Yang Qin. / "June 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 168-207). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Lau Kin Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 97-103). / Abstracts in English and Chinese. / Contents --- p.1 / Chapter Chapter 1 --- Introduction --- p.5 / Chapter 1.1 --- Calcium Signaling in Endothelial Cells --- p.5 / Chapter 1.1.1 --- Calcium and its functions --- p.5 / Chapter 1.1.2 --- "Second Messengers: Inositol-1,4,5-Triphosphate and Diacylglycerol" --- p.6 / Chapter 1.1.3 --- Propagation of Ca2+ Signals --- p.8 / Chapter 1.1.4 --- Ca2+-ATPases --- p.9 / Chapter 1.1.5 --- Regulation of Sarcoplasmic Reticulum --- p.10 / Chapter 1.1.6 --- Agonist-induced Ca2+ Entry --- p.11 / Chapter 1.2 --- Mechanism of Store-Operated Ca2+ Entry --- p.14 / Chapter 1.2.1 --- Signaling Mechanisms of SOC --- p.14 / Chapter 1.2.1.1 --- A Diffusible Messenger --- p.14 / Chapter 1.2.1.2 --- Conformational Coupling --- p.15 / Chapter 1.2.1.3 --- Vesicle Secretion --- p.16 / Chapter 1.3 --- Regulation of Ca2+ Entry by cGMP --- p.20 / Chapter 1.4 --- Molecular Structres of Store-operated Channels --- p.22 / Chapter 1.4.1 --- Drosophila Transient Receptor Potential (trp) Gene --- p.22 / Chapter 1.4.2 --- Trpl Gene --- p.23 / Chapter Chapter 2 --- Methods and Materials --- p.27 / Chapter 2.1 --- Materials --- p.27 / Chapter 2.1.1 --- Phosphate-buffered saline --- p.27 / Chapter 2.1.2 --- Culture Media and Materials --- p.27 / Chapter 2.2 --- Preparations and Culture of Cells --- p.28 / Chapter 2.2.1 --- Culture of Rat Aortic Endothelial Cells --- p.28 / Chapter 2.2.2 --- Culture of Human Bladder Epithelial Cell Line --- p.29 / Chapter 2.2.3 --- Culture of Human Embryonic Kidney Epithelial Cell Line --- p.29 / Chapter 2.3 --- Cell. Subculture and Marvest --- p.29 / Chapter 2.4 --- Intracellular Free Calcium Ions ([Ca2+]i) measurment --- p.30 / Chapter 2.4.1 --- Chemicals --- p.30 / Chapter 2.4.2 --- Bathing solutions --- p.31 / Chapter 2.4.3 --- Preparations of Cells for [Ca2+]i Measurement --- p.31 / Chapter 2.4.3.1 --- Plating cells on Glass Cover Slips for [Ca2+]i Measurement with PTI RatioMaster Fluorescence System --- p.31 / Chapter 2.4.3.2 --- Plating cells on Glass Cover Slips for [Ca2+]i Measurement with Confocal Imaging System and Confocal Laser Scanning Microscopy --- p.32 / Chapter 2.4.4 --- PTI RatioMaster Fluorescence System --- p.35 / Chapter 2.4.4.1 --- Experimental Setup --- p.35 / Chapter 2.4.4.2 --- Fura-2/AM Dye loading --- p.35 / Chapter 2.4.4.3 --- Background Fluorescence and [Ca ]i Measurement --- p.37 / Chapter 2.4.5 --- Confocal Imaging System --- p.37 / Chapter 2.4.5.1 --- Experimental Setup --- p.37 / Chapter 2.4.5.2 --- Fluo-3/AM Dye Loading --- p.39 / Chapter 2.4.5.3 --- [Ca2+]i Measurement --- p.39 / Chapter 2.4.6 --- Confocal Laser Scanning Microscopy --- p.40 / Chapter 2.4.6.1 --- Principles --- p.40 / Chapter 2.5 --- Cloning and expression of Trpl in HEK293 cell line --- p.43 / Chapter 2.5.1 --- Cloning of Htrpl Gene into pcDNA3 Vector --- p.43 / Chapter 2.5.1.1 --- Enzyme Digestion --- p.43 / Chapter 2.5.1.2 --- Gel electrophoresis and Isolation of Htrpl by GeneCIean II Kit --- p.44 / Chapter 2.5.1.3 --- Ligation of Trpl and pcDNA3 Vector --- p.44 / Chapter 2.5.1.4 --- Transformation --- p.47 / Chapter 2.5.1.5 --- Purification of cloned Trpl-pcDNA3 by QIAprep Spin Miniprep Kit --- p.47 / Chapter 2.5.2 --- Transfection of HEK293 Cells with Htrpl and pEGFP-Nl Vector --- p.48 / Chapter 2.5.2.1 --- Cell Preparation for Transfection --- p.48 / Chapter 2.5.2.2 --- Transfection --- p.48 / Chapter 2.5.3 --- Fluorescence Labeling of Expressed Htrpl Channel in HEK293 Cells --- p.49 / Chapter 2.5.3.1 --- Immunostaining with Anti-TRPCl Antibody --- p.49 / Chapter 2.5.3.2 --- Labeling with FITC2° Antibody --- p.50 / Chapter Chapter 3 --- Results --- p.51 / Chapter 3.1 --- Propagation of Ca2+ Signaling --- p.51 / Chapter 3.2. --- Effect of cGMP on SERCA --- p.55 / Chapter 3.2.1 --- ATP stimulated Ca2+ release from internal stores --- p.55 / Chapter 3.2.2 --- Effect of cGMP on the falling phase of [Ca2+]i --- p.55 / Chapter 3.2.3 --- Effect of CPA on the falling phase of [Ca2+]i --- p.58 / Chapter 3.2.4 --- Effect of KT5823 on cGMP --- p.63 / Chapter 3.3. --- Effect of cGMP on bradykinin-activated capacitative Ca2+ entry --- p.65 / Chapter 3.3.1 --- Bradykinin induced capacitative Ca2+ entry --- p.65 / Chapter 3.3.2 --- Effect of cGMP on Ca2+ entry activated by bradykinin --- p.67 / Chapter 3.3.3 --- Effect of KT5823 on the inhibitory effect of cGMP on Ca2+ entry activated by bradykinin --- p.67 / Chapter 3.3.4. --- Effect of cGMP and KT5823 on capacitative Ca2+ entry activated by a combination of different agonists. --- p.71 / Chapter 3.4 --- Cloning and expression of htrpl in HEK 293 cell line --- p.75 / Chapter 3.4.1 --- Optimizing transfection conditions using pEGFP-Nl --- p.78 / Chapter 3.4.2 --- Transient transfection of htrpl channel in HEK293 cells --- p.81 / Chapter 3.4.3 --- Channel properties of expressed htrpl channel --- p.84 / Chapter Chapter 4 --- Discussion --- p.88 / Chapter 4.1 --- Ptopagation of Ca2+ Signaling --- p.88 / Chapter 4.2 --- Effect of cGMP on[Ca2+]i of Vascular Endothelial Cells --- p.89 / Chapter 4.2.1 --- Effect of cGMP on SERCA --- p.89 / Chapter 4.2.2 --- Effect of cGMP on Regulation of Agonist-Activated Capacitative Ca2+ Entry --- p.92 / Chapter 4.2.3 --- Physiological Property of Expressed Htrpl in HEK293 cells --- p.95 / References --- p.97

Leung, Pan Cheung Catherine. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 105-120). / Abstracts in English and Chinese. / DECLARATION --- p.II / ACKNOWLEDGEMENTS --- p.III / ENGLISH ABSTRACT --- p.IV / CHINESE ABSTRACT --- p.VII / Chapter MODULE 1. --- INTRODUCTION --- p.1 / Chapter 1.1. --- Vascular Endothelium --- p.1 / Chapter 1.1.1. --- Vascular Endothelial Functions --- p.1 / Chapter 1.1.2. --- Calcium Signaling in Vascular Endothelial Cells --- p.2 / Chapter 1.2. --- The Founding Member of TRP Family: Drosophila TRP --- p.3 / Chapter 1.2.1. --- Discovery of Drosophila TRP and TRP-related Proteins --- p.3 / Chapter 1.2.2. --- Drosophila TRPs: Ca2+-permeable Channels? --- p.3 / Chapter 1.3. --- Mammalian TRP Superfamily --- p.5 / Chapter 1.3.1. --- The TRP Subfamily: TRPV --- p.9 / Chapter 1.3.2. --- The TRP Subfamily: TRPM --- p.9 / Chapter 1.3.3. --- The TRP Subfamily: TRPC --- p.11 / Chapter 1.4. --- Functional and Physiological Roles of Mammalian TRPCs --- p.12 / Chapter 1.4.1. --- TRPC1 --- p.15 / Chapter 1.4.2. --- TRPC2 --- p.16 / Chapter 1.4.3. --- "TRPC3, TRPC6 and TRPC7" --- p.17 / Chapter 1.4.4. --- TRPC4 and TRPC5 --- p.19 / Chapter 1.4.5. --- Over-expression of TRPCs: Physiologically Relevant Channels? --- p.20 / Chapter 1.4.6. --- Alternatives to Heterologous Expression Study --- p.21 / Chapter 1.5. --- Aims of the Study --- p.23 / Chapter MODULE 2. --- MATERIALS AND METHODS --- p.24 / Chapter 2.1. --- Functional Characterization of TRPCs by Antisense Technique --- p.24 / Chapter 