Endocannabinoid Signaling: Methods and Protocols

Description

This second edition provides new and updated chapters detailing all major elements of the ECB system. Chapters guide readers through identification of drug targets, electrophysiology, computational chemistry, and machine learning. Written in the 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 protocols, and notes on troubleshooting and avoiding known pitfalls. Comprehensive and cutting-edge, Endocannabinoid Signaling: Methods and Protocols, Second Edition is a valuable resource for all researchers interested in learning more about this important and developing field. Dedication Preface Contents Contributors Chapter 1: Need for Methods to Investigate Endocannabinoid Signaling 1 A Modern View of the Endocannabinoids 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 eCBs 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: Measuring the Content of Endocannabinoid-Like Compounds in Biological Fluids: A Critical Overview of Sample Prepara... Abbreviations 1 Introduction 1.1 Sample Preparation Prior to Introduction to Mass Spectrometric Detectors Is Critical for Optimizing eCB Measurements 1.2 Additional Factors to Consider for Optimization of eCB Measurement in Biological Fluids 1.3 Is a Standardized Sample Preparation for eCBs and Related Lipids Possible? Is It Even Important? References Chapter 4: LC-MS/MS Analysis of AEA and 2-AG 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 5: Analysis of Omega-3 Fatty Acid-Derived N-Acylethanolamines in Biological Matrices 1 Introduction 2 Materials 2.1 Analytical Standard Solutions 2.2 Solutions and Reagents 2.3 Synthesis of 13- and 16-HDHEA Standards (Optional) 2.4 Consumables 2.5 Equipment 3 Methods 3.1 Sample Collection 3.2 Sample Extraction 3.3 LC-MS Analysis 3.4 Data Processing and Results 3.5 Modifications to the Sample Preparation Protocol for Matrices Other Than Plasma 3.6 Modifications to the Sample Preparation Protocol to Analyse COX-2 Metabolites of DHEA 3.7 Factors that Influence Levels of n-3 PUFA-Derived Endocannabinoids and Their Metabolites 3.8 Choice of Target Analytes 4 Notes References Chapter 6: Endocannabinoid-Binding Receptors as Drug Targets 1 Cannabis Plant Ingredients and Endocannabinoid-Binding Receptors 2 CB1 Receptors 3 CB2 Receptors 4 GPR55 5 TRPV1 Receptors 6 PPAR Receptors 7 Conclusions and Future Perspectives References Chapter 7: Assay of CB1 Receptor Binding 1 Introduction 2 Materials 2.1 Labeled, Unlabeled Compounds 2.2 Buffers, Equipment 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 4 Notes References Chapter 8: 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 9: Fluorescence-Based Assay for TRPV1 Channels 1 Introduction 2 Materials 3 Methods 3.1 Cell Culture and Transfection TRPV1 Assay 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 10: DNA-Protein-Interaction (DPI)-ELISA Assay for PPAR-γ Receptor Binding 1 Introduction 2 Materials 2.1 Buffers, Reagents 2.2 Equipments 3 Methods 3.1 Pre-assay Preparation 3.2 Assay Protocol 4 Notes References Chapter 11: Scintillation Proximity Assay (SPA)-Based Radioligand Binding for PPARα, PPARγ, and PPARδ Receptors 1 Introduction 2 Representative Tool Compounds, Clinically Evaluated and Tritiated PPAR Ligands 2.1 Synthesis of [3H]Farglitazar 5b 2.2 Synthesis of [3H]L-165041 7b 2.3 Synthesis of [3H]GW501516 8b 3 Scintillation Proximity Assay 3.1 Materials 3.1.1 Buffers 3.1.2 Proteins 3.1.3 SPA Beads 3.2 Methods 3.2.1 General Protocol 3.2.2 