Stress-induced analgesia (SIA) is an important endogenous mechanism in response to stressful stimuli. Opioid peptides, as well as endocannabinoids, are known mediators of SIA. We were interested in whether the endocannabinoid tone and the resulting SIA could be influenced by changing the activity of cholecystokinin (CCK) in mice. This hypothesis is related to recent evidence showing a cellular colocalization of neuropeptide CCK and cannabinoid type 1 (CB(1)) receptors in many central nervous system structures. However, the physiological relevance of this colocalization is unknown. Our aim was to test whether abolishing the endogenous CCK tone might influence the cannabinoid-mediated form of SIA. As expected, the CB(1) antagonist rimonabant prevented the development of analgesia in response to footshock-induced stress in wild-type mice. In contrast, CCK type <em>2</em> (CCK(<em>2</em>)) receptor-deficient mice developed SIA regardless of rimonabant treatment. Naloxone, an opioid antagonist, antagonized SIA in both wild-type and CCK(<em>2</em>) receptor-deficient homozygous mice. This finding suggests that CCK, through CCK(<em>2</em>) receptors, modulates the action of endocannabinoids. Gene expression analysis revealed an up-regulation of CCK(<em>2</em>) receptor gene in the lumbar spinal cord and mesolimbic area in wild-type mice in response to stress. In addition, wild-type mice displayed up-regulation of genes implicated in endocannabinoid-mediated neurotransmission (elevation of CB(1) receptor, <em>2</em>-<em>arachidonoylglycerol</em> and the anandamide-synthesizing enzymes sn-1-diacylglycerol lipase alpha and N-acyl-phosphatidylethanolamine-hydrolysing phospholipase D) in response to stress in the lumbar spinal cord and mesolimbic area. We did not find any of these changes in CCK(<em>2</em>) receptor-deficient homozygous mice. Altogether, behavioural and gene expression studies reflect an involvement of CCK(<em>2</em>) receptors in the development of endocannabinoid-mediated SIA.

By means of retroviral insertional mutagenesis, we identified a novel common virus integration site (cVIS) (Evi11) in murine leukemias, and demonstrated that Cb<em>2</em>, encoding the peripheral cannabinoid receptor, is the potential proto-oncogene located in that region. Cb<em>2</em> is a 7-transmembrane G protein-coupled receptor (GPCR), which is normally expressed on B lymphocytes. Using transwell assays we observed strong migration of Cb<em>2</em>-expressing cells upon stimulation with <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), a potent endocannabinoid. Overexpression of Cb<em>2</em> receptor on murine myeloid precursor cells causes a block of neutrophilic differentiation, a major characteristic of myeloid leukemia. Intriguingly, we could not detect functional Cb<em>2</em> receptors on normal murine bone marrow precursor cells. Furthermore, analysis of human acute myeloid leukemia (AML) samples revealed the presence of CB<em>2</em> mRNA transcripts in several cases. Furthermore, migration could be induced by <em>2</em>-AG when analyzed in one of the patient samples. Our data suggest that the initially identified cVIS, Evi11, encodes for a murine onco-protein and that human CB<em>2</em> may be involved in certain cases of human AML as well.

Endocannabinoids are known as retrograde messengers, being released from the postsynaptic neuron and acting on specific presynaptic G-protein-coupled cannabinoid (CB) receptors to decrease neurotransmitter release. Also, at physiologically relevant concentrations cannabinoids can directly modulate the function of voltage-gated and receptor-operated ion channels. Using patch-clamp recording we analyzed the consequences of the direct action of an endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), on the functional properties of glycine receptor channels (GlyRs) and ionic currents in glycinergic synapses. At physiologically relevant concentrations (0.1-1 μM), <em>2</em>-AG directly affected the functions of recombinant homomeric α1H GlyR: it inhibited peak amplitude and dramatically enhanced desensitization. The action of <em>2</em>-AG on GlyR-mediated currents developed rapidly, within ∼300 ms. Addition of 1 μM <em>2</em>-AG strongly facilitated the depression of glycine-induced currents during repetitive (4-10 Hz) application of short (<em>2</em> ms duration) pulses of glycine to outside-out patches. In brainstem slices from CB1 receptor knockout mice, <em>2</em>-AG significantly decreased the extent of facilitation of synaptic currents in hypoglossal motoneurons during repetitive (10-<em>2</em>0 Hz) stimulation. These observations suggest that endocannabinoids can modulate postsynaptic metaplasticity of glycinergic synaptic currents in a CB1 receptor-independent manner.

