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PubMed2000

Oxygenation of the endocannabinoid, 2-arachidonylglycerol, to glyceryl prostaglandins by cyclooxygenase-2.

Cyclooxygenases (COX) play an important role in lipid signaling by oxygenating arachidonic acid to endoperoxide precursors of prostaglandins and thromboxane. Two cyclooxygenases exist which differ in tissue distribution and regulation but otherwise carry out identical chemical functions. The neutral arachidonate derivative, 2-arachidonylglycerol (2-AG), is one of two described endocannabinoids and appears to be a ligand for both the central (CB1) and peripheral (CB2) cannabinoid receptors. Here we report that 2-AG is a substrate for COX-2 and that it is metabolized as effectively as arachidonic acid. COX-2-mediated 2-AG oxygenation provides the novel lipid, prostaglandin H(2) glycerol ester (PGH(2)-G), in vitro and in cultured macrophages. PGH(2)-G produced by macrophages is a substrate for cellular PGD synthase, affording PGD(2)-G. Pharmacological studies reveal that macrophage production of PGD(2)-G from endogenous sources of 2-AG is calcium-dependent and mediated by diacylglycerol lipase and COX-2. These results identify a distinct function for COX-2 in endocannabinoid metabolism and in the generation of a new family of prostaglandins derived from diacylglycerol and 2-AG.

Animals↗
PubMed2001

Anandamide activates vanilloid receptor 1 (VR1) at acidic pH in dorsal root ganglia neurons and cells ectopically expressing VR1.

The vanilloid receptor type 1 (VR1) is a heat-activated ionophore preferentially expressed in nociceptive neurons of trigeminal and dorsal root ganglia (DRG). VR1, which binds and is activated by capsaicin and other vanilloid compounds, was noted to interact with the endocannabinoid anandamide (ANA) and certain inflammatory metabolites of arachidonic acid in a pH-dependent manner. At pH < or = 6.5 ANA induced (45)Ca(2+) uptake either in primary cultures of DRG neurons or cells ectopically expressing C-terminally tagged recombinant forms of VR1 with an EC(50) = approximately 10 microm at pH 5.5. Capsazepine, a potent antagonist of vanilloids, inhibited ANA-induced Ca(2+) transport in both cell systems. Vanilloids displaced [(3)H]ANA in VR1-expressing cells, suggesting competition for binding to VR1. Ratiometric determination of intracellular free calcium and confocal imaging of the VR1-green fluorescent fusion protein revealed that, at low pH (< or =6.5), ANA could induce an elevation of intracellular free Ca(2+) and consequent intracellular membrane changes in DRG neurons or transfected cells expressing VR1. These actions of ANA were similar to the effects determined previously for vanilloids. The ligand-induced changes in Ca(2+) at pH < or = 6.5 are consistent with the idea that ANA and other eicosanoids act as endogenous ligands of VR1 in a conditional fashion in vivo. The pH dependence suggests that tissue acidification in inflammation, ischemia, or traumatic injury can sensitize VR1 to eicosanoids and transduce pain from the periphery.

3T3 Cells↗
PubMed2001

Metabolism of prostaglandin glycerol esters and prostaglandin ethanolamides in vitro and in vivo.

