<em>2</em>-<em>Arachidonoylglycerol</em> is an endogenous ligand for the cannabinoid receptors (CB1 and CB<em>2</em>). Previously, we provided evidence that <em>2</em>-<em>arachidonoylglycerol</em>, but not anandamide (N-arachidonoylethanolamine), is the true natural ligand for the cannabinoid receptors. In the present study, we examined in detail the effects of <em>2</em>-<em>arachidonoylglycerol</em> on the production of chemokines in human promyelocytic leukemia HL-60 cells. We found that <em>2</em>-<em>arachidonoylglycerol</em> induced a marked acceleration in the production of interleukin 8. The effect of <em>2</em>-<em>arachidonoylglycerol</em> was blocked by treatment of the cells with SR1445<em>2</em>8, a cannabinoid CB<em>2</em> receptor antagonist, indicating that the effect of <em>2</em>-<em>arachidonoylglycerol</em> is mediated through the CB<em>2</em> receptor. Augmented production of interleukin 8 was also observed with CP55940, a synthetic cannabinoid, and an ether-linked analog of <em>2</em>-<em>arachidonoylglycerol</em>. On the other hand, neither anandamide nor the free arachidonic acid induced the enhanced production of interleukin 8. A similar effect of <em>2</em>-<em>arachidonoylglycerol</em> was observed in the case of the production of macrophage-chemotactic protein-1. The accelerated production of interleukin 8 by <em>2</em>-<em>arachidonoylglycerol</em> was observed not only in undifferentiated HL-60 cells, but also in HL-60 cells differentiated into macrophage-like cells. Noticeably, <em>2</em>-<em>arachidonoylglycerol</em> and lipopolysaccharide acted synergistically to induce the dramatically augmented production of interleukin 8. These results strongly suggest that the CB<em>2</em> receptor and its physiological ligand, i.e., <em>2</em>-<em>arachidonoylglycerol</em>, play important regulatory roles such as stimulation of the production of chemokines in inflammatory cells and immune-competent cells. Detailed studies on the cannabinoid receptor system are thus essential to gain a better understanding of the precise regulatory mechanisms of inflammatory reactions and immune responses.

Neuropathic pain elevates spinal anandamide (AEA) levels in a way further increased when URB597, an inhibitor of AEA hydrolysis by fatty acid amide hydrolase (FAAH), is injected intrathecally. Spinal AEA reduces neuropathic pain by acting at both cannabinoid CB1 receptors and transient receptor potential vanilloid-1 (TRPV1) channels. Yet, intrathecal URB597 is only partially effective at counteracting neuropathic pain. We investigated the effect of high doses of intrathecal URB597 on allodynia and hyperalgesia in rats with chronic constriction injury (CCI) of the sciatic nerve. Among those tested, the <em>2</em>00 µg/rat dose of URB597 was the only one that elevated the levels of the FAAH non-endocannabinoid and anti-inflammatory substrates, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), and of the endocannabinoid FAAH substrate, <em>2</em>-<em>arachidonoylglycerol</em>, and fully inhibited thermal and tactile nociception, although in a manner blocked almost uniquely by TRPV1 antagonism. Surprisingly, this dose of URB597 decreased spinal AEA levels. RT-qPCR and western blot analyses demonstrated altered spinal expression of lipoxygenases (LOX), and baicalein, an inhibitor of 1<em>2</em>/15-LOX, significantly reduced URB597 analgesic effects, suggesting the occurrence of alternative pathways of AEA metabolism. Using immunofluorescence techniques, FAAH, 15-LOX and TRPV1 were found to co-localize in dorsal spinal horn neurons of CCI rats. Finally, 15-hydroxy-AEA, a 15-LOX derivative of AEA, potently and efficaciously activated the rat recombinant TRPV1 channel. We suggest that intrathecally injected URB597 at full analgesic efficacy unmasks a secondary route of AEA metabolism via 15-LOX with possible formation of 15-hydroxy-AEA, which, together with OEA and PEA, may contribute at producing TRPV1-mediated analgesia in CCI rats.

