| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 2002: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2001: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
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, sGC
Our findings provide some of the first direct evidence that sGC is present in Muller's glia cel
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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 retina
One 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 hypothesis
Confounded 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 body
The 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
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