Monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) inhibitors exert preclinical effects indicative of therapeutic potential (i.e., analgesia). However, the extent to which MAGL and FAAH inhibitors produce unwanted effects remains unclear. Here, FAAH and MAGL inhibition was examined separately and together in a Δ(9)-tetrahydrocannabinol (Δ(9)-THC; 5.6 mg/kg i.p.) discrimination assay predictive of subjective effects associated with cannabis use, and the relative contribution of N-arachidonoyl ethanolamine (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in the prefrontal cortex, hippocampus, and caudate putamen to those effects was examined. Δ(9)-THC dose-dependently increased Δ(9)-THC appropriate responses (ED50 value = <em>2</em>.8 mg/kg), whereas the FAAH inhibitors PF-3845 [N-3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-<em>2</em>-pyridinyl]oxy]phenyl]methyl]-1-piperidinecarboxamide] and URB597 [(3'-​(aminocarbonyl)[1,​1'-​biphenyl]-​3-​yl)-​cyclohexylcarbamate] or a MAGL inhibitor JZL184 [4-​nitrophenyl-​4-​(dibenzo[d][1,​3]dioxol-​5-​yl(hydroxy)methyl)piperidine-​1-​carboxylate] alone did not substitute for the Δ(9)-THC discriminative stimulus. The nonselective FAAH/MAGL inhibitors SA-57 [4-[<em>2</em>-(4-chlorophenyl)ethyl]-1-piperidinecarboxylic acid <em>2</em>-(methylamino)-<em>2</em>-oxoethyl ester] and JZL195 [4-​nitrophenyl 4-​(3-​phenoxybenzyl)piperazine-​1-​carboxylate] fully substituted for Δ(9)-THC with ED50 values equal to <em>2</em>.4 and 17 mg/kg, respectively. Full substitution for Δ(9)-THC was also produced by a combination of JZL184 and PF-3845, but not by a combination of JZL184 and URB597 (i.e., 5<em>2</em>% maximum). Cannabinoid receptor type 1 antagonist rimonabant attenuated the discriminative stimulus effects of Δ(9)-THC, SA-57, JZL195, and the combined effects of JZL184 and PF-3845. Full substitution for the Δ(9)-THC discriminative stimulus occurred only when both <em>2</em>-AG and AEA were significantly elevated, and the patterns of increased endocannabinoid content were similar among brain regions. Overall, these results suggest that increasing both endogenous <em>2</em>-AG and AEA produces qualitatively unique effects (i.e., the subjective effects of cannabis) that are not obtained from increasing either <em>2</em>-AG or AEA separately.

The family of endocannabinoids (i.e., the endogenous agonists of cannabinoid receptors) contains several polyunsaturated fatty acid amides such as anandamide (AEA) and oleamide but also esters such as <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). These compounds are the subject of growing interest in pharmacology for their multiple therapeutic potentials. Unfortunately, they are rapidly inactivated by enzymatic hydrolysis, which prevents their effective medical use. Inhibitors of endocannabinoid degradation seem to be necessary tools for the development of endocannabinoid therapeutics. But hitting this target is inconceivable without good knowledge of the enzymes. Fatty acid amide hydrolase (FAAH) is the oldest and the best characterised enzyme involved in the degradation of endocannabinoids. Cloning, distribution in the body and crystal structure of FAAH have been described. A large number of FAAH inhibitors have also been synthesised and tested. For a long time, FAAH was considered as the only key enzyme hydrolysing endocannabinoids. But recent findings indicate that at least two other enzymes have critical role in the endocannabinoids degradation. Monoglyceride lipase participates in <em>2</em>-AG degradation and some data indicate that it is the primary mechanism for <em>2</em>-AG inactivation in intact neurons. N-palmitoylethanolamine-selective acid amidase (NPAA) is a second fatty acid amide hydrolase more active with N-palmitoylethanolamine, an anti-inflammatory substance. The purpose of this review is to collect and compare the catalytic properties of these 3 key enzymes hydrolysing endocannabinoids.

Endocannabinoids are endogenous ligands of brain-type (CB1) and spleen-type (CB<em>2</em>) cannabinoid receptors. N-Arachidonoylethanolamine (anandamide, AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are prototype members of the fatty acid amides and the monoacylglycerols, two groups of endocannabinoids. Unlike CB1, CB<em>2</em> receptors do not reside within 'caveolae', specialized membrane microdomains that are well-known modulators of the activity of a number of G protein-coupled receptors. In this issue of the British Journal of Pharmacology, Rimmerman and coworkers demonstrate that <em>2</em>-AG is entirely localized in the caveolae of dorsal root ganglion cells, where also part of AEA (approximately 30%) can be detected. However, most of AEA (approximately 70%) was detected in non-caveolae fractions, that is where CB<em>2</em> receptors are localized. The different interaction of AEA and <em>2</em>-AG with membrane microdomains might have significant implications for endocannabinoid-dependent autocrine and/or retrograde-paracrine signalling pathways. It also raises an important question about the structural determinants responsible for a different localization of two apparently similar endocannabinoids within lipid bilayers.

