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Anita K.L. Yiu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 93-112). / Acknowledgement --- p.i / List of Abbreviations --- p.ii / Abstract --- p.iv / Table of Contents --- p.vi / Chapter CHAPTER 1. --- Introduction / Chapter 1.1 --- The Renin-Angiotensin System (RAS) --- p.1 / Chapter 1.2 --- Physiology and Pathophysiology of Angiotensin --- p.3 / Chapter 1.3 --- Angiotensin Receptors / Chapter 1.3.1 --- Heterogeneity among Angiotensin Receptors --- p.10 / Chapter 1.3.2 --- Differential Distribution of Subtypes --- p.13 / Chapter 1.3.3 --- Molecular Structure of Subtypes --- p.15 / Chapter 1.3.4 --- Signal Transduction Mechanism --- p.20 / Chapter 1.3.5 --- Physiological Functional Correlates --- p.21 / Chapter 1.4 --- Aim of Study --- p.23 / Chapter CHAPTER 2. --- Preparation of Polyclonal Antibodies Against Angiotensin Receptors / Chapter 2.1 --- Introduction --- p.25 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Preparation of antisera / Chapter 2.2.1.1 --- Preparation of peptide conjugates --- p.25 / Chapter 2.2.1.2 --- Protein determination --- p.27 / Chapter 2.2.1.3 --- Immunization of rabbits with peptide conjugates --- p.27 / Chapter 2.2.1.4 --- Collection of rabbit sera --- p.28 / Chapter 2.2.1.5 --- Affinity purification of antisera --- p.28 / Chapter 2.2.2 --- Enzyme-linked immunosorbent assay (ELISA) / Chapter 2.2.2.1 --- Titer determination --- p.29 / Chapter 2.2.2.2 --- Specificity determination --- p.30 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Preparation of antisera --- p.30 / Chapter 2.3.2 --- Affinity purification of antisera --- p.30 / Chapter 2.3.3 --- ELISA / Chapter 2.3.3.1 --- Titer determination --- p.31 / Chapter 2.3.3.1.1 --- Thy-AT1 antiserum --- p.31 / Chapter 2.3.3.1.2 --- Thy-AT2 antiserum --- p.32 / Chapter 2.3.3.2 --- Specificity determination --- p.32 / Chapter 2.3.3.2.1 --- Thy-AT1 antibodies --- p.32 / Chapter 2.3.3.2.2 --- Thy-AT2 antibodies --- p.49 / Chapter 2.4 --- Discussions --- p.49 / Chapter CHAPTER 3. --- Application of Thy-AT1 Antiserumin Western Blot / Chapter 3.1 --- Introduction --- p.52 / Chapter 3.2 --- Methods / Chapter 3.2.1 --- Preparation of protein samples --- p.52 / Chapter 3.2.2 --- Protein determination --- p.53 / Chapter 3.2.3 --- SDS-PAGE --- p.53 / Chapter 3.2.3 --- Western blot --- p.54 / Chapter 3.2.5 --- Immunoblotting --- p.54 / Chapter 3.3 --- Results --- p.55 / Chapter 3.4 --- Discussions --- p.58 / Chapter CHAPTER 4. --- Evaluation of Pancreatic Response to Angiotensin II / Chapter 4.1 --- Introduction --- p.61 / Chapter 4.2 --- Methods / Chapter 4.2.1 --- Perfusion of pancreas --- p.62 / Chapter 4.2.2 --- Assay of amylase activity --- p.64 / Chapter 4.2.3. --- Calculations --- p.64 / Chapter 4.3 --- Results --- p.65 / Chapter 4.4 --- Discussions --- p.65 / Chapter CHAPTER 5. --- Application of Purified Thy-AT2 Antibodies in immunohistochemical studies / Chapter 5.1 --- Introduction --- p.74 / Chapter 5.2 --- Methods / Chapter 5.2.1 --- Preparation of adrenal sections --- p.75 / Chapter 5.2.2 --- Light-microscopic immunohistochemical study --- p.76 / Chapter 5.3 --- Results / Chapter 5.4 --- Discussions / Chapter CHAPTER 6. --- General Discussions --- p.84 / References --- p.93 / Appendix / Chapter A. --- Materials --- p.113 / Chapter B. --- Buffer Compositions --- p.121

