Prevention of Ornithine Cytotoxicity by Nonpolar Side Chain Amino Acids in Retinal Pigment Epithelial Cells

• Published in: Investigative ophthalmology & visual science Vol. 44; no. 11; pp. 5023 - 5028
• Main Authors: Nakauchi, Tadashi, Ando, Akira, Ueda-Yamada, Mami, Yamazaki, Yukari, Uyama, Masanobu, Matsumura, Miyo, Ito, Seiji
• Format: Journal Article
• Language: English
• Published: Rockville, MD ARVO 01-11-2003; Association for Research in Vision and Ophtalmology
• Subjects: Amino Acid Transport System y; Amino Acids - pharmacology; Amino Acids, Cyclic - pharmacology; Biological and medical sciences; Cell Survival; Cells, Cultured; Colorimetry; Cytoprotection - drug effects; Fusion Regulatory Protein 1, Heavy Chain - drug effects; Fusion Regulatory Protein 1, Heavy Chain - genetics; Fusion Regulatory Protein 1, Heavy Chain - metabolism; Fusion Regulatory Protein 1, Light Chains - drug effects; Fusion Regulatory Protein 1, Light Chains - genetics; Fusion Regulatory Protein 1, Light Chains - metabolism; Gyrate Atrophy - drug therapy; Gyrate Atrophy - metabolism; Humans; Large Neutral Amino Acid-Transporter 1 - drug effects; Large Neutral Amino Acid-Transporter 1 - genetics; Large Neutral Amino Acid-Transporter 1 - metabolism; Medical sciences; Ophthalmology; Ornithine - analogs & derivatives; Ornithine - toxicity; Ornithine-Oxo-Acid Transaminase - antagonists & inhibitors; Ornithine-Oxo-Acid Transaminase - deficiency; Pigment Epithelium of Eye - drug effects; Pigment Epithelium of Eye - metabolism; Pigment Epithelium of Eye - pathology; Retinopathies; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - metabolism; Tetrazolium Salts; Thiazoles; Prevention; Side chain; Aminoacid; Epithelial cell; Cytotoxicity; Retina; Ornithine; Retinal; Pigment cell
• ISSN: 0146-0404; 1552-5783; 1552-5783
• DOI: 10.1167/iovs.03-0403

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.
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.
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.
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.