RESUMO
Habits are inflexible behaviors that develop after extensive repetition, and overreliance on habits is a hallmark of many pathological states. The striatum is involved in the transition from flexible to inflexible responding, and interspersed throughout the striatum are patches, or striosomes, which make up ~15% of the volume of the striatum relative to the surrounding matrix compartment. Previous studies have suggested that patches are necessary for normal habit formation, but it remains unknown exactly how patches contribute to habit formation and expression. Here, using optogenetics, we stimulated striatal patches in Sepw1-NP67 mice during variable interval training (VI60), which is used to establish habitual responding. We found that activation of patches at reward retrieval resulted in elevated responding during VI60 training by modifying the pattern of head entry and pressing. Further, this optogenetic manipulation reduced subsequent responding following reinforcer devaluation, suggesting modified habit formation. However, patch stimulation did not generally increase extinction rates during a subsequent extinction probe, but did result in a small 'extinction burst', further suggesting goal-directed behavior. On the other hand, this manipulation had no effect in omission trials, where mice had to withhold responses to obtain rewards. Finally, we utilized fast-scan cyclic voltammetry to investigate how patch activation modifies evoked striatal dopamine release and found that optogenetic activation of patch projections to the substantia nigra pars compacta (SNc) is sufficient to suppress dopamine release in the dorsal striatum. Overall, this work provides novel insight into the role of the patch compartment in habit formation, and provides a potential mechanism for how patches modify habitual behavior by exerting control over dopamine signaling.
Assuntos
Corpo Estriado/fisiologia , Dopamina/metabolismo , Hábitos , Optogenética , Estimulação Física , Animais , Corpo Estriado/metabolismo , Aprendizagem , Locomoção , Camundongos , Camundongos Transgênicos , Optogenética/métodos , Substância Negra/fisiologiaRESUMO
Tiotropium is commonly used in the treatment of chronic obstructive pulmonary disease. Although largely considered to be a long-acting bronchodilator, its demonstrated efficacy in reducing the frequency of exacerbations and preliminary evidence from early studies indicating that it might slow the rate of decline in lung function suggested mechanisms of action in addition to simple bronchodilation. This hypothesis was examined in the recently published UPLIFT study and, although spirometric and other clinical benefits of tiotropium treatment extended to four years, the rate of decline in lung function did not appear to be reduced by the addition of tiotropium in this study. This article summarizes data from a variety of investigations that provide insights into possible mechanisms to account for the effects of tiotropium. The report summarizes the discussion on basic and clinical research in this field.
Assuntos
Broncodilatadores/farmacologia , Antagonistas Colinérgicos/farmacologia , Derivados da Escopolamina/farmacologia , Acetilcolina/fisiologia , Animais , Broncodilatadores/uso terapêutico , Antagonistas Colinérgicos/uso terapêutico , Tosse/tratamento farmacológico , Tosse/fisiopatologia , Humanos , Inflamação/patologia , Pulmão/inervação , Pulmão/fisiologia , Muco/metabolismo , Sistema Nervoso Parassimpático/efeitos dos fármacos , Sistema Respiratório/efeitos dos fármacos , Sistema Respiratório/patologia , Derivados da Escopolamina/uso terapêutico , Brometo de TiotrópioRESUMO
Inhaled air is contaminated with pathogens and particulates that may deposit in the airways and damage the host. In response to these invaders, the airway epithelium has developed innate immune responses that provide a defence against the invaders and protect the airway structure and function. Thus, the epithelium of conducting airways becomes the "battleground" between the invaders and the host. Recent evidence suggests that airway epithelial surface signalling through the epidermal growth factor receptor (EGFR) is a convergent pathway producing innate immune responses to a variety of infectious and noninfectious noxious stimuli. In the present review, the EGFR signalling pathways leading to airway mucin production, neutrophil recruitment (via interleukin-8 production) and airway epithelial repair were examined. The importance of these findings in human airway diseases was also investigated. The current authors suggest that the exaggerated innate immune responses found in chronic inflammatory airway diseases (e.g. chronic obstructive pulmonary disease, cystic fibrosis and severe asthma) contribute to the pathogenesis or the aggravation of these diseases. Potential therapies include inhibition of the various elements of the described epidermal growth factor receptor cascade. In considering each therapeutic intervention, the potential benefits must be considered in relation to potential deleterious effects.
