(Hypertension. 2001;37:1423.)
© 2001 American Heart Association, Inc.
Scientific Contributions |
Expression and Distribution of NOS1 and NOS3 in the Myocardium of Angiotensin II–Infused Rats
From the Department of Internal Medicine, School of Medicine, State University of Campinas (UNICAMP), Campinas, SP, Brazil.
Correspondence to Kleber G. Franchini, MD, Departamento de Clínica Médica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Cidade Universitária "Zefferino Vaz," 13081-970 Campinas, SP, Brasil. E-mail franchin{at}obelix.unicamp.br
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Abstract |
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Abstract—Studies have indicated a complex functional interaction between angiotensin (Ang) II and NO in the heart. The purpose of the present study was to examine the protein expression and tissue distribution of NO synthases 1 (NOS1) and 3 (NOS3) in the myocardium of rats that underwent continuous infusion of Ang II at 2 different rates (10 and 40 ng · kg-1 · min-1) for 6 days. Mean arterial pressure increased by
15 mm Hg in rats
infused with Ang II at 40 ng · kg-1 ·
min-1, but it remained close to the values
observed in saline-infused rats (110 mm Hg) when Ang II was
infused at 10 ng · kg-1 ·
min-1. The protein expression of a 160-kDa
NOS1 and a 135-kDa NOS3 were found to increase (200%) in the
myocardium of rats infused with both subpressor and pressor
doses of Ang II. Immunohistochemistry studies showed that NOS1 and NOS3
are differentially expressed in myocardial cells. NOS1 was detected in
cardiac myocytes and in smooth muscle cells of small and large
coronary arteries, whereas NOS3 was detected in the
endothelium and in perivascular and
interstitial tissues, but NOS3 was not detected in cardiac
or smooth muscle cells. Ang II infusion enhanced the tissue
immunoreactivity of both isoforms in their specific locations but did
not change the distribution throughout the myocardium.
Myocardium staining with anti–angiotensin type
1 (AT1) receptor antibody indicated that
AT1 receptor is expressed in cardiac myocytes,
coronary smooth muscle cells, and interstitial and
perivascular tissues. Ang II infusion did not change the protein
expression and distribution of AT1 receptor in
the myocardium. These results indicate that long-term
increases in the circulating levels of Ang II modulate the protein
expression of NOS1 and NOS3 and, consequently, the function of the
local myocardial NO system.
Key Words: angiotensin II • nitric oxide • nitric oxide synthase • heart • myocardium
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Introduction |
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Angiotensin (Ang) II modulates cardiac function and cellular growth in response to physiological and pathological processes.1 Many of the short- and long-term effects of Ang II on cardiac function and structure are due to its direct action on cardiac myocytes, vascular smooth muscle cells, and cardiac fibroblasts. These effects are mediated through at least 2 different types of receptors, which are broadly distributed in cardiac cells.2 However, some of the cardiac effects of Ang II occur through the induction and release of paracrine/autocrine factors, such as transforming growth factor-ß1 and endothelin-1.3 4 In this context, in recent years, experimental evidence has indicated that Ang II and NO influence each other by interacting at various levels of regulation.5 6 This may have implications not only for the cardiac functions directly influenced by these factors but also for the pathogenesis of processes such as myocardial ischemia and fibrosis.
In general, Ang II and NO exert antagonistic effects in cellular function and growth.6 7 The cellular mechanisms responsible for this antagonism are not clear. In some systems, this interaction seems to be a simple summation of the effects of Ang II and NO.7 8 Ang II is able to activate the NO system by inducing the secretion of NO in small and large coronary arteries. Because NO attenuates the vasoconstrictor effect of Ang II, this can cause a negative feedback system to limit the stimulation by Ang II.9 The antagonism of NO on Ang II effects is also seen in the growth effect of Ang II on cardiac fibroblasts.10
The mutual regulatory influence of Ang II and NO seems to extend to gene regulation. Studies performed in angiotensinogen gene–knockout mice and in rat adrenal medulla suggest that Ang II inhibits the expression of NO synthase (NOS)1.11 12 In rats, however, long-term infusion of high doses of Ang II increases the expression of NOS1 and NOS3 in the renal cortex but reduces NOS1 expression in the renal medulla.8 The influence of Ang II on the regulation of the constitutively expressed isoforms of NOS in the myocardium remains virtually unexplored.
