Endocannabinoids function as retrograde messengers and modulate synaptic transmission through presynaptic cannabinoid CB1 receptors. The magnitude and time course of endocannabinoid signaling are thought to depend on the balance between the production and degradation of endocannabinoids. The major endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is hydrolyzed by monoacylglycerol lipase (MGL), which is shown to be localized at axon terminals. In the present study, we investigated how MGL regulates endocannabinoid signaling and influences synaptic transmission in the hippocampus. We found that MGL inhibitors, methyl arachidonoyl fluorophosphonate and arachidonoyl trifluoromethylketone, caused a gradual suppression of cannabinoid-sensitive IPSCs in cultured hippocampal neurons. This suppression was reversed by blocking CB1 receptors and was attenuated by inhibiting <em>2</em>-AG synthesis, indicating that MGL scavenges constitutively released <em>2</em>-AG. We also found that the MGL inhibitors significantly prolonged the suppression of both IPSCs and EPSCs induced by exogenous <em>2</em>-AG and depolarization-induced suppression of inhibition/excitation, a phenomenon known to be mediated by retrograde endocannabinoid signaling. In contrast, inhibitors of other endocannabinoid hydrolyzing enzymes, fatty acid amide hydrolase and cyclooxygenase-<em>2</em>, had no effect on the <em>2</em>-AG-induced IPSC suppression. These results strongly suggest that presynaptic MGL not only hydrolyzes <em>2</em>-AG released from activated postsynaptic neurons but also contributes to degradation of constitutively produced <em>2</em>-AG and prevention of its accumulation around presynaptic terminals. Thus, the MGL activity determines basal endocannabinoid tone and terminates retrograde endocannabinoid signaling in the hippocampus.

After the discovery, in the early 1990s, of specific G-protein-coupled receptors for marijuana's psychoactive principle Delta(9)-tetrahydrocannabinol, the cannabinoid receptors, and of their endogenous agonists, the endocannabinoids, a decade of investigations has greatly enlarged our understanding of this altogether new signalling system. Yet, while the finding of the endocannabinoids resulted in a new effort to reveal the mechanisms regulating their levels in the brain and peripheral organs under physiological and pathological conditions, more endogenous substances with a similar action, and more molecular targets for the previously discovered endogenous ligands, anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, or for some of their metabolites, were being proposed. As the scenario becomes subsequently more complicated, and the experimental tasks to be accomplished correspondingly more numerous, we briefly review in this article the latest 'additions' to the endocannabinoid system together with earlier breakthroughs that have contributed to our present knowledge of the biochemistry and pharmacology of the endocannabinoids.

Anandamide was the first brain metabolite shown to act as a ligand of "central" CB1 cannabinoid receptors. Here we report that the endogenous cannabinoid potently and selectively inhibits the proliferation of human breast cancer cells in vitro. Anandamide dose-dependently inhibited the proliferation of MCF-7 and EFM-19 cells with IC50 values between 0.5 and 1.5 microM and 83-9<em>2</em>% maximal inhibition at 5-10 microM. The proliferation of several other nonmammary tumoral cell lines was not affected by 10 microM anandamide. The anti-proliferative effect of anandamide was not due to toxicity or to apoptosis of cells but was accompanied by a reduction of cells in the S phase of the cell cycle. A stable analogue of anandamide (R)-methanandamide, another endogenous cannabinoid, <em>2</em>-<em>arachidonoylglycerol</em>, and the synthetic cannabinoid HU-<em>2</em>10 also inhibited EFM-19 cell proliferation, whereas arachidonic acid was much less effective. These cannabimimetic substances displaced the binding of the selective cannabinoid agonist [3H]CP 55, 940 to EFM-19 membranes with an order of potency identical to that observed for the inhibition of EFM-19 cell proliferation. Moreover, anandamide cytostatic effect was inhibited by the selective CB1 receptor antagonist SR 141716A. Cell proliferation was arrested by a prolactin mAb and enhanced by exogenous human prolactin, whose mitogenic action was reverted by very low (0.1-0.5 microM) doses of anandamide. Anandamide suppressed the levels of the long form of the prolactin receptor in both EFM-19 and MCF-7 cells, as well as a typical prolactin-induced response, i.e., the expression of the breast cancer cell susceptibility gene brca1. These data suggest that anandamide blocks human breast cancer cell proliferation through CB1-like receptor-mediated inhibition of endogenous prolactin action at the level of prolactin receptor.

