Cell Calcium (2002) 340(0), 1–7
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N. M. Atucha, 1 D. Iyu, 1 M. De Rycker, 1,∗ A. Soler, 2 J. Garcı́a-Estañ 1
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Altered calcium regulation in freshly
isolated aortic smooth muscle cells
from bile duct-ligated rats: role of
nitric oxide
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Summary In the present study we have analyzed the mechanisms of calcium entry and mobilization in smooth muscle
cells (SMCs) freshly isolated from the abdominal aorta of rats with bile duct ligation (BDL). The SMCs were obtained in
the day of the experiment after collagenase digestion and loaded with fura-2. The intracellular calcium levels ([Ca]i ) were
determined in individual cells by fluorescence microscopy. Baseline [Ca]i was slightly but significantly lower in SMCs from
BDL rats (70.14 ± 2.02 nM, n = 51) than in controls (80.77 ± 3.52, n = 44). The application of the purinergic agonists
ATP and UTP induced a fast calcium peak and a slow return to baseline. But the calcium responses were significantly
smaller in the cells from the BDL rats. Also, the area under the curve (AUC) of the calcium responses elicited by the
agonists was always lower in the SMCs from BDL rats as compared to the controls. Similar results were obtained with
UTP, but the calcium response of the SMCs from the BDL rats was even lower than that observed with ATP. In experiments
performed in the absence of extracellular calcium, both agonists also elevated [Ca]i , although the responses were much
smaller than those obtained in the presence of calcium. Again, the peak and AUC responses of the SMCs from BDL
rats were significantly lower than those of the controls. Incubation with NNA, a non-specific nitric oxide synthase (NOS)
inhibitor, or with NIL, an inducible NOS inhibitor (iNOS), potentiated and normalized the calcium responses of the SMCs
obtained from BDL rats. These data indicate that, in SMCs from bile duct-ligated rats, both the entry of calcium and the
mobilization from internal stores is defective in response to purinergic agonists. NO, of an inducible origin, is involved in
this altered calcium regulation.
© 2002 Published by Elsevier Science Ltd.
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INTRODUCTION
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Calcium is a very important regulator of many physiological processes, among them muscle contraction. Specifically in vascular tissues, the vascular smooth muscle cells
(SMCs) use calcium as the trigger for contraction. Thus, a
number of vasoconstrictor and vasodilator hormones and
factors act to increase or decrease, respectively, the intra-
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Received 2 August 2002
Revised 22 October 2002
Accepted 4 November 2002
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Departamentos de Fisiologı́a, Facultades de Medicina de Murcia, Murcia, Spain
Departamentos de Fisiologı́a, Facultades de Medicina de, Granada, Granada, Spain
Present address: Department of Molecular Genetics, University of Cincinnati,
231 Albert Sabin Way, Cincinnati, OH 45267-0524, USA.
Correspondence to: Noemı́ M. Atucha, Departamentos de Fisiologı́a, Facultades de Medicina de Murcia, 30100 Murcia, Spain. Tel.: +34-968-364884;
fax: +34-968-364150; e-mail: [email protected]
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cellular calcium levels and, therefore, modulate the activity of the contractile apparatus of the muscle cells and,
hence, the diameter and resistance of the blood vessels
[1–3]. In diseases such as liver cirrhosis, it is well known
that vascular function is altered in many ways, resulting in
a lower than normal resistance to blood flow which contributes to the lower blood pressure characteristic of both
human and experimental liver cirrhosis [4,5].
One of the most studied manifestations of this altered
vascular function in cirrhosis is the phenomenon of
vascular hyporesponsiveness to vasoconstrictors [6–12].
Studies from this and other laboratories have clearly established the important role that nitric oxide (NO) plays
in this alteration. Specifically in the arterial mesenteric
bed of portal hypertensive and cirrhotic ascitic rats,
the excess of NO reduces the agonist-induced vascu1
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METHODS
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Male Sprague–Dawley rats born and raised in the Animal House of the Universidad de Murcia were used in the
present study. All the experiments were performed according to the ethical rules for the treatment of laboratory animals of the European Union.
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Experimental groups
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Animals weighing around 200 g were subjected to bile
duct ligation (BDL) and excision or sham operation (control) as previously described [7,13]. All the animals were
used in the fourth week after surgery (23–25 days).
