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Hexose transport
and phosphorylation
capillaries
isolated from rat brain
A. LORRIS BETZ, JUDIT CSEJTEY,
AND GARY
Departments
of Neurology and Pediatrics, University
San Francisco,
California
94143
by
W. GOLDSTEIN
of California School
of Medicine,
tubular segments of metabolically active microvessels
(19) exhibiting energy-dependent rubidium transport
(17) and Na+-dependent and Na+-independent amino
acid transport (9>. Thus, this preparation is useful for
the study of transport and metabolism in brain capillaries and provides a valuable tool for further characterization of the BBB.
Glucose transport across the BBB has been well
studied in vivo (3-8, 10-12, 20, 27-B). An earlier report
from our laboratory on sugar uptake into brain capillaries in vitro (18) described an apparent transport system
with much higher affinity and different stereospecificity
than is reported for the BBB transport of glucose in
vivo. However, this study did not adequately distinguish between transport and subsequent phosphorylation of the sugar. The present study further characterizes glucose transport into brain capillary endothelial
cells and examines the relationship between sugar
transport and phosphorylation.
blood-brain
barrier; glucose; Z-deoxyglucose;
3-O-methylglucase; endothelial
cells; cytechalasin
B; phloretin;
phlorizin;
accelerative exchange diffusion; glucose transport stereospecCity
Isolation of capillaries. The method used for isolation
of capillary segments from rat brain was a minor
modification of the one described previously
(19)
Twenty to forty Sprague-Dawley rats, 30-days old and
weighing loo-125 g, were killed by cervical dislocation.
The brains were immediately removed and placed in a
buffer consisting of oxygen-saturated Ringer solution
with 1.2 mM MgCl,!, 15 mM N-2-hydroxy-ethylpiperazine-N’-2-ethanesulfonic
acid (HEPES), pH 7.4, 5 mM
sodium pyruvate, and 1 % fraction V bovine serum
albumin. The brainstem cerebellum, and meninges
were discarded and cortical shells, free of choroid plexus
and ependyma, were minced in btier. A 10% (wt/vol>
homogenate was made using 20 up-and-down strokes in
a Teflon and glass homogenizer (0.25 mm clearance) at
390 rpm. The homogenate was centrifuged at 1,000 x g
for 10 min. To remove cellular debris and myelin, the
pellet was resuspended to a concentration of 16% (wt/
vol) in the same buffer now containing 25% bovine
serum albumin and centrifuged at 1,000 x g for 15 min.
The new pellet consisted of a mixture of free nuclei and
capillary segments of various sizes. To obtain a. more
uniform suspension of capillaries, the pellet was resuspended in buffer and then passed through a 118~pm
nylon mesh under gentle suction. The capil laries were
separated from nuclei by passing the suspension
of a selective permeability barrier between blood and brain is well established. Although the
cells or structures that constitute this blood-brain barrier (BBB) are not known with certainty, many investigators propose that the capillary endothelial cells
limit movement of solutes into the brain This hypothesis is based on ultrastructural
studies showing that
brain capillaries form a continuous layer of endothelial
cells joined together by tight junctions (31). Furthermore, when macromolecules such as horseradish peroxidase (31) or microperoxidase (30) are injected into the
bloodstream, they penetrate up to, but not through, the
tight junctional complexes. However, there is no direct
evidence that the endothelial cells are responsible for
the selective cerebral uptake of low-molecular-weight
compounds.
