216
Excess Membrane Cholesterol Alters Calcium
Movements, Cytosolic Calcium Levels, and
Membrane Fluidity in Arterial Smooth
Muscle Cells
Marie M. Gleason, Marvin S. Medow, and Thomas N. Tulenko
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The relations between membrane cholesterol content, basal (unstimulated) transmembrane
45Ca2' movements, cytosolic calcium levels, and membrane fluidity were investigated in cultured
rabbit aortic smooth muscle cells (SMCs) and isolated SMC plasma membrane microsomes.
SMCs were enriched with unesterified (free) cholesterol (FC) for 18-24 hours with medium
containing human low density lipoprotein and FC-rich phospholipid (PL) liposomes. This
procedure increased cholesterol mass without affecting PL mass, resulting in an increase in the
FC/PL molar ratio compared with controls in cells (67% FC increase, p<0.001; 43% FC/PL ratio
increase,p <0.01) and in SMC microsomes (52% FC increase,p <0.05; 43% FC/PL ratio increase,
p<0.05). Cholesterol enrichment also increased unstimulated 45Ca2' influx (p<0.001) and efflux
(p<0.05). Cellular cholesterol content correlated in a linear fashion with these changes (influx:
r=0.722,p<0.01; efflux: r=0.951,p<0.05). In addition, cytosolic calcium levels increased -34%
(p<0.01) with cholesterol enrichment. The cholesterol-induced increase in 45Ca21 influx was
reversible with time and demonstrated sensitivity to the channel blockers. Fluorescence anisotropy measured from 5°C to 40°C using the fluorophore diphenylhexatriene showed decreased
membrane fluidity in microsomal membranes obtained from cholesterol-enriched SMCs compared with controls (p<0.02). These results suggest that the SMC plasma membrane is very
sensitive to cholesterol enrichment with liposomes or human low density lipoprotein and that
increases in membrane cholesterol content increase cytosolic calcium levels in SMCs, are
associated with a decrease in membrane fluidity, and unmask a new, or otherwise silent,
dihydropyridine-sensitive calcium channel that may be involved in altered arterial wall properties with serum hypercholesterolemia. (Circuklaion Research 1991;69:216-227)
F ree (unesterified) cholesterol is a major lipid
class of mammalian plasma membranes in
general,' and arterial smooth muscle in particular,2 where it is thought to participate in the
regulation of the physical state or "fluidity" of the
From the Department of Physiology and Biochemistry (M.M.G.,
T.N.T.), The Medical College of Pennsylvania, Philadelphia, Pa.,
and the Department of Pediatrics (M.S.M.), Division of Pediatric
Gastroenterology and Nutrition, New York Medical College,
Valhalla, N.Y.
This publication was used in partial fulfillment of the requirements for the PhD degree (M.M.G.) from the Medical College of
Pennsylvania.
Support was provided, in part, by National Institutes of Health
grant HL-30497 and training grant HL-07443, a grant-in-aid from
the Delaware Affiliate of the American Heart Association, and a
predoctoral grant from the Southeastern Pennsylvania Affiliate of
the American Heart Association (M.M.G.).
Address for correspondence: Thomas N. Tulenko, PhD, Department of Physiology and Biochemistry, The Medical College of
Pennsylvania, 3300 Henry Avenue, Philadelphia, PA 19129.
Received April 16, 1990; accepted March 25, 1991.
phospholipid bilayer. The molar ratio of free cholesterol to phospholipid (FC/PL) in the membrane, an
index of cholesterol content, correlates with lipid
microviscosity.3,4 The amount of cholesterol in the
membrane is thought to be kept within narrow limits
in any given cell line and appears to be regulated by
a poorly understood balance of cholesterol influx,
efflux, esterification, deesterification, and synthesis.5
An increase in the cellular FC/PL has been shown to
alter the activity of several membrane-bound enzymes in a variety of cells, including hepatic cells,6
erythrocytes,7,8 intestinal epithelial cells,34 and renal
tubular cells.9 More recently, excess membrane cholesterol has been shown to influence the transmembrane movements of cations in intact arterial smooth
muscle,10 1' cultured arterial smooth muscle cells
(SMCs),12 isolated arterial SMC membranes,13 and
erythrocytes.14
The concept of excess membrane cholesterol is
important when considering the cellular pathobiol-
Gleason et al Effects of Cholesterol in Arterial Smooth Muscle
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ogy of arterial smooth muscle in atherosclerosis. For
example, aortic cells from atherosclerotic arteries are
enriched with unesterified cholesterol,15 and many
atherogenic processes are calcium dependent, raising
the question of whether atherosclerosis may be the
consequence of, or facilitated by, altered transmembrane calcium movements. Thus, excess calcium uptake could directly contribute to atherogenesis.
Taken together with the observations that calcium
antagonists retard atherogenesis1617 as well as block
cholesterol-induced 45calcium entry,10 excess membrane cholesterol may be an important mediator in
the development of atherosclerosis. In addition, since
vascular smooth muscle contraction is largely dependent on increases in cellular calcium, pathological
increases in calcium influx and/or cytosolic calcium
levels could also result in inappropriate vascular reactivity. The increased sensitivity of arterial tissue to
vasoconstrictor agents after chronic high cholesterol
diets18"19 or acute cholesterol exposure10'11 has been
reported previously, and cholesterol-induced hyperreactivity is mediated by calcium influx10"11 through
receptor-operated calcium channels that are themselves altered by excess membrane cholesterol.10
Although the causative role of calcium in atherogenesis has not yet clearly been established, the
above observations indicate that cholesterol enrichment augments calcium uptake by arterial smooth
muscle. The present study was undertaken to determine whether an increase in the cholesterol content,
as an isolated and independent variable, of arterial
smooth muscle results in increased cholesterol content of the SMC plasma membrane, whether this
enrichment increases calcium influx, efflux, and/or
cell calcium content, and the degree to which this
enrichment alters membrane lipid dynamics.
