Biosynthesis of N-Docosahexanoylethanolamine from Unesterified Docosahexaenoic Acid and Docosahexaenoyl-Lysophosphatidylcholine in Neuronal Cells.

We investigated the synthesis of N-docosahexaenoylethanolamine (synaptamide) in neuronal cells from unesterified docosahexaenoic acid (DHA) or DHA-lysophosphatidylcholine (DHA-lysoPC), the two major lipid forms that deliver DHA to the brain, in order to understand the formation of this neurotrophic and neuroprotective metabolite of DHA in the brain. Both substrates were taken up in Neuro2A cells and metabolized to N-docosahexaenoylphosphatidylethanolamine (NDoPE) and synaptamide in a time- and concentration-dependent manner, but unesterified DHA was 1.5 to 2.4 times more effective than DHA-lysoPC at equimolar concentrations. The plasmalogen NDoPE (pNDoPE) amounted more than 80% of NDoPE produced from DHA or DHA-lysoPC, with 16-carbon-pNDoPE being the most abundant species. Inhibition of N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD) by hexachlorophene or bithionol significantly decreased the synaptamide production, indicating that synaptamide synthesis is mediated at least in part via NDoPE hydrolysis. NDoPE formation occurred much more rapidly than synaptamide production, indicating a precursor-product relationship. Although NDoPE is an intermediate for synaptamide biosynthesis, only about 1% of newly synthesized NDoPE was converted to synaptamide, possibly suggesting additional biological function of NDoPE, particularly for pNDoPE, which is the major form of NDoPE produced.

Endocannabinoid Signaling in Embryonic Neuronal Motility and Cell-Cell Contact - Role of mGluR5 and TRPC3 Channels.

Cell-cell communication plays a central role in the guidance of migrating neuronal precursor cells during the development of the cerebral cortex. Endocannabinoids (eCBs) have previously been shown to be one of the central factors regulating neuronal migration. In this study the effects of eCBs on different parameters, expected to affect embryonic cortical neuronal motility have been analyzed in neurosphere-derived neuroblasts using time-lapse microscopy. Increased endogenous production of the endocannabinoid 2-arachidonyl glycerol (2-AG) causes bursts of neuroblast motility. The neuroblasts move longer distances and show a low frequency of turning, and the number of neuron-neuron contacts are reduced. Similar changes occur interfering with the function of the metabotropic glutamate receptor 5 (mGluR5) or its transducer canonical transient receptor potential channel 3 (TRPC3) or the neuregulin receptor ErbB4. Blocking of 2-AG production reverses these effects. The data suggest that eCB-regulated neuronal motility is controlled by mGluR5/TRPC3 activity possibly via NRG/ErbB4 signaling.

Self-administration of the anandamide transport inhibitor AM404 by squirrel monkeys.

RATIONALE: N-(4-hydroxyphenyl)-arachidonamide (AM404) is an anandamide transport inhibitor shown to reduce rewarding and relapse-inducing effects of nicotine in several animal models of tobacco dependence. However, the reinforcing/rewarding effects of AM404 are not clear. OBJECTIVES: We investigated whether AM404 maintains self-administration behavior or reinstates extinguished drug seeking in squirrel monkeys. METHODS AND RESULTS: In monkeys with a history of anandamide or cocaine self-administration, we substituted injections of AM404 (1-100 µg/kg/injection). Using a 10-response, fixed-ratio schedule, self-administration behavior was maintained by AM404. Dose-response curves had inverted U shapes, with peak response rates occurring at a dose of 10 µg/kg/injection. In anandamide-experienced monkeys, we also demonstrated self-administration of another anandamide transport inhibitor VDM11. In addition to supporting self-administration, priming injections of AM404 (0.03-0.3 mg/kg) reinstated drug-seeking behavior previously reinforced by cannabinoids (∆(9)-tetrahydrocannabinol (THC) or anandamide) or cocaine. Both AM404 self-administration behavior and reinstatement of drug seeking by AM404 were reduced by treatment with the cannabinoid CB1 receptor antagonist/inverse agonist rimonabant (0.3 mg/kg). Moreover, the reinforcing effects of AM404 were potentiated by the treatment with the fatty acid amide hydrolase (FAAH) inhibitor URB597 (0.3 mg/kg) suggesting a major role of anandamide in these effects. Finally, AM404 (0.3 mg/kg) potentiated the reinforcing effects of anandamide but not those of cocaine. CONCLUSIONS: In non-human primates, AM404 effectively reinforced self-administration behavior and induced reinstatement of drug-seeking behavior in abstinent monkeys. These effects appeared to be mediated by cannabinoid CB1 receptors. Therefore, compounds that promote actions of endocannabinoids throughout the brain by inhibiting their membrane transport may have a potential for abuse.

