS) with consequent loss of its protein product, FMRP, results in intellectual disability, hyperactivity, anxiety, seizure disorders, and autism-like behavior. In a mouse model (Fmr1 knockout (KO)) of FXS, a deficit in performance on the passive avoidance test of learning and memory is a robust phenotype. We report that drugs acting on the endocannabinoid (eCB) system can improve performance on this test. We present three lines of evidence: (1) Propofol (reported to inhibit fatty acid amide hydrolase (FAAH) activity) administered 30 min after training on the passive avoidance test improved performance in Fmr1 KO mice but had no effect on wild type (WT). FAAH catalyzes the metabolism of the eCB, anandamide, so its inhibition should result in increased anandamide levels. (2) The effect of propofol was blocked by prior administration of the cannabinoid receptor 1 antagonist AM-251. (3) Treatment with the FAAH inhibitor, URB-597, administered 30 min after training on the passive avoidance test also improved performance in Fmr1 KO mice but had no effect on WT. Our results indicate that the eCB system is involved in FXS and suggest that the eCB system is a promising target for treatment of FXS.

Topics: Amidohydrolases; Animals; Anxiety; Arachidonic Acids; Avoidance Learning; Benzamides; Cannabinoid Receptor Antagonists; Carbamates; Disease Models, Animal; Endocannabinoids; Enzyme Inhibitors; Fragile X Mental Retardation Protein; Fragile X Syndrome; Male; Memory; Mice, Inbred C57BL; Mice, Knockout; Piperidines; Polyunsaturated Alkamides; Propofol; Pyrazoles; Receptor, Cannabinoid, CB1; Receptors, GABA-A; Social Behavior

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

Increased endocannabinoid tonus by dual-action fatty acid amide hydrolase (FAAH) and substrate selective cyclooxygenase (COX-2) inhibitors is a promising approach for pain-relief. One such compound with this profile is 2-(2-fluorobiphenyl-4-yl)-N-(3-methylpyridin-2-yl)propanamide (Flu-AM1). These activities are shown by Flu-AM1 racemate, but it is not known whether its two single enantiomers behave differently, as is the case towards COX-2 for the parent flurbiprofen enantiomers. Further, the effects of the compound upon COX-2-derived lipids in intact cells are not known.. COX inhibition was determined using an oxygraphic method with arachidonic acid and 2-arachidonoylglycerol (2-AG) as substrates. FAAH was assayed in mouse brain homogenates using anandamide (AEA) as substrate. Lipidomic analysis was conducted in unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Both enantiomers inhibited COX-2 in a substrate-selective and time-dependent manner, with IC50 values in the absence of a preincubation phase of: (R)-Flu-AM1, COX-1 (arachidonic acid) 6 μM; COX-2 (arachidonic acid) 20 μM; COX-2 (2-AG) 1 μM; (S)-Flu-AM1, COX-1 (arachidonic acid) 3 μM; COX-2 (arachidonic acid) 10 μM; COX-2 (2-AG) 0.7 μM. The compounds showed no enantiomeric selectivity in their FAAH inhibitory properties. (R)-Flu-AM1 (10 μM) greatly inhibited the production of prostaglandin D2 and E2 in both unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Levels of 2-AG were not affected either by (R)-Flu-AM1 or by 10 μM flurbiprofen, either alone or in combination with the FAAH inhibitor URB597 (1 μM).. Both enantiomers of Flu-AM1 are more potent inhibitors of 2-AG compared to arachidonic acid oxygenation by COX-2. Inhibition of COX in lipopolysaccharide + interferon γ- stimulated RAW 264.7 cells is insufficient to affect 2-AG levels despite the large induction of COX-2 produced by this treatment.

Topics: Amides; Amidohydrolases; Animals; Arachidonic Acid; Arachidonic Acids; Benzamides; Brain; Carbamates; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Endocannabinoids; Flurbiprofen; Humans; Hydrolysis; Interferon-gamma; Ionomycin; Isoenzymes; Lipid Metabolism; Lipopolysaccharides; Male; Mice; Polyunsaturated Alkamides; Prostaglandins; RAW 264.7 Cells; Stereoisomerism

Marijuana exerts profound effects on human social behavior, but the neural substrates underlying such effects are unknown. Here we report that social contact increases, whereas isolation decreases, the mobilization of the endogenous marijuana-like neurotransmitter, anandamide, in the mouse nucleus accumbens (NAc), a brain structure that regulates motivated behavior. Pharmacological and genetic experiments show that anandamide mobilization and consequent activation of CB1 cannabinoid receptors are necessary and sufficient to express the rewarding properties of social interactions, assessed using a socially conditioned place preference test. We further show that oxytocin, a neuropeptide that reinforces parental and social bonding, drives anandamide mobilization in the NAc. Pharmacological blockade of oxytocin receptors stops this response, whereas chemogenetic, site-selective activation of oxytocin neurons in the paraventricular nucleus of the hypothalamus stimulates it. Genetic or pharmacological interruption of anandamide degradation offsets the effects of oxytocin receptor blockade on both social place preference and cFos expression in the NAc. The results indicate that anandamide-mediated signaling at CB1 receptors, driven by oxytocin, controls social reward. Deficits in this signaling mechanism may contribute to social impairment in autism spectrum disorders and might offer an avenue to treat these conditions.

Topics: Analysis of Variance; Animals; Arachidonic Acids; Autism Spectrum Disorder; Benzamides; Benzodiazepines; Camphanes; Carbamates; Clozapine; Cocaine; Endocannabinoids; Immunohistochemistry; Infusions, Intraventricular; Lipids; Male; Mice; Mice, Inbred C57BL; Nucleus Accumbens; Oxytocin; Piperazines; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Reward; Signal Transduction; Social Behavior

Recent evidence suggests that in addition to controlling emotional behavior in general, endocannabinoid signaling is engaged in shaping behavioral responses to challenges. This important function of endocannabinoids is still poorly understood.. Here we investigated the impact of blockade of fatty acid amide hydrolase (FAAH), the degrading enzyme of anandamide on behavioral responses induced by challenges of different intensity.. Mice treated with FAAH inhibitor URB597 were either manually restrained on their backs (back test) or received foot-shocks.. The behavior of mice showed bimodal distribution in the back test: they either predominantly showed escape attempts or equally distributed time between passivity and escape. URB597 increased escapes in animals with low escape scores. No effects were noticed in mice showing high escape scores, which is likely due to a ceiling effect. We hypothesized that stronger stressors would wash out individual differences in coping; therefore, we exposed mice to foot-shocks that decreased locomotion and increased freezing in all mice. URB597 ameliorated both responses. The re-exposure of mice to the shock cage 14 days later without delivering shocks or treatment was followed by reduced and fragmented sleep as shown by electrophysiological recordings. Surprisingly, sleep was more disturbed after the reminder than after shocks in rats receiving vehicle before foot-shocks. These reminder-induced disturbances were abolished by URB597 administered before shocks.. These findings suggest that FAAH blockade has an important role in the selection of behavioral responses under challenging conditions and-judging from its long-term effects-that it influences the cognitive appraisal of the challenge.

Topics: Adaptation, Psychological; Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Carbamates; Electrodes, Implanted; Electroencephalography; Electromyography; Electroshock; Endocannabinoids; Enzyme Inhibitors; Escape Reaction; Freezing Reaction, Cataleptic; Male; Mice, Inbred Strains; Motor Activity; Polyunsaturated Alkamides; Restraint, Physical; Sleep; Stress, Psychological

The role of endocannabinoids such as anandamide during atherogenesis remains largely unknown. Fatty acid amide hydrolase (FAAH) represents the key enzyme in anandamide degradation, and its inhibition is associated with subsequent higher levels of anandamide. Here, we tested whether selective inhibition of FAAH influences the progression of atherosclerosis in mice. Selective inhibition of FAAH using URB597 resulted in significantly increased plasma levels of anandamide compared to control, as assessed by mass spectrometry experiments in mice. Apolipoprotein E-deficient (ApoE(-/-)) mice were fed a high-fat, cholesterol-rich diet to induce atherosclerotic conditions. Simultaneously, mice received either the pharmacological FAAH inhibitor URB597 1mg/kg body weight (n=28) or vehicle (n=25) via intraperitoneal injection three times a week. After eight weeks, mice were sacrificed, and experiments were performed. Vascular superoxide generation did not differ between both groups, as measured by L012 assay. To determine whether selective inhibition of FAAH affects atherosclerotic plaque inflammation, immunohistochemical staining of the aortic root was performed. Atherosclerotic plaque formation, vascular macrophage accumulation, as well as vascular T cell infiltration did not differ between both groups. Interestingly, neutrophil cell accumulation was significantly increased in mice receiving URB597 compared to control. Vascular collagen structures in atherosclerotic plaques were significantly diminished in mice treated with URB597 compared to control, as assessed by picro-sirius-red staining. This was accompanied by an increased aortic expression of matrix metalloproteinase-9, as determined by quantitative RT-PCR and western blot analysis. Inhibition of fatty acid amide hydrolase does not influence plaque size but increases plaque vulnerability in mice.

Topics: Amidohydrolases; Animals; Apolipoproteins E; Arachidonic Acids; Benzamides; Carbamates; Cell Movement; Diet, High-Fat; Dietary Fats; Endocannabinoids; Enzyme Inhibitors; Gene Expression; Macrophages; Mice; Mice, Knockout; Neutrophils; Plaque, Atherosclerotic; Polyunsaturated Alkamides; Superoxides

Cannabinoid receptor (CBR) agonists produce antinociception in conventional preclinical assays of pain-stimulated behavior but are not effective in preclinical assays of pain-depressed behavior. Fatty acid amide hydrolase (FAAH) inhibitors increase physiological levels of the endocannabinoid anandamide, which may confer improved efficacy and safety relative to direct CBR agonists. To further evaluate FAAH inhibitors as candidate analgesics, this study assessed the effects of the FAAH inhibitor URB597 in assays of acute pain-stimulated and pain-depressed behavior in male Sprague-Dawley rats. Intraperitoneal injection of dilute lactic acid served as a noxious stimulus to stimulate a stretching response or depress positively reinforced operant behavior (intracranial self-stimulation), and URB597 was tested 1 and 4 h after administration. Consistent with FAAH inhibitor effects in other assays of pain-stimulated behavior, URB597 (1-10 mg/kg intraperitoneally) produced dose-related and CB1R-mediated decreases in acid-stimulated stretching. Conversely, in the assay of acid-depressed intracranial self-stimulation, URB597 produced a delayed, partial and non-CBR-mediated antinociceptive effect. The antinociceptive dose of URB597 (10 mg/kg) increased plasma and brain anandamide levels. These results suggest that URB597 produces antinociception in these models of 'pain stimulated' and 'pain depressed' behavior, but with different rates of onset and differential involvement of CBRs.

Topics: Amidohydrolases; Analgesics; Animals; Arachidonic Acids; Behavior, Animal; Benzamides; Brain; Carbamates; Conditioning, Operant; Depression; Dose-Response Relationship, Drug; Electric Stimulation; Endocannabinoids; Enzyme Inhibitors; Implantable Neurostimulators; Injections, Intraperitoneal; Lactic Acid; Male; Pain; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Self Administration

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

Endocannabinoid anandamide (N-arachidonoyl ethanolamide; AEA) has been shown to cause negative inotropic and antiarrhythmic effects in ventricular myocytes. In this study, using whole-cell patch clamp technique, we have investigated the effects of AEA on cardiac Na(+)/Ca(2+) exchanger (NCX1)-mediated currents. AEA suppressed NCX1 with an IC50 value of 4.7 μM. Both inward and outward components of exchanger currents were suppressed by AEA equally. AEA inhibition was mimicked by the metabolically stable analogue, methanandamide (metAEA, 10 μM) while it was not influenced by inhibition of fatty acid amide hydrolase with 1 μM URB597 incubation. The effect of AEA, was not altered in the presence of cannabinoid receptor 1 and 2 antagonists AM251 (1 μM) and AM630 (1 μM), respectively. In addition, inhibition by AEA remained unchanged after pertussis toxin (PTX, 2 μg/ml) treatment or following the inclusion of GDP-β-S (1 mM) in pipette solution. Currents mediated by NCX1 expressed in HEK-293 cells were also inhibited by 10 μM AEA a partially reversible manner. Confocal microscopy images indicated that the intensity of YFP-NCX1 expression on cell surface was not altered by AEA. Collectively, the results indicate that AEA directly inhibits the function of NCX1 in rat ventricular myocytes and in HEK-293 cells expressing NCX1.

Topics: Animals; Arachidonic Acids; Benzamides; Calcium Channel Blockers; Carbamates; Cells, Cultured; Endocannabinoids; HEK293 Cells; Humans; Male; Microscopy, Confocal; Myocytes, Cardiac; Patch-Clamp Techniques; Polyunsaturated Alkamides; Rats; Rats, Wistar; Sodium-Calcium Exchanger

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

Amelioration of neuropathic spinal cord injury (SCI) pain is a clinical challenge. Increasing the endocannabinoid anandamide and other fatty acid amides (FAA) by blocking fatty acid amide hydrolase (FAAH) has been shown to be antinociceptive in a number of animal models of chronic pain. However, an antinociceptive effect of blocking FAAH has yet to be demonstrated in a rat model of neuropathic SCI pain. Four weeks following a SCI, rats developed significantly decreased hind paw withdrawal thresholds, indicative of below-level cutaneous hypersensitivity. A group of SCI rats were systemically treated (i.p.) with either the selective FAAH inhibitor URB597 or vehicle twice daily for seven days. A separate group of SCI rats received a single dose (p.o.) of either the selective FAAH inhibitor PF-3845 or vehicle. Following behavioral testing, levels of the FAA N-arachidonoylethanolamide, N-oleoyl ethanolamide and N-palmitoyl ethanolamide were quantified in brain and spinal cord from SCI rats. Four weeks following SCI, FAA levels were markedly reduced in spinal cord tissue. Although systemic treatment with URB597 significantly increased CNS FAA levels, no antinociceptive effect was observed. A significant elevation of CNS FAA levels was also observed following oral PF-3845 treatment, but only a modest antinociceptive effect was observed. Increasing CNS FAA levels alone does not lead to robust amelioration of below-level neuropathic SCI pain. Perhaps utilizing FAAH inhibition in conjunction with other analgesic mechanisms could be an effective analgesic therapy.

Topics: Amidohydrolases; Analgesics; Animals; Arachidonic Acids; Benzamides; Benzoxazines; Brain; Carbamates; Endocannabinoids; Enzyme Inhibitors; Hindlimb; Male; Morpholines; Naphthalenes; Neuralgia; Pain Measurement; Pain Threshold; Piperidines; Polyunsaturated Alkamides; Pyridines; Rats, Sprague-Dawley; Spinal Cord; Spinal Cord Injuries; Treatment Outcome

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

Anandamide is an endocannabinoid involved in several physiological functions including neuroprotection. Anandamide is synthesized on demand and its endogenous level is regulated through its degradation, where fatty acid amide hydrolase plays a major role. The aim of this study was to characterize anandamide breakdown in physiological and pathological aging and its regulation by CB1 and CB2 receptor agonists. Fatty acid amide hydrolase activity was analyzed in an independent cohort of human cortical membrane samples from control and Alzheimer's disease patients, and in membrane and synaptosomes from adult and aged rat cerebral cortex. Our results demonstrate that fatty acid amide hydrolase activity decreases in the frontal cortex from human patients with Alzheimer's disease and this effect is mimicked by Aβ(1-40) peptide. This activity increases and decreases in aged rat cerebrocortical membranes and synaptosomes, respectively. Also, while the presence of JWH-133, a CB2 selective agonist, slightly increases anandamide hydrolysis in human controls, it decreases this activity in adults and aged rat cerebrocortical membranes and synaptosomes. In the presence of WIN55,212-2, a mixed CB1/CB2 agonist, anandamide hydrolysis increases in Alzheimer's disease patients but decreases in human controls as well as in adult and aged rat cerebrocortical membranes and synaptosomes. Although a similar profile is observed in fatty acid amide hydrolase activity between aged rat synaptic endings and human Alzheimer's disease brains, it is differently modulated by CB1/CB2 agonists. This modulation leads to a reduced availability of anandamide in Alzheimer's disease and to an increased availability of this endocannabinoid in aging.

