(Received for publication, February 14, 1995; and in revised form, May 18, 1995)
From the
Several subspecies of human high density lipoprotein (HDL) have
been shown to exist, and particle size is one parameter that can be
used to distinguish them. Recently, a small HDL subspecies has been
described that may be a particularly efficient acceptor of peripheral
cell unesterified (free) cholesterol (FC). To address the effects of
particle size on the ability of HDL to remove FC from cells,
homogeneous, well defined HDL particles were reconstituted (rHDL) that
varied in particle diameter within the size range of human HDL
particles (7-13 nm). The abilities of each of these particles to
remove cellular FC from mouse L-cells and rat Fu5AH hepatoma cells were
compared on the basis of their phospholipid (PL) content as well as on
a per particle basis. The effect of particle size was also examined
using small unilamellar vesicles (SUV) of 25 nm in diameter and large
unilamellar vesicles (LUVs) of 70-180 nm in diameter. The SUV
were prepared by sonication, and the LUVs were prepared by extrusion
techniques. The FC efflux efficiency of these particles (in order of
decreasing efficiency) was: rHDL > SUV > LUV when compared on the
basis of acceptor PL content across a range of concentrations (i.e. at a given PL concentration for these three acceptor classes,
smaller particles were more efficient). The FC efflux differences
between the rHDL and the vesicles were not due to the absence of
apolipoprotein in the vesicles. No difference was detected among the
rHDL of varying size, nor was a difference detected among the LUVs of
varying size when compared on the basis of PL content. When the FC
efflux data for rHDL and LUVs were normalized on the basis of the
number of acceptor particles present at a given PL concentration, a
correlation was found between acceptor particle radius and the ability
to accept cellular FC with larger particles being the most efficient.
However, the dependence of the rate of FC efflux on acceptor particle
size was not quantitatively the same within the rHDL and LUV classes of
acceptor particles. The dependence of FC efflux on acceptor particle
size may reflect differing abilities of the variously sized acceptor
particles to access the region very close to the cell plasma membrane
where most of the FC removal is expected to occur.
The concept of high density lipoprotein (HDL)
To study the effect of HDL structure
on the ability to remove cellular FC, we have taken the approach of
reconstituting well defined HDL particles (rHDL) that are homogeneous
in composition and structure and testing their abilities to remove
cultured cell FC. We have previously studied the role of HDL
apolipoprotein structure (13) and phospholipid composition (14) on the ability of the particles to remove cellular FC. The
current study focuses on the effect of HDL particle size on this
function. A previous study by Agnani and Marcel (15) took a
similar approach to this problem. In that work,
phosphatidylcholine-containing rHDL particles of increasing size that
contained 2, 3, and 4 molecules of apolipoprotein AI (apoAI) were used
in cultured cell FC efflux experiments. The authors concluded that the
larger rHDL were the most effective FC acceptors and that the FC efflux
to these particles could be correlated to the particle diameter, the
number of apoAI molecules per particle, and the apoAI:phospholipid
ratio. However, all of these parameters are interrelated, and the
important characteristic among them could not be distinguished because
the rHDL contained different amounts of apoAI per particle and
different PL:apoAI ratios. A different conclusion about the effect of
rHDL size on FC efflux was reported by Jonas et
al.(16) . They concluded that smaller rHDL particles were
more efficient acceptors of cell-derived and lipoprotein-derived FC
than larger particles. However, this conclusion was reached when the
data (initially based on apoAI content) were postexperimentally
normalized to the phospholipid content of the acceptor particles. Since
the dependence of FC efflux on the phospholipid content of HDL is not a
linear relationship(13, 14, 17) , such an
analysis is not straightforward and can lead to misleading results.
Close inspection of the data reveals that the FC efflux promoted by the
various particles in this study correlated to the amount of PL
contained in the particle, in agreement with Agnani and
Marcel(15) .
The objective in the current study was to test
the hypothesis that the size of a FC acceptor particle can affect its
ability to remove FC from cells. Since the size of a rHDL particle is
related to its PL:apoAI ratio, it is important to understand the
dependence of FC efflux to rHDL in terms of both the phospholipid
concentration and the number of particles present in a FC efflux
incubation. This can be done by preparing differently sized rHDL
particles that contain the same number of molecules of apoAI per
particle and comparing their abilities to remove cell FC when they are
added to the extracellular medium at equal phospholipid concentrations.
This allows a straightforward normalization of the FC efflux to the
acceptor PL content, and the FC efflux can then be expressed in terms
of the number of particles present at a given phospholipid
concentration. FC efflux experiments of this type were performed with
four highly homogeneous, well characterized rHDL that varied in
particle diameter across the size range of human plasma HDL
(7.3-12.8 nm). The smallest three of these particles contained
two molecules of apoAI, with the largest containing three molecules. In
addition, the ability of unilamellar vesicles ranging in diameter from
25 to 180 nm in diameter to remove FC was also assessed. The results
indicate that, among rHDL, larger particles are more effective than
smaller particles when compared on a per particle basis. A similar
relationship was found for the large unilamellar vesicles although the
vesicles were less efficient acceptors than rHDL. This effect was not
due to the absence of apolipoproteins in the unilamellar vesicles,
because vesicles that contained adsorbed apoAI were not able to remove
more cellular FC than unmodified vesicles. The results from this study
suggest that the dependence of FC efflux on acceptor particle size may
reflect differing abilities of the variously sized acceptor particles
to access the region very close to the cell plasma membrane where most
of the FC removal is expected to occur.
