Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 95-101).

Essential hypertension is defined as a chronic elevation of blood pressure of unknown cause. Though a definitive trigger for this change in blood pressure has not been established, there is a strong association with an upregulation of sympathetic output from the central nervous system. There are a number of central autonomic nuclei involved in the maintenance of blood pressure, including the brainstem regions of the nucleus tractus solitarii (NTS), caudal ventrolateral medulla (CVLM), rostral ventrolateral medulla (RVLM), the sympathetic preganglionic neurons (SPNs) within the intermediolateral cell column (IML) of the spinal cord, as well as forebrain regions such as the paraventricular nucleus (PVN) of the hypothalamus. Within these centers, a vast number of neurotransmitters have been identified that contribute to the control of blood pressure, including glutamate, angiotensin II, serotonin, neurotensin, neuropeptide Y, opioids and catecholamines. Recognition of the role of nitric oxide (NO) and its multiple influences over the neural control of blood pressure is gaining increasing significance. Nitric oxide is a unique modulatory molecule that acts as a non-conventional neurotransmitter. As NO is a gas with a short half-life of 4 – 6 seconds, its’ synthesising enzyme, nitric oxide synthase (NOS) is often used as a marker of location of production. Once activated, the best-known “receptor” for NO is soluble guanylate cyclase (sGC), which drives the production of cyclic guanosine monophosphate (cGMP). Identifying the presence of cGMP can therefore be used to determine sites receptive to NO. Previous studies examining the role of NO in the central autonomic control of blood pressure have focused predominantly upon application of either excitatory or inhibitory drugs into the key central autonomic regions and assessing pressor or depressor effects. This thesis aims instead to study the neuroanatomical relationship and functional significance of NO and cGMP expression in the brain and spinal cord of a hypertensive and normotensive rat model. In the first experimental chapter (Chapter 3), a comparative neuroanatomical analysis of neuronal NOS expression and its relationship with cGMP in the SPN of mature Spontaneously Hypertensive Rats (SHR) and their controls, Wistar Kyoto (WKY) was undertaken. Fluorescence immunohistochemistry confirmed the expression of nNOS in the majority of SPN located within the IML region of both strains. However, a strain specific anatomical arrangement of SPN cell clusters was evident and while there was no significant difference between the total number of SPN in each strain, there were significantly fewer nNOS positive SPN in the SHR animals. All nNOS positive SPN were found to express cGMP, and a novel subpopulation of nNOS negative, cGMP-positive SPN was identified. These cells were located in the medial edge of the IML SPN cell group. These results suggest that cGMP is a key signalling molecule in SPN, and that a reduced number of nNOS positive SPN in the SHR may be associated with the increase in sympathetic tone seen in essential hypertension. The second experimental chapter (Chapter 4) aimed to determine if reduced numbers of nNOS containing SPN translated into reduced detectable cGMP. The functional significance of cGMP signalling in the two strains was then examined. Based on previous work by our group, it was predicted that reduced nNOS in the SHR would translate into reduced cGMP and that intrathecal administration of exogenous cGMP in the spinal cord would drive a differential pressor response in the two animal strains. Immunohistochemical techniques confirmed that within each SPN, the relative level of cGMP expression was significantly reduced in the SHR when compared to the WKY. Intrathecal application of 8-bromo-cGMP, a drug analogous to cGMP, increased blood pressure in both strains and had a differential and dose dependent effect, causing only a small increase in blood pressure in anaesthetised WKY animals, while driving a significant pressor response in the SHR. This finding raised the novel hypothesis that in the SHR, reduced nNOS expression is not a driver of hypertension, but is instead a protective mechanism limiting the potent pressor effects of cGMP within SPN. The third experimental chapter (Chapter 5) examines the expression of neuronal and inducible isoforms of NOS (nNOS, iNOS) within the RVLM of SHR and WKY rats. Reverse transcription-polymerase chain reaction (RT-PCR) was used to analyse the level of mRNA expression and immunohistochemistry was then used to further analyse protein levels of nNOS. Total RNA was extracted and reverse transcribed from the RVLM of mature male WKY and SHR. Quantitative real-time PCR indicated that relative to WKY, mRNA levels for nNOS was significantly higher in RVLM of the SHR. This was confirmed immunohistochemically. When compared to iNOS, nNOS was expressed at