Assuntos
Asma/metabolismo , Fibrose Cística/metabolismo , Receptores ErbB/metabolismo , Imunidade Inata , Mucinas/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Animais , Asma/diagnóstico , Fibrose Cística/diagnóstico , Humanos , Interleucina-8/metabolismo , Ligantes , Modelos Biológicos , Neutrófilos/metabolismo , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Espécies Reativas de Oxigênio , Transdução de SinaisRESUMO
Formoterol is a selective long-acting beta2-adrenergic receptor agonist (LABA) that provides significant and sustained bronchodilatory effect for up to 12h following a single dose. The onset of effect is significantly faster with formoterol compared with an alternative LABA, salmeterol, although both have a similar duration of action. The overall efficacy of formoterol in improving lung function and controlling symptoms of chronic obstructive pulmonary disease (COPD) is comparable to that of salmeterol and potentially superior to that of ipratropium or theophylline. Formoterol provides additional benefit when administered in combination with other bronchodilators or inhaled corticosteroids. In clinical studies, formoterol was well tolerated and had an adverse-event profile similar to that of other beta2-adrenergic receptor agonists. Formoterol is a rapidly acting, well-tolerated, effective beta2-adrenergic receptor agonist that can be regularly used as a long-acting bronchodilator for patients with moderate to severe COPD, as per recommendations of the current treatment guidelines.
Assuntos
Agonistas Adrenérgicos beta/uso terapêutico , Broncodilatadores/uso terapêutico , Etanolaminas/uso terapêutico , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Administração por Inalação , Idoso , Broncodilatadores/efeitos adversos , Relação Dose-Resposta a Droga , Etanolaminas/efeitos adversos , Fumarato de Formoterol , Humanos , Pessoa de Meia-Idade , Guias de Prática Clínica como Assunto , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
Respiratory syncytial virus (RSV) persists as a significant human pathogen that continues to contribute to morbidity and mortality. In children, RSV is the leading cause of lower respiratory tract infections, and in adults RSV causes pneumonia and contributes to exacerbations of chronic lung diseases. RSV induces airway epithelial inflammation by activation of the epidermal growth factor receptor (EGFR), a tyrosine kinase receptor. Recently, EGFR inhibition was shown to decrease RSV infection, but the mechanism(s) for this effect are not known. Interferon (IFN) signaling is critical for innate antiviral responses, and recent experiments have implicated IFN-λ (lambda), a type III IFN, as the most significant IFN for mucosal antiviral immune responses to RSV infection. However, a role for RSV-induced EGFR activation to suppress airway epithelial antiviral immunity has not been explored. Here, we show that RSV-induced EGFR activation suppresses IFN regulatory factor (IRF) 1-induced IFN-λ production and increased viral infection, and we implicate RSV F protein to mediate this effect. EGFR inhibition, during viral infection, augmented IRF1, IFN-λ, and decreased RSV titers. These results suggest a mechanism for EGFR inhibition to suppress RSV by activation of endogenous epithelial antiviral defenses, which may be a potential target for novel therapeutics.
