Complementary genetic and pharmacological approaches to inhibit monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), the primary hydrolytic enzymes of the respective endogenous cannabinoids 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine, enable the exploration of potential therapeutic applications and physiologic roles of these enzymes. Complete and simultaneous inhibition of both FAAH and MAGL produces greatly enhanced cannabimimetic responses, including increased antinociception, and other cannabimimetic effects, far beyond those seen with inhibition of either enzyme alone. While cannabinoid receptor type 1 (CB1) function is maintained following chronic FAAH inactivation, prolonged excessive elevation of brain 2-AG levels, via MAGL inhibition, elicits both behavioral and molecular signs of cannabinoid tolerance and dependence. Here, we evaluated the consequences of a high dose of the MAGL inhibitor JZL184 [4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate; 40 mg/kg] given acutely or for 6 days in FAAH(-/-) and (+/+) mice. While acute administration of JZL184 to FAAH(-/-) mice enhanced the magnitude of a subset of cannabimimetic responses, repeated JZL184 treatment led to tolerance to its antinociceptive effects, cross-tolerance to the pharmacological effects of Δ(9)-tetrahydrocannabinol, decreases in CB1 receptor agonist-stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate binding, and dependence as indicated by rimonabant-precipitated withdrawal behaviors, regardless of genotype. Together, these data suggest that simultaneous elevation of both endocannabinoids elicits enhanced cannabimimetic activity but MAGL inhibition drives CB1 receptor functional tolerance and cannabinoid dependence.

Anandamide (N-arachidonoylethanolamine) and six fatty acid ethanolamides were synthesized and their pharmacological effects in mice were assessed using catalepsy, hypothermia and pentobarbital-induced sleep prolongation as indices. The effects of phenylmethylsulfonyl fluoride (PMSF) pretreatment on anandamide effects were also evaluated and discussed in relation to inhibition of anandamide amidohydrolase in mouse brain and liver. The cataleptogenic effect of anandamide (ED50=6.0 mg/kg, i.v.) was 4 to 6 times more active than those of N-oleoyl- (ED50=26.5 mg/kg, i.v.) and N-linoleoylethanolamine (ED50=37.5 mg/kg, i.v.), although the peak time in the effect was observed within 1 min after i.v. administration. None of the saturated fatty acid ethanolamides (N-myristoyl-, N-palmitoyl-, N-stearoyl- and N-arachidoylethanolamine) showed a positive response in the cataleptogenic effect even at a dose up to 40 mg/kg i.v. Anandamide, N-linoleoyl-, N-oleoyl- and N-myristoylethanolamine (10 mg/kg, i.v.) produced a significant hypothermia (0.19 to 0.59 degrees C) at 5 to 15 min after administration. The duration of the effects of these ethanolamides was also relatively short. Anandamide, N-linoleoyl-, N-oleoyl- and N-palmitoylethanolamine (5 or 10 mg/kg, i.v.) significantly prolonged pentobarbital-induced sleeping time by 148-207% of control sleeping time. The cataleptogenic effect of anandamide was markedly potentiated by pretreatment of mice with PMSF (100 mg/kg, i.p.). The ED50 (mg/kg, i.v.) of anandamide was 0.48 (0.24-0.96) in PMSF-pretreated mice. The pretreatment of mice with PMSF significantly decreased the metabolic clearance rate of anandamide in microsomal fractions of liver and brain. Thus, the Vmax/Km values of brain and hepatic microsomes were 26 and 10%, respectively, as compared with those of control mice. The present study demonstrated that anandamide and N-acylethanolamines of unsaturated fatty acids exhibited cannabinoid-like effects in mice, and that anandamide amidohydrolase has an important role in the pharmacological effects of anandamide in vivo.

