(Received for publication, April 11, 1995; and in revised form, May 17, 1995)
From the
In this study, we compared cholesterol efflux mediated by either
high density lipoproteins (HDL
The first step in reverse cholesterol transport is the efflux of
cellular cholesterol to a suitable acceptor, and this process is
thought to be mediated by HDL
In vitro studies have
demonstrated that exposure of fibroblasts to cyclodextrins can produce
cell toxicity, and that the extent of this toxicity is reduced by the
presence of serum(6) . Exposure of erythrocytes to
cyclodextrins results in hemolysis in the order of
Efflux
was calculated from the fraction of initial
[
Direct mass determinations of released cell
cholesterol and phospholipid were conducted on the media from L-cells
exposed to 10 mM 2OHp
The high efficacy of three types of
Figure 1:
Dose-response curve for L-cell
[
Figure 2:
Time course for efflux of L-cell
[
Figure 3:
Relation between cell sterol mass and
[
Figure 4:
Release of L-cell
[
Figure 5:
Efflux of
[
Figure 6:
Dose-response of cholesterol efflux from
Fu5AH hepatoma cells, L-cells, and human skin fibroblasts exposed to
HDL
Figure 7:
V
Figure 8:
Binding of
[
Because of their ability to encapsulate and solubilize
hydrophobic molecules, cyclodextrins have been used extensively as drug
delivery systems(6, 28) . The three general types of
cyclodextrins are distinguished by the number of glucose units in these
cyclic oligomers and are:
The
kinetics of cholesterol efflux to cyclodextrins are different from
those observed with a physiological cholesterol acceptor such as the
HDL
Of particular interest is
the observation that the cell specificity of the cholesterol release
rate that has been observed consistently with native and reconstituted
acceptor particles (29, 36) is not evident when
different cell types are exposed to cyclodextrins ( Fig. 6and Fig. 7). As illustrated in Fig. 6A, the rates of
release from the three cell types exposed to HDL are Fu5AH hepatoma
> L-cells > human skin fibroblasts, consistent with previously
published data(29) . In contrast, all of these cells exhibited
similar rates of release to either 2OHp
Two closely related
questions that arise from the present investigation on
cyclodextrin-mediated cholesterol efflux are as follows. 1) What is the
mechanism underlying efflux to phospholipid-containing acceptors whose
rate is specific for each cell type? 2) Is the mechanism for
cholesterol efflux to cyclodextrins similar to that of lipoproteins
such as HDL? We have previously proposed that the efflux or exchange of
cholesterol between cells and lipoproteins proceed primarily by an
aqueous diffusion mechanism in which cholesterol molecules desorb from
the cell membrane and are subsequently incorporated into the
lipoprotein acceptor particle(2, 15) . We also
proposed that the efficiency of the efflux process was influenced by
the presence of an unstirred water layer surrounding cells which
reduced diffusion and mixing of the desorbed cholesterol molecules with
acceptor particles, particularly at low concentrations of
acceptors(2, 15) . A prediction of this model was that
the rate-determining step in cholesterol efflux was the desorption of
cholesterol from the plasma membrane and thus: 1) at infinite acceptor
concentrations the V
The plasma membrane pool of
cholesterol leaves the cell by the aqueous diffusion mechanism which
involves the initial desorption of cholesterol molecules from the
membrane(2, 15) . At high acceptor concentrations
where V = V
Sufficient information is not currently available to permit
a definitive mechanistic explanation of how cyclodextrins remove cell
cholesterol. However, some possibilities can be eliminated. The fact
that there is a very large difference in the release of cholesterol and
phospholipid indicates that membrane solubilization or vesiculation is
not occurring. If this were the case, the relative release of
cholesterol and phospholipid should correspond to their abundance in
the plasma membrane. In addition, the very low association of
cyclodextrins with cells (Fig. 8) indicates that irreversible
binding and/or internalization events are probably not playing a
significant role in cholesterol efflux. Further studies will be
necessary to establish the mechanism(s) underlying the highly efficient
removal of cholesterol from cells by cyclodextrins.
In addition to
the use of cyclodextrins as valuable tools for studies on cholesterol
transport and membrane lipid flux, the cyclodextrins and related
compounds (39) have the potential to serve as model agents that
could impact on either the progression or regression of the
atherosclerotic plaque. Administration of large doses of these
compounds could shift the distribution of cholesterol between the
tissue and plasma compartments by having the acceptor molecules acting
as a sink for cholesterol. On the other hand, it is possible that low
concentrations of cyclodextrins could function as catalysts, shuttling
cholesterol between compartments and enhancing rates of exchange, while
not being present in sufficient quantities to shift equilibria. It is
this latter mode that is particularly intriguing and justifies further
investigation.