Thus, the present study was designed to examine the effect of long-term increases in circulating levels of Ang II on the expression, and the cardiac tissue distribution of the constitutive isoforms of NOS (ie, NOS1 and NOS3). Experiments were also performed to examine the protein expression and the tissue distribution of angiotensin type 1 (AT1) receptors in the left ventricle of rats treated or not treated with Ang II.
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Methods |
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The experiments were performed on male Wistar rats (270 to 300 g) obtained from animal facilities of the State University of Campinas (Campinas, SP, Brazil). All procedures followed the university’s guidelines for the use of animals in experimental studies.
Antibodies and Chemicals
Rabbit polyclonal antibodies raised against NOS1,
NOS3, and AT1 were purchased from Santa Cruz
Biotechnology. 125I-labeled protein A was
from Amersham. Ang II was from Calbiochem. All other reagent grade
chemicals were from Sigma.
Rat Instrumentation and
Arterial Pressure Monitoring
All surgical procedures were performed under aseptic
conditions. Rats were anesthetized with a mixture of
ketamine (70 mg/kg body wt IM) and diazepam (6 mg/kg body wt
IM) and maintained at 37°C. Tygon-tipped
polyvinyl cannulas were placed in the lower abdominal aorta and
inferior vena cava throughout the femoral artery and vein,
respectively. The cannulas were exteriorized at the back of the neck in
a 25-cm length of stainless steel spring (0.5-cm diameter) attached to
a swivel (Instech) at the top of an individual cage that allowed the
animal to move freely in its cage while being infused. The animals
received single doses of antibiotic (Pentabiótico
Veterinário, 100 mg/kg body wt) and were
allowed to recover for 5 days before the study. During this period,
0.9% saline was infused continuously through the venous catheter at a
rate of 0.5 mL/h. After this period, saline was substituted for Ang II
solutions in 2 different concentrations (10 and 40 ng ·
kg-1 ·
min-1) in the experimental animals,
whereas control animals continued to receive only saline.
Arterial pressure was monitored daily for 6 days for a 1-hour period from 3:00 to 4:00 PM. The amplified signal was beat-to-beat recorded and sampled at 100 Hz with WINDAQ-PRO data acquisition software (DATAQ Instruments).
Tissue
Homogenization
At the end of day 6 of Ang II infusion, the animals
were anesthetized, hearts were rapidly removed, and the
ventricles were minced coarsely and homogenized in 10
volumes of solubilization buffer (1% Triton-X 100; 100 mmol/L
Tris-HCl (pH 7.4); 100 mmol/L sodium pyrophosphate; 100
mmol/L sodium fluoride; 10 mmol/L EDTA; 10 mmol/L
sodium vanadate; 2 mmol/L PMSF; and 0.1 mg aprotinin/mL) at 4°C
with the polytron operated at maximum speed for 30 seconds. The
extracts were centrifuged at
10 000g at 4°C for 30
minutes, and the supernatant was used for the assay. Protein
concentrations were determined with the Bradford dye binding method.
The supernatant was treated with Laemmli’s sample buffer containing
100 mmol/L dithiothreitol and heated in a boiling water bath for 4
minutes and then subjected to SDS-PAGE (8% bis-acrylamide)
in a Bio-Rad miniature gel apparatus (Mini-Protean, Bio-Rad
Laboratories). An equal amount of total protein was used for all
samples. Electrotransfer of proteins from the gel to nitrocellulose
membrane was performed for 90 minutes at 120 V (constant).
