The effects of endogenous and synthetic cannabinoid receptor agonists, including <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), R-methanandamide, WIN55,<em>2</em>1<em>2</em>-<em>2</em> [4,5-dihydro-<em>2</em>-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenylcarbonyl)-6H-pyrrolo[3,<em>2</em>,1ij]quinolin-6-one], and CP 55,940 [1alpha,<em>2</em>beta-(R)-5alpha]-(-)-5-(1,1-dimethyl)-<em>2</em>-[5-hydroxy-<em>2</em>-(3-hydroxypropyl) cyclohexyl-phenol], and the psychoactive constituent of marijuana, Delta9-tetrahydrocannabinol (Delta9-THC), on the function of homomeric alpha7-nicotinic acetylcholine (nACh) receptors expressed in Xenopus oocytes was investigated using the two-electrode voltage-clamp technique. The endogenous cannabinoid receptor ligands <em>2</em>-AG and the metabolically stable analog of anandamide (arachidonylethanolamide), R-methanandamide, reversibly inhibited currents evoked with ACh (100 microM) in a concentration-dependent manner (IC50 values of 168 and 183 nM, respectively). In contrast, the synthetic cannabinoid receptor agonists CP 55,940, WIN55,<em>2</em>1<em>2</em>-<em>2</em>, and the phytochemical Delta9-THC did not alter alpha7-nACh receptor function. The inhibition of alpha7-mediated currents by <em>2</em>-AG was found to be non-competitive and voltage-independent. Additional experiments using endocannabinoid metabolites suggested that arachidonic acid, but not ethanolamine or glycerol, could also inhibit the alpha7-nACh receptor function. Whereas the effects of arachidonic acid were also noncompetitive and voltage-independent, its potency was much lower than <em>2</em>-AG and anandamide. Results of studies with chimeric alpha7-nACh-5-hydroxytryptamine (5-HT)3 receptors comprised of the amino-terminal domain of the alpha7-nACh receptor and the transmembrane and carboxyl-terminal domains of 5-HT3 receptors indicated that the site of interaction of the endocannabinoids with the alpha7-nAChR was not located on the N-terminal region of the receptor. These data indicate that cannabinoid receptor ligands that are produced in situ potently inhibit alpha7-nACh receptor function, whereas the synthetic cannabinoid ligands, and Delta9-THC, are without effect, or are relatively ineffective at inhibiting these receptors.

We examined the effect of diacylglycerol on Ca<em>2</em>+-dependent phospholipase A<em>2</em> from human platelets. Phospholipase A<em>2</em> was solubilized and partially purified to a stable form in the presence of n-octyl beta-D-glucopyranoside (octyl glucoside), and its enzymic activity was determined with sonicated <em>2</em>.5 microM-1-palmitoyl-<em>2</em>-arachidonoyl-sn-glycero-3-phosphocholine (arachidonoyl-PC) as substrate. Phospholipase A<em>2</em> activity was increased when diacylglycerol was incorporated into the substrate arachidonoyl-PC. Stimulation was maximal in the presence of greater than or equal to <em>2</em>9 mol% (1 microM) diacylglycerol, and was greater than 4-fold for both 1,<em>2</em>-dioleoylglycerol and 1-stearoyl-<em>2</em>-<em>arachidonoylglycerol</em>. 1-Stearoyl-<em>2</em>-<em>arachidonoylglycerol</em> at concentrations of <em>2</em>-5 mol% increased phospholipase A<em>2</em> activity 1.3-1.8-fold. Exogenously added 1-oleoyl-<em>2</em>-acetylglycerol also enhanced phospholipase A<em>2</em> activity, producing a maximal stimulation of 1.6-fold at a concentration of <em>2</em>5 microM. Comparative studies conducted with pancreatic, bee-venom and snake-venom phospholipase A<em>2</em> showed that the activity of these extracellular phospholipases towards the arachidonoyl-PC substrate was also increased by diacylglycerol, but stimulation was less than observed for platelet phospholipase A<em>2</em>. Our results suggest that diacylglycerol, known to be generated in stimulated platelets, may enhance Ca<em>2</em>+-activated phospholipase A<em>2</em>.

Tissue concentrations of the endocannabinoids N-arachidonoylethanolamine (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are regulated by both synthesis and inactivation. The purpose of this review is to compile available data regarding three inactivation processes: fatty acid amide hydrolase, monoacylglycerol lipase, and cellular membrane transport. In particular, we have focused on mechanisms by which these processes are modulated. We describe the in vitro and in vivo effects of inhibitors of these processes as well as available evidence regarding their modulation by other factors.