Diacylglycerol lipase disinhibits VTA dopamine neurons during chronic nicotine exposure.

Chronic nicotine exposure (CNE) alters synaptic transmission in the ventral tegmental area (VTA) in a manner that enhances dopaminergic signaling and promotes nicotine use. The present experiments identify a correlation between enhanced production of the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) and diminished release of the inhibitory neurotransmitter GABA in the VTA following CNE. To study the functional role of on-demand 2-AG signaling in GABAergic synapses, we used 1,2,3-triazole urea compounds to selectively inhibit 2-AG biosynthesis by diacylglycerol lipase (DAGL). The potency and selectivity of these inhibitors were established in rats in vitro (rat brain proteome), ex vivo (brain slices), and in vivo (intracerebroventricular administration) using activity-based protein profiling and targeted metabolomics analyses. Inhibition of DAGL (2-AG biosynthesis) rescues nicotine-induced VTA GABA signaling following CNE. Conversely, enhancement of 2-AG signaling in naïve rats by inhibiting 2-AG degradation recapitulates the loss of nicotine-induced GABA signaling evident following CNE. DAGL inhibition reduces nicotine self-administration without disrupting operant responding for a nondrug reinforcer or motor activity. Collectively, these findings provide a detailed characterization of selective inhibitors of rat brain DAGL and demonstrate that excessive 2-AG signaling contributes to a loss of inhibitory GABAergic constraint of VTA excitability following CNE.

ENP11, a potential CB1R antagonist, induces anorexia in rats.

Over the past decade, pharmacological manipulation of cannabinoid 1 receptor (CB1R) has become an interesting approach for the management of food ingestion disorders, among other physiological functions. Searching for new substances with similar desirable effects, but fewer side-effects we have synthesized a SR141716A (a cannabinoid receptor inverse agonist also called Rimonabant) analog, 1-(2,4-Difluorophenyl)-4-methyl-N-(1-piperidinyl)-5-[4-(trifluoromethyl)phenyl]-1H-pyrazole-3-carboxamide, ENP11, that so far, as we have previously shown, has induced changes in glucose availability, i.e. hypoglycemia, in rats. In this study we tested the effects, if any, of ENP11 (0.5, 1.0, and 3.0mg/kg) in food ingestion, core temperature, pain perception and motor control in adult Wistar rats. Results showed that ENP11 reduced food ingestion during the first hour immediately after administration. Likewise, ENP11 (1.0mg/kg) blocked anandamide (AEA)-induced hyperphagia during the first 4h of the dark phase of the light-dark cycle, and it also blocked AEA-induced hypothermia. However, none of the ENP11 doses used affected pain perception or motor control. We believe that ENP11 is a potential useful CB1R antagonist that reduces food ingestion and regulates core temperature.

Molecular analysis of the site for 2-arachidonylglycerol (2-AG) on the ß2 subunit of GABA(A) receptors.