) or
-cyclodextrins,
cyclic oligosaccharides that are able to dissolve lipids in their
hydrophobic core.
-Cyclodextrin,
2-hydroxypropyl-
-cyclodextrin, and methyl-
-cyclodextrin at 10
mM induced the release of 50-90% of L-cell
[
H]cholesterol after 8 h of incubation, with a
major portion of this cholesterol being released in the first 1-2
h of incubation. The cholesterol efflux kinetics are different if cells
are incubated with HDL
, which induces a relatively
constant rate of release of cholesterol throughout an 8-h incubation.
Cholesterol efflux to cyclodextrins was much greater than phospholipid
release. To test the hypothesis that maximal efflux rate constants for
a particular cell are independent of the type of acceptor, we estimated
the maximal rate constants for efflux (V
) of
cellular cholesterol from L-cells, Fu5AH cells, and GM3468A
fibroblasts. The rate constant for HDL
-mediated efflux
varied among cell lines in the order Fu5AH > L-cells >
fibroblasts. However, these differences were not evident when
cyclodextrins were used as cholesterol acceptors. The estimated V
values for cyclodextrin-mediated efflux were
3.5-70-fold greater than for HDL
for the three cell
lines. The very high efficiency of cyclodextrins in stimulating cell
cholesterol efflux suggests that these compounds can be used in two
general ways for studies of atherosclerosis: 1) as research tools to
probe mechanisms of cholesterol transport and aspects of membrane
structure or 2) as potential pharmacological agents that could modify in vivo cholesterol metabolism and influence the development
of the atherosclerotic plaque.
(
)or specific HDL
subclasses(1, 2, 3) . Although extensively
investigated, the exact mechanism underlying the movement of
cholesterol molecules from the plasma membrane to extracellular
acceptors remains controversial. Although the generally accepted
mechanism is now thought to involve the movement of cholesterol
molecules from the cell membrane through the aqueous phase to the
acceptor particle (i.e. aqueous diffusion
mechanism)(2) , other mechanisms have been proposed, including
collision (4) or receptor-mediated mechanisms(5) .
-Cyclodextrins, cyclic oligosaccharides consisting of 7
(1-4)-glucopyranose units, are water-soluble compounds with
a hydrophobic cavity capable of dissolving hydrophobic compounds and
thus enhancing their solubility in aqueous
solutions(6, 7) . Cyclodextrins have been used
extensively as drug delivery vehicles(6, 8) , and
derivatives of
-cyclodextrins have been made by chemical
modifications of the hydroxyl groups which greatly improve their
solubility and their ability to dissolve hydrophobic compounds, as well
as reducing their toxicity(9, 10, 11) . In vitro,
-cyclodextrins have a high affinity for sterols
as compared to other lipids(11, 12) , and, because of
the relatively high specificity of
-cyclodextrins for cholesterol,
it has been suggested that these compounds might be effective in
modifying cholesterol metabolism in
vivo(11, 13) . It can be proposed that
cyclodextrins can be used in two general ways for studies of
atherosclerosis: 1) as research tools to probe mechanisms of
cholesterol transport and aspects of membrane structure or 2) as
potential pharmacological agents that could modify in vivo cholesterol metabolism and influence the development of the
atherosclerotic plaque. Although there is extensive literature on
cyclodextrins, relatively little has been published on their ability to
influence cholesterol metabolism, either in vitro or in
vivo. It has been demonstrated by Frijlink et al.(10) that injection of either
-cyclodextrin or
2OHp
CD (200 mg/kg dose) resulted in transient decreases in both
serum-unesterified and total cholesterol. It has been proposed that the
cholesterol-lowering effect of the cyclodextrins was due to their
ability to efficiently cross the capillary wall and to function as
cholesterol carriers that redistribute cholesterol from the
interstitial space to the plasma compartment(10) , after which
it was rapidly cleared in the urine. It is believed that the
cyclodextrin-cholesterol complexes are processed within the kidneys.
Within the kidneys, the dissociation of cholesterol from the carrier
cyclodextrin can result in the deposition of cholesterol and the
associated nephrotoxicity sometimes associated with
cyclodextrins(10) .
>
>
(14) . This hemolysis may be attributed to the
removal of erythrocyte membrane components, particularly cholesterol.
