Thesis (M. Phil.)--University of Hong Kong, 1990.

Zugl.: Mainz, Univ., Diss., 2008

No description available.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal. / Angiotensinogen (ANG) is a single peptide glycoprotein that contains 452 amino acids in human (453 amino acids in rodents). ANG releases a 10-amino acids peptide known as angiotensin l (Ang I) from it's N-terminal when acted by renin, a acidic protease. Ang I can be fiirther converted into angiotensin II (Ang II) by angiotensin converting enzyme (ACE). Ang II is one of the most potent vasoconstrictors known. Ang II also acts on the brain to mcrease blood pressure. Ang II acts on the adrenal cortex to increase the secretion of aldosterone. The levels of ANG in plasma or local tissues diïectly contribute to the levels ofAng II. Therefore the studies of regulation ofANG gene expression is important to understand the molecular mechanisms of some related diseases, such as hypertension. Previous studies m which the transfection of the fusion genes that were generated with various lengths of 5'-flanking region of the rat ANG gene linked to a bacterial chloramphenicol acetyl transferase (CAT) gene as a reporter into mouse hepatoma (Hepa 1- 6) ceUs and opossum kidney (OK) cells, identified a putative cyclic AMP responsive element (CRE) in the rat ANG gene 5'-regulatory region (N-806/-779). Compared with palindromic CRE octamer (TGACGTCA), the putative CRE of the ANG gene (ANG-CRE) is almost identical except the last two bases were in reversed order (TGACGTAC). In the present study, we isolated a fuU-length cDNA of 1345 base pairs from mouse liver cDNA library, which encodes a nuclear DNA-binding protein consisting of 436 amino acids with an apparent molecular weight of 52 kilodalton (kDa). This protein binds to the ANG-CRE, and was designated as 52-kDa protein. Southwestern blot revealed that this 52 kDa protein was present in the following tissues, such as liver, kidney, testis, brain, but not in spleen. Analysis of the deduced amino acid sequence shows no apparent basic regionleucine zipper (bZIP) stmcture, indicatmg ANG-CREB is structurally distinct fi-om bZIP family members. The antiserum against 43-kDa-CREB or ATF-2 can not interact with this 52-kDa protein, further supporting that the 52-kDa protem is immunologically different fi-om the bZIP family. The 52-kDa protein may represent a new group of CRE-binding proteins. Competitive gel mobility shift assays and Southwestern blot analysis revealed that the ANG-CREB binds to ANG-CRE, and this bindmg is not displaceable by excess amounts of CREs fi-om somatostatin (SOM), phosphoenopymvate carboxyl kmase PEPCK), and tyrosine ammo transferase (TAT) genes in vitro. These data suggest that the 52-kDa protein binds specifically to ANG-CRE and may regulate the ANG gene expression. The biological function of the 52-kDa protein is not known at present. The primary data from transient gene transfection assays showed that this protein has represser activity on the ANG gene promoter when ANG gene was stimulated by isoproterenol, but showed no effect on the ANG basal level (without any stimulation). Further experiments will be needed to study the biological function of the 52-kDa protein. / L'angiotensinogène (ANG) est une glycoprotéine fonnée de 452 acide aminés chez l'humain (453 acide aminés chez les rongeurs). Une protéase acide, la rénine, clive l'ANG de son côté N-tenninal en libérant un peptide de 10 acide aminés connu sous le nom de l'angiotensine I (Angl). Ce dernier, peut être converti en angiotensine II (AngII) sous Faction d'une enzyme de conversion dite ACE. L'Ang II est un puissant vasocoiisticteur. Il agit au niveau du cerveau en augmentant la pression sanguine et au niveau du cortex rénal en augmentant la sécrétion de l'aldostérone. Les niveaux de l'ANG daiis le plasma ou dans les tissus locaux sont directement proportionnels aux niveaux de Ang II. Ainsi les études de regulation de l'expression du gène d'Ang II sont importantes pour comprendre les mécanismes moléculaires de certames maladies, comme l'hypertension. Des études antérieiu-es par transfection en utilisant des gènes de fusion générés avec différentes longueurs de région flanquées en 5' du gène ANG de rat lié au gène rapporteur de bactérie, le