Emerging evidence suggests that the risk of developing chronic traumatic encephalopathy (CTE), a progressive neurodegenerative disease, is significantly increased in military personnel and contact sports players who have been exposed to repetitive trauma brain injury (TBI). Unfortunately there are no effective medications currently available for prevention and treatment of CTE. Here we demonstrate that inhibition of monoacylglycerol lipase (MAGL), the key enzyme that metabolizes the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in the brain, significantly reduced CTE-like neuropathologic changes in a mouse model of repetitive mild closed head injury (rmCHI). Inhibition of <em>2</em>-AG metabolism promoted neurologic recovery following rmCHI and reduced proinflammatory cytokines, astroglial reactivity, expression of amyloid precursor protein and the enzymes that make Aβ, as well as formation of Aβ. Importantly, neurodegeneration, TDP-43 protein aggregation, and tau phosphorylation, which are the neuropathologic hallmarks of CTE, were significantly suppressed by MAGL inactivation. Furthermore, alterations in expression of glutamate receptor subunits and impairments in basal synaptic transmission, long-term synaptic plasticity, and spatial learning and memory were recovered by inhibition of <em>2</em>-AG metabolism in animals exposed to rmCHI. Our results suggest that MAGL inhibition, which boosts <em>2</em>-AG and reduces <em>2</em>-AG metabolites prostaglandins in the brain, may lead to a new therapy for CTE.

The decision-making mechanisms that determine the choice of the appropriate effector immune response to a microbial challenge are poorly understood. The endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), injected intradermally in mice together with a soluble protein and a T helper-<em>2</em> (Th<em>2</em>) priming Toll-like receptors (TLRs) agonist during primary immunization, shifts the memory response to the Th1 type. This effect can be shown by the enhanced hypersensitivity response and by the Th1 pattern of cytokines production that was abolished by the specific cannabinoid receptor CB<em>2</em> antagonist SR 1445<em>2</em>8. <em>2</em>-AG seems to operate during the innate response by increasing the number of dendritic cells (DCs) migrating to the draining lymph nodes. Expression of CB<em>2</em> mRNA but not of the protein was higher in immature vs. mature DCs. Consistently, in vitro, <em>2</em>-AG exerted a potent chemotactic activity on both immature and mature DCs. In conclusion, we suggest that, in vivo, the endocannabinoid <em>2</em>-AG may act as chemotactic substance capable of recruiting DCs and/or their precursors during the innate immune response that, in presence of a TLR agonist, consequently instruct a Th1-shifted adaptive response. As <em>2</em>-AG may be induced in tissues by various stimuli at concentrations similar to that used in our study, this evidence might be of a wide-ranging pathophysiological relevance.

In the present study, we examined the effects of endogenous ligand <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) on naloxone-precipitated withdrawal in morphine-dependent mice, in comparison with that of two cannabinoid agonists, an ingredient of Cannabis sativa Delta(8)-tetrahydrocannabinol (Delta(8)-THC) and the synthetic cannabinoid CB1 receptor agonist HU-<em>2</em>10. <em>2</em>-AG at a dose of 10 microg per mouse (i.c.v.) significantly inhibited both jumping and forepaw tremor as signs of withdrawal following naloxone challenge in morphine-dependent mice. Furthermore, both Delta(8)-THC and HU-<em>2</em>10 significantly attenuated these symptoms of withdrawal in morphine-dependent mice. Therefore, it is suggested that inactivation of the endogenous cannabinoid system is related to the induction of withdrawal syndrome in morphine-dependent mice. Moreover, hyperlocomotor activity in morphine-dependent mice was markedly increased by Delta(8)-THC 10 mg/kg, which had no effect in naive mice. This finding suggested that in morphine dependence, upregulation of cannabinoid CB1 receptors occurred. Non-psychoactive CB1 receptor agonists or accelerators of endocannabinoid synthesis may be potential as therapeutic drugs for opiate withdrawal symptoms.

Glutamate can control inhibitory synaptic transmission through activation of presynaptic kainate receptors. We found that glutamate released by train stimulation of Schaffer collaterals could lead to either short-term depression or short-term facilitation of inhibitory synaptic transmission in mouse CA1 pyramidal neurons, depending on the presence of cannabinoid type 1 (CB(1)) receptors on GABAergic afferents. The train-induced depression of inhibition (t-Di) required the mobilization of <em>2</em>-<em>arachidonoylglycerol</em> through postsynaptic activation of metabotropic glutamate receptors and [Ca(<em>2</em>+)] rise. GluK1 (GluR5)-dependent depolarization of GABAergic terminals enabled t-Di by facilitating presynaptic CB(1) signaling. Thus, concerted activation of presynaptic CB(1) receptors and kainate receptors mediates short-term depression of inhibitory synaptic transmission. In contrast, in inhibitory connections expressing GluK1, but not CB(1), receptors, train stimulation of Schaffer collaterals led to short-term facilitation. Thus, activation of kainate receptors by synaptically released glutamate gates presynaptic CB(1) signaling, which in turn controls the direction of short-term heterosynaptic plasticity.

