NAD & CD38 Form a Communication System in the Retina

这项研究建议受到我们的发现的刺激，CD38是一种最初被认为是专门定位于淋巴细胞的胞外酶，它在视网膜Muller细胞的细胞膜中存在显著水平。为了理解CD38的功能作用，我们从自己的实验中提出了一个假说，在这个假说中，我们提出了一个假设，即从细胞释放的外部NAD激活CD38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细胞的内部钙水平，cADPR通过激活ryanodine受体改变钙。兰尼定受体的激活会触发钙波，这种钙波可以被兰尼定、咖啡因和塔普西林阻断或修饰，后者会耗尽体内的钙储备。钙波可能通过释放物质改变米勒细胞的功能状态，如ATP，这可能会影响神经元和神经胶质细胞。此外，钙离子的变化也可能引发蛋白质表达的变化，从而改变米勒细胞的表型。这种变化可能有助于米勒细胞在应对机械或创伤性侮辱和疾病状态时的反应性质。这项建议超越了与通过外部NAD调节内部钙相关的问题；我们将使用成像和高效液相技术(On-Exchange HPLC)来检测NAD，彻底研究NAD从细胞释放的机制。我们将确定允许细胞释放NAD的细胞种类和细胞机制，例如缝隙连接半通道撕裂NAD允许途径的可能性。我们还将使用钙成像和外用cADPR来研究外部产生的cADPR是否可以作为旁分泌激素来改变神经细胞的行为。我们的一般假设，受到我们最近发现的CD38和NAD的作用的启发，认为CD38是内外沟通通路的震中，在该通路中，传入途径是通过细胞释放的NAD，通过CD38转化为内化的cADPR(可能还有NAADP)，这一途径提高了Muller细胞的内部钙水平，并触发了钙波。这一假说的传出途径是，cADPR，无论是由CD38外部合成的，还是从内部Muller细胞储存中释放出来的，都在细胞外循环，并被神经元内化，作为反馈途径，影响视网膜神经元和色素上皮细胞的钙水平。因此，这一被提出的途径是一种在Muller细胞中诱导的钙的大小和水平，提供了一种反馈的调节机制，参与并支持神经元钙的变化。如果这一假设能够在实验上得到证实，它将为一种全新的方法奠定基础，通过这种方法，神经元和胶质细胞相互支持，以稳定视网膜功能。

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.