This study was aimed at finding structural requirements for the interaction of the acyl chain of endocannabinoids with cannabinoid receptors, membrane transporter protein, and fatty acid amide hydrolase (FAAH). To this end, the flexibility of the acyl chain was restricted by introduction of an 1-hydroxy-<em>2</em>Z,4E-pentadiene system in anandamide (N-arachidonoylethanolamine, AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) at various positions using different lipoxygenases. This brought about selectivity and attenuated the binding potency of AEA and <em>2</em>-AG. Although the displacement constants were modest, 15(S)-hydroxy-eicosa-5Z,8Z,11Z,13E-tetraenoyl-N-(<em>2</em>-hydroxyethyl)amine was found to bind selectively to the CB(1) receptor, whereas its 1-arachidonoyl-sn-glycerol analogue and 13(S)-hydroxy-octadeca-9Z,11E-dienoyl-N-(<em>2</em>-hydroxyethyl)amine could selectively bind to the CB(<em>2</em>) receptor. 11(S)-Hydroxy-eicosa-5Z,8Z,1<em>2</em>E,14Z-tetraenoyl-N-(<em>2</em>-hydroxyethyl)amine did not bind to either receptor, whereas 1<em>2</em>(S)-hydroxy-eicosa-5Z,8Z,10E,14Z-tetraenoyl-N-(<em>2</em>-hydroxyethyl)amine did bind to both CB receptors with an affinity similar to that of AEA. All oxygenated anandamide derivatives were good inhibitors of FAAH (low micromolar K(i)) but were ineffective on the AEA transporter. <em>2</em>-AG rapidly isomerizes into 1(3)-arachidonoyl-sn-glycerol. Both 1- and 3-arachidonoyl-sn-glycerol did not bind to either CB receptor and did not interfere with AEA transport. Thus, after it is isomerized, <em>2</em>-AG is inactivated, thereby decreasing effective concentrations of <em>2</em>-AG. Analysis of (1)H NMR spectra revealed that chloroform did not induce notably different conformations in the acyl chain of 15(S)-hydroxy-eicosa-5Z,8Z,11Z,13E-tetraenoic acid as compared with water. Molecular dynamics (MD) simulations of AEA and its analogues in the presence of explicit water molecules revealed that a tightly folded conformation of the acyl chain is not the only requirement for CB(1) binding. Structural details of the C(<em>2</em>)-C(15) loop, such as an sp(<em>2</em>) carbon at position 11, are necessary for receptor binding. The MD simulations may suggest that the average orientations of the pentyl tail of AEA and 1<em>2</em>(S)-hydroxy-eicosa-5Z,8Z,10E,14Z-tetraenoyl-N-(<em>2</em>-hydroxyethyl)amine are different from that of the low-affinity, inactive ligands.

OBJECTIVE

Aim of this study was to evaluate a possible association between endocannabinoid (EC) plasma levels, such as anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), and coronary circulatory function in obesity.

RESULTS

Myocardial blood flow (MBF) responses to cold pressor test (CPT) and during pharmacological vasodilation with dipyridamole were measured with (13)N-ammonia PET/CT. Study participants (n = 77) were divided into three groups based on their body mass index (BMI, kg/m(<em>2</em>)): control group <em>2</em>0 ≤ BMI (<em>2</em>5 (n = <em>2</em>1); overweight group, <em>2</em>5 ≤ BMI <30 (n = <em>2</em>6); and obese group, BMI ≥ 30 (n = 30). Anandamide plasma levels, but not <em>2</em>-AG plasma levels, were significantly elevated in obesity as compared with controls, respectively [0.68 (0.53, 0.78) vs. 0.56 (0.47, 0.66) ng/mL, P = 0.0<em>2</em>0, and <em>2</em>.<em>2</em> (1.<em>2</em>1, 4.59) vs. <em>2</em>.0 (0.80, 5.90) ng/mL, P = 0.806)]. The endothelium-related change in MBF during CPT from rest (ΔMBF) progressively declined in overweight and obese when compared with control group [0.<em>2</em>1 (0.10, 0.<em>2</em>7) and 0.09 (-0.01, 0.15) vs. 0.<em>2</em>6 (0.<em>2</em>3, 0.39) mL/g/min; P = 0.010 and P = 0.0001, respectively). Compared with controls, hyperaemic MBFs were significantly lower in overweight and obese individuals [<em>2</em>.39 (1.97, <em>2</em>.6<em>2</em>) vs. 1.98 (1.69, <em>2</em>.<em>2</em>6) and <em>2</em>.10 (1.76, <em>2</em>.36); P = 0.007 and P = 0.04<em>2</em>, respectively)]. In obese individuals, AEA and <em>2</em>-AG plasma levels were inversely correlated with ΔMBF to CPT (r = -0.37, P = 0.046 and r = -0.48, P = 0.008) and hyperaemic MBFs (r = -0.38, P = 0.05<em>2</em> and r = -0.45, P = 0.017), respectively.

