Apoptosis of Corneal Epithelial Cells Caused by Ultraviolet B-induced Loss of K(+) is Inhibited by Ba(2.)

Abstract

UVB exposure at ambient outdoor levels triggers rapid K(+) loss and apoptosis in human corneal limbal epithelial (HCLE) cells cultured in medium containing 5.5 mM K(+), but considerably less apoptosis occurs when the medium contains the high K(+) concentration that is present in tears (25 mM). Since Ba(2+) blocks several K(+) channels, we tested whether Ba(2+)-sensitive K(+) channels are responsible for some or all of the UVB-activated K(+) loss and subsequent activation of the caspase cascade and apoptosis. Corneal epithelial cells in culture were exposed to UVB at 80 or 150 mJ/cm(2). Patch-clamp recording was used to measure UVB-induced K(+) currents. Caspase-activity and TUNEL assays were performed on HCLE cells exposed to UVB followed by incubation in the presence or absence of Ba(2+). K(+) currents were activated in HCLE cells following UVB-exposure. These currents were reversibly blocked by 5 mM Ba(2+). When HCLE cells were incubated with 5 mM Ba(2+) after exposure to UVB, activation of caspases-9, -8, and -3 and DNA fragmentation were significantly decreased. The data confirm that UVB-induced K(+) current activation and loss of intracellular K(+) leads to activation of the caspase cascade and apoptosis. Extracellular Ba(2+) inhibits UVB-induced apoptosis by preventing loss of intracellular K(+) when K(+) channels are activated. Ba(2+) therefore has effects similar to elevated extracellular K(+) in protecting HCLE cells from UVB-induced apoptosis. This supports our overall hypothesis that elevated K(+) in tears contributes to protection of the corneal epithelium from adverse effects of ambient outdoor UVB.

Keywords: K(+) channel; apoptosis; caspase; corneal epithelium; potassium; tears; ultraviolet.

Figure 1

K⁺ channel activation in an HCLE cell following exposure to 80 mJ/cm² UVB. Whole- cell currents were measured with 10 mV voltage steps given for 250 ms duration before exposure to UVB, after UVB exposure, during application of 5 mM BaCl₂, and following washout of Ba²⁺.

Figure 2

Normalized K⁺ channel current/voltage (I/V) relationships in HCLE cells exposed to UVB at 80 mJ/cm². K⁺ currents were measured before UVB exposure, after UVB exposure, during treatment with Ba²⁺, and after washout of Ba². UVB-activated K⁺ currents were reversibly inhibited by Ba²⁺. A. Response of a single cell to which 0.1, 1 and 5 mM Ba²⁺ were applied sequentially after exposure to UVB. This experiment was repeated in 3 cells with similar results. B. Effect of 5 mM Ba²⁺ alone on UVB-induced currents (n=9 cells).

Figure 3

Effect of Ba²⁺ on UVB-induced activation of caspase-9 in HCLE cells. Cells were exposed to 150 mJ/cm² UVB and incubated for 4 hr in the presence or absence of Ba²⁺. Increasing concentrations of Ba²⁺ caused a significant decrease in UVB-induced caspase-9 activity. *significantly different from cells exposed to UVB and incubated without Ba²⁺ (mean ± SD, n=6, One-way ANOVA and Dunnett’s test, P<.05).

Figure 4

Effect of Ba²⁺ on UVB-induced activation of caspase-8 in HCLE cells. Cells were exposed to 150 mJ/cm² UVB and incubated for 6 hr in the presence or absence of Ba²⁺. Increasing concentrations of Ba²⁺ caused a significant decrease in UVB-induced caspase-8 activity. *significantly different from cells exposed to UVB and incubated without Ba²⁺ (mean ± SD, n=6, One-way ANOVA and Dunnett’s test, P<.05).

Figure 5

Effect of Ba²⁺ on UVB-induced activation of caspase-3 in HCLE cells. Cells were exposed to 150 mJ/cm² UVB and incubated for 6 hr in the presence or absence of Ba²⁺. Increasing concentrations of Ba²⁺ caused a significant decrease in UVB-induced caspase-3 activity. *significantly different from cells exposed to UVB and incubated without Ba²⁺ (mean ± SD, n=6, One-way ANOVA and Dunnett’s test, P<.05).

Figure 6

Protection of HCLE cells from UVB-induced apoptosis by Ba²⁺. Cells were exposed to 150 mJ/cm² UVB, incubated for 6 hours and analyzed by the TUNEL assay and flow cytometry. Control cells were not exposed to UVB. Non-apoptotic cells have low fluorescence while UVB-induced apoptotic cells are shifted into a higher fluorescence window. During incubation in medium with 5 mM Ba²⁺ cells exposed to UVB remain the non-apoptotic window.

Figure 7

Decrease in UVB-induced apoptosis of HCLE cells, treated with Ba²⁺. HCLE cells were exposed to 150 mJ/cm² UVB and incubated in KSFM with 5 mM Ba²⁺ for 6 hr followed by measurement of apoptosis by the TUNEL assay and flow cytometry. Control cells were maintained for 6 hr in KSFM without Ba²⁺. Each bar represents an average of 2 samples of 10,000 cells. This experiment was repeated 3 times with similar results.