Characterization of blood-brain barrier L-arginine uptake using in situ brain perfusions in a female mouse model

Abstract Background L-arginine is a critical determinant of central nervous system (CNS) function through nitric oxide (NO) production. Its uptake from plasma into brain is dependent on carrier-mediated transport across the blood-brain barrier (BBB). Transport kinetics of L-arginine BBB uptake have been assessed in rat models, but saturation constants such as maximal transport rate ( V max ), half-saturation constant ( K M ), and diffusion constant ( K D ) in mice remain unknown. The aim of this study was to determine the transporter responsible for L-arginine BBB transport and to provide a complete kinetic profile, including whole brain and regional saturation kinetics of its transport, in a female mouse model. Methods BALBc mice were perfused with 3 H-L-arginine using the in situ brain perfusion technique. Linear and unidirectional uptake was determined by perfusion at increasing timepoints (15-60s). Saturation kinetics were identified regionally and in whole brain by adding unlabeled L-arginine to buffer and perfusing for 45s. Sodium sensitivity was evaluated by decreasing sodium levels with replacement of cesium to maintain physiologic osmolarity. Dependence of transport on hydrogen ions was determined across ranges of pH (5.5–8) by addition of hydrochloric acid or sodium hydroxide. The transport system responsible for L-arginine BBB transport was assessed by adding inhibitors such as harmaline, N-methylmaleimide (NMM), L-homoarginine, cimetidine, and 2-amino-2-norbornanecarboxylic acid (BCH), and was further evaluated for affinity to other cationic amino acids, including L-lysine and L-ornithine. Inhibitory constants ( K i ) were calculated to assess the affinity of inhibitors at the transporter. Results BBB arginine uptake showed both saturable and nonsaturable components, with a whole brain K in , K M and V max of 0.25 ± 0.02 × 10 −2 mL/s/g, 55 ± 10 µM and 5.9 ± 0.3 nmol/min/g, respectively. Whole brain diffusion constant, K D , was 2.7 ± 1.0 × 10 −4 mL/s/g. Furthermore, regional data showed cerebellar V max was significantly higher than in cortical tissue (5.5 ± 0.6 vs. 9.3 ± 0.9 nmol/min/g). L-arginine transport was insensitive to sodium depletion and was not inhibited at pH levels 7, 7.4, or 8, but was significantly inhibited at pH 5.5. Its transport was not significantly inhibited by BCH, harmaline, NMM, or cimetidine, but was sensitive to inhibition by L-homoarginine and other cationic amino acids, including lysine and ornithine. Conclusion The results indicate that mice predominantly use the y+ system, a cationic amino acid transporter, to transport L-arginine across the BBB. Our work supports previous characterization of BBB carrier-mediated transport of L-arginine yet extends the data by assessing complete Michaelis-Menten transport kinetics across regions and in whole brain in a female mouse model. Data further suggest species can influence BBB L-arginine transport function and there is differential need for L-arginine between brain regions. This data serves as a baseline for studies involving alterations in cationic amino acid homeostasis or altered L-arginine metabolism such as in cases of arginine auxotrophy.