Protection of the cornea from UVB radiation by elevated potassium in tears

项目摘要 眼泪，在大多数情况下，有电解质成分像其他细胞外液。眼泪 然而，在其他实施方案中，K+浓度是独特的，因为K+浓度（[K+]为约20-25 mM，相比之下，在其他实施方案中为约5 mM）是独特的。 细胞外液暴露于紫外线辐射引起细胞凋亡。UVB辐射剂量 与室外UVB暴露的环境水平相关的激活K+通道，导致细胞内 K+（K+i）。我们已经证明，角膜上皮细胞在暴露于25 mM K+的培养基中孵育， UVB防止K+i损失并抑制凋亡机制的激活。这些观察结果支持了我们的 总的假设是，眼泪中高浓度的钾离子有助于保护 角膜上皮免受环境UVB诱导的损伤是通过减少细胞内K+通道时的损失 被UVB激活。我们现在建议研究UVB通过细胞内信号传导机制， 激活角膜上皮细胞中的K+通道和凋亡信号。 本研究的具体目的是：1）阐明Fas配体- 独立的，UVB诱导的细胞凋亡信号通路的激活和K+通道的激活。这将 通过研究UVB对角膜细胞凋亡途径和K+通道激活的影响， 在Fas和FADD的siRNA敲低或caspase-8的抑制之后，研究上皮细胞是否 UVB诱导的K+通道激活是细胞凋亡途径激活的主要事件。K+通道 角膜上皮细胞将使用PCR阵列鉴定。然后将使用siRNA敲除特异性的 渠道随后将研究UVB诱导的细胞凋亡激活，以及 当特定通道被敲除时，UVB对K+电流和细胞内[K+]的影响; 分离的猪眼模型，UVB对完整角膜上皮的影响将在配置中进行研究 其允许以模拟洗浴的方式用生理盐水灌注眼表面 眼角膜破裂将检验升高的[K+]将保护浅层角膜的假设 紫外线诱导的细胞凋亡的细胞层。

Project summary The tears, for the most part, have an electrolyte composition like other extracellular fluids. The tears are unique, however, in that the K+ concentration ([K+] is about 20-25 mM, compared to about 5 mM in other extracellular fluids. Exposure to ultraviolet radiation causes apoptosis of cells. UVB radiation at doses relevant to ambient levels of outdoor UVB exposure activates K+ channels leading to rapid loss of intracellular K+ (K+i). We have shown that incubation of corneal epithelial cells in medium with 25 mM K+ after exposure to UVB prevents loss of K+i and inhibits activation of apoptotic mechanisms. These observations support our overall hypothesis that the high concentration of potassium ions in tears contributes to the protection of the corneal epithelium from ambient UVB-induced damage by reducing the loss of intracellular K+ when channels are activated by UVB. We now propose to investigate the intracellular signaling mechanisms by which UVB activates K+ channels and apoptotic signaling in corneal epithelial cells. The specific aims of the proposed research are: 1) to elucidate the relationships between Fas ligand- independent, UVB-induced activation of apoptotic signaling pathways and activation of K+ channels. This will be accomplished by studying the effects of UVB on apoptotic pathways and K+ channel activation in corneal epithelial cells after siRNA knockdown of Fas and FADD, or inhibition of caspase-8; 2) To investigate whether UVB-induced K+ channel activation is the primary event in activation of apoptotic pathways. K+ channels in corneal epithelial cells will be identified using a PCR array. siRNA will then be used to knock down specific channels. This will be followed by investigation of UVB-induced activation of apoptosis, as well as effects of UVB on K+ currents and intracellular [K+] when specific channels have been knocked down; 3) Using an isolated pig eye model, effects of UVB on the intact corneal epithelium will be investigated in a configuration that allows for superfusion of the ocular surface with physiologic saline in a manner that models the bathing of the cornea with tears. The hypothesis will be tested that elevated [K+] will protect the superficial corneal cell layers from UV-induced apoptosis.