A gestação é um período em que a fêmea é transitoriamente submetida a uma série de alterações em todo o organismo, resultando num quadro semelhante à síndrome metabólica, mas que são cuidadosamente reguladas de modo a fornecer o suprimento adequado de substratos para a mãe e para o feto. A gravidez caracteriza-se como modelo fisiológico e temporalmente definido de resistência à insulina. Já durante a lactação, observa-se hipersensibilidade sistêmica à ação da insulina. A ativação de vias de sinalização da insulina no cérebro, em particular no núcleo arqueado do hipotálamo (ARC), tem importante papel na regulação da homeostasia glicêmica. Já está bem estabelecido que a Ang-II é capaz de induzir a fosforilação em serina do IR, do IRS e da subunidade regulatória p85 da PI3K. No entanto, ainda não foi esclarecido se ocorrem alterações na via de sinalização da insulina em áreas do sistema nervoso central (SNC) responsáveis pelo controle da homeostasia glicêmica, como é o caso do ARC, do núcleo hipotalâmico ventromedial (VMH) e do núcleo paraventricular (PVN) durante a gestação, que poderia contribuir para o quadro de resistência à insulina nesta situação. O objetivo deste estudo foi avaliar a expressão das proteínas de sinalização intracelular da insulina IRS1-2, PI3K (p85<font face=\"Symbol\">&#945;, p55<font face=\"Symbol\">&#945;, p50<font face=\"Symbol\">&#945;), AKT, p-Ser473 AKT e dos receptores IR, e AT1a no hipotálamo total e no ARC, VMH e PVN na gravidez e na lactação. Investigou-se também o possível cross-talk da via de sinalização da insulina e da Angiotensina-II (Ang-II) no hipotálamo. Observou-se a redução da expressão do IRS-2, da AKT e da p-Ser473 AKT, concomitante com o aumento da expressão das subunidades regulatórias p85<font face=\"Symbol\">&#945;, p55<font face=\"Symbol\">&#945;, p50<font face=\"Symbol\">&#945; da PI3K e do receptor AT1a no hipotálamo na gravidez. No ARC, VMH e PVN observou-se co-localização da p85<font face=\"Symbol\">&#945; e do receptor AT1a, bem como aumento da expressão de ambos nessas regiões, nas ratas grávidas. O tratamento crônico com antagonista AT1 (losartan) produziu, nas grávidas redução significativa da expressão deste receptor, sem causar alteração na expressão da p85<font face=\"Symbol\">&#945;. A disfunção das vias da Ang-II e da insulina em graus variados pode levar a duas condições patológicas de alta prevalência e que muitas vezes são concomitantes: diabetes mellitus e hipertensão arterial. A melhor compreensão das alterações hipotalâmicas nas vias de sinalização da insulina e da Ang-II, bem como a caracterização molecular dessa interação, tanto nas situações fisiológicas (por exemplo na gravidez) como nas situações patológicas, poderá revelar alvos para futuras intervenções terapêuticas para diversas condições clínicas como a obesidade, o diabetes, a hipertensão arterial, a pré-eclâmpsia/eclâmpsia, etc... / Pregnancy is a period in which the female is temporarily exposed to a series of changes throughout the body, resulting in a condition similar to the metabolic syndrome, but that are carefully regulated to provide an adequate supply of substrates for the mother and the fetus. Pregnancy is characterized as defined in time and a physiological model of insulin resistance. Although during lactation, there is a systemic hypersensitivity to insulin. Activation of the insulin signaling pathways in the brain, particularly in the arcuate nucleus of the hypothalamus (ARC), plays an important role in glucose homeostasis. It is well established that angiotensin II (Ang-II) cross-talking to the insuling signaling pathway is capable of inducing serine phosphorylation of IR, IRS and the p85 regulatory subunit of PI3K. However, it is not clear whether there are changes in the insulin signaling pathway in areas of the central nervous system (CNS) responsible for homeostasis and glycemic control, such as the ARC, the ventromedial hypothalamic nucleus (VMH) and the paraventricular nucleus (PVN) during pregnancy, which could contribute to the symptoms of insulin resistance in this situation. The objective of this study was to evaluate the expression of intracellular insulin signaling proteins such as IRS1-2, PI3K (p85<font face=\"Symbol\">&#945;, p55<font face=\"Symbol\">&#945;, p50<font face=\"Symbol\">&#945;), AKT, p-Ser473 AKT and the expression of IR and AT1a receptors in the hypothalamus, and also specifically in the ARC, VMH and PVN during pregnancy and lactation. We also investigated the possible cross-talk of the insulin and Ang II signaling pathways within the hypothalamus. There was a reduction in IRS-2, AKT and p-Ser473AKT expression concomitant with increased expression of regulatory subunits p85<font