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BACKGROUND: Ethanol (EtOH) self-administration is particularly sensitive to the modulation of CB(1) signaling in the nucleus accumbens (NAc) shell, and EtOH consumption increases extracellular levels of the endogenous cannabinoid CB(1) receptor agonist 2-arachidonoyl glycerol (2-AG) in this brain region. Stimulation of CB(1) receptor with agonists increases EtOH consumption, suggesting that EtOH-induced increases in 2-AG might sustain motivation for EtOH intake. METHODS: In order to further explore this hypothesis, we analyzed the alterations in operant EtOH self-administration induced by intra-NAc shell infusions of 2-AG itself, the CB(1) inverse agonist SR141716A, the 2-AG clearance inhibitor URB602, anandamide, and the cyclooxygenase-2 (COX-2) inhibitor nimesulide. RESULTS: Surprisingly, self-administration of 10% EtOH was dose-dependently reduced by either intra-NAc shell SR141716A or 2-AG infusions. Similar effects were found by intra-NAc shell infusions of URB602, suggesting again a role for accumbal 2-AG on the modulation of EtOH intake. Intra-NAc shell anandamide did not alter EtOH self-administration, pointing to a specific role for 2-AG in the modulation of EtOH self-administration. Finally, the inhibitory effect of intra-NAc shell 2-AG on EtOH intake was significantly reversed by pretreatment with nimesulide, suggesting that oxidative metabolites of 2-AG might mediate these inhibitory effects on operant self-administration. CONCLUSIONS: We propose that 2-AG signaling in the NAc exerts an inhibitory influence on EtOH consumption through a non–CB1 receptor mechanism involving the COX-2 pathway.

Recurrent panic attacks, comprising emotional and cardiovascular aversive responses, are common features in panic disorder, a subtype of anxiety disorder. The underlying brain circuitry includes nuclei of the hypothalamus, such as the dorsomedial hypothalamus (DMH). The endocannabinoid system has been proposed to modulate several biological processes in the hypothalamus. Thus, we tested the hypothesis that hypothalamic endocannabinoid signalling controls aversive responses in an animal model of panic attacks. Local infusion of NMDA into the DMH of rats induced panic-like behaviour. This effect was prevented by local, but not intraperitoneal, injection of a 2-arachidonoylglycerol (2-AG) hydrolysis inhibitor (MAGL inhibitor, URB602). The anandamide hydrolysis inhibitor (FAAH inhibitor), URB597, was ineffective. The anti-aversive action of URB602 was reversed by CB1 and CB2 antagonists (AM251 and AM630, respectively), and mimicked by CB1 and CB2 agonists (ACEA and JWH133, respectively). URB602 also prevented the cardiovascular effects of DMH-stimulation in anaesthetised animals. None of the treatments modified blood corticosterone levels. In conclusion, facilitation of 2-AG-signalling in the DMH modulates panic-like responses. The possible mechanisms comprise activation of both CB1 and CB2 receptors in this brain region.

Objectives Monoacylglycerol lipase participates in organ protection by regulating the hydrolysis of the endocannabinoid 2-arachidonoylglycerol. This study investigated whether blocking monoacylglycerol lipase protects against postresuscitation myocardial injury and improves survival in a rat model of cardiac arrest and cardiopulmonary resuscitation. Design Prospective randomized laboratory study. Setting University research laboratory. Subjects Male Sprague-Dawley rat (n = 96). Interventions Rats underwent 8-minute asphyxia-based cardiac arrest and resuscitation. Surviving rats were randomly divided into cardiopulmonary resuscitation + URB602 group, cardiopulmonary resuscitation group, and sham group. One minute after successful resuscitation, rats in the cardiopulmonary resuscitation + URB602 group received a single dose of URB602 (5 mg/kg), a small-molecule monoacylglycerol lipase inhibitor, whereas rats in the cardiopulmonary resuscitation group received an equivalent volume of vehicle solution. The sham rats underwent all of the procedures performed on rats in the cardiopulmonary resuscitation and cardiopulmonary resuscitation + URB602 groups minus cardiac arrest and asphyxia. Measurements and main results Survival was recorded 168 hours after the return of spontaneous circulation (n = 22 in each group). Compared with vehicle treatment (31.8%), URB602 treatment markedly improved survival (63.6%) 168 hours after cardiopulmonary resuscitation. Next, we used additional surviving rats to evaluate myocardial and mitochondrial injury 6 hours after return of spontaneous circulation, and we found that URB602 significantly reduced myocardial injury and prevented myocardial mitochondrial damage. In addition, URB602 attenuated the dysregulation of endocannabinoid and eicosanoid metabolism 6 hours after return of spontaneous circulation and prevented the acceleration of mitochondrial permeability transition 15 minutes after return of spontaneous circulation. Conclusions Monoacylglycerol lipase blockade may reduce myocardial and mitochondrial injury and significantly improve the resuscitation effect after cardiac arrest and cardiopulmonary resuscitation.

Background Clinical reports suggest that rather than directly driving cocaine use, stress may create a biological context within which other triggers for drug use become more potent. We hypothesize that stress-induced increases in corticosterone “set the stage” for relapse by promoting endocannabinoid-induced attenuation of inhibitory transmission in the prelimbic cortex (PL). Methods We have established a rat model for these stage-setting effects of stress. In this model, neither a stressor (electric footshock) nor stress-level corticosterone treatment alone reinstates cocaine seeking following self-administration and extinction, but each treatment potentiates reinstatement in response to an otherwise subthreshold cocaine priming dose (2.5 mg/kg, intraperitoneal). The contributions of endocannabinoid signaling in the PL to the effects of stress-level corticosterone on PL neurotransmission and cocaine seeking were determined using intra-PL microinfusions. Endocannabinoid-dependent effects of corticosterone on inhibitory synaptic transmission in the rat PL were determined using whole-cell recordings in layer V pyramidal neurons. Results Corticosterone application attenuated inhibitory synaptic transmission in the PL via cannabinoid receptor type 1 (CB 1 R)– and 2-arachidonoylglycerol–dependent inhibition of gamma-aminobutyric acid release without altering postsynaptic responses. The ability of systemic stress-level corticosterone treatment to potentiate cocaine-primed reinstatement was recapitulated by intra-PL injection of corticosterone, the CB 1 R agonist WIN 55,212-2, or the monoacylglycerol lipase inhibitor URB602. Corticosterone effects on reinstatement were attenuated by intra-PL injections of either the CB 1 R antagonist, AM251, or the diacylglycerol lipase inhibitor, DO34. Conclusions These findings suggest that stress-induced increases in corticosterone promote cocaine seeking by mobilizing 2-arachidonoylglycerol in the PL, resulting in CB 1 R-mediated attenuation of inhibitory transmission in this brain region.