Assuntos
Citocinas/metabolismo , Mucosa Respiratória/fisiologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sinciciais Respiratórios/fisiologia , Antígenos Virais/imunologia , Linhagem Celular , Regulação para Baixo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Imunidade , Fator Regulador 1 de Interferon/metabolismo , Mucosa Respiratória/patologia , Mucosa Respiratória/virologia , Transdução de SinaisRESUMO
Glucocorticoids inhibit plasma extravasation induced in the rat tracheal mucosa by substance P and other tachykinins released from sensory nerves. This study was performed to determine whether this antiinflammatory effect of glucocorticoids is mediated by the tachykinin-degrading enzymes neutral endopeptidase (NEP) and kininase II (angiotensin converting enzyme, ACE). In addition, we studied the effect of dexamethasone on a nonpeptide inflammatory mediator, platelet-activating factor (PAF), which is not degraded by NEP or ACE. Adult male pathogen-free F344 rats were treated for 2 d with dexamethasone (0.5 mg/kg per d i.p.), or with the vehicle used to dissolve the steroid. The magnitude of plasma extravasation produced by an intravenous injection of substance P (5 micrograms/kg) or PAF (10 micrograms/kg) was then assessed by using Monastral blue pigment as an intravascular tracer. The role of NEP and ACE activities in the changes produced by dexamethasone was investigated by examining the effect of the selective inhibitors of these enzymes, phosphoramidon and captopril. Dexamethasone reduced the substance P-induced extravasation by 57% but did not affect the PAF-induced extravasation. The suppressive effect of dexamethasone on substance P-induced extravasation was completely reversed by simultaneously inhibiting NEP and ACE activities, but the inhibition of these enzymes had no effect on PAF-induced extravasation, regardless of whether the rats were pretreated with dexamethasone or not. These results suggest that NEP and ACE mediate a selective inhibitory effect of glucocorticoids on neurogenic plasma extravasation.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Dexametasona/farmacologia , Endopeptidases/fisiologia , Peptidil Dipeptidase A/fisiologia , Traqueia/efeitos dos fármacos , Animais , Captopril/farmacologia , Glicopeptídeos/farmacologia , Masculino , Fator de Ativação de Plaquetas/farmacologia , Ratos , Ratos Endogâmicos , Substância P/farmacologiaRESUMO
Capsaicin increases the permeability of blood vessels in the rat tracheal mucosa through a mechanism involving the release of tachykinins from sensory nerves. This capsaicin-induced increase in vascular permeability is potentiated by viral infections of the respiratory tract. The present study was done to determine whether this "neurogenic plasma extravasation" can be inhibited by glucocorticoids, to learn the time course of this inhibition, and to determine whether glucocorticoids can prevent the potentiating effect of viral respiratory infections on neurogenic plasma extravasation. Groups of pathogen-free F344 rats were treated with dexamethasone for 2 or 8 h (4 mg/kg i.p.) or 48 or 120 h (0.5-4 mg/kg per d i.p.). Another group of rats was treated with dexamethasone for 120 h following the intranasal inoculation of Sendai virus. The magnitude of plasma extravasation produced by capsaicin or substance P was assessed after this treatment by using Monastral blue pigment and Evans blue dye as intravascular tracers. We found that dexamethasone reduced, in a dose-dependent fashion, the magnitude of plasma extravasation produced in the rat trachea by capsaicin and substance P. Significant inhibition was produced by a dose of dexamethasone as small as 0.5 mg/kg i.p. The effect of dexamethasone had a latency of several hours and reached a maximum after 2 d of treatment. Furthermore, dexamethasone prevented the potentiation of neurogenic plasma extravasation usually present after 5 d of Sendai virus respiratory infection.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Capsaicina/farmacologia , Dexametasona/farmacologia , Infecções por Paramyxoviridae/fisiopatologia , Infecções Respiratórias/fisiopatologia , Traqueia/irrigação sanguínea , Animais , Peso Corporal , Cinética , Mucosa/patologia , Tamanho do Órgão , Vírus da Parainfluenza 1 Humana , Infecções por Paramyxoviridae/patologia , Ratos , Ratos Endogâmicos F344 , Infecções Respiratórias/patologia , Substância P/farmacologia , Traqueia/patologiaRESUMO
