Endocannabinoid Signaling: Methods and Protocols (Methods in Molecular Biology, 1412)

✍ Scribed by Mauro Maccarrone (editor)

• Publisher: Springer
• Year: 2016
• Tongue: English
• Leaves: 287
• Category: Library

✦ Synopsis

This volume encompasses all major methodologies to interrogate endocannabinoid systems (ECS) and endocannabinoids (eCBs) signaling. With increasing interest towards the manifold activities of eCBs, this book discusses the chemical, biochemical, and molecular biological assays, and activity of distinct elements of the ECS. These include membrane, nuclear receptors, biosynthetic and hydrolytic enzymes, and membrane transporters and oxidative enzymes. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Timely and cutting edge, Endocannabinoid Signaling: Methods and Protocols is a valuable resource and will help chemists, drug designers, biochemists, molecular biologists, cell biologists, pharmacologists, and (electro) physiologists navigate the mare magnum of endocannabinoid research.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Need for Methods to Investigate Endocannabinoid Signaling
1 A Modern View of the Endocannabinoid System
2 Conclusions
References
Chapter 2: Extraction and Simultaneous Quantification of Endocannabinoids and Endocannabinoid-Like Lipids in Biological Tissues
1 Introduction
2 Materials
2.1 Tissue Extraction Components
2.2 eCB Internal Standards and Calibration Standards
2.3 LC/MRM
2.4 BCA
3 Methods
3.1 Tissue Isolation and Pre-­processing
3.2 Preliminary Steps for Extraction of eCBs from Tissues
3.3 Tissue Weighing
3.4 Extraction of eCBs
3.5 LC/MRM
3.6 BCA Assay
4 Notes
References
Chapter 3: Determination of 2-Arachidonoylglycerol by μSPE-LC-MS/MS
1 Introduction
2 Materials
2.1 Specimens Collection
2.1.1 Tissue
2.1.2 Plasma
3 Methods
3.1 Extraction from Tissue
3.2 Extraction from Plasma
3.3 LC-MS/MS Analysis
4 Notes
References
Chapter 4: Analysis of Omega-3 Fatty Acid Derived N -Acylethanolamines in Biological Matrices
1 Introduction
2 Materials
2.1 Analytical Standard Solutions
2.1.1 Calibration Curve Solutions
2.1.2 Deuterated Standard Spiking Stock Solution
2.1.3 Reconstitution Solution
2.2 Solutions and Reagents
2.3 Consumables
2.4 Equipment
3 Methods
3.1 Sample Collection
3.2 Sample Extraction
3.3 LC-MS Analysis
3.4 Data Processing and Experimental Results
3.5 Modifications to the Sample Preparation Protocol for Matrices Other Than Plasma
3.6 Factors That Influence Levels of n-3 Endocannabinoids
3.7 Choice of Target Analytes
4 Notes
References
Chapter 5: Assay of CB1 Receptor Binding
1 Introduction
2 Materials
2.1 Labeled and Unlabeled Compounds
2.2 Buffers and Equipments
2.2.1 CB1 Binding Assay for Tissues
2.2.2 CB1 Binding Assay for Intact Cells
2.2.3 High-Throughput CB1 Binding Assay
3 Methods
3.1 Protocol 1: CB1 Binding Assay for Tissues
3.1.1 Preparation of Membrane Homogenates
3.1.2 Assay Using Vacuum Filtration Manifold Millipore® Model 1225
3.2 Protocol 2: CB1 Binding Assay for Adherent Living Cells
3.3 Protocol 3: CB1 Binding Assay for High-­Throughput Screening
3.4 Data Analysis
3.4.1 Protocols 1 and 2
3.4.2 Protocol 3
4 Notes
References
Chapter 6: The Displacement Binding Assay Using Human Cannabinoid CB2 Receptor-Transfected Cells
1 Introduction
2 Materials
2.1 Cell Culturing
2.2 Radioligand Displacement Binding Assay
3 Methods
3.1 Cell Culturing
3.2 Displacement Binding Assay
3.3 Analysis of the Results
4 Notes
References
Chapter 7: Assay of TRPV1 Receptor Signaling