Materials and Methods
Experimental Agents and Solutions
Sources of chemical supplies were as follows: collagenase (type IA), elastase, soybean trypsin inhibitor, cholesterol (chromatographically pure), egg
phosphatidylcholine (type Ill-E), norepinephrine
[(-)-arterenol], prazosin, diltiazem, verapamil, and
nifedipine were purchased from Sigma Chemical Co.,
St. Louis, Mo.; bovine serum albumin (BSA, fraction
V) was from Nutritional Biochemicals, Cleveland,
Ohio; 1,6-diphenyl-1,3,5 -hexatriene (DPH) was from
Aldrich Chemical Co., Milwaukee, Wis.; fetal bovine
serum (FBS) was from GIBCO Laboratories, Grand
Island, N.Y.; 15CaCl2 was from New England Nuclear, Boston, Mass.; 3-decyldimethylsilyl-N-2-(4methylphenyl)-1-phenylethylpropanamide (compound U58-035) was from Sandoz Pharmaceuticals
Corp., East Hanover, N.J.; EDTA and EGTA were
from Fisher Scientific Co., Pittsburgh, Pa.
All drugs were prepared as concentrated stock
solutions. Verapamil, diltiazem, and norepinephrine
were dissolved in physiological salt solution (PSS).
Prazosin was dissolved in ethanol. Nifedipine and
217
compound U58-035 were dissolved in dimethyl sulfoxide. When added to PSS, the final concentration of
the solvent was 0.1%.
Isolation and Culture of Smooth Muscle Cells
The entire thoracic aorta was aseptically removed
from male New Zealand rabbits immediately after
death by sodium pentobarbital. The vessel was transferred to warm (37°C), oxygenated (100%02) PSS at
pH 7.3, and adhering tissue and fat were carefully
dissected away. The PSS contained (mM) HEPES
2.5, sodium HEPES 2.5, NaCl 140, KCl 4.5, MgSO4
1.0, CaCI2 1.5, glucose 10.0, and EDTA 0.03. SMCs
were isolated as described previously by Campbell et
al.20 Briefly, the intimal and adventitial layers were
stripped away in Dulbecco's modified Eagle's medium (DMEM) buffered to pH 7.4 with 24 mM
NaHCO3. Medial smooth muscle strips were minced
and incubated for 1-2 hours in DMEM containing
375 units/ml collagenase, 0.425 units/ml elastase, and
0.12% soybean trypsin inhibitor (1 mg inhibits 2.5 mg
trypsin). This procedure yielded dispersed cells,
which were then washed in minimal essential medium (MEM) containing 15% FBS and pelleted (7
minutes at 350g). Cells isolated by this method demonstrated high viability (>95%) as determined by
trypan blue exclusion. The cells were incubated in
MEM containing 15% FBS for 4-5 days and subsequently maintained in MEM containing 10% FBS.
All experiments were performed using early passage
cells (passages 3-5) grown to confluence in 35-mm
Petri dishes. Cells buffered with bicarbonate were
incubated under a 95% air-5% CO2 atmosphere;
cells exposed to PSS were incubated in air. All tissue
culture solutions contained 50 units/ml penicillin, 50
ugg/ml streptomycin, and 50 gg/ml gentamicin.
Cholesterol Enrichment
SMCs were incubated for 18-24 hours in media with
or without FC/PL liposomes. Multilamellar liposomes
were prepared as previously described.10 This procedure yielded cholesterol-rich liposomes with a FC/PL
molar ratio of approximately 2: 1 as determined by gas
liquid chromatography and phospholipid-phosphorus
colorimetry before experimentation. Cholesterol-rich
medium was prepared by combining liposomes (500 ,ug
cholesterol/ml), human low density lipoprotein (LDL,
50 ,ug protein/ml), BSA (0.2% [wt/vol]), FBS (1%
[vol/vol]), and compound U58-035 (1.0 pag/ml) to inhibit acylcoenzyme A: cholesterol acyltransferase2' in
MEM. Control medium had the same composition,
with the exception that LDL and liposomes were
replaced with equal amounts of saline. Control and
cholesterol-rich media were determined to be isosmolar before experimentation. SMCs were incubated in
the presence of lipoprotein-deficient serum (human, 10
mg protein/ml MEM) for 2 days before cholesterol
enrichment to upregulate LDL receptors.5 In selected
experiments, control cells were incubated with lipopro-
218
Circulation Research Vol 69, No 1 July 1991
tein-deficient serum before incubation overnight with
control medium to control for any potential effects of
lipoprotein-deficient serum, but none were observed.
Lipoprotein-deficient serum was isolated from fresh
human plasma after sedimentation in a KBr gradient,
d>1.21,22 dialyzed against 0.9% saline, clotted with
thrombin, and filter-sterilized (0.45-,m filter, Millipore
Corp., Bedford, Mass.). LDL was isolated from fresh
human plasma, d <1.063 -1.019,22 dialyzed against
0.9% saline, and filter-sterilized. In some experiments,
cholesterol was depleted from SMCs by incubating cells
overmight with cholesterol-free liposomes in MEM (0: 1
FC/PL). To control for an effect of oxidized LDL or
cholesterol, the lipid soluble antioxidant butyrated hydroxytoluene (0.1 ,M) was included in the isolation
procedure in selected experiments.
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
45Ca2+ Uptake
Confluent cells were equilibrated in PSS for 60
minutes before experiments were begun. For assay,
PSS was aspirated and replaced with 45Ca2+-PSS (4
,Ci/ml), and the cultures were incubated with 45Ca21
at 37°C for time periods ranging from 1 second to 24
hours. 45Ca2+ uptake was terminated by placing the
dishes on a -6°C frosted cradle, followed by immediate, rapid washing of the cells with eight washes of
2 ml ice-cold (0-2°C) PSS (5 sec/wash). Preliminary
experiments demonstrated that this wash protocol
removed extracellular 45Ca21 while retaining intracellular 45Ca2+ after five washes over 25 seconds and was
equally effective in normal and cholesterol-enriched
cells. Beyond 25 seconds of wash with this protocol,
the calcium remaining in the cells was virtually
constant (data not shown). The cells were then lysed
with sodium dodecyl sulfate (1 mg/ml), and aliquots
of the lysate were analyzed for protein content and
radioactivity. Calcium uptake was determined from
counts derived from the cell lysate 45Ca2+ fraction
(cpm) divided by the specific activity of the 45Ca2+containing uptake media (cpm/,mol Ca2+). Unidirectional inward flux of calcium was estimated by exposing cells to 45Ca2+-PSS for 45 seconds. This
incubation time period is within the linear portion of
the calcium uptake curve. Calcium uptake is expressed as calcium content (nmol/mg protein) at
various times, and calcium influx is expressed as the
slope of uptake over time for several time points
(nmol/mg protein/sec).
