© 1997 American Heart Association, Inc.
Articles |
Pulmonary Vasoconstriction and Hypertension in Mice With Targeted Disruption of the Endothelial Nitric Oxide Synthase (NOS 3) Gene
From the Department of Anaesthesia and Critical Care (W.S., F.I., W.E.H., R.C.J., W.M.Z.) and the Cardiovascular Research Center (P.L.H., M.C.F.), Massachusetts General Hospital, Harvard Medical School, Boston, Mass, and the Department of Pharmacology (J.A.B.), University of Vermont College of Medicine, Burlington.
Correspondence to Warren M. Zapol, MD, Reginald Jenney Professor of Anaesthesia, Department of Anaesthesia and Critical Care, Massachusetts General Hospital, Harvard Medical School, 32 Fruit St, Boston, MA 02114.
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Abstract |
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Abstract NO, synthesized in endothelial cells by endothelial NO synthase (NOS 3), is believed to be an important endogenous pulmonary vasodilator substance that contributes to the normal low pulmonary vascular resistance. To selectively investigate the role of NOS 3 in the pulmonary circulation, mice with targeted disruption of the NOS 3 gene were studied. Pulmonary hemodynamics were studied by measuring pulmonary artery pressure, left ventricular end-diastolic pressure, and lower thoracic aortic flow by using a novel open-chest technique. Transient partial occlusion of the inferior vena cava was used to assess the pulmonary artery pressure-flow relationship. Tension developed by isolated pulmonary artery segments after acetylcholine stimulation was measured in vitro. The histological appearance of NOS 3–deficient and wild-type murine lungs was compared. NOS 3–deficient mice (n=27), when compared with wild-type mice (n=32), had pulmonary hypertension (pulmonary artery pressure, 19.0±0.8 versus 16.4±0.6 mm Hg [mean±SE]; P<.05) that was due to an increased total pulmonary resistance (62±6 versus 33±2 mm Hg·min·g·mL-1; P<.001). In vitro, acetylcholine induced vasodilation in the main pulmonary arteries of wild-type but not NOS 3–deficient mice. The morphology of the lungs of NOS 3–deficient mice did not differ from that of wild-type mice. We conclude that NOS 3 is a key enzyme responsible for providing basal pulmonary NO release. Congenital NOS 3 deficiency produces mild pulmonary hypertension in mice.
Key Words: targeted nitric oxide synthase 3 gene disruption • pulmonary vascular resistance • nitric oxide • mouse • acetylcholine
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Introduction |
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Endothelial NOS 3 is a constitutive calcium-dependent NOS isoform,1 which synthesizes NO from the terminal guanidino nitrogen of L-arginine. NO diffuses rapidly to adjacent vascular smooth muscle cells, where it binds to the ferrous heme moiety of soluble guanylate cyclase. The resulting nitrosyl heme activates soluble guanylate cyclase to produce cGMP, which relaxes vascular smooth muscle cells.2 3 In the human lung, NOS 3 has been detected in both endothelial and airway epithelial cells.4 5 It is believed that NO is responsible for maintaining the low normal PVR of children6 and adults.7 8
Pathological states in which NO production or availability is diminished appear to result in pulmonary hypertension.9 Primary pulmonary hypertension has been shown to be associated with decreased levels of NOS 3 in the lung.4 The therapeutic inhalation of NO reverses hypoxic pulmonary vasoconstriction and pulmonary hypertension and improves gas exchange in pulmonary injury by redistributing pulmonary blood flow toward ventilated lung regions.10 11 These observations have led to the hypothesis that NO produced by NOS 3 is critical for maintaining the normally low PVR. Therefore, a congenital absence of NOS 3 might result in increased PVR if other vasodilator substances were not upregulated or if vasoconstrictors were not reduced. With the recent generation of mice with a disrupted NOS 3 gene,12 an animal model has become available with which to study the role of NOS 3 in the pulmonary circulation. These NOS 3–deficient mice exhibit systemic arterial hypertension but grow and reproduce normally. Isolated aortic segments from NOS 3–deficient mice do not relax in response to ACh.12