2-arachidonyl glycerol (2-AG) allosterically potentiates GABA(A) receptors via a binding site located in transmembrane segment M4 of the ß2 subunit. Two amino acid residues have been described that are essential for this effect. With the aim to further describe this potential drug target, we performed a cysteine scanning of the entire M4 and part of M3. All four residues in M4 affecting the potentiation here and the two already identified residues locate to the same side of the α-helix. This side is exposed to M3, where further residues were identified. From the fact that the important residues span > 18 Å, we conclude that the hydrophobic tail of the bound 2-AG molecule must be near linear and that the site mainly locates to the inner leaflet but stretches far into the membrane. The influence of the structure of the head group of the ligand molecule on the activity of the molecule was also investigated. We present a model of 2-AG docked to the GABA(A) receptor.

A novel fluorophosphonate inhibitor of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol with potential anti-obesity effects.

BACKGROUND AND PURPOSE: The development of potent and selective inhibitors of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) via DAG lipases (DAGL) α and ß is just starting to be considered as a novel and promising source of pharmaceuticals for the treatment of disorders that might benefit from a reduction in endocannabinoid tone, such as hyperphagia in obese subjects. EXPERIMENTAL APPROACH: Three new fluorophosphonate compounds O-7458, O-7459 and O-7460 were synthesized and characterized in various enzymatic assays. The effects of O-7460 on high-fat diet intake were tested in mice. KEY RESULTS: Of the new compounds, O-7460 exhibited the highest potency (IC50 = 690 nM) against the human recombinant DAGLα, and selectivity (IC50 > 10 µM) towards COS-7 cell and human monoacylglycerol lipase (MAGL), and rat brain fatty acid amide hydrolase. Competitive activity-based protein profiling confirmed that O-7460 inhibits mouse brain MAGL only at concentrations ≥ 10 µM, and showed that this compound has only one major 'off-target', that is, the serine hydrolase KIAA1363. O-7460 did not exhibit measurable affinity for human recombinant CB1 or CB2 cannabinoid receptors (Ki > 10 µM). In mouse neuroblastoma N18TG2 cells stimulated with ionomycin, O-7460 (10 µM) reduced 2-AG levels. When administered to mice, O-7460 dose-dependently (0-12 mg·kg⁻¹, i.p.) inhibited the intake of a high-fat diet over a 14 h observation period, and, subsequently, slightly but significantly reduced body weight. CONCLUSIONS AND IMPLICATIONS: O-7460 might be considered a useful pharmacological tool to investigate further the role played by 2-AG both in vitro and in vivo under physiological as well as pathological conditions.

The anandamide transport inhibitor AM404 reduces the rewarding effects of nicotine and nicotine-induced dopamine elevations in the nucleus accumbens shell in rats.

BACKGROUND AND PURPOSE: The fatty acid amide hydrolase inhibitor URB597 can reverse the abuse-related behavioural and neurochemical effects of nicotine in rats. Fatty acid amide hydrolase inhibitors block the degradation (and thereby magnify and prolong the actions) of the endocannabinoid anandamide (AEA), and also the non-cannabinoid fatty acid ethanolamides oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). OEA and PEA are endogenous ligands for peroxisome proliferator-activated receptors alpha (PPAR-α). Since recent evidence indicates that PPAR-α can modulate nicotine reward, it is unclear whether AEA plays a role in the effects of URB597 on nicotine reward. EXPERIMENTAL APPROACH: A way to selectively increase endogenous levels of AEA without altering OEA or PEA levels is to inhibit AEA uptake into cells by administering the AEA transport inhibitor N-(4-hydroxyphenyl)-arachidonamide (AM404). To clarify AEA's role in nicotine reward, we investigated the effect of AM404 on conditioned place preference (CPP), reinstatement of abolished CPP, locomotor suppression and anxiolysis in an open field, and dopamine elevations in the nucleus accumbens shell induced by nicotine in Sprague-Dawley rats. KEY RESULTS: AM404 prevented the development of nicotine-induced CPP and impeded nicotine-induced reinstatement of the abolished CPP. Furthermore, AM404 reduced nicotine-induced increases in dopamine levels in the nucleus accumbens shell, the terminal area of the brain's mesolimbic reward system. AM404 did not alter the locomotor suppressive or anxiolytic effect of nicotine. CONCLUSIONS AND IMPLICATIONS: These findings suggest that AEA transport inhibition can counteract the addictive effects of nicotine and that AEA transport may serve as a new target for development of medications for treatment of tobacco dependence. LINKED ARTICLES: This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

Differential anti-cancer effects of purified EPA and DHA and possible mechanisms involved.