CONCLUSIONS

Increased EC plasma levels of AEA and <em>2</em>-AG are associated with coronary circulatory dysfunction in obese individuals. This observation might suggest increases in EC plasma levels as a novel endogenous cardiovascular risk factor in obesity, but needing further investigations.

The opioid and cannabinoid systems co-operate to regulate physiological processes such as nociception and reward. The endocannabinoid system may be a component of the brain reward circuitry and thus play a role not only in cannabinoid tolerance/dependence, but also in dependence/withdrawal for other misused drugs. We provide evidence of a cannabinoid mechanism in an animal model of morphine drug-seeking behaviour, referred to as behavioural sensitization. The present study was designed to test the effects of the CB1 cannabinoid receptor antagonist SR141716A in two different phases of morphine sensitization (induction and expression) and to measure the brain contents of arachidonoylethanolamide (anandamide, AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), the two main endogenous ligands for cannabinoid receptors in the different phases of morphine sensitization. The cannabinoid antagonist modified the signs of morphine sensitization when administered in the expression phase, whereas co-administration of SR141716A and morphine in the induction phase only slightly affected the behavioural responses, suggesting that CB1 receptor blockade attenuates the behavioural manifestations of morphine sensitization but not its development. AEA and <em>2</em>-AG were affected differently by morphine during the two phases of behavioural sensitization. The alterations were in opposite directions and specific for the cerebral area analysed (caudate putamen, nucleus accumbens, hippocampus and prefrontal cortex). The results suggest that the endocannabinoid system undergoes profound changes during the different phases of sensitization to morphine in rats, providing a possible neurochemical basis for the previously observed cross-sensitization between opiates and cannabinoids.

Anandamide (ANA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), two endogenous cannabinoids, can be generated by activated macrophages and platelets, respectively, in the context of endotoxic shock, and are proposed to play a crucial role in the induction of the shock-related hypotension. Taking advantage of our recently discovered function of polymyxin B (PMB) binding to ANA and <em>2</em>-AG, we developed a new method for measuring ANA and <em>2</em>-AG by applying PMB-immobilized beads to selectively adsorb them in biological fluids, instead of organic solvent extraction. The eluate from beads can be directly fractionated by reverse-phase high-performance liquid chromatography (HPLC), and the fractionations corresponding to authentic ANA and <em>2</em>-AG are collected and derivatized with fluorogenic reagent and subsequently quantified by HPLC with fluorometric detection. The calibration graphs of ANA and <em>2</em>-AG were linear over a range of 1 to 500 pmol/ml. The limits of detection for ANA and <em>2</em>-AG were <em>2</em>0 and 50 fmol, respectively. Intraassay precision was <em>2</em>.<em>2</em>4-4.<em>2</em>5 and 3.47-5.44%, and interassay was 4.05-6.14 and 4.9<em>2</em>-7.<em>2</em>8% for ANA and <em>2</em>-AG, respectively. Using this method, we first determined a 4-fold and 3-fold higher level of ANA and <em>2</em>-AG, respectively, in the sera of patients with endotoxic shock than in normal serum. This finding should help in elucidating the role of the endogenous cannabinoids in the hypotension of human endotoxic shock. This method is rapid, sensitive, and reliable for simultaneously quantifying ANA and <em>2</em>-AG in biological fluids, and has potential for clinical usage.

The possible involvement of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), an endogenous ligand for the cannabinoid receptors (CB1 and CB<em>2</em>), in contact dermatitis in mouse ear was investigated. We found that the level of <em>2</em>-AG was markedly elevated in the ear following a challenge with oxazolone in sensitized mice. Of note, the swelling following the challenge was suppressed by either the administration of SR1445<em>2</em>8, a CB<em>2</em> receptor antagonist, immediately after sensitization, or the administration of SR1445<em>2</em>8 upon the challenge. The effect of AM<em>2</em>51, a CB1 receptor antagonist, was marginal in either case. It seems apparent, therefore, that the CB<em>2</em> receptor and its endogenous ligand <em>2</em>-AG are closely involved in both the sensitization phase and the elicitation phase of oxazolone-induced contact dermatitis. In line with this, we found that Langerhans cells (MHC class II(+)) contain a substantial amount of CB<em>2</em> receptor mRNA, whereas keratinocytes (MHC class II(-)) do not. We also obtained evidence that the expression of mRNAs for proinflammatory cytokines following a challenge with oxazolone was markedly suppressed by treatment with SR1445<em>2</em>8. We next examined whether the CB<em>2</em> receptor and <em>2</em>-AG participate in chronic contact dermatitis accompanied by the infiltration of tissues by eosinophils. The amount of <em>2</em>-AG in mouse ear dramatically increased following repeated challenge with oxazolone. Importantly, treatment with SR1445<em>2</em>8 attenuated both the recruitment of eosinophils and ear swelling in chronic contact dermatitis induced by repeated challenge with oxazolone. These results strongly suggest that the CB<em>2</em> receptor and <em>2</em>-AG play important stimulative roles in the sensitization, elicitation, and exacerbation of allergic inflammation.

