by Lee Suk Kuen Zoe. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (p. 271-314). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstract in Chinese.

by Tung Sin Yi, Cindy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 117-132). / Acknowledgements --- p.I / List of Abbreviations --- p.II / Abstract --- p.IV / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Astrocytes in the Central Nervous System (CNS) l / Chapter 1.1.1 --- Characteristics of astrocytes / Chapter 1.1.2 --- Functions of astrocytes / Chapter 1.1.2.1 --- General functions of astrocytes / Chapter 1.1.2.2 --- Effects of neuroactive peptides on astrocytes / Chapter 1.1.3 --- Gliomas and the rat C6 glioma cells / Chapter 1.2 --- C-Type natriuretic peptide (CNP) in the CNS --- p.9 / Chapter 1.2.1 --- Structure and distribution of natriuretic peptides in the CNS / Chapter 1.2.2 --- Actions of CNP / Chapter 1.2.3 --- Natriuretic peptide receptors and signal transduction in astrocytes / Chapter 1.3 --- Endothelin-3 (ET-3) in the CNS --- p.18 / Chapter 1.3.1 --- Structure and distribution of endothelins (ETs) in the CNS / Chapter 1.3.2 --- Actions of ET-3 / Chapter 1.3.3 --- Endothelin receptors and signal transductionin astrocytes / Chapter 1.4 --- cGMP second messenger system in astrocytes --- p.28 / Chapter 1.4.1 --- Second messenger systems in astrocytes / Chapter 1.4.2 --- cGMP as second messenger in astrocytes / Chapter 1.4.3 --- Post cGMP cascade effects / Chapter 1.5 --- The aims of this project --- p.33 / Chapter Chapter 2 --- Methods / Chapter 2.1 --- In vitro culture of rat C6 glioma cells --- p.36 / Chapter 2.1.1 --- Preparation of reagents / Chapter 2.1.2 --- Culture of C6 glioma cells / Chapter 2.1.3 --- "Cell plating in 6-well, 24-well and 96-well plastic trays" / Chapter 2.2 --- Determination of cGMP --- p.40 / Chapter 2.2.1 --- Measurement of cGMP / Chapter 2.2.2 --- Data analysis / Chapter 2.3 --- Determination of the effect of CNP on cGMP productionin C6 cells --- p.41 / Chapter 2.4 --- Determination of the effect of ET-3 on the action of CNPin C6cells --- p.44 / Chapter 2.4.1 --- Measurement of intracellular cGMP levels affected by ET-3 / Chapter 2.4.2 --- Measurement of intracellular cGMP levels affected by CNP with ET-3 pretreatment / Chapter 2.5 --- Determination of the effects of PKC activator and inhibitor on CNP-treated C6 cells --- p.46 / Chapter 2.5.1 --- Measurement of intracellular cGMP levels affected by PKC activator or inhibitor / Chapter 2.5.2 --- Measurement of intracellular cGMP levels affected by CNP with PKC activator or inhibitor pretreatment / Chapter 2.5.3 --- Measurement of intracellular cGMP levels affected by CNP with PKC inhibitor antagonized PMA or ET-3 pretreatment / Chapter 2.6 --- Determination of the effect of arachidonic acid on the action of CNP in C6 cells --- p.49 / Chapter 2.7 --- Determination of the effects of ET-3 and CNP on calcium uptake in C6 cells --- p.50 / Chapter 2.8 --- Determination of the effects of CNP and ET-3 on cell volume change in C6 cells --- p.51 / Chapter 2.9 --- Determination of the effects of CNP and ET-3 on glucose and amino acids uptake in C6 cells --- p.53 / Chapter 2.9.1 --- Measurement of glucose uptake in CNP - and/or ET- 3-treated C6 cells / Chapter 2.9.2 --- Measurement of amino acids uptake in CNP - and/or ET-3-treated C6 cells / Chapter 2.10 --- "Determination of thymidine, uridine and leucine incorporation in CNP - and/or ET-3- treated C6 cells" --- p.55 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Effects of CNP and ET-3 on cGMP production in cultured rat C6 glioma cells --- p.56 / Chapter 3.1.1 --- Effect of CNP on cGMP production in cultured C6 glioma cells --- p.57 / Chapter 3.1.1.1 --- The time course of CNP on cGMP production / Chapter 3.1.1.2 --- Dosage-response of CNP on cGMP production / Chapter 3.1.2 --- Effect of ET-3 on cGMP production in C6 glioma cells --- p.61 / Chapter 3.1.2.1 --- Effect of ET-3 on basal cGMP production / Chapter 3.1.2.2 --- Effect of pre-exposure duration to ET-3 on CNP-induced cGMP formation / Chapter 3.1.2.3 --- Dosage-response of ET-3 on CNP-induced cGMP production / Chapter 3.1.3 --- Effect of PMA on cGMP production in C6 glioma cells --- p.65 / Chapter 3.1.3.1 --- Effect of PMA on basal cGMP production / Chapter 3.1.3.2 --- Effect of pre-exposure duration to PMA on CNP-induced cGMP formation / Chapter 3.1.3.3 --- Dosage-response of PMA on CNP-induced cGMP production / Chapter 3.1.4 --- Effects of PKC inhibitors on cGMP production in C6 glioma cells --- p.73 / Chapter 3.1.4.1 --- Effects of PKC inhibitors on basal cGMP production / Chapter 3.1.4.2 --- Effects of PKC inhibitors on CNP-induced cGMP formation / Chapter 3.1.4.3 --- Antagonism of PKC inhibitors on the action of PMA on CNP-induced cGMP formation / Chapter 3.1.4.4 --- Antagonism of PKC inhibitors on the action of ET-3 on CNP-induced cGMP formation / Chapter 3.1.5 --- Effect of arachidonic acid on CNP-induced cGMP production in C6 glioma cells --- p.82 / Chapter 3.2 --- Effects of CNP and ET-3 on cellular metabolism in cultured rat C6 glioma cells --- p.83 / Chapter 3.2.1 --- Effects of CNP and ET-3 on calcium uptake in C6 glioma cells --- p.86 / Chapter 3.2.2 --- Effects of CNP and ET-3 on cell volume changes in C6 glioma cells --- p.89 / Chapter 3.2.3 --- Effects of CNP and ET-3 on glucose and amino acids uptake in C6 glioma cells --- p.91 / Chapter 3.2.4 --- Effects of CNP and ET-3 on C6 cell proliferation --- p.98 / Chapter 3.2.5 --- Effects of CNP and ET-3 on RNA synthesis --- p.101 / Chapter 3.2.6 --- Effects of CNP and ET-3 on protein synthesis --- p.103 / Chapter Chapter 4 --- Discussion and Conclusion --- p.105 / References --- p.117

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.