The naturally occurring mammalian endocannabinoids possess biological attributes that extend beyond interaction with cannabinoid receptors. These extended biological properties are the result of oxidative metabolism of the principal mammalian endocannabinoids arachidonoyl ethanolamide (anandamide; A-EA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Both endocannabinoids are oxidized by cyclo-oxygenase-<em>2</em> (COX-<em>2</em>), but not by COX-1, to a series of prostaglandin derivatives (PGs) with quite different biological properties from those of the parent substrates. PG ethanolamides (prostamides, PG-EAs) and PG glyceryl esters (PG-Gs) are not only pharmacologically distinct from their parent endocannabinoids, they are distinct from the corresponding acidic PGs, and are differentiated from each other. Ethanolamides and glyceryl esters of the major prostanoids PGD<em>2</em>, PGE<em>2</em>, PGF<em>2</em>α, and PGI<em>2</em> are formed by the various PG synthases, and thromboxane ethanolamides and glyceryl esters are not similarly produced. COX-<em>2</em> is also of interest by virtue of its corollary central role in modulating endocannabinoid tone, providing a new therapeutic approach for treating pain and anxiety. Other major oxidative conversion pathways are provided for both A-EA and <em>2</em>-AG by several lipoxygenases (LOXs), resulting in the formation of numerous hydroxyl metabolites. These do not necessarily represent inactivation pathways for endocannabinoids but may mimic or modulate the endocannabinoids or even display alternative pharmacology. Similarly, A-EA and <em>2</em>-AG may be oxidized by P450 enzymes. Again a very diverse number of metabolites are formed, with either cannabinoid-like biological properties or an introduction of disparate pharmacology. The biological activity of epoxy and hydroxyl derivatives of the endocannabinoids remains to be fully elucidated. This review attempts to consolidate and compare the findings obtained to date in an increasingly important research area. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance".

Cocaine is a highly addictive drug that acts upon the brain's reward circuitry via the inhibition of monoamine uptake. Endogenous cannabinoids (eCB) are lipid molecules released from midbrain dopamine (DA) neurons that modulate cocaine's effects through poorly understood mechanisms. We find that cocaine stimulates release of the eCB, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), in the rat ventral midbrain to suppress GABAergic inhibition of DA neurons, through activation of presynaptic cannabinoid CB1 receptors. Cocaine mobilizes <em>2</em>-AG via inhibition of norepinephrine uptake and promotion of a cooperative interaction between Gq/11-coupled type-1 metabotropic glutamate and α1-adrenergic receptors to stimulate internal calcium stores and activate phospholipase C. The disinhibition of DA neurons by cocaine-mobilized <em>2</em>-AG is also functionally relevant because it augments DA release in the nucleus accumbens in vivo. Our results identify a mechanism through which the eCB system can regulate the rewarding and addictive properties of cocaine.

We examined the relative importance of G (Gi) protein-coupled brain-type (CB1-R) and spleen-type (CB<em>2</em>-R) cannabinoid receptors in preimplantation embryo development using agonists and antagonists specific to CB1-R and CB<em>2</em>-R. The results establish that endogenous cannabinoid ligands, anandamide and sn-<em>2</em> <em>arachidonoylglycerol</em>, arrest embryo development in vitro, and this effect is reversed by CB1-R antagonists SR141716A or AM <em>2</em>51, but not by SR1445<em>2</em>8, a CB<em>2</em>-R antagonist. A CB<em>2</em>-R selective agonist AM 663 failed to affect embryo development. These results suggest that cannabinoid effects on embryo development are mediated by CB1-R. We also observed that delta9-tetrahydrocannabinol ([-]THC) infused in the presence of cytochrome P450 inhibitors interfered with blastocyst implantation. This adverse effect was reversed by coinfusion of SR141716A. The less active stereoisomer (+)THC plus the inhibitors failed to affect implantation. Analysis of tissue levels demonstrated that uterine accumulation of (-)THC occurred when it was infused in the presence of the P450 inhibitors. These results demonstrate that the uterus and perhaps the embryo have the cytochrome P450 enzymes to metabolize (-)THC and neutralize its adverse effects on implantation. Collectively, the present study demonstrates that cannabinoid effects on embryo development and implantation are mediated by embryonic and/or uterine CB1-R, but not CB<em>2</em>-R.

