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

摘要

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. MethodsThe 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. ResultsThe 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. ConclusionsOur 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.