Amino acids
In order to put the importance of efflux of amino acids from brain parenchyma into context, it is necessary to consider not just the fluxes and transporters but also the need for fluxes.
Amino acids are required within the brain for protein synthesis (see Fig. 16) and for maintenance of pools of neurotransmitters, in particular glutamate and GABA (see Fig. 17). Amino acids are also needed for synthesis of many other substances, e.g. nucleosides, but when considering overall balance this demand has usually been ignored as being relatively minor and it will not be considered further here (compare [359]). The required amino acids must either be synthesized inside the brain or enter from outside primarily across the blood–brain barrier.
Fig. 16 Simplified overview of fates of amino acids in the brain parenchyma. Essential amino acids enter and leave the parenchyma across the blood–brain barrier. Non-essential amino-acids, e.g. glutamate (Glu), glutamine (Gln), and GABA can be synthesized within the brain. The amino groups for the synthesis are supplied either by transamination as shown for glutamate or to some extent [359] by incorporation of NH4+ by glutamate dehydrogenase. The latter route is believed to be minor [359, 641]. NH4+ is added to form the amide group of glutamine by glutamine synthetase (see Fig. 17). Within the parenchyma amino acids are used for synthesis of proteins and (not shown) formation of other nitrogen containing compounds, e.g. nucleotides. New amino acids must be supplied to replace those lost by metabolism. In the brain, input of amino acids is also required to provide amino groups to replace glutamate lost from the pool of amino acids involved in glutaminergic (and GABAergic, not shown) neurotransmission (see Fig. 17). α-KG α-ketoglutarate, e.a.a essential amino acids, t.a transaminase
Fig. 17 The glutamate/glutamine cycle shown in bold with indication of some of the losses and of replenishment of glutamate by denovo synthesis. Glutamate (Glu) in the presynaptic neuron is packaged into vesicles and released into the ISF during neurotransmission. Most of the glutamate is taken up into the astrocytes by the transporter Eaat1 (glast, Slc1a3) where it is converted to glutamine (Gln) by addition of an NH4+ by the enzyme glutamine synthase (g.s) [642, 643]. The glutamine is transported into the ISF by Snat3 and/or Snat5 (Slc38a3 and Slc38a5) from which it is taken up into the presynaptic terminals again by a transporter that may be a Snat. The glutamate is then regenerated by glutaminase (g.a). This cycle represents a large turnover of the amide group at the end of the side chain in glutamine, estimated to be 55% of the CMRglc (cerebral metabolic rate of glucose, see Sect. 5.3) for the entire brain in rats amounting to 490 nmol g−1 min−1 (estimated value in humans 280 nmol g−1 min−1) [644] (G. A. Dienel, personal communication). However the requirement for NH4+ consumed in the conversion of glutamate to glutamine within the astrocytes is balanced by an equal release of NH4+ in the reverse conversion in neurons. Whether diffusion of NH4+ itself is adequate to transfer the nitrogen from neurons to astrocytes as shown or some other form of N carrier is required remains controversial [385, 641, 645]. Regardless, if there were no losses of glutamine or glutamate from the cycle, there would be no need for any fluxes of amino acids into or out of the parenchyma to support glutaminergic neurotransmission. However, there are losses of glutamate and glutamine from the cycle [347, 646, 647]. At least in rodents, such losses are made good by de novo synthesis of glutamate in the astrocytes. Estimates of the total rate of loss and of de novo synthesis are around 11% of CMRglc ([648, 649] (G. A. Dienel, personal communication), i.e. about 0.11 × 0.9 µmol g−1 min−1 ≅ 100 nmol g−1 min−1. The carbon skeletons for the de novo synthesis are derived ultimately from glucose. Glucose is metabolized to two molecules of pyruvate one of which is carboxylated by pyruvate carboxylase (p.c) (thought to be present within the brain only in astrocytes) to form oxaloacetic acid (OAA) a component of the citric acid cycle. Addition of acetyl-CoA from