As the concepts of pharmaconutrition are receiving increasing attention, it seems essential to clearly assess the effects of specific dietary compounds in specific groups of patients or clinical conditions. We are herein interested in better defining the differential anti-neoplastic effects of the two major n-3 long chain polyunsaturated fatty acids present in fish oil, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The efficiency of these fatty acids represents a subject of intense interest and debate, and whereas plenty of preclinical studies have strongly demonstrated their preventive and therapeutic effect in different kinds of cancers, the results of the epidemiologic studies are still controversial, and only a few trials have been performed. It has been reported that EPA and DHA may act either through the same or different mechanisms, thus suggesting that a differential efficacy could exist. At present, however, this point has not been clarified, although its better comprehension would allow a more proper and effective use of these fatty acids in the human interventional studies. In an attempt to elucidate this aspect we have herein analyzed the data obtained in the studies which have directly compared the antitumor effects of separate treatments with EPA or DHA. Most of the in vitro data indicate DHA as the more powerful antineoplastic agent. However, an equivalent efficiency of EPA and DHA is suggested by the few in vivo studies. Possible reasons for this discrepancy are discussed and pathways of cell growth that could be differentially influenced by EPA and DHA are described.

[Anandamide inhibits the growth of colorectal cancer cells through CB1 and lipid rafts].

OBJECTIVE: To study the influences of endocannabinoid-anandamide (AEA) on the proliferation and apoptosis of the colorectal cancer cell line (CaCo-2) and to elucidate the effects of CB1 and lipid rafts, and to further elucidate the molecular mechanism and the effect of AEA on the generation and development of colorectal cancer. METHODS: Human colorectal cancer cell line CaCo-2 was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum in 5% CO(2) atmosphere at 37°C. CaCo-2 cells were divided into different groups and treated with different concentrations of AEA, AEA + SR141716A, AEA + AM630 and AEA + methyl-ß-cyclodextrin (MCD). MTT assay was used to determine the effects of AEA, its putative CB1, CB2 receptor antagonists (SR141716A and AM630) and MCD on the proliferation of CaCo-2 cells. Annexin V-PE/7AAD binding assay was used to detect apoptosis in the CaCo-2 cells. Western-blot was applied to check the expressions of CB1, CB2, p-AKT and caspase-3 proteins in different groups of CaCo-2 cells. RESULTS: AEA inhibited the proliferation of CaCo-2 cells in a concentration-dependent manner and the effect could be antagonized by SR141716A and MCD. The inhibiting rates were (21.52 ± 0.45)%, (42.16 ± 0.21)%, (73.64 ± 0.73)% and (83.28 ± 0.71)%, respectively, at different concentrations of AEA (5, 10, 20 and 40 µmol/L). The three groups (20 µmol/L AEA, 20 µmol/L AEA + 10 µmol/L SR141716A and 20 µmol/L AEA + 1 mmol/L MCD) showed different inhibiting rates [(73.64 ± 0.73)%, (16.15 ± 0.75)% and (12.58 ± 0.63)%], respectively. Annexin V-PE/7AAD binding assay showed that AEA induced apoptosis in the CaCo-2 cells and MCD could antagonize this effect. The apoptosis rates of the three groups (control, 20 µmol/L AEA and 20 µmol/L AEA + 1 mmol/L MCD) were (2.95 ± 0.73)%, (39.61 ± 0.73)% and (14.10 ± 0.64)%, respectively. The expressions of CB1, CB2, p-AKT and Caspase-3 proteins were all observed in the CaCo-2 cells. AEA inhibited p-AKT protein expression and induced caspase-3 protein expression. The two actions were also antagonized by MCD. CONCLUSIONS: AEA can strongly suppress the proliferation of colorectal cancer CaCo-2 cells via the CB1 receptor and membrane cholesterol-LRs and induce apoptosis via lipid rafts. Anandamide plays a very important role in the carcinogenesis and development of colorectal cancer. MCD is a critical member in this system.

