Format:
1 Online-Ressource (xxiv, 562 Seiten)
,
Illustrationen, Diagramme, Tabellen
Edition:
First edition
ISBN:
9780128121573
Series Statement:
Methods in Enzymology volume 593
Content:
Front Cover -- Cannabinoids and Their Receptors -- Copyright -- Contents -- Contributors -- Preface -- References -- Chapter One: Mouse Neuroblastoma CB1 Cannabinoid Receptor-Stimulated [35S]GTPɣS Binding: Total and Antibody-Targeted Gα P ... -- 1. Introduction -- 2. Membrane Preparations for Cultured Neuronal Cells -- 2.1. Equipment and Supplies -- 2.2. Buffers and Reagents -- 2.3. Neuroblastoma Cell Lines: Mouse N18TG2 Cell Culture Conditions -- 2.4. Procedure -- 3. Methods of Determining [35S]GTPɣS Binding to Total Gα in Membrane Preparations -- 3.1. Equipment and Supplies (PerkinElmer) -- 3.2. Buffers and Reagents -- 3.3. Procedures -- 4. Antibody-Targeted Scintillation Proximity Assay for [35S]GTPɣS Binding to Specific Gα Proteins in Membrane Preparations -- 4.1. Equipment and Supplies -- 4.2. Buffers and Reagents -- 4.3. Procedures -- 4.4. Antibody Verification by Western Blotting -- 5. Summary and Conclusion -- Acknowledgments -- References -- Chapter Two: Protocols and Good Operating Practices in the Study of Cannabinoid Receptors -- 1. Introduction -- 2. Receptor Assays -- 2.1. Competitive Radioligand Binding -- 2.1.1. Equipment -- 2.1.2. Buffers and Reagents -- 2.1.3. Note -- 2.1.4. Procedure -- 2.1.4.1. Membrane Protein Preparation -- 2.1.4.2. Siliconization of Assay Tubes -- 2.1.4.3. Radioligand Binding and Detection -- 2.2. Functional Receptor Assay-[35S]GTPγS -- 2.2.1. Equipment -- 2.2.2. Buffers and Reagents -- 2.2.3. Notes -- 2.2.4. Procedure -- 2.2.4.1. Crude Whole Cell Protein Preparation -- 2.2.4.2. Siliconization of Assay Tubes -- 2.2.4.3. Binding Assay -- 2.2.5. Notes -- 2.3. Principle Receptor Signaling Assay-cAMP -- 2.3.1. Equipment -- 2.3.2. Buffers and Reagents -- 2.3.3. Procedure -- 2.3.4. Notes -- 2.4. Non-G Protein Signaling Assay-β-Arrestin -- 2.4.1. Equipment -- 2.4.2. Buffers and Reagents -- 2.4.3. Protocol
Content:
2.4.4. Notes -- 2.5. Reporter Assays of Downstream Signaling-Serum Response Element (SRE) -- 2.5.1. Equipment -- 2.5.2. Buffers and Reagents -- 2.5.3. Procedure -- 2.5.4. Notes -- 2.6. Methods for Compound Suspension -- 2.6.1. In Vitro Cellular Assays -- 2.6.2. In Vivo Assays -- 2.6.3. Notes -- 3. Summary and Conclusions -- References -- Chapter Three: Real-Time Measurement of Cannabinoid Receptor-Mediated cAMP Signaling -- 1. Introduction -- 1.1. Canonical Cannabinoid Receptors Are GPCRs -- 1.2. Importance of cAMP Signaling -- 1.3. cAMP Signaling of Cannabinoid Receptors -- 1.4. Temporal Control of GPCR Signaling -- 1.5. Types of cAMP Biosensor -- 2. Detecting Cytoplasmic cAMP Using CAMYEL -- 2.1. Protocol -- 2.1.1. Equipment -- 2.1.2. Cell Line Considerations -- 2.1.3. Reagents -- 2.1.4. Preparation of Cells-Seeding, Transfection, Reseeding of Adherent Cells -- 2.1.5. Performing the Assay, Including Strategies for Seeing Gi vs Gs Signaling -- 2.1.6. Cell Suspension Assay Variation -- 2.2. Data Analysis -- 3. Design and Validation of V8-CAMYEL -- 3.1. Validation of V8-CAMYEL -- 3.1.1. Stable Cell Lines -- 4. Summary -- References -- Chapter Four: Techniques for the Cellular and Subcellular Localization