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Christina Locke, Michael W Dismuke, William Stamer, Brian S McKay; Effect of L-DOPA and Dopamine on exosome release from the RPE and Ciliary Body. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1526.
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Exosomes are nanovesicles, 40-100 nm in diameter, that function in cell-cell communication. We have shown that human RPE constitutively release exosomes, and that this release is reduced by stimulation of GPR143 with its agonist L-DOPA. The pigmented cells of the ciliary body (CB) also express GPR143 and other dopaminergic GPCRs. In this study, we test the hypothesis that both L-DOPA and dopamine, an antagonist of GPR143, alter exosome release from porcine RPE and CB.
Pig eyes were dissected to yield eyecups lined with RPE and CB tissue was cut into 5mm segments. The CB pieces were distributed into wells of a 6-well plate. Both tissues were incubated in either serum-free DMEM, or DMEM with 5υM L-DOPA or 5υM dopamine. Conditioned medium from the RPE was harvested after 25 minute incubation, while CB was incubated for 45 minutes. The exosomes released into the media were isolated by differential ultracentrifugation. Data from the CB tissue was standardized to protein content in each well to correct for variability, and experiments were conducted in triplicate. The effect of L-DOPA and dopamine stimulation on exosome release was analyzed using silver staining to characterize protein content of the exosomes released.
Porcine RPE and CB constitutively release exosomes in serum-free DMEM. In RPE, stimulation of GPR143 with L-DOPA showed a similar trend of reduced exosome release as previously seen in human tissues (n=3, p < 0.08), while stimulation with its antagonist dopamine results in similar reduction of release (n=3, p < 0.05). In CB, L-DOPA stimulation significantly decreased exosome release (n=3, p < 0.005), though dopamine stimulation had a variable response.
Our results suggest that human and pig RPE are similar with respect to constitutive release of exosomes, and L-DOPA lowers that release. Since GPR143 is the only known L-DOPA receptor, the response is likely due to this GPCR. We observed similar results in the CB experiments that also express GPR143, implicating this receptor. However, both RPE and CB express other dopaminergic receptors, complicating the response of exosome release. Dopamine is an antagonist of GPR143, and lowered exosome release from RPE, possibly suggesting activity from another receptor. Results using dopamine in CB were variable, even contradictory, perhaps suggesting multiple receptors involved in control of exosome release.
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