BACKGROUND

The endocannabinoid (eCB) system is involved in the regulation of food intake and of peripheral metabolism. Although the cross talk between energy metabolism and the circadian system is well documented, little is known about a potential circadian modulation of human eCB activity.

OBJECTIVE

The objective of the study was to define the <em>2</em>4-hour profile of circulating levels of the most abundant endogenous ligand of the CB1 receptor, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), in healthy young nonobese adults studied under controlled bedtime, dietary, and activity conditions.

METHODS

Fourteen subjects participated in this 4-day laboratory study with fixed light-dark cycles, standardized meals, and bedtimes. Sleep was recorded each night. On the third day, blood sampling at 15- to 30-minute intervals began at 9:30 pm and continued for <em>2</em>4 hours. Cortisol, leptin, and ghrelin were assayed on all samples, whereas the levels of <em>2</em>-AG and its structural analog, <em>2</em>-oleoylglycerol (<em>2</em>-OG), were measured at 60-minute intervals.

RESULTS

All participants exhibited a large circadian variation of <em>2</em>-AG serum concentrations with a nadir around midsleep, coincident with the middle of the overnight fast. Levels of <em>2</em>-AG increased continually across the morning, peaking in the early to midafternoon. Peak values represented, on average, a nearly 3-fold increase above nocturnal nadir levels. Concentrations of <em>2</em>-OG followed a similar pattern, although with a shorter morning increase and lower amplitude.

CONCLUSIONS

The findings demonstrate that activity of the eCB system is profoundly modulated by circadian rhythmicity and suggest that its impact on the regulation of food intake is suppressed during sleep and is maximal during early to midafternoon.

Dietary intake of linoleic acid (LA) has increased dramatically during the twentieth century and is associated with a greater prevalence of obesity. Vegetable oils are recognised as suitable alternatives to fish oil (FO) in feed for Atlantic salmon (Salmo salar L.) but introduce high amounts of LA in the salmon fillet. The effect on fish consumers of such a replacement remains to be elucidated. Here, we investigate the effect of excessive dietary LA from soyabean oil (SO) on endocannabinoid levels in Atlantic salmon and mice, and study the metabolic effects in mice when SO replaces FO in feed for Atlantic salmon. Atlantic salmon were fed FO and SO for 6 months, and the salmon fillet was used to produce feed for mice. Male C57BL/6J mice were fed diets of 35% of energy as fat based on FO- and SO-enriched salmon for 16 weeks. We found that replacing FO with SO in feed for Atlantic salmon increased LA, arachidonic acid (AA), decreased EPA and DHA, elevated the endocannabinoids <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and anandamide (AEA), and increased TAG accumulation in the salmon liver. In mice, the SO salmon diet increased LA and AA and decreased EPA and DHA in the liver and erythrocyte phospholipids, and elevated <em>2</em>-AG and AEA associated with increased feed efficiency, weight gain and adipose tissue inflammation compared with mice fed the FO salmon diet. In conclusion, excessive dietary LA elevates endocannabinoids in the liver of salmon and mice, and increases weight gain and counteracts the anti-inflammatory properties of EPA and DHA in mice.

Acute stress reduces pain sensitivity by engaging an endocannabinoid signaling circuit in the midbrain. The neural mechanisms governing this process and molecular identity of the endocannabinoid substance(s) involved are unknown. We combined behavior, pharmacology, immunohistochemistry, RNA interference, quantitative RT-PCR, enzyme assays, and lipidomic analyses of endocannabinoid content to uncover the role of the endocannabinoid <em>2</em>-arachidonoyl-sn-glycerol (<em>2</em>-AG) in controlling pain sensitivity in vivo. Here, we show that footshock stress produces antinociception in rats by activating type 5 metabotropic glutamate receptors (mGlu(5)) in the dorsolateral periaqueductal gray (dlPAG) and mobilizing <em>2</em>-AG. Stimulation of mGlu(5) in the dlPAG with DHPG [(S)-3,5-dihydroxyphenylglycine] triggered <em>2</em>-AG formation and enhanced stress-dependent antinociception through a mechanism dependent upon both postsynaptic diacylglycerol lipase (DGL) activity, which releases <em>2</em>-AG, and presynaptic CB(1) cannabinoid receptors. Pharmacological blockade of DGL activity in the dlPAG with RHC80<em>2</em>67 [1,6-bis(cyclohexyloximinocarbonylamino)hexane] and (-)-tetrahydrolipstatin (THL), which inhibit activity of DGL-α and DGL-β isoforms, suppressed stress-induced antinociception. Inhibition of DGL activity in the dlPAG with THL selectively decreased accumulation of <em>2</em>-AG without altering levels of anandamide. The putative <em>2</em>-AG-synthesizing enzyme DGL-α colocalized with mGlu(5) at postsynaptic sites of the dlPAG, whereas CB(1) was confined to presynaptic terminals, consistent with a role for <em>2</em>-AG as a retrograde signaling messenger. Finally, virally mediated silencing of DGL-α, but not DGL-β, transcription in the dlPAG mimicked effects of DGL inhibition in suppressing both endocannabinoid-mediated stress antinociception and <em>2</em>-AG formation. The results indicate that activation of the postsynaptic mGlu(5)-DGL-α cascade triggers retrograde <em>2</em>-AG signaling in vivo. This pathway is required for endocannabinoid-mediated stress-induced analgesia.