chloramphénicol acetyl transférase (CAT), dans les ceUules hépatome (Hepa 1-6) de souris et dans les ceUules rénales de sarigue, ont permis d'identifier un présumé élément réponse (CRE) d'AMP cyclique dans la région régulatrice en 5' du gène ANG de rat (N- 806/-779). En comparant l'octamère palindromique de CRE (TGACGTCA) avec le présumé CRE du gène ANG (ANG-CRE), il semble qu'ils sont identiques à l'exception des deux dernières bases qui sont dans un ordre inversé (TGACGTAC). Dans cette étude, nous avons isolé à partir d'une banque de cDNA du foie de souris, une cDNA de 1345 paire de bases qui code pour une protéine de liaison à l'ADN nucléaire. Cette protéine est fonnée de 436 acide aminés avec un poids moléculaire apparent de 52 kilodalton (kDa). En se liant à ANG-CER, cette protéme est désignée comme la 52-kDa protéine. Le "Southwestern blot" a révélé que la 52-kDa protéine de 52 kDa est présente dans les tissus suivants: foie, rein, testicule et cerveau; mais absente dans la rate. L'analyse de cette séquence déduite d'acide aminés ne montre pas du stmcture apparent à la région basique de la fermeture éclaire à leucine, bzip (basic region-leucme zipper), indiquant que la 52-kDa protéine est structurellement distincte des membres de la famille bzip. L'antisérum dérigé contre le 43- kDa-CREB ou contre ATF-2 n'intéragit pas avec la 52-kDa protéme confermant amsi que la 52-kDa protéine est immunologiquenient dififérente des membres de famille bzip. La 52-kDa protéine représente donc, un nouveau groupe des protéines de liaison à CRE. Les essais sur gel à mobilité compétitive et les analyses de "southwestern blot" ont révélé que la 52-kDa protéine lié ANG-CRE et que cette liaison n'était pas déplaçable par un excès du gène CRE de somatostatin (SOM), phosphoenopymvate carboxy kmase (PEPCK) et tyrosme ammo transférase (TAT) m vitro, suggérant que la 52-kDa protéine se lié spésifiquement à ANGCRE et qu'il peut réguler l'expression du gène ANG. Les fonctioiis biologiques de la 52- kDa protéine ne sont pas encore connus. Les premiers résultats obtenus à partir des essais de transfections des gènes transitoirs montrent que cette protéine a une activité represseur sur le promoteur du gène d'ANG quand ce dernier est stimulé par l'isoprotérénol, et que, sans stimulation, elle a aucun effet sur le niveau basai d'ANG. Les expériences futures seront nécessaires poiir étudier les fonctions biologiques d'AND.

Elevated circulating Ang II activates neurons in the forebrain cardiovascular regulatory areas to cause sympatho-excitation and hypertension. We hypothesized that circulating Ang II causes neuronal activation in the SFO and thereby activates efferent pathways to the PVN, and chronically causes activation of aldosterone production in magnocellular neurons in PVN and SON, which amplifies neuronal activation in the PVN and central sympatho-excitatory pathways. The aim of the present study was to determine the pattern of neuronal activation in forebrain nuclei by circulating Ang II and to elucidate where in the hypothalamus Ang II may stimulate aldosterone biosynthesis. Dose related effects of circulating Ang II on BP were first assessed. Wistar rats instrumented with telemetry probes were infused subcutaneously with Ang II 150 and 500 ng/kg/min for 14 days. The subcutaneous infusion of Ang II at 150 ng/kg/min increased blood pressure gradually up to 20 mmHg and at 500 ng/kg/min up to 60 mmHg. Ang II at 500 ng/kg/min increased plasma Ang II by 4-fold. To assess effects of circulating Ang II on CNS pathways, Wistar rats were implanted subcutaneously with minipumps infusing 150 and 500 ng/kg/min Ang II for 1, 4 and 14 days. Three patterns of neuronal activation were observed by sc infusion of Ang II. The SFO was activated during the first day and remained activated for 4 days, but at 14 days showed diminished activation. MnPO did not show significant activation during the first day but, after several days the activation was high and then less by 14 days. Parvocellular PVN (pPVN), magnocellular PVN (mPVN) and SON showed an initial activation that increased over time. Chronic intracerebroventricular infusion of an aldosterone synthase inhibitor or a mineralocorticoid receptor (MR) blocker attenuated the increase in Fra expression in PVN