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At least 883 records · Page 49Linked to original sources
PubMed2005

Inhibition of fatty acid amide hydrolase and monoacylglycerol lipase by the anandamide uptake inhibitor VDM11: evidence that VDM11 acts as an FAAH substrate.

There is some dispute concerning the extent to which the uptake inhibitor VDM11 (N-(4-hydroxy-2-methylphenyl) arachidonoyl amide) is capable of inhibiting the metabolism of the endocannabinoid anandamide (AEA) by fatty acid amide hydrolase (FAAH). In view of a recent study demonstrating that the closely related compound AM404 (N-(4-hydroxyphenyl)arachidonylamide) is a substrate for FAAH, we re-examined the interaction of VDM11 with FAAH. In the presence of fatty acid-free bovine serum albumin (BSA, 0.125% w v(-1)), both AM404 and VDM11 inhibited the metabolism of AEA by rat brain FAAH with similar potencies (IC(50) values of 2.1 and 2.6 microM, respectively). The compounds were about 10-fold less potent as inhibitors of the metabolism of 2-oleoylglycerol (2-OG) by cytosolic monoacylglycerol lipase (MAGL). The potency of VDM11 towards FAAH was dependent upon the assay concentration of fatty acid-free bovine serum albumin (BSA). Thus, in the absence of fatty acid-free BSA, the IC(50) value for inhibition of FAAH was reduced by a factor of about two (from 2.9 to 1.6 microM). A similar reduction in the IC(50) value for the inhibition of membrane bound MAGL by both this compound (from 14 to 6 microM) and by arachidonoyl serinol (from 24 to 13 microM) was seen. An HPLC assay was set up to measure 4-amino-m-cresol, the hypothesised product of FAAH-catalysed VDM11 hydrolysis. 4-Amino-m-cresol was eluted with a retention time of approximately 2.4 min, but showed a time-dependent degradation to compounds eluting at peaks of approximately 5.6 and approximately 8 min. Peaks with the same retention times were also found following incubation of the membranes with VDM11, but were not seen when the membranes were preincubated with the FAAH inhibitors URB597 (3'-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate) and CAY10401 (1-oxazolo[4,5-b]pyridin-2-yl-9-octadecyn-1-one) prior to addition of VDM11. The rate of metabolism of VDM11 was estimated to be roughly 15-20% of that for anandamide. It is concluded that VDM11 is an inhibitor of FAAH under the assay conditions used here, and that the inhibition may at least in part be a consequence of the compound acting as an alternative substrate.

Amidohydrolases↗
PubMed2005

Anandamide reduces infarct size in rat isolated hearts subjected to ischaemia-reperfusion by a novel cannabinoid mechanism.

Although the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide share a similar pharmacology, 2-AG reportedly limits myocardial ischaemia-reperfusion injury whereas anandamide does not. We therefore investigated whether or not anandamide reduces infarct size and which, if any, of the known cannabinoid-signalling pathways are involved. Rat isolated perfused hearts were subjected to global, no-flow ischaemia (30 min) and reperfusion (1 h). Agonists were present from 5 min before ischaemia until the end of reperfusion. Antagonists, where used, were present throughout the protocol. Recovery of left ventricular developed pressure and coronary flow was incomplete in control hearts and not significantly affected by any drug treatment. In vehicle-treated hearts, 26+/-3% (n=13) of the left ventricle was infarcted at the end of reperfusion. Infarction of the left ventricle was significantly reduced after 1 microM anandamide (10+/-1%, n=7) or 1 microM methanandamide (12+/-4%, n=6) but not 1 microM HU210. Neither ACPA (1 microM; CB1 receptor agonist) nor JWH133 (1 microM; CB2 receptor agonist), individually or combined significantly affected infarct size. Anandamide (1 microM) did not reduce infarct size in the presence of the CB1 receptor antagonist rimonabant (SR141716A, 1 microM) or the CB2 receptor antagonist, SR144528 (1 microM). Despite sensitivity to CB1 and CB2 receptor antagonists, the infarct-limiting action of anandamide was not mimicked by agonists selective for CB1 or CB2 receptors suggesting the involvement of a novel cannabinoid site of action.

Animals↗
PubMed2003

The endocannabinoid system, anandamide and the regulation of mammalian cell apoptosis.

