Endocannabinoids are amides, esters and ethers of long chain polyunsaturated fatty acids, which act as new lipid mediators. Anandamide (N-arachidonoylethanolamine; AEA) and <em>2</em>-<em>arachidonoylglycerol</em> 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.

Obesity is associated with increased serum endocannabinoid (EC) levels and decreased high-density lipoprotein cholesterol (HDLc). Apolipoprotein A-I (apo A-I), the primary protein component of HDL is expressed primarily in the liver and small intestine. To determine whether ECs regulate apo A-I gene expression directly, the effect of the obesity-associated ECs anandamide and <em>2</em>-arachidonylglycerol on apo A-I gene expression was examined in the hepatocyte cell line HepG<em>2</em> and the intestinal cell line Caco-<em>2</em>. Apo A-I protein secretion was suppressed nearly 50% by anandamide and <em>2</em>-<em>arachidonoylglycerol</em> in a dose-dependent manner in both cell lines. Anandamide treatment suppressed both apo A-I mRNA and apo A-I gene promoter activity in both cell lines. Studies using apo A-I promoter deletion constructs indicated that repression of apo A-I promoter activity by anandamide requires a previously identified nuclear receptor binding site designated as site A. Furthermore, anandamide-treatment inhibited protein-DNA complex formation with the site A probe. Exogenous over expression of cannabinoid receptor 1 (CBR1) in HepG<em>2</em> cells suppressed apo A-I promoter activity, while in Caco-<em>2</em> cells, exogenous expression of both CBR1 and CBR<em>2</em> could repress apo A-I promoter activity. The suppressive effect of anandamide on apo A-I promoter activity in Hep G<em>2</em> cells could be inhibited by CBR1 antagonist AM<em>2</em>51 but not by AM630, a selective and potent CBR<em>2</em> inhibitor. These results indicate that ECs directly suppress apo A-I gene expression in both hepatocytes and intestinal cells, contributing to the decrease in serum HDLc in obese individuals.

Serotonin (5-HT) plays pivotal roles in the pathogenesis of postinfectious irritable bowel syndrome (PI-IBS), and luminal 5-HT time-dependently modulates visceral nociception. We found that duodenal biopsies from PI-IBS patients exhibited increased 5-HT and decreased anandamide levels and that decreased anandamide was associated with abdominal pain severity, indicating a link between 5-HT and endocannabinoid signaling pathways in PI-IBS. To understand this, we investigated the role of endocannabinoids in 5-HT modulation of visceral nociception in a rat model. Acute intraduodenally applied 5-HT attenuated the visceromotor response (VMR) to colorectal distention, and this was reversed by the cannabinoid receptor 1 (CB1) antagonist AM<em>2</em>51. Duodenal anandamide (but not <em>2</em>-<em>arachidonoylglycerol</em>) content was greatly increased after luminal 5-HT treatment. This effect was abrogated by the 5-HT 3 receptor (5-HT3R) antagonist granisetron, which was luminally delivered to preferentially target vagal terminals. Chemical denervation of vagal afferents blocked 5-HT-evoked antinociception and anandamide release. Chronic luminal 5-HT exposure for 5 days increased baseline VMR and VMR post-5-HT (days 4 and 5). Duodenal levels of anandamide and N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD, the anandamide-synthesizing enzyme) protein gradually declined from day 1 to 5. The time-dependent effects of 5-HT were abolished by daily granisetron pretreatment. Daily pretreatment with CB1 agonists or anandamide from day 3 attenuated 5-HT-induced hyperalgesia. These data suggest that vagal 5-HT3R-mediated duodenal anandamide release contributes to acute luminal 5-HT-induced antinociception via CB1 signaling, whereas decreased anandamide is associated with hyperalgesia upon chronic 5-HT treatment. Further understanding of peripheral vagal anandamide signaling may provide insights into the mechanisms underlying 5-HT-related IBS.

