胰腺的胰島具有重要的生理功能，表現在系列的荷爾蒙，特別是能夠控制血糖穩態的胰島素的合成和分泌。胰島素的功能受到各種分子信號及環境的調節。在過去的十年裡，腎素血管緊張素系統(RAS)被發現除了調節血壓和體液穩態之外還具有局部性的生理功能。根據我們最近的發現，胰島存在自有的腎素血管緊張素系統並且可能在胰島生理作用和糖尿病方面發揮新穎的作用。同時，越來越多的研究發現一些與臨床相闊的調節因子在胰島的功能和糖尿病中起著關鍵的作用。這些調節因子促進胰島素分泌並且可以調節胰島細胞的生長和凋亡。其中一些調節因子顯示出極大的研究價值。胰高血糖素樣肽-1(GLP-1)能通過它在胰島上的受體改善胰島的功能和血糖的控制;另一方面， 維生素D 也可以通過它在胰島B細胞上的受體來起到調節胰島素分泌及控制糖尿病的作用。像胰島局部RAS一樣， GLP-1 和維生素D 都可以通過它們在同一個靶器官--胰島細胞上的受體來發揮它們的功能。因此，不難想象這三種調節因子之前具有潛在的聯系並且直接或間接地影響胰島功能。此研究可以分為三部分以闡述這三種調節因子在胰島上的新穎作用(1) GLP-l 和RAS 在胰島功能上的潛在協同作用; (2)維生素D 對於胰島RAS 表達的調節作用及對膜島功能的影響；(3) 維生素D 缺乏下的胰島RAS 表達以及胰島功能的改變。 / 在第一部分的研究裡，我們檢測了阻斷血管緊張素一型受體(纈沙坦)和增強GLP-l 作用(DPPIV 抑制劑LAF237) 的復合效應對二型糖尿病小鼠(db/db) 血糖控制和胰島功能方面的影響。我們比較了接受單一給藥和聯合給藥的db/db 小鼠的胰島功能。所有的藥物處理都改善了db/db 小鼠的血糖穩態，而聯合給藥組在增加胰島B細胞面積，減少細胞凋亡，促進增殖以及降低膜島氧化應激和膜島纖維化方面體現出復合效應。另外，短期的聯合給藥顯著促進分離出來的胰島細胞的胰島素分泌。這些結果顯示了血管緊張素型受體阻斷劑和DPPIV 抑制劑在改善胰島的結構與功能以及治療二型糖尿病方面具有復合效應。 / 據研究，維生素D 是種具有抗糖尿病和高血壓作用的荷爾蒙，而不適合的RAS活性能夠減少胰島功能和糖耐量。維生素D 對腎臟腎素的直接抑制作用表明維生素D 可能可以調節胰島得局部RAS 活性進而調節胰島的生理作用。因此第二部分的實驗旨在研究維生素D 是否能夠抑制分離培養的胰島中非正常表達的胰島局部RAS組分並且改善胰島且細胞功能。維生素D 受體存在於胰島且細胞的核與質中，計量依賴性地調節受體對活性維他命D-骨化三醇的反應。骨化三醇的刺激可以通過增加維生素D24羥化黣激發胰島局部維他命D 系統的反饋機制。在分離的胰島中，長期處於高糖的環境，胰島局部RAS 的異常表達可以一定濃度的骨化三醇治療和預防。然而，骨化三醇的送科治療效果，並沒有在生理正常糖濃度的情況下被發現。另外，在高糖環境下，骨化三醇增加胰島素前體合成以及葡萄糖刺激的服島素分泌。這些結果顯示骨化三醇能夠調節以及保護高糖環境引起的異常胰島RAS 組分表達並通過增加胰島素的合成與分泌來改善胰島的功能，為在高血糖和糖尿病情況下的維生素D 與胰島功能關系提供了新的機制。 / 循環中的維生素D 水平與血糖濃度以及糖尿病的患病風險成反比。第二部分的實驗結果現實了維生素D 具有潛在的調節胰島RAS 進而調節胰島功能的作用。因此，在第三部分的實驗裡，我們假設不充足的維生素D 水平可能引起異常的胰島RAS 表達進而引起胰島功能障薇。為了這個目的，我們使用了維生素D 受體缺失的基因敲除小鼠和維生素D 缺乏小鼠來檢測糖代謝，膜島形態以及局部RAS 組分的表達。結果顯示，在缺乏維生素D 以及正常的維生素D 作用的情況下，胰島局部RAS 組分異常表達。而這個維生素D 導致的RAS 異常表達的作用可能發生在高血糖現象之前，從而導致了胰島功能障礙，異常的糖代謝以及減弱的胰島且細胞本身的胰島素作用。這些結果為在生理情況下，維生素D 可以通過調節胰島局部RAS 的表達進而調節胰島功能提供了有力的支持。 / 總括來說，胰島局部RAS 在持續高糖環境下的胰島功能中有著關鍵的作用。GLP-l 和維生素D 都與胰島RAS 具有潛在的生物相關性並可以影響RAS 的表達，進而調節胰島功能和自細胞體積。我們的實驗數據顯示了這三種調節系統共同作用並調節目突島細胞功能以及血糖穩態，進一步提議了它們在二型糖尿病治療中的價值。 / Pancreatic islets perfonn critical biological activities by means of synthesizing and releasing islet peptide honnones, notably insulin that controls our glucose homeostasis. The insulin secretory function is, in turn, governed by various conditions and signaling molecules. In the past decade, it is recognized that the renin-angiotensin system (RAS) has local function rather than the maintenance of blood pressure and fluid homeostasis. With our recent recognition of an islet RAS, it is believed that it has novel roles in islet physiology and diabetes. Meanwhile, more and more clinically relevant regulators that have pivotal roles in islet function and diabetes have been well investigated; such regulators have positive action on insulin secretion, B-cell replication and cell apoptosis/proliferation balance. Of great interest in this context is the glucagon-like peptide I (GLP-I) that improves islet function and glycemic control via its islet specific receptors located on the islets. On the other hand,vitamin D also regulates islet insulin secretion and diabetes via its mediation of receptors on islet B-cells. Like islet RAS, GLPI and vitamin D exert their biological effects via mediation of respective receptors located on the common target, i.e. the islet beta-cells. As such, it is plausible to propose that all these three regulators have potential interactions so as to affect islet functions in a direct or an indirect manner. Accordingly, the primary objective of this study is to examine the potential roles oflocal RAS, GLP-I and vitamin D system in pancreatic islet function. The present study is thus divided into three main parts addressing the issues of these three novel regulators in islet function: (1) the potential synergism of GLP-I and RAS in islet function; (2) the modulatory effects of vitamin D on islet RAS expression and function; (3) The altered islet RAS and islet function under a hypovitaminosis D condition. / In the first part of our study, we examined the combined effect of blocking islet A Tl receptor (ATl receptor blocker: valsartan) and enhancing GLP-l actions (DPP IV inhibitor: LAF237) on islet function and glycemic control in a mouse model with type 2 diabetes, db/db mice. We compared the islet function in db/db mice with either valsartan or LAF237 mono treatment or combined treatment. Consistently, all these treatments improved glucose homeostasis in db/db mice while combined treatment resulted in a significant increase in islet B-cell area by decreasing cell apoptosis and increasing proliferation, together with marked decreases of islet oxidative stress and fibrosis. In addition, a short-term effect on stimulating insulin secretion was also observed in isolated islets with combined treatment. These results indicate that the combination treatments with ATl receptor blocker and DPP IV inhibitor has beneficial additive effects on islet structure and function in type 2 diabetes, compared with their monotherapeutic treatments. / It is reported that vitamin D is a hormone with anti-diabetic and anti-hypertension effects in human while inappropriate RAS activity has been known to reduce islet function and glucose tolerance. The direct suppressive effect of vitamin D on renal renin activity indicates vitamin D may acts as a regulator in RAS activity thus modulate islet physiology. In the second part of our study, it was aimed to study whether vitamin D vitamin D downregulation of abnormal islet RAS activity improves B-cell function using an isolated pancreatic islet model. VDR was localized in islet B-cell nuclei and cytoplasm, mediated responses to active form of vitamin D calcitriol in a dose-dependent manner. This islet local vitamin D system may have its own feedback system as a marked increase ofCYP24 transcription was triggered by calcitriol stimulation. In isolated islets exposed to prolonged high glucose environment, abnormal expressed islet RAS components could be reversed or protected