Anandamide assay

The assays (see above) for arachidonoyl ethanolamide hydrolysis (anandamide amidase) employed H-anandamide as the substrate... 

Radioligand-binding assays reveal that 12(5)-HETE-ethanolamide has Kj values at CB, and CBj receptors that are not dissimilar to those obtained for anandamide (Edgemond et al., 1997 van der Stelt et al., 2002). 12(R)-HETE-ethanolamide and 15-HETE(5)-ethanolaniide have K values of 416 nMand 700 nMfor the CB, receptor, but >1 pMfor the CBj receptor (Edgemond et al., 1997 van der Stelt et al., 2002). 5-HETE(5) ethanolamide does not bind to CB, or CBj receptors, whereas 15-HETE(R)-ethanolamide has low affinity for both receptors (K, 700 nM). 

SR141716A pretreatment eountered these inhibitory effects, but when administered alone it increased intestinal motility to the same extent in both control and croton-oil-exposed mice. Moreover, anan-damide and 2-AG level assay showed no significant difference between inflamed and noninflamed intestinal tissue. The lack of increase in endocannabinoid levels in the inflamed intestine could be due to a more rapid turnover of both 2-AG and anandamide, which could be due to upregulation of anandamide amidohydrolase as the activity of this enzyme was signilicantiy increased in the inflamed intestine. 

The transfected cells were suspended in DMEM, followed by distribution into 6-well plates. Cells were grown for 3 d and then assayed for their abilities to accumulate tritium-labeled anandamide [arachidonyl-5,6,8,9,ll,12,14,15- H]-, 160 Ci/mmol (New England Nuclear Research Products, Boston, MA). 

COS cells transfected with pcDNA3.1/V7-3 and the vector pCDNA3.1 served as positive and negative controls, respectively. Parallel incubations with 30 pM of unlabeled anandamide allowed estimation of nonspecific uptake and binding. Uptake assays were carried out for 5 min at 3TC, followed by two washes each with 2 mL of Krebs-Ringer-Henseleit buffer. 

Methods to Assay Anandamide Hydrolysis and Transport in Synaptosomes... 

Anandamide (AEA) is the most studied member of a new class of lipid mediators, collectively called endocannabinoids. The biological activity of AEA at cannabinoid and noncannabinoid receptors depends on its Hfe span in the extracellular space, which is regulated by a rapid cellular uptake, followed by intracellular degradation by the enzyme AEA hydrolase (fatty acid amide hydrolase). Here, we present the methodological details of the procedures that we have developed to assay fatty acid amide hydrolase activity and to characterize AEA transport through cell membranes in a new ideal ex vivo system like brain synaptosomes. 

The discovery of anandamide (arachidonoyl ethanolamide, AEA) and of its manifold roles in the central nervous system and in the periphery (reviewed in refs. 1 and 2) prompted several researchers to develop analytical methods to assay and characterize the activity of the enzymes responsible for AEA metabolism in various cells and tissues. Fatty acid amide hydrolase (arachidonoyl ethanolamide amidohydrolase, EC 3.5.1.4 FAAH) has emerged as the key AEA hydrolase, showing a molecular mass of approx 64 kDa and an optimum pH of around 9.0 (3). Recently, FAAH has been crystallized,and its three dimensional structure has been determined at 2.8A resolution (4). This enzyme cleaves the amide bond and releases arachidonic acid and ethanolamine. High-performance liquid chromatography (HPLC) is the most widely used method to determine FAAH activity from different sources. We developed a new method (5) based on reversed-phase (RP)-HPLC and on-line scintillation counting, which combines the need for high resolution, reproducibility, and sensitivity... 

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