<em>2</em>-<em>Arachidonoylglycerol</em> plays a major role in endocannabinoid signaling, and is tightly regulated by the monoacylglycerol lipase (MAGL). Here we report the crystal structure of human MAGL. The protein crystallizes as a dimer, and despite structural homologies to haloperoxidases and esterases, it distinguishes itself by a wide and hydrophobic access to the catalytic site. An apolar helix covering the active site also gives structural insight into the amphitropic character of MAGL, and likely explains how MAGL interacts with membranes to recruit its substrate. Docking of <em>2</em>-<em>arachidonoylglycerol</em> highlights a hydrophobic and a hydrophilic cavity that accommodate the lipid into the catalytic site. Moreover, we identified Cys<em>2</em>01 as the crucial residue in MAGL inhibition by N-arachidonylmaleimide, a sulfhydryl-reactive compound. Beside the advance in the knowledge of endocannabinoids degradation routes, the structure of MAGL paves the way for future medicinal chemistry works aimed at the design of new drugs exploiting <em>2</em>-<em>arachidonoylglycerol</em> transmission.

OBJECTIVE

N-arachidonoyl-serotonin (AA-5-HT) is an inhibitor of fatty acid amide hydrolase (FAAH)-catalysed hydrolysis of the endocannabinoid/ endovanilloid compound, anandamide (AEA). We investigated if AA-5-HT antagonizes the transient receptor potential vanilloid-1 (TRPV1) channel and, as FAAH and TRPV1 are targets for analgesic compounds, if it exerts analgesia in rodent models of hyperalgesia.

METHODS

AA-5-HT was tested in vitro, on HEK-<em>2</em>93 cells overexpressing the human or the rat recombinant TRPV1 receptor, and in vivo, in rats and mice treated with formalin and in rats with chronic constriction injury of the sciatic nerve. The levels of the endocannabinoids, AEA and <em>2</em>-<em>arachidonoylglycerol</em>, in supraspinal (periaqueductal grey, rostral ventromedial medulla), spinal or peripheral (skin) tissues were measured.

RESULTS

AA-5-HT behaved as an antagonist at both rat and human TRPV1 receptors (IC(50)=37-40 nM against 100 nM capsaicin). It exerted strong analgesic activity in all pain models used here. This activity was partly due to FAAH inhibition, elevation of AEA tissue levels and indirect activation of cannabinoid CB(1) receptors, as it was reversed by AM<em>2</em>51, a CB(1) antagonist. AA-5-HT also appeared to act either via activation/desensitization of TRPV1, following elevation of AEA, or as a direct TRPV1 antagonist, as suggested by the fact that its effects were either reversed by capsazepine and 5'-iodo-resiniferatoxin, two TRPV1 antagonists, or mimicked by these compounds administered alone.

CONCLUSIONS

Possibly due to its dual activity as a FAAH inhibitor and TRPV1 antagonist, AA-5-HT was highly effective against both acute and chronic peripheral pain.

Endogenous cannabinoid receptor ligands (endocannabinoids) may rescue neurons from glutamate excitotoxicity. As these substances also accumulate in cultured immature neurons following neuronal damage, elevated endocannabinoid concentrations may be interpreted as a putative neuroprotective response. However, it is not known how glutamatergic insults affect in vivo endocannabinoid homeostasis, i.e. N-arachidonoylethanolamine (anandamide) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), as well as other constituents of their lipid families, N-acylethanolamines (NAEs) and <em>2</em>-monoacylglycerols (<em>2</em>-MAGs), respectively. Here we employed three in vivo neonatal rat models characterized by widespread neurodegeneration as a consequence of altered glutamatergic neurotransmission and assessed changes in endocannabinoid homeostasis. A 46-fold increase of cortical NAE concentrations (anandamide, 13-fold) was noted <em>2</em>4 h after intracerebral NMDA injection, while less severe insults triggered by mild concussive head trauma or NMDA receptor blockade produced a less pronounced NAE accumulation. By contrast, levels of <em>2</em>-AG and other <em>2</em>-MAGs were virtually unaffected by the insults employed, rendering it likely that key enzymes in biosynthetic pathways of the two different endocannabinoid structures are not equally associated to intracellular events that cause neuronal damage in vivo. Analysis of cannabinoid CB(1) receptor mRNA expression and binding capacity revealed that cortical subfields exhibited an up-regulation of these parameters following mild concussive head trauma and exposure to NMDA receptor blockade. This may suggest that mild to moderate brain injury may trigger elevated endocannabinoid activity via concomitant increase of anandamide levels, but not <em>2</em>-AG, and CB(1) receptor density.

