Siu-Yin Sylvia Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 123-140). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iv / 英中譯名對照 --- p.vi / Acknowledgements --- p.vii / Table of Contents --- p.viii / Abbreviations --- p.xiii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Overview of Carotid Body --- p.1 / Chapter 1.1.1 --- Type I Cells --- p.3 / Chapter 1.1.2 --- Type II Cells --- p.4 / Chapter 1.1.3 --- Blood Vessels --- p.5 / Chapter 1.1.4 --- Innervation --- p.5 / Chapter 1.1.5 --- Biochemistry --- p.6 / Chapter 1.1.6 --- Physiology and Function --- p.7 / Chapter 1.2 --- The Renin-Angiotensin System (RAS) --- p.8 / Chapter 1.2.1 --- Circulating RAS --- p.8 / Chapter 1.2.1.1 --- Angiotensinogen --- p.10 / Chapter 1.2.1.2 --- Renin --- p.10 / Chapter 1.2.1.3 --- Angiotensin I --- p.11 / Chapter 1.2.1.4 --- Angiotensin Converting Enzyme --- p.12 / Chapter 1.2.1.5 --- Angiotensin II --- p.12 / Chapter 1.2.1.6 --- Angiotensin II Receptors --- p.13 / Chapter 1.2.1.7 --- Angiotensin IV and Angiotensin IV Receptor --- p.15 / Chapter 1.2.2 --- Tissue RAS --- p.16 / Chapter 1.3 --- Hypoxia and Carotid Body --- p.18 / Chapter 1.4 --- Hypoxia and RAS --- p.21 / Chapter 1.5 --- Hypoxia and RAS in Carotid Body --- p.23 / Chapter 1.6 --- Aims of Study --- p.24 / Chapter 1.6.1 --- Existence of Functional Angiotensin II Receptors --- p.24 / Chapter 1.6.2 --- Regulation and Function of Angiotensin II Receptors by Chronic Hypoxia --- p.24 / Chapter 1.6.3 --- Existence of an Intrinsic Angiotensin-generating System --- p.25 / Chapter 1.6.4 --- Regulation of Local RAS by Chronic Hypoxia --- p.25 / Chapter 1.6.5 --- Studies of AT4 Receptor --- p.26 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Experimental Animals and Rat Models --- p.27 / Chapter 2.1.1 --- Rat Model of Chronic Hypoxia --- p.27 / Chapter 2.1.2 --- Isolation of Carotid Body --- p.28 / Chapter 2.2 --- Semi-quantitative Reverse Transcriptase-polymerase Chain Reaction (RT-PCR) --- p.30 / Chapter 2.2.1 --- Total RNA Extraction and Quantification --- p.30 / Chapter 2.2.2 --- Reverse Transcription (RT) --- p.31 / Chapter 2.2.3 --- Polymerase Chain Reaction (PCR) --- p.31 / Chapter 2.2.4 --- Gel Electrophoresis --- p.34 / Chapter 2.2.5 --- Optimization of Semi-quantitative RT-PCR for RAS Gene Analysis --- p.34 / Chapter 2.3 --- Northern Blotting --- p.35 / Chapter 2.3.1 --- Transfer of Denatured RNA to Nitrocellulose Membrane By Capillary Elution --- p.35 / Chapter 2.3.2 --- Hybridization --- p.36 / Chapter 2.4 --- In-situ Hybridization --- p.38 / Chapter 2.4.1 --- Linearization of Angiotensinogen cDNA --- p.38 / Chapter 2.4.2 --- Riboprobe Preparation --- p.38 / Chapter 2.4.3 --- Quantification and Gel Electrophoresis of Riboprobes --- p.39 / Chapter 2.4.4 --- In-situ Hybridization Histochemistry --- p.39 / Chapter 2.5 --- Immunohistochemistry --- p.42 / Chapter 2.5.1 --- Preparation of Cryosection --- p.42 / Chapter 2.5.2 --- Indirect Immunoperoxidase Staining --- p.42 / Chapter 2.5.3 --- Immunofluorescent Double Staining --- p.43 / Chapter 2.6 --- Western Blot Analysis --- p.45 / Chapter 2.6.1 --- Preparation of Angiotensinogen Protein --- p.45 / Chapter 2.6.2 --- Quantification of Protein Concentration --- p.45 / Chapter 2.6.3 --- Sample Preparation --- p.45 / Chapter 2.6.4 --- Sodium Dodecyl-sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.46 / Chapter 2.6.5 --- Electroblotting and Immunodetection of Proteins --- p.46 / Chapter 2.7 --- Spectrofluorimetric Measurement