eNOS and prostanoid enzymes in lungs of
newborn piglets with chronic aortopulmonary shunts
Candice D.
Fike1,
Mark
R.
Kaplowitz1, and
Michael
Bousamra II2
2 Departments of Pediatrics and Cardiothoracic Surgery,
Medical College of Wisconsin, Milwaukee, Wisconsin 53226; and
1 Department of Pediatrics, Wake Forest University School of
Medicine, Winston-Salem, North Carolina 27157
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ABSTRACT |
Our purpose was to determine if abundance
of proteins underlying nitric oxide (NO) and prostanoid production is
altered in lungs of piglets with aortopulmonary shunts. We also
evaluated whether shunted piglets exhibit abnormal pulmonary vascular
responses to ACh, an endothelium-dependent agent that mediates dilation in part by NO and prostanoid release. At age 4-5 days, piglets underwent either a sham operation or placement of an aortopulmonary shunt. At age 5-6 wk, pulmonary arterial pressure (Ppa) and
cardiac output by the thermodilution technique were measured in
anesthetized piglets. Ppa responses to the endothelium-dependent agent,
ACh, and to a non-endothelium-dependent agent, papaverine, were
measured in perfused lungs. An immunoblot technique was applied to
homogenates of whole lung tissue and two size groups of pulmonary
arteries. In shunted piglets, Ppa and cardiac output were elevated, and Ppa responses to papaverine were reduced. ACh responses were not decreased when expressed relative to Ppa dilation with papaverine. Endothelial nitric oxide synthase (eNOS), cyclooxygenase-1,
cyclooxygenase-2, prostacyclin synthase, and thromboxane synthase
amounts were unaltered in all lung tissue homogenates. Altered
abundance of eNOS and/or prostanoid enzymes does not contribute to the
blunted dilation and the elevation in Ppa associated with
aortopulmonary shunts in newborn piglets.
neonatal pulmonary hypertension; acetylcholine responses; cyclooxygenase enzymes; prostacyclin synthase; thromboxane synthase
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INTRODUCTION |
IT IS WELL KNOWN
that infants with congenital heart lesions that are associated with
increased pulmonary blood flow develop pulmonary hypertension (2,
7, 15, 17, 23). The mechanisms underlying the development of
pulmonary hypertension in these infants remain uncertain but include
the possibility that production of factors known to regulate pulmonary
vascular pressure has been altered (2, 7, 11, 15, 28). For
example, decreased production of vasodilators, such as nitric oxide
(NO) and prostacyclin, or increased production of vasoconstrictors,
such as thromboxane, could contribute to the development of pulmonary
hypertension (2, 7, 11, 15, 28). In turn, a change in the
abundance of the proteins responsible for their production could
underlie altered production of these vasoactive factors. In other
words, reduced amounts of endothelial nitric oxide synthase (eNOS), an enzyme responsible for basal pulmonary vascular NO production, could
contribute to pulmonary hypertension. Similarly, altered amounts of
prostanoid enzymes underlying either prostacyclin and/or thromboxane
production could contribute to the pulmonary hypertension that develops
in infants with increased pulmonary blood flow.
Experimental attempts to produce increased-flow pulmonary hypertension
include placement of an aortopulmonary shunt in newborn piglets
(10, 26). Previous investigators reported that piglets studied 4-12 wk after placement of an aortopulmonary shunt
exhibited elevated pulmonary arterial pressure and thickened pulmonary
arteries (10, 26). The effect of prolonged aortopulmonary
shunts on amounts of eNOS and/or prostanoid enzymes in lungs of newborn piglets has not been reported. Our purpose was to test the hypothesis that abundance of either eNOS or prostanoid enzymes would be altered in
lungs of newborn piglets 5-6 wk after placement of an
aortopulmonary shunt. In addition, because both NO and prostanoids have
been shown to contribute to pulmonary vascular dilation to ACh
(4, 19, 31, 33), we tested the hypothesis that ACh-induced
dilation would be altered in lungs of piglets with chronic
aortopulmonary shunts.
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METHODS |
Animals.
