Anandamide Research Articles

The endocannabinoid system: emotion, learning and addiction

The identification of the cannabinoid receptor type 1 (CB1 receptor) was the milestone discovery in the elucidation of the behavioural and emotional responses induced by the Cannabis sativa constituent Delta(9)-tetrahydrocannabinol. The subsequent years have established the existence of the endocannabinoid system. The early view relating this system to emotional responses is reflected by the fact that N-arachidonoyl ethanolamine, the pioneer endocannabinoid, was named anandamide after the Sanskrit word 'ananda', meaning 'bliss'. However, the emotional responses to cannabinoids are not always pleasant and delightful. Rather, anxiety and panic may also occur after activation of CB1 receptors. The present review discusses three properties of the endocannabinoid system as an attempt to understand these diverse effects. First, this system typically functions 'on-demand', depending on environmental stimuli and on the emotional state of the organism. Second, it has a wide neuro-anatomical distribution, modulating brain regions with different functions in responses to aversive stimuli. Third, endocannabinoids regulate the release of other neurotransmitters that may have even opposing functions, such as GABA and glutamate. Further understanding of the temporal, spatial and functional characteristics of this system is necessary to clarify its role in emotional responses and will promote advances in its therapeutic exploitation.

Effects of cocaine in 5-lipoxygenase-deficient mice

5-Lipoxygenase (5-LOX), along with 12-lipoxygenase and cyclooxygenases, metabolizes arachidonic acid into eicosanoids. In rodents, 12-lipoxygenase deficiency alters behavioral responses to cocaine. We used 5-LOX-deficient mice and their controls to investigate cocaine's actions. After repeated cocaine injections, the increase in locomotor activity was greater in 5-LOX-deficient mice. Since the 5-LOX pathway may regulate the levels/metabolism of arachidonoylethanolamide (AEA) we assayed the AEA levels in the striatum, the binding of the endogenous AEA to the cannabinoid receptor CB1R, and anandamide hydrolase (FAAH) activity in the striatum, hippocampus, and cortex. Striatal AEA levels decreased after repeated cocaine injections. Cocaine also decreased CB1R binding in all brain regions studied and the only significant differences between 5-LOX-deficient and control mice was the greater hippocampal FAAH activity in 5-LOX-deficient mice. Our results demonstrated that a 5-LOX deficiency alters sensitivity to repeated cocaine. It should be investigated whether a human 5-LOX gene polymorphism affects cocaine's actions.

COX-2 oxidative metabolism of endocannabinoids augments hippocampal synaptic plasticity

Endocannabinoids (eCBs) are important endogenous lipid mediators in synaptic transmission and plasticity and are oxygenated by cyclooxygenase-2 (COX-2) to form new types of prostaglandins. However, little is known about whether COX-2 oxidative metabolism of eCBs and their metabolites alter synaptic signaling. Here we demonstrate that increased COX-2 expression significantly enhances basal synaptic transmission and augments long-term potentiation (LTP) in the mouse hippocampus. This augmentation was inhibited in the presence of a selective COX-2 inhibitor or with deletion of the COX-2 gene. The CB 1 receptor-mediated depolarization-induced suppression of inhibition (DSI) was diminished when COX-2 expression was increased either with lipopolysaccharide (LPS) stimulation or transgenic neuronal over-expression of COX-2. Conversely, DSI was potentiated when COX-2 activity was pharmacologically or genetically inhibited. Interestingly, COX-2 oxidative metabolites of eCBs elevated LTP, an effect opposite to that of their parent molecules 2-arachidonoylglycerol (2-AG) and arachidonoyl ethanolamide (AEA). In addition, the ERK/MAPK and IP 3 pathways were found to mediate PGE 2-G-induced enhancement of LTP. Our results indicate that COX-2 oxidative metabolism of eCBs is an important signaling pathway in modulation of synaptic transmission and plasticity.