Protein Analysis by
Immunoblotting
The nitrocellulose membrane was preincubated in
blocking buffer (5% nonfat dry milk, 10 mmol/L Tris, 150
mmol/L NaCl, and 0.02% Tween 20) overnight at 4°C. The membrane was
then incubated with anti-NOS1, anti-NOS3, or
anti-AT1 receptor antibodies diluted in 10 mL of
blocking buffer (3% BSA instead of nonfat dry milk) overnight at 4°C
and washed for 60 minutes in blocking buffer without milk or BSA. The
blots were subsequently incubated with 2 µCi of
125I-labeled protein A (30 µCi/µg) in 10
mL of blocking buffer for 2 hours at room temperature, and then washed
again for 30 minutes as described above.
125I-labeled protein A bound to the specific
antibodies was detected by autoradiography. Band
intensities were quantified by optical densitometry of the developed
autoradiographs.
Tissue Preparation for
Immunohistochemistry
Rats were heparinized, deeply anesthetized
with pentobarbital sodium, and euthanized with a lethal dose of
lidocaine. The ventricles were fixed by overnight immersion with 4%
paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.4,
and processed to inclusion in Histotec (Merck). Sections (5 µm) were
transferred to
poly-L-lysine–coated
glass slides. The endogenous peroxidase activity was
blocked by treatment with 0.03%
H2O2 in 0.1 mol/L PBS at
room temperature for 30 minutes. The sections were preincubated in
blocking buffer (5% nonfat dry milk on 0.1 mol/L PBS) for 45 minutes
at 37°C, followed by overnight incubation with the primary antibodies
anti-NOS1, anti-NOS3, and anti-AT1 (1:50) at
4°C. The sections were extensively rinsed in 0.05 mol/L PBS and
incubated with peroxidase-conjugated secondary antibodies (1:100) for 2
hours at 25°C. After washing as above, sections were subjected for 5
minutes to freshly prepared diaminobenzidine that contained
H2O2 (0.8%). Secondary
antibody specificity was tested in a series of positive and negative
control measurements. In the absence of primary antibodies, application
of secondary antibodies (negative controls) failed to produce any
significant staining.
Statistical Methods
Data are mean±SEM of absolute (arterial
pressure) or percent (blots) values. Differences among mean values were
tested with a 2-way ANOVA for repeated measurements. Bonferroni’s
multiple-range test was used as a post hoc analysis if the
probability from the F test was
<0.05.
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Results |
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Effect of Ang II Infusion on Arterial Pressure
Figure 1 summarizes the effects of saline and Ang II infusion on mean arterial pressure over the course of a 6-day period (saline, n=7; 10 ng · kg-1 · min-1 Ang II, n=8; 40 ng · kg-1 · min-1 Ang II, n=7). The mean arterial pressure of saline-infused rats remained stable at
110 mm Hg during the experimental period. No significant
change was observed in mean arterial pressure of rats
infused with Ang II at 10 ng · kg-1 ·
min-1, but rats infused with Ang II at 40
ng · kg-1 ·
min-1 showed a sustained increase in mean
arterial pressure of 15 mm Hg compared with
saline-infused rats.
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Effect of Ang II Infusion on Myocardial
Expression of NOS1, NOS3, and AT1
Representative Western blots and the
average values (n=5) of densitometric readings of blots obtained with
anti-NOS1 and anti-NOS3 antibodies are shown in
Figure 2A and 2B. Single bands of 160 and 135 kDa were
observed in blots of myocardial homogenates stained with
anti-NOS1 and anti-NOS3 antibodies, respectively. Infusion of Ang II
increased the protein expression levels of both NOS1 and NOS3 by
200%. Ang II infused at 10 and 40 ng ·
kg-1 ·
min-1 produced similar increases in the
protein expression levels of NOS1 and NOS3.
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A single 60-kDa band was observed in Western blots of the myocardium homogenates stained with anti-AT1 antibody (Figure 2C). Comparable protein expression levels of AT1 receptor were observed in saline- and Ang II–infused rats.