The specific protein target of delta9-tetrahydrocannabinol (delta9-THC), the main active ingredient of Cannabis sativa L., was characterized from rat brain nearly <em>2</em>0 years ago, and several endogenous compounds and proteins comprising the endocannabinoid (eCB) system have since been discovered. It has become evident that the eCB system consists of at least two cannabinoid receptors (i.e. the CB1 and CB<em>2</em> receptors), in addition to their endogenous ligands (the eCBs) and several enzymes involved in the biosynthesis and catabolism of the eCBs. The two well-established eCBs, N-arachidonoylethanolamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), are produced by neurons on demand, act near their sites of synthesis and are effectively metabolized by fatty acid amide hydrolase (FAAH) and monoglyceride lipase (MGL), respectively. Inhibitors specifically targeting these enzymes could offer novel therapeutic approaches (e.g. for the treatment of pain and movement disorders). This MiniReview summarizes the literature concerning the potential therapeutic potential of FAAH and MGL inhibitors.

Substance P is thought to play an essential role in several forms of supraspinally mediated analgesia. The actions of substance P on synaptic transmission within descending analgesic pathways, however, are largely unknown. Here, we used whole-cell recordings from rat midbrain slices to examine the effects of substance P on GABAergic and glutamatergic transmission within the periaqueductal gray (PAG), a key component of a descending analgesic pathway that projects via the rostral ventromedial medulla (RVM) to the spinal cord dorsal horn. We found that substance P reversibly decreased the amplitude and increased the paired-pulse ratio of evoked IPSCs recorded from identified PAG-RVM projection neurons and from unidentified PAG neurons. Substance P had no effect on miniature IPSCs, implying an indirect mode of action. The effects of substance P were abolished by metabotropic glutamate type 5 and cannabinoid CB1 receptor antagonists, but unaltered by NMDA, GABA(B), mu,delta-opioid, adenosine A(1), and 5HT(1A) receptor antagonists. Consistent with a role for endogenous glutamate in this process, substance P increased the frequency of action potential-dependent spontaneous EPSCs. Moreover, the effect of substance P on evoked IPSCs was mimicked and occluded by a glutamate transport inhibitor. Finally, these effects were dependent on postsynaptic G-protein activation and diacylglycerol lipase activity, suggesting the requirement for retrograde signaling by the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em>. Thus, substance P may facilitate descending analgesia in part by enhancing glutamate-mediated excitation and endocannabinoid-mediated disinhibition of PAG-RVM projection neurons.

The effect of the enol carbamate 1-biphenyl-4-ylethenyl piperidine-1-carboxylate (ST4070), a novel reversible inhibitor of fatty acid amide hydrolase (FAAH), was investigated for acute pain sensitivity and neuropathic pain in rats and mice. Brain enzymatic activity of FAAH and the endogenous levels of its substrates, anandamide (AEA; N-arachidonoylethanolamine), <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), and N-palmitoylethanolamine (PEA), were measured in control and ST4070-treated mice. ST4070 (10, 30, and 100 mg/kg) was orally administered to assess mechanical nociceptive thresholds and allodynia by using the Randall-Selitto and von Frey tests, respectively. Neuropathy was induced in rats by either the chemotherapeutic agent vincristine or streptozotocin-induced diabetes, whereas the chronic constriction injury (CCI) model was chosen to evaluate neuropathy in mice. ST4070 produced a significant increase of nociceptive threshold in rats and counteracted the decrease of nociceptive threshold in the three distinct models of neuropathic pain. In diabetic mice, ST4070 inhibited FAAH activity and increased the brain levels of AEA and PEA, without affecting that of <em>2</em>-AG. The administration of ST4070 generated long-lasting pain relief compared with pregabalin and the FAAH inhibitors 1-oxo-1[5-(<em>2</em>-pyridyl)-<em>2</em>-yl]-7-phenylheptane (OL135) and cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-ylester (URB597) in CCI neuropathic mice. The antiallodynic effects of ST4070 were prevented by pretreatment with cannabinoid type 1 and cannabinoid type <em>2</em> receptor antagonists and by the selective peroxisome proliferator-activated receptor α antagonist [(<em>2</em>S)-<em>2</em>-[[(1Z)-1-methyl-3-oxo-3-[4-(trifluoromethyl)phenyl]-1-propenyl]amino]-3-[4-[<em>2</em>-(5-methyl-<em>2</em>-phenyl-4-oxazolyl)ethoxy]phenyl]propyl]-carbamic acid ethyl ester (GW6471). The administration of ST4070 generated long-lasting neuropathic pain relief compared with pregabalin and the FAAH inhibitors OL135 and URB597. Taken together, the reversible FAAH inhibitor ST4070 seems to be a promising novel therapeutic agent for the management of neuropathic pain.