significantly higher levels overall, however there was no difference in iNOS mRNA expression between the two strains. This demonstration of differential expression levels of nNOS and iNOS in the RVLM raises the possibilities that (i) NO production is up-regulated in the RVLM in SHR in response to increased sympathetic activity in order to re-establish homeostatic balance or alternatively that (ii) an alteration in the balance between nNOS and iNOS activity may underlie the genesis of augmented sympathetic vasomotor tone during hypertension. The fourth experimental chapter (Chapter 6) extends the observations in Chapter 5 through examination of the expression of cGMP and sGC within the RVLM. There is strong functional evidence to suggest that NO signalling in the RVLM relies on cGMP as an intracellular signalling molecule and that this pathway is impaired in hypertension. Immunohistochemistry was used to assess cGMP expression as a marker of active NO signalling in the C1 region of the RVLM, again comparing SHR and WKY animals. Fluorescence immunohistochemistry on sections of the RVLM, double labelled for cGMP and either nNOS or phenylethylamine methyl-transferase (PNMT) failed to reveal cGMP positive neurons in the RVLM from aged animals of either strain, despite consistent detection of cGMP immunoreactivity neurons in the nucleus ambiguus from the same or adjacent sections. This was demonstrated both in the presence and absence of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) and in young vs. aged animals. In-vitro incubation of RVLM slices in the NO donor DETA-NO or NMDA did not reveal any additional cGMP neuronal staining within the RVLM. In all studies, cGMP was prominent within the vasculature. Soluble guanylate cyclase immunoreactivity was found throughout the RVLM, although it did not co-localise with the PNMT or nNOS neuronal populations. Overall, results suggest that within the RVLM, cGMP is not detectable in the resting state and cannot be elicited by phosphodiesterase inhibition, NMDA receptor stimulation or NO donor application. A short time course of cGMP signalling or degradation not inhibited by the phosphodiesterase inhibitor utilised (IBMX) in the RVLM cannot be excluded. The final experimental chapter (Chapter 7) examines cGMP expression in magnocellular and preautonomic parvocellular neurons of the PVN. Retrograde tracing techniques and immunohistochemistry were used to visualise cGMP immunoreactivity within functionally, neurochemically and topographically defined PVN neuronal populations in Wistar rats. Basal cGMP immunoreactivity was readily observed in the PVN, both in neuronal and vascular profiles. Cyclic GMP immunoreactivity was significantly higher in magnocellular compared to preautonomic neuronal populations. In preautonomic neurons, the level of cGMP expression was independent on their subnuclei location, innervated target or neurochemical phenotype. The data presented in this chapter indicates a highly heterogeneous distribution of basal cGMP levels within the PVN, and supports work by others indicating that constitutive NO inhibitory actions on preautonomic PVN neurons are likely mediated indirectly through activation of interneurons. Summary Together, these studies comprise a detailed analysis of the neuroanatomical expression of NO and its signalling molecule cGMP in key central autonomic regions involved in the regulation of blood pressure. Under resting or basal conditions, the studies demonstrate notable differences in the expression of NO synthesising enzymes between normotensive and hypertensive animals, and correlating changes in the downstream signalling molecule cGMP. In the spinal cord, novel functional differences in cGMP activity were also demonstrated. In the RVLM, although differences in nNOS were demonstrated, cGMP expression could not be readily detected in either the WKY or SHR, while in contrast within the PVN, cGMP was detected in both magnocellular and parvocellular neuronal populations. Conclusion This thesis gives insight into the physiological role of NO and cGMP as mediators of central blood pressure control. The results presented indicate that the NO-cGMP dependent signalling pathway may not be the dominant driver responsible for maintaining high blood pressure in the SHR model of essential hypertension, and that there is no globally consistent pattern of expression, and indeed the role of NO as a mediator of pressor and depressor function may vary between the autonomic regions examined. Further, it is possible that this pathway is only recruited during activation of reflex homeostatic pathways or during times of marked physiological stress, and that the differences we see in basal expression between the normotensive and SHR animals are instead a result of compensatory mechanisms.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / "December 2001." Typescript. Vita. Includes bibliographical references (leaves 95-113). Also available on the Internet.

