Table Of ContentAngiotensin Protocols
M E T H O D S I N M O L E C U L A R M E D I C I N ETM
Angiotensin Protocols
Edited by
Donna H. Wang
Department of Medicine, Michigan State University,
East Lansing, MI
Humana Press Totowa, New Jersey
© 2001 Humana Press Inc.
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Library of Congress Cataloging in Publication Data
Main entry under title:
Methods in molecular medicine™.
Angiotensin Protocols / edited by Donna H. Wang.
p. cm. -- (Methods in molecular medicine™51 )
Includes bibliographical references and index.
ISBN 0-89603-763-0 (alk. paper)
1. Angiotensin—Laboratory manuals. I. Wang, Donna H. II. Series.
[DNLM: 1. Renin–Angiotensin System—physiology. 2. Genetic Techniques.
3. Immunoassay. 4. Regional Blood Flow—physiology.
WG 106 A588 2000]
QP572.A54 A545 2000
612.'015756—dc21 00-027598
Preface
A qualitative leap in the understanding of cardiovascular and neu-
ral regulation by the renin–angiotensin system, and of the role of this sys-
tem in tissue damage, has occurred as a result of the many recent
advances in molecular genetic techniques. The cloning of the genes for
the components of the renin–angiotensin system, the design of specific
angiotensin receptor ligands, and the use of embryonic gene targeting tech-
niques for the creation of mutant strains have established that the renin–
angiotensin system is important in blood pressure regulation, ion and fluid
homeostasis, and tissue growth and remodeling
Further investigation of the mechanisms by which this system par-
ticipates in cardiovascular regulation may shed some light on the patho-
genesis of several cardiovascular diseases, e.g., hypertension, congestive
heart failure, and chronic renal failure. Despite the promise of this system
as a target for therapeutic interventions for these diseases, there are great
challenges in the integration of the attempts to close the gap between the
traditional literature of medicine and the explosion of information from
the new technologies.
This book’s title, Angiotensin Protocols, reflects the authors’ strong
efforts to translate expert knowledge into easy-to-follow practice. The book
opens with introductory chapters, and each specialty section provides
detailed methods covering a wide variety of techniques, ranging from
genetic manipulation of targeted genes to functional studies of the renin–
angiotensin system. An especially valuable dimension of the book is the
Notessection in each chapter that places particular emphasis on the most
critical steps of the procedure. With the ever-increasing advances in
molecular biological techniques, this book is not all-inclusive. The reader
is encouraged to seek out additional sources for the latest breakthroughs.
Wehave been very fortunate to enlist a group of renowned experts
in angiotensin research for this book. I am grateful to the dedication and
v
vi Preface
contribution of our authors. In addition, I would like to thank the series
editor, John Walker, for his constant guidance and support throughout the
preparation of the book. I am also thankful to Julie Doyle for providing
enormous effort toward the timely completion of this project. Finally, many
thanks are owed to Thomas Lanigan and Craig Adams at Humana Press
for their encouragement and assistance in the successful completion of
this book.
Donna H. Wang
Contents
Preface .............................................................................................................v
Contributors.....................................................................................................xi
PART I INTRODUCTION TO THE RENIN–ANGIOTENSIN SYSTEM
1 Historical Perspective of the Renin–Angiotensin System
John E. Hall............................................................................................3
2 Three Angiotensin Paradigms in One Patient: Etiology of
Hypertension, Glomerular Hemodynamics, and Long-Term
Glomerular Protection
Cheryl L. Laffer and Fernando Elijovich...........................................23
PART II TRANSGENIC AND GENE TARGETING METHODS
3 Production and Use of Chimeric Mice
Junji Takaya, Taiji Matsusaka, and Iekuni Ichikawa.......................41
4 Genetic Manipulation of the Renin–Angiotensin System Using
Cre-loxP-Recombinase
Curt D. Sigmund and David E. Stec..................................................53
5 Systemic Delivery of a Transgene in Intact Animals by a Retroviral
Vector
Michael J. Katovich, Hong-Wei Wang, Craig H. Gelband, and
