The endogenous cannabinoid anandamide (AEA) is transported into cells by a temperature-sensitive process of facilitated diffusion. This uptake process has been characterised both biochemically and pharmacologically, and shown to be regulated at least in part by the intracellular metabolism of the accumulated AEA by fatty acid amide hydrolase. In this review, the properties of this transport process are briefly reviewed together with the corresponding transport mechanisms for the related endogenous compounds <em>2</em>-<em>arachidonoylglycerol</em> and palmitoylethanolamide. In addition, the possibility that these transport mechanisms can be targets for therapeutic strategies aimed at prolonging the effects of the endocannabinoids is discussed.

Previous work in our laboratory demonstrated the existence of a membrane-bound diacylglycerol kinase highly selective for diacylglycerols containing arachidonate as the sn-<em>2</em> fatty acyl moiety (MacDonald, M. L., Mack K. F., Richardson, C. N., and Glomset, J. A. (1988) J. Biol. Chem. <em>2</em>63, 1575-1583). We now report the purification of arachidonoyl-diacylglycerol kinase 34,400-fold to apparent homogeneity from bovine testis. High concentrations of both salt and detergent were required to extract the enzyme from membranes and stabilize its activity, suggesting that in vivo the enzyme is part of a complex with other membrane or cytoskeletal proteins. Arachidonoyl-diacylglycerol kinase had an apparent M(r) of 58,000 both on SDS-polyacrylamide gels and by size exclusion chromatography. The enzyme appeared to be an integral membrane protein. In a mixed micellar assay, arachidonoyl-diacylglycerol kinase followed surface dilution kinetics with respect to diacylglycerol. The purified enzyme retained the arachidonate selectivity observed previously in membranes. Kinetic analyses indicated a Km for sn-1-stearoyl-<em>2</em>-<em>arachidonoylglycerol</em> of <em>2</em>.4 mol %, as compared to 43 mol % for sn-1-palmitoyl-<em>2</em>-oleoylglycerol. Calcium, an activator of some other diacylglycerol kinases, had no apparent effect on the arachidonate-specific enzyme. Guanosine triphosphate could effectively substitute for ATP as the phosphoryl donor and Mg<em>2</em>+ could be replaced by Mn<em>2</em>+ or Ca<em>2</em>+. Phosphatidylserine and, to a lesser extent, phosphatidylinositol inhibited the purified enzyme. Phosphatidylcholine and phosphatidylethanolamine had only small effects.

To date, several studies have demonstrated that phospholipase C-coupled receptors stimulate the production of endocannabinoids, particularly <em>2</em>-<em>arachidonoylglycerol</em>. There is now evidence that endocannabinoids are involved in phospholipase C-coupled serotonin 5-HT(<em>2</em>A) receptor-mediated behavioral effects in both rats and mice. The main objective of this study was to determine whether activation of the 5-HT(<em>2</em>A) receptor leads to the production and release of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em>. NIH3T3 cells stably expressing the rat 5-HT(<em>2</em>A) receptor were first incubated with [(3)H]-arachidonic acid for <em>2</em>4 h. Following stimulation with 10 mum serotonin, lipids were extracted from the assay medium, separated by thin layer chromatography, and analyzed by liquid scintillation counting. Our results indicate that 5-HT(<em>2</em>A) receptor activation stimulates the formation and release of <em>2</em>-<em>arachidonoylglycerol</em>. The 5-HT(<em>2</em>A) receptor-dependent release of <em>2</em>-<em>arachidonoylglycerol</em> was partially dependent on phosphatidylinositol-specific phospholipase C activation. Diacylglycerol produced downstream of 5-HT(<em>2</em>A) receptor-mediated phospholipase D or phosphatidylcholine-specific phospholipase C activation did not appear to contribute to <em>2</em>-<em>arachidonoylglycerol</em> formation in NIH3T3-5HT(<em>2</em>A) cells. In conclusion, our results support a functional model where neuromodulatory neurotransmitters such as serotonin may act as regulators of endocannabinoid tone at excitatory synapses through the activation of phospholipase C-coupled G-protein coupled receptors.

