Pinho Maria João, Serrão Maria Paula, Gomes Pedro, Hopfer Ulrich, Jose Pedro A, Soares-da-Silva P
Institute of Pharmacology and Therapeutics, Faculty of Medicine, Porto, Portugal.
Kidney Int. 2004 Jul;66(1):216-26. doi: 10.1111/j.1523-1755.2004.00722.x.
Spontaneously hypertensive rats (SHR) may have an increased renal production of dopamine. LAT2 promotes L-DOPA renal uptake, and this may determine the rate of dopamine synthesis. The present study evaluated L-DOPA inward and outward transfer in immortalized renal proximal tubular epithelial cells of SHR and Wistar-Kyoto rats (WKY).
Uptake of [(14)C]-L-DOPA was initiated by the addition of 1 mL Hanks' medium with a given concentration of the substrate. The apical fractional outflow of intracellular [(14)C]-L-DOPA was evaluated in cells loaded with [(14)C]-L-DOPA for 6 minutes, and then the corresponding efflux was monitored over 12 minutes. The presence of LAT1 and LAT2 transcripts and protein in WKY and SHR cells was examined, respectively, by reverse transcription-polymerase chain reaction (RT-PCR) and immunobloting.
LAT2 in WKY cells contributed almost exclusively for [(14)C]-L-DOPA uptake. In SHR cells [(14)C]-L-DOPA uptake was 25% through system B(0), 25% through LAT2 (resulting from inhibition by 1 mmol/L glycine, L-alanine, L-serine, and L-threonine), and the remaining 50% through LAT1. The efflux of [(14)C]-L-DOPA from WKY and SHR cells corresponded to approximately 65% and approximately 25%, respectively, of the amount accumulated in the cells. The LAT1 and LAT2 transcripts were present in both SHR and WKY cells, but the abundance of both LAT1 and LAT2 proteins in SHR cells was greater than in WKY cells.
Differences in L-DOPA handling between SHR and WKY cells may result from over-expression of LAT1 and LAT2 transporters in the former. The unique role of Na(+)-dependent transporters (system B(0)) in SHR cells also contributes to the enhanced L-DOPA uptake in these cells.
自发性高血压大鼠（SHR）的肾脏多巴胺生成可能增加。LAT2促进左旋多巴的肾脏摄取，这可能决定多巴胺的合成速率。本研究评估了SHR和Wistar-Kyoto大鼠（WKY）永生化肾近端小管上皮细胞中左旋多巴的内向和外向转运。
通过添加1 mL含有给定浓度底物的汉克斯培养基启动[(14)C]-左旋多巴的摄取。在加载[(14)C]-左旋多巴6分钟的细胞中评估细胞内[(14)C]-左旋多巴的顶端分数流出，然后在12分钟内监测相应的流出。分别通过逆转录-聚合酶链反应（RT-PCR）和免疫印迹检测WKY和SHR细胞中LAT1和LAT2转录本及蛋白质的存在情况。
WKY细胞中的LAT2几乎完全促成[(14)C]-左旋多巴的摄取。在SHR细胞中，[(14)C]-左旋多巴的摄取25%通过系统B(0)，25%通过LAT2（由1 mmol/L甘氨酸、L-丙氨酸、L-丝氨酸和L-苏氨酸抑制导致），其余50%通过LAT1。[(14)C]-左旋多巴从WKY和SHR细胞的流出分别对应于细胞中积累量的约65%和约25%。LAT1和LAT2转录本在SHR和WKY细胞中均存在，但SHR细胞中LAT1和LAT2蛋白的丰度均高于WKY细胞。
SHR和WKY细胞在左旋多巴处理上的差异可能源于前者中LAT1和LAT2转运体的过表达。SHR细胞中钠依赖性转运体（系统B(0)）的独特作用也有助于这些细胞中左旋多巴摄取的增强。
