Increasing evidence indicates that the gut peptide ghrelin facilitates learning behavior and memory tasks. The present study demonstrates a cellular signaling mechanism of ghrelin in the hippocampus. Ghrelin stimulated CREB (cAMP response-element binding protein) through the activation of cAMP, protein kinase A (PKA), and PKA-dependent phosphorylation of NR1 subunit of the NMDA receptor. Ghrelin increased phalloidin-binding to F-actin suggesting CREB-induced gene expression might include reorganization of cytoskeletal proteins. The effect was blocked by the antagonist of the ghrelin receptor in spite of the receptor's primary coupling to Gq proteins. We also discovered inhibitory effect of endocannabinoids on ghrelin-induced NR1 phosphorylation and CREB activity. <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) exerted its inhibitory effect in the Type 1 cannabinoid receptor (CB1R)-dependent manner, while anandamide's inhibitory effect persisted in the presence of antagonists of CB1R and the vanilloid receptor, suggesting that anandamide might directly inhibit NMDA receptor/channels. Our findings may explain how ghrelin and endocannabinoids regulate hippocampal appetitive learning and plasticity.

Intracellular signaling systems of G protein-coupled receptors are well established, but their role in paracrine regulation of adjacent cells is generally considered as a tissue-specific mechanism. We have shown previously that AT(1) receptor (AT(1)R) stimulation leads to diacylglycerol lipase-mediated transactivation of co-expressed CB(1)Rs in Chinese hamster ovary cells. In the present study we detected a paracrine effect of the endocannabinoid release from Chinese hamster ovary, COS7, and HEK<em>2</em>93 cells during the stimulation of AT(1) angiotensin receptors by determining CB(1) cannabinoid receptor activity with bioluminescence resonance energy transfer-based sensors of G protein activation expressed in separate cells. The angiotensin II-induced, paracrine activation of CB(1) receptors was visualized by detecting translocation of green fluorescent protein-tagged beta-arrestin<em>2</em>. Mass spectrometry analyses have demonstrated angiotensin II-induced stimulation of <em>2</em>-<em>arachidonoylglycerol</em> production, whereas no increase of anandamide levels was observed. Stimulation of G(q/11)-coupled M(1), M(3), M(5) muscarinic, V(1) vasopressin, alpha(1a) adrenergic, B(<em>2</em>) bradykinin receptors, but not G(i/o)-coupled M(<em>2</em>) and M(4) muscarinic receptors, also led to paracrine transactivation of CB(1) receptors. These data suggest that, in addition to their retrograde neurotransmitter role, endocannabinoids have much broader paracrine mediator functions during activation of G(q/11)-coupled receptors.

The hypothalamus-pituitary-adrenal-axis is strongly controlled by the endocannabinoid system. The specific impact of enhanced <em>2</em>-<em>arachidonoylglycerol</em> signaling on corticosterone plasma levels, however, was not investigated so far. Here we studied the effects of the recently developed monoacylglycerol lipase inhibitor JZL184 on basal and stress-induced corticosterone levels in male CD1 mice, and found that this compound dramatically increased basal levels without affecting stress responses. Since acute changes in corticosterone levels can affect behavior, JZL184 was administered concurrently with the corticosterone synthesis inhibitor metyrapone, to investigate whether the previously shown behavioral effects of JZL184 are dependent on corticosterone. We found that in the elevated plus-maze, the effects of JZL184 on "classical" anxiety-related measures were abolished by corticosterone synthesis blockade. By contrast, effects on the "ethological" measures of anxiety (i.e. risk assessment) were not affected by metyrapone. In the open-field, the locomotion-enhancing effects of the compound were not changed either. These findings show that monoacylglycerol lipase inhibition dramatically increases basal levels of corticosterone. This endocrine effect partly affects the anxiolytic, but not the locomotion-enhancing effects of monoacylglycerol lipase blockade.

