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-adrenergic vasorelaxation in aged rat carotid arteries
1Chair of Geriatrics and 2Chair of Internal Medicine, Department of Clinic Medicine, Cardiovascular, and Immunological Sciences, University of Naples "Federico II," 80131 Naples; and 3Department of Gerontology, Geriatrics, and Metabolic Diseases, II University of Naples, 80131 Naples, Italy
Submitted 14 January 2003 ; accepted in final form 6 March 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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-adrenergic receptor
(-AR) signaling and reduction in cardiovascular responses to -AR
stimulation. Because exercise can attenuate age-related impairment in
myocardial -AR signaling and function, we tested whether training could
also exert favorable effects on vascular -AR responses. We evaluated
common carotid artery responsiveness in isolated vessel ring preparations from
8 aged male Wistar-Kyoto (WKY) rats trained for 6 wk in a 5 days/wk swimming
protocol, 10 untrained age-matched rats, and 10 young WKY rats. Vessels were
preconstricted with phenylephrine (10-6 M), and vasodilation was
assessed in response to the -AR agonist isoproterenol
(10-10-3 x 10-8 M), the 
2-AR
agonist UK-14304 (10-9-10-6 M), the muscarinic receptor
agonist ACh (10-9-10-6 M), and nitroprusside
(10-8-10-5 M). -AR density and cytoplasmic
-AR kinase (-ARK) activity were tested on pooled carotid arteries.
-ARK expression was assessed in two endothelial cell lines from bovine
aorta and aorta isolated from a 12-wk WKY rat. -AR,
2-AR, and muscarinic responses, but not that to
nitroprusside, were depressed in untrained aged vs. young animals. Exercise
training restored -AR and muscarinic responses but did not affect
vasodilation induced by UK-14304 and nitroprusside. Aged carotid arteries
showed reduced -AR number and increased -ARK activity. Training
counterbalanced these phenomena and restored -AR density and -ARK
activity to levels observed in young rat carotids. Our data indicate that age
impairs -AR vasorelaxation in rat carotid arteries through -AR
downregulation and desensitization. Exercise restores this response and
reverts age-related modification in -ARs and -ARK. Our data
support an important role for -ARK in vascular -AR
vasorelaxation.
aging; beta-adrenergic receptor; exercise
-ADRENERGIC RECEPTOR (-AR) in the
regulation of the cardiovascular system is widely recognized
(7). During the past years, the
implications of impaired -AR signaling in the pathophysiology of several
cardiovascular disorders were explored in animals as well as in humans. Data
from these studies indicate that changes in -AR function are associated
with heart failure (6,
38), hypertension
(13,
14), and hypertension
complicated with ventricular hypertrophy
(2).
Upregulation of -AR kinase 1 (-ARK, also known as the G
protein-coupled receptor kinase 2) has been recognized as one of the
mechanisms responsible for -AR dysfunction
(29). Transgenic mice with
cardiac overexpression of -ARK show a depressed contractile response to
isoproterenol (Iso) (22). In
the failing human heart the uncoupling of cardiac 1- and
2-ARs from G proteins has been attributed to increased levels
of myocardial -ARK (38).
Abnormalities of -AR signaling due to increase in -ARK activity
have been observed in both human
(14) and animal
(10,
13) hypertension.
Interestingly, similar alterations in vascular and cardiac -AR
function were found with physiological aging
(8,
24). In the rat myocardium,
the age-related decline in -AR responsiveness was ascribed to -AR
desensitization and uncoupling of -ARs from stimulating G proteins
(30). Similarly, vascular
reactivity studies conducted in the aorta of aged rats
(4) or in the dorsal hand vein
and saphenous vein of elderly humans
(26) showed a reduced
vasorelaxant response to -AR agonists such as Iso. The recently
demonstrated -AR desensitization in aged rat aorta due to -ARK
upregulation has been proposed as one of the molecular mechanisms responsible
for age-related -AR dysfunction and decreased vasorelaxation
(31).
