血管內膜的進行性增厚是動脈粥樣硬化的重要標誌，並最終導致閉塞性血管病。血管內膜增生的一個主要因素是血管中膜的平滑肌細胞遷移至內膜層並增殖。大量研究證實，在動脈粥樣硬化的發生發展中，過量產生的活性氧簇（ROS）參與了血管壁的增厚。M型瞬時受體電位通道亞家族的成員TRPM2在血管平滑肌細胞中有表達，它能被ROS激活並對Ca²⁺通透，但其在血管平滑肌中的功能以及與心血管疾病的聯繫尚未見報道。 / 本論文著眼於探討TRPM2在鼠和人血管內膜增生中的作用。用血管外周套管法建立在體齧齒類動脈內膜增生模型。套管放置2周後，大鼠股動脈可見明顯的內膜增厚。免疫染色顯示新生內膜及其鄰近中膜區域內有大量增殖細胞核抗原陽性細胞，提示在增生的動脈中，細胞週期活動增強。動脈內膜和中膜内二氫乙錠螢光信號顯著增強，提示了ROS的過量生成。免疫染色和免疫印跡法均顯示，套管損傷導致TRPM2表達上調。免疫螢光雙標TRPM2與α-平滑肌肌動蛋白顯示內膜區域有大量TRPM2陽性的平滑肌細胞。與正常股動脈中膜平滑肌細胞相比，次黃嘌呤和黃嘌呤氧化酶在套管損傷的動脈來源的新生內膜平滑肌細胞中引起更大幅度的細胞內鈣離子濃度升高，而TRPM2抑制性抗體TM2E3預處理可消除這種差異。套管放置3周可引起小鼠頸動脈新生內膜形成，並伴隨著TRPM2表達上調。敲除TRPM2基因可顯著抑制內膜增生。取冠狀動脈搭橋術後殘餘的大隱靜脈，離體培養2周誘導內膜增生。免疫螢光雙標TRPM2與α-平滑肌肌動蛋白顯示新生內膜內含有大量TRPM2陽性的平滑肌細胞。TM2E3和另一TRPM2抑制劑2-氨乙氧基二苯酯硼酸處理均可有效降低內膜的增生。培養齧齒類主動脈平滑肌細胞，用劃痕試驗和MTT法檢測TRPM2阻斷劑和TRPM2基因敲除對過氧化氫誘導的細胞遷移和增殖的影響。結果顯示，暴露於過氧化氫48小時，細胞的遷移和增殖均明顯加快。TM2E3和2-氨乙氧基二苯酯硼酸處理有效抑制過氧化氫誘導的大鼠主動脈平滑肌細胞遷移和增殖；類似地，TRPM2基因敲除可顯著抑制過氧化氫誘導的小鼠主動脈平滑肌細胞遷移和增殖。 / 以上結果表明，血管內膜增生伴隨著TRPM2表達的上調；TRPM2參與了血管內膜增生以及血管平滑肌細胞的遷移、增殖；抑制TRPM2可能是對抗血管內膜增厚的潛在治療手段。 / A hallmark in atherosclerosis is progressive intimal thickening, which leads to occlusive vascular diseases. A causation of neointimal hyperplasia is the migration of medial smooth muscle cells (SMCs) to the intima where they proliferate. It is well recognized that excessive production of reactive oxide species (ROS) contributes to vascular wall thickening during arteriosclerotic development. TRPM2, a member of the melastatin-like transient receptor potential channel subfamily, is a Ca²⁺-permeable cation channel activated by ROS and is expressed in vascular smooth muscle cells (VSMCs). The functional properties of TRPM2 in vascular smooth muscle remain to be identified and an association between TRPM2 and cardiovascular diseases has not been reported. / In the present study, I investigated the involvement of TRPM2 in rodent and human neointimal hyperplasia. In vivo neointimal hyperplasia in rodent arteries was induced by perivascular cuff placement. After the cuff placement for 2 weeks, rat femoral arteries showed distinct intimal thickening. Immunostaining showed a great number of PCNA-positive proliferating cells in the neointima and its adjacent media region, indicating the enhanced cell cycle activity in the hyperplasic arteries. Dihydroethidium signal was markedly increased in the neointima and media of the cuffed arteries, suggesting that ROS is over-produced. Interestingly, both immunostaining and immunoblot showed that cuff-injury also led to an up-regulated expression of TRPM2. Double immunofluorescent labeling of TRPM2 and α-smooth muscle actin showed a large amount of TRPM2-positive SMCs in the neointimal region. Compared with the normal medial SMCs isolated from non-cuffed arteries, the neointimal SMCs from cuff-injured arteries displayed a greater [Ca²⁺] rise in response to hypoxanthine-xanthine oxidase, which was inhibited by pre-treatment with a TRPM2-specific blocking antibody TM2E3. In mouse carotid arteries, cuff placement for 3 weeks caused clear neointimal formation, accompanied by up-regulated expression of TRPM2. Trpm2 disruption dramatically reduced the neointimal growth. Human saphenous vein samples obtained during CABG surgery were organ-cultured for 2 weeks to allow growth of neointima. Double immunofluorescent labeling of TRPM2 and α-smooth muscle actin showed that the neointima contained numerous TRPM2-positive SMCs. Neointimal hyperplasia in the veins was effectively suppressed by in vitro treatment with TM2E3 or a chemical blocker 2-aminoethoxydiphenyl borate. Furthermore, the effect of TRPM2 blockers and Trpm2 disruption on hydrogen peroxide-induced migration and proliferation of cultured rodent aortic SMCs were evaluated by scratch wound healing assay and MTT assay, respectively. It was found that exposure to hydrogen peroxide for 48 hour substantially enhanced the migration and proliferation of rodent aortic SMCs. In rat aortic SMCs, both TM2E3 and 2-aminoethoxydiphenyl borate significantly inhibited the hydrogen peroxide-induced cell migration and proliferation. The hydrogen peroxide-induced cell migration and proliferation of SMCs was also reduced in Trpm2 knockout mice. / Taking together, these results provide strong evidences that in vivo neointimal hyperplasia is accompanied by an up-regulated expression of TRPM2 and that TRPM2 plays a key role in neointimal hyperplasia, VSMCs migration and proliferation. Blocking TRPM2 can be a potential therapeutic approach for protecting blood vessels against intimal thickening. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Ru, Xiaochen. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 125-151). / Abstracts also in Chinese. / Declaration of Originality --- p.i / Abstract --- p.ii / 論文摘要 --- p.iv / Acknowledgements --- p.vi / Abbreviations and Units --- p.vii / Table of Content --- p.x / List of Figures --- p.xvi / List of Tables --- p.xviii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Neointimal hyperplasia --- p.1 / Chapter 1.1.1 --- Definition of neointimal hyperplasia --- p.2 / Chapter 1.1.2 --- Medical significance of coronary neointimal hyperplasia --- p.3 / Chapter 1.1.3 --- Pathogenesis of neointimal hyperplasia --- p.5 / Chapter 1.1.3.1 --- “Response to injury“ hypothesis --- p.6 / Chapter 1.1.3.2 --- Role of VSMCs --- p.7 / Chapter 1.1.3.2.1 --- VSMC phenotypic switch --- p.7 / Chapter 1.1.3.2.2 --- Ca²⁺ channel modulation in VSMCs --- p.8 / Chapter 1.1.3.2.3 --- VSMC migration --- p.9 / Chapter 1.1.3.2.4 --- VSMC proliferation --- p.10 / Chapter 1.1.3.2.5 --- Extracellular matrix production by VSMCs --- p.11 / Chapter 1.1.3.3 --- Endothelial dysfunction --- p.11 / Chapter 1.1.3.4 --- Platelet adhesion --- p.12 / Chapter 1.1.3.5 --- Inflammation --- p.13 / Chapter 1.1.4 --- Role of ROS in neointimal hyperplasia --- p.14 / Chapter 1.1.4.1 --- Types of ROS --- p.15 / Chapter 1.1.4.1.1 --- Superoxide anion --- p.16 / Chapter 1.1.4.1.2 --- Hydroxyl radical --- p.16 / Chapter 1.1.4.1.3 --- Hydrogen peroxide --- p.16 / Chapter 1.1.4.1.4 --- Nitric oxide --- p.17 / Chapter 1.1.4.2 --- Sources of ROS in vessel wall --- p.17 / Chapter 1.1.4.3 --- ROS signaling in endothelial cells --- p.19 / Chapter 1.1.4.4 --- ROS signaling in VSMCs --- p.20 / Chapter 1.1.4.5 --- ROS and atherosclerosis --- p.21 / Chapter 1.1.5 --- Current therapeutic approaches to neointimal hyperplasia --- p.23 / Chapter 1.1.5.1 --- Pharmacological approaches --- p.23 / Chapter 1.1.5.2 --- Technical Approaches --- p.25 / Chapter 1.2 --- Transient receptor potential melastatin 2 (TRPM2) channel --- p.27 / Chapter 1.2.1 --- TRP Channels --- p.27 / Chapter 1.2.2 --- TRPM2 structure and expression --- p.29 / Chapter 1.2.2.1 --- Structure --- p.29 / Chapter 1.2.2.2 --- Alternative splicing isoforms --- p.30 / Chapter 1.2.2.3 --- Expression pattern --- p.32 / Chapter 1.2.3 --- TRPM2 channel properties --- p.32 / Chapter 1.2.4 --- TRPM2 activators and inhibitors --- p.32 / Chapter 1.2.4.1 --- Activators --- p.33 / Chapter 1.2.4.1.1 --- ADPR --- p.33 / Chapter 1.2.4.1.2 --- NAD, cADPR and NAADP --- p.33 / Chapter 1.2.4.1.3 --- H₂O₂ and oxidative stress --- p.34 / Chapter 1.2.4.1.4 --- Ca²⁺ --- p.34 / Chapter 1.2.4.1.5 --- Other regulators --- p.35 / Chapter 1.2.4.2 --- Inhibitors --- p.35 / Chapter 1.2.5 --- Biological relevance of TRPM2 --- p.36 / Chapter 1.2.5.1 --- TRPM2 in insulin release --- p.36 / Chapter 1.2.5.2 --- TRPM2 in inflammation --- p.36 / Chapter 1.2.5.3 --- TRPM2 in cell death --- p.37 / Chapter 1.2.5.4 --- TRPM2-mediated lysosomal Ca²⁺ release --- p.38 / Chapter 1.2.5.5 --- TRPM2 and cardiovascular diseases --- p.39 / Chapter Chapter 2 --- Objectives of the Present Study --- p.40 / Chapter Chapter 3 --- Materials and Methods --- p.42 / Chapter 3.1 --- Materials --- p.42 / Chapter 3.1.1 --- Chemicals --- p.42 / Chapter 3.1.2 --- Media, supplements and other reagents for cell/tissue culture --- p.44 / Chapter 3.1.3 --- Antibodies --- p.45 / Chapter 3.1.4 --- Solutions --- p.46 / Chapter 3.1.4.1 --- Solutions for immunohistochemical and immunocytochemical staining --- p.46 / Chapter 3.1.4.2 --- solutions for immunoblotting --- p.47 / Chapter 3.1.4.3 --- Solutions for Genotyping --- p.49 / Chapter 3.1.4.4 --- Solutions for hematoxylin and eosin (HE) staining --- p.50 / Chapter 3.1.4.5 --- Solutions for [Ca²⁺]i measurement --- p.51 / Chapter 3.1.4.6 --- Solutions for IgG purification --- p.51 / Chapter 3.1.5 --- Animals --- p.51 / Chapter 3.1.5.1 --- Rat --- p.51 / Chapter 3.1.5.2 --- Trpm2 knockout mice --- p.52 / Chapter 3.1.5.3 --- Rabbit --- p.52 / Chapter 3.1.5.4 --- Ethics --- p.52 / Chapter 3.1.6 --- Human Tissue --- p.52 / Chapter 3.2 --- Methods --- p.54 / Chapter 3.2.1 --- Rodent models of neointimal hyperplasia --- p.54 / Chapter 3.2.1.1 --- Cuff-induced vascular injury in rat femoral artery --- p.54 / Chapter 3.2.1.2 --- Cuff-induced vascular injury in mouse carotid artery --- p.54 / Chapter 3.2.2 --- Genotyping for Trpm2 knockout mice --- p.55 / Chapter 3.2.2.1 --- Genomic DNA extraction from tail --- p.55 / Chapter 3.2.2.2 --- Polymerase Chain Reaction (PCR) --- p.55 / Chapter 3.2.2.3 --- Agarose gel electrophoresis of DNA --- p.56 / Chapter 3.2.3 --- Human saphenous vein culture and treatment --- p.56 / Chapter 3.2.4 --- Generation of anti-TRPM2 antibody, TRPM2-specific blocking antibody TM2E3 and preimmune IgG --- p.57 / Chapter 3.2.5 --- Histological analysis and immunohistochemistry --- p.58 / Chapter 3.2.6 --- Western blotting --- p.59 / Chapter 3.2.7 --- Detection of ROS production by dihydroethidium fluorescence --- p.60 / Chapter 3.2.8 --- Isolation of rodent neointimal and medial smooth muscle cells --- p.60 / Chapter 3.2.9 --- Culture of rodent aortic smooth muscle cells --- p.61 / Chapter 3.2.9.1 --- Cell culture --- p.61 / Chapter 3.2.9.2 --- Cell identification --- p.61 / Chapter 3.2.10 --- [Ca²⁺]i measurement --- p.62 / Chapter 3.2.11 --- Cell proliferation assay --- p.63 / Chapter 3.2.12 --- Cell migration assay --- p.63 / Chapter 3.2.13 --- Statistical analysis --- p.64 / Chapter Chapter 4 --- ROS over-production and TRPM2 up-regulation in cuff-induced rodent neointimal hyperplasia --- p.65 / Chapter 4.1 --- Introduction --- p.65 / Chapter 4.2 --- Materials and Methods --- p.66 / Chapter 4.2.1 --- Cuff-induced vascular injury in rat femoral artery --- p.66 / Chapter 4.2.2 --- Preparation of anti-TRPM2 antibody, TM2E3 and preimmune IgG --- p.66 / Chapter 4.2.3 --- Histological analysis and immunohistochemistry --- p.66 / Chapter 4.2.4 --- Western blotting --- p.67 / Chapter 4.2.5 --- Detection of ROS production --- p.67 / Chapter 4.2.6 --- Isolation of rat neointimal and medial smooth muscle cells --- p.68 / Chapter 4.2.7 --- [Ca²⁺]i measurement --- p.68 / Chapter 4.2.8 --- Statistical analysis --- p.68 / Chapter 4.3 --- Results --- p.69 / Chapter 4.3.1 --- Cuff-induced neointimal hyperplasia in rat femoral arteries --- p.69 / Chapter 4.3.2 --- ROS over-production in neointimal region of cuff-injured rat femoral arteries --- p.69 / Chapter 4.3.3 --- TRPM2 up-regulation in neointimal region of cuff-injured rat femoral arteries --- p.69 / Chapter 4.3.4 --- Enhanced [Ca²⁺]i response to HX-XO in rat neointimal smooth muscle cells --- p.70 / Chapter 4.4 --- Discussion --- p.81 / Chapter Chapter 5 --- TRPM2 contributes to human and rodent neointimal hyperplasia --- p.86 / Chapter 5.1 --- Introduction --- p.86 / Chapter 5.2 --- Materials and Methods --- p.87 / Chapter 5.2.1 --- Cuff-induced vascular injury in mouse carotid artery --- p.87 / Chapter 5.2.2 --- Genotyping for Trpm2 knockout mice --- p.87 / Chapter 5.2.3 --- Organ culture of human saphenous vein --- p.87 / Chapter 5.2.4 --- Preparation of anti-TRPM2 antibody, TM2E3 and preimmune IgG --- p.88 / Chapter 5.2.5 --- Histological analysis and immunohistochemistry --- p.88 / Chapter 5.2.6 --- Western blotting --- p.88 / Chapter 5.2.7 --- Isolation of mouse neointimal and medial smooth muscle cells --- p.89 / Chapter 5.2.8 --- [Ca²⁺]i measurement --- p.89 / Chapter 5.2.9 --- Statistical analysis --- p.90 / Chapter 5.3 --- Results --- p.90 / Chapter 5.3.1 --- Cuff-induced neointimal hyperplasia was reduced in Trpm2 knockout mice --- p.90 / Chapter 5.3.2 --- [Ca²⁺]i response to HX-XO in mouse neointimal smooth muscle cells --- p.90 / Chapter 5.3.3 --- Inhibiting TRPM2 reduced the neointimal hyperplasia in in vitro cultured human saphenous vein --- p.91 / Chapter 5.4 --- Discussion --- p.99 / Chapter Chapter 6 --- Role of TRPM2 in H₂O₂-stimulated migration and proliferation of vascular smooth muscle cells --- p.103 / Chapter 6.1 --- Introduction --- p.103 / Chapter 6.2 --- Materials and Methods --- p.104 / Chapter 6.2.1 --- Culture of rodent aortic smooth muscle cells --- p.104 / Chapter 6.2.2 --- Immunocytochemistry --- p.104 / Chapter 6.2.3 --- Genotyping for Trpm2 knockout mice --- p.104 / Chapter 6.2.4 --- Preparation of anti-TRPM2 antibody, TM2E3 and