Vanin A F, Mordvintcev P I, Hauschildt S, Mülsch A
Institute of Chemical Physics, Russian Academy of Sciences, Moscow.
Biochim Biophys Acta. 1993 May 8;1177(1):37-42. doi: 10.1016/0167-4889(93)90154-h.
We identified the source of the nitrogen included into nitric oxide (NO) and studied the relationship between formation of NO, intracellular dinitrosyl ferrous iron complex (DNIC) and release of nitrite by murine bone-marrow-derived macrophages stimulated with E. coli lipopolysaccharide (LPS). NO was trapped in the cell membrane by iron-diethyldithiocarbamate complex (FeDETC) and was detected as a paramagnetic NOFe(DETC)2 complex by electron paramagnetic resonance (EPR) spectroscopy. Macrophages stimulated for 7 h up to 48 h with LPS and then incubated for 2 h with DETC exhibited an anisotropic EPR signal of axial symmetry with g-factor values g perpendicular = 2.035, g parallel = 2.02 and a triplet hyperfine structure (hfs) at g perpendicular characteristic for NOFe(DETC)2. In cells incubated with [15NG]L-arginine instead of [14NG]L-arginine the EPR signal of [15N]OFe(DETC)2 was detected with a doublet hfs at g perpendicular, indicating that NO was generated exclusively from the terminal guanidino-nitrogen of extracellular L-arginine. The ratio of NO formation and of nitrite release changed with time of exposure to LPS, nitrite exceeding NO at early stages of macrophage activation, and NO exceeding nitrite at later stages. DNIC with thiolate ligands (0.5 nmol/10(7) cells) was observed in stimulated macrophages not loaded with DETC. Furthermore, DNIC released from macrophages was trapped in the extracellular medium by bovine serum albumin (BSA) (1 nmol/10(7) cells per 2 h) by formation of a paramagnetic DNIC with BSA. DNIC release not only provides a route for iron loss from activated macrophages, but may also play a role in the cytotoxic and microbiostatic activity of macrophages.
我们确定了一氧化氮（NO）中氮的来源，并研究了用大肠杆菌脂多糖（LPS）刺激的小鼠骨髓来源巨噬细胞中NO的形成、细胞内二亚硝基亚铁络合物（DNIC）与亚硝酸盐释放之间的关系。NO通过铁-二乙基二硫代氨基甲酸盐络合物（FeDETC）捕获在细胞膜中，并通过电子顺磁共振（EPR）光谱检测为顺磁性NOFe(DETC)2络合物。用LPS刺激巨噬细胞7小时至48小时，然后与DETC孵育2小时，表现出具有轴对称性的各向异性EPR信号，g因子值g垂直 = 2.035，g平行 = 2.02，在g垂直处具有NOFe(DETC)2特有的三重态超精细结构（hfs）。在用[15N G]L-精氨酸代替[14N G]L-精氨酸孵育的细胞中，检测到[15N]OFe(DETC)2的EPR信号在g垂直处具有双峰hfs，表明NO仅由细胞外L-精氨酸的末端胍基氮产生。NO形成与亚硝酸盐释放的比率随暴露于LPS的时间而变化，在巨噬细胞激活的早期亚硝酸盐超过NO，而在后期NO超过亚硝酸盐。在未加载DETC的受刺激巨噬细胞中观察到具有硫醇盐配体的DNIC（0.5 nmol/10(7)个细胞）。此外，从巨噬细胞释放的DNIC通过与牛血清白蛋白（BSA）形成顺磁性DNIC而被捕获在细胞外培养基中（每2小时1 nmol/10(7)个细胞）。DNIC的释放不仅为活化巨噬细胞中的铁流失提供了一条途径，而且可能在巨噬细胞的细胞毒性和抑菌活性中发挥作用。
