Regulation of gamma-glutamylcysteine synthetase subunit gene expression inretinal Muller cells by oxidative stress

PURPOSE. To study regulation of gamma-glutamylcysteine synthetase (GCS) hea vy and light subunit gene expression in Muller cells under conditions of ox idative stress. METHODS. Experiments were carried out with an SV40 transformed cell line (r MC-1) that exhibits the phenotype of` rat retinal Muller cells. Endogenous glutathione levels were modified by treating cells with diethyl maleate (DE M), D,L-buthionine sulfoximine (BSO), or tert-butyllhydroquinone (TBH). In other experiments, cells were grown in either high (28 mM) or normal (5.5 m M) 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 norm alized to beta-actin or 18S rRNA. RESULTS. Treatment with DEM for 30 minutes depicted cell GSH to 20% to 30% of the normal value. GSH content recovered completely 6 hours after returni ng 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 ce lls. If cells were left in BSQ for 18 hours, however, GSH levels were reduc ed to < 1%. Treatment with TBH for 12 hours led to a 77% increase in cellul ar GSH level. Treatment with DEM, TBH, or BSO for 18 hours led to a signifi cant induction of the mRNA level of the GCS subunits, regardless of glucose concentration in thr medium. Shorter BSO treatment exerted no effect. Prol onged hyperglycemia resulted in 30% lower GSH level, 55% lower GCS heavy su bunit, and 30% lower GCS light subunit InRNA levels. CONCLUSIONS. Oxidative stress induced the gene expression of GCS heavy and light subunits in Muller cells. The effect of BSO on mRNA levels corrected with the degree of GSH depletion. Prolonged hyperglycemia lowered GCS subun it mRNA and GSH levels.