Molecular and functional characterization of an Na+-independent choline transporter in rat astrocytes
Molecular and functional characterization of an Na+-independent choline transporter in rat astrocytes
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DOI：
10.1111/j.1471-4159.2005.03299.x
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发表时间：
2005-09-01
影响因子：
4.7
通讯作者：
Matsumiya, T
中科院分区：
文献类型：
--
作者：
Inazu, M;Takeda, H;Matsumiya, T
在本研究中，我们研究了培养大鼠皮质星形胶质细胞摄取胆碱的分子和功能特征。星形胶质细胞对胆碱的摄取对细胞外Na+的依赖性较小。Na+非依赖性胆碱摄取是饱和的，由单一转运系统介导，表观Michaelis-Menten常数（K-m）为35.7 +/- 4.1 μ m，最大速度（V-max）为49.1 +/- 2.0 pmol/mg protein/min。细胞外培养基酸化和膜去极化使胆碱摄取明显减少。未标记的胆碱、乙酰胆碱和胆碱类似物-3可抑制Na+非依赖性胆碱摄取。典型有机阳离子四己基铵（TEA）和其他正四烷基铵化合物如四丁基铵（TBA）和四己基铵（THA）抑制Na+非依赖性胆碱摄取，其抑制作用顺序为THA > TBA > TEA。各种有机阳离子，如1-甲基-4-四氢吡啶（MPP+）、可乐定、奎宁、奎宁、胍、n -甲基烟酰胺、西咪替丁、地西帕明、苯海拉明和维拉帕米，也与Na+不依赖的胆碱运输系统相互作用。皮质酮和17 β -雌二醇是已知的有机阳离子转运体3 （OCT3）的抑制剂，没有引起任何显著的抑制作用。然而，抑制OCTs的decynium22明显抑制Na+非依赖性胆碱摄取。RT-PCR显示，星形胶质细胞表达低水平的OCT1、OCT2和OCT3 mRNA，但其胆碱摄取的功能特征与已知的这些oct的特性有很大不同。RT-PCR证实，高亲和力Na+依赖性胆碱转运体CHT1在星形胶质细胞中不表达。此外，大鼠星形胶质细胞中表达了胆碱转运蛋白样蛋白1 （CTL1）及其剪接变体CTL1a和CTL1b的mRNA，通过RNA干扰抑制CTL1的表达完全抑制Na+非依赖性胆碱摄取。我们得出结论，大鼠星形胶质细胞表达了一个中等亲和力的Na+不依赖的胆碱运输系统。该系统似乎通过CTL1发生，并负责这些细胞中胆碱和有机阳离子的摄取。
In this study, we examined the molecular and functional characterization of choline uptake into cultured rat cortical astrocytes. Choline uptake into astrocytes showed little dependence on extracellular Na+. Na+-independent choline uptake was saturable and mediated by a single transport system, with an apparent Michaelis-Menten constant (K-m) of 35.7 +/- 4.1 mu m and a maximal velocity (V-max) of 49.1 +/- 2.0 pmol/mg protein/min. Choline uptake was significantly decreased by acidification of the extracellular medium and by membrane depolarization. Na+-independent choline uptake was inhibited by unlabeled choline, acetylcholine and the choline analogue hemicholinium-3. The prototypical organic cation tetrahexylammonium (TEA), and other n-tetraalkylammonium compounds such as tetrabutylammonium (TBA) and tetrahexylammonium (THA), inhibited Na+-independent choline uptake, and their inhibitory potencies were in the order THA > TBA > TEA. Various organic cations, such as 1-methyl-4-tetrahydropyridinium (MPP+), clonidine, quinine, quinidine, guanidine, N-methylnicotinamide, cimetidine, desipramine, diphenhydramine and verapamil, also interacted with the Na+-independent choline transport system. Corticosterone and 17 beta-estradiol, known inhibitors of organic cation transporter 3 (OCT3), did not cause any significant inhibition. However, decynium22, which inhibits OCTs, markedly inhibited Na+-independent choline uptake. RT-PCR demonstrated that astrocytes expressed low levels of OCT1, OCT2 and OCT3 mRNA, but the functional characteristics of choline uptake are very different from the known properties of these OCTs. The high-affinity Na+-dependent choline transporter, CHT1, is not expressed in astrocytes as evidenced by RT-PCR. Furthermore, mRNA for choline transporter-like protein 1 (CTL1), and its splice variants CTL1a and CTL1b, was expressed in rat astrocytes, and the inhibition of CTL1 expression by RNA interference completely inhibited Na+-independent choline uptake. We conclude that rat astrocytes express an intermediate-affinity Na+-independent choline transport system. This system seems to occur through a CTL1 and is responsible for the uptake of choline and organic cations in these cells.