Orientadores: Fabíola Taufic Mónica Iglesias, Edson Antunes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-29T00:02:26Z (GMT). No. of bitstreams: 1 Fernandes_CamilaStefaniEstancial_M.pdf: 2289746 bytes, checksum: e67d3948b14883b515cd7e008bc69850 (MD5) Previous issue date: 2015 / Resumo: Duas classes de medicamentos denominadas estimuladores e ativadores da GCs foram desenvolvidas para uso terapêutico em situações patológicas, onde há menor biodisponibilidade NO ou tolerância farmacológica. A principal diferença entre os moduladores da GCs é que os ativadores atuam de maneira mais eficaz mesmo quando a enzima encontra-se no estado oxidado. O presente trabalho foi dividido em duas etapas, a saber: 1) caracterização farmacológica do ativador BAY 60-2770 em corpo cavernoso (CC) de coelho e 2) avaliação do efeito do ativador BAY 60-2770 e estimulador BAY 41-2272 da GCs em CC isolado de ratos espontaneamente hipertensos (SHR) e normotensos (WKY). O BAY 60-2770 promoveu relaxamento concentração dependente em corpo cavernoso de coelho (pEC50: 7,584 ± 0,1923), sendo este efeito potencializado na presença dos inibidores da GCs ODQ (10 µM; pEC50: 8,170 ± 0,1365) e da fosfodiesterase tipo 5 (PDE5), tadalafil (100 ?M; pEC50: 8,381 ± 0,1334). A contração induzida por estimulação elétrica foi reduzida em aproximadamente 50% na presença de BAY 60-2770, sendo este efeito potencializado na presença de ODQ. Em relação ao relaxamento induzido pela estimulação elétrica, o ODQ e BAY 60-2770 aboliu e aumentou, respectivamente o relaxamento. A co-incubação de BAY 60-2770 com ODQ não alterou o relaxamento em comparação com o ODQ sozinho. Curiosamente, quando BAY 60-2770 foi incubado 20 min antes da adição de ODQ, os relaxamentos induzidos por estimulação elétrica foram parcialmente restaurados quando comparado com o ODQ sozinho. Em relação à segunda etapa do trabalho, o relaxamento induzido pelos moduladores da GCs, BAY 60-2770 e BAY 41-2272 não diferiram em CC de ratos SHR comparados ao seu respectivo controle, porém, estes dados não corroboram com a quantificação de GMPc, no qual evidenciamos aumento dos seus níveis em CC de ratos SHR sendo estes níveis ainda maiores quando na presença de ODQ em comparação ao CC de WKY. Observamos também que o relaxamento induzido pela acetilcolina (ACh) encontrou-se reduzido em CC dos animais hipertensos, entretanto, a adição de BAY 60-2770 ou BAY 41-2272 restaurou este relaxamento. Não foi encontrada diferença na expressão das subunidades ?1 e ?1 da GCs em ratos SHR e WKY, porém quando analisamos a expressão da PDE-5, verificamos maior expressão em CC de ratos SHR. O BAY 60-2770 foi capaz de elevar a pressão intracavernosa em CC de ratos normotensos em todas as frequências estudadas, porém esta elevação somente foi significativa na frequência de 16 Hz. Considerando que 1) BAY 60-2770 induziu relaxamento em CC de coelho e de rato hipertenso de maneira mais potente que o estimulador BAY 41-2272, 2) a adição de ODQ potencializou o relaxamento do BAY 60-2770, mas não do BAY 41-2272 em CC de ambas espécies, 3) o aumento dos níveis de GMPc foi maior em CC de ratos SHR após estímulo com BAY 60-2770, mas não com BAY 41-2272 pode-se concluir que em situações patológicas onde há oxidação da GCs e/ou menor biodisponilidade de NO, os ativadores da GCs seriam mais vantajosos que os estimuladores ou inibidores da PDE5 para o tratamento da disfunção erétil / Abstract: Two classes of drugs referred to as stimulators and sGC activators have been developed for therapeutic use in pathological situations where there is less NO bioavailability and pharmacological tolerance. The main difference between the sGC activators modulators is that they act more effectively even when the enzyme is in the oxidized state. This study was divided into two stages: 1) pharmacological characterization of BAY 60-2770 activator in corpus cavernosum (CC) of rabbit and 2) evaluation of the effect of BAY 60-2770 BAY 41-2272 activator and stimulator of GCs isolated from spontaneously hypertensive rats (SHR) and normotensive (WKY). The BAY 60-2770 induced concentration-dependent relaxation in rabbit corpus cavernosum (pEC 50: 7.584 ± 0.1923), and this increased effect in the presence of GCs ODQ inhibitors (10 ?M; pEC 50: 8.170 ± 0.1365) and the phosphodiesterase type 5 (PDE5), tadalafil (100 ?m; pEC50: 8.381 ± 0.1334). The contraction induced by electric stimulation was reduced by approximately 50% in the presence of BAY 60-2770, and this increased effect in the presence of ODQ. In relation to the relaxation induced by electrical stimulation, ODQ and BAY 60-2770 abolished and increased, respectively the relaxation. Co-incubation with BAY 60-2770 with ODQ did not change compared to the relaxation with only ODQ. Curiously, when BAY 60-2770 was incubated 20 minutes before addition of ODQ, the relaxation induced by electric stimulation was partially restored when compared with only ODQ. In the second part, relaxation induced by modulators of GCs, BAY 60-2770 and BAY 41-2272 did not differ in SHR CC compared to their respective control, however, these data do not corroborate the quantification of cGMP, in which we observed an increase in their levels in SHR rats and these levels even higher in the presence of ODQ in comparing the CC of WKY. We also observed a reduction of the relaxation induced by acetylcholine (ACh) in CC of hypertensive animals, however, the addition of BAY 60-2770 or BAY 41-2272 restored this relaxation. There was no difference in the expression of ?1 and ?1 subunits of sGC in SHR and WKY rats, but when we analyzed the expression of PDE-5, we found greater expression in SHR. The BAY 60-2770 was able to raise up the intracavernous pressure in the normotensive rats in all studied frequencies, but this increase was only significant in the frequency of 16 Hz. Considering 1) BAY 60-2770 induced relaxation in rabbit and hypertensive rat so that the most potent stimulator BAY 41-2272, 2) addition of ODQ potentiated relaxation of BAY 60-2770, but not in BAY 41-2272 in CC of both, 3) increased levels cGMP was greater in SHR rats after stimulation with BAY 60-2770, but not in BAY 41-2272 so can be concluded that in pathologic situations where there is oxidation of GCs and / or low bioavailability of NO, would activators of sGC more advantageous than PDE5 inhibitors or stimulators for the treatment of erectile dysfunction / Mestrado / Farmacologia / Mestra em Farmacologia

