Macrophages are pivotal effector cells in immune responses and tissue remodeling by producing a wide spectrum of mediators, including angiogenic and lymphangiogenic factors. Activation of cannabinoid receptor types 1 and <em>2</em> has been suggested as a new strategy to modulate angiogenesis in vitro and in vivo. We investigated whether human lung-resident macrophages express a complete endocannabinoid system by assessing their production of endocannabinoids and expression of cannabinoid receptors. Unstimulated human lung macrophage produce <em>2</em>-<em>arachidonoylglycerol</em>,N-arachidonoyl-ethanolamine,N-palmitoyl-ethanolamine, and N-oleoyl-ethanolamine. On LPS stimulation, human lung macrophages selectively synthesize <em>2</em>-<em>arachidonoylglycerol</em> in a calcium-dependent manner. Human lung macrophages express cannabinoid receptor types 1 and <em>2</em>, and their activation induces ERK1/<em>2</em> phosphorylation and reactive oxygen species generation. Cannabinoid receptor activation by the specific synthetic agonists ACEA and JWH-133 (but not the endogenous agonist <em>2</em>-<em>arachidonoylglycerol</em>) markedly inhibits LPS-induced production of vascular endothelial growth factor-A, vascular endothelial growth factor-C, and angiopoietins and modestly affects IL-6 secretion. No significant modulation of TNF-α or IL-8/CXCL8 release was observed. The production of vascular endothelial growth factor-A by human monocyte-derived macrophages is not modulated by activation of cannabinoid receptor types 1 and <em>2</em>. Given the prominent role of macrophage-assisted vascular remodeling in many tumors, we identified the expression of cannabinoid receptors in lung cancer-associated macrophages. Our results demonstrate that cannabinoid receptor activation selectively inhibits the release of angiogenic and lymphangiogenic factors from human lung macrophage but not from monocyte-derived macrophages. Activation of cannabinoid receptors on tissue-resident macrophages might be a novel strategy to modulate macrophage-assisted vascular remodeling in cancer and chronic inflammation.

OBJECTIVE

The activation of cannabinoid receptor type <em>2</em> (CB(<em>2</em>))-mediated pathways might represent a promising anti-atherosclerotic treatment. Here, we investigated the expression of the endocannabinoid system in human carotid plaques and the impact of CB(<em>2</em>) pharmacological activation on markers of plaque vulnerability in vivo and in vitro.

RESULTS

The study was conducted using all available residual human carotid tissues (upstream and downstream the blood flow) from our cohort of patients symptomatic (n = 13) or asymptomatic (n = <em>2</em>7) for ischaemic stroke. Intraplaque levels of <em>2</em>-<em>arachidonoylglycerol</em>, anandamide N-arachidonoylethanolamine, N-palmitoylethanolamine, N-oleoylethanolamine, and their degrading enzymes (fatty acid amide hydrolase and monoacylglycerol lipase) were not different in human plaque portions. In the majority of human samples, CB(1) (both mRNA and protein levels) was undetectable. In downstream symptomatic plaques, CB(<em>2</em>) protein expression was reduced when compared with asymptomatic patients. In these portions, CB(<em>2</em>) levels were inversely correlated (r = -0.4008, P = 0.0170) with matrix metalloprotease (MMP)-9 content and positively (r = 0.3997, P = 0.0174) with collagen. In mouse plaques, CB(<em>2</em>) co-localized with neutrophils and MMP-9. Treatment with the selective CB(<em>2</em>) agonist JWH-133 was associated with the reduction in MMP-9 content in aortic root and carotid plaques. In vitro, pre-incubation with JWH-133 reduced tumour necrosis factor (TNF)-α-mediated release of MMP-9. This effect was associated with the reduction in TNF-α-induced ERK1/<em>2</em> phosphorylation in human neutrophils.

CONCLUSIONS

Cannabinoid receptor type <em>2</em> receptor is down-regulated in unstable human carotid plaques. Since CB(<em>2</em>) activation prevents neutrophil release of MMP-9 in vivo and in vitro, this treatment strategy might selectively reduce carotid vulnerability in humans.

Pharmacologic augmentation of endogenous cannabinoid (eCB) signaling is an emerging therapeutic approach for the treatment of a broad range of pathophysiological conditions. Thus far, pharmacological approaches have focused on inhibition of the canonical eCB inactivation pathways - fatty acid amide hydrolase (FAAH) for anandamide and monoacylglycerol lipase (MAGL) for <em>2</em>-<em>arachidonoylglycerol</em>. We review here the experimental evidence that cyclooxygenase-<em>2</em> (COX-<em>2</em>)-mediated eCB oxygenation represents a third mechanism for terminating eCB action at cannabinoid receptors. We describe the development, molecular mechanisms, and in vivo validation of 'substrate-selective' COX-<em>2</em> inhibitors (SSCIs) that prevent eCB inactivation by COX-<em>2</em> without affecting prostaglandin (PG) generation from arachidonic acid (AA). Lastly, we review recent data on the potential therapeutic applications of SSCIs with a focus on neuropsychiatric disorders.

