©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Neutral Amino Acid Transport Characterization of Isolated Luminal and Abluminal Membranes of the Blood-Brain Barrier (*)

Manuel M. Sánchez del Pino (§) , Darryl R. Peterson , Richard A. Hawkins

From the (1)Department of Physiology and Biophysics, Finch University of Health Sciences, The Chicago Medical School, North Chicago, Illinois 60064

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The neutral amino acid carrier composition of luminal and abluminal membranes of the blood-brain barrier has been studied using isolated membrane vesicles. Phenylalanine was carried almost exclusively by a high affinity (K = 10 ± 2 µM), Na-independent amino acid transport system, presumably L1 system, that was found to be symmetrically distributed between luminal and abluminal membranes. Inhibition of phenylalanine uptake was used to determine the affinities (K values) toward leucine (17 ± 3 µM), tryptophan (8 ± 1), 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BCH) (11 ± 2), alanine (628 ± 117), and glutamine (228 ± 51). Alanine was found to be transported by two Na-dependent transport systems that were located exclusively on the abluminal membrane. Kinetic and inhibition experiments indicated that one of these activities was due to system A, which is probably the main route for Na-dependent alanine transport (K = 0.6 ± 0.2 mM) under physiological conditions. The other Na-dependent activity was attributed to a B-like system based on its sensitivity toward BCH. This latter system showed greater affinity for large neutral amino acids. The affinities of these two transport systems for several other amino acids were also studied.


INTRODUCTION

The ease with which a solute moves across the BBB()is determined by the presence and abundance of carrier proteins in the endothelial cell membranes capable of transporting it. The presence of tight junctions that separate the plasma membrane into luminal and abluminal domains results in structural and functional polarity. Transport polarity is probably a key feature of BBB function. An asymmetrical distribution of transporters would permit vectorial transport capable of creating and sustaining ionic and metabolite gradients.

Although the importance of transport across the BBB is clear, the available information regarding the transport systems involved and the function of this barrier is small compared with that of the intestine and kidney epithelia. The reason for this lack of knowledge derives mainly from inadequate techniques available to study transport across the BBB. Most of the transport studies have been performed using animal models in vivo, which presents limitations for the detailed investigation of the BBB at the cellular level(1) .

Transport studies using isolated membrane vesicles offer considerable additional information that cannot be obtained with intact cells or tissues. This methodology provides an opportunity to study the carrier composition and kinetics of the respective membranes separately(2, 3) . We have recently shown that membrane vesicles can be isolated from brain endothelial cells and that they are adequate to study transport (4). Two membrane vesicle populations with different transport characteristics were separated. Transport of phenylalanine into one membrane population, probably derived from the luminal domain, showed similar transport characteristics as those observed in experiments in vivo, whereas Na-dependent transport of phenylalanine and MeAIB was observed in the other membrane population, apparently derived from the abluminal domain(4) .

System L for large neutral amino acids is often assumed to be present in both membranes of brain endothelial cells(4, 5, 6, 7) . To what extent this assumption is justified remains to be examined. Conversely, the Na-dependent systems are believed to be restricted to the abluminal side(4, 5, 8) . The purpose of the following experiments was to extend the characterization of neutral amino acid transport initiated in our laboratory (4) and to determine the distribution and kinetic properties of the pertinent neutral amino acid transport systems in luminal and abluminal membranes.


EXPERIMENTAL PROCEDURES

Materials

N-(Methylamino)[1-C]isobutyric acid (48.4-56.3 mCi/mmol), and [U-C]sucrose (475 mCi/mmol) were purchased from DuPont NEN. L-[U-C]Alanine (153-158 mCi/mmol) and L-[U-C]phenylalanine (513 mCi/mmol) were obtained from Amersham Corp. Collagenase type IA and cytochrome c type IV were obtained from Sigma. Bio-Rad protein assay was purchased from Bio-Rad.

Isolation of Endothelial Cell Membranes

Bovine brain microvessels and endothelial cell membranes were prepared as previously described(9) . The final product of the isolation procedure is five fractions obtained in a discontinuous Ficoll gradient. The fractions corresponding to the 0/5 and 10/15% Ficoll are considered as the luminal and abluminal membrane-rich fractions, respectively(9) .

