Na+-dependent transport of large neutral amino acids occurs at the abluminal membrane of the blood-brain barrier

Robyn L. O'Kane and Richard A. Hawkins

Department of Physiology and Biophysics, Finch University of Health Sciences/The Chicago Medical School, North Chicago, Illinois 60064

Submitted 28 April 2003 ; accepted in final form 18 August 2003


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 DISCLOSURES
 REFERENCES
 
Several Na+-dependent carriers of amino acids exist on the abluminal membrane of the blood-brain barrier (BBB). These Na+-dependent carriers are in a position to transfer amino acids from the extracellular fluid of brain to the endothelial cells and thence to the circulation. To date, carriers have been found that may remove nonessential, nitrogen-rich, or acidic (excitatory) amino acids, all of which may be detrimental to brain function. We describe here Na+-dependent transport of large neutral amino acids across the abluminal membrane of the BBB that cannot be ascribed to currently known systems. Fresh brains, from cows killed for food, were used. Microvessels were isolated, and contaminating fragments of basement membranes, astrocyte fragments, and pericytes were removed. Abluminal-enriched membrane fractions from these microvessels were prepared. Transport was Na+ dependent, voltage sensitive, and inhibited by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, a particular inhibitor of the facilitative large neutral amino acid transporter 1 (LAT1) system. The carrier has a high affinity for leucine (Km 21 ± 7 µM) and is inhibited by other neutral amino acids, including glutamine, histidine, methionine, phenylalanine, serine, threonine, tryptophan, and tyrosine. Other established neutral amino acids may enter the brain by way of LAT1-type facilitative transport. The presence of a Na+-dependent carrier on the abluminal membrane capable of removing large neutral amino acids, most of which are essential, from brain indicates a more complex situation that has implications for the control of essential amino acid content of brain.

active transport; brain extracellular fluid; capillaries; endothelial cells; essential amino acids


CEREBRAL CAPILLARIES have more extensive tight junctions between endothelial cells than other capillaries (7). This arrangement creates a blood-brain barrier (BBB) by blocking paracellular movement. It also prevents the movement of intrinsic proteins and lipids between the luminal and abluminal membranes (51, 52). Because of this, endothelial cells are divided into luminal and abluminal domains; different populations of both lipids and proteins exist on each side (2, 3, 48). Nutrients and other molecules must therefore pass two sheaths of membrane. It is the combined characteristics of these membranes that determine which molecules traverse the BBB and how quickly.

Early studies of transport in vivo revealed a distinct pattern of neutral amino acid uptake by brain; movement of essential neutral amino acids from blood to brain was greater than that of nonessential amino acids (1, 32); the latter were immaterial (33). A range of naturally occurring large neutral amino acids (asparagine, cysteine, glutamine, histidine, isoleucine, leucine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine) share a single transport system (33). This carrier is facilitative and Na+ independent, and L-amino acids are preferred (33); it seems to belong to the L system (leucine preferring), originally described by Oxender and Christensen (34). The carrier is most probably the high-affinity form (3, 45, 46), currently referred to as large neutral amino acid transporter 1 (LAT1) (4, 24, 25, 43). Fernstrom and Wurtman (16) demonstrated the important role of the LAT1 system. They showed that brain tryptophan and serotonin contents and serotonin concentrations were correlated with the ratio of tryptophan to neutral amino acids that exists in plasma. They concluded that competition between tryptophan and other large neutral amino acids for entry to brain was important.

Although Na+-dependent systems do not seem to exist at the luminal side (46), several have been found at the abluminal membrane by use of techniques in vitro. System A (alanine preferring) was first characterized and shown to actively transport small nonessential neutral amino acids (3). At least four other Na+-dependent carriers exist at the abluminal membrane: system ASC [alanine, serine, and cysteine preferring (17, 47, 50)], system Bo,+ [basic amino acids, e.g., lysine (30, 41)], system N [e.g., glutamine, asparagine, and histidine (27)], and excitatory amino acid carriers (EAAC) such as aspartate and glutamate (22, 31)]. These Na+-dependent carriers provide mechanisms for exporting nonessential amino acids that may be neurotransmitters, nitrogen-rich amino acids, and acidic (excitatory) amino acids that may be detrimental to brain function if they were allowed to accumulate in the extracellular fluid (44, 49). Thus the concept arose of the BBB being two complementary membranes working in concert to eliminate noxious molecules (18).