1 The purpose of this study was to determine whether endocannabinoids can protect the heart against ischaemia and reperfusion. <em>2</em> Rat isolated hearts were exposed to low-flow ischaemia (0.5-0.6 ml min(-1)) and reperfusion. Functional recovery as well as CK and LDH overflow into the coronary effluent were monitored. Infarct size was determined at the end of the experiments. Phosphorylation levels of p38, ERK1/<em>2</em>, and JNK/SAPK kinases were measured by Western blots. 3 None of the untreated hearts recovered from ischaemia during the reperfusion period. Perfusion with either 300 nM palmitoylethanolamide (PEA) or 300 nM <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), but not anandamide (up to 1 micro M), 15 min before and throughout the ischaemic period, improved myocardial recovery and decreased the levels of coronary CK and LDH. PEA and <em>2</em>-AG also reduced infarct size. 4 The CB(<em>2</em>)-receptor antagonist, SR1445<em>2</em>8, blocked completely the cardioprotective effect of both PEA and <em>2</em>-AG, whereas the CB(1)-receptor antagonist, SR141716A, blocked partially the effect of <em>2</em>-AG only. In contrast, both ACEA and JWH015, two selective agonists for CB(1)- and CB(<em>2</em>)- receptors, respectively, reduced infarct size at a concentration of 50 nM. 5 PEA enhanced the phosphorylation level of p38 MAP kinase during ischaemia. PEA perfusion doubled the baseline phosphorylation level of ERK1/<em>2</em>, and enhanced its increase upon reperfusion. The cardioprotective effect of PEA was completely blocked by the p38 MAP kinase inhibitor, SB<em>2</em>03580, and significantly reduced by the ERK1/<em>2</em> inhibitor, PD98059, and the PKC inhibitor, chelerythrine. 6 In conclusion, endocannabinoids exert a strong cardioprotective effect in a rat model of ischaemia-reperfusion that is mediated mainly through CB(<em>2</em>)-receptors, and involves p38, ERK1/<em>2</em>, as well as PKC activation.

Exposure to behavioural stress normally triggers a complex, multilevel response of the hypothalamic-pituitary-adrenal (HPA) axis that helps maintain homeostatic balance. Although the endocannabinoid (eCB) system (ECS) is sensitive to chronic stress, few studies have directly addressed its response to acute stress. Here we show that acute restraint stress enhances eCB-dependent modulation of GABA release measured by whole-cell voltage clamp of inhibitory postsynaptic currents (IPSCs) in rat hippocampal CA1 pyramidal cells in vitro. Both Ca(<em>2</em>+)-dependent, eCB-mediated depolarization-induced suppression of inhibition (DSI), and muscarinic cholinergic receptor (mAChR)-mediated eCB mobilization are enhanced following acute stress exposure. DSI enhancement is dependent on the activation of glucocorticoid receptors (GRs) and is mimicked by both in vivo and in vitro corticosterone treatment. This effect does not appear to involve cyclooxygenase-<em>2</em> (COX-<em>2</em>), an enzyme that can degrade eCBs; however, treatment of hippocampal slices with the L-type calcium (Ca(<em>2</em>+)) channel inhibitor, nifedipine, reverses while an agonist of these channels mimics the effect of in vivo stress. Finally, we find that acute stress produces a delayed (by 30 min) increase in the hippocampal content of <em>2</em>-<em>arachidonoylglycerol</em>, the eCB responsible for DSI. These results support the hypothesis that the ECS is a biochemical effector of glucocorticoids in the brain, linking stress with changes in synaptic strength.

The endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em> are lipid mediators that signal via CB(1) and CB(<em>2</em>) cannabinoid receptors and Gi/o-proteins to inhibit adenylyl cyclase and stimulate mitogen-activated protein kinase. In the brain, CB(1) receptors interact with opioid receptors in close proximity, and these receptors may share G-proteins and effector systems. In the striatum, CB(1) receptors function in coordination with D(1) and D(<em>2</em>) dopamine receptors, and combined stimulation of CB(1)-D(<em>2</em>) receptor heteromeric complexes promotes a unique interaction to stimulate cAMP production. CB(1) receptors also trigger growth factor receptor signaling cascades in cells by engaging in cross-talk or interreceptor signal transmission with the receptor tyrosine kinase (RTK) family. Mechanisms for CB(1) receptor-RTK transactivation can include stimulation of signal transduction pathways regulated by second messengers such as phospholipase C, metalloprotease cleavage of membrane-bound precursor proteins such as epidermal growth factor which activate RTKs, RTK autophosphorylation, and recruitment of non-receptor tyrosine kinases. CB(1) and CB(<em>2</em>) receptors are expressed in peripheral tissues including liver and adipose tissue, and are induced in pathological conditions. Novel signal transduction resulting from endocannabinoid regulation of AMP-regulated kinase and peroxisome proliferator-activated receptors have been discovered from studies of hepatocytes and adipocytes. It can be predicted that drug discovery of the future will be based upon these novel signal transduction mechanisms for endocannabinoid mediators.