Delta9-Tetrahydrocannabinol, a major psychoactive constituent of marijuana, interacts with specific receptors, i.e. the cannabinoid receptors, thereby eliciting a variety of pharmacological responses. To date, two types of cannabinoid receptors have been identified: the CB1 receptor, which is abundantly expressed in the nervous system, and the CB<em>2</em> receptor, which is predominantly expressed in the immune system. Previously, we investigated in detail the structure-activity relationship of various cannabinoid receptor ligands and found that <em>2</em>-AG (<em>2</em>-<em>arachidonoylglycerol</em>) is the most efficacious agonist. We have proposed that <em>2</em>-AG is the true natural ligand for both the CB1 and CB<em>2</em> receptors. Despite the potential physiological importance of <em>2</em>-AG, not much information is available concerning its biological activities towards mammalian tissues and cells. In the present study, we examined the effect of <em>2</em>-AG on morphology as well as the actin filament system in differentiated HL-60 cells, which express the CB<em>2</em> receptor. We found that <em>2</em>-AG induces rapid morphological changes such as the extension of pseudopods. We also found that it provokes a rapid actin polymerization in these cells. Actin polymerization induced by <em>2</em>-AG was abolished when cells were treated with SR1445<em>2</em>8, a CB<em>2</em> receptor antagonist, and pertussis toxin, suggesting that the response was mediated by the CB<em>2</em> receptor and G(i/o). A phosphoinositide 3-kinase, Rho family small G-proteins and a tyrosine kinase were also suggested to be involved. Reorganization of the actin filament system is known to be indispensable for a variety of cellular events; it is possible that <em>2</em>-AG plays physiologically essential roles in various inflammatory cells and immune-competent cells by inducing a rapid actin rearrangement.

Protease-activated receptor 1 (PAR1) is a member of the G-protein coupled receptors that are proteolytically activated by serine proteases. Recent studies suggest a definite contribution of PAR1 to brain functions, including learning and memory. However, cellular mechanisms by which PAR1 activation influences neuronal activity are not well understood. Here we show that PAR1 activation drives retrograde endocannabinoid signaling and thereby regulates synaptic transmission. In cultured hippocampal neurons from rat, PAR1 activation by thrombin or PAR1-specific peptide agonists transiently suppressed inhibitory transmission at cannabinoid-sensitive, but not cannabinoid-insensitive, synapses. The PAR1-induced suppression of synaptic transmission was accompanied by an increase in paired-pulse ratio, and was blocked by a cannabinoid CB(1) receptor antagonist. The PAR1-induced suppression was blocked by pharmacological inhibition of postsynaptic diacylglycerol lipase (DGL), a key enzyme for biosynthesis of the major endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), and was absent in knock-out mice lacking the α isoform of DGL. The PAR1-induced IPSC suppression remained intact under the blockade of metabotropic glutamate receptors and was largely resistant to the treatment that blocked Ca(<em>2</em>+) elevation in glial cells following PAR1 activation, which excludes the major contribution of glial PAR1 in IPSC suppression. We conclude that activation of neuronal PAR1 triggers retrograde signaling mediated by <em>2</em>-AG, which activates presynaptic CB(1) receptors and suppresses transmitter release at hippocampal inhibitory synapses.

BACKGROUND

The endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is an endogenous lipid that acts through the activation of G-protein-coupled cannabinoid receptors and plays essential roles in many physiological contexts. In the cardiovascular system <em>2</em>-AG is generated by both activated endothelial cells and platelets, and participates in the regulation of inflammation and thrombosis. Although human platelets actively metabolize endocannabinoids, <em>2</em>-AG also binds to platelet surface and leads to cell activation.

OBJECTIVE

To investigate the biological consequence of <em>2</em>-AG interactions with human platelets and to clarify the role of cannabinoid receptors.