Glucocorticoids secreted in response to stress activation of the hypothalamic-pituitary-adrenal axis feed back onto the brain to rapidly suppress neuroendocrine activation, including oxytocin and vasopressin secretion. Here we show using whole-cell patch clamp recordings that glucocorticoids elicit a rapid, opposing action on synaptic glutamate and gamma-aminobutyric acid (GABA) release onto magnocellular neurons of the hypothalamic supraoptic nucleus and paraventricular nucleus, suppressing glutamate release and facilitating GABA release by activating a putative membrane receptor. The glucocorticoid effect on both glutamate and GABA release was blocked by inhibiting postsynaptic G protein activity, suggesting a dependence on postsynaptic G protein signaling and the involvement of a retrograde messenger. Biochemical analysis of hypothalamic slices treated with dexamethasone revealed a glucocorticoid-induced rapid increase in the levels of the endocannabinoids anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). The glucocorticoid suppression of glutamate release was blocked by the type I cannabinoid receptor cannabinoid receptor antagonist, AM<em>2</em>51, and was mimicked and occluded by AEA and <em>2</em>-AG, suggesting it was mediated by retrograde endocannabinoid release. The glucocorticoid facilitation of GABA release was also blocked by AM<em>2</em>51 but was not mimicked by AEA, <em>2</em>-AG, or a synthetic cannabinoid, WIN 55,<em>2</em>1<em>2</em>-<em>2</em>, nor was it blocked by vanilloid or ionotropic glutamate receptor antagonists, suggesting that it was mediated by a retrograde messenger acting at an AM<em>2</em>51-sensitive, noncannabinoid/nonvanilloid receptor at presynaptic GABA terminals. The combined, opposing actions of glucocorticoids mediate a rapid inhibition of the magnocellular neuroendocrine cells, which in turn should mediate rapid feedback inhibition of the secretion of oxytocin and vasopressin by glucocorticoids during stress activation of the hypothalamic-pituitary-adrenal axis.

Central nervous system responses to cannabis are primarily mediated by CB(1) receptors, which couple preferentially to G(i/o) G proteins. Here, we used calcium photometry to monitor the effect of CB(1) activation on intracellular calcium concentration. Perfusion with 5 microM CB(1) aminoalkylindole agonist, WIN55,<em>2</em>1<em>2</em>-<em>2</em> (WIN), increased intracellular calcium by several hundred nanomolar in human embryonic kidney <em>2</em>93 cells stably expressing CB(1) and in cultured hippocampal neurons. The increase was blocked by coincubation with the CB(1) antagonist, SR141716A, and was absent in nontransfected human embryonic kidney <em>2</em>93 cells. The calcium rise was WIN-specific, being essentially absent in cells treated with other classes of cannabinoid agonists, including Delta(9)-tetrahydrocannabinol, HU-<em>2</em>10, CP55,940, <em>2</em>-<em>arachidonoylglycerol</em>, methanandamide, and cannabidiol. The increase in calcium elicited by WIN was independent of G(i/o), because it was present in pertussis toxin-treated cells. Indeed, pertussis toxin pretreatment enhanced the potency and efficacy of WIN to increase intracellular calcium. The calcium increases appeared to be mediated by G(q) G proteins and phospholipase C, because they were markedly attenuated in cells expressing dominant-negative G(q) or treated with the phospholipase C inhibitors U731<em>2</em><em>2</em> and ET-18-OCH(3) and were accompanied by an increase in inositol phosphates. The calcium increase was blocked by the sarco/endoplasmic reticulum Ca(<em>2</em>+) pump inhibitor thapsigargin, the inositol trisphosphate receptor inhibitor xestospongin D, and the ryanodine receptor inhibitors dantrolene and 1,1'-diheptyl-4,4'-bipyridinium dibromide, but not by removal of extracellular calcium, showing that WIN releases calcium from intracellular stores. In summary, these results suggest that WIN stabilizes CB(1) receptors in a conformation that enables G(q) signaling, thus shifting the G protein specificity of the receptor.

Endocannabinoids and their attending cannabinoid type 1 (CB1) receptor have been implicated in animal models of post-traumatic stress disorder (PTSD). However, their specific role has not been studied in people with PTSD. Herein, we present an in vivo imaging study using positron emission tomography (PET) and the CB1-selective radioligand [(11)C]OMAR in individuals with PTSD, and healthy controls with lifetime histories of trauma (trauma-exposed controls (TC)) and those without such histories (healthy controls (HC)). Untreated individuals with PTSD (N=<em>2</em>5) with non-combat trauma histories, and TC (N=1<em>2</em>) and HC (N=<em>2</em>3) participated in a magnetic resonance imaging scan and a resting PET scan with the CB1 receptor antagonist radiotracer [(11)C]OMAR, which measures the volume of distribution (VT) linearly related to CB1 receptor availability. Peripheral levels of anandamide, <em>2</em>-<em>arachidonoylglycerol</em>, oleoylethanolamide, palmitoylethanolamide and cortisol were also assessed. In the PTSD group, relative to the HC and TC groups, we found elevated brain-wide [(11)C]OMAR VT values (F(<em>2</em>,53)=7.96, P=0.001; 19.5% and 14.5% higher, respectively), which were most pronounced in women (F(1,53)=5.5<em>2</em>, P=0.0<em>2</em>3). Anandamide concentrations were reduced in the PTSD relative to the TC (53.1% lower) and HC (58.<em>2</em>% lower) groups. Cortisol levels were lower in the PTSD and TC groups relative to the HC group. Three biomarkers examined collectively--OMAR VT, anandamide and cortisol--correctly classified nearly 85% of PTSD cases. These results suggest that abnormal CB1 receptor-mediated anandamide signaling is implicated in the etiology of PTSD, and provide a promising neurobiological model to develop novel, evidence-based pharmacotherapies for this disorder.