The recent development of methods for isolating brain
capillaries (19) offers an opportunity for the in vitro
study of cells that are the probable site of the BBB in
vivo. The isolated capillary preparation consists of short
THE EXISTENCE
C96
MATERIALS
0363-6143/79/0000-0000$01.25
AND METHODS
l
Copyright
0
1979 the American
Physiological
Society
Downloaded from http://ajpcell.physiology.org/ by 10.220.32.247 on June 17, 2017
BETZ, A. LORRIS, JUDIT~SEJTEY,
ANDGARY
W. GOLDSTEIN.
Hexose transport and phosphoryla tion by capillaries
isolated
from
rat brain. Am. J. Physiol. 236(l): C96-ClO2, 1979 or
Am. J. Physiol.: Cell Physiol. 5(l): C96-C102, 1979, -Hexose
transport and phosphorylation
were studied in capillary segments isolated from rat brain. Uptake of 3-@methyl-n-glucose
(3MG) could be inhibited
by cytochalasin
13, phloretin,
and
phlorizin,
but not by 2,4-dinitrophenol
or ouabain. 2-Deoxy-nglucose (2DG), D-ghCOSe,
galactose, and mannose inhibited
3MG uptake, while L-glucose, fructose, and ribose did not.
Accelerative
exchange diffusion of 3MG was demonstrated.
At
equilibrium,
the intracellular
concentration
of hexose did not
exceed the external concentration,
and transport was, therefore, equilibrative
rather than accumulative.
Transport
of
2DG and n-glucose was not rate limiting
for metabolism.
When incubated
in 5 mM n-glucose, the endothelial
cells
contained
a large pool of free glucose. L-Glucose entered
capillaries
more slowly than other hexoses and served as a
marker
for simple diffusion
of sugars into the cells. Our
results suggest that sugar uptake into isolated brain capillaries occurs by a transport system similar to the one responsible
for glucose transport across the blood-brain
barrier in vivo.
SUGAR
TRANSPORT
INTO
ISOLATED
BRAIN
c97
CAPILLARIES
mM 3MG, 80 mM mannitol, and 50 @/ml
[:‘H]3MG.
Measurement
of metabolic cowersion
of sugars. Radiolabeled metabolites
of glucose were separated from
free glucose by ion-exchange
chromatography
(25). Immediately after terminating
glucose uptake and washing the cells, the filters were placed in 1.5 ml boiling
water for 5 min. The cooled samples were centrifuged at
1,000 x g for 10 min to remove denatured proteins. We
separated amino acids from the supernatant
by column
chromatography
using Dowex AG-SOW resin. The effluent was passed through a second column containing
Dowex AG-1X8 to remove carboxylic
acids and sugar
phosphates.
When 2-deoxy-D-glucose
(2DG) was used,
only the Dowex AG-1X8 column was necessary because
2DG is phosphorylated,
but not further
metabolized
(33) m
Determination
of the water space. A suspension
of
capillaries
was preincubated
for 15 min at 37OC and
then centrifuged
for 5 min at 200 x g. The pellet was
quickly resuspended
in iced buffer containing
2 x 10”
cpm/ml [14C]L-glucose, and aliquots were added to each
of four Pasteur pipettes with their narrow ends plugged
with parafilm. The loaded pipettes were centrifuged
for
3 min at 1,000 x g at 4°C. The pellet was separated from
the supernatant
by breaking
the pipette through the
pellet. The packed cells were added to preweighed
aluminum pans, weighed, dried to constant weight in a
vacuum oven at 4O”C, and reweighed.
The dried pellets
were dissolved in 0.1 N NaOH and the amount of 14C in
a neutralized
aliquot was determined. The intracellular
water space was calculated as the difference between
the wet and dry weights
minus the L-glucose water
space. It was assumed that L-glucose remained
completely extracellular
during this experiment.
We calculated the water space of the contaminating
erythrocytes
using the hemoglobin content of the preparation
(0.034
mg hemoglobin/mg
protein) and a mean corpuscular
hemoglobin
concentration
of 34 g/100 ml of packed
erythrocytes.
MuteriaZs.
The following
materials
were obtained
from New England Nuclear
Corp. (Boston,
MA): 23-O-[methyE-H]methyl-n-glu[“H(G)]deoxy-n-glucose,
case, D- [:6-:sH]glucose,
and L- [lJ*C]glucose.