Thesis (M.Sc.). / Written for the Dept. of Experimental Medicine. Title from title page of PDF (viewed 2008/05/14). Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 101-113).

Stroke is one of the leading causes of human death in the United States. The debilitating effects of an ischemic stroke are due to the fact that mammalian neurons are highly susceptible to hypoxia and subsequent oxygen reperfusion. From studies in Drosophila melanogaster, cGMP-dependent Protein Kinase (PKG) enzyme is thought to affect anoxia tolerance by modifying the electrical current through potassium ion channels. In this research, two animal models were employed: Drosophila melanogaster and mammalian neurons exposed to stroke-like conditions. First, in vivo studies using Drosophila were performed to further our knowledge about the differences between the naturally occurring variants of the Drosophila foraging gene, which shows different protein levels of PKG. Mitochondrial density and metabolic activity between two fly genotypes exposed to anoxia and reoxygenation were compared. It was found that flies with less enzyme potentially showed mitochondrial biogenesis and higher metabolic rates upon reoxygenation. Next, in vivo studies where PKG enzyme was activated pharmacologically were performed; it was found that the activation of the cGMP/PKG pathway led to neuroprotection upon anoxia and reoxygenation. Furthermore, this model was translated into the in vitro model using Drosophila cells. Instead of anoxia and reoxygenation, hypoxia mimetics and hydrogen peroxide were used to induce cellular injury. After showing the cGMP/PKG pathway activation-induced cell protection, the potential downstream targets of the molecular signaling as well as underlying biochemical changes were assessed. It was found that mitochondrial potassium ion channels were involved in the protective signaling and the signaling modulated metabolic function. Furthermore, it was found that acidosis protected Drosophila cells from cell death, metabolic disruption, and oxidative stress. Finally, this research was translated to a mammalian in vitro model of neuronal damage upon stroke-like conditions; there, it was demonstrated that the cGMP/PKG pathway activation in rat primary cortical neurons and human cortical neurons was protective from low oxygen and acute oxidative stress. The results of this study lead to a better understanding of molecular mechanisms taking place during low oxygen and oxidative stresses. Consequently, this knowledge may be used to identify potential therapeutic targets and treatments that may prevent detrimental neurological effects of an ischemic stroke in humans. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

by Tung Sin Yi, Cindy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 117-132). / Acknowledgements --- p.I / List of Abbreviations --- p.II / Abstract --- p.IV / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Astrocytes in the Central Nervous System (CNS) l / Chapter 1.1.1 --- Characteristics of astrocytes / Chapter 1.1.2 --- Functions of astrocytes / Chapter 1.1.2.1 --- General functions of astrocytes / Chapter 1.1.2.2 --- Effects of neuroactive peptides on astrocytes / Chapter 