RESUMO
BACKGROUND: Sputum eosinophil percentages are a strong predictor of airway inflammation and exacerbations and aid asthma management, whereas sputum neutrophil percentages indicate a different severe asthma phenotype that is potentially less responsive to TH2-targeted therapy. Variables, such as blood eosinophil counts, total IgE levels, fraction of exhaled nitric oxide (Feno) levels, or FEV1 percent predicted, might predict airway eosinophil percentages, whereas age, FEV1 percent predicted, or blood neutrophil counts might predict sputum neutrophil percentages. Availability and ease of measurement are useful characteristics, but accuracy in predicting airway eosinophil and neutrophil percentages either individually or combined is not established. OBJECTIVES: We sought to determine whether blood eosinophil counts, Feno levels, and IgE levels accurately predict sputum eosinophil percentages and whether age, FEV1 percent predicted, and blood neutrophil counts accurately predict sputum neutrophil percentages. METHODS: Subjects in the Wake Forest Severe Asthma Research Program (n = 328) were characterized by blood and sputum cell counts, health care use, lung function, Feno levels, and IgE levels. Multiple analytic techniques were used. RESULTS: Despite significant association with sputum eosinophil percentages, blood eosinophil counts, Feno levels, and total IgE levels did not accurately predict sputum eosinophil percentages, and combinations of these variables did not improve prediction. Age, FEV1 percent predicted, and blood neutrophil counts were similarly unsatisfactory for the prediction of sputum neutrophil percentages. Factor analysis and stepwise selection found Feno levels, IgE levels, and FEV1 percent predicted, but not blood eosinophil counts, correctly predicted 69% of sputum eosinophil percentages of less than 2% or 2% and greater. Likewise, age, asthma duration, and blood neutrophil counts correctly predicted 64% of sputum neutrophil percentages of less than 40% or 40% and greater. A model to predict both sputum eosinophil and neutrophil percentages accurately assigned only 41% of samples. CONCLUSION: Despite statistically significant associations, Feno levels, IgE levels, blood eosinophil and neutrophil counts, FEV1 percent predicted, and age are poor surrogates, both separately and combined, for accurately predicting sputum eosinophil and neutrophil percentages.
Assuntos
Asma/imunologia , Eosinófilos/fisiologia , Neutrófilos/fisiologia , Escarro/citologia , Adulto , Asma/fisiopatologia , Biomarcadores , Feminino , Volume Expiratório Forçado , Humanos , Imunoglobulina E/sangue , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Curva ROCRESUMO
Inhaled ß-agonists are effective airway smooth muscle (ASM)-relaxing agents that help reverse bronchoconstriction in asthma, but their ability to affect the aberrant ASM growth that also occurs with asthma is poorly understood. ß-Agonists exhibit PKA-dependent antimitogenic effects in several cell types. However, recent studies suggest that Epac, and not PKA, mediates the antimitogenic effect of cAMP in both ASM and fibroblasts. This study aims to clarify the role of PKA in mediating the effect of G(s)-coupled receptors on human ASM growth. Pretreatment of ASM cultures with ß-agonists albuterol, isoproterenol, or salmeterol (100 nM to 10 µM) caused a significant (â¼ 25-30%) inhibition of EGF-stimulated ASM thymidine incorporation and cell proliferation, whereas a much greater inhibition was observed from pretreatment with PGE(2) (75-80%). However, all agents were ineffective in cells expressing GFP chimeras of either PKI (a PKA inhibitor) or a mutant PKA regulatory subunit relative to the control cells expressing GFP. The antimitogenic efficacy of PGE(2) in inhibiting control cultures was associated with greater ability to stimulate sustained PKA activation and greater inhibition of late-phase promitogenic p42/p44 and PI3K activities. These findings suggest that therapeutic approaches enabling superior PKA activation in ASM will be most efficacious in deterring ASM growth.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Proliferação de Células/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dinoprostona/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Albuterol/análogos & derivados , Albuterol/farmacologia , Broncodilatadores/farmacologia , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Ativação Enzimática/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular , Isoproterenol/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Músculo Liso/citologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Miócitos de Músculo Liso/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Xinafoato de Salmeterol , Fatores de Tempo , Traqueia/citologia , TransfecçãoRESUMO