In this study, we examined whether inhalation of hypertonic saline aerosols increases vascular permeability in the rat trachea, and we examined the role of neurogenic inflammation in this response. Stereological point counting was performed to measure the percent area occupied by Monastral blue-labeled blood vessels as a means of quantifying the increase in vascular permeability in tracheal whole mounts. Hypertonic saline aerosols (3.6-14.4% NaCl) increased vascular permeability in a dose-dependent fashion compared with 0.9% NaCl. Thus, the area density of Monastral blue-labeled vessels after inhalation of 3.6% NaCl was greater (21.2 +/- 3.5% mean +/- SEM, n = 5) than after 0.9% NaCl aerosol (3.3 +/- 0.9%, n = 5, P less than 0.5). The neutral endopeptidase inhibitor phosphoramidon (2.5 mg/kg, i.v.) significantly potentiated the increase of vascular permeability caused by 3.6% NaCl. Desensitization of sensory nerve endings by pretreatment with capsaicin markedly reduced the usual increase in vascular permeability caused by 3.6% NaCl, but the increase in vascular permeability induced by aerosolized substance P (10(-4) M) was unchanged. These findings suggest that hypertonic saline increases vascular permeability in the rat trachea by stimulating the release of neuropeptides from sensory nerves.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Inflamação/induzido quimicamente , Neuropeptídeos/fisiologia , Solução Salina Hipertônica/farmacologia , Traqueia/irrigação sanguínea , Animais , Capsaicina/farmacologia , Relação Dose-Resposta a Droga , Glicopeptídeos/farmacologia , Técnicas In Vitro , Masculino , Neurônios Aferentes/fisiologia , Ratos , Ratos Endogâmicos F344 , Substância P/metabolismo , Traqueia/efeitos dos fármacosRESUMO
Inhalation of aerosols of citric acid, histamine phosphate, or carbon dust, or air cooled to - 20 degrees C or rapid respiratory maneuvers (inspiration or expiration) results in an increase in airway resistance in some patients with asthma or bronchitis. It has been shown previously in animals that stimulation of cough receptors results in bronchoconstriction through efferent cholinergic pathways. In the patients studied, the administration of atropine sulfate, which would block such pathways, abolished the bronchoconstrictor effects of all the stimuli except large doses of histamine, which may exert a direct effect on airway smooth muscle. These data suggest that sensitized cough receptors may be involved in triggering reflex airway constriction in such patients.
Assuntos
Asma , Atropina/farmacologia , Sistema Nervoso Autônomo/fisiologia , Bronquite , Tosse/induzido quimicamente , Adolescente , Adulto , Aerossóis , Carbono , Feminino , Histamina , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Skin mast cells release the neutral protease chymase along with histamine during degranulation. To test the hypothesis that chymase modulates histamine-induced plasma extravasation, we measured wheal formation following intradermal injection of purified mast cell chymase and histamine into the skin of ragweed-allergic dogs. We found that chymase greatly augments histamine-induced wheal formation. The magnitude of the potentiating effect increases with increasing doses of chymase and becomes maximal approximately 30 min after administration. Injection of chymase without histamine does not evoke wheal formation. The chymase potentiation of histamine-induced skin responses is prevented completely by pretreatment with the H1-receptor antagonist pyrilamine, and is prevented by inactivation of chymase with soybean trypsin inhibitor, suggesting that both histamine and preserved catalytic activity are required for the effects of chymase. To examine the effects of histamine and chymase released in situ in further experiments, we measured wheal size following local degranulation of mast cells by intradermal injection of ragweed antigen or compound 48/80. We found that pretreatment with either soybean trypsin inhibitor or pyrilamine markedly reduces ragweed antigen- or 48/80-induced wheal formation, supporting the results obtained by injection of exogenous chymase and histamine. These findings suggest a novel and important proinflammatory role for chymase in modulating the effects of histamine on vascular permeability during mast cell activation.
Assuntos
Alérgenos/imunologia , Histamina/fisiologia , Hipersensibilidade/fisiopatologia , Mastócitos/fisiologia , Serina Endopeptidases/fisiologia , Animais , Permeabilidade Capilar , Degranulação Celular , Quimases , Cães , Pirilamina/farmacologia , Inibidores de Serina Proteinase , Testes Cutâneos , Fatores de Tempo , Inibidores da Tripsina/farmacologia , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
To study the roles of substance P and endogenous neutral endopeptidase in mediating cough, we measured cough responses in awake guinea pigs in response to exogenous substance P and capsaicin aerosols in the presence and absence of the neutral endopeptidase inhibitors leucine-thiorphan and phosphoramidon. Substance P stimulated cough in very low concentrations (10(-17)-10(-16) M). In a second study where the investigator did not know whether substance P or diluent alone was aerosolized, substance P (10(-16) M) caused cough. Leucine-thiorphan (10(-5) M) and phosphoramidon (10(-5) M) potentiated substance P-induced cough; NEP inhibitors also potentiated capsaicin-induced cough significantly. These findings suggest that substance P is a potent stimulator of cough responses, that capsaicin-induced cough is mediated by substance P or another similar neuropeptide, and that cough responses are modulated by endogenous neutral endopeptidase.