Prostaglandin glycerol esters (PG-Gs) and prostaglandin ethanolamides (PG-EAs) are generated by the action of cyclooxygenase-2 on the endocannabinoids 2-arachidonylglycerol (2-AG) and arachidonylethanolamide, respectively. These novel eicosanoids may have unique pharmacological properties and/or serve as latent sources of prostaglandins at sites remote from their tissue of origin. Therefore, we investigated the metabolism of PG-Gs and PG-EAs in vitro and in vivo. PGE(2)-G was rapidly hydrolyzed in rat plasma to generate PGE(2) (t(1/2) = 14 s) but was only slowly metabolized in human plasma (t(1/2) > 10 min). An intermediate extent of metabolism of PGE(2)-G was observed in human whole blood (t(1/2) approximately 7 min). The parent arachidonylglycerol, 2-AG, and the more stable regioisomer, 1-AG, also were much more rapidly metabolized in rat plasma compared with human plasma. PGE(2)-EA was not significantly hydrolyzed in plasma, undergoing slow dehydration/isomerization to PGB(2)-EA. Both PGE(2)-G and PGE(2)-EA were stable in canine, bovine, and human cerebrospinal fluid. Human 15-hydroxyprostaglandin dehydrogenase, the enzyme responsible for the initial step in PG inactivation in vivo, oxidized both PGE(2)-G and PGE(2)-EA less efficiently than the free acid. The sterically hindered glyceryl prostaglandin was the poorest substrate examined in the E series. Minimal 15-hydroxyprostaglandin dehydrogenase oxidation of PGF(2 alpha)-G was observed. PGE(2)-G and PGE(2)-EA pharmacokinetics were assessed in rats. PGE(2)-G was not detected in plasma 5 min following an intravenous dose of 2 mg/kg. However, PGE(2)-EA was detectable up to 2 h following an identical dose, displaying a large apparent volume of distribution and a half-life of over 6 min. The results suggest that endocannabinoid-derived PG-like compounds may be sufficiently stable in humans to exert actions systemically. Furthermore, these results suggest that the rat is not an adequate model for investigating the biological activities of 2-arachidonylglycerol or glyceryl prostaglandins in humans.

Animals↗
PubMed2002

Astrocytes in culture produce anandamide and other acylethanolamides.

Anandamide (arachidonylethanolamide) is an endocannabinoid that belongs to the acylethanolamide lipid family. It is produced by neurons in a calcium-dependent manner and acts through cannabinoid CB1 receptors. Other members of the acylethanolamide lipid family are also produced by neurons and act through G-protein-coupled receptors: homo-gamma-linolenylethanolamide (HEA) and docosatetraenylethanolamide (DEA) act through CB1 receptors, palmitylethanolamide (PEA) acts through CB2-like receptors, and oleylethanolamide (OEA) acts through receptors that have not yet been cloned. Although it is clear that anandamide and other acylethanolamides play a major role in neuronal signaling, whether astrocytes also produce these lipids is unknown. We developed a chemical ionization gas chromatography/mass spectrometry method that allows femtomole detection and quantification of anandamide and other acylethanolamides. Using this method, we unambiguously detected and quantified anandamide, HEA, DEA, PEA, and OEA in mouse astrocytes in culture. Stimulation of mouse astrocytes with ionomycin, a calcium ionophore, enhanced the production of anandamide, HEA, and DEA, whereas PEA and OEA levels were unchanged. Endothelin-1, a peptide known to act on astrocytes, enhanced the production of anandamide, without affecting the levels of other acylethanolamides. These results show that astrocytes produce anandamide, HEA, and DEA in a calcium-dependent manner and that anandamide biosynthesis can be selectively stimulated under physiologically relevant conditions. The relative levels of acylethanolamides in astrocytes from rat and human were different from the relative levels of acylethanolamides in mouse astrocytes, indicating that the production of these lipids differs between species. Because astrocytes are known to express CB1 receptors and inactivate endocannabinoids, our finding strongly suggests the existence of a functional endocannabinoid signaling system in these cells.

Amides↗
PubMed2002

15-Lipoxygenase metabolism of 2-arachidonylglycerol. Generation of a peroxisome proliferator-activated receptor alpha agonist.