the second pyruvate then forms citrate, which is decarboxylated to form α-ketoglutarate (α-KG). Glutamate is then formed either a by transamination (t.a) of α-ketoglutarate using leucine or other amino acids as source (see e.g. [383, 641, 645, 650], or b by addition of NH4+ [366] catalyzed by glutamate dehydrogenase (g.d). The latter is believed to be a minor pathway [359, 366, 641]. The source of the amino groups for transamination is considered further in Sect. 5.5.3 and Fig. 18. Data for the pathways involved in glutamate synthesis are much less extensive for human than for rat. Rothman and colleagues [651, 652] have argued that the α-ketoglutarate is synthesized in astrocytes based on measurements of incorporation of 13C (see [653, 654]). However, the failure to find a key transaminase in human astrocytes by immunohistochemistry [655, 656] has cast some doubt on astrocytes being the major site for the conversion from α-ketoglutarate to glutamate. For recent reviews of glutamate synthesis see [386, 641, 645]
The need for amino acid input is different from the need for glucose input. Glucose is the basic fuel consumed in metabolism and must be supplied continually in large quantities. Amino acids are needed to allow the maintenance of cell structure and composition. But, the N containing constituents of the cells either are not consumed during metabolism or if they are they are partly replaced internally. The balance between influx and efflux across the blood–brain barrier need only provide sufficient amounts of amino acids to top up losses. Any metabolic losses that do occur will either be by efflux from the brain or by generation of NH4+ and carbon compounds. The latter become part of the carbon metabolism of cells. Possible fates of the NH4+ include: diffusion across the blood–brain barrier; reaction with glutamate to form glutamine, which is then exported from the parenchyma; and use in amino acid synthesis [359, 360]. Glutamate synthesis is considered further in Sect. 5.5.5.
For each amino acid at steady-state, its net fluxes across the blood–brain barrier and via perivascular routes and its net rate of synthesis must add to zero so that the concentrations in the brain parenchyma can remain constant. However, there are major complications in applying this principle to the interpretation of data: there are more than 20 different amino-acids, inter-conversions between them by transamination are common, and they compete with each other for the many amino acid transporters. Indeed the major application of this principle comes when considering overall N balance.
Allowing the fluxes that are required (see Sect. 5.5.1) while maintaining ISF concentrations of all amino acids except glutamine well below those in plasma (see Sect. 5.5.2) is a major challenge and it is not yet certain how the available transporters (see Sect. 5.5.4) achieve these objectives.

Requirements for amino acid fluxes (and NH4+)
While it is clear that there are losses of essential amino acids from brain parenchyma and thus that some influx of amino acids must occur, it is difficult to obtain a quantitative estimate of the influx required. Using radiolabelled amino acids in rats, Dunlop et al. [361–363] found a turnover rate for the protein content of rat brains to be about 0.6% h−1. Using a protein content of about 100 mg for each gram of brain and the molecular weight of an average amino acid, perhaps 125 Da, that corresponds to a rate of incorporation of amino acids of about 80 nmol g−1 min−1. Similarly amino groups required for de novo synthesis of glutamate amount to about 100 nmol g−1 min−1 (see legend to Fig. 17).
Many of the amino acids needed for protein synthesis are supplied either by de novo synthesis (which, however, still requires some source of amino groups, see Fig. 16) or by recycling those released during protein breakdown, which averaged over enough time must be occurring at the same rate as synthesis. Furthermore it may be possible to reuse some of the NH4+ lost from the glutamate/glutamine cycle in the de novo synthesis of glutamate. Thus the sum of the estimates above, 180 nmol g−1 min−1, is likely to exceed the actual requirement for amino-acid input.
