American journal of physiology. Lung cellular and molecular physiology
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Am. J. Physiol. Lung Cell Mol. Physiol. · Oct 2011
Regulatory domain phosphorylation to distinguish the mechanistic basis underlying acute CFTR modulators.
Modulator compounds intended to overcome disease-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) show significant promise in clinical testing for cystic fibrosis. However, the mechanism(s) of action underlying these compounds are not fully understood. Activation of CFTR ion transport requires PKA-regulated phosphorylation of the regulatory domain (R-D) and dimerization of the nucleotide binding domains. ⋯ In contrast, VRT-532 moderately activated CFTR short-circuit current and strongly potentiated forskolin-mediated current. Combined with the result that UC(CF)-152, but not VRT-532 or VX-770, acts by increasing CFTR R-D phosphorylation, these findings indicate that potentiation of endogenous cAMP-mediated activation of mutant CFTR is not due to a pathway involving augmented R-D phosphorylation. This study presents an assay useful to distinguish preclinical compounds by a crucial mechanism underlying CFTR activation, delineates two types of compound able to acutely augment CFTR activity (e.g., activators and potentiators), and demonstrates that a number of different mechanisms can be successfully employed to activate mutant CFTR.
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Am. J. Physiol. Lung Cell Mol. Physiol. · Oct 2011
Caveolin-1 in cytokine-induced enhancement of intracellular Ca(2+) in human airway smooth muscle.
Diseases such as asthma are characterized by airway hyperresponsiveness. Enhanced airway smooth muscle (ASM) intracellular Ca(2+) ([Ca(2+)](i)) response to agonist stimulation leading to increased airway constriction has been suggested to contribute to airway hyperresponsiveness. Caveolae are flask-shaped plasma membrane invaginations that express the scaffolding protein caveolin and contain multiple proteins important in [Ca(2+)](i) signaling (e.g., agonist receptors, ion channels). ⋯ However, disruption of caveolae using caveolin-1 suppression via small-interfering RNA resulted in significant blunting of agonist-induced [Ca(2+)](i) responses of vehicle and TNF-α-exposed cells. These functional data were correlated to the presence of TNFR(1) receptor (but not the IL-4/IL-13 receptor) within caveolae. Overall, these results indicate that caveolin-1 plays an important role in airway inflammation by modulating the effect of specific cytokines on [Ca(2+)](i).
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Am. J. Physiol. Lung Cell Mol. Physiol. · Oct 2011
Role of the Fas/FasL system in a model of RSV infection in mechanically ventilated mice.
Infection with respiratory syncytial virus (RSV) in children can progress to respiratory distress and acute lung injury necessitating mechanical ventilation (MV). MV enhances apoptosis and inflammation in mice infected with pneumonia virus of mice (PVM), a mouse pneumovirus that has been used as a model for severe RSV infection in mice. We hypothesized that the Fas/Fas ligand (FasL) system, a dual proapoptotic/proinflammatory system involved in other forms of lung injury, is required for enhanced lung injury in mechanically ventilated mice infected with PVM. ⋯ However, when PVM infection was allowed to progress one additional day, all of the lpr mice (7/7) died unexpectedly between 0.5 and 3.5 h after the onset of ventilation compared with three of the seven ventilated C57BL/6 mice. Parameters of lung injury were similar in nonventilated mice, as was the viral content in the lungs and other organs. Thus, the Fas/FasL system was partly required for the lung inflammatory response in ventilated mice infected with PVM, but attenuation of lung inflammation did not prevent subsequent mortality.
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Am. J. Physiol. Lung Cell Mol. Physiol. · Oct 2011
Inflammation and matrix remodeling during repair of ventilator-induced lung injury.
High-pressure ventilation triggers different inflammatory and matrix remodeling responses within the lung. Although some of them may cause injury, the involvement of these mediators in repair is largely unknown. To identify mechanisms of repair after ventilator-induced lung injury (VILI), mice were randomly assigned to baseline conditions (no ventilation), injury [90 min of high-pressure ventilation without positive end-expiratory pressure (PEEP)], repair (injury followed by 4 h of low-pressure ventilation with PEEP), and ventilated controls (low-pressure ventilation with PEEP for 90 and 330 min). ⋯ In conclusion, VILI can be reverted by normalizing ventilation pressures. An adequate inflammatory response and extracellular matrix remodeling are essential for recovery. MMP-2 could play a key role in epithelial repair after VILI and acute respiratory distress syndrome.
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Am. J. Physiol. Lung Cell Mol. Physiol. · Oct 2011
Cigarette smoke exposure aggravates air space enlargement and alveolar cell apoptosis in Smad3 knockout mice.
The concept of genetic susceptibility factors predisposing cigarette smokers to develop emphysema stems from the clinical observation that only a fraction of smokers develop clinically significant chronic obstructive pulmonary disease. We investigated whether Smad3 knockout mice, which develop spontaneous air space enlargement after birth because of a defect in transforming growth factor-β (TGF-β) signaling, develop enhanced alveolar cell apoptosis and air space enlargement following cigarette smoke exposure. We investigated Smad3(-/-) and Smad3(+/+) mice at different adult ages and determined air space enlargement, alveolar cell proliferation, and apoptosis. ⋯ Double immunofluorescence analysis revealed that endothelial apoptosis started before epithelial apoptosis. Our data indicate that balanced TGF-β signaling is not only important for regulation of extracellular matrix turnover, but also for alveolar cell homeostasis. Impaired signaling via the Smad3 pathway results in alveolar cell apoptosis and alveolar destruction, likely via increased Smad2 and reduced VEGF expression and might represent a predisposition for accelerated development of emphysema due to cigarette smoke exposure.