by calcitriol at a specific concentration. However, the inhibition effect of calcitriol on islet RAS were not observed at physiological glucose concentrations. In additon, calcitriol increased islet proinsulin synthesis and insulin secretion with hyperglycemia. These results indicated that calcitriol modulate or protect the abnormal isolated islet RAS component expression against hyperglycemia and improve islet function via increasing insulin synthesis and secretion, which might provide an alternative mechanism by which vitamin D availability enhances islet function in hyperglycemia or diabetes. / The circulating vitamin D level is inversely related to blood glucose level and risks of diabetes. Results in the second part of experiments suggested the potential RAS modulatory effect of vitamin D in isolated islets Therefore, in the third part of our study, we hypothesize that the insufficient vitamin D levels may lead to the inappropriate regulation of islet RAS expression and thus result in islet dysfunction. To achieve this, we examined the potential islet RAS-mediated effect of vitamin D on islet function by accessmg glucose homeostasis, islet histomorphology, and local RAS expression and function by means of using a vitamin D receptor knockout and diet-induced vitamin D deficiency mouse models. Results showed that the islet RAS components were abnormally expressed when lacking a sufficient vitamin D level and normal vitamin D action. These observed effects of insufficient vitamin D might occur prior to onset of hyperglycemia thus modulating islet RAS expression, which in turn lead to islet failure and dysfunctional glucose homeostasis, together with decreased insulin actions in islet B-cells. These results provide supports for the view that vitamin D physiologically exerts modulatory effects on islet function by downregulating islet RAS expression and function. / In summary, islet local RAS may have a central role in islet function under prolonged hyperglycemic stress. GLP-l and vitamin D have biological interactions with the islet RAS by downregulation of its expression and function, thereby affecting islet cell function and cell mass. Our data indicate that all three regulators work together in the regulation of pancreatic islet B-cell functions and glucose homeostasis, further suggestive of their potential values in the treatment of type 2 diabetes. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Cheng, Qianni. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves [205]-243). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / 摘要 --- p.v / Acknowledgements --- p.viii / List of Publications --- p.x / Table of Contents --- p.xii / List of Abbreviations --- p.xvi / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Endocrine Pancreas --- p.2 / Chapter 1.1.1 --- The structure and composition of endocrine pancreas --- p.3 / Chapter 1.1.2 --- Functions of endocrine pancreas --- p.4 / Chapter 1.1.3 --- Insulin structure and insulin receptors --- p.8 / Chapter 1.1.4 --- Mechanisms of insulin secretion --- p.11 / Chapter 1.1.5 --- Mechanisms of insulin actions --- p.18 / Chapter 1.1.6 --- Disorders of the endocrine pancreas --- p.22 / Chapter 1.2 --- Diabetes mellitus --- p.23 / Chapter 1.2.1 --- Type 1 diabetes mellitus (TlDM) --- p.24 / Chapter 1.2.2 --- Type 2 diabetes mellitus (T2DM) --- p.26 / Chapter 1.2.3 --- Other types of diabetes mellitus --- p.29 / Chapter 1.2.4 --- Islet dysfunction and T2DM --- p.30 / Chapter 1.3 --- Renin-angiotensin system (RAS) --- p.33 / Chapter 1.3.1 --- Components ofRAS --- p.33 / Chapter 1.3.2 --- Tissue local RAS --- p.42 / Chapter 1.3.3 --- Pancreatic local RAS --- p.43 / Chapter 1.4 --- Glucagon like peptide-l (GLP-l) and pancreatic islet function --- p.54 / Chapter 1.4.1 --- Gastrointestinal incretin honnones --- p.54 / Chapter 1.4.2 --- GLP-l and pancreatic islet function --- p.56 / Chapter 1.4.3 --- Incretin based therapies for T2DM --- p.59 / Chapter 1.4.4 --- GLP-lIRAS axis and pancreatic islet function --- p.62 / Chapter 1.5 --- Vitamin D and pancreatic islet function --- p.64 / Chapter 1.5.1 --- Vitamin D synthesis and metabolism --- p.65 / Chapter 1.5.2 --- Vitamin D physiological functions and pancreatic islets --- p.67 / Chapter 1.5.3 --- Vitamin D and diabetes mellitus --- p.68 / Chapter 1.5.4 --- Vitamin D and RAS --- p.70 / Chapter 1.6 --- Objectives --- p.71 / Chapter Chapter 2 --- Materials and Methods --- p.73 / Chapter 2.1 --- Experimental animal models --- p.74 / Chapter 2.1.1 --- Animal model ofT2DM --- p.74 / Chapter 2.1.2 --- Animal model for pancreatic islet isolation --- p.75 / Chapter 2.1.3 --- Vitamin D receptor knockout mice (VDRKO mice) --- p.75 / Chapter 2.1.4 --- Animal model for vitamin D deficiency --- p.76 / Chapter 2.2 --- Pancreatic islet isolation and culture --- p.76 / Chapter 2.2.1 --- Mice pancreatic islet and single B-cell isolation --- p.77 / Chapter 2.2.2 --- Primary culture of isolated pancreatic islets: --- p.78 / Chapter 2.3 --- Physiological assay for pancreatic islet function --- p.78 / Chapter 2.3.1 --- Measurement of blood glucose and glucose tolerance test --- p.78 / Chapter 2.3.2 --- Measurement of glucose-induced insulin secretion --- p.79 / Chapter 2.3.3 --- Measurement of (pro )insulin biosynthesis --- p.80 / Chapter 2.4 --- Detection ofmRNA expression --- p.80 / Chapter 2.4.1 --- Design of primers --- p.81 / Chapter 2.4.2 --- mRNA extraction and cDNA synthesis --- p.82 / Chapter 2.4.3 --- Detection of mRN A expression by conventional peR --- p.83 / Chapter 2.4.4 --- SYBR Green real-time peR --- p.83 / Chapter 2.4.5 --- Real-time peR analysis using the comparative eT method --- p.84 / Chapter 2.5 --- Detection of protein expression --- p.84 / Chapter 2.5.1 --- Western blot analysis --- p.84 / Chapter 2.5.2 --- Immunostaining