2038
Inhaled Nitric Oxide
A Selective Pulmonary Vasodilator Reversing Hypoxic
Pulmonary Vasoconstriction
Claes Frostell, MD, PhD; Marie-Dominique Fratacci, MD;
John C. Wain, MD; Rosemary Jones, PhD; and Warren M. Zapol, MD
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Background. We examined the effects of inhalation of 5-80 ppm nitric oxide (NO) gas on the
normal and acutely constricted pulmonary circulation in awake lambs.
Methods and Results. Spontaneous breathing of nitric oxide (an endothelium-derived relaxing
factor) at 40 ppm or more reversed acute pulmonary vasoconstriction within 3 minutes either
because of infusion of the stable thromboxane endoperoxide analogue U46619 or because of
pulmonary hypertension due to breathing a hypoxic gas mixture. Systemic vasodilation did not
occur. Pulmonary vasodilation by NO inhalation was produced during infusion of U46619 for
periods of 1 hour without observing evidence of short-term tolerance. Pulmonary hypertension
resumed within 3-6 minutes of ceasing NO inhalation. In the normal lamb, the pulmonary
vascular resistance, systemic vascular resistance, cardiac output, left atrial and central venous
pressures were unaltered by NO inhalation.
Conclusion. Breathing 80 ppm NO for 3 hours did not increase either methemoglobin or
extravascular lung water levels or modify lung histology compared with those in control lambs.
(Circulation 1991;83:2038-2047)
N itric Oxide (NO) was reported in 1987 to be
an important endothelium-derived relaxing
factor.12 The vascular relaxant effect of NO
on arterial strips is antagonized by substances such as
hemoglobin that bind NO extracellularly with high
affinity, thereby inactivating NO.3,4 The common
nitrovasodilators, nitroprusside and glyceryl trinitrate, act by releasing NO, probably intracellularly, to
mimic the effect of endogenous NO.4-6 We examined
the effects of inhalation of 5-80 ppm gaseous NO on
the normal and acutely constricted pulmonary circulation of awake lambs. We hypothesized that inhalation would allow NO to diffuse directly into the
smooth muscle of pulmonary vessels and produce
vasodilation while preventing NO from being exposed to hemoglobin. We believed an inhalation
strategy would surmount the central problem of
intravenous administration of vasodilators to dilate
the lung's vessels. The dosage of vasodilators is
From the Departments of Anesthesia (W.M.Z, C.F., M.D.F.)
and Surgery (J.C.W.), Harvard Medical School at the Massachusetts General Hospital, Boston, and the Department of Pathology
(R.J.), Children's Hospital Medical Center, Boston.
Supported by funds from the Department of Anesthesia, Massachusetts General Hospital, US Public Health Service grant HL42397. C.F. was supported by a Fogarty International Fellowship.
Address for correspondence: Dr. Warren M. Zapol, Department of Anesthesia, Massachusetts General Hospital, Boston, MA
02114.
Received June 25, 1990; revision accepted January 15, 1991.
limited during intravenous administration because of
concomitant dilation of the systemic circulation,
which causes peripheral hypotension, right ventricular ischemia, and consequent heart failure.7-9 Inhaled NO can potentially dilate pulmonary vessels
and yet because of its rapid reaction with hemoglobin, it would prevent systemic vasodilation.
There is evidence that hypoxic pulmonary vasoconstriction of isolated pulmonary artery rings is enhanced
by putative inhibitors of NO synthesis, including NG_
monomethyl-L-arginine.10,1' In addition, intravenous
aliquots of dissolved NO dilate the isolated angiotensin
II-primed rat lung perfused with an acellular Krebs'
solution containing meclofenamate.12
NO is the major oxide of nitrogen formed during a
variety of high-temperature combustion processes
and is a common air pollutant. NO is produced in the
hot reducing atmosphere near the glowing cone of a
cigarette and is inhaled in smoke at concentrations of
400-1,000 ppm.13 Thirty minutes of exposure of rats14
to 1,000 ppm NO and rabbits15 to 43 ppm NO for 6
days causes neither pulmonary pathological effects
(according to light and electron microscopy) nor
pulmonary edema (by gravimetry). Humans have
breathed up to 30 ppm NO for 15 minutes with minor
effects on gas exchange and respiratory function.16 17
The US Occupational Safety and Health Administration has set the time-weighted average NO value as
25 ppm.'8 Because NO has a much greater affinity for
Frostell et al Nitric Oxide: A Selective Pulmonary Vasodilator
hemoglobin than does carbon monoxide,19 NO has
been used clinically to measure the lung's diffusion
capacity.20,21
Our main goal was to examine the ability of NO
inhalation to reverse hypoxic pulmonary vasoconstriction because it is an important adaptation of lung
vessels, which can at times contribute to severe acute
pulmonary hypertension (e.g., high altitude pulmonary
edema). We studied awake unanesthetized lambs to
avoid general anesthesia, which can blunt hypoxic vasoconstriction.22 In addition, we planned to develop a
dose-response curve to inhaled NO during intravenous
infusion of a potent pulmonary vasoconstrictor, the
stable thromboxane analogue U46619.