Until recently, endocannabinoid (eCB) signalling was largely studied in the context of synaptic plasticity in the postnatal brain in the absence of detailed knowledge of the nature of the enzyme(s) responsible for the synthesis of the eCBs. However, the identification of two diacylglycerol lipases (DAGLα and DAGLβ) responsible for the synthesis of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) has increased the understanding of where this eCB is synthesised in relationship to the expression of cannabinoid receptor (CB)1 and CB<em>2</em>. Furthermore, the generation of knockout animals for each enzyme has allowed for the direct testing of their importance for established and emerging eCB functions. Based on this, we now know that DAGLα is enriched in dendritic spines that appose CB1-positive synaptic terminals, and that <em>2</em>-AG functions as a retrograde signal controlling synaptic strength throughout the nervous system. Consequently, we have built on the principle that expression of eCB components dictates function to identify additional physiological functions of this signalling cassette. A number of studies have now provided support for DAGL-dependent eCB signalling playing important roles in brain development and in cellular plasticity in the adult nervous system. In this article, we will review evidence based on the localisation of the enzymes, as well as from genetic and pharmacological studies, that show DAGL-dependent eCB signalling to play an important role in axonal growth and guidance during development, in retrograde synaptic signalling at mature synapses, and in the control of adult neurogenesis in the hippocampus and subventricular zone.

Although inhibitors of the enzymatic hydrolysis of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> are available, they are either rather weak in vitro (IC(50)>30 microM) or their selectivity towards other proteins of the endocannabinoid system has not been tested. Here we describe the synthesis and activity in vitro and in vivo of a tetrahydrolipstatin analogue, OMDM169, as a potent inhibitor of <em>2</em>-AG hydrolysis, capable of enhancing <em>2</em>-AG levels and of exerting analgesic activity via indirect activation of cannabinoid receptors. OMDM169 exhibited 0.13 microM<IC(50)<0.41 microM towards <em>2</em>-AG hydrolysing activities in COS-7 cells and rat cerebellum, and inhibited (IC(50)=0.89 microM) the human recombinant MAGL, whilst being inactive (K(i)>10 microM) at human CB(1) and CB(<em>2</em>) receptors. However, OMDM169 shared with tetrahydrolipstatin the capability of inhibiting the human pancreatic lipase (IC(50)=0.6 microM). OMDM169 inhibited fatty acid amide hydrolase and diacylglycerol lipase only at higher concentrations (IC(50)=3.0 and <em>2</em>.8 microM, respectively), and, accordingly, it increased by approximately 1.6-fold the levels of <em>2</em>-AG, but not anandamide, in intact ionomycin-stimulated N18TG<em>2</em> neuroblastoma cells. Acute intraperitoneal (i.p.) administration of OMDM169 to mice inhibited the second phase of the formalin-induced nocifensive response with an IC(50) of approximately <em>2</em>.5 mg/kg, and concomitantly elevated <em>2</em>-AG, but not anandamide, levels in the ipsilateral paw of formalin-treated mice. The antinociceptive effect of OMDM169 was antagonized by antagonists of CB(1) and CB(<em>2</em>) receptors, AM<em>2</em>51 and AM630, respectively (1 mg/kg, i.p.). OMDM69 might represent a template for the development of selective and even more potent inhibitors of <em>2</em>-AG hydrolysis.

OBJECTIVE

Endocannabinoids include anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Endocannabinoid-related molecules like oleoyl-ethanolamine (OEA) and palmitoyl-ethanolamine (PEA) have also been identified. AEA contributes to the pathogenesis of cardiovascular alterations in experimental cirrhosis, but data on the endocannabinoid system in human cirrhosis are lacking. Thus, we aimed to assess whether circulating and hepatic endocannabinoids are upregulated in cirrhotic patients and whether their levels correlate with systemic haemodynamics and liver function.

METHODS

The endocannabinoid levels were measured in peripheral and hepatic veins and liver tissue by isotope-dilution liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry. Systemic haemodynamics were assessed by the transthoracic electrical bioimpedance technique. Portal pressure was evaluated by hepatic venous pressure gradient.

RESULTS

Circulating AEA and, to a greater extent, PEA and OEA were significantly higher in cirrhotic patients than in controls. PEA and OEA were also increased in the cirrhotic liver tissue. AEA, OEA and PEA levels were significantly higher in peripheral than in the hepatic veins of cirrhotic patients, while the opposite occurred for <em>2</em>-AG. Finally, circulating AEA, OEA and PEA correlated with parameters of liver function, such as serum bilirubin and international normalized ratio. No correlations were found with systemic haemodynamics.

CONCLUSIONS

The endocannabinoid system is upregulated in human cirrhosis. Peripheral AEA is increased in patients with a high model of end-stage liver disease score and may reflect the extent of liver dysfunction. In contrast, the <em>2</em>-AG levels, the other major endocannabinoid, are not affected by cirrhosis. The upregulation of the endocannabinoid-related molecules, OEA and PEA, is even greater than that of AEA, prompting pharmacological studies on these compounds.

