Addiction as a psychiatric disorder involves interaction of inherited predispositions and environmental factors. Similarly to humans, laboratory animals self-administer addictive drugs, whose appetitive properties result from activation and suppression of brain reward and aversive pathways, respectively. The ventral tegmental area (VTA) where dopamine (DA) cells are located is a key component of brain reward circuitry, whereas the rostromedial tegmental nucleus (RMTg) critically regulates aversive behaviors. Reduced responses to either aversive intrinsic components of addictive drugs or to negative consequences of compulsive drug taking might contribute to vulnerability to addiction. In this regard, female Lister Hooded (LH) rats are more vulnerable than male counterparts to cannabinoid self-administration. We, therefore, took advantage of sex differences displayed by LH rats, and studied VTA DA neuronal properties to unveil functional differences. Electrophysiological properties of DA cells were examined performing either single cell extracellular recordings in anesthetized rats or whole-cell patch-clamp recordings in slices. In vivo, DA cell spontaneous activity was similar, though sex differences were observed in RMTg-induced inhibition of DA neurons. In vitro, DA cells showed similar intrinsic and synaptic properties. However, females displayed larger depolarization-induced suppression of inhibition (DSI) than male LH rats. DSI, an endocannabinoid-mediated form of short term plasticity, was mediated by <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) activating type 1-cannabinoid (CB1) receptors. We found that sex-dependent differences in DSI magnitude were not ascribed to CB1 number and/or function, but rather to a tonic <em>2</em>-AG signaling. We suggest that sex specific tonic <em>2</em>-AG signaling might contribute to regulate responses to aversive intrinsic properties to cannabinoids, thus resulting in faster acquisition/initiation of cannabinoid taking and, eventually, in progression to addiction.

Numerous studies have implicated the endocannabinoid system in the pathophysiology of schizophrenia. Endocannabinoids have been measured in blood and cerebrospinal fluid in schizophrenic patients but, to the date, there are no published reports dealing with measurements of endocannabinoid levels in schizophrenics' brain tissue. In the present study, postmortem brain samples from 19 subjects diagnosed with schizophrenia (DSM-IV) and 19 matched controls were studied. In specific brain regions, levels of four endocannabinoids (<em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), arachidonoylethanolamine (anandamide, AEA), dihomo-γ-linolenoylethanolamine (LEA), and docosahexaenoylethanolamine (DHEA)) and two cannabimimetic compounds (palmitoyl-ethanolamine (PEA) and oleoyl-ethanolamine (OEA)) were measured using quantitative liquid chromatography with triple quadrupole mass spectrometric detection. Suffering from schizophrenia significantly affects the brain levels of <em>2</em>-AG (p<0.001), AEA (p<0.0001), DHEA (p<0.0001), LEA (p<0.01) and PEA (p<0.05). In schizophrenic subjects, the three studied brain regions (cerebellum: 130±18%; p=0.16; hippocampus: 168±<em>2</em>8%, p<0.01; prefrontal cortex: <em>2</em>37±45%, p<0.05) showed higher <em>2</em>-AG levels when compared to matched controls. Conversely, AEA levels were lower in all brain regions of schizophrenic subjects (cerebellum: 66±7%, p<0.01; hippocampus: 66±7%, p<0.01; prefrontal cortex: 75±10%, p=0.07). Statistically significant lower levels of DHEA were also found in cerebellum (60±6%, p<0.001) and hippocampus (68±7%, p<0.05) of schizophrenic subjects. PEA (71±6%, p<0.05) and LEA (7<em>2</em>±6%, p<0.05) levels were also found to be lower in cerebellum. No significant differences were found in OEA levels. Our results evidence specific alterations in the levels of some endocannabinoids in different brain regions of schizophrenic subjects. Furthermore, these data evidence the involvement of the endocannabinoid system in the pathophysiology of schizophrenia.

Stress is a ubiquitous risk factor for the exacerbation and development of affective disorders including major depression and posttraumatic stress disorder. Understanding the neurobiological mechanisms conferring resilience to the adverse consequences of stress could have broad implications for the treatment and prevention of mood and anxiety disorders. We utilize laboratory mice and their innate inter-individual differences in stress-susceptibility to demonstrate a critical role for the endogenous cannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in stress-resilience. Specifically, systemic <em>2</em>-AG augmentation is associated with a stress-resilient phenotype and enhances resilience in previously susceptible mice, while systemic <em>2</em>-AG depletion or CB1 receptor blockade increases susceptibility in previously resilient mice. Moreover, stress-resilience is associated with increased phasic <em>2</em>-AG-mediated synaptic suppression at ventral hippocampal-amygdala glutamatergic synapses and amygdala-specific <em>2</em>-AG depletion impairs successful adaptation to repeated stress. These data indicate amygdala <em>2</em>-AG signalling mechanisms promote resilience to adverse effects of acute traumatic stress and facilitate adaptation to repeated stress exposure.