Monoacylglycerol lipase (MAGL) terminates the signaling function of the endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). During <em>2</em>-AG hydrolysis, MAGL liberates arachidonic acid, feeding the principal substrate for the neuroinflammatory prostaglandins. In cancer cells, MAGL redirects lipid stores toward protumorigenic signaling lipids. Thus MAGL inhibitors may have great therapeutic potential. Although potent and increasingly selective MAGL inhibitors have been described, their number is still limited. Here, we have characterized piperazine and piperidine triazole ureas that combine the high potency attributable to the triazole leaving group together with the bulky aromatic benzodioxolyl moiety required for selectivity, culminating in compound JJKK-048 that potently (IC50 < 0.4 nM) inhibited human and rodent MAGL. JJKK-048 displayed low cross-reactivity with other endocannabinoid targets. Activity-based protein profiling of mouse brain and human melanoma cell proteomes suggested high specificity also among the metabolic serine hydrolases.

The endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, and the anandamide-congener, palmitoylethanolamide, are all substrates for the enzyme fatty acid amide hydrolase, and are endowed with anti-inflammatory actions exerted via cannabinoid receptors or, in the case of palmitoylethanolamide, also via other targets. We investigated the role of the endocannabinoid system during granuloma formation, a model of chronic inflammation sustained by neoangiogenesis, in rats. Granuloma was induced by subcutaneous lambda-carrageenin-soaked sponge implants on the back of male Wistar rats. After 96h, granulomas were detached and tissue formation was evaluated as wet weight; the endocannabinoid system was evaluated by the measurement of endocannabinoid levels, by LC-MS, and of cannabinoid receptor expression, by western blot analysis. Moreover, angiogenesis was evaluated by the measurement of both hemoglobin content and CD31 protein expression. Arachidonoylserotonin (AA-5-HT, 1<em>2</em>.5-50mug/ml), an inhibitor of FAAH, and palmitoylethanolamide (PEA, <em>2</em>00-800mug/ml) were given locally only once at the time of implantation. Granuloma formation was accompanied by a significant decrease in endocannabinoid and palmitoylethanolamide levels paralleled by increased levels of the fatty acid amide hydrolase, responsible for their breakdown. Moreover, an increase of cannabinoid receptor expression was also observed. Pharmacological elevation of endocannabinoids and palmitoylethanolamide, obtained separately by arachidonoylserotonin and exogenous palmitoylethanolamide treatment, dose-dependently reduced inflammatory hallmarks including tumor necrosis factor-alpha as well as granuloma-dependent angiogenesis. The effect of arachidonoylserotonin was accompanied by near-normalization of <em>2</em>-<em>arachidonoylglycerol</em> and palmitoylethanolamide levels in the tissue. These findings suggest that chronic inflammation might develop also because of endocannabinoid and palmitoylethanolamide tissue concentration impairment, the correction of which might be exploited to develop new anti-inflammatory drugs.

The two putative endogenous ligands of cannabinoid receptors, anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, are synthesized by and released from neurons in a Ca<em>2</em>+-dependent fashion, and re-uptaken and catabolized by both neurons and astrocytes. These biochemical features of the endocannabinoids, as well as some of their pharmacological effects in both central and peripheral nervous systems, suggest a role as neuromodulators for these metabolites. This neuromodulatory role is supported by the brain regional distribution of anandamide, its biosynthetic precursor and its major inactivating enzyme, and by the existence of possible regulatory mechanisms for the biosynthesis and inactivation of endocannabinoids, which are reviewed in this article.

Agonists at cannabinoid receptors, such as the phytocannabinoid Δ(9)-tetrahydrocannabinol, exert a remarkable array of therapeutic effects but are also associated with undesirable psychoactive side effects. Conversely, targeting enzymes that hydrolyze endocannabinoids (eCBs) allows for more precise fine-tuning of cannabinoid receptor signaling, thus providing therapeutic relief with reduced side effects. Here, we report the development and characterization of an inhibitor of eCB hydrolysis, UCM710, which augments both N-arachidonoylethanolamine and <em>2</em>-<em>arachidonoylglycerol</em> levels in neurons. This compound displays a unique pharmacological profile in that it inhibits fatty acid amide hydrolase and α/β-hydrolase domain 6 but not monoacylglycerol lipase. Thus, UCM710 represents a novel tool to delineate the therapeutic potential of compounds that manipulate a subset of enzymes that control eCB signaling.

BACKGROUND

The contribution of two major endocannabinoids, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and anandamide (AEA), in the regulation of fear expression is still unknown.

OBJECTIVE

We analyzed the role of different players of the endocannabinoid system on the expression of a strong auditory-cued fear memory in male mice by pharmacological means.

