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The John B. Pierce Foundation Laboratory, Yale University School of Medicine, New Haven, Connecticut 06519
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Stitt, John T., Arthur B. DuBois, James S. Douglas, and
Steven G. Shimada. Exhalation of gaseous nitric oxide by rats in
response to endotoxin and its absorption by the lungs.
J. Appl. Physiol. 82(1): 305-316, 1997.
Rats injected with a lipopolysaccharide endotoxin produce
detectable concentrations of nitric oxide gas (NO) in the expired air
within 60 min. The concentration of NO reaches a plateau at 3 h. Production of the NO is dose dependent on
lipopolysaccharide, and at a dose of 1 mg/kg iv, lipopolysaccharide alveolar concentrations of >260 parts per billion are observed. NO
synthase inhibitors suppress this NO production in response to
endotoxin. Experiments were conducted to ascertain the site of origin
of this NO and to measure the capacity of the lungs to absorb NO from
alveolar air. Results indicate that the endotoxin-induced NO originates
from within the lungs themselves and that the lungs have the capacity
to absorb >60% of NO that is presented to them. Lung tissues absorb
~44-47% of the NO load, blood carries away between 15 and 19%,
while the remainder is exhaled in the expired air. It is proposed that
the exhalation of NO might prove useful as an early biomarker for acute
lung injury.
lung injury; neutrophils; endothelial cells
INTRODUCTION THE SEQUELAE OF ENDOTOXEMIA are often fatal, and two of
the commonest causes of death in septicemic patients are adult
respiratory distress syndrome (ARDS) and endotoxic shock
syndrome. The lung appears to be a sensitive shock organ
in endotoxemia, and, indeed, many distant-focus infections are first
detected as a result of pulmonary discomfort and dysfunction (15, 52).
The body responds to a variety of infections and injuries by mounting
the host defense response (HDR) (18). This multifaceted response
includes fever production, the acute-phase reactions, and immune
responses. The common stimulus to these three parts of the HDR is the
production of "early" cytokines, tumor necrosis factor- However, the HDR can also be a double-edged sword, and, when
overstimulated, it can lead to adverse effects on the host that often
prove fatal. Endotoxemia is a case in point, where the systemic distribution of endotoxins often elicits, first in the lungs and later
in the other organs, a concatenation of acute-phase inflammatory reactions that may often prove fatal to the host. Depending on the
severity of the neutrophil emigration and diapedesis, late-phase lung
injury is marked by edema and fibroblast proliferation that results in
fibrosis and it is often manifest by an impairment of gas exchange. It
also can impair the protective hypoxic pulmonary vasoconstriction
reflex that normally minimizes ventilation-perfusion mismatch. This
leads to a significant right-to-left shunt that produces hypoxemia (1,
15, 16). Diagnosable ARDS is now evident, but in many cases it will be
too late to treat it with much success (1, 15, 41).
Systemic shock in response to endotoxin is due to a decrease in
systemic vascular resistance, which, despite compensatory increases in
cardiac output, leads eventually to a low mean arterial pressure,
venous pooling, and left ventricular dysfunction (13, 26). Later
complications include disseminated intravascular coagulation with
hepatic, renal, and gastrointestinal ischemia, lactacidosis, and other
metabolic disorders that lead eventually to multiple-organ failure and
death (9, 12, 19, 26, 41). The etiology of endotoxic shock syndrome is
similar to acute lung injury. Neutrophil margination and activation may
be a primary event, and the initiation of the complement and cytokine
cascades is seminal to the production of endotoxic shock (29). Recent studies have shown that not only can most cardiovascular signs and
symptoms of shock be produced by TNF- Because the NO system appeared to be intimately bound to the effects of
endotoxin on the vasculature, and endotoxin is known to cause lung
injury, we decided to ascertain whether expired air from the lungs of
LPS-treated rats manifested any sign of NO production (47, 48). This
study had the following objectives: to document the relationship
between intravenously injected LPS and the exhalation of gaseous NO
from the lungs; to ascertain whether such NO had its origins in the
systemic or pulmonary divisions; and to investigate whether the NO was
related to the process of lung injury and whether it was NO synthase
(NOS) mediated. We also examined the role of the circulation and the
lung tissues in absorbing gaseous NO that entered the alveolar
compartment of the lungs. We observed that when rats were injected with
a LPS endotoxin, produced from Escherichia
coli, gaseous NO could be detected in their exhaled air
and that the probable source of this NO was the induction of NOS
somewhere from within the lungs themselves. We also ascertained that
both the lung tissues and pulmonary blood flow act as major sinks for
NO, by absorbing both inhaled NO as well as the NO that is produced in
the lungs in response to circulating endotoxin.
