Increased Sensitivity to Endothelial Nitric Oxide (NO)
Contributes to Arterial Normotension in Mice with Vascular Smooth
Muscle-selective Deletion of the Atrial Natriuretic Peptide (ANP)
Receptor*
Karim
Sabrane
,
Stepan
Gambaryan§,
Ralf P.
Brandes¶,
Rita
Holtwick
,
Melanie
Voss
, and
Michaela
Kuhn
From the
Institute of Pharmacology and
Toxicology, Universitätsklinikum Münster, D-48149
Münster, Germany, the § Institute of Clinical
Biochemistry and Pathobiochemistry, University of Würzburg, 97080 Würzburg, Germany, and the ¶ Institute of Cardiovascular
Physiology, Klinikum der J. W. Goethe-Universität, 60590 Frankfurt/Main, Germany
Received for publication, December 23, 2002, and in revised form, March 11, 2003
 |
ABSTRACT |
Atrial natriuretic peptide (ANP) plays a
key regulatory role in arterial blood pressure homeostasis. We recently
generated mice with selective deletion of the ANP receptor, guanylyl
cyclase-A (GC-A), in vascular smooth muscle (SMC GC-A knockout (KO)
mice) and reported that resting arterial blood pressure was completely normal in spite of clear abolition of the direct vasodilating effects
of ANP (Holtwick, R., Gotthardt, M., Skryabin, B., Steinmetz, M.,
Potthast, R., Zetsche, B., Hammer, R. E., Herz, J., and Kuhn M. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7142-7147).
The purpose of this study was to clarify mechanisms compensating for the missing vasodilator responses to ANP. In particular, we analyzed the effect of the endothelial, cGMP-mediated vasodilators C-type natriuretic peptide and nitric oxide (NO). In isolated arteries from
SMC GC-A KO mice, the vasorelaxing sensitivity to sodium nitroprusside
and the endothelium-dependent vasodilator, acetylcholine, was significantly greater than in control mice. There was no difference in responses to C-type natriuretic peptide or to the activator of
cGMP-dependent protein kinase I,
8-para-chlorophenylthio-cGMP. The aortic expression of soluble GC
(sGC), but not of endothelial NO synthase or cGMP-dependent
protein kinase I, was significantly increased in SMC GC-A KO mice.
Chronic oral treatment with the NO synthase inhibitor
Nw-nitro-L-arginine methyl ester increased
arterial blood pressure, the effect being significantly enhanced in SMC
GC-A KO mice. We conclude that SMC GC-A KO mice exhibit a higher
vasodilating sensitivity to NO. This can be attributed to an enhanced
expression of sGC, whereas the expression and/or activity levels of
downstream cGMP-effector pathways are not involved. Increased
vasodilating responsiveness to endothelial NO contributes to compensate
for the missing vasodilating effect of ANP in SMC GC-A KO mice.
 |
INTRODUCTION |
Cyclic GMP-dependent modulation of vascular tone is
fundamental to the regulation of blood pressure. The levels of cGMP in vascular smooth muscle cells
(SMC)1 are regulated by the
activities of three different guanylyl cyclases (GCs): soluble GC
(sGC), the intracellular receptor for endothelial nitric oxide (NO)
(1); particulate GC-A, a specific membrane receptor for the cardiac
natriuretic peptides, atrial (ANP) and B-type (BNP) natriuretic
peptides (2, 3); and particulate GC-B, a specific receptor for the
endothelial C-type natriuretic peptide (CNP) (4, 5). Stimulation of
either GC results in the conversion of GTP to the intracellular
messenger cGMP. Subsequent increases in cellular cGMP modulate the
activity of specific cGMP-effector molecules, i.e.
cGMP-dependent protein kinase I (PKG I), ultimately leading
to decreased cytosolic calcium levels and relaxation of vascular smooth
muscle cells (6, 7). Thereby, local factors released by the vascular
endothelium (NO and CNP) and circulating hormones (ANP and BNP)
cooperate in the cGMP-mediated regulation of vascular tone. In
addition, it has been suggested that the vasodilating effect of ANP
involves endothelial GC-A and is partly mediated by the stimulation of
the local release of NO and CNP (8-11).