1.1.3 --- Gliomas and the rat C6 glioma cells / Chapter 1.2 --- C-Type natriuretic peptide (CNP) in the CNS --- p.9 / Chapter 1.2.1 --- Structure and distribution of natriuretic peptides in the CNS / Chapter 1.2.2 --- Actions of CNP / Chapter 1.2.3 --- Natriuretic peptide receptors and signal transduction in astrocytes / Chapter 1.3 --- Endothelin-3 (ET-3) in the CNS --- p.18 / Chapter 1.3.1 --- Structure and distribution of endothelins (ETs) in the CNS / Chapter 1.3.2 --- Actions of ET-3 / Chapter 1.3.3 --- Endothelin receptors and signal transductionin astrocytes / Chapter 1.4 --- cGMP second messenger system in astrocytes --- p.28 / Chapter 1.4.1 --- Second messenger systems in astrocytes / Chapter 1.4.2 --- cGMP as second messenger in astrocytes / Chapter 1.4.3 --- Post cGMP cascade effects / Chapter 1.5 --- The aims of this project --- p.33 / Chapter Chapter 2 --- Methods / Chapter 2.1 --- In vitro culture of rat C6 glioma cells --- p.36 / Chapter 2.1.1 --- Preparation of reagents / Chapter 2.1.2 --- Culture of C6 glioma cells / Chapter 2.1.3 --- "Cell plating in 6-well, 24-well and 96-well plastic trays" / Chapter 2.2 --- Determination of cGMP --- p.40 / Chapter 2.2.1 --- Measurement of cGMP / Chapter 2.2.2 --- Data analysis / Chapter 2.3 --- Determination of the effect of CNP on cGMP productionin C6 cells --- p.41 / Chapter 2.4 --- Determination of the effect of ET-3 on the action of CNPin C6cells --- p.44 / Chapter 2.4.1 --- Measurement of intracellular cGMP levels affected by ET-3 / Chapter 2.4.2 --- Measurement of intracellular cGMP levels affected by CNP with ET-3 pretreatment / Chapter 2.5 --- Determination of the effects of PKC activator and inhibitor on CNP-treated C6 cells --- p.46 / Chapter 2.5.1 --- Measurement of intracellular cGMP levels affected by PKC activator or inhibitor / Chapter 2.5.2 --- Measurement of intracellular cGMP levels affected by CNP with PKC activator or inhibitor pretreatment / Chapter 2.5.3 --- Measurement of intracellular cGMP levels affected by CNP with PKC inhibitor antagonized PMA or ET-3 pretreatment / Chapter 2.6 --- Determination of the effect of arachidonic acid on the action of CNP in C6 cells --- p.49 / Chapter 2.7 --- Determination of the effects of ET-3 and CNP on calcium uptake in C6 cells --- p.50 / Chapter 2.8 --- Determination of the effects of CNP and ET-3 on cell volume change in C6 cells --- p.51 / Chapter 2.9 --- Determination of the effects of CNP and ET-3 on glucose and amino acids uptake in C6 cells --- p.53 / Chapter 2.9.1 --- Measurement of glucose uptake in CNP - and/or ET- 3-treated C6 cells / Chapter 2.9.2 --- Measurement of amino acids uptake in CNP - and/or ET-3-treated C6 cells / Chapter 2.10 --- "Determination of thymidine, uridine and leucine incorporation in CNP - and/or ET-3- treated C6 cells" --- p.55 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Effects of CNP and ET-3 on cGMP production in cultured rat C6 glioma cells --- p.56 / Chapter 3.1.1 --- Effect of CNP on cGMP production in cultured C6 glioma cells --- p.57 / Chapter 3.1.1.1 --- The time course of CNP on cGMP production / Chapter 3.1.1.2 --- Dosage-response of CNP on cGMP production / Chapter 3.1.2 --- Effect of ET-3 on