Newborn piglets arrived a few days before surgery at age 1-3
days and were placed in an environmental chamber so that they could
acclimate and learn to ad libitum feed sow milk replacer from a feeding
device attached to the chamber. At age 4-6 days, left
thoracotamies were performed in all piglets. An aortopulmonary shunt
was placed in five piglets, and six piglets underwent a sham operation.
For the thoracotamy, each piglet was preanesthetized with ketamine (10 mg/kg im) and acepromazine (1 mg/kg im) and then intubated. The piglet
was ventilated with a bellows-type anesthesia machine that delivered
supplemental oxygen as needed, a tidal volume of 10-15 ml/kg, and
a respiratory rate of 20-30 breaths/min. To maintain temperature
at 39-40°C, the piglet was placed on a heating blanket. After
percutaneous placement of an intravenous catheter in an ear vein, the
piglet received prophylactic antibiotic therapy (cefazolin, 25 mg/kg
iv) and was anesthetized with fentanyl (20 µg/kg iv). Throughout the
operation, the piglet received a continuous intravenous infusion of
normal saline (4-5 ml · kg
1 · h
1) and
additional fentanyl as needed. For the shunted piglets, a 5-mm Gortex
graft was sewn end to side to the aorta and then to the pulmonary
artery to create the aortopulmonary shunt. For the sham piglets, the
aorta and pulmonary artery were dissected and transiently clamped for
the same duration of time necessary to sew in a shunt. To minimize
movement during shunt placement, vecuronium (0.1 mg/kg) was given
during the time that the aorta and pulmonary artery were clamped.
Postoperatively, each piglet received prophylactic antibiotics
(cefazolin, 25 mg/kg im two times daily for 3 days) and analgesia
(bupemorphine, 10 µg/kg im two times daily for 1 day). Piglets were
maintained in the environmental chamber and fed ad libitum sow milk
replacer until 3 wk of age. Piglets were then weaned, placed in a pen,
and fed dry chow two times a day for an additional 2-3 wk.
Measurements in anesthetized animals.
After surgery (5-6 wk), all piglets were weighed,
preanesthetized with ketamine (30 mg/kg im), and then anesthetized with pentobarbital sodium (10 mg/kg iv). Additional intravenous
pentobarbital sodium was given as needed via an ear vein to maintain
anesthesia during placement of the catheters. First, the trachea of the
piglet was cannulated so that the animal could be ventilated if
necessary. Next, a catheter was placed in the right femoral artery for
monitoring systemic blood pressure and arterial blood gases. Then,
another catheter was placed through the right external jugular vein
into the pulmonary artery to monitor pulmonary arterial pressure. To obtain the pulmonary wedge pressure, the pulmonary arterial catheter was advanced into a distal pulmonary vessel. The zero reference for the
vascular pressures was the midthorax. To measure cardiac output by the
thermodilution technique (model 9520 thermodilution cardiac output
computer; Edwards Laboratory), a thermistor was placed in the aortic
arch via the left femoral artery, and a catheter that served as an
injection port was placed in the left ventricle via the left carotid
artery. Cardiac output was measured at end expiration as the mean of
three injections of 3 ml of 0.9% saline (0°C). For one of the
shunted piglets, equipment (Baxter Vigilance Monitor) became available
to measure oxygen saturations both proximal to the shunt, in the right
ventricle, and distal to the shunt in the pulmonary artery.
At the completion of the in vivo measurements, all animals were given
heparin (1,000 IU/kg iv) and additional anesthesia (3-5 mg/kg
pentobarbital sodium iv) and then were exsanguinated. Lungs were left
in situ and used for perfusion as described below.
Measurements in isolated perfused lungs.
For lung perfusion, the tracheal cannula was attached to a
large-animal piston-type ventilator, and the lungs were ventilated with
a normoxic gas mixture (17% O2-6% CO2-balance
N2) using a tidal volume of 15-20 ml/kg and a
respiratory rate of 15-20 breaths/min (mean airway pressure of
4-6 cmH2O). A midline sternotomy was performed, and,
for the shunted piglets, a clamp was placed across the shunt.