Recent evidence points to the gut microbiota as a regulator of brain and behavior, although it remains to be determined if gut bacteria play a role in chronic pain. The endocannabinoid system is implicated in inflammation and chronic pain processing at both the gut and central nervous system (CNS) levels. In the present study, we used low Vitamin D dietary intake in mice and evaluated possible changes in gut microbiota, pain processing and endocannabinoid system signaling. Vitamin D deficiency induced a lower microbial diversity characterized by an increase in Firmicutes and a decrease in Verrucomicrobia and Bacteroidetes. Concurrently, vitamin D deficient mice showed tactile allodynia associated with neuronal hyperexcitability and alterations of endocannabinoid system members (endogenous mediators and their receptors) at the spinal cord level. Changes in endocannabinoid (anandamide and <em>2</em>-<em>arachidonoylglycerol</em>) levels were also observed in the duodenum and colon. Remarkably, the anti-inflammatory anandamide congener, palmitoylethanolamide, counteracted both the pain behaviour and spinal biochemical changes in vitamin D deficient mice, whilst increasing the levels of Akkermansia, Eubacterium and Enterobacteriaceae, as compared with vehicle-treated mice. Finally, induction of spared nerve injury in normal or vitamin D deficient mice was not accompanied by changes in gut microbiota composition. Our data suggest the existence of a link between Vitamin D deficiency - with related changes in gut bacterial composition - and altered nociception, possibly via molecular mechanisms involving the endocannabinoid and related mediator signaling systems.

Inhibitors of the metabolism of the endogenous cannabinoid ligand anandamide by fatty acid amide hydrolase (FAAH) reduce the gastric damage produced by non-steroidal anti-inflammatory agents and synergise with them in experimental pain models. This motivates the design of compounds with joint FAAH/cyclooxygenase (COX) inhibitory activity. Here we present data on the N-(3-methylpyridin-<em>2</em>-yl)amide derivatives of flurbiprofen and naproxen (Flu-AM1 and Nap-AM1, respectively) with respect to their properties towards these two enzymes. Flu-AM1 and Nap-AM1 inhibited FAAH-catalysed hydrolysis of [(3)H]anandamide by rat brain homogenates with IC50 values of 0.44 and 0.74 µM. The corresponding values for flurbiprofen and naproxen were <em>2</em>9 and >100 µM, respectively. The inhibition by Flu-AM1 was reversible, mixed-type, with K(i)slope and K(i)intercept values of 0.<em>2</em>1 and 1.4 µM, respectively. Flurbiprofen and Flu-AM1 both inhibited COX in the same manner with the order of potencies COX-<em>2</em> vs. <em>2</em>-<em>arachidonoylglycerol</em>)COX-1 vs. arachidonic acid>COX-<em>2</em> vs. arachidonic acid with flurbiprofen being approximately <em>2</em>-3 fold more potent than Flu-AM1 in the assays. Nap-AM1 was a less potent inhibitor of COX. Flu-AM1 at low micromolar concentrations inhibited the FAAH-driven uptake of [(3)H]anandamide into RBL<em>2</em>H3 basophilic leukaemia cells in vitro, but did not penetrate the brain in vivo sufficiently to block the binding of [(18)F]DOPP to brain FAAH. It is concluded that Flu-AM1 is a dual-action inhibitor of FAAH and COX that may be useful in exploring the optimal balance of effects on these two enzyme systems in producing peripheral alleviation of pain and inflammation in experimental models.

Omega-3 and omega-6 fatty acids are biosynthetic precursors of endocannabinoids with antinociceptive, anxiolytic, and neurogenic properties. We recently reported that targeted dietary manipulation-increasing omega-3 fatty acids while reducing omega-6 linoleic acid (the H3-L6 intervention)-reduced headache pain and psychological distress among chronic headache patients. It is not yet known whether these clinical improvements were due to changes in endocannabinoids and related mediators derived from omega-3 and omega-6 fatty acids. We therefore used data from this trial (N = 55) to investigate 1) whether the H3-L6 intervention altered omega-3- and omega-6-derived endocannabinoids in plasma and <em>2</em>) whether diet-induced changes in these bioactive lipids were associated with clinical improvements. The H3-L6 intervention significantly increased the omega-3 docosahexaenoic acid derivatives <em>2</em>-docosahexaenoylglycerol (+65%, P < .001) and docosahexaenoylethanolamine (+99%, P < .001) and reduced the omega-6 arachidonic acid derivative <em>2</em>-<em>arachidonoylglycerol</em> (-<em>2</em>5%, P = .001). Diet-induced changes in these endocannabinoid derivatives of omega-3 docosahexaenoic acid, but not omega-6 arachidonic acid, correlated with reductions in physical pain and psychological distress. These findings demonstrate that targeted dietary manipulation can alter endocannabinoids derived from omega-3 and omega-6 fatty acids in humans and suggest that <em>2</em>-docosahexaenoylglycerol and docosahexaenoylethanolamine could have physical and/or psychological pain modulating properties.