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Isolation of SMCs
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Animals were anesthetized with thiobutabarbital (Inactin,
100 mg/kg, i.p., RBI, Massachusetts, USA) and the abdominal aorta was carefully excised and dissected free of
connective tissue and adventitia at 4 ◦ C in a physiological
buffer (composition, in mM: NaCl, 145; KCl, 5; MgCl2 , 2;
HEPES, 10; glucose, 10; CaCl2 , 2; KH2 PO4 , 0.5; NaH2 PO4 ,
0.5, pH 7.4). Once clean, the aorta was cut into pieces
of 2 mm length and transferred into a 5 ml glass tube
containing 1 ml of low-calcium buffer (0.16 mM) containing collagenase (100 ␮g, Liberase Blendzyme 3, Roche
Diagnostics, Barcelona, Spain), elastase (100 ␮g, type 1),
trypsin inhibitor (160 ␮g, from soybean), and bovine
serum albumin (500 ␮g). Then, the tube was placed in the
refrigerator (4 ◦ C) and stored overnight (at least 16 h). The
next morning, the tube was placed in an incubation bath
at 37 ◦ C for 30 min. Then, the enzyme-containing buffer
was discarded and the tissue washed three times with
fresh low-calcium buffer. The tissue was then transferred
to a 10 ml plastic tube and incubated at room temperature (25 ◦ C) for 45 min with 5 ␮M fura-2 AM (Molecular
Probes, Leiden, The Netherlands) and same amount of
pluronic acid 20% in 1 ml of low-calcium buffer containing 2 mg albumin. During incubation with the fluorophore, the tube was gently agitated to release the cells
from the tissue. Fura-2 incubation was terminated by addition of 9 ml of low-calcium buffer at 4 ◦ C and the tube
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Calcium measurements
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All the SMCs used in the present experiments had the
typical spindle-like appearance. All the round cells, supposed to be contracted or damaged, were not selected.
Calcium measurements were performed in individual
cells. The coverslips were placed in the optical field of a
40× oil-immersion fluorescence objective of an inverted
microscope (Olympus CK40). The cells were excited alternatively with light of 340 and 380 nm wavelength from
a monochromator (Model QA-100, Photon Technology
International (PTI), South Brunswick, NJ, USA). After passing signals through a barrier filter (510 nm), fluorescence
was acquired by a microphotometer (model D-104, PTI),
and stored and processed by a PC-compatible computer
equipped with Felix software (PTI). The calibration of
[Ca2+ ]i was based on the signal ratio at 340/380 (nm/nm)
and an established protocol as stated later. The [Ca2+ ]i
was calculated according to the formula:
(R − Rmin )S f
[Ca2+ ]i =
× Kd
(Rmax − R)Sb
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where R is the ratio of the 340/380 (nm/nm) fluorescence
signal. Rmax is the 340/380 ratio in the presence of saturating calcium (ionomycin, 10 ␮M), Rmin is the 340/380
ratio in calcium-free buffer (5 mM EGTA), Kd is the dissociation constant (225 nM), and Sf /Sb is the ratio of the
380 nm fluorescence measured in calcium-free conditions
to that in calcium repleted conditions. Background fluorescence was obtained after addition of MnCl2 (1 mM) and
subtracted from every value.
All drug and chemicals were added in small volumes (1
or 2 ␮l) to the droplet of cells (90 ␮l) on the surface of the
coverslip. Cells were not washed between additions in order not to dislodge them from their position on the glass
coverslip. Calculations of drug concentration were based
on the changing volumes of the droplet. During the time
necessary for each experiment (less than 6 min), we observed no significant changes in counts at 340 and 380 nm
as well as the ratio of the two.
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centrifuged at 500 rpm for 3.5 min at 4 ◦ C. After centrifugation, the supernatant was discarded and the cell suspension resuspended in 1 ml of low-calcium buffer. Then,
the cells were stored on ice until use, and calcium was
progressively added during 20 min to reach a final concentration of 2 mM. In the experiments performed in the
absence of extracellular calcium, this addition of calcium
was omitted and EGTA (200 ␮M) was added. Thirty minutes before starting the experiments, cells were allowed
to attach to poly-L-lysine (0.1 mg/ml)-coated coverslips
(Menzer-Glasser, 24 mm × 60 mm, #1.5, Germany) which
were covered with CoverWell perfusion chambers (Grace
Bio-Labs, Sigma).