Uptake Measurements

Membrane vesicle aliquots were incubated in ice-cold storage buffer (290 mM mannitol and 10 mM HEPES-Tris, pH 7.4), after which they were centrifuged at 37,500 g for 25 min at 4 °C. The supernatant was removed, and the pellet was resuspended in storage buffer at a concentration between 2.5 and 5 mg/ml. The membrane vesicle suspension was divided into 10-µl aliquots, one of which was saved to determine the protein concentration. The reaction was started by adding 10 µl of reaction media (see below) to the membrane suspension. After incubation at 37 °C for the specified time, the reaction was stopped by diluting with 1 ml of an ice-cold stopping solution containing 145 mM NaCl and 10 mM HEPES-Tris, pH 7.4, followed by filtration over a 0.45-µm Gelman Metricel filter. The filters were rinsed four times with 1 ml of the ice-cold stopping solution, and the retained radioactivity was counted by liquid scintillation spectroscopy. To correct for retention of radiolabeled material by the filter, control samples containing the same amount of radioactivity but without membranes were filtered as indicated above. In the kinetic and inhibition experiments, where the initial rate of uptake was measured, the incubation time was 15 s.

Solutions

The intravesicular solution used in all transport experiments was storage buffer (290 mM mannitol and 10 mM HEPES-Tris, pH 7.4). A transient swelling of membrane vesicles may occur when impermeant solutes in the medium are replaced by more permeant ones at the start of the experiment(10) . This transient change in vesicle volume would cause an artificial overshoot, where the substrate concentration appears to be temporally higher in the intravesicular space than in the extravesicular space. To prevent this effect, the extravesicular solution was storage buffer supplemented with KCl or NaCl and the appropriate radiolabeled substrate, as indicated in the figure legends, with no mannitol replacement.

Membrane Distribution of Transport Systems

The fraction of a transport system located on the luminal membrane, f, was calculated as previously described(9) .

Kinetic Analysis

The activity of each transport system was fitted to the following equation,

On-line formulae not verified for accuracy

where [R] is the concentration of the labeled substrate, [I] is the concentration of the competing amino acid, and V, K, and K have their usual meanings. In Na-independent transport experiments, the non-saturable component was determined by measuring the uptake in the presence of an excess of unlabeled substrate. In the case of Na-dependent transport experiments, the non-saturable component was determined by measuring the uptake in the absence of Na. The nature of the non-saturable component is not known, but it is probably due to diffusion as well as binding.

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(11) .


RESULTS

Membrane Vesicle Isolation

The characteristics of the isolated membrane vesicles have been previously reported(4, 9) . The luminal and abluminal membrane-rich fractions provided an adequate in vitro system for the assignment of transport activities associated with luminal and abluminal domains, as indicated in the accompanying paper(9) .

Diffusion

As a control for simple diffusion and to assess the integrity of the vesicles, the uptake of [C]sucrose was measured (). The initial rate of the permeability surface area product (clearance) for sucrose, in the absence of Na, was much lower than transport of metabolites, indicating that simple diffusion was a minor component of uptake at short incubation times and that the vesicles were not leaky (). However, the initial rate of the permeability surface area product for sucrose was similar to that of MeAIB in the absence of Na (). This result indicates that MeAIB is not carried by any transport system when Na is not present and that its uptake can be considered also as a measurement of diffusion.

NaDependence of Uptake

To determine the presence of Na-driven amino acid transport processes, time-course experiments in the presence and absence of inwardly directed Na gradients were performed (Fig. 1). Uptake of alanine, phenylalanine, and MeAIB was measured under these conditions. The initial rates of uptake were calculated and are shown in .


Figure 1: Na dependence of amino acid uptake. Uptake of 100 µM [C]MeAIB, 20 µM [C]alanine, and 10 µM [C]phenylalanine was measured in the presence of 100 mM KCl (opensymbols) or 100 mM NaCl (filledsymbols). Circles, luminal membrane-rich vesicles; triangles, abluminal membrane-rich vesicles. Uptake was normalized by the uptake at equilibrium, measured at 60 min. The data were fitted to a single exponential equation, U = A(1 - e), or a two exponential equation, U = A(1 - e



The data indicate that there was a clear stimulation of alanine and MeAIB uptake in the presence of a Na gradient. This stimulation was higher in the abluminal membrane-rich fractions where a clear overshoot, indicative of a cotransport process, was observed (Fig. 1). In the case of phenylalanine uptake, the Na effect was small. The results indicate that, under our experimental conditions, most alanine and MeAIB enter into the membrane vesicles by a concentrative, Na-driven transport system. Conversely, a facilitated diffusion process seems to be more important for phenylalanine transport, at least at low concentrations.