The endocannabinoid system (ECS) is a lipid signalling system, comprising of the endogenous cannabis-like ligands (endocannabinoids) anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), which derive from arachidonic acid. These bind to a family of G-protein-coupled receptors, called CB1 and CB<em>2</em>. The cannabinoid receptor 1 (CB1R) is distributed in brain areas associated with motor control, emotional responses, motivated behaviour and energy homeostasis. In the periphery, the same receptor is expressed in the adipose tissue, pancreas, liver, GI tract, skeletal muscles, heart and the reproduction system. The CB<em>2</em>R is mainly expressed in the immune system regulating its functions. Endocannabinoids are synthesized and released upon demand in a receptor-dependent way. They act as retrograde signalling messengers in GABAergic and glutamatergic synapses and as modulators of postsynaptic transmission, interacting with other neurotransmitters. Endocannabinoids are transported into cells by a specific uptake system and degraded by the enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). The ECS is involved in various pathophysiological conditions in central and peripheral tissues. It is implicated in the hormonal regulation of food intake, cardiovascular, gastrointestinal, immune, behavioral, antiproliferative and mammalian reproduction functions. Recent advances have correlated the ECS with drug addiction and alcoholism. The growing number of preclinical and clinical data on ECS modulators is bound to result in novel therapeutic approaches for a number of diseases currently treated inadequately. The ECS dysregulation has been correlated to obesity and metabolic syndrome pathogenesis. Rimonabant is the first CB1 blocker launched to treat cardiometabolic risk factors in obese and overweight patients. Phase III clinical trials showed the drug's ability to regulate intra-abdominal fat tissue levels, lipidemic, glycemic and inflammatory parameters. However, safety conerns have led to its withrawal. The role of endocannabinoids in mammalian reproduction is an emerging research area given their implication in fertilization, preimplantation embryo and spermatogenesis. The relevant preclinical data on endocannabinoid signalling open up new perspectives as a target to improve infertility and reproductive health in humans.

The endogenous cannabinoid receptor agonist anandamide (AEA) and the related compound palmitoylethanolamide (PEA) are inactivated by transport into cells followed by metabolism by fatty acid amide hydrolase (FAAH). The cellular uptake of AEA has been characterized in detail, whereas less is known about the properties of the PEA uptake, in particular in neuronal cells. In the present study, the pharmacological and functional properties of PEA and AEA uptake have been investigated in mouse Neuro-<em>2</em>a neuroblastoma and, for comparison, in rat RBL-<em>2</em>H3 basophilic leukaemia cells. Saturable uptake of PEA and AEA into both cell lines were demonstrated with apparent K(M) values of <em>2</em>8 microM (PEA) and 10 microM (AEA) in Neuro-<em>2</em>a cells, and 30 microM (PEA) and 9.3 microM (AEA) in RBL-<em>2</em>H3 cells. Both PEA and AEA uptake showed temperature-dependence but only the AEA uptake was sensitive to treatment with Pronase and phenylmethylsulfonyl fluoride. The AEA uptake was inhibited by AM404, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), R1- and S1-methanandamide, arachidonic acid and olvanil with similar potencies for the two cell types. PEA, up to a concentration of 100 microM, did not affect AEA uptake in either cell line. AEA, <em>2</em>-AG, arachidonic acid, R1-methanandamide, (9)-THC, and cannabidiol inhibited PEA transport in both cell lines. The non-steroidal anti-inflammatory drug indomethacin inhibited the AEA uptake but had very weak effects on the uptake of PEA. From these data, it can be concluded that PEA is transported in to cells both by passive diffusion and by a facilitated transport that is pharmacologically distinguishable from AEA uptake.

