Inducible NO synthase inhibition attenuates shear
stress-induced pulmonary vasodilation in the ovine fetus
Robyn L.
Rairigh1,
Laurent
Storme1,
Thomas A.
Parker1,
Timothy D.
le
Cras2,
John P.
Kinsella1,
Malathi
Jakkula2, and
Steven H.
Abman3
2 Pediatric Heart Lung Center
and Sections of 1 Neonatology and
3 Pulmonary and Critical Care
Medicine, Department of Pediatrics, University of Colorado School of
Medicine, Denver, Colorado 80218-1088
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ABSTRACT |
Recent studies have suggested that type II
(inducible) nitric oxide (NO) synthase (NOS II) is present in the fetal
lung, but its physiological roles are uncertain. Whether NOS II
activity contributes to the NO-mediated fall in pulmonary vascular
resistance (PVR) during shear stress-induced pulmonary vasodilation is
unknown. We studied the hemodynamic effects of two selective NOS II
antagonists [aminoguanidine (AG) and
S-ethylisothiourea (EIT)], a
nonselective NOS antagonist
[nitro-L-arginine
(L-NNA)], and
a nonselective vasoconstrictor (U-46619) on PVR during partial
compression of the ductus arteriosus (DA) in 20 chronically prepared
fetal lambs (mean age 132 ± 2 days, term 147 days). At surgery,
catheters were placed in the left pulmonary artery (LPA) for selective
drug infusion, an ultrasonic flow transducer was placed on the LPA to
measure blood flow, and an inflatable vascular occluder was placed
loosely around the DA for compression. On alternate days, a brief
intrapulmonary infusion of normal saline (control), AG, EIT,
L-NNA, or U-46619 was infused in
random order into the LPA. The DA was compressed to increase mean
pulmonary arterial pressure (MPAP) 12-15 mmHg above baseline
values and held constant for 30 min. In control studies, DA compression
reduced PVR by 42% from baseline values
(P < 0.01).
L-NNA treatment completely
blocked the fall in PVR during DA compression. AG and EIT attenuated
the decrease in PVR by 30 and 19%, respectively
(P < 0.05). Nonspecific elevation in
PVR by U-46619 did not affect the fall in PVR during DA
compression. Immunostaining for NOS II identified this isoform in
airway epithelium and vascular smooth muscle in the late-gestation ovine fetal lung. We conclude that selective NOS II antagonists attenuate but do not block shear stress-induced vasodilation in the
fetal lung. We speculate that stimulation of NOS II activity, perhaps
from smooth muscle cells, contributes in part to the NO-mediated fall
in PVR during shear stress-induced pulmonary vasodilation.
nitric oxide; pulmonary circulation; pulmonary hypertension; persistent pulmonary hypertension of the newborn; lung development
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INTRODUCTION |
NITRIC OXIDE (NO) modulates basal pulmonary vascular
tone in the late-gestation fetus (2, 10, 11) and mediates pulmonary vasodilation to several pharmacological and physiological stimuli such
as acetylcholine (ACh), oxygen, and shear stress (2, 10, 26, 31, 34).
At birth, the pulmonary circulation undergoes a dramatic fall in
resistance that is, in part, due to stimulation of NO synthase (NOS)
activity (2, 3, 10, 13, 23, 34). As shown by several studies (2, 10,
11, 13, 23, 26), inhibition of NOS with
L-arginine analogs increases
basal pulmonary vascular resistance (PVR), blocks the NO-mediated fall
in PVR during shear stress-induced pulmonary vasodilation, and
attenuates the fall in PVR at birth.
Three distinct isoforms of NOS have been identified: type I or neuronal
(NOS I), type II or inducible (NOS II), and type III or endothelial
(NOS III) (10). NOS I and NOS III are constitutive and regulated in
part by calcium and calmodulin. Changes in expression of these NOS
isoforms have been characterized during lung development in the rat and
sheep fetus (3, 6, 17, 28, 37). NOS II is distinct from the other
isoforms in that its activity is independent of intracellular calcium
and it is regulated at the transcriptional level primarily through
induction by endotoxin and cytokines (6). NOS II has been identified in
the endothelium, epithelium, and macrophages in human and rat fetal
lungs by immunostaining (22, 37). Although NOS II has been
predominantly associated with pathophysiological
conditions including septic shock, autoimmune disease, and
inflammation (8, 12, 15, 19, 21, 25), its role in normal physiology is unknown.
