Division of Geriatric Medicine, Department of Internal Medicine, and Institute of Gerontology, University of Michigan, and Geriatric Research, Education, and Clinical Center, Department of Veterans Affairs Medical Center, Ann Arbor, Michigan 48105
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ABSTRACT |
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We have previously demonstrated in normotensive
humans an age-associated increase in sympathetic nervous system (SNS)
activity combined with appropriate downregulation of -adrenergic
responsiveness. Impaired downregulation of
-adrenergic
responsiveness, despite a comparable level of SNS activity, could
contribute to higher blood pressure in older hypertensive humans. We
measured arterial plasma norepinephrine (NE) levels and the
extravascular NE release rate
(NE2) derived from
[3H]NE
kinetics (to assess systemic SNS activity), and platelet and forearm
arterial adrenergic responsiveness in 20 normotensive (N) and in 24 hypertensive (H), otherwise healthy, older subjects (60-75 yr). Although plasma NE levels were similar (N 357 ± 27 vs. H 322 ± 22 pg/ml; P = 0.37),
NE2 tended to be greater in the hypertensive group (H 2.23 ± 0.21 vs. N 1.64 ± 0.20 µg · min
1 · m
2;
P = 0.11), and the NE metabolic
clearance rate was greater (H 1,100 ± 30 vs. N 900 ± 50 ml/m2;
P = 0.004). In the hypertensive group,
there was a greater
-agonist-mediated inhibition of platelet
membrane adenylyl cyclase activity and a NE- but not ANG II-mediated
decrease in forearm blood flow. Compared with normotensive subjects, in
older hypertensive subjects 1) NE
metabolic clearance rate is increased,
2) systemic SNS activity tends to be
increased, and 3) arterial and
platelet
-adrenergic responsiveness is enhanced. These results
suggest that heightened SNS activity coupled with enhanced
-adrenergic responsiveness may contribute to elevated blood pressure
in older hypertensive humans.
norepinephrine; hypertension; aging
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INTRODUCTION |
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THERE IS AN AGE-RELATED increase in the prevalence of hypertension that is associated with significant morbidity and mortality from related cardiovascular and cerebrovascular disease (36). Although it is clear that hypertension is an important disease in elderly humans, there is a limited understanding of the factors that contribute either to the development or to the maintenance of elevated blood pressure in this population. The potential relationship between the level of sympathetic nervous system (SNS) activity and blood pressure elevation in the elderly has not been well characterized. There are many complexities involved in the definition of SNS activity. Additionally, the interaction between SNS activity and an elevated blood pressure is multifaceted, mediated in part through vascular adrenergic responsiveness to the SNS input.
A number of studies using various methodologies to measure systemic SNS activity {plasma norepinephrine (NE) levels (40); the rate of NE release from studies of [3H]NE kinetics (33, 48, 51); and microneurography (18)} have concluded that there is an age-related increase in SNS activity. There remains controversy about the level of SNS activity in patients with essential hypertension (11, 14, 30). It appears that significant increases in SNS activity are found in some young hypertensive patients (5, 28) but that a hypertensive-normotensive difference in SNS activity has not generally been identified in older hypertensive patients (11, 30, 43).
We have interpreted results from our previous studies of adrenergic
receptor responsiveness in aging, namely an age-associated decrease in
platelet (46, 49), venoconstriction (47), and arterial vasoconstrictor
(16) responses to -adrenergic stimulation, to represent
downregulation of
-adrenergic responsiveness, which is appropriate
given heightened SNS activity in aging. In support of this
interpretation, we found that when SNS activity is suppressed in older
normotensive humans there is appropriate upregulation of their vascular
-adrenergic responsiveness (16). Studies of adrenergic
responsiveness in hypertension have led to the suggestion that there
may be an increase in
-adrenergic responsiveness to catecholamine
stimulation, although to date these studies have not been conducted in
an older hypertensive subject population (1, 3, 4, 23).
This study was performed to address the hypothesis that there is
impaired desensitization of -adrenergic receptor responsiveness in
older hypertensive humans. Two primary research questions were addressed: compared with older normotensive subjects, do older hypertensive subjects have 1)
similar levels of systemic SNS activity (determined by the rate of NE
release into an extravascular compartment derived from compartmental
analysis of [3H]NE
kinetics studies) and 2) increased
platelet and vascular
-adrenergic receptor responsiveness?