2.1.1. --- Restriction Enzyme Digestion --- p.26 / Chapter 2.1.2. --- Purification of Released Inserts and Cut pcDNA3 Vectors --- p.27 / Chapter 2.1.3. --- "Ligation of TRPC Genes into Mammalian Vector, pcDNA3" --- p.27 / Chapter 2.1.4. --- Transformation for the Desired Clones --- p.28 / Chapter 2.1.5. --- Plasmid DNA Preparation for Transfection --- p.28 / Chapter 2.1.6. --- Confirmation of the Clones］ --- p.29 / Chapter 2.1.6.1. --- Restriction Enzymes Strategy --- p.29 / Chapter 2.1.6.2. --- Polymerase Chain Reaction (PRC) Check --- p.30 / Chapter 2.1.6.3. --- Automated Sequencing --- p.31 / Chapter 2.2. --- Establishing Stable Cell Lines --- p.33 / Chapter 2.2.1. --- Cell Culture --- p.33 / Chapter 2.2.2. --- Transfection Conditions Optimization --- p.33 / Chapter 2.2.3. --- Geneticin Selection --- p.35 / Chapter 2.3. --- Expression Pattern Studies of TRPC Genes in Vascular Tissues --- p.38 / Chapter 2.3.1. --- Immunofluorescence Staining in Cultured CPAE Cells --- p.38 / Chapter 2.3.2. --- Immunolocalization in Human Cerebral and Coronary Arteries --- p.40 / Chapter 2.3.2.1. --- Paraffin Section Preparation --- p.40 / Chapter 2.3.2.2. --- "Immunohistochemistry for TRPC1, 3, 4 and 6 Channels" --- p.40 / Chapter 2.3.2.3. --- Subcellular Localization of hTRPC1 and hTRPC3 Channels in Endothelial Cells --- p.42 / Chapter 2.4. --- Study of Bradykinin-induced Ca2+ Entry by Calcium Imaging --- p.47 / Chapter 2.4.1. --- Primary Aortic Endothelial Cell Culture --- p.47 / Chapter 2.4.2. --- Fura-2 Measurement of [Ca2+］］］ --- p.47 / Chapter 2.5. --- Study of Functional Role of TRPC6 in Stably Transfected H5V Cells … --- p.49 / Chapter 2.5.1. --- Protein Sample Preparation --- p.49 / Chapter 2.5.2. --- Western Blot Analysis --- p.50 / Chapter 2.5.3. --- Confocal Microscopy for Bradykinin-induced Calcium Entry --- p.51 / Chapter 2.6. --- Data Analysis --- p.52 / Chapter MODULE 3. --- RESULTS --- p.53 / Chapter 3.1. --- Bradykinin-induced Calcium Entry in Vascular Endothelial Cells --- p.53 / Chapter 3.1.1. --- Bradykinin-induced Calcium Entry --- p.53 / Chapter 3.1.2. --- Effects of cGMP and PKG on Bradykinin-induced Ca2+ Entry --- p.54 / Chapter 3.1.3. --- Effects of HOEUO on Bradykinin-induced Store-independent Ca2+ Entry --- p.55 / Chapter 3.1.4. --- Involvement of Phospholipase C Pathway in Bradykinin-induced Store-independent Ca2+ Entry --- p.55 / Chapter 3.2. --- Expression Pattern of TRPC Channels in Vascular Systems --- p.63 / Chapter 3.2.1. --- Immunolocalization of TRPC Homologues in CPAE Cells --- p.63 / Chapter 3.2.2. --- Immunolocalization of TRPC Homologues in Human Cerebral and Coronary Arteries --- p.66 / Chapter 3.2.3. --- Subcellular Localization of TRPC1 and TRPC3 Fused to Enhanced Green Fluorescence Protein (EGFP) --- p.77 / Chapter 3.3. --- Functional Role of TRPC6 Channels in Bradykinin-induced Calcium Entry --- p.81 / Chapter 3.3.1. --- Effect of Antisense TRPC6 Construct on Protein Expression --- p.81 / Chapter 3.3.2. --- Effect of Antisense TRPC6 on Bradykinin-induced Ca2+ Entry --- p.81 / Chapter 3.3.3. --- Effect of Antisense TRPC6 on Thapsigargin-depleted Ca2+ Stores --- p.82 / Chapter MODULE 4. --- DISCUSSION --- p.89 / Chapter 4.1. --- Characterization of Bradykinin-induced Ca2+ Entry in Endothelial Cells --- p.89 / Chapter 4.2. --- The Expression Pattern of TRPC Isoforms in Vascular Tissues --- p.93 / Chapter 4.3. --- Functional Characterization of TRPC6 Homologues in Bradykinin-induced Ca2+ Entry --- p.98 / Chapter 4.4. --- Perspectives --- p.103 / Chapter 4.5. --- Conclusion --- p.104 / Chapter MODULE 5. --- REFERENCES --- p.105

Cyclic nucleotide-gated (CNG) ion channels are Ca2+ permeable nonselective cation channels that are directly gated by binding of cAMP or cGMP, thus providing a linkage between two important signal molecules, cyclic nucleotides and calcium. They are known to play an important role in sensory transduction and in second-messenger modulation of synaptic neurotransmitter release. Previous studies showed that besides in neuronal cells, CNG were found also in non-neuronal tissues including heart, kidney, blood vessels and spleen, they are reported to be involved in a variety of cell function. / Ion channels play an indispensable role in endothelial cells, which is a unique signal-transducting surface in the vascular system that is responsible in altering vascular tone. The present study investigated the expression and functional roles of the cyclic nucleotide gated channels (CNG) in regulating the intracellular calcium level of vascular endothelial cells using molecular and calcium measurement techniques. / The present study provided evidence that the CNG channels, especially that of CNGA2, were expressed in vascular tissues. I used a variety of different methods, including RT-PCR, northern blot, in-situ hybridization, immunohistochemistry and western blot to study the localization of CNGA2 channels. RT-PCR amplify a CNGA2 fragment of 582bp from RNAs isolated from bovine vascular endothelial cell line, rat vascular smooth muscle cell line and rat aorta. Northern blot analysis detected a 2.3-kilobase (kb) CNGA2 transcript in rat aorta mRNA. The cellular distribution of CNGA2 was further studied by in-situ hybridization, which demonstrated expression of CNGA2 mRNA in human vascular endothelial and vascular smooth muscle cells. Immunohistochemistry data also agreed with those generated from in-situ hybridization. Western blot data also demonstrated proteins of CNG2 was expressed in both human vascular endothelial cells and vascular smooth cells layer. Subcellular localization of CNGA2 inside the vascular endothelial cells was also investigated with the use of a GFP linked CNGA2 channel gene. Taken together, the results showed that CNGA2 proteins were expressed on the plasma membrane of the vascular endothelial cells. (Abstract shortened by UMI.) / Cheng Kwong Tai Oscar. / "July 2005." / Adviser: Xiaoqiang Yao. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3531. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 216-243). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Yip Ham. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 132-141). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abbreviations --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Chapter Chapter 1: --- Introduction --- p.5 / Chapter 1.1 --- Calcium Signaling --- p.5 / Chapter 1.1.1 --- Importance of Calcium to Life Forms --- p.5 / Chapter 1.1.2 --- Calcium Channels in Excitable and Non-excitable Cells --- p.6 / Chapter 1.2 --- Vascular Endothelial Cells --- p.8 / Chapter 1.2.1 --- General Functions --- p.8 / Chapter 1.2.2 --- Calcium signaling in Endothelial Cells --- p.9 / Chapter 1.3 --- Capacitative Calcium Entry (CCE) or Store-operated Calcium Entry (SOC) --- p.10 / Chapter 1.3.1 --- Definition --- p.10 / Chapter 1.3.2 --- Endoplasmic Reticulum (ER) as the Main Intracellular Calcium Stores --- p.10 / Chapter 1.3.3 --- Types of Experiments leading to the Identification of SOCs --- p.11 / Chapter 1.3.4 --- Emptying the Internal Calcium Store --- p.11 / Chapter 1.3.4.1 --- Inhibition of Calcium ATPase --- p.11 / Chapter 1.3.4.2 --- IP3 Triggered Release of Calcium --- p.12 / Chapter 1.3.5 --- "Store-operated Calcium Current, Icrac" --- p.15 / Chapter 1.3.6 --- Different Types of SOCs in Animal Cells --- p.16 / Chapter 1.4 --- Transient Receptor Potential (Trp) Gene & Transient Receptor Potential Like (Trpl) Gene in Drosophila --- p.17 / Chapter 1.4.1 --- Discoverery of Trp and Trpl --- p.17 / Chapter 1.4.2 --- Expression Studies of Drosophila Trp and Trpl --- p.19 / Chapter 1.4.2.1 --- Trp and Trpl form Channels but only Trp is Store Operated --- p.19 / Chapter 1.4.2.2 --- Co-expression Studies of Trp and Trpl --- p.20 / Chapter 1.5 --- Molecular Cloning and Expression of Mammalian Trp Homologues --- p.21 / Chapter 1.5.1 --- Seven Human Homologus of Trp were found --- p.21 / Chapter 1.5.2 --- Expression Pattern of mammalian Trp Homologues in Different Tissues --- p.23 / Chapter 1.5.3 --- Expression Studies of Mammalian Trp Homologues Yields Contradictory Results --- p.27 / Chapter 1.5.3.1 --- Trpl --- p.27 / Chapter 1.5.3.2 --- Trp2 --- p.28 / Chapter 1.5.3.3 --- Trp3 --- p.29 / Chapter 1.5.3.4 --- Trp4 --- p.30 / Chapter 1.5.3.5 --- Trp5 --- p.31 / Chapter 1.5.3.6 --- Trp6 --- p.31 / Chapter 1.5.3.7 --- Trp7 --- p.31 / Chapter 1.5.3.8 --- "Activation of Trp3, Trp6 and Trp7 by Diacylglycerol (DAG)" --- p.32 / Chapter 1.5.3.9 --- Functional Consequence after Co-expression of Trp Homologues --- p.34 / Chapter 1.5.3.10 --- Antisense Strategy to Determine the Functional Subunits of Store-operated Channels --- p.35 / Chapter 1.5.3.11 --- Possible Reasons for the Contradictory Results of Trp Homologues When Expressed in a Heterologous System --- p.36 / Chapter 1.6 --- Aims Of Study --- p.37 / Chapter Chapter 2. --- Materials and Methods --- p.38 / Chapter 2.1 --- Cell Culture --- p.38 / Chapter 2.2 --- Total RNA extraction from HCAEC 5286 --- p.39 / Chapter 2.3 --- Reverse Transcription from Cultured Human Coronary Artery Endothelial Cell Line HCAEC 5286 --- p.40 / Chapter 2.4 --- Polymerase Chain Reaction (PCR) of Partial Trp Gene Fragments --- p.41 / Chapter 2.5 --- Separation and Purification of PCR Products --- p.43 / Chapter 2.5.1 --- Separation --- p.43 / Chapter 2.5.2 --- Purification --- p.43 / Chapter 2.6 --- Confirmation of PCR Products --- p.44 / Chapter 2.7 --- Molecular Cloning of Trp Gene Family --- p.45 / Chapter 2.7.1 --- "Cloning of HTrpl, HTrp3, HTrp4,HTrp5,HTrp6, HTrp7" --- p.45 / Chapter 2.7.1.1 --- Polishing the Purified PCR Products --- p.47 / Chapter 2.7.1.2 --- Determination of the Amount of Polished PCR Products --- p.47 / Chapter 2.7.1.3 --- Inserting the PCR Products into the pPCR-Script Amp SK(+)Cloning Vector (Ligation) --- p.48 / Chapter 2.7.1.4 --- Transformation --- p.48 / Chapter 2.7.1.5 --- Preparing Glycerol Stocks Containing the Bacterial Clones --- p.49 / Chapter 2.7.1.6 --- Plasmid DNA Preparation --- p.49 / Chapter 2.8.1.7 --- Clones Confirmation --- p.50 / Chapter 2.8 --- In situ Hybridization --- p.54 / Chapter 2.8.1 --- Probe Preparation --- p.54 / Chapter 2.8.1.1 --- Trp1 Probe --- p.54 / Chapter 2.8.1.2 --- Trp3 Probe --- p.58 / Chapter 2.8.1.3 --- Trp4 Probe --- p.61 / Chapter 2.8.1.4 --- Trp5 Probe --- p.62 / Chapter 2.8.1.5 --- Trp6 Probe --- p.63 / Chapter 2.8.1.6 --- Trp7 Probe --- p.65 / Chapter 2.8.1.7 --- Control Probe --- p.66 / Chapter 2.8.2 --- Testing of DIG-Labeled RNA Probes --- p.66 / Chapter 2.8.3 --- Paraffin Sections Preparation --- p.67 / Chapter 2.8.4 --- In Situ Hybridization: Pretreatment --- p.67 / Chapter 2.8.5 --- "Pre-hybridization, Hybridization and Post-hybridization" --- p.68 / Chapter 2.8.5.1 --- Pre-Hybridization --- p.68 / Chapter 2.8.5.2 --- Hybridization --- p.68 / Chapter 2.8.5.3 --- Post-Hybridization --- p.69 / Chapter 2.8.6 --- Colorimetric Detection of Human Trps mRNA --- p.69 / Chapter 2.9 --- Northern Hybridization --- p.70 / Chapter 2.9.2 --- Labelling of Riboprobe with 32P --- p.70 / Chapter 2.9.3 --- Prehybridization and Hybridization with Radiolabeled RNA Probes --- p.73 / Chapter Chapter 3. --- Results --- p.74 / Chapter 3.1 --- Polymerase Chain Reaction (PCR) of Partial Trp Gene Fragments --- p.74 / Chapter 3.2.1 --- Expression of TRPs RNA in Human Coronary Artery --- p.78 / Chapter 3.2.1.1 --- Expression of Trp Transcripts in Tunica Intima and Media --- p.79 / Chapter 3.2.1.2 --- Expression of Trp Transcripts in the Tunica Adventitia --- p.88 / Chapter 3.2.2 --- Expression of TRPs RNA in Human Cerebral Artery --- p.97 / Chapter 3.2.2.1 --- Expression of Trp Transcripts in Tunica Intima and Media --- p.97 / Chapter 3.3 --- Northern Blot Analysis of Human Trp5 RNA in Human Multiple Tissue Blot --- p.115 / Chapter Chapter 4: --- Discussion --- p.117 / Chapter 4.1 --- Co-expression of Trps in Vascular Tissues --- p.117 / Chapter 4.1.1 --- Expression of Trps in Endothelia --- p.117 / Chapter 4.1.2 --- In Smooth Muscle Cells --- p.118 / Chapter 4.2 --- Trp Channel and Store-operated Channel in Endothelial Cells --- p.119 / Chapter 4.3 --- Heteromultimerization of Trps Subtypes --- p.120 / Chapter 4.4 --- Northern Blot Analysis --- p.124 / Chapter 4.5 --- Potential Physiological Functions of Trps --- p.125 / Chapter 4.6 --- Trp Channels as a Therapeutic Target? --- p.128 / Chapter 4.7 --- Technical Aspects in the Present Studies --- p.129 / Chapter 4.8 --- Conclusion --- p.131 / Reference --- p.133

Zou Wei. / Thesis submitted in: December 2001. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 98-119). / Abstracts in English and Chinese. / Declaration --- p.i / Acknowledgements --- p.ii / Publication lists --- p.iii / Abstract --- p.ix / Abbreviations --- p.xiii / List of tables and figures --- p.xiv / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1. --- Endothelium-dependent relaxation in coronary and pulmonary circulation --- p.1 / Chapter 1.1.1. --- Endothelium-derived relaxing factors --- p.2 / Chapter 1.1.1.1. --- Nitric Oxide --- p.3 / Chapter 1.1.1.2. --- PGI2 --- p.5 / Chapter 1.1.1.3. --- EDHF --- p.6 / Chapter 1.1.2. --- EDHF in coronary and pulmonary circulation --- p.8 / Chapter 1.1.2.1. --- EDHF in coronary circulation --- p.8 / Chapter 1.1.2.2. --- EDHF in pulmonary circulation --- p.9 / Chapter 1.2. --- Effect of hyperkalemia on EDHF-mediated relaxation --- p.10 / Chapter 1.3. --- Organ Preservation Solutions --- p.13 / Chapter 1.3.1. --- Euro-Collins solution --- p.14 / Chapter 1.3.2. --- University of Wisconsin solution --- p.15 / Chapter Chapter 2: --- Objectives and research approaches --- p.16 / Chapter 2.1. --- Objectives --- p.16 / Chapter 2.1.1. --- "Endothelium-dependent relaxation resistant to INDO, L-NNA, and HbO in porcine and pulmonary coronary micro-arteries" --- p.16 / Chapter 2.1.2. --- "EET11,12 and EDHF-mediated function in porcine coronary micro-arteries" --- p.17 / Chapter 2.1.3. --- "Comparison of EC or UW solution on endothelium-dependent relaxation resistant to INDO, l-NNA, and HbO