MATERIALS AND METHODS The animals used in this study were male Sprague-Dawley rats weighing
between 250 and 350 g. They were anesthetized with pentobarbital sodium
(50 mg/kg ip) and then tracheotomized, and a catheter was inserted via
the left femoral vein and advanced to the inferior vena cava. The
animals were then paralyzed with gallamine triiodate (15.0 mg/kg iv)
and attached to a Harvard small-animal respirator, set for a minute
ventilation of 180 ml/min (tidal volume 3.0 ml and respiratory rate 60 breaths/min). Air supplied to the intake port of the respirator was
cleansed of any ambient NO by drawing it through a
permanganate-charcoal scrubbing canister, which was attached to the
intake. Anesthesia was maintained throughout the experiments by
intravenously infusing a cocktail of pentobarbital (6.0 mg/ml) and
gallamine (4.0 mg/ml) at a rate of 1.0 ml/h. Systemic arterial blood
pressure was measured by a Statham transducer, connected to a PE-60
catheter inserted into the left common carotid artery. In those
experiments in which pulmonary arterial pressure (PAP) was measured, a
J-shaped polyvinyl (PV 60) catheter, connected to a Statham transducer,
was advanced through the right jugular vein and the superior vena cava
into the right atrium. By employing a combination of exact
measurement and markings on the catheter and the blood pressure
profiles, the tip of the catheter was advanced through the right heart
and positioned just short of the bifurcation in the pulmonary artery.
Experiments were conducted at an ambient temperature of 22°C
between 0800 and 1600, and rectal temperature was monitored throughout each experiment and was kept above 37°C by the intermittent
application of infrared heat. Mixed expired air was withdrawn from the
ventilator outflow at a rate of 18 ml/min and passed through a Sievers
270B chemiluminescence NO detector that has a detection threshold and a
sensitivity of ~1.0 part per billion (ppb) gaseous NO. The fraction of the alveolar concentration of NO that was being measured on-line was
determined by securing direct samples of equilibrated lung gas and
passing them through the same analyzer. The respirator was briefly
disconnected, and a gastight syringe containing 3.0 ml of NO-free air
(inspired O2 fraction 0.3) was
"rebreathed" seven times into the rat's lung. This permitted its
equilibration with the concentration of NO in the lung alveoli, and
subsequent analysis of this sample determined the concentration of NO
in alveolar gas (44). The mixed expired NO concentration
([NO]e) values were
later converted to alveolar [NO] values. Using the ventilatory settings prescribed above, we then determined that on-line
mixed expired samples contained 58-72% of the alveolar NO level
that was measured directly in the lung by using this equilibrated
rebreathing technique. We also determined that the ratio of
[NO]e to rebreathed
alveolar [NO] did not change appreciably, as long as the
respirator ventilatory settings were kept constant throughout the
experiment. Because the calibration procedure was a quick and simple
task, it was repeated at regular intervals. Deeper sighs (twice normal,
i.e., tidal volume ~6 ml) were induced every 10-20 min to reduce
atelectasis.
The LPS endotoxin (from E. coli, batch
no. 82F-4012) and the NOS inhibitors
N The ability of the lungs to absorb NO was measured by connecting three
different concentrations of NO (at ~600, 1,200 and 1,800 ppb) carried
in air to the intake port of the respirator for 2 min and measuring the
respective concentrations of NO in the resulting mixed expired air.
This was performed on rats before endotoxemia, during endotoxemia, and
after circulatory arrest, and the plots of the
[NO]e vs. the inspired
concentration of NO ([NO]i) yielded
lines with slopes that described the mixed expired fraction of NO
( Statistical analyses of the data were carried out by using standard
unpaired t-tests, regression analysis,
and, where appropriate, analyses of variance. When data were averaged,
means ± SE are given, and when comparisons were made, values for
P < 0.05 were considered to be
significantly different.