In the past years, the development of several monogenetic mouse models
contributed to elucidate the role of these factors and their respective
receptor-GCs in the regulation of blood pressure. In particular, mice
lacking endothelial NO synthase (eNOS) (12), ANP (13), or the ANP
receptor, GC-A (14, 15), exhibit drastic arterial hypertension,
experimental observations that emphasize the importance of
cGMP-dependent vasodilation in cardiovascular homeostasis.
Because the soluble and particulate GC/cGMP systems have complementary
roles in blood pressure homeostasis, an interaction between these
pathways to regulate cGMP levels in vascular smooth muscle cells might
represent an important physiological mechanism to control vascular
tone. In this way, an excess or deficiency in one mediator could be
compensated by the other, or conversely, the interaction may constitute
a negative feedback system that prevents overactivation of cGMP
signaling in SMC by NO and/or natriuretic peptides. Indeed, acute or
chronic alterations of endothelial NO production in human and murine
arteries resulted in reciprocal changes in the vasorelaxing responses
to ANP (16). The authors suggested that the NO/sGC system modulates the
sensitivity and/or expression levels of GC-A in a
cGMP-dependent manner (16). Vice versa, it is not clear
whether changes in the vasodilating effects of ANP modulate the
activity and/or vasodilating effects of the NO/sGC and CNP/GC-B systems.
In a recent study, we developed a new genetic mouse model in which the
GC-A receptor is selectively deleted in vascular smooth muscle cells
(SMC GC-A KO mice) (17). Intriguingly, despite the clear abolition of
the direct vasorelaxing effects of ANP, the resting blood pressure of
conscious SMC GC-A KO mice is completely normal. This was unexpected
since the decisive role of the ANP/GC-A system in the chronic
regulation of arterial blood pressure has been clearly shown by the
hypertensive phenotype of mice with generalized GC-A gene deletion (14,
15). One possibility is that in the long term setting, other
cGMP-dependent vasodilating systems, such as NO/sGC or
CNP/GC-B, compensate for the missing vasodilating effects of ANP. To
address this possibility, in the current study, we evaluated the
responsiveness of SMC GC-A KO mice to different cGMP-mediated
vasodilators. In addition, we took advantage of the selective
inhibition of GC-A expression in vascular smooth muscle cells to
dissect the specific endothelium-mediated vasodilating effects of ANP
in intact vessel preparations.
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EXPERIMENTAL PROCEDURES |
Animals and Tissue Preparations--
Mice with selective
abolition of the ANP receptor, GC-A, in vascular smooth muscle (SMC
GC-A KO mice) were generated as described (loxP/Cre recombination
system) (17). Genotypings were by Southern blot (verification of the
floxed GC-A and the Cre-deleted GC-A alleles) and PCR analyses
(detection of the SM22-Cre transgene) of tail DNA. Studies were done
with 4-month-old floxed GC-A mice (which retain normal GC-A expression
levels) and SMC GC-A KO littermates (floxed GC-A mice harboring the
SM22-Cre transgene) (17). All experimental protocols included in this
manuscript were approved by the local animal care committee and conform
with the Guide for the Care and Use of Laboratory
Animals published by the U. S. National Institutes of Health
(32). For the in vitro studies, mice were killed by cervical
dislocation. The aorta was dissected free of surrounding tissue and
used for organ chamber studies, Western blot analysis, and
determination of cGMP content and sGC activity.
In Vitro Studies of Vascular Tone--
Ring segments of the
descending thoracic aorta (luminal diameter, Ø, 2000-2200 µm) were
mounted in a myograph (model 410A; J.P. Trading, Aarhus,
Denmark) for recording of isometric wall tension (17, 18). After
a 15-min equilibration in temperated (37 °C), oxygenated (95%
O2, 5% CO2) Krebs-Ringer bicarbonate buffer,
rings were contracted with phenylephrine (10 µM; Sigma), and the relaxant response to cumulative concentrations of ANP, CNP (both obtained from Calbiochem-Novabiochem), sodium nitroprusside (SNP), acetylcholine (both from Sigma), or 8-para-chlorophenylthio-cGMP (8p-CPT-cGMP; from Biolog, Bremen, Germany) was tested. ANP was tested
in the presence and absence of the NO synthase (NOS) inhibitor, Nw-nitro-L-arginine methyl ester
(100 µM L-NAME; Sigma).
