RESUMO
Plasma extravasation from postcapillary venules is one of the earliest steps of inflammation. Substance P (SP) and bradykinin (BK) mediate extravasation and cause hypotension. The cell-surface enzyme neutral endopeptidase (NEP) inactivates both peptides. Thus, absence of NEP may predispose development of inflammation and hypotension. We examined these possibilities in mice in which the NEP gene was deleted by homologous recombination. There was widespread basal plasma extravasation in postcapillary venular endothelia in NEP-/- mice, which was reversed by recombinant NEP and antagonists of SP (NK1) and BK (B2) receptors. Mean arterial blood pressure was 20% lower in NEP-/- animals, but this was unaffected by reintroduction of recombinant NEP and the kinin receptor antagonists. The hypotension was also independent of nitric oxide (NO), because NEP-/- mice treated with a NO synthase inhibitor remained hypotensive relative to the wild type. Thus, NEP has important roles in regulating basal microvascular permeability by degrading SP and BK, and may regulate blood pressure set point through a mechanism that is independent of SP, BK and NO. The use of NEP antagonists as candidate drugs in cardiovascular disease is suggested by the blood pressure data reported herein.
Assuntos
Pressão Sanguínea/fisiologia , Capilares/fisiologia , Permeabilidade Capilar/fisiologia , Neprilisina/fisiologia , Vênulas/fisiologia , Animais , Bradicinina/fisiologia , Antagonistas dos Receptores da Bradicinina , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neprilisina/genética , Antagonistas dos Receptores de Neurocinina-1 , Substância P/fisiologiaRESUMO
We recently demonstrated that gene-targeted disruption of the C5a anaphylatoxin receptor prevented lung injury in immune complex-mediated inflammation. In this study, we compare the effect of C5aR deficiency in immune complex-induced inflammation in the peritoneal cavity and skin with the results derived from our immune complex alveolitis model. C5aR- deficient mice exhibit decreased migration of neutrophils and decreased levels of TNF-alpha and interleukin 6 in the peritoneal reverse passive Arthus reaction compared to their wild-type littermates. In the reverse passive Arthus reaction in the skin the C5aR was also required for the full expression of neutrophil influx and edema formation; C5aR-deficient mice showed reduced neutrophil migration and microvascular permeability changes. In contrast to our studies in immune complex-induced lung inflammation, C5aR deficiency does not completely prevent injury in the peritoneal cavity and skin. These data indicate a dominant role for the C5aR and its ligand in the reverse passive Arthus reaction in the lung and a synergistic role together with other inflammatory mediators in immune complex-mediated peritonitis and skin injury.
Assuntos
Antígenos CD/imunologia , Reação de Arthus/imunologia , Doenças do Complexo Imune/imunologia , Inflamação/imunologia , Receptores de Complemento/imunologia , Animais , Anticorpos/imunologia , Antígenos CD/genética , Antígenos CD/metabolismo , Permeabilidade Capilar , Contagem de Células , Proteínas do Sistema Complemento/imunologia , Edema/imunologia , Marcação de Genes , Imunoglobulina G/imunologia , Inflamação/genética , Interleucina-6/análise , Pulmão/imunologia , Pulmão/patologia , Camundongos , Camundongos Knockout , Neutrófilos/imunologia , Ovalbumina/imunologia , Peritonite/imunologia , Peroxidase/metabolismo , Receptor da Anafilatoxina C5a , Receptores de Complemento/deficiência , Receptores de Complemento/genética , Receptores de Complemento/metabolismo , Receptores Fc/imunologia , Fator de Necrose Tumoral alfa/análiseRESUMO
The complement anaphylatoxin C5a and its seven-transmembrane segment (7TMS) receptor play an important role in host defense and in a number of inflammation-associated pathologies. The NH(2)-terminal domain of the C5a receptor (C5aR/CD88) contributes substantially to its ability to bind C5a. Here we show that the tyrosines at positions 11 and 14 of the C5aR are posttranslationally modified by the addition of sulfate groups. The sulfate moieties of each of these tyrosines are critical to the ability of the C5aR to bind C5a and to mobilize calcium. A C5aR variant lacking these sulfate moieties efficiently mobilized calcium in response to a small peptide agonist, but not to C5a, consistent with a two-site model of ligand association in which the tyrosine-sulfated region of the C5aR mediates the initial docking interaction. A peptide based on the NH(2) terminus of the C5aR and sulfated at these two tyrosines, but not its unsulfated analogue or a doubly sulfated control peptide, partially inhibited C5a association with its receptor. These observations clarify structural and mutagenic studies of the C5a/C5aR association and suggest that related 7TMS receptors are also modified by functionally important sulfate groups on their NH(2)-terminal tyrosines.