Location of the Amino Acid Transport Systems

The above results indicate that the Na-dependent transport activities are present to a greater extent in the abluminal membrane-rich fraction, whereas the luminal membrane-rich fraction contains a higher proportion of Na-independent transport activity. The f values, a measurement of the fraction of carriers located in the luminal membrane, for the major transport processes were (±S.E.) 0 ± 0.01 and 0.03 ± 0.01 for the Na-dependent uptake of MeAIB and alanine, respectively, and 0.44 ± 0.02 for the Na-independent uptake of phenylalanine. These results indicate that Na-dependent transport is present only in abluminal membranes, whereas a facilitated transport system is equally present in both membranes. The Na stimulation observed in luminal membrane-rich fractions was completely accounted for by abluminal vesicle contamination.

Characterization of the Na-independent Transport System

Kinetic studies were conducted on both membranes to determine whether the observed Na-independent transport systems in luminal and abluminal vesicles are the same. The Na-independent uptake of 10 µM [C]phenylalanine was measured in the presence of unlabeled phenylalanine, as well as other potential competing amino acids. To compare the two membrane populations, the percentage of the saturable transport activity remaining in the presence of inhibitor was expressed as a function of the inhibitor concentration (Fig. 2). No statistically significantly difference was observed between luminal and abluminal membrane-rich fractions (significance level of 0.05), indicating that both membranes contain the same transporter. The values of K and K, calculated for luminal and abluminal membranes together, are indicated in . The f value calculated from the maximum velocities (383 ± 81 and 106 ± 25 pmol min mg for luminal and abluminal, respectively) was 0.62 ± 0.03, indicating that the transport system was evenly distributed in both membranes.


Figure 2: Inhibition of the Na-independent transport of phenylalanine by competing amino acids. The initial rate of [C]phenylalanine uptake (10 µM) in the presence of the indicated competing amino acids was measured in the absence of Na. The activity is expressed as the percentage of the saturable activity measured in the absence of inhibitor. Opensymbols, luminal membrane-rich vesicles; filledsymbols, abluminal membrane-rich vesicles. The data were fitted to a single saturable component (Equation 1 under ``Experimental Procedures''). The parameters are indicated in Table II.



Characterization of the Na-dependent Transport System

Since the Na-dependent transport activity was located exclusively in abluminal membranes, the characterization experiments were carried out using only abluminal membrane-rich fractions.

To determine the contribution of known Na-dependent transport systems such as A, ASC, and B, the Na-dependent uptake of [C]alanine was measured in the presence of different concentrations of unlabeled MeAIB and BCH, an inhibitor of system B. The rationale of these experiments is that the MeAIB-sensitive and BCH-sensitive uptake of alanine can be attributed to systems A and B, respectively, and the remaining Na-dependent alanine uptake, not inhibited by MeAIB or BCH, can be ascribed to system ASC. Although MeAIB and BCH inhibited only partially alanine uptake, together they accounted for all Na-dependent uptake of alanine (Figs. 3A and 5), indicating the absence of system ASC. Since systems A and B were the two major components of uptake, the MeAIB-sensitive fraction in Fig. 3B was considered system A activity, whereas the remaining Na-dependent uptake was attributed to system B. The calculated V values for systems A and B were 2.9 ± 0.5 and 3.7 ± 0.9 nmol min mg, respectively. The K values for both systems are indicated in .


Figure 3: Alanine uptake into abluminal membrane-rich vesicles as a function of the substrate concentration. The initial rate of [C]alanine uptake into abluminal membrane-rich vesicles was measured in the presence of 100 mM KCl or NaCl. A, the transport activity inhibited by MeAIB and BCH was attributed to systems A and B, respectively. These activities account for all Na-dependent transport of alanine. B, the transport activity inhibited by MeAIB was attributed to system A, whereas the remaining Na-dependent, MeAIB-resistant activity was considered to be mediated by system B. The data were fitted to two Na-dependent saturable systems (Equation 1 under ``Experimental Procedures'') and a Na-independent non-saturable component. The parameters are indicated in Table II.



