CHARACTERISTICS OF L-CITRULLINE TRANSPORT TO THE BRAIN IN VITRO MODEL OF THE BRAIN CAPILLARY ENDOTHELIAL CELLS

Abstract

Objectives Brain capillary endothelial cells play a main role of the blood-brain barrier (BBB) which restricts to transport the various substances from blood to brain. L-Citrulline is a neutral amino acid and a major precursor of L-arginine in nitric oxide (NO) cycle. As L-arginine can be recycled from L-citrulline in citrulline-NO cycle, L-citrulline plays an important role for controlling NO metabolism disorders. Therefore, we aimed to clarify the mechanism of L-citrulline transport through the blood-brain barrier (BBB).

Methods The uptake study of [14C] L-citrulline was performed in the conditionally immortalized rat brain capillary endothelial cell lines (TR-BBB cells), as an in vitro model of the BBB. Inhibition studies in TR-BBB cells were conducted in the presence of L-amino acids and several compounds.

Results The uptake of [14C] L-citrulline was a time-dependent, but sodium and chloride ions-independent in TR-BBB cells. The transport process was two saturable components with Michaelis–Menten constant of 22.0±4.0µM (Km1) and 1.73 ±0.10 mM (Km2) in TR-BBB cells. In functional study, the uptake of [14C]L-citrulline in TR-BBB cells was inhibited significantly by various neutral amino acids and cationic amino acids such as arginine and lysine, but not by several anionic amino acids including L-glutamate and L-aspartate. In addition, [14C]L-citrulline uptake in the cells was inhibited markedly by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) which is the inhibitor of system L, B0, B0,+ and harmaline, the inhibitor of system b0,+. However, there was no inhibition effect for L-methylmaleimide, homoarginine, and N-(methylamino) isobutyric acid (MeAIB) which are the inhibitors for system y+L, y+, and A, respectively. Several drugs such as L-dopa, gabapentin, verpamil, and quinidine inhibited the uptake of L-citrulline, but donepzil, dopamine, riluzole, and tacrine had no effect on [14C] L-citrulline uptake in TR-BBB cells. IC50 values for L-dopa, gabapentin, L-phenylalanine and L-arginine were 501, 223, 68.9 µM and 33.4mM, respectively. In the Lineweaver–Burk plots of L-citrulline uptake for gabapentin and L-dopa as the substrates of system L in TR-BBB cells, gabapentin inhibited the uptake of [14C] L-citrulline competitively, but L-dopa inhibited uncompetitively.

Conclusions Our results suggest that L-citrulline transport to the brain may be mediated by system L and b0,+ in TR-BBB cells.