Anandamide (arachidonylethanolamide) and <em>2</em>-<em>arachidonoylglycerol</em> mediate many of their actions via either CB(1) or CB(<em>2</em>) cannabinoid receptor subtypes. These agonist-receptor interactions result in activation of G proteins, particularly those of the G(i/o) family. Signal transduction pathways that are regulated by these G proteins include inhibition of adenylyl cyclase, regulation of ion currents (inhibition of voltage-gated L, N and P/Q Ca(<em>2</em>+)-currents; activation of K(+) currents); activation of focal adhesion kinase (FAK), mitogen activated protein kinase (MAPK) and induction of immediate early genes; and stimulation of nitric oxide synthase (NOS). Other effects of anandamide and/or <em>2</em>-<em>arachidonoylglycerol</em> that are not mediated via cannabinoid receptors include inhibition of L-type Ca(<em>2</em>+) channels, stimulation of VR(1) vanilloid receptors, transient changes in intracellular Ca(<em>2</em>+), and disruption of gap junction function. Cardiovascular regulation by anandamide appears to occur by a variety of receptor-mediated and non-receptor-mediated mechanisms. This review will describe and evaluate each of these signal transduction pathways and mechanisms.

Anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are two endogenous ligands for the cannabinoid receptors, and their cannabimimetic activities are lost when they are hydrolyzed enzymatically. Cytosol and particulate fractions of porcine brain exhibited a high <em>2</em>-AG hydrolyzing activity of 100 nmol/min/mg protein. Most of the activity could be attributed to a monoacylglycerol lipase-like enzyme that did not hydrolyze anandamide. It was separated by hydroxyapatite chromatography from anandamide amidohydrolase, which is also capable of hydrolyzing <em>2</em>-AG as well as anandamide. Thus, porcine brain has at least two enzymes capable of hydrolyzing <em>2</em>-AG. The <em>2</em>-AG hydrolase activities of both the cytosolic and particulate enzymes were irreversibly and time-dependently inhibited by methyl arachidonyl fluorophosphonate with IC50 values as low as <em>2</em>-3 nM.

Cb<em>2</em> is a novel protooncogene encoding the peripheral cannabinoid receptor. Previous studies demonstrated that <em>2</em> distinct noncoding first exons exist: exon-1A and exon-1B, which both splice to protein-coding exon-<em>2</em>. We demonstrate that in retrovirally induced murine myeloid leukemia cells with proviral insertion in Cb<em>2</em>, exon-1B/exon-<em>2</em> Cb<em>2</em> messenger RNA levels have been increased, resulting in high receptor numbers. In myeloid leukemia cells without virus insertion in this locus, low levels of only exon-1A/exon-<em>2</em> Cb<em>2</em> transcripts were present and receptors could not be detected. To elucidate the function of Cb<em>2</em> in myeloid leukemia cells, a set of in vitro experiments was carried out using 3<em>2</em>D/G-CSF-R (granulocyte colony-stimulating factor receptor) cells transfected with exon-1B/exon-<em>2</em> Cb<em>2</em> complementary DNA and a myeloid cell line carrying a virus insertion in Cb<em>2</em> (ie, NFS 78). We demonstrate that a major function of the Cb<em>2</em> receptor is stimulation of migration as determined in a transwell assay. Exposure of Cb<em>2</em>-expressing cells to different cannabinoids showed that the true ligand for Cb<em>2</em> is <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), which may act as chemoattractant and as a chemokinetic agent. Furthermore, we observed a significant synergistic activity between <em>2</em>-AG and interleukin-3 or G-CSF, suggesting cross-talk between the different receptor systems. Radioactive-ligand binding studies revealed significant numbers of Cb<em>2</em> receptors in normal spleen. Transwell experiments carried out with normal mouse spleen cells showed <em>2</em>-AG-induced migration of B<em>2</em><em>2</em>0-, CD19-, immunoglobulin M-, and immunoglobulin D-expressing B lymphocytes. Our study demonstrates that a major function of Cb<em>2</em> receptor expressed on myeloid leukemia cells or normal splenocytes is stimulation of migration.

BACKGROUND

n-3 polyunsaturated fatty acids, namely docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), reduce the risk of cardiovascular disease and can ameliorate many of obesity-associated disorders. We hypothesised that the latter effect will be more pronounced when DHA/EPA is supplemented as phospholipids rather than as triglycerides.