of Endocannabinoid Receptors and Enzymes in the Ma ... -- 1. Introduction -- 2. Use of Conventional Fluorescence Microscopy to Study the ECS -- 3. Localization-Based Microscopy -- 3.1. STORM to Study the ECS -- 4. Transmission Electron Microscopy -- 5. Confocal, TEM, and STORM Microscopy Applied to Study the ECS: A Technical Comparison -- 6. FRET -- 7. Conclusions -- References -- Further Reading -- Chapter Five: Endocannabinoid Transport Proteins: Discovery of Tools to Study Sterol Carrier Protein-2 -- 1. Introduction -- 2. Materials and Methods -- 2.1. Materials -- 2.2. NBDS Displacement Assay
Content:
2.2.1. Binding Affinity of NBDS (Kd,NBDS) for SCP-2 Was Determined by Reverse and Forward Titrations -- 2.2.2. Displacement Assays Were Used to Determine Max % Displacement, EC50, and Ki -- 2.3. Computational Methods -- 3. Results and Discussion -- 3.1. Discovery of Inhibitor Probe Lead Compounds -- 3.2. SAR of Head Group-Substituted Fatty Acids -- 3.3. Hit-to-Lead Optimization -- 3.4. Hit Discovery by HTS In Silico -- 4. Conclusions -- References -- Further Reading -- Chapter Six: Lipidomics: A Corrective Lens for Enzyme Myopia -- 1. Introduction -- 2. Molecule Specifist vs Generalist -- 3. Lipoamines (aka Fatty Acid Amides -- Lipo Amino Acids -- N-Acyl Amides -- N-Acyl Amino Acids) -- 3.1. N-Acyl Ethanolamine, N-Acyl Glycine, and Novel N-Acyl Amino Acid Lipids in the Lipoamine Family -- 3.1.1. N-Acyl Ethanolamines -- 3.1.2. N-Acyl Glycines -- 3.1.3. Additional Lipoamines With Known Biological Significance -- 4. 2-Acyl Glycerols Beyond 2-AG -- 5. Prostaglandins Create Additional Complexity to the AA-Derived Signaling System -- 6. What Do Enzymes Tell Us About Lipoamines and What Do Lipoamines Tell Us about Enzymes? -- 6.1. The Many Faces of FAAH -- 7. Seeing Our Way Forward -- References -- Chapter Seven: Functional Analysis of Mitochondrial CB1 Cannabinoid Receptors (mtCB1) in the Brain -- 1. Introduction -- 2. Detection of mtCB1 -- 2.1. Immunoelectron Microscopy -- 2.1.1. Transcardial Perfusion of Animals -- 2.1.2. Preembedding Silver-Intensified Immunogold Method for Electron Microscopy -- 2.1.3. Semiquantification of Immunogold Staining -- 2.2. Immunoprecipitation and Western Blot -- 2.2.1. Isolation of Brain Mitochondria -- 2.2.2. Immunoprecipitation -- 2.2.3. Western Blotting -- 3. Impact of mtCB1 on Mitochondrial Respiration -- 3.1. Oxygen Consumption of Isolated Mitochondria -- 3.2. Oxygen Consumption of Intact Cells
Content:
3.2.1. Transfection of Cells: -- 3.2.2. Cellular Respiration Using the Oroboros Oxygraph-2k -- 4. Impact of mtCB1 on Complex I Activity -- 5. Impact of mtCB1 on ATP Levels -- 6. Impact of mtCB1 on Mitochondrial Mobility in Neurons -- 6.1. Preparation of Primary Hippocampal Cultures -- 6.2. Transfection of Primary Neurons -- 6.3. Image Acquisition -- 6.4. Analysis of Mitochondrial Mobility -- 7. Conclusions -- Acknowledgments -- References -- Chapter Eight: Modeling Neurodegenerative Disorders for Developing Cannabinoid-Based Neuroprotective Therapies -- 1. Introduction -- 2. The Biomedical Challenge of Neurodegenerative Disorders -- 3. Improving the Modeling of Neurodegenerative Disorders in Cells and Laboratory Animal Species -- 4. Cannabinoids as Neuroprotectant Agents -- 4.1. Cannabinoids