Recently, Na+-dependent transport of phenylalanine, an essential aromatic amino acid, was detected at the abluminal membrane (41). The purpose of the present experiments was to study the Na+-dependent phenylalanine carrier more closely. The results indicate a Na+-dependent carrier on the abluminal membrane of the BBB that preferentially transports aromatic and branched-chain amino acids and is in a position to remove these from the extracellular fluid (ECF) of brain.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 DISCLOSURES
 REFERENCES
 
Materials. L-[14C]leucine (292 mCi/mmol) and L-[4,5-3H]-isoleucine (40 Ci/mmol) were purchased from American Radiolabeled Chemicals (St. Louis, MO), amino acids, collagenase type IA, {alpha}-(methylamino)isobutyric acid (MeAIB), and 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) from Sigma (St. Louis, MO), and the Bio-Rad protein assay from Bio-Rad Laboratories (Hercules, CA).

Animals. Fresh bovine brains were bought from Aurora Packing (North Aurora, IL). The cows were killed for food under US Department of Agriculture supervision, and the meat was sold for human consumption.

Isolation and characterization of membrane vesicles. Membrane vesicles from brain endothelial cells were prepared as previously described (40). Briefly, isolated microvessels from bovine cerebral cortexes were obtained as described by Pardridge et al. (37). The microvessels were digested with collagenase type IA to remove the basement membrane, pericytes, and glial fragments. The refined microvessels were homogenized to release the membranes, which were then separated into five fractions at the interfaces of a discontinuous Ficoll gradient (0, 5, 10, 15, and 20%) (40). The amount of abluminal and luminal membrane in each fraction was determined by the activities of two markers previously demonstrated to be located exclusively on one of the membranes. System A transport of MeAIB was used as the marker of the abluminal membrane and {gamma}-glutamyl transpeptidase as the marker of the luminal membrane (40). The typical percentages of luminal membrane in each fraction were F1, 83%; F2, 47%; F3, 34%; F4, 24%; and F5, 34%. Fractions F3 and F4 were combined, resulting in a preparation with an abluminal membrane content of 70%. The contamination by luminal membranes could be ignored because Na+-dependent transport systems are not present on this side (40, 46). The vesicles were stored in aliquots at –70°C.

Measurement of transport rates. Experiments were performed by a rapid filtration method (41). Membrane vesicles were thawed, centrifuged at 37,500 g for 25 min at 4°C, and suspended in storage buffer (290 mM mannitol and 10 mM HEPES, pH 7.4). Vesicles were allowed to equilibrate overnight at 4°C. The final concentration of protein was between 2.5 and 5 µg protein/µl. The vesicle suspensions were divided into 5-µl aliquots that were preincubated at 37°C for 1 min before initiation of transport measurements. Reaction medium containing 3H- or 14C-labeled substrate, with or without competing substrate, was added to initiate the reaction. The concentration of extravesicular NaCl at the start of the measurements was 100 mM, and the internal concentration was 0 unless otherwise stated. Reaction times were 10 s except for time course studies. Reactions were stopped by adding 1 ml of an ice-cold stopping solution (145 mM NaCl and 10 mM HEPES, pH 7.4) and filtering immediately on a 0.45-µm Gelman Metricel filter (Ann Arbor, MI) under vacuum. The filtered membranes were immediately washed four times with 1-ml aliquots of stopping solution, after which the filters were counted by liquid scintillation spectroscopy. Unless otherwise indicated, all rates were corrected for nonspecific transport, binding, or trapping, as measured in the presence of a saturating dose of unlabeled substrate.

