RESUMO
T-kininogen (T-KG) is a precursor of T-kinin, the most abundant kinin in rat serum, and also acts as a strong and specific cysteine proteinase inhibitor. Its expression is strongly induced during aging in rats, and expression of T-KG in Balb/c 3T3 fibroblasts results in inhibition of cell proliferation. However, T-KG is a serum protein produced primarily in the liver, and thus, most cells are only exposed to the protein from the outside. To test the effect of T-KG on fibroblasts exposed to exogenous T-KG, we purified the protein from the serum of K-kininogen-deficient Katholiek rats. In contrast to the results obtained by transfection, exposure of Balb/c 3T3 fibroblasts to exogenously added T-KG leads to a dose-dependent increase in [3H]-thymidine incorporation. This response does not require kinin receptors, but it is clearly mediated by activation of the ERK pathway. As a control, we repeated the transfection experiments, using a different promoter. The results are consistent with our published data showing that, under these circumstances, T-KG inhibits cell proliferation. We conclude that T-KG exerts opposite effects on fibroblast proliferation, depending exclusively on the way that it is administered to the cells (transfection versus exogenous addition).
Assuntos
Proliferação de Células/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Cininogênios/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Envelhecimento/metabolismo , Animais , Células 3T3 BALB , Inibidores de Cisteína Proteinase/genética , Inibidores de Cisteína Proteinase/metabolismo , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Cininogênios/genética , Cininogênios/metabolismo , Camundongos , Ratos , TransfecçãoRESUMO
High molecular mass kininogen (HK) purified from Bothrops jararaca (Bj) plasma was tested on activities of the Bj venom in vivo and in vitro. Results showed that, when incubated with BjHK, the Bj venom presented inhibition on hemorrhagic, edema forming, myotoxic, and coagulant activities. It is well known that metalloproteinases are directly or indirectly involved in these activities. Similarly, human HK inhibits the hemorrhagic effect of the Bj venom as well as hemorrhagic and enzymatic effects of jararhagin, a hemorrhagic metalloproteinase isolated from Bj venom. Complex between HK and jararhagin was not detected by gel filtration. Nevertheless, the inhibitory effect of the hemorrhagic activity of the venom was only partial when HK was pre-incubated with 0.4mM ZnCl(2) or with 0.45mM CaCl(2). These data suggest that the inhibitory effect depends, at least partially, on the competition for ions between kininogen and metalloproteinases of the venom.
Assuntos
Bothrops/metabolismo , Cininogênios/farmacologia , Metaloproteases/antagonistas & inibidores , Venenos de Serpentes/enzimologia , Animais , Coagulação Sanguínea/efeitos dos fármacos , Cálcio/química , Cálcio/farmacologia , Creatina Quinase/metabolismo , Venenos de Crotalídeos/antagonistas & inibidores , Venenos de Crotalídeos/metabolismo , Venenos de Crotalídeos/farmacologia , Interações Medicamentosas , Edema/tratamento farmacológico , Hemorragia/induzido quimicamente , Hemorragia/tratamento farmacológico , Humanos , Cininogênios/química , Cininogênios/metabolismo , Masculino , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/metabolismo , Metaloendopeptidases/farmacologia , Camundongos , Peso Molecular , Inibidores da Agregação Plaquetária/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Coelhos , Venenos de Serpentes/química , Zinco/química , Zinco/farmacologia , Veneno de Bothrops jararacaRESUMO
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
This paper narrates Dr Héctor R Croxatto and collaborators' efforts over the past 50 years in search for peptidic hormones obtained by pepsin hydrolysis of blood plasma substrates. In the forties, Croxatto described three peptidic fractions characterized by their hypertensive, oxytocic and antidiuretic properties, designated as pepsitensin, pepsitocin and pepsanurin, respectively. While pepsitensin and pepsitocin were later identified as angiotensin I and metlys-bradykinin, pepsanurin was not identified and its research was halted for 35 years. During that time, Prof Croxatto and his group worked mostly on the renal kallikrein-kinin system, studying its physiological anti-hypertensive role, making significant contributions in the field of renovascular hypertension. After the discovery of atrial natriuretic peptide, Croxatto resumed his work with pepsanurin. In a series of papers from 1988 to 1998, it was shown that: 1) when injected intraperitoneally or in the intestinal lumen of anesthetized rats, or in the isolated perfused rat kidneys, pepsanurin is a potent inhibitor of the natriuretic effect of ANP; 2) plasma kininogens are identified as the substrates for pepsanurin formation; 3) bradykinin and prokinins exert the anti-ANP effect when injected either intravenously, intraperitoneally or intraduodenally, at small non-vasodilator doses; endogenous kinins also block ANP renal excretory effects; 4) a 20-amino acid peptide released by pepsin from domain 1 of purified LMW kininogen was isolated by Croxatto and collaborators, designed as PU-D1, and shown to exert similar anti-ANP effects as pepsanurin or kinins, but being more potent and longer lasting; 5) the anti-ANP effect of pepsanurin, kinins and PU-D1 is mediated by B2 kinin receptors, since it is blocked by a bradykinin receptor antagonist. Currently, Dr Croxatto is working on the hypothesis that intestinal-borne kinins and/or PU-D1 may reduce renal excretion during the prandial cycle.
