Chu Kit Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 142-168). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgement --- p.iv / Abbreviations --- p.v / Publications Based on Work in this thesis --- p.viii / 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 --- Heterotrimeric G proteins --- p.3 / Chapter 1.1.3 --- Second messenger systems --- p.4 / Chapter 1.1.4 --- Mechanism of GPCR activation --- p.6 / Chapter 1.2 --- Prostacyclin and its receptors --- p.9 / Chapter 1.2.1 --- General properties of prostacyclin --- p.9 / Chapter 1.2.1.1 --- Synthesis of prostacyclin --- p.9 / Chapter 1.2.1.2 --- Prostacyclin analogues --- p.10 / Chapter 1.2.2 --- Characterization of IP-receptors --- p.12 / Chapter 1.2.2.1 --- Distribution of IP-receptors --- p.12 / Chapter 1.2.2.2 --- Cloning of IP-receptors --- p.14 / Chapter 1.2.2.3 --- Structure of IP-receptors --- p.15 / Chapter 1.2.3 --- Coupling of IP-receptors to G proteins --- p.16 / Chapter 1.2.3.1 --- Interaction with Gs --- p.16 / Chapter 1.2.3.2 --- Interaction with Gq --- p.17 / Chapter 1.2.3.3 --- Interaction with Gi --- p.18 / Chapter 1.2.3.4 --- Interaction with PPARs --- p.20 / Chapter 1.2.4 --- Role of prostacyclin in mitogenesis/anti-mitogenesis --- p.20 / Chapter 1.3 --- Signal transduction network of MAPK family --- p.27 / Chapter 1.3.1 --- MAPK modules in mammalian cells --- p.29 / Chapter 1.3.1.1 --- Extracellular regulated kinase (ERK) cascade --- p.30 / Chapter 1.3.1.2 --- Stress-activated protein kinase (JNK and p38) cascades --- p.33 / Chapter 1.3.2 --- Activation ofERKl/2 through GPCRs --- p.35 / Chapter Chapter 2 --- Materials and solutions --- p.53 / Chapter 2.1 --- Materials --- p.53 / Chapter 2.2 --- "Culture media, buffer and solutions" --- p.58 / Chapter 2.2.1 --- Culture media --- p.58 / Chapter 2.2.2 --- Buffers --- p.59 / Chapter 2.2.3 --- Solutions --- p.62 / Chapter Chapter 3 --- Methods --- p.65 / Chapter 3.1 --- Maintenance of cell lines --- p.65 / Chapter 3.1.1 --- Chinese Hamster ovary (CHO) cells --- p.65 / Chapter 3.1.2 --- Human neuroblastoma (SK-N-SH) cells --- p.66 / Chapter 3.1.3 --- Rat/mouse neuroblastoma/glioma hybrid (NG108-15) cells --- p.66 / Chapter 3.2 --- Transient transfection of mammalian cells --- p.67 / Chapter 3.3 --- Measurement of ERK activity --- p.68 / Chapter 3.3.1 --- PathDetect® Elkl trans-Reporting System --- p.68 / Chapter 3.3.1.1 --- Introduction --- p.68 / Chapter 3.3.1.2 --- β-galactosidase assay --- p.72 / Chapter 3.3.1.3 --- Transient transfection of cells --- p.72 / Chapter 3.3.1.4 --- Cell assay --- p.73 / Chapter 3.3.1.5 --- Luciferase assay --- p.74 / Chapter 3.3.1.6 --- Micro β-gal assay --- p.74 / Chapter 3.3.1.7 --- Data analysis --- p.75 / Chapter 3.3.2 --- Western Blotting --- p.79 / Chapter 3.3.2.1 --- Introduction --- p.79 / Chapter 3.3.2.2 --- Transient transfection of cells --- p.79 / Chapter 3.3.2.3 --- Cell assay --- p.79 / Chapter 3.3.2.4 --- Protein electrophoresis and transfer --- p.80 / Chapter 3.3.2.5 --- Immunodetection --- p.80 / Chapter 3.4.1 --- Measurement of adenylyl cyclase activity --- p.83 / Chapter 3.4.1 --- wyo-[3H]-inositol labelling method --- p.83 / Chapter 3.4.1.1 --- Preparation of columns --- p.83 / Chapter 3.4.1.2 --- Incubation of cells --- p.84 / Chapter 3.4.1.3 --- Measurement of [3H]-cyclic AMP production --- p.84 / Chapter 3.4.1.4 --- Data analysis --- p.85 / Chapter 3.5 --- Measurement of phospholipase C activity --- p.85 / Chapter 3.5.1 --- wyo-[3H]-inositol labelling method --- p.85 / Chapter 3.5.1.1 --- Preparation of columns --- p.86 / Chapter 3.5.1.2 --- Incubation of cells --- p.86 / Chapter 3.5.1.3 --- Measurement of [3H]-inositol phosphate production --- p.87 / Chapter 3.5.1.4 --- Data analysis --- p.88 / Chapter Chapter 4 --- Results --- p.89 / Chapter 4.1 --- Validation of PathDetect® Elkl Trans-Reporting System --- p.89 / Chapter 4.1.1 --- Introduction --- p.89 / Chapter 4.1.2 --- Internal control --- p.89 / Chapter 4.1.3 --- Response to cicaprost and ATP --- p.91 / Chapter 4.1.4 --- Normalisation of ERK1/2 activity with transfection efficiency --- p.92 / Chapter 4.1.5 --- Cicaprost response in CHO cells in the absence of mIP- receptor --- p.93 / Chapter 4.1.6 --- Normalised luciferase activity reflecting ERK1/2 activation --- p.93 / Chapter 4.1.7 --- Conclusion --- p.95 / Chapter 4.2 --- Characterization of IP-receptors --- p.101 / Chapter 4.2.1 --- IP-receptor activation of adenylyl cyclase and phospholipase C --- p.101 / Chapter 4.2.2 --- IP-receptor activation ofERKl/2 in mIP-CHO cells --- p.102 / Chapter 4.2.2.1 --- PathDetect System --- p.102 / Chapter 4.2.2.2 --- Western Blotting --- p.103 / Chapter 4.2.2.3 --- Conclusion --- p.104 / Chapter 4.2.3 --- Role of the Gs-mediated pathway in cicaprost-stimulated ERK1/2 activation --- p.104 / Chapter 4.2.3.1 --- Role of cyclic AMP --- p.105 / Chapter 4.2.3.2 --- Role of protein kinase A --- p.106 / Chapter 4.2.4 --- Role of the Gq-mediated pathway in cicaprost-stimulated ERK1/2 activation --- p.106 / Chapter 4.2.4.1 --- Role of IP3 --- p.107 / Chapter 4.2.4.2 --- Role of protein kinase C --- p.108 / Chapter 4.2.4.3 --- Conclusion --- p.108 / Chapter 4.2.5 --- IP-receptor activation of ERKl/2 in hIP-CHO cells --- p.109 / Chapter 4.2.5.1 --- Activation ofERKl/2 in hIP-CHO cells --- p.109 / Chapter 4.2.5.2 --- Role of the Gq-mediated pathway in cicaprost- stimulated ERK 1/2 activation --- p.110 / Chapter 4.2.5.3 --- Role of the Gs-mediated pathway in cicaprost- stimulated ERK 1/2 activation --- p.111 / Chapter 4.2.5.4 --- Conclusions --- p.113 / Chapter 4.2.6 --- IP-receptor activation of ERX1/2 in neuroblastoma cells --- p.114 / Chapter 4.2.6.1 --- Rat/mouse neuroblastoma/glioma (NG108-15) cells --- p.114 / Chapter 4.2.6.2 --- Human neuroblastoma (SK-N-SH) cells --- p.115 / Chapter Chapter 5 --- General Discussion and Conclusions --- p.137 / References --- p.142

