From the
Luminal and abluminal membrane vesicles derived from bovine
brain endothelial cells, the site of the blood-brain barrier, were
fractionated in a discontinuous Ficoll gradient. A mathematical
analysis was developed to determine the membrane distribution of
membrane marker enzyme activities as well as the ratio of luminal to
abluminal membrane in each fraction of the gradient. The results of
this analysis indicate that
Isolated membrane vesicles provide a convenient tool to study
the function and characteristics of membranes at a molecular
level(1, 2) . Ideally, a pure and homogeneous membrane
vesicle population is desired. However, this is not possible in most
cases, and a mixture of membrane vesicles from different plasma
membrane domains is commonly obtained. The purity or origin of the
membranes is usually assessed by measuring the relative specific
activity (RSA)
A
variety of proteins has been associated with the blood-brain barrier
(BBB)(3, 4, 5) , including antigens of unknown
function, receptors, and enzymes. However, it is possible that no
molecular components exist that are exclusively located in BBB-specific
endothelial cells(5) . Traditionally, in the absence of
completely specific markers, the membrane-bound enzymes
Cytochemical studies located
Na
We have previously shown that membrane vesicles isolated from bovine
brain endothelial cells can be used to study transport(14) .
These results were interpreted according to the consensus opinion (3) that GGT and alkaline phosphatase were located on the
luminal membrane and Na
Isolated membranes were also prepared for transmission
electron microscopy. Samples of the Ficoll gradient fractions were
centrifuged at 37,500
Morphological analysis of isolated
membrane vesicles was performed on micrographs of the luminal and
abluminal membrane-rich fractions using the procedure described by
Weibel and Bolender(22) .
The activity of each plasma membrane marker was normalized
by the activity present in the whole gradient and expressed as a
percentage,
On-line formulae not verified for accuracy where M is the percentage of marker m in fraction i, and U is the activity units of marker m in fraction i. The array of markers (a-m)
in each fraction (1-i) was obtained as indicated in . Similarly, the amount of luminal and abluminal membrane
in each fraction was expressed as a percentage of the total amount
contained in the whole gradient. The content of luminal and abluminal
membranes in the gradient can be considered a good representation of
the total plasma membrane population since similar recoveries of the
plasma membrane markers were obtained at each step during the isolation
procedure. Thus, the membrane preparation that we loaded onto the
Ficoll gradient contained the same proportion of the plasma membrane
markers as the initial homogenate.
The fraction of plasma membrane
marker activity located on the luminal membrane, f, is the
activity of that marker located on the luminal membrane divided by the
total activity present in both luminal and abluminal membranes. This
fraction is 0 when all the activity is located on the abluminal
membrane and 1 when all the activity is located on the luminal
membrane. The fraction of marker activity located on the abluminal
membrane is 1 - f.
The percentage of marker present
in each fraction is given as
follows,
On-line formulae not verified for accuracy where L and A are the percentages of luminal and
abluminal membrane in fraction i, respectively. The array of
calculated marker activities is obtained from the definitions shown in . The percentage of marker in each fraction was determined
experimentally. Since the luminal (L) and abluminal (A) content was expressed as percentages, the values in one of
the fractions was a combination of the values in the other four
fractions. The number of unknown parameters were the luminal (L) and abluminal (A) content in only four fractions
and the f values for the markers. To determine these
parameters, Equation 2 was used to find the best least squares fit to
the measured amount of markers in each fraction.
The fitting was
carried out using the spreadsheet Microsoft Excel as follows. An
initial set of f values was provided to start the fitting
procedure. The L and A values were calculated as the
regression coefficients of f and (1 - f),
respectively, in a multiple linear regression analysis between rows in
the observed matrix and the f and (1 - f)
values, using a built-in function of the program. The measured M
With the information provided by the method,
specifically the luminal and abluminal percentages, the f values for any activity associated with the plasma membrane can be
readily determined. To establish the location of a particular marker,
its activity in terms of total activity units in only two fractions is
needed, as indicated below.
It is clear from Equation 1 that the
ratio of a marker activity in two fractions can be obtained using
either the percentage of activity (Equation 1) or the total activity.
