MOLECULAR MCHANISM OF CYTOSKELETON-MEDIATED STRESS RESPONSE IN GASTRIC MUCOSAL CELLS.

氧化应激启动培养的胃粘膜细胞中包括肌动蛋白在内的特定蛋白质的S硫基化。Actic的这种可逆修饰被认为在保护微丝在氧化应激下的动力学方面发挥了重要作用。为了验证细胞内谷胱甘肽可能调节应激反应的感知和信号转导的假设，我研究了谷胱甘肽耗竭的胃细胞中热休克蛋白(HSPs)的诱导。当豚鼠胃粘膜细胞原代培养暴露于高温(43ﾟC)、乙醇、过氧化氢或联胺时，它们迅速合成HSPs。热应激诱导HSP90、HSP72和HSC73，乙醇显著诱导HSP60。除了这些热休克蛋白，过氧化氢和联胺在几种未知蛋白质的合成中也增加了。当细胞内谷胱甘肽被DL-丁硫氨酸-[S，R]亚磺胺(BSO)预处理后，细胞内谷胱甘肽耗竭到对照的10%以下时，这些蛋白都不会被应激源诱导。用人工合成的编码HSP72热休克元件的寡核苷酸进行凝胶电泳率漂移试验，未发现热休克因子(HSF)在热暴露的谷胱甘肽耗竭细胞中的任何激活，随后在细胞中未检测到HSP72mRNA的积聚。ANT-HSF1免疫印迹分析表明，BSO处理后HSF1蛋白的量没有减少，但从BSO处理的细胞中提取的核蛋白中HSF1蛋白没有恢复。这些结果表明，谷胱甘肽可能通过在转录水平激活胃粘膜细胞中的热休克基因而发挥粘膜保护作用。

Oxodative stress initiates S-thiolation of specific proteins including actin in cultured gastric mucosal cells. This reversible modification of actic has been suggested to play an importent role in preserving microfilament dynamics under oxidative stress. To test a hypothesis that intracellular glutathione may regulate in sensing and signal transduction of stress response, I examined the induction of heat shock proteins (HSPs) in glutathione-depleted gastric cells.When primary cultures of gastric mucosal cells from guinea pigs were exposed to heat (43ﾟC), ethanol, hydrogen peroxide, or diamide, they rapidly synthesized HSPs. Heat stress induced HSP90, HSP72, and HSC73, and ethanol prominently induced HSP60. In addition to these HSPs, hydrogen peroxide and diamide increased in the syntheses of several undefined proteins. None of these proteins were induced by exposure to the stressors, when intracellular glutathione was depleted to less than 10% of control values by pretreatment of cells with DL-buthionine-[S,R]-sulfoximine (BSO). Gel morbility shift assay using a synthetic oligonucleotide coding HSP72 heat shock element did not show any activation of heat shock factor (HSF) in glutathione-depleted cells exposed to heat, and subsequently no accumulation of HSP72 mRNA was detected in the cells. Immunoblot analysis with ant-HSF1 showed that the amount of HSF1 protein was not decreased by treatment with BSO,but the protein was not recovered in nuclear proteins extracted from BSO-pretreated cells. These results suggest that glutathione may serve a role in mucosal protection through the transcriptional activation of heat shock genes in gastric mucosal cells.