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O. Strauss, R. Neussert, M.O. Karl, A.D. Marmorstein, S. Wimmers, G. Richard; ATP–Induced Ca2+ Increases in Freshly Isolated Mouse RPE Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5540.
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© ARVO (1962-2015); The Authors (2016-present)
Much of what is known about retinal pigment epitheium (RPE) function is based on cultured cells. How individual cells in a monolayer in vivo behave is not investigated. In order to study individual differences of RPE cell function in a monolayer we investigated effects of ATP application on intracellular free Ca2+ in freshly isolated sheets of mouse RPE.
Mouse RPE sheets were isolated after incubation in Ca2+ and Mg2+–free PBS solution. Cells were loaded with fura–2–AM and subjected for fluorescence microscopy. The concentration of intracellular free Ca2+ was calculated after cellular calibration according to the method by Grynkiewicz et al. (JBC; 1985: 3440). Changes in intracellular free Ca2+ were calculated for different individual cells.
Freshly isolated RPE cells in a monolayer showed a resting concentration of intracellular free Ca2+ of 84 ± 3 nM (mean ± SEM; n = 34). Application of 100 µM ATP resulted in two different pattern of intracellular Ca2+ rises: in 30 % of cells a sharp rise followed by a decrease in intracellular free Ca2+ below the resting level 74 % of control and in 70 % of cells a sharp rise followed by a transient decrease below the base level (79.9 % of control) followed by a long sustained increase to 142 % of control. In repeated ATP applications the initial rise became smaller whereas the later occurring Ca2+ changes remained. In contrast to freshly isolated cells, in ARPE–19 monolayers all cells (resting Ca2+: 98.9 ± 3.1 nM; n = 9) responded to ATP in the same fashion: an initial sharp increase followed by slight decrease below the resting level (73 % of control). Also in ARPE–19 cells the initial peak decreased with repeated ATP stimulation. RT–PCR analysis of ARPE–19 cells revealed expression of P2x receptors.
In the same monolayer RPE cells showed individual different responses to ATP stimulation due to activation of different receptors. The desensitization effects and RT–PCR data inidicate that the initial Ca2+ rise results from stimulation of a purinergic receptor which function as ligand–gated Ca2+ channel. The second, later occurring increase in cytosolic Ca2+ results from activation of receptors linked to a second–messenger cascade. These different patterns of ATP–induced Ca2+ rises indicate that individual RPE cells in a monolayer are specialised to different aspects of RPE function.
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