preimmune IgG --- p.104 / Chapter 6.2.5 --- [Ca²⁺]i measurement --- p.105 / Chapter 6.2.6 --- Cell proliferation assay --- p.105 / Chapter 6.2.7 --- Western blotting --- p.105 / Chapter 6.2.8 --- Cell migration assay --- p.106 / Chapter 6.2.9 --- Statistical analysis --- p.106 / Chapter 6.3 --- Results --- p.106 / Chapter 6.3.1 --- H₂O₂-induced [Ca²⁺]i rises in rodent aortic smooth muscle cells --- p.106 / Chapter 6.3.2 --- Role of TRPM2 in H₂O₂-stimulated smooth muscle cell proliferation --- p.107 / Chapter 6.3.3 --- Role of TRPM2 in H₂O₂-stimulated smooth muscle cell migration --- p.108 / Chapter 6.4 --- Discussion --- p.118 / Chapter Chapter 7 --- General Conclusion and Future Work --- p.121 / Chapter 7.1 --- Concluding remarks --- p.121 / Chapter 7.2 --- Future work --- p.123 / Chapter 7.2.1 --- Specific downstream signaling pathway of TRPM2 that mediates ROS-induced VSMC proliferation and migration --- p.123 / Chapter 7.2.2 --- Involvement of TRPM2 in leukocyte infiltration and inflammation in vascular wall --- p.124 / References --- p.125 / List of Publications --- p.152

壓力感受器在調節血壓的壓力感受性反射中的作用已是眾所周知。兩個動脈壓力感受器，分別為主動脈壓力感受器和頸動脈壓力感受器。它們作為重要的感應器以檢測主要動脈血壓，並和孤束核溝通，從而調節血壓。然而，壓力感受器的機械力敏感元件的分子身份仍是奧秘。因為機械敏感的陽離子通道受機械力刺激時會增加的神經元活動, 所以機械敏感的陽離子通道是合適的候選人。 / 在本研究中，通過使用膜片鉗和動作電位的測量，瞬时受体电位通道C5(TRPC5)被確定在主動脈壓力感受器的機械傳感器中。透過在壓力感受器神經元的鈣測量實驗，證實TRPC5參與由拉伸引起的鈣離子([Ca²⁺]i)上升。TRPC5基因敲除小鼠出現壓力感受器功能受損, 表明了TRPC5在血壓控制的重要性。 / 比較主動脈壓力感受器或頸動脈壓力感受器的不同敏感度現時存有不少爭論。在本研究中，我發現主動脈壓力感受器比頸動脈壓力感受器對於壓力變化更加敏感。此外，我還發現了主動脈壓力感受器神經元比頸動脈壓力感受器神經元有一個相對較高的瞬时受体电位通道V4(TRPV4)表達。鈣測量研究表明TRPV4通道在主動脈壓力感受器神經元的靈敏度可能發揮著重要作用。 / Baroreceptors have been well known for its role in the baroreflex regulation of blood pressure. Two arterial baroreceptors, aortic and carotid baroreceptors, serve as the important sensors to detect blood pressure in main arteries, and they communicate with the solitary nucleus tract for blood pressure regulation. However, the molecular identity of the mechano-sensitive components in the baroreceptors is still mysteries. Mechano-sensitive cation channels are the fascinating candidates as they increase neuronal activities when stimulated by stretch. In the present study, with the use of patch clamp and action potential measurement, TRPC5 channels were identified to be the mechanical sensor in the aortic baroreceptor. Calcium measurement studies demonstrated that TRPC5 was involved in the stretch-induced [Ca2+]i rise in baroreceptor neurons. The importance of TRPC5 in blood pressure control was also studied in TRPC5 knockout mice, which displayed an impaired baroreceptor function. / There have been controversies as to whether aortic baroreceptors or carotid baroreceptors are more sensitive to the change in blood pressure. In the present study, aortic baroreceptor was found to be more sensitive to the pressure change than the carotid baroreceptor. Furthermore, I also found a relative higher expression of TRPV4, a mechanosensitive channel, in the aortic baroreceptor neurons than in the carotid baroreceptor neurons. Moreover, calcium measurement studies showed that TRPV4 channels should play an important role in governing the differential pressure sensitivity in these two types of baroreceptor neurons. / Taken together, the present study provided novel information on the role of TRPC5 and TRPV4 in baroreceptor mechanosensing. In future, it will be of interest to explore whether TRPC5 and/or TRPV4 dysfunction could contribute to human diseases that are related to blood pressure control. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Lau, On Chai Eva. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 133-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / Declaration --- p.i / Abstract of the thesis entitled --- p.ii / Acknowledgement --- p.vii / Abbreviation --- p.ix / Table of content --- p.xii / List of figures --- p.xv / List of table --- p.xvii / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Baroreceptors --- p.1 / Chapter 1.1.2 --- Arterial baroreceptors --- p.2 / Chapter 1.1.2.1 --- Functions of arterial baroreceptors --- p.4 / Chapter 1.1.2.2 --- Sensitivity of the arterial baroreceptors --- p.6 / Chapter 1.1.3 --- Other baroreceptors --- p.8 / Chapter 1.1.4 --- The molecular identity of the mechanosensors in baroreceptor neurons --- p.9 / Chapter 1.2 --- Transient receptor potential ion channels (TRP channels) --- p.10 / Chapter 1.2.1 --- TRP channels superfamily --- p.10 / Chapter 1.2.2 --- Multimerization of TRP channels --- p.12 / Chapter 1.2.3 --- Physiological functions --- p.14 / Chapter 1.2.4 --- Mechanosensitive TRP channels --- p.16 / Chapter 1.2.5 --- Canonical transient receptor potential 5 (TRPC5) channels --- p.17 / Chapter 1.2.6 --- Vanilloid transient receptor potential 4 (TRPV4) channels Figures --- p.20 / Chapter Chapter 2: --- Objectives --- p.34 / Chapter Chapter 3: --- Materials and Methods --- p.35 / Chapter 3.1 --- Materials --- p.35 / Chapter 3.1.1 --- Chemicals and reagents --- p.35 / Chapter 3.1.2 --- Solutions --- p.36 / Chapter 3.1.2.1 --- Solutions for calcium imaging --- p.36 / Chapter 3.1.2.2 --- Solutions for electrophysiology study --- p.38 / Chapter 3.1.2.3 --- Solutions for agarose gel electrophoresis --- p.41 / Chapter 3.1.3 --- Primers for RT-PCR --- p.42 / Chapter 3.1.4 --- Animals --- p.43 / Chapter 3.2 --- Methods --- p.43 / Chapter 3.2.1 --- Total RNA isolation and RT-PCR --- p.43 / Chapter 3.2.2 --- Immunohistochemistry --- p.44 / Chapter 3.2.3 --- Neuron labeling by DiI --- p.45 / Chapter 3.2.4 --- Neuron culture --- p.46 / Chapter 3.2.5 --- [Ca²⁺]i measurement --- p.47 / Chapter 3.2.6 --- Electrophysiology --- p.48 / Chapter 3.2.7 --- Evaluation of baroreflex response --- p.49 / Chapter 3.2.8 --- Telemetric measurement of blood pressure --- p.50 / Chapter 3.2.9 --- Statistical analysis --- p.51 / Figures --- p.52 / Chapter Chapter 4: --- Functional role of TRPC5 channels in aortic baroreceptor --- p.56 / Chapter 4.1 --- Introduction --- p.56 / Chapter 4.2 --- Materials and Methods --- p.59 / Chapter 4.2.1 --- Animals --- p.59 / Chapter 4.2.2 --- Immunohistochemistry --- p.59 / Chapter 4.2.3 --- Neuron labeling by DiI --- p.61 / Chapter 4.2.4 --- Neuron culture --- p.62 / Chapter 4.2.5 --- [Ca²⁺]i measurement --- p.63 / Chapter 4.2.6 --- Electrophysiology --- p.63 / Chapter 4.2.7 --- Evaluation of baroreflex response --- p.64 / Chapter 4.2.8 --- Telemetric measurement of blood pressure --- p.66 / Chapter 4.2.9 --- Statistical analysis --- p.67 / Chapter 4.3 --- Results --- p.67 / Chapter 4.3.1 --- Endogenous expression of TRPC5 channels in aortic baroreceptor neurons --- p.67 / Chapter 4.3.2 --- Characterization on the pressure-sensitive component in aortic baroreceptors --- p.68 / Chapter 4.3.3 --- Involvement of TRPC5 in [Ca²⁺]i response in aortic baroreceptor neurons --- p.69 / Chapter 4.3.4 --- Participation of TRPC5 in pressure-induced action potential firing in cultured aortic baroreceptor neurons --- p.70 / Chapter 4.3.5 --- Role of TRPC5 in baroreceptor sensory nerve activity and baroreflex regulation --- p.71 / Chapter 4.3.6 --- Importance of TRPC5 in baroreceptor function --- p.72 / Chapter 4.4 --- Discussions --- p.74 / Figures --- p.79 / Table --- p.98 / Chapter Chapter --- 5: TRPV4 channels and baroreceptor sensitivity --- p.99 / Chapter 5.1 --- Introduction --- p.99 / Chapter 5.2 --- Materials and Methods --- p.101 / Chapter 5.2.1 --- Animals --- p.101 / Chapter 5.2.2 --- Neuron labeling by DiI --- p.101 / Chapter 5.2.3 --- Neuron culture --- p.102 / Chapter 5.2.4 --- Electrophysiology --- p.103 / Chapter 5.2.5 --- Immunohistochemistry --- p.104 / Chapter 5.2.6 --- [Ca²⁺]i measurement --- p.105 / Chapter 5.2.7 --- Statistical analysis --- p.105 / Chapter 5.3 --- Results --- p.106 / Chapter 5.3.1 --- Properties of the aortic and carotid baroreceptor neurons --- p.106 / Chapter 5.3.2 --- Stretch sensitivity of aortic and carotid baroreceptor neurons --- p.108 / Chapter 5.3.3 --- mRNA expression of mechanosensitive TRP channels in aortic and carotid baroreceptor neurons --- p.109 / Chapter 5.3.4 --- Protein expression of TRPV4 channels in aortic and carotid baroreceptor neurons --- p.109 / Chapter 5.3.5 --- Involvement of TRPV4 in stretch-induced [Ca²⁺]i response in baroreceptor neurons --- p.110 / Chapter 5.4 --- Discussions --- p.111 / Figures --- p.116 / Chapter Chapter 6: --- General conclusions and future directions --- p.124 / Figures --- p.128 / References --- p.133

Orientadora: Profa. Dra. Maria Camila Almeida / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Neurociência e Cognição, 2014.

