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.     

3-Alkenyl-2-azetidinones as fatty acid amide hydrolase inhibitors

A series of novel 2-azetidinones (β-lactams) bearing short alkenyl chains at C3 and N1 have been prepared and evaluated in vitro as inhibitors of human FAAH. Compound 9c (1-(4′-pentenoyl-3-(4′-pentenyl)-2-azetidinone)) featured an IC₅₀ value of 4.5 μM and a good selectivity for FAAH versus MGL.     

Piperidine and piperazine inhibitors of fatty acid amide hydrolase targeting excitotoxic pathology

FAAH inhibitors offer safety advantages by augmenting the anandamide levels “on demand” to promote neuroprotective mechanisms without the adverse psychotropic effects usually seen with direct and chronic activation of the CB1 receptor. FAAH is an enzyme implicated in the hydrolysis of the endocannabinoid N-arachidonoylethanolamine (AEA), which is a partial agonist of the CB1 receptor. Herein, we report the discovery of a new series of highly potent and selective carbamate FAAH inhibitors and their evaluation for neuroprotection. The new inhibitors showed potent nanomolar inhibitory activity against human recombinant and purified rat FAAH, were selective (>1000-fold) against serine hydrolases MGL and ABHD6 and lacked any affinity for the cannabinoid receptors CB1 and CB2. Evaluation of FAAH inhibitors 9 and 31 using the in vitro competitive activity-based protein profiling (ABPP) assay confirmed that both inhibitors were highly selective for FAAH in the brain, since none of the other FP-reactive serine hydrolases in this tissue were inhibited by these agents. Our design strategy followed a traditional SAR approach and was supported by molecular modeling studies based on known FAAH cocrystal structures. To rationally design new molecules that are irreversibly bound to FAAH, we have constructed “precovalent” FAAH-ligand complexes to identify good binding geometries of the ligands within the binding pocket of FAAH and then calculated covalent docking poses to select compounds for synthesis. FAAH inhibitors 9 and 31 were evaluated for neuroprotection in rat hippocampal slice cultures. In the brain tissue, both inhibitors displayed protection against synaptic deterioration produced by kainic acid-induced excitotoxicity. Thus, the resultant compounds produced through rational design are providing early leads for developing therapeutics against seizure-related damage associated with a variety of disorders.     

Synthesis and evaluation of potent and selective MGL inhibitors as a glaucoma treatment

Monoacylglycerol lipase (MGL) inhibition provides a potential treatment approach to glaucoma through the regulation of ocular 2-arachidonoylglycerol (2-AG) levels and the activation of CB1 receptors. Herein, we report the discovery of new series of carbamates as highly potent and selective MGL inhibitors. The new inhibitors showed potent nanomolar inhibitory activity against recombinant human and purified rat MGL, were selective (>1000-fold) against serine hydrolases FAAH and ABHD6 and lacked any affinity for the cannabinoid receptors CB1 and CB2. Protein-based ¹H NMR experiments indicated that inhibitor 2 rapidly formed a covalent adduct with MGL with a residence time of about 6?h. This interconversion process “intrinsic reversibility” was exploited by modifications of the ligand’s size (length and bulkiness) to generate analogs with “tunable’ adduct residence time (τ). Inhibitor 2 was evaluated in a normotensive murine model for assessing intraocular pressure (IOP), which could lead to glaucoma, a major cause of blindness. Inhibitor 2 was found to decrease ocular pressure by ～4.5?mmHg in a sustained manner for at least 12?h after a single ocular application, underscoring the potential for topically-administered MGL inhibitors as a novel therapeutic target for the treatment of glaucoma.     

