(Hypertension. 1997;29:1165-1172.)
© 1997 American Heart Association, Inc.
Articles |
Mechanical Load Opposes Angiotensin-Mediated Decrease in Vascular 
1-Adrenoceptors
From the Department of Physiology, The University of North Carolina, Chapel Hill.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract
1-Adrenergic receptor contraction of
vascular smooth muscle is augmented by increases in
angiotensin II and also in several forms of hypertension.
Whether angiotensin directly modulates
1-adrenoceptor subtype expression to contribute to this
effect is unknown. In a previous study, we demonstrated that increased
mechanical load (pressure) per se does not alter expression of
1B- and 1D-adrenoceptors in rat aortic
smooth muscle in cell culture, in vitro or in vivo. However, findings
in aortic coarctation hypertension suggested that a humoral factor,
possibly angiotensin, selectively reduces
1B-adrenoceptors and that increased mechanical load
opposes this decrease. The present study examined this hypothesis
by determining the effect of angiotensin alone and in the
presence of mechanical loading on the expression of 1D-
and 1B-adrenergic receptor mRNAs and
1-receptor density in cultured aortic smooth muscle
cells. 1D mRNA content, per smooth muscle cell,
concentration-dependently decreased after 3 hours of exposure to 0.3
nmol/L to 1 µmol/L angiotensin but by 24 hours had
returned to control levels. In contrast, 1B mRNA
concentration-dependently declined at a later time (24 hours) and
remained decreased at 48 hours to 27±6% of control with 1
µmol/L angiotensin. Angiotensin also
decreased 1-adrenoceptor density in a dose-dependent
manner. Angiotensin had no effect on cell number in these
confluent, quiescent cells but did increase cell protein and total RNA.
This cellular hypertrophy and the decreases in
1-adrenoceptor mRNAs were blocked by the
angiotensin type 1 receptor antagonist
losartan. Cyclic mechanical loading of smooth muscle cells
opposed the angiotensin-mediated hypertrophy
and decrease in 1B mRNA expression and
1-adrenergic receptor density. These data suggest that
angiotensin and intravascular pressure interact to affect
cell growth and expression of 1B-adrenergic receptors by
vascular smooth muscle.
Key Words: muscle, smooth, vascular • mechanical stretch • angiotensin • receptors, adrenergic, alpha
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Despite the importance of different
-AR subtypes (see Graham et al1 for a
description of the nomenclature for 1-ARs used in this
article) in the control of SMC contraction and growth, the
physiological regulation of their expression by
SMCs at the transcriptional and translational levels remains poorly
understood. SMCs can express multiple subtypes of both the
1- and 2-AR families, including
1B- and 1D-ARs (see Reference 22 and its
references). Indirect evidence suggests that Ang II, also a potent
stimulus for SMC contraction and cell growth, may influence SMC
1-AR density. Angiotensin enhances
norepinephrine release and inhibits reuptake by sympathetic
nerve endings, enhances central sympathetic outflow, and increases SMC
catecholamine responsiveness,3 4 mechanisms
that could indirectly alter 1-AR expression. Moreover,
the hypertrophic effect of Ang II on SMCs may be influenced in an as
yet ill-defined way by 1-ARs, since 1-AR
blockade inhibits Ang II–induced DNA synthesis in rat arteries in
vivo.5 In addition, vascular reactivity to
norepinephrine and 1-AR density are
augmented in certain hypertensive states that are characterized by
increased dependence on angiotensin.6 Aside
from these albeit indirect associations between 1-ARs
and Ang II, it is unclear whether Ang II can directly alter expression
of 1-AR subtypes by vascular smooth muscle.
Increased intravascular pressure, like norepinephrine and
Ang II, stimulates contraction and may itself influence SMC growth and
1-AR expression. Increased 1-AR density
has been noted in some types of hypertensive disease,6 but
the difficulty in isolating an influence of pressure per se from other
factors present in vivo has prevented identification of the
mechanism responsible for the increase. In a previous study, we
examined the direct effect of pressure (ie, mechanical loading) on
1-AR expression by SMCs.7 Mechanical
loading using rat aorta SMCs in cell culture, intact aorta maintained
in organ culture, and in vivo suprarenal aortic coarctation
hypertension had no effect on SMC expression of 1B- and
1D-AR mRNAs or 1-AR
density.7 However, in the Ang II–dependent coarctation
model, 1B-AR expression was reduced in the normotensive
subrenal aorta, whereas in the high-pressure suprarenal aorta, this
decrease was prevented. These affects were not dependent on differences
in vascular hypertrophy, since a similar amount of wall
hypertrophy was evident above and below the site of
stenosis. From these studies, we proposed that Ang II causes a
decrease in 1B-AR expression by SMCs that is opposed by
increased mechanical loading. The purpose of the present study was
to examine this hypothesis by first determining the direct effects of
Ang II on 1B- and 1D-AR mRNA levels and
1-AR density in cultured SMCs. Second, we sought to
identify whether mechanical loading of SMCs interacts with Ang II in
the regulation of 1-AR expression.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Cell Culture
Each primary culture of SMCs was obtained from the medial layer of thoracic aortas pooled from eight 200-g male rats (Sprague-Dawley, Sasko, Omaha, Neb) as previously described.7 8 Cells were maintained in medium 199 supplemented with 10% fetal bovine serum, 200 mg/mL L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, and 2.5 mg/L Fungizone when plated on Flex I plates (Flexcell International Corp). Medium was changed every 2 days. Cells were grown from primary isolation on plastic culture dishes, subcultured at 90% to 95% confluence every 5 to 7 days with 0.05% trypsin-EDTA, and seeded at a density of 5000 cells/cm2. To favor the differentiated phenotype and quiescence,7 8 9 cells from passages 4 and 5 were grown to 2 days postconfluence, and medium was changed to serum-free medium (50/50 Dulbecco's modified Eagle's medium and F-12 containing insulin, transferrin, ascorbic acid, and 2.5 mg/L penicillin/streptomycin/Fungizone) for 2 additional days before the experiment was started. Serum-free medium was also used to lessen degradation of Ang II (human, Bachem; dissolved in water) from peptidases.10 SMCs were exposed for either 3, 24, or 48 hours to 0.3, 3, and 3000 nmol/L Ang II (the approximate dissociation constant for SMC Ang II binding is 7±2 nmol/L11 12 ) and compared with levels in time-matched, vehicle-treated control cells. Medium was changed every 24 hours to minimize reductions in Ang II concentration. In agreement with our previous studies,7 cyclophilin mRNA was not significantly altered by Ang II and/or mechanical loading in these experiments and was used as an internal control for the
1-AR RNase protection assays. The
selective AT1 antagonist losartan was
added to serum-free medium with Ang II to determine whether effects of
Ang II were mediated by AT1 receptors. Aortic SMCs were seeded onto collagen-coated flexible membrane bottom plates (Flex) and studied 4 days after reaching confluence. A separate cell line was used in each experimental replicate. SMCs were treated with 3 nmol/L Ang II and subjected to 3 or 24 hours of cyclic stretch (1 Hz) and compared with time-matched control cells not exposed to stretch. Stretch was achieved by a computer-driven, vacuum-operated Flexercell strain unit that has been described in detail.13 This system was set to apply a vacuum level of 15 kPa to stretch the flexible membrane and attached SMCs by an average 18% elongation. The vacuum was then released, returning the membrane to its original conformation, resulting in one complete cycle per second. Viable cell number was determined by hemocytometry and trypan blue exclusion.
