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Patrycja Kaczara, Anna K. Pilat, Anja Herrnreiter, Christine M. Skumatz, Tadeusz J. Sarna, Janice M. Burke; Do Melanosomes Release or Bind Iron Ions inside RPE-Derived Cells?. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4777.
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We previously showed that melanosomes protect RPE cells against oxidative stress induced by hydrogen peroxide. The mechanism could depend upon the metal ion-binding property of melanin, which can theoretically protect against metal ion-dependent H2O2 decomposition. However, this property of the pigment has not been shown within cells. Here we investigated the interaction of iron ions with melanosomes inside RPE-derived cells.
Two experimental models were used. In one model, used to determine iron retention by granules within cells, control porcine melanosomes with endogenous iron content or melanosomes pre-loaded with higher iron were introduced into ARPE-19 cells by phagocytosis. A second model was used to determine iron binding by granules already within cells. Accordingly, ARPE-19 cells containing phagocytized control melanosomes were treated repeatedly with iron added to the culture medium. Comparable cytosolic iron levels in cells were controlled by western blot quantification of the iron-binding protein ferritin. Iron bound to melanosomes was then determined by two methods: in intact cells using ESR spectroscopy, or in re-isolated granules using the colorimetric bathophenanthrolene iron assay. Cytotoxicity after H2O2 treatment of cells containing control or preloaded-with-iron melanosomes was quantified by a real-time imaging assay using propidium iodide as a fluorescent reporter.
Melanosomes pre-loaded with iron exhibit higher iron than control granules for at least several days after delivery to ARPE cells indicating retention inside cells of bound-to-melanin iron ions. Melanosomes already within cells bind additional iron when exogenous iron is provided indicating that melanin within cells can accumulate iron. ARPE-19 cells containing melanosomes pre-loaded with higher iron do not differ from cells containing control granules in their cytotoxic response to H2O2-induced oxidative stress.
Melanosomes appear competent to bind and to retain bound iron ions within RPE-derived cells suggesting that the granules can participate in RPE iron homeostasis. Whether iron ion binding by melanosomes plays a role in their protection of cells against H2O2 is not yet clear, but this question is relevant because both oxidative stress to the RPE and elevated ocular iron have been implicated in AMD.
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