Phloretin
was purchased
from K & K Laboratories
(Plainview,
NY); 2,4-dinitrophenol
from Mallinckrodt
(St Louis,
MO); D-mannose
from SchwarzlMann
(Orangeburg,
NY); and insulin from Eli Lilly and Co. (Indianapolis,
IN). All other chemicals were obtained from Sigma
Chemical Co. (St. Louis, MO).
l
RESULTS
Properties
of 3-0-methylglucose
uptake. 3MG was
used as model substrate
for characterization
of the
glucose transport
system. This sugar is nonmetabolizable (13) and has been shown to compete with D-glucose
for BBS transport
in several species (5, 10, 12, 27, 29).
Figure 1 shows a time course for the uptake of 5 mM
3MG at 25°C and its inhibition by 0.05 mM cytochalasin
B uptake of the nontransported
sugar L-glucose was
used as a measure of the rate of simple diffusion into
the preparation.
Cytochalasin
B is an inhibitor
of glu-
Downloaded from http://ajpcell.physiology.org/ by 10.220.32.247 on June 17, 2017
through a 1.2 x 1.5 cm column containing 0.25mm glass
beads. Nuclei were not retained by the beads and could
be removed by washing
with buffer. The capillaries
remained attached to the beads and were recovered by
gentle agitation in buffer. After the beads settled, the
capillaries
were collected by centrifugation
at 500 x g
for 5 min. The quality of each preparation
was judged
by its appearance under phase microscopy.
We determined cell protein by the method of Lowry et al. (26)
after overnight
solubilization
in 1% sodium dodecyl
sulfate, using bovine serum albumin as a standard.
Measurement
of sugar uptake. The capillaries were
diluted with the preparative
buffer to a concentration
of
approximately
2 mg cell protein/ml,
kept on ice, and
used within
2 h. Assays for sugar uptake were performed at 37°C or 25OC in 1.5-ml plastic test tubes. Cell
suspension
(0.06 ml) was brought to temperature
and
the reaction was started by the rapid addition of 0.18 ml
buffer containing 3.3 or 6.7 &i of :jH-labeled sugar and
various amounts of unlabeled sugar. The reaction was
terminated
by the rapid addition of a 0.2-ml aliquot of
the incubation
mixture to 5 ml iced stopping solution
(see below) and immediate filtration
through a Whatman GF/A glass-fiber
filter. The filter and cells were
washed twice with 5 ml stopping solution and then
placed in liquid scintillation
vials containing
0.5 ml
water and 10 ml scintillation
fluid. Samples for background radioactivity
were obtained by adding 0.05 ml
cell suspension
to iced stopping
solution before the
addition of 0.15 ml of the isotope solution.
The stopping solution, based on a similar
solution
used to terminate sugar transport
in red blood cells (16),
contained Ringer solution, 1 mM HgCly, 0.1 mM phloretin, and 1% (vol/vol) ethanol. Preliminary
experiments
demonstrated
that use of this solution resulted in a 10%
increase in the amount of radioactivity
retained by the
cells. Quantitative
retention of capillaries by the glassfiber filters was also demonstrated.
These filters have
the advantage of permitting
rapid filtration
with low
nonspecific binding of labeled sugars. The entire procedure of stopping and washing could be completed in less
than 6 s.
Sugar uptake in the presence of inhibitors.
The effect
of glucose analogues
on 3-O-methyl-n-glucose
(3MG)
uptake
was determined
by incorporating
inhibiting
sugars into the isotope solution.
When uptake
was
measured in the presence of cytochalasin
B, phloretin,
or phlorizin,
0.01 ml inhibitor in 24% (vol/vol) ethanol
was added to 0.05 ml cell suspension before addition of
the isotope solution. Control incubations
contained the
same amount of ethanol without
inhibitor.
The final
ethanol concentration
was 1% (vol/vol) in these studies.