OBJECTIVE

The aim of this study was to evaluate the feasibility of targeted imaging of myocardial cannabinoid type 1 receptor (CB1-R) and its potential up-regulation in obese mice with translation to humans using [11C]-OMAR and positron emission tomography (PET)/computed tomography (CT).

BACKGROUND

Activation of myocardial CB1-R by endocannabinoids has been implicated in cardiac dysfunction in diabetic mice. Obesity may lead to an up-regulation of myocardial CB1-R, potentially providing a mechanistic link between obesity and the initiation and/or progression of cardiomyopathy.

METHODS

Binding specificity of [11C]-OMAR to CB1-R was investigated by blocking studies with rimonabant in mice. The heart was harvested from each mouse, and its radioactivity was determined by γ-counter. Furthermore, [11C]-OMAR dynamic micro-PET/CT was carried out in obese and normal-weight mice. Ex vivo validation was performed by droplet digital polymerase chain reaction (absolute quantification) and RNAscope Technology (an in situ ribonucleic acid analysis platform). Subsequently, myocardial CB1-R expression was probed noninvasively with intravenous injection of CB1-R ligand [11C]-OMAR and PET/CT in humans with advanced obesity and normal-weight human control subjects, respectively.

RESULTS

Rimonabant significantly blocked OMAR uptake in the heart muscle compared with vehicle, signifying specific binding of OMAR to the CB1-R in the myocardium. The myocardial OMAR retention quantified by micro-PET/CT in mice was significantly higher in obese compared with normal-weight mice. Absolute quantification of CB1-R gene expression with droplet digital polymerase chain reaction and in situ hybridization confirmed CB1-R up-regulation in all major myocardial cell types (e.g., cardiomyocytes, endothelium, vascular smooth muscle cells, and fibroblasts) of obese mice. Obese mice also had elevated myocardial levels of endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em> compared with lean mice. Translation to humans revealed higher myocardial OMAR retention in advanced obesity compared with normal-weight subjects.

CONCLUSIONS

Noninvasive imaging of cardiac CB1-R expression in obesity is feasible applying [11C]-OMAR and PET/CT. These results may provide a rationale for further clinical testing of CB1-R-targeted molecular imaging in cardiometabolic diseases.

OBJECTIVE

The hypothesis that endocannabinoids protect hearts against ventricular fibrillation (VF) induced by myocardial ischaemia and reperfusion was examined, and the concept that cannabinoids may represent a new class of anti-VF drug was tested.

METHODS

In rat isolated hearts (Langendorff perfusion), VF evoked by reperfusion after 60 min regional ischaemia is known to be exacerbated by inhibitors of endogenous protectants such as nitric oxide. This preparation was used to assay the effects of cannabinoid agonists and antagonists, and the protocols were varied to examine mechanisms.

RESULTS

Reperfusion-induced VF was not facilitated by relatively selective CB1 (1 μM AM<em>2</em>51) or CB<em>2</em> (1 μM AM630) antagonists. VF evoked during early (30 min) acute ischaemia was also unaffected. However, AM<em>2</em>51 significantly increased the incidence of VF and the duration of VF episodes occurring during the later stage of acute ischaemia (30-60 min). AM630 had no such effects. In a separate study, cannabinoid perfusion (anandamide or <em>2</em>-<em>arachidonoylglycerol</em>, both 0.01-1 μM) failed to reduce VF incidence concentration-dependently during 30 min ischaemia. In all these studies, changes in ancillary variables (QT, PR, heart rate) were unrelated to changes in VF.

CONCLUSIONS

Endocannabinoids are not endogenous anti-VF mediators during reperfusion, but may have a weak protective effect during the late stages of ischaemia, mediated via CB1 agonism. This does not suggest endocannabinoids are important endogenous protectants in these settings, or that CB1 (or CB<em>2</em>) receptors are useful novel targets for developing drugs for VF.