assessment --- p.85 / Chapter 2.6 --- In situ detection of oxidative stress, proliferation and apoptosis --- p.88 / Chapter 2.6.1 --- Detection of islet reactive oxygen species --- p.88 / Chapter 2.6.2 --- Detection of cell proliferation --- p.89 / Chapter 2.6.3 --- Measurement of cell apoptosis --- p.90 / Chapter 2.7 --- Statistical data analysis --- p.90 / Chapter Chapter 3 --- Combination of DPP-IV Inhibitor LAF237 with ATl Receptor Antagonist Valsartan Enhances Pancreatic Islet Morphology and Function in a Mouse Model of Type 2 Diabetes (This work has been published in J Pharmacal Exp Ther, 327: PI-9) --- p.91 / Chapter 3.1 --- Abstract --- p.92 / Chapter 3.2 --- Introduction --- p.94 / Chapter 3.3 --- Materials and Methods --- p.96 / Chapter 3.4 --- Results --- p.103 / Chapter 3.4.1 --- Effects of acute treatment with GLP-I and valsartan on insulin secretion in isolated islets --- p.103 / Chapter 3.4.2 --- Effects of LAF237 and valsartan on pancreatic --- p.105 / Chapter 3.4.3 --- Effects of LAF237 and valsartan on --- p.107 / Chapter 3.4.4 --- Effects ofLAF237 and valsartan on islet apoptosis --- p.109 / Chapter 3.4.5 --- Effects of LAF237 and valsartan on islet fibrosis --- p.110 / Chapter 3.4.6 --- Effects of LAF237 and valsartan on pancreatic islet superoxide and nitrotyrosine expression --- p.113 / Chapter 3.4.7 --- Effects of LAF237 and valsartan on bood glucose concentration and glucose tolerance in db/db diabetic mice --- p.116 / Chapter 3.5 --- Discussion --- p.119 / Chapter Chapter 4 --- The Role of Calcitriol in Modulating the Expression and Function of Islet Renin-Angiotensin System in Isolated Mouse Pancreatic Islets --- p.124 / Chapter 4.1 --- Abstract --- p.125 / Chapter 4.2 --- Introduction --- p.127 / Chapter 4.3 --- Materials and Methods --- p.130 / Chapter 4.4 --- Results --- p.135 / Chapter 4.4.1 --- The expression of islet VDR under different glucose conditions and the effects of calcitriol --- p.135 / Chapter 4.4.2 --- The effect of calcitriol on high glucose-modulated islet RAS component expression --- p.140 / Chapter 4.4.3 --- The protective effect of calcitriol against high glucose on islet RAS component expression --- p.144 / Chapter 4.4.4 --- The effect of calcitriol on (pro )insulin biosynthesis and insulin release in isolated islets --- p.148 / Chapter 4.5 --- Discussion --- p.151 / Chapter Chapter 5 --- Altered Islet Local Renin-Angiotensin System and Islet Function in Mice with Hypovitaminosis D --- p.158 / Chapter 5.1 --- Abstract --- p.159 / Chapter 5.2 --- Introduction --- p.160 / Chapter 5.3 --- Materials and methods --- p.163 / Chapter 5.4 --- Results --- p.168 / Chapter 5.4.1 --- Glucose homeostasis and islet morphology in VDR KO mice --- p.168 / Chapter 5.4.2 --- Expression of vitamin D receptor and major RAS components in the pancreatic islets of WT and VDR KO mice --- p.170 / Chapter 5.4.3 --- Vitamin D deficiency in mice on a vitamin D deficient diet --- p.172 / Chapter 5.4.4 --- Altered glucose homeostasis in vitamin D deficient mice --- p.174 / Chapter 5.4.5 --- Islet histomorphology in vitamin D deficient mice --- p.176 / Chapter 5.4.6 --- Regulation of islet RAS components expression in vitamin D deficient mice --- p.179 / Chapter 5.4.7 --- Transcriptional regulation of islet insulin receptor and its substrates in vitamin D deficient mice --- p.181 / Chapter 5.4.8 --- Effect of calcitriol treatment on glucose tolerance in vitamin D deficient mice --- p.183 / Chapter 5.5 --- Discussion --- p.185 / Chapter Chapter 6 --- General Discussion --- p.191 / Chapter 6.1 --- Combination effects of blocking islet RAS components and enhancing incretin activity on improving islet function in type 2 diabetes --- p.193 / Chapter 6.2 --- Potential modulatory effect of vitamin D on islet RAS expression and action --- p.196 / Chapter 6.3 --- The role of vitamin D in modulating islet RAS in glucose homeostasis and islet function --- p.199 / Chapter 6.4 --- The significance ofRAS, GLP-l and vitamin D in the management of T2DM --- p.201 / Chapter 6.5 --- Conclusion --- p.202 / Chapter 6.6 --- Future studies --- p.202 / Chapter Chapter 7 --- Bibliography --- p.205

La fibrillation auriculaire (FA) est l’arythmie cardiaque soutenue la plus fréquente et elle peut mener à des conséquences médicales sévères, comme les accidents vasculaires cérébraux (AVC). Des remodelages électrophysiologiques, c’est-à-dire dans les courants ioniques, ainsi que des remodelages structurels, comme l’hypertrophie et la fibrose, ont été associés avec la fibrillation auriculaire. Ces remodelages peuvent affecter la conduction auriculaire, augmentant les risques de fibrillation auriculaire. Plusieurs facteurs de risque ont été associés avec la fibrillation auriculaire, parmi ceux-ci, on compte les niveaux élevés d’angiotensine II. L’angiotensine II est l’effecteur principal du système rénine-angiotensine (SRA) et ses effets néfastes sont médiés par le récepteur de type I à l’angiotensine II (AT1R). La suractivation du SRA, par l’action d’AT1R peut entraîner, entre autres, l’hypertension, l’insuffisance cardiaque, ainsi que des arythmies, comme la fibrillation auriculaire. Cependant, les mécanismes par lesquelles l’angiotensine II affectent directement le cœur et prédispose à la fibrillation auriculaire sont encore peu connus. L’objectif de ce projet de recherche consiste à étudier les remodelages électriques et structurels chez des souris transgéniques surexprimant le récepteur AT1 de manière cardiomyocyte-spécifique. Ces souris, nommées souris AT1R, permettent de voir l’effet de la suractivation du SRA uniquement au niveau cardiaque, sans modification hémodynamique. Les souris AT1R ont été utilisés à deux âges afin de distinguer les effets directs d’AT1R sur l’électrophysiologie auriculaire (50 jours), de ceux