Compartmentalization of endocannabinoids into lipid rafts in a dorsal root ganglion cell line

N-arachidonoyl ethanolamine (AEA) and 2-arachidonoyl glycerol (2-AG) are endogenous cannabinoids binding to the cannabinoid receptors CB1 and CB2 to modulate neuronal excitability and synaptic transmission in primary afferent neurons. To investigate the compartmentalization of the machinery for AEA and 2-AG signalling, we studied their partitioning into lipid raft fractions isolated from a dorsal root ganglion X neuroblastoma cell line (F-11). F-11 cells were homogenized and fractionated using a detergent-free OptiPrep density gradient. All lipids were partially purified from methanolic extracts of the fractions on solid phase cartridges and quantified using liquid chromatography tandem mass spectrometry (LC/MS/MS). Protein distribution was determined by Western blotting. Under basal conditions, the endogenous cannabinoid AEA was present in both lipid raft and specific non-lipid raft fractions as was one of its biosynthetic enzymes, NAPE-PLD. The 2-AG precursor 1-stearoyl-2-arachidonoyl-sn-glycerol (DAG), diacylglycerol lipase alpha (DAGLalpha), which cleaves DAG to form 2-AG, and 2-AG were all co-localized with lipid raft markers. CB1 receptors, previously reported to partition into lipid raft fractions, were not detected in F-11 membranes, but CB2 receptors were detected at high levels and partitioned into non-lipid raft fractions. The biochemical machinery for the production of 2-AG via the putative diacylglycerol pathway is localized within lipid rafts, suggesting that 2-AG synthesis via DAG occurs within these microdomains. The observed co-localization of AEA, 2-AG, and their synthetic enzymes with the reported localization of CB1 raises the possibility of intrinsic-autocrine signalling within lipid raft domains and/or retrograde-paracrine signalling.

Endogenous cannabinoids and appetite.

Since pre-history, Cannabis sativa has been exploited for its potent and manifold pharmacological actions. Amongst the most renowned of these actions is a tendency to provoke ravenous eating. The characterization of the psychoactive principals in cannabis (exogenous cannabinoids) and, more recently, the discovery of specific brain cannabinoid receptors and their endogenous ligands (endocannabinoids) has stimulated research into the physiological roles of endocannabinoid systems. In this review, we critically discuss evidence from the literature that describe studies on animals and human subjects to support endocannabinoid involvement in the control of appetite. We describe the hyperphagic actions of the exogenous cannabinoid, Delta9-tetrahydrocannabinol, and the endogenous CB1 ligands, anandamide and 2-arachidonylglycerol, and present evidence to support a specific role of endocannabinoid systems in appetitive processes related to the incentive and reward properties of food. A case is made for more comprehensive and systematic analyses of cannabinoid actions on eating, in the anticipation of improved therapies for disorders of appetite and body weight, and a better understanding of the biopsychological processes underlying hunger.

Activated Protein C Inhibits Lipopolysaccharide-Induced Anandamide and 2-Arachidonoylglycerol Generation In Vitro and In Vivo.

Anandamide(ANA) and 2-arachidonoylglycerol(2-AG), two lipid mediators existing in the central nervous system and various peripheral tissues, have been shown to exert their diverse biological effects through cannabinoid receptors CB1 and CB2, and are therefore defined as endogenous cannabinoids. Recently, ANA and 2-AG have been shown to be generated by macrophages and platelets, respectively, by the stimulation with lipopolysaccharide(LPS) in vitro and in vivo in rats. We showed the endocannabinoids are increased in septic shock patients. Furthermore, the increased endocannabinoids are efficiently adsorbed onto polymyxin B immobilized column resulting the improvement of hypotensive shock. On the other hand it has been showed that activated protein C (APC) can ameliorate endotoxin induced-shock and -disseminated intravascular coagulation in experimental animals and patients, although the mechanisms governing its action are unknown. In the present study we found that APC inhibited the generation of ANA and 2-AG in vitro by lipopolysaccharide (LPS)-activated macrophages and platelets, respectively. Furthermore, APC significantly decreased the levels of ANA and 2-AG in the sera from rats preinfused with APC followed by LPS infusion. Consistent with the decrease of them, diminishment of hypotension and an increase in survival rate were confirmed in the APC pretreatment group. These findings demonstrate that APC can inhibit the generation of ANA and 2-AG in vitro and in vivo, and thereby suppress their induced hypotension. This action of APC may explain its efficiency in the treatment of septic shock in humans and experimental animals.