Deciphering the mechanism(s) of action of natural products: analgesic peroxide oil as example.

BACKGROUND: There are multiple reports of natural products having therapeutic effect. In an era of evidence-based medicine, clinical trials inform clinical decisions regarding use of the product, but prevailing preference is to identify and use a single 'active ingredient'. Yet, the clinical benefit of a natural product might derive from the fortuitous combination of its multiple components. Therefore, the elucidation of the mechanism(s) of action of natural products is important, but presents significant challenges. This article examines this issue using peroxide oil (essential oxygen oil) as an illustrative example. OBJECTIVE: To review the published literature of a natural product in an effort to elucidate postulated mechanism(s) of action of a complex mixture. METHODS: The clinical and preclinical literature was reviewed from the perspective of its contribution to elucidating a mechanism of analgesic action of a natural product. RESULTS: Peroxide oil contains ingredients that are associated with analgesic mechanisms, such inhibition of lipid peroxidation and arachidonic acid metabolism and non-opioid, glibenclamide-sensitive receptor-mediated and K(ATP) -NO-cGMP channel pathways. CONCLUSION: Although its exact mechanism remains unknown, peroxide oil provides an example of how a natural product can be evaluated for plausible mechanistic explanation of its purported therapeutic efficacy. Such an approach seems valuable, since, as in this case, the constituents appear to contribute in an additive or synergistic manner, something not possible with a single substance.

The selective anandamide transport inhibitor VDM11 attenuates reinstatement of nicotine seeking behaviour, but does not affect nicotine intake.

BACKGROUND AND PURPOSE: The endocannabinoid system appears to play a pivotal role in mediating the rewarding and reinforcing effects of nicotine. Recent studies have shown that the inhibition of fatty acid amide hydrolase (FAAH) attenuates reinstatement of nicotine-seeking induced by nicotine priming and nicotine-associated cues. FAAH hydrolyses the endogenous endocannabinoid anandamide, as well as other non-cannabinoid ligands such as oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). As OEA and PEA can attenuate both nicotine-taking and nicotine-seeking behaviour, the specific role of anandamide remains unclear. In this study, we have tested the selective anadamide uptake inhibitor, VDM11, which elevates anandamide levels without affecting levels of OEA/PEA, on nicotine-taking and nicotine-seeking behaviour. EXPERIMENTAL APPROACH: We used a nicotine intravenous self-administration model in rats to assess the effect of VDM11, given i.p., on nicotine taking using fixed and progressive ratio schedules of reinforcement as well as on reinstatement of nicotine-seeking induced by nicotine priming and nicotine-associated cues. KEY RESULTS: VDM11 did not affect levels of responding for nicotine under fixed-ratio and progressive-ratio schedules of reinforcement. In contrast, VDM11 dose-dependently attenuated reinstatement of nicotine-seeking behaviour induced by nicotine-associated cues and nicotine priming. CONCLUSIONS AND IMPLICATIONS: These results indicate that ligands elevating anandamide levels could have therapeutic value for preventing relapse into nicotine-seeking behaviour and should be tested in humans trying to quit smoking.

Cannabinoid-1 receptor activation induces reactive oxygen species-dependent and -independent mitogen-activated protein kinase activation and cell death in human coronary artery endothelial cells.

