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1.
Exp Lung Res ; 40(8): 380-91, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25058750

RESUMO

ABSTRACT During fetal development physiological stretching helps drive lung growth and maturation. At birth, the α-subunit of the alveolar epithelial sodium channel (α-ENaC) is a critical factor in helping to facilitate clearance of lung fluid during the perinatal period. The effects of stretch, however, on α-ENaC expression in the fetal lung have yet to be elucidated. In an effort to explore this question, we used both an in vitro cell culture model that exposes cells to repetitive cyclic stretch (CS) as well as an in vivo preterm animal model of mechanical ventilation (MV). We found that murine lung epithelial (MLE-12) cells exposed to repetitive CS showed a significant rise in α-ENaC mRNA expression. Total and cell-surface protein abundance of α-ENaC were also elevated after 24 h of CS. Stretch-induced increases in α-ENaC expression were suppressed in the presence of either actinomycin D or cycloheximide. Pharmacological inhibition of the extracellular signal-regulated protein kinase (ERK1/2) did not attenuate stretch-induced increases in α-ENaC protein, whereas inhibition of p38 MAPK or c-Jun NH2-terminal kinase (JNK) did. In 29-day preterm rabbits, alveolar stretching secondary to postnatal MV markedly elevated fetal lung α-ENaC expression compared to spontaneously breathing counterparts. In summary, our findings indicate that mechanical stretch promotes α-ENaC expression.


Assuntos
Canais Epiteliais de Sódio/metabolismo , Pulmão/embriologia , Mucosa Respiratória/metabolismo , Estresse Mecânico , Animais , Células Cultivadas , Feminino , Pulmão/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Gravidez , Coelhos , Distribuição Aleatória
2.
Lab Invest ; 91(10): 1530-9, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21769086

RESUMO

Respiratory dysfunction in adults has been correlated with neonatal Chlamydia trachomatis pneumonia in several studies, but a causal association has not been clearly demonstrated. In this study, we examined radial alveolar counts (RACs) by microscopy, and airway and parenchymal lung function using a small animal ventilator in juvenile (5 weeks age) and adult (8 weeks age) BALB/c mice challenged as neonates with Chlamydia muridarum (C. mur) on day 1 or day 7 after birth, representing saccular (human pre-term neonates) and alveolar (human term neonates) stages of lung development, respectively. Pups challenged with C. mur on either day 1 or 7 after birth demonstrated significantly enhanced airway hyperreactivity and lung compliance, both as juveniles (5 weeks age) and adults (8 weeks age), compared with mock-challenged mice. Moreover, mice challenged neonatally with Chlamydia displayed significantly reduced RACs, suggesting emphysematous changes. Antimicrobial treatment during the neonatal infection induced early bacterial clearance and partially ameliorated the Chlamydia-induced lung dysfunction as adults. These results suggest that neonatal chlamydial pneumonia, especially in pre-term neonates, is a cause of respiratory dysfunction continuing into adulthood, and that antimicrobial administration may be partially effective in preventing the adverse respiratory sequelae in adulthood. The results of our studies also emphasize the importance of prenatal screening and treatment of pregnant women for C. trachomatis in order to prevent the infection of neonates.


Assuntos
Envelhecimento , Animais Recém-Nascidos , Infecções por Chlamydia/patologia , Infecções por Chlamydia/fisiopatologia , Pneumonia Bacteriana/patologia , Pneumonia Bacteriana/fisiopatologia , Sistema Respiratório/patologia , Sistema Respiratório/fisiopatologia , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Antibacterianos/administração & dosagem , Hiper-Reatividade Brônquica/etiologia , Infecções por Chlamydia/complicações , Infecções por Chlamydia/tratamento farmacológico , Esquema de Medicação , Eritromicina/administração & dosagem , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/fisiopatologia , Complacência Pulmonar , Camundongos , Camundongos Endogâmicos BALB C , Pneumonia Bacteriana/complicações , Pneumonia Bacteriana/tratamento farmacológico , Alvéolos Pulmonares/crescimento & desenvolvimento
3.
J Cell Commun Signal ; 14(3): 303-314, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31659629