Phospholipase D Activity Is Essential for PKC-Mediated Phosphatidylserine Exposure.

The adverse effects of red blood cells (RBC) that expose phosphatidylserine (PS) in the sickle cell circulation are well recognized. However, the mechanism that underlies the formation of these cells is not well understood. We have shown previously that activation of protein kinase C (PKC) using phorbol myristate acetate (PMA) stimulates RBC to expose PS on a subset of cells. This process was dependent on Ca2+-influx induced by this stimulation. PKC inhibitors were shown to counteract this process and also reduce PS exposure induced by high levels of intracellular Ca2+. Furthermore, PIP2 administered to the RBC similarly induces PS exposure. Because both PKC and PIP2 can stimulate phospholipase D (PLD), we investigated the role of PLD in signal transduction initiated phospholipid scrambling. PLD cleaves phospholipids to produce phosphatidic acid (PA), which can be further metabolized to lysophosphatidic acid, an important lipid mediator, or to diacylglycerol (DAG), itself a PKC activator. In presence of primary alcohols such as ethanol or 1-butanol, PLD generates phosphatidylethanol or phosphatidylbutanol, respectively. Using annexin V, we measured the fraction of PS-exposing cells after treatment of RBC with PMA in presence of these primary alcohols. Treatment with 1% ethanol as well as 0.2% 1-butanol (at 0.4% hematocrit) completely abolished the generation of PS exposure in PMA-treated cells, indicating that the formation of PA by PLD was essential for scrambling. Anandamides are structural analogues of arachidonoyl ethanolamide, an endocannabinoid. Since these structures provide feedback inhibition of the PLD-isoform that produces these compounds in the brain and were found effective on plant PLD isoforms, we evaluated their effect on PLD-mediated PS scrambling in our assay. Short-chain lauroyl ethanolamide as well as arachidonoyl ethanolamide itself induced concentration-dependent inhibition of PMA-induced PS exposure. This suggests that these compounds indeed inhibit the isoforms of PLD present in the RBC, and confirms the importance of PLD activity for PKC-mediated PS exposure. Furthermore we evaluated the effect of propranolol, which inhibits phosphatidate phosphatase, the enzyme that converts PA to DAG. PMA-induced PS exposure was more than 80% inhibited by propranolol, indicating that the conversion of DAG from PLD-generated PA is essential for PKC-mediated PS exposure. PLD inhibitors did not inhibit Ca2+-induced scrambling initiated with Ca-ionophore, indicating that loading RBC with high levels of intracellular Ca2+ can override the requirement for PLD activation, possibly because DAG is formed through direct activation of phospholipase C. These results show that PLD activation is essential for PMA-induced Ca-influx and PS exposure, and that the subsequent formation of DAG from PA is a critical step in this process. Altered Ca2+ homeostasis, increased PA generation and increased PKC activation are observed in sickle RBC. Our data indicate that activation of signal transduction pathways plays an important role in generating PS exposure in sickle cells.

Role and regulation of acylethanolamides in energy balance: focus on adipocytes and β‐cells

