Lung Beractant Increases Free Cytosolic Levels Of Ca2+ In Human Lung Fibroblasts

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 mu M) and Ca2+ signals were recorded by microfluorimetric techniques. Beractant causes a concentration-dependent increase in [Ca2+] i with a EC50 of 0.82 mu g/ml. The application of beractant, at a concentration of 500 mu g/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 C beta (PLC beta), Ca2+ release from inositol 1,4,5-trisphosphate receptors (IP(3)Rs) 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 alpha(1)(I) procollagen expression. Therefore, beractant utilizes SOCE to exert its pro-apoptotic and antifibrinogenic effect on NHLF.