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Methods
All studies were approved by the Animal Studies
Committee of our hospital. Eight Suffolk lambs
weighing 25-35 kg underwent a sterile thoracotomy
to place a left atrial line, a tracheostomy, and a
femoral artery line under general endotracheal anesthesia with halothane and oxygen 3 days before the
study. After this recovery period, the lambs underwent sterile placement of a 7F thermodilution pulmonary artery monitoring catheter (Edwards Laboratories, Santa Ana, Calif.) through a jugular vein
under local anesthesia.
During the study, the tracheostomy was connected
to a non-rebreathing circuit consisting of a 5-1 reservoir bag and a one-way valve to separate inspired
from expired gas. Expired gas was scavenged and
discarded. The inspired gas was a precise mixture of
oxygen and nitrogen immediately diluted with NO to
produce the correct inspired concentration. With
volumetrically calibrated flowmeters, varying quantities of NO mixed with N2 were substituted for pure
N2 to obtain the desired inspired NO concentration
at an inspired oxygen concentration (FIo2) of 0.60.7. We chose 60-70% oxygen to avoid hypoxic
vasoconstriction. The reservoir bag was emptied after
each level of NO inhalation. NO was obtained from
Air Products and Chemicals, Inc., (Allentown,
Penn.), as a mixture of 235 ppm NO in pure N2.
Chemiluminescence analysis demonstrated less than
12 ppm NO2 in this mixture.23
Mean and phasic pulmonary artery pressure (PAP),
left atrial pressure (LAP), systemic arterial pressure
(SAP), and central venous pressure (CVP) were continuously monitored using calibrated pressure transducers (model 1280C, Hewlett-Packard, Palo Alto,
Calif.) zeroed at the left atrial level and a four-channel
recorder (model 7754A, Hewlett-Packard). Cardiac
output (CO) was measured by thermodilution as the
average of two determinations after injection of 5 ml
0°C Ringer's lactate. Pulmonary vascular resistance
(PVR) and systemic vascular resistance (SVR) were
computed by standard formulas.
Protocol
Eight lambs were studied awake, spontaneously
breathing, and eating ad libitum. The series of stud-
2039
ies (A-D) were completed, on separate days, on each
lamb when the following exclusion criteria did not
occur: a peripheral white blood cell count less than
4,000 or more than 12,000/mm3, mean PAP more
than 20 mm Hg, and a core temperature more than
40.10C.
A. Control NO inhalation. After baseline measurements, six of the eight lambs were allowed to breathe
80 ppm NO for 6 minutes at FIo2 0.6-0.7. Hemodynamic measurements were obtained at 3 and 6 minutes during NO inhalation and were repeatedly obtained 3 and 6 minutes after ceasing NO inhalation.
B. Dose-response study of intermittent NO inhalation
during U46619 infusion. Eight lambs breathing oxygen
at FIo2 0.6-0.7 were given an infusion of a potent
pulmonary vasoconstrictor, the stable endoperoxide
analogue of thromboxane (5Z=9a,13E,15S)-11,9-(Epoxymethano) prosta-5, 13-dien-l-oic acid (U46619, Upjohn, Kalamazoo, Mich.) at a rate of 0.4-0.8 ,ug/kg/min
to increase the mean PAP to 30 mm Hg.