and In-vitro Electrophysiology --- p.48 / Chapter 2.7.1 --- Dissociation of Carotid Body Type I Cells and Spectrofluorimetric Measurement --- p.48 / Chapter 2.7.2 --- In-vitro Electrophysiology --- p.49 / Chapter 2.8 --- Assay of ACE Activity --- p.51 / Chapter 2.8.1 --- Crude Membrane Preparation --- p.51 / Chapter 2.8.2 --- Basic Principle for ACE Activity Measurement --- p.51 / Chapter 2.8.3 --- Measurement of ACE Activity --- p.51 / Chapter 2.8.4 --- Fluorescence Measurement --- p.53 / Chapter 2.9 --- In-vitro Autoradiography and Fluorescence-labeled Binding Assay for Angiotensin IV --- p.54 / Chapter 2.9.1 --- Preparation of Frozen Tissue Sections --- p.54 / Chapter 2.9.2 --- Localization and Density of AT4 Receptor --- p.54 / Chapter 2.10 --- Statistics and Data Analysis --- p.57 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Functional Expression of Angiotensin II Receptors --- p.58 / Chapter 3.1.1 --- [Ca2+]i Response to Angiotensin II --- p.58 / Chapter 3.1.2 --- Antagonistic Blockade of Angiotensin II Receptor Subtypes --- p.58 / Chapter 3.1.3 --- Expression of AT1 Receptors mRNA --- p.61 / Chapter 3.1.4 --- Cellular Localization of AT1 Receptors Protein --- p.61 / Chapter 3.2 --- Effect of Chronic Hypoxia on the Expression and Function of Angiotensin II Receptors --- p.64 / Chapter 3.2.1 --- Effect of Chronic Hypoxia on the Expression of AT1 Receptors --- p.64 / Chapter 3.2.2 --- Effect of Chronic Hypoxia on the Expression of AT2 Receptors --- p.67 / Chapter 3.2.3 --- Cellular Localization of the AT1 Receptor by Chronic Hypoxia --- p.69 / Chapter 3.2.4 --- Increase of Afferent Nerve Activities of the Carotid Body In-vitro by Angiotensin II --- p.71 / Chapter 3.2.5 --- Inhibition of Angiotensin II-mediated Response in Chronically Hypoxic Carotid Body by Losartan --- p.73 / Chapter 3.3 --- Evidence for the Existence of an Intrinsic Angiotensin-generating System --- p.75 / Chapter 3.3.1 --- Expression and Localization of Angiotensinogen mRNA --- p.75 / Chapter 3.3.2 --- Expression and Localization of Angiotensinogen Protein --- p.78 / Chapter 3.3.3 --- Expression of Renin mRNA --- p.81 / Chapter 3.3.4 --- Expression of ACE mRNA --- p.81 / Chapter 3.4 --- Effect of Chronic Hypoxia on the Locally-generated Angiotensin System --- p.85 / Chapter 3.4.1 --- Effect of Chronic Hypoxia on the Expression of Angiotensinogen mRNA --- p.85 / Chapter 3.4.2 --- Effect of Chronic Hypoxia on the Localization of Angiotensinogen mRNA --- p.87 / Chapter 3.4.3 --- Effect of Chronic Hypoxia on the Expression of Angiotensinogen Protein --- p.89 / Chapter 3.4.4 --- Effect of Chronic Hypoxia on the Expression of ACE --- p.91 / Chapter 3.5 --- Time-course Effect of Chronic Hypoxia on ACE Activity --- p.93 / Chapter 3.6 --- Preliminary Studies of AT4 Receptor --- p.98 / Chapter 3.6.1 --- In-vitro Autoradiographic Study of AT4 Receptors --- p.98 / Chapter 3.6.2 --- Localization of AT4 Receptors --- p.100 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Functional Expression of Angiotensin II Receptors --- p.102 / Chapter 4.2 --- Upregulation and Function of Angiotensin II Receptors --- p.105 / Chapter 4.3 --- Existence of a Local RAS --- p.108 / Chapter 4.4 --- Regulation of the Local RAS --- p.112 / Chapter 4.5 --- Time-dependent Changes of ACE Activity --- p.155 / Chapter 4.6 --- Presence and Regulation of AT4 Receptor --- p.117 / Chapter 4.7 --- Conclusion --- p.120 / Chapter 4.8 --- Future Works --- p.121 / Chapter Chapter 5 --- References --- p.123