Kam Yiu-wing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 200-215). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.iii / Publications --- p.iv / Abbreviations --- p.v / Contents --- p.vii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- G protein-coupled receptors --- p.1 / Chapter 1.1.1 --- Introduction --- p.1 / Chapter 1.1.2 --- G protein-coupled receptors --- p.2 / Chapter 1.1.3 --- Heterotrimeric G-proteins --- p.4 / Chapter 1.1.4 --- Second messenger systems --- p.5 / Chapter 1.1.5 --- Mechanism of GPCR activation --- p.6 / Chapter 1.1.6 --- GPCR cross talk --- p.8 / Chapter 1.2 --- Receptor theory --- p.10 / Chapter 1.2.1 --- Introduction --- p.10 / Chapter 1.2.2 --- Two-state model --- p.11 / Chapter 1.2.3 --- Three-state model --- p.12 / Chapter 1.2.4 --- Extended ternary complex model --- p.12 / Chapter 1.2.5 --- Multiple receptor state --- p.13 / Chapter 1.2.6 --- Constitutively active mutant receptors --- p.14 / Chapter 1.3 --- Agonist trafficking --- p.15 / Chapter 1.3.1 --- Introduction --- p.15 / Chapter 1.3.2 --- Effect of agonist efficacy on receptor coupling --- p.17 / Chapter 1.3.3 --- Effect of receptor expression level on receptor coupling --- p.17 / Chapter 1.3.4 --- Receptor promiscuity --- p.18 / Chapter 1.3.5 --- Agonist-induced conformational changes --- p.19 / Chapter 1.3.5.1 --- Conformational induction --- p.19 / Chapter 1.3.5.2 --- Conformational selection --- p.19 / Chapter 1.3.6 --- Receptor/G-protein system --- p.20 / Chapter 1.3.7 --- Implication of agonist trafficking --- p.21 / Chapter 1.4 --- Receptor switching --- p.22 / Chapter 1.4.1 --- Introduction --- p.22 / Chapter 1.4.2 --- Receptor switching --- p.23 / Chapter Chapter 2 --- Prostacyclin receptors --- p.34 / Chapter 2.1 --- General properties of prostacyclin --- p.34 / Chapter 2.1.1 --- Synthesis of prostacyclin --- p.34 / Chapter 2.1.2 --- Prostacyclin and its mimetics --- p.34 / Chapter 2.1.3 --- Characterization of IP-receptors --- p.36 / Chapter 2.1.3.1 --- Classification of IP-receptors --- p.36 / Chapter 2.1.3.2 --- Distribution of IP-receptors in the peripheral system --- p.36 / Chapter 2.1.3.3 --- Distribution of IP-receptors in the central nervous system --- p.38 / Chapter 2.1.3.4 --- Structure of IP-receptors --- p.39 / Chapter 2.1.4 --- Anti-thrombotic role of prostacyclin --- p.40 / Chapter 2.1.5 --- Cytoprotective role of prostacyclin --- p.41 / Chapter 2.1.6 --- Role of prostacyclin in adipogenesis --- p.42 / Chapter 2.2 --- Coupling capacity of IP-receptors --- p.43 / Chapter 2.2.1 --- Introduction --- p.43 / Chapter 2.2.2 --- Interaction with Gs --- p.44 / Chapter 2.2.3 --- Interaction with Gi --- p.45 / Chapter 2.2.4 --- Interaction with Gq --- p.45 / Chapter 2.2.5 --- Interaction with PPARs --- p.43 / Chapter 2.2.6 --- IP-receptor isoprenylation --- p.49 / Chapter 2.3 --- Regulation of IP-receptors --- p.50 / Chapter 2.3.1 --- Mechanism of signal termination --- p.50 / Chapter 2.3.2 --- Desensitization of IP-receptors --- p.51 / Chapter 2.3.3 --- Internalization of IP-receptors --- p.53 / Chapter Chapter 3 --- Materials and solutions --- p.59 / Chapter 3.1 --- Materials --- p.59 / Chapter 3.2 --- "Culture media, buffers and solutions" --- p.53 / Chapter 3.2.1 --- Culture