In the present study, we investigated the consequences of congenital NOS 3 gene deficiency on pulmonary vascular hemodynamics and morphology in mice. Pulmonary hemodynamics were measured during anesthesia in both NOS 3–deficient and wild-type mice by using a newly developed open-chest technique that allows the simultaneous measurement of SAP, PAP, LTAF, and left ventricular pressure. The results obtained in NOS 3–deficient mice were compared with values measured in mice receiving a nonselective NOS inhibitor either acutely or chronically.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After institutional approval by the animal research committee, we studied NOS 3–deficient mice and SV 129 wild-type mice, of either sex, weighing 20 to 30 g. The generation of these NOS 3–deficient mice has been described in detail by Huang et al.12
Hemodynamic Studies
Anesthesia and Surgical Technique
Mice were anesthetized with 2% to 3% halothane
(Fluothane, Ayerst Laboratories) in oxygen in a 1-L chamber. While
spontaneously breathing, the mouse was placed supine on a heated
operating table; a 16-gauge thermistor was inserted into the rectum,
and body temperature was maintained between 37°C and 38.5°C.
Anesthesia was continued with halothane inhalation via a
small mask. All surgical procedures were observed with a dissecting
microscope (magnification, x7 to x20; Bausch & Lomb). A tracheotomy
was performed, and an endotracheal tube (20-gauge Angiocath, Becton
Dickinson) was inserted and sutured in place with a 4-0 silk ligature.
Volume-controlled ventilation (Harvard small animal ventilator model
683) was initiated at a respiratory rate of 85/min, a tidal volume of
0.025 mL/g body wt, and an inspired oxygen fraction of 1.0. Halothane
was discontinued, and anesthesia was maintained with
high-dose intraperitoneal fentanyl injections
(0.001 to 0.002 mg/g, Abbott Laboratories), with pancuronium (0.002
mg/g IP, Gensia Pharmaceuticals) added to produce muscle relaxation.
Airway pressure was continuously monitored, and the inspired volume was
adjusted to produce a peak inspiratory pressure of 15 mm Hg at a
positive end-expiratory pressure level of 1 cm H2O.
To obtain continuous SAP measurements and to sample blood gas tensions and pH, a right carotid artery catheter (a short length of PE-10 tubing connected to PE-50 tubing) was inserted via an arteriotomy. For fluid replacement, the left femoral vein was punctured with a 30-gauge intravenous needle and was adhered to the vessel with glue (Super-Glue, Melville Co). A midline skin incision from the lower xiphoid cartilage to the pretracheal region was performed, and the skin was retracted laterally. A substernal abdominal midline incision was used to access the abdominal surface of the diaphragm. The diaphragm was incised at its sternal insertion to induce a pneumothorax without injuring the lung or heart. A right parasternal thoracotomy from the xiphoid process to the manubrium was performed. The thoracic walls and the diaphragm were retracted with sutures to provide a wide view of the heart, lung, inferior vena cava, esophagus, and lower thoracic aorta. In order to access the lower thoracic aorta, the thoracic esophagus was transected behind the inferior vena cava. A 1-mm ultrasonic flow-probe (1RB, Transonic Instruments) was placed around the lower thoracic aorta. Hemostasis was obtained with electrocautery. Exhaled CO2 concentrations (Siemens Sirecust 960) were continuously measured, and arterial blood gas samples were intermittently analyzed. The ventilatory rate was adjusted (range, 75/min to 95/min), and sodium bicarbonate (8.4%, Abbott Laboratories) was infused to maintain an arterial pH of 7.35 to 7.40. Sampled blood volume was replaced with either an infusion of Ringer's lactate or 5% bovine albumin.
