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Experimental Eye Research


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.

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