Real-time amperometric analysis of reactive oxygen and nitrogen species released by single immunostimulated macrophages
Real-time amperometric analysis of reactive oxygen and nitrogen species released by single immunostimulated macrophages
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DOI：
10.1002/cbic.200700746
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发表时间：
2008-06-16
期刊：
影响因子：
3.2
通讯作者：
Koh, Alaric C. W.
中科院分区：
文献类型：
--
作者：
Amatore, Christian;Arbault, Stephane;Koh, Alaric C. W.
巨噬细胞是免疫系统的关键细胞。已知免疫活化的巨噬细胞释放活性氧和氮物质的混合物。在这项工作中，RAW 264.7巨噬细胞被γ-干扰素和脂多糖激活，并通过电流分析法在铂化碳微电极上真实的时间分析单细胞释放的反应混合物。与未处理的巨噬细胞相比，观察到活化的巨噬细胞的电流响应显著增加。一氧化氮（NO中心点），亚硝酸盐（NO2-），和过氧亚硝酸盐（ONOO-）的主要反应物种检测。定量这些反应性物质的量，并评价它们的超龄通量释放的单个活化巨噬细胞。ONOO-的检测特别令人感兴趣，因为它在各种生理条件下的作用和意义已被广泛讨论。在这里，直接证据ONOO-在刺激mocrophins的形成。最后，1400 W，一种选择性诱导型一氧化氮合酶（iNOS）抑制剂的存在下，导致激活RAW 264.7细胞的电流响应几乎完全衰减。因此，由巨噬细胞释放的大部分活性物质可能来源于NO中心点和由iNOS共同产生的超氧化物（O-2（中心点-））。
Macrophages are key cells of the immune system. Immunologically activated macrophages are known to release a cocktail of reactive oxygen and nitrogen species. In this work, RAW 264.7 macrophages were activated by interferon-gamma and lipopolysaccharide, and the reactive mixture released by single cells was analyzed, in real time, by amperometry at platinized carbon microelectrodes. In comparison with untreated macrophages, significant increases in amperometric responses were observed for activated macrophages. Nitric oxide (NO center dot), nitrite (NO2-), and peroxynitrite (ONOO-) were the main reactive species detected. The amounts of these reactive species were quantified, and their over-age fluxes released single, activated mocrophage were evaluated. The detection of ONOO- is of particular interest, as its role and implications in various physiological conditions have been widely debated. Herein, direct evidence for the formation of ONOO- in stimulated mocrophages is presented. Finally, the presence of 1400W, a selective inducible nitric oxide synthase (iNOS) inhibitor, led to an almost complete attenuation of the amperometric response of activated RAW 264.7 cells. The majority of the reactive species released by a macrophage are thus likely to be derived from NO center dot and superoxide (O-2(center dot-)) co-produced by iNOS.