Endocannabinoids participate in the control of neurogenesis, neural cell death and gliosis. The pharmacological effect of the fatty acid amide hydrolase (FAAH) inhibitor URB597, which limits the endocannabinoid degradation, was investigated in the present study. Cell proliferation (phospho-H3(+) or BrdU(+) cells) of the main adult neurogenic zones as well as apoptosis (cleaved caspase-3(+)), astroglia (GFAP(+)), and microglia (Iba1(+) cells) were analyzed in the hippocampus, hypothalamus and striatum of rats intraperitoneally treated with URB597 (0.3 mg/kg/day) at one dose/4-days resting or 5 doses (1 dose/day). Repeated URB597 treatment increased the plasma levels of the N-acylethanolamines oleoylethanolamide, palmitoylethanolamide and arachidonoylethanolamine, reduced the plasma levels of glucose, triglycerides and cholesterol, and induced a transitory body weight decrease. The hippocampi of repeated URB597-treated rats showed a reduced number of phospho-H3(+) and BrdU(+) subgranular cells as well as GFAP(+), Iba1(+) and cleaved caspase-3(+) cells, which was accompanied with decreased hippocampal expression of the cannabinoid CB1 receptor gene Cnr1 and Faah. In the hypothalami of these rats, the number of phospho-H3(+), GFAP(+) and 3-weeks-old BrdU(+) cells was specifically decreased. The reduced striatal expression of CB1 receptor in repeated URB597-treated rats was only associated with a reduced apoptosis. In contrast, the striatum of acute URB597-treated rats showed an increased number of subventricular proliferative, astroglial and apoptotic cells, which was accompanied with increased Faah expression. Main results indicated that FAAH inhibitor URB597 decreased neural proliferation, glia and apoptosis in a brain region-dependent manner, which were coupled to local changes in Faah and/or Cnr1 expression and a negative energy context.

Decidualization is essential for a successful pregnancy and is a tightly regulated process influenced by the local microenvironment. Lipid-based mediators, such as the endocannabinoid anandamide, and other compounds that have cannabimimetic actions may act on the decidua during early pregnancy. In this study, the levels of N-arachidonoylethanolamine (anandamide) and two other N-acylethanolamines, N-oleoylethanolamine and N-palmitoylethanolamine, were measured in rat plasma and maternal tissues between d 8 and 19 of pregnancy by ultraperformance liquid chromatography tandem mass spectrometry. The spatiotemporal expression of N-acylethanolamine metabolizing enzymes in implantation units were also determined by quantitative PCR, Western blot, and immunohistochemistry and shown to vary with gestation being mainly localized in decidual cells. The data also indicated that plasma and tissues levels of all three N-acylethanolamines fluctuate throughout pregnancy. Tissue levels of endocannabinoids did not correlate with plasma, suggesting that during pregnancy, maternal tissue levels of endocannabinoids are primarily regulated by in situ production and degradation to create endocannabinoid gradients conducive to successful pregnancy.

In the era of combined antiretroviral therapy (cART), human immunodeficiency virus type 1 (HIV-1) is considered a chronic disease that specifically targets the brain and causes HIV-1-associated neurocognitive disorders (HAND). Endocannabinoids (eCBs) elicit neuroprotective and anti-inflammatory actions in several central nervous system (CNS) disease models, but their effects in HAND remain unknown. HIV-1 does not infect neurons, but produces viral toxins, such as transactivator of transcription (Tat), that disrupt neuronal calcium equilibrium and give rise to synaptodendritic injuries and cell death, the former being highly correlated with HAND. Consequently, we tested whether the eCBs N-arachidonoylethanolamine (anandamide/AEA) and 2-arachidonoyl-glycerol (2-AG) offer neuroprotective actions in a neuronal culture model. Specifically, we examined the neuroprotective actions of these eCBs on Tat excitotoxicity in primary cultures of prefrontal cortex neurons (PFC), and whether cannabinoid receptors mediate this neuroprotection. Tat-induced excitotoxicity was reflected by increased intracellular calcium levels, synaptodendritic damage, neuronal excitability, and neuronal death. Further, upregulation of cannabinoid 1 receptor (CB1R) protein levels was noted in the presence of HIV-1 Tat. The direct application of AEA and 2-AG reduced excitotoxic levels of intracellular calcium and promoted neuronal survival following Tat exposure, which was prevented by the CB1R antagonist rimonabant, but not by the CB2R antagonist AM630. Overall, our findings indicate that eCBs protect PFC neurons from Tat excitotoxicity in vitro via a CB1R-related mechanism. Thus, the eCB system possesses promising targets for treatment of neurodegenerative disorders associated with HIV-1 infection.