face=\"Symbol\">&#945;, p55<font face=\"Symbol\">&#945;, PI3K p50<font face=\"Symbol\">&#945; and AT1a receptor in the hypothalamus in late pregnant rats. In the ARC, VMH and PVN it was observed co-localization of p85<font face=\"Symbol\">&#945; and AT1a receptor, and increased expression of both in these regions in pregnant rats. Chronic treatment with AT1 antagonist (losartan) produced a significant reduced expression of this receptor, without causing any changes in the expression of p85<font face=\"Symbol\">&#945;. The dysfunction of the Ang-II and insulin pathways can lead in different degrees to two conditions of high prevalence and often concomitant diseases such as diabetes mellitus and hypertension. A better understanding of the hypothalamic changes in insulin signaling pathways, the cross-talk with Ang-II and the molecular characterization of this interaction both in physiological (for example in pregnancy) and pathological situations may provide targets for future therapeutic intervention for various clinical conditions such as obesity, diabetes, hypertension, pre-eclampsia/eclampsia, etc...

Uma atividade funcional para uma via alternativa de geração de angiotensina II, como a elastase-2 foi sugerida em estudos realizados anteriormente em nosso laboratório no leito arterial mesentérico isolado de rato. No presente estudo, caracterizamos com o uso de substratos e inibidores seletivos a presença de via alternativa de geração de Ang II, independente da ECA, em carótida de ratos. Determinamos ainda a expressão do RNAm da elastase-2 nesta preparação arterial. Em anéis isolados de carótida de ratos analisamos o efeito vasoconstritor dos peptídeos Ang II, Ang I, TDP, [Pro11-D-Ala12]-Ang I (um substrato resistente a ECA) na ausência e presença de inibidores de proteases. Ang II e seus precursores produziram efeito vasoconstritor dependente da concentração em carótidas de ratos, de forma sensível ao losartan (1 M). Na presença de captopril (10 M) a resposta vasoconstritora produzida pela Ang I foi inibida, mas a resposta contrátil induzida pelo TDP e [Pro11-D-Ala12]-Ang I não foi alterada. Na presença de quimostatina (100 M) o efeito produzido pelo TDP e [Pro11-D-Ala12]-Ang I foi abolido enquanto que a curva cumulativa de Ang I foi significativamente deslocada para a direita. Inibidor Ac-AAPL-CK (seletivo para elastase-2) aboliu completamente a resposta contrátil induzida pelo PDA e não alterou o efeito vasoconstritor da Ang II. Na presença de captopril e quimostatina a resposta vasoconstritora dos peptídeos Ang I, TDP e [Pro11-D-Ala12]-Ang I foram inibidas, enquanto a resposta contrátil da Ang II não foi alterada em artéria carótida. A presença de RNAm da elastase-2 na carótida, juntamente com os dados funcionais apresentados aqui sugerem a participação desta enzima na via alternativa de geração de Ang II em carótidas de ratos. Embora a formação de Ang II a partir Ang I seja descrita como essencialmente dependente da ECA, nossos resultados sugerem a existência de vias alternativas de geração de Ang II sensível a quimostatina e Ac-AAPL-CK em artéria carótida de ratos. Muito provavelmente a elastase-2 seja a enzima responsável pela geração de Ang II nessa preparação. / We have recently described a chymostatin-sensitive elastase-2 as the major angiotensin (Ang) II-forming enzyme in the perfusate of rat mesenteric arterial bed (MAB). In the present study we investigated the role of this enzyme in generating Ang II in the isolated rat carotid artery rings by analyzing the vasoconstrictor effect of Ang II, Ang I, tetradecapetide renin-substrate (TDP), [Pro11-D-Ala12]-Ang I (an ACE-resistant substrate) in the absence and presence of proteases inhibitors. Ang II and its precursors produced a dose-dependent vasoconstrictor effect in vascular preparation that was blocked by losartan (1 M). In carotid rings, captopril (10M) abolished the responses induced by Ang I but did not affect those induced by TDP and [Pro11-D-Ala12]-Ang I. In the presence of chymostatin (100 M) alone, the effects induced by [Pro11-D-Ala12]-Ang I and TDP were abolished while the concentration-response curve to Ang I was shifted to the right. Ac-AAPL-CK (selective elastase-2 inhibitor) inhibited the responses induced by [Pro11-D-Ala12]-Ang I but did not affect Ang II-induced effects. In the presence of captopril and chymostatin, the vasoconstrictor effects of Ang I, TDP, and PDA were completely blocked while those induced by Ang II were not affected in rat artery carotid. Although Ang II formation from Ang I is essentially dependent on ACE in carotid artery, our results suggest the existence of an alternative chymostatin-sensitive pathway in rat arteries, most probably involving elastase-2.

