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Impressum
Inhibition of ATP-consuming proton pump activity and pH-increment in human RPE lysosomes by A2-E, a major lipofuscin fluorophore
1Holz F. G., 1Schütt F., 2Bergmann M., 2Kopitz J.
1Dept. of Ophthalmology, INF 400, 2Institute of Pathobiochemistry; INF 220; University of Heidelberg, D-69120 Heidelberg; Germany
Objective: Various types of macular degeneration including ARMD
are associated with an excessive accumulation of lipofuscin (LF) in RPE
cells. A major fluorophore of LF is toxic A2-E (N-retinylidene-Nretinylethanolamine).
We have shown that A2-E causes striking inhibition of intralysosomal catabolic
pathways of human RPE cells and excluded a direct interaction with lysosomal
enzymes. Lysosomal hydrolases have acidic pH-optima. We determined the
effect of A2-E on lysosomal pH and the ATP-consuming proton pumping activity.
Methods: Intralysosomal pH of A2-E loaded and control RPE cells
was determined using a fluorescent lysosomotropic pH indicator. RPE cell
homogenates were prepared using Potter-Elvejem-homogenization. ATPase
activity was determined by measuring the release of [33P]phosphate from
[y-33P]ATP and proton translocating activity was assayed by measurement
of the proton transport-dependent accumulation of [3H]methylamine in the
lysosome.
Results: A2-E treated cells showed an elevation of intralysosomal
pH near neutrality. A2-E induced a dose-dependent inhibition of proton
pump ATPase activity with a 50% inhibition in the presence of 1µM
A2E in comparison to untreated controls. Likewise increasing A2-E concentrations
decreased intralysosomal [3H]methylamine accumulation.
Conclusion: The results indicate that the inhibitory effect of
A2-E on RPE lysosomal catabolic function is mediated by a perturbation
of the acidic intralysosomal milieu, and that the mechanism for the pH-shift
is a strong inhibition of ATP-consuming proton pumping activity. The findings
implicate excessive LF accumulation in cell dysfunction such as occurs
in ageing and macular degenerations since build up of A2-E in RPE lysosomes
would result in progressively impaired digestion of phagocytosed outer
segments.
Support: DFG grant Ho 1926/2-1, DFG Priority Program AMD (1088); Ba.-Wü.
Research Fund 500/2000