If we calculate this ratio in terms of total activity and combine with
Equation 2, we have the following expression,
On-line formulae not verified for accuracy where U is the total units, L and A are
the percentages of luminal and abluminal membranes, respectively,
contained in each fraction, and i and j refer to two
different fractions. If we now solve for f,
On-line formulae not verified for accuracy and define R as follows,
On-line formulae not verified for accuracy then the following equation is obtained:
On-line formulae not verified for accuracy
The results
of the analysis also indicated that GGT activity is located almost
exclusively in the luminal membrane, whereas alkaline phosphatase and
5`-nucleotidase activities are evenly distributed in both luminal and
abluminal membranes (). K
A novel analytical approach was used to treat the enzyme
marker data and extract quantitative information concerning the
contribution of the individual membranes in transport experiments. The
analysis of the marker enzymes described here offers some advantages
over the more conventional approaches using RSA values. It is not
necessary to measure the activity in the initial homogenate, which, due
to the high content of cellular debris, may produce inconsistent
results. It allows the use of a broader variety of membrane markers,
even some with unknown location. Transport activities and receptor
binding sites are particularly appropriate markers to use with the
described protocol. It represents a quantitative, not qualitative,
approach so that once the distribution of the plasma membrane domains
(the fraction of luminal and abluminal) is known, the location of any
activity can be assigned by measuring the activity in only two
fractions, and the exact contribution of each membrane domain to the
activity being measured can be determined.
The presence of two
membrane vesicle populations (luminal and abluminal) was indicated by
the different distribution of plasma membrane markers. One population
was enriched in GGT, whereas the other was enriched in
Na
1)
Na
2) Na
3) Na
Although most of the K
GGT activity is located in
the luminal membrane. Since the distribution of GGT in the Ficoll
gradient is markedly different from that of the
Na
Alkaline phosphatase and 5`-nucleotidase
activities are, on the contrary, equally distributed between luminal
and abluminal membranes. The distribution of alkaline phosphatase found
here has been reported also by some authors (8) using
cytochemical techniques. The finding that 5`-nucleotidase is located on
both membranes is, however, in disagreement with some cytochemical
studies(3) . A possible explanation for this discrepancy is
that, as noted by Vorbrodt(3) , this enzyme is extremely
sensitive to the fixatives used to prepare the samples for cytochemical
examination. In most cytochemical experiments, the brains are perfused
with a fixative, probably creating a concentration gradient of fixative
from the luminal to the abluminal sides of the vessels. Thus, it is
possible that enzymes located on the luminal membrane are inactivated
but those located on the abluminal side are not.
The distribution of
membrane markers, schematized in Fig. 5, can be quantitatively
determined with the described analysis. The results reported here
indicate that luminal and abluminal membranes of brain endothelial
cells can be separated and that the properties of each membrane can be
studied separately. GGT and amino acid transport system A are suitable
markers for luminal and abluminal membranes, respectively. Our results
represent a confirmation of the concept, based on indirect
evidences(25, 29) , that transport system A is exposed
to the brain extracellular fluid. The results also validate the
interpretation of our previous results(14) , based on the
distribution of GGT and Na
The protein is
expressed in milligrams. The specific activity is expressed in nmol
min
The luminal and abluminal
content is expressed as percentages of the total amount present in the
gradient. The luminal and abluminal values represent the average
± S.D. of three isolations. L/A, luminal to abluminal ratio;
P+I, pellet plus material between interfaces.
The f values represent the average ± S.D. of three preparations
(alkaline phosphatase and system A measured in two preparations). A
membrane preparation is the combination of 4-5 vesicle
isolations, each obtained from 8-12 cow brains.
We thank Veronika Burmeister for preparing the samples
for electron microscopy.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-glutamyl transpeptidase and amino
acid transport system A are located on the luminal and abluminal
membranes, respectively. Conversely, 5`-nucleotidase and alkaline
phosphatase activities are evenly distributed between both membranes.
Although Na
/K
-ATPase activity is
primarily located on the abluminal membrane, approximately 25% of the
activity is of luminal origin.
Na
/K
-ATPase activities associated
with each membrane showed different ouabain sensitivities, suggesting
that different isoenzymes are located in luminal and abluminal
membranes. The analytical procedure used in this study provides a
quantitative means to determine the distribution of marker enzymes and
transport proteins in partially purified membrane vesicle populations.
