Functional Polarity of Microvascular Brain Endothelial Cells Supported by Neurovascular Unit Computational Model of Large Neutral Amino Acid Homeostasis

Abstract

The homeostatic regulation of large neutral amino acid (LNAA) concentration in the brain interstitial fluid (ISF) is essential for proper brain function. LNAA passage into the brain is primarily mediated by the complex and dynamic interactions between various solute carrier (SLC) transporters expressed in the neurovascular unit (NVU), among which SLC7A5/LAT1 is considered to be the major contributor in microvascular brain endothelial cells (MBEC). The LAT1-mediated trans-endothelial transport of LNAAs, however, could not be characterized precisely by available in vitro and in vivo standard methods so far. To circumvent these limitations, we have incorporated published in vivo data of rat brain into a robust computational model of NVU-LNAA homeostasis, allowing us to evaluate hypotheses concerning LAT1-mediated trans-endothelial transport of LNAAs across the blood brain barrier (BBB). We show that accounting for functional polarity of MBECs with either asymmetric LAT1 distribution between membranes and/or intrinsic LAT1 asymmetry with low intraendothelial binding affinity is required to reproduce the experimentally measured brain ISF response to intraperitoneal (IP) L-tyrosine and L-phenylalanine injection. On the basis of these findings, we have also investigated the effect of IP administrated L-tyrosine and L-phenylalanine on the dynamics of LNAAs in MBECs, astrocytes and neurons. Finally, the computational model was shown to explain the trans-stimulation of LNAA uptake across the BBB observed upon ISF perfusion with a competitive LAT1 inhibitor.

Keywords: SLC7A5/LAT1; amino acid transporter; blood brain barrier; large neutral amino acid; neurovascular unit.

Figure 1

Diagram of the dominant LNAA transporters expressed in cells of the neurovascular unit (NVU). The diagram represents the major compartments of the brain with the dominant NVU carrier-mediated LNAA transport pathways from brain capillary plasma (input) across blood brain barrier (BBB) microvascular endothelial cells (MVEC) into the interstitial fluid (ISF) and from there into astrocytes and neurons. The abbreviations used for the NVU-SLC transporters are LAT1 (SLC7A5) and LAT2 (SLC7A8), both Na⁺-independent large neutral amino acid antiporters, and B⁰AT2 (SLC6A15), a Na⁺-dependent large neutral amino acid symporter. The arrows indicate the transmembrane pathways of LNAAs via these transporters into and out of the NVU cells. TL and CL represent test and competing large neutral amino acids, respectively.

Figure 2

Plasma concentration and corresponding brain ISF concentration response after intraperitoneal injection of L-tyrosine and L-phenylalanine. (A) shows the plasma concentration of L-tyrosine (TL) and L-tyrosine competing LNAAs (CL) after intraperitoneal administration of 200 mg/kg L-tyrosine as measured by Bongiovanni et al. (2003) and used as input for the model calculation. (B,C) show the experimental data for the L-tyrosine (Tyr) post-stimulus response in the brain ISF, measured in the prefrontal cortex (PFC). (B) shows the model calculations for various ratios of the bi-directional kinetic constant of MBEC LAT1 (RK_LAT1, Equation 11) with symmetric distribution of LAT1 at both luminal and abluminal membranes of the BBB (RE_LAT1 = 1). (C) shows the model calculations for various abluminal to luminal expression distribution ratios of LAT1 (RE_LAT1, Equation 12) with symmetric bi-directional kinetics (RK_LAT1 = 1). The model results and experimental data are represented as percent of the baseline value. In (A), the plasma baseline value for L-tyrosine and L-tyrosine competing LNAAs (constant input) are 112 and 535 μM (Currie et al., ; Bongiovanni et al., 2003), respectively. In (B,C), the ISF baseline value for L-tyrosine is 1.0 and 1.1 μM (Supplementary Table 2), respectively. Each experimental data point represents the mean ± SD for three (plasma) and four to eight (ISF) animals (Bongiovanni et al., 2003). In (A), the CL refers to a mixture of L-tyrosine competing LNAAs (L-leucine, L-isoleucine, L-phenylalanine, L-tryptophan, L-valine, L-histidine, and L-methionine). The error bars associated with model calculations indicate standard deviation with respect to concentrations obtained with the nominal model parameter set (see Methods). (D) shows the measured plasma concentration of L-phenylalanine (TL) and L-phenylalanine competing LNAAs (CL) after intraperitoneal administration of 200 mg/kg L-phenylalanine as measured by Goldstein (1961) and Bongiovanni et al. (2010). (E,F) show the experimental data for the L-phenylalanine (Phe) post-stimulus response in the brain ISF, measured in the prefrontal cortex (PFC) vs. model calculations for different ratios for the bi-directional kinetic constant of MBEC LAT1 (RK_LAT1, Equation 11), assuming symmetric distribution for LAT1 at luminal and abluminal membranes of the BBB (RE_LAT1 = 1) and the model calculations for various abluminal to luminal expression distribution ratios of LAT1 (RE_LAT1, Equation 12), assuming symmetric bi-directional kinetics of MBEC LAT1 (RK_LAT1 = 1). In (E,F), the ISF baseline value for L-phenylalanine is 0.4 μM (Supplementary Table 2). The data are represented as percent of baseline. In (D), the plasma baseline value for L-phenylalanine and L-phenylalanine competing LNAAs (constant input) are 77 and 562 μM (Currie et al., ; Bongiovanni et al., 2003), respectively. In (D), the CL refers to a mixture of LNAAs competing with the test amino acid L-phenylalanine (L-leucine, L-isoleucine, L-tyrosine, L-tryptophan, L-valine, L-histidine, and L-methionine). In (B–E), the differences between the concentrations calculated with the symmetric model (RK_LAT1 = 1 and RE_LAT1 = 1) and the experimental measurements are statistically significant at all post-stimulus time points (p < 0.001, Supplementary Table 4). In contrast, there is no significant difference between the experimental measurements and the model calculations with RK_LAT1 = 160 and RE_LAT1 = 1 (B), RK_LAT1 = 1 and RE_LAT1 = 0.18 (C), RK_LAT1 = 80 and RE_LAT1 = 1 (E) and RK_LAT1 = 0.11 and RE_LAT1 = 1 (F) with the exception of the 30 min post-stimulus time point in (C,E,F) (Supplementary Table 4).