The endocannabinoid, arachidonoylethanolamide (AEA), and the peroxisome proliferator-activated receptor (PPAR)-alpha ligand, oleylethanolamide (OEA) produce opposite effects on lipogenesis. The regulation of OEA and its anti-inflammatory congener, palmitoylethanolamide (PEA), in adipocytes and pancreatic beta-cells has not been investigated. We report here the results of studies on acylethanolamide regulation in these cells during obesity and hyperglycaemia, and provide an overview of acylethanolamide role in metabolic control. We analysed by liquid chromatography-mass spectrometry OEA and PEA levels in: 1) mouse 3T3F442A adipocytes during insulin-induced differentiation, 2) rat insulinoma RIN m5F beta-cells kept in 'low' or 'high' glucose, 3) adipose tissue and pancreas of mice with high fat diet-induced obesity (DIO), and 4) in visceral fat or blood of obese or type 2 diabetes (T2D) patients. In adipocytes, OEA levels remain unchanged during differentiation, whereas those of PEA decrease significantly, and are under the negative control of both leptin and PPAR-gamma. PEA is significantly downregulated in subcutaneous adipose tissue of DIO mice. In RIN m5F insulinoma beta-cells, OEA and PEA levels are inhibited by 'very high' glucose, this effect being enhanced by insulin, whereas in cells kept for 24 h in 'high' glucose, they are stimulated by both glucose and insulin. Elevated OEA and PEA levels are found in the blood of T2D patients. Reduced PEA levels in hypertrophic adipocytes might play a role in obesity-related pro-inflammatory states. In beta-cells and human blood, OEA and PEA are down- or up-regulated under conditions of transient or chronic hyperglycaemia, respectively.

Discovery and Isolation of Anandamide and Other Endocannabinoids

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Discovery and Isolation of Anandamide and Other Endocannabinoids

Discovery and Isolation of Anandamide and Other Endocannabinoids

Evaluation of fatty acid amides in the carrageenan-induced paw edema model

While it has long been recognized that Δ9-tetrahydrocannabinol (THC), the primary psychoactive constituent of cannabis, and other cannabinoid receptor agonists possess anti-inflammatory properties, their well known CNS effects have dampened enthusiasm for therapeutic development. On the other hand, genetic deletion of fatty acid amide hydrolase (FAAH), the enzyme responsible for degradation of fatty acid amides, including endogenous cannabinoid N-arachidonoyl ethanolamine (anandamide; AEA), N-palmitoyl ethanolamine (PEA), N-oleoyl ethanolamine (OEA), and oleamide, also elicits anti-edema, but does not produce any apparent cannabinoid effects. The purpose of the present study was to investigate whether exogenous administration of FAAs would augment the anti-inflammatory phenotype of FAAH (−/−) mice in the carrageenan model. Thus, we evaluated the effects of the FAAs AEA, PEA, OEA, and oleamide in wild-type and FAAH (−/−) mice. For comparison, we evaluated the anti-edema effects of THC, dexamethasone (DEX), a synthetic glucocorticoid, diclofenac (DIC), a nonselective cyclooxygenase (COX) inhibitor, in both genotypes. A final study determined if tolerance to the anti-edema effects of PEA occurs after repeated dosing. PEA, THC, DEX, DIC elicited significant decreases in carrageenan-induced paw edema in wild-type mice. In contrast OEA produced a less reliable anti-edema effect than these other drugs, and AEA and oleamide failed to produce any significant decreases in paw edema. Moreover, none of the agents evaluated augmented the anti-edema phenotype of FAAH (−/−) mice, suggesting that maximal anti-edema effects had already been established. PEA was the most effective FAA in preventing paw edema and its effects did not undergo tolerance. While the present findings do not support a role for AEA in preventing carrageenan-induced edema, PEA administration and FAAH blockade elicited anti-edema effects of an equivalent magnitude as produced by THC, DEX, and DIC in this assay.

