MCT1-mediated transport of L-lactic acid at the inner blood-retinal barrier: A possible route for delivery of monocarboxylic acid drugs to the retina
MCT1-mediated transport of L-lactic acid at the inner blood-retinal barrier: A possible route for delivery of monocarboxylic acid drugs to the retina
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DOI：
10.1023/a:1013310210710
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发表时间：
2001-12-01
影响因子：
3.7
通讯作者：
Terasaki, T
中科院分区：
文献类型：
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作者：
Hosoya, K;Kondo, T;Terasaki, T
目的.本研究的目的是表征L-乳酸转运使用条件永生化大鼠视网膜毛细血管内皮细胞系（TR-iBRB 2）作为模型的体外内血视网膜屏障（iBRB），以获得更好地了解在iBRB的运输机制。在33 ℃培养TR-iBRB 2细胞，通过在37 ℃测量[（14）C] L-乳酸来监测L-乳酸摄取。分别采用逆转录聚合酶链反应（RT-PCR）和实时荧光定量RT-PCR检测单羧酸转运体（MCT）1和MCT 2的表达和mRNA水平。TR-iBRB 2细胞的[（14）C] L-乳酸摄取增加至pH 5.0，并在10 mM L-乳酸存在下受到抑制。在pH 6.0时，[（14）C] L-乳酸的摄取具有温度和浓度依赖性，米氏常数为1.7 mM，最大摄取速率为15 nmol/（30 s .蛋白质）。该过程被羰基氰化物对三氟甲氧基苯腙（质子载体）、α-氰基-4-羟基肉桂酸酯和对氯汞苯磺酸盐（H-偶联单羧酸转运的典型抑制剂）还原，表明TR-iBRB 2细胞摄取L-乳酸是一种与H（+）梯度偶联的载体介导的转运过程。[（14）C] L-乳酸的摄取被一元羧酸显著抑制，但不被二元羧酸和氨基酸抑制。此外，水杨酸和丙戊酸竞争性抑制这一过程的抑制常数分别为4.7 mM和5.4 mM。尽管发现MCT 1和MCT 22 mRNA在TR-iBRB 2细胞中表达，但使用定量实时PCR发现MCT 1 mRNA的浓度是MCT 2 mRNA的33倍。5-（N，N-D）-amiloride在pH7.4时可显著降低TR-iBRB 2细胞内[（14）C] L-乳酸的含量，并可诱导TR-iBRB 2细胞内Na +/H+交换体1 mRNA的表达。iBRB上的L-乳酸转运是一种H（+）偶联和载体介导的机制，通过MCT 1进行，可被单羧酸类药物竞争性抑制。
Purpose. The aim of this study was to characterize L-lactic acid transport using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) as a model of in vitro inner blood-retinal barrier (iBRB) to obtain a better understanding of the transport mechanism at the iBRB.Methods. TR-iBRB2 cells were cultured at 33 degreesC, and L-lactic acid uptake was monitored by measuring [(14)C]L-lactic acid at 37 degreesC. The expression and mRNA level of monocarboxylate transporter (MCT) 1 and MCT2 were determined by reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR with specific primers, respectively.Results. The [(14)C]L-lactic acid uptake by TR-iBRB2 cells increased up to a pH of 5.0 and was inhibited in the presence of 10 mM L-lactic acid. The [(14)C]L-lactic acid uptake at pH 6.0 was both temperature- and concentration-dependent with a Michaelis-Menten constant of 1.7 mM and a maximum uptake rate of 15 nmol/(30 s . mg of protein). This process was reduced by carbonylcyanide p-trifluoromethoxy-phenylhydrazone (protonophore), alpha -cyano-4-hydroxycinnamate, and p-chloromercuribenzenesulfonate (typical inhibitors for H-Coupled monocarboxylic acid transport), suggesting that L-lactic acid uptake by TR-iBRB2 cells is a carrier-mediated transport process coupled with an H(+) gradient. [(14)C]L-Lactic acid uptake was markedly inhibited by monocarboxylic acids but not dicarboxylic acids and amino acids. Moreover, salicylic and valproic acids competitively inhibited this process with an inhibition constant of 4.7 mM and 5.4 mM, respectively. Although MCT1 and MCT22 mRNA were found to be expressed in TR-iBRB2 cells, MCT1 mRNA was found to be present at a concentration 33-fold greater than that of MCT2 mRNA using quantitative real-time PCR. [(14)C]L-Lactic acid was significantly reduced by 5-(N,N-hexamethylene)-amiloride at pH 7.4 and Na(+)/H(+) exchanger 1 mRNA was expressed in TR-iBRB2 cells.Conclusion. L-Lactic acid transport at the iBRB is an H(+)-coupled and carrier-mediated mechanism via MCT1 that is competitively inhibited by monocarboxylate drugs.