A major finding--that (-)-trans-Delta(9)-tetrahydrocannabinol (Delta(9)-THC) is largely responsible for the psychotropic effects of cannabis--prompted research in the 1970s and 1980s that led to the discovery that this plant cannabinoid acts through at least two types of cannabinoid receptor, CB(1) and CB(<em>2</em>), and that Delta(9)-THC and other compounds that target either or both of these receptors as agonists or antagonists have important therapeutic applications. It also led to the discovery that mammalian tissues can themselves synthesize and release agonists for cannabinoid receptors, the first of these to be discovered being arachidonoylethanolamide (anandamide) and <em>2</em>-<em>arachidonoylglycerol</em>. These 'endocannabinoids' are released onto their receptors in a manner that appears to maintain homeostasis within the central nervous system and sometimes either to oppose or to mediate or exacerbate the unwanted effects of certain disorders. This review provides an overview of the pharmacology of cannabinoid receptors and their ligands. It also describes actual and potential clinical uses both for cannabinoid receptor agonists and antagonists and for compounds that affect the activation of cannabinoid receptors less directly, for example by inhibiting the enzymatic hydrolysis of endocannabinoids following their release.

BACKGROUND

CB(1) cannabinoid receptors in the brain are known to participate in the regulation of reward-based behaviors. However, the contribution of each of the endocannabinoid transmitters, anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), to these behaviors remains undefined. To address this question, we assessed the effects of URB597, a selective anandamide deactivation inhibitor, as a reinforcer of drug-seeking and drug-taking behavior in squirrel monkeys.

METHODS

We investigated the reinforcing effects of the fatty acid amide hydrolase (FAAH) inhibitor URB597 in monkeys trained to intravenously self-administer Delta(9)-tetrahydrocannabinol (THC), anandamide, or cocaine and quantified brain endocannabinoid levels using liquid chromatography/mass spectrometry. We measured brain FAAH activity using an ex vivo enzyme assay.

RESULTS

URB597 (.3 mg/kg, intravenous) blocked FAAH activity and increased anandamide levels throughout the monkey brain. This effect was accompanied by a marked compensatory decrease in <em>2</em>-AG levels. Monkeys did not self-administer URB597, and the drug did not promote reinstatement of extinguished drug-seeking behavior previously maintained by THC, anandamide, or cocaine. Pretreatment with URB597 did not modify self-administration of THC or cocaine, even though, as expected, it significantly potentiated anandamide self-administration.

CONCLUSIONS

In the monkey brain, the FAAH inhibitor URB597 increases anandamide levels while causing a compensatory down-regulation in <em>2</em>-AG levels. These effects are accompanied by a striking lack of reinforcing properties, which distinguishes URB597 from direct-acting cannabinoid agonists such as THC. Our results reveal an unexpected functional heterogeneity within the endocannabinoid signaling system and suggest that FAAH inhibitors might be used therapeutically without risk of abuse or triggering of relapse to drug abuse.

The endocannabinoid anandamide (AEA) is shown to induce apoptotic bodies formation and DNA fragmentation, hallmarks of programmed cell death, in human neuroblastoma CHP100 and lymphoma U937 cells. RNA and protein synthesis inhibitors like actinomycin D and cycloheximide reduced to one-fifth the number of apoptotic bodies induced by AEA, whereas the AEA transporter inhibitor AM404 or the AEA hydrolase inhibitor ATFMK significantly increased the number of dying cells. Furthermore, specific antagonists of cannabinoid or vanilloid receptors potentiated or inhibited cell death induced by AEA, respectively. Other endocannabinoids such as <em>2</em>-<em>arachidonoylglycerol</em>, linoleoylethanolamide, oleoylethanolamide, and palmitoylethanolamide did not promote cell death under the same experimental conditions. The formation of apoptotic bodies induced by AEA was paralleled by increases in intracellular calcium (3-fold over the controls), mitochondrial uncoupling (6-fold), and cytochrome c release (3-fold). The intracellular calcium chelator EGTA-AM reduced the number of apoptotic bodies to 40% of the controls, and electrotransferred anti-cytochrome c monoclonal antibodies fully prevented apoptosis induced by AEA. Moreover, 5-lipoxygenase inhibitors 5,8,11,14-eicosatetraynoic acid and MK886, cyclooxygenase inhibitor indomethacin, caspase-3 and caspase-9 inhibitors Z-DEVD-FMK and Z-LEHD-FMK, but not nitric oxide synthase inhibitor Nomega-nitro-l-arginine methyl ester, significantly reduced the cell death-inducing effect of AEA. The data presented indicate a protective role of cannabinoid receptors against apoptosis induced by AEA via vanilloid receptors.