BACKGROUND: Investigative bronchoscopy was performed in a subset of participants in the Severe Asthma Research Program to gain insights into the pathobiology of severe disease. We evaluated the safety aspects of this procedure in this cohort with specific focus on patients with severe asthma. OBJECTIVE: To evaluate prospectively changes in lung function and the frequency of adverse events related to investigative bronchoscopy. METHODS: Bronchoscopy was performed by using a common manual of procedures. A subset of very severe asthma was defined by severe airflow obstruction, chronic oral corticosteroid use, and recent asthma exacerbations. Subjects were monitored for changes in lung function and contacted by telephone for 3 days after the procedure. RESULTS: A total of 436 subjects underwent bronchoscopy (97 normal, 196 not severe, 102 severe, and 41 very severe asthma). Nine subjects were evaluated in hospital settings after bronchoscopy; 7 of these were respiratory-related events. Recent emergency department visits, chronic oral corticosteroid use, and a history of pneumonia were more frequent in subjects who had asthma exacerbations after bronchoscopy. The fall in FEV1 after bronchoscopy was similar in the severe and milder asthma groups. Prebronchodilator FEV1 was the strongest predictor of change in FEV1 after bronchoscopy with larger decreases observed in subjects with better lung function. CONCLUSION: Bronchoscopy in subjects with severe asthma was well tolerated. Asthma exacerbations were rare, and reduction in pulmonary function after the procedure was similar to that in subjects with less severe asthma. With proper precautions, investigative bronchoscopy can be performed safely in severe asthma.
Assuntos
Asma/fisiopatologia , Broncoscopia/efeitos adversos , Adolescente , Adulto , Asma/etiologia , Asma/patologia , Brônquios/patologia , Brônquios/fisiopatologia , Testes de Provocação Brônquica , Broncoscopia/métodos , Feminino , Volume Expiratório Forçado , Humanos , Masculino , Testes de Função Respiratória , Índice de Gravidade de Doença , Adulto JovemRESUMO
Irreversible airflow obstruction or limitation occurs in some patients with asthma, can develop early in life, and becomes more common as asthma becomes more severe. Efforts to understand irreversible airflow obstruction or limitation have been hampered by the lack of a standardized definition of the phenotype and by the lack of appropriate research models. Unfortunately, it appears that currently available asthma treatments do not prevent this important asthma complication. Herein, the evidence of an irreversible component of asthma, its underlying pathology, and the limitations of current asthma treatments are reviewed.
Assuntos
Asma/complicações , Asma/fisiopatologia , Doença Pulmonar Obstrutiva Crônica/etiologia , Corticosteroides/uso terapêutico , Antiasmáticos/uso terapêutico , Asma/tratamento farmacológico , Ensaios Clínicos como Assunto , Humanos , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológicoRESUMO
Glucocorticoids (GCs) and protein kinase A (PKA)-activating agents (beta-adrenergic receptor agonists) are mainstream asthma therapies based on their ability to prevent or reverse excessive airway smooth muscle (ASM) constriction. Their abilities to regulate another important feature of asthma--excessive ASM growth--are poorly understood. Recent studies have suggested that GCs render agents of inflammation such as IL-1 beta and TNF-alpha mitogenic to ASM, via suppression of (antimitogenic) induced cyclooxygenase-2-dependent PKA activity. To further explore the mechanistic basis of these observations, we assessed the effects of epidermal growth factor and IL-1 beta stimulation, and the modulatory effects of GC treatment and PKA inhibition, on the ASM transcriptome by microarray analysis. Results demonstrate that ASM stimulated with IL-1 beta, in a manner that is often cooperative with stimulation with epidermal growth factor, exhibit a profound capacity to function