Regulation of pHi and Fluid Flux in Corneal Endothelium

描述（由申请人提供）：角膜是眼睛的主要屈光元件，因此它必须光滑透明，以获得良好的视力。透明度受基质结缔组织水合水平的影响最大。水合作用依赖于角膜上皮和内皮的代谢活动；然而，内皮主要负责泵送离子和液体，维持基质的稳态水合作用。该项目的长期目标是了解角膜内皮是如何完成这项工作的，这样就可以开发出医学疗法来增强因疾病或创伤而受损的角膜的内皮功能。内皮离子和流体泵的功能最依赖于HCOa。我们的方法是识别、表征并将膜泵、转运体和通道整合到培养内皮细胞系统中跨内皮HCCV和流体运输的模型中。在项目的这个阶段，我们的重点是了解顶端HCOa外排的机制，特别是碳酸酐酶（CAs）和阴离子交换剂的作用，以及它们如何缓冲和促进乳酸的运输。为此，我们将识别和定位乳酸：H+共转运体，并确定乳酸通量是否会因CAs和HCO3”转运体的药理学抑制剂而减少，以及随后用小干扰RNA治疗以减少CA或转运体的表达。我们还研究了腺苷受体刺激和可溶性腺苷酸环化酶在凝固细胞中的作用[cAMP]，它对内皮功能有刺激作用。本研究还将使用siRNA方法，检测cAMP/PKA介导的转运蛋白、肌球蛋白轻链和CREB转录因子的磷酸化，以及对HCO3“净转运的影响，作为PKA相对活性的测量指标。最后，我们将研究用体外培养的细胞在兔体内角膜中构建的HCOa转运模型。我们的方法是使用慢病毒递送干扰RNA分子来减少体内特异性转运蛋白的表达，并检查对角膜厚度和内皮功能的影响。本实验的结果将告诉我们各种转运体在维持体内角膜水化中的相对重要性。

DESCRIPTION (provided by applicant): The cornea is the primary refractive element of the eye and therefore it must be smooth and transparent for good vision. Transparency is most affected by the hydration level of the stromal connective tissue. Hydration is dependent on the metabolic activities of the corneal epithelium and endothelium; however it is the endothelium that is primarily responsible for pumping ions and fluid that maintains the steady-state hydration of the stroma. The long-term goal of this project is to understand how the corneal endothelium does this job, so that medical therapies can be developed to enhance endothelial function in corneas that have been compromised by disease or trauma. The endothelial ion and fluid pump is most dependent on HCOa" for its function. Our approach has been to identify, characterize, and integrate the membrane pumps, transporters and channels into a model for transendothelial HCCV and fluid transport in a cultured endothelial cell system. In this period of the project we focus on understanding the mechanisms for apical HCOa' efflux, particularly the roles of carbonic anhydrases (CAs) and anion exchangers and how they may buffer and facilitate the transport of lactic acid. To do this, we will identify and localize lactate: H+ cotransporters and determine if lactate fluxes are reduced by pharmacological inhibitors for CAs and HCO3" transporters as well as following treatment with small interfering RNA to reduce CA or transporter expression. We also examine the roles of adenosine receptor stimulation and soluble adenylyl cyclase in setting cell [cAMP], which has a stimulatory effect on endothelial function. This will also use the siRNA approach and examine cAMP/PKA mediated phosphorylation of transporters, myosin light chain, and CREB transcription factor as well as the effects on net HCO3" transport as measures of relative PKA activity. Lastly, we will examine the model of HCOa" transport that is being built in vitro with cultured cells in the in vivo rabbit cornea. Our approach is to use lentiviral delivery of interfering RNA molecules to reduce specific transporter expression in vivo and examine the effect on corneal thickness and endothelial function. The results from this Aim will tell us the relative importance of the various transporters in maintaining corneal hydration in the in vivo cornea.