Preliminary Observations on
Abnormalities of Membrane Structure and
Function in Essential Hypertension
ALLEN J. NAFTILAN, VICTOR J. DZAU, AND JOSEPH LOSCALZO
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SUMMARY To test the hypothesis that structural abnormalities exist in the cell membrane in
persons with essential hypertension and that these abnormalities affect membrane-related cellular
functions, we examined several membrane-dependent phenomena and membrane lipid composition in
the blood cells of subjects with essential hypertension. We analyzed platelet aggregability, membrane
fluidity, membrane fatty acid composition, and erythrocyte deforinability in four normolipidemic
subjects with untreated essential hypertension and in five age-matched normotensive controls. As
compared with the controls, the subjects with essential hypertension had platelets that aggregated at
lower concentrations of adenosine 5-diphosphate, platelet membranes that were less fluid, and
erythrocytes that were more deformable. Lipid analysis of the membranes of platelets from the two
study groups showed that although the cholesterol content was identical, the membranes from the
essential hypertension group contained significantly less linoleic acid (18:2) than did those from the
normotensive controls. Given the known effects of c/s-unsaturated fatty acyl composition on membrane fluidity and membrane-related cellular functions, these data suggest that one factor contributing to essential hypertension is an inherent structural membrane abnormality that alters the physical
and functional properties of the cell membrane. (Hypertension 8 [Suppl II]: 11-174—11-179, 1986)
KEY WORDS
platelets • membrane fluidity • fatty acids • atherosclerosis
• essential
hypertension
E
SSENTIAL hypertension has been associated
with various functional membrane abnormalities in humans 15 and animals.6"8 This diversity
of membrane-associated disturbances suggests that an
intrinsic structural abnormality of the cell membrane
that is important for a variety of disparate membrane
protein activities may be a key etiological factor. One
such global membrane property is microenvironmental
fluidity, variations in which have been shown to affect
a number of different membrane functions, including
lymphocyte capping, 9 platelet aggregation,10 macrophage phagocytic activity," and sodium-potassium
adenosine triphosphatase (ATPase) activity.12 Factors
that have been shown to influence membrane fluidity
include the cholesterol content and fatty acid composi-
From the Division of Vascular Medicine and Atherosclerosis and
the Cardiology Division, Department of Medicine, Brigham and
Women's Hospital and Harvard Medical School, Boston, Massachusetts.
Supported in part by National Institutes of Health Grant
HL19295. V.J. Dzau is an Established Investigator of the American
Heart Association, and J. Loscalzo is supported in part by a Clinician Scientist Award from the American Heart Association.
Address for reprints: Dr. Joseph Loscalzo, Division of Vascular
Medicine and Atherosclerosis, Department of Medicine, Brigham
and Women's Hospital, 75 Francis Street, Boston, MA 02115.
tion of the membrane bilayer; the fluidity increases
with decreasing cholesterol content or increasing cisunsaturated fatty acyl composition.13
To test the hypothesis that a structural membrane
abnormality exists in the cells of persons with essential
hypertension and that this abnormality affects the
physical state and functional properties of the membrane, we examined two membrane-dependent cell
functions, platelet aggregability and erythrocyte deformability, in normolipidemic subjects with untreated
hypertension and in age-matched normotensive controls. We also measured membrane fluidity in the
platelets of these individuals, as well as the cholesterol
content and fatty acyl composition of the platelet membranes, in an attempt to correlate functional abnormalities with structural alterations. The preliminary results
from a small group of individuals demonstrate that
inherent abnormalities of membrane composition and
function are present in the blood cells of persons with
essential hypertension. Structural membrane abnormalities that affect the physical state and function of
cell membranes may be a generalized phenomenon in
essential hypertension, and these abnormalities may
be either primary or secondary to the hypertensive
process.
11-174
MEMBRANE ABNORMALITIES IN ESSENTIAL HYPERTENSION/JVq/r//an et al.
Materials and Methods
Materials
l,6-Diphenyl-l,3»5-hexatriene (DPH) and adenosine 5'-diphosphate (ADP) were obtained from Sigma
Chemical (St. Louis, MO, USA). High pressure liquid
chromatography-grade hexane, 2-propanol, chloroform, methanol, petroleum ether, diethyl ether, and
acetyl chloride were purchased from Aldrich Chemical
(Milwaukee, WI, USA). Fatty acid methyl ester standards were obtained from Nu Chek Prep (Elysian,
MN, USA). All other chemicals used were reagent
grade or better. Deionized water was used throughout.
