Pae H O, Yoo J C, Choi B M, Paik S G, Kim Y H, Jin H S, Chung H T
Department of Microbiology and Immunology, Wonkwang University School of Medicine, Medicinal Resources Research Center of Wonkwang University, Iksan, Chonbug, Korea.
Immunol Invest. 1999 Mar-May;28(2-3):149-63. doi: 10.3109/08820139909061144.
A previous study has demonstrated that both interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) were needed to induce the production of nitric oxide (NO) in BNL CL.2 cells, murine embryonic liver cells. We here demonstrate that when BNL CL.2 cells were cultured with serum-free medium, they were induced to produce NO by the stimulation of IFN-gamma alone. BNL CL.2 cells were cultured with serum-free or serum-containing medium for 1-3 days and then stimulated to synthesize NO by IFN-gamma. Surprisingly, only serum-starved cells showed significant amount of nitrite accumulation and iNOS protein expression in response to IFN-gamma in dose- and time-dependent manners, but serum-supplied cells did not. When the cells were stimulated with IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), or LPS in combinations, only the combination of IFN-gamma and LPS produced more NO than that produced by IFN-gamma alone. The production of NO by the cells stimulated with IFN-gamma or IFN-gamma plus LPS was blocked by the addition of N(G)-monomethyl-L-arginine (N(G)MMA), a NO synthesis inhibitor. To address the intracellular signal pathway responsible for the production of NO by the cells stimulated with IFN-gamma aloneor IFN-gamma plus LPS, we examined the effects of several protein kinase inhibitors on the production of NO from the cells. The production of NO was significantly inhibited by protein tyrosine kinase (PTK) inhibitors, genistein and herbimycin A, but not by protein kinase A or C inhibitors. These results suggest that the deprivation of serum from BNL CL.2 cell culture medium might prime the cells to induce NO synthesis when the cells are triggered by IFN-gamma and the involvement of PTK signal transduction pathway in the expression of inducible NO synthase gene in murine hepatoma cells.
先前的一项研究表明，干扰素-γ（IFN-γ）和脂多糖（LPS）都需要才能诱导小鼠胚胎肝细胞BNL CL.2细胞产生一氧化氮（NO）。我们在此证明，当BNL CL.2细胞在无血清培养基中培养时，仅通过IFN-γ刺激就能诱导它们产生NO。将BNL CL.2细胞在无血清或含血清的培养基中培养1至3天，然后用IFN-γ刺激以合成NO。令人惊讶的是，只有血清饥饿的细胞以剂量和时间依赖性方式对IFN-γ产生显著量的亚硝酸盐积累和诱导型一氧化氮合酶（iNOS）蛋白表达，而补充血清的细胞则没有。当细胞用IFN-γ、肿瘤坏死因子-α（TNF-α）或LPS联合刺激时，只有IFN-γ和LPS的联合产生的NO比单独IFN-γ产生的更多。用IFN-γ或IFN-γ加LPS刺激的细胞产生NO可被添加NO合成抑制剂N（G）-单甲基-L-精氨酸（N（G）MMA）阻断。为了探究单独用IFN-γ或IFN-γ加LPS刺激的细胞产生NO的细胞内信号通路，我们研究了几种蛋白激酶抑制剂对细胞产生NO的影响。蛋白酪氨酸激酶（PTK）抑制剂染料木黄酮和赫曲霉素A显著抑制了NO的产生，但蛋白激酶A或C抑制剂没有。这些结果表明，从BNL CL.2细胞培养基中去除血清可能会使细胞在被IFN-γ触发时引发NO合成，并且PTK信号转导通路参与小鼠肝癌细胞中诱导型NO合酶基因的表达。