Stimulation of human fibroblasts with bradykinin (BK) results in the generation of diacylglycerol (DG) and phosphatidic acid (PA). Prelabeling of the cells with [3H]arachidonic acid and [14C]palmitic acid allowed us to quantitate these lipid second messengers and to determine their origin, i.e. DGi and PAi from 3H-enriched inositol phospholipids, and DGc and PAc from 14C-enriched phosphatidylcholine, respectively. BK elicited a biphasic DG response: a first peak at 10-15 s, containing DGi, followed by a second peak at 10-30 min, which is mainly DGc. The latter did not result from de novo lipid biosynthesis. BK also generated free [3H]arachidonate and, to a lesser extent, mono[3H]<em>arachidonoylglycerol</em>. BK stimulation rapidly increased PAi, much more so than PAc, suggesting that DGi, rather than DGc, is the preferred substrate for the enzyme DG kinase. Short pretreatment of the cells with phorbol 1<em>2</em>-myristate 13-acetate (PMA) abolished the BK-induced breakdown of phosphoinositides, but did not affect the second-phase DGc level. PMA alone also elicited DGc formation, but more slowly, suggesting a different mechanism. Down-regulation of protein kinase C (PKC) by long term treatment with phorbol ester, prior to BK stimulation, resulted in (i) enhanced DGi and decreased PAi formation, suggesting that DG kinase activity is positively controlled by PKC; (ii) the unexpected manifestation of rapidly formed DGc; (iii) no change in the DGc levels obtained after 30-min BK stimulation, but complete suppression of PMA-induced DGc formation. In contrast, two inhibitors of PKC, staurosporin and 1-O-hexadecyl-<em>2</em>-O-methylglycerol, inhibited both BK- and PMA-induced DGc formation at 30 min, leaving the rapid response towards BK unaffected. The results suggest that the BK-induced rapid and later-phase DG formation and the PMA-induced DG formation are differentially controlled by PKC via mechanisms that differ in the susceptibility to down-regulation or inhibition of PKC.

Diacylglycerol lipases (DAGLα and DAGLβ) convert diacylglycerol to the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em>. Our understanding of DAGL function has been hindered by a lack of chemical probes that can perturb these enzymes in vivo. Here, we report a set of centrally active DAGL inhibitors and a structurally related control probe and their use, in combination with chemical proteomics and lipidomics, to determine the impact of acute DAGL blockade on brain lipid networks in mice. Within <em>2</em> h, DAGL inhibition produced a striking reorganization of bioactive lipids, including elevations in DAGs and reductions in endocannabinoids and eicosanoids. We also found that DAGLα is a short half-life protein, and the inactivation of DAGLs disrupts cannabinoid receptor-dependent synaptic plasticity and impairs neuroinflammatory responses, including lipopolysaccharide-induced anapyrexia. These findings illuminate the highly interconnected and dynamic nature of lipid signaling pathways in the brain and the central role that DAGL enzymes play in regulating this network.

OBJECTIVE

Endocannabinoids (anandamide and <em>2</em>-arachidonoylgycerol) and related N-acylethanolamines (N-oleoylethanolamine) exhibit opposite effects in the control of appetite. The purpose of this review is to highlight the similarities and differences of three major lipid-signaling molecules by focusing on their mode of action and the proteins involved in the control of food intake and energy metabolism.

RESULTS

Anandamide and <em>2</em>-<em>arachidonoylglycerol</em> promote food intake and are the main endogenous ligands of the cannabinoid receptors. One of them, the cannabinoid receptor 1, is responsible for the control of food intake and energy expenditure both at a central and a peripheral level, affecting numerous anorexigenic and orexigenic mediators (leptin, neuropeptide Y, ghrelin, orexin, endogenous opioids, corticotropin-releasing hormone, alpha-melanocyte stimulating hormone, cocaine and amphetamine-related transcript). In the gut, N-oleoylethanolamine plays an opposite role in food regulation, by interacting with two molecular targets different from the cannabinoid receptors: the nuclear receptor peroxisome proliferator-activated receptor alpha and a G-protein coupled receptor GPR119.

CONCLUSIONS

Recent findings on the molecular mechanisms underlying the promotion of food intake or, in contrast, the suppression of food intake by anandamide and N-oleoylethanolamine, are summarized. Potential strategies for treating overweight, metabolic syndrome, and type II diabetes are briefly outlined.