Transient receptor potential (TRP) channels comprise a superfamily of cation-selective ion channels that are largely calcium (Ca2+) permeable and that play diverse physiological roles ranging from nociception in primary afferent neurons to the absorption of dietary Ca2+. The 28 mammalian TRP channels are categorized into 6 subfamilies. The vanilloid subfamily is named for its founding member, TRPV1, the capsaicin receptor, and has 6 members. TRPV1-4 are all heat sensitive ion channels whereas TRPV5 and TRPV6 are involved in renal Ca2+ reabsorption and Ca2+ absorption in the intestine, respectively. In our structural studies, we have focused on TRPV3 and TRPV6. TRPV6 is a highly Ca2+ selective TRP channel (PCa/PNa ~ 130) that functions in active Ca2+ absorption in the intestine. Its expression is upregulated by vitamin D and is, on the molecular level, regulated by PIP2 and calmodulin (CaM). Previously, the structure of TRPV6 was solved using X-ray crystallography. Using the crystal structure, a negatively charged extracellular vestibule was identified and anomalous diffraction was used to identify ion binding sites in the pore. Also, at the top of the selectivity filter, four aspartates were identified that coordinate Ca2+ entering the pore and confer to TRPV6 its selectivity for Ca2+. However, only the structure of the rat orthologue was solved and only in the closed, apo state. We used cryo-electron microscopy (cryo-EM) to solve structures of the human orthologue of TRPV6 in the open and closed (we used the mutation R470E to close the channel) states. The closed-to-open TRPV6 transition is accompanied by the formation of short π-helices in the middle of the pore-lining S6 helices, which in turn results in their turning and a different set of residues facing the pore. Additionally, the formation of the π-helices results in kinking of the S6 helices, which further widens the pore. TRPV6 is constitutively active when expressed heterologously. In other words, the addition of external stimuli is not necessary for the activation of the channel. Therefore, its activity needs to be regulated to prevent toxic Ca2+ overload. One mechanism by which this occurs is through its regulation by CaM. CaM has been shown to bind TRPV6 and regulate its function, however, the way it binds to and regulates TRPV6 remained unknown. To uncover this mechanism, we solved the structure of TRPV6 bound to CaM. We found that CaM binds TRPV6 in a 1:1 stoichiometric ratio and that CaM directly blocks the TRPV6 pore by inserting a positively charged lysine into a tera-tryptophan cage at the bottom of the pore. As a result, the channel adopts an inactivated conformation; although the pore-lining S6 helices still contain local π-helices, they are pulled closer together, narrowing the pore and further blocking it with hydrophobic side chains. We have also conducted studies of TRPV3. Unlike TRPV6, TRPV3 is a heat-activated vanilloid TRP channel. TRPV3 is expressed highly in keratinocytes where it has been implicated in wound healing and maintenance of the skin barrier, and in the regulation of hair growth. We solved the structure of apo TRPV3 in a closed state, and the structure of a TRPV3 mutant bound to 2-APB in an open state. Like TRPV6, the opening of TRPV3 is accompanied by the formation of local π-helices in the middle of the pore-lining S6 helices. The formation of the π-helices results in the lining of the ion permeation pathway with a different set of residues, resulting in a largely negatively charged pathway. Unlike TRPV6, TRPV3 is only slightly selective for Ca2+ and correspondingly, during gating state transitions, rearrangements were not only observed only in its pore-lining helices, but also in the cytosolic domain and the selectivity filter. Based on a comparison of our structures, we proposed a model of TRPV3 regulation by 2-APB. Together, our studies provide insight into the regulatory and gating mechanisms of the vanilloid subtype TRP channels and can provide the foundation for future studies.

Background and aim: The calcium ion (Ca2+) is an important ion involved in intracellular signalling. An increase in the free intracellular calcium concentration ([Ca2+]i) is essential for triggering insulin secretion from pancreatic beta cells. Beta cell death or disturbed insulin secretion are key factors in the pathogenesis of type 1 and type 2 diabetes respectively. A number of Ca2+ channels located on the plasma membrane or on the endoplasmic reticulum (ER) mediate Ca2+ increase in beta cells. Among the plasma membrane Ca2+ channels, members of the Transient Receptor Potential (TRP) family are currently of great interest. Transient Receptor Potential Vanilloid subtype 1 (TRPV1) is one of the 28 members of the TRP family. This ion channel is activated by heat and pungent chemicals like capsaicin. The main aim of this study was to investigate if functional TRPV1 channels are present in insulin secreting cells. Further more we examined if TRP channels could be studied by using microfluorometry in single cells. A third objective was to investigate if members of the TRP family could be identified by western blot. Methods: We used S5 cells, a highly differentiated rat insulinoma cell line, as a model of beta cells. A ratiometric fluorescence technique was used for measurement of [Ca2+]i concentration from single Fura-2 loaded cells. [Ca2+]i was measured continuously using microscope based fluorometry with the time resolution of 1 Hz. For western blot we used proteins extracted from S5 cells and human islets. The blots were probed with antibodies directed against both the N-terminal and the C-terminal end of the protein. Results: Capsaicin, an activator of TRPV1, increased [Ca2+]i in a dose-dependent manner with a half maximal effective concentration (EC50) ~ 100 nM. In nominally Ca2+ free buffer the capsaicin-induced [Ca2+]i increase was completely lost, while the intracellular depots of Ca2+ were not emptied as shown by administration of carbachol. The capsaicin-induced [Ca2+]i increase was completely blocked by capsazepine, an antagonist of TRPV1. An increase in temperature in the range of 43 – 49 °C increased [Ca2+]i, whereas temperatures < 42 °C did not. In nominally Ca2+ free medium the response to heat was reduced. Subsequent administration of carbachol showed that intracellular depots of Ca2+ were not emptied. Ruthenium red, an antagonist of TRPV1, also reduced the heat induced [Ca2+]i response. Another heat-sensitive, Ca2+ permeable protein Transient Receptor Potential Melastatin-like subtype 2 (TRPM2) was detected in S5 cells and human islets by western blot. The 171 kDa band represents the full length TRPM2 and is clearly visible in human islets, while the 95 KDa band represents the truncated form of TRPM2 and is more prominent in S5 cells. Interpretation and conclusions: Microscope based fluorometry is a powerful method for studying ion channels of the TRP family in single living cells. We found that pancreatic beta cells express functional TRPV1 channels that were activated by capsaicin and heat. TRPV1 channels of beta cells are located on the plasma membrane and not on the ER. TRP channel proteins can also be detected by the western blot technique. The ease of studying TRP channels by microfluorometry and our demonstration of functionalTRPV1 channels in beta cells paves the way for studying the role of these channels in insulin secretion and in the pathogenesis of diabetes.

