Thesis (M. Med. Sc.)--University of Hong Kong, 2005.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A guanilina e a uroguanilina foram recentemente descobertas, respectivamente, no intestino e na urina, (Currie et al., 1992; Hamra et al., 1993). Fazem parte da famÃlia de peptÃdeos que ativam a guanilato ciclase de membrana (GC-C), aumentando os nÃveis intracelulares de cGMP (Schulz et al., 1990). EstÃo presentes em diversos tecidos, como respiratÃrio, linfonodos, testÃculos, cÃrebro e medula adrenal (Field et a.l., 1978; Forte et al., 1988, 1989; Hamra et al., 1993; Schulz et al., 1992). Foi comprovado que adicionando uma lisina na porÃÃo N-terminal, obtÃm-se um anÃlogo mais estÃvel e potente que a guanilina. O objetivo desse estudo à pesquisar os efeitos renais de um novo anÃlogo, ser-thr-lys-guanilina em sistema de perfusÃo. Os rins foram perfundidos com a soluÃÃo de Krebs-Henseleit modificada com 6g% de albumina bovina. Os dados foram comparados atravÃs de teste t de Student e ANOVA, com significÃncia p<0,05. Na dose de 0,1 Âg/mL, esse peptÃdeo apresentou efeitos similares aos da uroguanilina, na dose de 0,5 Âg/mL, em todos os parÃmetros testados. Ambas causaram aumento na pressÃo de perfusÃo (PP: de 101,5Â3,7 para 111Â2,9mmHg; de 101,2Â2,6 para 113,4Â2,5mmHg), no fluxo urinÃrio (FU: de 0,158Â0,016 para 0,223Â0,01 mL.g-1.min-1; de 0,16Â0,016 para 0,226Â0,2mL.g-1.min-1) e diminuiÃÃo no transporte tubular total e proximal de sÃdio (%TNa+: de 0,774Â0,06 para 0,724Â0,035; de 0,735Â0,065 para 0,773Â0,084), potÃssio (%TK+: de 66,89Â2,77 para 47,29Â3,34; de 63,54Â3,82 para 42,54Â8,14) e cloreto (%TCl-: de 85,69Â1,19 para 73,59Â2,63). Esses resultados foram similares aos previamente descritos apÃs a administraÃÃo da toxina termo-estÃvel da Escherichia coli (STa), guanilina, uroguanilina e lys-guanilina no mesmo sistema (Lima et al., 1992; Fonteles et al., 1996 e 1998). A dose maior (1 Âg/mL) causou aÃÃo antidiurÃtica (FU: de 0,165Â0,004 para 0,111Â0,009mL.g-1.min-1) e nenhum efeito sobre o transporte de sÃdio, embora a diminuiÃÃo na reabsorÃÃo tubular de potÃssio (%TK+: de 72,29Â1,2 para 49,73Â6,75) e cloreto (%TCl-: de 85,96Â0,79 para 81,9Â1,47) continuassem presentes. Nesta dose, nÃo apenas bloqueou o efeito diurÃtico da uroguanilina, como continuou causando um efeito antidiurÃtico significativo (FU: de 0,168Â0,004 para 0,116Â0,006). No entanto, nÃo foi capaz de bloquear os efeitos natriurÃticos da uroguanilina (%TNa+: de 85,35Â2,55 para 79,92Â1,05). O mecanismo de aÃÃo renal preciso dos peptÃdeos da famÃlia das guanilinas ainda nÃo foi completamente esclarecido. Sabe-se que esses peptÃdeos se ligam aos receptores GC-C (Schulz et al., 1990), porÃm hà indÃcios de que existam outras vias de aÃÃo renal, independentes desse receptor. Hà ainda a possibilidade de que haja duas entidades agindo de modo antagÃnico no sistema. Talvez haja a necessidade de isolÃ-los. A descoberta dos peptÃdeos da famÃlia das guanilinas promoveu avanÃos significativos na compreensÃo da regulaÃÃo endÃgena dos transportes de Ãgua e eletrÃlitos. O completo esclarecimento do seu mecanismo de aÃÃo renal oferece perspectivas reais para o tratamento de doenÃas como a hipertensÃo arterial. / Guanylin and uroguanylin are members of a family of peptides