A PA catheter (30-gauge intravenous needle connected to PE-10 and PE-50 tubing) was placed by direct puncture of the PA and adhered with glue. The tubing was connected to the pressure transducer, which was mounted on a micromanipulator. In experiments assessing LVEDP, a 24-gauge plastic intravenous catheter (Angiocath, Becton Dickinson) was passed across the lateral wall of the left ventricle and directly connected to a pressure transducer.
Blood Pressure and Flow Measurements
For all blood pressure measurements, saline-filled membrane
transducers (Argon, Maxxim Medical; natural frequency, >100 Hz) were
used. In order to reduce damping and improve the frequency response of
the system, connecting tubing was short and of large diameter. Mean
SAP, mean PAP, and, in some experiments, left ventricular
pressure were continuously monitored using biomedical amplifiers
(Hewlett Packard 8805C and Siemens Sirecust 960). Mean LTAF was
measured with a small-vessel flow probe connected to a flowmeter (T106,
Transonic Instruments). All signals were transferred to an
analog-to-digital convertor and displayed on a computer screen (see Fig 1
) and recorded at 600 Hz (for LVEDP measurements at
1500 Hz) using a data acquisition system (DI 220, Dataq Instruments).
All monitoring equipment was calibrated before each experiment.
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Pulmonary Pressure-Flow Relationships
The inferior vena cava was partially occluded with a
circumferential 4-0 silk ligature to transiently reduce the LTAF by a
maximum of 60% to 80% for 2 to 3 seconds to obtain
pressure-flow relationships. The slope and intercept of the PAP/LTAF,
SAP/LTAF, and (PAP-LVEDP)/LTAF relationships (see definitions below)
were calculated using a two-point analysis, reporting the
steady state values before occlusion and the values obtained after a
transient 50% LTAF reduction (see Fig 2).
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The following hemodynamic values were obtained:
Before thoracotomy, values were determined for initial
(mm Hg), which is the initial mean SAP after
insertion of the arterial line, and for the initial heart
rate (min -1), which is the initial heart rate
after insertion of the arterial line. After thoracotomy,
values were determined for (mm Hg), which is mean
SAP; TSR (mm Hg·min·g·mL-1),
which is incremental total systemic (vascular) resistance (slope of
/
relationship);
(mL·min-1·g-1),
which is mean LTAF/body weight; 
(mm Hg), which is
mean PAP; TPR (mm Hg·min·g·mL-1),
which is incremental total pulmonary (vascular) resistance
(slope of / relationship); and
CP (mm Hg), which is pulmonary vascular CP (zero flow
intercept of / relationship).
In experiments assessing LVEDP, values were determined for LVEDP
(mm Hg); PVR (mm Hg·min·g·mL-1),
which is incremental PVR [slope of the
(-LVEDP)/ relationship]; and
CP (mm Hg), which is pulmonary vascular CP [zero flow
intercept of the (-LVEDP)/
relationship].
Acute and Chronic Nonspecific NOS Inhibition
Acute NOS inhibition was produced in wild-type mice (n=7)
by intravenous injection of 0.1 mg/g body wt L-NAME
(Sigma Chemical Co) dissolved in 0.1 mL normal saline. To more
chronically inhibit NOS, wild-type mice (n=10) were
anesthetized with halothane 5 days before central
hemodynamic measurements were obtained. A micro-osmotic
pump (Alzet 1077D), which continuously delivered 0.1 mg/g body wt per
day of L-NAME, was implanted under sterile conditions into the
peritoneal cavity. The abdominal wall was closed with sutures. The
anesthetic was discontinued, and after mice recovered from surgery,
they were returned to their cages for 5 days.