Endocannabinoids are amides, esters and ethers of long chain polyunsaturated fatty acids, which act as new lipid mediators. Anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol are the main endogenous agonists of cannabinoid receptors, able to mimic several pharmacological effects of Delta(9)-tetrahydrocannabinol, 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 specific membrane transporter, followed by intracellular degradation by a 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 therapeutic drugs of potential value for the treatment of pathologies in the central nervous system and in the periphery. Here, the potential applications of these inhibitors for human disease will be reviewed, with an emphasis on the properties of hydro(pero)xy-anandamides. In fact, these oxygenated derivatives of AEA are the most powerful inhibitors of FAAH of natural origin as yet discovered. In addition, new insights into the promoter region of FAAH gene will be presented, and the therapeutic potential of mimetics of transcription factors of this gene in the management of human infertility will be discussed.

It has been suggested recently that the endocannabinoid system might be a component of the brain reward circuitry and thus play a role not only in cannabinoid tolerance/dependence, but also in dependence/withdrawal to other drugs of abuse. Here we have examined the changes in endocannabinoid ligands and their receptors in different brain regions, with particular attention to those areas related to reinforcement processes, during dependence on the powerful addictive drug, morphine. Thus, we analysed the brain contents of N-arachidonoylethanolamine (anandamide, AEA), the first discovered endocannabinoid, in rats subjected to daily injections of increasing doses of morphine, according to a schedule designed to render the animals opiate-dependent. Although evidence of physical dependence was assured by the appearance of somatic and neurovegetative responses in these animals after an acute challenge with naloxone, there were no changes in the contents of this endocannabinoid in any of the brain regions analysed. By contrast, we observed a significant decrease in the specific binding for CB(1) receptors in the midbrain and the cerebral cortex of morphine-dependent rats, with no changes in the other regions. The decrease in the cerebral cortex was, however, accompanied by a rise in the activation of signalling mechanisms by CB(1) receptor agonists, as revealed by WIN-55,212-2-stimulated [(35)S]GTPgammaS binding, whereas a reduction in this parameter was measured in the brainstem of morphine-dependent rats. In summary, the present data are indicative of the existence of an alteration of the endocannabinoid transmission during morphine dependence in rats, although the changes observed were region-dependent and affected exclusively CB(1) receptors with no changes in endocannabinoid levels. Because the changes occurred in regions of the midbrain, the cerebral cortex and the brainstem, which have been implicated in drug dependence, our data suggest that pharmacological manipulation of the endocannabinoid system might be a novel tool to reduce morphine addiction.

Endocannabinoids are bioactive lipids, that comprise amides, esters and ethers of long chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol (2-AG) are the best studied endocannabinoids, and act as agonists of cannabinoid receptors. Thus, AEA and 2-AG mimic several pharmacological effects of the exogenous cannabinoid delta9-tetrahydrocannabinol, the psychoactive principle of hashish and marijuana. It is known that the activity of endocannabinoids at their receptors is limited by cellular uptake through specific membrane transporters, followed by intracellular degradation by a fatty acid amide hydrolase (for AEA and partly 2-AG) or by a monoacylglycerol lipase (for 2-AG). Together with AEA, 2-AG and congeners, the proteins that bind, transport and metabolize these lipids form the "endocannabinoid system". This new system will be briefly presented in this review, in order to put in a better perspective the role of the endocannabinoid pathway in neurodegenerative disorders, like Parkinson's disease, Huntington's disease, and multiple sclerosis. In addition, the potential exploitation of antagonists of endocannabinoid receptors, or of inhibitors of endocannabinoid metabolism, as next-generation therapeutics will be discussed.

