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In this thesis a detailed study of the kinetics of GnRH-stimulated LH release was made. GnRH stimulated LH release in a biphasic manner. During the first 3 minutes of stimulation, there was a transient spike phase of release followed by plateau phase of lower amplitude. Both phases of release are largely dependent on extracellular Ca²⁺. The spike phase of release is dependent on Ca²⁺ entry via a receptor-operated Ca²⁺ channel (ROCC) (about 90%) and on the mobilization of intracellular Ca²⁺ stores. The role of ROCC were examined by using ruthenium red which inhibits both ROCC and voltage-sensitive Ca²⁺ channels (VSCC). VSCC are not involved in the spike phase of GnRH-stimulated LH release since D600 and nifedipine, inhibitors of VSCC, have no effect on the spike phase. The plateau phase of release is dependent on Ca²⁺ entry via VSCC (about 50%) and ROCC (about 50%). Forskolin, an activator of adenylate cyclase, was used to investigate the role of cAMP in LH release. Forskolin stimulated an increase in both LH release and cellular cAMP levels. GnRH was also able to elevate the cellular CAMP concentration. GnRH interacted synergistically with forskolin to stimulate LH release. The synergism between GnRH and forskolin was not due to an interaction at (1) the GnRH receptor, (2) the level of intracellular Ca²⁺ mobilization, or (3) inositol phosphate metabolism. However, forskolin was able to synergistically interact with secretagogues that increase the cytosolic Ca²⁺ concentration and activators of protein kinase C. This suggested that forskolin was interacting with GnRH at a site distal to the activation of the Ca²⁺ second messenger system and protein kinase C. The data suggest that the initial response to GnRH is largely Ca²⁺-dependent and that other second messengers, if active, play a minor role. cAMP is thought to play a modulatory role and may be involved in the maintenance of secretion.

Kwok, Po Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 124-130). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.i / Abstract --- p.ii / 中文摘要 --- p.v / Acknowledgements --- p.vii / List of Figures --- p.viii / List of Tables --- p.x / Table of Abbreviations --- p.xii / Contents --- p.xix / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- The Biology of Human Umbilical Cord Perivascular Cells (HUCPVs) and Their Potential Applications in Tissue Regeneration / Chapter 2.1 --- INTRODUCTION --- p.5 / Chapter 2.1.1 --- Stem cells --- p.5 / Chapter 2.1.2.1 --- Embryonic stem cells --- p.6 / Chapter 2.1.2.2 --- iPS cells --- p.7 / Chapter 2.1.2.3 --- Somatic stem cells --- p.8 / Chapter 2.1.3 --- Mesenchymal stem cells --- p.9 / Chapter 2.1.4 --- Pericytes --- p.11 / Chapter 2.1.5 --- CD146 positive MSCs --- p.12 / Chapter 2.1.6 --- Human umbilical cord perivascular cells (HUCPVs) --- p.13 / Chapter 2.1.7 --- The biology of stem cell microenvironment (niche) --- p.14 / Chapter 2.1.8 --- Current applications of HUCPVs --- p.17 / Chapter 2.1.9 --- Regenerative medicine --- p.17 / Chapter 2.1.10 --- Applications of stem cells in bone regeneration --- p.19 / Chapter 2.2 --- MATERIALS AND METHODS --- p.22 / Chapter 2.2.1 --- Cell culture --- p.22 / Chapter 2.2.2 --- Preparation of Human Umbilical Cord Perivascular (HUCPV) cells --- p.22 / Chapter 2.2.2.1 --- Isolation of Human Umbilical Cord Perivascular (HUCPV) cells from human umbilical cord --- p.22 / Chapter 2.2.2.2 --- Purification of HUCPV cells --- p.23 / Chapter 2.2.3 --- Immunocytochemsitry --- p.24 / Chapter 2.2.4 --- Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) --- p.25 / Chapter 2.2.4.1 --- Isolation of total cellular RNA --- p.25 / Chapter 2.2.4.2 --- Complementary DNA (cDNA) synthesis --- p.26 / Chapter 2.2.4.3 --- Polymerase chain reaction (PCR) --- p.26 / Chapter 2.2.5 --- Quantitative real-time reverse transcriptionpolymerase chain reaction (qRT-PCR) --- p.30 / Chapter 2.2.6 --- In vitro differentiation assays --- p.33 / Chapter 2.2.6.1 --- Osteogenic differentiation --- p.33 / Chapter 2.2.6.2 --- Adipogenic differentiation --- p.33 / Chapter 2.2.6.3 --- Chondrogenic differentiation --- p.34 / Chapter 2.2.6.4 --- In vitro chondrogenic differentiation on gelfoam® --- p.34 / Chapter 2.2.7 --- Cytochemistry