Conditions and Radioligands Used 3.2.3 Competitive Binding Assay 3.2.4 PPAR Binding Data for Representative Sets of Ligands References Chapter 12: Homogeneous Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Cofactor Recruitment Assay for PPARα an... 1 Introduction 2 Materials 2.1 Buffer 2.2 Protein 2.3 Cofactors 2.4 Agonists 2.5 Detection Reagents 3 Methods 3.1 General Protocol 3.2 Optimization of Protein Concentration 3.3 Cofactor Profiling 3.4 Optimization of Cofactor Concentration 3.5 Optimization of Detection Reagents 3.6 Compound-Dependent Cofactor Recruitment Profiling 4 Notes References Chapter 13: 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 14: Assay of GTPγS Binding in Autoradiography 1 Introduction 2 Materials 2.1 Slides 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 15: Cellular Assay to Study β-Arrestin Recruitment by the Cannabinoid Receptors 1 and 2 1 Introduction 2 Materials 2.1 Cell Culture 2.2 Assay 2.3 Detection and Data Processing 3 Methods 3.1 Cell Culture 3.2 Ligands Under Investigation 3.3 Cell Seeding 3.4 Cell Stimulation and Incubation 3.4.1 Antagonistic Assay 3.4.2 Agonistic Assay 3.4.3 Inverse Agonistic Assay 3.5 Detection and Measurements 3.6 Data Analysis 3.7 Results 4 Notes References Chapter 16: Endocannabinoid Metabolism and Transport as Drug Targets 1 Introduction 2 Targeting eCB Biosynthesis 2.1 DAGLα and DAGLβ 2.2 NAPE-PLD 3 Targeting eCB Degradation 3.1 FAAH 3.2 MAGL 4 Targeting eCB Transporters 5 Conclusions References Chapter 17: Assay of NAT Activity 1 Introduction 2 Materials 2.1 Partial Purification of Ca-NAT from Rat Brain 2.2 Purification of Recombinant cPLA2ε and PLAAT-2 2.3 Enzyme Assay 3 Methods 3.1 Partial Purification of Ca-NAT from Rat Brain 3.2 Purification of Recombinant cPLA2ε and PLAAT-2 3.3 Enzyme Assay 4 Notes References Chapter 18: Radiometric Assay of NAPE-PLD Activity 1 Introduction 2 Materials 2.1 Radiolabeled Components 2.2 Unlabeled Components 2.3 Equipment 3 Methods 3.1 Preparation of Substrates 3.2 Preparation of Tissue and Cell Samples 3.3 Enzyme Assay 3.4 TLC 4 Notes References Chapter 19: Fluorescence-Based NAPE-PLD Activity Assay 1 Introduction 2 Materials 2.1 Cell Culture 2.2 Lysate Preparation 2.3 In Vitro Activity Assay 3 Methods 3.1 Cell Culture and Transfection 3.2 Membrane Lysate Preparation 3.3 NAPE-PLD PED6 Activity Assay 3.4 Data Analysis 4 Notes References Chapter 20: Radiometric 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 21: Fluorimetric Assay of FAAH Activity 1 Introduction 2 Materials 2.1 Enzymatic Assay Components 2.2 Equipment 3 Methods 3.1 Enzyme and Substrate Preparation 3.2 Enzymatic Reaction 4 Notes References Chapter 22: 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 23: 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 24: Assay of Monoacylglycerol Lipase Activity 1 Introduction 2 Materials 2.1 Reagents 2.1.1 Lipids 2.1.2 Buffers 2.1.3 Solvents and Chemicals 2.2 Equipment 2.3 Supplies and Apparatus 3 Methods 3.1 Preparation of Enzyme Source 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 25: Radiometric Assay of ABHD2 Activity 1 Introduction 2 Materials 3 Methods 3.1 Cell Culture and Transfection 3.2 Substrate Mix Preparation 3.3 Enzymatic Reaction 4 Notes References Chapter 26: 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 27: 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 28: 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 3.4 LC-ESI-MS/MS Quantification 4 Notes References Chapter 29: Assessing Gene Expression of the Endocannabinoid System Components by Real-Time Quantitative Reverse Transcription... 