Assuntos
Capsaicina/farmacologia , Tosse/induzido quimicamente , Neprilisina/antagonistas & inibidores , Substância P/farmacologia , Animais , Sinergismo Farmacológico , Glicopeptídeos/farmacologia , Cobaias , Masculino , Tiorfano/análogos & derivados , Tiorfano/farmacologiaRESUMO
We examined the effects of acute exposure to cigarette smoke on the airway responses to substance P in anesthetized guinea pigs and on the activity of airway neutral endopeptidase (NEP). After exposure to air or to cigarette smoke we measured the change in total pulmonary resistance (RL) induced by increasing concentrations of aerosolized substance P in the absence or presence of the NEP inhibitor phosphoramidon. In the absence of phosphramidon the bronchoconstrictor responses to substance P were greater in cigarette smoke-exposed guinea pigs than in air-exposed animals. Phosphoramidon did not further potentiate the responses to substance P in smoke-exposed guinea pigs, whereas it did so in air-exposed animals. In the presence of phosphoramidon, bronchoconstrictor responses to substance P in animals exposed to air or to cigarette smoke were not different. Aerosols of SOD delivered before cigarette smoke exposures dramatically reduced smoke-induced hyperresponsiveness to substance P, whereas heat-inactivated SOD had no effect on smoke-induced hyper-responsiveness to substance P. Cigarette smoke solution inhibited NEP activity from tracheal homogenate in a concentration-dependent fashion, an inhibitory effect that was mostly due to the gas phase of the smoke, but not to nicotine. The mild chemical oxidant N-chlorosuccinimide mimicked the concentration-dependent inhibitory effect of smoke solution on airway NEP activity. We conclude that cigarette smoke causes enhanced airway responsiveness to substance P in vivo by inactivating airway NEP. We suggest that cigarette smoke-induced inhibition of airway NEP is due to effects of free radicals.
Assuntos
Resistência das Vias Respiratórias , Brônquios/enzimologia , Neprilisina/metabolismo , Fumar/efeitos adversos , Substância P/administração & dosagem , Resistência das Vias Respiratórias/efeitos dos fármacos , Animais , Testes de Provocação Brônquica , Capsaicina/administração & dosagem , Ativação Enzimática , Radicais Livres , Glicopeptídeos/administração & dosagem , Cobaias , Masculino , Nicotina/administração & dosagem , Soluções , Succinimidas/administração & dosagem , Superóxido Dismutase/administração & dosagemRESUMO
To investigate the hypothesis that neutrophil proteases stimulate airway gland secretion, we studied the effect of human cathepsin G and elastase on secretion of 35S-labeled macromolecules from cultured bovine airway gland serous cells. Both proteases stimulated secretion in a concentration-dependent fashion with a threshold of greater than or equal to 10(-10) M. Elastase was more potent than cathepsin G, causing a maximal secretory response of 1,810 +/- 60% over baseline at 10(-8) M. The maximal response to cathepsin G (1,810 +/- 70% over baseline at 10(-7) M) was similar to the maximal response to elastase. These responses were greater than 10-fold larger than the response to other agonists such as histamine. Protease-induced secretion was noncytotoxic and required catalytically active enzymes. The predominant sulfated macromolecule released by proteases was chondroitin sulfate proteoglycan. Immunocytochemical staining demonstrated chondroitin sulfate in cytoplasmic granules and decreased granular staining after stimulation of cells with elastase. The neutrophil proteases also degraded the proteoglycan released from serous cells. Cathepsin G and elastase in supernatant obtained by degranulation of human peripheral neutrophils also caused a secretory response. Thus, neutrophil proteases stimulate airway gland serous cell secretion of chondroitin sulfate proteoglycan and degrade the secreted product. These findings suggest a potential role for neutrophil proteases in the pathogenesis of increased and abnormal submucosal gland secretions in diseases associated with inflammation and neutrophil infiltration of the airways.
