Apoptosis of Corneal Epithelial Cells Caused by Ultraviolet B-induced Loss of K+ is Inhibited by Ba2+

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 Ba2+ blocks several K+ channels, we tested whether Ba2+-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/cm2. 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 Ba2+. K+ currents were activated in HCLE cells following UVB-exposure. These currents were reversibly blocked by 5 mM Ba2+. When HCLE cells were incubated with 5 mM Ba2+ 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 Ba2+ inhibits UVB-induced apoptosis by preventing loss of intracellular K+ when K+ channels are activated. Ba2+ 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.

The effect of K+ on caspase activity of corneal epithelial cells exposed to UVB

“…This study supports our overall hypothesis that the relatively high [K + ] in tears reduces the electrochemical gradient for loss of intracellular K + in corneal epithelial cells in response to UVB (Singleton et al, 2009; Ubels et al, 2011, 2016; Glupker et al, 2016). This may contribute to protection of the corneal epithelium from ambient UVB by inhibiting apoptotic pathways that are normally suppressed by the relatively high intracellular K + concentration.…”

“…UVC and UVB radiation increased caspase-3 activity in rabbit corneal epithelial cells (Lu et al, 2003) and a human corneal epithelial cell line (Shimmura et al, 2004), respectively, as well as in keratinocytes (Daher et al, 2006). Finally, we have shown that HCLE cells exposed to UVB at doses relevant to ambient outdoor exposure show increased caspase-9, caspase-8, and caspase-3 activity (Singleton et al, 2009; Glupker et al, 2016). …”

“…We have published several reports showing that apoptosis of human corneal limbal epithelial (HCLE) cells can be inhibited if loss of intracellular K + in response to exposure to UVB radiation is prevented (Singleton et al, 2009; Ubels et al, 2010, 2016; Glupker et al, 2016). The mechanism by which the relatively high concentration of intracellular K + inhibits apoptosis in these cells has not been investigated.…”

“…Exposure of HCLE cells to 150 mJ/cm 2 UVB activates K + channels, allowing for efflux of intracellular K + , resulting in apoptosis (Singleton et al, 2009; Ubels et al, 2011). After UVB exposure, incubating HCLE cells in medium with elevated extracellular [K + ] (25–100 mM) as compared to the usual [K + ] of 5 mM in culture medium, or with the K + channel blocker, Ba 2+ , prevents UVB-induced loss of K + from the cells (Ubels et al, 2011, Glupker et al, 2016), which in turn significantly decreases the activation of caspase-3, capsase-8, and caspase-9 by UVB (Singleton et al, 2009; Glupker et al, 2016). …”

TNF-R1 and FADD mediate UVB-Induced activation of K+ channels in corneal epithelial cells

“…Ubels et al (2011) demonstrated that culture medium with elevated extracellular K + (25 mM), similar to that in tear fluid, reduces K + loss following UVB-induced K + channel activation by diminishing the electrochemical gradient between the intracellular and extracellular fluids (Singleton et al, 2009), reducing UVB-induced apoptosis. Likewise, K + channel blockers Ba 2+ and BDS-1 reduce UVB-induced K + currents and subsequent apoptosis (Singleton et al, 2009; Ubels et al, 2010, 2011; Glupker et al, 2016). …”