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Janet R. Sparrow, Craig A. Parish, Masaru Hashimoto, Koji Nakanishi; A2E, a Lipofuscin Fluorophore, in Human Retinal Pigmented Epithelial Cells in Culture. Invest. Ophthalmol. Vis. Sci. 1999;40(12):2988-2995.
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purpose. To study A2E, a component of retinal pigmented epithelial (RPE) cell
lipofuscin, after its internalization by cultured human RPE cells.
methods. A2E was synthesized and incubated with an adult RPE cell line
devoid of native lipofuscin. To investigate the cellular
compartmentalization of A2E, cells were incubated simultaneously with
A2E and a fluorescent acidotropic probe, (Lysotracker Red DND-99;
Molecular Probes, Eugene, OR). Plasma membrane integrity was evaluated
by assaying for leakage of the cytoplasmic enzyme lactate dehydrogenase
(LDH), by fluorescence nuclear staining with a membrane-impermeant dye
and by morphologic criteria. The emission spectrum of internalized A2E
was also determined. The levels of A2E accumulated by the cultured
cells were quantified by high-performance liquid chromatography and
compared with amounts present in RPE isolated from human eyes.
results. Internalization of A2E by the RPE cells was evidenced by the
acquisition of intracellular granules detectable by fluorescence
confocal imaging. Internalized A2E had an emission maxima of 565 to 570
nm. The levels of A2E accumulating in cells incubated with 10 to 25μ
M A2E were comparable to the amounts of A2E present in equal numbers
of RPE cells harvested from human eyes. Colocalization of A2E and the
Lysotracker probe revealed a preferential accumulation in acidic
organelles. The elevated LDH levels that were measured after exposure
to 50 and 100 μM A2E were attributable to membrane damage in a
subpopulation of the A2E-accumulating cells, determined by fluorescence
conclusions. Internalized A2E has an affinity for acidic organelles. The membrane
damage exhibited by A2E-accumulating RPE is dependent on the
concentration of A2E and reflects the ability of this amphiphilic
compound to exert detergent-like effects.
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