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L.A. Voloboueva, S.S. Miller; Involvement of Mitochondrial Metabolism in the Regulation of the RPE Phagocytosis of Photoreceptor Outer Segments . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1642.
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Purpose: To investigate the role of mitochondrial metabolism in the regulation of photoreceptor outer segments (OS) phagocytosis by retinal pigment epithelium (RPE). We also examined the effects of oxidative stress on the rates of the RPE phagocytosis, and the alteration in oxidant-induced changes by mitochondrial protective compounds. Methods: Second or third passage confluent monolayers of human fetal RPE (hfRPE) cell cultures and quantitative fluorescence scanning and microscopic observations were used to determine the phagocytic binding and internalization of FITC-labeled outer segments. Bound and internalized OS were distinguished by trypan blue quenching of external OS fluorescence. Mitochondrial potential was evaluated using the mitochondrial specific fluorescence probe, JC-1. Results: Confluent hfRPE cells (occludin localized to tight junctions) demonstrated significantly higher number of mitochondria with high mitochondrial potential (ΔΨ) in comparison with partially confluent hfRPE cells (occludin labeling throughout the cell). The mitochondrial uncoupler, FCCP (4 µM), and the electron transport inhibitor antimycin A (1µM) reduced the rates of OS internalization by ≈35% and ≈25%, respectively, and decreased ΔΨ values in confluent hfRPE cells. In partially confluent hfRPE cells FCCP application caused an ≈15% decrease in the rates of OS internalization. Application of an oxidative agent, tert-butyl hydroperoxide, caused lower ΔΨ values and a dose-dependant decrease in the phagocytic OS internalization by confluent hfRPE cells. This oxidant-induced decrease was partially reversed by preincubation of the hfRPE cells with the mitochondrial protective agent acetyl-L-Carnitine (2 mM). Conclusions: A decrease in mitochondrial potential reduces the ability of RPE cells to internalize OS, and the results of oxidative stress experiments suggest a link between oxidative stress, mitochondrial potential, and RPE phagocytic capability.
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