Western Blot Analysis--
To determine the expression
levels of eNOS, sGC, and PKG I, frozen aortas were homogenized and
analyzed by Western blot. Samples (20 µg of protein/lane) were
separated on 8% SDS-polyacrylamide gels and then blotted onto
nitrocellulose membrane. Membranes were first incubated with specific
antisera against PKG I (19, 20) (diluted 1:3000), sGC
1,
or sGC
1 (21) (both diluted 1:1000) or eNOS (BD
Bioscience; diluted 1:500) and then with a peroxidase-labeled anti-rabbit antibody in an ECL detection system (Amersham
Biosciences) (19, 20). For quantitative analysis, the blots were
scanned and quantified using Amersham Biosciences ImageQuant software. The antibody against PKG I was a generous gift from Dr. Suzanne Lohmann
(19).
Determination of cGMP Content in Isolated Blood
Vessels--
Aortic rings were incubated in temperated (37 °C)
Dulbecco's modified Eagle's medium (Invitrogen) containing the
phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 1 mM). After 15 min, rings were treated with either vehicle
(Dulbecco's modified Eagle's medium) or the NO donor,
3-morpholinosydnonimine (SIN-1, 10 or 100 µM; Sigma) for
an additional 5 min. Thereafter, rings were frozen in liquid nitrogen
and homogenized, and cGMP was extracted with ice-cold 70% (v/v)
ethanol. After centrifugation (13000 × g, 10 min,
4 °C), the supernatants were dried in a speed vacuum concentrator,
resuspended in sodium acetate buffer (50 mM, pH 6.0), and
acetylated, and then the cGMP contents were quantified by
radioimmunoassay (17, 20). The pellets of the ethanol extracts were
used for determination of protein content according to the method of
Bradford (17, 20).
Determination of Guanylyl Cyclase
Activity--
NO-dependent guanylyl cyclase
activity in aortic homogenates was determined (17) according to
Li et al. (22). Individual aortas were homogenized in 1 ml
of ice-cold buffer containing 50 mM Tris·HCl (pH 7.6), 1 mM EDTA, 1 mM dithiothreitol, 2 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Extracts were centrifuged for 10 min at 500 × g (4 °C) (23). To initiate cyclase activity, supernatants
(4 µg of protein) were incubated in assay buffer containing 50 mM Tris·HCl, pH 7.5, 4 mM MgCl2,
2 mM IBMX, 1 mM GTP, 7.5 mM
creatine phosphate, and 200 µg/ml creatine phosphokinase (185 units/mg) at 37 °C in the presence of the NO donor SIN-1 (10 µM). At 5 min of incubation, the reaction was stopped by
addition of ice-cold 100% (v/v) ethanol (final concentration, 70%),
and cGMP was extracted and measured as described above.
In Vivo Experiments--
The animals were housed under a 12-h
day/night cycle and fed a standard diet containing 0.6% NaCl (normal
salt conditions). Floxed GC-A mice (n = 10) and SMC
GC-A KO littermates (n = 9) were treated with the
inhibitor of NO synthase, L-NAME (~100 mg/kg/day), via drinking water
for 21 days according to published studies (24). Arterial blood
pressure and heart rate were measured in conscious mice by tail-cuff
plethysmography (Softron, Tokyo) as described previously (17, 25),
before and during oral L-NAME treatment.
Data Analysis--
Results are expressed as means ± S.E.
