Very little is known about the processes regulating the cellular uptake of the endogenous cannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). In the present study, we investigated whether inhibition of <em>2</em>-AG hydrolysis reduced its uptake, i.e. whether this compound behaves in a manner analogous to the related endocannabinoid anandamide. The selective fatty acid amide hydrolase inhibitor URB597 (3'-(aminocarbamoyl)[1,1'-biphenyl]-3-yl)-cyclohexylcarbamate) completely blocked the hydrolysis of anandamide and reduced its uptake by about half in RBL<em>2</em>H3 basophilic leukaemia cells. In contrast, in these cells, in PC3 and R33<em>2</em>7AT-1 prostate cancer cells and in Neuro-<em>2</em>a neuroblastoma cells, the compound had more modest effects upon the hydrolysis of <em>2</em>-AG and did not affect its cellular uptake at all, indicating that in these cells fatty acid amide hydrolase does not regulate the uptake of <em>2</em>-AG. The serine hydrolase inhibitor methylarachidonoyl fluoronophosphonate behaved like URB597 with respect to anandamide uptake by RBL<em>2</em>H3 and Neuro-<em>2</em>a cells, and inhibited the hydrolysis of <em>2</em>-AG with IC50 values of 0.014, 0.05<em>2</em>, 0.41 and approximately 1 microM for RBL<em>2</em>H3, PC3, AT-1 and Neuro-<em>2</em>a cells, respectively. MAFP (1 microM) did not significantly reduce the uptake of <em>2</em>-AG by RBL<em>2</em>H3, PC3 and AT-1 cells but did reduce the uptake of this endocannabinoid by Neuro-<em>2</em>a cells. Arachidonoyl trifluoromethyl ketone and URB60<em>2</em> ([1,1'-biphenyl]-3-yl-carbamic acid, cyclohexyl ester) reduced the uptake of <em>2</em>-AG by both RBL<em>2</em>H3 and Neuro-<em>2</em>a cells, but at the high concentrations needed, the compound also blocked the retention of these ligands by wells. It is concluded that unlike the situation for anandamide, hydrolysis of <em>2</em>-AG does not regulate its cellular uptake in RBL<em>2</em>H3, AT-1 and PC3 cells, but may gate the uptake in Neuro-<em>2</em>a cells.

Arrestin translocation and signaling have come to the fore of the G protein-coupled receptor molecular pharmacology field. Some receptor-arrestin interactions are relatively well understood and considered responsible for specific therapeutic or adverse outcomes. Coupling of arrestins with cannabinoid receptors 1 (CB₁) and <em>2</em> (CB_<em>2</em>) has been reported, though the majority of studies have not systematically characterized the differential ligand dependence of this activity. In addition, many prior studies have utilized bovine (rather than human) arrestins, and the most widely applied assays require reporter-tagged receptors, which prevent meaningful comparison between receptor types. We have employed a bioluminescence resonance energy transfer (BRET) method that does not require the use of tagged receptors and thereby allows comparisons of arrestin translocation between receptor types, as well as with cells lacking the receptor of interest - an important control. The ability of a selection of CB₁ and CB_<em>2</em> agonists to stimulate cell surface translocation of human and bovine β-arrestin-1 and -<em>2</em> was assessed. We find that some CB₁ ligands induce moderate β-arrestin-<em>2</em> translocation in comparison with vasopressin V_<em>2</em> receptor (a robust arrestin recruiter); however, CB₁ coupling with β-arrestin-1 and CB_<em>2</em> with either arrestin elicited low relative efficacies. A range of efficacies between ligands was evident for both receptors and arrestins. Endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> stood out as a high efficacy ligand for translocation of β-arrestin-<em>2</em> via CB₁. Δ⁹-tetrahydrocannabinol was generally unable to elicit translocation of either arrestin subtype via CB₁ or CB_<em>2</em>; however, control experiments revealed translocation in cells not expressing CB₁/CB_<em>2</em>, which may assist in explaining some discrepancy with the literature. Overexpression of GRK<em>2</em> had modest influence on CB₁/CB_<em>2</em>-induced arrestin translocation. Results with bovine and human arrestins were largely analogous, but a few instances of inconsistent rank order potencies/efficacies between bovine and human arrestins raise the possibility that subtle differences in receptor conformation stabilized by these ligands manifest in disparate affinities for the two arrestin species, with important potential consequences for interpretation in ligand bias studies. As well as contributing important information regarding CB₁/CB_<em>2</em> ligand-dependent arrestin coupling, our study raises a number of points for consideration in the design and interpretation of arrestin recruitment assays.

