Adrenergic receptors are important modulators in the sympathetic c- trol of various metabolic processes in the central and peripheral nervous s- tems. These receptors are localized at multiple sites throughout the central nervous system (CNS) and serve as important regulators of CNS-mediated behavior and neural functions, including mood, memory, neuroendocrine c- trol, and stimulation of autonomic function. Adrenergic Receptor Protocols consists of 35 chapters dealing with va- ous aspects of adrenergic receptor analyses, including the use of genetic, RNA, protein expression, transactivator, second messenger, immunocytochemical, electrophysiological, transgenic, and in situ hybridization approaches. This volume details the use of various methods to examine the adrenergic receptor system, using aspects of the genetic flow of information as a guide (DNA? RNA ? transactivator ? protein expression ? second messenger analyses ? cellular analyses ? transgenic whole animal approaches). Adrenergic Receptor Protocols displays step-by-step methods for s- cessful replication of experimental procedures, and would be useful for both experienced investigators and newcomers in the field, including those beg- ning graduate study or undergoing postdoctoral training. The Notes section contained in each chapter provides valuable troubleshooting guides to help develop working protocols for your laboratory. With Adrenergic Receptor Protocols, it has been my intent to develop a comprehensive collection of modern molecular methods for analyzing adrenergic receptors. I would like to thank the many chapter authors for their contributions.
Part I. Genetic Analysis. Construction of Libraries for Isolation of Adrenergic Receptor Genes, Margaret A. Scofield, Jean D. Deupree, and David B. Bylund. Isolation of Adrenergic Receptor Genes, Margaret A. Scofield, Jean D. Deupree, and David B. Bylund. Analyses of Adrenergic Receptor Sequences, Jean D. Deupree, Margaret A. Scofield, and David B. Bylund. Polymerase Chain Reaction Screening of Genomic Libraries for Adrenergic Receptor Genes, Dianne M. Perez and Michael J. Zuscik. Solution-Phase Library Screening for Identification of Rare Adrenergic Receptor Clones, Dianne M. Perez and Michael J. Zuscik. Genetic Polymorphisms of Adrenergic Receptors, Ian P. Hall, Amanda P. Wheatley, and Jane C. Dewar. Part II. RNA Analysis. Northern Blot Analyses Detecting Adrenergic Receptor mRNAs, Judith C. W. Mak. Ribonuclease Protection Assay for the Detection of b1-Adrenergic Receptor RNA, Yong-Feng Yang and Curtis A. Machida. Determination of Adrenergic Receptor mRNAs by Quantitative Reverse Transcriptase-Polymerase Chain Reactions, Stefan Engelhardt and Martin J. Lohse. Nuclear Run-On Assays for Measurement of Adrenergic Receptor Transcription Rate, Zhuo-Wei Hu and Brian B. Hoffman. Primer Extension Methods for Determination of b1-Adrenergic Receptor mRNA Start Sites, Yi-Tang Tseng and James F. Padbury. Part III. Expression Analysis. Use of Eukaryotic Vectors for the Expression of Adrenergic Receptors, Stéphane Schaak, Jean-Christophe Devedjian, and Hervé Paris. Expression of b-Adrenergic Receptors in Recombinant Baculovirus-Infected Insect Cells, A. Donny Strosberg and Jean-Luc Guillaume. Expression of b-Adrenergic Receptors in E. coli, Ralf Jockers and A. Donny Strosberg. Use and Pharmacological Analysis of Established and Transfected Cell Lines Expressing Adrenergic Receptors, Hongying Zhong and Kenneth P. Minneman. Transient Transfection and Adrenergic Receptor Promoter Analysis, Yi-Tang Tseng and James F. Padbury. Antisense RNA/DNA-Based Techniques to Probe Adrenergic Receptor Function, Hsien-yu Wang, Fubao Lin, and Craig C. Malbon. Targeted Disruption of Adrenergic Receptor Genes, Daniel K. Rohrer. Part IV. Receptor and Second Messenger Analysis. Development of Antibodies to Adrenergic Receptors, Suleiman W. Bahouth. Western Blot Detection of Adrenergic Receptors, Suleiman W. Bahouth. UV Crosslinking of Adrenergic Receptors and Ligands: Detection by SDS-PAGE, Michael K. Sievert, John F. Resek, Zhongren Wu, Yajing Rong, and Arnold E. Ruoho. Detection of b-Adrenergic Receptors by Radioligand Binding, Cheryl D. Dunigan, Patricia K. Curran, and Peter H. Fishman. Assessing Adrenergic Receptor Conformation Using Chemically Reactive Fluorescent Probes, Anne Dam Jensen and Ulrik Gether. Biochemical Methods for Detection and Measurement of Cyclic AMP and Adenylyl Cyclase Activity, Steven R. Post, Rennolds S. Ostrom, and Paul A. Insel. Assay of Arachidonic Acid Release Coupled to a1- and a2-Adrenergic Receptors, Lincoln Edwards and Paul Ernsberger. Patch-Clamp Recording Methods for Examining Adrenergic Regulation of Potassium Currents in Ocular Epithelial Cells, Jennifer S. Ryan, Chanjuan Shi, and Melanie E. M. Kelly. Part V. Transactivator Analysis. Southwestern Blots for Detection of a DNA Binding Protein Recognizing the a1B-Adrenergic Receptor Gene Promoter, Bin Gao and George Kunos. DNase I Footprinting Analysis of Transcription Factors Recognizing Adrenergic Receptor Gene Promoter Sequences, Bin Gao and George Kunos. Electrophoretic Mobility Shift Assay for Detection of DNA Binding Proteins Recognizing b-Adrenergic Receptor Gene Sequences, Philbert Kirigiti and Curtis A. Machida. Determination of mRNA Stability and Characterization of Proteins Interacting with Adrenergic Receptor mRNAs, Burns C. Blaxall and J. David Port. Part VI. Microscopy Analysis. Use of Immunohistochemistry and Confocal Microscopy in the Detection of Adrenergic Receptors, Ruth L. Stornetta. Distribution of a2A- and a2C-Adrenergic Receptor Immunoreactivity in the Central Nervous System, Diane L. Rosin. Quantitative Light Microscopic Autoradiography of a2-Adrenergic Radioligand Binding Sites, Ursula H. Winzer-Serhan and Frances M. Leslie. In Situ Hybridization of Adrenergic Receptor mRNA in Brain, Mark Stafford Smith and Debra A. Schwinn. Use of Electron Microscopy in the Detection of Adrenergic Receptors, Chiye Aoki, Sarina Rodrigues, and Hitoshi Kurose. Index