RESUMO
Human H-kininogen (120 kDa) plays a role in many pathophysiological processes and interacts with the cell surface through protein receptors and proteoglycans, which mediate H-kininogen endocytosis. In the present work we demonstrate that H-kininogen containing bradykinin domain is internalized and different endogenous kininogenases are present in CHO-K1 cells. We used CHO-K1 (wild type) and CHO-745 (mutant deficient in proteoglycans biosynthesis) cell lines. H-kininogen endocytosis was studied using confocal microscopy, and its hydrolysis by cell lysate fraction was determined by immunoblotting. Bradykinin release was also measured by radioimmunoassay. H-kininogen interaction with the cell surface of CHO-745 cells resulted in bradykinin release by serine proteases. In CHO-K1 cells, which produce heparan and chondroitin sulfate proteoglycans, internalization of H-kininogen through its bradykinin domain can occur on lipid raft domains/caveolae. Nevertheless bradykinin-free H-kininogen was not internalized by CHO-K1 cells. The H-kininogen present in acidic endosomal vesicles in CHO-K1 was approximately 10-fold higher than the levels in CHO-745. CHO-K1 lysate fractions were assayed at pH 5.5 and intact H-kininogen was totally hydrolyzed into a 62 kDa fragment. By contrast, at an assay pH 7.4, the remained fragments were 115 kDa, 83 kDa, 62 kDa and 48 kDa in size. The antipain-Sepharose chromatography separated endogenous kininogenases from CHO-K1 lysate fraction. No difference was detected in the assays at pH 5.5 or 7.4, but the proteins in the fraction bound to the resin released bradykinin from H-kininogen. However, the proteins in the unbound fraction cleaved intact H-kininogen at other sites but did not release bradykinin. H-kininogen can interact with extravascular cells, and is internalized dependent on its bradykinin domain and cell surface proteoglycans. After internalization, H-kininogen is proteolytically processed by intracellular kininogenases. The present data also demonstrates that serine or cysteine proteases in lipid raft domains/caveolae on the CHO cell can hydrolyze H-kininogen, thus releasing kinins.
Assuntos
Bradicinina/metabolismo , Endocitose/fisiologia , Cininogênio de Alto Peso Molecular/metabolismo , Animais , Células CHO , Cavéolas/metabolismo , Linhagem Celular , Cricetulus , Endossomos/metabolismo , Hidrólise , Calicreínas/metabolismo , Proteoglicanas/metabolismo , Serina Proteases/metabolismoRESUMO
In this study, we analyzed the influence of proteoglycans on the interaction between human high molecular weight kininogen (HK) and the cell surface. We found that D5- related peptide inhibits HK-biotin cellular uptake. Confocal microscopy showed that HK colocalizes with heparan sulfate proteoglycan (HSPG) at the cell surface. When biotin-HK is incubated with rabbit aorta endothelial cells (RAECs) and CHO-K1 cells, it is internalized into acidic intracellular vesicles, whereas when incubated with CHO-745 cells, which express reduced levels of glycosaminoglycans, HK is not internalized. To further verify the hypothesis that HSPG-dependent mechanisms are involved in HK uptake and proteolytic processing in lysosomes, we tested chloroquine, which blocks Alexa 488- HK colocalization with Lyso Tracker in acidic endosomal vesicles. The process of HK internalization was blocked by low temperatures, methyl-beta-cyclodextrin, FCCP and 2-deoxy-D-glucose, implying that HK uptake into acidic vesicles is energy-dependent and most likely involves binding to HSPG structures localized in cholesterol-rich domains present in the plasma membrane. Kinin generation at the cell surface was much higher in tumorigenic cells (CHO-K1) when compared to endothelial cells (RAECs). The present data indicate that the process of HK endocytosis involving HSPG is a novel additional mechanism which may control kinin generation at the cell surface.