To further characterize the properties of the two Na-dependent transport systems, the uptake of radiolabeled MeAIB or alanine was measured in the presence of competing amino acids ( Fig. 4and Fig. 5). In the absence of Na, no saturable uptake of MeAIB or alanine was detected (Fig. 3, 4A, and 5). The Na-dependent uptake of MeAIB was competitively and completely inhibited by alanine (Fig. 4A, inset, and Fig. 4B). Conversely, uptake of alanine was only partially inhibited by MeAIB or BCH (Fig. 5), indicating that system A is not the only route for alanine uptake. The alanine uptake blocked by MeAIB and BCH, as also shown in Fig. 3A, accounted for all Na-dependent transport of alanine. At the alanine concentration used in these experiments (50 µM), about 65% of alanine uptake is MeAIB-sensitive, 24% is BCH-sensitive, and 11% is non-saturable Na-independent uptake. The K and K values obtained are shown in .


Figure 4: Inhibition of MeAIB uptake by competing amino acids. A, the initial rate of [C]MeAIB uptake (100 µM) was determined in abluminal membrane-rich vesicles in the presence of the indicated concentrations of unlabeled MeAIB and 0 (filledcircles), 0.375 (opentriangles), 0.75 (filledtriangles), and 1.5 (opensquares) mM alanine in the presence of 100 mM NaCl or KCl (opencircles). Inset, Lineweaver-Burk plot of the Na-dependent component. B, the initial rate of Na-dependent uptake of [C]MeAIB (100 µM) was determined in abluminal membrane-rich vesicles in the presence of different concentrations of the indicated competing amino acids. The activity is expressed as the percentage of the Na-dependent activity measured in the absence of inhibitor. The data were fitted to a single saturable component (Equation 1 under ``Experimental Procedures''). The parameters are indicated in Table II.




Figure 5: Inhibition of alanine uptake by competing amino acids. The initial rate of [C]alanine uptake (50 µM) was determined in abluminal rich membrane vesicles in the presence of 100 mM NaCl or KCl (brokenline). The activity is expressed as the percentage of the activity measured in the absence of inhibitor. The activity inhibited by MeAIB and BCH accounted for 65 and 24%, respectively. The data were fitted to two Na-dependent saturable systems (Equation 1 under ``Experimental Procedures'') and a Na-independent non-saturable component. The parameters are indicated in Table II.




DISCUSSION

The results establish the following points: 1) transport of neutral amino acids across luminal membranes of brain endothelial cells is Na independent; 2) the high affinity form of system L, the so-called L1 form(12, 13) , is symmetrically distributed between the luminal and abluminal membranes of the BBB; 3) the Na-dependent system A is present only at the abluminal side of the endothelial cell and represents the major component of alanine uptake; and 4) a minor Na-dependent component is also present in abluminal membranes, attributed to a B-like system based on its sensitivity to the amino acid analog BCH.

A Na-independent transport system is symmetrically distributed between luminal and abluminal membranes of the BBB. The results presented here as well as those previously reported (4) indicate that neutral amino acids cross the luminal membranes of brain endothelial cells through a facilitative diffusion system. The preference of this carrier for large amino acids suggests that the measured activity may be attributed to system L, which agrees with the reported results using techniques in vivo(13, 14, 15) . It has been proposed that system L is also present in abluminal membranes, although no direct evidence exists(16) . The results obtained in this study indicate that system L activity is symmetrically distributed between the two membranes of brain endothelial cells.

The high affinity system L1 is present at the BBB. The same substrate specificity was observed in luminal and abluminal membranes. The K values found for phenylalanine, tryptophan, and leucine were 10 ± 2, 8 ± 1, and 17 ± 3 µM, respectively. These values are close to those reported in experiments performed in vivo(13, 15) . This high affinity is the reason to postulate that the L1 form is present in brain endothelial cells (13), and the results presented here support this idea. The affinity for alanine and glutamine, however, is higher than that observed by Smith et al.(13) . This discrepancy may be explained by the more complicated situation in vivo where the kinetics measured represent two membranes in series.