The ability of these particles to remove
tritiated FC from mouse L-cells was determined. Previous studies
demonstrated that decreases in cellular FC mass closely paralleled
decreases in cellular FC radioactivity upon exposure to
acceptor-containing medium, indicating that the radiolabel correctly
reflected movement of FC mass(13) . In addition, the PL
concentration of the extracellular medium has been shown to be constant
over the 6-h time course for apoAI/POPC rHDL particles, indicating that
the particles did not precipitate out of solution during the
experiment(14) . Gel filtration chromatography of test medium
recovered after an efflux incubation showed that the FC radioactivity
appearing in the medium eluted at a volume that was characteristic of
the discoidal rHDL particle that was present initially in the
medium(14) . This indicated the following: (a) the
acceptor particles sequestered cellular FC, and (b) the
acceptor particles did not significantly change size during the efflux
assay (i.e. there was no fusion or aggregation of rHDL). Fig. 1shows the fraction of cellular FC remaining in the cell
over a 6-h FC efflux incubation with each of the four rHDL particles
present at 50 µg/ml of PL. The t
Figure 1:
Time course of
Figure 2:
The concentration dependence of the rate
for cholesterol efflux from mouse L-cell fibroblasts to apoAI
Figure 3:
The concentration dependence of the rate
of cholesterol efflux from mouse L-cell fibroblasts and rat FU5AH
hepatoma cells to a discoidal rHDL particle and unilamellar vesicles of
varying size. The incubation conditions were the same as those for Fig. 1except that the acceptor concentration is varied as shown.
The particles shown are as follows: 90:1 (PL:apoAI, mol ratio)
POPC/apoAI discoidal rHDL particle (
The difference in V
Figure 4:
The
concentration dependence of the rate constants for cholesterol efflux
from mouse L-cell fibroblasts to a discoidal rHDL particle and SUV
alone or with adsorbed apoAI. The incubation conditions were the same
as those for Fig. 1except that the acceptor concentration was
varied as indicated below. The SUV and rHDL were the same as shown in Fig. 2. The SUV-apoAI contained five molecules of apoAI per
vesicle. The openbars represent the FC efflux at 50
µg POPC/ml, and the filledbars represent the FC
efflux at 500 µg of POPC/ml. Each point represents three cell
wells, and the errorbars represent one S.D. The
values shown are corrected for the FC efflux to control
medium.
Figure 5:
The
dependence on acceptor particle number of the rate of FC efflux from
mouse L-cell fibroblasts to discoidal rHDL particles and unilamellar
vesicles of increasing hydrodynamic diameter. The incubation conditions
were the same as those for Fig. 1. The data from the L-cell
experiments in Fig. 2and Fig. 3have been expressed in
terms of the number of particles present at each PL concentration (see Table 1and Table 2). PanelA shows efflux
to the discoidal rHDL of various size compared with the SUV. The
acceptors shown are POPC:apoAI 37:1 (
Figure 6:
The
relationship of rHDL and unilamellar vesicle size and FC efflux
efficiency at a fixed number of acceptor particles. The FC efflux rate
was estimated for each type of acceptor particle used in this study at
a particle number of 4.4
There is general agreement that the PM pool of FC leaves the
cell via the aqueous diffusion mechanism(5, 32) . FC
transfer by this mechanism occurs by a two step process that involves
the desorption of a FC molecule from the PM and subsequent diffusion
through the aqueous phase until it incorporates into a PL-containing
acceptor particle (33) . At dilute acceptor concentrations, the
rate of cellular FC transfer to an extracellular particle is first
order with respect to the concentration of acceptor particle
[A] and depends on the rate constants for (a)
desorption from the PM (k
Apart from acceptor particle-induced changes in k
In summary, the differences in FC efflux efficiency noted for the
variously sized acceptor particles in this study may be explained by
two factors. The first is the differential effective FC-acceptor
collision frequencies induced by the PL packing characteristics of the
various particles. The second factor is the different concentrations of
the variously sized particles at the cell surface (or at specific cell
surface regions) due to the complexity of the cell surface. It appears
that particles in the size range of human plasma HDL can traverse the
cell surface barrier to a similar extent and that, on a per particle
basis, large rHDL particles are more efficient than smaller ones
because they provide a larger target for diffusing FC molecules.
Interestingly, large (11-25-nm) discoidal complexes have been
documented in the peripheral lymph of dogs in response to
hypercholesterolemia(46) . The presence of such particles in
this locus, which is important for FC removal from peripheral cells,
may be significant for the process of reverse cholesterol transport.