Assuntos
Catepsinas/farmacologia , Neutrófilos/enzimologia , Elastase Pancreática/farmacologia , Traqueia/metabolismo , Animais , Catepsina G , Bovinos , Degranulação Celular/efeitos dos fármacos , Células Cultivadas , Sulfatos de Condroitina/metabolismo , Glicoconjugados/metabolismo , Humanos , Serina Endopeptidases , Traqueia/efeitos dos fármacosRESUMO
In reticulocytes, the enzyme 15-lipoxygenase (15-LO) is believed to contribute to cellular differentiation, and in leukocytes and airway cells 15-LO generates inflammatory mediators. The recent availability of antibodies to 15-LO now allows us to determine which specific cells contain the enzyme, to characterize its subcellular localization, and to determine its expression at the translational level. A polyclonal antibody to recombinant human reticulocyte 15-LO was used with a standard immunofluorescent technique. In rabbit red blood cells, fluorescence appeared during the course of anemia. Early reticulocytes did not fluoresce, but more mature reticulocytes showed increased fluorescent intensity. Late reticulocytes contained little fluorescence. Among human leukocytes, only eosinophils fluoresced. In human trachea, 15-LO immunofluorescence was localized to epithelial cells, and both basal and ciliated cells fluoresced. In all cells studied, fluorescence was localized to the cytoplasm and was variable in degree among cells in each preparation. We conclude that the 15-LO of airway cells and eosinophils is immunologically related to the reticulocyte 15-LO. Furthermore, the variable fluorescence among cells (e.g., in epithelium) and during development (e.g., reticulocytes) suggests a role of 15-LO in cell growth and development.
Assuntos
Araquidonato 15-Lipoxigenase/metabolismo , Eritrócitos/enzimologia , Leucócitos/enzimologia , Pulmão/enzimologia , Animais , Araquidonato 15-Lipoxigenase/imunologia , Citoplasma/enzimologia , Eosinófilos/enzimologia , Imunofluorescência , Humanos , Coelhos , Proteínas Recombinantes/imunologia , Traqueia/enzimologiaRESUMO
Substance P and related tachykinins contribute to the airway hyperresponsiveness caused by toluene diisocyanate (TDI) in guinea pigs. Neutral endopeptidase (NEP) is an important modulator of substance P-induced responses. To test the hypothesis that exposure to TDI would increase responsiveness to substance P by inhibiting activity of this enzyme, we determined the dose of substance P required to increase pulmonary resistance by 200% above baseline (PD200) before and after administration of the pharmacologic inhibitor phosphoramidon in guinea pigs studied 1 h after a 1-h exposure to air or 3 ppm TDI. TDI exposure increased responsiveness to substance P significantly. However, phosphoramidon caused a significantly greater leftward shift of the substance P dose-response curve in air-exposed animals than it did in TDI-exposed animals, so that after phosphoramidon, mean values of PD200 in animals exposed to air or TDI did not differ. Tracheal NEP activity was significantly less after exposure to TDI than after exposure to air, whereas activity in the esophagus was the same in both groups. These results suggest that TDI exposure increases the bronchoconstrictor responsiveness of guinea pigs to substance P, in large part through inhibition of airway NEP.
Assuntos
Resistência das Vias Respiratórias/efeitos dos fármacos , Cianatos/farmacologia , Inibidores de Proteases , Substância P/farmacologia , Tolueno 2,4-Di-Isocianato/farmacologia , Animais , Endopeptidases , Esôfago/enzimologia , Glicopeptídeos/farmacologia , Cobaias , Técnicas In Vitro , Pulmão/fisiologia , Neprilisina , Traqueia/enzimologiaRESUMO
Supernatants obtained by degranulation of dog mastocytoma cells greatly increased the sensitivity and the magnitude of the contractile response of isolated dog bronchial smooth muscle to histamine. The enhanced contractile response was reversed completely by H1-receptor antagonists and was prevented by an inhibitor of tryptase (a major protease released with histamine from secretory granules of mast cells). The potentiation of histamine-induced contractions was reproduced by active tryptase in pure form. The contractions due to the combination of histamine and purified tryptase were abolished by the Ca2+ channel blockers nifedipine and verapamil. The bronchoconstricting effects of KCl and serotonin, which, like histamine, contract airway smooth muscle by a mechanism predominantly involving membrane potential-dependent Ca2+ transport, were also potentiated by tryptase. However, the contractile effects of acetylcholine, which contracts dog airway smooth muscle by a mechanism independent of Ca2+ channels, were unaffected by tryptase. These findings show a striking promotion of agonist-induced bronchial smooth muscle contraction by mast cell tryptase, via direct or indirect effects on Ca2+ channels, and the findings therefore suggest a novel potential mechanism of hyperresponsiveness in dog bronchi.
