Mechanism of thiamine transport in neuroblastoma cells. Inhibition of a high affinity carrier by sodium channel activators and dependence of thiamine uptake on membrane potential and intracellular ATP.
Mechanism of thiamine transport in neuroblastoma cells. Inhibition of a high affinity carrier by sodium channel activators and dependence of thiamine uptake on membrane potential and intracellular ATP.
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神经母细胞瘤细胞中硫胺素的转运机制。
DOI：
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发表时间：
1994
影响因子：
4.8
通讯作者：
P. Wins
中科院分区：
文献类型：
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作者：
L. Bettendorff;P. Wins
神经细胞对硫胺素缺乏特别敏感。我们研究了小鼠神经母细胞瘤 (Neuro 2a) 细胞中的硫胺素转运。在低外部浓度下，[14C]硫胺素通过可饱和的高亲和力机制被吸收（Km = 35 nM）。这种现象被低浓度的 Na+ 通道激活剂藜芦定 (IC50 = 7 +/- 4 microM) 和箭毒毒素 (IC50 = 0.9 microM) 阻断。这些效应不会被河豚毒素拮抗，并且在缺乏 Na+ 通道的细胞系中也观察到，表明这些通道不参与抑制机制。在高细胞外浓度下，硫胺素的摄取主要通过低亲和力载体 (Km = 0.8 mM) 进行，对藜芦定不敏感，但被二价阳离子阻断。在这两种情况下，摄取均独立于外部钠，部分被去极化抑制（10-35%），并且对代谢抑制剂敏感。发现硫胺素转运速率与细胞内 ATP 浓度之间存在线性关系。当在低硫胺素浓度 (6 nM) 的培养基中生长的细胞暴露于 100 nM 细胞外硫胺素时，2 小时后观察到细胞内硫胺素二磷酸增加了 3 倍，而随之而来的细胞内游离硫胺素的增加几乎不显着。这些数据表明硫胺素的二次主动转运，主要驱动力是硫胺素磷酸化而不是钠梯度。
Nerve cells are particularly sensitive to thiamine deficiency. We studied thiamine transport in mouse neuroblastoma (Neuro 2a) cells. At low external concentration, [14C]thiamine was taken up through a saturable high affinity mechanism (Km = 35 nM). This was blocked by low concentrations of the Na+ channel activators veratridine (IC50 = 7 +/- 4 microM) and batrachotoxin (IC50 = 0.9 microM). These effects were not antagonized by tetrodotoxin and were also observed in cell lines devoid of Na+ channels, suggesting that these channels are not involved in the mechanism of inhibition. At high extracellular concentrations, thiamine uptake proceeds essentially via a low affinity carrier (Km = 0.8 mM), insensitive to veratridine but blocked by divalent cations. In both cases, the uptake was independent on external sodium, partially inhibited (10-35%) by depolarization and sensitive to metabolic inhibitors. A linear relationship between the rate of thiamine transport and intracellular ATP concentration was found. When cells grown in a medium of low thiamine concentration (6 nM) were exposed to 100 nM extracellular thiamine, a 3-fold increase in intracellular thiamine diphosphate was observed after 2 h while the concomitant increase in intracellular free thiamine was barely significant. These data suggest a secondary active transport of thiamine, the main driving force being thiamine phosphorylation rather than the sodium gradient.