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lar contraction mostly through the formation of cGMP
[11,12]. Recently, in an study using the mesenteric arterial
bed of bile duct-ligated rats, we have observed that this
excess of NO interferes with several calcium entry and
mobilization pathways [13]. Then, in the present study we
aimed at directly measuring intracellular calcium levels in
SMCs isolated from animals with experimental cirrhosis,
in order to analyze some of the mechanisms that regulate the intracellular calcium concentration and the role
of NO.
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Calcium signaling in cirrhosis
Once a cell was selected, baseline values were obtained
for 30 s, and then the appropriate drug concentration was
added and the fluorescence recorded for 300 s. Then, ionomycin, EGTA, and MnCl2 (at the concentrations reported
earlier and in this same order) were added and the experiment was finished. The cells were studied in the presence
and in the absence of extracellular calcium, and were challenged by addition of the purinoceptor agonists ATP and
UTP (1, 10, 30, 100 ␮M). Experiments were also performed
in the presence of N␻ -nitro-L-arginine (NNA, 100 ␮M), a
non-specific nitric oxide synthase (NOS) inhibitor, or with
N6 -(1-iminoethyl)-L-lysine hydrochloride (NIL), a specific
inducible NOS (iNOS) inhibitor (30 ␮M, RBI, Natick, MA,
USA).
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Drugs
Fura-2 AM was dissolved in DMSO. The rest of products
used were from Sigma Chemical (Madrid, Spain), except
where indicated. Drug stock solutions were prepared in
distilled water and maintained frozen (−20 ◦ C). Appropriate dilutions were prepared freshly every day in normal
Krebs.
Statistical analysis
Data are expressed as the mean ± S.E. In order to compare
the calcium responses between control and BDL, the individual calcium responses were normalized and the area
under the curve (AUC) calculated by summation of all
experimental values (300 s) minus the averaged baseline
All the BDL rats used in the present study showed, at
inspection in the moment of the experiment, the typical features of this model: jaundice, enlarged liver and
spleen, and mesenteric edema. Ascites was not present in
any animal.
Basal calcium levels in the presence of extracellular calcium was slightly but significantly lower in SMCs from
BDL rats (70.14 ± 2.02 nM, n = 51) than in control cells
(80.77 ± 3.52, n = 44).
Application of 100 ␮M ATP resulted in a very fast calcium peak and then a slow return to baseline. However, the
response was significantly lower in the cells from the BDL
animals (Fig. 1). The peak averaged 1280.8 ± 288.0 nM in
the controls and 559.6 ± 121.5 in the BDL, 4 s after the
agonist application. Thereafter, calcium levels returned to
baseline but were always higher in the control cells, so
that the AUC was significantly lower in the BDL. Similar responses in shape, but lower in magnitude, were observed
with 10 and 30 ␮M ATP, and the response of the cells from
the BDL animals were significantly lower than those of the
controls (Fig. 2 and Table 1). The dose of 1 ␮M ATP induced a very small response and there was no difference
between groups (Fig. 2).
Application of UTP revealed also an altered calcium response in the SMCs from BDL rats (Fig. 3). In this case, the
differences were much more important, since the concentration of 10 ␮M was almost maximal for the control cells
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(30 s). The resulting values as well as baseline calcium values were compared by unpaired Student’s t-test.
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Fig. 1 Changes of intracellular calcium levels in response to 100 ␮M ATP in smooth muscle cells from control and BDL rats.
© 2002 Published by Elsevier Science Ltd.
Cell Calcium (2002) 340(0), 1–7
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NM Atucha, D Iyu, MD Rycker, A Soler, J Garcı́a-Estañ
Control
BDL
55.5 ± 19.9 (3)
190.9 ± 32.8 (4)
353.0 ± 26.3 (5)
575.9 ± 92.4 (11)
127.7 ± 32.4 (8)
267.8 ± 71.3 (5)
350.4 ± 64.8 (4)
398.2 ± 119.7 (5)
418.2 ± 65.3 (7)
245.6 ± 70.9 (9)
56.6 ± 14.4 (6)
58.3 ± 19.6 (7)*
164.3 ± 37.9 (4)*
331.2 ± 47.7 (14)*
41.1 ± 23.9 (10)*
11.3 ± 17.4 (5)*
97.8 ± 51.9 (4)*
53.3 ± 20.3 (3)*
407.9 ± 46.6 (8)
48.8 ± 25.4 (7)*
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ATP 10 ␮M
ATP 30 ␮M
ATP 100 ␮M
ATP 100 ␮M, 0 Ca2+
UTP 1 ␮M
UTP 10 ␮M
UTP 30 ␮M
UTP 100 ␮M
UTP 100 ␮M, 0 Ca2+
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Table 1 Area under the curve (units) of the normalized calcium response
Number in parentheses is number of cells studied.
