在表达神经递质转运体的HEK - 293细胞摄取过程中，由于扩散导致底物大量损失。

Substantial loss of substrate by diffusion during uptake in HEK-293 cells expressing neurotransmitter transporters.

作者信息

Scholze P, Sitte H H, Singer E A

机构信息

Pharmacological Institute, University of Vienna, Waehringer Strasse 13a, 1090 Vienna, Austria.

出版信息

Neurosci Lett. 2001 Aug 31;309(3):173-6. doi: 10.1016/s0304-3940(01)02058-4.

DOI:10.1016/s0304-3940(01)02058-4

PMID:11514069

Abstract

Human embryonic kidney 293 (HEK-293) cells stably transfected with the human serotonin (5-HT) or dopamine transporter (hSERT, hDAT), or the rat GABA transporter GAT-1 were incubated with saturating concentrations of transporter substrates (hSERT: [(3)H]5-HT, [(3)H]N-methyl-phenyl-pyridinium (MPP+); hDAT: [(3)H]dopamine, [(3)H]MPP(+); rGAT: [(3)H]GABA). Uptake velocities decreased significantly over time for [(3)H]5-HT and [(3)H]dopamine (already visible at 1 min), but not for [(3)H]MPP(+) or [(3)H]GABA. In efflux experiments cells were preloaded and substrate diffusion into the medium was studied following the addition of appropriate uptake inhibitors. Fractional effluxes were (% min(-1)) 1.27, 0.72, 0.27 and 0.08 for [(3)H]5-HT, [(3)H]dopamine, [(3)H]MPP(+) and [(3)H]GABA, respectively. The results suggest that in uptake experiments the more lipophilic substrates [(3)H]5-HT and [(3)H]dopamine leave the cells by diffusion already after a short time (1 min) of accumulation.

摘要

将稳定转染人血清素（5-HT）或多巴胺转运体（hSERT、hDAT），或大鼠γ-氨基丁酸转运体GAT-1的人胚肾293（HEK-293）细胞，与转运体底物的饱和浓度（hSERT：[³H]5-HT、[³H]N-甲基苯基吡啶鎓（MPP⁺）；hDAT：[³H]多巴胺、[³H]MPP⁺；rGAT：[³H]γ-氨基丁酸）一起孵育。对于[³H]5-HT和[³H]多巴胺，摄取速度随时间显著降低（在1分钟时就已可见），但对于[³H]MPP⁺或[³H]γ-氨基丁酸则不然。在流出实验中，细胞预先加载，加入适当的摄取抑制剂后研究底物向培养基中的扩散。[³H]5-HT、[³H]多巴胺、[³H]MPP⁺和[³H]γ-氨基丁酸的分数流出率分别为（% min⁻¹）1.27、0.72、0.27和0.08。结果表明，在摄取实验中，亲脂性更强的底物[³H]5-HT和[³H]多巴胺在积累短时间（1分钟）后就已通过扩散离开细胞。