We thank Faye Baldwin, Sheila Benowitz, and Margaret
Nickel for expert technical assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)heterogeneity has become a topic of great interest
because of recent reports by several investigators that HDL subspecies
may play a particularly important role in the various stages of reverse
cholesterol transport(1, 2, 3, 4) ,
the postulated process by which excess peripheral cell cholesterol is
transported to the liver for catabolism(5) . HDL subspecies
have been characterized according to a variety of physical properties
including apolipoprotein composition (6) , charge
characteristics(7) , and particle size(8) . Of
particular interest are the studies of Fielding et
al.(9, 10) , which have demonstrated that a minor
charge subspecies of human HDL may be a particularly important factor
in the first step of reverse cholesterol transport, the removal of
cellular unesterified (free) cholesterol (FC) from cells. One
characteristic of this particle is its small size (70 kDa) (9) with respect to the bulk of human plasma HDL (180-390
kDa) (11) . It is conceivable that the size of an HDL particle
may be an important mediator of reverse cholesterol transport because
smaller particles are expected to more freely infiltrate the
interstitial space, the site of the majority of peripheral cell FC
removal(12) . Currently, the effect of particle size on this
function of HDL has not been studied in detail. Such studies of HDL
size subspecies have been hindered by the high degree of structural and
compositional heterogeneity that is inherent in even highly purified
preparations from human plasma.
Materials
Sodium cholate and bovine serum albumin were purchased from
Sigma. 1-Palmitoyl, 2-oleoyl phosphatidylcholine (POPC) was purchased
from Avanti Polar Lipids (Birmingham, AL) (+99% grade).
[1,2-H]cholesterol was obtained from DuPont NEN.
Minimal essential medium and bovine calf and fetal serum were from Life
Technologies, Inc. Media were supplemented with 50 µg/ml of
gentamicin (Sigma). All other reagents were analytical grade.
Methods
Purification of Human Apolipoproteins
Human HDL
was isolated from the fresh plasma of normolipidemic subjects by
sequential ultracentrifugation as described previously(18) .
HDL was delipidated (apoHDL) in ethanol:diethyl ether as described by
Scanu and Edelstein (19) , and purified apoAI was isolated by
anion exchange chromatography on Q-Sepharose (20) and stored in
lyophilized form at -70 °C. Prior to use, apoAI was resolubilized
in 6 M guanidine HCl and dialyzed extensively against standard
Tris buffer (10 mM Tris, 150 mM NaCl, 1.0 mM EDTA, pH 8.2).
Preparation and Characterization of Discoidal
Reconstituted HDL Particles
All discoidal particles were
reconstituted using the sodium cholate removal method described in
detail elsewhere (21) using the starting PL to protein
stoichiometries shown in Table 1. Intact rHDL were separated from
unreacted lipid and protein by gel filtration chromatography on a (2.5
100 cm) Superose 6 column eluted with Tris buffer (0.7 ml/min
at 25 °C). The homogeneity and hydrodynamic diameters of rHDL
particles were estimated by Phast native polyacrylamide gradient gel
electrophoresis using precast, 8-25% gradient gels (Pharmacia
Biotech Inc.)(21) . The particle migrations were determined to
within 0.1 mm by digitizing the gel with a Mustek 105 scanner followed
by computer image analysis (Jandel Scientific, San Rafael, CA). Intact
rHDL particles were chemically analyzed using the Markwell modification
of the Lowry protein assay(22) , while PL were determined as
inorganic phosphorus by the method of Sokoloff and
Rothblat(23) . The number of protein molecules per particle was
determined by cross-linking the apoAI with dimethyl
suberimidate(24) . The electrophoretic mobilities of native and
reconstituted HDL were measured on Beckman Paragon preformed 0.5%
agarose gels, and the potential at the particle surface of shear
(surface potential, S) was calculated as described by Sparks
and Phillips(25) . The average percentage of
-helix
content was determined by circular dichroism (CD) spectroscopy on a
Jasco J41A spectropolarimeter at 25 °C in a 0.1-cm path length
quartz cuvette. The percent
-helix was determined from the molar
ellipticities at 222 nm(21) .
Preparation and Characterization of PL Unilamellar
Vesicles
POPC small unilamellar vesicles (SUV) with estimated
diameters of 25 ± 5 nm were made using the sonication technique
of Barenholz et al.(26) . The SUV that had been
reisolated by ultracentrifugation at 40,000 rpm in a 50-Ti rotor for 2
h at 25 °C appeared homogeneous by gel filtration chromatography.
SUV preparations in Tris buffer were mixed with apoAI to make SUV with
adsorbed apoAI (SUV-apoAI) as described by Yokoyama et
al.(27) . The initial preparation contained SUV and apoAI
at a 170:1 mol POPC:mol apoAI ratio, and this mixture was incubated for
16 h at 25 °C. Using gel filtration chromatography, SUV-apoAI
fractions that were similar in size to protein-free SUV (25 nm) were
combined and concentrated. Analysis of the PL and protein content of
four independent preparations resulted in SUV-apoAI that contained
4-5 molecules of apoAI per SUV. The isolated SUV-apoAI appeared
to be stable for at least 3 days at 4 °C; no lipid-free apoAI was
detectable by gel filtration chromatography during this period (data
not shown). LUVs were prepared by the extrusion technique of Hope et al.(28) . Briefly, POPC multilamellar liposomes
were placed into an extrusion device (Lipex Biomembranes Inc.,
Vancouver, B.C.), which passed the mixture through polycarbonate
filters of varying pore size under pressures of 100-500
lb/in. The resulting LUVs were classified according to the
pore size of the filter through which they were passed (for example,
LUV
s were created by passage through a 50-nm filter). The
characteristics of the LUVs made with 50-, 100-, and 200-nm filters are
shown in Table 2.
