Figure 4. D5 agonists reduce levels of photoreceptor outer segment autofluorescence.

A. Cultured ARPE-19 cells were examined by confocal fluorescence microscopy following 7 days of incubation without (i) or with (ii) unlabeled photoreceptor outer segments (POS). Lipofuscin-like cellular autofluorescence (green) was detected in panel ii using a fluorescein filter set (ex 480 nm, em 535 nm). Nuclei were visualized by DAPI staining blue. Scale bar = 10 μm. Autofluorescence associated with POS incubation (iii, pseudocolored green), and the signal from LysoTracker Red (ex 540 nm, em >570) showed considerable overlap (vi), implying the majority of POS were in acidic organelles 2 hrs after outer segments were removed from the bath. (v – DIC image). Scale bar = 10 μm.

B. SKF 81297 reduced the autofluorescence from internalized POS. Cells were fed POS for three hours, washed, and two hours chase period were allowed for outer segment delivery to the lysosomes. At this point, 10 μM SKF 81297 was added to the cells (adding the drug after the two hour interval ensured effects were restricted to outer segment digestion and did not alter binding or phagocytosis). This two stage treatment was repeated every 1–2 days for one week, with a total of three treatments. Cells were dissociated and the autofluorescence excited at 488 nm was determined using flow cytometry. Compared to control cells (blue), exposure to POS shifted the fluorescence to the right (red), indicating an increased fluorescence. Treatment with SKF 81297 shifted the curve back to the left (green) as autofluorescence was reduced.

C. Quantification of autofluorescence reduction by SKF 81297. The mean autofluorescence was increased by incubation with POS but restored to low levels by SKF 81297. SKF 81297 alone did not alter autofluorescence levels. Bars represent the mean ± SEM fluorescence in each sample and are representative of results in three separate experiments. Data were normalized to peak levels in untreated cells to control for variation between trials. * p <0.05 vs. control; ** p<0.05 vs. POS.

D. Bodipy-pepstatin A binding is improved by SKF 81297. While binding of the probe was reduced by treating cells with 10 μM chloroquine for 48 hrs, concurrent exposure to 10 μM SKF 81297 restored fluorescence. These results are consistent with chloroquine decreasing activity of pH sensitive lysosomal enzyme cathepsin D, and of SKF 81297 restoring enzyme activity.