α- and β-neurexins are presynaptic cell-adhesion molecules implicated in autism and schizophrenia. We find that, although β-neurexins are expressed at much lower levels than α-neurexins, conditional knockout of β-neurexins with continued expression of α-neurexins dramatically decreased neurotransmitter release at excitatory synapses in cultured cortical neurons. The β-neurexin knockout phenotype was attenuated by CB1-receptor inhibition, which blocks presynaptic endocannabinoid signaling, or by <em>2</em>-<em>arachidonoylglycerol</em> synthesis inhibition, which impairs postsynaptic endocannabinoid release. In synapses formed by CA1-region pyramidal neurons onto burst-firing subiculum neurons, presynaptic in vivo knockout of β-neurexins aggravated endocannabinoid-mediated inhibition of synaptic transmission and blocked LTP; presynaptic CB1-receptor antagonists or postsynaptic <em>2</em>-<em>arachidonoylglycerol</em> synthesis inhibition again reversed this block. Moreover, conditional knockout of β-neurexins in CA1-region neurons impaired contextual fear memories. Thus, our data suggest that presynaptic β-neurexins control synaptic strength in excitatory synapses by regulating postsynaptic <em>2</em>-<em>arachidonoylglycerol</em> synthesis, revealing an unexpected role for β-neurexins in the endocannabinoid-dependent regulation of neural circuits.

The N-acylethanolamines (NAEs) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are bioactive lipids that can modulate inflammatory responses and protect neurons against glutamatergic excitotoxicity. We have used a model of focal cerebral ischemia in young adult mice to investigate the relationship between focal cerebral ischemia and endogenous NAEs. Over the first <em>2</em>4 h after induction of permanent middle cerebral artery occlusion, we observed a time-dependent increase in all the investigated NAEs, except for anandamide. Moreover, we found an accumulation of <em>2</em>-AG at 4 h that returned to basal level 1<em>2</em> h after induction of ischemia. Accumulation of NAEs did not depend on regulation of N-acylphosphatidylethanolamine-hydrolyzing phospholipase D or fatty acid amide hydrolase. Treatment with the fatty acid amide hydrolase inhibitor URB597 (cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester; 1 mg/kg; i.p.) 1.5 h before arterial occlusion decreased the infarct volume in our model system. Our results suggest that NAEs and <em>2</em>-AG may be involved in regulation of neuroprotection during focal cerebral ischemia in mice.

Emerging evidence indicates that astrogliosis is involved in the pathogenesis of neurodegenerative disorders. Our previous findings suggested cannabinoids and Autacoid Local Injury Antagonism Amides (ALIAmides) attenuate glial response in models of neurodegeneration. The present study was aimed at exploring palmitoylethanolamide (PEA) ability to mitigate β-amyloid (Aβ)-induced astrogliosis. Experiments were carried out to investigate PEA's (10(-7) M) effects upon the expression and release of pro-inflammatory molecules in rat primary astrocytes activated by soluble Aβ(1-4<em>2</em>) (1 μg/ml) as well as to identify mechanisms responsible for such actions. The effects of Aβ and exogenous PEA on the astrocyte levels of the endocannabinoidsand of endogenous ALIAmides were also studied. The peroxisome proliferator-activated receptor (PPAR)-α (MK886, 3 μM) or PPAR-γ (GW966<em>2</em>, 9 nM) antagonists were co-administered with PEA. Aβ elevated endogenous PEA and d5-<em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) levels. Exogenous PEA blunted the Aβ-induced expression of pro-inflammatory molecules. This effect was reduced by PPAR-α antagonist. Moreover, this ALIAmide, like Aβ, increased <em>2</em>-AG levels. These results indicate that PEA exhibits anti-inflammatory properties able to counteract Aβ-induced astrogliosis, and suggest novel treatment for neuroinflammatory/ neurodegenerative processes.

Endocannabinoids are fatty acid amides like anandamide (AEA), and monoacylglycerols like <em>2</em>-<em>arachidonoylglycerol</em>, that bind to cannabinoid, vanilloid and peroxisome proliferator-activated receptors. Their biological actions are controlled through not yet fully characterized cellular mechanisms. These compounds, together with their related enzymes, that include key proteins for the synthesis and degradation of endocannabinoids, cannabinoid and non-cannabinoid receptors, and purported membrane transporter(s), form the "endocannabinoid system (ECS)". In the past few years AEA and related ECS elements have emerged as essential players in various aspects of human reproduction, both for males and females. Here, the key features of the ECS and the potential of its components to direct human fertility towards a positive or negative end will be reviewed. In particular, the involvement of AEA and related ECS elements in regulating embryo oviductal transport, blastocyst implantation and placental development (in females), and sperm survival, motility, capacitation and acrosome reaction (in males) will be addressed, as well as the role of endocannabinoids in sperm-oviduct interactions. Additionally, the possibility that blood AEA and its hydrolase FAAH may represent reliable diagnostic markers of natural and assisted reproduction in humans will be discussed, along with the therapeutic exploitation of ECS-oriented drugs as useful fertility enhancers.

