Determination of p-glycoprotein ATPase activity using luciferase.
Determination of p-glycoprotein ATPase activity using luciferase.
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DOI：
10.1248/bpb.29.560
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发表时间：
2006-03
影响因子：
2
通讯作者：
T. Matsunaga;E. Kose;S. Yasuda;H. Ise;Uichi Ikeda;S. Ohmori
中科院分区：
文献类型：
--
作者：
T. Matsunaga;E. Kose;S. Yasuda;H. Ise;Uichi Ikeda;S. Ohmori
我们研究了用荧光素-荧光素酶反应测定细胞膜上的P-糖蛋白(P-gp)ATPase活性是否可以用来测量细胞膜上的ATP消耗，以及这一结果是否有助于评估P-gp与药物之间的相互作用。用荧光素-荧光素酶反应快速、灵敏地测定了Caco-2细胞膜对钒敏感的ATPase活性。环孢菌素A、维拉帕米、地高辛和奎尼丁对ATPase活性的刺激作用呈浓度依赖性，Km值分别为5.3、0.9、1.2和4.1微米。除地高辛外，这些数值与以前的报道相当。全反式维甲酸、地高辛和左旋甲状腺素可诱导Caco-2细胞ATPase活性和P-gp基因表达，但不能诱导地塞米松和利福平。该方法可用于评价P-gp与药物的相互作用，并可用于阐明左旋甲状腺素与地高辛的相互作用机制。
We investigated whether P-glycoprotein (P-gp) ATPase activity of Caco-2 cell membranes could be estimated by measuring consumption of ATP using luciferin-luciferase reaction, and whether the results would be useful for assessment of the interactions between P-gp and drugs. The vanadate-sensitive ATPase activity of Caco-2 cell membranes was measured rapidly with high sensitivity using luciferin-luciferase reaction. Cyclosporin A, verapamil, digoxin and quinidine stimulated the ATPase activity concentration-dependently with Km values of 5.3, 0.9, 1.2 and 4.1 microM, respectively. These values except for digoxin were comparable with previous reports. The ATPase activity and P-gp mRNA expression in Caco-2 cells were induced by all-trans-retinoic acid, digoxin and levothyroxine, but not dexamethasone or rifampicin. This method was useful to assess interactions with P-gp and drugs, and was used to elucidate the mechanisms of interaction of levothyroxine and digoxin.