Determination of kinetic characteristics. Apparent Km and Vmax values for leucine were determined by measuring the initial rates of transport at various concentrations. From these data, a nonlinear regression analysis was performed using Sigma Plot (SPSS, Chicago, IL), and apparent Km and Vmax were calculated. The permeability-to-surface area product (PSA) was calculated by dividing the Vmax by the Km.

Inhibition studies: Ki calculations. In some experiments, amino acids or inhibitors were included in the reaction medium. The velocity with and without the putative substrates was measured and the percent inhibition determined.

Transmembrane potential effect on transport. To create different initial transmembrane potentials, valinomycin (12.5 µg/mg protein), a K+-specific ionophore, was used to increase the permeability of vesicles to K+. Different ratios of internal and external concentrations of K+ were used to establish potential differences ranging from –80.7 to +10.7 mV. The vesicles were prepared overnight with equilibrating solutions containing 25 or 100 mM KCl, 10 mM HEPES, and mannitol to bring the final osmolality to 300 mosmol/kgH2O.

The reaction was initiated by the addition of an appropriate volume and composition of extravesicular solution to create the desired initial transmembrane potential as calculated by the Nernst equation

Protein determination. Protein concentrations were determined using the Bio-Rad Protein Microassay, with bovine serum albumin as the standard, based on the method of Bradford (6).

Statistical analyses. Curves were fit by Sigma Plot, and data were analyzed with StatView (SAS, Cary, NC) using ANOVA and Fisher's least significant difference test. Values were considered significant at P < 0.05.


    RESULTS AND DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 DISCLOSURES
 REFERENCES
 
Absence of BCH inhibition on cationic amino acid transport. Sánchez del Pino et al. (41) demonstrated Na+-dependent phenylalanine transport that was inhibited by BCH. System Bo,+ is a Na+-dependent carrier that recognizes neutral amino acids and is inhibited by BCH (53). Because of this characteristic and the observed inhibition, those authors thought that System Bo,+ was likely to be responsible for the transport activity. Another characteristic of System Bo,+ is the ability to transport cationic amino acids (53). In view of this, we measured the initial rate of lysine transport (80 µM) in the presence of varying concentrations of BCH up to 10 mM, but no inhibition was detected (Fig. 1). Because BCH did not interfere with the transport of a cationic amino acid, it seemed plausible that another system was responsible for the BCH-inhibited, Na+-dependent phenylalanine transport reported by Sánchez del Pino et al.



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Fig. 1. Lysine transport is not inhibited by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). [14C]lysine (80 µM) transport was measured in the presence of an inwardly directed Na+ gradient (100 mM external, internal, nil). BCH did not inhibit at any of the indicated concentrations. Each observation was expressed as the mean of 3 determinations ± SE.

 

System ASC and aromatic amino acid transport. The presence of System ASC (which has a preference for alanine, serine, and cysteine) on the BBB has been suggested (17, 47), and we confirmed its activity in the BBB (30). To determine whether the ASC system could have been responsible for the Na+-dependent phenylalanine transport observed by Sánchez del Pino et al. (41), [3H]alanine (0.4 µM) transport was measured in the presence of three aromatic amino acids (phenylalanine, tryptophan, and tyrosine). No significant inhibition was observed, indicating that System ASC could not explain the aromatic amino acid transport (Fig. 2).



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Fig. 2. Na+-dependent alanine transport in the presence of aromatic amino acids. Net [3H]alanine (0.4 µM) transport was measured in the presence of an inwardly directed Na+ gradient (100 mM external, internal, nil). Measurements were corrected for influx measured in the absence of a Na+ gradient. {alpha}-(Methylamino)isobutyric acid (MeAIB, 20 mM) was included to block System A (alanine preferring). Three aromatic amino acids, 2 mM phenylalanine, 2 mM tryptophan, and 2 mM tyrosine, were tested. No significant inhibition was observed. Each observation was expressed as the mean of 3 determinations ± SE.