Effect of Ang II Infusion on Myocardial
Distribution of NOS1, NOS3, and AT1
Figure 3 shows the NOS1 staining in the left ventricle of
saline-
(Figure 3A and 3C) and 10 ng ·
kg-1 ·
min-1 Ang II–infused rats
(Figure 3B and 3C). NOS1 staining was detected in cardiac
myocytes
(Figure 3A and 3B, arrows) and in the smooth muscle cells of
small and large coronary vessels
(Figure 3C and 3D, arrows and asterisks). No significant
staining for NOS1 was detected in the endothelium or in
the perivascular tissues
(Figure 3C and 3D). Remarkable increases of NOS1 staining
intensity were observed in cardiac myocytes and in the smooth muscle
cells of coronary arteries of rats infused with Ang II. This
effect was similar for Ang II infused at rates of 10 and 40 ng ·
kg-1 ·
min-1 (data not shown).
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NOS3 staining was detected (Figure 4A and 4B) in the endothelium (arrowheads) and in perivascular and interstitial tissues (asterisks) of myocardial small and large coronary arteries. Virtually no reaction product was detected within the media of coronary arteries (Figure 4A and 4B) or cardiac myocytes (data not shown). Infusion of Ang II increased the staining intensity of both the perivascular and interstitial tissues and apparently left the endothelium staining unchanged. Again, this effect was similar for both Ang II infusion rates (data not shown).
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Figure 5A and B shows the AT1 receptor staining in the myocardium of saline- and Ang II–infused rats. AT1 receptor was detected in cardiac myocytes (arrows), in the smooth muscle cells of small and large coronary arteries (arrowheads), and in the perivascular and interstitial tissues. No remarkable difference in the intensity or the distribution of the signal was detected in saline- or Ang II–infused rats (data not shown).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The present study showed that long-term infusion of Ang II enhances the protein expression of NOS1 and NOS3 in the rat myocardium. This effect was not dependent on the pressor effect of Ang II because similar increases in the myocardial NOS1 and NOS3 protein levels were found in rats infused with subpressor and pressor doses of this hormone. Myocardial staining with specific antibodies against NOS1 and NOS3 confirmed and extended the observation of Western blot experiments to show that NOS1 and NOS3 are differentially expressed in myocardial cells. NOS1 was detected in cardiac myocytes and in smooth muscle cells of small and large coronary arteries, but it could not be detected in the endothelium or perivascular and interstitial tissues. However, NOS3 was detected in the endothelium and perivascular and interstitial tissues, but it was not detected in cardiac myocytes or in coronary smooth muscle cells. Ang II infusion enhanced the tissue immunoreactivity for both NOS1 and NOS3 in their specific locations, but it did not change the distribution of these isoforms in myocardial structures. Finally, the myocardium staining with anti-AT1 antibody indicated that the AT1 receptor, the major effector of cardiac effects of Ang II, is expressed in cardiac myocytes, smooth muscle cells of coronary vessels, and perivascular and interstitial tissue. These data are compatible with the notion that Ang II modulates the protein expression of NOS1 and NOS3 and, consequently, the function of the NO system in the myocardial cells.
Although the results of the present study indicate that Ang II may regulate the expression of NOS1 and NOS3 in the rat myocardium, the mechanisms responsible for this phenomenon were not explored in the present study. However, because the effects of Ang II on NOS1 and NOS3 protein expression were demonstrated to be independent of the pressor effect of this hormone, it is reasonable to ascribe the enhanced expression of these NOS isoforms to the action of Ang II on myocardial cells, either directly or indirectly through the local release of autocrine/paracrine factors.
The mechanisms for the regulation of NOS1 and NOS3
expression in the myocardium are virtually unknown.
