Abstract
Purpose :
Photoreceptors undergo a daily renewal process by shedding the distal 10% of their outer segments (POS), which are phagocytosed and degraded by the retinal pigment epithelium (RPE). Inefficient digestion of POS may lead to the buildup of lipids and proteins within or under the RPE and play a role in retinopathies such as age-related macular degeneration (AMD). In previous work, we found that L-DOPA, the ligand of the GPCR GPR143, had a significant effect on POS degradation between hours 4 and 12, likely related to efficient endosomal trafficking to the lysosomal compartment. Here we test whether dopamine, a molecular relative of L-DOPA, has the same effects between hours 4 and 12.
Methods :
We isolated POS from bovine retinas as previously described. POS were labeled with a pH-sensitive dye that increases fluorescence intensity as pH decreases, such that the POS fluoresces more brightly in the acidic lysosomal compartment. Primary porcine RPE was challenged with POS for 4 hours to allow endocytosis with and without L-DOPA or dopamine. We removed the excess POS, then added fresh medium with 1μM L-DOPA or 1μM dopamine. Phase-contrast and fluorescent images were captured every 4 hours for 12 hours. 4 experiments were conducted with 4 replicates in each. The number and area of fluorescent POS were analyzed using ImageJ and statistical analysis was performed using Prism Graph.
Results :
L-DOPA, the agonist of GPR143, increases the efficiency of POS degradation, but not POS uptake. Surprisingly, similar results were shown with dopamine, a GPR143 antagonist. 4 hours after POS introduction, the number of fluorescent particles was similar. Particle counts were similar for L-DOPA 270.5±29.95 and dopamine 276.9±36 (p-value > 0.5) treatments. At 12 hours, the same was true for both L-DOPA and dopamine. Both treatment groups displayed increased fluorescence relative to the 4-hour time point (L-DOPA 444.3±61, n=20 and dopamine 396±48, n=20), but were not significantly different from each other.
Conclusions :
Our results suggest that both L-DOPA and dopamine had similar effects on POS digestion throughout the 12-hour time course. Since the effects were similar, but the effects on GPR143 are opposite, we hypothesize a different explanation. Both L-DOPA and dopamine bind to and activate GPCRs, which are then endocytosed. Perhaps the effects are not GPCR specific, but rather nonspecific activation of the endosomal trafficking system in the cells.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.