but not SON, and prevented the decrease in SFO after 14 days infusion of Ang II. A significant increase in mRNA expression of steroidogenic acute regulatory protein (StAR), a rate limiting enzyme in aldosterone production was found in glia cells of PVN and SFO assessed by rt-PCR after 3 days subcutaneous infusion of Ang II at 500 ng/kg/min. Total expression of aldosterone synthase (CYP11B2) mRNA was increased in SFO, MnPO, SON and PVN after 3 days of infusion of Ang II. After 14 days no significant changes were observed in the expression of StAR or CYP11B2 mRNA. In comparison, in adrenal StAR mRNA expression increased after 3 days but no longer after 14 days. In contrast, CYP11B2 mRNA expression in adrenal increased after both 3 and 14 days of infusion. These findings may support our hypothesis that chronic elevation of circulating Ang II increases neuronal activity in CVOs, presumably leading to activation of the PVN and SON to induce an increase in aldosterone production in magnocelular PVN and SON. In the second phase activation of CVOs appears to diminish, but an aldosterone-dependent amplifying mechanisms, causes sustained activation of the PVN and thereby hypertension.

Angiotensin II (Ang II) plays a very important role in regulating cardiac and vascular contraction and proliferation/hypertrophy via stimulation of AT1 receptors. A few studies have demonstrated that 5-lipoxygenase (5-LO) derived cysteinyl leukotrienes (CysLT) contribute to Ang II evoked tension responses in rat aortic rings. Whether CysLT would contribute to Ang II evoked Ca2+ mobilization in neonatal rat cardiomyocytes (NRC) and rat aortic smooth muscle cells (ASMC) has not been investigated. In the present study, using primary cultures of NRC and minimally passaged cultures of rat ASMC, an effort was made to address this key issue. The agonists evoked increase in cytosolic free calcium ([Ca2+]i) level was determined by fura-2 fluorescence measurement in NRC and ASMC. Total CysLT levels in the culture medium were determined using an ELISA kit. CysLT1/CysLT2 receptor mRNA levels of NRC and ASMC were quantified by Northern blot analysis. In NRC, the AT1 but not the AT2 selective antagonist, attenuated the elevations in [Ca2+]i and CysLT levels evoked by Ang II. Vasopressin (AVP) and endothelin-1 (ET-1) increased [Ca2+]i but not CysLT levels. The 5-LO inhibitor, AA861, and the CysLT1 selective antagonist, MK-571, reduced the maximal [Ca2+]i responses (Emax) to Ang II but not to AVP and ET-1. While CysLT1 antagonist reduced the Emax to leukotriene D4, (LTD4), the dual CysLT1/CysLT2 antagonist, BAY u9773, completely blocked the [Ca2+]i elevation to both LTD4 and leukotriene C4 (LTC4). Both CysLT1 and CysLT2 mRNA were detected in NRC. The inositol 1,4,5 triphosphate (InsP3) antagonist, 2-aminoethoxyphenyl borate (2-APB), attenuated the [Ca2+]i responses to Ang II and LTD4. In ASMC, Ang II, ET-1 and AVP evoked [Ca2+]i responses were significantly higher in the cultured ASMC isolated from spontaneously hypertensive rats (SHR) compared to ASMC derived from age-matched normotensive Wistar-Kyoto (WKY) strain. Addition of either MK571 or BAY u9773, reduced the Emax values to Ang II (but not to ET-1and AVP) in both strains. While BAY u9773 abolished the [Ca2+]i responses evoked by both LTD4 and LTC4, MK571, the CysLT1 antagonist reduced the responses evoked by LTD4 but not LTC4. The basal CysLT levels were higher in the ASMC of SHR. Ang II but not ET-1 and AVP evoked time and concentration dependent increases in CysLT levels in ASMC of both WKY and SHR strains. The AT1 selective antagonist, losartan, but not the AT2 antagonist, PD123319, attenuated the increases in [Ca2+]i and CysLT levels evoked by Ang II. The InsP3 antagonist, attenuated the [Ca2+]i responses to Ang II, LTD4 and LTC4. Both CysLT1 and CysLT2 mRNA were detected in the ASMC of either strain; but they were significantly higher in SHR. These data suggest that AT1 mediated CysLT production contributes to Ang II evoked Ca2+ mobilization in NRC and that elevated CysLT production along with increased expression of both CysLT1/CysLT2 receptors may account for the exaggerated [Ca2+]i responses to Ang II in ASMC of SHR due to enhanced mobilization of Ca2+ from InsP3 sensitive intracellular Ca2+ stores.