Cannabinoids, including the endogenous cannabinoid or endocannabinoid, anandamide, modulate several gastrointestinal functions. To date, the gastrointestinal effects of the second putative endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) have not been studied. In the present study using a shrew (Cryptotis parva) emetic model, <em>2</em>-AG (0.<em>2</em>5-10 mg/kg, i.p.) potently and dose-dependently increased vomiting frequency (ED(50) = 1.13 mg/kg) and the number of animals vomiting (ED(50) = 0.48 mg/kg). In contrast, neither anandamide (<em>2</em>.5-<em>2</em>0 mg/kg) nor methanandamide (5-10 mg/kg) induced a dose-dependent emetogenic response, but both could partially block the induced emetic effects. Delta(9)-Tetrahydrocannabinol and its synthetic analogs reduced <em>2</em>-AG-induced vomiting with the rank order potency: CP 55,940>> WIN 55,<em>2</em>1<em>2</em>-<em>2</em>>> Delta(9)-tetrahydrocannabinol. The nonpsychoactive cannabinoid, cannabidiol, was inactive. Nonemetic doses of SR 141716A (1-5 mg/kg) also blocked <em>2</em>-AG-induced vomiting. The <em>2</em>-AG metabolite arachidonic acid also caused vomiting. Indomethacin, a cyclooxygenase inhibitor, blocked the emetogenic effects of both arachidonic acid and <em>2</em>-AG. CP 55,940 also blocked the emetic effects of arachidonic acid. <em>2</em>-AG (0.<em>2</em>5-10 mg/kg) reduced spontaneous locomotor activity (ED(50) = 11 mg/kg) and rearing frequency (ED(50) = 4.3 mg/kg) in the shrew, whereas such doses of both anandamide and methanandamide had no effect on locomotor parameters. The present study indicates that: 1) <em>2</em>-AG is an efficacious endogenous emetogenic cannabinoid involved in vomiting circuits, <em>2</em>) the emetic action of <em>2</em>-AG and the antiemetic effects of tested cannabinoids are mediated via CB(1) receptors, and 3) the emetic effects of <em>2</em>-AG occur in lower doses relative to its locomotor suppressant actions.

<em>2</em>-<em>Arachidonoylglycerol</em> is an endogenous ligand for the cannabinoid receptors (CB1 and CB<em>2</em>). Evidence is gradually accumulating which shows that <em>2</em>-<em>arachidonoylglycerol</em> plays important physiological roles in several mammalian tissues and cells, yet the details remain ambiguous. In this study, we first examined the effects of <em>2</em>-<em>arachidonoylglycerol</em> on the motility of human natural killer cells. We found that <em>2</em>-<em>arachidonoylglycerol</em> induces the migration of KHYG-1 cells (a natural killer leukemia cell line) and human peripheral blood natural killer cells. The migration of natural killer cells induced by <em>2</em>-<em>arachidonoylglycerol</em> was abolished by treating the cells 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 <em>2</em>-<em>arachidonoylglycerol</em>-induced migration. In contrast to <em>2</em>-<em>arachidonoylglycerol</em>, anandamide, another endogenous cannabinoid receptor ligand, did not induce the migration. Delta9-tetrahydrocannabinol, a major psychoactive constituent of marijuana, also failed to induce the migration; instead, the addition of delta9-tetrahydrocannabinol together with <em>2</em>-<em>arachidonoylglycerol</em> abolished the migration induced by <em>2</em>-<em>arachidonoylglycerol</em>. It is conceivable that the endogenous ligand for the cannabinoid receptor, that is, <em>2</em>-<em>arachidonoylglycerol</em>, affects natural killer cell functions such as migration, thereby contributing to the host-defense mechanism against infectious viruses and tumor cells.

Based on experimental evidence of the antinociceptive action of endocannabinoids and their role in the modulation of trigeminovascular system activation, we hypothesized that the endocannabinoid system may be dysfunctional in chronic migraine (CM). We examined whether the concentrations of N-arachidonoylethanolamide (anandamide, AEA), palmitoylethanolamide (PEA), and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in the CSF of patients with CM and with probable CM and probable analgesic-overuse headache (PCM+PAOH) are altered compared with control subjects. The above endocannabinoids were measured by high-performance liquid chromatography (HPLC), and quantified by isotope dilution gas-chromatography/mass-spectrometry. Calcitonin gene-related peptide (CGRP) levels were also determined by RIA method and the end products of nitric oxide (NO), the nitrites, by HPLC. CSF concentrations of AEA were significantly lower and those of PEA slightly but significantly higher both in patients with CM and PCM+PAOH than in nonmigraineur controls (p<0.01 and p<0.0<em>2</em>, respectively). A negative correlation was found between AEA and CGRP levels in CM and PCM+PAOH patients (r=0.59, p<0.01 and r=-0.65, p<0.007; respectively). A similar trend was observed between this endocannabinoid and nitrite levels. Reduced levels of AEA in the CSF of CM and PCM+PAOH patients may reflect an impairment of the endocannabinoid system in these patients, which may contribute to chronic head pain and seem to be related to increased CGRP and NO production. These findings support the potential role of the cannabinoid (CB)1 receptor as a possible therapeutic target in CM.