Multiple pathways involved in the biosynthesis of anandamide

Endocannabinoids, including anandamide (arachidonoyl ethanolamide) have been implicated in the regulation of a growing number of physiological and pathological processes. Anandamide can be generated from its membrane phospholipid precursor N-arachidonoyl phosphatidylethanolamine (NAPE) through hydrolysis by a phospholipase D (NAPE-PLD). Recent evidence indicates, however, the existence of two additional, parallel pathways. One involves the sequential deacylation of NAPE by α,β-hydrolase 4 (Abhd4) and the subsequent cleavage of glycerophosphate to yield anandamide, and the other one proceeds through phospholipase C-mediated hydrolysis of NAPE to yield phosphoanandamide, which is then dephosphorylated by phosphatases, including the tyrosine phosphatase PTPN22 and the inositol 5′ phosphatase SHIP1. Conversion of synthetic NAPE to AEA by brain homogenates from wild-type and NAPE-PLD−/− mice can proceed through both the PLC/phosphatase and Abdh4 pathways, with the former being dominant at shorter (<10min) and the latter at longer (60min) incubations. In macrophages, the endotoxin-induced synthesis of anandamide proceeds uniquely through the phospholipase C/phosphatase pathway.

Self-Assembling Cannabinomimetics: Supramolecular Structures of N-Alkyl Amides

Certain fatty acid N-alkyl amides from the medicinal plant Echinacea activate cannabinoid type-2 (CB2) receptors. In this study we show that the CB2-binding Echinacea constituents dodeca-2E,4E-dienoic acid isobutylamide (1) and dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (2) form micelles in aqueous medium. In contrast, micelle formation is not observed for undeca-2E-ene-8,10-diynoic acid isobutylamide (3), which does not bind to CB2, or structurally related endogenous cannabinoids, such as arachidonoyl ethanolamine (anandamide). The critical micelle concentration (CMC) range of 1 and 2 was determined by fluorescence spectroscopy as 200-300 and 7400-10000 nM, respectively. The size of premicelle aggregates, micelles, and supermicelles was studied by dynamic light scattering. Microscopy images show that compound 1, but not 2, forms globular and rod-like supermicelles with radii of approximately 75 nm. The self-assembling N-alkyl amides partition between themselves and the CB2 receptor, and aggregation of N-alkyl amides thus determines their in vitro pharmacological effects. Molecular mechanics by Monte Carlo simulations of the aggregation process support the experimental data, suggesting that both 1 and 2 can readily aggregate into premicelles, but only 1 spontaneously assembles into larger aggregates. These findings have important implications for biological studies with this class of compounds.

Exploration of natural and synthetic N-alkyl amides as source for new cannabinoid receptor type-2 (CB2) selective ligands

We recently reported that certain anti-inflammatory N-alkyl amides from purple coneflower (Echinacea spp.) constitute a new class of cannabinomimetics, which selectively bind to and activate the cannabinoid type-2 (CB2) receptor [1]. In the present study, we have investigated whether chain length and substitution of the headgroup of this class of natural products can result in new compounds with nM affinities to CB2 receptors. More than 30 N-alkyl amide derivatives were synthesized. A comparison of the preliminary structure-activity relationship of N-alkyl amides with the endogenous cannabinoid arachidonoyl ethanolamine (anandamide) clearly indicates that these compounds are different pharmacophores. Moreover, unlike anandamide, N-alkyl amides and 2-arachidonoyl glycerol (2-AG) trigger CB2-receptor dependent intracellular calcium transients in myelo-monocytic cells. In dodecanoic acid derivatives, the 2E,4E double bonds were found to be crucial for optimal binding to CB2 while only the 2E double bond appears to be required for the moderate CB1 affinity. The most active compounds were isobutylamides (Ki ˜ 60 nM). We show that certain derivatives segregate and form micelles, which are no longer able to interact with the receptor. Thus, the self-assembling of these compounds directly influences CB2 affinity.

Modafinil enhances extracellular levels of dopamine in the nucleus accumbens and increases wakefulness in rats

Modafinil (MOD) is a wakefulness-promoting drug that improves the alertness levels in narcolepsy; however, the molecular mechanism of action remains to be elucidated. We found that after a single icv injection of MOD (10 μg/5 μl) the extracellular levels of dopamine (DA) and l-DOPA collected from the nucleus accumbens were increased and decreased, respectively. Separately, the icv administration of MOD (10 μg/5 μl) to rats enhanced wakefulness (W) whereas diminished sleep during 4 h. Lastly, the alertness induced by MOD was partially antagonized by the sleep-inducing endocannabinoid anandamide (ANA). We conclude that MOD enhances the extracellular levels of DA, promotes W and its effects on sleep are partially blocked by ANA.