Abstract not available

Long-term stimuli of many systems leads to decreased cellular responsiveness, or desensitization. We characterized the desensitization of angiotensin II (Ang 11)-mediated inositol phospholipid (IP) hydrolysis in cultured human aortic smooth muscle cells (HASMC). Although it has been suggested that the desensitization induced by long-term Mg II exposure may result partially from down-regulation of Ang II receptor, this is not sufficient to explain fully desensitization in many systems. Post-receptor desensitization of IP hydrolysis may also result from phosphorylation or changes in protein levels of the effector enzyme, PLC-β. We identified the major PLC-β isoenzymes expressed by HASMC as PLC-β1 and PLC-β3. Ang II pretreatment reduced IP accumulation induced by Ang II (1μM) in a time-dependent manner. Phorbol ester-12-myristrate-13-acetate (PMA), a protein kinase C (PKC) activator, also reduced Ang II-stimulated IP accumulation. These results suggest that PKC activation may negatively regulate Ang II-stimulated IP signaling in HASMC, similar to rat cells. In addition, PKC also reduced IP accumulation stimulated by A1F₄⁻, directly activating the G protein. It suggests that the majority of PKC-induced desensitization of Ang II-stimulated IP signaling occurs downstream of the Ang II receptor in HASMC. However, both PLC-β1 and PLC-β3, expected candidates for PKC phosphorylation, were phosphorylated independently of PKC activation or inhibition, indicating that PKC might not be involved in direct phosphorylation of PLC-β1 and PLC-β3. Furthermore, PLC-β1, but not PLC-β3, was highly phosphorylated under basal conditions, suggesting that PLC-β1 and PLC-β3 may play different roles in IP signaling in HASMC. / Graduation date: 2003

The renin-angiotensin system (RAS) has been implicated in vascular inflammation and atherosclerosis. Angiotensin II via the ATR1 can activate monocytes to produce inflammatory factors and increase adhesion. ATR1 expression is partly regulated by alternate splicing of the ATR1 gene. The RAS may also regulate immune function as part of the stress response: a model is proposed. ATR1 expression in two monocyte cell lines (U937 and THP-1) compared to a human microvascular endothelial cell line (HMEC1) was investigated. Western blot showed ATR1 protein expression in all cell types. PCR protocols targeted to the terminal protein-coding exon common to all transcript variants confirmed mRNA expression of the ATR1 gene in EC and monocytes. The 5 known splice variants were not identified in monocytes. 5-RLM RACE was used to identify the 5 untranslated ATR1 exons in monocytes. These data suggest a novel monocyte-specific splice variant, which may function in the cardiovascular disease process.

Angiotensin-converting enzyme 2 (ACE2) is an important component of the renin-angiotensin system (RAS). ACE2 is thought to have a renoprotective effect in diabetic nephropathy because it is capable of degrading profibrotic angiotensin II to potentially protective angiotensin-(1-7). Podocyte death and detachment is a key component of diabetic nephropathy. ACE2 is localized in the podocyte and during a diabetic state, podocyte ACE2 expression is reduced. The purpose of this study was to determine the effects of podocyte-specific ACE2 overexpression on the course of diabetic nephropathy. Diabetes was induced using streptozotocin in transgenic mice with podocyte-specific overexpression of human ACE2. The following parameters were assessed: systolic blood pressure, glomerular filtration rate, urinary albumin excretion, mesangial and glomerular area, and podocyte number. Transgenic diabetic mice showed a significant transient attenuated increase in albuminuria, an attenuated increase in mesangial area, decreased glomerular area, and preserved podocyte number, compared to wildtype diabetic mice. This was independent of a change in blood pressure. This study showed that the podocyte-specific overexpression of human ACE2 attenuates the development of diabetic nephropathy.