Pharmacological Interventions
NO was delivered from an 800 ppm tank (BOC Gases Inc) via a
rotameter and mixed with oxygen. Inspired concentrations were sampled
at a side port before entering the respirator and measured by
chemiluminescence (Eco Physics CLD 700 AL). The inspired oxygen
fraction was maintained constant throughout each study at 0.8 by adding
nitrogen to the inspired gas mixture. A soda lime filter within the
inspiratory limb removed NO2. SNP (Elkins-Sinn) was
dissolved in lactated Ringer's solution to obtain a concentration of
10 mg/mL and injected as a 0.005 mL/g body wt (0.05 mg/g) bolus via the
PA catheter. Indomethacin (Sigma) was dissolved in an
aqueous solution containing nine parts lactated Ringer's solution and
one part 8.4% sodium bicarbonate to achieve a concentration of 2
mg/mL; 0.005 mL/g body wt (0.01 mg/g) of the solution was injected
intravenously. ACh (Kodak) was dissolved in lactated
Ringer's solution at a concentration of
1.8x10-5 mg/mL and injected as a bolus of
0.005 mL/g body wt (9x10-8 mg/g) via the PA
catheter.
In Vitro PA Segment Studies
The main PA was dissected free from wild-type (n=6) and NOS
3–deficient (n=6) mice after rapid decapitation and a midline
thoracotomy and was placed in 37°C physiological
saline bubbled with 95% O2 and 5% CO2.
Segments (2 mm) were mounted on tungsten wires (outer diameter,
0.002 mm) in conventional myographs connected to a pen
recorder and maintained at optimum resting tension (1000 mg) for 60
minutes. After preconstriction with 5-hydroxytryptamine
(10-6 to 10-5 mol/L),
ACh (10-7 to 10-5
mol/L) or SNP (10-8 to
3x10-7 mol/L) was added. The same
pharmacological interventions were repeated in wild-type PA segments
(n=5) 30 minutes after exposing the tissue to
physiological saline containing
10-5 mol/L L-NA.
Morphometric Analysis
After pentobarbital euthanasia (0.1 mg/g IP), lungs obtained
from three wild-type mice and lungs from three NOS 3–deficient mice
were analyzed. The lungs were fixed,13 processed,
and embedded in Historesin Plus (Leica). The degree of alveolar
distension, the distribution of macrophages, erythrocytes, and
leukocytes, and the size of the vessel walls were examined in each of
three tissue sections. The abundance of vessels was assessed by
calculation of the vessel-to-alveolar ratio in sections through the
middle of the apical region of the left lung. A vessel was defined as a
structure >20 µm in diameter, containing erythrocytes, with
typical lining structures identifiable as smooth muscle cells or
endothelial cells. We used a Histostain SP Kit (Zymed)
and a monoclonal antibody against 
-smooth muscle actin (Sigma) at a
dilution of 1:400 with aminoethylcarbazole as the chromogen to stain
smooth muscle cells.
Statistical Analysis
The slope and intercept of each pressure-flow relationship was
calculated using a two-point analysis (Fig 2
). At baseline, two
partial inferior vena caval occlusions were performed. The
calculated slope and intercept of both occlusions were averaged to
obtain a single baseline value for TPR (slope) and CP (zero flow
intercept) per mouse. In a similar manner, two partial
inferior vena caval occlusions were performed before and
after each pharmacological intervention.
Differences between groups were determined using an unpaired Student's
t test. The effects of interventions were analyzed
using a paired t test and by multiple-regression
analysis. The dose-response data were examined using a repeated
measures analysis and an unpaired t test.
Dichotomous qualitative group differences were assessed using a
2 test. A statistically significant difference
was assumed with a value of P<.05. All data are expressed
as mean±SE.