Anandamide is a ligand of the endocannabinoid system. Animals show a depletion following repeated Δ(9)-tetrahydrocannabinol (THC) administration but the effect of cannabis use on central nervous system levels of endocannabinoids has not been previously examined in humans. Cerebrospinal fluid (CSF) levels of the endocannabinoids anandamide, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and related lipids were tested in 33 volunteers (<em>2</em>0 cannabis users). Lower levels of CSF anandamide and higher levels of <em>2</em>-AG in serum were observed in frequent compared with infrequent cannabis users. Levels of CSF anandamide were negatively correlated with persisting psychotic symptoms when drug-free. Higher levels of anandamide are associated with a lower risk of psychotic symptoms following cannabis use.

Endocannabinoid signaling is terminated by enzymatic hydrolysis, a process that, for <em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG), is mediated by monoacylglycerol lipase (MAGL). The piperidine carbamate, 4-nitrophenyl- 4-(dibenzo[d] [1,3]dioxol-5-yl (hydroxy) methyl) piperidine- 1-carboxylate (JZL184), is a drug that inhibits MAGL and presents high potency and selectivity. Thus, JZL184 increases the levels of <em>2</em>-AG, an endocannabinoid that acts on the CB1 and CB<em>2</em> cannabinoid receptors. Here, we investigated the effects of MAGL inhibition, with a single dose (16 mg/kg, intraperitoneally (i.p.)) of JZL184, in a murine model of lipopolysaccharide (LPS) -induced acute lung injury (ALI) 6, <em>2</em>4 and 48 hours after the inflammatory insult. Treatment with JZL184 decreased the leukocyte migration into the lungs as well as the vascular permeability measured through the bronchoalveolar lavage fluid (BAL) and histological analysis. JZL184 also reduced the cytokine and chemokine levels in the BAL and adhesion molecule expression in the blood and BAL. The CB1 and CB<em>2</em> receptors were considered involved in the anti-inflammatory effects of JZL184 because the AM<em>2</em>81 selective CB1 receptor antagonist (1-(<em>2</em>,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide) and the AM630 selective CB<em>2</em> receptor antagonist ([6-iodo-<em>2</em>-methyl-1-[<em>2</em>-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)-methanone) blocked the anti-inflammatory effects previously described for JZL184. It was concluded that MAGL inhibition, and consequently the increase in <em>2</em>-AG levels, produced anti-inflammatory effects in a murine model of LPS-induced ALI, a finding that was considered a consequence of the activation of the CB1 and CB<em>2</em> receptors.

Endocannabinoids control hippocampal inhibitory synaptic transmission through activation of presynaptic CB(1) receptors. During depolarization-induced suppression of inhibition (DSI), endocannabinoids are synthesized upon postsynaptic depolarization. The endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) may mediate hippocampal DSI. Currently, the best studied pathway for biosynthesis of <em>2</em>-AG involves the enzyme diacylglycerol lipase (DAGL). However, whether DAGL is necessary for hippocampal DSI is controversial and was not systematically addressed. Here, we investigate DSI at unitary connections between CB(1) receptor-containing interneurons and pyramidal neurons in CA1. We found that the novel DAGL inhibitor OMDM-188, as well as the established inhibitor RHC-80<em>2</em>67, did not affect DSI. As reported previously, effects of the DAGL inhibitor tetrahydrolipstatin depended on the application method: postsynaptic intracellular application left DSI intact, while incubation blocked DSI. We show that all DAGL inhibitors tested block slow self-inhibition in neocortical interneurons, which involves DAGL. We conclude that DAGL is not involved in DSI at unitary connections in hippocampus.