Efflux of Plasma Membrane Cholesterol
Mouse L-cell
fibroblast or rat Fu5AH hepatoma cells were used to monitor the FC
efflux efficiencies of the various vesicles and rHDL. The methods for
the efflux assay as well as the postexperiment work-up were as
previously reported (13) with the following exceptions. The
L-cells were plated 6 days prior to the experiment in 12-well cell
plates (22 mm) at 75-100 10
cells/well.
1.0-1.5 µCi/well [
H]FC in
bicarbonate-buffered minimal essential medium with 3% fetal calf serum
was used for the cell-labeling procedure. After a brief wash, the
efflux measurements were initiated by the application of 1.0 ml/well of
the test medium containing 0.5% bovine serum albumin and the rHDL
complex at the appropriate concentration. Typical cell protein values
were between 250 and 300 µg of protein/well. Efflux experiments
that used rat Fu5AH hepatoma cells were done exactly the same as the
L-cell efflux experiments except that only 0.5-1.0 µCi of
labeled FC was used to label the cells.
Data Analysis
The fractional release of cellular
FC determined experimentally was analyzed as described in detail for
these systems previously(13) . Briefly, the kinetic analysis
assumes a closed system in which FC exists in one of two kinetic pools,
either the cellular FC pool or the acceptor FC pool. The equilibration
of FC between these pools is fit to the single exponential equation Y = He
+ H
(see (5) for a detailed
description of this equation), which has been shown to fit the data
better than a double exponential equation by comparison of the F statistic (13) . Y represents the fraction of
radiolabeled FC remaining in the cells, t is the incubation
time in h, H
is a preexponential term that
reflects the fraction of cellular FC that exists in the medium at
equilibrium, g is the sum of the rate constants for efflux (k
) and influx (k
), and H
is a constant that represents the fraction of
labeled cell FC that remains associated with the cells at equilibrium
due to a constant retrograde flux of FC from the extracellular acceptor
to the cell. H
, g, and H
are variables that can be fit to the experimental data by
computer (Jandel Scientific, San Rafael, CA). The apparent rate
constant for the efflux process (k
) is the product
of H
and g. This value should be
considered ``apparent'' because it is dependent on acceptor
particle concentration under some conditions. The apparent t
value in hours is then calculated as follows: t
= ln 2/k
.
FC Efflux to Discoidal rHDL Particles Compared on the
Basis of PL Concentration
To determine the effect of particle
size on FC efflux within the size range of human HDL particles,
homogeneous, discoidal rHDL that varied in size from 7 to 13 nm in
diameter were reconstituted with apoAI and POPC. Table 1shows
clear increases in the particle hydrodynamic diameter as the rHDL
phospholipid to protein ratio was increased. All rHDL contained 2
molecules of apoAI except for the 12.8-nm particle, which contained 2
or 3 molecules. The particles appeared homogeneous on native
polyacrylamide gradient gel electrophoresis after reisolation by gel
filtration chromatography (data not shown). All rHDL exhibited surface
potential values in the pre- migration range according to the
classification scheme proposed by Sparks and Phillips (-7.0 to
-10.5 mV)(25) . In addition, the
-helix content of
the associated apoAI molecules increased with the increasing PL:apoAI
ratio in agreement with previous studies in this laboratory (refer to (21) for a detailed discussion of the effects of PL:apoAI ratio
on the structure of rHDL particles). These data show that the
conformation of apoAI changes in response to increases in the PL
content of rHDL particles.
values for the
7.3-, 9.3-, 10.0-, and 12.8-nm rHDL were 12.6 ± 1.3, 12.0
± 2.0, 12.5 ± 1.6 and 13.5 ± 2.2 h, respectively,
indicating that the FC efflux to each of these complexes was similar
despite the differences in PL:apoAI ratio and apoAI conformation. To
determine the dependence of FC efflux on acceptor particle
concentration, the FC efflux rate constants were measured for each
particle over a range of 10-200 µg of POPC/ml (Fig. 2). No significant differences were detectable at low or
high acceptor particle concentrations. Analysis of the kinetic data
provides information about the predicted equilibrium distribution of FC
between the cell and acceptor pools. At low rHDL concentrations (10
µg of PL/ml) the distribution was similar for all four rHDL with
7-10% of cell FC present in the acceptor pool at equilibrium. At
200 µg of PL/ml this increased to 75-80% of cellular FC in
the medium (data not shown). To estimate the maximal FC efflux rate
that can be attained by these particles, the initial rates of cellular
FC efflux were plotted as a function of particle concentration as
proposed by Hofstee(29) . This linearizes the data so that a
maximal velocity (V
) can be estimated by
extrapolating the data to the condition of infinitely high acceptor
concentration(13, 14) . It should be noted that the V
values as expressed below in percentage of FC
released per h are actually the maximum k
.
However, in the experiments reported in this study, all of the cell FC
behaved as a single kinetic pool for efflux. Thus, if the cell
concentration of FC is taken as unity throughout, then V
equals the maximum k
. The
actual V
can be obtained by multiplying the
maximal k
by the FC mass contained in the cells at
the initiation of the experiment (e.g. at 40 µg of FC/mg
of cell protein a V
value of 10%/h corresponds
to a mass release of 4 µg of FC/h). All rHDL particles exhibited a
similar V
of 10-11% of cell FC released/h.