Epinephrine-mediated inhibition of in vitro platelet membrane adenylyl
cyclase activity and the decrease in forearm blood flow resulting from
in vivo intra-arterial NE infusion were determined to characterize
platelet and vascular
-adrenergic receptor responsiveness,
respectively. We report that compared with older normotensive subjects,
older hypertensive subjects tended to have a further increase in
systemic SNS activity, and despite equal or greater SNS activity, an
increase in both platelet and vascular
-adrenergic receptor responsiveness.
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METHODS |
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Subjects.
Twenty normotensive and twenty-four hypertensive older (>60 yr)
subjects in otherwise good general health were recruited through the
Human Subjects Core of the University of Michigan Geriatrics Center, as
well as through newspaper advertisement. Descriptive characteristics of
each subject group are provided in Table 1. Subjects were screened before study entry with a medical history, physical examination, and laboratory tests, including a complete blood
count, routine chemistries, and an electrocardiogram (ECG). Subjects
were excluded from participation in either group if they exceeded 150%
of ideal body weight (Metropolitan Life Insurance tables, 1983), were
taking any medication known to interact with SNS function, or had
evidence from either history, physical exam, or laboratory results of
significant underlying illness. Normotensive subjects gave no prior
history of hypertension and had a resting seated blood pressure <160
mmHg systolic and <90 mmHg diastolic at the screening visit. Subjects
with mild to moderate hypertension whose blood pressure was well
controlled on monotherapy were recruited for the hypertensive group
such that their blood pressure would likely remain below a limit of 200 mmHg systolic and 110 mmHg diastolic throughout a 4-wk antihypertensive
medication washout period. Individuals whose blood pressure exceeded
these limits during the washout discontinued their participation in the
study and resumed their antihypertensive medication. At the completion of the 4-wk antihypertensive washout period, hypertensive subjects were
required to demonstrate a resting seated diastolic blood pressure of
>90 mmHg. Each subject gave written informed consent that was
approved by the University of Michigan Human Use Committee.
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Study Protocol.
All subjects reported to the General Clinical Research Center of the
University of Michigan Medical Center at 0730 to control for any
diurnal variation in NE metabolism (41) or arterial -adrenergic tone
(39). Subjects were instructed to fast from 2200 the night before and
to abstain from cigarettes, caffeine, and other known modulators of
catecholamines for 12 h before each study began. Subjects were studied
in the supine position in a quiet room maintained at a constant
temperature of 23-25°C, to facilitate achieving an adequate
baseline forearm blood flow (FABF). The proportion of body fat was
determined by bioelectrical impedance (RJL Systems, Mt. Clemens, MI)
(26), and the waist-to-hip ratio was determined from an individual's
waist and hip circumferences taken at the level of the umbilicus and
the largest gluteal circumference, respectively.
FABF protocol.
After the tracer
[3H]NE infusion
protocol, FABF was measured using venous occlusion plethysmography
during an intra-arterial infusion protocol we have previously described
(15, 16). To establish a stable baseline, FABF readings were taken
until three consecutive readings representing similar FABF were
obtained. To determine the effect of intra-arterial infusions of NE on
FABF, NE (Levophed bitartrate, Sterling Drug) was diluted in 5%
dextrose to achieve stepwise increasing infusion doses of 1.25, 5, 20, 80 and 240 ng · 100 ml
FAV1 · min
1.
Each NE dose was administered by an infusion pump (Harvard model 970T;
Harvard Apparatus, South Natick, MA) for 4 min before
FABF was recorded during the 5th
minute of each infusion. After the FABF measurement at the 240-ng dose,
the NE infusion was stopped.
Platelet membrane preparation.
Platelet membrane lysates were prepared from
50-200 ml whole venous blood as previously described (46, 49). An
aliquot of the freshly prepared membrane lysate was used for adenylyl cyclase assays. The remainder of the sample was quick-frozen in liquid
nitrogen and stored at 70°C; radioligand binding studies were performed within 2 wk of membrane preparation.
Radioligand equilibrium binding assays.
