Kim Y M, Son K, Hong S J, Green A, Chen J J, Tzeng E, Hierholzer C, Billiar T R
Department of Surgery, University of Pittsburgh, Pennsylvania, USA.
Mol Med. 1998 Mar;4(3):179-90.
Nitric oxide (NO) is cytostatic for proliferating cells, inhibits microbial growth, and down-regulates the synthesis of specific proteins. Studies were undertaken to determine the mechanism by which NO inhibits total protein synthesis and whether the inhibition correlates with established cytostatic activities of NO.
In in vitro experiments, various cell types were exposed to NO using either donors or expression of inducible NO synthase (iNOS). The capacity of NO to suppress total protein synthesis, measured by incorporation of 35S-methionine into protein, was correlated with the capacity of NO to suppress cell proliferation, viral replication, or iNOS expression. Phosphorylation of eIF-2 alpha was examined as a possible mechanism for the suppressed protein synthesis by NO.
Both NO donors and expression of the iNOS suppressed total protein synthesis in L929 cells and A2008 human ovarian tumor cells in parallel with decreased cell proliferation. Suppressed protein synthesis was also shown to correlate with decreased vaccinia virus proliferation in murine peritoneal macrophages in an iNOS-dependent manner. Furthermore, iNOS expression in pancreatic islets or RAW264.7 cells almost completely inhibited total protein synthesis, suggesting that nonspecific inhibition of protein synthesis may be the mechanism by which NO inhibited the synthesis of specific proteins such as insulin or iNOS itself. This possibility was confirmed in RAW264.7 cells where the inhibition of total protein synthesis correlated with the decreased iNOS protein. The decrease in protein levels occurred without changes in iNOS mRNA levels, implicating an inhibition of translation. Mechanistic studies revealed that iNOS expression in RAW264.7 cells resulted in the phosphorylation of eIF-2 alpha and inhibition of the 80S ribosomal complex formation.
These results suggest that NO suppresses protein synthesis by stimulating the phosphorylation of eIF-2 alpha. Furthermore, our observations indicate that nonspecific inhibition of protein synthesis may be a generalized response of cells exposed to high levels of NO and that inhibition of protein synthesis may contribute to many of the described cytostatic actions of NO.
一氧化氮（NO）对增殖细胞具有细胞生长抑制作用，可抑制微生物生长，并下调特定蛋白质的合成。本研究旨在确定NO抑制总蛋白质合成的机制，以及这种抑制作用是否与NO既定的细胞生长抑制活性相关。
在体外实验中，使用供体或诱导型一氧化氮合酶（iNOS）的表达使各种细胞类型暴露于NO。通过将35S-甲硫氨酸掺入蛋白质来测量NO抑制总蛋白质合成的能力，该能力与NO抑制细胞增殖、病毒复制或iNOS表达的能力相关。研究了真核细胞起始因子2α（eIF-2α）的磷酸化，作为NO抑制蛋白质合成的一种可能机制。
NO供体和iNOS的表达均抑制了L929细胞和A2008人卵巢肿瘤细胞中的总蛋白质合成，同时细胞增殖也减少。在鼠腹膜巨噬细胞中，蛋白质合成的抑制也被证明与痘苗病毒增殖的减少呈iNOS依赖性相关。此外，胰岛或RAW264.7细胞中iNOS的表达几乎完全抑制了总蛋白质合成，这表明蛋白质合成的非特异性抑制可能是NO抑制胰岛素或iNOS本身等特定蛋白质合成的机制。这一可能性在RAW264.7细胞中得到证实，其中总蛋白质合成的抑制与iNOS蛋白的减少相关。蛋白质水平的降低在iNOS mRNA水平没有变化的情况下发生，这意味着翻译受到抑制。机制研究表明，RAW264.7细胞中iNOS的表达导致eIF-2α的磷酸化，并抑制80S核糖体复合物的形成。
这些结果表明，NO通过刺激eIF-2α的磷酸化来抑制蛋白质合成。此外，我们的观察结果表明，蛋白质合成的非特异性抑制可能是暴露于高水平NO的细胞的一种普遍反应，并且蛋白质合成的抑制可能有助于NO所描述的许多细胞生长抑制作用。