To obtain a pulmonary vasodilator dose-response
curve during U46619 infusion, after a stable hemodynamic period, each of the eight lambs breathed a
series of NO and 02 mixtures of 5, 10, 20, 40, and 80
ppm NO for 6 minutes. Each level of NO exposure
was followed by 6 minutes of breathing the oxygen
mixture without NO. Then, a second exposure to 5,
10, and 20 ppm NO for 6 minutes was performed, and
each lamb was examined for the occurrence of acute
tolerance. Subsequently, each lamb was studied during a control period of breathing the oxygen mixture
6 minutes after ceasing U46619 infusion. Methemoglobin levels were measured before and after the
study.24
C. Study of tolerance after 1 hour of NO inhalation
during U46619 infusion. Four awake lambs were given
a U46619 infusion to raise their mean PAP to 30
mm Hg. Pulmonary and systemic hemodynamic measurements were obtained. Then, the lambs breathed
80 ppm NO at FIo2 0.6-0.7 for 1 hour. Repeated
measurements were obtained at 3, 6, 15, 30, and 60
minutes of NO breathing and again at 3 and 6
minutes after ceasing NO breathing. Then, U46619
infusion was discontinued, and repeated hemodynamic measurements were obtained after 10 minutes.
D. NO breathing during hypoxic exposure. Five of the
awake lambs were studied during a period of breathing a hypoxic gas mixture to induce acute hypoxic
pulmonary hypertension. We used a nonrebreathing
circuit containing a 25-1 reservoir bag, and the FIo2
was reduced to 0.06-0.08 to produce a mean PAP
near 25 mm Hg at a Pao2 near 30 mm Hg. Then,
either 40 or 80 ppm NO was added to the inspired gas
mixture at the same FIo2. Total gas flows were
maintained at 35 1/min to prevent rebreathing due to
hyperventilation. The inspired FIo2 was monitored
with an electrode, and pure CO2 was added to the
inspired gas to maintain the end-tidal CO2 concentration (model LB-2, Beckman, Schiller Park, Ill.) at
4.5-6%. Measurements of central hemodynamics
and gas exchange were obtained at baseline, while
2040
Circulation Vol 83, No 6 June 1991
-20
20
PAP
x PVR
*
18-
05
18
16-
16
14-
-14
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2
mean
n-
1
0
.
4
6
-1
8
12
±
-2
SE
14
n
16
Time (min)
FIGURE 1. Plot of effects of inhaling 80 ppm nitric oxide for 6 minutes on the mean pulmonary arterial pressure (PAP) and
pulmonary vascular resistance (PVR) of normal lambs without infusion of U46619 (n=6, mean ±SEM).
breathing air, during hypoxia, and at 3-6 minutes of
NO breathing during hypoxia.
E. Toxicology at 1 and 3 hours of 80 ppm NO
inhalation. Thirteen additional lambs were included in
this study to examine the effects of longer NO exposure. Twenty-four hours before study, a tracheostomy
was performed during a brief halothane and 02 anesthesia. Then, lambs were allowed to breathe either a
gas mixture of room air and 02 at FIo2 0.3-0.4 for 3
hours (control group, n = 5) or air and at the same
FIo2 containing 80 ppm NO for 1 hour (n=5) or 3
hours (n=3). Each animal was then killed with potassium chloride administered intravenously during a
brief halothane anesthesia. The lungs were quickly
excised and separated. The right lung was dissected to
remove bronchi greater than 5 mm in diameter and
was used to determine the percentage of extravascular
lung water content by the method described by Peterson et al.25 Five appropriate tissue blocks were selected from each lobe of the right lung and placed in
Trumps' fixative (1% glutaraldehyde and 4% formaldehyde in phosphate buffer, pH 7.2) and then processed for pathological examination. Five-micron resinembedded sections were stained with hematoxylin and
eosin. Methemoglobin levels were measured before
and after 1 and 3 hours of NO inhalation.
02
Statistical Analysis
All data are expressed as mean+SEM. Data were
compared by a two-way analysis of variance
(ANOVA, version 5.16, SAS Institute, Cary, N.C.) or
a paired t test (dose-response slopes). A p value less
than 0.05 was considered to indicate statistical significance. For the dose-response study, the dose-response slopes, and the ANOVA were calculated with
the values from both the early and the late series for
the 5, 10, and 20 ppm NO doses. By averaging these
values, the effect of time was minimized. For the
hypoxic exposure study, comparisons were performed
with paired t tests.
Results
A. Control NO Inhalation
In the six lambs spontaneously breathing 80 ppm
NO for 6 minutes, we measured no change of mean
PAP, PVR, SAP, CO or SVR (Figure 1).
