Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 9 de 9
Filtrar
1.
Am J Respir Cell Mol Biol ; 43(6): 703-11, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20097831

RESUMO

Reactive airway disease is mediated by smooth muscle contraction initiated through several agonist-dependent pathways. Activation of type 1 N-methyl-D-aspartate receptors (NMDA-R1s) by plasminogen activators (PAs) has been linked to control of vascular tone, but their effect on airway smooth muscle contractility has not previously been studied to our knowledge. We observed that NMDA-R1s are expressed by human airway smooth muscle cells and constitutively inhibit the contraction of isolated rat tracheal rings in response to acetylcholine (Ach). Both tissue-type PA (tPA) and urokinase-type PA (uPA) bind to NMDA-R1 and reverse this effect, thereby enhancing Ach-induced tracheal contractility. Tracheal contractility initiated by Ach is reduced in rings isolated from tPA(-/-) and uPA(-/-) mice compared with their wild-type counterparts. The procontractile effect of uPA or tPA was mimicked and augmented by the nitric oxide synthase inhibitor, l-NAME. uPA and tPA further enhanced the contractility of rings denuded of epithelium, an effect that was inhibited by the NMDA-R antagonist, MK-801. Binding of PAs to NMDA-R1 and the subsequent activation of the receptor were inhibited by PA inhibitor type 1, by a PA inhibitor type 1-derived hexapeptide that recognizes the tPA and uPA docking domains, as well as by specific mutations within the docking site of tPA. These studies identify involvement of PAs and NMDA-R1 in airway contractility, and define new loci that could lead to the development of novel interventions for reactive airway disease.


Assuntos
Contração Muscular/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Traqueia/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Sítios de Ligação , Biocatálise/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Contração Muscular/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Inibidor 1 de Ativador de Plasminogênio/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Ativador de Plasminogênio Tecidual/farmacologia , Traqueia/citologia , Traqueia/efeitos dos fármacos , Ativador de Plasminogênio Tipo Uroquinase/farmacologia
2.
Br J Pharmacol ; 141(6): 971-8, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14993107

RESUMO

1. The renal medulla is a major source of plasminogen activators (PA), recently shown to induce vasodilation in vitro. Treatment with PA inhibitors has been associated with renal dysfunction, suggesting compromised renal microvasculature. We investigated the impact of the PA inhibitor epsilon amino-caproic acid (EACA) upon vascular tone in vitro, and studied the effect of both tPA and EACA upon intrarenal hemodynamics in vivo. 2. In vitro experiments were carried out in isolated aortic rings and with cultured vascular smooth muscle cells. Studies of renal microcirculation and morphology were conducted in anesthetized Sprague-Dawley rats. 3. In isolated aortic rings, EACA (but not the other inhibitors of the fibrinolytic system PAI-1 or alpha-2 antiplasmin) reduced the half-maximal effective concentration of phenylephrine (PE) required to induce contraction (from 32 nm in control solution to 2 and 0.1 nm at EACA concentrations of 1 and 10 microm, respectively). Using reteplase (retavase) in the same model, we also provide evidence that the vasoactivity of tPA is in part kringle-dependent. In cultured vascular smooth muscle cells, Ca(2+) internalization following PE was enhanced by EACA, and retarded by tPA. 4. In anesthetized rats, EACA (150 mg x kg(-1)) did not affect systemic blood pressure, total renal or cortical blood flow. However, the outer medullary blood flow declined 12+/-2% below the baseline (P<0.03). By contrast, tPA (2 mg x kg(-1)), transiently increased outer medullary blood flow by 8+/-5% (P<0.02). Fibrin microthrombi were not found within the renal microvasculature in EACA-treated animals. 5. In conclusion, both fibrinolytic and antifibrinolytic agents modulate medullary renal blood flow with reciprocal effects of vasodilation (PA) and vasoconstriction (EACA). In vitro studies suggest that these hemodynamic responses are related to direct modulation of the vascular tone.


