HOMEOSTATIC ABILITY OF THE RETINAL PIGMENT EPITHELIUM

视网膜色素上皮的稳态能力

• 批准号: 2158380
• 负责人: SHELDON S MILLER
• 金额: $ 24.95万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-02-01 至 1996-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2158380
• 关键词: acetazolamide; acid base balance; adenosinetriphosphatase; animal tissue; antiport; apical membrane; basolateral membrane; biological fluid transport; biological signal transduction; calcium flux; carbonates; electrophysiology; epinephrine; eye pharmacology; fluorescence microscopy; growth factor; growth media; homeostasis; hormones; human fetus tissue; immunofluorescence technique; ion transport; membrane channels; membrane transport proteins; neurotransmitters; pharmacokinetics; protein purification; receptor coupling; retina; retinal pigment epithelium; second messengers; transport proteins; vitreous body

The experiments in this proposal are designed to analyze the regulation and function of the plasma membrane and intracellular mechanisms that determine ion and fluid transport across the bovine and human RPE. This should provide the basis for a pharmacology of the RPE and hopefully lead to the therapeutic alleviation of retina/RPE diseases that cause fluid accumulation in the subretinal space. Focus is on the extracellular signals, membrane receptors, and intracellular signalling pathways that help regulate: (1) ion, metabolite, and fluid movement across the RPE; and (2) the chemical composition of the cells and their environment. The choice of signals, receptors and pathways is restricted by functional considerations. For example, epinephrine is a potent stimulator of transepithelial fluid absorption (in vitro) and acetazolamide has been apparently successful in increasing fluid absorption from the subretinal space of patients with cystoid macular edema. In both cases, the membrane and cellular mechanisms that determine the efficacy of these drugs will to be determined. The preliminary data demonstrates that fresh explant tissue from human donors (adult or fetal) can be maintained and studied in-vitro for hours. The first step will be to determine the channels, cotransporters, exchangers and pumps at each membrane. The mechanisms that regulate pHi and [Ca++] will be determined. The hormones, neurotransmitters, growth factors and other substances that are known to affect human cultured RPE cells will be also studied with particular emphasis on those drugs or hormones already known to alter solute-linked fluid transport. Fetal tissue of varying gestational age will be used to follow the development of a key transport protein. The HCO3-dependent mechanisms seem to be absent in fetal RPE (~20 weeks). This could be because the proteins have not yet been assembled or if assembled have not yet been routed to the plasma membrane. Another possibility is that they are in place but not yet turned on. These possibilities will be studied by using polyclonal antibodies to a conserved COOH-terminal peptide of mouse band 3, which contains the catalytic site for anion exchange activity. Immunofluorescence microscopy will be used to localize this anion exchange- related polypeptide to intracellular or plasma membrane sites.

本提案中的实验旨在分析法规和 质膜和细胞内机制的功能 离子和液体横穿牛和人类RPE。 这应该 为RPE的药理学提供基础，并希望导致 导致液体的视网膜/RPE疾病的治疗性缓解 在视网膜下空间中积聚。 专注于细胞外 信号，膜受体和细胞内信号通路 帮助调节：（1）离子，代谢物和横跨RPE的流体运动；和 （2）细胞及其环境的化学组成。 这 信号，受体和途径的选择受功能限制 考虑因素。 例如，肾上腺素是 thransepithitial液体吸收（体外）和乙酰唑胺一直是 显然可以成功地增加视网膜下的液体吸收 囊状黄斑水肿患者的空间。 在这两种情况下，膜 以及确定这些药物功效的细胞机制将 可以确定。 初步数据证明了新鲜的外植物组织 可以从人类捐助者（成人或胎儿）中进行维护和研究 几个小时。 第一步是确定频道， 每个膜上的共转运剂，交换器和泵。 这些机制 将确定调节PHI和[Ca ++]。 激素， 神经递质，生长因子和其他已知的物质 还将研究影响人培养的RPE细胞 强调已经已知可以改变溶质连接的药物或激素 流体传输。 不同胎龄的胎儿组织将使用 遵循关键运输蛋白的开发。 HCO3依赖性 在胎儿RPE中似乎没有机制（约20周）。 可能是 因为蛋白质尚未组装，或者如果组装尚未 但是被路由到质膜。 另一个可能是他们 已经到位，但尚未打开。 这些可能性将由 使用对小鼠保守的COOH末端肽的多克隆抗体 带3，其中包含用于阴离子交换活性的催化位点。 免疫荧光显微镜将用于定位这种阴离子交换 - 相关的多肽与细胞内或质膜位点。