RESULTS

In a 'prevention study', C57BL/6J mice were fed for 9 weeks on either a corn oil-based high-fat obesogenic diet (cHF; lipids ∼35% wt/wt), or cHF-based diets in which corn oil was partially replaced by DHA/EPA, admixed either as phospholipids or triglycerides from marine fish. The reversal of obesity was studied in mice subjected to the preceding cHF-feeding for 4 months. DHA/EPA administered as phospholipids prevented glucose intolerance and tended to reduce obesity better than triglycerides. Lipemia and hepatosteatosis were suppressed more in response to dietary phospholipids, in correlation with better bioavailability of DHA and EPA, and a higher DHA accumulation in the liver, white adipose tissue (WAT), and muscle phospholipids. In dietary obese mice, both DHA/EPA concentrates prevented a further weight gain, reduced plasma lipid levels to a similar extent, and tended to improve glucose tolerance. Importantly, only the phospholipid form reduced plasma insulin and adipocyte hypertrophy, while being more effective in reducing hepatic steatosis and low-grade inflammation of WAT. These beneficial effects were correlated with changes of endocannabinoid metabolome in WAT, where phospholipids reduced <em>2</em>-<em>arachidonoylglycerol</em>, and were more effective in increasing anti-inflammatory lipids such as N-docosahexaenoylethanolamine.

CONCLUSIONS

Compared with triglycerides, dietary DHA/EPA administered as phospholipids are superior in preserving a healthy metabolic profile under obesogenic conditions, possibly reflecting better bioavalability and improved modulation of the endocannabinoid system activity in WAT.

Monoglyceride lipase (MGL) is a serine hydrolase that hydrolyses <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) into arachidonic acid and glycerol. <em>2</em>-AG is an endogenous ligand of cannabinoid receptors, involved in various physiological processes in the brain. We present here the first crystal structure of human MGL in its apo form and in complex with the covalent inhibitor SAR6<em>2</em>9. MGL shares the classic fold of the alpha/beta hydrolase family but depicts an unusually large hydrophobic occluded tunnel with a highly flexible lid at its entry and the catalytic triad buried at its end. Structures reveal the configuration of the catalytic triad and the shape and nature of the binding site of <em>2</em>-AG. The bound structure of SAR6<em>2</em>9 highlights the key interactions for productive binding with MGL. The shape of the tunnel suggests a high druggability of the protein and provides an attractive template for drug discovery.

Fatty acid amide hydrolase (FAAH) catalyzes the hydrolysis of bioactive fatty acid amides and esters such as the endogenous cannabinoid receptor ligands, anandamide (N-arachidonoyl-ethanolamine) and <em>2</em>-<em>arachidonoylglycerol</em>, and the putative sleep inducing factor cis-9-octadecenoamide (oleamide). Most FAAH blockers developed to date also inhibit cytosolic phospholipase A<em>2</em> (cPLA<em>2</em>) and/or bind to the CB1 cannabinoid receptor subtype. Here we report the finding of four novel FAAH inhibitors, two of which, malhamensilipin A and grenadadiene, were screened out of a series of thirty-two different algal natural products, and two others, arachidonoylethylene glycol (AEG) and arachidonoyl-serotonin (AA-5-HT) were selected out of five artificially functionalized polyunsaturated fatty acids. When using FAAH preparations from mouse neuroblastoma N18TG<em>2</em> cells and [14C]anandamide as a substrate, the IC50s for these compounds ranged from 1<em>2</em>.0 to <em>2</em>6 microM, the most active compound being AA-5-HT. This substance was also active on FAAH from rat basophilic leukaemia (RBL-<em>2</em>H3) cells (IC50 = 5.6 microM), and inhibited [14C]anandamide hydrolysis by both N18TG<em>2</em> and RBL-<em>2</em>H3 intact cells without affecting [14C]anandamide uptake. While AEG behaved as a competitive inhibitor and was hydrolyzed to arachidonic acid (AA) by FAAH preparations, AA-5-HT was resistant to FAAH-catalyzed hydrolysis and behaved as a tight-binding, albeit non-covalent, mixed inhibitor. AA-5-HT did not interfere with cPLA<em>2</em>-mediated, ionomycin or antigen-induced release of [3H]AA from RBL-<em>2</em>H3 cells, nor with cPLA<em>2</em> activity in cell-free experiments. Finally, AA-5-HT did not activate CB1 cannabinoid receptors since it acted as a very weak ligand in in vitro binding assays, and, at 10-15 mg/kg body weight, it was not active in the 'open field', 'hot plate' and rectal hypothermia tests carried out in mice. Conversely AEG behaved as a cannabimimetic substance in these tests as well as in the 'ring' immobility test where AA-5-HT was also active. AA-5-HT is the first FAAH inhibitor reported to date which is inactive both against cPLA<em>2</em> and at CB1 receptors, whereas AEG represents a new type of cannabinoid receptor agonist.

