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Shelly C. Lu, Yuzhou Bao, Zong–Zhi Huang, Vijay P. Sarthy, Ram Kannan; Regulation of γ-Glutamylcysteine Synthetase Subunit Gene Expression in Retinal Müller Cells by Oxidative Stress. Invest. Ophthalmol. Vis. Sci. 1999;40(8):1776-1782.
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purpose. To study regulation of γ-glutamylcysteine synthetase (GCS) heavy and
light subunit gene expression in Müller cells under conditions of
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 β-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.
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