Saline-filled cannulas were placed in the pulmonary artery and left
atrium through incisions in the right and left ventricles. The
diaphragm and all abdominal contents were removed. The vascular
cannulas were connected to the perfusion circuit described previously
(13, 14). The perfusion circuit was filled with 200 ml of
the animal's own blood collected during exsanguination (as described
in Measurements in anesthetized animals), mixed with 100 ml
of 3% albumin-saline. The perfusion circuit included a rotary pump
(model 7523-00; Cole-Palmer Masterflex) that continuously circulated
the perfusate from a reservoir through a bubble trap into the pulmonary
arterial cannula, through the lungs to the left atrial cannula, and
back to the reservoir. Pulmonary arterial, left atrial, and airway
pressures were continuously monitored. The most dependent edge of the
lung was used as the zero reference for vascular pressures.
After connection to the perfusion circuit, the lungs were
perfused for 30-60 min until a stable pulmonary arterial pressure was achieved. The perfusate flow and left atrial pressure were adjusted
to respective levels of 50 ml · min
1 · kg
1 and 0 cmH2O and were maintained constant for the remainder of the study.
Changes in pulmonary arterial pressure in response to ACh were
then measured. Because we knew from our first few studies (see RESULTS) that baseline pulmonary arterial pressure is
greater in shunted than in sham animals, the pulmonary arterial
pressure of the sham animals was elevated to a level comparable to that of the shunted animals by adding 1 M KCl to the perfusate of the sham
lungs. ACh was added cumulatively to the reservoir to achieve concentrations of 10
9 to 10
6 M. The ACh
responses were transient so that the pulmonary arterial pressure was
allowed to return to baseline between doses. After the response to ACh
was measured, the endothelium-independent agent papaverine was added to
the reservoir (300 µg/ml of perfusate) and allowed to circulate for
20 min to determine the contribution of vasomotor tone to pulmonary
arterial pressure.
For the shunted piglets, patency of the shunt was confirmed by
visual examination at the end of perfusion. For all piglets, a piece of
lung was immediately frozen in liquid nitrogen and stored at
80°C
for later analysis of eNOS and the prostanoid enzymes cyclooxygenase
(COX)-1, COX-2, prostacyclin synthase, and thromboxane synthase in
whole lung homogenate samples as described below. For lungs of some
piglets (n = 4 shunted, n = 4 sham), the remainder of the lungs was then submersed in saline and stored overnight at 4°C. The next morning, two size groups of pulmonary arteries (small pulmonary arteries = 100-600 µm in diameter
and larger pulmonary arteries = 700-1,200 µm in diameter)
were dissected from the stored lungs and then immediately frozen in
liquid nitrogen for later analysis of eNOS and prostanoid enzymes
(COX-1, COX-2, prostacyclin synthase, and thromboxane synthase) in
pulmonary artery homogenate samples as described below.
Immunoblot analysis.
We performed preliminary studies with different amounts of total
protein to determine the dynamic range of the immunoblot analysis for
each protein and tissue homogenate. An amount of protein that was
within the dynamic range of the immunoblot analysis was then used to
compare protein abundance between homogenates from shunted and
sham-operated piglets as described below. For example, Fig.
1 shows an immunoblot for eNOS using
small pulmonary artery homogenate samples containing total protein
amounts of 1, 2.5, 5, 7.5, and 10 µg. On the basis of these findings,
to compare eNOS abundance in small pulmonary artery homogenates between shunted and sham-operated piglets, we followed methods as described below using 2.5-µg total protein samples.

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Fig. 1.
Immunoblot for endothelial nitric oxide synthase (eNOS)
using small pulmonary artery (Pa) homogenate samples from sham-operated
and shunted piglets containing total protein amounts of 1, 2.5, 5, 7.5, and 10 µg. Note that for 1, 2.5, 5, and 7.5 µg of total protein,
the intensity of staining for eNOS increases with increasing amounts of
protein.
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Immunoblot analysis for whole lung homogenate samples.