induit par la présence de remodelage structurel (6 mois). Ceci a été validé par la quantification de la fibrose auriculaire par marquage histologique de type rouge Sirius. L’utilisation des deux groupes est importante puisque la fibrose est connue pour affecter l’électrophysiologie cardiaque et altérer la conduction auriculaire. L’hypothèse de ce projet de recherche est que la surexpression d’AT1R induit un remodelage électrique auriculaire qui altère la conduction et mène au développement de fibrillation auriculaire. La présence de remodelage structurel amplifierait ces effets néfastes, favorisant davantage la survenue de fibrillation auriculaire. Les données obtenues montrent une diminution du courant sodique d’environ 60% comparativement aux souris contrôles (CTL) dès l’âge de 50 jours. Cette diminution est associée avec une augmentation de l’expression protéique sarcolemmale de la PKCα, une protéine kinase capable de phosphoryler le canal Nav1.5 menant à la réduction du courant sodique. L’expression génique du canal Nav1.5 (encodé par le gène Scn5a) n’était pas modifiée à 50 jours, suggérant une absence de régulation transcriptionnel pour ce canal. De plus, une diminution de l’expression génique des connexines 40 et 43 a été observée chez les souris AT1R dès 50 jours. Une prolongation de la durée de l’onde P, qui correspond au temps nécessaire à la dépolarisation des oreillettes, est observée à 50 jours sur les électrocardiogrammes des souris AT1R par rapport aux CTL. À 6 mois, la présence de remodelage structurel n’aggrave pas les remodelages au niveau du courant sodique et des connexines, mais prolonge encore davantage la durée de l’onde P. La susceptibilité à la fibrillation auriculaire est légèrement augmentée à 50 jours et nettement plus à 6 mois. En conclusion, la surexpression d’AT1R induit directement des remodelages électriques qui affectent la conduction dans l’oreillette, et ce indépendamment de la présence de remodelage structurel. Le remodelage structurel affecte la conduction auriculaire, mais n’amplifie pas les altérations du courant sodique et des connexines observées dans ce projet. Ainsi, ce projet apporte des connaissances sur les mécanismes par lesquels l’angiotensine II peut altérer la conduction auriculaire et mener à la fibrillation auriculaire, indépendamment des effets hémodynamiques. / Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and is associated with an increased risk of strokes and other morbidities. Atrial fibrillation has been associated with ionic currents remodeling, as well as structural remodeling, such as fibrosis and hypertrophy. These atrial remodeling can slow down atrial conduction velocity increasing the risk of atrial fibrillation. Several risk factors have been associated with atrial fibrillation and elevated levels of angiotensin II are one of them. Angiotensin II is the primary effector of the renin-angiotensin system (RAS). Angiotensin II type 1 receptors (AT1R) are responsible for the several pathologies induced by RAS overactivity, such as hypertension, heart failure and atrial fibrillation. However, the direct effects of angiotensin II on the heart and its role in atrial fibrillation pathophysiology remain largely unexplored. The objective of this research project is to study electrical and structural remodeling in transgenic mice overexpressing AT1R specifically in cardiomyocytes. These mice, identified as AT1R mice, do not have hemodynamic changes and are therefore suitable for studying the direct effect of RAS overactivation on the heart. Two age groups were used to distinguish the direct effects of AT1R on atrial electrophysiology (50-day-old mice) from those induced by structural remodeling (6-month-old mice). The presence of atrial fibrosis solely in 6 months old AT1R mice was confirmed using Picrosirius Red histological method. Characterizing electrical remodeling with and without structural remodeling is important since fibrosis is known to modulate cardiac electrophysiology and impair atrial conduction. The hypothesis of this project is that AT1R overexpression induces atrial electrical remodeling which alters conduction, leading to atrial fibrillation. Structural remodeling is expected to worsen conduction defects, further increasing the risk of atrial fibrillation. In this project, we measured an approximately 60% decrease in sodium current in 50 days old AT1R mice compared to controls (CTL). This change in sodium current was associated with an increase of PKCα protein expression in the sarcolemmal fraction. PKCα is a protein kinase able to phosphorylate Nav1.5 channels, resulting in a decrease in its function. Scn5a gene expression, encoding for Nav1.5 channels, was not changed at 50 days, suggesting an absence of transcriptional regulation of the sodium current. Moreover, a decrease in connexins 40 and 43 gene expression was observed in AT1R mice from the age of 50 days. Accordingly, a prolongation in P wave duration, which corresponds to atrial depolarization, is observed at 50 days in AT1R mice compared to CTL. At 6 months, structural remodeling did not worsen the reduction in sodium current or connexins induced by AT1R overexpression but was associated with a further prolongation of P wave duration. Susceptibility to AF was slightly increased in 50 days old AT1R mice and even more at 6 months. In conclusion, AT1R overexpression directly induces electrical remodeling, which slows down atrial conduction, independently of structural remodeling. Structural remodeling further alters atrial conduction without changes in sodium current or connexins expression. This project provides knowledge on mechanisms by which angiotensin II alters atrial conduction and leads to atrial fibrillation, even in absence of angiotensin II-induced hemodynamic changes.