45Ca2+ EfflUx
Confluent cells were rinsed with PSS and incubated to isotopic equilibrium in 45Ca2+-PSS for 2
hours as determined by preliminary studies in normal
and cholesterol-enriched cells. For assay, 45Ca2+-PSS
was aspirated, and the cells were washed three times
(5 seconds each) with ice-cold PSS (2 ml) to remove
superficially bound 45Ca2+. Each dish was incubated
with 1 ml efflux solution (PSS at 37°C), which was
removed every 2 minutes. At the termination of the
experiment, cells were lysed with sodium dodecyl
sulfate as described above, and an aliquot was taken
for protein analysis. The radioactivity of the remaining cell lysate and of the efflux solutions was determined by liquid scintillation counting. Cellular calcium content and calcium loss were calculated as
described above. Efflux curves were generated by
adding the activity in the final cell lysate and various
efflux wash media in reverse order. Normalized efflux
curves (fractional loss) were then obtained by dividing activities at each time point by the initial activity
at zero time. Rate constants (k) for calcium efflux for
each time interval were calculated as described in
detail for cation efflux studies in arterial smooth
muscle23 using the following equation:
k=ln (A1/A2)/(t2-tl)
where A, and A2 represent the total tissue counts at
time points t, and t2, respectively.
Cytosolic Calcium
The concentration of free cytosolic calcium in
SMCs was determined using the fluorescent calcium
indicator fura 2. After cholesterol enrichment, fura 2
was loaded into suspended SMCs by incubation in 2
1LM fura 2-AM in HEPES-PSS supplemented with
0.1% BSA. Cells were then centrifuged and reincubated with HEPES-PSS-BSA without fura 2-AM (20
minutes) to allow complete hydrolysis of entrapped
ester. Aliquots of cells (2 ml, 106 cells/ml) were then
placed in a cuvette, incubated at 37°C (5 minutes),
and assayed for fluorescence in a spectrofluorometer
(SPEX Cation Measurement System, SPEX Industries Inc., Edison, N.J.). Each sample was excited
alternatively at 340 and 380 nm, and fluorescence was
monitored at 505 nm. Fluorescence intensity was
measured for each excitation wavelength, and the
ratio (R) of the 340/380 emission signal was calculated for steady-state fluorescence. At the end of the
measurement, the cells were permeabilized with ionomycin (1 mM) for maximum fluorescence (Rma,)
followed by the addition of EGTA (2 mM) for
minimum fluorescence (R,,fi) and used to calculate
intracellular cytosolic Ca21 levels in the various experiments, using the following equation24:
[Cai] =Kd(R-Rmin/Rmax-R)(F2B2)
where the dissociation constant (Kd) is 224 nmol for
Ca2' binding to fura 2 at 37°C, F2 is the 380 emission
intensity in the presence of EGTA, and B2 is the 380
emission intensity in the presence of ionomycin.
Preparation of Microsomes
Microsomal membranes were isolated as previously described.25 Cells were removed with a rubber
policeman and pelleted at 700g for 10 minutes.
Sixteen 100-mm plates of confluent SMCs produced
a cell pellet weighing -0.3 g wet weight and containing -4x 107 cells. The cells were suspended in cold
(4°C), hypo-osmolar sucrose (0.1 M) containing 10
mM Tris (pH 7.4) for 6 minutes, followed by disrup-
Gleason et al Effects of Cholesterol in Arterial Smooth Muscle
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tion with 20 strokes of a type B pestle in a small
Dounce homogenizer (Kontes, Vineland, N.J.). An
equal volume of 0.4 M sucrose solution containing 10
mM Tris, pH 7.4, was then added to the disrupted
cells, and the resultant crude homogenate was centrifuged at 700g for 10 minutes after removal of an
aliquot for chemical and enzymatic analysis. The
resulting nuclear pellet was resuspended in 0.25 M
sucrose, 10 mM Tris, pH 7.4 (as were all subsequent
pellets), and the supernatant was centrifuged at
9,750g for 10 minutes. The resulting mitochondrial/
lysosomal pellet was resuspended, and the supernatant was centrifuged at 110,OOOg for 30 minutes. The
resulting microsomal pellet was resuspended after
removal of the postmicrosomal supernatant (soluble
fraction). All fractions were frozen for chemical and
enzymatic analysis at a later time, with the exception
of cytochrome c oxidase activity, which was measured
on the day of cell fractionation (see "Analytical
Determinations").
The following markers for the various subcellular
fractions were assayed according to the method of the
indicated references: alkaline phosphodiesterase (plasma membrane),26 cytochrome c oxidase (mitochondria),27 8-N-acetylglucosaminidase (lysosomes),25 and
DNA (nuclei).28 Protein (soluble fraction), cholesterol
(plasma membrane), and phospholipid (total membrane) masses were determined as described below.
Fluorescence Polarization
Preparation of the lipid soluble fluorescence probe
1,6-diphenyl-1,3,5-hexatriene (DPH) and fluorescence polarization studies were performed as previously described.29 At the time of study, 2 ,M DPH in
tetrahydrofuran was dispersed in phosphate buffered
saline that contained (mM) NaCl 145, KCI 4.0,
NaH2PO4 5.0, and Na2HPO2 5.0; pH was adjusted to
7.2 with NaOH. This dispersion was stirred vigorously until no odor of tetrahydrofuran could be
detected (2-3 hours). Arterial SMC membrane protein (100 gg) was then added to 3 ml DPH suspension and incubated for 30 minutes at 37°C.