[methyl-3H]yohimbine
(72.5-90.0 Ci/mmol; Amersham, Arlington Heights, IL), a specific
2-adrenergic receptor
antagonist, was used to determine platelet membrane
2-adrenergic receptor
antagonist binding properties, and the imidazoline full
2-adrenergic receptor agonist,
3H-labeled
5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine ({imidazolyl-4,5-3H}UK-14,304
or [3H]bromoxidine;
60.0-93.9 Ci/mmol, New England Nuclear, Boston, MA) was used to
determine platelet membrane
2-adrenergic receptor agonist
binding properties using methods we have previously described (46). In
human platelet membranes
[3H]bromoxidine has
been demonstrated to bind to one high-affinity site (37), which is
functionally coupled to adenylyl cyclase (50). We have previously shown
that analysis of
[3H]bromoxidine
specific binding over the concentration range and assay conditions
utilized in these experiments identifies the high-affinity binding
state (46).
Adenylyl cyclase assays.
Platelet membrane basal adenylyl cyclase activity was
determined using freshly prepared membranes at the beginning
(time 0) and at the conclusion of a
15-min incubation at 30°C as previously described (46, 49).
Stimulation of adenylyl cyclase activity was achieved with the addition
of 25 mM NaF and its
2-adrenergic receptor-mediated
inhibition by
10
8-10
4
M epinephrine (Epi). The concentration of cAMP in the assay tubes was
measured by radioimmunoassay (49). The concentration of cAMP in the
time 0 basal activity condition was
subtracted from the 15-min basal, NaF-stimulated, and Epi-inhibition
conditions so that the values would reflect only the accumulation of
cAMP over the 15-min incubation period. The extent of Epi-mediated inhibition at each Epi concentration was determined as the percent decrease in cAMP accumulation in the presence of Epi and NaF from the
NaF-stimulated activity without Epi.
SDS-PAGE and Western immunoblotting for
Gi-binding protein content.
On the day of platelet membrane preparation, 0.1 ml of
resuspended platelet membranes was added to an equal volume of a 4% cholate buffer [50 mM Tris · HCl, 1 mM
Na2EDTA, 2 mM dithiothreitol (DTT), pH 7.6]. This solution was shaken on ice for 1 h and then subjected to ultracentrifugation at 38,000 rpm in a 60 Ti
rotor for 1 h (Beckman L8-70M ultracentrifuge, Beckman
Instruments, Fullerton, CA). A 0.1-ml aliquot of the supernatant was
added to 0.9 ml of buffer containing 0.05% Lubrol (20 mM
Tris · HCl, 1 mM
Na2EDTA, 1 mM DTT, pH 8.0). Two
aliquots of 0.5 ml each were then snap-frozen in liquid nitrogen and
stored at 70°C.
Plasma catecholamine analytic methods.
Arterial or arterialized-venous blood samples were
collected into chilled plastic tubes containing EGTA and reduced
glutathione. The tubes were kept on ice until centrifugation at
4°C. Plasma samples were stored at 70°C until assayed.
Plasma NE and Epi were quantified by a single-isotope radioenzymatic
assay, with all samples from a given subject analyzed in the same assay
(6). The intra-assay coefficient of variation for NE in this assay is
5%. Alumina extraction of plasma samples and measurement of [3H]NE levels were
carried out as previously described (25, 33).
Data and statistical analysis. Steady-state, one-compartment kinetic parameters [the rate of NE appearance into the circulation (NEAP) and clearance from the circulation (NECL)] were calculated from steady-state plasma levels of [3H]NE and NE as previously described (25). Compartmental analysis of NE kinetics was performed using the previously described minimal two-compartment model (25). The quantity of NE in each compartment [NE mass in the intravascular compartment (Q1) and in the extravascular compartment (Q2)], the rate of NE appearance into each compartment (R12 into compartment 1 and NE2 into compartment 2), the NE metabolic clearance rate from compartment 1 (MCR1), the NE spillover fraction (NESF), and the volume of distribution of NE in compartment 1 (V1) were calculated from the two-compartment model as functions of the estimated transfer rate coefficients as previously described (25).