Os Hormônios Tiroideanos (HT) representam um fenótipo de cardioproteção e influenciam no estado trófico do tecido cardíaco através de diversos mecanismos, dentre eles a modulação dos componentes do Sistema Renina Angiotensina- a Angiotensina II e seus receptores (AT1 e AT2). Este estudo teve como objetivos avaliar o papel do receptor AT2 na cardioproteção induzida pelo HT e a participação da proteína quinase ativada por AMP (AMPK) nesse processo. O modelo de isquemia e reperfusão (I/R) foi desenvolvido em corações de ratos submetidos ao hipertiroidismo experimental na presença ou ausêcia do antagonista do receptor AT2 (PD123319), utilizando-se o aparto de Langendorff. Os resultados mostraram que o HT exerce efeito cardioprotetor com aumento na expressão protéica do receptor AT2 e da AMPK fosforilada, que foi prevenido com a administração do PD, seguido de piora da função cardíaca. Estes dados sugerem que parte da cardioproteção induzida pelo HT é mediada pelo receptor AT2, e que a AMPK também está envolvida proteção do miocárdio após injúria por I/R. / Thyroid Hormones (TH) is a phenotype of cardioprotection and may influence the trophic state of cardiac tissue through several mechanisms, including the modulation of the components of renin-angiotensin system - angiotensin II and its receptors (AT1 and AT2). The present study aimed to evaluate the role of AT2 receptor in cardioprotection mediated by the TH and the involvement of AMP-activated protein kinase (AMPK) in this context. A model of Ischemia and Reperfusion (I/R) was performed in isolated hearts of rats subjected to experimental hyperthyroidism, in the presence or absence of the AT2 receptor antagonist (PD123319), using the Langendorff system. The results showed that TH exerts cardioprotective effect with increased levels of AT2 and phosphorylated AMP protein expression, which was prevented by the administration of PD, with a loss in cardiac function. These data suggest that part of the TH-induced cardioprotection is mediated by the AT2 receptor with the involvement of AMPK in protection of the myocardium after I/R injury.

Neste estudo avaliou-se a ação da Angiotensina II (Ang II) e do bloqueio dos seus receptores AT1 e AT2 no remodelamento da matriz extracelular (MEC) e traçamos o perfil da sinalização intracelular envolvida no processo. O estudo foi feito in vivo em camundongos isogênicos C57Bl/6J submetidos a tratamento durante 7 e 14 dias com doses subpressoras de Ang II, bloqueador do receptor AT1 (Losartan) e uma combinação destes. Os animais foram sacrificados e procedeu-se a coleta de tecidos de artérias (aorta, carótida e femoral), coração, rins e pulmão para análise da síntese e degradação de componentes da MEC. Foram feitas avaliações hemodinâmicas, morfológicas em microscopia de luz, morfométricas, imunohistoquímicas para os componentes da matriz extracelular: colágeno (tipos I, III, IV e VI), fibronectina, tenascina-C, elastina, metaloproteinases (tipos 2 e 9), e quantificação de algumas proteínas ligadas à sinalização intracelular da via das Proteínas quinases ativadas por mitógenos (MAPK - Mitogen-activated protein kinases) usando-se Western Blotting. / In this study we evaluated the action of Angiotensin II (Ang II) and the blockade of AT1 and AT2 receptors in the remodeling of extracellular matrix (ECM) and outlines the process involved in intracellular signaling. The study was done in vivo in C57Bl/6J inbred mice undergoing treatment for 7 and 14 days subpressor doses of Ang II, AT1 receptor blocker (Losartan) and a combination thereof. The animals were sacrificed and proceeded to collect tissues of arteries (aorta, carotid and femoral arteries), heart, kidneys and lungs for analysis of the synthesis and degradation of ECM components. We assessed hemodynamic, morphological light microscopy, morphometry, immunohistochemistry for extracellular matrix components: collagen (types I, III, IV and VI), fibronectin, tenascin-C, elastin, metalloproteinases (types 2 and 9) and quantification of some proteins related to intracellular signaling pathways of the mitogen-activated protein kinase (MAPK) using Western Blotting.

Os hormônios peptídicos Angiotensina II (Ang II) e bradicinina (BK) ativam transdução de sinal através da ligação a Receptores Acoplados a Proteínas G (GPCR). Este trabalho propõe o estudo de propriedades conformacionais, através de espectroscopia de fluorescência da Ang II e BK e de seus análogos contendo o marcador de spin ácido 2,2,6,6-tetrametilpiperidina-1-oxil-4-amino-4-carboxílico, TOAC (TOAC1-Ang II, TOAC3-Ang II, TOAC0-BK, TOAC3-BK). Os peptídeos foram estudados em solução (efeito do pH) e também na presença de membranas modelo, micelas e bicamadas, formadas por anfifílicos zwitteriônicos ou aniônicos. Foi monitorada a fluorescência intrínseca dos resíduos aromáticos (Tyr4 na Ang II e Phe5 e Phe8 na BK). O efeito de supressão da fluorescência pelo TOAC foi utilizado para obter informação sobre a proximidade desse resíduo aos grupos fluoróforos. Foi observada dependência da fluorescência com o pH e regiões de pKs dos grupamentos ionizáveis. Os espectros evidenciaram também a interação peptídeo-membrana modelo. Interações mais fortes ocorreram entre os peptídeos e membranas com carga superficial negativa, evidenciando a importância de interações eletrostáticas para a ligação. Porém, interações hidrofóbicas também estão envolvidas, como verificado pela ligação dos peptídeos a membranas zwitteriônicas. Estudos com variação de pH também mostraram o papel dessa variável na interação peptídeo-membrana e a alteração de pKs de resíduos ionizáveis decorrentes da interação. A titulação com concentrações crescentes de membranas permitiu o cálculo das constantes de associação. A ligação a membranas é função da conformação dos peptídeos. Em particular, a presença de TOAC na posição 3 parece diminuir a afinidade desses análogos por membranas. Supressão de fluorescência foi efetuada empregando três diferentes abordagens: 1) supressão pela molécula aquossolúvel acrilamida, 2) supressão da fluorescência de fosfolipídeos contendo o fluoróforo NBD em diferentes posições da molécula pelos análogos marcados com TOAC, 3) supressão da fluorescência dos peptídeos por ésteres metílicos do ácido esteárico contendo o grupamento nitróxido em diferentes posições da cadeia. Esses estudos permitiram determinar a localização dos peptídeos na interface água-membrana. Medidas de anisotropia de fluorescência também evidenciaram a ligação dos peptídeos a membranas, revelando maior imobilidade dos mesmos nessas condições. Foi ainda estudado um peptídeo que contém os resíduos 92-100 (fEL1) do receptor AT1 de Ang II humano. Predições baseadas na estrutura cristalina da rodopsina estimam que essa seqüência localiza-se na primeira alça extra-celular do receptor. A seqüência contém a Tyr92, considerada um resíduo importante para a ligação hormônio-receptor. Resultados preliminares sugeriram que fEL1 interage com Ang II e TOAC1-Ang II, mas não com TOAC3-Ang II. Este último resultado provavelmente deve-se à dobra causada por TOAC que restringe a liberdade de movimento do esqueleto peptídico. Essa característica provavelmente determina a falta de atividade biológica de TOAC3-Ang II e TOAC3-BK, enquanto os análogos marcados no N-terminal retém atividade parcial (Nakaie et al., 2002). Tem sido proposto que peptídeos ligantes de GPCR se ligariam à bicamada lipídica e atingiriam seu