Inhibition of glutamine synthetase decreases proliferation of cultured rat intestinal epithelial cells
Inhibition of glutamine synthetase decreases proliferation of cultured rat intestinal epithelial cells
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DOI：
10.1093/jn/129.1.57
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发表时间：
1999-01-01
影响因子：
4.2
通讯作者：
Neu, J
中科院分区：
文献类型：
--
作者：
DeMarco, V;Dyess, K;Neu, J
通过在不同的细胞外谷氨酰胺 (Q) 浓度下用 10 mmol/L 蛋氨酸亚磺酰亚胺 (MS) 抑制其活性，在大鼠肠隐窝细胞 (IEC-6) 中检查了谷氨酰胺合成酶 (GS) 对细胞增殖的重要性。在未抑制的培养物中，细胞数量、蛋白质、DNA 积累和合成在 0.06 至 1.06 mmol/L 的浓度范围内表现出对细胞外 Q 的依赖性，表观半最大响应为 0.46 mmol/L 细胞外 Q。相反，GS 抑制培养物的增殖需要大于或等于 1.06 mmol/L 细胞外 Q，表观半最大响应为 2 mmol/L。 MS 抑制洗涤细胞提取物中 GS 活性 >97%，并且似乎具有特异性，因为它对增殖的影响被 4.06 mmol/L Q 克服，并且是可逆的。当 GS 被抑制时，IEC-6 细胞对细胞外 Q 的依赖性增加，表明源自 GS 的 Q (GS-Q) 在细胞外 Q 的生理水平 (0.6 mmol/L) 下对细胞增殖有重要贡献。相对于报道的 Q 转运到细胞中的 K-m，在 OS 抑制期间挽救最大增殖所需的细胞外 Q 浓度出人意料地高，表明源自细胞外介质 (exo-Q) 的细胞内 Q 未被有效利用。在之前的研究中，我们发现GS蛋白和mRNA在体内集中在小肠的增殖性隐窝区域，并预测GS活性对于隐窝细胞增殖具有重要意义。在这里，我们表明，在此细胞培养模型中，在生理浓度的 Q 下，酶活性对于细胞增殖非常重要。最后，我们推测 exo-Q 和 GS-Q 在细胞中的利用方式不同。
The importance of glutamine synthetase (GS) for cell proliferation was examined in rat intestinal crypt cells (IEC-6) by inhibiting its activity with 10 mmol/L methionine sulfoximine (MS) at varying extracellular glutamine (Q) concentrations. In uninhibited cultures, cell number, protein, and DNA accumulation and synthesis showed a dependence on extracellular Q over a concentration range of 0.06 to 1.06 mmol/L, with apparent half-maximal responses of 0.46 mmol/L extracellular Q. In contrast, proliferation of GS-inhibited cultures required greater than or equal to 1.06 mmol/L extracellular Q, with an apparent half-maximal response of 2 mmol/L. MS inhibited GS activity >97% in extracts of washed cells and appeared to be specific because its effects on proliferation were overcome by 4.06 mmol/L Q and were reversible. The increased dependence of IEC-6 cells on extracellular Q when GS was inhibited suggests that Q derived from GS (GS-Q) contributes importantly to cell proliferation at physiologic levels of extracellular Q (0.6 mmol/L). The unexpectedly high concentration of extracellular Q required to rescue maximal proliferation during OS-inhibition, relative to a reported K-m for Q-transport into the cell, indicates that intracellular Q derived from the extracellular medium (exo-Q) is inefficiently utilized. In a previous study, we found that GS-protein and mRNA are concentrated in the proliferative crypt region of the small intestine in vivo, and predicted that GS activity is important for crypt cell proliferation. Here, we show that enzyme activity is important for cell proliferation at physiologic concentrations of Q in this cell culture model. Finally, we speculate that exo-Q and GS-Q are utilized differently in the cell.