The functions of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), the most abundant endocannabinoid found in the brain, remain largely unknown. Here we show that two previously unknown inhibitors of monoacylglycerol lipase, a presynaptic enzyme that hydrolyzes <em>2</em>-AG, increase <em>2</em>-AG levels and enhance retrograde signaling from pyramidal neurons to GABAergic terminals in the hippocampus. These results establish a role for <em>2</em>-AG in synaptic plasticity and point to monoacylglycerol lipase as a possible drug target.

N -arachidonoylethanolamine (anandamide) was the first endogenous cannabinoid receptor ligand to be discovered. Dual synthetic pathways for anandamide have been proposed. One is the formation from free arachidonic acid and ethanolamine, and the other is the formation from N -arachidonoyl phosphatidylethanolamine (PE) through the action of a phosphodiesterase. These pathways, however, do not appear to be able to generate a large amount of anandamide, at least under physiological conditions. The generation of anandamide from free arachidonic acid and ethanolamine is catalyzed by a degrading enzyme anandamide amidohydrolase/fatty acid amide hydrolase operating in reverse and requires large amounts of substrates. As for the second pathway, arachidonic acids esterified at the 1-position of glycerophospholipids, which are mostly esterified at the <em>2</em>-position, are utilized for the formation of N -arachidonoyl PE, a stored precursor form of anandamide. In fact, the actual levels of anandamide in various tissues are generally low except in a few cases. <em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG) was the second endogenous cannabinoid receptor ligand to be discovered. <em>2</em>-AG is a degradation product of arachidonic acid-containing glycerophospholipids such as inositol phospholipids. Several investigators have demonstrated that <em>2</em>-AG is produced in a variety of tissues and cells upon stimulation. <em>2</em>-AG acts as a full agonist at the cannabinoid receptors (CB1 and CB<em>2</em>). Evidence is gradually accumulating and indicates that <em>2</em>-AG is the most efficacious endogenous natural ligand for the cannabinoid receptors. In this review, we summarize the tissue levels, biosynthesis, degradation and possible physiological significance of two endogenous cannabimimetic molecules, anandamide and <em>2</em>-AG.

Activation of group I metabotropic glutamate (mGlu) receptors recruits the endocannabinoid system to produce both short- and long-term changes in synaptic strength in many regions of the brain. Although there is evidence that the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) mediates this process, the molecular mechanism underlying <em>2</em>-AG mobilization remains unclear. In the present study, we used a combination of genetic and targeted lipidomic approaches to investigate the role of the postsynaptic membrane-associated lipase, diacylglycerol lipase type-alpha (DGL-alpha), in mGlu receptor-dependent <em>2</em>-AG mobilization. DGL-alpha overexpression in mouse neuroblastoma Neuro-<em>2</em>a cells increased baseline <em>2</em>-AG levels. This effect was accompanied by enhanced utilization of the <em>2</em>-AG precursor 1-stearoyl,<em>2</em>-arachidonoyl-sn-glycerol and increased accumulation of the <em>2</em>-AG breakdown product arachidonic acid. A similar, albeit less marked response was observed with other unsaturated and polyunsaturated monoacylglycerols, 1,<em>2</em>-diacylglycerols, and fatty acids. Silencing of DGL-alpha by RNA interference elicited lipidomic changes opposite those of DGL-alpha overexpression and abolished group I mGlu receptor-dependent <em>2</em>-AG mobilization. Coimmunoprecipitation and site-directed mutagenesis experiments revealed that DGL-alpha interacts, via a PPxxF domain, with the coiled-coil (CC)-Homer proteins Homer-1b and Homer-<em>2</em>, two components of the molecular scaffold that enables group I mGlu signaling. DGL-alpha mutants that do not bind Homer maintained their ability to generate <em>2</em>-AG in intact cells but failed to associate with the plasma membrane. The findings indicate that DGL-alpha mediates group I mGlu receptor-induced <em>2</em>-AG mobilization. They further suggest that the interaction of CC-Homer with DGL-alpha is necessary for appropriate function of this lipase.

