Current Protocols in Pharmacology
Table of Contents

Foreword by Sir James Black
Preface
Contributors

1 Receptor Binding
1.1 Receptors as Drug Targets
1.2 Receptor Theory
1.3 Practical Aspects of Radioligand Binding
1.4 Characterization of Opioid Receptors
1.5 Characterization of Adrenoceptors
1.6 Characterization of Dopamine Receptors
1.7 Characterization of GABA Receptors
1.8 Characterization of Neuronal Nicotinic Acetylcholine Receptors
1.9 Characterization of P1 (Adenosine) Purinoceptors
1.10 Characterization of Angiotensin II Receptors
1.11 Characterization of Neuropeptide Y Receptors
1.12 Characterization of Cholecystokinin Recptors
1.13 Characterization of CRF Receptors
1.14 Characterization of Wild-Type Excitatory Amino Acid Ion Channels Receptors
1.15 Characterization of Tachykinin Receptors
1.16 Characterization of Benzodiazepine Binding to GABA_A Receptors
1.17 Characterization of Potassium Channel Binding
1.18 Characterization of the Picrotoxin Site of GABA_A Receptors
1.19 Characterization of Histaminergic Receptors
1.20 Radioligand Assay of Drug Activity at the Glycine Site of the NMDA Receptor
1.21 Overview of Receptor Allosterism
1.22 Quantification of Allosteric Interactions at G Protein Coupled Receptors Using Radioligand Binding Assays
1.23 Characterization of 5-HT_1A,B and 5-HT_2A,C Serotonin Receptor Binding
1.24 Characterization of Chemokine Receptors
1.25 Characterization of Calcium Channel Binding 
1.26 Characterization of Cannabinoid Receptors 
1.27 Radioligand Binding Assays for the Glycine Site on N-Methyl-D-Aspartate Receptors
1.28 Characterization of Melanocortin Receptors
1.29 Characterization of Neurotensin Receptors
1.30 Characterization of CGRP Receptor Binding
1.31 Overview of Approaches to the Identification of Inhibitors of Cytokine Action

2 Signal Transduction
2.1 Overview of Signal Transduction
2.2 Adenylate and Guanylate Cyclase Assays
2.3 Overview of Phosphoinositide Hydrolysis
2.4 Nitric Oxide Synthase Assays
2.5 Analysis of Early Gene Responses
2.6 Agonist-Stimulated [³⁵S]GTPgS Binding
2.7 Assay of Receptor-Stimulated Phosphoinositide Turnover

3 Enzyme Assays
3.1 Cyclooxygenase Assays
3.2 HIV Protease Assays
3.3 Topoisomerase Assays
3.4 Protein Farnesyltransferase Assays
3.5 Protein Tyrosine Kinase Activity Assays
3.6 Monoamine Oxidase Assays
3.7 Characterization of Matrix Metalloproteinase Inhibitors: Enzymatic Assays 
3.8 In Vitro Enzymatic Assays of Protein Tyrosine Phosphatase 1B
3.9 Cytochrome P450 Assays 
3.10 Measurement of Glutamate Carboxypeptidase II (NAALADase) Enzyme Activity by the Hydrolysis of [³H]-N Acetylaspartylglutamate (NAAG) 
3.11 In Vitro Enzymatic Assays for Ser/Thr-Selective Protein Kinases 

4 Isolated Tissues
4.1 Overview of Receptor Interactions of Agonists and Antagonists
4.2 Overview of Length-Tension Relationships in Isolated Tissues
4.3 Isolated Cardiac Muscle Assays
4.4 Isolated Blood Vessel Assays
4.5 a- Adrenoceptor Assays
4.6 b- Adrenoceptor Assays
4.7 P1 (Adenosine) Purinoceptor Assays
4.8 Opioid Receptor Assays
4.9 Angiotensin II Receptor Assays
4.10 Tachykinin Receptor Assays
4.11 Neuropeptide Y (NPY) Receptor Assays
4.12 Nicotinic Acetylcholine Receptor Assays
4.13 Cholecystokinin (CKK) Assays
4.14 Dopamine 2 (DA-2) Receptor Assays Using Sympathetic Nerve Terminals
4.15 Muscarinic Receptor Assays
4.16 Cysteinyl Leukotriene Receptor Assays
4.17 Histamine Receptor Assays 
4.18 Prostanoid Receptor Assays
4.19 5-Hydroxytryptamine Receptor Assays7 