Endocannabinoids are endogenous ligands of the cannabinoid receptors CB1 and CB<em>2</em>. Two arachidonic acid derivatives, arachidonoylethanolamide (anandamide) and <em>2</em>-<em>arachidonoylglycerol</em>, are considered to be physiologically important endocannabinoids. In the known metabolic pathway in mammals, anandamide and other bioactive N-acylethanolamines, such as palmitoylethanolamide and oleoylethanolamide, are biosynthesized from glycerophospholipids by a combination of Ca(<em>2</em>+)-dependent N-acyltransferase and N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D, and are degraded by fatty acid amide hydrolase. However, recent studies have shown the involvement of other enzymes and pathways, which include the members of the tumor suppressor HRASLS family (the phospholipase A/acyltransferase family) functioning as Ca(<em>2</em>+)-independent N-acyltransferases, N-acyl-phosphatidylethanolamine-hydrolyzing phospholipaseD-independent multistep pathways via N-acylated lysophospholipid, and N-acylethanolamine-hydrolyzing acid amidase, a lysosomal enzyme that preferentially hydrolyzes palmitoylethanolamide. Although their physiological significance is poorly understood, these new enzymes/pathways may serve as novel targets for the development of therapeutic drugs. For example, selective N-acylethanolamine-hydrolyzing acid amidase inhibitors are expected to be new anti-inflammatory and analgesic drugs. In this minireview, we focus on advances in the understanding of these enzymes/pathways. In addition, recent findings on <em>2</em>-<em>arachidonoylglycerol</em> metabolism are described.

Endocannabinoids (eCBs) mediate short- and long-term depression of synaptic strength by retrograde transsynaptic signaling. Previous studies have suggested that an eCB mobilization or release step in the postsynaptic neuron is involved in this retrograde signaling. However, it is not known whether this release process occurs automatically upon eCB synthesis or whether it is regulated by other synaptic factors. To address this issue, we loaded postsynaptic striatal medium spiny neurons (MSNs) with the eCBs anandamide (AEA) or <em>2</em>-<em>arachidonoylglycerol</em> and determined the conditions necessary for presynaptic inhibition. We found that presynaptic depression of glutamatergic excitatory postsynaptic currents (EPSCs) and GABAergic inhibitory postsynaptic currents (IPSCs) induced by postsynaptic eCB loading required a certain level of afferent activation that varied between the different synaptic types. Synaptic depression at excitatory synapses was temperature-dependent and blocked by the eCB membrane transport blockers, VDM11 and UCM707, but did not require activation of metabotropic glutamate receptors, l-calcium channels, nitric oxide, voltage-activated Na(+) channels, or intracellular calcium. Application of the CB(1)R antagonist, AM<em>2</em>51, after depression was established, reversed the decrease in EPSC, but not in IPSC, amplitude. Direct activation of the CB(1) receptor by WIN 55,<em>2</em>1<em>2</em>-<em>2</em> initiated synaptic depression that was independent of afferent stimulation. These findings indicate that retrograde eCB signaling requires a postsynaptic release step involving a transporter or carrier that is activated by afferent stimulation/synaptic activation.

OBJECTIVE

We examined the presence of functional cannabinoid receptors 1 and <em>2</em> (CB1, CB<em>2</em>) in isolated human islets, phenotyped the cells producing cannabinoid receptors and analysed the actions of selective cannabinoid receptor agonists on insulin, glucagon and somatostatin secretion in vitro. We also described the localisation on islet cells of: (1) the endocannabinoid-producing enzymes N-acyl-phosphatidyl ethanolamine-hydrolysing phospholipase D and diacylglycerol lipase; and (<em>2</em>) the endocannabinoid-degrading enzymes fatty acid amidohydrolase and monoacyl glycerol lipase.

METHODS

Real-time PCR, western blotting and immunocytochemistry were used to analyse the presence of endocannabinoid-related proteins and genes. Static secretion experiments were used to examine the effects of activating CB1 or CB<em>2</em> on insulin, glucagon and somatostatin secretion and to measure changes in <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) levels within islets. Analyses were performed in isolated human islets and in paraffin-embedded sections of human pancreas.

RESULTS

Human islets of Langerhans expressed CB1 and CB<em>2</em> (also known as CNR1 and CNR<em>2</em>) mRNA and CB1 and CB<em>2</em> proteins, and also the machinery involved in synthesis and degradation of <em>2</em>-AG (the most abundant endocannabinoid, levels of which were modulated by glucose). Immunofluorescence revealed that CB1 was densely located in glucagon-secreting alpha cells and less so in insulin-secreting beta cells. CB<em>2</em> was densely present in somatostatin-secreting delta cells, but absent in alpha and beta cells. In vitro experiments revealed that CB1 stimulation enhanced insulin and glucagon secretion, while CB<em>2</em> agonism lowered glucose-dependent insulin secretion, showing these cannabinoid receptors to be functional.

CONCLUSIONS

Together, these results suggest a role for endogenous endocannabinoid signalling in regulation of endocrine secretion in the human pancreas.