Elevated circulating Ang II activates neurons in the forebrain cardiovascular regulatory areas to cause sympatho-excitation and hypertension. We hypothesized that circulating Ang II causes neuronal activation in the SFO and thereby activates efferent pathways to the PVN, and chronically causes activation of aldosterone production in magnocellular neurons in PVN and SON, which amplifies neuronal activation in the PVN and central sympatho-excitatory pathways. The aim of the present study was to determine the pattern of neuronal activation in forebrain nuclei by circulating Ang II and to elucidate where in the hypothalamus Ang II may stimulate aldosterone biosynthesis. Dose related effects of circulating Ang II on BP were first assessed. Wistar rats instrumented with telemetry probes were infused subcutaneously with Ang II 150 and 500 ng/kg/min for 14 days. The subcutaneous infusion of Ang II at 150 ng/kg/min increased blood pressure gradually up to 20 mmHg and at 500 ng/kg/min up to 60 mmHg. Ang II at 500 ng/kg/min increased plasma Ang II by 4-fold. To assess effects of circulating Ang II on CNS pathways, Wistar rats were implanted subcutaneously with minipumps infusing 150 and 500 ng/kg/min Ang II for 1, 4 and 14 days. Three patterns of neuronal activation were observed by sc infusion of Ang II. The SFO was activated during the first day and remained activated for 4 days, but at 14 days showed diminished activation. MnPO did not show significant activation during the first day but, after several days the activation was high and then less by 14 days. Parvocellular PVN (pPVN), magnocellular PVN (mPVN) and SON showed an initial activation that increased over time. Chronic intracerebroventricular infusion of an aldosterone synthase inhibitor or a mineralocorticoid receptor (MR) blocker attenuated the increase in Fra expression in PVN but not SON, and prevented the decrease in SFO after 14 days infusion of Ang II. A significant increase in mRNA expression of steroidogenic acute regulatory protein (StAR), a rate limiting enzyme in aldosterone production was found in glia cells of PVN and SFO assessed by rt-PCR after 3 days subcutaneous infusion of Ang II at 500 ng/kg/min. Total expression of aldosterone synthase (CYP11B2) mRNA was increased in SFO, MnPO, SON and PVN after 3 days of infusion of Ang II. After 14 days no significant changes were observed in the expression of StAR or CYP11B2 mRNA. In comparison, in adrenal StAR mRNA expression increased after 3 days but no longer after 14 days. In contrast, CYP11B2 mRNA expression in adrenal increased after both 3 and 14 days of infusion. These findings may support our hypothesis that chronic elevation of circulating Ang II increases neuronal activity in CVOs, presumably leading to activation of the PVN and SON to induce an increase in aldosterone production in magnocelular PVN and SON. In the second phase activation of CVOs appears to diminish, but an aldosterone-dependent amplifying mechanisms, causes sustained activation of the PVN and thereby hypertension.