For these experiments, estimates of relative membrane fluidity were calculated after fluorescence polarization measurements using a recording spectrofluorophotometer (model RF- 540, Shimadzu
Scientific Instruments, Columbia, Md.) fitted with a
thermoregulated sample chamber and automatic polarizing filters (C.N. Wood Manufacturing, Newtown,
Pa.). The term "fluidity" is used to describe the
motional freedom of lipid soluble molecular probes
(i.e., DPH), within a membrane bilayer, subject to
limitations discussed in previous publications.29'30
Determination of absolute fluidity is limited in an
anisotropic membrane because of the inability to
accurately reproduce the three-dimensional structure of the hydrophobic bilayer. Therefore, the
steady-state fluorescence anisotropy (rf, reciprocal of
fluidity) is used to estimate relative degrees of fluidity, after probe incorporation into the membrane
bilayer. Values for rf were calculated from fluores-
219
cence polarization measurements using the Perrin
equation31:
rf = (Il-Il)/(Iii +2Il)
where I1 and I equal fluorescence intensities parallel
and perpendicular, respectively, to the excitation
plane (excitation wavelength=365 nm; emission
wavelength=430 nm). In preliminary studies, the
sum of the contributions of scattered light
(membranes+buffer-probe) and fluorescence of the
ambient medium (after pelleting membranes) to the
total fluorescence intensity was found to be <5% of
the total fluorescence intensity throughout the temperature range used in these studies. Arrhenius plots
of membrane fluidity (log rf versus [1/temperature
('K)] x 103) were constructed to provide information
regarding apparent thermotropic transitions (i.e., liquid-crystal to gel) of the membrane lipid.
Analytical Determinations
Parallel dishes of SMCs were analyzed for free
cholesterol, phospholipid, and protein content. Before lipid analyses, cells were incubated for 2 hours in
0.5% BSA after exposure to control or cholesterolrich media to solubilize surface bound LDL and
liposomes. The cultures were then rinsed two times
with warm (37°C) PSS. Cellular lipids were extracted
with isopropyl alcohol, which was evaporated to
dryness under a nitrogen atmosphere. Free cholesterol and total cholesterol (esterified and free) were
quantitated by gas liquid chromatography using coprostanol as an internal standard, and phospholipid
mass was assessed using a phospholipid phosphorus
assay as previously described.10 Cellular protein was
extracted by incubation with 1 ml sodium dodecyl
sulfate (1 mg/ml) and assayed using the method of
Lowry et al.32
Statistical Analysis
Data are shown as mean+ 1 SEM. The null hypothesis was examined through nonpaired Student's t test
and was rejected at p<O0.05.
Results
Cholesterol Enrichment of Cells and Microsomes
In preliminary experiments, SMCs incorporated
more free cholesterol from the combination of LDL
and cholesterol-rich liposomes (89% increase) than
from liposomes alone (78% increase) or from LDL
alone (29% increase) compared with control medium
(Table 1). Enrichment with cholesterol resulted in an
increased FC/PL ratio in all cases. These results
demonstrate the ability of LDL alone to deliver
excess free cholesterol to SMCs in culture. However,
to maximize cholesterol enrichment, the combination
of human LDL and cholesterol-rich liposomes (cholesterol-rich medium) was used throughout.
U58-035 inhibits cellular esterification of cholesterol by acylcoenzyme A: cholesterol acyltransferase.21 In cells exposed to control medium, cholesteryl
220
Circulation Research Vol 69, No 1 July 1991
TABLE 1. Free Cholesterol Content and Free Cholesterol/Phospholipid Molar Ratios in Smooth Muscle Cells After
Various Protocols for Delivery of Exogenous Free Cholesterol
FC content
Experimental condition
Control
+LDL
+FC/PL liposomes
+LDL and FC/PL liposomes
(,ug FC/mg
protein)
16.91±0.99
21.80±1.95*
30.15+2.73t
n
8
8
7
7
31.97±1.414
Increase (%)
0
29
78
89
FC/PL
0.201±0.026
0.278±0.026§
0.335±0.18211
0.291±0.028¶
Increase (%)
0
38
66
44
Values are mean±SEM. n, Number of culture plates; FC, free cholesterol; PL, phospholipid; LDL, low density
lipoprotein. Liposomes are cholesterol rich (2:1 FC/PL molar ratio); percent increase is compared with control.
*p<0.01, tp<0.001, tp<0.0001, §p<0.054, 11p<0.086, and ¶p<0.038 vs. corresponding control value.
ttg/mg protein
(n=12), -1.5% of the total cellular cholesterol content. Cholesteryl ester formation was reduced in the
presence of U58-035 to 0.098±0.047 ,ug/mg protein
(n = 26), -.0.45% of total cellular cholesterol. For this
reason, U58-035 was routinely used to avoid esterification of excess cholesterol and to enhance availability of free cholesterol for solubilization into the
plasma membrane. U58-035 alone had no effect on
calcium movements or cytosolic calcium levels.
For subsequent experiments to determine 45Ca2+
movements, cells were exposed to cholesterol-rich or
control medium. In this series of experiments, cellular cholesterol mass increased (67%, p<0.001) after
exposure to cholesterol-rich medium compared with
control (Table 2). There was no difference in cholesteryl ester or phospholipid content in control
versus cholesterol-enriched cells. Enrichment in free
cholesterol with no concomitant change in phospholipid mass resulted in an increase (43%, p<0.01) in
the cellular FC/PL molar ratio. Microsomal membranes prepared from the SMCs demonstrated a
fivefold enrichment in alkaline phosphodiesterase
and a sixfold enrichment in cholesterol, the plasma
membrane markers. This same fraction was not enriched in DNA, f3-N-acetylglucosaminidase, or cytochrome c oxidase and was therefore taken to be
enriched with plasmalemmal membrane. After incubation with cholesterol-rich medium, the microsomal
ester content measured 0.335±0.157
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fraction revealed an increase in free cholesterol content
of 52% (p<0.05) compared with control, with no
difference in phospholipid content. Enrichment in free
cholesterol with no concomitant change in phospholipid mass resulted in an increase (43%,p<0.05) in the
microsomal FC/PL molar ratio. Cholesteryl esters were
not detectable in the microsomal fraction isolated from
control or cholesterol-enriched cells. Table 2 summarizes the alterations in lipid values in intact SMCs and
SMC microsomal membranes.
Effect of Cholesterol on Unstimulated
Calcium Uptake
Unstimulated 45Ca2' uptake in control and cholesterol enriched SMCs is shown in Figure 1. Calcium
influx from 7 to 45 seconds in control cells was 16.9
pmol/mg protein/sec (Figure 1). After cholesterol
enrichment, calcium influx over this period increased
3.1-fold to 52.4 pmol/mg protein/sec (p<0.001).