B. Dose-Response Study of Intermittent NO
Inhalation During U46619 Infusion
At all dose levels, NO inhalation produced a
prompt reduction of the pulmonary hypertension
caused by U46619 infusion. The onset of pulmonary
vasodilation occurred within seconds after beginning
NO inhalation, and the vasodilator effect was nearly
maximal within 3 minutes (Figure 2). Termination of
NO inhalation caused a return to the prior level of
vasoconstriction within 3-6 minutes. The inhaled
NO pulmonary vasodilator dose-response curve of
eight lambs is shown in Figures 3 and 4. Note that 5
ppm NO (an inhaled lung dosage of approximately
0.9 ,g/kg/min) significantly reduced the PAP, and an
almost complete vasodilator response occurred by
inhalation of 40 or 80 ppm NO. Mean methemoglo-
1-
Frostell et al Nitric Oxide: A Selective Pulmonary Vasodilator
20)41
20
~ Cr
N 40
X
1''W
E20
0~~~~~~~~~~~8 PPM NO -........0|
8
EE1,50
s
0-
CO
/lr.
E
PPM O.
=__
4
.1min
2.48
2.7 f
2,56
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FIGURE 2. Plot of effects of inhaling 80 ppm nitric oxide (NO) for 6 minutes on the left atrial pressure (LAP), pulmonary artery
pressure (PAP), and systemic arterial pressure (SAP) of a lamb receiving an infusion of U46619. Cardiac output (CO) is noted.
bin levels were 1.06 ±0.13% before and 1.29+0.33%
after (not significant) the U46619 infusion during
inhalation of the various NO mixtures (n=7). After
consideration of the minor reduction over time of
baseline PAP during U46619 infusion, the vasodilator response of the second exposure was compared
with breathing 5, 10, and 20 ppm NO; no significant
reduction from the prior series of exposures occurred. Regression analyses of NO concentration
during U46619 infusion with SVR, CO, or SAP
showed that the slopes were not significantly different from zero.
C. Study of Tolerance After 1 Hour of NO Inhalation
During U46619 Infusion
In four lambs, inhalation of 80 ppm NO for 1 hour
during U46619 infusion produced sustained pulmonary
vasodilation to a normal PAP and PVR for 1 hour, and
pulmonary hypertension promptly recurred after ceasing NO inhalation (Figure 5). There was no short-term
tolerance to NO vasodilation. Methemoglobin levels
ranged from 0.6% to 2.0% (1.10±0.25%) after 1 hour
of 80 ppm NO inhalation and did not differ significantly
from the control value (0.68+0.14%).
D. NO Breathing During Hypoxic Exposure
In all five lambs, acute hypoxia produced pulmonary
hypertension and a marked increase of cardiac output.
In each instance when 40 or 80 ppm NO was added to
the inspired hypoxic gas mixture, pulmonary hypertension was relieved and pulmonary artery pressure returned to control levels while the lambs breathed air
despite a maintenance of elevated CO (Table 1).
E. Toxicology of 1 and 3 Hours of 80 ppm
NO Inhalation
Effects of 1 and 3 hours of 80 ppm NO inhalation
are summarized in Table 2. Five lambs were killed
after breathing 80 ppm NO through the tracheo-
stomy for 1 hour. The extravascular lung water level
of lambs breathing 80 ppm NO for 1 hour was
74.8+0.9% and did not differ from the control value
of 74.2± 1.4% obtained in five lambs. The extravascular lung water level of three lambs breathing 80
ppm NO for 3 hours was 73.9+1.2%, and these
values were not significantly different from control
levels. Methemoglobin levels after 1 or 3 hours of
breathing 80 ppm NO did not change from control
values. All the lungs from NO breathing and control
lambs showed microscopic evidence of a scattered
low-grade inflammatory response. There were no
consistent histological differences between the control and NO breathing groups.
Discussion
Our studies demonstrate that inhaled NO can act
as a selective local pulmonary vasodilator without
causing systemic vasodilation. NO inhalation reversed the pulmonary hypertension caused by infusing the stable endoperoxide analogue U46619 without decreasing systemic arterial pressure (Figure 2).
