氧化应激对视网膜Müller细胞中γ-谷氨酰半胱氨酸合成酶亚基基因表达的调控

Regulation of gamma-glutamylcysteine synthetase subunit gene expression in retinal Müller cells by oxidative stress.

作者信息

Lu S C, Bao Y, Huang Z Z, Sarthy V P, Kannan R

机构信息

Department of Medicine, USC Liver Disease Research Center, University of Southern California School of Medicine, Los Angeles, USA.

出版信息

Invest Ophthalmol Vis Sci. 1999 Jul;40(8):1776-82.

PMID:10393048

Abstract

PURPOSE

To study regulation of gamma-glutamylcysteine synthetase (GCS) heavy and light subunit gene expression in Müller cells under conditions of oxidative stress.

METHODS

Experiments were carried out with an SV40 transformed cell line (rMC-1) that exhibits the phenotype of rat retinal Müller cells. Endogenous glutathione levels were modified by treating cells with diethyl maleate (DEM), D,L-buthionine sulfoximine (BSO), or tert-butylhydroquinone (TBH). In other experiments, cells were grown in either high (28 mM) or normal (5.5 mM) glucose medium for 1 week to examine the effects of hyperglycemia. Cells were processed for reduced glutathione (GSH) measurement, RNA extraction, cell count, and, in some cases, lactate dehydrogenase activity. The steady state mRNA levels of GCS heavy and light subunits were measured by northern blot analysis using specific cDNA probes. Changes in mRNA levels were normalized to beta-actin or 18S rRNA.

RESULTS

Treatment with DEM for 30 minutes depleted cell GSH to 20% to 30% of the normal value. GSH content recovered completely 6 hours after returning to normal medium. BSO treatment for 12 hours followed by a medium change for 6 hours resulted in a cell GSH level that was 26% that of untreated cells. If cells were left in BSO for 18 hours, however, GSH levels were reduced to < 1%. Treatment with TBH for 12 hours led to a 77% increase in cellular GSH level. Treatment with DEM, TBH, or BSO for 18 hours led to a significant induction of the mRNA level of the GCS subunits, regardless of glucose concentration in the medium. Shorter BSO treatment exerted no effect. Prolonged hyperglycemia resulted in 30% lower GSH level, 55% lower GCS heavy subunit, and 30% lower GCS light subunit mRNA levels.

CONCLUSIONS

Oxidative stress induced the gene expression of GCS heavy and light subunits in Müller cells. The effect of BSO on mRNA levels correlated with the degree of GSH depletion. Prolonged hyperglycemia lowered GCS subunit mRNA and GSH levels.

摘要

目的

研究氧化应激条件下缪勒细胞中γ-谷氨酰半胱氨酸合成酶（GCS）重链和轻链亚基基因表达的调控。

方法

使用表现出大鼠视网膜缪勒细胞表型的SV40转化细胞系（rMC-1）进行实验。通过用马来酸二乙酯（DEM）、D，L-丁硫氨酸亚砜胺（BSO）或叔丁基对苯二酚（TBH）处理细胞来改变内源性谷胱甘肽水平。在其他实验中，将细胞在高糖（28 mM）或正常葡萄糖（5.5 mM）培养基中培养1周，以研究高血糖的影响。对细胞进行还原型谷胱甘肽（GSH）测量、RNA提取、细胞计数，在某些情况下还进行乳酸脱氢酶活性检测。使用特异性cDNA探针通过Northern印迹分析测量GCS重链和轻链亚基的稳态mRNA水平。将mRNA水平的变化标准化为β-肌动蛋白或18S rRNA。

结果

用DEM处理30分钟可使细胞内GSH降至正常值的20%至30%。恢复到正常培养基6小时后，GSH含量完全恢复。用BSO处理12小时，然后更换培养基6小时，导致细胞内GSH水平为未处理细胞的26%。然而，如果将细胞留在BSO中18小时，GSH水平会降至<1%。用TBH处理12小时导致细胞内GSH水平增加77%。用DEM、TBH或BSO处理18小时导致GCS亚基的mRNA水平显著上调，无论培养基中的葡萄糖浓度如何。较短时间的BSO处理没有效果。长期高血糖导致GSH水平降低30%，GCS重链亚基降低55%，GCS轻链亚基mRNA水平降低30%。