N-Arachidonoylethanolamine (anandamide; AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), the two proposed endogenous agonists of cannabinoid receptors, and the putative AEA biosynthetic precursor, N-arachidonoylphosphatidylethanolamine (NArPE), were identified in bovine retina by means of gas chromatography-electron impact mass spectrometry (GC-EIMS). This technique also allowed us to identify N-docosahexanoylethanolamine (DHEA) and <em>2</em>-docosahexanoylglycerol (<em>2</em>-DHG), two derivatives of docosahexaenoic acid (DHA), one of the most abundant fatty acids esterified in retina phospholipids and necessary for optimal retinal function. N-Docosahexaenoylphosphatidylethanolamine (NDHPE), the potential biosynthetic precursor for DHEA, was also found. The fatty acid composition of the sn-1 and sn-<em>2</em> positions of bovine retina's most abundant phospholipid classes, also determined here, were in agreement with a phospholipid-dependent mechanism for <em>2</em>-AG, <em>2</em>-DHG, AEA, and DHEA biosynthesis, as very high levels of polyunsaturated fatty acids, including DHA, were found on the sn-<em>2</em> position of phosphatidylcholine (PC) and -ethanolamine (PE), and measurable amounts of di-docosahexanoyl-PC and -PE, two potential biosynthetic precursors of NDHPE, were detected. Accordingly, we found that isolated particulate fractions from bovine retina could release AEA and DHEA in a time-dependent fashion. Finally, a fatty acid amide hydrolase (FAAH)-like activity with subcellular distribution and pH dependency similar to those reported for the brain enzyme was also detected in bovine retina. This activity was inhibited by FAAH inhibitors, phenylmethylsulfonyl fluoride and arachidonoyltrifluoromethylketone, and appeared to recognize DHEA with a lower efficiency than AEA. These data indicate that AEA and its congeners may play a physiological role in the mammalian eye.

Cannabinoid CB(1) receptors are involved in ocular physiology and may regulate intraocular pressure (IOP). However, endocannabinoid levels in human ocular tissues of cornea, iris, ciliary body, retina, and choroid from normal and glaucomatous donors have not been investigated. Anandamide (N-arachidonoylethanolamine; AEA), <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), and the anandamide congener, palmitoylethanolamide (PEA), were detected in all the human tissues examined. In eyes from patients with glaucoma, significantly decreased <em>2</em>-AG and PEA levels were detected in the ciliary body, an important tissue in the regulation of IOP. The findings suggest that these endogenous compounds may have a role in this disease, particularly with respect to regulation of IOP.

We have previously shown that krill oil (KO), more efficiently than fish oil, was able to downregulate the endocannabinoid system in different tissues of obese zucker rats.We therefore aimed at investigating whether an intake of <em>2</em> g/d of either KO or menhaden oil (MO), which provides 309 mg/d of EPA/DHA <em>2</em>:1 and 390 mg/d of EPA/DHA 1:1 respectively, or olive oil (OO) for four weeks, is able to modify plasma endocannabinoids in overweight and obese subjects.The results confirmed data in the literature describing increased levels of endocannabinoids in overweight and obese with respect to normo-weight subjects. KO, but not MO or OO, was able to significantly decrease <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), although only in obese subjects. In addition, the decrease of <em>2</em>-AG was correlated to the plasma n-6/n-3 phospholipid long chain polyunsaturated fatty acid (LCPUFA) ratio. These data show for the first time in humans that relatively low doses of LCPUFA n-3 as KO can significantly decrease plasma <em>2</em>-AG levels in obese subjects in relation to decrease of plasma phospholipid n-6/n-3 LCPUFA ratio. This effect is not linked to changes of metabolic syndrome parameters but is most likely due to a decrease of <em>2</em>-AG biosynthesis caused by the replacement of <em>2</em>-AG ultimate precursor, arachidonic acid, with n-3 PUFAs, as previously described in obese Zucker rats.

The endocannabinoid system modulates neurotransmission at inhibitory and excitatory synapses in brain regions relevant to the regulation of pain, emotion, motivation, and cognition. This signaling system is engaged by the active component of cannabis, Delta9-tetrahydrocannabinol (Delta9-THC), which exerts its pharmacological effects by activation of G protein-coupled type-1 (CB1) and type-<em>2</em> (CB<em>2</em>) cannabinoid receptors. During frequent cannabis use a series of poorly understood neuroplastic changes occur, which lead to the development of dependence. Abstinence in cannabinoid-dependent individuals elicits withdrawal symptoms that promote relapse into drug use, suggesting that pharmacological strategies aimed at alleviating cannabis withdrawal might prevent relapse and reduce dependence. Cannabinoid replacement therapy and CB1 receptor antagonism are two potential treatments for cannabis dependence that are currently under investigation. However, abuse liability and adverse side-effects may limit the scope of each of these approaches. A potential alternative stems from the recognition that (i) frequent cannabis use may cause an adaptive down-regulation of brain endocannabinoid signaling, and (ii) that genetic traits that favor hyperactivity of the endocannabinoid system in humans may decrease susceptibility to cannabis dependence. These findings suggest in turn that pharmacological agents that elevate brain levels of the endocannabinoid neurotransmitters, anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), might alleviate cannabis withdrawal and dependence. One such agent, the fatty-acid amide hydrolase (FAAH) inhibitor URB597, selectively increases anandamide levels in the brain of rodents and primates. Preclinical studies show that URB597 produces analgesic, anxiolytic-like and antidepressant-like effects in rodents, which are not accompanied by overt signs of abuse liability. In this article, we review evidence suggesting that (i) cannabis influences brain endocannabinoid signaling and (ii) FAAH inhibitors such as URB597 might offer a possible therapeutic avenue for the treatment of cannabis withdrawal.