staining --- p.35 / Chapter 2.2.7.1 --- Alkaline Phosphatase staining --- p.35 / Chapter 2.2.7.2 --- Alizarin Red S staining --- p.35 / Chapter 2.2.7.3 --- Oil Red O staining --- p.36 / Chapter 2.2.7.4 --- Alcian Blue staining --- p.36 / Chapter 2.2.8 --- Scanning electron microscopy (SEM) --- p.37 / Chapter 2.2.9 --- Transmission electron microscopy (TEM) --- p.37 / Chapter 2.2.10 --- Paraffin tissue embedding --- p.38 / Chapter 2.2.10 --- Haematoxylin and Eosin staining --- p.38 / Chapter 2.3 --- RESULTS --- p.40 / Chapter 2.3.1 --- Isolation and purification of HUCPVs --- p.40 / Chapter 2.3.2 --- Osteogenic differentiation of HUCPVs under normoxia --- p.41 / Chapter 2.3.3 --- Osteogenic differentiation of HUCPVs under hypoxia --- p.42 / Chapter 2.3.4 --- Adipogenic differentiation of HUCPVs --- p.43 / Chapter 2.3.5 --- Chondrogenic differentiation of HUCPVs --- p.43 / Chapter 2.3.6 --- Chondrogenic differentiation of HUCPVs on gelfoam® --- p.44 / Chapter 2.4 --- DISCUSSION --- p.59 / Chapter Chapter 3 --- Calcium and Calcium-sensing Receptor (CaSR) in osteogenesis / Chapter 3.1 --- INTRODUCTION --- p.62 / Chapter 3.1.1 --- Metabolism of calcium --- p.62 / Chapter 3.1.2 --- Calcium-sensing receptor --- p.64 / Chapter 3.1.2.1 --- The molecular structure of calcium-sensing Receptor (CaSR) --- p.64 / Chapter 3.1.2.2 --- The expression pattern of calciumsensing receptor (CaSR) --- p.67 / Chapter 3.1.2.3 --- The physiological function of calcium-sensing receptor in different tissues or organs --- p.68 / Chapter 3.1.2.4 --- Regulatory role of calcium-sensing receptor in calcium sensing and homeostasis --- p.71 / Chapter 3.1.2.5 --- The role of calcium-sensing receptor in diseases --- p.72 / Chapter 3.1.2.6 --- Genetic animal models targeting calciumsensing receptor --- p.73 / Chapter 3.1.2.7 --- Calcium-sensing receptor in mesenchymal lineage Differentiation --- p.76 / Chapter 3.1.2.8 --- The role of calcium-sensing receptor in the skeleton --- p.76 / Chapter 3.1.3 --- Calcium-sensing receptor related pathway --- p.78 / Chapter 3.1.3.1 --- Cyclic AMP pathway --- p.78 / Chapter 3.1.3.2 --- Cyclic AMP response element-binding protein (CREB) --- p.80 / Chapter 3.2 --- MATERIALS AND METHODS --- p.83 / Chapter 3.2.1 --- Preparation of primary mouse osteoblasts (MOB) from long bone --- p.83 / Chapter 3.2.2 --- Preparation of primary mouse osteoblasts (CMOB) from calvaria --- p.84 / Chapter 3.2.3 --- Immunocytochemistry --- p.84 / Chapter 3.2.4 --- Osteogenic differentiation --- p.85 / Chapter 3.2.3 --- Quantitative real-time reverse transcriptionpolymerase chain reaction (qRT-PCR) --- p.85 / Chapter 3.2.4 --- Cell proliferation measurement by BrdU ELISA (colorimetric) assay --- p.85 / Chapter 3.2.5 --- Western blotting analysis --- p.86 / Chapter 3.2.5.1 --- Preparation of the protein lysate --- p.86 / Chapter 3.2.5.2 --- Protein quantitation --- p.86 / Chapter 3.2.5.3 --- SDS-PAGE --- p.87 / Chapter 3.2.5.4 --- Protein transfer --- p.87 / Chapter 3.2.5.5 --- Immunodetection --- p.88 / Chapter 3.2.6 --- cAMP EIA assay --- p.89 / Chapter 3.3 --- RESULTS --- p.91 / Chapter 3.3.1 --- "Expression of CD 146 and CaSR in HUCPVs, primary mouse long bone osteoblasts and MC3T3-E1 cell line" --- p.91 / Chapter 3.3.2 --- The effect of calcium treatment on the osteogenic differentiation potential of MC3T3-E1 cells under normoxia --- p.91 / Chapter 3.3.3 --- The effect of calcium treatment on the osteogenic differentiation potential of MC3T3-E1 cells under hypoxia --- p.92 / Chapter 3.3.4 --- The effect of calcium treatment on cell proliferation in primary mouse long bone osteoblasts --- p.93 / Chapter 3.3.5 --- The effect of calcium treatment on calcium-sensing receptor expression in primary mouse long bone osteoblasts --- p.94 / Chapter 3.3.6 --- The effect of calcium treatment on calcium-sensing receptor expression in HUCPVs --- p.95 / Chapter 3.3.7 --- The effect of calcium treatment on calcium-sensing receptor expression in primary mouse calvarian osteoblasts --- p.96 / Chapter 3.3.8 --- The effect of calcium treatment on cyclic AMP levels in primary mouse long bone osteoblasts --- p.97 / Chapter 3.4 --- DISCUSSION --- p.117 / Chapter Chapter 4 --- General Discussions --- p.121 / References --- p.124 / Appendices --- p.131