During a single bout of aerobic exercise, blood flow is shunted to exercising tissues while blood flow is maintained or reduced to less metabolically active areas (i.e. splanchnic area and non-working muscles). Although increased sympathetic activation and multiple metabolic factors participate in redistributing blood flow during aerobic exercise, the precise mechanism is not entirely known. The renin-angiotensin system (RAS), specifically the local vascular wall RAS, has been hypothesized to participate in the redistribution of blood flow during exercise. This study aimed to investigate whether vascular wall RAS in the femoral arteries (an artery which feeds active tissues during exercise) was altered by acute exercise, and if these vascular RAS alterations led to specific changes in vasomotor function. Male Sprague Dawley rats were exercised on a motorized treadmill for 1h at 21m/min with 15% grade. Immediately following exercise femoral arteries were excised, cleaned of surrounding connective tissue, and vascular RAS was evaluated. There was a decrease in femoral ACE activity (~40%) and expression (~20%) following a single bout of exercise. No change was observed in AT1 and AT2 receptor expression. To evaluate the effect of acute exercise and vascular RAS on vessel reactivity, vasomotor properties of the femoral arteries were assessed via vasoconstrictor and vasodilatory dose-response curves. No changes were observed in femoral artery responses to potassium chloride (KCl), signifying that electromechanical coupling was not affected by exercise or RAS pharmacological interventions. However, a significant decrease in maximum phenylephrine (PE) constriction was observed for acutely exercise animals (~13%). Paired with the observed maintenance KCl-mediated constriction, it appears an acute bout of exercise is able to attenuate α-adrenergic receptor-mediated vasoconstriction in the femoral artery. The decrease in maximum α-adrenergic vasoconstriction may be attributed to vascular RAS. The decrease in ACE activity supports the production of local vasodilating factors. Blocking AT1 receptors with telmisartan decreased PE constriction in control and exercised animals. Combining AT1 and AT2 receptor blockade (with PD123319) eliminated the attenuating effect of telmisartan alone on PE constriction. This data suggests that the attenuating effect of AT1 receptor blockade, on PE constriction, may depend on AT2 receptor activation. In addition, combined AT1 receptor blockade and nitric oxide synthase inhibition eliminated both the lone AT1 receptor blockade effect and exercise effect on PE constriction. Together, this data suggests that reduced PE constriction following acute exercise, and AT1 receptor blockade, is dependent on nitric oxide production. Vasodilation to the nitric oxide donor sodium nitroprusside (SNP) was not altered following exercise or RAS pharmacological intervention, signifying no change in signaling downstream of NO production/release. Endothelium-dependent vasodilation to acetylcholine (ACh) was not affected by acute exercise. However, responses to ACh were modulated by RAS pharmacological interventions supporting the responses seen in PE constriction and signifying the participation of vascular RAS in vasomotor function.

During a single bout of aerobic exercise, blood flow is shunted to exercising tissues while blood flow is maintained or reduced to less metabolically active areas (i.e. splanchnic area and non-working muscles). Although increased sympathetic activation and multiple metabolic factors participate in redistributing blood flow during aerobic exercise, the precise mechanism is not entirely known. The renin-angiotensin system (RAS), specifically the local vascular wall RAS, has been hypothesized to participate in the redistribution of blood flow during exercise. This study aimed to investigate whether vascular wall RAS in the femoral arteries (an artery which feeds active tissues during exercise) was altered by acute exercise, and if these vascular RAS alterations led to specific changes in vasomotor function. Male Sprague Dawley rats were exercised on a motorized treadmill for 1h at 21m/min with 15% grade. Immediately following exercise femoral arteries were excised, cleaned of surrounding connective tissue, and vascular RAS was evaluated. There was a decrease in femoral ACE activity (~40%) and expression (~20%) following a single bout of exercise. No change was observed in AT1 and AT2 receptor expression. To evaluate the effect of acute exercise and vascular RAS on vessel reactivity, vasomotor properties of the femoral arteries were assessed via vasoconstrictor and vasodilatory dose-response curves. No changes were observed in femoral artery responses to potassium chloride (KCl), signifying that electromechanical coupling was not affected by exercise or RAS pharmacological interventions. However, a significant decrease in maximum phenylephrine (PE) constriction was observed for acutely exercise animals (~13%). Paired with the observed maintenance KCl-mediated constriction, it appears an acute bout of exercise is able to attenuate α-adrenergic receptor-mediated vasoconstriction in the femoral artery. The decrease in maximum α-adrenergic vasoconstriction may be attributed to vascular RAS. The decrease in ACE activity supports the production of local vasodilating factors. Blocking AT1 receptors with telmisartan decreased PE constriction in control and exercised animals. Combining AT1 and AT2 receptor blockade (with PD123319) eliminated the attenuating effect of telmisartan alone on PE constriction. This data suggests that the attenuating effect of AT1 receptor blockade, on PE constriction, may depend on AT2 receptor activation. In addition, combined AT1 receptor blockade and nitric oxide synthase inhibition eliminated both the lone AT1 receptor blockade effect and exercise effect on PE constriction. Together, this data suggests that reduced PE constriction following acute exercise, and AT1 receptor blockade, is dependent on nitric oxide production. Vasodilation to the nitric oxide donor sodium nitroprusside (SNP) was not altered following exercise or RAS pharmacological intervention, signifying no change in signaling downstream of NO production/release. Endothelium-dependent vasodilation to acetylcholine (ACh) was not affected by acute exercise. However, responses to ACh were modulated by RAS pharmacological interventions supporting the responses seen in PE constriction and signifying the participation of vascular RAS in vasomotor function.

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No description available.