Phosphodiesterase Methods and Protocols

1. Front Matter

   Pages i-xii

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2. Study of Cyclic Adenosine Monophosphate Microdomains in Cells

   • Marco Mongillo, Anna Terrin, Sandrine Evellin, Valentina Lissandron, Manuela Zaccolo

   Pages 1-13

3. High-Resolution Measurements of Cyclic Adenosine Monophosphate Signals in 3D Microdomains

   • Jeffrey W. Karpen, Thomas C. Rich

   Pages 15-26

4. Cygnets

   • Akira Honda, Carolyn L. Sawyer, Sharon M. Cawley, Wolfgang R. G. Dostmann

   Pages 27-43

5. High-Throughput Screening of Phosphodiesterase Activity in Living Cells

   • Thomas C. Rich, Jeffrey W. Karpen

   Pages 45-61

6. Assessment of Phosphodiesterase Isozyme Contribution in Cell and Tissue Extracts

   • Thérèse Keravis, Rima Thaseldar-Roumié, Claire Lugnier

   Pages 63-74

7. Localization of the Cyclic Guanosine 3′,5′-Monophosphate- Hydrolyzing Phosphodiesterase Type 9 in Rat Brain by Nonradioactive In Situ Hybridization

   • Wilma C. G. van Staveren, Marjanne Markerink-van Ittersum

   Pages 75-84

8. Determination of Ca2+/Calmodulin-Stimulated Phosphodiesterase Activity in Intact Cells

   • Chen Yan

   Pages 85-92

9. Adenovirus-Mediated Overexpression of Murine Cyclic Nucleotide Phosphodiesterase 3B

   • Faiyaz Ahmad, Linda Härndahl, Yan Tang, Lena Stenson Holst, Vincent C. Manganiello

   Pages 93-107

10. Identification of Promoter Elements in the 5′-Flanking Region of Murine Cyclic Nucleotide Phosphodiesterase 3B Gene

    • Hanguan Liu, Jing Rong Tang, Eva Degerman, Vincent C. Manganiello

    Pages 109-124

11. Purification of PDE6 Isozymes From Mammalian Retina

    • Dana C. Pentia, Suzanne Hosier, Rachel A. Collupy, Beverly A. Valeriani, Rick H. Cote

    Pages 125-140

12. Cyclic Guanosine 5′-Monophosphate Binding to Regulatory GAF Domains of Photoreceptor Phosphodiesterase

    • Rick H. Cote

    Pages 141-154

13. Renaturation of the Catalytic Domain of PDE4A Expressed in Escherichia coli as Inclusion Bodies

    • Wito Richter, Thomas Hermsdorf, Dietrich Dettmer

    Pages 155-165

14. Determining the Subunit Structure of Phosphodiesterases Using Gel Filtration and Sucrose Density Gradient Centrifugation

    • Wito Richter

    Pages 167-180

15. Crystallization of Cyclic Nucleotide Phosphodiesterases

    • Hengming Ke, Qing Huai, Robert X. Xu

    Pages 181-190

16. Generation of PDE4 Knockout Mice by Gene Targeting

    • S.-L. Catherine Jin, Anne M. Latour, Marco Conti

    Pages 191-210

17. Immunoprecipitation of PDE2 Phosphorylated and Inactivated by an Associated Protein Kinase

    • J. Kelley Bentley

    Pages 211-223

18. Investigation of Extracellular Signal-Regulated Kinase 2 Mitogen-Activated Protein Kinase Phosphorylation and Regulation of Activity of PDE4 Cyclic Adenosine Monophosphate-Specific Phosphodiesterases

    • Elaine V. Hill, Miles D. Houslay, George S. Baillie

    Pages 225-237

19. Radiolabeled Ligand Binding to the Catalytic or Allosteric Sites of PDE5 and PDE11

    • James L. Weeks II, Mitsi A. Blount, Alfreda Beasley, Roya Zoraghi, Melissa K. Thomas, Konjeti Raja Sekhar et al.

    Pages 239-262

20. Analysis of Dimerization Determinants of PDE6 Catalytic Subunits

    • Khakim G. Muradov, Kimberly K. Boyd, Nikolai O. Artemyev

    Pages 263-275

The multigenic family of cyclic nucleotide phosphodiesterases (PDEs) is now known to offer many new therapeutic possibilities through their influence over intracellular signaling, though their precise cell mechanisms to modulating that process remain uncertain. In Phosphodiesterase Methods and Protocols, a panel of research leaders in the PDE field describes new concepts and techniques for investigating the role of PDEs in orchestrating normal and pathophysiological responses. Presented in step-by-step detail, these readily reproducible methods allow the measurement of cyclic nucleotide variations in living cells, as well as their visualization in a spatiotemporal manner, the localization and characterization of their activities in tissues and living cells, and the assessment of targeted PDEs in creating specific tools and drugs. Additional chapters discuss the generation of PDE4 knockout mice to demonstrate not only the potential role of targeted PDEs, but also their use in further studies of the central role of PDE regulation in intracellular signaling control. These techniques offer clinicians a way to find new therapies for numerous pathologies whose molecular origins are unknown and whose treatment is symptomatic. Alterations of intracellular signaling related to PDE deregulation in such pathologies as inflammation, neurodegeneration, and cancer may explain the difficulties observed in their prevention and treatment. The protocols follow the successful Methods in Molecular Biology™ series format, each offering step-by-step laboratory instructions, an introduction outlining the principle behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.
Path-breaking and highly practical, Phosphodiesterase Methods and Protocols offers cell biologists, molecular biologists, pharmacologists, and medicinal chemists a broad ranging survey of the advanced tools and concepts needed to understand therole of PDEs in physiological functions, their implications in several critical pathologies, and the opportunities they offer for new drug design.

• Calcium
• Escherichia coli
• Filtration
• Lipid
• Nucleotide
• Promoter
• Termination
• genes
• hybridization

"...interesting and thought provoking, but technically challenging...excellent guide to current research techniques in the field of phosphodiesterase." - Doody's Health Sciences Book Review Journal

• Pharmacolgoie et Physico-Chimie des Interactions Celluaires et Moléculaires, Centre National de la Recherche Scientifique, Université Louis Pasteur de Strasbourg, Faculté de Pharmacie, Illkirch, France

  Claire Lugnier

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