Figure 3

The post-stimulus response in MBECs, ISF, astrocytes and neurons after intraperitoneal administration (IP) of L-tyrosine and L-phenylalanine for asymmetric bi-directional kinetics of LAT1 in MBECs. (A–H) show the model calculations for the post-stimulus responses in the NVU individual compartments after IP administration of L-tyrosine (RK_LAT1 = 160 and RE_LAT1 = 1) and L-phenylalanine (RK_LAT1 = 80 and RE_LAT1 = 1), respectively. The error bars associated with model calculations indicate standard deviation with respect to concentrations obtained with the nominal model parameter set. In (A–D), CL refers to a mixture of L-tyrosine competing LNAAs (L-leucine, L-isoleucine, L-phenylalanine, L-tryptophan, L-valine, L-histidine, and L-methionine). In (E–H), CL indicates a mixture of L-phenylalanine competing LNAAs (L-leucine, L-isoleucine, L-tyrosine, L-tryptophan, L-valine, L-histidine, and L-methionine). The ISF post-stimulus response for TL in (B,F) are replotted from Figures 2B,E, respectively. In all panels, the baseline concentration for L-tyrosine, L-tyrosine competing LNAAs, L-phenylalanine and L-phenylalanine competing LNAAs are reported in Supplementary Table 2.

Figure 4

The post-stimulus response in MBECs, ISF, astrocytes and neurons after intraperitoneal administration (IP) of L-tyrosine and L-phenylalanine for asymmetric expression distribution of LAT1 at luminal and abluminal membranes of the BBB. (A–H) show the model calculations for the post-stimulus responses in the NVU individual compartments after IP administration of L-tyrosine (RE_LAT1 = 0.18 and RK_LAT1 = 1) and L-phenylalanine (RE_LAT1 = 0.11 and RK_LAT1 = 1), respectively. The error bars associated with model calculations indicate standard deviation with respect to concentrations obtained with the nominal model parameter set. In (A–D), CL refers to a mixture of L-tyrosine competing LNAAs (L-leucine, L-isoleucine, L-phenylalanine, L-tryptophan, L-valine, L-histidine, and L-methionine). In (E–H), CL indicates a mixture of L-phenylalanine competing LNAAs (L-leucine, L-isoleucine, L-tyrosine, L-tryptophan, L-valine, L-histidine, and L-methionine). The ISF post-stimulus response for TL in B,F are replotted from Figures 2C,F. In all panels, the baseline concentration for L-tyrosine, L-tyrosine competing LNAAs, L-phenylalanine and L-phenylalanine competing LNAAs are reported in Supplementary Table 2.

Figure 5

Trans-stimulation of the test LNAA uptake across the BBB during ISF perfusion with BCH. This figure shows the ISF concentration of the test LNAAs during ISF perfusion with 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) started at time zero. In all panels, the experimental data are measured by Dolgodilina et al. (2015) for trans- stimulation of test LNAA (L-valine) during 170 min continues ISF perfusion with 20 mM BCH into a group of freely moving mice (four animals). (A,B) show the model calculations for L-tyrosine trans-stimulations upon perfusion of BCH with different global concentration levels. In (A,B), the bidirectional kinetic constant and the expression ratio of LAT1 are considered, (RK_LAT1 = 160, RE_LAT1 = 1) and (RK_LAT1 = 1, RE_LAT1 = 0.18), respectively. (C,D) show the model calculations for L-phenylalanine trans-stimulations during perfusion of BCH with different global concentration levels in the entire brain ISF compartment. In (A,B), the bi-directional kinetic constant and the expression ratio of LAT1 are considered, (RK_LAT1 = 80, RE_LAT1 = 1) and (RK_LAT1 = 1, RE_LAT1 = 0.11), respectively. The model simulations and the experimental data are represented as percent of the baseline value. The error bars associated with model calculations indicate standard deviation with respect to concentrations obtained with the nominal model parameter set. For all panels, the calculated baseline concentrations of the test LNAAs are reported in Supplementary Table 2. The differences between the experimental measurements and model calculations with BCH = 10 and 100 μM (A,B) as well as BCH = 5 and 100 μM (C,D) are statistically significant at all post-stimulus time points (p < 0.001, Supplementary Table 4). In contrast, model calculations with BCH = 30 μM (A,B) and BCH = 17 μM (C,D) are not significantly different from the experimental measurements with the exception of the 20 min post-stimulus time point (Supplementary Table 4).