Crohn's disease and ulcerative colitis are two major forms of inflammatory bowel diseases (IBD), which are chronic inflammatory disorders of the gastrointestinal tract. These pathologies are currently under investigation to both unravel their etiology and find novel treatments. Anandamide and <em>2</em>-<em>arachidonoylglycerol</em> are endogenous bioactive lipids that bind to and activate the cannabinoid receptors, and together with the enzymes responsible for their biosynthesis and degradation [fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL)] constitute the endocannabinoid system (ECS). The ECS is implicated in gut homeostasis, modulating gastrointestinal motility, visceral sensation, and inflammation, as well as being recently implicated in IBD pathogenesis. Numerous subsequent studies investigating the effects of cannabinoid agonists and endocannabinoid degradation inhibitors in rodent models of IBD have identified a potential therapeutic role for the ECS.

The endocannabinoid system (ECS), comprising two G protein-coupled receptors (the cannabinoid receptors 1 and <em>2</em> [CB1 and CB<em>2</em>] for marijuana's psychoactive principle ∆(9)-tetrahydrocannabinol [∆(9)-THC]), their endogenous small lipid ligands (namely anandamide [AEA] and <em>2</em>-<em>arachidonoylglycerol</em> [<em>2</em>-AG], also known as endocannabinoids), and the proteins for endocannabinoid biosynthesis and degradation, has been suggested as a pro-homeostatic and pleiotropic signaling system activated in a time- and tissue-specific way during physiopathological conditions. In the brain activation of this system modulates the release of excitatory and inhibitory neurotransmitters and of cytokines from glial cells. As such, the ECS is strongly involved in neuropsychiatric disorders, particularly in affective disturbances such as anxiety and depression. It has been proposed that synthetic molecules that inhibit endocannabinoid degradation can exploit the selectivity of endocannabinoid action, thus activating cannabinoid receptors only in those tissues where there is perturbed endocannabinoid turnover due to the disorder, and avoiding the potential side effects of direct CB1 and CB<em>2</em> activation. However, the realization that endocannabinoids, and AEA in particular, also act at other molecular targets, and that these mediators can be deactivated by redundant pathways, has recently led to question the efficacy of such approach, thus opening the way to new multi-target therapeutic strategies, and to the use of non-psychotropic cannabinoids, such as cannabidiol (CBD), which act via several parallel mechanisms, including indirect interactions with the ECS. The state of the art of the possible therapeutic use of endocannabinoid deactivation inhibitors and phytocannabinoids in mood disorders is discussed in this review article.

Endocannabinoids are amides, esters and ethers of long chain polyunsaturated fatty acids, which act as new lipidic mediators. Anandamide (N-arachidonoylethanolamine; AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are the main endogenous agonists of cannabinoid receptors, able to mimic several pharmacological effects of (-)-Delta9-tetrahydrocannabinol (THC), the active principle of Cannabis sativa preparations like hashish and marijuana. The activity of AEA and <em>2</em>-AG at their receptors is limited by cellular uptake through an anandamide membrane transporter (AMT), followed by intracellular degradation. A fatty acid amide hydrolase (FAAH) is the main AEA hydrolase, whereas a monoacylglycerol lipase (MAGL) is critical in degrading <em>2</em>-AG. Here, we will review growing evidence that demonstrates that these hydrolases are pivotal regulators of the endogenous levels of AEA and <em>2</em>-AG in vivo, overall suggesting that specific inhibitors of AMT, FAAH or MAGL may serve as attractive therapeutic targets for the treatment of human disorders. Recently, the N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD), which synthesizes AEA from N-arachidonoylphosphatidylethanolamine (NArPE), and the diacylglycerol lipase (DAGL), which generates <em>2</em>-AG from diacylglycerol (DAG) substrates, have been characterized. The role of these synthetic routes in maintaining the endocannabinoid tone in vivo will be discussed. Finally, the effects of inhibitors of endocannabinoid degradation in animal models of human disease will be reviewed, with an emphasis on their ongoing applications in anxiety, cancer and neurodegenerative disorders.

BACKGROUND

Endocannabinoids (ECs) and related N-acyl-ethanolamides (NAEs) play important roles in stress response regulation, anxiety and traumatic memories. In view of the evidence that circulating EC levels are elevated under acute mild stressful conditions in humans, we hypothesized that individuals with traumatic stress exposure and post-traumatic stress disorder (PTSD), an anxiety disorder characterized by the inappropriate persistence and uncontrolled retrieval of traumatic memories, show measurable alterations in plasma EC and NAE concentrations.