Endocannabinoids are known to mediate retrograde suppression of synaptic transmission, modulate synaptic plasticity, and influence learning and memory. The <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) produced by diacylglycerol lipase α (DGLα) is regarded as the major endocannabinoid that causes retrograde synaptic suppression. To determine how <em>2</em>-AG signaling influences learning and memory, we subjected DGLα knock-out mice to two learning tasks. We tested the mice using habituation and odor-guided transverse patterning tasks that are known to involve the dentate gyrus and the CA1, respectively, of the hippocampus. We found that DGLα knock-out mice showed significantly faster habituation to an odor and a new environment than wild-type littermates with normal performance in the transverse patterning task. In freely moving animals, long-term potentiation (LTP) induced by theta burst stimulation was significantly larger at perforant path-granule cell synapses in the dentate gyrus of DGLα knock-out mice. Importantly, prior induction of synaptic potentiation at this synapse caused a significant retardation of habituation in DGLα knock-out but not in wild-type littermates. The excitability of granule cells became higher in DGLα knock-out mice after they generated action potentials. Since no differences were found in intrinsic membrane properties and responses to odor stimuli in granule cells, the elevated excitability is considered to result from enhanced activity of an excitatory recurrent network composed of granule cells and mossy cells. These results suggest that retrograde <em>2</em>-AG signaling negatively regulates habituation by suppressing excitatory recurrent network activity and reducing LTP in the dentate gyrus.

The endocannabinoid system has been considered as a target for pharmacological intervention. Accordingly, inhibition of fatty acid amide hydrolase (FAAH), a degrading enzyme of the endocannabinoids N-arachidonoylethanolamine (anandamide; AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) as well as of the endocannabinoid-like substances N-oleoylethanolamine (OEA) and N-palmitoylethanolamine (PEA), can cause augmented endogenous cannabinoid tone. Using liquid chromatography coupled with positive electrospray ionisation mass spectrometry, we herein describe a method to simultaneously quantify levels of AEA, OEA, PEA and <em>2</em>-AG in cultured cells. The procedure was developed according to the FDA guidelines for bioanalytical methods validation. The limits of quantification (LOQs) were 0.05 pmol for AEA, 0.09 pmol for OEA, 0.10 pmol for PEA and 0.80 pmol for <em>2</em>-AG when molecular ion monitoring was used. In H460 human lung carcinoma cells, basal levels of all four analytes ranged between <em>2</em> and 17 pmol mg(-1) protein with PEA showing the lowest and OEA the highest concentrations. Endocannabinoid levels observed in mesenchymal stem cells were of the same order of magnitude when compared to those in H460 human lung carcinoma cells.

The Cys-loop ligand-gated ion channel (LGIC) family comprises a group of membrane ion channel receptors that play a crucial role in fast synaptic neurotransmission in the central and peripheral nervous system. The members of this superfamily include gamma-aminobutyric acid type A (GABA(A)), neuronal nicotinic acetylcholine (nACh), 5-HT(3), and glycine receptors. These receptors serve as therapeutic sites for general anesthetic, antipsychoactive, antinociceptive, and anxiolytic drugs in the brain. These receptors are also thought to be primary targets of alcohol and other drugs of abuses. A number of studies reported that fatty acids affected the function of GABA(A) receptors in the early nineties. Accumulating evidence has suggested that the derivatives of arachidonic acid (AA), such as anandamide (N-arachidonoylethanolamine, AEA) and <em>arachidonoylglycerol</em> (<em>2</em>-AG), can critically regulate the other members of the Cys-loop LGIC superfamily through a cannabinoid receptor-independent mechanism. This chapter focuses on the results of recent studies showing that the Cys-loop LGICs could be additional molecular targets for fatty acid and endocannabinoid action in the central and peripheral nervous system. Some of these targets may mediate behavioral effects for cannabinoids to alter neuronal function.

The cerebellar cortex exhibits a strikingly high expression of type 1 cannabinoid receptor (CB1), the cannabinoid binding protein responsible for the psychoactive effects of marijuana. CB1 is primarily found in presynaptic elements in the molecular layer. While the functional importance of cerebellar CB1 is supported by the effect of gene deletion or exogenous cannabinoids on animal behavior, evidence for a role of endocannabinoids in synaptic signaling is provided by in vitro experiments on superfused acute rodent cerebellar slices. These studies have demonstrated that endocannabinoids can be transiently released by Purkinje cells and signal at synapses in a direction opposite to information transfer (retrograde). Here, following a description of the reported expression pattern of the endocannabinoid system in the cerebellum, I review the accumulated in vitro data, which have addressed the mechanism of retrograde endocannabinoid signaling and identified <em>2</em>-<em>arachidonoylglycerol</em> as the mediator of this signaling. The mechanisms leading to endocannabinoid release, the effects of CB1 activation, and the associated synaptic plasticity mechanisms are discussed and the remaining unknowns are pointed. Notably, it is argued that the spatial specificity of this signaling and the physiological conditions required for its induction need to be determined in order to understand endocannabinoid function in the cerebellar cortex.

