A2E, a lipofuscin fluorophore, in human retinal pigmented epithelial cellsin culture

PURPOSE. To study A2E, a component of retinal pigmented epithelial (RPE) ce ll lipofuscin, after its internalization by cultured human RPE cells. METHODS. A2E was synthesized and incubated with an adult RPE cell line devo id of native lipofuscin. To investigate the cellular compartmentalization o f A2E, cells were incubated simultaneously with A2E and a fluorescent acido tropic probe, (Lysotracker Red DND-99; Molecular Probes, Eugene, OR). Plasm a membrane integrity was evaluated by assaying for leakage of the cytoplasm ic enzyme lactate dehydrogenase (LDH), by fluorescence nuclear staining wit h a membrane-impermeant dye and by morphologic criteria. The emission spect rum of internalized A2E was also determined. The levels of A2E accumulated by the cultured cells were quantified by high-performance liquid chromatogr aphy and compared with amounts present in RPE isolated from human eyes. RESULTS. Internalization of A2E by the RPE cells was evidenced by the acqui sition of intracellular granules detectable by fluorescence confocal imagin g. Internalized A2E had an emission maxima of 565 to 570 nm. The levels of A2E accumulating in cells incubated with 10 to 25 mu 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 pref erential accumulation in acidic organelles. The elevated LDH levels that we re measured after exposure to 50 and 100 mu M A2E were attributable to memb rane damage in a subpopulation of the A2E-accumulating cells, determined by fluorescence nuclear labeling. CONCLUSIONS. Internalized A2E has an affinity for acidic organelles. The me mbrane damage exhibited by A2E-accumulating RPE is dependent on the concent ration of A2E and reflects the ability of this amphiphilic compound to exer t detergent-like effects.