Preimplantation embryo development to the blastocyst stage and uterine differentiation to the receptive state are prerequisites for embryo implantation. Burgeoning evidence suggests that endocannabinoid signaling is critical to early pregnancy events. Anandamide (N-arachidonoylethanolamine) and <em>2</em>-AG (<em>2</em>-<em>arachidonoylglycerol</em>) are two major endocannabinoids that bind to and activate G-protein coupled cannabinoid receptors CB1 and CB<em>2</em>. We have previously shown that a physiological tone of anandamide is critical to preimplantation events in mice, since either silencing or amplification of anandamide signaling causes retarded development and oviductal retention of embryos via CB1, leading to deferred implantation and compromised pregnancy outcome. Whether <em>2</em>-AG, which also influences many biological functions, has any effects on early pregnancy remains unknown. Furthermore, mechanisms by which differential uterine endocannabinoid gradients are established under changing pregnancy state is not clearly understood. We show here that <em>2</em>-AG is present at levels one order of magnitude higher than those of anandamide in the mouse uterus, but with similar patterns as anandamide, i.e. lower levels at implantation sites and higher at interimplantation sites. We also provide evidence that region- and stage-specific uterine expression of N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD) and fatty acid amide hydrolase (FAAH), and sn-1-diacylglycerol (DAG) lipase alpha (DAGLalpha) and monoacylglycerol lipase (MAGL) for synthesis and hydrolysis of anandamide and <em>2</em>-AG, respectively, creates endocannabinoid gradients conducive to implantation. Our genetic evidence suggests that FAAH is the major degrading enzyme for anandamide, whereas COX-<em>2</em>, MAGL and to some extent COX-1 participate in metabolizing <em>2</em>-AG in the pregnant uterus. The results suggest that aberrant functioning of these pathways impacting uterine anandamide and/or <em>2</em>-AG levels would compromise pregnancy outcome.

The serine hydrolase monoacylglycerol lipase (MGL) modulates endocannabinoid signaling in vivo by inactivating <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), the main endogenous agonist for central CB1 and peripheral CB<em>2</em> cannabinoid receptors. To characterize this key endocannabinoid enzyme by mass spectrometry-based proteomics, we first overexpressed recombinant hexa-histidine-tagged human MGL (hMGL) in Escherichia coli and purified it in a single chromatographic step with high yield (approximately 30 mg/L). With <em>2</em>-AG as substrate, hMGL displayed an apparent V max of <em>2</em>5 micromol/(microg min) and K m of 19.7 microM, an affinity for <em>2</em>-AG similar to that of native rat-brain MGL (rMGL) (Km=33.6 microM). hMGL also demonstrated a comparable affinity (Km approximately 8-9 microM) for the novel fluorogenic substrate, arachidonoyl, 7-hydroxy-6-methoxy-4-methylcoumarin ester (AHMMCE), in a sensitive, high-throughput fluorometric MGL assay. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) unequivocably demonstrated the mass (34,1<em>2</em>6 Da) and purity of this hMGL preparation. After in-solution tryptic digestion, hMGL full proteomic characterization was carried out, which showed (1) an absence of intramolecular disulfide bridges in the functional, recombinant enzyme and (<em>2</em>) the post-translational removal of the enzyme's N-terminal methionine. Availability of sufficient quantities of pure, well-characterized hMGL will enable further molecular and structural profiling of this key endocannabinoid-system enzyme.