Dose-response data for NE and ANG II were analyzed as the percent change in FABF from the baseline value obtained before the infusion of each drug, to control for potential differences between groups in baseline FABF utilizing linear mixed-effects analysis (using SAS/PROC MIXED; SAS Institute, Cary, NC). This analysis was chosen to adjust group differences for gender, to accommodate unbalanced data, and to permit dose to be tested either as a linear effect or as a factor with several levels (19). Mean arterial pressure (MAP) was determined from the electronically integrated area under the intra-arterial blood pressure curve from the Marquette telemetry system (series 7700, Marquette Electronics, Milwaukee, Wisconsin) just before each FABF measurement. Forearm vascular resistance (FAVR) was calculated as the MAP divided by the FABF and is presented in arbitrary units. An unweighted nonlinear least-squares fit of the specific binding data for [3H]yohimbine and [3H]bromoxidine was made to a hyperbolic binding curve, Y = (A × X)/(B + X), where Y is specific binding (in fmol/mg protein), X is free [3H]yohimbine or bromoxidine concentration (in nM), A is the maximum receptor density Bmax, and B is the apparent dissociation constant Kd (InPlot 3.1, GraphPAD Software, San Diego, CA). Values are presented as means ± SE. Statistical analysis was performed using SAS (SAS Institute). A value of P < 0.05 was selected to indicate statistical significance. One-tailed tests were employed to test for the hypothesized increase in ![]() |
RESULTS |
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Subject characteristics. Characteristics of the normotensive and hypertensive subject groups are compared in Table 1. The groups were similar with respect to age and gender distribution. Although subjects who exceeded 150% of their ideal body weight were excluded, the BMI and percent body fat of the hypertensive group, after we adjusted for gender, were significantly greater than for the normotensive group. There was no statistically significant group difference in waist-to-hip ratio.
Plasma catecholamine levels and NE kinetics.
As summarized in Table 2,
there were no normotensive-hypertensive group differences for either
arterial plasma NE or Epi levels. NE kinetics results were not obtained
from one hypertensive subject because of technical difficulties.
One-compartment model analysis indicated a significantly greater
NECL in the hypertensive group and
no group difference in the NEAP.
The analogous results from the two-compartment model analysis were
similar; MCR1 was significantly
greater in the hypertensive group, and there was no group difference in
R12. The extravascular release
rate NE2 tended to be greater in
the hypertensive group, although this difference was not statistically
significant (P = 0.11). The
NESF from compartment 2 into
1 was significantly less in the
hypertensive group. There were no normotensive-hypertensive group
differences for NE mass in either compartment
1 or 2 (although
Q2 tended to be greater in the
hypertensive group) or for the NE volume of distribution in
compartment 1,
V1. There were no relationships identified between BMI and NE2 in
the combined or either individual subject group
(r values of 0.050 for combined,
0.053 for normotensive, and
0.088 for hypertensive;
P > 0.68 for each).
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FABF during vasoactive infusions.
There were no normotensive-hypertensive group
differences in baseline FABF measured before the intra-arterial
infusion protocol (Table 1). There was also no significant difference
between groups in FAVR before the infusion protocol
(normotensive 31 ± 4 vs. hypertensive 36 ± 3 U;
P = 0.35). There were modest, although statistically significant, increases in MAP during the NE-infusion protocol in each group (MAP measured during the highest NE-infused dose
was 103 ± 1 mmHg in the normotensive group and 130 ± 3 mmHg in
the hypertensive group, each change P < 0.001). The change in FABF from baseline in response to each NE
intra-arterial infusion dose is shown for each of the subject groups in
Fig. 1 as the percent decrease from
baseline FABF. Complete NE dose-response results were not available for
one hypertensive subject. In addition to a significant effect of NE
dose (P < 0.0001), the linear
mixed-effects analysis indicated a significant group difference in the
NE-mediated decrease in FABF, with the dose response for the
hypertensive subject group being significantly shifted to the left of
the normotensive group (P < 0.05).
The addition of gender as a covariate in the linear mixed-effects model
revealed a significant gender effect, with males having enhanced
response compared with females (P = 0.0001). The gender-adjusted normotensive-hypertensive group difference was similar but no longer achieved statistical significance
(P = 0.12).
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Platelet membrane adenylyl cyclase activity.