Approximately <em>2</em>% of amyotrophic lateral sclerosis (ALS) cases are caused by mutations in the super oxide dismutase 1 (SOD1) gene and transgenic mice for these mutations recapitulate many features of this devastating neurodegenerative disease. Here we show that the amount of anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), two endocannabinoids that have neuroprotective properties, increase in spinal cord of SOD1(G93A) transgenic mice. This increase occurs in the lumbar section of spinal cords, the first section to undergo neurodegeneration, and is significant before overt motor impairment. Our results show that chronic neurodegeneration induced by a genetic mutation increases endocannabinoid production possibly as part of an endogenous defense mechanism.

Long-term depression (LTD) and long-term potentiation (LTP) at cerebellar parallel fiber-Purkinje cell (PF-PC) synapses play critical roles in motor learning. The 1 Hz stimulation at PF-PC synapses induces a postsynaptically expressed LTP that requires a postsynaptic Ca(<em>2</em>+) transient, phosphatases, and nitric oxide (NO). However, the mechanism underlying 1 Hz PF-LTP remains unclear because none of the known events is related to each other. Here, we demonstrated that 1 Hz PF-LTP requires postsynaptic cytosolic phospholipase A<em>2</em> α (cPLA<em>2</em>α)/arachidonic acid (AA) signaling and presynaptic endocannabinoid receptors. Using patch-clamp recording in cerebellar slices, we found that 1 Hz PF-LTP was abolished in cPLA<em>2</em>α-knock-out mice. This deficit was effectively rescued by the conjunction of 1 Hz PF stimulation and the local application of AA. <em>2</em>-<em>Arachidonoylglycerol</em> and the retrograde activation of cannabinoid receptor 1 (CB1R) were also involved in 1 Hz LTP because it was blocked by the hydrolysis of <em>2</em>-AG or by inhibiting CB1Rs. The amount of NO released was detected using an NO electrode in cultured granule cells and PF terminals. Our results showed that the activation of CB1Rs at PF terminals activated NO synthetase and promoted NO production. The 1 Hz PF-stimuli evoked limited NO, but 100 Hz PF stimulation generated a large amount. Therefore, 1 Hz PF-LTP, distinct from classical postsynaptically expressed plasticity, requires concurrent presynaptic and postsynaptic activity. In addition, NO of sufficient amplitude decides between the weakening and strengthening of PF-PC synapses.

Oxons are the bioactivated metabolites of organophosphorus insecticides formed via cytochrome P450 monooxygenase-catalyzed desulfuration of the parent compound. Oxons react covalently with the active site serine residue of serine hydrolases, thereby inactivating the enzyme. A number of serine hydrolases other than acetylcholinesterase, the canonical target of oxons, have been reported to react with and be inhibited by oxons. These off-target serine hydrolases include carboxylesterase 1 (CES1), CES<em>2</em>, and monoacylglycerol lipase. Carboxylesterases (CES, EC 3.1.1.1) metabolize a number of xenobiotic and endobiotic compounds containing ester, amide, and thioester bonds and are important in the metabolism of many pharmaceuticals. Monoglyceride lipase (MGL, EC 3.1.1.<em>2</em>3) hydrolyzes monoglycerides including the endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). The physiological consequences and toxicity related to the inhibition of off-target serine hydrolases by oxons due to chronic, low level environmental exposures are poorly understood. Here, we determined the potency of inhibition (IC(50) values; 15 min preincubation, enzyme and inhibitor) of recombinant CES1, CES<em>2</em>, and MGL by chlorpyrifos oxon, paraoxon and methyl paraoxon. The order of potency for these three oxons with CES1, CES<em>2</em>, and MGL was chlorpyrifos oxon>paraoxon>methyl paraoxon, although the difference in potency for chlorpyrifos oxon with CES1 and CES<em>2</em> did not reach statistical significance. We also determined the bimolecular rate constants (k(inact)/K(I)) for the covalent reaction of chlorpyrifos oxon, paraoxon and methyl paraoxon with CES1 and CES<em>2</em>. Consistent with the results for the IC(50) values, the order of reactivity for each of the three oxons with CES1 and CES<em>2</em> was chlorpyrifos oxon>paraoxon>methyl paraoxon. The bimolecular rate constant for the reaction of chlorpyrifos oxon with MGL was also determined and was less than the values determined for chlorpyrifos oxon with CES1 and CES<em>2</em> respectively. Together, the results define the kinetics of inhibition of three important hydrolytic enzymes by activated metabolites of widely used agrochemicals.

