Adrenergic Receptor Protocols

562 Pages·2000·3.54 MB·English

by  Margaret A. Scofield, Jean D. Deupree, David B. Bylund (auth.), Curtis A. Machida (eds.)

Adrenergic Receptor Protocols
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M E T H O D S I N M O L E C U L A R B I O L O G Y™
Adrenergic Receptor
Protocols
Edited by
Curtis A. Machida
Oregon Regional Primate Research Center
Oregon Health Sciences University
Beaverton, OR
Humana Press Totowa, New Jersey
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Preface
Adrenergic receptors are important modulators in the sympathetic con-
trol of various metabolic processes in the central and peripheral nervous sys-
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 con-
trol, and stimulation of autonomic function.
Adrenergic Receptor Protocols consists of 35 chapters dealing with vari-
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 suc-
cessful replication of experimental procedures, and would be useful for both
experienced investigators and newcomers in the field, including those begin-
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.
They are all experts in various aspects of adrenergic receptors, and I appreci-
ate their efforts and hard work in developing comprehensive chapters. As
Volume Editor, it was a privilege to preview the development of Adrenergic
Receptor Protocols, and to acquire insight on the various methodological
approaches from different contributors. I would like to thank Professor John
Walker, Series Editor for Methods in Molecular Biology, for his guidance and
help in the development of this volume, and Thomas Lanigan, President of
Humana Press. I would also like to thank Carol Houser for her administrative
assistance in the preparation of manuscripts, and for members of my labora-
v
vi Preface
tory group, who have been helpful in the early stages of identification of
potential chapter topics and contributors. Special thanks are extended to my
wife, Dr. Cindy Machida, and my daughter, Cerina, for their support during
the long hours involved in the compilation and editing of this volume.
Curtis A. Machida
Contents
Preface .............................................................................................................v
List of Contributors .......................................................................................... xi
PART I. GENETIC ANALYSIS ............................................................................... 1
1 Construction of Libraries for Isolation of Adrenergic Receptor Genes
Margaret A. Scofield, Jean D. Deupree, and David B. Bylund ........ 3
2 Isolation of Adrenergic Receptor Genes
Margaret A. Scofield, Jean D. Deupree, and David B. Bylund ...... 37
3 Analyses of Adrenergic Receptor Sequences
Jean D. Deupree, Margaret A. Scofield, and David B. Bylund ...... 53
4 Polymerase Chain Reaction Screening of Genomic Libraries
for Adrenergic Receptor Genes
Dianne M. Perez and Michael J. Zuscik ........................................... 73
5 Solution-Phase Library Screening for Identification
of Rare Adrenergic Receptor Clones
Dianne M. Perez and Michael J. Zuscik ........................................... 91
6 Genetic Polymorphisms of Adrenergic Receptors
Ian P. Hall, Amanda P. Wheatley, and Jane C. Dewar ................. 117
PART II. RNA ANALYSIS ................................................................................ 127
7 Northern Blot Analyses Detecting Adrenergic Receptor mRNAs
Judith C. W. Mak ............................................................................... 129
8 Ribonuclease Protection Assay for the Detection of β1-Adrenergic
Receptor RNA
Yong-Feng Yang and Curtis A. Machida ....................................... 139
9 Determination of Adrenergic Receptor mRNAs by Quantitative
Reverse Transcriptase-Polymerase Chain Reactions
Stefan Engelhardt and Martin J. Lohse ......................................... 155
10 Nuclear Run-On Assays for Measurement of Adrenergic Receptor
Transcription Rate
Zhuo-Wei Hu and Brian B. Hoffman ............................................... 169
vii
viii Contents
11 Primer Extension Methods for Determination of β -Adrenergic
1
Receptor mRNA Start Sites
Yi-Tang Tseng and James F. Padbury ........................................... 181
PART III. EXPRESSION ANALYSIS ...................................................................... 187
12 Use of Eukaryotic Vectors for the Expression of Adrenergic Receptors
Stéphane Schaak, Jean-Christophe Devedjian,
and Hervé Paris ............................................................................ 189
13 Expression of β-Adrenergic Receptors in Recombinant
Baculovirus-Infected Insect Cells
A. Donny Strosberg and Jean-Luc Guillaume .............................. 207
14 Expression of β-Adrenergic Receptors in E. coli
Ralf Jockers and A. Donny Strosberg ........................................... 215
15 Use and Pharmacological Analysis of Established
and Transfected Cell Lines Expressing Adrenergic Receptors
Hongying Zhong and Kenneth P. Minneman ................................ 221
16 Transient Transfection and Adrenergic Receptor Promoter Analysis
Yi-Tang Tseng and James F. Padbury ........................................... 235
17 Antisense RNA/DNA-Based Techniques to Probe Adrenergic
