The 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

摘要

Intracellular calcium concentration ([Ca²⁺]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 [Ca²⁺]i and its control mechanisms in process of hydrogen peroxide (H2O2)-induced apoptosis of primary cultured Sprague-Dawley (SD) rat retinal cells and 17β-estradiol (βE2) anti-apoptosis. Fluo-3AM was used as a Ca²⁺ indicator to detect [Ca²⁺]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 H2O2-induced retinal cell apoptosis occurred at 4 h after H2O2 stress and increased in a time-dependent manner, but [Ca²⁺]i increased earlier at 2 h, sustained to 12 h, and then recovered at 24 h after H2O2 stress; b) 10 μM βE2 treatment for 0.5-24 hrs increased cell viability by transiently increasing [Ca²⁺]i, which appeared only at 0.5 h after βE2 application; c) increased [Ca²⁺]i under 100 µM H2O2 treatment for 2 hrs or 10 µM βE2 treatment for 0.5 hrs was, at least partly, due to extracellular Ca²⁺ stores; d) importantly, the transiently increased [Ca²⁺]_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 Ca²⁺ channels (L-VGCC), but the increased [Ca²⁺]_i induced by 100 µM H₂O₂ 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 H₂O₂, which was also associated with its transient [Ca²⁺]_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.