Escherichia coli endotoxin inhibits agonist-mediated cytosolic Ca2+ mobilization and nitric oxide biosynthesis in cultured endothelial cells.
Escherichia coli endotoxin inhibits agonist-mediated cytosolic Ca2+ mobilization and nitric oxide biosynthesis in cultured endothelial cells.
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大肠杆菌内毒素抑制培养内皮细胞中激动剂介导的胞浆 Ca2 动员和一氧化氮生物合成。
DOI：
10.1161/01.res.75.4.659
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发表时间：
1994
影响因子：
20.1
通讯作者：
Parker,JL
中科院分区：
文献类型：
--
作者：
Graier,WF;Myers,PR;Rubin,LJ;Adams,HR;Parker,JL
内皮来源的松弛因子/一氧化氮（EDRF/NO）的释放改变被认为是革兰氏阴性脂多糖（内毒素）异常血管舒张反应的最终共同途径。然而，脂多糖诱导内皮细胞中EDRF/NO释放变化的机制尚未明确。我们评估了大肠杆菌内毒素对激动剂刺激的细胞质Ca2+动员和培养牛和猪主动脉内皮细胞（ECs） NO生物合成的直接影响。NO的测定采用两种方法：(1)分析NO诱导的内皮细胞cGMP水平，作为NO生成的生物学指标；(2)直接定量测量NO释放（化学发光法）。使用fura 2荧光法（340/380 nm比激发和500 nm发射）评估胞质游离Ca2+ ([Ca2+]i)。内皮细胞内毒素孵育（0.5微克/毫升，1小时加1小时洗涤）显著抑制缓激肽（100 nmol/L）和ADP （10 μ mol/L）介导的内皮细胞cGMP升高，分别为对照反应的37%和22%。相比之下，内毒素未能抑制非受体依赖性Ca2+离子载体A23187 （1 mmol/L）或硝普钠（1 mmol/L）产生的cGMP的增加。同样，内毒素孵育抑制adp刺激的NO释放和EDRF生物活性分别增加到对照值的55%和56%，但不影响a23187刺激的NO释放和EDRF生物活性的增加。内毒素显著降低了ECs对缓激肽和ADP的瞬时和持续[Ca2+]i反应。例如，牛EC [Ca2+]i在缓激肽作用下的初始快速增加减少到对照细胞初始增加的31%，而二次平台期减少到各自对照反应的3%。与内毒素(10（-3)~ 10(0)微克/mL）的浓度反应关系表明，其对cGMP和[Ca2+]i的抑制作用具有高度相关性，IC50值相近（分别为0.025和0.021微克/mL）。内毒素对缓激肽诱导的肌醇三磷酸形成（[3H]肌醇掺入）和细胞内Ca2+释放（[Ca2+]i在无Ca（2+）培养基中的反应）没有影响。然而，激动剂刺激的Mn2+猝灭（Ca2+内流指数）被内毒素处理显著减弱。这些研究表明，内毒素直接减少激动剂（缓激肽和ADP）介导的ECs中EDRF/NO的生物合成和释放。这些影响可以通过改变[Ca2+]i动员机制来解释，这反过来又导致Ca(2+)-钙调素依赖的一氧化氮合酶组成异构体活性的降低，最终导致激动剂介导的一氧化氮释放和内皮依赖性血管舒张的损害。
Altered release of endothelium-derived relaxing factor/nitric oxide (EDRF/NO) has been proposed as a final common pathway underlying the abnormal vasodilator responses to gram-negative lipopolysaccharide (endotoxin). However, mechanisms responsible for lipopolysaccharide-induced changes in EDRF/NO release from endothelial cells have not been clarified. We evaluated direct effects of Escherichia coli endotoxin on agonist-stimulated cytosolic Ca2+ mobilization and NO biosynthesis in cultured bovine and porcine aortic endothelial cells (ECs). Two methods were used to assay for NO: (1) analysis of NO-induced endothelial levels of cGMP as a biological indicator of NO generation and (2) direct quantitative measurement of NO release (chemiluminescence method). Cytosolic free Ca2+ ([Ca2+]i) was evaluated using fura 2 fluorescence methodology (340/380-nm ratio excitation and 500-nm emission). Incubation of ECs with endotoxin (0.5 microgram/mL, 1 hour plus 1-hour wash) significantly inhibited bradykinin (100 nmol/L)- and ADP (10 mumol/L)-mediated increases in endothelial cell cGMP to 37% and 22% of control responses, respectively. In contrast, endotoxin failed to inhibit the increase in cGMP produced by the non-receptor-dependent Ca2+ ionophore A23187 (1 mumol/L) or sodium nitroprusside (1 mmol/L). Similarly, incubation with endotoxin inhibited ADP-stimulated increases in NO release and EDRF bioactivity to 55% and 56% of control values, respectively, but did not affect A23187-stimulated increases in NO release or EDRF bioactivity. Endotoxin produced significant decreases in both transient and sustained [Ca2+]i responses of ECs to bradykinin and ADP. For example, the initial rapid increase in bovine EC [Ca2+]i in response to bradykinin was reduced to 31% of the initial increases in control cells, and the secondary plateau phase was reduced to only 3% of respective control responses. Concentration-response relation to endotoxin (10(-3)) to 10(0) micrograms/mL) indicated high correlation and similar IC50 values (0.025 and 0.021 micrograms/mL, respectively) for inhibitory effects on cGMP and [Ca2+]i. Endotoxin had no effect on inositol trisphosphate formation ([3H]myo-inositol incorporation) and intracellular Ca2+ release ([Ca2+]i responses in Ca(2+)-free medium) induced by bradykinin. However, agonist-stimulated Mn2+ quenching (index of Ca2+ influx) was significantly attenuated by endotoxin treatment. These studies demonstrate that endotoxin directly decreases agonist (bradykinin and ADP)-mediated biosynthesis and release of EDRF/NO from ECs. These effects can be explained by altered [Ca2+]i mobilization mechanisms, which in turn produce subsequent decreases in activity of the Ca(2+)-calmodulin-dependent constitutive isoform of NO synthase and, ultimately, impairment of agonist-mediated NO release and endothelium-dependent vasodilation.