Topics: Aged; Aged, 80 and over; Aging; Alzheimer Disease; Amidohydrolases; Amyloid beta-Peptides; Animals; Arachidonic Acids; Benzamides; Benzoxazines; Cannabinoid Receptor Agonists; Cannabinoids; Carbamates; Case-Control Studies; Cerebral Cortex; Endocannabinoids; Enzyme Inhibitors; Female; Frontal Lobe; Humans; In Vitro Techniques; Male; Middle Aged; Morpholines; Naphthalenes; Polyunsaturated Alkamides; Rats; Rats, Wistar; Synaptosomes

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

The endogenous cannabinoid system plays an important role in motivation, stress, and drug abuse. Pharmacologically, the endocannabinoid system can be stimulated by either agonists of CB1 receptors or inhibition of metabolic degradation of endogenous cannabinoids and consequent increases in their brain levels.. Here, we investigated whether chronic administration during a period of withdrawal of the fatty acid amide hydrolase inhibitor URB597, which increases anandamide levels, would decrease the risks of relapse to cocaine seeking. Rats were allowed to self-administer cocaine and then they underwent forced withdrawal for 28 days, during which they were treated with URB597 or vehicle. One day after the last injection, we investigated cocaine seeking in one 6h extinction session and relapse triggered by re-exposure to drug-associated cues or a pharmacological stressor.. We found that administration of URB597 significantly decreases cocaine-seeking behavior and cue- and stress-induced relapse.. These results suggest that stimulation of the endocannabinoid system could be helpful to prevent relapse to cocaine addiction.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Carbamates; Cocaine; Cocaine-Related Disorders; Cues; Disease Models, Animal; Dopamine Uptake Inhibitors; Drug-Seeking Behavior; Endocannabinoids; Enzyme Inhibitors; Male; Polyunsaturated Alkamides; Rats, Sprague-Dawley; Recurrence; Self Administration; Stress, Physiological; Yohimbine

The neuronal mechanisms underlying social withdrawal, one of the core negative symptoms of schizophrenia, are not well understood. Recent studies suggest an involvement of the endocannabinoid system in the pathophysiology of schizophrenia and, in particular, of negative symptoms. We used biochemical, pharmacological, and behavioral approaches to investigate the role played by the endocannabinoid system in social withdrawal induced by sub-chronic administration of phencyclidine (PCP). Pharmacological enhancement of endocannabinoid levels via systemic administration of URB597, an inhibitor of endocannabinoid degradation, reversed social withdrawal in PCP-treated rats via stimulation of CB1 receptors, but reduced social interaction in control animals through activation of a cannabinoid/vanilloid-sensitive receptor. In addition, the potent CB agonist CP55,940 reversed PCP-induced social withdrawal in a CB₁-dependent manner, whereas pharmacological blockade of CB₁ receptors by either AM251 or SR141716 reduced the time spent in social interaction in control animals. PCP-induced social withdrawal was accompanied by a decrease of anandamide (AEA) levels in the amygdala and prefrontal cortex, and these deficits were reversed by URB597. As CB₁ receptors are predominantly expressed on GABAergic interneurons containing the anxiogenic peptide cholecystokinin (CCK), we also examined whether the PCP-induced social withdrawal resulted from deficient CB₁-mediated modulation of CCK transmission. The selective CCK2 antagonist LY225910 blocked both PCP- and AM251-induced social withdrawal, but not URB597 effect in control rats. Taken together, these findings indicate that AEA-mediated activation of CB₁ receptors is crucial for social interaction, and that PCP-induced social withdrawal results from deficient endocannabinoid transmission.

Topics: Amidohydrolases; Amygdala; Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Carbamates; Cyclohexanols; Dose-Response Relationship, Drug; Endocannabinoids; Male; Phencyclidine; Piperidines; Polyunsaturated Alkamides; Prefrontal Cortex; Pyrazoles; Quinazolinones; Rats; Receptor, Cannabinoid, CB1; Receptor, Cholecystokinin B; Rimonabant; Schizophrenia; Social Behavior

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

Opioids do not effectively manage pain in many patients with advanced cancer. Because anandamide (AEA) activation of cannabinoid type-1 receptors (CB1R) on nociceptors reduces nociception, manipulation of AEA metabolism in the periphery may be an effective alternative or adjuvant therapy in the management of cancer pain. AEA is hydrolyzed by the intracellular enzyme fatty acid amide hydrolase (FAAH), and this enzyme activity contributes to uptake of AEA into neurons and to reduction of AEA available to activate CB1R. We used an in vitro preparation of adult murine dorsal root ganglion (DRG) neurons co-cultured with fibrosarcoma cells to investigate how tumors alter the uptake of AEA into neurons. Evidence that the uptake of [(3)H]AEA into dissociated DRG cells in the co-culture model mimicked the increase in uptake that occurred in DRG cells from tumor-bearing mice supported the utility of the in vitro model to study AEA uptake. Results with the fluorescent AEA analog CAY10455 confirmed that an increase in uptake in the co-culture model occurred in neurons. One factor that contributed to the increase in [(3)H]AEA uptake was an increase in total cellular cholesterol in the cancer condition. Treatment with the FAAH inhibitor URB597 reduced CAY10455 uptake in the co-culture model to the level observed in DRG neurons maintained in the control condition (i.e., in the absence of fibrosarcoma cells), and this effect was paralleled by OMDM-1, an inhibitor of AEA uptake, at a concentration that had no effect on FAAH activity. Maximally effective concentrations of the two drugs together produced a greater reduction than was observed with each drug alone. Treatment with BMS309403, which competes for AEA binding to fatty acid binding protein-5, mimicked the effect of OMDM-1 in vitro. Local injection of OMDM-1 reduced hyperalgesia in vivo in mice with unilateral tumors in and around the calcaneous bone. Intraplantar injection of OMDM-1 (5μg) into the tumor-bearing paw reduced mechanical hyperalgesia through a CB1R-dependent mechanism and also reduced a spontaneous nocifensive behavior. The same dose reduced withdrawal responses evoked by suprathreshold mechanical stimuli in naive mice. These data support the conclusion that OMDM-1 inhibits AEA uptake by a mechanism that is independent of inhibition of FAAH and provide a rationale for the development of peripherally restricted drugs that decrease AEA uptake for the management of cancer pain.

Topics: Animals; Arachidonic Acids; Benzamides; Brain Neoplasms; Cannabinoid Receptor Antagonists; Carbamates; Cells, Cultured; Coculture Techniques; Disease Models, Animal; Endocannabinoids; Enzyme Inhibitors; Fibrosarcoma; Fluorescent Dyes; Ganglia, Spinal; Hyperalgesia; Indoles; Lactones; Male; Mice; Mice, Inbred C3H; Pain; Pain Threshold; Polyunsaturated Alkamides; Sensory Receptor Cells; Statistics, Nonparametric; Tritium

Coping styles are fundamental characteristics of behavior that affect susceptibility to, and resilience during, mental and physical illness. Shifts from passive to active coping are considered therapeutic goals in many stress-related disorders, but the neural control of coping is poorly understood. Based on earlier findings, we hypothesized that coping styles are influenced by endocannabinoids.. Here, we tested whether FAAH inhibition by URB597 affects behaviors aimed at controlling a critical situation and the degree to which environmental stimuli influence behavior i.e., we studied the impact of URB597 on the two main attributes of coping styles.. Rats were tested in the tail-pinch test of coping and in the elevated plus-maze test that was performed under highly divergent conditions.. Under the effects of URB597, rats focused their behavior more on the discomfort-inducing clamp in the tail-pinch test, i.e., they coped with the challenge more actively. In the elevated plus-maze, URB597-treated rats demonstrated an autonomous behavioral control by reducing both "wariness" induced by aversive conditions and "carelessness" resulting from favorable conditions.. URB597 treatment-induced behavioral changes indicated a shift towards active coping with challenges. This behavioral change appears compatible with the previously suggested role of endocannabinoids in emotional homeostasis. Albeit further studies are required to characterize the role of endocannabinoids in coping, these findings suggest that the enhancement of endocannabinoid signaling may become a therapeutic option in emotional disorders characterized by passive coping (e.g., anxiety and depression) and in physical diseases where active coping is therapeutically desirable.

Topics: Adaptation, Psychological; Amidohydrolases; Animals; Arachidonic Acids; Behavior, Animal; Benzamides; Carbamates; Endocannabinoids; Male; Maze Learning; Polyunsaturated Alkamides; Rats; Rats, Wistar; Signal Transduction

(1) The aim of the present study was to evaluate the effects of intracerebroventricular administration of anandamide (AEA), an inhibitor of fatty acid amide hydrolase activity (URB597) and a CB1 receptor (CB1 R) antagonist (AM251) on the homeostatic responses elicited by extracellular volume expansion (EVE) in male adult rats. (2) Pretreatment with AEA (100 ng/4 μL) significantly reduced the effect of hypertonic (H-) EVE on plasma concentrations of prolactin (PRL), oxytocin (OT) and corticosterone, but not vasopressin (AVP). Administration of URB597 (20 μg/5 μL) alone significantly reduced PRL, OT, AVP and corticosterone in the H-EVE group. Conversely, URB597 and AEA had no significant effect on basal hormone concentrations. Pretreatment with AM251 (200 ng/2 μL) potentiated OT but did not change AVP plasma levels in the H-EVE group. (3) Hypertonic EVE significantly increased AVP and OT mRNA expression in the supraoptic nucleus (SON), an effect that was blunted in AEA-pretreated rats. Pretreatment with AEA did not change the percentage of vasopressinergic or oxytocinergic neurons colocalizing c-Fos in the SON, but increased nitrate concentrations in the median eminence of animals subjected to H-EVE. (4) The present data suggest that: (i) vasopressinergic and oxytocinergic neurons may be differentially affected by AEA; (ii) activation of CB1 R may restrain the response of the neurohypophyseal system (NHS) to EVE; (iii) the hypothalamic-pituitary-adrenal axis, PRL and the NHS may still be sensitive to AEA after EVE, with these effects probably not dependent on AEA metabolism; and (iv) AEA and nitric oxide could interact in vivo as modulators to directly control stress-induced responses.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Carbamates; Endocannabinoids; Gene Expression Regulation; Infusions, Intraventricular; Male; Median Eminence; Neuroprotective Agents; Nitrates; Oxytocin; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; RNA, Messenger; Saline Solution, Hypertonic; Vasopressins

In contextual fear conditioning animals have to integrate various elemental stimuli into a coherent representation of the condition and then associate context representation with punishment. Although several studies indicated the modulating role of endocannabinoid system (ECS) on the associative learning, ECS effect on contextual fear conditioning requires further investigations. The present study assessed the effects of the increased endocannabinoid anandamide (AEA) tone on acquisition, retrieval and extinction of the contextual fear conditioning. Given that AEA may bind to cannabinoid type 1 (CB1) receptors as well as to postsynaptic ionotropic Transient Receptor Potential Vanilloid type 1 (TRPV1) channels, particular attention was paid in determining how the increased AEA tone influenced fear responses. Furthermore, it was investigated how the ECS modulated the effects of stress-sensitization on fear response. Thus, mice submitted or not to a social defeat stress protocol were treated with drugs acting on ECS, CB1 receptors or TRPV1 channels and tested in a contextual fear conditioning whose conditioning, retrieval and extinction phases were analyzed. ECS activation influenced the extinction process and contrasted the stress effects on fear memory. Furthermore, CB1 receptor antagonist blocked and TRPV1 channel antagonist promoted short- and long-term extinction. The present study indicates that ECS controls the extinction of aversive memories in the contextual fear conditioning.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Carbamates; Endocannabinoids; Enzyme Inhibitors; Extinction, Psychological; Fear; Freezing Reaction, Cataleptic; Male; Memory; Mice, Inbred C57BL; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Social Dominance; Stress, Psychological; Time Factors; TRPV Cation Channels

Endocannabinoids are important regulators of organ homeostasis. Although their role in systemic vasculature has been extensively studied, their impact on pulmonary vessels remains less clear. Herein, we show that the endocannabinoid anandamide (AEA) is a key mediator of hypoxic pulmonary vasoconstriction (HPV) via fatty acid amide hydrolase (FAAH)-dependent metabolites. This is underscored by the prominent vasoconstrictive effect of AEA on pulmonary arteries and strongly reduced HPV in FAAH(-/-) mice and wild-type mice upon pharmacological treatment with FAAH inhibitor URB597. In addition, mass spectrometry measurements revealed a clear increase of AEA and the FAAH-dependent metabolite arachidonic acid in hypoxic lungs of wild-type mice. We have identified pulmonary vascular smooth muscle cells as the source responsible for hypoxia-induced AEA generation. Moreover, either FAAH(-/-) mice or wild-type mice treated with FAAH inhibitor URB597 are protected against hypoxia-induced pulmonary hypertension and the concomitant vascular remodeling in the lung. Thus, the AEA/FAAH pathway is an important mediator of HPV and is involved in the generation of pulmonary hypertension.

Topics: Amidohydrolases; Analysis of Variance; Animals; Arachidonic Acids; Benzamides; Blotting, Western; Carbamates; Chromatography, Liquid; DNA Primers; Endocannabinoids; Hypertension, Pulmonary; Hypoxia; Immunohistochemistry; Lung; Mice; Mice, Knockout; Myocytes, Smooth Muscle; Polyunsaturated Alkamides; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Vasoconstriction

High-fat diet (HFD)-induced obesity and insulin resistance are associated with increased activity of the endocannabinoid/CB1 receptor (CB1R) system that promotes the hepatic expression of lipogenic genes, including stearoyl-CoA desaturase-1 (SCD1). Mice deficient in CB1R or SCD1 remain lean and insulin-sensitive on an HFD, suggesting a functional link between the two systems. The HFD-induced increase in the hepatic levels of the endocannabinoid anandamide [i.e., arachidonoylethanolamide (AEA)] has been attributed to reduced activity of the AEA-degrading enzyme fatty acid amide hydrolase (FAAH). Here we show that HFD-induced increased hepatic AEA levels and decreased FAAH activity are absent in SCD1(-/-) mice, and the monounsaturated fatty acid (MUFA) products of SCD1, palmitoleic and oleic acid, inhibit FAAH activity in vitro at low micromolar concentrations. HFD markedly increases hepatic SCD1 activity in WT mice as well as in CB1R(-/-) mice with transgenic reexpression of CB1R in hepatocytes, but not in global CB1R(-/-) mice. Treatment of HFD-fed mice with the SCD1 inhibitor A939572 prevents the diet-induced reduction of hepatic FAAH activity, normalizes hepatic AEA levels, and improves insulin sensitivity. SCD1(-/-) mice on an HFD remain insulin-sensitive, but develop glucose intolerance and insulin resistance in response to chronic treatment with the FAAH inhibitor URB597. An HFD rich in MUFA or feeding mice pure oleic acid fail to inhibit hepatic FAAH activity. We conclude that MUFAs generated via SCD1 activity, but not diet-derived MUFAs, function as endogenous FAAH inhibitors mediating the HFD-induced increase in hepatic AEA, which then activates hepatic CB1R to induce insulin resistance.

Topics: Amidohydrolases; Analysis of Variance; Animals; Arachidonic Acids; Benzamides; Carbamates; Endocannabinoids; Fatty Acids, Monounsaturated; Feedback, Physiological; Insulin Resistance; Liver; Male; Mice; Mice, Inbred C57BL; Obesity; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Stearoyl-CoA Desaturase

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

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

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

Endocannabinoids such as anandamide (AEA) are important lipid ligands regulating cell proliferation, differentiation and apoptosis. Their levels are regulated by hydrolase enzymes, the fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL). Here, we investigated whether FAAH or AEA are involved in NF (erythroid-derived 2)-like 2 (Nrf2)/antioxidant responsive element (ARE) pathway.. The aim of this study was to analyse the effects of AEA or FAAH inhibition by the URB597 inhibitor or FAAH/siRNA on the activation of Nrf2-ARE signalling pathway and heme oxygenase-1 (HO-1) induction and transcription.. Endogenous AEA was detected in the immortalized human mammary epithelial MCF-10A cells (0.034 ng per 10(6) cells) but not in MCF-7 or MDA-MB-231 breast cancer cells. Because breast tumour cells express FAAH abundantly, we examined the effects of FAAH on Nrf2/antioxidant pathway. We found that inhibition of FAAH by the URB597 inhibitor induced antioxidant HO-1 in breast cancer cells and MCF-10A cells. RNAi-mediated knockdown of FAAH or treatment with AEA-activated ARE-containing reporter induced HO-1 mRNA and protein expression, independent of the cannabinoid receptors, CB1, CB2 or TRPV1. Furthermore, URB597, AEA and siRNA-FAAH treatments induced the nuclear translocation of Nrf2, while siRNA-Nrf2 treatment and Keap1 expression blocked AEA, URB597 and si-FAAH from activation of ARE reporter and HO-1 induction. siRNA-HO-1 treatment decreased the viability of breast cancer cells and MCF-10A cells.. These data uncovered a novel mechanism by which inhibition of FAAH or exposure to AEA induced HO-1 transcripts and implicating AEA and FAAH as direct modifiers in signalling mediated activation of Nrf2-HO-1 pathway, independent of cannabinoid receptors.

Topics: Active Transport, Cell Nucleus; Amidohydrolases; Antineoplastic Agents; Arachidonic Acids; Benzamides; Breast Neoplasms; Carbamates; Cell Survival; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Induction; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Heme Oxygenase-1; Humans; MCF-7 Cells; NF-E2-Related Factor 2; Polyunsaturated Alkamides; RNA Interference; Signal Transduction; Transcription, Genetic; Transfection

The endocannabinoid system regulates nociception and aversion and mediates fear-conditioned analgesia (FCA). We investigated the effects of the fatty acid amide hydrolase (FAAH) inhibitor URB597, which inhibits the catabolism of the endocannabinoid anandamide and related N-acylethanolamines, on expression of FCA and fear and pain related behaviour per se in rats. We also examined associated alterations in the expression of the signal transduction molecule phospho-Akt in the periaqueductal grey (PAG) by immunoblotting. FCA was modelled by assessing formalin-evoked nociceptive behaviour in an arena previously paired with footshock. URB597 (0.3 mg/kg, i.p.) enhanced FCA and increased fear-related behaviour in formalin-treated rats. Conditioned fear per se in non-formalin-treated rats was associated with increased expression of phospho-Akt in the PAG. URB597 reduced the expression of fear-related behaviour in the early part of the trial, an effect that was accompanied by attenuation of the fear-induced increase in phospho-Akt expression in the PAG. Intra-plantar injection of formalin also reduced the fear-induced increase in phospho-Akt expression. These data provide evidence for a role of FAAH in FCA, fear responding in the presence or absence of nociceptive tone, and fear-evoked increases in PAG phospho-Akt expression. In addition, the results suggest that fear-evoked activation of Akt signalling in the PAG is abolished in the presence of nociceptive tone.