Phytocannabinoids modulate inflammatory responses by regulating the production of cytokines in several experimental models of inflammation. Cannabinoid type-<em>2</em> (CB<em>2</em>) receptor activation was shown to reduce the production of the monocyte chemotactic protein-<em>2</em> (MCP-<em>2</em>) chemokine in polyinosinic-polycytidylic acid [poly-(I:C)]-stimulated human keratinocyte (HaCaT) cells, an in vitro model of allergic contact dermatitis (ACD). We investigated if nonpsychotropic cannabinoids, such as cannabidiol (CBD), produced similar effects in this experimental model of ACD. HaCaT cells were stimulated with poly-(I:C), and the release of chemokines and cytokines was measured in the presence of CBD or other phytocannabinoids (such as cannabidiol acid, cannabidivarin, cannabidivarinic acid, cannabichromene, cannabigerol, cannabigerolic acid, cannabigevarin, tetrahydrocannabivarin, and tetrahydrocannabivarinic acid) and antagonists of CB1, CB<em>2</em>, or transient receptor potential vanilloid type-1 (TRPV1) receptors. HaCaT cell viability following phytocannabinoid treatment was also measured. The cellular levels of endocannabinoids [anandamide (AEA), <em>2</em>-<em>arachidonoylglycerol</em>] and related molecules (palmitoylethanolamide, oleoylethanolamide) were quantified in poly-(I:C)-stimulated HaCaT cells treated with CBD. We show that in poly-(I:C)-stimulated HaCaT cells, CBD elevates the levels of AEA and dose-dependently inhibits poly-(I:C)-induced release of MCP-<em>2</em>, interleukin-6 (IL-6), IL-8, and tumor necrosis factor-α in a manner reversed by CB<em>2</em> and TRPV1 antagonists 6-iodopravadoline (AM630) and 5'-iodio-resiniferatoxin (I-RTX), respectively, with no cytotoxic effect. This is the first demonstration of the anti-inflammatory properties of CBD in an experimental model of ACD.

α/β-Hydrolase domain containing 6 (ABHD6) is a transmembrane serine hydrolase that hydrolyzes the endogenous cannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) to regulate certain forms of cannabinoid receptor-dependent signaling in the nervous system. The full spectrum of ABHD6 metabolic activities and functions is currently unknown and would benefit from selective, in vivo-active inhibitors. Here, we report the development and characterization of an advanced series of irreversible (<em>2</em>-substituted)-piperidyl-1,<em>2</em>,3-triazole urea inhibitors of ABHD6, including compounds KT18<em>2</em> and KT<em>2</em>03, which show exceptional potency and selectivity in cells (<5 nM) and, at equivalent doses in mice (1 mg kg(-1)), act as systemic and peripherally restricted ABHD6 inhibitors, respectively. We also describe an orally bioavailable ABHD6 inhibitor, KT185, that displays excellent selectivity against other brain and liver serine hydrolases in vivo. We thus describe several chemical probes for biological studies of ABHD6, including brain-penetrant and peripherally restricted inhibitors that should prove of value for interrogating ABHD6 function in animal models.

Little is known about the expression and function of cannabinoid receptor type 1 (CB1) in the human pituitary gland. The aim of this study was to investigate CB1 expression in human normal and tumoral pituitaries by in situ hybridization and immunohistochemistry using an antibody against CB1. CB1 was found in corticotrophs, mammotrophs, somatotrophs, and folliculostellate cells in the anterior lobe of normal pituitary. After examination of 4<em>2</em> pituitary adenomas, CB1 was detected in acromegaly-associated pituitary adenomas, Cushing's adenomas, and prolactinomas, whereas faint or no expression was found in nonfunctioning pituitary adenomas. Experiments with cultured pituitary adenoma cells showed that the CB1 agonist WIN 55,<em>2</em>1<em>2</em>--<em>2</em> inhibited GH secretion in most of acromegaly-associated pituitary adenomas tested and that the CB1 antagonist SR 141716A was generally able to reverse this effect. Moreover, WIN 55,<em>2</em>1<em>2</em>--<em>2</em> was able to suppress GHRH-stimulated GH release, and this effect was not blocked by coincubation with SR 141716A, possibly indicating a non-CB1-mediated effect. In contrast, WIN 55,<em>2</em>1<em>2</em>--<em>2</em> was ineffective on GH-releasing peptide-stimulated GH release. In four Cushing's adenomas tested, WIN 55,<em>2</em>1<em>2</em>--<em>2</em> was not able to modify basal ACTH secretion. However, simultaneous application of CRF and WIN 55,<em>2</em>1<em>2</em>--<em>2</em> resulted in a synergistic effect on ACTH secretion, and this effect could be abolished by SR 141716A, demonstrating a CB1-mediated effect. In the single case of prolactinomas tested, WIN 55,<em>2</em>1<em>2</em>--<em>2</em> was able to inhibit basal secretion of PRL. Finally, the presence of endocannabinoids (anandamide and <em>2</em>-<em>arachidonoylglycerol</em>) was investigated in normal and tumoral pituitaries. All tumoral samples had higher contents of anandamide and <em>2</em>-<em>arachidonoylglycerol</em> compared with the normal hypophysis. Moreover, endocannabinoid content in the different pituitary adenomas correlated with the presence of CB1, being elevated in the tumoral samples positive for CB1 and lower in the samples in which no or low levels of CB1 were found. The results of this study point to a direct role of cannabinoids in the regulation of human pituitary hormone secretion.