System T was not detected at the BBB. System T is a transport system specific for aromatic amino acids(17, 18) . This system does not carry leucine and is not inhibited by leucine(17, 18) . The ability of leucine to inhibit completely the uptake of phenylalanine (Fig. 2) indicates that system T, if present, is not a major component of the brain endothelial cell membranes.

Na-dependent transport of neutral amino acids takes place exclusively at the abluminal membrane of brain endothelial cells. The ability of abluminal membrane vesicles to concentrate amino acids in the presence of an inwardly directed Na gradient is demonstrated in Fig. 1by the presence of an overshoot. When the analysis described in the accompanying paper (9) was applied to the Na-dependent transport of alanine and MeAIB, the results indicate that the transporters responsible for these processes are detected only at the abluminal side of the BBB.

The major Na-driven transport of alanine can be attributed to system A. Our results indicate that alanine and MeAIB share a transport system with similar kinetic properties to those reported by Christensen et al.(19) for system A. First, alanine competitively inhibited transport of MeAIB. Second, the K values for alanine and MeAIB are similar to their K values for inhibition of the transport of each other. Third, the phenylalanine and glutamine Kvalues for MeAIB transport are similar to the values for the MeAIB-sensitive alanine transport. At an alanine concentration of 50 µM, system A accounts for 65% of the uptake.

A second Na cotransporter, a B-like system, is also present in abluminal membranes of brain endothelial cells. Transport by system B is Na dependent and BCH sensitive and has been observed in endothelial cells(20) . There are some differences, however, between the transport system observed in this study and that described by Van Winkle et al.(21) . For instance, the affinity for alanine is in the millimolar range, whereas that reported by Van Winkle et al.(21, 22) for system B is in the micromolar range. The affinity for BCH is higher than that for alanine, contrary to what was observed in mouse conceptuses(22) . It seems that the system observed in abluminal membrane-rich membrane vesicles favors large neutral amino acids. The concentration of small neutral amino acids in the cerebral extracellular fluid is in the submicromolar range(23) . Thus, our results suggest that system A is the main carrier of these amino acids under physiological conditions, unless a very high affinity and low capacity system, which might have been undetected in our experiments, is present.

System ASC, if present, does not represent a significant component of the Na-dependent uptake of alanine. MeAIB- and BCH-sensitive activities accounted for all Na-dependent uptake of alanine. This result is different from that reported by Tayarani et al.(8) using isolated rat brain capillaries. However, these authors attributed to system ASC all MeAIB-resistant activity, which is not always correct. In fact, they showed almost as much inhibition of the Na-dependent alanine uptake by BCH as by alanine itself, indicating that an activity similar to system B was present. Nevertheless, the transport properties observed by these authors (8) differ from those presented here, which may be due to different animal species. Tovar et al.(24) also described the presence of the ASC system in experiments in vivo, based on its MeAIB resistance. However, Cornford et al.(25) were unable to detect any ASC system activity using the same technique. These discrepant results might be explained if a small component of the uptake measured by Tovar et al.(24) would be due to uptake into red blood cells for which they did not present any control.

In conclusion, the results indicate that transport system L1, characterized by its Na independence and high affinity toward large neutral amino acids, is symmetrically distributed between luminal and abluminal membranes of the brain endothelial cells. In contrast, two Na-dependent transport systems are located exclusively on the abluminal membrane. Transport system A is characterized by its preference toward small neutral amino acids and its ability to transport the analog MeAIB, whereas the transport activity of a B-like system is inhibited by the analog BCH and seems to favor large neutral amino acids.

  
Table: Initial rate of permeability-surface area product

The initial rate of uptake (±S.E.) was calculated as the value of the derivative of the equations indicated in Fig. 1: V= Ak or V = Ak - Ak. The permeability-surface area product (clearance) values were obtained by dividing the initial rate of uptake by the substrate concentration.


  
Table: K or K values (±S.E.) for the different transport systems

When substrate (in parenthesis) and inhibitor are the same amino acid, K values are given. All others indicate K values. The values were obtained from the experiments shown in Figs. 2-5.



FOOTNOTES

*
This work was supported by Grants NS 16389 and DK42331 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: MRC Unit for Protein Function and Design, Dept. of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, United Kingdom.

The abbreviations used are: BBB, blood-brain barrier; BCH, 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid; MeAIB, N-(methylamino)-isobutyric acid.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.