Assuntos
Ácido Aminocaproico/farmacologia , Fibrinolíticos/farmacologia , Medula Renal/irrigação sanguínea , Músculo Liso Vascular/fisiologia , Inativadores de Plasminogênio/farmacologia , Ativador de Plasminogênio Tecidual/farmacologia , Animais , Aorta , Cálcio/metabolismo , Células Cultivadas , Hemodinâmica , Humanos , Técnicas In Vitro , Contração Isométrica/efeitos dos fármacos , Medula Renal/efeitos dos fármacos , Microcirculação/efeitos dos fármacos , Perfusão , Ratos , Ratos Sprague-Dawley , Circulação Renal/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos
3.
Atherosclerosis ; 194(2): 452-7, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16989837

RESUMO

BACKGROUND: Several markers of inflammation predict the risk of thrombotic cardiovascular events in patients with atherosclerosis. However, the mechanism by which vascular inflammation promotes atherothrombotic disease is incompletely understood. Human neutrophil peptides 1-3, also known as alpha-defensins, are found in human atherosclerotic arteries, inhibit LDL metabolism and fibrinolysis and promote Lp(a) binding. We asked, therefore, if alpha-defensins are risk factors for the presence and severity of atherosclerosis. METHODS: alpha-Defensin was measured in skin biopsies taken from 53 male patients (age 58.7+/-11.3 years, mean+/-S.D.) immediately prior to coronary artery catheterization. Other established risk factors were measured concurrently. The correlation between alpha-defensin deposition in the skin and the severity of the coronary artery disease (CAD) was examined. RESULTS: A statistically significant correlation was observed between the amount of alpha-defensin in skin and the severity of CAD (R=0.40, p=0.003). Multiple regression analysis showed that skin alpha-defensin is an independent predictor for CAD severity (F=4.68, p=0.035). Logistic regression analysis confirmed that skin alpha-defensin independently predicted the likelihood for CAD (p=0.016, odds estimate 5.97, 95% CL 1.4-24.2). CONCLUSIONS: The deposition of alpha-defensin in the skin is a strong independent predictor of CAD in men. These results suggest a link between neutrophil activation and progression of atherosclerosis and provide a novel approach to assessment of risk factors for CAD.


Assuntos
Doença da Artéria Coronariana , Pele/química , alfa-Defensinas/análise , Idoso , Aterosclerose/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Fatores de Risco , Pele/imunologia
4.
Am J Physiol Heart Circ Physiol ; 291(3): H1351-9, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16489109

RESUMO

Tissue-type plasminogen activator (tPA) regulates vascular contractility through the low-density lipoprotein-related receptor (LRP), and this effect is inhibited by plasminogen activator inhibitor type 1 (PAI-1). We now report that tPA-mediated vasocontraction also requires the integrin alphavbeta3. tPA-induced contraction of rat aortic rings is inhibited by the Arg-Gly-Asp (RGD) peptide and by monoclonal anti-alphavbeta3 antibody. tPA induces the formation of a complex between LRP and alphavbeta3 in vascular smooth muscle cells. The three proteins are internalized within 10 min, causing the cells to become refractory to the readdition of tPA. LRP and alphavbeta3 return to the cell surface by 90 min, restoring cell responsiveness to tPA. PAI-1 and the PAI-1-derived hexapeptide EEIIMD abolish the vasocontractile activity of tPA and inhibit the tPA-mediated interaction between LRP and alphavbeta3. tPA induces calcium mobilization from intracellular stores in vascular smooth muscle cells, and this effect is inhibited by PAI-1, RGD, and antibodies to both LRP and alphavbeta3. These data indicate that tPA-mediated vasocontraction involves the coordinated interaction of LRP with alphavbeta3. Delineating the mechanism underlying these interactions and the nature of the signals transduced may provide new tools to regulate vascular tone and other consequences of tPA-mediated signaling.


Assuntos
Integrina alfaVbeta3/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Ativador de Plasminogênio Tecidual/fisiologia , Vasoconstrição/efeitos dos fármacos , Animais , Cálcio/metabolismo , Regulação da Expressão Gênica/fisiologia , Humanos , Integrina alfaVbeta3/genética , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Músculo Liso Vascular/metabolismo , Oligopeptídeos/farmacologia , Inibidor 1 de Ativador de Plasminogênio/farmacologia , Ligação Proteica , Ratos , Vasoconstrição/fisiologia
5.
Blood ; 105(3): 1021-8, 2005 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-15353482