(n = number of animals). The EC50 for
agent-induced relaxation in aortic rings was calculated by nonlinear
regression analysis of mean contraction (% of relaxation) versus log [agent] (GraphPad Prism 1.00; GraphPad, San
Diego, CA). Statistical comparison of floxed GC-A mice (as controls) and SMC GC-A KO mice was performed by unpaired Student's t
test. The serial changes in arterial blood pressure and heart rate
before (control period) and after L-NAME treatment were analyzed by a repeated measures analysis of variance followed by Student-Newman-Keuls multiple comparisons test (GraphPad InStat software). p
values of less than 0.05 were considered statistically significant.
 |
RESULTS |
Organ Chamber Studies--
The contracting responses of aortic
rings to phenylephrine (10 µM) were not different between
genotypes. ANP (100 pM to 1 µM) induced the
complete relaxation of preconstricted aortic rings obtained from floxed
GC-A mice (controls, with normal GC-A expression levels) but had only
marginal relaxing effects on arteries from SMC GC-A KO mice
(Emax, 88 ± 3% versus 26 ± 5%)
(Fig. 1). Pretreatment of aortic rings
with the NOS inhibitor L-NAME (100 µM) completely abolished the small relaxing responses to ANP observed in aortas from
SMC GC-A KO mice, whereas relaxations in floxed GC-A mice were
unaffected. The EC50 for ANP relaxation in floxed GC-A
aortas was 6.9 ± 1.4 nM in the absence
versus 4.8 ± 1.4 nM in the presence of L-NAME (no significant difference) (Fig. 1). L-NAME pretreatment totally prevented the relaxing responses to the
endothelium-dependent vasodilator acetylcholine (1 nM to 10 µM) in both genotypes
(Emax for acetylcholine, 7 ± 3% in the presence
versus 66 ± 4% in the absence of L-NAME,
p < 0.05).

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Fig. 1.
Relaxations of isolated aortas in
response to ANP. Aortic rings from floxed GC-A (top)
and SMC GC-A KO mice (bottom) were contracted with
phenylephrine (10 µM), and then cumulative concentrations
of ANP were added. Experiments were performed in the presence
(white dots) or absence (black dots, controls) of
the NOS inhibitor L-NAME (100 µM). The vasorelaxing
effects are presented as a percentage of the phenylephrine-induced
contraction (n = 6 in each group; *, p < 0.05 versus rings in the presence of L-NAME).
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Relaxations in response to acetylcholine (1 nM to 10 µM) and to the NO donor SNP (100 pM to 10 µM) were both significantly greater in aortas from SMC
GC-A KO mice as compared with floxed GC-A mice (Fig.
2). The EC50 for
acetylcholine relaxation in floxed GC-A mice was 346 ± 60 nM, and in SMC GC-A KO mice, it was 158 ± 21 nM (p < 0.05). The EC50 for
SNP was 17.4 ± 3.8 in floxed GC-A and 6.5 ± 1.3 in SMC GC-A
KO mice (p < 0.05). In contrast, CNP (100 pM to 1 µM) and the PKG-activator 8p-CPT-cGMP
(100 nM to 100 µM) had similar effects in
aortas from both genotypes (Fig. 2). The EC50 for CNP was
93.2 ± 19 nM in floxed GC-A and 136 ± 23 nM in SMC GC-A KO mice, and for 8p-CPT-cGMP, it was
33.7 ± 13 µM versus 32.6 ± 6.6 µM (no significant difference). The lower potency of CNP
relative to that of ANP is similar to previous reports (25).

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Fig. 2.
Relaxations of isolated aortas in response to
acetylcholine (ACh), SNP, CNP, and 8p-CPT-cGMP.
Aortic rings from floxed GC-A and SMC GC-A KO mice were contracted with
phenylephrine (10 µM), and then cumulative concentrations
of test agents were added. The vasorelaxing effects are presented as a
percentage of the phenylephrine-induced contraction (n = 9 in each group; *, p < 0.05 versus
floxed GC-A).
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Western Blot Analysis--
To investigate the mechanisms mediating
the enhanced vasorelaxing responses to SNP and acetylcholine,
downstream effectors were studied. As shown in Fig.