Cannabinoids have long been shown to have a range of potential therapeutic effects, including antiemetic actions, analgesia, and anxiolysis. However, psychomimetic and memory disruptive side effects, as well as the potential for abuse and dependence, have restricted their clinical development. Endogenous cannabinoids (i.e., endocannabinoids; eCBs), such as anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), are produced throughout the limbic system and other brain regions associated with emotionality and are believed to modulate behavioral responses to stress-related conditions. AEA and <em>2</em>-AG are rapidly metabolized by the respective enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). Accordingly, inhibition of each enzyme increases brain levels of the appropriate eCB. Although FAAH inhibition has been established to decrease anxiety-like behavior, the role of <em>2</em>-AG has been difficult to ascertain until the recent synthesis of JZL184, a potent and selective MAGL inhibitor. In the present study, we investigated the effects of inhibiting FAAH or MAGL on anxiety-like behavior in marble burying, a model of repetitive, compulsive behaviors germane to anxiety disorders such as obsessive-compulsive disorder. The FAAH inhibitor PF-3845, the MAGL inhibitor JZL184, and the benzodiazepine diazepam decreased marble burying at doses that did not affect locomotor activity. In contrast, Δ9-tetrahydrocannabinol (THC), the primary psychoactive constituent of marijuana, did not consistently reduce marble burying without also eliciting profound decreases in locomotor behavior. The CB1 cannabinoid receptor antagonist rimonabant blocked the reduction in marble burying caused by FAAH and MAGL inhibitors, but not by diazepam, indicating a CB1 receptor mechanism of action. These data indicate that elevation of AEA or <em>2</em>-AG reduces marble burying behavior and suggest that their catabolic enzymes represent potential targets for the development of new classes of pharmacotherapeutics to treat anxiety-related disorders.

Direct stimulation of cannabinoid CB1 receptors exerts a protective function in animal models of inflammatory bowel diseases (IBDs). However, it is not known whether endocannabinoids are up-regulated during IBDs in animals or humans, nor whether pharmacological elevation of endocannabinoid levels can be exploited therapeutically in these disorders. In this study we addressed these questions. Colon inflammation was induced in mice and rats with <em>2</em>,4-dinitrobenzene- and <em>2</em>,4,6-trinitrobenzene sulfonic acids (DNBS and TNBS), respectively. DNBS-treated mice were treated chronically (for 3 or 7 days) with inhibitors of anandamide enzymatic hydrolysis (N-arachidonoyl-serotonin, AA-5-HT) or reuptake (VDM11), 10 or 5 mg/kg, s.c., or with 5-amino-salicilic acid (5-ASA, 1.4 mg/kg, i.r.). Endocannabinoids (anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, <em>2</em>-AG) were quantified in mouse colon, or in rat colon mucosa and submucosa, and in bioptic samples from the colon of patients with untreated ulcerative colitis, by liquid chromatography-mass spectrometry. A strong elevation of anandamide, but not <em>2</em>-AG, levels was found in the colon of DNBS-treated mice, in the colon submucosa of TNBS-treated rats, and in the biopsies of patients with ulcerative colitis. VDM-11 significantly elevated anandamide levels in the colon of DNBS-treated mice and concomitantly abolished inflammation, whereas AA-5-HT did not affect endocannabinoid levels and was significantly less efficacious at attenuating colitis. 5-ASA also increased anandamide levels and abolished colitis. Thus, anandamide is elevated in the inflamed colon of patients with ulcerative colitis, as well as in animal models of IBDs, to control inflammation, and elevation of its levels with inhibitors of its cellular reuptake might be used in the treatment of IBDs.

The endocannabinoid system is implicated in a variety of physiological and pathological conditions (inflammation, immunomodulation, analgesia, cancer and others). The main active ingredient of cannabis, Δ(9) -tetrahydrocannabinol (Δ(9) -THC), produces its effects through activation of CB(1) and CB(<em>2</em>) receptors. CB(1) receptors are expressed at high levels in the central nervous system (CNS), whereas CB(<em>2</em>) receptors are concentrated predominantly, although not exclusively, in cells of the immune system. Endocannabinoids are endogenous lipid-signalling molecules that are generated in the cell membrane from phospholipid precursors. The two best characterized endocannabinoids identified to date are anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Here we review the relationship between the endocannabinoid system and anti-tumour actions (inhibition of cell proliferation and migration, induction of apoptosis, reduction of tumour growth) of the cannabinoids in different types of cancer. This review will focus on examining how activation of the endocannabinoid system impacts breast, prostate and bone cancers in both in vitro and in vivo systems. The therapeutic potential of cannabinoids for cancer, as identified in clinical trials, is also discussed. Identification of safe and effective treatments to manage and improve cancer therapy is critical to improve quality of life and reduce unnecessary suffering in cancer patients. In this regard, cannabis-like compounds offer therapeutic potential for the treatment of breast, prostate and bone cancer in patients. Further basic research on anti-cancer properties of cannabinoids as well as clinical trials of cannabinoid therapeutic efficacy in breast, prostate and bone cancer is therefore warranted.