Acylethanolamides such as anandamide (AEA), and monoacylglycerols like <em>2</em>-<em>arachidonoylglycerol</em> are endocannabinoids that bind to cannabinoid, vanilloid and peroxisome proliferator-activated receptors. These compounds, their various receptors, the purported membrane transporter(s), and related enzymes that synthesize and degrade them are collectively referred to as the "endocannabinoid system (ECS)". Poorly defined cellular and molecular mechanisms control the biological actions of the ECS. Over the last decade evidence has been emerging to suggest that the ECS plays a significant role in various aspects of human reproduction. In this review, we summarize our current understanding of this role especially the involvement of AEA and related ECS elements in regulating oogenesis, embryo oviductal transport, blastocyst implantation, placental development and pregnancy outcomes, and sperm survival, motility, capacitation and acrosome reaction. Additionally, the possibility that plasma and tissue AEA and other cannabinoids may represent reliable diagnostic markers of natural and assisted reproduction and pregnancy outcomes in women will be discussed.

The cannabinoid receptor type 1 (CB1) is a G protein-coupled receptor that is activated in an autocrine fashion by the endocannabinoids (EC), N-arachidonoylethanolamine (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). The CB1 and its endogenous and synthetic agonists are emerging as therapeutic targets in several cancers due to their ability to suppress carcinoma cell invasion and migration. However, the mechanisms that the CB1 regulates cell motility are not well understood. In this study, we examined the molecular mechanisms that diminish cell migration upon the CB1 activation in prostate carcinoma cells. The CB1 activation with the agonist WIN55<em>2</em>1<em>2</em> significantly diminishes the small GTPase RhoA activity but modestly increases the Rac1 and Cdc4<em>2</em> activity. The diminished RhoA activity is accompanied by the loss of actin/myosin microfilaments, cell spreading, and cell migration. Interestingly, the CB1 inactivation with the selective CB1 antagonist AM<em>2</em>51 significantly increases RhoA activity, enhances microfilament formation and cell spreading, and promotes cell migration. This finding suggests that endogenously produced EC activate the CB1, resulting in chronic repression of RhoA activity and cell migration. Consistent with this possibility, RhoA activity is significantly diminished by the exogenous application of AEA but not by <em>2</em>-AG in PC-3 cells (cells with very low AEA hydrolysis). Pretreatment of cells with a monoacylglycerol lipase inhibitor, JZL184, which blocks <em>2</em>-AG hydrolysis, decreases the RhoA activity. These results indicate the unique CB1 signaling and support the model that EC, through their autocrine activation of CB1 and subsequent repression of RhoA activity, suppress migration in prostate carcinoma cells.

Microglia are a population of macrophage-like cells in the central nervous system (CNS) which, upon infection by the human immunodeficiency virus (HIV), secrete a plethora of inflammatory factors, including the virus-specified trans-activating protein Tat. Tat has been implicated in HIV neuropathogenesis since it elicits chemokines, cytokines, and a chemotactic response from microglia. It also harbors a β-chemokine receptor binding motif, articulating a mode by which it acts as a migration stimulus. Since select cannabinoids have anti-inflammatory properties, cross the blood-brain barrier, and target specific receptors, they have potential to serve as agents for dampening untoward neuroimmune responses. The aim of this study was to investigate the effect of select cannabinoids on the migration of microglial-like cells toward Tat. Using a mouse BV-<em>2</em> microglial-like cell model, it was demonstrated that the exogenous cannabinoids Delta-9-tetrahydrocannabinol (THC) and CP55940 exerted a concentration-related reduction in the migration of BV-<em>2</em> cells towards Tat. A similar inhibitory response was obtained when the endogenous cannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) was used. The CB(<em>2</em>) receptor (CB<em>2</em>R) antagonist SR1445<em>2</em>8, but not the CB(1) receptor (CB1R) antagonist SR141716A, blocked this inhibition of migration. Similarly, CB<em>2</em>R knockdown with small interfering RNA reversed the cannabinoid-mediated inhibition. In addition, the level of the β-chemokine receptor CCR-3 was reduced and its intracellular compartmentation was altered. These results indicate that cannabinoid-mediated inhibition of BV-<em>2</em> microglial-like cell migration to Tat is linked functionally to the CB<em>2</em>R. Furthermore, the results indicate that activation of the CB<em>2</em>R leads to altered expression and compartmentation of the β-chemokine receptor CCR-3.