Assuntos
Antígenos CD/metabolismo , Complemento C5a/metabolismo , Processamento de Proteína Pós-Traducional , Receptores de Complemento/metabolismo , Sulfatos/metabolismo , Tirosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cálcio/metabolismo , Linhagem Celular Transformada , Humanos , Dados de Sequência Molecular , Peptídeos/metabolismo , Receptor da Anafilatoxina C5aRESUMO
Neutral endopeptidase (NEP; EC. 3.4.24.11) is a type 2 cell surface metalloprotease known by a variety of eponyms, including enkephalinase, common acute lymphoblastic leukemia antigen, and CD10. Identified substrates are largely neural or humoral oligopeptide agonists, and the enzyme functions to terminate signaling by degrading the ligand, analogously to acetylcholine/acetylcholinesterase. Targeted disruption of the NEP locus in mice results in enhanced lethality to endotoxin shock with a pronounced gene dosage effect. The site(s) of action appears downstream from release of tumor necrosis factor and interleukin-1 since NEP-deficient animals demonstrate increased sensitivity to these mediators as well. This unexpected finding indicates an important protective role for NEP in septic shock.
Assuntos
Neprilisina/genética , Neprilisina/metabolismo , Choque Séptico/fisiopatologia , Animais , Células Cultivadas , Quimera , Clonagem Molecular , Morte , Feminino , Biblioteca Genômica , Humanos , Interleucina-1/farmacologia , Lipopolissacarídeos , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neprilisina/deficiência , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Mapeamento por Restrição , Salmonella enteritidis , Choque Séptico/enzimologia , Choque Séptico/patologia , Células-Tronco , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Chemokines provide signals for activation and recruitment of effector cells into sites of inflammation, acting via specific G protein-coupled receptors. However, in vitro data demonstrating the presence of multiple ligands for a given chemokine receptor, and often multiple receptors for a given chemokine, have led to concerns of biologic redundancy. Here we show that acute cardiac allograft rejection is accompanied by progressive intragraft production of the chemokines interferon (IFN)-gamma-inducible protein of 10 kD (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T cell alpha chemoattractant (I-TAC), and by infiltration of activated T cells bearing the corresponding chemokine receptor, CXCR3. We used three in vivo models to demonstrate a role for CXCR3 in the development of transplant rejection. First, CXCR3-deficient (CXCR3(-/)-) mice showed profound resistance to development of acute allograft rejection. Second, CXCR3(-/)- allograft recipients treated with a brief, subtherapeutic course of cyclosporin A maintained their allografts permanently and without evidence of chronic rejection. Third, CXCR(+/+) mice treated with an anti-CXCR3 monoclonal antibody showed prolongation of allograft survival, even if begun after the onset of rejection. Taken in conjunction with our findings of CXCR3 expression in rejecting human cardiac allografts, we conclude that CXCR3 plays a key role in T cell activation, recruitment, and allograft destruction.
