Regulation of pHi and Fluid Flux in Corneal Endothelium

本研究的目的是了解角膜内皮液体“泵”的工作原理。的 “泵”保持角膜水合和透明度。当“泵”因创伤而失效时， 炎症、老化或角膜营养不良、角膜水肿、透明性丧失， 视力明显下降。通常的治疗方法是移植， 重大的妥协和并发症。了解“泵”的工作原理是一种方法， 开发可以延迟或取代移植需求的医学疗法。为 人口年龄和内皮功能障碍的患病率增加， 内皮治疗也将增加。离子传输是“泵”的关键特征。到目前为止 “泵”已被建模为经典的离子分泌机制， 和碳酸酐酶敏感。以前的研究和我们在前一时期的工作 但是，这表明需要研究替代模型。我们的总体假设是 角膜内皮是乳酸盐清除“泵”。因为角膜的糖酵解作用很强 必须去除最终产物，即乳酸，我们建议， 乳酸盐与水运动耦合;乳酸盐通量通过单羧酸转运蛋白 HCO 3-、CA活性、膜pHi调节剂和HCO 3-的缓冲作用 转运蛋白协同作用以促进乳酸盐的流动。通过缓冲质子的运输， MCT、乳酸盐：H+和水通量通过防止用于 持续乳酸：H+转运。使用多种体外和体内互补方法， 将在三个目标中进行测试。目标1将研究缓冲能力对水的作用， 乳酸通量可以估计通量比，即mMoles乳酸盐/ul水，以确定 这种渗透耦合的张力。如果与乳酸盐结合的水是显著的，我们预期等渗的（300 mEq/L）转运。目的2将研究初级和次级主动转运的作用 (Na 1 Na+：2 HCO 3-共转运和Na+/H+交换）促进乳酸通量 通过测定乳酸通量比的变化，当这些转运蛋白 感到不安目的3将研究MCT在易化乳酸转运中的作用。使用 药理学抑制剂和shRNA方法在体内，我们预测，抑制MCT将 对角膜水合作用有显著影响。如果假设是正确的，我们将有一个更大的 完整的内皮功能模型，将允许进一步开发诊断和 医学治疗或具有必需的转运特性的内皮样细胞的工程化。

The goal of this study is to understand how the corneal endothelial fluid "pump" works. The "pump" maintains corneal hydration and transparency. When the "pump" fails due to trauma, inflammation, ageing, or corneal dystrophy, corneal edema ensues, transparency is lost and vision is significantly degraded. The usual therapy is transplantation, which is not without significant compromises and complications. Knowing how the "pump" works is one approach to developing medical therapies that could delay or supplant the need for transplantation. As the population ages and the prevalence of endothelial dysfunction increases, demand for endothelial therapy will also increase. Ion transport is a key feature of the "pump". Up to now the "pump" has been modeled as a classic ion secretory mechanism that is bicarbonate dependent and carbonic anhydrase sensitive. Previous studies and our work in the preceding period however, indicate that alternate models need to be investigated. Our overall hypothesis is that the corneal endothelium is a lactate removal "pump". Because the cornea is very glycolytic and must remove the end product, which is lactic acid, we propose that the transcellular flux of lactate is coupled to water movement; that lactate flux is via monocarboxylic acid transporters (MCTs); and that the buffering action of HCO3-, CA activity, membrane pHi regulators & HCO3- transporters act in concert to facilitate the flux of lactate. By buffering the protons transported by the MCTs, lactate:H+ & water flux is maximized by preventing reductions in the driving force for continued lactate:H+ transport. Using multiple in vitro & in vivo complementary approaches this will be tested in three aims. Aim 1 will investigate the role of buffering capacity on water and lactate fluxes. The flux ratio, i.e. mMoles lactate/ul water can be estimated to determine the tonicity of this osmotic coupling. If water coupled to lactate is significant, we expect isotonic (300 mEq/L) transport. Aim 2 will investigate the role of primary and secondary active transport (Na+,K+ ATPase,1Na+:2HCO3- cotransport, and Na+/H+ exchange) in facilitating lactate flux across the endothelium by determining the change in lactate flux ratio when these transporters are disturbed. Aim 3 will investigate the role of MCTs in facilitated lactate transport. Using pharmacological inhibitors and shRNA approaches in vivo we predict that inhibition of MCTs will have significant effects on corneal hydration. If the hypothesis is correct, we will have a more complete model of endothelial function that will allow further development of diagnostic and medical therapies or engineering of endothelial-like cells with the requisite transport properties.