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748
Lipid Bilayer in Genetic Hypertension
Anna F. Dominiczak, Dan F. Lazar, Arun K. Das, and David F. Bohr
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Membrane microviscosity, phospholipid composition, and turnover were measured in cultured
vascular smooth muscle cells isolated from mesenteric arteries of stroke-prone spontaneously
hypertensive and age-matched, normotensive Wistar-Kyoto rats. Membrane microviscosity,
measured with fluorescence polarization, revealed greater microviscosity (lower fluidity) of the
membranes isolated from smooth muscle cells from hypertensive as compared with those
isolated from normotensive rats (p<0.01). Preincubation of membranes from hypertensive rats
with 5 mM calcium reduced membrane microviscosity in "core" and in "surface" regions of the
bilayer toward values observed in Wistar-Kyoto rats. Phospholipid composition did not differ
between intact aortas and cultured mesenteric cells or between those tissues obtained from
normotensive and from hypertensive rats. The total lipid-associated radioactivity was significantly lower in cells from stroke-prone spontaneously hypertensive rats than in those from
Wistar-Kyoto controls (/?<0.01). Phosphatidylcholine incorporated 70% and phosphatidylinositol 16% of total lipid-associated radioactivity, with no difference between cells from hypertensive and normotensive animals. Turnover of phosphatidylethanolamine was greater in cells
from Wistar-Kyoto rats (p=0.02), whereas turnover of phosphatidylserine was greater in cells
from stroke-prone spontaneously hypertensive rats (p=0.04). The greater microviscosity of the
lipid bilayer in hypertension is a generalized defect of the matrix in which the transport
proteins function. We hypothesize that this defect is responsible for the multiple abnormalities
of membrane transport systems that have been described in genetic hypertension. (Hypertension
1991;18:748-757)
H
ypertension has been associated with abnormalities in many membrane transport systems including channels for sodium and
potassium1"3 and calcium,4 exchangers for sodium/
hydrogen56 and for sodium/calcium,7 as well as active
pumps for sodium/potassium78 and for calcium.910
The diversity of the membrane proteins that have
been found to have abnormal functions in hypertension led to the hypothesis that this generalized dysfunction of diverse protein transport systems is
caused by a fault in the matrix in which they all
function, the lipid bilayer.11
There is extensive evidence that the functions of
membrane proteins are influenced by properties of
the lipid bilayer.12 For example, Remmers et al13
have observed that the opioid receptor can be modulated by specific fatty acids through mechanisms
From the Departments of Physiology (A.F.D., D.F.B.), Biological Chemistry (D.F.L.), and Neuroscience Laboratory (A.K.D.),
University of Michigan, Ann Arbor, Mich.
Supported by grant HL-18575 from the National Institutes of
Health. This work was done during the tenure of the British
American Research Fellowship (to A.F.D.) supported by the
British Heart Foundation and American Heart Association.
Address for correspondence: Dr. A.F. Dominiczak, MRC Blood
Pressure Unit, Western Infirmary, Glasgow, Gil 6NT, Scotland, UK.
Received March 12, 1991; accepted in revised form August 16,
1991.
involving membrane fluidity. In addition, the coupling of the /3-adrenergic receptor to adenylate cyclase is enhanced by decreasing membrane microviscosity,14 and Na+, K+-ATPase activity is accelerated
when the membrane microviscosity is low.15
Fluorescence polarization studies in erythrocytes
and platelets from patients with essential hypertension and from spontaneously hypertensive rats
(SHR) showed increased microviscosity as compared
with normotensive controls.16-19 Tsuda et al,20 using
an electron spin resonance and spin-labeling method,
confirmed decreased membrane fluidity in erythrocytes and cultured vascular smooth muscle cells
(VSMC) of SHR. The physicochemical properties
such as microviscosity of biomembranes are affected
by the content and physiological states of cholesterol,
phospholipids, and their fatty acids.15 Okamoto et
al21 have shown that renal membrane phospholipids,
especially phosphatidylcholine and phosphatidylethanolamine, decrease with age in SHR. This decrease
is associated with increased phospholipase A2 activity
and phospholipid degradation.
The majority of studies of lipid bilayer composition
and its functional properties in hypertension have
been performed on circulating blood cells. There is
very little information on lipid bilayer in VSMC. This
tissue is relevant since it is directly involved in the
Dominiczak et al
TABLE 1. Data on Blood Pressure, Weight, and Age of Rats
Variable
n
Blood pressure
(mm Hg)
Weight (g)
Age (days)
SHRSP
WKY
40
40
122±4*
104 ±6
45 ±5
88±3
111±9
44±4
SHRSP, stroke-prone spontaneously hypertensive rats; WKY,
Wistar-Kyoto rats.
*/?<0.001.
increased vascular reactivity and medial hypertrophy,
hallmarks of the hypertensive process.
The purpose of this study is to compare membrane
microviscosity, phospholipid composition, and metabolism in VSMC from stroke-prone SHR (SHRSP)
and from Wistar-Kyoto normotensive rats (WKY).