No description available.

No description available.

INTRODUÇÃO. O termo Lesão Pulmonar Aguda (LPA) é usado para descrever a resposta pulmonar à lesão que ocorre diretamente ou indiretamente nos pulmões. A quebra da barreira alvéolo-capilar determina o influxo de líquido rico em proteínas para dentro dos espaços alveolares, sendo necessária a reabsorção desse líquido no processo de resolução da LPA. A infusão intravenosa de Ácido Oleico (AO) em ratos provoca agudamente edema alveolar difuso e focos hemorrágicos intra-alveolares, sendo um bom modelo de indução. Estudos relatam que o Azul de Metileno (AM) atenua tais lesões, com efeito protetor, no tecido pulmonar, e reduz o edema presente na LPA em animais com sepse através da inibição da guanilato ciclase solúvel (GCs), uma enzima ativadora da via NO-GMPc. OBJETIVO. Estudar a repercussão da inibição da GCs pelo AM na permeabilidade capilar pulmonar ministrando-o antes e após a indução da lesão pulmonar por AO. MÉTODO. Ratos Wistar foram divididos em 5 grupos: Sham com infusão de salina em bolus; AM com infusão de AM por 2h; AO com infusão de AO em bolus, AM/AO com infusão de AM por 2h, sendo que, após 5 min do início, recebeu AO simultaneamente em bolus e AO/AM com infusão de AO em bolus e, após 2h, infusão de AM por mais 2h. Após 4h foi realizada a coleta de materiais (sangue, lavado bronco-alveolar e tecido pulmonar) para análise do NO plasmático e tecidual, gasometria arterial, cálculo do peso úmido/peso seco (PU/PS) e histologia do tecido pulmonar de todos os grupos. A estatística utilizada foi a análise de variância (one-way ANOVA) com p<0.05. RESULTADO. Não foi encontrado hipoxemia grave após 4h de lesão. O grupo AO apresenta um aumento no número de proteínas no lavado bronco-alveolar e na relação PU/PS comparado aos grupos controle: Sham e AM, confirmando a presença de lesão e alteração de permeabilidade pulmonar. Os grupos tratados com AM apresentaram melhora na permeabilidade pulmonar, porém, apenas o grupo pré-tratamento (AM/AO) apresentou diferença estatística na redução do extravasamento de proteínas no lavado. Não foram encontradas diferenças significativas no NO plasmático e tecidual. Na microscopia, a congestão capilar foi intensa, acompanhada de múltiplos focos de edema alveolar, exsudato intra-alveolar proteico, áreas de hemorragia e infiltrado inflamatório neutrofílico, tanto no interstício quanto nos septos alveolares.Os grupos tratados com AM apresentaram diminuição das áreas de edema, exsudação e hemorragia, porém, com maior evidência no grupo AM/AO. CONCLUSÃO. O AM diminui a permeabilidade pulmonar quando administrado de maneira precoce amenizando os danos causados pela LPA induzida por AO. / BACKGROUND. The term Acute Lung Injury (ALI) is used to describe the response to lung injury that occurs directly or indirectly in the lungs. The rupture of the alveolar - capillary barrier determines the influx of protein-rich fluid into the alveolar spaces, the reabsorption of this fluid in the process of resolving the ALI is required. Intravenous infusion of oleic acid (OA) in rats acutely causes diffuse alveolar edema and intra-alveolar hemorrhagic foci, being a good role model induction. Studies have reported that Methylene Blue (MB) attenuates such injuries, with a protective effect in lung tissue and reduce edema present in the ALI present in with sepsis by inhibition of soluble guanylate cyclase (sGC), an activator of the enzyme NO- cGMP pathway. OBJECTIVE. To study the effect of inhibition of sGC by MB in pulmonary capillary permeability ministering to the before and after induction of lung injury by OA. METHOD. Wistar rats were divided into 5 groups: Sham infused with saline bolus, MB infused with MB for 2hrs, OA infused with OA bolus, MB/OA infused with MB for 2hrs, and after 5 min from the beginning, simultaneously received OA bolus