Because the brain parenchyma must be in N balance and there must be net inputs of essential amino acids, there must also be a route or routes for N removal. As the brain normally doesn’t produce urea as a means of disposing of NH4+ [364–366], the two main routes for exit to be considered are efflux of NH4+ and efflux of glutamine. Fluxes of NH4+ are easily demonstrated to occur in both directions across the blood–brain barrier and are almost certainly by diffusion across the membranes of NH3 combined with transport either of H+ in the same direction or, more likely, of HCO3− in the opposite direction [4, 359, 367]. Because concentrations of NH4+ in brain, 150–300 µM, and CSF, 100–300 µM, normally exceed those in arterial plasma, 50–250 µM [359], it is likely that there is some net NH4+ efflux. However, an arterio-venous difference in NH4+ concentration and thus its net transport have only been demonstrated in the brain when plasma NH4+ concentration is raised as in hepatic insufficiency [359, 368]. There is then net NH4+ entry, rapid incorporation of the NH4+ into glutamine by reaction with glutamate [359], and efflux of the resultant glutamine. Glutamine efflux is considered further in Sect. 5.5.4.
Lee et al. [360] made the interesting suggestion that much of the NH4+ that moves from brain microvascular endothelial cells to plasma is produced within the endothelial cells by glutaminase acting on glutamine. However, that taken alone would suggest that there should also be a substantial efflux of glutamate, which has not been observed. Alternatively the NH4+ effluxed may derive from metabolism of both glutamine and glutamate. This is considered further in Sect. 5.5.4.

Concentrations of amino acids in CSF and ISF
Values of amino acid concentrations measured in blood plasma, CSF and ISF are summarized in Table 3. There is agreement in all studies that, with the exception of glutamine, the concentrations of all other amino acids in CSF and ISF are substantially less than those in plasma. This could arise if the rates of consumption were to reduce the concentrations greatly or if there were active transport of amino acids from brain fluids to blood. Whether or not there is a substantial difference in amino acid concentrations between CSF and ISF is less clear.17
Table 3 Amino acid concentrations in plasma, CSF and ISF
Plasma concentration/µM CSF concentration/µM ISF concentration/µM
Human1 Rat2 Human3 Rabbit4 Rat5 Mice6 Human1 Human7 Rat2, c Rat1, 2 Human3 Rabbit4 Rat5 Mice6 Rabbit4 Rabbit8 Rabbit9 Rat5 Mice6
Gln 619 641 868 834 598 863 780 552 583 524 517 250 547 159 193 80
Asn 112 55 Trace 14 16 3 7.8 4.3 1.4
Ala 330 489a 382 430 408 27 18.6 34a 39 85.4 57 15 19 15 7.7 9.3
Ser 149 196 140 263 247 109 28.9 30.4 79 80 30 116 62.3 37 34 24.9 9.8
Gly 249 221 283 246 1.3 6.4 20 15 6 34 7.8
Pro 212 60 Trace 0.6 4.2 1.8
Thr 142 166 113 28.5 27.7 36 22 3.5
Val 222 430 309 191 173 14 16.1 78 47 20 14 9.4 13 12.5 7.4 2.9
Leu 109 432 155 81 127 13.1 11.9 66 42 15 7 6.6 9 1.5
Ile 61 77 101 5.2 4.7 6 7 9
Tyr 70 73 43 54 7.3 8.1 10 7 7.2 5 0.7
His 85 80 59 10.8 12.0 12 8.9 1.2
Lys 158 290 171 137 321 19.7 22.0 120 87 21 8 46 19 10
Arg 80 94 81 227 103 17 18.6 55 46 22 32.5 18 6.9 3.2
Glu 83 61 56 159.6 37 7.2 8.7 26 10 11.4 21 4 4.3 3.4 2.9 4.1
Asp 7 33.8 2.4 1.5 5.8 0.6 0.5 1.7
Phe 71 64 45 8.3 8.2 10 7 6
Met 41 28 30 2.8 2.6 3 3
Trp 62
Concentrations measured in CSF are, with the exception of glutamine always substantially less, than the concentrations in plasma. Concentrations in ISF are measured by microdialysis with extrapolation to zero flow (see text). If these are correct, ISF concentrations are substantially lower than those in CSF. c cisternal, l lumbar
1Plum et al. [576]
2Franklin et al. [577]
3McGale et al. [578]
4Hamberger et al. [579]
5Lerma et al. [580]
6Dolgodilina et al. [581]
7Table 8.15 in Davson and Segal [56]
8Jacobson et al. [582] microdialysis by concentration profile
9Jacobson et al. [582] microdialysis by recovery of samples

The relative importance of perivascular supply and removal for amino acid turnover in ISF
Excluding glutamine, concentrations of each of the amino acids in CSF and ISF are usually < 1/5th of those in plasma (see below) and in total < 1 mM. With a perivascular clearance of 1 µL g−1 min−1, and an amino acid concentration at the high end of the observed range, 100 µM, the rate of loss or gain of any particular amino acid by the perivascular route is expected to be of the order of 0.1 nmol g−1 min−1 or less, which is likely to be negligible. Amino acid loss from the brain by outflow of CSF at 0.25 µL g−1 min−1 (500 mL day−1 for a 1400 g brain) at 100 µM would be 0.025 nmol g−1 min−1 which again is likely to be negligible.

