Table Of ContentMethods in
Molecular Biology 1020
Thomas Krieg
Robert Lukowski Editors
Guanylate
Cyclase
and Cyclic GMP
Methods and Protocols
M M B ™
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hat fi eld, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651
Guanylate Cyclase
and Cyclic GMP
Methods and Protocols
Edited by
Thomas Krieg
Department of Medicine, Addenbrooke’s Hospital
University of Cambridge, Cambridge, UK
Robert Lukowski
Department of Pharmacology, Toxicology and Clinical Pharmacy
Universität Tübingen, Tübingen, Germany
Editors
Thomas Krieg Robert Lukowski
Department of Medicine Department of Pharmacology
Addenbrooke’s Hospital Toxicology and Clinical Pharmacy
University of Cambridge Universität Tübingen
Cambridge, UK Tübingen, Germany
ISSN 1064-3745 ISSN 1940-6029 (electronic)
ISBN 978-1-62703-458-6 ISBN 978-1-62703-459-3 (eBook)
DOI 10.1007/978-1-62703-459-3
Springer New York Heidelberg Dordrecht London
Library of Congress Control Number: 2013937718
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Preface
Since the Nobel Prize in Physiology or Medicine was awarded to Robert Furchgott, Louis
Ignarro, and Ferid Murad in 1998 for the discovery of nitric oxide (NO) as an important
signalling molecule, the downstream pathway of NO has attracted considerable interest in
various physiological and pathophysiological conditions. To date, soluble guanylate
cyclase(s), cyclic guanosine 3’-5’-monophosphate (cGMP), protein kinase G (PKG) (also
known as cGMP-dependent protein kinase), and cGMP-activated or -inactivated phospho-
diesterases (PDEs) are by far the best-characterized elements of the downstream signalling.
In the past years each of these structures has been of intense scientifi c interest not only as
important signalling molecules but also as highly promising drug targets.
It is immensely challenging to measure NO and the spatiotemporal profi le of its down-
stream effectors and targets in vitro or in vivo to unravel their roles in physiological condi-
tions as well as various diseases. Recently, many groundbreaking steps have been made
towards a better understanding of the NO/cGMP/PKG pathway, its components, sub-
strates, and its localization within a given cell. These advances were possible only due to the
development of sophisticated new techniques in the fi eld.
This book seeks to provide an overview of novel techniques to identify various elements
of the NO/cGMP/PKG pathway and further characterize their function, signalling, local-
ization, and importance on a cellular level and in whole animal models providing a higher
patho-/physiological integration and relevance.
The fi rst two chapters briefl y review the current state of research and methodology in
the fi eld and might serve as a reminder for the expert or an introduction for anybody new
in this fast-evolving, exciting area.
The following 14 chapters offer detailed step-by-step instructions of each method,
including a full list of materials and reagents, as well as useful tips to avoid common pitfalls.
We hope that readers will fi nd Guanylate Cyclase and Cyclic GMP: Methods and Protocols a
comprehensive overview of current methods and a useful guide towards the possibility to
apply these techniques to their own research. In addition, some of the chapters use disease
models in order to demonstrate the applicability of the method in a currently ongoing
research area.
Finally, we would like to thank all the authors for their excellent contributions to this
volume and all the time and effort that went into it. We are particularly grateful for the
guidance and support from the senior editor of the M ethods in Molecular Biology series,
John Walker.
Cambridge, UK Thomas Krieg
Tübingen, Germany Robert Lukowski
v
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 NO/cGMP: The Past, the Present, and the Future . . . . . . . . . . . . . . . . . . . . . . 1
Michael Russwurm, Corina Russwurm, Doris Koesling,
and Evanthia Mergia
2 cGMP-Dependent Protein Kinases (cGK). . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Franz Hofmann and Jörg W. Wegener
3 Enzyme Assays for cGMP Hydrolyzing Phosphodiesterases. . . . . . . . . . . . . . . . 51
Sergei D. Rybalkin, Thomas R. Hinds, and Joseph A. Beavo
4 Radioimmunoassay for the Quantification of cGMP Levels
in Cells and Tissues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Ronald Jäger, Dieter Groneberg, Barbara Lies, Noomen Bettaga,
Michaela Kümmel, and Andreas Friebe
5 Hyperspectral Imaging of FRET-Based cGMP Probes. . . . . . . . . . . . . . . . . . . . 73
Thomas C. Rich, Andrea L. Britain, Tiffany Stedman,
and Silas J. Leavesley
