REGULATION OF OXYGEN METABOLISM IN BLOOD NEUTROPHILS

血液中性粒细胞氧代谢的调节

• 批准号: 3133823
• 负责人: LINDA C. McPHAIL
• 金额: $ 8.67万
• 依托单位: WAKE FOREST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 1990-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3133823
• 关键词: antimetabolites; arachidonate; autoradiography; biophysics; blood fractionation; calcium; cell free system; cell growth regulation; electrophoresis; esters; fatty acids; fluorescence spectrometry; human tissue; membrane lipids; neutrophil; phorbols; phospholipids; phosphorylation; protein kinase C; respiratory oxygen; scintillation counter; spectrometry

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.

人体血液中性粒细胞产生氧自由基至关重要 对于宿主防御的成功，但也可能对宿主不利 某些炎症过程，即成人呼吸窘迫 综合症、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 产品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、化学引诱剂、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 氧化酶激活的途径很复杂， 刺激特异性和共享成分，以及共享路径 由至少两个单独调节的步骤组成。 最近的观察 表明蛋白激酶 C 在 受体介导的细胞调节。 多个事件由以下事件触发 刺激中性粒细胞，可能导致蛋白激酶 C 激活， 包括增加钙浓度、释放 二酰基甘油，以及花生四烯酸的释放。 此外，蛋白质 激酶 C 似乎是佛波酯的受体，佛波酯是有效的 中性粒细胞兴奋剂。 该项目的具体目标 1 将测试 假设蛋白激酶 C 是该过程中的核心、共享中间体 NADPH氧化酶激活途径。 初步数据表明 刺激中性粒细胞会增加蛋白激酶 C 的活性 细胞的颗粒部分，并且增加与 氧化酶激活。 进一步的实验来测试之间的相关性 将执行这两个事件。 首席研究员最近 显示花生四烯酸激活亚细胞组分中的 NADPH 氧化酶 来自未刺激的人类中性粒细胞，并分离出非活性部分 可以重组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 以确定特定的生化指标 机制（即蛋白质磷酸化、膜磷脂 与 NADPH 氧化酶相关的变化（膜生物物理变化） 激活并确定所需的辅助因子。