PHYSIOLOGIC PHOSPHOLIPASE A2 ACTIVATION IN NEUTROPHILS

中性粒细胞中磷脂酶 A2 的生理激活

• 批准号: 3455649
• 负责人: SUE A BAULDRY
• 金额: $ 11.72万
• 依托单位: WAKE FOREST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3455649
• 关键词: G protein; arachidonate; biological signal transduction; calcium; chemoattractants; densitometry; enzyme mechanism; gas chromatography; hematopoietic growth factor; high performance liquid chromatography; interleukin 1; leukocyte activation /transformation; leukotrienes; membrane lipids; membrane permeability; neutrophil; peptides; phospholipase A2; phospholipids; radioimmunoassay; radiotracer; receptor; receptor coupling; second messengers; stainings; staphylococcal exotoxin; thin layer chromatography

Polymorphonuclear neutrophils (PMN) produce the important lipid inflammatory mediators arachidonic acid (AA), 5-hydroxyeicosatetraenoic acid (5-HETE), leukotriene B4 (LTB4), and platelet activating factor (PAF). Pharmacologic activation of human PMN with calcium ionophore has demonstrated that the initial step in formation of these molecules involves phospholipase A2 (PLA2) catalyzed release of AA from membrane phospholipids; however, progress in studying formation of these lipid mediators has been limited by lack of information on the physiological control of PLA2. In the proposed studies we will employ a newly developed model of permeabilized PMN to study PLA2 activation initiated with soluble stimuli and will test the hypothesis that receptor-mediated PLA2 activation involves a G-protein mediated event which alters enzyme requirements for specific cofactors, in particular, Ca2+. We also will define the optimum intracellular concentrations of cofactors and/or ions necessary for PLA2 activation in stimulated cells. Requirements for PLA2 activity will be assessed in PMN permeabilized with the plasma membrane specific agent, Staphylococcus aureus alpha-toxin. This model allows manipulation of intracellular concentration of small molecules or ions which are potential cofactors for PLA2 activation while keeping enzymes, proteins and other intracellular organelles intact. PLA2 activation will be assayed by determining the release and metabolism of radiolabeled AA from membrane phospholipids and by monitoring radiolabeled lyso-PAF and PAF formation. Thin layer chromatographic techniques and high pressure liquid chromatography (HPLC) will be utilized for lipid identification. Mass determinations of AA, LTB4, 5-HETE, lyso-PAF and PAF will be performed using techniques of gas chromatography, radioimmunoassay, HPLC and coomassie blue staining followed by densitometry. While stimulation of human PMN with a single physiologic agonist evokes only limited PLA2 activity, receptor-mediated activation of PLA2 is greatly enhanced during "primed-stimulation". In this process, neutrophils are sequentially treated with non-stimulatory concentrations of agonists; in primed PMN exposed to a second agonist, PLA2 activity is initiated and release of AA is much greater than expected from an additive response to the individual stimulus. After investigating the mechanism(s) involved in direct PLA2 activation in permeabilized PMN, the molecular basis leading to enzyme activation during "primed-stimulation" will be determined.

多形核中性粒细胞（PMN）产生重要的脂质 炎症介质蛛网膜酸（AA），5-羟基乙酸酯 酸（5-HETE），白三烯B4（LTB4）和血小板激活因子（PAF）。 用钙离子载体对人PMN的药理激活具有 证明这些分子形成的第一步涉及 磷脂酶A2（PLA2）催化AA从膜上释放 磷脂；但是，研究这些脂质的形成进展 介体因缺乏生理信息而受到限制 PLA2的控制。 在拟议的研究中，我们将采用新开发的 透化PMN的模型研究了用可溶性启动的PLA2激活 刺激并将测试受体介导的PLA2激活的假设 涉及G蛋白介导的事件，该事件改变了酶的要求 特定的辅助因子，特别是Ca2+。 我们还将定义最佳 PLA2所需的辅因子和/或离子的细胞内浓度 刺激细胞的激活。 PMN通透性将评估PLA2活性的要求 质膜特异性剂，金黄色葡萄球菌α-毒素。 该模型允许操纵小细胞内浓度 分子或离子是潜在的辅助因子，用于PLA2激活 保持酶，蛋白质和其他细胞内细胞器完整。 PLA2 通过确定释放和代谢，将测定激活 从膜磷脂和通过监测放射性标记的放射性标记的AA Lyso-PAF和PAF组。 薄层色谱技术和高 压力液相色谱（HPLC）将用于脂质 鉴别。 AA，LTB4，5-HETE，LYSO-PAF和PAF的质量确定 将使用气相色谱技术，放射免疫测定技术进行 HPLC和Coomassie蓝色染色，然后是光密度法。 而用单个生理激动剂刺激人PMN唤起 只有有限的PLA2活性，受体介导的PLA2激活极大地是 在“启动刺激”期间增强。 在此过程中，中性粒细胞是 用非刺激浓度的激动剂依次处理；在 暴露于第二个激动剂的PMN启动PLA2活性，并 AA的释放远大于预期的添加响应 个体刺激。 研究了涉及的机制之后 透化PMN中的直接PLA2激活，这是分子基础 将确定“启动刺激”期间的酶激活。