by Tung Wai Lin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 110-123. / ACKNOWLEDGEMENT / LIST OF ABBREVIATIONS / ABSTRACT --- p.1 / Chapter CHAPTER 1 --- INTRODUCTION --- p.4 / Chapter 1.1 --- TAURINE IN THE CNS --- p.4 / Chapter 1.1.1 --- General metabolism of taurine --- p.4 / Chapter 1.1.2 --- Release and uptake of taurine by neurons and glia --- p.5 / Chapter 1.1.2.1 --- Taurine uptake --- p.5 / Chapter 1.1.2.2 --- Taurine release --- p.6 / Chapter 1.2 --- FUNCTIONS OF ASTROCYTES --- p.7 / Chapter 1.2.1 --- Potassium homeostasis --- p.8 / Chapter 1.2.2 --- Water homeostasis --- p.9 / Chapter 1.3 --- NEURON-GLIA COMMUNICATION : NEUROTRANSMITTER RECEPTORS AND SECOND MESSENGER SYSTEMS IN ASTROCYTES --- p.11 / Chapter 1.3.1 --- Adenylate cyclase pathway --- p.12 / Chapter 1.3.2 --- Inositol lipid pathway --- p.12 / Chapter 1.3.2.1 --- "Ins(l,4,5)P3/Ca2+ pathway" --- p.13 / Chapter 1.3.2.2 --- DAG/PKC pathway --- p.15 / Chapter 1.3.2.3 --- Dual actions of PKC --- p.16 / Chapter 1.3.3 --- Interaction between second messenger systems --- p.17 / Chapter 1.4 --- SUBSTANCE P RECEPTOR ON ASTROCYTES --- p.18 / Chapter 1.4.1 --- SP and SP receptors --- p.18 / Chapter 1.4.2 --- Evidence for the existence of SP receptor in glial cell --- p.20 / Chapter CHAPTER 2 --- METHODS --- p.23 / Chapter 2.1 --- IN VITRO CULTURE OF HUMAN ASTROCYTOMA CELLS (U-3 7 3 MG) --- p.23 / Chapter 2.1.1 --- Preparation of reagents --- p.23 / Chapter 2.1.2 --- Culture of astrocytoma cells --- p.24 / Chapter 2.1.3 --- Cell plating in 24-well plastic trays --- p.25 / Chapter 2.2 --- DETERMINATION OF [3H]-TAURINE RELEASE --- p.26 / Chapter 2.2.1 --- Physiological salt solution (PSS) --- p.26 / Chapter 2.2.2 --- r> Preparation of working [ H]-taurine solution --- p.26 / Chapter 2.2.3 --- Assay of [ H]-taurine release --- p.26 / Chapter 2.2.4 --- Drug pretreatment --- p.27 / Chapter 2.2.5 --- Data analysis --- p.27 / Chapter 2.3 --- DOWN REGULATION OF PKC ACTIVITY BY PROLONGED PMA PRETREATMENT --- p.29 / Chapter 2.3.1 --- Preparation of working Phorbol-12-myristate-13-acetate (PMA) solution --- p.29 / Chapter 2.3.2 --- Pretreatment of cells with PMA --- p.29 / Chapter 2.4 --- DETERMINATION OF CAMP --- p.29 / Chapter 2.4.1 --- Drugs effects on cAMP --- p.30 / Chapter 2.4.2 --- Data analysis --- p.30 / Chapter 2.5 --- MEASUREMENT OF INOSITOL PHOSPHATES --- p.31 / Chapter 2.5.1 --- Downx column preparation --- p.31 / Chapter 2.5.2 --- Determination of total inositol phosphates accumulation --- p.31 / Chapter 2.5.3 --- Column separation --- p.33 / Chapter 2.5.4 --- Determination of inositol trisphosphate (IP3) --- p.34 / Chapter 2.6 --- MEASUREMENT OF PKC --- p.34 / Chapter 2.6.1 --- Preparation of cytosolic and membrane bound PKC --- p.34 / Chapter 2.6.1.1 --- Reagent preparation --- p.34 / Chapter 2.6.1.2 --- Preparation of DEAE-cellulose column --- p.35 / Chapter 2.6.1.3 --- Protocol of preparing cytosolic and membrane bound PKC --- p.36 / Chapter 2.6.2 --- PKC assay --- p.37 / Chapter 2.6.2.1 --- Reagent preparation --- p.37 / Chapter 2.6.2.2 --- PKC activity assay protocol --- p.38 / Chapter 2.7 --- STATISTICAL METHOD --- p.39 / Chapter CHAPTER 3 --- RESULTS --- p.40 / Chapter 3.1 --- SOME CHARACTERISTICS OF TAURINE TRANSPORT IN U-373 MG ASTROCYTOMA CELLS --- p.40 / Chapter 3.1.1 --- Time and sodium dependence of taurine uptake --- p.40 / Chapter 3.1.2 --- Time dependence of SP-stimulated taurine release --- p.42 / Chapter 3.1.3 --- Effect of ions on SP-stimulated taurine release --- p.42 / Chapter 3.1.3.1 --- Effect of sodium on taurine release --- p.42 / Chapter 3.1.3.2 --- Effect of potassium on taurine release --- p.42 / Chapter 3.1.3.3 --- Effect of calcium and magnesium on taurine release --- p.46 / Chapter 3.1.4 --- Temperature-dependence of [H]-taurine release --- p.49 / Chapter 3.1.5 --- Comparison of chemical-induced release of taurine and GABA from U-373 MG astrocytoma cells --- p.49 / Chapter 3.2 --- PHARMACOLOGICAL STUDIES OF SP-STIMULATED TAURINE RELEASE --- p.49 / Chapter 3.2.1 --- Effect of mammalian tachykinins and their analogues on the release of [3H]-taurine --- p.49 / Chapter 3.2.2 --- Antagonistic effect of spantide on SP-stimulated [3H]-taurine release --- p.51 / Chapter 3.2.3 --- The interaction between SP- and IPR- stimulated taurine release --- p.55 / Chapter 3.2.3.1 --- Concentration dependence of IPR-induced release of [ H]-taurine --- p.55 / Chapter 3.2.3.2 --- Effect of 100 nM IPR on SP-stimulated taurine release --- p.55 / Chapter 3.3 --- SECOND MESSENGER SYSTEMS INVOLVED IN THE REGULATION OF TURINE RELEASE BY SP AND IPR --- p.55 / Chapter 3.3.1 --- The role of cAMP --- p.58 / Chapter 3.3.2 --- The role of phosphatidylinositol bisphosphate (PIP2) metabolism --- p.58 / Chapter 3.3.2.1 --- Time course of SP-induced inositol phosphates accumulation --- p.61 / Chapter 3.3.2.2 --- Pharmacological studies of SP-induced IPs accumulation --- p.61 / Chapter 3.3.2.3 --- Effect of SP on inositol trisphosphate --- p.65 / Chapter 3.3.2.4 --- Effect of IPR on IPs accumulation --- p.65 / Chapter 3.3.3 --- The role of Ca2+ mobilization --- p.65 / Chapter 3.3.4 --- The role of protein kinase C (PKC) --- p.69 / Chapter 3.3.4.1 --- Concentration dependence of PMA-stimulated taurine release --- p.70 / Chapter 3.3.4.2 --- Effect of H7 on the release of taurine --- p.70 / Chapter 3.3.4.3 --- Effect of staurosporine on taurine release --- p.73 / Chapter 3.3.4.4 --- Effect of chronic PMA pretreatment --- p.76 / Chapter 3.3.4.5 --- Measurement of PKC activity --- p.80 / Chapter CHAPTER 4 --- DISCUSSIONS --- p.90 / Chapter 4.1 --- A COMPARISON OF THE CHARACTERISTICS OF TAURINE RELEASE FROM ASTROCYTOMA CELLS AND NEURONS --- p.90 / Chapter 4.2 --- SP-INDUCED [3H]-TAURINE RELEASE FROM U-3 7 3 MG ASTROCYTOMA CELLS --- p.93 / Chapter 4.3 --- THE ROLE OF SECOND MESSENGER SYSTEMS IN THE SP-INDUCED [3H]-TAURINE RELEASE --- p.95 / Chapter 4.4 --- BIOLOGICAL SIGNIFICANCE OF SP-INDUCED TAURINE RELEASE --- p.103 / CONCLUSIONS --- p.107 / REFERENCES --- p.110

