Joint Thesis Supervision between UNIVERSITÉ PARIS-SUD DOCTORAL SCHOOL: INNOVATION THÉRAPEUTIQUE: DU FONDAMENTAL A L’APPLIQUÉ PÔ LE: PHYSIOPATHOLOGIE MOLECULAIRE ET CELLULAIRE DISCIPLINE: PHYSIOPATHOLOGIE MOLECULAIRE ET CELLULAIRE & INSTITUTE OF BIOPHYSICS (CHINESE ACADEMY OF SCIENCES) YEAR 2012 - 2013 SÉRIE DOCTORAT N° DOCTORAL THESIS defended on November 20th, 2012 by Kui ZHAI Cyclic Nucleotide Phosphodiesterases (PDEs) in Smooth Muscle: Expression, Function and Mechanism Director of thesis: Véronique LEBLAIS Professeur des Universités (Université Paris-Sud, France) Co-director of thesis: Guangju JI Professeur (Chinese Academy of Science, Beijing, Chine) Composition of the jury: President and reviewer: Qing-Hua LIU Professeur (South-Central University for Nationalities, Wuhan, Chine) Reviewer: Claire LUGNIER DR émérite CNRS (Université de Strasbourg) Examiners: Rodolphe FISCHMEISTER DR Inserm (Université Paris-Sud, France) Zengqiang YUAN Professeur (Chinese Academy of Science, Beijing, Chine) Acknowledgements Acknowledgements This thesis was based on experimental work done in INSERM UMR-S 769, LabEx LERMIT, Faculté de Pharmacie, Université Paris-Sud and in National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences. In 2008, I was fortunate to receive a doctoral grant from French Embassy which I am truly grateful to. Additional support was provided by Université Paris-Sud and Institute of Biophysics, which are highly appreciable. My deepest gratitude goes firstly and foremost to my directors Prof. Véronique LEBLAIS and Prof. Guangju JI. Their professional guidance, constant encouragement and support kept me all the way on track during my scientific research. I address my special thanks to them for their supervision and guidance over the past years. I would also express heartfelt gratitude to Dr. Rodolphe FISCHMEISTER, Director of Inserm UMR-S 769, for giving me the opportunity to work in his lab, sharing his endless knowledge in physiology and pharmacology, and creating a pleasant working atmosphere. I would like to thank all my wonderful colleagues for teaching me techniques, offering experimental tips. I sincerely thank Fabien HUBERT for his help in all experiments. I would like to express gratitude to Dr. Grégoire VANDECASTEELE, Dr. Jérôme LEROY and Patrick LECHÈNE for their help on FRET and path clamp experiments; to Valérie NICOLAS for her help on confocal microscope; to Yassine SASSI on cell isolation and culture; to Delphine MIKA, Françoise BOUSSAC, Cristina MOLINA-ESPINOSA, Zeineb HAJ SLIMANE, Philippe MATEO, Julia SCHITTL, Audrey VARIN, Sophie SEURON, Yanyun WU, Congyan PAN, Bin WEI, Xu ZHANG, Lin MIAO, Yan CHANG, Qi YUAN, Yingxiao CHEN, Yinglong TANG, Lei GU and Zhiguang YANG for their all kind help. It gave me many pleasures to work with them. I sincerely thank Dominique FORTIN for her help. It is my great pleasure to spend Christmas with your family and to visit Royan. I am also grateful to Jérôme Acknowledgements PIQUEREAU and Stéphanie RIMBAUD for giving me a chance to visit their beautiful hometown. Special thanks to my friends Jinguo WANG, Sibo LI, Dejiu ZHANG, Xiaoli TIAN and Lin XIA for their help and encouragements. Last but not least, I am especially indebted to my beloved parents and wife for everything they have done for me. Without their love and encourage, I would never have the chance to finish the thesis. Kui ZHAI Beijing, 2012.10 Index Index Index ........................................................................................................... I List of figures .......................................................................................... IV List of tables.............................................................................................. V Glossary and abbreviations ................................................................... VI 1 Introduction ............................................................................................ 1 1.1 Physiology of the smooth muscle ................................................................... 2 1.1.1 Characteristics of the smooth muscle cells............................................................... 2 1.1.2 Cardiovascular system ............................................................................................. 5 1.1.2.1 Overview ....................................................................................................... 5 1.1.2.2 Morphology of vascular smooth muscle (VSM) ........................................... 5 1.1.2.3 Sympathetic innervation ................................................................................ 6 1.1.3 Lower urinary tract ................................................................................................... 7 1.1.3.1 Overview ....................................................................................................... 7 1.1.3.2 Morphology of urinary bladder ..................................................................... 8 1.1.3.3 Spontaneous activity of the bladder .............................................................. 9 1.1.4 Excitation-contraction-coupling (ECC) in the SM ................................................. 11 1.1.4.1 Mechanisms of contraction in the SMC ...................................................... 11 1.1.4.2 The electromechanical coupling .................................................................. 13 1.1.4.3 The pharmacomechanical coupling ............................................................. 13 1.2 The cAMP/β-adrenergic signaling pathway ............................................... 15 1.2.1 β-ARs ..................................................................................................................... 16 1.2.2 G protein ................................................................................................................ 16 1.2.3 Adenylyl cyclase (AC) ........................................................................................... 18 1.2.4 cAMP targets .......................................................................................................... 18 1.2.4.1 PKA ............................................................................................................. 18 1.2.4.2 Epac ............................................................................................................. 20 1.2.4.3 Cyclic nucleotide-activated ion channels .................................................... 20 1.2.5 Mechanisms of cyclic nucleotide-induced relaxation ............................................ 22 2+ 1.2.5.1 Cytosolic Ca concentration modulation in the SMC ................................ 22 I Index 1.2.5.2 Hyperpolarization of the SMC .................................................................... 25 1.2.5.3 Decrease in MLC20 phosphorylation.......................................................... 27 1.3 PDEs family ................................................................................................... 29 1.3.1 PDE1 ...................................................................................................................... 30 1.3.2 PDE2 ...................................................................................................................... 33 1.3.3 PDE3 ...................................................................................................................... 36 1.3.4 PDE4 ...................................................................................................................... 39 1.3.5 PDE5 ...................................................................................................................... 42 1.3.6 PDE6 ...................................................................................................................... 44 1.3.7 PDE7 ...................................................................................................................... 45 1.3.8 PDE8 ...................................................................................................................... 46 1.3.9 PDE9 ...................................................................................................................... 47 1.3.10 PDE10 .................................................................................................................. 48 1.3.11 PDE11 .................................................................................................................. 49 1.4 Cyclic AMP compartmentation ................................................................... 50 1.4.1 Overview ................................................................................................................ 50 1.4.2 Innovative methods for subcellular compartmentation analysis ............................ 52 1.4.3 Role of PDEs in cAMP compartmentation ............................................................ 54 1.4.3.1 PDEs and cAMP compartmentation in cardiac myocyte ............................ 54 1.4.3.2 cAMP compartmentation in SMC ............................................................... 58 2 Objectives of the thesis ........................................................................ 60 3 Materials and methods ........................................................................ 61 3.1 Materials ........................................................................................................ 61 3.1.1 Drugs and reagents ................................................................................................