Functional expression of a proton-coupled organic cation (H+/OC) antiporter in human brain capillary endothelial cell line hCMEC/D3, a human blood–brain barrier model

Keita Shimomura,Sayaka Kato,Tetsuya Terasaki,Jean-Michel Schermann,Yoshiharu Deguchi,Takashi Okura,Pierre-Olivier Couraud

doi:10.1186/2045-8118-10-8

Abstract

BackgroundKnowledge of the molecular basis and transport function of the human blood–brain barrier (BBB) is important for not only understanding human cerebral physiology, but also development of new central nervous system (CNS)-acting drugs. However, few studies have been done using human brain capillary endothelial cells, because human brain materials are difficult to obtain. The purpose of this study is to clarify the functional expression of a proton-coupled organic cation (H+/OC) antiporter in human brain capillary endothelial cell line hCMEC/D3, which has been recently developed as an in vitro human BBB model.MethodsDiphenhydramine, [3H]pyrilamine and oxycodone were used as cationic drugs that proved to be H+/OC antiporter substrates. The in vitro uptake experiments by hCMEC/D3 cells were carried out under several conditions.ResultsDiphenhydramine and [3H]pyrilamine were both transported into hCMEC/D3 cells in a time- and concentration-dependent manner with Km values of 59 μM and 19 μM, respectively. Each inhibited uptake of the other in a competitive manner, suggesting that a common mechanism is involved in their transport. The diphenhydramine uptake was significantly inhibited by amantadine and quinidine, but not tetraethylammonium and 1-methyl-4-phenylpyridinium (substrates for well-known organic cation transporters). The uptake was inhibited by metabolic inhibitors, but was insensitive to extracellular sodium and membrane potential. Further, the uptake was increased by extracellular alkalization and intracellular acidification. These transport properties are completely consistent with those of previously characterized H+/OC antiporter in rat BBB.ConclusionsThe present results suggest that H+/OC antiporter is functionally expressed in hCMEC/D3 cells.

Highlights

Knowledge of the molecular basis and transport function of the human blood–brain barrier (BBB) is important for understanding human cerebral physiology, and development of new central nervous system (CNS)-acting drugs
Uptake kinetics of diphenhydramine and [3H]pyrilamine by hCMEC/D3 cells The uptake of diphenhydramine (30 μM) increased in proportion with time until 60 sec at 37°C, and reached equilibrium with the cell-to-medium (C/M) ratio of 97.7 – 103 μL/mg protein at 60 – 180 sec (Figure 1A). [3H]Pyrilamine uptake (74 kBq/μL, 90 nM) by hCMEC/D3 cells increased linearly with time until 30 sec, and the C/M ratio was 161 – 186 μL/mg protein at 30 – 60 sec (Figure 2A)
We previously showed that diphenhydramine, pyrilamine and oxycodone are taken up via a pH-sensitive, energy-dependent, protoncoupled antiport system in TR-BBB13 cells, which are an in vitro rat BBB model [9,10]

Summary

Introduction

Knowledge of the molecular basis and transport function of the human blood–brain barrier (BBB) is important for understanding human cerebral physiology, and development of new central nervous system (CNS)-acting drugs. The purpose of this study is to clarify the functional expression of a proton-coupled organic cation (H+/OC) antiporter in human brain capillary endothelial cell line hCMEC/D3, which has been recently developed as an in vitro human BBB model. The uptake was increased by extracellular alkalization and intracellular acidification These transport properties are completely consistent with those of previously characterized H+/OC antiporter in rat BBB. Knowledge of the molecular basis and transport function of the human BBB is important for understanding human cerebral physiology, and for development of new central nervous system (CNS)-acting drugs. The development of simple in vitro BBB models is highly desirable

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