that stimulates cGMP production in several organic tissues, as intestine, kidney, airway, linfonodes, testis, brain and adrenal medulla (Field et a.l., 1978; Forte et al., 1988, 1989; Hamra et al., 1993; Schulz et al., 1992). Their 15 amino acid structures have been identified from rat intestine and opossum urine, respectively (Currie et al., 1992; Hamra et al., 1993), and they seem to be the link between intestine and kidney functions in controling blood pressure, as the âintestinal natriuretic hormoneâ suggested by some authors (Carey, 1978; Lennane et al., 1975). It was demonstrated that a Lysine-1 analog of guanylin is a more potent natriuretic and kaliuretic peptide. The aim of this study was to evaluate the renal effects of a novel analog of guanylin: ser-thr-lys-guanylin. Its effects were examined using isolated perfused kidneys from Wistar rats. All experiments were preceded by a 30 minutes internal control period and an external control group (C), in which the kidneys were perfused only with Krebs-Henseleit solution containing 6g% of a previously dialysed bovine albumine serum. The data was analyzed by Student t-test and ANOVA. The level of significance was set at p<0,05. Ser-thr-lys-guanylin, at the lowest dose (0.1 Âg/mL) and uroguanylin (0.5Âg/mL) caused similar effects. Both groups were able to increase perfusion presure (PP: 101.5Â3.7 to 111Â2.9mmHg; 101.2Â2.6 to 113.4Â2.5 mmHg), urinary flow (UF: 0.158Â0.016 to 0.223Â0.019 mL.g-1.min-1; 0.16Â0.016 to 0.226Â0.2mL.g-1.min-1) and to decrease sodium (%TNa+: 0.774Â0.06 to 0.724Â0.035; 0.735Â0.065 to 0.773Â0.084), potassium (%TK+: 66.89Â2.77 to 47.29Â3.34; 63.54Â3.82 to 42.54Â8.14) and cloride (%TCl-: 85.69Â1.19 to 73.59Â2.63) tubular reabsorption. Similar effects were also found in response to the Escherichia coli heat-stable enterotoxin (STa), guanylin, uroguanylin and lys-guanylin in the same system (Lima et al., 1992; Fonteles et al., 1996 e 1998). However, a greater dose (1Âg/mL), not only caused signifcantly decrease in the urinary flow (UF: 0.165Â0.004 to 0.111Â0.009 mL.g-1.min-1), but was also able to block the diuretic effects of uroguanylin (UF: 0.168Â0.004 to 0.116Â0.006 mL.g-1.min-1), although it still decreased potassium (%TK+: 72.29Â1.2 to 49.73Â6.75) and cloride(%TCl-: 85.96Â0.79 to 81.9Â1.47) tubular reabsorption. The precise renal mecanism of action of this family of peptides has not yet been fully elucidated. Deletion of GC-C genes in transgenic mice reveals that intestinal fluid secretion responses to STa are completely lost (Schulz et al., 1997 & Mann et al., 1997), but the natriuretic responses to STa and uroguanylin are retained (Carrithers et al., 1999), suggesting that other receptors are envolved. There is a possibility that there are to peptides causing antagonic effects. Further isolation may be necessary. Further studies are required to elucidate the specific renal mechanism of action of this new peptide. The discovery of guanylin and its family has promoted significant advances in the understanding of endogenous control of salt, water and eletrolites. The study of its analogs in perfused rat kidneys could help in elucidating their specific renal mecanism of action and bring great perspectives in the control of blood pressure.

