Gonadotropin-releasing hormone (GnRH) neurons are controlled by 17β-estradiol (E<em>2</em>) contributing to the steroid feedback regulation of the reproductive axis. In rodents, E<em>2</em> exerts a negative feedback effect upon GnRH neurons throughout the estrus-diestrus phase of the ovarian cycle. The present study was undertaken to reveal the role of estrogen receptor subtypes in the mediation of the E<em>2</em> signal and elucidate the downstream molecular machinery of suppression. The effect of E<em>2</em> administration at low physiological concentration (10 pM) on GnRH neurons in acute brain slices obtained from metestrous GnRH-green fluorescent protein (GFP) mice was studied under paradigms of blocking or activating estrogen receptor subtypes and interfering with retrograde <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) signaling. Whole-cell patch clamp recordings revealed that E<em>2</em> significantly diminished the frequency of spontaneous postsynaptic currents (sPSCs) in GnRH neurons (49.6<em>2</em> ± 7.6%) which effect was abolished by application of the estrogen receptor (ER) α/β blocker Faslodex (1 μM). Pretreatment of the brain slices with cannabinoid receptor type 1 (CB1) inverse agonist AM<em>2</em>51 (1 μM) and intracellularly applied endocannabinoid synthesis blocker THL (10 μM) significantly attenuated the effect of E<em>2</em> on the sPSCs. E<em>2</em> remained effective in the presence of tetrodotoxin (TTX) indicating a direct action of E<em>2</em> on GnRH cells. The ERβ specific agonist DPN (10 pM) also significantly decreased the frequency of miniature postsynaptic currents (mPSCs) in GnRH neurons. In addition, the suppressive effect of E<em>2</em> was completely blocked by the selective ERβ antagonist PHTPP (1 μM) indicating that ERβ is required for the observed rapid effect of the E<em>2</em>. In contrast, the ERα agonist PPT (10 pM) or the membrane-associated G protein-coupled estrogen receptor (GPR30) agonist G1 (10 pM) had no significant effect on the frequency of mPSCs in these neurons. AM<em>2</em>51 and tetrahydrolipstatin (THL) significantly abolished the effect of E<em>2</em> whereas AM<em>2</em>51 eliminated the action of DPN on the mPSCs. These data suggest the involvement of the retrograde endocannabinoid mechanism in the rapid direct effect of E<em>2</em>. These results collectively indicate that estrogen receptor beta and <em>2</em>-AG/CB1 signaling mechanisms are coupled and play an important role in the mediation of the negative estradiol feedback on GnRH neurons in acute slice preparation obtained from intact, metestrous mice.

Seasonal (circannual) rhythms play an important role for the control of body functions (reproduction, metabolism, immune responses) in nearly all living organisms. Also humans are affected by the seasons with regard to immune responses and mental functions, the seasonal affective disorder being one of the most prominent examples. The hypophysial pars tuberalis (PT), an important interface between the hypophysial pars distalis and neuroendocrine centers in the brain, plays an essential role in the regulation of seasonal functions and may even be the seat of the circannual clock. Photoperiodic signals provide a major input to the PT. While the perception of these signals involves extraocular photoreceptors in non-mammalian species (birds, fish), mammals perceive photoperiodic signals exclusively in the retina. A multisynaptic pathway connects the retina with the pineal organ where photoperiodic signals are translated into the neurohormone melatonin that is rhythmically produced night by night and encodes the length of the night. Melatonin controls the functional activity of the mammalian PT by acting upon MT1 melatonin receptors. The PT sends its output signals via retrograde and anterograde pathways. The retrograde pathway targetting the hypothalamus employs TSH as messenger and controls a local hypothalamic T3 system. As discovered in Japanese quail, TSH triggers molecular cascades mediating thyroid hormone conversion in the ependymal cell layer of the infundibular recess of the third ventricle. The local accumulation of T3 in the mediobasal hypothalamus (MBH) appears to activate the gonadal axis by affecting the neuro-glial interaction between GnRH terminals and tanycytes in the median eminence. This retrograde pathway is conserved in photoperiodic mammals (sheep and hamsters), and even in non-photoperiodic laboratory mice provided that they are capable to synthesize melatonin. The anterograde pathway is implicated in the control of prolactin secretion, targets cells in the PD and supposedly employs small molecules as signal substances collectively denominated as "tuberalins". Several "tuberalin" candidates have been proposed, such as tachykinins, the secretory protein TAFA and endocannabinoids (EC). The PT-intrinsic EC system was first demonstrated in Syrian hamsters and shown to respond to photoperiodic changes. Subsequently, the EC system was also demonstrated in the PT of mice, rats and humans. To date, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) appears as the most important endocannabinoid from the PT. Likely targets for the EC are folliculo-stellate cells that contain the CB1 receptor and appear to contact lactotroph cells. The CB1 receptor was also found on corticotroph cells which appear as a further target of the EC. Recently, the CB1 receptor was also localized to CRF-containing nerve fibers running in the outer zone of the median eminence. This finding suggests that the EC system of the PT contributes not only to the anterograde, but also to the retrograde pathway. Taken together, the results support the concept that the PT transmits its signals via a "cocktail" of messenger molecules which operate also in other brain areas and systems rather than through PT-specific "tuberalins". Furthermore, they may attribute a novel function to the PT, namely the modulation of the stress response and immune functions.

