VOLUME REGULATION IN CORNEAL ENDOTHELIUM

这项建议的主要焦点是从溶质-溶剂偶联和信号通路方面研究支持角膜内皮液体运输的离子转运机制。该项目将分三步进行。在第一步中，将通过观察细胞体积和细胞内离子活动的变化来研究受到急性非等渗扰动的内皮细胞的细胞体积调节机制。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。眼睛稀释技术的指标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，将确定第二信使和蛋白激酶参与的信号通路对细胞体积调节的调节。最后，我们将利用细胞信号转导机制来研究它们对完整角膜内皮液体转运功能的影响。这三个步骤的方法源于一个假设，即驱动向量流体运输的离子运输活性和溶质-溶剂耦合与短期细胞体积调节过程中涉及的那些基本相同。这项研究的结果对阐明液体运输的机制及其对长期合同晶状体佩戴、老化、糖尿病和眼内手术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.