RESUMO

Intra-amniotic exposure to proinflammatory cytokines such as interleukin-1 (IL-1) correlates with a decreased incidence of respiratory distress syndrome (RDS) in infants following premature birth. At birth, inadequate absorption of fluid from the fetal lung contributes to the onset RDS. Lung fluid clearance is coupled to Na+ transport via epithelial sodium channels (ENaC). In this study, we assessed the effects of IL-1 on the expression of ENaC, particularly the α-subunit which is critical for fetal lung fluid clearance at birth. Cultured mouse lung epithelial (MLE-12) cells were treated with either IL-1α or IL-1ß to determine their effects on α-ENaC expression. Changes in IL-1-induced α-ENaC levels in the presence of IL-1 receptor antagonist (IL-1ra), cycloheximide, NF-κB inhibitor, and MAP kinase inhibitors were investigated. IL-1α and IL-1ß independently induced a significant increase of α-ENaC mRNA and protein after 24 h compared to untreated cells. IL-1-dependent increases in α-ENaC protein were mitigated by IL-1ra and cycloheximide. IL-1 exposure induced NF-κB binding activity. Attenuation of IL-1-induced NF-κB activation by its inhibitor SN50 decreased α-ENaC protein abundance. Inhibition of ERK 1,2 MAPK significantly decreased both IL-1α and ß-induced α-ENaC protein expression whereas inhibition of p38 MAPK only blocked IL-1ß-induced α-ENaC protein levels. In contrast, IL-1-induced α-ENaC protein levels were unaffected by a c-Jun N-terminal kinase (JNK) inhibitor. Our results suggest that in MLE-12 cells, IL-1-induced elevation of α-ENaC is mediated via NF-κB activation and in part involves stimulation of the ERK 1,2 and p38 MAPK signaling pathways.

4.
J Cell Physiol ; 217(3): 632-42, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18615584

RESUMO

Alpha-ENaC expression and activity is regulated by a variety of hormones including beta-adrenergic agonists via the second messenger cAMP. We evaluated the early intermediate pathways involved in the up-regulation of SGK1 by DbcAMP and whether SGK1 is a prerequisite for induction of alpha-ENaC expression. Submandibular gland epithelial (SMG-C6) cells treated with DbcAMP (1 mM) induced both SGK1 mRNA and protein expression. DbcAMP-stimulated SGK1 mRNA expression was decreased by actinomycin D and mRNA and protein expressions were attenuated by PKA inhibitors (H-89 and KT5720). Inhibition of PI3-K with either LY294002 or dominant negative PI3-K reduced DbcAMP-stimulated SGK1 protein and mRNA levels, attenuated the phosphorylation of CREB (a cAMP-activated transcription factor) and decreased alpha-ENaC protein levels and Na(+) transport. In addition, the combination of PKA inhibitors with dominant negative PI3-K synergistically inhibited DbcAMP-induced Na(+) transport. Inhibition of SGK1 expression by siRNA decreased but did not obliterate DbcAMP-induced alpha-ENaC expression. Thus, in a cell line which endogenously exhibits minimal alpha-ENaC expression, induction of SGK1 by DbcAMP occurs via the PI3-K and PKA pathways. Increased alpha-ENaC levels and function are partly dependent upon the early induction of SGK1 expression.