Cannabinoid receptors and their endogenous ligands have been recently identified in the brain as potent inhibitors of neurotransmitter release. Here we show that, in a rat model of Parkinson's disease induced by unilateral nigral lesion with 6-hydroxydopamine (6-OHDA), the striatal levels of anandamide, but not that of the other endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em>, were increased. Moreover, we observed a decreased activity of the anandamide membrane transporter (AMT) and of the anandamide hydrolase [fatty acid amide hydrolase (FAAH)], whereas the binding of anandamide to cannabinoid receptors was unaffected. Spontaneous glutamatergic activity recorded from striatal spiny neurons was higher in 6-OHDA-lesioned rats. Inhibition of AMT by N-(4-hydroxyphenyl)-arachidonoylamide (AM-404) or by VDM11, or stimulation of the cannabinoid CB1 receptor by HU-<em>2</em>10 reduced glutamatergic spontaneous activity in both naive and 6-OHDA-lesioned animals to a similar extent. Conversely, the FAAH inhibitors phenylmethylsulfonyl fluoride and methyl-arachidonoyl fluorophosphonate were much more effective in 6-OHDA-lesioned animals. The present study shows that inhibition of anandamide hydrolysis might represent a possible target to decrease the abnormal cortical glutamatergic drive in Parkinson's disease.

The endogenous cannabinoid system is an ubiquitous lipid signalling system that appeared early in evolution and which has important regulatory functions throughout the body in all vertebrates. The main endocannabinoids (endogenous cannabis-like substances) are small molecules derived from arachidonic acid, anandamide (arachidonoylethanolamide) and <em>2</em>-<em>arachidonoylglycerol</em>. They bind to a family of G-protein-coupled receptors, of which the cannabinoid CB(1) receptor is densely distributed in areas of the brain related to motor control, cognition, emotional responses, motivated behaviour and homeostasis. Outside the brain, the endocannabinoid system is one of the crucial modulators of the autonomic nervous system, the immune system and microcirculation. Endocannabinoids are released upon demand from lipid precursors in a receptor-dependent manner and serve as retrograde signalling messengers in GABAergic and glutamatergic synapses, as well as modulators of postsynaptic transmission, interacting with other neurotransmitters, including dopamine. Endocannabinoids are transported into cells by a specific uptake system and degraded by two well-characterized enzymes, the fatty acid amide hydrolase and the monoacylglycerol lipase. Recent pharmacological advances have led to the synthesis of cannabinoid receptor agonists and antagonists, anandamide uptake blockers and potent, selective inhibitors of endocannabinoid degradation. These new tools have enabled the study of the physiological roles played by the endocannabinoids and have opened up new strategies in the treatment of pain, obesity, neurological diseases including multiple sclerosis, emotional disturbances such as anxiety and other psychiatric disorders including drug addiction. Recent advances have specifically linked the endogenous cannabinoid system to alcoholism, and cannabinoid receptor antagonism now emerges as a promising therapeutic alternative for alcohol dependence and relapse.

The endogenous cannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is produced by neurons and other cells in a stimulus-dependent manner and undergoes rapid biological inactivation through transport into cells and catalytic hydrolysis. The enzymatic pathways responsible for <em>2</em>-AG degradation are only partially understood. We have shown previously that overexpression of monoacylglycerol lipase (MGL), a cytosolic serine hydrolase that cleaves 1- and <em>2</em>-monoacylglycerols to fatty acid and glycerol, reduces stimulus-dependent <em>2</em>-AG accumulation in primary cultures of rat brain neurons. We report here that RNA interference-mediated silencing of MGL expression greatly enhances <em>2</em>-AG accumulation in HeLa cells. After stimulation with the calcium ionophore ionomycin, <em>2</em>-AG levels in MGL-silenced cells were comparable with those found in cells in which <em>2</em>-AG degradation had been blocked using methyl arachidonyl fluorophosphonate, a nonselective inhibitor of <em>2</em>-AG hydrolysis. The results indicate that MGL plays an important role in the degradation of endogenous <em>2</em>-AG in intact HeLa cells. Furthermore, immunodepletion experiments show that MGL accounts for at least 50% of the total <em>2</em>-AG-hydrolyzing activity in soluble fractions of rat brain, suggesting that this enzyme also contributes to <em>2</em>-AG deactivation in the central nervous system.