In this study, we investigated the role of the endocannabinoid system (ECS) in the emotional and cognitive alterations associated with osteoarthritis pain. The monosodium iodoacetate model was used to evaluate the affective and cognitive manifestations of osteoarthritis pain in type 1 (CB1R) and type <em>2</em> (CB<em>2</em>R) cannabinoid receptor knockout and wild-type mice and the ability of CB1R (ACEA) and CB<em>2</em>R (JWH133) selective agonists to improve these manifestations during a 3-week time period. The levels of the endocannabinoids anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) were measured in plasma and brain areas involved in the control of these manifestations. Patients with knee osteoarthritis and healthy controls were recruited to evaluate pain, affective, and cognitive symptoms, as well as plasma endocannabinoid levels and cannabinoid receptor gene expression in peripheral blood lymphocytes. The affective manifestations of osteoarthritis were enhanced in CB1R knockout mice and absent in CB<em>2</em>R knockouts. Interestingly, both ACEA and JWH133 ameliorated the nociceptive and affective alterations, whereas ACEA also improved the associated memory impairment. An increase of <em>2</em>-AG levels in prefrontal cortex and plasma was observed in this mouse model of osteoarthritis. In agreement, an increase of <em>2</em>-AG plasmatic levels and an upregulation of CB1R and CB<em>2</em>R gene expression in peripheral blood lymphocytes were observed in patients with osteoarthritis compared with healthy subjects. Changes found in these biomarkers of the ECS correlated with pain, affective, and cognitive symptoms in these patients. The ECS plays a crucial role in osteoarthritis and represents an interesting pharmacological target and biomarker of this disease.

Medicine continues to struggle in its approaches to numerous common subjective pain syndromes that lack objective signs and remain treatment resistant. Foremost among these are migraine, fibromyalgia, and irritable bowel syndrome, disorders that may overlap in their affected populations and whose sufferers have all endured the stigma of a psychosomatic label, as well as the failure of endless pharmacotherapeutic interventions with substandard benefit. The commonality in symptomatology in these conditions displaying hyperalgesia and central sensitization with possible common underlying pathophysiology suggests that a clinical endocannabinoid deficiency might characterize their origin. Its base hypothesis is that all humans have an underlying endocannabinoid tone that is a reflection of levels of the endocannabinoids, anandamide (arachidonylethanolamide), and <em>2</em>-<em>arachidonoylglycerol</em>, their production, metabolism, and the relative abundance and state of cannabinoid receptors. Its theory is that in certain conditions, whether congenital or acquired, endocannabinoid tone becomes deficient and productive of pathophysiological syndromes. When first proposed in <em>2</em>001 and subsequently, this theory was based on genetic overlap and comorbidity, patterns of symptomatology that could be mediated by the endocannabinoid system (ECS), and the fact that exogenous cannabinoid treatment frequently provided symptomatic benefit. However, objective proof and formal clinical trial data were lacking. Currently, however, statistically significant differences in cerebrospinal fluid anandamide levels have been documented in migraineurs, and advanced imaging studies have demonstrated ECS hypofunction in post-traumatic stress disorder. Additional studies have provided a firmer foundation for the theory, while clinical data have also produced evidence for decreased pain, improved sleep, and other benefits to cannabinoid treatment and adjunctive lifestyle approaches affecting the ECS.

We have previously shown that 1,<em>2</em>,3-triazole ureas (1,<em>2</em>,3-TUs) act as versatile class of irreversible serine hydrolase inhibitors that can be tuned to create selective probes for diverse members of this large enzyme class, including diacylglycerol lipase-β (DAGLβ), a principal biosynthetic enzyme for the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Here, we provide a detailed account of the discovery, synthesis, and structure-activity relationship (SAR) of (<em>2</em>-substituted)-piperidyl-1,<em>2</em>,3-TUs that selectively inactivate DAGLβ in living systems. Key to success was the use of activity-based protein profiling (ABPP) with broad-spectrum and tailored activity-based probes to guide our medicinal chemistry efforts. We also describe an expanded repertoire of DAGL-tailored activity-based probes that includes biotinylated and alkyne agents for enzyme enrichment coupled with mass spectrometry-based proteomics and assessment of proteome-wide selectivity. Our findings highlight the broad utility of 1,<em>2</em>,3-TUs for serine hydrolase inhibitor development and their application to create selective probes of endocannabinoid biosynthetic pathways.

