ABSTRACT
L-Type Amino Acid Transporter 1 (LAT1/Lat1) is responsible for carrying large, neutral L-amino acids as well as several drugs and prodrugs across the blood-brain barrier (BBB). However, the BBB is not the only barrier that hinders drugs acting effectively within the brain; the brain parenchymal cell membranes represent a secondary barrier for the drugs with intracellular target sites. In this study, expression and function of Lat1 was quantified in mouse primary neuron, astrocyte and immortalized microglia (BV2) cultures. Moreover, ability of Lat1 to carry prodrugs inside these brain cells was evaluated. The results showed that Lat1 was localized at the similar level in all studied cells (3.07 ± 0.92-3.77 ± 0.91 fmol/µg protein). The transporter was also functional in all three cell types, astrocytes having the highest transport capacity and affinity for the LAT1/Lat1-substrate, [14C]-L-leucine, followed by neurons and microglia. The designed prodrugs (1-6) were able to utilize Lat1 for their cellular uptake and it was mainly much higher than the one of their parent drugs. Interestingly, improved cellular uptake was also achieved in cells representing Alzheimer's Disease phenotype. Therefore, improved delivery and intra-brain targeting of drugs can be attained by utilizing LAT1/Lat1 and prodrug approach.
Subject(s)
Astrocytes/metabolism , Drug Delivery Systems/methods , Large Neutral Amino Acid-Transporter 1/metabolism , Microglia/metabolism , Neurons/metabolism , Prodrugs/administration & dosage , Alzheimer Disease/metabolism , Animals , Animals, Newborn , Astrocytes/cytology , Biological Transport , Blood-Brain Barrier/metabolism , Brain/cytology , Brain/metabolism , Cell Line , Cells, Cultured , Mice, Inbred C57BL , Mice, Transgenic , Microglia/cytology , Neurons/cytology , Prodrugs/chemistry , Prodrugs/pharmacokineticsABSTRACT
PURPOSE: The study aim was to evaluate the effect of Alzheimer's disease (AD) and inflammatory insult on the function of L-type amino acid transporter 1 (Lat1) at the mouse blood-brain barrier (BBB) as well as Lat1 function and expression in mouse primary astrocytes. METHODS: The Lat1 function and expression was determined in wildtype astrocytes with and without lipopolysaccharide (LPS)-induced inflammation and in LPS treated AD APP/PS1 transgenic astrocytes. The function of Lat1 at the BBB was evaluated in wildtype mice with and without LPS-induced neuroinflammation and APP/PS1 transgenic mice by in situ brain perfusion. RESULTS: There were 2.1 and 1.6 -fold decreases in Lat1 mRNA and protein expression in LPS-treated wildtype astrocytes compared to vehicle-treated astrocytes. In contrast, Lat1 mRNA and protein expression were increased by 1.7 and 1.2 -fold (not statistically significant) in the transgenic cells. A similar trend was observed in the cell uptake of [14C]-L-leucine. There were no statistically significant differences in [14C]-L-leucine BBB permeation between the groups. CONCLUSIONS: The results showed that neither LPS-induced inflammation or the presence of APP/PS1 mutations alters Lat1 function at the mouse BBB as well as Lat1 protein expression and function in mouse primary astrocytes.
