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Caracterização da atividade cininogenásica no líquido pericárdico humanoLíquido pericárdico.Cininogenase.Cininas.Pericardial fluid.Kininogenase.Kinins.Ciências BiológicasO líquido pericárdico está em contato com o coração e reflete seu status fisiológico. Durante os processos patológicos cardíacos, o nível de fatores bioquímicos no fluido pericárdico é significativamente alterado, sendo considerado um ótimo material para pesquisas cardiovasculares. Atividade de enzimas das classes de serino, aspartil e cisteíno peptidases já foram descritas no fluido pericárdico. Como estas classes enzimáticas têm sido incluídas como cininogenases, e a bradicinina exerce um importante papel de proteção do sistema cardiovascular pela sua potente ação vasodilatadora, a caracterização de atividade cininogenásica no fluido pericárdico é de grande interesse no entendimento dos processos de regulação do tônus vascular neste local. O objetivo deste trabalho foi investigar a capacidade de enzimas presentes no líquido pericárdio gerar cininas. Vinte e três (23) amostras de fluido pericárdico humano, obtidas durante procedimentos cirúrgicos de revascularização coronária, cirurgia de aorta torácica e substituição valvar, foram analisadas quanto às atividades de aspartil, serino e cisteíno proteases. Para determinação das atividades enzimáticas foram utilizados os substratos sintéticos, com apagamento intramolecular de fluorescência, Abz-KPIEFFRLQ-Eddnp (suscetível à hidrólise por aspartil peptidases), Abz-AIKFFRLQ-Eddnp (suscetível à hidrólise por aspartil e cisteíno peptidases), Abz-MISLMKRPQ-Eddnp e Abz-FRSSRQ-Eddnp (sequências N e C terminais em torno da bradicinina), na faixa de pH de 2,0 a 9,0. Inibidores (Pepstatina A, PMSF e E-64) foram utilizados para caracterizar as classes enzimáticas. As hidrólises foram avaliadas através da variação de fluorescência. Os pontos de clivagem nos substratos com sequências do cininogênio foram monitorados através de cromatografia líquida de alta eficiência (CLAE) e da análise de aminoácidos dos fragmentos liberados. Todas as amostras de fluido pericárdico utilizadas neste estudo hidrolisaram os substratos testados. O pH ótimo de hidrólise dos peptídeos pelas enzimas do fluido pericárdico para cada um dos substratos foi 4,0 (Abz-KPIEFFRLQ-Eddnp, Abz-AIKFFRLQ-Eddnp, Abz-MISLMKRPQ-Eddnp e Abz-FRSSRQ-Eddnp), inibida pela Pepstatina A; 6,0 (Abz-AIKFFRLQ-Eddnp) inibida por E-64; e 8,0 (Abz-MISLMKRPQ-Eddnp e Abz-FRSSRQ-Eddnp) inibida por PMSF. Os resultados mostram que o fluido pericárdico humano hidrolisou os substratos relacionados ao cininogênio. Para o substrato Abz-MISLMKRPQ-Eddnp os dados obtidos foram 75±31µM.min-1( média ± DP) em pH ácido, e 334±161 µM.min-1( média ± DP) em pH 8,0. Os inibidores enzimáticos foram úteis para mostrar que aspartil e serino proteases são as classes enzimáticas com potencial atividade cininogenásica no fluido pericárdico. A análise cromatográfica dos peptídeos e de aminoácidos dos fragmentos liberados após as hidrólises permitiu observar que cada um dos substratos foi clivado em uma única ligação. Em pH 4,0 os produtos fluorescentes foram similares aos fragmentos obtidos da hidrólise por uma protease ácida renal que libera MLBK do cininogênio humano, com clivagem das ligações Leu-Met e Arg-Ser nos substratos Abz-MISLMKRPQ-Eddnp e Abz-FRSSRQ-Eddnp, respectivamente. Em pH 8,0 os produtos de hidrólise apresentaram tempos de retençao comparáveis aos obtidos para a calicreína plasmática e a hidrólise ocorreu nas ligações Lys-Arg e Arg-Ser para os mesmos substratos, sugerindo a liberação de bradicinina. Nas condições de ensaio utilizadas não foi possível identificar atividade cininogenásica relacionada a cisteíno peptidases. Nossos resultados mostram que o fluido pericárdico humano contém enzimas com potencial atividade cininogenásica, corroborando com outros estudos que sugerem que o conteúdo de peptídeos do fluido pericárdico não deve ser consequência apenas do fluxo do fluido intersticial miocárdico. Ainda não podemos afirmar se este fluido contém substratos para a atividade cininogenásica e se as cininas liberadas localmente tem significância fisiológica.The pericardial fluid is in contact with the heart and reflects its physiological status. During