Intravenous application of high dose of capsaicin to the rat stimulates C-fiber neurons that innervate the airways to release tachykinins that produce acute inflammation in the mucosal tissue. Large amount of extravasated plasma is retained underneath the tracheal epithelium to form edema. Substance P (SP) is the most important inflammation-producing peptide of tachykinin family. The present study was to investigate time-dependent formation and remission of edema induced by SP (3 µg/ml/kg) by the use of digital morphometric analysis of montages of tracheal cross sections. Furthermore, 3-dimensional reconstruction of serial tracheal sections was carried out to analyze the relative distribution of subepithelial edematous loci. Two edema indexes were designated for evaluation of the status of edema. Edema length ratio was the ratio of the total length of edematous loci to the circumference of a tracheal section. Edema area ratio was the ratio of the total area of edematous loci to the area of tracheal epithelium and associated edema. The degree of edematous status in the mucosa exhibited a time-dependent change. Five min after application of SP, edema length ratio and edema area ratio in the trachea were 35.80¡Ó1.42% and 16.28¡Ó2.51%, that were 7.6 and 7.9 times, respectively, the values of vehicle control group. At 1 h after SP, edema length ratio and edema area ratio declined to 16.40¡Ó2.46% and 8.00¡Ó1.60%, 2.2 and 2.8 times the values of control, but still significantly different (P < 0.05). At 24 or 72 h after SP, the values of edema were not significantly different (P > 0.05) from the control values. Three- dimensional reconstruction study showed that, in the trachea of rats 5 min after receiving SP, there were many subepithelial edematous loci, evenly distributed along the inner circumference of trachea. They were interconnected. The number of edematous loci decreased drastically by 1 h after SP. Loci of edema were rarely found 24 or 72 h after SP. The close association of edema to the tracheal epithelium suggests that the mucosal surface may be the site for elimination of edema fluid.