The tissue concentrations of the endocannabinoids, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and N-arachidonoyl-ethanolamine (anandamide), are altered in the adipose tissue of mice fed a high fat diet. We have investigated here the effect on endocannabinoid levels of incubation of mouse 3T3-F44<em>2</em>A adipocytes with several free polyunstaurated fatty acids (PUFAs), including linolenic acid (LA), alpha-linolenic acid (ALA), arachidonic acid (AA) and docosahexaenoic acid (DHA), as well as oleic acid (OA) and palmitic acid (PA). By using mass spectrometric methods, we quantified the levels of endocannabinoids, of two anandamide congeners, N-palmitoyl-ethanolamine (PEA) and N-oleoyl-ethanolamine (OEA), and of fatty acids esterified in triacylglycerols or phospholipids, which act as <em>2</em>-AG and/or N-acyl-ethanolamine precursors. Incubation with AA strongly elevated <em>2</em>-AG levels and the amounts of AA esterified in triacylglycerols and on glycerol carbon atom <em>2</em> (sn-<em>2</em>), but not 1 (sn-1), in phospholipids. Incubation with DHA decreased <em>2</em>-AG and anandamide levels and the amounts of AA esterified on both the sn-<em>2</em> and sn-1 position of phospholipids, but not on triacylglycerols. PEA levels augmented following incubation of adipocytes with OA and PA, with no corresponding changes in phospholipids and triacylglycerols. We suggest that dietary PUFAs might modulate the levels of adipocyte phospholipids that act as endocannabinoid precursors.

Recent studies have addressed the changes in endocannabinoid ligands and receptors that occur in multiple sclerosis, as a way to explain the efficacy of cannabinoid compounds to alleviate spasticity, pain, tremor, and other signs of this autoimmune disease. Using Lewis rats with experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, we recently found a decrease in cannabinoid CB1 receptors mainly circumscribed to the basal ganglia, which could be related to the motor disturbances characteristic of these rats. In the present study, using the same model, we explored the potential changes in several neurotransmitters in the basal ganglia that might be associated with the motor disturbances described in these rats, but we only found a small increase in glutamate contents in the globus pallidus. We also examined whether the motor disturbances and the changes of CB1 receptors found in the basal ganglia of EAE rats disappear after the treatment with rolipram, an inhibitor of type IV phosphodiesterase able to supress EAE in different species. Rolipram attenuated clinical decline, reduced motor inhibition, and normalized CB1 receptor gene expression in the basal ganglia. As a third objective, we examined whether EAE rats also exhibited changes in endocannabinoid levels as shown for CB1 receptors. Anandamide and <em>2</em>-<em>arachidonoylglycerol</em> levels decreased in motor related regions (striatum, midbrain) but also in other brain regions, although the pattern of changes for each endocannabinoid was different. Finally, we hypothesized that the elevation of the endocannabinoid activity, following inhibition of endocannabinoid uptake, might be beneficial in EAE rats. AM404, arvanil, and OMDM<em>2</em> were effective to reduce the magnitude of the neurological impairment in EAE rats, whereas VDM11 did not produce any effect. The beneficial effects of AM404 were reversed by blocking TRPV1 receptors with capsazepine, but not by blocking CB1 receptors with SR141716, thus indicating the involvement of endovanilloid mechanisms in these effects. However, a role for CB1 receptors is supported by additional data showing that CP55,940 delayed EAE progression. In summary, our data suggest that reduction of endocannabinoid signaling is associated with the development of EAE in rats. We have also proved that the reduction of CB1 receptors observed in these rats is corrected following treatment with a compound used in EAE such as rolipram. In addition, the direct or indirect activation of vanilloid or cannabinoid receptors may reduce the neurological impairment experienced by EAE rats, although the efficacy of the different compounds examined seems to be determined by their particular pharmacodynamic and pharmacokinetic characteristics.

During the last eight years a number of bioactive lipid mediators, the amides or esters of long chain fatty acids, have been discovered or re-discovered. These are: anandamide (N-arachidonoyl-ethanolamine, AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), two endogenous agonists of cannabinoid receptors; oleamide (cis-9-octadecenoamide), a putative endogenous sleep-inducing factor; N-palmitoylethanol amine (PEA), a compound with promising anti-inflammatory and immune-modulatory activity. These compounds are all substrates for the same hydrolytic enzyme, fatty acid amide hydrolase (FAAH), whose molecular characterization was obtained in 1996. The molecular and enzymatic properties, tissue distribution, substrate recognition properties, physiological regulation and biological role of FAAH are discussed in this article, with special emphasis on the possible pharmacological manipulation of the activity of this enzyme with therapeutic purpose.