cardiac disease processes, the level of biochemical factors in the pericardial fluid is significantly changed, being considered a great material for cardiovascular research. Enzyme activity class of serine, aspartyl and cysteine peptidases have been described in the pericardial fluid. As these enzyme classes have been included as kininogenases, and the bradykinin plays an important role in the cardiovascular system protection, for its potent vasodilator action, the characterization of kininogenase activity in the pericardial fluid is of great interest in understanding the regulatory processes of vascular tone at this location. The aim of this study was to investigate the ability of enzymes present in the pericardial fluid generate kinins. Twenty-three (23) human pericardial fluid samples obtained during surgical procedures for coronary revascularization, thoracic aortic surgery and valve replacement were analyzed for aspartyl, serine and cysteine proteases activities. To determine the enzymatic activity were used Fluorescence Resonance Energy Transfer substrates, Abz-KPIEFFRL-EDDnp (susceptible to hydrolysis by aspartyl peptidases), Abz-AIKFFRLQ-EDDnp (susceptible to hydrolysis by aspartyl and cysteine peptidases), Abz-MISLMKRPQ-EDDnp and Abz-FRSSRQ-EDDnp (N- and C-terminal sequences around the bradykinin) in the pH range 2.0 to 9.0. Inhibitors (Pepstatin A, and E-64 PMSF) were used to characterize the enzymatic classes. The hydrolysis were assessed by fluorescence variation. The cleavage sites in substrates with kininogen sequences were monitored by high-performance liquid chromatography (HPLC) and amino acid analysis of the released fragments. All the pericardial fluid samples used in this study hydrolyzed substrates tested. The optimum pH for hydrolysis of peptides by enzymes in the pericardial fluid to each of the substrates was 4.0 (Abz-KPIEFFRL-EDDnp, Abz-AIKFFRLQ-EDDnp, Abz-MISLMKRPQ-EDDnp and Abz-FRSSRQ-EDDnp) inhibited by Pepstatin A; 6.0 (Abz-AIKFFRLQ-EDDnp) inhibited by E-64; and 8.0 (Abz-MISLMKRPQ-EDDnp and Abz-FRSSRQ-EDDnp) inhibited by PMSF. The results show that the human pericardial fluid hydrolyzed substrates related kininogen. For Abz-MISLMKRPQ-EDDnp the data obtained were 75 ± 31μM.min-1 (mean ± SD) at acid pH, and 334 ± 161 μM.min-1 (mean ± SD) at pH 8.0. Enzyme inhibitors are useful for showing that aspartyl and serine proteases are enzyme classes with potential kininogenase activity in the pericardial fluid. Chromatographic analysis of peptides and amino acids of fragments released after the hydrolysis allowed observing that each of the substrates was cleaved into a single connection. At pH 4.0 fluorescent products were similar to fragments obtained from hydrolysis by renal acid protease that releases MLBK the human kininogen with cleavage of Leu-Met and Arg-Ser bonds in substrates Abz-MISLMKRPQ-EDDnp and Abz-FRSSRQ-EDDnp, respectively. At pH 8.0 the hydrolysis products had retention times comparable to those obtained for the plasma kallikrein and hydrolysis has occurred in the Lys-Arg and Arg-Ser bonds for the same substrates, suggesting the release of bradykinin. At these assay conditions it was not possible to identify kininogenase activity related to cysteine peptidases. Our results show that the human pericardial fluid contains enzymes with the potential kininogenase activity, which corroborates with studies suggesting that the peptide content of pericardial fluid must not only be a consequence of myocardial interstitial fluid. If pericardial fluid contains substrates to kininogenase activity, and kinins released in this locally has physiological significance, remains to be determined.Fundação de Amparo à Pesquisa do Estado de Minas GeraisFundação de Ensino e Pesquisa de UberabaUniversidade Federal de São PauloUniversidade Federal do Triângulo MineiroInstituto de Ciências da Saúde - ICS::Curso de MedicinaBrasilUFTMPrograma de Pós-Graduação em Ciências FisiológicasGOMES, Roseli Aparecida da Silva44940874672http://lattes.cnpq.br/6327703613785635CIELLO, Giani del2018-04-25T18:06:11Z2015-09-15info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfapplication/pdfCIELLO, Giani del. Caracterização da atividade cininogenásica no líquido pericárdico humano. 2015. 47f. 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