Department of Biopharmaceutics, Nagoya City University, Mizuho-ku, Japan.
Drug Metab Dispos. 2010 Apr;38(4):715-21. doi: 10.1124/dmd.109.030221. Epub 2010 Jan 4.
Multidrug and toxin extrusion protein 1 (MATE1) and MATE2-K are organic cation/H(+) antiporters that have recently been identified and suggested to be responsible for the brush border secretory transport of many cationic drugs in renal tubules. We here report our finding that 4',6-diamidino-2-phenylindole (DAPI) can be used as a probe substrate for rapid assays of the functionality of the human MATEs, hMATE1, and hMATE2-K, by taking advantage of its fluorescent nature. The specific cellular uptakes of DAPI by cloned hMATE1 and hMATE2-K, which were assessed by fluorescence intensity, were found to be rapid and saturable with the Michaelis constants of 1.13 and 3.16 microM, respectively, indicating that DAPI is a good substrate of both hMATEs. It was found that many organic cations inhibit the specific uptake of DAPI by hMATE1 and hMATE2-K, and the extents of inhibition are in good correlation with those of inhibition of the specific uptake of [(3)H]cimetidine as a typical substrate, indicating comparable performances of both substrates as probes in identifying inhibitors. Thus, DAPI can be an alternative probe substrate that enables fluorometric rapid assays of the functionality of both hMATEs. It was also found that the other major renal organic cation transporters, human organic cation transporter 2 (hOCT2), hOCT3, human novel organic cation transporter 1 (hOCTN1), and hOCTN2, cannot transport DAPI, although hOCT1, which is mainly expressed in the liver, can. Therefore, the DAPI uptake assay can be a method specific to the hMATEs among organic cation transporters in the human kidney.
多药和毒素外排蛋白 1（MATE1）和 MATE2-K 是有机阳离子/H+反转运蛋白，最近已被鉴定出来，并被认为负责肾脏小管中许多阳离子药物的刷状缘分泌转运。我们在此报告我们的发现，4'，6-二脒基-2-苯基吲哚（DAPI）可以通过利用其荧光性质，用作快速测定人 MATE 人 MATE1 和 hMATE2-K 功能的探针底物。通过荧光强度评估，克隆的 hMATE1 和 hMATE2-K 对 DAPI 的特定细胞摄取被发现是快速和饱和的，米氏常数分别为 1.13 和 3.16μM，表明 DAPI 是两者的良好底物。发现许多有机阳离子抑制 hMATE1 和 hMATE2-K 对 DAPI 的特异性摄取，并且抑制的程度与作为典型底物的 [(3)H]西咪替丁的特异性摄取的抑制程度很好地相关，表明两种底物作为探针在识别抑制剂方面具有相当的性能。因此，DAPI 可以作为替代探针底物，用于荧光法快速测定两者的功能。还发现其他主要的肾脏有机阳离子转运体，人有机阳离子转运体 2（hOCT2）、hOCT3、人新型有机阳离子转运体 1（hOCTN1）和 hOCTN2，不能转运 DAPI，尽管主要在肝脏中表达的 hOCT1 可以。因此，DAPI 摄取测定可以是一种特定于人肾脏中有机阳离子转运体的 hMATE 方法。
