(Hypertension. 2001;37:1141.)
© 2001 American Heart Association, Inc.
Scientific Contributions |
Angiotensin-(1-7) Downregulates the Angiotensin II Type 1 Receptor in Vascular Smooth Muscle Cells
From The Hypertension and Vascular Disease Center, Wake Forest University School of Medicine, Winston-Salem, NC.
Correspondence to E. Ann Tallant, PhD, The Hypertension and Vascular Disease Center, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1032. E-mail atallant{at}wfubmc.edu
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Abstract—Angiotensin (Ang)-(1-7) is a biologically active peptide of the renin-angiotensin system that has both vasodilatory and antiproliferative activities that are opposite the constrictive and proliferative effects of angiotensin II (Ang II). We studied the actions of Ang-(1-7) on the Ang II type 1 (AT1) receptor in cultured rat aortic vascular smooth muscle cells to determine whether the effects of Ang-(1-7) are due to its regulation of the AT1 receptor. Ang-(1-7) competed poorly for [125I]Ang II binding to the AT1 receptor on vascular smooth muscle cells, with an IC50 of 2.0 µmol/L compared with 1.9 nmol/L for Ang II. The pretreatment of vascular smooth muscle cells with Ang-(1-7) followed by treatment with acidic glycine to remove surface-bound peptide resulted in a significant decrease in [125I]Ang II binding; however, reduced Ang II binding was observed only at micromolar concentrations of Ang-(1-7). Scatchard analysis of vascular smooth muscle cells pretreated with 1 µmol/L Ang-(1-7) showed that the reduction in Ang II binding resulted from a loss of the total number of binding sites [Bmax 437.7±261.5 fmol/mg protein in Ang-(1-7)–pretreated cells compared with 607.5±301.2 fmol/mg protein in untreated cells, n=5, P<0.05] with no significant effect on the affinity of Ang II for the AT1 receptor. Pretreatment with the AT1 receptor antagonist L-158,809 blocked the reduction in [125I]Ang II binding by Ang-(1-7) or Ang II. Pretreatment of vascular smooth muscle cells with increasing concentrations of Ang-(1-7) reduced Ang II–stimulated phospholipase C activity; however, the decrease was significant (81.2±6.4%, P<0.01, n=5) only at 1 µmol/L Ang-(1-7). These results demonstrate that pharmacological concentrations of Ang-(1-7) in the micromolar range cause a modest downregulation of the AT1 receptor on vascular cells and a reduction in Ang II–stimulated phospholipase C activity. Because the antiproliferative and vasodilatory effects of Ang-(1-7) are observed at nanomolar concentrations of the heptapeptide, these responses to Ang-(1-7) cannot be explained by competition of Ang-(1-7) at the AT1 receptor or Ang-(1-7)–mediated downregulation of the vascular AT1 receptor.
Key Words: angiotensin II • muscle, smooth, vascular • receptors, angiotensin-(1-7)
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Introduction |
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Angiotensin (Ang)-(1-7), the amino-terminal angiotensin heptapeptide, is an important biologically active peptide of the renin-angiotensin system (RAS).1 Ang-(1-7) plays a role in vascular regulation that is distinct and often opposite that of angiotensin II (Ang II). Furthermore, these opposing effects of Ang II and Ang-(1-7) occur at similar concentrations of the 2 angiotensin peptides. For example, in contrast to the mitogenic effects of nanomolar Ang II, Ang-(1-7) inhibits vascular smooth muscle cell (VSMC) growth with an IC50 value in the nanomolar range.2 Ang-(1-7) reduces neointimal formation after vascular injury and increases urinary prostaglandin release at concentrations of the heptapeptide 2- to 3-fold higher than the endogenous circulating concentration of Ang-(1-7) or Ang II.3 4 Nanomolar concentrations of Ang-(1-7) increased arachidonic acid release and prostacyclin production in rabbit VSMCs, with maximal effects observed at 100 nmol/L Ang-(1-7).5 Ang-(1-7) stimulated prostaglandin (PG)E2 and prostacyclin release from both porcine and rat VSMCs with EC50 values in the nanomolar range.6 7 Finally, Ang-(1-7) caused the vasodilation of rabbit renal afferent arterioles at nanomolar concentrations of the heptapeptide.8 In addition, the actions of endogenous Ang-(1-7) unmasked after converting enzyme inhibition9 or a low-salt diet10 appear to occur at submicromolar concentrations of the heptapeptide. These results indicate that Ang-(1-7) has both vasodilatory and antiproliferative effects that are observed at subpharmacological concentrations of the heptapeptide.