METHODS

We determined plasma concentrations of the ECs anandamide (ANA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and the NAEs palmitoylethanolamide (PEA), oleoylethanolamide (OEA), stearoylethanolamine (SEA), and N-oleoyldopamine (OLDA) by HPLC-MS-MS in patients with PTSD (n = 10), trauma-exposed individuals without evidence of PTSD (n = 9) and in healthy control subjects (n = <em>2</em>9). PTSD was diagnosed according to DSM-IV criteria by administering the Clinician Administered PTSD Scale (CAPS), which also assesses traumatic events.

RESULTS

Individuals with PTSD showed significantly higher plasma concentrations of ANA (0.48 ± 0.11 vs. 0.36 ± 0.14 ng/ml, p = 0.01), <em>2</em>-AG (8.93 ± 3.<em>2</em>0 vs. 6.<em>2</em>6±<em>2</em>.10 ng/ml, p<0.01), OEA (5.90 ± <em>2</em>.10 vs. 3.88 ± 1.85 ng/ml, p<0.01), SEA (<em>2</em>.70 ± 3.37 vs. 0.83 ± 0.47, ng/ml, p<0.05) and significantly lower plasma levels of OLDA (0.1<em>2</em> ± 0.05 vs. 0.45 ± 0.59 ng/ml, p<0.05) than healthy controls. Moreover, PTSD patients had higher <em>2</em>-AG plasma levels (8.93 ± 3.<em>2</em>0 vs. 6.01 ± 1.3<em>2</em> ng/ml, p = 0.03) and also higher plasma concentrations of PEA (4.06 ± 1.87 vs. <em>2</em>.63±1.34 ng/ml, p<0.05) than trauma-exposed individuals without evidence of PTSD. CAPS scores in trauma-exposed individuals with and without PTSD (n = 19) correlated positively with PEA (r = 0.55, p = 0.0<em>2</em>) and negatively with OLDA plasma levels (r = -0.68, p<0.01). CAPS subscores for intrusions (r = -0.65, p<0.01), avoidance (r = -0.60, p<0.01) and hyperarousal (r = -0.66, p<0.01) were all negatively related to OLDA plasma concentrations.

CONCLUSIONS

PTSD appears to be associated with changes in plasma EC/NAE concentrations. This may have pathophysiological and diagnostic consequences but will need to be reproduced in larger cohorts.

Cannabinoid administration suppresses pain by acting at spinal, supraspinal and peripheral levels. Intrinsic analgesic pathways also exploit endocannabinoids; however, the underlying neurobiological substrates of endocannabinoid-mediated analgesia have remained largely unknown. Compelling evidence shows that, upon exposure to a painful environmental stressor, an endocannabinoid molecule called <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is mobilized in the lumbar spinal cord in temporal correlation with stress-induced antinociception. We therefore characterized the precise molecular architecture of <em>2</em>-AG signaling and its involvement in nociception in the rodent spinal cord. Nonradioactive in situ hybridization revealed that dorsal horn neurons widely expressed the mRNA of diacylglycerol lipase-alpha (DGL-alpha), the synthesizing enzyme of <em>2</em>-AG. Peroxidase-based immunocytochemistry demonstrated high levels of DGL-alpha protein and CB(1) cannabinoid receptor, a receptor for <em>2</em>-AG, in the superficial dorsal horn, at the first site of modulation of the ascending pain pathway. High-resolution electron microscopy uncovered postsynaptic localization of DGL-alpha at nociceptive synapses formed by primary afferents, and revealed presynaptic positioning of CB(1) on excitatory axon terminals. Furthermore, DGL-alpha in postsynaptic elements receiving nociceptive input was colocalized with metabotropic glutamate receptor 5 (mGluR(5)), whose activation induces <em>2</em>-AG biosynthesis. Finally, intrathecal activation of mGluR(5) at the lumbar level evoked endocannabinoid-mediated stress-induced analgesia through the DGL-<em>2</em>-AG-CB(1) pathway. Taken together, these findings suggest a key role for <em>2</em>-AG-mediated retrograde suppression of nociceptive transmission at the spinal level. The striking positioning of the molecular players of <em>2</em>-AG synthesis and action at nociceptive excitatory synapses suggests that pharmacological manipulation of spinal <em>2</em>-AG levels may be an efficacious way to regulate pain sensation.

BACKGROUND

The hippocampus is strongly implicated in memory processes and contains high concentrations of both cannabinoid receptors and their endogenous ligands. Chronic alcohol consumption impairs a variety of cognitive and performance tasks, including memory and learning. As the activation of cannabinoid receptors by their endogenous ligands modulates hippocampal neurotransmission, we hypothesized that the impaired memory and learning in alcoholism may be due to alterations in the hippocampal endocannabinoid system.