Orientadores: Tatiana de Sousa da Cunha, Fernanda Klein Marcondes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-20T06:59:33Z (GMT). No. of bitstreams: 1 Sanches_Andrea_M.pdf: 1126813 bytes, checksum: 30cc93581a60db907dc7c938e7d40caf (MD5) Previous issue date: 2012 / Resumo: O estresse crônico é um fator de risco para o desenvolvimento de hipertensão, aterosclerose e diabetes. O protocolo de estresse crônico moderado e imprevisível (ECMI) é um modelo animal de estresse crônico. Em estudo prévio, foi observado que o ECMI induziu resistência à insulina, dislipidemia e disfunção endotelial, em ratos. Considerando que o aumento na atividade do sistema renina-angiotensina (SRA) tem sido associado à disfunção endotelial e à resistência à insulina, o objetivo deste estudo foi investigar a participação do SRA sobre os efeitos cardiovasculares e metabólicos induzidos pelo ECMI, em ratos. Foram utilizados 62 ratos machos Sprague-Dawley, com dois meses de idade. A duração do período experimental foi de 7 semanas. No Experimento 1, os animais foram divididos em 2 grupos: controle e estresse. O ECMI foi aplicado nas semanas 3, 4 e 5 e os animais foram eutanasiados 1 e 15 dias após a aplicação do protocolo de ECMI. O ECMI aumentou a atividade sistêmica da renina e da enzima conversora de angiotensina (ECA), da ECA na aorta torácica e as concentrações plasmáticas de angiotensina II e angiotensina (1-7). Com base nestes resultados, que mostraram aumento da atividade do SRA induzido pelo ECMI, o Experimento 2 foi delineado com o objetivo de avaliar a participação da angiotensina II e do seu receptor AT1 nos efeitos desencadeados pelo ECMI sobre a captação de glicose e sobre o sistema cardiovascular. Outros animais foram divididos em 4 grupos experimentais: controle, estresse, controle losartan (antagonista do receptor AT1 - 50 mg/Kg/dia, v.o.) e estresse losartan. O ECMI induziu aumento da área sob a curva, no teste de tolerância à glicose (TTG), diminuição da resposta vasodilatadora à acetilcolina na aorta torácica in vitro e aumento da pressão arterial in vivo, em comparação com o grupo controle, sem haver diferença entre os grupos controle, controle losartan e estresse losartan. Estes resultados mostram que os efeitos do ECMI levaram ao processo de disfunção endotelial em ratos, os quais foram associados positivamente à hiperatividade do SRA, bem como foram cancelados pelo tratamento com losartan. Assim, este estudo esclarece parte dos mecanismos fisiológicos envolvidos nas alterações metabólicas e cardiovasculares decorrentes do estresse crônico, demonstrando que estas alterações são mediadas pelo receptor AT1, provavelmente pela ligação da angiotensina II ao mesmo / Abstract: Chronic stress is a risk factor for the development of hypertension, atherosclerosis and diabetes. The protocol of chronic mild and unpredictable stress (CMUS) is an animal model of chronic stress. Previously, it has been shown that CMUS induced insulin resistance, dyslipidemia and endothelial dysfunction in rats. Considering that endothelial dysfunction and insulin resistance have been associated with high activity of renin-angiotensin system (RAS), the aim of this study was to investigate the involvement of RAS components on cardiovascular effects induced by CMUS in rats. Sixty two male Sprague-Dawley rats, (2 months old) were used. The experiment period was 7 weeks. In experiment 1, animals were divided into 2 groups: control and stress. The CMUS was applied on weeks 3, 4 and 5 and animals were euthanized 1 and 15 days after the CMUS. The CMUS increased systemic renin and angiotensin converting enzyme (ACE) activity, ACE activity in the thoracic aorta and plasma angiotensin II and angiotensin (1-7) concentrations. Based on these results, showing increased activity of the RAS induced by ECMI, the second experiment was designed to evaluate the involvement of angiotensin II and its AT1 receptor in the effects triggered by CMUS on glucose uptake and on cardiovascular system. Other animals were divided into 4 experimental groups: control, stress, losartan control (AT1 receptor antagonist, losartan - 50 mg /kg/day, orally) and losartan stress. The CMUS induced an increase in area under the curve in the glucose tolerance test (GTT), decreased the in vitro vasodilator response to acetylcholine in the thoracic aorta and increased blood pressure, compared to control group, without difference among control, losartan control and losartan stress group. These results show that the effects of CMUS led to endothelial dysfunction in rats, which was positively associated with hyperactivity of the RAS and was canceled by the treatment with losartan. Thus, this study explains part of the physiological mechanisms involved in cardiovascular and metabolic changes resulting from chronic stress, demonstrating that these changes are mediated by the AT1 receptor, probably by angiotensin II binding to it / Mestrado / Fisiologia Oral / Mestre em Odontologia