in porcine pulmonary arteries" --- p.17 / Chapter 2.2. --- Research approaches --- p.18 / Chapter 2.2.1. --- "Endothelium-dependence of the relaxation by BK or EET11,12" --- p.18 / Chapter 2.2.2. --- Effect of hypothermic storage with EC and UW solution on EDHF-related relaxation --- p.18 / Chapter 2.2.3. --- Time-dependent alteration of endothelium-dependent relaxation in pulmonary micro-arteries by EC and UW solution --- p.19 / Chapter 2.2.4. --- Effect of HbO in endothelium-dependent relaxation --- p.19 / Chapter Chapter 3: --- Material and Methods --- p.21 / Chapter 3.1. --- General Methods --- p.21 / Chapter 3.1.1. --- Porcine heart and lung collection and transportion / Chapter 3.1.2. --- Myograph --- p.21 / Chapter 3.1.3. --- Myosight --- p.24 / Chapter 3.1.4. --- Anatomizing blood vessel --- p.24 / Chapter 3.1.5. --- Mounting --- p.24 / Chapter 3.1.6 --- Normalization --- p.26 / Chapter 3.1.6.1. --- Normalization of coronary micro-artery --- p.27 / Chapter 3.1.6.2. --- Normalization of pulmonary micro-artery --- p.28 / Chapter 3.1.7. --- Precontraction --- p.30 / Chapter 3.1.8. --- Endothelium-dependent relaxation --- p.31 / Chapter 3.2. --- Coronary artery studies --- p.32 / Chapter 3.2.1. --- Porcine heart harvest and anatomy --- p.32 / Chapter 3.2.2. --- Characteristic of histology of porcine coronary micro-artery --- p.32 / Chapter 3.3. --- Pulmonary artery studies --- p.35 / Chapter 3.3.1. --- Porcine lung harvest and anatomy --- p.35 / Chapter 3.3.2. --- Characteristic of histology of porcine pulmonary micro- artery --- p.36 / Chapter 3.4. --- Drugs --- p.41 / Chapter 3.4.1. --- Drugs --- p.41 / Chapter 3.4.2. --- Preparation of oxyhemoglobin solution --- p.41 / Chapter 3.5. --- Statistical Analysis --- p.42 / Chapter 3.5.1. --- Calculation of EC50 --- p.42 / Chapter 3.5.2. --- Statistical analysis --- p.42 / Chapter Chapter 4: --- "Epoxyeicosatrienoic Acids (EET11,12) May Partially Restore EDHF-Mediated Function in Coronary Micro-Arteries" --- p.43 / Chapter 4.1. --- Abstract --- p.43 / Chapter 4.2. --- Introduction --- p.44 / Chapter 4.3. --- Experimental Protocol --- p.45 / Chapter 4.3.1. --- Precontraction --- p.45 / Chapter 4.3.2. --- "EDHF-mediated (INDO, L-NNA, and HbO-resistant) relaxation" --- p.45 / Chapter 4.3.3. --- "EET11,12-mediated relaxation after exposure to hyperkalemia" --- p.46 / Chapter 4.3.4. --- "The effect of incubation with EET11,12 on the BK-induced, EDHF-mediated relaxation" --- p.46 / Chapter 4.4. --- Results --- p.47 / Chapter 4.4.1. --- Resting force --- p.47 / Chapter 4.4.2. --- HbO and U46619-induced contraction force --- p.48 / Chapter 4.4.3. --- "EET11,12-induced relaxation in coronary micro-arteries after exposure to hyperkalemia" --- p.49 / Chapter 4.4.4. --- "The EDHF-mediated relaxation to BK resistant to INDO, l- NNA,and HbO" --- p.51 / Chapter 4.4.4.1. --- Incubated in either hyperkalemic solution (K+ 20mmol/L) or Krebs' solution (control) --- p.51 / Chapter 4.4.4.2. --- "Incubated in either hyperkalemic solution (K+ 20mmol/L) plus EET11,12 or Krebs' solution (control)" --- p.53 / Chapter 4.5. --- Discussion --- p.57 / Chapter 4.5.1. --- EDHF plays an important role in the coronary micro-arteries --- p.57 / Chapter 4.5.2. --- "EDHF-mediated (INDO, l-NNA, and HbO-resistant) relaxation in the coronary micro-arteries" --- p.58 / Chapter 4.5.3. --- "EET11,12 may partially mimic the EDHF-mediated relaxation in the porcine coronary micro-artery" --- p.59 / Chapter 4.5.4. --- "Effect of EET11,12 added in hyperkalemia may partially restore the EDHF-mediated relaxation in the porcine coronary micro-arteries" --- p.59 / Chapter Chapter 5: --- Impaired EDHF-Mediated Relaxationin Porcine Pulmonary Micro-arteries by Cold Store with Euro-Collin's and University of Wisconsin Solution --- p.61 / Chapter 5.1. --- Abstract --- p.61 / Chapter 5.2. --- Introduction --- p.62 / Chapter 5.3. --- Experimental Protocol --- p.64 / Chapter 5.3.1. --- Precontraction --- p.64 / Chapter 5.3.2. --- "Role of EDHF-mediated (INDO, L-NNA and HbO-resistant) relaxation in porcine pulmonary micro-arteries by BK orA23187" --- p.64 / Chapter 5.3.3. --- Effect of hyperkalemia or preservation solutions (EC or UW) on the EDHF-mediated relaxation by BK or A23187 --- p.65 / Chapter 5.3.3.1. --- The effect of hyperkalemia --- p.65 / Chapter 5.3.3.2. --- Effect of EC solution on the EDHF-mediated relaxation --- p.65 / Chapter 5.3.3.3. --- Effect of UW solution on the EDHF-mediated relaxation --- p.66 / Chapter 5.3.3.4. --- The effect of UW and EC solutions on the contractility of the pulmonary micro-artery --- p.66 / Chapter 5.4. --- Results --- p.66 / Chapter 5.4.1. --- Resting force --- p.66 / Chapter 5.4.2. --- U46619-induced contraction force --- p.67 / Chapter 5.4.3. --- Role of EDHF-mediated relaxation induced by BK or A23187 --- p.67 / Chapter 5.4.4. --- The effect of hyperkalemia --- p.71 / Chapter 5.4.5. --- Effect of EC solution on the EDHF-mediated relaxation --- p.72 / Chapter 5.4.6. --- Effect of UW solution on the EDHF-mediated relaxation --- p.73 / Chapter 5.4.7. --- The effect of UW and EC solution on the contractility of the pulmonary micro-artery --- p.73 / Chapter 5.5. --- Discussion --- p.77 / Chapter 5.5.1. --- EDHF-mediated endothelial function exists in the pulmonary micro-circulation --- p.77 / Chapter 5.5.2. --- Hyperkalemia exposure reduces EDHF-related relaxation and possible mechanism --- p.78 / Chapter 5.5.3. --- The effect of EC and UW solutions on the EDHF-media relaxation in the pulmonary micro-arteries --- p.79 / Chapter Chapter 6: --- General Discussion --- p.82 / Chapter 6.1. --- Endothelium-dependent vasodilators: BK and A23187 --- p.82 / Chapter 6.2. --- EDHF in porcine coronary and pulmonary micro-arteries --- p.84 / Chapter 6.2.1. --- EDHF in porcine coronary micro-arteries --- p.84 / Chapter 6.2.2. --- EDHF in porcine pulmonary micro-arteries --- p.87 / Chapter 6.2.3. --- Vascular stretch and release of endothelium-derived vasodilators --- p.87 / Chapter 6.2.4. --- "EET11,12" --- p.88 / Chapter 6.3. --- "Endothelium-dependent relaxation resistant to INDO, L- NNA, and HbO in porcine coronary and pulmonary microcirculation" --- p.89 / Chapter 6.4. --- "Alteration of endothelium-dependent relaxation resistant to INDO, l-NNA, and HbO after exposure to hyperkalemia" --- p.90 / Chapter 6.5. --- "Alteration of endothelium-dependent contraction resistant to INDO, L-NNA, and HbO after exposure to EC or UW solutions" --- p.91 / Chapter 6.6. --- Clinical implications --- p.92 / Chapter 6.7. --- Limitations --- p.93 / Chapter 6.7.1. --- Common limitations --- p.93 / Chapter 6.7.2. --- Limitation of in vitro study --- p.93 / Chapter 6.8. --- Future work --- p.94 / Chapter Chapter 7: --- Conclusion --- p.96 / References --- p.98 / Appendies / "Wei Zou, Qin Yang, Anthony PC Yim, & Guo-Wei He Epoxyeicosatrienoic acids (EET11,12) may partially restore endothelium- derived hyperpolarizing factor-mediated function in coronary micro- arteries. Annals of Thoracic Surgery. 2001; 72(12): 1970~1976."