DDHD2 has been reported to exhibit phospholipase A1, triacylglycerol (TG) lipase and diacylglycerol (DG) lipase activities. However, the detailed enzymatic properties of DDHD2 have not yet been elucidated. In the current study, the substrate specificity of DDHD2 towards DG, TG and phosphatidic acid (PA) has been examined using highly purified recombinant rat DDHD2 (rDDHD2) with a liquid chromatography mass spectrometer. The k cat/Km value for DG (18:0/20:4) was much higher than those for TG (18:1/18:1/18:1), and PA (18:0/20:4) in the presence of sodium deoxycholate. The enzyme activity of rDDHD2 towards DG (18:0/20:4) was highest among all of the substrates tested. In addition, rDDHD2 was highly specific to DG substrates with a polyunsaturated fatty acid at their sn-2 position. The levels of 2-arachidonoylglycerol (2-AG) in CHO cells were quantified by gas chromatography-tandem mass spectrometry, showing that CHO cells expressing recombinant rDDHD2 contained higher levels of 2-AG when cells were treated with a monoacylglycerol lipase inhibitor, URB602. These results therefore support the idea that DDHD2 functions as a DG lipase in vivo and produces 2-AG.

Lung ischemia-reperfusion injury (LIRI) is a common and severe postoperative pathologic complication that often occurs when the oxygen supply disrupted to the lung tissue fallowed by reperfusion period, in most cases after lung transplantation and cardiopulmonary bypass. Endocannabinoids such as 2-arachidonoylglycerol (2-AG) have very important role as regulators of inflammation. Monoacylglycerol lipase (MAGL) is the main 2-AG-degrading enzyme, and the downstream metabolites of 2-AG play a role in the inflammation. Ischemia reperfusion (IR) was induced by clamping the left pulmonary hilum for 60 min, followed by 120 min of reperfusion in male C57BL/6 mice. Effects of URB602, a MAGL inhibitor, were evaluated in a preventive or therapeutic regimen (5 min before ischemia or reperfusion, respectively). Oxygenation index, wet-to-dry weight ratio and lung injury score were analyzed. Endocannabinoids including 2-AG, anandamide (AEA) and arachidonic acid (AA) levels, metabolites such as Prostaglandin I2 (PGI2), Thromboxane B2 (TXB2) and Leukotrienes B4 (LTB4) and inflammatory markers (Interleukin 6 (IL-6) andTumor necrosis factor-α (TNF-α)) in lung tissues were measured by using mass spectrometry or ELISA analyses. We found that IR increased the wet-to-dry weight ratio of lung and lung injury score and decreased oxygenation index as compared to the sham group. Moreover, treatment with URB602 in preventive or therapeutic regimen reduced the wet-to-dry weight ratio and lung injury score while increased oxygenation index when compared with the IR group, with a more improvement in the preventive regimen group. In addition, treatment with URB602 before ischemia increased 2-AG level but decreased metabolites (AA, PGI2, TXB2, LTB4) and inflammatory markers (IL-6, TNF-α). Thus, our study demonstrated that a pretreatment with URB602 significantly reduced IR-induced lung injury and inflammation. URB602 inhibited LIRI and inflammation by increasing 2-AG level and reducing downstream metabolites from AA to PGI2, TXB2 and LTB4 in lung tissues.

Monoacylglycerol lipase (MAGL) has an essential role in the catabolic pathway of the endocannabinoid 2-arachidonoylglycerol, which makes it a potential target for highly specific inhibitors for the treatment of a number of diseases. We designed and synthesized a series of carbamate analogues of URB602. We evaluated their inhibitory activity toward human MAGL in vitro both in cell culture and lysates. The target compounds exhibited moderate to excellent inhibitory activity against MAGL. The most promising compound 2b showed good inhibitory activity with IC50 value of 4.5 ± 0.70 μM reducing MAGL activity to 82% of controls at 10 μM compared to 66% for the parent compound URB602. Interestingly, compounds 2b and 2c induce cell death through the inhibition of MAGL. Molecular modelling approaches and docking studies, used to investigate inhibitory profiles, indicated that trifluoromethyl substitutions of the aryl group and the benzene ring present at the oxygen side of the carbamate molecule had a significant impact on the activity.

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

Background Drugs that modulate endocannabinoid signalling are effective in reducing nociception in animal models of pain and may be of value in the treatment of migraine. Methods We investigated the anti-nociceptive effects of inhibition of monoacylglycerol lipase (MGL), a key enzyme in the hydrolysis of the 2-arachidonoylglycerol, in a rat model of migraine based on nitroglycerin (NTG) administration. We evaluated c-fos expression in specific brain areas and nociceptive behavior in trigeminal and extra-trigeminal body areas. Results URB602, a reversible MGL inhibitor, did not show any analgesic effect in the tail flick test, but it inhibited NTG-induced hyperalgesia in both the tail flick test and the formalin test applied to the hind paw or to the orofacial area. Quite unexpectedly, URB602 potentiated formalin-induced hyperalgesia in the trigeminal area when used alone. The latter result was also confirmed using a structurally distinct, irreversible MGL inhibitor, JZL184. URB602 did not induce neuronal activation in the area of interest, but significantly reduced the NTG-induced neuronal activation in the ventrolateral column of the periaqueductal grey and the nucleus trigeminalis caudalis. Conclusions These findings support the hypothesis that modulation of the endocannabinoid system may be a valuable approach for the treatment of migraine. The topographically segregated effect of MGL inhibition in trigeminal/extra-trigeminal areas calls for further mechanistic research.

