Kim Ji Young, Lee Kwang Hoon, Lee Bong Ki, Ro Jai Youl
Department of Pharmacology,Center for Molecular Medicine, SBRI,Sungkyunkwan University School of Medicine, Suwon, Korea.
Int Arch Allergy Immunol. 2005 Jun;137(2):104-14. doi: 10.1159/000085465. Epub 2005 Apr 25.
Peroxynitrite (ONOO-), the product of the reaction between the superoxide anion (*O2-) and nitric oxide (NO), is produced during inflammatory disease and may be a major cytotoxic agent. No reports are available as to whether ONOO- generates or modulates inflammatory mediator release from activated guinea pig lung mast cells. In this study, we explored the modulatory role of intracellular ONOO- on inflammatory mediator release (histamine and leukotrienes) from activated mast cells.
Guinea pig lung mast cells were purified by the enzyme digestion, and by using the rough and discontinuous Percoll density gradients. Mast cells were sensitized with IgG1 (anti-ovalbumin) antibody and challenged with ovalbumin (OVA). The intracellular ROS formation was determined by following the oxidative production of 2', 7'-dichlorofluorescein diacetate (DCFH-DA), dihydrorhodamine 123 (DHR), and anti-nitrotyrosine antibody immunofluorescence. Histamine was assayed using a fluorometric analyzer, leukotrienes by radioimmunoassay, intracellular Ca2+ levels by confocal scanning microscopy, and PLA(2) activity using prelabeling of [3H]arachidonic acid.
ROS detected by DCFH-DA weakly increased in mast cells activated with OVA (1.0 g/ml), and the ROS so generated was inhibited by ebselen (50 microM). However, the ROS detected by DHR increased 3-fold under the same conditions. Peroxynitrite scavengers sL-MT, DMTU, and inhibitor FeTPPS inhibited ROS formation but the NADPH oxidase inhibitor diphenyleneiodonium (DPI) only partially inhibited this formation. Dimethyl thiourea (DMTU) and seleno-L-methionine (sL-MT) inhibited the tyrosine nitration of cytosolic proteins, the release of histamine and leukotrienes, Ca2+ influx, and the PLA(2) activity evoked by mast cell activation.
The data obtained suggests that the ROS generated by the antigen/antibody reaction activated mast cells is ONOO-, and that this modulates the release of inflammatory mediators via Ca2+ -dependent PLA(2) activity.
过氧亚硝酸根（ONOO-）是超氧阴离子（*O2-）与一氧化氮（NO）反应的产物，在炎症性疾病过程中产生，可能是一种主要的细胞毒性因子。关于ONOO-是否会引发或调节活化的豚鼠肺肥大细胞释放炎症介质，目前尚无相关报道。在本研究中，我们探讨了细胞内ONOO-对活化肥大细胞释放炎症介质（组胺和白三烯）的调节作用。
通过酶消化法，并使用粗制且不连续的Percoll密度梯度对豚鼠肺肥大细胞进行纯化。肥大细胞用IgG1（抗卵清蛋白）抗体致敏，并用卵清蛋白（OVA）进行刺激。通过跟踪2',7'-二氯荧光素二乙酸酯（DCFH-DA）、二氢罗丹明123（DHR）的氧化产物以及抗硝基酪氨酸抗体免疫荧光来测定细胞内活性氧的形成。使用荧光分析仪测定组胺，通过放射免疫分析法测定白三烯，通过共聚焦扫描显微镜测定细胞内Ca2+水平，并使用[3H]花生四烯酸预标记法测定磷脂酶A2（PLA(2)）活性。
用OVA（1.0 μg/ml）激活的肥大细胞中，DCFH-DA检测到的活性氧略有增加，且所产生的活性氧被依布硒啉（50 μM）抑制。然而，在相同条件下，DHR检测到的活性氧增加了3倍。过氧亚硝酸根清除剂sL-MT、DMTU和抑制剂FeTPPS抑制活性氧的形成，但NADPH氧化酶抑制剂二苯基碘鎓（DPI）仅部分抑制这种形成。二甲基硫脲（DMTU）和硒代-L-甲硫氨酸（sL-MT）抑制细胞溶质蛋白的酪氨酸硝化、组胺和白三烯的释放、Ca2+内流以及肥大细胞活化所引发的PLA(2)活性。
所获得的数据表明，抗原/抗体反应激活的肥大细胞产生的活性氧是ONOO-，并且它通过Ca2+依赖性PLA(2)活性调节炎症介质的释放。
