The involvement of PI3K-mediated and L-VGCC-gated transient Ca2+ influx in 17β-estradiol-mediated protection of retinal cells from H 2O2-induced apoptosis with Ca2+ overload

Abstract

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 (H2O 2)-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 H2O 2 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 H 2O2 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. © 2013 Feng et al.