Effects of hyperglycemia and oxidative stress on the glutamate transporters GLAST and system xc in mouse retinal Müller glial cells

Barbara A Mysona, Ying Dun, Jennifer Duplantier, Vadivel Ganapathy, Sylvia B Smith

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)477-488
Number of pages12
JournalCell and Tissue Research
Volume335
Issue number3
DOIs
StatePublished - Mar 1 2009

Fingerprint

Amino Acid Transport System X-AG
Neuroglia
Hyperglycemia
Oxidative Stress
Glutamic Acid
Diabetic Retinopathy
Xanthine
Xanthine Oxidase
Protein Isoforms
Cystine
Glutathione
Cultured Cells
Proteins
Antioxidants
Cell Membrane
Glucose
Messenger RNA

Keywords

  • Cell culture
  • Cystine-glutamate exchanger
  • Mouse (C57BL/6)
  • Transporter kinetics

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Histology
  • Cell Biology

Cite this

Effects of hyperglycemia and oxidative stress on the glutamate transporters GLAST and system xc in mouse retinal Müller glial cells. / Mysona, Barbara A; Dun, Ying; Duplantier, Jennifer; Ganapathy, Vadivel; Smith, Sylvia B.

In: Cell and Tissue Research, Vol. 335, No. 3, 01.03.2009, p. 477-488.

Research output: Contribution to journalArticle

@article{689467cd4f884139a5f644bed8634538,
title = "Effects of hyperglycemia and oxidative stress on the glutamate transporters GLAST and system xc − in mouse retinal M{\"u}ller glial cells",
abstract = "Elevated glutamate levels have been reported in humans with diabetic retinopathy. Retinal M{\"u}ller 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 M{\"u}ller 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 M{\"u}ller cells subjected to conditions associated with diabetic retinopathy may be beneficial, as this exchanger is important for the synthesis of the antioxidant glutathione.",
keywords = "Cell culture, Cystine-glutamate exchanger, Mouse (C57BL/6), Transporter kinetics",
author = "Mysona, {Barbara A} and Ying Dun and Jennifer Duplantier and Vadivel Ganapathy and Smith, {Sylvia B}",
year = "2009",
month = "3",
day = "1",
doi = "10.1007/s00441-008-0742-1",
language = "English (US)",
volume = "335",
pages = "477--488",
journal = "Cell and Tissue Research",
issn = "0302-766X",
publisher = "Springer Verlag",
number = "3",

}

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 A

AU - Dun, Ying

AU - Duplantier, Jennifer

AU - Ganapathy, Vadivel

AU - Smith, Sylvia B

PY - 2009/3/1

Y1 - 2009/3/1

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

UR - http://www.scopus.com/inward/record.url?scp=67651163925&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=67651163925&partnerID=8YFLogxK

U2 - 10.1007/s00441-008-0742-1

DO - 10.1007/s00441-008-0742-1

M3 - Article

VL - 335

SP - 477

EP - 488

JO - Cell and Tissue Research

JF - Cell and Tissue Research

SN - 0302-766X

IS - 3

ER -