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Tae-im Kim, Hun Lee, Ji Won Jung, Eung Kweon Kim; Expression and secreting mechanism of mucins induced by DA-6034 in cultured human conjunctival epithelial cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):912. doi: https://doi.org/.
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The purpose of this study was to investigate the mechanism of ocular mucin secretion mediated by DA-6034 in cultured human conjunctival epithelial cells.
After application of 100 μM DA-6034, in cultured human conjunctival epithelial cells, expressions of MUC1, MUC4, MUC5AC, MUC16, P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors were measured by reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, and Western blot analysis. The effects of 100 μM DA-6034 on expression of mucin-secreting cells were measured by the counting of Periodic Acid-Schiff (PAS) positive cells. The effects of DA-6034 on Cl- currents were measured by perforated patch clamp. The changes of Intracellular Ca2+ concentrations ([Ca2+]i) mediated by 100 μM DA-6034 were measured by loading cultured human conjunctival epithelial cells with fluorescent calcium indicator Fluo-4/AM and 0.05% pluronic F-127. The level of intracellular Ca2+ was monitored by fluorescence monitoring camera
RT-PCR and real-time PCR showed that DA-6034 increased MUC1, MUC4, MUC5AC, MUC16, P2Y2, P2Y4, and P2Y6 receptor gene expression. Western blot analysis showed that DA-6034 increased MUC5AC and MUC16 protein expression. DA-6034 induced the expression of mucin-secreting cells as demonstrated by PAS staining. DA-6034 stimulated not only active Cl- transport but also [Ca2+]i increases in cultured human conjunctival epithelial cells. [Ca2+]i increases were demonstrated by emitted fluorescence.
We concluded that DA-6034 stimulated mucin production and secretion accompanied with the enhanced expression of P2Y2, P2Y4 and P2Y6 receptor in cultured human conjunctival epithelial cells. In addition, DA-6034 stimulated active Cl- transport and [Ca2+]i increases.
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