METHODS

We used the rat chronic intermittent ethanol (CIE) model for alcohol withdrawal and dependence which involves intermittent episodes of ethanol intoxication (60 doses) and withdrawal (approximating binge drinking episodes in humans). We measured the levels of cannabinoid 1 receptor (CB1R) protein (Western blot using a C-terminal-directed antibody), CB1R mRNA (real-time RT-PCR), CB1R localization (immunocytochemistry), tissue levels of the endocannabinoids N-arachidonoylethanolamine/anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), and function (patch-clamp recordings of depolarization-induced suppression of inhibition (DSI), as well as effects of CB1R agonist WIN 55,<em>2</em>1<em>2</em>-<em>2</em> on inhibitory currents) in the hippocampus of CIE rats and their saline-treated controls.

RESULTS

Results were obtained in saline and CIE-treated rats after <em>2</em> and 40 days of withdrawal (DW) from their respective treatments. In <em>2</em> DW CIE rats, CB1R mRNA and protein levels were decreased by <em>2</em>7% (p<0.05) compared with saline controls. Surprisingly, in 40 DW CIE rats, CB1R mRNA increased by 100% and protein increased by <em>2</em>1%, confirmed by immunohistochemistry. Hippocampal [<em>2</em>-AG] increased in both <em>2</em> and 40 DW CIE rats; [AEA] increased only at 40 DW. Hippocampal DSI of CIE rats was significantly reduced at <em>2</em> DW but not at 40 DW. The CB1R agonist WIN 55,<em>2</em>1<em>2</em>-<em>2</em> (0.5 microM) produced a significantly greater decrease in the frequency of spontaneous inhibitory currents from saline-treated rats compared with CIE rats at <em>2</em> DW, but not at 40 DW.

CONCLUSIONS

These data demonstrate that CIE treatment and withdrawal transiently down-regulates hippocampal CB1 Rs followed by a long-term up-regulation, including increased levels of endogenous cannabinoids. These findings are consistent with our hypothesis and suggest that long-term up-regulation of hippocampal CB1Rs may contribute to the long-term cognitive impairments in alcoholism. The data further suggest that the effectiveness of CB1R blockade in decreasing alcohol consumption may be greater after protracted abstinence from alcohol.

Endocannabinoids were first defined in 1995 as 'endogenous substances capable of binding to and functionally activating the cannabinoid receptors'. To date, two well-established endocannabinoids, N-arachidonoylethanolamine (anandamide) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), as well as a few other putative ligands, all derived from long-chain polyunsaturated fatty acids, have been identified in animal tissues. The biosynthetic and metabolic pathways for anandamide and <em>2</em>-AG have been elucidated, and most of the enzymes therein involved have been cloned. We now know that CB1 receptors, and endocannabinoids in tissue concentrations sufficient to activate them, are more widely distributed than originally thought, and are found in brain and peripheral organs involved in the control of energy intake and processing, including the hypothalamus, nucleus accumbens, brainstem, vagus nerve, gastrointestinal tract, adipose tissue and liver. Endocannabinoid biosynthetic and inactivating pathways are under the regulation of neuropeptides and hormones involved in energy homeostasis, and endocannabinoid levels are directly affected by the diet. Endocannabinoids, in turn, regulate the expression and action of mediators involved in nutrient intake and processing. These cross-talks are at the basis of the proposed role of endocannabinoid signalling in the control of food intake, from invertebrates to lower vertebrates and mammals, and their perturbation appears to contribute to the development of eating disorders.

The endocannabinoid (eCB) system regulates mood, emotion, and stress coping, and dysregulation of the eCB system is critically involved in pathophysiology of depression. The eCB ligand <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is inactivated by monoacylglycerol lipase (MAGL). Using chronic unpredictable mild stress (CUS) as a mouse model of depression, we examined how <em>2</em>-AG signaling in the hippocampus was altered in depressive-like states and how this alteration contributed to depressive-like behavior. We report that CUS led to impairment of depolarization-induced suppression of inhibition (DSI) in mouse hippocampal CA1 pyramidal neurons, and this deficiency in <em>2</em>-AG-mediated retrograde synaptic depression was rescued by MAGL inhibitor JZL184. CUS induced depressive-like behaviors and decreased mammalian target of rapamycin (mTOR) activation in the hippocampus, and these biochemical and behavioral abnormalities were ameliorated by chronic JZL184 treatments. The effects of JZL184 were mediated by cannabinoid CB1 receptors. Genetic deletion of mTOR with adeno-associated viral (AAV) vector carrying the Cre recombinase in the hippocampus of mTORf/f mice recapitulated depressive-like behaviors induced by CUS and abrogated the antidepressant-like effects of chronic JZL184 treatments. Our results suggest that CUS decreases eCB-mTOR signaling in the hippocampus, leading to depressive-like behaviors, whereas MAGL inhibitor JZL184 produces antidepressant-like effects through enhancement of eCB-mTOR signaling.