RESULTS Figure
1A shows
the time course of the NO exhaled from the lungs after endotoxin
treatment in rats over a range of LPS from 1 µg/kg to 1 mg/kg iv. NO
was first detected ~60 min after the injection, and it gradually rose
to a peak during the next 2 h. The NO level then plateaued and
gradually declined over the ensuing 3 h, which was as long as we
observed the response. The increases in lung [NO] values
shown in Fig. 1A are corrected to
alveolar levels from mixed expired values measured on-line as described in MATERIALS AND METHODS. In control
rats that were injected with 0.9% saline (iv) and monitored for 6 h,
lung [NO] values never exceeded 3 ppb during the
experiments. Figure 1B illustrates the dose-response relationship between peak lung [NO] and
endotoxin over the dose range 1 µg/kg to 1 mg/kg iv. The threshold
for detecting NO appears to be ~1 µg/kg iv, and there is a linear
relationship between peak lung [NO] and the log of LPS dose
over the range of doses investigated in this study.
Figure 2 demonstrates the effect of two NOS
inhibitors, L-NAME and
aminoguanidine, given intravenously, on LPS-treated rats after NO
production reached a plateau at 3 h. The results show the reduction of
ongoing NO exhalation as a function of time after NOS inhibitor
infusion, and they indicate that the exhaled NO derives from the action
of NOS and can be reduced to 33 and 8% within 60 min of
L-NAME and aminoguanidine
treatment, respectively.
Having established that endotoxin induced NO exhalation from the lungs,
and realizing that it has been also demonstrated that NO is a potent
vasodilator, we then wished to ascertain what happened to the systemic
and pulmonary blood pressures in the rat. The results of such studies
are shown in Fig. 3. Approximately 30 min
after injection of 10 µg/kg LPS iv, the mean PAP began to rise and
reached a significant increase of ~30% above control values by 1 h,
where it remained for the next hour. As NO began to appear in the
exhaled air after 1 h and as [NO] gradually rose during the
next 2 h, PAP began to drop, and mean PAP had returned to a normal
level at 3 h, when lung [NO] was at its height. Throughout this period there were no significant changes in systemic arterial pressure by using the unpaired t-test.
However, at 3 h after LPS there was a tendency for pressure to be lower
than its initial control values, and this difference attained
significance when analyzed by using a paired
t-test
(P < 0.04, results not shown).
An important factor in understanding the origins of the NO that is
exhaled by the lungs after endotoxin treatment is the role of the
pulmonary circulation. Two possibilities existed. Either NO could be
produced at remote sites from the lungs and be carried to and unloaded
into the lungs by the circulation or NO could be produced from within
the lungs, and, in that case, the pulmonary circulation might even
carry a portion of this NO away from the lungs. During these studies we
observed that on occasions after rats were killed by induction of
cardiac arrest, while ventilation still continued, there was an
immediate and large increase in the concentration of NO being exhaled
from the lungs. This indicated that before death the blood
might have been carrying away any NO generated in the lungs. Therefore,
we conducted experiments to evaluate the possibility that the pulmonary
blood flow acts as a sink to any NO that is presented by the lungs or
to them.
To reversibly reduce pulmonary blood flow, we used acute hemorrhage.
Rats were injected with 30 µg/kg LPS (iv) and the endotoxemia was
allowed to develop for 3 h while lung
[NO]e was monitored. At that time, the animals were rendered hypovolemic by rapid withdrawal of 9.0 ml of blood (~30% of the circulating blood volume) via the
inferior vena caval catheter within a period of 3 min. Reduction in
venous return to the heart induced a precipitous drop in the right
heart cardiac output perfusing the lungs. The hypovolemia lasted ~4
min, and during that time changes in lung
[NO]e were observed.
Cardiac output was then restored by rapid reinfusion of the blood. An
example from one experiment is illustrated in Fig.