Endocannabinoids are a new class of lipid mediators, which include amides, esters and ethers of long-chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol (2-AG) are the main endogenous agonists of cannabinoid receptors able to mimic several pharmacological effects of Delta-9-tetrahydrocannabinol, the active principle of Cannabis sativa preparations like hashish and marijuana. The pathways leading to the synthesis and release of AEA and 2-AG from neuronal and non-neuronal cells are still rather uncertain. Instead, it is known that the activity of AEA is limited by cellular uptake through a specific membrane transporter, followed by intracellular degradation by a fatty acid amide hydrolase. Together with AEA and congeners these proteins form the 'endocannabinoid system'. Here, the involvement of AEA in apoptosis and the underlying signal transduction pathways will be reviewed, along with the metabolic routes and the molecular targets of this endocannabinoid. Also, recent findings on the apoptotic potential of AEA for neuronal cell differentiation and brain development will be discussed.

Animals↗
PubMed2007

Circulating endocannabinoid levels, abdominal adiposity and related cardiometabolic risk factors in obese men.

OBJECTIVE: The link between excess intra-abdominal adiposity (IAA) and metabolic complications leading to type 2 diabetes and cardiovascular disease is well recognized. Blockade of endocannabinoid action at cannabinoid CB(1) receptors was shown to reduce these complications. Here, we investigated the relationship between IAA, circulating endocannabinoid levels and markers of cardiometabolic risk in male obese subjects. DESIGN, SUBJECTS AND MEASUREMENTS: Fasting plasma levels of the endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), were measured by liquid chromatography-mass spectrometry in a study sample of 62 untreated asymptomatic men with body mass index (BMI) from 18.7 to 35.2 kg/m(2). RESULTS: Plasma 2-AG, but not AEA, levels correlated positively with BMI, waist girth, IAA measured by computed tomography, and fasting plasma triglyceride and insulin levels, and negatively with high-density lipoprotein cholesterol and adiponectin levels. Obese men with similar BMI values (> or =30 kg/m(2)) but who markedly differed in their amount of IAA (< vs > or = 130 cm(2), n=17) exhibited higher 2-AG levels in the presence of high IAA. No difference in 2-AG concentrations was observed between obese men with low levels of IAA vs nonobese controls. CONCLUSIONS: These results provide evidence for a relationship in men between a key endocannabinoid, 2-AG, and cardiometabolic risk factors, including IAA.

Adiponectin↗
PubMed2006

Role of endocannabinoids in alcohol consumption and intoxication: studies of mice lacking fatty acid amide hydrolase.

Endocannabinoid signaling plays the important role in regulation of ethanol intake. Fatty acid amide hydrolase (FAAH) is a key membrane protein for metabolism of endocannabinoids, including anandamide, and blockade of FAAH increases the level of anandamide in the brain. To determine if FAAH regulates ethanol consumption, we studied mutant mice with deletion of the FAAH gene. Null mutant mice showed higher preference for alcohol and voluntarily consumed more alcohol than wild-type littermates. There was no significant difference in consumption of sweet or bitter solutions. To determine the specificity of FAAH for ethanol intake, we studied additional ethanol-related behaviors. There were no differences between null mutant and wild-type mice in severity of ethanol-induced acute withdrawal, conditioned taste aversion to alcohol, conditioned place preference, or sensitivity to hypnotic effect of ethanol. However, null mutant mice showed shorter duration of loss of righting reflex induced by low doses of ethanol (3.2 and 3.4 g/kg) and faster recovery from motor incoordination induced by ethanol. All three behavioral phenotypes (increased preference for ethanol, decreased sensitivity to ethanol-induced sedation, and faster recovery from ethanol-induced motor incoordination) seen in mutant mice were reproduced in wild-type mice by injection of a specific inhibitor of FAAH activity--URB597. These data suggest that increased endocannabinoid signaling increased ethanol consumption owing to decreased acute ethanol intoxication.

Alcohol-Induced Disorders, Nervous System↗
PubMed2000

Down-regulation of anandamide hydrolase in mouse uterus by sex hormones.

Endocannabinoids are an emerging class of lipid mediators, which mimic several effects of cannabinoids. Anandamide (arachidonoylethanolamide) is a major endocannabinoid, which has been shown to impair pregnancy and embryo development. The activity of anandamide is controlled by cellular uptake through a specific transporter and intracellular degradation by the enzyme anandamide hydrolase (fatty acid amide hydrolase, FAAH). We characterized FAAH in mouse uterus by radiochromatographic and immunochemical techniques, showing that the enzyme is confined to the epithelium and its activity decreases appreciably during pregnancy or pseudopregnancy because of lower gene expression at the translational level. Ovariectomy prevented the decrease in FAAH, and both progesterone and estrogen further reduced its basal levels, suggesting hormonal control of the enzyme. Anandamide was shown to induce programmed cell death in mouse blastocysts, through a pathway independent of type-1 cannabinoid receptor. Blastocysts, however, have a specific anandamide transporter and FAAH, which scavenge this lipid. Taken together, these results provide evidence of an interplay between endocannabinoids and sex hormones in pregnancy. These findings may also be relevant for human fertility, as epithelial cells from healthy human uterus showed FAAH activity and expression, which in adenocarcinoma cells was increased fivefold.

