Hemodynamic profile, responsiveness to anandamide, and baroreflex sensitivity of mice lacking fatty acid amide hydrolase

The endocannabinoid anandamide exerts neurobehavioral, cardiovascular, and immune-regulatory effects through cannabinoid receptors (CB). Fatty acid amide hydrolase (FAAH) is an enzyme responsible for the in vivo degradation of anandamide. Recent experimental studies have suggested that targeting the endocannabinergic system by FAAH inhibitors is a promising novel approach for the treatment of anxiety, inflammation, and hypertension. In this study, we compared the cardiac performance of FAAH knockout (FAAH-/-) mice and their wild-type (FAAH+/+) littermates and analyzed the hemodynamic effects of anandamide using the Millar pressure-volume conductance catheter system. Baseline cardiovascular parameters, systolic and diastolic function at different preloads, and baroreflex sensitivity were similar in FAAH-/- and FAAH+/+ mice. FAAH-/- mice displayed increased sensitivity to anandamide-induced, CB1-mediated hypotension and decreased cardiac contractility compared with FAAH(+/+) littermates. In contrast, the hypotensive potency of synthetic CB1 agonist HU-210 and the level of expression of myocardial CB1 were similar in the two strains. The myocardial levels of anandamide and oleoylethanolamide, but not 2-arachidonylglycerol, were increased in FAAH-/- mice compared with FAAH+/+ mice. These results indicate that mice lacking FAAH have a normal hemodynamic profile, and their increased responsiveness to anandamide-induced hypotension and cardiodepression is due to the decreased degradation of anandamide rather than an increase in target organ sensitivity to CB1 agonists.     

Human adipose tissue binds and metabolizes the endocannabinoids anandamide and 2-arachidonoylglycerol

Endocannabinoids are a group of biologically active endogenous lipids that have recently emerged as important mediators in energy balance control. The two best studied endocannabinoids, anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG) are the endogenous ligands of the central and peripheral cannabinoid receptors. Furthermore, AEA binds to the transient receptor potential vanilloid type-1 (TRPV1), a capsaicin-sensitive, non-selective cation channel. The synthesis of these endocannabinoids is catalyzed by the N-acylphosphatidylethanolamine-selective phospholipase D (NAPE-PLD) and the sn-1-selective diacylglycerol lipase (DAGL), whereas their degradation is accomplished by the fatty acid amide hydrolase (FAAH) and the monoglyceride lipase (MGL), respectively. We investigated the presence of a functional endocannabinoid system in human adipose tissue from seven healthy subjects. Subcutaneous abdominal adipose tissue underwent biochemical and molecular biology analyses, aimed at testing the expression of this system and its functional activity. AEA and 2-AG levels were detected and quantified by HPLC. Real time PCR analyzed the expression of the endocannabinoid system and immunofluorescence assays showed the distribution of its components in the adipose tissue. Furthermore, binding assay for the cannabinoid and vanilloid receptors and activity assay for each metabolic enzyme of the endocannabinoid system gave clear evidence of a fully operating system. The data presented herein show for the first time that the human adipose tissue is able to bind AEA and 2-AG and that it is endowed with the biochemical machinery to metabolize endocannabinoids.     

Hydrolysis of prostaglandin glycerol esters by the endocannabinoid-hydrolyzing enzymes, monoacylglycerol lipase and fatty acid amide hydrolase

Cyclooxygenase-2 (COX-2) can oxygenate the endocannabinoids, arachidonyl ethanolamide (AEA) and 2-arachidonylglycerol (2-AG), to prostaglandin-H2-ethanolamide (PGH2-EA) and -glycerol ester (PGH2-G), respectively. Further metabolism of PGH2-EA and PGH2-G by prostaglandin synthases produces a variety of prostaglandin-EA's and prostaglandin-G's nearly as diverse as those derived from arachidonic acid. Thus, COX-2 may regulate endocannabinoid levels in neurons during retrograde signaling or produce novel endocannabinoid metabolites for receptor activation. Endocannabinoid-metabolizing enzymes are important regulators of their action, so we tested whether PG-G levels may be regulated by monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH). We found that PG-Gs are poor substrates for purified MGL and FAAH compared to 2-AG and/or AEA. Determination of substrate specificity demonstrates a 30-100- and 150-200-fold preference of MGL and FAAH for 2-AG over PG-Gs, respectively. The substrate specificity of AEA compared to those of PG-Gs was approximately 200-300 fold higher for FAAH. Thus, PG-Gs are poor substrates for the major endocannabinoid-degrading enzymes, MGL and FAAH.     

