RESUMO
The main objective of this study was to determine if the components of the kallikrein-kinin system are released into the venous effluent from isolated perfused rat hearts. To assess the contribution of kinins and the vascular and cardioprotective effects of the ACE inhibitor ramipril, we determined the status of cardiac kallikrein (CKK), potent kinin-generating enzyme, in rats with right ventricular hypertrophy induced by chronic volume overload and left ventricular hypertrophy by aortic banding. CKK was measured as previously described (Nolly, H.L., Carbini, L., Carretero, O.A., Scicli, A.G., 1994). Kininogen by a modification of the technique of Dinitz and Carvalho (1963) and kinins were extracted with a Sep-Pak C18 cartridge and measured by RIA. CKK (169 +/- 9 pg Bk/30 min), kininogen (670 +/- 45 pg Bk/30 min) and immunoreactive kinins (62 +/- 10 pg Bk/30 min) were released into the perfusate. The release was almost constant over a 120 min period. Pretreatment with the protein synthesis inhibitor puromycin (10 mg i.p.) lowered the release of kallikrein (42 +/- 12 pg Bk/30 min, p < 0.001) and kininogen (128 +/- 56 pg Bk/30 min, p < 0.001). Addition of ramiprilat (10 micrograms/ml) increased kinin release from 54 +/- 18 to 204 +/- 76 pg Bk/30 min (p < 0.001). Aortic banding of rats increased their blood pressure (BP) (p < 0.001), relative heart weight (RHW) (p < 0.001) and CKK (p < 0.001). Ramipril treatment induced a reduction in BP (p < 0.05) and RHW (p < 0.005) while CKK remained elevated. Aortocaval shunts increased their ANF plasma levels (p < 0.05), RHW (p < 0.001) and CKK (p < 0.01). Ramipril treatment induced a reduction in RHW (p < 0.05), while CKK and ANF increased significantly (p < 0.05). The present data show that the components of the kallikrein-kinin system are continuously formed in the isolated rat heart and that ramipril reduces bradykinin breakdown with subsequent increase in bradykinin outflow. The experiments with aorta caval shunt and aortic banding show that cardiac tissues increase their kinin-generating activity and this was even higher in ramipril-treated animals. This may suggest that the actual level of kinins is finely tuned to the local metabolic demands. In this experimental model of cardiac hypertrophy. ACE inhibitors potentiate the actions of kinins and probably try to normalise endothelial cell function.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Bradicinina/metabolismo , Coração/efeitos dos fármacos , Sistema Calicreína-Cinina/efeitos dos fármacos , Ramipril/farmacologia , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Animais , Derivação Arteriovenosa Cirúrgica , Fator Natriurético Atrial/sangue , Pressão Sanguínea/efeitos dos fármacos , Modelos Animais de Doenças , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/fisiopatologia , Hipertensão/tratamento farmacológico , Hipertensão/fisiopatologia , Hipertrofia Ventricular Esquerda/tratamento farmacológico , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/fisiopatologia , Hipertrofia Ventricular Direita/tratamento farmacológico , Hipertrofia Ventricular Direita/metabolismo , Hipertrofia Ventricular Direita/fisiopatologia , Sistema Calicreína-Cinina/fisiologia , Calicreínas/metabolismo , Cininogênios/isolamento & purificação , Cininogênios/metabolismo , Masculino , Miocárdio/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Puromicina/farmacologia , Radioimunoensaio , Ramipril/uso terapêutico , Ratos , Ratos WistarRESUMO
Kininogens are the major mammalian plasma cysteine proteinase inhibitors; a kininogen-like protein was also found in the snake Bothrops jararaca plasma. This communication describes a kininogen-like protein in plasma of Caiman crocodilus vacare. Caiman crude plasma, unlike snake plasma, contains a detectable cysteine proteinase inhibitor. The inhibitor was purified by DEAE-Sephadex ion-exchange chromatography and chromatography on carboxy-methylated-papain-Sepharose. The estimated molecular weight of Caiman cysteine proteinase inhibitor is 70,000. Caiman plasma also hydrolyzes plasma kallikrein synthetic substrates and inhibits trypsin. Reptilian kininogen may lack the site for interaction with plasma prokallikrein, and the sequence of the released kinin may be distinct from bradykinin. The poor effectiveness of bradykinin on reptile smooth muscle shows that the reptile kinin receptors may be adapted to a specific kinin.