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Experimental Groups
The populations studied included four normolipidemic subjects with newly diagnosed essential hypertension (two white men and two white women, three of
whom had family histories of hypertension) who had
received no prior medical therapy, and five agematched normotensive controls (three white men and
two white women). Various characteristics of the two
groups are listed in Table 1. Individuals were defined
as hypertensive if on three consecutive occasions the
systolic blood pressure was higher than 140 mm Hg or
if the diastolic blood pressure was higher than 90 mm
Hg. The subjects were on their usual diets, which
appeared to be comparable, on the basis of informal
dietary histories.
Platelet and Erythrocyte Preparation
Fifty milliliters of venous blood was obtained from
each subject and anticoagulated with 13 mM sodium
citrate. Platelet-rich plasma was prepared by centrifuging the anticoagulated blood at 160 g for 10 minutes.
Washed platelets were prepared by centrifuging the
platelets from platelet-rich plasma at 800 g for 20 minutes, resuspending the platelet pellet in platelet washing buffer (10 mM sodium citrate, 0.15 MNaCl, 1 mM
ethylenediaminetetraacetate, pH 7.0), centrifuging the
platelets at 800 g for 20 minutes, repeating the suspension in washing buffer and the centrifugation, and finally resuspending the platelets at 2 to 3 x IOV/AI in
modified Tyrode's solution (0.13 M NaCl, 2.7 mM
KC1, 12 mM NaHCO 3 , 0.42 mM NaH2PO4, pH 7.4).
Platelet counts were determined with a Coulter counter
TABI.K I. Characteristics of the Normotensive and Hypertensive
Study Groups
Normotensive
(n = 5)
Hypertensive
(n = 4)
Height (cm)
170 + 8
Weight (kg)
67 + 9
Body surface area (rrr)
1.78 + 0.08
Age (yr)
29 + 5
Total plasma cholesterol (mg/dl)
182± 17
Platelet count (x 10"Vmm')
278±3I
Blood pressure (mm Hg)
118±8/74±6
Values are means ± SD.
180 + 5
85±I5
2.05±0.08
27±6
I84±28
272±20
145±5/99±4
Parameter
11-175
(Model F; Coulter Electronics, Hialeah, FL, USA).
Erythrocytes were centrifuged at 800 g for 20 minutes, resuspended in modified Tyrode's solution, centrifuged, and resuspended twice more. The packed
erythrocytes were then diluted with modified Tyrode's
solution for the filtration experiments described
below.
Platelet Aggregation
Platelet aggregation was monitored by a standard
nephelometric technique14 in which 0.4-ml aliquots of
platelet-rich plasma were incubated at 37 °C and stirred
at 900 rpm in a Payton dual-channel aggregometer
(Payton Associates, Buffalo, NY, USA). Aggregation
was induced by the addition of ADP (over a range of
concentrations), and changes in light transmittance
were recorded with an Omniscribe recorder (Houston
Instruments, Austin, TX, USA). Aggregation was
quantitated by measuring the maximal rate of change
in light transmittance.
Fluorescence Measurements
DPH13-13 at a final concentration of 2 fiM was incubated at room temperature with a suspension of washed
platelets in modified Tyrode's solution at 1 x lO^/yu-l
for 20 minutes, after which the platelets were centrifuged and resuspended in modified Tyrode's solution
twice.
Fluorescence measurements were performed with a
Perkin-Elmer Model 44B spectrofluorometer (PerkinElmer, Norwalk, CT, USA) equipped with a thermostat-controlled cell holder and Hitachi polarizers. The
steady-state fluorescence polarization was measured
by exciting the cell suspension at 360 nm and recording the emission at 430 nm. The polarization of fluorescence emission was calculated from the equation
P = (/w - G/VHVC/W + G/VH), where P is polarization, / is the fluorescence intensity, the first and second
subscripts refer to the plane of polarization of the excitation and emission beams, respectively (V = vertical,
H = horizontal), and G = IHyJIm. For each measurement of P, the emission intensities were corrected for
contributions due to scattering by measurement of
these intensities in a blank containing all the solution
constituents except the fluorophore.
Erythrocyte Deformability Measurements
Erythrocyte deformability was assessed by measuring the flow time of a suspension of erythrocytes
through 4.7-micron Nucleopore filters (Hemafil polycarbonate membranes; Nucleopore, Pleasanton, CA,
USA) under a constant negative pressure of 20 cm
H2O, according to the method of Reid and colleagues.16
Lipid Extraction and Analysis
Platelet membrane lipids were extracted from
washed platelets with hexane-2-propanol, as described
by Hara and Radin.17
Cholesterol was determined with o-phthalaldehyde,
by the method of Rudel and Morris.18
11-176
1985 BLOOD PRESSURE COUNCIL
Platelet membrane phospholipids were transesterified with methanolic-HCl. 19 The fatty acid methyl esters were extracted into petroleum ether and separated
and identified by gas chromatography with a Chrompack CP Sil 88 column (Chrompack, Bridgewater, NJ,
USA) in a Varian chromatograph (Varian Associates,
Palo Alto, CA, USA) equipped with a solid injector
and a flame ionization detector connected to a computer integrator. Fatty acid methyl esters were identified
by comparison with known standards and were quantitated by integration of peak areas.
Statistical Analysis
The data are expressed as means ± SD. Statistical
comparisons were performed with the nonpaired Student's t test, and the null hypothesis was rejected forp
values less than 0.05.
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Results
Platelet Aggregation
The platelets from subjects with essential hypertension aggregated at lower concentrations of ADP than
did those from the normotensive controls. The doseresponse data presented in Figure 1 indicate that the
50% effective concentration (ECJO) for ADP-induced
aggregation was reduced approximately 2.5-fold, from
5.0 /iM to 2.0 /xM in the hypertensive group
(p < 0.01). The difference between the two groups was
not uniform throughout the range of concentrations of
ADP tested but appeared to be more marked at higher
concentrations of the agonist.
Platelet Membrane Fluidity
The fluorescence polarization of DPH incorporated
in the platelet membrane bilayer was measured as a
function of temperature in suspensions of platelets prepared from the hypertensive and normotensive subjects. DPH fluorescence polarization decreases with
increasing microenvironmental fluidity. The data in
Figure 2 demonstrate that at temperatures higher than
20°C, the fluorescence polarization of DPH was sig-
FIGURE 1. Relative maximal rale of
platelet aggregation as a function of the
agonist concentration in platelet-rich
plasma from hypertensive (o) and normotensive (*) subjects. Each point represents the mean value (± SD) of the
rate of aggregation relative to the maximal rate at the highest concentration of
ADP used.
SUPPL
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JUNE
1986
nificantly greater in platelets from the hypertensive
subjects than in those from the normotensive controls
(p<0.001 at temperatures higher than 35°C). This
difference in polarization became more pronounced as
the temperature was increased, suggesting that the microenvironment of DPH in platelet membranes from
hypertensive persons is considerably less fluid than
that in platelets from normotensive persons.
Erythrocyte Deformability
The deformability of erythrocytes from the two
groups was compared by measuring the flow rate of a
range of concentrations of cells through polycarbonate
membranes of 4.7-micron pore size (Figure 3). The
more deformable the erythrocyte, the faster it will flow
through the membrane. The data demonstrate that
erythrocytes from the hypertensive subjects were less
deformable than those from the normotensive controls
(p<0.0\ at hematocrits > 0 . 1 ) . Over a range of erythrocyte concentrations, the flow rate of the cell suspension was slower for cells from hypertensive subjects
than for those from controls, with differences between
the two groups being most marked at hematocrits
above 0.1 (i.e., 10%).
Platelet Lipid Analysis
Since the structural elements of bilayer membranes
that influence fluidity most dramatically are cholesterol and phospholipid fatty acyl chain saturation, we
analyzed the cholesterol content and fatty acid composition of platelet membranes in the two experimental
groups. The cholesterol content of platelets did not
differ significantly between the two groups (12 ± 2
jig/10* platelets in the hypertensive subjects and
13 ± 2 ptg/108 platelets in the normotensive controls).
