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H. Liu, J. Peterson, M. Zhao, G. Zhan, S. Ohia, C. Opere; Regulation of Apoptosis by Isoprostanes in RGC-5 Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4191.
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There is evidence that isoprostanes (IsoPs) can exert a dual regulatory effect on glutamate release from bovine retinae, in vitro (Opere et al., 2005). However, it remains to be determined whether IsoPs can affect apoptosis induced by glutamate retinal ganglion cells (RGCs). In the present study, we examined the effect of IsoPs on (1) cell viability and (2) glutamate-induced apoptosis in RGC-5 cells.
CellTiter-Blue® Cell Viability Assay (Promega, Madision, WI) was utilized in these experiments. Briefly, 5,000 RGC-5 cells were plated in each of 96 wells containing DMEM medium supplemented with 10% serum. After overnight incubation the medium was replaced with serum-free DMEM and pretreated with 8-isoprostane E2 (8-isoPGE2) and 8-isoPGF2α (1nM - 10 µM) for 2 hours prior to 24 hour exposure to 6mM glutamate (pH = 7.4). The cells were then treated for 4 hours with 20 µl of CellTiter-Blue® reagent, followed by 100 µl of stop solution. Fluorescence was measured at 560/590 nm using a Spectra max/Gemini EM fluorescent plate reader (Molecular Device Corp, Sunnyvale, CA).
8-isoPGE2 (100µM) induced 66% (p<0.01) apoptosis in RGC-5 cells while lower concentrations (1 nM-10µM) of the IsoP had no effect on cell viability. It was interesting to note that 8-isoPGE2 (10 - 100 µM) potentiated the apoptotic effect induced by glutamate (6mM). On the contrary, 8-isoPGE2 (0.1µM) was protective against glutamate (6mM -16mM)-induced apoptosis. For instance, 8-isoPGE2 (0.1 µM) attenuated glutamate (6mM)-induced apoptosis by 29% (n=16, p<0.05), an effect that was comparable to that of MK801 (1 µM). Although 8-isoPGF2α (1 nM to 100µM) had an inhibitory effect on cell viability, in the same concentration range, the IsoP had no potentiating effect on glutamate (6 mM)-induced apoptosis.
We conclude that isoprostanes can regulate cell viability and modulate glutamate-induced apoptosis in RGC-5 cells.
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