Caracterização da atividade cininogenásica no líquido pericárdico humano

Abstract

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.