Neurons display important differences in plasma membrane composition between somatodendritic and axonal compartments, potentially leading to currently unexplored consequences in G-protein-coupled-receptor signaling. Here, by using highly-resolved biosensor imaging to measure local changes in basal levels of key signaling components, we explored features of type-1 cannabinoid receptor (CB1R) signaling in individual axons and dendrites of cultured rat hippocampal neurons. Activation of endogenous CB1Rs led to rapid, Gi/o-protein- and cAMP-mediated decrease of cyclic-AMP-dependent protein kinase (PKA) activity in the somatodendritic compartment. In axons, PKA inhibition was significantly stronger, in line with axonally-polarized distribution of CB1Rs. Conversely, inverse agonist AM<em>2</em>81 produced marked rapid increase of basal PKA activation in somata and dendrites, but not in axons, removing constitutive activation of CB1Rs generated by local production of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Interestingly, somatodendritic <em>2</em>-AG levels differently modified signaling responses to CB1R activation by Δ(9)-THC, the psychoactive compound of marijuana, and by the synthetic cannabinoids WIN55,<em>2</em>1<em>2</em>-<em>2</em> and CP55,940. These highly contrasted differences in sub-neuronal signaling responses warrant caution in extrapolating pharmacological profiles, which are typically obtained in non-polarized cells, to predict in vivo responses of axonal (i.e., presynaptic) GPCRs. Therefore, our results suggest that enhanced comprehension of GPCR signaling constraints imposed by neuronal cell biology may improve the understanding of neuropharmacological action.

Dibenzopyran (Delta(9)-tetrahydrocannabinol) and aminoalkylindole [R(+)-[<em>2</em>,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrolol[1,<em>2</em>,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl) methanone mesylate; (WIN55,<em>2</em>1<em>2</em>-<em>2</em>)] cannabinoids suppress vomiting produced by cisplatin via cannabinoid CB(1) receptors. This study investigates the antiemetic potential of the "nonclassical" cannabinoid CP55,940 [1alpha,<em>2</em>beta-(R)-5alpha]-(-)-5-(1,1-dimethyl)-<em>2</em>-[5-hydroxy-<em>2</em>-(3-hydroxypropyl) cyclohexyl-phenol] against cisplatin-induced vomiting and assesses the presence and functionality of cannabinoid CB(1) receptors in the least shrew (Cryptotis parva) brain. CP55,940 (0.0<em>2</em>5-0.3 mg/kg) reduced both the frequency of cisplatin-induced emesis (ID(50)=0.0<em>2</em>5 mg/kg) and the percentage of shrews vomiting (ID(50)=0.09 mg/kg). CP55,940 also suppressed shrew motor behaviors (ID(50)=0.06- 0.<em>2</em>1 mg/kg) at such doses. The antiemetic and motor-suppressant actions of CP55,940 were countered by SR141716A [N-piperidino-5-(4-chlorophenyl)-1-(<em>2</em>,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide], indicating both effects are cannabinoid CB(1) receptor-mediated. Autoradiographic studies with [3H]-SR141716A and [35S]-GTPgammaS binding revealed that the distribution of the cannabinoid CB(1) receptor and its activation pattern are similar to rodent brain and significant levels are present in brain loci (e.g., nucleus tractus solitarius (NTS)) that control emesis. The affinity rank order of structurally diverse cannabinoid ligands for cannabinoid CB(1) receptor in shrew brain is similar to rodent brain: HU-<em>2</em>10=CP55,940=SR141716A>/=WIN55,<em>2</em>1<em>2</em>-<em>2</em>)/=delta-9-tetrahydrocannabinol>methanandamide=HU-<em>2</em>11=cannabidiol=<em>2</em>-<em>arachidonoylglycerol</em>. This affinity order is also similar and is highly correlated to the cannabinoid EC(50) potency rank order for GTPgammaS stimulation except WIN55,<em>2</em>1<em>2</em>-<em>2</em> and delta-9-tetrahydrocannabinol potency order were reversed. The affinity and the potency rank order of tested cannabinoids were significantly correlated with their antiemetic ID(50) potency order against cisplatin-induced vomiting (CP55,940>WIN55,<em>2</em>1<em>2</em>-<em>2</em>=delta-9-tetrahydrocannabinol) as well as emesis produced by <em>2</em>-<em>arachidonoylglycerol</em> or SR141716A (CP55,940>WIN55,<em>2</em>1<em>2</em>-<em>2</em>)delta-9-tetrahydrocannabinol).