Assuntos
Rejeição de Enxerto/imunologia , Transplante de Coração/imunologia , Receptores de Quimiocinas/imunologia , Doença Aguda , Animais , Rejeição de Enxerto/genética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutagênese , Receptores CXCR3 , Receptores de Quimiocinas/genética , Linfócitos T/imunologia , Transplante HomólogoRESUMO
The chemokine eotaxin is unusual in that it appears to be a highly specific chemoattractant for eosinophils. Ligand-binding studies with radiolabeled eotaxin demonstrated a receptor on eosinophils distinct from the known chemokine receptors CKR-1 and -2. The distinct eotaxin binding site on human eosinophils also bound RANTES (regulated on activation T expressed and secreted) and monocyte chemotactic protein (MCP)3. We have now isolated a cDNA from eosinophils, termed CKR-3, with significant sequence similarity to other well characterized chemokine receptors. Cells transfected with CKR-3 cDNA bound radiolabeled eotaxin specifically and with high affinity, comparable to the binding affinity observed with eosinophils. This receptor also bound RANTES and MCP-3 with high affinity, but not other CC or CXC chemokines. Furthermore, receptor transfectants generated in a murine B cell lymphoma cell line migrated in transwell chemotaxis assays to eotaxin, RANTES, and MCP-3, but not to any other chemokines. A monoclonal antibody recognizing CKR-3 was used to show that eosinophils, but not other leukocyte types, expressed this receptor. This pattern of expression was confirmed by Northern blot with RNA from highly purified leukocyte subsets. The restricted expression of CKR-3 on eosinophils and the fidelity of eotaxin binding to CKR-3, provides a potential mechanism for the selective recruitment and migration of eosinophils within tissues.
Assuntos
Quimiocinas CC , Citocinas/farmacologia , Eosinófilos/imunologia , Receptores de Quimiocinas , Receptores de Citocinas/fisiologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Sequência de Bases , Quimiocina CCL11 , Quimiocina CCL4 , Quimiocina CCL5/farmacologia , Quimiocina CCL7 , Fatores Quimiotáticos de Eosinófilos/metabolismo , Quimiotaxia de Leucócito , Clonagem Molecular , Sequência Conservada , Citocinas/metabolismo , Primers do DNA , Humanos , Linfoma de Células B , Proteínas Inflamatórias de Macrófagos , Camundongos , Dados de Sequência Molecular , Proteínas Quimioatraentes de Monócitos/farmacologia , Monocinas/farmacologia , Reação em Cadeia da Polimerase , Receptores CCR3 , Receptores de Citocinas/biossíntese , Receptores de Citocinas/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Transfecção , Células Tumorais CultivadasRESUMO
CD4+ T lymphocyte depletion in human immunodeficiency virus type 1 (HIV-1)-infected humans underlies the development of acquired immune deficiency syndrome. Using a model in which rhesus macaques were infected with chimeric simian-human immunodeficiency viruses (SHIVs), we show that both the level of viremia and the structure of the HIV-1 envelope glycoprotein ectodomains individually contributed to the efficiency with which CD4(+) T lymphocytes were depleted. The envelope glycoproteins of recombinant SHIVs that efficiently caused loss of CD4(+) T lymphocytes exhibited increased chemokine receptor binding and membrane-fusing capacity compared with those of less pathogenic viruses. These studies identify the HIV-1 envelope glycoprotein ectodomains as determinants of CD4(+) T lymphocyte loss in vivo and provide a foundation for studying pathogenic mechanisms.
Assuntos
Linfócitos T CD4-Positivos/imunologia , HIV-1/imunologia , Depleção Linfocítica , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Proteínas do Envelope Viral/fisiologia , Animais , Antivirais/imunologia , Linfócitos T CD4-Positivos/virologia , Quimera/imunologia , Células Gigantes/virologia , HIV-1/genética , HIV-1/patogenicidade , Humanos , Linfonodos/virologia , Contagem de Linfócitos , Macaca mulatta , Testes de Neutralização , Estrutura Terciária de Proteína , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/imunologia , Vírus da Imunodeficiência Símia/patogenicidade , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Replicação Viral/genética , Replicação Viral/imunologiaRESUMO
The formation of intrapulmonary immune complexes in mice generates a vigorous inflammatory response characterized by microvascular permeability and polymorphonuclear neutrophil influx. Gene-targeted disruption of the substance P receptor (NK-1R) protected the lung from immune complex injury, as did disruption of the C5a anaphylatoxin receptor. Immunoreactive substance P was measurable in fluids lining the lung at time points before neutrophil influx and may thus be involved in an early step in the inflammatory response to immune complexes in the lung.