Extensive release of erythrocyte cholesterol can be achieved upon
incubation of the cells with the acceptor(11) . What is
particularly intriguing about the interaction of cyclodextrins with
erythrocytes is the rapidity of the movement of cholesterol from the
cells to the incubation medium, with half-times (t) of
apparently less than 1 min(11) . This rapid exchange is in
contrast to that previously reported for the exchange or transfer of
cholesterol from erythrocytes to serum or isolated lipoproteins, which
is in the range of 1-8 h (for a review, see (15) ). It
was this very fast exchange rate that prompted us to conduct the
present detailed study of the characteristics of cholesterol movement
between cells in culture and cyclodextrins.
Cell Culture
MEM, DMEM, and Ham's F12 were
obtained from Mediatech (Herndon, VA). Calf serum, heat-inactivated
fetal bovine serum (FBS), and gentamicin were purchased from Sigma.
Media were buffered with sodium bicarbonate (2 g/liter), and cells were
cultured in a humidified incubator at 37 °C with 5% CO.
All media were supplemented with 2 mML-glutamine and
50 µg of gentamicin/ml. Mouse L-cell fibroblasts and GM3468A human
fibroblasts were cultured in MEM with 10% FBS, and Fu5AH cells were
cultured in MEM supplemented with 5% calf serum.
Efflux of Cell
[
Cells were plated
in 16- or 22-mm wells 3 to 7 days prior to efflux experiments (1
H]Cholesterol
10
cells per well for 12-well plates and 0.5
10
per well for 24-well plates). Pretreatments were
started when the cells were about 90% confluent, except for GM3468A
fibroblasts, which were used 2 to 4 days after reaching confluency.
Cells were labeled for 24 h with medium containing 1 or 2 µCi of
[
H]cholesterol/ml (0.1% ethanol final
concentration) and 2.5% FBS (1 ml/well).
[1,2-
H]Cholesterol was obtained from DuPont NEN
and checked for purity by thin layer chromatography prior to
use(16) . Subsequently, cells were incubated with medium
containing 0.2% bovine serum albumin for 24 h to allow for
equilibration of the radioactive isotope in the various cellular
cholesterol pools. An acyl-CoA:cholesterol O-acyltransferase
inhibitor, Sandoz 58-035, was added during both labeling and
equilibration (1 µg/ml, 0.1% dimethyl sulfoxide final
concentration, Sandoz compound 58-035 was a gift from Dr. John
Heider). After labeling and equilibration, cells were rinsed three
times with medium supplemented with only gentamicin and were then
switched to medium containing various cholesterol acceptors. This
medium was buffered with 50 mM Hepes (Life Technologies, Inc.)
instead of sodium bicarbonate. Cells were incubated for 10 min to 8 h
in a shaking water bath (40 rpm) at 37 °C, and 50-µl aliquots
were taken out for the liquid scintillation counter at various time
points. Cholesterol acceptors used in these studies were varying
concentrations of either HDL
,
-cyclodextrin (
CD,
Sigma, C-4805), 2-hydroxypropyl-
-cyclodextrin (2OHp
CD, Sigma,
C-0926), or methyl-
-cyclodextrin (M
CD, Sigma, C-4555).
HDL
(d = 1.125-1.21 g/ml) was
isolated by sequential ultracentrifugation of human plasma according to
Hatch and Lees(17) . Cell monolayers (before and after the
incubation with acceptors) were rinsed three times with
phosphate-buffered saline, and lipids were extracted with 2-propanol
overnight(18) . An internal standard (cholesteryl methyl ether,
Sigma) was added to the wells prior to extraction, and cholesterol was
measured by gas-liquid chromatography using a 50% phenylmethyl
polysiloxane column(19) . An extra set of cells was harvested
at the beginning of the efflux period to determine initial cellular
[
H]cholesterol and cholesterol mass. Cell protein
was determined on the remaining monolayer using a modification of the
method of Markwell et al.(20) . Cell protein was
dissolved in a solution of 0.1 N NaOH and 1% SDS, and
duplicate aliquots were taken out for protein determinations.
H]cholesterol remaining in the cells at each
time point. For time course experiments, these data were fitted to a
single exponential model using nonlinear regression as described
previously (21) . The equation used was y = A
e
+ C,
in which y is the fraction of initial cell
[
H]cholesterol remaining in the cells and t is the incubation time in hours. The apparent rate constant for
efflux of cellular cholesterol, k
, is the
initial slope of the efflux curve and can be calculated using k
= A
B,
and the values are the average of triplicate determinations.
Approximations of maximal efflux rate constants were derived from the k
versus
k
/[S] plot, in analogy with a
Hofstee plot used to determine maximal velocities for enzyme reactions.
