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S. Parameswaran, C. Spee, S. J. Ryan, R. Kannan, D. R. Hinton; Decreased Cellular Glutathione (GSH) and Increased GSH Efflux in -Crystallin Knockout Retina. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1831.
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© ARVO (1962-2015); The Authors (2016-present)
Knockout of -crystallins renders retinal cells more susceptible to apoptotic cell death. Decreased retinal antioxidant status could contribute to increased vulnerability to apoptosis. We sought to determine GSH regulation in -crystallin knockout retina and -crystallin-overexpressing ARPE-19 cells.
Neural retina and RPE/choroid were dissected from 6- to 8-wk-old wild-type (WT), A crystallin (-/-) and B crystallin (-/-) mice. GSH levels were measured by colorimetric assay. Gene and protein expression of γ-glutamylcysteine synthetase (GCS) was determined by real-time PCR and Western blot. Localization of GSH expression in -crystallin (-/-) mice was evaluated by confocal microscopy. Overexpression of -crystallin in ARPE-19 cells was achieved by transfection with pcDNA3.1 -crystallin plasmid. The effect of H2O2(150µM, 24h) on these cells was examined. GSH levels in cytosolic and mitochondrial fractions in crystallin-overexpressing cells were also measured. GSH efflux was measured in RPE derived from B crystallin (-/-) mice in serum-free medium 24 h after H2O2.
Knockout of -crystallins significantly (p<0.05) decreased GSH in neural retina and RPE/choroid. Confocal microscopy revealed the intensity of GSH immunostaining in -crystallin (-/-) mice was markedly lower vs WT retina. Overexpression of -crystallins in ARPE-19 cells significantly increased GSH levels (p<0.05 vs vector control) with 2.2-fold upregulation of the catalytic subunit of GCS. The GSH increase was more pronounced in mitochondrial fraction vs cytosolic fraction with oxidant stress. Upon H2O2 challenge, cell death in -crystallin overexpressing cells was attenuated and active caspase 3 expression was decreased vs controls. GSH efflux in RPE of B crystallin (-/-) mice was twofold higher than vs WT RPE.
Absence of -crystallins is accompanied by a decrease in GSH levels in mouse retina and a significant increase in GSH efflux from RPE. The protection from oxidant-induced cell death in -crystallin-overexpressed cells could be due to increased mitochondrial levels of GSH.
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