Protein and RNA Determinations
Since Ang II can cause SMC growth,14 15 16 17 18 19 20 21 22 23 24 cell number
and total per-cell protein and RNA were determined. Total soluble
protein concentration was obtained by BCA assay (Pierce Chemical).
Total cellular RNA was extracted, and RNA integrity and concentration
were assessed by gel electrophoresis and spectrophotometry. mRNA levels
were measured with RPAs. Details, [32P]CTP-labeled probe
construction, and specificity of our RPAs have been previously
described.7 8 The 1D-AR riboprobe consisted
of a cRNA probe of 159 bases against a region within the putative third
intracellular loop that protects 117 bp when hybridized to
1D-AR mRNA. A fragment of the 1B-AR cDNA
between the putative second and third intracellular loops yielded a
cRNA probe of 342 bases and a 306-bp protected hybrid. A riboprobe
specific for the rat cyclophilin gene (Ambion) was used as an internal
control for the 1D- and 1B-AR RPAs. In
agreement with previous studies,7 preliminary studies with
Ang II, and also as demonstrated herein (see figures below), Ang II had
no effect on cyclophilin mRNA in the absence or presence of load.
To control for the effects of interventions on cell growth (see "Results"), we conducted each RPA on the same number of SMCs from treated and control groups to allow estimation of mRNA per cell. Total cellular RNA was incubated with specific riboprobes in solution, nonhybrids were digested with RNase (RNase A and T1, Boehringer Mannheim), and hybrids were separated by electrophoresis. Gels were dried and exposed to autoradiographic film (X-OMAT, Eastman Kodak). mRNA levels were measured according to film densitometry (laser scanner [UMAX, UC630] and the Image program [National Institutes of Health]), with exposure times adjusted to keep signal densities within the linear range of the film.
Determination of Receptor Density
Receptor number was determined by radioligand
binding using methods described in detail previously.7 8
Briefly, groups of 4-day postconfluent SMCs (15 to 20 plastic culture
dishes [100-mm] or 20 to 25 Flex I plates) were treated with Ang II
for 24 hours in the absence or presence of cyclic load as described
above. Cells were washed twice with ice-cold phosphate-buffered
saline, scraped in 5 mmol/L Tris (pH 7.5) and 5 mmol/L EDTA
buffer, homogenized, and centrifuged at 18 000 rpm
for 10 minutes at 4°C. The resulting pellets were frozen at -80°C
and, within 2 weeks, thawed on ice, rehomogenized in 5
mmol/L Tris (pH 7.5) and 5 mmol/L EDTA buffer, and
centrifuged at 18 000 rpm for 10 minutes at 4°C. The crude
membrane was resuspended in incubation buffer (5 mmol/L Tris [pH
7.5], 5 mmol/L MgCl2) and protein concentration
determined by BCA assay. Membrane protein was diluted to 1 mg/mL and
saturation binding determined with [3H]prazosin (New
England Nuclear) diluted with incubation buffer in borosilicate tubes
to achieve a final concentration of 0.01 to 3.0 nmol/L. Each reaction
was assembled at 4°C in triplicate in polypropylene tubes in a final
volume of 125 µL: 100 µL (100 µg) membrane, 12.5 µL
[3H]prazosin, and either 12.5 µL incubation buffer
(total binding) or 12.5 µL phentolamine at a final
concentration of 10 µmol/L (nonspecific binding). Binding
reactions were incubated with shaking at 25°C for 45 minutes, and
then tubes were placed on ice. Reactions were diluted with 3 mL
incubation buffer and immediately filtered through Whatman GF/C filters
using a Millipore filter manifold and washed three times with
incubation buffer at 4°C. Filters were dried, placed in Ecoscint H
(National Diagnostics), and counted in a liquid
scintillation counter.