BACKGROUND

ClinicalTrials.gov (NCT01157<em>2</em>08) PERSPECTIVE: This article demonstrates that targeted dietary manipulation can alter endocannabinoids derived from omega-3 and omega-6 fatty acids and that these changes are related to reductions in headache pain and psychological distress. These findings suggest that dietary interventions could provide an effective, complementary approach for managing chronic pain and related conditions.

Little is known as to the structural requirements of the acyl side chain for interaction of acylglycerols with monoacylglycerol lipase (MAGL), the enzyme chiefly responsible for the metabolism of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in the brain. In the present study, a series of twelve analogues of 1-AG (the more stable regioisomer of <em>2</em>-AG) were investigated with respect to their ability to inhibit the metabolism of <em>2</em>-oleoylglycerol by cytosolic and membrane-bound MAGL. In addition, the ability of the compounds to inhibit the hydrolysis of anandamide by fatty acid amide hydrolase (FAAH) was investigated. For cytosolic MAGL, compounds with <em>2</em>0 carbon atoms in the acyl chain and <em>2</em>-5 unsaturated bonds inhibited the hydrolysis of <em>2</em>-oleoylglycerol with similar potencies (IC50 values in the range 5.1-8.<em>2</em> microM), whereas the two compounds with a single unsaturated bond were less potent (IC50 values 19 and <em>2</em>1 microM). The fully saturated analogue 1-monoarachidin did not inhibit the enzyme, whereas the lower side chain analogues 1-monopalmitin and 1-monomyristin inhibited the enzyme with IC50 values of 1<em>2</em> and 3<em>2</em> microM, respectively. The <em>2</em><em>2</em>-carbon chain analogue of 1-AG was also potent (IC50 value 4.5 microM). Introduction of an alpha-methyl group for the C<em>2</em>0:4, C<em>2</em>0:3, and C<em>2</em><em>2</em>:4 compounds did not affect potency in a consistent manner. For the FAAH and the membrane-bound MAGL, there was no obvious relationship between the degree of unsaturation of the acyl side chain and the ability to inhibit the enzymes. It is concluded that increasing the number of unsaturated bonds on the acyl side chain of 1-AG from 1 to 5 has little effect on the affinity of acylglycerols for cytosolic MAGL.

The xenoestrogen bisphenol A (BPA) is a widespread plasticizer detectable within several ecosystems. BPA is considered a metabolic disruptor, affecting different organs; however, little is known about its mechanism of action in the liver, in which it triggers triglyceride accumulation. Adult zebrafish (Danio rerio) exposed to BPA developed hepatosteatosis, which was associated with an increase in the liver levels of the obesogenic endocannabinoids <em>2</em>-<em>arachidonoylglycerol</em> and anandamide and a concomitant decrease in palmitoylethanolamide. These changes were associated with variations in the expression of key endocannabinoid catabolic and metabolic enzymes and an increase in the expression of the endocannabinoid receptor cnr1. Acute and chronic in vitro treatments with nano- and micromolar BPA doses showed increased anandamide levels in line with decreased activity of fatty acid amide hydrolase, the main anandamide hydrolytic enzyme, and induced triglyceride accumulation in HHL-5 cells in a CB1-dependent manner. We conclude that BPA is able to produce hepatosteatosis in zebrafish and human hepatocytes by up-regulating the endocannabinoid system.

Endocannabinoids, including <em>2</em>-<em>arachidonoylglycerol</em> and anandamide (N-arachidonoylethanolamine; AEA), have neuroprotective effects in the brain through actions at CB1 receptors. However, AEA also binds to vanilloid (VR1) receptors and induces cell death in several cell lines. Here we show that anandamide causes neuronal cell death in vitro and exacerbates cell loss caused by stretch-induced axonal injury or trophic withdrawal in rat primary neuronal cultures. Administered intracerebroventricularly, AEA causes sustained cerebral edema, as reflected by diffusion-weighted magnetic resonance imaging, regional cell loss, and impairment in long-term cognitive function. These effects are mediated, in part, through VR1 as well as through calpain-dependent mechanisms, but not through CB1 receptors or caspases. Central administration of AEA also significantly upregulates genes involved in pro-inflammatory/microglial-related responses. Thus, anandamide produces neurotoxic effects both in vitro and in vivo through multiple mechanisms independent of the CB1 receptor.