Assuntos
Resistência das Vias Respiratórias/efeitos dos fármacos , Mastócitos/enzimologia , Músculo Liso/efeitos dos fármacos , Peptídeo Hidrolases/metabolismo , Animais , Cães , Sinergismo Farmacológico , Histamina/farmacologia , Contração Muscular/efeitos dos fármacos , Nifedipino/farmacologia , Cloreto de Potássio/farmacologia , Serotonina/farmacologia , Verapamil/farmacologiaRESUMO
To determine whether recombinant enkephalinase (neutral endopeptidase, EC 3.4.24.11) prevents cough induced by exogenously applied and endogenously released neuropeptides, we measured cough responses to aerosolized solutions of substance P or of capsaicin for 2 min in random-source guinea pigs before or after exposing them to aerosolized recombinant human enkephalinase. Substance P (10(-16) M) increased coughing compared with its vehicle. Enkephalinase (120 micrograms) inhibited cough induced by subsequent exposure to substance P compared with the response to substance P alone, but after further exposure to the enkephalinase inhibitor leucine-thiorphan (10(-5) M), substance P increased cough significantly. Similar results were obtained for capsaicin-induced cough. In pathogen-free guinea pigs, after they inhaled inactive recombinant enkephalinase (33 micrograms), capsaicin (10(-13) M) increased cough significantly. In contrast, after they inhaled active recombinant enkephalinase (33 micrograms), capsaicin increased cough only slightly. These results suggest that aerosolized enkephalinase reaches the sites of release or actions of endogenous neuropeptides and, by degrading them, prevents cough induced by their release. Furthermore, these studies suggest that recombinant enkephalinase might be useful in the treatment of cough and other symptoms of diseases involving peptides cleaved by this enzyme.
Assuntos
Antitussígenos/farmacologia , Tosse/prevenção & controle , Neprilisina/farmacologia , Taquicininas , Aerossóis , Animais , Antitussígenos/administração & dosagem , Capsaicina , Cobaias , Humanos , Lipossomos , Masculino , Neprilisina/administração & dosagem , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologia , Cloreto de Sódio , Substância PRESUMO
Because high concentrations of IL-8 are found in the sputum of cystic fibrosis patients, we hypothesized that Pseudomonas aeruginosa (PA) induces the production of IL-8 in airway epithelial cells and in monocytes. Therefore, we incubated the supernatant from PA culture with human transformed bronchial epithelial cells (16-HBE) or with monocytes. The culture medium of 16-HBE cells that had been incubated with PA supernatant for 6 h had chemotactic activity that was inhibited by an antibody to human IL-8. The PA supernatant induced IL-8 production by primary bronchial epithelial cells, by 16-HBE cells, and by monocytes. After incubation with PA supernatant, 16-HBE cells showed a marked increase in the levels of IL-8 gene expression. The PA product responsible for IL-8 production resisted freezing, boiling, and proteolysis. This product was not lipid extractable and was present in a 1-kD filtrate. We conclude that a small molecular mass product of PA stimulates IL-8 production by 16-HBE cells and by monocytes, and that the chemotactic activity produced by 16-HBE cells after exposure to PA is due principally to IL-8.
