September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
The role of K+ and Ba2+ in inhibiting apoptosis in corneal epithelial cells exposed to UVB
Author Affiliations & Notes
  • Courtney D Glupker
    Biology, Calvin College, Grand Rapids, Michigan, United States
  • Peter M Boersma
    Biology, Calvin College, Grand Rapids, Michigan, United States
  • John R Leerar
    Biology, Calvin College, Grand Rapids, Michigan, United States
  • Mark Schotanus
    Biology, Calvin College, Grand Rapids, Michigan, United States
  • Loren D Haarsma
    Physics, Calvin College, Grand Rapids, Michigan, United States
  • John L Ubels
    Biology, Calvin College, Grand Rapids, Michigan, United States
  • Footnotes
    Commercial Relationships   Courtney Glupker, None; Peter Boersma, None; John Leerar, None; Mark Schotanus, None; Loren Haarsma, None; John Ubels, None
  • Footnotes
    Support  NIH Grant R15EY023836 and West Michigan Optometric Scholarship
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 4373. doi:
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      Courtney D Glupker, Peter M Boersma, John R Leerar, Mark Schotanus, Loren D Haarsma, John L Ubels; The role of K+ and Ba2+ in inhibiting apoptosis in corneal epithelial cells exposed to UVB. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4373.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

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