To date, N-arachidonoylethanolamine (anandamide) and <em>2</em>-<em>arachidonoylglycerol</em> are the best studied endocannabinoids and are thought to act as retrograde messengers in the central nervous system (CNS). By activating presynaptic cannabinoid CB1 receptors, they can reduce glutamate release in dorsal and ventral striatum (nucleus accumbens) and alter synaptic plasticity, thereby modulating neurotransmission in the basal ganglia and in the mesolimbic reward system. In this review, we will focus on the role of the endocannabinoid system within these neuronal pathways and describe its effect on dopaminergic transmission and vice versa. The endocannabinoid system is unlikely to directly affect dopamine release, but can modify dopamine transmission trough trans-synaptic mechanisms, involving gamma-aminobutyric acid (GABA)-ergic and glutamatergic synapses, as well as by converging signal transduction cascades of the cannabinoid and dopamine receptors. The dopamine and endocannabinoid systems exert a mutual control on each other. Cannabinergic signalling may lead to release of dopamine, which can act via dopamine D1-like receptors as a negative feedback mechanism to counteract the effects of activation of the cannabinoid CB1 receptor. On the other hand, dopaminergic signalling via dopamine D<em>2</em>-like receptors may lead to up-regulation of cannabinergic signalling, which is likely to represent a negative feedback on dopaminergic signalling. The consequences of these interactions become evident in pathological conditions in which one of the two systems is likely to be malfunctioning. We will discuss neurological and psychiatric disorders such as Parkinson's and Huntington's disease, drug addiction and schizophrenia. Furthermore, the possible role of the endocannabinoid system in disorders not necessarily depending on the dopaminergic system, such as eating disorders and anxiety, will be described.

Selective activation of the peripheral cannabinoid receptor 1 (CB1R) has been shown to suppress neuropathic pain symptoms in rodents. However, relatively little is known about changes in CB1R and its endogenous ligands during development or maintenance of neuropathic pain. Using immunohistochemistry, Western blot, real-time reverse transcription polymerase chain reaction, as well as liquid chromatography/mass spectrometry, we studied the changes in CB1Rs and endocannabinoids N-arachidonoylethanolamine/anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in rat lumbar (L4 and L5) dorsal root ganglia (DRG) after neuropathic pain induction (L5 spinal nerve ligation: SNL). Immunohistochemistry revealed that in control rats, CB1R is expressed in the majority (76-83%) of nociceptive neurons as indicated by co-labeling with isolectin B4 (IB4) or antibodies recognizing transient receptor potential vanilloid (TRPV1), calcitonin gene related peptide (CGRP), and the NR<em>2</em>C/<em>2</em>D subunits of the N-methyl-D-aspartate receptor. After L5 SNL, CB1R mRNA and protein increases in the ipsilateral uninjured L4 DRG whereas the percentages of CB1R immunoreactive (CB1R-ir) neurons remain unchanged in L4 and L5 DRG. However, for these CB1R-ir neurons, we observe significant increases in percentage of TRPV1-ir cells in ipsilateral L4 DRG, and decreases in percentage of IB4- and CGRP-co-labeled cells in ipsilateral L5 DRG. Levels of both AEA and <em>2</em>-AG increase significantly only in the ipsilateral L5 DRG. These results are consistent with the preserved analgesic effects of cannabinoids in neuropathic pain and provide a rational framework for the development of peripherally acting endocannabinoid-based therapeutic interventions for neuropathic pain.

Dysregulation in signaling of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is implicated in hyperresponsiveness to stress. We hypothesized that blockade of monoacylglycerol lipase (MGL), the primary enzyme responsible for <em>2</em>-AG deactivation in vivo, would produce context-dependent anxiolytic effects in rats. Environmental aversiveness was manipulated by varying illumination of an elevated plus maze. Percentage open arm time and numbers of open and closed arm entries were measured in rats receiving a single intraperitoneal (i.p.) injection of either vehicle, the MGL inhibitor JZL184 (1-8mg/kg), the benzodiazepine diazepam (1mg/kg), the cannabinoid CB(1) receptor antagonist rimonabant (1mg/kg), or JZL184 (8mg/kg) coadministered with rimonabant (1mg/kg). JZL184 (8mg/kg) produced anxiolytic-like effects (i.e., increased percentage open arm time and number of open arm entries) under high, but not low, levels of environmental aversiveness. Diazepam produced anxiolytic effects in either context. Rimonabant blocked the anxiolytic-like effects of JZL184, consistent with mediation by CB(1). Anxiolytic effects of JZL184 were preserved following chronic (8mg/kg per day×6 days) administration. Chronic and acute JZL184 treatment similarly enhanced behavioral sensitivity to an exogenous cannabinoid (WIN55,<em>2</em>1<em>2</em>-<em>2</em>; <em>2</em>.5mg/kg i.p.) <em>2</em>4 or 7<em>2</em>h following the terminal injection, suggesting a pervasive effect of MGL inhibition on the endocannabinoid system. We attribute our results to alterations in emotion rather than locomotor activity as JZL184 did not alter the number of closed arm entries in the plus maze or produce motor ataxia in the bar test. Our results demonstrate that JZL184 has beneficial, context-dependent effects on anxiety in rats, presumably via inhibition of MGL-mediated hydrolysis of <em>2</em>-AG. These data warrant further testing of MGL inhibitors to elucidate the functional role of <em>2</em>-AG in controlling anxiety and stress responsiveness. Our data further implicate a role for <em>2</em>-AG in the regulation of emotion and validate MGL as a therapeutic target.