Fatty acid amide hydrolase (EC 3.5.1.4.) is the enzyme responsible for the rapid degradation of lipid-derived chemical messengers such as anandamide, oleamide, and <em>2</em>-<em>arachidonoylglycerol</em>. The pharmacological characterization of this enzyme in vivo has been hampered by the lack of selective and bioavailable inhibitors. We have developed a simple, radioactive, high-throughput-compatible assay for this enzyme based on the differential absorption of the substrate and its products to activated charcoal. The assay was validated using known inhibitors. It may be applied for the identification of new inhibitors from a compound library.

Hypoxic-ischemic (HI) insult during the perinatal period remains as one of the most common causes of brain injury and produces long-term neurological deficits, and there is a growing need for effective therapies. The aim of the present work was to perform a prospective study designed to assess the possible protector effect of two endocannabinoids: <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>AG) and anandamide (AEA) in the brain after HI injury in perinatal rat model. We evaluate their effects on cell death and check several cellular parameters. 7-days-old Wistar rats were assigned to four different experimental groups (n=7-10): Sham, HI, and HI treated with <em>2</em>AG or AEA. The injury was induced by the left carotid artery ligature and subsequent exposure to 8% O(<em>2</em>) for 1<em>2</em>0 min. Immediately after the injury, treated groups received a single dose of <em>2</em>AG (1mg/kg) or AEA (5mg/kg) and then animals were sacrificed <em>2</em>4, 7<em>2</em> h or 7 days after the HI event. Brains fixed by perfusion were stained with Nissl for morphological studies, and non-fixed brains were dissociated and analyzed by flow cytometry to quantify apoptosis, mitochondrial state, intracellular calcium and reactive oxygen species. Our results show that both <em>2</em>AG and AEA have beneficial effects after HI injury in this rat model, producing a remarkable amelioration of brain injury, reducing apoptotic cell death, contributing to the maintenance of mitochondrial functionality, and improving cellular parameters such as the influx of calcium and ROS production.

The endocannabinoid system (ECS) plays an important role in the regulation of physiological functions,from stress and memory regulation to vegetative control and immunity. The ECS is considered a central and peripheral stress response system to emotional or physical challenges and acts through endocannabinoids (ECs), which bind to .their receptors inducing subsequent effecting mechanisms. In our studies, the ECS responses have been assessed through blood concentrations of the ECs anandamide and <em>2</em>-<em>arachidonoylglycerol</em>. In parallel, saliva cortisol was determined and the degree of perceived stress was quantified by questionnaires. This report summarizes the reactivity of the ECS in humans subjected to brief periods of kinetic stress and weightlessness during parabolic flights and to prolonged stress exposure during life onboard the International Space Station (ISS). Both conditions resulted in a significant increase in circulating ECs. Under the acute stress during parabolic flights, individuals who showed no evidence of motion sickness were in low-stress conditions and had a significant increase of plasma ECs. In contrast,highly stressed individuals with severe motion sickness had an absent EC response and a massive increase in hypothalamic-pituitary-adrenal axis activity. Likewise, chronic but well-tolerated exposure to weightlessness and emotional and environmental stressors on the ISS for 6 months resulted in a sustained increase in EC blood concentrations,which returned to baseline values after the cosmonauts'return. These preliminary results suggest that complex environmental stressors result in an increase of circulating ECs and that enhanced EC signaling is probably required for adaptation and tolerance under stressful conditions.