receptor através da difusão pela bicamada. Em solução aquosa essas moléculas são flexíveis, existindo um equilíbrio dinâmico entre várias conformações. A ligação à bicamada lipídica estabilizaria uma ou algumas conformações, entre elas aquela que o ligante adota ao ligar-se ao receptor. O presente estudo contribui para a compreensão, a nível molecular, do processo de interação entre os hormônios peptídicos e membranas lipídicas. / The peptide hormones Angiotensin II (Ang II) and bradykinin (BK) trigger signal transduction by binding to G Protein Coupled Receptors (GPCR). This work proposes the study of conformational properties of Ang II and BK, as well as their analogues containing the spin label 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid, TOAC (TOAC1-Ang II, TOAC3-Ang II, TOAC0-BK, TOAC3-BK) making use of fluorescence spectroscopy. Studies were performed in solution (effect of pH) and also of the interaction between the peptides and model membranes - micelles and bilayers - formed by amphiphiles, either zwitterionic or negatively charged. The intrinsic fluorescence of aromatic residues (Tyr4 in Ang II and Phe5 and Phe8 in BK) was monitored. Fluorescence quenching by the TOAC-carrying analogues provided information about the proximity between TOAC and the fluorophores. The fluorescence was pH-dependent and evinced regions corresponding to pKs of ionizable groups. Peptide-model membrane interactions were also examined. Stronger interactions were detected between the peptides and membranes formed by negatively charged amphiphiles, pointing to the importance of electrostatic interactions for binding. However, hydrophobic interactions were also involved, as suggested by the fact that the peptides also bound to zwitterionic membranes. Variable pH studies showed the effect of this parameter on peptide-membrane interaction. The peptide-membrane interactions promoted changes in the pKs of ionizable residues. Titrations with increasing membrane concentrations allowed calculation of binding constants. Binding to membranes is a function of peptide conformation. In particular, TOAC at position 3 seems to decrease the affinity of both Ang II and BK for membranes. Fluorescence quenching studies made use of three different approaches: 1) quenching by water soluble acrylamide, 2) quenching of the fluorescence of phospholipids carrying the fluorescent group NBD in different positions by the spin-labeled TOAC-bearing analogues, 3) quenching of the peptides fluorescence by methyl esters of stearic acid containing the nitroxide moiety at different positions in the acyl chain. These studies indicated that the peptides are located at the water-membrane interface. Measurements of fluorescence anisotropy also evinced binding of the peptides to the membranes and showed that the peptides undergo more restricted motion under these conditions. A peptide containing residues 92-100 (fEL1) of the Ang II AT1 human receptor was also studied. Predictions based on rhodopsin crystalline structure estimate that this sequence is located in the receptor´s first extra-cellular loop. Preliminary results suggest that fEL1 interacts with Ang II and TOAC1-Ang II, but not TOAC3-Ang II. This latter result is probably due to the TOAC-induced bend that restricts the freedom of motion of the peptide backbone. This feature is probably the cause of lack of biological activity of TOAC3-Ang II and TOAC3-BK, while the N-terminally labeled analogues retain partial activity (Nakaie et al., 2002). GPCR-binding peptides have been proposed to bind to the lipid bilayer and reach their receptors by diffusion in the bilayer. In aqueous solution these molecules exist as a dynamic equilibrium between various flexible conformations, among them, the receptor-bound conformation. The present study provides contributions for the understanding, at the molecular level, of the interaction between the peptide hormones and lipid membranes.

Os inibidores do receptor de angiotensina II do tipo 1 (AT1R), fármacos da classe das sartanas, são muito utilizados na terapêutica da insuficiência cardíaca. Apesar de serem eficientes por baixarem a pressão arterial, esses inibidores diminuem a contratilidade do músculo cardíaco, acentuando a patologia. Nesse sentido, os agonistas enviesados para β-arrestina do AT1R surgem como uma solução para esse problema. Estudos com o mais promissor peptídeo com ação agonista enviesada (TRV120027) mostram que ele é capaz de diminuir a pressão arterial sem causar o efeito inotrópico negativo no coração. Tendo em vista esse novo e promissor mecanismo de ação e a característica peptídica do novo agonista enviesado que restringe sua utilização, o presente trabalho visou à busca de ligantes não peptídicos com potencial ação enviesada. Foram realizados estudos de ancoramento seguidos de dinâmica molecular, no AT1R, de sete peptídeos agonistas e agonistas enviesados descritos na literatura, empregando-se os programas Surflex-Dock 2.0 e o GROMACS 4.5, além de análises de campos de interação molecular no programa GRID. Os dados das interações intermoleculares retirados da dinâmica e dos campos de interação guiaram a construção de um farmacóforo que foi utilizado posteriormente em uma busca virtual na base de dados ZINC, com o módulo UNITY 3D do pacote Sybyl-X Suite 2.0. Após ancoramento e análise visual das moléculas selecionadas na busca, foram identificadas 15 moléculas promissoras, sendo cinco delas consideradas de maior interesse. As moléculas selecionadas na busca poderão ser futuramente testadas quanto ao perfil de ação enviesada em receptores AT1R. Os resultados obtidos nesse estudo podem levar à descoberta de um novo protótipo mais eficiente e seguro para o tratamento de doenças cardiovasculares, como a insuficiência cardíaca. / Angiotensin II type 1 receptor (AT1R) inhibitors, the sartans, are widely used in the treatment of heart failure. Although they are effective for lowering blood pressure, these inhibitors decrease the contractility of the heart muscle, accentuating the pathology. Accordingly, β-arrestin biased agonists for AT1R emerge as a solution to this problem. Studies with the most promising biased agonist peptide (TRV120027) show that it is able to lower blood pressure without causing negative inotropic effect on the heart. Given this promising new mechanism of action and the peptide feature of the new agonist that restricts its use, this work aims the search for non-peptide ligands with a potential biased action. Docking studies, followed by molecular dynamics simultions, were performed for seven full and biased agonists in the AT1R, using the Surflex-Dock 2.0 and 4.5 GROMACS programs, besides molecular interaction fields analysis with GRID software. The data of intermolecular interactions from the molecular dynamic\'s analysis and the molecular interaction fields guided to the construction of a pharmacophore model which was subsequently used in a virtual screening from ZINC database, employing the 3D UNITY module from Sybyl-X Suite 2.0 package. After a docking study and visual analysis of the primary selected molecules, 15 promising molecules have been identified, five of them considered of most interest. The molecules selected in the search can be further tested for biased action on AT1R. The results of this study may lead to the discovery of a more efficient and secure lead for the treatment of cardiovascular diseases, such as heart failure.

