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