This review evaluates the cellular mechanisms of constitutive activity of the cannabinoid (CB) receptors, its reversal by inverse agonists, and discusses the pitfalls and problems in the interpretation of the research data. The notion is presented that endogenously produced anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) serve as autocrine or paracrine stimulators of the CB receptors, giving the appearance of constitutive activity. It is proposed that one cannot interpret inverse agonist studies without inference to the receptors' environment vis-à-vis the endocannabinoid agonists which themselves are highly lipophilic compounds with a preference for membranes. The endocannabinoid tone is governed by a combination of synthetic pathways and inactivation involving transport and degradation. The synthesis and degradation of <em>2</em>-AG is well characterized, and <em>2</em>-AG has been strongly implicated in retrograde signalling in neurons. Data implicating endocannabinoids in paracrine regulation have been described. Endocannabinoid ligands can traverse the cell's interior and potentially be stored on fatty acid-binding proteins (FABPs). Molecular modelling predicts that the endocannabinoids derived from membrane phospholipids can laterally diffuse to enter the CB receptor from the lipid bilayer. Considering that endocannabinoid signalling to CB receptors is a much more likely scenario than is receptor activation in the absence of agonist ligands, researchers are advised to refrain from assuming constitutive activity except for experimental models known to be devoid of endocannabinoid ligands.

The hydrolysis of endocannabinoids has profound effects on the function of the endocannabinoid signaling system in the regulation of prostate carcinoma cells. Prostate carcinoma cells exhibit a wide range of hydrolysis activity for <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), the major endocannabinoid. However, enzyme(s) responsible for <em>2</em>-AG hydrolysis and their functions in prostate cancer have not been characterized. In this study, we demonstrated that fatty acid amide hydrolase (FAAH) was differentially expressed in normal and prostate carcinoma cells. In PC-3 cells, overexpression of FAAH resulted in increased FAAH protein, <em>2</em>-AG hydrolysis, cell invasion and cell migration. Conversely, small-interfering RNA (siRNA) knockdown of FAAH in LNCaP cells decreased FAAH protein, <em>2</em>-AG hydrolysis and cell invasion. Furthermore, CAY10401, a FAAH inhibitor, decreased cell invasion and it enhanced the reduction of invasion in FAAH siRNA-transfected LNCaP cells. Immunohistochemistry staining of commercial tissue microarrays (TMAs) demonstrated FAAH staining in 109 of 157 cores of prostate adenocarcinomas but weak staining in 1 of 8 cores of normal prostate tissues. These results suggest that FAAH regulates <em>2</em>-AG hydrolysis and invasion of prostate carcinoma cells and is potentially involved in prostate tumorigenesis.

OBJECTIVE

Endocannabinoids have both anti-inflammatory and neuroprotective properties against harmful stimuli. We previously demonstrated that the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) protects hippocampal neurons by limiting the inflammatory response via a CB(1) receptor-dependent MAPK/NF-κB signalling pathway. The purpose of the present study was to determine whether PPARγ, an important nuclear receptor, mediates <em>2</em>-AG-induced inhibition of NF-κB phosphorylation and COX-<em>2</em> expression, and COX-<em>2</em>-enhanced miniature spontaneous excitatory postsynaptic currents (mEPSCs).

METHODS

By using a whole-cell patch clamp electrophysiological recording technique and immunoblot analysis, we determined mEPSCs, expression of COX-<em>2</em> and PPARγ, and phosphorylation of NF-kB in mouse hippocampal neurons in culture.

RESULTS

Exogenous and endogenous <em>2</em>-AG-produced suppressions of NF-κB-p65 phosphorylation, COX-<em>2</em> expression and excitatory synaptic transmission in response to pro-inflammatory interleukin-1β (IL-1β) and LPS were inhibited by GW966<em>2</em>, a selective PPARγ antagonist, in hippocampal neurons in culture. PPARγ agonists 15-deoxy-Δ(1<em>2</em>,14) -prostaglandin J(<em>2</em>) (15d-PGJ(<em>2</em>)) and rosiglitazone mimicked the effects of <em>2</em>-AG on NF-κB-p65 phosphorylation, COX-<em>2</em> expression and mEPSCs, and these effects were eliminated by antagonism of PPARγ. Moreover, exogenous application of <em>2</em>-AG or elevation of endogenous <em>2</em>-AG by inhibiting its hydrolysis with URB60<em>2</em> or JZL184, selective inhibitors of monoacylglycerol lipase (MAGL), prevented the IL-1β- and LPS-induced reduction of PPARγ expression. The <em>2</em>-AG restoration of the reduced PPARγ expression was blocked or attenuated by pharmacological or genetic inhibition of the CB(1) receptor.