Statistics
Data are expressed as mean±SEM, where n sizes represent
independent replicates from different cell lines. Values were compared
with ANOVA plus Bonferroni multiple comparison tests and/or Student's
t test for unpaired observations. Values for receptor number
(Bmax) and antagonist dissociation
constant (Kd) obtained in
radioligand binding assays were determined by nonlinear
regression analysis (GraphPad). Differences were considered
significant at a value of P<.05.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Angiotensin Acting via AT1 Receptors Decreases Expression of
1D- and
1B-ARsSince Ang II may induce SMC proliferation and/or hypertrophy, depending on the conditions of cell culture (see Reference 1717 ), RPAs were performed on total RNA from a constant number of cells for groups receiving vehicle, Ang II, or Ang II plus mechanical loading. Because of differences in mRNA abundance, 6x106 SMCs (70 to 140 µg RNA) were used for determination of
1B-AR and 2.5x106 SMCs (25
to 45 µg RNA) for 1D-AR message levels. Cell number
and total cellular protein and RNA were measured before RPAs as
controls for changes in cell proliferation and growth to permit
interpretation of changes in mRNA on a per-cell basis. Compared with
vehicle-treated, time-matched control SMCs (used in all experiments),
cell number for these 4-day postconfluent quiescent cells maintained in
serum-free medium was unaffected by 3 hours of exposure to Ang II but
was decreased after 24 hours of 3 nmol/L Ang II (75±2% of control,
P<.01) and 1 µmol/L Ang II (85±4% of control,
P<.01) and after 48 hours of exposure to 0.3 nmol/L Ang II
(78±9% of control, P<.01). The reason for this small
decrease in cell number was not apparent but has been reported by other
researchers18 and may be due to a loss of cell adhesion as
cells undergo hypertrophy (see below) or contraction.
However, a similar time course and magnitude of hypertrophy
produced by 1-AR stimulation was not accompanied by a
decrease in number of passage 4 SMCs studied under identical
conditions.25
Ang II increased cell protein (Fig 1
). Three hours of 0.3
nmol/L to 1 µmol/L Ang II increased protein 1.2- to 1.5-fold,
respectively. Longer exposure to Ang II produced additional increases
in total protein per cell for the lower Ang II concentrations. Three
hours of 0.3 nmol/L to 1 µmol/L Ang II resulted in 1.1- to
1.3-fold increases, respectively, in total cell RNA (Fig 1).
Twenty-four and 48 hours of Ang II produced additional increases in
total RNA per cell. These parallel increases in cell protein and RNA
have been shown to correlate with Ang II–induced increases in cell
volume.26
|
After exposure to 3 hours of 0.3 nmol/L Ang II, 1D-AR
mRNA levels were 73±8% of control levels (Fig 2). Ang II
at 3 nmol/L and 1 µmol/L further reduced 1D mRNA
levels to 55% and 56%, respectively. By 24 and 48 hours of Ang II
treatment, 1D mRNA had returned to control levels.
Densitometry values for 1D (and 1B, Fig 3) were normalized to cyclophilin mRNA, which, in agreement
with our previous study,7 was unaffected by Ang II,
stretch, or Ang II plus stretch. Thus, 1D mRNA per SMC
declined initially but then returned to control levels as cells
accumulated additional protein and RNA with time. Unlike
1D-AR mRNA, per-cell 1B mRNA was not
decreased by 3 hours of Ang II (Fig 3). However, 24 hours of 3 nmol/L
and 1 µmol/L Ang II decreased 1B mRNA levels to
72% and 42% of control, respectively. This decline was sustained over
48 hours of 1 µmol/L Ang II (27% of control). The
AT1-specific antagonist losartan
abolished the increases in per-cell total RNA and protein induced by
Ang II (Fig 1
). Losartan also prevented the decrease in
1D mRNA after 3 hours of Ang II and the decrease in
1B mRNA produced by 24 hours of Ang II (Fig 4; losartan was not tested against 48 hours of Ang
II treatment). Losartan alone had no effect on total RNA or
protein or on 1D or 1B mRNA (n=3, data
not shown). 1A-ARs are not expressed by these
SMCs,7 and none of the Ang II concentrations induced
1A transcripts detectable by RPA after 3, 24, or 48
hours (data not shown).
|
|
|
To determine whether the decrease in 1D and
1B mRNAs was accompanied by a decrease in
1-AR density, we performed radioligand
binding assays with [3H]prazosin on membrane protein from
SMCs that had been exposed to Ang II for 24 hours and compared them
with assays on membrane protein from time-matched, vehicle-treated
control cells. Ang II decreased Bmax in a
concentration-dependent manner (-53% at 1 µmol/L Ang II),
whereas the Kd value with prazosin was
unaffected (Table). Nonspecific binding at the
Kd value was less than 10% in all assays. The
data in Figs 1 through 4 and the Table indicate that Ang II, acting
through an AT1 receptor, produces a time- and
concentration-dependent decrease in the number of 1D and
1B mRNA transcripts and 1 receptors per
cell as the cells undergo hypertrophy.
|
Cyclic Load Opposes Ang II–Mediated Decrease in
1B-AR Expression
In a previous study,7 suprarenal aortic coarctation
caused a decrease in 1B-AR mRNA in the subrenal
normotensive aorta that was prevented in the suprarenal hypertensive
aorta. To determine whether this derives from an effect of increased
load to attenuate the Ang II–induced reduction in 1B
expression, we exposed SMCs to cyclic load in the presence of 3 nmol/L
Ang II. The first experiment examined 1D expression
after 3 hours of exposure to the following three interventions (because
the transient decrease in 1D by 3 nmol/L Ang II was
at 3 hours; Fig 2): 3 nmol/L Ang II alone, mechanical loading alone,
and Ang II plus mechanical loading. As in the previous experiments,
cell number tended to decrease to 73±17% of control by Ang II
(P=.2); cell number was 86±14% in load conditions
(P=.4) and 104±3% in Ang II plus load conditions
(P=.2) (n=3 for each). Also, similar to data shown in Fig 2, Ang II (3 nmol/L) increased total protein and RNA (the former
nonsignificantly) per cell, and load inhibited these increases (Fig 5). Unlike the previous experiment with cells grown on
plastic (Fig 2), in the present experiments with cells in all
groups maintained on collagen (as required for cell attachment to the
flexible plate membrane), 3 nmol/L Ang II alone did not decrease total
1D mRNA per cell. This may reflect an effect of plating
surface, although we have found no effect of plastic, collagen, or
fibronectin plating surfaces on baseline levels of
1B-AR, 1D-AR, -actin, or cyclophilin
mRNAs nor on cell number and RNA or protein per cell (n=2). Stretch
alone had no significant effect on per-cell total RNA (119±4% of
control), protein (124±11%), or 1D mRNA (114+10%),
confirming our previous studies.7
|
A second experiment examined the effects of load on 1B
expression during 24 hours of exposure to 3 nmol/L Ang II. In a
previous study,7 24 hours of load alone had no effect on
1B mRNA. In the present experiment, cell number
after 24 hours of Ang II was 110±16% of control and after Ang II plus
load was 119±21% of control. As shown in Figs 1 and 3, 24 hours of 3
nmol/L Ang II increased per-cell total protein and RNA
(P=.07 for RNA) and decreased 1B mRNA per
cell (Fig 5). Load abolished these Ang II effects. Specific prazosin
binding was also examined after 3 nmol/L Ang II in the presence of 24
hours of cyclic load. Like the decrease in 1B-AR mRNA,
load abolished the decrease in 1-AR density induced by
Ang II (Table).