The serine hydrolase α/β-hydrolase domain 6 (ABHD6) hydrolyzes the most abundant endocannabinoid (eCB) in the brain, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), and controls its availability at cannabinoid receptors. We show that ABHD6 inhibition decreases pentylenetetrazole (PTZ)-induced generalized tonic-clonic and myoclonic seizure incidence and severity. This effect is retained in Cnr1(-/-) or Cnr<em>2</em>(-/-) mice, but blocked by addition of a subconvulsive dose of picrotoxin, suggesting the involvement of GABAA receptors. ABHD6 inhibition also blocked spontaneous seizures in R6/<em>2</em> mice, a genetic model of juvenile Huntington's disease known to exhibit dysregulated eCB signaling. ABHD6 blockade retained its antiepileptic activity over chronic dosing and was not associated with psychomotor or cognitive effects. While the etiology of seizures in R6/<em>2</em> mice remains unsolved, involvement of the hippocampus is suggested by interictal epileptic discharges, increased expression of vGLUT1 but not vGAT, and reduced Neuropeptide Y (NPY) expression. We conclude that ABHD6 inhibition may represent a novel antiepileptic strategy.

Steroids regulate cell proliferation, tissue development, and cell signaling via two pathways: a nuclear receptor mechanism and genome-independent signaling. Sperm activation, egg maturation, and steroid-induced anesthesia are executed via the latter pathway, the key components of which remain unknown. Here, we present characterization of the human sperm progesterone receptor that is conveyed by the orphan enzyme α/β hydrolase domain-containing protein <em>2</em> (ABHD<em>2</em>). We show that ABHD<em>2</em> is highly expressed in spermatozoa, binds progesterone, and acts as a progesterone-dependent lipid hydrolase by depleting the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>AG) from plasma membrane. The <em>2</em>AG inhibits the sperm calcium channel (CatSper), and its removal leads to calcium influx via CatSper and ensures sperm activation. This study reveals that progesterone-activated endocannabinoid depletion by ABHD<em>2</em> is a general mechanism by which progesterone exerts its genome-independent action and primes sperm for fertilization.

Emerging evidence suggests that cannabinoids play an important role in the modulation of fatty liver, which appears to be mediated via activation of cannabinoid receptors. Steatogenic agents such as ethanol and high-fat diet can upregulate the activity of cannabinoid 1 (CB1) receptors via increasing synthesis of endocannabinoids, <em>2</em>-<em>arachidonoylglycerol</em>, and anandamide. CB1 receptors can also be upregulated by obesity. CB1 receptor activation results in upregulation of lipogenic transcription factor, sterol regulatory element-binding protein 1c and its target enzymes, acetyl-CoA carboxylase-1, and fatty acid synthase and concomitantly, downregulation of carnitine palmitoyltransferase-1. This leads to increased de novo fatty acid synthesis as well as decreased fatty acid oxidation, culminating into the development of fatty liver. High-fat diet, in addition to CB1 receptor activation, appears to activate CB<em>2</em> receptors that may also contribute to fatty liver. In non-alcoholic fatty liver disease, CB<em>2</em> receptor activation is associated with the development of fatty liver. Cannabis smoking can increase the severity of fatty liver in hepatitis C patients although the precise mechanism is unknown. As the mechanisms involved in endocannabinoid receptor signaling are being increasingly well understood and the biosynthetic regulatory elements elucidated, these present good opportunity for the pharmaceutical scientists to design drugs to treat liver diseases, including steatosis, based on the cannabinoids, endocannabinoids, and related templates.

We have previously shown that treatment of Zucker rats and mice with diet-induced obesity with dietary docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids in the form of krill oil reduces peripheral levels of endocannabinoids, ectopic fat formation and hyperglycemia. We reported that such treatment reduces plasma endocannabinoid levels also in overweight and obese human individuals, in whom high triglycerides may correlate with high circulating endocannabinoid levels. In this study, we report the effects of krill powder, which contains proteins (34%) in addition to krill oil (61.8%), on these two parameters. We submitted 11 obese men (average BMI of 3<em>2</em>.3 kg/m², age of 4<em>2</em>.6 years and plasma triglycerides of 19<em>2</em>.5 ± 96.3 mg/dl) to a <em>2</em>4 week dietary supplementation with krill powder (4 g/day per os) and measured anthropometric and metabolic parameters, as well as blood endocannabinoid (anandamide and <em>2</em>-<em>arachidonoylglycerol</em>) and esterified DHA and EPA levels. Six subjects were included as control subjects and not given any supplements. The treatment produced, after 1<em>2</em> and <em>2</em>4 weeks, a significant increase in DHA and EPA in total plasma, a 59 and 84% decrease in anandamide plasma levels, and a <em>2</em><em>2</em>.5 and <em>2</em>0.6% decrease in triglyceride levels, respectively. There was also a significant decrease in waist/hip ratio and visceral fat/skeletal muscle mass ratio at <em>2</em>4 weeks, but no change in body weight. These data confirm that dietary krill powder reduces peripheral endocannabinoid overactivity in obese subjects, and might ameliorate some parameters of the metabolic syndrome.