In contrast, various effects of Ang-(1-7) were reported at pharmacological doses of the heptapeptide. We observed a rapid transient increase in pressure preceding a more prolonged depressor response when high doses of Ang-(1-7) were injected into pithed rats.11 The pressor, but not the depressor, component of this response was blocked by the AT1 receptor antagonist losartan, suggesting that high concentrations of Ang-(1-7) may stimulate the AT1 receptor to increase blood pressure. Mahon et al12 reported that micromolar concentrations of Ang-(1-7) caused a rightward shift in the dose-contractile response to Ang II in rabbit aortic rings and a concentration-dependent decrease in the maximal response, with a pA2 of 5.5, and suggested that Ang-(1-7) was an antagonist at the AT1 receptor at micromolar concentrations of the heptapeptide. Roks et al13 also demonstrated that Ang-(1-7) at a concentration of 10 µmol/L reduced Ang I– and Ang II–induced contraction of human internal mammary arteries, suggesting that micromolar Ang-(1-7) was an antagonist at the AT1 receptor. Because Ang-(1-7) competes for binding to the vascular AT1 receptor at micromolar concentrations,7 pharmacological concentrations of Ang-(1-7) may oppose or counteract the actions of Ang II through competitive antagonism. Alternatively, Ang-(1-7) could oppose the vasoconstrictive and proliferative effects of Ang II through downregulation or desensitization of the AT1 receptor. This study was designed to determine whether Ang-(1-7) causes a downregulation of the AT1 receptor or desensitizes responses to Ang II by direct actions at the AT1 receptor.
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Methods |
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Materials
[125I]Sodium iodide was obtained from Amersham Pharmacia, and myo-[3H]inositol was obtained from American Radiolabeled Chemicals, Inc. DMEM/Ham’s F-12 (DMEM/F12), inositol-free DMEM, FBS, penicillin, streptomycin, and trypsin/EDTA were obtained from GIBCO. Ang II and Ang-(1-7) were purchased from Bachem, Inc. The AT1 receptor antagonists losartan, EXP 3174, and L-158,809 were kindly provided by Merck and Co, Inc, and the AT2 receptor antagonist PD 123319 was a gift from Parke-Davis. All other chemicals were purchased from Sigma Chemical Co.
Cell Culture Procedures
Aortic VSMCs were isolated from 10- to 12-week-old
male Hannover Sprague-Dawley rats bred and raised at the
ALACC-accredited animal facility of Wake Forest University School of
Medicine. Primary cultures of VSMCs were obtained from aortic explants
as previously described.2 The
cells were cultured in DMEM/F12 supplemented with 10% FBS, 100 µg/mL
penicillin, and 100 U/mL streptomycin and maintained at 37°C in a
humidified CO2 incubator (5%
CO2 and 95% air). Confluent monolayers of VSMCs
from passages 4 to 9 were used in all
experiments.
Measurement of
[125I]Ang II Binding
Ang II was iodinated according to a
modified chloramine T procedure, and the monoiodinated form
was purified through HPLC according to previously established
procedures.14
[125I]Ang II binding was measured by
incubating confluent monolayers of VSMCs in binding buffer (PBS:
50 mmol/L NaHPO4, 0.15 mol/L NaCl, pH 7.4,
with 0.2% fatty acid–free BSA, 2.5 mmol/L EGTA, and 5
mmol/L MgCl2) containing
[125I]Ang II. Nonspecific binding was
determined in the presence of 10 µmol/L unlabeled Ang II. After a
1-hour incubation at room temperature, binding reactions were
terminated by aspirating the binding buffer from individual wells and
washing each well with ice-cold PBS (2 mL/wash). Cellular proteins were
solubilized in 2N NaOH, and cell-associated radioactivity was
quantified with 
-spectroscopy. Protein was measured according to the
Lowry method15 with BSA used
as the standard.