Although past studies (17, 28, 37) have emphasized the potential role
of the NOS III isoform as the source of vascular NO production in the
perinatal lung, arginine analogs that were used to inhibit NOS activity
in physiological studies were not isoform selective (2, 10, 11, 13, 23,
26). Whether other NOS isoforms, including NOS II, contribute to NO
production in the normal perinatal pulmonary circulation is uncertain.
A recent study (29) demonstrated that NOS II is present in fetal sheep
lungs, and brief intrapulmonary infusions of selective NOS II
antagonists increase basal PVR in the ovine fetus. These findings suggest that NOS II may also play a "constitutive" role and
contribute to NO production in the normal fetal lung.
Although NOS II activity contributes to basal PVR, the effects of
physiological studies on NOS II activity are unknown. A past study (10)
demonstrated that inhibition of NOS blocks pulmonary vasodilation in
response to increased shear stress. To test whether NOS II contributes
in part to NO release during shear stress-induced pulmonary
vasodilation, we studied the hemodynamic effects of two selective NOS
II antagonists, aminoguanidine (AG) and
S-ethylisothiourea (EIT) (5, 9, 14,
16, 20, 27, 35, 38), during partial compression of the ductus
arteriosus (DA) in chronically prepared fetal lambs. We compared these
responses with the effects of
nitro-L-arginine
(L-NNA), a nonselective NOS
antagonist, and the effects of nonspecific elevation in basal PVR by
U-46619 treatment. Furthermore, to determine the the localization of
NOS II, we performed immunostaining on nonsurgical late-gestation ovine
fetal lungs.
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METHODS |
Surgical preparation. All procedures
and protocols were previously reviewed and approved by the Animal Care
and Use Committee at the University of Colorado Health Sciences Center
(Denver). Twenty mixed-breed (Columbia-Rambouillet, Nebekar
Ranch) pregnant ewes between 125 and 128 days gestation
(term 147 days) were fasted 24 h before surgery. The ewes were sedated
with intravenous pentobarbital sodium (2-4 g) and anesthetized
with 1% tetracaine hydrochloride (3 mg) by lumbar puncture. The ewes
were kept sedated with pentobaribital sodium but breathed spontaneously
throughout the surgery. Penicillin (500 mg) and gentamicin (2 g) were
administered to the ewes at surgery. Under sterile conditions, the
fetal lamb's left forelimb was delivered through a uterine incision. A
skin incision was made under the left forelimb after local infiltration
with lidocaine (2-3 ml, 1% solution). Polyvinyl catheters were
inserted into the axillary artery and vein and advanced into the
ascending aorta (Ao) and superior vena cava, respectively. A left
axillary to sternal thoracotomy exposed the heart and great arteries.
Polyvinyl catheters were inserted into the left pulmonary artery (LPA), main pulmonary artery (MPA), and left atrium (LA) by direct puncture and secured in position by purse-string catheters. The catheters were
guided into position with a 14- or 16-gauge intravenous placement unit
(Angiocath, Travenol, Deerfield, IL). The LPA catheter was inserted at
the bifurcation of the MPA and the DA and guided through the common
pulmonary artery into the LPA. The MPA catheter was inserted between
the DA and the pulmonic valve. A 10-mm inflatable occluder (In Vivo
Metric, Heldsburg, CA) was loosely placed around the DA to avoid any
compression before the study period (2). A 6-mm ultrasonic flow
transducer (Transonics Systems, Ithaca, NY) was placed around the LPA
to measure blood flow to the left lung. The thoracotomy and skin
incision were closed. The uteroplacental circulation was kept intact,
and the fetus was gently placed in the uterus, with exposed surfaces
bathed in warm towels. A catheter was placed in the amniotic space,
ampicillin (500 mg) was administered into the amniotic cavity, and the
hysterotomy was closed. The catheters and flow transducer cable were
tunneled subcutaneouly to an external pouch on the ewe. Ampicillin (500 mg) was infused daily in the fetal vein and amniotic cavity during the
first 3 days after surgery. The ewe was allowed to recover, and no
further anesthesia was given. The animals were allowed at least
48-72 h for recovery from the surgery before studies were
performed. Catheter patency was ensured by daily infusions of
heparinized saline (100 U/ml).
