LEUKOTRIENES AND MESSENGERS IN PHOTORECEPTOR RENEWAL

白三烯和光感受器更新中的信使

• 批准号: 2159302
• 负责人: Nicolas G. Bazan
• 金额: $ 11.36万
• 依托单位: LSU HEALTH SCIENCES CENTER
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-03-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2159302
• 关键词: Xenopus; alternatives to animals in research; calcium; cell cell interaction; diacylglycerols; excitatory aminoacid; eye pharmacology; eye regeneration; gene expression; glycerophosphorylcholine; inhibitor /antagonist; inositol phosphates; leukotrienes; phagocytosis; phospholipase A2; phospholipase C; photoactivation; platelet activating factor; retinal pigment epithelium; rod cell; second messengers; stimulant /agonist; tissue /cell culture; visual photoreceptor

This proposal is the continuation of our studies on second messenger systems derived from membrane lipids involved in retinal pigment epithelium - visual cell interactions during photoreceptor renewal. We have developed new approaches and a hypothesis that entails a chain of events triggered at the initiation of shedding and phagocytosis. The focus is on the testing of the hypothesis that an initial step in shedding and phagocytosis is the activation of a phospholipase C that generates inositol trisphosphate and diacylglycerol in the retinal pigment epithelial cells. These second messengers then promote intracellular Ca2+ mobilization and protein kinase C activation, respectively, which in turn modulate several cell functions. The ionized Ca2+ then activates both a phospholipase A2, leading to arachidonic acid release, and lipoxygenase enzymes, resulting in formation of leukotrienes (e.g. leukotriene C4) and other metabolites (e.g. 12-hydroxyeicosatetraenoic acid, lipoxins). These messengers diffuse to the interphotoreceptor matrix and are prime candidates as intercellular signals possibly acting on photoreceptor cells. Phospholipase A2 also gives rise to another lipid mediator, platelet-activating factor, which may contribute to the regulation of long-term events in the retinal pigment epithelium. The proposed experimental design will use powerful now analytical procedures, such as high-performance liquid chromatography-particle beam-mass spectrometry and high-performance liquid chromatography-capillary gas-liquid chromatography. These methods will also allow the isolation and identification of storage sites at membranes of lipid mediators, such as 1 -alkyl-2-- arachidonoyl-glycerophosphorylcholine (the precursor of platelet-activating factor) and of the second messengers of the enzymatic oxygenation of arachidonic acid. Frogs will be used because RPE - visual cell interaction can be experimentally manipulated by altering the light-dark cycle, and shedding can be triggered at the onset of light. A rapid procedure to isolate intact retinal pigment epithelial cells will also allow the direct biochemical study in vivo of each step of the hypothesis. This information will be correlated with the histological assessment of shedding and phagocytosis. Moreover, eyecups will be incubated in vitro with selective antagonists or inhibitors of the second messenger systems involved in these events, as well as receptor antagonists to study the shedding response. In addition, human retinal pigment epithelial cells in culture will be used to explore the various steps of the hypothesis. The results will define the involvement of second messengers in the interactions between visual cells and retinal pigment epithelium during photoreceptor renewal.

这个提议是我们对第二信使研究的延续 源自参与视网膜色素的膜脂的系统 上皮-感光细胞更新过程中视觉细胞的相互作用。 我们 开发了新的方法和假设，其中涉及一系列 在脱落和吞噬作用开始时触发的事件。 这 重点是检验假设的第一步 脱落和吞噬作用是磷脂酶 C 的激活， 在视网膜中生成肌醇三磷酸和二酰甘油 色素上皮细胞。 这些第二信使然后促进 细胞内 Ca2+ 动员和蛋白激酶 C 激活， 分别，进而调节多种细胞功能。 电离的 Ca2+ 然后激活磷脂酶 A2，产生花生四烯酸 释放和脂氧合酶，导致白三烯的形成 （例如白三烯 C4）和其他代谢物（例如 12-羟基二十碳四烯酸，脂氧素）。 这些使者扩散到 光感受器间矩阵，是细胞间质的主要候选者 信号可能作用于感光细胞。 磷脂酶 A2 也 产生另一种脂质介质，血小板激活因子， 可能有助于视网膜长期事件的调节 色素上皮。 拟议的实验设计将使用强大的 现在的分析程序，例如高性能液体 色谱-粒子束-质谱和高效能 液相色谱-毛细管气相-液相色谱。 这些方法 还将允许隔离和识别储存地点 脂质介质的膜，例如 1 -烷基-2-- 花生四烯酰甘油磷酰胆碱（ 血小板活化因子）和酶的第二信使 花生四烯酸的氧化。 青蛙将被使用，因为 RPE - 视觉 细胞相互作用可以通过改变 光暗循环，并且在光照开始时可以触发脱落。 一个 分离完整视网膜色素上皮细胞的快速程序将 还允许对每个步骤进行直接体内生化研究 假设。 该信息将与组织学相关 脱落和吞噬作用的评估。 此外，眼罩将 在体外与第二种选择性拮抗剂或抑制剂一起孵育 参与这些事件的信使系统以及受体 拮抗剂来研究脱落反应。 此外，人类视网膜 培养中的色素上皮细胞将用于探索各种 假设的步骤。 结果将定义参与 视觉细胞和视网膜相互作用的第二信使 光感受器更新期间的色素上皮。