Assuntos
Complexo Antígeno-Anticorpo/imunologia , Complemento C5a/fisiologia , Doenças do Complexo Imune/metabolismo , Pneumopatias/metabolismo , Substância P/fisiologia , Animais , Antígenos CD/genética , Antígenos CD/fisiologia , Líquido da Lavagem Broncoalveolar/química , Permeabilidade Capilar , Feminino , Marcação de Genes , Doenças do Complexo Imune/imunologia , Doenças do Complexo Imune/patologia , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Pulmão/química , Pulmão/patologia , Pneumopatias/imunologia , Pneumopatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos , Receptor da Anafilatoxina C5a , Receptores de Complemento/genética , Receptores de Complemento/fisiologia , Receptores da Neurocinina-1/genética , Receptores da Neurocinina-1/fisiologia , Substância P/análise , Fator de Necrose Tumoral alfa/análiseRESUMO
Amyloid beta peptide (Abeta), the pathogenic agent of Alzheimer's disease (AD), is a physiological metabolite in the brain. We examined the role of neprilysin, a candidate Abeta-degrading peptidase, in the metabolism using neprilysin gene-disrupted mice. Neprilysin deficiency resulted in defects both in the degradation of exogenously administered Abeta and in the metabolic suppression of the endogenous Abeta levels in a gene dose-dependent manner. The regional levels of Abeta in the neprilysin-deficient mouse brain were in the distinct order of hippocampus, cortex, thalamus/striatum, and cerebellum, where hippocampus has the highest level and cerebellum the lowest, correlating with the vulnerability to Abeta deposition in brains of humans with AD. Our observations suggest that even partial down-regulation of neprilysin activity, which could be caused by aging, can contribute to AD development by promoting Abeta accumulation.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Neprilisina/metabolismo , Envelhecimento/metabolismo , Doença de Alzheimer/etiologia , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Animais , Encéfalo/enzimologia , Cromatografia Líquida de Alta Pressão , Regulação para Baixo , Elementos Facilitadores Genéticos , Ensaio de Imunoadsorção Enzimática , Dosagem de Genes , Hipocampo/enzimologia , Hipocampo/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Mutação , Neprilisina/genética , Neurônios/enzimologia , Fragmentos de Peptídeos/metabolismo , Presenilina-1 , Elementos de Resposta , Regulação para CimaRESUMO
CCR5 and CD4 are coreceptors for immunodeficiency virus entry into target cells. The gp120 envelope glycoprotein from human immunodeficiency virus strain HIV-1(YU2) bound human CCR5 (CCR5hu) or rhesus macaque CCR5 (CCR5rh) only in the presence of CD4. The gp120 from simian immunodeficiency virus strain SIVmac239 bound CCR5rh without CD4, but CCR5hu remained CD4-dependent. The CD4-independent binding of SIVmac239 gp120 depended on a single amino acid, Asp13, in the CCR5rh amino-terminus. Thus, CCR5-binding moieties on the immunodeficiency virus envelope glycoprotein can be generated by interaction with CD4 or by direct interaction with the CCR5 amino-terminus. These results may have implications for the evolution of receptor use among lentiviruses as well as utility in the development of effective intervention.
Assuntos
Antígenos CD4/fisiologia , Proteína gp120 do Envelope de HIV/metabolismo , Glicoproteínas de Membrana , Receptores CCR5/metabolismo , Vírus da Imunodeficiência Símia/metabolismo , Proteínas do Envelope Viral , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Linhagem Celular , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/química , HIV-2/imunologia , Humanos , Macaca mulatta , Macrófagos/virologia , Mutação , Receptores CCR5/química , TransfecçãoRESUMO
The responsiveness of isolated guinea pig lung parenchymal strips to substance P was enhanced by at least 100-fold in the presence of the endopeptidase inhibitors phosphoramidon (1 microM) or thiorphan (1 microM), but not with the converting enzyme inhibitor, captopril, or an inhibitor of serum carboxypeptidase N (both 1 microM). Responses of guinea pig tracheal rings to substance P were also markedly potentiated by phosphoramidon. The increase in tissue responsiveness by these inhibitors was relatively specific for substance P among several other spasmogenic peptides, including formyl-methionyl-leucyl-phenylalanine and the complement peptides C3a and C5a. The enhanced responses appear to result from a decrease in the rate of substance P degradation in the presence of neutral endopeptidase inhibitors. Specific binding of substance P to its receptor on bronchial membranes was increased by three- to fourfold in the presence of phosphoramidon. These data demonstrate an enhanced potential for substance P to contract lung tissues when degradation by a neutral endopeptidase-like enzyme is blocked.