A similar independence of FC efflux on rHDL particle size was found in
experiments using rat Fu5AH hepatoma cells as the FC donors (data not
shown). These results indicate that, when compared on the basis of PL
content, no difference in FC efflux is observed between rHDL of varying
PL:apoAI ratio.
H-labeled
free cholesterol efflux from mouse L-cell fibroblasts to apoAI/POPC
rHDL of increasing particle hydrodynamic diameter. Mouse L-cell
fibroblasts that had been trace-labeled with
H-labeled free
cholesterol in 22-mm tissue culture wells were incubated for 6 h at 37
°C in a humidified incubator (5% CO
) with 1.0 ml of
test medium containing 0.5% bovine serum albumin, 1.0 µg/ml Sandoz
compound 58035 (to prevent intracellular esterification of the label),
and the apoAI/POPC rHDL (see Table 1) at 50 µg of POPC/ml.
The acceptors shown are POPC:apoAI 37:1 (
), 75:1 (
), 107:1
(▾), and 156:1 (
) (mol:mol). The verticalaxis indicates the fraction of initial labeled FC that remained in the
cell at the designated times. Each point represents the mean of six
cell wells. The errorbars represent one S.D. All curves were obtained by fitting the entire time course to the
model for tracer equilibration between two pools (see
``Methods'').
POPC
complexes of increasing particle hydrodynamic diameter. The incubation
conditions were the same as those for Fig. 1except that the
acceptor concentration was varied as shown. The acceptor particles are
the same as in Fig. 1with the same symbols. Each point
represents six cell wells, and the errorbars represent one S.D. The values shown are corrected for the FC
efflux to control medium.
FC Efflux to Unilamellar Vesicles Compared on the Basis
of PL Concentration
To further examine the effect of acceptor
particle size on FC efflux efficiency, unilamellar vesicles of varying
diameters (25-185 nm) were produced by sonication and extrusion
techniques (Table 2). The abilities of these vesicles to accept
cellular FC from L-cells and rat Fu5AH hepatoma cells were compared
over a range of acceptor PL concentrations (Fig. 3). As for the
rHDL, all of the unilamellar vesicles were shown by gel filtration
chromatography to not significantly change size or precipitate out of
solution during a 6-h FC efflux incubation (data not shown). The FC
efflux to a 90:1 (mol:mol) PL:apoAI discoidal rHDL is also shown. In
all cases, the rHDL was substantially more efficient than the same
amount of PL present as an SUV. This was consistent with previous
observations in this laboratory and
others(13, 14, 16) . Furthermore, the SUV
were more efficient than the various LUVs, which were not significantly
different from each other. The FC efflux results were similar for both
cell types studied (Fig. 3, A and B). The
equilibrium distribution of FC at each concentration was similar for
all unilamellar vesicles; about 75-80% of the FC was in the
acceptor pool at equilibrium at high concentrations. The equilibrium
distributions exhibited a different concentration dependence in the
case of the unilamellar vesicles compared with the discoidal rHDL; the
vesicles exhibited a higher FC capacity at low acceptor concentrations
(data not shown). This result is likely due to differences in the
retrograde FC flux from the acceptor to the cell. The tvalues for FC desorption from rHDL, SUV, and LUV
particles are about 15, 45, and 235 min,
respectively(5, 30, 50) . As an additional
comparison, isolated human LDL was used as an acceptor of L-cell FC (Fig. 3A). LDL is of comparable diameter to the SUV
(20-35 nm), but it contains a nonexchangeable apolipoprotein
(apoB-100). Despite this, the LDL and SUV exhibited similar abilities
to remove labeled FC (the FC mass distributions were different because
LDL contains FC initially). The order of FC efflux efficiency as
reflected in the k
values was rHDL > SUV
= LDL > LUV, indicating a general correlation between FC
efflux efficiency and particle size, with smaller particles being more
efficient than larger ones when compared on the basis of PL
concentration. However, this relationship does not hold for particles
with diameters larger than 70 nm and, as shown in the previous section,
also does not hold for rHDL particles below 12 nm in diameter.
), SUV (
), LDL
isolated from human plasma at d = 1.019-1.066
g/ml (filledhexagon), LUV
(
),
LUV
(
), and LUV
(
). PanelA shows the results from a FC efflux incubation with
mouse L-cells, and panelB shows the same experiment
performed with rat Fu5AH hepatoma cells. Except for the rHDL data with
the L-cells (n = 9 wells), each point represents three
cell wells, and the errorbars represent one S.D. The
values shown are corrected for the FC efflux to control
medium.