Critical enzymes involved in endocannabinoid metabolism

Investigations of the pathways involved in the metabolism of endocannabinoids have grown exponentially in recent years following the discovery of cannabinoid receptors (CB) and their endogenous ligands, such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG). The in vivo biosynthesis of AEA has been shown to occur through several pathways mediated by N-acylphosphatidylethanolamide-phospholipase D (NAPE-PLD), a secretory PLA(2) and PLC. 2-AG, a second endocannabinoid is generated through the action of selective enzymes such as phosphatidic acid phsophohydrolase, diacylglycerol lipase (DAGL), phosphoinositide-specific PLC (PI-PLC) and lyso-PLC. A putative membrane transporter or facilitated diffusion is involved in the cellular uptake or release of endocannabinoids. AEA is metabolized by fatty acid amidohydrolase (FAAH) and 2-AG is metabolized by both FAAH and monoacylglycerol lipase (MAGL). The author presents an integrative overview of current research on the enzymes involved in the metabolism of endocannabinoids and discusses possible therapeutic interventions for various diseases, including addiction.     

The postmortal accumulation of brain N-arachidonylethanolamine (anandamide) is dependent upon fatty acid amide hydrolase activity

N-arachidonylethanolamine (AEA) accumulates during brain injury and postmortem. Because fatty acid amide hydrolase (FAAH) regulates brain AEA content, the purpose of this study was to determine its role in the postmortal accumulation of AEA using FAAH null mice. As expected, AEA content in immediately frozen brain tissue was significantly greater in FAAH-deficient (FAAH-/-) than in wild-type mice. However, AEA content was significantly lower in brains from FAAH-/- mice at 5 and 24 h postmortem. Similarly, wild-type mice treated in vivo with a FAAH inhibitor (URB532) had significantly lower brain AEA content 24 h postmortem compared with controls. These data indicate that FAAH contributes significantly to the postmortal accumulation of AEA. In contrast, the accumulations of two other N-acylethanolamines, N-oleoylethanolamine (OEA) and N-palmitoylethanolamine (PEA), were not reduced at 24 h postmortem in either the FAAH-/- mice or mice treated with URB532. FAAH-/- mice accumulated significantly less ethanolamine at 24 h postmortem compared with wild-type mice, suggesting that FAAH activity plays a role in the accumulation of ethanolamine postmortem. These data demonstrate that FAAH activity differentially affects AEA and OEA/PEA contents postmortem and suggest that AEA formation specifically occurs via an ethanolamine-dependent route postmortem.     

Lipid Droplets Are Novel Sites of N-Acylethanolamine Inactivation by Fatty Acid Amide Hydrolase-2

Anandamide (AEA) and other bioactive N-acylethanolamines (NAEs) are primarily inactivated by the enzyme fatty acid amide hydrolase (FAAH). Recently, FAAH-2 was discovered in humans, suggesting an additional enzyme can mediate NAE inactivation in higher mammals. Here, we performed a biochemical characterization of FAAH-2 and explored its capacity to hydrolyze NAEs in cells. In homogenate activity assays, FAAH-2 hydrolyzed AEA and palmitoylethanolamide (PEA) with activities ∼6 and ∼20% those of FAAH, respectively. In contrast, FAAH-2 hydrolyzed AEA and PEA in intact cells with rates ∼30–40% those of FAAH, highlighting a potentially greater contribution toward NAE catabolism in vivo than previously appreciated. In contrast to endoplasmic reticulum-localized FAAH, immunofluorescence revealed FAAH-2 was localized on lipid droplets. Supporting this distribution pattern, the putative N-terminal hydrophobic region of FAAH-2 was identified as a functional lipid droplet localization sequence. Lipid droplet localization was essential for FAAH-2 activity as chimeras excluded from lipid droplets lacked activity and/or were poorly expressed. Lipid droplets represent novel sites of NAE inactivation. Therefore, we examined substrate delivery to these organelles. AEA was readily trafficked to lipid droplets, confirming that lipid droplets constitute functional sites of NAE inactivation. Collectively, these results establish FAAH-2 as a bone fide NAE-catabolizing enzyme and suggest that NAE inactivation is spatially separated in cells of higher mammals.     