3, the expression levels of both sGC
subunits
1 and
1 were significantly
enhanced in aortas from SMC GC-A KO mice as compared with floxed GC-A
mice. In contrast, the expression levels of eNOS and of a downstream
target for cGMP, PKG I, were not different between genotypes (Fig.
3).

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Fig. 3.
Aortic sGC, PKG I, and eNOS protein
expression. Aortic extracts from floxed GC-A and SMC GC-A KO mice
were subjected to SDS-PAGE and Western blot analysis. Top,
Western blots. Bottom, relative expression levels of sGC,
PKG I, and eNOS (fold increase of floxed GC-A). sGC 1 (80 kDa), sGC 1 (68 kDa), PKG I (78 kDa), and endothelial NOS
(135 kDa) were detected using specific antisera and a
peroxidase-labeled anti-rabbit antibody in an ECL detection system. The
expression levels of sGC (both subunits) but not PKG I or eNOS were
significantly enhanced in the aortas from SMC GC-A KO mice
(n = 9 per genotype, *, p < 0.05 versus floxed GC-A).
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cGMP Content of Aortic Rings--
The basal cGMP content in aortic
rings was slightly greater in floxed GC-A as compared with SMC GC-A KO
mice (235 ± 17 versus 179 ± 14 pmol/mg of
protein, n = 5; p < 0.05). Incubation
with SIN-1 increased cGMP content in aortic rings from floxed GC-A (at
100 µM SIN-1) and SMC GC-A KO mice (at 10 and 100 µM SIN-1) (Fig. 4). When
compared with the respective basal cGMP content of each individual
aorta, the increases of cGMP in response to SIN-1 were significantly
greater in SMC GC-A KO (6.6 ± 0.7-fold increase at 100 µM SIN-1 versus untreated controls) as
compared with floxed GC-A mice (3.4 ± 0.5-fold increase at 100 µM SIN-1; n = 5; p < 0.05) (Fig. 4).

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Fig. 4.
Effect of SIN-1 (10 or 100 µM) on cGMP content of aortic rings from
floxed GC-A and SMC GC-A KO mice. Rings were pretreated with the
phosphodiesterase inhibitor IBMX (1 mM, 15 min) and then
incubated with vehicle (Dulbecco's modified Eagle's medium) or SIN-1
for an additional 5 min in the presence of IBMX. Responses to SIN-1 are
expressed as fold increase of cGMP content as compared with parallel
vehicle-treated rings prepared from the same individual aortas
(n = 5 per genotype, *, p < 0.05 versus basal; §, p < 0.05 versus floxed GC-A).
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sGC Activity--
First, to test the linearity of the assay,
SIN-1-stimulated sGC activity was compared in samples containing 1, 2, or 4 µg of protein extracted from wild-type mouse aortas. As shown in Fig. 5A, doubling the protein
content of the samples (and thereby the amount of sGC protein) resulted
in a proportional increase of SIN-1-stimulated cGMP formation
(n = 3). Fig. 5B demonstrates that SIN-1 (10 µM)-stimulated sGC activity in protein extracts from SMC
GC-A KO aortas was significantly higher than in floxed GC-A aortas
by ~ 170% (n = 5; p < 0.05).

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Fig. 5.
Determination of vascular sGC activity.
A, linearity of the sGC assay showing correlation
between increasing amounts of aortic protein and SIN-1 (10 µM)-stimulated, NO-dependent sGC activity.
Enzymatic activity is expressed as picomoles of cGMP formed per minute
(n = 3). B, SIN-1 (10 µM)-stimulated activity of sGC in aortic homogenates
obtained from floxed GC-A and SMC GC-A KO mice. Enzymatic activity is
expressed as picomoles of cGMP formed per milligram of protein per
minute (n = 5 mice per genotype, with sGC activity
determinations performed in duplicate; *, p < 0.05 versus floxed GC-A).
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Effects of L-NAME on Blood Pressure--
To ascertain whether
increased sensitivity to endothelial NO affects chronic blood pressure
levels, conscious homozygous floxed GC-A (n = 10) and
SMC GC-A KO mice (n = 9) were treated with the NOS-inhibitor L-NAME. Systolic blood pressure was measured in awake
mice by tail-cuff plethysmography. As reported previously (17), initial
blood pressures and heart rates were not significantly different
between floxed GC-A and SMC GC-A KO mice (Fig.