cGMP production in C6 glioma cells --- p.61 / Chapter 3.1.2.1 --- Effect of ET-3 on basal cGMP production / Chapter 3.1.2.2 --- Effect of pre-exposure duration to ET-3 on CNP-induced cGMP formation / Chapter 3.1.2.3 --- Dosage-response of ET-3 on CNP-induced cGMP production / Chapter 3.1.3 --- Effect of PMA on cGMP production in C6 glioma cells --- p.65 / Chapter 3.1.3.1 --- Effect of PMA on basal cGMP production / Chapter 3.1.3.2 --- Effect of pre-exposure duration to PMA on CNP-induced cGMP formation / Chapter 3.1.3.3 --- Dosage-response of PMA on CNP-induced cGMP production / Chapter 3.1.4 --- Effects of PKC inhibitors on cGMP production in C6 glioma cells --- p.73 / Chapter 3.1.4.1 --- Effects of PKC inhibitors on basal cGMP production / Chapter 3.1.4.2 --- Effects of PKC inhibitors on CNP-induced cGMP formation / Chapter 3.1.4.3 --- Antagonism of PKC inhibitors on the action of PMA on CNP-induced cGMP formation / Chapter 3.1.4.4 --- Antagonism of PKC inhibitors on the action of ET-3 on CNP-induced cGMP formation / Chapter 3.1.5 --- Effect of arachidonic acid on CNP-induced cGMP production in C6 glioma cells --- p.82 / Chapter 3.2 --- Effects of CNP and ET-3 on cellular metabolism in cultured rat C6 glioma cells --- p.83 / Chapter 3.2.1 --- Effects of CNP and ET-3 on calcium uptake in C6 glioma cells --- p.86 / Chapter 3.2.2 --- Effects of CNP and ET-3 on cell volume changes in C6 glioma cells --- p.89 / Chapter 3.2.3 --- Effects of CNP and ET-3 on glucose and amino acids uptake in C6 glioma cells --- p.91 / Chapter 3.2.4 --- Effects of CNP and ET-3 on C6 cell proliferation --- p.98 / Chapter 3.2.5 --- Effects of CNP and ET-3 on RNA synthesis --- p.101 / Chapter 3.2.6 --- Effects of CNP and ET-3 on protein synthesis --- p.103 / Chapter Chapter 4 --- Discussion and Conclusion --- p.105 / References --- p.117

Chan Ho Yu, Richard. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 115-122). / Abstracts in English and Chinese. / Declaration --- p.i / Acknowledgements --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Table of Contents --- p.vii / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Potassium Channels --- p.1 / Chapter 1.1.1 --- Potassium Ions --- p.1 / Chapter 1.1.2 --- Potassium Channels --- p.1 / Chapter 1.1.3 --- Structure of K Channels --- p.2 / Chapter 1.1.4 --- Classification ofK Channels --- p.3 / Chapter 1.1.5 --- Mechanisms Contributed to K Channel Functions and Diversity --- p.5 / Chapter 1.1.5.1 --- RNA Editing --- p.5 / Chapter 1.1.5.2 --- Alternative Splicing --- p.6 / Chapter 1.1.5.3 --- Heteromultimeric Assembly of Principal Subunits --- p.6 / Chapter 1.1.5.4 --- Auxiliary Subunits --- p.7 / Chapter 1.1.5.5 --- Posttranslational Modifications --- p.7 / Chapter 1.2 --- Voltage-gated Potassium (Kv) Channels --- p.9 / Chapter 1.2.1 --- Diversity of Kv Channel Structure --- p.9 / Chapter 1.2.2 --- Early Origin of the Kv Family --- p.10 / Chapter 1.2.3 --- Structural Diversity of Kv Channels in Drosophila --- p.11 / Chapter 1.2.4 --- Structural Diversity of Kv Channels in Mammals --- p.11 / Chapter 1.2.5 --- Phylogenetic Tree of Kv Family --- p.13 / Chapter 1.2.6 --- Tissue Expression of Kv Channels --- p.13 / Chapter 1.2.7 --- "Three Main