Although it is well established that many glutamatergic neurons sequester Zn(<em>2</em>+) within their synaptic vesicles, the physiological significance of synaptic Zn(<em>2</em>+) remains poorly understood. In experiments performed in a Zn(<em>2</em>+)-enriched auditory brainstem nucleus--the dorsal cochlear nucleus--we discovered that synaptic Zn(<em>2</em>+) and GPR39, a putative metabotropic Zn(<em>2</em>+)-sensing receptor (mZnR), are necessary for triggering the synthesis of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). The postsynaptic production of <em>2</em>-AG, in turn, inhibits presynaptic probability of neurotransmitter release, thus shaping synaptic strength and short-term synaptic plasticity. Zn(<em>2</em>+)-induced inhibition of transmitter release is absent in mutant mice that lack either vesicular Zn(<em>2</em>+) or the mZnR. Moreover, mass spectrometry measurements of <em>2</em>-AG levels reveal that Zn(<em>2</em>+)-mediated initiation of <em>2</em>-AG synthesis is absent in mice lacking the mZnR. We reveal a previously unknown action of synaptic Zn(<em>2</em>+): synaptic Zn(<em>2</em>+) inhibits glutamate release by promoting <em>2</em>-AG synthesis.

OBJECTIVE

Animal studies suggest that endocannabinoids could contribute to the development of nonalcoholic fatty liver disease (NAFLD). In addition, NAFLD has been shown to be associated with multiple changes in lipid concentrations in liver biopsies. There are no data on splanchnic free fatty acid (FFA), glycerol, ketone body, endocannabinoid, and lipid fluxes in vivo in subjects with NAFLD.

METHODS

We performed hepatic venous catheterization studies in combination with [(<em>2</em>)H(<em>2</em>)]palmitate infusion in the fasting state and during a low-dose insulin infusion in 9 subjects with various degrees of hepatic steatosis as determined using liver biopsy. Splanchnic balance of endocannabinoids and individual lipids was determined using ultra performance liquid chromatography coupled to mass spectrometry.

RESULTS

Concentrations of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> were higher in arterialized (91 ± 33 μg/L basally) than in hepatic venous (51 ± 19 μg/L; P < .05) plasma. Fasting arterial (r = 0.7<em>2</em>; P = .031) and hepatic venous (r = 0.70; P = .037) concentrations of <em>2</em>-<em>arachidonoylglycerol</em> were related positively to liver fat content. Analysis of fluxes of 85 different triglycerides showed that the fatty liver overproduces saturated triglycerides. In the plasma FFA fraction in the basal state, the relative amounts of palmitoleate and linoleate were lower and those of stearate and oleate were higher in the hepatic vein than in the artery. Absolute concentrations of all nontriglyceride lipids were comparable in arterialized venous plasma and the hepatic vein both in the basal and insulin-stimulated states.

CONCLUSIONS

The human fatty liver takes up <em>2</em>-<em>arachidonoylglycerol</em> and overproduces triacylglycerols containing saturated fatty acids, which might reflect increased de novo lipogenesis.

Among a variety of phytocannabinoids, Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are the most promising therapeutic compounds. Besides the well-known palliative effects in cancer patients, cannabinoids have been shown to inhibit in vitro growth of tumor cells. Likewise, the major endocannabinoids (eCBs), anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), induce tumor cell death. The purpose of the present study was to characterize cannabinoid elements and evaluate the effect of cannabinoids in endometrial cancer cell viability. The presence of cannabinoid receptors, transient receptor potential vanilloid 1 (TRPV1), and endocannabinoid-metabolizing enzymes were determined by qRT-PCR and Western blot. We also examined the effects and the underlying mechanisms induced by eCBs and phytocannabinoids in endometrial cancer cell viability. Besides TRPV1, both EC cell lines express all the constituents of the endocannabinoid system. We observed that at concentrations higher than 5 μM, eCBs and CBD induced a significant reduction in cell viability in both Ishikawa and Hec50co cells, whereas THC did not cause any effect. In Ishikawa cells, contrary to Hec50co, treatment with AEA and CBD resulted in an increase in the levels of activated caspase -3/-7, in cleaved PARP, and in reactive oxygen species generation, confirming that the reduction in cell viability observed in the MTT assay was caused by the activation of the apoptotic pathway. Finally, these effects were dependent on TRPV1 activation and intracellular calcium levels. These data indicate that cannabinoids modulate endometrial cancer cell death. Selective targeting of TPRV1 by AEA, CBD, or other stable analogues may be an attractive research area for the treatment of estrogen-dependent endometrial carcinoma. Our data further support the evaluation of CBD and CBD-rich extracts for the potential treatment of endometrial cancer, particularly, that has become non-responsive to common therapies.

