From the
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
The ease with which a solute moves across the BBB
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
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
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
To determine the contribution of known
Na
The results establish the following points: 1) transport of
neutral amino acids across luminal membranes of brain endothelial cells
is Na
A
Na
The high affinity
system L1 is present at the BBB. The same substrate specificity was
observed in luminal and abluminal membranes. The K
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
The major
Na
A second Na
System ASC, if present, does not represent
a significant component of the Na
In conclusion, the results
indicate that transport system L1, characterized by its Na
The initial rate of uptake (±S.E.) was calculated as
the value of the derivative of the equations indicated in Fig. 1: V
When substrate (in parenthesis) and
inhibitor are the same amino acid, 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.
(
)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.
-dependent transport of
phenylalanine and MeAIB was observed in the other membrane population,
apparently derived from the abluminal domain(4) .
-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.
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, , 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) .
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.
Na
To determine the presence of
NaDependence of
Uptake
-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
Kinetic studies were conducted on both membranes to
determine whether the observed Na-independent Transport
System
-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
Since the Na-dependent Transport
System
-dependent transport
activity was located exclusively in abluminal membranes, the
characterization experiments were carried out using only abluminal
membrane-rich fractions.
-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.
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.
-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.
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.
-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.
-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 K
values 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.
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.
-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.
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
= Ak or V
= A
k
- A
k
. 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
values are given. All others indicate K
values. The values were obtained from the experiments shown
in Figs. 2-5.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.