A biosynthetic pathway for anandamide

The endocannabinoid arachidonoyl ethanolamine (anandamide) is a lipid transmitter synthesized and released "on demand" by neurons in the brain. Anandamide is also generated by macrophages where its endotoxin (LPS)-induced synthesis has been implicated in the hypotension of septic shock and advanced liver cirrhosis. Anandamide can be generated from its membrane precursor, N-arachidonoyl phosphatidylethanolamine (NAPE) through cleavage by a phospholipase D (NAPE-PLD). Here we document a biosynthetic pathway for anandamide in mouse brain and RAW264.7 macrophages that involves the phospholipase C (PLC)-catalyzed cleavage of NAPE to generate a lipid, phosphoanandamide, which is subsequently dephosphorylated by phosphatases, including PTPN22, previously described as a protein tyrosine phosphatase. Bacterial endotoxin (LPS)-induced synthesis of anandamide in macrophages is mediated exclusively by the PLC/phosphatase pathway, which is up-regulated by LPS, whereas NAPE-PLD is down-regulated by LPS and functions as a salvage pathway of anandamide synthesis when the PLC/phosphatase pathway is compromised. Both PTPN22 and endocannabinoids have been implicated in autoimmune diseases, suggesting that the PLC/phosphatase pathway of anandamide synthesis may be a pharmacotherapeutic target.

Endocannabinoid Biosynthesis Proceeding through Glycerophospho-N-acyl Ethanolamine and a Role for α/β-Hydrolase 4 in This Pathway

N-Acyl ethanolamines (NAEs) are a large class of signaling lipids implicated in diverse physiological processes, including nociception, cognition, anxiety, appetite, and inflammation. It has been proposed that NAEs are biosynthesized from their corresponding N-acyl phosphatidylethanolamines (NAPEs) in a single enzymatic step catalyzed by a phospholipase D (NAPE-PLD). The recent generation of NAPE-PLD(-/-) mice has revealed that these animals possess lower brain levels of saturated NAEs but essentially unchanged concentrations of polyunsaturated NAEs, including the endogenous cannabinoid anandamide. These findings suggest the existence of additional enzymatic routes for the production of NAEs in vivo. Here, we report evidence for an alternative pathway for NAE biosynthesis that proceeds through the serine hydrolase-catalyzed double-deacylation of NAPE to generate glycerophospho-NAE, followed by the phosphodiesterase-mediated cleavage of this intermediate to liberate NAE. Furthermore, we describe the functional proteomic isolation and identification of a heretofore uncharacterized enzyme alpha/beta-hydrolase 4 (Abh4) as a lysophospholipase/phospholipase B that selectively hydrolyzes NAPEs and lysoNAPEs. Abh4 accepts lysoNAPEs bearing both saturated and polyunsaturated N-acyl chains as substrates and displays a distribution that closely mirrors lysoNAPE-lipase activity in mouse tissues. These results support the existence of an NAPE-PLD-independent route for NAE biosynthesis and suggest that Abh4 plays a role in this metabolic pathway by acting as a (lyso)NAPE-selective lipase.