as immunomodulatory cells. Moreover, results implicate an important role for induced autocrine/paracrine factors (many whose regulation was minimally affected by GCs or PKA inhibition) as regulators of both airway inflammation and ASM growth. Induction of numerous chemokines, in conjunction with regulation of proteases and agents of extracellular matrix remodeling, is suggested as an important mechanism promoting upregulated G protein-coupled receptor signaling capable of stimulating ASM growth. Additional functional assays suggest that intracellular PKA plays a critical role in suppressing the promitogenic effects of induced autocrine factors in ASM. Finally, identification and comparison of GC- and PKA-sensitive genes in ASM provide insight into the complementary effects of beta-agonist/GC combination therapies, and suggest specific genes as important targets for guiding the development of new generations of GCs and adjunct asthma therapies.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Androstadienos/farmacologia , Antiasmáticos/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Glucocorticoides/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Traqueia/efeitos dos fármacos , Comunicação Autócrina/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Análise por Conglomerados , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/genética , Fator de Crescimento Epidérmico/metabolismo , Fluticasona , Perfilação da Expressão Gênica/métodos , Humanos , Interleucina-1beta/metabolismo , Miócitos de Músculo Liso/enzimologia , Análise de Sequência com Séries de Oligonucleotídeos , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/genética , Fatores de Tempo , Traqueia/enzimologia , TransfecçãoRESUMO
Pulmonary rehabilitation is a core component of management of patients with chronic lung disease that have exercise or functional limitations. Causes of these limitations are manifold but include loss of skeletal muscle mass, power and endurance, diminished respiratory capacity owing to respiratory muscle weakness, inefficient gas exchange, and increased work of breathing, and impaired cardiovascular functioning. Besides physical limitations, patients with chronic lung disease have high rates of depression and anxiety leading to social isolation and increased health care use. Pulmonary rehabilitation uses a comprehensive and holistic approach that has been shown to ameliorate most effects of chronic lung disease.
Assuntos
Terapia por Exercício/métodos , Doença Pulmonar Obstrutiva Crônica/reabilitação , Assistência Ambulatorial , Terapia por Exercício/educação , Tolerância ao Exercício , Humanos , Seleção de Pacientes , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , AutocuidadoRESUMO
Although G protein-coupled receptor (GPCR) kinases (GRKs) have been shown to mediate desensitization of numerous GPCRs in studies using cellular expression systems, their function under physiological conditions is less well understood. In the current study, we employed various strategies to assess the effect of inhibiting endogenous GRK2/3 on signaling and function of endogenously expressed G s-coupled receptors in human airway smooth muscle (ASM) cells. GRK2/3 inhibition by expression of a Gbetagamma sequestrant, a GRK2/3 dominant-negative mutant, or siRNA-mediated knockdown increased intracellular cAMP accumulation mediated via beta-agonist stimulation of the beta-2-adrenergic receptor (beta 2AR). Conversely, neither 5'-( N-ethylcarboxamido)-adenosine (NECA; activating the A2b adenosine receptor) nor prostaglandin E2 (PGE 2; activating EP2 or EP4 receptors)-stimulated cAMP was significantly increased by GRK2/3 inhibition. Selective knockdown using siRNA suggested the majority of PGE 2-stimulated cAMP in ASM was mediated by the EP2 receptor. Although a minor role for EP3 receptors in influencing PGE 2-mediated cAMP was determined, the GRK2/3-resistant nature of EP2 receptor signaling in ASM was confirmed using the EP2-selective agonist butaprost. Somewhat surprisingly, GRK2/3 inhibition did not augment the inhibitory effect of the beta-agonist on mitogen-stimulated increases in ASM growth. These findings demonstrate that with respect to G s-coupled receptors in ASM, GRK2/3 selectively attenuates beta 2AR signaling, yet relief of GRK2/3-dependent beta 2AR desensitization does not influence at least one important physiological function of the receptor.