CONCLUSIONS

Our results suggest that CB(1) receptor-dependent PPARγ expression is an important and novel signalling pathway in endocannabinoid <em>2</em>-AG-produced resolution of neuroinflammation in response to pro-inflammatory insults.

The endocannabinoid system is currently defined as the ensemble of the two 7-transmembrane-domain and G protein-coupled receptors for Δ(9)-tetrahydrocannabinol (but not for most other plant cannabinoids or phytocannabinoids)-cannabinoid receptor type-1 (CB1R) and cannabinoid receptor type-<em>2</em> (CB<em>2</em>R); their two most studied endogenous ligands, the "endocannabinoids" N-arachidonoylethanolamine (anandamide) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG); and the enzymes responsible for endocannabinoid metabolism. However, anandamide and <em>2</em>-AG, and also the phytocannabinoids, have more molecular targets than just CB1R and CB<em>2</em>R. Furthermore, the endocannabinoids, like most other lipid mediators, have more than just one set of biosynthetic and degrading pathways and enzymes, which they often share with "endocannabinoid-like" mediators that may or may not interact with the same proteins as Δ(9)-tetrahydrocannabinol and other phytocannabinoids. In some cases, these degrading pathways and enzymes lead to molecules that are not inactive and instead interact with other receptors. Finally, some of the metabolic enzymes may also participate in the chemical modification of molecules that have very little to do with endocannabinoid and cannabinoid targets. Here, we review the whole world of ligands, receptors, and enzymes, a true "endocannabinoidome", discovered after the cloning of CB1R and CB<em>2</em>R and the identification of anandamide and <em>2</em>-AG, and its interactions with phytocannabinoids.

OBJECTIVE

The endocannabinoid and eicosanoid lipid signaling pathways have important roles in inflammatory syndromes. Monoacylglycerol lipase (MAGL) links these pathways, hydrolyzing the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> to generate the arachidonic acid precursor pool for prostaglandin production. We investigated whether blocking MAGL protects against inflammation and damage from hepatic ischemia/reperfusion (I/R) and other insults.

METHODS

We analyzed the effects of hepatic I/R in mice given the selective MAGL inhibitor JZL184, in Mgll(-/-) mice, fatty acid amide hydrolase(-/-) mice, and in cannabinoid receptor type 1(-/-) (CB1-/-) and cannabinoid receptor type <em>2</em>(-/-) (CB<em>2</em>-/-). Liver tissues were collected and analyzed, along with cultured hepatocytes and Kupffer cells. We measured endocannabinoids, eicosanoids, and markers of inflammation, oxidative stress, and cell death using molecular biology, biochemistry, and mass spectrometry analyses.

RESULTS

Wild-type mice given JZL184 and Mgll(-/-) mice were protected from hepatic I/R injury by a mechanism that involved increased endocannabinoid signaling via CB<em>2</em> and reduced production of eicosanoids in the liver. JZL184 suppressed the inflammation and oxidative stress that mediate hepatic I/R injury. Hepatocytes were the major source of hepatic MAGL activity and endocannabinoid and eicosanoid production. JZL184 also protected from induction of liver injury by D-(+)-galactosamine and lipopolysaccharides or CCl4.

CONCLUSIONS

MAGL modulates hepatic injury via endocannabinoid and eicosanoid signaling; blockade of this pathway protects mice from liver injury. MAGL inhibitors might be developed to treat conditions that expose the liver to oxidative stress and inflammatory damage.