The goal of this review is to summarize studies in which concentrations of circulating endocannabinoids in humans have been examined in relationship to physiological measurements and pathological status. The roles of endocannabinoids in the regulation of energy intake and storage have been well studied and the data obtained consistently support the hypothesis that endocannabinoid signaling is associated with increased consumption and storage of energy. Physical exercise mobilizes endocannabinoids, which could contribute to refilling of energy stores and also to the analgesic and mood-elevating effects of exercise. Circulating concentrations of <em>2</em>-<em>arachidonoylglycerol</em> are very significantly circadian and dysregulated when sleep is disrupted. Other conditions under which circulating endocannabinoids are altered include inflammation and pain. A second important role for endocannabinoid signaling is to restore homeostasis following stress. Circulating endocannabinoids are stress-responsive and there is evidence that their concentrations are altered in disorders associated with excessive stress, including post-traumatic stress disorder. Although determination of circulating endocannabinoids can provide important information about the state of endocannabinoid signaling and thus allow for hypotheses to be defined and tested, the large number of physiological factors that contribute to their circulating concentrations makes it difficult to use them in isolation as a biomarker for a specific disorder.

Endocannabinoids like <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) exert neuroprotective effects after brain injuries. According to current concepts, these neuroprotective effects are due to interactions between <em>2</em>-AG and cannabinoid (CB)1 receptors on neurons. Moreover, <em>2</em>-AG modulates migration and proliferation of microglial cells which are rapidly activated after brain lesion. This effect is mediated via CB<em>2</em>- and abnormal-cannabidiol (abn-CBD)-sensitive receptors. In the present study, we investigated whether the abn-CBD-sensitive receptor on microglial cells contributes to <em>2</em>-AG-mediated neuroprotection in organotypic hippocampal slice cultures (OHSCs) after excitotoxic lesion induced by NMDA (50 microM) application for 4 h. This lesion caused neuronal damage and accumulation of microglial cells within the granule cell layer. To analyze the role of abn-CBD-sensitive receptors for neuroprotection and microglial cell accumulation, two agonists of the abn-CBD-sensitive receptor, abn-CBD or <em>2</em>-AG, two antagonists, 1,3-dimethoxy-5-methyl-<em>2</em>-[(1R,6R)-3-methyl-6-(1-methylethenyl)-<em>2</em>-cyclohexen1-yl]-benzene (O-1918) or cannabidiol (CBD), and the CB1 receptor antagonist AM<em>2</em>51, were applied to NMDA-lesioned OHSC. Propidium iodide (PI) labeling was used as a marker of degenerating neurons and isolectin B(4) (IB(4)) as a marker of microglial cells. Application of both, abn-CBD or <em>2</em>-AG to lesioned OHSC significantly decreased the number of IB(4)(+) microglial cells and PI(+) neurons in the dentate gyrus. In contrast to AM<em>2</em>51, application of O-1918 or CBD antagonized these effects. When microglial cells were depleted by preincubation of OHSC with the bisphosphonate clodronate (100 microg/mL) for 5 days before excitotoxic lesion, <em>2</em>-AG and abn-CBD lost their neuroprotective effects. We therefore propose that the endocannabinoid <em>2</em>-AG exerts its neuroprotective effects via activation of abn-CBD-sensitive receptors on microglial cells.

The molecular species compositions of monoacylglycerols obtained from various rat tissues were examined by reverse-phase high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) analyses. We confirmed that <em>2</em>-<em>arachidonoylglycerol</em>, an endogenous cannabinoid receptor agonist, is one of the most abundant molecular species of monoacylglycerols in the brain. Substantial amounts of <em>2</em>-<em>arachidonoylglycerol</em> were also found in the liver, spleen, lung and kidney, but the levels were considerably lower than that in the brain. We found that a small amount of <em>2</em>-<em>arachidonoylglycerol</em> was generated in a brain homogenate during incubation in the absence of Ca<em>2</em>+. Importantly, the generation of <em>2</em>-<em>arachidonoylglycerol</em> was markedly augmented in the presence of Ca<em>2</em>+, suggesting that Ca<em>2</em>+ plays a key role in regulation of the generation of <em>2</em>-<em>arachidonoylglycerol</em> in this tissue.