Abstract Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are cardiac hormones, which are involved in the regulation of blood pressure and fluid homeostasis. The major determinant for ANP and BNP release are atrial and ventricular wall stretch, but also some vasoactive factors such as endothelin-1 (ET-1) can enhance cardiac hormone secretion. The mechanical stretch rapidly activates multiple signal transduction pathways in cardiac cells, but the cellular mechanisms mediating stretch-induced ANP secretion are still unknown. The aim of the present study was to examine the cellular mechanisms of autocrine/paracrine factors and stretch-induced ANP secretion. Genistein, a potent protein tyrosine kinase (PTK) inhibitor, rapidly increased cardiac contractile force and ANP secretion in perfused rat heart. This effect of genistein may be unrelated to the inhibition of PTKs since this stimulation was blocked by a L-type calcium channel antagonist and Ca2+/calmodulin-dependent protein kinase II inhibitor. Pregnancy hormone relaxin increased heart rate and ANP secretion in perfused spontaneously beating heart, suggesting that relaxin may have a role in modulating cardiac function. Cellular mechanisms of atrial wall stretch-induced ANP secretion were also studied. This enhanced secretion was blocked by sarcoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin and PTK inhibitor lavendustin A, indicating that thapsigargin sensitive Ca2+ pools and activation of PTK orPTK cascade have an important role in the regulation of stretch-secretion coupling. In addition, protein phosphatase inhibitor okadaic acid accelerated stretch-induced ANP secretion, suggesting that precise balance of protein kinase and phosphatase activity plays a role in mechanical stretch-induced ANP secretion. Finally interactions of endothelial factors regulating ANP exocytosis were studied. The potent nitric oxide synthase inhibitor L-NAME increased basal and atrial wall stretch-induced ANP secretion in the presence of ET-1, suggesting that nitric oxide may tonically inhibit ANP secretion.

Abstract Cardiac pathologies, including ventricular hypertrophy, are the primary cause of death in industrialized countries. Cardiac hypertrophy is often the consequence of work overload on the heart and characterizes several cardiovascular diseases, including atherosclerosis and hypertension. Cardiac hypertrophy is accompanied by genetic reprogramming characterized by the reexpression of several embryonic and growth response genes. Two of these genes encode A- and B-type natriuretic peptides (ANP and BNP), two cardiac-specific hormones secreted by myocytes, which play an important role in blood pressure regulation. The aim of the present study was to study the effect of acute pressure overload on BNP gene expression in the hearts of normal and hypertensive rats and then to examine the role of a passerine factor, angiotensin II (Ang II), on volume and pressure overload -induced ANP and BNP secretion and synthesis. Further, the aim was to characterize elements on the BNP promoter mediating hemodynamic stress in vivo. BNP gene expression was studied in conscious spontaneously hypertensive (SHR) rats and together with ANP in two hypertensive, ream Transgenic rat models. The increased workload of the heart was produced by the infusion of vasopressin (AVP), phenylephrine (PHE) or bolus saline infusion. The increased workload caused rapid increases in cardiac BNP mRNA levels. Daring both AVP and PHE infusions, substantial increases in ventricular BNP mRNA levels were already evident after I h, and peak levels of BNP mRNA were reached at 4 h. Transgenic rats carrying one extra mouse renin gene showed impaired secretion and synthesis of ANP and BNP, while double transgenic rats carrying both human angiotensinogen and human renin genes showed augmentation of left atrial, but not ventricular BNP gene expression in response ta acute pressure overload. To characterize the elements mediating hemodynamic stress, bi-lateral nephrectomy was performed. GATA motif transduced the hemodynamic stress stimulus 26–28 hrs postnephrectomy in BNP gene expression.In conclusion, these results show that pressure overload abruptly stimulates the cardiac expression of a noncontractile protein gene BNP, suggesting that it may be used as a myocyte-specific marker of mechanical loading. BNP gene expression was augmented in atria hut nut in ventricles in response to pressure overload in an experimental model of hypertension, suggesting that high local levels of Ang II may differentially regulate cardiac gene expression in atrial and ventricular myocytes in double transgenic rats. At the transcriptional level, acute hemodynamic stress produced by nephrectomy increases BNP reporter expression through a GATA-dependent pathway.