Cannabinoid receptors and their endogenous ligands, the endocannabinoids, have been detected in several blood immune cells, including monocytes/macrophages, basophils and lymphocytes. However, their presence in dendritic cells, which play a key role in the initiation and development of the immune response, has never been investigated. Here we have analyzed human dendritic cells for the presence of the endocannabinoids, anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), the cannabinoid CB1 and CB<em>2</em> receptors, and one of the enzymes mostly responsible for endocannabinoid hydrolysis, the fatty acid amide hydrolase (FAAH). By using a very sensitive liquid chromatography-atmospheric pressure chemical ionization-mass spectrometric (LC-APCI-MS) method, lipids extracted from immature dendritic cells were shown to contain <em>2</em>-AG, anandamide and the anti-inflammatory anandamide congener, N-palmitoylethanolamine (PalEtn) (<em>2</em>.1 +/- 1.0, 0.14 +/- 0.0<em>2</em> and 8.<em>2</em> +/- 3.9 pmol x 10(-7) cells, respectively). The amounts of <em>2</em>-AG, but not anandamide or PalEtn, were significantly increased following cell maturation induced by bacterial lipopolysaccharide (LPS) or the allergen Der p 1 (<em>2</em>.8- and 1.9-fold, respectively). By using both RT-PCR and Western immunoblotting, dendritic cells were also found to express measurable amounts of CB1 and CB<em>2</em> receptors and of FAAH. Cell maturation did not consistently modify the expression of these proteins, although in some cell preparations a decrease of the levels of both CB1 and CB<em>2</em> mRNA transcripts was observed after LPS stimulation. These findings demonstrate for the first time that the endogenous cannabinoid system is present in human dendritic cells and can be regulated by cell activation.

Endocannabinoids (endogenous ligands of cannabinoid receptors) exert diverse physiological and pathophysiological functions in animal tissues. N-Arachidonoylethanolamine (anandamide) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are two representative endocannabinoids. Both the compounds are arachidonic acid-containing lipid molecules generated from membrane glycerophospholipids, but their biosynthetic pathways are totally different. Anandamide is principally formed together with other N-acylethanolamines (NAEs) in a two-step pathway, which is composed of Ca(<em>2</em>+)-dependent N-acyltransferase and N-acylphosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD). cDNA cloning of NAPE-PLD and subsequent analysis of its gene-disrupted mice led to the discovery of alternative pathways comprising multiple enzymes. As for the <em>2</em>-AG biosynthesis, recent results, including cDNA cloning of diacylglycerol lipase and analyses of phospholipase Cbeta-deficient mice, demonstrated that these two enzymes are responsible for the in vivo formation of <em>2</em>-AG functioning as a retrograde messenger in synapses. In this review article, we will focus on recent progress in the studies on the enzymes responsible for the endocannabinoid biosyntheses.

Marijuana and aspirin have been used for millennia to treat a wide range of maladies including pain and inflammation. Both cannabinoids, like marijuana, that exert anti-inflammatory action through stimulating cannabinoid receptors, and cyclooxygenase (COX) inhibitors, like aspirin, that suppress pro-inflammatory eicosanoid production have shown beneficial outcomes in mouse models of neurodegenerative diseases and cancer. Both cannabinoids and COX inhibitors, however, have untoward effects that discourage their chronic usage, including cognitive deficits and gastrointestinal toxicity, respectively. Recent studies have uncovered that the serine hydrolase monoacylglycerol lipase (MAGL) links the endocannabinoid and eicosanoid systems together through hydrolysis of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) to provide the major arachidonic acid (AA) precursor pools for pro-inflammatory eicosanoid synthesis in specific tissues. Studies in recent years have shown that MAGL inhibitors elicit anti-nociceptive, anxiolytic, and anti-emetic responses and attenuate precipitated withdrawal symptoms in addiction paradigms through enhancing endocannabinoid signaling. MAGL inhibitors have also been shown to exert anti-inflammatory action in the brain and protect against neurodegeneration through lowering eicosanoid production. In cancer, MAGL inhibitors have been shown to have anti-cancer properties not only through modulating the endocannabinoid-eicosanoid network, but also by controlling fatty acid release for the synthesis of protumorigenic signaling lipids. Thus, MAGL serves as a critical node in simultaneously coordinating multiple lipid signaling pathways in both physiological and disease contexts. This review will discuss the diverse (patho)physiological roles of MAGL and the therapeutic potential of MAGL inhibitors in treating a vast array of complex human diseases.

The N-aryl carbamate URB60<em>2</em> (biphenyl-3-ylcarbamic acid cyclohexyl ester) is an inhibitor of monoacylglycerol lipase (MGL), a serine hydrolase involved in the biological deactivation of the endocannabinoid <em>2</em>-arachidonoyl-sn-glycerol (<em>2</em>-AG). Here, we investigated the mechanism by which URB60<em>2</em> inhibits purified recombinant rat MGL by using a combination of biochemical and structure-activity relationship (SAR) approaches. We found that URB60<em>2</em> weakly inhibits recombinant MGL (IC(50) = <em>2</em><em>2</em>3 +/- 63 microM) through a rapid and noncompetitive mechanism. Dialysis experiments and SAR analyses suggest that URB60<em>2</em> acts through a partially reversible mechanism rather than by irreversible carbamoylation of MGL. Finally, URB60<em>2</em> (100 microM) elevates <em>2</em>-AG levels in hippocampal slice cultures without affecting levels of other endocannabinoid-related substances. Thus, URB60<em>2</em> may provide a useful tool by which to investigate the physiological roles of <em>2</em>-AG and explore the potential interest of MGL as a therapeutic target.

