REGULATION OF OXYGEN METABOLISM IN BLOOD NEUTROPHILS

人体血液中性粒细胞产生氧自由基是至关重要的 对于宿主防御的成功，也可能对宿主不利， 某些炎症过程，即成人呼吸窘迫 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，对于 开发控制各种疾病状态的方法。 酶 中性粒细胞中负责将氧气转化为有毒物质的系统 产物似乎是一种可活化的膜结合NADPH氧化酶， 激活机制尚不清楚。 多种刺激剂 (i.e.吞噬颗粒、化学引诱物、钙离子载体，以及 佛波醇酯）激活NADPH氧化酶。 此前的数据显示， 导致NADPH氧化酶活化的途径是复杂的， 刺激特异性和共享成分，共享途径 包括至少两个单独调节的步骤。 最近的观察 暗示蛋白激酶C在 受体介导的细胞调节。 几个事件是由 刺激中性粒细胞，可能导致蛋白激酶C激活， 包括钙浓度的增加， 甘油二酯和花生四烯酸的释放。 此外，蛋白质 激酶C似乎是佛波酯的受体，佛波酯是有效的 刺激中性粒细胞。 该项目的具体目标1将测试 假设蛋白激酶C是细胞凋亡的中心、共享中间体， NADPH氧化酶活化途径。 初步数据表明 中性粒细胞的刺激增加蛋白激酶C活性， 细胞的颗粒部分，并且增加与 氧化酶活化 进一步的实验来测试 这两项活动将举行。 首席研究员最近 表明花生四烯酸在亚细胞组分中激活NADPH氧化酶 从未受刺激的人类中性粒细胞中分离出无活性的部分， 可以被重组以形成活性系统。 这种无细胞激活 系统将用于特定目标2，以确定特定的生化 机制（即蛋白质磷酸化、膜磷脂 变化，膜生物物理变化），与NADPH氧化酶 激活并识别所需的辅助因子。

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.