In the not too distant future, humankind will embark on one of its greatest adventures, the travel to distant planets. However, deep space travel is associated with an inevitable exposure to radiation fields. Space-relevant doses of protons elicit persistent disruptions in cognition and neuronal structure. However, whether space-relevant irradiation alters neurotransmission is unknown. Within the hippocampus, a brain region crucial for cognition, perisomatic inhibitory control of pyramidal cells (PCs) is supplied by two distinct cell types, the cannabinoid type 1 receptor (CB1)-expressing basket cells (CB1BCs) and parvalbumin (PV)-expressing interneurons (PVINs). Mice subjected to low-dose proton irradiation were analyzed using electrophysiological, biochemical and imaging techniques months after exposure. In irradiated mice, GABA release from CB1BCs onto PCs was dramatically increased. This effect was abolished by CB1 blockade, indicating that irradiation decreased CB1-dependent tonic inhibition of GABA release. These alterations in GABA release were accompanied by decreased levels of the major CB1 ligand <em>2</em>-<em>arachidonoylglycerol</em>. In contrast, GABA release from PVINs was unchanged, and the excitatory connectivity from PCs to the interneurons also underwent cell type-specific alterations. These results demonstrate that energetic charged particles at space-relevant low doses elicit surprisingly selective long-term plasticity of synaptic microcircuits in the hippocampus. The magnitude and persistent nature of these alterations in synaptic function are consistent with the observed perturbations in cognitive performance after irradiation, while the high specificity of these changes indicates that it may be possible to develop targeted therapeutic interventions to decrease the risk of adverse events during interplanetary travel.

Knee osteoarthritis (KOA) is a highly prevalent, chronic joint disorder, which can lead to chronic pain. Although electroacupuncture (EA) is effective in relieving chronic pain in the clinic, the involved mechanisms remain unclear. Reduced diffuse noxius inhibitory controls (DNIC) function is associated with chronic pain and may be related to the action of endocannabinoids. In the present study, we determined whether EA may potentiate cannabinoid receptor-mediated descending inhibitory control and inhibit chronic pain in a mouse model of KOA. We found that the optimized parameters of EA inhibiting chronic pain were the low frequency and high intensity (<em>2</em> Hz + 1 mA). EA reversed the reduced expression of CB1 receptors and the <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) level in the midbrain in chronic pain. Microinjection of the CB1 receptor antagonist AM<em>2</em>51 into the ventrolateral periaqueductal gray (vlPAG) can reversed the EA effect on pain hypersensitivity and DNIC function. In addition, CB1 receptors on GABAergic but not glutamatergic neurons are involved in the EA effect on DNIC function and descending inhibitory control of 5-HT in the medulla, thus inhibiting chronic pain. Our data suggest that endocannabinoid (<em>2</em>-AG)-CB1R-GABA-5-HT may be a novel signaling pathway involved in the effect of EA improving DNIC function and inhibiting chronic pain.

To evaluate the association between circulating levels of endocannabinoids (eCBs) and non-alcoholic fatty liver disease (NAFLD).

The serum levels of the main eCBs, anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), and their endogenous precursor and breakdown product, arachidonic acid (AA), were analyzed by liquid chromatography/tandem mass spectrometry in 105 volunteers screened for NAFLD. Hepatic ultrasound, fasting blood tests, and anthropometrics were assessed. Liver fat was quantified by the hepato-renal-ultrasound index representing the ratio between the brightness level of the liver and the kidney.

Patients with NAFLD had higher levels (pmol/mL) of AA (<em>2</em>,7<em>2</em>1 ± 1,11<em>2</em> vs. <em>2</em>,<em>2</em>48 ± 977, P = 0.0<em>2</em><em>2</em>) and <em>2</em>-AG (46.5 ± <em>2</em>5.8 vs. 33.5 ± 13.6, P = 0.003), but not AEA. The trend for higher levels of AA and <em>2</em>-AG in the presence of NAFLD was observed in both genders and within subgroups of overweight and obesity. The association of AA and <em>2</em>-AG with NAFLD was maintained with adjustment for age, gender, and BMI (OR = 1.001, 1.000-1.001 95% CI, P = 0.008 and OR = 1.05, 1.01-1.09, P = 0.006, respectively) or waist circumference.

This study is the first to show high circulating levels of <em>2</em>-AG and AA in NAFLD patients compared with controls, independent of obesity. The findings may suggest an independent role of eCBs in the pathogenesis of NAFLD.

