Arias-Díaz J, Vara E, García C, Villa N, Balibrea J L
Department of Biochemistry (Facultad de Medicina), Universidad Complutense, Madrid, Spain.
Arch Surg. 1995 Dec;130(12):1287-93. doi: 10.1001/archsurg.1995.01430120041006.
The second messenger cyclic guanosine 3',5'-monophosphate (cGMP) seems to be implicated in the release of tumor necrosis factor alpha (TNF-alpha) by activated macrophages. There is controversy regarding the potential of human macrophages to produce nitric oxide (NO). Since guanylate cyclase can be activated also by carbon monoxide (CO) and this gas may be formed endogenously, we examined the ability of human pulmonary macrophages to produce CO in the presence of lipopolysaccharide (LPS) or LPS+interferon gamma (IFN-gamma). In addition, the source and the relative contribution of this molecule to the LPS-induced increase in cell cGMP content and TNF-alpha release were explored.
Interstitial macrophages were obtained from multiple organ donor lungs by enzymatic digestion. After 24-hour preculture, purified macrophages were cultured for 24 hours in the presence or absence of LPS, LPS+IFN-gamma, CO (250 and 500 mumol/L), sodium nitroprusside, 8-Br-cGMP, hemoglobin, methylene blue, zinc-protoporphyrin IX, hemin, S-adenosylmethionine, deferoxamine mesylate, or combinations. The cGMP content of the cells and TNF-alpha, CO, and NO release to the medium were determined.
In the presence of LPS, TNF-alpha production was not accompanied by any detectable increase in the NO release to the medium. However, an increase in medium CO concentration (mean +/- SEM) (5.81 +/- 0.20 vs 3.74 +/- 0.08 pmol/microgram protein; n = 11; P < .01) and cell cGMP content (0.273 +/- 0.021 vs 0.138 +/- 0.019 pmol/microgram protein; n = 10; P < .01) was observed. These changes were more pronounced in the presence of LPS+IFN-gamma. Release of TNF-alpha also was induced by both sodium nitroprusside and 8-Br-cGMP. In contrast, methylene blue, a guanylate cyclase inhibitor, inhibited LPS-, LPS+IFN-gamma-, and sodium nitroprusside-induced TNF-alpha production and cGMP increase; hemoglobin, which traps CO, had a similar effect.
Intracellular cGMP increase, secondary to an endogenous production of CO, participates in the release of TNF-alpha by activated human pulmonary macrophages.
第二信使环磷酸鸟苷（cGMP）似乎与活化巨噬细胞释放肿瘤坏死因子α（TNF-α）有关。关于人类巨噬细胞产生一氧化氮（NO）的潜力存在争议。由于鸟苷酸环化酶也可被一氧化碳（CO）激活，且这种气体可能内源性生成，我们研究了人类肺巨噬细胞在脂多糖（LPS）或LPS +干扰素γ（IFN-γ）存在的情况下产生CO的能力。此外，还探讨了该分子对LPS诱导的细胞cGMP含量增加和TNF-α释放的来源及相对贡献。
通过酶消化从多个器官供体肺中获取间质巨噬细胞。经过24小时预培养后，将纯化的巨噬细胞在有或无LPS、LPS + IFN-γ、CO（250和500 μmol/L）、硝普钠、8-溴-cGMP、血红蛋白、亚甲蓝、锌原卟啉IX、血红素、S-腺苷甲硫氨酸、去铁胺甲磺酸盐或其组合的条件下培养24小时。测定细胞的cGMP含量以及TNF-α、CO和NO向培养基中的释放量。
在LPS存在的情况下，TNF-α的产生并未伴随着培养基中NO释放量的任何可检测到的增加。然而，观察到培养基中CO浓度增加（平均值±标准误）（5.81±0.20对3.74±0.08 pmol/μg蛋白质；n = 11；P <.01）以及细胞cGMP含量增加（0.273±0.021对0.138±0.019 pmol/μg蛋白质；n = 10；P <.01）。在LPS + IFN-γ存在的情况下，这些变化更为明显。硝普钠和8-溴-cGMP也诱导了TNF-α的释放。相反，鸟苷酸环化酶抑制剂亚甲蓝抑制了LPS、LPS + IFN-γ和硝普钠诱导的TNF-α产生和cGMP增加；捕获CO的血红蛋白也有类似作用。
内源性产生CO导致的细胞内cGMP增加参与了活化的人类肺巨噬细胞释放TNF-α的过程。
