The Involvement of PI3K-Mediated and L-VGCC-Gated Transient Ca(2+) Influx in 17β-Estradiol-Mediated Protection of Retinal Cells from H(2)O(2)-Induced Apoptosis with Ca(2+) Overload

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.