The pulmonary vasodilator effect of NO inhalation
for 1 hour did not exhibit short-term tolerance irn
lambs (Figure 5). Intravenous infusion of nitroprusside and nitroglycerine can also reduce the pulmonary vasoconstriction produced in dogs by U46619
infusion, but such infusion causes marked concomitant systemic arterial vasodilation and hypotension.26
NO directly combines with heme to form the paramagnetic species, nitrosyl-heme, which is the active
species responsible for the activation of guanylate
cyclase by NO and, thereby, for stimulating the
accumulation of intracellular cyclic GMP in vascular
smooth muscle.3 5 We believe rapid combination
with hemoglobin in red blood cells inactivates inhaled NO, restricting inhaled NO vasodilation to
vessels in the lung and preventing systemic vasodilation. Thus, in normal lambs breathing 80 ppm for 6
2042
Circulation Vol 83, No 6 June 1991
36-
U4661 9 infusion
3432
30'
28
E
26-
CL
24-
2220-
18169
RA
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FIGURE 3. Plots of mean pulmonary artery
(PAP) and pulmonary vascular resistance (PVR) during a continous infusion
of U46619. Lambs breathed various levels of
nitric oxide (5-80 ppm) at FIo2 0.6 for 6
minutes, then breathed a gas mixture at FIo2
0.6 for 6 minutes without nitric oxide (n= 8,
mean ±SEM).
pressure
,4. -
20
40
60
80
1do
120
100
120
10
9[men tSE
8
7c
6
E
5
G
4
3
2
0
20
60
40
lime
80
(min)
minutes, PAP, PVR, SAP, or SVR did not change
(Figure 1). The normally low-resistance pulmonary
vasculature had no dilatory response during NO
breathing, and the SVR was not altered.
Hypoxic pulmonary constriction is an important
regulatory mechanism in both the normal and diseased lung. Pulmonary hypertension due to alveolar
hypoxia occurs at high altitude and contributes to
high-altitude pulmonary edema and right ventricular
failure. Pulmonary hypertension due to alveolar hypoxia is common in the Adult Respiratory Distress
Syndrome, augmenting pulmonary edema and leading to hypoxia and death.7 Others have shown that
both intravenous infusion of nitroprusside and nitroglycerin can reverse acute pulmonary vasoconstriction and hypertension due to breathing 12.5%0 in
normal humans.27 However, there was marked con2
comitant systemic vasodilation and systemic hypotension. The ability of NO inhalation at 40 and 80 ppm
to antagonize hypoxic pulmonary vasoconstriction
without reducing systemic blood pressure or SVR
may allow a new therapeutic strategy for the treatment of these types of acute pulmonary hypertension,
provided the toxicity of NO does not preclude prolonged pulmonary exposure. This topic will be discussed considering: 1) oxidation of NO to NO2,
2) pulmonary effects of inhaling NO and NO2 at high
and low levels, 3) NO and hemoglobin interaction,
and 4) the metabolic fate of NO.
Oxidation of NO to NO2
The inhalation toxicology of inhaled NO and NO2
is difficult to ascertain separately because of oxidation of NO to NO2 in the presence of oxygen, the rate
Frostell et al Nitric Oxide: A Selective Pulmonary Vasodilator
321
n=8
m
moan ± SE
+
30.
28
+ p < .001 vs 0 ppm
* p < .001 vs 80 ppm
*
+
26-
*
+
24.
10
2043
+
*
22.
+
20-
j
18-
is,'
.
.
,
,
20
4o
60
NO (ppm)
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n=8
mean + SE
+ p < .001 vs 0 ppm
* p < .001 vs 80 ppm
+
-20+
FIGURE 4.
Pulmonary arterypressure (PAP)
dose -response curves (n=8, mean +SEM) for
nitric oxide (NO) inhalation in lambs given an
infusion of U46619. Top panel: Mean PAP vs.
NO concentration. Bottom panel: Percent
maximal change of PAP vs. NO concentration.
PAP (% max) was computed as: PAP (%
max) =lOOx (PAHT-PAPNO)/(PAPHT-PAPC),
where PAPHT is the elevated mean PAP level
recorded immediately before NO inhalation,
PAPNO is the mean PAP measured after 6
minutes of NO inhalation, and PAPc is the
mean control value obtained before infusion of
U46619.
