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

Cyclic guanosine 3', 5'-monophosphate (cGMP) has recently emerged as an endogenous regulator for controlling or reversing cardiac hypertrophy. Increased protein accumulation is a key feature of cardiac hypertrophy; thus, our study investigates the effects of a cGMP analog on protein accumulation in primary culture of adult rat cardiomyocytes and dissects out the mechanisms involved. We confirmed that a cGMP analog, 8-bromo-cGMP, inhibits phenylephrine (PE)-increased accumulation of newly synthesized proteins in cultured adult rat ventricular cardiomyocytes. Firstly, we have obtained data showing that 8-bromo-cGMP does not inhibit phosphorylation of S6K1 by PE during short time treatment (10 min to 2 h), but blocks phosphorylation of S6K1 by PE at 6 h; moreover this blocking effect is completely abolished by phosphatase inhibitor Tautomycin. Then, we have demonstrated that PE and cGMP induce sustained and transient increased phosphorylation of ERK, respectively. Moreover, cGMP inhibits PE-induced phosphorylation of ERK during long term treatment (3 and 6h). We have also shown that 8-bromo-cGMP inhibits ROS generation induced by PE. Other effects of PE that could be related to hypertrophy (i.e. increased concentration of upstream binding factor mRNA and decreased concentration of the mRNAs of Atrogin and muscle specific RING finger) were not abolished by 8-bromo-cGMP. We conclude that cGMP analog blocks protein accumulation by inhibiting the sustained phosphorylation of S6K1 via the activation of phosphatases.

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Diss. (sammanfattning) Linköping : Univ., 2001. / Härtill 4 uppsatser.