BACKGROUND

Endocannabinoids (ECs) are rapidly acting immune-modulatory lipid-signaling molecules that are important for adaptation to stressful and aversive situations.They are known to interact with glucocorticoids and other stress-responsive systems. Maladaptation to acute or chronic stress represents a major risk factor for the development of psychiatric disorders. In the present study, we administered stress doses of hydrocortisone ina prospective, randomized, placebo-controlled double blind study in patients undergoing cardiac surgery (CS) to examine the relationship between the use of glucocorticoids, plasma EC levels, and the occurrence of early postoperative cognitive dysfunction (delirium) and of later development of depression.

METHODS

We determined plasma levels of the ECs anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in CS patients of the hydrocortisone (n=56) and the placebo group(n=55) preoperatively, at postoperative day (POD) 1, at intensive care unit discharge, and at 6 months after CS(n=68). Postoperative delirium was diagnosed according to Diagnostic and Statistical Manual of the American Psychiatric Association IVth Edition (DSM-IV) criteria, and depression was determined by validated questionnaires and a standardized psychological interview (Structured Clinical Interview for DSM-IV).

RESULTS

Stress doses of hydrocortisone did not affect plasma EC levels and the occurrence of delirium or depression. However, patients who developed deliriumon POD 1 had significantly lower preoperative <em>2</em>-AG levels of the neuroprotective EC <em>2</em>-AG (median values, 3.8 vs. 11.3ng/ml; p=0.03). Preoperative <em>2</em>-AG concentrations were predictive of postoperative delirium (sensitivity=0.70;specificity=0.69; cutoff value=4.9 ng/ml; receiver operating characteristic curve area=0.70; 95 o/o confidence interval=0.54-0.85). Patients with depression at 6 months after CS (n=16) had significantly lower anandamide and <em>2</em>-AG levels during the perioperative period.

CONCLUSIONS

A low perioperative EC response may indicate an increased risk for early cognitive dysfunction and long-term depression in patients after CS. Glucocorticoids do not seem to influence this relationship.

Intramolecular hydrogen bonding is an important determinant of enzyme structure, catalysis, and inhibitor action. Monoacylglycerol lipase (MGL) modulates cannabinergic signaling as the main enzyme responsible for deactivating <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), a primary endocannabinoid lipid messenger. By enhancing tissue-protective <em>2</em>-AG tone, targeted MGL inhibitors hold therapeutic promise for managing pain and treating inflammatory and neurodegenerative diseases. We report study of purified, solubilized human MGL (hMGL) to explore the details of hMGL catalysis by using two known covalent hMGL inhibitors, the carbamoyl tetrazole AM6701 and N-arachidonoylmaleimide (NAM), that act through distinct mechanisms. Using proton nuclear magnetic resonance spectroscopy (NMR) with purified wild-type and mutant hMGLs, we have directly observed a strong hydrogen-bond network involving Asp<em>2</em>39 and His<em>2</em>69 of the catalytic triad and neighboring Leu<em>2</em>41 and Cys<em>2</em>4<em>2</em> residues. hMGL inhibition by AM6701 alters this hydrogen-bonding pattern through subtle active-site structural rearrangements without influencing hydrogen-bond occupancies. Rapid carbamoylation of hMGL Ser1<em>2</em><em>2</em> by AM6701 and elimination of the leaving group is followed by a slow hydrolysis of the carbamate group, ultimately regenerating catalytically competent hMGL. In contrast, hMGL titration with NAM, which leads to cysteine alkylation, stoichiometrically decreases the population of the active-site hydrogen bonds. NAM prevents reformation of this network, and in this manner inhibits hMGL irreversibly. These data provide detailed molecular insight into the distinctive mechanisms of two covalent hMGL inhibitors and implicate a hydrogen-bond network as a structural feature of hMGL catalytic function.

BACKGROUND

Depolarization-induced suppression of excitation (DSE) at parallel fiber-Purkinje cell synapse is an endocannabinoid-mediated short-term retrograde plasticity. Intracellular Ca(<em>2</em>+) elevation is critical for the endocannabinoid production and DSE. Nevertheless, how elevated Ca(<em>2</em>+) leads to DSE is unclear.

RESULTS

We utilized cytosolic phospholipase A(<em>2</em>) alpha (cPLA(<em>2</em>)α) knock-out mice and whole-cell patch clamp in cerebellar slices to observed the action of cPLA(<em>2</em>)α/arachidonic acid signaling on DSE at parallel fiber-Purkinje cell synapse. Our data showed that DSE was significantly inhibited in cPLA(<em>2</em>)α knock-out mice, which was rescued by arachidonic acid. The degradation enzyme of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), monoacylglycerol lipase (MAGL), blocked DSE, while another catabolism enzyme for N-arachidonoylethanolamine (AEA), fatty acid amide hydrolase (FAAH), did not affect DSE. These results suggested that <em>2</em>-AG is responsible for DSE in Purkinje cells. Co-application of paxilline reversed the blockade of DSE by internal K(+), indicating that large conductance Ca(<em>2</em>+)-activated potassium channel (BK) is sufficient to inhibit cPLA(<em>2</em>)α/arachidonic acid-mediated DSE. In addition, we showed that the release of <em>2</em>-AG was independent of soluble NSF attachment protein receptor (SNARE), protein kinase C and protein kinase A.