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Sympathetic nerve stimulation of
1-ARs, transmural
pressure, and circulating Ang II each regulate vascular SMC contraction
and blood flow and exert effects on SMC growth. Moreover, each can
modulate the effects of the others, presumably through intracellular
second messenger interactions and/or secretion of autocrine modulators.
However, little is known concerning whether Ang II receptor activation
and pressure-induced myogenic stimulation modify 1-AR
expression itself. In a previous study,7 we found that
exposure of rat aortic SMCs and intact aorta in organ culture to
mechanical loading for up to 48 hours had no effect on
1B- and 1D-AR mRNA levels or
1-AR density. However, in in vivo renin-dependent
coarctation hypertension, 1B-AR message levels were
selectively decreased (1D-AR, -actin, and ß-actin
were unchanged) in the normotensive aorta below the stenosis
but not in the hypertensive suprarenal aorta.7 To begin to
examine the mechanisms involved, in the present study we
tested the hypothesis that Ang II directly decreases
1B-AR expression and that elevated wall tension opposes
this decrease. Findings in the present study support this
hypothesis. Ang II stimulation of AT1 receptors induced a
concentration-dependent reduction in 1D- and
1B-AR mRNA expression and 1-AR density in
aortic SMCs. The decline in 1D mRNA detected at 3 hours
was transient, returning to control levels within 24 hours. In
contrast, reduction of 1B mRNA was not detected until 24
hours, when receptor density was also decreased, and remained depressed
after 48 hours of exposure to 1 µmol/L Ang II. This decrease in
1B mRNA and 1-AR density was prevented
when cells were concomitantly exposed to mechanical load. These results
in cell culture suggest that Ang II and mechanical load
(arterial pressure) may modulate 1-AR
expression in vivo.
Consistent with the growth-associated effects of Ang II
shown in Fig 1, Ang II has been reported to increase SMC protein
synthesis and content, - and ß-actin expressions, and rRNA in
association with cellular hypertrophy.17 18 19 20 21 22 23 24 26
For example, Bunkenburg et al18 found that 1 to 100 nmol/L
Ang II concentration-dependently increased DNA and protein synthesis
without affecting cell number in confluent rat aortic SMCs from
passages 2 through 5. AT1 but not AT2 receptor
blockade inhibited the effects, consistent with the presence of
only AT1 receptors on the cells. Similar results were
reported by Berk and Rao26 for rat aortic SMCs treated
with Ang II for 24 hours. Ang II at the maximally effective
concentration in their study (100 nmol/L) increased cell protein and
RNA synthesis by 25%, protein content by 30%, and cell size by 40%
and did not increase cell number. This hypertrophy, which
was dependent on new transcription and associated with release of
PDGF-like immunoreactivity, was blocked by a PDGF-A chain antibody and
by hydrocortisone, whereas the induction of c-fos and
c-myc were unaffected.26 Under similar
conditions, Ang II increased -actin, myosin heavy chain,
tropomyosin, and vimentin by twofold to eightfold.17 Wang
et al27 found that rat aortic hypertrophy was
produced by chronic in vivo Ang II infusion, together with increased
expression of PDGF and proliferating cell nuclear antigen. Exposure of
rat aortic rings to 1 µmol/L Ang II for 16 hours increased
protein synthesis by 35% without any change in DNA
synthesis.21 Thus, our cultured SMCs responded to Ang II
similarly to the response described in previous in vivo, in vitro, and
cell culture studies. However, Holycross et al21 found
that the Ang II–induced increase in protein synthesis was not
inhibited by concomitant application of 0.6 g/mm of tonic load.
However, this contrasts with our results, in which phasic load
inhibited increases in cell protein and RNA that were produced by a
lower (3 nmol/L) concentration of Ang II.
In the present study, Ang II hypertrophy was evidenced
as an AT1 receptor–dependent increase in per-cell protein
and RNA (Fig 1). Because of this, we assayed 1D- and
1B-AR mRNA levels from a constant number of cells
instead of a constant amount of total RNA to allow changes in mRNA to
be compared on a per-cell basis (Figs 2 and 3). When -AR mRNA values
are normalized for a constant amount of RNA (ie, dividing the mRNA
values by the increase in per-cell RNA [Fig 1]), 0.3, 3, and 1000
nmol/L Ang II decreased 1D mRNA cellular
"concentration" by 34%, 53%, and 55%, respectively, at 3
hours; by 18%, 36%, and 44% at 24 hours; and by 14%, 27%, and 52%
at 48 hours. And, accordingly, 1B transcript
"concentration" decreased by 18%, 19%, and 6% at 3 hours; by
10%, 58%, and 72% at 24 hours; and by 31%, 32%, and 85% at 48
hours. The delayed decrease in 1B-AR mRNA may be
influenced by the longer half-life of 1B- versus
1D-AR mRNA (8 versus 4 hours).8 Thus, when
viewed together with the increase in total cell RNA, Ang II induced a
sustained decrease in cellular "concentrations" of both messages.