Este estudo tem o objetivo de investigar a participação da espironolactona, um antagonista dos receptores MR, sobre as respostas renais induzidas pela hiperatividade do SRAA. Utilizamos 2 grupos de animais tratados com Ang II ( 15 e 28 dias). Foram avaliados o fluxo plasmático renal (FPR) e Ritmo de filtração glomerular (RFG), eletrólitos no plasma e urina, expressão proteica e morfologia renal. Nossos resultados indicam que, a Ang II (15 e 28 dias), induziu à hipertensão, queda do FPR, RFG, fluxo urinário, e aumentou a fração de filtração (FF), a expressão de NHE3, a área glomerular e alterações morfológicas no túbulo proximal, incluindo perda da borda em escova e morte celular. A espironolactona corrigiu a maioria dos efeitos da Ang II. A Ang II e/ou espironolactona não modificaram a carga excretada de NH4, Na+ ou K+. Os resultados indicam que a hiperatividade do SRAA induz aumento na concentração plasmática de aldosterona, e esta contribui para os efeitos deletérios do SRAA sobre a função e morfologia renal. / This study investigated the effect of spironolactone (MR antagonist) on renal function and morphology in hypertensive rats with chronic Ang II infusion for 15 or 28 days. Blood pressure (BP) was monitored. Renal plasmatic flow (RPF) and glomerular filtration rate (GFR) were determined. Plasmatic levels of renin and aldosterone, urinary excretion of ions, protein expression and renal morphology were analyzed. Our results showed that Ang II used was enough to induce hypertension in both periods. The RPF, GFR and urinary flux decreased. Ang II increased the filtration fraction (FF). Spironolactone impaired all effect of Ang II. The plasma renin and aldosterone levels increased and spironolactone amplified both. Urinary Na+ and K+ did not changed in Ang II and/or spironolactone rats treated. Ang II increased the NHE3 expression, induced proximal tubule injury and cellular apoptosis. Spironolactone impaired the effect of Ang II. In conclusion, spironolactone has a beneficial effect on renal injury induced by hyperactivity of renin-angiotensin-aldosterone system.

A angiotensina II (AngII) pode ativar as vias de sinalização das proteínas quinases ativadas por mitógenos (MAPKs). Investigamos o papel da obesidade e das MAPKs na resposta à AngII em ratos obesos por injeção de glutamato monossódico (Ob). Artérias mesentéricas de resistência com endotélio intacto e não as artérias sem endotélio, isoladas de Ob, respondem menos à AngII. As respostas à noradrenalina e ao cloreto de potássio estavam inalteradas. Aumento da expressão do receptor AT2 (AT2R), da óxido nítrico sintase (eNOS) e da ERK1/2 podem estar envolvidos na menor resposta pois a inibição do AT2R, da eNOS e da ERK1/2 corrigiram-na. A maior ativação da ERK1/2 nos Ob levou à maior ativação da eNOS e maior geração de NO, diminuindo a resposta à Ang II. Concluímos que na obesidade, a resposta contrátil à Ang II é menor, como possível mecanismo adaptativo frente ao aumento da ativação do sistema renina-angiotensina. Esse mecanismo envolve a participação do endotélio com maior liberação de NO, aumento do número de AT2R, e da fosforilação da eNOS e da ERK1/2. / Angiotensin II (AngII) can activate mitogen-activated protein kinases (MAPKs) pathways. We investigated the role of obesity and MAPKs in AngII response in monosodium glutamate-induced obese rats (Ob). Endothelium-intact but not endothelium-denuded mesenteric resistance arteries isolated from Ob exhibited a lower response to AngII. The response to nordrenaline and potassium chloride were unaltered. Increased expression of AT2 receptor, nitric oxide synthase (eNOS) and ERK1/2 might be involved in the reduced response since inhibition of AT2R, eNOS and ERK1/2 corrected it. Increased activation of ERK 1/2 in Ob might activate eNOS, generating more NO and vasodilation that contributed to reduce the contraction to AngII. We concluded that, in obesity, the lower response to AngII might be an adaptive mechanism against the increased activation of the renin-angiotensin system. This mechanism involves the participation of the endothelium through a greater release of NO, increased AT2R, eNOS and ERK1/2 expressions.