Les maladies cardiovasculaires sont la première cause de mortalité dans le monde et voient leur incidence augmenter avec l’expansion de leurs principaux facteurs de risque, tels que le vieillissement, l’obésité et le diabète. Parmi ces maladies l’anévrisme de l’aorte abdominale (AAA), dont on sait aujourd’hui que le système immunitaire favorise le développement. Il est parmi les pathologies les plus courantes de l’aorte abdominale et représente un problème de santé publique. Le rôle des macrophages dans l’AAA a été récemment mis en évidence. Ces cellules inflammatoires sont les plus représentées dans l’adventice anévrismale, et ont tendance à s’accumuler au cours du temps dans l’AAA. Des données récentes montrent que leur accumulation est particulièrement accrue dans les premiers jours de la formation de l’AAA chez la souris, et que l’inhibition de la voie CCR2/MCP1 ou la déplétion précoce des monocytes circulants ont un effet protecteur. Il semble donc essentiel de comprendre les mécanismes d’infiltration des monocytes lors de l’initiation et du développement de l’AAA. Mon travail de thèse se focalise sur le rôle des sous-types monocytaires et l’impact de leur trafic sur le développement de l’AAA. En utilisant un modèle d’induction d’anévrisme chez des souris ApoE-/-, nous avons montré que l’angiotensine-II (Ang II) entraine la mobilisation des monocytes ‘Ly-6Chigh’, et des monocytes ‘Ly-6Clow’ à un moindre degré, à partir de la rate, puis leur recrutement au niveau de l’aorte. La splénectomie ou la déficience en lymphocyte B inhibe cette mobilisation monocytaire et protège de l’anévrisme. En revanche, la reconstitution de souris immuno-déficientes Apoe-/-Rag2-/- par des splénocytes totaux, contrairement aux splénocytes déplétés en lymphocytes B, restaure la mobilisation monocytaire et restitue la susceptibilité de ces souris à l’AAA. Cette thèse apporte de nouveaux indices sur les événements précoces impliqués dans la formation d’AAA, en mettant l’accent sur le rôle du réservoir monocytaire splénique dans ce processus. Elle identifie également une fonction jusque-là méconnue des lymphocytes B dans la mobilisation rapide et transitoire des monocytes en réponse à l’Ang II. Il conviendra par la suite d’identifier les mécanismes moléculaires sous-jacents à ces interactions, ce qui devrait permettre d’envisager de nouvelles voies thérapeutiques visant à moduler l’effet de ces types cellulaires dans l’AAA. / Cardiovascular diseases are the first cause of mortality around the industrialized world with a continuous increase in their incidence along with the expansion of the major risk factors such as aging, obesity and diabetes. In abdominal aortic aneurysm (AAA), the presence of inflammatory infiltrates, and particularly monocytes/macrophages, has underscored the contribution and importance of immuno-inflammatory responses in aneurysmal degeneration. It is one of the most common diseases of the abdominal aorta and presents a major health problem. The importance of macrophages has recently been highlighted by a number of evidence, which are the main population observed within the site of aneurysm, are believed to derive from circulating monocytes although no direct evidence has been provided to date. Recent evidence has shown that their accumulation is particularly enhanced in the early onset of AAA in mice, and that the inhibition of CCR2/ MCP1 signaling as well as the early depletion of circulating monocytes, are protective. Hence, it seems crucial to understand the mechanisms of monocyte recruitment in the initiation and progression of AAA. In this PhD project with a particular interest in abdominal aortic aneurysm, we were particularly interested in understanding the trafficking behavior of monocyte subsets in AAA and their role in disease pathogenesis. Using a mouse model of aneurysm induction in ApoE-/- mice, we showed that Ang II triggered the mobilization of Ly-6Chigh, and to a lesser extent Ly-6Clow monocytes, from the spleen and their consequent recruitment in the aorta. Spleen removal or B lymphocyte deficiency in Apoe-/- mice similarly impaired early monocyte mobilization in response to Ang II and protected against AAA development, independently of blood pressure. Reconstitution of Apoe-/- Rag-/- mice with total splenocytes but not with B-Cell depleted splenocytes restored monocyte mobilization in response to Ang II and enhanced susceptibility to AAA. Taken together, this study provides novel mechanistic insights on the early events involved in AngII-induced AAA formation. It highlights the role of the splenic monocyte reservoir in this process and identifies an intriguing role for B lymphocytes in mediating AngII-induced early and transient mobilization of splenic monocytes. Nevertheless, further understanding of the molecular mechanisms that underlie such interactions is likely to lead to the identification of effective therapeutic targets.