1 Introduction
2 Materials
3 Methods
3.1 Cell Culture and Transfection
3.2 Fluo-4 AM Loading
3.3 Fura-2 AM Loading
3.4 Assay of TRP-­Mediated Elevation of Intracellular Ca2+ in Transfected HEK-293 Cells
3.5 Measuring Ca2+ in TRP Transfected HEK-293 Cells
3.6 Study of Other TRPs Using the Calcium Assay
4 Notes
References
Chapter 8: A Functional Assay for GPR55: Envision Protocol
1 Introduction
2 Materials
3 Methods
3.1 ERK1/2 Phosphorylation Assay
3.2 Data Analysis
4 Notes
References
Chapter 9: The Cyclic AMP Assay Using Human Cannabinoid CB2 Receptor-Transfected Cells
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Cyclic AMP Assay
3 Methods
3.1 Cell Culture
3.2 Cyclic AMP Assay
3.3 Analysis of the Results
4 Notes
References
Chapter 10: Assay of GTPγS Binding in Autoradiography
1 Introduction
2 Materials
2.1 Slide Preparation
2.2 Tissue Preparation
2.3 Assay Components
3 Methods
3.1 Gelatine-­Coated Slides
3.2 Tissue Preparation
3.3 Assay
3.4 Image Analysis
4 Notes
References
Chapter 11: Protocol to Study β-Arrestin Recruitment by CB1 and CB2 Cannabinoid Receptors
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Assay
2.3 Detection and Analysis
3 Methods
3.1 Cell Culture
3.2 Ligands Under Investigation
3.3 PathHunter® Assay Protocol
3.3.1 Plating Cells
3.3.2 Treatment of Cells and Incubation
3.3.3 Detection and Measurements
3.4 Data Analysis
3.5 Results
3.6 Conclusions
4 Notes
References
Chapter 12: Assay of NAT Activity
1 Introduction
2 Materials
2.1 Partial Purification of Ca-NAT from Rat Brain
2.2 Purification of Recombinant PLA/AT-2
2.3 Enzyme Assay
3 Methods
3.1 Partial Purification of Ca-NAT from Rat Brain
3.2 Purification of Recombinant PLA/AT-2
3.3 Enzyme Assay
4 Notes
References
Chapter 13: Assay of NAPE-PLD Activity
1 Introduction
2 Materials
2.1 Radiolabeled Components
2.2 Unlabeled Components
2.3 Equipment
3 Methods
3.1 Substrate Preparation
3.2 Preparation of Tissue and Cell Samples
3.3 Enzyme Assay
3.4 TLC
4 Notes
References
Chapter 14: Assay of FAAH Activity
1 Introduction
2 Materials
2.1 Chemicals
2.2 Equipment
3 Methods
3.1 Sample Preparation
3.2 Substrate Mix Preparation
3.3 Enzymatic Reaction
4 Notes
References
Chapter 15: Assay of NAAA Activity
1 Introduction
2 Materials
2.1 Preparation of NAAA from Rat Lung
2.2 Preparation of Recombinant NAAA
2.3 Synthesis of Radiolabeled Substrate
2.4 Enzyme Assay
3 Methods
3.1 Preparation of NAAA from Rat Lung
3.2 Preparation of Recombinant NAAA
3.3 Synthesis of Radiolabeled Substrate
3.4 Enzyme Assay
4 Notes
References
Chapter 16: Assay of DAGLα/β Activity
1 Introduction
2 Materials
2.1 Enzymatic Assay Components
2.2 Components for TLC
3 Methods
3.1 Enzymatic Assay
3.2 TLC Preparation and Development
4 Notes
References
Chapter 17: Assay of Monoacylglycerol Lipase Activity
1 Introduction
2 Materials
2.1 Reagents
2.1.1 Lipids
2.1.2 Buffers and Growth Medium
2.1.3 Solvents and Chemicals
2.2 Equipment
2.3 Supplies and Apparatuses
3 Methods
3.1 Enzyme Preparation
3.1.1 Cell Homogenate
3.1.2 Tissue Homogenate
3.1.3 Homogenate of MGL-­Overexpressing HeLa Cells
3.2 Setting Up the Enzyme Reaction
3.3 Lipid Extraction
3.4 LC/MS Analysis
3.5 Calculation of MGL Activity
3.5.1 Standard Curve
3.5.2 Calculations
4 Notes
References
Chapter 18: A Sensitive and Versatile Fluorescent Activity Assay for ABHD6
1 Introduction
2 Materials
2.1 Basic Incubation Cocktail
2.2 Components of the Coupled-­Enzyme System
2.3 Other Reagents
2.4 Glycerol Standards and Glycerol Quality Control
2.5 Reagents for Cell Culture
