Induction of arginase II mRNA by nitric oxide using an in vitro model of gyrate atrophy of choroid and retina.
OBJECTIVE
The authors previously reported ornithine cytotoxicity in ornithine-δ-aminotransferase (OAT)-deficient human retinal pigment epithelial (RPE) cells as an in vitro model of gyrate atrophy of the choroid and retina (GA). Given that RPE cells are severely damaged by arginine combined with ornithine, they investigated the role of arginine metabolism using that in vitro model.


METHODS
Human telomerase reverse transcriptase (hTERT)-RPE cells were incubated with ornithine or other agents in the presence of 5-fluoromethylornithine (5-FMO), an OAT-specific inhibitor. mRNA expression was determined by quantitative real-time polymerase chain reaction, and the concentration of nitric oxide (NO) was quantified using a Griess assay. Furthermore, cytotoxicity was examined by morphologic observations and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assays, with the effect of arginase II examined using short interfering (si) RNA for arginase II and S-(2-boronoethyl)-L-cysteine (BEC), an arginase inhibitor.


RESULTS
NO production in 5-FMO-treated hTERT-RPE cells was increased by ornithine, and the NO donors S-nitroso-N-acetyl-DL-penicillamine (SNAP) and S-nitrosoglutathione induced cytotoxicity. Ornithine increased the expression of arginase II mRNA in 5-FMO-treated cells. Arginase II upregulation was partially inhibited by an NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester, which was mimicked by SNAP. Arginase II siRNA and BEC enhanced ornithine cytotoxicity, and arginase II silencing resulted in a further increase in NO production.


CONCLUSIONS
These results demonstrate that NO is produced in our in vitro GA model, which induced cytotoxicity of RPE cells and upregulation of arginase II. NO may be involved in RPE degeneration in GA through the regulation of arginase II mRNA expression.
Ohnaka
Masayuki
M
Department of Ophthalmology, University of Houston, Houston, Texas.
Okuda-Ashitaka
Emiko
E
Kaneko
Shiho
S
Ando
Akira
A
Maeda
Masahide
M
Furuta
Kyoji
K
Suzuki
Masaaki
M
Takahashi
Kanji
K
Ito
Seiji
S
D000818:Animals; D001119:Arginase / Q000037:antagonists & inhibitors / Q000235:genetics*; D001897:Boronic Acids / Q000494:pharmacology; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015799:Gyrate Atrophy / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D009569:Nitric Oxide / Q000378:metabolism*; D009952:Ornithine / Q000031:analogs & derivatives / Q000494:pharmacology; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis*; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000201:enzymology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism; D015854:Up-Regulation
C425062:(2-boronoethyl)-cysteine; D001897:Boronic Acids; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D013778:Tetrazolium Salts; D013844:Thiazoles; C057405:5-fluoromethylornithine; D009569:Nitric Oxide; D009952:Ornithine; C477631:ARG2 protein, human; D001119:Arginase; C022616:thiazolyl blue