RESULTS

The cannabinoid receptor type 1 (CB1) antagonist SR141716 (3 mg/kg) caused an increase in conditioned freezing upon repeated tone presentation on three consecutive days. The cannabinoid receptor type <em>2</em> (CB<em>2</em>) antagonist AM630 (3 mg/kg), in contrast, had opposite effects during the first tone presentation, with no effects of the transient receptor potential vanilloid receptor type 1 (TRPV1) antagonist SB366791 (1 and 3 mg/kg). Administration of the CB<em>2</em> agonist JWH133 (3 mg/kg) failed to affect the acute freezing response, whereas the CB1 agonist CP55,940 (50 μg/kg) augmented it. The endocannabinoid uptake inhibitor AM404 (3 mg/kg), but not VDM11 (3 mg/kg), reduced the acute freezing response. Its co-administration with SR141716 or SB366791 confirmed an involvement of CB1 and TRPV1. AEA degradation inhibition by URB597 (1 mg/kg) decreased, while <em>2</em>-AG degradation inhibition by JZL184 (4 and 8 mg/kg) increased freezing response. As revealed in conditional CB1-deficient mutants, CB1 on cortical glutamatergic neurons alleviates whereas CB1 on GABAergic neurons slightly enhances fear expression. Moreover, <em>2</em>-AG fear-promoting effects depended on CB1 signaling in GABAergic neurons, while an involvement of glutamatergic neurons remained inconclusive due to the high freezing shown by vehicle-treated Glu-CB1-KO.

CONCLUSIONS

Our findings suggest that increased AEA levels mediate acute fear relief, whereas increased <em>2</em>-AG levels promote the expression of conditioned fear primarily via CB1 on GABAergic neurons.

OBJECTIVE

To measure the circulating levels of endocannabinoids and related molecules at fasting, after acute hyperinsulinemia and after weight loss in insulin sensitive vs. insulin resistant obese postmenopausal women.

METHODS

The sample consisted of 30 obese postmenopausal women (age: 58.9 ± 5.<em>2</em> yrs; BMI: 3<em>2</em>.9 ± 3.6 kg/m(<em>2</em>) ). Subjects underwent a 3-hour hyperinsulinaemic-euglycaemic clamp (HEC) (glucose disposal rate (M-value): 10.7 ± 3.3 mg min(-1) kg(-1) FFM) and 6-month weight loss intervention. Participants were classified as insulin sensitive obese (ISO) or insulin resistant obese (IRO) based on a predefined cutoff. Plasma levels of the endocannabinoids, anandamide (AEA), <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), and of the AEA-related compounds, palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), were measured by liquid chromatography-mass spectrometry.

RESULTS

IRO presented higher levels of <em>2</em>-AG (P < 0.05) independently of the HEC and weight loss, whereas the HEC had an independent inhibitory effect on AEA, PEA, and OEA levels (P < 0.05) in both groups. Furthermore, there was an independent stimulatory effect of weight loss only on PEA levels in both groups (P < 0.05).

CONCLUSIONS

This study is the first to show that higher circulating levels of the endocannabinoid <em>2</em>-AG are found in IRO compared to ISO postmenopausal women, and that weight loss is associated with an increase in PEA, a PPAR-α ligand.

In the hypothalamic arcuate nucleus (ARC), proopiomelanocortin (POMC) neurons and the POMC-derived peptide α-melanocyte-stimulating hormone (α-MSH) promote satiety. POMC neurons receive orexin-A (OX-A)-expressing inputs and express both OX-A receptor type 1 (OX-1R) and cannabinoid receptor type 1 (CB1R) on the plasma membrane. OX-A is crucial for the control of wakefulness and energy homeostasis and promotes, in OX-1R-expressing cells, the biosynthesis of the endogenous counterpart of marijuana's psychotropic and appetite-inducing component Δ(9)-tetrahydrocannabinol, i.e., the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), which acts at CB1R. We report that OX-A/OX-1R signaling at POMC neurons promotes <em>2</em>-AG biosynthesis, hyperphagia, and weight gain by blunting α-MSH production via CB1R-induced and extracellular-signal-regulated kinase 1/<em>2</em> activation- and STAT3 inhibition-mediated suppression of Pomc gene transcription. Because the systemic pharmacological blockade of OX-1R by SB334867 caused anorectic effects by reducing food intake and body weight, our results unravel a previously unsuspected role for OX-A in endocannabinoid-mediated promotion of appetite by combining OX-induced alertness with food seeking. Notably, increased OX-A trafficking was found in the fibers projecting to the ARC of obese mice (ob/ob and high-fat diet fed) concurrently with elevation of OX-A release in the cerebrospinal fluid and blood of mice. Furthermore, a negative correlation between OX-A and α-MSH serum levels was found in obese mice as well as in human obese subjects (body mass index>> 40), in combination with elevation of alanine aminotransferase and γ-glutamyl transferase, two markers of fatty liver disease. These alterations were counteracted by antagonism of OX-1R, thus providing the basis for a therapeutic treatment of these diseases.

