May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Elevated Extracellular K+ Inhibits Apoptosis of Corneal Epithelial Cells Exposed to UV-B Radiation
Author Affiliations & Notes
  • J. L. Ubels
    Dept of Biology, Calvin College, Grand Rapids, Michigan
  • K. R. De Young
    Dept of Biology, Calvin College, Grand Rapids, Michigan
  • M. P. Schotanus
    Dept of Biology, Calvin College, Grand Rapids, Michigan
  • Footnotes
    Commercial Relationships  J.L. Ubels, None; K.R. De Young, None; M.P. Schotanus, None.
  • Footnotes
    Support  NIH Grant EY018100
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 2934. doi:https://doi.org/
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      J. L. Ubels, K. R. De Young, M. P. Schotanus; Elevated Extracellular K+ Inhibits Apoptosis of Corneal Epithelial Cells Exposed to UV-B Radiation. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2934. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : The tears differ from most extracellular fluids in that the K+ concentration is about 20 mM compared to about 4.2 mM in other extracellular fluids. The function of the high [K+] in tears is unknown, especially with respect to the health of the cells of the ocular surface. Previous studies have shown that UV-C causes K+ channels in corneal epithelial cells to open, activating apoptotic pathways (IOVS 44:5095, 2003), and that elevated extracellular [K+] prevents chemically induced apoptosis in lymphocytes (J Biol Chem 272:32436, 1997). The hypothesis of this study was that high [K+]o will inhibit the activation of apoptotic pathways when human corneal limbal epithelial (HCLE) cells are exposed to UV-B.

Methods: : HCLE cells were exposed to 50-200 mJ/cm2 UV-B followed by incubation in culture media containing isosmotic 5.5, 25, 50 or 100 mM K+ for 6 hours. Activation of apoptosis was determined by measurement of caspase-3 activity, a DNA laddering assay and a TUNEL assay with flow cytometry. Controls were cells not exposed to UV-B and incubated in 5.5 mM K+ for 6 hours.

Results: : In cells exposed to 50 or 100 mJ/cm2 UV-B, the highest level of caspase-3 activity was observed in cells incubated in 5.5 mM K+ after UV-B exposure, while increased extracellular [K+] caused dose-dependent caspase-3 inhibition, reaching control levels at 100 mM. DNA laddering in cells exposed to 200 mJ/cm2 UV-B was similarly reduced in the presence of elevated [K+]o. In the TUNEL assay, the percentage of apoptotic cells was 41.7% in cultures incubated in 5.5 mM K+ after exposure to 200 mJ/cm2. UV-B (200 mJ/cm2) induced apoptosis decreased to 35% of cells incubated in 25 mM K+ and 14.2% in the presence of 100 mM K+, as compared to 4.1% in controls. After exposure of cells to 150 mJ/cm2 UV-B, the percentage of apoptotic cells was 9.5% of cells cultured in 5.5 mM K+, versus 1.9% in controls. This decreased to 5.5% and 2.9% of cells incubated in 25 mM or 100 mM K+, respectively. Exposure of cells to 100 mJ/cm2 UV-B followed by incubation in elevated [K+]o yielded similar results.

Conclusions: : The data show that high [K+]o reduces the activation of apoptotic pathways in corneal epithelial cells exposed to UV-B radiation at levels relevant to ambient UV exposure. It is suggested that the relatively high [K+] in tears reduces the loss of intracellular K+ from ocular surface cells in response to UV exposure, preventing apoptotic damage.

Keywords: cornea: epithelium • radiation damage: light/UV • apoptosis/cell death 
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