5 Animal Models of Disease
5.1 Models of Cardiovascular Disease: Measurement of Arrhythmogenic/Antiarrhythmic Activity in the Guinea Pig and Antihypertensive Activity in the Conscious Rat
5.2 Models of Pulmonary Disease: Acute and Chronic Allergic Asthma in the Monkey and Acute and Chronic Viral Pulmonitis in the Mouse
5.3 Gastrointestinal Models: Intestinal Transit and Ulcerogenic Activity in the Rat
5.4 Models of Inflammation: Carrageenan-Induced Paw Edema in the Rat
5.5 Models of Inflammation: Adjuvant-Induced Arthritis in the Rat
5.6 Models of Inflammation: Carrageenan Air Pouch in the Rat
5.7 Models of Pain: Hot-Plate and Formalin Tests in Rodents
5.8 Models of Affective Illness: Forced Swimming and Tail Suspension Tests in Rodents
5.9 Affective Illness: Chronic Mild Stress
5.10 Urogenital Dysfunction: Benign Prostatic Hyperplasia
5.11 Models of Urogenital Dysfunction: Urinary Urge Incontinence (UUI)
5.12 Models of Urogenital Dysfunction: Volume-Evoked Micturition Reflex (VEMR) in the Conscious Rat
5.13 Models of Dimentia: Delayed Alternation in Aged Rats
5.14 Spatial Discrimination Water Maze Test in Septal-Lesioned Rats
5.15 Models of Schizophrenia: PPI of Startle in Rats
5.16 Models of Anxiety: Stress-Induced Hyperthermia in Group-Housed Mice
5.17 Models for Assessing Antipsychotics: Amphetamine-Induced Hyperactivity and Stereotypies in Mice
5.18 Models of Anxiety: Ultrasonic Vocalizations of Isolated Rat Pups
5.19 Models for Environmentally Induced Eating Disorders: Dietary Hyperphagia and Anorexia Nervosa
5.20 Overview of the Use of Transgenic Animals in CNS Drug Discovery
5.21 Models of Renal Insufficiency: The Anti-Thy-1.1 Model of Acute Proliferative Glomerulonephritis
5.22 Models of Epilepsy: Electroshock and Chemical Induced Convulsions in the Mouse
5.23 Characterization of Matrix Metalloproteinase Inhibitors: Angiogenesis and Tumor Models 
5.24 Classic In Vivo Cancer Models: Three Examples of Mouse Models Used in Experimental Therapeutics 
5.25 Overview of Animal Models of Asthma 
5.26 Guinea Pig Models of Asthma 
5.27 Models of Cardiac Ischemia-Reperfusion Injury in Dogs and Rats 
5.28 Intraperitoneal and Subcutaneous Tumor Models for Assessing Anti-Neoplastic Agents in Rodents 
5.29 Electrocardiographic Toxicity in the Guinea Pig 
5.30 Social Recognition Task in the Rat
5.31 Emetic Liability Testing in Ferrets
5.32 Models of Neuropathic Pain in the Rat
5.33 Electrical Amygdala Kindling
5.34 A Rat Model of Postoperative Pain
5.35 Models of Muscle Pain: Carrageenan Model and Acidic Saline Model
5.36 Models of Visceral Pain: Colorectal Distension (CRD)
5.37 Model of Attention Deficit Hyperactivity Disorder: Five-Trial, Repeated Acquisition Inhibitory Avoidance in Spontaneously Hypertensive Rat Pups
5.38 Animal Tests of Anxiety

6 Molecular Biology
6.1 Overview of Receptor Cloning
6.2 Receptor Reporter Systems
6.3 Expression of Cloned Receptors in Mammalian Cell Lines
6.4 Isotopic Assays for Reporter Gene Activity
6.5 Nonisotopic Assays for Reporter Gene Activity
6.6 Use of the A. Victoria Green Fluorescent Protein to Study Protein Dynamics In Vivo
6.7 Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
6.8 Mobility Shift DNA-Binding Assay Using Gel Electrophoresis 
6.9 Tissue Expression Profiling Using Real-Time PCR 
6.10 Overview of Pharmacogenetics