Regular physical activity may counteract the decline of cardiovascular
function observed with age
(11,
33). Improvements in left
ventricular performance, including higher maximal stroke volume, ejection
fraction, and cardiac output and lower resting and exercise peripheral
vascular resistances, are beneficial effects of exercise in the elderly
related to enhanced cardiovascular -AR responsiveness
(1,
32).
The ability of exercise to attenuate the age-related alterations in
-AR function was demonstrated in experimental studies conducted on aged
rat myocardium (30). Whether
physical activity may improve -AR responsiveness by modulating
-ARK levels in aging vasculature has not yet been explored. To
investigate this issue, we conducted a study exploring the vasorelaxant
responses to -AR stimulation of rat carotids from aged trained and
untrained animals. Vascular reactivity data were correlated with
exercise-induced changes in vascular -ARK activity. Because -AR
vasorelaxation in the rat carotid is largely endothelium dependent, we
confirmed for the first time that -ARK is indeed expressed in the
endothelium.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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-ARK expression. Two
endothelial cell lines from bovine aorta (BAEC; American Type Culture
Collection) and aorta isolated from a 12-wk-old Wistar-Kyoto (WKY) rat as
previously described (20) were
assessed for -ARK expression. Cells were cultured to 75% confluence in
DMEM-10% FBS, washed twice in phosphate-buffered saline, and then Dounce
homogenized with an insulin syringe in 25 mM Tris · HCl (pH 7.5), 5 mM
EDTA, 5 mM EGTA, 10 µg/ml leupeptin, 20 µg/ml aprotinin, and 1 mM PMSF.
Soluble cytosol fractions were separated from membrane fractions by
centrifugation. Alternatively, cells were solubilized with ice-cold 50 mM Tris
· HCl (pH 8.0), 5 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% SDS, 10 mM NaF, 5 mM EGTA, 10 mM sodium pyrophosphate, and
1 mM PMSF and -ARK was immunoprecipitated from 200 µg of protein from
clarified extracts with 1:2,000 of a monoclonal anti--ARK1/2 (C5/1)
antibody (19) and 35 µl of
a 50% slurry of protein A agarose conjugate agitated for 1 h at 4°C.
Cytosol fractions or immune complexes were resolved on 10% polyacrylamide
Tris-glycine gels and transferred to nitrocellulose. The 80-kDa -ARK
protein was visualized with a commercially available polyclonal antibody
(SC-562; Santa Cruz) and a standard chemiluminescence technique (Renaissance;
NEN). Rat strain and training protocol. The study protocol was designed in accordance with the American Physiological Society's "Guiding Principles for Research Involving Animals and Human Beings" and approved by the ethics committee of our institution. Ten aged male WKY rats (age = 24 mo; wt = 490 ± 40 g) were trained according to Orenstein et al. (25). The training program lasted 6 wk, starting with a 5 min/day, 5 days/wk swimming exercise that was gradually increased over the period until the rats were swimming continuously for 40 min/day. The rats swam in groups of four in a 60-cm-deep tub with water temperature maintained constant at 35°C and were air-dried after each session. Of 10 trained animals, 2 did not complete the exercise protocol because of injuries (n = 1) or death (n = 1) unrelated to swimming.
Ten aged male WKY rats (age = 24 mo; wt = 480 ± 30 g) maintained under sedentary conditions were dipped in a 10-cm-deep tub containing water at temperature maintained constant at 35°C for 30 min for 5 days/wk. Ten young male WKY rats (age = 4 mo; weight = 340 ± 40 g), under the same protocol used for aged sedentary animals, completed the animal population of this study. Care was taken to avoid sedentary rats swimming or making any physical effort during the bath. Total protocol duration was 6 wk, and all sedentary animals completed this period in good condition.
Vascular reactivity studies. All animals were anesthetized with a mixture of ketamine (50 mg/kg) and xylazine (0.5 mg/kg) and killed by decapitation. Right and left common carotid arteries were rapidly dissected free and cut into 4-mm-long rings. Vascular reactivity studies were conducted as previously described (20).