 

System y+L is a broad-scope amino acid transporter that was first identified in human erythrocytes (11, 12, 35). System y+L has two distinctive properties: it can bind and translocate cationic and neutral amino acids, and its specificity varies depending on the ionic composition of the medium. In a Na+ medium, it transports a variety of neutral amino acids, with a particular affinity for leucine, which binds most strongly. We measured the Na+-dependent transport of [14C]leucine in the presence of a range of neutral, basic, and acidic amino acids (Table 1). Eight large neutral amino acids and two small (alanine and glycine) significantly inhibited Na+-dependent leucine transport and may be considered putative substrates. However, neither lysine nor arginine, both transported by System y+L with affinities in the low micromolar range (11, 12, 35), had detectable effects. Therefore, participation by System y+L in the Na+-dependent transport of phenylalanine or leucine is dubious.


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Table 1. Inhibition of Na+-dependent transport of leucine

 

Na+ dependence of leucine and isoleucine transport. Because Systems Bo,+, ASC, and y+L were considered unlikely candidates for Na+-dependent transport of phenylalanine and leucine, and the other known Na+-dependent systems, A, N, and the various excitatory amino acid transporters (EAATs) have different substrate preferences (they do not transport large neutral essential amino acids), we conducted additional experiments. It seemed possible that an as-yet-unidentified system may be present and active on the BBB. One possibility was a Na+-dependent system for the branched-chain amino acids leucine, isoleucine, and valine similar to that described by Jorgensen et al. (23).

If a process is Na+ dependent, the concentration of substrate within vesicles may transiently exceed the equilibrium value in the presence of an inwardly directed Na+ gradient, a so-called "overshoot" (19). Therefore, the demonstration of an overshoot is evidence of a Na+-dependent process. The transport of tracer concentrations of [14C]leucine and [3H]isoleucine was measured in the presence of initial external concentrations of 100 mM Na+ or 100 mM choline (Fig. 3). The concentrations of both branched-chain amino acids were substantially increased by the inwardly directed Na+ gradient, and an overshoot, indicating a Na+-driven process, was observed for both amino acids.



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Fig. 3. Na+-dependent leucine and isoleucine transport. The accumulation of [3H]isoleucine (1.3 µM; A) and [14C]leucine (33 µM; B) was measured over time in the presence of an inwardly directed Na+ gradient (100 mM external, internal, nil, {bullet}), or in the presence of choline (100 mM external, internal, nil, {blacktriangleup}). The Na+ gradient augmented the rate of transport and caused an overshoot (see text), indicating a Na+-dependent component of transport. Each observation was expressed as the mean ± SE of 3–4 determinations.

 

Effect of transmembrane potential on Na+-dependent leucine transport. The initial rate of Na+-dependent [14C]leucine (33 µM) was measured over a range of voltage from +10.7 to –80.7 mV (Fig. 4). The data indicated voltage sensitivity and suggested that a positive charge was translocated with leucine.



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Fig. 4. Effect of transmembrane potential on the rate of leucine transport. The initial rate of [14C]leucine transport (33 µM), normalized to the maximal rate, was measured over a range of calculated transmembrane potentials. The background level of uptake was determined in the presence of 10 mM BCH and subtracted. Each observation was expressed as the mean ± SE of 3–4 determinations.

 

Inhibition of isoleucine transport. Isoleucine transport was measured in the presence of other branchedchain amino acids (valine and leucine), MeAIB, a specific substrate of System A, and BCH, a substrate of System L (Fig. 5). Valine, leucine, and BCH inhibited isoleucine transport nearly completely, suggesting a carrier that recognizes branched-chain amino acids. MeAIB did not inhibit, providing further evidence that System A, which is inhibited by MeAIB, was not involved.



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Fig. 5. Inhibition of Na+-isoleucine transport by various substrates. Net Na+-dependent [3H]isoleucine (1.3 µM) transport (corrected for transport in the absence of a Na+ gradient) was measured in the presence of 20 mM BCH, valine, leucine, and MeAIB. MeAIB had no significant effect. Each observation was expressed as the mean ± SE of 3–4 determinations.

 

Kinetics of leucine transport. The initial rate of Na+-dependent leucine transport (10 s) was measured over a range of leucine concentrations up to 1 mM. The data showed the apparent Km to be 21 ± 7 µM with a Vmax value of 114 ± 6 pmol · mg–1 · min–1 and a PSA value of 5.4 µl · mg–1 · min–1 (Fig. 6). The apparent Km was similar to that found by Smith et al. (32) for the facilitative carrier (Na+ independent) of the BBB.



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Fig. 6. Kinetics of leucine transport. Na+-dependent [14C]leucine transport (corrected for transport in the absence of a Na+ gradient) was measured as a function of concentration. The initial transmembrane potential was 0 mV. Each observation was expressed as the mean ± SE of 3–4 determinations.

 

Concluding comments. Amino acids and other nutrients may enter brain through the endothelial cells making up the BBB, the choroid plexus, or the circumventricular organs that have fenestrated capillaries, the last being minute and inconsequential with regard to cerebral nutrition. Pardridge (36) noted that the BBB has a surface area >1,000 times that of the choroid plexus, which forms the blood-cerebrospinal fluid barrier. The large BBB surface area means that the extent to which a given molecule enters brain is determined almost exclusively by the permeability characteristics of the BBB (36).

Oldendorf and Szabo (33) demonstrated that three facilitative amino acid transport systems exist in the BBB: one for basic amino acids, one for large neutral amino acids, and one for acidic amino acids. For many years it was thought that the content of amino acids in brain was controlled by these facilitative systems. But with the ability to examine the membranes of the BBB separately, it has become clear that the situation is far more complex.

The concentrations of all amino acids, with the exception of glutamine, are 6–30 times less in the ECF of brain than in plasma (Table 2). On the other hand, arteriovenous differences of most amino acids are very small or undetectable (13, 15, 39). In other words, amino acids are leaving the brain against a concentration gradient at about the same rate at which they enter. The efflux of amino acids similar to influx cannot be explained by facilitative transport systems: energy must be involved. It must be concluded that the active (e.g., Na+-dependent) systems on the abluminal membrane have an important role in maintaining both homeostasis of brain amino acid content and the lower concentration in the ECF. On the basis of similar observations, Bradbury (5) wrote, "[t]here is a strong indirect argument in favor of the hypothesis that most amino acids must be moved against a concentration gradient from interstitial fluid to blood."


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Table 2. Amino acid concentrations in plasma and brain

 

Several Na+-dependent carriers are now known to be located in the abluminal membranes of the BBB in a position to export most naturally occurring amino acids, thereby explaining the significantly lower concentrations of amino acids in the ECF of brain. These systems include A (3), ASC (17, 30, 47), N (27), EAAT1–3 (31), and the large neutral amino acids described herein.

The present results establish that Na+-dependent transport of large neutral amino acids exists in the abluminal membrane of the BBB. The spectrum of inhibitors is similar to the substrates of the facilitative system LAT1 (Table 1). BCH, as well as a wide range of amino acids that may be considered putative substrates, inhibits this Na+-dependent system. It seems likely, on the basis of the spectrum of substrates, that the Na+-dependent carrier on the abluminal membrane is similar to carriers reported in rabbit kidney (23) and bacteria (21). The system also has some similarities to the ASC/Bo described by Pollard et al. (38), but it is different in that alanine (a substrate of ASC/Bo) transport is not inhibited by phenylalanine, tryptophan, or tyrosine in the BBB Na+-dependent system.

Although the BBB determines the availability, and thereby the brain content, of essential amino acids, astrocytes and neurons participate in maintaining the extracellular concentrations. Astrocytes and neurons have Na+-dependent transport systems capable of transporting neutral and acidic amino acids (8, 9, 29, 42). These various systems are actively involved in regulating the concentrations in ECF and are especially important in the maintenance of low concentrations of neurotransmitter amino acids such as glutamate, aspartate, and glycine. On the other hand, it now seems clear that the BBB also participates in the active regulation of brain ECF and that the abluminal membrane is especially important in this role.

The emerging view is that cerebral endothelial cells participate actively in regulating the composition of brain ECF and the amino acid content of brain. The two membranes seem to be working in a complementary fashion, with Na+-dependent transport of amino acids occurring at the abluminal membrane and facilitative transport at the luminal or, in the case of large neutral amino acids, at both membranes (18).


    DISCLOSURES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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This work was supported by National Institute of Neurological Disorders and Stroke Grant no. NS-041405.


    ACKNOWLEDGMENTS
 
We thank M. R. DeJoseph and A. Mokashi for excellent technical assistance. We are also grateful to Drs. Juan Viña, Ian Simpson, and Héctor Rasgado-Flores for constructive criticism.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. A. Hawkins, Dept. of Physiology and Biophysics, Finch University of Health Sciences/The Chicago Medical School, 3333 Green Bay Rd., North Chicago, IL 60064–3095 (E-mail: RAH{at}finchcms.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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 REFERENCES
 

  1. Battistin L, Grynbaum A, and Lajtha A. The uptake of various amino acids by the mouse brain in vivo. Brain Res 29: 85–99, 1971.[ISI][Medline]
  2. Betz AL, Firth JA, and Goldstein GW. Polarity of the blood-brain barrier: distribution of enzymes between the luminal and antiluminal membranes of brain capillary endothelial cells. Brain Res 192: 17–28, 1980.[ISI][Medline]
  3. Betz AL and Goldstein GW. Polarity of the blood-brain barrier: neutral amino acid transport into isolated brain capillaries. Science 202: 225–226, 1978.[ISI][Medline]
  4. Boado RJ, Li JY, Nagaya M, Zhang C, and Pardridge WM. Selective expression of the large neutral amino acid transporter at the blood-brain barrier. Proc Natl Acad Sci USA 96: 12079–12084, 1999.[Abstract/Free Full Text]
  5. Bradbury M. The Concept of a Blood-Brain Barrier. New York: Wiley, 1979.
  6. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of dye binding. Anal Biochem 72: 248–254, 1976.[ISI][Medline]
  7. Brightman MW and Reese TW. Junctions between intimately apposed cell membranes in the vertebrate brain. J Cell Biol 40: 648–677, 1969.[Abstract/Free Full Text]
  8. Broer S. Molecular identification of astroglial neutral amino acid transport systems. Dev Neurosci 18: 484–491, 1996.[ISI][Medline]
  9. Broer S and Brookes N. Transfer of glutamine between astrocytes and neurons. J Neurochem 77: 705–719, 2001.[ISI][Medline]
  10. Davson H and Welch K. The relations of blood, brain and cerebrospinal fluid. In: Ion Homeostasis of the Brain, edited by Siesjo BK and Sorensen SC. Copenhagen: Munksgaard, 1971, p. 9–21.
  11. Deves R, Angelo S, and Rojas AM. System y+L: the broad scope and cation modulated amino acid transporter. Exp Physiol 83: 211–220, 1998.[Abstract]
  12. Deves R, Chavez P, and Boyd CA. Identification of a new transport system (y+L) in human erythrocytes that recognizes lysine and leucine with high affinity. J Physiol 454: 491–501, 1992.[Abstract]
  13. Drewes LR, Conway WP, and Gilboe DD. Net amino acid transport between plasma and erythrocytes and perfused dog brain. Am J Physiol Endocrinol Metab Gastrointest Physiol 233: E320–E325, 1977.[Free Full Text]
  14. Farinelli SE and Nicklas WJ. Glutamate metabolism in rat cortical astrocyte cultures. J Neurochem 58: 1905–1915, 1992.[ISI][Medline]
  15. Felig P, Wahren J, and Ahlborg G. Uptake of individual amino acids by the human brain. Proc Soc Exp Biol Med 142: 230–231, 1973.
  16. Fernstrom JD and Wurtman RJ. Brain serotonin content: physiological regulation by plasma neutral amino acids. Science 178: 414–416, 1972.[ISI][Medline]
  17. Hargreaves KM and Pardridge WM. Neutral amino acid transport at the human blood-brain barrier. J Biol Chem 263: 19392–19397, 1988.[Abstract/Free Full Text]
  18. Hawkins RA, Peterson DR, and Viña JR. The complementary membranes forming the blood-brain barrier. IUBMB Life 54: 101–107, 2002.[ISI][Medline]
  19. Heinz E and Weinstein AM. The overshoot phenomenon in cotransport. Biochim Biophys Acta 776: 83–91, 1984.[ISI][Medline]
  20. Hille B. Ionic Channels of Excitable Membranes. Sunderland, MA: Sinauer Associates, 1992.
  21. Hoshino T and Kageyama M. Sodium-dependent transport of L-leucine in membrane vesicles prepared from Pseudomonas aeruginosa. J Bacteriol 137: 73–81, 1979.[ISI][Medline]
  22. Hutchison HT, Eisenberg HM, and Haber B. High-affinity transport of glutamate in rat brain microvessels. Exp Neurol 87: 260–269, 1985.[ISI][Medline]
  23. Jorgensen KE, Kragh-Hansen U, and Sheikh MI. Transport of leucine, isoleucine, and valine by luminal membrane vesicles from rabbit proximal tubule. J Physiol 422: 41–54, 1990.[Abstract]
  24. Kanai Y, Segawa H, Miyamoto K, Uchino H, Takeda E, and Endou H. Expression cloning and characterization of a transporter for large neutral amino acids activated by the heavy chain of 4F2 antigen (CD98). J Biol Chem 273: 23629–23632, 1998.[Abstract/Free Full Text]
  25. Killian DM and Chikhale PJ. Predominant functional activity of the large, neutral amino acid transporter (LAT1) isoform at the cerebrovasculature. Neurosci Lett 306: 1–4, 2001.[ISI][Medline]
  26. Kruse T, Reiber H, and Neuhoff V. Amino acid transport across the human blood-CSF barrier. J Neurol Sci 70: 129–138, 1985.[ISI][Medline]
  27. Lee WJ, Hawkins RA, Viña JR, and Peterson DR. Glutamine transport by the blood-brain barrier: a possible mechanism for nitrogen removal. Am J Physiol Cell Physiol 274: C1101–C1107, 1998.[Abstract/Free Full Text]
  28. Martinez M, Arnalich F, Vazquez JJ, and Hernanz A. Altered cerebrospinal fluid amino acid pattern in the anorexia of aging: relationship with biogenic amine metabolism. Life Sci 53: 1643–1650, 1993.[ISI][Medline]
  29. Miralles VJ, Martinez-Lopez I, Zaragoza R, Borras E, Garcia C, Pallardo FV, and Viña JR. Na+ dependent glutamate transporters (EAAT1, EAAT2, and EAAT3) in primary astrocyte cultures: effect of oxidative stress. Brain Res 922: 21–29, 2001.[ISI][Medline]
  30. O'Kane R. A Study on the Amino Acid Transporters of the Blood-Brain Barrier (PhD thesis). North Chicago, IL: Finch University of Health Sciences/The Chicago Medical School, 2000.
  31. O'Kane RL, Martinez-Lopez I, DeJoseph MR, Viña JR, and Hawkins RA. Na(+)-dependent glutamate transporters (EAAT1, EAAT2, and EAAT3) of the blood-brain barrier. A mechanism for glutamate removal. J Biol Chem 274: 31891–31895, 1999.[Abstract/Free Full Text]
  32. Oldendorf WH. Uptake of radiolabeled essential amino acids by brain following arterial injection. Proc Soc Exp Biol Med 136: 385–386, 1971.
  33. Oldendorf WH and Szabo J. Amino acid assignment to one of three blood-brain barrier amino acid carriers. Am J Physiol 230: 94–98, 1976.[Abstract/Free Full Text]
  34. Oxender DL and Christensen HN. Distinct mediating systems for the transport of neutral amino acids by the Ehrlich cell. J Biol Chem 238: 3686–3699, 1963.[Free Full Text]
  35. Palacin M, Estevez R, Bertran J, and Zorzano A. Molecular biology of mammalian plasma membrane amino acid transporters. Physiol Rev 78: 969–1054, 1998.[Abstract/Free Full Text]
  36. Pardridge WM. Brain Drug Targeting. Cambridge, UK: Cambridge Univ. Press, 2001.
  37. Pardridge WM, Eisenberg J, and Yamada T. Rapid sequestration and degradation of somatostatin analogues by isolated brain microvessels. J Neurochem 44: 1178–1184, 1985.[ISI][Medline]
  38. Pollard M, Meredith D, and McGivan JD. Characterisation and cloning of a Na(+)-dependent broad-specificity neutral amino acid transporter from NBL-1 cells: a novel member of the ASC/B(0) transporter family. Biochim Biophys Acta 1561: 202–208, 2002.[ISI][Medline]
  39. Sacks W, Sacks S, Brebbia DR, and Fleischer A. Cerebral uptake of amino acids in human subjects and rhesus monkeys in vivo. J Neurosci Res 7: 431–436, 1982.[ISI][Medline]
  40. Sánchez del Pino MM, Hawkins RA, and Peterson DR. Neutral amino acid transport by the blood-brain barrier: membrane vesicle studies. J Biol Chem 267: 25951–25957, 1992.[Abstract/Free Full Text]
  41. Sánchez del Pino MM, Peterson DR, and Hawkins RA. Neutral amino acid transport characterization of isolated luminal and abluminal membranes of the blood-brain barrier. J Biol Chem 270: 14913–14918, 1995.[Abstract/Free Full Text]
  42. Schousboe A. Role of astrocytes in the maintenance and modulation of glutamatergic and GABAergic neurotransmission. Neurochem Res 28: 347–352, 2003.[ISI][Medline]
  43. Segawa H, Fukasawa Y, Miyamoto K, Takeda E, Endou H, and Kanai Y. Identification and functional characterization of a Na+-independent neutral amino acid transporter with broad substrate selectivity. J Biol Chem 274: 19745–19751, 1999.[Abstract/Free Full Text]
  44. Shaw PJ, Forrest V, Ince PG, Richardson JP, and Wastell HJ. CSF and plasma amino acid levels in motor neuron disease: elevation of CSF glutamate in a subset of patients. Neurodegeneration 4: 209–216, 1995.[ISI][Medline]
  45. Smith QR, Momma S, Aoyagi M, and Rapoport SI. Kinetics of neutral amino acid transport across the blood-brain barrier. J Neurochem 49: 1651–1658, 1987.[ISI][Medline]
  46. Smith QR and Stoll J. Blood-brain barrier amino acid transport. In: Introduction to the Blood-Brain Barrier: Methodology, Biology, and Pathology, edited by Pardridge WM. Cambridge, UK: Cambridge Univ. Press, 1998, p. 188–197.
  47. Tayarani I, Lefauconnier J-M, Roux F, and Bourrer J-M. Evidence for alanine, serine, and cystine system of transport in isolated brain capillaries. J Cereb Blood Flow Metab 7: 585–591, 1987.[ISI][Medline]
  48. Tewes BJ and Galla H-J. Lipid polarity in brain capillary endothelial cells. Endothelium 8: 207–220, 2001.[Medline]
  49. Tossman U, Jonsson G, and Ungerstedt U. Regional distribution and extracellular levels of amino acids in rat central nervous system. Acta Physiol Scand 127: 533–545, 1986.[ISI][Medline]
  50. Tovar A, Tews JK, Torres N, and Harper AE. Some characteristics of the threonine transport across the blood-brain barrier of the rat. J Neurochem 51: 1285–1293, 1988.[ISI][Medline]
  51. Van Meer G, Gumbiner B, and Simons K. The tight junction does not allow lipid molecules to diffuse from one epithelial cell to the next. Nature 322: 639–641, 1986.[ISI][Medline]
  52. Van Meer G and Simons K. The function of tight junctions in maintaining differences in lipid composition between the apical and the basolateral cell surface domains of MDCK cells. EMBO J 5: 1455–1464, 1986.[Abstract]
  53. Van Winkle L, Christensen H, and Campione A. Na+-dependent transport of basic, zwitterionic, and bicyclic amino acids by a broad-scope system in mouse blastocysts. J Biol Chem 260: 12118–12123, 1985.[Abstract/Free Full Text]