Potassium ion fluxes in corneal epithelial cells exposed to UVB

Abstract

The goal of this study was to investigate the efflux of K+ from human corneal limbal epithelial cells (HCLE) exposed to ambient levels of UVB, which is known to cause apoptosis, and to examine the effect of K+ channel blockers on loss of potassium induced by UVB. HCLE cells were exposed to 100–200 mJ/cm2 UVB, followed by incubation in culture media with 5.5–100 mM K+, BDS-1, Ba2+ or ouabain. To measure intracellular cations, cells were washed in 280 mM sucrose and lysed in DI water. K+ and Na+ levels in lysates were measured by ion chromatography. HCLE cells showed maximal loss of K+i 10 min after exposure to UVB and 5.5 mM K+ media, with recovery of normal K+ levels after 90 min. Treatment with 1 μM BDS-1 following UVB exposure reduced the loss of K+ by HCLE cells. Exposure to 0.1–5 mM Ba2+ inhibited UVB-induced K+ loss in a time and dose-dependent manner. These results confirm that blocking K+ channels in HCLE cells exposed to UVB prevents efflux of K+, confirming that UVB activates K+ channels in these cells. Electrophysiology data show that K+ channels remain highly active at least 90 min after UVB exposure. HCLE cells exposed to UVB and incubated in 0.01–1 μM ouabain did not recover from UVB-induced K+ loss. These data suggest that the Na/K pump may act to restore [K+]i to control levels in HCLE cells following UVB exposure and that the pump is not damaged by exposure to UVB. Incubation of HCLE cells exposed to UVB in medium with 25–100 mM K+ media prevented K+ efflux at extracellular concentrations as low as 25 mM (the concentration in tear fluid), maintaining control levels of K+i. In all experiments inward fluxes and intracellular Na+ levels mirrored K+ changes, albeit at the expected lower concentrations. The prevention of UVB-induced Ki+ loss by 25 mM Ko+ is consistent with the possible contribution of the relatively high K+ concentration in tears to protection of the corneal epithelium from ambient UVB.