Assuntos
Células Endoteliais/metabolismo , Proteoglicanas de Heparan Sulfato/farmacologia , Cininogênio de Alto Peso Molecular/metabolismo , Animais , Aorta/citologia , Aorta/metabolismo , Células CHO , Linhagem Celular , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Endocitose , Células Endoteliais/efeitos dos fármacos , Feminino , Humanos , Concentração de Íons de Hidrogênio , Imuno-Histoquímica , Proteoglicanas , CoelhosRESUMO
Human neutrophils play a pivotal role in acute inflammation. However, their capacity to generate bioactive kinin peptides has not been established as yet. We have examined the ability of neutrophil enzymes to release biologically active kinins in vitro from purified human H- and L-kininogens. Neutrophils isolated from human blood were stimulated with f-Met-Leu-Phe, thrombin, or human immunoglobulin G adsorbed to silica particles. Supernatants were incubated with iodinated kininogens, and polyacrylamide gel electrophoresis analyzed aliquots taken after a range of incubation times. A time-course analysis demonstrated that supernatants from stimulated neutrophils caused a rapid hydrolysis of both substrates, resulting in an accumulation of fragments ranging from 20 to less than 10 kDa. Radioimmunoassay (RIA) revealed that all supernatants were able to generate kinins in vitro. High-performance liquid chromatography of the generated peptides indicated that they had a retention time similar to that of bradykinin and Met-Lys-bradykinin, clearly recognized as kinin peptides when the corresponding fractions were tested by RIA. The kinin-immunoreactive fractions produced lowering of blood pressure and a dramatic increase in venular permeability. Biological activity of the neutrophil-generated kinins was completely abolished by the B2 receptor antagonist HOE140, indicating that over the time-course of the experiments, only kinin B2 agonists appeared to have been generated and that cellular actions of these were mediated by kinin B2 receptors. Together, our results demonstrate that human neutrophil proteases can release kinins from both plasma kininogens, suggesting that these peptides may participate actively during acute inflammation.
Assuntos
Bradicinina/análogos & derivados , Cininogênio de Alto Peso Molecular/metabolismo , Cininogênio de Baixo Peso Molecular/metabolismo , Cininas/metabolismo , Neutrófilos/metabolismo , Bradicinina/metabolismo , Antagonistas de Receptor B2 da Bradicinina , Humanos , Imunoglobulina G/farmacologia , Inflamação/metabolismo , Inflamação/fisiopatologia , Mediadores da Inflamação/metabolismo , Microscopia Eletrônica , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/ultraestrutura , Receptor B2 da Bradicinina/metabolismo , Vesículas Secretórias/efeitos dos fármacos , Vesículas Secretórias/metabolismo , Vesículas Secretórias/ultraestrutura , Trombina/farmacologiaRESUMO
Human plasma kallikrein (huPK) is a serine proteinase involved in many biological processes including those of the kallikrein-kinin system. The action of huPK on kininogen results in bradykinin (BK) release, a potent mediator of inflammatory responses. BK generation may be influenced by several agents, and the aim of this work was to investigate the effect of glycosaminoglycans (GAGs) on human high-molecular-weight kininogen (HK) hydrolysis by huPK and on inflammation. huPK was pre-incubated in the absence and presence of different GAGs, followed by the addition of kininogen. Bradykinin released at different times was measured by radioimmunoassay, and KM and kcat were calculated. Tuna and bovine dermatan sulfates, the most potent GAGs studied, reduced by 80% and 68%, respectively, the catalytic efficiency of huPK (control = 4. x 10(4) M(-1) s(-1) in BK release. The effect of bovine dermatan sulfate (BDS) on inflammatory response was studied in rat paw edema induced by carrageenin and hourly determined (1-4 h) by plethysmography. BDS significantly reduced the inflammatory response in the first and second hours of measurements (24% and 28%, respectively), p < 0.05. GAGs were shown to reduce bradykinin release "in vitro" and in an inflammation model. This reduction may play a role in the control or maintenance of some pathological and physiological processes.
Assuntos
Dermatan Sulfato/uso terapêutico , Edema/tratamento farmacológico , Inflamação/tratamento farmacológico , Calicreína Plasmática/química , Animais , Bradicinina/metabolismo , Carragenina , Dermatan Sulfato/farmacologia , Edema/induzido quimicamente , Edema/metabolismo , Humanos , Hidrólise , Inflamação/induzido quimicamente , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Cinética , Cininogênio de Alto Peso Molecular/química , Cininogênio de Alto Peso Molecular/metabolismo , Masculino , Calicreína Plasmática/metabolismo , Ratos , Ratos WistarRESUMO
Investigations determined if extracellular matrix of endothelial cells (EC) is a platform for HK assembly and PK activation. In buffers containing bovine serum albumin, biotin-HK binding to ECV304 cells or their matrix requires > or = 50 microM added Zn2+. Ortho-phenanthroline or a HK domain 5 peptide blocks HK binding. Binding to umbilical vein EC or matrix, but not ECV304 cells or matrix, is mediated by cytokeratin 1. Biotin-HK binds to ECV304 cells or matrix with a Kd of 15.8 or 9.0 nM and a Bmax of 2.6 x 10(7) or 2.4 x 10(7) sites/cell, respectively. PK activation on ECV304 cells or matrix is blocked by antipain or SBTI and corn trypsin inhibitor partially inhibits kallikrein formation. PK activation occurs on ECV304 cells or matrix prepared without serum or in human factor XII deficient serum, indicating that the PK activator is not factor XIIa. EC matrix promotes plasminogen activation after the assembly of HK, PK and pro-urokinase. These studies indicate that matrix of various EC has the ability to assemble HK allowing for PK activation and subsequent activities.