No description available.

L'angiotensine II (Ang II) joue un rôle important dans la régulation du système cardiovasculaire par l’activation de plusieurs voies de signalisation. L’activation de ces voies passe par le récepteur de l'angiotensine II de type 1 (AT1R). Ce récepteur fait partie de la famille des récepteurs couplés aux protéines G (GPCRs). De plus, il est maintenant connu que certains ligands peuvent lier le récepteur et induire une conformation qui permet d'activer certaines voies de signalisation tout en n’étant pas favorable à l'activation d'autres voies. Il est alors question de sélectivité fonctionnelle, aussi appelée signalisation biaisée. Ainsi, avec cette approche, il est possible de cibler les voies qui produiront les effets thérapeutiques désirés sans toutefois activer les voies qui seraient responsables des effets indésirables. Nous avons émis l’hypothèse que de cycliser des ligands va restreindre les conformations possibles lors du couplage avec AT1R et induire un agonisme biaisé. Ainsi, des analogues cycliques de l’AngII substitués aux positions 3 et 5 par des cystéines et des homocystéines ont été synthétisés: [Sar1Hcy3,5]AngII, [Sar1Cys3Hcy5]AngII et [Sar1Cys3,5]AngII. D’abord, la capacité de ces analogues cycliques à activer la voie Gq a été évaluée par la mesure de la production des inositol phosphates. Puis, la capacité à activer les voies G12, le recrutement des β-arrestines (1 et 2) ainsi que l’activation de ERK1/2 a également été évaluée. Nos travaux ont montré que l’analogue cyclique [Sar1Hcy3,5]AngII a une puissance et une efficacité maximales sur toutes les voies testées à l'exception de la voie Gq. Des simulations de dynamique moléculaire ont été effectuées pour nous permettre de comprendre comment la conformation du ligand influence la structure d’AT1R et donc l’activation des différentes voies de signalisation. Les simulations en dynamique moléculaire ont montré que la barrière énergétique associée à l'insertion du résidu Phe8 de l’AngII dans le coeur hydrophobe d'AT1R est augmentée avec [Sar1Hcy3,5]AngII, pouvant expliquer que cet analogue active moins bien la voie Gq. D’autres analogues cyclisés aux positions 3 et 5 de l’AngII ont été synthétisés; [Sar1Hcy3Ile4Hcy5]AngII, [Sar1Hcy3,5Ile8]AngII et [Sar1Hcy3Cys5]AngII. Leur capacité à activer les voies Gq, ERK1/2 et le recrutement des β-arrestines (1 et 2) a été évaluée. L’analogue [Sar1Hcy3Cys5]AngII semblait bien activer la voie ERK1/2, mais pas les voies G12 et β-arrestines. Ces résultats suggèrent que le fait de contraindre les mouvements des déterminants moléculaires d’un ligand en introduisant des structures cycliques peut entraîner un biais dans la signalisation en stabilisant différentes structures du récepteur. / Abstract: Angiotensin II (Ang II) has an important role in the regulation of the cardiovascular system by its ability to activate several signaling pathways. The activation of these pathways occurs via the angiotensin II receptor type 1 (AT1R). This receptor belongs to the family of G protein-coupled receptors (GPCRs). Moreover, it is now known that certain ligands can bind to the receptor and induce a conformation that allow the activation of certain signaling pathways while not promoting the activation of other pathways. This concept is known as functional selectivity or biased signaling. With this approach, it is possible to target the signaling pathways that produce the desired therapeutic effects rather than activating the pathways responsible for adverse effects. We hypothesized that cyclizing ligands would restrict possible conformations when coupled with AT1R and induce biased agonism. Thus, cyclic AngII analogs substituted at positions 3 and 5 by cysteines and homocysteines were synthesized: [Sar1Hcy3,5]AngII, [Sar1Cys3Hcy5]AngII and [Sar1Cys3,5]AngII. First, the ability of these cyclic