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Methods
Animals
SHRSP and WKY rats were obtained from colonies in the Department of Anatomy and Cell Biology at the University of Michigan. These are inbred
colonies, derived from a stock supplied by the
National Institutes of Health (NIH), Bethesda, Md.
Data on age, weight, and blood pressure of rats are
given in Table 1. Rats were decapitated and the
mesenteric arteries and thoracic aortas were removed under aseptic conditions and were placed in
ice-cold Hanks' balanced salt solution (HBSS).
Aortas (five per preparation) were cleaned of fat
and adventitia and immediately were frozen by
placing in aluminum foil over a mixture of dry ice
and acetone. Aortas were stored in -70°C before
the lipid extraction procedure.
Isolation and Culture of Rat Mesenteric Artery
Smooth Muscle Cells
VSMC were harvested from enzymatically dissociated rat mesenteric arteries by modifications of the
methods described by Gunther et al22 and Lyall et
al.23 All procedures were carried out under aseptic
conditions. The superior mesenteric artery with its
major branches was excised, en bloc, from its origin
at the aorta to the mesenteric border of the intestine
and was placed in a petri dish containing ice-cold
HBSS with 0.2 mM added Ca2+. Fat, adventitia, and
veins were removed by blunt dissection, the branches
were cut off close to the main trunk of the superior
mesenteric artery, and the artery was cut open
lengthwise to expose the endothelial layer. The
cleaned mesenteric arteries (five per preparation)
were transferred into a petri dish containing 5 ml
enzyme dissociation mixture: HBSS with 0.2 mM
added Ca2+, 1.25 mg/ml collagenase, 0.25 mg/ml
elastase, 0.5 mg/ml soybean trypsin inhibitor, and 2
mg/ml bovine serum albumin. The arteries were
incubated at 37°C in a humidified 5% CO2-95% air
atmosphere for approximately 30 minutes. Then the
arteries were transferred to fresh HBSS and tritu-
Lipid Bilayer in Hypertension
749
rated 10 times through a plastic Pasteur pipette. They
were cut into small pieces, transferred to fresh enzyme dissociation mixture, and incubated at 37°C
with periodic trituration until a single cell suspension
was obtained (usually 45-60 minutes). The tissue
suspension was sieved through 106-^.m stainless steel
mesh to separate dispersed cells from undigested
vessel wall fragments and debris. The filtered suspension was diluted in 20 ml Dulbecco's modified Eagles
medium (DMEM) supplemented with 10% (vol/vol)
fetal bovine serum, 10% (vol/vol) equine serum, 2
mM L-glutamine, 100 units/ml penicillin, and 100
jig/ml streptomycin and then was centrifuged (400g,
5 minutes). The cell pellet was resuspended in 5 ml
DMEM supplemented as described above. The dispersed cells were plated in a 25-cm2 culture flask and
were maintained in a humidified atmosphere of 5%
CO2-95% air at 37°C.
After the first 24 hours, the medium was replaced to
remove nonadherent cells. Plating efficiency was consistently above 50%. Medium was changed at 48-72hour intervals. VSMC had usual growth characteristics,
took 7 days to reach confluence after plating, and at
confluence, had the typical "hill and valley" pattern.22
Cells up to the 10th passage were harvested once a
week with trypsin-EDTA and passaged at a 1:3 ratio in
162-cm2flasks.Before experiments, cells were deprived
of serum by a 24-48-hour incubation in fresh DMEM
supplemented with 0.4% fetal calf serum. The cells
were positively identified as smooth muscle by indirect
immunofluorescent staining for a-actin. To detect the
presence of smooth muscle-specific a-actin, the cells
grown to 50% confluence on sterilized round cover
slips, 12-mm diameter, werefixedin acetone: methanol
(1:1, vol/vol) for 2 minutes at -20°C. They were then
incubated with monoclonal anti-a-smooth muscle actin
antibody diluted 1:400 in phosphate-buffered saline
(PBS) with 1% bovine serum albumin for 60 minutes at
37°C. After washing three times in PBS of the same
composition, the cells were incubated with secondary
antibody-anti-mouse immunoglobulin G (IgG) (whole
molecule)fluoresceinisothiocyanate (FITC) conjugate
in the dark for 45 minutes at 37°C. After being washed
three times in PBS, cover slips were mounted onto
microscope slides on glycerol 9:1 (vol/vol) in PBS and
examined under afluorescencemicroscope at a magnification of x400 and x630. NIH-3T3 cells (mesenchymal cell line; gift from Dr. Donald MacCallum) and
VSMC incubated with secondary antibody only were
used as controls. An example of the immunofluorescence study of mesenteric cells at the stage of second
passage is shown in Figure 1.
Membrane Preparation
Confluent monolayers of VSMC were washed
three times with ice-cold HBSS (Ca2+, Mg2+-free
formula) and detached by gentle scraping with the
aid of a "rubber policeman." The suspension was
centrifuged for 5 minutes at 400g, and the pellet was
suspended in hypotonic buffer of the following composition (mM): Na2HPO4 0.61, KH2PO4 0.38, MgSO4
750
Hypertension
Vol 18, No 6 December 1991
FIGURE 1.