OBJECTIVE

The endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) inhibit cancer cell proliferation by acting at cannabinoid receptors (CBRs). We studied (1). the levels of endocannabinoids, cannabinoid CB(1) and CB(<em>2</em>) receptors, and fatty acid amide hydrolase (FAAH, which catalyzes endocannabinoid hydrolysis) in colorectal carcinomas (CRC), adenomatous polyps, and neighboring healthy mucosa; and (<em>2</em>). the effects of endocannabinoids, and of inhibitors of their inactivation, on human CRC cell proliferation.

METHODS

Tissues were obtained from <em>2</em>1 patients by biopsy during colonoscopy. Endocannabinoids were measured by liquid chromatography-mass spectrometry (LC-MS). CB(1), CB(<em>2</em>), and FAAH expression were analyzed by RT-PCR and Western immunoblotting. CRC cell lines (CaCo-<em>2</em> and DLD-1) were used to test antiproliferative effects.

RESULTS

All tissues and cells analyzed contain anandamide, <em>2</em>-AG, CBRs, and FAAH. The levels of the endocannabinoids are 3- and <em>2</em>-fold higher in adenomas and CRCs than normal mucosa. Anandamide, <em>2</em>-AG, and the CBR agonist HU-<em>2</em>10 potently inhibit CaCo-<em>2</em> cell proliferation. This effect is blocked by the CB(1) antagonist SR141716A, but not by the CB(<em>2</em>) antagonist SR1445<em>2</em>8, and is mimicked by CB(1)-selective, but not CB(<em>2</em>)-selective, agonists. In DLD-1 cells, both CB(1) and CB(<em>2</em>) receptors mediate inhibition of proliferation. Inhibitors of endocannabinoid inactivation enhance CaCo-<em>2</em> cell endocannabinoid levels and block cell proliferation, this effect being antagonized by SR141716A. CaCo-<em>2</em> cell differentiation into noninvasive cells results in increased FAAH expression, lower endocannabinoid levels, and no responsiveness to cannabinoids.

CONCLUSIONS

Endocannabinoid levels are enhanced in transformed colon mucosa cells possibly to counteract proliferation via CBRs. Inhibitors of endocannabinoid inactivation may prove useful anticancer agents.

The amounts, in nine different rat brain regions, of the two endocannabinoids, anandamide (arachidonoylethanolamide, AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), and of the putative AEA precursor N-arachidonoyl-phosphatidylethanolamine (NArPE), were determined by isotope-dilution gas chromatography-mass spectrometry and compared to the number of cannabinoid binding sites in each region. The distribution of NArPE, reported here for the first time, exhibited a good correlation with that of AEA, the former metabolite being 3-13 times more abundant than the endocannabinoid in all regions. The highest amounts of both metabolites (up to 358.5 and 87 pmol/g wet weight tissue, respectively) were found in the brainstem and striatum, and the lowest in the diencephalon, cortex, and cerebellum. These data support the hypothesis that, in the brain, AEA is a metabolic product of NArPE and may reach levels compatible with its proposed neuromodulatory function. The brain distribution of <em>2</em>-AG, also described in this study for the first time, was found to correlate with that of AEA with levels ranging from <em>2</em>.0 to 14.0 nmol/g (in the diencephalon and brainstem, respectively). The distribution of the endocannabinoids did not match exactly with that of cannabinoid binding sites, suggesting either that these compounds are not necessarily produced near their molecular targets, or that they play functional roles additional to the activation of cannabinoid receptors. Regional differences in the ligand/receptor ratios may also lead to predict corresponding differences in the efficiency of receptor activation, as shown by previous studies.

The endocannabinoid system is a neuroactive lipid signaling system that functions to gate synaptic transmitter release. Accumulating evidence has demonstrated that this system is responsive to modulation by both stress and glucocorticoids within the hypothalamus and limbic structures; however, the nature of this regulation is more complex than initially assumed. The aim of the current review is to summarize the research to date which examines the effects of acute stress and glucocorticoid administration on endocannabinoid signaling in limbic-hypothalamic-pituitary-adrenal (LHPA) axis, and in turn the role endocannabinoid signaling plays in the neurobehavioural responses to acute stress and glucocorticoid administration. The majority of research suggests that acute stress produces a mobilization of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) while concurrently reducing the tissue content of the other endocannabinoid ligand anandamide. Genetic and pharmacological studies demonstrate that the reduction in anandamide signaling may be involved in the initiation of HPA axis activation and the generation of changes in emotional behaviour, while the increase in <em>2</em>-AG signaling may be involved in terminating the stress response, limiting neuronal activation and contributing to changes in motivated behaviours. Collectively, these studies reveal a complex interplay between endocannabinoids and the HPA axis, and further identify endocannabinoid signaling as a critical regulator of the stress response.

