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Hons), National University of Singapore MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF THE ROLE OF HYDROGEN SULPHIDE IN SEXUAL MEDICINE ROESWITA LEONO LIAW B.Sc. (Hons), National University of Singapore A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF OBSTETRICS & GYNAECOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2011 i ii ACKNOWLEDGEMENTS First and foremost, I would like to express my heartfelt gratitude to my project supervisor, Professor Ganesan P Adaikan for the great opportunity to work on this interesting project and also for his invaluable advice, patient guidance and encouragement throughout the course of this project. I would also like to thank Dr Balasubramanian Srilatha, my co-supervisor, for her helpful input and constructive suggestions which were instrumental to the development of the project. My sincere thanks also go out to Dr Jun Meng for the friendship, advice, support, bantering of ideas along the way as well as for sharing his expertise and setting aside time for consultation on troubleshooting problems with regards to real time PCR and western blot. I would also like to thank Miss Maryam Jameelah, past member of this lab who has done a good job in maintaining an orderly lab environment and also for providing assistance. I would like to extend my appreciation to both the academic and non-academic staff of the Department of Obstetrics & Gynaecology, NUS for the kind help they rendered along the way. I would also like to express my gratitude to the National University of Singapore for granting me the graduate research scholarship and hence allowing me to pursue my interest in research. The project was made possible under the NMRC grant (R-174-000-104-213) awarded to my supervisors. i Last but not least, I would like to express my heartfelt appreciation to my parents and family members. Without their strong support and loving encouragements, this project would not have reached fruition. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS...................................................................................................... i TABLE OF CONTENTS........................................................................................................ iii SUMMARY ............................................................................................................................. vi LIST OF FIGURES .............................................................................................................. viii LIST OF TABLES ................................................................................................................... x LIST OF ABBREVIATIONS ................................................................................................ xi 1. INTRODUCTION ............................................................................................................ 1 1.1 Penile structure and innervation ....................................................................................... 1 1.2 Erectile dysfunction ......................................................................................................... 3 1.2.1 Pathophysiology of erectile dysfunction ................................................................... 4 1.2.2 Management of erectile dysfunction ......................................................................... 5 1.3 Gasotransmitters .............................................................................................................. 7 1.3.1 Hydrogen sulphide .................................................................................................... 8 1.3.1.1 Overview of H2S ................................................................................................ 8 1.3.1.2 Biosynthesis of H2S............................................................................................ 9 1.3.1.3 Metabolism of H2S ........................................................................................... 11 1.3.1.4 Roles of H2S in erectile function ...................................................................... 12 1.3.2 Nitric oxide ............................................................................................................. 16 1.3.2.1 Overview of NO ............................................................................................... 16 1.3.2.2 Biosynthesis of NO .......................................................................................... 16 1.3.2.3 Metabolism of NO ........................................................................................... 19 1.3.2.4 Roles of NO in erectile function ...................................................................... 20 1.3.2.5 RhoA/Rho-kinase in contractile mechanism ................................................... 21 1.3.3 Cross talk between H2S and NO ............................................................................. 22 2. RESEARCH INTEREST AND OBJECTIVES .......................................................... 25 3. MATERIALS AND METHODS .................................................................................. 26 3.1 Materials ........................................................................................................................ 26 3.1.1 Drugs ....................................................................................................................... 26 3.1.2 Chemicals ................................................................................................................ 26 3.2 Experimental Methods ................................................................................................... 28 3.2.1 Cell culture .............................................................................................................. 29 3.2.1.1 Media preparation ............................................................................................ 29 iii 3.2.1.2 Isolation of rat erectile tissue ........................................................................... 29 3.2.1.3 Primary culture of rat corpus cavernosum smooth muscle .............................. 29 3.2.1.4 Trypan blue exclusion assay ............................................................................ 31 3.2.2 Experimental protocol to investigate the involvement of second messenger cGMP and cAMP in H2S action .................................................................................................. 31 3.2.2.1 Measurement of cGMP and cAMP concentration ........................................... 32 3.2.3 Experimental protocol to investigate effects of H2S on erectile function in vivo ... 33 3.2.3.1 Measurement of intracavernosal pressure ........................................................ 34 3.2.4 Experimental protocol to investigate effects of H2S on biochemical parameters in vivo ................................................................................................................................... 37 3.2.4.1 Measurement of H2S production (CBS/CSE activity) in corpus cavernosum . 37 3.2.4.2 Measurement of plasma H2S concentration ..................................................... 38 3.2.4.3 Measurement of NO concentration in plasma and corpus cavernosum ........... 38 3.2.5 Experimental protocol to investigate effects of H2S on expression of targeted mRNAs in vitro ................................................................................................................ 39 3.2.5.1 Extraction of total RNA from rat corpus cavernosum ..................................... 39 3.2.6 Reverse transcription of RNA to cDNA ................................................................. 41 3.2.7 Real Time (Quantitative) RT-PCR.......................................................................... 41 3.2.8 Experimental protocol to investigate the effects of H2S on expression of target proteins in vitro ................................................................................................................ 44 3.2.8.1 Protein extraction from rat corpus cavernosum tissue ..................................... 44 3.2.8.2 Isolation of cytoplasmic and total membrane protein ...................................... 44 3.2.8.3 Western blot ..................................................................................................... 45 3.2.9 Experimental protocol to investigate the involvement of testosterone in H2S’ effects ............................................................................................................................... 46 3.2.9.1 Castration procedure in rat model .................................................................... 47 3.2.9.2 Measurement of testosterone concentration ..................................................... 47 3.2.10 Statistical analysis ................................................................................................. 48 4. RESULTS ....................................................................................................................... 49 4.1 Effects of treatments in vivo ........................................................................................... 49 4.2 Effects of treatments on NO level in plasma and corpus cavernosum in vivo ............... 51 4.3 Effects of treatments on H2S level in plasma and H2S production in corpus cavernosum in vivo ................................................................................................................................... 53 4.4 Effects of NaHS on cGMP and cAMP level in vitro ....................................................
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