Prevention of ornithine cytotoxicity by nonpolar side chain amino acids in retinal pigment epithelial cells
Prevention of ornithine cytotoxicity by nonpolar side chain amino acids in retinal pigment epithelial cells
复制标题
DOI：
10.1167/iovs.03-0403
复制
发表时间：
2003-11-01
影响因子：
4.4
通讯作者：
Ito, S
中科院分区：
文献类型：
--
作者：
Nakauchi, T;Ando, A;Ito, S
目的.研究氨基酸对鸟氨酸-δ-氨基转移酶（OAT）缺陷的人视网膜色素上皮（RPE）细胞作为脉络膜和视网膜的回旋状萎缩（GA）的体外模型中的鸟氨酸细胞毒性的影响。RPE细胞用0.5 mM 5-氟甲基-鸟氨酸（5-FMOrn）（一种特异性和不可逆的OAT抑制剂）处理。将OAT缺陷型RPE细胞与10 mM鸟氨酸在20 mM 18种氨基酸中的1种或10 mM 2-氨基-2-降冰片烷-羧酸（BCH）（氨基酸转运系统L的常规抑制剂）存在下孵育。在OAT缺陷型RPE细胞中用鸟氨酸处理72小时后，用3-（4，5-二甲基噻唑-2-基）-2，5-二苯基溴化四唑（MTT）比色测定法评价每种氨基酸的鸟氨酸细胞毒性和细胞保护作用。使用DL-[C-14]鸟氨酸. MTT法评价鸟氨酸掺入RPE细胞。MTT比色分析显示，小的和大的两性离子氨基酸，但不是酸性或碱性氨基酸，降低鸟氨酸的细胞毒性在OAT缺陷的RPE细胞。在与20 mM亮氨酸（最有效的细胞保护氨基酸）孵育48小时后，RPE细胞对DL-[C-14]鸟氨酸的掺入降低至对照水平的79%。此外，BCH以剂量依赖性方式防止鸟氨酸细胞毒性。L型氨基酸转运蛋白（LAT）-1、LAT 2、y（+）LAT 1和4F 2 hc的轻链和重链在RPE细胞中均有表达。目前的结果表明，L-型氨基酸转运蛋白可能参与保护人RPE细胞免受鸟氨酸细胞毒性。因此，RPE细胞中的氨基酸转运可能是GA以及其他类型的脉络膜视网膜变性的新疗法的良好靶点。
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-fluoromethyl-ornithine (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-[C-14] 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-[C-14] 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.