Observed fluxes of amino acids
Quantitative measurements of fluxes of amino acids have been either of influx or net flux. Influx is measured by adding a tracer to the blood perfusing the brain and measuring the amount that enters the brain over a short period. Net flux of an amino acid is calculated as10 net flux=A-V difference×blood flowby using measurements of the blood flow and the A − V difference equal to the difference between the concentrations in arterial blood entering and venous blood leaving the brain. Direct measurements of efflux have proved difficult. In practice efflux into the blood has been calculated as the difference between influx and net flux from the blood.
Influx of amino acids into brain parenchyma across the blood–brain barrier has been studied in rats. In a highly influential early study, rates were compared to that for water using 14C-labeled amino acids and 3HOH added together as a single bolus arterial injection. The results were reported as the brain uptake index (BUI), defined as a ratio of ratios ((uptake of 14C-aa)/[14C-aa])/((uptake of 3HOH)/[3HOH]) [300]. When added one at a time, the influxes of the amino acids varied greatly, with BUI for phenylalanine or leucine found to be more than 50% (i.e. each enters about half as easily as water) while at the other extreme influxes of proline, glutamate, asparagine and glycine were below the background limit of detection by the technique, BUI < ~ 3%. Influx of each of the essential amino acids (those not able to be formed within the brain) was easily measurable.
All of the influxes that were clearly above baseline were inhibited when the radiolabeled amino acids were added using serum rather than a simple buffer suggesting competition for transport with the amino acids present in serum. Competition was investigated further and confirmed by measuring uptake of tracer in the presence of an excess of individual unlabelled amino acids [300].18 Quantitative estimates of the influxes of various amino acids in rats when plasma concentrations of tracers were held constant by controlled infusions [369] or during perfusion of isolated brains [43, 370, 371] have confirmed the pattern seen using BUI measurements [300, 372] (see Table 4).
Table 4 Influx and net flux of amino acids in the indicated species
Rat1 Rat2 Rat3 Rat4 Rat5 Rat6 Dog7 Dog7 Cat8 Sheep9 Human10 Human11 Human12 Human13 Human14
Influx* Influx Influx Influx Influx Net Net* Net* Net Net Net Net Net Net Net
Phenylalanine 6.5 7 8 13.2 − 0.6 0.12 0.01 2.2 0.06 0.3 − 0.5
Leucine 6.2 15 14.5  9.7 1.56 1.28 1.9 6 5.2 3.2 2.2 0.6
Isoleucine 1.8 4  5.7 0.78 0.46 1.5 3.1 1.2 1.4 0.7 0.3
Valine 1.3 1.8  2.9 1.26 0.37 2.7 11.0 1.7 0.8 − 1.7
Tyrosine 5.3 7 4.1 − 1.1 0 0.06 1.6 0.1 − 0.1 − 0.1
Methionine 1.6 1.7 − 1.1 0 − 0.12 1.0 0.6 0.2 0.2
Tryptophan − 1.1
Histidine 2.5 2.5 − 1.7 − 0.23 − 0.7 0.7 − 0.3 − 1.3
threonine 1.2 0.8 − 3.4 0.62 − 0.1 5.0 2.6 0.1
Arginine 1 − 7.4 − 0.2 − 0.05 2 0.7 − 0.5 − 0.1
Lysine 6.2 9 − 1.1 0.58 − 1.6 2.5 − 0.3 − 0.9
Glutamate  1.7 − 0.24 − 0.18 − 0.05 1.4 − 0.5 − 6
Glutaminea 11.6 1 − 15.4 − 6.6 − 4.3 − 1.94 − 7.4 11.0 − 20 − 20.4
nerv. stim.b glutamate − 1.26
nerv. stim.b glutamine − 6.69
Values are stated in nmol g−1 min−1. Influxes have been measured only in rats. The only available measurements of net fluxes in rats, shown in the italicized column, failed to reach statistical significance
* Data used by Pardridge [379] in his comparison of influxes and net fluxes
aIn some columns the values are for glutamine + asparagine as the assay used detected both
bRelease measured during nervous stimulation
1Banos et al. [369]
2Hawkins et al. [583]
3Mans et al. [584]
4Ennis et al. [415]
5Smith and Stoll [43]
6Brosnan et al. [378]
7Betz and Gilboe [365]
8Abdul-Ghani et al. [394]
9Pell and Bergman [585]
10Felig et al. [586]
11Lying-Tunell et al. [587]
12Eriksson et al. [588]
13Grill et al. [589]
14Strauss et al. [590]