Angiotensin II (Ang II) plays a very important role in regulating cardiac and vascular contraction and proliferation/hypertrophy via stimulation of AT1 receptors. A few studies have demonstrated that 5-lipoxygenase (5-LO) derived cysteinyl leukotrienes (CysLT) contribute to Ang II evoked tension responses in rat aortic rings. Whether CysLT would contribute to Ang II evoked Ca2+ mobilization in neonatal rat cardiomyocytes (NRC) and rat aortic smooth muscle cells (ASMC) has not been investigated. In the present study, using primary cultures of NRC and minimally passaged cultures of rat ASMC, an effort was made to address this key issue. The agonists evoked increase in cytosolic free calcium ([Ca2+]i) level was determined by fura-2 fluorescence measurement in NRC and ASMC. Total CysLT levels in the culture medium were determined using an ELISA kit. CysLT1/CysLT2 receptor mRNA levels of NRC and ASMC were quantified by Northern blot analysis. In NRC, the AT1 but not the AT2 selective antagonist, attenuated the elevations in [Ca2+]i and CysLT levels evoked by Ang II. Vasopressin (AVP) and endothelin-1 (ET-1) increased [Ca2+]i but not CysLT levels. The 5-LO inhibitor, AA861, and the CysLT1 selective antagonist, MK-571, reduced the maximal [Ca2+]i responses (Emax) to Ang II but not to AVP and ET-1. While CysLT1 antagonist reduced the Emax to leukotriene D4, (LTD4), the dual CysLT1/CysLT2 antagonist, BAY u9773, completely blocked the [Ca2+]i elevation to both LTD4 and leukotriene C4 (LTC4). Both CysLT1 and CysLT2 mRNA were detected in NRC. The inositol 1,4,5 triphosphate (InsP3) antagonist, 2-aminoethoxyphenyl borate (2-APB), attenuated the [Ca2+]i responses to Ang II and LTD4. In ASMC, Ang II, ET-1 and AVP evoked [Ca2+]i responses were significantly higher in the cultured ASMC isolated from spontaneously hypertensive rats (SHR) compared to ASMC derived from age-matched normotensive Wistar-Kyoto (WKY) strain. Addition of either MK571 or BAY u9773, reduced the Emax values to Ang II (but not to ET-1and AVP) in both strains. While BAY u9773 abolished the [Ca2+]i responses evoked by both LTD4 and LTC4, MK571, the CysLT1 antagonist reduced the responses evoked by LTD4 but not LTC4. The basal CysLT levels were higher in the ASMC of SHR. Ang II but not ET-1 and AVP evoked time and concentration dependent increases in CysLT levels in ASMC of both WKY and SHR strains. The AT1 selective antagonist, losartan, but not the AT2 antagonist, PD123319, attenuated the increases in [Ca2+]i and CysLT levels evoked by Ang II. The InsP3 antagonist, attenuated the [Ca2+]i responses to Ang II, LTD4 and LTC4. Both CysLT1 and CysLT2 mRNA were detected in the ASMC of either strain; but they were significantly higher in SHR. These data suggest that AT1 mediated CysLT production contributes to Ang II evoked Ca2+ mobilization in NRC and that elevated CysLT production along with increased expression of both CysLT1/CysLT2 receptors may account for the exaggerated [Ca2+]i responses to Ang II in ASMC of SHR due to enhanced mobilization of Ca2+ from InsP3 sensitive intracellular Ca2+ stores.

Argument: Hypertension is a main contributing risk factor to many cardiovascular diseases and may be the cause or the result of cardiovascular dysfunction. Black Africans, especially, suffer from hypertension because of lifestyle changes that occur during westernisation, which may lead to sympatho-adrenal hyperactivity. Vascular endothelial growth factor-A (VEGF-A) and angiopoietin-2 (Ang-2) are regulators of angiogenesis and are significantly up regulated during states of vascular dysfunction. Levels of angiogenic factors are unknown for African people and may not be the same as levels thus far reported for Caucasians. Aims: The aim of this study is firstly, to determine whether differences exist regarding the levels of VEGF-A and Ang-2 in urbanised compared to rural black Africans and secondly, to determine whether increased levels of VEGF-A and Ang-2 factors are related to hypertension in black Africans. Methodology: This is a sub study that is based upon the Prospective Urban and Rural Epidemiological (PURE) study. Apparently healthy, fasting African men and women (N=272, aged 35 to 50 years) from the North-West province of South Africa were selected by a medical doctor to participate in this study. Groups were stratified according to gender and urbanisation status based upon information derived from sociodemographic questionnaires. Cardiovascular parameters (Omron HEM-757), pulse wave velocity (PWV) (Compiler SP), plasma angiogenic factor levels (ELISA) and anthropometric measures were determined. An independent t-test and Pearson Chi-square test were used to compare urban and rural data, followed by an analysis of covariance (ANCOVA) while correcting for confounders (age, body mass index, physical