DeMeester S L, Qiu Y, Buchman T G, Hotchkiss R S, Dunnigan K, Karl I E, Cobb J P
Department of Surgery, Washington University, St. Louis, MO 63110, USA.
Crit Care Med. 1998 Sep;26(9):1500-9. doi: 10.1097/00003246-199809000-00016.
To determine a mechanism by which nitric oxide alters induction of stress-induced endothelial cell apoptosis in vitro. Apoptosis is a form of cellular suicide that has been implicated in the pathogenesis of multiple organ dysfunction syndrome.
Prospective, controlled trial.
Research laboratory of a large, academic medical center.
Cultured primary porcine aortic endothelial cells.
Cells were treated with a range of doses of agents that either spontaneously generate nitric oxide (S-nitroso-N-acetyl-D,L-penicillamine [SNAP] or (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1,2-diolate [DETA-NO]) or block nitric oxide production (Nomega-methyl-L-arginine [L-NMA]). The ability of these agents to alter the rate of cell death by apoptosis (induced by the sequence stimuli lipopolysaccharide [LPS] followed by sodium arsenite) was measured. Mechanistic studies included examining the ability of: a) nitric oxide "donors" to alter nuclear factor kappa B (NF-kappaB) DNA binding activity and the level of IkappaBalpha accumulation; and b) a stable cyclic guanosine monophosphate (cGMP) analog (8-bromo-cGMP) to mimic the effect of nitric oxide donors.
The sequence LPS/sodium arsenite increased the rate of endothelial cell apoptosis (47.4%, p< .05 vs. control), as measured by fluorescent-activated cell scanning using annexin V/propidium iodide staining. DETA-NO generated nitric oxide (as indicated by an increase in the concentration of the stable end-products of nitric oxide metabolism) and decreased the rate of endothelial cell apoptosis (20.6% at a dose of 2 mM, p=.0001 vs. control). DETA-NO also decreased NF-kappaB DNA binding activity and the apparent accumulation of its endogenous inhibitor, IkappaBalpha. The 8-bromo-cGMP did not mimic the effects of nitric oxide donors (DETA-NO) on apoptosis.
These data suggest that exogenous nitric oxide can block stress-induced endothelial cell apoptosis in vitro. The mechanistic studies are consistent with our hypothesis that inhibitors of NF-kappaB DNA binding activity are associated with protection against apoptosis-inducing stimuli. The results do not support a role for cGMP in mediating the protective effect of DETA-NO in our model.
确定一氧化氮在体外改变应激诱导的内皮细胞凋亡的诱导机制。凋亡是一种细胞自杀形式，与多器官功能障碍综合征的发病机制有关。
前瞻性对照试验。
大型学术医学中心的研究实验室。
培养的原代猪主动脉内皮细胞。
用一系列剂量的试剂处理细胞，这些试剂要么自发产生一氧化氮（S-亚硝基-N-乙酰-D,L-青霉胺[SNAP]或(Z)-1-[2-(2-氨基乙基)-N-(2-氨乙基)氨基]重氮-1-鎓-1,2-二醇盐[DETA-NO]），要么阻断一氧化氮的产生（Nω-甲基-L-精氨酸[L-NMA]）。测量这些试剂改变细胞凋亡导致的细胞死亡率的能力（由脂多糖[LPS]后接亚砷酸钠的序列刺激诱导）。机制研究包括检查：a）一氧化氮“供体”改变核因子κB（NF-κB）DNA结合活性和IκBα积累水平的能力；b）稳定的环磷酸鸟苷（cGMP）类似物（8-溴-cGMP）模拟一氧化氮供体作用的能力。
通过使用膜联蛋白V/碘化丙啶染色的荧光激活细胞扫描测量，LPS/亚砷酸钠序列增加了内皮细胞凋亡率（47.4%，与对照组相比p<0.05）。DETA-NO产生了一氧化氮（如一氧化氮代谢稳定终产物浓度增加所示）并降低了内皮细胞凋亡率（2 mM剂量下为20.6%，与对照组相比p = 0.0001）。DETA-NO还降低了NF-κB DNA结合活性及其内源性抑制剂IκBα的明显积累。8-溴-cGMP没有模拟一氧化氮供体（DETA-NO）对凋亡的影响。
这些数据表明外源性一氧化氮可在体外阻断应激诱导的内皮细胞凋亡。机制研究与我们的假设一致，即NF-κB DNA结合活性抑制剂与抵抗凋亡诱导刺激的保护作用相关。结果不支持cGMP在介导DETA-NO在我们模型中的保护作用方面的作用。