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

Colorectal cancer (CRC) is a major health problem in Western countries. The endocannabinoid 2-arachidonoyl-glycerol (2-AG) exerts antiproliferative actions in a number of tumoral cell lines, including CRC cells. Monoacylglycerol lipase (MAGL), a serine hydrolase that inactivates 2-AG, is highly expressed in aggressive human cancer cells. Here, we investigated the role of MAGL in experimental colon carcinogenesis. The role of MAGL was assessed in vivo by using the xenograft and the azoxymethane models of colon carcinogenesis; MAGL expression was evaluated by RT-PCR and immunohistochemistry; 2-AG levels were measured by liquid chromatography mass spectrometry; angiogenesis was evaluated in tumor tissues [by microvessel counting and by investigating the expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) proteins] as well as in human umbilical vein endothelial cells (HUVEC); cyclin D1 was evaluated by RT-PCR. MAGL and 2-AG were strongly expressed in tumor tissues. The MAGL inhibitor URB602 reduced xenograft tumor volume, this effect being associated to down-regulation of VEGF and FGF-2, reduction in the number of vessels and down-regulation of cyclin D1. In HUVEC, URB602 exerted a direct antiangiogenic effect by inhibiting FGF-2 induced proliferation and migration, and by modulating pro/anti-angiogenic agents. In experiments aiming at investigating the role of MAGL in chemoprevention, URB602 attenuated azoxymethane-induced preneoplastic lesions, polyps and tumors. MAGL, possibly through modulation of angiogenesis, plays a pivotal role in experimental colon carcinogenesis. Pharmacological inhibition of MAGL could represent an innovative therapeutic approach to reduce colorectal tumor progression. (C) 2017 Elsevier Ltd. All rights reserved.

We have synthesised an extensive series of URB602 analogues as inhibitors of monoacylglycerol lipase (MAGL), which is the major enzyme responsible for metabolising the endocannabinoid 2-arachidonylglycerol. The recently identified crystal structure of MAGL was used in the design strategy and revealed three possible binding sites for URB602 and the proposed analogues. A test series of carbamate analogues were docked into the identified sites to predict the most favourable binding location. The synthesised analogues of URB602 explored the biological effects of isosteric replacement, ring size and substitution, para substitution of the biphenyl moiety and the incorporation of a bicyclic element. The compounds were tested for their ability to inhibit human MAGL. The carbamate analogue 16 displayed the most significant inhibitory activity, reducing MAGL activity to 26% of controls at 100 μM compared to 73% for the parent compound URB602.

BACKGROUND AND PURPOSE Endocannabinoids have both anti-inflammatory and neuroprotective properties against harmful stimuli. We previously demonstrated that the endocannabinoid 2-arachidonoylglycerol (2-AG) protects hippocampal neurons by limiting the inflammatory response via a CB1 receptor-dependent MAPK/NF-κB signalling pathway. The purpose of the present study was to determine whether PPARγ, an important nuclear receptor, mediates 2-AG-induced inhibition of NF-κB phosphorylation and COX-2 expression, and COX-2-enhanced miniature spontaneous excitatory postsynaptic currents (mEPSCs). EXPERIMENTAL APPROACH By using a whole-cell patch clamp electrophysiological recording technique and immunoblot analysis, we determined mEPSCs, expression of COX-2 and PPARγ, and phosphorylation of NF-kB in mouse hippocampal neurons in culture. KEY RESULTS Exogenous and endogenous 2-AG-produced suppressions of NF-κB-p65 phosphorylation, COX-2 expression and excitatory synaptic transmission in response to pro-inflammatory interleukin-1β (IL-1β) and LPS were inhibited by GW9662, a selective PPARγ antagonist, in hippocampal neurons in culture. PPARγ agonists 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and rosiglitazone mimicked the effects of 2-AG on NF-κB-p65 phosphorylation, COX-2 expression and mEPSCs, and these effects were eliminated by antagonism of PPARγ. Moreover, exogenous application of 2-AG or elevation of endogenous 2-AG by inhibiting its hydrolysis with URB602 or JZL184, selective inhibitors of monoacylglycerol lipase (MAGL), prevented the IL-1β- and LPS-induced reduction of PPARγ expression. The 2-AG restoration of the reduced PPARγ expression was blocked or attenuated by pharmacological or genetic inhibition of the CB1 receptor. CONCLUSIONS AND IMPLICATIONS Our results suggest that CB1 receptor-dependent PPARγ expression is an important and novel signalling pathway in endocannabinoid 2-AG-produced resolution of neuroinflammation in response to pro-inflammatory insults. LINKED ARTICLES This article is part of a themed issue on Cannabinoids in Biology and Medicine. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.163.issue-7

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

The intrathecal (i.t.) injection of 50 and 100 nmol anandamide to urethane anesthetized rats induced a dose-dependent decrease in the mean blood pressure (-10.6+/-1.6 mmHg and -15.0+/-1.7 mmHg, respectively; n=6) whereas a lower dose of this endocannabinoid (25 nmol) was devoid of effect. Similar responses were obtained both with the non-metabolizable analog methanandamide and with the endocannabinoid N-arachidonoyldopamine. When the sub-effective dose (25 nmol) of each compound was co-injected with palmitoylethanolamide (100 nmol), significant decreases in the blood pressure were observed (-12.3+1.3 mmHg for anandamide; -12.1+/-0.8 mmHg for methanandamide; -12.1+/-0.8 mmHg for N-arachidonoyldopamine; n=4-6). Palmitoylethanolamide also enhanced the hypotensive responses to the 50 nmol-dose of both anandamide and methanandamide. The hypotensive response induced by co-administration of palmitoylethanolamide and 25 nmol anandamide was prevented both by the cannabinoid CB(1) receptor antagonist SR 144716A (20 nmol; i.t.) and by the vanilloid TRPV1 receptor antagonist capsazepine (20 nmol; i.t.) and enhanced by pretreatment with URB602 (3.5 nmol; i.t.), a putative inhibitor of palmitoylethanolamide degradation. These results suggest that in the spinal cord palmitoylethanolamide acts as an entourage compound for the hypotensive effects of i.t. administered endocannabinoids. The facilitative action of palmitoylethanolamide affects the vanilloid TRPV1 as well as the cannabinoid CB(1) receptor-mediated effects of endocannabinoids on the blood pressure control.

