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V. F. Roche, H. Liu, J. L. Peterson, M. Zhao, C. A. Opere, G. L. Zhan, S. E. Ohia; Further Studies on the Effect of Bicyclic Hexahydroaporphines on Apoptosis of Retinal Ganglion Cells (RGC-5). Invest. Ophthalmol. Vis. Sci. 2007;48(13):4204.
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We have previously shown that bicyclic hexahydroaporphines (HHAs) including nor-HHA (1) and N-methyl HHA (2), attenuate ischemia-induced excitatory neurotransmitter release from isolated bovine retinae and inhibit glutamate-induced apoptosis in retinal ganglion cells (RGC-5). Previous RGC-5 studies employed 32 mM glutamate and a strongly acidic pH. In the present study, we investigated the effect of HHAs on glutamate-induced apoptosis in RGC-5 at pH 7.45 utilizing a lower concentration of glutamate (6 mM) to induce apoptosis.
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 HHAs (1 nM - 10 µM) for 1 hour prior to 24 hour exposure to 6mM glutamate (pH = 7.45). 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). The effect of HHAs on cell viability is reported as percent reversal of glutamate-induced apoptosis.
Glutamate (5 - 32 mM) induced apoptosis in RGC-5 cells in a concentration-dependent manner. Glutamate (6 mM) caused a 32% decrease in cell viability, which was attenuated by 2 at contentrations of 1 and 10 µM. Compound 2 (1 and 10 µM) reversed 6 mM glutamate-induced apoptosis by 6% and 15%, respectively (p<0.05). Compound 2 (10 µM) was more potent than MK801 (1 µM) and memantine (1 µM) in attenuating 6 mM glutamate-induced apoptosis. Interesting, MK 801 (1 µM) and both concentrations of 1 (1 and 10 µM) had no significant effect on apoptosis induced by glutamate (6 mM) in this experiment (p>0.05).
Our results demonstrate that the lower concentration of glutamate-induced apoptosis was prevented by N-methyl HHA (2) but not by nor-HHA (1). We conclude that the HHAs are capable of preventing cell death in glutamate-treated RGC-5 cells depending on the nature of the stimuli.
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