(
)of marker enzymes, which
identify specific membrane populations. Some problems are associated
with this practice. First, starting with a tissue containing several
cell types, with which the activity of the final preparation is
compared, may produce a misleading high value of RSA. Second, if the
protein density of a particular membrane domain is higher than the
others, the RSA may be lower, although the same enrichment is achieved.
Thus, only limited quantitative conclusions can be obtained.
-glutamyl
transpeptidase (GGT), alkaline phosphatase, 5`-nucleotidase, and
Na
/K
-ATPase are the most commonly
used markers. All studies have shown alkaline phosphatase on the
luminal membrane of brain endothelial cells, most of them exclusively.
Some reports indicate, however, that alkaline phosphatase is on both
membranes (reviewed in Ref. 3). GGT is probably the most widely used
marker for the BBB(5) ; yet, there are only two cytochemical
studies showing GGT localization at the electron microscopic level. The
first indicates that the enzyme is exclusively on the luminal
membrane(6) , whereas the second ascribes an entirely abluminal
position(7) . Biochemical results (8) suggest that
alkaline phosphatase and GGT are located on both luminal and abluminal
sides of the brain endothelial cells. GGT and alkaline phosphatase
colocalize in the luminal membrane of epithelial cells, and, in fact,
the expression of both enzymes parallel the appearance of epithelial
characteristics in tissue culture(9) . If brain endothelial
cells can be considered as a specialized endothelium, with epithelial
properties mediating the transport of substrates between blood and
brain(10) , one might expect the same distribution observed in
epithelial cells.
/K
-ATPase exclusively (8) or
primarily (11) on the abluminal membrane. However, a more recent
report claims that enzyme activity is equally distributed on both
membranes(12) . Vorbrodt (3) noted that when the incubation time
with the Na
/K
-ATPase substrate is
increased, some reaction product appears on the luminal side as well as
the abluminal surface, which suggests heterogeneity in
Na
/K
-ATPase activity. A multiplicity
of isoforms (3
and 2
subunits of the
Na
/K
-ATPase) has been shown recently
using Western blot analysis(13) , indicating that six different
isoenzymes may be expressed in brain endothelial cells. Collectively,
these results suggest that while abluminal membranes contain most of
the Na
/K
-ATPase activity, or the most
active isoform, some activity may also be present in luminal membranes.
/K
-ATPase was
on the abluminal membrane. However, as indicated above, contradictory
results concerning the location of markers have been reported that may
affect the interpretation of transport experiments. The following
experiments were designed to show that the marker enzyme information
can be analyzed in such a way that their location on each membrane
domain can be deduced. Furthermore, the relative composition of luminal
and abluminal membranes in each fraction can be determined, and the
contribution of each membrane to the measured transport may be
quantified.
Materials
N-(Methylamino)-[1-C]isobutyric
acid (48.4-56.3 mCi/mmol) and [G-
H]ouabain
(15.4 Ci/mmol) were purchased from DuPont NEN. 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
Brain microvessels were isolated essentially as
described by Pardridge et al. (15) with only minor
modifications(14) . Brain endothelial cell membranes were
isolated by a modification (14) of the procedure described by
Betz et al.(8) . Briefly, stored microvessels were
incubated at 37 °C for 25 min in isolation buffer (101 mM NaCl, 4.6 mM KCl, 2.5 mM CaCl2H
O, 1.2 mM KH
PO
, 1.2 mM MgSO
,
and 14.5 mM HEPES, pH 7.4) containing 1800 units of
collagenase type IA per gram of capillary. The collagenase-treated
capillaries were homogenized in TSEM buffer (250 mM sucrose,
0.1 mM EGTA, 0.5 mM MgCl
, and 10 mM Tris-HCl, pH 7.4) using a Tissuemizer (Tekmar, Cincinnati, OH),
and the cellular debris was removed by centrifugation. To the membrane
fraction contained in the supernatant, solid MgSO
was added
to a final concentration of 10 mM. The suspension was
centrifuged at 3,200
g for 15 min at 4 °C, and the
supernatant was centrifuged at 90,000
g for 1 h at 4
°C in a Ty 35 Beckman rotor. The pellet was resuspended in TSEM and
layered on top of a discontinuous Ficoll gradient (5, 10, 15, and 20%
Ficoll). After centrifuging at 162,500
g for 2.5 h at
4 °C in a 70 Ti Beckman rotor, five fractions corresponding to the
interfaces between the different Ficoll layers and the pellet were
collected. All fractions were diluted in storage buffer (290
mM mannitol and 10 mM HEPES-Tris, pH 7.4) and left
overnight in an ice bath. Samples were centrifuged at 90,000
g for 30 min at 4 °C in a Ty 35 Beckman rotor, and the
pellet was resuspended in 2-3 ml of the same buffer. Aliquots
were stored at -80 °C.
