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，包括液体和离子从视网膜下空间的运输，以及盘膜的周期性吞噬。从杆外段脱落。最近的证据表明，在RPE细胞中加入嘌呤、ATP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，以及它们的可用性如何被修改以影响吞噬和液体流动。有三个具体目标。首先，ATP释放的生理触发因素将被确定。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/荧光素酶测定系统来确定。我们将使用ATP转运蛋白的阻滞剂，包括在这些细胞中表达的囊性纤维化跨膜传导调节因子，来探索ATP的退出途径。第二个目的是确定释放的ATP是否在细胞外转化为腺苷。腺苷水平将在触发ATP释放时直接测量。负责细胞外ATP转化为腺苷的酶的活性将被测量和药理学改变。这些酶的特定同种异构体将通过聚合酶链反应进行鉴定。这些酶的特定同种异构体将通过聚合酶链反应进行鉴定。第三，这条路线所产生的生理后果将被检查。对吞噬作用的影响将通过检测RPE细胞对荧光棒外段和珠的摄取来直接监测。c1传输速率的变化将通过测量组织中c1相关的短路电流来评估。本研究将通过识别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的影响，为视网膜下空间嘌呤能信号的内源性协调提供信息。

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.