OBJECTIVE

To test the hypothesis that the two nonsynonymous single nucleotide polymorphisms at the CB<em>2</em> cannabinoid receptor gene may have functional consequences on human CB<em>2</em>.

METHODS

Q63R, H316Y, and Q63R/H316 mutations were made in recombinant human CB<em>2</em> by the method of site-directed mutagenesis. After these mutant CB<em>2</em> receptors were stably transfected into HEK<em>2</em>93 cells, ligand binding, ligand-induced activity, and constitutive activity assays were performed to test the functional significance of these mutations.

RESULTS

In general, our results showed that the CB<em>2</em> polymorphic receptors are able to bind cannabinoid ligands and mediate signal transduction. However, in ligand-induced cyclic AMP accumulation assays, the cannabinoid agonists WIN55<em>2</em>1<em>2</em>-<em>2</em> and <em>2</em>-<em>arachidonoylglycerol</em> had reduced efficacy in cells expressing the polymorphic receptors as compared with the CB<em>2</em> wild-type receptor. Furthermore, in constitutive activity assays, the H316Y and Q63R/H316Y polymorphic receptors exhibited higher constitutive activity than the CB<em>2</em> wild-type receptor.

CONCLUSIONS

Our data shows that the presence of the polymorphisms at both positions 63 and 316 produce alterations in the CB<em>2</em> receptor functions. Moreover, these findings strengthen the idea that the CB<em>2</em> polymorphic receptors may contribute to the etiology of certain diseases.

Research into the endocannabinoid 'system' has grown exponentially in recent years, with the discovery of cannabinoid receptors and their endogenous ligands, such as anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Important advances have been made in our understanding of endocannabinoid transduction mechanisms, their metabolic pathways, and of the biological processes in which they are implicated. A decade of endocannabinoid studies has promoted new insights into neural regulation and mammalian physiology that are as revolutionary as those arising from the discovery of the endogenous opioid peptides in the 1970s. Thus, endocannabinoids have been found to act as retrograde signals: released by postsynaptic neurons, they bind to presynaptic heteroceptors to modulate the release of inhibitory and excitatory neurotransmitters through multiple G-protein-coupled receptor (GPCR)-linked effector mechanisms. The metabolic pathways of anandamide and <em>2</em>-AG have now been been characterised in great detail, and we can anticipate that these pathways -- together with endocannabinoid uptake mechanisms -- will complement cannabinoid receptors as targets for the pharmacological analysis of the physiological functions of these substances. Specific insights into the potential role of endocannabinoid-CB1 receptor systems in central appetite control, peripheral metabolism and body weight regulation herald the clinical application of CB1 receptor antagonists in the management of obesity and its associated disorders.

Chronic kidney disease, secondary to renal fibrogenesis, is a burden on public health. There is a need to explore new therapeutic pathways to reduce renal fibrogenesis. To study this, we used unilateral ureteral obstruction (UUO) in mice as an experimental model of renal fibrosis and microarray analysis to compare gene expression in fibrotic and normal kidneys. The cannabinoid receptor 1 (CB1) was among the most upregulated genes in mice, and the main endogenous CB1 ligand (<em>2</em>-<em>arachidonoylglycerol</em>) was significantly increased in the fibrotic kidney. Interestingly, CB1 expression was highly increased in kidney biopsies of patients with IgA nephropathy, diabetes, and acute interstitial nephritis. Both genetic and pharmacological knockout of CB1 induced a profound reduction in renal fibrosis during UUO. While CB<em>2</em> is also involved in renal fibrogenesis, it did not potentiate the role of CB1. CB1 expression was significantly increased in myofibroblasts, the main effector cells in renal fibrogenesis, upon TGF-β1 stimulation. The decrease in renal fibrosis during CB1 blockade could be explained by a direct action on myofibroblasts. CB1 blockade reduced collagen expression in vitro. Rimonabant, a selective CB1 endocannabinoid receptor antagonist, modulated the macrophage infiltrate responsible for renal fibrosis in UUO through a decrease in monocyte chemoattractant protein-1 synthesis. Thus, CB1 has a major role in the activation of myofibroblasts and may be a new target for treating chronic kidney disease.

BACKGROUND

The endocannabinoid system is a potential pharmacotherapy target for obesity. However, the role of this system in human food intake regulation is currently unknown.