The basal rate of cAMP production in the platelet
membrane lysates over the 15-min incubation period (after the cAMP
concentration present in the membrane samples at time
0 of the assay was subtracted) was significantly less
in the hypertensive subject group (normotensive 690 ± 40 vs.
hypertensive 553 ± 45 pmol · mg1 · min
1;
P = 0.04), although there was no
difference in the NaF-stimulated rate of cAMP production (normotensive
2,230 ± 110 vs. hypertensive 2,190 ± 100 pmol · mg
1 · min
1;
P = 0.81). The dose-response effect
for Epi-mediated inhibition of NaF-stimulated adenylyl cyclase activity
is presented in Fig. 3. In addition to a
significant overall dose effect for Epi-mediated inhibition
(P < 0.0001), the linear
mixed-effects analysis indicated a significant group effect observed
for the percent inhibition from the NaF-stimulated level, with greater
inhibition in the hypertensive group
(P = 0.006). The hypertensive group
was also found to have significantly greater maximal percent inhibition (normotensive 41 ± 2 vs. hypertensive 53 ± 2%;
P < 0.001).
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Equilibrium binding studies.
The receptor binding densities from equilibrium binding
studies for the antagonist
[3H]yohimbine and the
agonist
[3H]bromoxidine and
the ratio of the bromoxidine to yohimbine binding density for the
subject groups are presented in Fig. 4. There was no
group difference in either receptor density (normotensive 128 ± 9 vs. hypertensive 128 ± 9 fmol/mg;
P = 0.99) or antagonist binding
affinity (normotensive 2.6 ± 0.2 vs. hypertensive 2.1 ± 0.2 nM;
P = 0.18) detected for
[3H]yohimbine. An
insufficient platelet protein yield prevented [3H]bromoxidine
studies from being done in 4 normotensive and 16 hypertensive subjects.
The [3H]bromoxidine
receptor density tended to be greater in the hypertensive group
(normotensive 29 ± 4 vs. hypertensive 42 ± 6 fmol/mg;
P = 0.08), although there was no group
difference noted for its binding affinity (normotensive 2.4 ± 0.3 vs. hypertensive 3.1 ± 0.8 nM; P = 0.34). Analysis of covariance indicated that subject group (P = 0.07)
and yohimbine Bmax (P = 0.002)
were each significantly associated with bromoxidine
Bmax.
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Gi protein content.
Western immunoblot studies utilizing an
anti-Gi2 antibody demonstrated
a single band at an approximate molecular mass of 41 kDa for the known
Gi standard and the cholate
extracts from the subjects' platelet membrane lysates. There was no
cross-reactivity against a known
Gs standard. Protein content could
be determined in 11 of the normotensive and 22 of the hypertensive
subjects. There was significantly less
Gi protein content detected in
cholate extracts from hypertensive compared with normotensive subjects (normotensive 84 ± 10 vs. hypertensive 64 ± 4% of
control; P = 0.03).
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DISCUSSION |
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The results from this study in older hypertensive humans provide
evidence for enhanced platelet and vascular -adrenergic receptor
responsiveness despite systemic SNS activity equal to or greater than
that of normotensive age-matched subjects. These results support the
hypothesis that there is impaired desensitization of
-adrenergic
receptor responsiveness in older hypertensive humans and suggest that
altered SNS function may contribute in part to the pathophysiology of
hypertension in the elderly.
The role of enhanced SNS activity in the pathogenesis of essential human hypertension remains controversial (28). A significant component of this controversy is related in part to limitations in each of the available methods to assess SNS activity in humans. Studies that have measured normotensive-hypertensive differences in plasma catecholamines have been reviewed in detail by Goldstein (10). The overall conclusion derived from these studies is that although there are some hypertensive patients who exhibit significant increases in plasma NE, namely young patients with persistent hypertension (5), the hypertensive-normotensive difference in plasma NE is generally not apparent in older hypertensive patients (11, 30, 43). One report that studied 24-h plasma catecholamine levels found that the mean plasma NE level over a 24-h period was lower in older hypertensive compared with normotensive patients (45). Consistent with these previous reports, we did not detect an increase of arterial plasma NE levels in our older hypertensive subject population.