Endocannabinoids including N-acylethanolamides (NAEs) are a family of lipid-related signaling molecules implicated in many physiological and disease states which elicit their activities via the cannabinoid receptors. Anandamide (N-arachidonoylethanolamine, AEA) is the most characterized endocannabinoid and has been detected in many tissues and bio-fluids including human plasma and the central nervous system. The endocannabinoid-like NAEs, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) are described as entourage compounds because they illicit similar physiological effects to AEA but have little or no affinity for cannabinoid receptors. As entourage compounds, levels of these NAEs can greatly influence the efficacy of AEA yet there are few studies which measure these compounds in bio-fluids. Here we describe a rapid, highly sensitive, specific and highly reproducible ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the analysis of AEA, OEA, and PEA in human bio-fluids including plasma, serum, breast milk, and amniotic fluids. This validated method using deuterated (AEA-d(8), OEA-d(2), and PEA-d(4)) internal standards, represents an improvement over previous analyses in terms of run time (4 min), limit of detection (0.9 fmol on column for AEA and PEA and 4.4 fmol on column for OEA), precision (relative standard deviations of peak areas: 3.1% (AEA), 2.9% (OEA), and 5.4% (PEA) for 133 fmol on column) and accuracy (95.1-104.9%). The sensitivity and precision of the validated method described here suggests that this method is suitable for the analysis of AEA, OEA, and PEA in clinical samples and may be utilized for the investigation of bio-matrices containing limited amounts of NAEs.

A fluorimetric determination method for N-arachidonoylethanolamine (anandamide) was developed using a precolumn fluorescence derivatization followed by coupled-column high-performance liquid chromatography (HPLC). Anandamide extracted from the rat brain tissue was derivatized with 4-N-chloroformylmethyl-N-methylamino-7-N, N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-COCl), purified by a solid-phase extraction (Emporetrade mark), and assayed by the coupled-column HPLC. The HPLC consisted of phenyl (100 x 4.6 mm i.d. ) and octadecylsilica columns (250 x 4.6 mm i.d.), both connected by a six-port valve. The concentration of anandamide in rat brain was 3. 37 +/- 0.73 pmol/g with 6.47 and 3.57% of intra- and inter-day precisions, respectively. Using this method, we investigated the alteration of anandamide concentration in rat brain 30 min after administration of anandamide (2 mg/kg, i.p.) to rats pretreated with or without phenylmethylsulfonyl fluoride (PMSF; 30 mg/kg, i.p.), an inhibitor of amidohydrolase. In rats pretreated with PMSF, the brain concentration of anandamide was approx. 16-fold higher than that of rats without PMSF (p < 0.01).

Endocannabinoids are a new class of lipids, which include amides, esters and ethers of long chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol are the main endogenous agonists of cannabinoid receptors able to mimic several pharmacological effects of Delta(9)-tetrahydrocannabinol, the active principle of Cannabis sativa preparations like hashish and marijuana. AEA is released "on demand" from membrane lipids, and its activity at the receptors is limited by cellular uptake followed by intracellular hydrolysis. Together with AEA and congeners, the proteins which bind, synthesize, transport and hydrolyze AEA form the "endocannabinoid system". Endogenous cannabinoids are present in the central nervous system and in peripheral tissues, suggesting a physiological role as broad spectrum modulators. This review summarizes the main features of the endocannabinoid system, and the latest advances on its involvement in ageing of central and peripheral cells. In addition, the therapeutic potential of recently developed drugs able to modulate the endocannabinoid tone for the treatment of ageing and age-related human pathologies will be reviewed.

The endocannabinoid (eCB) system can promote food intake by increasing odor detection in mice. The eCB system is over-active in human obesity. Our aim is to measure circulating eCB concentrations and olfactory capacity in a human sample that includes people with obesity and explore the possible interaction between olfaction, obesity and the eCB system. The study sample was made up of 161 females with five groups of body mass index sub-categories ranging from under-weight to morbidly obese. We assessed olfactory capacity with the "Sniffin´Sticks" test, which measures olfactory threshold-discrimination-identification (TDI) capacity. We measured plasma concentrations of the eCBs 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine or anandamide (AEA), and several eCB-related compounds, 2-acylglycerols and N-acylethanolamines. 2-AG and other 2-acylglycerols fasting plasma circulating plasma concentrations were higher in obese and morbidly obese subjects. AEA and other N-acylethanolamine circulating concentrations were lower in under-weight subjects. Olfactory TDI scores were lower in obese and morbidly obese subjects. Lower TDI scores were independently associated with higher 2-AG fasting plasma circulating concentrations, higher %body fat, and higher body mass index, after controlling for age, smoking, menstruation, and use of contraceptives. Our results show that obese subjects have a lower olfactory capacity than non-obese ones and that elevated fasting plasma circulating 2-AG concentrations in obesity are linked to a lower olfactory capacity. In agreement with previous studies we show that eCBs AEA and 2-AG, and their respective congeners have a distinct profile in relation to body mass index. The present report is the first study in humans in which olfactory capacity and circulating eCB concentrations have been measured in the same subjects.