and OA/MB infused with OA bolus and after 2hrs, MB infusion for 2hrs. After 4hrs the collection of materials was performed (blood, bronchoalveolar lavage and lung tissue) for analysis of plasma and tissue NO, arterial blood gases, calculation of the wet weight/dry weight (WW/DW) and histology of lung tissue from all groups. The statistic used was analysis of variance (one-way ANOVA) with p<0.05. RESULTS. Not found severe hypoxemia after 4hrs of injury. The OA group shows an increase in the number of proteins in bronchoalveolar lavage and in WW/DW ratio compared to the control groups: Sham and MB, confirming the presence of injury and alterations of lung permeability. The groups treated with MB showed improvement in lung permeability, however, only the pretreatment group (MB/OA) showed statistical significance in reducing the leakage of protein in the lavage. No significant differences were found in plasma and tissue NO. In microscopy, capillary congestion was intense, accompanied by multiple foci of alveolar edema, intra-alveolar proteinaceous exudates, areas of hemorrhage and neutrophilic inflammatory infiltrate in both the interstitium and in the alveolar septa. The groups treated with MB showed reduction in areas of edema, exudation and hemorrhage, however, most obviously in MB/OA group. CONCLUSION. The MB decreases lung permeability when administered as early as possible, mitigating the damage caused by OA-induced ALI.

Abstract not available

Primary cilia are evolutionarily conserved organelles required in a number of signalling pathways influencing the development and behaviour of a diverse range of organisms. More recently, studies into a new class of human diseases known as ciliopathies have helped to shed light on the critical role of this once-ignored signalling centre. Bardet-Biedl syndrome (BBS) proteins localize to the primary cilium and participate in cilium biogenesis and function. BBS is a pleiotropic human disorder with variable severity that is suitable as a disease model for investigating the pathogenesis of a number of common ciliopathy features such as photoreceptor degeneration, renal cysts, and obesity. The C. elegans genome encodes a number of BBS proteins which undergo intraflagellar transport (IFT) at the primary cilium. Given the conservation between C. elegans and human BBS proteins, I hypothesize the existence of unidentified conserved genetic pathways related to the functions of these proteins. Using C. elegans, I characterize novel features of bbs mutants while identifying sources of genomic variation that may elucidate the variability of human BBS features. I show that C. elegans bbs mutants exhibit smaller body size, delayed development, and decreased exploration behaviour. Moreover, I identify a role for the soluble guanylate cyclases GCY-35/GCY-36 in modifying these bbs phenotypes. I conclude that BBS proteins non-cell autonomously influence a set of body cavity neurons in which GCY-35/GCY-36 function genetically upstream of a cGMP-dependent protein kinase (PKG), EGL-4, to control body size. Furthermore, the role of GCY-35/GCY-36 is unique amongst a large number of guanylate cyclases and BBS proteins may influence body size via an IFT-independent function. I explore the biological functions of EGL-4 and conclude that it may regulate body size through multiple cellular mechanisms. I also examine potential candidate genes related to cGMP production and turnover, confirming that additional cGMP-related factors can influence body size although not necessarily in body cavity neurons. In conclusion, I propose a model where BBS-expressing sensory neurons influence body size and development through cGMP-PKG signalling in body cavity neurons while functioning in parallel with additional sensory neurons (possibly BBS-independent) that use similar cGMP-PKG signalling dynamics.

