ABSTRACT
PURPOSE: A prior study showed inactivation of ornithine-delta-aminotransferase (OAT)-deficient human retinal pigment epithelial (RPE) cells by a specific irreversible inhibitor (5-fluoromethylornithine; 5-FMO) leading to cell death, in an in vitro model of gyrate atrophy (GA) of the choroid and retina. In the present study, the cytotoxicity of metabolites of ornithine, especially spermine, in RPE cells was investigated, to clarify the mechanism of ornithine cytotoxicity in RPE cells. METHODS: RPE cells were incubated with ornithine or compounds involved in ornithine metabolic pathways. The effects on RPE cell viability and proliferative activity were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric and [(3)H]thymidine incorporation assays. Incorporation of spermine into RPE cells was examined by using [(14)C]spermine and dansyl-spermine. To assess spermine-induced RPE cell death, cells were double stained with annexin V and propidium iodide and subjected to flow cytometry. RESULTS: Ornithine, arginine, glutamate, proline, creatine, glycine, and putrescine exhibited no effects on the viability and proliferative activities of RPE cells, whereas spermidine and spermine (10 mM) inhibited [(3)H]thymidine incorporation by 13% and 89%, respectively. The inhibition of [(3)H]thymidine incorporation by spermine was dose dependent and was observed as early as 4 hours after addition. Further, spermine was incorporated and accumulated in the perinuclear region of RPE cells. Apoptotic RPE cell death was induced by spermine in a dose-dependent manner. CONCLUSIONS: The present results demonstrated that excessive spermine is cytotoxic to RPE cells and suggest that metabolites of ornithine, especially spermine, may be involved in the mechanism of RPE degeneration in GA.
Subject(s)
Apoptosis/drug effects , Pigment Epithelium of Eye/drug effects , Spermine/toxicity , Animals , Annexin A5/metabolism , Cattle , Cell Culture Techniques , Cell Proliferation/drug effects , Colorimetry , DNA/biosynthesis , Dose-Response Relationship, Drug , Flow Cytometry , Microscopy, Confocal , Ornithine/toxicity , Pigment Epithelium of Eye/pathology , Propidium/metabolism , Tetrazolium Salts , Thiazoles , Thymidine/metabolismABSTRACT
PURPOSE: 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.
