Monthly Archives: June 2016

Analysis of Omega-3 Fatty Acid Derived N-Acylethanolamines in Biological Matrices.

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“The adequate quantification of endocannabinoids can be complex due to their low endogenous levels and structural diversity. Therefore, advanced analytical approaches, such as LC-MS, are used to measure endocannabinoids in plasma, tissues, and other matrices. Recent work has shown that endocannabinoids that are synthesized from n-3 fatty acids, such as docosahexaenoylethanolamide (DHEA) and eicosapentaenoylethanolamide (EPEA), have anti-inflammatory and anti-tumorigenic properties and stimulate synapse formation in neurites. Here, an LC-MS based method for the quantification of n-3 endocannabinoids DHEA and EPEA which is also suited to measure a wider spectrum of endocannabinoids is described. The chapter contains a step-by-step protocol for the analysis of n-3 endocannabinoids in plasma, including sample collection and solid phase extraction, LC-MS analysis, and data processing. Modifications to the protocol that allow quantifying n-3 endocannabinoids in tissues and cell culture media will also be discussed. Finally, conditions that alter endocannabinoid concentrations are briefly discussed.”

http://www.ncbi.nlm.nih.gov/pubmed/27245889

Measuring ECS Interaction with Biomembranes.

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“Understanding the correct interaction among the different components of the endocannabinoid system (ECS) is fundamental for a proper assessment of the function of endocannabinoids (eCBs) as signaling molecules. The knowledge of how membrane environment is able to modulate intracellular trafficking of eCBs and their interacting proteins holds a huge potential in unraveling new mechanisms of ECS modulation.Here, fluorescence resonance energy transfer (FRET) technique is applied to measure the binding affinity of ECS proteins to model membranes (i.e., large unilamellar vesicles, LUVs). In particular, we describe in details the paradigmatic example of the interaction of recombinant rat FAAH-ΔTM with LUVs constituted by 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC).”

http://www.ncbi.nlm.nih.gov/pubmed/27245912

Modulation of cellular redox homeostasis by the endocannabinoid system.

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“The endocannabinoid system (ECS) and reactive oxygen species (ROS) constitute two key cellular signalling systems that participate in the modulation of diverse cellular functions. Importantly, growing evidence suggests that cross-talk between these two prominent signalling systems acts to modulate functionality of the ECS as well as redox homeostasis in different cell types. Herein, we review and discuss evidence pertaining to ECS-induced regulation of ROS generating and scavenging mechanisms, as well as highlighting emerging work that supports redox modulation of ECS function. Functionally, the studies outlined reveal that interactions between the ECS and ROS signalling systems can be both stimulatory and inhibitory in nature, depending on cell stimulus, the source of ROS species and cell context. Importantly, such cross-talk may act to maintain cell function, whereas abnormalities in either system may propagate and undermine the stability of both systems, thereby contributing to various pathologies associated with their dysregulation.”

http://www.ncbi.nlm.nih.gov/pubmed/27248801

Western Blotting of the Endocannabinoid System.

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“Measuring expression levels of G protein-coupled receptors (GPCRs) is an important step for understanding the distribution, function, and regulation of these receptors. A common approach for detecting proteins from complex biological systems is Western blotting. In this chapter, we describe a general approach to Western blotting protein components of the endocannabinoid system using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nitrocellulose membranes, with a focus on detecting type 1 cannabinoid (CB1) receptors. When this technique is carefully used, specifically with validation of the primary antibodies, it can provide quantitative information on protein expression levels. Additional information can also be inferred from Western blotting such as potential posttranslational modifications that can be further evaluated by specific analytical techniques.”

http://www.ncbi.nlm.nih.gov/pubmed/27245910

Quantitation of Plasma Membrane (G Protein-Coupled) Receptor Trafficking in Cultured Cells.

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“Measuring the functional behavior of G protein-coupled receptors (GPCRs) has been a major focus of academic and pharmaceutical research for many decades. These efforts have led to the development of many assays to measure the downstream effects of ligand binding on receptor activity. In this chapter, we describe an internalization/recycling assay that can be used to track changes in receptor number at the plasma membrane. Used in concert with other assays, this antibody-based technique can provide important information on GPCR activation by receptor-specific ligands.”

http://www.ncbi.nlm.nih.gov/pubmed/27245911

Assay of Endocannabinoid Oxidation by Cyclooxygenase-2.