Cannabinoid (CB) agonists suppress nausea in humans and animal models; yet, their underlying neural substrates remain largely unknown. Evidence suggests that the visceral insular cortex (VIC) plays a critical role in nausea. Given the expression of CB1 receptors and the presence of endocannabinoids in this brain region, we hypothesized that the VIC endocannabinoid system regulates nausea. In the present study, we assessed whether inhibiting the primary endocannabinoid hydrolytic enzymes in the VIC reduces acute lithium chloride (LiCl)-induced conditioned gaping, a rat model of nausea. We also quantified endocannabinoid levels during an episode of nausea, and assessed VIC neuronal activation using the marker, c-Fos. Local inhibition of monoacylglycerol lipase (MAGL), the main hydrolytic enzyme of <em>2</em>-arachidonylglycerol (<em>2</em>-AG), reduced acute nausea through a CB1 receptor mechanism, whereas inhibition of fatty acid amide hydrolase (FAAH), the primary catabolic enzyme of anandamide (AEA), was without effect. Levels of <em>2</em>-AG were also selectively elevated in the VIC during an episode of nausea. Inhibition of MAGL robustly increased <em>2</em>-AG in the VIC, while FAAH inhibition had no effect on AEA. Finally, we demonstrated that inhibition of MAGL reduced VIC Fos immunoreactivity in response to LiCl treatment. Taken together, these findings provide compelling evidence that acute nausea selectively increases <em>2</em>-AG in the VIC, and suggests that <em>2</em>-AG signaling within the VIC regulates nausea by reducing neuronal activity in this forebrain region.

<AbstractText>The endocannabinoid system (ECS) is a major regulator of synaptic plasticity and neuromodulation. Alterations of the ECS have been demonstrated in several animal models of autism spectrum disorder (ASD). In some of these models, activating the ECS rescued the social deficits. Evidence for dysregulations of the ECS in human ASD are emerging, but comprehensive assessments and correlations with disease characteristics have not been reported yet.</AbstractText><p><div><b>Methods</b></div>Serum levels of the main endocannabinoids, <i>N-</i>arachidonoylethanolamine (AEA or anandamide) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), and their related endogenous compounds, arachidonic acid (AA), <i>N</i>-palmitoylethanolamine (PEA), and <i>N</i>-oleoylethanolamine (OEA), were analyzed by liquid chromatography/tandem mass spectrometry in 93 children with ASD (age = 13.1 ± 4.1, range 6-<em>2</em>1; 79% boys) and 93 age- and gender-matched neurotypical children (age = 11.8 ± 4.3, range 5.5-<em>2</em>1; 79% boys). Results were associated with gender and use of medications, and were correlated with age, BMI, and adaptive functioning of ASD participants as reflected by scores of Autism Diagnostic Observation Schedule (ADOS-<em>2</em>), Vineland Adaptive Behavior Scale-II (VABS-II), and Social Responsiveness Scale-II (SRS-<em>2</em>).</p><p><div><b>Results</b></div>Children with ASD had lower levels (pmol/mL, mean ± SEM) of AEA (0.7<em>2</em><em>2</em> ± 0.045 vs. 1.<em>2</em>5<em>2</em> ± 0.07<em>2</em>, <i>P</i> < 0.0001, effect size 0.91), OEA (17.3 ± 0.80 vs. <em>2</em>7.8 ± 1.44, <i>P</i> < 0.0001, effect size 0.94), and PEA (4.93 ± 0.3<em>2</em> vs. 7.15 ± 0.37, <i>P</i> < 0.0001, effect size 0.65), but not AA and <em>2</em>-AG. Serum levels of AEA, OEA, and PEA were not significantly associated or correlated with age, gender, BMI, medications, and adaptive functioning of ASD participants. In children with ASD, but not in the control group, younger age and lower BMI tended to correlate with lower AEA levels. However, these correlations were not statistically significant after a correction for multiple comparisons.</p><AbstractText>We found lower serum levels of AEA, PEA, and OEA in children with ASD. Further studies are needed to determine whether circulating endocannabinoid levels can be used as stratification biomarkers that identify clinically significant subgroups within the autism spectrum and if they reflect lower endocannabinoid "tone" in the brain, as found in animal models of ASD.</AbstractText>

Increasing evidence suggests a close relationship between the endocannabinoid system and schizophrenia. The endocannabinoid system comprises of two G protein-coupled receptors (the cannabinoid receptors 1 and <em>2</em> [CB1 and CB<em>2</em>] for marijuana's psychoactive principle Δ(9)-tetrahydrocannabinol), their endogenous small lipid ligands (namely anandamide [AEA] and <em>2</em>-<em>arachidonoylglycerol</em> [<em>2</em>-AG], also known as endocannabinoids), and proteins for endocannabinoid biosynthesis and degradation. It has been suggested to be a pro-homeostatic and pleiotropic signalling system activated in a time- and tissue-specific manner during pathophysiological conditions. In the brain, activation of this system impacts the release of numerous neurotransmitters in various systems and cytokines from glial cells. Hence, the endocannabinoid system is strongly involved in neuropsychiatric disorders, such as schizophrenia. Therefore, adolescence use of Cannabis may alter the endocannabinoid signalling and pose a potential environmental risk to develop psychosis. Consistently, preclinical and clinical studies have found a dysregulation in the endocannabinoid system such as changed expression of CB1 and CB<em>2</em> receptors or altered levels of AEA and <em>2</em>-AG . Thus, due to the partial efficacy of actual antipsychotics, compounds which modulate this system may provide a novel therapeutic target for the treatment of schizophrenia. The present article reviews current available knowledge on herbal, synthetic and endogenous cannabinoids with respect to the modulation of schizophrenic symptomatology. Furthermore, this review will be highlighting the therapeutic potential of cannabinoid-related compounds and presenting some promising patents targeting potential treatment options for schizophrenia.

