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The Role of the Secondary Messenger NAADP in Physiological and Pathological Calcium Signalling in Pancreatic Acinar Cells

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The Role of the Secondary Messenger NAADP in Physiological and Pathological Calcium Signalling in Pancreatic Acinar Cells The role of the secondary messenger NAADP in physiological and pathological calcium signalling in pancreatic acinar cells Thesis submitted in accordance with the requirements of Cardiff University for the degree of Doctor of Philosophy Martyn Richard Charlesworth April, 2017 To my Mother For the inspiration she provided and the endless support, encouragement and faith she has given me throughout. 2 Abstract Acute Pancreatitis (AP) is inflammatory disease characterised by the pathological activation of the digestive enzymes and extensive necrosis of the pancreases and surrounding tissue, which currently has no effective treatment. Pathological agents like the bile acid taurolithocholic acid-3-sulfate (TLC-S) have been shown to induce necrosis of pancreatic acinar cells by causing intracellular calcium (Ca2+) to reach cytotoxic concentrations. TLC-S acts on elements of the secondary Ca2+ messenger pathways normally used in physiological signalling to achieve these Ca2+ increases. Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most novel of these secondary Ca2+ messengers to be identified and several recent advancements have been made regarding its activity. This study has used these advancements to better characterise NAADP-induced Ca2+ release in PAC, and its role in both physiological and pathological Ca2+ signalling. NAADP-induced Ca2+ release was found to involve the activity of both Two-pore channel (TPC) and Ryanodine receptor (RyR) Ca2+ channels; with a varying involvement of the 2 TPC and 3 RyR isoforms. The NAADP antagonist Ned-19 completely inhibited Ca2+ responses to a physiological concentration of the secretagogue cholecystokinin (CCK) and had a protective effect against a pathological concentration of CCK. The size of the Ca2+ response to TLC-S was significantly inhibited in the presence of Ned-19, and the antagonist significantly reduced the amount of necrosis induced. Another related bile acid, cholate, was found to have a similar necrotic effect to PAC as a result of pathological increases in cytosolic Ca2+. While neither Ned-19 or GSK-7975A (an inhibitor of store- 3 operated Ca2+ entry) alone had a significant effect on cholate-induced necrosis, a combination of the two did have a protective effect. These findings suggest that Ned-19 may provide a potential therapeutic solution to AP, though it may need to be used in combination with inhibitors of other Ca2+ signalling to be effective. 4 Acknowledgments First I would like to thank my supervisors’ Dr Oleg Gerasimenko and Dr Julia Gerasimenko for their support and advice throughout my time under their supervision. Together their theoretical, practical and technical knowledge helped guide my project and develop my skills as a scientist. I am also thankful to my other supervisor Professor Ole Petersen for his role in reviewing the data I produced and providing advice on the directions my project should take. Thank you to my student advisor Professor David Lloyd for critiquing my annual reports and contributing a perspective from someone outside my specific area of research. I received much help from the other members of my group: Dr Bill Williams, Dr Monika Jakubowska and especially Dr Pawel Ferdek for all the assistance they provided. I would also like to thank my fellow PhD students Eloise Stapleton and Shuang Peng for sharing the experience with me and acting as my sounding boards. Thanks to Dr Parrington for providing the TPC2 knockout mice and Professor Barry Potter for providing the BZ194 compound used in this study. Also thank you to Dr Katsuhiko Mikoshiba for providing access to his colony of RyR3 knockout mice. Many thanks to Cardiff University for providing me with the opportunity to perform this research project and all the assistance that was provide by the members of its postgraduate team, academic staff, animal unit and fellow PhD students. Finally, thank you to the Medical Research Council for providing the funding that allowed this project to occur. 5 Table of Content Abstract……………………………………………………………………………………….3 Acknowledgments…………………………………………………………………………….5 Table of Content……………………………………………………………………………...6 Table of Figures………………………………………………………………….………….10 Abbreviations………………………………………………………………………………..14 Chapter 1: Introduction...…………………………………………………………………..18 1.1 Calcium signalling………………...………………………………...………………...19 1.1.1 Calcium as a signal………………….………………………...………………..19 1.1.2 Calcium Homeostasis……………………….………………...………………..20 1.1.3 Secondary messengers...………...………………. …………...………………..23 1.1.3.1 IP3-induced calcium release…...………………………...……………….24 1.1.3.2 cADPR-induced calcium release…………………...