Although liver fatty acid binding protein (FABP1, L-FABP) is not detectable in the brain, Fabp1 gene ablation (LKO) markedly increases endocannabinoids (EC) in brains of male mice. Since the brain EC system of females differs significantly from that of males, it was important to determine if LKO differently impacted the brain EC system. LKO did not alter brain levels of arachidonic acid (ARA)-containing EC, i.e. arachidonoylethanolamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), but decreased non-ARA-containing N-acylethanolamides (OEA, PEA) and <em>2</em>-oleoylglycerol (<em>2</em>-OG) that potentiate the actions of AEA and <em>2</em>-AG. These changes in brain potentiating EC levels were not associated with: (1) a net decrease in levels of brain membrane proteins associated with fatty acid uptake and EC synthesis; (<em>2</em>) a net increase in brain protein levels of cytosolic EC chaperones and enzymes in EC degradation; or (3) increased brain protein levels of EC receptors (CB1, TRVP1). Instead, the reduced or opposite responsiveness of female brain EC levels to loss of FABP1 (LKO) correlated with intrinsically lower FABP1 level in livers of WT females than males. These data show that female mouse brain endocannabinoid levels were unchanged (AEA, <em>2</em>-AG) or decreased (OEA, PEA, <em>2</em>-OG) by complete loss of FABP1 (LKO).

α/β-Hydrolase domain 6 (ABHD6) is a monoacylglycerol hydrolase that degrades the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Although complete or peripheral ABHD6 loss of function is protective against diet-induced obesity and insulin resistance, the role of ABHD6 in the central control of energy balance is unknown. Using a viral-mediated knockout approach, targeted endocannabinoid measures, and pharmacology, we discovered that mice lacking ABHD6 from neurons of the ventromedial hypothalamus (VMHKO) have higher VMH <em>2</em>-AG levels in conditions of endocannabinoid recruitment and fail to physiologically adapt to key metabolic challenges. VMHKO mice exhibited blunted fasting-induced feeding and reduced food intake, energy expenditure, and adaptive thermogenesis in response to cold exposure, high-fat feeding, and dieting (transition to a low-fat diet). Our findings identify ABHD6 as a regulator of the counter-regulatory responses to major metabolic shifts, including fasting, nutrient excess, cold, and dieting, thereby highlighting the importance of ABHD6 in the VMH in mediating energy metabolism flexibility.

Various studies performed in cultured cells and in <i>in vivo</i> models of neuronal damage showed that cannabinoids exert a neuroprotective effect. The increase in cannabinoids and cannabinoid like substances after stroke has been postulated to limit the content of neuronal injury. As well-accepted, inflammation, and neuronal damage are coupled processes and microglial cells as the main intrinsic immunological effector within the brain play a central role in their regulation. Treatment with the endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) or the endocannabinoid-like substance, palmitoylethanolamide (PEA) affected microglial cells and led to a decrease in the number of damaged neurons after excitotoxical lesion in organotypic hippocampal slice cultures (OHSC). <em>2</em>-AG activated abnormal cannabidiol (abn-CBD) receptor, PEA was shown to mediate neuroprotection via peroxisome proliferator-activated receptor (PPAR)α. Despite the known neuroprotective and anti-inflammatory properties, the potential synergistic effect, namely possible entourage effect after treatment with the combination of these two protective cannabinoids has not been examined yet. After excitotoxical lesion OHSC were treated with PEA, <em>2</em>-AG or a combination of both and the number of damaged neurons was evaluated. To investigate the role of microglial cells in PEA and <em>2</em>-AG mediated protection, primary microglial cell cultures were treated with lipopolysaccharide (LPS) and <em>2</em>-AG, PEA or a combination of those. Thereafter, we measured NO production, ramification index, proliferation and PPARα distribution in microglial cells. While PEA or <em>2</em>-AG alone were neuroprotective, their co-application vanished the protective effect. This behavior was independent of microglial cells. Furthermore, PEA and <em>2</em>-AG had contrary effects on ramification index and on NO production. No significant changes were observed in the proliferation rate of microglial cells after treatment. The expression of PPARα was not changed upon stimulation with PEA or <em>2</em>-AG, but the distribution was significantly altered. <em>2</em>-AG and PEA mediated neuroprotection was abolished when co-applied. Both cannabinoids exert contrary effects on morphology and function of microglial cells. Co-application of both cannabinoids with different targets did not lead to a positive additive effect as expected, presumably due to the contrary polarization of microglial cells.

The endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) can be considered a true agonist as it is able to activate human platelets stimulating arachidonic acid release, thromboxane B<em>2</em> formation and calcium intracellular elevation. Recently we have shown that <em>2</em>-AG induces a rapid myosin light chain (MLC) phosphorylation/activation, early mediated by RhoA kinase (ROCK) signalling pathway and later by myosin light chain kinase. The aim of the present study was to investigate the role of phosphatidylinositol 3 kinase (PI3K)/AKT pathway in MLC phosphorylation and some downstream events such as actin polymerization, ATP secretion and aggregation. We demonstrated that PI3K in particular the isoforms α and β and AKT have a role in MLC phosphorylation. The stimulation of PI3K/AKT pathway activates ROCK. ROCK is directly involved in the early phase of MLC activation stimulating thr18 phosphorylation. MLC activation is strengthened through the MLC phosphatase inhibition, that is accomplished through the phosphorylation of MYPT1, catalytic subunit of MLC phosphatase, overall mediated by ROCK. In addition we have found that the PI3Kα/β isoforms and AKT are involved in the downstream mechanisms leading to actin polymerization, ATP secretion and aggregation of human platelets stimulated by <em>2</em>-AG.

Cannabinoids and modulators of the endocannabinoid system affect specific mechanisms that are critical to the establishment and development of endometriosis. The aim of this study was to measure the systemic levels of endocannabinoids and related mediators in women with and without endometriosis and to investigate whether such levels correlated with endometriosis-associated pain. Plasma and endometrial biopsies were obtained from women with a laparoscopic diagnosis of endometriosis (n = <em>2</em>7) and no endometrial pathology (n = <em>2</em>9). Plasma levels of endocannabinoids (N-arachidonoylethanolamine [AEA] and <em>2</em>-<em>arachidonoylglycerol</em> [<em>2</em>-AG]) and related mediators (N-oleoylethanolamine [OEA] and N-palmitoylethanolamine [PEA]), messenger RNA expression of some of their receptors (cannabinoid receptor type 1 [CB1], CB<em>2</em>, transient receptor potential vanilloid type [TRPV1]), and the enzymes involved in the synthesis (N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D [NAPE-PLD]) and degradation (fatty acid amide hydrolase 1 [FAAH]) of AEA, OEA, and PEA were evaluated in endometrial stromal cells. The systemic levels of AEA, <em>2</em>-AG, and OEA were elevated in endometriosis in the secretory phase compared to controls. The expression of CB1 was higher in secretory phase endometrial stromal cells of controls versus endometriosis. Similar expression levels of CB<em>2</em>, TRPV1, NAPE-PLD, and FAAH were detected in controls and endometriosis. Patients with moderate-to-severe dysmenorrhea and dyspareunia showed higher AEA and PEA levels than those with low-to-moderate pain symptoms, respectively. The association of increased circulating AEA and <em>2</em>-AG with decreased local CB1 expression in endometriosis suggests a negative feedback loop regulation, which may impair the capability of these mediators to control pain. These preliminary data suggest that the pharmacological manipulation of the action or levels of these mediators may offer an alternative option for the management of endometriosis-associated pain.

The endocannabinoid (eCB) system is a modulatory system that is both altered by stress and mediates the effects of acute stress, including contributing to restoration of homeostasis. Earlier studies suggest that circulating eCBs are dysregulated in adults with post-traumatic stress disorder (PTSD); however, it is not known whether circulating eCBs remain responsive to stress. The purpose of this study was to examine eCB and psychological responses to physical (exercise) and psychosocial (Trier Social Stress Test) stressors, using a randomized, counterbalanced procedure in adults with PTSD and healthy controls (N = <em>2</em>0, mean age = <em>2</em>4, SD = 7 yrs). Results from mixed-design, repeated measures ANOVAs revealed significant increases (p < .05) in N-arachidonoylethanolamine (AEA) and oleoylethanolamide (OEA) following exercise and psychosocial stress in both groups. However, only the control group exhibited a significant increase (p < .05) in <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) following exercise and psychosocial stress exposure. These data extend our current understanding of circulating eCB responsiveness in PTSD, and provide preliminary evidence to suggest that the eCB system is hypoactive in PTSD following exposure to physical and psychosocial stressors.

Huntington's disease (HD) is an inherited progressive neurodegenerative disease characterized by motor, cognitive, and behavioural changes. One of the earliest changes to occur in HD is a reduction in cannabinoid 1 receptor (CB₁) levels in the striatum, which is strongly correlated with HD pathogenesis. CB₁ positive allosteric modulators (PAM) enhance receptor affinity for, and efficacy of activation by, orthosteric ligands, including the endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em>. The goal of this study was to determine whether the recently characterized CB₁ allosteric modulators GAT<em>2</em>11 (racemic), GAT<em>2</em><em>2</em>8 (R-enantiomer), and GAT<em>2</em><em>2</em>9 (S-enantiomer), affected the signs and symptoms of HD. GAT<em>2</em>11, GAT<em>2</em><em>2</em>8, and GAT<em>2</em><em>2</em>9 were evaluated in normal and HD cell models, and in a transgenic mouse model of HD (7-week-old male R6/<em>2</em> mice, 10 mg/kg/d, <em>2</em>1 d, i.p.). GAT<em>2</em><em>2</em>9 was a CB₁ PAM that improved cell viability in HD cells and improved motor coordination, delayed symptom onset, and normalized gene expression in R6/<em>2</em> HD mice. GAT<em>2</em><em>2</em>8 was an allosteric agonist that did not enhance endocannabinoid signaling or change symptom progression in R6/<em>2</em> mice. GAT<em>2</em>11 displayed intermediate effects between its enantiomers. The compounds used here are not drugs, but probe compounds used to determine the potential utility of CB₁ PAMs in HD. Changes in gene expression, and not protein, were quantified in R6/<em>2</em> HD mice because HD pathogenesis is associated with dysregulation of mRNA levels. The data presented here provide the first proof of principle for the use of CB₁ PAMs to treat the signs and symptoms of HD.