Physiological measurements. The Ao,
MPA, LA, and amniotic cavity catheters were connected to a
computer-driven pressure transducer and recorder (Biopac Systems, Santa
Barbara, CA). Pressures were referenced to the amniotic cavity
pressure. The pressure transducer was calibrated with a mercury column
manometer. The flow transducer cable was attached to an internally
calibrated flowmeter (Transonics Systems) for continuous measurement of
LPA flow. The absolute values of flow were determined from phasic blood
flow signals as previously described (18, 24). PVR in the left lung was calculated by the following equation: PVR (in
mmHg · ml
1 · min) = (mean MPA pressure
mean LA pressure)/LPA flow. Arterial blood
gas tensions, pH, hemoglobin, and oxygen saturation were measured from
blood samples that were drawn from the Ao catheter and measured at
39.5°C with a blood gas analyzer and hemoximeter (OSM-3,
Radiometer, Copenhagen, Denmark).
Drug preparation. All drugs were
freshly prepared on the day of study. Aminoguanidine hydrochloride
(Tocris Cookson, St. Louis, MO) at a dose of 140 mg was dissolved in 2 ml of sterile saline. EIT (Tocris Cookson) at a dose of 0.12 mg was
dissolved in 2 ml of sterile saline.
L-NNA (Sigma Pharmaceuticals,
St. Louis, MO) at a dose of 20 mg was dissolved with 1 M HCl, then 1 ml
of normal saline was added. NaOH (1 M) was added to titrate the pH to
7.40. U-46619 (9,11-dideoxy-9
,11
-epoxymethanoprostaglandin
F2
; Sigma Pharmaceuticals) at a
dose of 0.01-0.25 µg/min (diluted in sterile
saline) was titrated to increase basal PVR by 50%.
Experimental design. Studies were
performed after at least 48 h of recovery from surgery. Study drugs
were infused in random order on separate days, with a 1-day recovery
between different drug studies. The identical protocol was followed for
each drug: control (sterile saline; n = 4 animals, mean gestational age 130 days);
L-NNA (20 mg;
n = 4 animals, mean gestational age
130 days); AG (140 mg; n = 4 animals,
mean gestational age 130 days); EIT (0.12 mg;
n = 4 animals, mean
gestational age 131 days); U-46619 (0.01-0.25 µg/min;
n = 4 animals, mean gestational age 129 days). Hemodynamic
measurements were recorded at 10-min intervals throughout the study
period (baseline, drug infusion, ductal compression, and recovery).
After 30 min of stable baseline hemodynamic measurements, one of the
study drugs was infused into the LPA over 10 min. Immediately after the
drug infusion was complete, the DA was compressed by inflation of the
vascular occluder with saline, increasing mean pulmonary arterial
pressure (MPAP) by 12-15 mmHg from baseline values, and held
constant for 30 min. The selection of this level of MPAP was based on
our previous experience in which pulmonary blood flow doubles by 30 min
wthout causing changes in arterial blood gas tensions, pH, or aortic
pressure (AoP) (1). The increased MPAP was kept constant during the
study period by gently adjusting the degree of inflation as needed.
Fluctuations in pressure were minimized by continuously monitoring
MPAP. Heart rate, LPA blood flow, AoP, and LA and amniotic cavity
pressures were similarly recorded. After 30 min of ductal compression,
the occluder was rapidly deflated. Arterial blood gas tensions, pH, and
oxygen saturation were measured at baseline, after 30 min of partial ductal compression, and after 30 min of recovery.
The dosage for each drug was based on previous studies and those
published in the literature (9, 10, 29). The dose of
L-NNA was based on a past study
(2) that demonstrated a sustained pulmonary and systemic hypertension
and a decrease in pulmonary blood flow with inhibition of ACh-induced
pulmonary vasodilation. Selection of the doses for AG and EIT was based on a past study (29) demonstrating an increase in pulmonary and
systemic pressures and a decrease in pulmonary blood flow without
affecting ACh-induced pulmonary vasodilation, suggesting that AG and EIT selectively inhibit NOS II. U-46619 infusion was titrated to increase basal PVR by 50%.