There was no difference in cell-associated 45Ca2+
after 1 or 7 seconds of exposure to the isotope, and
the initial rapid uptake from 0 to 7 seconds likely
reflects a pool of 45Ca2' that rapidly associates with
the membrane.33 45Ca2' uptake increased linearly
from 7 to 45 seconds in control (r=0.991) and
cholesterol-enriched cells (r=0.996). The linearity of
the uptake curve is consistent with a unidirectional
and inwardly directed flux of calcium over this time
TABLE 2. Free Cholesterol, Cholesteryl Ester, and Phospholipid Contents and Free Cholesterol/Phospholipid Molar
Ratios From Control and Cholesterol-Enriched Smooth Muscle Cells and Smooth Muscle Cell Microsomal Membranes
Lipid
Cellular
FC (ug/mg protein)
CE (grg/mg protein)
PL (,ug/mg protein)
n
28
26
20
20
FC/PL
Microsomal
8
FC (,ug/mg protein)
7
PL (,ug/mg protein)
7
FC/PL
Values are mean+SEM. n, Number of culture plates;
cholesteryl ester; PL, phospholipid; NS, not significant.
*p<0.001, tp=NS, *p<0.01, and §p<0.05 vs. control.
Control
SMCs
Cholesterol-enriched SMCs
20.81±0.88
0.098+0.047
181.65±+ 18.59
0.144±0.060t
193.11 + 21.09t
0.279+0.020
0.399+0.031t
131.50±+12.08
34.89+1.46*
199.93+31.67§
568+104
538+59t
0.428+0.085
0.613+0.074§
SMCs, smooth muscle cells; FC, free cholesterol; CE,
Gleason et al Effects of Cholesterol in Arterial Smooth Muscle
A
221
y = 1.0513 + 3.7272e-2x
R = 0.722 p<.01
CD
o- 3
2.0-
En En
-5
.9
1.5
CL 2
=
E
0
1.0-
E
Control
*~~~~~~
Cholesterol-enriched
c ,,'
0.5
CD
20
10
0
0.0
0
5
10
15
20
25
30
35
40
45
50
Time (seconds)
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
FIGURE 1. Graph showing unstimulated 45Ca2' uptake in
control and cholesterol-enriched aortic smooth muscle cells
from 0 to 45 seconds. Cells were incubated ovemight with
cholesterol-rch or control medium, followed by incubation in
physiological salt solution (2 hours) to which 45Ca21 (4
,iCi/ml) was added at time 0. The cells were washed at the
indicated times as described in the text. Each point represents
mean +SEM offive to eight culture plates.
period.34 The effect of cholesterol enrichment on
calcium content at 60 and 120 minutes was not
different between control and cholesterol-enriched
groups as assessed with isotopic 45Ca2+ (3.96±0.51
versus 4.35 4.35 nmol/mg protein at 60 minutes and
4.91±0.31 versus 4.55±0.46 nmol/mg protein at 120
minutes, respectively); however, net calcium content
after 24 hours incubation with 45Ca2+ was increased
71±5% (3.518±1.306 versus 6.029±0.428 nmol/mg
protein, respectively, p<0.05). No effects of the antioxidant butyrated hydroxytoluene were observed in
either control or cholesterol-enriched cells.
To further assess whether cholesterol enrichment
altered nonspecific association of 45Ca2+ to SMCs, the
effects of washing with ice-cold calcium-free PSS
containing 2 mM EGTA (EGTA-PSS) was compared
with the PSS wash solution described above. We
observed no difference in 45Ca2, uptake during a
45-second pulse with 45Ca2' between control cells
washed with PSS and those washed with EGTA-PSS
(1.510±0.185 versus 1.522±0.150 nmol/mg protein,
respectively, n=5). There was also no difference in
cholesterol-enriched cells washed with PSS compared with those washed with EGTA-PSS (2.923±
0.142 versus 3.058±0.379 nmol/mg protein, respectively, n=5). Thus, cholesterol enrichment of SMCs
did not alter nonspecific binding of 45Ca2+.
In many studies, exposure of SMCs to the same
concentration of LDL/liposomal cholesterol resulted
in varying degrees of cholesterol incorporation into
cell membranes. However, when plotted together,
the relation between cell cholesterol content and
calcium influx always correlated in a linear fashion
(r=0.722,p<0.01) (Figure 2).
±
. . .
.
60
50
30
40
Cell Cholesterol
(Igimg protein)
70
FIGURE 2. Graph showing correlation between basal calcium uptake and cholesterol content in cultured arterial
smooth muscle cells. Each point represents mean + SEM of at
least three dishes for cholesterol measurement and at leastfour
dishes for calcium influx measurement.
Effect of Cholesterol on Calcium Efflux
To characterize the effect of cholesterol enrichment
on Ca2' loss from SMCs, unstimulated 45Ca2, efflux was
evaluated. Plotted as fractional loss, significantly less
45Ca2+ remained in the cholesterol-enriched cells compared with controls (p<0.05) under basal conditions
(Figure 3). Rate constants for unstimulated calcium
efflux were thus greater in cholesterol-enriched versus
control cells (0.0249+0.0023 versus 0.0178±0.0018
min-1, respectively, n =16;p<0.02). These data demon-
Basal
100
^90
,,,+ Control (5)
50 70
2
60
_)5
*
CL
~~~~~Cholesterol-
.:
enriched
(6)
E 40
0
+
C
*p<0.05
0 30
6
8
10
12
14
Time (minutes)
FIGURE 3. Graph showing fractional loss of cellular 45Ca2'
during basal (unstimulated) conditions in control (solid circles) and cholesterol-enriched (open circles) aortic smooth
muscle cells. Numbers in parentheses indicate number of
culture plates studied. Each point represents mean+±SEM.
*p<O0.05 compared with control.
Circulation Research Vol 69, No 1 July 1991
222
A.
y = 5.931e-3 + 7.790e-4x
R = 0.951 p<.05
c
0
Free Cholesterol Content
40
0
O Control
*
0.020
-T-
M CholestEterol-enriched
0.018 -
O.
t
30
c
0.016-
00
E
U
-0.
0.014
0
10
20
15
25
20
U-.
30
10
Cell Cholesterol
(lgg/mg protein)
FIGURE 4. Graph showing correlation between basal
-
rate
constants for calcium efflux and cholesterol content in control,
0 -
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
cholesterol-enriched, and cholesterol-depleted cells. Cholesterol enrichment was accomplished by incubating cells overnight with medium containing liposomes at a free cholesterol!
phospholipid molar ratio of 2:1, and cholesterol depletion was
accomplished by ovemight incubation at a molar ratio of 0:1.
