K 27Inhibition of lysosomal functions of human RPE cells by a retinoid component of lipofuscin (A2-E)
F. Schütt, M. Bergmann, J. Kopitz, F. G. Holz
Background: We have shown that a fluorescent retinoid compound of lipofuscin (A2-E, N-retinylidene-N-retinylethanolamine) affect lysosomal degradative functions of human RPE cells, and that it possess phototoxic properties. Lysosomal function is essential for degrading phagozytosed outer segment disks. Herein we sought to determine possible mechanisms apart from pH-elevation for the inhibitory effect on lysosomal function.
Methods: Potter-Elvejem-homogenization was used to prepare RPE cell homogenates. The activities of 24 different lysosomal enzymes were measured in RPE cell homogenates before and after incubation with various A2-E concentrations ranging from 0.1 to 10 uM. The protein content was determined, and the activity was calculated as U/mg protein. Further investigations included measurements of lysosomal membrane stability after incubation with various concentrations of A2-E (0.01-10 m M).
Results: Activities of isolated lysosomal enzymes were unaffected by the various A2-E concentration tested. A2-E concentrations above 2 m M induced a rupture of the lysosomal membrane as indicated by the release of the marker enzyme ß-hexosaminidase.
Conclusions: The results indicate that the inhibitory effect on lysosomal degradation in human RPE cells is not mediated via a direct inhibitory effect on lysosomal enzymes. A previously described elevation of the acidic intralysosomal milieu may be mediated by interaction with proton pumps in the lysosomal membrane. A diminished lysosomal enzymatic action by A2-E would be expected to result in accumulation of undegraded material. Rupture of lysosomal membranes indicates detergent properties of A2-E. Exposure of intralysosomal enzymes within the cytosol would lead to lethal RPE cell damage. A better understanding of the molecular mechanisms are important for the development of new therapeutic strategies for ARMD.
Department of Ophthalmology, University of Heidelberg, Im Neuenheimer Feld 400, D-69120 Heidelberg;
supported by DFG (Ho 1926 2-1)