-AR-mediated vasorelaxation was assayed after phenylephrine (PE;
10-6 M) by the addition of increasing amounts of Iso
(10-10-3 x 10-8 M). Dose-response curves to Iso
were also evaluated after nitric oxide (NO) synthase inhibition with
N
-monomethyl-L-arginine
(L-NMMA; 10-5 M).
Endothelium-dependent vasodilation was assessed with the muscarinic
receptor agonist ACh and the 2-AR agonist UK-14304
(10-9-10-6 M)
(34). Endothelium-independent
vasodilation was evaluated in response to nitroprusside
(10-8-10-5 M).
-AR density and cytosolic -ARK activity in rat
carotids. Rat carotids were pulverized in a liquid nitrogen-chilled
stainless steel Dounce homogenizer and resuspended in 250 µl of ice-cold
lysis buffer [25 mM Tris · HCl (pH 7.5), 5 mM EDTA, 5 mM EGTA, 10
µg/ml leupeptin, 20 µg/ml aprotinin, and 1 mM PMSF]. Cytosolic and
membrane fractions were separated by serial centrifugation
(19).
Receptor binding on pooled rat carotid membranes was performed with the
nonselective -AR ligand 125I-labeled cyanopindolol.
Nonspecific binding was determined in the presence of 10 µM alprenolol.
Reactions were conducted in 100 µl of binding buffer at 37°C for 1 h
and then terminated by vacuum filtration through glass fiber filters. All
assays were performed in triplicate, and receptor density (in fmol) was
normalized to milligrams of membrane protein. -ARK activity was tested
by rhodopsin phosphorylation assays on rat carotid cytosolic extracts as
previously described (19).
Statistical analysis. All values are presented as means ±
SE. A one-way ANOVA was performed to separately test the main effects of age
and exercise training in adult and sedentary and trained aged rats.
Dose-response curves to all drugs used were analyzed by a two-way ANOVA for
repeated measures with Bonferroni's post hoc analysis. -AR density and
-ARK activity values from different groups were compared by a one-way
ANOVA analysis. A P value <0.05 was considered statistically
significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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-ARK is expressed in endothelium. Although -ARK
expression has been postulated to be ubiquitous, the presence of -ARK in
the endothelium has never been demonstrated before. In rat and bovine vascular
endothelium we showed -ARK expression by Western blot on whole cell
lysate or cytosol immunoprecipitation (Fig.
1).
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Effect of age and training on whole body and left ventricle weight. Whole body and left ventricle weight and left ventricle-to-body weight ratio of study groups are shown in Table 1. Both trained and untrained aged animals had significantly greater body and left ventricular weights than young rats. A significant training effect was observed between untrained and trained aged rats, trained being leaner than untrained aged rats. Accordingly, the left ventricle-to-body weight ratio was smaller in aged trained than young and aged untrained animals.
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Vasoreactivity studies. Maximal vasoconstriction induced by PE (10-6 M) was 572.5 ± 49.9 mg in young, 537.5 ± 44.3 mg in untrained aged, and 577.5 ± 54.7 mg in trained aged animals (P = not significant between groups, ANOVA).
ISO-induced vasorelaxation was lower in untrained aged than in young and trained aged animals (Fig. 2A); differences between groups were abolished by L-NMMA to demonstrate that the age-impaired vasorelaxation in untrained aged animals was mainly related to a defect in the endothelium-mediated component of vasodilation induced by Iso (Fig. 2B).
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ACh-induced vasodilation was lower in untrained aged than in trained aged and young animals (Fig. 2C). These data indicate that exercise induces a favorable effect on muscarinic response.
In contrast, exercise was not able to restore the
2-AR-mediated vasorelaxation to UK-14304, which was
depressed in untrained and trained aged animals compared with young animals
(Fig. 2D). Responses
to nitroprusside were not different between groups
(Fig. 2E). This
demonstrates that the endothelium-independent component of vasodilation was
unaffected by age and physical conditioning.