The endogenous cannabinoid system plays an important role in the regulation of gastrointestinal function in health and disease. Endocannabinoid levels are regulated by catabolic enzymes. Here, we describe the presence and localization of monoacylglycerol lipase (MGL), the major enzyme responsible for the degradation of 2-arachidonoylglycerol. We used molecular, biochemical, immunohistochemical, and functional assays to characterize the distribution and activity of MGL. MGL mRNA was present in rat ileum throughout the wall of the gut. MGL protein was distributed in the muscle and mucosal layers of the ileum and in the duodenum, proximal colon, and distal colon. We observed MGL expression in nerve cell bodies and nerve fibers of the enteric nervous system. There was extensive colocalization of MGL with PGP 9.5 and calretinin- immunoreactive neurons, but not with nitric oxide synthase. MGL was also present in the epithelium and was highly expressed in the small intestine. Enzyme activity levels were highest in the duodenum and decreased along the gut with lowest levels in the distal colon. We observed both soluble and membrane-associated enzyme activities. The MGL inhibitor URB602 significantly inhibited whole gut transit in mice, an action that was abolished in cannabinoid 1 receptor-deficient mice. In conclusion, MGL is localized in the enteric nervous system where endocannabinoids regulate intestinal motility. MGL is highly expressed in the epithelium, where this enzyme may have digestive or other functions yet to be determined. Copyright © 2008 the American Physiological Society.

Diisopropylfluorophosphate (DFP) elicits cholinergic toxicity by inhibiting acetylcholinesterase, leading to accumulation of the neurotransmitter acetylcholine and excessive stimulation of cholinergic receptors throughout the body. Endocannabinoids inhibit the release of neurotransmitters including acetylcholine via a widely distributed retrograde signaling pathway. Endocannabinoid signaling is therefore a potential therapeutic target for the management of OP poisoning. We first evaluated the relative in vitro and in vivo (2.5 mg/kg, sc) effects of DFP on cholinesterase, fatty acid amide hydrolase (FAAH, an endocannabinoid degrading enzyme), monoacylglycerol lipase (MAGL, another endocannabinoid degrading enzyme) and cannabinoid receptor (CB1) binding in rat hippocampus. The effects of WIN 55212-2 (cannabinoid receptor agonist, 1.5 mg/kg), URB597 (FAAH inhibitor, 3 mg/kg), URB602 (MAGL inhibitor, 10 mg/kg) or AM404 (endocannabinoid uptake inhibitor, 10 mg/kg) on DFP toxicity were then examined. Adult male rats (n=5/treatment group) were given either peanut oil or DFP followed immediately by vehicle or one of the four cannabinomimetic drugs. Functional signs of toxicity were evaluated for 24 hours and then rats were sacrificed for neurochemical measurements. DFP inhibited cholinesterase, FAAH, MAGL and CB1 receptor binding in vitro in a concentration-dependent manner, with highest and lowest potency against cholinesterase and FAAH, respectively. In vivo, DFP inhibited hippocampal cholinesterase (89%) and FAAH (42%), but had no significant effect on MAGL or CB1 binding. Rats treated with DFP alone showed typical signs of cholinergic toxicity including involuntary movements and excessive secretions (SLUD signs). WIN 55212-2, URB597, URB602 and AM404 all significantly reduced involuntary movements following DFP exposure in a time-dependent manner, and most (URB597, URB602 and AM404) also significantly reduced DFP-induced SLUD signs. These results suggest that enhancing endocannabinoid signaling can attenuate the acute toxicity of DFP and provide rationale for further investigations on the role of endocannabinoids in cholinergic toxicity.

Background and purpose: There are limited options for the treatment of neuropathic pain. Endocannabinoids, such as anandamide and 2-arachidonoyl glycerol (2-AG), are promising pain modulators and there is recent evidence of interactions between anandamide and 2-AG biosynthesis and metabolism. It has been clearly demonstrated that 2-AG degradation is mainly catalysed not only by monoacylglycerol lipase (MGL) but also by a fatty acid amide hydrolase (FAAH). Inhibitors specifically targeting these two enzymes have also been described: URB602 and URB597, respectively. However, the anti-nociceptive effects of the combination of peripherally injected 2-AG, URB602 and URB597 in a neuropathic pain model have not yet been determined. This was performed in the presence or absence of cannabinoid CB1 (AM251) and CB2 (AM630) receptor antagonists. Experimental approach: Mechanical allodynia and thermal hyperalgesia were evaluated in 213 male Wistar rats allocated to 32 different groups. Drugs were injected subcutaneously in the dorsal surface of the hind paw (50 μL) 15 min before pain tests. Key results: 2-AG, URB602 and URB597 significantly decreased mechanical allodynia and thermal hyperalgesia with ED50 of 1.6±1.5 and 127±83 μg for 2-AG and URB602, respectively. These effects were mediated locally and were mostly inhibited by the two cannabinoid antagonists. Conclusions and implications: The combination of the three compounds did not produce any greater anti-allodynic or anti-hyperalgesic effects, suggesting that FAAH inhibition could reduce or limit the anti-nociceptive effects of 2-AG. Peripheral administration of endocannabinoids or MGL/FAAH inhibitors is a promising analgesic approach requiring further investigation. British Journal of Pharmacology (2008) 155, 913–924; doi:10.1038/bjp.2008.322; published online 11 August 2008

Microglial cell activation and migration play an important role in neuroinflammation propagation. While it is known that the lipid transmitter palmitoylethanolamide (PEA) regulates microglial migration by interacting with a cannabinoid-like receptor, the production and inactivation of this lipid by microglia has never been addressed directly. Here we show that the mouse microglial cell line BV-2 produces and hydrolyzes PEA. The carbamate compound URB602 inhibits PEA hydrolysis in BV-2 cell homogenates and increases PEA levels in intact cells, whereas the FAAH inhibitor URB597 and serine-hydrolase inhibitor MAFP do not affect PEA levels in intact cells. This unique pharmacological profile of inhibitors on PEA hydrolysis suggests the involvement of a previously undescribed enzyme that degrades PEA. This enzyme expressed by microglia constitutes a promising target for controlling the propagation of neuroinflammation.