BACKGROUND AND PURPOSE: Impaired endothelial activity and/or cell death play a critical role in the development of vascular dysfunction associated with congestive heart failure, diabetic complications, hypertension, coronary artery disease and atherosclerosis. Increasing evidence suggests that cannabinoid 1 (CB(1)) receptor inhibition is beneficial in atherosclerosis and cardiovascular inflammation both in experimental models, as well as in humans. Here, we investigated the effects of CB(1) receptor activation with the endocannabinoid anandamide (AEA) or synthetic agonist HU210 on cell death and interrelated signal transduction pathways in human primary coronary artery endothelial cells (HCAECs). EXPERIMENTAL APPROACH: Cell death, CB(1) receptor expression, reactive oxygen species (ROS) generation and activation of signal transduction pathways in HCAECs were determined by flow cytometry and molecular biology tools. KEY RESULTS: In HCAECs expressing CB(1) receptors (demonstrated by Western immunoblot and flow cytometry) AEA (5-15 microM) or HU210 (30-1000 nM) triggered concentration- and time-dependent activation of p38 and c-Jun NH(2)-terminal protein kinase (JNK)-mitogen-activated protein kinases (MAPKs), cell death and ROS generation. The AEA- or HU210-induced cell death and MAPK activation were attenuated by CB(1) antagonists [SR141716 (rimonabant) and AM281], inhibitors of p38 and JNK-MAPKs or the antioxidant N-acetylcysteine. N-acetylcysteine alone prevented AEA- or HU210-induced ROS generation, but only partially attenuated MAPK activation and cell death. In contrast, in combination with CB(1) antagonists, N-acetylcysteine completely prevented these effects. CONCLUSIONS AND IMPLICATIONS: CB(1) receptor activation in endothelial cells may amplify the ROS-MAPK activation-cell death pathway in pathological conditions when the endocannabinoid synthetic or metabolic pathways are dysregulated by excessive inflammation and/or oxidative/nitrosative stress, thereby contributing to the development of endothelial dysfunction and pathophysiology of multiple cardiovascular diseases.

N-arachidonoyl glycine, an endogenous lipid that acts as a vasorelaxant via nitric oxide and large conductance calcium-activated potassium channels.

BACKGROUND AND PURPOSE: N-arachidonoyl glycine (NAGly) is an endogenous lipid that is structurally similar to the endocannabinoid, N-arachidonoyl ethanolamide (anandamide). While NAGly does not activate cannabinoid receptors, it exerts cannabimimetic effects in pain regulation. Here, we have determined if NAGly, like anandamide, modulates vascular tone. EXPERIMENTAL APPROACH: In rat isolated small mesenteric arteries, the relaxant responses to NAGly were characterized. Effects of N-arachidonoyl serine and N-arachidonoyl gamma-aminobutyric acid were also examined. KEY RESULTS: In endothelium-intact arteries, NAGly-induced relaxation (pEC(50%)= 5.7 +/- 0.2; relaxation at 30 microM = 98 +/- 1%) was attenuated by l-NAME (a nitric oxide synthase inhibitor) or iberiotoxin [selective blocker of large conductance Ca(2+)-activated K(+) channels (BK(Ca))], and abolished by high extracellular K(+) concentration. Endothelial removal reduced the potency of NAGly, and the resultant relaxation was inhibited by iberiotoxin, but not l-NAME. NAGly responses were sensitive to the novel cannabinoid receptor antagonist O-1918 independently of endothelial integrity, whereas pertussis toxin, which uncouples G(i/o) proteins, attenuated NAGly relaxation only in endothelium-intact arteries. Treatments with antagonists for CB(1), CB(2) and TRPV1 receptors, or inhibitors of fatty acid amide hydrolase and COX had no effect. The two other arachidonoyl amino acids also induced iberiotoxin- and L-NAME-sensitive relaxations. CONCLUSION AND IMPLICATIONS: NAGly acts as a vasorelaxant predominantly via activation of BK(Ca) in rat small mesenteric arteries. We suggest that NAGly activates an unknown G(i/o)-coupled receptor, stimulating endothelial release of nitric oxide which in turn activates BK(Ca) in the smooth muscle. In addition, NAGly might also activate BK(Ca) through G(i/o)- and nitric oxide-independent mechanisms.

17,18-epoxyeicosatetraenoic acid targets PPARγ and p38 mitogen-activated protein kinase to mediate its anti-inflammatory effects in the lung: role of soluble epoxide hydrolase.