However, plasma NE levels provide at best only an indirect index of systemic SNS activity. The plasma NE concentration reflects the net balance between NE appearance and removal mechanisms and provides no information concerning the complex metabolic fate of NE after its release from presynaptic sympathetic nerve terminals. Tracer NE kinetics studies utilizing isotope dilution methods have been developed to estimate systemic rates for NEAP into and NECL from plasma. Esler et al. (5) have examined the effect of age on [3H]NE kinetic parameters in 34 patients with essential hypertension from age 22 to 74 yr using the isotope dilution technique (5). In this study, although an increase in NEAP was noted overall in the hypertensive group compared with controls, the NEAP of the older (i.e., >40 yr) hypertensive subjects was similar to their age-matched controls, suggesting that SNS activity of older hypertensive subjects is similar to that of age-matched controls. The NE kinetics results from the present study are consistent with this interpretation because there was no normotensive-hypertensive group difference identified in NEAP or in the analogous NE kinetic parameter from the two-compartment model analysis, the rate of NE appearance into the vascular compartment, R12.
A minimal two-compartment model developed to describe NE kinetics was used in the present study to determine the rate of NE release into the extravascular compartment (NE2) as a more proximate index of systemic SNS activity (25). We have previously demonstrated in normotensive subjects an age-associated increase in NE2 (48). Compared with older normotensives, the rate of NE release into the extravascular compartment tended to be greater in the older hypertensive group, although this gender-adjusted difference was not statistically significant (P = 0.11). There was also a trend toward greater NE mass in the extravascular compartment in the hypertensive group. These results suggest that systemic SNS activity in older hypertensive subjects is equal to or greater than that of older normotensive subjects. Our results are consistent with those of Yamada et al. (52), who reported an increase in muscle SNS activity (assessed using tibial nerve microneurography) in older (51-67 yr) hypertensive subjects compared with age-matched normotensive controls. Thus the age-associated increase in systemic SNS activity is present in older humans with established hypertension and may be exaggerated in this population.
Only a minority (<25%) of NE released into the synapse appears or spills over into the circulation; the majority of NE reenters the presynaptic terminal by neuronal reuptake mechanisms. In addition to providing an estimate of the rate of NE release into the extravascular compartment, the two-compartment model analysis also permits estimation of the spillover fraction. This analysis demonstrated that the spillover fraction was significantly lower in the hypertensive subject group, suggesting that NE reuptake mechanisms may be enhanced. The NE kinetics results also demonstrated significantly greater NE metabolic clearance rate in the hypertensive group. This finding coupled with the decrease in NE spillover fraction may explain why plasma NE levels were not elevated in the hypertensive group despite the trend for increased NE2. Therefore, at least in part because of the increase in NE clearance rate, the plasma NE level appears to underestimate the level of systemic SNS activity in older hypertensive subjects.
Vascular adrenergic tone represents the integration of SNS activity and
vascular - and
-adrenergic receptor responsiveness. A number of
studies have suggested that there is an impairment in
-adrenergic-mediated vasodilation among hypertensive subjects (7,
35, 44). The present study focused on characterizing
-adrenergic
receptor responsiveness because of our results in previous studies in
older normotensive humans, which demonstrated that there is appropriate
regulation of vascular
-adrenergic receptor responsiveness to
perturbations in SNS activity (15, 16). We concluded from these
observations that the age-associated decrease in platelet and vascular
-adrenergic receptor responsiveness is appropriate given the
age-associated increase in SNS activity. In the present study, vascular
-adrenergic responsiveness was assessed concurrently with measures
of systemic SNS activity in a population of older hypertensive subjects
to permit interpretation of adrenergic responsiveness in the context of
the prevailing level of SNS activity. In contrast to our observations
in normotensive elderly subjects, the hypertensive elderly subject
group demonstrated enhanced NE-mediated vasoconstriction despite
evidence for an equal or a greater level of systemic SNS activity.
Moreover, because there was no normotensive-hypertensive group
difference noted in the decrease in FABF mediated by the nonadrenergic
vasoconstrictor ANG II, the enhanced vasoconstrictor response appears
to be specific for NE. This argues against the possibility of a
structural or other nonadrenergic mechanism producing a nonspecific
increase in vascular reactivity in the hypertensive group as an
explanation for their enhanced response to NE.
