EFFECT OF CYCLIC AMP MEDIATORS ON THE CYTOSKELETON

环AMP介质对细胞骨架的影响

• 批准号: 3426474
• 负责人: DENNIS HAN
• 金额: $ 1.02万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-08-01 至 1989-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3426474
• 关键词: actins; beta adrenergic receptor; cell migration; chemotaxis; cyclic AMP; cytoskeleton; eye pharmacology; fibroblasts; fluorescence microscopy; fluorescent dye /probe; forskolin; human tissue; immunofluorescence technique; membrane proteins; microfilaments; monoclonal antibody; retina disorder; retinal pigment epithelium; retinaldehyde; vinculin

The long-term objective of the study is to determine the role of the cytoskeleton in retinal pigment epithelial (RPE) cell migration. By defining changes in the cytoskeletal configuration which occur during cell migration, the pathophysiologic mechanisms of proliferative vitreoretinopathy can be better understood, leading to more effective treatments of this condition. The specific aim of this study is to define the effect of mediators of intracellular cyclic-3, '5'-AMP (cAMP) on the frequency and distribution of the contractile protein F-actin and the membrane-associated protein vinculin with respect to microfilament bundles which have been implicated in cell migration and chemotaxis. Mediators of intracellular cAMP have been demonstrated to inhibit migration and chemotaxis in human RPE cells, and have also been shown to promote formation and inhibit contraction of stress fibers (microfilament bundles consisting primarily of F-actin) in fibroblasts. Thus, these mediators which are both inhibitory of RPE migration and affect F-actin arrangements in fibroblasts are ideal agents to determine cytoskeletal changes associated with inhibition of cell migration of human RPE. Following adherence and spreading of human RPE cells in vitro, incubation with test reagents (dibutyryl-cAMP, forskolin, L- epinephrine, L-norepinephrine, L-isoproterenol, and caffeine) will be performed, followed by immunofluorescent labeling of vinculin using mouse monoclonal anti-chicken vinculin antibody and fluorescein-conjugated goat anti-mouse IgG antibody. Double labeling with the F-actin fluorescent probe, rhodamine-phalloidin, will then be performed. The frequency and distribution of actin and vinculin and their relationship to microfilament bundles will be documented by fluorescence microscopy and compared with controls. In the event that adrenergic agents induce cytoskeletal changes, pharmacologic blockade (timolol) will be used to determine the role of beta adrenergic receptors in the mediation of such changes.

该研究的长期目标是确定 视网膜色素上皮（RPE）细胞的细胞骨架 迁移。 通过定义细胞骨架构型的变化 发生在细胞迁移过程中，病理生理学 增殖性玻璃体视网膜病变的机制可以更好 理解，从而可以更有效地治疗这种疾病 健康）状况。 本研究的具体目的是确定 细胞内环状 3,'5'-AMP (cAMP) 的介质 收缩蛋白 F-肌动蛋白的频率和分布 膜相关蛋白纽蛋白 与细胞有关的微丝束 迁移和趋化性。 细胞内 cAMP 的介质有 已被证明可以抑制人类的迁移和趋化性 RPE 细胞，也被证明可以促进形成和 抑制应力纤维（微丝束 主要由成纤维细胞中的 F-肌动蛋白组成。 因此，这些 既抑制 RPE 迁移又影响 RPE 的介质 成纤维细胞中的 F-肌动蛋白排列是确定的理想试剂 与细胞迁移抑制相关的细胞骨架变化 人类 RPE。 人类 RPE 细胞在体外粘附和扩散后， 与测试试剂（二丁酰-cAMP、毛喉素、L- 肾上腺素、L-去甲肾上腺素、L-异丙肾上腺素和咖啡因） 进行，然后进行纽蛋白的免疫荧光标记 使用小鼠单克隆抗鸡纽蛋白抗体和 荧光素标记的山羊抗小鼠 IgG 抗体。 双倍的 用 F-肌动蛋白荧光探针、罗丹明-鬼笔环肽进行标记， 然后将被执行。 肌动蛋白的频率和分布 和纽蛋白及其与微丝束的关系将 通过荧光显微镜记录并与 控制。 如果肾上腺素能药物诱导细胞骨架 变化时，将使用药物阻断（噻吗洛尔） 确定β肾上腺素能受体在介导中的作用 此类变化。