RESUMO

Activation of plasminogen by urokinase plasminogen activator (uPA) plays important roles in several physiologic and pathologic conditions. Cells secrete uPA as a single-chain molecule (scuPA). scuPA can be activated by proteolytic cleavage to a 2-chain enzyme (tcuPA). scuPA is also activated when it binds to its receptor (uPAR). The mechanism by which the enzymatic activity of the scuPA/suPAR complex is regulated is only partially understood. We now report that the plasminogen activator activity of the scuPA/suPAR complex is inhibited by Glu- and Lys-plasminogen, but not by mini-plasminogen. In contrast, neither Glunor Lys-plasminogen inhibits the activation of plasminogen by 2-chain uPA. Inhibition of scuPA/suPAR activity was evident at a Glu-plasminogen concentration of approximately 100 nM, and at physiologic plasma concentrations inhibition was nearly complete. A plasminogen fragment containing kringles 1-3 inhibited the enzymatic activity of scuPA/suPAR with an inhibition constant (Ki) equal to 1.9 microM, increased the Michaelis constant (Km) of scuPA/suPAR from 18 nM to 49 nM, and decreased the catalytic constant (Kcat) approximately 3-fold from 0.035 sec(-1) to 0.011 sec(-1). Inhibition of scuPA/suPAR by plasminogen was completely abolished in the presence of fibrin clots. These studies provide insight into the regulation of uPA-mediated plasminogen activation and identify a novel mechanism for its fibrin specificity.


Assuntos
Fibrina/fisiologia , Plasminogênio/fisiologia , Receptores de Superfície Celular/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Primers do DNA , Ativação Enzimática , Camundongos , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Proteínas Recombinantes/metabolismo
6.
J Biol Chem ; 277(43): 40499-504, 2002 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-12171938

RESUMO

Urokinase plasminogen activator (uPA) is a multifunctional protein that has been implicated in several physiological and pathological processes involving cell adhesion and migration in addition to fibrinolysis. In a previous study we found that two-chain urokinase plasminogen activator (tcuPA) stimulates phenylephrine-induced vasoconstriction of isolated rat aortic rings. In the present paper we report that uPA(-/-) mice have a significantly lower mean arterial blood pressure than do wild type mice and that aortic rings from uPA(-/-) mice show an attenuated contractile response to phenylephrine. In contrast, the blood pressure of urokinase receptor knockout (uPAR(-/-)) mice and the response of their isolated aortic rings to phenylephrine were normal, indicating that the effect of uPA on vascular contraction is independent of uPAR. Addition of mouse and human uPA almost completely reversed both the impaired vascular contractility and the lower arterial blood pressure in vivo. The in vitro and in vivo effects of infused uPA on aortic contractility and the restoration of normal blood pressure in uPA(-/-) mice were prevented by antibody to low-density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor (LRP). A modified form of uPA that lacks the kringle failed to restore the blood pressure in uPA(-/-) mice, notwithstanding having a longer half-life in the circulation. Ligands that regulate the interaction of uPA with LRP, such as PAI-1 or the PAI-1-derived peptide (EEIIMD), abolished the vasoactivity of tcuPA in vitro and in vivo. These studies identify a novel signal transducing cellular receptor pathway involved in the regulation of vascular contractility.


Assuntos
Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/fisiologia , Músculo Liso Vascular/citologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Pressão Sanguínea , Técnicas In Vitro , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Ligação Proteica , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/fisiologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Vasoconstrição/fisiologia
7.
Blood ; 100(12): 4026-32, 2002 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-12393692

RESUMO

We have previously identified alpha-defensin in association with medial smooth muscle cells (SMCs) in human coronary arteries. In the present paper we report that alpha-defensin, at concentrations below those found in pathological conditions, inhibits phenylephrine (PE)-induced contraction of rat aortic rings. Addition of 1 microM alpha-defensin increased the half-maximal effective concentration (EC(50)) of PE on denuded aortic rings from 32 to 630 nM. The effect of alpha-defensin was dose dependent and saturable, with a half-maximal effect at 1 microM. alpha-Defensin binds to human umbilical vein SMCs in a specific manner. The presence of 1 microM alpha-defensin inhibited the PE-mediated Ca(++) mobilization in SMCs by more than 80%. The inhibitory effect of alpha-defensin on contraction of aortic rings and Ca(++) mobilization was completely abolished by anti-low-density lipoprotein receptor-related protein/alpha(2-)macroglobulin receptor (LRP) antibodies as well as by the antagonist receptor-associated protein (RAP). alpha-Defensin binds directly to isolated LRP in a specific and dose-dependent manner; the binding was inhibited by RAP as well as by anti-LRP antibodies. alpha-Defensin is internalized by SMCs and interacts with 2 intracellular subtypes of protein kinase C (PKC) involved in muscle contraction, alpha and beta. RAP and anti-LRP antibodies inhibited the binding and internalization of alpha-defensin by SMCs and its interaction with intracellular PKCs. These observations suggest that binding of alpha-defensin to LRP expressed in SMCs leads to its internalization; internalized alpha-defensin binds to PKC and inhibits its enzymatic activity, leading to decreased Ca(++) mobilization and SMC contraction in response to PE.