Physiological concentrations of progesterone stimulate the activity of the endocannabinoid-degrading enzyme anandamide hydrolase (fatty acid amide hydrolase, FAAH) in human lymphocytes. At the same concentrations, the membrane-impermeant conjugate of progesterone with BSA was ineffective, suggesting that binding to an intracellular receptor was needed for progesterone activity. Stimulation of FAAH occurred through up-regulation of gene expression at transcriptional and translational level, and was partly mediated by the Th<em>2</em> cytokines. In fact, lymphocyte treatment with IL-4 or with IL-10 had a stimulating effect on FAAH, whereas the Th1 cytokines IL-1<em>2</em> and IFN-gamma reduced the activity and the protein expression of FAAH. Human chorionic gonadotropin or cortisol had no effect on FAAH activity. At variance with FAAH, the lymphocyte anandamide transporter and cannabinoid receptors were not affected by treatment with progesterone or cytokines. Good FAAH substrates such as anandamide and <em>2</em>-<em>arachidonoylglycerol</em> inhibited the release of leukemia-inhibitory factor from human lymphocytes, but N-palmitoylethanolamine, a poor substrate, did not. A clinical study performed on 100 healthy women showed that a low FAAH activity in lymphocytes correlates with spontaneous abortion, whereas anandamide transporter and cannabinoid receptors in these cells remain unchanged. These results add the endocannabinoids to the hormone-cytokine array involved in the control of human pregnancy.

Hydra (Cnidaria) is the first animal organism to have developed a neural network, which has been proposed to control, inter alia, the "feeding response", i.e. a mechanism through which the coelenterate opens and then closes its mouth in the presence of prey and/or glutathione. Here, we report that Hydra contains: (i) selective cannabinoid binding sites; (ii) the endogenous cannabinoid receptor ligand, anandamide (arachidonoylethanolamide); (iii) a fatty acid amide hydrolase-like activity catalysing anandamide hydrolysis; and (iv) the putative biosynthetic precursor of anandamide, N-arachidonoylphosphatidylethanolamine. We suggest that this "endogenous cannabinoid system" is involved in the modulation of the "feeding response". Anandamide (1 nM-1 microM) potently inhibited (up to 45%) the glutathione-induced "feeding response" by accelerating Hydra vulgaris mouth closure. The effect was maximal at 100 nM anandamide and was reversed by the selective antagonist of the CB1 subtype of mammalian cannabinoid receptors, SR 141716A (50-100 nM). Specific cannabinoid binding sites were detected in membranes from Hydra polyps by using [3H]SR 141716A (Kd= 1.87 nM, Bmax = <em>2</em>6.7 fmol/mg protein), and increasing anandamide concentrations were found to displace the binding of [3H]SR 141716A to these membranes (Ki = .505 nM). Hydra polyps were also found to contain amounts of anandamide (15.6 pmol/g) and N-arachidonoylphosphatidylethanolamine (3<em>2</em>.4 pmol/g), as well as the other "endocannabinoid" <em>2</em>-<em>arachidonoylglycerol</em> (11.<em>2</em> nmol/g), comparable to those described previously for mammalian brain. Finally, a fatty acid amide hydrolase activity (Vmax = 3.4 nmol/min/mg protein), with subcellular distribution, pH dependency and sensitivity to inhibitors similar to those reported for the mammalian enzyme, but with a lower affinity for anandamide (Km = 400 microM), was also detected in Hydra polyps. These data suggest that the endocannabinoid signalling system plays a physiological role in Hydra that is to control the feeding response. Hydra is the simplest living organism described so far to use this recently discovered regulatory system.

Chronic alcohol exposure leads to significant changes in the levels of endocannabinoids and their receptors in the brains of humans and laboratory animals, as well as in cultured neuronal cells. However, little is known about the effects of short-term periods of alcohol exposure. In the present study, we examined the changes in endocannabinoid levels (anandamide and <em>2</em>-<em>arachidonoylglycerol</em>), as well as four additional N-acylethanolamines, in four brain regions of rats exposed to alcohol through the liquid diet for a period of <em>2</em>4h. The levels of N-acylethanolamines were diminished <em>2</em>4h after the onset of alcohol exposure. This was particularly evident for anandamide in the hypothalamus, amygdala and caudate-putamen, for N-palmitoylethanolamine in the caudate-putamen, for N-oleoylethanolamine in the hypothalamus, caudate-putamen and prefrontal cortex, and for N-stearoylethanolamine in the amygdala. The only exception was N-linoleoylethanolamine for which the levels increased in the amygdala after the exposure to alcohol. The levels of the other major endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em>, were also reduced with marked effects in the prefrontal cortex. These results support the notion that short-term alcohol exposure reduces endocannabinoid levels in the brain accompanied by a reduction in several related N-acylethanolamines.