This interpretation is consistent with the receptor density
findings. 1-AR density, determined for a constant amount
of cell membrane protein, was concentration-dependently decreased by
Ang II, averaging 47% of control with 1 µmol/L Ang II (Table)
at a time (24 hours) when normalized 1D and
1B mRNA concentrations were reduced to 44% and 72%,
respectively. Since our previous binding studies with
chloroethylclonidine alkylation estimated that 1B-ARs
make up approximately 15% to 20% of the total 1-AR
density in these SMCs,8 the 47% decrease in total
1-AR density would predictably require reductions in
both 1D- and 1B-AR densities. Also, the
transient decrease in total 1D-AR mRNA per cell may not
imply a transient decline in receptor density, since other researchers
have reported a sustained decrease in 1-ARs despite
transient decreases in SMC 1-AR
transcripts.28 Overall, these data suggest that prolonged
Ang II exposure causes a maintained decrease in expression of both
1D- and 1B-ARs. Quantitative confirmation
of this suggestion awaits development of highly selective antibodies or
subtype antagonists.
While hypertrophy and reduced 1-AR
expression were induced by the two lowest Ang II concentrations tested
(0.3 and 3 nmol/L), it is likely that the actual concentrations in the
serum-free culture medium declined over the test interval. The presence
of confluent SMCs causes an unavoidable approximately 100-fold
reduction of Ang II in the medium by the end of a 24-hour
incubation.18 26 Thus, the responses we measured likely
resulted from exposure of SMCs to concentrations within the
physiological plasma range (0.01 to 0.05
nmol/L).29 It is also possible that vascular wall Ang
II levels may be considerably higher than circulating
levels.30 The intrinsic vascular wall
renin-angiotensin system has been proposed to be an
important regulator of vessel wall growth.31
The mechanisms by which Ang II reduces SMC expression of
1-ARs remain to be determined, but several possibilities
are noteworthy. Increases in DNA synthesis in rat carotid artery and
aorta induced by chronic infusion of Ang II were prevented by
1-AR blockade,5 suggesting an interaction
between Ang II and 1-ARs that may lead to downregulation
of the latter by Ang II receptor stimulation. Interestingly, Ang II
reduces hepatocyte 1B- and
1D-ARs by a protein kinase C–dependent
mechanism.32 33 The growth-promoting agonists
norepinephrine, endothelin, prostaglandin
F2, and phorbol ester reduce
1B- and variably affect 1D-AR mRNA levels
in cultured cardiac myocytes.34 Conversely,
norepinephrine reduces Ang II receptor density in neuronal
cultures.35 Furthermore, the mitogens epidermal growth
factor, basic fibroblast growth factor, and PDGF-BB induce a sustained
(24 hours) decrease in SMC expression of AT1 that is
evident as early as 4 hours after exposure and that is associated with
a reduction in AT1 transcription and mRNA
half-life.36 In contrast to these and the current studies,
Hu et al37 reported that Ang II increased SMC
1B- and 1D-AR mRNA levels and increased
the rate of return of 1-ARs after receptor alkylation
with phenoxybenzamine, effects that were blocked by actinomycin D.
However, mechanisms activated after pharmacological depletion
of 1-ARs (and other amine receptors) may differ from
those regulating native receptor density.
Exposure of SMCs to phasic mechanical loading to simulate an increase
in arterial pressure inhibited Ang II–mediated
increases in SMC protein and RNA content and decreases in
1B transcripts and 1-AR density. The
decrease in 1-AR expression may not be secondary to Ang
II–induced SMC growth. We previously reported that exposure of these
cells to 24 and 48 hours of norepinephrine increased cell
protein and RNA (and -actin mRNA) by amounts similar to those
produced by Ang II in the present study, yet 1B- and
1D-AR mRNAs were not decreased as in the present
study.25 Although speculative, interactions between
postreceptor signaling pathways or growth factor release by SMC
stretch and Ang II receptor activation may underlie the ability of SMC
loading to inhibit Ang II reduction in 1-AR expression.
AT1 receptor stimulation of SMCs activates various
intracellular pathways, including the
phosphoinositide–protein kinase C pathway. Increased
protein kinase C activity decreases expression of 1B-
and 1D-ARs in rat aortic SMCs and the
DDT1-MF-2 SMC cell line,38 39 40 thus providing a
possible link to AT1 stimulation and reduced
1-AR expression. Cyclic mechanical loading of porcine
coronary SMCs using the same frequency, degree of stretch, and
duration as in the current study reduces Gs
and cAMP levels.41 Additional studies are needed to
determine whether these changes are responsible for interfering with
the ability of Ang II to decrease 1-AR expression.
Data in Fig 5 suggest that phasic load opposes Ang II–induced SMC
growth, as indexed by per-cell protein and RNA levels. These results
seem at variance with evidence that both Ang II17 18 19 20 21 22 23 24 26
and load42 individually can promote SMC growth. Thus, an
additive or synergistic, rather than antagonistic,
interaction between Ang II and load might have been expected.
Holycross et al21 found that 0.6 g/mm tonic load versus no
load applied to rat aortic rings over 16 hours did not inhibit Ang
II–induced increases in protein and DNA synthesis. In vivo experiments
also do not seem to support antagonism of Ang II growth by load;
arterial hypertrophy produced by Ang II
infusion was lessened, not enhanced, when rats were infused with
hydralazine.43 Also, the abdominal aorta does not
hypertrophy more than the thoracic aorta in suprarenal
aortic coarctation (eg, Fig 4). It remains unclear whether these
distinctions extend from differences in the type of load (tonic versus
phasic), serum presence or absence (serum was required for the load
effect on growth in Bardy et al42 ; we did not use serum so
as to obviate Ang II degradation), time course of Ang II and load
exposure, or load-independent effects of agents such as
hydralazine on protein synthesis. It is noteworthy that load
applied over 48 hours to aortic SMCs in both cell and organ cultures,
although increasing -actin mRNA, did not cause SMC
growth,7 in agreement with Holycross et al21
for 16 hours of load but in disagreement with Bardy et
al42 for 72 hours of load. It should also be mentioned
that the present experiments were conducted with cells grown on a
collagen matrix required for cell adhesion to the flexible membrane and
that some responses can be greatly affected by culture plate surface
chemistry.
