RESUMO
In the diaphragms of chronic obstructive pulmonary disease (COPD) patients, the nature of oxidatively modified proteins and superoxide anion production were explored. Diaphragm specimens were obtained through thoracotomy because of localised lung lesions in COPD patients (16 severe and eight moderate) and 10 control subjects. Lung and respiratory muscle functions were evaluated. Oxidised proteins were identified using immunoblotting and mass spectrometry. Protein and activity levels of the identified proteins were determined using immunoblotting and activity assays. Lucigenin-derived chemiluminescence signals in a luminometer were used to determine superoxide anion levels in muscle compartments (mitochondria, membrane and cytosol) using selective inhibitors. In severe COPD patients compared with controls, respiratory muscle function was impaired; creatine kinase, carbonic anhydrase III, actin and myosin were oxidised; myosin carbonylation levels were increased five-fold; creatine kinase content and activity and myosin protein were reduced; superoxide anion levels were increased in both mitochondria and membrane compartments; and the percentage of superoxide anion inhibition achieved by rotenone was significantly greater. In severe COPD patients, oxidation of diaphragm proteins involved in energy production and contractile performance is likely to partially contribute to the documented respiratory muscle dysfunction. Furthermore, generation of the superoxide anion was increased in the diaphragms of these patients.
Assuntos
Diafragma/metabolismo , Proteínas Musculares/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Superóxidos/metabolismo , Actinas/metabolismo , Idoso , Dióxido de Carbono/metabolismo , Estudos de Casos e Controles , Creatina Quinase/metabolismo , Diafragma/fisiopatologia , Diafragma/cirurgia , Humanos , Immunoblotting , Luminescência , Neoplasias Pulmonares/cirurgia , Masculino , Espectrometria de Massas , Miosinas/metabolismo , Oxirredução , Estresse Oxidativo , Carbonilação Proteica , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Índice de Gravidade de Doença , ToracotomiaRESUMO
Angiopoietin 1 (Ang-1) is the main ligand for endothelial cell-specific tyrosine kinase (Tie-2) receptors and it promotes migration and proliferation and inhibits apoptosis and vascular leakage. The exact mechanisms through which the Ang-1 exerts these effects remain unclear. The authors exposed human umbilical vein endothelial cells (HUVECs) to Ang-1 (300 ng/mL) for 4 h and conducted gene expression profiling using oligonucleotide microarrays. Real-time polymerase chain reaction (PCR) was also conducted to verify several of the genes that were regulated by Ang-1. Exposure to Ang-1 resulted in induction of 86 genes that are involved in endothelial cell (EC) proliferation, differentiation, migration, and survival. Thirty-six of these genes, including stanniocalcin, cyclin D1, vascular endothelial growth factor C, fms-related tyrosine kinase 1, interleukin 8, and CXCR4 have previously been shown to be induced by vascular endothelial growth factor (VEGF), suggesting significant similarities between VEGF and Ang-1 pathways. Ang-1 exposure also inhibited mRNA expressions of 49 genes, most of which are involved in cell cycle arrest, apoptosis, and suppression of transcription. These results indicate that Ang-1 triggers coordinated responses in endothelial cells designed to inhibit the expression of proapoptotic and antiproliferative genes and up-regulate proproliferative, proangiogenic, and antiapoptotic pathways. Moreover, we also found that the Erk1/2, phosphatidylinositol (PI) 3-kinase, and the mTOR pathways are involved in Ang-1-induced gene expression in HUVECs.
Assuntos
Angiopoietina-1/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Perfilação da Expressão Gênica , Veias Umbilicais/efeitos dos fármacos , Veias Umbilicais/metabolismo , Androstadienos/farmacologia , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Células Endoteliais/enzimologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Flavonoides/farmacologia , Redes Reguladoras de Genes , Humanos , Interleucina-8/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação/efeitos dos fármacos , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Veias Umbilicais/enzimologia , Regulação para Cima/efeitos dos fármacos , WortmaninaRESUMO
We hypothesized that resistive breathing of moderate to high intensity might increase diaphragm oxidative stress, which could be partially attenuated by antioxidants. Our objective was to assess the levels of oxidative stress in the dog diaphragm after respiratory muscle training of a wide range of intensities and whether N-acetyl-cysteine (NAC) might act as an antioxidant. Twelve Beagle dogs were anesthetized with 1% propophol, tracheostomized, and subjected to continuous inspiratory resistive breathing (IRB) (2 h/day for 2 wk). They were further divided into two groups (n = 6): NAC group (oral NAC administration/24 h for 14 days) and control group (placebo). Diaphragm biopsies were obtained before (baseline biopsy) and after (contralateral hemidiaphragm) IRB and NAC vs. placebo treatment. Oxidative stress was evaluated in all diaphragm biopsies through determination of 3-nitrotyrosine immunoreactivity, protein carbonylation, hydroxynoneal protein adducts, Mn-SOD, and catalase, using immunoblotting and immunohistochemistry. Both protein tyrosine nitration and protein carbonylation were directly related to the amount of the respiratory loads, and NAC treatment abrogated this proportional rise in these two indexes of oxidative stress in response to increasing inspiratory loads. A post hoc analysis revealed that only the diaphragms of dogs subjected to high-intensity loads showed a significant increase in both protein tyrosine nitration and carbonylation, which were also significantly reduced by NAC treatment. These results suggest that high-intensity respiratory loading-induced oxidative stress may be neutralized by NAC treatment during IRB in the canine diaphragm.