In this plot, the intercept with the y axis is the maximal k
, and the substrate concentration at
which half of the maximal rate constant for efflux is reached, the K
, is the slope of the regression line.
The nonparametric Mann-Whitney test was used to compare treatments.
Efflux of Cell Phospholipid
L-cells were plated at
1 10
cells per well (35-mm wells) in 2 ml of MEM
containing 10% FBS and 1 µCi of
[methyl-
H]choline chloride/ml (NET-109)
and grown to confluency for 4 days. Subsequently, cells were rinsed
three times with MEM and incubated with MEM plus 0.2% bovine serum
albumin for 1 h. The cells were then rinsed again, and efflux was
started by incubating the cells with 1 ml of medium per well for 2 h at
37 °C. Separate wells were used to determine initial cell
H-phospholipid and cell phospholipid mass. Incubation media
were MEM alone as a control, MEM containing HDL
(0.2 or 0.5
mg of phospholipid/ml), or cyclodextrins (
CD, 2OHp
CD, or
M
CD at 2 or 5 mM). At the end of the incubation, media
were collected and 800 µl was used for phospholipid extraction. For
both the t
cellular
H-phospholipid and
the medium
H-phospholipid counts at t = 2
h, samples were extracted according to Bielicki et al.(22) to remove non-phospholipid
[
H]choline counts. Phospholipid efflux was
expressed as the percentage release of initial cell
H-phospholipid to the medium after 2 h of incubation.
Initial cell phospholipid mass was used to calculate the specific
activity of the labeled phospholipids to predict the released
phospholipid mass.
CD for 2 h at 37 °C. There was a
tendency of the cyclodextrin to co-extract with lipids if the
traditional chloroform-methanol extraction procedures were employed,
and the presence of cyclodextrins complicated the subsequent mass
determinations. To circumvent this problem, media containing the
2OHp
CD was extracted twice with 2
volumes of methylene
chloride, followed by 2 additional extractions with 1
volume of
ethyl ether. After combining and drying the extraction solvents, the
cholesterol was determined as described previously(23) .
Phospholipid mass was determined by the method described by Sokoloff
and Rothblat (24) and was sensitive to at least 0.1 µg of
phosphorus. Isotopic amounts of radiolabeled cholesterol and
phospholipid were added to the media prior to extraction to monitor
lipid recoveries. The recovery of cholesterol was 105% ± 3%, the
recovery of phospholipid from the incubation media was 81% ± 6%.
Toxicity Assay
The release of cellular adenine, as
described by Reid et al.(25) , was used to monitor
membrane integrity. To label the intracellular adenosine pool of
L-cells, cells were pretreated the same as described above for efflux
experiments, and, in addition, 0.5 µCi of
[8-C]adenine (Amersham) per ml was added to the
equilibration medium. Before efflux medium was added, the wells were
rinsed extensively with MEM/gentamicin to remove extracellularly bound
[
C]adenine. The release of cellular adenine was
monitored by removing 75-µl medium aliquots, filtering through
0.45-µm Multiscreen filtration plates (Millipore), and counting
50-µl aliquots. The initial amount of cellular
[
C]adenine was quantified by dissolving the
cells in 1 ml of 0.5% Triton X-100.
Binding of
The total
cell association of
[-Cyclodextrins to Cells
C]2-hydroxypropyl-
-cyclodextrin
([
C]2OHp
CD; CTD Inc., Gainesville, FL) to
L-cells was determined by incubation with 0-20 mM
2OHp
CD (specific activity between 70 and 1960 dpm/nmol) for 2 h at
37 °C. The cells were then rinsed, and cell monolayers were
dissolved in 0.1 N NaOH. Aliquots were used for liquid
scintillation counting and protein determinations.
-cyclodextrin for
stimulating the efflux of L-cell [
H]cholesterol
is shown in Fig. 1.
-Cyclodextrin (
CD),
2-hydroxypropyl-
-cyclodextrin (2OHp
CD), and
methyl-
-cyclodextrin (M
CD) all induced a
concentration-dependent release of cellular cholesterol. M
CD was
more efficient than the two other cyclodextrins. Nearly 90% of cellular
[
H]cholesterol was released after 8 h of
incubation with 10 mM M
CD. Table 1gives the sterol
mass of L-cells after 8 h of incubation with 2OHp
CD and M
CD.
Since L-cells cannot synthesize cholesterol(26) , the
fractional release of labeled cholesterol is equal to the decrease of
cellular cholesterol mass (compare Fig. 1and Table 1).
