MITOCHONDRIAL SUPEROXIDE GENERATION IN HYPEROXIA

高氧条件下线粒体超氧化物的生成

• 批准号: 3087848
• 负责人: David Marshall Guidot
• 金额: $ 8.36万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3087848
• 关键词: NAD(P)H dehydrogenase; Saccharomyces cerevisiae; cellular respiration; cytochrome b; electron transport; enzyme activity; genetic manipulation; genetic models; hyperoxia; southern blotting; superoxide dismutase; superoxides; western blottings

The Problem: Medical management of many critically ill patients necessitates delivery of high concentrations of inspired oxygen to maintain tissue oxygenation. However, hyperoxic therapy creates a dilemma as it directly damages cells. While extensive research has implicated superoxide anion (O2-) or O2- derived products as key mediators of hyperoxic injury, the precise locations of O2- generation and its specific molecular targets are not known. Our hypothesis is that mitochondrial electron transport contributes to increased O2- production in hyperoxia and that O2- damages both mitochondrial and cytosolic targets. We have chosen a yeast model to initially test our hypothesis. Yeast are eukaryotic cells with important similarities to mammalian cells. However, unlike mammalian cells, yeast can be mutated in precise ways and can grow facultatively if electron transport is disrupted. Our preliminary data support our premise. First, we found that yeast which lack the mitochondrial form of superoxide dismutase (Mn-SOD) are sensitive to hyperoxia despite normal levels of the cytosolic form of SOD (Cu,Zn- SOD). Oxygen sensitivity is increased if electron transport activity is increased with non-fermentable carbon sources. Complete genetic disruption of electron transport in the Mn-SOD deficient cell restores oxygen resistance. These data strongly implicate mitochondrial electron transport as an important source of O2- in hyperoxia. Second, we found that in enriched media with glucose, yeast lacking Cu,Zn-SOD but not Mn-SOD grow normally in room air but fail to grow when electron transport is increased by supplying a non-fermentable carbon source in place of glucose. These data imply that O2- generated in the mitochondria leaks into and causes damage in the cytosol. My immediate specific objectives are to identify a) the relevant source(s) of O2- in the electron transport chain, b) the magnitude of O2- release from mitochondria in hyperoxia, c) the molecular targets of O2- within mitochondria, d) the role of Cu,Zn-SOD in scavenging O2- released from mitochondria, e) if mitochondria can be uncoupled by back diffusion of protonated O2- and f) if extramitochondrial SOD can protect the mitochondria from hyperoxic damage. The significance of this project is to provide new information about the role of mitochondrial electron transport in hyperoxic injury. The site(s) of O2- production in the electron transport chain and its principal molecular targets must be identified in order to better understand how O2- mediates hyperoxic injury and how the poor outcome of patients with catastrophic illnesses who require hyperoxic therapy can be improved.

问题：许多重症患者的医疗管理 必须递送高浓度的灵感氧气以维持 组织氧合。 但是，高氧化疗法会产生困境 直接损坏细胞。 虽然广泛的研究暗示了超氧化物 阴离子（O2-）或O2衍生产物作为高氧损伤的关键介体， O2生成及其特定分子靶标的精确位置 不知道。 我们的假设是线粒体电子传输有助于 增加了O2的生产高氧，O2-两者都会损害 线粒体和胞质靶标。 我们选择了酵母模型 最初检验我们的假设。 酵母是具有重要的真核细胞 与哺乳动物细胞相似。 但是，与哺乳动物细胞不同，酵母 可以以精确的方式突变，如果电子 运输受到干扰。 我们的初步数据支持我们的前提。 首先，我们发现酵母 缺乏超氧化物歧化酶（MN-SOD）的线粒体形式很敏感 尽管SOD的胞质形式正常（Cu，Zn- 草皮）。 如果电子传输活性为 不可发酵的碳源增加。 完全遗传破坏 Mn-Sod缺乏细胞中的电子传输可恢复氧气 反抗。 这些数据强烈暗示线粒体电子传输 作为高氧中O2-的重要来源。 其次，我们发现 富含葡萄糖的培养基，酵母缺乏Cu，Zn-Sod，但Mn-Sod不生长 通常在房间空气中，但当电子传输增加时无法生长 通过提供不可发酵的碳源代替葡萄糖。 这些 数据暗示线粒体中产生的O2泄漏到并导致原因 细胞质的损伤。 我的直接特定目标是确定a）相关来源 o2-在电子传输链中，b）O2释放的幅度 从高氧中的线粒体中，c）O2-内部的分子靶标 线粒体，d）Cu，Zn-Sod在清除O2中的作用 - 从 线粒体，e）如果线粒体可以通过向后扩散解开 质子化的O2-和f）如果胶外软糖可以保护 线粒体因高氧损伤。 该项目的意义是提供有关 线粒体电子转运在高氧损伤中的作用。 网站 电子运输链中的O2生产及其本金 必须确定分子靶标，以便更好地了解O2- 介导高氧损伤以及患者的不良预后如何 需要改善需要高氧化治疗的灾难性疾病。