Alteration of endocannabinoid system in human gliomas

Endocannabinoids are neuromodulatory lipids that mediate the central and peripheral neural functions. Endocannabinoids have demonstrated their anti-proliferative, anti-angiogenic and pro-apoptotic properties in a series of studies. In the present study, we investigated the levels of two major endocannabinoids, anandamide and 2-arachidonylglycerol (2-AG), and their receptors, CB1 and CB2, in human low grade glioma (WHO grade I-II) tissues, high grade glioma (WHO grade III-IV) tissues, and non-tumor brain tissue controls. We also measured the expressions and activities of the enzymes responsible for anandamide and 2-AG biosynthesis and degradation, that is, N-acylphosphatidylethanolamine-hydrolysing phospholipase D (NAPE-PLD), fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MGL), and diacylglycerol lipase-alpha (DGL), in the same samples. Liquid chromatography-mass spectometry analysis showed that the levels of anandamide decreased, whereas the levels of 2-AG increased in glioma tissues, comparing to the non-tumor controls. The expression levels and activities of NAPE-PLD, FAAH and MGL also decreased in glioma tissues. Furthermore, quantitative-PCR analysis and western-blot analysis revealed that the expression levels of cananbinoid receptors, CB1 and CB2, were elevated in human glioma tissues. The changes of anandamide and 2-AG contents in different stages of gliomas may qualify them as the potential endogenous biomarkers for glial tumor malignancy.     

Conformationally constrained analogues of 2-arachidonoylglycerol

Novel monocyclic analogues of 2-arachidonoylglycerol (2-AG) were designed in order to explore the pharmacophoric conformations of this endocannabinoid ligand at the key cannabinergic proteins. All 2-arachidonoyl esters of 1,2,3-cyclohexanetriol [meso-7 (AM5504), (+/-)-8 (AM5503), and meso-9 (AM5505)] were synthesized by regioselective acylation of 2,3-dihydroxycyclohexanone followed by selective reductions. The optically active isomers (+)-8 (AM4434) and (-)-8 (AM4435) were synthesized from (2S,3S)- and (2R,3R)-2,3-dihydroxycyclohexanone, respectively, via a chemoenzymatic route. These head group constrained and conformationally restricted analogues of 2-AG as well as the 1-keto precursors were evaluated as substrates for the endocannabinoid deactivating hydrolytic enzymes monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH), and also were tested for their affinities for CB1 and CB2 cannabinoid receptors. The observed biochemical differences between these ligands can help define the conformational requirements for 2-AG activity at each of the above endocannabinoid protein targets.     

Hyperglycemia induces apoptosis via CB1 activation through the decrease of FAAH 1 in retinal pigment epithelial cells

Fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of the main endocannabinoid, anandamide, and related fatty acid amides, has emerged as a regulator of endocannabinoid signaling. Retinal pigment epithelial (RPE) cells are believed to be important cells in the pathogenesis of diabetic retinopathy. However, the pathophysiology of FAAH in diabetic retinopathy has not been determined. Thus, we examined the effect of high glucose (HG) on the expression of FAAH and CB(1)R in the ARPE-19 human RPE cells. We found that HG downregulated the expression of FAAH 1 mRNA and protein in ARPE-19 cells. In contrast, it upregulated the expression of CB(1)R mRNA and protein. HG-induced internalization of CB(1)R in HEK 293 cells and ARPE-19 cells was blocked by overexpression of FAAH 1 and treatment with the CB(1)R blocker, AM 251. HG-induced generation of reactive oxygen species and lipid peroxide formation were blocked by the overexpression of FAAH 1. FAAH 1 overexpression also blocked HG-induced expression of CB(1)R in the cytosolic fraction. We also investigated whether the overexpression of FAAH 1 protected against HG-induced apoptosis. High glucose increased the Bax/Bcl-2 ratio and levels of cleaved PARP, cleaved caspase-9 and caspase-3, and reduced cell viability. HG-induced apoptotic effects were reduced by the overexpression of FAAH 1, treatment with the CB(1)R-specific antagonist AM 251 and CB(1)R siRNA transfection. In conclusion, HG-induced apoptosis in ARPE-19 cells by inducing CB(1)R expression through the downregulation of FAAH 1 expression. Our results provide evidence that CB(1)R blockade through the recovery of FAAH 1 expression may be a potential anti-diabetic therapy for the treatment of diabetic retinopathy.     