Advanced liver cirrhosis is associated with hyperdynamic circulation consisting of systemic hypotension, decreased peripheral resistance, and cardiac dysfunction, termed cirrhotic cardiomyopathy. Previous studies have revealed the role of endocannabinoids and vascular CB(1) receptors in the development of generalized hypotension and mesenteric vasodilation in animal models of liver cirrhosis, and CB(1) receptors have also been implicated in the decreased beta-adrenergic responsiveness of isolated heart tissue from cirrhotic rats. Here we document the cardiac contractile dysfunction in vivo in liver cirrhosis and explore the role of the endocannabinoid system in its development. Rats with CCl(4)-induced cirrhosis developed decreased cardiac contractility, as documented through the use of the Millar pressure-volume microcatheter system, low blood pressure, and tachycardia. Bolus intravenous injection of the CB(1) antagonist AM<em>2</em>51 (3 mg/kg) acutely increased mean blood pressure, as well as both load-dependent and -independent indexes of systolic function, whereas no such changes were elicited by AM<em>2</em>51 in control rats. Furthermore, tissue levels of the endocannabinoid anandamide increased <em>2</em>.7-fold in the heart of cirrhotic compared with control rats, without any change in <em>2</em>-<em>arachidonoylglycerol</em> levels, whereas, in the cirrhotic liver, both <em>2</em>-<em>arachidonoylglycerol</em> (6-fold) and anandamide (3.5-fold) were markedly increased. CB(1)-receptor expression in the heart was unaffected by cirrhosis, as verified by Western blotting. Activation of cardiac CB(1) receptors by endogenous anandamide contributes to the reduced cardiac contractility in liver cirrhosis, and CB(1)-receptor antagonists may be used to improve contractile function in cirrhotic cardiomyopathy and, possibly, in other forms of heart failure.

Adenosine A(<em>2</em>A) receptor antagonists are psychomotor stimulants that also hold therapeutic promise for movement disorders. However, the molecular mechanisms underlying their stimulant properties are not well understood. Here, we show that the robust increase in locomotor activity induced by an A(<em>2</em>A) antagonist in vivo is greatly attenuated by antagonizing cannabinoid CB(1) receptor signaling or by administration to CB(1)(-/-) mice. To determine the locus of increased endocannabinoid signaling, we measured the amount of anandamide [AEA (N-arachidonoylethanolamine)] and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in brain tissue from striatum and cortex. We find that <em>2</em>-AG is selectively increased in striatum after acute blockade of A(<em>2</em>A) receptors, which are highly expressed by striatal indirect-pathway medium spiny neurons (MSNs). Using targeted whole-cell recordings from direct- and indirect-pathway MSNs, we demonstrate that A(<em>2</em>A) receptor antagonists potentiate <em>2</em>-AG release and induction of long-term depression at indirect-pathway MSNs, but not direct-pathway MSNs. Together, these data outline a molecular mechanism by which A(<em>2</em>A) antagonists reduce excitatory synaptic drive on the indirect pathway through CB(1) receptor signaling, thus leading to increased psychomotor activation.

Human vascular endothelial cells were found to generate and release <em>2</em>-<em>arachidonoylglycerol</em>, an endogenous cannabinoid receptor ligand, upon stimulation with thrombin or A<em>2</em>3187. We confirmed that vascular smooth muscle cells as well as endothelial cells possess cannabinoid CB1 receptor mRNA. <em>2</em>-<em>Arachidonoylglycerol</em>, generated in vascular tissues, may play an important role in modulating vascular tone through acting on the cannabinoid CB1 receptor expressed on vascular smooth muscle cells, endothelial cells as well as peripheral nerve terminals.

BACKGROUND

Omega-3 polyunsaturated fatty acids (ω-3-PUFA) are known to ameliorate several metabolic risk factors for cardiovascular disease, and an association between elevated peripheral levels of endogenous ligands of cannabinoid receptors (endocannabinoids) and the metabolic syndrome has been reported. We investigated the dose-dependent effects of dietary ω-3-PUFA supplementation, given as krill oil (KO), on metabolic parameters in high fat diet (HFD)-fed mice and, in parallel, on the levels, in inguinal and epididymal adipose tissue (AT), liver, gastrocnemius muscle, kidneys and heart, of: 1) the endocannabinoids, anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), <em>2</em>) two anandamide congeners which activate PPARα but not cannabinoid receptors, N-oleoylethanolamine and N-palmitoylethanolamine, and 3) the direct biosynthetic precursors of these compounds.

METHODS

Lipids were identified and quantified using liquid chromatography coupled to atmospheric pressure chemical ionization single quadrupole mass spectrometry (LC-APCI-MS) or high resolution ion trap-time of flight mass spectrometry (LC-IT-ToF-MS).

RESULTS

Eight-week HFD increased endocannabinoid levels in all tissues except the liver and epididymal AT, and KO reduced anandamide and/or <em>2</em>-AG levels in all tissues but not in the liver, usually in a dose-dependent manner. Levels of endocannabinoid precursors were also generally down-regulated, indicating that KO affects levels of endocannabinoids in part by reducing the availability of their biosynthetic precursors. Usually smaller effects were found of KO on OEA and PEA levels.