* P < 0.05 vs. control.
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DISCUSSION
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Previous studies from our and other laboratories demonstrated the involvement of an excess of NO as an important mediator of the well known phenomenon of vascular
hyporesponsiveness in experimental liver cirrhotic models
[6–13]. We have also shown recently that this enhanced
NO production can interact with one of the crucial steps
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while the response of the BDL cells was not significant until the concentration of 100 ␮M (Fig. 4 and Table 1).
In the absence of extracellular calcium, 100 ␮M ATP
also induced an increase in calcium levels, albeit of lower
magnitude than in the presence of calcium (Fig. 5 and
Table 1). The peak in the control cells was 188.0 ± 25.3
and 111.5 ± 20.7 in the cells from the BDL rats. Again,
the response of the control cells was significantly greater
than that of the cells from the BDL animals. Similarly, the
administration of 100 ␮M UTP, in the absence of extracellular calcium, induced a much lower response than in
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the presence of calcium, and also the response of the cells
from the BDL rats was significantly lower than that of the
control rats (Fig. 6 and Table 1).
Incubation of SMCs with NNA or NIL potentiated the
calcium responses of the BDL rats, both with (Figs. 7 and 8
and Table 2) or without calcium (Figs. 5 and 6 and Table 2).
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Fig. 2 Area under the curve of the integrated calcium responses
after ATP administration.
Fig. 3 Changes of intracellular calcium levels in response to 10 ␮M UTP in smooth muscle cells from control and BDL rats.
Cell Calcium (2002) 340(0), 1–7
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Fig. 4 Area under the curve of the integrated calcium responses
after UTP administration.
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Fig. 6 Area under the curve in response to 100 ␮M UTP in the
absence of extracellular calcium before and after inhibiting NO
synthesis with NNA.
involved in the contraction of smooth muscle [13], that is,
the regulation of intracellular calcium levels. In order to directly analyze this question, we aimed at the measurement
of intracellular calcium levels by means of microscopy of
fluorescence in isolated SMCs.
The issue of calcium signaling in cirrhosis has not been
studied in detail. Very few studies have directly analyzed
this issue and the results are not completely homogeneous. A recent review, thoroughly discusses this and
other related topics [14]. Thus, in aortic rings from portal vein-ligated rats, the blunted hyporesponsiveness to
norepinephrine was associated with a defect in the mo-
Fig. 7 Area under the curve in response to 100 ␮M ATP, in basal
conditions and after inhibition of NO synthesis with NNA and NIL.
Table 2 Area under the curve (units) of the normalized calcium response in the cells pretreated with NNA or NIL to inhibit NO synthesis
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Fig. 5 Area under the curve in response to 100 ␮M ATP, in basal
conditions and in the absence of extracellular calcium before and
after inhibiting NO synthesis with NNA.
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ATP 100 ␮M + NNA
ATP 10 ␮M + NIL
ATP 100 ␮M + NIL
ATP 100 ␮M, 0 Ca + NNA
UTP 10 ␮M + NNA
UTP 100 ␮M + NNA
UTP 10 ␮M + NIL
UTP 100 ␮M, 0 Ca + NNA
Control
BDL
334.07 ± 44.9 (6)
n.d.
n.d.
423.9 ± 11.7 (4)
423.3 ± 31.8 (4)
268.6 ± 100.5 (4)
n.d.
329.1 ± 74.1 (3)
590.9 ± 81.1 (6)*
402.5 ± 36.7 (4)
512.3 ± 65.9 (5)*
343.7 ± 63.7 (5)*
467.6 ± 46.9 (5)*
403.8 ± 62.4 (8)
326.9 ± 70.4 (5)*
360.9 ± 90.5 (8)*
Number in parentheses is number of cells studied; n.d.: not determined.
* P < 0.05 vs. BDL not treated with NO inhibitors (values in Table 1).