Background: Regulation of vascular tone is complex. Various complementary signaling pathways causing contraction and relaxation of vascular smooth muscle take place to ensure proper blood flow within the vasculature. Mitogen activated protein kinase (MAPK) signaling cascade is observed to be one of the many signaling pathways that regulate vascular tone. Aim: This study examines the role of the following MAPK: mitogen-activated extracellular-regulated protein kinase kinase (MEK), extracellular signal-regulated kinase (ERK), and p38 MAPK in the regulation of relaxation in the endothelium and smooth muscle. Method: Isometric tension of isolated porcine coronary artery rings were measured with organ chamber setup. The effects of MEK inhibitor, PD98059 (30 μM), ERK inhibitor, U0126 (10 μM) and p38 MAPK inhibitor, SB203580 (10 μM), on relaxations induced by bradykinin (a vasodilating peptide), SKA-31 [an activator of small and intermediate conductance calcium-activated potassium channels (SKCa and IKCa,, respectively)], Deta NONOate (a nitric oxide donor) and forskolin (an adenylate cyclase activator) were examined in arteries with and without endothelium, contracted with an thromboxane A2 analog, U46619 (300 nM – 1 μM). In some experiments, rings were also incubated with the following pharmacological inhibitors, indomethacin (cyclooxygenase inhibitor, 10 μM), L-NAME (nitric oxide synthase inhibitor, 300 μM), TRAM34 (IKCa blocker, 1 μM), and UCL1684 (SKCa blocker, 1 μM), alone or in combination. Results: 1. Bradykinin-induced relaxation was potentiated by MEK and ERK inhibition but not by p38 MAPK inhibition. 2. SKA-31-induced relaxation was potentiated by MEK and p38 MAPK inhibition but not by ERK inhibition. 3. Deta NONOate-induced relaxation was potentiated by MEK, p38 MAPK inhibition, but not by ERK inhibition except in the presence of indomethacin, TRAM-34 plus UCL1684. 4. Forskolin-induced relaxation was potentiated by MEK and p38 MAPK inhibition, but not by ERK inhibition. Discussion: MAPK plays a role in regulating the vascular tone in both the endothelium and smooth muscle of porcine coronary arteries. MEK appears to have an inhibitory action on relaxation that is downstream of the generation of endothelium-derived nitric oxide, activation of IKCa and SKCa and activation of adenylate cyclase. ERK are unlikely to be the downstream target of MEK for inhibiting relaxation, in view of the lack of effects of its inhibitor on endothelium-derived hyperpolarizing factor (EDHF)-mediated and endothelium-independent relaxations. The involvement of ERK in relaxation pathways in the endothelium appears to be complicated, since U0126 caused opposing effects (inhibition and potentiation) on bradykinin-induced relaxation in the presence of indomethacin without and with L-NAME or TRAM-34 plus UCL1684. As inhibition of p38 MAPK results in potentiation of relaxations to all relaxing agents tested except bradykinin, this MAPK may have opposing action in the endothelium and smooth muscle; endothelial p38 MAPK may facilitate relaxation while smooth muscle p38 MAPK attenuates it. In conclusion, this study provided additional information on the influences of MEK, ERK and p38 MAPK on relaxation; this knowledge may contribute to the understanding of the mechanisms underlying the development of vascular disorders. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Medical Sciences

Thesis (Ph. D.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on June 19, 2009) Includes bibliographical references.

No description available.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Master / Master of Philosophy

No description available.

No description available.

Thesis (M.S. in Cancer Biology)--Vanderbilt University, May 2007. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 130-153).