NAD & CD38 Form a Communication System in the Retina

辅酶A

• 批准号: 6623070
• 负责人: Robert Francis Miller
• 金额: $ 28.81万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6623070
• 关键词: CD38 molecule; Chelonia; Muller's cell; Primates; Urodela; adenosine triphosphate; calcium flux; cats; cell cell interaction; confocal scanning microscopy; gap junctions; goldfish; high performance liquid chromatography; immunocytochemistry; immunofluorescence technique; laboratory rabbit; laboratory rat; neurons; nicotinamide adenine dinucleotide; organ culture; retina; retinal ganglion; ribose; western blottings

This research proposal has been stimulated by our discovery that CD38, an ectoenzyme originally thought to be exclusively localized to lymphocytes, is present in significant levels in the cell membranes of retinal Muller cells. To understand the functional role of CD38, we have developed a hypothesis, derived from our own experiments, in which we propose that external NAD, released from cells, activates CD38 to produce internalized second-messengers, such as cyclic ADP- ribose (cADPR)and/or nicotinic acid adenine dinucleotide phosphate (NAADP). Our working hypothesis is that cADPR and/or NAADP modulate the internal levels of calcium in Muller cells and that cADPR alters calcium through activation of ryanodine receptors. Activation of ryanodine receptors triggers calcium waves which can be blocked or modified by ryanodine, caffeine and thapsigargin, the latter of which depletes internal calcium stores. Calcium waves may alter the functional state of the Muller cells by releasing agents, such as ATP, which can affect neurons and glial cells. In addition, the changes in calcium may also trigger changes in the expression of proteins which alter the phenotype of the Muller cell. Such changes may contribute to the reactive nature of Muller cells in responding to mechanical or traumatic insults and in response to disease states. This proposal goes beyond issues related to the regulation of internal calcium through external NAD; we will thoroughly examine the mechanisms by which NAD is released from cells using both imaging and HPLC techniques (on-exchange HPLC) to assay NAD. We will determine the kinds of cells and the cellular mechanisms which permit cellular release of NAD, such as the possibility that gap junction hemi channels torn an NAD permissive pathway. We will also investigate whether externally produced cADPR could serve as a paracrine hormone to modify the behavior of nerve cells, using calcium imaging and external application of cADPR. Our general hypothesis, stimulated by our recent discoveries of CD38 and the action of NAD, is that CD38 serves as the epicenter for an external/internal communication pathway in which the afferent path is through NAD released from cells which, through CD38 is converted to internalized cADPR (and possibly NAADP)and that this pathway enhances the internal calcium levels of Muller cells and triggers calcium waves. The efferent pathway of this hypothesis is that cADPR, whether synthesized externally by CD38 or released from internal Muller cell storage circulates extracellularly and is internalized by neurons to serve as a feedback pathway to after calcium levels in retinal neurons and perhaps the pigment epithelial cells. Thus, this proposed pathway is one in which the magnitude and levels of calcium induced in Muller cells, provides a feedback, regulatory mechanism which engages and supports changes in neuronal calcium. If this hypothesis can be experimentally established, it will form the basis of an entirely new method by which neurons and glia support each other to stabilize retinal function.

本研究的建议已经刺激了我们的发现，CD 38，一种胞外酶，最初被认为是专门定位于淋巴细胞，是目前在视网膜穆勒细胞的细胞膜中的显着水平。为了理解CD 38的功能作用，我们从我们自己的实验中提出了一个假设，其中我们提出从细胞释放的外部NAD激活CD 38以产生内化的第二信使，如环状ADP-核糖（cADPR）和/或烟酸腺嘌呤二核苷酸磷酸（NAADP）。我们的工作假设是cADPR和/或NAADP调节Muller细胞中的内部钙水平，并且cADPR通过激活ryanodine受体来改变钙。Ryanodine受体的激活触发钙波，钙波可以被Ryanodine、咖啡因和毒胡萝卜素阻断或改变，后者消耗内部钙储存。钙波可以通过释放能够影响神经元和神经胶质细胞的物质（如ATP）来改变Muller细胞的功能状态。此外，钙的变化也可能触发改变Muller细胞表型的蛋白质表达的变化。这些变化可能有助于Muller细胞对机械或创伤性损伤以及对疾病状态的反应性。该建议超越了通过外部NAD调节内部钙的相关问题;我们将彻底研究NAD从细胞中释放的机制，使用成像和HPLC技术（交换HPLC）来测定NAD。我们将确定允许细胞释放NAD的细胞类型和细胞机制，例如间隙连接半通道撕裂NAD允许通路的可能性。我们还将研究是否外部产生的cADPR可以作为一种旁分泌激素来改变神经细胞的行为，使用钙成像和cADPR的外部应用。我们的一般假设，由我们最近发现的CD 38和NAD的作用刺激，是CD 38作为外部/内部通信途径的中心，其中传入路径是通过从细胞释放的NAD，通过CD 38转化为内化的cADPR（可能还有NAADP），并且该途径增强Muller细胞的内部钙水平并触发钙波。该假说的传出途径是cADPR，无论是由CD 38外部合成还是从内部Muller细胞储存释放，都在细胞外循环，并被神经元内化，以作为视网膜神经元和可能的色素上皮细胞中钙水平的反馈途径。因此，该提出的途径是其中在Muller细胞中诱导的钙的幅度和水平提供了参与和支持神经元钙变化的反馈调节机制的途径。如果这一假设能够在实验中得到证实，那么它将成为一种全新方法的基础，通过这种方法，神经元和神经胶质相互支持以稳定视网膜功能。