sn-1,<em>2</em>-diacylglycerol (DAG), a key intermediate in lipid metabolism, activates protein kinase C and is a fusogen. Phosphoinositides, the main sources of DAG in cell signaling, contain mostly stearoyl and arachidonoyl in the sn-1 and -<em>2</em> positions, respectively. The polymorphic behavior of sn-1-stearoyl-<em>2</em>-<em>arachidonoylglycerol</em> (SAG) was studied by differential scanning calorimetry, x-ray powder diffraction, and solid state magic angle spinning (MAS) (13)C NMR. Three alpha phases were found in the dry state. X-ray diffraction indicated that the acyl chains packed in a hexagonal array in the alpha phase, and the two sub-alpha phases packed with pseudo-hexagonal symmetry. In the narrow angle range strong diffractions of approximately 31 and approximately 6<em>2</em> A were present. High power proton-decoupled MAS (13)C NMR of isotropic SAG gave 16 distinct resonances of the <em>2</em>0 arachidonoyl carbons and 5 distinct resonances of the 18 stearoyl carbons. Upon cooling, all resonances of stearoyl weakened and vanished in the sub-alpha(<em>2</em>) phase, whereas arachidonoyl carbons from 8/9 to <em>2</em>0 gave distinct resonances in the frozen phases. Remarkably, the omega-carbon of the two acyl chains had different chemical shifts in alpha, sub-alpha(1), and sub-alpha(<em>2</em>) phases. Large differences in spin lattice relaxation of the stearoyl and arachidonoyl methene and methyl groups were demonstrated by contact time (cross-polarization) MAS (13)C NMR experiments in the solid phases alpha, sub-alpha(1), and sub-alpha(<em>2</em>). This shows that stearoyl and arachidonoyl in SAG have different environments in the solid states (alpha, sub-alpha(1), and sub-alpha(<em>2</em>) phases) and may segregate during cooling. The NMR and long spacing x-ray diffraction results suggest that SAG does not pack in a conventional double layer with the two acyls in a hairpin fashion. Our findings thus provide a physicochemical basis for DAG hexagonal phase domain separation within membrane bilayers.

Carboxylesterases (CES, EC 3.1.1.1) are members of a superfamily of serine hydrolases that hydrolyze ester, amide, and carbamate bonds. Several different CES genes exist in mammalian species with evidence of multiple gene duplication events occurring throughout evolutionary history. There are five CES genes reported in the Human Genome Organization database, although CES1 and CES<em>2</em> are the two best characterized human genes. An emerging picture of the CES family suggests that these enzymes have dual roles in the metabolism of xenobiotic and endobiotic compounds. Pesticides, such as the pyrethroids, are important xenobiotic substrates that are metabolized by CES, whereas cholesteryl esters, triacylglycerols, and <em>2</em>-<em>arachidonoylglycerol</em> are examples of endobiotics known to be substrates for CES. Functional studies using selective chemical inhibitors, siRNA, and gene knockout models are providing valuable insights into the physiological functions of CES, and suggest that CES may be a novel target for the treatment of diseases such as diabetes and atherosclerosis. This review will examine the known physiological functions of CES, the interactions between xenobiotics (primarily pesticides) and lipids that occur with CES enzymes, and where possible the implications that these findings may have in terms of health and disease.

The retrograde suppression of the synaptic transmission by the endocannabinoid sn-<em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is mediated by the cannabinoid CB1 receptors and requires the elevation of intracellular Ca(<em>2</em>+) and the activation of specific <em>2</em>-AG synthesizing (i.e., DAGLα) enzymes. However, the anatomical organization of the neuronal substrates that express <em>2</em>-AG/CB1 signaling system-related molecules associated with selective Ca(<em>2</em>+)-binding proteins (CaBPs) is still unknown. For this purpose, we used double-label immunofluorescence and confocal laser scanning microscopy for the characterization of the expression of the <em>2</em>-AG/CB1 signaling system (CB1 receptor, DAGLα, MAGL, and FAAH) and the CaBPs calbindin D<em>2</em>8k, calretinin, and parvalbumin in the rat hippocampus. CB1, DAGLα, and MAGL labeling was mainly localized in fibers and neuropil, which were differentially organized depending on the hippocampal CaBPs-expressing cells. CB(+) 1 fiber terminals localized in all hippocampal principal cell layers were tightly attached to calbindin(+) cells (granular and pyramidal neurons), and calretinin(+) and parvalbumin(+) interneurons. DAGLα neuropil labeling was selectively found surrounding calbindin(+) principal cells in the dentate gyrus and CA1, and in the calretinin(+) and parvalbumin(+) interneurons in the pyramidal cell layers of the CA1/3 fields. MAGL(+) terminals were only observed around CA1 calbindin(+) pyramidal cells, CA1/3 calretinin(+) interneurons and CA3 parvalbumin(+) interneurons localized in the pyramidal cell layers. Interestingly, calbindin(+) pyramidal cells expressed FAAH specifically in the CA1 field. The identification of anatomically related-neuronal substrates that expressed <em>2</em>-AG/CB1 signaling system and selective CaBPs should be considered when analyzing the cannabinoid signaling associated with hippocampal functions.