Adenocarcinoma↗
PubMed1999

Biosynthesis and inactivation of the endocannabinoid 2-arachidonoylglycerol in circulating and tumoral macrophages.

The stimulus-induced biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) in intact mouse J774 macrophages and the inactivation of 2-AG by the same cells or by rat circulating macrophages was studied. By using gas chromatography-mass spectrometry, we found that ionomycin (5 microM) and lipopolysaccharide (LPS, 200 microg x mL-1) cause a 24-fold and 2.5-fold stimulation of 2-AG levels in J774 cells, respectively, thus providing unprecedented evidence that this cannabimimetic metabolite can be synthesized by macrophages. In J774 cells, LPS also induced a 7.8-fold increase of the levels of the other endocannabinoid, anandamide, and, in rat circulating macrophages, an almost twofold increase of 2-AG levels. Extracellular [3H]2-AG was cleared from the medium of intact J774 macrophages (t1/2 = 19-28 min) and esterified to phospholipids, diacylglycerols and triglycerides or hydrolyzed to [3H]arachidonic acid and glycerol. These catabolic processes were attenuated differentially by various enzyme inhibitors. Rat circulating macrophages were shown to contain enzymatic activities for the hydrolysis of 2-AG, including: (a) fatty acid amide hydrolase (FAAH), the enzyme responsible for anandamide breakdown and previously shown to catalyse also 2-AG hydrolysis, and (b) a 2-AG hydrolase activity different from FAAH and down-regulated by LPS. High levels of FAAH mRNA were found in circulating macrophages but not platelets, which, however, contain a 2-AG hydrolase. Both platelets and macrophages were shown to express the mRNA for the CB1 cannabinoid receptor. A macrophage 2-AG hydrolase with apparent Km = 110 microM and Vmax = 7.9 nmol x min-1 x (mg protein)-1 was partially characterized in J774 cells and found to exhibit an optimal pH of 6-7 and little or no sensitivity to typical FAAH inhibitors. These findings demonstrate for the first time that macrophages participate in the homeostasis of the hypotensive and immunomodulatory endocannabinoid 2-AG through metabolic mechanisms that are subject to regulation.

Animals↗
PubMed2003

Postsynaptic M1 and M3 receptors are responsible for the muscarinic enhancement of retrograde endocannabinoid signalling in the hippocampus.

The cholinergic system is crucial for higher brain functions including learning and memory. These functions are mediated primarily by muscarinic acetylcholine receptors (mAChRs) that consist of five subtypes (M(1)-M(5)). A recent study suggested a novel role of acetylcholine as a potent enhancer of endocannabinoid signalling that acts retrogradely from postsynaptic to presynaptic neurons. In the present study, we further investigated the mechanisms of this cholinergic effect on endocannabinoid signalling. We made paired whole-cell recordings from cultured hippocampal neurons, and monitored inhibitory postsynaptic currents (IPSCs). The postsynaptic depolarization induced a transient suppression of IPSCs (DSI), a phenomenon known to involve retrograde signalling by endocannabinoids. The cholinergic agonist carbachol (CCh) markedly enhanced DSI at 0.01-0.3 microM without changing the presynaptic cannabinoid sensitivity. The facilitating effect of CCh on DSI was mimicked by the muscarinic agonist oxotremorine-M, whereas it was eliminated by the muscarinic antagonist atropine. It was also blocked by a non-hydrolizable analogue of GDP (GDP-beta-S) that was applied intracellularly to postsynaptic neurons. The muscarinic enhancement of DSI persisted to a substantial degree in the neurons prepared from M1-knockout and M3-knockout mice, but was virtually eliminated in the neurons from M1/M3-compound-knockout mice. CCh still enhanced DSI significantly under the blockade of postsynatpic K(+) conductance, and did not significantly influence the depolarization-induced Ca(2+) transients. These results indicate that the activation of postsynaptic M1 and M3 receptors facilitates the depolarization-induced release of endocannabinoids.

