Holz F G, Schütt F, Kopitz J, Eldred G E, Kruse F E, Völcker H E, Cantz M
Department of Ophthalmology, University of Heidelberg, Germany.
Invest Ophthalmol Vis Sci. 1999 Mar;40(3):737-43.
To investigate the effect of the lipofuscin component N-retinylidene-N-retinylethanolamine (A2-E) on degradative functions of lysosomes in human retinal pigment epithelial (RPE) cells and to evaluate its mechanism of action.
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 lysosomes, cells were fractionated in a Percoll gradient, and protein degradation was determined by metabolic labeling and measurement of the release of low-molecular-weight radioactivity. Lysosomal degradation was distinguished from nonlysosomal degradation by inclusion of NH4Cl in the medium. The metabolism of sulfated glycosaminoglycans was studied by radiosulfate incorporation in pulse-chase experiments. Intralysosomal pH was determined using a fluorescent lysosomotropic pH indicator.
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 demonstrated by its lack of effect on degradation of extralysosomal protein. Lysosomal glycosaminoglycan catabolism of RPE cells was also strongly inhibited by A2-E. Lysosomal pH was increased by A2-E.
The findings indicate that accumulation of A2-E in RPE cells interferes with lysosomal functions as exemplified by its inhibitory effect on protein and glycosaminoglycan catabolic pathways. The quaternary amine character of the A2-E apparently causes a perturbation of the acidic intralysosomal milieu, resulting in diminished hydrolase action and consequent accumulation of undegraded material. Such mechanism could be operative in retinal diseases associated with excessive lipofuscin accumulation including age-related macular degeneration.
研究脂褐素成分N - 视黄叉基 - N - 视黄基乙醇胺（A2 - E）对人视网膜色素上皮（RPE）细胞溶酶体降解功能的影响，并评估其作用机制。
将A2 - E与低密度脂蛋白（LDL）偶联。用人RPE细胞培养物加载A2 - E/LDL复合物，对照组仅用含LDL的培养基培养。为确定A2 - E是否在溶酶体中积累，将细胞在Percoll梯度中进行分级分离，并通过代谢标记和测量低分子量放射性物质的释放来测定蛋白质降解。通过在培养基中加入氯化铵来区分溶酶体降解和非溶酶体降解。在脉冲追踪实验中，通过放射性硫酸盐掺入研究硫酸化糖胺聚糖的代谢。使用荧光溶酶体pH指示剂测定溶酶体内pH。
A2 - E几乎完全积累在溶酶体区室中。在A2 - E处理的细胞中，溶酶体蛋白质降解以剂量依赖性方式降低。A2 - E对溶酶体功能的选择性通过其对溶酶体外蛋白质降解无影响得以证明。A2 - E也强烈抑制RPE细胞的溶酶体糖胺聚糖分解代谢。A2 - E使溶酶体pH升高。
研究结果表明，A2 - E在RPE细胞中的积累会干扰溶酶体功能，如对蛋白质和糖胺聚糖分解代谢途径的抑制作用所示。A2 - E的季胺特性显然会导致溶酶体内酸性环境受到干扰，从而导致水解酶作用减弱，进而导致未降解物质积累。这种机制可能在与脂褐素过度积累相关的视网膜疾病中起作用，包括年龄相关性黄斑变性。
