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 [14c] 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 [14c] 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 [14c] l-citrulline in tr-bbb cells was significantly inhibited by neutral and cationic amino acids,but not by anionic amino acids. in addition,[14c]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 [14c] 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 [14c] 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. © 2017 the author(s).