Monoxide-sensing in a neural tissue: Retinal Muller cells as a sensor

在这个项目中，我们提出了一个新的假说来解释关于双原子气体分子，即一氧化氮(NO)和一氧化碳(CO)在体内对可溶性鸟苷环化酶(SGC)的调节作用的长期争议。我们认为，CO对sGC活性的调节作用不是静态的，而是动态的，低组织可利用性的NO使CO成为sGC的刺激性调节器，而高组织可利用度的NO使CO成为抑制性调节器。我们的研究旨在评估NO是sGC的主要激活剂的假说，但内源性CO在完善NO介导的sGC功能调节中起作用。我们选择大鼠视网膜作为实验系统，是因为它由特定细胞类型组成的明确的解剖层使我们能够检查无或共生成酶与其受体蛋白之间的空间关系。sGCOur的发现提供了一些第一批直接证据，证明sGC存在于穆勒的胶质细胞…中更多的LS(MGCS)和ON型双极细胞，其活性受局部产生的NO和CO控制。此外，这些气体调节sGC的机制似乎不是统一执行的，而是在视网膜的不同层上特定部位执行的。在锌原苯乙酸锌抑制CO看家水平的条件下，所有视网膜细胞层都在最大程度上表现出不依赖于sGC的激活。另一方面，在无抑制条件下，内源CO的抑制以层特异性的方式抑制了光纤层和外限膜的sGC激活。这些结果表明，内源性CO通过抑制sGC功能的最大反应以及适度上调特定视网膜层的最低反应，在微调sGC功能的动态范围中发挥作用。本研究的一个亮点涉及一种独特的抗sGC抗体的特征，该抗体可以检测这种酶的活性。抗体mAb3221根据酶的激活状态相应地改变其亲和力；即它与激活的sGC变得高亲和力，而在酶未被激活时保持低亲和力；因此，它是“功能感应”。使用这种抗体使我们能够可视化sGC在体内的活动。因此，该方法是评估假设的最直接的实验方法。文献中存在关于CO控制sGC功能的一致结果；几个小组假设CO实际上调节cGMP水平，而其他小组未能证明CO的作用。这些相互矛盾的数据大部分可能是由于缺乏评估NO和CO产生的相对可用性的实验系统而产生的，可能可以用我们的假设来解释。大多数调控sGC功能的分子机制的研究都是在培养细胞或试管中进行的，因为它们为蛋白质含量和GAS含量的分析提供了足够的细胞材料。然而，这些系统可能不能充分描述调节完整器官中sGC功能的机制。我们的发现可能会使我们重新理解这两种气体与体内sGC功能之间的联系，以及我们体内感知NO和CO的机制。具有相似结构的不同气体(例如O2、NO、CO)相互作用以改变细胞中的一种蛋白质功能的想法是一个有趣的、潜在的非常重要的概念，我们认为未来不值得继续研究

In this project, we propose a novel hypothesis to explain the long standing controversy concerning the contribution of diatomic gas molecules, namely nitric oxide (NO) and carbon monoxide (CO), to the regulation of soluble guanylate cyclase (sGC) in vivo. We suggest that the effect of CO on modulating sGC activity is not static but dynamic in that low tissue availability of NO makes CO a stimulatory modulator of sGC while high tissue availability of NO makes CO an inhibitory modulator. Our study was designed to evaluate the hypothesis that NO is the dominant activator of sGC but endogenous CO plays a role on refining the NO-mediated regulation of sGC function. We chose the rat retina as an experimental system because its well-defined anatomical layers consisting of specific cell types enabled us to examine spatial relationships between NO- or CO-generating enzyme and its receptor protein, sGCOur findings provide some of the first direct evidence that sGC is present in Muller's glia cel … More ls (MGCs) and on-type bipolar cells and that its activity is controlled by locally produced NO and CO. Furthermore, mechanisms for sGC regulation by these gases appear to be executed not uniformly but site-specifically over the different layers of retina. Under conditions where house-keeping levels of CO were suppressed by zinc protophophyrin IX (ZnPP), all retinal cell layers homogeneously exhibited NO-dependent activation of sGC to the greatest extent. On the other hand, under NO-suppressing conditions, inhibition of endogenous CO abrogated the sGC activation in a layer-specific manner at the optic fiber layer and external limiting membrane. These results suggest that endogenous CO plays a role in fine-tuning dynamic ranges of the NO-dependent regulation of sGC functbn by suppressing the maximum response as well as by modestly elevating the minimum response in particular layers of retinaOne of the highlights of this study involves the characterization of a unique antibody against sGC that can detect the activities of this enzyme. The antibody, mAb3221, changes its affinity accordingly to the activation-state of the enzyme; i.e. it becomes high affinity to the activated sGC, while it maintains low affinity when the enzyme is not activated; thus, it is "function-sensing". Using this antibody enabled us to visualize sGC activities in vivo. Thus, the method is the most direct experimental approach to evaluate the hypothesisConfounded results exist in the literature as to roles of CO to control sGC functions; several groups have postulated that CO actually mediates cGMP levels, while other groups have failed to demonstrate a role for CO. Much of these conflicting data may have arisen from the lack of experimental systems to evaluate the relative availability of NO and CO production, and may be explained by our hypothesis. Most investigations of the molecular mechanisms regulating the sGC function have been carried out in cultured cells or in test tubes because they provide sufficient cellular material for the analysis of protein content and gas content. However, these systems may not adequately describe the mechanisms that regulate sGC functions in intact organs. Our findings might enable a new understanding of the link between the two gases and the sGC function in vivo and, furthermore, of the mechanisms whereby NO and CO are sensed in our bodyThe idea that different gases possessing similar structure (e.g. O2, NO, CO) interact with one another to alter one protein function and thus biochemical and physiological functions in cells is an interesting and potentially highly important concept that we believe worth pursueing in the future Less