Dopamine Synthesis and Secretion by Cultured Human Retinal Pigment Epithelium and the Effects of Melatonin

Abstract

Abstract: : Purpose: The aims of this study are to determine whether cultured human retinal pigment epithelium (HRPE) synthesizes and secretes dopamine (DA), and to examine the effect of melatonin on DA synthesis and secretion. Methods: Cultured HRPE were grown in the presence or absence of 750 nM melatonin. Whole cell lysates and conditioned medium samples were taken at varying time points in a 12-hour light/12-hour dark cycle. Cell samples were analyzed for cell count, total protein, tyrosine hydroxylase (TH) and dopa-decarboxylase (DD) immunoreactivity on Western blots, and TH mRNA expression by RT-PCR. Cell and conditioned medium paired samples were analyzed for DA content by [³H]-radioenzymatic catecholamine assay. Results: For samples grown without melatonin, immunoblotting performed on HRPE whole cell lysates shows immunoreactive labeling by anti-TH as well as anti-DD. RT-PCR using gene-specific primers confirms TH mRNA expression. Cell samples analyzed for DA content show peak DA synthesis of 25 pg/million cells within 4 hours of light onset with a baseline level of 5 pg/million cells. Conditioned medium shows peak DA secretion at 15 pg/million cells 2 hours after light onset, followed by a plateau at 7 pg/million cells. For samples incubated with melatonin, immunoreactivity on immunoblots for anti-TH and anti-DD is abolished. DA content in cell samples is reduced to a level of 1 pg/million cells. Conditioned medium shows peak DA secretion at 4 pg/million cells 2 hours after light onset, followed by a fall to 1 pg/million cells. Conclusions: Cultured HRPE expresses TH and DD, the two enzymes required for DA synthesis. DA synthesis and secretion peaks within 4 hours of light onset followed by a return to baseline levels. Melatonin inhibits TH and DD expression with subsequent loss of DA synthesis and secretion. Experiments are in progress to further elucidate the details of this process. Dopamine synthesis and secretion by HRPE with modulation by melatonin may function in the circadian control of rod outer segment disc shedding and phagocytosis of shed discs by HRPE.

Keywords: dopamine • retinal pigment epithelium • melatonin