Each point represents mean ±SEM of six dishes for cholesterol
or calcium efflux rate constant measurements.
was enhanced
after cholesterol enrichment.
Like calcium influx, unstimulated 45Ca2' efflux
correlated linearly with cellular cholesterol content
achieved by enrichment and depletion procedures
(r=0.951,p<0.05) as illustrated in Figure 4.
-1In
B.
i t ia
Reversal
45-Ca Uptake
strate that unstimulated calcium efflux
Effect of Cholesterol on Cytosolic Calcium Levels
To determine the effects of cholesterol enrichment on cytosolic calcium levels, several experiments were performed using the calcium indicator
fura 2. After overnight exposure to the cholesterol
enrichment medium, an increase in cellular free
calcium levels was observed, averaging 33.8+4.9%
(p<0.01, n=7), as illustrated for a representative
set of experiments in Figure 5.
350
-
300
-
250
-
E
200
iv
150
0
100
-
O
Control
*
Cholesterol-enriched
.
p<.01
50
01
FIGURE 5. Bar graph showing cytosolic calcium levels in
control and cholesterol-enriched arterial smooth muscle cells
as measured with the calcium indicator fura 2. A 31.3%
increase in cytosolic calcium (219±19 vs. 288±18 nM) after
cholesterol enrichment is illustrated for a representative series
of experiments. Each point represents mean+±SEM (n=3).
*p <0.01 control vs. cholesterol-enriched.
-0
CL
.n
Oi
Initial
Reversal
FIGURE 6. Bar graphs showing effect of incubation of
control and cholesterol-enriched aortic smooth muscle cells in
reversal medium (described in 'Results') for 4 days on free
cholesterol content (panel A) and 45Ca2' influx (panel B).
Each determination is the mean +SEM of eight to 12 culture
plates. *p <0. Ol compared with initial control, tp <0. 0 compared with initial cholesterol-enriched.
Reversal of Cholesterol Effect on Unstimulated
Calcium Influx
To determine whether the cholesterol-induced increase in 45Ca2' influx was a reversible alteration, SMCs
were enriched with cholesterol as described and then
exposed to MEM containing 10% FBS, but without
liposomes or added LDL, for 4 days (reversal medium).
45Ca2' influx and cholesterol content were determined
after initial enrichment and again after 4 days in the
reversal medium. Figure 6A shows a significant reduction in cholesterol content after 4 days on reversal
medium. The initial cholesterol-induced increase in
45Ca2' influx was also reduced significantly after exposure to reversal medium, such that there was no significant difference from control after 4 days (Figure 6B).
Protein content after the 4-day reversal was not dif-
Gleason et al Effects of Cholesterol in Arterial Smooth Muscle
Fluorescence Anisotropy
o Control
G Diltiazem
'1
M Verapamil
ER Nifedipine
c
.0.
40
0.3
-
0.2
-
-30
1o
20
i
oc
i
.
_-~~~~~~;~
em
T
-
_
223
CL
O DPH CONTROL
*-* DPH CHOLESTEROL-ENRICHED
-
E 00
E
.3.1
3.2
3.3
5.4
3.5
3.6
17
l/T (°K) x 103
0.0
,
*
Control
Cholesterol-enriched
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
FIGURE 7. Bar graphs showing effect ofdiltiazem (1.0 MM),
verapamil (1.0 MM), and nifedipine (0.1 pM) on unstimulated
45Ca2' influx in control and cholesterol-enriched aortic
smooth muscle cells. Cells were incubated with the blocker 30
minutes before the addition of 45Ca2' (45 seconds). Each
determination is the mean-+SEM (n25). *p<0.05 compared
with cholesterol-enriched vehicle control; tp<0.01.
ferent from initial content in control (0.268±0.026
0.256±0.016 mg protein/dish, respectively,
n=11) or cholesterol-enriched (0.264±0.022 versus
0.279±0.019 mg protein/dish, respectively, n= 12) cells,
indicating that the reversal of cholesterol content and
cholesterol-induced calcium influx were not a function
of changes in cell numbers.
versus
Effect of Calcium Channel Blockers on
Cholesterol-Induced Calcium Influx
The effects of various calcium channel blockers on
unstimulated calcium influx in control and cholesterolenriched cells were tested. Thirty-minute preincubation with the benzothiazepine, diltiazem (1.0 ,uM),
abolished the cholesterol-induced increase in calcium
influx back to control levels (53% reduction, p<0.01)
(Figure 7). Likewise, the arylalkylamine, verapamil (1.0
,uM), as well as the dihydropyridine, nifedipine (0.1
,uM), antagonized the cholesterol-induced increase in
calcium influx by 29% (p<0.05) and 40% (p<0.01),
respectively. There was no effect of any of these agents
on unstimulated calcium influx in control cells.
Effects of Cholesterol on Membrane Fluidity
Estimates of membrane fluidity were derived from
steady-state fluorescence polarization studies using
TABLE 3. Fluorescence Anisotropy Values for 1,6-Diphenyl-1,3,5hexatriene at 25°C and 37°C in Microsomal Membranes Isolated
From Control and Cholesterol-Enriched Arterial Smooth
Muscle Cells
Fluorescence anisotropy
At 250C
At 370C
Smooth muscle cells
0.1623+0.0039
Control
0.1861±+0.0032
0.1980 + 0.0024*
Cholesterol-enriched
0.1765±+-0.0029*
Values are mean+-SEM; n>5.
*p<0.02 vs. control.
FIGURE 8. Representative Arrhenius plots of fluorescence
anisotropy (rf, reciprocal of fluidity) of the lipid soluble
fluorophore 1,6-diphenyl-1,3,5-hexatriene (DPH) in microsomal membranes isolated from control and cholesterolenriched smooth muscle cells.
the fluorescent probe DPH. Table 3 summarizes the
effects of cholesterol enrichment on rf, a measure of
fluorescence anisotropy, determined at 25°C and
37°C. Higher values of rf indicate a lower membrane
fluidity. Cholesterol enrichment of microsomes resulted in an increased value of rf or lower membrane
fluidity compared with control at both of these temperatures (p<0.02) and is in agreement with similar
changes in fluorescence anisotropy values obtained in
SMCs after exposure to free cholesterol-rich medium by Bolotina et al.13 A typical Arrhenius plot,
which depicts the temperature dependence of fluorescence anisotropy in control and cholesterol-enriched SMC microsomal membranes, is shown in
Figure 8. Cholesterol enrichment decreased membrane fluidity at all temperatures measured between
40°C and 25°C, with no apparent phase transition or
change in the physical state of the membrane.