The results demonstrating enhanced NE-mediated vasoconstriction in the
older hypertensive group are consistent with other studies that have
concluded that there is enhanced adrenergic receptor responsiveness in
younger hypertensive subjects. Systemic NE infusions have been shown to
decrease brachial artery diameter and blood flow to a greater extent in
hypertensive compared with normotensive subjects (23). Additional
studies have demonstrated augmentation of pressor responses to systemic
infusions of adrenergic agonists (3), augmented vasoconstriction
response of postjunctional 2-receptors to epinephrine (1),
and an increase in vascular
-adrenergic tone (4) in hypertensives.
Taken together, these studies support a conclusion that enhanced
vascular adrenergic responsiveness may contribute to an increase in
vascular resistance and blood pressure in hypertensive subjects. The
increase in vascular adrenergic responsiveness may be of even greater
physiological significance among older hypertensive subjects given the
age-associated increased level of SNS activity. Although FAVR was
higher among the hypertensive subject group, this difference was not
statistically significant. The combination of equal or greater SNS
activity and enhanced FABF responsiveness to NE infusion might be
expected to result in greater FAVR. However, the regulation of FAVR is complex, involving a number of compensatory systems in addition to the
adrenergic system, such that enhanced forearm vascular responses to NE
may not directly translate to greater FAVR.
In parallel with enhanced NE-mediated vasoconstriction, our results
also indicated enhanced platelet
2-adrenergic responsiveness in
the hypertensive subject group. The extent to which platelet
-adrenergic receptor response provides a valid marker of vascular
-adrenergic receptor responsiveness has been questioned. Given the
inaccessibility of human vascular
-adrenergic receptors, platelet
membrane
2-adrenergic receptors
have been utilized as a surrogate model system (31). The majority of
investigations have reported only yohimbine (antagonist) binding
properties in hypertensive and control subject populations. The results
from these studies have not been consistent, demonstrating either
increased (2, 32), decreased (20), or similar (17, 34, 38) total
2-receptor binding density.
Several limitations need to be considered in interpreting results from
platelet
2-adrenergic receptor
antagonist binding studies (31). In particular, alterations in total
receptor binding density may not convey any functional significance
because only those receptors in the high-affinity (coupled) agonist
binding state mediate inhibition of adenylyl cyclase. For this reason,
we included measures of agonist binding properties and
receptor-mediated adenylyl cyclase inhibition in the present study. Our
results uniquely demonstrate a parallel increase in vascular and
platelet
-adrenergic responsiveness. In the present study, platelet
membranes from the older hypertensive subjects demonstrated greater
Epi-mediated inhibition of adenylyl cyclase activity relative to the
older normotensive group. This finding is consistent with results from
a study that reported a defect in the ability of adrenergic agonists to
desensitize platelet
2-adrenergic receptors from
younger hypertensive subjects (17). Given that there was not a
normotensive-hypertensive group difference in plasma NE levels, the
difference in platelet
2-receptor response cannot be
accounted for by differences in plasma NE. This difference in response
may be due to another sympathetic mechanism, such as platelet rather
than plasma NE concentration (21), or a nonsympathetic mechanism. The
enhanced response in platelet membranes from older hypertensive
subjects appears not to be due to a greater density of platelet
2-adrenergic receptors but
rather to an increase in the proportion of receptors in the high-affinity, or coupled, binding state. In the present study, the
binding density for the direct
-agonist bromoxidine tended to be
higher among the hypertensive subjects. Receptor agonist affinity state
has been examined in only one previous study; using analysis of agonist
competition binding for
[3H]yohimbine, no
significant normotensive-hypertensive group difference was found for
the proportion of receptors in the high-affinity binding state (17).
Enhanced Epi-mediated inhibition of adenylyl cyclase activity in the
hypertensive group could not be explained by an increase in
Gi binding protein content
inasmuch as there was less Gi2 protein content detected in cholate extracts of the platelet membranes from the hypertensive subjects. This finding is consistent with another
study that reported lower G
i2
protein levels in hypertensive subjects (27), although in another
report no differences in G
i2
protein levels were noted (29). Our study was not designed to determine
whether there were differences in content of other Gi binding protein subtypes,
Gs
or its subtypes, or the
functional activities of these proteins. Stimulatory G protein labeling
by cholera toxin (but not by immunoblotting) has been shown to be reduced in lymphocyte preparations from younger hypertensive subjects in conjunction with a reduction in
-agonist-mediated stimulation of
adenylyl cyclase activity (8). This reduction in lymphocyte
-adrenergic receptor responsiveness in hypertensive subjects has
recently been associated with an increase in G protein-coupled receptor
kinase activity (12). Therefore, additional studies need to be
conducted to more completely examine the
-adrenergic receptor-effector coupling pathway and its regulation in older hypertensive humans to attempt to elucidate the mechanism responsible for the apparent lack of appropriate desensitization.