Endocannabinoids are a new class of lipids, which include amides, esters and ethers of long chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine; AEA) and <em>2</em>-<em>arachidonoylglycerol</em> are the main endogenous agonists of cannabinoid receptors, able to mimic several pharmacological effects of Delta(9)-tetrahydrocannabinol, the active principle of Cannabis sativa preparations like hashish and marijuana. It is known that the activity of AEA is limited by cellular uptake through a specific membrane transporter, followed by intracellular degradation by a fatty acid amide hydrolase. Together with AEA and congeners these proteins form the "endocannabinoid system". The endogenous cannabinoids were identified in brain, and also in neuronal and endothelial cells, suggesting a potential role as modulators in the central nervous system and in the periphery. This review summarises the metabolic routes for the synthesis and degradation of AEA, and the latest advances in the involvement of this lipid in neurovascular biology. In addition, the therapeutic potential of the modulation of endocannabinoid metabolism for neuronal and vascular system will be also reviewed.

Considerable progress has been made in recent years in developing selective, potent monoacylglycerol lipase (MAGL) inhibitors. In the investigations of measures to inhibit this enzyme, less attention has been paid to improving our understanding of its catalytic mechanisms or substrate preferences. In our study, we used site-directed mutagenesis, and we show via versatile activity assays combined with molecular modeling that Cys<em>2</em>4<em>2</em> and Tyr194, the two opposing amino acid residues in the catalytic cavity of MAGL, play important roles in determining the rate and the isomer preferences of monoacylglycerol hydrolysis. In contrast to wild-type enzymes that hydrolyzed 1- and <em>2</em>-monoacylglycerols at similar rates, mutation of Cys<em>2</em>4<em>2</em> to alanine caused a significant reduction in overall activity (maximal velocity, Vmax), particularly skewing the balanced hydrolysis of isomers to favor the <em>2</em>-isomer. Molecular modeling studies indicate that this was caused by structural features unfavorable toward 1-isomers as well as impaired recognition of OH-groups in the glycerol moiety. Direct functional involvement of Cys<em>2</em>4<em>2</em> in the catalysis was found unlikely due to the remote distance from the catalytic serine. Unlike C<em>2</em>4<em>2</em>A, mutation of Tyr194 did not bias the hydrolysis of 1- and <em>2</em>-monoacylglycerols but significantly compromised overall activity. Finally, mutation of Cys<em>2</em>4<em>2</em> was also found to impair inhibition of MAGL, especially that by fluorophosphonate derivatives (13- to 63-fold reduction in potency). Taken together, this study provides new experimental and modeling insights into the molecular mechanisms of MAGL-catalyzed hydrolysis of the primary endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> and related monoacylglycerols.

The endocannabinoids, anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), are arachidonic acid (AA) derivatives that are known to regulate human cardiovascular functions. CYP<em>2</em>J<em>2</em> is the primary cytochrome P450 in the human heart and is most well known for the metabolism of AA to the biologically active epoxyeicosatrienoic acids. In this study, we demonstrate that both <em>2</em>-AG and AEA are substrates for metabolism by CYP<em>2</em>J<em>2</em> epoxygenase in the model membrane bilayers of nanodiscs. Reactions of CYP<em>2</em>J<em>2</em> with AEA formed four AEA-epoxyeicosatrienoic acids, whereas incubations with <em>2</em>-AG yielded detectable levels of only two <em>2</em>-AG epoxides. Notably, <em>2</em>-AG was shown to undergo enzymatic oxidative cleavage to form AA through a NADPH-dependent reaction with CYP<em>2</em>J<em>2</em> and cytochrome P450 reductase. The formation of the predominant AEA and <em>2</em>-AG epoxides was confirmed using microsomes prepared from the left myocardium of porcine and bovine heart tissues. The nuances of the ligand-protein interactions were further characterized using spectral titrations, stopped-flow small-molecule ligand egress, and molecular modeling. The experimental and theoretical data were in agreement, which showed that substitution of the AA carboxylic acid with the <em>2</em>-AG ester-glycerol changes the binding interaction of these lipids within the CYP<em>2</em>J<em>2</em> active site, leading to different product distributions. In summary, we present data for the functional metabolomics of AEA and <em>2</em>-AG by a membrane-bound cardiovascular epoxygenase.