In contrast, the data listed in Table 2 indicate that there
was significantly less linoleic acid (18:2) in the platelet membranes from the hypertensive subjects than in
those from the normotensive controls, with 44% less
cis-unsaturated fatty acid in the former group
(/?<0.01). In addition, less oleic acid (18:1) was de-
(ADFj CpMj
MEMBRANE ABNORMALITIES IN ESSENTIAL HYPERTENSION/Ata/h/an et al.
FIGURE 2.
0.4
11-177
1,6-Diphenyl-l ,3,5-hexa-
triene fluorescence polarization as a
function of temperature (T) for fluorophore incorporated in intact platelets
from hypertensive (O) and normotensive
(*) subjects. Each point represents the
mean value (± SD)for steady-state fluorescence polarization, derived as described in Materials and Methods.
0.3
02
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10
20
TCC)
30
4O
50
FIGURE 3.
Erythrocyte deformability
assessed by measuring the flow rate of
erythrocyte suspensions through 4.7micron-pore polycarbonate filters as a
function of the erythrocyte concentration in hypertensive (O) and normotensive (•) subjects. The flow rate is normalized to that for buffer (modified
Tyrode's solution). Each point represents the mean value (± SD)for the entire group of hypertensive or normotensive measurements.
TABI>: 2. Platelet Membrane Fatty Acid Composition in Normotensive and Hypertensive Subjects
Normotensive
Hypertensive
(9b of total)
Fatty acid
16:0
14.3 + 2.3
18.8+10.9
18:0
19.8±4.1
18.5 ± 1.3
18 1
17.5±9.5
ll.9±2.7
18:2
IO.5±5.8
5.9±1.4*
18:3
1.01 ±0.55
0.62 + 0.66
20:4
26.9 ±0.5
28.9± 1.0
Values are means ± SD.
V<0.0l.
tected in the platelets from the hypertensive subjects,
but this was only significant to thep<0.05 level. This
relative deficiency of c/5-unsaturated fatty acids appeared to be accommodated by a slight but nonsignificant increase in palmitic acid (16:0) in platelets from
the hypertensive subjects, as compared with those
from the normotensive controls.
Discussion
Few data exist on cellular membrane structure and
function in essential hypertension. Two groups have
demonstrated increased microviscosity in platelet and
erythrocyte ghosts in the spontaneously hypertensive
11-178
1985 BLOOD PRESSURE COUNCIL
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rat model of essential hypertension.20"22 In addition,
McGregor and colleagues22 showed that platelets from
spontaneously hypertensive rats aggregated in response to lower concentrations of thrombin or ADP
than did platelets from Wistar-Kyoto controls and that
this difference was potentiated by feeding the animals
diets rich in saturated fats. A decrease in erythrocyte
deformability and an increase in erythrocyte membrane microviscosity have been reported in patients
with essential hypertension.23-24 Nara and colleagues25
have also recently demonstrated that platelets aggregated in response to lower concentrations of ADP in
men with a family history of essential hypertension
than in those with no such history.
Our data support the concept that the cell membranes of persons with essential hypertension are structurally abnormal. This alteration in structure produces
a stiffer, less fluid membrane that modulates surfacedependent cellular physical properties and functions.
Platelets with surface membranes made less fluid by
exogenous increases in the cholesterol content26-27 or
the saturated fatty acid composition10 become hyperaggregable. Similarly, less fluid erythrocyte membranes
produce less deformable erythrocytes, as we have
noted in this study. An alternative explanation for the
decreased deformability of erythrocytes in essential
hypertension is, of course, that they have lower Na + K + -dependent ATPase activity, thereby increasing
cell volume. One study28 failed to demonstrate any
correlation between Na + -K + -dependent ATPase activity and erythrocyte filterability in hypertensive persons. Conversely, Yeagle12 has shown that erythrocyte
membrane lipid composition markedly affects Na + K + -dependent ATPase activity, with increased cholesterol resulting in reduced ATPase activity and diminished membrane fluidity.
From the data presented here, we cannot determine
whether the observed changes in membrane composition are causative or a consequence of the shear stress
engendered by elevated blood pressure in hypertensive
individuals. Two recent studies,8-23 however, support
the former hypothesis. Bianchi and colleagues8 have
shown that an alteration in erythrocyte Na + -K + cotransport observed in the Milan hypertensive rat strain
could be transferred to normotensive controls by bone
marrow transplantation, and Nara and co-workers25
have reported increased platelet aggregability in response to ADP in healthy normotensive men with a
family history of hypertension.
Interpreted in the context of these studies, our data
support the hypothesis that one factor contributing to
or potentially causing essential hypertension is an inherent membrane structural abnormality that alters the
physical state of the membrane and thereby modifies
membrane-related cellular functions. Although we
have studied only platelets and erythrocytes, this abnormality may be global, affecting the membranes of
all cells, including endothelial and smooth muscle
cells and the surface effectors of their functions that are
important for blood pressure regulation. If a single
genetic defect is responsible for this abnormality, one
SUPPL
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HYPERTENSION, VOL
8, No 6,
JUNE
1986
might speculate that a defective fatty acid desaturase or
elongase29-w may be a critical determinant of the hypertensive state. Failure to incorporate fatty acyl
chains of the appropriate length and degree of unsaturation would lead to changes in membrane fluidity and
consequent changes in membrane protein conformation and function.
Two critical lipid determinants of fluidity in cell
membranes have been defined, cholesterol composition (and the cholesterol/phospholipid ratio) an cisunsaturated fatty acyl group content (and the saturated/
c/5-unsaturated fatty acyl group ratio). Exogenous
modification of cholesterol content has been shown to
alter the membrane bilayer fluidity and membrane protein function in several systems. A 41 % decrease in the
cellular cholesterol/phospholipid ratio produces a 57%
reduction in lymphocyte capping. 9 A 54% increase in
the platelet cholesterol/phospholipid ratio leads to a
47% reduction in the EC*, of thrombin-induced platelet
aggregation, with a concomitant 73% increase in highaffinity thrombin receptors.27 In contrast, a 30% depletion of the erythrocyte cholesterol content induces a
40% increase in the apparent maximal rate of sodium
efflux, a 45% decrease in the apparent dissociation
constant of K + from the pump, and an 88% increase in
the apparent dissociation constant of Na + from the
pump.31 Similar kinds of studies using cell membrane
fatty acyl group modifications have been performed
less frequently. A 12 to 15% increase in macrophage
stearate content reduces erythrophagocytic activity by
76%, as compared with control cells. Similarly, an
approximate 5% increase in platelet membrane linoleate content is associated with a 10% decrease in DPH
fluorescence polarization and an 80% inhibition of
platelet aggregation.10 These data indicate that although changes in cholesterol content must be rather
extensive to produce marked changes in membrane
protein function, similar effects may be produced by
much less dramatic alterations in fatty acyl group composition. The biophysical reasons for this differential
sensitivity are not known but may relate to the notion
that specific annular32 or boundary33 lipids relatively
inexchangeably bound to membrane proteins form local microdomains34 that can dramatically alter local
protein conformation or function without markedly
changing global membrane properties. This principle
was espoused as early as 1963, when Jurtshuk et al.35
showed that optimal activation of mitochondrial
d{ — )/3-hydroxybutyric acid dehydrogenase requires
an unsaturated acyl lecithin in its microenvironment.
It is this kind of mechanism that may be invoked to
explain the myriad of transport abnormalities and other
surface membrane changes that have been described in
hypertension. Some of the transport abnormalities
themselves could also lead to changes in the amount
and type of fatty acyl chains incorporated in the membrane phospholipids,36 thereby further altering microdomain composition, fluidity, and membrane protein
function. Further studies are under way to address
these complex issues concerning cellular structurefunction relationships in hypertension.
MEMBRANE ABNORMALITIES IN ESSENTIAL HYPERTENSION/Afa/n/a/i et al.
Acknowledgments
The authors wish to acknowledge the excellent technical assistance provided by Ms. Jane Freedman and Mr. Nicholas Triano.
20.
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Preliminary observations on abnormalities of membrane structure and function in
essential hypertension.
A J Naftilan, V J Dzau and J Loscalzo
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Hypertension. 1986;8:II174
doi: 10.1161/01.HYP.8.6_Pt_2.II174
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