Background and purpose: 2-arachidonoylglycerol (2-AG) is an endocannabinoid whose hydrolysis is predominantly catalysed by the enzyme monoacylglycerol lipase (MAGL). The development of MAGL inhibitors could offer an opportunity to investigate the anti-inflammatory and anti-nociceptive role of 2-AG, which have not yet been elucidated. On these bases, URB602, a MAGL inhibitor, was tested in a murine model of inflammation/inflammatory pain. Experimental approach: Acute inflammation was induced by intraplantar injection of λ-carrageenan into mice. The highest dose to be employed has been selected performing the tetrad assays for cannabimimetic activity in mice. URB602 anti-inflammatory and anti-nociceptive efficacy (assessed by plethysmometer and plantar test, respectively) was evaluated both in a preventive regimen (drug administered 30 min before carrageenan) and in a therapeutic regimen (URB602 administered 30 min after carrageenan). To elucidate the cannabinoid receptor involvement, rimonabant and SR144528, CB1 and CB2 selective antagonists, respectively, were given 15 min before URB602. Key results: Systemic administration of URB602 elicited a dose-dependent anti-oedemigen and anti-nociceptive effect that was reversed exclusively by the CB2 receptor antagonist. The efficacy of URB602 persisted also when the compound was administered in a therapeutic regimen, suggesting the ability of URB602 to improve established disease. Conclusions and implications: The present report highlighted the ability of the selective MAGL inhibitor, URB602, to prevent and treat an acute inflammatory disease without producing adverse psychoactive effects. The data presented herein also contributed to clarify the physiological role of 2-AG in respect to inflammatory reactions, suggesting its protective role in the body. British Journal of Pharmacology (2007) 152, 787–794; doi:10.1038/sj.bjp.0707425; published online 13 August 2007

Background and purpose: 2-arachidonoyl glycerol (2-AG) is an endogenous cannabinoid with central antinociceptive properties. Its degradation is catalysed by monoacylglycerol lipase (MGL) whose activity is inhibited by URB602, a new synthetic compound. The peripheral antinociceptive effects of 2-AG and URB602 in an inflammatory model of pain are not yet determined. We have evaluated these effects with and without the cannabinoid CB1 (AM251) and CB2 (AM630) receptor antagonists. Experimental approach: Inflammation was induced in rat hind paws by intraplantar injection of formalin. Nociception was assessed behaviourally over the next 60 min, in 19 experimental groups: (1) control; (2-6) 2-AG (0.01-100 μg); (7) AM251 (80 μg); (8) AM251+2-AG (10 μg); (9) AM630 (25 μg); (10) AM630+2-AG (10 μg); (11-16) URB602 (0.1-500 μg); (17) 2-AG+URB602 (ED50); (18) AM251+URB602 (ED50); (19) AM630+URB602 (ED50). Drugs were injected s.c. in the dorsal surface of the hind paw (50 μl), 15 min before formalin injection into the same paw. Key results: 2-AG and URB602 produced dose-dependent antinociceptive effects for the late phases of the formalin test with ED50 of 0.65±0.455 μg and 68±14.3 μg, respectively. Their combination at ED50 doses produced an additive antinociceptive effect. These effects were inhibited by AM630 but not by AM251 for 2-AG and by the two cannabinoid antagonists for URB602. Conclusions and implications: Locally injected 2-AG and URB602 decreased pain behaviour in a dose-dependent manner in an inflammatory model of pain. The antinociceptive effect of 2-AG was mediated by the CB2 receptor. British Journal of Pharmacology (2007) 150, 693–701. doi:10.1038/sj.bjp.0706990

Lack of selectivity of URB602 for 2-oleoylglycerol compared to anandamide hydrolysis in vitro.

Human monoacylglycerol lipase (MAGL), a soluble serine hydrolase that belongs to the α/β hydrolase fold superfamily, regulates 2-arachidonoyl glycerol level in the endocannabinoid system, which is implicated in a number of severe diseases, and therefore, inhibition of MAGL activity is crucial in the treatment of these diseases. We have synthesized a red fluorogenic substrate, 7-hydroxyresorufinyl-arachidonate (7-HRA), for a new MAGL assay. This assay is simple, sensitive, and reliable and useful for identifying compounds that modulate MAGL activity. In addition, the assay emits red fluorescence, which can significantly reduce interference due to compound fluorescence and dust or lint, all of which fluoresce in the blue wavelength. MAGL catalyzes the hydrolysis of 7-HRA to generate arachidonic acid and a highly red fluorescent resorufin, excitation at 571 nm and emission at 588 nm, with a Km of 0.87 μM and Vmax of 26 nmol min(-1) mg protein(-1). The known MAGL inhibitors URB602, methyl arachidonyl fluorophosphonate, and JZL184 were used to validate the test assay. The assay was highly reproducible with an overall average Z' value of 0.80. This new red fluorogenic substrate and the resulting enzyme assay could be used in high-throughput screening to identify and develop new potential MAGL inhibitors.

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

Aims URB602 is a selective inhibitor of monoacylglycerol lipase (MAGL), a serine hydrolase involved in the biological deactivation of the endocannabinoid 2-arachidonoyl glycerol (2-AG). It has been described that URB602 significantly enhances depolarization-induced increases in 2-AG. A high level of homocysteine (Hcy) is a modifiable risk factor for developing Alzheimer's disease (AD). The aim of this study was to investigate the protective effects of URB602 on Hcy-induced impairments underlying its cellular and molecular mechanism in primary cultured caudate nucleus (CN) neurons. Main methods The expressions of cyclooxygenase-2 (COX-2), ERK1/2, NF-κB and IκB-α as well as cleaved caspase-3 and p-Bcl-2 in Hcy-, URB602 or SR1 (a selective inhibitor of CB1 receptor)-treated primary cultured neurons in CN were measured by immunoblotting technique and neurotoxicity assays were performed by using Hoechst staining. Key findings The MAGL inhibitor URB602 exerted a neuroprotective effect on Hcy-induced impairment through suppression of cyclooxygenase-2 (COX-2) elevation and ERK1/2 and NF-κB phosphorylation as well as suppressions of IκB-α degradation in a CB1 receptor-dependent way. Moreover, anti-neuronal impairments of URB602 were mediated by modulating down-regulation of cleaved caspase-3 expression and up-regulation of p-Bcl-2 expression in a CB1 receptor-dependent manner in primary cultured CN neurons. Significance These data suggest that the MAGL inhibitor is a promising therapeutic target for some neurodegenerative disorders, such as AD, via the COX-2 signaling pathway.