Yau Ho Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 131-144). / Abstracts in English and Chinese. / DECLARATION --- p.I / ACKNOWLEDGEMENTS --- p.II / ENGLISH ABSTRACT --- p.III / CHINESE ABSTRACT --- p.VI / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Oxidative Stress --- p.1 / Chapter 1.1.1 --- Historical Background of reactive oxygen/nitrogen species --- p.1 / Chapter 1.1.2 --- What is Oxidative Stress? --- p.3 / Chapter 1.1.3 --- Reactive Oxygen Species (ROS) --- p.4 / Chapter 1.1.3.1 --- Superoxide anion (02-) --- p.4 / Chapter 1.1.3.2 --- Hydrogen peroxide (H202) --- p.5 / Chapter 1.1.3.3 --- Hydroxyl radical --- p.6 / Chapter 1.1.3.4 --- Nitric oxide (NO) --- p.7 / Chapter 1.2 --- Cardiovascular System --- p.8 / Chapter 1.2.1 --- Enzymatic and Non-enzymatic Sources of ROS in Cardiovascular System --- p.8 / Chapter 1.2.1.1 --- NADPH oxidase --- p.8 / Chapter 1.2.1.2 --- Hypoxanthine-Xanthine oxidase (HX-XO) --- p.9 / Chapter 1.2.1.3 --- Nitric oxide synthase (NOS) --- p.10 / Chapter 1.2.1.4 --- Mitochondrial electron transport chain (ETC) --- p.11 / Chapter 1.2.1.5 --- Cyclooxygenase --- p.11 / Chapter 1.2.1.6 --- Lipoxygenae --- p.12 / Chapter 1.2.1.7 --- Endoplasmic reticulum --- p.12 / Chapter 1.2.2 --- ROS/RNS Scavenging Systems --- p.13 / Chapter 1.2.2.1 --- Superoxide dismutase (SOD) --- p.13 / Chapter 1.2.2.2 --- Catalase --- p.14 / Chapter 1.2.2.3 --- Glutathione peroxidase --- p.15 / Chapter 1.2.2.4 --- Non-enzymatic antioxidants --- p.15 / Chapter 1.2.3 --- Factors that stimulate ROS production in cardiovascular system --- p.18 / Chapter 1.2.3.1 --- Oxygen tension --- p.18 / Chapter 1.2.3.2 --- "Flow, Shear, and Stretch as an initial stimulus for endothelial oxidant signalling" --- p.18 / Chapter 1.2.3.3 --- Activation of rennin-angiotensin system promote oxidative stress in cardiovascular system --- p.19 / Chapter 1.2.3.4 --- Regulation of vascular ROS production by vasoactive substances --- p.19 / Chapter 1.2.4 --- Regulation of vascular tone in Cardiovascular System by ROS/RNS --- p.20 / Chapter 1.2.4.1 --- Regulation of vascular tone --- p.20 / Chapter 1.2.5 --- Pathophysiological Effects of ROS --- p.23 / Chapter 1.2.5.1 --- Cellular injury by lipid peroxidation --- p.23 / Chapter 1.2.5.2 --- Role of ROS in immune defence --- p.23 / Chapter 1.2.5.3 --- Redox regulation of cell adhesion --- p.24 / Chapter 1.2.6 --- Evidences from Clinical Studies of Oxidative Stress-Related Vascular Diseases --- p.25 / Chapter 1.2.6.1 --- Hyperlipidaemia --- p.25 / Chapter 1.2.6.2 --- Hypertension --- p.25 / Chapter 1.2.6.3 --- Chronic heart failure (CHF) --- p.26 / Chapter 1.2.6.4 --- Chronic renal failure (CRF) --- p.26 / Chapter 1.2.6.5 --- Atherosclerosis --- p.27 / Chapter 1.2.6.6 --- Ischemia/reperfusion (I/R) injury --- p.27 / Chapter 1.2.7 --- Role of Vascular Endothelium in Oxidative Stress --- p.29 / Chapter 1.2.8 --- Role of Ca in oxidative stress in cardiovascular system --- p.29 / Chapter 1.2.8.1 --- Calcium Signaling in Vascular Endothelial Cells --- p.30 / Chapter 1.2.9 --- ROS effect on endothelial Ca2+ --- p.31 / Chapter 1.2.9.1 --- Multiple targets of ROS on intracellular Ca2+ mobilization --- p.32 / Chapter 1.2.9.2 --- Reports of H202-induced Ca2+ release in various cell types --- p.33 / Chapter 1.2.9.3 --- Reported effects of H202 on agonist-induced Ca2+ signal --- p.34 / Chapter 1.2.9.4 --- Differences between macrovessels and microvessels --- p.34 / Chapter 1.3 --- TRP Channel --- p.41 / Chapter 1.3.1 --- Discovery of Drosophila TRP --- p.41 / Chapter 1.3.2 --- Mammalian TRP subfamily --- p.41 / Chapter 1.3.3 --- General topology of TRP channel --- p.42 / Chapter 1.3.4 --- Interactions of oxidative stress with TRP channels --- p.44 / Chapter 1.3.5 --- The role of TRPC3 and TRPC4 in oxidative stress --- p.44 / Chapter 1.3.6 --- TRPM subfamily --- p.44 / Chapter 1.3.6.1 --- Expression of TRPM2 --- p.45 / Chapter 1.3.6.2 --- Dual Role of TRPM´2ؤChannel and Enzyme --- p.45 / Chapter 1.3.6.3 --- Regulatory mechanisms of TRPM2 --- p.46 / Chapter 1.3.6.3.1 --- ADP-ribose (ADPR) directly regulating --- p.46 / Chapter 1.3.6.3.2 --- NAD regulating --- p.46 / Chapter 1.3.6.3.3 --- Oxidative stress regulating independent of ADPR or NAD --- p.47 / Chapter 1.4 --- Cell Death Induced by Oxidative Stress --- p.48 / Chapter 1.4.1 --- Redox status as a factor to determine cell death --- p.48 / Chapter 1.4.2 --- Role of TRPM2 in oxidative stress-induced cell death --- p.48 / Chapter 1.5 --- Aims of the Study --- p.49 / Chapter Chapter 2: --- Materials and Methods --- p.50 / Chapter 2.1 --- Functional Characterization of TRPM2 by Antisense Technique --- p.50 / Chapter 2.1.1 --- Restriction Enzyme Digestion --- p.50 / Chapter 2.1.2 --- Purification of Released Inserts and Cut pcDNA3 Vectors --- p.51 / Chapter 2.1.3 --- "Ligation of TRPM2 Genes into Mammalian Vector, pcDNA3" --- p.52 / Chapter 2.1.4 --- Transformation for the Desired Clones --- p.52 / Chapter 2.1.5 --- Plasmid DNA Preparation for Transfection --- p.53 / Chapter 2.1.6 --- Confirmation of the Clones --- p.53 / Chapter 2.1.6.1 --- Restriction Enzymes Strategy --- p.53 / Chapter 2.1.6.2 --- Polymerase Chain Reaction (PCR) Check --- p.54 / Chapter 2.1.6.3 --- Automated Sequencing --- p.55 / Chapter 2.2 --- Establishing Stable Cell Lines --- p.56 / Chapter 2.2.1 --- Cell Culture --- p.56 / Chapter 2.2.2 --- Geneticin Selection --- p.57 / Chapter 2.3 --- Expression of TRPM2 in Transfected and non-Transfected H5V Cells --- p.57 / Chapter 2.3.1 --- Protein Sample Preparation --- p.57 / Chapter 2.3.2 --- Western Blot Analysis --- p.58 / Chapter 2.3.3 --- Protein Expression Analysis --- p.59 / Chapter 2.4 --- "Immunolocalization of TRPM2 in Human Heart, Cerebral Artery, Renal, Hippocampus and Liver" --- p.59 / Chapter 2.4.1 --- Paraffin Section Preparation --- p.59 / Chapter 2.4.2 --- Immunohistochemistry --- p.60 / Chapter 2.5 --- [Ca2+ ］i Measurement in Confocal Microscopy --- p.62 / Chapter 2.5.1 --- Cytosolic Ca2+ measurement --- p.62 / Chapter 2.5.2 --- Measuring the Ca2+ in the Internal Calcium Stores --- p.63 / Chapter 2.5.3 --- Data Analysis --- p.64 / Chapter 2.6 --- Examining Cell Death Induced by H2O2 by DAPI Staining --- p.65 / Chapter 2.6.1 --- DAPI Staining --- p.65 / Chapter Chapter 3: --- Results --- p.66 / Chapter 3.1 --- Superoxide Anion-Induced [Ca 2+]i rise in H5V Mouse Heart Microvessel Endothelial Cells --- p.66 / Chapter 3.1.1 --- Superoxide Anion-induced [Ca2+ ]i Rise --- p.66 / Chapter 3.1.2 --- Effect of Catalase on the Superoxide Anion-induced [Ca2+]i］］ Rise --- p.66 / Chapter 3.1.3 --- IP3R inhibitor Inhibits Superoxide anion-induced [Ca 2+]i Rise --- p.67 / Chapter 3.1.4 --- Effect of Phospholipase A2 Inhibitor on Superoxide anion- induced [Ca2+]i Rise --- p.67 / Chapter 3.1.5 --- Effect of Hydroxyl Radical Scavenger on Superoxide Anion- induced [Ca2+]i Rise --- p.68 / Chapter 3.2 --- Hydrogen Peroxide-induced Ca2+ Entry in Mouse Heart Microvessel Endothelial Cells --- p.74 / Chapter 3.2.1 --- Hydrogen Peroxide Induces [Ca2 +]i rise in H5V Mouse Heart Microvessel Endothelial Cells --- p.74 / Chapter 3.2.2 --- Hydrogen Peroxide Induces [Ca 2+]i rise in two phases (Rapid and Slow response) --- p.74 / Chapter 3.2.3 --- Hydrogen Peroxide Induces [Ca 2+]i rise in a Extracellular Ca + Concentration Dependent Manner --- p.77 / Chapter 3.3 --- Hydrogen Peroxide Reduces Agonist-induced [Ca2+]i rise --- p.79 / Chapter 3.3.1 --- Hydrogen Peroxide Reduces ATP-induced [Ca2+ ]i rise in a H2O2 Concentration Dependent Manner --- p.79 / Chapter 3.3.2 --- Hydrogen Peroxide Reduces ATP-induced [Ca 2+]i rise in a H2O2 Incubation Time Dependent Manner --- p.79 / Chapter 3.3.3 --- Hydrogen Peroxide Reduces the ATP-induced Intracellular Ca2+ Release --- p.80 / Chapter 3.3.4 --- XeC Inhibited H202-induced [Ca2+]i rise --- p.80 / Chapter 3.3.5 --- Hydrogen Peroxide Partially Depletes Internal Ca2+ Stores --- p.81 / Chapter 3.4 --- Dissecting Signal Transduction Pathways in H202-induced [Ca2+]i rise --- p.82 / Chapter 3.4.1 --- Effect of Phospholipase C Inhibitor on H202-induced [Ca2 +]i rise --- p.82 / Chapter 3.4.2 --- Effect of Phospholipase A2 Inhibitor on H202-induced [Ca 2+]i rise --- p.83 / Chapter 3.4.3 --- Effect of hydroxyl radical scavenger on H2O2-induced [Ca 2+]i rise --- p.83 / Chapter 3.5 --- Functional Role of TRPM2 Channel in H202-induced [Ca2+]i Rise in H5V Cells --- p.92 / Chapter 3.5.1 --- Expression of TRPM2 and the Effect of TRPM2 Antisense Construct on TRPM2 Protein Expression --- p.92 / Chapter 3.5.2 --- Effect of Antisense TRPM2 on H202-induced Ca2+ Entry --- p.94 / Chapter 3.6 --- H202-induced Cell Death --- p.101 / Chapter 3.7 --- Expression Pattern of TRPM2 Channel in Vascular System --- p.104 / Chapter 3.7.1 --- Immunolocalization of TRPM2 in Human Cerebral Arteries --- p.104 / Chapter 3.7.2 --- Immunolocalization of TRPM2 in Human Cardiac Muscles --- p.105 / Chapter 3.7.3 --- Immunolocalization of TRPM2 in Human Kidney --- p.105 / Chapter Chapter 4: --- Discussion --- p.113 / Chapter 4.1 --- Oxidative modification of Ca2+ homeostasis --- p.113 / Chapter 4.2 --- Pathophysiological effects of ROS on endothelium --- p.113 / Chapter 4.3 --- Effects of ROS on microvascular endothelial Ca2+ reported by other investigators --- p.115 / Chapter 4.4 --- Studies of the effect of HX-XO on cytosolic [Ca2+]i --- p.116 / Chapter 4.4.1 --- Role of 0´2Ø- and H202 in HX-XO-induced [Ca2+]i elevation --- p.116 / Chapter 4.4.2 --- IP3R involvement in HX-XO-evoked Ca + movements in H5V cells --- p.118 / Chapter 4.4.3 --- PLA2 involvement in HX-XO experiment --- p.119 / Chapter 4.5 --- Studies of the effect of direct H202 application on cytosolic [Ca2+]i --- p.120 / Chapter 4.5.1 --- Hydrogen Peroxide Induced [Ca2 +]i rise in a Extracellular Ca2 + Concentration Dependent Manner --- p.120 / Chapter 4.5.2 --- Hydrogen Peroxide Induced [Ca 2+]i rise in two phases (Rapid and Slow response) --- p.121 / Chapter 4.6 --- Effect of H202 on ATP-induced Ca2+ response --- p.121 / Chapter 4.6.1 --- H202 inhibited ATP-induced Ca2+ release in a concentration and time dependent manner --- p.121 / Chapter 4.6.2 --- IP3R involvement and store depletion in H202 experiment --- p.123 / Chapter 4.7 --- Dissecting Signal Transduction Pathways in H202-induced [Ca2+]i rise --- p.124 / Chapter 4.7.1 --- PLC involvement in H2O2 experiment --- p.124 / Chapter 4.7.2 --- PLA2 involvement in H2O2 experiment --- p.125 / Chapter 4.7.3 --- Hydroxyl radical did not involve in H2O2 experiment --- p.125 / Chapter 4.8 --- Functional Studies of TRPM2 --- p.127 / Chapter 4.8.1 --- Expression of TRPM2 in H5V on protein level --- p.127 / Chapter 4.8.2 --- TRPM2 involvement in the Ca2+ signalling in response to H2O2 in H5V cells --- p.127 / Chapter 4.9 --- H202 concentration in my projec´tؤphysiological or pathological? --- p.128 / Chapter 4.10. --- H20´2ؤTRPM´2ؤCell death --- p.129 / Chapter 4.11 --- Expression of TRPM2 in human blood vessels and other tissues --- p.130 / References --- p.131

Based on the results aforementioned, I went on in the second part of the study to examine the impact of aging on EDCF-mediated contractions - the alterations of COX-2-mediated endothelium-dependent contractions and the associated release of prostaglandin(s) in the aortae from aged (>18 month-old) hamsters. Endothelium-dependent contractions in the presence of NG-nitro-L-arginine methyl ester (L-NAME) were significantly greater in the aortae from aged hamsters and contractions could also be observed without L-NAME, which were sensitive to COX-2 inhibitors and TP receptor antagonists. The levels of COX-2 expression, the release of PGF2alpha and vascular sensitivity to PGF 2alpha were augmented in aortae of aged hamsters. The present results indicate a positive impact of aging on COX-2-derived PGF2alpha-mediated endothelium-dependent contractions. / In the first part of the study, I investigated whether COX-2 participated in the occurrence of endothelium-dependent contractions in the aortae from young (-3 month-old) hamsters and identified the most possible EDCF. Endothelium-dependent contractions were elicited by acetylcholine and abolished by COX-2 inhibitors (NS-398, DuP-697 and celecoxib) and thromboxane-prostanoid (TP) receptor antagonists (S 18886, L-655,240 and GR 32191), but not by COX-1 inhibitors (valeryl salicylate and sc 560). RT-PCR and Western blot analysis using aortae with and without endothelium revealed that the COX-2 expression was localized mainly in the endothelium. Levels of prostangladin F2alpha (PGF2alpha ) and prostacyclin (PGI2) increased in response to acetylcholine and the release of both prostaglandins was inhibited by COX-2 but not COX-1 inhibitors. Exogenous PGF2alpha but not PGI2 caused contractions at a concentration that corresponded to the amount released endogenously. The release of PGF2alpha was not affected by the presence of nitric oxide (NO). The results of the present study suggest that a novel constitutive role of COX-2 in endothelium-dependent contractions, with its metabolites PGF2alpha acting as a physiological EDCF in the young hamster aortae. / In the third part of the study, I investigated the relationship and the intracellular signaling cascades linking two pro-inflammatory factors Ang II and COX-2, and tested whether COX-2 mediated the Ang II-induced vascular pathogenesis. Eight hour-incubation with 100 nmol/L Ang II resulted in maximal COX-2 expression in primary rat endothelial cells and it was inhibited by losartan and RNA synthesis inhibitor (actinomycin-D). Inhibitors of either p38 MAPK or ERK1/2 (respectively SB 202190 and PD 98059) decreased the COX-2 expression, and co-treatment with both inhibitors caused an additive effect, suggesting a joint mediation through both kinases. Protein kinase C (PKC) inhibitor (GF109203X), and particularly, the specific PKCdelta inhibitor (rottlerin), prevented Ang II-induced phosphorylation of ERK1/2 and COX-2 expression, indicating an upstream regulation of ERK1/2 by PKC delta. A pivotal role of PKCdelta in Ang II-induced COX-2 expression was further supported by a similar stimulatory effect of PKC activator, signified by the Ang II-stimulated translocation of PKCdelta to the membrane and confirmed by its phosphorylation (Tyr311). Small interfering RNA targeting PKCdelta (siPKCdelta) diminished COX-2 expression, which was abrogated in siPKCdelta-treated cells treated with SB 202190, confirming the parallel pathways of PKC delta-ERK1/2 and p38 MAPK. Aortae and renal arteries from Ang II-infused rats exhibited an increased endothelial COX-2 expression and impaired acetylcholine-induced relaxation that was normalized by celecoxib. Human mesenteric arteries incubated with Ang II demonstrated elevated endothelial COX-2 and MCP-1 expressions, of which the former was inhibited by SB 202190 plus rottlerin and the latter prevented by COX-2 inhibitor celecoxib. Renal arteries from hypertensive or diabetic patients revealed an exaggerated expression of COX-2 and MCP-1 in the endothelium. The present novel findings indicate that the activation of PKCdelta-ERK1/2 and p38 MAPK is critical in Ang II-induced COX-2 up-regulation in endothelial cells, and identify a COX-2-dependent pro-atherosclerotic cytokine MCP-1. (Abstract shortened by UMI.) / Wong, Siu Ling. / Adviser: Huang Yu. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 192-228). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Wu Min. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 87-114). / Abstracts in English and Chinese. / Declaration --- p.i / Acknowledgement --- p.ii / Publication list --- p.iii / Abstract (English) --- p.xi / Abstract (Chinese) --- p.xiv / Abbreviations --- p.xvi / List of figures / tables --- p.xviii / Chapter Chapter 1. --- General Introduction --- p.1 / Chapter 1.1 --- Endothelial function in the regulation of vascular tone --- p.1 / Chapter 1.1.1 --- NO --- p.2 / Chapter 1.1.2 --- PGI2 --- p.5 / Chapter 1.1.3 --- EDHF --- p.6 / Chapter 1.2 --- Alteration of endothelial functions after preservation with cardioplegia /organ preservation solutions in the coronary and pulmonary microcirculations --- p.18 / Chapter 1.2.1 --- Cardioplegia/organ preservation solutions --- p.21 / Chapter 1.2.2 --- Effect of Cardioplegia/organ preservation solutions on endothelial function --- p.22 / Chapter 1.2.2.1 --- Effect of K+ on endothelial function --- p.23 / Chapter 1.2.2.2 --- Effect of other components on endothelial function --- p.24 / Chapter Chapter 2. --- Materials and Methods --- p.26 / Chapter 2.1 --- Isometric force study in coronary/pulmonary resistance arteries --- p.26 / Chapter 2.1.1 --- Preparation of vessels --- p.26 / Chapter 2.1.1.1 --- Preparation of porcine coronary small arteries --- p.26 / Chapter 2.1.1.2 --- Preparation of porcine pulmonary small arteries --- p.26 / Chapter 2.1.2 --- Technique of setting up --- p.29 / Chapter 2.1.2.1 --- Mounting of small vessels --- p.29 / Chapter 2.1.2.2 --- Normalization procedure for small vessels --- p.29 / Chapter 2.1.3 --- EDHF-mediated vasorelaxation --- p.30 / Chapter 2.1.3.1 --- Precontraction and stimuli of EDHF --- p.30 / Chapter 2.1.3.2 --- """True"" response of EDHF" --- p.31 / Chapter 2.1.4 --- Data acquisition and analysis --- p.32 / Chapter 2.2 --- Electrophysiological study --- p.32 / Chapter 2.2.1 --- Preparation of small porcine coronary/pulmonary arteries --- p.32 / Chapter 2.2.2 --- Preparation of microelectrode --- p.32 / Chapter 2.2.3 --- Impaling of microelectrode --- p.33 / Chapter 2.2.4 --- Recording of membrane potential --- p.33 / Chapter 2.3 --- Statistical analysis --- p.34 / Chapter 2.4 --- Chemicals --- p.34 / Chapter Chapter 3. --- Effects of Celsior Solution on Endothelial Function in Resistance Coronary Arteries Compared to St. Thomas' Hospital Solution --- p.37 / Chapter 3.1 --- Abstract --- p.37 / Chapter 3.2 --- Introduction --- p.38 / Chapter 3.3 --- Experimental design and analysis --- p.40 / Chapter 3.3.1 --- Vessel preparation --- p.40 / Chapter 3.3.2 --- Normalization --- p.40 / Chapter 3:3.3 --- "Relaxation study: BK-induced, EDHF-mediated relaxation" --- p.41 / Chapter 3.3.4 --- Cellular electrophysiological study: EDHF-mediated cellular hyperpolarization and associated relaxation --- p.41 / Chapter 3.3.5 --- Data analysis --- p.42 / Chapter 3.4 --- Results --- p.43 / Chapter 3.4.1 --- Relaxation study --- p.43 / Chapter 3.4.1.1 --- Resting force --- p.43 / Chapter 3.4.1.2 --- U46619-induced precontraction --- p.43 / Chapter 3.4.1.3 --- EDHF-mediated relaxation --- p.43 / Chapter 3.4.2 --- Electrophysiological studies --- p.44 / Chapter 3.4.2.1 --- Resting membrane potential --- p.44 / Chapter 3.4.2.2 --- EDHF-mediated cellular hyperpolarization --- p.45 / Chapter 3.4.2.3 --- Cellular hyperpolarization-associated relaxation --- p.45 / Chapter 3.5 --- Discussion --- p.46 / Chapter 3.5.1 --- Effects of Celsior solution on endothelial function --- p.47 / Chapter 3.5.2 --- Effects of ST solution on EDHF-mediated function --- p.48 / Chapter 3.5.3 --- Comparison between Celsior and ST solutions on EDHF-mediated function --- p.48 / Chapter 3.5.4 --- Clinical implications --- p.49 / Chapter Chapter 4. --- Effects of Perfadex and Celsior Solution on Endothelial Function in Resistance Pulmonary Arteries --- p.57 / Chapter 4.1 --- Abstract --- p.57 / Chapter 4.2 --- Introduction --- p.58 / Chapter 4.3 --- Experimental design and analysis --- p.59 / Chapter 4.3.1 --- Vessel Preparation --- p.59 / Chapter 4.3.2 --- Normalization --- p.60 / Chapter 4.3.3 --- Isometric force study --- p.60 / Chapter 4.3.4 --- Electrophysiological studies --- p.61 / Chapter 4.3.5 --- Data analysis --- p.61 / Chapter 4.4 --- Results --- p.62 / Chapter 4.4.1 --- Relaxation study: EDHF-mediated relaxation --- p.62 / Chapter 4.4.1.1 --- Resting force --- p.62 / Chapter 4.4.1.2 --- U46619-induced precontraction --- p.62 / Chapter 4.4.1.3 --- EDHF-mediated relaxation --- p.62 / Chapter 4.4.2 --- Electrophysiological studies --- p.63 / Chapter 4.4.2.1 --- Resting membrane potential --- p.63 / Chapter 4.4.2.2 --- EDHF-mediated cellular hyperpolarization --- p.64 / Chapter 4.4.2.3 --- Cellular hyperpolarization-associated relaxation --- p.64 / Chapter 4.5 --- Discussion --- p.65 / Chapter 4.5.1 --- Effects of Celsior solution on endothelial function during cardiopulmonary surgery --- p.65 / Chapter 4.5.2 --- Effects of Perfadex solution on EDHF-mediated endothelial function --- p.66 / Chapter 4.5.3 --- Comparison between Celsior and Perfadex solutions on EDHF-mediated function --- p.66 / Chapter 4.5.4 --- Clinical implications --- p.67 / Chapter Chapter 5. --- Exploration of the Nature of EDHF - the Effect of H2O2 on the Membrane Potential in the Rat Small Mesenteric Arteries --- p.73 / Chapter Chapter 6. --- General Discussion --- p.75 / Chapter 6.1 --- EDHF-mediated endothelial function in porcine coronary and pulmonary circulation --- p.75 / Chapter 6.1.1 --- Role of EDHF in the regulation of porcine coronary arterial tone --- p.75 / Chapter 6.1.2 --- Role of EDHF in the regulation of porcine pulmonary arterial tone --- p.76 / Chapter 6.2 --- Alteration of EDHF-mediated endothelial functions after exposure to organ preservation solutions --- p.77 / Chapter 6.2.1 --- Effects of hyperkalemic solution on EDHF-mediated endothelial function in coronary and pulmonary circulation --- p.78 / Chapter 6.2.2 --- Effects of low-potassium-based preservation solution on EDHF-mediated endothelial function in pulmonary circulation --- p.79 / Chapter 6.2.3 --- Comparison between hyperkalemic solution and low-potassium-based preservation solution on EDHF-mediated endothelial function --- p.80 / Chapter 6.2.4 --- Effects of other component of organ preservation solutions on EDHF-mediated endothelial function --- p.81 / Chapter 6.3 --- Clinical implications --- p.82 / Chapter 6.4 --- The effect of H202 on the membrane potential in rat small mesenteric arteries --- p.83 / Chapter 6.5 --- Limitation of the study --- p.84 / Chapter 6.6 --- Future investigations --- p.85 / Chapter 6.7 --- Conclusions --- p.85 / References --- p.87

