[논문]Regulation of Choline Transport by Oxidative Stress at the Blood-Brain Barrier In Vitro Model

Kang, Young-Sook; Lee, Hyun-Ae; Lee, Na-Young

doi:10.4062/biomolther.2008.16.1.014

[국내논문] Regulation of Choline Transport by Oxidative Stress at the Blood-Brain Barrier In Vitro Model 원문보기

Biomolecules & therapeutics, v.16 no.1, 2008년, pp.14 - 20

Kang, Young-Sook (College of Pharmacy and Research Institute of Pharmaceutical Science, Sookmyung Women's University) , Lee, Hyun-Ae (College of Pharmacy and Research Institute of Pharmaceutical Science, Sookmyung Women's University) , Lee, Na-Young (College of Pharmacy and Research Institute of Pharmaceutical Science, Sookmyung Women's University)

Abstract ▼ AI-Helper

In the present study, we examined how the transport of choline is regulated at the blood-brain barrier (BBB) under the central nervous system (CNS) cellular damages by oxidative stress using a conditionally immortalized rat brain capillary endothelial cells (TR-BBB), in vitro the BBB model. It was also tested whether the choline uptake is influenced by membrane potential, extracellular pH, protonophore (FCCP) and amiloride in TR-BBB cells. In result, $[^3H]choline$ uptake was inhibited by FCCP and dependent on extracellular pH. The treatment of TR-BBB cells with 20 ng/mL tumor necrosis $factor-{\alpha}$ $(TNF-{\alpha})$, 10 ng/mL lipopolysaccharide (LPS), 100 ${\mu}M$ diethyl maleate (DEM) and 100 ${\mu}M$ glutamate resulted in 3.0-fold, 2.6-fold, 1.8-fold and 2.0-fold increases of $[^3H]choline$ uptake at the respective peak time, respectively. In contrast, hydrogen peroxide and raffinose did not show any significant effects on choline uptake. In addition, choline efflux was significantly inhibited by $TNF-{\alpha}$, LPS and DEM producing cell damage states. In conclusion, the influx and efflux transport system for choline existed in TR-BBB cell line and this process was affected by several oxidative stress inducing agents.

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문제 정의

The purpose of this study is to clarify the driving force of choline transport through 0CT2 using TR-BBB cells as an in vitro BBB model. Furthermore, we examined the regual-tory mechanisms of choline transport under the oxidative stress condition induced by tumor necrosis factor-a (TNF-α), lipopolysaccharide (LPS), diethyl maleate (DEM) and glutamate.

제안 방법

4 or ECF buffer with Ba2+, a K+ channel blocker. To investigate the change of choline uptake under the oxidative stress condition, the TR-BBB cells were pretreated with 20 ng/mL TNF-α, 10 ng/mL LPS, 100 p, M DEM or 100 μM glutamate for 3, 6, 12 and 24 h and the uptake study was performed as described above. Then, the cells were s_이ubilized in 750 pL of 1 M NaOH-PBS buffer overnight.
The ECF buffer at 37℃ was added to the cells and incubated for the designated time periods, followed by the removal of the incubation medium from the cells. To test the change of choline effhjx under the oxidative stress condition, the cells were pretreated with 20 ng/mL TNF-α, 10 ng/mL LPS, or 1001μM DEM for 3 h and the efflux study was performed as described above. The cells were then solubilized in 1 N NaOH overnight at room temperature and an aliq_나ot was taken for the measurements of the amount of [3H]choline remained in the cells.

대상 데이터

UK). Lipopolysaccharide (LPS) from Salmonella Minnesota R595 (Re) was obtained from List Biological Laboratories (Campbel, CA, USA). Tumor necrosis factor-a (TNF-α, human recombinant (Escherichia coli) solution, sterile, 10 μg/mL), diethyl maleate (DEM), glutamate, amiloride and carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) were purchased from Sigma Chemical (St.
Lipopolysaccharide (LPS) from Salmonella Minnesota R595 (Re) was obtained from List Biological Laboratories (Campbel, CA, USA). Tumor necrosis factor-a (TNF-α, human recombinant (Escherichia coli) solution, sterile, 10 μg/mL), diethyl maleate (DEM), glutamate, amiloride and carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) were purchased from Sigma Chemical (St. Louis, MO, USA). Donepezil hydrochloride, (±)-2-[(1-benzylpipe-ridin-4-yl) methyl]-5, 6-dimethoxyindan-1 -one monohydrochloride was kindly provided by Daewoong Co.

데이터처리

values. An unpaired, two-tailed Student's t-test was used to determine the significance of differences between two group means and pv0.05 was considered statistically significant.

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