-40+
0~
E~
-60-80-
_r^A
-nn2
40
20
- do
i
6
6.0
80
NO (ppm)
of oxidation being dependent on the initial concentration of NO and the FIo2.28 For example, in air at
20°C, a 10,000 ppm NO mixture undergoes 50%
conversion from NO to NO2 in 24 seconds, whereas a
10 ppm NO mixture undergoes 50% conversion to
NO2 in about 7 hours.29
The level of NO2 in our gas mixtures was less than
5% of the NO concentration as measured by
chemiluminescence.23 We believe there was minimal
conversion of NO to NO2 during the brief mean
transit time (<30 seconds) from the gas reservoir bag
to alveoli. Thus, our lambs were exposed to no more
than 4 ppm NO2 at the highest dose (80 ppm NO); we
did not believe it was necessary to chemically absorb
NO2 from the inspired gas.30
Airway and Pulmonary Effects of Inhaling NO and
NO2 at High Levels
Inhalation of gas mixtures containing high concentrations of NO and NO2 can be rapidly lethal in
humans and can cause severe acute lung damage with
pulmonary edema and marked methemoglobinemia.31 Greenbaum et a132 exposed anesthetized dogs
to 5,000-20,000 ppm NO, and the exposure produced
rapid death due to a critical reduction of arterial
oxygen content caused by methemoglobinemia, a
right to left shunt due to severe alveolar edema, and
acidemia with a rightward shift of the oxyhemoglobin
dissociation curve. A rapid reduction of lung compliance was measured during exposure to 20,000 ppm
NO. The investigators believed32 that conversion of
high levels of inhaled NO to NO2 followed by subsequent transformation to nitric and nitrous acid33
resulted in an acid pneumonitis.
Pulmonary Effects of Inhaling NO and NO2 at
Low Levels
More recent studies examined exposure to lower
levels of NO. Stavert and Lehnert14 reported that
rats exposed to 15 minutes of 1,500 ppm NO showed
no increases of extravascular lung water and no
evidence of histopathological changes. In their study,
the conversion of NO to NO2 was minimized by using
high flow rates to reduce gas residency time in the
exposure chamber as well as partially filling the gas
mixing chamber with soda lime to chemically absorb
2044
Circulation Vol 83, No 6 June 1991
35-
-16
30-
-14
* PAP
x PVR
-12
25-10
E
20-
gCL
-U
-8
3
:E
15-6
10-
.4
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2
114
-10
0
.
bT
. . . 20
30
,
40
.
50
60
70
n
80
Time (min)
* p
<
.001 values differ before and after NO Inhalaton
FIGURE 5. Plot of effects of] hour of 80 ppm nitric oxide (NO) inhalation on the mean pulmonary arterial pressure (PAP) and
pulmonary vascular resistance (PVR) during a continous infusion of U46619 (n=4, mean +SEM). U46619 was infused from 1 to
73 minutes.
NO2.30 They reported that NO2 contamination during
NO exposure was less than 15 ppm. In other studies,
exposure of rats to 25-100 ppm NO2 for 30 minutes
resulted in histological evidence of lung injury.14 At
TABLE 1. Alterations of Hemodynamics and Gas Exchange
Hypoxia +
40-80
ppm NO
0.06-0.08
31.1+1.7*
19.8+3.2
40.0±2.7
7.40+0.03
Control
Hypoxia
0.21
0.06-0.08
FIo2
28.2+1.4*
Pao2 (mm Hg)
70.8+4.4
PvO2 (mm Hg)
16.6+1.8*
36.8+2.5
33.9+1.4
38.6+2.6
Paco2 (mm Hg)
7.47+0.01 7.42+0.03
pHa
PAP (mm Hg)
16.7+0.6
28.3±2.2*
18.7+1.1t
LAP (mm Hg)
5.2+0.8
4.2±1.0
6.4±0.5
CO (1/min)
4.55±0.13 7.08+0.22*
7.56±0.79*
PVR (mm Hg/i/min) 2.51±0.11 3.07±0.25
2.01±0.35t
SAP (mm Hg)
103±6
113±+-7
106±5t
CVP (mmHg)
3.0+1.3
3.5+0.8
2.8±1.6
SVR (mm Hg/i/min) 21.7±1.4
16.2+0.9*
13.7+1.0*
Data are mean±SEM; n=5 lambs.
NO, nitric oxide; FIo2, inspired oxygen concentration; PAP,
pulmonary arterial pressure; LAP, left atrial pressure; CO, cardiac
output; PVR, pulmonary vascular resistance; SAP, systemic arterial pressure; CVP, central venous pressure; SVR, systemic vascular resistance.
