Purinergic Regulation of the Retinal Pigment Epithelium

视网膜色素上皮的嘌呤能调节

• 批准号: 6431297
• 负责人: CLAIRE H MITCHELL
• 金额: $ 23.11万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6431297
• 关键词: adenosine; adenosine triphosphate; animal tissue; circadian rhythms; enzyme activity; fluid flow; growth factor; ion transport; phagocytosis; polymerase chain reaction; protein isoforms; purinergic receptor; purines; receptor expression; retinal pigment epithelium; tissue /cell culture; visual photoreceptor

The retinal pigment epithelium controls a number of functions critical for the visual process, including the transport of fluid and ions out of subretinal space, and the periodic phagocytosis of disc membranes. shed from rod outer segments. Recent evidence suggests that the addition of purines ATP and adenosine to RPE cells can modify both of these functions. The goal of the current proposal is to determine the source of these purines and how their availability can be modified to affect phagocytosis and fluid flow. There are three Specific Aims. First, physiologic triggers for ATP release will be identified. In particular, the ability of neurochemicals showing circadian fluctuation to stimulate ATP release from fresh bovine RPE cells will be determine with the luciferin/luciferase assay system. The pathway for ATP exit will be probed using blockers for putative ATP transporters, including the cystic fibrosis transmembrane conductance regulator expressed in these cells. The second aim is to determine if this released ATP is converted extracellularly into adenosine. Adenosine levels will be measured directly in response to triggering the release of ATP. The activity of the enzymes responsible for the extracellular conversion of ATP to adenosine will be measured and altered pharmacologically. The specific isoforms of these enzymes will be identified using the PCR reaction. The specific isoforms of these enzymes will be identified using the PCR reaction. Third, physiologic ramifications produced by this route will be examined. Effects on phagocytosis will be monitored directly by examining the uptake of fluorescent rod outer segments and beads by RPE cells. Changes in the rate of C1-transport will be assessed by measured the C1-dependent short circuit current across the tissue. This research will provide information about the endogenous coordination of purinergic signals in the sub-retinal space by identifying the source of ATP and adenosine capable of stimulating purinergic receptors and the consequent effects of this stimulation on ion flow and phagocytosis.

视网膜色素上皮控制着许多对视觉过程至关重要的功能，包括将流体和离子从视网膜下空间传输出来，以及椎间盘膜的周期性吞噬作用。从杆外段脱落。最近的证据表明，将嘌呤ATP和腺苷添加到RPE细胞中可以改变这两个功能。当前建议的目的是确定这些嘌呤的来源以及如何修改其可用性以影响吞噬作用和流体流动。有三个特定的目标。首先，将确定用于ATP释放的生理触发器。特别是，通过荧光素/荧光素酶测定系统，将确定神经化学物质显示昼夜节律刺激ATP从新鲜牛RPE细胞中释放的能力。将使用定义ATP转运蛋白的阻滞剂进行探测ATP出口的途径，包括这些细胞中表达的囊性纤维化跨膜电导调节剂。第二个目的是确定该释放的ATP是否被细胞外转化为腺苷。腺苷水平将直接测量以触发ATP的释放。负责ATP细胞外转化为腺苷的酶的活性将通过药理测量和改变。这些酶的特异性同工型将使用PCR反应鉴定。这些酶的特异性同工型将使用PCR反应鉴定。第三，将检查该路线产生的生理分析。通过检查RPE细胞对荧光杆外部段和珠的摄取，将直接监测对吞噬作用的影响。 C1-Transport速率的变化将通过在整个组织中测量的C1依赖性短路电流来评估。这项研究将通过确定能够刺激嘌呤能受体的ATP和腺苷的来源以及这种刺激对离子流量和吞噬作用的影响，提供有关视网膜下空间中嘌呤能信号的内源性协调的信息。