…...………………..25 1.1.3.3 NAADP-induced calcium release………………...………..…………….28 1.1.3.4 NAADP synthesis……………..………………………...……………….33 1.1.3.5 NAADP receptors……………..………………………...……………….38 1.1.3.5.1 Ryanodine receptors…………………….………...………………..39 1.1.3.5.2 TRP-ML channels……………..……………..…...………………..41 1.1.3.5.3 Two-pore channels……………..…………...……….……………..45 1.1.3.5.4 P2Y11 receptor…………...…...…………………...………………..55 1.1.3.6 Pharmacological regulation of NAADP signalling…......………..…………..56 1.1.4 Store-operated calcium entry…………...……..……..….…….....……………..58 1.1.5 Visualizing calcium signalling…….…...……..……..….…….....……………..63 1.2 The pancreas…….………………...………………………………...……………..…66 1.2.1 The structure and function of the pancreas…………………....……………….66 6 1.2.2 Pancreatic acinar cells...............…….………………………...………………..70 1.2.2.1 Structure and function of acinar cells…...……………………..……...……...70 1.2.2.2 Physiological calcium signalling in acinar cells………..…...……………..…72 1.2.3 Diseases of the pancreas............…….………………………...………………..77 1.2.3.1 Acute pancreatitis……….………….…...……..……………………...……...82 1.2.3.1.1 Causes of acute pancreatitis…….....………………………………...….….82 1.2.3.1.2 Bile acids……………………….....………………………………...……...86 1.2.3.1.3 Pathological calcium signalling in acute pancreatitis………………...…….91 1.3 Aims of study…………..……………..……..……………………...………..………93 Chapter 2: Materials and Methods………………………………………………………...95 2.1 Reagents………………………...………………………………………………..96 2.2 Animals…..……………………...……………………………………………….97 2.3 Genotyping……………………...………………………………………………..98 2.4 Perpetration of NaHEPES solution………………………...……………..…….100 2.5 Perpetration of K-HEPES solution…………….…………...……………..…….100 2.6 Preparation of Collagenase…………………….…………...…………..……….100 2.7 Preparation of fluorescent calcium indicators………..…………………………102 2.8 Isolation of pancreatic acinar cells…………….…………...…………..……….102 2.9 Calcium measurements in intact cells………………………...………..……….103 2.10 Calcium measurements in permeabilized cells………………………......…....103 2.11 Two-photon permeabilization……………………...……...……………….….104 2.12 Cell death assay…………………………………………...…………...………105 2.13 Equipment………………….……………………………...………….……….105 2.14 Data analysis…………………….………………………...………….……….107 2.15 Software………………...….……………………………...………….……….108 7 Chapter 3: NAADP-induced calcium release from intracellular organelle stores requires the activity of both ryanodine receptors and two-pore channels………….….109 3.1 Overview of NAADP-induced calcium release in pancreatic acinar cells……...110 3.2 TPC and RyR isoform requirements for NAADP activity in pancreatic acinar cells……………....….……………………………...…………………...119 3.3 Discussion……………....….…..…………………………...…………………...132 Chapter 4: The NAADP antagonist ned-19 has an inhibitory effect on cholecystokinin-induced calcium release in pancreatic acinar cells……………..….….138 4.1 Overview of the effect of Ned-19……………...…………...…………………...139 4.2. The effect of Ned-19 on CCK-induced calcium oscillations in pancreatic acinar cells……………………………..……...…………...…………………...147 4.3 Discussion…………..…………………..……...…………...…………………..159 Chapter 5: The NAADP antagonist Ned-19 has an inhibitory effect on pathological calcium release induced by the bile acid TLC-S in pancreatic acinar cells..………......168 5.1: Overview of TLC-S-induced calcium release in pancreatic acinar cells….…...169 5.2: The effect of Ned-19 on TLC-S-induced calcium release in pancreatic acinar cells…………...…………....…...…...…...………...……….……….…..175 5.3 Discussion…………...……..……....…...…...…...………...……….…………..182 Chapter 6: Inhibition of pancreatic acinar cell necrosis induced by the bile acid cholate requires inhibition of both NAADP-induced calcium release and store operated calcium entry…………………..….….……………..….….………….…..….…190 6.1: Overview of calcium entry and GSK-7975A…...………...……….…………...191 8 6.2: The effect of the bile acid cholate on pancreatic acinar cells.…………..……...194 6.3 Discussion…...………...……….………….………….………….……………203 Chapter 7: The synthetic NAADP antagonist BZ194 shows no effect in pancreatic acinar cells…………………..….….…………………………..….….………….…..….….208 7.1: Overview of BZ194...……….………….……………………….……………...209 7.2: The effect of BZ194 in pancreatic acinar cells…...……..…...……….………..214 7.3 Discussion...……….………….………….………….…………………...……..216 Chapter 8: Concluding remarks…………………..….….……………..….….………….221 8.1 Summary...……….…………………..….……………………….……………..222 8.2 NAADP-induced calcium release from organelle stores in pancreatic acinar cells requires the involvement of both TPC and RyR channels…………..…....224 8.3 The activity of NAADP is essential for the physiological response to CCK in pancreatic acinar cells…………………………..………………….………..227 8.4 Inhibition of NAADP-induced calcium release by Ned-19 is a potential therapeutic solution to acute pancreatitis…………….……………….………..229 Bibliography…………………..….….…………………………………...….….…………232 9 Table of Figures Fig. 1.1.2 The cell’s ‘Calcium Signalling Toolkit’…………………………………………..21
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