A Angiotensina II (Ang II), principal peptídeo efetor do Sistema Renina Angiotensina (SRA), tem sido relatada como importante mediador de processos inflamatórios. Considera-se de extrema relevância detectar esse sistema e compreender a modulação da Ang II nas células da papila apical (CPA) considerando que esta população celular é a chave no sucesso na revascularização pulpar. Vale também considerar que a infecção do sistema de canais radiculares pode alterar as funções destas células. Sendo assim, o presente trabalho in vitro teve como objetivo elucidar o papel do lipopolissacarídeo (LPS) nas células da papila apical, detectar os componentes do Sistema Renina Angiotensina (SRA) e compreender a modulação da Ang II nas CPA. Para vencer o desafio científico, culturas de células da papila foram estabelecidas e caracterizadas fenotipicamente por citometria de fluxo e funcionalmente por Vermelho de Alizarina. Quando estimuladas por LPS, foi feita análise da citotoxicidade por MTT e produção de citocinas e Osteoprotegerina (OPG) por ELISA. Em seguida, a detecção da expressão gênica dos componentes do Sistema Renina Angiotensina foi realizada por Transcrição Reversa seguida de Reação em Cadeia da Polimerase quantitativa (RT-qPCR) e do peptídeo Ang II por ELISA. As células foram estimuladas com LPS, a própria Ang II e um antagonista do receptor de Angiotensina II tipo 1 (AT1) e avaliadas quanto à citotoxicidade por ensaio de MTT, capacidade de formação de nódulos mineralizados por Vermelho de Alizarina e produção de citocinas inflamatórias e OPG por ELISA. Os resultados demonstraram que culturas de CPA expressaram níveis típicos de marcadores de células-tronco mesenquimais CD146, CD24 e STR0-1 e formaram nódulos mineralizados com o meio de diferenciação. O estímulo com LPS a 0,1; 1 e 10?g/mL no decorrer de 1, 3, 5, 7 e 14 dias não afetou a viabilidade das CPA. As células estimuladas com LPS apresentaram níveis maiores da quimiocina MCP-1/ CCL-2 a 1?g/mL em uma e 24 horas e a expressão de OPG diminui na presença de LPS a 0,1 e 10?g/mL. Na concentração de 1?g/mL, foi detectada a expressão gênica de Angiotensinogênio, Renina, Enzima Conversora de Angiotensina (ECA) e AT1. Houve maior expressão de ECA no período experimental de 1 hora com LPS e em 24 horas, apenas AT1 e ECA continuaram presentes. Sendo a produção da proteína soberana, Ang II foi encontrada nas células pelo ELISA. Os ensaios funcionais demonstraram que a Ang II aumentou a proliferação celular em 24, 48 e 72 horas. Em 24 horas, a presença do peptídeo efetor do SRA aumentou a produção da quimiociona MCP-1/ CCL-2 e do ligante do ativador do receptor do fator nuclear- ?B (RANKL). Concluímos que o LPS foi capaz de alterar funcionalmente as CPA, que o Sistema Renina Angiotensina está presente e a Ang II tem capacidade de modulação pró-inflamatória, proliferativa e de reabsorção óssea nesta população celular. / Angiotensin II (Ang II), the main effector peptide of the Renin Angiotensin System (SRA), has been reported as an important mediator of inflammatory processes. It is considered of extreme relevance to detect this system and understand the modulation of Ang II in the apical papilla cells (CPA) considering that this cellular population is the key in the success in pulpar revascularization. It is also worth noting that infection of the root canal system can alter the function of these cells. The aim of the present work was to elucidate the role of lipopolysaccharide (LPS) in the apical papilla cells, to detect the components of the Renin Angiotensin System (SRA) and to understand the modulation of Ang II in the CPA. To overcome the scientific challenge, cultures of papilla cells were established and characterized phenotypically by flow cytometry and functionally by Alizarin Red. When stimulated by LPS, cytotoxicity by MTT and cytokine and Osteoprotegerin (OPG) were analyzed by ELISA. Next, the detection of the gene expression of the components of the Renin Angiotensin System was performed by Reverse Transcription followed by quantitative Polymerase Chain Reaction (RT-qPCR) and the Ang II peptide by ELISA. The cells were stimulated with LPS, Ang II itself and an Angiotensin II receptor antagonist type 1 (AT1) and evaluated for cytotoxicity by MTT assay, alizarin red mineralized nodule formation capacity, and production of inflammatory cytokines and OPG by ELISA. The results demonstrated that CPA cultures expressed typical levels of CD146, CD24 and STR0-1 mesenchymal stem cell markers and formed mineralized nodules with the differentiation medium. The stimulus with LPS at 0.1; 1 and 10?g/mL in the period of 1, 3, 5, 7 and 14 days did not affect the viability of CPA. LPS-stimulated cells had higher levels of 1?g/mL chemokine MCP-1/CCL-2 at one and 24 hours and OPG expression decreased in the LPS presence at 0.1 and 10?g/mL. At the concentration of 1 ?g/mL, the gene expression of Angiotensinogen, Renin, Angiotensin Converting Enzyme (ECA) and AT1 was detected. There was greater expression of ACE in the experimental period of 1 hour with LPS and in 24 hours, only AT1 and ACE remained present. Being the protein production sovereign, Ang II was found in the cells by the ELISA. Functional assays demonstrated that Ang II increased cell proliferation at 24, 48 and 72 hours. In 24 hours, the presence of the effector peptide of SRA increased the production of the chemokine MCP-1/CCL-2 and the activator of the nuclear factor receptor-KB (RANKL). We conclude that LPS was able to functionally alter CPA, that the Renin Angiotensin System is present and Ang II has the capacity for pro-inflammatory, proliferative and bone resorption modulation in this cell population.

Uma das causas da hipertensão está relacionada a alterações de núcleos autonômicos responsáveis pelo controle dos reflexos cardiovasculares, destacando-se o PVN, o NTS e o RVLM. A ANG II, atuando nestes núcleos, participa do controle da PA. Estudos in vitro, demonstraram uma via de sinalização da ANG II que envolve a ativação da PI3K e da Akt. Neste estudo avaliamos as diferenças na atividade da PI3K, através da quantificação da subunidade p85 (PI3K) e da p-Akt por meio de immunoblotting desses núcleos centrais de animais SHR, WKY e Wistar, além da interação da PI3K com os efeitos cardiovasculares da ANG II injetada no PVN de Wistar acordados. A expressão da p85 não diferiu entre os núcleos estudados. No entanto, a p-Akt estava 1,5x mais expressa no PVN de SHR. Ainda, o aumento da PAM promovido pela injeção de ANG II no PVN foi reduzido 5 min após a injeção do inibidor da PI3K, com recuperação da resposta 30 min mais tarde, sugerindo que a via da PI3K está mais ativa no PVN de animais SHR e que a interação das vias ANG II-PI3K está relacionada com a hipertensão. / One cause of the hypertension is associated to changes in autonomic nuclei that control the cardiovascular reflexes. Among these nuclei are PVN, NST and RVLM. The ANG II, acting in these nuclei, contributes to the control of the AP. In vitro studies had demonstrated a signaling pathway of the ANG II that involves the activation of PI3K and Akt. In the present study we evaluated the PI3K activity via expression of p85 (PI3K) and p-Akt, through the immunoblotting of these central nuclei of SHR, WKY and Wistar. Moreover, we evaluated the interaction of PI3K to the cardiovascular effects of ANG II injected into the PVN of awake Wistar. The p85 expression was not different at all autonomic nuclei studied. However, the p-Akt expression was 1.5x higher in the PVN of SHR. Besides, the increase in the MAP elicited by the injection of ANG II into the PVN was reduced 5 min after the blockade of the PI3K, and restored 30 min later, suggesting that PI3K pathway is more activate at the PVN level of SHR and that the interaction between ANG II-PI3K is related to hypertension.