The recent demonstrations that cyclooxygenase-2 and leukocyte-type 12-lipoxygenase (LOX) efficiently oxygenate 2-arachidonylglycerol (2-AG) prompted an investigation into related oxygenases capable of metabolizing this endogenous cannabinoid receptor ligand. We evaluated the ability of six LOXs to catalyze the hydroperoxidation of 2-AG. Soybean 15-LOX, rabbit reticulocyte 15-LOX, human 15-LOX-1, and human 15-LOX-2 oxygenate 2-AG, providing 15(S)-hydroperoxyeicosatetraenoic acid glyceryl ester. In contrast, potato and human 5-LOXs do not efficiently metabolize this endocannabinoid. Among a series of structurally related arachidonyl esters, arachidonylglycerols serve as the preferred substrates for 15-LOXs. Steady-state kinetic analysis demonstrates that both 15-LOX-1 and 15-LOX-2 oxygenate 2-AG comparably or preferably to arachidonic acid. Furthermore, 2-AG treatment of COS-7 cells transiently transfected with human 15-LOX expression vectors or normal human epidermal keratinocytes results in the production and extracellular release of 15-hydroxyeicosatetraenoic acid glyceryl ester (15-HETE-G), establishing that lipoxygenase metabolism of 2-AG occurs in an eukaryotic cellular environment. Investigations into the potential biological actions of 15-HETE-G indicate that this lipid, in contrast to its free-acid counterpart, acts as a peroxisome proliferator-activated receptor alpha agonist. The results demonstrate that 15-LOXs are capable of acting on 2-AG to provide 15-HETE-G and elucidate a potential role for endocannabinoid oxygenation in the generation of peroxisome proliferator-activated receptor alpha agonists.

Animals↗
PubMed2002

The endocannabinoid anandamide inhibits neuronal progenitor cell differentiation through attenuation of the Rap1/B-Raf/ERK pathway.

Endocannabinoids are neuromodulators that act as retrograde synaptic messengers inhibiting the release of different neurotransmitters in cerebral areas such as hippocampus, cortex, and striatum. However, little is known about other roles of the endocannabinoid system in brain. In the present work we provide substantial evidence that the endocannabinoid anandamide (AEA) regulates neuronal differentiation both in culture and in vivo. Thus AEA, through the CB(1) receptor, inhibited cortical neuron progenitor differentiation to mature neuronal phenotype. In addition, human neural stem cell differentiation and nerve growth factor-induced PC12 cell differentiation were also inhibited by cannabinoid challenge. AEA decreased PC12 neuronal-like generation via CB(1)-mediated inhibition of sustained extracellular signal-regulated kinase (ERK) activation, which is responsible for nerve growth factor action. AEA thus inhibited TrkA-induced Rap1/B-Raf/ERK activation. Finally, immunohistochemical analyses by confocal microscopy revealed that adult neurogenesis in dentate gyrus was significantly decreased by the AEA analogue methanandamide and increased by the CB(1) antagonist SR141716. These data indicate that endocannabinoids inhibit neuronal progenitor cell differentiation through attenuation of the ERK pathway and suggest that they constitute a new physiological system involved in the regulation of neurogenesis.

Animals↗
PubMed2003

Leptin activates the anandamide hydrolase promoter in human T lymphocytes through STAT3.

Physiological concentrations of leptin stimulate the activity of the endocannabinoid-degrading enzyme anandamide hydrolase (fatty acid amide hydrolase, FAAH) in human T lymphocytes up to approximately 300% over the untreated controls. Stimulation of FAAH occurred through up-regulation of gene expression at transcriptional and translational levels and involved binding of leptin to its receptor with an apparent dissociation constant (K(d)) of 1.95 +/- 0.14 nm and maximum binding (B(max)) of 392 +/- 8 fmol x mg protein(-1). Leptin binding to the receptor triggered activation of STAT3 but not STAT1 or STAT5 or the mitogen-activated protein kinases p38, p42, and p44. Peripheral lymphocytes of leptin knock-out (ob/ob) mice showed decreased FAAH activity and expression (approximately 25% of the wild-type littermates), which were reversed to control levels by exogenous leptin. Analysis of the FAAH promoter showed a cAMP-response element-like site, which is a transcriptional target of STAT3. Consistently, mutation of this site prevented FAAH activation by leptin in transient expression assays. Electrophoretic mobility shift and supershift assays further corroborated the promoter activity data. Taken together, these results suggest that leptin, by up-regulating the FAAH promoter through STAT3, enhances FAAH expression, thus tuning the immunomodulatory effects of anandamide. These findings might also have critical implications for human fertility.

Acute-Phase Proteins↗
PubMed2003

Molecular identification of a functional homologue of the mammalian fatty acid amide hydrolase in Arabidopsis thaliana.