Topics: Amidohydrolases; Analgesia; Animals; Arachidonic Acids; Behavior, Animal; Benzamides; Cannabinoid Receptor Modulators; Carbamates; Conditioning, Psychological; Endocannabinoids; Ethanolamines; Exploratory Behavior; Fear; Formaldehyde; Hippocampus; Male; Motor Activity; Nociception; Pain; Periaqueductal Gray; Polyunsaturated Alkamides; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction

Activation of cannabinoid CB(1) receptor is shown to inhibit marble-burying behavior (MBB), a behavioral model for assessing obsessive-compulsive disorder (OCD). Anandamide, an endogenous agonist at CB(1) receptor also activates the transient receptor potential vanilloid type 1 (TRPV1) channels but at a higher concentration. Furthermore, anandamide-mediated TRPV1 effects are opposite to that of the CB(1) receptor. Therefore, the present study was carried out to investigate the influence of low and high doses of anandamide on MBB in CB(1) and TRPV1 antagonist pre-treated mice. The results revealed that i.c.v. administration of lower doses of anandamide (1-10 μg/mouse) or its analogues (AM404 or URB597; 1-5 μg/mouse) inhibited MBB indicating the anticompulsive activity. Conversely, at higher doses (40 or 20 μg/mouse) these compounds increased MBB similar to capsaicin (TRPV1 agonist, 100 μg/mouse) exhibiting a pro-compulsive effect. Pretreatment with AM251 (CB(1) antagonist, 1 μg/mouse) antagonized the anticompulsive effect of these compounds, while their pro-compulsive effect at higher doses was attenuated by inactive dose of capsazepine (TRPV1 antagonist, 10 μg/mouse). However, capsazepine per se at a higher dose (100 μg/mouse) inhibited MBB. When given daily for 14 days, the anticompulsive effect of anandamide and its analogues gradually disappeared, whereas capsazepine either alone or with URB597 produced consistent inhibition of MBB comparable to fluoxetine. Thus, the study indicates the biphasic influence of anandamide on MBB, and chronic administration of capsazepine either alone or with URB597 might be an effective tool in the treatment of OCD.

Topics: Animals; Arachidonic Acids; Behavior, Animal; Benzamides; Cannabinoids; Carbamates; Endocannabinoids; Mice; Motor Activity; Polyunsaturated Alkamides; TRPV Cation Channels

We hypothesized that arachidonic acid produced by anandamide breakdown contributes to the vascular effects of anandamide.. Isolated, pressurized rat skeletal muscle arteries, which possess spontaneous myogenic tone, were treated with anandamide, arachidonic acid, capsaicin (vanilloid receptor agonist), WIN 55-212-2 (cannabinoid receptor agonist), URB-597 (FAAH inhibitor), baicalein (lipoxygenase inhibitor), PPOH (cytochrome P450 inhibitor), and indomethacin (cyclooxygenase inhibitor). Changes in the arteriolar diameter in response to the various treatments were measured. To assess the effect of anandamide metabolism, anandamide was applied for 20 min followed by washout for 40 min. This protocol was used to eliminate other, more direct effects of anandamide in order to reveal how anandamide metabolism may influence vasodilation.. Anandamide at a low dose (1μM) evoked a loss of myogenic tone, while a high dose (30 μM) not only attenuated the myogenic response but also evoked acute dilation. Both of these effects were inhibited by the FAAH inhibitor URB-597 and were mimicked by arachidonic acid. The CB1 and CB2 agonist R-WIN 55-212-2 and the vanilloid receptor agonist capsaicin were without effect on the myogenic response. The inhibition of the myogenic response by anandamide was blocked by indomethacin and PPOH, but not by baicalein or removal of the endothelium. FAAH expression in the smooth muscle cells of the blood vessels was confirmed by immunohistochemistry.. Anandamide activates the arachidonic acid pathway in the microvasculature, affecting vascular autoregulation (myogenic response) and local perfusion.

Topics: Analysis of Variance; Animals; Arachidonic Acids; Arterioles; Benzamides; Benzoxazines; Blood Pressure; Caproates; Capsaicin; Carbamates; Dose-Response Relationship, Drug; Endocannabinoids; Flavanones; Immunohistochemistry; In Vitro Techniques; Indomethacin; Morpholines; Muscle Contraction; Muscle, Smooth, Vascular; Naphthalenes; Polyunsaturated Alkamides; Rats

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

The incidence of melanoma is considerably increasing worldwide. Frequent failing of classical treatments led to development of novel therapeutic strategies aiming at managing advanced forms of this skin cancer. Additionally, the implication of the endocannabinoid system in malignancy is actively investigated.. We investigated the cytotoxicity of endocannabinoids and their hydrolysis inhibitors on the murine B16 melanoma cell line using a MTT test. Enzyme and receptor expression was measured by RT-PCR and enzymatic degradation of endocannabinoids using radiolabeled substrates. Cell death was assessed by Annexin-V/Propidium iodine staining. Tumors were induced in C57BL/6 mice by s.c. flank injection of B16 melanoma cells. Mice were injected i.p. for six days with vehicle or treatment, and tumor size was measured each day and weighted at the end of the treatment. Haematoxylin-Eosin staining and TUNEL assay were performed to quantify necrosis and apoptosis in the tumor and endocannabinoid levels were quantified by HPLC-MS. Tube formation assay and CD31 immunostaining were used to evaluate the antiangiogenic effects of the treatments.. The N-arachidonoylethanolamine (anandamide, AEA), 2-arachidonoylglycerol and N- palmitoylethanolamine (PEA) reduced viability of B16 cells. The association of PEA with the fatty acid amide hydrolase (FAAH) inhibitor URB597 considerably reduced cell viability consequently to an inhibition of PEA hydrolysis and an increase of PEA levels. The increase of cell death observed with this combination of molecules was confirmed in vivo where only co-treatment with both PEA and URB597 led to decreased melanoma progression. The antiproliferative action of the treatment was associated with an elevation of PEA levels and larger necrotic regions in the tumor.. This study suggests the interest of targeting the endocannabinoid system in the management of skin cancer and underlines the advantage of associating endocannabinoids with enzymatic hydrolysis inhibitors. This may contribute to the improvement of long-term palliation or cure of melanoma.

Topics: Amides; Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Modulators; Carbamates; Cell Death; Cell Line, Tumor; Cell Survival; Disease Models, Animal; Drug Synergism; Endocannabinoids; Ethanolamines; Male; Mass Spectrometry; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Palmitic Acids; Polyunsaturated Alkamides; Random Allocation; Reverse Transcriptase Polymerase Chain Reaction; Skin Neoplasms

Inhibitors of fatty acid amide hydrolase (FAAH) and anandamide (AEA) uptake, which limit the degradation of endogenous cannabinoids, have received interest as potential therapeutics for pain. There is also evidence that endogenous cannabinoids mediate the antinociceptive effects of opioids. Assays of pain-elicited and pain-suppressed behavior have been used to differentiate the effects of drugs that specifically alter nociception from drugs that alter nociception caused by nonspecific effects such as catalepsy or a general suppression of activity. Using such procedures, this study examines the effects of the direct cannabinoid type 1 (CB1) agonist (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55940), the FAAH inhibitor cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-yl ester (URB597), and the AEA uptake inhibitor N-(4-hydroxyphenyl) arachidonylamide (AM404). Additional experiments examined these compounds in combination with morphine. CP55940 produced antinociception in assays of pain-elicited, but not pain-suppressed, behavior and disrupted responding in an assay of schedule-controlled behavior. URB597 and AM404 produced antinociception in assays of pain-elicited and pain-suppressed behavior in which acetic acid was the noxious stimulus, but had no effect on the hotplate and schedule-controlled responding. CP55940 in combination with morphine resulted in effects greater than those of morphine alone in assays of pain-elicited and scheduled-controlled behavior but not pain-suppressed behavior. URB597 in combination with morphine resulted in enhanced morphine effects in assays of pain-elicited and pain-suppressed behavior in which diluted acetic acid was the noxious stimulus, but did not alter morphine's effects on the hotplate or schedule-controlled responding. These studies suggest that, compared with direct CB1 agonists, manipulations of endogenous cannabinoid signaling have enhanced clinical potential; however, their effects depend on the type of noxious stimulus.

Topics: Amidohydrolases; Analgesics; Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Modulators; Carbamates; Cyclohexanols; Endocannabinoids; Male; Mice; Mice, Inbred C57BL; Morphine; Nociception; Pain; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1

Painful peripheral neuropathy is a dose-limiting complication of chemotherapy. Cisplatin produces a cumulative toxic effect on peripheral nerves, and 30-40% of cancer patients receiving this agent experience pain. By modeling cisplatin-induced hyperalgesia in mice with daily injections of cisplatin (1 mg/kg, i.p.) for 7 d, we investigated the anti-hyperalgesic effects of anandamide (AEA) and cyclohexylcarbamic acid 3'-carbamoyl-biphenyl-3-yl ester (URB597), an inhibitor of AEA hydrolysis. Cisplatin-induced mechanical and heat hyperalgesia were accompanied by a decrease in the level of AEA in plantar paw skin. No changes in motor activity were observed after seven injections of cisplatin. Intraplantar injection of AEA (10 μg/10 μl) or URB597 (9 μg/10 μl) transiently attenuated hyperalgesia through activation of peripheral CB₁ receptors. Co-injections of URB597 (0.3 mg/kg daily, i.p.) with cisplatin decreased and delayed the development of mechanical and heat hyperalgesia. The effect of URB597 was mediated by CB₁ receptors since AM281 (0.33 mg/kg daily, i.p.) blocked the effect of URB597. Co-injection of URB597 also normalized the cisplatin-induced decrease in conduction velocity of Aα/Aβ-fibers and reduced the increase of ATF-3 and TRPV1 immunoreactivity in dorsal root ganglion (DRG) neurons. Since DRGs are a primary site of toxicity by cisplatin, effects of cisplatin were studied on cultured DRG neurons. Incubation of DRG neurons with cisplatin (4 μg/ml) for 24 h decreased the total length of neurites. URB597 (100 nM) attenuated these changes through activation of CB₁ receptors. Collectively, these results suggest that pharmacological facilitation of AEA signaling is a promising strategy for attenuating cisplatin-associated sensory neuropathy.

Topics: Activating Transcription Factor 3; Animals; Antineoplastic Agents; Arachidonic Acids; Benzamides; Cannabinoid Receptor Modulators; Carbamates; Cells, Cultured; Cisplatin; Disease Models, Animal; Drug Interactions; Endocannabinoids; Enzyme Inhibitors; Ganglia, Spinal; Hyperalgesia; Male; Mice; Mice, Inbred C3H; Morpholines; Motor Activity; Neurites; Neurotoxicity Syndromes; Peripheral Nervous System Diseases; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; TRPV Cation Channels

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

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

While the etiology of depression is not clearly understood at the present time, this mental disorder is thought be a complex and multifactorial trait with important genetic and environmental contributing factors.. The role of the endocannabinoid (eCB) system in depressive behavior was examined in Wistar Kyoto (WKY) rat strain, a genetic model of depression. Our findings revealed selective abnormalities in the eCB system in the brains of WKY rats compared to Wistar (WIS) rats. Immunoblot analysis indicated significantly higher levels of fatty acid amide hydrolase (FAAH) in frontal cortex and hippocampus of WKY rats with no alteration in the level of N-arachidonyl phosphatidyl ethanolamine specific phospholipase-D (NAPE-PLD). Significantly higher levels of CB1 receptor-mediated G-protein coupling and lower levels of anandamide (AEA) were found in frontal cortex and hippocampus of WKY rats. While the levels of brain derived neurotropic factor (BDNF) were significantly lower in frontal cortex and hippocampus of WKY rats compared to WIS rats, pharmacological inhibition of FAAH elevated BDNF levels in WKY rats. Inhibition of FAAH enzyme also significantly increased sucrose consumption and decreased immobility in the forced swim test in WKY rats.. These findings suggest a critical role for the eCB system and BDNF in the genetic predisposition to depressive-like behavior in WKY rats and point to the potential therapeutic utility of eCB enhancing agents in depressive disorder.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Brain; Brain-Derived Neurotrophic Factor; Cannabinoid Receptor Modulators; Carbamates; Depressive Disorder; Disease Models, Animal; Endocannabinoids; Enzyme Inhibitors; Frontal Lobe; Genetic Predisposition to Disease; Hippocampus; Male; Phospholipase D; Polyunsaturated Alkamides; Rats; Rats, Inbred WKY; Rats, Wistar; Receptor, Cannabinoid, CB1; Species Specificity; Swimming

Endocannabinoids exhibit vasodilatory properties and reduce blood pressure in vivo. However, the influence and mechanism of action of the prominent endocannabinoid, anandamide (AEA), in pulmonary arteries are not known. The present study determined the vascular response to AEA in isolated rat pulmonary arteries. AEA relaxed rat pulmonary arteries that were pre-constricted with U-46619. This relaxation was reduced by the following conditions:removal of the endothelium; in KCl pre-constricted preparations; in the presence of the potassium channel (K(Ca)) blockers, tetraethylammonium and the combination of charybdotoxin and apamin, and the prostacyclin receptor antagonist, RO1138452. Inhibitors of cyclooxygenase (indomethacin), nitric oxide (NO) synthase (N(G)-nitro-l-arginine methyl ester) and fatty acid amide hydrolase (URB597) alone or in combination diminished AEA-induced relaxation in endothelium-intact vessels. The remaining experiments were performed in the presence of URB597 to eliminate the influence of AEA metabolites. Antagonists of the endothelial cannabinoid receptor (CB(x)), O-1918 and cannabidiol, attenuated the AEA-induced response. Antagonists of CB(1), CB(2) and TRPV1 receptors, AM251, AM630 and capsazepine, respectively, did not modify the AEA-induced response. A reference activator of CB(x) receptors, abnormal cannabidiol, mimicked the receptor-mediated AEA effects. The present study demonstrated that AEA relaxed rat pulmonary arteries in an endothelium-dependent fashion via the activation of the O-1918-sensitive CB(x) receptor and/or prostacyclin-like vasoactive products of AEA. One or both of these mechanisms may involve K(Ca) or the NO pathway.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Amidohydrolases; Animals; Anisoles; Apamin; Arachidonic Acids; Benzamides; Benzyl Compounds; Cannabinoid Receptor Antagonists; Capsaicin; Carbamates; Charybdotoxin; Cyclohexanes; Endocannabinoids; Endothelium, Vascular; Imidazoles; Indoles; Indomethacin; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Piperidines; Polyunsaturated Alkamides; Potassium Channel Blockers; Potassium Chloride; Prostaglandin-Endoperoxide Synthases; Pulmonary Artery; Pyrazoles; Rats; Rats, Wistar; Receptors, Cannabinoid; Receptors, Epoprostenol; Tetraethylammonium; TRPV Cation Channels; Vasodilation

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

The functional significance of adult neural stem and progenitor cells in hippocampal-dependent learning and memory has been well documented. Although adult neural stem and progenitor cells in the subventricular zone are known to migrate to, maintain and reorganize the olfactory bulb, it is less clear whether they are functionally required for other processes. Using a conditional transgenic mouse model, selective ablation of adult neural stem and progenitor cells in the subventricular zone induced a dramatic increase in morbidity and mortality of central nervous system disorders characterized by excitotoxicity-induced cell death accompanied by reactive inflammation, such as 4-aminopyridine-induced epilepsy and ischaemic stroke. To test the role of subventricular zone adult neural stem and progenitor cells in protecting central nervous system tissue from glutamatergic excitotoxicity, neurophysiological recordings of spontaneous excitatory postsynaptic currents from single medium spiny striatal neurons were measured on acute brain slices. Indeed, lipopolysaccharide-stimulated, but not unstimulated, subventricular zone adult neural stem and progenitor cells reverted the increased frequency and duration of spontaneous excitatory postsynaptic currents by secreting the endocannabinod arachidonoyl ethanolamide, a molecule that regulates glutamatergic tone through type 1 cannabinoid receptor (CB(1)) binding. In vivo restoration of cannabinoid levels, either by administration of the type 1 cannabinoid receptor agonist HU210 or the inhibitor of the principal catabolic enzyme fatty acid amide hydrolase, URB597, completely reverted the increased morbidity and mortality of adult neural stem and progenitor cell-ablated mice suffering from epilepsy and ischaemic stroke. Our results provide the first evidence that adult neural stem and progenitor cells located within the subventricular zone exert an 'innate' homeostatic regulatory role by protecting striatal neurons from glutamate-mediated excitotoxicity.