The presence of the elements of the endocannabinoid system (ECS) in sperm isolated from several species (from invertebrates to mammals, humans included) has supported the "evolutionary theory" that proposes endocannabinoids as check points in reproductive events like capacitation. In this study, we characterized the ECS elements at the mRNA, protein and functional levels in mouse sperm before and after capacitation. We found that the latter process increases the endogenous levels of the two major endocannabinoids (anandamide and <em>2</em>-<em>arachidonoylglycerol</em>), through a decreased degradation and increased biosynthesis, respectively. Additionally, we found that the binding activity of cannabinoid receptors was not affected by sperm capacitation, whereas that of vanilloid receptor was reduced. Overall, our data demonstrate that mouse sperm have a fully functional ECS, and that capacitation alters the endogenous tone of the major endocannabinoids through distinct mechanisms.

<AbstractText>We have previously reported that endocannabinoids modulate permeability in Caco-<em>2</em> cells under inflammatory conditions and hypothesised in the present study that endocannabinoids could also modulate permeability in ischemia/reperfusion.</AbstractText><p><div><b>EXPERIMENTAL APPROACH</b></div>Caco-<em>2</em> cells were grown on cell culture inserts to confluence. Trans-epithelial electrical resistance (TEER) was used to measure permeability. To generate hypoxia (0% O_<em>2</em>), a GasPak™ EZ anaerobe pouch system was used. Endocannabinoids were applied to the apical or basolateral membrane in the presence or absence of receptor antagonists.</p><p><div><b>KEY RESULTS</b></div>Complete hypoxia decreased TEER (increased permeability) by ~35% after 4 h (recoverable) and ~50% after 6 h (non-recoverable). When applied either pre- or post-hypoxia, apical application of N-arachidonoyl-dopamine (NADA, via TRPV1), oleamide (OA, via TRPV1) and oleoylethanolamine (OEA, via TRPV1) inhibited the increase in permeability. Apical administration of anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) worsened the permeability effect of hypoxia (both via CB₁). Basolateral application of NADA (via TRPV1), OA (via CB₁ and TRPV1), noladin ether (NE, via PPARα), and palmitoylethanolamine (PEA, via PPARα) restored permeability after 4 h hypoxia, whereas OEA increased permeability (via PPARα). After 6 h hypoxia, where permeability does not recover, only basolateral application PEA sustainably decreased permeability, and NE decreased permeability.</p><p><div><b>CONCLUSIONS AND IMPLICATIONS</b></div>A variety of endocannabinoids and endocannabinoid-like compounds modulate Caco-<em>2</em> permeability in hypoxia/reoxygenation, which involves multiple targets, depending on whether the compounds are applied to the basolateral or apical membrane. CB₁ antagonism and TRPV1 or PPARα agonism may represent novel therapeutic targets against several intestinal disorders associated with increased permeability.</p>

Dopamine and the endocannabinoids, anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, interact at several levels in the brain, with the involvement of both cannabinoid CB(1) receptors and transient receptor potential vanilloid type-1 (TRPV1) channels (which are alternative anandamide receptors). Using pharmacological, immunohistochemical and analytical approaches, we investigated the response of dopamine D(3) receptor null (D3R((-/-))) mice in models of epilepsy and anxiety, in relation to their brain endocannabinoid and endovanilloid tone. Compared to wild-type mice, D3R((-/-)) mice exhibited a delayed onset of clonic seizures, enhanced survival time, reduced mortality rate and more sensitivity to anticonvulsant effects of diazepam after intraperitoneal administration of picrotoxin (7 mg/kg), and a less anxious-like behaviour in the elevated plus maze test. D3R((-/-)) mice also exhibited different endocannabinoid and TRPV1, but not CB(1), levels in the hippocampus, nucleus accumbens, amygdala and striatum. Given the role played by CB(1) and TRPV1 in neuroprotection and anxiety, and based on data obtained here with pharmacological tools, we suggest that the alterations of endocannabinoid and endovanilloid tone found in D3R((-/-)) mice might account for part of their altered responses to excitotoxic and anxiogenic stimuli.