The endogenous cannabinoid (endocannabinoid) system regulates a diverse array of physiological processes and unsurprisingly possesses considerable potential targets for the potential treatment of numerous disease states, including two receptors (i.e., CB1 and CB2 receptors) and enzymes regulating their endogenous ligands N-arachidonoylethanolamine (anandamide) and 2-arachidonyl glycerol (2-AG). Increases in brain levels of endocannabinoids to pathogenic events suggest this system plays a role in compensatory repair mechanisms. Traumatic brain injury (TBI) pathology remains mostly refractory to currently available drugs, perhaps due to its heterogeneous nature in etiology, clinical presentation, and severity. Here, we review pre-clinical studies assessing the therapeutic potential of cannabinoids and manipulations of the endocannabinoid system to ameliorate TBI pathology. Specifically, manipulations of endocannabinoid degradative enzymes (e.g., fatty acid amide hydrolase, monoacylglycerol lipase, and α/β-hydrolase domain-6), CB1 and CB2 receptors, and their endogenous ligands have shown promise in modulating cellular and molecular hallmarks of TBI pathology such as; cell death, excitotoxicity, neuroinflammation, cerebrovascular breakdown, and cell structure and remodeling. TBI-induced behavioral deficits, such as learning and memory, neurological motor impairments, post-traumatic convulsions or seizures, and anxiety also respond to manipulations of the endocannabinoid system. As such, the endocannabinoid system possesses potential drugable receptor and enzyme targets for the treatment of diverse TBI pathology. Yet, full characterization of TBI-induced changes in endocannabinoid ligands, enzymes, and receptor populations will be important to understand that role this system plays in TBI pathology. Promising classes of compounds, such as the plant-derived phytocannabinoids, synthetic cannabinoids, and endocannabinoids, as well as their non-cannabinoid receptor targets, such as TRPV1 receptors, represent important areas of basic research and potential therapeutic interest to treat TBI.

In this study, we analyzed the components of the endocannabinoid system (ECS) in R6/2 mice, a widely used model of Huntington's disease (HD). We measured the endogenous content of N-arachidonoylethanolamine and 2-arachidonoylglycerol and the activity of their biosynthetic enzymes (N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D and diacylglycerol lipase, respectively) and hydrolytic enzymes [fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase, respectively] and of their target receptors (type 1 cannabinoid receptor, type 2 cannabinoid receptor, and transient receptor potential vanilloid-1) in the brains of wild-type and R6/2 mice of different ages, as well as in the striatum and cortex of 12-week-old animals. In addition, we measured FAAH activity in lymphocytes of R6/2 mice. In the whole brains of 12-week-old R6/2 mice, we found reductions in N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D activity, diacylglycerol lipase activity and cannabinoid receptor binding, mostly associated with changes in the striatum but not in the cortex, as well as an increase in 2-arachidonoylglycerol content as compared with wild-type littermates, without any other change in ECS elements. Then, our analysis was extended to HD43 cells, an inducible cellular model of HD derived from rat ST14A cells. In both induced and noninduced conditions, we demonstrated a fully functional ECS. Overall, our data suggest that the ECS is differently affected in mouse and human HD, and that HD43 cells are suitable for high-throughput screening of FAAH-oriented drugs affecting HD progression.

Anandamide (N-arachidonoylethanolamine), an arachidonic acid derivative, is an endogenous ligand for both the brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors. To investigate the possible effects of anandamide on embryo implantation in the mouse, we used a co-culture system in which mouse embryos are cultured with a monolayer of uterine epithelial cells. Our results indicate that 14 nM anandamide significantly promotes the attachment and outgrowth of the blastocysts on the monolayer of uterine epithelial cells, and those effects could be blocked by CB1-R antagonists SR141716A, but not by SR144528, a CB2-R antagonist. It suggests that the effects of anandamide on embryo attachment and outgrowth are mediated by CB1-R. However, 56 nM anandamide is capable of inhibiting the blastocyst attachment and outgrowth, we, therefore, conclude that anandamide may play an essential role at the outset of implantation.