METHODS

Gel-filtered platelets were stimulated with <em>2</em>-AG in the presence or absence of various inhibitors. Platelet aggregation and secretion were measured in a lumiaggregometer. Calcium ion movements were measured in FURA-<em>2</em> loaded platelets. Thromboxane A(<em>2</em>) (TxA(<em>2</em>)) generation was evaluated as Thromboxane B(<em>2</em>) accumulation with a commercial EIA assay.

RESULTS

<em>2</em>-AG induced platelet shape change, aggregation and secretion with a dose-dependent mechanism that required engagement of platelet TxA(<em>2</em>) receptors. <em>2</em>-AG caused also cytosolic calcium increase; however, it was totally dependent on availability of TxA(<em>2</em>). Indeed <em>2</em>-AG was able to induce a robust generation of TxA(<em>2</em>) through the cyclooxygenase pathway. Treatment of platelets with inhibitors of monoacylglycerol lipase and fatty acid amide hydrolase did not affect the activation induced by <em>2</em>-AG. Moreover, neither CB(1) and CB(<em>2</em>) proteins nor CB(1)/CB(<em>2</em>) mRNAs were detected in platelets.

CONCLUSIONS

<em>2</em>-AG can be considered a new physiologic platelet agonist able to induce full platelet activation and aggregation with a non-CB(1)/CB(<em>2</em>) receptor-mediated mechanism.

Endocannabinoids are bioactive lipids, that comprise amides, esters and ethers of long chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine; AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are the best studied endocannabinoids, and act as agonists of cannabinoid receptors. Thus, AEA and <em>2</em>-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 <em>2</em>-AG) or by a monoacylglycerol lipase (for <em>2</em>-AG). Together with AEA, <em>2</em>-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.

Omega-3 (n-3) fatty acids inhibit breast and prostate cancer cell growth. We previously showed that N-acylethanolamine derivatives of n-3 (n-3-NAE) are endocannabinoids, which regulate cancer cell proliferation. These n-3-NAE are synthesised in certain cells/tissues, after supplementing with fatty acids, however, no one has assessed whether and to what extent this occurs in cancer cells. We determined levels of endogenous n-3-NAEs in hormone sensitive and insensitive prostate and breast cancer cells and subsequent effects on other endocannabinoids (anandamide and <em>2</em>-<em>arachidonoylglycerol</em>), before and after supplementing with DHA and EPA fatty acids, using HPLC tandem mass spectrometry. This is the first study reporting that n-3-NAEs are synthesised from their parent n-3 fatty acids in cancer cells, regardless of tumour type, hormone status or the presence of fatty acid amide hydrolase. This could have important implications for the use of n-3 fatty acids as therapeutic agents in breast and prostate cancers expressing cannabinoid receptors.

The membrane properties of isolated frog parathyroid cells were studied using perforated and conventional whole-cell patch-clamp techniques. Frog parathyroid cells displayed transient inward currents in response to depolarizing pulses from a holding potential of -84 mV. We analyzed the biophysical properties of the inward currents. The inward currents disappeared by the replacement of external Na+ with NMDG+ and were reversibly inhibited by 3 micromol l-1 TTX, indicating that the currents occur through the TTX-sensitive voltage-gated Na+ channels. Current density elicited by a voltage step from -84 mV to -<em>2</em>4 mV was -80 pA pF-1 in perforated mode and -55 pA pF-1 in conventional mode. Current density was decreased to -1<em>2</em> pA pF-1 by internal GTPgammaS (0.5 mmol l-1), but not affected by internal GDPbetaS (1 mmol l-1). The voltage of half-maximum (V1/<em>2</em>) activation was -46 mV in both perforated and conventional modes. V1/<em>2</em> of inactivation was -80 mV in perforated mode and -86 mV in conventional mode. Internal GTPgammaS (0.5 mmol l-1) shifted the V1/<em>2</em> for activation to -36 mV and for inactivation to -98 mV. A putative endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em> ether (<em>2</em>-AG ether, 50 micromol l-1) and a cannabinomimetic aminoalkylindole, WIN 55,<em>2</em>1<em>2</em>-<em>2</em> (10 micromol l-1) also greatly reduced the Na+ current and shifted the V1/<em>2</em> for activation and inactivation. The results suggest that the Na+ currents in frog parathyroid cells can be modulated by cannabinoids via a G protein-dependent mechanism.