A functional endocannabinoid system is present in several mammalian organs and tissues. Recently, endocannabinoids and their receptors have been reported in the skeleton. Osteoblasts, the bone forming cells, and osteoclasts, the bone resorbing cells, produce the endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em> and express CB<em>2</em> cannabinoid receptors. Although CB<em>2</em> has been implicated in pathological processes in the central nervous system and peripheral tissues, the skeleton appears as the main system physiologically regulated by CB<em>2</em>. CB<em>2</em>-deficient mice show a markedly accelerated age-related bone loss and the CNR<em>2</em> gene (encoding CB<em>2</em>) in women is associated with low bone mineral density. The activation of CB<em>2</em> attenuates ovariectomy-induced bone loss in mice by restraining bone resorption and enhancing bone formation. Hence synthetic CB<em>2</em> ligands, which are stable and orally available, provide a basis for developing novel anti-osteoporotic therapies. Activation of CB1 in sympathetic nerve terminals in bone inhibits norepinephrine release, thus balancing the tonic sympathetic restrain of bone formation. Low levels of CB1 were also reported in osteoclasts. CB1-null mice display a skeletal phenotype that is dependent on the mouse strain, gender and specific mutation of the CB1 encoding gene, CNR1.

We used a model of neuropathic pain consisting of rats with chronic constriction injury (CCI) of the sciatic nerve, in order to investigate whether endocannabinoid levels are altered in the dorsal raphe (DR) and to assess the effect of repeated treatment with (R)-(+)-[<em>2</em>,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,<em>2</em>,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate, a synthetic cannabinoid agonist, or N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (AM404), an inhibitor of endocannabinoid reuptake, on DR serotonergic neuronal activity and on behavioural hyperalgesia. CCI resulted in significantly elevated anandamide but not <em>2</em>-<em>arachidonoylglycerol</em> levels in the DR. Furthermore, as well as thermal and mechanical hyperalgesia, CCI caused serotonergic hyperactivity (as shown by the increase of basal activity of serotonergic neurones, extracellular serotonin levels and expression of 5-HT1A receptor gene). Repeated treatment with either (R)-(+)-[<em>2</em>,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,<em>2</em>,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate or AM404 reverted the hyperalgesia and enhanced serotonergic activity induced by CCI in a way attenuated by N-piperidino-5-(4-chlorophenyl)-1-(<em>2</em>,4dichlorophenyl)-4-methyl-3-pyrazolecarboxamide, a selective cannabinoid subtype 1 (CB1) receptor antagonist. Despite the elevated levels of anandamide following CCI, N-piperidino-5-(4-chlorophenyl)-1-(<em>2</em>,4dichlorophenyl)-4-methyl-3-pyrazolecarboxamide did not produce hyperalgesia or any other effect on serotonergic neuronal activity when administered alone. Furthermore, the effects of AM404 were not accompanied by an increase in endocannabinoid levels in the DR. In conclusion, following CCI of the sciatic nerve, the endocannabinoid and serotonergic systems are activated in the DR, where repeated stimulation of CB1 receptors with exogenous compounds restores DR serotonergic activity, as well as thermal and mechanical nociceptive thresholds, to pre-surgery levels. However, an elevated level of endogenous anandamide in the DR does not necessarily contribute to the CB1-mediated tonic control of analgesia and serotonergic neuronal activity.

<em>2</em>-<em>Arachidonoylglycerol</em> is an endogenous ligand for the cannabinoid receptors (CB1 and CB<em>2</em>) and has been shown to exhibit a variety of cannabimimetic activities in vitro and in vivo. Recently, we proposed that <em>2</em>-<em>arachidonoylglycerol</em> is the true endogenous ligand for the cannabinoid receptors, and both receptors (CB1 and CB<em>2</em>) are primarily <em>2</em>-<em>arachidonoylglycerol</em> receptors. The CB1 receptor is assumed to be involved in the attenuation of neurotransmission. On the other hand, the physiological roles of the CB<em>2</em> receptor, which is abundantly expressed in several types of leukocytes such as macrophages, still remain unknown. In this study, we examined the effects of <em>2</em>-<em>arachidonoylglycerol</em> on the motility of HL-60 cells differentiated into macrophage-like cells. We found that <em>2</em>-<em>arachidonoylglycerol</em> induces the migration of differentiated HL-60 cells. The migration induced by <em>2</em>-<em>arachidonoylglycerol</em> was blocked by treatment of the cells with either SR1445<em>2</em>8, a CB<em>2</em> receptor antagonist, or pertussis toxin, suggesting that the CB<em>2</em> receptor and Gi/Go are involved in the <em>2</em>-<em>arachidonoylglycerol</em>-induced migration. Several intracellular signaling molecules such as Rho kinase and mitogen-activated protein kinases were also suggested to be involved. In contrast to <em>2</em>-<em>arachidonoylglycerol</em>, anandamide, another endogenous cannabinoid receptor ligand, failed to induce the migration. The <em>2</em>-<em>arachidonoylglycerol</em>-induced migration was also observed for two other types of macrophage-like cells, the U937 cells and THP-1 cells, as well as human peripheral blood monocytes. These results strongly suggest that <em>2</em>-<em>arachidonoylglycerol</em> induces the migration of several types of leukocytes such as macrophages/monocytes through a CB<em>2</em> receptor-dependent mechanism thereby stimulating inflammatory reactions and immune responses.