Photomicrograph of
mesenteric smooth muscle cells
that reacted with anti-a smooth
muscle actin antibody followed by
an anti-mouse immunoglobulin G
fluorescein isothiocyanate conjugate as described in "Methods."
Magnification, x 1,800. Bar (1
cm) =5 fim.
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0.2, dithiothreitol 1, pH 7.4. The pellet was then
dispersed in a Dounce homogenizer and was centrifuged for 15 minutes at 20,000g, 4°C. The resulting
pellet was resuspended in 50 mM Tris HC1, pH 7.4, at
a protein concentration of 1 mg/ml.
Determination of Protein
The methods used were according to Lowry et al24
and Bradford25 with bovine serum albumin as a
standard. The membrane protein was solubilized
initially with 1M NaOH- for 30 minutes at 25°C.
Measurement of Membrane Microviscosity
A previously described method13 was applied, except in our study both fluorophores were measured at
the same wavelengths for excitation and emission,
respectively. The fluorescent probes diphenylhexatriene (DPH) (solution in Af,JV-dimethylformamide) and its charged trimethylammonium derivative
(TMA-DPH) (solution in tetrahydrofuran) were incubated with membranes at 25°C for 30 minutes and
60 minutes, respectively. The molar ratio of probe to
membrane phospholipid was approximately 1:200 in
all experiments. It was determined that this ratio of
probe to membrane phospholipid did not perturb
membrane microviscosity. Fluorescence polarization
was measured in a spectrofluorometer (SLM Instruments, Inc., Urbana, 111.) at wavelengths of 340 nm
for excitation and 450 nm for emission. The results
are expressed as anisotropy units (r), where
r=(lfl-lgfl)(lfl+2190)
and IQ and 1% represent the intensities of light when
polarizers were in parallel or perpendicular orienta-
tion, respectively. Differences in the efficiency of
transmitting vertically and horizontally polarized
light were corrected for by determining the polarization ratio (l0—lgo) using horizontally rather than vertically polarized excitation light. The advantage of
expressing fluorescence polarization as anisotropy is
the additive nature of the latter.13
Phospholipid Composition of Cultured Vascular
Smooth Muscle Cells and Intact Aortas
Confluent monolayers of VSMC were washed three
times with ice-cold physiological salt solution (PSS) of
the following composition (mM): NaCl 130, KC1 4-7,
KH2PO4 1.18, MgSO4-7H2O 1.17, CaCl2-2H2O 1.6,
NaHCO314.9, dextrose 5.5, CaNa2-EDTA 0.026. The
cells were scraped into 15-ml ice-cold PSS and were
centrifuged at 400g for 5 minutes. Lipid extraction was
performed as described by Lee and Hajra.26 In
brief, cell pellets and intact aortas were homogenized
three times for 30 seconds each at 2.5 setting, in
chloroform: methanol (1:1, 12 ml/g of tissue) with a
Polytron homogenizer (Brinkmann Instruments, Inc.,
Westbury, N.Y.). The homogenate was centrifuged
(l,000g, 10 minutes), and the supernatant was saved.
The residue was dispersed in water (0.5 ml/g tissue) by
agitation in a sonic bath, and this dispersed residue was
rehomogenized in chloroform: methanol (1:1, 6 ml/g
original tissue). This second homogenate was centrifuged (l,000g, 10 minutes), and the supernatant was
combined with the first supernatant. To the combined
supernatants, an additional amount of chloroform was
added (9 ml/g tissue) and mixed well, and 0.9% aqueous NaQ (5.5 ml/g tissue) was added to the extract to
facilitate the separation. The mixture was vortex-mixed
Dominiczak et al
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and centrifuged (l,000g, 5 minutes), and the upper
layer was removed. The lower layer was dried under a
stream of nitrogen at 40°C and resuspended in 100 fi\
chloroform: methanol (2:1, vol/vol). The phospholipids
were resolved by monodimensional thin-layer chromatography (TLC) using the following solvent systems: 1)
chloroform:methanol:0.25% aqueous KC1:ethyl
acetate: isopropanol (60:18:12:36:50, vol/vol); 2) chloroform : methanol: acetic acid:H2O (100:40:12:4, vol/
vol); and 3) chloroform: methanol: 40% methylamine
(63:35:10, vol/vol). The individual phospholipids, cochromatographed with standards and visualized with
primuline, were identified and scraped for phosphate
determination performed according to Ames.27 Samples of scraped phospholipids and K2HPO4 standards
were mixed with 10% Mg(NO3)2 in 95% ethanol,
dried and heated in a flame until brown vapor
disappeared. After cooling, 0.5N HC1 (0.3 ml) was
added, and samples were heated in a heating block at
100°C for 15 minutes. This was followed by incubation with 0.7 ml of a mixture of 10% ascorbic
acid: 0.42% ammonium molybdate in IN H2SO4 (1:6,
vol/vol) for 30 minutes at 45°C. The concentration of
phosphate was determined spectrophotometrically
(Beckmann, DU70, Arlington Heights, 111.) at 820
nm. The results are expressed as a percentage of total
phospholipid content in each lipid extract.