The content of the marine n-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is far lower in lean than in fatty seafood. Cod filets contain less than 2g fat per kg, whereof approximately 50% is EPA and DHA. However, a large fraction of these n-3 PUFAs is present in the phospholipid (PL) fraction and may have high bioavailability and capacity to change the endocannabinoid profile. Here we investigated whether exchanging meat from a lean terrestrial animal with cod in a background Western diet would alter the endocannabinoid tone in mice and thereby attenuate obesity development and hepatic lipid accumulation. Accordingly, we prepared iso-caloric diets with 15.1 energy (e) % protein, 39.1 e% fat and 45.8 e% carbohydrates using freeze-dried meat from cod filets or pork sirloins, and using a combination of soybean oil, corn oil, margarine, milk fat, and lard as the fat source. Compared with mice receiving diets containing pork, mice fed cod gained less adipose tissue mass and had a lower content of hepatic lipids. This was accompanied by a lower n-6 to n-3 ratio in liver PLs and in red blood cells (RBCs) in the mice. Furthermore, mice receiving the cod-containing diet had lower circulating levels of the two major endocannabinoids, N-arachidonoylethanolamine and 2-arachidonoylglycerol. Together, our data demonstrate that despite the relatively low content of n-3 PUFAs in cod fillets, the cod-containing diet could exert beneficial metabolic effects.

N-Acylethanolamine phospholipids (NAPEs) serve as endogenous precursors of N-acylethanolamines (NAEs), e.g. N-arachidonoylethanolamine (anandamide) and N-palmitoylethanolamine that are endogenous ligands of cannabinoid receptors. Under physiological conditions, NAPE is found in very low concentrations in mammalian tissue (3-12 nmol g(-1)). However, pathophysiological conditions may increase the endogenous NAPE levels, which again may cause an increase in endocannabinoid concentrations. This paper presents a simple and selective method for the determination of NAPE standards using negative electrospray ionization mass spectrometry (ESI-MS). The procedure provides complete positioning of all acyl and alkenyl groups contained in each NAPE species. The calibration curve for standard NAPE was linear over the range 100 fmol-50 pmol (0.1-50 ng) per injection. The lower limit of detection (signal-to-noise ratio of 3) was 100 fmol, implying that this method is superior to previous methods for the determination of NAPE. These results suggest that this ESI-MS method can be used to identify and quantify NAPE species in mammalian tissues and provide information on the corresponding NAEs to be released from the endogenous NAPE pool.

Class A G-protein coupled receptors (GPCRs) are thought to have a common topology that includes seven transmembrane alpha helices (TMHs) that are arranged to form a closed bundle. This bundle forms the ligand binding pocket into which ligands are commonly thought to enter via the extracellular milieu. This ligand approach direction makes sense for GPCRs that have small positively charged ligands, such as the beta-2-adrenergic or the dopamine D2 receptor. However, there is a growing sub-group of Class A GPCRs that bind lipid-derived endogenous ligands, such as the cannabinoid CB1 and CB2 receptors (with endogenous ligands, N-arachidonoylethanolamine (anandamide) and sn-2-arachidonylglycerol (2-AG)) and the S1P1-5 receptors (with endogenous ligand, sphingosine-1-phosphate). Even the widely studied Class A GPCR, rhodopsin, binds a highly lipophillic chromophore (11-cis-retinal). For these receptors, ligand approach from the extracellular milieu has seemed unlikely given that the ligands of these receptors readily partition into lipid or are actually synthesized in the lipid bilayer. The recent X-ray-crystal structure of the sub-type 1 sphingosine-1-phosphate receptor (S1P1) provides important information on the key structural variations that may be the hallmarks for a Class A GPCR that binds lipid-derived ligands. These include an extracellular domain that is closed off to the extracellular milieu and the existence of an opening between transmembrane helices that may serve as a portal for ligand entry via the lipid bilayer. This review examines structural aspects that the cannabinoid receptors may share with the S1P1 receptor based upon sequence homology. This review also examines experimental and simulation results that suggest ligand entry via a lipid portal is quite likely for this emerging sub-group.