analogs to activate the Gq pathway was measured by the inositol phosphates production. Then, the G12 pathway activation, β-arrestin (1 and 2) recruitment and the ability of these analogs to activate the ERK1/2 pathway was evaluated. Our work has shown that [Sar1Hcy3,5]AngII has maximum potency and efficacy on all of the evaluated pathways, except for the Gq pathway. Molecular dynamic simulations were used to understand how a distinct ligand conformation influences the AT1R structure and the activation of signaling pathways. These studies have shown that the energy barrier associated with the insertion of the Phe8residue of AngII within the hydrophobic core of AT1R is increased with [Sar1Hcy3,5]AngII, possibly explaining why this analog is less potent in activating the Gq pathway. Other analogues cyclized at positions 3 and 5 of AngII were synthesized; [Sar1Hcy3Ile4Hcy5]AngII, [Sar1Hcy3,5Ile8]AngII and [Sar1Hcy3Cys5]AngII. Their ability to activate Gq, ERK1/2 and recruitment of β-arrestins (1 and 2) was evaluated. The analog [Sar1Hcy3Cys5]AngII appeared to activate the ERK1/2 pathway but not the G12 and β-arrestin pathways. These results suggest that constraining the movements of molecular determinants of a ligand by introducing cyclic structures can lead to a signaling bias by stabilizing different structures of the receptor.

Master of Science / Department of Electrical and Computer Engineering / Masaaki Tamura / Steven Warren / Lung cancer mortality is the highest among all cancer–associated deaths. Despite early detection and treatment, prognosis of this disease remains poor. Therefore, development of new therapeutic agents and effective treatment procedures are urgently needed. Endogenous Angiotensin II (Ang II) type 2 receptor (AT[subscript]2), one of two isoforms of Ang II, has been shown to mediate apoptosis. Nanoparticle delivery systems make possible targeted drug delivery and controlled release of therapeutic molecules and genes. Thus, the aim of this study was to determine the anti-cancer effect of the over-expressed AT[subscript]2 gene on lung adenocarcinoma cells in vitro using adenoviral vector (Ad-) and nanoparticle (NP-) based gene delivery systems. This study showed that over-expression of Ad-AT[subscript]2 induced cancer cell-specific apoptosis in several human lung adenocarcinoma cell lines with minimal effect on normal lung epithelial cells. Ad-AT[subscript]2 significantly attenuated multiple human lung cancers' cell growth (A549 and H358) in vitro compared to the control viral vector, Ad-[Beta]-galactosidase (Ad-LacZ) when examined by direct cell count. The growth attenuation effect was detected as early as 24 hours after Ad-AT[subscript]2 transfection and lasted 12 days. Western Blot analysis revealed the activation of the caspase pathway. Examination for Annexin V by flow cytometry also confirmed activation of the apoptotic pathway via AT[subscript]2 over-expression. Similarly, AT[subscript]2 cDNA encapsulated poly(DL-lactide-co-glycolide) (PLGA) biodegradable nanoparticles (NPs) were shown to be effectively taken up into lung cancer cells. Surface conjugation of the angiotensin II peptide significantly stimulated uptake of the particles. This PLGA vector-dependent AT[subscript]2 transfection was effective in sustained gene expression and resultant cell death. These results indicate that the AT[subscript]2 over-expression effectively attenuated growth of lung adenocarcinoma cells through activation of intrinsic apoptosis. Since PLGA safety has been proven, whereas adenoviral vectors have several drawbacks in safety, the Ang II conjugated PLGA nanoparticles may be a better therapeutic gene delivery system. Therefore, it is concluded that the discovery of AT[subscript]2 DNA encapsulated PLGA conjugated with the Ang II peptide is a potentially useful tool for lung cancer gene therapy.