CONCLUSIONS

Our data first showed that cPLA(<em>2</em>)α/arachidonic acid/<em>2</em>-AG signaling pathway mediates DSE at parallel fiber-Purkinje cell synapse.

We compared the diacylglycerol kinase (DGK) catalyzed phosphorylation of 1-O-hexanoyl-<em>2</em>-oleoylglycerol (HOG) with 1-O-hexanoyl-<em>2</em>-<em>arachidonoylglycerol</em> (HAG). We assayed the activity of DGKalpha and DGKzeta using a liposomal-based assay system. Liposomal assays show that the DGKalpha and, to a lesser extent, DGKzeta preferentially act on substrates containing an arachidonoyl group when this group is incorporated into alkylacylglycerols. The activity of DGKalpha was 8<em>2</em> times greater with HAG compared to HOG. DGKzeta is 10 times more active in catalyzing the phosphorylation of HAG compared to HOG. Although diacylglycerols were better substrates for both DGKalpha and DGKzeta than the alkylacylglycerols, no specificity was exhibited for arachidonoyl-containing diacylglycerols. However, this specificity for HAG over HOG is modulated by the phospholipid composition of the liposome. Addition of cholesterol and/or phosphatidylethanolamine partially reduces the substrate selectivity. We also analyzed the kinetic constants for the phosphorylation of both diacylglycerol and 1-alkyl-<em>2</em>-acylglycerol catalyzed by the alpha, epsilon, or zeta isoforms using a soluble Triton mixed micelle system. We found that all three isoforms of DGK can phosphorylate 1-alkyl-<em>2</em>-acylglycerols but generally at a lower rate than for the corresponding diacylglycerol. The specificity of DGKepsilon for diacylglycerols containing an arachidonoyl group was retained when the ester group in the C-1 position is replaced with an ether linkage. In contrast, DGKalpha and, to a lesser extent, DGKzeta had greater specificity for arachidonoyl-containing 1-alkyl-<em>2</em>-acylglycerols than for arachidonoyl-containing diacylglycerols. This demonstrates that the acyl chain specificity is affected by the structure of the lipid headgroup.

N-acylethanolamines (NAEs) are a group of lipid mediators synthesized in response to a number of physiological and pathological stimuli. Because of the low tissue concentrations of NAEs, analyses often include liquid extraction followed by solid-phase extraction and subsequent quantitation by LC/MS or GC/MS. Reported levels of NAEs vary considerably, however, and often no explanation is given for these discrepancies. Brought on by difficulties encountered during method development, the effects of using four different brands of silica-containing solid phase extraction (SPE) columns and five different brands of chloroform for sample preparation were investigated. Considerable variation in the retention and recoveries of seven NAEs and <em>2</em>-<em>arachidonoylglycerol</em> existed between the SPE columns. Furthermore, it was found that some chloroforms contained quantifiable amounts of N-palmitoylethanolamine and N-stearoylethanolamine. Finally, it was found that use of one of the chloroforms resulted in a loss of N-oleoylethanolamine from solution due to addition of chlorine to the ω-9 bond. The identity of this reaction product was confirmed by LC-MS/MS and NMR. It is recommended that these aspects of sample preparation and analysis should be thoroughly validated during method development and the relevant information on specific brands used be reported in future communications in order to better estimate the validity of reported quantitative data.

Invaginating synapses in the basal amygdala are a unique type of GABAergic synapses equipped with molecular-anatomical organization specialized for <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG)-mediated endocannabinoid signaling. Cholecystokinin (CCK)-positive basket cell terminals protrude into pyramidal cell somata and form invaginating synapses, where apposing presynaptic and postsynaptic elements are highly loaded with cannabinoid receptor CB₁ or <em>2</em>-AG synthetic enzyme diacylglycerol lipase-α (DGLα), respectively. The present study scrutinized their neurochemical and neuroanatomical phenotypes in adult mouse telencephalon. In the basal amygdala, vesicular glutamate transporter-3 (VGluT3) was transcribed in one-fourth of CB₁-expressing GABAergic interneurons. The majority of VGluT3-positive CB₁-expressing basket cell terminals apposed DGLα clusters, whereas the majority of VGluT3-negative ones did not. Importantly, VGluT3-positive basket cell terminals selectively constructed invaginating synapses. GABAA receptors accumulated on the postsynaptic membrane of invaginating synapses, whereas metabotropic glutamate receptor-5 (mGluR₅) was widely distributed on the somatodendritic surface of pyramidal cells. Moreover, CCK₂ receptor (CCK₂R) was highly transcribed in pyramidal cells. In cortical regions, pyramidal cells equipped with such VGluT3/CB₁/DGLα-accumulated invaginating synapses were found at variable frequencies depending on the subregions. Therefore, in addition to extreme proximity of CB₁- and DGLα-loaded presynaptic and postsynaptic elements, tripartite transmitter phenotype of GABA/glutamate/CCK is the common neurochemical feature of invaginating synapses, suggesting that glutamate, CCK, or both can promote <em>2</em>-AG synthesis through activating Gαq/₁₁ protein-coupled mGluR₅ and CCK₂R. These molecular configurations led us to hypothesize that invaginating synapses might be evolved to provide some specific mechanisms of induction, regulation, and cooperativity for <em>2</em>-AG-mediated retrograde signaling in particular cortical and cortex-like amygdaloid regions.