Recently, we have shown that treatment of rat C6 glioma cells with the raft disruptor methyl-beta-cyclodextrin (MCD) doubles the binding of anandamide (AEA) to type-1 cannabinoid receptors (CB1R), followed by CB1R-dependent signaling via adenylate cyclase and p4<em>2</em>/p44 MAPK activity. In the present study, we investigated whether type-<em>2</em> cannabinoid receptors (CB<em>2</em>R), widely expressed in immune cells, also are modulated by MCD. We show that treatment of human DAUDI leukemia cells with MCD does not affect AEA binding to CB<em>2</em>R, and that receptor activation triggers similar [35S]guanosine-5'-O-(3-thiotriphosphate) binding in MCD-treated and control cells, similar adenylate cyclase and MAPK activity, and similar MAPK-dependent protection against apoptosis. The other AEA-binding receptor transient receptor potential channel vanilloid receptor subunit 1, the AEA synthetase N-acyl-phosphatidylethanolamine-phospholipase D, and the AEA hydrolase fatty acid amide hydrolase were not affected by MCD, whereas the AEA membrane transporter was inhibited (approximately 55%) compared with controls. Furthermore, neither diacylglycerol lipase nor monoacylglycerol lipase, which respectively synthesize and degrade <em>2</em>-<em>arachidonoylglycerol</em>, were affected by MCD in DAUDI or C6 cells, whereas the transport of <em>2</em>-<em>arachidonoylglycerol</em> was reduced to approximately 50%. Instead, membrane cholesterol enrichment almost doubled the uptake of AEA and <em>2</em>-<em>arachidonoylglycerol</em> in both cell types. Finally, transfection experiments with human U937 immune cells, and the use of primary cells expressing CB1R or CB<em>2</em>R, ruled out that the cellular environment could account per se for the different modulation of CB receptor subtypes by MCD. In conclusion, the present data demonstrate that lipid rafts control CB1R, but not CB<em>2</em>R, and endocannabinoid transport in immune and neuronal cells.

Endocannabinoids are lipid mediators thought to modulate central and peripheral neural functions. We report here gas chromatography-electron impact mass spectrometry analysis of human brain, showing that lipid extracts contain anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), the most active endocannabinoids known to date. Human brain also contained the endocannabinoid-like compounds N-oleoylethanolamine, N-palmitoylethanolamine and N-stearoylethanolamine. Anandamide and <em>2</em>-AG (0.16 +/- 0.05 and 0.10 +/- 0.05 nmol/mg protein, respectively) represented 7.7% and 4.8% of total endocannabinoid-like compounds, respectively. N-Palmitoyethanolamine was the most abundant (50%), followed by N-oleoyl (<em>2</em>3.6%) and N-stearoyl (13.9%) ethanolamines. A similar composition in endocannabinoid-like compounds was found in human neuroblastoma CHP100 and lymphoma U937 cells, and also in rat brain. Remarkably, human meningioma specimens showed an approximately six-fold smaller content of all N-acylethanolamines, but not of <em>2</em>-AG, and a similar decrease was observed in a human glioblastoma. These ex vivo results fully support the purported roles of endocannabinoids in the nervous system.

BACKGROUND

<em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG), an endogenous ligand for the cannabinoid receptors (CB1 and CB<em>2</em>), has been shown to exhibit a variety of cannabimimetic activities in vitro and in vivo. Recently, we found that human eosinophilic leukemia EoL-1 cells and human peripheral blood eosinophils express the CB<em>2</em> receptor. We also found that <em>2</em>-AG induces the migration of these cells in a CB<em>2</em> receptor-dependent manner. In this study, we investigated whether the <em>2</em>-AG-induced migration of human eosinophils is due to chemotaxis or chemokinesis. We also compared the ability of <em>2</em>-AG to induce the migration of eosinophils with those of other eosinophil chemoattractants.