From the patterns of competition between amino acids for influx across the blood–brain barrier it appeared that there were four separate systems of transport (see e.g. [43, 44]).System L primarily for neutral amino acids, which can be inhibited by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH);
System ASC primarily for neutral amino acids, which is not inhibited by BCH;
System y+ (sometimes called system Lys+) primarily for basic amino acids;
System N primarily for the nitrogen-rich amino acids glutamine, histidine and asparagine.
A number of amino acids fit into more than one of these groups. Most of the amino acids with large BUI values are substrates for system L.
Studies with isolated brain microvessels, which provide access to the abluminal membranes of the endothelial cells, identified two more systems.A Na+-linked transport system for small neutral amino acids (system A, with identifying substrate N-methyl-a-aminoisobutyric acid, MeAIB) [373].
Another system for glutamate [374].
The ability to prepare vesicles enriched in membranes from either the luminal or abluminal membranes of the endothelial cells [375] allowed localization of transport activities to the separate membranes with the generalization (since revised, see Sect. 5.5.6) that transporters in the luminal membrane are not Na+-linked and hence bidirectional while those in the abluminal membrane are Na+-linked favouring transport from ISF into the endothelial cells [44]. There are now known to be many more types of transporter present at the blood–brain barrier than initially suggested by identification of these systems (see Sect. 5.5.6).
Large rates of efflux of amino acids from CSF to blood were detected in cats [376] and rabbits [377] undergoing ventriculo-cisternal or ventriculo-cortical subarachnoid space perfusions. However, it was not possible in these studies to determine how much of the efflux was going via the choroid plexuses and how much via the parenchyma and the blood–brain barrier. Evidence that the latter route is important derives from the observation that transfer was much more rapid in ventriculo-subarachnoid infusion than in ventriculo-cisternal infusion. Both types of perfusion expose the infused fluid to the choroid plexuses, but in the former a much larger surface area of parenchyma is exposed to the fluid [376].
The net flux into a region can be calculated if the blood flow to that region and the concentrations of the solute in arterial blood and the venous outflow can be measured (see Sect. 5.5.6). (Equating net flux out of blood with net flux into the brain ignores possible metabolism within the endothelial cells, see the end of Sect. 5.5.6). Net flux measurements have been attempted using rats [378], but all except one of the A − V differences were not statistically significant. The rest of the net flux data in Table 4 are for larger species.
Pardridge [379] compared the influx data for rats obtained by Banos et al. [369] with the net flux data for dogs obtained by Betz et al. [365] (see Table 4) and noted that the net fluxes are much smaller than the unidirectional influxes. With the assumption that the fluxes are similar in various species, this comparison implies that there must be large effluxes, comparable in size to the influxes. Measurements of net fluxes in dogs, sheep, and humans have produced data broadly comparable with each other (see Table 4) favouring the assumption that when expressed per gram of tissue the fluxes are the same in all species.19
At present there are strong indications that the net flux of glutamine is outwards. This was seen in five out of six studies. There is also indication that the combined net flux of the branched chain amino acids, leucine + isoleucine + valine, is inwards. This was seen in six out of seven studies. But as described in the next section there is no evidence for a sufficiently large inwards net flux of neutral amino acids to provide for all of the transamination invoked in the explanations of glutamate turnover, at least in rats.