Assuntos
Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Quinase 3 de Receptor Acoplado a Proteína G/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Músculo Liso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/genética , Quinase 3 de Receptor Acoplado a Proteína G/genética , Humanos , Músculo Liso/enzimologia , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
Excessive smooth muscle growth occurs within the context of inflammation associated with certain vascular and airway diseases. The inflammatory cytokines interleukin (IL)-1beta and tumor necrosis factor-alpha (TNF-alpha) have been shown previously to inhibit mitogen-stimulated smooth muscle growth through a mechanism presumed to be dependent on the induction of cyclooxygenase-2, prostaglandins, and activation of the cAMP-dependent protein kinase (PKA). Using both molecular and pharmacological strategies, we demonstrate that the mitogenic effects of IL-1beta and TNF-alpha on cultured human airway smooth muscle (ASM) cells are tightly regulated by PKA activity. Suppression of induced PKA activity by either corticosteroids or cyclooxygenase inhibitors converts the cytokines from inhibitors to enhancers of mitogen-stimulated ASM growth, and biological variability in the capacity to activate PKA influences the modulatory effect of cytokines. Promitogenic effects of IL-1beta are associated with delayed increases in p42/p44 and phosphoinositide-3 kinase activities, suggesting a role for induced autocrine factors. These findings suggest a mechanism by which mainstream therapies such as corticosteroids or cyclooxygenase inhibitors could fail to address or exacerbate the pathogenic smooth muscle growth that occurs in obstructive airway and cardiovascular diseases.
Assuntos
Corticosteroides/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Inibidores de Ciclo-Oxigenase/farmacologia , Citocinas/fisiologia , Mitógenos/fisiologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/genética , Citocinas/genética , Humanos , Interleucina-1beta/genética , Interleucina-1beta/fisiologia , Mitógenos/genéticaRESUMO
Leukotrienes are important lipid mediators of asthma that contribute to airway inflammation and bronchoconstriction. Critical mechanisms for physiological regulation of the main G protein-coupled receptor (GPCR) mediating the leukotriene responses in asthma, cysteinyl leukotriene type 1 receptor (CysLT1R), have not been delineated. Although desensitization of GPCRs is a well-established phenomenon, studies demonstrating its physiological relevance are lacking. Here, we demonstrate that relief of PKC-mediated desensitization of endogenous CysLT1Rs augments multiple LTD4-stimulated cellular functions, with associated increases in intracellular signaling events. In analyses of airway smooth muscle contraction ex vivo, PKC inhibition augmented LTD4-stimulated contraction, and increased phosphoinositide hydrolysis and calcium flux in both murine and human airway smooth muscle cells. Similarly, for human monocytes, PKC inhibition augmented LTD4-stimulated calcium flux and cell migration assessed in transwell chamber experiments and also augmented LTD4-induced production of monocyte chemotactic protein assessed by ELISA. In contrast, PKC inhibition had no effect or slightly attenuated these cell functions and signaling events promoted by other receptor agonists, suggesting that despite antithetical effects on downstream events, desensitization of the CysLT1R is the principal mechanism by which PKC regulates the functional consequences of CysLT1R activation.
Assuntos
Proteínas de Membrana/fisiologia , Contração Muscular/fisiologia , Proteína Quinase C/metabolismo , Receptores de Leucotrienos/fisiologia , Animais , Cálcio/sangue , Quimiocina CCL2/fisiologia , Quimiotaxia de Leucócito/fisiologia , Humanos , Inalação/fisiologia , Leucotrienos/fisiologia , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Monócitos/fisiologia , Músculo Liso/fisiologia , Fosfatidilinositóis/metabolismo , Reação em Cadeia da Polimerase , Receptores de Leucotrienos/genética , Traqueia/fisiologiaAssuntos
Asma/tratamento farmacológico , Asma/genética , Aldeído Oxirredutases/metabolismo , Antibacterianos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Asma/complicações , Asma/imunologia , Ensaios Clínicos como Assunto , Surtos de Doenças , Suscetibilidade a Doenças , Refluxo Gastroesofágico/complicações , Refluxo Gastroesofágico/tratamento farmacológico , Humanos , Imunomodulação , Vírus da Influenza A Subtipo H1N1 , Vacinas contra Influenza , Influenza Humana/epidemiologia , Interleucina-5/antagonistas & inibidores , Guias de Prática Clínica como Assunto , Inibidores da Bomba de Prótons/uso terapêutico , Células Th2/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Vitamina D/sangueRESUMO
OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death in the United States and is estimated to be responsible for 119,000 deaths in the year 2000 alone. Additionally, COPD places a tremendous burden on the health care system, with estimated annual costs of US 24 billion dollars in 2000, and it is generally expected that costs will continue to rise as more individuals are diagnosed. COPD was responsible for approximately 8 million physician outpatient visits, 1.5 million emergency department visits and 726,000 hospitalizations, also in the year 2000. The objective of this article is to review current, pertinent clinical issues in the management of patients with COPD, with estimates of their relative utility and efficacy. SUMMARY: COPD is a disease characterized by airflow limitation that is not fully reversible. Patients with COPD may frequently experience symptoms of chronic cough with sputum production, dyspnea, and reduced exercise capacity. They may frequently experience exacerbations characterized by increased symptoms that often require medical intervention. The diagnosis of COPD is usually fairly straightforward and made in a cigarette smoker, with the aforementioned symptoms and airflow obstruction measured by spirometry. Spirometry should be performed in all patients in whom COPD is suspected, as it provides useful prognostic information and may be used to stage the disease. The Global Initiative for Chronic Obstructive Lung Disease (GOLD) has provided evidenced-based management guidelines for COPD. GOLD guidelines advocate staging COPD by spirometry and make specific treatment recommendations based on COPD stage. The most important risk factor for the development of COPD is cigarette smoking, and smoking cessation has been shown to reduce all-cause mortality and to alter the natural history of COPD. Smoking cessation strategies that employ both counseling and medications like buproprion and nicotine replacement are most effective, but relapse rates remain high. It has not been shown that medications like bronchodilators or inhaled steroids change the natural history of COPD, nor do they reduce mortality, but they can affect other important outcomes. Long-acting bronchodilators, including beta- 2-adrenergic agonists such as salmeterol and formoterol, and the anticholinergic agent tiotropium, improve lung function and exercise tolerance, reduce symptoms, and modestly reduce exacerbation rates. Long-acting bronchodilators are indicated for all COPD patients with chronic symptoms. Short-acting bronchodilators are indicated for rescue when acute symptoms occur. Inhaled corticosteroids minimally improve lung function, but, importantly, reduce exacerbation rates and are indicated in severe COPD or when exacerbations are frequent. Continuous oxygen therapy has been shown to reduce mortality when severe hypoxemia is present and can improve quality of life when moderate hypoxia is present. Finally, well-designed, multidisciplinary disease management programs and pulmonary rehabilitation can improve important disease outcomes in a cost-effective manner. CONCLUSION: COPD is a common, preventable disease that affects a significant number of people. It may be managed by utilizing various readily available medical therapies, as well as other nonpharmacologic interventions, such as pulmonary rehabilitation. Proper coordination of care is important in this disease, and efforts should be focused on improving quality of life and reduction of symptoms.
Assuntos
Assistência ao Paciente/métodos , Doença Pulmonar Obstrutiva Crônica/terapia , Educação Continuada/métodos , Humanos , Assistência ao Paciente/economia , Assistência ao Paciente/tendências , Guias de Prática Clínica como Assunto , Doença Pulmonar Obstrutiva Crônica/economiaRESUMO
Asthma is a common disease encountered in internal medicine practice. In most cases, the diagnosis and management of asthma is straightforward; however, the management of severe asthma may require subspecialty consultation. Abnormal responses of resident cells to infections and antigens may cause asthma in early life and may chronically perpetuate asthma. The natural history of asthma remains poorly understood. In this article, the authors focus on the diagnosis and management of asthma in adults.
Assuntos
Asma/diagnóstico , Asma/terapia , Antiasmáticos/uso terapêutico , Asma/tratamento farmacológico , Asma/fisiopatologia , Asma/prevenção & controle , Gerenciamento Clínico , Progressão da Doença , Humanos , Testes de Função Respiratória , Medição de Risco , Espirometria , Linfócitos T/fisiologiaRESUMO
Current asthma treatments are effective for most but not all patients. Asthma is classified as a complex genetic disease with its pathogenesis and expression (severity) determined by the interaction of many genes and environmental factors. Asthma is characterized by its heterogeneity in terms of its clinical and inflammatory phenotypes and their responses to therapy. This disease heterogeneity likely has played a role in variable results from clinical trials that evaluate specific inhibitors of inflammatory mediators ('biologics') in asthma. Moreover, although existing treatments are effective and safe in most asthma patients they may be less effective or potentially harmful in others. In addition, if an individual with asthma is less responsive to standard therapies such as corticosteroids because of specific pharmacogenetic interactions then that patient with asthma will be classified as having more severe or difficult-to-treat asthma. Pharmacogenetic approaches hold the promise of matching individualized treatments to specific genotypes in a way that minimizes side effects while improving therapeutic outcomes.
