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。透明度最受间质结缔组织水合水平的影响。水合作用取决于角膜上皮和内皮的代谢活动;然而，主要负责泵送离子和液体的内皮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如何完成这项工作，以便开发药物治疗方法来增强因疾病或创伤而受损的角膜的内皮功能。内皮离子和液体泵的功能最依赖于HCO 2+。我们的方法已经确定，表征，并整合成一个模型的膜泵，转运蛋白和通道的跨内皮HCCV和流体运输在培养的内皮细胞系统。在该项目的这一阶段，我们专注于了解顶端HCOa流出的机制，特别是碳酸酐酶（CA）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，我们将鉴定和定位乳酸：H+协同转运蛋白，并确定CA和HCO 3+转运蛋白的药理学抑制剂以及小干扰RNA治疗后降低CA或转运蛋白表达是否会降低乳酸通量。我们还研究了腺苷受体刺激和可溶性腺苷酸环化酶在设置细胞[cAMP]中的作用，这对内皮功能有刺激作用。这也将使用siRNA方法并检查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净HCO 3-转运的影响，作为相对PKA活性的量度。最后，我们将检查HCOa运输模型，该模型是在体外用体内兔角膜中的培养细胞建立的。我们的方法是使用干扰RNA分子的慢病毒递送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。该目的的结果将告诉我们各种转运蛋白在体内角膜中维持角膜水合作用的相对重要性。

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.