Observed fluxes of neutral amino acids compared with their requirement in glutamate synthesis
A major difficulty is revealed by comparison of the small net fluxes for the large, essential neutral amino acids and the large provision of these amino acids required for transamination to convert α-ketoglutarate into glutamate (see Figs. 16 and 17). For this requirement to be satisfied by influx across the blood–brain barrier of leucine, isoleucine and valine, their combined net influx would need to be > 100 nmol min−1 g−1 (see Sect. 5.5.1). For a cerebral blood flow of 0.57 mL min−1 g−1 (see e.g. Sect. 5.3) that would correspond to an A − V difference > 175 µM. Given that the total of the arterial plasma concentrations for these amino acids is only 392 µM (see Table 3), this A − V difference and hence net rate of transport should have been well above the “noise” in all of the studies, even that in rats (see Table 4).
If, as indicated by all available studies, sufficient net inward flux of amino acids does not in fact exist, the amino groups for synthesis of glutamate in the astrocytes must be obtained from sources within the brain. Independent evidence that such a source is available comes from studies comparing isotope dilution in the brain when plasma leucine was labeled with 13C or 15N. 62% of the N in brain leucine was derived from reverse transamination [380–383].
One detailed suggestion (see Fig. 18) is that loss of the branched chain α-ketoacids (BCKA), e.g. α-ketoisocaproate, generated in the transamination in the astrocytes is prevented by using a branched chain amino acid (BCAA) shuttle ([382, 384], reviewed in [385]). In this scheme instead of being further metabolized within the astrocytes as shown in Fig. 17, the BCKA are transferred to neurons where the branched chain amino acids (BCAA), e.g. leucine, can be regenerated by transamination from glutamate producing α-ketoglutarate. The leucine is then exported back to the astrocytes while the glutamate within the neuron is regenerated by glutamate dehydrogenase from NH4+ and the α-ketoglutarate [384, 386]. In this scheme NH4+ is taken from the neuron where it is released from glutamine and will be at relatively high concentration. This is shifted to the astrocyte by the BCAA shuttle where it can be combined with new α-ketoglutarate to complete the de novo synthesis of glutamate. This scheme greatly reduces the need for net flux of BCAA across the blood–brain barrier.
Fig. 18 The branched chain amino acid shuttle for provision of branched chain amino acids (BCAA) in the astrocytes to allow de novo synthesis of glutamate. Leucine (Leu) is used as example of a BCAA. α-KG α-ketoglutarate, α-KIC α-ketoisocaproic acid, Gln glutamine, Glu glutamate, g.a glutaminase, g.d glutamate dehydrogenase, g.s glutamine synthetase, t.a transaminase. Losses of Gln, primarily by efflux, and of Glc, primarily by catabolism are replaced by de novo synthesis of α-KG in astrocytes and transamination using Leu producing α-KIC. Leu is regenerated from α-KIC in the neuron by transamination from Glu producing α-KG. The Glu is in turn regenerated from the α-KG and NH4+ by gdh. Loss of N via efflux of Gln, Glu, and Leu is made good by net inward flux of Leu and NH4+. The BCAA shuttle greatly reduces the need for net inward flux of Leu as this is only required to make good the metabolic loss of α-KIC
(Based on Figure 1 in Hutson [384])

Amino acid transporters at the blood–brain barrier
The transporters currently thought to be involved in amino acid transport across the blood–brain barrier are indicated in Fig. 19. These will be discussed below according to the categories of amino acids transported.