Using two distinct anti-CB<em>2</em> receptor Abs, we investigated the expression patterns of the peripheral cannabinoid receptor CB<em>2</em> in human secondary lymphoid organs. Immunohistochemical analysis using an N-terminal specific anti-CB<em>2</em> Ab revealed high protein expression in the germinal centers (GCs) of secondary follicles. A C-terminal specific anti-CB<em>2</em> Ab, which only recognizes a nonphosphorylated inactive receptor, showed positivity in the mantle zones (MZs) and marginal zones (MGZs) of the secondary follicles where resting cells reside, and in the primary follicles. In contrast, no positivity was observed in GCs using the C-terminal Ab, suggesting that active CB<em>2</em> receptors are mainly present on cells in the GCs. Dual immunohistochemical analysis revealed that B lymphocytes express the CB<em>2</em> protein abundantly. In contrast to B cells in the MZ or MGZ, CB<em>2</em>-expressing cells in the GCs coexpress the costimulatory membrane protein CD40, which is mainly expressed in the GCs and at very low levels in the MZs and MGZs and the proliferation marker Ki-67. Using the human Raji B cell line as a model, we demonstrate in a transwell assay that moderate migration occurs upon stimulation of the CB<em>2</em> receptor with the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em>, which is enhanced by CD40 costimulation. Our findings, that GC-related cells express active CB<em>2</em> and that CB<em>2</em>-dependent migration requires CD40 costimulation, suggest that CB<em>2</em> is involved in B cell activation.

Although adverse effects of cannabinoids on pregnancy have been indicated for many years, the mechanisms by which they exert their actions were not clearly understood. Only recently, molecular and biochemical approaches have led to the identification of two types of cannabinoid receptors, brain-type receptors (CB1-R) and spleen-type receptors (CB<em>2</em>-R), which mediate cannabinoid effects. These findings were followed by the discovery of endocannabinoids, anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). The natural cannabinoids and endocannabinoids exert their effects via cannabinoid receptors and share similar pharmacological and physiological properties. Recent demonstration of expression of functional CB1-R in the preimplantation embryo and synthesis of anandamide in the pregnant uterus of mice suggests that cannabinoid ligand-receptor signaling is operative in the regulation of preimplantation embryo development and implantation. This review describes recent observations and their significance in embryo-uterine interactions during implantation and future research directions in this emerging area of interest.

The chemotherapeutic agent cisplatin may produce emesis via release of several neurotransmitters such as serotonin (5-HT), substance P and/or dopamine as well as production of prostaglandins (PGs). Administration of synthetic <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) but not of anandamide, which are two putative endocannabinoids, causes vomiting via its downstream metabolites such as arachidonic acid (AA) and PGs in the least shrew (Cryptotis parva). We report here that cisplatin (0, 5, 10 and <em>2</em>0 mg/kg, i.p.) causes dose- and time-dependent increases in brain tissue levels of <em>2</em>-AG but not anandamide in this vomiting species. Concomitantly, intestinal tissue levels of both endocannabinoids are relatively reduced. Selective inhibitors [arachidonoyl-serotonin (AA-5-HT) and URB597, 0-5 and 0-10 mg/kg, i.p.] of one of the major endocannabinoid metabolic enzymes, the intracellular fatty acid amide hydrolase (FAAH), do not significantly prevent vomiting produced by emetic doses of i.p.-administered <em>2</em>-AG, cisplatin or the dopamine receptor agonist apomorphine. At large doses (10 and <em>2</em>0 mg/kg, respectively), both FAAH inhibitors caused emesis per se. Administration of one selective uptake inhibitor of endocannabinoids, OMDM1 (0-5 mg/kg, i.p.), also did not significantly prevent emesis by the direct and indirect emetic stimuli, and likewise caused emesis by itself at a high (10 mg/kg) dose. However, another selective uptake inhibitor, VDM11, did not produce significant emesis per se and prevented emesis caused by apomorphine. Both the corticosteroid dexamethasone, and the cyclooxygenase inhibitor indomethacin, reduced vomiting produced by cisplatin. These data: (a) provide the first evidence that cisplatin causes a selective increase in <em>2</em>-AG levels in the brain, and (b) support the established notion that <em>2</em>-AG may produce some of its effects, including emesis, via downstream metabolites produced independently of FAAH.