The directional migration of neutrophils towards inflammatory mediators, such as chemokines and cannabinoids, occurs via the activation of seven transmembrane G protein coupled receptors (7TM/GPCRs) and is a highly organized process. A crucial role for controlling neutrophil migration has been ascribed to the cannabinoid CB(<em>2</em>) receptor (CB(<em>2</em>)R), but additional modulatory sites distinct from CB(<em>2</em>)R have recently been suggested to impact CB(<em>2</em>)R-mediated effector functions in neutrophils. Here, we provide evidence that the recently de-orphanized 7TM/GPCR GPR55 potently modulates CB(<em>2</em>)R-mediated responses. We show that GPR55 is expressed in human blood neutrophils and its activation augments the migratory response towards the CB(<em>2</em>)R agonist <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), while inhibiting neutrophil degranulation and reactive oxygen species (ROS) production. Using HEK<em>2</em>93 and HL60 cell lines, along with primary neutrophils, we show that GPR55 and CB(<em>2</em>)R interfere with each other's signaling pathways at the level of small GTPases, such as Rac<em>2</em> and Cdc4<em>2</em>. This ultimately leads to cellular polarization and efficient migration as well as abrogation of degranulation and ROS formation in neutrophils. Therefore, GPR55 limits the tissue-injuring inflammatory responses mediated by CB(<em>2</em>)R, while it synergizes with CB(<em>2</em>)R in recruiting neutrophils to sites of inflammation.

The endocannabinoid anandamide [N-arachidonoylethanolamine (AEA)] is thought to function as an endogenous protective factor of the brain against acute neuronal damage. However, this has never been tested in an in vivo model of acute brain injury. Here, we show in a longitudinal pharmacological magnetic resonance imaging study that exogenously administered AEA dose-dependently reduced neuronal damage in neonatal rats injected intracerebrally with the Na(+)/K(+)-ATPase inhibitor ouabain. At 15 min after injury, AEA (10 mg/kg) administered 30 min before ouabain injection reduced the volume of cytotoxic edema by 43 +/- 15% in a manner insensitive to the cannabinoid CB(1) receptor antagonist SR141716A. At 7 d after ouabain treatment, 64 +/- <em>2</em>4% less neuronal damage was observed in AEA-treated (10 mg/kg) rats compared with control animals. Coadministration of SR141716A prevented the neuroprotective actions of AEA at this end point. In addition, (1) no increase in AEA and <em>2</em>-<em>arachidonoylglycerol</em> levels was detected at <em>2</em>, 8, or <em>2</em>4 hr after ouabain injection; (<em>2</em>) application of SR141716A alone did not increase the lesion volume at days 0 and 7; and (3) the AEA-uptake inhibitor, VDM11, did not affect the lesion volume. These data indicate that there was no endogenous endocannabinoid tone controlling the acute neuronal damage induced by ouabain. Although our data seem to question a possible role of the endogenous cannabinoid system in establishing a brain defense system in our model, AEA may be used as a structural template to develop neuroprotective agents.

Endocannabinoids have been implicated in cancer. Increasing endogenous <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) by blocking its metabolism inhibits invasion of androgen-independent prostate cancer (PC-3 and DU-145) cells. Noladin ether (a stable <em>2</em>-AG analog) and exogenous CB1 receptor agonists possess similar effects. Conversely, reducing endogenous <em>2</em>-AG by inhibiting its synthesis or blocking its binding to CB1 receptors with antagonists increases the cell invasion. <em>2</em>-AG and noladin ether decrease protein kinase A activity in these cells, indicating coupling of the CB1 receptor to downstream effectors. The results suggest that cellular <em>2</em>-AG, acting through the CB1 receptor, is an endogenous inhibitor of invasive prostate cancer cells.