Suppressing hyperactive endocannabinoid tone is a critical target for reducing obesity. The backbone of both endocannabinoids <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and anandamide (AEA) is the ω-6 fatty acid arachidonic acid (AA). Here we posited that excessive dietary intake of linoleic acid (LA), the precursor of AA, would induce endocannabinoid hyperactivity and promote obesity. LA was isolated as an independent variable to reflect the dietary increase in LA from 1 percent of energy (en%) to 8 en% occurring in the United States during the <em>2</em>0th century. Mice were fed diets containing 1 en% LA, 8 en% LA, and 8 en% LA + 1 en% eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) in medium-fat diets (35 en% fat) and high-fat diets (60 en%) for 14 weeks from weaning. Increasing LA from 1 en% to 8 en% elevated AA-phospholipids (PL) in liver and erythrocytes, tripled <em>2</em>-AG + 1-AG and AEA associated with increased food intake, feed efficiency, and adiposity in mice. Reducing AA-PL by adding 1 en% long-chain ω-3 fats to 8 en% LA diets resulted in metabolic patterns resembling 1 en% LA diets. Selectively reducing LA to 1 en% reversed the obesogenic properties of a 60 en% fat diet. These animal diets modeled <em>2</em>0th century increases of human LA consumption, changes that closely correlate with increasing prevalence rates of obesity. In summary, dietary LA increased tissue AA, and subsequently elevated <em>2</em>-AG + 1-AG and AEA resulting in the development of diet-induced obesity. The adipogenic effect of LA can be prevented by consuming sufficient EPA and DHA to reduce the AA-PL pool and normalize endocannabinoid tone.

OBJECTIVE

Endocannabinoids in tissues controlling energy homeostasis are altered in obesity, thus contributing to metabolic disorders. Here we evaluate endocannabinoid dysregulation in the small intestine of mice with diet-induced obesity (DIO) and in peripheral tissues of Zucker and lean rats following food deprivation and re-feeding.

METHODS

Intestinal transit, evaluated using rhodamine-B-labelled dextran, and small intestinal endocannabinoid levels, measured by liquid chromatography mass spectrometry, were measured in mice fed normal or high-fat diets (HFDs). Endocannabinoid levels were measured also in various tissues of lean and Zucker rats fed ad libitum or following overnight food deprivation with and without subsequent re-feeding.

RESULTS

After 8 weeks of HFD, baseline intestinal transit was increased in DIO mice and enhanced by cannabinoid CB(1) receptor antagonism less efficaciously than in lean mice. Small intestinal anandamide and <em>2</em>-<em>arachidonoylglycerol</em> levels were reduced and increased respectively. In Zucker rats, endocannabinoids levels were higher in the pancreas, liver and duodenum, and lower in the subcutaneous adipose tissue. Food deprivation increased endocannabinoid levels in the duodenum and liver of both rat strains, in the pancreas of lean rats and in adipose tissues of Zucker rats.

CONCLUSIONS

Reduced anandamide levels might account for increased intestinal motility in DIO mice. Regulation of endocannabinoid levels in rat peripheral tissues, induced by food deprivation and re-feeding, might participate in food intake and energy processing and was altered in Zucker rats. These data, together with previous observations, provide further evidence for dysregulation of peripheral endocannabinoids in obesity.

The only endogenous substances isolated and characterised so far that are capable of mimicking the pharmacological actions of the active principle of marijuana, (-)-Delta9-tetrahydrocannabinol, are amides and esters of fatty acids. Some of these compounds, like anandamide (N-arachidonoylethanolamine) and <em>2</em>-<em>arachidonoylglycerol</em>, act as true 'endogenous cannabinoids' by binding and functionally activating one or both cannabinoid receptor subtypes present on nervous and peripheral cell membranes. The metabolic pathways and molecular mode of actions of these metabolites, as well as their possible implication in physiopathological responses, are reviewed here.

