Category Archives: Endocannabinoid System

Cannabinoid receptor-2 stimulation suppresses neuroinflammation by regulating microglial M1/M2 polarization through the cAMP/PKA pathway in an experimental GMH rat model.

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“Excessive inflammatory responses are involved in secondary brain injury during germinal matrix hemorrhage (GMH). The process of microglial polarization to the pro-inflammatory M1 or anti-inflammatory M2 phenotypes is considered to occur in a major immunomodulatory manner during brain inflammation.

We previously found that cannabinoid receptor-2 (CB2R) stimulation attenuated microglial accumulation and brain injury following experimental GMH.

Herein, we investigated the effects of CB2R stimulation on neuroinflammation after experimental GMH and the potential mechanisms that mediate M1/M2 microglial phenotype regulation.

This is the first study to propose that promotion of microglial M2 polarization through the cAMP/PKA pathway participates in the CB2R-mediated anti-inflammatory effects after GMH induction.

The results will help to further understand the mechanisms that underlie neuroprotection by CB2R in GMH and promote clinical translational research for CB2R agonists.”

http://www.ncbi.nlm.nih.gov/pubmed/27261088

Anandamide transporter-mediated regulation of the micturition reflex in urethane-anesthetized rats.

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“The aim of this study was to investigate the effects of an anandamide transporter inhibitor that can increase endogenous anandamide concentration on the micturition reflex in urethane-anesthetized rats.

These results suggest that anandamide, an endogenous CB ligand, can modulate the micturition reflex and that anandamide transporters play an important role in this modulation. In urethane-anesthetized rats, inhibition of the uptake of anandamide can inhibit the micturition reflex and these inhibitory effects of VDM11 are at least in part mediated by the CB1 receptor.”

http://www.ncbi.nlm.nih.gov/pubmed/27256398

Clinical Effects of Synthetic Cannabinoid Receptor Agonists Compared with Marijuana in Emergency Department Patients with Acute Drug Overdose.

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“Synthetic cannabinoid receptor agonists (SCRAs) are heterogeneous compounds originally intended as probes of the endogenous cannabinoid system or as potential therapeutic agents.

In the first clinical study comparing the adverse effects of SCRA overdose vs. marijuana controls in an ED population, we found that SCRA overdoses had significantly pronounced neurotoxicity and cardiotoxicity compared with marijuana.”

http://www.ncbi.nlm.nih.gov/pubmed/27255136

Visualization of Endocannabinoids in the Cell.

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“A still unsolved, although critical, issue in endocannabinoid research is the mechanism by which the lipophilic compound anandamide (AEA) moves from its site of synthesis, crosses the aqueous milieu, and reaches the different intracellular compartments, where its metabolic and signaling pathways take place. The difficulty of studying intracellular AEA transport and distribution results from the lack of specific probes and techniques to track and visualize this bioactive lipid within the cell. Here, we describe the use of a biotinylated, non-hydrolyzable derivative of AEA (biotin-AEA, b-AEA) for visualizing the subcellular distribution of this endocannabinoid by means of confocal fluorescence microscopy.”

http://www.ncbi.nlm.nih.gov/pubmed/27245913

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