METHODS

Eosinophils were separated from the peripheral blood of healthy donors. The migration of eosinophils to various stimulants was examined using Transwell inserts. In view of the fact that <em>2</em>-AG is rapidly metabolized by cells, we employed <em>2</em>-AG ether, an ether-linked nonhydrolyzable analog of <em>2</em>-AG, instead of <em>2</em>-AG to determine whether the <em>2</em>-AG-induced migration is due to chemotaxis or chemokinesis.

RESULTS

<em>2</em>-AG ether induced the migration of human eosinophils, like <em>2</em>-AG. The <em>2</em>-AG ether-induced migration was reduced by the coincubation of eosinophils with <em>2</em>-AG ether in the upper compartment of the Transwell inserts, indicating that the migration is attributable to chemotaxis. The concentration of <em>2</em>-AG required to induce the eosinophil migration appears to be pathophysiologically relevant, although the order of the pharmacologically effective concentration of <em>2</em>-AG was approximately ten times lower than those of platelet-activating factor, RANTES and eotaxin.

CONCLUSIONS

These results strongly suggest that <em>2</em>-AG is involved in the infiltration of eosinophils during allergic inflammation.

Cb<em>2</em>, the gene encoding the peripheral cannabinoid receptor, is located in a common virus integration site and is overex-pressed in retrovirally induced murine myeloid leukemias. Here we show that this G protein-coupled receptor (GPCR) is also aberrantly expressed in a high percentage of human acute myeloid leukemias. We investigated the mechanism of transformation by Cb<em>2</em> and demonstrate that aberrant expression of this receptor on hematopoietic precursor cells results in distinct effects depending on the ligand used. Cb<em>2</em>-expressing myeloid precursors migrate upon stimulation by the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> and are blocked in neutrophilic differentiation upon exposure to another ligand, CP55940. Both effects depend on the activation of G(alphai) proteins and require the mitogen-induced extracellular kinase/extracellular signal-regulated kinase (MEK/ERK) pathway. Down-regulation of cyclic adenosine monophosphate (cAMP) levels upon G(alphai) activation is important for migration induction but is irrelevant for the maturation arrest. Moreover, the highly conserved G protein-interacting DRY motif, present in the second intracellular loop of GPCRs, is critical for migration but unimportant for the differentiation block. This suggests that the Cb<em>2</em>-mediated differentiation block requires interaction of G(alphai) proteins with other currently unknown motifs. This indicates a unique mechanism by which a transforming GPCR, in a ligand-dependent manner, causes <em>2</em> distinct oncogenic effects: altered migration and block of neutrophilic development.

The endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-Delta(4)Ach-Gro) activates human platelets in platelet-rich plasma at physiological concentrations. The activation was inhibited by selective antagonists of CB(1) and CB(<em>2</em>) cannabinoid receptors, but not by acetylsalicylic acid. Human platelets can metabolize <em>2</em>-Delta(4)Ach-Gro by internalization through a high affinity transporter (K(m) = 300 +/- 30 nM, V(max) = 10 +/- 1 pmol.min(-1).mg protein(-1)), followed by hydrolysis by a fatty acid amide hydrolase (K(m) = 8 +/- 1 microM, V(max) = 400 +/- 50 pmol.min(-1).mg protein(-1)). The anandamide transport inhibitor AM404, and anandamide itself, were ineffective on <em>2</em>-Delta(4)Ach-Gro uptake by platelets, whereas anandamide competitively inhibited <em>2</em>-Delta(4)Ach-Gro hydrolysis (inhibition constant = 10 +/- 1 microM). Platelet activation by <em>2</em>-Delta(4)Ach-Gro was paralleled by an increase of intracellular calcium and inositol-1,4,5-trisphosphate, and by a decrease of cyclic AMP. Moreover, treatment of preloaded platelet-rich plasma with <em>2</em>-Delta(4)Ach-Gro induced an approximately threefold increase in [(3)H]<em>2</em>-Delta(4)Ach-Gro release, according to a CB receptor-dependent mechanism. On the other hand, ADP and collagen counteracted the activation of platelets by <em>2</em>-Delta(4)Ach-Gro, whereas 5-hydroxytryptamine (serotonin) enhanced and extended its effects. Remarkably, ADP and collagen also reduced [(3)H]<em>2</em>-Delta(4)Ach-Gro release from <em>2</em>-Delta(4)Ach-Gro-activated platelets, whereas 5-hydroxytryptamine further increased it. These findings suggest a so far unnoticed interplay between the peripheral endocannabinoid system and physiological platelet agonists.