Endocannabinoids, including <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), activate presynaptic cannabinoid type 1 receptors (CB1R) on inhibitory and excitatory neurons, resulting in a decreased release of neurotransmitters. The event-specific activation of the endocannabinoid system by inhibition of the endocannabinoid degrading enzymes may offer a promising strategy to selectively activate CB1Rs at the site of excessive neuronal activation with the overall goal to prevent the development epilepsy. The aim of this study was to investigate the impact of monoacylglycerol lipase (MAGL) inhibition on the development and progression of epileptic seizures in the kindling model of temporal lobe epilepsy. Therefore, we selectively blocked MAGL by JZL184 (8mg/kg, i.p.) in mice to analyze the effects of increased <em>2</em>-AG levels on kindling acquisition and to exclude an anticonvulsive potential. Our results showed that JZL184 treatment significantly delayed the development of generalized seizures (p=0.0066) and decreased seizure (p<0.0001) and afterdischarge duration (p<0.001) in the kindling model of temporal lobe epilepsy, but caused only modest effects in fully kindled mice. Moreover, we proved that JZL184 treatment had no effects in conditional CB1R knockout mice lacking expression of the receptor in principle neurons of the forebrain. In conclusion, the data demonstrate that indirect CB1R agonism delays the development of generalized epileptic seizures but has no relevant acute anticonvulsive effects. Furthermore, we confirmed that the effects of JZL184 on kindling progression are CB1R mediated. Thus, the data indicate that the endocannabinoid <em>2</em>-AG might be a promising target for an anti-epileptogenic approach.

Impairments in fear extinction are thought to be central to the psychopathology of posttraumatic stress disorder, and endocannabinoid (eCB) signaling has been strongly implicated in extinction learning. Here we utilized the monoacylglycerol lipase inhibitor JZL184 to selectively augment brain <em>2</em>-AG levels combined with an auditory cue fear-conditioning paradigm to test the hypothesis that <em>2</em>-AG-mediated eCB signaling modulates short-term fear extinction learning in mice. We show that systemic JZL184 impairs short-term extinction learning in a CB1 receptor-dependent manner without affecting non-specific freezing behavior or the acquisition of conditioned fear. This effect was also observed in over-conditioned mice environmentally manipulated to re-acquire fear extinction. Cumulatively, the effects of JZL184 appear to be partly due to augmentation of <em>2</em>-AG signaling in the basolateral nucleus of the amygdala (BLA), as direct microinfusion of JZL184 into the BLA produced similar results. Moreover, we elucidate a short ~3-day temporal window during which <em>2</em>-AG augmentation impairs extinction behavior, suggesting a preferential role for <em>2</em>-AG-mediated eCB signaling in the modulation of short-term behavioral sequelae to acute traumatic stress exposure.

Accumulation of activated eosinophils in tissue is a hallmark of allergic inflammation. The endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) has been proposed to elicit eosinophil migration in a CB<em>2</em> receptor/Gi/o -dependent manner. However, it has been claimed recently that this process may also involve other mechanisms such as cytokine priming and the metabolism of <em>2</em>-AG into eicosanoids. Here, we explored the direct contribution of specific CB<em>2</em> receptor activation to human and mouse eosinophil effector function in vitro and in vivo.

In vitro studies including CB<em>2</em> expression, adhesion and migratory responsiveness, respiratory burst, degranulation, and calcium mobilization were conducted in human peripheral blood eosinophils and mouse bone marrow-derived eosinophils. Allergic airway inflammation was assessed in mouse models of acute OVA-induced asthma and directed eosinophil migration.

CB<em>2</em> expression was significantly higher in eosinophils from symptomatic allergic donors. The selective CB<em>2</em> receptor agonist JWH-133 induced a moderate migratory response in eosinophils. However, short-term exposure to JWH-133 potently enhanced chemoattractant-induced eosinophil shape change, chemotaxis, CD11b surface expression, and adhesion as well as production of reactive oxygen species. Receptor specificity of the observed effects was confirmed in eosinophils from CB<em>2</em> knockout mice and by using the selective CB<em>2</em> antagonist SR1445<em>2</em>8. Of note, systemic application of JWH-133 clearly primed eosinophil-directed migration in vivo and aggravated both AHR and eosinophil influx into the airways in a CB<em>2</em> -specific manner. This effect was completely absent in eosinophil-deficient ∆dblGATA mice.

Our data indicate that CB<em>2</em> may directly contribute to the pathogenesis of eosinophil-driven diseases. Moreover, we provide new insights into the molecular mechanisms underlying the CB<em>2</em> -mediated priming of eosinophils. Hence, antagonism of CB<em>2</em> receptors may represent a novel pharmacological approach for the treatment of allergic inflammation and other eosinophilic disorders.

Elements of the endocannabinoid system (ECS) are expressed by islet endocrine cells and activation of CB1 and CB<em>2</em> cannabinoid receptors regulates insulin secretion from mouse and human β-cells. The current study aimed to investigate the expression and function, in mouse and human β-cells, of monoacylglycerol lipase (MGL), an enzyme that facilitates degradation of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). We found that MGL mRNA is expressed by MIN6 β-cells, mouse islets, human islets and enriched human islet β-cells, and immunohistochemistry indicated that MGL localisation in human islets is consistent with its expression by some β- and -α-cells. Blockade of MGL activity with the pharmacological inhibitor URB60<em>2</em> led to increased [Ca(<em>2</em>+)](i )and enhanced insulin secretion from MIN6 β-cells, and MGL inhibition also elevated insulin and glucagon secretion from isolated human islets in vitro. These data imply a stimulatory role for endogenous <em>2</em>-AG in islets that is amplified when its degradation is blocked.