Assuntos
Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/fisiologia , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , alfa-Defensinas/farmacocinética , Animais , Aorta , Sinalização do Cálcio/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Radioisótopos do Iodo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Masculino , Músculo Liso Vascular/citologia , Fenilefrina/antagonistas & inibidores , Ligação Proteica , Proteína Quinase C/metabolismo , Ratos , Ratos Sprague-Dawley , Veias Umbilicais , Vasoconstritores/antagonistas & inibidores , alfa-Defensinas/metabolismo , alfa-Defensinas/farmacologia
8.
Blood ; 103(3): 897-902, 2004 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-14512309

RESUMO

Tissue type plasminogen activator (tPA) is a key enzyme in the fibrinolytic cascade. In this paper we report that tPA contains 2 independent epitopes that exert opposite effects on blood vessel tone. Low concentrations of tPA (1 nM) inhibit the phenylephrine (PE)-induced contraction of isolated aorta rings. In contrast, higher concentrations (20 nM) stimulate the contractile effect of PE. The 2 putative vasoactive epitopes of tPA are regulated by the plasminogen activator inhibitor-1 (PAI-1) and by a PAI-1-derived hexapeptide that binds tPA. TNK-tPA, a tPA variant in which the PAI-1 docking site has been mutated, stimulates PE-induced vasoconstriction at all concentrations used. The stimulatory, but not the inhibitory, effect of tPA on the contraction of isolated aorta rings was abolished by anti-low-density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor (LRP) antibodies. Administering tPA or TNK-tPA to rats regulates blood pressure and cerebral vascular resistance in a dose-dependent mode. In other in vivo experiments we found that the vasopressor effect of PE is more pronounced in tPA knockout than in wild-type mice. Our findings draw attention to a novel role of tPA and PAI-1 in the regulation of blood vessel tone that may affect the course of ischemic diseases.


Assuntos
Inibidor 1 de Ativador de Plasminogênio/farmacologia , Ativador de Plasminogênio Tecidual/farmacologia , Vasoconstrição/efeitos dos fármacos , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/fisiologia , Sítios de Ligação/genética , Pressão Sanguínea/efeitos dos fármacos , Relação Dose-Resposta a Droga , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Ativador de Plasminogênio Tecidual/química , Ativador de Plasminogênio Tecidual/deficiência , Ativador de Plasminogênio Tecidual/genética , Resistência Vascular/efeitos dos fármacos
9.
J Biol Chem ; 278(8): 6187-93, 2003 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-12466273

RESUMO

Accumulation of low-density lipoprotein (LDL)-derived cholesterol by macrophages in vessel walls is a pathogenomic feature of atherosclerotic lesions. Platelets contribute to lipid uptake by macrophages through mechanisms that are only partially understood. We have previously shown that platelet factor 4 (PF4) inhibits the binding and degradation of LDL through its receptor, a process that could promote the formation of oxidized LDL (ox-LDL). We have now characterized the effect of PF4 on the binding of ox-LDL to vascular cells and macrophages and on the accumulation of cholesterol esters. PF4 bound to ox-LDL directly and also increased ox-LDL binding to vascular cells and macrophages. PF4 did not stimulate ox-LDL binding to cells that do not synthesize glycosaminoglycans or after enzymatic cleavage of cell surface heparan and chondroitin sulfates. The effect of PF4 on binding ox-LDL was dependent on specific lysine residues in its C terminus. Addition of PF4 also caused an approximately 10-fold increase in the amount of ox-LDL esterified by macrophages. Furthermore, PF4 and ox-LDL co-localize in atherosclerotic lesion, especially in macrophage-derived foam cells. These observations offer a potential mechanism by which platelet activation at sites of vascular injury may promote the accumulation of deleterious lipoproteins and offer a new focus for pharmacological intervention in the development of atherosclerosis.


Assuntos
Plaquetas/fisiologia , Endotélio Vascular/fisiologia , Lipoproteínas LDL/metabolismo , Fator Plaquetário 4/farmacologia , Substituição de Aminoácidos , Animais , Arteriosclerose/patologia , Células CHO , Células Cultivadas , Cricetinae , Endotélio Vascular/patologia , Variação Genética , Humanos , Imuno-Histoquímica , Microscopia Confocal , Mutagênese Sítio-Dirigida , Fator Plaquetário 4/química , Fator Plaquetário 4/genética , Ligação Proteica , Proteoglicanas/farmacologia , Proteínas Recombinantes/metabolismo , Transfecção , Veias Umbilicais
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA