Alteration of Lipids, G Proteins, and PKC in Cell
Membranes of Elderly Hypertensives
Pablo V. Escribá, José M. Sánchez-Dominguez, Regina Alemany, Javier S. Perona, Valentina Ruiz-Gutiérrez
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
Abstract—In this study, we quantified the levels of lipids and signaling proteins in erythrocyte membranes from elderly
normotensive and hypertensive subjects. In hypertensive subjects, the cholesterol/phospholipid ratio increased
significantly in erythrocyte membranes, owing to the reduction of phospholipid levels concomitant with a rise in the
levels of cholesterol. In addition, differences were also found in the amount of fatty acids in both phospholipid and
cholesterol esters. Erythrocyte membranes from hypertensive subjects contained higher levels of monounsaturated and
lower levels of polyunsaturated fatty acids. On the other hand, signaling proteins such as G proteins and protein kinase
C have been implicated in the control of blood pressure. Previous studies have shown that the cellular localization and
the activity of these proteins are modulated by the type and the abundance of membrane lipids. For this reason, we
assessed the levels of these signaling molecules in the membrane. We found that the levels of membrane-associated
(active/preactive) G proteins (G␣i, G␣o, and G␤) and protein kinase C were significantly reduced in hypertensive
subjects. We believe that these alterations could be related to the etiopathology of hypertension in elderly subjects or
alternatively may correspond to adaptive compensatory mechanisms. (Hypertension. 2003;41:176-182.)
Key Words: blood pressure 䡲 G proteins 䡲 elderly 䡲 protein kinases 䡲 membranes 䡲 lipids
C
ardiovascular pathologies constitute the main cause of
death in industrialized countries.1 Among the different
manifestations and symptoms that arise during the development of cardiovascular illnesses, hypertension is a major risk
factor,2 the control of which is one of the main aims of
cardiovascular therapies.3 Multiple metabolic abnormalities
accompany essential hypertension, and these include alterations in serum lipoprotein levels (HDL, LDL and VLDL),
hypertriglyceridemia, hypercholesterolemia, insulin resistance, and so forth.4 – 6 In addition, alterations in the composition and properties of membranes have been reported both
in hypertensive humans7–11 and animal models of hypertension.12–14 Modifications of membrane lipids change the physical and functional properties of the cell barrier, and such
modifications could be reflected in alterations of cellular
physiology in hypertensive subjects. Indeed, the cellular
localization and activity of G proteins and protein kinase C
(PKC) are modulated by membrane lipids and drugs that
modulate the membrane structure.15,16
G proteins and PKC are signaling elements critical in the
propagation of messages from G-protein– coupled receptors
(GPCRs). The relevance of G proteins and PKC in the control
of blood pressure is reflected in the fact that a wide variety of
GPCRs are involved in this physiological process. In this
context, ␣1-, ␣2-, and ␤-adrenoceptors have been shown to
control blood pressure at different levels. The ␣ 2 -
adrenoceptor controls blood pressure centrally by activating
Gi proteins, whose ␣-subunit inhibits the production of
cAMP by adenylyl cyclase.17,18 In contrast, ␣1- and
␤-adrenoceptors control blood pressure peripherally, activating phospholipase C and adenylyl cyclase through G␣o/q and
G␣s proteins, respectively. The effector proteins phospholipase C and adenylyl cyclase regulate intracellular levels of
second messengers, such as diacylglycerol, inositol phosphate, and cAMP. These second messengers, along with
G␤␥-protein subunits, modulate the activity of other messengers, including various protein kinases (PKC, PKA, and
GRK) that phosphorylate and modulate the activity of several
proteins. Some of the targets of these kinases are activated
GPCRs that, on phosphorylation, become desensitized.19 –21
In this scenario, alterations in membrane lipids and of
signaling membrane proteins could be relevant to the pathophysiology of hypertension.22,23 On one hand, hypertension
has been associated with multiple alterations in the physical
properties of the plasma membrane.24 On the other hand, an
impairment of the adrenoceptor/G protein/adenylyl cyclase
system has been consistently observed in hypertensive humans and animals, although the molecular basis of these
alterations remains largely unknown.25–29
This study was designed to evaluate the status of serum and
membrane lipids as well as membrane-associated signaling
proteins (G proteins and PKC) in erythrocytes of elderly
Received June 14, 2002; first decision July 10, 2002; revision accepted November 7, 2002.
From the Laboratory of Molecular and Cellular Biomedicine, Department of Biology, University of the Balearic Islands (P.V.E., R.A.), Palma de
Mallorca, Spain; and Instituto de la Grasa, Consejo Superior de Investigaciones Científicas (J.M.S.-D., J.S.P., V.R.-G.), Sevilla, Spain.
Correspondence to Pablo V. Escribá, Molecular and Cellular Biomedicine, Department of Biology, University of the Balearic Islands, Ctra.
Valldemossa Km 7.5, E-07071 Palma de Mallorca, Spain. E-mail [email protected].
© 2003 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
DOI: 10.1161/01.HYP.0000047647.72162.A8
176
Escribá et al
TABLE 1.
Characteristics of Hypertensive and Control Subjects
Hypertensive
Subjects
Control
Subjects
Age, y
83⫾1
86⫾1
Male/female, n
7/21
6/22
28.1⫾1.1
28.1⫾0.8
Characteristics
Body mass index, kg/m2
Diastolic blood pressure, mm Hg
76.6⫾1.9
72.4⫾2.1‡
Systolic blood pressure, mm Hg
145.2⫾4.2
140.4⫾4.0‡
Total serum cholesterol, mg/dL
184.8⫾7.7
185.7⫾7.3
LDL-cholesterol, mg/dL
114.2⫾5.5
113.4⫾6.6
HDL-cholesterol, mg/dL
51.7⫾3.2
56.8⫾3.5‡
Triglycerides, mg/dL
94.6⫾6.4
77.8⫾5.3†
Phospholipids, mg/dL
178.5⫾7.1
182.0⫾4.8*
Values are expressed as mean⫾SEM. LDL indicates low-density lipoproteins
in serum; HDL, high-density lipoproteins in serum.
*P⬍0.05, †P⬍0.01, ‡P⬍0.001 vs hypertensive subjects.
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
normotensive and hypertensive subjects. Between these two
groups, we found significant differences in the levels of
membrane lipids, G proteins, and PKC. In erythrocytes from
hypertensive subjects, an increase in the levels of membrane
cholesterol and a decrease in total phospholipids was found,
as were alterations in the relative abundance of their fatty acid
moieties. Immunochemical quantification of membranebound G proteins revealed decreases in different G-protein
subunits. Finally, the amount of PKC␣ was significantly
lower in hypertensive subjects with respect to normotensive
subjects.
In summary, this work presents relevant data that could
explain the alterations of GPCR signaling associated with
hypertension in elderly (and possibly other) subjects. It is
likely that alterations in membrane lipid levels in hypertensive subjects affected the localization and activity of G
proteins, PKC, and possibly other signaling proteins. These
changes could explain the observed impairment in signal
propagation from GPCRs in hypertensive subjects.
Methods
Subjects
Elderly hypertensive and normotensive (control) subjects were
studied. The hypertensive and control groups each consisted of 28
subjects (Table 1). Blood pressure was measured with a mercurygauge sphygmomanometer on the right brachial artery of each
subject. At each visit, 3 blood pressure readings were recorded, and
the average was used to determine the eligibility of the subject. In
addition, the subject’s blood pressure was recorded at the beginning
and end of each period of study. The criterion for hypertension was
a systolic blood pressure ⱖ140 mm Hg and a diastolic value of
ⱖ90 mm Hg on at least 3 different occasions. Blood pressure was
recorded after the subject had been at rest in a supine position for 10
minutes.30 Before recruitment, the medical histories of all the
participants were reviewed comprehensively, and a physical examination and clinical chemical analysis were performed to exclude
possible secondary causes of hypertension. None of the subjects used
in the study had diabetes mellitus or hypothyroidism, and no history
of alcohol abuse or cigarette smoking was seen. All subjects gave
their informed consent before participating in the study. Hypertensive subjects were treated with enalapril, felodipine, spironolactone,
or amiloride, but none of these drugs has been reported to alter the
levels of the proteins studied here. Biochemical and physiological
Cell Membrane Signaling in Elderly Hypertensives
177
parameters indicated that the control group was healthy. All the
protocols used in this study were approved by the Institutional
Committee of Human Research (Comité de Ensayos Clínicos,
Hospital Universitario Virgen del Rocío, Sevilla, Spain).
Biochemical and Plasma Lipid Measurements
Levels of blood glucose, creatinine, and uric acid were measured by
conventional enzymatic methods, with the use of venous blood
obtained on the day of the examination after overnight fasting.