Discussion
The object of this study was to characterize the
effects of cholesterol enrichment on membrane cholesterol content and transmembrane calcium movements in cultured arterial smooth muscle cells. We
found that cholesterol enrichment of the plasma
membrane increased unstimulated calcium influx,
efflux, and cytosolic calcium levels. Furthermore, the
effect of cholesterol enrichment on calcium uptake
could be abolished by calcium channel blockers and
was shown to be reversible after 4 days in normal
growth medium.
Platelets,35 macrophages,36 Fu5AH rat hepatoma,37 and arterial smooth muscle10 have been
shown to accumulate excess cholesterol from LDL
and/or liposomes. In our study, we demonstrated that
SMCs could be enriched with cholesterol using LDL
alone (29%) or cholesterol-rich liposomes alone
(78%) (Table 1). Using a combination of LDL and
liposomes, free cholesterol enrichment was greatest
(89%). Uptake of liposomal cholesterol can occur
through internalization of the entire particle,36 by
direct transfer of molecular cholesterol at the surface
224
Circulation Research Vol 69, No 1 July 1991
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of the plasma membrane,38 or by transfer of molecular cholesterol to lipoproteins,37 which are then
internalized by receptor-mediated endocytosis.5
Since the cellular content of total phospholipids did
not increase after cholesterol enrichment (Table 2),
it is unlikely that the entire liposome or an LDLliposome complex was internalized, suggesting that
selective uptake of cholesterol occurred in our system. An increase in free cholesterol, with no change
in the phospholipid content, resulted in an increase
in the FC/PL molar ratio. An increase in this ratio
was observed after enrichment with cholesterol in
whole cells as well as in microsomes prepared from
the SMCs. Cholesterol enrichment of SMCs resulted
in the accumulation of excess free cholesterol in a
membrane fraction enriched with plasmalemma. This
finding is consistent with the demonstration that
>90% of the cellular free cholesterol is contained in
the plasma membrane of eukaryotic cells.1 Thus, the
cellular FC/PL molar ratio accurately reflects incorporation of cholesterol into the plasma membrane of
SMCs as illustrated in Table 2.
Many investigators have demonstrated that cholesterol incorporation into cell membranes alters the
physical state of the phospholipid bilayer, an alteration that can affect function of integral membrane
proteins and cellular function as well. An increase in
the FC/PL molar ratio has been correlated with
decreased adenylate cyclase,6 Na+,K+-ATPase,4 and
alkaline phosphatase3 activities. Increased platelet
aggregation35 and altered arterial smooth muscle
contractile function10"139 were also observed after
cholesterol exposure. In our studies, cholesterol enrichment of smooth muscle cell membranes was
associated with a significant increase in the unstimulated calcium influx rate (Figure 1). Similar elevations in unstimulated calcium influx have been reported after perfusion with cholesterol-rich
liposomes in intact carotid arterial segments10 and
red blood cells.14 These data suggest that cholesterol
enrichment of the plasma membrane alters the lipid
bilayer and affects calcium channel proteins. Accordingly, cholesterol-induced alterations in the SMC
plasma membrane may change the conformation or
position of calcium channel proteins in the bilayer,10
thereby exposing or activating previously silent ion
channels. Diltiazem, verapamil, and nifedipine all
reduced unstimulated calcium influx in cholesterolenriched SMCs back to control levels, but they had
no effect on unstimulated Ca`+ influx in control cells
(Figure 7). This finding confirms a previous report
from this laboratory that the cholesterol-induced
increase in unstimulated calcium influx in intact
carotid arteries occurs through a dihydropyridinesensitive pathway.10 In red blood cells, Neyses et a140
have shown that the cholesterol-induced increase in
calcium influx is sensitive to nitrendipine. Taken together with the well-known inability of calcium channel
blockers to inhibit unstimulated calcium influx,4' this
finding suggests that the unstimulated calcium "leak"
pathway is not affected by cholesterol enrichment but
that cholesterol enrichment resulted in the appearance
of a new, or otherwise "silent," calcium channel (present, but inactive under normal conditions) with pharmacological characteristics similar to L-type channels,
that is, dihydropyridine sensitivity.
Because the organic calcium channel blockers are
relatively specific for voltage-operated calcium influx,
the similarity in sensitivity of the cholesterol-induced
calcium influx to these structurally different agents
may suggest that cholesterol enrichment of the bilayer results in membrane depolarization. However,
the observation that cholesterol-enriched arterial
ring segments do not relax after exposure to calciumfree or diltiazem-containing buffers and that they
demonstrate sensitivity to depolarizing concentration
of KCI identical to that of control ring segments10
indicates that there is no preexisting tension mediated by calcium influx through a voltage-dependent
pathway. Thus, it is unlikely that excess free cholesterol results in membrane depolarization, although
this possibility remains to be tested directly with
electrophysiological techniques.
In addition to the increased calcium influx, we also
observed that cholesterol enrichment increased unstimulated cytosolic calcium levels in SMCs by -34%
(p <0.01), as measured with the calcium indicator fura
2 (Figure 5). Consistent with the increase in calcium
influx and cytosolic calcium levels, unstimulated calcium efflux was also increased (Figures 3 and 4) in the
cholesterol-enriched cells. It is reasonable to assume
that, in the steady state, influx and efflux should be
equal in magnitude. Thus, we suggest that calcium
efflux is increased secondary to elevated calcium influx
and/or cytosolic calcium levels. However, we cannot
rule out the possibility that cholesterol enrichment
directly affected calcium efflux by altering either Na+Ca`s exchange or Ca-ATPase activity, as has been
suggested in isolated cardiac sarcolemmal membranes
after cholesterol enrichment.4243
In support of our observation of increased cytosolic calcium content with cholesterol enrichment,
vasoconstrictor sensitivity to norepinephrine has
been shown to be increased severalfold in cholesterol-enriched rabbit femoral artery'1 and carotid arterial segments.10 In addition, we12 recently reported
that basal and norepinephrine-activated 86Rb+ efflux
through K' channels is also significantly elevated in
cholesterol-enriched arterial tissue and cultured aortic SMCs. In the latter study, the cholesterol-induced
increase in K' efflux under both basal and norepinephrine stimulation was also reversed by diltiazem,
again consistent with the finding that calcium influx
and cytosolic calcium levels are increased by excess
membrane cholesterol.