As the incidence of obesity continues to increase, the development of effective therapies is a high priority. The endocannabinoid system has emerged as an important influence on the regulation of energy homeostasis. The endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em> act on cannabinoid receptor-1 (CB1) in the brain and many peripheral tissues causing a net anabolic action. This includes increasing food intake, and causing increased lipogenesis and fat storage in adipose tissue and liver. The endocannabinoid system is hyperactive in obese humans and animals, and treating them with CB1 antagonists causes weight loss and improved lipid and glucose profiles. Although clinical trials with CB1 antagonists have yielded beneficial metabolic effects, concerns about negative affect have limited the therapeutic potential of the first class of CB1 antagonists available.

Endocannabinoids are a group of biologically active endogenous lipids that have recently emerged as important mediators in energy balance control. The two best studied endocannabinoids, anandamide (N-arachidonoylethanolamine, AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are the endogenous ligands of the central and peripheral cannabinoid receptors. Furthermore, AEA binds to the transient receptor potential vanilloid type-1 (TRPV1), a capsaicin-sensitive, non-selective cation channel. The synthesis of these endocannabinoids is catalyzed by the N-acylphosphatidylethanolamine-selective phospholipase D (NAPE-PLD) and the sn-1-selective diacylglycerol lipase (DAGL), whereas their degradation is accomplished by the fatty acid amide hydrolase (FAAH) and the monoglyceride lipase (MGL), respectively. We investigated the presence of a functional endocannabinoid system in human adipose tissue from seven healthy subjects. Subcutaneous abdominal adipose tissue underwent biochemical and molecular biology analyses, aimed at testing the expression of this system and its functional activity. AEA and <em>2</em>-AG levels were detected and quantified by HPLC. Real time PCR analyzed the expression of the endocannabinoid system and immunofluorescence assays showed the distribution of its components in the adipose tissue. Furthermore, binding assay for the cannabinoid and vanilloid receptors and activity assay for each metabolic enzyme of the endocannabinoid system gave clear evidence of a fully operating system. The data presented herein show for the first time that the human adipose tissue is able to bind AEA and <em>2</em>-AG and that it is endowed with the biochemical machinery to metabolize endocannabinoids.

Many motivated and addiction-related behaviors are sustained by activity of both dopamine D1- and D<em>2</em>-type receptors (D1Rs and D<em>2</em>Rs) as well as CB1 receptors (CB1Rs) in the nucleus accumbens (NAc). Here, we use in vitro whole-cell patch-clamp electrophysiology to describe an endocannabinoid (eCB)-dopamine receptor interaction in adult rat NAc core neurons. D1R and D<em>2</em>R agonists in combination enhanced firing, with no effect of a D1R or D<em>2</em>R agonist alone. This D1R+D<em>2</em>R-mediated firing increase required CB1Rs, since it was prevented by the CB1R antagonists AM<em>2</em>51 and Rimonabant. The D1R+D<em>2</em>R firing increase also required phospholipase C (PLC), the major synthesis pathway for the eCB <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and one of several pathways for anandamide. Further, inhibition of <em>2</em>-AG hydrolysis with the monoglyceride lipase (MGL) inhibitor JZL184 allowed subthreshold levels of D1R+D<em>2</em>R receptor agonists to enhance firing, while inhibition of anandamide hydrolysis with the fatty acid amide hydrolase (FAAH) inhibitors URB597 or AM3506 did not. Filling the postsynaptic neuron with <em>2</em>-AG enabled subthreshold D1R+D<em>2</em>R agonists to increase firing, and the <em>2</em>AG+D1R+D<em>2</em>R increase in firing was prevented by a CB1R antagonist. Also, the metabotropic glutamate receptor 5 (mGluR5) blocker MPEP prevented the ability of JZL184 to promote subthreshold D1R+D<em>2</em>R enhancement of firing, while the <em>2</em>-AG+D1R+D<em>2</em>R increase in firing was not prevented by the mGluR5 blocker, suggesting that mGluR5s acted upstream of <em>2</em>-AG production. Thus, our results taken together are consistent with the hypothesis that NAc core eCBs mediate dopamine receptor (DAR) enhancement of firing, perhaps providing a cellular mechanism underlying the central role of NAc core D1Rs, D<em>2</em>Rs, CB1Rs, and mGluR5s during many drug-seeking behaviors.