Monoacylglycerol lipase (MAGL) is one of the key enzymes of the endocannabinoid system (ECS). It hydrolyzes one of the major endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), an endogenous full agonist at G protein coupled cannabinoid receptors CB1 and CB<em>2</em>. Numerous studies showed that MGL inhibitors are potentially useful for the treatment of pain, inflammation, cancer and CNS disorders. These provocative findings suggested that pharmacological inhibition of MAGL function may confer significant therapeutic benefits. In this study, we presented hybrid ligand and structure-based approaches to obtain a novel set of virtual leads as MAGL inhibitors. The constraints used in this study, were Glide score, binding free energy estimates and ADME properties to screen the ZINC database, containing approximately <em>2</em>1 million compounds. A total of seven virtual hits were obtained, which showed significant binding affinity towards MAGL protein. Ligand, ZINC<em>2</em>409<em>2</em>691 was employed in complex form with the protein MAGL, for molecular dynamics simulation study, because of its excellent glide score, binding free energy and ADME properties. The RMSD of ZINC<em>2</em>409<em>2</em>691 was observed to stay at 0.1 nm (1 Å) in most of the trajectories, which further confirmed its ability to inhibit the protein MAGL. The hits were then evaluated for their ability to inhibit human MAGL. The compound ZINC<em>2</em>409<em>2</em>691 displayed the noteworthy inhibitory activity reducing MAGL activity to <em>2</em>1.15% at 100 nM concentration, with an IC50 value of 10 nM.

Early-life stress modulates the development of cortico-limbic circuits and increases vulnerability to adult psychopathology. Given the important stress-buffering role of endocannabinoid (eCB) signaling, we performed a comprehensive investigation of the developmental trajectory of the eCB system and the impact of exposure to early life stress induced by repeated maternal separation (MS; 3 h/day) from postnatal day <em>2</em> (PND<em>2</em>) to PND1<em>2</em>. Tissue levels of the eCB molecules anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) were measured after MS exposures, as well under basal conditions at juvenile (PND14), adolescent (PND40) and adult (PND70) timepoints in the prefrontal cortex (PFC), amygdala and hippocampus. We also examined the effects of MS on CB1 receptor binding in these three brain regions at PND40 and PND70. AEA content was found to increase from PND<em>2</em> into adulthood in a linear manner across all brain regions, while <em>2</em>-AG was found to exhibit a transient spike during the juvenile period (PND1<em>2</em>-14) within the amygdala and PFC, but increased in a linear manner across development in the hippocampus. Exposure to MS resulted in bidirectional changes in AEA and <em>2</em>-AG tissue levels within the amygdala and hippocampus and produced a sustained reduction in eCB function in the hippocampus at adulthood. CB1 receptor densities across all brain regions were generally found to be downregulated later in life following exposure to MS. Collectively, these data demonstrate that early life stress can alter the normative ontogeny of the eCB system, resulting in a sustained deficit in eCB function, particularly within the hippocampus, in adulthood.

The N-acylethanolamines (NAEs) exert important behavioral, physiological, and immunological effects through actions at cannabinoid and other receptors. We measured concentrations of three NAEs, the Km and Vmax for fatty acid amide hydrolysis (FAAH), FAAH protein and FAAH mRNA in prefrontal cortex, hippocampus, hypothalamus, amygdala, striatum, and cerebellum at 4 h intervals, starting at 03:00. Significant differences in N-arachidonylethanolamine contents among the times examined occur in the prefrontal cortex (PFC), hippocampus, hypothalamus, and striatum. N-Oleoylethanolamine concentrations exhibit large fluctuations over the day in the cerebellum, including a threefold decrease between 19:00 and <em>2</em>3:00. N-Palmitoylethanolamine and N-oleoylethanolamine were significantly, positively correlated in all regions examined except the hypothalamus. FAAH Km values are significantly affected by time of day in PFC, hippocampus and amygdala and FAAH Vmax values are significantly affected in PFC, hippocampus and cerebellum. However, correlational data indicate that FAAH does not play a primary role in the circadian regulation of the NAE concentrations. FAAH protein expression is not significantly different among the harvest times in any brain region examined. Concentrations of <em>2</em>-<em>arachidonoylglycerol</em> are significantly affected by time of harvest in the striatum and cerebellum, but not in other brain regions. Together, these data indicate that the NAEs exhibit diverse patterns of change with time of day that are likely the result of alterations in biosynthesis, and support the hypothesis that N-arachidonylethanolamine is a tonic activator of cannabinoid receptor signaling.

