Effect of Elevated Extracellular K+ on UVB-induced Efflux of K+ from Human Corneal Epithelial Cells

Abstract

Purpose: : Previous research has shown that UVB activates K⁺ channels in human corneal limbal epithelial (HCLE) cells, causing efflux of K⁺ and initiation of apoptosis. Extracellular K⁺ (K⁺_o) at 25 mM, the concentration in tear fluid, inhibits this UVB-induced apoptosis (Singleton et al, IOVS 89:140, 2009), presumably by preventing loss of intracellular K⁺ (K⁺_i). Our goal was to further investigate this efflux of K⁺ by measuring the levels and regulation of K⁺_i in HCLE cells following exposure to UVB at a dose relevant to ambient levels.

Methods: : HCLE cells were exposed to 150 mJ/cm² UVB followed by incubation for 10-90 minutes in media with 5.5-100 mM K⁺, the K⁺ channel blockers BDS-1 and Ba²⁺, or the Na/K ATPase inhibitor ouabain. K⁺_i was determined by washing cells in 280 mM sucrose, lysis by freeze/thaw in DI water and analysis of [K⁺] in lysates by ion chromatography. Controls were cells not exposed to UVB, incubated in medium with 5.5 mM K⁺.

Results: : Exposure of cells to UVB and incubation in 5.5 mM K⁺_o caused an initial drop in [K⁺]_i to 54% of control at 10-30 minutes, followed by full recovery by 90 min. Incubation in media with 25-100 mM K⁺ for 20 min after UVB exposure prevented the UVB-induced loss of K⁺_i. BDS-1 (1 uM) significantly reduced the UVB-induced K⁺_i loss, while K⁺ efflux was blocked by 5 mM Ba²⁺. The recovery of K⁺_i that followed UVB-induced efflux was inhibited by 0.01-1µM ouabain in a dose-dependent manner.

Conclusions: : The data confirm that high [K⁺]_o reduces the amount of K⁺ lost by HCLE cells after UVB exposure, probably due to the reduced the concentration gradient for K⁺. The effect of K⁺ channel blockers on UVB-induced K⁺_i loss confirms the activation of K⁺ channels by UVB. The inhibition of K⁺_i recovery by ouabain in UVB-treated cells shows that the Na/K pump is responsible for this recovery and is not damaged by UVB and activation of apoptotic pathways. The prevention of UVB-induced K⁺_i loss by 25 mM K⁺_o is consistent with the possible contribution of high K⁺ in tears to protection of the corneal epithelium from ambient UVB.

Keywords: cornea: tears/tear film/dry eye • cornea: epithelium • apoptosis/cell death