OBJECTIVE

Chronic liver disease is associated with endotoxemia, oxidative stress, increased endocannabinoids and decreased cardiac responsiveness. Endocannabinoids activate the tumor necrosis factor-alpha (TNFalpha)-nuclear factor kappaB (NFkappaB) pathway. However, how they interact with each other remains obscure. We therefore aimed to clarify the relationship between the TNFalpha-NFkappaB pathway and endocannabinoids in the pathogenesis of cardiodepression of cholestatic bile duct ligated (BDL) mice.

METHODS

BDL mice with TNFalpha knockout (TNFalpha-/-) and infusion of anti-TNFalpha antibody were used. Cardiac mRNA and protein expression of NFkappaBp65, c-Jun-N-terminal kinases (JNK), p38 mitogen-activated protein kinase (p38MAPK), extracelullar-signal- regulated kinase (ERK), inducible nitric oxide synthase (iNOS), Copper/Zinc and Magnesium-superoxide dismutase (Cu/ Zn- and Mn-SOD), cardiac anandamide, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), nitric oxide (NOx) and glutathione, and plasma TNFalpha were measured. The effects of TNFalpha, cannabinoid receptor (CB1) antagonist AM<em>2</em>51 and the endocannabinoid reuptake inhibitor UCM707, on the contractility of isolated cardiomyocytes, were assessed.

RESULTS

In BDL mice, cardiac mRNA and protein expression of NFkappaBp65, p38MAPK, iNOS, NOx, anandamide, and plasma TNFa were increased, whereas glutathione, Cu/Zn-SOD, and Mn-SOD were decreased. Cardiac contractility was blunted in BDL mice. Anti-TNFa treatment in BDL mice decreased cardiac anandamide and NOx, reduced expression of NFkappaBp65, p38MAPK, and iNOS, enhanced expression of Cu/Zn-SOD and Mn-SOD, increased reductive glutathione and restored cardiomyocyte contractility. TNFa-depressed contractility was worsened by UCM707, whereas AM<em>2</em>51 improved contractility.

CONCLUSIONS

Increased TNFalpha, acting via NFkappaB-iNOS and p38MAPK signaling pathways, plays an important role in the pathogenesis of cardiodepression in BDL mice. TNFalpha also suppressed contractility by increasing oxidative stress and endocannabinoid activity.

The endocannabinoid system comprises amides, esters and ethers of long chain polyunsaturated fatty acids. Narachidonoylethanolamide (anandamide; ANA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are endogenous cannabinoids (endocannabinoids) ligands for the cannabinoid family of G-protein-coupled receptors named CB1 and CB<em>2</em>. Endocannabinoids are released upon demand from lipid precursors in a receptor-dependent manner and behave as retrograde signaling messengers, as well as modulators of postsynaptic transmission, interacting with other neurotransmitters systems. The two principal enzymes that are responsible for the metabolism of ANA and <em>2</em>-AG are fatty acid amide hydrolase and monoacylglycerol lipase, respectively. Pharmacological experiments have shown that the administration of endocannabinoids induce cannabimimetic effects, including sleep promotion. This review will focus on some of the current evidence of the pharmacological potential of the endocannabinoid system on sleep modulation.

Episodic memory, a fundamental component of human cognition, is significantly impaired in autism. We believe we report the first evidence for this problem in the Fmr1-knockout (KO) mouse model of Fragile X syndrome and describe potentially treatable underlying causes. The hippocampus is critical for the formation and use of episodes, with semantic (cue identity) information relayed to the structure via the lateral perforant path (LPP). The unusual form of synaptic plasticity expressed by the LPP (lppLTP) was profoundly impaired in Fmr1-KOs relative to wild-type mice. Two factors contributed to this defect: (i) reduced GluN1 subunit levels in synaptic NMDA receptors and related currents, and (ii) impaired retrograde synaptic signaling by the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Studies using a novel serial cue paradigm showed that episodic encoding is dependent on both the LPP and the endocannabinoid receptor CB1, and is strikingly impaired in Fmr1-KOs. Enhancing <em>2</em>-AG signaling rescued both lppLTP and learning in the mutants. Thus, two consequences of the Fragile-X mutation converge on plasticity at one site in hippocampus to prevent encoding of a basic element of cognitive memory. Collectively, the results suggest a clinically plausible approach to treatment.