M
CD also produced a larger fractional release of cellular
desmosterol than the other cyclodextrins.
H]cholesterol efflux by
-cyclodextrin,
2-hydroxypropyl-
-cyclodextrin and methyl-
-cyclodextrin.
[
H]Cholesterol-labeled L-cells were incubated
with
CD (
), 2OHp
CD (
), and M
CD (
) at
0, 2, 5, or 10 mM. The percent of
[
H]cholesterol released to the medium was
measured after 8 h of incubation at 37 °C. Values are means
± S.D. (n = 3). Error bars are within
the markers if not apparent. FC, cell-free
cholesterol.
A comparison of the time
course of cellular [H]cholesterol efflux mediated
by HDL
or by various concentrations of 2OHp
CD is shown
in Fig. 2. There was no significant efflux of
[
H]cholesterol when cells were incubated with
medium without any cholesterol acceptor. HDL
induced efflux
of cholesterol at a relatively constant rate during the 8-h time
course. However, 2OHp
CD induced a more rapid rate of cholesterol
release than HDL
, even when present at the lowest
concentration of 2 mM. The initial fast rate of
[
H]cholesterol release diminished after 1 to 2 h,
until an apparent equilibrium was reached between the labeled
cholesterol in the medium and in the cells. Thus, the kinetics for the
efflux of cellular cholesterol are very different when cyclodextrin is
compared to HDL
.
H]cholesterol by MEM, HDL
, or
2OHp
CD. [
H]Cholesterol-labeled L-cells were
incubated with MEM alone (
), 0.2 mg of
HDL
-phospholipid/ml (
), or 2OHp
CD (2 mM (
), 5 mM (
), 10 mM (
)). Medium
aliquots were removed at the indicated times, counted by a liquid
scintillation counter, and efflux is expressed as the fraction of
[
H]cholesterol remaining in the cells. Values are
means ± S.D. for triplicate wells. Error bars are
within the markers if not apparent.
The kinetics of the cholesterol efflux
time course studies (Fig. 2) suggested that incubation of
L-cells with cyclodextrin resulted in the rapid equilibration of
labeled cholesterol between cells and medium. Table 2shows the
estimated molar ratio of cell free cholesterol to cyclodextrin in the
incubation medium after 8 h exposure of L-cells to the cholesterol
acceptors. The molar cell-free cholesterol:cyclodextrin ratio was
approximately 1:1900 for 2OHpCD, and 1:1300 for
CD,
respectively. These ratios were obtained at both 2 mM and 5
mM concentrations of the cyclodextrins. These results indicate
that cholesterol equilibrates between the cells and the available
cyclodextrin molecules in the medium, and that the final ratio is
dependent on the affinity of cholesterol for the type of cyclodextrin
that is present.
The cyclodextrin-mediated release of a major
portion of cell cholesterol mass, as shown in Table 1, might be
expected to deplete plasma membrane cholesterol and result in cell
toxicity. To test for the presence of CD-induced cell toxicity, cells
were preincubated with labeled adenine, the subsequent release of which
serves as a measure of cell membrane
integrity(25, 27) . The release of cellular
[C]adenine after an 8-h incubation of L-cells
with MEM alone or various concentrations of cyclodextrins was inversely
correlated with the cell total sterol content (Fig. 3). The
higher the concentrations of cyclodextrins the greater the depletion of
cell sterol and the greater the leak of
[
C]adenine (Fig. 3). At equivalent
concentrations, M
CD was more toxic than 2OHp
CD, consistent
with its greater ability to promote cholesterol efflux. The results
shown in Fig. 3were obtained from studies in which cells were
extensively depleted of cell cholesterol by long incubations with CD,
therefore, a time course for the release of labeled adenine was done
using various acceptors. Fig. 4shows the results for
incubations of 2 and 8 h. The pattern for the release of labeled
adenine obtained after 8 h of incubation was similar to that previously
observed (Fig. 3); however, release of adenine above background
levels was not evident after a shorter exposure of cell to the
acceptors (2 h), even at the highest concentration of
-cyclodextrins (10 and 20 mM). This indicates that
toxicity occurs only after prolonged incubations with cyclodextrins,
but not during the first initial rapid phase of efflux.
C]adenine release after 8 h of incubation of
L-cells with various acceptors.
[
C]Adenine-labeled L-cells were incubated with
MEM (
), 2OHp
CD (2 mM (
), 4 mM (
), 10 mM (
)), or M
CD (2 mM
(
)) for 8 h. After the incubation, the amount of cellular
[
C]adenine release was measured in the efflux
media, and monolayers were used to assay L-cell protein and sterol
(cholesterol plus desmosterol) mass. The correlation between toxicity
and total cell sterol mass is presented. Each point represents a single
well.