Central endocannabinoid signaling is known to be responsive to stressful stimuli; however, there is no research to date characterizing the effects of stress on peripheral endocannabinoid content. The current study examined serum content of the endocannabinoid ligands N-arachidonylethanolamide (anandamide; AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), and the non-cannabinoid N-acyl ethanolamine (NAE) molecules palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) under basal conditions, immediately following the Trier Social Stress Test (TSST), and 30 min thereafter, in 15 medication-free women diagnosed with major depression, and 15 healthy matched controls. Basal serum concentrations of AEA and <em>2</em>-AG, but not PEA or OEA, were significantly reduced in women with major depression relative to matched controls, indicating a deficit in peripheral endocannabinoid activity. Immediately following the TSST, serum <em>2</em>-AG concentrations were increased compared to baseline; serum AEA concentration was unchanged at this time point. Serum concentrations of PEA and OEA were significantly lower than baseline 30 min following the cessation of the TSST. The magnitude of these responses did not differ between depressed and control subjects. These are the first data to demonstrate that the peripheral endocannabinoid/NAE system is responsive to exposure to stress.

Recent isothiocyanate covalent labeling studies have suggested that a classical cannabinoid, (-)-7'-isothiocyanato-11-hydroxy-1',1'dimethylheptyl-hexahydrocannabinol (AM841), enters the cannabinoid CB<em>2</em> receptor via the lipid bilayer (Pei, Y., Mercier, R. W., Anday, J. K., Thakur, G. A., Zvonok, A. M., Hurst, D., Reggio, P. H., Janero, D. R., and Makriyannis, A. (<em>2</em>008) Chem. Biol. 15, 1<em>2</em>07-1<em>2</em>19). However, the sequence of steps involved in such a lipid pathway entry has not yet been elucidated. Here, we test the hypothesis that the endogenous cannabinoid sn-<em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) attains access to the CB<em>2</em> receptor via the lipid bilayer. To this end, we have employed microsecond time scale all-atom molecular dynamics (MD) simulations of the interaction of <em>2</em>-AG with CB<em>2</em> via a palmitoyl-oleoyl-phosphatidylcholine lipid bilayer. Results suggest the following: 1) <em>2</em>-AG first partitions out of bulk lipid at the transmembrane alpha-helix (TMH) 6/7 interface; <em>2</em>) <em>2</em>-AG then enters the CB<em>2</em> receptor binding pocket by passing between TMH6 and TMH7; 3) the entrance of the <em>2</em>-AG headgroup into the CB<em>2</em> binding pocket is sufficient to trigger breaking of the intracellular TMH3/6 ionic lock and the movement of the TMH6 intracellular end away from TMH3; and 4) subsequent to protonation at D3.49/D6.30, further <em>2</em>-AG entry into the ligand binding pocket results in both a W6.48 toggle switch change and a large influx of water. To our knowledge, this is the first demonstration via unbiased molecular dynamics that a ligand can access the binding pocket of a class A G protein-coupled receptor via the lipid bilayer and the first demonstration via molecular dynamics of G protein-coupled receptor activation triggered by a ligand binding event.

Recent studies have shown that activation of the cannabinoid CB(1) receptor by synthetic agonists, and pharmacological elevation of endocannabinoid levels, suppress hyperalgesia and allodynia in animal models of neuropathic pain. However, the concentrations of endocannabinoids in the nervous tissues involved in pain transmission during neuropathic pain have never been measured. Here we have determined the levels of anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), as well as of the analgesic anandamide congener, palmitoylethanolamide (PEA), in three brain areas involved in nociception, i.e. the dorsal raphe (DR), periaqueductal grey (PAG) and rostral ventral medulla (RVM), as well as in the spinal cord (SC), following chronic constriction injury (CCI) of the sciatic nerve in the rat, in comparison with sham-operated rats. After 3 days from CCI, anandamide or <em>2</em>-AG levels were significantly enhanced only in the SC or PAG, respectively. After 7 days from CCI, when thermal hyperalgesia and mechanical allodynia are maximal, a strong (1.3-3-fold) increase of both anandamide and <em>2</em>-AG levels was observed in the PAG, RVM and SC. At this time point, anandamide, but not <em>2</em>-AG, levels were also enhanced in the DR. PEA levels were significantly decreased in the SC after 3 days, and in the DR and RVM after 7 days from CCI. These data indicate that anandamide and <em>2</em>-AG, operating at both spinal and supra-spinal levels, are up-regulated during CCI of the sciatic nerve, possibly to inhibit pain. Yet to be developed substances that inhibit both endocannabinoid and PEA inactivation might be useful for the treatment of neuropathic pain.