CONCLUSIONS

Our data suggest that KO may promote therapeutic benefit by reducing endocannabinoid precursor availability and hence endocannabinoid biosynthesis.

The cannabinoid CB1 and CB<em>2</em> receptors are Class A G protein-coupled receptors (GPCRs). While many Class A GPCRs have endogenous ligands that are hydrophilic cations (e.g., the serotonin and dopamine receptors), the cannabinoid receptors have neutral, highly lipophilic ligands derived from the fatty acid, arachidonic acid. The most well-studied of these are N-arachidonoylethanolamine (anandamide, AEA) and sn-<em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). This review focuses on the experimental and computational studies that have been used to probe the nature of endocannabinoid interaction with the cannabinoid receptors. These studies include mutation, SAR and NMR studies, as well as, QSAR, docking and molecular dynamics simulations. Gaps in our knowledge are identified. The review begins more generally, however, by discussing the entire endocannabinoid system, of which the cannabinoid receptors are part. For in order to understand endocannabinoid action, one needs an appreciation for the environments for which these ligands have been designed and the conformational changes these ligands must undergo in order to act on the cannabinoid receptors.

Since the discovery of endocannabinoids and their receptors, two major members of the endocannabinoid family, anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), have been regarded almost as twin brothers. Pharmacological properties were initially considered to be similar, as these molecules were believed mutually exchangeable and almost indistinguishable in the regulation of synaptic functions, such as long- and short-term synaptic plasticity, and in behavioral aspects, such as learning and memory, reward and addiction, antinociception, and anxiety. In recent years, however, endocannabinoid signaling specificity began to emerge, in particular, due to the production of genetically engineered mice lacking key enzymes in endocannabinoid synthesis or degradation, together with the development of selective inhibitors of AEA or <em>2</em>-AG catabolic enzymes. Evidence now suggests that AEA and <em>2</em>-AG possess specific pharmacological properties, are engaged in different forms of synaptic plasticity, and take part in different behavioral functions. In this review, we provide an overview on similarities and specificities of the two endocannabinoids in the CNS and on the unresolved questions concerning their role in synaptic signaling.

Colorectal cancer is an increasingly important cause of death in Western countries. Endocannabinoids inhibit colorectal carcinoma cell proliferation in vitro. In this paper, we investigated the involvement of endocannabinoids on the formation of aberrant crypt foci (ACF, earliest preneoplastic lesions) in the colon mouse in vivo. ACF were induced by azoxymethane (AOM); fatty acid amide hydrolase (FAAH) and cannabinoid receptor messenger ribonucleic acid (mRNA) levels were analyzed by the quantitative reverse transcription polymerase chain reaction (RT-PCR); endocannabinoid levels were measured by liquid chromatography-mass spectrometry; caspase-3 and caspase-9 expressions were measured by Western blot analysis. Colonic ACF formation after AOM administration was associated with increased levels of <em>2</em>-<em>arachidonoylglycerol</em> (with no changes in FAAH and cannabinoid receptor mRNA levels) and reduction in cleaved caspase-3 and caspase-9 expression. The FAAH inhibitor N-arachidonoylserotonin increased colon endocannabinoid levels, reduced ACF formation, and partially normalized cleaved caspase-3 (but not caspase-9) expression. Notably, N-arachidonoylserotonin completely prevented the formation of ACF with four or more crypts, which have been show to be best correlated with final tumor incidence. The effect of N-arachidonoylserotonin on ACF formation was mimicked by the cannabinoid receptor agonist HU-<em>2</em>10. No differences in ACF formation were observed between CB(1) receptor-deficient and wild-type mice. It is concluded that pharmacological enhancement of endocannabinoid levels (through inhibition of endocannabinoid hydrolysis) reduces the development of precancerous lesions in the mouse colon. The protective effect appears to involve caspase-3 (but not caspase-9) activation.

<em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG) is a monoacylglycerol (MAG) molecule containing an esterified arachidonic acid chain at sn-<em>2</em> position of the glycerol backbone. Together with structurally similar N-arachidonoylethanolamine (anandamide), <em>2</em>-AG has been extensively studied as an endogenous ligand of cannabinoid receptors (an endocannabinoid) in brain and other mammalian tissues. Accumulating evidence demonstrates that the endocannabinoid system, including the central-type cannabinoid receptor CB1 and <em>2</em>-AG, is responsible for synaptic retrograde signaling in the central nervous system. As <em>2</em>-AG is rapidly formed from membrane phospholipids on cellular stimuli and degraded to arachidonic acid and glycerol, the enzymes catalyzing its biosynthesis and degradation are believed to play crucial roles in the regulation of its tissue levels. The major biosynthetic pathway appears to consist of sequential hydrolyses of inositol phospholipids via diacylglycerol (DAG) by β-type phospholipase C and DAG lipase, while MAG lipase is a principal enzyme in the degradation. In this short review, we will briefly outline rapid advances in enzymological research on the biosynthetic and degradative pathways of <em>2</em>-AG.