Marker Enzymes
GGT, alkaline phosphatase,
5`-nucleotidase, and Na/K
-ATPase
activities were used as plasma membrane markers(3) . Amino acid
transport system A was also used as a plasma membrane marker. GGT and
alkaline phosphatase activities were measured in the presence of 0.1%
(v/v) Triton X-100 (16). 5`-Nucleotidase activity was determined in the
presence of 0.02% (v/v) Triton X-100(17) . The
Na
/K
-ATPase activity was determined
by measuring the K
-dependent, ouabain-sensitive
phosphatase activity (17) or the binding of radiolabeled
ouabain(14) . The activity of amino acid transport system A was
determined by measuring the initial rate of uptake of 100
µM [
C]MeAIB in the presence of NaCl
or KCl as indicated in the accompanying paper(18) . The specific
activity was calculated by subtracting the activity in the presence of
KCl from the activity in the presence of NaCl and expressed as pmol
MeAIB min
mg
of protein. N-Acetyl-
-D-glucosaminidase activity was used as
a lysosomal marker and measured in the presence of 0.2% (v/v) Triton
X-100(19) . The activity of cytochrome c-oxidase, a
mitochondrial enzyme, was measured in the presence of 0.1% (v/v) Triton
X-100(20) .
Electron Microscopy
Samples of isolated
capillaries were critical point dried after dehydration. They were then
positioned on an aluminum stub and sputter coated with gold. The
specimen was examined in a JEOL scanning electron microscope (JSM-35)
at 20 kV.
g for 25 min at 4 °C, and
the pelleted membranes were fixed in modified Tyrode's
solution(21) , post-fixed in osmium tetroxide, dehydrated in an
ethanol series, cleared in propylene oxide, and embedded in Embed 812
(Electron Microscopy Sciences, Fort Washington, PA). Thin sections were
stained with lead citrate and uranyl acetate and examined under a Zeiss
EM 10C electron microscope.
Protein Determination
Protein
concentration was determined using the Bio-Rad protein microassay, with
bovine serum albumin as the standard, based on the method of
Bradford(23) .
Analysis of Marker Enzyme Data
The
procedure is based on two assumptions. First, the markers are located
exclusively on the plasma membrane, either luminal or abluminal, which
is the case for those used (reviewed in Ref. 3). Second, contamination
by cells other than capillary endothelial cells is negligible. The
second assumption is supported by the results described below. To know
the content of luminal and abluminal membranes in each fraction and in
which membrane the marker enzymes are located, the following analysis
was used.
values were compared with those
obtained using Equation 2 with a first set of estimated parameters. The
agreement between measured and calculated results was given by least
squares. An iterative procedure (quasi-Newton method) was used to find
the f values that produced the best agreement with the
observed results. The L and A values were updated
after each iteration. The standard errors of the estimated values of L and A were obtained from the multiple linear
regression analysis indicated above. At the end of the fitting
procedure, a multiple linear regression analysis between columns of the
observed matrix and the L and A values was performed.
This analysis provided the standard errors of f and (1 - f), the regression coefficients of L and A,
respectively.
Isolation of Brain Microvessels
The
isolated microvessel preparation appeared to consist, at the light
microscope level, of branching capillary segments with only occasional
small arterioles and venules and without contamination by other cell
types. Scanning electron microscopy showed that the isolated
microvessels were devoid of any nerve or glial cells (Fig. 1a). The average diameter of the isolated
microvessels was 3.5 ± 0.6 µm (±S.D.), in agreement
with the size observed in other capillary beds (24). The absence of
blood cell contamination was supported by the results shown in the
accompanying paper (18) where no ASC transport system activity
present in red blood cells was detected. Thus, the protocol to isolate
brain microvessels produced a nearly pure capillary preparation in
which endothelial cells constituted the predominant cell type.