activity and tobacco usage). ANCOVAs (corrected for confounders) were applied where hypertensive and normotensive groups were compared within the whole group and urbanised groups. Correlations, correcting for confounders, between cardiovascular variables and angiogenic factors were determined within the whole group and urbanised groups. Results and conclusion: Plasma VEGF-A values for all black Africans were very low while the ANG-2 levels were elevated compared to control values for Caucasians (normotensive and hypertensive) in literature. Urbanised men were more overweight and indicated a higher incidence of hypertension (42.47%) and elevated VEGF-A levels, but lower Ang-2 levels compared to rural men. Urbanised women were generally overweight, physically less active and smoked less, but indicated higher diastolic blood pressure (BP), VEGF-A levels and lower PWV compared with their rural counterparts. Ang-2 levels indicate a negative relationship to diastolic BP data in rural women. No relationships between hypertensive individuals and high angiogenic factor levels were uncovered. Conclusive evidence suggested that angiogenic factor levels were affected more by urbanisation than by the state of hypertension. If low levels of VEGF-2 occur, ANG-2 stimulation and properties may be altered, thereby switching ANG-2 from an anti-angiogenic to a pro-angiogenic molecule, inferring blood vessel destabilisation and vascular dysfunction, such as is observed in hypertensive urbanised men. Higher ANG-2 levels may result in Tie-2 receptor down regulation, hence causing VEGF-A levels to be lower. Further study is needed to ascertain this mechanism since Tie-2 receptor activity was not determined in this study. / Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2009.

Elevated circulating Ang II activates neurons in the forebrain cardiovascular regulatory areas to cause sympatho-excitation and hypertension. We hypothesized that circulating Ang II causes neuronal activation in the SFO and thereby activates efferent pathways to the PVN, and chronically causes activation of aldosterone production in magnocellular neurons in PVN and SON, which amplifies neuronal activation in the PVN and central sympatho-excitatory pathways. The aim of the present study was to determine the pattern of neuronal activation in forebrain nuclei by circulating Ang II and to elucidate where in the hypothalamus Ang II may stimulate aldosterone biosynthesis. Dose related effects of circulating Ang II on BP were first assessed. Wistar rats instrumented with telemetry probes were infused subcutaneously with Ang II 150 and 500 ng/kg/min for 14 days. The subcutaneous infusion of Ang II at 150 ng/kg/min increased blood pressure gradually up to 20 mmHg and at 500 ng/kg/min up to 60 mmHg. Ang II at 500 ng/kg/min increased plasma Ang II by 4-fold. To assess effects of circulating Ang II on CNS pathways, Wistar rats were implanted subcutaneously with minipumps infusing 150 and 500 ng/kg/min Ang II for 1, 4 and 14 days. Three patterns of neuronal activation were observed by sc infusion of Ang II. The SFO was activated during the first day and remained activated for 4 days, but at 14 days showed diminished activation. MnPO did not show significant activation during the first day but, after several days the activation was high and then less by 14 days. Parvocellular PVN (pPVN), magnocellular PVN (mPVN) and SON showed an initial activation that increased over time. Chronic intracerebroventricular infusion of an aldosterone synthase inhibitor or a mineralocorticoid receptor (MR) blocker attenuated the increase in Fra expression in PVN but not SON, and prevented the decrease in SFO after 14 days infusion of Ang II. A significant increase in mRNA expression of steroidogenic acute regulatory protein (StAR), a rate limiting enzyme in aldosterone production was found in glia cells of PVN and SFO assessed by rt-PCR after 3 days subcutaneous infusion of Ang II at 500 ng/kg/min. Total expression of aldosterone synthase (CYP11B2) mRNA was increased in SFO, MnPO, SON and PVN after 3 days of infusion of Ang II. After 14 days no significant changes were observed in the expression of StAR or CYP11B2 mRNA. In comparison, in adrenal StAR mRNA expression increased after 3 days but no longer after 14 days. In contrast, CYP11B2 mRNA expression in adrenal increased after both 3 and 14 days of infusion. These findings may support our hypothesis that chronic elevation of circulating Ang II increases neuronal activity in CVOs, presumably leading to activation of the PVN and SON to induce an increase in aldosterone production in magnocelular PVN and SON. In the second phase activation of CVOs appears to diminish, but an aldosterone-dependent amplifying mechanisms, causes sustained activation of the PVN and thereby hypertension.