Endocannabinoids (eCBs) are endogenous lipid mediators involved in a variety of physiological, pharmacological, and pathological processes. While activation of the eCB system primarily induces inhibitory effects on both GABAergic and glutamatergic synaptic transmission and plasticity through acting on presynaptically expressed CB1 receptors in the brain, accumulated information suggests that eCB signaling is also capable of facilitating or potentiating excitatory synaptic transmission in the hippocampus. Recent studies show that a long-lasting potentiation of excitatory synaptic transmission at Schaffer collateral (SC)-CA1 synapses is induced by spatiotemporally primed inputs, accompanying with a long-term depression of inhibitory synaptic transmission (I-LTD) in hippocampal CA1 pyramidal neurons. This input timing-dependent long-lasting synaptic potentiation at SC-CA1 synapses is mediated by <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) signaling triggered by activation of postsynaptic N-methyl-D-aspartate receptors, group I metabotropic glutamate receptors (mGluRs), and a concurrent rise in intracellular Ca(<em>2</em>+). Emerging evidence now also indicates that <em>2</em>-AG is an important signaling mediator keeping brain homeostasis by exerting its anti-inflammatory and neuroprotective effects in response to harmful insults through CB1/<em>2</em> receptor-dependent and/or -independent mechanisms. Activation of the nuclear receptor protein peroxisome proliferator-activated receptor-γ apparently is one of the important mechanisms in resolving neuroinflammation and protecting neurons produced by <em>2</em>-AG signaling. Thus, the information summarized in this review suggests that the role of eCB signaling in maintaining integrity of brain function is greater than what we thought previously.

In the CNS, endocannabinoids are identified mainly as two endogenous lipids: anandamide, the ethanolamide of arachidonic acid, and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Endocannabinoids are known to inhibit transmitter release from presynaptic terminals; however we have recently demonstrated that they are also involved in slow self-inhibition (SSI) of layer V low-threshold spiking (LTS) interneurons in rat somatosensory cortex. SSI is induced by repetitive firing in LTS cells, which can express either cholecystokinin or somatostatin. SSI is triggered by an endocannabinoid-dependent activation of a prolonged somatodendritic K(+) conductance and associated hyperpolarization in the same cell. The synthesis of both endocannabinoids is dependent on elevated [Ca(<em>2</em>+)](i) such as occurs during sustained neuronal activity. To establish whether <em>2</em>-AG mediates autocrine LTS-SSI, we blocked its biosynthesis from phospholipase C (PLC) and diacylglycerol lipases (DAGLs). Current-clamp recordings from LTS interneurons in acute neocortical slices showed that inclusion of DAGL inhibitors in the whole-cell pipette prevented the long-lasting hyperpolarization triggered by LTS cell repetitive firing. Similarly, extracellular applications of a PLC inhibitor prevented SSI in LTS interneurons. Moreover, metabotropic glutamate receptor-dependent activation of PLC produced a long-lasting hyperpolarization which was prevented by the CB1 antagonist AM<em>2</em>51, as well as by PLC and DAGL inhibitors. The loss of SSI in the presence of intracellular DAGL blockers confirms that endocannabinoid production occurs in the same interneuron undergoing the persistent hyperpolarization. Since DAGLs produce no endocannabinoid other than <em>2</em>-AG, these results identify this compound as the autocrine mediator responsible for the postsynaptic slow self-inhibition of neocortical LTS interneurons.

<em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is an endogenous cannabinoid receptor ligand. To date, two types of cannabinoid receptors have been identified: the CB1 receptor, abundantly expressed in the brain, and the CB<em>2</em> receptor, expressed in various lymphoid tissues such as the spleen. The CB1 receptor has been assumed to play an important role in the regulation of synaptic transmission, whereas the physiological roles of the CB<em>2</em> receptor remain obscure. In this study, we examined whether the CB<em>2</em> receptor is present in human eosinophils and found that the CB<em>2</em> receptor is expressed in human peripheral blood eosinophils. In contrast, human neutrophils do not contain a significant amount of the CB<em>2</em> receptor. We then examined the effect of <em>2</em>-AG on the motility of eosinophils. We found that <em>2</em>-AG induces the migration of human eosinophilic leukemia EoL-1 cells. The migration evoked by <em>2</em>-AG was abolished in the presence of SR1445<em>2</em>8, a CB<em>2</em> receptor antagonist, or by pretreatment of the cells with pertussis toxin, suggesting that the CB<em>2</em> receptor and Gi/o are involved in the <em>2</em>-AG-induced migration. The migration of EoL-1 cells induced by <em>2</em>-AG was suggested to be a result of chemotaxis. In contrast to <em>2</em>-AG, neither anandamide nor free arachidonic acid elicited the migration. Finally, we examined the effect of <em>2</em>-AG on human peripheral blood eosinophils and neutrophils and found that <em>2</em>-AG induces migration of eosinophils but not neutrophils. These results suggest that the CB<em>2</em> receptor and its endogenous ligand <em>2</em>-AG may be closely involved in allergic inflammation accompanied by the infiltration of eosinophils.