The endogenous cannabinoid 2-arachidonoylglycerol (2-AG) is produced by neurons and other cells in a stimulus-dependent manner and undergoes rapid biological inactivation through transport into cells and catalytic hydrolysis. The enzymatic pathways responsible for 2-AG degradation are only partially understood. We have shown previously that overexpression of monoacylglycerol lipase (MGL), a cytosolic serine hydrolase that cleaves 1- and 2-monoacylglycerols to fatty acid and glycerol, reduces stimulus-dependent 2-AG accumulation in primary cultures of rat brain neurons. We report here that RNA interference-mediated silencing of MGL expression greatly enhances 2-AG accumulation in HeLa cells. After stimulation with the calcium ionophore ionomycin, 2-AG levels in MGL-silenced cells were comparable with those found in cells in which 2-AG degradation had been blocked using methyl arachidonyl fluorophosphonate, a nonselective inhibitor of 2-AG hydrolysis. The results indicate that MGL plays an important role in the degradation of endogenous 2-AG in intact HeLa cells. Furthermore, immunodepletion experiments show that MGL accounts for at least 50% of the total 2-AG-hydrolyzing activity in soluble fractions of rat brain, suggesting that this enzyme also contributes to 2-AG deactivation in the central nervous system.

2-Arachidonoylglycerol (2-AG) is an endogenous cannabinoid (endocannabinoid) lipid whose functions remain poorly understood. Guindon and colleagues report the novel finding that exogenous application of 2-AG induces peripheral antinociceptive effects that are mediated, at least in part, by actions at peripheral cannabinoid CB2 receptors. URB602, a recently described inhibitor of monoacylglycerol lipase, an enzyme that catalyzes 2-AG hydrolysis in vivo, also induced peripheral antinociceptive effects and enhanced the actions of 2-AG. Peripheral analgesic mechanisms represent promising therapeutic targets for suppressing pain in the absence of unwanted central nervous system side-effects (e.g. psychoactivity) associated with activation of central CB1 receptors. The therapeutic potential of inhibitors of 2-AG deactivation for the treatment of inflammatory pain is discussed.

The present study was designed to investigate the effect of pharmacological inhibition of endocannabinoid degradation on behavioural actions of the dopamine D2/D3 receptor agonist quinpirole in male C57Bl/6J mice. In addition, we studied the effects of endocannabinoid degradation inhibition on both cocaine-induced psychomotor activation and behavioural sensitization. We analysed the effects of inhibition of the two main endocannabinoid degradation enzymes: fatty acid amide hydrolase (FAAH), using inhibitor URB597 (1 mg/kg); monoacylglycerol lipase (MAGL), using inhibitor URB602 (10 mg/kg). Administration of quinpirole (1 mg/kg) caused a temporal biphasic response characterized by a first phase of immobility (0–50 min), followed by enhanced locomotion (next 70 min) that was associated with the introduction of stereotyped behaviours (stereotyped jumping and rearing). Pretreatment with both endocannabinoid degradation inhibitors did not affect the hypoactivity actions of quinpirole. However, this pretreatment resulted in a marked decrease in quinpirole-induced locomotion and stereotyped behaviours. Administration of FAAH or MAGL inhibitors did not attenuate the acute effects of cocaine. Furthermore, these inhibitors did not impair the acquisition of cocaine-induced behavioural sensitization or the expression of cocaine-induced conditioned locomotion. Only MAGL inhibition attenuated the expression of an already acquired cocaine-induced behavioural sensitization. These results suggest that pharmacological inhibition of endocannabinoid degradation might exert a negative feedback on D2/D3 receptor-mediated hyperactivity. This finding might be relevant for therapeutic approaches for either psychomotor disorders (dyskinesia, corea) or disorganized behaviours associated with dopamine-mediated hyperactivity. Fil: Luque Rojas, María Jesús. Fundación IMABIS; España Fil: Galeano, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Cardiológicas (i); Argentina Fil: Suarez, Juan . Fundación IMABIS; España Fil: Araos, Pedro. Fundación IMABIS; España Fil: Santín Nuñez, Luis Javier. Universidad de Malaga; España Fil: Rodríguez de Fonseca, Fernando. Fundación IMABIS; España Fil: Blanco Calvo, Eduardo. Fundación IMABIS; España. Universidad de Malaga; España

Lipid signals derived from lipolysis and membrane phospholipids play an important role in glucose-stimulated insulin secretion (GSIS), though the exact secondary signals remain unclear. Previous reports have documented a stimulatory role of exogenously added mono-acyl-glycerol (MAG) on insulin secretion from cultured β-cells and islets. In this report we have determined effects of increasing intracellular MAG in the β-cell by inhibiting mono-acyl-glycerol lipase (MGL) activity, which catalyzes the final step in triacylglycerol breakdown, namely the hydrolysis of MAG to glycerol and free fatty acid (FA). To determine the role of MGL in GSIS, we used three different pharmacological agents (JZL184, MJN110 and URB602). All three inhibited GSIS and depolarization-induced insulin secretion in INS-1 (832/13). JZL184 significantly inhibited both GSIS and depolarization-induced insulin secretion in rat islets. JZL184 significantly decreased lipolysis and increased both mono- and diacyglycerol species in INS-1 cells. Analysis of the kinetics of GSIS showed that inhibition was greater during the sustained phase of secretion. A similar pattern was observed in the response of Ca2+ to glucose and depolarization but to a lesser degree suggesting that altered Ca2+ handling alone could not explain the reduction in insulin secretion. In addition, a significant reduction in long chain-CoA (LC-CoA) was observed in INS-1 cells at both basal and stimulatory glucose following inhibition of MGL. Our data implicate an important role for MGL in insulin secretion.