<em>2</em>-<em>Arachidonoylglycerol</em> is an endogenous ligand for the cannabinoid receptors. Two types of cannabinoid receptors have been identified to date. The CB1 receptor is abundantly expressed in the brain, and assumed to be involved in the attenuation of neurotransmission. On the other hand, the physiological roles of the CB<em>2</em> receptor, mainly expressed in several types of inflammatory cells and immunocompetent cells, have not yet been fully elucidated. In this study, we investigated possible pathophysiological roles of the CB<em>2</em> receptor and <em>2</em>-<em>arachidonoylglycerol</em> in acute inflammation in mouse ear induced by the topical application of 1<em>2</em>-O-tetradecanoylphorbol-13-acetate. We found that the amount of <em>2</em>-<em>arachidonoylglycerol</em> was markedly augmented in inflamed mouse ear. In contrast, the amount of anandamide, another endogenous cannabinoid receptor ligand, did not change markedly. Importantly, 1<em>2</em>-O-tetradecanoylphorbol-13-acetate-induced ear swelling was blocked by treatment with SR1445<em>2</em>8, a CB<em>2</em> receptor antagonist, suggesting that the CB<em>2</em> receptor is involved in the swelling. On the other hand, the application of AM<em>2</em>51, a CB1 receptor antagonist, exerted only a weak suppressive effect. The application of SR1445<em>2</em>8 also reduced the 1<em>2</em>-O-tetradecanoylphorbol-13-acetate-induced production of leukotriene B(4) and the infiltration of neutrophils in the mouse ear. Interestingly, the application of <em>2</em>-<em>arachidonoylglycerol</em> to the mouse ear evoked swelling, which was abolished by treatment with SR1445<em>2</em>8. Nitric oxide was suggested to be involved in the ear swelling induced by <em>2</em>-<em>arachidonoylglycerol</em>. These results suggest that the CB<em>2</em> receptor and <em>2</em>-<em>arachidonoylglycerol</em> play crucial stimulative roles during the course of inflammatory reactions.

This review examines pharmacological and biochemical evidence that suggests the existence of an as yet undefined endothelial receptor that mediates endocannabinoid-induced vasodilation. The signaling mechanisms triggered through this receptor and its potential physiological role are also discussed. Since vasodilation is often associated with hypotension, mechanisms involved in the hypotensive actions of cannabinoids, including the endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, are also briefly reviewed.

Cannabinoids and their derivatives are group of more than 60 biologically active chemical agents, which have been used in natural medicine for centuries. The major agent of exogenous cannabinoids is Delta(9)-tetrahydrocannabinol (Delta(9)-THC), natural psychoactive ingredient of marijuana. However, psychoactive properties of these substances limited their use as approved medicines. Recent discoveries of endogenous cannabinoids (e.g. arachidonoylethanolamide, <em>2</em>-<em>arachidonoylglycerol</em> or palmithyloethanolamide) and their receptors initiated discussion on the role of cannabinoid system in physiological conditions as well as in various diseases. Based on the current knowledge, it could be stated that cannabinoids are important mediators in the skin, however their role have not been well elucidated yet. In our review, we summarized the current knowledge about the significant role of the cannabinoid system in the cutaneous physiology and pathology, pointing out possible future therapeutic targets.