Endogenous <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) inhibits invasion of androgen-independent prostate cancer cells. Blocking cellular hydrolysis of <em>2</em>-AG to increase its endogenous concentration results in a decrease in cell invasion. A series of compounds containing a trifluoromethyl ketone (TFK) moiety or the methyl analog (known to inhibit carboxylesterases) were investigated for their ability to inhibit <em>2</em>-AG hydrolysis and prostate cancer cell invasion. Compounds containing a thioether beta to a TFK moiety inhibited <em>2</em>-AG hydrolysis as well as cell invasion in a concentration-dependent manner. Inhibition of <em>2</em>-AG hydrolysis increased concomitantly with inhibitor alkyl chain length from 4- to 1<em>2</em>-carbons while inhibition of cell invasion exhibited a maximum at 8- to 10-carbons of the compounds. These results demonstrate a new series of <em>2</em>-AG hydrolysis inhibitors as a potential therapeutic approach for prostate cancer.

The endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, as well as several anandamide-related N-acylethanolamines, belong to a family of lipid transmitter that regulate fundamental physiological processes, including neurotransmission and neuroinflammation. Their precise quantification in biological matrices can be achieved by gas chromatography-mass spectrometry (GC-MS), but this method typically requires multiple time-consuming purification steps such as solid-phase extraction followed by HPLC. Here we report a novel solid-phase extraction procedure allowing for single-step, and thus higher throughput, purification of endocannabinoids and N-acylethanolamines before GC-MS quantification. We determined the minimal amount of mouse brain tissue required to reliably detect endocannabinoids and N-acylethanolamines when using this approach and provide direct evidence for quantification accuracy by using radioactive and deuterated standards spiked into mouse brain samples. Using this method, we found that mouse brain contains much higher levels of anandamide (>1 nmol/g tissue) than previously reported, whereas levels of <em>2</em>-<em>arachidonoylglycerol</em> and other N-acylethanolamines are well within the range of previous reports. In addition, we show that mouse brain amounts of endocannabinoids and N-acylethanolamines differ depending on animal gender as well as on whether the tissue was fixed or not. Our study shows that endocannabinoid and N-acylethanolamine levels quantified in mouse brain by GC-MS depend closely on tissue amount and preparation as well as on animal gender and that, depending on such parameters, anandamide levels could be underestimated.

Chronic migraine (CM) is frequently associated with medication overuse headache (MOH). The endocannabinoid system plays a role in modulating pain including headache and is involved in the common neurobiological mechanism underlying drug addiction and reward system. Anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> are the most biologically active endocannabinoids, which bind to both central and peripheral cannabinoid receptors. The level of AEA in the extracellular space is controlled by cellular uptake via a specific AEA membrane transporter (AMT), followed by intracellular degradation by the enzyme AEA hydrolase (fatty acid amide hydrolase, FAAH). AMT and FAAH have also been characterized in human platelets. We assayed the activity of AMT and of FAAH in platelets isolated from four groups of subjects: MOH, CM without MOH, episodic migraine and controls. AMT and FAAH were significantly reduced in CM and MOH, compared to either controls or episodic migraine group. This latter finding was observed in both males and females with CM and MOH. Changes observed in the biochemical mechanisms degrading endogenous cannabinoids may reflect an adaptative behaviour induced by chronic headache and/or drug overuse.

BACKGROUND

The "classic" endocannabinoid (eCB) system includes the cannabinoid receptors CB1 and CB<em>2</em>, the eCB ligands anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), and their metabolic enzymes. An emerging literature documents the "eCB deficiency syndrome" as an etiology in migraine, fibromyalgia, irritable bowel syndrome, psychological disorders, and other conditions. We performed a systematic review of clinical interventions that enhance the eCB system--ways to upregulate cannabinoid receptors, increase ligand synthesis, or inhibit ligand degradation.

RESULTS

We searched PubMed for clinical trials, observational studies, and preclinical research. Data synthesis was qualitative. Exclusion criteria limited the results to 184 in vitro studies, 10<em>2</em> in vivo animal studies, and 36 human studies. Evidence indicates that several classes of pharmaceuticals upregulate the eCB system, including analgesics (acetaminophen, non-steroidal anti-inflammatory drugs, opioids, glucocorticoids), antidepressants, antipsychotics, anxiolytics, and anticonvulsants. Clinical interventions characterized as "complementary and alternative medicine" also upregulate the eCB system: massage and manipulation, acupuncture, dietary supplements, and herbal medicines. Lifestyle modification (diet, weight control, exercise, and the use of psychoactive substances--alcohol, tobacco, coffee, cannabis) also modulate the eCB system.