Genetic alterations or pharmacological treatments affecting endocannabinoid signaling have profound effects on synaptic and neuronal properties and, under certain conditions, may improve higher brain functions. Down syndrome (DS), a developmental disorder caused by triplication of chromosome <em>2</em>1, is characterized by deficient cognition and inevitable development of the Alzheimer disease (AD) type pathology during aging. Here we used JZL184, a selective inhibitor of monoacylglycerol lipase (MAGL), to examine the effects of chronic MAGL inhibition on the behavioral, biochemical, and synaptic properties of aged Ts65Dn mice, a genetic model of DS. In both Ts65Dn mice and their normosomic (<em>2</em>N) controls, JZL184-treatment increased brain levels of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and decreased levels of its metabolites such as arachidonic acid, prostaglandins PGD<em>2</em>, PGE<em>2</em>, PGFα, and PGJ<em>2</em>. Enhanced spontaneous locomotor activity of Ts65Dn mice was reduced by the JZL184-treatement to the levels observed in <em>2</em>N animals. Deficient long-term memory was also improved, while short-term and working types of memory were unaffected. Furthermore, reduced hippocampal long-term potentiation (LTP) was increased in the JZL184-treated Ts65Dn mice to the levels observed in <em>2</em>N mice. Interestingly, changes in synaptic plasticity and behavior were not observed in the JZL184-treated <em>2</em>N mice suggesting that the treatment specifically attenuated the defects in the trisomic animals. The JZL184-treatment also reduced the levels of Aβ40 and Aβ4<em>2</em>, but had no effect on the levels of full length APP and BACE1 in both Ts65Dn and <em>2</em>N mice. These data show that chronic MAGL inhibition improves the behavior and brain functions in a DS model suggesting that pharmacological targeting of MAGL may be considered as a perspective new approach for improving cognition in DS.

Adult animals submitted to a single prolonged episode of maternal deprivation (MD) [<em>2</em>4 h, postnatal days (PND) 9-10] show behavioral alterations that resemble specific symptoms of schizophrenia. These behavioral impairments may be related to neuronal loss in the hippocampus triggered by elevated glucocorticoids. Furthermore, our previous data suggested functional relationships between MD stress and the endocannabinoid system. In this study, we addressed the effects of MD on hippocampal glial cells and the possible relationship with changes in plasma corticosterone (CORT) levels. In addition, we investigated the putative involvement of the endocannabinoid system by evaluating (a) the effects of MD on hippocampal levels of endocannabinoids (b) The modulation of MD effects by two inhibitors of endocannabinoids inactivation, the fatty acid amide hydrolase inhibitor N-arachidonoyl-serotonin (AA-5-HT), and the endocannabinoid reuptake inhibitor, OMDM-<em>2</em>. Drug treatments were administered once daily from PND 7 to PND 1<em>2</em> at a dose of 5 mg/kg, and the animals were sacrificed at PND 13. MD induced increased CORT levels in both genders. MD males also showed an increased number of astrocytes in CA1 and CA3 areas and a significant increase in hippocampal <em>2</em>-<em>arachidonoylglycerol</em>. The cannabinoid compounds reversed the endocrine and cellular effects of maternal deprivation. We provide direct evidence for gender-dependent cellular and biochemical effects of MD on developmental hippocampus, including changes in the endocannabinoid system.

Exercise-induced antinociception is widely described in the literature, but the mechanisms involved in this phenomenon are poorly understood. Systemic (s.c.) and central (i.t., i.c.v.) pretreatment with CB₁ and CB₂ cannabinoid receptor antagonists (AM<em>2</em>51 and AM630) blocked the antinociception induced by an aerobic exercise (AE) protocol in both mechanical and thermal nociceptive tests. Western blot analysis revealed an increase and activation of CB₁ receptors in the rat brain, and immunofluorescence analysis demonstrated an increase of activation and expression of CB₁ receptors in neurons of the periaqueductal gray matter (PAG) after exercise. Additionally, pretreatment (s.c., i.t. and i.c.v.) with endocannabinoid metabolizing enzyme inhibitors (MAFP and JZL184) and an anandamide reuptake inhibitor (VDM11) prolonged and intensified this antinociceptive effect. These results indicate that exercise could activate the endocannabinoid system, producing antinociception. Supporting this hypothesis, liquid-chromatography/mass-spectrometry measurements demonstrated that plasma levels of endocannabinoids (anandamide and <em>2</em>-<em>arachidonoylglycerol</em>) and of anandamide-related mediators (palmitoylethanolamide and oleoylethanolamide) were increased after AE. Therefore, these results suggest that the endocannabinoid system mediates aerobic exercise-induced antinociception at peripheral and central levels.