<p>From insects to mammals, the ability to constantly sense environmental stimuli is essential for the survival of most living organisms. Most animals have nocifensive behaviors towards extreme temperatures, mechanical stimuli or irritant chemicals that are considered to be noxious. Nociception is defined as the neural encoding and processing of noxious stimuli. This process starts from the activation of pain detecting peripheral sensory neurons (nociceptors) that can detect noxious mechanical, thermal or chemical stimuli. On the other hand, animals also have the ability to discriminate innocuous temperatures and to direct their locomotions to their favorable environmental temperatures and this behavior is called thermotaxis. </p><p>In this study, I used <italic>Drosophila melanogaster</italic>as a genetic model organism to study the molecular and cellular basis of nociception and thermotaxis. <italic>Drosophila</italic> larvae exhibit a stereotyped defensive behavior in response to nociceptive stimuli (termed nocifensive escape locomotion behavior, NEL). Using this behavior as a readout, we manipulated the neuronal activities of periphery sensory Type II multidendritic neurons and have identified a specific class of neurons, class IV multidendritic neurons, to function as nociceptors in <italic>Drosophila</italic> larvae. </p><p>After identifying the nociceptors, I next investigated several ion channels that are critical molecular components for larval nociception. The Degenerin Epithelial Sodium Channel (DEG/ENaC) protein called pickpocket (ppk) is required specifically for larval mechanical nociception but not for thermal nociception. Being specifically expressed in class IV multidendritic neurons (the nociceptors), pickpocket is likely to function as a first detector of mechanical stimuli and upstream of general neuronal action potential propagation. In addition, I have found that the <italic>Drosophila</italic> orthologue of mammalian TRPA1 gene, <italic>TrpA1</italic>, is required for both mechanical and thermal nociception in <italic>Drosophila</italic> larvae. I have cloned a new isoform of dTRPA1 and have found it to be specifically expressed in class IV md neurons. Unlike the known dTRPA1 isoform that is warmth activated, this new isoform is not directly activated by temperatures between 15-42 °C. Instead, it may function downstream of sensory transduction step in the nociceptors. </p><p>Interestingly, <italic>dTrpA1</italic> mutants are also defective in their thermotaxis behavior within innocuous temperature ranges. In addition to the previously reported defects in avoiding warm temperatures, I have found these flies also failed to avoid cool temperatures between 16-19.5 °C. This defect is likely to be mediated by temperature sensing neurons in the antennae. I have detected antennal expression using a GAL4 reporter of dTrpA1. Significantly, these neurons exhibit elevated calcium levels in response to cooling. dTrpA1 mutants have a premature decay of the cooling response at temperatures below 22 °C during a cooling process. I have also identified another population of cells in the antennae that can respond to temperature changes. These neurons express the olfactory co-receptor Or83b and are known to be olfactory neurons. Calcium oscillations triggered by cooling were detected in these neurons and they were terminated by warming. Severe behavioral defects in avoiding cool temperatures were found in animals lacking <italic>Or83b</italic>. Our results suggest that there are multiple pathways regulating cooling sensation in the fly antennae.</p><p>Taken together, I have shown that <italic>Drosophila</italic> serves as a great model system to study nociception and thermosensation at molecular, cellular and behavioral levels.</p> / Dissertation

<p>Background and aim: The calcium ion (Ca2+) is an important ion involved in intracellular signalling. An increase in the free intracellular calcium concentration ([Ca2+]i) is essential for triggering insulin secretion from pancreatic beta cells. Beta cell death or disturbed insulin secretion are key factors in the pathogenesis of type 1 and type 2 diabetes respectively. A number of Ca2+ channels located on the plasma membrane or on the endoplasmic reticulum (ER) mediate Ca2+ increase in beta cells. Among the plasma membrane Ca2+ channels, members of the Transient Receptor Potential (TRP) family are currently of great interest. Transient Receptor Potential Vanilloid subtype 1 (TRPV1) is one of the 28 members of the TRP family. This ion channel is activated by heat and pungent chemicals like capsaicin. The main aim of this study was to investigate if functional TRPV1 channels are present in insulin secreting cells. Further more we examined if TRP channels could be studied by using microfluorometry in single cells. A third objective was to investigate if members of the TRP family could be identified by western blot.</p><p>Methods: We used S5 cells, a highly differentiated rat insulinoma cell line, as a model of beta cells. A ratiometric fluorescence technique was used for measurement of [Ca2+]i concentration from single Fura-2 loaded cells. [Ca2+]i was measured continuously using microscope based fluorometry with the time resolution of 1 Hz. For western blot we used proteins extracted from S5 cells and human islets. The blots were probed with antibodies directed against both the N-terminal and the C-terminal end of the protein.</p><p>Results: Capsaicin, an activator of TRPV1, increased [Ca2+]i in a dose-dependent manner with a half maximal effective concentration (EC50) ~ 100 nM. In nominally Ca2+ free buffer the capsaicin-induced [Ca2+]i increase was completely lost, while the intracellular depots of Ca2+ were not emptied as shown by administration of carbachol. The capsaicin-induced [Ca2+]i increase was completely blocked by capsazepine, an antagonist of TRPV1. An increase in temperature in the range of 43 – 49 °C increased [Ca2+]i, whereas temperatures < 42 °C did not. In nominally Ca2+ free medium the response to heat was reduced. Subsequent administration of carbachol showed that intracellular depots of Ca2+ were not emptied. Ruthenium red, an antagonist of TRPV1, also reduced the heat induced [Ca2+]i response. Another heat-sensitive, Ca2+ permeable protein Transient Receptor Potential Melastatin-like subtype 2 (TRPM2) was detected in S5 cells and human islets by western blot. The 171 kDa band represents the full length TRPM2 and is clearly visible in human islets, while the 95 KDa band represents the truncated form of TRPM2 and is more prominent in S5 cells.</p><p>Interpretation and conclusions: Microscope based fluorometry is a powerful method for studying ion channels of the TRP family in single living cells. We found that pancreatic beta cells express functional TRPV1 channels that were activated by capsaicin and heat. TRPV1 channels of beta cells are located on the plasma membrane and not on the ER. TRP channel proteins can also be detected by the western blot technique. The ease of studying TRP channels by microfluorometry and our demonstration of functionalTRPV1 channels in beta cells paves the way for studying the role of these channels in insulin secretion and in the pathogenesis of diabetes.</p>

Thesis (Ph.D.)--University of Central Florida, 2008. / Adviser: Sic L. Chan. Includes bibliographical references (p. 74-84).

