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

Kyeong-Eun Lee, Young-Sook Kang

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. The uptake study of [(14)C] L-citrulline, quantitative real-time polymerase chain reaction (PCR) analysis, and rLAT1, system b(0,+), and CAT1 small interfering RNA study were performed in TR-BBB cells. 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), B(0), B(0,+) and harmaline, the inhibitor of system b(0,+). 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 b(0,+) in TR-BBB cells. In the studies of LAT1, system b(0,+) 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. 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.