Endocannabinoids are endogenous agonists of cannabinoid receptors, and comprise amides, esters and ethers of long chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine) and <em>2</em>-<em>arachidonoylglycerol</em> are the best-studied members of this class of lipid mediators, and it is now widely accepted that their in vivo concentration and biological activity are largely dependent on a "metabolic control." Therefore, the proteins that synthesize, transport and degrade endocannabinoids, and that together with the target receptors form the so-called "endocannabinoid system," are the focus of intense research. This new system will be presented in this review, in order to put in a better perspective the impact of its modulation on Huntington's disease. In particular, the effect of agonists/antagonists of endocannabinoid receptors, or of inhibitors of endocannabinoid metabolism, will be discussed in the context of onset and progression of Huntington's disease, and will be compared with other neurodegenerative diseases like Parkinson's disease, Alzheimer's disease, and amyotropic lateral sclerosis. Also the plastic changes of endocannabinoids in multiple sclerosis will be reviewed, as a paradigm of their impact in neuroinflammatory disorders.

The effects of delta9-tetrahydrocannabinol and <em>2</em>-<em>arachidonoylglycerol</em> on the intracellular free Ca<em>2</em>+ concentration ([Ca<em>2</em>+]i) in NG108-15 cells were examined in detail. We found that delta9-tetrahydrocannabinol induces a rapid, modest increase in [Ca<em>2</em>+]i. The response was detectable with 3 nM delta9-tetrahydrocannabinol. We also found that very low concentrations of <em>2</em>-<em>arachidonoylglycerol</em> elicit a rapid, more prominent increase in [Ca<em>2</em>+]i. Such a response was observed not only in NG108-15 cells but also in N18TG<em>2</em> cells. The response induced by <em>2</em>-<em>arachidonoylglycerol</em> in either NG108-15 cells or N18TG<em>2</em> cells was abolished by pretreatment of the cells with a cannabinoid CB1 receptor specific antagonist, SR141716A, suggesting that <em>2</em>-<em>arachidonoylglycerol</em> interacts with the CB1 receptor to induce the response. The results of an experiment involving a phospholipase C inhibitor suggested that phospholipase C is involved in the rapid increase in [Ca<em>2</em>+]i induced by <em>2</em>-<em>arachidonoylglycerol</em>. We also found that 1(3)-<em>arachidonoylglycerol</em> exhibits similar activity to that of <em>2</em>-<em>arachidonoylglycerol</em>, although its activity at low concentrations was somewhat weak compared with that of <em>2</em>-<em>arachidonoylglycerol</em>. We further confirmed that several structural analogues of <em>2</em>-<em>arachidonoylglycerol</em> were less active compared with <em>2</em>-<em>arachidonoylglycerol</em>. These results suggest that the structure of <em>2</em>-<em>arachidonoylglycerol</em> is strictly recognized by the CB1 receptor, which raises the possibility that the CB1 receptor is originally a <em>2</em>-<em>arachidonoylglycerol</em> receptor.

The cannabinoid receptors for Δ(9)-THC, and particularly, the CB(1) receptor, as well as its endogenous ligands, the endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, are deeply involved in all aspects of the control of energy balance in mammals. While initially it was believed that this endocannabinoid signaling system would only facilitate energy intake, we now know that perhaps even more important functions of endocannabinoids and CB(1) receptors in this context are to enhance energy storage into the adipose tissue and reduce energy expenditure by influencing both lipid and glucose metabolism. Although normally well controlled by hormones and neuropeptides, both central and peripheral aspects of endocannabinoid regulation of energy balance can become dysregulated and contribute to obesity, dyslipidemia, and type <em>2</em> diabetes, thus raising the possibility that CB(1) antagonists might be used for the treatment of these metabolic disorders. On the other hand, evidence is emerging that some nonpsychotropic plant cannabinoids, such as cannabidiol, can be employed to retard β-cell damage in type 1 diabetes. These novel aspects of endocannabinoid research are reviewed in this chapter, with emphasis on the biological effects of plant cannabinoids and endocannabinoid receptor antagonists in diabetes.

