Establishment and application of cell models with stable expression of hOCTN1/2
10.16438/j.0513-4870.2015-0949
- VernacularTitle:稳定表达人OCTN1/OCTN2的细胞模型构建及应用
- Author:
Ya-yun WENG
1
;
Li-sha JIN
1
;
Yu-qing WANG
1
;
Fei-feng SONG
1
;
Li-ping LI
1
;
Hui ZHOU
1
;
Su ZENG
1
;
Hui-di JIANG
1
Author Information
1. Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
- Publication Type:ORIGINAL ARTICLES
- Keywords:
human carnitine/organic cation transporter 1 and 2;
ergothioneine;
mildronate;
inhibition;
Madin-Darby canine kidney cell
- From:
Acta Pharmaceutica Sinica
2016;51(6):931-
- CountryChina
- Language:Chinese
-
Abstract:
Human carnitine/organic cation transporter 1 and 2(hOCTN1 and hOCTN2) mediate transport of endogenous and exogenous compounds. The present study aimed to establish cell models with stable expression of hOCTN1 or hOCTN2 to study interactions with compounds and transporters. MDCK cells were transfected with pcDNA3.1(+) plasmid vector containing hOCTN1 or hOCTN2(pcDNA3.1(+)-hOCTN1/2), several stable transfected clones were obtained after G418 screening. hOCTN1 and hOCTN2 clones were screened with ergothioneine and mildronate respectively as substrates to identify the best candidates. We explored interactions of endogenous substances, alkaloids, flavonoids and ACEIs with hOCTN1/2. As a result, the cellular accumulation of ergothioneine in MDCK-hOCTN1 or mildronate in MDCK-hOCTN2 was 122 and 108 folds of the control cells, respectively. The kinetic parameters, Km and Vmax of ergothioneine, mediated by MDCK-hOCTN1, were 8.19±0.61 μmol·L-1 and 1427±49 pmol·mg-1(protein)·min-1; while Km and Vmax of mildronate by MDCKhOCTN2 were 52.3±4.3 μmol·L-1 and 2454±64 pmol·mg-1(protein)·min-1. Dopamine, glutamine, piperine, berberine, nuciferine, lisinopril and fosinopril could inhibit ergothioneine or mildronate uptake by MDCKhOCTN1/2. In conclusion, cell models with good stable hOCTN1 and hOCTN2 functions have been established successfully, which can be applied to the study of interactions between compounds and transporters of hOCTN1 and hOCTN2.
