Navegando por Palavras-chave "Receptor B1"
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- ItemAcesso aberto (Open Access)Clonagem, expressão e purificação de BMP-2 recombinante como ferramenta para avaliar o papel do receptor B1 das cininas na diferenciação osteoblástica induzida por BMP2(Universidade Federal de São Paulo (UNIFESP), 2015-05-27) Silva, Marina Rodrigues e [UNIFESP]; Pesquero, Joao Bosco [UNIFESP]; Nagem, Ronaldo Alves Pinto; http://lattes.cnpq.br/7305428791149745; http://lattes.cnpq.br/0856630824759511; http://lattes.cnpq.br/5900039099570404; Universidade Federal de São Paulo (UNIFESP)O tecido ósseo é um tecido dinâmico, constantemente remodelado, que depende de diversos fatores para manutenção do equilíbrio entre formação e reabsorção óssea. Perdas e danos ocorridos neste tecido constituem um grave problema de saúde pública. Milhões de brasileiros sofrem, atualmente, com problemas relacionados a lesões musculoesqueléticas. O tecido ósseo é acometido por várias injúrias, entre as quais se destacam doenças congênitas e metabólicas, além de fatores externos, como fraturas. Todos esses comprometimentos despertam grande interesse na busca por um mecanismo capaz de reparar tecido ósseo lesado. Atualmente as técnicas de engenharia de tecido têm sido indicadas em tratamentos de lesões em tecido ósseo, por serem das mais promissoras terapias alternativas para reparo de danos ósseos. Existem vários mediadores envolvidos no processo de diferenciação osteogênica que buscam reparar e formar o tecido ósseo, sendo alguns desses mediadores as proteínas morfogenéticas ósseas (BMPs). Além desses, alguns sistemas estão sendo relacionados ao processo de diferenciação óssea, como é o caso do sistema calicreína-cininas. Esse sistema tem sido relacionado com a destruição da matriz óssea, porém, pouco se sabe até o momento do verdadeiro papel dos diversos componentes do sistema no referido processo patológico. Com a expressão e utilização da BMP-2 recombinante, o presente trabalho buscou elucidar o envolvimento do receptor B1 de cininas na indução do processo de diferenciação óssea. Para alcançar esse objetivo, a proteína BMP-2 foi clonada para a expressão da proteína recombinante fusionada a uma cauda de histidina em sua porção N-terminal, capaz de se ligar a colunas de afinidade. Posteriormente, a viabilidade funcional da proteína expressa foi testada em ensaios de diferenciação no intuito de avaliar o papel do receptor B1 de cininas na osteogênese in vitro como perspectiva para futura avaliação in vivo.
- ItemAcesso aberto (Open Access)Influência da deleção genética de receptores de cininas no metabolismo de óxido nítrico vascular(Universidade Federal de São Paulo (UNIFESP), 2011-09-28) Loiola, Rodrigo Azevedo [UNIFESP]; Pesquero, João Bosco [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Activation of B1 receptor in the vascular endothelium triggers diverse signaling pathways that results in elevation of intracellular Ca2+ and Nitric Oxide Synthase (NOS) activation, followed by NO production and vasodilation. Although much has been investigated about the B1-induction and functionality during inflammation, the importance of B1 subtype in normal vessels remains unclear. To clarify this question, the present study analyzed endothelial function and endothelial NO generation in B1 receptor knockout (B1 -/-) and Wild Type (WT) mice. Mesenteric arteriolar bed was perfused with Krebs solution and vascular responses to Acetilcholine (ACh), sodium nitroprusside (SNP) and norepinephrine (NE) were evaluated by a data acquisition system. Plasmatic NO levels (μmol/L) were analyzed by NO derivatives nitrate and nitrite using NO Analyzer (NOATM280, Sievers Instruments) and vascular NO generation was assessed in mesenteric arterioles slices using DAF -2 DA, a fluorescent cell permeable dye for NO (arbitrary units, a.u.). NOS activity (pmol/mg.min) was measured by the biochemical conversion of L-[3H] arginine to L-[3H] citrulline in homogenates of mesenteric vessels in the presence of optimal levels of substrate and co-factors. Primary endothelial cells were incubated with DAF-2 DA and images obtained in a confocal microscope were analyzed by optic densitometry (a.u.). Cells were stimulated with ACh [1 mmol/L] in presence or absence of the NOS substrate Larginine, or the