This study sought to assess putative pathways involved in the anti-inflammatory effects of 17,18-epoxyeicosatetraenoic acid (17,18-EpETE), as measured by a decrease in the contractile reactivity and Ca(2+) sensitivity of TNF-α-pretreated human bronchi. Tension measurements performed in the presence of 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), a soluble epoxide hydrolase (sEH)-specific inhibitor, demonstrated that 17,18-EpETE reduced the reactivity of TNF-α-pretreated tissues. The overexpression of sEH detected in patients with asthma and TNF-α-treated bronchi contributed to the maintenance of hyperresponsiveness in our models, which involved intracellular proinflammatory cascades. The inhibition of peroxisome proliferator-activated receptor (PPAR)γ by GW9662 abolished 17,18-EpETE + AUDA-mediated anti-inflammatory effects by inducing IκBα degradation and cytokine synthesis, indicating that PPARγ is a molecular target of epoxy-eicosanoids. Western blot analysis revealed that 17,18-EpETE pretreatment reversed the phosphorylation of p38 mitogen-activated protein kinase (p38-MAPK) induced by TNF-α in human bronchi. The Ca(2+) sensitivity of human bronchial explants was also quantified on ß-escin permeabilized preparations. The presence of SB203580, a p38-MAPK inhibitor, reversed the effect induced by epoxy-eicosanoid in the presence of AUDA on TNF-α-triggered Ca(2+) hypersensitivity by increasing the phosphorylation level of PKC Potentiated Inhibitor Protein-17 (CPI-17) regulatory protein. Moreover, PPARγ ligands, such as rosiglitazone and 17,18-EpETE, decreased the expression of CPI-17, both at the mRNA and protein levels, whereas this effect was countered by GW9662 treatment in TNF-α-treated bronchi. These results demonstrate that 17,18-EpETE is a potent regulator of human lung inflammation and concomitant hyperresponsiveness, and may represent a valuable asset against critical inflammatory bronchial disorder.

Cannabinoid receptor type 1- and 2-mediated increase in cyclic AMP inhibits T cell receptor-triggered signaling.

The aim of this study was to characterize inhibitory mechanisms on T cell receptor signaling mediated by the cannabinoid receptors CB1 and CB2. Both receptors are coupled to G(i/o) proteins, which are associated with inhibition of cyclic AMP formation. In human primary and Jurkat T lymphocytes, activation of CB1 by R(+)-methanandamide, CB2 by JWH015, and both by Delta9-tetrahydrocannabinol induced a short decrease in cyclic AMP lasting less than 1 h. However, this decrease was followed by a massive (up to 10-fold) and sustained (at least up to 48 h) increase in cyclic AMP. Mediated by the cyclic AMP-activated protein kinase A and C-terminal Src kinase, the cannabinoids induced a stable phosphorylation of the inhibitory Tyr-505 of the leukocyte-specific protein tyrosine kinase (Lck). By thus arresting Lck in its inhibited form, the cannabinoids prevented the dephosphorylation of Lck at Tyr-505 in response to T cell receptor activation, which is necessary for the subsequent initiation of T cell receptor signaling. In this way the cannabinoids inhibited the T cell receptor-triggered signaling, i.e. the activation of the zeta-chain-associated protein kinase of 70 kDa, the linker for activation of T cells, MAPK, the induction of interleukin-2, and T cell proliferation. All of the effects of the cannabinoids were blocked by the CB1 and CB2 antagonists AM281 and AM630. These findings help to better understand the immunosuppressive effects of cannabinoids and explain the beneficial effects of these drugs in the treatment of T cell-mediated autoimmune disorders like multiple sclerosis.

Development of a potent inhibitor of 2-arachidonoylglycerol hydrolysis with antinociceptive activity in vivo.