Immunolocalization of NOS II protein in fetal lamb
lung. To determine the sites of NOS II protein
localization, we performed immunostaining of late-gestation fetal lung
tissue that was obtained from nonsurgical fetal lambs
(n = 4) at the same gestational age as
the animals used for physiological studies. At autopsy, lungs were
inflated by slow infusion of agarose (17). The pulmonary vasculature
was perfused at 30-40 cmH2O
pressure with saline, followed by buffered Formalin.
Five-micrometer-thick paraffin sections were serially mounted onto
Superfrost Plus slides (Fisher Scientific, Fairlawn, NJ). For NOS II
immunostaining, the slides were dewaxed in 100% xylene and then
rehydrated by immersion in 100% ethanol, 95% ethanol-5% water, 70%
ethanol-30% water, and then 100% water. Antigen retrieval was
performed by boiling the slides in 0.01 M citric acid, pH 6.0. The
slides were washed in PBS (1× PBS is 2.7 mM KCl, 1.2 mM
KH2PO4,
138 mM NaCl, and 8.1 mM
Na2HPO4).
Endogenous biotin in the tissue sections was blocked by glucose-glucose
oxidase treatment [0.2 M glucose and 1.5 U/ml of glucose oxidase
(Boehringer Mannheim Biochemicals) in 1× PBS]. The slides
were washed in 1× PBS. The sections were blocked with Super Block
(diluted 1:10 vol/vol in 1× PBS; Sky Tek, Logan, UT) and then
incubated with an anti-NOS II primary antibody (Santa Cruz
Biotechnology, Santa Cruz, CA) diluted 1:100 or an
IgG1-negative control (Jackson
Laboratories, West Grove, PA) in 1× PBS, 2% (wt/vol) bovine serum albumin (BSA), and 0.1% (wt/vol)
NaN3. After incubation with the
primary antibodies, the sections were washed in 1× PBS. Biotin-labeled anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA) was incubated with the sections at a dilution of 1:200
in 1× PBS, 2% (wt/vol) BSA, and 0.1% (wt/vol)
NaN3. Again, the slides were
washed in 1× PBS. The slides were incubated in streptavidin-biotin-horseradish peroxidase and developed with diaminobenzidine and hydrogen peroxide, with NiCl for enhancement (Vector). The NiCl enhancement-diaminobenzidine color development reaction was stopped by washing with water, and then the slides were
dehydrated in 70% ethanol-30% water, 95% ethanol-5% water, 100%
ethanol, and finally 100% xylene before coverslips were applied.
Data analysis. Data are presented as
means ± SE. Statistical analysis was performed with the Statview
4.5 software package (Abacus Concepts, Berkeley, CA) and the analysis
of variance SuperANOVA software package (Abacus Concepts). Comparisons
were made by using univariate repeated-measures by linear-contrast
analysis. P < 0.05 was considered significant.
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RESULTS |
Hemodynamic effects of partial compression of the
DA. In control studies, infusions of normal saline (0.2 ml/min for 10 min) had no effects on MPAP, AoP, or LPA blood flow.
After baseline measurements, the DA occluder was partially inflated to
rapidly increase MPAP by 15 mmHg above baseline values. This pressure was kept constant for 30 min. In control (saline-treated) animals, LPA
blood flow progressively increased above baseline by twofold during
partial DA compression (P < 0.01).
AoP did not change during the study period. Arterial blood gas
tensions, pH, oxygen saturation, and heart rate did not change during
or after partial compression of the DA (Table
1).
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Table 1.
Effects of nonselective NOS blockade, selective NOS II blockade, and
nonspecific elevation in PVR on arterial blood gas tensions, pH,
hemoglobin, O2 saturation, and heart rate
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Hemodynamic effects of a nonselective NOS antagonist
on pulmonary vasodilation during partial compression of the
DA. L-NNA infusion (0.2 ml/min for 10 min) into the LPA did not
affect MPAP, AoP, LPA blood flow, or the pressure
gradient between the pulmonary artery and the Ao. After infusions of
L-NNA, the DA occluder was partially inflated to elevate MPAP to the same level achieved during
the control study. In contrast with the twofold increase in LPA blood
flow in control studies, L-NNA
treatment inhibited the progressive pulmonary vasodilation after DA
compression (Fig. 1). LPA blood flow at
baseline was not different from measurements at 30 min of DA
compression after L-NNA
treatment [P = not
significant (NS); Fig. 1]. At 30 min of DA compression, LPA blood
flow in the L-NNA treatment
group was nearly one-half of the blood flow in the control group
(P < 0.001; Fig. 1). AoP, arterial
blood gas tensions, pH, oxygen saturation, and heart rate did not
change in this study group (Table 1).

