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Os receptores acoplados à proteína G (GPCRs), também chamados de receptores 7TM, são conhecidos por regular virtualmente todos os processos fisiológicos em mamíferos e cerca de 40% de todas as drogas comerciais agem através destes receptores. A sinalização mediada por eles é classicamente atribuída à proteína G, que é ativada pela troca de GDP por GTP, promovendo a separação das subunidades G e G, e leva à produção de mensageiros secundários como cAMP, Ca2+ e DAG. Após a resposta os GPCRs são fosforilados pelas quinases de GPCRs (GRKs), sinalizando para recrutamento das -arrestinas citoplasmáticas, que por sua vez desencadeiam a formação de endossomos internalizando e dessensibilizando o receptor. Entretanto, estudos mostram que este endossomo, contendo o complexo ligante-receptor--arrestina, pode interagir com proteínas sinalizadoras no citoplasma desencadeando vias de sinalização independentes de proteína G. Recentemente foram descritos para diferentes receptores, ligantes capazes de ativar seletivamente uma das duas vias, proteína G ou -arrestina, chamados agonistas seletivos. O receptor AT1 é um GPCR particularmente interessante no estudo do agonismo seletivo, tanto por sua vasta expressão em tecidos quanto pelo conhecimento de agonistas seletivos já estabelecidos, tais como os ligantes SII e TRV120027. O objetivo deste trabalho foi analisar comparativamente os perfis de sinalização decorrente da ativação de AT1 por SII ou TRV120027 através do uso de arranjos de quinases e da modulação de genes relacionados a sinalização de GPCRs. Ang II que é ligante natural e total (ativa via dependente de proteína G e de -arrestina) neste receptor foi usada como controle para fins de comparação. Nossos dados mostraram que o perfil da sinalização mediada pelo receptor AT1 varia não só entre AngII e os agonistas seletivos, mas também entre os dois ligantes seletivos SII e TRV120027, mostrando que a interação receptor-ligante pode influenciar a sinalização em um grau mais refinado, além da ativação dependente de -arrestina ou proteína G. Estes dados mostram que existem perspectivas para o desenvolvimento futuro de ligantes com ainda maior grau de seletividade. / G protein coupled receptors (GPCRs), also known as 7TM receptors, are known to regulate virtually all physiological processes in mammals and approximately 40% of all current clinical drugs act by modulating such receptors. The signaling mediated by them is classically by coupling to G protein, which is activated by exchanging bound GDP for GTP, dissociation of G and G subunits, then leading to production of second messengers such as cAMP, Ca2+, and DAG. After the signal transduction, GPCR are phosphorylated by GPCR kinases (GRKs), followed by recruitment of cytoplasmic -arrestins, which initiate the endosome formation with consequent internalization and desensitization of the receptor. However, is has been demonstrated that the endosome assembling the ligand-receptor--arrestin complex can interact with cytoplasmic signaling proteins, therefore activating signaling pathways independently of G protein coupling. Recently, for different receptors, it has been described ligands capable of selectively activating one of these signaling pathways, G protein or -arrestin, called biased agonists. The AT1 receptor is a particularly interesting GPCR for the study of biased agonism, either due to its wide tissue expression as well as also due the existence of known and established biased ligands, such as SII and TRV120027. The aim of our study was to comparatively analyze the AT1 receptor signaling pathways profiles after activation by SII or TRV120027, using kinases arrays, and expression modulation of genes related to GPCRs signaling. AngII is the natural and full agonist of this receptor (activates both G protein and -arrestin signaling pathways) was used for comparison. Our data show that the signaling profile mediated by AT1 receptor can be distinct not only when comparing the profiles from AngII and the biased agonists, but also when comparing the profiles from the two biased ligands SII and TRv120027; revealing that the complex ligand-receptor can influence the downstream signaling pathways in a fine-tune way, further to the activation of -arrestin or G-protein. This data show that there are perspectives for the future development of ligands with even higher degree of selectivity.

