Prevention of ornithine cytotoxicity by nonpolar side chain amino acids in retinal pigment epithelial cells.
OBJECTIVE
To investigate the effect of amino acids on ornithine cytotoxicity in ornithine-delta-aminotransferase (OAT)-deficient human retinal pigment epithelial (RPE) cells as an in vitro model of gyrate atrophy (GA) of the choroid and retina.


METHODS
RPE cells were treated with 0.5 mM 5-fluoromethylornithine (5-FMOrn), a specific and irreversible OAT inhibitor. OAT-deficient RPE cells were incubated with 10 mM ornithine in the presence of 20 mM of 1 of 18 amino acids or 10 mM 2-amino-2-norbornane-carboxylic acid (BCH), a conventional inhibitor of the amino acid transporter system L. Ornithine cytotoxicity and cytoprotective effects of each amino acid was evaluated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay 72 hours after treatment with ornithine in OAT-deficient RPE cells. Ornithine incorporation into RPE cells was evaluated using DL-[14C]ornithine.


RESULTS
An MTT colorimetric assay revealed that small and large zwitterionic amino acids, but not acidic or basic amino acids, decreased ornithine cytotoxicity in OAT-deficient RPE cells. Incorporation of DL-[14C]ornithine by RPE cells decreased to 79% of the control level after incubation for 48 hours with 20 mM leucine, the most effective cytoprotective amino acid. Further, BCH prevented ornithine cytotoxicity in a dose-dependent manner. Both light and heavy chains of L-type amino acid transporter (LAT)-1, LAT2, y+LAT1, and 4F2hc were expressed in RPE cells.


CONCLUSIONS
The present results demonstrate that L-type amino acid transporter(s) may be involved in protection against ornithine cytotoxicity in human RPE cells. Thus, amino acid transportation in RPE cells may be a good target for a new therapy for GA as well as other kinds of chorioretinal degeneration.
Nakauchi
Tadashi
T
Department of Ophthalmology, Kansai Medical University, Osaka, Japan.
Ando
Akira
A
Ueda-Yamada
Mami
M
Yamazaki
Yukari
Y
Uyama
Masanobu
M
Matsumura
Miyo
M
Ito
Seiji
S
D027182:Amino Acid Transport System y+; D000596:Amino Acids / Q000494:pharmacology*; D000598:Amino Acids, Cyclic / Q000494:pharmacology; D002470:Cell Survival; D002478:Cells, Cultured; D003124:Colorimetry; D019610:Cytoprotection / Q000187:drug effects*; D027282:Fusion Regulatory Protein 1, Heavy Chain / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D027301:Fusion Regulatory Protein 1, Light Chains / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D015799:Gyrate Atrophy / Q000188:drug therapy / Q000378:metabolism; D006801:Humans; D027283:Large Neutral Amino Acid-Transporter 1 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D009952:Ornithine / Q000031:analogs & derivatives* / Q000633:toxicity*; D009953:Ornithine-Oxo-Acid Transaminase / Q000037:antagonists & inhibitors / Q000172:deficiency; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013778:Tetrazolium Salts; D013844:Thiazoles
D027182:Amino Acid Transport System y+; D000596:Amino Acids; D000598:Amino Acids, Cyclic; D027282:Fusion Regulatory Protein 1, Heavy Chain; D027301:Fusion Regulatory Protein 1, Light Chains; D027283:Large Neutral Amino Acid-Transporter 1; D012333:RNA, Messenger; C420646:SLC7A8 protein, human; D013778:Tetrazolium Salts; D013844:Thiazoles; C057405:5-fluoromethylornithine; C006504:2-aminobicyclo(2,2,1)heptane-2-carboxylic acid; D009952:Ornithine; D009953:Ornithine-Oxo-Acid Transaminase; C022616:thiazolyl blue