The effects of 2,4-dinitrophenol,
ouabain, and insulin
were examined after a 30-min preincubation
at 37°C in
the presence of these compounds. Accelerative
exchange
diffusion of 3MG was demonstrated
by suspending capillaries in either 100 mM 3MG or 100 mM mannitol. The
cells were preincubated
at 37°C for 30 min to allow
equilibration
and then brought
to 25°C. Uptake
of
[sH]3MG was initiated by adding isotope solutions containing unlabeled 3MG and mannitol at concentrations
desiffned to result in final external concentrations
of 20
BETZ,
CSEJTEY,
AND
GOLDSTEIN
1
1.o
0.5
mm
TIME (min)
case transport in several other cell systems including
Even at 25”C, 3MG uptake was nearly half-equilithe BBB (15). In isolated brain capillaries it clearly
brated by 10 s. Because this was the earliest time point
inhibited 3MG transport and reduced the initial uptake
that could be reliably measured using the filt&ion
assay, it was not possible to determine transport kinetto nearly that of the diffusion marker (Fig. 1).
The final equilibrium water space for 3MG was 2.2 ics based on initial velocities. Although a kinetic anal~1 water/mg capillary protein. This is less than the ysis was not possible, the relative inhibition by sugar
total intracellular water space of 4.2 $/mg protein t 0.7 transport inhibitors and glucose analogues could be
(SD) that was determined as the difference between wet determined. Table 1 shows that 3MG uptake was more
and dry weights with L-glucose used as a correction for effectively inhibited by phloretin than by phlorizin.
extracellular water. A similar discrepancy has been Cytochalasin B was the most potent inhibitor tested.
There was no inhibition of 3MG uptake in the presence
reported by Kimmich (22) for isolated intestinal epithelial cells. For these cells, the equilibrium 3MG space of ouabain or 2,4-dinitrophenol ,, and insulin did not
enhance uptake. Table 2 shows that 3MG uptake was
was 2.3 $/mg protein while the total intracellular
water space calculated by the difference between wet inhibited by high concentrations of ZDG, D-glucose,
galactose, mannose, and to a lesser extent, xylose.
and dry weights minus the polyethylene glycol water
space was 5.0 ,ul/mg protein. The reason for a difference
There was no significant inhibition by L-glucose, frucbetween the equilibrium sugar space and the total cell tose, or ribose.
The data in Fig. 2 indicate that 3MG uptake could be
water space is not clear. It is possible that the sugar is
excluded from intracellular
organelles, such as the stimulated by preloading the cells with a high concennucleus or mitochondria. On the other hand, it may be tration of 3MG. This phenomenon, accelerative exchange diffusion has been described for several other
that some of the intracellular [“H]3MG is lost during
filtration and washing of the cells; however, we have carrier-mediated glucose transport systems, including
the BBB (6).
not been able to demonstrate this effect experimentally
Relationship between sugar uptake and phosphoryl(results not shown). Furthermore, using the filtration
method, we have previously demonstrated that the ation. 2DG is-a glucose analogue that can be phosphoequilibrium water space for amino acids that are not rylated by mammalian cells, but not further metaboconcentrated by the capillaries is 4.2 pl/mg protein (9). lized (33). It is also an effective inhibitor of glucose
transport across the BBB (4,27,29), and thus is a useful
The erythrocyte water space was less than O-1 pl/mg
protein in our preparation and thus red blood cells made model substrate for a study of the relationship between
sugar transport and phosphorylation in isolated capila negligible contribution to the total 3MG uptake.
Downloaded from http://ajpcell.physiology.org/ by 10.220.32.247 on June 17, 2017
FIG. 1. Time course
for uptake
at 25°C of 5 mM
3-0-methylglucose
(a), 5 mM 3-0-methylglucose
in
presence
of 0.05 mM cytochalasin
B (0) and 5 mM
L-glucose
(A). Insert:
expanded
view of 1st min of
uptake.
Data shown are averages
of 3 determinations -+ SD.