6). L-NAME (100 mg/kg of body weight/day,
orally) provoked a significant rise in systolic blood pressure
levels by 13 ± 3.7 mm Hg in floxed GC-A mice (from 118 ± 2.6 mm Hg at baseline to 131 ± 3.8* mm Hg after L-NAME) and by
28 ± 3.3 mm Hg in SMC GC-A KO mice (113 ± 2.7 mm Hg at
baseline; 141 ± 1.6* mm Hg after L-NAME; *, p < 0.05 versus baseline). This was associated with a
significant decrease in heart rate (floxed GC-A mice, 623 ± 12 bpm at baseline and 541 ± 17* bpm after L-NAME; SMC GC-A KO mice,
632 ± 18 bpm at baseline and 549 ± 21* bpm after L-NAME; *,
p < 0.05 versus baseline). As shown in Fig.
6, the hypertensive but not the bradycardic response to L-NAME was
significantly greater in SMC GC-A KO mice as compared with floxed GC-A
mice.

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Fig. 6.
Systolic blood pressure
(top) and heart rate (bottom) in
floxed GC-A (n = 10) and SMC GC-A KO mice
(n = 9) before and after 21 days of oral treatment
with L-NAME (~100 mg/kg of body weight/day). Measurements were
obtained in awake mice using a tail-cuff method. Significant changes
are indicated (*, p < 0.05 versus baseline;
§, p < 0.05 versus floxed GC-A
mice).
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DISCUSSION |
In the present study, we demonstrate that mice with
selective deletion of the ANP receptor in vascular smooth muscle
exhibit an increased sensitivity to nitrovasodilators and endothelial NO but not to downstream targets of the cGMP pathway (PKG I) and other
cGMP-mediated vasodilators, such as CNP. The results indicate a close
interaction between the ANP/GC-A and NO/sGC pathways, which does not
affect the CNP/GC-B pathway.
Using the Cre/loxP gene recombination strategy, we recently generated
mice with selective deletion of the ANP receptor, guanylyl cyclase-A
(GC-A), in vascular smooth muscle cells (SMC GC-A KO mice) and reported
that chronic blood pressure was completely normal in spite of clear
abolition of the direct vasodilating effects of ANP (17). This was
unexpected because the decisive role of the ANP/GC-A system in the
chronic regulation of arterial blood pressure has been clearly shown by
the hypertensive phenotype of mice with generalized GC-A gene deletion
(14, 15). We hypothesized that the chronic reduction of blood pressure
by ANP might be mainly mediated by the other known endocrine actions of
the hormone such as the inhibition of the sympathetic and
renin-angiotensin-aldosteron systems as well as the stimulation of
renal function (2, 3). Alternatively other vasodilating systems might
be able to compensate for the missing vasodilating effects of ANP when
all other cardiovascular actions of the peptide are preserved. To
address this possibility, in the current study, we compared the
vascular expression levels of eNOS as well as the responsiveness of
floxed GC-A mice (which exhibit normal GC-A expression levels) and SMC
GC-A KO mice to different cGMP-mediated vasodilators. As shown, the
vascular expression levels of eNOS were similar in both genotypes.
Also, the vasorelaxing responses to CNP and to the membrane-permeable
cGMP analog, 8p-CPT-cGMP, were identical in aortas of floxed GC-A and
SMC GC-A KO mice. However, the latter exhibited an increased
vasorelaxing sensitivity to the endothelium-dependent
vasodilator, acetylcholine, and to the NO donor, SNP.
