The role of K⁺ and Ba²⁺ in inhibiting apoptosis in corneal epithelial cells exposed to UVB

Abstract

Purpose : Exposure of corneal epithelial (HCLE) cells to ambient UVB causes activation of K⁺ channels, loss of 50% of intracellular K⁺ (K⁺_i) within 10 min, activation of caspases and apoptosis. Extracellular K⁺ at 25-100 mM inhibits these responses to UVB, and K⁺_i is restored by the Na/K pump within 90 min (Ubels et al. Exp Eye Res. 92:145; 145:26). The latter is unexpected since the pump is inhibited during apoptosis in lymphocytes. In the context of our hypothesis that high [K⁺] in tears protects the corneal epithelium from UVB by reducing loss of K⁺_i, this study investigated effects of Ba²⁺ on UVB-induced K⁺ current and apoptosis of HCLE cells, effects of UVB on Na/K ATPase activity and effects of [K⁺] on UVB-induced caspase-3 activity.

Methods : HCLE cells were exposed to UVB at 80-150 mJ/cm² and incubated with or without Ba²⁺ for 4-6 hrs. The effect of Ba²⁺ on UVB-induced K⁺ currents was measured by patch-clamp recording. Caspase-activity assays and a TUNEL assay were used to determine whether Ba²⁺ inhibits activation of UVB-induced apoptotic pathways. Na/K ATPase activity was measured in cells exposed to UVB. Effect of [K⁺] on UVB-induced caspase-3 activity was measured by addition of 100 mM K⁺ to reaction medium.

Results : UVB-induced K⁺ currents were reduced in the presence of 5 mM Ba²⁺. UVB-exposed cells incubated with 1-5 mM Ba²⁺ showed significant decreases in activation of caspases -9, -8, and -3 compared to control cells not exposed to Ba²⁺. Apoptosis of cells exposed to UVB, as measured by the TUNEL assay, was also inhibited in the presence of 5 mM Ba²⁺. UVB had no effect on Na/K ATPase activity. Addition of 100 mM K⁺ to the reaction medium significantly decreased UVB-induced caspase-3 activity.

Conclusions : The data confirm that K⁺ current activation by UVB leads to activation of the caspase cascade and apoptosis. Extracellular Ba²⁺ inhibits this activation by preventing loss of intracellular K⁺, inhibiting activation of downstream apoptotic pathways. The lack of an effect of UVB on Na/K ATPase suggests that the pump is active in recovery of intracellular K⁺, contributing to the protection of the cornea from UVB. The data also suggest that UVB-induced caspase-3 activity requires low intracellular K⁺. This supports our hypothesis that high extracellular K⁺ in tears protects the cornea from UVB-induced damage by limiting K⁺ efflux following UVB exposure.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.