Ornithine transport via cationic amino acid transporter-1 is involved in ornithine cytotoxicity in retinal pigment epithelial cells.
Ornithine transport via cationic amino acid transporter-1 is involved in ornithine cytotoxicity in retinal pigment epithelial cells.
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DOI：
10.1167/iovs.06-0398
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发表时间：
2007
影响因子：
4.4
通讯作者：
S. Kaneko;A. Ando;E. Okuda‐Ashitaka;Masahide Maeda;K. Furuta*;Masaaki Suzuki;M. Matsumura;S. Ito
中科院分区：
文献类型：
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作者：
S. Kaneko;A. Ando;E. Okuda‐Ashitaka;Masahide Maeda;K. Furuta*;Masaaki Suzuki;M. Matsumura;S. Ito
先前的报道显示在脉络膜和视网膜的回旋状萎缩的体外模型中，鸟氨酸-δ-氨基转移酶（OAT）缺陷的人视网膜色素上皮（RPE）细胞中的鸟氨酸细胞毒性。本研究旨在阐明鸟氨酸细胞毒性的机制，并确定负责的氨基酸转运蛋白。方法采用逆转录聚合酶链反应（RT-PCR）和北方印迹法检测人端粒酶逆转录酶（hTERT）-RPE细胞中氨基酸转运体mRNA的表达。通过短干扰（si）RNA介导的基因沉默评价了通过L型氨基酸转运蛋白（LAT）1、LAT 2、阳离子氨基酸转运蛋白（CAT）-1和y（+）LAT 2系统的载体介导的鸟氨酸转运。CAT-1特异性siRNA对鸟氨酸细胞毒性的细胞保护作用在OAT缺陷型RPE细胞中用鸟氨酸处理后24小时使用细胞三磷酸腺苷（ATP）的定量分析来测量。结果：hTERT-RPE细胞中LAT 1、LAT 2、CAT-1和y（+）LAT 2 mRNA表达均为北方杂交结果; RT-PCR结果显示，hTERT-RPE细胞中LAT 1、LAT 2、y（+）LAT 1、y（+）LAT 2、CAT-1和B（0，+）AT mRNA与异源二聚体糖蛋白4F 2 hc和rBAT一起表达。CAT-1和γ（+）LAT 2 siRNA分别使hTERT-RPE细胞中的1-[（14）C]鸟氨酸摄取减少46.6%和22.0%，而LAT 1和LAT 2 siRNA没有显著影响。此外，通过siRNA沉默CAT-1降低了OAT缺陷型RPE细胞中的鸟氨酸细胞毒性。结论：鸟氨酸通过CAT-1转运可能在hTERT-RPE细胞的细胞毒性中起重要作用。通过CAT-1减少鸟氨酸转运可能是治疗脑回萎缩的新靶点。
PURPOSE 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.