Analysis of L-leucine amino acid transporter species activity and gene expression by human blood brain barrier hCMEC/D3 model reveal potential LAT1, LAT4, B⁰AT2 and y⁺LAT1 functional cooperation

In the CNS, amino acid (AA) neurotransmitters and neurotransmitter precursors are subject to tight homeostatic control mediated by blood-brain barrier (BBB) solute carrier amino acid transporters (AATs). Since the BBB is composed of multiple closely apposed cell types and opportunities for human <i>in vivo</i> studies are limited, we used <i>in vitro</i> and computational approaches to investigate human BBB AAT activity and regulation. Quantitative real-time PCR (qPCR) of the human BBB endothelial cell model hCMEC/D3 (D3) was used to determine expression of selected AAT, tight junction (TJ), and signal transduction (ST) genes under various culture conditions. L-leucine uptake data were interrogated with a computational model developed by our group for calculating AAT activity in complex cell cultures. This approach is potentially applicable to <i>in vitro</i> cell culture drug studies where multiple "receptors" may mediate observed responses. Of 7 Leu AAT genes expressed by D3 only the activity of SLC7A5-SLC3A2/LAT1-4F2HC (LAT1), SLC43A2/LAT4 (LAT4) and sodium-dependent AATs, SLC6A15/B⁰AT2 (B⁰AT2), and SLC7A7/y⁺LAT1 (y⁺LAT1) were calculated to be required for Leu uptake. Therefore, D3 Leu transport may be mediated by a potentially physiologically relevant functional cooperation between the known BBB AAT, LAT1 and obligatory exchange (y⁺LAT1), facilitative diffusion (LAT4), and sodium symporter (B⁰AT2) transporters.