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Fig. 1.
Effects of nonselective nitric oxide (NO) synthase (NOS) blockade on
left pulmonary arterial (LPA) blood flow during 30 min of partial
compression of ductus arteriosus (DA) in chronically prepared
late-gestation fetus. Mean pulmonary arterial pressure (MPAP;
top) and LPA blood flow
(bottom) are shown during control
and nitro-L-arginine
(L-NNA) treatment periods. Brief
infusions of L-NNA (20 mg)
inhibited progressive increase in LPA blood flow, suggesting that NO
mediates pulmonary vasodilation during partial compression of
DA.
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Hemodynamic effects of selective NOS II antagonists on
pulmonary vasodilation during partial compression of the
DA. AG infusion (0.2 ml/min for 10 min) into the LPA
increased MPAP from 45 ± 2 to 52 ± 3 mmHg at 10 min
(P < 0.05) and caused a parallel
rise in mean AoP (41 ± 1 to 44 ± 1 mmHg;
P < 0.05; Fig.
2). The pressure gradient between the
pulmonary artery and the Ao did not change after the infusion of AG
(Table 2). In comparison with control studies, AG attenuated the progressive pulmonary vasodilation after DA
compression. After the infusion of AG, LPA blood flow increased from 76 ± 17 ml/min at baseline to 149 ± 25 ml/min after 30 min of DA
compression (P < 0.01; Fig. 2). At
30 min of DA compression, LPA blood flow in the AG treatment group was
1.5-fold lower than blood flow in the control group
(P < 0.01; Fig. 2). AoP, arterial blood gas tensions, pH, oxygen saturation, and heart rate did not
change during partial compression of the DA (Table 1).

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Fig. 2.
Effects of selective NOS II blockade on LPA blood flow during 30 min of
partial compression of DA in chronically prepared late-gestation fetus.
MPAP (top) and LPA blood flow
(bottom) are shown during control
and aminoguanidine (AG) treatment periods. Brief infusions of AG (120 mg) increased MPAP before DA compression. AG treatment
attenuated but did not block progressive increase in LPA
blood flow, suggesting that NOS II contributes to NO-mediated pulmonary
vasodilation during partial compression of DA.
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Table 2.
Effects of nonselective NOS blockade, selective NOS II blockade, and
nonspecific elevation in PVR on pressure gradient between pulmonary
artery and aorta
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EIT infusion (0.2 ml/min for 10 min) into the LPA increased MPAP from
47 ± 2 to 51 ± 2 mmHg (P < 0.05) before inflation of the DA occluder, and there was no change in
mean AoP (P = NS; Fig.
3). The pressure gradient between the
pulmonary artery and the Ao did not change significantly after infusion
of EIT (Table 2). As observed with AG, EIT attenuated the progressive
pulmonary vasodilation during DA compression. DA compression after EIT
increased LPA blood flow from 80 ± 6 to 126 + 19 ml/min after 30 min (P < 0.01; Fig. 3). At 30 min of
DA compression, LPA blood flow in the EIT treatment group was 1.6-fold
lower than blood flow in the control group
(P < 0.01; Fig. 3). AoP, arterial
blood gas tensions, pH, oxygen saturation, and heart rate did not
change during partial compression of the DA (Table 1).

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Fig. 3.
Effects of selective NOS II blockade on LPA blood flow during 30 min of
partial compression of DA in chronically prepared late-gestation fetus.
MPAP (top) and LPA blood flow
(bottom) are shown during control
and S-ethylisothiourea (EIT) treatment periods. Brief
infusions of EIT (0.12 mg) increased MPAP before DA compression. EIT
treatment attenuated but did not block progressive increase in LPA
blood flow, suggesting that NOS II contributes to NO-mediated pulmonary
vasodilation during partial compression of DA.