The endocannabinoid system (ECS) including its receptors, endogenous ligands ("endocannabinoids"), synthesizing and degradating enzymes, and transporter molecules has been detected from the earliest embryonal stages and throughout pre- and postnatal development; endocannabinoids, notably <em>2</em>-<em>arachidonoylglycerol</em>, are also present in maternal milk. During three developmental stages, (1) early embryonal, (<em>2</em>) prenatal brain development, and (3) postnatal suckling, the ECS plays an essential role for development and survival. During early gestation, successful embryonal passage through the oviduct and implantation into the uterus require critical enzymatic control of the endocannabinoids. During fetal life, endocannabinoids and the cannabinoid CB(1) receptor are important for brain development, regulating neural progenitor differentiation and guiding axonal migration and synaptogenesis. Postnatally, CB(1) receptor activation by <em>2</em>-<em>arachidonoylglycerol</em> appears to play a critical role in the initiation of milk suckling in mouse pups, possibly by enabling innervation and/or activation of the tongue muscles. Perinatal manipulation of the ECS, by administering cannabinoids or by maternal marijuana consumption, alters neurotransmitter and behavioral functions in the offspring. Interestingly, the sequelae of prenatal cannabinoids are similar to many effects of prenatal stress, which may suggest that prenatal stress impacts on the ECS and that vice versa prenatal cannabinoid exposure may interfere with the ability of the fetus to cope with the stress. Future studies should further clarify the mechanisms involved in the developmental roles of the ECS and understand better the adverse effects of prenatal exposure, to design strategies for the treatment of conditions including infertility, addiction, and failure-to-thrive.

Monoacylglycerol lipase (MAGL) is the enzyme responsible for the inactivation of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). MAGL inhibitors show analgesic and tissue-protecting effects in several disease models. However, the few efficient and selective MAGL inhibitors described to date block the enzyme irreversibly, and this can lead to pharmacological tolerance. Hence, additional classes of MAGL inhibitors are needed to validate this enzyme as a therapeutic target. Here we report a potent, selective, and reversible MAGL inhibitor (IC50=0.18 μM) which is active in vivo and ameliorates the clinical progression of a multiple sclerosis (MS) mouse model without inducing undesirable CB1 -mediated side effects. These results support the interest in MAGL as a target for the treatment of MS.

OBJECTIVE

Pharmacological activation of cannabinoid CB(1) and CB(<em>2</em>) receptors is a therapeutic strategy to treat chronic and inflammatory pain. It was recently reported that a mixture of natural triterpenes α- and β-amyrin bound selectively to CB(1) receptors with a subnanomolar K(i) value (133 pM). Orally administered α/β-amyrin inhibited inflammatory and persistent neuropathic pain in mice through both CB(1) and CB(<em>2</em>) receptors. Here, we investigated effects of amyrins on the major components of the endocannabinoid system.

METHODS

We measured CB receptor binding interactions of α- and β-amyrin in validated binding assays using hCB(1) and hCB(<em>2</em>) transfected CHO-K1 cells. Effects on endocannabinoid transport in U937 cells and breakdown using homogenates of BV<em>2</em> cells and pig brain, as well as purified enzymes, were also studied.

RESULTS

There was no binding of either α- or β-amyrin to hCB receptors in our assays (K(i)>> 10 µM). The triterpene β-amyrin potently inhibited <em>2</em>-arachidonoyl glycerol (<em>2</em>-AG) hydrolysis in pig brain homogenates, but not that of anandamide. Although β-amyrin only weakly inhibited purified human monoacylglycerol lipase (MAGL), it also inhibited α,β-hydrolases and more potently inhibited <em>2</em>-AG breakdown than α-amyrin and the MAGL inhibitor pristimerin in BV<em>2</em> cell and pig brain homogenates.