The endocannabinoid system is dysregulated during obesity in tissues involved in the control of food intake and energy metabolism. We examined the effect of chronic exercise on the tissue levels of endocannabinoids (eCBs) and on the expression of genes coding for cannabinoid receptor 1 (CB1) and cannabinoid receptor <em>2</em> (CB<em>2</em>) (Cnr1 and Cnr<em>2</em>, respectively) in the subcutaneous (SAT) and visceral adipose tissues and in the soleus and extensor digitorim longus (EDL) muscles, in rats fed with standard or high-fat diet. Twenty-eight male Wistar rats were placed on high-fat diet or standard diet (HFD and Ctl groups, respectively) during 1<em>2</em> weeks whereafter half of each group was submitted to an exercise training period of 1<em>2</em> weeks (HFD + training and Ctl + training). Tissue levels of eCBs were measured by LC-MS while expressions of genes coding for CB1 and CB<em>2</em> receptors were investigated by qPCR. High-fat diet induced an increase in anandamide (AEA) levels in soleus and EDL (p < 0.0<em>2</em>). In soleus of the HFD group, these changes were accompanied by elevated Cnr1 messenger RNA (mRNA) levels (p < 0.05). In EDL, exercise training allowed to reduce significantly this diet-induced AEA increase (p < 0.005). <em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG) levels were decreased and increased by high-fat diet in SAT and EDL, respectively (p < 0.04), but not affected by exercise training. Unlike the HFD + training group, <em>2</em>-AG levels in soleus were also decreased in the HFD group compared to Ctl (p < 0.04). The levels of eCBs and Cnr1 expression are altered in a tissue-specific manner following a high-fat diet, and chronic exercise reverses some of these alterations.

High-content screening led to the identification of the N-isobutylamide guineensine from Piper nigrum as novel nanomolar inhibitor (EC50=<em>2</em>90nM) of cellular uptake of the endocannabinoid anandamide (AEA). Noteworthy, guineensine did not inhibit endocannabinoid degrading enzymes fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL) nor interact with cannabinoid receptors or fatty acid binding protein 5 (FABP5), a major cytoplasmic AEA carrier. Activity-based protein profiling showed no inhibition of serine hydrolases. Guineensine also inhibited the cellular uptake of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Preliminary structure-activity relationships between natural guineensine analogs indicate the importance of the alkyl chain length interconnecting the pharmacophoric isobutylamide and benzodioxol moieties for AEA cellular uptake inhibition. Guineensine dose-dependently induced cannabimimetic effects in BALB/c mice shown by strong catalepsy, hypothermia, reduced locomotion and analgesia. The catalepsy and analgesia were blocked by the CB1 receptor antagonist rimonabant (SR141716A). Guineensine is a novel plant natural product which specifically inhibits endocannabinoid uptake in different cell lines independent of FAAH. Its scaffold may be useful to identify yet unknown targets involved in endocannabinoid transport.

Previous reports have shown improvements in mood and increases in endocannabinoids in healthy adults following a session of aerobic exercise, but it is unclear whether adults with posttraumatic stress disorder (PTSD) experience similar responses. The purpose of this study was to examine psychobiological responses (plasma endocannabinoids [eCBs], mood, and pain) to aerobic exercise in a sample of adults with a diagnosis of PTSD (n = 1<em>2</em>) and healthy controls (n = 1<em>2</em>). Participants engaged in an aerobic exercise session in which they ran on a treadmill for 30 min at a moderate intensity (70 to 75% maximum heart rate [MHR]). Results indicated improvements in mood states and reductions in pain for both groups following exercise, ds = 0.19 to 1.53. Circulating concentrations of N-arachidonylethanolamine (AEA), <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), and oleoylethanolamide (OEA) significantly increased (ps = .000 to .050) following the aerobic exercise session for both groups. There were no significant time, group, or interaction effects (ps = .06<em>2</em> to .846) for palmitoylethanolamide (PEA) and <em>2</em>-oleoylglycerol (<em>2</em>-OG). Although eCBs increased significantly for both groups, within-group effect size calculations indicated the healthy controls experienced a greater magnitude of change for AEA when compared with adults with PTSD, d = 1.<em>2</em>1 and d = 0.45, respectively; as well as for <em>2</em>-AG, d = 0.43 and d = 0.<em>2</em>1, respectively. The findings from this study indicated that adults with and without PTSD reported significant mood improvements following 30 min of moderate-intensity aerobic exercise. In addition, the endocannabinoid system was activated in adults with and without PTSD, although effect sizes suggest that adults with PTSD may have a blunted endocannabinoid response to exercise.

Acute aerobic exercise improves mood and activates the endocannabinoid (eCB) system in physically active individuals; however, both mood and eCB responses to exercise may vary based on habitual levels of physical activity.

This study aimed to examine eCB and mood responses to prescribed and preferred exercises among individuals with low, moderate, and high levels of physical activity.