Although inhibitors of the enzymatic hydrolysis of the endocannabinoid 2-arachidonoylglycerol 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 2-AG hydrolysis, capable of enhancing 2-AG levels and of exerting analgesic activity via indirect activation of cannabinoid receptors. OMDM169 exhibited 0.13 microM10 microM) at human CB(1) and CB(2) 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 2.8 microM, respectively), and, accordingly, it increased by approximately 1.6-fold the levels of 2-AG, but not anandamide, in intact ionomycin-stimulated N18TG2 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 2.5 mg/kg, and concomitantly elevated 2-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(2) receptors, AM251 and AM630, respectively (1 mg/kg, i.p.). OMDM69 might represent a template for the development of selective and even more potent inhibitors of 2-AG hydrolysis.

New tetrazole-based selective anandamide uptake inhibitors.

A new series of 1,5- and 2,5-disubstituted tetrazoles have been synthesized and evaluated as inhibitors of anandamide cellular uptake. Some of them inhibit the uptake process with a relatively high potency (IC(50)=2.3-5.1microM) and selectively over other proteins involved in endocannabinoid action and metabolism.

RNA interference-mediated knockdown of dynamin 2 reduces endocannabinoid uptake into neuronal dCAD cells.

The precise mechanism by which the cellular uptake of the endocannabinoid anandamide (AEA) occurs has been the source of much debate. In the current study, we show that neuronal differentiated CAD (dCAD) cells accumulate anandamide by a process that is inhibited in a dose-dependent manner by N-(4-hydroxyphenyl)arachidonylamide (AM404). We also show that dCAD cells express functional fatty acid amide hydrolase, the enzyme primarily responsible for anandamide metabolism. Previous data from our laboratory indicated that anandamide uptake occurs by a caveolae-related endocytic mechanism in RBL-2H3 cells. In the current study, we show that anandamide uptake by dCAD cells may also occur by an endocytic process that is associated with detergent-resistant membrane microdomains or lipid rafts. Nystatin and progesterone pretreatment of dCAD cells significantly inhibited anandamide accumulation. Furthermore, RNA interference (RNAi)-mediated knockdown of dynamin 2, a protein involved in endocytosis, blocked the internalization of the fluorescently labeled anandamide analog SKM 4-45-1 ([3',6'-bis(acetyloxy)-3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthen-5-yl]-2-[[1-oxo-5Z,8Z,11Z,14Z-eicosatetraenyl]amino]ethyl ester carbamic acid). RNAi-mediated knockdown of the beta2 subunit of the clathrin-associated activator protein 2 complex had no effect on SKM 4-45-1 internalization. We were surprised to find that dynamin 2 knockdown in dCAD cells did not affect [3H]AEA uptake. However, dynamin 2 knockdown caused a significant increase in the overall levels of intact [3H]AEA associated with the cells, suggesting that trafficking of [3H]AEA to FAAH had been disrupted. This finding may be the result of an accumulation of the anandamide carrier protein in detergent-resistant membranes after dynamin 2 knockdown. Our studies provide evidence that the cellular uptake of anandamide may occur by a dynamin 2-dependent, caveolae-related endocytic process in dCAD cells.

Carbamoyl tetrazoles as inhibitors of endocannabinoid inactivation: a critical revisitation.

We have synthesized a series of 18 1,5- and 2,5-disubstituted carbamoyl tetrazoles, including LY2183240 (1) and LY2318912 (7), two compounds previously described as potent inhibitors of the cellular uptake of the endocannabinoid anandamide, and their regioisomers 2 and 8. We confirm that compound 1 is a potent inhibitor of both the cellular uptake and, like the other new compounds synthesized here, the enzymatic hydrolysis of anandamide. With the exception of 9, 12, 15, and the 2,5-regioisomer of LY2183240 2, the other compounds were all found to be weakly active or inactive on anandamide uptake. Several compounds also inhibited the enzymatic hydrolysis of the other main endocannabinoid, 2-arachidonoylglycerol, as well as its enzymatic release from sn-1-oleoyl-2-arachidonoyl-glycerol, at submicromolar concentrations. Four of the novel compounds, i.e. 3, 4, 17, and 18, inhibited anandamide hydrolysis potently (IC50=2.1-5.4nM) and selectively over all the other targets tested (IC50 >or= 10microM), thus representing new potentially useful tools for the inhibition of fatty acid amide hydrolase.