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Results |
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Pulmonary and Systemic Hemodynamic Measurements in NOS 3–Deficient Mice
Mice congenitally deficient in NOS 3 had mild pulmonary hypertension with a PAP of 19.0±0.8 mm Hg, which was significantly greater (P<.05) than the PAP in wild-type mice (16.4±0.6 mm Hg, Table
). The elevated PAP in
NOS 3–deficient mice was due to an elevated TPR (62±6
mm Hg·min·g·mL-1), which was
substantially higher (P<.001) than the TPR of wild-type
mice (33±2
mm Hg·min·g·mL-1). Determination
of PVR (including LVEDP measurements, Fig 3) confirmed
this elevation: the PVR of 63±12 mm Hg 
min·g·mL-1 in NOS 3–deficient mice
was significantly increased (P<.05) compared with the PVR
of 27±5 mm Hg·min·g·mL-1 in
wild-type mice. The LVEDP was low and did not differ between the two
groups. The pulmonary vascular CP (Fig 3) in NOS
3–deficient mice did not differ from that in wild-type mice. NOS
3–deficient mice had a significantly (P<.01) decreased
LTAF of 0.21±0.02
mL·min-1·g-1
compared with wild-type mice (0.31±0.02
mL·min-1·g-1).
In 78% of the NOS 3–deficient mice, but in only 22% of the wild-type
mice, LTAF was <0.25
mL·min-1·g-1
(P<.01, Fig 4). Before thoracotomy, NOS
3–deficient mice exhibited a significantly (P<.01)
increased heart rate (586±13 versus 542±8
min-1) and SAP (156±5 versus 119±4
mm Hg, P<.001) compared with wild-type mice. After
thoracotomy, systemic arterial hypertension persisted and
was noted to be due to an increased TSR (283±29 versus
203±14 mm Hg·min·g·mL-1,
P<.001).
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Pulmonary and Systemic Hemodynamic
Measurements After Chronic and Acute NOS Inhibition
Administration of L-NAME for 5 days was associated with a
small increase of TPR (38±2 versus 33±2
mm Hg·min·g·mL-1,
P<.05) but not pulmonary hypertension (Table).
Treatment of wild-type mice with L-NAME for 5 days produced significant
(P<.05) systemic arterial hypertension (138±7
versus 119±4 mm Hg) due to an elevated TSR (293±43 versus
203±14 mm Hg·min·g·mL-1,
P<.05). Acute NOS inhibition with L-NAME injection
increased SAP (134±6 versus 111±3 mm Hg) and TSR (280±33
versus 203±14
mm Hg·min·g·mL-1) in wild-type
mice (P<.05). However, there was no significant increase in
PAP or TPR after an acute L-NAME injection. None of the mice treated
acutely with L-NAME had an LTAF of <0.25
mL·min-1·g-1,
whereas 8 of 10 (P<.001) of the mice treated for 5 days
with L-NAME had an LTAF of <0.25
mL·min-1·g-1
(Fig 4).
Effects of NO Inhalation and SNP Administration
When 80 ppm NO was inhaled for 5 minutes by NOS 3–deficient
mice (n=5), L-NAME–treated mice (n=5), or untreated wild-type mice
(n=4), no measured pulmonary or systemic
hemodynamic parameter changed significantly
(data not shown). Intra-PA injection of SNP (0.05 mg/g body wt) caused
a significant (P<.001) and transient reduction of SAP by
59±8, 51±5, and 44±6 mm Hg in NOS 3–deficient mice (n=6),
L-NAME–treated mice (n=7), and wild-type mice (n=6), respectively, due
to a marked reduction of TSR (P<.05). SNP did not produce
any changes in PAP and TPR in L-NAME–treated or NOS 3–deficient mice.
In wild-type mice, PAP decreased from 15.4±2.0 to 13.8±2.2
mm Hg after injection of SNP (P<.05).
In vitro, after exposure to increasing concentrations of SNP, a
similar degree of vasodilation was observed in preconstricted PA
segments of NOS 3–deficient mice (n=6), untreated wild-type mice
(n=6), and wild-type mice acutely treated with an NOS
inhibitor (n=5). Maximum dilation (100%) was obtained at
an SNP concentration of 
10-7 mol/L in all
segments.