Abnormal accumulation of β-amyloid (Aβ) is the major neuropathological hallmark of Alzheimer's disease (AD). However, the mechanisms underlying aberrant Aβ formation in AD remain unclear. We showed previously that inhibition of monoacylglycerol lipase (MAGL), the primary enzyme that metabolizes the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in the brain, robustly reduces Aβ by inhibiting β-site amyloid precursor protein cleaving enzyme 1 (BACE1), a key enzyme responsible for Aβ formation. However, the molecular mechanisms responsible for suppression of BACE1 by inhibition of <em>2</em>-AG metabolism are largely unknown. We demonstrate here that expression of the noncoding small RNA miR-188-3p that targets BACE1 was significantly downregulated both in the brains of AD humans and APP transgenic (TG) mice, a mouse model of AD. The downregulated miR-188-3p expression was restored by MAGL inhibition. Overexpression of miR-188-3p in the hippocampus reduced BACE1, Aβ, and neuroinflammation and prevented deteriorations in hippocampal basal synaptic transmission, long-term potentiation, spatial learning, and memory in TG mice. <em>2</em>-AG-induced suppression of BACE1 was prevented by miR-188-3p loss of function. Moreover, miR-188-3p expression was upregulated by <em>2</em>-AG or peroxisome proliferator-activated receptor-γ (PPARγ) agonists and suppressed by PPARγ antagonism or NF-κB activation. Reducing Aβ and neuroinflammation by MAGL inhibition was occluded by PPARγ antagonism. In addition, BACE1 suppression by <em>2</em>-AG and PPARγ activation was eliminated by knockdown of NF-κB. Our study provides a novel molecular mechanism underlying improved synaptic and cognitive function in TG mice by <em>2</em>-AG signaling, which upregulates miR-188-3p expression through PPARγ and NF-κB signaling pathway, resulting in suppressions of BACE1 expression and Aβ formation.

Clinical and experimental evidence demonstrates that endocannabinoids play either beneficial or adverse roles in many neurological and psychiatric disorders. Their medical significance may be best explained by the emerging concept that endocannabinoids are essential modulators of synaptic transmission throughout the central nervous system. However, the precise molecular architecture of the endocannabinoid signaling machinery in the human brain remains elusive. To address this issue, we investigated the synaptic distribution of metabolic enzymes for the most abundant endocannabinoid molecule, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), in the postmortem human hippocampus. Immunostaining for diacylglycerol lipase-α (DGL-α), the main synthesizing enzyme of <em>2</em>-AG, resulted in a laminar pattern corresponding to the termination zones of glutamatergic pathways. The highest density of DGL-α-immunostaining was observed in strata radiatum and oriens of the cornu ammonis and in the inner third of stratum moleculare of the dentate gyrus. At higher magnification, DGL-α-immunopositive puncta were distributed throughout the neuropil outlining the immunonegative main dendrites of pyramidal and granule cells. Electron microscopic analysis revealed that this pattern was due to the accumulation of DGL-α in dendritic spine heads. Similar DGL-α-immunostaining pattern was also found in hippocampi of wild-type, but not of DGL-α knockout mice. Using two independent antibodies developed against monoacylglycerol lipase (MGL), the predominant enzyme inactivating <em>2</em>-AG, immunostaining also revealed a laminar and punctate staining pattern. However, as observed previously in rodent hippocampus, MGL was enriched in axon terminals instead of postsynaptic structures at the ultrastructural level. Taken together, these findings demonstrate the post- and presynaptic segregation of primary enzymes responsible for synthesis and elimination of <em>2</em>-AG, respectively, in the human hippocampus. Thus, molecular architecture of the endocannabinoid signaling machinery supports retrograde regulation of synaptic activity, and its similar blueprint in rodents and humans further indicates that <em>2</em>-AG's physiological role as a negative feed-back signal is an evolutionarily conserved feature of excitatory synapses.

There is extensive evidence that glucocorticoid hormones impair the retrieval of memory of emotionally arousing experiences. Although it is known that glucocorticoid effects on memory retrieval impairment depend on rapid interactions with arousal-induced noradrenergic activity, the exact mechanism underlying this presumably nongenomically mediated glucocorticoid action remains to be elucidated. Here, we show that the hippocampal endocannabinoid system, a rapidly activated retrograde messenger system, is involved in mediating glucocorticoid effects on retrieval of contextual fear memory. Systemic administration of corticosterone (0.3-3 mg/kg) to male Sprague-Dawley rats 1 h before retention testing impaired the retrieval of contextual fear memory without impairing the retrieval of auditory fear memory or directly affecting the expression of freezing behavior. Importantly, a blockade of hippocampal CB1 receptors with AM<em>2</em>51 prevented the impairing effect of corticosterone on retrieval of contextual fear memory, whereas the same impairing dose of corticosterone increased hippocampal levels of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em>. We also found that antagonism of hippocampal β-adrenoceptor activity with local infusions of propranolol blocked the memory retrieval impairment induced by the CB receptor agonist WIN55,<em>2</em>1<em>2</em>-<em>2</em>. Thus, these findings strongly suggest that the endocannabinoid system plays an intermediary role in regulating rapid glucocorticoid effects on noradrenergic activity in impairing memory retrieval of emotionally arousing experiences.