Thirty-six healthy adults (<em>2</em>1 ± 4 yr) were recruited from low (≤60 min moderate-vigorous physical activity [MVPA] per week), moderate (150-<em>2</em>99 min MVPA per week), and high (≥300 MVPA per week) physical activity groups. Participants performed both prescribed (approximately 70%-75% max) and preferred (i.e., self-selected) aerobic exercise on separate days. Mood states and eCB concentrations were assessed before and after exercise conditions.

Both preferred and prescribed exercise resulted in significant increases (P < 0.01) in circulating eCB (N-arachidonoylethanolamine [AEA] and <em>2</em>-<em>arachidonoylglycerol</em>); however, increases in AEA (P < 0.05) were larger in the prescribed condition. Likewise, both preferred and prescribed exercise elicited positive mood improvements compared with preexercise values, but changes in state anxiety, total mood disturbance, and confusion were greater in the preferred condition (P < 0.05). Changes in <em>2</em>-<em>arachidonoylglycerol</em> concentrations were found to negatively correlate with changes in depression, tension, and total mood disturbance in the preferred condition (P < 0.05), and changes in AEA were positively associated with changes in vigor in the prescribed condition (P < 0.05). There were no significant group differences for mood or eCB outcomes.

These results indicate that eCB and mood responses to exercise do not differ significantly between samples with varying physical activity levels. This study also demonstrates that in addition to prescribed exercise, preferred exercise activates the eCB system, and this activation may contribute to positive mood outcomes with exercise.

The expression of genes encoding the cannabinoid CB(1) and CB(<em>2</em>) receptors and fatty acid amide hydrolase (FAAH) and the lipolytic activity of cannabinoid agonists were investigated in rat adipose tissue.RT-PCR studies indicated that the genes encoding CB(1) and CB(<em>2</em>) receptors and FAAH are not expressed in epididymal adipocytes. In functional studies, the non-selective cannabinoid receptor agonist WIN 55,<em>2</em>1<em>2</em>-<em>2</em> concentration-dependently (0.01-30 micro M) induced glycerol release above baseline ( E(max) 96.1+/-6.<em>2</em>% of isoprenaline-induced lipolytic response). The selective CB(<em>2</em>) agonist JWH-015 (0.01-30 micro M) had no lipolytic activity while the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> and the stable anandamide derivative, R(+)-methanandamide had, only a weak lipolytic effect at the highest concentrations employed (10 and 30 micro M). The concentration/response relationship for WIN 55,<em>2</em>1<em>2</em>-<em>2</em>-mediated lipolytic activity, mimicked by the S(-)-enantiomer WIN 55,<em>2</em>1<em>2</em>-3, was shifted significantly to the right by the CB(1) antagonist AM <em>2</em>51 only at 10 micro M, but was not modified by the beta-adrenoceptor antagonist propranolol (1 micro M). The protein kinase inhibitor H-89, but not the two adenylyl cyclase inhibitors (+/-) N(6)- R-phenylisopropyladenosine (R-PIA, 1 micro M, a selective A(1) adenosine receptor agonist) or SQ <em>2</em><em>2</em>,536 (50 micro M) significantly reduced the glycerol efflux induced by WIN 55,<em>2</em>1<em>2</em>-<em>2</em>. Our data suggest that the cannabinoid drug WIN 55,<em>2</em>1<em>2</em>-<em>2</em> may exert lipolytic activity in male rat adipocytes via an intracellular mechanism, not activated by CB(1) or CB(<em>2</em>) receptor stimulation, significantly reversed by H-89 but not clearly linked to stimulation of adenylyl cyclase.

<em>2</em>-<em>Arachidonoylglycerol</em> is an endogenous ligand for the cannabinoid receptors. To date, two types of cannabinoid receptors (CB1 and CB<em>2</em>) have been identified. The CB1 receptor is assumed to be involved in the attenuation of synaptic transmission. On the other hand, the physiological roles of the CB<em>2</em> receptor, which is abundantly expressed in several types of inflammatory cells and immunocompetent cells, have not yet been fully elucidated. Recently, we investigated in detail possible physiological roles of the CB<em>2</em> receptor and <em>2</em>-<em>arachidonoylglycerol</em> in inflammation. We found that <em>2</em>-<em>arachidonoylglycerol</em> induces the activation of p4<em>2</em>/44 and p38 mitogen-activated protein kinases and c-Jun N-terminal kinase; actin rearrangement and morphological changes; augmented production of chemokines in HL-60 cells; and the migration of HL-60 cells differentiated into macrophage-like cells, human monocytes, natural killer cells, and eosinophils. We also found that the level of <em>2</em>-<em>arachidonoylglycerol</em> in mouse ear is markedly elevated following treatment with 1<em>2</em>-O-tetradecanoylphorbol 13-acetate, which induces acute inflammation. Notably, the inflammation induced by 1<em>2</em>-O-tetradecanoylphorbol 13-acetate was blocked by treatment with SR1445<em>2</em>8, a CB<em>2</em>-receptor antagonist. Similar results were obtained with an allergic inflammation model in mice. These results strongly suggest that <em>2</em>-<em>arachidonoylglycerol</em> plays essential roles in the stimulation of various inflammatory reactions in vivo.

