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.

人类血液中性粒细胞生产氧自由基是至关重要的 为了获得主持人防御的成功，但也可能对主持人有害 某些炎症过程，即在成人呼吸窘迫中 综合征，关节炎等。了解氧气的调节 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。 酶 负责氧转化为有毒的中性粒细胞的系统 产品似乎是一种可活化的膜结合的NADPH氧化酶，它的 激活机制尚不清楚。 各种刺激剂 （即吞噬颗粒，趋化因子，钙离子载体和 佛波酯）激活NADPH氧化酶。 以前的数据表明 导致NADPH氧化酶激活的途径很复杂，两者都 刺激特定和共享组件，以及共享途径 由至少两个单独调节的步骤组成。 最近的观察 将蛋白激酶C牵涉到在 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的激活 包括钙浓度的增加，释放 二酰基甘油和蛛网膜酸的释放。 另外，蛋白质 激酶C似乎是凤凰酯的受体，这是有效的 中性粒细胞的兴奋剂。 该项目的具体目标1将测试 假设蛋白激酶C是中心，共享的中间体 NADPH氧化酶激活的途径。 初步数据表明 中性粒细胞的刺激增加了蛋白激酶C活性 细胞的颗粒分数，增加与 氧化酶激活。 进一步测试相关性的实验 这两个事件将进行。 主要调查员最近有 表明蛛网酸会激活亚细胞级分中的NADPH氧化酶 来自未刺激的人类嗜中性粒细胞和分开的，无活跃的部分 可以重构以形成一个活动系统。 这种无细胞激活 系统将在特定目标2中使用以确定特定的生化 机理（即蛋白质的磷酸化，膜磷脂 变化，膜生物物理变化）与NADPH氧化酶相关 激活并识别所需的共同因素。