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

Diphenhydramine, [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.Diphenhydramine 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.The present results suggest that H+/OC antiporter is functionally expressed in hCMEC/D3 cells.The human brain is protected by the existence of the blood–brain barrier (BBB), which consists of brain capillary endothelial cells linked with tight junctions [1]. It is well established that the polarized expression of numerous transporters and receptors at the brain capillary endothelial cells controls the blood–brain exchange of nutrients, waste products produced from neurotransmitter substances, and drugs [2]. Therefore, knowledge of the molecular basis and transport function of the human BBB is important for not only understanding human cerebral physiology, but also for 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. In addition, isolation and primary culture of brain capillary endothelial ce