Potassium Channel Blockers Inhibit UV-B Induced Apoptosis of Corneal Epithelial Cells: A Possible Role for Elevated K+ in Tears

Abstract

Purpose: : The tears differ from most extracellular fluids in that the K⁺ concentration is 20-25 mM compared to about 4.2 mM in other extracellular fluids. The overall hypothesis of this study is that high [K⁺] in tears can protect the corneal epithelium from ambient UV-B radiation. We previously reported that UV-B activates K⁺ channels in corneal epithelial cells in vitro, leading to apoptosis, and that high [K⁺]_o blocks K⁺ efflux and reduces UV-induced apoptosis (IOVS 2008;49 E-abstracts 2934, 5304 ). The purpose of this study is to show that K⁺ channel blockers can also block K⁺ efflux and inhibit apoptosis of corneal epithelial cells exposed to UV-B.

Methods: : Activation of K⁺ channels in cells exposed to 80 mJ/cm² UV-B was measured by whole cell perforated patch-clamp recording, followed by exposure to 4-AP, quinidine or BDS-1 to determine effects of K⁺ channel blockers on UV-activated currents. Cells were also exposed to 100-150 mJ/cm² UV-B followed by incubation in culture media containing 4-AP, quinidine or BDS-1 for 6 hr. Apoptosis was determined by measuring caspase-3 activity and by a TUNEL assay with flow cytometry.

Results: : Following activation of K⁺ currents by UV, 4-AP (4 mM) and quinidine (0.6 mM) slowed the activation of the K⁺ currents in response to voltage steps. The specific K_v3.4 channel blocker, BDS-1 (1 uM), blocked most of the UV-activated current. 4-AP had no effect on UV-induced (150 mJ/cm²) caspase-3 activity or DNA fragmentation (TUNEL assay). Quinidine (0.05-1 mM) and BDS-1 (0.01-0.1 uM) caused dose-dependent inhibition of caspase-3, reducing activity to 45% and 67% of UV-induced levels, respectively, at the maximum dosages. Quinidine (0.01-0.05 mM) reduced the percentage of apoptotic cells measured by the TUNEL assay after 100 mJ/cm² UV exposure from 15% (no quinidine) to 7% at the maximum dosage. After 150 mJ/cm² UV apoptotic cells were reduced from 19% to 6% by quinidine. BDS-1 (0.03 uM) reduced the number of apoptotic cells induced by UV (150 mJ/cm²) from 11% (no BDS-1) to 3.6%. Cultures not exposed to UV contained only 0.7% apoptotic cells.

Conclusions: : The data show that K⁺ channel blockers, like high [K⁺]_o, reduce UV-activated K⁺ currents and inhibit the activation of apoptotic pathways in corneal epithelial cells in culture exposed to UV-B radiation at levels relevant to ambient UV exposure. This suggests that the relatively high [K⁺] in tears may also inhibit UV-B induced efflux of K⁺ from corneal epithelial cells in the intact cornea, reducing apoptotic damage.

Keywords: cornea: tears/tear film/dry eye • cornea: epithelium • lacrimal gland