ANP may alter the NO-sGC-cGMP transduction cascade in several ways,
affecting the release of endothelial NO (10) or the expression as well
as the activity of sGC and targets further downstream. Since the
vasorelaxing responses to CNP and to 8p-CPT-cGMP were identical in
aortas with and without deletion of GC-A in smooth muscle cells,
alterations of downstream effectors or modulators of cGMP, such as PKG
I or phosphodiesterases (6, 7), are excluded as a cause for the
increased NO sensitivity in SMC GC-A KO mice. Indeed, Western blot
analysis showed that the vascular expression levels of PKG I, a common
downstream target for all cGMP-dependent vasodilators (6,
7), are not different in floxed GC-A and SMC GC-A KO mice. In contrast,
the vascular expression levels of the sGC
1 and
1 subunit proteins were significantly enhanced in aortas
from SMC GC-A KO as compared with floxed GC-A mice. This was concordant
with an increased effect of the NO donor, SIN-1, on both the cGMP
content of aortic rings and the enzymatic activity of sGC in aortic
homogenates from SMC GC-A KO mice. Thus, deletion of the GC-A gene in
SMC leads to increased expression of sGC, which renders the arteries
more susceptible to increases in cGMP and to vasodilation by NO.
Notably, despite the 2-fold increase in vascular sGC expression levels,
basal cGMP contents in aortas from SMC GC-A KO mice were slightly lower
as compared with floxed GC-A littermates. However, basal cGMP levels in
vascular tissues are regulated by the activity of both soluble and
particulate guanylyl cyclases (GC-A and GC-B). Therefore, it is likely
that the deletion of GC-A in smooth muscle cells accounts for the lower cGMP levels observed in unstimulated aortic rings from SMC GC-A KO mice
under in vitro conditions. The increased protein expression of sGC was also observed in aortas obtained from younger, 6-8-week-old SMC GC-A KO mice (not shown), indicating that the compensatory changes
in the NO/sGC/cGMP pathway are present already at early stages.
To determine whether the studies with isolated arteries reflected
in vivo effects on blood pressure, adult (4-month-old) mice were subjected to chronic treatment with the NO synthase inhibitor L-NAME. As shown, L-NAME significantly increased the blood pressure levels of floxed GC-A and SMC GC-A KO littermates. Remarkably, the
magnitude of blood pressure increases was significantly more pronounced
in the latter group, suggesting that increased vasodilator responsiveness to endogenous, endothelial NO indeed contributes to the
maintenance of physiological arterial blood pressure levels in mice
with abolished vasodilator responses to ANP.
Our observations somehow differ from the results by Melo et
al. (26) in ANP-deficient mice (ANP
/
). In this study, the hypertensive responses to L-NAME were not different in ANP
/
and
wild-type mice, suggesting that the synthesis of or responsiveness to
endothelial NO was not enhanced by chronic ANP deficiency. The
following are possible explanations for the divergent results: absence
of ANP (ANP
/
) may result in an increased expression of vascular SMC
GC-A, and the consequent increase in basal activity of this receptor
may prevent compensatory changes of the NO/sGC system; also,
circulating BNP levels may still activate vascular GC-A receptors in
ANP
/
mice.
Why did the deletion of SMC GC-A enhance the vasorelaxing
responsiveness to NO and not to CNP? We cannot exclude that the CNP/GC-B system was up-regulated at the level of the local, endothelial synthesis and/or secretion of CNP (11). However, we and others have
shown previously that the potency of CNP for vasodilation or reduction
of blood pressure is much lower as compared with ANP (2, 25,
27). Even more, CNP-deficient mice do not exhibit arterial hypertension
(28).2 Taken together, these
data might indicate that the CNP/GC-B system is not as crucial as the
ANP/GC-A and NO/sGC systems in the regulation of vascular tone.