Topics: 4-Aminopyridine; Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Carbamates; Corpus Striatum; Disease Models, Animal; Dronabinol; Endocannabinoids; Epilepsy; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Ganciclovir; Glutamic Acid; Lateral Ventricles; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neural Stem Cells; Neuroprotective Agents; Polyunsaturated Alkamides; Stem Cells; Stroke

Obsessive-compulsive disorder (OCD) is a common psychiatric disorder characterized by the occurrence of obsessions and compulsions. Glutamatergic abnormalities have been related to the pathophysiology of OCD. Cannabinoids inhibit glutamate release in the central nervous system, but the involvement of drugs targeting the endocannabinoid system has not yet been tested in animal models of repetitive behavior. Thus, the aim of the present study was to verify the effects of the CB1 receptor agonist WIN55,212-2, the inhibitor of anandamide uptake AM404 and the anandamide hydrolysis inhibitor URB597, on compulsive-associate behavior in male C57BL/6J mice submitted to the marble burying test (MBT), an animal model used for anti-compulsive drug screening. WIN55,212-2 (1 and 3 mg/kg), AM404 (1 and 3 mg/kg) and URB597 (0.1, 0.3 and 1 mg/kg) induced a significant decrease in the number of buried marbles compared to controls. Pretreatment with the CB1 receptor antagonist, AM251, prevented both WIN55,212-2 and URB597 effects. These results suggest a potential role for drugs acting on the cannabinoid system in modulating compulsive behavior.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Behavior, Animal; Benzamides; Benzoxazines; Calcium Channel Blockers; Cannabinoids; Carbamates; Drug Interactions; Endocannabinoids; Male; Mice; Mice, Inbred C57BL; Morpholines; Motor Activity; Naphthalenes; Obsessive-Compulsive Disorder; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Synaptic Transmission

The fatty acid amide hydrolase inhibitor URB 597 increases brain anandamide levels, suggesting that URB 597 could enhance the behavioural effects of anandamide. The goal of the current study was to examine and characterize the in vivo pharmacology of URB 597 alone and in combination with anandamide and Δ⁹-tetrahydrocannabinol (Δ⁹ -THC) in two drug discrimination assays in rhesus monkeys.. The effects of URB 597 alone and in combination with anandamide were investigated in one group of monkeys (n= 4) that discriminated Δ⁹-THC (0.1 mg·kg⁻¹ i.v.) from vehicle, and in another group (n= 5) receiving chronic Δ⁹-THC (1 mg·kg⁻¹ 12 h⁻¹ s.c.) that discriminated the cannabinoid antagonist rimonabant (1 mg·kg⁻¹ i.v.).. Intravenous anandamide fully substituted for, and had infra-additive effects with, Δ⁹-THC. URB 597 (up to 3.2 mg·kg⁻¹ i.v.) did not substitute for or modify the effects of Δ⁹-THC but markedly increased the potency (32-fold) and duration of action of anandamide. The rimonabant discriminative stimulus in Δ⁹-THC-treated monkeys (i.e. Δ⁹-THC withdrawal) was attenuated by both Δ⁹-THC (at doses larger than 1 mg·kg⁻¹ per 12 h) and anandamide but not by URB 597 (3.2 mg·kg⁻¹). URB 597 did not increase the potency of anandamide to attenuate the rimonabant-discriminative stimulus.. URB 597 enhanced the behavioural effects of anandamide but not other CB₁ agonists. However, URB 597 did not significantly enhance the attenuation of Δ⁹-THC withdrawal induced by anandamide. Collectively, these data suggest that endogenous anandamide in primate brain does not readily mimic the behavioural effects of exogenously administered anandamide.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Carbamates; Discrimination, Psychological; Dronabinol; Drug Interactions; Drug Therapy, Combination; Endocannabinoids; Female; Macaca mulatta; Male; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rimonabant

Endocannabinoid analogues exhibit antidepressant and anti-compulsive like effects similar to that of serotonin selective reuptake inhibitors (SSRIs) indicating a parallelism between the effects of serotonin and endocannabinoids. Therefore, the present study was designed to investigate the role of endocannabinoids in the antidepressant and anti-compulsive like effect of fluoxetine using mice model of forced swim test (FST) and marble-burying behavior (MBB). The results revealed that intracerebroventricular injections of endocannabinoid analogues, anandamide, a CB(1) agonist (AEA: 1-20 μg/mouse); AM404, an anandamide transport inhibitor (0.1-10 μg/mouse); and URB597, a fatty acid amide hydrolase inhibitor (0.05-10 μg/mouse) produced antidepressant-like effect dose-dependently, whereas influenced the MBB in a biphasic manner (produced a U-shaped dose-response curve). Fluoxetine (2.5-20 mg/kg, i.p.) dose dependently decreased the immobility time as well as burying behavior. Co-administration of sub-effective dose of fluoxetine (2.5 mg/kg, i.p.) potentiated the effect of sub-effective dose of AEA (0.5 μg/mouse, i.c.v.), AM404 (0.05 μg/mouse, i.c.v) or URB597 (0.01 μg/mouse, i.c.v) in both the paradigms. Interestingly, pretreatment with AM251, a CB(1) antagonist, blocked the effect of fluoxetine in FST and MBB at a dose (1 μg/mouse, i.c.v) that per se had no effect on either parameter. Similar effects were obtained with endocannabinoid analogues in AM251 pretreated mice. However, AM251 increased the burying behavior in MBB at a highest dose tested (5 μg/mouse). None of the treatments had any influence on locomotor activity. Thus, the study indicates an interaction between endocannabinoid and serotonergic system in regulation of depressive and compulsive-like behavior.

Topics: Animals; Antidepressive Agents; Arachidonic Acids; Benzamides; Cannabinoid Receptor Modulators; Carbamates; Compulsive Behavior; Depression; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Drug Therapy, Combination; Endocannabinoids; Fluoxetine; Injections, Intraventricular; Male; Mice; Motor Activity; Piperidines; Polyunsaturated Alkamides; Pyrazoles

Nitric oxide production, catalyzed by nitric oxide synthase (NOS), should be strictly regulated to allow embryo implantation. Thus, our first aim was to study NOS activity during peri-implantation in the rat uterus. Day 6 inter-implantation sites showed lower NOS activity (0.19±0.01 pmoles L-citrulline mg prot(-1) h(-1)) compared to days 4 (0.34±0.03) and 5 (0.35±0.02) of pregnancy and to day 6 implantation sites (0.33±0.01). This regulation was not observed in pseudopregnancy. Both dormant and active blastocysts maintained NOS activity at similar levels. Anandamide (AEA), an endocannabinoid, binds to cannabinoid receptors type 1 (CB1) and type 2 (CB2), and high concentrations are toxic for implantation and embryo development. Previously, we observed that AEA synthesis presents an inverted pattern compared to NOS activity described here. We adopted a pharmacological approach using AEA, URB-597 (a selective inhibitor of fatty acid amide hydrolase, the enzyme that degrades AEA) and receptor selective antagonists to investigate the effect of AEA on uterine NOS activity in vitro in rat models of implantation. While AEA (0.70±0.02 vs 0.40±0.04) and URB-597 (1.08±0.09 vs 0.83±0.06) inhibited NOS activity in the absence of a blastocyst (pseudopregnancy) through CB2 receptors, AEA did not modulate NOS on day 5 pregnant uterus. Once implantation begins, URB-597 decreased NOS activity on day 6 implantation sites via CB1 receptors (0.25±0.04 vs 0.40±0.05). While a CB1 antagonist augmented NOS activity on day 6 inter-implantation sites (0.17±0.02 vs 0.27±0.02), a CB2 antagonist decreased it (0.17±0.02 vs 0.12±0.01). Finally, we described the expression and localization of cannabinoid receptors during implantation. In conclusion, AEA levels close to and at implantation sites seems to modulate NOS activity and thus nitric oxide production, fundamental for implantation, via cannabinoid receptors. This modulation depends on the presence of the blastocyst. These data establish cannabinoid receptors as an interesting target for the treatment of implantation deficiencies.

Topics: Animals; Arachidonic Acids; Benzamides; Blastocyst; Cannabinoid Receptor Modulators; Carbamates; Embryo Implantation; Endocannabinoids; Female; Immunohistochemistry; Nitric Oxide Synthase; Polymerase Chain Reaction; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Uterus

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

Fatty acid amide hydrolase (FAAH) is the key hydrolytic enzyme for the endogenous cannabinoid receptor ligand anandamide. The synthesis and evaluation for their FAAH inhibitory activities of a series of 18 paracetamol esters are described. Structure-activity relationship studies indicated that the ester (33) with a 2-(4-(2-(trifluoromethyl)pyridin-4-ylamino)phenyl)acetic acid substituent was the most potent analogue in this series. The compound inhibited FAAH activity in a competitive manner with a K(i) value of 0.16 microM. The compound was also able to inhibit the FAAH activity in rat basophilic leukemia cells as assessed by measuring either the hydrolysis of anandamide, the FAAH-dependent cellular accumulation of anandamide, or the FAAH-dependent recycling of tritium to the cell membranes. The compound also inhibited the activity of monoacylglycerol lipase (MGL), the enzyme responsible for the hydrolysis of the endogenous cannabinoid receptor ligand 2-arachidonoylglycerol, with an IC(50) value of 1.9 microM. It is concluded that the compound may be a useful template for the design of potent novel inhibitors of FAAH.

Topics: Amidohydrolases; Analgesics; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acids; Benzoates; Cell Line, Tumor; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Inhibitory Concentration 50; Kinetics; Magnetic Resonance Spectroscopy; Polyunsaturated Alkamides; Pyridines; Rats; Rats, Sprague-Dawley; Rats, Wistar; Spectrophotometry, Infrared; Structure-Activity Relationship

The endocannabinoid, anandamide (AEA), modulates the activity of the dopamine transporter (DAT) in heterologous cells and synaptosomal preparations. The cellular mechanisms mediating this effect are unknown. The present studies employed live cell imaging techniques and the fluorescent, high affinity DAT substrate, 4-(4-(dimethylamino)-styryl)-N-methylpyridinium (ASP(+)), to address this issue. AEA addition to EM4 cells expressing yellow fluorescent protein-tagged human DAT (hDAT) produced a concentration-dependent inhibition of ASP(+) accumulation (IC(50): 3.2 +/- 0.8 microM). This effect occurred within 1 min after AEA addition and persisted for 10 min thereafter. Pertussis toxin did not attenuate the effects of AEA suggesting a mechanism independent of G(i)/G(o) coupled receptors. The amidohydrolase inhibitor, phenylmethylsulfonyl fluoride (0.2 mM), failed to alter the AEA-evoked inhibition of ASP(+) accumulation. Methanandamide (10 microM), a metabolically stable analogue of AEA inhibited accumulation but arachidonic acid (10 microM) was without effect suggesting that the effects of AEA are not mediated by its metabolic products. The extent of AEA inhibition of ASP(+) accumulation was not altered in cells pre-treated with 1 microM URB597, a specific and potent fatty acid amide hydrolase inhibitor, and the cyclooxygenase inhibitor, indomethacin (5 microM) Live cell imaging revealed a significant redistribution of hDAT from the membrane to the cytosol in response to AEA treatment (10 microM; 10 min). Similarly biotinylation experiments revealed that the decrease in DAT function was associated with a reduction in hDAT cell surface expression. These results demonstrate that AEA modulates DAT function via a cannabinoid receptor-independent mechanism and suggest that AEA may produces this effect, in part, by modulating DAT trafficking.

Topics: Alanine; Arachidonic Acids; Benzamides; Calcium; Cannabinoid Receptor Modulators; Carbamates; Cell Line, Transformed; Dopamine Plasma Membrane Transport Proteins; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Humans; Luminescent Proteins; Microscopy, Confocal; Phenylmethylsulfonyl Fluoride; Polyunsaturated Alkamides; Receptors, Cannabinoid; Receptors, Scavenger; Silicone Elastomers; Time Factors; Transfection; Tritium; Tropanes

This study investigated the participation of the hypothalamic endocannabinoid system in the response to lipopolysaccharide (LPS) challenge evaluating oxytocin (OXT) and tumor necrosis factor-alpha (TNF-alpha) plasma levels in vivo and their release from hypothalamic fragments in vitro. LPS increased OXT and TNF-alpha release through anandamide-activation of hypothalamic cannabinoid receptor CB(1,) since the antagonist AM251 blocked this effect. Anandamide, through its receptors, also increased hypothalamic nitric oxide (NO) which inhibited OXT release, ending the stimulatory effect of the endocannabinoid. Our findings reveal a hypothalamic interaction between oxytocin, endocannabinoid and NO-ergic systems providing a regulation of the hypothalamic-neurohypophyseal axis under basal and stress conditions.

Topics: Analysis of Variance; Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Modulators; Carbamates; Endocannabinoids; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Hypothalamus; Indoles; Lipopolysaccharides; Male; Nitric Oxide; Oxytocin; Polyunsaturated Alkamides; Radioimmunoassay; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Tumor Necrosis Factor-alpha

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

High levels of anandamide are located in the basal ganglia. The subthalamic nucleus (STN) is considered to be an important modulator of basal ganglia output. The present study aims at characterizing the modulation of the electrical activity of STN neurons by exogenous anandamide or endocannabinoids. Single-unit extracellular recordings in anesthetized rats and patch-clamp techniques in rat brain slices containing the STN were performed. Immunohistochemical assays were used. In vivo, anandamide administration produced two opposite effects (inhibition or stimulation) on STN neuron firing rates, depending of the precise location of the neuron within the nucleus. These effects were enhanced by prior inhibition of fatty acid amide hydrolase with URB597, but not by the inhibitor of carrier-mediated anandamide transport AM404. Rimonabant, a specific CB(1) receptor antagonist, also produced inhibition or stimulation of STN neuron activity when administered alone or after anandamide. These effects seem to be mediated by indirect mechanisms since: (1) STN neuron activity is not modified by the cannabinoid agonist Delta(9)-tetrahydrocannabinol (Delta(9)-THC) in vitro; (2) no depolarization-induced suppression of inhibition phenomena were observed; and (3) CB(1) receptor immunolabeling was not detected in the STN, but was abundant in areas which project efferents to this nucleus. Moreover, chemical lesion of the globus pallidus abolished the stimulatory effect of anandamide and microinfusion of anandamide into the prefrontal cortex led to inhibition of STN neuron activity. The present results show that endocannabinoids exert a tonic control on STN activity via receptors located outside the nucleus. These findings may contribute to enhance our understanding of the role of the endocannabinoid system in motor control.

Topics: Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Modulators; Carbamates; Cerebral Cortex; Dose-Response Relationship, Drug; Dronabinol; Electrophysiology; Endocannabinoids; Globus Pallidus; Immunohistochemistry; Injections, Intraventricular; Male; Microinjections; Neurons; Piperidines; Polyunsaturated Alkamides; Prefrontal Cortex; Pyrazoles; Rats; Rats, Sprague-Dawley; Rimonabant; Subthalamic Nucleus

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

Secretion of glucocorticoid hormones during stress produces an array of physiological changes that are adaptive and beneficial in the short term. In the face of repeated stress exposure, however, habituation of the glucocorticoid response is essential as prolonged glucocorticoid secretion can produce deleterious effects on metabolic, immune, cardiovascular, and neurobiological function. Endocannabinoid signaling responds to and regulates the activity of the hypothalamic-pituitary-adrenal (HPA) axis that governs the secretion of glucocorticoids; however, the role this system plays in adaptation of the neuroendocrine response to repeated stress is not well characterized. Herein, we demonstrate a divergent regulation of the two endocannabinoid ligands, N-arachidonylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG), following repeated stress such that AEA content is persistently decreased throughout the corticolimbic stress circuit, whereas 2-AG is exclusively elevated within the amygdala in a stress-dependent manner. Pharmacological studies demonstrate that this divergent regulation of AEA and 2-AG contribute to distinct forms of HPA axis habituation. Inhibition of AEA hydrolysis prevented the development of basal hypersecretion of corticosterone following repeated stress. In contrast, systemic or intra-amygdalar administration of a CB(1) receptor antagonist before the final stress exposure prevented the repeated stress-induced decline in corticosterone responses. The present findings demonstrate an important role for endocannabinoid signaling in the process of stress HPA habituation, and suggest that AEA and 2-AG modulate different components of the adrenocortical response to repeated stressor exposure.