METHODS

To test whether circulating endocannabinoids might functionally respond to food intake and verify whether these orexigenic signals are deregulated in obesity alongside with anorexigenic ones, we measured plasma anandamide (AEA), <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and peptide YY (PYY) changes in response to a meal in 1<em>2</em> normal-weight and 1<em>2</em> non-diabetic, insulin-resistant obese individuals.

RESULTS

Both normal-weight and obese subjects had a significant preprandial AEA peak. Postprandially, AEA levels significantly decreased in normal-weight, whereas no significant changes were observed in obese subjects. Similarly, PYY levels significantly increased in normal-weight subjects only. No meal-related changes were found for <em>2</em>-AG. Postprandial AEA and PYY changes inversely correlated with waist circumference, and independently explained <em>2</em>0.7 and <em>2</em>1.3% of waist variance. Multiple regression analysis showed that postprandial AEA and PYY changes explained 34% of waist variance, with 8.<em>2</em>% of the variance commonly explained.

CONCLUSIONS

These findings suggest that AEA might be a physiological meal initiator in humans and furthermore show that postprandially AEA and PYY are concomitantly deregulated in obesity.

Cannabinoids have been always identified as harmful drugs because of their negative effects on male and female reproduction. The discovery of the 'endocannabinoid system (ECS)', composed of bioactive lipids (endocannabinoids), their receptors and their metabolic enzymes, and the generation of mouse models missing cannabinoid receptors or other elements of the ECS, has enabled a wealth of information on the significance of endocannabinoid signalling in multiple reproductive events: Sertoli cell survival, spermatogenesis, placentation, fertilization, preimplantation embryo development, implantation and postimplantation embryonic growth. These studies have also opened new perspectives in clinical applications, pointing to the ECS as a new target for correcting infertility and for improving reproductive health in humans. This review will focus on the involvement of type-<em>2</em> cannabinoid (CB<em>2</em>) receptors in reproductive biology, covering both the male and female sides. It will also discuss the potential relevance of the immunological activity of CB<em>2</em> at the maternal/foetal interface, as well as the distinctiveness of CB<em>2</em> versus type-1 cannabinoid (CB1) receptors that might be exploited for a receptor subtype-specific regulation of fertility. In this context, the different signalling pathways triggered by CB1 and CB<em>2</em> (especially those controlling the intracellular tone of nitric oxide), the different activation of CB1 and CB<em>2</em> by endogenous agonists (like anandamide and <em>2</em>-<em>arachidonoylglycerol</em>) and the different localization of CB1 and CB<em>2</em> within membrane subdomains, termed 'lipid rafts', will be discussed. It is hoped that CB<em>2</em>-dependent endocannabinoid signalling might become a useful target for correcting infertility, in both men and women.

Rich evidence has shown that cannabis products exert a broad gamut of effects on emotional regulation. The main psychoactive ingredient of hemp, Δ9-tetrahydrocannabinol (THC), and its synthetic cannabinoid analogs have been reported to either attenuate or exacerbate anxiety and fear-related behaviors in humans and experimental animals. The heterogeneity of cannabis-induced psychological outcomes reflects a complex network of molecular interactions between the key neurobiological substrates of anxiety and fear and the endogenous cannabinoid system, mainly consisting of the arachidonic acid derivatives anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and two receptors, respectively termed CB1 and CB<em>2</em>. The high degree of interindividual variability in the responses to cannabis is contributed by a wide spectrum of factors, including genetic and environmental determinants, as well as differences in the relative concentrations of THC and other alkaloids (such as cannabidiol) within the plant itself. The present article reviews the currently available knowledge on the herbal, synthetic and endogenous cannabinoids with respect to the modulation of anxiety responses, and highlights the challenges that should be overcome to harness the therapeutic potential of some of these compounds, all the while limiting the side effects associated with cannabis consumption. In addition the article presents some promising patents on cannabinoid-related agents.