N-Acylethanolamines (NAEs) are endogenous constituents of plant and animal tissues, and in vertebrates their hydrolysis terminates their participation as lipid mediators in the endocannabinoid signaling system. The membrane-bound enzyme responsible for NAE hydrolysis in mammals has been identified at the molecular level (designated fatty acid amide hydrolase, FAAH), and although an analogous enzyme activity was identified in microsomes of cotton seedlings, no molecular information is available for this enzyme in plants. Here we report the identification, the heterologous expression (in Escherichia coli), and the biochemical characterization of an Arabidopsis thaliana FAAH homologue. Candidate Arabidopsis DNA sequences containing a characteristic amidase signature sequence (PS00571) were identified in plant genome data bases, and a cDNA was isolated by reverse transcriptase-PCR using Arabidopsis genome sequences to develop appropriate oligonucleotide primers. The cDNA was sequenced and predicted to encode a protein of 607 amino acids with 37% identity to rat FAAH within the amidase signature domain (18% over the entire length). Residues determined to be important for FAAH catalysis were conserved between the Arabidopsis and rat protein sequences. In addition, a single transmembrane domain near the N terminus was predicted in the Arabidopsis protein sequence, similar to that of the rat FAAH protein. The putative plant FAAH cDNA was expressed as an epitope/His-tagged fusion protein in E. coli and solubilized from cell lysates in the nonionic detergent, dodecyl maltoside. Affinity-purified recombinant protein was indeed active in hydrolyzing a variety of naturally occurring N-acylethanolamine types. Kinetic parameters and inhibition data for the recombinant Arabidopsis protein were consistent with these properties of the enzyme activity characterized previously in plant and animal systems. Collectively these data now provide support at the molecular level for a conserved mechanism between plants and animals for the metabolism of NAEs.

Amidohydrolases↗
PubMed2004

A role for caveolae/lipid rafts in the uptake and recycling of the endogenous cannabinoid anandamide.

The mechanisms responsible for the uptake and cellular processing of the endogenous cannabinoid anandamide are not well understood. We propose that anandamide uptake may occur via a caveola/lipid raft-related endocytic process in RBL-2H3 cells. Inhibitors of caveola-related (clathrin-independent) endocytosis reduced anandamide transport by approximately 50% compared with the control. Fluorescein derived from fluorescently labeled anandamide colocalized with protein markers of caveolae at early time points following transport. In this study, we have also identified a yet unrecognized process involved in trafficking events affecting anandamide following its uptake. Following uptake of [(3)H]anandamide by RBL-2H3 cells, we found an accumulation of tritium in the caveolin-rich membranes. Inhibitors of both anandamide uptake and metabolism blocked the observed enrichment of tritium in the caveolin-rich membranes. Mass spectrometry of subcellular membrane fractions revealed that the tritium accumulation observed in the caveolin-rich membrane fraction was not representative of intact anandamide, suggesting that following metabolism by the enzyme fatty acid amide hydrolase (FAAH), anandamide metabolites are rapidly enriched in caveolae. Furthermore, HeLa cells, which do not express high levels of FAAH, showed an accumulation of tritium in the caveolin-rich membrane fraction only when transfected with FAAH cDNA. Western blot and immunocytochemistry analyses of RBL-2H3 cells revealed that FAAH was localized in intracellular compartments distinct from caveolin-1 localization. Together, these data suggest that following uptake via caveola/lipid raft-related endocytosis, anandamide is rapidly metabolized by FAAH, with the metabolites efficiently recycled to caveolin-rich membrane domains.

Amidohydrolases↗
PubMed2006

Endocannabinoid biosynthesis proceeding through glycerophospho-N-acyl ethanolamine and a role for alpha/beta-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.

AlkB Homolog 4, Lysine Demethylase↗
PubMed2006

Targeted cyclooxygenase gene (ptgs) exchange reveals discriminant isoform functionality.