Functions of Kv Channels as Signaling Proteins: Ion Permeation, Gating and Sensing" --- p.16 / Chapter 1.2.7.1 --- Ion Permeation --- p.16 / Chapter 1.2.7.2 --- Gating --- p.18 / Chapter 1.2.7.2.1 --- Gating at the S6 Bundle Crossing --- p.18 / Chapter 1.2.7.2.2 --- Ball-and-Chain Gating --- p.19 / Chapter 1.2.7.2.3 --- Gating at the Selectivity Filter --- p.19 / Chapter 1.2.7.3 --- Sensing Mechanisms --- p.20 / Chapter 1.2.7.3.l --- Voltage Sensor --- p.20 / Chapter 1.2.7.3.2 --- Gating Sensors for Ligands --- p.21 / Chapter 1.3 --- KCNA10 --- p.22 / Chapter 1.3.1 --- "Rabbit Homologue of KCNA10, Kcnl" --- p.22 / Chapter 1.3.2 --- Genomic Localization of Human KCNA10 --- p.23 / Chapter 1.3.3 --- Human Gene for KCNA10 --- p.23 / Chapter 1.3.4 --- Basic Kinetic and Pharmacological Properties of KCNA10 --- p.25 / Chapter 1.3.5 --- "Regulation of KCNAlO by KCNA4B, a β -subunit" --- p.27 / Chapter 1.4 --- Aim of the Present Study --- p.30 / Chapter Chapter2: --- Materials and Methods --- p.31 / Chapter 2.1 --- Molecular Sub-Cloning ofKCNAlO --- p.31 / Chapter 2.1.1 --- Polymerase Chain Reaction (PCR) ofKCNA10 Fragment from KCNA Clone --- p.10 / Chapter 2.1.2 --- Separation and Purification of PCR Products --- p.32 / Chapter 2.1.2.1 --- Separation --- p.32 / Chapter 2.1.2.2 --- Purification --- p.33 / Chapter 2.1.3 --- Polishing the Purified PCR Products --- p.33 / Chapter 2.1.4 --- Ligation of PCR Products and pPCR-Script Amp SK(+) Cloning Vector --- p.34 / Chapter 2.1.5 --- Transformation --- p.34 / Chapter 2.1.6 --- Preparing Glycerol Stocks Containing the Bacterial Clones --- p.35 / Chapter 2.1.7 --- Plasmid DNA Preparation --- p.35 / Chapter 2.1.8 --- Clones Confirmation --- p.36 / Chapter 2.1.8.1 --- Restriction Enzyme Digestion --- p.36 / Chapter 2.1.8.2 --- Automatic Sequencing --- p.37 / Chapter 2.2 --- In situ Hybridization --- p.39 / Chapter 2.2.1 --- Probe Preparation --- p.39 / Chapter 2.2.1.1 --- Antisense KCNA10 RNA Probe --- p.39 / Chapter 2.2.1.2 --- Sense KCNA10 RNA Probe (Control Probe) --- p.40 / Chapter 2.2.2 --- Testing of DIG-Labeled RNA Probes --- p.43 / Chapter 2.2.3 --- Paraffin Sections Preparation --- p.43 / Chapter 2.2.4 --- In situ Hybridization: Pretreatment --- p.44 / Chapter 2.2.5 --- "Pre-hybridization, Hybridization and Post-hybridization" --- p.45 / Chapter 2.2.5.1 --- Pre-hybridization --- p.45 / Chapter 2.2.5.2 --- Hybridization --- p.45 / Chapter 2.2.5.3 --- Post-hybridization --- p.46 / Chapter 2.2.6 --- Colourimetnc Detection of Human KCNA10 --- p.46 / Chapter 2.3 --- Cell Culture --- p.47 / Chapter 2.3.1 --- Human Kidney Proximal Epithelial Cell Line (OK) --- p.47 / Chapter 2.3.2 --- Mouse Micro-vessel Endothelial Cell Line (H5V) --- p.48 / Chapter 2.3.3 --- Mouse Neuroblastoma Cell Line (NG108-15) --- p.48 / Chapter 2.3.4 --- Human Bladder Epithelial Cell Line (ECV304) --- p.48 / Chapter 2.3.5 --- Human T Cell Leukemia Cell Line (Jurkat) --- p.49 / Chapter 2.4 --- Total RNA Extraction --- p.49 / Chapter 2.5 --- Reverse Transcription from Cell Line --- p.51 / Chapter 2.6 --- Polymerase Chain Reaction (PCR) ofKCNAl 0 Fragment from Frist Strand cDNA --- p.51 / Chapter 2.7 --- Northern Hybridization --- p.52 / Chapter 2.7.1 --- Probe