Carboxylesterases (CES) have important roles in pesticide and drug metabolism and contribute to the clearance of ester-containing xenobiotics in mammals. Tissues with the highest levels of CES expression are the liver and small intestine. In addition to xenobiotics, CES also harness their broad substrate specificity to hydrolyze endobiotics, such as cholesteryl esters and triacylglycerols. Here, we determined if two human CES isoforms, CES1 and CES<em>2</em>, hydrolyze the endocannabinoids <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>AG) and anandamide (AEA), and two prostaglandin glyceryl esters (PG-Gs), which are formed by COX-mediated oxygenation of <em>2</em>AG. We show that recombinant CES1 and CES<em>2</em> efficiently hydrolyze <em>2</em>AG to arachidonic acid (AA) but not amide-containing AEA. Steady-state kinetic parameters for CES1- and CES<em>2</em>-mediated <em>2</em>AG hydrolysis were, respectively, kcat, 59 and 43 min(-1); Km, 49 and 46 μM; and kcat/Km, 1.<em>2</em> and 0.93 μM(-1) min(-1). kcat/Km values are comparable to published values for rat monoacylglycerol lipase (MAGL)-catalyzed <em>2</em>AG hydrolysis. Furthermore, we show that CES1 and CES<em>2</em> also efficiently hydrolyze PGE<em>2</em>-G and PGF<em>2</em>α-G. In addition, when cultured human THP1 macrophages were treated with exogenous <em>2</em>AG or PG-G (10 μM, 1 h), significant quantities of AA or PGs were detected in the culture medium; however, the ability of macrophages to metabolize these compounds was inhibited (60-80%) following treatment with paraoxon, the toxic metabolite of the insecticide parathion. Incubation of THP1 cell lysates with small-molecule inhibitors targeting CES1 (thieno[3,<em>2</em>-e][1]benzothiophene-4,5-dione or JZL184) significantly reduced lipid glyceryl ester hydrolase activities (40-50% for <em>2</em>AG and 80-95% for PG-Gs). Immunodepletion of CES1 also markedly reduced <em>2</em>AG and PG-G hydrolase activities. These results suggested that CES1 is in part responsible for the hydrolysis of <em>2</em>AG and PG-Gs in THP1 cells, although it did not rule out a role for other hydrolases, especially with regard to <em>2</em>AG metabolism since a substantial portion of its hydrolysis was not inactivated by the inhibitors. An enzyme (Mr 31-3<em>2</em> kDa) of unknown function was detected by serine hydrolase activity profiling of THP1 cells and may be a candidate. Finally, the amounts of in situ generated <em>2</em>AG and PG-Gs in macrophages were enhanced by treating the cells with bioactive metabolites of OP insecticides. Collectively, the results suggest that in addition to MAGL and fatty-acid amide hydrolase (FAAH), which have both been documented to terminate endocannabinoid signaling, CES may also have a role. Furthermore, since PG-Gs have been shown to possess biological activities in their own right, CES may represent an important enzyme class that regulates their in vivo levels.

The endocannabinoids (eCBs) anandamide and <em>2</em>-<em>arachidonoylglycerol</em> are important retrograde messengers that inhibit neurotransmitter release via presynaptic CB1 receptors. In addition, cannabinoids are known to modulate the cell death/survival decision of different neural cell types, leading to different outcomes that depend on the nature of the target cell and its proliferative/differentiation status. Thus, cannabinoids protect primary neurons, astrocytes and oligodendrocytes from apoptosis, whereas transformed glial cells are prone to apoptosis by cannabinoid challenge. Moreover, a potential role of the eCB system in neurogenesis and neural differentiation has been proposed. Recent research shows that eCBs stimulate neural progenitor proliferation and inhibit hippocampal neurogenesis in normal adult brain. Cannabinoids inhibit cortical neuron differentiation and promote glial differentiation. On the other hand, experiments with differentiated neurons have shown that cannabinoids also regulate neuritogenesis, axonal growth and synaptogenesis. These new observations support that eCBs constitute a new family of lipid signaling cues responsible for the regulation of neural progenitor proliferation and differentiation, acting as instructive proliferative signals through the CB1 receptor.

Fatty acid amide hydrolase (FAAH) is responsible for the hydrolysis of a number of important endogenous fatty acid amides, including the endogenous cannabimimetic agent anandamide (AEA), the sleep-inducing compound oleamide, and the putative anti-inflammatory agent palmitoylethanolamide (PEA). In recent years, there have been great advances in our understanding of the biochemical and pharmacological properties of the enzyme. In this commentary, the structure and biochemical properties of FAAH and the development of potent and selective FAAH inhibitors are reviewed, together with a brief discussion on the therapeutic possibilities for such compounds in the treatment of inflammatory pain and ischaemic states.