The prostaglandin G/H synthase enzymes, commonly termed COX-1 and COX-2, differ markedly in their responses to regulatory stimuli and their tissue expression patterns. COX-1 is the dominant source of "housekeeping" prostaglandins, whereas COX-2 synthesizes prostaglandins of relevance to pain, inflammation, and mitogenesis. Despite these distinctions, the two enzymes are remarkably conserved, and their subcellular distributions overlap considerably. To address the functional interchangeability of the two isozymes, mice in which COX-1 is expressed under COX-2 regulatory elements were created by a gene targeting "knock-in" strategy. In macrophages from these mice, COX-1 was shown to be lipopolysaccharide-inducible in a manner analogous to COX-2 in wild-type macrophages. However, COX-1 failed to substitute effectively for COX-2 in lipopolysaccharide-induced prostaglandin E2 synthesis at low concentrations of substrate and in the metabolism of the endocannabinoid 2-arachidonylglycerol. The marked depression of the major urinary metabolite of prostacyclin in COX-2 null mice was only partially rescued by COX-1 knock-in, whereas the main urinary metabolite of prostaglandin E2 was rescued totally. Replacement with COX-1 partially rescued the impact of COX-2 deletion on reproductive function. The renal pathology consequent to COX-2 deletion was delayed but not prevented, whereas the corresponding peritonitis was unaltered. Insertion of COX-1 under the regulatory sequences that drive COX-2 expression indicated that COX-1 can substitute for some COX-2 actions and rescue only some of the consequences of gene disruption. Manipulation of COX-2 also revealed a preference for coupling with distinct downstream prostaglandin synthases in vivo. These mice will provide a valuable reagent with which to elucidate the distinct roles of the COX enzymes in mammalian biology.

3' Untranslated Regions↗
PubMedDate not supplied

2-Arachidonoylglycerol increases in ischemia-reperfusion injury of the rat liver.

Several studies have implicated endocannabinoids in various forms of shock. However, the role of endocannabinoids in hepatic ischemia-reperfusion injury remains unclear. The purpose of this study was to evaluate the changes of two endocannabinoidsin hepatic ischemia-reperfusion injury: anandamide (ANA) and 2-arachidonoylglycerol (2-AG). Male Sprague-Dawley rats were divided into 2 groups: the short (15 min) ischemic group and the long (60 min)ischemic group in the segmental (70%) hepatic tissue. Blood levels of aspartate transaminase (AST), alanine transaminase (ALT), lactate dehydrogenase (LDH), ANA, and 2-AG were examined. Serum lev-els of AST, ALT, and LDH were significantly higher in the long-ischemia group than in the short-ischemia group. Plasma levels of 2-AG showed similar augmentation prior to and after reperfusion in both the short- and long-ischemia groups, although plasma 2-AG lev-els tended to be higher in the long-ischemia group than in the short-ischemia group. Plasma levels of ANA were augmented in the early phase of reperfusion in the short-ischemia group and did not differ significantly from the normal level with time after reperfusion in the long-ischemia group. These results suggest that the endocannabinoid 2-AG increases in hepatic ischemia-reperfusion injury of rats, rather than ANA.

Alanine Transaminase↗
PubMed2003

Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain.

Diacylglycerol (DAG) lipase activity is required for axonal growth during development and for retrograde synaptic signaling at mature synapses. This enzyme synthesizes the endocannabinoid 2-arachidonoyl-glycerol (2-AG), and the CB1 cannabinoid receptor is also required for the above responses. We now report on the cloning and enzymatic characterization of the first specific sn-1 DAG lipases. Two closely related genes have been identified and their expression in cells correlated with 2-AG biosynthesis and release. The expression of both enzymes changes from axonal tracts in the embryo to dendritic fields in the adult, and this correlates with the developmental change in requirement for 2-AG synthesis from the pre- to the postsynaptic compartment. This switch provides a possible explanation for a fundamental change in endocannabinoid function during brain development. Identification of these enzymes may offer new therapeutic opportunities for a wide range of disorders.

Animals↗
PubMed2005

Characterization and comparison of recombinant human and rat TRPV1 receptors: effects of exo- and endocannabinoids.