Hemodynamic Effects of
Cyclooxygenase Inhibition by Indomethacin
Indomethacin injection did not significantly alter
any measured pulmonary or systemic hemodynamic
parameter in NOS 3–deficient (n=5) or wild-type (n=5) mice
within 5 minutes of administration (data not shown).
Effects of ACh Administration In Vivo and In Vitro
In vivo, intra-PA injection of 9x10-8
mg/g body wt ACh (Fig 5) induced profound systemic
arterial hypotension in wild-type mice. The SAP decrease
after ACh injection was less (P<.05) in NOS 3–deficient
mice (
SAP, 21±5 mm Hg) and in mice chronically treated with
L-NAME (SAP, 15±5 mm Hg) compared with wild-type mice
(SAP, 47±7 mm Hg). TSR decreased after ACh injection in
wild-type (P<.001) and L-NAME–treated mice
(P<.01), whereas it did not significantly change in NOS
3–deficient mice. The PAP in NOS 3–deficient mice was significantly
increased (P<.05) after ACh injection by 1.25±0.3
mm Hg because of an increased TPR, whereas PAP and TPR
remained unchanged in both wild-type and L-NAME–treated mice.
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, n=5),
wild-type mice (
, n=6), and mice treated for 5 days with L-NAME
(
, n=9). ACh decreased the TPR significantly in wild-type mice
and increased PAP and TPR in NOS 3–deficient mice. C indicates control
before ACh; ACh was recorded 1 minute after ACh injection.
*P<.05, **P<.01, and ***P<.001 vs
control value. 
Effect of ACh injection (increase vs decrease) differs
significantly from effect in wild-type mice (P<.05).
In vitro, endothelium-dependent vasodilation produced
by ACh addition (Fig 6) was detectable in wild-type
murine PA segments up to an ACh concentration of
10-5 mol/L. In contrast to untreated wild-type
PA segments, ACh-induced vasoconstriction was noted in wild-type PA
segments after NOS inhibition. PA segments obtained from NOS
3–deficient mice were unaffected by the addition of ACh.
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Pulmonary Morphology
The gross morphology of bronchial, alveolar, and pulmonary
vascular structures was similar in wild-type (n=3) and NOS 3–deficient
(n=3) mice. There was no difference in the number or muscularity of
pulmonary vessels, as demonstrated by immunocytochemical
-smooth muscle actin localization. Actin was present in both
genotypes in the walls of bronchi and large bronchioles, as
well as in the vessels close to larger bronchioles (bronchial
arteries). Intrapulmonary inflammatory cells were not evident
in either wild-type or NOS 3–deficient animals.
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Discussion |
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The major finding of the present study is that the congenital absence of NOS 3 in anesthetized mice causes mild pulmonary hypertension, associated with a significant decline in LTAF, but no increase in LVEDP. The elevation of PAP is attributed to the increased PVR, which was more than doubled in NOS 3–deficient compared with wild-type mice. Acute and chronic NOS inhibition in wild-type mice caused significant systemic arterial hypertension and vasoconstriction without significant pulmonary hypertension. Although preconstricted PA segments of NOS 3–deficient mice dilated when exposed to low concentrations of SNP in vitro, indicating normal soluble guanylate cyclase activity and intact NO signal transduction, in vivo PAP and TPR were not significantly diminished by 80 ppm NO inhalation or SNP injection. No evidence of structural changes in the pulmonary vasculature was found in NOS 3–deficient mice. ACh-induced pulmonary and systemic vasodilation was present in wild-type mice but absent in NOS 3–deficient mice.
Effects of NOS Inhibition and Congenital NOS 3 Deficiency on the
Pulmonary Vasculature
NO, synthesized by endothelial cells in the
vasculature of the lung, is believed to contribute to the low-pressure
and resistance characteristics of the normal pulmonary
circulation.6 7 8 14 Studies in humans and animals have
examined the role of basal NO release in pulmonary vascular
tone.6 7 8 14 15 16 17 18 19 20 21 22 23 24 25 26 A common approach is to decrease NO
synthesis by the administration of L-arginine analogues,
which inhibit all three isoforms of NOS with varying isoform
selectivity. Stamler et al7 demonstrated in healthy human
volunteers that a short-term infusion of the NOS inhibitor
L-NMMA acutely increased the PVR by 40% and decreased cardiac output
by 28%, associated with a decrease of plasma NO concentrations.