The Angiotensin II Type 1 (AT1) receptor is the main receptor responsible for the effects of the renin-angiotensin system, and its expression pattern is altered in several diseases. [11C]Methyl-Losartan has been developed based on the clinically used AT1 receptor antagonist Losartan. The aim of this work is to characterize the pharmacokinetics, repeatability and reliability of measurements, binding specificity and selectivity of [11C]Methyl-Losartan in rats using in vivo small animal positron emission tomography (PET) imaging, ex vivo biodistribution and in vitro autoradiography methods. Also, we aim to measure the presence of metabolites in the kidney and plasma using high-performance liquid chromatography. We have demonstrated in vivo that [11C]Methyl-Losartan is taken up in the AT1 receptor-rich kidneys and that it is displaceable by selective AT1 receptor antagonists. Using ex vivo biodistribution, we have confirmed these results and demonstrated that [11C]Methyl-Losartan binds selectively to the AT1 receptor over the AT2, Mas and β-adrenergic receptors. In vitro autoradiography results confirmed these renal binding selectivity studies. [11C]Methyl-Losartan was also shown to have one and two C-11 labeled metabolites in the plasma and kidneys, respectively. In conclusion, [11C]Methyl-Losartan is a promising agent for studying the AT1 receptor in rat models with normal and altered AT1 receptor expression using small animal PET imaging.

High salt intake contributes to the development of hypertension in salt-sensitive humans and animals and the mechanistic causes are poorly understood. In Dahl salt-sensitive (S) but not salt-resistant (R) rats, high salt diet increases cerebrospinal fluid (CSF) [Na+] and activates an aldosterone-mineralocorticoid receptor-epithelial sodium channel-endogenous ouabain (MR-ENaC-EO) neuromodulatory pathway in the brain that enhances the activity of sympatho-excitatory angiotensinergic and glutamatergic pathways, leading to an increase in sympathetic nerve activity (SNA) and blood pressure (BP). We hypothesize that high salt diet in Dahl S rats enhances Ang II release in the paraventricular nucleus (PVN), causing a decrease in local nitric oxide (NO) action and an increase in local glutamate release thereby elevating SNA, BP and heart rate (HR). The present study evaluated the effects of agonists or blockers of MR, ENaC, EO, nitric oxide synthase (NOS) or glutamate and AT1-receptors on the BP and HR responses to acute infusions of Na+ rich aCSF, intracerebroventricularly (icv), or in the PVN of Dahl S, R or Wistar rats or to high salt diet in Dahl S and R rats. In Wistar rats, aldosterone in the PVN enhanced the BP and HR responses to infusion of Na+ rich aCSF in the PVN, but not in the CSF, and only the enhancement was prevented by blockers of MR, ENaC and EO in the PVN. AT1-receptor blockers in the PVN fully blocked the enhancement by aldosterone and the responses to infusion of Na+ rich aCSF icv, or in the PVN. Na+ rich aCSF in the PVN caused larger increases in BP and HR in Dahl S vs. R rats and the responses to Na+ were fully blocked by an AT1-receptor blocker in the PVN. BP and HR responses to a NOS blocker in the PVN were the same, but L-NAME enhanced Na+ effects more in Dahl R than S rats. High salt diet attenuated increases in BP from L-NAME in the PVN of Dahl S but not R rats. AT1 and glutamate receptor blockers candesartan and kynurenate in the PVN decreased BP in Dahl S but not R rats on high salt diet. At the peak BP response to candesartan, kynurenate in the PVN further decreased BP whereas candesartan did not further decrease BP at the peak BP response to kynurenate. Our findings indicate that both an acute increase in CSF [Na+] and high salt intake in Dahl S rats increases AT1-receptor activation and decreases NO action in the PVN thereby contributing to the pressor responses to Na+ and presumably, to dietary salt-induced hypertension. The increased BP response to AT1-receptor activation in the PVN of Dahl S is mediated by enhanced local glutamate receptor activation. An MR-ENaC-EO pathway in the PVN can be functionally active and further studies need to assess its role in Dahl S rats on high salt intake.

