Ornithine transport via cationic amino acid transporter-1 is involved in ornithine cytotoxicity in retinal pigment epithelial cells.
OBJECTIVE
A prior report showed ornithine cytotoxicity in ornithine-delta-aminotransferase (OAT)-deficient human retinal pigment epithelial (RPE) cells in an in vitro model of gyrate atrophy of the choroid and retina. This study was intended to clarify the mechanism of ornithine cytotoxicity and to determine the responsible amino acid transporters.


METHODS
The mRNA expression of amino acid transporters in human telomerase reverse transcriptase (hTERT)-RPE cells was examined by reverse transcription polymerase chain reaction (RT-PCR) and Northern blot analysis. Carrier-mediated ornithine transport via the L-type amino acid transporter (LAT)1, LAT2, cationic amino acid transporter (CAT)-1, and y(+)LAT2 systems was evaluated by short interfering (si)RNA-mediated gene silencing. The cytoprotective effect of CAT-1-specific siRNA on ornithine cytotoxicity was measured using quantitative analysis of cellular adenosine triphosphate (ATP) at 24 hours after treatment with ornithine in OAT-deficient RPE cells.


RESULTS
LAT1, LAT2, CAT-1, and y(+)LAT2 mRNA expression was detected by Northern blot analysis, whereas RT-PCR revealed that LAT1, LAT2, y(+)LAT1, y(+)LAT2, CAT-1, and b(0,+)AT mRNAs were expressed together with the heterodimeric glycoproteins 4F2hc and rBAT in hTERT-RPE cells. l-[(14)C]ornithine uptake in hTERT-RPE cells was decreased by 46.6% and 22.0% by CAT-1 and y(+)LAT2 siRNA, respectively, whereas LAT1 and LAT2 siRNA had no significant effect. Further, CAT-1 silencing by siRNA reduced ornithine cytotoxicity in OAT-deficient RPE cells.


CONCLUSIONS
The results suggest that ornithine transport via CAT-1 may play a crucial role in ornithine cytotoxicity in hTERT-RPE cells. Reduction of the ornithine transport via CAT-1 may be a new target for treatment of gyrate atrophy.
Kaneko
Shiho
S
Department of Ophthalmology, Kansai Medical University, Osaka, Japan.
Ando
Akira
A
Okuda-Ashitaka
Emiko
E
Maeda
Masahide
M
Furuta
Kyoji
K
Suzuki
Masaaki
M
Matsumura
Miyo
M
Ito
Seiji
S
D000255:Adenosine Triphosphate / Q000378:metabolism; D001692:Biological Transport; D015152:Blotting, Northern; D027201:Cationic Amino Acid Transporter 1 / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D020868:Gene Silencing; D006801:Humans; D007930:Leucine / Q000378:metabolism; D009952:Ornithine / Q000378:metabolism* / Q000633:toxicity*; D009953:Ornithine-Oxo-Acid Transaminase / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000378:metabolism*; D010957:Plasmids; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D027201:Cationic Amino Acid Transporter 1; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D000255:Adenosine Triphosphate; D009952:Ornithine; D009953:Ornithine-Oxo-Acid Transaminase; D007930:Leucine