Isolation of Platelets
Washed platelets were prepared as described previously.28 Briefly, platelet-rich plasma was prepared
by centrifuging citrated (1 ml of 3.9% [wt/vol] sodium
citrate/9 ml of blood) whole blood at 400g for 5
minutes at room temperature. EGTA (5 mM) was
added to the platelet-rich plasma, which was centrifuged at 40Qg for 10 minutes. The supernatant was
removed, and the pellet was suspended in calciumpoor medium containing (mM) NaCl 130, KC1 4.7,
MgCl2 1, glucose 10, HEPES 20, pH 7.4. The platelets were counted in a Coulter counter (Coulter
Corp., Hialeah, Fla.) and the concentration of platelets adjusted to 1.5-2xlO8/ml.
Lipid Bilayer in Hypertension
751
solvent system of chloroform: methanol: 40% methylamine (63:35:10, vol/vol). Individual phospholipids
were localized by overnight (12-14 hours) autoradiography (Kodak X-OMAT film), and 32P incorporation was quantified by scraping the phospholipid
spots from the silica plate and counting in the
scintillation counter.
Statistical Analysis
Data are presented as the mean±SEM of n experiments (five animals were used for each tissue/cell
culture preparation). Statistical analysis was performed
using the unpaired Student's t test with Bonferroni
correction for multiple comparisons. A value ofp < 0.05
was regarded as a significant difference.
Materials
Phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidic acid, sphingomyelin, collagenase (type II), elastase
(type IV), soybean trypsin inhibitor (type I-S), fetal
bovine serum, equine serum, monoclonal anti-asmooth muscle actin, and anti-mouse IgG (whole molecule) FITC conjugate were obtained from Sigma
Chemical Co., St. Louis, Mo. DPH and its charged
derivative TMA-DPH were purchased from Molecular
Probes, Junction City, Ore. Carrier-free 32P (1 mCi/ml)
was bought from Amersham Corp., Arlington Heights,
111. DMEM, penicillin, streptomycin, glutamine, trypsin-EDTA, and HBSS (Ca2+, Mg2+-free formula) were
obtained from Irvine Scientific, Santa Ana, Calif.;
20x20 cm TLC plates (silica gel 60, 0.25 mm thick)
were purchased from Merck, Cherry Hill, N.J. All
other chemicals were of analytical grade.
Results
Membrane Microviscosity in Vascular Smooth
Muscle Cells
Measurements with the membrane "core" probe
DPH and "surface" probe TMA-DPH revealed sigLabeling of Phospholipids in Vascular Smooth Muscle nificant difference in microviscosity between memCells and Platelets
branes obtained from SHRSP cells and those obVSMC were trypsinized and pelleted at 400g for 10
tained from WKY cells (Figure 2). This difference
minutes. The pellet was resuspended in HEPES
was slightly greater for core than for surface probe
buffer of the following composition (mM): NaCl 130,
measurements. Preincubation of VSMC membranes
KC1 4.7, MgCl2 1, glucose 10, HEPES 20, CaCl2 1.6,
with 5 mM calcium, added to Tris buffer 30 minutes
pH 7.4. Aliquots of the VSMC (or platelet) suspenbefore fluorescent probes, significantly reduced
sion (0.58 ml each) were incubated with 0.02 mCi
membrane microviscosity in VSMC from SHRSP for
phosporus-32 for 0, 30, 60, 120, and 180 minutes at
DPH and TMA-DPH measurements. Membrane mi37°C in a shaking water bath. Platelets were incucroviscosity
in VSMC from WKY was also slightly
bated in calcium-poor medium as described above.
reduced,
but
only the value for TMA-DPH reached
The reaction was terminated by addition of 2.25 ml
statistical
significance
(Figure 2).
chloroform: methanol (1:2, vol/vol). The phospholipids were extracted under acidic conditions according
Phospholipid Composition
to the method of Bligh and Dyer (as modified by
LaBelle and Hajra29). The lower layer was evapoPhosphatidylcholine was the major phospholipid in
rated to dryness under a stream of nitrogen at 40cC,
cultured vascular smooth muscle cells and in intact
and phospholipids were separated by TLC using
aortas (approximately 40% of total phospholipid con-
752
Hypertension
Vol 18, No 6 December 1991
SHRSP
ft
WKY
•
TMA DPH
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FIGURE 2. Bar graph shows microviscosities of the lipid
bilayer from cultured vascular smooth muscle cells from
Wistar-Kyoto rats (WKY) and stroke-prone spontaneously
hypertensive rats (SHRSP); effect of 5 mM calcium. Measurements were made using fluorescent probes: diphenylhexatriene
(DPH) for the core of the bilayer and the charged trimethylammonium derivative (TMA-DPH) with which the microviscosity of the surface region is monitored. Data are
mean±SEM from seven studies comparing cells from SHRSP
and WKY and from four studies in 5 mM Ca2+. **p<0.01
SHRSP vs. WKY and SHRSP vs. SHRSP in 5 mM Ca2+;
*p<0.05 WKY vs. WKY in 5 mM Ca2+.
tent). No significant differences were observed in
phospholipid composition between mesenteric VSMC
and intact aortas. There was also no difference between vascular tissue or cells obtained from SHRSP
and those obtained from WKY (Figure 3).
