TY - JOUR
T1 - Effects of hyperglycemia and oxidative stress on the glutamate transporters GLAST and system xc − in mouse retinal Müller glial cells
AU - Mysona, Barbara
AU - Dun, Ying
AU - Duplantier, Jennifer
AU - Ganapathy, Vadivel
AU - Smith, Sylvia B.
N1 - Funding Information:
This work was supported by NIH R01 EY014560. B.Mysona.Y.Dun.J.Duplantier.S.B.Smith(*) Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, GA 30912, USA e-mail: sbsmith@mail.mcg.edu
Publisher Copyright:
© Springer-Verlag 2009.
PY - 2009/3
Y1 - 2009/3
N2 - Elevated glutamate levels have been reported in humans with diabetic retinopathy. Retinal Müller glial cells regulate glutamate levels via the GLAST transporter and system xc − (cystine-glutamate exchanger). We have investigated whether transporter function and gene and/or protein expression are altered in mouse Müller cells cultured under conditions of hyperglycemia or oxidative stress (two factors implicated in diabetic retinopathy). Cells were subjected to hyperglycemic conditions (35 mM glucose) over an 8-day period or to oxidative stress conditions (induced by exposure to various concentrations of xanthine:xanthine oxidase) for 6 h. The Na+-dependent and –independent uptake of [3H] glutamate was assessed as a measure of GLAST and system xc − function, respectively. Hyperglycemia did not alter the uptake of [3H] glutamate by GLAST or system xc −; neither gene nor protein expression decreased. Oxidative stress (70:14 or 100:20 μM xanthine:mU/ml xanthine oxidase) decreased GLAST activity by ~10% but increased system xc − activity by 43% and 89%, respectively. Kinetic analysis showed an oxidative-stress-induced change in Vmax, but not Km. Oxidative stress caused a 2.4-fold increase in mRNA encoding xCT, the unique component of system xc −. Of the two isoforms of xCT (40 and 50 kDa), oxidative stress induced a 3.6-fold increase in the 40-kDa form localized to the plasma membrane. This is the first report of the differential expression and localization of xCT isoforms as caused by cellular stress. Increased system xc − activity in Müller cells subjected to conditions associated with diabetic retinopathy may be beneficial, as this exchanger is important for the synthesis of the antioxidant glutathione.
AB - Elevated glutamate levels have been reported in humans with diabetic retinopathy. Retinal Müller glial cells regulate glutamate levels via the GLAST transporter and system xc − (cystine-glutamate exchanger). We have investigated whether transporter function and gene and/or protein expression are altered in mouse Müller cells cultured under conditions of hyperglycemia or oxidative stress (two factors implicated in diabetic retinopathy). Cells were subjected to hyperglycemic conditions (35 mM glucose) over an 8-day period or to oxidative stress conditions (induced by exposure to various concentrations of xanthine:xanthine oxidase) for 6 h. The Na+-dependent and –independent uptake of [3H] glutamate was assessed as a measure of GLAST and system xc − function, respectively. Hyperglycemia did not alter the uptake of [3H] glutamate by GLAST or system xc −; neither gene nor protein expression decreased. Oxidative stress (70:14 or 100:20 μM xanthine:mU/ml xanthine oxidase) decreased GLAST activity by ~10% but increased system xc − activity by 43% and 89%, respectively. Kinetic analysis showed an oxidative-stress-induced change in Vmax, but not Km. Oxidative stress caused a 2.4-fold increase in mRNA encoding xCT, the unique component of system xc −. Of the two isoforms of xCT (40 and 50 kDa), oxidative stress induced a 3.6-fold increase in the 40-kDa form localized to the plasma membrane. This is the first report of the differential expression and localization of xCT isoforms as caused by cellular stress. Increased system xc − activity in Müller cells subjected to conditions associated with diabetic retinopathy may be beneficial, as this exchanger is important for the synthesis of the antioxidant glutathione.
KW - Cell culture
KW - Cystine-glutamate exchanger
KW - Mouse (C57BL/6)
KW - Transporter kinetics
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U2 - 10.1007/s00441-008-0742-1
DO - 10.1007/s00441-008-0742-1
M3 - Article
C2 - 19156441
AN - SCOPUS:67651163925
SN - 0302-766X
VL - 335
SP - 477
EP - 488
JO - Cell and Tissue Research
JF - Cell and Tissue Research
IS - 3
ER -