Previous studies have shown an impairment of the endocannabinoid system in experimental models of Huntington's disease. In transgenic R6/<em>2</em> mice, created by inserting exon 1 of the human IT15 mutant gene into the mouse, and exhibiting 150 CAG repeats as well as signs of HD, a progressive decline of CB(1) receptor expression and an abnormal sensitivity to CB(1) receptor stimulation have been reported. Here, by using isotope-dilution liquid chromatography-mass spectrometry, we investigated whether the levels of three endogenous neuroprotective substances, the endocannabinoids anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), and palmitoylethanolamide (PEA), are altered in different brain areas of transgenic R6/<em>2</em> versus wild-type (WT) mice at two different disease phases, i.e. in pre-symptomatic (4.5 weeks) or overtly symptomatic (10 weeks) R6/<em>2</em> mice versus age-matched WT mice (n=4/group). Except for a approximately <em>2</em>5% decrease in <em>2</em>-AG levels in the cortex, no significant changes in endocannabinoid and PEA levels were observed in pre-symptomatic R6/<em>2</em> versus WT mice. By contrast, in symptomatic R6/<em>2</em> mice the levels of all three compounds were significantly (approximately 30-60%) decreased in the striatum, whereas little changes were observed in the hippocampus, and a approximately <em>2</em>8% decrease of <em>2</em>-AG levels, accompanied by a approximately 50% increase of AEA levels, was found in the cortex. These findings show that endocannabinoid levels change in a disease phase- and region-specific way in the brain of R6/<em>2</em> mice and indicate that an impaired endocannabinoid system is a hallmark of symptomatic HD, thus suggesting that drugs inhibiting endocannabinoid degradation might be used to treat this disease.

<em>2</em>-<em>Arachidonoylglycerol</em> is an arachidonic acid-containing monoacylglycerol isolated from the rat brain and canine gut as an endogenous ligand for the cannabinoid receptors (CB1 and CB<em>2</em>). <em>2</em>-<em>Arachidonoylglycerol</em> binds to both the CB1 receptor, abundantly expressed in the nervous system, and the CB<em>2</em> receptor, mainly expressed in the immune system, with high affinity, and exhibits a variety of cannabimimetic activities. Notably, anandamide, another endogenous ligand for the cannabinoid receptors, acts as a partial agonist at these cannabinoid receptors, whereas <em>2</em>-<em>arachidonoylglycerol</em> acts as a full agonist. The results of structure-activity relationship experiments strongly suggested that <em>2</em>-<em>arachidonoylglycerol</em> rather than anandamide is the true natural ligand for both the CB1 and the CB<em>2</em> receptors. Evidence is gradually accumulating which shows that <em>2</em>-<em>arachidonoylglycerol</em> plays physiologically and pathophysiologically essential roles in various mammalian tissues and cells.

The endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is a retrograde lipid messenger that modulates synaptic function, neurophysiology, and behavior. <em>2</em>-AG signaling is terminated by enzymatic hydrolysis-a reaction that is principally performed by monoacylglycerol lipase (MAGL). MAGL is broadly expressed throughout the nervous system, and the contributions of different brain cell types to the regulation of <em>2</em>-AG activity in vivo remain poorly understood. Here, we genetically dissect the cellular anatomy of MAGL-mediated <em>2</em>-AG metabolism in the brain and show that neurons and astrocytes coordinately regulate <em>2</em>-AG content and endocannabinoid-dependent forms of synaptic plasticity and behavior. We also find that astrocytic MAGL is mainly responsible for converting <em>2</em>-AG to neuroinflammatory prostaglandins via a mechanism that may involve transcellular shuttling of lipid substrates. Astrocytic-neuronal interplay thus provides distributed oversight of <em>2</em>-AG metabolism and function and, through doing so, protects the nervous system from excessive CB1 receptor activation and promotes endocannabinoid crosstalk with other lipid transmitter systems.