Topics: Adaptation, Physiological; Analysis of Variance; Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Modulators; Carbamates; Corticosterone; Endocannabinoids; Male; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Signal Transduction; Stress, Physiological

Inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for the metabolism of the endogenous cannabinoid (CB) receptor ligand anandamide (AEA), are effective in a number of animal models of pain. Here, we investigated a series of isoflavones with respect to their abilities to inhibit FAAH.. In vitro assays of FAAH activity and affinity for CB receptors were used to characterize key compounds. In vivo assays used were biochemical responses to formalin in anaesthetized mice and the 'tetrad' test for central CB receptor activation.. Of the compounds tested, biochanin A was adjudged to be the most promising. Biochanin A inhibited the hydrolysis of 0.5 microM AEA by mouse, rat and human FAAH with IC(50) values of 1.8, 1.4 and 2.4 microM respectively. The compound did not interact to any major extent with CB(1) or CB(2) receptors, nor with FAAH-2. In anaesthetized mice, URB597 (30 microg i.pl.) and biochanin A (100 microg i.pl.) both inhibited the spinal phosphorylation of extracellular signal-regulated kinase produced by the intraplantar injection of formalin. The effects of both compounds were significantly reduced by the CB(1) receptor antagonist/inverse agonist AM251 (30 microg i.pl.). Biochanin A (15 mg.kg(-1) i.v.) did not increase brain AEA concentrations, but produced a modest potentiation of the effects of 10 mg.kg(-1) i.v. AEA in the tetrad test.. It is concluded that biochanin A, in addition to its other biochemical properties, inhibits FAAH both in vitro and peripherally in vivo.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Behavior, Animal; Benzamides; Brain; Cannabinoid Receptor Antagonists; Carbamates; Cell Line, Transformed; Chlorocebus aethiops; COS Cells; Drug Interactions; Endocannabinoids; Extracellular Signal-Regulated MAP Kinases; Formaldehyde; Genistein; Humans; Isoflavones; Liver; Mice; Mice, Inbred ICR; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats

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

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

Endocannabinoids are lipid signaling molecules that regulate a wide range of mammalian behaviors, including pain, inflammation, and cognitive/emotional state. The endocannabinoid anandamide is principally degraded by the integral membrane enzyme fatty acid amide hydrolase (FAAH), and there is currently much interest in developing FAAH inhibitors to augment endocannabinoid signaling in vivo. Here, we report the discovery and detailed characterization of a highly efficacious and selective FAAH inhibitor, PF-3845. Mechanistic and structural studies confirm that PF-3845 is a covalent inhibitor that carbamylates FAAH's serine nucleophile. PF-3845 selectively inhibits FAAH in vivo, as determined by activity-based protein profiling; raises brain anandamide levels for up to 24 hr; and produces significant cannabinoid receptor-dependent reductions in inflammatory pain. These data thus designate PF-3845 as a valuable pharmacological tool for in vivo characterization of the endocannabinoid system.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Brain; Crystallography, X-Ray; Endocannabinoids; Enzyme Inhibitors; Humans; Male; Pain; Piperazine; Piperazines; Piperidines; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Structure-Activity Relationship; Urea

Anandamide binds to cannabinoid receptors and plays several central and peripheral functions. The aim of this work was to study the possible role for this endocannabinoid in controlling sperm-oviduct interaction in mammals. We observed that bull sperm and bovine oviductal epithelial cells express cannabinoid receptors, CB1 and CB2, and fatty acid amide hydrolase, the enzyme that controls intracellular anandamide levels. A quantitative assay to determine whether anandamide was involved in bovine sperm-oviduct interaction was developed. R(+)-methanandamide, a non-hydrolysable anandamide analog, inhibited sperm binding to and induced sperm release from oviductal epithelia. Selective CB1 antagonists (SR141716A or AM251) completely blocked R(+)-methanandamide effects. However, SR144528, a selective CB2 antagonist, did not exert any effect, indicating that only CB1 was involved in R(+)-methanandamide effect. This effect was not caused by inhibition of the sperm progressive motility or by induction of the acrosome reaction. Overall, our findings indicate for the first time that the endocannabinoid system is present in bovine sperm and oviductal epithelium and that anandamide modulates the sperm-oviduct interaction, by inhibition of sperm binding and induction of sperm release from oviductal epithelial cells, probably by activating CB1 receptors.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Blotting, Western; Camphanes; Carbamates; Cattle; Endocannabinoids; Epithelium; Fallopian Tubes; Female; Immunohistochemistry; Male; Microscopy, Fluorescence; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Sperm Motility; Sperm-Ovum Interactions; Spermatozoa

Despite the alarming increment in the use and abuse of cannabis preparations among young people, little is known about possible long-term consequences of targeting the endocannabinoid system during the critical developmental period of adolescence. Therefore, we aimed to analyze possible long-lasting neurobiological consequences of enhancing endocannabinoid signalling during adolescence, by means of blocking anandamide (AEA) hydrolysis. Adolescent Wistar male rats were administered an inhibitor of AEA hydrolysis, i.e. URB597 (0, 0.1 or 0.5 mg/kg/day from postnatal days 38 to 43). The expression of brain cannabinoid receptor type 1 (CB1R) was then analyzed by [(3)H]CP-55,940 auto-radiographic binding at adulthood. Repeated URB597 administration during adolescence persistently modified CB1R binding in a region-dependent manner. A long-lasting decrease of CB1R binding levels was found in caudate-putamen, nucleus accumbens, ventral tegmental area and hippocampus, while an opposite increment was observed in the locus coeruleus. Present results provide evidence for long-lasting effects of adolescent URB597 administration. Activation of endocannabinoid transmission during the still plastic phase of adolescence may have implications for the maturational end-point of the endocannabinoid system itself, which could lead to permanent alterations in neuronal brain circuits and behavioural responses. Insights into the developmental trajectories of this neuromodulatory system may help us to better understand and prevent outcomes of neonatal and adolescent cannabis exposure.

Topics: Aging; Analysis of Variance; Animals; Arachidonic Acids; Autoradiography; Benzamides; Brain; Carbamates; Endocannabinoids; Male; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1

The cardiovascular effects of cannabinoids can be influenced by anaesthesia and can differ in chronic hypertension, but the extent to which they are influenced by acute hypertension in conscious animals has not been determined.. We examined cardiovascular responses to intravenous administration of anandamide and the synthetic cannabinoid, (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55212-2), in conscious male Wistar rats made acutely hypertensive by infusion of angiotensin II (AII) and arginine vasopressin (AVP). Rats were chronically instrumented for measurement of arterial blood pressure and vascular conductances in the renal, mesenteric and hindquarters beds.. Anandamide dose-dependently decreased the mean arterial blood pressure of rats made hypertensive by AII-AVP infusion, but not normotensive rats. Interestingly, acute hypertension also revealed a hypotensive response to WIN55212-2, which caused hypertension in normotensive animals. The enhanced depressor effects of the cannabinoids in acute hypertension were associated with increased vasodilatation in hindquarters, renal and mesenteric vascular beds. Treatment with URB597, which inhibits anandamide degradation by fatty acid amide hydrolase, potentiated the depressor and mesenteric vasodilator responses to anandamide. Furthermore, haemodynamic responses to WIN55212-2, but not to anandamide, were attenuated by the CB(1) receptor antagonist, AM251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophen yl)-4-methyl-1H-pyrazole-3-carboxamide].. These results broadly support the literature showing that the cardiovascular effects of cannabinoids can be exaggerated in hypertension, but highlight the involvement of non-CB(1) receptor-mediated mechanisms in the actions of anandamide.

Topics: Acute Disease; Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Benzoxazines; Blood Pressure; Cannabinoids; Carbamates; Consciousness; Endocannabinoids; Hindlimb; Hypertension; Infusions, Intravenous; Injections, Intravenous; Male; Morpholines; Naphthalenes; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Renal Circulation; Splanchnic Circulation; Vascular Resistance; Vasodilation

Considerable evidence implicates the endocannabinoid system as a neuromodulator of nausea and vomiting. The action of anandamide (AEA) can be prolonged by inhibiting its degradation, through the use of URB597 (URB), a Fatty Acid Amide Hydrolase (FAAH) enzyme inhibitor. Here we present evidence that the FAAH inhibitor, URB, interferes with cisplatin- and nicotine-induced vomiting in the Suncus murinus. In Experiment 1, shrews were injected with URB (0.9 mg/kg) or vehicle 120 min prior to the behavioral testing. They received a second injection of AEA (5 mg/kg) or vehicle 15 min prior to being injected with cisplatin (20 mg/kg) or saline and the number of vomiting episodes were counted for 60 min. In Experiment 2, shrews were injected with vehicle or URB (0.9 mg/kg) 120 min prior to receiving an injection of nicotine (5 mg/kg) or saline and the number of vomiting episodes were counted for 15 min. Experiment 3 evaluated the potential of the CB(1) antagonist, SR141716, to reverse the effect of URB on nicotine-induced vomiting. URB attenuated vomiting produced by cisplatin and nicotine and the combination of URB+AEA suppressed vomiting produced by cisplatin. The effect of URB on nicotine-induced vomiting was reversed by SR141716. These data suggest that the EC system plays a tonic role in the regulation of toxin-induced vomiting.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Carbamates; Cisplatin; Drug Interactions; Endocannabinoids; Female; Male; Nicotine; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; Shrews; Vomiting

N-arachidonoyl glycine (NAGly) is an endogenous signaling lipid with a wide variety of biological activity whose biosynthesis is poorly understood. Two primary biosynthetic pathways have been proposed. One suggests that NAGly is formed via an enzymatically regulated conjugation of arachidonic acid (AA) and glycine. The other suggests that NAGly is an oxidative metabolite of the endogenous cannabinoid, anandamide (AEA), through an alcohol dehydrogenase. Here using both in vitro and in vivo assays measuring metabolites with LC/MS/MS we test the hypothesis that both pathways are present in mammalian cells.. The metabolic products of deuterium-labeled AEA, D4AEA (deuterium on ethanolamine), indicated that NAGly is formed by the oxidation of the ethanolamine creating a D2NAGly product in both RAW 264.7 and C6 glioma cells. Significantly, D4AEA produced a D0NAGly product only in C6 glioma cells suggesting that the hydrolysis of AEA yielded AA that was used preferentially in a conjugation reaction. Addition of the fatty acid amide (FAAH) inhibitor URB 597 blocked the production of D0NAGly in these cells. Incubation with D8AA in C6 glioma cells likewise produced D8NAGly; however, with significantly less efficacy leading to the hypothesis that FAAH-initiated AEA-released AA conjugation with glycine predominates in these cells. Furthermore, the levels of AEA in the brain were significantly increased, whereas those of NAGly were significantly decreased after systemic injection of URB 597 in rats and in FAAH KO mice further supporting a role for FAAH in endogenous NAGly biosynthesis. Incubations of NAGly and recombinant FAAH demonstrated that NAGly is a significantly less efficacious substrate for FAAH with only ~50% hydrolysis at 30 minutes compared to 100% hydrolysis of AEA. Co-incubations of AEA and glycine with recombinant FAAH did not, however, produce NAGly.. These data support the hypothesis that the signaling lipid NAGly is a metabolic product of AEA by both oxidative metabolism of the AEA ethanolamine moiety and through the conjugation of glycine to AA that is released during AEA hydrolysis by FAAH.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Brain; Brain Chemistry; Carbamates; Cell Line, Tumor; Endocannabinoids; Glycine; Lipid Metabolism; Male; Metabolic Networks and Pathways; Mice; Mice, Knockout; Oxidation-Reduction; Polyunsaturated Alkamides; Rats; Recombinant Proteins

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

Anandamide (N-arachidonoylethanolamine, AEA) or its metabolites participate in energy balance mainly through feeding modulation. In addition, AEA has been found to increase 3T3-L1 adipocyte differentiation process. In this study, the effect of AEA, R(+)-methanandamide (R(+)-mAEA), URB597, and indomethacin on primary rat adipocyte differentiation was evaluated by a flow cytometry method and by Oil Red-O staining. Reverse transcription-PCR and western blotting analysis were performed in order to study the effect of AEA on peroxisome proliferator-activated receptor (PPAR)gamma2, cannabinoid receptors (CBRs), fatty acid amidohydrolase (FAAH), and cyclooxygenase-2 (COX-2) expression, during the differentiation process. AEA increased adipocyte differentiation in primary cell cultures in a concentration- and time-dependent manner and induced PPARgamma2 gene expression, confirming findings with 3T3-L1 cell line. CB1R, FAAH, and COX-2 expression was also increased while CB2R expression was decreased. Inhibition of FAAH and COX-2 attenuated the AEA-induced differentiation. Our findings indicate that AEA regulates energy homeostasis not only by appetite modulation but may also regulate adipocyte differentiation and phenotype.

Topics: Adipocytes; Adipose Tissue; Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Blotting, Western; Carbamates; Cell Differentiation; Cyclooxygenase 2; Endocannabinoids; Enzyme Inhibitors; Flow Cytometry; Indomethacin; Male; Polyunsaturated Alkamides; PPAR gamma; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Reverse Transcriptase Polymerase Chain Reaction; Up-Regulation

Endocannabinoids inhibit hypothalamic-pituitary-adrenal (HPA) axis activity; however, the neural substrates and pathways subserving this effect are not well characterized. The amygdala is a forebrain structure that provides excitatory drive to the HPA axis under conditions of stress. The aim of this study was to determine the contribution of endocannabinoid signaling within distinct amygdalar nuclei to activation of the HPA axis in response to psychological stress. Exposure of rats to 30-min restraint stress increased the hydrolytic activity of fatty acid amide hydrolase (FAAH) and concurrently decreased content of the endocannabinoid/CB(1) receptor ligand N-arachidonylethanolamine (anandamide; AEA) throughout the amygdala. In stressed rats, AEA content in the amygdala was inversely correlated with serum corticosterone concentrations. Pharmacological inhibition of FAAH activity within the basolateral amygdala complex (BLA) attenuated stress-induced corticosterone secretion; this effect was blocked by co-administration of the CB(1) receptor antagonist AM251, suggesting that stress-induced decreases in CB(1) receptor activation by AEA contribute to activation of the neuroendocrine stress response. Local administration into the BLA of a CB(1) receptor agonist significantly reduced stress-induced corticosterone secretion, whereas administration of a CB(1) receptor antagonist increased corticosterone secretion. Taken together, these findings suggest that the degree to which stressful stimuli reduce amygdalar AEA/CB(1) receptor signaling contributes to the magnitude of the HPA response.

Topics: Amidohydrolases; Amygdala; Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Modulators; Carbamates; Corticosterone; Dronabinol; Drug Interactions; Endocannabinoids; Hypothalamo-Hypophyseal System; Male; Microinjections; Piperidines; Pituitary-Adrenal System; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Restraint, Physical; Signal Transduction; Stress, Psychological

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

To study the effects of N-arachidonylethanolamide (anandamide [AEA]) on aqueous humor outflow and to investigate the existence and activity of fatty acid amide hydrolase (FAAH), an AEA metabolic enzyme in trabecular meshwork (TM) tissue.. The effects of AEA on aqueous humor outflow were measured using a porcine anterior segment-perfused organ culture model. Western blot analysis was used to study the expression of FAAH, and a thin-layer chromatography-based approach was used to measure the enzymatic activity of FAAH in TM tissue.. Administration of AEA caused a transient enhancement of aqueous humor outflow facility. In the presence of 100 nM URB597, an FAAH inhibitor, the effect of 10 nM AEA on outflow facility was prolonged by at least 4 hours. The AEA-induced enhancement of outflow facility was blocked by SR141716A, a CB1 antagonist, and was partially blocked by SR144528, a CB2 antagonist. In Western blot studies, positive signals were detected on TM tissues with an anti-FAAH antibody. In the enzyme activity studies, the enzymatic activity of AEA hydrolysis was detected in TM tissues, and this activity was reduced with the addition of 100 nM URB597.. Results from this study demonstrate that the administration of AEA increases aqueous humor outflow facility and that this effect of AEA involves CB1 and CB2 cannabinoid receptors. In addition, this study reveals the existence and the activity of FAAH, an AEA-metabolizing enzyme, in the TM tissues.

Topics: Amidohydrolases; Animals; Anterior Eye Segment; Aqueous Humor; Arachidonic Acids; Benzamides; Blotting, Western; Camphanes; Cannabinoid Receptor Antagonists; Cannabinoid Receptor Modulators; Carbamates; Chromatography, Thin Layer; Endocannabinoids; Organ Culture Techniques; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Rimonabant; Swine; Trabecular Meshwork

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

There are contradictory results concerning the effects of systemic injections of cannabinoid agonists in anxiety-induced behavioral changes. Direct drug administration into brain structures related to defensive responses could help to clarify the role of cannabinoids in these changes. Activation of cannabinoid CB(1) receptors in the dorsolateral periaqueductal gray induces anxiolytic-like effects in the elevated plus maze. The aim of this work was to verify if facilitation of endocannabinoid-mediated neurotransmission in this region would also produce anxiolytic-like effects in another model of anxiety, the Vogel conflict test. Male Wistar rats (n=5-9/group) with cannulae aimed at the dorsolateral periaqueductal gray were water deprived for 24 h and pre-exposed to the apparatus where they were allowed to drink for 3 min. After another 24 h-period of water deprivation, they received the microinjections and, 10 min later, were placed into the experimental box. In this box an electrical shock (0.5 mA, 2 s) was delivered in the spout of a drinking bottle at every twenty licks. The animals received a first microinjection of vehicle (0.2 microl) or AM251 (a cannabinoid CB(1) receptor antagonist; 100 pmol) followed, 5 min later, by a second microinjection of vehicle, anandamide (an endocannabinoid, 5 pmol), AM404 (an inhibitor of anandamide uptake, 50 pmol) or URB597 (an inhibitor of Fatty Acid Amide Hydrolase, 0.01 or 0.1 nmol). Anandamide, AM404 and URB597 (0.01 nmol) increased the total number of punished licks. These effects were prevented by AM251. The results give further support to the proposal that facilitation of CB(1) receptor-mediated endocannabinoid neurotransmission in the dorsolateral periaqueductal gray modulates defensive responses.