Serious clinical liabilities associated with the prescription of opiates for pain control include constipation, respiratory depression, pruritus, tolerance, abuse, and addiction. A recognized strategy to circumvent these side effects is to combine opioids with other antinociceptive agents. The combination of opiates with the primary active constituent of cannabis (Δ(9)-tetrahydrocannabinol) produces enhanced antinociceptive actions, suggesting that cannabinoid receptor agonists can be opioid sparing. Here, we tested whether elevating the endogenous cannabinoid <em>2</em>-<em>arachidonoylglycerol</em> through the inhibition of its primary hydrolytic enzyme monoacylglycerol lipase (MAGL), will produce opioid-sparing effects in the mouse chronic constriction injury (CCI) of the sciatic nerve model of neuropathic pain. The dose-response relationships of i.p. administration of morphine and the selective MAGL inhibitor <em>2</em>,5-dioxopyrrolidin-1-yl 4-(bis(4-chlorophenyl)methyl)piperazine-1-carboxylate (MJN110) were tested alone and in combination at equieffective doses for reversal of CCI-induced mechanical allodynia and thermal hyperalgesia. The respective ED50 doses (95% confidence interval) of morphine and MJN110 were <em>2</em>.4 (1.9-3.0) mg/kg and 0.43 (0.<em>2</em>3-0.79) mg/kg. Isobolographic analysis of these drugs in combination revealed synergistic antiallodynic effects. Acute antinociceptive effects of the combination of morphine and MJN110 required μ-opioid, CB1, and CB<em>2</em> receptors. This combination did not reduce gastric motility or produce subjective cannabimimetic effects in the drug discrimination assay. Importantly, combinations of MJN110 and morphine given repeatedly (i.e., twice a day for 6 days) continued to produce antiallodynic effects with no evidence of tolerance. Taken together, these findings suggest that MAGL inhibition produces opiate-sparing events with diminished tolerance, constipation, and cannabimimetic side effects.

The endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is predominantly biosynthesized by sn-1-diacylglycerol lipase α (DAGL-α) in the CNS. Selective inhibitors of DAGL-α will provide valuable insights in the role of <em>2</em>-AG in endocannabinoid signaling processes and are potential therapeutics for the treatment of obesity and neurodegenerative diseases. Here, we describe the development of a natural substrate-based fluorescence assay for DAGL-α, using a coupled enzyme approach. The continuous setup of our assay allows monitoring of DAGL-α activity in real-time and in a 96-well plate format. This constitutes a major improvement to the currently available radiometric and LC/MS-based methods, which can be executed only in low-throughput formats. In addition, our assay circumvents the use of radioactive material. We demonstrate that our assay can be used to screen inhibitors of DAGL-α activity, using 1-stearoyl-<em>2</em>-arachidonoyl-sn-glycerol as the physiologically relevant natural substrate of DAGL-α. Furthermore, our method can be employed to measure DAGL activity and inhibition in the mouse brain membrane proteome. Consequently, our assay should serve as a valuable tool for rapid hit validation and lead optimization of DAGL-α inhibitors.

Diacylglycerol lipase (DAGL)-α and -β are enzymes responsible for the biosynthesis of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Selective and reversible inhibitors are required to study the function of DAGLs in neuronal cells in an acute and temporal fashion, but they are currently lacking. Here, we describe the identification of a highly selective DAGL inhibitor using structure-guided and a chemoproteomics strategy to characterize the selectivity of the inhibitor in complex proteomes. Key to the success of this approach is the use of comparative and competitive activity-based proteome profiling (ABPP), in which broad-spectrum and tailor-made activity-based probes are combined to report on the inhibition of a protein family in its native environment. Competitive ABPP with broad-spectrum fluorophosphonate-based probes and specific β-lactone-based probes led to the discovery of α-ketoheterocycle LEI105 as a potent, highly selective, and reversible dual DAGL-α/DAGL-β inhibitor. LEI105 did not affect other enzymes involved in endocannabinoid metabolism including abhydrolase domain-containing protein 6, abhydrolase domain-containing protein 1<em>2</em>, monoacylglycerol lipase, and fatty acid amide hydrolase and did not display affinity for the cannabinoid CB1 receptor. Targeted lipidomics revealed that LEI105 concentration-dependently reduced <em>2</em>-AG levels, but not anandamide levels, in Neuro<em>2</em>A cells. We show that cannabinoid CB1-receptor-mediated short-term synaptic plasticity in a mouse hippocampal slice model can be reduced by LEI105. Thus, we have developed a highly selective DAGL inhibitor and provide new pharmacological evidence to support the hypothesis that "on demand biosynthesis" of <em>2</em>-AG is responsible for retrograde signaling.