SUGAR
TRANSPORT
TABLE
1, Effect
3-0-methylglucose
INTO
of inhibitors
BRAIN
B
on
Uptake,
nmollmg
0.05
0.50
0.50
1.00
0.50
0.10 U/ml
0.24
1.64
2.90
4.33
4.31
k
*
1
f
f
3.60
2 0.64
Percent
Control
0.64
0.33
0.57
0.73
0.75
6
37
67
97
96
80
2. Inhibition
by glucose analugues
Added
Sugar
2-Deoxy-n-glucose
D-Glucose
3-@methyl-D-glucose
n-Galactose
D-Mannose
D-Xylose
D-Fructose
D-Ribose
L-Glucose
Control
of 3-0-methylglucose
Uptake,
nmollmg
0.94
1.22
1.23
1.57
1.92
2.30
2.96
3.31
3.39
3.26
k
*
4
k
-Ik
k
+
4
k
0.37
0.36
0.35
0.45
0.71
0.33
0.78
0.32
0.38
0.38
uptake
Percent
Control
tion of 2DG-PO4 have been reported for rat kidney slices
(23)
Figure 3 shows the time course for 2DG uptake and
phosphorylation
by capillaries
with 0.1 mM 2DG at
37°C. This concentration
of 2DG was used to more
clearly define the relationship
between transport
and
phosphorylation
as well as to permit comparison
with
our previous results (18). The rapid equilibrat.ion of free
2DG with an intracellular
water space of 2.2 $/mg
protein was followed by a slower accumulation
of 2DGPO,. These data suggest that transport
was not rate
limiting for metabolism
at a 2DG concentration
of 0.1
mM. Figure 4 shows similar results with D-glucose at
the physiologic
concentration
of 5 mM. The free sugar
equilibrated
with a water space of 2.1 pl/mg protein and
this process was more rapid than metabolism.
DISCUSSION
The movement
of glucose between blood and brain
has been extensively
studied in vivo. It occurs via a
mediated transport
system (3-8, 10-12, 20, 27-29) with
very little free diffusion
(8). The kinetic constants
for
transmort
fall within
a relatively
narrow
range from
29
38
38
48
59
70
91
101
104
100
Values
are averages
of 4 determinations
* SD. We corrected
for
diffusion
using
the uptake
of [:iH]L-glucose
(Fig.
1). Results
are
expressed
as percent
of control
containing
100 mM D-mannitol
to
correct for osmotic effects. Uptake
of [“H]3MG
was determined
after
a 30-s incubation
in the presence
of 5 mM 3MG
and 100 mM
inhibiting
sugar.
lary endothelial cells.
Capillaries
were incubated with [:‘H]2DG for various
periods of time and then the reaction was stopped, the
cells washed,
and the retained radioactivity
fractionated into free 2DG and 2DG-PO4 by ion-exchange
chromatography.
Preliminary
studies
showed
that the
method by which the radioactivity
was released from
the cells was very important
(Table 3). When cells were
incubated for 30 min at 37OC in 0.1 mM 2DG, washed,
and then sonicated
to release radioactivity,
we recovered only 10.4% of the intracellular
label as 2DGPO,. However,
release of intracellular
radioactivity
by
sonication, as had been done in our previous study (la),
would not necessarily
protect 2DG-PO4 from dephosphorylation by glucose-6-phosphatase
and other phosphatases, e.g., alkaline phosphatase,
which are known to
be present in brain capillaries
(19). When the washed
cells were immediately
placed in boiling water to inactivati enzymes and then sonicated, 94.8% of the intracellular radioactivity
was recovered as 2DG-PO4 (Table
3). Thus, the use of boiling water to inactivate phosphatases avoids dephosphorylation
of intracellular
2DGPO,. Similar artifacts caused by rapid dephosphoryla-
10
0.25
0.50
0.75
TIME
1.0
(min)
FIG. 2. Accelerative
exchange
diffusion
of 30methylglucose.