Studies of the endocannabinoid system in the CNS have been mostly focused on endocannabinoid receptors and inactivating mechanisms. Until recently, very little was known about the role of biosynthetic enzymes in endocannabinoid signaling. New data from the recent development of pharmacological and genetic tools for the study of these enzymes point to their fundamental role in determining where and when endocannabinoids function, and raise the possibility of new intriguing and previously unsuspected concepts in the general strategy of endocannabinoid signaling. However, even with these new tools, the cross-talk between anandamide and <em>2</em>-<em>arachidonoylglycerol</em> biosynthesis makes it difficult to dissect one from the other, and data will need to be interpreted with this in mind.

The endocannabinoid system is known to have positive effects on depression partly through its actions on neurotrophins, such as Brain-Derived Neurotrophic Factor (BDNF). As BDNF is also considered the major candidate molecule for exercise-induced brain plasticity, we hypothesized that the endocannabinoid system represents a crucial signaling system mediating the beneficial antidepressant effects of exercise. Here we investigated, in 11 healthy trained male cyclists, the effects of an intense exercise (60 min at 55% followed by 30 min at 75% W(max)) on plasma levels of endocannabinoids (anandamide, AEA and <em>2</em>-<em>arachidonoylglycerol</em>, <em>2</em>-AG) and their possible link with serum BDNF. AEA levels increased during exercise and the 15 min recovery (P<0.001), whereas <em>2</em>-AG concentrations remained stable. BDNF levels increased significantly during exercise and then decreased during the 15 min of recovery (P<0.01). Noteworthy, AEA and BDNF concentrations were positively correlated at the end of exercise and after the 15 min recovery (r>0.66, P<0.05), suggesting that AEA increment during exercise might be one of the factors involved in exercise-induced increase in peripheral BDNF levels and that AEA high levels during recovery might delay the return of BDNF to basal levels. AEA production during exercise might be triggered by cortisol since we found positive correlations between these two compounds and because corticosteroids are known to stimulate endocannabinoid biosynthesis. These findings provide evidence in humans that acute exercise represents a physiological stressor able to increase peripheral levels of AEA and that BDNF might be a mechanism by which AEA influences the neuroplastic and antidepressant effects of exercise.

Focal cerebral ischemia (FCI) induces rapid neuronal death in the ischemic core, which gradually expands toward the penumbra, partly as the result of a neuroinflammatory response. It is known that propagation of neuroinflammation involves microglial cells, the resident macrophages of the brain, which are highly motile when activated by specific signals. However, the signals that increase microglial cell motility in response to FCI remain mostly elusive. Here, we tested the hypothesis that endocannabinoids mediate neuroinflammation propagation by increasing microglial cell motility. We found that, in mouse cerebral cortex, FCI greatly increases palmitoylethanolamide (PEA), only moderately increases anandamide [arachidonylethanolamide (AEA)], and does not affect <em>2</em>-<em>arachidonoylglycerol</em> levels. We also found that PEA potentiates AEA-induced microglial cell migration, without affecting other steps of microglial activation, such as proliferation, particle engulfment, and nitric oxide production. This potentiation of microglial cell migration by PEA involves reduction in cAMP levels. In line with this, we provide evidence that PEA acts through Gi/o-coupled receptors. Interestingly, these receptors engaged by PEA are pharmacologically distinct from CB1 and CB<em>2</em> cannabinoid receptors, as well as from the WIN and abn-CBD (abnormal-cannabidiol) receptors, two recently identified cannabinoid receptors. Our results show that PEA and AEA increase after FCI and synergistically enhance microglial cell motility. Because such a response could participate in the propagation of the FCI-induced neuroinflammation within the CNS, and because PEA is likely to act through its own receptor, a better understanding of the receptor engaged by PEA may help guide the search for improved therapies against neuroinflammation.

Alzheimer's disease (AD) is the most common cause of dementia among older people. There are no effective medications currently available to prevent and treat AD and halt disease progression. Monoacylglycerol lipase (MAGL) is the primary enzyme metabolizing the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> in the brain. We show here that inactivation of MAGL robustly suppressed production and accumulation of β-amyloid (Aβ) associated with reduced expression of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) in a mouse model of AD. MAGL inhibition also prevented neuroinflammation, decreased neurodegeneration, maintained integrity of hippocampal synaptic structure and function, and improved long-term synaptic plasticity, spatial learning, and memory in AD animals. Although the molecular mechanisms underlying the beneficial effects produced by MAGL inhibition remain to be determined, our results suggest that MAGL, which regulates endocannabinoid and prostaglandin signaling, contributes to pathogenesis and neuropathology of AD, and thus is a promising therapeutic target for the prevention and treatment of AD.