Os receptores acoplados à proteína G (GPCRs) são proteínas integrais de membrana caracterizados por possuírem sete alfa-hélices transmembranares. Esses receptores são importantes alvos de estudos biomédicos e aproximadamente 40% dos medicamentos atualmente comercializados agem sobre estes receptores. O receptor de Angiotensina II do tipo 1 (AT1) é um GPCR e o principal mediador do Sistema Renina-Angiotensina que tem como principal efetor o octopeptídeo Angiotensina II (AngII). Recentemente foi descrito que as mutações A244S e I103T-A244S no receptor AT1 podem estar relacionadas com a predisposição à fibrilação atrial. Neste trabalho foi realizada a construção, caracterização bioquímica e farmacológica destes mutantes, bem como do mutante I103T, com o objetivo de compreender como a funcionalidade desses receptores mutantes poderiam contribuir para a predisposição à fibrilação atrial. Os mutantes I103T e I103T-A244S revelaram ser mais eficientes e potentes que o receptor selvagem em aumentar os níveis de cálcio intracelular em resposta à AngII. Todos os mutantes estudados apresentaram baixa eficiência quanto à ativação da via das MAPKs e apresentaram comportamento diferente do receptor selvagem quando bloqueados com o antagonista Losartan, seletivo para o receptor AT1 e muito usado na clínica como medicamento anti-hipertensivo. Esses dados ressaltam a relevância do estudo tanto em termos de melhor compreender as bases moleculares da relação entre as mutações e a doença, bem como possível prevenção ao uso de medicamentos que possam interagir e agir diferentemente em receptores com essas mutações. / G-protein coupled receptors (GPCRs) are integral membrane proteins characterized by having seven transmembrane alpha-helices. These receptors are important targets of biomedical studies and approximately 40% of currently marketed drugs act on such receptors. The angiotensin II type 1 receptor (AT1) is a GPCR and the main mediator of the Renin-Angiotensin System whose main effector is the octapeptide Angiotensin II (Ang II). It was recently described that I103T and A244S mutations in the AT1 receptor may be related to the susceptibility to atrial fibrillation. In this study we carried out the construction of these mutants and their biochemical and functional characterization. The I103T and I103T/A244S mutants were shown to be more efficient and potent than the wild-type receptor on the increase of intracellular calcium levels. All mutants showed lower efficcacy for MAPK pathway activation and showed different behavior when compared to the wild-type receptor after antagonism with Losartan. These data highlight the relevance of the present study concerning a better understanding of the molecular basis of cardiovascular diseases and showing that conventional therapies for certain diseases may lead to adverse effects on patients carrying point mutations on the receptor sequence.

The insulin resistance syndrome [INSR] is associated with increased cardiovascular risk and affects up to 25% of the Australian population. The mechanism underlying the relationship between the INSR and increased cardiovascular risk is controversial. We postulated that perturbations in the renin-angiotensin system [RAS] and endothelium-derived NO may be implicated in the development of early vascular changes in the INSR. Repeated measurements of arterial stiffness [using digital photoplethysmography] and haemodynamic parameters in response to vasoactive medications were used to demonstrate the functional expression of angiotensin II [Ang II] receptors and NO synthase [NOS]. Ang II acts via two main receptor sub-types: the Ang II type 1 [AT1] and Ang II type 2 [AT2] receptors. The AT1 receptor is central to the development of arterial stiffness and endothelial dysfunction. The role of AT2 receptors in humans is controversial but is postulated to counter-act AT1 receptor mediated effects in diseased vascular beds. We demonstrated increased AT1 and AT2 receptor-mediated effects in small to medium-sized arteries of subjects with early INSR [Chapter 6]. In addition, functional expression of AT2 receptors in adult insulin resistant humans [Chapter 5], but not in healthy volunteers [Chapter 4] was demonstrated. AT1 receptor blockade in subjects with early INSR resulted in improvements in vascular function, with a consequent functional down-regulation of AT2 receptors [Chapter 7]. Functional NOS expression was demonstrated to be increased in subjects with early INSR compared with healthy controls [Chapter 6]. This was postulated to be a homeostatic response to counteract early vascular changes in subjects with early INSR. AT1 receptor blockade in these subjects reduced functional NOS expression [Chapter 8]. In conclusion, patients with early INSR represent a model of early disease where early intervention may be able to reverse the process incited by the initial exposure to multiple cardiovascular risk factors. Early vascular changes in these individuals are mediated at least in part, by increased AT1 receptor activity and/or expression, and may be detected by changes in arterial stiffness indices and non-invasive vascular reactivity studies. There is a compensatory increase in AT2 receptor and NOS expression/activity to counter-act these vascular changes.

The insulin resistance syndrome [INSR] is associated with increased cardiovascular risk and affects up to 25% of the Australian population. The mechanism underlying the relationship between the INSR and increased cardiovascular risk is controversial. We postulated that perturbations in the renin-angiotensin system [RAS] and endothelium-derived NO may be implicated in the development of early vascular changes in the INSR. Repeated measurements of arterial stiffness [using digital photoplethysmography] and haemodynamic parameters in response to vasoactive medications were used to demonstrate the functional expression of angiotensin II [Ang II] receptors and NO synthase [NOS]. Ang II acts via two main receptor sub-types: the Ang II type 1 [AT1] and Ang II type 2 [AT2] receptors. The AT1 receptor is central to the development of arterial stiffness and endothelial dysfunction. The role of AT2 receptors in humans is controversial but is postulated to counter-act AT1 receptor mediated effects in diseased vascular beds. We demonstrated increased AT1 and AT2 receptor-mediated effects in small to medium-sized arteries of subjects with early INSR [Chapter 6]. In addition, functional expression of AT2 receptors in adult insulin resistant humans [Chapter 5], but not in healthy volunteers [Chapter 4] was demonstrated. AT1 receptor blockade in subjects with early INSR resulted in improvements in vascular function, with a consequent functional down-regulation of AT2 receptors [Chapter 7]. Functional NOS expression was demonstrated to be increased in subjects with early INSR compared with healthy controls [Chapter 6]. This was postulated to be a homeostatic response to counteract early vascular changes in subjects with early INSR. AT1 receptor blockade in these subjects reduced functional NOS expression [Chapter 8]. In conclusion, patients with early INSR represent a model of early disease where early intervention may be able to reverse the process incited by the initial exposure to multiple cardiovascular risk factors. Early vascular changes in these individuals are mediated at least in part, by increased AT1 receptor activity and/or expression, and may be detected by changes in arterial stiffness indices and non-invasive vascular reactivity studies. There is a compensatory increase in AT2 receptor and NOS expression/activity to counter-act these vascular changes.