Retrograde synaptic signaling by endogenous cannabinoids (endocannabinoids) is a recently discovered form of neuromodulation in various brain regions. In hippocampus, it is well known that endocannabinoids suppress presynaptic inhibitory neurotransmitter release in CA1 region. However, endocannabinoid signaling in CA3 region remains to be examined. Here we investigated whether presynaptic inhibition can be caused by activation of postsynaptic group I metabotropic glutamate receptors (mGluRs) and following presynaptic cannabinoid receptor type 1 (CB1 receptor) using mechanically dissociated rat hippocampal CA3 pyramidal neurons with adherent functional synaptic boutons. Application of group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) reversibly suppressed spontaneous inhibitory postsynaptic currents (IPSCs). In the presence of tetrodotoxin (TTX), frequency of miniature IPSCs was significantly reduced by DHPG, while there were no significant changes in minimum quantal size and sensitivity of postsynaptic GABA(A) receptors to the GABA(A) receptor agonist muscimol, indicating that this suppression was caused by a decrease in GABA release from presynaptic nerve terminals. Application of CB1 synthetic agonist WIN55<em>2</em>1<em>2</em>-<em>2</em> (mesylate(R)-(+)-[<em>2</em>,3-dihydro-5-methyl-3-[4-morpholino)methyl]pyrrolo-[1,<em>2</em>,3-de]-1,4-benzoxazin-6-yl](1-naphthyl)methanone) or endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> also suppressed the spontaneous IPSC. The inhibitory effect of DHPG on spontaneous IPSCs was abolished by SR-141716 (5-(4-chlorophenyl)-1-(<em>2</em>,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide), a CB1 receptor antagonist. Furthermore, postsynaptic application of GDP-betaS blocked the DHPG-induced inhibition of spontaneous IPSCs, indicating the involvement of endcannabinoid-mediated retrograde synaptic signaling. These results provide solid evidence for retrograde signaling from postsynaptic group I mGluRs to presynaptic CB1 receptors, which induces presynaptic inhibition of GABA release in rat hippocampal CA3 region.

In recent decades, there has been increased interest in the physiological roles of the endocannabinoid (eCB) system and its receptors, the cannabinoid receptor types 1 (CB1R) and <em>2</em> (CB<em>2</em>R). Exposure to cannabinoids during development results in neurofunctional alterations, which implies that the eCB system is involved in the developmental processes of the brain. Because of their lipophilic nature, eCBs are synthesized on demand and are not stored in vesicles. Consequently, the enzymes responsible for their synthesis and degradation are key regulators of their physiological actions. Therefore, knowing the localization of these enzymes during development is crucial for a better understanding of the role played by eCBs during the formation of the central nervous system. In this study, we investigated the developmental protein localization of the synthesizing and catabolic enzymes of the principal eCB, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in the retinas of young and adult rats. The distribution of the enzymes responsible for the synthesis (DAGLα) and the degradation (MAGL) of <em>2</em>-AG was determined for every retinal cell type from birth to adulthood. Our results indicate that DAGLα is present early in postnatal development. It is highly expressed in photoreceptor, horizontal, amacrine, and ganglion cells. MAGL appears later during the development of the retina and its presence is limited to amacrine and Müller cells. Overall, these results suggest that <em>2</em>-AG is strongly present in early retinal development and might be involved in the regulation of the structural and functional maturation of the retina.

Monoacylglycerol lipase is a serine hydrolase that play a major role in the degradation of <em>2</em>-<em>arachidonoylglycerol</em>, an endocannabinoid neurotransmitter implicated in several physiological processes. Recent studies have shown the possible role of MAGL inhibitors as anti-inflammatory, anti-nociceptive and anti-cancer agents. The use of irreversible MAGL inhibitors determined an unwanted chronic MAGL inactivation, which acquires a functional antagonism function of the endocannabinoid system. However, the application of reversible MAGL inhibitors has not yet been explored, mainly due to the scarcity of known compounds possessing efficient reversible inhibitory activities. In this study we reported the first virtual screening analysis for the identification of reversible MAGL inhibitors. Among the screened compounds, the (4-(4-chlorobenzoyl)piperidin-1-yl)(4-methoxyphenyl)methanone (CL6a) is a promising reversible MAGL inhibitor lead (Ki=8.6μM), which may be used for the future development of a new class of MAGL inhibitors. Furthermore, the results demonstrate the validity of the methodologies that we followed, encouraging additional screenings of other commercial databases.