Redox Activity of the Pulmonary Endothelial Surface

描述(申请人提供)：肺内皮细胞影响血液中氧化还原活性内源性、药理、毒理和饮食化合物的氧化还原状态。它通过充当一个巨大的化学反应表面来实现这一点，该表面由位于静脉和动脉循环之间的细胞表面和细胞内的氧化还原酶组成。内皮细胞对这些化合物氧化还原状态的影响会影响它们的生物活性，包括促氧化或抗氧化作用、组织渗透和化学反应活性。喹酮类化合物是一类重要的氧化还原活性化合物，具有广泛的生理、药理和毒理活性。这一假设的基础是，肺内皮细胞对苯二酚的氧化还原代谢决定了在肺和内皮细胞、血浆以及下游血管和器官系统中的苯醌还原产物及其生物活性。这项拟议研究的目标是确定和量化与细胞和对苯二酚的性质有关的因素，这些因素决定了与肺内皮细胞接触的给定对苯二酚的命运。其具体目的是：1.鉴定和量化参与一系列具有不同物理化学性质的苯醌类化合物归宿的肺内皮醌还原酶和氢氧化酶介导的反应：2.确定影响某一对苯二酚归宿的苯醌还原酶活性与肺内皮细胞氧化还原状态的依赖关系：3.确定肺内皮细胞对高氧的适应对影响苯醌归宿的氧化还原过程的影响。为了达到这些目的，一般的方法包括：利用培养的完整的常氧肺动脉内皮细胞和适应高氧的细胞作为氧化应激的模型，测量细胞内和细胞表面的苯二酚还原、对苯二酚的氧化动力学及其与细胞成分的关系。一组抑制剂和动力学模型将为功能和定量鉴定所涉及的对苯二酚还原酶和对苯二酚氧化酶的贡献提供手段。细胞的氧化还原状态将被实验操纵，以确定它在与内皮细胞接触的苯二酚的命运中所扮演的角色。最后，将分离和鉴定对照细胞和高氧适应细胞中的苯醌还原酶，以进一步表征它们对两种细胞类型对苯醌命运的净影响的贡献。由于对苯二酚还原酶和对苯二酚氧化酶在许多其他氧化还原活性化合物的新陈代谢中起重要作用，这一结果也将对物质的内皮加工产生影响。这些结果将揭示肺内皮细胞参与处理血源性氧化还原活性物质的机制，以及它们对氧化应激的适应，并对它们在全身的药理、生理和毒理活动产生影响。

DESCRIPTION (provided by applicant): The pulmonary endothelium influences the redox status of blood borne redox active endogenous, pharmacological, toxicological and dietary compounds. It does so by acting as a large chemical reactor surface comprised of cell surface and intracellular redox enzymes situated between the venous and arterial circulations. The impact of the endothelium on the redox status of these compounds affects their bioactivity, including pro-or anti-oxidant effects, tissue permeation, and chemical reactivity. Quinones represent 1 important class of redox-active compounds with a range of physiological, pharmacological, and toxicological activities. The hypothesis underlying this proposal is that redox metabolism of quinones by pulmonary endothelial cells determines the quinone reduction products, and their bioactivity, within the lung and endothelial cells, the plasma, and downstream vessels and organ systems. The goal of the proposed research is to identify and quantify the factors, with regard to properties of both the cells and quinones, that determine the fate of a given quinone that comes in contact with the pulmonary endothelium. The specific aims are to: 1. Identify and quantify the pulmonary endothelial quinone reductase and hydroquinone oxidase mediated reactions contributing to the fate of a series of quinones with varying physical and chemical properties: 2. Determine the dependence of the activities of quinone reductases contributing to the fate of a given quinone on the pulmonary endothelial redox status: 3. Determine the influence of adaptation of the pulmonary endothelium to hyperoxia on the redox processes contributing to quinone fate. To achieve these aims, the general approaches include: measuring the kinetics of intracellular and cell surface quinone reduction, hydroquinone oxidation and associations with cellular constituents using intact normoxic pulmonary arterial endothelial cells in culture and cells adapted to hyperoxia, as a model of oxidant stress. A panel of inhibitors and kinetic modeling will provide the means for functional and quantitative identification of the contributions of the quinone reductases and hydroquinone oxidases involved. Cell redox status will be manipulated experimentally to determine its role in the fate of the quinones that come in contact with the endothelial cells. Finally, the quinone reductases in the control and hyperoxia-adapted cells will be isolated and identified to further characterize their contributions to the net effect of both cell types to quinone fate. Since quinone reductases and hydroquinone oxidases are important in metabolism of a wide range of other redox active compounds, the results will have implications for endothelial processing of substances as well. The results will reveal pulmonary endothelial mechanisms involved in processing blood borne redox active substances, and their adaptation to oxidative stress, with implications for their pharmacological, physiological and toxicological activities throughout the body.