However, the NOS1 promoter region has
cis-acting elements such as
AP-2, transcriptional enhancer factor-1, cAMP response binding element,
and nuclear factor-
B, whereas NOS3 promoter region has Sp1, GATA,
and cAMP-responsive
element.13 Accordingly, Ang
II regulates the activity of transcription factors such as nuclear
factor-B and GATA-4 in vascular smooth muscle cells and cardiac
myocytes,
respectively.14 15
These mechanisms could be implicated in the regulation of NOS1 and NOS3
expression during Ang II infusion. In addition, Ang II upregulates the
cardiac expression of various growth factors such as transforming
growth factor-ß1, platelet-derived growth
factor, and fibroblast growth factor, which potentially can induce gene
expression, including the constitutive isoforms of
NOS.13
Although NOS1 and NOS3 are known to be expressed in the heart, the cellular distribution of these 2 isoforms is still not clear.13 The anatomic distribution and physiological roles of NOS1 in the heart have been the subjects of relatively few reports to date. Early reports indicated that NOS1 is expressed exclusively in the myocardial neurons.16 More recent studies, however, demonstrated that cardiac myocytes, in addition to the neurons, indeed stain for NOS1.17 Our present data show that in addition to cardiac myocytes, NOS1 is also expressed in smooth muscle cells of coronary arteries. This localization of NOS1 in cardiac and smooth muscle cells may indicate a tissue-specific regulation. Accordingly, it is now well accepted that cardiac, skeletal, and smooth muscle cells express µNOS1, an elongated splice variant of NOS1.18 19 Whether or not the product detected in the present study is the µNOS1 isoform needs further study.
The results of the present study show a remarkable difference in the distribution of NOS1 and NOS3 in the myocardial cells. In addition to the expected endothelial location of NOS3, it was also detected in the interstitial and perivascular tissues. Increases in the staining produced by Ang II infusion were easily detected in the interstitial and perivascular tissues, but no staining could be detected in the endothelium. The reason for the absence of detectable changes in the NOS3 staining in the endothelium could be related to the narrow space occupied by the endothelial cell, which makes conclusions difficult in regard to changes in NOS3 expression by immunohistochemical analysis. The increases of NOS3 protein expression in cardiac interstitial and perivascular tissues induced by Ang II could be due to the well-known effect of angiotensin II on interstitial tissue proliferation and fibrosis.
NOS3 was not detected in significant amounts in cardiac myocytes. Although this result agrees with some of the early studies, it contrasts with more recent studies showing that cardiac myocytes indeed express NOS3.17 The reason for this discrepancy is not clear but could be related to antibody specificity against certain isoforms of NOS3 in cardiac myocytes.
Finally, we have shown that AT1 receptor is also expressed in cardiac and vascular smooth muscles, as well as in interstitial and perivascular tissues, the structures in which the increases in protein expression of NOS1 and NOS3 were detected. This suggests that Ang II could enhance NOS 1 and NOS3 expression via AT1 receptor. However, the mediation via the angiotensin type 2 receptors is also possible.
In conclusion, the present study demonstrates that long-term infusion of Ang II is accompanied by an increase in the protein expression of NOS1 and NOS3 in the cells of rat myocardium, independent of changes in arterial pressure. The present data also provide evidence that NOS1 and NOS3 are differentially distributed in cardiac myocytes, coronary vessels, and interstitial tissue and that these NOS isoforms are upregulated by Ang II in these specific locations. The Ang II upregulation of NOS1 and NOS3 in myocardial cells could account for the impairment of the direct contractile and growth effects of this hormone on vascular smooth muscle cells, cardiac myocytes, and fibroblasts when plasma or tissue levels of Ang II are increased. This may favor functions such as local blood flow, modulation of oxygen consumption, and inhibition of fibroblasts, vascular smooth muscle cells, and cardiac myocyte growth effects of Ang II.
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Acknowledgments |
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This study was sponsored by grants from Fundação de Auxílio à Pesquisa do Estado de São Paulo (FAPESP) (Proc. 98/11403–7) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Proc. 521098/97-1).
Received September 7, 2000; first decision October 2, 2000; accepted December 4, 2000.
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