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

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 (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.

17β-Estradiol up-regulates Nrf2 via PI3K/AKT and estrogen receptor signaling pathways to suppress light-induced degeneration in rat retina

“…(Feng et al, 2013;Li et al, 2013a;Mo et al, 2013). Moreover, pretreatment with 10 µM LY was sufficient to inhibit pAKT protein expression in rats.…”

“…The PI3K/AKT signaling pathway is activated when retinal cells are exposed to oxidative stress, which can subsequently alter downstream signaling cascades (Wang et al, 2008). We additionally demonstrated that PI3K/AKT regulates downstream signaling, such as the nuclear factor kappa B (NF-κB) (Mo et al, 2013), mitochondrial apoptosis signaling (Li et al, 2013a), and Ca 2+ -regulated signaling pathways (Feng et al, 2013). We additionally demonstrated that PI3K/AKT regulates downstream signaling, such as the nuclear factor kappa B (NF-κB) (Mo et al, 2013), mitochondrial apoptosis signaling (Li et al, 2013a), and Ca 2+ -regulated signaling pathways (Feng et al, 2013).…”

Calcium-engaged Mechanisms of Nongenomic Action of Neurosteroids

“…17-βE triggered rapid Ca 2+ influx in hippocampal neurons with subsequent activation of Src/ERK and CREB cascades, followed by an up-regulation of Bcl-2 expression [ 115 ] which was, in turn, blocked by 10 µM nifedipine. Neuroprotective effect of 17-βE has also been demonstrated in primary cultures of Sprague-Dawley rat retinal cells after hydrogen peroxide-induced apoptosis [ 116 ]. The transient Ca 2+ increase induced by 10 μM 17-βE treatment for 0.5 h was mediated by the PI3K and gated by the L-type VGCC.…”

VEGF as a Survival Factor in Ex Vivo Models of Early Diabetic Retinopathy

“…28 In our preliminary experiments, treatments with 75 mM glucose were necessary to induce significant increases of apoptotic markers in retinal explants maintained in serum-free medium up to 10 days. Oxidative stress treatments were performed adding H 2 O 2 to reach a concentration of 10 lM or 100 lM, 29 while AGE treatments were performed adding 10 lg/mL or 100 lg/ mL AGE-BSA (BioVision, Milpitas, CA, USA) to culture medium. 30 Control experiments were performed incubating the explants in the culture medium with the addition of 69 mM mannitol (HG control) or of 100 lg/mL BSA (AGE control).…”