“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.”
Category Archives: Endocannabinoid System
Assay of DAGLα/β Activity.
“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.”
Protocol to Study β-Arrestin Recruitment by CB1 and CB2 Cannabinoid Receptors.
“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.”
The Cyclic AMP Assay Using Human Cannabinoid CB2 Receptor-Transfected Cells.
“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.”
A Functional Assay for GPR55: Envision Protocol.
“AlphaScreen(®) SureFire(®) assay is a novel technology that combines luminescent oxygen channeling technology, nano-beads, and monocloncal antibodies to detect the level of a selected protein in a volume lower than 5 μl. This method is more sensitive compared with the traditional enzyme-linked immunosorbent assays (ELISA), and can detect an increasing number of new targets. Here, we described a method for AlphaScreen(®) SureFire(®) assay that targets ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys activation of GPR55 by L-α-lysophosphatidylinositol (LPI) and certain cannabinoids.”
The Displacement Binding Assay Using Human Cannabinoid CB2 Receptor-Transfected Cells.
“Displacement binding assays are nonfunctional assays mostly used with the aim of determining whether a certain compound (plant-derived or synthetic) is able to bind to a specific receptor with high affinity. Here, we describe the displacement binding assay that is carried out with a radioligand and CHO (Chinese Hamster Ovarian) cells stably transfected with the human cannabinoid CB2 receptor.”
Assay of CB1 Receptor Binding.
“Type-1 cannabinoid receptor (CB1), one of the main targets of endocannabinoids, plays a key role in several pathophysiological conditions that affect both central nervous system and peripheral tissues. Today, its biochemical identification and pharmacological characterization, as well as the screening of thousands of novel ligands that might be useful for developing CB1-based therapies, are the subject of intense research. Among available techniques that allow the analysis of CB1 binding activity, radioligand-based assays represent one of the best, fast, and reliable methods.Here, we describe radioligand binding methods standardized in our laboratory to assess CB1 binding in both tissues and cultured cells. We also report a high-throughput radioligand binding assay that allows to evaluate efficacy and potency of different compounds, which might represent the basis for the development of new drugs that target CB1 receptor-dependent human diseases.”
Need for Methods to Investigate Endocannabinoid Signaling.
“Endocannabinoids (eCBs) are endogenous lipids able to activate cannabinoid receptors, the primary molecular targets of the cannabis (Cannabis sativa) active principle Δ(9)-tetrahydrocannabinol. During the last 20 years, several N-acylethanolamines and acylesters have been shown to act as eCBs, and a complex array of receptors, metabolic enzymes, and transporters (that altogether form the so-called eCB system) has been shown to finely tune their manifold biological activities. It appears now urgent to develop methods and protocols that allow to assay in a specific and quantitative manner the distinct components of the eCB system, and that can properly localize them within the cell. A brief overview of eCBs and of the proteins that bind, transport, and metabolize these lipids is presented here, in order to put in a better perspective the relevance of methodologies that help to disclose molecular details of eCB signaling in health and disease. Proper methodological approaches form also the basis for a more rationale and effective drug design and therapeutic strategy to combat human disorders.”
Endocannabinoid system: a promising therapeutic target for the treatment of haematological malignancies?
“The therapeutic properties of cannabinoids are well-known since ancient years.
Growing evidence exist on endocannabinoid system (ECS) modulation related with human tumorigenesis.
Taking into account the substantial role of ECS on immune cell regulation, the present review is aimed to summarize the emerging evidence concerning cannabinoid receptor (CBR) expression and cannabinoid ligand effects on haematological malignancies.
CONCLUSIONS:
Most of cannabinoid actions, mainly CB2R-mediated against haematopoietic malignant cells, seems promising, as inhibition of cell proliferation and apoptosis and paraptosis induction have been documented.
Cannabinoid ligands appear to activate rudimentary pathways for cell survival, such as ERK, JNK, p38 MAPK, and to induce caspase synthesis, in vitro. Such data are strongly recommended to be confirmed by in vivo experiments with emphasis on cannabinoid ligands’ bioavailability and phytocannabinoid psychotropic properties.
The preliminary antitumoral ECS effects and their relative lack of important side effects render ECS a promising therapeutic target for the treatment of haematological malignancies.”
Endocannabinoid Modulation of Orbitostriatal Circuits Gates Habit Formation.
“Everyday function demands efficient and flexible decision-making that allows for habitual and goal-directed action control. An inability to shift has been implicated in disorders with impaired decision-making, including obsessive-compulsive disorder and addiction. Despite this, our understanding of the specific molecular mechanisms and circuitry involved in shifting action control remains limited. Here we identify an endogenous molecular mechanism in a specific cortical-striatal pathway that mediates the transition between goal-directed and habitual action strategies. Deletion of cannabinoid type 1 (CB1) receptors from cortical projections originating in the orbital frontal cortex (OFC) prevents mice from shifting from goal-directed to habitual instrumental lever pressing. Activity of OFC neurons projecting to dorsal striatum (OFC-DS) and, specifically, activity of OFC-DS terminals is necessary for goal-directed action control. Lastly, CB1 deletion from OFC-DS neurons prevents the shift from goal-directed to habitual action control. These data suggest that the emergence of habits depends on endocannabinoid-mediated attenuation of a competing circuit controlling goal-directed behaviors.”