We acknowledge several limitations inherent in our study primarily related to the heterogeneity of SNS activity and to the heterogeneous nature of hypertension in an older subject population. The NE kinetics methodology we employed assesses systemic SNS activity. Consequently, it is not possible to infer from our results whether there is greater regional, organ-specific (e.g., cardiac or renal) SNS activity in older hypertensive humans in addition to the systemic, whole body assessment. Future studies need to focus attention on this important question. Also, despite the similarity in vasoconstriction response to ANG II, our results cannot exclude the possibility of impaired withdrawal of SNS activity in the hypertensive group in the context of increased vasoconstriction due to a nonadrenergic (e.g., insulin resistance or impaired endothelial function) mechanism. We also recognize that despite our efforts to characterize a uniform older study population with mild to moderate essential hypertension who were studied after a 4-wk antihypertensive medication withdrawal period, the heterogeneous nature of this disorder (with respect to, for example, obesity, duration of illness, physical activity, racial background, ethnicity, insulin resistance, and sodium sensitivity of blood pressure) in this population may have influenced our results. An effect of gender was noted in the vasoconstriction response to NE (men having enhanced response), but no gender effects were identified for any of the other parameters we investigated. Several recent studies have reported an effect of estrogen supplementation to improve endothelium-dependent vasodilation (22) and to decrease NE-mediated vasoconstriction (24) in postmenopausal women, raising the possibility that these effects of estrogen may contribute to the gender difference we observed in the vasoconstriction response to NE. Among the other potential factors, we are able to comment only indirectly with respect to the influence of obesity. Studies that have observed associations between body mass index and percent body fat with muscle SNS activity have suggested that body fat may be an important regulator of SNS activity (13, 42). In our subject population, the hypertensive group had significantly greater body mass index and higher percent body fat but not waist-to-hip ratio. However, because there were no associations between body mass index and NE2 in our subject population, it seems unlikely that the trend toward increased NE2 in the hypertensive group may be ascribed to their higher body mass index. Future studies are needed to address potential interactions between other factors, including gender, obesity, and others cited above, and SNS function in older hypertensive populations.
In summary, these studies demonstrate that, compared with older
normotensive subjects, older hypertensive subjects tended to have a
further increase in systemic SNS activity and, despite equal or greater
SNS activity, an increase in both platelet and vascular -adrenergic
receptor responsiveness. These results suggest that heightened level of
SNS activity in conjunction with enhanced
-adrenergic receptor
response may contribute toward the increase in peripheral vascular
resistance and blood pressure in older hypertensive humans.
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ACKNOWLEDGEMENTS |
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We thank Marla Smith and Eric Leiendecker for technical assistance and the nursing staff of the Univ. of Michigan General Clinical Research Center for care of our subjects during this study. Angiotensin II was donated by Ciba-Geigy (Summit, NJ).
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FOOTNOTES |
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This work was supported in part by National Institutes of Health Grants RR-00042 (to the University of Michigan General Clinical Research Center), AG-08808 (to the Claude D. Pepper Geriatric Research and Training Center at the University of Michigan), and AG-00433 and AG-10053 (to M. A. Supiano); by the Geriatric Research, Education, and Clinical Center and the Medical Research Service of the Ann Arbor Dept. of Veterans Affairs Medical Center (R. V. Hogikyan and M. A. Supiano); and by a grant from the John A. Hartford Foundation (to J. L. Krueger).
Portions of this work were presented at the National Meeting of the American Federation for Clinical Research in 1993 and 1994 and the American Geriatrics Society in 1994.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address reprint requests to M. A. Supiano.
Received 27 January 1998; accepted in final form 10 November 1998.
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