180
120
60
30
30
180
FIGURE 4. Autoradiogram of thin-layer chromatogram in
which the phospholipids of cultured vascular smooth muscle
cells had been labeled by incubation with phosphorus-32 for
30, 60, 120, or 180 minutes. Labeling of phospholipids from
the Wistar-Kyoto rat (WKY) cells is greater than that of
stroke-prone spontaneously hypertensive rat (SHRSP) cells.
Discrete spot above phosphatidylethanolamine probably represents diphosphatidylglycerol (cardiolipin); it accounted for
less than 1% of total radioactivity. O indicates the origin; PI,
phosphatidylinositol; PS, phosphatidylserine; PC, phosphatidylcholine; PE, phosphatidylethanolamine.
ity of each spot was determined by scraping and
scintillation counting. VSMC from SHRSP showed
less 32P labeling of phosphatidylcholine and phosphatidylethanolamine as compared with 32P labeling of
the same phospholipids from WKY cells (Figures 5B
32
Phosphorus-32 Labeling of Phospholipids in Vascular and 5C). Incorporation of P into phosphatidylinositol and phosphatidylserine also tended to be lower
Smooth Muscle Cells
in cells from SHRSP than in those from WKY, but
32
A representative autoradiogram showing P labelthese differences did not reach statistical significance
ing of phospholipids in VSMC is shown in Figure 4.
at any time point (Figure 5D).
After chromatographic separation, the 32P radioactivThe total lipid-associated 32P radioactivity was significantly lower in cells from SHRSP than in those
from WKY after 60, 120, and 180 minutes of incubation (Figure 5A). The deficit in total lipid-associated
32
P observed in VSMC from SHRSP indicates that this
tissue has a generalized defect in its mechanism for
incorporating exogenous phosphate into lipids. To
determine whether there were also differences among
the phospholipids in their uptakes of 32P, the results
for individual phospholipid classes were expressed as
percentages of total lipid-associated radioactivity after
2 hours of incubation (Table 2).
The phosphatidylcholine incorporated approximately 70% of the 32P, and there was no significant
SPh
PA
PC
PE
PI
difference in 32P labeling between cells from SHRSP
and WKY (Table 2). Phosphatidylinositol incorporated
FIGURE 3. Bar graph shows phospholipid composition of
approximately 16% of the 32P, and again no significant
vascular smooth muscle cells (VSMC) and aortas. Values
difference was observed between the two cell types.
represent mean±SEM for eight studies on VSMC and four
Phosphatidylethanolamine that incorporated relatively
studies on aortas in each group. No difference was found
much less radiolabeled phosphorus retained the origieither between VSMC and aortas or between these tissues from
nal differential labeling with cells from WKY incorpostroke-prone spontaneously hypertensive rats (SHRSP) and
rating 6.2±0.7% 32P into phosphatidylethanolamine
Wistar-Kyoto rats (WKY). PI, phosphatidylinositol; PA, phosand cells from SHRSP incorporating 4 ±0.4% 32P into
phatidic acid; PS, phosphatidylserine; SPh, sphingomyelin;
phosphatidylethanolamine (p=0.02; Table 2). In conPC, phosphatidylcholine; PE, phosphatidylethanolamine.
Dominiczak et al Lipid Bilayer in Hypertension
110
•o
110
110
753
ISO
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i
120
100
0
SO
120
TIME(mln)
110
TIHE(mln)
FIGURE 5. Line graphs show time course of phosphorus-32 incorporation in phospholipids of vascular smooth muscle cells from
Wistar-Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP). PL, total phospholipids; PC, phosphatidylcholine;
PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine. Rate of incorporation of 32P into each phospholipid
from WKY is faster than in that from SHRSP. Each value is the mean±SEMfor six or eight determinations. *p<0.05; **p<0.01.
trast, labeling of phosphatidylserine showed slightly but
significantly more 32P in this phospholipid class extracted from cells obtained from SHRSP than in those
obtained from WKY (11.3±1.3% and 7.8±0.8%, respectively;/? =0.04, Table 2).
TABLE 2.
PE
VSMC (%
total lipid
radioactivity
at 2 hrs)
# 1
WKY
70.4±1.9
6.1
7.4
SHRSP
67.7±1.7
10.1
11.1
1.0
Strain
WKY
SHRSP
PS
WKY
PIP2
Platelets
#2
6.2±0.7
1.1
4.0±0.4*
1.1
1.6
7.8 ±0.8
7.3
12.2
11.3±1.3*
11.8
12.1
WKY
16.0±1.9
67.0
51.7
SHRSP
16.9±1.0
SHRSP
PI
Related comparisons of 32P uptake in WKY and
SHR have been made previously on platelets.30-31 We
Phosphorus-32 Labeling of Phospholipids in Smooth Muscle Cells and Platelets
Phospholipid
PC
Phosphorus-32 Labeling of Phospholipids in Platelets
From SHRSP and WKY
62.6
53.6
WKY
18.1
27.7
SHRSP
14.3
21.6
Results for vascular smooth muscle cells are mean±SEM of eight studies; data for platelets represent two single
experiments. VSMC, vascular smooth muscle cells; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS,
phosphatidylserine; PI, phosphatidylinositol; PIP 2 , phosphatidylinositol 4,5-bisphosphate; WKY, Wistar-Kyoto rats;
SHRSP, stroke-prone spontaneously hypertensive rats.