2.6 Equipment
3 Methods
3.1 Enzyme Preparation
3.2 Activity Assay
3.3 Guidelines for Calculations
4 Notes
References
Chapter 19: A Sensitive and Versatile Fluorescent Activity Assay for ABHD12
1 Introduction
2 Materials
2.1 Basic Incubation Cocktail
2.2 Components of the Coupled-­Enzyme System
2.3 Other Reagents
2.4 Glycerol Standards and Glycerol Quality Control
2.5 Reagents for Cell Culture
2.6 Equipment
3 Methods
3.1 Enzyme Preparation
3.2 Activity Assay
3.3 Guidelines for Calculations
4 Notes
References
Chapter 20: Assay of Endocannabinoid Uptake
1 Introduction
2 Materials
2.1 Components and Reagents
2.2 Cell Culture Material
2.3 Buffers and Stock Solutions
3 Methods
3.1 Cell Culture Maintenance
3.2 Three-Phase AEA Uptake Assay
3.3 Three-Phase 2-AG Uptake Assay
4 Notes
References
Chapter 21: Assay of Endocannabinoid Oxidation by Cyclooxygenase-2
1 Introduction
2 Materials
2.1 General
2.2 In Vitro Assays
2.3 Cell Assay
2.4 LC-MS/MS
3 Methods
3.1 In Vitro Assay
3.2 Cell Assay
3.3 LC-MS Analysis
4 Notes
References
Chapter 22: Oxygenation of Anandamide by Lipoxygenases
1 Introduction
2 Materials
2.1 Buffers and Solutions
2.2 Preparation of Enzymes
3 Methods
3.1 Production of Hydroperoxy- AEA
3.2 Reduction of Hydroperoxy-­AEA to Hydroxy-AEA
3.3 Purification of Hydroxy-AEA
3.4 Analytical Characterization
3.5 Chemical Data of the Oxygenated Derivatives
4 Notes
References
Chapter 23: Assay of Endocannabinoid Oxidation by Cytochrome P450
1 Introduction
2 Materials
2.1 Components for Metabolic Reactions
2.2 Components for Metabolite Extraction and Analysis
3 Methods
3.1 General Protocol for the Preparation of Tissue Mitochondria and Microsomes
3.2 Metabolic Reactions
3.3 Extraction of Metabolites and Known Standards
3.4 Liquid Chromatography-Mass Spectrometry Analysis
3.5 Data Analysis
4 Notes
References
Chapter 24: Assessing Gene Expression of the Endocannabinoid System
1 Introduction
2 Materials
2.1 RNA Isolation
2.2 Determination of RNA Yield and Integrity
2.3 RT Reaction
2.4 qRT-PCR Reaction
3 Methods
3.1 RNA Isolation
3.2 Determination of RNA Yield and Integrity
3.3 RT Reaction
3.4 qRT-PCR
3.4.1 Comparative Ct Method (ΔΔCt)
4 Notes
References
Chapter 25: Western Blotting of the Endocannabinoid System
1 Introduction
2 Materials
2.1 Buffers and Solutions
2.2 Gel Apparatus Assembly
2.3 Gel Electrophoresis and Blotting Equipment
3 Methods
3.1 Typical Tissue Preparation
3.2 Preparing Cultured Cells
3.3 Running the Gel
3.4 Transferring the Gel
3.5 Western Blot
4 Notes
Chapter 26: Quantitation of Plasma Membrane (G Protein-Coupled) Receptor Trafficking in Cultured Cells
1 Introduction
2 Materials
2.1 Plasmids and Cell Lines
2.2 Buffers and Solutions
2.3 Plate Preparation
2.4 Cell Line Preparation
2.5 Detection Equipment
3 Methods
3.1 Making Stable Cell Lines
3.2 Live Cell Screening for Expression Lines
3.3 Internalization Assay
3.4 Recycling Assay
4 Notes
References
Chapter 27: Measuring ECS Interaction with Biomembranes
1 Introduction
2 Materials
2.1 Components for Liposome Preparation
2.2 Components for Isolation of Rat Liver Membranes
2.3 Protein Solution
2.4 FRET Components
3 Methods
3.1 Liposome Preparation
3.2 Fluorescence Measurements
4 Notes
References
Chapter 28: Visualization of Endocannabinoids in the Cell
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Cover Slips
2.3 Solutions and Imaging Equipment
3 Methods
3.1 Cell Culture
3.2 Incubation with b-AEA
3.3 Labeling with b-AEA
3.4 Cell Examination
4 Notes
References
Index