Assuntos
Brônquios/metabolismo , Quimiotaxia de Leucócito , Interleucina-8/biossíntese , Monócitos/fisiologia , Neutrófilos/fisiologia , Pseudomonas aeruginosa/fisiologia , Northern Blotting , Linhagem Celular , Linhagem Celular Transformada , Células Cultivadas , Meios de Cultura , Epitélio/metabolismo , Humanos , Pseudomonas aeruginosa/classificação , Pseudomonas aeruginosa/isolamento & purificação , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Especificidade da EspécieRESUMO
We studied the metabolism of sulfated cell-surface macromolecules in dog tracheal epithelial cells in primary culture. To examine the time-course and rate of appearance of sulfated macromolecules at the cell surface, the cells were pulsed with 35SO4 for short periods (5-15 min), and the incubation medium was sampled for spontaneously released macromolecules (basal secretions) and for release induced by trypsin (trypsin-accessible secretions). Trypsin-accessible 35S-labeled macromolecules appeared on the cell surface within 5-10 min, increased linearly, and plateaued by 40 min; the median transit time for 35S-labeled macromolecules to reach the cell surface was 21 min. 35S-labeled macromolecules in basal secretions increased with a similar time-course, reaching a plateau by 40 min. Incorporation of [3H]serine into the protein moiety of trypsin-accessible macromolecules occurred more slowly; trypsin-accessible 3H-labeled macromolecules were barely detectable at 1 h and increased to a maximum after 2 h, suggesting the presence of a preformed pool of nonsulfated core protein. Pretreatment with cycloheximide, an inhibitor of protein synthesis, decreased trypsin-accessible 35S-labeled macromolecules log-linearly depending on the duration of pretreatment providing an estimate of the rate of depletion of the core protein pool (t1/2 = 32 min). During continuous exposure to 35SO4, 35S-labeled macromolecules accumulated on the cell surface (trypsin-accessible compartment) for 16 h, at which point the cell-surface pool was saturated (t1/2 = 7.5 h). After pulse-labeling the cells with 35SO4 for 15 min, the 35S-labeled macromolecules disappeared continuously from the cell surface (t1/2 = 4.6 h), and 79% of the radioactivity was recovered in the medium as nondialyzable macromolecules. Release of the 35S-labeled macromolecules from the cell surface was abolished at 4 degrees C, indicative of an energy-dependent process, but multiple proteinase inhibitors did not affect the release. We conclude that sulfate is metabolized rapidly into epithelial cell-surface macromolecules, which accumulate continuously into a relatively large cell-surface pool, before they are released by an undefined energy-dependent mechanism.
Assuntos
Membrana Celular/metabolismo , Traqueia/metabolismo , Animais , Membrana Celular/enzimologia , Células Cultivadas , Cães , Células Epiteliais , Epitélio/enzimologia , Epitélio/metabolismo , Cinética , Substâncias Macromoleculares , Proteínas de Membrana/metabolismo , Inibidores de Proteases , Serina/metabolismo , Sulfatos/metabolismo , Radioisótopos de Enxofre , Temperatura , Traqueia/citologia , Traqueia/enzimologia , TripsinaRESUMO
We isolated and characterized a chymotryptic serine proteinase from dog mastocytomas. Chymotryptic activity extracted at high ionic strength from mastocytomas propagated in nude mice was separated from tryptic activity by gel filtration and rapidly purified by sequential high-performance hydrophobic interaction and cation-exchange chromatography. The purified enzyme had an Mr of 27,000-30,000 by both analytical gel filtration and SDS-polyacrylamide gel electrophoresis, and a single amino-terminal sequence by automated Edman degradation. Like chymases from rat and human mast cells, the mastocytoma enzyme exhibited a high kcat/Km (1.1.10(5) M-1.s-1) employing succinyl-L-Val-Pro-Phe-p-nitroanilide, the best of several p-nitroanilide substrates screened. It was inhibited by diisopropyl fluorophosphate and soybean trypsin inhibitor, but not by aprotinin, distinguishing it from the otherwise closely related neutrophil enzyme, cathepsin G. The amino-terminal 25 residues of mastocytoma chymase were found to be 72 and 68% identical to the corresponding sequences of chymases from rat peritoneal and mucosal mast cells, respectively; they were also closely related to human cathepsin G and to proteinase sequences from mouse cytotoxic T-lymphocytes. The mastocytoma chymotryptic enzyme contained an octapeptide sequence which is common to all chymotryptic leukocyte proteinases sequenced to date from four mammalian species; this feature distinguishes chymases and other chymotryptic leukocyte proteinases from serine proteinases of coagulation and digestion.