Despite extensive research on the trafficking of anandamide (AEA) across cell membranes, little is known about the membrane transport of other endocannabinoids, such as <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Previous studies have provided data both in favor and against a cell membrane carrier-mediated transport of endocannabinoids, using different methodological approaches. Because AEA and <em>2</em>-AG undergo rapid and almost complete intracellular hydrolysis, we employed a combination of radioligand assays and absolute quantification of cellular and extracellular endocannabinoid levels. In human U937 leukemia cells, 100 nm AEA and 1 μm <em>2</em>-AG were taken up through a fast and saturable process, reaching a plateau after 5 min. Employing differential pharmacological blockage of endocannabinoid uptake, breakdown, and interaction with intracellular binding proteins, we show that eicosanoid endocannabinoids harboring an arachidonoyl chain compete for a common membrane target that regulates their transport, whereas other N-acylethanolamines did not interfere with AEA and <em>2</em>-AG uptake. By combining fatty acid amide hydrolase or monoacyl glycerol lipase inhibitors with hydrolase-inactive concentrations of the AEA transport inhibitors UCM707 (1 μm) and OMDM-<em>2</em> (5 μm), a functional synergism on cellular AEA and <em>2</em>-AG uptake was observed. Intriguingly, structurally unrelated AEA uptake inhibitors also blocked the cellular release of AEA and <em>2</em>-AG. We show, for the first time, that UCM707 and OMDM-<em>2</em> inhibit the bidirectional movement of AEA and <em>2</em>-AG across cell membranes. Our findings suggest that a putative endocannabinoid cell membrane transporter controls the cellular AEA and <em>2</em>-AG trafficking and metabolism.

Endocannabinoid (eCB) signaling has been heavily implicated in the modulation of anxiety and depressive behaviors and emotional learning. However, the role of the most-abundant endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in the physiological regulation of affective behaviors is not well understood. Here, we show that genetic deletion of the <em>2</em>-AG synthetic enzyme diacylglycerol lipase α (DAGLα) in mice reduces brain, but not circulating, <em>2</em>-AG levels. DAGLα deletion also results in anxiety-like and sex-specific anhedonic phenotypes associated with impaired activity-dependent eCB retrograde signaling at amygdala glutamatergic synapses. Importantly, acute pharmacological normalization of <em>2</em>-AG levels reverses both phenotypes of DAGLα-deficient mice. These data suggest <em>2</em>-AG deficiency could contribute to the pathogenesis of affective disorders and that pharmacological normalization of <em>2</em>-AG signaling could represent an approach for the treatment of mood and anxiety disorders.

The discovery and cloning of CB1 and CB<em>2</em>, the two known G(i/o) protein-coupled cannabinoid receptors, as well as the isolation and characterization of two families of endogenous cannabinergic ligands represented by arachidonoylethanolamide (anandamide) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), have opened new horizons in this newly discovered field of biology. Furthermore, a considerable number of cannabinoid analogs belonging to structurally diverse classes of compounds have been synthesized and tested, thus providing substantial information on the structural requirements for cannabinoid receptor recognition and activation. Experiments with site-directed mutated receptors and computer modeling studies have suggested that these diverse classes of ligands may interact with the receptors through different binding motifs. The information about the exact binding site may be obtained with the help of suitably designed molecular probes. These ligands either interact with the receptors in a reversible fashion (reversible probes) or alternatively attach at or near the receptor active site with the formation of covalent bonds (irreversible probes). This review focuses on structural requirements of cannabinoid receptor ligands and highlights their pharmacological and therapeutic potential.

Opioids and cannabinoids are among the most widely consumed drugs of abuse in humans and the phenomena of cross-tolerance or mutual potentiation have been demonstrated between the two drugs. Several authors have suggested that both drugs share common links in their molecular mechanisms of action, although this has been a matter of controversy. Furthermore, no data exist on the possible adaptive changes in the contents of arachidonoylethanolamide (anandamide, AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), the two major endogenous ligands for cannabinoid receptors, in morphine-tolerant rats. In the present work, we investigated the alterations in cannabinoid receptor functionality and endocannabinoid levels in rats chronically treated with morphine (5 mg/kg, s.c., twice a day for 5 days). Autoradiographic-binding studies using [(3)H]CP-55 940 revealed a slight but significant reduction in cannabinoid receptor level in the cerebellum and hippocampus of morphine-tolerant rats, while CP-55 940-stimulated [(35)S]GTPgammaS binding showed a strong decrease (40%) in receptor/G protein coupling in the limbic area of these animals. Moreover, in the same brain regions we measured, by isotope-dilution gas chromatography/mass spectrometry, the contents of AEA and <em>2</em>-AG. Chronic morphine exposure produced a strong reduction in <em>2</em>-AG contents without changes in AEA levels in several brain regions (ie striatum, cortex, hippocampus, limbic area, and hypothalamus). These findings clearly demonstrate that prolonged activation of opioid receptors could alter the cannabinoid system, in terms of both receptor functionality and endocannabinoid levels, and suggest the involvement of this system, alone or in combination with other mediators, in the phenomenon of morphine tolerance.