Tissue pieces that did not contain large airways or large vessels were
selected from frozen perfused lungs of sham and shunted piglets. The
tissue pieces were homogenized in 10 mM HEPES buffer containing 250 mM
sucrose, 3 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride, pH 7.4, on
ice using three 15-s pulses of a Polytron blender, taking care to avoid
foaming of the homogenate. Protein concentration of the lung homogenate
was determined by the Bio-Rad protein assay. Each lung homogenate was
diluted with PBS to obtain a protein concentration of 1 mg/ml. Forty
microliters of each protein concentration were solubilized in 40 µl
of denaturing, reducing sample buffer [0.25 M Tris · HCl, 5%
(wt/vol) SDS, 2.5% (vol/vol)
-mercaptoethanol, 10% glycerol, and
0.05% bromphenol blue, pH 6.8; Novex], heated to 80°C for 15 min,
and centrifuged for 3 min at 5,600 g in a microfuge. Equal
volumes of these supernatants were then applied to Tris-glycine precast
8% polyacrylamide gels (Novex) so that 15-µg protein whole lung
homogenate samples were loaded on both gels. We used 15-µg protein
samples for eNOS. Electrophoresis was carried out in 25 mM Tris, 192 mM
glycine, and 0.1% SDS (pH = 8.3) at 125 volts for 1.7 h. The
proteins were transferred from the gel to a nitrocellulose membrane
(Novex) at 100 volts for 1 h in 25 mM Tris, 192 mM glycine, and
20% methanol (pH = 8.3). The membrane was incubated overnight at
4°C in PBS containing 10% nonfat dried milk and 0.1% Tween 20 to
block nonspecific protein binding. To detect eNOS, the nitrocellulose
membrane was incubated for 1 h at room temperature with the
primary antibody (Transduction Laboratories) diluted 1:500 in PBS
containing 0.1% Tween 20 and 1% nonfat dried milk (carrier buffer)
followed by incubation for 30 min at room temperature with a
biotinylated secondary antibody (Vector Elite, ABC Kit; Vector
Laboratories) diluted 1:5,000 in the carrier buffer, followed by
incubation for 30 min at room temperature with streptavidin-horseradish
peroxidase conjugate (Amersham) diluted 1:1,500 in PBS containing 0.1%
Tween 20. The nitrocellulose membrane was washed three times between
the first two incubations with the carrier buffer, three times with the carrier buffer, and one time with PBS containing 0.1% Tween 20 after
the final incubation. To visualize the biotinylated antibody, the
membranes were developed using enhanced chemiluminescence (ECL)
reagents (Amersham), and the chemiluminescent signal was captured on
X-ray film (ECL Hyperfilm; Kodak). The bands for eNOS were quantified
using densitometry.
Similar procedures were followed using primary antibodies for COX-1
(Cayman), COX-2 (Oxford), prostacyclin synthase (Cayman), thromboxane
synthase (from Dr. W. B. Campbell, Medical College of Wisconsin),
and appropriate horseradish peroxidase-conjugated secondary antibodies
(Sigma or Zymed). For determinations of COX-1, prostacyclin synthase,
and thromboxane synthase we used 15-µg protein samples. For COX-2
analysis, we used 40-µg protein samples.
After analyses for eNOS or the prostanoid enzymes, the membranes were
reprobed for the smooth muscle cell marker actin (Sigma). All of the
reprobed membranes contained whole lung homogenate total protein
amounts outside the dynamic range of the immunoblot analysis for actin.
Therefore, we performed additional studies to compare actin abundance
between whole lung homogenates from shunted and sham-operated piglets.
For these studies, we used samples containing total protein amounts
within the dynamic range for actin analysis, i.e., 2.5-µg protein samples.
Immunoblot analysis for pulmonary artery homogenate samples.
For both size groups of pulmonary artery homogenate samples, procedures
similar to those described above for whole lung homogenate samples were
applied to frozen samples of 100- to 600-µm-diameter (small) or 700- to 1,200-µm-diameter (larger) pulmonary arteries. For eNOS analysis,
2.5-µg total protein samples were used for both size groups of
pulmonary artery homogenates. For all of the prostanoid enzymes,
7.5-µg total protein samples were used for the pulmonary artery
homogenates. After analyses for eNOS or the prostanoid enzymes, the
membranes were reprobed for the smooth muscle cell marker actin
(Sigma). All of the reprobed membranes contained pulmonary artery
homogenate total protein amounts outside the dynamic range of the
immunoblot analysis for actin. Therefore, we performed additional
studies to compare actin abundance between pulmonary artery homogenates
from shunted and sham-operated piglets using samples containing total
protein amounts within the dynamic range for actin analysis, i.e.,
0.5-µg protein samples.