To investigate the underlying structural features of the neuropeptides α-melanotropin (α-MSH) and substance P (SP), which are responsible for their biological actions, the following study was undertaken. By means of side-chain, fragment and conformational restriction analysis, several α-MSH peptides were prepared by solid-phase synthesis and evaluated by the frog and lizard skin bioassays. Using conformational restriction and fragment methods, several SP peptides were synthesized and examined for biological activity on the guinea-pig isolated ileum, rat brain binding and intrathecal injection assay systems. The results with the new α-MSH analogues show that the histidine-6 side-chain is not needed for signal transduction, but is very important for full potency. The tryptophan-9 side-chain is similarly not needed for signal transduction, but is critically important for full potency. The data also indicate that the positions 6 and 9 side-chains are important for full potency because they likely interact with the melanophore receptor, rather than playing a role in conformationally folding the MSH peptide into a pseudocyclic structure. The results also show that the arginine side-chain at position 8 is not particularly important for signal transduction or full potency, but on the lizard skin bioassay this side-chain is implicated in the previously reported prolongation of Nle⁴, D-Phe⁷-α-MSH. The data provided by the SP peptides suggest that the previously postulated pseudocyclic structure of the 5-11 sequence may not be as fundamental to SP activity as heretofore believed. The data suggest that this type of turn conformation may be important for signal transduction, but is apparently not the only requirement for receptor recognition. Finally, the data show that part of the signal transduction message of SP is contained within the 5-8 region of the peptide, but that most of the receptor recognition elements are probably located outside this sequence.

Pterygium is an ocular surface disorder characterised by centripetal invasion of the cornea by altered limbal cells, accompanied by fibrosis and neovascularisation. One of the enigmatic features of pterygium is its wing-like shape and the mechanism(s) supporting its centripetal growth remain to be elucidated. As the growth pattern of pterygia mirrors the radial arrangement of corneal nerves, we hypothesised that neuropeptides may facilitate its directional growth. In this thesis, we investigated the roles that the sensory neuropeptide substance P (SP) may play in the pathogenesis of pterygia given its known functions in corneal cell migration, proliferation, wound healing and neurogenic inflammation. Using a modified Boyden chamber method, SP was shown to act as a chemoattractant to pterygium fibroblasts and vascular endothelial cells, and this activity was diminish by blockade of its receptor (NK1). 3H-thymidine incorporation assays confirmed that our cell migration results were unrelated to SP-stimulated proliferation. A bead-based multiplex cytokine array detected secretion of pro-inflammatory cytokines (IL-6, IL-8 and CCL2) from SP stimulated pterygium and limbal epithelial cells. Using real-time RT-PCR and immunoblotting, we show that UVB stimulated transcription of the TAC1 gene followed by secretion of SP in ocular surface epithelial cell cultures. Finally, SP and NK1receptor immunoreactivity was identified in pterygium tissue, where overall, NK1receptors were up-regulated in pterygia. Furthermore, we identified a population of NK1 receptor positive mononuclear cells in pterygia that did not express lineage markers for T or B-Iymphocytes, macrophages or mast cells, but may represent immature haemopoietic cells that may have migrated in from the blood since these cells were also present in autologous conjunctival tissue. In summary, SP may contribute to the shape of pterygia by facilitating migration of fibroblasts and vascular endothelial cells into the normally avascular cornea. Additionally, UVB stimulates SP production in epithelial cells and the presence of SP contributes to inflammation in pterygia by inducing pro-inflammatory cytokine release. Finally, we identified a population of relatively immature, NK1 receptor positive cells in pterygia that may have been attracted by the presence of SP. Collectively, these results imply that SP may contribute to the pathogenesis of pterygia.

Thesis (M.S.)--Youngstown State University, 2008. / Includes bibliographical references (leaves 43-51). Also available via the World Wide Web in PDF format.

Theses (M.A.)--Marshall University, 2006. / Title from document title page. Includes abstract. Document formatted into pages: contains v, 17 p. Bibliography: p. 14-17.

Thesis (doctoral)--Karolinska Institutet, Stockholm, 1983. / Extra t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Karolinska Institutet, Stockholm, 1983. / Extra t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (Ph. D.)--University of Chicago, Dept. of Organismal Biology and Anatomy. / Includes bibliographical references. Also available on the Internet.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.