Preparation --- p.52 / Chapter 2.7.2 --- Separating RNA on an Agarose Gel --- p.52 / Chapter 2.7.3 --- RNA Transfer and Fixation --- p.52 / Chapter 2.7.4 --- Hybridization --- p.54 / Chapter 2.7.5 --- Post-hybridization --- p.54 / Chapter 2.7.6 --- Chemiluminescent Detection --- p.55 / Chapter 2.8 --- Intracellular Free Calcium Ion ([Ca2+］i) Measurement by Confocal Imaging System --- p.56 / Chapter 2.8.1 --- Bathing Solutions --- p.56 / Chapter 2.8.2 --- Preparation of Cells for [Ca2+]i Measurement --- p.56 / Chapter 2.8.3 --- Confocal Imaging System --- p.57 / Chapter 2.8.3.1 --- Fluo-3/AM Dye Loading --- p.57 / Chapter 2.8.3.2 --- [Ca2+]i Measurement --- p.57 / Chapter Chapter3: --- Results --- p.59 / Chapter 3.1 --- Phylogenetic Tree Reconstruction ofKCNAl0 --- p.59 / Chapter 3.2 --- Hydropathy Analysis ofKCNAl0 --- p.60 / Chapter 3.3 --- Molecular Sub-Cloning ofKCNAl0 --- p.61 / Chapter 3.3.1 --- Polymerase Chain Reaction (PCR) ofKCNAl0 Fragment from KCNA10 Clone --- p.61 / Chapter 3.3.2 --- Clones Confirmation --- p.63 / Chapter 3.4 --- In situ Hybridization Analysis ofKCNAl0 mRNAExpression --- p.65 / Chapter 3.4.1 --- Expression ofKCNAl0 in Human Kidney (Nephron) --- p.66 / Chapter 3.4.2 --- Expression ofKCNAl0 in Human Cerebral Artery --- p.69 / Chapter 3.4.3 --- Expression ofKCNAl0 in Human Cerebellum --- p.71 / Chapter 3.4.4 --- Expression ofKCNAl0 in Human Hippocampus --- p.73 / Chapter 3.4.5 --- Expression ofKCNAl0 in Human Occipital Cortex --- p.75 / Chapter 3.4.6 --- Expression ofKCNAl0 in Human Esophagus --- p.77 / Chapter 3.4.7 --- Expression ofKCNAl0 in Human Lung --- p.79 / Chapter 3.4.8 --- Expression ofKCNAl0 in Human Thyroid Glands --- p.81 / Chapter 3.4.9 --- Expression ofKCNAl0 in Human Adrenal Glands --- p.83 / Chapter 3.4.10 --- Expression ofKCNAl0 in Human Spleen --- p.86 / Chapter 3.5 --- RT-PCR ofKCNAl0 Fragment from Different Tissues --- p.88 / Chapter 3.6 --- Northern Blot Analysis of KCNA10 in Different Tissues --- p.90 / Chapter 3.7 --- Effects of Blocking KCNA10 on Ca2+ influx in Human Renal Proximal Tubule Epithelial Cells --- p.91 / Chapter Chapter4: --- Discussion --- p.97 / Chapter 4.1 --- Phylogency ofKCNAlO --- p.97 / Chapter 4.2 --- Hydropathy Plot for KCNA10 --- p.97 / Chapter 4.3 --- Expression ofKCNAl0 --- p.98 / Chapter 4.3.1 --- In situ Hybridization --- p.98 / Chapter 4.3.2 --- RT-PCR & Northern Blot Analysis --- p.99 / Chapter 4.4 --- Functional Implication of KCNA10 Expression in Different Human Tissues --- p.100 / Chapter 4.4.1 --- Unique Functional Properties ofKCNAlO --- p.100 / Chapter 4.4.2 --- Role ofKCNAlO in Renal Proximal Tubule --- p.101 / Chapter 4.4.2.1 --- Functions ofK+ Channels in Kidney --- p.101 / Chapter 4.4.2.2 --- The Function ofKCNAlO --- p.104 / Chapter 4.4.3 --- Role ofKCNAl0 in Blood Vessels --- p.106 / Chapter 4.4.3.1 --- Endothelial Cells --- p.106 / Chapter 4.4.3.2 --- Smooth Muscle Cells --- p.108 / Chapter 4.4.4 --- Role ofKCNA10 in CNS --- p.109 / Chapter 4.4.5 --- Role ofKCNAl0 in Secretory Cells --- p.111 / Chapter 4.4.6 --- Role ofKCNAl0 in Lung --- p.112 / Chapter 4.5 --- Conclusion --- p.114 / Chapter Chapter5： --- Reference --- p.115

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