The perivascular sensory nerve (PvN) Ca(<em>2</em>+)-sensing receptor (CaR) is implicated in Ca(<em>2</em>+)-induced relaxation of isolated, phenylephrine (PE)-contracted mesenteric arteries, which involves the vascular endogenous cannabinoid system. We determined the effect of inhibition of diacylglycerol (DAG) lipase (DAGL), phospholipase A(<em>2</em>) (PLA(<em>2</em>)), and cytochrome P-450 (CYP) on Ca(<em>2</em>+)-induced relaxation of PE-contracted rat mesenteric arteries. Our findings indicate that Ca(<em>2</em>+)-induced vasorelaxation is not dependent on the endothelium. The DAGL inhibitor RHC 80<em>2</em>675 (1 microM) and the CYP and PLA(<em>2</em>) inhibitors quinacrine (5 microM) (EC(50): RHC 80<em>2</em>675 <em>2</em>.8 +/- 0.4 mM vs. control 1.4 +/- 0.3 mM; quinacrine 4.8 +/- 0.4 mM vs. control <em>2</em>.0 +/- 0.3 mM; n = 5) and arachidonyltrifluoromethyl ketone (AACOCF(3), 1 microM) reduced Ca(<em>2</em>+)-induced relaxation of mesenteric arteries. Synthetic <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and glycerated epoxyeicosatrienoic acids (GEETs) induced concentration-dependent relaxation of isolated arteries. <em>2</em>-AG relaxations were blocked by iberiotoxin (IBTX) (EC(50): control 0.96 +/- 0.14 nM, IBTX 1.3 +/- 0.5 microM) and miconazole (48 +/- 3%), and 11,1<em>2</em>-GEET responses were blocked by IBTX (EC(50): control 55 +/- 9 nM, IBTX 690 +/- 96 nM) and SR-141716A. The data suggest that activation of the CaR in the PvN network by Ca(<em>2</em>+) leads to synthesis and/or release of metabolites of the CYP epoxygenase pathway and metabolism of DAG to <em>2</em>-AG and subsequently to GEETs. The findings indicate a role for <em>2</em>-AG and its metabolites in Ca(<em>2</em>+)-induced relaxation of resistance arteries; therefore this receptor may be a potential target for the development of new vasodilator compounds for antihypertensive therapy.

The levels of <em>2</em>-<em>arachidonoylglycerol</em>, an endogenous cannabinoid receptor ligand, and other molecular species of monoacylglycerols in rat brain were examined. In this study, we sacrificed the animals in liquid nitrogen to minimize postmortem changes. We found that rat brain contains 0.<em>2</em>3 nmol/g tissue of <em>2</em>-<em>arachidonoylglycerol</em>, which accounts for 10.5% of the total monoacylglycerol present in this tissue. We next investigated the level of <em>2</em>-<em>arachidonoylglycerol</em> after in vivo stimulation with picrotoxinin. We found that the level of <em>2</em>-<em>arachidonoylglycerol</em> was elevated markedly in picrotoxinin-administered rat brain (4- to 6-fold over the control level). Changes in the levels of other molecular species were relatively small or negligible. Several cannabimimetic molecules as well as Delta(9)-tetrahydrocannabinol are known to depress neurotransmission and to exert anticonvulsant activities; endogenous <em>2</em>-<em>arachidonoylglycerol</em> produced during neural excitation may play a regulatory role in calming the enhanced synaptic transmission.

The endocannabinoid (eCB) <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) produced by diacylglycerol lipase α (DGLα) is one of the best-characterized retrograde messengers at central synapses. It has been thought that <em>2</em>-AG is produced 'on demand' upon activation of postsynaptic neurons. However, recent studies propose that <em>2</em>-AG is pre-synthesized by DGLα and stored in neurons, and that <em>2</em>-AG is released from such 'pre-formed pools' without the participation of DGLα. To address whether the <em>2</em>-AG source for retrograde signalling is the on-demand biosynthesis by DGLα or the mobilization from pre-formed pools, we examined the effects of acute pharmacological inhibition of DGL by a novel potent DGL inhibitor, OMDM-188, on retrograde eCB signalling triggered by Ca(<em>2</em>+) elevation, Gq/11 protein-coupled receptor activation or synergy of these two stimuli in postsynaptic neurons. We found that pretreatment for 1 h with OMDM-188 effectively blocked depolarization-induced suppression of inhibition (DSI), a purely Ca(<em>2</em>+)-dependent form of eCB signalling, in slices from the hippocampus, striatum and cerebellum. We also found that at parallel fibre-Purkinje cell synapses in the cerebellum OMDM-188 abolished synaptically induced retrograde eCB signalling, which is known to be caused by the synergy of postsynaptic Ca(<em>2</em>+) elevation and group I metabotropic glutamate receptor (I-mGluR) activation. Moreover, brief OMDM-188 treatments for several minutes were sufficient to suppress both DSI and the I-mGluR-induced retrograde eCB signalling in cultured hippocampal neurons. These results are consistent with the hypothesis that <em>2</em>-AG for synaptic retrograde signalling is supplied as a result of on-demand biosynthesis by DGLα rather than mobilization from presumptive pre-formed pools.