Various types of binding studies were conducted to determine competition by peptides and antagonists, the effect of pretreatment with angiotensin peptides on subsequent Ang II binding, and saturation isotherms to calculate binding constants, as described here.
First, competition studies were performed by incubating VSMCs with increasing concentrations (from 10-11 to 10-5 mol/L) of each peptide or with 10 µmol/L losartan, EXP 3174, or PD 123319 in the presence of 0.3 nmol/L [125I]Ang II.
Second, the effect of pretreatment with Ang-(1-7) or Ang II on subsequent binding of [125I]Ang II to VSMCs was determined through preincubation with increasing concentrations of peptide in Hanks’ balanced salt solution for 30 minutes at room temperature. After this preincubation, surface-bound Ang-(1-7) or Ang II was removed with a 5-minute treatment with ice-cold 50 mmol/L glycine/50 mmol/L HCl, pH 3.0, before the measurement of [125I]Ang II binding.
Third, saturation isotherms were constructed after treatment (30 minutes at room temperature) of VSMCs with vehicle or 1 µmol/L Ang-(1-7). Surface-bound Ang-(1-7) was removed with acidic glycine, and acid-stripped cells were incubated with increasing concentrations of [125I]Ang II (0.075 to 2.4 nmol/L) for 1 hour at room temperature.
Measurement of
Phosphoinositide-Specific Phospholipase C
Phospholipase C (PLC) activity was assayed by the
production of 3H-labeled inositol
phosphates in VSMCs prelabeled with
myo-3H-inositol,
as previously described.16
Confluent monolayers of VSMCs in 6-well culture plates were labeled for
72 hours with
myo-3H-inositol
(2 µCi/well) in inositol-free DMEM that contained 0.5% FBS. The
radiolabeled VSMCs were preincubated with increasing concentrations of
Ang-(1-7) (0.1 to 10 µmol/L) in HEPES-buffered Krebs-Ringer solution
(125 mmol/L NaCl, 5 mmol/L KCl, 1.2 mmol/L
MgSO4, 6 mmol/L glucose, 1 mmol/L
CaCl2, and 25 mmol/L HEPES, pH 7.4) and
10 mmol/L LiCl2 for 30 minutes at 37°C.
The cells were then treated with 10 nmol/L Ang II, and the reaction was
continued for 30 minutes at 37°C. Inositol phosphates were extracted
with ice-cold perchloric acid, separated through ion exchange
chromatography, and quantified with liquid
scintillation spectroscopy as previously
described.16
Statistics
All data are expressed as the mean±SEM of 
3
experiments. Competition binding data were analyzed by
nonlinear regression with the computer program Prism (GraphPad).
Saturation isotherms were analyzed with the EBDA/LIGAND
computer program (Elsevier-BIOSOFT). Repeated measures 1-way ANOVA or
ANOVA with Dunnett’s post hoc test were used to compare
treatment groups. The criterion for statistical significance was
P<0.05.
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Results |
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Competition for the AT1 Receptor by Ang-(1-7)
VSMCs were incubated with increasing concentrations of Ang II and Ang-(1-7) to determine whether Ang-(1-7) competed for [125I]Ang II binding to VSMCs. As shown in Figure 1, Ang-(1-7) competed poorly for binding to the AT1 receptor on VSMCs isolated from the thoracic aorta of Sprague-Dawley rats with an IC50 value of 2.0 µmol/L compared with an IC50 value of 1.9 nmol/L for Ang II. The [125I]Ang II binding to VSMCs was characterized as AT1 based on
95% competition of specific binding by either 10 µmol/L
concentration of the AT1 receptor
antagonist losartan or EXP 3174 and no competition
by 10 µmol/L PD 123319, an AT2 receptor
antagonist
(Figure 1).