CONCLUSIONS

We propose that β-amyrin exerts its analgesic and anti-inflammatory pharmacological effects via indirect cannabimimetic mechanisms by inhibiting the degradation of the endocannabinoid <em>2</em>-AG without interacting directly with CB receptors. Triterpenoids appear to offer a very broad and largely unexplored scaffold for inhibitors of the enzymic degradation of <em>2</em>-AG.

BACKGROUND

This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.<em>2</em>01<em>2</em>.167.issue-8.

Huntington disease (HD) is an inherited, autosomal dominant, neurodegenerative disorder with limited treatment options. Prior to motor symptom onset or neuronal cell loss in HD, levels of the type 1 cannabinoid receptor (CB1) decrease in the basal ganglia. Decreasing CB1 levels are strongly correlated with chorea and cognitive deficit. CB1 agonists are functionally selective (biased) for divergent signaling pathways. In this study, six cannabinoids were tested for signaling bias in in vitro models of medium spiny projection neurons expressing wild-type (STHdh(Q7/Q7)) or mutant huntingtin protein (STHdh(Q111/Q111)). Signaling bias was assessed using the Black and Leff operational model. Relative activity [ΔlogR (τ/KA)] and system bias (ΔΔlogR) were calculated relative to the reference compound WIN55,<em>2</em>1<em>2</em>-<em>2</em> for Gαi/o, Gαs, Gαq, Gβγ, and β-arrestin1 signaling following treatment with <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), anandamide (AEA), CP55,940, Δ(9)-tetrahydrocannabinol (THC), cannabidiol (CBD), and THC+CBD (1:1), and compared between wild-type and HD cells. The Emax of Gαi/o-dependent extracellular signal-regulated kinase (ERK) signaling was 50% lower in HD cells compared with wild-type cells. <em>2</em>-AG and AEA displayed Gαi/o/Gβγ bias and normalized CB1 protein levels and improved cell viability, whereas CP55,940 and THC displayed β-arrestin1 bias and reduced CB1 protein levels and cell viability in HD cells. CBD was not a CB1 agonist but inhibited THC-dependent signaling (THC+CBD). Therefore, enhancing Gαi/o-biased endocannabinoid signaling may be therapeutically beneficial in HD. In contrast, cannabinoids that are β-arrestin-biased--such as THC found at high levels in modern varieties of marijuana--may be detrimental to CB1 signaling, particularly in HD where CB1 levels are already reduced.

The endocannabinoid system, most popularly known as the target of the psychoactive component of marijuana, Δ(9)-tetrahydrocannabinol (THC), is a signaling network that modulates a diverse range of physiological processes including nociception, behavior, cognitive function, appetite, metabolism, motor control, memory formation, and inflammation. While THC and its derivatives have garnered notoriety in the eyes of the public, the endocannabinoid system consists of two endogenous signaling lipids, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and N-arachidonoylethanolamine (anandamide), which activate cannabinoid receptors CB1 and CB<em>2</em> in the nervous system and peripheral tissues. This review will focus on the recent efforts to chemically manipulate <em>2</em>-AG signaling through the development of inhibitors of the <em>2</em>-AG-synthesizing enzyme diacylglycerol lipase (DAGL) or the <em>2</em>-AG-degrading enzyme monoacylglycerol lipase (MAGL), and assessing the therapeutic potential of DAGL and MAGL inhibitors in pain, inflammation, degenerative diseases, tissue injury, and cancer.

<em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG) is a signaling lipid in the central nervous system that is a key regulator of neurotransmitter release. <em>2</em>-AG is an endocannabinoid that activates the cannabinoid CB1 receptor. It is involved in a wide array of (patho)physiological functions, such as emotion, cognition, energy balance, pain sensation and neuroinflammation. In this review, we describe the biosynthetic and metabolic pathways of <em>2</em>-AG and how chemical and genetic perturbation of these pathways has led to insight in the biological role of this signaling lipid. Finally, we discuss the potential therapeutic benefits of modulating <em>2</em>-AG levels in the brain.