Assuntos
Endopeptidases/fisiologia , Contração Muscular/efeitos dos fármacos , Substância P/farmacologia , Inibidores da Enzima Conversora de Angiotensina , Animais , Captopril/farmacologia , Complemento C3/farmacologia , Complemento C3a , Complemento C5/farmacologia , Complemento C5a , Interações Medicamentosas , Glicopeptídeos/farmacologia , Cobaias , Pulmão , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Peptidil Dipeptidase A/fisiologia , Receptores da Neurocinina-1 , Receptores de Neurotransmissores/metabolismo , Substância P/metabolismo , Tiorfano , Tiopronina/análogos & derivados , Tiopronina/farmacologia , TraqueiaRESUMO
The effects of the angiotensin converting enzyme (ACE) inhibitor captopril and the neutral endopeptidase (NEP) inhibitors thiorphan and SCH 32615 on the changes in airway opening pressure (PaO) and the recovery of offered peptide were studied after intratracheal administration of substance P (SP) and neurokinin A (NKA) in isolated guinea pig lungs superfused through the trachea. Pao changes and the recovery of offered peptide were significantly greater in NEP inhibitor-treated lungs than in control lungs. Captopril did not cause a significant change in the physiological effects or the recovery of SP and NKA. HPLC analysis of [3H]Pro2,4-SP and 125I-Histidyl1-NKA perfused through the airways showed major cleavage products consistent with NEP action. We conclude that there is significant degradation of both SP and NKA after tracheal infusion of peptides by NEP-like but not by ACE activity; this effect significantly influences the physiological effects of these peptides.
Assuntos
Pulmão/fisiologia , Neurocinina A/farmacologia , Substância P/farmacologia , Traqueia/fisiologia , Animais , Captopril/farmacologia , Dipeptídeos/farmacologia , Cobaias , Cinética , Pulmão/efeitos dos fármacos , Masculino , Neurocinina A/metabolismo , Peptídeo Hidrolases , Perfusão , Substância P/metabolismo , Tiorfano/farmacologiaRESUMO
beta-Chemokines and their receptors mediate the trafficking and activation of a variety of leukocytes including the lymphocyte and macrophage. An array of no less than eight beta-chemokine receptors has been identified, four of which are capable of recognizing the chemokines MIP1alpha and RANTES. Genetic deletion of one of the MIP1alpha and RANTES receptors, CCR5, is associated with protection from infection with HIV-1 in humans, while deletion of the ligand MIP1alpha protects against Coxsackie virus-associated myocarditis. In this report we show that the deletion of another receptor for MIP1alpha and RANTES, the CCR1 receptor, is associated with protection from pulmonary inflammation secondary to acute pancreatitis in the mouse. The protection from lung injury is associated with decreased levels of TNF-alpha in a temporal sequence indicating that the activation of the CCR1 receptor is an early event in the systemic inflammatory response syndrome.
Assuntos
Pancreatite/complicações , Receptores de Quimiocinas/genética , Síndrome do Desconforto Respiratório/prevenção & controle , Animais , Permeabilidade Capilar , Ceruletídeo/farmacologia , Quimiocina CCL4 , Quimiocina CCL5/metabolismo , Deleção de Genes , Pulmão/química , Proteínas Inflamatórias de Macrófagos/metabolismo , Camundongos , Microcirculação , Pâncreas/química , Pâncreas/patologia , Pancreatite/induzido quimicamente , Receptores CCR1 , Síndrome do Desconforto Respiratório/etiologia , Síndrome do Desconforto Respiratório/genética , Fator de Necrose Tumoral alfa/análise , Água/análiseRESUMO
Toxin A, a 308,000-Mr enterotoxin from Clostridium difficile, mediates antibiotic-associated diarrhea and colitis in humans. Injection of toxin A into animal intestine triggers an acute inflammatory response characterized by activation of sensory neurons and immune cells of the intestinal lamina propria, including mast cells and macrophages, and migration of circulating neutrophils in the involved intestinal segment. In this study we show that mice genetically deficient in the neurokinin-1 receptor are protected from the secretory and inflammatory changes as well as from epithelial cell damage induced by toxin A. The protective effect of neurokinin-1R deletion correlates with diminished intestinal levels of the cytokine TNF-alpha and its mRNA and the leukocyte enzyme myeloperoxidase. These results demonstrate a major requirement for substance P receptors in the pathogenesis of acute inflammatory diarrhea.