Decreased age-related cardiac dysfunction, myocardial nitrative stress, inflammatory gene expression, and apoptosis in mice lacking fatty acid amide hydrolase

Recent studies have uncovered important cross talk between inflammation, generation of reactive oxygen and nitrogen species, and lipid metabolism in the pathogenesis of cardiovascular aging. Inhibition of the endocannabinoid anandamide metabolizing enzyme, the fatty acid amide hydrolase (FAAH), is emerging as a promising novel approach for the treatment of various inflammatory disorders. In this study, we have investigated the age-associated decline of cardiac function and changes in inflammatory gene expression, nitrative stress, and apoptosis in FAAH knockout (FAAH(-/-)) mice and their wild-type (FAAH(+/+)) littermates. Additionally, we have explored the effects of anandamide on TNF-alpha-induced ICAM-1 and VCAM-1 expression and monocyte-endothelial adhesion in human coronary artery endothelial cells (HCAECs). There was no difference in the cardiac function (measured by the pressure-volume conductance catheter system) between 2- to 3-mo-old (young) FAAH(-/-) and FAAH(+/+) mice. In contrast, the aging-associated decline in cardiac function and increased myocardial gene expression of TNF-alpha, gp91phox, matrix metalloproteinase (MMP)-2, MMP-9, caspase-3 and caspase-9, myocardial inducible nitric oxide synthase protein expression, nitrotyrosine formation, poly (ADP-ribose)polymerase cleavage and caspase-3/9 activity, observed in 28- to 31-mo-old (aging) FAAH(+/+) mice, were largely attenuated in knockouts. There was no difference in the myocardial cannabinoid CB(1) and CB(2) receptor gene expression between young and aging FAAH(-/-) and FAAH(+/+) mice. Anandamide dose dependently attenuated the TNF-alpha-induced ICAM-1 and VCAM-1 expression, NF-kappaB activation in HCAECs, and the adhesion of monocytes to HCAECs in a CB(1)- and CB(2)-dependent manner. These findings suggest that pharmacological inhibition of FAAH may represent a novel protective strategy against chronic inflammation, oxidative/nitrative stress, and apoptosis associated with cardiovascular aging and atherosclerosis.     

Expression, purification, and characterization of histidine-tagged mouse monoglyceride lipase from baculovirus-infected insect cells

Monoglyceride lipase (MGL) has been produced with the baculovirus-insect cell system. The mouse MGL cDNA was subcloned into a baculovirus transfer vector in frame with a sequence encoding an N-terminal stretch of six histidine residues. Purification to apparent homogeneity was obtained by nickel-chelating chromatography. The final yield was 3 mg of pure enzymatically active MGL per liter of Sf9 cell suspension culture. Analysis by SDS-PAGE and mass spectrometry showed that the recombinant histidine-tagged enzyme had the expected molecular mass. With monoolein as substrate, the specific activity and the apparent K(m) were close to those of rat MGL of adipose tissue.     

Identification of sialoadhesin as a dominant lymph node counter-receptor for mouse macrophage galactose-type C-type lectin 1

In the sensitization phase of contact hypersensitivity in mice, dermal macrophages (MOs) expressing MO galactose-type C-type lectin1 (MGL1) are known to migrate from the dermis to lymph nodes (LNs) where they accumulate in the subcapsular sinus, interfollicular regions, and areas surrounding high endothelial venules. We hypothesize that the interactions between MGL1 and its ligands determine the localizations of MGL1-positive cells within the LNs. In the present study, our major aim was to isolate MGL1 counter-receptor(s) from lysates of LNs using affinity chromatography with immobilized recombinant MGL1. Fractions bound and eluted with EDTA were analyzed by SDS-PAGE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. One of the predominant components was sialoadhesin (Sn, Siglec-1). Sn from lysates of LNs was immobilized on microtiter plates precoated with anti-Sn monoclonal antibody, and binding of recombinant MGL1 and adhesion of cells expressing MGL1 were tested. The binding of recombinant MGL1 to Sn was shown to be dependent on Ca2+ and N-glycans on Sn. MGL1-transfected Chinese hamster ovary cells adhered to the Sn-coated plates, whereas mock transfectants did not. Immunohistochemical localization of anti-Sn monoclonal antibody in LN coincided with the subcapsular sinus area to which recombinant MGL1 was bound. Furthermore, the distribution of MGL1+ cells after sensitization with FITC was demonstrated to overlap with that of Sn within the subcapsular sinus of draining LNs. These results suggest that Sn acts as an endogenous counter-receptor for MGL1.     