Recent work in our laboratories has demonstrated that an opioid-independent form of stress-induced analgesia (SIA) is mediated by endogenous cannabinoids [Hohmann et al., <em>2</em>005. Nature 435, 1108]. Non-opioid SIA, induced by a 3-min continuous foot shock, is characterized by the mobilization of two endocannabinoid lipids--<em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and anandamide--in the midbrain periaqueductal gray (PAG). The present studies were conducted to examine the contributions of spinal endocannabinoids to nonopioid SIA. Time-dependent increases in levels of <em>2</em>-AG, but not anandamide, were observed in lumbar spinal cord extracts derived from shocked relative to non-shocked rats. Notably, <em>2</em>-AG accumulation was of smaller magnitude than that observed previously in the dorsal midbrain following foot shock. <em>2</em>-AG is preferentially degraded by monoacylglycerol lipase (MGL), whereas anandamide is hydrolyzed primarily by fatty-acid amide hydrolase (FAAH). This metabolic segregation enabled us to manipulate endocannabinoid tone at the spinal level to further evaluate the roles of <em>2</em>-AG and anandamide in nonopioid SIA. Intrathecal administration of the competitive CB1 antagonist SR141716A (rimonabant) failed to suppress nonopioid SIA, suggesting that supraspinal rather than spinal CB1 receptor activation plays a pivotal role in endocannabinoid-mediated SIA. By contrast, spinal inhibition of MGL using URB60<em>2</em>, which selectively inhibits <em>2</em>-AG hydrolysis in the PAG, enhanced SIA through a CB1-selective mechanism. Spinal inhibition of FAAH, with either URB597 or arachidonoyl serotonin (AA-5-HT), also enhanced SIA through a CB1-mediated mechanism, presumably by increasing accumulation of tonically released anandamide. Our results suggest that endocannabinoids in the spinal cord regulate, but do not mediate, nonopioid SIA.

OBJECTIVE

Recently, an activation of the endocannabinoid system during obesity has been reported. More particularly, it has been demonstrated that hypothalamic levels of both endocannabinoids, <em>2</em>-<em>arachidonoylglycerol</em> and anandamide (N-arachidonoylethanolamine), are up-regulated in genetically obese rodents. Circulating levels of both endocannabinoids were also shown to be higher in obese compared with lean women. Yet, the direct production of endocannabinoids by human adipocytes has never been demonstrated. Our aim was to evaluate the ability of human adipocytes to produce endocannabinoids.

METHODS

The production of endocannabinoids by human adipocytes was investigated in a model of human white subcutaneous adipocytes in primary culture. The effects of leptin, adiponectin, and peroxisome proliferator-activated receptor (PPAR)-gamma activation on endocannabinoid production by adipocytes were explored. Endocannabinoid levels were determined by high-performance liquid chromatography (HPLC)-atmospheric pressure chemical ionization (APCI)-mass spectrometry (MS) analysis, leptin and adiponectin secretion measured by enzyme-linked immunosorbent assay (ELISA), and PPAR-gamma protein expression examined by Western blotting.

RESULTS

We show that <em>2</em>-<em>arachidonoylglycerol</em>, anandamide, and both anandamide analogs, N-palmitoylethanolamine and N-oleylethanolamine, are produced by human white subcutaneous adipocytes in concentrations ranging from 0.04<em>2</em>+/-0.004 to 0.531+/-0.048 pM/mg lipid extract. N-palmitoylethanolamine is the most abundant cannabimimetic compound produced by human adipocytes, and its levels are significantly down-regulated by leptin but not affected by adiponectin and PPAR-gamma agonist ciglitazone. N-palmitoylethanolamine itself does not affect either leptin or adiponectin secretion or PPAR-gamma protein expression in adipocytes.

CONCLUSIONS

This study has led to the identification of human adipocytes as a new source of endocannabinoids and related compounds. The biological significance of these adipocyte cannabimimetic compounds and their potential implication in obesity should deserve further investigations.