Assuntos
Acetilcisteína/farmacologia , Diafragma/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Trabalho Respiratório/fisiologia , Acetilcisteína/administração & dosagem , Animais , Antioxidantes/administração & dosagem , Antioxidantes/farmacologia , Diafragma/metabolismo , Cães , Masculino , Fadiga Muscular , Carbonilação Proteica , Proteínas/metabolismo , Respiração , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
In a porcine model of endotoxic shock, we evaluated the circulatory and respiratory effects of NO synthase (NOS) blockade. Twenty anaesthetised pigs were divided into three groups and studied for 240 min after induction of endotoxic shock with lipopolysaccharides of Escherichia coli (LPS). After 180 min of endotoxic shock, one group (n = 6) received aminoguanidine, another group (n = 6) received N(G)-nitro-L -arginine methyl ester (L -NAME) and a third group (n = 8) received only LPS. A sham group (n = 3) was also studied. LPS decreased systemic arterial pressure and cardiac output (CO) and increased mean pulmonary arterial pressure (MPAP), pulmonary vascular resistance (PVR) and heart rate. Significant changes were also observed in compliance (-18.4%) and resistance (+33.6%) of the respiratory system. Aminoguanidine did not modify LPS-dependent effects, while, after L -NAME, a significant increase in MPAP, PVR and SVR and a decrease in CO were observed. In conclusion, aminoguanidine does not play a significant cardiocirculatory and pulmonary role in the short-term dysfunction of endotoxic shock, while L -NAME has a detrimental effect on haemodynamics, suggesting a protective role of constitutive NO production at vascular level during the early stages of endotoxaemia.
Assuntos
Sistema Cardiovascular/efeitos dos fármacos , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Respiração/efeitos dos fármacos , Choque Séptico/fisiopatologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Débito Cardíaco/efeitos dos fármacos , Sistema Cardiovascular/fisiopatologia , Pressão Venosa Central/efeitos dos fármacos , Escherichia coli , Guanidinas/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Hemodinâmica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Choque Séptico/induzido quimicamente , Suínos , Fatores de Tempo , Resistência Vascular/efeitos dos fármacosAssuntos
Insuficiência Respiratória/etiologia , Músculos Respiratórios/fisiopatologia , Sepse/fisiopatologia , Animais , Hemodinâmica , Humanos , Camundongos , Camundongos Knockout , Insuficiência de Múltiplos Órgãos/etiologia , Músculo Esquelético/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio , Pesquisa , Músculos Respiratórios/enzimologia , Músculos Respiratórios/metabolismo , Sepse/metabolismoRESUMO
The antioxidant N-acetylcysteine (NAC) prevented sepsis-induced diaphragmatic dysfunction. As an indirect antioxidant NAC was shown to induce superoxide dismutase (SOD) activity in immune cells from endotoxaemic mice. The aim of this study was to assess whether NAC acts as an indirect antioxidant by inducing manganese (Mn)-SOD activity in the diaphragms of endotoxaemic rats, while preventing muscle dysfunction. A controlled study was conducted, in which protein carbonylation, Mn-SOD, catalase, and 3-nitrotyrosine immunoreactivity were detected using immunoblotting and immunohistochemistry in rat diaphragms. Six groups were studied for 24 h after a saline (control) or lipopolysaccharide (LPS; 20 mg.kg-1) i.p. injection in the absence and presence of NAC pre-treatment (either 1.5 or 3 mmol.kg(-1).24 h-1 for 7 days, oral administration). Diaphragm mitochondrial Mn-SOD activity and respiratory muscle function were also determined. Within 24 h, LPS induced maximal inspiratory pressure reduction, increasing diaphragmatic protein carbonylation and nitration. Pre-treatment with 3 mmol.kg-1 NAC clearly increased muscle Mn-SOD protein content and activity in both LPS- and saline-injected animals, while reducing protein carbonylation and nitration, and partially preventing the LPS-induced respiratory muscle dysfunction. Data produced from this study indicate that high doses of N-acetylcysteine induces manganese superoxide dismutase, as well as preserves its activity, possibly by preventing nitration of critical tyrosine residues of the enzyme.
Assuntos
Acetilcisteína/farmacologia , Sepse/tratamento farmacológico , Sepse/enzimologia , Superóxido Dismutase/metabolismo , Análise de Variância , Animais , Biópsia por Agulha , Diafragma/efeitos dos fármacos , Diafragma/patologia , Modelos Animais de Doenças , Imuno-Histoquímica , Masculino , Probabilidade , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Valores de Referência , Fatores de Risco , Sensibilidade e Especificidade , Sepse/patologia , Superóxido Dismutase/efeitos dos fármacosRESUMO
In this study, we evaluated the differential influence of chronic treadmill training (30 m/min, 15% incline, 1 h/day, 5 days/wk) on nitric oxide (NO) production and NO synthase (NOS) isoform expression as well as 3-nitrotyrosine formation (footprint of peroxynitrite) both in limb (gastrocnemius) and ventilatory (diaphragm) muscles. A group of exercise-trained rats and a control group (no training) were examined after a 4-wk experimental period. Exercise training elicited an approximate fourfold rise in gastrocnemius NOS activity and augmented protein expression of the endothelial (eNOS) and neuronal (nNOS) isoforms of NOS to approximately 480% and 240%, respectively. Qualitatively similar but quantitatively smaller elevations in NOS activity and eNOS and nNOS expression were observed in the diaphragm. No detectable inducible NOS (iNOS) protein expression was found in any of the muscle samples. Training increased the intensity of 3-nitrotyrosine only in the gastrocnemius muscle. We conclude that whole body exercise training enhances both limb and ventilatory muscle NO production and that constitutive and not iNOS isoforms are responsible for increased protein tyrosine nitration in trained limb muscles.