Figure 1:
a, scanning electron
micrograph of isolated brain microvessels. b and c,
phase-contrast micrographs of isolated brain capillaries. b,
before collagenase treatment, some rounded particles (arrowheads) can be seen at the external surface of
capillaries. c, after collagenase treatment and washing of the
capillaries, almost no such particles are present at the capillary
walls. The bars are 10 µm in
length.
Collagenase Treatment
Before collagenase
digestion, some rounded bodies were observed (Fig. 1b)
scattered along the isolated capillaries. Their location at the outer
surface of capillaries suggested that these bodies were pericytes or
fragments of glial endfeet. Collagenase treatment, which digested the
basement membrane, released them. After centrifugation and resuspension
of the collagenase-digested capillaries, none of these particles were
seen (Fig. 1c). It is interesting to note that if the
observed structures were indeed pericytes, the described protocol might
be useful to isolate pericytes associated with the blood-brain barrier.
Isolation of Membrane Vesicles
As we
previously showed, the membrane preparations consist of sealed vesicles
without detectable contamination by other organelles(14) .
Morphological analysis (22) of transmission electron micrographs
(not shown) indicated that the average radius of the vesicles was 164
± 15 (± S.D.) and 125 ± 12 nm for luminal and
abluminal membrane-rich fractions, respectively.
Marker Enzyme Distribution
When the RSA
for each of the plasma membrane marker enzymes was calculated, a
maximum enrichment in the 0/5% Ficoll fraction was observed. The
enhancement declined in the remaining fractions (I). The
achieved enrichments were between 7 and 20, except for the
K-dependent, ouabain-sensitive phosphatase activity.
However, when the activities of the markers were expressed as
percentages, as indicated under ``Experimental Procedures,''
the difference between markers was apparent (Fig. 2). The GGT
activity was concentrated in the first fraction, which corresponds to
the 0/5% Ficoll interface, whereas the other markers were concentrated
in denser fractions (5/10 and 10/15% Ficoll interfaces). As an
additional marker, the Na
-dependent transport activity
of the amino acid analog MeAIB was used. This analog is specifically
transported by system A, which was centered in fraction 10/15. There
was good correlation between the measured marker activities and those
predicted by Equation 2 (Fig. 3), supporting the validity of the
estimated parameters.
Figure 2:
Distribution of marker enzymes within the
gradient. Enzyme activity is expressed as the percentage of the total
activity recovered in the gradient. The markers located in only one
membrane are indicated by the solidlines. The brokenlines indicate markers present in both
membranes. AP, alkaline phosphatase; 5N,
5`-nucleotidase; KP, K-dependent,
ouabain-sensitive phosphatase; A, system A transport activity; P+I, pellet plus material between
interfaces.
Figure 3:
Correlation between measured and predicted
marker activity. The marker activity, expressed as the percentage of
the total activity recovered in the gradient, was calculated using
Equation 2, and the result was plotted as a function of the measured
activity. The line is the regression fit, which has a slope of
0.986 and a correlation coefficient of 0.986. AP, alkaline
phosphatase; 5N, 5`-nucleotidase; KP,
K-dependent, ouabain-sensitive phosphatase; A, system A transport activity.
The results obtained using the mathematical
analysis described above indicated that fraction 0/5 was enriched in
one membrane domain and fractions 10/15 and 15/20 in the other (Tables
IV and V). As discussed below, the low and high density membrane
vesicles were considered to be enriched in luminal and abluminal
membranes, respectively. Thus, fraction 0/5 and 10/15 can be considered
as luminal and abluminal membrane-rich fractions (),
respectively, which is a similar distribution to that found by Betz et al.(8) . Although fraction 15/20 has a higher
abluminal/luminal ratio than fraction 10/15, the latter was selected as
most suitable for transport studies for two reasons. First, the amount
of protein obtained in fraction 10/15 is much higher than that in
15/20, allowing many more experiments. Second, contamination by
lysosomes and mitochondria is lower in fraction 10/15.
