Elevated Extracellular K+ Inhibits Apoptosis of Corneal Epithelial Cells Exposed to UV-B Radiation

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