N-Arachidonoylethanolamine (AEA) is a proposed endogenous ligand of the central cannabinoid receptor (CB1). Previous studies indicate that AEA is translocated across membranes via a process that has the characteristics of carrier-mediated facilitated diffusion. To date, studies of this mechanism have relied on [(3)H]AEA as a substrate for the carrier. We have synthesized an analog of AEA, SKM 4-45-1, that is nonfluorescent in the extracellular environment. When SKM 4-45-1 is exposed to intracellular esterases, it is de-esterified and becomes fluorescent. We have carried out studies to demonstrate that SKM 4-45-1 accumulation in cells occurs via the AEA carrier. SKM 4-45-1 is accumulated by both cerebellar granule cells and C6 glioma cells. Uptake of SKM 4-45-1 into C6 glioma is inhibited by AEA (IC(50)=53.8 +/- 1.8 microM), arachidonoyl-3-aminopyridine amide (IC(50)=10.1 +/- 1.4 microM), and arachidonoyl-4-hydroxyanilineamide (IC(50)=6.1 +/- 1.3 microM), all of which also inhibit [(3)H]AEA accumulation. Conversely, [(3)H]AEA accumulation by cerebellar granule cells is inhibited by SKM 4-45-1 with an IC(50) of 7.8 +/- 1. 3 microM. SKM 4-45-1 is neither a substrate nor inhibitor of fatty acid amide hydrolase, an enzyme that catabolizes AEA. SKM 4-45-1 does not bind the CB1 cannabinoid receptor at concentrations <10 microM. In summary, the cellular accumulation of SKM 4-45-1 occurs via the same pathway as AEA uptake and provides an alternative substrate for the study of this important cellular process.

Endocannabinoids are endogenous ligands of brain-type (CB1) and spleen-type (CB2) cannabinoid receptors. N-Arachidonoylethanolamine (anandamide, AEA) and 2-arachidonoylglycerol (2-AG) are prototype members of the fatty acid amides and the monoacylglycerols, two groups of endocannabinoids. Unlike CB1, CB2 receptors do not reside within 'caveolae', specialized membrane microdomains that are well-known modulators of the activity of a number of G protein-coupled receptors. In this issue of the British Journal of Pharmacology, Rimmerman and coworkers demonstrate that 2-AG is entirely localized in the caveolae of dorsal root ganglion cells, where also part of AEA (approximately 30%) can be detected. However, most of AEA (approximately 70%) was detected in non-caveolae fractions, that is where CB2 receptors are localized. The different interaction of AEA and 2-AG with membrane microdomains might have significant implications for endocannabinoid-dependent autocrine and/or retrograde-paracrine signalling pathways. It also raises an important question about the structural determinants responsible for a different localization of two apparently similar endocannabinoids within lipid bilayers.

The endocannabinoid, N-arachidonoylethanolamine (anandamide; AEA) is known to interact with voltage-gated K(+) (Kv) channels in a cannabinoid receptor-independent manner. AEA modulates the functional properties of Kv channels, converting channels with slowly inactivating current into apparent fast inactivation. In this study, we characterize the mechanism of action and binding site for AEA on Kv1.5 channels expressed on HEK-293 cells using the patch-clamp techniques. AEA exhibited high-potency block (IC(50) approximately 200 nM) from the cytoplasmic membrane surface, consistent with open-channel block. Alanine-scanning mutagenesis revealed that AEA interacts with two crucial beta-branching amino acids, Val505 and Ile508 within the S6 domain. Both residues face toward the central cavity and constitute a motif that forms a hydrophobic ring around the ion conduction pathway. This hydrophobic ring motif may be a critical determinant of cannabinoid receptor-independent AEA modulation in other K(+) channel families.

BACKGROUND

Depolarization-induced suppression of excitation (DSE) at parallel fiber-Purkinje cell synapse is an endocannabinoid-mediated short-term retrograde plasticity. Intracellular Ca(2+) elevation is critical for the endocannabinoid production and DSE. Nevertheless, how elevated Ca(2+) leads to DSE is unclear.