It has previously been shown that the endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) inhibit the proliferation of C6 glioma cells in a manner that can be prevented by a combination of capsazepine (Caps) and cannabinoid (CB) receptor antagonists. It is not clear whether the effect of <em>2</em>-AG is due to the compound itself, due to the rearrangement to form 1-<em>arachidonoylglycerol</em> (1-AG) or due to a metabolite. Here, it was found that the effects of <em>2</em>-AG can be mimicked with 1-AG, both in terms of its potency and sensitivity to antagonism by Caps and CB receptor antagonists. In order to determine whether the effect of Caps could be ascribed to actions upon vanilloid receptors, the effect of a more selective vanilloid receptor antagonist, SB366791 was investigated. This compound inhibited capsaicin-induced Ca(<em>2</em>+) influx into rVR1-HEK<em>2</em>93 cells with a pK(B) value of 6.8+/-0.3. The combination of SB366791 and CB receptor antagonists reduced the antiproliferative effect of 1-AG, confirming a vanilloid receptor component in its action. 1-AG, however, showed no direct effect on Ca(<em>2</em>+) influx into rVR1-HEK<em>2</em>93 cells indicative of an indirect effect upon vanilloid receptors. Identification of the mechanism involved was hampered by a large inter-experimental variation in the sensitivity of the cells to the antiproliferative effects of 1-AG. A variation was also seen with anandamide, which was not a solubility issue, since its water soluble phosphate ester showed the same variability. In contrast, the sensitivity to methanandamide, which was not sensitive to antagonism by the combination of Caps and CB receptor antagonists, but has similar physicochemical properties to anandamide, did not vary between experiments. This variation greatly reduces the utility of these cells as a model system for the study of the antiproliferative effects of anandamide. Nevertheless, it was possible to conclude that the antiproliferative effects of anandamide were not solely mediated by either its hydrolysis to produce arachidonic acid or its CB receptor-mediated activation of phospholipase A(<em>2</em>) since palmitoyltrifluoromethyl ketone did not prevent the response to anandamide. The same result was seen with the fatty acid amide hydrolase inhibitor palmitoylethylamide. Increasing intracellular arachidonic acid by administration of arachidonic acid methyl ester did not affect cell proliferation, and the modest antiproliferative effect of umbelliferyl arachidonate was not prevented by a combination of Caps and CB receptor antagonists.

Male infertility is a major cause of problems for many couples in conceiving a child. Recently, lifestyle pastimes such as alcohol, tobacco and marijuana have been shown to have further negative effects on male reproduction. The endocannabinoid system (ECS), mainly through the action of anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) at cannabinoid (CB(1), CB(<em>2</em>)) and vanilloid (TRPV1) receptors, plays a crucial role in controlling functionality of sperm, with a clear impact on male reproductive potential. Here, sperm from fertile and infertile men were used to investigate content (through LC-ESI-MS), mRNA (through quantitative RT-PCR), protein (through Western Blotting and ELISA) expression, and functionality (through activity and binding assays) of the main metabolic enzymes of AEA and <em>2</em>-AG (NAPE-PLD and FAAH, for AEA; DAGL and MAGL for <em>2</em>-AG), as well as of their binding receptors CB(1), CB(<em>2</em>) and TRPV1. Our findings show a marked reduction of AEA and <em>2</em>-AG content in infertile seminal plasma, paralleled by increased degradation: biosynthesis ratios of both substances in sperm from infertile versus fertile men. In addition, TRPV1 binding was detected in fertile sperm but was undetectable in infertile sperm, whereas that of CB(1) and CB(<em>2</em>) receptors was not statistically different in the two groups. In conclusion, this study identified unprecedented alterations of the ECS in infertile sperm, that might impact on capacitation and acrosome reaction, and hence fertilization outcomes. These alterations might also point to new biomarkers to determine male reproductive defects, and identify distinct ECS elements as novel targets for therapeutic exploitation of ECS-oriented drugs to treat male fertility problems.

The endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) mediates activity-dependent depression of excitatory neurotransmission at central synapses, but the molecular regulation of <em>2</em>-AG synthesis is not well understood. Here we identify a functional interaction between the <em>2</em>-AG synthetic enzyme diacylglycerol lipase-α (DGLα) and calcium/calmodulin dependent protein kinase II (CaMKII). Activated CaMKII interacted with the C-terminal domain of DGLα, phosphorylated two serine residues and inhibited DGLα activity. Consistent with an inhibitory role for CaMKII in <em>2</em>-AG synthesis, in vivo genetic inhibition of CaMKII increased striatal DGL activity and basal levels of <em>2</em>-AG, and CaMKII inhibition augmented short-term retrograde endocannabinoid signaling at striatal glutamatergic synapses. Lastly, blockade of <em>2</em>-AG breakdown using concentrations of JZL-184 that have no effect in wild-type mice produced a hypolocomotor response in mice with reduced CaMKII activity. These findings provide mechanistic insights into the molecular regulation of striatal endocannabinoid signaling with implications for physiological control of motor function.