V<0.05 WKY vs. SHRSP.
754
Hypertension
Vol 18, No 6 December 1991
SHRSP
WKY
PE
PC
•
*
mm
120
60
I
PS
PI
30
I
PIP2
o
30
60
120
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FIGURE 6. Autoradiogram of thin-layer chromatogram in
which the phospholipids of platelets from Wistar-Kyoto rats
(WKY) and stroke-prone spontaneously hypertensive rats
(SHRSP) had been labeled with phosphorus-32 for 30, 60, or
120 minutes. O, indicates the origin. Phosphatidylinositol
4,5-bisphosphate (PIP2), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylethanolamine (PE) were all labeled in platelets from both
strains of rats. Spot identified as PIP2 might contain lyso PI as
complete separation was not possible.
therefore repeated studies as we had done on VSMC
in two experiments on platelets. Results of these two
experiments were similar (Table 2). The autoradiogram of one such experiment is presented in Figure
6. Spots unambiguously identified as phosphatidylcholine were present on autoradiograms of platelet
phospholipids from SHRSP and WKY. The 32P incorporation into phosphatidylcholine was greater in
platelets from SHRSP than in those from WKY
(Figure 6 and Table 2). The pattern of 32P uptake by
the individual phospholipids in platelets is entirely
different from that in VSMC. This difference is
evident in Table 2 in which 32P uptake of the two
platelet experiments is compared with the mean
uptake values for eight VSMC studies. The uptake
value for each phospholipid is expressed as the
percentage of total lipid-associated radioactivity.
Discussion
The major finding of this study is that membrane
microviscosity, as measured with DPH the core or
TMA-DPH the surface probe, was significantly
greater in membranes from cultured VSMC obtained
from SHRSP than in those obtained from the WKY
normotensive control strain. Moreover, membrane
microviscosity was modified in vitro by incubating the
membranes with 5 mM calcium. This treatment significantly lowered microviscosity of SHRSP membranes
in core and surface regions of the bilayer, making
these membranes very similar to those obtained from
VSMC from normotensive controls.
Early studies on membrane microviscosity in genetic hypertension were limited to membranes of
circulating blood cells (red blood cells and platelets)
and to measurements with the membrane core probe
DPH. Studies with red blood cells involved only the
plasma membrane, whereas in our studies with
VSMC, both plasma membrane and intracellular
membranes were isolated. Nevertheless, these earlier
studies of red blood cells of SHR1617-32 or of patients
with essential hypertension1819 demonstrated increased microviscosities of the same order of magnitude as that in our current data.
Devynck et al33 reported similar changes in microviscosity in the plasma membranes of hepatocytes,
synaptosomes, and cardiomyocytes of SHR. Tsuda et
al,20 using an entirely different methodology, an
electron spin resonance found that values of outer
hyperfine splitting and of the order parameter determined from electron spin resonance spectra for fatty
acid spin-label agent (5-nitroxy stereate) were significantly higher in red blood cells and cultured VSMC
from SHR than in those from WKY rats. These data
were interpreted as indicating a decreased membrane fluidity in the SHR. The same group34 showed
that the red blood cells pretreated with the calcium
ionophore A23187 and 1 mM calcium chloride had
increased microviscosity as compared with the untreated cells. These changes were more prominent in
the red blood cells from hypertensive rats and patients with essential hypertension than in their respective normotensive controls. The authors argued
that an abnormality of cellular calcium handling
might affect physicochemical properties of the biomembranes in hypertension.34
The current study is the first fluorescence polarization study showing increased microviscosity of
VSMC membranes from genetically hypertensive
rats. Our findings demonstrate that the microviscosity of the hydrophobic core of the lipid bilayer of
cultured VSMC from genetically hypertensive rats is
greater than that of these cells from normotensive
controls. This is the first experimental evidence indicating that there is a similarly greater microviscosity
in the surface region of the bilayer of any cell isolated
from genetically hypertensive rats. The hydrophilic
surface of the bilayer consists of the polar heads of
the phospholipids, and in all membranes it has a
greater viscosity (lower fluidity) than does the central
core, which consists of the acyl chains. Our observation that the microviscosity is greater in both the
surface and the core regions suggests that the abnormality of the lipid bilayer in hypertension is generalized and therefore has the potential for affecting the
function of transport proteins. In line with these
findings is the frequently reported observation that
membrane permeability to Na+, K+, and Ca2+ is
increased in hypertension.1-4
The possibility that the increased microviscosity
may be the cause of these greater cation fluxes in
hypertension is supported by observations of the
effects of increased extracellular calcium concentration on membrane microviscosity and on cation
fluxes. In the current study, we observed that an
increase in calcium concentration decreases microviscosity of the lipid bilayer of VSMC from SHRSP.
