Soares-da-Silva P, Serrão M P
Institute of Pharmacology and Therapeutics, Faculty of Medicine, Porto, Portugal.
Amino Acids. 2005 Nov;29(3):213-9. doi: 10.1007/s00726-005-0242-5. Epub 2005 Sep 5.
The present study aimed to examine the presence and define the role of 4F2hc, a glycoprotein associated with the LAT2 amino acid transporter, in L-DOPA handling by LLC-PK1 cells. For this purpose we have measured the activity of the apical and basolateral inward and outward transport of [14C] L-DOPA in cell monolayers and examined the influence of 4F2hc antisense oligonucleotides on [14C] L-DOPA handling. The basal-to-apical transepithelial flux of [14C] L-DOPA progressively increased with incubation time and was similar to the apical-to-basal transepithelial flux. The spontaneous and the L-DOPA-stimulated apical fractional outflow of [14C] L-DOPA were identical to that through the basal cell side. The L-DOPA-induced fractional outflow of [14C] L-DOPA through the apical or basal cell side was accompanied by marked decreases in intracellular levels of [14C] L-DOPA. In cells treated with an antisense oligonucleotide complementary to 4F2hc mRNA for 72 h, [14C] L-DOPA inward transport and 4F2hc expression were markedly reduced. Treatment with the 4F2hc antisense oligonucleotide markedly decreased the spontaneous fractional outflow of [14C] L-DOPA through the apical or the basal cell side. It is likely that the Na+-independent and pH-sensitive uptake of L-DOPA include the hetero amino acid exchanger LAT2/4F2hc, which facilitates the trans-stimulation of L-DOPA and its outward transfer at both the apical and basal cell sides.
本研究旨在检测与LAT2氨基酸转运体相关的糖蛋白4F2hc的存在，并确定其在LLC-PK1细胞处理左旋多巴（L-DOPA）过程中的作用。为此，我们测量了细胞单层中[14C]L-DOPA的顶端和基底外侧内向和外向转运活性，并研究了4F2hc反义寡核苷酸对[14C]L-DOPA处理的影响。[14C]L-DOPA的基底到顶端的跨上皮通量随孵育时间逐渐增加，且与顶端到基底的跨上皮通量相似。[14C]L-DOPA的自发和L-DOPA刺激的顶端分数流出与通过基底细胞侧的流出相同。L-DOPA诱导的[14C]L-DOPA通过顶端或基底细胞侧的分数流出伴随着细胞内[14C]L-DOPA水平的显著降低。在用与4F2hc mRNA互补的反义寡核苷酸处理72小时的细胞中，[14C]L-DOPA的内向转运和4F2hc的表达明显降低。用4F2hc反义寡核苷酸处理显著降低了[14C]L-DOPA通过顶端或基底细胞侧的自发分数流出。L-DOPA的不依赖于钠且对pH敏感的摄取可能包括异源氨基酸交换体LAT2/4F2hc，它促进L-DOPA的反式刺激及其在顶端和基底细胞侧的外向转运。