Differential regulation of endocannabinoid synthesis and degradation in the uterus during embryo implantation

Preimplantation embryo development to the blastocyst stage and uterine differentiation to the receptive state are prerequisites for embryo implantation. Burgeoning evidence suggests that endocannabinoid signaling is critical to early pregnancy events. Anandamide (N-arachidonoylethanolamine) and 2-AG (2-arachidonoylglycerol) are two major endocannabinoids that bind to and activate G-protein coupled cannabinoid receptors CB1 and CB2. We have previously shown that a physiological tone of anandamide is critical to preimplantation events in mice, since either silencing or amplification of anandamide signaling causes retarded development and oviductal retention of embryos via CB1, leading to deferred implantation and compromised pregnancy outcome. Whether 2-AG, which also influences many biological functions, has any effects on early pregnancy remains unknown. Furthermore, mechanisms by which differential uterine endocannabinoid gradients are established under changing pregnancy state is not clearly understood. We show here that 2-AG is present at levels one order of magnitude higher than those of anandamide in the mouse uterus, but with similar patterns as anandamide, i.e. lower levels at implantation sites and higher at interimplantation sites. We also provide evidence that region- and stage-specific uterine expression of N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD) and fatty acid amide hydrolase (FAAH), and sn-1-diacylglycerol (DAG) lipase alpha (DAGLalpha) and monoacylglycerol lipase (MAGL) for synthesis and hydrolysis of anandamide and 2-AG, respectively, creates endocannabinoid gradients conducive to implantation. Our genetic evidence suggests that FAAH is the major degrading enzyme for anandamide, whereas COX-2, MAGL and to some extent COX-1 participate in metabolizing 2-AG in the pregnant uterus. The results suggest that aberrant functioning of these pathways impacting uterine anandamide and/or 2-AG levels would compromise pregnancy outcome.     

Release of Endocannabinoids into the Cerebrospinal Fluid during the Induction of the Trigemino-Hypoglossal Reflex in Rats

The endocannabinoid system (ECS) plays an important role in pain processing and modulation. Since the specific effects of endocannabinoids within the orofacial area are largely unknown, we aimed to determine whether an increase in the endocannabinoid concentration in the cerebrospinal fluid (CSF) caused by the peripheral administration of the FAAH inhibitor URB597 and tooth pulp stimulation would affect the transmission of impulses between the sensory and motor centers localized in the vicinity of the third and fourth cerebral ventricles. The study objectives were evaluated on rats using a method that allowed the recording of the amplitude of evoked tongue jerks (ETJ) in response to noxious tooth pulp stimulation and URB597 treatment. The amplitude of ETJ was a measure of the effect of endocannabinoids on the neural structures. The concentrations of the endocannabinoids tested (AEA and 2-AG) were determined in the CSF, along with the expression of the cannabinoid receptors (CB1 and CB2) in the tissues of the mesencephalon, thalamus, and hypothalamus. We demonstrated that anandamide (AEA), but not 2-arachidonoylglycerol (2-AG), was significantly increased in the CSF after treatment with a FAAH inhibitor, while tooth pulp stimulation had no effect on the AEA and 2-AG concentrations in the CSF. We also found positive correlations between the CSF AEA concentration and cannabinoid receptor type 1 (CB1R) expression in the brain, and between 2-AG and cannabinoid receptor type 2 (CB2R), and negative correlations between the CSF concentration of AEA and brain CB2R expression, and between 2-AG and CB1R. Our study shows that endogenous AEA, which diffuses through the cerebroventricular ependyma into CSF and exerts a modulatory effect mediated by CB1Rs, alters the properties of neurons in the trigeminal sensory nuclei, interneurons, and motoneurons of the hypoglossal nerve. In addition, our findings may be consistent with the emerging concept that AEA and 2-AG have different regulatory mechanisms because they are involved differently in orofacial pain. We also suggest that FAAH inhibition may offer a therapeutic approach to the treatment of orofacial pain.     