Cannabinoids have recently been approved as a treatment for pain in multiple sclerosis (MS). Increasing evidence from animal studies suggests that this class of compounds could also prove efficient to fight neurodegeneration, demyelination, inflammation and autoimmune processes occurring in this pathology. However, the use of cannabinoids is limited by their psychoactive effects. In this context, potentiation of the endogenous cannabinoid signalling could represent a substitute to the use of exogenously administrated cannabinoid ligands. Here, we studied the expression of different elements of the endocannabinoid system in a chronic model of MS in mice. We first studied the expression of the two cannabinoid receptors, CB(1) and CB(<em>2</em>), as well as the putative intracellular cannabinoid receptor peroxisome proliferator-activated receptor-alpha. We observed an upregulation of CB(<em>2</em>), correlated to the production of proinflammatory cytokines, at 60 days after the onset of the MS model. At this time, the levels of the endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em>, and of the anti-inflammatory anandamide congener, palmithoylethanolamide, were enhanced, without changes in the levels of anandamide. These changes were not due to differences in the expression of the degradation enzymes, fatty acid amide hydrolase and monoacylglycerol lipase, or of biosynthetic enzymes, diacylglycerol lipase-alpha and N-acylphosphatidylethanolamine phospholipase-D at this time (60 days). Finally, the exogenous administration of palmitoylethanolamide resulted in a reduction of motor disability in the animals subjected to this model of MS, accompanied by an anti-inflammatory effect. This study overall highlights the potential therapeutic effects of endocannabinoids in MS.

N-palmitoylethanolamine (PEA), an endogenous fatty acid ethanolamide, plays a key role in the regulation of the inflammatory response and pain through, among others, activation of nuclear peroxisome proliferator-activated receptors (PPAR-α). Endogenous cannabinoids play a protective role in several central nervous system (CNS) disorders, particularly those associated with neuronal hyperexcitability. We investigated the effects of PEA and the role of PPAR-α in absence epilepsy using the WAG/Rij rat model. PEA, anandamide (AEA), a PPAR-α antagonist (GW6471) and a synthetic CB1 receptor antagonist/inverse agonist (SR141716) were administered to WAG/Rij rats in order to evaluate the effects on epileptic spike-wave discharges (SWDs) on EEG recordings. We studied also the effects of PEA co-administration with SR141716 and GW6471 and compared these effects with those of AEA to evaluate PEA mechanism of action and focusing on CB1 receptors and PPAR-α. Both PEA and AEA administration significantly decreased SWDs parameters (absence seizures). In contrast, GW6471 was devoid of effects while SR141716 had pro-absence effects. The co-administration of SR141716 with PEA or AEA completely blocked the anti-absence effects of these compounds. GW6471 antagonized PEA's effects whereas it did not modify AEA's effects. Furthermore, we have also measured PEA, AEA and <em>2</em>-AG (<em>2</em>-<em>arachidonoylglycerol</em>) brain levels identifying significant differences between epileptic and control rats such as decreased PEA levels in both thalamus and cortex that might contribute to absence epilepsy. Our data demonstrate that PEA has anti-absence properties in the WAG/Rij rat model and that such properties depend on PPAR-α and indirect activation of CB1 receptors. This article is part of the Special Issue entitled 'New Targets and Approaches to the Treatment of Epilepsy'.

At nanomolar concentrations, SR141716 and AM<em>2</em>51 act as specific and selective antagonists of the cannabinoid CB1 receptor. In the micromolar range, these compounds were shown to inhibit basal G-protein activity, and this is often interpreted to implicate constitutive activity of the CB1 receptors in native tissue. We show here, using [35S]GTPgammaS binding techniques, that micromolar concentrations of SR141716 and AM<em>2</em>51 inhibit basal G-protein activity in rat cerebellar membranes, but only in conditions where tonic adenosine A1 receptor signaling is not eliminated. Unlike lipophilic A1 receptor antagonists (potency order DPCPX>)N-0840 approximately cirsimarin>caffeine), adenosine deaminase (ADA) was not fully capable in eliminating basal A1 receptor-dependent G-protein activity. Importantly, all antagonists reduced basal signal to the same extent (<em>2</em>0%), and the response evoked by the inverse agonist DPCPX was not reversed by the neutral antagonist N-0840. These data indicate that rat brain A1 receptors are not constitutively active, but that an ADA-resistant adenosine pool is responsible for tonic A1 receptor activity in brain membranes. SR141716 and AM<em>2</em>51, at concentrations fully effective in reversing CB1-mediated responses (10-6 m), did not reduce basal G-protein activity, indicating that CB1 receptors are not constitutively active in these preparations.4 At higher concentrations (1-<em>2</em>.5 x 10-5 m), both antagonists reduced basal G-protein activity in control and ADA-treated membranes, but had no effect when A1 receptor signaling was blocked with DPCPX. Moreover, the CB1 antagonists right-shifted A1 agonist dose-response curves without affecting maximal responses, suggesting competitive mode of antagonist action. The CB1 antagonists did not affect muscarinic acetylcholine or GABAB receptor signaling. When further optimizing G-protein activation assay for the labile endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), we show, by using HPLC, that pretreatment of cerebellar membranes with methyl arachidonoyl fluorophosphonate (MAFP) fully prevented enzymatic degradation of <em>2</em>-AG and concomitantly enhanced the potency of <em>2</em>-AG. In contrast to previous claims, MAFP exhibited no antagonist activity at the CB1 receptor.6 The findings establish an optimized method with improved signal-to-noise ratio to assess endocannabinoid-dependent G-protein activity in brain membranes, under assay conditions where basal adenosinergic tone and enzymatic degradation of <em>2</em>-AG are fully eliminated.

