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M. P. Langford, P. Redmond, R. Chanis, P. Zan, T. B. Redens, C. Liang; EAAT, Xc- Exchanger, Glutamine Synthetase (GS), -Glutamyltranspeptidase (GGT) and Glutamate in Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5529.
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© ARVO (1962-2015); The Authors (2016-present)
To demonstrate the cellular distribution of immunoreactive cross-linked glutamate (xGlu) and determine the Na+-dependent, high-affinity excitatory amino acid transporter (EAAT) subtypes, xCT (light chain of Glu/Cys exchanger; system Xc-), GS and GGT levels and/or activity in transformed human corneal epithelial cells (HCoEC).
Cross-linked Glu (xGlu), EAAT subtypes, xCT, GS and GGT in HCoEC were detected by western blot and IFA analyses. EAAT and Xc- activity was characterized by the uptake of radiolabeled L-Glu, D-aspartate (D-Asp) and L-cystine (Cys) in the presence and absence of Na+. Predominant EAAT and Xc- exchanger activities were assessed by substrate-specific inhibition of radiolabeled Glu/D-Asp and Cys by Glu/Asp/Cys analogs. GS GGT, and glutathione (GSH) were detected by western blot, IFA and/or colorimetic assaays.
xGlu was detected in the cytoplasm of most cells, but was highly concentrated in the cytoplasm of some cells. All five EAATs as well as xCT of the Xc- exchanger system were detected in HCoEC. However, intracellular EAAT2, extrinsic EAAT3, and xCT were predominantly expressed by most HCoEC. Uptake of radiolabeled D-Asp and L-Glu was Na+-dependent and inhibited by Glu/Asp analogs consistent with EAAT1 and 3 (i.e., not inhibited by kainate or D-Glu), but EAAT4 and EAAT5 activity can not be ruled out. Radiolabeled L-Cys uptake by HCoEC was Na+-independent and inhibited by L-Cys and L-Glu. GS was detected in mitochondrial rich perinuclear region of the HCoEC, while GGT was detected along the intercellular surfaces of adjacent cells. Four hour incubation with arachidonic acid inhibited HCoEC Glu transport by 37% and reduced GSH by 15%.
The distribution of xGlu, EAATs, xCT, GS, and GGT in HCoEC is presented. The results suggest that xGu is accumulated and that Glu/Asp is transported by EAAT and Xc- exchanger. Inhibition of Glu transport by arachidonic acid reduced GSH levels in HCoEC. HCoEC express GS and GGT supporting intracellular conversion and extracellular generation of Glu. Taken together, the results suggest that HCoEC express enzymes and transporters critical to Glu regulation and support the importance of EAAT activity in maintaining physiological GSH levels in the inflamed eye.
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