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M.J. Hoegger, L.A. Levin; Mitochondria From Brain and the Retinal Ganglion Cell–Like RGC–5 Line Differ in Superoxide Production . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2571.
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© ARVO (1962-2015); The Authors (2016-present)
We previously showed that superoxide production after retinal ganglion cell (RGC) axotomy is an early step in signaling apoptosis. To better understand the source of superoxide, we compared the production of superoxide with different substrates in mitochondria isolated from rat cerebral cells and the RGC–like RGC–5 line in the presence of specific mitochondrial electron transport chain (METC) substrates and inhibitors.
Mitochondria were isolated from 200 mg portions of rat cerebral cortex or cultured RGC–5 cells using standard protocols. Mitochondria were then suspended in PBS, and concentration normalized using the mitochondria–specific fluorescent probe MitoTracker Green FM. Assays were carried out in triplicate in 96–well assay plates using the H2O2 fluorescent probe Amplex Red to indirectly measure superoxide, with fluorescence readings were taking approximately every 15 seconds for 10 minutes using a Victor2 1420 multi–label counter. Substrates were glutamate and malate (Glu/Mal) or succinate (Succ), and inhibitors rotenone (Rot) or antimycin A (Anti A). The rate of H2O2 production in presence of substrates or inhibitors was adjusted to the basal production, and are expressed in fluorescent units / second.
In the presence of the Glu/Mal, the rate of H2O2 production in brain and RGC–5 was 0.621± 0.221 and 0.036±0.017 (p = 0.025), respectively. Rot increased the rate of H2O2 production primarily in RGC–5 cells to 0.751±0.267 and 0.088±0.046, respectively (p = 0.035). In the presence of Succ, the brain and RGC–5 mitochondrial H2O2 production rate was 0.458±0.159 and 0.065±0.034, (p = 0.037). Anti A increased the rate in brain but not RGC–5 cells to 0.974± 0.355 and 0.063±0.033 (p = 0.028).
Brain and RGC–5 mitochondria produce superoxide at different rates in the presence of METC substrates and inhibitors. This difference between these cell types may relate to RGC–selective death seen in diseases such as Leber’s hereditary optic neuropathy.
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