The nervous system, including the brain, is a complex network with billions of complex neurons. Ion channels mediate the electrical signals that neurons use to integrate input and produce appropriate output, and could thus be thought of as key instruments in the neuronal orchestra. In the field of neuroscience we are not only curious about how our brains work, but also strive to characterize and develop treatments for neural disorders, in which the neuronal harmony is distorted. By modulating ion channel activity (pharmacologically or otherwise) it might be possible to effectively restore neuronal harmony in patients with various types of neural (including channelopathic) disorders. However, this exciting strategy is impeded by the gaps in our understanding of ion channels and neurons, so more research is required. Thus, the aim of this thesis is to improve the understanding of how specific ion channel types contribute to shaping neuronal dynamics, and in particular, neuronal integration, excitability and memory. For this purpose I have used computational modeling, an approach which has recently emerged as an excellent tool for understanding dynamically complex neurophysiological phenomena. In the first of two projects leading to this thesis, I studied how neurons in the brain, and in particular their dendritic structures, are able to integrate synaptic inputs arriving at low frequencies, in a behaviorally relevant range of ~8 Hz. Based on recent experimental data on synaptic transient receptor potential channels (TRPC), metabotropic glutamate receptor (mGluR) dynamics and glutamate decay times, I developed a novel model of the ion channel current ITRPC, the importance of which is clear but largely neglected due to an insufficient understanding of its activation mechanisms. We found that ITRPC, which is activated both synaptically (via mGluR) and intrinsically (via Ca2+) and has a long decay time constant (τdecay), is better suited than the classical rapidly decaying currents (IAMPA and INMDA) in supporting low-frequency temporal summation. It was further concluded that τdecay varies with stimulus duration and frequency, is linearly dependent on the maximal glutamate concentration, and might require a pair-pulse protocol to be properly assessed. In a follow-up study I investigated small-amplitude (a few mV) long-lasting (a few seconds) depolarizations in pyramidal neurons of the hippocampal cortex, a brain region important for memory and spatial navigation. In addition to confirming a previous hypothesis that these depolarizations involve an interplay of ITRPC and voltage-gated calcium channels, I showed that they are generated in distal dendrites, are intrinsically stable to weak excitatory and inhibitory synaptic input, and require spatial and temporal summation to occur. I further concluded that the existence of multiple stable states cannot be ruled out, and that, in spite of their small somatic amplitudes, these depolarizations may strongly modulate the probability of action potential generation. In the second project I studied the axonal mechanisms of unmyelinated peripheral (cutaneous) pain-sensing neurons (referred to as C-fiber nociceptors), which are involved in chronic pain. To my knowledge, the C-fiber model we developed for this purpose is unique in at least three ways, since it is multicompartmental, tuned from human microneurography (in vivo) data, and since it includes several biologically realistic ion channels, Na+/K+ concentration dynamics, a Na-K-pump, morphology and temperature dependence. Based on simulations aimed at elucidating the mechanisms underlying two clinically relevant phenomena, activity-dependent slowing (ADS) and recovery cycles (RC), we found an unexpected support for the involvement of intracellular Na+ in ADS and extracellular K+ in RC. We also found that the two major Na+ channels (NaV1.7 and NaV1.8) have opposite effects on RC. Furthermore, I showed that the differences between mechano-sensitive and mechano-insensitive C-fiber types might reside in differing ion channel densities. To conclude, the work of this thesis provides key insights into neuronal mechanisms with relevance for memory, pain and neural disorders, and at the same time demonstrates the advantage of using computational modeling as a tool for understanding and discovering fundamental properties of central and peripheral neurons. / <p>QC 20120914</p>

Irritable Bowel Syndrome (IBS) is one of the most common functional disorders of the gastrointestinal tract. Visceral hypersensitivity is the most commonly reported symptom of IBS, yet is the least adequately treated. Mechanosensitive information from the colon is relayed to the CNS by extrinsic colonic primary afferent nerves which have their cell bodies within dorsal root ganglia (DRG). This thesis aims to identify the contribution of several putatively mechanosensitive ion channels (ASIC1, 2 and 3, TRPV4 and TRPA1) toward detection of mechanical stimuli in the colon. This involvement is assessed by both molecular and functional means. The abundance of each of these channels was assessed by comparing expression within whole DRG against that in specifically colonic DRG neurons using an in situ hybridization methodology developed as part of this PhD. The functional role TRPV4 and TRPA1 impart toward colonic mechanosensation was investigated by recording responses to mechanical stimuli from colonic primary afferent fibres and comparing the results from mice genetically modified to lack either TRPV4 or TRPA1 with those of their intact littermates. The results from these studies indicate expression patterns within whole DRG do not provide accurate representation of the organ of interest, with abundances of each of the channels investigated differing between colonic DRG cells and the whole DRG. In particular ASIC3 and TRPV4 are preferentially expressed in colonic DRG neurons, unlike ASIC2 and TRPA1. Further, TRPV4 is functionally restricted to detection of noxious mechanical stimuli in the colon, while expression of TRPA1 is more widespread and functionally less restricted. Each of these channels are each potential targets for the treatment of IBS as each affects specific aspects of colonic mechanotransduction. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1347202 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Sciences, 2008