BACKGROUND

Food palatability increases food intake and may lead to overeating. The mechanisms behind this observation are still largely unknown.

OBJECTIVE

The aims of this study were the following: 1) to elucidate the plasma responses of endocannabinoids, N-acylethanolamines, and gastrointestinal peptides to a palatable (sweet), unpalatable (bitter), and sensory-acceptable (tasteless control) food, and <em>2</em>) to verify whether some of these bioactive compounds can serve as plasma biomarkers of food liking in humans.

METHODS

Three puddings providing 60 kcal (35% from proteins, 6<em>2</em>% from carbohydrates, and 3% from fats) but with different taste were developed. Twenty healthy subjects (11 women and 9 men; mean age <em>2</em>8 y and BMI <em>2</em><em>2</em>.7 kg/m(<em>2</em>)), selected because they liked the puddings in the order sweet>> control>> bitter, participated in a randomized crossover study based on a modified sham feeding (MSF) protocol. Blood samples at baseline and every 5 min up to <em>2</em>0 min after the MSF were analyzed for gastrointestinal peptides, endocannabinoids, and N-acylethanolamines. Thirty minutes after the MSF, energy intake at an ad libitum breakfast was measured.

RESULTS

After the MSF, no response was observed in 7 of 9 gastrointestinal peptides measured. The plasma ghrelin concentration at <em>2</em>0 min after the sweet and bitter puddings was <em>2</em>5% lower than after the control pudding (P = 0.04), and the pancreatic polypeptide response after the sweet pudding was <em>2</em>3% greater than after the bitter pudding (P = 0.0<em>2</em>). The plasma response of <em>2</em>-<em>arachidonoylglycerol</em> after the sweet pudding was 37% and 15% higher than after the bitter (P < 0.001) and control (P = 0.03) puddings, respectively. Trends for greater responses of anandamide (P = 0.06), linoleoylethanolamide (P = 0.07), palmitoylethanolamide (P = 0.06), and oleoylethanolamide (P = 0.09) were found after the sweet pudding than after the bitter pudding. No differences in subsequent energy intake were recorded.

CONCLUSIONS

The data demonstrated that food palatability influenced some plasma endocannabinoid and N-acylethanolamine concentrations during the cephalic phase response and indicated that <em>2</em>-<em>arachidonoylglycerol</em> and pancreatic polypeptide can be used as biomarkers of food liking in humans.

The anti-inflammatory agent palmitoylethanolamide (PEA) reduces cyclooxygenase (COX) activity in vivo in a model of inflammatory pain. It is not known whether the compound reduces prostaglandin production in RAW<em>2</em>64.7 cells, whether such an action is affected by compounds preventing the breakdown of endogenous PEA, whether other oxylipins are affected, or whether PEA produces direct effects upon the COX-<em>2</em> enzyme. RAW<em>2</em>64.7 cells were treated with lipopolysaccharide and interferon-γ to induce COX-<em>2</em>. At the level of mRNA, COX-<em>2</em> was induced >1000-fold following <em>2</em>4 h of the treatment. Coincubation with PEA (10 μmol/L) did not affect the levels of COX-<em>2</em>, but reduced the levels of prostaglandins D<em>2</em> and E<em>2</em> as well as 11- and 15-hydroxyeicosatetraenoic acid, which can also be synthesised by a COX-<em>2</em> pathway in macrophages. These effects were retained when hydrolysis of PEA to palmitic acid was blocked. Linoleic acid-derived oxylipin levels were not affected by PEA. No direct effects of PEA upon the oxygenation of either arachidonic acid or <em>2</em>-<em>arachidonoylglycerol</em> by COX-<em>2</em> were found. It is concluded that in lipopolysaccharide and interferon-γ-stimulated RAW<em>2</em>64.7 cells, PEA reduces the production of COX-<em>2</em>-derived oxylipins in a manner that is retained when its metabolism to palmitic acid is inhibited.

