Purinergic Regulation of the Retinal Pigment Epithelium

视网膜色素上皮细胞控制着许多对视觉过程至关重要的功能，包括将液体和离子运输出视网膜下腔，以及对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，加入嘌呤ATP和腺苷的RPE细胞可以修改这两个功能。目前建议的目标是确定这些嘌呤的来源，以及如何改变它们的可用性以影响吞噬作用和液体流动。有三个具体目标。首先，将识别ATP释放的生理触发器。特别地，将用荧光素酶/荧光素酶测定系统确定显示昼夜节律波动的神经化学物质刺激ATP从新鲜牛RPE细胞释放的能力。将使用假定的ATP转运蛋白（包括这些细胞中表达的囊性纤维化跨膜传导调节因子）的阻断剂探测ATP退出途径。第二个目的是确定这种释放的ATP是否在细胞外转化为腺苷。腺苷水平将在触发ATP释放后直接测量。将测量负责ATP到腺苷的细胞外转化的酶的活性，并将其改变。将使用PCR反应鉴定这些酶的特异性同种型。将使用PCR反应鉴定这些酶的特异性同种型。第三，将检查由该途径产生的生理分支。通过检查RPE细胞对荧光棒外段和珠的摄取，直接监测对吞噬作用的影响。将通过测量组织中的C1依赖性短路电流来评估C1转运速率的变化。这项研究将提供信息的内源性协调的嘌呤信号在视网膜下的空间，通过确定ATP和腺苷的来源能够刺激嘌呤受体和随后的影响，这种刺激对离子流和吞噬作用。

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.