Abstract
Beractant, a natural surfactant, induces an antifibrogenic phenotype and apoptosis in normal human lung fibroblasts (NHLF). As intracellular Ca2+ signalling has been related to programmed cell death, we aimed to assess the effect of beractant on intracellular Ca2+ concentration ([Ca2+]i) in NHLF in vitro. Cultured NHLF were loaded with Fura-2 AM (3 μM) and Ca2+ signals were recorded by microfluorimetric techniques. Beractant causes a concentration-dependent increase in [Ca2+]i with a EC50 of 0.82 μg/ml. The application of beractant, at a concentration of 500 μg/ml, which has been shown to exert an apoptotic effect in human fibroblasts, elicited different patterns of Ca2+ signals in NHLF: a) a single Ca2+ spike which could be followed by b) Ca2+ oscillations, c) a sustained Ca2+ plateau or d) a sustained plateau overlapped by Ca2+ oscillations. The amplitude and pattern of Ca2+ transients evoked by beractant were dependent on the resting [Ca2+]i. Pharmacological manipulation revealed that beractant activates a Ca2+ signal through Ca2+ release from intracellular stores mediated by phospholipase Cβ (PLCβ), Ca2+ release from inositol 1,4,5-trisphosphate receptors (IP3Rs) and Ca2+ influx via a store-operated pathway. Moreover, beractant-induced Ca2+ release was abolished by preventing membrane depolarization upon removal of extracellular Na+ and Ca2+. Finally, the inhibition of store-operated channels prevented beractant-induced NHLF apoptosis and downregulation of α1(I) procollagen expression. Therefore, beractant utilizes SOCE to exert its pro-apoptotic and antifibrinogenic effect on NHLF.
Highlights
IntroductionPulmonary surfactant is a liquid layer covering the alveolar network of mammalian lungs and composed of approximately 90% lipids (mainly phospholipids) and 10% proteins (mainlyPLOS ONE | DOI:10.1371/journal.pone.0134564 July 31, 2015Ca2+ Signal Evoked by Lung Surfactant in Normal Lung Fibroblasts surfactant-associated proteins or SAPs) [1]
Pulmonary surfactant is a liquid layer covering the alveolar network of mammalian lungs and composed of approximately 90% lipids and 10% proteins [1]
The intracellular Ca2+ signal evoked by beractant consisted in a rapid Ca2+ spike (158/568, 27.82%; Fig 1A), that could be followed by Ca2+ oscillations (75/568, 13.20%; Fig 1B), a sustained plateau (252/568, 44.37%; Fig 1C), or a plateau overlapped by Ca2+ oscillations
Summary
Pulmonary surfactant is a liquid layer covering the alveolar network of mammalian lungs and composed of approximately 90% lipids (mainly phospholipids) and 10% proteins (mainlyPLOS ONE | DOI:10.1371/journal.pone.0134564 July 31, 2015Ca2+ Signal Evoked by Lung Surfactant in Normal Lung Fibroblasts surfactant-associated proteins or SAPs) [1]. IPF is characterized by the accumulation of excessive numbers of fibroblasts and myofibroblasts, exaggerated deposition of extracellular matrix proteins, such as fibrillar collagens, and distortion of normal tissue architecture [7] The pathogenesis of this disease is still unclear, and the hypothesis of unremitting chronic inflammation as the primary explanation of the pathophysiology of IPF has been challenged by the epithelial injury and activation hypothesis. This hypothesis suggests that chronic noxious stimuli to the alveolar epithelium causes an aberrant activation of the alveolar epithelial cells, as well as abnormalities in the basement membrane integrity, allowing the migration of fibroblasts from interstitium to the alveolar regions of the injured lung, leading to excessive accumulation of extracellular matrix and irreversible loss of the structure of lung parenchyma [8,9]. In accordance with this hypothesis, at some point during the pathogenesis of IPF, fibroblasts come in close contact with the components of the pulmonary surfactant system
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