The endocannabinoid signaling system is believed to play a down-regulatory role in the control of cell functions. However, little is known about the factors activating endocannabinoid synthesis and which of two known endocannabinoids, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) or N-arachidonoylethanolamine (<em>2</em>0:4n-6 NAE, anandamide), is of physiological importance. We approached these questions by studying a possible link between cell activation with 1-O-alkyl-<em>2</em>-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor, PAF) and the generation of <em>2</em>-AG and anandamide in human platelets and mouse P388D1 macrophages. Human platelets responded to stimulation with the production of various 1- and <em>2</em>-monoacylglycerols, including <em>2</em>-AG, whereas stimulation of P388D1 macrophages induced the rapid and selective generation of <em>2</em>-AG, which was immediately released into the medium. The effect of PAF was receptor mediated, as PAF receptor antagonist BN5<em>2</em>0<em>2</em>1 blocked the effect. The treatment did not change the content of anandamide in either macrophages or platelet-rich plasma. The inhibitors of PI- and PC-specific phospholipases C (U731<em>2</em><em>2</em> and D609) as well as PI3-kinase inhibitor (wortmannin) attenuated PAF-induced <em>2</em>-AG production in macrophages. These data suggest a direct role for the endocannabinoid system in controlling immune cell activation status and indicate that <em>2</em>-AG rather than anandamide is the endocannabinoid rapidly produced in response to proinflammatory stimulation of immune cells.

The family of endocannabinoids contains several polyunsaturated fatty acid amides such as anandamide (AEA), but also esters such as <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). These compounds are the main endogenous agonists of cannabinoid receptors, able to mimic several pharmacological effects of Delta9-tetrahydrocannabinol (Delta9-THC), the active principle of Cannabis sativa preparations like hashish and marijuana. The activity of AEA at its receptors is limited by cellular uptake, through a putative membrane transporter, followed by intracellular degradation by fatty acid amide hydrolase (FAAH). Growing evidence demonstrates that FAAH is the critical regulator of the endogenous levels of AEA, suggesting that it may serve as an attractive therapeutic target for the treatment of human disorders. In particular, FAAH inhibitors may be next generation therapeutics of potential value for the treatment of pathologies of the central nervous system, and of peripheral tissues. Investigations into the structure and function of FAAH, its biological and therapeutic implications, as well as a description of different families of FAAH inhibitors, are the topic of this chapter.

CD1 mice lacking the CB1 receptors (knockout, KO) were compared with wild-type littermates for their ability to degrade N-arachidonoylethanolamine (anandamide, AEA) through a membrane transporter (AMT) and a fatty acid amide hydrolase (FAAH). The regional distribution and age-dependence of AMT and FAAH activity were investigated. Anandamide membrane transporter and FAAH increased with age in knockout mice, whereas they showed minor changes in wild-type animals. Remarkably, they were higher in all brain areas of 6-month-old knockout versus wild-type mice, and even higher in 1<em>2</em>-month-old animals. The molecular mass (approximately 67 kDa) and isoelectric point (approximately 7.6) of mouse brain FAAH were determined and the FAAH protein content was shown to parallel the enzyme activity. The kinetic constants of AMT and FAAH in the cortex of wild-type and knockout mice at different ages suggested that different amounts of the same proteins were expressed. The cortex and hippocampus of wild-type and knockout mice contained the following N-acylethanolamines: AEA (8% of total), <em>2</em>-<em>arachidonoylglycerol</em> (5%), N-oleoylethanolamine (<em>2</em>0%), N-palmitoylethanolamine (53%) and N-stearoylethanolamine (14%). These compounds were twice as abundant in the hippocampus as in the cortex. Minor differences were observed in AEA or <em>2</em>-<em>arachidonoylglycerol</em> content in knockout versus wild-type mice, whereas the other compounds were lower in the hippocampus of knockout versus wild-type animals.