-AR density and -ARK activity. Carotid
-AR density was significantly affected by age as demonstrated by its
reduction in untrained aged compared with young animals. Exercise increased
-AR density to values not statistically different from those measured in
young rats (Fig.
3A).
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-ARK activity in cytosol carotid extracts from young, untrained aged,
and trained aged animals was assessed by rhodopsin phosphorylation. In
untrained aged animals total carotid -ARK activity was increased nearly
twofold over that recorded in young animals. Physical exercise significantly
reduced the kinase activity to levels similar to those observed in young
animals (Fig. 3B).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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-AR response caused by receptor downregulation and
desensitization with functional consequences of the senescent vasculature.
Physical training shows a favorable effect on vascular reactivity of aged rat
carotids and corrects age-impaired vascular -AR vasodilation by
increasing -AR density and reducing cytoplasmic -ARK activity.
To our knowledge, this is the first study to investigate the relationship
between abnormalities of the -AR pathway and impaired vasorelaxation in
aged rat carotid artery. Our data clearly indicate that carotid responsiveness
to -AR stimulation is blunted in aged sedentary rats compared with young
rats and that the reduced vasorelaxation is associated with a decrease of
-AR density in whole artery preparation. The presence of -AR
downregulation in the senescent vasculature is controversial because different
studies exploring the age-related changes in vascular -AR pathway
provide opposite results (15,
37). Our study confirms that
the vascular -AR number is reduced in aged sedentary rats and extends
previous evidences by showing that exercise training corrects this
abnormality.
Our data also point to -ARK upregulation as a possible mechanism of
-AR dysfunction in aged rat carotids. The important role of increased
cardiac -ARK in the pathophysiology of -AR signaling has been
consolidated in animal and human models of heart failure
(29). At the vascular level,
increased -ARK protein expression was demonstrated in spontaneously
hypertensive rats, suggesting that increased -ARK activity might account
for the impairment of -AR-mediated vasodilation observed in hypertension
(13,
14). Consistent with this, a
recent report showed impaired -AR vasorelaxation and hypertension in
transgenic mice with selective overexpression of -ARK in vascular smooth
muscle cells (10). Our results
indicate that vascular -ARK is increased and participates in the
deterioration of vasodilation to -AR stimulation also in aging. The
present study extends a previous report showing the relationship between the
increase in -ARK activity and the decline in -AR-mediated
signaling occurring with age
(31) and for the first time
demonstrates that exercise reduces vascular -ARK activity and exerts a
beneficial effect on -AR vasorelaxation.
We also provide a demonstration that -ARK is expressed in the
endothelium and suggest that endothelial -ARK has physiological
implications in the control of the vascular tone. Indeed, in rat carotids
-AR vasorelaxation is largely endothelium dependent
(20). The physiological
relevance of endothelial -ARs is supported by their distribution in the
vasculature. Evidence is mounting that -AR vasorelaxation is largely
endothelium dependent in a wide range of vascular regions that actively
participate in the determination of total peripheral resistances
(9,
16,
23). Furthermore, in vivo
studies in cat hindlimb (12),
canine coronary artery (27),
and newborn pial arteries (28)
suggest that the endothelium dependence of -AR vasorelaxant responses is
generalized.
Although the role of -ARK in -AR phosphorylation and
desensitization is well recognized, several studies show a possible
implication in the regulation of other receptor signaling. To this regard, the
recent observation of cross talk between M3 muscarinic receptors
and -AR in terms of receptor phosphorylation and desensitization
(3,
36) and the demonstration that
-ARK may phosphorylate M3 and M2 muscarinic
receptors (18) provide a
possible explanation for our results on the reduced ACh-mediated
vasorelaxation in carotids of untrained aged animals. The reduction in
vascular -ARK activity obtained in trained aged rats could also
contribute to improve muscarinic receptor responsiveness and explain our and
previous results on the enhanced ACh-mediated vasodilation after training
(5,
21).