Cannabidiol, a constituent of Cannabis sativa, modulates sleep in rats

Δ 9-tetrahydrocannabinol (Δ 9-THC) and cannabidiol (CBD) are two major constituents of Cannabis sativa. Δ 9-THC modulates sleep, but no clear evidence on the role of CBD is available. In order to determine the effects of CBD on sleep, it was administered intracerebroventricular (icv) in a dose of 10 μg/5 μl at the beginning of either the lights-on or the lights-off period. We found that CBD administered during the lights-on period increased wakefulness (W) and decreased rapid eye movement sleep (REMS). No changes on sleep were observed during the dark phase. Icv injections of CBD (10 μg/5 μl) induced an enhancement of c-Fos expression in waking-related brain areas such as hypothalamus and dorsal raphe nucleus (DRD). Microdialysis in unanesthetized rats was carried out to characterize the effects of icv administration of CBD (10 μg/5 μl) on extracellular levels of dopamine (DA) within the nucleus accumbens. CBD induced an increase in DA release. Finally, in order to test if the waking properties of CBD could be blocked by the sleep-inducing endocannabinoid anandamide (ANA), animals received ANA (10 μg/2.5 μl, icv) followed 15 min later by CBD (10 μg/2.5 μl). Results showed that the waking properties of CBD were not blocked by ANA. In conclusion, we found that CBD modulates waking via activation of neurons in the hypothalamus and DRD. Both regions are apparently involved in the generation of alertness. Also, CBD increases DA levels as measured by microdialysis and HPLC procedures. Since CBD induces alertness, it might be of therapeutic value in sleep disorders such as excessive somnolence.

Synthesis, cannabinoid receptor activity, and enzymatic stability of reversed amide derivatives of arachidonoyl ethanolamide

Retroanandamide ( 2f) and its 10 analogues ( 1a– e, 2a– e) were synthesized and evaluated for the cannabinoid receptor activation by a [ 35S]GTPγS binding assay using rat cerebellar membranes, and Chinese hamster ovary cell membranes expressing human CB2 receptors. The primary goal of the study was to develop cannabinoid receptor agonists having improved enzymatic stability compared to endogenous N-arachidonoyl ethanolamide (AEA). Furthermore, by reversing the amide bond of AEA, the formation of arachidonic acid would be prevented. Finally, an effect of the carbonyl carbon position on the cannabinoid receptor activity was explored by synthesizing retroanandamide analogues having different chain lengths ( 1a– e, C 19; 2a– f, C 20). All the synthesized compounds, except 2c, behaved as partial agonists for the both cannabinoid receptors. In rat brain homogenate, the reversed amides possessed significantly higher stability against FAAH induced degradation than AEA. Therefore, the reversed amide analogues of AEA may serve as enzymatically stable structural basis for the drug design based on the endogenous cannabinoids.

PGE2 glycerol ester, a COX‐2 oxidative metabolite of 2‐arachidonoyl glycerol, modulates inhibitory synaptic transmission in mouse hippocampal neurons

The oxygenation of endogenous cannabinoids (eCBs) 2-arachidonoyl glycerol (2-AG) and arachidonoyl ethanolamide by cyclooxygenase-2 (COX-2) produces novel types of prostanoids: prostaglandin glycerol esters (PG-Gs) and prostaglandin ethanolamides (PG-EAs). However, the physiological function of COX-2 oxidative metabolites of eCBs is still unclear. Here we demonstrate that PGE2-G, a COX-2 oxidative metabolite of 2-AG, induced a concentration-dependent increase in the frequency ofminiature inhibitory postsynaptic currents (mIPSCs) in primary cultured hippocampal neurons, an effect opposite to that of 2-AG. This increase was not inhibited by SR141716, a CB1 receptor antagonist, but was attenuated by an IP3 or MAPK inhibitor. In addition, we also examined the effects of other prostanoids derived from COX-2 oxygenation of eCBs on mIPSCs. PGD2-G, PGF2alpha-G and PGD2-EA, but not PGE2-EA or PGF2alpha-EA, also increased the frequency of mIPSCs. The eCB-derived prostanoid-induced responses appeared to be different from those of corresponding arachidonic acid-derived prostanoids, implying that these effects are not mediated via known prostanoid receptors. We further discovered that the inhibition of COX-2 activity reduced inhibitory synaptic activity and augmented depolarization-induced suppression of inhibition (DSI), whereas the enhancement of COX-2 augmented the synaptic transmission and abolished DSI. Our results, which show that COX-2 oxidative metabolites of eCBs exert opposite effects to their parent molecules on inhibitory synaptic transmission, suggest that alterations in COX-2 activity will have significant impact on endocannabinoid signalling in hippocampal synaptic activity.