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Hemodynamic effects of nonspecific elevation of basal
PVR during partial compression of the DA. Continuous
infusion of U-46619 (0.01-0.25 µg/min) increased MPAP from 49 ± 3 to 56 ± 1 mmHg after 10 min
(P < 0.01) and caused a parallel
rise in mean AoP from 45 ± 2 to 49 ± 3 mmHg
(P < 0.01; Fig.
4). After a 50% increase in basal PVR was
acheived, the DA occluder was partially inflated to elevate MPAP to the
same level achieved during the control study. In contrast to the NOS
antagonists, U-46619 did not affect progressive pulmonary
vasodilation after DA compression. During DA compression, LPA blood
flow increased from 117 ± 16 to 188 ± 9 ml/min
(P < 0.01; Fig. 4). At 30 min of DA
compression, LPA blood flow in the U-46619 treatment group was not
significantly different from blood flow in the control group
(P = NS; Fig. 4). AoP, arterial blood
gas tensions, pH, oxygen saturation, and heart rate did not change
during partial compression of the DA (Table 1).

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Fig. 4.
Effects of nonspecific elevation in basal pulmonary vascular resistance
(PVR) on LPA blood flow during 30 min of partial compression of DA
in chronically prepared late-gestation fetus. MPAP
(top) and LPA blood flow
(bottom) are shown during control
and U-46619 treatment periods. Continuous infusion of U-46619
(0.01-0.25 µg/min) increased MPAP and decreased LPA
blood flow to achieve an increase in basal PVR of 50% before DA
compression. Nonspecific elevation of PVR by U-46619 did not affect
progressive increase in LPA blood flow during partial compression of
DA.
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Comparisons of the effects on PVR during partial DA compression with
the selective NOS II antagonists, the nonselective NOS antagonist, and
the vasoconstrictor U-46619 are shown in Figs. 5-8.
In control animals, PVR decreased during partial DA compression by 40%
(P < 0.01). After the infusion of
L-NNA, AG, EIT, and U-46619, basal PVR increased by 23, 32, 15, and 47%, respectively, before DA
compression (P < 0.01). After 30 min
of partial DA compression, PVR decreased by 40% in control animals
(P < 0.01). AG and EIT attenuated
the decrease in PVR (30 and 19%, respectively;
P < 0.05) after 30 min of partial DA
compression, whereas L-NNA
completely blocked the decrease in PVR. Nonspecific vasoconstriction by
U-46619 had no effect on pulmonary vasodilation during DA
compression (Fig. 9).

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Fig. 5.
Effects of nonselective NOS blockade on PVR during partial compression
of DA in chronically prepared late-gestation fetus. Brief infusions of
L-NNA (20 mg) increased basal
PVR by 23% before DA compression.
L-NNA inhibited progressive fall
in PVR during partial compression of DA.
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Fig. 6.
Effects of selective NOS II blockade on PVR during partial compression
of DA in chronically prepared late-gestation fetus. Brief infusions of
AG (120 mg) increased basal PVR by 32% before DA compression. AG
attenuated but did not block progressive fall in PVR during partial
compression of DA.
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Fig. 7.
Effects of selective NOS II blockade on PVR during partial compression
of DA in chronically prepared late-gestation fetus. Brief infusions of
EIT (0.12 mg) increased basal PVR by 15% before DA compression. EIT
attenuated but did not block progressive fall in PVR during partial
compression of DA.
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Fig. 8.
Effects of nonspecific elevation in basal PVR on PVR during partial
compression of DA in chronically prepared late-gestation fetus.
Continuous infusion of U-46619 (0.01-0.25 µg/min) increased
basal PVR by 50% before DA compression. Nonspecific elevation of PVR
did not affect progressive fall in PVR during partial compression of
DA.
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Fig. 9.
Comparison of effects of nonselective NOS antagonist, selective NOS II
antagonists, and nonspecific vasoconstriction during partial
compression of DA. After 30 min of partial DA compression, AG and EIT
attenuated decrease in PVR, whereas
L-NNA completely blocked
decrease in PVR. Nonspecific vasoconstriction by U-46619 had no effect
on pulmonary vasodilation during partial compression of DA.