Assuntos
Antiasmáticos/farmacologia , Asma/tratamento farmacológico , Farmacogenética , Antiasmáticos/efeitos adversos , Asma/genética , Asma/fisiopatologia , Ensaios Clínicos como Assunto , Glucocorticoides/efeitos adversos , Glucocorticoides/farmacologia , Humanos , Medicina de Precisão/métodos , Índice de Gravidade de DoençaRESUMO
Exposure of airway smooth muscle (ASM) cells to the cytokine IL-1beta results in an induction of PGE2 synthesis that affects numerous cell functions. Current dogma posits induction of COX-2 protein as the critical, obligatory event in cytokine-induced PGE2 production, although PGE2 induction can be inhibited without a concomitant inhibition of COX-2. To explore other putative regulatory features we examined the role of phospholipase A2 (PLA2) and PGE synthase (PGES) enzymes in IL-1beta-induced PGE2 production. Treatment of human ASM cultures with IL-1beta caused a time-dependent induction of both cytosolic PLA2 (cPLA2) and microsomal PGES (mPGES) similar to that observed for COX-2. Regulation of COX-2 and mPGES induction was similar, being significantly reduced by inhibition of p42/p44 or p38, whereas cPLA2 induction was only minimally reduced by inhibition of p38 or PKC. COX-2 and mPGES induction was subject to feed-forward regulation by PKA, whereas cPLA2 induction was not. SB-202474, an SB-203580 analog lacking the ability to inhibit p38 but capable of inhibiting IL-1beta-induced PGE2 production, was effective in inhibiting mPGES but not COX-2 or cPLA2 induction. These data suggest that although COX-2, cPLA2, and mPGES are all induced by IL-beta in human ASM cells, regulatory features of cPLA2 are dissociated, whereas those of COX-2 and mPGES are primarily associated, with regulation of PGE2 production. mPGES induction and, possibly, cPLA2 induction appear to cooperate with COX-2 to determine IL-1beta-mediated PGE2 production in human ASM cells.
Assuntos
Dinoprostona/biossíntese , Interleucina-1/farmacologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Traqueia/efeitos dos fármacos , Traqueia/metabolismo , Células Cultivadas , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Citosol/enzimologia , Inibidores Enzimáticos/farmacologia , Humanos , Microssomos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Músculo Liso/citologia , Fosfolipases A/metabolismo , Fosfolipases A2 , Prostaglandina-Endoperóxido Sintases/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Traqueia/citologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Airway inflammation, airflow obstruction, and bronchial hyperresponsiveness are characteristic phenotypic features of asthma. Clinically, airflow obstruction in asthma often is not fully reversible, and many asthmatic subjects experience an accelerated and progressive loss of lung function over time. Histopathologic studies of the asthmatic airway have demonstrated stereotypic changes that might explain the loss of lung function that many patients with asthma experience. The notion of airway remodeling in asthma postulates that the alteration of the structure and function of key airway constituents, including airway smooth muscle, epithelium, blood vessels, and mucus glands, might explain, at least in part, the progressive loss of lung function that is observed clinically. Inflammation driven by CD4(+) lymphocytes and mediated by effector cells, particularly the eosinophil, appears to modulate the function of mesenchymal cells, including fibroblasts and myofibroblasts, changing the composition of the airway wall matrix. Changes in the airway epithelium might alter the function of the underlying smooth muscle and the composition of the matrix and could drive inflammation. Alterations in the structure and function of airway smooth muscle change the mechanical properties of the airway wall and might also affect the function of other airway constituents. A variety of experimental models have identified candidate mechanisms and mediators for these observed changes, which are thus potential therapeutic targets. However, clinical studies to date have been disappointing, and it remains to be seen whether targeted therapies will prevent the progressive loss of lung function seen in asthma.