BACKGROUND: TRPV1 is a ligand-gated ion channel whose activation by capsaicin increases intracellular Ca(2+) ([Ca(2+)](i)). TRPV1 and cannabinoid CB(1) receptor activation are capable of eliciting analgesia. In this study, using recombinant human (h) and rat (r) TRPV1 receptors expressed in HEK293 cells, we have performed a comparison of both TRPV1 species at 22 and 37 degrees C and compared endo- and exocannabinoid activity at both receptors. METHODS: [Ca(2+)](i) was measured in Fura-2-loaded HEK293(hTRPV1) and HEK293(rTRPV1) cells. To assess native CB(1) receptor activity, [(35)S]GTPgammaS binding to membranes prepared from rat cerebellum was measured. RESULTS: Both capsaicin (pEC(50) rat approximately 6.9 and pEC(50) human approximately 6.8 at 37 degrees C) and anandamide (pEC(50) rat approximately 5.3 and pEC(50) human approximately 5.8 at 37 degrees C) produced a concentration-dependent increase in [Ca(2+)](i) in rat and human systems and at 22 and 37 degrees C. In HEK293(rTRPV1) cells, anandamide appeared to be a partial agonist. Capsazepine demonstrated competitive antagonism at both human and rat TRPV1 receptors and at both temperatures studied. Capsazepine effects were not temperature dependent: pK(B) at rTRPV1 was 5.98 at 22 degrees C and 6.02 at 37 degrees C, and pK(B) at hTRPV1 was 6.76 at 22 degrees C and 6.75 at 37 degrees C. However, there was a consistent 6-fold increase in capsazepine potency for hTRPV1 relative to rTRPV1. The exocannabinoid Delta(9)-tetrahydrocannabinol failed to increase [Ca(2+)](i), although its solvent ethanol was an effective TRPV1 activator. In the [(35)S]GTPgammaS binding assay using rat cerebellar membranes, anandamide (pEC(50) approximately 5.8) and Delta(9)-tetrahydrocannabinol (pEC(50) approximately 7.1), but not capsaicin, stimulated binding. Delta(9)-tetrahydrocannabinol was a partial agonist. pEC(50) values for anandamide at rTRPV1 and rCB(1) were similar. CONCLUSIONS: There were small differences in the pharmacology of rat and human TRPV1 receptors. Whilst capsaicin activated TRPV1 and Delta(9)-tetrahydrocannabinol activated CB(1), anandamide is an endogenous agonist for both receptor systems.

Animals↗
PubMed2004

Reduced cellular expression and activity of the P129T mutant of human fatty acid amide hydrolase: evidence for a link between defects in the endocannabinoid system and problem drug use.

Fatty acid amide hydrolase (FAAH) inactivates the endogenous cannabinoid (endocannabinoid) anandamide and related lipid transmitters in vivo. A single nucleotide polymorphism (SNP) in the human FAAH gene (385C to A) has recently been described that, in homozygous form, is over-represented in subjects with problem drug use. This SNP, which converts a conserved proline residue in FAAH to threonine (P129T), suggests a potential role for the FAAH-endocannabinoid system in regulating addictive behavior. Nonetheless, the impact of the 385A mutation on the biochemical and cellular function of FAAH remains unknown. Here, we report that T-lymphocytes isolated from patients homozygous for the P129T-FAAH variant express less than half of the FAAH protein and activity observed in wild-type (WT) lymphocytes. Transfected COS-7 cells also expressed significantly lower levels of P129T-FAAH compared with WT-FAAH, indicating that the aberrant expression of the former protein is not a cell type-specific phenomenon. A comparison of the transcription/translation efficiencies and cellular stabilities of WT- and P129T-FAAH proteins revealed that the reduced expression of the mutant enzyme is due to a post-translational mechanism that precedes productive folding. These findings indicate that the natural 385A SNP in the human FAAH gene produces a mutant enzyme with reduced cellular stability, thus fortifying a potential link between functional abnormalities in the endocannabinoid system and drug abuse and dependence.

Amidohydrolases↗
PubMed2003

Anandamide enhances extracellular levels of adenosine and induces sleep: an in vivo microdialysis study.