Activation of hepatic cannabinoid 1 receptor (Cb1r) signaling has been implicated in the development of phenotypes associated with fatty liver, hypertriglyceridemia, and insulin resistance. In the current study, we have elucidated the critical role of endoplasmic reticulum-bound transcription factor cyclic AMP-response element-binding protein H (Crebh) in mediating activated Cb1r signaling in inducing phosphatidic acid phosphatase Lipin1 gene expression and subsequently deregulating hepatic insulin receptor signaling. Cb1r agonist (<em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG)) treatment induced Lipin1 gene expression in a Crebh-dependent manner via recruiting CREBH to the endogenous Lipin1 gene promoter. Adenoviral overexpression of Crebh or <em>2</em>-AG treatment in mice induced Lipin1 gene expression to increase the hepatic diacylglycerol (DAG) level and phosphorylation of protein kinase Cε (PKCε). This in turn inhibited hepatic insulin receptor signaling. Knockdown of Crebh or Cb1r antagonism attenuated <em>2</em>-AG-mediated induction of Lipin1 gene expression and decreased DAG production in mouse liver and subsequently restored insulin receptor signaling. Similarly, knockdown of Lipin1 attenuated the <em>2</em>-AG-induced increase in the DAG level and PKCε phosphorylation. Finally, shRNA-mediated knockdown of Crebh partially but significantly blunted Lipin1 expression and the DAG level in db/db mice. These results demonstrate a novel mechanism by which Cb1r signaling induces Lipin1 gene expression and increases DAG production by activating Crebh, thereby deregulating insulin receptor signaling pathway and lipid homeostasis.

Several G protein-associated receptors and synaptic proteins function within lipid rafts, which are subdomains of the plasma membranes that contain high concentrations of cholesterol. In this study we addressed the possible role of lipid rafts in the control of endocannabinoid system in striatal slices. Disruption of lipid rafts following cholesterol depletion with methyl-beta-cyclodestrin (MCD) failed to affect synthesis and degradation of anandamide, while it caused a marked increase in the synthesis and concentration of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), as well as in the binding activity of cannabinoid CB1 receptors. Surprisingly, endogenous <em>2</em>-AG-mediated control of GABA transmission was not potentiated by MCD treatment and, in contrast, neither basal nor 3,5-Dihydroxyphenylglycine-stimulated <em>2</em>-AG altered GABA synapses in cholesterol-depleted slices. Synaptic response to the cannabinoid CB1 receptor agonist HU<em>2</em>10 was however intact in MCD-treated slices, indicating that reduced sensitivity of cannabinoid CB1 receptors does not explain why endogenous <em>2</em>-AG is ineffective in inhibiting striatal GABA transmission after cholesterol depletion. Confocal microscopy analysis suggested that disruption of raft integrity by MCD might uncouple metabotropic glutamate 5-CB1 receptor interaction by altering the correct localization of both receptors in striatal neuron elements. In conclusion, our data indicate that disruption of raft integrity causes a complex alteration of the endocannabinoid signalling in the striatum.

The pharmacological (and recreational) effects of cannabis have been known for centuries. However, it is only recently that one has identified two subtypes of G-protein-coupled receptors, namely CB1 and CB<em>2</em>-receptors, which mediate the numerous effects of delta9-tetrahydrocannabinol and other cannabinoids. Logically, the existence of cannabinoid-receptors implies that endogenous ligands for these receptors (endocannabinoids) exist and exert a physiological role. Hence, arachidonoylethanolamide (anandamide) and sn-<em>2</em> <em>arachidonoylglycerol</em>, the first two endocannabinoids identified, are formed from plasma membrane phospholipids and act as CB1 and/or CB<em>2</em> agonists. The presence of both CB1 and CB<em>2</em>-receptors in the rat heart is noteworthy. This endogenous cardiac cannabinoid system is involved in several phenomena associated with cardioprotective effects. The reduction in infarct size following myocardial ischemia, observed in rats exposed to either LPS or heat stress <em>2</em>4 hours before, is abolished in the presence of a CB<em>2</em>-receptor antagonist. Endocannabinoids and synthetic cannabinoids, the latter through either CB1 or CB<em>2</em>-receptors, exert direct cardioprotective effects in rat isolated hearts. The ability of cannabinoids to reduce infarct size has been confirmed in vivo in anesthetized mice and rats. This latter effect appears to be mediated through CB<em>2</em>-receptors. Thus, the endogenous cardiac cannabinoid system, through activation of CB<em>2</em>-receptors, appears to be an important mechanism of protection against myocardial ischemia.

