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.
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 (H2O2)-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 H2O2-induced retinal cell apoptosis occurred at 4 h after H2O2 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 H2O2 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 H2O2 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 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 [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.
Department of Genetics and Molecular Biology, School of Medicine, Xi'an Jiaotong University, Xi'an, China.
Yan
Y
Feng
Baoying
B
Wang
Fangying
F
Du
Hongbo
H
Li
Shaolan
S
Wang
Chenghu
C
Hu
Chunhui
C
Zhu
Xiaorui
X
Yu
D000818:Animals; D017209:Apoptosis; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D020013:Calcium Signaling; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D019610:Cytoprotection; D004958:Estradiol / Q000494:pharmacology*; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D009025:Morpholines / Q000494:pharmacology; D018696:Neuroprotective Agents / Q000494:pharmacology*; D009543:Nifedipine / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D061251:Primary Cell Culture; D051381:Rats; D017207:Rats, Sprague-Dawley; D055351:Retinal Neurons / Q000502:physiology
D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D002867:Chromones; D004958:Estradiol; D006861:Hydrogen Peroxide; D009025:Morpholines; D018696:Neuroprotective Agents; D009543:Nifedipine; D016877:Oxidants; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one