Cannabinoids act via cell surface G protein-coupled receptors (CB(1) and CB(<em>2</em>)) and the ion channel receptor TRPV1. Evidence has now emerged suggesting that an additional target is the peroxisome proliferator-activated receptor (PPAR) family of nuclear receptors. There are three PPAR subtypes alpha, delta (also known as beta) and gamma, which regulate cell differentiation, metabolism and immune function. The major endocannabinoids, anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, and ajulemic acid, a structural analogue of the phytocannabinoid Delta(9)-tetrahydrocannabinol (THC), have anti-inflammatory properties mediated by PPARgamma. Other cannabinoids which activate PPARgamma include N-arachidonoyl-dopamine, THC, cannabidiol, HU<em>2</em>10, WIN55<em>2</em>1<em>2</em>-<em>2</em> and CP55940. The endogenous acylethanolamines, oleoylethanolamide and palmitoylethanolamide regulate feeding and body weight, stimulate fat utilization and have neuroprotective effects mediated through PPARalpha. Other endocannabinoids that activate PPARalpha include anandamide, virodhamine and noladin ether. There is, as yet, little direct evidence for interactions of cannabinoids with PPARdelta. There is a convergence of effects of cannabinoids, acting via cell surface and nuclear receptors, on immune cell function which provides promise for the targeted therapy of a variety of immune, particularly neuroinflammatory, diseases.

The endocannabinoid system was revealed following the understanding of the mechanism of action of marijuana's major psychotropic principle, Δ(9)-tetrahydrocannabinol, and includes two G-protein-coupled receptors (GPCRs; the cannabinoid CB1 and CB<em>2</em> receptors), their endogenous ligands (the endocannabinoids, the best studied of which are anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG)), and the proteins that regulate the levels and activity of these receptors and ligands. However, other minor lipid metabolites different from, but chemically similar to, anandamide and <em>2</em>-AG have also been suggested to act as endocannabinoids. Thus, unlike most other GPCRs, cannabinoid receptors appear to have more than one endogenous agonist, and it has been often wondered what could be the physiological meaning of this peculiarity. In 1999, it was proposed that anandamide might also activate other targets, and in particular the transient receptor potential of vanilloid type-1 (TRPV1) channels. Over the last decade, this interaction has been shown to occur both in peripheral tissues and brain, during both physiological and pathological conditions. TRPV1 channels can be activated also by another less abundant endocannabinoid, N-arachidonoyldopamine, but not by <em>2</em>-AG, and have been proposed by some authors to act as ionotropic endocannabinoid receptors. This article will discuss the latest discoveries on this subject, and discuss, among others, how anandamide and <em>2</em>-AG differential actions at TRPV1 and cannabinoid receptors contribute to making this signalling system a versatile tool available to organisms to fine-tune homeostasis.

Macrophage-derived endocannabinoids have been implicated in endotoxin (lipopolysaccharide (LPS))-induced hypotension, but the endocannabinoid involved and the mechanism of its regulation by LPS are unknown. In RAW<em>2</em>64.7 mouse macrophages, LPS (10 ng/ml) increases anandamide (AEA) levels >10-fold via CD14-, NF-kappaB-, and p44/4<em>2</em>-dependent, platelet-activating factor-independent activation of the AEA biosynthetic enzymes, N-acyltransferase and phospholipase D. LPS also induces the AEA-degrading enzyme fatty acid amidohydrolase (FAAH), and inhibition of FAAH activity potentiates, whereas actinomycin D or cycloheximide blocks the LPS-induced increase in AEA levels and N-acyltransferase and phospholipase D activities. In contrast, cellular levels of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are unaffected by LPS but increased by platelet-activating factor. LPS similarly induces AEA, but not <em>2</em>-AG, in mouse peritoneal macrophages where basal AEA levels are higher, and the LPS-stimulated increase in AEA is potentiated in cells from FAAH-/- as compared with FAAH+/+ mice. Intravenous administration of 107 LPS-treated mouse macrophages to anesthetized rats elicits hypotension, which is much greater in response to FAAH-/- than FAAH+/+ cells and is susceptible to inhibition by SR141716, a cannabinoid CB1 receptor antagonist. We conclude that AEA and <em>2</em>-AG synthesis are differentially regulated in macrophages, and AEA rather than <em>2</em>-AG is a major contributor to LPS-induced hypotension.