Assuntos
Inibidores de Cisteína Proteinase , Cininogênios , Peptídeos , Animais , Fator Natriurético Atrial , Inibidores de Cisteína Proteinase/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular , Mucosa Intestinal/metabolismo , Calicreínas , Rim/metabolismo , Cininogênios/farmacologia , Cininas , Pepsina A , Fragmentos de Peptídeos/farmacologia , Peptídeos/farmacologia , RatosRESUMO
This paper describes long term research efforts which have lead: 1) to the identification of peptides present in pepsanurin, a peptidic fraction obtained by pepsin hydrolysis of plasma globulins that inhibits the renal excretory action of atrial natriuretic peptide (ANP) and 2) to the discovery of an unexpected role of glucose, as a requisite for these inhibitory effects. The active peptides identified in pepsanurin are derived from plasma kininogens, substrates of the kallikrein-kinin system. Pro-kinins of 15, 16 and 18 amino acids, and bradykinin itself, block ANP-induced diuresis and natriuresis when injected i.v., i.p. or into the duodenal lumen of anesthetized rats in picomol doses. Furthermore, a novel 20 amino acids fragment derived from kininogen dominium-1, named PU-D1, is the most potent and longer lasting blocker of ANP renal effects. The anti-ANP effects of those peptides are prevented by B2-kinin receptor antagonists. The inhibition of ANP by kinins and PU-D1 was evident only in rats infused with isotonic glucose; whereas the excretory effect of ANP was not affected in fasted rats not infused, or infused with saline. These findings provide evidence that glucose facilitates liquid retention through a kinin-mediated inhibition of ANP excretory action that may be related to the prandial cycle.
Assuntos
Fator Natriurético Atrial/efeitos dos fármacos , Diurese/efeitos dos fármacos , Glucose/farmacologia , Cininogênios/farmacologia , Natriurese/efeitos dos fármacos , Animais , Ratos , Retenção Urinária/metabolismoAssuntos
Animais , Ratos , Inibidores de Cisteína Proteinase , Cininogênios , Peptídeos , Fator Natriurético Atrial , Inibidores de Cisteína Proteinase/farmacologia , Hidrólise , Intestinos/metabolismo , Calicreínas , Rim/metabolismo , Cininogênios/farmacologia , Cininas , Pepsina A , Peptídeos/farmacologiaRESUMO
rSMT3, a tonin like angiotensin II generating enzyme present in rSMG presents a potent oxytocic effect on the isolated rat uterus, which was blocked by a B2 bradykinin receptor antagonist. Under optimal conditions of pH, rSMT3 liberates kinin at rate 19-fold greater than angiotensin II.
Assuntos
Angiotensina II/isolamento & purificação , Calicreínas/metabolismo , Peptidil Dipeptidase A/metabolismo , Glândula Submandibular/enzimologia , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Animais , Bioensaio , Pressão Sanguínea/efeitos dos fármacos , Bradicinina/farmacologia , Cromatografia Líquida de Alta Pressão , Feminino , Técnicas In Vitro , Cinética , Cininogênios/farmacologia , Masculino , Ratos , Ratos Wistar , Saralasina/farmacologia , Contração Uterina/efeitos dos fármacos , Útero/efeitos dos fármacos , Útero/fisiologiaRESUMO
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.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Inibidores de Cisteína Proteinase/sangue , Cininogênios/farmacologia , Serpentes/sangue , Animais , Feminino , Humanos , Cininogênios/sangue , Masculino , Mamíferos , Contração Muscular/efeitos dos fármacos , Papaína/antagonistas & inibidoresRESUMO
A kininogen-like protein was purified from Bothrops jararaca plasma by DEAE-Sephadex ion-exchange and carboxy-methul-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 hosrse urinary kallikrein. After incubation with rrypsin, 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 ogf plasma from Waglerophis merremii, a nonpoisonous snake; the same type of inhibitor was foind in this nonpoisonous snake. The dissociation cosntant (Ki) for the papain-inhibitor complex is approximately 1.6 nM