Fig. 19 Amino acid transporters thought to exist at the blood–brain barrier. Based on Nalecz [200]; Broer [393]; Mann et al. [520]; O’Kane et al. [657]; and Hawkins et al. [44]. #See [44, 398] but contrast [399, 400]
Anionic amino acids, in particular glutamate, are transported by EAATs 1, 2 and/or 3 (coded by SLC1A3, 2, 1 respectively) which are found only in the abluminal membrane of the endothelial cells [387]. These EAATs mediate co-transport of the anionic amino acid together with 3 Na+ ions and 1 H+ ion followed by return transport of 1 K+ ion [388–390]. Because the electrochemical gradient for Na+ is directed from ISF into the endothelial cells and 3 Na+ ions are transported, this coupling renders the amino acid transport effectively unidirectional into the cells. Glutamate is also produced within the endothelial cells from breakdown of glutamine mediated by glutaminase [360]. Glutamate in the endothelial cells can then either be metabolized releasing NH4+, as argued by Helms and colleagues [391, 392], or be transported to blood plasma by a transporter other than an EAAT. Glutamate metabolism within endothelial cells is analogous to the extensive metabolism known to occur within gut epithelial cells (see e.g. [393]). Glutamate transport from brain endothelial cells to plasma has been demonstrated after sensory stimulation in vivo, which increases glutamate production [394]. This transport is likely to be via the glutamate/cystine exchanger, Xc− (SLC7A11 + SLC3A2), [200, 395]), though there is also evidence for a transporter, yet to be identified, that functions in the absence of cystine [396].
Cationic amino acids such as arginine and lysine are transported by CAT-1 (SLC7A1), which is known to exist in the abluminal membrane of the endothelial cells. Transport of these amino acids across the luminal membrane is less well-characterized but may be also via CAT-1 or possibly ATB0,+ (SLCA14). Transport of cationic amino acids by CAT-1 can involve exchange of one amino acid for another (trans-stimulation see Sect. 5.3.1), but this is not essential [397]. There may be at least one more transporter for cationic amino acids at the abluminal membrane (but see [398]). Hawkins et al. [44] reported that cationic amino-acid transport across both membranes can be inhibited by a number of neutral amino acids in the presence of Na+. CAT-1 is thought not to be so affected [397, 399, 400]. The additional transporter may be y+L [4F2hc (SLC3A2) + either y+LAT2 (SLC7A6) or y+LAT1 (SLC7A7)] [399, 400].
Neutral amino acids are transported by several systems as indicated in Fig. 19.System L, primarily the heterodimer 4F2hc/Lat1 (Slc3a2 + Slc7a5) which is present in both membranes and functions independently of Na+;
System A, primarily ATA2 (Slc38a2) in the abluminal membrane which because it is a Na+-linked transporter is biased towards transport from ISF into the endothelial cells;
ASC, primarily ASCT2 (Slc1a5), an obligatory exchanger that requires the presence of Na+-but is not driven by the Na+ gradient;
System Na+-LNAA a Na+-linked system whose molecular basis is still unknown;
ATB0,+ (SLC6A14) which allows net fluxes without exchange;
And possibly the y+L transporter [4F2hc (SLC3A2) + either y+LAT2 (SLC7A6) or y+LAT1 (SLC7A7)].
The large influxes of neutral amino acids from blood-to-brain seen in the early work and ascribed to system L have subsequently been shown to be mediated by 4F2hc/Lat1 [401–403]. The discovery that not only can this system mediate exchanges of amino acids [404, 405] but the exchange is obligatory [406–408] has far reaching consequences for amino acid transport at the blood–brain barrier [409]. It provides an important part of the explanation for how it is that there are large unidirectional fluxes (influx and efflux) but only small net fluxes. In order for system L to mediate a net inward flux of one amino acid, it must have net outward flux of another. An exchanger of neutral solutes, like system L, tends to equilibrate the concentration ratios for all of its substrates. Thus predicting the flux of any one of the amino acids across a membrane requires knowledge of the concentrations of all of the substrates on both sides of the membrane.20 Consumption of any system L substrate within the parenchyma will by reducing its ISF concentration tend to lead to net inward flux of that substrate and net outward flux of others. Similarly production of any system L substrate will tend to lead to its net outward flux together with net inward flux of others.
