Kai Hong Kwok. / Thesis submitted in: December 1997. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 90-98). / Abstract also in Chinese. / Chapter Chapter 1 --- Introduction / Chapter 1.1. --- Classification of β-adrenoceptor in cardiovascular system --- p.1 / Chapter 1.2. --- Vasodilator effects of β-adrenoceptor-agonists and their mechanisms --- p.4 / Chapter 1.3. --- Role of endothelium in β-adrenoceptor-mediated vasodilation --- p.7 / Chapter 1.4. --- Role of K+ channels in β-adrenoceptor-mediated relaxation --- p.11 / Chapter 1.5. --- Other aspect regarding the vascular response to stimulation of B-adrenoceptor --- p.15 / Chapter 1.6. --- Clinical aspect of B-adrenoceptor agents --- p.15 / Chapter Chapter 2 --- Methods and Materials / Chapter 2.1. --- Tissue Preparation --- p.19 / Chapter 2.1.1. --- Preparation of the isolated rat mesenteric artery --- p.19 / Chapter 2.1.2. --- Removal of the functional endothelium --- p.19 / Chapter 2.1.3. --- Organ bath set-up --- p.20 / Chapter 2.1.4. --- Length-tension relationship and an optimal resting tension --- p.22 / Chapter 2.2. --- Experimental Procedure --- p.22 / Chapter 2.2.1. --- Relaxant effects of the B-adrenoceptor agonists --- p.24 / Chapter 2.2.2. --- Effects of putative K+ channel blockers --- p.24 / Chapter 2.2.3. --- Effects of inhibitors of nitric oxide activity --- p.25 / Chapter 2.2.4. --- Effect of indomethacin --- p.25 / Chapter 2.2.5. --- "Effects of K+ channel opener, nitric oxide donor and forskolin" --- p.26 / Chapter 2.3. --- Chemicals and Solutions --- p.26 / Chapter 2.3.1. --- Chemicals and drugs --- p.26 / Chapter 2.3.2. --- Preparation of drug stock solutions --- p.26 / Chapter 2.3.3. --- Solutions --- p.28 / Chapter 2.4. --- Statistical Analysis --- p.28 / Chapter Chapter 3 --- Results / Chapter 3.1. --- Relaxant Effect of Isoprenaline --- p.29 / Chapter 3.1.1. --- Relaxant effect of isoprenaline --- p.29 / Chapter 3.1.2. --- Effects of inhibitors of nitric oxide activity --- p.29 / Chapter 3.1.3. --- Effect of charybdotoxin on the vasorelaxant response to isoprenaline --- p.32 / Chapter 3.1.4. --- Effect of glibenclamide on the vasorelaxant response to isoprenaline --- p.32 / Chapter 3.1.5. --- Effect of TPA+ on isoprenaline-induced relaxation --- p.36 / Chapter 3.1.6. --- Effect of TPA+ in the presence of iberiotoxin or glibenclamide --- p.36 / Chapter 3.1.7. --- Effect of Ba2+ on the vasorelaxant effect of isoprenaline --- p.41 / Chapter 3.1.8. --- Effect of raising extracellular K+ on isoprenaline-mediated relaxation --- p.41 / Chapter 3.2. --- Relaxant Effect of Dobutamine --- p.44 / Chapter 3.2.1. --- Effects of inhibitors of endothelium-derived factors on the relaxant effect of dobutamine --- p.44 / Chapter 3.2.2. --- Antagonism of the effect of dobutamine by β1-adrenoceptor antagonist --- p.44 / Chapter 3.2.3. --- Effects of putative Kca channel blockers on the relaxant effect of dobutamine --- p.51 / Chapter 3.2.4. --- Effect of TPA+ on the relaxant effect of dobutamine --- p.55 / Chapter 3.2.5. --- Effect of raising extracellular K+ on the relaxant effect of dobutamine --- p.55 / Chapter 3.3. --- Relaxant Effect of Fenoterol --- p.57 / Chapter 3.3.1. --- Effect of inhibitors of nitric oxide activity on the relaxant effect of fenoterol --- p.57 / Chapter 3.3.2. --- Effect of charybdotoxin on the relaxant effect of fenoterol --- p.57 / Chapter 3.3.3. --- Effect of TPA+ on the relaxant effect of fenoterol --- p.64 / Chapter 3.3.4. --- Effect of glibenclamide on the relaxant effect of fenoterol --- p.64 / Chapter 3.3.5. --- Effect of raising extracellular K+ on fenoterol-mediated relaxation --- p.64 / Chapter 3.4. --- Effects of cAMP- and cGMP-elevating agents --- p.69 / Chapter 3.4.1. --- Effects of inhibitors of endothelium-derived factors on the relaxation induced by nitroprusside and forskolin --- p.69 / Chapter 3.4.2 --- Effect of charybdotoxin on relaxant effect of forskolin --- p.69 / Chapter 3.4.3 --- Effect of Ba2+ on the vasorelaxant effect of forskolin --- p.76 / Chapter 3.4.4 --- Effect of TPA+ on the relaxant effect of forskolin --- p.76 / Chapter 3.4.5 --- Effect of glibenclamide on the relaxant effects of forskolin and cromakalim --- p.76 / Chapter Chapter 4 --- Discussion / Chapter 4.1. --- Effect of Isoprenaline and Fenoterol --- p.77 / Chapter 4.2. --- Effect of Dobutamine --- p.83 / Chapter 4.3. --- Conclusion --- p.88 / References --- p.90 / Publications --- p.98

Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / "August 2002." Typescript. Vita. Includes bibliographical references (leaves 132-150).

Vasoconstrictor tone in the splanchnic circulation redistributes blood flow during hemorrhage and resuscitation. A metabolic sensor, 5’adenosine monophosphate-activated protein kinase (AMPK), has been proposed to relax arteries by inhibiting myosin light chain (MLC) kinase (MLCK) and rho kinase (ROCK) activities. Because AMPK activation might be beneficial in re-establishing splanchnic blood flow during resuscitation, we sought to explore the relative ability of AMPK activators (AICAR, A769662, berberine (BBR) and simvastatin (SIMV)) to relax mesenteric artery (MA) contraction. Our data revealed that these drugs caused vasorelaxation when tissues were stimulated either with KCl (producing primarily a Ca2+ dependent contraction) or phenylephrine (PE; producing a primarily Ca2+ independent contraction). We further investigated the potential mechanisms by which BBR induced mesenteric artery relaxation. We found that BBR did not inhibit MLC phosphorylation, nor did it phosphorylate AMPK, and therefore is likely working through another mechanism to cause vasorelaxation. Notably, PE induced an increase in AMPK phosphorylation and, of all the AMPK activators examined, only AICAR phosphorylated AMPK in rabbit mesenteric artery, which provides a guide for future studies.

Vários mecanismos controlam os processos de contração e relaxamento das células musculares lisas vasculares (CMLVs). Receptores e moléculas sinalizadoras intracelulares, os quais controlam os processos de contração e relaxamento das CMLVs, são alvo de modificações pós-traducionais, como a O-GlcNAcilação que modula respostas vasculares. O aumento de proteínas modificadas por O-GlcNAc apresenta efeito ambíguo sobre as CMLVs, sendo protetor em situações de aumento agudo, mas lesivo quando mantido cronicamente. O aumento crônico de O-GlcNAc em animais está associado a repostas contráteis mais intensas e redução do relaxamento vascular, assim como o aumento da produção de espécies reativas de oxigênio (EROs), denominado estresse oxidativo, alterações constantemente descritas em doenças crônicas como diabetes e hipertensão arterial. Considerando que algumas proteínas que controlam a contratilidade vascular são modificadas por O-GlcNAc e que pouco se sabe a respeito da via de Biossíntese das Hexosaminas (VBH) e sua relação com o sistema vascular em humanos, nós investigamos a hipótese de que modificações de proteínas por O-GlcNAc estão relacionadas a alterações vasculares na hipertensão arterial em humanos. Durante a realização de nossos experimentos, demonstramos que os principais componentes da VBH estão presentes em CMLVs humanas. O tratamento com Thiamet G (TMG) por 24 h aumentou os níveis de proteínas modificadas por O-GlcNAc nas CMLVs pela redução da atividade de OGA, assim como induziu efeito compensatório de aumento da expressão dessa enzima. TMG reduziu a atividade de OGA em CMLVs no grupo Controle, mas não promoveu alteração na geração de EROs. Após tratamento com TMG, artérias de grupo Controle apresentaram maior sensibilidade à noradrenalina (NA) e maior relaxamento ao nitroprussiato de sódio (NPS); enquanto o grupo Hipertenso não apresentou alterações na contratilidade ou