RESULTS

We utilized cytosolic phospholipase A(2) alpha (cPLA(2)α) knock-out mice and whole-cell patch clamp in cerebellar slices to observed the action of cPLA(2)α/arachidonic acid signaling on DSE at parallel fiber-Purkinje cell synapse. Our data showed that DSE was significantly inhibited in cPLA(2)α knock-out mice, which was rescued by arachidonic acid. The degradation enzyme of 2-arachidonoylglycerol (2-AG), monoacylglycerol lipase (MAGL), blocked DSE, while another catabolism enzyme for N-arachidonoylethanolamine (AEA), fatty acid amide hydrolase (FAAH), did not affect DSE. These results suggested that 2-AG is responsible for DSE in Purkinje cells. Co-application of paxilline reversed the blockade of DSE by internal K(+), indicating that large conductance Ca(2+)-activated potassium channel (BK) is sufficient to inhibit cPLA(2)α/arachidonic acid-mediated DSE. In addition, we showed that the release of 2-AG was independent of soluble NSF attachment protein receptor (SNARE), protein kinase C and protein kinase A.

CONCLUSIONS

Our data first showed that cPLA(2)α/arachidonic acid/2-AG signaling pathway mediates DSE at parallel fiber-Purkinje cell synapse.

A radiochromatographic method has been set up to assay the activity of N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD), based on reversed-phase high-performance liquid chromatography (HPLC) and online scintillation counting. The anandamide (N-arachidonoylethanolamine, AEA), product released by NAPE-PLD from the N-arachidonoyl-phosphatidylethanolamine (NArPE) substrate, was separated using a C18 column eluted with methanol-water-acetic acid and was quantified with an external standard method. Baseline separation of AEA and NArPE was completed in less than 15 min, with a detection limit of 0.5 fmol AEA at a signal-to-noise ratio of 4:1. The sensitivity and accuracy of the radiochromatographic procedure allowed detection and characterization of NAPE-PLD activity in very tiny tissue samples or in samples where the enzymatic activity is very low. With this method, we could determine the kinetic constants (i.e., apparent Michaelis-Menten constant (Km) of 40.0+/-5.6 microM and maximum velocity (Vmax) of 22.2+/-3.5 pmol/min per milligram protein toward NArPE) and the distribution of NAPE-PLD activity in brain areas and peripheral tissues of mouse. In addition, we could collect unprecedented evidence that compounds widely used in studies of the endocannabinoid system (e.g., AEA and congeners, receptor a(nta)gonists and inhibitors of AEA degradation) can also affect NAPE-PLD activity.

<em>N</em>egative emotional states that are associated with excessive alcohol intake, particularly anxiety-like states, have been linked to opponent processes in the central nucleus of the amygdala (CeA), affecting stress-related transmitters and monoamines. This study extends these observations to include endocannabinoid signaling in alcohol-dependent animals. Rats and mice were exposed to chronic intermittent alcohol with vapor inhalation or liquid diet to induce dependence. In vivo microdialysis was used to estimate interstitial concentrations of endocannabinoids [<em>N</em>-<em>arachidonoylethanolamine</em> (anandamide; AEA) and 2-arachidonoylglycerol (2-AG)] and amino acids (glutamate and GABA) in rat CeA. Additionally, we evaluated the inhibition of endocannabinoids clearance enzymes [monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase] on anxiety-like behavior and alcohol consumption in alcohol-dependent rats and mice. Results revealed that alcohol dependence produced decreases in baseline 2-AG dialysate levels and increases in baseline levels of glutamate and GABA. Acute alcohol abstinence induced an enhancement of these dependence-induced effects and the levels of 2-AG and GABA were restored upon alcohol re-exposure. Additional studies showed that the increased CeA 2-AG levels induced by restraint stress and alcohol self-administration were blunted in alcohol-dependent rats. Pharmacological studies in rats and mice showed that anxiety-like behavior and alcohol consumption were increased in alcohol-dependent animals, and these behavioral effects were attenuated mainly by MAGL inhibitors [MJ<em>N</em>110 (10 and 20 mg/kg) in rats and JZL184 (1 and 3 mg/kg) in mice]. The present results suggest a key role for endocannabinoid signaling in motivational neuroadaptations during alcohol dependence, in which a deficiency in CeA 2-AG signaling in alcohol-dependent animals is linked to stress and excessive alcohol consumption.