Characterization of glutathione uptake, synthesis, and efflux pathways in the epithelium and endothelium of the rat cornea.
OBJECTIVE
To compare and contrast glutathione (GSH) uptake, synthesis, and efflux pathways in the epithelium and endothelium of the rat cornea.


METHODS
Whole eyes were cryosectioned in an equatorial orientation and sections fixed in either 0.75% paraformaldehyde alone or 0.75% paraformaldehyde plus 0.01% glutaraldehyde. Sections were then labeled with GSH, γ-glutamylcysteine synthetase (γ-GCS), cysteine, xCT, or multidrug resistance-associated proteins (MRP1, 2, 4, and 5 isoforms) antibodies and then with secondary antibodies to visualize labeling patterns. Cornea morphology was visualized using propidium iodide. Reverse transcriptase-polymerase chain reaction was used to determine which of the 3 putative GSH transporters, NaDC3, C-terminal organic anion transporter 1 (OAT1), and/or N-terminal organic anion transporter 3 (OAT3), were present at the transcript level in the cornea. Colocalization of OAT3 and sodium dependent dicarboxylate transporter 3 (NaDC3) was performed by labeling with OAT3 and NaDC3 primary antibodies that were visualized with secondary antibodies and then mounted in 4'6-diamidino-2-phenylindole to visualize cell morphology.


RESULTS
Although immunohistochemical labeling showed GSH to be localized throughout the cornea, labeling for cysteine, γ-GCS, xCT, MRP4, and MRP5 was strongest in the epithelium. In contrast, although GSH labeling was strong in the endothelium, xCT and MRP labeling was absent and cysteine and γ-GCS labeling was weak relative to the epithelium. Reverse transcriptase-polymerase chain reaction revealed OAT3 and NaDC3, but not OAT1, to be present at the transcript level. Immunohistochemical labeling showed OAT3 and NaDC3 to be localized to the endothelium but absent from the epithelium.


CONCLUSIONS
The corneal epithelium and endothelium exhibit differences in GSH uptake, synthesis, and efflux pathways. The corneal epithelium seems to be the region where GSH synthesis and GSH efflux occurs. However, in the endothelium, GSH accumulation is likely to be predominantly by direct uptake of GSH from the aqueous humor.
Department of Optometry and Vision Science, University of Auckland, Auckland, New Zealand.
Bo
B
Li
Monica S
MS
Lee
Rebecca S Y
RS
Lee
Paul J
PJ
Donaldson
Julie C
JC
Lim
D027182:Amino Acid Transport System y+ / Q000378:metabolism*; D026922:Amino Acid Transport Systems, Acidic; D000818:Animals; D003545:Cysteine / Q000378:metabolism*; D029382:Dicarboxylic Acid Transporters / Q000378:metabolism; D004728:Endothelium, Corneal / Q000378:metabolism*; D019573:Epithelium, Corneal / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D005721:Glutamate-Cysteine Ligase / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism*; D027362:Organic Anion Transport Protein 1 / Q000378:metabolism; D029363:Organic Anion Transporters, Sodium-Dependent / Q000378:metabolism; D029362:Organic Anion Transporters, Sodium-Independent / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D027981:Symporters / Q000378:metabolism
D027182:Amino Acid Transport System y+; D026922:Amino Acid Transport Systems, Acidic; D003545:Cysteine; D029382:Dicarboxylic Acid Transporters; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D027362:Organic Anion Transport Protein 1; D029363:Organic Anion Transporters, Sodium-Dependent; D029362:Organic Anion Transporters, Sodium-Independent; D027981:Symporters; C490478:Slc13a2 protein, rat; C421043:Slc22a6 protein, rat; C114338:organic anion transport protein 3; C000723960:Slc7a11 protein, rat