Topics: Amidohydrolases; Animals; Anti-Anxiety Agents; Arachidonic Acids; Benzamides; Carbamates; Conflict, Psychological; Data Interpretation, Statistical; Drinking; Endocannabinoids; Enzyme Inhibitors; Male; Pain Measurement; Periaqueductal Gray; Polyunsaturated Alkamides; Rats; Rats, Wistar; Reaction Time; Receptor, Cannabinoid, CB1

Emerging evidence suggests that the rewarding, abuse-related effects of nicotine are modulated by the endocannabinoid system of the brain. For example, pharmacological blockade or genetic deletion of cannabinoid CB(1) receptors can reduce or eliminate many abuse-related behavioral and neurochemical effects of nicotine. Furthermore, doses of Delta(9)-tetrahydrocannabinol and nicotine that are ineffective when given alone can induce conditioned place preference when given together. These previous studies have used systemically administered CB(1) receptor agonists and antagonists and gene deletion techniques, which affect cannabinoid CB(1) receptors throughout the brain. A more functionally selective way to alter endocannabinoid activity is to inhibit fatty acid amide hydrolase (FAAH), thereby magnifying and prolonging the effects of the endocannabinoid anandamide only when and where it is synthesized and released on demand. Here, we combined behavioral and neurochemical approaches to evaluate whether the FAAH inhibitor URB597 (cyclohexyl carbamic acid 3'-carbamoyl-3-yl ester) could alter the abuse-related effects of nicotine in rats. We found that URB597, at a dose (0.3 mg/kg) that had no behavioral effects by itself, prevented development of nicotine-induced conditioned place preference (CPP) and acquisition of nicotine self-administration. URB597 also reduced nicotine-induced reinstatement in both CPP and self-administration models of relapse. Furthermore, in vivo microdialysis showed that URB597 reduced nicotine-induced dopamine elevations in the nucleus accumbens shell, the terminal area of the brain's mesolimbic reward system. These findings suggest that FAAH inhibition can counteract the addictive properties of nicotine and that FAAH may serve as a new target for development of medications for treatment of tobacco dependence.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Carbamates; Conditioning, Psychological; Dopamine; Endocannabinoids; Hydrolysis; Male; Motor Activity; Nicotine; Nucleus Accumbens; Polyunsaturated Alkamides; Rats; Rats, Long-Evans; Rats, Sprague-Dawley; Reward; Self Administration; Tobacco Use Disorder

Lack of involvement of the opioid system with the endocannabinoid, arachidonylethanolamide (anandamide) was possibly due to hydrolysis by fatty acid amide hydrolase (FAAH). Cyclohexylcarbamic acid 3'-carbamoyl-biphenyl-3-yl ester (URB597) is an inhibitor of FAAH, increases brain anandamide levels and enhances anandamide-induced antinociception in male ICR mice (25-30 g). The combination of URB597 (10 mg/kg, i.p.) and anandamide (40 mg/kg, i.p.) produced maximal antinociception in the mouse tail-flick test [68.7+/-16.8 percent maximum possible effect (%MPE)], versus either substance alone (27.3+/-7.9%MPE and 4.6+/-2.3%MPE, respectively) and is significantly blocked (p<0.05) by the cannabinoid CB(1) receptor antagonist, SR141716A (rimonabant), the kappa opioid receptor-selective antagonist, nor-Binaltorphimine (10 microg i.t.; 12.7+/-4.0%MPE) and the mu opioid receptor antagonist, naloxone (1 mg/kg, s.c.; 6.0+/-3.8%MPE), but not by the delta opioid receptor-selective antagonist, naltrindole (2 mg/kg, s.c.; 29.7+/-8.2%MPE) or the cannabinoid CB(2) receptor antagonist, SR144528. In addition, nor-BNI (10 microg i.t) administration to FAAH(-/-) knockout mice produced a nociceptive response. The URB597/anandamide combination was not active in the CB(1)(-/-) knockout mice, but retained activity in the MOR(-/-) knockout mice. The sub-active combination of (URB597 10 mg/kg, i.p/anandamide 10 mg/kg, i.p.; 15.5+/-4.3%MPE) shifted the dose response curve of morphine to the left (morphine alone ED(50)=4.6 mg/kg [3.7-5.6] versus morphine/URB597/anandamide (ED(50)=2.5 mg/kg [1.9-3.4]). These data are the first demonstration that anandamide, if protected from degradation, acts via the CB(1) receptor to interact with kappa opioid receptor systems in opioid analgesia.

Topics: Amidohydrolases; Analgesics, Opioid; Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Modulators; Carbamates; Dose-Response Relationship, Drug; Drug Therapy, Combination; Endocannabinoids; Enzyme Inhibitors; Male; Mice; Mice, Inbred ICR; Mice, Knockout; Morphine; Pain; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Receptors, Opioid, kappa

CB(1) cannabinoid receptors in the brain are known to participate in the regulation of reward-based behaviors. However, the contribution of each of the endocannabinoid transmitters, anandamide and 2-arachidonoylglycerol (2-AG), to these behaviors remains undefined. To address this question, we assessed the effects of URB597, a selective anandamide deactivation inhibitor, as a reinforcer of drug-seeking and drug-taking behavior in squirrel monkeys.. We investigated the reinforcing effects of the fatty acid amide hydrolase (FAAH) inhibitor URB597 in monkeys trained to intravenously self-administer Delta(9)-tetrahydrocannabinol (THC), anandamide, or cocaine and quantified brain endocannabinoid levels using liquid chromatography/mass spectrometry. We measured brain FAAH activity using an ex vivo enzyme assay.. URB597 (.3 mg/kg, intravenous) blocked FAAH activity and increased anandamide levels throughout the monkey brain. This effect was accompanied by a marked compensatory decrease in 2-AG levels. Monkeys did not self-administer URB597, and the drug did not promote reinstatement of extinguished drug-seeking behavior previously maintained by THC, anandamide, or cocaine. Pretreatment with URB597 did not modify self-administration of THC or cocaine, even though, as expected, it significantly potentiated anandamide self-administration.. In the monkey brain, the FAAH inhibitor URB597 increases anandamide levels while causing a compensatory down-regulation in 2-AG levels. These effects are accompanied by a striking lack of reinforcing properties, which distinguishes URB597 from direct-acting cannabinoid agonists such as THC. Our results reveal an unexpected functional heterogeneity within the endocannabinoid signaling system and suggest that FAAH inhibitors might be used therapeutically without risk of abuse or triggering of relapse to drug abuse.

Topics: Amidohydrolases; Analysis of Variance; Animals; Arachidonic Acids; Behavior, Animal; Benzamides; Brain; Cannabinoid Receptor Modulators; Carbamates; Chromatography, Liquid; Cocaine; Dose-Response Relationship, Drug; Dronabinol; Endocannabinoids; Male; Polyunsaturated Alkamides; Rats; Rats, Wistar; Reinforcement Schedule; Reinforcement, Psychology; Saimiri; Self Administration

The opioid and endocannabinoid systems mediate analgesia expressed upon re-exposure to a contextually aversive stimulus (fear-conditioned analgesia; FCA), and modulate the mitogen-activated protein kinase (MAPK) pathway. However, an interaction between the opioid and endocannabinoid systems during FCA has not been investigated at the behavioural or molecular level. FCA was modeled in male Lister-hooded rats by assessing formalin-evoked nociceptive behaviour in an arena previously paired with footshock. Administration of the fatty acid amide hydrolase and endocannabinoid catabolism inhibitor, URB597 (0.3 mg/kg, i.p.), enhanced expression of FCA. The opioid receptor antagonist, naloxone, attenuated FCA and attenuated the URB597-induced enhancement of FCA. SR141716A (CB(1) antagonist) and SR144528 (CB(2) antagonist) also attenuated the URB597-mediated enhancement of FCA. Expression of FCA was associated with increased relative phospho-ERK2 expression in the amygdala, an effect blocked by naloxone, SR141716A, and SR144528. Furthermore, URB597-mediated enhancement of FCA was associated with reduced phospho-ERK1 and phospho-ERK2 in the amygdala. Phospho-ERK1/2 expression in the hippocampus, prefrontal cortex, and thalamus was unchanged following FCA and drug treatment. None of the drugs affected formalin-evoked nociceptive behaviour or phospho-ERK1/2 expression in non-fear-conditioned rats. These data suggest that endocannabinoid-mediated enhancement of FCA is abolished by pharmacological blockade of opioid receptors as well as CB(1) or CB(2) receptors. Both pharmacological enhancement (with URB597) and attenuation (with naloxone) of this form of endogenous analgesia were associated with reduced expression of phospho-ERK1/2 in the amygdaloid complex arguing against a causal role for ERK1/2 signaling in the amygdala during expression of FCA or its modulation by opioids or cannabinoids.

Topics: Amidohydrolases; Amygdala; Analgesia; Animals; Arachidonic Acids; Avoidance Learning; Benzamides; Cannabinoid Receptor Modulators; Carbamates; Conditioning, Psychological; Endocannabinoids; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Fear; Male; Narcotic Antagonists; Neural Inhibition; Pain; Pain Measurement; Polyunsaturated Alkamides; Rats; Receptor, Cannabinoid, CB1; Receptors, Opioid; Signal Transduction

Synthetic and plant-derived cannabinoid CB(1) receptor agonists have consistently been shown to impair sexual behavior in male rodents; however, the role of the endocannabinoid system in regulating copulatory processes is largely unknown. The aim of this experiment was to determine the effect of pharmacological facilitation or antagonism of endocannabinoid signaling on male rat sexual behavior.. Male Long-Evans rats were administered a single injection of either the cannabinoid CB(1) receptor antagonist AM251 (1, 2, or 5 mg/kg), the fatty acid amide hydrolase (FAAH) inhibitor URB597 (0.1, 0.3, or 0.5 mg/kg), or the anandamide uptake inhibitor/FAAH inhibitor AM404 (1, 2, and 5 mg/kg), or their respective vehicles, and examined on parameters of appetitive and consummatory sexual behavior.. Inhibition of anandamide metabolism through URB597 had no effect on any parameter of sexual behavior. However, the highest dose of AM404 increased the latency to engage in intromitting behavior, but had no other effect on sexual behavior, suggesting that this effect may be due to the sedative-suppressive effects of this drug. AM251 produced a dose-dependent facilitation of ejaculation, such that the number of intromissions required to achieve ejaculation and the ejaculation latency were reduced by AM251 administration.. These data suggest that antagonism of the CB(1) receptor facilitates ejaculatory processes, an effect which may be due to interactions with neuropeptidergic systems in the hypothalamus, and further, suggest a novel target for pharmacological agents aimed at treating ejaculatory-based sexual dysfunction.

Topics: Amidohydrolases; Animals; Appetitive Behavior; Arachidonic Acids; Benzamides; Cannabinoid Receptor Modulators; Carbamates; Consummatory Behavior; Dose-Response Relationship, Drug; Ejaculation; Endocannabinoids; Enzyme Inhibitors; Male; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Long-Evans; Receptor, Cannabinoid, CB1; Sexual Behavior, Animal

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

Converging evidence suggests that the endocannabinoid system is an important constituent of neuronal substrates involved in brain reward processes and emotional responses to stress. Here, we evaluated motivational effects of intravenously administered anandamide, an endogenous ligand for cannabinoid CB1-receptors, in Sprague-Dawley rats, using a place-conditioning procedure in which drugs abused by humans generally produce conditioned place preferences (reward). Anandamide (0.03-3 mg/kg intravenous) produced neither conditioned place preferences nor aversions. However, when rats were pre-treated with the fatty acid amide hydrolase (FAAH) inhibitor URB597 (cyclohexyl carbamic acid 3'-carbamoyl-3-yl ester; 0.3 mg/kg intraperitoneal), which blocks anandamide's metabolic degradation, anandamide produced dose-related conditioned place aversions. In contrast, URB597 alone showed no motivational effects. Like URB597 plus anandamide, the synthetic CB1-receptor ligand WIN 55,212-2 (50-300 microg/kg, intravenous) produced dose-related conditioned place aversions. When anxiety-related effects of anandamide and URB597 were evaluated in a light/dark box, both a low anandamide dose (0.3 mg/kg) and URB597 (0.1 and 0.3 mg/kg) produced anxiolytic effects when given alone, but produced anxiogenic effects when combined. A higher dose of anandamide (3 mg/kg) produced anxiogenic effects and depressed locomotor activity when given alone and these effects were potentiated after URB597 treatment. Finally, anxiogenic effects of anandamide plus URB597 and development of place aversions with URB597 plus anandamide were prevented by the CB1-receptor antagonist AM251 (3 mg/kg intraperitoneal). Thus, additive interactions between the effects of anandamide on brain reward processes and on anxiety may account for its aversive effects when intravenously administered during FAAH inhibition with URB597.

Topics: Amidohydrolases; Analgesics; Analysis of Variance; Animals; Anxiety; Arachidonic Acids; Avoidance Learning; Behavior, Animal; Benzamides; Benzoxazines; Carbamates; Conditioning, Operant; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Male; Morpholines; Motivation; Motor Activity; Naphthalenes; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Time Factors

In the present study we explored with a multidisciplinary approach, the role of anandamide (AEA) in the modulation of anxiety behavior at the level of the prefrontal cortex (PFC). Low doses of the metabolically stable AEA analog, methanandamide, microinjected into the PFC, produced an anxiolytic-like response in rats, whereas higher doses induced anxiety-like behaviors. Pretreatment with the selective antagonist of CB1 or TRPV1 receptors (AM251 and capsazepine, respectively) suggested that the anxiolytic effect evoked by AEA might be due to the interaction with the CB1 cannabinoid receptor, whereas vanilloid receptors seem to be involved in AEA anxiogenic action. When AEA contents in the PFC were increased by microinjecting the selective inhibitor of fatty acid amide hydrolase (FAAH), URB597, we observed an anxiolytic response only at low doses of the compound and no effect or even an anxiogenic profile at higher doses. In line with this, a marked decrease of AEA levels in the PFC, achieved by lentivirus-mediated local overexpression of FAAH, produced an anxiogenic response. These findings support an anxiolytic role for physiological increases in AEA in the PFC, whereas more marked increases or decreases of this endocannabinoid might lead to an anxiogenic response due to TRPV1 stimulation or the lack of CB1 activation, respectively.

Topics: Animals; Anxiety; Arachidonic Acids; Benzamides; Cannabinoid Receptor Modulators; Carbamates; Dose-Response Relationship, Drug; Endocannabinoids; Male; Polyunsaturated Alkamides; Prefrontal Cortex; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1

A major clinical concern with the use of cannabinoid receptor 1 (CB1) direct agonists is that these compounds increase alcohol drinking and drug abuse-related behaviours. As an alternative approach, CB1-receptor-mediated activity can be facilitated by increasing anandamide levels with the use of hydrolase fatty acid amide hydrolase (FAAH) inhibitors.. Using the selective FAAH inhibitor URB597, we investigated whether activation of the endogenous cannabinoid tone increases alcohol abuse liability, as what happens with the CB1 receptor direct agonists.. URB597 was tested on alcohol self-administration in Wistar rats and on homecage alcohol drinking in genetically selected Marchigian Sardinian alcohol-preferring (msP) rats. In Wistar rats, URB597 effects on alcohol-induced anxiety and on stress-, yohimbine- and cue-induced reinstatement of alcohol seeking were also evaluated. For comparison, the effect of the CB1 receptor antagonist rimonabant on ethanol self-administration was also tested.. Under our experimental condition, intraperitoneal (IP) administration of URB597 (0.0, 0.3 and 1.0 mg/kg) neither increased voluntary homecage alcohol drinking in msP rats nor facilitated fixed ratio 1 and progressive ratio alcohol self-administration in nonselected Wistars. In the reinstatement tests, the compound did not have effects on cue-, footshock stress- and yohimbine-induced relapse. Conversely, URB597 completely abolished the anxiogenic response measured during withdrawal after an acute IP administration of alcohol (3.0 g/kg). Rimonabant (0.0, 0.3, 1.0 and 3.0 mg/kg) significantly reduced ethanol self-administration.. Results demonstrate that activation of the endocannabinoid anandamide system by selective inhibition of FAAH does not increase alcohol abuse risks but does reduce anxiety associated to alcohol withdrawal. We thus can speculate that medication based on the use of endocannabinoid system modulators such as URB597 may offer important advantages compared to treatment with direct CB1 receptor activators.