The
differences in FC efflux efficiency noted at low acceptor particle
concentrations persisted at high concentrations. The average V values for the L-cell FC efflux experiment (in
terms of percentage of cellular FC released per h) for the discoidal
rHDL, SUV, LDL, LUV
, LUV
, and LUV
were 12.0 ± 2.2, 3.0 ± 0.1, 3.0 ± 0.1, 1.0
± 0.1, 1.0 ± 0.1, and 1.0 ± 1.0, respectively. The V
values from the rat Fu5AH hepatoma cell FC
efflux experiment for the discoidal rHDL, SUV, LUV
,
LUV
, and LUV
were 20.0 ± 1.3, 10.4
± 0.2, 1.2 ± 0.1, 1.5 ± 0.1, 1.0 ± 0.1, and
1.2 ± 0.1%/h, respectively. Thus, the V
values for the rHDL, SUV, and LUV acceptor particles did not
converge to a common value.
between an SUV and a rHDL made with the same PL has been reported
previously(13, 14) , and it was hypothesized that the
rHDL apolipoproteins were capable of interacting with the cell plasma
membrane (PM) to increase the intrinsic rate of FC desorption from the
lipid surface into the aqueous medium(13) . Since discoidal
rHDL and SUV have significantly different particle diameters (and,
therefore, different diffusion coefficients (see Tables I and II)), a
direct test of this hypothesis requires the comparison of acceptor
particles of similar size and composition that either do or do not
contain apolipoproteins(31) . To this end, SUV were prepared
with five molecules of apoAI adsorbed to the vesicle surface without
significantly changing the size of the original SUV (see
``Methods''). The abilities of the SUV and SUV-apoAI to
remove cellular FC from mouse L-cells and Fu5AH cells were compared on
the basis of acceptor PL content. Gel filtration chromatography
demonstrated that the FC counts appearing in the SUV-apoAI particles
over a 6-h FC incubation had a similar elution volume to that of the
unmodified SUV, indicating that they were stable during the time course
of the incubation and were of similar size to the SUV. Fig. 4shows the FC efflux rate constants for efflux from mouse
L-cells found with a low (50 µg of acceptor particle POPC/ml) and a
high (500 µg of POPC/ml) concentration of SUV, SUV-apoAI, and a
90:1 (POPC:apoAI (mol:mol)) discoidal rHDL particle. The discoidal rHDL
was substantially more efficient at both concentrations than the same
amount of PL present in an SUV. It is clear that the presence of five
molecules of apoAI on the surface of particles of otherwise similar
size and PL content did not result in an increase in the particle FC
efflux efficiency. Similar results were obtained using Fu5AH hepatoma
cells (data not shown).
FC Efflux to Discoidal rHDL and Unilamellar Vesicles
Compared on a Per Particle Basis
Since the PL:apoAI ratios
differed among the rHDL (Table 1), the number of particles per ml
of extracellular medium at a given concentration of PL also varied. At
a given PL concentration, for every one 12.8-nm rHDL particle there
were 5.0 times as many 7.3-nm particles, 2.2 times as many 9.3-nm
particles, and 1.4 times as many 10.0-nm particles. When the data from Fig. 2are replotted correcting for the particle number, it is
clear that the FC efflux efficiency increased with particle size (Fig. 5A). These results are consistent with those of
Agnani and Marcel(15) . A similar analysis is shown in Fig. 5B for the unilamellar vesicles based on the
number of PL molecules present in each vesicle (Table 2). The
efflux efficiency slightly increased with vesicle size. Fig. 6shows the rate constant for FC efflux for all of the
particles in this study at a constant number of acceptor particles (4.4
10
particles/ml). The rate constant for FC efflux
exhibited a linear dependence on the radius of both the rHDL (Fig. 6, inset) and the LUV (Fig. 6). The slope
of the regression line through the rHDL data was significantly steeper
than that for the LUV, indicating that a change in size of the rHDL
improved the ability of the particle to remove cell FC to a greater
extent than the same change in size of the LUV. The FC efflux to the
SUV did not fit either of these relationships. Relative to the rHDL
discs, the SUV were less efficient than predicted by their size,
whereas, relative to the LUV, the SUV were more efficient than
predicted by their size. Apparently, the rate of FC efflux is
proportional to acceptor particle size when particles of similar
structure are compared but not when structurally dissimilar particles
are compared.
), 75:1 (
), 107:1
(▾), 156:1 (
), and SUV (
). PanelB compares efflux to the LUV; the particles shown are as follows:
LUV
(
), LUV
(
), and LUV
(
). The data in panelA are from six
wells, whereas the data in panelB are from three
cell wells. The errorbars represent one S.D. The
values shown are corrected for the FC efflux to control
medium.
10
per ml of medium by
analyzing the data in Fig. 2and 3 by the method proposed by
Hofstee (29) and extrapolating to the appropriate PL
concentration for each of the different particles at this particle
number. This particle number was selected because it was the maximum
number of the LUV
preparation studied (i.e. at
10,000 µg of POPC/ml there are 4.4
10
LUV
particles/ml present). The symbols are
the same as in Fig. 5.
) and (b)
collision with and incorporation into the acceptor particle (k
). Under these conditions, the rate of FC efflux (v) = k
[A] where k
is a function of the rate constants k
and k
(see the Appendix in (14) ). In contrast, at high acceptor concentrations, the FC
efflux rate depends on the rate constant for FC desorption from the PM (k
) as well as the concentration of PM FC that is
available for desorption [DC] so that v = V
= k
[DC](14) . If it is assumed
that [DC] is the same for any two given acceptor particles,
it follows that both particles should exhibit a common V
at sufficiently high acceptor particle
concentrations. The possible explanations for the observed differences
in FC efflux for variously sized FC acceptor particles at low and high
acceptor concentrations are considered below.
FC Transfer under Conditions of Low Acceptor Particle
Concentration
At low acceptor particle concentrations (<100
µg of POPC/ml), the rate of FC efflux depends on k, which includes the term describing the
propensity of FC to collide with and incorporate into the acceptor
particle (k
). Under these conditions, this term
can be affected by (a) different diffusion coefficients of the
various acceptor particles and/or (b) changes in the
percentage of FC-acceptor collisions that result in successful
incorporation into the acceptor particle(14) . The collision
frequency between dissolved FC molecules and extracellular acceptor
particles per unit volume is directly proportional to the product
(number of acceptor particles present
particle
radius)(31) . Thus, a given number of LUV make better
``targets'' for diffusing FC molecules than the same number
of small rHDL, despite slower LUV diffusion rates (see Tables I and II
for the respective diffusion coefficients). However, at a given PL
concentration, the relative number of particles can differ by up to 2
orders of magnitude, with many more rHDL particles present than LUV
particles (see Table 1and Table 2). Relative to LUV, the
small target size of rHDL is offset by their greater numbers at a given
PL concentration, explaining the ranking of acceptor efficiencies
depicted in Fig. 3. As predicted by DeLamatre et
al.(31) , when compared at a fixed particle number
concentration, rHDLs of varying sizes exhibit a linear relationship
between the degree of FC efflux and particle radius, with larger rHDL
being more efficient than smaller ones (Fig. 6). A linear
relationship between these parameters also exists for the various LUVs.
Interestingly, the slope of the rHDL line is significantly steeper than
that of the LUV line, indicating that small increases in rHDL size have
larger effects on the number of rHDL-FC effective collisions than
similar increases in LUV size. Furthermore, the FC efflux to the SUV
does not appear to fit on either the rHDL or the LUV line. These
observations may be due to differences in the ability of colliding FC
molecules to incorporate into the particle (effective collision
frequency). We have proposed that FC acceptor particles containing more
loosely packed PL molecules exhibit a higher effective collision rate
than those with relatively ordered, tightly packed PL(14) . The
PL bilayer of an LUV is essentially a planar surface in which PL acyl
chains are motionally restricted (34) compared with the curved
surface of the SUV(33, 35) . An increased FC-SUV
effective collision frequency relative to the LUV may explain the
higher FC efflux efficiency of the SUV than predicted from its size by
the LUV data (Fig. 6). In the case of the rHDL particles, the
presence of the apolipoprotein may facilitate the incorporation of
incoming FC molecules by creating PL packing defects in the particle
surface(30) , contributing to the increased efficiency of the
rHDL over the vesicles. Although the presence of apoAI on the surface
of an SUV does not increase its ability to accept cell FC (Fig. 4), it may be that apoAI facilitates FC incorporation into
discoidal particles more efficiently than into spherical particles.
More FC efflux studies using spherical and discoidal acceptor particles
are required to address this issue.
FC Transfer under Conditions of High Acceptor Particle
Concentrations
At high concentrations of acceptor particle
(>500 µg of POPC/ml), the FC efflux differences already noted
between the various particle types persist, resulting in a range of V values. This result is unexpected because the V
for different particles should converge to a
single value that reflects the rate-limiting step of FC desorption from
the cell PM. The factors responsible for the V
differences can be examined by assuming two limiting situations:
a) the experimentally derived V
values represent
the true maximal rate of FC efflux to the various acceptor particles
or, b) these V
values do not represent the true
maximal rate of FC efflux to the acceptor particles. Each of these
situations is addressed in turn below.
V
If the observed VValues Represent True Maximal FC
Efflux Rate
values are
reliable expressions of the true maximal FC efflux velocity (v) to the various particles then, as discussed above, v = V
= k
[DC](14) . It follows that
particles exhibiting different V
values must
differentially affect either the intrinsic tendency of FC to desorb
from the PM (k
) or the concentration of cell PM FC
that is available for desorption [DC] (or both). k
can be affected by a modification of the PM
either via the exchange of acceptor-derived PL into the PM or through
an interaction of acceptor particle-derived apolipoproteins with the
cell PM(13, 14) . Since all of the particles in this
study contain the same type of PL and it has been demonstrated that the
exchange rate of PL is significantly slower than that for
FC(32) , it is unlikely that exchange of acceptor PL can
account for the V
differences. Similarly, the
demonstration that SUV of otherwise similar structure and differing
only in the presence or absence of apoAI, can remove cellular FC at
similar rates (Fig. 4) suggests that the observed V
differences between discoidal rHDL and SUV (13, 14) are not due to an apolipoprotein-PM
interaction. Although the possibility exists that SUV-associated apoAI
is not in the correct conformation to interact with the cell surface,
the observation in Fig. 3that unilamellar vesicles that lack
apolipoproteins also exhibit different V
values (e.g. LUV versus SUV) argues that the V
differences are not due to the presence of an
apolipoprotein.
