May 2006
Volume 47, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2006
ATP–Induced Ca2+ Increases in Freshly Isolated Mouse RPE Cells
Author Affiliations & Notes
  • O. Strauss
    University Medical Center Hamburg–Eppendorf, Hamburg, Germany
    Experimental Ophthalmology,
  • R. Neussert
    University Medical Center Hamburg–Eppendorf, Hamburg, Germany
    Experimental Ophthalmology,
  • M.O. Karl
    University Medical Center Hamburg–Eppendorf, Hamburg, Germany
    Experimental Ophthalmology,
  • A.D. Marmorstein
    Department of Ophthalmology, University of Arizona, Tucson, AZ
  • S. Wimmers
    University Medical Center Hamburg–Eppendorf, Hamburg, Germany
    Experimental Ophthalmology,
  • G. Richard
    University Medical Center Hamburg–Eppendorf, Hamburg, Germany
    Eye Hospital,
  • Footnotes
    Commercial Relationships  O. Strauss, None; R. Neussert, None; M.O. Karl, None; A.D. Marmorstein, None; S. Wimmers, None; G. Richard, None.
  • Footnotes
    Support  DFG STR480/9–1
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 5540. doi:
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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)

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Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: retinal pigment epithelium • calcium • second messengers: pharmacology/physiology 
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