Similarly, LDL, HDL, and total cholesterol, phospholipids, and
triglycerides in serum were measured by enzymatic methods.31,32
Preparation of Erythrocytes
Erythrocyte membranes were prepared as described previously.4 – 8
Briefly, blood samples obtained after overnight fasting were collected in heparinized tubes and centrifuged at 1750g at 4°C for 10
minutes. The plasma and buffy coat were removed, and the erythrocyte pellet was washed twice with 110 mmol/L MgCl2. Aliquots of
this preparation were used to simultaneously determine the membrane lipid and protein content.
Analysis of Lipid Classes and Fatty Acid
Methyl Esters
Quantitative extraction of total erythrocyte membrane lipids from 5
mL of blood was carried out as described elsewhere.33–35 Lipid
extracts were dissolved in chloroform/methanol (2:1 vol:vol) and
passed through 0.2-␮m filters. They were then analyzed by liquid
chromatography, as described previously, with the use of standard
solutions for both identification and quantification.4 –11 Lipids were
also separated by thin-layer chromatography on silica gel plates
(Kieselgel 60 F254, Merck). The mobile phase was a mixture of
hexane/diethylether/acetic acid (80:20:1 vol/vol/vol), as described.36
The phospholipid and cholesteryl-ester fractions were transmethylated, and the resulting fatty acid methyl esters were analyzed by gas
chromatography.36 Individual fatty acid methyl esters were identified
by comparison with known standards or by gas chromatography–
mass spectrometry.
Immunoblot Analysis and Quantification of G
Proteins and PKC␣
Immunoblotting of G proteins and PKC␣ from erythrocyte membranes of elderly normotensive (control) or hypertensive subjects
was performed as described elsewhere.37 Briefly, a pellet of erythrocytes from 1 mL of blood was combined with 1 mL of homogenization buffer (50 mmol/L Tris-HCl [pH 7.5], 1 mmol/L EDTA,
2 mmol/L MgCl2, 1 mmol/L PMSF, and 5 mmol/L iodoacetamide)
and homogenized with a blade-type homogenizer. The samples were
then centrifuged at 600g and 4°C for 5 minutes. The pellets that
contained whole cells were discarded, and the supernatants were then
centrifuged twice at 40 000g and 4°C for 20 minutes. These final
pellets contained the erythrocyte membranes, and they were resuspended in 500 ␮L of homogenization buffer and 250 ␮L of
solubilization buffer (160 mmol/L Tris-HCl [pH 6.8], 8% sodium
dodecyl sulfate). The protein content of these samples was then
determined by means of the bicinchoninic acid method.38 Finally, the
sample proteins were separated by electrophoresis and immunoblotted with the following specific primary antibodies, as described
elsewhere37: anti-G␣i1/2 (1:7000 dilution), anti-G␣s (1:5000; used to
measure the 52-kDa band),37,39 anti-G␣o (1:4000), and anti-G␤
(1:5000) were from New England Nuclear Corp, and anti-PKC␣
(1:1000) was from BD Transduction Laboratories. The primary
antibodies were detected with horseradish peroxidase–linked secondary antibodies (1:2000 dilution; Amersham Pharmacia) and visualized with the enhanced chemiluminescence Western blot detection
system (Amersham Pharmacia) exposed to enhanced chemiluminescence hyperfilm (Amersham Pharmacia). Quantification was performed by image analysis as described.37
178
Hypertension
January 2003
Statistical Analysis
The results of the statistical analyses are expressed as mean⫾SEM.
One-way ANOVA, followed by the Scheffé test, was used for
statistical evaluations. Differences between experimental groups
were considered statistically significant at a value of P⬍0.05.
TABLE 2. Relative Percentages of the Individual Lipid
Composition Extracted From Erythrocyte Plasma Membrane of
Hypertensive and Control Subjects
Lipid Species
Hypertensive
Subjects
Control
Subjects
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
Results
CE
7.2⫾0.9
6.7⫾0.6
The demographic characteristics, blood pressure, and serum
lipid/lipoprotein levels of the subjects used in this study are
shown in Table 1. The mean⫾SEM levels of glucose,
creatinine, and uric acid in control subjects were 101⫾3
mg/dL, 0.97⫾0.18 mg/dL, and 4.9⫾1.4 mg/dL, respectively.
In hypertensive subjects (n⫽28), these values were 95⫾13
mg/dL, 1.1⫾0.24 mg/dL, and 5.5⫾1.5 mg/dL, respectively.
In the control group, mean⫾SEM blood pressure was
140⫾4.0 mm Hg (n⫽28), a normal value for subjects of this
age (86⫾1 years). The hypertensive group had a significantly
higher systolic blood pressure value (145.2⫾4.2 mm Hg,
P⬍0.001, n⫽28) (Table 1). Diastolic blood pressure was also
significantly higher in elderly hypertensive subjects
(72.4⫾2.1 mm Hg and 76.6⫾2.2 mm Hg for normotensives
and hypertensives, respectively; P⬍0.001) (Table 1).
TG
7.1⫾1.2
6.3⫾0.5
Serum Lipoproteins in Elderly
Hypertensive Subjects
Although no significant differences in total cholesterol were
observed, a significant decrease in the levels of HDL-cholesterol was found in elderly hypertensives (56.8⫾3.5 mg/dL
and 51.7⫾3.2 mg/dL in normotensive and hypertensive
subjects, respectively, P⬍0.001) (Table 1). Increased levels
of serum triglycerides were also found in this group
(77.8⫾5.3 mg/dL and 94.6⫾6.4 mg/dL for normotensive and
hypertensive subjects, respectively; P⬍0.01). In contrast, the
hypertensive group had lower levels of phospholipids
(182⫾5 mg/dL and 178⫾7 mg/dL in normotensives and
hypertensives, respectively, P⬍0.05) (Table 1). These alterations are characteristic features of hypertensive subjects,
further evidence of their pathological status.4 – 8
Erythrocyte Membrane Lipids in Elderly
Hypertensive Subjects
Erythrocyte membranes from elderly hypertensive subjects
contained a higher percentage of cholesterol with respect to
the total lipid content in normotensive and hypertensive
subjects, respectively (23.5⫾1.5% and 25.8⫾0.9%; P⬍0.05;
Table 2). In contrast, the percentage of phospholipids was
lower in hypertensive subjects than in control subjects
(63.6⫾1.5% and 59.9⫾1.9% in control and hypertensive
subjects, respectively, P⬍0.05) (Table 2). These changes
resulted in a marked and significant (P⬍0.05) increase of the
cholesterol/phospholipid ratio in hypertensive subjects
(0.37⫾0.1 and 0.44⫾0.0 in normotensive and hypertensive
subjects, respectively, P⬍0.05).
Differences were also found in the levels of the fatty acid
moieties of phospholipids and cholesterol esters from erythrocyte membranes. Apart from particular changes in the
levels of distinct fatty acid species, monounsaturated fatty
acids were more abundant in hypertensive subjects, whereas,
the content of polyunsaturated fatty acids was higher in blood
cell membranes from control subjects (Table 3).
C
25.8⫾0.9
23.5⫾1.5*
PL
59.9⫾1.9
63.6⫾1.5*
C/PL
0.44⫾0.0
0.37⫾0.1*
Values are expressed as mean relative percentage⫾SEM (n⫽28). CE
indicates cholesterol esters; TG, triglycerides; C, cholesterol; PL, phospholipids;
and C/PL, cholesterol/phospholipid ratio.
*P⬍0.05 vs hypertensive subjects.
Density of G-Protein Subunits and PKC␣ in the
Erythrocyte Membranes of Elderly
Hypertensive Subjects
The precise quantification of membrane proteins in normotensive and hypertensive subjects was achieved through
quantitative immunoblotting. The correlation between the
amount of protein loaded on the gel and integrated optical
density (IOD) values was linear in the ranges used. In
addition, the steep slope of the curve ensured the reliability of
the data. These analyses showed that pertussis toxin–sensitive
G-protein ␣-subunit levels were reduced in blood cell membranes of hypertensive subjects. Specifically, the levels of the
adenylyl cyclase inhibitory G␣i1/2 proteins decreased in elderly hypertensive subjects (decreases of 31⫾10%, P⬍0.01;
Figure 1). Similarly, the levels of G␣o proteins were significantly and markedly reduced in the hypertensive group
(decreases of 38⫾11%, P⬍0.01; Figure 1). However, the
decrease observed in the levels of cholera toxin–sensitive G
protein (52-kDa G␣s protein) was not statistically significant
in hypertensive subjects (decreases of 16⫾7%, P⬎0.05,
Figure 1). With respect to the common G-protein ␤-subunit,
the levels of this protein were also downregulated in blood
cells of elderly hypertensive patients (decreases of 21⫾8%,
P⬍0.05, Figure 2). Finally, the levels of PKC␣ also showed
a marked and significant decrease in the membranes of red
blood cells from elderly hypertensive subjects (decreases of
32⫾8%, P⬍0.01, Figure 2).
Discussion
Cells exert an exquisite control of lipid levels both in plasma
and organelle membranes.40 The characteristic properties of
membranes are dictated by the specific combinations of
membrane lipids and proteins that they contain. In this
context, the changes in membrane lipid and protein levels in
erythrocyte membranes reported here are most probably
associated with alterations in cell behavior in elderly hypertensive subjects. These results are of special relevance to the
field of cell signaling because a direct relation between
membrane lipid levels in human erythrocytes and neurons has
been recently discovered.41 Therefore, the changes found in
blood cells could reflect alterations in other cell types that
control blood pressure.
Escribá et al
Cell Membrane Signaling in Elderly Hypertensives
179
TABLE 3. Fatty Acid Composition of Phospholipids and Cholesterol Esters in
Erythrocytes Membranes (mg/100 mg)
Phospholipids
Fatty Acid
Species
C14:0
C14:1n-5
Cholesterol Esters
Hypertensives
Controls
Hypertensives
Controls
0.4⫾0.1
0.2⫾0.1
0.9⫾0.3
1.0⫾0.2
0.7⫾0.2
n.d.‡
23.7⫾0.6
23.1⫾0.5
15.1⫾0.6
14.3⫾0.5*
C16:1n-9
0.4⫾0.0
0.3⫾0.0
3.1⫾0.6
2.5⫾0.3†
C16:1n-7
0.5⫾0.0
0.5⫾0.1
2.4⫾0.4
1.7⫾0.1‡
C16:0
C16:4n-3
C18:0
1.7⫾0.2
2.2⫾0.3†
2.6⫾0.3
2.5⫾0.5
n.d.
n.d.
16.6⫾0.3
17.1⫾0.6
3.2⫾0.5
2.9⫾0.5
C18:1n-9t
0.9⫾0.1
C18:1n-9
16.3⫾0.6
0.6⫾0.0‡
16.0⫾0.8
n.d.
n.d.
18.4⫾1.2
16.7⫾0.6†
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C18:1n-7
1.8⫾0.1
2.0⫾0.2†
1.5⫾0.1
1.7⫾0.2†
C18:2 n-6
12.5⫾0.7
13.4⫾0.7*
45.8⫾2.8
51.1⫾1.6‡
C18:3n-6
0.4⫾0.0
n.d.‡
0.9⫾0.2
0.8⫾0.1†
C18:3n-3
0.3⫾0.0
0.4⫾0.1
n.d.
n.d.
C20:2n-6
2.1⫾0.1
2.1⫾0.2
0.9⫾0.1
0.9⫾0.1
C20:4n-6
17.0⫾0.4
16.5⫾0.4
7.0⫾0.6
6.4⫾0.9*
C22:4n-6
0.7⫾0.3
0.6⫾0.1
n.d.
n.d.
C22:6n-3
0.7⫾0.1
0.8⫾0.2
n.d.
n.d.
C24:1n-9
1.5⫾0.1
1.6⫾0.1
n.d.
n.d.
Total SFA
41.2⫾1.1
41.1⫾0.7
19.3⫾1.2
18.2⫾1.1*
Total MUFA
22.6⫾0.6
21.7⫾0.7*
25.9⫾1.6
22.7⫾0.8‡
Total PUFA
36.2⫾1.2
38.2⫾0.9†
54.8⫾2.4
59.1⫾1.6‡
PUFA:SFA
0.8⫾0.04
0.9⫾0.03
2.7⫾0.3
3.4⫾0.3‡
PUFA:MUFA
1.6⫾0.1
1.7⫾0.1
2.2⫾0.2
2.6⫾0.2‡
Values are expressed as mean⫾SEM (n⫽28). SFA indicates saturated fatty acids; MUFA,
monounsaturated fatty acids; and PUFA, polyunsaturated fatty acids.
*P⬍0.05, †P⬍0.01, ‡P⬍0.001 vs hypertensives; n.d., not detected.
