P2 Receptors in Airway Smooth Muscle Thesis submitted for the degree of Doctor of Philosophy at the University of Leicester by Adam Luke Smith, MBiolSci Department of Infection, Immunity and Inflammation University of Leicester Feb 2019 Supervisors: Dr Catherine Vial Prof Peter Bradding Prof Andy Wardlaw 1 Abstract The hypertrophic airway smooth muscle (ASM) of asthmatic airways is hyperresponsive compared to that of healthy airways, although the underlying causes for this are unknown. P2 receptors, receptors for extracellular nucleotides including ATP, contribute to the regulation of smooth muscle contraction and proliferation. However, the roles of P2 receptors in ASM have not been fully elucidated. As the extracellular ATP concentration is increased in asthmatic airways, this could provide a mechanism by which P2 receptors contribute to the pathogenesis of ASM dysfunction in asthma. In this thesis, I have demonstrated using qPCR the presence of P2 receptor transcripts in murine airways, and in ASM cells cultured from murine airways and those of both healthy and asthmatic donors. Biochemical and immunohistochemical approaches have revealed the expression of P2X1, P2X4 and P2X7 receptor proteins in murine airways which localises to the ASM specifically, and in ASM cells cultured from murine airways and those of healthy and asthmatic individuals. Using Ca2+ imaging, ASM cells cultured from murine airways were shown to express functional P2X7, P2Y1 and P2Y2 receptors. Additionally, ASM cells cultured from healthy airways expressed only functional P2Y2 receptors whilst those from asthmatic airways expressed both functional P2X4 and P2Y2 receptors. The application of extracellular nucleotides did not contract ASM of either murine or human airways. Yet, the pre-treatment of ASM from mouse, but not humans, with extracellular nucleotides appeared to enhance its contraction. In addition, extracellular nucleotides did not stimulate human ASM cell proliferation, although UTP (100 µM) induced the proliferation of murine ASM cells. These data suggest that functional P2 receptors are expressed in ASM from mice and humans, but that P2 receptor function is comparable between healthy and asthmatic human ASM. The functional role of P2 receptors expressed by the ASM in asthma therefore remains unclear. 2 Acknowledgements I must first thank my supervisory team, Dr Catherine Vial and Professors Peter Bradding and Andy Wardlaw, for sticking with me and this project throughout the good and difficult times of this journey. I have learned a lot from you and, despite any difficulties encountered along the way, I am genuinely thankful and appreciative to all. I must also thank my colleagues at Glenfield for sharing this experience with me and for their contributions to my project, and particularly those who helped with the project by performing bronchoscopies and culturing and caring for the cHASM cells. Next, I have to thank my lab group in the HWB. Alistair was always available to provide comedic relief and support (particularly with western blotting experiments) whilst Anastasios always had an appropriate gif to share with the group. I am grateful to have had Hong to share the experience with me. I also never expected to find such a true friend in Manijeh, and I’m so thankful to have met her. As an extension of this group, I have to thank Rich Rainbow and his group for adopting me for the majority of my second year whilst performing my Ca2+ imaging experiments. I would probably still be struggling to see responses without your help and support! I am also so extremely proud of my close friends Alina and Claire. I’m glad that we could share this together. Next, I’d like to thank my ‘gay group’ in Leicester including Shay, Tom and Scruggs for accepting me even when I had no friends, for being so supportive and for always providing the opportunity to sesh. Sam and Elliot, thank you in particular for gracefully housing me for the duration of my write up. I don’t know what I would have done without you. Last of the Leicester group, of course I have to thank Dan. You’ve become so special to me during these last few years and I’m glad that you were able to see me through the final hurdles and beyond. I hope your future is as bright as you deserve. Finally, I must thank my family, and particularly my Mum. You’ve always been there for me and I honestly couldn’t have done this without you. Over the last few years I’ve particularly enjoyed our carvery outings with Pete! I also have to thank my loving but crazy brother, Jack, and his partner Rosealean for giving me the best present I could have ever asked for. Temperance has brought such a light to our lives that I honestly can’t imagine our family without her. I’m so excited to see her blossom into a big sister for Blair. 