Statistics.
Data are presented as means ± SE. An unpaired t-test
was used to compare the data between shunted and sham piglets.
P < 0.05 was indicative of statistical significance.
 |
RESULTS |
On the day of surgery, the ages and weights of the animals did not
differ between piglets used for the sham (age = 5 ± 0.4 days, wt = 1,926 ± 118 g) and shunt (age = 5 ± 0.3 days, wt = 2,302 ± 117 g) operations. Likewise,
on the day of the in vivo hemodynamic measurements, there was no
difference between ages, weights, or hematocrits of the sham compared
with the shunt-operated piglets (Table
1). The measured values of blood pH,
PO2, and PCO2 obtained
during in vivo hemodynamic measurements in anesthetized animals were
similar between the sham and shunted piglets except for a slightly
higher PO2 in the sham piglets (Table 1).
Measurements of pulmonary arterial pressure, pulmonary wedge pressure,
cardiac output, and aortic pressure are summarized in Table 1.
Pulmonary arterial pressure was doubled, and cardiac output was 75%
greater in shunted than in sham piglets. For the one shunted piglet in which we have measurements, the right ventricle oxygen saturation was
60%, and the distal pulmonary arterial pressure saturation was 86%,
such that the pulmonary-to-systemic blood flow was 2.8 to 1 in this
piglet.
For the perfused lungs, there was no difference in perfusate hematocrit
nor in values of pH, PO2, or
PCO2 measured in the perfusate of isolated
lungs from sham and shunted piglets (Table 2). In the perfused lungs, left atrial
pressure was zero, and perfusate flow was the same for both groups
(Table 2) and was maintained constant throughout the study so that
changes and differences in pulmonary arterial pressure, respectively,
represent changes and differences in pulmonary vascular resistance. The
baseline pulmonary arterial pressure was greater in isolated perfused
lungs of the shunted piglets compared with the sham piglets (Table 2). After the addition of KCl, the pulmonary arterial pressure in the sham
group was elevated to a level comparable to the baseline pulmonary
arterial pressure in the shunted group (Table 2). When calculated as an
absolute change from comparable pulmonary arterial pressures (Table 2),
the decrease in pulmonary arterial pressure with all doses of ACh was
less in the shunted lungs than in the sham lungs (Fig.
2). In addition, the change in pulmonary
arterial pressure with papaverine was less in lungs of shunted than in lungs of sham-operated piglets (Fig. 3).
Thus, when the change in pulmonary arterial pressure with ACh was
calculated relative to the change in pulmonary arterial pressure with
the non-endothelium-dependent dilator papaverine, responses to all but
the lowest dose of ACh were the same in shunted and in sham lungs (Fig.
4). Of note, after the addition of
papaverine and removal of vasomotor tone, the pulmonary arterial
pressure in the lungs of shunted piglets was greater than the pulmonary
arterial pressure in the lungs of sham piglets (Table 2).

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Fig. 2.
ACh-induced changes in pulmonary arterial pressure in
perfused lungs of sham-operated and shunted piglets. Data are
means ± SE. *Different from sham, P < 0.05.
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Fig. 3.
Papaverine-induced changes in pulmonary arterial pressure
in perfused lungs of sham-operated and shunted piglets. Data are
means ± SE. *Different from sham, P < 0.05.
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Fig. 4.
ACh-induced changes in pulmonary arterial pressure
expressed relative to dilation to the non-endothelium-dependent agent
papaverine. Data are means ± SE. *Different from sham,
P < 0.05.
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Immunoblot analyses for eNOS in the whole lung homogenates and both
size groups of small pulmonary artery homogenates (small pulmonary
arteries 100-600 µm in diameter and larger pulmonary arteries
700-1,200 µm in diameter) are shown in Fig.
5. The antibody to eNOS detected eNOS in
all homogenates at an apparent molecular mass of 135 kDa as determined
from an exponential fit of molecular mass standards. The absorbance of
the eNOS bands as determined by densitometry did not differ between
shunted and sham piglets for either whole lung homogenates or for
homogenates of either size group of small pulmonary arteries (Table
3).