Assuntos
Matriz Extracelular/fisiologia , Cininogênio de Alto Peso Molecular/química , Pré-Calicreína/química , Sequência de Aminoácidos , Sítios de Ligação , Sistema Livre de Células , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Ativação Enzimática , Fibrinólise , Humanos , Técnicas In Vitro , Queratinas/imunologia , Queratinas/metabolismo , Cininogênio de Alto Peso Molecular/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologia , Pré-Calicreína/metabolismo , Ligação ProteicaRESUMO
We have isolated and purified two cysteine proteinases of molecular weights 25 and 26 kDa, secreted by Fasciola hepatica adult worm. Their 15 N-terminal residues were found to be identical to those of earlier described cathepsin L-like enzymes, isolated from the same source, reported as CL1 and CL2. Radioimmunoassay experiments have shown that these CL1- (25 kDa) and CL2-like (26 kDa) cysteine proteinases mediated kinin release from high molecular weight kininogen (HMWK). Lys-bradykinin (KRPPGFSPFR) was characterized as the kinin released from a synthetic fragment of HMWK from Leu373 to Ile393 (Abz-LGMISLMKRPPGFSPFRSSRI-NH2) labeled with the fluorescent group Abz (ortho-aminobenzoic acid). We examined the activity of CL1- and CL2-like on internally quenched fluorescent peptides containing HMWK sequences, in which Met379-Lys380 or Arg389-Ser390 bonds were present in the middle of the molecules. These peptides were flanked by the fluorescent donor-acceptor pair Abz and EDDnp (N-[2,4-dinitrophenyl] ethylenediamine). Peptidyl-methylcoumarin amides (MCA) were used to study the substrate specificity requirements. The enzymes presented significantly lower Km values at pH 8.0. The inverse was observed with the kcat values, which were higher at pH 5.0.
Assuntos
Cisteína Endopeptidases/metabolismo , Fasciola hepatica/enzimologia , Calicreínas/metabolismo , Amidas , Sequência de Aminoácidos , Animais , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/isolamento & purificação , Hidrólise , Cininogênio de Alto Peso Molecular/metabolismo , Cininas/metabolismo , Dados de Sequência Molecular , Coloração e Rotulagem , Especificidade por Substrato , ortoaminobenzoatosRESUMO
Cephalic stimulation by food elicits, among other responses, dilatation of mesenteric blood vessels preparatory for digestion. The possible participation of bradykinin (BK), a powerful endogenous vasodilator, in this response was studied in fasted rats prior and following stimulation by sight and scent of food (sensory stimulation, SS), actual ingestion being denied to the animals. BK content of plasma high (HK) and low molecular weight kininogen (LK) was determined by bioassay on the atropinized, antihistamine-treated isolated guinea-pig ileum following release by trypsin from heat/acid denatured plasma. BK corresponding to LK was estimated in plasma which prior to denaturation had been incubated with kaolin, a process which leads to quantitative release and inactivation of BK from HK, but does not affect LK. BK corresponding to (HK + LK) was determined in plasma not exposed to kaolin. BK contained in HK was the difference between BK of (HK + LK) and of BK of LK. Plasma and glandular kallikreins were estimated by fluorimetry, using specific synthetic substrates. A 40.6+/-4.0% decrease (P<0.001) of BK in HK occurred in rats after 90 s of SS; LK remained unaffected. Ten minutes of SS did not result in further change. Atropine inhibited the effect of SS. Return of HK to pretreatment levels occurred when, following 90s of SS, rats were allowed to rest for 60 min in the absence of food. Renewed capacity to respond to SS was then observed. Plasma kallikrein, but not glandular kallikrein, increased in plasma of rats after SS. Increased free BK was detected in the circulation of Enalapril-protected rats after SS. Electrical stimulation of the distal sector of the sectioned left abdominal vagus nerve of Nembutal-anesthetized fasted rats reproduced the effect of SS on HK. It is concluded that visuo-olfactory stimulation by food generates nerve impulses, possibly carried by the vagus nerve, which by activating plasma kallikrein lead to cleavage of circulatory HK and release of BK in the rat.