在MDCK细胞和干细胞衍生的人脑血管内皮细胞（BC1-hBMECs）中对氯解磷定转运及乙酰胆碱酯酶复活作用的体外特性研究。
In vitro characterization of pralidoxime transport and acetylcholinesterase reactivation across MDCK cells and stem cell-derived human brain microvascular endothelial cells (BC1-hBMECs).
作者信息
Gallagher Erin, Minn Il, Chambers Janice E, Searson Peter C
机构信息
Institute for Nanobiotechnology Johns Hopkins University, 3400 North Charles Street, Baltimore, MD, 21218, USA.
Department of Materials Science and Engineering, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD, 21218, USA.
出版信息
Fluids Barriers CNS. 2016 Jul 11;13(1):10. doi: 10.1186/s12987-016-0035-0.
BACKGROUND
Current therapies for organophosphate poisoning involve administration of oximes, such as pralidoxime (2-PAM), that reactivate the enzyme acetylcholinesterase. Studies in animal models have shown a low concentration in the brain following systemic injection.
METHODS
To assess 2-PAM transport, we studied transwell permeability in three Madin-Darby canine kidney (MDCKII) cell lines and stem cell-derived human brain microvascular endothelial cells (BC1-hBMECs). To determine whether 2-PAM is a substrate for common brain efflux pumps, experiments were performed in the MDCKII-MDR1 cell line, transfected to overexpress the P-gp efflux pump, and the MDCKII-FLuc-ABCG2 cell line, transfected to overexpress the BCRP efflux pump. To determine how transcellular transport influences enzyme reactivation, we developed a modified transwell assay where the inhibited acetylcholinesterase enzyme, substrate, and reporter are introduced into the basolateral chamber. Enzymatic activity was inhibited using paraoxon and parathion.
RESULTS
The permeability of 2-PAM is about 2 × 10(-6) cm s(-1) in MDCK cells and about 1 × 10(-6) cm s(-1) in BC1-hBMECs. Permeability is not influenced by pre-treatment with atropine. In addition, 2-PAM is not a substrate for the P-gp or BCRP efflux pumps.
CONCLUSIONS
The low permeability explains poor brain penetration of 2-PAM and therefore the slow enzyme reactivation. This elucidates one of the reasons for the necessity of sustained intravascular (IV) infusion in response to organophosphate poisoning.
背景
目前用于有机磷中毒的治疗方法包括使用肟类药物，如氯解磷定（2 - PAM），其可使乙酰胆碱酯酶重新激活。动物模型研究表明，全身注射后脑中的浓度较低。
方法
为评估2 - PAM的转运情况，我们研究了其在三种麦迪逊 - 达比犬肾（MDCKII）细胞系和干细胞衍生的人脑微血管内皮细胞（BC1 - hBMECs）中的跨膜通透性。为确定2 - PAM是否为常见脑外排泵的底物，在转染过表达P - gp外排泵的MDCKII - MDR1细胞系和转染过表达BCRP外排泵的MDCKII - FLuc - ABCG2细胞系中进行了实验。为确定跨细胞转运如何影响酶的重新激活，我们开发了一种改良的跨膜测定法，将被抑制的乙酰胆碱酯酶、底物和报告物引入基底外侧腔室。使用对氧磷和对硫磷抑制酶活性。
结果
2 - PAM在MDCK细胞中的通透性约为2×10⁻⁶ cm s⁻¹，在BC1 - hBMECs中的通透性约为1×10⁻⁶ cm s⁻¹。通透性不受阿托品预处理的影响。此外，2 - PAM不是P - gp或BCRP外排泵的底物。
结论
低通透性解释了2 - PAM脑穿透性差的原因，因此酶重新激活缓慢。这阐明了有机磷中毒时持续静脉输注的必要性的原因之一。
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