Abstract The natriuretic peptides are a family of hormones secreted by the heart. They play a fundamental role in salt and water balance and blood pressure regulation. Atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are the known members of the mammalian natriuretic peptide family. A major stimulus for the secretion of cardiac natriuretic peptides is myocyte stretch. Therefore, the secretion of natriuretic peptides is increased in response to elevated blood volume. Natriuretic peptide production and release is also affected by several other factors, such as endothelin-1 (ET-1), acting in paracrine fashion. The aim of this study was to elucidate factors regulating the novel cardiac peptide hormone, salmon cardiac peptide (sCP), belonging to the family of natriuretic peptides. The role of mechanical load, temperature and ET-1 in sCP secretion and production was studied using in vitro (isolated perfused ventricle preparation) and in vivo methods. Comparisons between the natriuretic peptide systems in fish and mammals were done to clarify functional evolution of this hormone family. Salmon (Salmo salar) was selected as a model, since it has an outstanding adaptability to wide variations in environmental salinity and has developed defence mechanisms against volume or salt load. The results showed that salmon ventricle stores large amounts of the prohormone of sCP, whereas the secreted form is the mature 29-amino acid form. The N-terminal fragment of pro-sCP is co-secreted with sCP in equimolar amounts. sCP is released rapidly in response to appropriate stimulus, whereas induction of its gene expression is slower. Mechanical load is an important regulator of sCP secretion. Temperature also plays a major role in regulating sCP plasma concentration by affecting its elimination from circulation. Additionally, ET-1 is a potent secretagogue of the sCP system and an inotropic agent in salmon heart. Furthermore, the present results reveal remarkable synergism between the cardiac effects of ET-1 and β-adrenergic stimulation. In conclusion, the sCP system in salmon ventricle largely resembles the ANP system in mammalian atrium, while also having specific characteristics, such as a regulated ventricular natriuretic peptide secretion pathway. Therefore, the sCP system offers a unique model for studying mechanisms of natriuretic peptide biology.

Abstract Natriuretic peptides have emerged as important candidates for the development of diagnostic tools in cardiovascular disease. Their increased concentrations have been found to be useful for ruling out disease of cardiac origin, as prognostic indicators, and in the follow-up of patients with heart failure. In order for natriuretic peptides to be efficient biomarkers, analytical problems in assay specificity and calibration need to be resolved. The aim of the present study was to elucidate circulating molecular components of N-terminal fragments of A- and B-type natriuretic peptides (NT-proANP and NT-proBNP) in human blood, and to develop reliable and novel assays for their measurement with clinical application. Reliable immunoassays for NT-proANP and NT-proBNP were set up based on recombinant calibrators and antisera against different epitopes. A novel immunoassay for detecting the activation of A- and/or B-type natriuretic peptide systems, referred to as NT-proXNP, was also developed. The chromatographic results of human plasma and serum samples indicated that NT-proANP and especially NT-proBNP are heterogeneous in human circulation. They are truncated at both termini, causing a serious risk of preanalytical errors. Further studies with recombinant peptides confirmed that the central parts of NT-proANP and NT-proBNP are stable in plasma and serum even at harsh storage conditions. Thus the most reliable assays are directed at the central portions of the molecule only. All developed assays were applicable to clinical samples of cardiac patients. NT-proXNP showed a diagnostic efficiency equal to or slightly better compared to individual NT-proANP and NT-proBNP assays. Furthermore, the prognostic value of NT-proANP and NT-proBNP was investigated in a population-based sample of men. Both peptides were strong predictors of mortality and its co-morbidities, adding to the prognostic value of conventional risk factors.