Animals↗
PubMed1999

Phosphatidic acid as the biosynthetic precursor of the endocannabinoid 2-arachidonoylglycerol in intact mouse neuroblastoma cells stimulated with ionomycin.

In mouse neuroblastoma N18TG2 cells prelabeled with [3H]arachidonic acid ([3H]AA) the biosynthesis of 2-arachidonoylglycerol (2-AG) is induced by ionomycin in a fashion sensitive to an inhibitor of diacylglycerol (DAG) lipase, RHC 80267, but not to four different phospholipase C (PLC) blockers. Pulse experiments with [3H]AA showed that ionomycin stimulation leads to the sequential formation of [3H]phosphatidic acid ([3H]PA), [3H]DAG, and [3H]2-AG. [3H]2-AG biosynthesis in N18TG2 cells prelabeled with [3H]AA was counteracted by propranolol and N-ethylmaleimide, two inhibitors of the Mg2+/Ca2(+)-dependent brain PA phosphohydrolase. Pretreatment of cells with exogenous phospholipase D (PLD) led to a strong potentiation of ionomycin-induced [3H]2-AG formation. These data indicate that DAG precursors for 2-AG in intact N18TG2 cells are obtained from the hydrolysis of PA and not through the activation of PLC. The presence of 2% ethanol during ionomycin stimulation failed to elicit the synthesis of [3H]phosphatidylethanol and did not counteract the formation of [3H]PA, thus arguing against the activation of PLD by the Ca2+ ionophore. Selective inhibitors of secretory phospholipase A2 and the acyl-CoA acylase inhibitor thimerosal significantly reduced [3H]2-AG biosynthesis. The implications of these latter findings, and of the PA-dependent pathways of 2-AG formation described here, are discussed.

Animals↗
PubMed2003

Membrane cholesterol but not putative receptors mediates anandamide-induced hepatocyte apoptosis.

The endogenous cannabinoid anandamide, a lipid mediator, induces various physiologic events such as vascular relaxation, inhibition of gap-junctions formation, tumor proliferation, neurologic analgesia, and apoptosis. Although increased concentration of anandamide in plasma has been implicated in pathophysiologic states including endotoxin-induced hypotension, the effects of anandamide on hepatocytes still remain unclear. In this study, we present evidence that plasma anandamide concentration is highly increased in severe hepatitis and cirrhosis patients. In addition, concentrations of anandamide within the pathophysiologic range potently induced apoptosis of hepatoma cell line (Hep G2) and primary hepatocytes, suggesting a possible link between increased anandamide level and hepatocyte damage. Anandamide-induced cell death was preceded by G0/G1 cell-cycle arrest, activation of proapoptotic signaling (i.e., p38 MAPK and JNK), and inhibition of antiapoptotic signaling (i.e., PKB/Akt) pathways. Moreover, anandamide increased susceptibility to oxidative stress-induced hepatocyte damage. In this context, methyl-beta-cyclodextrin (MCD), a membrane cholesterol depletor, or mevastatin, an HMG-CoA reductase inhibitor, or N-acetyl cysteine, an antioxidant, potently inhibited the anandamide-induced proapoptotic events and cell death, whereas putative cannabinoid receptor antagonists did not exhibit an inhibitory effect on anandamide-induced cell death. Furthermore, binding assay using polymyxin beads revealed that anandamide could interact with cholesterol. In conclusion, our data suggest that cholesterol present in the cell membrane determines the fate of hepatocytes exposed to anandamide, possibly functioning as an anandamide receptor.

Animals↗
PubMedDate not supplied

The fatty acid amide hydrolase (FAAH).

The fatty acid amide hydrolase (FAAH), is the enzyme responsible for the hydrolysis of anandamide, an endocannabinoid. The FAAH knockout, the assays for FAAH, the activity of its substrates, its reversibility and its cloning from rat, mouse, human, and pig are covered in this review. The conserved regions of FAAH are described in terms of sequence and function, including the domains that contains the serine catalytic nucleophile, the hydrophobic domain important for self-association, the proline rich domain region which may be important for subcellular localization and the fatty acid chain binding domain. The FAAH mouse promoter region was characterized in terms of its transcription start site and its activity in different cell types. The distribution of FAAH in the major organs in the body is described as well as regional distribution in the brain and its correlation with cannabinoid receptors. Since FAAH is recognized as a drug target, a large number of inhibitors have been synthesized and tested since 1994 and these are reviewed in terms of reversibility, potency, and specificity for FAAH.

Amidohydrolases↗
PubMed2004

Alcohol inhibits luteinizing hormone-releasing hormone release by activating the endocannabinoid system.