Endogenous cannabinoids (endocannabinoids, eCBs) are ubiquitous regulators of synaptic transmission in the brain, mediating numerous forms of short- and long-term plasticity, and having strong influences on synapse formation and neurogenesis. Their roles as retrograde messengers that suppress both excitatory and inhibitory transmission are well-established. Yet, despite intensive investigation, many basic aspects of the eCB system are not understood. This brief review highlights recent advances, problems that remain unresolved, and avenues for future exploration. While <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is probably the major eCB for intercellular CB1R-dependent signalling, anandamide (AEA) has come to the forefront in several novel contexts, both as a dual endovanilloid/endocannabinoid that regulates synaptic transmission acutely and as the source of a steady eCB tone in hippocampus. Complexities in the cellular processing of <em>2</em>-AG are receiving renewed attention, as they are increasingly recognized as major determinants of how <em>2</em>-AG affects cells. Long-standing fundamental issues such as the synthesis pathway for AEA and the molecular mechanism(s) underlying cellular uptake and release of eCBs remain problematical.

The purpose of this study was to examine opioid and endocannabinoid mechanisms of exercise-induced hypoalgesia (EIH). Fifty-eight men and women (mean age = <em>2</em>1 years) completed 3 sessions. During the first session, participants were familiarized with the temporal summation of heat pain and pressure pain protocols. In the exercise sessions, following double-blind administration of either an opioid antagonist (50 mg naltrexone) or placebo, participants rated the intensity of heat pulses and indicated their pressure pain thresholds and pressure pain ratings before and after 3 minutes of submaximal isometric exercise. Blood was drawn before and after exercise. Results indicated that circulating concentrations of <em>2</em> endocannabinoids, N-arachidonylethanolamine and <em>2</em>-<em>arachidonoylglycerol</em>, as well as related lipids oleoylethanolamide, palmitoylethanolamide, N-docosahexaenoylethanolamine, and <em>2</em>-oleoylglycerol, increased significantly (P < .05) following exercise. Pressure pain thresholds increased significantly (P < .05), whereas pressure pain ratings decreased significantly (P < .05) following exercise. Also, temporal summation ratings were significantly lower (P < .05) following exercise. These changes in pain responses did not differ between the placebo and naltrexone conditions (P>> .05). A significant association was found between EIH and docosahexaenoylethanolamine. These results suggest involvement of a nonopioid mechanism in EIH following isometric exercise.

CONCLUSIONS

Currently, the mechanisms responsible for EIH are unknown. This study provides support for a potential endocannabinoid mechanism of EIH following isometric exercise.

Proinflammatory macrophages are key mediators in several pathologies; thus, controlling their activation is necessary. The endocannabinoid system is implicated in various inflammatory processes. Here we show that in macrophages, the newly characterized enzyme α/β-hydrolase domain 6 (ABHD6) controls <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) levels and thus its pharmacological effects. Furthermore, we characterize a unique pathway mediating the effects of <em>2</em>-AG through its oxygenation by cyclooxygenase-<em>2</em> to give rise to the anti-inflammatory prostaglandin D<em>2</em>-glycerol ester (PGD<em>2</em>-G). Pharmacological blockade of cyclooxygenase-<em>2</em> or of prostaglandin D synthase prevented the effects of increasing <em>2</em>-AG levels by ABHD6 inhibition in vitro, as well as the <em>2</em>-AG-induced increase in PGD<em>2</em>-G levels. Together, our data demonstrate the physiological relevance of the interaction between the endocannabinoid and prostanoid systems. Moreover, we show that ABHD6 inhibition in vivo allows for fine-tuning of <em>2</em>-AG levels in mice, therefore reducing lipopolysaccharide-induced inflammation, without the characteristic central side effects of strong increases in <em>2</em>-AG levels obtained following monoacylglycerol lipase inhibition. In addition, administration of PGD<em>2</em>-G reduces lipopolysaccharide-induced inflammation in mice, thus confirming the biological relevance of this <em>2</em>-AG metabolite. This points to ABHD6 as an interesting therapeutic target that should be relevant in treating inflammation-related conditions, and proposes PGD<em>2</em>-G as a bioactive lipid with potential anti-inflammatory properties in vivo.