Role of carbon monoxide derived from heme-oxygenase 2 on the regulation of neural function

作为该项目的第一部分，我们提出了一个新的假说来解释长期存在的争议，即体内代谢气体分子，即一氧化氮(NO)和一氧化碳(CO)对可溶性鸟苷环化酶(SGC)的调节作用。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。我们的发现提供了M…中存在sGC的一些第一个直接证据更多的Uller‘s胶质细胞(MGCs)和ON型双极细胞，其活动受局部产生的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功能的最大反应和适度升高视网膜各层的最小反应，在微调sGC功能依赖的动态范围中发挥作用。作为项目的第二部分，我们提供了第一个直接证据，证明HO产生的CO可以通过干扰NO的产生来减弱成年大鼠软脑膜小动脉的血管扩张反应。在这项研究中，脑微循环中CO产生的生理学相关性得到了两种独立实验方法的结果的支持：在内源性CO或NO产生不同的情况下测量软脑膜小动脉直径，以及在正常和CO抑制条件下使用荧光探针直接原位检测NO产生。虽然显示NO+敏感荧光的确切细胞类型尚未确定，但使用共聚焦系统量化荧光强度并结合NO产生酶的免疫组织化学检测首次提供了原位大脑微循环中NO产生的时空信息。

As the first part of the project, we propose a novel hypothesis to explain the long standing controversy concerning the contribution of diatoimic 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, sGC.Our findings provide some of the first direct evidence that sGC is present in M … More uller's glia cells(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 function by suppressing the maximum response as well as by modestly elevating the minimum response in particular layers of retina.As the second part of the project, we provide the first direct evidence that CO produced by HO can attenuate vasodilatory responses of pial arterioles by interfering with the NO production in the adult rat. In this study, the physiological relevance of CO production in cerebral microcirculation is supported by the results of two independent experimental approaches : measurement of pial arteriolar diameter under conditions where endogenous CO or NO production was varied, and direct detection of NO production in situ using the fluoroprobe under normal and CO-suppressed conditions. Although exact cell types that exhibit the NO^+-sensitive fluorescence are yet to be identified, the use of the confocal system to quantify fluorescence intensity together with the immunohistochemical detection of NO-producing enzymes provides spatial and temporal information of NO production of cerebral microcirculation in situ for the first time.0 Less