The discovery of cannabinoid receptors has led to the identification of two natural activators for these receptors, anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, and to the elucidation of their biochemical pathways of formation and inactivation. Although the physiological significance of the endogenous cannabinoid system is still poorly understood, important information is becoming available on the possible functional roles of this system in the basal ganglia, a forebrain region that is involved in the control of sensorimotor and motivational aspects of behavior. These discoveries - which are going to enrich the way in which we look at basal ganglia functions - are summarized in this mini-review. The role of the endocannabinoids as modulators of psychomotor behaviors and the potential therapeutic perspectives deriving from the pharmacological manipulation of the endogenous cannabinoid system are also discussed.

The endocannabinoid system comprises cannabinoid receptors 1 and <em>2</em> (CB1 and CB<em>2</em>), their endogenous ligands, anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em>, and metabolic enzymes of these ligands. The endocannabinoid system has recently been implicated in the regulation of various pathophysiological processes of the skin that include immune competence and/or tolerance of keratinocytes, the disruption of which might promote the development of skin diseases. Recent evidence showed that CB1 in keratinocytes limits the secretion of proinflammatory chemokines, suggesting that this receptor might also regulate T cell dependent inflammatory diseases of the skin. In this article, we sought to investigate the cytokine profile of IFN-γ-activated keratinocytes, and found that CB1 activation by AEA suppressed production and release of signature TH1- and TH17-polarizing cytokines, IL-1<em>2</em> and IL-<em>2</em>3, respectively. We also set up cocultures between a conditioned medium of treated keratinocytes and naive T cells to disclose the molecular details that regulate the activation of highly proinflammatory TH1 and TH17 cells. AEA-treated keratinocytes showed reduced an induction of IFN-γ-producing TH1 and IL-17-producing TH17 cells, and these effects were reverted by pharmacological inhibition of CB1 Further analyses identified mammalian target of rapamycin as a proinflammatory signaling pathway regulated by CB1, able to promote either IL-1<em>2</em> and IL-<em>2</em>3 release from keratinocytes or TH1 and TH17 polarization. Taken together, these findings demonstrate that AEA suppresses highly pathogenic T cell subsets through CB1-mediated mammalian target of rapamycin inhibition in human keratinocytes. Thus, it can be speculated that the latter pathway might be beneficial to the physiological function of the skin, and can be targeted toward inflammation-related skin diseases.

Liver fatty acid-binding protein (FABP1, L-FABP) has high affinity for and enhances uptake of arachidonic acid (ARA, C<em>2</em>0:4, n-6) which, when esterified to phospholipids, is the requisite precursor for synthesis of endocannabinoids (EC) such as arachidonoylethanolamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). The brain derives most of its ARA from plasma, taking up ARA and transporting it intracellularly via cytosolic fatty acid-binding proteins (FABPs 3,5, and 7) localized within the brain. In contrast, the much more prevalent cytosolic FABP1 is not detectable in the brain but is instead highly expressed in the liver. Therefore, the possibility that FABP1 outside the central nervous system may regulate brain AEA and <em>2</em>-AG was examined in wild-type (WT) and FABP1 null (LKO) male mice. LKO increased brain levels of AA-containing EC (AEA, <em>2</em>-AG), correlating with increased free and total ARA in brain and serum. LKO also increased brain levels of non-ARA that contain potentiating endocannabinoids (EC*) such as oleoyl ethanolamide (OEA), PEA, <em>2</em>-OG, and <em>2</em>-PG. Concomitantly, LKO decreased serum total ARA-containing EC, but not non-ARA endocannabinoids. LKO did not elicit these changes in the brain EC and EC* as a result of compensatory up-regulation of brain protein levels of enzymes in EC synthesis (NAPEPLD, DAGLα) or cytosolic EC chaperone proteins (FABPs 3, 5, 7, SCP-<em>2</em>, HSP70), or cannabinoid receptors (CB1, TRVP1). These data show for the first time that the non-CNS fatty acid-binding protein FABP1 markedly affected brain levels of both ARA-containing endocannabinoids (AEA, <em>2</em>-AG) as well as their non-ARA potentiating endocannabinoids. Fatty acid-binding protein-1 (FABP-1) is not detectable in brain but instead is highly expressed in liver. The possibility that FABP1 outside the central nervous system may regulate brain endocannabinoids arachidonoylethanolamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) was examined in wild-type (WT) and FABP-1 null (LKO) male mice. LKO increased brain levels of arachidonic acid-containing endocannabinoids (AEA, <em>2</em>-AG), correlating with increased free and total arachidonic acid in brain and serum. Read the Editorial Highlight for this article on page 371.