co-factor tetrahydrobiopterin (BH4), or the antioxidant compound ascorbic acid. Production of superoxide anion (a.u.) was assessed in endothelial cells incubated with dihydroethidine, a fluorescent cell permeable dye for superoxide anion, in the presence or absence of BH4 or ascorbic acid. Mesenteric arterioles from B1 -/- exhibited a severe impairment of ACh-vasodilation for all tested doses, with no changes in the response to SNP and NE. Circulating NO was markedly decreased in B1 -/- (49.6 ± 10.5*; n=6) vs WT (141.9 ± 17.3; n=6 ), accompanied by reduced basal NO release in mesenteric arterioles from B1 -/- (0.16 ± 0.03*; n=6) when compared to WT (0.58 ± 0.08; n=4). NOS activity was elevated in mesenteric homogenates from B1 -/- (3.4 ± 0.58*; n=4) in comparison to WT (1.9 ± 0.05; n=5). ACh-induced NO release was markedly reduced in primary cultured endothelial cells from B1 -/- (35.8 ± 3.1*; n=4) in comparison to WT cells (66.9 ± 3.2; n=4). NO release in endothelial cells from B1 -/- was reversed by incubation with BH4 (54.3 ± 1.7; n=4) and ascorbic acid (101.8 ± 6.0; n=4), but not by L-arginine, while incubation of endothelial cells from WT with BH4, ascorbic acid or L-arginine had no effect. Elevated production of superoxide anion in endothelial cells from B1 -/- (77,1 ± 2,5*; n=4) in comparison to WT (29,3 ± 6,9; n=4) was reversed by incubation with ascorbic acid (35,3 ± 6,4; n=3). The severe impairment in the endothelial-mediated vasodilation accompanied by decreased NO bioavailability, despite the augmented NOS activity, strongly indicates an exacerbation of NO inactivation. Reduced NO availability may be preceded by exacerbation of NO inactivation by superoxide anion, which can leads to inactivation of BH4 in vascular endothelium, resulting in NOS uncoupling and NOS derived production of superoxide anion.
- ItemSomente MetadadadosParticipação Do Receptor B1 De Cininas No Metabolismo Hepático De Gllcose(Universidade Federal de São Paulo (UNIFESP), 2017-05-30) Gregnani, Marcos Antonio Fernandes Da Silva [UNIFESP]; Araujo, Ronaldo De Carvalho [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Objective: To analyze Kinin 81 receptor participation in hepatic rnetabolisrn using maximal exercise test as strategy. Methods: 81 KO and WT mice was submited to maximal test protocol in treadmill in conditions "feeding" and "fasting". Measures of glycemia was done in basal state and during exercise protocol, as well after a challenge with piruvate to induce gluconeogenesis. Also, we done a measure of urinary glucose excretion after a big injection with these compound. In ex vivo analysis, we evaluated hepatic glycogen content, enzymatic activity of citrate synthase, tecidual oxygen consumption, and gene expression of proteins involved with glucose metabolism. Results: The 81 KO mice presented reduced performance in exercise protocol, coupled a reduced glycemic response in fed state, this phenotype was absent in fasting state.Furthermore,81 KO has a reduced glucose urinary excretion, and a reduced content of hepatic glycogen. The enzymes that are responsible for synthesis and degradation of glycogen has upregulated expression in 81 KO mice. Which respect to regulation of glucose flux inside hepatocytes, the enzyme glucose-6-phospatase, involved in gluconeogenesis presented a reduction in fed state. In addition, PPARalpha and two targets genes of these protein was also reduced, suggesting a modulation directed to glycolysis. This data was corroborated by a increassd rate of oxigen consumption in 81 KO mice in hepatic tissue, whereas there was not differences in muscle. It was observed also a increased expression of 82 kinin receptor in 81 knockout mice. Conclusion: The 81 kinin receptor seems able to modulate maximal exercise tolerance and this outcomes probably are related to his hepatic activity. In this tissue 81 kinin receptor can be able to maintain a equilibrated distribution in between glycolisis and glycogenesis, having a important role in glycemic homeostasis in fed state. These effects seems to be mediated by transcription factor PPAR-alpha and your target genes. Its translates in a global frame of adaptations that resu s in increased 02 consumption in hepatic tissue, negatively influencing glycogen 5 esis, en resulting in poor performance in acute maximal exercise.