SYNERGISTIC STIMULATION AND PRIMING OF NEUTROPHILS

中性粒细胞的协同刺激和启动

• 批准号: 3142793
• 负责人: JOHN A BADWEY
• 金额: $ 16.99万
• 依托单位: BOSTON BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3142793
• 关键词: antibody formation; binding proteins; blood donor; calcium flux; calcium transporting ATPase; cell membrane; cytoskeleton; eicosanoids; flavins; guinea pigs; human tissue; laboratory rabbit; leukocyte oxidative burst; leukopoietic factor; magnesium; neutrophil; phospholipase A2; phosphoproteins; phosphorylation; protein biosynthesis; protein kinase C; protein sequence; protein signal sequence; superoxides; synthetic peptide; tissue /cell culture

The long term objectives of this proposal are to elucidate the exact sequence of biochemical events that are involved in the priming and stimulation of superoxide (02-) production by neutrophils. Superoxide is a key component of the oxygen-dependent antimicrobial mechanisms of these cells which provide a major defense against infectious organisms. Certain cytokines prime neutrophils to release increased amounts of 02-. Therapeutic value of these factors is now being tested clinically in a variety of cancer and AIDS patients with promising results. Thus, experiments outlined herein are relevant to an understanding of host- defense mechanisms and immune deficiency diseases. Specifically, this project addresses two largely unknown areas. These are: (1) the mechanism(s) of action of the hydroxylated eicosatetraenoic acids (HETE) in modulating 02- production, and (2) the link between protein phosphorylation and 02- generation. Neutrophils produce 5- and 15-HETE under various circumstances. 5-HETE dramatically potentiates 02- release, whereas 15-HETE inhibits it. We propose that 5- HETE primes neutrophils by increasing the flux of Ca2+ across the plasmalemma, perhaps by opening a channel for this cation. This will be measuring the rates of 45Ca2+ uptake by these cells in the presence of different concentrations of 5-HETE. The effects of a variety of calcium channel antagonists on this process will be evaluated. The relationships between the rates of 45Ca2+ uptake and 02- generation will be defined, and possible antagonistic effects of 15-HETE will be sought. With regard to protein phosphorylation, we have observed that stimulation of 02- release by neutrophils is always accompanied by an intense phosphorylation of two proteins with molecular weights of ca. 47 and 49 kDa. Quantitatively, these are the two major proteins which are phosphorylated upon stimulation of these cells. While the 47 kDa protein has been extensively characterized, virtually nothing is known of the 49 kDa species. Experiments are detailed herein to identify, characterize and purify this protein. Techniques of biochemistry and cell biology will be employed. We will determine whether the 49 kDa protein is a component of the 02- generating system, or is involved in some other fashion in the mechanism of stimulation of these cells (e.g., phospholipose D). Changes in the activity of the 49 kDa protein after phosphorylation will be sought to forge a link between this modification reaction and cell stimulation.

该提案的长期目标是阐明确切的 参与启动和启动的生化事件序列 刺激中性粒细胞产生超氧化物 (02-)。 超氧化物是 这些药物的氧依赖性抗菌机制的关键组成部分 细胞提供针对传染性生物体的主要防御。 肯定 细胞因子促使中性粒细胞释放更多的 02-。 这些因素的治疗价值现在正在临床上进行测试 多种癌症和艾滋病患者的治疗效果有希望。 因此， 本文概述的实验与理解宿主相关 防御机制和免疫缺陷疾病。 具体来说，该项目涉及两个很大程度上未知的领域。 这些 是： (1) 羟基化二十碳四烯酸的作用机制 酸 (HETE) 在调节 02- 生产中的作用，以及 (2) 之间的联系 蛋白质磷酸化和02-生成。 中性粒细胞产生 5- 和 15-HETE在各种情况下。 5-HETE 显着 增强 02- 释放，而 15-HETE 抑制它。 我们建议 5- HETE 通过增加 Ca2+ 穿过细胞膜的通量来启动中性粒细胞 质膜，也许是通过为该阳离子打开通道来实现的。 这将是 测量存在时这些细胞对 45Ca2+ 的摄取率 不同浓度的5-HETE。 各种效果 钙通道拮抗剂对该过程的影响将被评估。 这 45Ca2+摄取率与02-生成率之间的关系 将被定义，并且 15-HETE 可能的拮抗作用将被确定 寻求。 关于蛋白质磷酸化，我们观察到刺激 中性粒细胞释放的 02- 总是伴随着强烈的 分子量约为 2 的两种蛋白质的磷酸化。 47 和 49 kDa。 从数量上讲，这是两种主要蛋白质 这些细胞受到刺激后被磷酸化。 而 47 kDa 的蛋白质 已被广泛表征，但实际上对这 49 个一无所知 kDa 物种。 本文详细描述了实验来识别、表征 并纯化该蛋白质。 生物化学和细胞技术 将采用生物学。 我们将确定 49 kDa 蛋白质是否是 02-生成系统的一个组成部分，或涉及其他一些 这些细胞的刺激机制的时尚（例如， 磷脂 D). 49 kDa 蛋白活性的变化 将寻求磷酸化以在这种修饰之间建立联系 反应和细胞刺激。