Lung Beractant Increases Free Cytosolic Levels of Ca2+ in Human Lung Fibroblasts

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.

Phosphatidylethanolamine Induces an Antifibrotic Phenotype in Normal Human Lung Fibroblasts and Ameliorates Bleomycin-Induced Lung Fibrosis in Mice

“…NHLF were loaded with the Ca 2+ -sensitive fluorochrome, Fura-2 and then stimulated with PE at increasing doses and in the absence and in the presence of specific inhibitors of the Ca 2+ signaling toolkit. PE induced an increase in [Ca 2+ ] i similar to that evoked by Beractant ( Figure 5 ) [ 8 ]. At 50 µg· mL −1 , around 20% of cells responded with a transient Ca 2+ spike ( Figure 5 A, left); 500 µg·mL −1 PE induced a biphasic Ca 2+ signal in all the cells, which consisted in an initial Ca 2+ spike followed by a plateau phase of intermediate amplitude ( Figure 5 A, right) or Ca 2+ oscillations ( Figure 5 B, left).…”

“…We have previously shown that Beractant elicits an intracellular Ca 2+ signal in NHLF and that this Ca 2+ response was not triggered either by DPPC or PG [ 8 ]. Thus, we tested if PE mimics this effect.…”

“…At 50 µg· mL −1 , around 20% of cells responded with a transient Ca 2+ spike ( Figure 5 A, left); 500 µg·mL −1 PE induced a biphasic Ca 2+ signal in all the cells, which consisted in an initial Ca 2+ spike followed by a plateau phase of intermediate amplitude ( Figure 5 A, right) or Ca 2+ oscillations ( Figure 5 B, left). The Ca 2+ response to Beractant was mediated by the interaction between inositol-1,4,5-trisphosphate (InsP 3 )-dependent Ca 2+ release from the endoplasmic reticulum (ER) and store-operated Ca 2+ entry (SOCE) [ 8 ]. Likewise, upon removal of extracellular Ca 2+ (0Ca 2+ ) PE induced a transient increase in [Ca 2+ ] i which lacked the plateau or oscillation phase ( Figure 5 C).…”

“…In a previous study, we showed that pulmonary surfactant promotes programmed cell death of normal human lung fibroblasts and induces the upregulation of matrix metalloproteinase-1 and the downregulation of type I collagen [ 7 ]. In that study, we used Beractant, a natural bovine lung extract containing phospholipids, neutral lipids, fatty acids and surfactant-associated hydrophobic proteins B and C. In other study [ 8 ], we found that Beractant promoted apoptosis and reduced the expression levels of type I collagen through an increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) that was initiated by the recruitment of phospholipase C (PLC). Intriguingly, neither DPPC nor PG were able to induce an increase in [Ca 2+ ] i , which suggests that other surfactant components induce apoptosis and regulate gene expression in normal lung fibroblasts (NHLF).…”

Protective effects of β‐sheet breaker α/β‐hybrid peptide against amyloid β‐induced neuronal apoptosis in vitro

“…The intracellular free Ca 2+ levels in suspension of SH‐SY5Y cells were measured by Ca 2+ ‐sensitive dye Fura‐2AM by previously defined method . Shortly, after treatment with respective peptides, 5 μm of Fura‐2AM (Cat.…”