3 Probably the most difficult part of this thesis to write is this paragraph. I have to thank those family members who have left us recently and can’t see me complete my PhD: my Nan and Grandad, Annie and Bernie Burns, and my Aunt Sheila. Corny or not, I dedicate this thesis to you. CF will love you always. 4 Abstracts and prizes arising from this thesis Abstracts: Smith A, Bradding P, Wardlaw A, Vial C. Mouse airway smooth muscle cells express P2X receptors. Presented at ILH Joint Respiratory Research Day conference, Leicester, 2016 and the Italian-German Purine Club meeting, Rome, 2017. Smith A, Wardlaw A, Bradding P, Vial C. Cultured Mouse Tracheal Airway Smooth Muscle Cells Express Functional P2 Receptors. Presented at ILH Joint Respiratory Research Day conference, Nottingham, UK, 2017 and University of Leicester Department of Infection, Immunity and Inflammation 9th Annual Postgraduate conference, 2017. Prizes: Highly commended award for Best Presentation at University of Leicester Department of Infection, Immunity and Inflammation 9th Annual Postgraduate conference, 2017. The Physiological Society Travel Award to Italian-German Purine Club meeting, Rome, 2017. 5 Contents 1.1 The airways and respiratory diseases ................................................................... 24 1.1.1 The structure of the airways .......................................................................... 24 1.1.2 Airway diseases .............................................................................................. 24 1.1.3 The definition of asthma and its subtypes .................................................... 26 1.1.4 The pathogenesis of asthma .......................................................................... 27 1.1.4.1 The onset of asthma and its risk factors ................................................. 27 1.1.4.2 The pathophysiology of asthma ............................................................. 30 1.1.4.2.1 The inflammatory response in asthma pathogenesis ..................... 32 1.1.4.2.2 Airway remodeling ........................................................................... 35 1.1.4.2.2.1 Epithelial injury and shedding .................................................. 35 1.1.4.2.2.2 Mucous hypersecretion ............................................................ 35 1.1.4.2.2.3 ASM dysfunction in asthma ...................................................... 36 1.1.5 The treatment of asthma ............................................................................... 38 1.2 Airway Smooth Muscle biology ............................................................................ 41 1.2.1 The origins of ASM ......................................................................................... 41 1.2.2 The physiological roles of ASM in healthy airways ........................................ 41 1.2.3 Calcium flux in smooth muscle ...................................................................... 45 1.2.3.1 Routes of calcium influx .......................................................................... 47 1.2.3.2 Calcium release from intracellular stores ............................................... 48 1.2.3.2.1 RYR-mediated calcium release from the SR in smooth muscle ....... 48 1.2.3.2.2 IP3-induced calcium release in smooth muscle ............................... 51 1.2.4 Smooth muscle contraction ........................................................................... 52 1.2.4.1 Regulation of ASM resting tone .............................................................. 53 1.2.4.2 Mechanisms of smooth muscle contraction .......................................... 53 1.2.4.3 Mechanisms of smooth muscle relaxation ............................................. 54 1.2.4.4 The regulation of smooth muscle contraction ....................................... 57 1.2.4.4.1 Calcium-dependent regulation of smooth muscle contraction ...... 58 1.2.4.4.2 Calcium-independent regulation of smooth muscle contraction ... 59 6 1.2.5 ASM in asthma ............................................................................................... 60 1.2.5.1 Possible mechanisms behind the hypercontractility of asthmatic ASM 60 1.2.5.2 Possible mechanisms behind the hyper-proliferation of asthmatic ASM ............................................................................................................................ 63 1.2.6 ASM cells in culture........................................................................................ 64 1.3 P2 receptors .......................................................................................................... 67 1.3.1 Nucleotide structures and functions ............................................................