Lung Beractant Increases Free Cytosolic Levels of Ca(2+) in Human Lung Fibroblasts

Beractant, a natural surfactant, induces an antifibrogenic phenotype and apoptosis in normal human lung fibroblasts (NHLF). As intracellular Ca(2+) signalling has been related to programmed cell death, we aimed to assess the effect of beractant on intracellular Ca(2+) concentration ([Ca(2+)](i)) in NHLF in vitro. Cultured NHLF were loaded with Fura-2 AM (3 μM) and Ca(2+) signals were recorded by microfluorimetric techniques. Beractant causes a concentration-dependent increase in [Ca(2+)](i) with a EC(50) 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 Ca(2+) signals in NHLF: a) a single Ca(2+) spike which could be followed by b) Ca(2+) oscillations, c) a sustained Ca(2+) plateau or d) a sustained plateau overlapped by Ca(2+) oscillations. The amplitude and pattern of Ca(2+) transients evoked by beractant were dependent on the resting [Ca(2+)](i). Pharmacological manipulation revealed that beractant activates a Ca(2+) signal through Ca(2+) release from intracellular stores mediated by phospholipase Cβ (PLCβ), Ca(2+) release from inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and Ca(2+) influx via a store-operated pathway. Moreover, beractant-induced Ca(2+) release was abolished by preventing membrane depolarization upon removal of extracellular Na(+) and Ca(2+). Finally, the inhibition of store-operated channels prevented beractant-induced NHLF apoptosis and downregulation of α(1)(I) procollagen expression. Therefore, beractant utilizes SOCE to exert its pro-apoptotic and antifibrinogenic effect on NHLF.