These findings have several potential physiological
implications. Regulation of growth and increased expression of
contractile proteins by contractile agonists induce structural vascular
remodeling. During hypertension, this could serve as an adaptive
response that might, however, concomitantly favor additional increases
in peripheral resistance.44 Both Ang II and
1-adrenergic G protein–coupled receptor types cause
contraction and SMC growth. For example, Ang II and 1-AR
stimulation increase the mitogen PDGF by SMCs.45 46 Load
itself increases PDGF,47 which has been suggested along
with several other growth factors as potential mediators of
pressure-induced vascular wall hypertrophy.26
Also, Ang II,48 norepinephrine acting at the
1D-AR,49 and phasic load50
each activate the extracellular signal–regulated kinase
(ERK/MAPK) growth cascade in muscle cells. Thus, in agreement with our
current findings, downregulation of 1-ARs during
prolonged stimulation of AT1 receptors, which are well
known to amplify the contractile actions of
norepinephrine,3 4 in the absence of an
increase in arterial pressure, may limit excessive
constriction and SMC growth. That is, Ang II–induced reduction in
1-AR expression may provide a "compensatory"
mechanism to lessen catecholamine-induced contraction and
hypertrophy in high-renin states. On the other hand, during
renin-dependent hypertension, blockade of Ang II–induced
downregulation of 1-ARs by increased SMC load may
prevent a decrease in adrenergic reactivity and preserve or enhance
adrenergic control of vascular resistance and SMC
hypertrophy in the face of greater afterload. Vascular
smooth muscle in many types of hypertension, including renin-dependent
forms, evidences increases in -AR reactivity arising from multiple
mechanisms, including vessel wall hypertrophy, altered
membrane and intracellular signaling components, and changes in -AR
density.6 Although speculative, the present results
may provide an additional level of interaction. Studies of vascular
catecholamine contractile sensitivity using the aortic
coarctation model, with vessel segments taken above and below the
coarctation, but preloaded at hypertensive and normotensive levels,
respectively (unlike previous studies51 52 ), would provide
an approach to the testing of this hypothesis.
Additional studies are also required to determine whether our findings
have significance in arterial restenosis after
angioplasty. Restenosis is largely due to excessive SMC growth
and matrix secretion. Ang II, pressure, and 1-ARs have
been implicated, albeit along with many other factors. Converting
enzyme inhibitors or Ang II antagonists, which
decrease Ang II receptor stimulation, pressure, and Ang II
amplification3 4 of adrenergic SMC tone, greatly attenuate
neointimal formation in rat carotid artery and aorta after
balloon angioplasty.53 54 Also, chronic Ang II infusion
increases neointimal and medial growth.54 55 56
Van Kleef et al5 found that chronic Ang II infusion
increased arterial media DNA synthesis, an effect abolished
by prazosin. We have shown that 1D-AR stimulation
induces hypertrophy in rat aortic SMCs in cell and organ
cultures.25 49 Thus, Ang II and 1-AR
activation, together with a modulatory influence of
arterial pressure suggested by our present study, may
participate in restenosis complications that limit the efficacy
of angioplasty for revascularization.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This study was supported by National Heart, Lung, and Blood Institute grant HL-52610.
|
Footnotes |
|---|
Reprint requests to James E. Faber, PhD, Department of Physiology, 474 Medical Sciences Research Building, University of North Carolina, Chapel Hill, NC 27599-7545.
Received August 20, 1996; first decision October 15, 1996; accepted November 7, 1996.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Graham RM, Perez DM, Hwa J, Piascik MT.
1-Adrenergic receptor subtypes: molecular structure,
function and signaling. Circ Res. 1996;78:737-749.
2.
Leech CJ, Faber JE. Different -adrenoceptor
subtypes mediate constriction of arterioles and venules.
Am J Physiol. 1966;270:H710-H722.
3. Zimmerman BG, Gomez J. Increased response to sympathetic stimulation in the cutaneous vasculature in the presence of angiotensin. Int J Neuro Pharmacol. 1965;4:185-193.
4. Johnson EM, Marshall GR, Needleman P. Modification of responses to sympathetic nerve stimulation by the renin-angiotensin system in rats. Br J Pharmacol. 1974;51:541-547.[Medline] [Order article via Infotrieve]
5.
van Kleef EM, Smits JFM, DeMey JGR, Cleutjens JPM,
Lombardi DM, Schwartz SM, Daemen MJAP.
1-adrenoreceptor blockade reduces the
angiotensin II-induced vascular smooth muscle cell DNA
synthesis in the rat thoracic aorta and carotid artery.
Circ Res. 1992;70:1122-1127.
6.
Michel MC, Brodde O-E, Insel PA.
Peripheral adrenergic receptors in hypertension.
Hypertension. 1990;16:107-120.
7.
Clements ML, Banes AJ, Faber JE. Effect of
mechanical loading on vascular 1D- and
1B-adrenergic receptor expression.
Hypertension. 1997;29:1156-1164.
8.
Eckhart AD, Zhu Z, Arendshorst WJ, Faber JE.
Oxygen modulates 1B-adrenoceptor gene expression by
arterial but not venous vascular smooth muscle.
Am J Physiol. 1996;271:H1599-H1608.
9.
Monical PL, Owens GK, Murphy RA. Expression of
myosin regulatory light-chain isoforms and regulation of
phosphorylation in smooth muscle. Am
J Physiol. 1993;264:C1466-C1472.
10.
Bagby SP, Kirk EA, Mitchell LH, O'Reilly MM, Holden
WE, Stenberg PE, Bakke AC. Proliferative synergy of Ang II and
EGF in porcine aortic vascular smooth muscle cells.
Am J Physiol. 1993;265:F239-F249.
