N-demethylation and N-oxidation of imipramine in rat thoracic aortic endothelial cells.

Yukari Ueda,Eiko Sakurai,Toshihiko Yaginuma,Eiichi Sakurai

doi:10.1007/s11626-014-9739-0

Abstract

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.

Highlights

Endothelial cells have unique and efficient protective systems for controlling the passage of materials
We examined whether cultured rat thoracic aortic endothelial cells (TAECs) have the ability to form N-demethylate by cytochrome P450 (CYP) and N-oxide by flavincontaining monooxygenase (FMO) from the tertiary amine, imipramine, using enzyme inhibition or inactivation techniques, and investigated the significance of drug metabolic ability in rat TAECs
The inhibitory effect of anti-CYP antibodies on the N-demethylation activity in rat TAECs

Summary

Introduction

Endothelial cells have unique and efficient protective systems for controlling the passage of materials. The expression of FMO1, FMO2, and FMO5 proteins was confirmed in rat brain microvascular endothelial cells (BMECs) by western blotting analysis, suggesting that N-oxide of d-chlorpheniramine was formed in rat BMECs (Sakurai et al 2013). Our previous data showed that for detoxification in rat lung microvascular endothelial cells (LMECs), nicotine was biotransformed into cotinine and nicotine N′-oxide by CYPs (CYP2C11 and CYP3A2) and FMO, respectively (Ochiai et al 2006). Because aortic endothelial cells construct the systemic circulation of the blood, the metabolic ability of drug in thoracic aortic endothelial cells (TAECs) may be different from that in BMECs and LMECs. But Borlak et al (2003) already showed that the genes and proteins of major CYP monooxygenases, such as CYP2C8 and CYP2E1, are expressed in cultures of primary human coronary endothelial cells, and the endothelium has the ability to metabolize verapamil, a commonly and widely prescribed calcium antagonist. We examined whether cultured rat TAECs have the ability to form N-demethylate by CYPs and N-oxide by FMO from the tertiary amine, imipramine, using enzyme inhibition or inactivation techniques, and investigated the significance of drug metabolic ability in rat TAECs

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