In agreement with the highly lipophilic nature of (-)-delta9-tetrahydrocannabinol, all the endogenous ligands of cannabinoid receptors identified so far are derivatives of long chain fatty acids. N- Arachidonoylethanolamine (anandamide) and some of its polyunsaturated congeners have been found in mammalian brain and shown to activate the CB1 and, with a lower efficacy, CB<em>2</em> cannabinoid receptor subtypes. More recently, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), a widespread intermediate in the metabolism of phosphoglycerides, diacylglycerols and triglycerides, was also found to activate the cannabinoid receptors. The capability of palmitoylethanolamide, an anti-inflammatory metabolite, to activate CB<em>2</em>-like receptors is still being debated. Here we review: 1) the metabolic pathways suggested so far to underlie the biosynthesis and inactivation of anandamide and <em>2</em>-AG, and <em>2</em>) the current knowledge of the chemical bases for the interactions of anandamide and <em>2</em>-AG with proteins of the endogenous cannabinoid system characterized so far, i.e. the CB1 and CB<em>2</em> receptor subtypes, the membrane anandamide carrier , which facilitates anandamide diffusion into cells, and the enzyme fatty acid amide hydrolase , which catalyzes anandamide and, to a certain extent, <em>2</em>-AG hydrolysis in vivo.

The development of sensitive analytical methods for measurement of endocannabinoids, their metabolites, and related lipids, has underlined the complexity of the endocannabinoid system. A case can be made for an 'endocannabinoid soup' (akin to the inflammatory soup) whereby the net effect of a pathological state and/or a pharmacological intervention on this system is the result not only of changes in endocannabinoid levels but also of their metabolites and related compounds that affect their function. With respect to the metabolism of anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, the main hydrolytic enzymes involved are fatty acid amide hydrolase and monoacylglycerol lipase. However, other pathways can come into play when these are blocked. Cyclooxygenase-<em>2</em> derived metabolites of anandamide and <em>2</em>-<em>arachidonoylglycerol</em> have a number of properties, including effects upon cell viability, contraction of the cat iris sphincter (an effect mediated by a novel receptor), mobilization of calcium and modulation of synaptic transmission. Nonsteroidal anti-inflammatory agents, whose primary mode of action is the inhibition of cyclooxygenase, can also interact with the endocannabinoid system both in vitro and in vivo. Other enzymes, such as the lipoxygenase and cytochrome P450 oxidative enzymes, can also metabolize endocannabinoids and produce biologically active compounds. It is concluded that sensitive analytical methods, which allow for measurement of endocannabinoids and related lipids, should provide vital information as to the importance of these alternative metabolic pathways when the primary hydrolytic endocannabinoid metabolizing enzymes are inhibited.

In mouse neuroblastoma N18TG<em>2</em> cells prelabeled with [3H]arachidonic acid ([3H]AA) the biosynthesis of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is induced by ionomycin in a fashion sensitive to an inhibitor of diacylglycerol (DAG) lipase, RHC 80<em>2</em>67, but not to four different phospholipase C (PLC) blockers. Pulse experiments with [3H]AA showed that ionomycin stimulation leads to the sequential formation of [3H]phosphatidic acid ([3H]PA), [3H]DAG, and [3H]<em>2</em>-AG. [3H]<em>2</em>-AG biosynthesis in N18TG<em>2</em> cells prelabeled with [3H]AA was counteracted by propranolol and N-ethylmaleimide, two inhibitors of the Mg<em>2</em>+/Ca<em>2</em>(+)-dependent brain PA phosphohydrolase. Pretreatment of cells with exogenous phospholipase D (PLD) led to a strong potentiation of ionomycin-induced [3H]<em>2</em>-AG formation. These data indicate that DAG precursors for <em>2</em>-AG in intact N18TG<em>2</em> cells are obtained from the hydrolysis of PA and not through the activation of PLC. The presence of <em>2</em>% ethanol during ionomycin stimulation failed to elicit the synthesis of [3H]phosphatidylethanol and did not counteract the formation of [3H]PA, thus arguing against the activation of PLD by the Ca<em>2</em>+ ionophore. Selective inhibitors of secretory phospholipase A<em>2</em> and the acyl-CoA acylase inhibitor thimerosal significantly reduced [3H]<em>2</em>-AG biosynthesis. The implications of these latter findings, and of the PA-dependent pathways of <em>2</em>-AG formation described here, are discussed.

OBJECTIVE

The non-psychotropic cannabinoid cannabichromene is known to activate the transient receptor potential ankyrin-type1 (TRPA1) and to inhibit endocannabinoid inactivation, both of which are involved in inflammatory processes. We examined here the effects of this phytocannabinoid on peritoneal macrophages and its efficacy in an experimental model of colitis.

METHODS

Murine peritoneal macrophages were activated in vitro by LPS. Nitrite levels were measured using a fluorescent assay; inducible nitric oxide (iNOS), cyclooxygenase-<em>2</em> (COX-<em>2</em>) and cannabinoid (CB1 and CB<em>2</em> ) receptors were analysed by RT-PCR (and/or Western blot analysis); colitis was induced by dinitrobenzene sulphonic acid (DNBS). Endocannabinoid (anandamide and <em>2</em>-<em>arachidonoylglycerol</em>), palmitoylethanolamide and oleoylethanolamide levels were measured by liquid chromatography-mass spectrometry. Colonic inflammation was assessed by evaluating the myeloperoxidase activity as well as by histology and immunohistochemistry.

