NONHEME IRON NITROSYL COMPLEX-FORMATION IN RAT HEPATOCYTES - DETECTION BY ELECTRON-PARAMAGNETIC RESONANCE SPECTROSCOPY
NONHEME IRON NITROSYL COMPLEX-FORMATION IN RAT HEPATOCYTES - DETECTION BY ELECTRON-PARAMAGNETIC RESONANCE SPECTROSCOPY
复制标题
DOI：
10.1006/abbi.1993.1173
复制
发表时间：
1993-04-01
影响因子：
3.9
通讯作者：
LANCASTER, JR
中科院分区：
文献类型：
--
作者：
STADLER, J;BERGONIA, HA;LANCASTER, JR
在体外暴露于炎症刺激（干扰素-γ [IFN-γ]、肿瘤坏死因子-α [TNF-α]、白细胞介素-1β [IL-1β] 和脂多糖 [LPS]）后，在小棒状杆菌体内免疫激活后，以及在暴露于 · Ndouble bond0 和硝普钠（硝基铁氰化物）（一种 NO 供体）后，通过 EPR 光谱法检查分离的大鼠肝细胞。硝基血管扩张剂。暴露于IFN-γ、TNF-α、IL-1β和LPS的肝细胞表现出ag=2.04轴向EPR信号的出现，表明非血红素铁-亚硝酰复合物的形成。与N双键O合酶的竞争性抑制剂L-NG-单甲基精氨酸(L-NMMA)同时孵育可防止信号的出现。 g2.04 信号位于肝细胞提取物的胞质部分中。新鲜分离自C.的肝细胞。 parvum处理的大鼠表现出适度的g = 2.04信号，在随后在没有额外刺激的培养基中培养24小时后，该信号增加了约2.5倍。这种增加被培养基中的 L-NMMA 以及添加到培养物中的大鼠红细胞所阻止。在红细胞存在的情况下，几乎所有产生的·N双键O都通过与红细胞内的细胞内血红蛋白反应而被氧化，这通过检测到的亚硝酸盐和硝酸盐的相对量来判断。这些结果表明，在该模型系统中，N双键O足够稳定且可扩散，可以从肝细胞逃逸并扩散到红细胞中，而无需首先与氧或在其在肝细胞内形成的位点处与细胞内铁反应。用外源·N双键O或硝普钠处理肝细胞产生相同的g=2.04信号，其强度比用细胞因子加LPS强得多。用硝普钠处理还引起来自五氰基亚硝基高铁酸根离子的信号的出现，验证了先前报道的通过氰化物离子和·N双键O的还原和释放来进行这种硝基血管舒张剂的代谢。这些结果表明，与细胞毒性激活的巨噬细胞相比，肝细胞中的细胞内非血红素铁亚硝基化存在显着差异，这可能与这两个系统中·N双键O的生理功能差异相关。
Isolated rat hepatocytes were examined by EPR spectroscopy after exposure to inflammatory stimuli (interferon-γ [IFN-γ], tumor necrosis factor-α [TNF-α], interleukin-1β [IL-1β], and lipopolysaccharide [LPS])in vitro, afterin vivoimmune activation by Corynebacterium parvum, and after exposure to · Ndouble bond0 and to nitroprusside (nitroferricyanide), an NO-donating nitrovasodilator. Hepatocytes exposed to IFN-γ, TNF-α, IL-1β, and LPS demonstrated the appearance of ag= 2.04 axial EPR signal indicative of the formation of nonheme iron-nitrosyl complexes. Concurrent incubation with L-NG-monomethylarginine (L-NMMA), a competitive inhibitor of · Ndouble bondO synthase, prevented the appearance of the signal. Theg2.04 signal was localized in the cytosolic fraction of hepatocyte extracts. Hepatocytes freshly isolated fromC. parvum-treated rats exhibited a modestg= 2.04 signal, which was increased by a factor of approximately 2.5-fold upon subsequent 24-h culture in media without additional stimuli. This increase was prevented by L-NMMA in the culture medium and also by the presence of rat erythrocytes added to the culture. In the presence of erythrocytes, virtually all of the · Ndouble bondO produced was oxidized by reaction with intracellular hemoglobin within the erythrocyte, as judged by the relative amounts of nitrite and nitrate detected. These results suggest that in this model system · Ndouble bondO is sufficiently stable and diffusible to escape from the hepatocyte and diffuse into the erythrocyte without first reacting with oxygen or with intracellular iron at the site of its formation within the hepatocyte. Treatment of hepatocytes with exogenous · Ndouble bondO or nitroprusside generated an identicalg= 2.04 signal of much greater intensity than with cytokines plus LPS. Treatment with nitroprusside also caused the appearance of a signal from pentacyanonitrosylferrate ion, verifying the previously reported metabolism of this nitrovasodilator by reduction and liberation of cyanide ion and · Ndouble bondO. These results indicate significant differences in intracellular nonheme iron nitrosylation in hepatocytes compared to cytotoxic activated macrophages, which may correlate with the differences in physiological function of · Ndouble bondO in these two systems.