Dominiczak et al
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Jones and Hart35 and Furspan and Bohr3 studied the
effects of the same magnitude of increase in extracellular calcium concentration on potassium efflux
from vascular smooth muscle and from lymphocytes,
respectively. They found that the increase in extracellular calcium concentration caused a decrease in
potassium efflux (i.e., calcium affected the membrane
in such a way that the protein channel responsible for
potassium efflux became less active). Their studies
demonstrated that a concentration of 5 mM calcium
had clear depressing effects on monovalent cation
fluxes and on vascular reactivity. We therefore chose
this concentration (5 mM) for our study of the effect
of calcium on membrane microviscosity. These observations suggest that under conditions of hypertension or high extracellular calcium, membrane microviscosity may have an effect on cation fluxes. The
increased microviscosity of hypertension is accompanied by an increase in cation fluxes, whereas when
microviscosity is reduced by increasing extracellular
calcium, the cation fluxes are depressed.
Bialecki and Tulenko36 have studied the effects of
another intervention and reported observations that
give further support to this relation between membrane microviscosity and permeability. They observed that increased incorporation of cholesterol
into VSMC membrane caused an increase in membrane permeability to calcium. Cholesterol is known
to increase membrane microviscosity.3738 Thus, cholesterol, which increases microviscosity, causes an
increase in permeability as in hypertension; whereas
calcium, which reduces microviscosity, causes a decrease in permeability to levels similar to those of
normotensive rat.
Data from two laboratories documented that the
cell membrane of various tissues from hypertensive
humans and from the SHR binds less calcium than do
tissues from normotensive controls.3339-41 The molecular basis for this abnormality might be related to
deficiency in calcium binding proteins or abnormalities of negatively charged phospholipids. Kowarski et
al42 have reported that various tissues from SHR
contain less of an integral calcium binding protein
than those from WKY. However, the affinity of this
protein for calcium is too high: the binding sites would
be saturated at physiological concentrations of extracellular calcium. This leaves the phospholipids of the
lipid bilayer, particularly the negatively charged phosphatidic acid, phosphatidylserine, and phosphatidylinositol as the most important components that bind
calcium, especially at low affinity sites.38-43
Contrary to our observation that membrane microviscosity is decreased by an elevated calcium concentration, several investigators have reported that calcium causes an increased microviscosity.44-46 For
example, Sauerheber et al44 reported that incubation
of rat epididymal adipocyte ghosts with calcium
chloride decreased membrane fluidity in a concentration-dependent manner. We have no clear explanation for the difference between these results and
ours except the very different cell type studied and
Lipid Bilayer in Hypertension
755
different methodology of fluidity measurements:
whole cell electron spin resonance versus membranes
and polarization spectrofluorometry. The interaction
of calcium with the membrane has been shown to
affect phase separation, bilayer-nonbilayer transition,
changes in membrane permeability, and fusion.37-38
Calcium may also provide bridging between proteins
and lipids, which could exert a marked effect on the
membrane assembly.38 It is clear that there are many
tenable alternatives to our speculation relating calcium "stabilization" to bilayer microviscosity.
We carried out studies to determine whether these
clear differences in membrane microviscosity and permeability between VSMC of WKY and SHRSP could
be caused by differences in phospholipid composition
or their dynamic properties within the lipid bilayer.
Direct measurements of phospholipid composition in
cultured VSMC and in intact aortas demonstrated a
similarity between the two vascular preparations, suggesting that this property of the membrane had not
undergone a phenotypic change in culture. There was
no difference in the content of major classes of phospholipids between VSMC from SHRSP as compared
with those from WKY rats. This is in agreement with
data on phospholipid composition in platelets30 and
thoracic aortas47 from SHR and WKY. The authors of
the latter study measured only phosphatidylethanolamine and phosphatidylcholine content.
Okamoto et al21 presented evidence for increased
activity of phospholipase A2 in renal membranes of
SHRSP as compared with WKY. This phospholipase
activity increased with age in SHRSP and was associated with parallel decreases of phosphatidylcholine,
phosphatidylethanolamine, and of the phosphatidylcholine-to-sphingomyelin ratio. Phosphatidylcholine
and phosphatidylethanolamine act as membrane fluidizers, whereas proteins, cholesterol, and sphingomyelin act as rigidifiers.37 Okamoto et al21 speculated
therefore that the renal membrane of SHRSP became
"rigid" with age due to the changes in phospholipid
composition.
The method used in the current study permitted
the determination of phosphate content with precision to 0.01 /umol,27 and no difference was observed
between those measurements in VSMC from WKY
and from SHRSP. It should be stressed, however,
that under physiological conditions, lipid constituents
of biological membranes diffuse freely in the plane of
the membrane. Another feature that adds to this
complexity is the asymmetric distribution of phospholipids in the endofacial and the exofacial sides of
the membrane.37 It might therefore be more relevant,
for pathophysiological considerations, to measure
lipid microviscosity rather than the chemical composition of the phospholipids within the bilayer.