Capillaries
were preincubated
with either 100 mM 3MG (a) or 100
mM mannitol
(0). Uptake
of 3MG was then determined
in presence
of 20 mM 3MG
and 80 mM mannitol
at 25°C. Data
shown
are
averages
of 3 determinations
&SD.
TABLE
3, Importance
of boiling in recovery of
phosphorylated
2-deoxyglucose
from capillary cells
Release
by
Sonication
Boiling,
sonication
Percent
2DG
89.5 k 8.2
5.2
k 0.5
Values
are averages
of 3 determinations
* SD.
incubated
at 37°C with 0.1 mM 2DG for 30 min.
filtered,
washed,
and intracellular
radioactivity
either
sonication
alone or by treating
with boiling
sonication.
2DG and 2DG-6-Po4
were separated
chromatography
on Dowex
AG-1X8.
Total sugar
nmollmg
k 0.10 SD.
Percent
10.5
94.8
2DG-6-PO,
2 3.0
4 3.0
Capillaries
were
Cells were then
was released
by
water
and then
by ion-exchange
uptake
was 4.34
Downloaded from http://ajpcell.physiology.org/ by 10.220.32.247 on June 17, 2017
Values
are averages
of 4 determinations
-+- SD; we corrected
for
diffusion
using uptake
of [iJH]L-glucose
(Fig. 1). Uptake
of [:$HJ3MG
was determined
after a 30-s incubation
at 25°C in the presence
of 5
mM 3MG. Control
uptake
for studies with cytochalasin
B, phloretin,
and phlorizin
was 4.34 nmollmg
* 0.32 SD. There
was a 30-min
preincubation
at 37°C at the stated
concentration
of inhibitor
for
those experiments
in which
ouabain,
2,4-dinitrophenol,
or insulin
was present.
Corresponding
control
sample was also preincubated
and had an uptake
of 4.47 nmollmg
* 0.81 SD.
TABLE
c99
CAPILLARIES
uptake
Concentration,
mM
Inhibitor
Cytochalasin
Phloretin
Phlorizin
Ouabain
2,4-Dinitrophenol
Insulin
ISOLATED
BETZ,
0.5
0.1
DP
/
/D I
1
I
I
2
TIME
3
I
4
1
5
(An)
3. Time course for uptake
of 0. I mM 2-deoxyglucose.
After
incubation
at 37”C, intracellular
radioactivity
was separated
into
free 2DG (a) and 2DG-6-P04
(0) by ion-exchange
chromatography.
Data shown are averages
of 3 determinations
*SD.
FIG.
species to species. Reported values for the apparent K,,
are within a range of 6-9 mM while the apparent V,,,,,
varies from 1.2 to 3.0 (pmol/g whole brain)/min
(3, 8,
11, 20, 28, 29). The stereospecificity
for BBB glucose
transport
is well described
in rat (29) and dog (4).
Although phlorizin and phloretin are both competitive
inhibitors
of glucose transport
into brain, phloretin
is
more potent than phlorizin (4). In contrast, Na+-dependent sugar transport
in intestine
(14) and kidney (1) is
much more sensitive to phlorizin than phloretin.
Cytochalasin B, which is a potent noncompetitive
inhibitor
of glucose transport
into dog brain (E), has no effect on
Na+-dependent
glucose transport
in the intestine
(21).
Sugar transport
into brain is unaffected by ouabain (29)
or insulin (8) and exhibits accelerative
exchange diffusion when the brain glucose concentration
is increased
(6). Further more, transport
of glucose into brain is not
rate limiting for subsequent
brain metabolism
(7). In
general, BBB glucose transport
is thought to be mediated by a Na+-independent
facilitated
diffusion system similar to the one in the erythrocyte
(7).