Topics: Adrenergic alpha-Antagonists; Alcohol Drinking; Alcoholism; Amidohydrolases; Animals; Anxiety; Arachidonic Acids; Benzamides; Brain Chemistry; Carbamates; Conditioning, Operant; Cues; Electroshock; Endocannabinoids; Enzyme Inhibitors; Extinction, Psychological; Male; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Recurrence; Reinforcement Schedule; Self Administration; Stress, Psychological; Yohimbine

The endogenous cannabinoid system plays a vital role in the control of nausea and emesis. Because of the rapid breakdown and hydrolysis of endocannabinoids, such as anandamide, the therapeutic effects may be enhanced by prolonging their duration of action.. The present experiment evaluated the potential of various doses of URB597, a fatty acid amide hydrolase (FAAH) inhibitor, alone and in combination with systemic administration of anandamide to modulate the establishment of lithium-induced conditioned taste reactivity responses in rats.. In experiment 1, on the conditioning day, rats first received an injection of 0.3 mg/kg URB597, 0.15 mg/kg URB597, or vehicle and then received a second injection of anandamide (5 mg/kg) or vehicle, before a 3-min exposure of 0.1% saccharin by intraoral infusion. Immediately after the saccharin exposure, the rats were injected with lithium chloride. On each of three test days, rats received a 3-min intraoral infusion of saccharin solution, and the taste reactivity responses were videotaped and monitored. In experiment 2, the effects of pretreatment with the CB(1) antagonist, AM-251, on URB597 and anandamide-induced suppressed aversion was evaluated.. Administration of URB597 alone and in combination with anandamide reduced active rejection reactions elicited by a LiCl-paired saccharin solution; both effects were reversed by pretreatment with AM-251, suggesting that they were CB(1) receptor mediated.. The results suggest that prolonging the action of anandamide by pretreatment with the FAAH inhibitor, URB597, suppresses lithium-induced nausea in the rat.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Association Learning; Avoidance Learning; Benzamides; Carbamates; Conditioning, Classical; Dose-Response Relationship, Drug; Endocannabinoids; Injections, Intraperitoneal; Lithium Chloride; Male; Nausea; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Long-Evans; Receptor, Cannabinoid, CB1; Vomiting

Anandamide is an endogenous ligand for brain cannabinoid CB(1) receptors, but its behavioral effects are difficult to measure due to rapid inactivation. Here we used a drug-discrimination procedure to test the hypothesis that anandamide, given i.v. or i.p., would produce discriminative effects like those of delta-9-tetrahydrocannabinol (THC) in rats when its metabolic inactivation was inhibited. We also used an in vivo microdialysis procedure to investigate the effects of anandamide, given i.v. or i.p., on dopamine levels in the nucleus accumbens shell in rats. When injected i.v., methanandamide (AM-356), a metabolically stable anandamide analog, produced clear dose-related THC-like discriminative effects, but anandamide produced THC-like discriminative effects only at a high 10-mg/kg dose that almost eliminated lever-press responding. Cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester (URB-597), an inhibitor of fatty acid amide hydrolase (FAAH), the main enzyme responsible for metabolic inactivation of anandamide, produced no THC-like discriminative effects alone but dramatically potentiated discriminative effects of anandamide, with 3 mg/kg anandamide completely substituting for the THC training dose. URB-597 also potentiated the ability of anandamide to increase dopamine levels in the accumbens shell. The THC-like discriminative-stimulus effects of anandamide after URB-597 and methanandamide were blocked by the CB1 receptor antagonist rimonabant, but not the vanilloid VR1 receptor antagonist capsazepine. Surprisingly, the anandamide transport inhibitors N-(4-hydroxyphenyl)-eicosa-5,8,11,14-tetraenamide (AM-404) and N-(3-furylmethyl)eicosa-5,8,11,14-tetraenamide (UCM-707) did not potentiate THC-like discriminative effects of anandamide or its dopamine-elevating effects. Thus, anandamide has THC-like discriminative and neurochemical effects that are enhanced after treatment with a FAAH inhibitor but not after treatment with transport inhibitors, suggesting brain area specificity for FAAH versus transport/FAAH inactivation of anandamide.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Biological Transport, Active; Brain Chemistry; Carbamates; Discrimination Learning; Discrimination, Psychological; Dose-Response Relationship, Drug; Dronabinol; Endocannabinoids; Enzyme Inhibitors; Furans; Hallucinogens; Injections, Intraperitoneal; Injections, Intravenous; Male; Microdialysis; Nucleus Accumbens; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Reinforcement Schedule; TRPV Cation Channels

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

This study investigates the actions of N-(2-hydroxyethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (anandamide) on blood flow of the rat knee joint. Topical bolus administration of anandamide (10-1000 nmol) onto the exposed knee joint capsules produced dose-dependent increases in the knee joint blood flow. Various antagonists were tested on the vasodilator response to 100 nmol anandamide. Capsazepine (N-[2-(4-chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide), an antagonist of the transient receptor potential vanilloid type 1 (TRPV1) receptor, given at 10 and 100 nmol, suppressed the response by a maximum of 71%. A cannabinoid CB(1) receptor antagonist AM281 (10 nmol) and a CB(2) receptor antagonist AM630 (10 nmol) shortened its duration from 15 min to 5 min. O-1918 (1 nmol), an antagonist of the putative endothelial anandamide/abnormal-cannabidiol receptor, on its own or combined with capsazepine and the two cannabinoid receptor antagonists produced 38% and 24% inhibition on the peak vasodilator response to anandamide, respectively. URB597 (1 nmol), an inhibitor of fatty acid amide hydrolase (FAAH) suppressed the response by 40%, and an anandamide transporter inhibitor [N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide] (AM404; 1 nmol) or a cyclo-oxygenase (COX) inhibitor flurbiprofen (20 nmol) abolished the response. These findings suggest the vasodilator action of anandamide in the rat knee joint involved hydrolysis of the compound by FAAH, production of COX-derived eicosanoid(s), activation of TRPV1 receptors, and a small component involved activation of endothelial anandamide/abnormal-cannabidiol receptors; a minor delayed dilator response was mediated by activation of conventional cannabinoid receptors.

Topics: Amidohydrolases; Analysis of Variance; Animals; Arachidonic Acids; Benzamides; Blood Vessels; Cannabinoid Receptor Antagonists; Cannabinoid Receptor Modulators; Capsaicin; Carbamates; Cyclooxygenase Inhibitors; Dose-Response Relationship, Drug; Endocannabinoids; Flurbiprofen; Hindlimb; Indoles; Joints; Morpholines; Polyunsaturated Alkamides; Pyrazoles; Rats; Regional Blood Flow; TRPV Cation Channels; Vasodilation

Manipulations of the endocannabinoid/fatty acid amide hydrolase (FAAH) signaling systems result in conflicting and paradoxical effects in rodent models of emotional reactivity.. In the present study, we tested the hypothesis that the inhibition of FAAH would elicit significant effects in murine models used to screen anxiolytic and antidepressant drugs.. FAAH (-/-) mice and wild-type mice treated with FAAH inhibitors (URB597 and OL-135) were evaluated in standard behavioral screening models for antidepressant (i.e., tail suspension and forced-swim tests) and anxiolytic (i.e., elevated plus maze) agents. The doses of URB597 and OL-135 selected were based on their ability to augment the pharmacological effects (i.e., analgesia, catalepsy, and hypothermia) of exogenously administered anandamide.. FAAH (-/-) mice, anandamide-injected FAAH (-/-) mice, or wild-type mice injected with FAAH inhibitors or anandamide failed to exhibit significant effects in standard tests of emotional reactivity, although the antidepressant desipramine and the anxiolytic agent midazolam were active in the appropriate assays. FAAH- (-/-) and URB597-treated mice finally displayed significant effects in the tail suspension test when substantial methodological changes were made (i.e., altered ambient light and increased sample sizes).. Although FAAH suppression can elicit significant effects under some instances in which consequential procedural modifications are made, the present results indicate that the pharmacological inhibition or genetic deletion of FAAH is ineffective in standard mouse models of emotional reactivity. It remains to be established whether the effects of FAAH inhibition in these modified tasks are predictive of their efficacy in treating emotional disorders.

Topics: Amidohydrolases; Animals; Anxiety; Arachidonic Acids; Behavior, Animal; Benzamides; Cannabinoid Receptor Modulators; Carbamates; Depression; Disease Models, Animal; Dose-Response Relationship, Drug; Emotions; Endocannabinoids; Enzyme Inhibitors; Lighting; Maze Learning; Mice; Mice, Knockout; Polyunsaturated Alkamides; Pyridines; Signal Transduction

The cellular uptake of anandamide is reduced by inhibitors of fatty acid amide hydrolase (FAAH) and by agents disrupting endocytotic mechanisms. However, it is not clear if these events occur over the same time frame and if they occur to the same extent in different cells. We have therefore investigated the effects of such compounds in three cell lines of different origins using different assay incubation times and temperatures.. FAAH activity and cellular uptake of anandamide was measured using anandamide, radio-labelled either in the ethanolamine or arachidonoyl part of the molecule.. The FAAH inhibitor URB597 inhibited the uptake of anandamide into C6 glioma, RBL2H3 basophilic leukaemia cells and P19 embryonic carcinoma cells at incubation time 4 min. However, a time-dependent and temperature-sensitive residual uptake remained after URB597 treatment. The combination of progesterone and nystatin reduced the uptake, but also decreased the amount of anandamide retained by the wells. Genistein inhibited anandamide uptake in a manner that was not additive to that of URB597. However, genistein was a potent competitive inhibitor of FAAH (K(i) value 8 microM).. The reduction of anandamide uptake by genistein can be explained by its ability to inhibit FAAH with a potency which overlaps that for inhibition of tyrosine kinase. The FAAH- resistant but time-dependent uptake of anandamide is seen in all three cell lines studied and is thus presumably a generally occurring process.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Brain; Carbamates; Cell Line, Tumor; Endocannabinoids; Genistein; Hydrolysis; Mice; Nystatin; Polyunsaturated Alkamides; Progesterone; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Rats

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

To evaluate whether high intraocular pressure (IOP)-induced ischemia is associated with modifications in the retinal endocannabinoid metabolism and to ascertain whether drugs that interfere with the endocannabinoid system may prevent retinal damage due to ischemic insult.. Anandamide (AEA) synthesis, transport, hydrolysis, and AEA endogenous levels were assessed by means of high-performance liquid chromatography in the retinas of rats undergoing 45 minutes of ischemia followed by 12 hours of reperfusion. Under these experimental conditions, binding to cannabinoid (CB1R) and vanilloid (TRPV1) receptor was assessed with rapid-filtration assays. AEA-hydrolase (FAAH, fatty acid amide hydrolase), CB1R and TRPV1 protein content was determined by enzyme-linked immunosorbent assay. Finally, to characterize the neuroprotective profile of drugs that interfere with the endocannabinoid system, cell counting in the retinal ganglion cell (RGC) layer and real-time polymerase chain reactions for Thy-1 mRNA expression were used.. In rat retina, ischemic insult followed by reperfusion resulted in enhanced FAAH activity and protein expression paralleled by a significant decrease in the endogenous AEA tone, whereas the AEA-membrane transporter or the AEA-synthase NAPE-PLD (N-acyl-phosphatidylethanolamine-hydrolyzing-phospholipase-d) were not affected. Retinal ischemia-reperfusion decreased the expression of cannabinoid (CB1) and vanilloid (TRPV1) receptors. Systemic administration of a specific FAAH inhibitor (e.g., URB597) reduced enzyme activity and minimized the retinal damage observed in ischemic-reperfused samples. Similarly, intravitreal injection of the AEA stable analogue, R(+)-methanandamide, reduced cell loss in the RGC layer, and this was prevented by systemic administration of a CB1 or TRPV1 selective antagonist (e.g., SR141716 and capsazepine, respectively).. The original observation that retinal ischemia-reperfusion reduces endogenous AEA via enhanced expression of FAAH supports the deduction that this is implicated in retinal cell loss caused by high IOP in the RGC layer.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Modulators; Capsaicin; Carbamates; Cell Count; Chromatography, High Pressure Liquid; Endocannabinoids; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Hydrolysis; Intraocular Pressure; Male; Ocular Hypertension; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Reperfusion Injury; Retinal Diseases; Retinal Ganglion Cells; Retinal Vessels; Reverse Transcriptase Polymerase Chain Reaction; Rimonabant; RNA, Messenger; Thy-1 Antigens; TRPV Cation Channels

Genistein, a tyrosine kinase inhibitor used to block caveolae dependent endocytosis, reduces the cellular uptake of anandamide in RBL2H3 basophilic leukaemia cells. However, genistein is also a competitive inhibitor of fatty acid amide hydrolase, the enzyme responsible for anandamide hydrolysis. Here we have investigated whether inhibition of fatty acid amide hydrolase rather than inhibition of endocytosis is the primary determinant of genistein actions upon anandamide uptake.. Cellular uptake of anandamide, labelled in the arachidonoyl part of the molecule was assessed in four different cell lines using a standard method. Fatty acid amide hydrolase activity in homogenates and intact cells was measured using anandamide labelled in the ethanolamine part of the molecule.. The fatty acid amide hydrolase inhibitor URB597 inhibited anandamide uptake into RBL2H3 cells and R3327 AT-1 prostate cancer cells, but not into 3T3-L1 preadipocytes or PC-3 prostate cancer cells. An identical pattern was seen with genistein. The related compound daidzein inhibited anandamide hydrolysis in homogenates and intact cells, and reduced its uptake into RBL2H3 and R3327 AT-1, but not PC-3 cells. Anandamide hydrolysis by cell homogenates was in the order RBL2H3 > R3327 AT-1 > PC-3 approximately 3T3-L1.. The ability of genistein to inhibit anandamide uptake is mimicked by daidzein (which does not affect tyrosine kinase), and is only seen in cells that show sensitivity to URB597. This indicates that blockade of fatty acid amide hydrolase is the primary determinant of the effects of genistein on cellular anandamide uptake.

Topics: 3T3-L1 Cells; Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Biological Transport; Carbamates; Cell Line, Tumor; Dose-Response Relationship, Drug; Endocannabinoids; Genistein; Humans; Hydrogen-Ion Concentration; Hydrolysis; Isoflavones; Male; Mice; Molecular Structure; Polyunsaturated Alkamides; Protein Kinase Inhibitors; Rats; Tritium

Studies in isolated preparations of vascular tissue (mainly resistance vessels) provide evidence that anandamide exerts vasorelaxation. The aim of the present work was to further characterize the mechanisms involved in the vascular response induced by anandamide in a conduit vessel, rat aorta.. Isometric tension changes in response to a cumulative concentration-response curve of anandamide (1 nM-100 micro M) were recorded in aortic rings from male Wistar rats. The involvement of a number of factors in this relaxation was investigated including endothelium-derived vasorelaxant products, cannabinoid and vanilloid receptors (transient potential vanilloid receptor-1 (TRPV1)), release of calcitonin gene-related peptide (CGRP), anandamide metabolism and the membrane transporter for anandamide.. Anandamide caused a significant concentration-dependent vasorelaxation in rat aorta. This vasorelaxation was significantly inhibited by Pertussis toxin, by a non-CB1/non-CB2 cannabinoid receptor antagonist, by endothelial denudation, by inhibition of nitric oxide synthesis or inhibition of prostanoid synthesis via cyclooxygenase-2 (COX-2), by blockade of prostaglandin receptors EP4 and by a fatty acid amino hydrolase inhibitor. Antagonists for CB1, CB2, TRPV1 or CGRP receptors, an inhibitor of the release of endothelium-derived hyperpolarizing factor, and an inhibitor of anandamide transport did not modify the vascular response to anandamide.. Our results demonstrate, for the first time, the involvement of the non-CB1/non-CB2 cannabinoid receptor and an anandamide-arachidonic acid-COX-2 derived metabolite (which acts on EP4 receptors) in the endothelial vasorelaxation caused by anandamide in rat aorta.

Topics: Animals; Aorta, Abdominal; Apamin; Arachidonic Acids; Benzamides; Calcitonin Gene-Related Peptide; Camphanes; Cannabinoid Receptor Modulators; Capsaicin; Carbamates; Charybdotoxin; Dose-Response Relationship, Drug; Endocannabinoids; Endothelium, Vascular; In Vitro Techniques; Indomethacin; Isoindoles; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Peptide Fragments; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; Sulfonamides; Vasodilation

Levodopa is the most commonly prescribed drug for Parkinson's disease (PD). Although levodopa improves PD symptoms in the initial stages of the disease, its long-term use is limited by the development of side effects, including abnormal involuntary movements (dyskinesias) and psychiatric complications. The endocannabinoid system is emerging as an important modulator of basal ganglia functions and its pharmacologic manipulation represents a promising therapy to alleviate levodopa-induced dyskinesias. Rats with 6-OHDA lesions that are chronically treated with levodopa develop increasingly severe axial, limb, locomotor and oro-facial abnormal involuntary movements (AIMs). Administration of the cannabinoid agonist WIN 55,212-2 attenuated levodopa-induced axial, limb and oral AIMs dose-dependently via a CB(1)-mediated mechanism, whereas it had no effect on locomotive AIMs. By contrast, systemic administration of URB597, a potent FAAH inhibitor, did not affect AIMs scoring despite its ability to increase anandamide concentration throughout the basal ganglia. Unlike WIN, anandamide can also bind and activate transient receptor potential vanilloid type-1 (TRPV1) receptors, which have been implicated in the modulation of dopamine transmission in the basal ganglia. Interestingly, URB597 significantly decreased all AIMs subtypes only if co-administered with the TRPV1 antagonist capsazepine. Our data indicate that pharmacological blockade of TRPV1 receptors unmasks the anti-dyskinetic effects of FAAH inhibitors and that CB(1) and TRPV1 receptors play opposite roles in levodopa-induced dyskinesias.