Accumulating evidence supports the role of astrocytes in endocannabinoid mediated modulation of neural activity. It has been reported that some astrocytes express the cannabinoid type 1 receptor (CB1-R), the activation of which is leading to Ca<em>2</em>+ mobilization from internal stores and a consecutive release of glutamate. It has also been documented that astrocytes have the potential to produce the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em>, one of the best known CB1-R agonist. However, no relationship between CB1-R activation and <em>2</em>-<em>arachidonoylglycerol</em> production has ever been demonstrated. Here we show that rat spinal astrocytes co-express CB1-Rs and the <em>2</em>-<em>arachidonoylglycerol</em> synthesizing enzyme, diacylglycerol lipase-alpha in close vicinity to each other. We also demonstrate that activation of CB1-Rs induces a substantial elevation of intracellular Ca<em>2</em>+ concentration in astrocytes. Finally, we provide evidence that the evoked Ca<em>2</em>+ transients lead to the production of <em>2</em>-<em>arachidonoylglycerol</em> in cultured astrocytes. The results provide evidence for a novel cannabinoid induced endocannabinoid release mechanism in astrocytes which broadens the bidirectional signaling repertoire between astrocytes and neurons.

The endocannabinoid system (ECS) represents one of the most important physiologic systems involved in organism homeostasis, having various implications upon individual behavior and metabolic phenotype. It is composed of cannabinoid receptors CB1 and CB<em>2</em>, and their genes (CNR1 and CNR<em>2</em>), their endogenous ligands and the enzymes which mediate endogenous ligands' biosynthesis and degradation. Anandamide and <em>2</em>-<em>arachidonoylglycerol</em> are two endogenous agonists of the cannabinoid receptors. It is considered that ECS connects physical and emotional response to stress with appetite and energy balance, functioning like an after stress recovery system which remains inactive in repose physiologic conditions. It is involved in several physiologic processes like nociception, motor control, memory, learning, appetite, food intake and energy balance. This review analyzes the implication of 11 polymorphisms of CNR1 gene in the modulation of the ECS metabolic and central effects. A lot of studies show that rs1<em>2</em>7<em>2</em>0071, rs1049353, rs806381, rs10485170, rs6454674, rs<em>2</em>0<em>2</em>3<em>2</em>39 polymorphisms are associated with metabolic effects. From them rs1<em>2</em>7<em>2</em>0071, rs104935, rs6454674, rs<em>2</em>0<em>2</em>3<em>2</em>39 polymorphisms are also associated with central effects of ECS (substance addiction, impulsivity, resistance to antidepressive treatment). Other studies indicate that rs806368, rs1535<em>2</em>55, (AAT)9,(AAT)1<em>2</em> and (AAT)n are correlated only with central effects (schizophrenia, substance addiction, impulsivity, Parkinson syndrome). The discovery of ECS and its signaling pathways opens a door towards the understanding of several important physiologic processes regarding appetite, food intake, metabolism, weight gain, motor control, memory, learning, drug addiction and nociception. The detailed analysis and validation of the ECS functioning can bring us very close to the discovery of new diagnosis and treatment methods for obesity, drugs abuse and numerous psychic diseases.

Neuropathologies that affect our population include ischemic stroke and neurodegenerative diseases of immune origin, including multiple sclerosis. The endocannabinoid system in the brain, including agonists anandamide (arachidonyl ethanolamide) and <em>2</em>-<em>arachidonoylglycerol</em>, and the CB1 and CB<em>2</em> cannabinoid receptors, has been implicated in the pathophysiology of these disease states, and can be a target for therapeutic interventions. This review concentrates on cellular signal transduction pathways believed to be involved in the cellular damage.

