[Detergent-like effects of the lipofuscin retinoid component A2-E in retinal pigment epithelial cells]

Several lines of evidence suggest that excessive accumulation of lipofuscin in postmitotic retinal pigment epithelial (RPE) cells with age and in various hereditary retinal diseases, plays a pathogenetic role. The lipofuscin retinoid component A2-E (N-retinylidene-N-retinylethanolamine) inhibits lysosomal degradation. Here we sought to evaluate additional toxic mechanisms of A2-E, whereby possible detergent-like effects on various membranes in human RPE cells were investigated by latency measurements.

A postnuclear supernatant prepared from cultured human RPE cells was used to isolate intact lysosomes by fractionation of cellular organelles in two sequential gradients. Destabilization of the lysosomal membrane was tested by incubating the purified lysosomal fraction in the presence of A2-E and subsequent measurement of the latency of the lysosomal luminal marker beta-hexosaminidase. In order to compare the effect of A2-E on other cellular membranes, latencies of the specific markers succinate dehydrogenase and UDP-galactosyltransferase were assessed using partially purified mitochondria and microsomes. Intactness of the plasma membrane was tested by including A2-E in the culture medium before leakage of lactate dehydrogenase into the medium was determined.

A more than 100-fold purification of the lysosomal fraction was achieved. Except for a minor activity of the mitochondrial marker, no contamination with other cell fractions was observed. Intactness of the purified lysosomes was well preserved during incubation in isotonic media and provided the basis for investigations on a possible detergent-like action of A2-E on lysosomal integrity. At concentrations above 2 microM A2-E, progressive leakage of the lysosomal marker was observed. In comparison leakage of the mitochondrial marker was induced at significantly lower concentrations (1 microM), whereas ER/Golgi membranes and the plasma membrane were relatively insensitive to a detergent effect of the retinoid.

The described practical and fast methodology to obtain highly purified and intact lysosomes from RPE cells, provides a very suitable tool for investigations on compounds affecting the lysosomal structure. The results suggest that A2-E causes disintegration of the lysosomal membrane at relatively low concentrations which may implicate an involvement of such a mechanism in triggering lipofuscin-induced dysfunction of aged RPE in vivo. Secondary to disintegration of the lysosomal membrane, damage to mitochondria might be an additional pathogenic mechanism. Our data provide evidence for surfactant-like properties of A2-E on biomembranes which might be operative in retinal diseases associated with excessive lipofuscin accumulation including age-related macular degeneration.