The stimulus-induced biosynthesis of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in intact mouse J774 macrophages and the inactivation of <em>2</em>-AG by the same cells or by rat circulating macrophages was studied. By using gas chromatography-mass spectrometry, we found that ionomycin (5 microM) and lipopolysaccharide (LPS, <em>2</em>00 microg x mL-1) cause a <em>2</em>4-fold and <em>2</em>.5-fold stimulation of <em>2</em>-AG levels in J774 cells, respectively, thus providing unprecedented evidence that this cannabimimetic metabolite can be synthesized by macrophages. In J774 cells, LPS also induced a 7.8-fold increase of the levels of the other endocannabinoid, anandamide, and, in rat circulating macrophages, an almost twofold increase of <em>2</em>-AG levels. Extracellular [3H]<em>2</em>-AG was cleared from the medium of intact J774 macrophages (t1/<em>2</em> = 19-<em>2</em>8 min) and esterified to phospholipids, diacylglycerols and triglycerides or hydrolyzed to [3H]arachidonic acid and glycerol. These catabolic processes were attenuated differentially by various enzyme inhibitors. Rat circulating macrophages were shown to contain enzymatic activities for the hydrolysis of <em>2</em>-AG, including: (a) fatty acid amide hydrolase (FAAH), the enzyme responsible for anandamide breakdown and previously shown to catalyse also <em>2</em>-AG hydrolysis, and (b) a <em>2</em>-AG hydrolase activity different from FAAH and down-regulated by LPS. High levels of FAAH mRNA were found in circulating macrophages but not platelets, which, however, contain a <em>2</em>-AG hydrolase. Both platelets and macrophages were shown to express the mRNA for the CB1 cannabinoid receptor. A macrophage <em>2</em>-AG hydrolase with apparent Km = 110 microM and Vmax = 7.9 nmol x min-1 x (mg protein)-1 was partially characterized in J774 cells and found to exhibit an optimal pH of 6-7 and little or no sensitivity to typical FAAH inhibitors. These findings demonstrate for the first time that macrophages participate in the homeostasis of the hypotensive and immunomodulatory endocannabinoid <em>2</em>-AG through metabolic mechanisms that are subject to regulation.

The monoacylglycerol <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) has been recently suggested as a possible endogenous agonist at cannabinoid receptors both in brain and peripheral tissues. Here we report that a widely used model for neuronal cells, mouse N18TG<em>2</em> neuroblastoma cells, which contain the CB1 cannabinoid receptor, also biosynthesize, release and degrade <em>2</em>-AG. Stimulation with ionomycin (1-5 microM) of intact cells prelabelled with [3H]arachidonic acid ([3H]AA) led to the formation of high levels of a radioactive component with the same chromatographic behaviour as synthetic standards of <em>2</em>-AG in TLC and HPLC analyses. The amounts of this metabolite were negligible in unstimulated cells, and greatly decreased in cells stimulated in the presence of the Ca<em>2</em>+-chelating agent EGTA. The purified component was further characterized as <em>2</em>-AG by: (1) digestion with Rhizopus arrhizus lipase, which yielded radiolabelled AA; (<em>2</em>) gas chromatographic-MS analyses; and (3) TLC analyses on borate-impregnated plates. Approx. <em>2</em>0% of the <em>2</em>-AG produced by stimulated cells was found to be released into the incubation medium when this contained 0.1% BSA. Subcellular fractions of N18TG<em>2</em> cells were shown to contain enzymic activity or activities catalysing the hydrolysis of synthetic [3H]<em>2</em>-AG to [3H]AA. Cell homogenates were also found to convert synthetic [3H]sn-1-acyl-<em>2</em>-<em>arachidonoylglycerols</em> (AcAGs) into [3H]<em>2</em>-AG, suggesting that <em>2</em>-AG might be derived from AcAG hydrolysis. When compared with ionomycin stimulation, treatment of cells with exogenous phospholipase C, but not with phospholipase D or A<em>2</em>, led to a much higher formation of <em>2</em>-AG and AcAGs. However, treatment of cells with phospholipase A<em>2</em> 10 min before ionomycin stimulation caused a <em>2</em>.5-3-fold potentiation of <em>2</em>-AG and AcAG levels with respect to ionomycin alone, whereas preincubation with the phospholipase C inhibitor neomycin sulphate did not inhibit the effect of ionomycin on <em>2</em>-AG and AcAG levels. These results suggest that the Ca<em>2</em>+-induced formation of <em>2</em>-AG proceeds through the intermediacy of AcAGs but not necessarily through phospholipase C activation. By showing for the first time the existence of molecular mechanisms for the inactivation and the Ca<em>2</em>+-dependent biosynthesis and release of <em>2</em>-AG in neuronal cells, the present paper supports the hypothesis that this cannabimimetic monoacylglycerol might be a physiological neuromodulator.