Dong Yingying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 99-125). / Abstracts in English and Chinese. / Declaration --- p.i / Acknowledgement --- p.ii / Publication list --- p.iii / Abstract (English) --- p.ix / Abstract (Chinese) --- p.xii / Abbreviations --- p.xiv / List of figures / tables --- p.xvi / Chapter Chapter 1. --- General Introduction / Chapter 1.1 --- The role of endothelium in regulating vascular tone --- p.1 / Chapter 1.1.1 --- Nitric oxide (NO) --- p.2 / Chapter 1.1.2 --- Endothelium-derived hyperpolarizing factor (EDHF) --- p.7 / Chapter 1.1.3 --- Prostacyclin (PGI2) --- p.20 / Chapter 1.2 --- EDHF-mediated endothelial function in coronary circulation --- p.22 / Chapter 1.2.1 --- Role of EDHF in coronary microarteries --- p.23 / Chapter 1.2.2 --- Role of EDHF in cardiac veins --- p.24 / Chapter 1.3 --- Effect of ischemia-reperfusion on endothelial function in coronary circulation --- p.25 / Chapter 1.3.1 --- Ischemia-reperfusion injury --- p.26 / Chapter 1.3.2 --- Effect of ischemia-reperfusion on endothelial function in coronary microarteries --- p.28 / Chapter 1.3.3 --- Effect of ischemia-reperfusion on endothelial function in cardiac veins --- p.29 / Chapter 1.4 --- Alteration of endothelial function during cardiac surgery / Chapter 1.4.1 --- Cardioplegia and organ preservation solutions --- p.31 / Chapter 1.4.2 --- Combined effects of hypoxia-reoxygenation and ST solution on endothelial function in coronary microarteries/cardiac veins --- p.34 / Chapter 1.4.3 --- Effect of nicorandil on endothelial function --- p.34 / Chapter Chapter 2. --- Materials and Methods --- p.37 / Chapter 2.1 --- Isometric force study in micro arteries/veins --- p.37 / Chapter 2.1.1 --- Preparation of vessels --- p.37 / Chapter 2.1.1.1 --- Preparation of porcine coronary microarteries --- p.37 / Chapter 2.1.1.2 --- Preparation of porcine cardiac veins --- p.37 / Chapter 2.1.2 --- Technique of setting up --- p.39 / Chapter 2.1.2.1 --- Mounting of microvessels --- p.39 / Chapter 2.1.2.2 --- Normalization procedure for microvessels --- p.39 / Chapter 2.1.3 --- EDHF-mediated vasorelaxation --- p.40 / Chapter 2.1.3.1 --- Precontraction and stimuli of EDHF --- p.40 / Chapter 2.1.3.2. --- “Truéحresponse of EDHF --- p.40 / Chapter 2.1.4 --- Data acquisition and analysis --- p.41 / Chapter 2.2 --- Hypoxia and reoxygenation --- p.41 / Chapter 2.2.1 --- Calibration of 02-special electrode --- p.41 / Chapter 2.2.2 --- Measurement of --- p.02 / Chapter 2.3 --- Statistical analysis --- p.42 / Chapter 2.4 --- Chemicals --- p.43 / Chapter Chapter 3. --- Hypoxia-Reoxygenation in Coronary Microarteries: Combined Effect with St Thomas Cardioplegia and Temperature on the Endothelium- derived Hyperpolarizing Factor and Protective Effect of Nicorandil --- p.44 / Chapter 3.1 --- Abstract --- p.44 / Chapter 3.2 --- Introduction --- p.45 / Chapter 3.3 --- Experimental design and analysis --- p.47 / Chapter 3.3.1 --- Vessel Preparation --- p.47 / Chapter 3.3.2 --- Normalization --- p.48 / Chapter 3.3.3 --- Hypoxia --- p.48 / Chapter 3.3.4 --- Effect of H-R on EDHF-mediated relaxation in coronary microarteries --- p.49 / Chapter 3.3.5 --- Combined effects ofH-R and ST solution on EDHF-mediated relaxation in coronary microarteries --- p.49 / Chapter 3.3.6 --- Effect of addition of nicorandil Krebs or ST solution under H-R on EDHF-mediated relaxation in coronary microarteries --- p.49 / Chapter 3.3.7 --- Data analysis --- p.50 / Chapter 3.4 --- Results --- p.51 / Chapter 3.4.1 --- Resting force --- p.51 / Chapter 3.4.2 --- U46619-induced contraction force --- p.51 / Chapter 3.4.3 --- Partial pressure of oxygen in hypoxia --- p.51 / Chapter 3.4.4 --- EDHF-mediated relaxation in coronary microarteries --- p.51 / Chapter 3.4.4.1 --- Effect of H-R --- p.51 / Chapter 3.4.4.2 --- Combined effects ofH-R and ST solution on EDHF-mediated relaxation --- p.52 / Chapter 3.4.4.3 --- Effects of addition of nicorandil to Krebs or ST solution under H-R on EDHF-mediated relaxation --- p.52 / Chapter 3.5 --- Discussion --- p.53 / Chapter 3.5.1 --- EDHF-mediated relaxation after exposure to H-R --- p.53 / Chapter 3.5.2 --- EDHF-mediated relaxation after H-R in ST solution at different temperature --- p.54 / Chapter 3.5.3 --- Effect of addition of nicorandil to Krebs or ST solution during H-R on EDHF-mediated relaxation --- p.55 / Chapter 3.5.4 --- Clinical implications --- p.56 / Chapter Chapter 4. --- Hypoxia-Reoxygenation in Cardiac Microveins: Combined Effect with Cardioplegia and Temperature on the Endothelial Function --- p.68 / Chapter 4.1 --- Abstract --- p.68 / Chapter 4.2 --- Introduction --- p.69 / Chapter 4.3 --- Experimental design and analysis --- p.73 / Chapter 4.3.1 --- Vessel Preparation --- p.73 / Chapter 4.3.2 --- Normalization --- p.73 / Chapter 4.3.3 --- Hypoxia --- p.73 / Chapter 4.3.4 --- Effect of H-R on EDHF-mediated relaxation in cardiac micro veins --- p.74 / Chapter 4.3.5 --- Combined effects of H-R and ST solution on EDHF-mediated relaxation in cardiac microveins --- p.74 / Chapter 4.3.6 --- Data analysis --- p.75 / Chapter 4.4 --- Results --- p.75 / Chapter 4.4.1 --- Resting force --- p.75 / Chapter 4.4.2 --- U46619-induced contraction force --- p.76 / Chapter 4.4.3 --- Partial pressure of oxygen in hypoxia --- p.76 / Chapter 4.4.4 --- EDHF-mediated relaxation after H-R in Krebs solution at 37°C --- p.76 / Chapter 4.4.5 --- EDHF-mediated relaxation after exposure to H-R in ST solution at different temperatures --- p.77 / Chapter 4.5 --- Discussion --- p.78 / Chapter 4.5.1 --- Effect of H-R on EDHF-mediated relaxation --- p.78 / Chapter 4.5.2 --- Combined effects of H-R with ST solution on EDHF-mediated relaxation --- p.80 / Chapter 4.5.3 --- Clinical implications / Chapter Chapter 5. --- General Discussion --- p.89 / Chapter 5.1 --- EDHF-mediated endothelial function in porcine coronary circulation --- p.89 / Chapter 5.1.1 --- EDHF in porcine coronary microarteries --- p.92 / Chapter 5.1.2 --- EDHF in porcine cardiac veins --- p.90 / Chapter 5.2 --- Alteration of EDHF-mediated function after exposure to H-R --- p.91 / Chapter 5.2.1 --- In coronary microarteries --- p.91 / Chapter 5.2.2 --- In cardiac veins --- p.92 / Chapter 5.3 --- Alteration of EDHF-mediated function after exposure to ST solution under H-R --- p.92 / Chapter 5.3.1 --- In coronary microarteries --- p.93 / Chapter 5.3.2 --- In cardiac veins --- p.93 / Chapter 5.4 --- EDHF-mediated function in nicorandil-supplemented ST solution under H-R in coronary microarteries --- p.93 / Chapter 5.5 --- Clinical implications / Chapter 5.5.1 --- H-R injury --- p.94 / Chapter 5.5.2 --- H-R injury and cardioplegic solution --- p.95 / Chapter 5.5.2 --- Nicorandil-supplementation in cardioplegic solution --- p.95 / Chapter 5.6 --- Limitation of the study --- p.96 / Chapter 5.7 --- Future investigations --- p.96 / Chapter 5.8 --- Conclusions --- p.97 / References --- p.99