Topics: Amidohydrolases; Animals; Antiparkinson Agents; Arachidonic Acids; Basal Ganglia; Benzamides; Benzoxazines; Cannabinoids; Capsaicin; Carbamates; Drug Therapy, Combination; Dyskinesia, Drug-Induced; Endocannabinoids; Levodopa; Male; Morpholines; Naphthalenes; Oxidopamine; Parkinson Disease, Secondary; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; TRPV Cation Channels

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

Fatty acid amides constitute a large and diverse class of lipid transmitters that includes the endogenous cannabinoid anandamide and the sleep-inducing substance oleamide. The magnitude and duration of fatty acid amide signaling are controlled by enzymatic hydrolysis in vivo. Fatty acid amide hydrolase (FAAH) activity in mammals has been primarily attributed to a single integral membrane enzyme of the amidase signature (AS) family. Here, we report the functional proteomic discovery of a second membrane-associated AS enzyme in humans that displays FAAH activity. The gene that encodes this second FAAH enzyme was found in multiple primate genomes, marsupials, and more distantly related vertebrates, but, remarkably, not in a number of lower placental mammals, including mouse and rat. The two human FAAH enzymes, which share 20% sequence identity and are referred to hereafter as FAAH-1 and FAAH-2, hydrolyzed primary fatty acid amide substrates (e.g. oleamide) at equivalent rates, whereas FAAH-1 exhibited much greater activity with N-acyl ethanolamines (e.g. anandamide) and N-acyl taurines. Both enzymes were sensitive to the principal classes of FAAH inhibitors synthesized to date, including O-aryl carbamates and alpha-keto heterocycles. These data coupled with the overlapping, but distinct tissue distributions of FAAH-1 and FAAH-2 suggest that these proteins may collaborate to control fatty acid amide catabolism in primates. The apparent loss of the FAAH-2 gene in some lower mammals should be taken into consideration when extrapolating genetic or pharmacological findings on the fatty acid amide signaling system across species.

Topics: Amidohydrolases; Amino Acid Sequence; Animals; Arachidonic Acids; Cell Line, Tumor; Chlorocebus aethiops; COS Cells; Endocannabinoids; Fatty Acids; Humans; Hydrolysis; Mice; Molecular Sequence Data; Placenta; Polyunsaturated Alkamides; Rats; Sequence Homology, Amino Acid; Signal Transduction; Tissue Distribution

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

A binding assay for human fatty acid amide hydrolase (FAAH) using the scintillation proximity assay (SPA) technology is described. This SPA uses the specific interactions of [3H]R(+)-methanandamide (MAEA) and FAAH expressing microsomes to evaluate the displacement activity of FAAH inhibitors. We observed that a competitive nonhydrolyzed FAAH inhibitor, [3H]MAEA, bound specifically to the FAAH microsomes. Coincubation with an FAAH inhibitor, URB-597, competitively displaced the [3H]MAEA on the FAAH microsomes. The released radiolabel was then detected through an interaction with the SPA beads. The assay is specific for FAAH given that microsomes prepared from cells expressing the inactive FAAH-S241A mutant or vector alone had no significant ability to bind [3H]MAEA. Furthermore, the binding of [3H]MAEA to FAAH microsomes was abolished by selective FAAH inhibitors in a dose-dependent manner, with IC50 values comparable to those seen in a functional assay. This novel SPA has been validated and demonstrated to be simple, sensitive, and amenable to high-throughput screening.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Binding Sites; Calcium Channel Blockers; Carbamates; CHO Cells; Cricetinae; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Endocannabinoids; Enzyme Inhibitors; Humans; Inhibitory Concentration 50; Ligands; Microsomes; Polyunsaturated Alkamides; Scintillation Counting; Wheat Germ Agglutinins

Male Wistar rats were trained to discriminate (-)-nicotine (0.4 mg/kg) from saline under a two-lever, fixed-ratio 10 schedule of water reinforcement. During test sessions the following drugs were coadministered with saline (substitution studies) or nicotine (0.025-0.4 mg/kg; combination studies): the alpha4beta2 nicotinic acetylcholine receptor subtype antagonist dihydro-beta-erythroidine (DHbetaE), the non-selective nicotinic acetylcholine receptor subtype antagonist mecamylamine, the alpha7 nicotinic acetylcholine receptor subtype antagonist methyllycaconitine (MLA), the alpha4beta2 nicotinic acetylcholine receptor subtype agonist 5-iodo-3-(2(S)-azetidinylmethoxy)pyridine (5-IA), the cannabinoid CB1 receptor antagonist/partial agonist rimonabant, the cannabinoid CB2 receptor antagonist N-[(1S)-endo-1,3,3-trimethylbicyclo-[2.2.1]heptan-2-yl]5-(4-chloro-3-methyl-phenyl)-1-(4-methybenzyl)pyrazole-3-carboxamide (SR 144528), the cannabinoid CB1/2 receptor agonists (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]-trans-4-(3-hydroxy-propyl)cyclohexanol (CP 55,940) or R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]-pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-(1-naphthalenyl)-methanone mesylate (WIN 55,212-2), the endogenous cannabinoid agonist and non-competitive alpha7 nicotinic acetylcholine receptor subtype antagonist anandamide, the anandamide uptake and fatty acid amide hydrolase inhibitor N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (AM-404), the fatty acid amide hydrolase inhibitor cyclohexylcarbamic acid 3'-carbamoyl-biphenyl-3-yl ester (URB 597), AM-404+anandamide or URB 597+anandamide. 5-IA (0.01 mg/kg) fully substituted for nicotine, while other drugs were inactive. In combination studies, DHbetaE and mecamylamine dose-dependently attenuated the discriminative stimulus effects of nicotine and the full substitution of 5-IA, while MLA, rimonabant, SR 144528, CP 55,940, WIN 55,212-2, and URB 597 did not alter the nicotine cue. Pretreatment with AM-404+anandamide or URB 597+anandamide weakly enhanced nicotine-lever responding. Our pharmacological analyses demonstrates that the expression of nicotine discrimination is under the control of nicotinic acetylcholine receptor subtypes composed of alpha4beta2 (but not of alpha7) subunits. Furthermore, we excluded the involvement of either cannabinoid CB1 and CB2 receptors or increases in the endocannabinoid tone in the nicotine discrimination.

Topics: Aconitine; Animals; Arachidonic Acids; Azetidines; Benzamides; Benzoxazines; Camphanes; Cannabinoid Receptor Antagonists; Carbamates; Cyclohexanols; Dihydro-beta-Erythroidine; Discrimination Learning; Dose-Response Relationship, Drug; Drug Synergism; Endocannabinoids; Male; Mecamylamine; Morpholines; Naphthalenes; Nicotine; Nicotinic Agonists; Nicotinic Antagonists; Polyunsaturated Alkamides; Pyrazoles; Pyridines; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Nicotinic

Although endogenous cannabinoid systems have been implicated in the modulation of the rewarding effects of abused drugs and food, little is known about the direct effects of endogenous ligands for cannabinoid receptors on brain reward processes. Here we show for the first time that the intravenous administration of anandamide, an endogenous ligand for cannabinoid receptors, and its longer-lasting synthetic analog methanandamide, increase the extracellular dopamine levels in the nucleus accumbens shell of awake, freely moving rats, an effect characteristic of most drugs abused by humans. Anandamide produced two distinctly different effects on dopamine levels: (1) a rapid, transient increase that was blocked by the cannabinoid CB1 receptor antagonist rimonabant, but not by the vanilloid VR1 receptor antagonist capsazepine, and was magnified and prolonged by the fatty acid amide hydrolase (FAAH) enzyme inhibitor, URB597; (2) a smaller delayed and long-lasting increase, not sensitive to CB1, VR1 or FAAH blockade. Both effects were blocked by infusing either tetrodotoxin (TTX, 1 microm) or calcium-free Ringer's solution through the microdialysis probe, demonstrating that they were dependent on the physiologic activation of dopaminergic neurotransmission. Thus, these results indicate that anandamide, through the activation of the mesolimbic dopaminergic system, participates in the signaling of brain reward processes.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Calcium; Cannabinoids; Carbamates; Data Interpretation, Statistical; Dopamine; Endocannabinoids; Enzyme Inhibitors; Male; Microdialysis; Nucleus Accumbens; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Synaptic Transmission; Tetrodotoxin; TRPV Cation Channels

The endogenous cannabinoid system is responsive to the neurobiological actions of Delta9-tetrahydrocannabinol (THC) and other cannabinoid ligands. While numerous studies have focused on the behavioral and pharmacological effects of THC and cannabinoid agonists in experimental animals, most recent work focuses on compounds that modulate endocannabinoid neurotransmission. However, the relevant studies concerning the ability of endocannabinoid modulators to modify reward processes in experimental animals remain rather scarce.. The present study examined the effects of drugs modulating endocannabinoid neurotransmission on brain reward function using the rate-frequency curve shift paradigm of intracranial self-stimulation (ICSS).. Animals were implanted with electrodes into the medial forebrain bundle (MFB). After brain stimulation reward thresholds stabilized, rats received intraperitoneal injections of the fatty acid amide hydrolase (FAAH) inhibitors phenylmethylsulfonyl fluoride (PMSF) (0, 15, 30, and 60 mg/kg) and URB-597 (0, 0.3, 1, and 3 mg/kg) and the selective anandamide reuptake inhibitor OMDM-2 (0, 3, 10, and 30 mg/kg).. The highest dose of URB-597 and OMDM-2 significantly increased the threshold frequency required for MFB ICSS, while PMSF increased the threshold frequency in all doses tested. The cannabinoid 1 (CB1) receptor antagonist SR141716A reversed the actions of URB-597 and OMDM-2, but not PMSF, without affecting reward thresholds by itself.. These results indicate that under the present experimental conditions endocannabinoid modulators do not exhibit reinforcing properties, but rather have inhibitory influence on reward processes. The anhedonic effects of URB-597 and OMDM-2, but not PMSF, observed at the highest doses in this study are probably mediated through direct CB1 receptor stimulation.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Benzyl Compounds; Brain; Cannabinoid Receptor Modulators; Carbamates; Conditioning, Operant; Dose-Response Relationship, Drug; Electric Stimulation; Endocannabinoids; Injections, Intraperitoneal; Male; Medial Forebrain Bundle; Phenylmethylsulfonyl Fluoride; Piperidines; Polyunsaturated Alkamides; Protease Inhibitors; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Reinforcement Schedule; Reward; Rimonabant; Self Stimulation; Synaptic Transmission

The aim of the present study was to investigate the relationship between TRPV1 stimulation and endocannabinoid-driven CB(1) receptor-mediated inhibition of activity in adult rat dorsal root ganglion (DRG) neurons, a model of primary afferent nociceptors. Calcium-imaging studies were performed to compare the effects of the fatty acid amide hydrolase (FAAH) inhibitor URB597 (1 microm) vs. the anandamide (AEA) uptake inhibitor UCM707 (1 microm) on capsaicin (100 nm) and N-arachidonoyl dopamine (NADA; 1 microm)-evoked changes in intracellular calcium [Ca(2+)](i) in DRG neurons. The ability of the CB(1) receptor antagonist AM251 (1 microm) to modulate the effects of URB597 and UCM707 was also determined. Suprafusion of NADA and capsaicin evoked robust increases in [Ca(2+)](i) in DRG neurons (89 +/- 4% and 132 +/- 6% of the depolarizing KCl response, respectively). Co-incubation with URB597 significantly attenuated both NADA and capsaicin-evoked increases in [Ca(2+)](i) (39 +/- 3% and 79 +/- 4% of KCl response, respectively). Similarly, co-incubation with UCM707 significantly attenuated both NADA and capsaicin-evoked increases in [Ca(2+)](i) (59 +/- 7% and 72 +/- 4% of KCl response, respectively). The CB(1) receptor antagonist AM251 significantly attenuated the effects of URB597 on NADA-evoked increases in [Ca(2+)](i) but not the effects of URB597 on capsaicin-evoked increases in [Ca(2+)](i). By contrast, AM251 significantly attenuated the inhibitory effects of UCM707 on both NADA and capsaicin-evoked increases in [Ca(2+)](i.) These data suggest that transport of both NADA and capsaicin into DRG neurons and the subsequent activation of TRPV1 is partly governed by FAAH-dependent mechanisms as well as via the putative AEA membrane transporter.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Calcium; Capsaicin; Carbamates; Cells, Cultured; Dopamine; Drug Interactions; Endocannabinoids; Fura-2; Furans; Ganglia, Spinal; Male; Membrane Potentials; Membrane Transport Proteins; Neurons; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; TRPV Cation Channels

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

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

Bioactive N-acylethanolamines including the endocannabinoid anandamide are known to be hydrolyzed to fatty acids and ethanolamine by fatty acid amide hydrolase (FAAH). In addition, we recently cloned an isozyme termed "N-acylethanolamine-hydrolyzing acid amidase (NAAA)", which is active only at acidic pH [Tsuboi, Sun, Okamoto, Araki, Tonai, Ueda, J. Biol. Chem. 285 (2005) 11082-11092]. However, physiological roles of NAAA remained unclear. Here, we examined a possible contribution of NAAA to the degradation of various N-acylethanolamines in macrophage cells. NAAA mRNA as well as FAAH mRNA was detected in several macrophage-like cells, including RAW264.7, and mouse peritoneal macrophages. The homogenates of RAW264.7 cells showed both the NAAA and FAAH activities which were confirmed with the aid of their respective specific inhibitors, N-cyclohexanecarbonylpentadecylamine (CCP) and URB597. As analyzed with intact cells, RAW264.7 cells and peritoneal macrophages degraded anandamide, N-palmitoylethanolamine, N-oleoylethanolamine, and N-stearoylethanolamine. Pretreatment of the cells with CCP or URB597 partially inhibited the degradation, and a combination of the two compounds caused more profound inhibition. In contrast, the anandamide hydrolysis in mouse brain appeared to be principally attributable to FAAH despite the expression of NAAA in the brain. These results suggested that NAAA and FAAH cooperatively degraded various N-acylethanolamines in macrophages.

Topics: Amides; Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Brain; Carbamates; Cell Line; Cell Line, Tumor; Endocannabinoids; Enzyme Inhibitors; Ethanolamines; Gene Expression; Humans; Kidney; Liver; Lung; Macrophages; Macrophages, Peritoneal; Male; Mice; Mice, Inbred C57BL; Polyunsaturated Alkamides; Rats; Rats, Wistar; U937 Cells

Although anecdotal reports suggest that cannabis may be used to alleviate symptoms of depression, the psychotropic effects and abuse liability of this drug prevent its therapeutic application. The active constituent of cannabis, delta9-tetrahydrocannabinol, acts by binding to brain CB1 cannabinoid receptors, but an alternative approach might be to develop agents that amplify the actions of endogenous cannabinoids by blocking their deactivation. Here, we show that URB597, a selective inhibitor of the enzyme fatty-acid amide hydrolase, which catalyzes the intracellular hydrolysis of the endocannabinoid anandamide, exerts potent antidepressant-like effects in the mouse tail-suspension test and the rat forced-swim test. Moreover, URB597 increases firing activity of serotonergic neurons in the dorsal raphe nucleus and noradrenergic neurons in the nucleus locus ceruleus. These actions are prevented by the CB1 antagonist rimonabant, are accompanied by increased brain anandamide levels, and are maintained upon repeated URB597 administration. Unlike direct CB1 agonists, URB597 does not exert rewarding effects in the conditioned place preference test or produce generalization to the discriminative effects of delta9-tetrahydrocannabinol in rats. The findings support a role for anandamide in mood regulation and point to fatty-acid amide hydrolase as a previously uncharacterized target for antidepressant drugs.

Topics: Animals; Antidepressive Agents; Arachidonic Acids; Behavior, Animal; Benzamides; Brain; Cannabinoid Receptor Agonists; Carbamates; Dronabinol; Endocannabinoids; Hydrolysis; Male; Mice; Mice, Inbred C57BL; Neurons; Norepinephrine; Polyunsaturated Alkamides; Rats; Receptors, Cannabinoid; Serotonin

Endocannabinoids are novel lipid mediators with hypotensive and cardiodepressor activity. Here, we examined the possible role of the endocannabinergic system in cardiovascular regulation in hypertension.. In spontaneously hypertensive rats (SHR), cannabinoid-1 receptor (CB1) antagonists increase blood pressure and left ventricular contractile performance. Conversely, preventing the degradation of the endocannabinoid anandamide by an inhibitor of fatty acid amidohydrolase reduces blood pressure, cardiac contractility, and vascular resistance to levels in normotensive rats, and these effects are prevented by CB1 antagonists. Similar changes are observed in 2 additional models of hypertension, whereas in normotensive control rats, the same parameters remain unaffected by any of these treatments. CB1 agonists lower blood pressure much more in SHR than in normotensive Wistar-Kyoto rats, and the expression of CB1 is increased in heart and aortic endothelium of SHR compared with Wistar-Kyoto rats.. We conclude that endocannabinoids tonically suppress cardiac contractility in hypertension and that enhancing the CB1-mediated cardiodepressor and vasodilator effects of endogenous anandamide by blocking its hydrolysis can normalize blood pressure. Targeting the endocannabinoid system offers novel therapeutic strategies in the treatment of hypertension.

Topics: Amidohydrolases; Angiotensin II; Animals; Arachidonic Acids; Benzamides; Benzyl Compounds; Blood Pressure; Camphanes; Carbamates; Dronabinol; Endocannabinoids; Endothelium, Vascular; Hypertension; Male; Models, Cardiovascular; Myocardial Contraction; Myocardium; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Inbred Dahl; Rats, Inbred SHR; Rats, Inbred WKY; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Rimonabant; Up-Regulation; Vascular Resistance; Vasodilation; Ventricular Function, Left