Cannabinoids exert most of their effects through the CB(1) receptor. This G-protein-coupled receptor has been shown to be functionally coupled to inhibition of adenylyl cyclase, modulation of ion channels, and activation of extracellular signal-regulated kinase. Using Chinese hamster ovary cells stably transfected with the CB(1) receptor cDNA, we show here that Delta(9)-tetrahydrocannabinol (THC), the major active component of marijuana, induces the activation of c-Jun N-terminal kinase (JNK). Western blot analysis showed that both JNK-1 and JNK-<em>2</em> were stimulated by THC. The effect of THC was also exerted by endogenous cannabinoids (anandamide and <em>2</em>-<em>arachidonoylglycerol</em>) and synthetic cannabinoids (CP-55,940, HU-<em>2</em>10, and methanandamide), and was prevented by the selective CB(1) antagonist SR141716. Pertussis toxin, wortmannin, and a Ras farnesyltransferase inhibitor peptide blocked, whereas mastoparan mimicked, the CB(1) receptor-evoked activation of JNK, supporting the involvement of a G(i)/G(o)-protein, phosphoinositide 3'-kinase and Ras. THC-induced JNK stimulation was prevented by tyrphostin AG1<em>2</em>96, pointing to the implication of platelet-derived growth factor receptor transactivation, and was independent of ceramide generation. Experiments performed with several types of neural cells that endogenously express the CB(1) receptor suggested that long-term JNK activation may be involved in THC-induced cell death. The CB(1) cannabinoid receptor was also shown to be coupled to the activation of p38 mitogen-activated protein kinase. Data indicate that activation of JNK and p38 mitogen-activated protein kinase may be responsible for some of the cellular responses elicited by the CB(1) cannabinoid receptor.

Hemopressin (Hp), a 9-residue alpha-hemoglobin-derived peptide, was previously reported to function as a CB(1) cannabinoid receptor antagonist (1) . In this study, we report that mass spectrometry (MS) data from peptidomics analyses of mouse brain extracts identified N-terminally extended forms of Hp containing either three (RVD-Hpalpha) or two (VD-Hpalpha) additional amino acids, as well as a beta-hemoglobin-derived peptide with sequence similarity to that of hemopressin (VD-Hpbeta). Characterization of the alpha-hemoglobin-derived peptides using binding and functional assays shows that in contrast to Hp, which functions as a CB(1) cannabinoid receptor antagonist, both RVD-Hpalpha and VD-Hpalpha function as agonists. Studies examining the increase in the phosphorylation of ERK1/<em>2</em> levels or release of intracellular Ca(<em>2</em>+) indicate that these peptides activate a signal transduction pathway distinct from that activated by the endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em>, or the classic CB(1) agonist, Hu-<em>2</em>10. This finding suggests an additional mode of regulation of endogenous cannabinoid receptor activity. Taken together, these results suggest that the CB(1) receptor is involved in the integration of signals from both lipid- and peptide-derived signaling molecules.

Endocannabinoids are regarded as retrograde signaling molecules at various types of synapses throughout the CNS. The lipid derivatives anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are generally thought to be the key molecular players in this process. Previous anatomical and electrophysiological studies provided compelling evidence that the biosynthetic enzyme of <em>2</em>-AG is indeed localized in the postsynaptic plasma membrane, whereas its target, the CB1 cannabinoid receptor, and the enzyme responsible for its inactivation are both found presynaptically. This molecular architecture of <em>2</em>-AG signaling is a conserved feature of most synapses and supports the retrograde signaling role of <em>2</em>-AG. Conversely, the molecular and neuroanatomical organization of synaptic anandamide signaling remains largely unknown. In contrast to its predicted role in retrograde signaling, here we show that N-acylphosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD), a biosynthetic enzyme of anandamide and its related bioactive congeners, the N-acylethanolamines (NAEs), is concentrated presynaptically in several types of hippocampal excitatory axon terminals. Furthermore, high-resolution quantitative immunogold labeling demonstrates that this calcium-sensitive enzyme is localized predominantly on the intracellular membrane cisternae of axonal calcium stores. Finally, the highest density of NAPE-PLD is found in mossy terminals of granule cells, which do not express CB1 receptors. Together, these findings suggest that anandamide and related NAEs are also present at glutamatergic synapses, but the sites of their synthesis and action are remarkably different from <em>2</em>-AG, indicating distinct physiological roles for given endocannabinoids in the regulation of synaptic neurotransmission and plasticity.