Monoacylglycerol lipase (MAGL) is a serine hydrolase that plays an important role in the degradation of the endocannabinoid neurotransmitter <em>2</em>-<em>arachidonoylglycerol</em>, which is implicated in many physiological processes. Beyond the possible utilization of MAGL inhibitors as anti-inflammatory, antinociceptive, and anticancer agents, their application has encountered obstacles due to the unwanted effects caused by the irreversible inhibition of this enzyme. The possible application of reversible MAGL inhibitors has only recently been explored, mainly due to the deficiency of known compounds possessing efficient reversible inhibitory activities. In this work, we report a new series of reversible MAGL inhibitors. Among them, compound <em>2</em>6 showed to be a potent MAGL inhibitor (IC50 = 0.51 μM, Ki = 41<em>2</em> nM) with a good selectivity versus fatty acid amide hydrolase (FAAH), α/β-hydrolase domain-containing 6 (ABHD6), and 1<em>2</em> (ABHD1<em>2</em>). Interestingly, this compound also possesses antiproliferative activities against two different cancer cell lines and relieves the neuropathic hypersensitivity induced in vivo by oxaliplatin.

OBJECTIVE

The endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) bind to CB1 and CB<em>2</em> cannabinoid receptors in the brain and modulate the mesolimbic dopaminergic pathway. This neurocircuitry is engaged by psychostimulant drugs, including cocaine. Although CB1 receptor antagonism and CB<em>2</em> receptor activation are known to inhibit certain effects of cocaine, they have been investigated separately. Here, we tested the hypothesis that there is a reciprocal interaction between CB1 receptor blockade and CB<em>2</em> receptor activation in modulating behavioural responses to cocaine.

METHODS

Male Swiss mice received i.p. injections of cannabinoid-related drugs followed by cocaine, and were then tested for cocaine-induced hyperlocomotion, c-Fos expression in the nucleus accumbens and conditioned place preference. Levels of endocannabinoids after cocaine injections were also analysed.

RESULTS

The CB1 receptor antagonist, rimonabant, and the CB<em>2</em> receptor agonist, JWH133, prevented cocaine-induced hyperlocomotion. The same results were obtained by combining sub-effective doses of both compounds. The CB<em>2</em> receptor antagonist, AM630, reversed the inhibitory effects of rimonabant in cocaine-induced hyperlocomotion and c-Fos expression in the nucleus accumbens. Selective inhibitors of anandamide and <em>2</em>-AG hydrolysis (URB597 and JZL184, respectively) failed to modify this response. However, JZL184 prevented cocaine-induced hyperlocomotion when given after a sub-effective dose of rimonabant. Cocaine did not change brain endocannabinoid levels. Finally, CB<em>2</em> receptor blockade reversed the inhibitory effect of rimonabant in the acquisition of cocaine-induced conditioned place preference.

CONCLUSIONS

The present data support the hypothesis that CB1 and CB<em>2</em> receptors work in concert with opposing functions to modulate certain addiction-related effects of cocaine.

OBJECTIVE

The inflammatory response plays an important role in the pathogenesis of many diseases in the central nervous system. Cannabinoids exhibit diverse pharmacological actions including anti-inflammatory activity. In this study, we tried to elucidate possible effects of cannabinoids on lipopolysaccharide (LPS)-induced expression of inflammatory cytokine mRNAs in rat cerebellar granule cells.

METHODS

Inhibitory effects of cannabinoids on cytokine induction in cerebellar granule cells were determined by RT-PCR method.

RESULTS

In these cells, both mRNA and protein of cannabinoid receptor 1 (CB(1) ), but not CB(<em>2</em>) , were expressed. LPS (1 µg/ml) produced a marked increase in the induction of inflammatory cytokines, including interleukin-1β, interleukin-6 and tumour necrosis factor-α. CP55940, a synthetic cannabinoid analogue, concentration-dependently inhibited inflammatory cytokine expression induced by LPS. On the other hand, the endocannabinoids <em>2</em>-<em>arachidonoylglycerol</em> and anandamide were not able to inhibit this inflammatory response. Notably, a CB(1) /CB(<em>2</em>) antagonist NESS03<em>2</em>7 (3 µm) did not reverse the inhibition of cytokine mRNA expression induced by CP55940. GPR55, a putative novel cannabinoid receptor, mRNA was also expressed in cerebellar granule cells. Although it has been suggested that G(q) associates with GPR55, cannabinoids including CP55940 did not promote phosphoinositide hydrolysis and consequent elevation of intracellular Ca([<em>2</em>+]) concentration. Furthermore, a putative GPR55 antagonist, cannabidiol, also showed a similar inhibitory effect to that of CP55940.

CONCLUSIONS

These results suggest that the synthetic cannabinoid CP55940 negatively modulates cytokine mRNA expression in cerebellar granule cells by a CB and GPR55 receptor-independent mechanism.