11. Lassegue B, Alexander RW, Nickenig G, Clark M, Murphy TJ, Griendling KK. Angiotensin II down-regulates the vascular smooth muscle AT1 receptor by transcriptional and post-transcriptional mechanisms: evidence for homologous and heterologous regulation. Mol Pharmacol. 1995;48:601-609.[Abstract]
12. Grover AK, Fomin VP, Samson SE. Angiotensin II contractions in coronary artery. Mol Cell Biochem. 1994;135:11-19.[Medline] [Order article via Infotrieve]
13. Banes AJ, Gilbert J, Taylor D, Monbureau O. A new vacuum-operated stress-providing instrument that applies static or variable duration cyclic tension or compression to cells in vitro. J Cell Sci. 1985;75:35-42.[Abstract]
14. Lyall F, Morton JJ, Lever AF, Cragoe EJ. Angiotensin II activates Na+-H+ exchange and stimulates growth in cultured vascular smooth muscle cells. J Hypertens. 1988;6:S438-S441.
15. Millett D, Desgranges D, Campan M, Gadeau A, Costerousse O. Effects of angiotensins on cellular hypertrophy and c-fos expression in cultured arterial smooth muscle cells. Eur J Biochem. 1992;206:367-372.[Medline] [Order article via Infotrieve]
16. Owens GK. Determinants of angiotensin II-induced hypertrophy versus hyperplasia in vascular smooth muscle. Drug Dev Res. 1993;29:83-87.
17.
Turla MB, Thompson MM, Corjay MH, Owens GK.
Mechanisms of angiotensin II- and arginine
vasopressin-induced increases in protein synthesis and content in
cultured rat aortic smooth muscle cells. Circ Res. 1991;68:288-299.
18.
Bunkenburg B, van Amelsvoort T, Rogg H, Wood JM.
Receptor-mediated effect of angiotensin II on growth of
vascular smooth muscle cells from spontaneously hypertensive
rats. Hypertension. 1992;20:746-754.
19.
Berk BC, Vekshtein V, Gordon HM, Tsuda T.
Angiotensin II-stimulated protein synthesis in cultured
vascular smooth muscle cells. Hypertension. 1989;13:305-314.
20.
Geisterfer AAT, Peach MJ, Owens GK.
Angiotensin II induces hypertrophy, not
hyperplasia, of cultured rat aortic smooth muscle cells.
Circ Res. 1988;62:749-756.
21. Holycross BJ, Peach MJ, Owens GK. Angiotensin II stimulates increased protein synthesis, not increased DNA synthesis, in intact rat aortic segments, in vitro. J Vasc Res. 1993;30:80-86.[Medline] [Order article via Infotrieve]
22.
Ullian ME, Hutchison FN, Hazen-Martin DJ, Morinelli
TA. Angiotensin II-aldosterone
interactions on protein synthesis in vascular smooth muscle
cells. Am J Physiol. 1993;264:C1525-C1531.
23. Natarajan R, Gonzales N, Lanting L, Nadler J. Role of the lipoxygenase pathway in angiotensin II-induced vascular smooth muscle cell hypertrophy. Hypertension. 1994;23(suppl I):I-142-I-147.
24.
Hershey JC, Hautmann M, Thompson NN, Rothblum LI,
Haystead TAJ, Owens GK. Angiotensin II-induced
hypertrophy of rat vascular smooth muscle is associated
with increased 18 S rRNA synthesis and phosphorylation
of the rRNA transcription factor, upstream binding factor.
J Biol Chem. 1995;270:25096-25101.
25.
Chen LQ, Xin X, Eckhart AD, Yang N, Faber JE.
Regulation of vascular smooth muscle growth by
1-adrenoceptor subtypes in vitro and in situ.
J Biol Chem. 1995;270:30980-30988.
26. Berk BC, Rao GN. Angiotensin II-induced vascular smooth muscle cell hypertrophy: PDGF A-chain mediates the increase in cell size. J Cell Physiol. 1993;154:368-380.[Medline] [Order article via Infotrieve]
27.
Wang DH, Prewitt RL, Beebe SJ. Regulation of
PDGF-A: a possible mechanism for angiotensin II-induced
vascular growth. Am J Physiol. 1995;269:H356-H364.
28.
Izzo NJ, Seidman CE, Collins S, Colucci WS.
1-Adrenergic receptor mRNA level is regulated by
norepinephrine in rabbit aortic smooth muscle
cells. Proc Natl Acad Sci U S A. 1990;87:6268-6271.
29. Safar ME, London GM, Simon A, Chau NP. Volume factors, total exchangeable sodium, and potassium in hypertensive disease. In: Genest J, Kuchel O, Hamet P, Cantin M, eds. Hypertension. 2nd ed. New York, NY: McGraw-Hill; 1983: chap 3, 46.
30. Campbell DJ. Circulating and tissue angiotensin systems. J Clin Invest. 1987;79:1-6.
31. Dzau V. Multiple pathways of angiotensin production in the blood vessel wall: evidence, possibilities and hypotheses. J Hypertens. 1989;7:933-937.[Medline] [Order article via Infotrieve]
32.
Garcia-Sainz JA, Tussie-Luna MI, Hernandez-Sotomayor
SMT. Phorbol esters, vasopressin and angiotensin II
block 1-adrenergic action in rat hepatocytes: possible
role of protein kinase C. Biochim Biophys Acta. 1986;887:69-72.[Medline]
[Order article via Infotrieve]
33.
Garcia-Sainz JA, Hernandez-Sotomayor SMT, Tussie-Luna
MI. Homologous and heterologous desensitization of one of the
pathways of the 1-adrenergic action: effects of epinephrine,
vasopressin, angiotensin II and phorbol
12-myristate 13-acetate. Biochim Biophys
Acta. 1986;887:73-79.[Medline]
[Order article via Infotrieve]
34.
Rokosh DG, Stewart AFR, Chang KC, Bailey BA, Karliner
JS, Camacho SA, Long CS, Simpson PC. 1-adrenergic
receptor subtype mRNAs are differentially regulated by
1-adrenergic and other hypertrophic stimuli in cardiac
myocytes in culture and in vivo. J Biol Chem. 1996;271:5839-5843.
35.
Sumners C, Watkins LL, Raizada MK.