Different properties of the membrane lipid fraction modulate the activity of membrane proteins. In this context, it has
been demonstrated that the membrane cholesterol content
(also defined by the cholesterol/phospholipid ratio) regulates
the membrane fluidity or microviscosity.42,43 Thus, an increase in this ratio is associated with a decrease in membrane
fluidity.43 In addition to cholesterol, the type and abundance
of fatty acids (free or esterified) also contribute to the
modulation of membrane fluidity.44,45 We found that erythrocyte membranes from hypertensives have a higher cholesterol/phospholipid ratio (0.44⫾0.0) than those from normotensive subjects (0.37⫾0.1). This result is in agreement with
previous studies showing that the hydrophobic core of erythrocyte membranes is less fluid in hypertensive rats.46 Moreover, we found differences in the fatty acid composition of
erythrocyte membranes that may further alter the plasma
membrane fluidity in hypertensive subjects. Also, in line with
these results, vascular cells of spontaneously hypertensive
rats exhibit differences in membrane fatty acids that may
contribute to a lower membrane fluidity.47
Previous studies have also associated hypertension with
multiple plasma membrane alterations.4,23,24 Since membrane
fluidity regulates the activity of several membrane proteins,15,48 the lipid alterations observed in membranes of
elderly hypertensive subjects could be involved in altering
membrane protein function. Membrane lipids also regulate
the formation of hexagonal (HII) phases,16,44 nonlamellar
membrane structures that organize into tubular micelles.49 –51
HII phases are critical in cells,15,16 where they influence the
localization and activity of membrane proteins.15,48 G proteins and PKC are signaling molecules capable of translocating from the cytosol to membranes. They propagate and
amplify messages from membrane GPCRs (eg, those which
control the blood pressure) to other signaling proteins, and
their localization is modulated by the membrane lipid composition and HII-phase propensity.15,16 In the knockouts of the
␣2- and ␤1/2-adrenoceptors, it has been shown that these
receptor types are implicated in the central and peripheral
control of blood pressure, respectively.52,53 These receptors
use G␣i, G␣o, and G␣s proteins, further evidence that the
results presented here are associated with the control of blood
pressure. The levels of G␣i1/2 and G␣o were seen here to be
markedly and significantly lower in erythrocyte membranes
from hypertensive subjects (decreases of 31⫾10% and
38⫾11% for G␣i and G␣o, respectively, P⬍0.01), possibly
as the result of membrane lipid alterations. Although the
common G␤-subunit also appeared to be reduced in hypertensive subjects (21⫾8% decreases, P⬍0.05), G␣s levels
180
Hypertension
January 2003
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Figure 1. A, G-protein levels (mean⫾SEM) in erythrocyte membranes from elderly normotensive (open bars, n⫽10) and hypertensive (solid bars, n⫽10) subjects. Levels of G-protein ␣i1/2 (Gi),
␣o (Go), ␣s (Gs), and ␤ (Gb) subunits are shown. *P⬍0.05;
**P⬍0.01. B, Representative immunoblots for erythrocyte G proteins from a control (C) and a hypertensive subject (H). About 36
␮g of total protein was loaded for both subjects.
were not significantly decreased in hypertensive subjects
(16⫾7%, P⬎0.05). These alterations in the levels of membrane G proteins could be associated with the reduced
G-protein function and/or decreased receptor–G protein coupling described elsewhere, which may alter vasodilator function in hypertensive subjects.25
Figure 2. A, PKC␣ levels (mean⫾SEM) in erythrocyte membranes from elderly normotensive (control, n⫽10) and hypertensive subjects (n⫽10). **P⬍0.01. B, Representative immunoblot
for erythrocyte PKC␣ from a control (C) and a hypertensive subject (H). About 36 ␮g of total protein was loaded for both
subjects.