The classical cannabinoid receptors CB1 and CB<em>2</em> as well as the cannabinoid-sensitive receptor GPR55 are widely distributed throughout the mammalian body. In the cardiovascular field, CB1 and CB<em>2</em> crucially impact on diseases characterized by inflammatory processes, such as atherosclerosis and acute myocardial infarction. Both receptors and their endogenous ligands anandamide and <em>2</em>-<em>arachidonoylglycerol</em> are up-regulated in the ischaemic heart in humans and animal models. Pharmacological and genetic interventions with CB1 and CB<em>2</em> vitally affect acute ischaemia-induced cardiac inflammation. Herein, CB1 rather aggravates the inflammatory response whereas CB<em>2</em> mitigates inflammation via directly affecting immune cell attraction, macrophage polarization and lymphocyte clusters in the pericardial adipose tissue. Furthermore, cannabinoids and their receptors affect numerous cardiac risk factors. In this context, cannabis consumption is debated to trigger arrhythmias and even myocardial infarction. Moreover, CB1 activation is linked to impaired lipid and glucose metabolism and therefore obesity and diabetes, while its antagonism leads to the reduction of plasma triglycerides, low-density lipoprotein cholesterol, leptin, insulin and glucose. On the other hand, activation of cannabinoid-sensitive receptors can also counteract unfavourable predictors for cardiovascular diseases. In particular, hypertension can be mitigated via CB1 agonism and impaired adrenoceptor responsiveness prevented by functional GPR55. Taken together, current insights identify the cannabinoid system as promising target not only to therapeutically interfere with the vasculature, but also to affect the heart as target organ. This review discusses current knowledge regarding a direct cardiac role of the cannabinoid system and points out its feasible therapeutic manipulation in the ischaemic myocardium. This article is part of a Special Issue entitled: Cardiomyocyte biology: new pathways of differentiation and regeneration edited by Marijke Brinkm, Marcus C. Schaub, and Christian Zuppinger.

The endocannabinoid system has been demonstrated to be active in the pancreatic β-cell. However the effects of the endocannabinoids (ECs) on insulin secretion are not well defined and may vary depending on the metabolic state of the β-cell. Specifically it is not known whether the effects of the ECs occur by activation of the cannabinoid receptors or via their direct interaction with the ion channels of the β-cell. To begin to delineate the effects of ECs on β-cell function, we examined how the EC, <em>2</em>-AG influences β-cell ion channels in the absence of glucose stimulation. The mouse insulinoma cell line R7T1 was used to survey the effects of <em>2</em>-AG on the high voltage activated (HVA) calcium, the delayed rectifier (K(v)), and the ATP-sensitive K (K(ATP)) channels by whole cell patch clamp recording. At <em>2</em>mM glucose, <em>2</em>-AG inhibited the HVA calcium (the majority of which are L-type channels), K(v), and K(ATP) channels. The channel exhibiting the most sensitivity to <em>2</em>-AG blockade was the K(ATP) channel, where the IC(50) for <em>2</em>-AG was 1 μM. Pharmacological agents revealed that the blockade of all these channels was independent of cannabinoid receptors. Our results provide a mechanism for the previous observations that CB1R agonists increase insulin secretion at low glucose concentrations through CB1R independent blockade of the K(ATP) channel.

In this report, we describe the localization of diacylglycerol lipase-α (DAGLα) in nuclei from adult cortical neurons, as assessed by double-immunofluorescence staining of rat brain cortical sections and purified intact nuclei and by western blot analysis of subnuclear fractions. Double-labeling assays using the anti-DAGLα antibody and NeuN combined with Hoechst staining showed that only nuclei of neuronal origin were DAGLα positive. At high resolution, DAGLα-signal displayed a punctate pattern in nuclear subdomains poor in Hoechst's chromatin and lamin B1 staining. In contrast, SC-35- and NeuN-signals (markers of the nuclear speckles) showed a high overlap with DAGLα within specific subdomains of the nuclear matrix. Among the members of the phospholipase C-β (PLCβ) family, PLCβ1, PLCβ<em>2</em>, and PLCβ4 exhibited the same distribution with respect to chromatin, lamin B1, SC-35, and NeuN as that described for DAGLα. Furthermore, by quantifying the basal levels of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) by liquid chromatography and mass spectrometry (LC-MS), and by characterizing the pharmacology of its accumulation, we describe the presence of a mechanism for <em>2</em>-AG production, and its PLCβ/DAGLα-dependent biosynthesis in isolated nuclei. These results extend our knowledge about subcellular distribution of neuronal DAGLα, providing biochemical grounds to hypothesize a role for <em>2</em>-AG locally produced within the neuronal nucleus.

The nucleus accumbens (NAc) plays a key role in drug-related behavior and natural reward learning. Synaptic plasticity in dopamine D1 and D<em>2</em> receptor medium spiny neurons (MSNs) of the NAc and the endogenous cannabinoid (eCB) system have been implicated in reward seeking. However, the precise molecular and physiological basis of reward-seeking behavior remains unknown. We found that the specific deletion of metabotropic glutamate receptor 5 (mGluR5) in D1-expressing MSNs (D1^miRmGluR5 mice) abolishes eCB-mediated long-term depression (LTD) and prevents the expression of drug (cocaine and ethanol), natural reward (saccharin), and brain-stimulation-seeking behavior. In vivo enhancement of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) eCB signaling within the NAc core restores both eCB-LTD and reward-seeking behavior in D1^miRmGluR5 mice. The data suggest a model where the eCB and glutamatergic systems of the NAc act in concert to mediate reward-seeking responses.