The amino acids involved in the distinct carbohydrate specificities between macrophage galactose-type C-type lectins 1 and 2 (CD301a and b) of mice

Binding specificities of mouse macrophage galactose-type C-type lectin 1 (MGL1/CD301a) and 2 (MGL2/CD301b) toward various oligosaccharides were compared by frontal affinity chromatography. MGL1 preferentially bound oligosaccharides containing Lewis(X) (Le(X)) trisaccharides among 111 oligosaccharides tested, whereas MGL2 preferentially bound globoside Gb4. The important amino acids for the preferential bindings were investigated by pair-wise site-directed mutagenesis at positions 61, 89, 97, 100, 110-113, 115, 124, and 125 in the soluble recombinant carbohydrate recognition domains (CRD) prepared in Escherichia coli and purified with galactose-Sepharose. Mutations of Val, Ala, Thr, and Phe at positions 61, 89, 111 and 125 on MGL1 CRD caused reductions in Le(X) binding. Mutations of MGL2 CRD at Leu, Arg, Arg, and Tyr at positions 61, 89, 115 and 125 were implicated in the preference for beta-GalNAc. Le(X) binding was observed with MGL2 mutants of Arg89Ala and Arg89Ala/Ser111Thr. MGL1 mutants of Ala89Arg and Ala89Arg/Pro115Arg showed beta-GalNAc bindings. Molecular modeling illustrated potential direct molecular interactions of Leu61, Arg89, and His109 in MGL2 CRD with GalNAc.     

Full mass spectrometric characterization of human monoacylglycerol lipase generated by large-scale expression and single-step purification

The serine hydrolase monoacylglycerol lipase (MGL) modulates endocannabinoid signaling in vivo by inactivating 2-arachidonoylglycerol (2-AG), the main endogenous agonist for central CB1 and peripheral CB2 cannabinoid receptors. To characterize this key endocannabinoid enzyme by mass spectrometry-based proteomics, we first overexpressed recombinant hexa-histidine-tagged human MGL (hMGL) in Escherichia coli and purified it in a single chromatographic step with high yield (approximately 30 mg/L). With 2-AG as substrate, hMGL displayed an apparent V max of 25 micromol/(microg min) and K m of 19.7 microM, an affinity for 2-AG similar to that of native rat-brain MGL (rMGL) (Km=33.6 microM). hMGL also demonstrated a comparable affinity (Km approximately 8-9 microM) for the novel fluorogenic substrate, arachidonoyl, 7-hydroxy-6-methoxy-4-methylcoumarin ester (AHMMCE), in a sensitive, high-throughput fluorometric MGL assay. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) unequivocably demonstrated the mass (34,126 Da) and purity of this hMGL preparation. After in-solution tryptic digestion, hMGL full proteomic characterization was carried out, which showed (1) an absence of intramolecular disulfide bridges in the functional, recombinant enzyme and (2) the post-translational removal of the enzyme's N-terminal methionine. Availability of sufficient quantities of pure, well-characterized hMGL will enable further molecular and structural profiling of this key endocannabinoid-system enzyme.     

Carbohydrate binding mechanism of the macrophage galactose-type C-type lectin 1 revealed by saturation transfer experiments

Macrophage galactose-type C-type lectins 1 and 2 (MGL1/2) are expressed on the surfaces of macrophages and immature dendritic cells. Despite the high similarity between the primary sequences of MGL1 and MGL2, they display different ligand specificities. MGL1 shows high affinity for the LewisX trisaccharide, whereas MGL2 shows affinity for N-acetylgalactosamine. To elucidate the structural basis for the ligand specificities of the MGLs, we performed NMR analyses of the MGL1-LewisX complex. To identify the LewisX binding site on MGL1, a saturation transfer experiment for the MGL1-LewisX complex where sugar-CH/CH2-selective saturation was applied was carried out. To obtain sugar moiety-specific information on the interface between MGL1 and the LewisX trisaccharide, saturation transfer experiments where each of galactose-H5-, fucose-CH3-, and N-acetylglucosamine-CH3-selective saturations was applied to the MGL1-LewisX complex were performed. Based on these results, we present a LewisX binding mode on MGL1 where the galactose moiety is bound to the primary sugar binding site, including Asp-94, Trp-96, and Asp-118, and the fucose moiety interacts with the secondary sugar binding site, including Ala-89 and Thr-111. Ala-89 and Thr-111 in MGL1 are replaced with arginine and serine in MGL2, respectively. The hydrophobic environment formed by a small side chain of Ala-89 and a methyl group of Thr-111 is a requisite for the accommodation of the fucose moiety of the LewisX trisaccharide within the sugar binding site of MGL1.     