, changes in the concentration of cell FC
available for efflux ([DC]) could also affect the V
if the acceptor particles have limited access
to regions of the plasma membrane that may be particularly important
for FC efflux. It can be proposed that small particles have access to
PM regions that may be obscured by extracellular matrix or to highly
curved surfaces located within cell surface folds or
invaginations(36) , whereas large particles may be restricted
from these sites. The observation of monoexponential FC efflux kinetics
appears to preclude the existence of such regions, although the time
frame of the FC efflux studies may not be sufficient for their
detection. PM FC heterogeneity has been proposed in a number of cell
types(37) , and kinetic studies have suggested that certain
regions of the membrane may release FC faster than others(38) ,
although the structural characteristics of these regions remain
unclear. This issue will require further study.
V
Alternatively, it can be proposed that a
complication inherent in the cell FC efflux assay prevents the
measurement of VValues Do Not Represent True Maximal
FC Efflux Rate
values that represent true
maximal rates of FC efflux to the various acceptor particles. Since the
FC efflux differences that are noted at low acceptor concentrations
persist at high concentrations, a simple explanation is that the same
rationale is responsible for the observations at both concentrations (14) . It was proposed above that different effective collision
frequencies between dissolved FC molecules and the various acceptor
particles can account for the observed differences in FC efflux under
conditions of low acceptor concentrations. Ordinarily, this explanation
would not be expected to account for FC efflux differences at high
acceptor particle concentrations because a surplus of acceptor
particles should compensate for a low effective collision frequency (5) . Since FC efflux differences are observed at high acceptor
particle concentrations, it follows that the concentration of acceptor
particles at the surface of the cell PM (where most of the FC transfer
is expected to occur) is limited to a concentration that is a) distinct
from the bulk medium concentration and b) below that required to make
FC desorption from the PM rate-limiting(14) . In this case, the
rate of FC efflux is described by a similar equation as that for low
acceptor particle concentrations. Thus, the FC efflux rate is governed
by the k
term as well as the concentration of
acceptor at the cell surface [A
]. The k
term can be affected by the effective
collision rate of the various acceptor particles as in the case of low
acceptor concentration. Furthermore, since small rHDL exhibit more
efficient FC efflux than LUV, it is logical to propose that the cell
surface exhibits a characteristic that limits the
[A
] of large particles at the cell
surface and allows a higher [A
] for
smaller particles. Thus the V
differences among
the various particles may arise from different FC-acceptor effective
collision frequencies as well as different concentrations of the
various sized particles at the cell surface. An observation that
supports this idea is that, in L-cells, the rHDL exhibits a 4-fold
higher V
value than the SUV. The same comparison
in rat Fu5AH hepatoma cells yields only a 2-fold difference. These
discrepancies may be explained by differential partitioning of the
particles into the proximal and distal regions of the extracellular
medium, presumably in response to the variations in cell surface
complexity between the two cell types.
Restricted Access of Acceptor Particles to the Cell
Surface
It is not yet clear whether the surface structure of
living cells restricts large acceptor vesicles from specific regions of
the PM that are important for FC efflux (i.e. effectively
changing [DC]) or if it restricts them from closely
approaching the cell surface altogether (change in
[A]). The most obvious cellular
characteristics that may restrict acceptor particle access to the cell
surface include the unstirred water layer (USWL) at the cell
surface(39) , the convoluted nature of the PM(36) , and
the extracellular matrix. The USWL is a layer of fluid over a planar
cell surface, which is not subject to convective currents found in the
bulk medium, and passage though it occurs solely by passive
diffusion(40) ; its thickness for most cells present in a
planar cell monolayer is between 300 and 800 µm(39) . The
USWL is known to dramatically alter the cell surface concentration of
solutes that are destined for passage through the cell PM(39) .
However, in the case of an extracellular FC acceptor particle that is
not metabolized by the cell, the concentration of acceptor particles at
the cell surface should ultimately equilibrate with the bulk medium
particle concentration. The t
for this
equilibration is proportional to the square of the USWL thickness and
inversely proportional to the diffusion coefficient of the particular
solute (39) (see Table 1and Table 2). Assuming an
USWL thickness of 300 µm, it can be calculated that the t
for equilibration of the the largest
particles (LUV
s) across the USWL is about 3 h, whereas
the t
for the smallest rHDL is about 6 min.
Therefore, during the 6-h FC efflux experiments the bulk medium
concentration of LUV
does not fully equilibrate across
the USWL because only two half-times have elapsed. This is a possible
explanation for the different V
values obtained
for the different size particles. Another cell surface complication is
the presence of an extracellular matrix composed of
proteoglycans(41) . This matrix is 50-200 nm thick in
vascular endothelial cells (42) and may be a selective barrier
for the passage of various materials(43) . It is possible that
this matrix can exclude extracellular particles on the basis of size,
operating like a filter over the cell surface. For example, it has been
demonstrated that arterial endothelial cells can take up LDL faster
when sialic acid residues are removed from the cell
glycocalyx(44) , and albumin uptake by cells is more efficient
at cell surface sites that are less glycosylated than at heavily
glycosylated sites(45) . In addition to the cell glycocalyx,
the convoluted nature of the cell surface may also contribute to the
exclusion of large acceptor particles from the cell surface. Bovine
aortic endothelial cells have been shown to exhibit ridges and
depressions that can vary from the plane of the cell surface by
50-100 nm(36) . Determination of the cellular
characteristic(s) that are responsible for this effect requires further
studies in which the complexity of the cell surface is manipulated.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.