Assuntos
Inibidores de Cisteína Proteinase/sangue , Cininogênios/sangue , Serpentes/sangue , Animais , Cromatografia em Agarose , Cromatografia por Troca Iônica , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/isolamento & purificação , Feminino , Técnicas In Vitro , Cinética , Cininogênios/química , Cininogênios/isolamento & purificação , Masculino , Peso Molecular , Papaína/antagonistas & inibidores , Especificidade da EspécieAssuntos
Inibidores de Cisteína Proteinase/sangue , Animais , Bromelaínas/antagonistas & inibidores , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Inibidores de Cisteína Proteinase/isolamento & purificação , Feminino , Cinética , Cininogênios/sangue , Cininogênios/isolamento & purificação , Cininas/análise , Masculino , Papaína/antagonistas & inibidores , SerpentesRESUMO
The isolation procedure for horse urinary kallikrein was considerably improved by the introduction of two new purification steps: a) removal of mucoproteins and concentration of the urine by ultrafiltration and b) affinity chromatography on benzamidine-Sepharose conjugate. The homogeneity of the enzyme preparations, regarding their protein moiety, was demonstrated by: 1) a single symmetric peak on DEAE-Sephadex chromatography, with constant values for A280/A260 ratios, esterolytic and amidolytic specific activities; 2) a single band, although dispersed, on gel-electrophoresis at pH 8.3, also in the presence of sodium dodecyl sulfate, and 3) a unique sequence for the six amino-terminal residues. The isolated enzyme was shown to be a single chain glycoprotein (alpha-kallikrein), similar to human urinary and porcine-pancreatic kallikreins regarding the protein moiety molecular mass, amino-acid composition, and partial amino-terminal sequence; differences were found in their total sugar content and even more conspicuously in their carbohydrate composition. In contrast to porcine pancreatic beta-kallikrein, horse urinary kallikrein was not substrate-activated and unlike other alpha-kallikreins, did not present the biphasic time-course in benzoyl-L-arginine ethyl ester hydrolysis. The specificity constants (kcat/Km) for ester and 4-nitroanilide substrates were lower for horse urinary than for pancreatic beta-kallikrein and as observed with the latter enzyme, were affected by NaCl.
Assuntos
Calicreínas/urina , Aminoácidos/análise , Animais , Carboidratos/análise , Cromatografia de Afinidade , Cromatografia em Gel , Cromatografia por Troca Iônica , Cavalos , Indicadores e Reagentes , Calicreínas/isolamento & purificação , Cinética , Cininogênios/sangue , Cininogênios/isolamento & purificação , Peso MolecularRESUMO
Plasma supernatant in which kallikrein has been activated and removed by glass powder whilst kininogen I (HMW) has been consumed by the activated kallikrein, was used for the preparation of kininogen II. It was purified by chromatography on DEAE-cellulose followed by gel filtration on G-200 Sephadex. The purification of kininogen II was assessed from determinations of the amount of kinin released (expressed as bradykinin) as measured on the isolated guinea pig ileum, using samples incubated with human salivary kallikrein or trypsin. A preparation of kininogen II containing an activity equivalent to 8 microgram Br/mg protein, was obtained. Salivary kallikrein released approximately three times more kinin from the substrate as compared to trypsin.
Assuntos
Cininogênios/isolamento & purificação , Cromatografia DEAE-Celulose , Cromatografia em Gel , Humanos , Calicreínas/metabolismo , Cininogênios/sangue , Cininas/sangue , Peso Molecular , Saliva/enzimologia , Tripsina/metabolismoRESUMO
Kininogens I (HMW) and II (LMW) were isolated and partially purified from human plasma. The disappearance of kininogen I was prevented by the use of hexadimethrine bromide, which inhibits the activation of plasma prekallikrein. The two kininogens were separated by chromatography on DEAE-cellulose. Further purification of kininogen I and II was achieved by separate chromatographic steps of the partially purified kininogens on SP-Sephadex. The purification of the kininogens was controlled by incubation of the respective samples with different kininogenases: human plasma kallikrein, human salivary kallikrein and trypsin. As determined by gel filtration, a molecular weight of 250 000 daltons was found for kininogen I (HMW) and 48 000 daltons for kininogen II (LMW).