The profiles of serine hydrolases in human and mouse macrophages are similar yet different. For instance, human macrophages express high levels of carboxylesterase 1 (CES1), whereas mouse macrophages have minimal amounts of the orthologous murine CES1. On the other hand, macrophages from both species exhibit limited expression of the canonical <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) hydrolytic enzyme, MAGL. Our previous study showed CES1 was partly responsible for the hydrolysis of <em>2</em>-AG (50%) and prostaglandin glyceryl esters (PG-Gs) (80-95%) in human THP1 monocytes and macrophages. However, MAGL and other endocannabinoid hydrolases, FAAH, ABHD6, and ABHD1<em>2</em>, did not have a role because of limited expression or no expression. Thus, another enzyme was hypothesized to be responsible for the remaining <em>2</em>-AG hydrolysis activity following chemical inhibition and immunodepletion of CES1 (previous study) or CES1 gene knockdown (this study). Here we identified two candidate serine hydrolases in THP1 cell lysates by activity-based protein profiling (ABPP)-MUDPIT and Western blotting: cathepsin G and palmitoyl protein thioesterase 1 (PPT1). Both proteins exhibited electrophoretic properties similar to those of a serine hydrolase in THP1 cells detected by gel-based ABPP at 31-3<em>2</em> kDa; however, only PPT1 exhibited lipolytic activity and hydrolyzed <em>2</em>-AG in vitro. Interestingly, PPT1 was strongly expressed in THP1 cells but was significantly less reactive than cathepsin G toward the activity-based probe, fluorophosphonate-biotin. KIAA1363, another serine hydrolase, was also identified in THP1 cells but did not have significant lipolytic activity. On the basis of chemoproteomic profiling, immunodepletion studies, and chemical inhibitor profiles, we estimated that PPT1 contributed 3<em>2</em>-40% of <em>2</em>-AG hydrolysis activity in the THP1 cell line. In addition, pure recombinant PPT1 catalyzed the hydrolysis of <em>2</em>-AG, PGE<em>2</em>-G, and PGF<em>2</em>α-G, although the catalytic efficiency of hydrolysis of <em>2</em>-AG by PPT1 was ~10-fold lower than that of CES1. PPT1 was also insensitive to several chemical inhibitors that potently inhibit CES1, such as organophosphate poisons and JZL184. This is the first report to document the expression of PPT1 in a human monocyte and macrophage cell line and to show PPT1 can hydrolyze the natural substrates <em>2</em>-AG and PG-Gs. These findings suggest that PPT1 may participate in endocannabinoid metabolism within specific cellular contexts and highlights the functional redundancy often exhibited by enzymes involved in lipid metabolism.

Clinical and laboratory studies suggest that the endocannabinoid system is involved in schizophrenia disorders. Recent evidence indicates that cannabinoid receptor (CB1) antagonists have a pharmacological profile similar to antipsychotic drugs. We investigated the behavioural and biochemical effects of the CB1 antagonist AM<em>2</em>51 in a phencyclidine (PCP) animal paradigm modelling the cognitive deficit and some negative symptoms of schizophrenia. Chronic AM<em>2</em>51 (0.5 mg/kg for 3 wk) improved the PCP-altered recognition memory, as indicated by a significant amelioration of the discrimination index compared to chronic PCP alone (<em>2</em>.58 mg/kg for 1 month). AM<em>2</em>51 also reversed the PCP-induced increase in immobility in the forced swim test resembling avolition, a negative sign of schizophrenia. In order to analyse the mechanisms underlying these behaviours, we studied the effects of AM<em>2</em>51 on the endocannabinoid system (in terms of CB1 receptor density and functional activity and endocannabinoid levels) and c-Fos protein expression. The antagonist counteracted the alterations in CB1 receptor function induced by PCP in selected cerebral regions involved in schizophrenia. In addition, in the prefrontal cortex, the key region in the integration of cognitive and negative functions, AM<em>2</em>51 markedly raised anandamide levels and reversed the PCP-induced increase of <em>2</em>-<em>arachidonoylglycerol</em> concentrations. Finally, chronic AM<em>2</em>51 fully reversed the PCP-elicited expression of c-Fos protein in the prefrontal cortical region. These findings suggest an antipsychotic-like profile of the CB1 cannabinoid receptor antagonist which, by restoring the function of the endocannabinoid system, might directly or indirectly normalize some of the neurochemical maladaptations present in this schizophrenia-like animal model.