Purinergic Regulation of the Retinal Pigment Epithelium

视网膜色素上皮控制着许多对视觉过程至关重要的功能，包括将液体和离子输送出视网膜下间隙，以及周期性地吞噬视盘膜。从杆件外段脱落。最近的证据表明，在RPE细胞中加入嘌呤、三磷酸腺苷和腺苷可以改变这两种功能。当前提案的目标是确定这些嘌呤的来源，以及如何修改它们的可用性以影响吞噬和液体流动。有三个具体目标。首先，将确定ATP释放的生理触发因素。特别是，显示昼夜节律波动的神经化学物质刺激新鲜牛RPE细胞释放ATP的能力将通过荧光素/荧光素酶检测系统来确定。将使用假定的ATP转运体的阻滞剂来探索ATP退出的途径，包括在这些细胞中表达的囊性纤维化跨膜电导调节因子。第二个目的是确定这种释放的三磷酸腺苷是否在细胞外转化为腺苷。将直接测量腺苷水平，以响应触发ATP的释放。负责细胞外将三磷酸腺苷转化为腺苷的酶的活性将被测量和药理学改变。这些酶的特定亚型将通过聚合酶链式反应进行鉴定。这些酶的特定亚型将通过聚合酶链式反应进行鉴定。第三，我们将研究这条路线产生的生理后果。通过检查RPE细胞对荧光棒外段和珠子的摄取，将直接监测对吞噬功能的影响。通过测量组织中依赖于c1的短路电流来评估c1转运速率的变化。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动和吞噬作用的后续影响，来提供关于视网膜下间隙中嘌呤能信号的内源性协调的信息。

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.