4. It will be noted that as the hypovolemia
developed, both pulmonary and systemic pressures dropped profoundly
because of the large decrease in cardiac output. During this time, lung
[NO]e rose slowly,
whereas, after cardiac output was restored by reinfusion of the blood,
lung [NO]e decreased
quickly back to its previous control level. Experiments in which
hypovolemia was induced in control animals did not detect any NO in the
exhaled air during this maneuver. These experiments show that the
pulmonary blood flow must have been removing NO from the lungs rather
than delivering it. However, because of the transient and reversible
nature of the experiments, the full extent of the removal of lung NO by the pulmonary blood flow could not be assessed. Therefore, a second series of experiments was conducted in which a complete cessation of
pulmonary blood flow was produced by the induction of cardiac arrest.
Rats were injected with 1 mg/kg LPS iv, and the endotoxemia was
permitted to develop for 4 h while lung
[NO]e was monitored. Then, the heart was arrested by injection of 0.4 ml lidocaine into the
inferior vena caval catheter, ventilation was continued, and lung
[NO] was monitored for a further 90 min. The results are
shown in Fig. 5, where it can be seen that
[NO]e rose from 170 ± 33 ppb before cardiac arrest to a peak value of 618 ± 51 ppb
15 min after death. Also illustrated in Fig. 5 is the normal 5-h
progression of lung [NO], without the intervention of
cardiac arrest, as well as the effect of cardiac arrest induced in
control rats that were not treated with endotoxin.
These data indicated that nearly three-quarters of the NO, which became
apparent 15 min after cardiac arrest, must have been cleared previously
from the lungs by the pulmonary circulation. Thus, in the live
endotoxemic rat, only a very small fraction of the total NO production
within the lungs is evident as exhaled NO gas. This led us to wonder,
What is the capacity of the lungs of normal rats to absorb NO that is
administered by inhalation, how is it modified by the induction of
endotoxemia, and what is the effect of circulatory arrest on NO
absorption? Animals were prepared as before, and the pulmonary
absorption of NO was determined by measuring the lung
Figure 6 displays the combined results of
these experiments. The slopes of the lines expressing
[NO]e as a function of
[NO]i (herein referred
to as the exhaled fraction of NO or
Table 1.
NO absorption coefficients of lungs with respect to inhaled exogenous
NO in rats
(TNF-), and the
interleukins (IL) IL-1, IL-6 and IL-8 (3, 18, 36, 39, 40,
51). They cause neutrophil aggregation and adhesion within
the lung vasculature (11, 27, 53) and alter the vascular permeability
and airway smooth muscle reactivity in the lungs (25, 29, 45). Among
other events, these cytokines also activate and induce neutrophils to
generate eicosanoids, oxygen free radicals, and several proteolytic
enzymes (8, 29, 45). More recently, endotoxins have been demonstrated
to upregulate the inducible isoform of nitric oxide (NO) synthase
(iNOS) throughout the body (32), particularly in the lung within the
resident macrophage population as well as in airway epithelial cells
(33).
infusions in
animals, but also, more importantly, both the severity and lethality of lipopolysaccharide (LPS)-induced shock can be abrogated in animals that
have been pretreated with anti-TNF- monoclonal
antibody (4, 5, 51). In the case of reduced vascular resistance, there
is evidence that an endothelial cell-derived relaxing factor (most
likely NO) is a mediator of the vascular collapse that is observed in
endotoxic shock (14, 19, 33, 43, 50). It appears that endotoxic shock
is in large measure due to inappropriate overproduction of NO by the
systemic vasculature that leads to the widespread vasodilatation and
profound systemic hypotension that characterizes shock.
-nitro-L-arginine methyl ester
(L-NAME) and aminoguanidine were purchased from Sigma Chemical (St. Louis, MO). They were dissolved in
sterile 0.9% saline, and all injections were made via the femoral-vena caval catheter. Cardiac arrest was induced in some rats by injecting 0.4 ml iv of a 4% solution of lidocaine (Astra Laboratories). Bronchoalveolar lavages were performed, by using standard techniques, on both endotoxemic (1 mg/kg iv) and sham-injected control rats after
they had been ventilated on the respirator 3 h, and viability tests and
differential cell counts were performed on the aspirated cells. Lungs
of similarly matched pairs of rats were examined histologically after
they were embedded in a 50% OCT-phosphate-buffered saline medium,
frozen at 
50°C, and sectioned at 5 µm in a
cryostat. OCT embedding medium was obtained from Baxter
Scientific (NJ). The sections were stained in hematoxylin with an eosin
counterstain.