The function of 4F2hc/Lat1 (Slc3a2/Slc7a5), the principal component of system L, was explored in mice by Tarlungeanu et al. [410]. They compared the concentrations of amino acids in brain (amount per unit weight of brain) between a conditional Slc7a5 knockout [411] and normal controls. In adult mice they found that the levels of methionine, leucine and isoleucine in the knockouts were about 0.66 times the levels in normals, i.e. a reduction of about 35%. This suggests that there is normally a net inward flux of these amino acids via 4F2hc/Lat1 but that there are other routes at least as important. By contrast levels of phenylalanine, proline, glycine, threonine, and serine in the knockouts were about 1.3 times higher than in normals, i.e. an increase of about 30%. This suggests that for these amino acids there is normally a net outward flux via Lat1 but that there are other important routes for their elimination. More dramatically with histidine the level in knockouts was sevenfold higher, a 600% increase compared to normals. This suggests that 4F2hc/Lat1 is normally the main route for eliminating histidine from the parenchyma and that a net inward flux of histidine occurs by some route other than 4F2hc/Lat1. However, it is important to note that while these results show that 4F2hc/Lat1 is very important for the fluxes of histidine, they do not in themselves show that histidine efflux is a large fraction of the total efflux carried by 4F2hc/Lat1. Further evidence for exchanges involving histidine have been obtained using 4F2hc/Lat1 expressed in proteoliposomes. High concentrations of cysteine inside the vesicles can allow or drive influx of histidine and high concentrations of many amino acids outside of the vesicles can allow or drive efflux of histidine [412].
It has been tempting to propose that the combined net flux of neutral amino acids, inward or outward, is determined by their fluxes via systems other than system L and by their synthesis and breakdown in the parenchyma. System L is, however, still important, because it is the combined action of system L with the other transporters that determines which of the neutral amino acids move inwards and which outwards. A coherent overall account of the transport of neutral amino acids across the blood–brain barrier is still awaited.
With regard to glutamine, which is synthesized within the parenchyma, it has been tempting to propose that a substantial part of its efflux occurs via a system L mediated exchange for the essential large neutral amino acids such as leucine, isoleucine, valine and phenylalanine entering the parenchyma. Indeed such exchanges can be observed with isolated microvessels under experimental conditions [413, 414]. However, there is no evidence for this effect under conditions that exist in vivo.
The observation that there is a net efflux of glutamine is especially important because it is present at high concentration in plasma and ISF and it is the obvious sink for excess NH4+ in the brain. Glutamine is a substrate not only of 4F2hc/LAT1 (system L) as outlined above but also of Snat3 (SLC38A3) (system N), ATA2 (SLC38A2) (system A), and CAT (SLC7A1) (system y+) [200]. Of these the principal transport that has been observed is mediated by system N. Localization of system N has been controversial. Lee et al. [360] (see also [44]) found that vesicles prepared from abluminal membranes displayed a Na+-linked transport for glutamine while vesicles prepared from luminal membranes had only Na+-independent transport. This combination would explain net outward flux of glutamine from the brain. However, Ennis et al. [415] found marked Na+-dependent tracer influx of glutamine. While there are alternatives (see footnote 3 on p. 9 in [4]) the simplest interpretation is that there are Na+-linked transporters in both membranes. More recently immunohistochemical localization studies [416] have shown Snat3 primarily on the abluminal membrane but also on the luminal membrane of brain capillaries. It should be noted that while linking transport of glutamine to that of a single Na+ confers a bias towards transport into the endothelial cells, it does not preclude flux in the opposite direction via the same transporter and thus it is possible that Snat3 is responsible for the transport across both membranes.
As already mentioned, Lee et al. [360] found that brain endothelial cells have glutaminase activity, and thus following glutamine transport from ISF into the cells, at least some of the glutamine will be broken down to glutamate and NH4+. Helms et al. [391, 392] have suggested that some of the glutamate can be metabolized further releasing more NH4+. As a consequence of metabolism within the endothelial cells, glutamine removal from the parenchyma and glutamine appearance in plasma need not be the same. Glutamine net flux cannot be assessed in isolation.