Effects of cannabinoids on LPS-stimulated inflammatory mediator release from macrophages: involvement of eicosanoids

Delta(9)-Tetrahydrocannabinol (Delta(9)-THC) is the major psychoactive component of marijuana and elicits pharmacological actions via cannabinoid receptors. Anandamide (AEA) and 2-arachidonoyl-glycerol (2-AG) are endogenous ligands for cannabinoid receptors, which because of their structural similarities to arachidonic acid (AA), AEA, and 2-AG could serve as substrates for lipoxygenases and cyclooxygenases (COXs) that metabolize polyunsaturated fatty acids to potent bioactive molecules. In this study, we have compared the effects of Delta(9)-THC, AEA, 2-AG, and another cannabinoid agonist, indomethacin morpholinylamide (IMMA), on lipopolysaccharide (LPS)-induced NO, IL-6, and PGE(2) release from J774 macrophages. Delta(9)-THC, IMMA, and AEA diminish LPS-induced NO and IL-6 production in a concentration-dependent manner. 2-AG inhibits the production of IL-6 but slightly increases iNOS-dependent NO production. Delta(9)-THC and IMMA also inhibit LPS-induced PGE(2) production and COX-2 induction, while AEA and 2-AG have no effects. These discrepant results of 2-AG on iNOS and COX-2 induction might be due to its bioactive metabolites, AA and PGE(2), whose incubation cause the potentiation of both iNOS and COX-2 induction. On the contrary, the AEA metabolite, PGE(2)-ethanolamide, influences neither the LPS-induced NO nor IL-6 production. Taken together, direct cannabinoid receptor activation leads to anti-inflammatory action via inhibition of macrophage function. The endogenous cannabinoid, 2-AG, also serves as a substrate for COX-catalyzing PGE(2) production, which in turn modulates the action of CB2.     

Symptom-related changes of endocannabinoid and palmitoylethanolamide levels in brain areas of R6/2 mice, a transgenic model of Huntington's disease

Previous studies have shown an impairment of the endocannabinoid system in experimental models of Huntington's disease. In transgenic R6/2 mice, created by inserting exon 1 of the human IT15 mutant gene into the mouse, and exhibiting 150 CAG repeats as well as signs of HD, a progressive decline of CB(1) receptor expression and an abnormal sensitivity to CB(1) receptor stimulation have been reported. Here, by using isotope-dilution liquid chromatography-mass spectrometry, we investigated whether the levels of three endogenous neuroprotective substances, the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG), and palmitoylethanolamide (PEA), are altered in different brain areas of transgenic R6/2 versus wild-type (WT) mice at two different disease phases, i.e. in pre-symptomatic (4.5 weeks) or overtly symptomatic (10 weeks) R6/2 mice versus age-matched WT mice (n=4/group). Except for a approximately 25% decrease in 2-AG levels in the cortex, no significant changes in endocannabinoid and PEA levels were observed in pre-symptomatic R6/2 versus WT mice. By contrast, in symptomatic R6/2 mice the levels of all three compounds were significantly (approximately 30-60%) decreased in the striatum, whereas little changes were observed in the hippocampus, and a approximately 28% decrease of 2-AG levels, accompanied by a approximately 50% increase of AEA levels, was found in the cortex. These findings show that endocannabinoid levels change in a disease phase- and region-specific way in the brain of R6/2 mice and indicate that an impaired endocannabinoid system is a hallmark of symptomatic HD, thus suggesting that drugs inhibiting endocannabinoid degradation might be used to treat this disease.     

