REGULATION OF OXYGEN METABOLISM IN BLOOD NEUTROPHILS

人血中性粒细胞产生氧自由基是至关重要的 对于成功的主机防御，也可能对主机不利 某些炎症过程，即在成人呼吸窘迫中 综合征、关节炎等。了解氧自由基的调节 生产是该项目的长期目标，对 开发控制各种疾病状态的方法。这种酶 中性粒细胞中负责将氧气转化为有毒物质的系统 产物似乎是一种可激活的膜结合NADPH氧化酶，其 激活机制尚不清楚。多种刺激剂 (即吞噬颗粒、化学诱导剂、钙离子载体和 佛波酯)激活NADPH氧化酶。之前的数据表明， 导致NADPH氧化酶激活的途径是复杂的，两者都 刺激特定的和共享的组件，并且共享路径 由至少两个单独监管的步骤组成。最近的观察结果 已经暗示蛋白激酶C在 受体介导的细胞调节。由触发的多个事件 对中性粒细胞的刺激可能导致蛋白激酶C的激活， 包括钙浓度的增加，释放 甘油二酯和花生四烯酸的释放。此外，蛋白质 激酶C似乎是佛波醇酯的受体，佛波醇酯是有效的 中性粒细胞的兴奋剂。本项目的具体目标1将测试 假设蛋白激酶C是中心的，共享的中间产物 NADPH氧化酶的激活途径。初步数据显示， 中性粒细胞的刺激增加了蛋白激酶C的活性 细胞中的颗粒物分数，这种增加与 氧化物酶活性。进一步的实验来测试两者之间的相关性 这两个项目将被执行。首席调查员最近 显示花生四烯酸可激活亚细胞组分中的NADPH氧化酶 来自未受刺激的人中性粒细胞和分离的、非活性的部分 可以重组以形成一个活跃的系统。这种无细胞激活 系统将用于特定目标2以确定特定生化 机制(即蛋白质、膜磷脂的磷酸化 变化、膜生物物理变化)与NADPH氧化酶相关 激活并确定所需的辅助因素。

The production of oxygen radicals by human blood neutrophils is critical for the success of host defense, but also may be detrimental to the host in certain inflammatory processes, i.e. in the adult respiratory distress syndrome, arthritis, etc. Understanding the regulation of oxygen radical production is the long-term goal of this project and is important for developing methods of controlling various disease states. The enzyme system in neutrophils responsible for the conversion of oxygen to toxic products appears to be an activatable membrane-bound NADPH oxidase, and its mechanism of activation is not yet known. A variety of stimulating agents (i.e. phagocytic particles, chemoattractants, calcium ionophores, and phorbol esters) activate NADPH oxidase. Previous data suggest that the pathways leading to NADPH oxidase activation are complex, with both stimulus-specific and shared components, and that the shared pathway consists of at least two separately regulated steps. Recent observations have implicated protein kinase C as having a major role in receptor-mediated cellular regulation. Several events are triggered by stimulation of neutrophils that could cause activation of protein kinase C, including an increase in calcium concentration, the release of diacylglycerol, and the release of arachidonate. In addition, protein kinase C appears to be the receptor for phorbol esters, which are potent stimulants for neutrophils. Specific Aim 1 of this project will test the hypothesis that protein kinase C is the central, shared intermediate in the pathway of NADPH oxidase activation. Preliminary data indicate that stimulation of neutrophils increases protein kinase C activity in a particulate fraction of the cell and that the increase correlates with oxidase activation. Further experiments to test the correlation between the two events will be performed. The principal investigator has recently shown that arachidonate activates NADPH oxidase in subcellular fractions from unstimulated human neutrophils and that separate, inactive fractions can be reconstituted to form an active system. This cell-free activation system will be utilized in Specific Aim 2 to determine specific biochemical mechanisms (i.e. phosphorylation of proteins, membrane phospholipid changes, membrane biophysical changes) which correlate with NADPH oxidase activation and to identify required co-factors.