A basal tone of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) enhances late oligodendrocyte progenitor cell (OPC) differentiation. Here, we investigated whether endogenous <em>2</em>-AG may also promote OPC proliferation in earlier stages. We found that the blockade of <em>2</em>-AG synthesizing enzymes, sn-1-diacylglycerol lipases α and β (DAGLs), with RHC-80<em>2</em>67 or the antagonism of either CB1 or CB<em>2</em> cannabinoid receptors with AM<em>2</em>81 and AM630, respectively, impaired early OPC proliferation stimulated by platelet-derived growth factor (PDGF-AA) and basic fibroblast growth factor (bFGF). On the contrary, increasing the levels of endogenous <em>2</em>-AG by blocking the degradative enzyme monoacylglycerol lipase (MAGL) with JZL-184, significantly increased OPC proliferation as did agonists of cannabinoid receptor CB1 (ACEA), CB<em>2</em> (JWH133) or both (HU-<em>2</em>10). To elucidate signaling pathways underlying OPC proliferation, we studied the involvement of phosphatidylinositol 3-kinase (PI3K)/Akt and its downstream target mammalian target of rapamycin (mTOR). We show that phosphorylation of Akt and mTOR is required for OPC proliferation stimulated by growth factors (PDGF-AA and bFGF) or by CB1/CB<em>2</em> agonists (ACEA/JWH133), since it was strongly decreased after LY<em>2</em>9400<em>2</em> or rapamycin treatment. In line with this, blockade of CB1 (AM<em>2</em>81), CB<em>2</em> (AM630) or DAGLs (RHC-80<em>2</em>67), decreased phosphorylation of Akt, mTOR and 4E-BP1, diminished cyclin E-cdk<em>2</em> complex association and increased p<em>2</em>7(kip1) levels. Our data suggest that proliferation of early OPCs stimulated by PDGF-AA and bFGF depends on the tonic activation of cannabinoid receptors by endogenous <em>2</em>-AG and provide further evidence on the role of endocannabinoids in oligodendrocyte development, being important for the maintenance and self-renewal of the OPCs. The results highlight the therapeutic potential of the endocannabinoid signaling in the emerging field of brain repair.

Nicotine has been reported to exert certain protective effect in the Parkinson's and Alzheimer's diseases. Whether it has a similar action in focal cerebral ischemia was unclear. In the present study, rats received either an injection of (-)-nicotine hydrogen tartrate salt (1.<em>2</em> mg/kg, i.p.) or the vehicle <em>2</em> h before the 1<em>2</em>0 min middle cerebral artery occlusion. Neurological deficits and histological injury were assessed at <em>2</em>4 h after reperfusion. The content of endocannabinoids and the expression of cannabinoid receptor CB1 in brain tissues were determined at different time points after nicotine administration. Results showed that nicotine administration ameliorated neurological deficits and reduced infarct volume induced by cerebral ischemia in the rats. The neuroprotective effect was partially reversed by CB1 blockage. The content of the endocannabinoids N-arachidonylethanolamine and <em>2</em>-<em>arachidonoylglycerol</em>, as well as the expression of cannabinoid receptor CB1 were up-regulated in brain tissues after nicotine delivery. These results suggest that endogenous cannabinoid system is involved in the nicotine-induced neuroprotection against transient focal cerebral ischemia.

The main endocannabinoids (EC) identified in mammalian tissues are N-arachidonoylethanolamide (AEA, anandamide), and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). AEA levels are critical in pregnancy, especially during implantation, decidualization, and placental development. As <em>2</em>-AG functions in pregnancy are still largely undefined, we hypothesized that it may also have a role during fetoplacental development. We showed that <em>2</em>-AG is not only present in the rat mesometrial decidua and plasma during fetoplacental development, but that both <em>2</em>-AG synthesizing (diacylglycerol lipase) and degradation (monoacylglycerol lipase) enzymes are expressed by decidual cells. While lower concentrations of <em>2</em>-AG induced apoptosis of rat primary decidual cells, via the CB1 receptor, higher concentrations induced a dramatic effect on cell morphology, cell viability and lactate dehydrogenase release, triggered through a mechanism independent of CB1. This study provides evidences that <em>2</em>-AG fluctuation in maternal tissues throughout normal pregnancy is primarily regulated by its metabolizing enzymes. Together, these data supports the hypothesis that a deregulation of the endocannabinoid system through aberrant cannabinoid signalling may impact normal uterine remodelling process and consequently normal pregnancy.

We previously demonstrated that socially isolated rats at weaning showed a significant decrease in corticosterone and adrenocorticotropic hormone (ACTH) levels, associated with an enhanced response to acute stressful stimuli. Here we shown that social isolation decreased levels of total corticosterone and of its carrier corticosteroid-binding globulin, but did not influence the availability of the free active fraction of corticosterone, both under basal conditions and after acute stress exposure. Under basal conditions, social isolation increased the abundance of glucocorticoid receptors, while it decreased that of mineralocorticoid receptors. After acute stress exposure, socially isolated rats showed long-lasting corticosterone, ACTH and corticotrophin releasing hormone responses. Moreover, while in the hippocampus and hypothalamus of group-housed rats glucocorticoid receptors expression increased with time and reached a peak when corticosterone levels returned to basal values, in socially isolated rats expression of glucocorticoid receptors did not change. Finally, social isolation also affected the hypothalamic endocannabinoid system: compared to group-housed rats, basal levels of anandamide and cannabinoid receptor type 1 were increased, while basal levels of <em>2</em>-<em>arachidonoylglycerol</em> were decreased in socially isolated rats and did not change after acute stress exposure. The present results show that social isolation in male rats alters basal HPA axis activity and impairs glucocorticoid-mediated negative feedback after acute stress. Given that social isolation is considered an animal model of several neuropsychiatric disorders, such as generalized anxiety disorder, depression, post-traumatic stress disorder and schizophrenia, these data could contribute to better understand the alterations in HPA axis activity observed in these disorders.

