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Mary C. McGahan, Steven Nagar, Malgorzata Goralska, Lloyd N. Fleisher, Jill Harned; Transepithelial Iron Transport and Polarized Iron Reuptake in Retinal Pigmented Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5347.
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Our earlier studies have shown that iron levels in the intraocular fluids and the crystalline lens are carefully controlled by a functioning blood ocular barrier (BOB) system. However, the mechanisms by which iron is taken up into the eye and how intraocular levels of iron are regulated are not known. The purpose of this study was to determine how iron is transported across the retinal pigmented epithelium (RPE), which forms an important part of the BOB's.
Canine RPE were grown in transwell culture plates where the apical and basolateral surfaces are separate. Formation of tight junctions was determined by transepithelial resistance and polarization of cells determined by directional secretion of glutamate. Tight junctional, polarized RPE were incubated with 59Fe labeled transferrin on either the apical or basolateral sides and samples of media collected from the opposite side at 1-24h were counted. At 24h, RPE were washed with fresh media and efflux of iron determined on both sides at 6 and 24h.
At 24h transepithelial iron movement was greater in the apical to basolateral direction (5.6% of total iron loaded appeared in the basolateral compartment) vs. basolateral to apical (1.6% of the total iron loaded appeared in the apical compartment). Efflux measurements at 6h demonstrated a higher amount of iron secreted into the basolateral (18.7% of total cell lysate iron) vs. apical compartment (5.5%) of cells loaded with iron on the apical side. Interestingly, at 24h there is a loss of greater than 95% of the counts present at 6h in the basolateral compartment of cells loaded with iron on the apical side, while there is a 5-fold increase in iron in the apical compartment of these same cells at 24h.
There is polarized movement of iron across the RPE monolayer, with greater movement of iron in the apical to basolateral direction. Most interesting are the results of the efflux experiments where RPE loaded with iron from the apical side initially (6h) had more iron enter the basolateral than apical compartment. However, at 24h there is almost no measurable iron present in the basolateral compartment, while iron continues to accumulate in apical compartment. This result indicates that there is an increase in transferrin receptor on the basolateral surface after iron loading which is responsible for the removal of iron from the surrounding medium. Iron movement into and out of cells is a complex process involving a number of different proteins. It is apparent from these results that there is a dynamic polarized movement and localization of these components that is altered by iron.
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