Folate transport and binding by cultured human proximal tubule cells.
Folate transport and binding by cultured human proximal tubule cells.
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培养的人近端小管细胞的叶酸转运和结合。
DOI：
10.1152/ajprenal.1992.263.5.f841
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
Sens,DA
中科院分区：
文献类型：
--
作者：
McMartin,KE;Morshed,KM;Hazen-Martin,DJ;Sens,DA
由于叶酸排泄的肾脏调节机制知之甚少，因此使用代表人近端小管（HPT）的细胞培养系统研究肾脏叶酸转运。HPT细胞的汇合培养物与3 H-标记的叶酸（PteGlu）孵育后，通过酸去除程序确定与顶端膜的结合，随后在溶解的细胞中测量运输。虽然PteGlu结合几乎都是特异性的（被过量未标记的PteGlu抑制），但HPT细胞通过特异性和非特异性过程转运PteGlu。特异性PteGlu结合和转运都是可饱和的过程，分别达到0.5和0.1 pmol/mg蛋白的最大值，半最大常数分别为12和50 nM。PteGlu类似物甲氨蝶呤和5-甲基四氢叶酸（5-CH 3-H4 PteGlu）抑制PteGlu的结合和转运，其中5-CH 3-H4 PteGlu更有效（最大抑制浓度的一半较低）。相比之下，5-甲酰四氢叶酸在对结合没有影响的浓度（100-250 nM）下显著降低PteGlu转运。这些数据表明，HPT细胞将作为一个很好的模型，研究肾脏叶酸重吸收。HPT细胞对叶酸转运的初步表征表明了两个不同的过程，即与高亲和力膜叶酸结合蛋白结合，然后结构特异性地转移到细胞中。
Because mechanisms for the renal regulation of folate excretion are poorly understood, a cell culture system representative of the human proximal tubule (HPT) was used for studies of renal folate transport. After confluent cultures of HPT cells were incubated with 3H-labeled folic acid (PteGlu), binding to the apical membrane was determined by an acid removal procedure, and transport was subsequently measured in solubilized cells. Although PteGlu binding was almost all specific (suppressed by excess unlabeled PteGlu), HPT cells transported PteGlu by specific and nonspecific processes. Specific PteGlu binding and transport were both saturable processes, reaching maxima of 0.5 and 0.1 pmol/mg protein, respectively, with half-maximal constants of 12 and 50 nM, respectively. The PteGlu analogues methotrexate and 5-methyltetrahydrofolic acid (5-CH3-H4PteGlu) inhibited both the binding and transport of PteGlu, with 5-CH3-H4PteGlu being more potent (lower half-maximal inhibitory concentration). In contrast, 5-formyltetrahydrofolic acid significantly reduced PteGlu transport at concentrations (100–250 nM) that had no effect on binding. These data suggest that the HPT cells will serve as a good model for studies of renal folate reabsorption. Initial characterization of the transport of folate by HPT cells suggests two distinct processes, binding to a high-affinity membrane folate-binding protein followed by a structurally specific transfer into the cell.