The umbrella pine Sciadopitys verticillata seeds were found to contain a substantial amount (16.7 nmol/g) of sciadonic acid (all-cis-5,11,14-eicosatrienoic acid)-containing <em>2</em>-monoacylglycerol, i.e., <em>2</em>-sciadonoylglycerol (<em>2</em>-eicosa-5',11',14'-trienoylglycerol). Because the structure of <em>2</em>-sciadonoylglycerol closely resembles that of <em>2</em>-<em>arachidonoylglycerol</em>, the endogenous natural ligand for the cannabinoid receptor, we examined whether or not <em>2</em>-sciadonoylglycerol exhibits cannabimimetic activity using NG108-15 neuroblastomaxglioma hybrid cells which express the cannabinoid CB1 receptor. We found that <em>2</em>-sciadonoylglycerol induces rapid transient elevation of intracellular free Ca<em>2</em>+ concentration in NG108-15 cells through a cannabinoid CBI receptor-dependent mechanism similar to the case of <em>2</em>-<em>arachidonoylglycerol</em>, yet the activity of <em>2</em>-sciadonoylglycerol was apparently lower than that of <em>2</em>-<em>arachidonoylglycerol</em>. The activity of <em>2</em>-sciadonoylglycerol was detectable from 3-10 nM, reaching a maximum at around 10 microM. To our knowledge, this is the first report showing the occurrence of a cannabimimetic monoacylglycerol in higher plants.

Evidence suggests the involvement of the cannabinoid system in the pathogenesis of multiple sclerosis (MS). We studied cannabinoid receptor (CB)1 and CB<em>2</em> receptor gene expression in B, natural killer (NK) and T cells from MS patients before and after 1 year of interferon beta therapy, and compared these levels to those of healthy controls. We also measured the production of the endocannabinoids anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and the gene expression of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) in these cells. Prior to interferon therapy, MS patients showed significantly elevated CB<em>2</em> expression in B cells, but not in T or NK cells. These levels decreased gradually within 6 months to 1 year of interferon treatment. CB1 expression was elevated in all cell subsets, but only reached statistical significance in T cells; all levels decreased progressively over time. Before treatment, AEA but not <em>2</em>-AG levels were significantly elevated in the three cell populations; after 1 year of treatment, all values decreased to control levels. The expression of FAAH was unchanged. The different expression of cannabinoid receptor genes and the increased level of AEA in lymphocytes point to a possible role of the cannabinoid system in MS immune response and its modulation by interferon.

Endocannabinoids are a new class of lipids, which include amides, esters and ethers of long chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine; AEA) and <em>2</em>-<em>arachidonoylglycerol</em> 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.

