Regulation of pHi and Fluid Flux in Corneal Endothelium

角膜内皮 pHi 和液体流量的调节

• 批准号: 7212665
• 负责人: Joseph Aurelio Bonanno
• 金额: $ 30.3万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 2012-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7212665
• 关键词: Adenosine; Adenylate Cyclase; Affect; Age; Anions; Apical; Bicarbonates; Binding; Biological Assay; Buffers; CREB1 gene; Carbonic Anhydrase Inhibitors; Carboxyamidotriazole; Carrier Proteins; Cells; Chronic; Condition; Connective Tissue; Cornea; Corneal Endothelium; Corneal edema; Cultured Cells; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cystic Fibrosis Transmembrane Conductance Regulator; Diabetes Mellitus; Disease; Disruption; Dorzolamide; Drug usage; Elements; Endothelial Cells; Endothelium; Eye; Fuchs' Endothelial Dystrophy; Functional disorder; Goals; Hydration status; In Vitro; Inflammation; Inflammatory; Ion Pumps; Ion Transport; Ions; Keratoplasty; Lactic acid; Lead; Lentivirus Vector; Link; Liquid substance; Localized; Location; Maintenance; Measurement; Measures; Mediating; Medical; Membrane; Metabolic; Metabolism; Modeling; Molecular; Myosin Light Chains; Na(+)-K(+)-Exchanging ATPase; Occupations; Operative Surgical Procedures; Oryctolagus cuniculus; Patients; Permeability; Phosphorylation; Population; Prevalence; Production; Pump; Purinergic P1 Receptors; RNA; Receptor Activation; Regulation; Relative (related person); Research Personnel; Resistance; Rest; Role; Small Interfering RNA; Swelling; System; Systemic disease; Testing; Thick; Time; Transplantation; Trauma; Vision; carbonate dehydratase; corneal epithelium; in vivo; in vivo Model; protein expression; receptor; research study; transcription factor

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.

描述（由申请人提供）：角膜是眼睛的主要折射元素，因此它必须平稳且透明。透明度受基质结缔组织的水合水平影响最大。水合取决于角膜上皮和内皮的代谢活性。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。该项目的长期目标是了解角膜内皮如何完成这项工作，以便可以开发出医疗疗法以增强因疾病或创伤而损害的角膜的内皮功能。内皮离子和流体泵的功能最依赖于HCOA。我们的方法是识别，表征和将膜泵，转运蛋白和转运蛋白和渠道整合到跨内皮HCCV和流体转运的模型中，并在项目的内皮细胞系统中。在项目的这一时期。阴离子交换器及其如何缓冲和促进乳酸的运输，我们将识别和定位乳酸：H+共转运蛋白，并确定CAS和HCO3“ Transporter的药理学抑制剂以及使用小型干扰RNA降低Ca或Transporter Ca”的处理。我们还研究了腺苷受体刺激和可溶性腺苷环酶在设置细胞[CAMP]中的作用，该细胞对内皮功能具有刺激作用。这还将使用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Net HCO3的影响“作为相对PKA活性的量度。我们的方法是使用干扰RNA分子的慢病毒递送来降低体内特定的转运蛋白表达，并检查对角膜厚度和内皮功能的影响。该目标的结果将告诉我们各种转运蛋白在维持体内角膜中保持角膜水合的相对重要性。