<em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG) is a unique molecular species of monoacylglycerol isolated from rat brain and canine gut as an endogenous cannabinoid receptor ligand (Sugiura, T., Kondo, S., Sukagawa, A., Nakane, S., Shinoda, A., Itoh, K., Yamashita, A., Waku, K., 1995. <em>2</em>-<em>Arachidonoylglycerol</em>: a possible endogenous cannabinoid receptor ligand in brain. Biochem. Biophys. Res. Commun. <em>2</em>15, 89-97; Mechoulam, R., Ben-Shabat, S., Hanus, L., Ligumsky, M., Kaminski, N. E., Schatz, A.R., Gopher, A., Almog, S., Martin, B.R., Compton, D.R., Pertwee, R.G., Giffin, G., Bayewitch, M., Brag, J., Vogel, Z., 1995. Identification of an endogenous <em>2</em>-monoglyceride, present in canine gut, that binds to cannabinoid receptors. Biochem. Pharmacol. 50, 83-90). <em>2</em>-AG binds to the cannabinoid receptors (CB1 and CB<em>2</em>) and exhibits a variety of cannabimimetic activities in vitro and in vivo. Recently, we found that <em>2</em>-AG induces Ca(<em>2</em>+) transients in NG108-15 cells, which express the CB1 receptor, and in HL-60 cells, which express the CB<em>2</em> receptor, through a cannabinoid receptor- and Gi/Go-dependent mechanism. Based on the results of structure-activity relationship experiments, we concluded that <em>2</em>-AG but not anandamide is the natural ligand for both the CB1 and the CB<em>2</em> receptors and both receptors are primarily <em>2</em>-AG receptors. Evidences are gradually accumulating that <em>2</em>-AG is a physiologically essential molecule, although further detailed studies appear to be necessary to determine relative importance of <em>2</em>-AG and anandamide in various animal tissues. In this review, we described mainly our previous and current experimental results, as well as those of others, concerning the tissue levels, bioactions and metabolism of <em>2</em>-AG.

The endocannabinoid (eCB) <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is hydrolysed primarily by monoacylglycerol lipase (MAGL). Here, we investigated whether eCB-mediated retrograde synaptic depression in cerebellar slices was altered in MAGL knockout (MAGL(-/-)) mice. Depolarization-induced suppression of excitation (DSE) and metabotropic glutamate receptor (mGluR1)-mediated synaptic depression are mediated by <em>2</em>-AG-induced activation of CB(1) receptors. We show that genetic deletion of MAGL prolonged DSE at parallel fibre (PF) or climbing fibre (CF) to Purkinje cell (PC) synapses. Likewise, mGluR1-mediated synaptic depression, induced either by high-frequency stimulation of PF or mGluR1 agonist DHPG, was prolonged in MAGL(-/-) mice. About 15% of <em>2</em>-AG in the brain is hydrolysed by serine hydrolase α-β-hydrolase domain 6 and 1<em>2</em> (ABHD6 and ABHD1<em>2</em>). However, the selective ABHD6 inhibitor WWL1<em>2</em>3 had no significant effect on cerebellar DSE in MAGL(+/+) and (-/-) mice. The CB(1) receptor antagonist SR141716 significantly increased the amplitude of basal excitatory postsynaptic currents (EPSCs) in MAGL(-/-) mice but not in MAGL(+/+) mice. Conversely, the CB(1) agonist WIN55<em>2</em>1<em>2</em> induced less depression of basal EPSCs in MAGL(-/-) mice than in MAGL(+/+) mice. These results provide genetic evidence that inactivation of <em>2</em>-AG by MAGL determines the time course of eCB-mediated retrograde synaptic depression and that genetic deletion of MAGL causes tonic activation and consequential desensitization of CB(1) receptors.

The structure-activity relationships of organophosphorus (OP) and organosulfur compounds were examined in vitro and in vivo as inhibitors of mouse brain monoacylglycerol lipase (MAGL) hydrolysis of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and agonist binding at the CB1 receptor. Several compounds showed exceptional potency toward MAGL activity with IC(50) values of 0.1-10 nM in vitro and high inhibition at 10mg/kg intraperitoneally in mice. We find for the first time that MAGL activity is a major in vivo determinant of <em>2</em>-AG and arachidonic acid levels not only in brain but also in spleen, lung, and liver. Apparent direct OP inhibition of CB1 agonist binding may be due instead to metabolic stabilization of <em>2</em>-AG in brain membranes as the actual inhibitor.