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Regulation of the AT1
Receptor by Ang-(1-7)
Ang II binds to AT1 receptors on
VSMCs and induces a rapid internalization of the Ang
II-AT1 receptor complex. Because we observed
that Ang-(1-7) binds to the vascular AT1
receptor, albeit at a 1000-fold higher concentration than Ang II,
Ang-(1-7) binding to the AT1 receptor could
reduce subsequent responses to Ang II by causing internalization and
subsequent loss of membrane AT1 receptors. To
test this hypothesis, VSMCs were pretreated with increasing
concentrations of Ang-(1-7) before the measurement of
[125I]Ang II binding to determine whether
treatment with Ang-(1-7) downregulates the AT1
receptor. As a control, cells were also pretreated with Ang II. After a
30-minute pretreatment with either Ang-(1-7) or Ang II, surface-bound
peptide was removed through treatment with acidic glycine and
[125I]Ang II binding was measured. Ang II
caused a dose-dependent reduction in total binding, with a significant
reduction of 51.4±10.5%
(P<0.05) of total binding at
10 nmol/L
(Figure 2). Ang-(1-7) also caused a similar reduction in Ang
II binding, with significant inhibition of 36.1±7.8% of
[125I]Ang II binding at 1 µmol/L
Ang-(1-7). A higher concentration of Ang-(1-7), up to 10 µmol/L,
caused no further reduction in [125I]Ang
II binding.
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To determine whether Ang-(1-7) reduced binding to the vascular AT1 receptor by increasing receptor affinity or by reducing the total number of AT1 receptors, VSMCs were pretreated with 1 µmol/L Ang-(1-7) for 30 minutes. The cells were subsequently treated with acidic glycine to remove surface-bound Ang-(1-7) and incubated with increasing concentrations of [125I]Ang II to saturate the receptor. Specific [125I]Ang II binding was measured and receptor density and affinity were determined through Scatchard analysis. Pretreatment with Ang-(1-7) had no significant effect on the affinity of Ang II for the AT1 receptor (Table). In contrast, pretreatment with 1 µmol/L Ang-(1-7) for 30 minutes caused a significant 66±8% decrease in the Bmax of Ang II receptors on VSMCs.
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Effect of AT1 Receptor
Antagonism on AT1 Receptor
Downregulation
The AT1 receptor
antagonist L-158,809 was used to further demonstrate that
the reduction in [125I]Ang II binding by
Ang-(1-7) was due to a direct interaction of the heptapeptide with the
AT1 receptor. VSMCs were treated with 10 nmol/L
L-158,809 before the addition of 1 µmol/L Ang-(1-7) or 100 nmol/L Ang
II. After 30 minutes at room temperature, the cells were treated with
acidic glycine to remove surface-bound peptide and
antagonist, and the amount of
[125I]Ang II binding was measured.
Pretreatment with 1 µmol/L Ang-(1-7) reduced Ang II binding to the
AT1 receptor to 84.3±2.2% of total binding.
The inclusion of 10 nmol/L L158,809 prevented the Ang-(1-7)–induced
reduction in [125I]Ang II binding
(Figure 3). Pretreatment with 100 nmol/L Ang II also reduced
subsequent binding to the AT1 receptor to
74.2±7.0% of total binding, which was prevented by the inclusion of
the AT1 receptor antagonist
L-158,809. Pretreatment with the antagonist alone followed
by treatment with acidic glycine had no significant effect on
[125I]Ang II binding to the
AT1 receptor, as would be expected with this
antagonist.
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Effect of Ang-(1-7) on Ang II–Induced PLC
Activity
Ang II significantly stimulated inositol phosphate
release from VSMCs, measured as the release of total inositol
phosphates from cells prelabeled with
myo-3H-inositol.
A 30-minute incubation of VSMCs with Ang II (10 nmol/L) increased
phosphoinositide-specific PLC activity by 102.6±19.6%
above basal levels (P<0.05,
n=5). A similar incubation with 1 µmol/L Ang-(1-7) had no effect on
inositol phosphate release (85.8±8.7% of basal level, n=5). However,
a 30-minute pretreatment with concentrations of Ang-(1-7) of 0.1 to 10
µmol/L, followed by a 30-minute incubation with 10 nmol/L Ang II,
resulted in a dose-dependent decrease in Ang II–stimulated PLC
activity, with a significant decrease to 81.2±6.4% above basal levels
(P<0.01, n=5) at 1 µmol/L
Ang-(1-7) and to 38.4±7.0% above basal levels
(P<0.01, n=5) at 10
µmol/L Ang-(1-7) as shown in
Figure 4.