A reduction in G-protein activity in blood cells of older and
hypertensive subjects has been reported previously.29 Although these changes were not accompanied by modulations
in whole-cell G-protein levels, they may possibly have been
associated with the decreases in membrane G proteins described here. Our study was carried out in cell membranes, so
that the levels of G proteins found corresponded to active
and/or preactive signal transduction components and may
account for alterations in signal propagation. In line with this
study and previous works, it has been observed that high
blood pressure is associated with downregulation or loss of
responsiveness of ␣-adrenoceptors in elderly hypertensive
subjects.54
PKC was also found to be reduced in the membranes
isolated from hypertensive subjects. As for G proteins, the
cellular localization of this signaling enzyme is modulated by
the membrane lipid composition and HII-phase propensity.15,48 This enzyme is a key element in many GPCR-related
signals, including the control of blood pressure.55 Adrenoceptors (and other GPCRs) can activate PKC through phospholipase C–induced diacylglycerol production. Activated PKC
translocates to the plasma membrane,56 where it phosphorylates the serine/threonine residues of a wide variety of
proteins, including adrenoceptors and other GPCRs.20,21,57–59
The half-life of activated PKC is less than 1 hour, as the result
of its degradation by proteases.60 Therefore, lower PKC
levels could either be the result of decreased mRNA expression or increased enzyme activation (and subsequent degradation). The former possibility would be parallel to a decrease
in receptor phosphorylation, whereas the latter would be
associated with increased receptor phosphorylation and desensitization. Various facts favor the latter hypothesis. First, it
has been shown that PKC activation is followed by its
degradation, so that protein levels appear to be reduced
despite of an elevation in mRNA expression.61,62 Second, an
increase in PKC activation would result in increased receptor
phosphorylation and reduced adrenoceptor-associated signaling.60 Third, a greater degree of receptor desensitization
coupled with lower G-protein levels is in agreement with
previous studies demonstrating impaired adrenoceptor signaling. However, this matter requires further investigation because the relevance of this protein in the control of blood
pressure and atherosclerosis has also been associated with
other signaling events, such as the regulation of endothelin-1,
the cytosolic levels of Ca2⫹, and so forth.63,64 Moreover, it is
possible that PKC may exert different or even opposite
effects in central and peripheral systems.
In summary, we have found alterations in the levels of
erythrocyte membrane lipids in elderly hypertensive subjects
that could account for the alterations in the levels of
membrane-associated G proteins and PKC also observed
here. The decreases in G protein and PKC levels in erythrocyte membranes probably alters GPCR-related signaling in
these hypertensive subjects. GPCR-associated signaling alterations could be involved in the etiopathology of hypertension
in elderly subjects or may constitute an adaptive mechanism
in response to other alterations.
Escribá et al
Perspectives
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In industrialized countries, cardiovascular pathologies are
involved in about one half of all deaths. In fact, if we want to
live longer and healthier, it is essential to control cardiovascular, tumoral, and neurodegenerative risk factors. In increasingly aging societies, the knowledge of the molecular alterations underlying high blood pressure in elderly hypertensive
subjects and its control are crucial issues. In this study, we
highlight some molecular alterations associated with hypertension in elderly subjects. We found that the levels of certain
membrane lipids are altered in elderly hypertensive subjects.
Specifically, we have shown that the cholesterol/phospholipid
ratio and the levels of monounsaturated fatty acids were
higher in this group compared with normotensive control
subjects, who in turn had lower levels of polyunsaturated
fatty acids. We have previously demonstrated that the levels
and the types of membrane lipids modulate membrane structure and regulate G protein–membrane and PKC-membrane
interactions. When we studied the membrane levels of these
proteins, we found significant and marked changes in elderly
subjects with high blood pressure. It is feasible that the
changes we found in signaling proteins in the membrane were
related to alterations in the protein-lipid interactions that
resulted from the altered lipid levels. If this hypothesis is
correct, it may also be possible to control high blood pressure
by modulating the membrane lipid structure. In this context,
we have developed molecules capable of acting in this
manner, and we have called this therapeutic approach “lipid
therapy.” We believe that such an approach will become more
widespread during the next decades and hence provide many
benefits to human health.
Acknowledgments
This work was supported in part by grants FEDER 1FD97–2288
(European Community) and CAO001– 002 (Junta de Andalucía) (to
V.R.-G.) and by grants FIS00/1029 (Ministerio de Sanidad y
Consumo, Spain) and SAF2001– 0839 (Ministerio de Ciencia y
Tecnología, Spain) (to P.V.E.). We thank the Marathon Foundation
for its financial support. RA is a Ramón y Cajal Fellow. We are
grateful to the director, nurses, and medical personnel of the
Residencia de la Tercera Edad Heliópolis for their cooperation
during the period of the study.
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Alteration of Lipids, G Proteins, and PKC in Cell Membranes of Elderly Hypertensives
Pablo V. Escribá, José M. Sánchez-Dominguez, Regina Alemany, Javier S. Perona and
Valentina Ruiz-Gutiérrez
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Hypertension. 2003;41:176-182; originally published online December 9, 2002;
doi: 10.1161/01.HYP.0000047647.72162.A8
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