The hypothalamic-pituitary-adrenal (HPA) axis regulates the outflow of glucocorticoid hormones under basal conditions and in response to stress. Within the last decade, a large body of evidence has mounted indicating that the endocannabinoid system is involved in the central regulation of the stress response; however, the specific role endocannabinoid signaling plays in phases of HPA axis regulation, and the neural sites of action mediating this regulation, were not mapped out until recently. This review aims to collapse the current state of knowledge regarding the role of the endocannabinoid system in the regulation of the HPA axis to put together a working model of how and where endocannabinoids act within the brain to regulate outflow of the HPA axis. Specifically, we discuss the role of the endocannabinoid system in the regulation of the HPA axis under basal conditions, activation in response to acute stress, and glucocorticoid-mediated negative feedback. Interestingly, there appears to be some anatomical specificity to the role of the endocannabinoid system in each phase of HPA axis regulation, as well as distinct roles of both anandamide and <em>2</em>-<em>arachidonoylglycerol</em> in these phases. Overall, the current level of information indicates that endocannabinoid signaling acts to suppress HPA axis activity through concerted actions within the prefrontal cortex, amygdala, and hypothalamus.

The endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) has been implicated as a key retrograde mediator in the nervous system based on pharmacological studies using inhibitors of the <em>2</em>-AG biosynthetic enzymes diacyglycerol lipase alpha and beta (DAGL-alpha/beta). Here, we show by competitive activity-based protein profiling that the DAGL-alpha/beta inhibitors, tetrahydrolipstatin (THL) and RHC80<em>2</em>67, block several brain serine hydrolases with potencies equal to or greater than their inhibitory activity against DAGL enzymes. Interestingly, a minimal overlap in target profiles was observed for THL and RHC80<em>2</em>67, suggesting that pharmacological effects observed with both agents may be viewed as good initial evidence for DAGL-dependent events.

An endogenous cannabimimetic molecule, <em>2</em>-<em>arachidonoylglycerol</em>, induces a rapid, transient increase in intracellular free Ca<em>2</em>+ concentrations in NG108-15 cells through a cannabinoid CB1 receptor-dependent mechanism. We examined the activities of <em>2</em>4 relevant compounds (<em>2</em>-<em>arachidonoylglycerol</em>, its structural analogues, and several synthetic cannabinoids). We found that <em>2</em>-<em>arachidonoylglycerol</em> is the most potent compound examined so far: its activity was detectable from as low as 0.3 nM, and the maximal response induced by <em>2</em>-<em>arachidonoylglycerol</em> exceeded the responses induced by others. Activities of HU-<em>2</em>10 and CP55940, potent cannabinoid receptor agonists, were also detectable from as low as 0.3 nM, whereas the maximal responses induced by these compounds were low compared with <em>2</em>-<em>arachidonoylglycerol</em>. Anandamide was also found to act as a partial agonist in this assay system. We confirmed that free arachidonic acid failed to elicit a response. Furthermore, we found that a metabolically stable ether-linked analogue of <em>2</em>-<em>arachidonoylglycerol</em> possesses appreciable agonistic activity, although its activity was apparently lower than that of <em>2</em>-<em>arachidonoylglycerol</em>. We also confirmed that pretreating cells with various cannabinoid receptor agonists nullified the response induced by <em>2</em>-<em>arachidonoylglycerol</em>, whereas pretreating cells with other neurotransmitters or neuromodulators did not affect the response. These results strongly suggested that the cannabinoid CB1 receptor is originally a <em>2</em>-<em>arachidonoylglycerol</em> receptor, and <em>2</em>-<em>arachidonoylglycerol</em> is the intrinsic physiological ligand for the cannabinoid CB1 receptor.

Dietary (n-3) long-chain PUFA [(n-3) LCPUFA] ameliorate several metabolic risk factors for cardiovascular diseases, although the mechanisms of these beneficial effects are not fully understood. In this study, we compared the effects of dietary (n-3) LCPUFA, in the form of either fish oil (FO) or krill oil (KO) balanced for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) content, with a control (C) diet containing no EPA and DHA and similar contents of oleic, linoleic, and alpha-linolenic acids, on ectopic fat and inflammation in Zucker rats, a model of obesity and related metabolic dysfunction. Diets were fed for 4 wk. Given the emerging evidence for an association between elevated endocannabinoid concentrations and metabolic syndrome, we also measured tissue endocannabinoid concentrations. In (n-3) LCPUFA-supplemented rats, liver triglycerides and the peritoneal macrophage response to an inflammatory stimulus were significantly lower than in rats fed the control diet, and heart triglycerides were lower, but only in KO-fed rats. These effects were associated with a lower concentration of the endocannabinoids, anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, in the visceral adipose tissue and of anandamide in the liver and heart, which, in turn, was associated with lower levels of arachidonic acid in membrane phospholipids, but not with higher activity of endocannabinoid-degrading enzymes. Our data suggest that the beneficial effects of a diet enriched with (n-3) LCPUFA are the result of changes in membrane fatty acid composition. The reduction of substrates for inflammatory molecules and endocannabinoids may account for the dampened inflammatory response and the physiological reequilibration of body fat deposition in obese rats.