Receptor Function
Hsien-Yu Wang, Fubao Lin, and Craig C. Malbon ........................ 241
18 Targeted Disruption of Adrenergic Receptor Genes
Daniel K. Rohrer ................................................................................ 259
PART IV. RECEPTOR AND SECOND MESSENGER ANALYSIS ................................... 279
19 Development of Antibodies to Adrenergic Receptors
Suleiman W. Bahouth ....................................................................... 281
20 Western Blot Detection of Adrenergic Receptors
Suleiman W. Bahouth ....................................................................... 301
21 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 ............................................ 315
22 Detection of β-Adrenergic Receptors by Radioligand Binding
Cheryl D. Dunigan, Patricia K. Curran, and Peter H. Fishman ... 329
23 Assessing Adrenergic Receptor Conformation Using Chemically
Reactive Fluorescent Probes
Anne Dam Jensen and Ulrik Gether ............................................... 345
24 Biochemical Methods for Detection and Measurement
of Cyclic AMP and Adenylyl Cyclase Activity
Steven R. Post, Rennolds S. Ostrom, and Paul A. Insel ............. 363
Contents ix
25 Assay of Arachidonic Acid Release Coupled
to α - and α -Adrenergic Receptors
1 2
Lincoln Edwards and Paul Ernsberger .......................................... 375
26 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 ........... 391
PART V. TRANSACTIVATOR ANALYSIS ............................................................... 409
27 Southwestern Blots for Detection of a DNA Binding Protein
Recognizing the α -Adrenergic Receptor Gene Promoter
1B
Bin Gao and George Kunos ............................................................ 411
28 DNase I Footprinting Analysis of Transcription Factors Recognizing
Adrenergic Receptor Gene Promoter Sequences
Bin Gao and George Kunos ............................................................ 419
29 Electrophoretic Mobility Shift Assay for Detection of DNA Binding
Proteins Recognizing β-Adrenergic Receptor Gene Sequences
Philbert Kirigiti and Curtis A. Machida .......................................... 431
30 Determination of mRNA Stability and Characterization of Proteins
Interacting with Adrenergic Receptor mRNAs
Burns C. Blaxall and J. David Port ................................................. 453
PART VI. MICROSCOPY ANALYSIS ..................................................................... 467
31 Use of Immunohistochemistry and Confocal Microscopy
in the Detection of Adrenergic Receptors
Ruth L. Stornetta............................................................................... 469
32 Distribution of α - and α -Adrenergic Receptor Immunoreactivity
2A 2C
in the Central Nervous System
Diane L. Rosin ................................................................................... 475
33 Quantitative Light Microscopic Autoradiography of α -Adrenergic
2
Radioligand Binding Sites
Ursula H. Winzer-Serhan and Frances M. Leslie .......................... 507
34 In Situ Hybridization of Adrenergic Receptor mRNA in Brain
Mark Stafford Smith and Debra A. Schwinn ................................. 519
35 Use of Electron Microscopy in the Detection of Adrenergic Receptors
Chiye Aoki, Sarina Rodrigues, and Hitoshi Kurose ..................... 535
Index ............................................................................................................ 565
Contributors
CHIYE AOKI • Center for Neural Science, New York University, New York, NY
SULEIMAN W. BAHOUTH • Department of Pharmacology, University
of Tennessee, Memphis, TN
BURNS C. BLAXALL • Department of Pharmacology, University of Colorado
Health Sciences Center, Denver, CO
DAVID B. BYLUND • Department of Pharmacology, University of Nebraska
Medical Center, Omaha, NE
PATRICIA K. CURRAN • Membrane Biochemistry Section, Laboratory of
Molecular and Cellular Neurobiology, National Institute of Neurological
Disorders and Stroke, National Institutes of Health, Bethesda, MD
JEAN D. DEUPREE • Department of Pharmacology, University of Nebraska
Medical Center, Omaha, NE
JEAN-CHRISTOPHE DEVEDJIAN • Laboratoire d’Ingénierie des Systèmes
Macromoléculaires, Marseille, France
JANE C. DEWAR • Division of Therapeutics, University Hospital,
Nottingham, UK
CHERYL D. DUNIGAN • Membrane Biochemistry Section, Laboratory of
Molecular and Cellular Neurobiology, National Institute of Neurological
Disorders and Stroke, National Institutes of Health, Bethesda, MD
LINCOLN EDWARDS • Departments of Nutrition and Pharmacology, Case
Western Reserve University School of Medicine, Cleveland, OH
STEFAN ENGELHARDT • Institut für Pharmakologie, Universität Würzburg,
Germany
PAUL ERNSBERGER • Departments of Nutrition and Pharmacology, Case
Western Reserve University School of Medicine, Cleveland, OH
PETER H. FISHMAN • Membrane Biochemistry Section, Laboratory of
Molecular and Cellular Neurobiology, National Institute of Neurological
Disorders and Stroke, National Institutes of Health, Bethesda, MD
BIN GAO • Department of Pharmacology and Toxicology, Medical College
of Virginia, Richmond, VA
ULRIK GETHER • Division of Molecular and Cellular Physiology,
Department of Medical Physiology, The Panum Institute,
University of Copenhagen, Denmark
xi