Pulmonary hypertension was not reported in that study of 11
volunteers.
In isolated/perfused lungs of pigs, sheep, and humans,14 L-NAME increased the PAP-flow slope, but it did not have this effect in isolated/perfused dog or rat lungs.14 15 26 In anesthetized dogs, however, L-NMMA significantly increased the PVR.27 Anesthetized rabbits developed PA hypertension and an increase in PVR after L-NAME administration.21 Awake newborn lambs also demonstrated pulmonary hypertension after acute L-NA administration.19 Hampl et al16 investigated the effects of long-term NOS inhibition on the pulmonary circulation of the rat. They reported that oral intake of L-NAME for 3 weeks caused systemic hypertension and vasoconstriction, an increased TPR, and a decreased cardiac output, but no pulmonary hypertension or pulmonary vascular remodeling. In isolated lungs of these NOS-inhibited rats, these investigators reported a shift of the PAP-flow relationship toward a higher pressure, but the slope (incremental resistance) did not increase.
In summary, acute and chronic NOS inhibition increased PVR in many species but did not consistently cause pulmonary hypertension in vivo. NOS inhibition in humans, dogs, rats, and sheep was also associated with a decreased cardiac output and stroke volume.7 16 28 29 It appears that cardiac output might be decreased secondary to an increased afterload or a decreased preload or an effect of altered NOS activity on contractility. Our results in L-NAME–treated mice confirm these aspects of previous NOS inhibitor studies, whereas in NOS 3– deficient mice, the increased PAP at baseline and the substantially increased (incremental) PVR suggest that congenital absence NOS 3 affects the pulmonary circulation and raises its tone to a greater degree than does pharmacological NOS inhibition.
Cyclooxygenase Inhibition in NOS
3–Deficient Mice
The congenital absence of NOS 3 activity might have an
effect on other pulmonary vascular tone mediators that are
involved in the regulation of basal PVR. Examples of such vasoactive
mediators in the pulmonary circulation are
thromboxane and prostacyclin, a vasoconstrictor and
vasodilator, respectively. In vitro, endothelial NO
release is associated with both prostacyclin and
thromboxane liberation.30 This is attenuated
by NOS inhibitors and hemoglobin, suggesting that NO
interacts with and activates prostanoid formation. It is
unlikely, however, that important upregulation of prostanoid formation
occurred in NOS 3–deficient mice, since pulmonary
hemodynamics were unchanged after injection of the
prostanoid synthesis inhibitor,
indomethacin.
NO-Mediated Vasodilation In Vivo and In Vitro
The hypothesis that pulmonary hypertension in NOS
3–deficient mice is due to the absence of continuous pulmonary
endothelial NO synthesis led us to examine whether the
increased PVR could be reduced by exogenous NO administration.
Selective delivery of NO to the pulmonary vasculature by
inhalation did not reduce the pulmonary vascular tone in the
congenitally NOS 3–deficient mouse. The intravascular administration
of SNP, which is known to dilate both small resistance arteries and
larger capacitance pulmonary arteries and veins,31
as well as systemic vessels, produced systemic vasodilation in NOS
3–deficient mice but also had no effect on PAP or TPR. Although our
results provide evidence that both NOS-inhibited and NOS
3–deficient PAs can be stimulated by NO, resulting in
pulmonary vasodilation in vitro, there is no certain
explanation as to why inhaled NO administration did not reverse the
increased pulmonary tone in vivo. It is known that inhaled NO
does not dilate the pulmonary vasculature under basal
conditions.10 Chronic inhibition or absence of NOS
activity may reset the basal pulmonary tone at an increased
level, resulting in unresponsiveness to NO inhalation.