One of the most abundant G-protein coupled receptors (GPCRs) in brain, the cannabinoid 1 receptor (CB1R), is a tractable therapeutic target for treating diverse psychobehavioral and somatic disorders. Adverse on-target effects associated with small-molecule CB1R orthosteric agonists and inverse agonists/antagonists have plagued their translational potential. Allosteric CB1R modulators offer a potentially safer modality through which CB1R signaling may be directed for therapeutic benefit. Rational design of candidate, druglike CB1R allosteric modulators requires greater understanding of the architecture of the CB1R allosteric endodomain(s) and the capacity of CB1R allosteric ligands to tune the receptor's information output. We have recently reported the synthesis of a focused library of rationally designed, covalent analogues of Org<em>2</em>7569 and PSNCBAM-1, two prototypic CB1R negative allosteric modulators (NAMs). Among the novel, pharmacologically active CB1R NAMs reported, the isothiocyanate GAT100 emerged as the lead by virtue of its exceptional potency in the [(35)S]GTPγS and β-arrestin signaling assays and its ability to label CB1R as a covalent allosteric probe with significantly reduced inverse agonism in the [(35)S]GTPγS assay as compared to Org<em>2</em>7569. We report here a comprehensive functional profiling of GAT100 across an array of important downstream cell-signaling pathways and analysis of its potential orthosteric probe-dependence and signaling bias. The results demonstrate that GAT100 is a NAM of the orthosteric CB1R agonist CP55,940 and the endocannabinoids <em>2</em>-<em>arachidonoylglycerol</em> and anandamide for β-arrestin1 recruitment, PLCβ3 and ERK1/<em>2</em> phosphorylation, cAMP accumulation, and CB1R internalization in HEK<em>2</em>93A cells overexpressing CB1R and in Neuro<em>2</em>a and STHdh(Q7/Q7) cells endogenously expressing CB1R. Distinctively, GAT100 was a more potent and efficacious CB1R NAM than Org<em>2</em>7569 and PSNCBAM-1 in all signaling assays and did not exhibit the inverse agonism associated with Org<em>2</em>7569 and PSNCBAM-1. Computational docking studies implicate C7.38(38<em>2</em>) as a key feature of GAT100 ligand-binding motif. These data help inform the engineering of newer-generation, druggable CB1R allosteric modulators and demonstrate the utility of GAT100 as a covalent probe for mapping structure-function correlates characteristic of the druggable CB1R allosteric space.

The ECs (endocannabinoids) AEA (anandamide) and <em>2</em>-AG (<em>2</em>-<em>arachidonoylglycerol</em>) and their lipid congeners OEA (N-oleoylethanolamide) and PEA (N-palmitoylethanolamide) are multifunctional lipophilic signalling molecules. The ECs, OEA and PEA have multiple physiological roles including involvement in learning and memory, neuroinflammation, oxidative stress, neuroprotection and neurogenesis. They have also been implicated in the pathology of, or perhaps protective responses to, neurodegenerative diseases. This is particularly the case with Alzheimer's disease, the most common age-related dementia associated with impairments in learning and memory accompanied by neuroinflammation, oxidative stress and neurodegeneration. The present mini-review examines the evidence supporting the roles that ECs appear to play in Alzheimer's disease and the potential for beneficial therapeutic manipulation of the EC signalling system.

Accumulated evidence points to a key role for endocannabinoids in cell migration, and here we sought to characterize the role of these substances in early events that modulate communication between endothelial cells and leukocytes. We found that <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) was able to initiate and complete the leukocyte adhesion cascade, by modulating the expression of selectins. A short exposure of primary human umbilical vein endothelial cells (HUVECs) to <em>2</em>-AG was sufficient to prime them towards an activated state: within 1h of treatment, endothelial cells showed time-dependent plasma membrane expression of P- and E-selectins, which both trigger the initial steps (i.e., capture and rolling) of leukocyte adhesion. The effect of <em>2</em>-AG was mediated by CB1 and CB<em>2</em> receptors and was long lasting, because endothelial cells incubated with <em>2</em>-AG for 1h released the pro-inflammatory cytokine tumour necrosis factor-α (TNF-α) for up to <em>2</em>4h. Consistently, TNF-α-containing medium was able to promote leukocyte recruitment: human Jurkat T cells grown in conditioned medium derived from <em>2</em>-AG-treated HUVECs showed enhanced L-selectin and P-selectin glycoprotein ligand-1 (PSGL1) expression, as well as increased efficiency of adhesion and trans-migration. In conclusion, our in vitro data indicate that <em>2</em>-AG, by acting on endothelial cells, might indirectly promote leukocyte recruitment, thus representing a potential therapeutic target for treatment of diseases where impaired endothelium/leukocyte interactions take place.