Properties of blocking and non-blocking monoclonal antibodies specific for human macrophage galactose-type C-type lectin (MGL/ClecSF10A/CD301)

Monoclonal antibodies (mAbs) specific for the human macrophage galactose-type calcium-type lectin (MGL) were established. The recombinant extracellular domain of MGL was used to immunize a mouse, and 10 hybridoma clones were obtained. Binding of recombinant MGL to asialo-bovine submaxillary mucin was shown to be blocked by mAbs MLD-1, 4 and 6. Immunoprecipitation of MGL from lysates of COS-1 cells transfected with MGL cDNA (form 6A) was achieved with mAbs MLD-1, 4, 7, 8 and 16. Chimeric recombinant proteins between human MGL and mouse MGL1 were used to determine the location of the epitopes for these mAbs. mAbs MLD-8, 13, 15 and 16 interacted with the amino terminal side of the conserved WVDGTD sequence immediately upstream of QPD, whereas mAbs MLD-7, 12 and 17 interacted with the other side. mAbs MLD-1, 4, and 6 apparently required both sides of this boundary. mAbs MLD-15 and 16 were shown to recognize the protein products of alternatively spliced mRNA 6A/8A and 6C/8A, having deletions at the boundary of exons 7 and 8, in addition to full length and other spliced forms of MGL (6A, 6B and 6C), whereas the other mAbs bound only full length and forms 6A, 6B and 6C.     

The C-type lectin macrophage galactose-type lectin impedes migration of immature APCs

Dendritic cells (DCs) are the most potent APCs of the immune system that seed the peripheral tissues and lymphoid organs. In an immature state, DCs sample their surroundings for incoming pathogens. Upon Ag encounter, DCs mature and migrate to the lymph node to induce adaptive immune responses. The C-type macrophage galactose-type lectin (MGL), expressed in immature DCs, mediates binding to glycoproteins carrying GalNAc moieties. In the present study, we demonstrate that MGL ligands are present on the sinusoidal and lymphatic endothelium of lymph node and thymus, respectively. MGL binding strongly correlated with the expression of the preferred MGL ligand, alpha-GalNAc-containing glycan structures, as visualized by staining with the alpha-GalNAc-specific snail lectin Helix pomatia agglutinin. MGL(+) cells were localized in close proximity of the endothelial structures that express the MGL ligand. Strikingly, instead of inducing migration, MGL mediated retention of human immature DCs, as blockade of MGL interactions enhanced DC trafficking and migration. Thus, MGL(+) DCs are hampered in their migratory responses and only upon maturation, when MGL expression is abolished; these DCs will be released from their MGL-mediated restraints.     

阴道毛滴虫蛋氨酸裂解酶的基因克隆及重组酶性质分析

为研究重组阴道毛滴虫蛋氨酸裂解酶的活性,从阴道毛滴虫提取RNA,经过RT-PCR克隆蛋氨酸γ-裂解酶基因,与pET-15b质粒连接构建原核表达载体pET-15b-mgl1,并测序。转化宿主菌BL21后进行诱导表达,分离纯化目的蛋白并研究其酶学性质。以L-蛋氨酸、DL-同型半胱氨酸、L-半胱氨酸为底物测定重组酶Km值为0.318、2.14、1.62mmol/L,比活性分别为20.17、318.69、56.96μmol/min/mg protein。MGL最适pH在5.0到6.0之间,在50℃可稳定30min无活性损失。实验结果显示重组酶具有较高的活性和较好的热稳定性,为临床应用研究奠定基础。     

Cyclic changes in endometrial echotexture of cows using a computer-assisted program for the analysis of first- and second-order grey level statistics of B-Mode ultrasound images