RESULTS

LPS caused a significant production of nitrites, associated to up-regulation of anandamide, iNOS, COX-<em>2</em>, CB1 receptors and down-regulation of CB<em>2</em> receptors mRNA expression. Cannabichromene significantly reduced LPS-stimulated nitrite levels, and its effect was mimicked by cannabinoid receptor and TRPA1 agonists (carvacrol and cinnamaldehyde) and enhanced by CB1 receptor antagonists. LPS-induced anandamide, iNOS, COX-<em>2</em> and cannabinoid receptor changes were not significantly modified by cannabichromene, which, however, increased oleoylethanolamide levels. In vivo, cannabichromene ameliorated DNBS-induced colonic inflammation, as revealed by histology, immunohistochemistry and myeloperoxidase activity.

CONCLUSIONS

Cannabichromene exerts anti-inflammatory actions in activated macrophages - with tonic CB1 cannabinoid signalling being negatively coupled to this effect - and ameliorates experimental murine colitis.

Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate generates diacylglycerol, inositol 1,4,5-trisphosphate and protons, all of which can regulate TRPV1 activity via different mechanisms. Here we explored the possibility that the diacylglycerol metabolites <em>2</em>-<em>arachidonoylglycerol</em> and 1-<em>arachidonoylglycerol</em>, and not metabolites of these monoacylglycerols, activate TRPV1 and contribute to this signaling cascade. <em>2</em>-<em>Arachidonoylglycerol</em> and 1-<em>arachidonoylglycerol</em> activated native TRPV1 on vascular sensory nerve fibers and heterologously expressed TRPV1 in whole cells and inside-out membrane patches. The monoacylglycerol lipase inhibitors methylarachidonoyl-fluorophosphonate and JZL184 prevented the metabolism of deuterium-labeled <em>2</em>-<em>arachidonoylglycerol</em> and deuterium-labeled 1-<em>arachidonoylglycerol</em> in arterial homogenates, and enhanced TRPV1-mediated vasodilator responses to both monoacylglycerols. In mesenteric arteries from TRPV1 knock-out mice, vasodilator responses to <em>2</em>-<em>arachidonoylglycerol</em> were minor. Bradykinin and adenosine triphosphate, ligands of phospholipase C-coupled membrane receptors, increased the content of <em>2</em>-<em>arachidonoylglycerol</em> in dorsal root ganglia. In HEK<em>2</em>93 cells expressing the phospholipase C-coupled histamine H1 receptor, exposure to histamine stimulated the formation of <em>2</em>-AG, and this effect was augmented in the presence of JZL184. These effects were prevented by the diacylglycerol lipase inhibitor tetrahydrolipstatin. Histamine induced large whole cell currents in HEK<em>2</em>93 cells co-expressing TRPV1 and the histamine H1 receptor, and the TRPV1 antagonist capsazepine abolished these currents. JZL184 increased the histamine-induced currents and tetrahydrolipstatin prevented this effect. The calcineurin inhibitor ciclosporin and the endogenous "entourage" compound palmitoylethanolamide potentiated the vasodilator response to <em>2</em>-<em>arachidonoylglycerol</em>, disclosing TRPV1 activation of this monoacylglycerol at nanomolar concentrations. Furthermore, intracerebroventricular injection of JZL184 produced TRPV1-dependent antinociception in the mouse formalin test. Our results show that intact <em>2</em>-<em>arachidonoylglycerol</em> and 1-<em>arachidonoylglycerol</em> are endogenous TRPV1 activators, contributing to phospholipase C-dependent TRPV1 channel activation and TRPV1-mediated antinociceptive signaling in the brain.

In mammals, including humans, bone metabolism is manifested as an ongoing modelling/remodelling process whereby the bone mineralised matrix is being continuously renewed. Recently, the main components of the endocannabinoid system have been reported in the skeleton. Osteoblasts, the bone forming cells, and other cells of the osteoblastic lineage, as well as osteoclasts, the bone resorbing cells, and their precursors, synthesise the endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). CB(1) cannabinoid receptors are present in sympathetic nerve terminals in close proximity to osteoblasts. Activation of these CB(1) receptors by elevated bone <em>2</em>-AG levels communicates brain-to-bone signals as exemplified by traumatic brain injury-induced stimulation of bone formation. In this process, the retrograde CB(1) signalling inhibits noradrenaline release and alleviates the tonic sympathetic restrain of bone formation. CB(<em>2</em>) receptors are expressed by osteoblasts and osteoclasts. Their activation stimulates bone formation and suppresses bone resorption. CB(<em>2</em>)-deficient mice display a markedly accelerated age-related bone loss. Ovariectomy-induced bone loss can be both prevented and rescued by a CB(<em>2</em>) specific agonist. Hence, synthetic CB(<em>2</em>) ligands, which are stable and orally available, provide a basis for developing novel anti-osteoporotic therapies, free of psychotropic effects. The CNR<em>2</em> gene (encoding CB(<em>2</em>)) in women is associated with low bone mineral density, offering an assay for identifying females at risk of developing osteoporosis.