Primary cilia are evolutionarily conserved organelles required in a number of signalling pathways influencing the development and behaviour of a diverse range of organisms. More recently, studies into a new class of human diseases known as ciliopathies have helped to shed light on the critical role of this once-ignored signalling centre. Bardet-Biedl syndrome (BBS) proteins localize to the primary cilium and participate in cilium biogenesis and function. BBS is a pleiotropic human disorder with variable severity that is suitable as a disease model for investigating the pathogenesis of a number of common ciliopathy features such as photoreceptor degeneration, renal cysts, and obesity. The C. elegans genome encodes a number of BBS proteins which undergo intraflagellar transport (IFT) at the primary cilium. Given the conservation between C. elegans and human BBS proteins, I hypothesize the existence of unidentified conserved genetic pathways related to the functions of these proteins. Using C. elegans, I characterize novel features of bbs mutants while identifying sources of genomic variation that may elucidate the variability of human BBS features. I show that C. elegans bbs mutants exhibit smaller body size, delayed development, and decreased exploration behaviour. Moreover, I identify a role for the soluble guanylate cyclases GCY-35/GCY-36 in modifying these bbs phenotypes. I conclude that BBS proteins non-cell autonomously influence a set of body cavity neurons in which GCY-35/GCY-36 function genetically upstream of a cGMP-dependent protein kinase (PKG), EGL-4, to control body size. Furthermore, the role of GCY-35/GCY-36 is unique amongst a large number of guanylate cyclases and BBS proteins may influence body size via an IFT-independent function. I explore the biological functions of EGL-4 and conclude that it may regulate body size through multiple cellular mechanisms. I also examine potential candidate genes related to cGMP production and turnover, confirming that additional cGMP-related factors can influence body size although not necessarily in body cavity neurons. In conclusion, I propose a model where BBS-expressing sensory neurons influence body size and development through cGMP-PKG signalling in body cavity neurons while functioning in parallel with additional sensory neurons (possibly BBS-independent) that use similar cGMP-PKG signalling dynamics.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 98-113).

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 118-135).

Soluble guanylyl cyclase (sGC) is the receptor for nitric oxide and a highly sought-after therapeutic target for the management of cardiovascular diseases. New compounds that stimulate sGC show clinical promise, but where these stimulator compounds bind and how they function remains unknown. Here, using a photolyzable diazirine derivative of a novel stimulator compound, IWP-051, and MS analysis, we localized drug binding to the 1 heme domain of sGC proteins from the hawkmoth Manduca sexta and from human. Covalent attachments to the stimulator were also identified in bacterial homologs of the sGC heme domain, referred to as H-NOX domains, including those from Nostoc sp. PCC 7120, Shewanella oneidensis, Shewanella woodyi, and Clostridium botulinum, indicating that the binding site is highly conserved. The identification of photoaffinity-labeled peptides was aided by a signature MS fragmentation pattern of general applicability for unequivocal identification of covalently attached compounds. Using NMR, we also examined stimulator binding to sGC from M. sexta and bacterial H-NOX homologs. These data indicated that stimulators bind to a conserved cleft between two subdomains in the sGC heme domain. L12W/T48W substitutions within the binding pocket resulted in a 9-fold decrease in drug response, suggesting that the bulkier tryptophan residues directly block stimulator binding. The localization of stimulator binding to the sGC heme domain reported here resolves the longstanding question of where stimulators bind and provides a path forward for drug discovery.