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Publico-12890b.pdf: 1540336 bytes, checksum: 7786d4d28a3b5762f4bd7f7137c3c3c0 (MD5) / A ativacao de receptores B1 de cininas no endotelio vascular desencadeia vias de sinalizacao que resultam na elevacao do Ca+2 intracelular e ativacao da enzima oxido nitrico sintase (NOS), seguido por producao de NO e vasodilatacao. Embora a inducao do receptor B1 e sua funcao durante a inflamacao tenha sido abordada por diversos estudos, a importancia de receptores B1 na homeostase vascular em condicoes fisiologicas nao esta totalmente elucidada. Para esclarecer essa questao, o presente estudo analisou a funcao endotelial e a producao de NO em camundongos nocaute do receptor B1 (B1 -/-) e selvagens (WT). O leito arteriolar mesenterico foi perfundido por solucao Krebs e respostas vasculares para Acetil-colina (ACh), nitroprussiato de sodio (SNP) e norepinefrina (NE) foram analisadas por um sistema de aquisicao de dados. Niveis plasmaticos de NO (ƒÊmol/L) foram analisados por deteccao dos derivados nitritos/nitratos atraves de metodo de quimioluminescencia e a producao vascular de NO foi avaliada em cortes histologicos de arteriolas mesentericas incubadas com DAF-2 DA, um marcador fluorescente de NO (unidades arbitrarias, u.a.). A atividade da NOS (pmol/mg.min) foi mensurada atraves da conversao bioquimica de L-[3H]arginina para L-[3H]citrulina em homogenatos de vasos mesentericos na presenca de substrato e co-fatores. Celulas endoteliais primarias foram incubadas com DAF-2 DA e as imagens obtidas em microscopio confocal foram analisadas por densitometria optica (u.a.). Celulas foram estimuladas com ACh [1 mmol/L] na presenca ou ausencia de Larginina, o substrato da NOS, ou tetrahidrobiopterina (BH4), co-fator da NOS, ou acido ascorbico, composto antioxidante. Producao de anion superoxido (u.a.) foi avaliada em celulas endoteliais incubadas com di-hidroetidina, um marcador fluorescente de anion superoxido, na presenca ou ausencia de BH4 ou acido ascorbico. Arteriolas mesentericas de B1 -/- exibiram severo comprometimento da vasodilatacao mediada por ACh, sem alteracoes na resposta ao NPS e NE. Os niveis circulantes de NO foram consideravelmente reduzidos em B1 -/- (49,6 } 10,5*; n=6) vs WT (141,9 } 17,3; n=6 ), acompanhado por reducao da producao basal de NO em arteriolas mesentericas de B1 -/- (0,16 } 0,03*; n=6) quando comparado a WT (0,58 } 0,08; n=4). A atividade da NOS foi elevada em amostras de B1 -/- (3,4 } 0,58*; n=4) em comparacao a WT (1,9 } 0,05; n=5). A producao de NO mediada por ACh foi significantemente reduzida em celulas endoteliais de B1 -/- (35,8 } 3,1*; n=4) quando comparado a celulas de WT (66,9 } 3,2; n=4). A producao de NO em celulas endoteliais de B1 -/- foi revertida por incubacao com BH4 (54,3 } 1,7; n=4) e acido ascorbico (101,8 } 6,0; n=4), mas nao por L-arginina, enquanto incubacao de celulas endoteliais de WT com BH4, acido ascorbico ou L-arginina nao teve efeito. A producao elevada de anion superoxido em celulas endoteliais de B1 -/- (77,1 } 2,5*; n=4) quando comparado a WT (29,3 } 6,9; n=4) foi revertida pela incubacao com acido ascorbico (35,3 } 6,4; n=3). O severo comprometimento da vasodilatacao mediada pelo endotelio acompanhado por reducao da biodisponibilidade de NO, apesar do aumento da atividade da NOS, sugere a exacerbacao da inativacao de NO em endotelio de B1 -/-. A producao elevada de anion superoxido em endotelio de B1 -/- provavelmente e responsavel pela exacerbacao da inativacao de NO nestes animais. Adicionalmente, a inativacao de BH4 por peroxinitrito pode acarretar em desacoplamento da NOS e producao de anion superoxido pela enzima. / 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. / TEDE
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.unifesp.br:11600/9747 |
| Date | 28 September 2011 |
| Creators | Loiola, Rodrigo Azevedo [UNIFESP] |
| Contributors | Universidade Federal de São Paulo (UNIFESP), Pesquero, João Bosco [UNIFESP] |
| Publisher | Universidade Federal de São Paulo (UNIFESP) |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | 78 p. |
| Source | reponame:Repositório Institucional da UNIFESP, instname:Universidade Federal de São Paulo, instacron:UNIFESP |
| Rights | info:eu-repo/semantics/openAccess |
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