Lee Kyeong-Eun, Kang Young-Sook
College of Pharmacy and Research Center for Cell Fate Control, Sookmyung Women's University, 52, Hyochangwon-gil, Yongsan-gu, Seoul, 140-742, South Korea.
J Biomed Sci. 2017 May 10;24(1):28. doi: 10.1186/s12929-017-0336-x.
-Citrulline is a neutral amino acid and a major precursor of -arginine in the nitric oxide (NO) cycle. Recently it has been reported that -citrulline prevents neuronal cell death and protects cerebrovascular injury, therefore, -citrulline may have a neuroprotective effect to improve cerebrovascular dysfunction. Therefore, we aimed to clarify the brain transport mechanism of -citrulline through blood-brain barrier (BBB) using the conditionally immortalized rat brain capillary endothelial cell line (TR-BBB cells), as an in vitro model of the BBB.
The uptake study of [C] L-citrulline, quantitative real-time polymerase chain reaction (PCR) analysis, and rLAT1, system b, and CAT1 small interfering RNA study were performed in TR-BBB cells.
The uptake of [C] -citrulline was a time-dependent, but ion-independent manner in TR-BBB cells. The transport process involved two saturable components with a Michaelis-Menten constant of 30.9 ± 1.0 μM (Km) and 1.69 ± 0.43 mM (Km). The uptake of [C] -citrulline in TR-BBB cells was significantly inhibited by neutral and cationic amino acids, but not by anionic amino acids. In addition, [C]-citrulline uptake in the cells was markedly inhibited by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), which is the inhibitor of the large neutral amino acid transporter 1 (LAT1), B, B and harmaline, the inhibitor of system b. Gabapentin and -dopa as the substrates of LAT1 competitively inhibited the uptake of [C] -citrulline. IC values for -dopa, gabapentin, -phenylalanine and -arginine were 501 μM, 223 μM, 68.9 μM and 33.4 mM, respectively. The expression of mRNA for LAT1 was predominantly increased 187-fold in comparison with that of system b in TR-BBB cells. In the studies of LAT1, system b and CAT1 knockdown via siRNA transfection into TR-BBB cells, the transcript level of LAT1 and [C] -citrulline uptake by LAT1 siRNA were significantly reduced compared with those by control siRNA in TR-BBB cells.
Our results suggest that transport of -citrulline is mainly mediated by LAT1 in TR-BBB cells. Delivery strategy for LAT1-mediated transport and supply of L-citrulline to the brain may serve as therapeutic approaches to improve its neuroprotective effect in patients with cerebrovascular disease.
瓜氨酸是一种中性氨基酸，是一氧化氮（NO）循环中精氨酸的主要前体。最近有报道称，瓜氨酸可预防神经元细胞死亡并保护脑血管损伤，因此，瓜氨酸可能具有神经保护作用，以改善脑血管功能障碍。因此，我们旨在使用条件永生化大鼠脑微血管内皮细胞系（TR-BBB细胞）作为血脑屏障（BBB）的体外模型，阐明瓜氨酸通过血脑屏障的脑转运机制。
在TR-BBB细胞中进行了[C] L-瓜氨酸的摄取研究、定量实时聚合酶链反应（PCR）分析以及rLAT1、系统b和CAT1小干扰RNA研究。
在TR-BBB细胞中，[C] -瓜氨酸的摄取呈时间依赖性，但不依赖离子。转运过程涉及两个可饱和成分，米氏常数分别为30.9±1.0 μM（Km）和1.69±0.43 mM（Km）。TR-BBB细胞中[C] -瓜氨酸的摄取受到中性和阳离子氨基酸的显著抑制，但不受阴离子氨基酸的抑制。此外，细胞中[C]-瓜氨酸的摄取受到2-氨基双环-（2,2,1）-庚烷-2-羧酸（BCH）的显著抑制，BCH是大中性氨基酸转运体1（LAT1）的抑制剂，以及系统b的抑制剂B、B和哈马灵。作为LAT1底物的加巴喷丁和-多巴竞争性抑制[C] -瓜氨酸的摄取。-多巴、加巴喷丁、-苯丙氨酸和-精氨酸的IC值分别为501 μM、223 μM、68.9 μM和33.4 mM。与TR-BBB细胞中的系统b相比，LAT1的mRNA表达主要增加了187倍。在通过siRNA转染到TR-BBB细胞中对LAT1、系统b和CAT1进行敲低的研究中，与TR-BBB细胞中的对照siRNA相比，LAT1的转录水平和LAT1 siRNA对[C] -瓜氨酸的摄取显著降低。
我们的结果表明，在TR-BBB细胞中，瓜氨酸的转运主要由LAT1介导。LAT1介导的转运和向大脑供应L-瓜氨酸的递送策略可能作为改善脑血管疾病患者神经保护作用的治疗方法。
