Fg. Holz et al., Inhibition of lysosomal degradative functions in RPE cells by a retinoid component of lipofuscin, INV OPHTH V, 40(3), 1999, pp. 737-743
PURPOSE. To investigate the effect of the lipofuscin component N-retinylide
ne-N-retinylethanolamine (A2-E) on degradative functions of lysosomes in hu
man retinal pigment epithelial (RPE) cells and to evaluate its mechanism of
action.
METHODS. A2-E was coupled to low-density lipoprotein (LDL). Human RPE cell
cultures were loaded with the A2-E/LDL complex, and controls were run with
medium containing LDL alone. To determine whether A2-E accumulated in lysos
omes, cells were fractionated in a Percoll gradient, and protein degradatio
n was determined by metabolic labeling and measurement of the release of lo
w-molecular-weight radioactivity. Lysosomal degradation was distinguished f
rom nonlysosomal degradation by inclusion of NH4Cl in the medium. The metab
olism of sulfated glycosaminoglycans was studied by radiosulfate incorporat
ion in pulse-erase experiments. Intralysosomal pH was determined using a fl
uorescent lysosomotropic pH indicator.
RESULTS. A2-E accumulated almost exclusively in the lysosomal compartment.
Lysosomal protein degradation was reduced in a dose-dependent fashion in A2
-E-treated cells. The selectivity of A2-E on lysosomal function was demonst
rated by its lack of effect on degradation of extralysosomal protein. Lysos
omal glycosaminoglycan catabolism of RPE cells was also strongly inhibited
by A2-E. Lysosomal pH was increased by A2-E.
CONCLUSIONS. The findings indicate that accumulation of A2-E in RPE cells i
nterferes with lysosomal functions as exemplified by its inhibitory effect
on protein and glycosaminoglycan catabolic pathways. The quaternary amine c
haracter of the A2-E apparently causes a perturbation of the acidic intraly
sosomal milieu, resulting in diminished hydrolase action and consequent acc
umulation of undegraded material. Such mechanism could be operative in reti
nal diseases associated with excessive lipofuscin accumulation including ag
e-related macular degeneration.