CONCLUSIONS

Few clinical trials have assessed interventions that upregulate the eCB system. Many preclinical studies point to other potential approaches; human trials are needed to explore these promising interventions.

<em>2</em>-Eicosa-5',8',11',14'-tetraenylglycerol (<em>2</em>-AG ether, HU310, noladin ether) is a metabolically stable ether-linked analogue of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), an endogenous cannabinoid receptor ligand. <em>2</em>-AG ether has been used as a valuable experimental tool by a number of investigators. Recently, several groups reported that <em>2</em>-AG ether is present in mammalian brains. We examined in detail whether <em>2</em>-AG ether actually exists in the brains of various mammalian species. We found that <em>2</em>-AG ether is not present, at least in an appreciable amount, in the rat brain by gas chromatography-mass spectrometry analysis and fluorometric high performance liquid chromatography analysis. The level of <em>2</em>-AG ether in the rat brain was below 0.<em>2</em> pmol/g brain, if at all present. Similar results were obtained for the mouse brain, hamster brain, guinea-pig brain and pig brain. The fact that <em>2</em>-AG ether was not detected in the brains of various mammalian species is consistent with the fact that an ether bond is formed through enzymatic replacement of the fatty acyl moiety of 1-acyl dihydroxyacetone phosphate by a fatty alcohol, the resultant 1-O-alkyl dihydroxyacetone phosphate being a common intermediate of the biosynthesis of ether-linked lipids in mammalian tissues. It is rather questionable whether <em>2</em>-AG ether is present in appreciable amounts in the brain and acts as an 'endogenous' cannabinoid receptor ligand.

The endocannabinoid (eCB) system is involved in processes as diverse as control of appetite, perception of pain and the limitation of cancer cell growth and invasion. The enzymes responsible for eCB breakdown are attractive pharmacological targets, and fatty acid amide hydrolase inhibitors, which potentiate the levels of the eCB anandamide, are now undergoing pharmaceutical development. 'Drugable' selective inhibitors of monoacylglycerol lipase, a key enzyme regulating the levels of the other main eCB, <em>2</em>-<em>arachidonoylglycerol</em>, were however not identified until very recently. Their availability has resulted in a large expansion of our knowledge concerning the pharmacological consequences of monoacylglycerol lipase inhibition and hence the role(s) played by the enzyme in the body. In this review, the pharmacology of monoacylglycerol lipase will be discussed, together with an analysis of the therapeutic potential of monoacylglycerol lipase inhibitors as analgesics and anticancer agents.

Cannabinoids include not only plant-derived compounds (of which delta9-tetrahydrocannabinol is the primary psychoactive ingredient of cannabis), but also synthetic agents and endogenous substances termed endocannabinoids which include anandamide (<em>2</em>-arachidonoylethanolamide) and <em>2</em>-<em>arachidonoylglycerol</em>. Cannabinoids act on specific, G-protein-coupled, receptors which are currently divided into two types, CB1 and CB<em>2</em>. Relatively selective agonists and antagonists for these receptors have been developed, although one agent (SR141716A) widely used as an antagonist at CB1 receptors has non-cannabinoid receptor-mediated effects at concentrations which are often used to define the presence of the CB1 receptor. Both cannabinoid receptors are primarily coupled to Gi/o proteins and act to inhibit adenylyl cyclase. Stimulation of CB1 receptors also modulates the activity of K+ and Ca<em>2</em>+ channels and of protein kinase pathways including protein kinase B (Akt) which might mediate effects on apoptosis. CB, receptors may activate the extracellular signal-regulated kinase cascade through ceramide signalling. Cannabinoid actions on the cardiovascular system have been widely interpreted as being mediated by CB1 receptors although there are a growing number of observations, particularly in isolated heart and blood vessel preparations, that suggest that other cannabinoid receptors may exist. Interestingly, the currently identified cannabinoid receptors appear to be related to a wider family of lipid receptor, those for the lysophospholipids, which are also linked to Gi/o protein signalling. Anandamide also activates vanilloid VR1 receptors on sensory nerves and releases the vasoactive peptide, calcitonin gene-related peptide (CGRP), which brings about vasodilatation through its action on CGRP receptors. Current evidence suggests that endocannabinoids have important protective roles in pathophysiological conditions such as shock and myocardial infarction. Therefore, their cardiovascular effects and the receptors mediating them are the subject of increasing investigative interest.