Endogenous <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is antiinvasive in androgen-independent prostate carcinoma (PC-3) cells. Invasion of PC-3 cells is also inhibited by exogenously added noladin ether, a non-hydrolyzable analog of <em>2</em>-AG. In contrast, exogenous <em>2</em>-AG has the opposite effect. Cell invasion significantly increased with high concentrations of exogenous <em>2</em>-AG. In PC-3 cells, arachidonic acid (AA) and 1<em>2</em>-hydroxyeicosatetraenoic acid (1<em>2</em>-HETE) concentrations increased along with exogenously added <em>2</em>-AG, and 1<em>2</em>-HETE concentrations increased with exogenously added AA. Invasion of PC-3 cells also increased with exogenously added AA and 1<em>2</em>(S)-HETE but not 1<em>2</em>(R)-HETE. The exogenous <em>2</em>-AG-induced invasion of PC-3 cells was inhibited by 3-octylthio-1,1,1-trifluoropropan-<em>2</em>-one (OTFP, an inhibitor of <em>2</em>-AG hydrolysis) and baicalein (a 1<em>2</em>-LO inhibitor). Western blot and RT-PCR analyses indicated expression of 1<em>2</em>-HETE producing lipoxygenases (LOs), platelet-type 1<em>2</em>-LO (P-1<em>2</em>-LO) and leukocyte-type 1<em>2</em>-LO (L-1<em>2</em>-LO), in PC-3 cells. These results suggest that exogenous <em>2</em>-AG induced, rather inhibited, cell invasion because of its rapid hydrolysis to free AA, and further metabolism by 1<em>2</em>-LO of AA to 1<em>2</em>(S)-HETE, a promoter of PC cell invasion. The results also suggest that PC-3 cells and human prostate stromal (WPMY-1) cells released free AA, <em>2</em>-AG, and 1<em>2</em>-HETE. In the microenvironment of the PC cells, this may contribute to the cell invasion. The <em>2</em>-AG hydrolysis and concentration of <em>2</em>-AG in microenvironment are critical for PC cell's fate. Therefore, inhibitors of <em>2</em>-AG hydrolysis could potentially serve as therapeutic agents for the treatment of prostate cancer. (c) <em>2</em>007 Wiley-Liss, Inc.

Monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) are two enzymes from the serine hydrolase superfamily that degrade the endocannabinoids <em>2</em>-<em>arachidonoylglycerol</em> and anandamide, respectively. We have recently discovered that MAGL and FAAH are both inhibited by carbamates bearing an N-piperidine/piperazine group. Piperidine/piperazine carbamates show excellent in vivo activity, raising brain endocannabinoid levels and producing CB1-dependent behavioral effects in mice, suggesting that they represent a promising class of inhibitors for studying the endogenous functions of MAGL and FAAH. Herein, we disclose a full account of the syntheses, structure-activity relationships, and inhibitory activities of piperidine/piperazine carbamates against members of the serine hydrolase family. These scaffolds can be tuned for MAGL-selective or dual MAGL-FAAH inhibition by the attachment of an appropriately substituted bisarylcarbinol or aryloxybenzyl moiety, respectively, on the piperidine/piperazine ring. Modifications to the piperidine/piperazine ring ablated inhibitory activity, suggesting a strict requirement for a six-membered ring to maintain potency.

Delta9-Tetrahydrocannabinol, a major psychoactive component of marijuana, has been shown to interact with specific cannabinoid receptors, thereby eliciting a variety of pharmacological responses in experimental animals and human. In 1990, the gene encoding a cannabinoid receptor (CB1) was cloned. This prompted the search for endogenous ligands. In 199<em>2</em>, N-arachidonoylethanolamine (anandamide) was isolated from pig brain as an endogenous ligand, and in 1995, <em>2</em>-<em>arachidonoylglycerol</em> was isolated from rat brain and canine gut as another endogenous ligand. Both anandamide and <em>2</em>-<em>arachidonoylglycerol</em> exhibit various cannabimimetic activities. The results of structure-activity relationship experiments, however, revealed that <em>2</em>-<em>arachidonoylglycerol</em>, but not anandamide, is the intrinsic natural ligand for the cannabinoid receptor. <em>2</em>-<em>Arachidonoylglycerol</em> is a degradation product of inositol phospholipids that links the function of cannabinoid receptors with the enhanced inositol phospholipid turnover in stimulated tissues and cells. The possible physiological roles of cannabinoid receptors and <em>2</em>-<em>arachidonoylglycerol</em> in various mammalian tissues such as those of the nervous system are discussed.

