VOLUME REGULATION IN CORNEAL ENDOTHELIUM

该建议的主要重点是研究支持角膜内皮在溶质-溶剂偶联和信号传导途径方面的液体转运的离子转运机制。该项目将分三个步骤进行。在第一步中，将通过以下细胞体积和细胞内离子活性的变化来研究受到急性各向异性扰动的内皮细胞中细胞体积调节的机制。细胞体积的变化将使用光散射、染料稀释和动态荧光猝灭的原理来测量。眼稀释技术的指示剂将包括瞬时表达的绿色荧光蛋白或极性荧光染料。在第二步中，将确定涉及第二信使和蛋白激酶的信号通路对细胞体积调节的调节。最后，将采用诱导细胞体积调节发生深刻变化的细胞信号传导策略来研究其对完整角膜内皮细胞液体转运功能的影响。这种三步方法源于这样的假设，即驱动矢量流体转运的离子转运活性和溶质-溶剂偶联与短期细胞体积调节期间所涉及的那些基本相同。本研究的结果对阐明液体转运的潜在机制及其对长期接触透镜配戴、老化、糖尿病和眼内手术操作的调节具有直接意义。

The principal focus of this proposal is to investigate the ion transport mechanisms that support fluid transport by the corneal endothelium in terms of solute-solvent coupling and signaling pathways. The project will be carried out in three steps. In the first step, the mechanisms of cell volume regulation in endothelial cells subjected to acute anisosmotic perturbations will be investigated by following changes in cell volume and intracellular ionic activities. The changes in cell volume will be measured using the principles of light scattering, dye dilution and dynamic fluorescence quenching. The indicators for eye dilution technique will include transiently expressed green fluorescent protein or polar fluorescent dyes. In the second step, the modulation of cell volume regulation by signaling pathways involving second messengers and protein kinases will be identified. Finally, the maneuvers of cell signaling that induce profound changes in cell volume regulation will be employed to investigate their influence on fluid transport function by the endothelium in intact corneas. This three-step approach stems from the hypothesis that the ion transport activity and solute-solvent coupling that drive the vectorial fluid transport are mostly identical to those involved during short-term cell volume regulation. The results from this study have directly implications towards the elucidation of the mechanisms underlying fluid transport and their modulation upon long-term contract lens wear, aging, diabetes and intra-ocular surgical maneuvers.