title: EAAT and Xc⁻ Exchanger Inhibition Depletes Glutathione in the Transformed Human Lens Epithelial Cell Line SRA 01/04.
abstract: OBJECTIVE
  Maintaining the high glutathione (GSH; tripeptide of glutamate, cysteine and glycine) levels in the lens cortex promotes lens health. The role of glutamate/aspartate (Glu/Asp) transporters and the cystine (Cys)/Glu exchanger (Xc(-) exchanger) in maintaining GSH in transformed human lens epithelial cells (SRA 01/04) was investigated.
  
  
  METHODS
  Detection and differentiation of excitatory amino acid transporters (EAAT1-5) and the Xc(-) exchanger was performed by the uptake of radiolabeled l-Glu, d-Asp and l-Cys in the presence and absence of Na(+), substrate-specific inhibition studies and Western-blot analysis. Reductions in GSH levels post-inhibition of Xc(-) exchanger and EAAT activities by substrate inhibitors demonstrated the roles of EAAT and Xc(-) exchanger in maintaining GSH.
  
  
  RESULTS
  Glu and d-Asp uptake in HLEC was Na(+)-dependent. Strong inhibition by substrate-specific Glu/Asp uptake inhibitors and weak inhibition by kainic acid (KA) was consistent with Na(+)-dependent EAAT1/3/4/5 activity and weak EAAT2 activity, respectively. Na(+)-independency and Glu inhibition of Cys uptake were consistent with Xc(-) exchanger activity, but inhibition of Na(+)-dependent Cys uptake by N-acetylcysteine suggests Cys uptake by EAAT3. EAAT1-5 and xCT (Xc(-) exchanger light chain) immunoreactive peptides were detected by Western-blot analysis of HLEC lysates. EAAT and Xc(-) exchanger inhibition by substrate antagonists depleted GSH concentrations by 15-28% (p's ≤ 0.02), while GSH synthesis inhibition by buthionine sulfoximine depleted GSH by 33% (p = 0.008).
  
  
  CONCLUSIONS
  Inhibition of Glu and Cys uptake by EAAT and Xc(-) exchanger antagonists depletes GSH in human lens epithelial cells. These in vitro results support pivotal roles for EAAT and Xc(-) exchanger activities in maintaining GSH and protection against oxidative stress in cortical lens epithelium.
authors.affiliation: a Department of Ophthalmology , Louisiana State University Health Sciences Center , Shreveport , LA , USA.
authors.forename: Marlyn P
authors.initials: MP
authors.lastname: Langford
authors.affiliation: a Department of Ophthalmology , Louisiana State University Health Sciences Center , Shreveport , LA , USA.
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Redens
authors.affiliation: a Department of Ophthalmology , Louisiana State University Health Sciences Center , Shreveport , LA , USA.
authors.forename: Chanping
authors.initials: C
authors.lastname: Liang
authors.affiliation: a Department of Ophthalmology , Louisiana State University Health Sciences Center , Shreveport , LA , USA.
authors.forename: A Scott
authors.initials: AS
authors.lastname: Kavanaugh
authors.affiliation: a Department of Ophthalmology , Louisiana State University Health Sciences Center , Shreveport , LA , USA.
authors.forename: Donald E
authors.initials: DE
authors.lastname: Texada
mesh_terms: D027182:Amino Acid Transport System y+ / Q000037:antagonists & inhibitors*; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D015153:Blotting, Western; D002461:Cell Line, Transformed; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D050491:Glutamate Plasma Membrane Transport Proteins / Q000037:antagonists & inhibitors*; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D007608:Kainic Acid / Q000494:pharmacology; D007908:Lens, Crystalline / Q000166:cytology*
chemical_list: D027182:Amino Acid Transport System y+; D018691:Excitatory Amino Acid Antagonists; D050491:Glutamate Plasma Membrane Transport Proteins; C118895:SLC7A11 protein, human; C120673:benzyloxyaspartate; C011274:3-hydroxyaspartic acid; D001224:Aspartic Acid; D005978:Glutathione; D007608:Kainic Acid
keywords: Cystine; EAAT; Xc− exchanger; epithelial cells; glutamate; glutathione; lens