OBJECTIVE

Endocannabinoids and neuropeptide Y (NPY) promote energy storage via central and peripheral mechanisms. In the hypothalamus, the two systems were suggested to interact. To investigate such interplay also in non-hypothalamic tissues, we evaluated endocannabinoid levels in obese OE-NPY(DβH) mice, which overexpress NPY in the noradrenergic neurons in the sympathetic nervous system and the brain.

METHODS

The levels of the endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) were measured in key regulatory tissues, that is, hypothalamus, pancreas, epididymal white adipose tissue (WAT), liver and soleus muscle, over the development of metabolic dysfunctions in OE-NPY(DβH) mice. The effects of a 5-week treatment with the CB1 receptor inverse agonist AM<em>2</em>51 on adiposity and glucose metabolism were studied.

RESULTS

<em>2</em>-AG levels were increased in the hypothalamus and epididymal WAT of pre-obese and obese OE-NPY(DβH) mice. Anandamide levels in adipose tissue and pancreas were increased at 4 months concomitantly with higher fat mass and impaired glucose tolerance. CB1 receptor blockage reduced body weight gain and glucose intolerance in OE-NPY(DβH) to the level of vehicle-treated wild-type mice.

CONCLUSIONS

Altered endocannabinoid tone may underlie some of the metabolic dysfunctions in OE-NPY(DβH) mice, which can be attenuated with CB1 inverse agonism suggesting interactions between endocannabinoids and NPY also in the periphery. CB1 receptors may offer a target for the pharmacological treatment of the metabolic syndrome with altered NPY levels.

Monoacylglycerol lipase (MAGL) has been recently proposed as the main enzymatic activity responsible for the in vivo hydrolysis of the most abundant endocannabinoid in the brain, the <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). The endocannabinoids, mainly anandamide (AEA) and <em>2</em>-AG, are a class of lipid messengers that modulate a broad number of physiological processes both in the central nervous system and in the periphery. To date, AEA has been by far the most studied endocannabinoid, although increasing evidence is pointing out the prominent, and sometimes underestimated, role of <em>2</em>-AG in the regulation of different functions. Therefore, it is of outmost importance to dissect the specific cellular pathways in which these two endocannabinoids are involved. Nonetheless, little is known about the structural require-ments of MAGL. Here we review the current knowledge on MAGL, with special focus on its structure and catalytic mechanism as the rational basis for the design of potent and selective compounds able to interact with it; the inhibitors that have been described to date, and the therapeutic applications that make MAGL an attractive therapeutic target.

Several linear fatty acid dopamides (N-acyldopamines) have been identified recently in the brain. Among them, N-arachidonoyldopamine (NADA) is an endogenous lipid mediator sharing endocannabinoid and endovanilloid biological activities. We have reported previously that NADA exerts some of its biological activities through inhibition of the NF-kappaB pathway and, because this transcription factor plays a key role in HIV-1-long terminal repeat (LTR) trans activation, we have evaluated the anti-HIV-1 activity of NADA. In this study, we show that NADA inhibits vesicular stomatitis virus-pseudotyped HIV-1 infection in the human leukemia T cell line Jurkat, in primary T cells, and in the human astrocytic cell line U373-MG. Other endocannabinoids such as anandamide, <em>2</em>-<em>arachidonoylglycerol</em>, and noladin ether did not show inhibitory activity in the HIV-1 replication assays. The anti-HIV-1 activity of NADA was independent of known cannabinoid and vanilloid receptor activation. In addition, NADA did not affect reverse transcription and integration steps of the viral cycle, and its inhibitory effect was additive with that of the reverse transcriptase inhibitor azidothymidine. NADA inhibited both TNF-alpha and HIV-1 trans activator protein-induced HIV-1-LTR activation. We also show that NADA counteracts the TNF-alpha-mediated trans activation capacity of the p65 NF-kappaB subunit without affecting its physical association to the HIV-1-LTR promoter. Moreover, NADA inhibited the p65 transcriptional activity by specifically targeting the phosphorylation of this NF-kappaB subunit at Ser(536). These findings provide new mechanistic insights into the biological activities of NADA, and highlight the potential of lipid mediators for the management of AIDS.

The endocannabinoid system is dysregulated in schizophrenia. Mice with heterozygous deletion of neuregulin 1 (Nrg1 HET mice) provide a well-characterised animal model of schizophrenia, and display enhanced sensitivity to stress and cannabinoids during adolescence. However, no study has yet determined whether these mice have altered brain endocannabinoid concentrations. Nrg1 application to hippocampal slices decreased <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) signalling and disrupted long-term depression, a form of synaptic plasticity critical to spatial learning. Therefore we specifically aimed to examine whether Nrg1 HET mice exhibit increased <em>2</em>-AG concentrations and disruption of spatial learning. As chronic stress influences brain endocannabinoids, we also sought to examine whether Nrg1 deficiency moderates adolescent stress-induced alterations in brain endocannabinoids. Adolescent Nrg1 HET and wild-type (WT) mice were submitted to chronic restraint stress and brain endocannabinoid concentrations were analysed. A separate cohort of WT and Nrg1 HET mice was also assessed for spatial learning performance in the Morris Water Maze. Partial genetic deletion of Nrg1 increased anandamide concentrations in the amygdala and decreased <em>2</em>-AG concentrations in the hypothalamus. Further, Nrg1 HET mice exhibited increased <em>2</em>-AG concentrations in the hippocampus and impaired spatial learning performance. Chronic adolescent stress increased anandamide concentrations in the amygdala, however, Nrg1 disruption did not influence this stress-induced change. These results demonstrate for the first time in vivo interplay between Nrg1 and endocannabinoids in the brain. Our results demonstrate that aberrant Nrg1 and endocannabinoid signalling may cooperate in the hippocampus to impair cognition, and that Nrg1 deficiency alters endocannabinoid signalling in brain stress circuitry.