1-adrenergic receptor-mediated down-regulation of
angiotensin II receptors in neuronal cultures.
J Neurochem. 1986;47:1117-1126.[Medline]
[Order article via Infotrieve]
36. Nickenig G, Murphy TJ. Down-regulation by growth factors of vascular smooth muscle angiotensin receptor gene expression. Mol Pharmacol. 1994;46:653-659.[Abstract]
37.
Hu Z, Shi X, Okazaki M, Hoffman BB.
Angiotensin II induces transcription and expression of
1-adrenergic receptors in vascular smooth muscle
cells. Am J Physiol. 1995;268:H1006-H1014.
38.
Izzo NJ, Tulenko TN, Colucci WS. Phorbol esters
and norepinephrine destabilize 1B-adrenergic
receptor mRNA in vascular smooth muscle cells. J
Biol Chem. 1994;269:1705-1710.
39.
Hu Z-W, Shi X-Y, Sakaue M, Hoffman BB. Prolonged
activation of protein kinase C induces transcription and expression of
the 1B adrenergic receptor gene in DDT1 MF-2
cells. J Biol Chem. 1993;268:3610-3615.
40. Hadcock JR, Malbon CC. Agonist regulation of gene expression of adrenergic receptors and G proteins. J Neurochem. 1993;60:1-9.[Medline] [Order article via Infotrieve]
41. Wiersbitsky M, Mills I, Gewirtz H. Cyclic stretch reduces adenylate cyclase activity and stimulatory G-protein subunit levels in coronary smooth muscle cells. Exp Cell Res. 1994;210:52-55.[Medline] [Order article via Infotrieve]
42.
Bardy N, Karillon GJ, Merval R, Samuel J-L, Tedgui
A. Differential effects of pressure and flow on DNA and protein
synthesis and on fibronectin expression by arteries in a novel organ
culture system. Circ Res. 1996;79:70-78.
43.
Griffin SA, Brown WCB, MacPherson F, McGrath JC, Wilson
VG, Korsgaard N, Mulvany MJ, Lever AF. Angiotensin
II causes vascular hypertrophy in part by a non-pressor
mechanism. Hypertension. 1991;17:626-635.
44. Folkow B, Hallback M, Lundgren R, Sivertsson R, Weiss L. Importance of adaptive changes in vascular design for establishment of primary hypertension, studied in man and in spontaneously hypertensive rats. Circ Res. 1973;32:2-16.
45.
Fath KA, Alexander RW, Delafontain P. Abdominal
coarctation increases insulin-like growth factor I mRNA in rat
aorta. Circ Res. 1993;72:271-277.
46.
Majesky MW, Daemen MJAP, Schwartz SM.
1-Adrenergic stimulation of platelet-derived growth
factor A-chain gene expression in rat aorta. J Biol
Chem. 1990;265:1082-1088.
47.
Wilson E, Qing M, Sudhir K, Weiss RH, Ives HE.
Mechanical strain induces growth of vascular smooth muscle cells via
autocrine action of PDGF. J Cell Biol. 1993;123:741-747.
48.
Lucchesi PA, Bell JM, Willis LS, Byron KL, Corson MA,
Berk BC. Ca2+-dependent mitogen-activated
protein kinase activation in spontaneously hypertensive rat vascular
smooth muscle defines a hypertensive signal transduction
phenotype. Circ Res. 1996;78:962-970.
49.
Xin X, Yang N, Eckhart AD, Faber JE.
1A-Adrenergic receptors and mitogen-activated
protein kinase mediate increased protein synthesis by
arterial smooth muscle. Mol Pharmacol.
In press.
50. Komuro I, Kudo S, Yamazaki T, Zou Y, Shiojima I, Yazaki Y. Mechanical stretch activates the stress-activated protein kinases in cardiac myocytes. FASEB J. 1996;10:631-636.[Abstract]
51. Husken BCP, Mertens MJF, Pfaffendorf M, Van Zwieten PA. The influence of coarctation hypertension on the pharmacodynamic behavior of rat isolated conduit vessels. Blood Pressure. 1994;3:255-259.[Medline] [Order article via Infotrieve]
52.
Bell DR, Bohr DF. Endothelium in
functional aortic changes of coarctation hypertension.
Am J Physiol. 1991;260:H1187-H1193.
53.
Powell JS, Clozel J-P, Mueller RKM, Huhn H, Hefti F,
Hosang M, Baumgartner HR. Inhibitors of
angiotensin converting enzyme prevent myointimal
proliferation after vascular injury. Science. 1989;245:186-188.
54. Osterrieder W, Muller RKM, Powell JS, Clozel J-P, Hefti F, Baumgartner HR. Role of angiotensin II in injury-induced neointima formation in rats. Hypertension. 1991;18(suppl II):II-60-II-64.
55.
Daemen MJAP, Lombardi DM, Bosman FT, Schwartz
SM. Angiotensin II induces smooth muscle cell
proliferation in the normal and injured rat arterial
wall. Circ Res. 1991;68:450-456.
56.
Janiak P, Pillon A, Prost J-F, Vilaine J-P. Role
of angiotensin subtype 2 receptor in neointima
formation after vascular injury. Hypertension. 1992;20:737-745.
This article has been cited by other articles:
 |
|
 |
|
  T. Bleeke, H. Zhang, N. Madamanchi, C. Patterson, and J. E. Faber Catecholamine-Induced Vascular Wall Growth Is Dependent on Generation of Reactive Oxygen Species Circ. Res., January 9, 2004; 94(1): 37 - 45. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Xin, N. Yang, and J. E. Faber Platelet-Derived Growth Factor Inhibits alpha 1D-Adrenergic Receptor Expression in Vascular Smooth Muscle Cells In Vitro and Ex Vivo Mol. Pharmacol., December 1, 1999; 56(6): 1143 - 1151. [Abstract] [Full Text]
|
|
|
|
 |
|
 S. S. Salvi {alpha}1-Adrenergic Hypothesis for Pulmonary Hypertension Chest, June 1, 1999; 115(6): 1708 - 1719. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||