Cellular cholesterol enrichment was reversed after
exposure to medium containing 10% serum (no
liposomes) for 4 days (Figure 6A), as was the cholesterol-induced effect on calcium influx (Figure 6B). In
their studies of cardiac sarcolemma, Ortega and
Mas-Oliva43 also demonstrated a reversible inhibi-
Gleason et al Effects of Cholesterol in Arterial Smooth Muscle
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tion of the Ca'+-ATPase, as did others,44 when excess
cholesterol was removed. The reversibility of SMC
cholesterol enrichment is consistent with the belief
that cells tightly regulate the free cholesterol content
of the plasma membrane through a variety of complex and poorly understood mechanisms.5 Further,
this observation suggests that, in the absence of a
high cholesterol environment, SMCs can rectify an
experimentally induced elevation in membrane cholesterol content.
The positive correlation between membrane cholesterol content and calcium influx (Figure 2) and
efflux (Figure 4) further supports our hypothesis that
transmembrane calcium movements are strongly influenced by membrane cholesterol content and that
alterations in membrane cholesterol in either direction disturb transmembrane calcium movements.
Moreover, vascular smooth muscle calcium permeability is sensitive to the dynamics of the lipid microenvironment. Exactly how cholesterol influences
calcium permeability is unclear at this time. Cholesterol could influence calcium channel protein activity
by interfering with protein-lipid interactions through
a cholesterol-induced reorganization in the lateral
distribution of membrane lipids, as suggested by
Yeagle.8 Integral membrane proteins require specific
lipid environments for proper structure and function.44 45 When phospholipids in the lipid anulus
surrounding an isolated Ca2+-ATPase were replaced
with cholesterol, calcium transport activity was inhibited.44 These investigators suggested that the structure of the membrane is designed to exclude cholesterol from the phospholipid anulus surrounding
integral membrane proteins to avoid inhibition of
protein function by this membrane sterol. Cholesterol could also alter protein-protein interactions. In
this regard, excess membrane cholesterol has been
shown to stimulate Na+-Ca'+ exchange42 and inhibit
unstimulated42 and calmodulin-activated Ca2ATPase in isolated cardiac sarcolemma.43
A cholesterol-induced alteration in the physical
state (fluidity) of the bilayer is another possible
mechanism for cholesterol-mediated alterations in
membrane protein function. Lipid fluidity describes
the motional freedom of lipid molecules or lipid
soluble probes within a membrane bilayer. Since
determination of absolute fluidity is limited in anisotropic structures like cell membranes, fluorescence
polarization measurements estimate relative degrees
of fluidity after incorporation of fluorescent probes
such as DPH into the bilayer.314647 Cholesterol is
thought to condense and rigidify the plasma membrane by restricting the random motion and mean
cross-sectional area occupied by the neighboring
phospholipid acyl chains.48,49 After enrichment of
SMC membranes with cholesterol, fluorescence anisotropy was increased, and membrane fluidity decreased at all temperatures measured (Table 3 and
Figure 8). This decreased membrane fluidity was
associated with an increased membrane FC/PL ratio,
as was shown in platelets,35 rat intestinal microvillus
225
membranes,3,4 liver surface membranes,50 and quail
erythrocytes.7 Taken together, these and our results
are consistent with the hypothesis that arterial SMC
calcium permeability is sensitive to changes in the
lipid dynamics of the plasma membrane bilayer. We
suggest, therefore, that cholesterol-induced alterations in the lipid microenvironment, and consequently membrane fluidity, perturb the plasma membrane, resulting in the appearance of new or
otherwise silent cholesterol-induced calcium channels in SMCs.
In conclusion, we have demonstrated that acquisition of excess membrane cholesterol increases transmembrane calcium movements and cytosolic calcium
levels, which were temporally associated with
changes in membrane fluidity in arterial smooth
muscle cells. These cholesterol-induced alterations in
calcium handling by SMCs are significant in view of
the fact that elevated serum cholesterol levels have
long been associated with atherogenesis and many
atherogenic processes are calcium dependent.5' In
addition, a functional consequence of cholesterol
enrichment in SMCs is increased contractile responses.10 4' Strickberger et a152 recently reported indirect
evidence that smooth muscle calcium levels are elevated in atherosclerotic rabbit arteries after a high
cholesterol diet. The functional significance is elevated contraction during a-adrenergic stimulation.
Hyperreactivity of arterial tissue to norepinephrine
after a hypercholesterolemic diet has been reported
in dogs19 and monkeys.18 It is also of interest to
correlate the reversibility of the cholesterol effect on
SMC calcium movements with the reversibility of
hypercontractility associated with the return to normal diet and reduced serum cholesterol levels in
atherosclerotic monkeys53 and pigS.54
We present the hypothesis, based on work from
this laboratory,'0-12 that cholesterol enrichment alters lipid dynamics in the arterial smooth muscle
plasma membrane and results in the appearance of a
new, or an unmasking of an otherwise silent, dihydropyridine-sensitive calcium channel. This alteration in the SMC phospholipid bilayer may represent
an early smooth muscle cell defect important in the
development of atherosclerosis and could explain the
ability of calcium channel blockers to retard atherogenesis.16 Our studies can also provide an underlying
explanation for the hyperreactivity of vascular tissue
in hypercholesterolemia.
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KEY WoRDs * transmembrane Ca2' fluxes * fura 2 * liposomes
* fluorescence anisotropy * calcium channels * membrane
lipid dynamics * arterial smooth muscle * low density lipoprotein
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Excess membrane cholesterol alters calcium movements, cytosolic calcium levels, and
membrane fluidity in arterial smooth muscle cells.
M M Gleason, M S Medow and T N Tulenko
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Circ Res. 1991;69:216-227
doi: 10.1161/01.RES.69.1.216
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