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Fig. 5.
eNOS immunoblots in whole lung homogenates (A)
and the following homogenates of two size groups of small pulmonary
arteries: smaller pulmonary arteries = 100-600 µm in
diameter (B) and larger pulmonary arteries = 700-1,200 µm in diameter (C). +, Positive control for
eNOS (obtained from Transduction Laboratories). Note that there is no
apparent difference in the intensity of the eNOS bands between
sham-operated and shunted piglets for any of the lung tissue
homogenates.
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Immunoblot analyses for the prostanoid enzymes COX-1, COX-2,
prostacyclin synthase, and thromboxane synthase in the whole lung
homogenates and both size groups of small pulmonary artery homogenates
(small pulmonary arteries 100-600 µm in diameter and larger
pulmonary arteries 700-1,200 µm in diameter) are shown in Figs.
6 and 7.
The antibody to COX-1 detected COX-1 in all homogenates at an apparent
molecular mass of 70 kDa (Fig. 6); the antibody to COX-2 detected COX-2
in all homogenates at an apparent molecular mass of 72 kDa (Fig. 6);
the antibody to prostacyclin synthase detected prostacyclin synthase in
all homogenates at an apparent molecular mass of 52 kDa (Fig. 7); and
the antibody to thromboxane synthase detected thromboxane synthase in
all homogenates at an apparent molecular mass of 60 kDa (Fig. 7).
Similar to findings for eNOS, as determined by densitometry, there was
no difference in the absorbance of bands for any of the prostanoid
enzymes for whole lung homogenates or for homogenates of either size
group of small pulmonary arteries from shunted compared with sham
piglets (Table 3).

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Fig. 6.
A-C: cyclooxygenase (COX)-1 in whole lung
homogenates (A) and homogenates of the following two size
groups of small pulmonary arteries: small pulmonary arteries = 100-600 µm in diameter (B) and larger pulmonary
arteries = 700-1,200 µm in diameter (C). +,
Positive control for COX-1 (obtained from Cayman Laboratories).
D-F: COX-2 immunoblots in whole lung homogenates
(D) and homogenates of small pulmonary arteries 100-600
µm in diameter (E) and larger pulmonary arteries
700-1,200 µm in diameter (F). +, Positive control for
COX-2 (obtained from Cayman Laboratories). Note that there is no
apparent difference in the intensity of the COX-1 or the COX-2 bands
between sham-operated and shunted piglets for any of the lung tissue
homogenates.
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Fig. 7.
A-C: prostacyclin (PGI2) synthase
immunoblots in whole lung homogenates (A) and homogenates of
small pulmonary arteries 100-600 µm in diameter (B)
and larger pulmonary arteries 700-1,200 µm in diameter
(C). +, Positive control for PGI2 synthase
(obtained from Cayman Laboratories). D-F: thromboxane
A2 (TXA2) synthase immunoblots in whole lung
homogenates (D) and homogenates of small pulmonary arteries
100-600 µm in diameter (E) and larger pulmonary
arteries 700-1,200 µm in diameter (F). +, Positive
control for TXA2 synthase (platelet lysate). Note that
there is no apparent difference in the intensity of the
PGI2 synthase bands or the TXA2 synthase bands
between sham-operated and shunted piglets for any of the lung tissue
homogenates.
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Figure 8, A-C, shows
representative immunoblot analyses for actin in reprobed whole lung and
reprobed pulmonary artery homogenates. Figure 8, D-F,
shows immunoblot analyses for actin abundance in the whole lung
homogenates and both size groups of small pulmonary artery homogenates
using samples containing total protein amounts within the dynamic range
of the actin immunoblot analyses. The antibody to actin detected actin
in all homogenates at an apparent molecular mass of 42 kDa as
determined from an exponential fit of molecular mass standards. For the
reprobed homogenate samples, the absorbance of the actin bands did not
differ between shunted and sham piglets for either reprobed whole lung
homogenates or for either size group of reprobed small pulmonary
arteries (Table 3). Moreover, there was no difference in the absorbance
of bands for actin for whole lung homogenates or for homogenates of
either size group of small pulmonary arteries from samples of shunted compared with sham piglets containing total protein amounts within the
dynamic range for actin analysis (Table 3). Thus actin abundance was
unaltered in lung tissue samples from shunted piglets, and normalization of the eNOS and prostanoid enzyme data for actin amounts
does not change the results (Table 3).