<em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is an endogenous ligand of the cannabinoid CB1 receptor. This endocannabinoid and its hydrolyzing enzyme, monoacylglycerol lipase (MAGL), are present in encephalic regions related to psychiatric disorders, including the midbrain dorsolateral periaqueductal grey (dlPAG). The dlPAG is implicated in panic disorder and its stimulation results in defensive responses proposed as a model of panic attacks. The present work verified if facilitation of <em>2</em>-AG signalling in the dlPAG counteracts panic-like responses induced by local chemical stimulation. Intra-dlPAG injection of <em>2</em>-AG prevented panic-like response induced by the excitatory amino acid N-methyl-d-aspartate (NMDA). This effect was mimicked by the <em>2</em>-AG hydrolysis inhibitor (MAGL preferring inhibitor) URB60<em>2</em>. The anti-aversive effect of URB60<em>2</em> was reversed by the CB1 receptor antagonist, AM<em>2</em>51. Additionally, a combination of sub-effective doses of <em>2</em>-AG and URB60<em>2</em> also prevented NMDA-induced panic-like response. Finally, immunofluorescence assay showed a significant increase in c-Fos positive cells in the dlPAG after local administration of NMDA. This response was also prevented by URB60<em>2</em>. These data support the hypothesis that <em>2</em>-AG participates in anti-aversive mechanisms in the dlPAG and reinforce the proposal that facilitation of endocannabinoid signalling could be a putative target for developing additional treatments against panic and other anxiety-related disorders.

The effect of <em>2</em>-<em>arachidonoylglycerol</em>, a cannabimimetic eicosanoid, was studied on mucosa-free longitudinal muscle strips isolated from the guinea-pig distal colon. In the presence of indomethacin (3 microM) and N(G)-nitro-L-arginine (100 microM), <em>2</em>-<em>arachidonoylglycerol</em> (10 nM-10 microM) produced concentration-dependent and tetrodotoxin (1 microM)-sensitive contractions of the longitudinal muscle strips. The contractions were markedly attenuated in the presence of atropine (0.<em>2</em> microM), and partially by hexamethonium (100 microM) pretreatment. The response to <em>2</em>-<em>arachidonoylglycerol</em> was mimicked with N-arachidonoylethanolamine (anandamide, 0.1-30 microM), another cannabimimetic eicosanoid, but the cannabinoid CB(1)/CB(<em>2</em>) receptor agonist, R-[<em>2</em>,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,<em>2</em>,3,-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55,<em>2</em>1<em>2</em>-<em>2</em>) (0.1-10 microM), and the vanilloid receptor agonist, (all Z)-(4-hydroxyphenyl)-5,8,11,14-eicosatetraenamide (AM 404) (10-30 microM), were without effect. The cannabinoid CB(1) receptor antagonist, N-piperidino-5-(4-chlorophenyl)-l-(<em>2</em>,4-dichlorophenyl)-4-methyl-3-pyrazole-caroxamide (SR141716A) (1 microM), the cannabinoid CB(<em>2</em>) receptor antagonist, [N-[1S]-endo-1,3,3-trimethyl bicyclo [<em>2</em>.<em>2</em>.1] heptan-<em>2</em>-yl]-5-(4-chloro-3-methylphenyl)-l-(4-methylbenzyl)-pyrazole-3-carboxamide (SR1445<em>2</em>8) (1 microM), and the vanilloid receptor antagonist, capsazepine (10 microM), did not shift the concentration-response curve for <em>2</em>-<em>arachidonoylglycerol</em> to the right. The contractile action of <em>2</em>-<em>arachidonoylglycerol</em> was also partially attenuated in the presence of nordihydroguaiaretic acid (10 microM), a lipoxygenase inhibitor. These results indicate that <em>2</em>-<em>arachidonoylglycerol</em> produces contraction of longitudinal muscle of the guinea-pig distal colon via mainly stimulation of myenteric cholinergic neurones, and that neither cannabinoid CB(1)/CB(<em>2</em>) receptors nor vanilloid receptors contributed to the response. The present results suggest the possibility that lipoxygenase metabolites may also contribute, at least in part, to the contractile action of <em>2</em>-<em>arachidonoylglycerol</em>.