The endocannabinoid system (ECS) represents one of the major determinants of metabolic disorders. We investigated potential changes in the endogenous levels of anandamide (AEA), <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), N-oleoylethanolamine (OEA) and N-palmitoylethanolamine (PEA) in some peripheral organs and tissues of obese Zucker(fa/fa) and lean Zucker(fa/+) rats by qPCR, liquid chromatography mass spectrometry, western blot and enzymatic activity assays. At 10-1<em>2</em> weeks of age AEA levels were significantly lower in BAT, small intestine and heart and higher in soleus of Zucker(fa/fa) rats. In this tissue, also the expression of CB1 receptors was higher. By contrast in Zucker(fa/fa) rats, <em>2</em>-AG levels were changed (and lower) solely in the small and large intestine. Finally, in Zucker(fa/fa), PEA levels were unchanged, whereas OEA was slightly lower in BAT, and higher in the large intestine. Interestingly, these differences were accompanied by differential alterations of the genes regulating ECS tone. In conclusion, the levels of endocannabinoids are altered during obesity in a way partly correlating with changes of the genes related to their metabolism and activity.

Evidence for the role of the cannabimimetic fatty acid derivatives (CFADs), i.e. anandamide (arachidonoylethanolamide, AEA), <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and palmitoylethanolamide (PEA), in the control of inflammation and of the proliferation of tumor cells is reviewed here. The biosynthesis of AEA, PEA, or <em>2</em>-AG can be induced by stimulation with either Ca(<em>2</em>+) ionophores, lipopolysaccharide, or platelet activating factor in macrophages, and by ionomycin or antigen challenge in rat basophilic leukemia (RBL-<em>2</em>H3) cells (a widely used model for mast cells). These cells also inactivate CFADs through re-uptake and/or hydrolysis and/or esterification processes. AEA and PEA modulate cytokine and/or arachidonate release from macrophages in vitro, regulate serotonin secretion from RBL-<em>2</em>H3 cells, and are analgesic in some animal models of inflammatory pain. However, the involvement of endogenous CFADs and cannabinoid CB(1) and CB(<em>2</em>) receptors in these effects is still controversial. In human breast and prostate cancer cells, AEA and <em>2</em>-AG, but not PEA, potently inhibit prolactin and/or nerve growth factor (NGF)-induced cell proliferation. Vanillyl-derivatives of anandamide, such as olvanil and arvanil, exhibit even higher anti-proliferative activity. These effects are due to suppression of the levels of the 100 kDa prolactin receptor or of the high affinity NGF receptors (trk), are mediated by CB(1)-like cannabinoid receptors, and are enhanced by other CFADs. Inhibition of adenylyl cyclase and activation of mitogen-activated protein kinase underlie the anti-mitogenic actions of AEA. The possibility that CFADs act as local inhibitors of the proliferation of human breast cancer is discussed here.

N-arachidonoylethanolamine (anandamide, AEA), is a full agonist at both cannabinoid CB(1) receptors and "transient receptor potential vanilloid" type 1 (TRPV1) channels, and N-palmitoylethanolamine (PEA) potentiates these effects. In neurons of the rat dorsal root ganglia (DRG), TRPV1 is activated and/or sensitised by AEA as well as upon activation of protein kinases C (PKC) and A (PKA). We investigated here the effect on AEA levels of PKC and PKA activators in DRG neurons. AEA levels were significantly enhanced by both phorbol-miristoyl-acetate (PMA), a typical PKC activator, and forskolin (FSK), an adenylate cyclase stimulant, as well as by thrombin, which also activates PKC by stimulating protease-activated receptors (PARs). The levels of the other endocannabinoid and TRPV1-inactive compound, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), were enhanced only by thrombin and to a lesser extent than AEA, whereas PEA was not affected by any of the treatments. Importantly, FSK- and PMA-induced elevation of AEA levels was not sensitive to intracellular Ca<em>2</em>+ chelation with BAPTA-acetoxymethyl (AM) ester. In human embryonic kidney (HEK-<em>2</em>93) cells, which constitutively express PARs, thrombin, PMA and FSK elevated AEA levels, and the effects of the two former compounds were counteracted by the PKC inhibitor, RO318<em>2</em><em>2</em>0, whereas the effect of FSK was reduced by the PKA inhibitor RpcAMPs. In conclusion, we report that AEA levels are stimulated by both PKC, either directly or after thrombin receptor activation, and PKA, possibly in a way independent from intracellular calcium. Since AEA activates TRPV1, these findings may suggest the existence of an amplificatory cascades on this receptor in sensory neurons.