Characteristics of L -citrulline transport through blood-brain barrier in the brain capillary endothelial cell line (TR-BBB cells).

BACKGROUND: L -Citrulline is a neutral amino acid and a major precursor of L -arginine in the nitric oxide (NO) cycle. Recently it has been reported that L -citrulline prevents neuronal cell death and protects cerebrovascular injury, therefore, L -citrulline may have a neuroprotective effect to improve cerebrovascular dysfunction. Therefore, we aimed to clarify the brain transport mechanism of L -citrulline through blood-brain barrier (BBB) using the conditionally immortalized rat brain capillary endothelial cell line (TR-BBB cells), as an in vitro model of the BBB.

METHODS: The uptake study of [14 C] L-citrulline, quantitative real-time polymerase chain reaction (PCR) analysis, and rLAT1, system b0,+ , and CAT1 small interfering RNA study were performed in TR-BBB cells.

RESULTS: The uptake of [14 C] L -citrulline was a time-dependent, but ion-independent manner in TR-BBB cells. The transport process involved two saturable components with a Michaelis-Menten constant of 30.9 ± 1.0 μM (Km1 ) and 1.69 ± 0.43 mM (Km2 ). The uptake of [14 C] L -citrulline in TR-BBB cells was significantly inhibited by neutral and cationic amino acids, but not by anionic amino acids. In addition, [14 C]L -citrulline uptake in the cells was markedly inhibited by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), which is the inhibitor of the large neutral amino acid transporter 1 (LAT1), B0 , B0,+ and harmaline, the inhibitor of system b0,+ . Gabapentin and L -dopa as the substrates of LAT1 competitively inhibited the uptake of [14 C] L -citrulline. IC50 values for L -dopa, gabapentin, L -phenylalanine and L -arginine were 501 μM, 223 μM, 68.9 μM and 33.4 mM, respectively. The expression of mRNA for LAT1 was predominantly increased 187-fold in comparison with that of system b0,+ in TR-BBB cells. In the studies of LAT1, system b0,+ and CAT1 knockdown via siRNA transfection into TR-BBB cells, the transcript level of LAT1 and [14 C] L -citrulline uptake by LAT1 siRNA were significantly reduced compared with those by control siRNA in TR-BBB cells.

CONCLUSIONS: Our results suggest that transport of L -citrulline is mainly mediated by LAT1 in TR-BBB cells. Delivery strategy for LAT1-mediated transport and supply of L-citrulline to the brain may serve as therapeutic approaches to improve its neuroprotective effect in patients with cerebrovascular disease.