Mohan K. Raizada............................................................................67
6 Antisense Inhibition of the Renin–Angiotensin System
Dagmara Mohuczy and M. Ian Phillips.............................................83
PART III ANALYSIS OF TRANSCRIPTIONAL AND TRANSLATIONAL MECHANISMS
7 Analysis of Transcriptional Control Mechanisms I: Techniques for
Characterization of cis-Regulatory Elements
Christopher T. Sherman and Allan R. Brasier...............................107
8 Analysis of Transcriptional Control Mechanisms II: Techniques
for Characterization of trans-Acting Factors
Allan R. Brasier, Christopher T. Sherman, and
Mohammad Jamaluddin...............................................................127
vii
viii Contents
9 Analysis of Cytosolic Proteins that Bind to the 5' Leader Sequence
of the Angiotensin AT Receptor by RNA Electromobility
1
Shift Assay
Zheng Wu, Kamakshi Krishnamurthi, Koby Mok, and
Kathryn Sandberg.........................................................................151
10 In Vitro Translation of the Angiotensin AT Receptors in Wheat-
1
Germ Extracts
Hong Ji, Kamakshi Krishnamurthi, Zheng Wu, and
Kathryn Sandberg.........................................................................171
PART IV ASSAYS FOR DETERMINATION OF MRNA LEVELS
11 Quantitation of Angiotensin II Receptors by Competitive Reverse-
Transcription Polymerase Chain Reaction
Wei Wu, Aqing Yao, and Donna H. Wang.......................................195
12 Analysis of RNA by Northern-Blot Hybridization
Patricia E. Gallagher and Debra I. Diz............................................205
13 Use of the Ribonuclease Protection Assay for the Analysis and
Characterization of Target mRNAs
Scott C. Supowit and Donald J. DiPette ........................................215
14 In Situ Hybridization
Susan Riggs Runge, Zhaoyong Hu, and Marschall Runge .........223
PART V ASSAYS FOR DETERMINATION OF PROTEIN CONTENTS AND
ENZYME ACTIVITY
15 Immunohistochemical Detection of Angiotensin II Receptor
Zhi-Qin Wang and Robert M. Carey.................................................237
16 Western Blotting
Nageswara R. Madamanchi and Marschall S. Runge ..................245
17 Quantification of Angiotensin-Converting Enzyme (ACE) Activity
Qing Cheng Meng and Kathleen H. Berecek.................................257
18 Quantification of Angiotensin Peptides
Qing Cheng Meng and Kathleen H. Berecek.................................267
19 Radiolabeling of Angiotensin Peptides
Robert C. Speth and Joseph W. Harding........................................275
Contents ix
20 Assays for Radiolabel-Photoaffinity Labeling of Angiotensin
Receptors
Jacqueline Pérodin, Antony A. Boucard, Richard Leduc,
Emanuel Escher, and Gaétan Guillemette................................297
21 Radioligand Binding Assay
Gaétan Thibault and Ernesto L. Schiffrin ......................................305
22 Autoradiographic Localization and Quantification of Components
of the Renin–Angiotensin System in Tissues
Siew Yeen Chai, Andrew M. Allen, Jialong Zhuo, Ingrid Moeller,
and Frederick A. O. Mendelsohn................................................315
PART VI BIOCHEMICAL STUDIES OF THE RENIN–ANGIOTENSIN SYSTEM
23 Measurement of Intracellular Free Calcium Ion Concentration
in Vascular Smooth Muscle Cells: Fluorescence Imaging
of Cytosolic Calcium
Rhian M. Touyz and Ernesto L. Schiffrin ......................................341
24 Measurement of Phospholipidase D Activation in Vascular
Smooth Muscle Cells
Rhian M. Touyz and Ernesto L. Schiffrin ......................................355
25 Measurement of Cyclic AMP and Cyclic GMP in XenopusOocytes
Stimulated with Angiotensin II and Atrial Natriuretic Factor
Hong Ji ...............................................................................................363
26 Measurement of Nitric Oxide Formation
Christian Thorup and Leon C. Moore.............................................379
27 Measuring Renin Secretion from Juxtaglomerular Cells
Armin Kurtz and Frank Schweda.....................................................391
PART VII FUNCTIONAL STUDIES OF THE RENIN–ANGIOTENSIN SYSTEM
28 Measurement of Regional Blood Flow in the Kidney Using
Laser–Doppler Flowmetry
Richard J. Roman, David L. Mattson,
and Allen W. Cowley, Jr. .............................................................407
29 Measurement of Renal Tubular Angiotensin II
John D. Imig and L. Gabrial Navar..................................................427