During neuropathic pain, caspases are activated in the limbic cortex. We investigated the role of TRPV1 channels and glial caspases in the mouse prelimbic and infralimbic (PL-IL) cortex after spared nerve injury (SNI). Reverse transcriptase-polymerase chain reaction, western blots, and immunfluorescence showed overexpression of several caspases in the PL-IL cortex 7 days postinjury. Caspase-3 release and upregulation of AMPA receptors in microglia, caspase-1 and IL-1β release in astrocytes, and upregulation of Il-1 receptor-1, TRPV1, and VGluT1 in glutamatergic neurons, were also observed. Of these alterations, only those in astrocytes persisted in SNI Trpv1(-/-) mice. A pan-caspase inhibitor, injected into the PL-IL cortex, reduced mechanical allodynia, this effect being reduced but not abolished in Trpv1(-/-) mice. Single-unit extracellular recordings in vivo following electrical stimulation of basolateral amygdala or application of pressure on the hind paw, showed increased excitatory pyramidal neuron activity in the SNI PL-IL cortex, which also contained higher levels of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em>. Intra-PL-IL cortex injection of mGluR5 and NMDA receptor antagonists and AMPA exacerbated, whereas TRPV1 and AMPA receptor antagonists and a CB(1) agonist inhibited, allodynia. We suggest that SNI triggers both TRPV1-dependent and independent glutamate- and caspase-mediated cross-talk among IL-PL cortex neurons and glia, which either participates or counteracts pain.

The brain produces at least five compounds that possess sub-micromolar affinity for cannabinoid receptors: anandamide, <em>2</em>-<em>arachidonoylglycerol</em>, noladin ether, virodhamine, and N-arachidonoyldopamine (NADA). One function of these and/or related compounds is to suppress pain sensitivity. Much evidence supports a role of endocannabinoids in pain modulation in general, and some evidence points to the role of particular endocannabinoids. Related endogenous fatty acid derivatives such as oleamide, palmitoylethanolamide, <em>2</em>-lineoylglycerol, <em>2</em>-palmitoylglycerol, and a family of arachidonoyl amino acids may interact with endocannabinoids in the modulation of pain sensitivity.

Insulin controls fatty acid (FA) release from white adipose tissue (WAT) through direct effects on adipocytes and indirectly through hypothalamic signaling by reducing sympathetic nervous system outflow to WAT. Uncontrolled FA release from WAT promotes lipotoxicity, which is characterized by inflammation and insulin resistance that leads to and worsens type <em>2</em> diabetes. Here we tested whether early diet-induced insulin resistance impairs the ability of hypothalamic insulin to regulate WAT lipolysis and thus contributes to adipose tissue dysfunction. To this end we fed male Sprague-Dawley rats a 10% lard diet (high fat diet (HFD)) for 3 consecutive days, which is known to induce systemic insulin resistance. Rats were studied by euglycemic pancreatic clamps and concomitant infusion of either insulin or vehicle into the mediobasal hypothalamus. Short term HFD feeding led to a 37% increase in caloric intake and elevated base-line free FAs and insulin levels compared with rats fed regular chow. Overfeeding did not impair insulin signaling in WAT, but it abolished the ability of mediobasal hypothalamus insulin to suppress WAT lipolysis and hepatic glucose production as assessed by glycerol and glucose flux. HFD feeding also increased hypothalamic levels of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> after only 3 days. In summary, overfeeding impairs hypothalamic insulin action, which may contribute to unrestrained lipolysis seen in human obesity and type <em>2</em> diabetes.

OBJECTIVE

Evidence from recent studies suggests that the endocannabinoid system participates in the regulation of lipid metabolism and body composition. We hypothesize that the system is activated by oxidized low-density lipoprotein (oxLDL) and regulates cellular cholesterol metabolism in macrophages.

RESULTS

Primary peritoneal macrophages isolated from Sprague-Dawley rats and RAW<em>2</em>64.7 mice macrophages were cultured. A liquid chromatography/mass spectrometry (LC/MS) system was used to measure the endocannabinoid anandamide (AEA), <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), and cellular cholesterol levels in macrophages. The regulatory mechanisms of cellular cholesterol metabolism were also investigated by molecular biology methods. The results showed that the endocannabinoid system in macrophages was activated by oxLDL through elevation of the AEA and <em>2</em>-AG levels and the up-regulation of the cannabinoid CB1 and CB<em>2</em> receptor expression. Win55,<em>2</em>1<em>2</em>-<em>2</em>, a synthetic cannabinoid, promotes cellular cholesterol accumulation in macrophages, which was associated with an increase in the expression of CD36 and a decrease in the expression of ATP-binding cassette protein A1 (ABCA1) as mediated by an up-regulated peroxisome proliferator-activated receptor gamma (PPARgamma). AM<em>2</em>51, a selective cannabinoid CB1 receptor antagonist, impaired the abilities of Win55,<em>2</em>1<em>2</em>-<em>2</em>-treated macrophages to accumulate cholesterol by down-regulating CD36 receptor expression and up-regulating ABCA1 expression.

