Abstract
Purpose :
Interphotoreceptor retinoid binding protein (IRBP) is the physiological carrier that transports the visual pigment chromophore 11-cis retinal from the retinal pigment epithelium (RPE) to rod photoreceptor outer segments. In the outer segments, 11-cis retinal combines with available opsin to form rhodopsin, but can also generate precursors of lipofuscin that eventually accumulate in RPE lysosomes. We tested the effects of different extracellular concentrations of 11-cis retinal and IRBP on the formation of lipofuscin precursors in the outer segments of isolated human rod photoreceptors.
Methods :
Fresh human donor eyes procured through the National Disease Resource Interchange were dissected and the retinas isolated within 48 hrs after death. Isolated retinas were bleached to destroy residual rhodopsin, and stored in physiological solution at 4 °C. IRBP was purified from bovine retinas and its concentration was determined by absorption spectroscopy and amino-acid analysis. Metabolically active rod photoreceptors were isolated from the peripheral regions and incubated in the presence of either IRBP with 11-cis retinal to regenerate rhodopsin, or IRBP alone. After incubation, cells were washed with physiological saline and placed on a fluorescence microscope stage at 37 °C. Cells were then exposed to different concentrations of 11-cis retinal and IRBP for 5 or 10 min. The levels of rod outer segment lipofuscin precursors were determined from their fluorescence (λex, 490 nm; λem >520 nm).
Results :
Exposure to 11-cis retinal resulted in a significant increase in lipofuscin precursor levels, which was much higher for cells that contained rhodopsin. Lipofuscin precursor formation increased with the concentration of 11-cis retinal and time of exposure. Higher concentrations of IRBP resulted in smaller increases in lipofuscin precursor formation. The presence of IRBP however could not fully prevent lipofuscin precursor formation from 11-cis retinal.
Conclusions :
11-cis retinal can generate lipofuscin precursors in the outer segment of human rod photoreceptors. Available opsin limits the formation of lipofuscin precursors, as do higher extracellular concentrations of IRBP. Although not able to fully prevent it, IRBP probably mitigates lipofuscin formation by pacing the delivery of 11-cis retinal to the outer segment.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.