6 Visualization of cGMP with cGi Biosensors. . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Martin Thunemann, Natalie Fomin, Christian Krawutschke,
Michael Russwurm, and Robert Feil
7 Advances and Techniques to Measure cGMP in Intact Cardiomyocytes. . . . . . . 121
Konrad R. Götz and Viacheslav O. Nikolaev
8 Real-Time Monitoring the Spatiotemporal Dynamics
of Intracellular cGMP in Vascular Smooth Muscle Cells. . . . . . . . . . . . . . . . . . . 131
Kara F. Held and Wolfgang R. Dostmann
9 Methods for Identification of cGKI Substrates. . . . . . . . . . . . . . . . . . . . . . . . . . 147
Katharina Salb and Jens Schlossmann
10 Approaches for Monitoring PKG1a Oxidative Activation . . . . . . . . . . . . . . . . . 163
Joseph Robert Burgoyne and Philip Eaton
11 Analysis of cGMP Signaling in Adipocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Katja Jennissen, Bodo Haas, Michaela M. Mitschke, Franziska Siegel,
and Alexander Pfeifer
12 A Genetic Strategy for the Analysis of Individual Axon Morphologies
in cGMP Signalling Mutant Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Hannes Schmidt, Gohar Ter-Avetisyan, and Fritz G. Rathjen
vii
viii Contents
13 Receptor Binding Assay for NO-Independent Activators
of Soluble Guanylate Cyclase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Peter M. Schmidt and Johannes-Peter Stasch
14 Direct Intrathecal Drug Delivery in Mice for Detecting
In Vivo Effects of cGMP on Pain Processing. . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Ruirui Lu and Achim Schmidtko
15 The Geisler Method: Tracing Activity-Dependent cGMP Plasticity
Changes upon Double Detection of mRNA and Protein on Brain Slices . . . . . . 223
Wibke Singer, Hyun-Soon Geisler, and Marlies Knipper
16 Detection of cGMP in the Degenerating Retina . . . . . . . . . . . . . . . . . . . . . . . . 235
Stylianos Michalakis, Jianhua Xu, Martin Biel, and Xi-Qin Ding
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
Contributors
JOSEPH A. BEAVO (cid:129) Department of Pharmacology, University of Washington, Seattle , USA
NOOMEN BETTAGA (cid:129) Physiologisches Institut I, Universität Würzburg, Würzburg, Germany
MARTIN BIEL (cid:129) Center for Integrated Protein Science Munich (CIPSM),
Ludwig-Maximilians-Universität, Munich, Germany; D epartment of Pharmacy,
Center for Drug Research, Ludwig-Maximilians-Universität, Munich, Germany
ANDREA L. B RITAIN (cid:129) Department of Pharmacology, University of South Alabama,
Mobile , USA
JOSEPH ROBERT BURGOYNE (cid:129) Cardiovascular Division, The Rayne Institute,
St. Thomas’ Hospital, King’s College London, London , UK
XI-QIN DING (cid:129) Department of Cell Biology , U niversity of Oklahoma Health Sciences Center,
Oklahoma City , USA
WOLFGANG R. DOSTMANN (cid:129) Department of Pharmacology, University of Vermont,
Burlington, USA
PHILIP EATON (cid:129) Cardiovascular Division, The Rayne Institute, St. Thomas’ Hospital, King’s
College London, London, UK
ROBERT FEIL (cid:129) Interfakultäres Institut für Biochemie, Universität Tübingen, Tübingen,
Germany
NATALIE FOMIN (cid:129) Interfakultäres Institut für Biochemie, Universität Tübingen, Tübingen,
Germany; G raduate School of Cellular and Molecular Neuroscience, Universität Tübingen ,
Tübingen, Germany
ANDREAS FRIEBE (cid:129) Physiologisches Institut I, Universität Würzburg, Würzburg, Germany
HYUN-SOON GEISLER (cid:129) Department of Otolaryngology , Hearing Research Centre Tübingen
(THRC), Molecular Physiology of Hearing, Universität Tübingen, Tübingen, Germany
KONRAD R. GÖTZ (cid:129) Emmy Noether Group of the DFG, Department of Cardiology and
Pneumology, European Heart Research Institute Göttingen, Universität Göttingen ,
Göttingen, Germany
DIETER GRONEBERG (cid:129) Physiologisches Institut I, Universität Würzburg, Würzburg, Germany
BODO HAAS (cid:129) Institute of Pharmacology and Toxicology, Universität Bonn, Bonn , Germany;
Institute for Drugs and Medical Devices, Bonn , Germany
KARA F. HELD (cid:129) Department of Pharmacology, Yale University, New Haven, USA
THOMAS R. HINDS (cid:129) Department of Pharmacology, University of Washington, Seattle , USA
FRANZ HOFMANN (cid:129) FOR 923, Institut für Pharmakologie und Toxikologie, der Technischen
Universität München, Munich, Germany
RONALD JÄGER (cid:129) Physiologisches Institut I, Universität Würzburg, Würzburg, Germany
KATJA JENNISSEN (cid:129) Institute of Pharmacology and Toxicology, Universität Bonn, Bonn ,
Germany
MARLIES KNIPPER (cid:129) Department of Otolaryngology , Hearing Research Centre Tübingen
(THRC), Molecular Physiology of Hearing, Universität Tübingen, Tübingen, Germany
ix