CONCLUSIONS

We have demonstrated, for the first time, that the endocannabinoid system in macrophages is activated by oxLDL and that the activated endocannabinoid system promotes cellular cholesterol accumulation in macrophages. The results also indicate that selectively blocking the CB1 receptor can reduce oxLDL accumulation in macrophages, which might represent a promising therapeutic strategy for atherosclerosis.

Although the endocannabinoids <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and anandamide share a similar pharmacology, <em>2</em>-AG reportedly limits myocardial ischaemia-reperfusion injury whereas anandamide does not. We therefore investigated whether or not anandamide reduces infarct size and which, if any, of the known cannabinoid-signalling pathways are involved. Rat isolated perfused hearts were subjected to global, no-flow ischaemia (30 min) and reperfusion (1 h). Agonists were present from 5 min before ischaemia until the end of reperfusion. Antagonists, where used, were present throughout the protocol. Recovery of left ventricular developed pressure and coronary flow was incomplete in control hearts and not significantly affected by any drug treatment. In vehicle-treated hearts, <em>2</em>6+/-3% (n=13) of the left ventricle was infarcted at the end of reperfusion. Infarction of the left ventricle was significantly reduced after 1 microM anandamide (10+/-1%, n=7) or 1 microM methanandamide (1<em>2</em>+/-4%, n=6) but not 1 microM HU<em>2</em>10. Neither ACPA (1 microM; CB1 receptor agonist) nor JWH133 (1 microM; CB<em>2</em> receptor agonist), individually or combined significantly affected infarct size. Anandamide (1 microM) did not reduce infarct size in the presence of the CB1 receptor antagonist rimonabant (SR141716A, 1 microM) or the CB<em>2</em> receptor antagonist, SR1445<em>2</em>8 (1 microM). Despite sensitivity to CB1 and CB<em>2</em> receptor antagonists, the infarct-limiting action of anandamide was not mimicked by agonists selective for CB1 or CB<em>2</em> receptors suggesting the involvement of a novel cannabinoid site of action.

Monoacylglycerol lipase (MAGL) is a principal metabolic enzyme responsible for hydrolyzing the endogenous cannabinoid (endocannabinoid) <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Selective inhibitors of MAGL offer valuable probes to further understand the enzyme's function in biological systems and may lead to drugs for treating a variety of diseases, including psychiatric disorders, neuroinflammation, and pain. N-Hydroxysuccinimidyl (NHS) carbamates have recently been identified as a promising class of serine hydrolase inhibitors that shows minimal cross-reactivity with other proteins in the proteome. Here, we explore NHS carbamates more broadly and demonstrate their potential as inhibitors of endocannabinoid hydrolases and additional enzymes from the serine hydrolase class. We extensively characterize an NHS carbamate 1a (MJN110) as a potent, selective, and in-vivo-active MAGL inhibitor. Finally, we demonstrate that MJN110 alleviates mechanical allodynia in a rat model of diabetic neuropathy, marking NHS carbamates as a promising class of MAGL inhibitors.

Anandamide (N-arachidonoylethanolamine) and <em>2</em>-<em>arachidonoylglycerol</em> are the two endogenous agonists of cannabinoid receptors discovered to date. Like other eicosanoids, and unlike classical neuromodulators, these two compounds are synthesized by neurons on demand, i.e., their biosynthesis, rather than release, is stimulated by Ca<em>2</em>+ influx and cell membrane depolarization. Both endocannabinoids can be produced from membrane phosphoglycerides through the action of phospholipases, although de novo pathways have also been suggested. Once released by cells, the action of both anandamide and <em>2</em>-<em>arachidonoylglycerol</em> is terminated--after their diffusion through the cell membrane--by the hydrolysis of the amide or ester bonds to yield arachidonic acid, which is then immediately reincorporated into phospholipids. One enzyme, fatty acid amide hydrolase, catalyzes the hydrolysis of both endocannabinoids in nervous and nonnervous cells. This enzyme also recognizes N-palmitoylethanolamine, an antiinflammatory congener of anandamide, with a catalytic efficiency that depends on the cell type under study. However, the existence of different isozymes with different affinity for anandamide and N-palmitoylethanolamine has not been investigated. Moreover, little work has been performed on the regulation of anandamide formation and breakdown, and several open questions remain as to the possible biosynthetic and degradative mechanisms of cannabimimetic <em>2</em>-<em>arachidonoylglycerol</em> in nucleated blood cells such as macrophages. Finally, the co-existence of both endocannabinoids in invertebrates has not been fully established. Here we briefly review the state of the art, and present new data from our laboratory, on these four largely unexplored aspects of endocannabinoid metabolism.