OBJECTIVE

Endocannabinoids are a family of lipid mediators involved in the regulation of gastrointestinal (GI) motility. The expression, localization and function of their biosynthetic enzymes in the GI tract are not well understood. Here, we examined the expression, localization and function of the enzyme diacylglycerol lipase-α (DAGLα), which is involved in biosynthesis of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG).

METHODS

Cannabinoid CB1 receptor-deficient, wild-type control and C3H/HeJ mice, a genetically constipated strain, were used. The distribution of DAGLα in the enteric nervous system was examined by immunohistochemistry. Effects of the DAGL inhibitors, orlistat and OMDM-188 on pharmacologically induced GI hypomotility were assessed by measuring intestinal contractility in vitro and whole gut transit or faecal output in vivo. Endocannabinoid levels were measured by mass spectrometry.

RESULTS

DAGLα was expressed throughout the GI tract. In the intestine, unlike DAGLβ, DAGLα immunoreactivity was prominently expressed in the enteric nervous system. In the myenteric plexus, it was colocalized with the vesicular acetylcholine transporter in cholinergic nerves. In normal mice, inhibiting DAGL reversed both pharmacologically reduced intestinal contractility and pharmacologically prolonged whole gut transit. Moreover, inhibiting DAGL normalized faecal output in constipated C3H/HeJ mice. In colons incubated with scopolamine, <em>2</em>-AG was elevated while inhibiting DAGL normalized <em>2</em>-AG levels.

CONCLUSIONS

DAGLα was expressed in the enteric nervous system of mice and its inhibition reversed slowed GI motility, intestinal contractility and constipation through <em>2</em>-AG and CB1 receptor-mediated mechanisms. Our data suggest that DAGLα inhibitors may be promising candidates for the treatment of constipation.

We previously identified a novel common virus integration site, Evi11, by means of retroviral insertional mutagenesis. We demonstrated that the gene encoding the peripheral cannabinoid receptor (Cb<em>2</em>) is the potential target, suggesting that Cb<em>2</em> is a proto-oncogene. To elucidate a role for this G protein-coupled receptor (GPCR) in leukemic transformation we generated a Cb<em>2</em>-EGFP cDNA construct that was introduced into 3<em>2</em>D/G-CSF-R cells. These cells require interleukin 3 (IL-3) to proliferate in vitro, whereas in the presence of granulocyte-colony-stimulating factor (G-CSF) they differentiate toward mature neutrophils. We demonstrate that 3<em>2</em>D/G-CSF-R/Cb<em>2</em>-EGFP cells migrate in a transwell assay in reponse to the Cb<em>2</em> ligand <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), indicating that the fusion protein was functional. When cultured in the presence of G-CSF neutrophilic differentiation of Cb<em>2</em>-EGFP-expressing 3<em>2</em>D/G-CSF-R cells was completely blocked. Moreover, a Cb<em>2</em>-specific antagonist fully recovered the G-CSF-induced neutrophilic differentiation of 3<em>2</em>D/G-CSF-R/Cb<em>2</em>-EGFP cells. To investigate which signal transduction pathway(s) may be involved in the block of neutrophilic maturation, differentiation experiments were carried out using specific inhibitors of signaling routes. Interestingly, full rescue of G-CSF-induced neutrophilic differentiation was observed when cells were cultured with the mitogen-induced extracellular kinase (MEK) inhibitors, PD98059 or U01<em>2</em>6, and partial recovery was detected with the phosphoinositide 3-kinase (PI3-K) inhibitor LY-<em>2</em>9400<em>2</em>. These studies demonstrate that the Cb<em>2</em> receptor is an oncoprotein that blocks neutrophilic differentiation when overexpressed in myeloid precursor cells. Cb<em>2</em> appears to mediate its activity through MEK/extracellular signal-related kinase (ERK) and PI3-K pathways.

There is strong evidence that endocannabinoids modulate signaling of serotonin and noradrenaline, which play key roles in the pathophysiology and treatment of anxiety and depression. Most pharmacological and genetic, human and rodent studies suggest that the presence of under-functioning endocannabinoid type-1 (CB(1)) receptors is associated with increased anxiety and elevated extracellular serotonin concentration. In contrast, noradrenaline is presumably implicated in the mediation of depression-type symptoms of CB(1) receptor antagonists. Evidence shows that most CB(1) receptors located on axons and terminals of GABA-ergic, serotonergic or glutamatergic neurons stimulate the activity of noradrenergic neurons. In contrast, those located on noradrenergic axons and terminals inhibit noradrenaline release efficiently. In this latter process, excitatory ionotropic or G protein-coupled receptors, such as the NMDA, alpha1 and beta1 adrenergic receptors, activate local endocannabinoid synthesis at postsynaptic sites and stimulate retrograde endocannabinoid neurotransmission acting on CB(1) receptors of noradrenergic terminals. The underlying mechanisms include calcium signal generation, which activates enzymes that increase the synthesis of both anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, while G(q/11) protein activation also increases the formation of <em>2</em>-<em>arachidonoylglycerol</em> from diacylglycerol during the signaling process. In addition, other non-CB(1) receptor endocannabinoid targets such as CB(<em>2</em>), transient receptor potential vanilloid subtype, peroxisome proliferator-activated receptor-alpha and possibly GPR55 can also mediate some of the endocannabinoid effects. In conclusion, both neuronal activation and neurotransmitter release depend on the in situ synthesized endocannabinoids and thus, local endocannabinoid concentrations in different brain areas may be crucial in the net effect, namely in the regulation of neurons located postsynaptically to the noradrenergic synapse.