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Immunolocalization of NOS II in the fetal lung is shown in Fig.
10. As illustrated, NOS II protein was
detected in the airway epithelium and vascular smooth muscle. There was
no staining of vascular endothelium or airway smooth muscle in any of
the animals. Staining with an IgG control was negative.

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Fig. 10.
Immunolocalization of NOS II in late-gestation fetal lung tissue that
was obtained from nonsurgical fetal lambs at same gestational age as
animals used for physiological studies.
A: low-power view
illustrating distribution of NOS II in distal lung. Magnification,
×40. B: terminal bronchioli and
accompanying small pulmonary artery. Magnification, ×100.
C: high-power view of medium-size
airway and accompanying artery. NOS II protein was detected in airway
epithelium (solid arrow), with weaker staining of vascular smooth
muscle (open arrow). Magnification, ×200.
D: staining with an isotype-matched
control antibody (IgG) was negative. Magnification, ×100.
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DISCUSSION |
A past study (1) showed that acute compression of the DA progressively
increases pulmonary blood flow and decreases PVR in the late-gestation
fetus. The nonselective NOS antagonist
L-NNA inhibits the rise in
pulmonary blood flow and the decrease in PVR (10, 26), suggesting that
an abrupt increase in flow or shear stress stimulated NO formation by
one of the three known NOS isoforms. Although the endothelial or NOS
III isoform is the likely source of NO in response to elevated shear
stress flow, a recent study (29) demonstrated that NOS II mRNA is
present in the ovine fetal lung and that selective NOS II antagonists increase basal PVR in the fetus. To determine whether physiological stimuli such as shear stress can stimulate NOS II activity, we studied
the effects of selective NOS II antagonists on shear stress-induced fetal pulmonary vasodilation. We now report that two different selective NOS II antagonists (AG and EIT) attenuate the rise in pulmonary blood flow and decrease in PVR during partial DA compression. These findings suggest that shear stress stimulation of NO release is
in part due to the activation of the NOS II isoform.
A previous study (4) demonstrated that maternal administration of
selective NOS II antagonists caused contriction of the great vessels,
including the DA, in the fetal rat. It is uncertain, however, whether
these hemodynamic effects were due to direct blockade in the fetal lung
or secondary to effects on the maternal or placental circulation, such
as severe hypoxia or hypertension. A recent study (29) demonstrated
that brief intrapulmonary infusions of three different selective NOS II
antagonists did not increase the pressure gradient between the
pulmonary artery and the Ao, suggesting a lack of significant effect on
basal tone of the DA. We found that two selective NOS II antagonists
(AG and EIT) did not increase the pressure gradient between the
pulmonary artery and the Ao before partial compression of the DA.
We also report that NOS II protein is present in the late-gestation
ovine fetal lung and predominantly localizes to airway epithelium and
vascular smooth muscle. The strong immunostaining for NOS II was
detected in the airway epithelium, with weaker staining of the vascular
smooth muscle. The physiological data presented in this study do not
allow the specific identification of the cell type responsible for the
NOS II activity that caused NO release in response to shear stress.
However, it is most likely that the hemodynamic changes caused by
partial DA compression are more likely to be activation of NOS II in
the vascular smooth muscle than in the airway epithelium. Shear stress
predominantly acts on the vascular endothelium to stimulate NO release
(35, 37). Whether shear stress or other physiological stimuli such as
cyclic stretch or increased wall tension or pressure can act on
vascular smooth muscle to release NO is uncertain.
These findings are interesting because little is known about the role
of NOS II in the regulation of vascular tone in the normal fetal lung.
Although past studies (17, 28, 37) have emphasized the role of the NOS
III isoform as the source of vascular NO production in the perinatal
lung, arginine analogs that were used to inhibit NOS activity in
physiological studies were not isoform selective (2, 10, 11, 13, 23,
26). Whether other NOS isoforms including NOS II contribute to NO
production in the normal perinatal pulmonary circulation is uncertain.
Although NOS II has been shown to play a role in the pathophysiology of shock, autoimmune disease, and chronic inflammation (8, 12, 19, 21),
few studies have expanded its potential physiological roles in normal
circulation. A recent study (29) showed that NOS II may contribute to
the regulation of basal vascular tone in the normal developing fetal
lung. These data provide further support for the hypothesis that the
NOS II ("inducible") isoform is constitutively expressed and that
it actively produces NO under basal and stimulated conditions in the
fetal pulmonary circulation.
