Potassium ion fluxes in corneal epithelial cells exposed to UVB.

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/cm(2) UVB, followed by incubation in culture media with 5.5-100 mM K(+), BDS-1, Ba(2+) 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 Ba(2+) 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 K(i)(+) loss by 25 mM K(o)(+) is consistent with the possible contribution of the relatively high K(+) concentration in tears to protection of the corneal epithelium from ambient UVB.

Inhibition of UV-B induced apoptosis in corneal epithelial cells by potassium channel modulators.

The goal of this study was to determine whether prevention of K(+) loss can protect human corneal-limbal epithelial (HCLE) cells from UV-B induced apoptosis. Immunostaining for activated caspase-3 of HCLE cells exposed to 150-200 mJ/cm(2) UV-B demonstrated induction of apoptosis 6 h after exposure. The number of apoptotic cells was decreased by incubation in medium with 25 or 100 mM K(+). If this protection is due to a reduction of UV-induced K(+) loss then K(+) channel blockers should also protect HCLE cells from UV-B. Caspase-8 activity induced by exposure to UV-B at 150 mJ/cm(2) was significantly reduced when the cells were incubated in 0.3 microM BDS-I or 0.05-1 mM quinidine. Caspase-3 was also activated by UV-B and a reduction in activity was observed after incubation in 0.1-0.3 microM BDS-I and 0.1-1 mM quinidine. Induction of DNA fragmentation, as measured by the TUNEL assay, was decreased by treatment with 0.3 microM BDS-I and 0.01-0.05 mM quinidine. Patch-clamp recording showed activation of K(+) channels after exposure to UV-B and a decrease in outward K(+) current was observed following application of BDS-I. Quinidine did not block K(+) currents in HCLE cells, suggesting that the protective effect of quinidine occurs by a mechanism other than via K(+) channels. The effect of the K(+) channel blocker BDS-1 on HCLE cells exposed to UV-B confirms that preventing K(+) efflux protects corneal epithelial cells from apoptosis. This suggests the elevated [K(+)] in tears may protect the corneal epithelium from effects of ambient UV-B.