Open AccessThe Involvement of PI3K-Mediated and L-VGCC-Gated Transient Ca2+ Influx in 17β-Estradiol-Mediated Protection of Retinal Cells from H2O2-Induced Apoptosis with Ca2+ Overload
Author(s) -
Yan Feng,
Baoying Wang,
Fuxin Du,
Hongbo Li,
Shaolan Wang,
Cheng-Hu Hu,
Zhu C,
Xiaorui Yu
Publication year - 2013
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0077218
Subject(s) - apoptosis , propidium iodide , viability assay , annexin , microbiology and biotechnology , mtt assay , intracellular , programmed cell death , chemistry , biology , biochemistry
Intracellular calcium concentration ([Ca 2+ ] i ) plays an important role in regulating most cellular processes, including apoptosis and survival, but its alterations are different and complicated under diverse conditions. In this study, we focused on the [Ca 2+ ] i and its control mechanisms in process of hydrogen peroxide (H 2 O 2 )-induced apoptosis of primary cultured Sprague-Dawley (SD) rat retinal cells and 17β-estradiol (βE2) anti-apoptosis. Fluo-3AM was used as a Ca 2+ indicator to detect [Ca 2+ ] i through fluorescence-activated cell sorting (FACS), cell viability was assayed using MTT assay, and apoptosis was marked by Hoechst 33342 and annexin V/Propidium Iodide staining. Besides, PI3K activity was detected by Western blotting. Results showed: a) 100 μM H 2 O 2 -induced retinal cell apoptosis occurred at 4 h after H 2 O 2 stress and increased in a time-dependent manner, but [Ca 2+ ] i increased earlier at 2 h, sustained to 12 h, and then recovered at 24 h after H 2 O 2 stress; b) 10 μM βE2 treatment for 0.5-24 hrs increased cell viability by transiently increasing [Ca 2+ ] i , which appeared only at 0.5 h after βE2 application; c) increased [Ca 2+ ] i under 100 µM H 2 O 2 treatment for 2 hrs or 10 µM βE2 treatment for 0.5 hrs was, at least partly, due to extracellular Ca 2+ stores; d) importantly, the transiently increased [Ca 2+ ] i induced by 10 µM βE2 treatment for 0.5 hrs was mediated by the phosphatidylinositol-3-kinase (PI3K) and gated by the L-type voltage-gated Ca 2+ channels (L-VGCC), but the increased [Ca 2+ ] i induced by 100 µM H 2 O 2 treatment for 2 hrs was not affected; and e) pretreatment with 10 µM βE2 for 0.5 hrs effectively protected retinal cells from apoptosis induced by 100 µM H 2 O 2 , which was also associated with its transient [Ca 2+ ] i increase through L-VGCC and PI3K pathway. These findings will lead to better understanding of the mechanisms of βE2-mediated retinal protection and to exploration of the novel therapeutic strategies for retina degeneration.