Vasoconstriction can be initiated via the interaction of a number of chemicals with specific "receptive sites" known as the receptors. This thesis examines two distinctly different modes by which drugs initiate a contractile response, namely, (i) the interaction of angiotensin analogues with a heterogeneous population of angiotensin receptors in vascular smooth muscles, and (ii) the conditions whereby B-adrenoceptor antagonists interact with a-adrenoceptor antagonists thereby causing a pressor response. Conscious, unrestrained, instrumented-rats were used for the study. It has been suggested that angiotensin receptors in vascular and non-vascular tissues may not be of a homogeneous population. The first study examined whether a heterogeneous population of angiotensin receptors was responsible for increasing vascular tone. Dose-response curves were constructed for angiotensin II (ANG II) and des Asp¹ angiotensin II (ANG III) on mean arterial pressure (MAP) and mean circulatory filling pressure (MCFP), an index of total body venous tone, in the presence or absence of [Sar¹, Ile⁸]ANG II. The i.v. infusion of ANG II or ANG III caused dose-dependent increases in MAP and MCFP. In the presence of [Sar¹, Ile⁸]ANG II, the MAP and MCFP curves for ANG II were displaced to the right with pA₂ values of 9.2 and 8.4 for the arterioles and veins, respectively. However, the antagonist displaced dose-MCFP but not the dose-MAP response curve of ANG III. This suggests that ANG II and ANG III act on the same receptor in veins but not arterioles. This concept was further investigated by obtaining dose-MAP and dose-MCFP response curves for ANG II in the presence of ANG II or ANG III. Dose-MAP response curve to ANG II was displaced to the right in the presence of ANG II but not ANG III. Dose-MCFP response curve for ANG II was displaced to the right in the presence of ANG III but not ANG II. These results again suggest that ANG III acts on the same receptors as ANG II in the veins but not arterioles. In the last series of experiments two analogues of angiotensin III were compared as antagonists of the pressor response to ANG II and ANG III. In the presence of [Ile⁷]ANG III, the dose-MAP response curves for ANG II and ANG III were displaced to the right while in the presence of [Sar¹, Ile⁷]ANG III, the dose-MAP response curve for ANG III but not ANG II was displaced. This suggests that [Sar¹, Ile⁷]ANG III is a selective antagonist of ANG III in the arterioles. In summary, the results indicate that ANG III acts on a different sub-class of angiotensin receptors than ANG II in the arterioles but it may act as a partial agonist on the same type of receptors as ANG II in the venous bed. Thus, ANG II receptors in the arterioles appear to be different from those in veins. The administration of a non-selective β-antagonist propranolol into animals subjected to non-selective α-blockade has been observed to cause a paradoxical pressor response. This second study examines whether the paradoxical pressor response to β-antagonists was due to: (i) an interaction of a β-antagonist with an α-antagonist, (ii) blockade of vasodilator β₂-adrenoceptors or (iii) an increase in the release of catecholamines. Cumulative dose-response curves for propranolol, atenolol (β₁-antagonist) and ICI 118,551 (β₂-antagonist) were obtained in rats subjected to a continuous i.v. infusion of phentolamine, a non-selective α-antagonist. The administration of each of the β-antagonists caused a dose-dependent increase in MAP suggesting that the pressor response was not due to the blockade of vasodilator β₂-adrenoceptors. Another four groups of phentolamine-treated rats were given a single i.v. bolus injection of saline, propranolol, atenolol or ICI 118,551, and sampling of arterial blood for the determination of adrenaline (A) and noradrenaline (NA) concentration by HPLC/ec. Phentolamine caused a decrease in MAP and an increase in the plasma levels of A and NA. Subsequent injection of propranolol, atenolol and ICI 118,551 but not saline increased MAP. Neither saline nor any of the β-antagonists increased plasma NA or A levels suggesting that the pressor response was not associated with an acute increase in the release of catecholamines. It was also shown that prior injection of a β-antagonist partially antagonized the hypotensive effect of phentolamine suggesting that the pressor response was related to an interaction between α- and β-antagonists. It was further shown that a continuous infusion of either prazosin or rauwolseine caused a small but not significant decrease in MAP which was reversed by propranolol. Concurrent infusions of prazosin and rauwolscine caused a large decrease in MAP. Subsequent injection of propranolol caused a large pressor response. On the contrary, sodium nitroprusside or metha-choline each decreased MAP but the hypotension was not antagonized by propranolol. These results were consistent with the existence of a specific interaction between α- and β-antagonists. These experiments demonstrated that although the mechanisms involved in the initiation of a change in vascular tone did not share a common pathway, the final outcome shared a common denomination. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate