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
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ABSTRACT |
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active transport; brain extracellular fluid; capillaries; endothelial cells; essential amino acids
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.
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MATERIALS AND METHODS |
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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 -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
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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.
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RESULTS AND DISCUSSION |
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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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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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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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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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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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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 · mg1 · min1 and a PSA value of 5.4 µl · mg1 · min1 (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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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 630 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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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), EAAT13 (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).
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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