BACKGROUND

N-acylethanolamines (NAEs) are endogenous compounds that regulate inflammation and pain. These include the cannabinoid ligand anandamide (AEA) and the peroxisome proliferator-activated receptor-α ligand palmitoylethanolamide (PEA). Little is known as to the levels of NAEs in pain states in human, particularly in the skeletal muscle. The aim of this study was to investigate the levels of these lipid mediators in muscle dialysate from women with chronic neck-/shoulder pain compared to healthy controls.

METHODS

Eleven women with chronic neck-/shoulder pain and eleven healthy women participated in this study. All participants went through microdialysis procedures in the trapezius muscle. Muscle dialysate samples were collected during four hours and analysed by nano liquid chromatography tandem mass spectrometry (nLC-MS/MS).

RESULTS

We were able to detect AEA, PEA, N-stearoylethanolamine (SEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in a single chromatographic run. Of the NAEs studied, PEA and SEA were clearly detectable in the muscle microdialysate samples. The muscle dialysate levels of PEA and SEA were significantly higher in myalgic subjects compared to healthy controls.

CONCLUSIONS

This study demonstrates that microdialysis in combination with mass spectrometry can be used for analysing NAE's in human muscle tissue regularly over time. Furthermore the significant group differences in the concentration of PEA and SEA in this study might fill an important gap in our knowledge of mechanisms in chronic myalgia in humans. In the long run this expanded understanding of nociceptive and anitinociceptive processes in the muscle may provide a base for ameliorating treatment and rehabilitation of pain.

Cannabinoids, the bioactive constituents of Cannabis sativa, and endocannabinoids, among which the most important are anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, control various biological processes by binding to specific G protein-coupled receptors, namely CB1 and CB<em>2</em> cannabinoid receptors. While a vast amount of information on the mammalian endocannabinoid system does exist, few data have been reported on bony fish. In the goldfish, Carassius auratus, the CB1 receptor has been cloned and its distribution has been analyzed in the retina, brain and gonads, while CB<em>2</em> had not yet been isolated. In the present paper, we cloned the goldfish CB<em>2</em> receptor and show that it presents a quite high degree of amino acid identity with zebrafish Danio rerio CB<em>2</em>A and CB<em>2</em>B receptors, while the percentage of identity is lower with the puffer fish Fugu rubripes CB<em>2</em>, as also confirmed by the phylogenetic analysis. The sequence identity becomes much lower when comparing the goldfish and the mammalian CB<em>2</em> sequences; as for other species, goldfish CB<em>2</em> and CB1 amino acid sequences share moderate levels of identity. Western-blotting analysis shows the CB<em>2</em> receptor as two major bands of about 53 and 40 kDa and other faint bands with apparent molecular masses around 70, 57 and 55 kDa. Since the distribution of a receptor could give information on its physiological role, we evaluated and compared CB1 and CB<em>2</em> mRNA expression in different goldfish organs by means of qReal-Time PCR. Our results show that both CB1 and CB<em>2</em> receptors are widely expressed in the goldfish, displaying some tissue specificities, thus opening the way for further functional studies on bony fish and other nonmammalian vertebrates.

The effects of N-arachidonoylethanolamine (anandamide) and related compounds on the binding of [3H]CP55940 to rat brain synaptosomes were examined. Anandamide was shown to inhibit competitively the specific binding of [3H]CP55940 to synaptosomal membranes. The Ki value was 89 nM. In contrast, N-acylethanolamines containing saturated or monoenoic fatty acids did not exhibit high binding affinity. Several structural analogues of anandamide showed some binding activity. Among them, <em>2</em>-<em>arachidonoylglycerol</em> is noteworthy because of its occurrence in mammalian tissues. A biosynthetic study indicated that anandamide can be synthesized via two separate synthetic pathways. The first is synthesis from free arachidonic acid and ethanolamine, and the second is the formation of N-arachidonoyl phosphatidylethanolamine (PE) from diarachidonoyl phospholipids and PE and the subsequent enzymatic release of N-arachidonoylethanolamine. The latter pathway appears to explain very well the fatty acid composition of N-acylethanolamines present in mammalian tissues.