Philosophiae Doctor - PhD / Plant natriuretic peptides (PNPs) are a novel class of extracellular, systemically mobile molecules that elicit a number of plant responses important in homeostasis and growth. Natriuretic peptides were first identified in vertebrates where they play a role in the regulation of salt and water balance. Subsequent experimental investigations have identified the presence of a natriuretic peptide hormone system in plants. While PNPs have been implicated in various physiological responses such as stomatal guard cell movements and regulation of net water uptake, its biological role has remained elusive. Here we have used co-expression and promoter content analysis tools to understand the biological role of the Arabidopsis thaliana PNP (AtPNP-A). The analysis of AtPNP-A and its co-expressed genes revealed that genes annotated as part of the systemic acquired resistance (SAR) pathway were over-represented, thus suggesting that AtPNP-A may function as a component of plant defense responses and specifically, SAR. The results further show that AtPNP-A shares many characteristics with pathogenesis related (PR) proteins in that its transcription is strongly induced in response to pathogen challenges, thus implying a newly described role for AtPNP-A in pathogen attack. Additional tissue expression analysis also indicated distinct localization of PNP activity in sepals and transcriptional meta-analysis showed that AtPNP-A may play a role in starch breakdown. Therefore, together with the finding that AtPNP-A plays a role in regulating phloem transport, we also hypothesize that AtPNP-A may play a role in phloem unloading in sepals to assist processes such as seed formation in plants. In plants, the second messenger, guanosine 3’,5’-cyclic monophosphate (cGMP) mediates a whole range of important processes including salinity tolerance, disease resistance, drought tolerance and responses to light. Since PNPs regulate water and salt homeostasis via a cGMP-dependent signaling pathways, it is thus important to analyse the transcriptome induced by the second messenger (cGMP) in Arabidopsis thaliana to give a better understanding of its mechanism of action. This study was also supplemented by the analysis of the gibberellic acid (GA) dependent transcriptome, since cGMP also plays a role its transcription pathway. This data analysis, together with promoter content investigation, revealed that genes upregulated after cGMP treatment and down-regulated in the GA insensitive mutant (ga1-3) were enriched with a GA response element (GARE), while no GARE enrichment were observed in genes up-regulated in the ga1-3 mutant. These findings suggest that GARE is indicative of GA-induced and cGMP-dependent transcriptional up-regulation. Gene ontology analysis confirmed previous reports that cGMP is involved in ion homeostasis and indicated that the transcriptional cGMP response is bi-polar in the sense that both genes up- and down-regulated in response to cGMP is involved in cation transport. Additionally, ab initio analysis of genes transcriptionally dependent on cGMP identified CHX8 as a hub gene and promoter content of CHX8 co-expressed genes show enrichment of the GARE motif. The fact that CHX8 has its highest expression levels during male gametogenesis and pollen tube growth, together with our findings, suggest that GA-induced and cGMP- dependent genes may play a key role in ion and water homeostasis in the male gametophyte. Finally, we propose that the type of analysis undertaken here can yield new insights into gene regulation networks and inform experimental strategies to unravel complex transcription regulatory systems under different developmental and stimulus specific conditions. / South Africa

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19964号 / 医博第4154号 / 新制||医||1017(附属図書館) / 33060 / 京都大学大学院医学研究科医学専攻 / (主査)教授 柳田 素子, 教授 横出 正之, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13206号 / 論医博第2160号 / 新制||医||1031(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 木村 剛, 教授 岩田 想, 教授 秋山 芳展 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Objective: We sought to determine N-terminal proatrial natriuretic peptide concentrations [Nt-proANP] in plasma from cats with hypertrophic cardiomyopathy (HCM). Secondarily, we wished to evaluate the relationship between [Nt-proANP] and echocardiographic variables. Methods: Venous blood samples were obtained from seventeen cats with HCM and from nineteen healthy cats. Plasma [Nt-proANP] was determined using an ELISA assay. The relationship between plasma [Nt-proANP] and M-mode, 2-dimensional and Doppler echocardiographic variables was evaluated. Cats that were hyperthyroid or had evidence of renal disease were excluded from the study. Results: The mean plasma [Nt-proANP] was higher in cats with HCM (3.81 +/- 1.23 pmol/l) than in control cats (3.08 +/- 1.41 pmol/l); however, this difference was not statistically significant (p=0.17). There was a significant correlation between plasma [Nt-proANP] and left ventricular posterior wall thickness (r = 0.42; p=0.01). Additionally, plasma [Nt-proANP] was correlated with left atrial size (r = 0.35; p=0.03). A linear regression model was developed to further explore these relationships. LAs2D and LVPWd had an interactive effect on plasma [Nt-proANP] (R2 = 0.2737; p= 0.02). There was no correlation between any other echocardiographic variable and plasma [Nt-proANP]. There was no correlation between plasma [Nt-proANP] and heart rate (HR), body-weight, or age. Conclusions: Cats with HCM do not have significantly higher plasma [Nt-proANP] than normal cats. There was a significant linear relationship between [Nt-proANP] and LAs2D, LVPWd and the model that described their interaction. / Master of Science