Bi-directional effects of the elevation of intracellular calcium on the expression of inducible nitric oxide synthase in J774 macrophages exposed to low and to high concentrations of endotoxin
Bi-directional effects of the elevation of intracellular calcium on the expression of inducible nitric oxide synthase in J774 macrophages exposed to low and to high concentrations of endotoxin
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DOI：
10.1042/0264-6021:3540351
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发表时间：
2001-03-01
影响因子：
4.1
通讯作者：
Moilanen, E
中科院分区：
文献类型：
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作者：
Korhonen, R;Kankaanranta, H;Moilanen, E
诱导型一氧化氮合酶（inducible Nitric oxide synthase，iNOS）是机体免疫反应的重要介质。各种细胞外因子，包括炎症刺激，影响细胞内游离Ca 2+水平（[Ca 2 +]（i）），调节细胞信号传导和基因表达。在本研究中，我们研究了增加[Ca 2 +]（i）对J774巨噬细胞通过iNOS途径产生NO的影响。以Ca 2 +-ATP酶抑制剂毒胡萝卜素（TG）和Ca 2+离子载体A23187为工具，诱导胞浆[Ca 2 +]（i）增加。Fura 2方法证实了这种增加。采用Western blotting和RT-PCR方法检测iNOS蛋白和mRNA的表达。用电泳迁移率改变法检测核因子-κ B（NF-κ B）的活化。TG（100 nM）诱导iNOS mRNA，iNOS蛋白和NO的显着合成的细胞用低浓度的内毒素[脂多糖（LPS）1 ng/ml]，其本身诱导几乎检测不到NO的合成。高浓度LPS（100 ng/ml）刺激可诱导iNOS的表达和NO的产生。在此条件下，TG处理通过加速iNOSmRNA的降解来抑制iNOS蛋白的合成和NO的产生。用TG（100 nM）处理不影响低浓度（1 ng/ml）或高浓度（100 ng/ml）LPS诱导的NF-κ B活性。通过2，3-双[2-甲氧基-4-硝基-5-磺基苯基]-2H-四唑鎓-5-羧基苯胺（“XTT”）方法确认细胞的活力;通过碘化丙啶染色和流式细胞术排除细胞凋亡。A23187（1 μ M）也瞬时增加[Ca ~（2+）]（i），并对NO的产生有相反的作用，这取决于LPS浓度。我们的研究结果表明，增加[Ca 2 +]（i）诱导的刺激或抑制NO的生产，通过诱导型一氧化氮合酶在巨噬细胞取决于细胞活化状态。这些发现表明，受体介导的[Ca 2 +]（i）增加可能是控制炎症基因（包括编码iNOS的基因）上调和下调之间平衡的重要因素，这取决于炎症反应的阶段。
Nitric oxide produced through the action of inducible nitric oxide synthase (iNOS) is an important mediator in immune responses of the host. Various extracellular factors, including inflammatory stimuli, affect intracellular free Ca2+ levels ([Ca2+](i)), modulating cellular signalling and gene expression. In the present study we investigated the effects of increased [Ca2+](i) on NO production through the iNOS pathway in J774 macrophages. Thapsigargin (TG), a Ca2+-ATPase inhibitor, and the Ca2+ ionophore A23187 were used as tools to induce an increase in [Ca2+](i) in the cytosol. This increase was confirmed by the fura 2 method. The production of NO was measured as accumulated nitrite in the cell culture medium; iNOS protein and iNOS mRNA were detected by Western blotting and reverse-transcriptase-mediated PCR respectively. The activation of nuclear factor kappaB (NF-kappaB) was investigated by electrophoretic mobility-shift assay. TG (100 nM) induced a marked synthesis of iNOS mRNA, iNOS protein and NO in cells primed with a low concentration of endotoxin [lipopolysaccharide (LPS) 1 ng/ml], which on its own induced barely detectable NO synthesis. Stimulation by a high concentration of LPS (100 ng/ml) induced a marked expression of iNOS and NO production. Under these conditions, treatment with TG hindered the synthesis of iNOS protein and NO production by accelerating the degradation of iNOS mRNA. Treatment with TG (100 nM) did not affect the NF-kappaB activity induced by low (1 ng/ml) or high (100 ng/ml) concentrations of LPS. Viability of the cells was confirmed by the 2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxyaniline ('XTT') method; apoptosis was ruled out by propidium iodide staining and flow cytometry. A23187 (1 muM) also transiently increased [Ca2+](i) and had opposite effects on NO production depending on the LPS concentration. Our results show that increased [Ca2+](i) induced the stimulation or suppression of NO production through iNOS in macrophages depending on the state of cell activation. These findings suggest that the receptor-mediated increase in [Ca2+](i) might be an important factor in the control of the balance between the upregulation and down-regulation of inflammatory genes, including that encoding iNOS, depending on the phase of the inflammatory response.