Have a Broad-Spectrum Neuroprotective Profile -- 4.1.1. Effects Mediated by Endocannabinoid-Related Targets -- 4.1.2. Effects Mediated by Nonendocannabinoid Targets -- 4.2. Cannabinoid Targets Have a Key Cellular Location -- 4.3. Cannabinoids Mimic an Endogenous Protective Response -- 5. Concluding Remarks and Future Perspectives -- Acknowledgments -- References -- Chapter Nine: Metabolic Profiling of CB1 Neutral Antagonists -- 1. Introduction -- 2. Background -- 2.1. Constitutive Activity of CB1R -- 3. Computational Modeling -- 4. Chemistry and Structure Proof and Stability Ki CB1 CB2 -- 5. In Vivo Metabolic Effects -- 5.1. Methods -- 5.1.1. Animals -- 5.1.2. Experimental Protocol -- 5.1.3. Tissue Levels of PIMSR -- 5.1.4. Blood Chemistry -- 5.1.5. Glucose Tolerance (ipGTT) and Insulin Sensitivity Tests (ipIST) -- 5.1.6. Hepatic Triglyceride (TG) Content -- 5.1.7. Statistics -- 5.2. Results -- 5.2.1. PIMSR Is a Brain-Penetrant Neutral CB1R Antagonist -- 5.2.2. PIMSR Improves Metabolic Profile in Diet-Induced Obese Mice
Content:
6. PIMSR and Binge Alcoholic Hepatic Steatosis -- 6.1. Materials and Methods -- 6.1.1. Binge Alcohol-Induced Liver Steatosis -- 6.2. Results -- 6.2.1. Acute Alcohol-Induced Hepatic Steatosis and Prevention by PIMSR -- 7. Conclusions -- Acknowledgments -- References -- Chapter Ten: Ligand-Assisted Protein Structure (LAPS): An Experimental Paradigm for Characterizing Cannabinoid-Receptor L ... -- 1. Introduction -- 2. Ligand-Assisted Protein Structure -- 3. Application of LAPS to Endocannabinoid-System GPCRs -- 3.1. Contextual and Strategic Precedents -- 3.2. Proof-of-Principal Studies With a Classical Cannabinoid Probe, AM841 -- 3.3. Applied LAPS Methodology for Characterizing Cannabinoid-Receptor-Binding Motifs -- 3.3.1. hCB2R Biarylpyrazole Antagonist/Inverse Agonist Binding Motif -- 3.3.2. hCB2R Classical Cannabinoid Agonist Binding Motif -- 4. Conclusions -- Acknowledgments -- References -- Chapter Eleven: New Methods for the Synthesis of Cannabidiol Derivatives -- 1. Introduction -- 2. Synthesis of CBD Derivatives -- 2.1. Synthesis of CBD -- 2.2. Synthesis of Selected CBD Derivatives -- 2.3. Synthesis of N-Heterocyclic Derivatives of CBD -- 3. Experimental Details for Schemes 13 and 14 -- 3.1. Materials -- 3.2. Methods -- 4. Conclusions -- Acknowledgments -- References -- Chapter Twelve: Approaches to Assess Biased Signaling at the CB1R Receptor -- 1. Introduction -- 2. Quantifying CB1R Ligand Bias -- 3. Assays Used to Examine CB1R Ligand Bias -- 3.1. G Protein Coupling -- 3.2. β-Arrestin Recruitment -- 3.3. Inhibition of cAMP Accumulation -- 3.4. Changes in Ca2+ -- 3.5. Receptor Internalization -- 3.6. Protein Phosphorylation and Changes in Gene Expression -- 4. Important Considerations and Caveats -- 4.1. Cell Type and Receptor Density -- 4.2. Proximal vs Distal Effects to the Receptor -- 4.3. Ligand Kinetics
Content:
4.4. Applying Measurements of Bias to Allosteric Ligands
Additional Edition:
ISBN 9780128121566
Additional Edition:
Erscheint auch als Druck-Ausgabe Reggio, Patricia H Cannabinoids and Their Receptors Saint Louis : Elsevier Science,c2017 ISBN 9780128121566
Language:
English
Keywords:
Cannabinoide
;
Rezeptor
;
Aufsatzsammlung
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