media --- p.63 / Chapter 3.2.2 --- Buffers --- p.64 / Chapter 3.2.3 --- Solutions --- p.65 / Chapter Chapter 4 --- Methods --- p.67 / Chapter 4.1 --- Cell culture --- p.67 / Chapter 4.2 --- Mammalian cell transfection --- p.68 / Chapter 4.2.1 --- Preparation of plasmid DNA --- p.68 / Chapter 4.2.2 --- Transient transfection of mammalian cells --- p.71 / Chapter 4.2.3 --- β-galactosidase assay --- p.73 / Chapter 4.2.3.1 --- Introduction --- p.73 / Chapter 4.2.3.2 --- Preparation of cell lysate --- p.73 / Chapter 4.2.3.3 --- Micro β-galactosidase assay --- p.73 / Chapter 4.3 --- Measurement of adenylate cyclase activity --- p.74 / Chapter 4.3.1 --- [3H]-adenine prelabelling method --- p.74 / Chapter 4.3.1.1 --- Preparation of columns --- p.74 / Chapter 4.3.1.2 --- Incubation of cells --- p.75 / Chapter 4.3.1.3 --- Measurement of [3H]-cyclic AMP production --- p.75 / Chapter 4.3.1.4 --- Data analysis --- p.76 / Chapter 4.3.2 --- cAMP [125I]-scintillation proximity assay (SPA) --- p.77 / Chapter 4.3.2.1 --- Introduction --- p.77 / Chapter 4.3.2.2 --- Cell lysis method --- p.77 / Chapter 4.3.2.3 --- cAMP [I25I]-scintillation proximity assay --- p.78 / Chapter 4.4 --- Measurement of phospholipase C activity --- p.78 / Chapter 4.4.1 --- Introduction --- p.78 / Chapter 4.4.1.1 --- Preparation of columns --- p.78 / Chapter 4.4.1.2 --- [3H]-inositol phosphate assay --- p.79 / Chapter 4.4.1.3 --- Measurement of [3H]-inositol phosphate production --- p.79 / Chapter 4.4.1.4 --- Data analysis --- p.80 / Chapter 4.4.2 --- "D-myo-Inositol 1,4,5-trisphosphate (IP3) [3H] assay system" --- p.81 / Chapter 4.4.2.1 --- Introduction --- p.81 / Chapter 4.4.2.2 --- Sample preparation --- p.81 / Chapter 4.4.2.3 --- "D-myo-Inositol 1,4,5-trisphosphate (IP3) [3H] assay system" --- p.82 / Chapter 4.5 --- Measurement of changes in intracellular Ca2+ concentration --- p.82 / Chapter 4.5.1 --- Introduction --- p.82 / Chapter 4.5.2 --- Cell preparation --- p.83 / Chapter 4.5.3 --- Measurement of Fura-2 fluorescence --- p.83 / Chapter 4.6 --- Radioligand binding --- p.84 / Chapter 4.6.1 --- Introduction --- p.84 / Chapter 4.6.2 --- [3H]-iloprost ligand binding --- p.85 / Chapter 4.6.3 --- Data analysis --- p.86 / Chapter 4.7 --- Cytotoxicity test using trypan blue exclusion test --- p.86 / Chapter Chapter 5 --- Multiple coupling capacity of prostacyclin receptors in CHO cells --- p.88 / Chapter 5.1 --- Introduction --- p.88 / Chapter 5.2 --- Method --- p.88 / Chapter 5.3 --- Results and Discussion --- p.89 / Chapter 5.3.1 --- IP agonist log concentration-response curves for [3H]-cAMP and [3H]-inositol phosphate production in mIP-CHO cells --- p.89 / Chapter 5.3.2 --- Effect of varying Gαs cDNA concentration on cicaprost-stimulated [3H]-cAMP and [3H]-inositol phosphate production in mlP-CHO cells --- p.90 / Chapter 5.3.3 --- Effect of varying Gαq cDNA concentration on cicaprost-stimulated [3H]-cAMP and [3H]-inositol phosphate production in mlP-CHO cells --- p.92 / Chapter 5.4 --- Conclusion --- p.95 / Chapter Chapter 6 --- Multiple coupling capacity of prostacyclin receptors in neuroblastoma cells --- p.113 / Chapter 6.1 --- Introduction --- p.113 / Chapter 6.2 --- Method --- p.114 / Chapter 6.3 --- Results and Discussion --- p.114 / Chapter 6.3.1 --- Characterization of prostanoid receptors in SK-N-SH cells --- p.114 / Chapter 6.3.2 --- Property of IP-receptor signaling in SK-N-SH cells --- p.116 / Chapter 6.3.3 --- Effect of Gαq subunits on PLC activation in SK-N-SH cells --- p.117 / Chapter 6.3.4 --- Coupling capacity of IP-receptorin rat/mouse neuroblastoma (NG108-15) cells --- p.119 / Chapter 6.4 --- Conclusion --- p.123 / Chapter Chapter 7 --- Agonist trafficking --- p.133 / Chapter 7.1 --- Introduction --- p.133 / Chapter 7.2 --- Method --- p.134 / Chapter 7.3 --- Results and Discussion --- p.134 / Chapter 7.3.1 --- Simultaneous measurement of AC and PLC activation in metabolically-labelled mIP-CHO cells --- p.134 / Chapter 7.3.1.1 --- Effect of IBMX on PLC activation --- p.135 / Chapter 7.3.1.2 --- Effect of Li+ ion on AC activation --- p.135 / Chapter 7.3.1.3 --- Separation of [3H]-adenine and [3H]-inositol using column chromatography method --- p.136 / Chapter 7.3.2 --- Measurement of AC and PLC activation using different assay systems --- p.137 / Chapter 7.3.2.1 --- cAMP 125I-Scintillation Proximity Assay System --- p.137 / Chapter 7.3.2.2 --- "D-myo-Inositol 1,4,5-trisphosphate (IP3) [3H]-assay system" --- p.138 / Chapter 7.4 --- Conclusion --- p.139 / Chapter Chapter 8 --- Regulation of prostacyclin receptor coupling --- p.147 / Chapter 8.1 --- Introduction --- p.147 / Chapter 8.2 --- Methods --- p.149 / Chapter 8.3 --- Results and Discussion --- p.150 / Chapter 8.3.1 --- Role of protein kinases on IP-receptor activity --- p.150 / Chapter 8.3.2 --- Effect of SQ22536 on IP-receptor activity --- p.155 / Chapter 8.3.3 --- Role of PKA phosphorylation site in mIP-receptor activity --- p.156 / Chapter 8.4 --- Conclusion --- p.157 / Chapter Chapter 9 --- Prostacyclin receptor isoprenylation --- p.171 / Chapter 9.1 --- Introduction --- p.171 / Chapter 9.2 --- Method --- p.172 / Chapter 9.3 --- Results and Discussion --- p.173 / Chapter 9.4 --- Conclusion --- p.175 / Chapter Chapter 10 --- IP/DP chimeric receptors --- p.178 / Chapter 10.1 --- Introduction --- p.178 / Chapter 10.2 --- Method --- p.179 / Chapter 10.3 --- Results and Discussion --- p.180 / Chapter 10.3.1 --- Property of IP/DP chimeric receptors --- p.180 / Chapter 10.3.2 --- "ID1 chimeric receptor mutant receptors (M4, M5, M6)" --- p.182 / Chapter 10.3.3 --- "Mutant mIP-receptors (Ml, M2, M3)" --- p.183 / Chapter 10.3.4 --- Comparison between M5 and ID1 receptors --- p.184 / Chapter 10.4 --- Conclusion --- p.184 / Chapter Chapter 11 --- General Discussion and Conclusions --- p.193 / Chapter 11.1 --- Introduction --- p.193 / Chapter 11.2 --- Multiple coupling capacity of prostacyclin receptors --- p.193 / Chapter 11.3 --- Factors influencing prostacyclin receptor coupling --- p.196 / Chapter 11.4 --- Prostacyclin receptor cross talk and regulation --- p.198 / References --- p.200
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_323639 |
| Date | January 2001 |
| Contributors | Kam, Yiu-wing., Chinese University of Hong Kong Graduate School. Division of Pharmacology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xii, 215 leaves : ill. ; 30 cm. |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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