Enhanced anandamide degradation is associated with neuronal apoptosis induced by the HIV-1 coat glycoprotein gp120 in the rat neocortex

Human immunodeficiency virus type-1 coat glycoprotein gp120 causes delayed apoptosis in rat brain neocortex. Here, we investigated the possible role of the endocannabinoid system in this process. It is shown that gp120 causes a time-dependent increase in the activity and immunoreactivity of the anandamide (AEA)-hydrolyzing enzyme fatty acid amide hydrolase (FAAH), paralleled by increased activity of the AEA membrane transporter and decreased endogenous levels of AEA. The AEA-synthesizing phospholipase D and the AEA-binding receptors were not affected by gp120. None of the changes induced by gp120 in the cortex were induced by bovine serum albumin, nor were they observed in the hippocampus of the same animals. Also, the activity of 5-lipoxygenase, which generates AEA derivatives able to inhibit FAAH, decreased down to approximately 25% of the control activity upon gp120 treatment, due to reduced protein level ( approximately 45%). In addition, the FAAH inhibitor methyl-arachidonoyl fluorophosphonate significantly reduced gp120-induced apoptosis in rat brain neocortex, whereas selective blockers of AEA membrane transporter or of AEA-binding receptors were ineffective. Taken together, these results suggest that gp120, by activating FAAH, decreases endogenous levels of AEA, and the latter effect seems instrumental in the execution of delayed neuronal apoptosis in the brain neocortex of rats.     

An endocannabinoid system is localized to the hypophysial pars tuberalis of Syrian hamsters and responds to photoperiodic changes

The hypophysial pars tuberalis (PT), an important interface between neuroendocrine brain centers (hypothalamus, pineal organ) and the pars distalis (PD) of the hypophysis, plays a central role in regulating seasonal reproduction and prolactin release. However, the signaling molecules that transmit photoperiodic information from the PT to the PD and control prolactin release (the so-called “tuberalins”) have not yet been identified, despite an intense search for more than three decades. Here, we demonstrate an endocannabinoid system in the PT of the Syrian hamster, a photoperiodic species. By means of in situ hybrization, the PT was found to express N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD), fatty acid amide hydrolase (FAAH), sn-1-selective diacylglycerol lipases (DAGLα and DAGLβ), and monoacylglycerol lipase (MAGL), enzymes involved in endocannabinoid synthesis and degradation. The expression of NAPE-PLD, FAAH, and DAGLα was confirmed by immunohistochemistry. Expression and protein levels of DAGLs controlling the synthesis of 2-arachidonoyl glycerol (2-AG), a major endocannabinoid, were upregulated in the PT of Syrian hamsters kept under long-day conditions. Consequently, 2-AG levels were increased in the PT of these hamsters. A primary target of 2-AG, the cannabinoid receptor 1 (CB1), was expressed in the PD. Double-immunolabeling revealed that most of the CB1-immunoreactive cells in the PD were folliculostellate cells that were also immunoreactive for S-100 protein. Thus, the PT comprises an endocannabinoid system, and 2-AG may act as a photoperiodic messenger from the PT to the PD for the regulation of hypophysial hormonal secretion.     

Endocannabinoids,hormone-cytokine networks and human fertility

Anandamide (N -arachidonoylethanolamine, AEA) is a major endocannabinoid, shown to impair mouse pregnancy and embryo development and to induce apoptosis in blastocysts. Here, we review the roles of AEA, of the AEA-binding cannabinoid (CB) receptors, of the selective AEA membrane transporter (AMT), and of the AEA-hydrolyzing enzyme fatty acid amide hydrolase (FAAH), in human gestation. In particular, we discuss the interplay between the endocannabinoid system and the hormone-cytokine array involved in the control of human pregnancy, showing that the endocannabinoids take part in the immunological adaptation occurring during early pregnancy. In this line, we discuss the critical role of FAAH in human peripheral lymphocytes, showing that the expression of this enzyme is regulated by progesterone, Th1 and Th2 cytokines, which also regulate fertility. Moreover, we show that AEA and the other endocannabinoid, 2-arachidonoylglycerol, inhibit the release of the fertility-promoting cytokine leukemia inhibitory factor from human lymphocytes. Taken together, low FAAH and consistently high blood levels of AEA, but not CB receptors or AMT, can be early (<8 weeks of gestation) markers of spontaneous abortion, potentially useful as diagnostic tools for large-scale, routine monitoring of gestation in humans.     