The aim of this study was to test the suitability of a computer-assisted echotexture analysis program for analysing first- and second-order grey level statistics of grey levels of B-Mode ultrasound images to examine morphologic changes in the endometrium during oestrous cycle in cows. Four Simmental cows were examined for two consecutive oestrous cycles. Echotexture of the endometrium was assessed by mean grey level (MGL) and homogeneity (HOM) of digitised B-Mode images of the uterine body and both uterine horns. As there were no differences (P>0.05) in MGL and HOM, respectively, between the images of the uterine body and the uterine horns, the mean values of all endometrial images were used for subsequent analyses of MGL and HOM. The factor 'day of oestrous cycle' showed a highly significant (P<0.0001) effect and the factor 'cow' showed a significant (P<0.05) effect on MGL and HOM. No differences (P>0.05) in both echotexture parameters were measured between oestrous cycles within cows. MGL was negatively related to HOM (r=-0.66; P<0.0001). Low MGL and high HOM occurred on Day 0 (=ovulation) and between Days -3 and -1. While MGL was consistently (P>0.05) low between Days -3 and -1, significant changes of HOM with maximum levels on Day -2 (P<0.05) were observed during this time. MGL was consistently (P>0.05) high between Days 4 and 13 while HOM was consistently (P>0.05) low between Days 2 and 13. From Day -3 to Day -1 (r=0.48; P<0.05) plasma oestrogen levels were correlated with HOM, but not with MGL (P<0.05). The results of this study show that changes in endometrial morphology of cows can be measured using a computer-assisted texture analysis program.     

Lipoxygenase-mediated oxidation of polyunsaturated N-acylethanolamines in Arabidopsis

N-acylethanolamines (NAEs) are bioactive fatty acid derivatives that occur in all eukaryotes. In plants, NAEs have potent negative growth-regulating properties, and fatty acid amide hydrolase (FAAH)-mediated hydrolysis is a primary catabolic pathway that operates during seedling establishment to deplete these compounds. Alternatively, polyunsaturated (PU)-NAEs may serve as substrates for lipid oxidation. In Arabidopsis, PU-NAEs (NAE 18:2 and NAE 18:3) were the most abundant NAE species in seeds, and their levels were depleted during seedling growth even in FAAH tDNA knock-out plants. Therefore, we hypothesized that lipoxygenase (LOX) participated in the metabolism of PU-NAEs through the formation of NAE-oxylipins. Comprehensive chromatographic and mass spectrometric methods were developed to identify NAE hydroperoxides and -hydroxides. Recombinant Arabidopsis LOX enzymes expressed in Escherichia coli utilized NAE 18:2 and NAE 18:3 as substrates with AtLOX1 and AtLOX5 exhibiting 9-LOX activity and AtLOX2, AtLOX3, AtLOX4, and AtLOX6 showing predominantly 13-LOX activity. Feeding experiments with exogenous PU-NAEs showed they were converted to hydroxide metabolites indicating that indeed Arabidopsis seedlings had the capacity for LOX-mediated metabolism of PU-NAEs in planta. Detectable levels of endogenous NAE-oxylipin metabolites were identified in FAAH fatty acid amide hydrolase seedlings but not in wild-type or FAAH overexpressors, suggesting that NAE hydroxide pools normally do not accumulate unless flux through hydrolysis is substantially reduced. These data suggest that Arabidopsis LOXs indeed compete with FAAH to metabolize PU-NAEs during seedling establishment. Identification of endogenous amide-conjugated oxylipins suggests potential significance of these metabolites in vivo, and FAAH mutants may offer opportunities to address this in the future.     

A novel scintillation proximity assay for fatty acid amide hydrolase compatible with inhibitor screening

A binding assay for human fatty acid amide hydrolase (FAAH) using the scintillation proximity assay (SPA) technology is described. This SPA uses the specific interactions of [3H]R(+)-methanandamide (MAEA) and FAAH expressing microsomes to evaluate the displacement activity of FAAH inhibitors. We observed that a competitive nonhydrolyzed FAAH inhibitor, [3H]MAEA, bound specifically to the FAAH microsomes. Coincubation with an FAAH inhibitor, URB-597, competitively displaced the [3H]MAEA on the FAAH microsomes. The released radiolabel was then detected through an interaction with the SPA beads. The assay is specific for FAAH given that microsomes prepared from cells expressing the inactive FAAH-S241A mutant or vector alone had no significant ability to bind [3H]MAEA. Furthermore, the binding of [3H]MAEA to FAAH microsomes was abolished by selective FAAH inhibitors in a dose-dependent manner, with IC50 values comparable to those seen in a functional assay. This novel SPA has been validated and demonstrated to be simple, sensitive, and amenable to high-throughput screening.