-dependent,
ouabain-sensitive phosphatase activity appeared to be located mainly on
the abluminal side, although some activity was present also in the
luminal side (about 25%). When ouabain binding was measured, the
affinity for ouabain was lower in the luminal than in the abluminal
membrane-rich fractions (Fig. 4), indicating that activity may be
indeed present in both membranes but due to different isoforms. The
transport activity of system A appeared to be located exclusively on
the abluminal membrane, which makes it a good marker for abluminal
membranes.
Figure 4:
Binding of ouabain to luminal and
abluminal membrane-rich vesicles. An Eadie-Hofstee plot of the
fractional saturation of ouabain binding sites present in luminal and
abluminal membrane-rich fractions shows two dissociation constants
(± S.E.) for ouabain.
/K
-ATPase and system A activities.
The designation of luminal and abluminal has been made on the basis of
the following considerations.
/K
-ATPase activity has always been
found on the abluminal membrane. In the cases where some activity was
also present on the luminal membrane, the same or more activity was on
the abluminal side, indicating that this enzyme is, at least primarily,
an abluminal membrane-bound enzyme(3, 11, 12) .
-dependent transport of amino acids has been
measured only using isolated brain microvessels where the abluminal
membrane is the most accessible membrane, suggesting that this
transport system is most probably located on the abluminal side (25,
26). A luminal location of system A would predict a significant
transport of small neutral amino acids, the preferred substrates of
this system, across the BBB. However, transport of these amino acids is
undetectable in experiments in vivo, where substrate is
presented to luminal membranes, supporting the abluminal location of
this carrier.
/K
-ATPase
activity and system A transport activity co-localize primarily to a
single membrane population in the current study, implying that both
proteins can be considered markers for the same plasma membrane domain
( and Fig. 2). This distribution is opposite that of
GGT activity. These observations strengthen the previous conclusions,
supporting an abluminal location of both
Na
/K
-ATPase and system A activities.
-dependent, ouabain-sensitive
phosphatase activity appeared to be located on the abluminal side, some
activity was detected also in the luminal side. The ouabain binding
results presented here, along with the results reported by other
authors(11, 12) , support the idea that different
isoforms are located in each membrane. A multiplicity of isoforms (3
and 2
subunits of the
Na
/K
-ATPase) has been shown recently
using Western blot analysis(13) , indicating that as many as six
different isoenzymes could be expressed in brain endothelial cells.
These results indicate that polarity exists in the sense that abluminal
membranes, representing the majority of the activity, contain an
isoform with a higher affinity for ouabain, whereas luminal membranes
contain a minor component of the activity, which has a lower ouabain
affinity. This distribution may favor a net sodium transport from blood
to brain, as suggested by some authors (27), because more sodium is
removed by the Na
/K
-ATPase isoform
located in abluminal membranes. A polarized distribution of different
isoforms of membrane proteins such as glucose transporters has also
been shown in other tissues(28) .
/K
-ATPase and system A activities,
which are located on the abluminal membranes, it has been concluded
that GGT activity is located on the luminal membrane. This location
agrees with the results of Ghandour et al.(6) ,
identifying this enzyme activity with the luminal membrane of brain
endothelial cells.
/K
-ATPase
activities, and establish the conditions to further characterize the
constituting membranes of the blood-brain barrier.
Figure 5:
Schematic representation of the location
of plasma membrane markers. The marker distribution is based on data
shown in Table V.
Table: Array of markers measured in the gradient
fractions
Table: Array of calculated marker activities
Table: Relative specific activity of the
marker enzymes in the Ficoll gradient fractions
mg
, except for COX, which is
A
min
mg
. H,
microvessel homogenate; AP, alkaline phosphatase; 5`N, 5`-nucleotidase;
KP, K
-dependent, ouabain-sensitive phosphatase; COX,
cytochrome-c-oxidase; NAG, N-acetyl-
-D-glucosaminidase, P+I, pellet
plus material between interfaces. Specific activity is indicated in
parentheses.
Table: Distribution of luminal and abluminal
membranes in the Ficoll gradient fractions
Table: Marker enzyme distribution
-glutamyl
transpeptidase.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.