Within the vascular system, not only SMC but also endothelial cells are
rich in GC-A and respond to ANP with increased production of cGMP (8,
9). In cultured endothelial cells, elevation of cGMP levels inhibits
endothelin-1 (29) and stimulates NO (10) and CNP synthesis (11). Thus,
it has been reported that ANP-induced vasodilation is partly mediated
by the endothelial release of NO (30). By selective disruption of GC-A
in smooth muscle cells (17), we generated an elegant mouse model
allowing us to dissect the specific endothelial effects of ANP in
intact vessel preparations. As shown, the vasorelaxing effect of ANP was almost completely abolished in SMC GC-A-deficient aortas. We
observed a small relaxation at higher ANP concentrations, which was
totally prevented by the inclusion of L-NAME in the organ chambers,
indicating that these responses were mediated by the ANP/GC-A-stimulated release of endothelial NO. However, the NO-mediated vasorelaxing effect of ANP observed in SMC GC-A KO arteries was rather
small as compared with ANP effects on arteries with intact SMC GC-A
expression levels. Even more, inhibition of endothelial NO synthesis by
L-NAME did not affect the vasorelaxing responses of the latter, floxed
GC-A arteries to ANP. Taken together, these observations indicate that
under normal conditions, the contribution of endothelial NO to the
vasodilating effects of ANP is minor, if any. Thus, the role of the
endothelium in the maintenance of blood pressure and volume homeostasis
by ANP remains intriguing. Many studies have shown that ANP modulates
endothelial permeability (31), an effect that might be essential for
the physiological regulation of blood volume. In our future studies, we
will take advantage of this new mouse model with SMC-specific deletion
of GC-A to elucidate how the endothelial GC-A receptor contributes to
the known actions of ANP on vascular permeability and, overall, to the
regulatory effects of this peptide on blood volume homeostasis.
In summary, we demonstrated that isolated arteries from SMC
GC-A KO mice exhibit a higher sensitivity to nitrovasodilators and
endothelial NO as compared with arteries with normal GC-A expression
levels. This effect can be attributed to an enhanced expression of sGC,
whereas the expression and/or activity levels of eNOS and of downstream
cGMP-effector pathways are not involved. Increased vasodilating
responsiveness to endogenous endothelial NO contributes to compensate
for the missing vasodilating effect of ANP in SMC GC-A KO mice. Our
study adds an important piece of information to the local, reciprocal
interactions between the ANP/GC-A and NO/sGC systems within the
vascular wall. Given that blood pressure is elevated in mice with
generalized deletion of ANP (13), GC-A (14), or eNOS (12), neither
system is able to fully compensate for the complete loss of the other.
However, their mutual interactions may moderate the hypertensive
phenotype and prevent an otherwise lethal form of hypertension.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge the excellent
technical assistance of Bernd Zetsche. The antibody against PKG I was a
generous gift from Dr. Suzanne Lohmann, Institute of Clinical
Biochemistry and Pathobiochemistry, University of Würzburg.
 |
FOOTNOTES |
*
This work was supported by the Bundesministerium für
Bildung und Forschung (Grant BMBF 01EC9801), the University of
Münster (Interdisziplinäre Klinische Forschung, Grant IZKF
B12), and the Deutsche Forschungsgemeinschaft (Grant DFG KU 1037/3) (to M. K.).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.
To whom correspondence should be addressed: Institute of
Pharmacology and Toxicology, Universitätsklinikum Münster,
Domagkstrasse 12, D-48149 Münster, Germany. Tel.:
49-251-83-52597; Fax: 49-251-83-55501; E-mail:
mkuhn@uni-muenster.de.
Published, JBC Papers in Press, March 11, 2003, DOI 10.1074/jbc.M213113200
2
Y. Ogawa, personal communication.
 |
ABBREVIATIONS |
The abbreviations used are:
SMC, smooth
muscle cells;
ANP, atrial natriuretic peptide;
BNP, B-type natriuretic
peptide;
CNP, C-type natriuretic peptide;
NO, nitric oxide;
NOS, NO
synthase;
eNOS, endothelial NO synthase;
GC-A, guanylyl cyclase-A;
sGC, soluble guanylyl cyclase;
KO, knockout;
PKG, cGMP-dependent
protein kinase;
8p-CPT-cGMP, 8-para-chlorophenylthio-cGMP;
SNP, sodium
nitroprusside;
L-NAME, Nw-nitro-L-arginine methyl ester;
IBMX, 3-isobutyl-1-methylxanthine;
SIN-1, 3-morpholinosydnonimine;
bpm, beats per minute.
 |
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Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.