FR 2.4 - Experimental and Clinical Pharmacology and Toxicology Faculty II – Medicine: Theoretical Medicine and Biosciences Universität des Saarlandes, Homburg / Saar Expression and function of TRPM3 proteins in the pituitary gland of the mouse Dissertation submitted to fulfill the requirements for the degree of Doctor rerum naturalium at UNIVERSITÄT DES SAARLANDES 2014 Submitted by: Nouma Hasan Born on: 08.3.1983 in Latakia, Syria First referee PD. Dr. Stephan Philipp Second referee Univ. Prof. Dr. Peter Lipp Homburg, August 2014 To my husband, my son my parents and beloved sister and brothers Acknowledgments I would like to express my great thanks to my supervisor PD. Dr. Stephan Philipp for the unlimited support throughout my PhD and the correction of this thesis. I thank him for his professional guidance and useful discussions and for challenging me to learn and to create my own ideas. I am also deeply grateful for his encouragement, patience and understanding. I am extremely grateful to Prof. Dr. Veit Flockerzi for providing me with his help and valuable discussions and ideas. I also thank him to give me the opportunity to work in his lab. I wish to thank Dr. Andreas Beck for performing the electrophysiological experiments involved in this work and for his help and discussion. I would also like to take the chance to thank Prof. Dr. Frank Kirchhof, Prof. Dr. Frank Schmitt and Prof. Dr. Ulrich Boehm to give me the opportunity to learn some techniques in their lab. Special thanks to Ute Soltek, Heidi Löhr, Karin Wolske and Martin Simon-Thomas for their technical assistance and help. I thank Sandeep Dembla and Julia Camacho Londoño for introducing me to various techniques I used to perform this work. I wish to thank all my colleges in the Experimental and Clinical Pharmacology and Toxicology and specially Thabet Belcacemi and Anour Belcacemi for their help and support. I wish to thank the graduate research school Calcium-signaling and cellular nanodomains at the University of Saarland for providing the opportunity to join the program. My sincere thanks go on my dear friend Dalia Al-ansary for her continuous and unlimited support and help and for being part of my family here in Germany. I thank Mahnaz Amini for being real friend for me. Last but not least, I thank my beloved Ossama for being always there to surround me with love, patience and optimism and my sweet Ammar who made our life always happy. Table of Content Table of content 1 Summary ........................................................................................................................................... 1 2 Introduction ........................................................................................................................................ 3 2.1 The pituitary gland .......................................................................................................................... 3 2.1.1 Anatomy and histology of the pituitary gland .............................................................................. 3 2.1.2 Regulation and function of hormone-releasing pituitary cells ..................................................... 5 2.1.2.1 Somatotrophs ........................................................................................................................... 5 2.1.2.2 Lactrotrophs ............................................................................................................................. 6 2.1.2.3 Gonadotrophs ........................................................................................................................... 6 2.1.2.4 Thyrotrophs .............................................................................................................................. 7 2.1.2.5 Corticotrophs ............................................................................................................................ 7 2.1.2.6 Melanotrophs ............................................................................................................................ 8 2.1.2.7 Oxytocin-releasing hypothalamic nerve terminals ................................................................... 9 2.1.2.8 Vasopressin-releasing hypothalamic nerve terminals .............................................................. 9 2.1.3 Function and regulation of non-endocrine pituitary cells ............................................................. 9 2.1.3.1 Pituicytes .................................................................................................................................. 9 2.1.3.2 Folliculostellate cells ............................................................................................................... 10 2.2 Calcium signaling in pituitary cells and regulation of hormone release ....................................... 11 2.3 TRP ion channels ......................................................................................................................... 12 2.3.1 Structure of TRP channel complexes ........................................................................................ 14 2.3.2 Activation and functional properties of TRP channels .............................................................. 17 2.4 Transient receptor potential Melastatin 3 (TRPM3) ..................................................................... 20 2.4.1 The Trpm3 gene encodes a variety of different TRPM3 proteins ............................................. 20 2.4.2 Expression and function of TRPM3 proteins ............................................................................. 23 2.4.3 Pharmacology of TRPM3 channels ........................................................................................... 25 2.5 Aim of the work ............................................................................................................................. 26 3 Materials .......................................................................................................................................... 27 3.1 Chemicals ..................................................................................................................................... 27 3.2 Reagent systems (Kits) ................................................................................................................ 27 3.3 Vectors ......................................................................................................................................... 27 3.4 Oligonucleotides ........................................................................................................................... 28 3.5 Primary and secondary antibodies ............................................................................................... 29 3.6 Bacterial strains ............................................................................................................................ 31 3.7 Mice .............................................................................................................................................. 31 Table of Content 4 Methods ........................................................................................................................................... 33 4.1 Molecular biological methods ....................................................................................................... 33 4.1.1 Cultivation and transformation of eukaryotic cells ..................................................................... 33 4.1.1.1 Cultivation of HEK 293 cells ................................................................................................... 33 4.1.1.2 Preparation and cultivation of primary pituitary cells .............................................................. 33 4.1.1.3 Transformation of eukaryotic cells ......................................................................................... 34 4.1.2 Cultivation and transformation of Escherichia coli .................................................................... 34 4.1.2.1 Cultivation of Escherichia coli ................................................................................................ 34 4.1.2.2 Preparation of electrocompetent E. coli cells ......................................................................... 34 4.1.2.3 Transformation of plasmid DNA into electrocompetent bacterial cells .................................. 35 4.1.3 Isolation and purification of nucleic acids .................................................................................. 35 4.1.3.1 Isolation of total RNA .............................................................................................................. 35 4.1.3.2 Analytical quick preparation of plasmid DNA for sequencing ................................................ 36 4.1.3.3 Electroelution of DNA fragments from agarose gels .............................................................. 36 4.1.3.4 Ethanol precipitation of nucleic acids ..................................................................................... 36 4.1.4 Analysis of nucleic acids ........................................................................................................... 37 4.1.4.1 Photometry ............................................................................................................................. 37 4.1.4.2 Gel electrophoresis of DNA .................................................................................................... 37 4.1.4.3 Gel electrophoretic analysis