The aim of the present study is to examine the relationship between preference for HFD and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), endogenous cannabinoid. The 3-day HFD intake induced preference for HFD, which was suppressed by CB1 antagonist, O-<em>2</em>050. Moreover, hypothalamic <em>2</em>-AG was increased after 3-day HFD intake. Our results show that preference for HFD is induced by activation of CB1 receptors via an increment of <em>2</em>-AG in hypothalamus.

Nuclear factor (NF)-κB is the key transcription factor involved in the inflammatory responses, and its activation aggravates tumors. Peptidoglycan (PGN), a main cell wall component of Gram-positive bacteria, stimulates Toll-like receptor <em>2</em> (TLR-<em>2</em>) and activates a number of inflammatory pathways, including NF-κB. Cannabinoids have been reported to exert anti-inflammatory and antitumor effects. The mechanisms underlying these actions, however, are largely unknown. The purpose of this study was to investigate whether cannabinoids can suppress the PGN-induced activation of NF-κB and cell growth via cannabinoid receptors in U87MG human malignant glioma cells. PGN treatment induced the phosphorylation of NF-κB and cell proliferation in a concentration-dependent manner. The main endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em>, prevented the PGN-induced phosphorylation of NF-κB, which was reversed by the CB1 cannabinoid receptor antagonist, AM<em>2</em>81. The synthetic cannabinoid, WIN55,<em>2</em>1<em>2</em>-<em>2</em>, abolished the PGN-activated cell growth, and this effect was reversed by AM<em>2</em>81. The preferential expression of CB1 rather than CB<em>2</em> receptors in these cells was confirmed by reverse transcription-mediated polymerase chain reaction experiments and the observation that the WIN55,<em>2</em>1<em>2</em>-<em>2</em>-induced morphological changes were completely reversed by AM<em>2</em>81 but not by the CB<em>2</em> antagonist, AM630. Our finding that cannabinoids suppress the NF-κB inflammatory pathway and cell growth via CB1 receptors in glioma cells provides evidence for the therapeutic potential of targeting cannabinoid receptors for the treatment of inflammation-dependent tumor progression.

Anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are the two main endocannabinoids, exerting their effects by activating type 1 (CB1r) and type <em>2</em> (CB<em>2</em>r) cannabinoid receptors. Anandamide inhibits anxiety-like responses through the activation of CB1r in certain brain regions, including the dorsolateral periaqueductal gray (dlPAG). <em>2</em>-AG also attenuates anxiety-like responses, although the neuroanatomical sites for these effects remained unclear. Here, we tested the hypothesis that enhancing <em>2</em>-AG signaling in the dlPAG would induce anxiolytic-like effects. The mechanisms involved were also investigated. Male Wistar rats received intra-dlPAG injections of <em>2</em>-AG, URB60<em>2</em> (inhibitor of the <em>2</em>-AG hydrolyzing enzyme, mono-acylglycerol lipase--MGL), AM<em>2</em>51 (CB1r antagonist) and AM630 (CB<em>2</em>r antagonist). The behavior was analyzed in the elevated plus maze after the following treatments. Exp. 1: vehicle (veh) or <em>2</em>-AG (5 pmol, 50 pmol, and 500 pmol). Exp. <em>2</em>: veh or URB60<em>2</em> (30 pmol, 100 pmol or 300 pmol). Exp. 3: veh or AM<em>2</em>51 (100 pmol) followed by veh or <em>2</em>-AG (50 pmol). Exp. 4: veh or AM630 (1000 pmol) followed by veh or <em>2</em>-AG. Exp. 5: veh or AM<em>2</em>51 followed by veh or URB60<em>2</em> (100 pmol). Exp. 6: veh or AM630 followed by veh or URB60<em>2</em>. <em>2</em>-AG (50 pmol) and URB60<em>2</em> (100 pmol) significantly increased the exploration of the open arms of the apparatus, indicating an anxiolytic-like effect. These behavioral responses were prevented by CB1r (AM<em>2</em>51) or CB<em>2</em>r (AM630) antagonists. Our results showed that the augmentation of <em>2</em>-AG levels in the dlPAG induces anxiolytic-like effects. The mechanism seems to involve both CB1r and CB<em>2</em>r receptors.