Opposite effects of methanandamide on lipopolysaccharide-induced prostaglandin E2 and F2α synthesis in uterine explants from pregnant mice

Prostaglandins (PG) are effective abortifacients and are important mediators of lipopolisaccharide (LPS)-induced embryonic resorption (ER). Besides, anandamide (AEA) has been described as one of the major endocannabinoids present in the uterus suggesting that it might play a role in reproduction. It has been reported that high levels of AEA are associated with pregnancy failure and that LPS increases AEA production. Also, it has been observed that AEA modulates PG production in different tissues. In this sense, we studied whether LPS-induced PG production is modulated by AEA and we also assessed the effect of this endocannabinoid on PG metabolism in an in vitro model. Uterine explants from BALB/c implantation sites were cultured in the presence of LPS plus cannabinoid receptor (CB) specific antagonists and PG production was assessed. Then, we studied the effect of exogenous AEA on different steps of PG metabolic pathway. We showed that AEA is involved in LPS-induced PG biosynthesis. Also, we observed that AEA exerts opposite effects on PGE(2) and PGF(2α) biosynthesis, by inhibiting PGE(2) production and increasing PGF(2α) levels. We suggest that AEA could be involved in the mechanisms implicated in LPS-induced ER. A better understanding of how AEA could be affecting ER could help developing specific interventions to prevent this pathology.     

Fatty acid amide hydrolase determines anandamide-induced cell death in the liver

The endocannabinoid anandamide (AEA) induces cell death in many cell types, but determinants of AEA-induced cell death remain unknown. In this study, we investigated the role of the AEA-degrading enzyme fatty acid amide hydrolase (FAAH) in AEA-induced cell death in the liver. Primary hepatocytes expressed high levels of FAAH and were completely resistant to AEA-induced cell death, whereas primary hepatic stellate cells (HSCs) expressed low levels of FAAH and were highly sensitive to AEA-induced cell death. Hepatocytes that were pretreated or with the FAAH inhibitor URB597 isolated from FAAH(-/-) mice displayed increased AEA-induced reactive oxygen species (ROS) formation and were susceptible to AEA-mediated death. Conversely, overexpression of FAAH in HSCs prevented AEA-induced death. Since FAAH inhibition conferred only partial AEA sensitivity in hepatocytes, we analyzed additional factors that might regulate AEA-induced death. Hepatocytes contained significantly higher levels of glutathione (GSH) than HSCs. Glutathione depletion by dl-buthionine-(S,R)-sulfoximine rendered hepatocytes susceptible to AEA-mediated ROS production and cell death, whereas GSH ethyl ester prevented ROS production and cell death in HSCs. FAAH inhibition and GSH depletion had additive effects on AEA-mediated hepatocyte cell death resulting in almost 70% death after 24 h at 50 microm AEA and lowering the threshold for cell death to 500 nm. Following bile duct ligation, FAAH(-/-) mice displayed increased hepatocellular injury, suggesting that FAAH protects hepatocytes from AEA-induced cell death in vivo. In conclusion, FAAH and GSH are determinants of AEA-mediated cell death in the liver.     

Inhibition of Endocannabinoid Metabolism by the Metabolites of Ibuprofen and Flurbiprofen

Background

In addition to their effects upon prostaglandin synthesis, the non-steroidal anti-inflammatory drugs ibuprofen and flurbiprofen inhibit the metabolism of the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (AEA) by cyclooxygenase-2 (COX-2) and fatty acid amide hydrolase (FAAH), respectively. Here, we investigated whether these effects upon endocannabinoid metabolism are shared by the main metabolites of ibuprofen and flurbiprofen.