1 Introduction 2 Materials 2.1 RNA Isolation 2.2 Determining Yield and Integrity of RNA 2.3 Reverse Transcription (RT) Reaction 2.4 qRT-PCR 3 Methods 3.1 Total RNA Isolation Procedure 3.2 Determining Yield and Integrity of Total RNA 3.3 Reverse Transcription (RT) 3.4 qRT-PCR 3.4.1 Comparative Ct Method (ΔΔCt) for Relative Quantitation of Gene Expression 4 Notes References Chapter 30: Bioinformatics of the Endocannabinoid System: Study of DNA Methylation at Rat Cnr1 Gene Promoter 1 Introduction 2 Materials 3 Methods 3.1 Find the Sequence of the Gene 3.2 Identify the Promoter Region 3.3 Identify CpG Islands in the Promoter Region 3.4 Find the Transcription Factors That Potentially Bind to the CpG Motifs in the Island 3.5 Experimentally Verify the Binding by In Vitro Assays 4 Notes References Chapter 31: DNA Methylation Analysis of Cnr1 Gene Promoter 1 Introduction 2 Materials 2.1 DNA Isolation 2.2 Bisulfite Conversion of DNA 2.3 Real-Time Methylation-Specific PCR (RT-MSP) 2.4 Pyrosequencing 3 Methods 3.1 Genomic DNA Isolation and Determination of DNA Yield 3.2 Bisulfite Conversion of DNA 3.3 Real-Time Methylation-Specific PCR (RT-MSP) 3.4 Pyrosequencing 4 Notes References Chapter 32: 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 Blotting 4 Notes Chapter 33: 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 34: Assessing CB1 Expression in the Brain by Immunohistochemical Methods: Light, Confocal, and Electron Microscopy 1 Introduction 2 Materials 2.1 Immunoperoxidase Method 2.2 Immunofluorescence Method 2.3 Electron Microscopy Method 3 Methods 3.1 Immunoperoxidase Protocol 3.2 Immunofluorescence Protocol 3.3 Pre-embedding Immunogold Technique and Electron Microscopy 4 Notes References Chapter 35: Measuring Endocannabinoid System 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 36: STORM Super-Resolution Imaging of CB1 Receptors in Tissue Preparations 1 Introduction 2 Materials 2.1 Brain Section Collection 2.2 Immunostaining 2.3 STORM Image Acquisition and Analysis 3 Methods 3.1 Brain Section Collection 3.2 Immunostaining 3.3 STORM Image Acquisition 3.4 STORM Image Analysis 4 Notes References Chapter 37: Visualization of Endocannabinoids in the Cell 1 Introduction 2 Materials 2.1 Cell Culture 2.2 Coverslips 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 Chapter 38: Electrophysiology of Endocannabinoid Signaling 1 Introduction 1.1 Electrophysiological Approaches for Dissecting eCB Signaling 2 Evaluation of CB1-Mediated Synaptic Signaling by Exogenous Stimulation 3 Evaluation of CB1-Mediated Synaptic Signaling by Endogenous Release 4 Evaluation of Tonic eCB Signaling 5 Non-CB1s Signaling and Functional Cross-Talk Between eCBs 6 Indirect Pathways for eCB-Mediated Neuronal Transmission: Neuron-Astrocyte Communication 7 Conclusions References Chapter 39: Machine Learning and Computational Chemistry for the Endocannabinoid System 1 Introduction 1.1 Machine Learning and Computational Chemistry for Drug Design 1.2 Endocannabinoid System 2 Structure-Based Drug Design 3 Virtual Screening 4 Quantitative Structure-Activity Relationship (QSAR) Models 5 De Novo Design 6 Conclusions and Outlook References Chapter 40: Virtual Reverse Screening Approach to Target Type 2 Cannabinoid Receptor 1 Introduction 2 Materials 3 Methods 3.1 Virtual Screening Data 3.2 Reverse Screening Details 3.3 Output Analysis 4 Notes References Index