During transit through the epididymis, spermatozoa are normally kept immotile and do not attain the ability to become motile until they reach the caudal epididymis. This study was undertaken to determine whether endocannabinoids play a role in the epididymis and in particular in suppressing the ability of spermatozoa to become motile. We show that the levels of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are high in mouse spermatozoa isolated from the caput (head) of the epididymis, where these cells do not move (or possess sluggish and irregular motility) and decrease dramatically in spermatozoa isolated from the cauda (tail). The subsequent gradient regulates, via autocrine communication, the activity of cannabinoid receptor CNR1 (previously known as CB1) present on the sperm cell membrane and induces caudal spermatozoa to acquire the potential to become motile ("start-up"). Accordingly, the genetic or pharmacological inactivation of CNR1 increases number of motile spermatozoa in caput. Also, blockers of endocannabinoid cellular uptake inhibit the potential to move of spermatozoa and destroy the <em>2</em>-AG gradient throughout the epididymis. This gradient-regulated mechanism may encourage further research for future therapies related to male infertility.

Cannabinoid receptors and their endogenous ligands (endocannabinoids) have been implicated in cocaine and amphetamine reward. Their role in psychostimulant-induced behavioural sensitization still has to be determined. The purpose of the present study was, for one, to compare the effects of a pharmacological and genetic manipulation of CB(1) cannabinoid receptors on amphetamine-induced locomotor sensitization in mice, and, secondly, to quantify the concentration of anandamide and <em>2</em>-<em>arachidonoylglycerol</em> in different forebrain areas of behaviourally sensitized animals. The results can be summarized as follows: CB(1) knockout mice failed to sensitize to the locomotor stimulant effects of amphetamine. On the contrary, administration of the CB(1) receptor antagonist SR141716A (rimonabant; 3mg/kg; i.p.) increased amphetamine sensitization in wild-type animals, indicating that the difference between CB(1) knockouts and SR141716A treated animals could be due to the 'chronic' versus 'acute' loss of CB(1) receptor function, or, alternatively, that SR141716A could exert pharmacological effects beyond its proposed CB(1) antagonistic action. Furthermore, sensitized wild-type mice and animals, which had received a single amphetamine injection on the challenge day, both had increased anandamide concentrations in the dorsal striatum and decreased anandamide levels in the ventral striatum, comprising nucleus accumbens. <em>2</em>-<em>Arachidonoylglycerol</em> levels were decreased in the ventral striatum of sensitized animals only. Together, these findings suggest that prolonged activation of dopamine receptors could alter endocannabinoid levels and support the proposed involvement of the CB(1) receptor in amphetamine sensitization.

Monoacylglycerol lipase (MAGL) represents a primary degradation enzyme of the endogenous cannabinoid (eCB), <em>2</em>-arachidonoyglycerol (<em>2</em>-AG). This study reports a potent covalent MAGL inhibitor, SAR1<em>2</em>7303. The compound behaves as a selective and competitive inhibitor of mouse and human MAGL, which potently elevates hippocampal levels of <em>2</em>-AG in mice. In vivo, SAR1<em>2</em>7303 produces antinociceptive effects in assays of inflammatory and visceral pain. In addition, the drug alters learning performance in several assays related to episodic, working and spatial memory. Moreover, long term potentiation (LTP) of CA1 synaptic transmission and acetylcholine release in the hippocampus, two hallmarks of memory function, are both decreased by SAR1<em>2</em>7303. Although inactive in acute seizure tests, repeated administration of SAR1<em>2</em>7303 delays the acquisition and decreases kindled seizures in mice, indicating that the drug slows down epileptogenesis, a finding deserving further investigation to evaluate the potential of MAGL inhibitors as antiepileptics. However, the observation that <em>2</em>-AG hydrolysis blockade alters learning and memory performance, suggests that such drugs may have limited value as therapeutic agents.

