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Excitatory Amino Acid Transporters, Xc− Antiporter, γ-Glutamyl Transpeptidase, Glutamine Synthetase, and Glutathione in Human Corneal Epithelial Cells

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Studies on the Cornea and Lens

Abstract

Glutamate (Glu) and cystine (Cys) modulating enzymes and transport systems were identified in human corneal epithelial cells and their importance to maintaining glutathione (GSH; potent antioxidant tripeptide of Glu, Cys, and glycine) investigated. Glu, Na-dependent excitatory amino acid transporters (EAAT), Xc− antiporter, γ-glutamyltranspeptidase (GGT; cleaves GSH) and glutamine synthetase (GS; amidates Glu to form glutamine) were identified by immunofluorescent antibody analysis, characterized by inhibitor sensitivity and their importance to GSH levels assessed pharmacologically. Immunoreactive Glu was detected in the cytoplasm of most cells, but was highly concentrated in the mitochondria-rich cytoplasm. All five EAATs and the Xc− antiporter light chain (xCT) were detected, but cells predominantly expressed EAAT1, 2, 3, and Xc− antiporter. Uptake of radiolabeled d-Asp was Na-dependent and inhibited by Glu/Asp analogs consistent with EAAT1 and EAAT3 activity. l-Cys uptake was Na+-independent consistent with Xc− antiporter activity. Extrinsic membrane-bound GGT was concentrated between cells and GS was detected in perinuclear cytoplasm of most cells. Inhibition of EAAT, Xc− antiporter, GGT, GS and GSH synthesis reduced GSH by 21 %, 24 %, 19 %, 32.7 %, and 54 %, respectively. The results support the dependence of GSH on Glu and Cys uptake by EAAT and Xc− exchanger activity, extracellular Glu and Cys generation by GGT and regulation of intracellular Glu by GS. The results suggest that Glu and Cys transport systems, GGT and GS activities maintain physiological Glu, Cys, and GSH levels and protect human cornea epithelial cells against oxidative stress.

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Abbreviations

Cys:

Cystine

EAAT:

Excitatory amino acid transporter

GGT:

Gamma–glutamyl transpeptidase

Gln:

Glutamine

Glu:

Glutamate

GS:

Glutamine synthetase

HCEC:

Human conjunctival epithelial cells

NAC:

N-acetylcysteine

ROS:

Reactive oxygen species

xCT:

Xc-exchanger C-terminal light chain

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Acknowledgements

The authors thank Christopher Duggan for excellent technical support. The authors have no conflicts of interest. The authors acknowledge the support of the LSUHSC, Department of Ophthalmology Faculty Improvement Fund.

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Authors and Affiliations

  1. Department of Ophthalmology, Louisiana State University Health Sciences Center, Shreveport, LA, 71130-3932, USA

    Marlyn P. Langford Ph.D., Thomas B. Redens & Donald E. Texada

Authors
  1. Marlyn P. Langford Ph.D.
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  2. Thomas B. Redens
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  3. Donald E. Texada
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Corresponding author

Correspondence to Marlyn P. Langford Ph.D. .

Editor information

Editors and Affiliations

  1. Innovative Vision Products Inc., Moscow, Russia, Innovative Vision Products, Inc.,, County of New Castle, Delaware, USA

    Mark A. Babizhayev

  2. Department of Ophthalmology and Visual Sciences, Truhlsen Eye Institute, College of Medicine, University of Nebraska Medical Center; Key Laboratory of Protein Chemistry and Developmental Biology of Education Ministry of China, College of Life Sciences, Hunan Normal University, Changsha, Hunan, China, Omaha, Nebraska, USA

    David Wan-Cheng Li

  3. Department of Pharmaceutical Sciences and Oklahoma Center for Neuroscience, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA

    Anne Kasus-Jacobi

  4. Department of Ophthalmology, University of Pristina, Faculty of Medicine, Kosovska Mitrovica, Serbia

    Lepša Žorić

  5. Ophthalmology, Miguel Hernandez University, Alicante, Spain

    Jorge L. Alió

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Langford, M.P., Redens, T.B., Texada, D.E. (2015). Excitatory Amino Acid Transporters, Xc− Antiporter, γ-Glutamyl Transpeptidase, Glutamine Synthetase, and Glutathione in Human Corneal Epithelial Cells. In: Babizhayev, M., Li, DC., Kasus-Jacobi, A., Žorić, L., Alió, J. (eds) Studies on the Cornea and Lens. Oxidative Stress in Applied Basic Research and Clinical Practice. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1935-2_4

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