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Fig. 8.
A-C: actin immunoblots in reprobed whole
lung homogenates (A) and homogenates of small pulmonary
arteries 100-600 µm in diameter (B) and larger
pulmonary arteries 700-1,200 µm in diameter (C).
D-F: actin immunoblots using total protein amounts
within the dynamic range for actin analysis in whole lung protein
homogenates (D) and homogenates of small pulmonary arteries
100-600 µm in diameter (E) and larger pulmonary
arteries 700-1,200 µm in diameter (F). Note that
there is no apparent difference in the intensity of the actin bands
between sham-operated and shunted piglets for either the reprobed lung
tissue homogenates or for homogenates using protein amounts within the
dynamic range of actin immunoblot analysis.
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DISCUSSION |
The major findings in this study were that 5-6 wk after
placement of an aortopulmonary shunt in newborn piglets, amounts of eNOS and of the prostanoid enzymes COX-1, COX-2, prostacyclin synthase,
and thromboxane synthase were unaltered in distal lung tissue and in
two different size groups of small pulmonary arteries (100-600
µm in diameter and 700-1,200 µm in diameter). Other important findings were that pulmonary vascular resistance was greater and pulmonary vascular dilation to both an endothelium-dependent and an
endothelium-independent agent was less in isolated lungs of shunted
piglets than in isolated lungs of sham-operated piglets. Moreover, in
vivo measurements in anesthetized piglets showed that pulmonary
arterial pressure was elevated in the shunted animals. Together these
findings suggest that mechanisms other than altered amounts of either
eNOS or the prostanoid enzymes COX-1, COX-2, prostacyclin synthase, and
thromboxane synthase underlie the elevated pulmonary arterial pressure
and the blunted dilation that develop in lungs of newborn piglets
5-6 wk after aortopulmonary shunt placement.
Other investigators have studied animals with aortopulmonary shunts to
investigate the development of pulmonary hypertension resulting from
high pulmonary blood flow. Previous investigators surgically placed
aortopulmonary shunts in piglets aged 1-3 mo and studied them
1-3 mo later (10, 22, 26). Their findings provided
evidence that pulmonary hypertension develops when aortopulmonary shunts are placed in growing piglets (10, 22, 26). In
contrast, experimental attempts to induce high-flow pulmonary
hypertension with surgically placed shunts in adult animals have met
with less success (16, 20). The presence of low pulmonary
vascular resistance at surgical placement, making the animals
susceptible to developing fatal congestive heart failure, is believed
to be one of the reasons that pulmonary hypertension is difficult to
induce with shunts in adult animals. We chose to surgically place
aortopulmonary shunts in piglets at an age before their pulmonary
vascular resistance decreases to adult levels (25) but at
an age allowing for acclimation to the experimental environment before surgery.
Similar to previous findings with shunted growing piglets (10,
22, 26), we found that pulmonary arterial pressure was significantly elevated in anesthetized piglets studied 5-6 wk after aortopulmonary shunt placement. A limitation of our study is that
pulmonary blood flow was not measured in vivo. Our finding that cardiac
output was 75% greater in shunted piglets than in sham-operated
piglets supports the presence of elevated pulmonary blood flow in the
shunted piglets. That pulmonary hypertension developed is supported by
the elevated pulmonary vascular resistance that we measured in
isolated, perfused lungs of the shunted piglets. Structural remodeling
in the shunted lungs (22, 26) may contribute to the
elevated pulmonary arterial pressure in isolated, perfused lungs of the
shunted piglets that persists after removal of vasomotor tone with papaverine.
Our finding of reduced responses to both the endothelium-dependent
agent ACh and the endothelium-independent agent papaverine suggests
that impaired smooth muscle dilation and not pulmonary vascular
endothelial dysfunction underlies altered pulmonary vascular responses
in the shunted piglets. By comparison, impaired responses to
endothelium-dependent agents but preserved responses to
endothelium-independent agents have been demonstrated both in children
aged 3-12 yr with left-to-right shunts and increased pulmonary
blood flow (7) and in 4-wk-old newborn lambs that had
undergone in utero surgical placement of aortopulmonary shunts
(24).