Several studies suggest that the main mechanism by which exercise improves impaired vasodilation is increased endothelial NO bioavailability (17, 35). Our data are only in apparent contrast with these previous observations. In fact, we agree on the crucial role played by increased endothelial NO release in the enhanced endothelium-dependent vasorelaxation induced by exercise.
Nevertheless, we also affirm that activation of the NO pathway is related
to the specific improvement in -AR signaling observed in trained
animals. If the only mechanism by which exercise exerted its favorable effects
on endothelium-dependent vasorelaxation was the enhancement of NO
availability, all the vascular responses implying NO as final mediator would
have been enhanced after training. This is not the case, because our data
indicate that vasorelaxant response to 2-AR stimulation was
still impaired in trained aged animals. Therefore, we propose that exercise
may improve age-impaired endothelium-dependent vasorelaxation by interfering
with specific receptor signal transduction pathways. Our data suggest the
importance of the integrity of vascular -AR signaling for maintaining
adequate levels of endothelial NO production and preserving the endothelial
control of vascular tone. This observation is supported by the recent
demonstration that the overexpression of 2-ARs, via
adenovirus-mediated gene transfer to the vascular endothelium of the carotid
artery, is able to correct impaired -AR vasorelaxation in hypertensive
rats (20).
The favorable effects of exercise on vascular age-related changes in
-AR responsiveness and in the postreceptor adrenergic pathway reported
in this study are similar to those previously observed in aged rat myocardium
(33). In fact, chronic dynamic
exercise attenuates the age-determined alterations in postreceptor elements of
cardiac signal transduction. This suggests the potential role of physical
activity in a global cardiovascular improvement in -AR function.
Our study may contribute to a better understanding of the mechanisms
underlying the physiological aging of the cardiovascular system, providing
important insights about the clinical manifestation of cardiovascular disease
in the elderly population. -AR downregulation and desensitization
phenomena in senescent vasculature may partly explain the reduced
cardiovascular adaptations to different stressors occurring with age and may
contribute to the vulnerability of the cardiovascular system typical of the
aging process. Changes in -AR signaling could explain the progression
with age of endothelial dysfunction, which is a common finding in pathological
conditions such as coronary and peripheral artery disease, hypertensive
status, and heart failure.
It seems reasonable to affirm that physiological aging is characterized by
an altered vascular -AR reactivity, which may be one of the molecular
backgrounds for the development of cardiac and vascular diseases. The ability
of physical activity to counteract the biochemical abnormalities related to
endothelial dysfunction and impaired vascular reactivity suggests its
potentially useful role in the prevention, treatment, and prognosis of
cardiovascular diseases in the elderly.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
* D. Leosco and G. Iaccarino contributed equally to this work. 
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  D. Leosco, G. Rengo, G. Iaccarino, L. Golino, M. Marchese, F. Fortunato, C. Zincarelli, E. Sanzari, M. Ciccarelli, G. Galasso, et al. Exercise promotes angiogenesis and improves {beta}-adrenergic receptor signalling in the post-ischaemic failing rat heart Cardiovasc Res, May 1, 2008; 78(2): 385 - 394. [Abstract] [Full Text] [PDF]
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 D. Leosco, G. Rengo, G. Iaccarino, A. Filippelli, A. Lymperopoulos, C. Zincarelli, F. Fortunato, L. Golino, M. Marchese, G. Esposito, et al. Exercise training and beta-blocker treatment ameliorate age-dependent impairment of beta-adrenergic receptor signaling and enhance cardiac responsiveness to adrenergic stimulation Am J Physiol Heart Circ Physiol, September 1, 2007; 293(3): H1596 - H1603. [Abstract] [Full Text] [PDF]
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F = 2.409, P < 0.05; 
F = 5.593,
P < 0.01 (2-way ANOVA).