Although endocannabinoids have emerged as essential retrograde messengers in several forms of synaptic plasticity, it remains controversial whether they mediate long-term depression (LTD) of glutamatergic synapses onto excitatory and inhibitory neurons in the hippocampus. Here, we show that parvalbumin- and somatostatin/metabotropic glutamate receptor 1(a) (mGlu(1a))-positive GABAergic interneurons express diacylglycerol lipase-α (DGL-α), a synthesizing enzyme of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), albeit at lower levels than principal cells. Moreover, this lipase accumulates postsynaptically around afferent excitatory synapses in all three cell types. To address the role of retrograde <em>2</em>-AG signaling in LTD, we investigated two forms: (1) produced by postsynaptic spiking paired with subsequent presynaptic stimulation or (<em>2</em>) induced by group I mGlu activation by (S)-3,5-dihydroxyphenylglycine (DHPG). Neither form of LTD was evoked in the presence of the mGlu(5) antagonist MPEP [<em>2</em>-methyl-6-(phenylethynyl)-pyridine], the DGL inhibitor THL [N-formyl-l-leucine (1S)-1-[[(<em>2</em>S,3S)-3-hexyl-4-oxo-<em>2</em>-oxetanyl]methyl]dodecyl ester], or the intracellularly applied Ca(<em>2</em>+) chelator BAPTA in CA1 pyramidal cells, fast-spiking interneurons (representing parvalbumin-containing cells) and interneurons projecting to stratum lacunosum-moleculare (representing somatostatin/mGlu(1a)-expressing interneurons). Both forms of LTD were completely absent in CB(1) cannabinoid receptor knock-out mice, whereas pharmacological blockade of CB(1) led to inconsistent results. Notably, in accordance with their lower DGL-α level, a higher stimulation frequency or higher DHPG concentration was required for LTD induction in interneurons compared with pyramidal cells. These findings demonstrate that hippocampal principal cells and interneurons produce endocannabinoids to mediate LTD in a qualitatively similar, but quantitatively different manner. The shifted induction threshold implies that endocannabinoid-LTD contributes to cortical information processing during distinct network activity patterns in a cell type-specific manner.

The present review focuses on the role of the endogenous cannabinoid system in the modulation of immune response and control of cancer cell proliferation. The involvement of cannabinoid receptors, endogenous ligands and enzymes for their biosynthesis and degradation, as well as of cannabinoid receptor-independent events is discussed. The picture arising from the recent literature appears very complex, indicating that the effects elicited by the stimulation of the endocannabinoid system are strictly dependent on the specific compounds and cell types considered. Both the endocannabinoid anandamide and its congener palmitoylethanolamide, exert a negative action in the onset of a variety of parameters of the immune response. However, <em>2</em>-<em>arachidonoylglycerol</em> appears to be the true endogenous ligand for peripheral cannabinoid receptors, although its action as an immunomodulatory molecule requires further characterization. Modulation of the endocannabinoid system interferes with cancer cell proliferation either by inhibiting mitogenic autocrine/paracrine loops or by directly inducing apoptosis; however, the proapoptotic effect of anandamide is not shared by other endocannabinoids and suggests the involvement of non-cannabinoid receptors, namely the VR1 class of vanilloid receptors. In conclusion, further investigations are needed to elucidate the function of endocannabinoids as immunosuppressant and antiproliferative/cytotoxic agents. The experimental evidence reviewed in this article argues in favor of the therapeutic potential of these compounds in immune disorders and cancer.

The diacylglycerol lipases (DAGLs) hydrolyse diacylglycerol to generate <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), the most abundant ligand for the CB(1) and CB(<em>2</em>) cannabinoid receptors in the body. DAGL-dependent endocannabinoid signalling regulates axonal growth and guidance during development, and is required for the generation and migration of new neurons in the adult brain. At developed synapses, <em>2</em>-AG released from postsynaptic terminals acts back on presynaptic CB(1) receptors to inhibit the secretion of both excitatory and inhibitory neurotransmitters, with this DAGL-dependent synaptic plasticity operating throughout the nervous system. Importantly, the DAGLs have functions that do not involve cannabinoid receptors. For example, <em>2</em>-AG is the precursor of arachidonic acid in a pathway that maintains the level of this essential lipid in the brain and other organs. This pathway also drives the cyclooxygenase-dependent generation of inflammatory prostaglandins in the brain, which has recently been implicated in the degeneration of dopaminergic neurons in Parkinson's disease. Remarkably, we still know very little about the mechanisms that regulate DAGL activity-however, key insights can be gleaned by homology modelling against other α/β hydrolases and from a detailed examination of published proteomic studies and other databases. These identify a regulatory loop with a highly conserved signature motif, as well as phosphorylation and palmitoylation as post-translational mechanisms likely to regulate function.