[The proliferation-promoting effects of calcitonin gene-related peptide on type II alveolar epithelial cell exposed to hyperoxia mediated by protein kinase C alpha pathway].

OBJECTIVE

To explore the effects of calcitonin gene-related peptide (CGRP) on type II alveolar epithelial cell (AECII) exposed to hyperoxia, and to determine whether the mechanism is mediated by protein kinase C alpha/nuclear factor-KappaB (PKC alpha/NF-KappaB) signal pathway.

METHODS

AECII were isolated from the lung of 21 days fetal rat and cultured for 15 hours to coalesce. Then AECII were randomly assigned into four groups: air, hyperoxia, O(2)/CGRP, and O(2)/CGRP8-37 (a receptor antagonist against CGRP). AECII were exposed to FiO(2) 21% (air) or 85% (hyperoxia) for 24 hours respectively. In O(2)/CGRP and O(2)/CGRP8-37 groups CGRP or both CGRP and CGRP8-37 were added into cultural fluid before placing the plate into 85% oxygen. Cell proliferation ability was determined by methyl thiazolyl tetrazolium (MTT) assay and cell cycles by flow cytometry. Western blotting was employed to detect the fraction of PKC alpha in membrane and cytosol, and translocation of NF-KappaB was observed under laser confocal microscopy.

RESULTS

AECII in hyperoxia group showed a decreased viability of AECII [(68.752+/-5.766)% vs. (100.000+/-6.682)%] and had an enhanced percentage of G0/G1 phase [(80.652+/-6.253)% vs. (45.825+/-2.899)%] with a corresponding decline in percentage of S phase [(14.198+/-4.785)% vs. (27.470+/-2.775)%] and G2/M phases [(5.148+/-1.688)% vs. (26.708+/-1.863)%] compared with AECII in air (all P<0.01). Addition with CGRP before hyperoxia exposure promoted AECII proliferation [(94.813+/-6.102)%] and enhanced the cell proportions in S and G2/M phases [(30.547+/-9.861)% and (17.668+/-9.509)%, all P<0.01]. The ratio of membrane to cytoplasm fraction of PKC alpha declined (0.63+/-0.10 vs. 1.00+/-0.09) and the fluorescence of NF-KappaB in nucleus enhanced (22.98+/-2.20 vs. 14.54+/-2.35) in hyperoxia compared with that in air, while both the ratio of PKC alpha and intensity of NF-KappaB were increased in O(2)/CGRP group (1.41+/-0.23, 35.38+/-3.37) compared with those in hyperoxia (0.63+/-0.10, 22.98+/-2.20) and O(2)/CGRP8-37 groups (0.74+/-0.10, 24.88+/-1.81, all P<0.01).

CONCLUSION

CGRP could promote proliferation of AECII when exposed to high oxygen tension. PKC alpha participates in the signal transduction process and NF-KappaB is a downstream molecular of PKC alpha, executing in part the function of PKC alpha signal.