RESUMO
Serum levels of T-kininogen increase dramatically as rats approach the end of their lifespan. Stable expression of the protein in Balb/c 3T3 fibroblasts leads to a dramatic inhibition of cell proliferation, as well as inhibition of the ERK signaling pathway. T-kininogen is a potent inhibitor of cysteine proteinases, and we have described that the inhibition of ERK activity occurs, at least in part, via stabilization of the MAP kinase phosphatase, MKP-1. Since fibroblasts are not a physiological target of T-kininogen, we have now purified the protein from rat serum, and used it to assess the effect of T-kininogen on endothelial cells. Adding purified T-kininogen to EAhy 926 hybridoma cells resulted in inhibition of basal ERK activity levels, as estimated using appropriate anti-phospho ERK antibodies. Furthermore, exogenously added T-kininogen inhibited the activation of the ERK pathway induced by either bradykinin or T-kinin. We conclude that the age-related increase in hepatic T-kininogen gene expression and serum levels of the protein could have dramatic consequences on endothelial cell physiology, both under steady state conditions, and after activation by cell-specific stimuli. Our results are consistent with T-kininogen being an important modulator of the senescent phenotype in vivo.
Assuntos
Bradicinina/análogos & derivados , Inibidores de Cisteína Proteinase/farmacologia , Endotélio Vascular/enzimologia , Cininogênios/farmacologia , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Fatores Etários , Animais , Western Blotting , Bradicinina/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Inibidores de Cisteína Proteinase/sangue , Endotélio Vascular/citologia , Ativação Enzimática/efeitos dos fármacos , Cininogênios/sangue , Cininas/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Ratos , Ratos Endogâmicos BN , Transdução de SinaisRESUMO
Pepsanurin is a peptidic fraction resulting from pepsin digestion of plasma globulins, that inhibits ANP renal excretory actions. We studied whether kinin-like peptides mediate the anti-ANP effect by testing if pepsanurin: 1) was blocked by the kinin B2 receptor antagonist HOE-140, 2) was produced from kininogen, and 3) was mimicked by bradykinin. Anti-ANP activity was assessed in anesthetized female rats by comparing the excretory response to two ANP boluses (0.5 microgram i.v.) given before and after i.p. injection of test samples. Pepsanurin from human or rat plasma (1-5 mL/kg), and bradykinin (5-20 micrograms/kg), dose-relatedly inhibited ANP-induced water, sodium, potassium and cyclic GMP urinary excretion, without affecting arterial blood pressure. The same effect was exerted by pepsin hydrolysates of purified kininogen, whereas hydrolysates of kininogen-free plasma had no effect. HOE-140 (5 micrograms, i.v.) did not alter baseline, or ANP-induced excretion, but blocked the anti-ANP effects of pepsanurin. Histamine (15 micrograms/kg) plus seroalbumin hydrolysates did not affect ANP response, despite inducing larger peritoneal fluid accumulation as compared with pepsanurin or bradykinin. We concluded that kinins cleaved from kininogen mediate the anti-ANP effects of pepsanurin by activation of kinin B2 receptors, independently of changes in systemic arterial pressure or peritoneal fluid sequestration.
Assuntos
Fator Natriurético Atrial/antagonistas & inibidores , Diuréticos/farmacologia , Cininas/fisiologia , Peptídeos/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Bradicinina/análogos & derivados , GMP Cíclico/urina , Inibidores de Cisteína Proteinase/sangue , Diurese , Feminino , Peptídeos e Proteínas de Sinalização Intercelular , Cininogênios/sangue , Ratos , Ratos Sprague-DawleyRESUMO
Pepsanurin is a peptidic fraction resulting from pepsin digestion of plasma globulins, that inhibits ANP renal excretory actions. We studied whether kinin-like peptides mediate the anti-ANP effect by testing if pepsanurin: 1) was blocked by the kinin B12 receptor antagonist HOE-140, 2) was produced from kininogen, and 3) was mimicked by bradykinin. Anti-ANP activity was assessed in anesthetized female rats by comparing the excretory response to two ANP boluses (0.5 mug iv) given before and after ip injection of test samples. Pepsanurin from human or rat plasma (1-5 mL/Kg), and bradykinin (5-20 mug/Kg), dose-relatedly inhibited ANP-induced water, sodium, potassium and cyclic GMP urinary excretion, without affecting arterial blood pressure. The same effect was exerted by pepsin hydrolysates of purified kininogen, whereas hydrolysates of kininogen-free plasma had no effect. HOE-140 (5 mug, iv) did not alter baseline, or ANP-induced excretion, but blocked the anti-ANP effects of pepsanurin. Histamine (15 mug/Kg) plus seroalbumin hydrolysates did not affect ANP response, despite inducing larger peritoneal fluid accumulation as compared with pepsanurin or bradykinin. We concluded that kinins cleaved from kininogen mediate the anti-ANP effects of pepsanurin by activation of kinin B2 receptors, independently of changes in systemic arterial pressure or peritoneal fluid sequestration.
Assuntos
Animais , Feminino , Ratos , Fator Natriurético Atrial/antagonistas & inibidores , Diuréticos/farmacologia , Cininas/farmacologia , Peptídeos/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Bradicinina/análogos & derivados , GMP Cíclico/urina , Inibidores de Cisteína Proteinase/sangue , Diurese , Cininogênios/sangue , Ratos Sprague-DawleyRESUMO
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 EspécieRESUMO
A kininogen-like protein was purified from Bothrops jararaca plasma by DEAE-Sephadex ion-exchange and carboxy-methyl-papain-Sepharose affinity chromatography. The molecular weight, estimated by SDS-gel electrophoresis, is about 100,000 and a species of about 75,000 is formed after incubation with horse urinary kallikrein. After incubation with trypsin, only traces of biological activity were detected in tests on guinea pig ileum. The purified protein inhibits papain and bromelain, does not correct the clotting time of a kininogen-depleted human plasma, and does not affect the clotting time of plasma from Waglerophis merremii, a nonpoisonous snake; the same type of inhibitor was found in this nonpoisonous snake. The dissociation constant (Ki) for the papain-inhibitor complex is approximately 1.6 nM.