The mechanisms underlying the diminished dilation to papaverine, i.e.,
impaired smooth muscle dilation, in the shunted piglets are not clear.
Papaverine is a nonselective phosphodiesterase inhibitor
(21). It is possible that the ability to elevate cAMP and/or cGMP is impaired in pulmonary vascular smooth muscle of the
shunted piglets. However, papaverine may also cause dilation by
nonselective inhibition of voltage-activated calcium channels (18). Either possibility is consistent with our findings
of unaltered amounts of either eNOS or prostanoid enzymes in lung and
vessel homogenates of the shunted piglets. In other words, our findings
suggest that a mechanism downstream from altered synthesis of NO,
prostacyclin, and thromboxane underlies the blunted dilation that
develops in newborn piglets with shunt-induced pulmonary hypertension.
Like us, other investigators found no change in lung tissue eNOS
amounts resultant from altered flow in studies with 6-wk-old piglets
(22), adult rats (12), and fetal lambs
(29). In comparison, eNOS abundance was increased in
distal lung tissue and in fifth-generation pulmonary arteries of
4-wk-old newborn lambs who had undergone in utero surgical placement of
aortopulmonary shunts (6). The different results could be
due to use of different species and ages of animals and differences in
experimental conditions, such as the magnitude and duration of changes
in pulmonary blood flow.
Although a number of studies have evaluated eNOS abundance in both high
flow-induced (6, 12, 22) and other models of pulmonary
hypertension (14, 27, 32), the influence of prostanoid enzymes has received little attention (8, 30). Yet, it has long been hypothesized that altered production of prostanoid
metabolites could contribute to the pathogenesis of pulmonary
hypertension in both newborns and adults (1-3, 5, 8, 9, 15,
30). In this regard, it was recently reported that amounts of
COX-2, but not of COX-1, were increased in lungs of adult rats with
chronic hypoxia-induced pulmonary hypertension (8). Other
investigators recently reported decreased prostacyclin synthase
expression in lungs from patients with severe pulmonary hypertension
(30). Our findings do not support an important role for
either a decrease or an increase in the abundance of either COX-1,
COX-2, prostacyclin synthase, or thromboxane synthase and the elevation
in pulmonary arterial pressure found in newborn piglets with
aortopulmonary shunts for 5-6 wk.
Limitations in use of the immunoblot technique to assess protein
abundance must be acknowledged. For example, potential changes in lung
tissue composition in remodeled lungs make it difficult to know what
protein to use as an internal control. It is important that the
internal control remain unaltered with the experimental conditions. Our
finding that actin did not differ between lung tissue samples from
shunted and sham lungs supports the use of actin as an internal
control. Unfortunately, the amount of total protein needed to detect
eNOS and prostanoid enzymes was outside the dynamic range for actin
immunoblot analysis. Thus it is possible, although unlikely, that
differences in actin amounts were undetected in the "reprobed" samples.
In summary, our findings suggest that blunted pulmonary vascular
responses and pulmonary hypertension develop in piglets 5-6 wk
after placement of an aortopulmonary shunt because of impaired smooth
muscle dilation and not because of altered abundances of eNOS or the
prostanoid enzymes COX-1, COX-2, prostacyclin synthase, and thromboxane
synthase. Future studies are needed to clarify the mechanisms for the
impaired smooth muscle dilation.
 |
ACKNOWLEDGEMENTS |
We thank W. B. Campbell for the generous gift of the
thromboxane synthase antibody.
 |
FOOTNOTES |
This work was funded in part by a Children's Hospital Foundation grant
(C. Fike).
Address for reprint requests and other correspondence:
C. D. Fike, Dept. of Pediatrics, Wake Forest Univ. School of
Medicine, Medical Center Blvd., Winston-Salem, NC 27157 (E-mail:
cfike{at}wfubmc.edu).
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.
Received 3 November 2000; accepted in final form 22 March 2001.
 |
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