NOS 3 Releases Endothelium-Derived Relaxing Factor
In Vivo and In Vitro
ACh-induced endothelium-dependent
vasodilation due to release of NO by endothelium was
the basic observation leading to the identification of
endothelium-derived relaxing factor as
NO32 33 synthesized by NOS 3.12 Our results
confirm this in vivo: ACh injection did not induce systemic or
pulmonary vasodilation in NOS 3–deficient mice. ACh exposure
caused vasoconstriction of PA segments in vitro in acutely
NOS-inhibited but not in NOS 3–deficient mice, suggesting that other
inhibitable isoforms of NOS were present in the pulmonary
vascular preparation. These isoforms of NOS may contribute NO in NOS
3–deficient PA segments to counterbalance ACh-induced
vasoconstriction.
Study Limitations
We recognize that the invasive measurement techniques used in the
present study provide limitations. It is likely that open-chest
measurements of PAP in the mouse underestimate the awake or
closed-chest PAP. The SAP was lower in anesthetized than in
awake mice12 because anesthetic drugs and surgical
procedures reduce vascular tone and alter circulatory homeostasis.
Hemodynamic studies continuously assessing PAP and
cardiac output in awake mice would be preferable, but these techniques
are not yet available. The measurement of LTAF only approximates
cardiac output, since flow into proximal branches of the aorta was not
measured. Whereas it is technically possible to measure ascending
aortic flow, the presence of a flow probe in this position interferes
with simultaneous PA catheterization. LVEDP
measurements (and therefore PVR assessment) were limited to a brief
period, since direct puncture of the left ventricle can cause
ventricular injury and bleeding. Placement of a catheter
into the left ventricle via the carotid artery obstructs the aortic
outflow tract and is limited by a poor frequency response due to the
narrow caliber of the catheter.
Summary and Conclusions
The targeted disruption of the NOS 3 gene in mice causes mild
pulmonary hypertension due to a substantially increased PVR
with an increased systemic vascular resistance and reduced LTAF. NOS
inhibition, acutely or chronically, only partially mimicked these
pulmonary vascular changes, suggesting that the duration and
specificity of NOS 3 deficiency affect the nature and extent of the
pulmonary hemodynamic changes. Our results
provide evidence that NOS 3 contributes to maintaining the normal low
pulmonary vascular tone in mice.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by US Public Health Service grant HL-42397 (Drs Zapol and Hurford), by National Institute of Neurological Disorders and Stroke grants NS-33335 and NS-10828 (Dr Huang), and by US Public Health Service grant HL-32985 (Dr Bevan). Dr Steudel is a Research Fellow supported by the Deutsche Forschungsgemeinschaft (German Research Association, STE 835/1-1). Dr Huang is an Established Investigator of the American Heart Association. The authors would like to thank Maria del Mar Aris, Melehat Kavosi, Margaretha Jacobson, Alison Croke, and Heather Burbank for technical help; Jeffrey B. Cooper, PhD, and Kevin Stanek, Department of Anaesthesia and Critical Care, Massachusetts General Hospital, for assistance with the data acquisition systems and measurements of frequency response; and Kenneth D. Bloch, MD, Cardiovascular Research Center, Massachusetts General Hospital, for critically reviewing the manuscript. We are further indebted to Dr YuChiao Chang, Medical Practice Evaluation Center, Massachusetts General Hospital, for statistical consultation and Wolfgang Scholz, Siemens Medical Systems, Electromedical Corporation, Danvers, Mass, who generously developed and installed the mouse capnograph.
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Footnotes |
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This manuscript was sent to Bradford C. Berk, Corresponding Editor, for review by expert referees, editorial decision, and final disposition.
Received November 12, 1996; accepted May 1, 1997.
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