C]adenine after 2 or 8 h of incubation with
various acceptors. [
C]Adenine-labeled L-cells
were incubated with either MEM alone, HDL
(0.2 mg of
phospholipid/ml), or cyclodextrins at the indicated concentrations. The
amount of cellular [
C]adenine released to the
medium was measured by removing and counting medium aliquots after 2
and 8 h of incubation.
Efflux of Cellular Phospholipids
Although
-cyclodextrins are reported to have a relatively high specificity
for sterols compared to
phospholipids(11, 12, 28) , it is important
to know the amount of cyclodextrin-mediated release of cellular
phospholipids in order to acquire information on the mechanism by which
cyclodextrins promote cellular cholesterol efflux. To quantitate the
fractional release of cell phospholipids, L-cells were grown in
[
H]choline as described under ``Materials
and Methods'' to label phosphatidylcholine and sphingomyelin, the
2 major phospholipids in cell plasma membranes. Subsequent incubation
in medium supplemented with cyclodextrins or HDL
provided
an estimate of the fractional release of phospholipids from the cells (Fig. 5). The fractional release of cellular phospholipids was
small; maximally, 2% of cellular phospholipids were released after 4 h
of incubation with 5 mM M
CD. This value is similar to
that obtained with high concentrations of HDL
and
considerably lower than that observed for cholesterol efflux. An
attempt was made to quantitate the actual mass of phospholipid present
in the medium after a 2-h incubation of L-cells with 10 mM 2OHp
CD. Under conditions where from 38% to 64% of the cell
pool of sterol (average 9.5 µg of sterol/well; 80% cholesterol, 20%
desmosterol) was recovered, no measurable phospholipid was detected in
the media (sensitivity of assay = 2.5 µg of phospholipid)
from monolayers having 115 ± 8 µg of phospholipid/well.
H]choline-labeled L-cell phospholipids. L-cell
phospholipids were labeled with [
H]choline, and
efflux of cellular
H-phospholipids was measured after a 2-h
incubation of the cells with 1 ml of MEM per well containing HDL
(0.2 and 0.5 mg of phospholipid/ml), 2OHp
CD, or M
CD (at
2 and 5 mM). Cellular lipids at the beginning of the
experiments and medium aliquots after 2 h of incubation at 37 °C
were extracted to determine phospholipid-derived
H counts.
Efflux is expressed as the percent of initial cell
H-phospholipid released to the medium. Values are means
± S.D. of triplicate wells.
Cyclodextrin-mediated Cholesterol Efflux from Different
Cell Types
Previously, it has been observed that the rate of
cholesterol release from various cells differs considerably depending
on the individual tissue culture cell line(29, 30) .
The differences in the relative rates of cholesterol release among
cells is observed with any phospholipid-containing acceptor including
vesicles, reconstituted and native lipoproteins, and whole
serum(29, 31, 32, 33) . To determine
if cell-specific fractional efflux values persisted when the very
efficient and phospholipid-free cyclodextrins served as cholesterol
acceptors, a series of acceptor-dose experiments were conducted
comparing L-cell mouse fibroblasts, Fu5AH rat hepatoma cells, and
GM3468A human skin fibroblasts. HDL, 2OHp
CD, and
M
CD served as acceptors. Incubation times of 10 min were used for
the cyclodextrins and 30 min for the HDL
in order to
approximate the initial rates of cell cholesterol release. Fig. 6shows the dose-response curves for the three cell types on
three different acceptors. It is clear from the data in Fig. 6A that with HDL as the acceptor the differences
in k
among cell types was as previously
reported (Fu5AH L-cell > fibroblasts)(29) . However, in
contrast to the HDL data, the same cell specificity, in terms of
cholesterol efflux, was not apparent when either 2OHp
CD (Fig. 6B) or M
CD (Fig. 6C) served
as acceptors. With the cyclodextrins, cholesterol efflux from the human
fibroblasts and Fu5AH hepatoma cells was similar and slightly greater
than that for L-cells.
, M
CD, and 2OHp
CD.
[
H]Cholesterol-labeled cells in 12-well plates
were incubated with 2 ml of medium containing one of the following:
HDL
(A), 2OHp
CD (B), or M
CD (C). Incubation with HDL was for 30 min, and incubations with
cyclodextrins were for 10 min. Medium aliquots were removed and
counted, and efflux is expressed as the fraction of initial cell
[
H]cholesterol released from the cells and
presented as fractions/h. Data are mean ± S.D. of triplicate
wells.