Background: The endocannabinoids are emerging as natural brain protective substances that exert potentially beneficial effects in several neurological disorders by virtue of their hypothermic, immunomodulatory, vascular, antioxidant, and antiapoptotic actions. This study was undertaken to assess whether preventing the deactivation of the endocannabinoid 2- arachidonoylglycerol (2-AG) with the monoacylglycerol lipase (MAGL) inhibitor URB602 can provide neuroprotective effects in hypoxia-ischemia (HI)-induced brain injury. Methods: URB602 was administered into the right lateral ventricle 30 min before 7-day-old pup rats were subjected to HI. The neuroprotective effect was evaluated on postnatal day (PN) 14 or at adulthood (PN80) using behavioral and histological analyses. Activated caspase-3 expression and propidium iodide labeling were assessed as indexes of apoptotic and necrotic cell death, respectively. Results: Pretreatment with URB602 reduced apoptotic and necrotic cell death, as well as the infarct volume measured at PN14. At adulthood, URB602-treated HI animals performed better at the T-maze and the Morris maze, and also showed a significant reduction of brain damage. Conclusion: These results demonstrate that a pretreatment with URB602 significantly reduces brain damage and improves functional outcome, indicating that endocannabinoid-degrading enzymes may represent an important target for neuroprotection in neonatal ischemic brain injury. © 2012 International Pediatric Research Foundation, Inc.

Elements of the endocannabinoid system (ECS) are expressed by islet endocrine cells and activation of CB1 and CB2 cannabinoid receptors regulates insulin secretion from mouse and human β-cells. The current study aimed to investigate the expression and function, in mouse and human β-cells, of monoacylglycerol lipase (MGL), an enzyme that facilitates degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG). We found that MGL mRNA is expressed by MIN6 β-cells, mouse islets, human islets and enriched human islet β-cells, and immunohistochemistry indicated that MGL localisation in human islets is consistent with its expression by some β- and -α-cells. Blockade of MGL activity with the pharmacological inhibitor URB602 led to increased [Ca(2+)](i )and enhanced insulin secretion from MIN6 β-cells, and MGL inhibition also elevated insulin and glucagon secretion from isolated human islets in vitro. These data imply a stimulatory role for endogenous 2-AG in islets that is amplified when its degradation is blocked.

Cannabinoid system plays an important role in controlling neuronal excitability and brain function. On the other hand, modulation of gamma-aminobutyric acid (GABA) transmission is one of the initial strategies for the treatment of seizure. The aim of the present study was to evaluate possible interaction between cannabinoidergic and GABAergic systems in pentylenetetrazole (PTZ)-induced acute seizure in rat. Drugs were administered by intracerebroventricular (i.c.v.) administration 20 min before a single intraperitoneal (i.p.) injection of PTZ and the latency to the first generalized tonic-clonic seizure was measured. Both the cannabinoid receptor agonist WIN55212-2 (10, 30, 50 and 100 μg/rat) and the GABA-A receptor agonist isoguvacine (IGN; 10, 30 and 50 μg/rat) significantly increased the latency of seizure occurrence. Moreover, the fatty acid amide hydrolase inhibitor URB597 showed no anticonvulsive effect while the monoacyl glycerol lipase (MAGL) inhibitor URB602 (10, 50 and 100 μg/rat) protected rats against PTZ-induced seizure. Moreover, co-administration of IGN and cannabinoid compounds attenuated the anticonvulsant action of both WIN55212-2 and IGN in this model of seizure. Our data suggests that exogenous cannabinoid WIN55212-2 and MAGL inhibitor URB602 imply their antiseizure action in part through common brain receptorial system. Moreover, the antagonistic interaction of cannabinoids and IGN in protection against PTZ-induced seizure could suggest the involvement of GABAergic system in their anticonvulsant action.

The comparative effects of atropine and the indirect cannabinomimetics URB597 (a fatty acid amide hydrolase inhibitor) and URB602 (a monoacylglycerol lipase inhibitor) on functional and neurobehavioral endpoints following acute diisopropylfluorophosphate intoxication were studied. Male Sprague-Dawley rats were treated with vehicle or DFP (2.5 mg/kg, sc), immediately post-treated with either vehicle, atropine (16 mg/kg), URB597 (3 mg/kg), URB602 (10 mg/kg) or a combination of URB597 and URB602, and functional signs of toxicity as well as nocturnal motor activity were measured daily for seven consecutive days. Performance in the elevated plus maze (for anxiety-like behavior) and the forced swimming test (for depression-like behavior) was measured at days 6-8 and 27-29 after dosing. Twenty-four hours after dosing, DFP markedly reduced cholinesterase activity in selected brain regions and peripheral tissues (diaphragm and plasma). Substantial recovery of cholinesterase activity was noted at both 8 and 29 days after dosing but significant inhibition was still noted in some brain regions at the latest time-point. DFP elicited body weight reductions and typical signs of cholinergic toxicity, and reduced nocturnal ambulation and rearing. Atropine and the cannabinomimetics (alone and in combination) partially attenuated DFP-induced functional signs of toxicity. None of the post-treatments reversed the DFP-induced reduction in ambulation or rearing, however. No significant treatment-related effects on elevated plus maze performance were noted. DFP-treated rats exhibited decreased swimming and increased immobility in the forced swimming test at both time-points. None of the post-treatments had any effect on DFP-induced changes in immobility or swimming at day 8. At day 29, atropine and the combination of URB597/URB602 significantly blocked DFP-induced changes in immobility, while URB597 and the combination reversed DFP-induced changes in swimming. The results suggest that early blockade of muscarinic receptors and enhancement of eCB signaling can attenuate both acute and delayed effects elicited by DFP.