Assuntos
Clostridioides difficile/patogenicidade , Enterocolite Pseudomembranosa/etiologia , Receptores da Neurocinina-1/metabolismo , Animais , Toxinas Bacterianas/toxicidade , Enterocolite Pseudomembranosa/metabolismo , Enterocolite Pseudomembranosa/patologia , Enterotoxinas/toxicidade , Humanos , Íleo/efeitos dos fármacos , Íleo/metabolismo , Íleo/patologia , Técnicas In Vitro , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores da Neurocinina-1/deficiência , Receptores da Neurocinina-1/genética , Substância P/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Isoquinoline sulfonamides have recently been shown to exert novel inhibitory effects on mammalian protein kinases by competitively binding to the ATP substrate site (Hidaka, H., M. Inagaki, S. Kawamoto, and Y. Sasaki, 1984, Biochemistry, 23: 5036-5041). We synthesized a unique analog of the previously reported compounds, 1-(5-isoquinolinesulfonyl) piperazine (C-I), in order to assess the role of protein kinases in modulating the agonist-stimulated oxidative burst of human polymorphonuclear leukocytes (PMN). Compound C-I, at micromolar concentration, markedly inhibited the release of superoxide anion from human PMN stimulated with phorbol myristate acetate or the synthetic diacylglycerol, 1-oleoyl-2-acetyl glycerol. These data are consonant with previously reported data which indicate that the calcium and phospholipid-dependent protein kinase, protein kinase C, serves as the intracellular receptor for these agonists. In contrast, superoxide anion production stimulated by the complement anaphylatoxin peptide C5a or the synthetic chemotaxin formyl-methionyl-leucyl-phenylalanine were not inhibited by C-I. These data suggest that parallel pathways exist for the agonist-stimulated respiratory burst of human neutrophils, only one of which utilizes the calcium and phospholipid-dependent protein kinase.
Assuntos
Isoquinolinas/farmacologia , Neutrófilos/metabolismo , Oxigênio/metabolismo , Piperazinas/farmacologia , Proteína Quinase C/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina , Complemento C5/farmacologia , Complemento C5a , Humanos , Isoquinolinas/isolamento & purificação , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Consumo de Oxigênio/efeitos dos fármacos , Piperazinas/isolamento & purificação , Proteína Quinase C/fisiologia , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The past few years have witnessed the convergence of research into inflammation and research on the G protein linked receptors; the receptors encoded by this gene family recognize the ligands C5a, interleukin 8 and related chemokines, such as platelet activating factor and formyl peptide. The theme that is emerging suggests that distinct signals at the cell surface feed into common pathways of cell activation. Different agonist functions may relate to G protein heterogeneity, kinetics of activation, receptor phosphorylation, and non-selective coupling of receptors with multiple varieties of G proteins.
Assuntos
Inflamação/imunologia , Leucócitos/fisiologia , Receptores Imunológicos/fisiologia , Animais , Proteínas de Ligação ao GTP/fisiologia , Humanos , Leucócitos/química , Ligantes , Receptores Imunológicos/química , Transdução de SinaisRESUMO
The existence of a third non-coding exon in the human neprilysin-encoding gene (h-NEP), positionally located as exon 3, has been demonstrated by reverse transcription of RNA from human kidney and lung, coupled with the polymerase chain reaction. Comparison of nucleotide sequences between h-NEP and the rat NEP (r-NEP) genes shows a high degree of sequence conservation within noncoding exons 1 and 2. In contrast, the region of the gene containing exon 3 is highly divergent. Two transcripts derived from exon 2 by alternative splicing, type-2a and type-2b, were demonstrated in human kidney and lung. In contrast, only the type-2b transcript was present in these same tissues in the rat. The type-1 transcript was detected in human kidney, lung and brain, this transcript appearing to be the major species in brain.