Monoglyceride lipase (MGL) plays a major role in the metabolism of the lipid transmitter <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). This endocannabinoid is known to mediate a large number of physiological processes, and its regulation is thought to be of great therapeutic potential. However, the number of available monoglyceride lipase inhibitors is limited, mostly due to the lack of rapid and accurate pharmacological assays for the enzyme. We have developed a 96-well-format assay for MGL using a nonradiolabeled substrate, 4-nitrophenylacetate. The IC(50) values that were obtained for known inhibitors of MGL using 4-nitrophenylacetate were similar to those reported by using the radiolabeled form of an endogenous substrate, <em>2</em>-oleoylglycerol. In a first small-scale screening, we identified CAY10499 as a novel monoglyceride lipase inhibitor. Thus, we report here the characterization of this submicromolar inhibitor, which acts on MGL through an unprecedented mechanism for inhibitors of this enzyme.

Endocannabinoids, such as anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, are synthesized from membrane phospholipids in the heart and other cardiovascular tissues. They activate cannabinoid CB1 and CB<em>2</em> receptors, transient receptor potential V1 (TRPV1), peroxisome proliferator-activated receptors, and perhaps a novel vascular G-protein-coupled receptor. Inactivation is by cellular uptake and fatty acid amide hydrolase. Endocannabinoids relax coronary and other arteries and decrease cardiac work but seem not to be involved in tonic regulation of cardiovascular function. They act as a stress response system, which is activated, for example, in myocardial infarction and circulatory shock. Endocannabinoids are largely protective; they decrease tissue damage and arrhythmia in myocardial infarction and may reduce progression of atherosclerosis (CB<em>2</em> receptor stimulation inhibits lesion progression), and fatty acid amide hydrolase knockout mice (which have enhanced endocannabinoid levels) show decreased cardiac dysfunction with age compared with wild types. However, endocannabinoids may mediate doxorubicin-induced cardiac dysfunction. Their signaling pathways are not fully elucidated but they can lead to changed expression of a variety of genes, including those involved in inflammatory responses. There is potential for therapeutic targeting of endocannabinoids and their receptors, but their apparent involvement in both protective and deleterious actions on the heart means that careful risk assessment is needed before any treatment can be introduced.

Monoacylglycerols (MAGs) are short-lived intermediates of glycerolipid metabolism. Specific molecular species, such as <em>2</em>-<em>arachidonoylglycerol</em>, which is a potent activator of cannabinoid receptors, may also function as lipid signaling molecules. In mammals, enzymes hydrolyzing MAG to glycerol and fatty acids, resembling the final step in lipolysis, or esterifying MAG to diacylglycerol, are well known; however, despite the high level of conservation of lipolysis, the corresponding activities in yeast have not been characterized yet. Here we provide evidence that the protein Yju3p functions as a potent MAG hydrolase in yeast. Cellular MAG hydrolase activity was decreased by more than 90% in extracts of Yju3p-deficient cells, indicating that Yju3p accounts for the vast majority of this activity in yeast. Loss of this activity was restored by heterologous expression of murine monoglyceride lipase (MGL). Since yju3Delta mutants accumulated MAG in vivo only at very low concentrations, we considered the possibility that MAGs are re-esterified into DAG by acyltransferases. Indeed, cellular MAG levels were further increased in mutant cells lacking Yju3p and Dga1p or Lro1p acyltransferase activities. In conclusion, our studies suggest that catabolic and anabolic reactions affect cellular MAG levels. Yju3p is the functional orthologue of mammalian MGL and is required for efficient degradation of MAG in yeast.