We hypothesized that ethanol (EtOH) might act through the endocannabinoid system to inhibit luteinizing hormone-releasing hormone (LHRH) release. Therefore, we examined the mechanism by which EtOH and anandamide (AEA), an endogenous cannabinoid, inhibit LHRH release from incubated medial basal hypothalamic explants. In previous work, we demonstrated that EtOH inhibits the N-methyl-D-aspartic acid-stimulated release of LHRH by increasing the release of two neurotransmitters: beta-endorphin and gamma-aminobutyric acid (GABA). In the present work, bicuculline, a GABAergic antagonist, completely prevented the inhibition of AEA (10(-9)M) on N-methyl-D-aspartic acid-induced LHRH release, but naltrexone, a micro-opioid receptor antagonist, had no effect. AEA also significantly increased GABA release but had no effect on beta-endorphin release. Therefore, AEA could inhibit LHRH release by increasing GABA but not beta-endorphin release. Because EtOH and AEA acted similarly to inhibit LHRH release, we investigated whether both substances would affect the adenylate cyclase activity acting through the same GTP-coupled receptors, the cannabinoid receptors 1 (CB1-rs). AEA and EtOH (10(-1)M) reduced the forskolin-stimulated accumulation of cAMP, but AM251, a specific antagonist of CB1-r, significantly blocked that inhibition. Additionally we investigated whether CB1-r is involved in the inhibition of LHRH by EtOH and AEA. AEA and EtOH reduced forskolin-stimulated LHRH release, but AM251 significantly blocked that inhibition. Also, we demonstrated that EtOH did not act by increasing AEA synthase activity to inhibit LHRH release in our experimental conditions. Therefore, our results indicate that EtOH inhibits the release of LHRH acting through the endocannabinoid system.

Animals↗
PubMed2004

Anandamide transport is independent of fatty-acid amide hydrolase activity and is blocked by the hydrolysis-resistant inhibitor AM1172.

The endogenous cannabinoid anandamide is removed from the synaptic space by a high-affinity transport system present in neurons and astrocytes, which is inhibited by N-(4-hydroxyphenyl)-arachidonamide (AM404). After internalization, anandamide is hydrolyzed by fatty-acid amide hydrolase (FAAH), an intracellular membrane-bound enzyme that also cleaves AM404. Based on kinetic evidence, it has recently been suggested that anandamide internalization may be mediated by passive diffusion driven by FAAH activity. To test this possibility, in the present study, we have investigated anandamide internalization in wild-type and FAAH-deficient (FAAH(-/-)) mice. Cortical neurons from either mouse strain internalized [(3)H]anandamide through a similar mechanism, i.e., via a rapid temperature-sensitive and saturable process, which was blocked by AM404. Moreover, systemic administration of AM404 to either wild-type or FAAH(-/-) mice enhanced the hypothermic effects of exogenous anandamide, a response that was prevented by the CB(1) cannabinoid antagonist rimonabant (SR141716A). The results indicate that anandamide internalization in mouse brain neurons is independent of FAAH activity. In further support of this conclusion, the compound N-(5Z, 8Z, 11Z, 14Z eicosatetraenyl)-4-hydroxybenzamide (AM1172) blocked [(3)H]anandamide internalization in rodent cortical neurons and human astrocytoma cells without acting as a FAAH substrate or inhibitor. AM1172 may serve as a prototype for novel anandamide transport inhibitors with increased metabolic stability.

Amidohydrolases↗
PubMed2002

A missense mutation in human fatty acid amide hydrolase associated with problem drug use.

Problem drug use and dependence are neurobehavioral disorders of complex origin. Although environmental factors contribute to drug abuse and addiction, genetic factors also play a significant role estimated at 40-60% of the total risk. Nonetheless, the precise identities of human genes that confer vulnerability to problem drug use remain mostly unknown. Here, we describe a natural single nucleotide polymorphism in the human gene that encodes the principal endocannabinoid-inactivating enzyme, fatty acid amide hydrolase (FAAH), that in homozygous form is strongly associated with both street drug use and problem drug/alcohol use. This single nucleotide polymorphism results in a missense mutation (385C-->A) that converts a conserved proline residue to threonine (Pro129-->Thr), producing a FAAH variant that displays normal catalytic properties but an enhanced sensitivity to proteolytic degradation. Collectively, these results suggest that genetic mutations in FAAH may constitute important risk factors for problem drug use and support a potential link between functional abnormalities in the endogenous cannabinoid system and drug abuse and dependence.

Alcohol Drinking↗