7 Pharmacokinetics
7.1 Overview of Pharmacokinetics
7.2 Estimating Intestinal Mucosal Permeation of Compounds Using Caco-2 Cell Monolayers
7.3 Overview of Drug Product Development 
7.4 In Vivo Measurement of Blood-Brain Barrier Permeability 
7.5 Determination of Compound Binding to Plasma Proteins 
7.6 Determination of the Stability of Drugs in Plasma
7.7 Uptake Studies for Evaluating Activity of Efflux Transporters in a Cell Line Representative of the Blood-Brain Barrier
7.8 In Vitro Drug Metabolism Using Liver Microsomes
7.9 Hepatic Clearance and Drug Metabolism Using Isolated Perfused Rat Liver  

8 Receptor/Enzyme Localization
8.1 In Vitro Autoradiography
8.2 Autoradiographic Localization of Growth Factor Receptors in Neuronal Tissues

9 Drug Discovery Technologies
9.1 Overview of Chemical Diversity
9.2 Cell-Based Assays Using the Fluorometric Imaging Plate Reader (FLIPR)
9.3 Overview of Combinatorial Chemistry
9.4 Overview of High-Throughput Screening
9.5 Screening in Constitutively Active Receptor  Systems
9.6 Overview of Microarrays in Drug Discovery and Development
9.7 Overview on the Use of Therapeutic Antibodies in Drug Discovery

10 Safety Pharmacology/Toxicology
10.1 Overview of Safety Pharmacology
10.2 Models of Inflammation: Gastrointestinal Ulceration in the Rat
10.3 Toxicology in Drug Discovery and Development
10.4 Place Reference Test in Rodents 
10.5 Substance Abuse: Self-Administration in Monkeys
10.6 Vigilance-Controlled Quantified EEG in Safety Pharmacology
10.7 An Overview of QT Interval Assessment in Safety  Pharmacology
10.8 Patch-Clamp Studies of Human Cardiac Ion Channels in the Evaluation of Cardiac Electrophysiological Effects of Compounds
10.9 Respiratory Function Assessment in Safety Pharmacology
10.10 Primary Observation (Irwin) Test in Rodents for Assessing Acute Toxicity of a Test Agent and its Effects on Behavior and Physiological Function

11 Electrophysiological Techniques
11.1 Overview of Electrophysiology
11.2 Electrophysiological Analysis of G Protein-Coupled Receptors in Mammalian Neurons
11.3 The Action Potential of the Purkinje Fiber: An In Vitro Model for Evaluation of the Proarrhythmic Potential of Cardiac and Noncardiac Drugs 
11.4 Use of Electrophysiological Methods in the Study of Recombinant and Native Neuronal Ligand-Gated Ion Channels
11.5 Electrophysiological Analysis of Heterologously Expressed Kv and SK/IK Potassium Channels (New, March 2003)
11.6 Electrophysiological Analysis of ATP-Sensitive Potassium Channels in Mammalian Cells and Xenopus Oocytes
11.7 Overview of Electrophysiological Characterization of Neuronal Nicotinic Acetylcholine Receptors
11.8 Electrophysiological Analysis of Tetrodotoxin-Resistant Sodium Channel Pharmacology
11.9 Electrophysiological Characterization of Recombinant and Native P2X Receptors

12 In Vitro Cellular Assays
12.1 Overview of Cell and Tissue Culture Techniques
12.2 Chondrocyte Culture and Assay
12.3 Preparation of Nuclear and Cytoplasmic Extracts from Mammalian Cell
12.4 Cellular Assays of Chemokine Receptor Activation
12.5 Production and Use of HIV-1 Luciferase Reporter Viruses
12.6 Methods of Measuring Internalization of G Protein—Coupled Receptors
12.7 Measurement of VLA-4/CS-1 and VLA-4/VCAM Adhesion Inhibition
12.8 Measurement of Cell Death in Mammalian Cells  

Appendices
A.1 NOMENCLATURE AND USEFUL DATA