A novel fluorescence-based assay of monoacylglycerol lipase (MAGL) activity that is simple, sensitive, and amenable to the screening of small molecule inhibitors is described. Purified recombinant human MAGL protein and 7-hydroxycoumarinyl-arachidonate (7-HCA), a fluorogenic substrate for MAGL, were employed in the assay. MAGL protein catalyzes the hydrolysis of 7-HCA to generate arachidonic acid and the highly fluorescent 7-hydroxyl coumarin (7-HC). Release of 7-HC was measured using a fluorometer. MAGL protein catalyzed the hydrolysis of 7-HCA with an apparent K(m) of 9.8 microM and V(max) of 1.7 mmol/min/mg of protein. The assay is specific for MAGL as assay buffer alone or heat-denatured MAGL protein had no significant activity against 7-HCA. Furthermore, MAGL activity was inhibited in a dose-dependent manner by the specific inhibitor URB602 as well as N-arachidonyl maleimide with 50% inhibitory concentration values of 3.1 microM and 155 nM, respectively. The assay was further optimized under different conditions, including pH range and bovine serum albumin protein and dimethyl sulfoxide concentrations. The assay was found to be reproducible, having Z' values ranging from 0.7 to 0.9, and is therefore suitable for high-throughput screening.

The endocannabinoids (eCBs) anandamide and 2-arachidonoyl glycerol (2-AG) are inactivated by a two-step mechanism. First, they are carried into cells, and then anandamide is hydrolyzed by fatty acid amide hydrolase (FAAH) and 2-AG by monoacylglycerol lipase (MGL). Here we provide evidence for a previously undescribed MGL activity expressed by microglial cells. We found that the mouse microglial cell line BV-2 does not express MGL mRNA and yet efficiently hydrolyzes 2-AG. URB597 (3′-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate) reduces this hydrolysis by 50%, suggesting the involvement of FAAH. The remaining activity is blocked by classic MGL inhibitors [[1,1-biphenyl]-3-yl-carbamic acid, cyclohexyl ester (URB602) and MAFP (methylarachidonyl fluorophosphate)] and is unaffected by inhibitors of COXs (cyclooxygenases), LOXs (lipooxygenases), and DGLs (diacylglycerol lipases), indicating the involvement of a novel MGL activity. Accordingly, URB602 leads to selective accumulation of 2-AG in intact BV-2 cells. Although MGL expressed in neurons is equally distributed between the cytosolic, mitochondrial, and nuclear fractions, the novel MGL activity expressed by BV-2 cells is enriched in mitochondrial and nuclear fractions. A screen for novel inhibitors of eCB hydrolysis identified several compounds that differentially block MGL, FAAH, and the novel MGL activity. Finally, we provide evidence for expression of the novel MGL by mouse primary microglia in culture. Our results suggest the presence of a novel, pharmacologically distinct, MGL activity that controls 2-AG levels in microglia.

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 CB1antagonist SR141716A (rimonabant) failed to suppress nonopioid SIA, suggesting that supraspinal rather than spinal CB1receptor 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. © 2005 Elsevier Ltd. All rights reserved.

The functions of 2-arachidonoylglycerol (2-AG), the most abundant endocannabinoid found in the brain, remain largely unknown. Here we show that two previously unknown inhibitors of monoacylglycerol lipase, a presynaptic enzyme that hydrolyzes 2-AG, increase 2-AG levels and enhance retrograde signaling from pyramidal neurons to GABAergic terminals in the hippocampus. These results establish a role for 2-AG in synaptic plasticity and point to monoacylglycerol lipase as a possible drug target. © 2005 Nature Publishing Group.

Acute stress suppresses pain by activating brain pathways that engage opioid or non-opioid mechanisms. Here we show that an opioid-independent form of this phenomenon, termed stress-induced analgesia, is mediated by the release of endogenous marijuana-like (cannabinoid) compounds in the brain. Blockade of cannabinoid CB(1) receptors in the periaqueductal grey matter of the midbrain prevents non-opioid stress-induced analgesia. In this region, stress elicits the rapid formation of two endogenous cannabinoids, the lipids 2-arachidonoylglycerol (2-AG) and anandamide. A newly developed inhibitor of the 2-AG-deactivating enzyme, monoacylglycerol lipase, selectively increases 2-AG concentrations and, when injected into the periaqueductal grey matter, enhances stress-induced analgesia in a CB1-dependent manner. Inhibitors of the anandamide-deactivating enzyme fatty-acid amide hydrolase, which selectively elevate anandamide concentrations, exert similar effects. Our results indicate that the coordinated release of 2-AG and anandamide in the periaqueductal grey matter might mediate opioid-independent stress-induced analgesia. These studies also identify monoacylglycerol lipase as a previously unrecognized therapeutic target.

The endocannabinoids such as 2-AG modulate immune response. It is known that the levels of 2-AG are increased by monoacylglycerol lipase (MAGL) inhibition. The aim of this work was to investigate the effects of URB602 and JZL184 (MAGL inhibitors) in a murine model of lung injury. Fifty-nine C57BL/6 male mice were used URB602 treatment: mice were randomly divided into 6 groups (n = 6–8), vehicle + saline, vehicle + LPS, and URB602 + LPS (10, 15, 20 or 25 mg/kg). JZL184 treatment: mice were randomly divided into 5 groups, vehicle + saline, vehicle + LPS, and JZL184 + LPS (4, 16 or 40 mg/kg). URB602 or JZL184 treatments were made (i.p.) 30 or 60 min before an intranasal instillation of LPS or sterile saline. Twenty-four hours later, mice were euthanized and bronchoalveolar lavage fluid (BAL) was harvested. Mice that received intranasal LPS presented more neutrophils in the BAL than mice that received intranasal saline. Animals treated with all doses of URB602 and JZL184 (16 and 40 mg/kg) presented a reduction in the number of inflammatory cells induced by LPS. No differences were found among the doses of URB602 used. Further analysis showed that JZL184 induced more relevant effects in the total number of cells and neutrophils in the BAL than URB602. The number of lymphocytes and macrophages were not changed by the drugs used. Altogether, present data show that increment on 2-AG induced by MAGL inhibition decrease LPS-induced lung inflammation in mice.