no relaxamento arterial. Artérias do grupo Hipertenso apresentaram maior sensibilidade à NA que o grupo Controle antes de qualquer tratamento, além de deficiência no relaxamento, com menor sensibilidade e menor resposta máxima ao NPS em comparação ao grupo Controle. O grupo Hipertenso apresentou aumento da pressão arterial média de internação (PAMi), assim como da idade, quando comparado com o grupo Controle. Em conclusão, a VBH está presente nas CMLVs humanas. A inibição da atividade da OGA por TMG aumenta os níveis de proteínas modificadas por O-GlcNAc, a expressão de OGA e modula a reatividade vascular no grupo Controle, mas não no grupo Hipertenso. Os resultados demonstram que pacientes hipertensos apresentam respostas vasculares a drogas vasoativas diferentes daquelas observadas em pacientes controle, antes e após o aumento dos níveis de proteínas modificadas por O-GlcNAc nas CMLVs. Novos estudos serão necessários para determinar se as alterações observadas são decorrentes da hipertensão arterial e/ou do tratamento farmacológico aos quais os pacientes estão submetidos. / Several mechanisms control the contraction and relaxation processes in the vascular smooth muscles cells (VSMC). Intracellular receptors and signaling molecules involved in contraction and relaxation mechanisms are targets of post-translational modifications (PTM), such as O-GlcNAcylation, which modulates vascular responses. Augmented levels of O-GlcNAc-modified proteins show a dual effect in VSMC, being protective during acute stressful events and deleterious when O-GlcNAc is chronically augmented. In animals, chronic increases in O-GlcNAc-modified proteins are linked to increased vascular responses to constrictor agents, to reduced vascular relaxation in response to dilator drugs, and to increased production of reactive oxygen species (ROS), named oxidative stress. All these changes are frequently described in chronic diseases such as diabetes and arterial hypertension. Since proteins involved in vascular contractility are modified by O-GlcNAc, and our knowledge on the influence of the hexosamine biosynthesis pathway (HBP) in the human vascular system is limited, we tested the hypothesis that proteins modified by O-GlcNAc contribute to vascular changes in hypertensive patients. Our data show that human VSMC express the main components of the HBP; the treatment of human VSMC with Thiamet G (TMG) for 24 h augmented O-GlcNAc levels, decreased OGA activity and induced a compensatory increase in OGA\'s expression. TMG reduced OGA activity, increased levels of O-GlcNAc-modified proteins but did not change ROS generation in human arteries from the Control group. After treatment with TMG, arteries from the Control group exhibited increased sensitivity to norepinephrine (NE) and to sodium nitroprusside (SNP), as well as increased maximum relaxation to SNP. Augmented O-GlcNAc levels produced no changes in contractile or relaxation responses in the Hypertensive group. Arteries from the Hypertensive group exhibited an increased sensitivity to NE as well as decreased sensitivity and maximum relaxation to SNP when compared to arteries from the Control group. Mean arterial blood pressure (hMABP) and the average age were increased in patients from the Hypertensive group. In conclusion, the HBP is present in human VSMC and the inhibition of OGA activity with TMG increases O-GlcNAc levels, increases OGA expression and modifies vascular responses to constrictor and dilator stimuli in human arteries from the Control group, but not from the Hypertensive group. These results indicate that hypertensive patients have altered vascular responses to vasoactive drugs both in the absence and in the presence of augmented O-GlcNAc levels. Further research is needed to explain whether these differences are due to the hypertensive disease and/or to the patient\'s medical treatment.