Assuntos
Bucladesina/farmacologia , Canais Epiteliais de Sódio/metabolismo , Proteínas Imediatamente Precoces/biossíntese , Proteínas Serina-Treonina Quinases/biossíntese , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Cicloeximida/farmacologia , Ativação Enzimática/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Canais Epiteliais de Sódio/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Imediatamente Precoces/genética , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Sódio/metabolismo
5.
J Cell Physiol ; 215(1): 101-10, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17960568

RESUMO

A major mechanism for Na+ transport across epithelia occurs through epithelial Na+ channels (ENaC). ENaC is a multimeric channel consisting of three subunits (alpha, beta, and gamma). The alpha-subunit is critical for ENaC function. In specific culture conditions, the rat submandibular gland epithelial cell line (SMG-C6) demonstrates minimal Na+ transport properties and exposure to dibutyryl cAMP (DbcAMP) for up to 48 h caused an elevation of alpha-ENaC mRNA and protein expression and amiloride-sensitive short-circuit current (I(SC)). Here we examined the early signaling pathways evoked by DbcAMP which contribute to the eventual increase in Na+ transport is present. Treatment with either of the protein kinase A (PKA) inhibitors KT5720 or H-89 followed by exposure to 1 mM DbcAMP for 24 h markedly attenuated DbcAMP-induced alpha-ENaC protein formation and I(SC). Exposure of SMG-C6 cells to 1 mM DbcAMP induced a rapid, transient phosphorylation of the cAMP response element binding protein (CREB). This response was attenuated in the presence of either KT5720 or H-89. Dominant-negative CREB decreased DbcAMP-induced alpha-ENaC expression. Suppression of the extracellular signal-regulated protein kinase (ERK 1,2) with PD98059 or the p38 mitogen-activated protein kinase (MAPK) pathway with SB203580 reduced DbcAMP-induced alpha-ENaC protein levels in SMG-C6 cells. DbcAMP-induced phosphorylation of CREB was markedly attenuated by PD98059 or SB203580. DbcAMP-induced activation of the either the p38 or the ERK 1,2 MAPK pathways was abolished by either of the PKA inhibitors, H-89 or KT5720. Cross talk between these signaling pathways induced by DbcAMP via the activation of CREB appears to contribute to increased levels of alpha-ENaC observed after 24 h of treatment in SMG-C6 epithelial cells.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Canais Epiteliais de Sódio/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Animais , Bucladesina/farmacologia , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Canais Epiteliais de Sódio/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
6.
Am J Physiol Lung Cell Mol Physiol ; 291(2): L166-74, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16461433

RESUMO

The treatment of severe lung disease often requires the use of high concentrations of oxygen coupled with the need for assisted ventilation, potentially exposing the pulmonary epithelium to both reactive oxygen species and nonphysiological cyclic stretch. Whereas prolonged hyperoxia is known to cause increased cell injury, cyclic stretch may result in either cell proliferation or injury depending on the pattern and degree of exposure to mechanical deformation. How hyperoxia and cyclic stretch interact to affect the pulmonary epithelium in vitro has not been previously investigated. This study was performed using human alveolar epithelial A549 cells to explore the combined effects of cyclic stretch and hyperoxia on cell proliferation and viability. Under room air conditions, cyclic stretch did not alter cell viability at any time point and increased cell number after 48 h compared with unstretched controls. After exposure to prolonged hyperoxia, cell number and [(3)H]thymidine incorporation markedly decreased, whereas evidence of oxidative stress and nonapoptotic cell death increased. The combination of cyclic stretch with hyperoxia significantly mitigated the negative effects of prolonged hyperoxia alone on measures of cell proliferation and viability. In addition, cyclic stretch resulted in decreased levels of oxidative stress over time in hyperoxia-exposed cells. Our results suggest that cyclic stretch, as applied in this study, can minimize the detrimental effects of hyperoxia on alveolar epithelial A549 cells.


Assuntos
Proliferação de Células , Sobrevivência Celular , Células Epiteliais/fisiologia , Hiperóxia , Alvéolos Pulmonares/citologia , Apoptose , Linhagem Celular Tumoral , Forma Celular , Células Epiteliais/citologia , Humanos , Espécies Reativas de Oxigênio/metabolismo , Estresse Mecânico , Superóxidos/metabolismo
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