Assuntos
Asma/fisiopatologia , Pulmão/fisiopatologia , Animais , Humanos , Inflamação/fisiopatologia , Músculo Liso/fisiopatologia , Mucosa Respiratória/fisiopatologiaRESUMO
Cysteinyl leukotrienes activate the cysteinyl leukotriene type 1 receptor (CysLT1R) to regulate numerous cell functions important in inflammatory processes and diseases such as asthma. Despite its physiologic importance, no studies to date have examined the regulation of CysLT1R signaling or trafficking. We have established model systems for analyzing recombinant human CysLT1R and found regulation of internalization and signaling of the CysLT1R to be unique among G protein-coupled receptors. Rapid and profound LTD4-stimulated internalization was observed for the wild type (WT) CysLT1R, whereas a C-terminal truncation mutant exhibited impaired internalization yet signaled robustly, suggesting a region within amino acids 310-321 as critical to internalization. Although overexpression of WT arrestins significantly increased WT CysLT1R internalization, expression of dominant-negative arrestins had minimal effects, and WT CysLT1R internalized in murine embryonic fibroblasts lacking both arrestin-2 and arrestin-3, suggesting that arrestins are not the primary physiologic regulators of CysLT1Rs. Instead, pharmacologic inhibition of protein kinase C (PKC) was shown to profoundly inhibit CysLT1R internalization while greatly increasing both phosphoinositide (PI) production and calcium mobilization stimulated by LTD4 yet had almost no effect on H1 histamine receptor internalization or signaling. Moreover, mutation of putative PKC phosphorylation sites within the CysLT1R C-tail (CysLT1RS(313-316)A) reduced receptor internalization, increased PI production and calcium mobilization by LTD4, and significantly attenuated the effects of PKC inhibition. These findings characterized the CysLT1R as the first G protein-coupled receptor identified to date in which PKC is the principal regulator of both rapid agonist-dependent internalization and rapid agonist-dependent desensitization.
Assuntos
Proteínas de Membrana/fisiologia , Receptores de Leucotrienos/fisiologia , Sequência de Aminoácidos , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Humanos , Rim , Cinética , Leucotrieno D4/farmacologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Fases de Leitura Aberta , Conformação Proteica , Transporte Proteico , Receptores de Leucotrienos/genética , Receptores de Leucotrienos/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
Beta2AR desensitization in airway smooth muscle (ASM) mediated by airway inflammation has been proposed to contribute to asthma pathogenesis and diminished efficacy of beta-agonist therapy. Mechanistic insight into this phenomenon is largely conceptual and lacks direct empirical evidence. Here, we employ molecular and genetic strategies to reveal mechanisms mediating cytokine effects on ASM beta2AR responsiveness. Ectopic expression of inhibitory peptide (PKI-GFP) or a mutant regulatory subunit of PKA (RevAB-GFP) effectively inhibited intracellular PKA activity in cultured human ASM cells and enhanced beta2AR responsiveness by mitigating both agonist-specific (beta-agonist-mediated) desensitization and cytokine (IL-1beta and TNF-alpha)-induced heterologous desensitization via actions on multiple targets. In the absence of cytokine treatment, PKA inhibition increased beta2AR-mediated signaling by increasing both beta2AR-G protein coupling and intrinsic adenylyl cyclase activity. PKI-GFP and RevAB-GFP expression also conferred resistance to cytokine-promoted beta2AR-G protein uncoupling and disrupted feed-forward mechanisms of PKA activation by attenuating the induction of COX-2 and PGE2. Cytokine treatment of tracheal ring preparations from wild-type mice resulted in a profound loss of beta-agonist-mediated relaxation of methacholine-contracted rings, whereas rings from EP2 receptor knockout mice were largely resistant to cytokine-mediated beta2AR desensitization. These findings identify EP2 receptor- and PKA-dependent mechanisms as the principal effectors of cytokine-mediated beta2AR desensitization in ASM.