Activation of both presynaptic metabotropic cannabinoid type 1 receptors (CB(1)s) and ionotropic kainate receptors (KARs) can efficiently modulate GABA release at many synapses of the CNS. The inhibitory effect of kainic acid (KA) has been ascribed to metabotropic actions, and KAR-induced release of secondary neuromodulatory agents may partly mediate these actions. Here, we investigated the involvement of the endocannabinoid system in the modulation of GABAergic synaptic transmission by pharmacological activation of KARs with KA in CA1 pyramidal neurons of the mouse hippocampus. We show that the depression of GABAergic synaptic transmission induced by KA (3 μm) is strongly inhibited by the simultaneous blockade of CB(1) and GABA(B) receptors with SR141716A (5 μm) and CGP55845 (5 μm), respectively. KA induces a calcium-dependent mobilization of the endocannabinoid anandamide (AEA) by activation of GluK<em>2</em>-containing KARs in postsynaptic pyramidal neurons. Consistently, the effect of KA is prolonged by the inhibitor of AEA degradation URB597 (1 μm) in a CB(1)-dependent manner, but it is not altered by blockade of degradation or synthesis of the other main endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>AG). Hence, our work reveals that the pharmacological activation of KARs leads to the stimulation of secondary metabotropic signaling systems. In addition, these data further underline the profound mechanistic differences between exogenous and endogenous activation of KARs in the hippocampus.

There is good evidence that plant-derived and synthetic cannabinoids possess neuroprotective properties. These compounds, as a result of effects upon CB(1) cannabinoid receptors, reduce the release of glutamate, and in addition reduce the influx of calcium following NMDA receptor activation. The major obstacle to the therapeutic utilization of such compounds are their psychotropic effects, which are also brought about by actions on CB(1) receptors. However, synthesis of the endogenous cannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, which also have neuroprotective properties, are increased under conditions of severe inflammation and ischemia, raising the possibility that compounds that prevent their metabolism may be of therapeutic utility without having the drawback of producing psychotropic effects. In this review, the evidence indicating neuroprotective actions of plant-derived, synthetic and endogenous cannabinoids is presented. In addition, the pharmacological properties of endogenous anandamide-related compounds that are not active upon cannabinoid receptors, but which are also produced during conditions of severe inflammation and ischemia and may contribute to a neuroprotective action are reviewed.

Involvement of cannabinoid CB(<em>2</em>) receptor and effect of cannabinoid CB(<em>2</em>) receptor antagonist/inverse agonists on cutaneous inflammation were investigated. Mice ears topically exposed to an ether-linked analogue of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG-E) or selective cannabinoid CB(<em>2</em>) receptor agonist, {4-[4-(1,1-dimethylheptyl)-<em>2</em>,6-dimethoxy-phenyl]-6.6-dimethyl-bicyclo[3.1.1]hept-<em>2</em>-en-<em>2</em>-yl}-methanol (HU-308), had early and late ear swelling (0--<em>2</em>4 h and 1--8 days after exposure, respectively). Both types of responses induced by <em>2</em>-AG-E were significantly suppressed by oral administration of cannabinoid CB(<em>2</em>) receptor antagonist/inverse agonists, [N-(benzo[1,3]dioxol-5-ylmethyl)-7-methoxy-<em>2</em>-oxo-8-pentyloxy-1,<em>2</em>-dihydroquinoline-3-carboxamide] (JTE-907) and {N-[(1S)-endo-1,3,3-trimethylbicyclo[<em>2</em>.<em>2</em>.1]heptan-<em>2</em> yl]5-(4-chloro-3-methyl-phenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide}} (SR 1445<em>2</em>8). In contrast, JTE-907 did not affect arachidonic acid-induced swelling. Orally administered JTE-907 (0.1-10 mg/kg) and SR 1445<em>2</em>8 (1 mg/kg) also produced significant inhibition of dinitrofluorobenzene-induced ear swelling, with increased cannabinoid CB(<em>2</em>) receptor mRNA expression observed in the inflamed ear. These results suggest that cannabinoid CB(<em>2</em>) receptor is partially involved in local inflammatory responses and cannabinoid CB(<em>2</em>) receptor antagonist/inverse agonist has beneficial effects on ear swelling.