Endogenous ligands of cannabinoid receptors have been discovered recently and include some N-acylethanolamines (NAEs; e.g., N-arachidonoylethanolamine) and some <em>2</em>-acylglycerols (e.g., sn-<em>2</em>-<em>arachidonoylglycerol</em>). Previously, we found these compounds to be active biologically when administered per os in large quantities to mice. In the present work, piglets were fed diets with and without <em>2</em>0:4n-6 and <em>2</em><em>2</em>:6n-3 fatty acid precursors of NAEs, in levels similar to those found in porcine milk, during the first 18 days of life, and corresponding brain NAEs were assessed. In piglets fed diets containing <em>2</em>0:4n-6 and <em>2</em><em>2</em>:6n-3, there were increases in several biologically active NAEs in brain homogenates-<em>2</em>0:4n-6 NAE (4-fold), <em>2</em>0:5n-3 NAE (5-fold), and <em>2</em><em>2</em>:5n-3 and <em>2</em><em>2</em>:6n-3 NAE (9- to 10-fold). These results support a mechanism we propose for dietary long-chain polyunsaturated fatty acids influences on brain biochemistry with presumed functional sequelae. This paradigm will enable targeted investigations to determine whether and why specific populations such as infants, elderly, or persons suffering from certain clinical conditions may benefit from dietary long-chain polyunsaturated fatty acids.

Endocannabinoid (eCB) signaling is tightly regulated by eCB biosynthetic and degradative enzymes. The eCB <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is hydrolyzed primarily by monoacylglycerol lipase (MAGL). Here, we investigated whether eCB signaling, synaptic function, and learning behavior were altered in MAGL knock-out mice. We report that MAGL⁻/⁻ mice exhibited prolonged depolarization-induced suppression of inhibition (DSI) in hippocampal CA1 pyramidal neurons, providing genetic evidence that the inactivation of <em>2</em>-AG by MAGL determines the time course of the eCB-mediated retrograde synaptic depression. CB₁ receptor antagonists enhanced basal IPSCs in CA1 pyramidal neurons in MAGL⁻/⁻ mice, while the magnitude of DSI or CB₁ receptor agonist-induced depression of IPSCs was decreased in MAGL⁻/⁻ mice. These results suggest that <em>2</em>-AG elevations in MAGL⁻/⁻ mice cause tonic activation and partial desensitization of CB₁ receptors. Genetic deletion of MAGL selectively enhanced theta burst stimulation (TBS)-induced long-term potentiation (LTP) in the CA1 region of hippocampal slices but had no significant effect on LTP induced by high-frequency stimulation or long-term depression induced by low-frequency stimulation. The enhancement of TBS-LTP in MAGL⁻/⁻ mice appears to be mediated by <em>2</em>-AG-induced suppression of GABA(A) receptor-mediated inhibition. MAGL⁻/⁻ mice exhibited enhanced learning as shown by improved performance in novel object recognition and Morris water maze. These results indicate that genetic deletion of MAGL causes profound changes in eCB signaling, long-term synaptic plasticity, and learning behavior.

Anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), two endogenous ligands of the CB1 and CB<em>2</em> cannabinoid receptor subtypes, inhibit the proliferation of PRL-responsive human breast cancer cells (HBCCs) through down-regulation of the long form of the PRL receptor (PRLr). Here we report that 1) anandamide and <em>2</em>-AG inhibit the nerve growth factor (NGF)-induced proliferation of HBCCs through suppression of the levels of NGF Trk receptors; <em>2</em>) inhibition of PRLr levels results in inhibition of the proliferation of other PRL-responsive cells, the prostate cancer DU-145 cell line; and 3) CB1-like cannabinoid receptors are expressed in HBCCs and DU-145 cells and mediate the inhibition of cell proliferation and Trk/PRLr expression. Beta-NGF-induced HBCC proliferation was potently inhibited (IC50 = 50-600 nM) by the synthetic cannabinoid HU-<em>2</em>10, <em>2</em>-AG, anandamide, and its metabolically stable analogs, but not by the anandamide congener, palmitoylethanolamide, or the selective agonist of CB<em>2</em> cannabinoid receptors, BML-190. The effect of anandamide was blocked by the CB1 receptor antagonist, SR141716A, but not by the CB<em>2</em> receptor antagonist, SR1445<em>2</em>8. Anandamide and HU-<em>2</em>10 exerted a strong inhibition of the levels of NGF Trk receptors as detected by Western immunoblotting; this effect was reversed by SR141716A. When induced by exogenous PRL, the proliferation of prostate DU-145 cells was potently inhibited (IC50 = 100-300 nM) by anandamide, <em>2</em>-AG, and HU-<em>2</em>10. Anandamide also down-regulated the levels of PRLr in DU-145 cells. SR141716A attenuated these two effects of anandamide. HBCCs and DU-145 cells were shown to contain 1) transcripts for CB1 and, to a lesser extent, CB<em>2</em> cannabinoid receptors, <em>2</em>) specific binding sites for [3H]SR141716A that could be displaced by anandamide, and 3) a CB1 receptor-immunoreactive protein. These findings suggest that endogenous cannabinoids and CB1 receptor agonists are potential negative effectors of PRL- and NGF-induced biological responses, at least in some cancer cells.

In recent years, cannabinoid receptors and their endogenous ligands (endocannabinoids) have been identified within the brain. The high density of CB1 cannabinoid receptors within the basal ganglia suggests a potential role for endocannabinoids in the control of voluntary movement and in basal ganglia-related movement disorders such as Parkinson's disease. However, whether endocannabinoids play a role in regulating motor behavior in health and disease is unknown. Here we report the presence in two regions of the basal ganglia, the globus pallidus and substantia nigra, of the endocannabinoids <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>AG) and anandamide. The levels of the latter compound are approximately threefold higher than those previously reported in any other brain region. In the reserpine-treated rat, an animal model of Parkinson's disease, suppression of locomotion is accompanied by a sevenfold increase in the levels of the <em>2</em>AG in the globus pallidus, but not in the other five brain regions analyzed. Stimulation of locomotion in the reserpine-treated rat by either of the two selective agonists of D<em>2</em> and D1 dopamine receptors, quinpirole and R-(+/-)-3-allyl-6-chloro-7, 8-dihydroxy-1-phenyl-<em>2</em>,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (Cl-APB), respectively, results in the reduction of both anandamide and <em>2</em>AG levels in the globus pallidus. Finally, full restoration of locomotion in the reserpine-treated rat is obtained by coadministration of quinpirole and the selective antagonist of the cannabinoid CB1 receptor subtype, SR141716A. These findings indicate a link between endocannabinoid signaling in the globus pallidus and symptoms of Parkinson's disease in the reserpine-treated rat, and suggest that modulation of the endocannabinoid signaling system might prove useful in treating this or other basal ganglia-related movement disorders.

OBJECTIVE

Certain cognitive deficits in individuals with schizophrenia have been linked to disturbed gamma-aminobutyric acid (GABA) and glutamate neurotrans-mission in the prefrontal cortex. Thus, it is important to understand how the mechanisms that regulate GABA and glutamate neurotransmission are altered in schizophrenia. For example, group I metabo-tropic glutamate receptors (mGluR1α, mGluR5) modulate both GABA and gluta-mate systems. In addition, regulator of G protein signaling 4 (RGS4) reduces intra-cellular signaling through several different G protein-coupled receptors, including group I mGluRs. Finally, the endocannabinoid system plays an important role in regulating GABA and glutamate neurotrans-mission. The status of endocannabinoid ligands, such as <em>2</em>-<em>arachidonoylglycerol</em>, can be inferred in part through measures of diacylglycerol lipase and monoglyceride lipase, which synthesize and degrade <em>2</em>-<em>arachidonoylglycerol</em>, respectively.

METHODS

Quantitative polymerase chain reaction was used to measure mRNA levels for group I mGluRs, RGS4, and markers of the endocannabinoid system in the prefrontal cortex Brodmann's area 9 of 4<em>2</em> schizophrenia subjects and matched normal comparison subjects. Similar analyses in monkeys chronically exposed to haloperidol, olanzapine, or placebo were also conducted.

RESULTS

Schizophrenia subjects had higher mRNA levels for mGluR1α and lower mRNA levels for RGS4, and these differences did not appear to be attributable to antipsychotic medications or other potential confounds. In contrast, no differences between subject groups were found in mRNA levels for endocannabinoid synthesizing and metabolizing enzymes.

CONCLUSIONS

Together, higher mGluR1α and lower RGS4 mRNA levels may represent a disturbed "molecular hub" in schizophrenia that may disrupt the function of prefrontal cortical networks, including both GABA and glutamate systems.