A2E, a lipofuscin fluorophore, in human retinal pigmented epithelial cells in culture.
OBJECTIVE
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 25 microM 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 microM A2E were attributable to membrane damage in a subpopulation of the A2E-accumulating cells, determined by fluorescence nuclear labeling.


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.
Sparrow
J R
JR
Department of Ophthalmology, Columbia University, New York, New York 10032, USA. jrs88@columbia.edu
Parish
C A
CA
Hashimoto
M
M
Nakanishi
K
K
D000368:Aged; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004705:Endocytosis / Q000502:physiology*; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008062:Lipofuscin / Q000378:metabolism*; D018613:Microscopy, Confocal; D008875:Middle Aged; D015388:Organelles / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D012168:Retinal Pigments / Q000378:metabolism*; D012176:Retinoids / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
C112040:A2-E (N-retinylidene-N-retinylethanolamine); D005456:Fluorescent Dyes; D008062:Lipofuscin; D012168:Retinal Pigments; D012176:Retinoids; D007770:L-Lactate Dehydrogenase