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Discussion |
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The pretreatment of VSMCs with Ang-(1-7) followed by treatment with acidic glycine to remove surface-bound peptide reduced subsequent binding of Ang II to the AT1 receptor. However, the decrease in Ang II binding was detected only at micromolar concentrations of the heptapeptide, which is in agreement with the micromolar affinity of Ang-(1-7) for the AT1 receptor. The reduction in Ang II binding by Ang-(1-7) resulted from a decrease in the number of receptors available for subsequent binding with no significant effect on receptor affinity. The downregulation of the Ang II receptor by Ang-(1-7) was blocked by pretreatment with the AT1 receptor antagonist L-158,809, demonstrating that micromolar concentrations of Ang-(1-7) have direct effects at the AT1 receptor to reduce the number of receptors available for subsequent binding. Finally, Ang-(1-7), at a concentration of 1 µmol/L, significantly attenuated the Ang II–mediated stimulation of PLC to reduce the production of inositol phosphates. These results clearly demonstrate that the short-term treatment of VSMCs with pharmacological concentrations of Ang-(1-7) downregulates the vascular AT1 receptor.
Although 1 µmol/L Ang-(1-7) alone did not activate
PLC in VSMCs, it did reduce subsequent binding of Ang II to the
AT1 receptor and attenuated activation of
AT1-coupled cellular responses by Ang II. The
AT1 receptor on VSMCs is rapidly internalized on
stimulation by Ang II, a phenomenon accompanied by a reduction in
subsequent responses. This suggests that Ang-(1-7), at micromolar
concentrations, binds to the AT1 receptor to
induce receptor internalization without activating subsequent cellular
responses. Previous work with substance P and serotonin
receptors clearly indicates that receptor internalization can occur
independent of changes in functional
responsiveness.17 18 19
Studies by several investigators suggest that different motifs in the
structure of the AT1 receptor are required for
receptor internalization compared with intracellular signal generation
by Ang
II.20 21 22
The truncation of a section of the carboxyl terminus of the
AT1A receptor prevented receptor internalization
without affecting coupling to
PLC.21 23 A point
mutation in the AT1 receptor that eliminates Ang
II coupling to PLC (Asp74
Asn) did
not block receptor
internalization.22 In
addition, the potent antagonist
[Sar1,Ile8]Ang
II, a sarcosine analog of Ang II, was internalized but did not
activate PLC in Chinese hamster ovary cells transfected with
the AT1 receptor, as well as in adrenal
medullary
cells.22 24 This
suggests that Ang-(1-7), as well as sarcosine analogs of Ang II, bind
to and activate the AT1 receptor to
produce receptor internalization without coupling to G proteins to
activate PLC. In contrast, the nonpeptide
antagonist L-158,809 did not cause internalization in our
study. Thus, it is possible that high concentrations of Ang-(1-7) and
the peptide antagonists produce receptor internalization
through weak agonist properties. This would also be consistent
with our observation of a weak pressor response to high doses of
Ang-(1-7) in the pithed rat, an effect that was mediated by an
AT1
receptor.11
Mahon et al12 suggested that Ang-(1-7) is a noncompetitive antagonist at AT1 receptors in rabbit aortic rings, causing a rightward shift in the dose-response contraction curve to Ang II administered 60 minutes after Ang-(1-7). However, the concentrations of Ang-(1-7) used in their study ranged from 1 to 30 µmol/L. Roks et al13 also suggested that 10 µmol/L Ang-(1-7) was antagonistic at the AT1 receptor in human VSMCs. This would be consistent with our observation that Ang-(1-7) had an IC50 value of 2 µmol/L at the AT1 receptor in VSMCs. Our evidence suggests that short-term exposure of cells to micromolar levels of Ang-(1-7) downregulates the AT1 receptor over a time frame consistent with receptor internalization. Thus, when Ang II is given after a prior exposure to Ang-(1-7) (as we did in our study) or in the presence of a large excess of Ang-(1-7) (as in studies described by Roks et al13 and Mahon et al12 ), one might expect antagonist