Remmal et al30 and Marche et al31 presented
evidence for enhanced turnover of phosphatidylcholine in platelets of SHR, SHRSP, and inbred Dahl
salt-sensitive rats as compared with their respective
normotensive control strains. In contrast, platelets
from deoxycorticosterone acetate (DOCA)-salt
756
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Vol 18, No 6 December 1991
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hypertensive rats showed similar 32P phosphatidylcholine labeling as their normotensive controls. It
was argued that the enhanced turnover of the most
abundant membrane phospholipid may constitute a
primary membrane alteration, ultimately resulting in
increased free cytosolic calcium and increased sensitivity to agonists.31
The current experiments, examining 32P labeling of
individual phospholipids in VSMC, gave results that
differed from those obtained in platelets.3031 VSMC
from both sources exhibited a major radioactive spot
identified as phosphatidylcholine. The total lipidassociated radioactivity was greater in VSMC from
WKY than in those from SHRSP. The possible role
of the different metabolic pools of ATP or lack of the
isotopic equilibrium with the y-phosphate of the
ATP, or both, cannot be excluded as the basis for this
difference. Insight into differences that may occur
between WKY and SHRSP phospholipids can be
obtained by expressing the 32P labeling of the individual phospholipids as a percentage of total lipidassociated radioactivity after incubation with the
isotope.3048 After 2 hours of incubation, phosphatidylcholine labeling was similar in cells from SHRSP
and WKY, whereas 32P incorporation into phosphatidylethanolamine was significantly greater in cells
from WKY than in those from SHRSP. Phosphatidylethanolamine has been shown to act as membrane
fluidizer37 and to affect the activity of Ca2+ATPase
possibly through interactions between the bilayer
surface and the extramembranous portions of the
calcium pump protein.15 There was also a small but
significant difference in 32P labeling of phosphatidylserine, which similar to phosphatidylethanolamine, is
located in the inner half of the bilayer. It might be
relevant that the calcium phospholipid-dependent
protein kinase C requires phosphatidylserine for
activation and that mesenteric arteries from SHRSP
have been shown to be more responsive to the
contractile effects of protein kinase C activators than
those from WKY.49
The observed discrepancies in 32P labeling between
VSMC in this study and platelets30-31 may be related
to individualities of the cell types. However, because
of this difference in labeling between VSMC in our
laboratory and platelets as studied by Marche et al,31
we felt that it was incumbent on us to repeat the
platelet studies in our laboratory. Marche observed
that after 2 hours of incubation with 32P, platelets
from SHRSP but not those from WKY exhibited
radioactive spots identified as phosphatidylcholine.
In contrast to this observation, our autoradiograms
showed radioactive spots identified as phosphatidylcholine in platelets from SHRSP and WKY at each
time point studied (see Figure 6). Counting of the
radioactivity in these spots confirmed Marche's observation that more 32P was incorporated into phosphatidylcholine in SHRSP, but the differences observed in the current study were less pronounced
than the 10- to 16-fold difference reported previously.31 The explanation for these discrepancies is not
clear; the possibilities to consider are: 1) betweenstrain differences in genetically hypertensive rats
obtained from commercial suppliers as compared
with inbred colonies used in this study50 and 2) time
of exposure of the x-ray film to TLC plates; the total
lipid-associated radioactivity per microgram of protein tends to be low in platelets and longer exposure
(up to 48 hours) is needed to visualize the phosphatidylcholine in platelets from WKY.
In conclusion, this is the first fluorescence polarization study to establish that membrane microviscosity of VSMC is elevated in SHRSP as compared
with membranes from WKY. It confirms observations on membranes from red blood cells and
platelets suggesting that this bilayer abnormality
may be generalized. More importantly, the current
observation was made in a tissue that is involved
directly in vascular contractility and thus in regulation of blood pressure. Our observations also demonstrate for the first time that the abnormality
affects similarly the core and the surface regions of
the lipid bilayer. In addition, we observed that an
increase in calcium concentration causes a decrease
in membrane microviscosity in both regions. Since
an increase in extracellular calcium concentration
also has been shown to decrease membrane permeability, we hypothesize that the increased microviscosity of the cell membrane in hypertension causes
the increase in membrane permeability to K+, Na+,
and Ca2+ observed in hypertension.
Acknowledgments
We thank Dr. Fedor Medzihradsky and Dr. Amiya
K. Hajra for sharing their insight into lipid biochemistry. We are grateful to Dr. Hajra for the use of his
laboratory and supplies for our studies on phospholipid
composition and turnover. We thank Dr. Donald MacCallum for guidance in immunofluorescent techniques,
and Betty Bunnell for expert secretarial assistance.
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KEY WORDS • vascular smooth muscle • cell culture • blood
platelets • membranes • phospholipids • membrane fluidity •
spontaneously hypertensive rats
Lipid bilayer in genetic hypertension.
A F Dominiczak, D F Lazar, A K Das and D F Bohr
Hypertension. 1991;18:748-757
doi: 10.1161/01.HYP.18.6.748
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