Ueda Yukari, Yaginuma Toshihiko, Sakurai Eiko, Sakurai Eiichi
Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Yamashiro-cho, Tokushima, 770-8514, Japan.
In Vitro Cell Dev Biol Anim. 2014 Jun;50(6):496-501. doi: 10.1007/s11626-014-9739-0. Epub 2014 Mar 20.
The aim of this study was to examine whether cultured rat thoracic aortic endothelial cells (TAECs) have the ability to metabolize the tertiary amine, imipramine. In rat TAECs, imipramine was biotransformed into N-demethylate and N-oxide by cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO), respectively. The intrinsic clearance (V max/K m) for the N-oxide formation was approximately five times as high as that for the N-demethylate formation, indicating that oxidation by CYP was much higher than that by FMO. Moreover, we suggest that CYP2C11 and CYP3A2 are key players in the metabolism to N-demethylate in rat TAECs using the respective anti-rat CYP antibodies (anti-CYP2C11 and anti-CYP3A2). The presence of CYP2C11 and CYP3A2 proteins was also confirmed in cultured rat TAECs using a polyclonal anti-CYP antibody and immunofluorescence microscopy. In contrast, the formation rate of N-oxide at pH 8.4 was higher than that at pH 7.4. Inhibition of N-oxide formation by methimazole was found to be the best model of competitive inhibition yielding an apparent K i value of 0.80 μmol/L, demonstrating that N-oxidation was catalyzed by FMO in rat TAECs. These results suggest that rat TAEC enzymes can convert substrates of exogenous origin such as imipramine, indicating that TAECs have an important function for metabolic products, besides hepatic cells.
本研究的目的是检验培养的大鼠胸主动脉内皮细胞（TAECs）是否具有代谢叔胺丙咪嗪的能力。在大鼠TAECs中，丙咪嗪分别通过细胞色素P450（CYP）和含黄素单加氧酶（FMO）生物转化为N-去甲基化产物和N-氧化物。N-氧化物形成的内在清除率（V max/K m）约为N-去甲基化产物形成的内在清除率的五倍，这表明CYP介导的氧化作用远高于FMO。此外，我们使用各自的抗大鼠CYP抗体（抗CYP2C11和抗CYP3A2）表明，CYP2C11和CYP3A2是大鼠TAECs中N-去甲基化代谢的关键参与者。使用多克隆抗CYP抗体和免疫荧光显微镜也证实了培养的大鼠TAECs中存在CYP2C11和CYP3A2蛋白。相反，在pH 8.4时N-氧化物的形成速率高于pH 7.4时。发现甲巯咪唑对N-氧化物形成的抑制是竞争性抑制的最佳模型，其表观K i值为0.80 μmol/L，这表明大鼠TAECs中的N-氧化是由FMO催化的。这些结果表明，大鼠TAEC酶可以转化外源性底物如丙咪嗪，这表明除肝细胞外，TAECs对代谢产物具有重要作用。