Assuntos
Hominidae/genética , Neprilisina/biossíntese , Neprilisina/genética , Ratos/genética , Processamento Alternativo , Animais , Sequência de Bases , Encéfalo/enzimologia , Éxons , Expressão Gênica , Humanos , Rim/enzimologia , Pulmão/enzimologia , Dados de Sequência Molecular , Especificidade de Órgãos , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Transcrição GênicaRESUMO
The tachykinin neurokinin-1 (NK(1)) receptor mediates the vasoactive effects of substance P and related members of the tachykinin family. Substance P acts via the NK(1) receptor to mediate increased microvascular permeability leading to oedema formation as confirmed in NK(1) receptor knockout mice. In addition there is evidence that neuropeptides such as substance P can have a modulatory effect on the wound-healing process. In this study male and female wild-type and NK(1) knockout mice were investigated for their comparative ability to induce acute oedema formation in response to topical application of capsaicin, as measured by the extravasation of intravenous radiolabelled-albumin, and wound healing in response to a cut, as measured by area of wound over the following days. Significant (P<0.001) oedema, approximately three-fold over basal, was induced by capsaicin in both male and female wild-type mice, an indicator of a similar responsiveness irrespective of sex. However, as expected, the oedema was not observed in the knockout mice. Wounding was achieved through a 1-cm full-thickness cut into the interscapular area of dorsal skin. Wound healing was then followed in two different protocols. The wound was left to heal naturally over 14 days in the first protocol and no significant changes in healing were observed in wild-type compared to knockout. In the second protocol, the skin was sutured open for the first 48 h, to prevent the elasticity of the skin from initiating a natural healing process through flap formation. This caused a significant increase in the area of the wound. Despite this, wounds in both wild-type and knockout mice healed in an identical manner that was complete after 17 days. In conclusion, it is shown that deletion of a functional NK(1) receptor has little effect on wound healing in response to a simple cut in mouse skin.
Assuntos
Receptores da Neurocinina-1/genética , Pele/lesões , Cicatrização/fisiologia , Animais , Capsaicina , Edema/induzido quimicamente , Edema/fisiopatologia , Deleção de Genes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores da Neurocinina-1/metabolismo , Pele/inervação , Pele/metabolismo , Substância P/metabolismo , SuturasRESUMO
1. Scald injury in Sv129+C57BL/6 mice induced a temperature and time dependent oedema formation as calculated by the extravascular accumulation of [(125)I]-albumin. Oedema formation was suppressed in NK(1) knockout mice compared to wildtypes at 10 (P<0.01) and 30 min (P<0.001). However, at 60 min a similar degree of extravasation was observed in the two groups. 2. Kinin B(1) (des-Arg(10) Hoe 140; 1 micromol kg(-1)) and B(2) (Hoe 140; 100 nmol kg(-1)) antagonists caused an inhibition of oedema in wildtype mice at 10 and 30 min (P<0.001), but not at 60 min or at 30 min in NK(1) receptor knockout mice. 3. The inhibition of thermic oedema by des-Arg(10) Hoe 140 was reversed by des-Arg(9) bradykinin (0.1 micromol kg(-1); P<0.01) and also observed with a second B(1) receptor antagonist (des-Arg(9) Leu(8) bradykinin; 3 micromol kg(-1); P<0.01). Furthermore des-Arg(10) Hoe 140 had no effect on capsaicin (200 microg ear(-1)) ear oedema, but this was significantly reduced with Hoe 140 (P<0.05). 4. Scalding induced a large neutrophil accumulation at 4 h, as assessed by myeloperoxidase assay (P<0.001). This was not suppressed by NK(1) receptor deletion or kinin antagonists. 5. These results confirm an essential role for the NK(1) receptor in mediating the early, but not the delayed phase of oedema formation or neutrophil accumulation in response to scalding. The results also demonstrate a pivotal link between the kinins and sensory nerves in the microvascular response to burn injury, and for the first time show a rapid involvement of the B(1) receptor in murine skin.