, Fu5AH cells;
, L-cells;
,
fibroblasts.
The data presented in Fig. 6can be
used to obtain estimates of the maximum rate of cellular cholesterol
release for each cell type-acceptor combination. This estimate of V for cholesterol efflux is derived from V
versus V/[S] plots, where V is the fractional
release of cholesterol per h and [S] is the concentration of
acceptor, in analogy with a Hofstee plot used to determine maximal
velocities for enzyme reactions(34) . Estimated V
values for the 3 cell types are shown in Fig. 7and illustrate that in contrast to the results with HDL as
an acceptor, the 2 cyclodextrins yielded similar V
values that did not differ between the fibroblasts and hepatoma
cells (>3 fractions/h, Fig. 7). L-cells were somewhat slower
(2.4-2.8 fractions/h). Thus: 1) the maximum efflux from cells
exposed to cyclodextrins is extremely rapid with values of
approximately 3 fractions/h, 2) V
values for
cells exposed to OHp
CD and M
CD are the same, 3) there is
little or no difference between V
for
cholesterol efflux among cell types exposed to cyclodextrins, and 4) V
values differ considerably among cells when
HDL is the acceptor, and, even with the fastest cell type (Fu5AH, 1
fraction/h), the maximum rate of release to HDL is much slower than to
cyclodextrins.
values for
cholesterol efflux from Fu5AH hepatoma cells, L-cells, and human skin
fibroblasts exposed to HDL
, M
CD, and 2OHp
CD.
Cholesterol efflux values from the dose experiment shown in Fig. 6were used to estimate the V
for
cholesterol release.
, M
CD;
, 2OHp
CD;
, HDL
.
Binding of Cyclodextrin to Cells
To test whether
efflux mediated by cyclodextrins was linked to the direct association
of the cyclodextrins (i.e. binding and/or internalization)
with cells, we measured the association of
[C]2OHp
CD to L-cells over a wide
concentration range (1 to 20 mM). Subsequently, the cell
association data were compared with the dose-response curve for
cholesterol efflux at the same concentrations (Fig. 8). As was
previously demonstrated, cholesterol efflux approached a maximal value,
consistent with a saturable process; however, binding/internalization
of labeled 2OHp
CD at 37 °C was linear over the whole
concentration range. In addition, the amount of labeled cyclodextrin
associated with the cells was consistently very low (
0.5% of the
amount present in the extracellular medium).
C]2OHp
CD to L-cells. L-cells were
incubated with the indicated concentrations of
[
C]2OHp
CD for 2 h at 37 °C. After
washing the cell monolayers, the amount of cell-associated radiolabeled
cyclodextrin was determined. Efflux of cholesterol was determined from
separate wells using unlabeled 2OHp
CD as described under
``Materials and Methods.'' Values are the means of 3 wells
± S.D.
= 6,
= 7, and
= 8 glucose units, respectively. The physicochemical properties
of the cyclodextrins can be influenced by a number of different
structural modifications involving the additions of functional groups
to the molecule(7, 28) . The ability to solubilize
guest molecules is, in part, a function of the size of the hydrophobic
cavity within cyclodextrins, and this internal cavity has been
estimated to range in diameter between 5 Å and 8 Å,
depending on the specific cyclodextrin(28) . Clearly, the three
cyclodextrins used in this investigation have an enormous capacity for
removing cholesterol from cells in culture. The efflux of cholesterol
is concentration-dependent, and, at the end of an 8-h incubation period
with 10 mM cyclodextrin, from 50% to 80% of the labeled
cholesterol has been removed from L-cell mouse fibroblasts (Fig. 1). In this experimental system in which the cyclodextrins
are present in the culture medium in the absence of any source of
exogenous cholesterol, efflux of labeled cholesterol is an accurate
reflection of the depletion of cellular sterol (Table 1). The
extensive removal of cell sterol results in membrane instability, as
measured by the leakage of [
C]adenine, and a
strong inverse correlation is obtained between cell sterol content and
adenine release (Fig. 3). Although this toxic response could be
a complicating factor in some cell culture studies, shorter incubations
which do not result in as extensive cell sterol depletion do not result
in any detectable increase in adenine leakage (Fig. 4).
used in the current experiment. Previously we have
demonstrated that cell cholesterol efflux generally follows first order
kinetics (2) and that in some cells cholesterol appears to be
released from 2 kinetic pools(35, 36, 37) .
