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B. G. Kennedy, S. Brown, N. J. Mangini; Effect of Ammonium Chloride on Pigmentation in Primary Cultured Human Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2008;49(13):482. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
: Cultured human retinal pigment epithelium (HRPE) are widely employed as a model system to examine RPE function. Both primary cultures and established cell lines differ in the extent of differentiation, in particular in the extent of pigment formation. The purpose of the present work is to determine the effect of ammonium chloride treatment, an agent shown to increase pigmentation in skin melanocytes, on cultured HRPE.
: Experiments were performed in primary RPE cultures established from four adult human donors (age range 47 - 82 years). Cells were maintained in Eagle’s modified MEM to which was added 17 mM additional sodium bicarbonate, 5% calf serum and 10% fetal bovine serum. Confluent HRPE monolayers were exposed to ammonium chloride concentrations ranging from 5 to 20 mM for periods ranging from 3 days to 2 weeks. Cells were examined by phase contrast microscopy, immunofluorescence and western blotting. Melanin content was assayed spectrophotometrically.
: Treatment of primary HRPE cultures with ammonium chloride markedly increased morphological differentiation. Effects were evident after 3 days exposure and observed at the lowest (5 mM) ammonium concentration tested. Control cultures frequently contained spindle-shaped mesenchymal-appearing cells, while ammonium treated cells had well-differentiated epitheloid morphology, with a tightly packed cobblestone appearance and sharply defined cell borders. In line with the observed morphological changes, immunostaining of the tight junction protein Z-01 was increased by ammonium treatment. Ammonium chloride treatment also caused translocation of the calcium sensing receptor from a predominantly cytoplasmic to a plasma membrane localization. Pigmentation was dramatically increased by ammonium treatment, with pigment level increased above that found in control cultures within six days of ammonium exposure. Tyrosinase (the rate limiting enzyme in pigment formation) expression, assayed by western blotting, was more than doubled by ammonium treatment.
: Ammonium chloride dramatically increases pigmentation and alters cell morphology in primary cultured HRPE. This treatment can be used as a tool to readily produce pigmented HRPE cells in vitro. Examination of the mechanism by which ammonia exerts its effects will shed light on factors involved in the control of melanization and differentiation in HRPE.
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