It is well known that certain active ingredients of the plants of Cannabis genus, i.e., the "phytocannabinoids" [pCBs; e.g., (-)-trans-Δ9-tetrahydrocannabinol (THC), (-)-cannabidiol, etc.] can influence a wide array of biological processes, and the human body is able to produce endogenous analogs of these substances ["endocannabinoids" (eCB), e.g., arachidonoylethanolamine (anandamide, AEA), <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), etc.]. These ligands, together with multiple receptors (e.g., CB1 and CB<em>2</em> cannabinoid receptors, etc.), and a complex enzyme and transporter apparatus involved in the synthesis and degradation of the ligands constitute the endocannabinoid system (ECS), a recently emerging regulator of several physiological processes. The ECS is widely expressed in the human body, including several members of the innate and adaptive immune system, where eCBs, as well as several pCBs were shown to deeply influence immune functions thereby regulating inflammation, autoimmunity, antitumor, as well as antipathogen immune responses, etc. Based on this knowledge, many in vitro and in vivo studies aimed at exploiting the putative therapeutic potential of cannabinoid signaling in inflammation-accompanied diseases (e.g., multiple sclerosis) or in organ transplantation, and to dissect the complex immunological effects of medical and "recreational" marijuana consumption. Thus, the objective of the current article is (i) to summarize the most recent findings of the field; (ii) to highlight the putative therapeutic potential of targeting cannabinoid signaling; (iii) to identify open questions and key challenges; and (iv) to suggest promising future directions for cannabinoid-based drug development.

Cyclooxygenase-<em>2</em> (COX-<em>2</em>) mediates inflammation and contributes to neurodegeneration. Best known for its pathological up-regulation, COX-<em>2</em> is also constitutively expressed within the brain and mediates synaptic transmission through prostaglandin synthesis. Along with arachidonic acid, COX-<em>2</em> oxygenates the endocannabinoids anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> in vitro. Inhibition of COX-<em>2</em> enhances retrograde signaling in the hippocampus, suggesting COX-<em>2</em> mediates endocannabinoid tone in healthy brain. The degree to which COX-<em>2</em> may regulate endocannabinoid metabolism in vivo is currently unclear. Therefore, we explored the effect of COX-<em>2</em> inhibition on [(3)H]AEA metabolism in mouse brain. Although AEA is hydrolyzed primarily by fatty acid amide hydrolase (FAAH), ex vivo autoradiography revealed that COX-<em>2</em> inhibition by nimesulide redirected [(3)H]AEA substrate from COX-<em>2</em> to FAAH in the cortex, hippocampus, thalamus, and periaqueductal gray. These data indicate that COX-<em>2</em> possesses the capacity to metabolize AEA in vivo and can compete with FAAH for AEA in several brain regions. Temporal fluctuations in COX-<em>2</em> expression were observed in the brain, with an increase in COX-<em>2</em> protein and mRNA in the hippocampus at midnight compared with noon. COX-<em>2</em> immunolocalization was robust in the hippocampus and several cortical regions. Although most regions exhibited no temporal changes in COX-<em>2</em> immunolocalization, increased numbers of immunoreactive cells were detected at midnight in layers II and III of the somatosensory and visual cortices. These temporal variations in COX-<em>2</em> distribution reduced the enzyme's contribution toward [(3)H]AEA metabolism in the somatosensory cortex at midnight. Taken together, our findings establish COX-<em>2</em> as a mediator of regional AEA metabolism in mouse brain.

Recent work in our laboratories has demonstrated that an opioid-independent form of stress-induced analgesia (SIA) is mediated by endogenous ligands for cannabinoid receptors-anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) [A.G. Hohmann, R.L. Suplita, N.M. Bolton, M.H. Neely, D. Fegley, R. Mangieri, J.F. Krey, J.M. Walker, P.V. Holmes, J.D. Crystal, A. Duranti, A. Tontini, M. Mor, G. Tarzia, D. Piomelli, An endocannabinoid mechanism for stress-induced analgesia, Nature 435 (<em>2</em>005) 1108-111<em>2</em>]. The present study was conducted to examine the contribution of cannabinoid CB1 receptors in the basolateral nucleus of the amygdala (BLA) and central nucleus of the amygdala (CeA) to nonopioid SIA. SIA was induced by continuous footshock (3 min 0.9 mA) and quantified behaviorally using the tail-flick test. Microinjection of the CB1 antagonist/inverse agonist rimonabant (SR141716A) into the BLA, a limbic forebrain region with high densities of CB1 receptors, suppressed SIA relative to control conditions. By contrast, the same dose administered into the CeA, where CB1 immunoreactivity is largely absent, or outside the amygdala did not alter SIA. To examine the contribution of endocannabinoids in the BLA to SIA, we used selective pharmacological inhibitors of the anandamide-degrading enzyme fatty-acid amide hydrolase (FAAH) and the <em>2</em>-<em>arachidonoylglycerol</em>-degrading enzyme monoacylglycerol lipase (MGL). The FAAH inhibitor URB597 and MGL inhibitor URB60<em>2</em>, at doses that enhanced SIA following microinjection in the midbrain periaqueductal gray, did not alter SIA relative to control conditions. Our findings suggest that CB1 receptors in the BLA but not the CeA contribute to SIA, but pharmacological inhibition of endocannabinoid degradation at these sites does not affect the expression of stress antinociception.