Methodology/Principal Findings

COX activities were measured via changes in oxygen consumption due to oxygenation of arachidonic acid (for COX-1) and arachidonic acid and 2-AG (for COX-2). FAAH activity was quantified by measuring hydrolysis of tritium labelled AEA in rat brain homogenates. The ability of ibuprofen and flurbiprofen to inhibit COX-2-catalysed oxygenation of 2-AG at lower concentrations than the oxygenation of arachidonic acid was seen with 4′-hydroxyflurbiprofen and possibly also 3′-hydroxyibuprofen, albeit at lower potencies than the parent compounds. All ibuprofen and flurbiprofen metabolites retained the ability to inhibit FAAH in a pH-dependent manner, although the potency was lower than seen with the parent compounds.

Conclusions/Significance

It is concluded that the primary metabolites of ibuprofen and flurbiprofen retain some of the properties of the parent compound with respect to inhibition of endocannabinoid metabolism. However, these effects are unlikely to contribute to the actions of the parent compounds in vivo.     

Anandamide and 2-arachidonoylglycerol inhibit fatty acid amide hydrolase by activating the lipoxygenase pathway of the arachidonate cascade

Treatment of intact human neuroblastoma CHP100 cells with anandamide (arachidonoylethanolamide, AEA) or 2-arachidonoylglycerol (2-AG) inhibits intracellular fatty acid amide hydrolase (FAAH). This effect was not associated with covalent modifications of FAAH, since specific inhibitors of farnesyltransferase, kinases, phosphatases, glycosyltransferase or nitric oxide synthase were ineffective. Electrophoretic analysis of (33)P-labelled proteins, Western blot with anti-phosphotyrosine antibodies, and glycan analysis of cellular proteins confirmed the absence of covalent modifications of FAAH. The inhibition by AEA was paralleled by an increased arachidonate release, which was not observed upon treatment of cells with linoleoylethanolamide, palmitoylethanolamide, or oleoylethanolamide. Moreover, cell treatment with AEA or 2-AG increased the activity of cyclooxygenase and 5-lipoxygenase, and the hydro(pero)xides generated from arachidonate by lipoxygenase were shown to inhibit FAAH, with inhibition constants in the low micromolar range. Consistently, inhibitors of 5-lipoxygenase, but not those of cyclooxygenase, significantly counteracted the inhibition of FAAH by AEA or 2-AG.     

Comparison of anandamide transport in FAAH wild-type and knockout neurons: evidence for contributions by both FAAH and the CB1 receptor to anandamide uptake

The cellular inactivation of the endogenous cannabinoid (endocannabinoid) anandamide (AEA) represents a controversial and intensely investigated subject. This process has been proposed to involve two proteins, a transporter that promotes the cellular uptake of AEA and fatty acid amide hydrolase (FAAH), which hydrolyzes AEA to arachidonic acid. However, whereas the role of FAAH in AEA metabolism is well-characterized, the identity of the putative AEA transporter remains enigmatic. Indeed, the indirect pharmacological evidence used to support the existence of an AEA transporter has been suggested also to be compatible with a model in which AEA uptake is driven by simple diffusion coupled to FAAH metabolism. Here, we have directly addressed the contribution of FAAH to AEA uptake by examining this process in neuronal preparations from FAAH(-/-) mice and in the presence of the uptake inhibitor UCM707. The results of these studies reveal that (i) care should be taken to avoid the presence of artifacts when studying the cellular uptake of lipophilic molecules like AEA, (ii) FAAH significantly contributes to AEA uptake, especially with longer incubation times, and (iii) a UCM707-sensitive protein(s) distinct from FAAH also participates in AEA uptake. Interestingly, the FAAH-independent component of AEA transport was significantly reduced by pretreatment of neurons with the cannabinoid receptor 1 (CB1) antagonist SR141716A. Collectively, these results indicate that the protein-dependent uptake of AEA is largely mediated by known constituents of the endocannabinoid system (FAAH and the CB1 receptor), although a partial contribution of an additional UCM707-sensitive protein is also suggested.