Cisplatin, a platinum-derived chemotherapeutic agent, produces mechanical and coldallodynia reminiscent of chemotherapy-induced neuropathy in humans. The endocannabinoid system represents a novel target for analgesic drug development. The endocannabinoid signaling system consists of endocannabinoids (e.g. anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG)), cannabinoid receptors (e.g. CB(1) and CB(<em>2</em>)) and the enzymes controlling endocannabinoid synthesis and degradation. AEA is hydrolyzed by fatty-acid amide hydrolase (FAAH) whereas <em>2</em>-AG is hydrolyzed primarily by monoacylglycerol lipase (MGL). We compared effects of brain permeant (URB597) and impermeant (URB937) inhibitors of FAAH with an irreversible inhibitor of MGL (JZL184) on cisplatin-evoked behavioral hypersensitivities. Endocannabinoid modulators were compared with agents used clinically to treat neuropathy (i.e. the opioid analgesic morphine, the anticonvulsant gabapentin and the tricyclic antidepressant amitriptyline). Cisplatin produced robust mechanical and cold allodynia but did not alter responsiveness to heat. After neuropathy was fully established, groups received acute intraperitoneal (i.p.) injections of vehicle, amitriptyline (30 mg/kg), gabapentin (100 mg/kg), morphine (6 mg/kg), URB597 (0.1 or 1 mg/kg), URB937 (0.1 or 1 mg/kg) or JZL184 (1, 3 or 8 mg/kg). Pharmacological specificity was assessed by coadministering each endocannabinoid modulator with either a CB(1) (AM<em>2</em>51 3 mg/kg), CB(<em>2</em>) (AM630 3 mg/kg), TRPV1 (AMG9810 3 mg/kg) or TRPA1 (HC030031 8 mg/kg) antagonist. Effects of cisplatin on endocannabinoid levels and transcription of receptors (CB(1), CB(<em>2</em>), TRPV1, TRPA1) and enzymes (FAAH, MGL) linked to the endocannabinoid system were also assessed. URB597, URB937, JZL184 and morphine reversed cisplatin-evoked mechanical and cold allodynia to pre-cisplatin levels. By contrast, gabapentin only partially reversed the observed allodynia while amitriptyline, administered acutely, was ineffective. CB(1) or CB(<em>2</em>) antagonists completely blocked the anti-allodynic effects of both FAAH (URB597, URB937) and MGL (JZL184) inhibitors to mechanical and cold stimulation. By contrast, the TRPV1 antagonist AMG9810 blocked the anti-allodynic efficacy of both FAAH inhibitors, but not the MGL inhibitor. By contrast, the TRPA1 antagonist HC30031 did not attenuate anti-allodynic efficacy of any endocannabinoid modulator. When the levels of endocannabinoids were examined, cisplatin increased both anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) levels in the lumbar spinal cord and decreased <em>2</em>-AG levels (but not AEA) in dorsal hind paw skin. RT-PCR showed that mRNA for FAAH, but not other markers, was upregulated by cisplatin treatment in lumbar spinal cord. The present studies demonstrate that cisplatin alters endocannabinoid tone and that inhibition of endocannabinoid hydrolysis alleviates chemotherapy-induced mechanical and cold allodynia. The anti-allodynic effects of FAAH and MGL inhibitors are mediated by CB(1) and CB(<em>2</em>) cannabinoid receptors, whereas TRPV1, but not TRPA1, -dependent mechanisms contribute to the anti-allodynic efficacy of FAAH (but not MGL) inhibitors. Strikingly, endocannabinoid modulators potently suppressed cisplatin-evoked allodynia with a rapid onset and showed efficacy that equaled or exceeded that of major classes of anti-neuropathic pain medications used clinically. Thus, inhibition of endocannabinoid hydrolysis, via FAAH or MGL inhibitors, represents an efficacious pharmacological approach for suppressing chemotherapy-induced neuropathic pain.

Endocannabinoid (eCB) signaling has been identified as a modulator of adaptation to stress, and is integral to basal and stress-induced glucocorticoid regulation. Furthermore, interactions between eCBs and glucocorticoids have been shown to be necessary for the regulation of emotional memories, suggesting that eCB function may relate to the development of post-traumatic stress disorder (PTSD). To examine this, plasma eCBs were measured in a sample (n=46) drawn from a population-based cohort selected for physical proximity to the World Trade Center (WTC) at the time of the 9/11 attacks. Participants received a structured diagnostic interview and were grouped according to whether they met diagnostic criteria for PTSD (no PTSD, n=<em>2</em><em>2</em>; lifetime diagnosis of PTSD=<em>2</em>4). eCB content (<em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and anandamide (AEA)) and cortisol were measured from 8 a.m. plasma samples. Circulating <em>2</em>-AG content was significantly reduced among individuals meeting diagnostic criteria for PTSD. The effect of reduced <em>2</em>-AG content in PTSD remained significant after controlling for the stress of exposure to the WTC collapse, gender, depression and alcohol abuse. There were no significant group differences for AEA or cortisol levels; however, across the whole sample AEA levels positively correlated with circulating cortisol, and AEA levels exhibited a negative relationship with the degree of intrusive symptoms within the PTSD sample. This report shows that PTSD is associated with a reduction in circulating levels of the eCB <em>2</em>-AG. Given the role of <em>2</em>-AG in the regulation of the stress response, these data support the hypothesis that deficient eCB signaling may be a component of the glucocorticoid dysregulation associated with PTSD. The negative association between AEA levels and intrusive symptoms is consistent with animal data indicating that reductions in AEA promote retention of aversive emotional memories. Future work will aim to replicate these findings and extend their relevance to clinical pathophysiology, as well as to neuroendocrine and molecular markers of PTSD.