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“The endocannabinoids, 2-arachidonoylglycerol (2-AG) and arachidonylethanolamide (AEA), are endogenous ligands for the cannabinoid receptors (CB1 and CB2) and are implicated in a wide array of physiological processes. These neutral arachidonic acid (AA) derivatives have been identified as efficient substrates for the second isoform of the cyclooxygenase enzyme (COX-2). A diverse family of prostaglandin glycerol esters (PG-Gs) and prostaglandin ethanolamides (PG-EAs) is generated by the action of COX-2 (and downstream prostaglandin synthases) on 2-AG and AEA. As the biological importance of the endocannabinoid system becomes more apparent, there is a tremendous need for robust, sensitive, and efficient analytical methodology for the endocannabinoids and their metabolites. In this chapter, we describe methodology suitable for carrying out oxygenation of endocannabinoids by COX-2, and analysis of products of endocannabinoid oxygenation by COX-2 and of endocannabinoids themselves from in vitro and cell assays.”

http://www.ncbi.nlm.nih.gov/pubmed/27245906

Assay of Monoacylglycerol Lipase Activity.

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“Monoacylglycerol lipase (MGL) is a serine hydrolase involved in the biological deactivation of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). 2-AG is one of the main endogenous lipid agonists for cannabinoid receptors in the brain and elsewhere in the body. In the central nervous system (CNS), MGL is localized to presynaptic nerve terminals of both excitatory and inhibitory synapses, where it helps control the regulatory actions of 2-AG on synaptic transmission and plasticity. In this chapter, we describe an in vitro method to assess MGL activity by liquid chromatography/mass spectrometry (LC/MS)-based quantitation of the reaction product. This method may be used to determine the basal or altered MGL activity in various cells or animal tissues after pharmacological, genetic, or biological manipulations. In addition, this assay can be used for MGL inhibitor screening using purified recombinant enzyme or MGL-overexpressing cells.”

http://www.ncbi.nlm.nih.gov/pubmed/27245902

Assay of DAGLα/β Activity.

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“The endocannabinoid 2-arachidonoylglycerol (2-AG) exerts its physiological action by binding to and functionally activating type-1 (CB1) and type-2 (CB2) cannabinoid receptors. It is thought to be produced through the action of sn-1 selective diacylglycerol lipase (DAGL) that catalyzes 2-AG biosynthesis from sn-2-arachidonate-containing diacylglycerols. Since 2-AG biosynthetic enzymes have been identified only recently, little information on methodological approaches for measuring DAGL activity is as yet available. Here, a highly sensitive radiometric assay to measure DAGL activity by using 1-oleoyl[1-(14)C]-2-arachidonoylglycerol as the substrate is reported. All the steps needed to perform lipid extraction, fractionation by thin-layer chromatography (TLC), and quantification of radiolabeled [(14)C]-oleic acid via scintillation counting are described in detail.”

http://www.ncbi.nlm.nih.gov/pubmed/27245901

Protocol to Study β-Arrestin Recruitment by CB1 and CB2 Cannabinoid Receptors.

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“Cannabinoid CB1 and CB2 receptors are G-protein-coupled receptors (GPCRs) that recruit β-arrestins upon activation by (partial) agonists. β-Arrestin recruitment is induced by phosphorylation of their C-terminal tails, and is associated with the termination of GPCR signaling; yet, it may also activate cellular signaling pathways independent of G-proteins. Here, we describe a detailed protocol to characterize the potency and efficacy of ligands to induce or inhibit β-arrestin recruitment to the human CB1 and CB2 receptors, by using the PathHunter(®) assay. The latter is a cellular assay that can be performed in plates with 384-wells. The PathHunter(®) assay makes use of β-galactosidase complementation, and has a chemiluminescent readout. We used this assay to characterize a set of reference ligands (both agonists and antagonists) on human CB1 and CB2 receptors.”

http://www.ncbi.nlm.nih.gov/pubmed/27245896

The Cyclic AMP Assay Using Human Cannabinoid CB2 Receptor-Transfected Cells.

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“The cyclic AMP assay is a functional assay that is commonly used to determine the pharmacological behavior (agonists, antagonists, inverse agonists) of G-protein-coupled receptor (GPCR) ligands. Here, we describe the cyclic AMP assay that is carried out with commercially available non-radioligand ready-to-use kits and Chinese hamster ovarian (CHO) cells stably transfected with the human cannabinoid CB2 receptor.”

http://www.ncbi.nlm.nih.gov/pubmed/27245894