Vários mecanismos controlam os processos de contração e relaxamento das células musculares lisas vasculares (CMLVs). Receptores e moléculas sinalizadoras intracelulares, os quais controlam os processos de contração e relaxamento das CMLVs, são alvo de modificações pós-traducionais, como a O-GlcNAcilação que modula respostas vasculares. O aumento de proteínas modificadas por O-GlcNAc apresenta efeito ambíguo sobre as CMLVs, sendo protetor em situações de aumento agudo, mas lesivo quando mantido cronicamente. O aumento crônico de O-GlcNAc em animais está associado a repostas contráteis mais intensas e redução do relaxamento vascular, assim como o aumento da produção de espécies reativas de oxigênio (EROs), denominado estresse oxidativo, alterações constantemente descritas em doenças crônicas como diabetes e hipertensão arterial. Considerando que algumas proteínas que controlam a contratilidade vascular são modificadas por O-GlcNAc e que pouco se sabe a respeito da via de Biossíntese das Hexosaminas (VBH) e sua relação com o sistema vascular em humanos, nós investigamos a hipótese de que modificações de proteínas por O-GlcNAc estão relacionadas a alterações vasculares na hipertensão arterial em humanos. Durante a realização de nossos experimentos, demonstramos que os principais componentes da VBH estão presentes em CMLVs humanas. O tratamento com Thiamet G (TMG) por 24 h aumentou os níveis de proteínas modificadas por O-GlcNAc nas CMLVs pela redução da atividade de OGA, assim como induziu efeito compensatório de aumento da expressão dessa enzima. TMG reduziu a atividade de OGA em CMLVs no grupo Controle, mas não promoveu alteração na geração de EROs. Após tratamento com TMG, artérias de grupo Controle apresentaram maior sensibilidade à noradrenalina (NA) e maior relaxamento ao nitroprussiato de sódio (NPS); enquanto o grupo Hipertenso não apresentou alterações na contratilidade ou no relaxamento arterial. Artérias do grupo Hipertenso apresentaram maior sensibilidade à NA que o grupo Controle antes de qualquer tratamento, além de deficiência no relaxamento, com menor sensibilidade e menor resposta máxima ao NPS em comparação ao grupo Controle. O grupo Hipertenso apresentou aumento da pressão arterial média de internação (PAMi), assim como da idade, quando comparado com o grupo Controle. Em conclusão, a VBH está presente nas CMLVs humanas. A inibição da atividade da OGA por TMG aumenta os níveis de proteínas modificadas por O-GlcNAc, a expressão de OGA e modula a reatividade vascular no grupo Controle, mas não no grupo Hipertenso. Os resultados demonstram que pacientes hipertensos apresentam respostas vasculares a drogas vasoativas diferentes daquelas observadas em pacientes controle, antes e após o aumento dos níveis de proteínas modificadas por O-GlcNAc nas CMLVs. Novos estudos serão necessários para determinar se as alterações observadas são decorrentes da hipertensão arterial e/ou do tratamento farmacológico aos quais os pacientes estão submetidos. / Several mechanisms control the contraction and relaxation processes in the vascular smooth muscles cells (VSMC). Intracellular receptors and signaling molecules involved in contraction and relaxation mechanisms are targets of post-translational modifications (PTM), such as O-GlcNAcylation, which modulates vascular responses. Augmented levels of O-GlcNAc-modified proteins show a dual effect in VSMC, being protective during acute stressful events and deleterious when O-GlcNAc is chronically augmented. In animals, chronic increases in O-GlcNAc-modified proteins are linked to increased vascular responses to constrictor agents, to reduced vascular relaxation in response to dilator drugs, and to increased production of reactive oxygen species (ROS), named oxidative stress. All these changes are frequently described in chronic diseases such as diabetes and arterial hypertension. Since proteins involved in vascular contractility are modified by O-GlcNAc, and our knowledge on the influence of the hexosamine biosynthesis pathway (HBP) in the human vascular system is limited, we tested the hypothesis that proteins modified by O-GlcNAc contribute to vascular changes in hypertensive patients. Our data show that human VSMC express the main components of the HBP; the treatment of human VSMC with Thiamet G (TMG) for 24 h augmented O-GlcNAc levels, decreased OGA activity and induced a compensatory increase in OGA\'s expression. TMG reduced OGA activity, increased levels of O-GlcNAc-modified proteins but did not change ROS generation in human arteries from the Control group. After treatment with TMG, arteries from the Control group exhibited increased sensitivity to norepinephrine (NE) and to sodium nitroprusside (SNP), as well as increased maximum relaxation to SNP. Augmented O-GlcNAc levels produced no changes in contractile or relaxation responses in the Hypertensive group. Arteries from the Hypertensive group exhibited an increased sensitivity to NE as well as decreased sensitivity and maximum relaxation to SNP when compared to arteries from the Control group. Mean arterial blood pressure (hMABP) and the average age were increased in patients from the Hypertensive group. In conclusion, the HBP is present in human VSMC and the inhibition of OGA activity with TMG increases O-GlcNAc levels, increases OGA expression and modifies vascular responses to constrictor and dilator stimuli in human arteries from the Control group, but not from the Hypertensive group. These results indicate that hypertensive patients have altered vascular responses to vasoactive drugs both in the absence and in the presence of augmented O-GlcNAc levels. Further research is needed to explain whether these differences are due to the hypertensive disease and/or to the patient\'s medical treatment.

Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 132-150). Also available on the Internet.

Traumatic brain injury (TBI) is the most frequent cause of death in children and young adults in the United States. Besides emergency neurosurgical procedures, there are few medical treatment options to improve recovery in people who have experienced a TBI. Management of patients who survive TBI is complicated by both central nervous system and peripheral systemic effects. The pathophysiology of systemic inflammation and coagulopathy following TBI has been attributed to trauma-induced endothelial cell dysfunction; however, there is little knowledge of the mechanisms by which trauma might impact the functions of the vascular endothelium at sites remote from the injury. The endothelium lining these small vessels normally produces nitric oxide (NO), arachidonic acid metabolites, and endothelial-dependent hyperpolarizing factors to relax the surrounding vascular smooth muscle. For this research study we investigated the effects of fluid-percussion-induced TBI on endothelial-dependent vasodilatory functions in a remote tissue bed (the mesenteric circulation) 24 hours after injury. We hypothesized that TBI causes changes in the mesenteric artery endothelium that result in a loss of endothelial-dependent vasodilation. We found that vasodilations induced by the muscarinic-receptor agonist, acetylcholine, are attenuated following TBI. While the endothelial-derived hyperpolarizing component of vasodilation was preserved, the NO component was severely impaired. Therefore, we tested whether the loss of NO component was due to a decrease in bioavailablity of the NO synthase (NOS) cofactor BH4, the NOS substrate L-arginine, or to changes in expression/activity of the enzyme arginase, which competes with NOS for L-arginine. We found that supplementation of L-arginine and inhibition of the enzyme arginase rescues endothelial-dependent vasodilations in TBI arteries. This study demonstrates that there are pathological systemic effects outside the point of injury following TBI leading to a dysfunctional endothelial vasodilatory pathway. These data provide insight into the pathophysiology of endothelial dysfunction after trauma and may lead to new potential targets for drug therapy.

Several studies suggest that diabetes affects male and female vascular beds differently. However, the mechanisms underlying the interaction of sex and diabetes remain to be investigated. This study investigates whether there are 1) sex differences in the development of abnormal vascular responses and 2) changes in the relative contributions of endothelium-derived relaxing factors (EDRFs) in modulating vascular reactivity of mesenteric arteries taken from streptozotocin (STZ)-induced diabetic rats at early and intermediate stages of the disease (one and eight weeks, respectively). We also investigated the mesenteric expression of the mRNAs for endothelial nitric oxide synthase (eNOS) and NADPH oxidase (Nox) in STZ-induced diabetes in both sexes. Vascular responses to acetylcholine (ACh) in mesenteric arterial rings pre-contracted with phenylephrine were measured before and after pretreatment with indomethacin (cyclooxygenase inhibitor), L -NAME (NOS inhibitor), or barium chloride (K IR blocker) plus ouabain (Na + -K + ATPase inhibitor). We demonstrated that ACh-induced relaxations were significantly impaired in mesenteric arteries from both male and female diabetic rats at one and eight weeks. However, at eight weeks the extent of impairment was significantly greater in diabetic females than diabetic males. Our data also showed that in females, the levels of eNOS and Nox2- and Nox4-dependent NADPH oxidase mRNA expression and the relative importance of NO to the regulation of vascular reactivity were substantially enhanced, while the importance of endothelium-derived hyperpolarizing factor (EDHF) was significantly reduced at both one and eight weeks after the induction of diabetes. This study reveals the predisposition of female rat mesenteric arteries to vascular injury after the induction of diabetes, may be due to a shift away from a putative EDHF, initially the major vasodilatory factor, towards a greater reliance on NO, and the interaction of oxidative stress with elevated NO.