*p<0.01 value differs from control; tp<0.01 NO + hypoxia
value differs from hypoxia.
than 50 ppm NO2, 5 minutes of inhalation
resulted in significant lung injury, with increases of
extravascular lung water, extravasated erythrocytes,
type II pneumocyte hyperplasia and accumulation of
fibrin, polymorphonuclear cells, and alveolar macrophages in the alveoli.
Little evidence for NO toxicity exists with exposures less than 100 ppm in normal rats14 and rabbits.15 Hugod15 reported on exposure of rabbits for 6
days to air containing a mixture of 3.6 ppm NO2 and
43 ppm NO. Using two different procedures for
tissue fixation from control and exposed animals and
a blind evaluation of light and electron microscopy
results, Hugod reported no evidence of acute NO
toxicity.
Evans et a134 studied young rats exposed to 2.0 and
17 ppm NO2 for up to 360 days. Early evidence of cell
proliferation in terminal bronchioles and alveoli was
found in both groups after 2-3 days but was normalized after 5 days. Epithelial hyperplasia of terminal
bronchioles and an increased turnover rate of type II
alveolar cells were noted. The same investigative
group35 noted that after exposure to 2 ppm NO2 for
24-72 hours pulmonary changes included loss of
cilia, hypertrophy, and focal epithelial hyperplasia of
the terminal bronchioles with an apparent return to
normal (adaptation to exposure) after 21 days of
continuous exposure to NO2. Species differences may
exist in pulmonary susceptibility to NO2 toxicity as
more
Frostell et al Nitric Oxide: A Selective Pulmonary Vasodilator
TABLE 2. Effects of 1 and 3 Hours of 80 ppm Nitric
Oxide Inhalation
EVLW (%)
MetHb (%)
Control (n=5)
74.2±1.4
0.8+0.2
80 ppm nitric oxide, 1 hr (n=5)
74.8+0.9
1.22+0.23
73.9+1.2
1.53+0.53
80 ppm nitric oxide, 3 hr (n=3)
Data are mean+SEM.
EVLW, extravascular lung water; MetHb, methemoglobin.
EVLW and MetHB values after nitric oxide breathing do not
differ from control values.
Downloaded from http://circ.ahajournals.org/ by guest on June 14, 2017
demonstrated by Erlich,36 who noted that squirrel
monkeys and hamsters tolerated inhaling a dose of
NO2 10-fold higher than that tolerated by mice.
von Nieding and coworkers16 examined pulmonary
mechanics and gas exchange in more than 100
healthy humans and in 132 patients with respiratory
disease breathing 1-30 ppm NO for up to 15 minutes.
At concentrations of 15 ppm NO or greater, they
reported a small reversible decrease in arterial oxygen tension and a minor increase of airway resistance. They also measured a decreased arterial oxygen tension and a reduced diffusion capacity for CO
after the subjects had been breathing 5 ppm NO2 or
more for 15 minutes. An altered diffusion capacity
for CO did not occur after breathing 40 ppm NO.
In our lamb studies, the frequent finding of endemic pneumonia with regional bronchopneumonia
in control lambs did not allow us to histologically
differentiate lambs breathing 80 ppm NO for 3 hours
from control lambs. In the future, inhalation toxicology studies should be pursued in an experimental
animal species without endemic low-grade pulmonary inflammation. We found no increase of extravascular lung water levels in these animals after breathing 80 ppm NO for 1 and 3 hours (Table 2).
NO and Hemoglobin Interaction
The heme structure of hemoglobin has great affinity for NO, and we believe that inhaled NO is
inactivated by hemoglobin, thereby restricting its
vasodilator activity to the lung. NO combines with
hemoglobin forming nitrosyl hemoglobin with an
affinity 1,500 times higher than that measured for
carbon monoxide.19 Nitrosyl hemoglobin is oxidized
to methemoglobin when oxygen is present, by a
poorly understood mechanism.37 Methemoglobin, in
turn, is metabolized into nitrate. In the reaction
process of NO with hemoglobin, damage to erythrocyte membranes has been observed.38,39 Oda and
coworkers4041 studied mice after 6 months of exposure to 10 ppm NO and reported that the mice
displayed signs of increased red blood cell turnover
with enlarged spleens and increased bilirubin levels.