A cannabinoid receptor orthologue (CiCBR) has been described in the sea squirt Ciona intestinalis. Here we report that CiCBR mRNA expression is highest in cerebral ganglion, branchial pharynx, heart and testis of C. intestinalis, and that this organism also contains cannabinoid receptor ligands and some of the enzymes for ligand biosynthesis and inactivation. Using liquid chromatography-mass spectrometry, the endocannabinoid anandamide was found in all tissues analysed (0.063-5.4<em>2</em>3 pmol/mg of lipid extract), with the highest concentrations being found in brain and heart. The endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) was fivefold more abundant than anandamide, and was most abundant in stomach and intestine and least abundant in heart and ovaries (<em>2</em>.677-50.607 pmol/mg of lipid extract). Using phylogenomic analysis, we identified orthologues of several endocannabinoid synthesizing and degrading enzymes. In particular, we identified and partly sequenced a fatty acid amide hydrolase (FAAH) orthologue, showing 44% identity with human FAAH and containing nearly all the amino acids necessary for a functional FAAH enzyme. Ciona intestinalis also contained specific binding sites for cannabinoid receptor ligands, and an amidase enzyme with pH-dependency and subcellular/tissue distribution similar to mammalian FAAHs. Finally, a typical C. intestinalis behavioural response, siphon reopening after closure induced by mechanical stimulation, was inhibited by the cannabinoid receptor agonist HU-<em>2</em>10, and this effect was significantly attenuated by mammalian cannabinoid receptor antagonists.

It is generally accepted that the endocannabinoid system plays important roles in spinal pain processing. Although it is documented that cannabinoid-1 receptors are strongly expressed in the superficial spinal dorsal horn, the cellular distribution of enzymes that can synthesize endocannabinoid ligands is less well studied. Thus, using immunocytochemical methods at the light and electron microscopic levels, we investigated the distribution of diacylglycerol lipase-alpha (DGL-α) and N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD), enzymes synthesizing the endocannabinoid ligands, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and anandamide, respectively. Positive labeling was revealed only occasionally in axon terminals, but dendrites displayed strong immunoreactivity for both enzymes. However, the dendritic localization of DGL-α and NAPE-PLD showed a remarkably different distribution. DGL-α immunolabeling in dentrites was always revealed at membrane compartments in close vicinity to synapses. In contrast to this, dendritic NAPE-PLD labeling was never observed in association with synaptic contacts. In addition to dendrites, a substantial proportion of astrocytic (immunoreactive for GFAP) and microglial (immunoreactive for CD11b) profiles were also immunolabeled for both DGL-α and NAPE-PLD. Glial processes immunostained for DGL-α were frequently found near to synapses in which the postsynaptic dendrite was immunoreactive for DGL-α, whereas NAPE-PLD immunoreactivity on glial profiles at the vicinity of synapses was only occasionally observed. Our results suggest that both neurons and glial cells can synthesize and release <em>2</em>-AG and anandamide in the superficial spinal dorsal horn. The <em>2</em>-AG can primarily be released by postsynaptic dendrites and glial processes adjacent to synapses, whereas anandamide can predominantly be released from nonsynaptic dendritic and glial compartments.

Anandamide (arachidonoylethanolamide) is an endogenous ligand for cannabinoid receptors, and its cannabimimetic activities are lost when the compound is hydrolyzed to arachidonic acid and ethanolamine by an enzyme referred to as anandamide amidohydrolase. We cloned a cDNA for the enzyme of porcine brain, and the cDNA encoded a protein of 579 amino acids with a molecular mass of 6<em>2</em>.9 kDa. The amino acid sequence was 81, 80 and 85% identical with the enzymes previously cloned from the liver of rat, mouse, and human, respectively. When the enzyme protein was overexpressed in COS-7 cells, the particulate fraction of the cells showed an anandamide hydrolyzing activity and also catalyzed the reverse reaction synthesizing anandamide from arachidonic acid and ethanolamine both with a specific activity of 0. <em>2</em>-0.3 micromol/min/mg protein at 37 degrees C. The brain enzyme exhibited a wide substrate specificity hydrolyzing oleamide, <em>2</em>-<em>arachidonoylglycerol</em>, and methyl arachidonate. The point mutation of Ser-<em>2</em>17, Asp-<em>2</em>37, Ser-<em>2</em>41, or Cys-<em>2</em>49 completely abolished the hydrolyses of all the above-mentioned substrates as well as the synthesis of anandamide in the reverse reaction.