As can be seen from Fig. 2, the efflux observed with
cyclodextrins is much more rapid than with HDL and appears to reach
equilibrium quickly between cells and medium. That equilibrium
conditions are approached is demonstrated by the fact the estimated
molar ratio of cholesterol to cyclodextrin molecules after 8 h of
incubation with 2 mM concentrations of
CD and 2OHp
CD
are approximately 1:1300 and 1:2000, and these ratios are similar when
cyclodextrin concentrations are raised to 5 mM (Table 2). Although it has been suggested that cyclodextrins
can form 1:1 molar complexes with cholesterol(28) , the high
molar ratio observed in this study probably reflects an equilibrium
distribution between cell and acceptor, as well as the fact that other
compounds derived from cells or medium may compete with cholesterol for
encapsulation within the cyclodextrins.
CD or M
CD (Fig. 6, B and C). The data from the
dose-response studies (Fig. 6) were used to calculate an
approximate V
value (see ``Materials and
Methods'' and (33) ) for each acceptor/cell combination.
When we compare the fastest (Fu5AH) and slowest cells (fibroblasts) (Fig. 7), cyclodextrin produced V
values
for cholesterol efflux that were 3.5-fold greater than HDL for the
Fu5AH and 70-fold greater for fibroblasts.
for efflux (i.e. minimum t) would be the same for all types of acceptor
particles (2, 15) and 2) the differences in efflux
among cell types was a function of the composition of the plasma
membrane that modulated the rate of desorption of cholesterol
molecules(2, 15) . This latter prediction was
confirmed by studies demonstrating that the difference in efflux
between 2 cell types was maintained when efflux was measured using
plasma membrane vesicles as cholesterol donors(30) . Although
we have consistently observed a difference in efflux among cell
types(29, 36) , a series of recent studies has
indicated that the estimated maximum rate of cell cholesterol release
is not the same for all acceptor particles and can be modified by
acceptor composition and size. For example, the estimated V
value for cholesterol efflux from L-cells to
reconstituted HDL particles containing phospholipid
(dioleoylphosphatidylcholine or 1-palmitoyl,
2-oleoylphosphatidylcholine) in the liquid crystalline state ranged
between 0.12 and 0.14 fraction/h, whereas V
with
reconstituted particles with phospholipid in the gel phase
(dipalmitoylphosphatidylcholine or distearoylphosphatidylcholine)
yielded values of 0.03 fraction/h(33) . Another recent
investigation of the effect of acceptor size has demonstrated that the
estimated V
values for efflux from L-cells were:
0.12 fraction/h for reconstituted HDL (13 nm diameter), 0.03 fraction/h
for 100 nm large unilamellar vesicles and 0.01 fraction/h for 200 nm
large unilamellar vesicles(40) . The very high efficiency of
cyclodextrins for cholesterol efflux extends these observations and
re-emphasizes the importance of acceptor size on the V
for cell cholesterol efflux.
, the cholesterol
efflux rate depends on the rate constant for cholesterol desorption
from the plasma membrane (k
) as well as the
concentration of plasma membrane cholesterol that is available for the
desorption ([DC]) so that V = V
= k
[DC].
It follows that the high values of V
observed
with the cyclodextrins could arise because of alterations in k
and/or [DC]. At this time is not
possible to distinguish these possibilities. Increases in k
could perhaps arise from cyclodextrin-membrane
interactions that facilitate cholesterol desorption. Alternatively,
increases in the concentration of plasma membrane cholesterol available
for desorption could arise because, relative to HDL particles, small
cyclodextrin molecules can access more of the cell surface. The
modulation of acceptor concentration at the cell surface based on size
could be a reflection of the complexity of the surface in terms of
either the unstirred water layer, the cell glycocalyx or membrane
domains caused by plasma membrane ridges, depressions, or vacuoles. Of
particular importance is the observation that very high rates of
cholesterol release can be obtained from all cells with the appropriate
acceptor. It has previously been assumed that one of the major
rate-limiting steps in cell cholesterol efflux was the desorption of
cholesterol from the plasma membrane, since in most cells efflux
appeared to be slower than intracellular transport
steps(2, 19, 38) . If physiological acceptors
with efflux potentials approaching that of cyclodextrins exist, efflux
would no longer be rate-limiting, and intracellular metabolic events
would become more important in determining overall rates of cholesterol
release.
CD,
-cyclodextrin; M
CD,
methyl-
-cyclodextrin; 2OHp
CD,
2-hydroxypropyl-
-cyclodextrin.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.