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

Guzmán-Silva A, Vázquez de Lara LG, Torres-Jácome J, Vargaz-Guadarrama A, Flores-Flores M, Pezzat Said E, Lagunas-Martínez A, Mendoza-Milla C, Tanzi F, Moccia F, Berra-Romani R - PLoS ONE (2015)

Bottom Line: 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.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.Therefore, beractant utilizes SOCE to exert its pro-apoptotic and antifibrinogenic effect on NHLF.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedicine, School of Medicine, Benemérita Universidad Autónoma de Puebla, Puebla, Puebla, México.

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.

No MeSH data available.


Related in: MedlinePlus

Effect of beractant on the initial Ca2+ spike amplitude and plateau phase amplitude in NHLF.A) ΔR/Ri for the peak response to beractant in presence of designated drugs. B) ΔR/Ri for the plateau phase of beractant-evoked Ca2+ increase. See the text for drugs concentrations. Data expressed as means ± SE were analysed statistically by Student’s t-test. * p <0.05, *** p <0.001,**** p <0.0001.
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pone.0134564.g004: Effect of beractant on the initial Ca2+ spike amplitude and plateau phase amplitude in NHLF.A) ΔR/Ri for the peak response to beractant in presence of designated drugs. B) ΔR/Ri for the plateau phase of beractant-evoked Ca2+ increase. See the text for drugs concentrations. Data expressed as means ± SE were analysed statistically by Student’s t-test. * p <0.05, *** p <0.001,**** p <0.0001.

Mentions: Beractant effect in NHLF was reversible: the Ca2+ signal ceased when the agonist was removed from the bath and a similar Ca2+ transient was evoked on beractant restoration. As shown in Fig 3A, the second application of a supramaximal concentration of beractant (500 μg/ml) produced a Ca2+ response with similar kinetics and peak amplitude of that evoked by the first application of beractant (n = 55). The same results were obtained when the Ca2+ response to beractant consisted in the onset of repetitive Ca2+ oscillations (Fig 3B, n = 45). There was a slight reduction in the mean peak amplitude of the second Ca2+ transient (13.46%), but the decrease was not statistically significant (Fig 4A, compare Beractant 1st vs Beractant 2nd, p > 0.05).


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

Guzmán-Silva A, Vázquez de Lara LG, Torres-Jácome J, Vargaz-Guadarrama A, Flores-Flores M, Pezzat Said E, Lagunas-Martínez A, Mendoza-Milla C, Tanzi F, Moccia F, Berra-Romani R - PLoS ONE (2015)

Effect of beractant on the initial Ca2+ spike amplitude and plateau phase amplitude in NHLF.A) ΔR/Ri for the peak response to beractant in presence of designated drugs. B) ΔR/Ri for the plateau phase of beractant-evoked Ca2+ increase. See the text for drugs concentrations. Data expressed as means ± SE were analysed statistically by Student’s t-test. * p <0.05, *** p <0.001,**** p <0.0001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4521834&req=5

pone.0134564.g004: Effect of beractant on the initial Ca2+ spike amplitude and plateau phase amplitude in NHLF.A) ΔR/Ri for the peak response to beractant in presence of designated drugs. B) ΔR/Ri for the plateau phase of beractant-evoked Ca2+ increase. See the text for drugs concentrations. Data expressed as means ± SE were analysed statistically by Student’s t-test. * p <0.05, *** p <0.001,**** p <0.0001.
Mentions: Beractant effect in NHLF was reversible: the Ca2+ signal ceased when the agonist was removed from the bath and a similar Ca2+ transient was evoked on beractant restoration. As shown in Fig 3A, the second application of a supramaximal concentration of beractant (500 μg/ml) produced a Ca2+ response with similar kinetics and peak amplitude of that evoked by the first application of beractant (n = 55). The same results were obtained when the Ca2+ response to beractant consisted in the onset of repetitive Ca2+ oscillations (Fig 3B, n = 45). There was a slight reduction in the mean peak amplitude of the second Ca2+ transient (13.46%), but the decrease was not statistically significant (Fig 4A, compare Beractant 1st vs Beractant 2nd, p > 0.05).

Bottom Line: 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.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.Therefore, beractant utilizes SOCE to exert its pro-apoptotic and antifibrinogenic effect on NHLF.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedicine, School of Medicine, Benemérita Universidad Autónoma de Puebla, Puebla, Puebla, México.

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.

No MeSH data available.


Related in: MedlinePlus