The insulin resistance syndrome [INSR] is associated with increased cardiovascular risk and affects up to 25% of the Australian population. The mechanism underlying the relationship between the INSR and increased cardiovascular risk is controversial. We postulated that perturbations in the renin-angiotensin system [RAS] and endothelium-derived NO may be implicated in the development of early vascular changes in the INSR. Repeated measurements of arterial stiffness [using digital photoplethysmography] and haemodynamic parameters in response to vasoactive medications were used to demonstrate the functional expression of angiotensin II [Ang II] receptors and NO synthase [NOS]. Ang II acts via two main receptor sub-types: the Ang II type 1 [AT1] and Ang II type 2 [AT2] receptors. The AT1 receptor is central to the development of arterial stiffness and endothelial dysfunction. The role of AT2 receptors in humans is controversial but is postulated to counter-act AT1 receptor mediated effects in diseased vascular beds. We demonstrated increased AT1 and AT2 receptor-mediated effects in small to medium-sized arteries of subjects with early INSR [Chapter 6]. In addition, functional expression of AT2 receptors in adult insulin resistant humans [Chapter 5], but not in healthy volunteers [Chapter 4] was demonstrated. AT1 receptor blockade in subjects with early INSR resulted in improvements in vascular function, with a consequent functional down-regulation of AT2 receptors [Chapter 7]. Functional NOS expression was demonstrated to be increased in subjects with early INSR compared with healthy controls [Chapter 6]. This was postulated to be a homeostatic response to counteract early vascular changes in subjects with early INSR. AT1 receptor blockade in these subjects reduced functional NOS expression [Chapter 8]. In conclusion, patients with early INSR represent a model of early disease where early intervention may be able to reverse the process incited by the initial exposure to multiple cardiovascular risk factors. Early vascular changes in these individuals are mediated at least in part, by increased AT1 receptor activity and/or expression, and may be detected by changes in arterial stiffness indices and non-invasive vascular reactivity studies. There is a compensatory increase in AT2 receptor and NOS expression/activity to counter-act these vascular changes.

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.

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.

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.

The insulin resistance syndrome [INSR] is associated with increased cardiovascular risk and affects up to 25% of the Australian population. The mechanism underlying the relationship between the INSR and increased cardiovascular risk is controversial. We postulated that perturbations in the renin-angiotensin system [RAS] and endothelium-derived NO may be implicated in the development of early vascular changes in the INSR. Repeated measurements of arterial stiffness [using digital photoplethysmography] and haemodynamic parameters in response to vasoactive medications were used to demonstrate the functional expression of angiotensin II [Ang II] receptors and NO synthase [NOS]. Ang II acts via two main receptor sub-types: the Ang II type 1 [AT1] and Ang II type 2 [AT2] receptors. The AT1 receptor is central to the development of arterial stiffness and endothelial dysfunction. The role of AT2 receptors in humans is controversial but is postulated to counter-act AT1 receptor mediated effects in diseased vascular beds. We demonstrated increased AT1 and AT2 receptor-mediated effects in small to medium-sized arteries of subjects with early INSR [Chapter 6]. In addition, functional expression of AT2 receptors in adult insulin resistant humans [Chapter 5], but not in healthy volunteers [Chapter 4] was demonstrated. AT1 receptor blockade in subjects with early INSR resulted in improvements in vascular function, with a consequent functional down-regulation of AT2 receptors [Chapter 7]. Functional NOS expression was demonstrated to be increased in subjects with early INSR compared with healthy controls [Chapter 6]. This was postulated to be a homeostatic response to counteract early vascular changes in subjects with early INSR. AT1 receptor blockade in these subjects reduced functional NOS expression [Chapter 8]. In conclusion, patients with early INSR represent a model of early disease where early intervention may be able to reverse the process incited by the initial exposure to multiple cardiovascular risk factors. Early vascular changes in these individuals are mediated at least in part, by increased AT1 receptor activity and/or expression, and may be detected by changes in arterial stiffness indices and non-invasive vascular reactivity studies. There is a compensatory increase in AT2 receptor and NOS expression/activity to counter-act these vascular changes.