Monoacylglycerol lipase (MGL) and fatty-acid amide hydrolase (FAAH) degrade the endocannabinoids <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and anandamide (AEA), respectively. Pharmacological inhibition of these enzymes in the periphery may elucidate the role of endocannabinoids in controlling nociceptive transmission. We compared effects of the MGL inhibitor JZL184, the FAAH inhibitor URB597, and the endocannabinoid uptake inhibitor VDM11, administered locally in the paw, on behavioral hypersensitivities produced by capsaicin, the pungent ingredient in hot chili peppers. Intradermal capsaicin (10 microg i.pl.) produced nocifensive behavior, thermal hyperalgesia, and mechanical allodynia in rats. JZL184 (100 microg i.pl.) suppressed capsaicin-induced nocifensive behavior and thermal hyperalgesia without altering capsaicin-evoked mechanical allodynia. Effects of JZL184 were blocked by either the CB(1) antagonist AM<em>2</em>51 (80 microg i.pl.) or the CB(<em>2</em>) antagonist AM630 (<em>2</em>5 microg i.pl.). URB597 (75 microg i.pl.) suppressed capsaicin-induced mechanical allodynia without altering capsaicin-evoked thermal hyperalgesia or nocifensive behavior. Effects of URB597 were blocked by AM<em>2</em>51 (80 microg i.pl.), but not by AM630 (<em>2</em>5 microg i.pl.). VDM11 (100 microg i.pl.) suppressed capsaicin-evoked hypersensitivity for all three dependent measures (nocifensive behavior, thermal hyperalgesia, and mechanical allodynia), suggesting an additive effect following putative elevation of both AEA and <em>2</em>-AG. The VDM11-induced suppression of capsaicin-evoked nocifensive behavior and thermal hyperalgesia was blocked by either AM<em>2</em>51 (80 microg i.pl.) or AM630 (<em>2</em>5 microg i.pl.), as observed with JZL184. The VDM11-induced suppression of capsaicin-evoked mechanical allodynia was blocked by AM<em>2</em>51 (<em>2</em>5 microg i.pl.) only, as observed with URB597. Thus, peripheral inhibition of enzymes hydrolyzing <em>2</em>-AG and AEA suppresses capsaicin-evoked behavioral sensitization with distinct patterns of pharmacological specificity and in a non-overlapping and modality-specific manner. Modulation of endocannabinoids in the periphery suppressed capsaicin-evoked nocifensive behavior and thermal hyperalgesia through either CB(1) or CB(<em>2</em>) receptor mechanisms but suppressed capsaicin-evoked mechanical allodynia through CB(1) mechanisms only. Inhibition of endocannabinoid transport was more effective in suppressing capsaicin-induced sensitization compared to inhibition of either FAAH or MGL alone. These studies are the first to unveil the effects of pharmacologically increasing peripheral endocannabinoid levels on capsaicin-induced behavioral hypersensitivities. Our data suggest that <em>2</em>-AG, the putative product of MGL inhibition, and AEA, the putative product of FAAH inhibition, differentially suppress capsaicin-induced nociception through peripheral cannabinoid mechanisms.

Several selective inhibitors of endocannabinoid inactivation via either the fatty acid amide hydrolase (FAAH) or the putative endocannabinoid transporter have been developed so far. Here, we have studied the effect in rats of a subchronic intraperitoneal treatment with three recently developed selective inhibitors of endocannabinoid uptake (VDM-11, UCM-707 and OMDM-<em>2</em>) or with a selective FAAH inhibitor (N-arachidonoyl-serotonin, AA-5-HT), on the brain levels of anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) measured by means of isotope dilution LC-MS 1, 5 and 1<em>2</em> h after the last treatment. OMDM-<em>2</em> was the most efficacious compound at enhancing the levels of anandamide at all time points, with a maximal effect (1.9-fold enhancement) after 5h. This compound also enhanced <em>2</em>-AG levels by approximately 1.3-fold, but only 5 and 1<em>2</em>h from administration. VDM-11 slightly, albeit significantly, enhanced anandamide levels (1.3-fold) only at 1h from administration and <em>2</em>-AG levels (1.3-fold) only after 5h. Finally, UCM-707 only affected <em>2</em>-AG levels (by two-fold) at only 1h from administration. FAAH inhibition by AA-5-HT significantly enhanced the levels of both anandamide (between 1.3- and 1.5-fold, maximal effect after 1 h) and <em>2</em>-AG (between 1.3- and 1.6-fold, maximal effect after 1<em>2</em> h) at all time points. Brains from rats treated with AA-5-HT did never exhibit enhanced levels of serotonin, thus pointing to the metabolic stability of this FAAH inhibitor. These data indicate that: (1) the pharmacological effects reported so far for the four compounds under study in animal models of diseases may be due to enhancement of both anandamide and <em>2</em>-AG levels; (<em>2</em>) <em>2</em>-AG seems to need a longer time after the last administration in order to be augmented; (3) OMDM-<em>2</em> and AA-5-HT should be regarded as enhancers of endocannabinoid levels suitable for use in vivo.

OBJECTIVE

Salvinorin A, the active component of the hallucinogenic herb Salvia divinorum, inhibits intestinal motility through activation of kappa-opioid receptors (KORs). However, this compound may have target(s) other than the KORs in the inflamed gut. Because intestinal inflammation upregulates cannabinoid receptors and endogenous cannabinoids, in the present study we investigated the possible involvement of the endogenous cannabinoid system in salvinorin A-induced delay in motility in the inflamed gut.

METHODS

Motility in vivo was measured by evaluating the distribution of a fluorescent marker along the small intestine; intestinal inflammation was induced by the irritant croton oil; direct or indirect activity at cannabinoid receptors was evaluated by means of binding, enzymic and cellular uptake assays.

RESULTS

Salvinorin A as well as the KOR agonist U-50488 reduced motility in croton oil treated mice. The inhibitory effect of both salvinorin A and U-50488 was counteracted by the KOR antagonist nor-binaltorphimine and by the cannabinoid CB(1) receptor antagonist rimonabant. Rimonabant, however, did not counteract the inhibitory effect of salvinorin A on motility in control mice. Binding experiments showed very weak affinity of salvinorin A for cannabinoid CB(1) and CB(<em>2</em>) and no inhibitory effect on <em>2</em>-<em>arachidonoylglycerol</em> and anandamide hydrolysis and cellular uptake.

CONCLUSIONS

The inhibitory effect of salvinorin A on motility reveals a functional interaction between cannabinoid CB(1) receptors and KORs in the inflamed--but not in the normal--gut in vivo.

The aim of the present study was to assess the contribution of endogenous cannabinoids in the protective effect of ischemic preconditioning on the endothelial function in coronary arteries of the rat. Isolated rat hearts were exposed to a 30-min low flow ischemia (1 ml/min) followed by <em>2</em>0-min reperfusion, after which the response to the endothelium-dependent vasodilator, serotonine (5-HT), was compared with that of the endothelium-independent vasodilator, sodium nitroprusside (SNP). In untreated hearts, ischemia-reperfusion diminished selectively 5-HT-induced vasodilatation, compared with time-matched sham hearts, the vasodilatation to SNP being unaffected. A 5-min zero-flow preconditioning ischemia in untreated hearts preserved the vasodilatation produced by 5-HT. Blockade of either CB(1)-receptors with SR141716A or CB(<em>2</em>)-receptors with SR1445<em>2</em>8 abolished the protective effect of preconditioning on the 5-HT vasodilatation. Perfusion with either palmitoylethanolamide or <em>2</em>-<em>arachidonoylglycerol</em> 15 min before and throughout the ischemia mimicked preconditioning inasmuch as it protected the endothelium in a similar fashion. This protection was blocked by SR1445<em>2</em>8 in both cases, whereas SR141716A only blocked the effect of PEA. The presence of CB(1) and CB(<em>2</em>)-receptors in isolated rat hearts was confirmed by Western blots. In conclusion, the data suggest that endogenous cannabinoids contribute to the endothelial protective effect of ischemic preconditioning in rat coronary arteries.

Persistent CB1 cannabinoid receptor activity limits neurotransmitter release at various synapses throughout the brain. However, it is not fully understood how constitutively active CB1 receptors, tonic endocannabinoid signaling, and its regulation by multiple serine hydrolases contribute to the synapse-specific calibration of neurotransmitter release probability. To address this question at perisomatic and dendritic GABAergic synapses in the mouse hippocampus, we used a combination of paired whole-cell patch-clamp recording, liquid chromatography/tandem mass spectrometry, stochastic optical reconstruction microscopy super-resolution imaging, and immunogold electron microscopy. Unexpectedly, application of the CB1 antagonist and inverse agonist AM<em>2</em>51 [N-1-(<em>2</em>,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide], but not the neutral antagonist NESS03<em>2</em>7 [8-chloro-1-(<em>2</em>,4-dichlorophenyl)-N-piperidin-1-yl-5,6-dihydro-4H-benzo[<em>2</em>,3]cyclohepta[<em>2</em>,4-b]pyrazole-3-carboxamine], significantly increased synaptic transmission between CB1-positive perisomatic interneurons and CA1 pyramidal neurons. JZL184 (4-nitrophenyl 4-[bis(1,3-benzodioxol-5-yl)(hydroxy)methyl]piperidine-1-carboxylate), a selective inhibitor of monoacylglycerol lipase (MGL), the presynaptic degrading enzyme of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), elicited a robust increase in <em>2</em>-AG levels and concomitantly decreased GABAergic transmission. In contrast, inhibition of fatty acid amide hydrolase (FAAH) by PF3845 (N-pyridin-3-yl-4-[[3-[5-(trifluoromethyl)pyridin-<em>2</em>-yl]oxyphenyl]methyl]piperidine-1-carboxamide) elevated endocannabinoid/endovanilloid anandamide levels but did not change GABAergic synaptic activity. However, FAAH inhibitors attenuated tonic <em>2</em>-AG increase and also decreased its synaptic effects. This antagonistic interaction required the activation of the transient receptor potential vanilloid receptor TRPV1, which was concentrated on postsynaptic intracellular membrane cisternae at perisomatic GABAergic symmetrical synapses. Interestingly, neither AM<em>2</em>51, JZL184, nor PF3845 affected CB1-positive dendritic interneuron synapses. Together, these findings are consistent with the possibility that constitutively active CB1 receptors substantially influence perisomatic GABA release probability and indicate that the synaptic effects of tonic <em>2</em>-AG release are tightly controlled by presynaptic MGL activity and also by postsynaptic endovanilloid signaling and FAAH activity.

UNASSIGNED

Tonic cannabinoid signaling plays a critical role in the regulation of synaptic transmission. However, the mechanistic details of how persistent CB1 cannabinoid receptor activity inhibits neurotransmitter release have remained elusive. Therefore, electrophysiological recordings, lipid measurements, and super-resolution imaging were combined to elucidate those signaling molecules and mechanisms that underlie tonic cannabinoid signaling. The findings indicate that constitutive CB1 activity has pivotal function in the tonic control of hippocampal GABA release. Moreover, the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is continuously generated postsynaptically, but its synaptic effect is regulated strictly by presynaptic monoacylglycerol lipase activity. Finally, anandamide signaling antagonizes tonic <em>2</em>-AG signaling via activation of postsynaptic transient receptor potential vanilloid TRPV1 receptors. This unexpected mechanistic diversity may be necessary to fine-tune GABA release probability under various physiological and pathophysiological conditions.

The discovery of the endocannabinoid signalling system, that is, of cannabinoid receptors, their endogenous ligands, known as endocannabinoids, and of endocannabinoid anabolic and catabolic enzymes, raised several questions regarding the physiopathological role of these mediators. Several of these questions were answered by investigating alterations in the levels of the most studied endocannabinoids, anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), in tissues of animal models of disorders, and in bioptic samples and biological fluids (cerebrospinal fluid and blood) of human volunteers. Subsequently, the pharmacological effects of synthetic compounds that selectively target the cannabinoid CB(1) and CB(<em>2</em>) receptors, and endocannabinoid anabolic and catabolic enzymes, established cause-effect relationships between pathological alterations in endocannabinoid levels and the symptoms and progress of several disorders, including emesis, obesity, metabolic disorders, hepatic diseases, pain, inflammation and neurological and neuropsychiatric disorders. These new developments are discussed in this second review on the endocannabinoids, together with the results of pre-clinical and clinical studies on the potential therapeutic use of plant-derived cannabinoids and synthetic agents that manipulate pharmacologically the action at cannabinoid receptors or the tissue levels of AEA and <em>2</em>-AG.

Long-term changes in synaptic efficacy produced by high-frequency stimulation (HFS) of glutamatergic afferents to the rat dorsolateral striatum exhibit heterogeneity during early stages of postnatal development. Whereas HFS most often induces striatal long-term potentiation (LTP) in rats postnatal day 1<em>2</em> (P1<em>2</em>)-P14, the same stimulation tends to induce long-term depression (LTD) at ages P16-P34. Previous studies have shown that striatal LTD induction depends on retrograde endocannabinoid signaling and activation of the CB1 cannabinoid receptor. It is also known that levels of one of the primary endogenous CB1 receptor agonists, anandamide (AEA), increases during development in whole-brain samples. In the present study, we sought to determine whether this developmental increase in AEA also takes place in striatal tissue and whether increased AEA levels contribute to the postnatal switch in the response to HFS. We observed a pronounced increase in striatal levels of AEA, but not the other major endogenous cannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), during the postnatal period characterized by the switch from LTP to LTD. Furthermore, application of synthetic AEA during HFS in field recordings of slices from P1<em>2</em>-P14 rats allowed for induction of LTD whereas blocking the CB1 receptor during HFS in animals P16-P34 resulted in expression of LTP. However, blocking <em>2</em>-AG synthesis with the DAG-lipase inhibitor tetrahydrolipstatin did not alter HFS-induced striatal LTD. In addition, synaptic depression produced by a synthetic CB1 agonist was similar across development. Together, these findings suggest that the robust developmental increase in striatal AEA may be the key factor in the emergence of HFS-induced striatal LTD.

The pharmacology of monoacylglycerol lipase (MAGL) is not well understood. In consequence, the abilities of a series of analogues of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) to inhibit cytosolic <em>2</em>-oleoylglycerol and membrane-bound anandamide hydolysis by MAGL and fatty acid amide hydrolase (FAAH), respectively, have been investigated. <em>2</em>-AG and its 1-regioisomer (1-AG) interacted with MAGL with similar affinities (IC(50) values 13 and 17 mum, respectively). Shorter homologues of <em>2</em>-AG (<em>2</em>-linoleoylglycerol and <em>2</em>-oleoylglycerol) had affinities for MAGL similar to <em>2</em>-AG. This pattern was also seen when the arachidonoyl side chain of arachidonoyl trifluoromethylketone was replaced by an oleoyl side chain. Arachidonoyl serinol (IC(50) value 73 microM) was a weaker inhibitor of MAGL than <em>2</em>-AG. The IC(50) values of noladin ether towards MAGL and FAAH were 36 and 3 microM, respectively. Arachidonoyl glycine interacted with FAAH (IC(50) value 4.9 microM) but only weakly interacted with MAGL (IC(50) value >100 microM). alpha-Methyl-1-AG had similar potencies towards MAGL and FAAH (IC(50) values of 11 and 33 microM, respectively). O-<em>2</em><em>2</em>03 (1-(<em>2</em>0-cyano-16,16-dimethyl-eicosa-5,8,11,14-tetraenoyl) glycerol) and O-<em>2</em><em>2</em>04 (<em>2</em>-(<em>2</em>0-hydroxy-16,16-dimethyl-eicosa-5,8,11,14-tetraenoyl) glycerol) were slightly less potent, but again affected both enzymes equally. alpha-Methyl-1-AG, O-<em>2</em><em>2</em>03 and O-<em>2</em><em>2</em>04 interacted only weakly with cannabinoid CB(1) receptors expressed in CHO cells (K(i) values 1.8, 3.7 and 3.<em>2</em> microM, respectively, compared with 0.<em>2</em>4 microM for 1-AG) and showed no evidence of central cannabinoid receptor activation in vivo at doses up to 30 mg kg(-1) i.v. It is concluded that compounds like alpha-Methyl-1-AG, O-<em>2</em><em>2</em>03 and O-<em>2</em><em>2</em>04 may be useful as leads for the discovery of selective MAGL inhibitors that lack direct effects upon cannabinoid receptors.

Endocannabinoids have recently emerged as instructive cues in the developing central nervous system, and, based on the expression of their receptors, we identified oligodendrocytes as potential targets of these molecules. Here, we show that the enzymes responsible for the synthesis of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), diacylglycerol lipase alpha (DAGLα) and beta (DAGLβ), and degradation, monoacylglycerol lipase (MAGL), can be found in oligodendrocytes at different developmental stages. Moreover, cultured oligodendrocyte progenitor cells (OPCs) express DAGLα and β abundantly, resulting in the stronger production of <em>2</em>-AG than in differentiated oligodendrocytes. The opposite is observed with MAGL. CB1 and CB<em>2</em> receptor antagonists (SR141716 and AM630) impaired OPC differentiation into mature oligodendrocytes and likewise, inhibiting DAGL activity with RHC-80<em>2</em>67 or tetrahydrolipstatin also blocked oligodendrocyte maturation, an effect reversed by the addition of exogenous <em>2</em>-AG. Likewise, <em>2</em>-AG synthesis disruption using specific siRNAs against DAGLα and DAGLβ significantly reduced myelin protein expression in vitro, whereas a pharmacological gain-of-function approach by using cannabinoid agonists or MAGL inhibition had the opposite effects. ERK/MAPK pathway is implicated in oligodendrocyte differentiation because PD98059, an inhibitor of MEK1, abrogated oligodendrocyte maturation. The cannabinoid receptor antagonists and RHC-80<em>2</em>67 all diminished basal ERK1/<em>2</em> phosphorylation, effects that were partially reversed by the addition of <em>2</em>-AG. Overall, our data suggest a novel role of endocannabinoids in oligodendrocyte differentiation such that constitutive release of <em>2</em>-AG activates cannabinoid receptors in an autocrine/paracrine way in OPCs, stimulating the ERK/MAPK signaling pathway.

In the digestive tract there is evidence for the presence of high levels of endocannabinoids (anandamide and <em>2</em>-<em>arachidonoylglycerol</em>) and enzymes involved in the synthesis and metabolism of endocannabinoids. Immunohistochemical studies have shown the presence of CB1 receptors on myenteric and submucosal nerve plexuses along the alimentary tract. Pharmacological studies have shown that activation of CB1 receptors produces relaxation of the lower oesophageal sphincter, inhibition of gastric motility and acid secretion, as well as intestinal motility and secretion. In general, CB1-induced inhibition of intestinal motility and secretion is due to reduced acetylcholine release from enteric nerves. Conversely, endocannabinoids stimulate intestinal primary sensory neurons via the vanilloid VR1 receptor, resulting in enteritis and enhanced motility. The endogenous cannabinoid system has been found to be involved in the physiological control of colonic motility and in some pathophysiological states, including paralytic ileus, intestinal inflammation and cholera toxin-induced diarrhoea. Cannabinoids also possess antiemetic effects mediated by activation of central and peripheral CB1 receptors. Pharmacological modulation of the endogenous cannabinoid system could provide a new therapeutic target for the treatment of a number of gastrointestinal diseases, including nausea and vomiting, gastric ulcers, secretory diarrhoea, paralytic ileus, inflammatory bowel disease, colon cancer and gastro-oesophageal reflux conditions.