Our results for 3MG uptake by brain capillaries
in
vitro are entirely consistent
with studies of BBB sugar
transport
in vivo. The stereospecificity
data shown in
Table 2 are nearly identical to the in vivo data from rat
(29) and dog (4). The relative potency of cytochalasin
B,
phloretin,
and phlorizin are also the same (4, X5), and
there was no effect of ouabain, 2,4-dinitrophenol
(29), or
insulin (8). Both isolated capillaries
(Fig. 2) and the
BBB in vivo (6) show accelerative
exchange diffusion
AND
GOLDSTEIN
for sugars. Furthermore,
in isolated capillaries,
transport is equilibrative
and is not rate limiting for metabolism. Kolber and Morel1 (24) have recently presented
preliminary
data that also show equilibrative
3MG
uptake into isolated brain capillaries.
It would be useful to compare the kinetics of glucose
transport
into isolated capillaries
with the kinetics of
transport
across the BBB. This kind of data could not
be obtained with
our studies
because the rate of
D-glucose uptake by capillaries
is too rapid at 37°C.
Thus, at 5 mM, D-glucose, uptake was half-equilibrated
by 10 s. Reducing the temperature
would lower the rate
of uptake, but also significantly
alter both the K,, and
V H-liis (32). An alternate
approach utilizes integrated
rate equations and time courses of uptake to describe
the kinetics of transport
(16). Unfortunately,
the significant rate of simple diffusion into the isolated capillary
preparation
(Fig. 1, L-glucose) makes such equations
prohibitively
complex.
The first study of sugar transport
into isolated brain
capillaries
by Goldstein
et al. (18) presented
results
inconsistent
with data from in vivo studies or with the
results of the present study. The time course for 2DG
uptake was linear for more than 5 min and the apparent
K,,, for this process was 0.093 mM. In addition, a study
of stereospecificity
showed inhibition by D-fructose, but
not by D-galactose.
These properties
are opposite to
those found for BBB glucose transport
in vivo (4), and
are, in fact, similar to the properties
of mammalian
hexokinase
(35). Other investigators
have recently reported that 2DG transport
into isolated capillaries
is
12
10
2
TIME
(min)
4. Time course for uptake
of 5 mM D-glucose.
After
incubation at 37”C, intracellular
radioactivity
was separated
into D-glucose
(a) and glucose
metabolites
(0) by ion-exchange
chromatography.
Data shown are averages
of 3 determinations
*SD.
FIG.
Downloaded from http://ajpcell.physiology.org/ by 10.220.32.247 on June 17, 2017
0.4
.-c
aI
‘0
&
f 0.3
\
;2
0
E
= 0.2
CSEJTEY,
SUGAR
TRANSPORT
INTO
ISOLATED
BRAIN
Cl01
CAPILLARIES
require little energy because it is not concentrative or
Na+-dependent and does not require phosphorylation of
the sugar. Our data also indicate that there is a sizable
pool of free glucose in endothelial cells under physiological conditions (Fig. 4). Furthermore, sugar transport
into capillaries and, presumably, also out of capillaries,
occurs very rapidly. This would permit rapid response
of the glucose supply to changes in cerebral metabolism.
In conclusion, this study demonstrates the usefulness
of isolated brain capillaries for the investigation of
transport phenomena thought to occur at the BBB. Our
observation that hexose transport into isolated brain
capillaries is mechanistically similar to glucose transport across the BBB does not by itself prove that the
BBB is located at the capillary endothelial cell. However, we anticipate that further studies with the isolated capillary preparation will more firmly establish
this relationship.
We thank A. Ste. Marie for her excellent
technical
assistance
and
I. Diamond
and A. Gordon
for their helpful review of the manuscript.
This work
was supported
by the National
Foundation
of the
March
of Dimes,
and Grants
ES-01164
and HL-22088
from
the
National
Institutes
of Health.
A. L. Betz is the recipient
of the
National
Institutes
of Health
postdoctoral
fellowship
NS-05807.
G.
W. Goldstein
is the recipient
of the National
Institutes
of Health
Research
Career Development
Award
NS-00278.
Received
30 May
1978; accepted
in final
form
17 August
1978.
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