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Rachel E. Van Dyken, Julienne R. Louters, Mark P. Schotanus, John L. Ubels; Effect of Elevated Extracellular K+ on UVB-induced Efflux of K+ from Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):301.
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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.
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+.
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.
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.
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