STUDY OBJECTIVES: The principal component of marijuana, delta-9-tetrahydrocannabinol increases sleep in humans. Endogenous cannabinoids, such as N-arachidonoylethanolamine (anandamide), also increase sleep. However, the mechanism by which these molecules promote sleep is not known but might involve a sleep-inducing molecule such as adenosine. Microdialysis samples were collected from the basal forebrain in order to detect levels of adenosine before and after injection of anandamide. DESIGN: Rats were implanted for sleep studies, and a cannula was placed in the basal forebrain to collect microdialysis samples. Samples were analyzed using high-performance liquid chromatography. SETTINGS: Basic neuroscience research laboratory. PARTICIPANTS AND INTERVENTIONS: Three-month-old male F344 rats. At the start of the lights-on period, animals received systemic injections of dimethyl sulfoxide (vehicle), anandamide, SR141716A (cannabinoid receptor 1 [CB1] antagonist), or SR141716A and anandamide. One hour after injections, microdialysis samples were collected (5 microL) from the basal forebrain every hour over a 20-minute period for 5 hours. The samples were immediately analyzed via high-performance liquid chromatography for adenosine levels. Sleep was also recorded continuously over the same period. MEASUREMENTS AND RESULTS: Anandamide increased adenosine levels compared to vehicle controls with the peak levels being reached during the third hour after drug injection. There was a significant increase in slow-wave sleep during the third hour. The induction in sleep and the rise in adenosine were blocked by the CB1-receptor antagonist, SR141716A. CONCLUSIONS: Anandamide increased adenosine levels in the basal forebrain and also increased sleep. The soporific effects of anandamide were mediated by the CB1 receptor, since the effects were blocked by the CB1-receptor antagonist. These findings identify a potential therapeutic use of endocannabinoids to induce sleep in conditions where sleep may be severely attenuated.

Adenosine↗
PubMed2001

Activation of PAF receptors results in enhanced synthesis of 2-arachidonoylglycerol (2-AG) in immune cells.

The endocannabinoid signaling system is believed to play a down-regulatory role in the control of cell functions. However, little is known about the factors activating endocannabinoid synthesis and which of two known endocannabinoids, 2-arachidonoylglycerol (2-AG) or N-arachidonoylethanolamine (20:4n-6 NAE, anandamide), is of physiological importance. We approached these questions by studying a possible link between cell activation with 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor, PAF) and the generation of 2-AG and anandamide in human platelets and mouse P388D1 macrophages. Human platelets responded to stimulation with the production of various 1- and 2-monoacylglycerols, including 2-AG, whereas stimulation of P388D1 macrophages induced the rapid and selective generation of 2-AG, which was immediately released into the medium. The effect of PAF was receptor mediated, as PAF receptor antagonist BN52021 blocked the effect. The treatment did not change the content of anandamide in either macrophages or platelet-rich plasma. The inhibitors of PI- and PC-specific phospholipases C (U73122 and D609) as well as PI3-kinase inhibitor (wortmannin) attenuated PAF-induced 2-AG production in macrophages. These data suggest a direct role for the endocannabinoid system in controlling immune cell activation status and indicate that 2-AG rather than anandamide is the endocannabinoid rapidly produced in response to proinflammatory stimulation of immune cells.

Animals↗
PubMed2002

Long-lasting increase in voluntary ethanol consumption and transcriptional regulation in the rat brain after intermittent exposure to alcohol.

Prolonged exposure of the brain to ethanol is a prerequisite for developing ethanol dependence, but the underlying neural adaptations are unknown. Here we demonstrate that rats subjected to repeated cycles of intoxication and withdrawal develop a marked and long-lasting increase in voluntary ethanol intake. Exposure-induced but not spontaneous alcohol intake is antagonized by acamprosate, a compound clinically effective in human alcoholism. Expression analysis of cingulate cortex and amygdala reveals a set of long-term up-regulated transcripts in this model. These include members of pathways previously implicated in alcohol dependence (glutamatergic, endocannabinoid, and monoaminergic neurotransmission), as well as pathways not previously thought to be involved in this disorder (e.g., members of the mitogen-activated protein kinase pathway). Thus, alternating periods of ethanol intoxication and withdrawal are sufficient to induce an altered functional brain state, which is likely to be encoded by long-term changes in gene expression. These observations may have important implications for how alcoholism is managed clinically. Novel clinically effective treatments may be possible to develop by targeting the products of genes found to be regulated in our model.

Acamprosate↗