Cannabinoid-based interventions are being explored for central nervous system (CNS) pathologies such as neurodegeneration, demyelination, epilepsy, stroke, and trauma. As these disease states involve dysregulation of myelin integrity and/or remyelination, it is important to consider effects of the endocannabinoid system on oligodendrocytes and their precursors. In this review, we examine research reports on the effects of the endocannabinoid system (ECS) components on oligodendrocytes and their precursors, with a focus on therapeutic implications. Cannabinoid ligands and modulators of the endocannabinoid system promote cell signaling in oligodendrocyte precursor survival, proliferation, migration and differentiation, and mature oligodendrocyte survival and myelination. Agonist stimulation of oligodendrocyte precursor cells (OPCs) at both CB1 and CB<em>2</em> receptors counter apoptotic processes via Akt/PI3K, and promote proliferation via Akt/mTOR and ERK pathways. CB1 receptors in radial glia promote proliferation and conversion to progenitors fated to become oligodendroglia, whereas CB<em>2</em> receptors promote OPC migration in neonatal development. OPCs produce <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), stimulating cannabinoid receptor-mediated ERK pathways responsible for differentiation to arborized, myelin basic protein (MBP)-producing oligodendrocytes. In cell culture models of excitotoxicity, increased reactive oxygen species, and depolarization-dependent calcium influx, CB1 agonists improved viability of oligodendrocytes. In transient and permanent middle cerebral artery occlusion models of anoxic stroke, WIN55<em>2</em>1<em>2</em>-<em>2</em> increased OPC proliferation and maturation to oligodendroglia, thereby reducing cerebral tissue damage. In several models of rodent encephalomyelitis, chronic treatment with cannabinoid agonists ameliorated the damage by promoting OPC survival and oligodendrocyte function. Pharmacotherapeutic strategies based upon ECS and oligodendrocyte production and survival should be considered.

The probability of suffering the mood disorder depression is up to 30% in women and 15% in men during their life span. Pharmacological options for depression are limited: conventional antidepressants have low efficacy and a delayed onset of action (several weeks). Here we investigate the antidepressant actions of inhibitors of monoacylglycerol lipase (MAGL), the major degradative enzyme of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em>. A low-dose of MAGL inhibitors produces antidepressant effects on acute stress-exposed mice, through glutamatergic synaptic long-term depression (LTD), without significant effects on chronic corticosterone-exposed mice. In contrast, a high-dose of MAGL inhibitors produces pro- or antidepressant effects on acute stress- or chronic corticosterone-exposed mice, respectively, through GABAergic synaptic disinhibition. In the hippocampus, in vivo inhibition of MAGL induces a CB1 cannabinoid receptor (CB1R)-dependent suppression of inhibitory GABAergic synapses and an in vivo LTD of excitatory glutamatergic synapses. LTD induction requires CB1R in astroglial cells (but not in GABAergic or glutamatergic neurons) and postsynaptic glutamate receptors. The conventional antidepressant fluoxetine produces rapid or delayed antidepressant effects in acute stress- or chronic corticosterone-exposed mice, respectively. We propose that depression-like behavior of animals in response to acute stress is the normal behavioral response, and thus, MAGL inhibitors, which produce antidepressant effects in chronic corticosterone-exposed animals through GABAergic synaptic disinhibition, represent a new class of rapidly-acting and long-lasting antidepressants.

We synthesized <em>2</em>-<em>arachidonoylglycerol</em> (1), an endogenous cannabinoid receptor ligand, and its metabolically stable ether-linked analogues. Compound 1 was synthesized from 1,3-benzylideneglycerol (6) and arachidonic acid in the presence of N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine followed by treatment with boric acid and trimethyl borate. An ether-linked analogue of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>) was synthesized from 6 and 5,8,11,14-eicosatetraenyl iodide (9). The ether-linked analogues of <em>2</em>-palmitoylglycerol (4) and <em>2</em>-oleoyglycerol (5) were synthesized from 6 and hexadecyl iodide (1<em>2</em>) and 9-octadecenyl iodide (14), respectively. We confirmed that 1 stimulates NG108-15 cells to induce rapid transient elevation of the intracellular free Ca<em>2</em>+ concentrations through a CB1 receptor-dependent mechanism. Noticeably, <em>2</em> exhibited appreciable agonistic activity, although its activity was significantly lower than that of 1. Compound <em>2</em> would be a useful tool in exploring the physiological significance of 1, because this compound is resistant to hydrolyzing enzymes in contrast to 1. On the other hand, the ether-linked analogues of either 4 or 5 failed to act as a CB1 receptor agonist. Compounds 4 and 5 would also be valuable as control molecules in experiments where <em>2</em> is employed.