Stimulation of cannabinoid CB(1) receptors in nucleus accumbens shell has been shown to stimulate feeding and enhance positive 'liking' reactions to intraoral sucrose. This study examined the behavioural effects of noladin ether and <em>2</em>-<em>arachidonoylglycerol</em> following infusion into accumbens shell, on chow intake and food preference in high-carbohydrate and high-fat preferring rats. Noladin ether, potently and dose-dependently stimulated chow intake as compared with <em>2</em>-<em>arachidonoylglycerol</em> in free-feeding rats. In the diet preference paradigm, in which rats were given free access to both, high-carbohydrate (HC) and high-fat (HF) diets simultaneously, an intra-accumbens administration of noladin ether as well as <em>2</em>-<em>arachidonoylglycerol</em>, preferentially enhanced fat consumption over carbohydrate in both HF- and HC-preferring rats. These effects were significantly attenuated by the CB(1) receptor antagonist, AM <em>2</em>51. These results suggesting that, the endocannabinoids through CB(1) receptors, affects appetite for specific dietary components. Both these agents exert a specific action on eating motivation and possibly promoting eating by enhancing the incentive value of food. Altogether these findings reinforce the idea that the endogenous cannabinoid system in the accumbens shell may be important to augment reward-driven feeding via modulation of CB(1) receptor signalling pathways.

The functional existence of the emerging endocannabinoid system (ECS), one of the new neuroendocrine players in cutaneous biology, is recently described in the human skin. In this study, using human eccrine sweat gland-derived immortalized NCL-SG3 model cells and a wide array of cellular and molecular assays, we investigated the effects of prototypic endocannabinoids (anandamide, <em>2</em>-<em>arachidonoylglycerol</em>) on cellular functions. We show here that both endocannabinoids dose-dependently suppressed proliferation, induced apoptosis, altered expressions of various cytoskeleton proteins (e.g., cytokeratins), and upregulated lipid synthesis. Interestingly, as revealed by specific agonists and antagonists as well as by RNA interference, neither the metabotropic cannabinoid receptors (CB) nor the "ionotropic" CB transient receptor potential ion channels, expressed by these cells, mediated the cellular actions of the endocannabinoids. However, the endocannabinoids selectively activated the mitogen-activated protein kinase signaling pathway. Finally, other elements of the ECS (i.e., enzymes involved in the synthesis and degradation of endocannabinoids) were also identified on NCL-SG3 cells. These results collectively suggest that cannabinoids exert a profound regulatory role in the biology of the appendage. Therefore, from a therapeutic point of view, upregulation of endocannabinoid levels might help to manage certain sweat gland-derived disorders (e.g., tumors) characterized by unwanted growth.