Shear stress- or flow-induced vasodilation is mediated by the release
of vasoactive substances such as NO, prostacyclin, and endothelium-dependent hyperpolarization factor from the endothelium (1,
7, 10, 30). Previous studies (1, 2, 10) that looked at shear
stress-induced pulmonary vasodilation in the fetus were based on
nonselective NOS blockade, thus inhibiting all three isoforms of NOS.
With nonselective blockade of NOS with L-NNA, shear stress-induced
pulmonary vasodilation is completely blocked. Two different selective
NOS II antagonists attenuated shear stress-induced vasodilation,
suggesting that NOS II activity may contribute to NO production during
shear stress-induced vasodilation. Shear stress (3-24 h) has been
shown to enhance expression of NOS III mRNA in a dose-dependent manner
and increase the capacity of endothelial cells to release NO
chronically (36). In cell culture studies of the acute endothelial cell
response to the onset of flow, there is an abrupt increase in NO
release (33), with an increase of several orders of magnitude in NO
release over the shear stress range. This provides evidence of shear
stress as a very sensitive regulator of NO. During acute partial
compression of the DA, an abrupt increase in blood flow is more likely
due to acute NO release than to transcriptional upregulation of NOS III. The increase in blood flow (shear stress) along the endothelial surface causes the release of NO from NOS III, which may contribute to
pulmonary vasodilation (10). This same increase in blood flow may also
stimulate NOS II in the vascular smooth muscle to release NO,
contributing to NO-mediated pulmonary vasodilation in an autocrine
pathway. Alternatively, diffusion of NO produced from airway epithelium
could act on smooth muscle cells of adjacent pulmonary arteries, but
this seems less plausible. We speculate, however, that perhaps
ventilation-induced release of NO (10) could directly stimulate
epithelial NOS II activity.
Each of the NOS II antagonists studied is competitive with
L-arginine, but the exact
mechanism of action remains unclear. AG, a non-amino acid inhibitor, is
thought to inhibit NOS II by binding as a ligand to the heme iron at
the catalytic site because AG deactivates other iron- or
copper-containg enzymes in this manner (32). The isothiourea EIT has
been shown to alter the heme spectra of NOS, suggesting binding to or
interaction with the heme center (14, 32). Each agent has the potential
at high doses to also inhibit NOS I and NOS III activities (27, 35). AG
has been previously shown to have no effect on ACh-induced vasodilation, suggesting that AG inhibits NOS II selectively without blocking NOS III (9, 16, 20, 38). EIT has been described as a very
potent and selective inhibitor of NOS II (5, 14, 27, 34). In vitro
studies (27, 35) showed that AG and EIT are 80- and 30-fold more potent
for NOS II than for NOS III, respectively. The
IC50 for EIT was 13 nM, indicating
that EIT is ~1,000-fold more potent than AG
(IC50 value of 12 µM) (27, 35).
A recent study (29) showed that AG and EIT elevated fetal PVR without
blocking ACh-induced pulmonary vasodilation, suggesting that these
inhibitors blocked NOS II selectively without affecting NOS III. The
doses of AG and EIT used in our study were previously described to have
no effect on ACh-induced pulmonary vasodilation, suggesting that they
are selective for NOS II inhibition (29).
We conclude that nonselective blockade of NOS inhibits shear
stress-induced pulmonary vasodilation in the late-gestation fetal lamb.
Selective blockade of NOS II (inducible) attenuates shear stress-induced vasodilation. Nonspecific pharmacological elevation in
basal PVR does not block shear stress-induced vasodilation. These
findings support the hypothesis that NOS II activity contributes to the
NO-mediated shear stress-induced pulmonary vasodilation. NOS II located
in the smooth muscle cell and airway epithelium may contribute to the
release of NO and the marked decrease in PVR at birth.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. L. Rairigh, Neonatology, Box B070, The Children's Hospital,
1056 E. Nineteenth Ave., Denver, CO 80218-1088.
Received 17 August 1998; accepted in final form 9 December 1998.
 |
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