Cell Calcium (2002) 340(0), 1–7
NM Atucha, D Iyu, MD Rycker, A Soler, J Garcı́a-Estañ
from the extracellular milieu and at the same time, by
interacting with the signal transduction cell mechanism,
are able to release calcium from the internal stores, a
situation that can be studied simply removing the extracellular calcium. These conditions are filled by the
purinoceptor agonists used here. Thus, ATP and UTP are
able to simultaneously increase the concentration of cell
calcium by both entrance from the outside of the cell and
release from the internal stores [22,23]. It is important to
note that we use the term internal stores instead of using
other more specific terms, such as sarcoplasmic reticulum,
to designate all the intracellular organelles able to store
calcium and to release it under appropriate conditions.
As observed, ATP and UTP, at the concentrations used,
produced a dose-dependent change in calcium levels in
SMCs from the control rats. In the vascular cells obtained
from the BDL rats, however, this dose-dependency was
clearly of lower magnitude and also displaced to higher
doses (with UTP), so a lower calcium response was clearly
detectable. Essentially, the same results were obtained in
the experiments performed in the absence of calcium,
although the magnitude of the integrated response was
much lower. Thus, part of this altered response is due to
a lower mobilization from the internal stores. A number
of causes may explain this alteration, from the binding of
the agonist to the membrane receptor to the signal transduction mechanism, although a lower production of IP3
has been found by other investigators [24] in conditions
similar to those used here. Also, the defective calcium
response observed in SMCs from the BDL rats seems to
be due to a lower entrance of calcium from the extracellular space, as it indicates the difference between the
total calcium mobilized with and without calcium. This
is specially evident with ATP. But, in the case of UTP the
response was so abnormal that it is not easy to make a
clear conclusion. In any case, both purinoceptor agonists
activate different kind of receptors, both ionotropic and
metabotropic, and there could be a different proportion
of each one in the cells of the BDL rats.
The present results also show that this defective regulation of calcium in vascular cells from cirrhotic animals
is dependent on an excess of NO. Thus, incubation either
with a non-specific NOS inhibitor, NNA, or with a specific
iNOS inhibitor, NIL, potentiated the responses of the cells
from the BDL animals, either with or without calcium, thus
suggesting that the inducible isoform of NOS is upregulated in the smooth muscle layer of the aorta, but not in
the cells from the control animals, whose responses were
unchanged after treatment with NIL or NNA. This is not
the first demonstration that iNOS can be present in vascular [25] or non-vascular [26] cells from cirrhotic animals,
but we believe that our experiments clearly demonstrate
that this excess of NO interferes directly with several of the
mechanisms that control intracellular calcium regulation.
Fig. 8 Area under the curve in response to 10 ␮M UTP, in basal
conditions and after inhibition of NO synthesis with NNA and NIL.
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bilization of intracellular calcium [15,16]. A reduced calcium mobilization in cultured SMCs or in vascular rings
or strips has been also suggested by several groups together with different alterations in the phosphoinositide
signaling system [17–21]. To the best of our knowledge,
the approach we have used in the present study has
never been utilized in cirrhosis. The use of freshly isolated SMCs, therefore, not cultured, should allow a more
physiologic study of the diverse signaling pathways in cirrhosis, an area in need of new information. We decided to
work with cells that have been obtained in the same day
of the experiment because the culture conditions and the
successive passages necessary to obtain a viable cell culture could change the phenotypical alterations that SMCs
may possess while in vivo. A second important difference
with experiments performed in cultured cells is that the
present experiments have been performed in individual
cells. Thus, cells have been studied one at a time so cells
that did not have the spindle-like morphology characteristic of SMCs or that did not respond properly to the
calibration method, as stated in METHODS Section, were
rejected. In the study by Castro et al. [21], for instance,
cells were studied in a conventional cuvette fluorimeter where the signal is the average of many thousands
of cells.
The results obtained by using these freshly isolated
SMCs completely agree with our previous observation of
an important interaction between NO and intracellular
calcium which may be the basis for the lower contractile
response in the vessels of the cirrhotic animals [13]. In
order to study this interaction, we have used a simple
approach, to monitor the changes in intracellular calcium
levels in response to agonists that increase the entrance
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This work has been performed with grants from Fundación Séneca de Murcia (PB/FS/99) and Comisión Interministerial de Investigación Cient´ıfica y Técnica of Spain
(SAF2000-0157).
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In conclusion, SMCs freshly isolated from the abdominal aorta of bile duct-ligated rats show an altered intracellular calcium response to purinoceptor agonists which
is corrected after treatment with a selective inhibitor of
iNOS. These results indicate that the phenomenon of vascular hyporesponsiveness in this experimental model of
liver cirrhosis is related to a direct effect of NO on the muscle layer.
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