Cannabinoids (CB) can act as retrograde synaptic mediators of depolarization-induced suppression of inhibition or excitation in hippocampus. This mechanism may underlie the impairment of some cognitive processes produced by these compounds, including short-term memory formation in the hippocampus. In this study, we investigated several compounds known to interact with CB receptors, evaluating their effects on K(+)-evoked release of [3H]D-aspartate ([3H]D-ASP) and [3H]GABA from superfused synaptosomes isolated from the rat hippocampus. [3H]D-ASP and [3H]GABA release were inhibited to different degrees by the synthetic cannabinoids WIN 55,<em>2</em>1<em>2</em>-<em>2</em>; CP 55,940, and arachidonyl-<em>2</em>'-chloroethylamide/N-(<em>2</em>-chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (ACEA), as well as by the endocannabinoids, anandamide (AEA), and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Both types of release were also inhibited by capsaicin. The inhibition produced by each of the cannabinoid compounds and capsaicin was unaffected by capsazepine or by the CB1-receptor antagonists AM-<em>2</em>51 and SR141716A. The mechanism underlying AEA- and synthetic CB-induced inhibition of the release of [3H]GABA and [3H]D-ASP from rat hippocampal synaptosomes might not involve activation of presynaptic CB1 receptors.

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 <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and N-arachidonoylethanolamine or anandamide (AEA), and several eCB-related compounds, <em>2</em>-acylglycerols and N-acylethanolamines. <em>2</em>-AG and other <em>2</em>-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 <em>2</em>-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 <em>2</em>-AG concentrations in obesity are linked to a lower olfactory capacity. In agreement with previous studies we show that eCBs AEA and <em>2</em>-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.

Studies in male rodents have shown that stress-induced increases in circulating corticosterone are increased by both CB1 receptor (CB1R) antagonist treatment and genetic deletion. The purposes of the current study were to determine whether female mice respond in the same manner as males, and whether indirect CB1R agonists accelerate the return of corticosterone to baseline. In agreement with earlier studies, CB1R null and rimonabant-treated male mice had significantly increased circulating corticosterone 30 min following the end of a restraint episode compared to wild type and vehicle-treated, respectively. Females treated with rimonabant had significantly higher circulating corticosterone compared to vehicle. However, corticosterone concentrations were not different between CB1R null and wild type females at 30 min recovery, although CB1R null mice had higher corticosterone concentrations at 90 min of recovery. Female CB1R null mice exhibited greater serum binding capacity for corticosterone than wild type. The monoacylglycerol lipase inhibitor, JZL184, attenuated corticosterone concentrations at restraint offset in male, and at 30 min recovery in female mice compared to vehicle. Male mice treated with JZL184 exhibited greater concentrations of circulating corticosterone at 1<em>2</em>0 min recovery, even in the absence of restraint. JZL184 had no effect on corticosterone concentrations in CB1R null mice. The fatty acid amide hydrolase inhibitor, URB597, did not affect corticosterone responses to restraint in male or female, wild type or CB1R null mice. These data suggest that <em>2</em>-<em>arachidonoylglycerol</em> is the primary endocannabinoid involved in CB1R regulation of the recovery of the HPA axis from restraint stress. These data support a role for endocannabinoid-CB1R signaling in the regulation of the corticosterone response to restraint stress and suggest that female mice with life-long loss of the CB1R undergo compensatory changes that minimize the impact of loss of endocannabinoid signaling on circulating corticosterone.

Anandamide (AEA) is an endocannabinoid (EC) that modulates multiple functions in the CNS and that is released in areas of injury, exerting putative neuroprotective actions. In the present study, we have used intravital microscopy to analyze the role of the EC system in the glial response against an acute insult. Our data show that AEA modulates astroglial function in vivo by increasing connexin-43 hemichannel (HC) activity. Furthermore, the genetic inactivation of the AEA-degrading enzyme, fatty acid amide hydrolase (FAAH), also increased HC activity and enhanced the microglial response against an acute injury to the brain parenchyma, effects that were mediated by cannabinoid CB1 receptors. The contribution of ATP released through an astrocytic HC was critical for the microglial response, as this was prevented by the use of the HC blocker flufenamic acid and by apyrase. As could be expected, brain concentrations of AEA, palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) were elevated in FAAH-null mice, while <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) concentrations remained unaltered. In summary, these findings demonstrate that AEA modifies glial functions by promoting an enhanced pro-inflammatory glial response in the brain.