actions of Ang-(1-7). However, endogenous circulating levels of Ang-(1-7), as well as tissue concentrations of the peptide, are in the picomolar range.25 26 27 Although treatment with ACE inhibitors increased circulating levels of Ang-(1-7) from 9- to 25-fold,25 28 there is no evidence showing an increase in endogenous Ang-(1-7) levels to the micromolar range. In addition, the majority of the in vivo and in vitro actions of Ang-(1-7) occur at doses lower than 1 to 30 µmol/L. Because Ang-(1-7) downregulated Ang II binding and receptor activation only at micromolar concentrations, the vasodilatory and antiproliferative responses that occur at nanomolar concentrations of the heptapeptide cannot be explained by antagonist effects of Ang-(1-7) at the AT1 receptor. Ueda et al29 recently reported that the infusion of Ang-(1-7) antagonized the Ang II–mediated vasoconstriction of human forearm resistance vessels, suggesting that Ang-(1-7) is an endogenous antagonist of the human AT1 receptor. However, the authors reported that Ang-(1-7) significantly reduced the response to Ang II at a dose of 10 nmol/L, a concentration significantly lower than the IC50 value for the AT1 receptor (>1 µmol/L). In light of our observations, the responsiveness to Ang-(1-7) in the study by Ueda et al29 may be due to activation of a non-AT1 receptor by Ang-(1-7) and the release of a compensatory vasodilator such as NO or prostaglandins.
In addition, evidence from several studies clearly shows that the actions of Ang-(1-7) are independent of AT1 receptor activation. The vasodilation of canine or porcine coronary arteries induced by Ang-(1-7) was prevented by removal of the endothelium or by blockade of NO release,30 31 suggesting that Ang-(1-7) stimulated NO production to counter the vasoconstrictor effects of Ang II. These effects were not blocked by AT1 or AT2 receptor antagonists. Muthalif et al5 showed that Ang-(1-7) stimulated arachidonic acid release from rabbit VSMCs, an effect that was partially prevented by [D-Ala7]Ang-(1-7), the selective Ang-(1-7) receptor antagonist, or by the AT2 antagonist PD 123319 but not by an AT1 receptor antagonist. Ang-(1-7) caused the vasodilation of renal afferent arterioles, an effect blocked by [D-Ala7]Ang-(1-7).8 Further, Ang-(1-7) blocked mitogen-stimulated growth of rat VSMCs, responses that were not reversed by an AT1 or AT2 receptor antagonist2 but were prevented by the selective Ang-(1-7) receptor antagonist [D-Ala7]Ang-(1-7),32 suggesting that Ang-(1-7) activates a unique receptor on VSMCs to counteract the mitogenic response to Ang II. These in vitro studies are further supported by observations in combined ACE inhibitor/losartan–treated rats where a monoclonal antibody to Ang-(1-7)9 or [D-Ala7]Ang-(1-7)33 unmasked a role for endogenous Ang-(1-7), working in part through prostaglandins, in the blood pressure–lowering effects.34 Furthermore, there is evidence for a [D-Ala7]Ang-(1-7)–sensitive, non-AT1, non-AT2 receptor in the aorta32 and mesentery34 of the animals treated with ACE inhibitors and losartan. Thus, Ang-(1-7) has unique effects in the vasculature that are coupled to a non-AT1 receptor and do not result from antagonism of the AT1 receptor.
In the present study, we showed that prior treatment of VSMCs with Ang-(1-7) attenuated [125I]Ang II binding to the AT1 receptor and reduced Ang II–stimulated PLC activation. However, significant downregulation of Ang II binding and Ang II–mediated responsiveness occurred only at micromolar concentrations of Ang-(1-7), in agreement with its IC50 value for competition at the AT1 receptor. These results suggest that at micromolar concentrations, Ang-(1-7) acts as a weak agonist at the AT1 receptor to downregulate the receptor and to reduce subsequent cellular responses to Ang II.
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Acknowledgments |
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This work was supported in part by grants HL-51952 and NS-31664 from the National Institutes of Health and a Grant-in-Aid from the American Heart Association, North Carolina Affiliate.
Received September 7, 2000; first decision October 2, 2000; accepted October 9, 2000.
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