Spleen weights increased significantly after 2 weeks
of exposure. Nitrosyl hemoglobin levels remained low
(0.13%); methemoglobin levels were 0.2% in both
control and exposed mice.41
It is uncertain what the critical duration and level of
NO exposure in humans will be before red blood cell
2045
degenerative processes become significant. In studies
by von Nieding et al17 of humans breathing 20-30 ppm
NO for 15 minutes, the methemoglobin levels increased
by a mean of 0.52% from an initial mean value of
0.72%. Our lambs had no significant increase of methemoglobin levels when measured before and after 1
and 3 hours of breathing 80 ppm NO (Table 2).
Metabolic Fate of NO
Hemoglobin and related hemoproteins bind NO
with great affinity3,4'42 and form methemoglobin. NO
gas is unstable and undergoes spontaneous oxidation
to NO2 and higher oxides of nitrogen as outlined
above. As a dilute solution exposed to oxygen, NO
has a half-life of less than 10 seconds because of
rapid oxidation to inorganic nitrite and nitrate.43 In
the presence of superoxide, the half-life of NO is
greatly reduced whereas in the presence of superoxide dismutase, its half-life is significantly increased.'
NO is cleared from inhaled air by approximately 80%
during quiet breathing and by up to 90% during deep
breathing.44 Goldstein et a145 examined the distribution in monkeys of an inhaled dose of 0.3-0.9 ppm
`3NO2. They reported that after a few minutes 5060% of the inhaled NO2 was found in the lung and
spread to other organs through the blood stream.
Yoshida and Kasama46 measured a high nitrogen-15
content in blood serum and urine of rats and mice
after inhalation of 138 to 880 ppm 15NO. Within 24
hours, about 40% of the inhaled "5N was excreted
into urine.
It is possible that inhalation of NO will not dilate
chronically constricted pulmonary vessels if pulmonary vascular musculature is too distant from the
alveolus to allow diffusion in sufficient concentrations
or if downstream venous muscle is protected by
intravascular hemoglobin. In a report of seven patients with primary pulmonary hypertension breathing 40 ppm NO, only a minor dilatory effect on PAP
and reduction of PVR was noted.47 A dose-response
curve was not reported. These patients were awaiting
heart and lung transplantation and suffered from
chronic obstructive pulmonary hypertension rendering their vascular cross-sectional area fixed and restricted. Because NO inhalation is readily performed
and vasodilator effects commence within minutes
after beginning administration, brief periods of NO
inhalation may provide a simple and selective test of
pulmonary vasodilatory potential. Patients with
chronic pulmonary hypertension vasodilating during
NO breathing could then be tested with intravenous
agents that are suitable for long-term administration.
In this study, we did not measure the effects of NO
inhalation on airway mechanics. However, we note
with interest that there are experimental data48 indicating that NO may be one of the recently described
epithelium-derived relaxing factors49 causing local
bronchodilation somewhat analogous to what has
been demonstrated in the blood vessel wall.
In conclusion, inhalation of low levels of NO for
brief periods warrants further study as a selective
2046
Circulation Vol 83, No 6 June 1991
pulmonary vasodilator in acute and chronic lung
disease associated with pulmonary hypertension. NO
inhalation, thus, represents a practical application of
basic vascular research. This mode of direct vasodilator delivery to the lung may allow novel diagnostic
and therapeutic applications advancing our understanding and treatment of pulmonary vascular diseases. However, a number of toxicological studies
remain to be performed; for example, toxicity to the
lungs caused by long-term breathing of NO has not
been examined and therefore, NO for long-term
inhalation therapy cannot be recommended. Longterm toxicological studies of NO inhalation by animals with preexisting acute or chronic lung disease
are also necessary. If long-term inhalation of low
levels of NO or NO-releasing compounds proves
both safe and effective at reducing pulmonary hypertension, a new era in the selective treatment of
pulmonary vascular disease will have begun.
Downloaded from http://circ.ahajournals.org/ by guest on June 14, 2017
Acknowledgments
We thank Dr. A. Zaslavsky for statistical assistance, Dr. T. Y. Chen and E. Greene for technical
assistance, Drs. W. Jung and L. Reid for assistance
with pathological examination of lung tissue, and R.
Kalil and Upjohn Company for providing U46619.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
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KEY WORDS * endothelium-derived relaxing factor * pulmonary
hypertension * thromboxane analogue * hypoxia
Inhaled nitric oxide. A selective pulmonary vasodilator reversing hypoxic pulmonary
vasoconstriction.
C Frostell, M D Fratacci, J C Wain, R Jones and W M Zapol
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Circulation. 1991;83:2038-2047
doi: 10.1161/01.CIR.83.6.2038
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