Regulation of pHi and Fluid Flux in Corneal Endothelium

这项研究的目的是了解角膜内皮液体“泵”是如何工作的。这个 “Pump”维持角膜的水化和透明度。当“泵”因创伤而失效时， 炎症、老化或角膜营养不良，继而出现角膜水肿，透明度丧失， 视力明显下降。通常的治疗方法是移植，但也不是没有 重大妥协和复杂情况。了解“泵”是如何工作的是一种方法 开发可以延缓或取代移植需求的医学疗法。作为 人口老龄化和内皮功能障碍的患病率增加，需求 血管内皮细胞治疗也将增加。离子传输是“泵”的一个关键特征。到目前为止， “泵”被模拟为一种依赖于碳酸氢盐的经典离子分泌机制。 对碳酸酐酶敏感。前人的研究和前期的工作 然而，这表明需要对替代模式进行调查。我们的总体假设是 角膜内皮是一个清除乳酸的“泵”。因为角膜有很强的糖酵解作用 必须去除最终产物，也就是乳酸，我们建议跨细胞通量 乳酸与水的运动相耦合；乳酸的流动是通过单羧酸转运体实现的 (MCTs)；HCO3-、CA活性、膜PHI调节剂和HCO3-的缓冲作用 转运体协同作用促进乳酸的流动。通过缓冲被传输的质子 MCTS、乳酸：H+和水的通量通过防止驱动力的降低而最大化 持续乳酸：H+转运。使用多种体外和体内互补方法 将在三个目标上进行测试。目标1将调查缓冲能力对水和 乳酸盐通量。可以估计通量比，即摩尔乳酸/微升水，以确定 这种渗透耦合的调性。如果水与乳酸的偶联很重要，我们预计等张(300 商务部/L)交通运输。目标2将调查初级和次级主动运输的作用 (Na+，K+ATPase，1Na+：2HCO3-共转运和Na+/H+交换)在促进乳酸通量中的作用 通过测定乳酸流量比的变化来确定这些转运体 都受到了干扰。目的3研究MCTs在促进乳酸转运中的作用。vbl.使用 药理抑制剂和shRNA方法在体内我们预测，对MCTs的抑制将 对角膜水化有显著影响。如果假设是正确的，我们将有更多的 完整的内皮功能模型，将允许进一步开发诊断和 具有必要运输特性的内皮样细胞的医学疗法或工程。

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.