) that was
exhaled and, therefore, not absorbed by the lungs, under each of these
different conditions.
Fig. 1.
A: time course of nitric oxide (NO)
appearance in exhaled air of rats treated with doses of
lipopolysaccharride (LPS) over range of 1 µg/kg to 1 mg/kg iv.
Alveolar lung NO concentration ([NO]) is expressed in parts
per billion (ppb). B: log
dose-response curve constructed for peak lung [NO] by using
data shown in A. Values are means ± SE; n = 6 rats for each dose of
LPS. 
, change.
[View Larger Version of this Image (48K GIF file)]
Fig. 2.
Effects of administration of NO synthase inhibitors
N-nitro-L-arginine methyl ester
(L-NAME) and aminoguanidine
administered intravenously at doses indicated when lung
[NO] was at its height, 3 h after LPS treatment of 100 µg/kg iv. Values are means ± SE expressed as percent reduction in
lung [NO] from initial peak value;
n = 5 rats for each inhibitor
tested.
[View Larger Version of this Image (47K GIF file)]
Fig. 3.
Changes in lung [NO] (
) and changes in mean pulmonary
arterial pressure
(
;
) observed after rats were injected with 10 µg/kg LPS iv. Values
are means ± SE expressed as mean changes with respect to values
determined at time 0, when LPS was
injected; n = 4. * Increased
values are significantly different from value at time
0, P < 0.05.
at time 0 was 17.7 ± 3.3 Torr.
[View Larger Version of this Image (22K GIF file)]
Fig. 4.
Portion of an experimental record illustrating effect of rapid
reversible hemmorhage on level of lung [NO] in a rat
treated with 30 µg/kg LPS iv 3 h earlier.
A: mixed expired lung [NO]
measured on-line. B: heart rate.
C: systemic arterial blood pressure
(BP). D: mean pulmonary arterial (Pulm
art) blood pressure. E: undamped pulmonary arterial blood pressure, showing full systolic and diastolic values. Blood (9 ml) was withdrawn during period indicated by 1st
horizontal line in E (bleed 9 ml) and
was reinfused ~1 min later, during period indicated by 2nd horizontal
line in E (inject).
[View Larger Version of this Image (15K GIF file)]
Fig. 5.
Effect of complete circulatory arrest on mixed expired lung
[NO] of rats treated with 1 mg/kg LPS iv at
time 0 (LPS treated). Arrest was
performed at time 240. Also shown is
normal progression of lung [NO] without circulatory arrest
(control) and effect of circulatory arrest on control animals that were
sham injected with 0.9% saline at time
0 (control arrested). Values are means ± SE;
n = 5 rats for LPS-treated arrested
group, n = 6 rats for control rats, and n = 4 for
arrested rats. All values of LPS-treated arrested lung [NO]
after 240 min are significantly different from control,
P < 0.01.
[View Larger Version of this Image (18K GIF file)]
that resulted
2 min after each of three different concentrations of exogenous NO were
administered to the respirator intake port in random order. The animals
were then treated with LPS (1 mg/kg iv), and endotoxemia was permitted to develop for a period of 3 h. Absorption of inhaled NO was
remeasured, and finally at 4 h after the LPS treatment the circulation
was arrested. A final measurement of NO absorption was again performed 10-15 min after circulatory arrest, when exhaled lung
[NO] was at its height.
) describe
the fraction of the inspired NO that was not absorbed by the
lungs. Therefore, the fraction of inspired NO that was absorbed by the lungs is equal to 1 . Table
1 lists that fraction of inspired NO that
was absorbed by the lungs in each of four conditions: control animals,
LPS-treated animals, circulatory arrested LPS-treated animals, and
circulatory-arrested control animals. From these values,
we partitioned the absorption of
[NO]i. Control animals
absorbed 0.588 of inspired NO. After the animals had been treated with
1 mg/kg LPS (iv), lung NO absorption rose to 0.666, although these rats
were now exhaling 171 ppb NO/breath from lung sources. After the
pulmonary blood flow was arrested, lung NO absorption declined to
0.473, whereas the
[NO]e rose to 612 ppb
NO/breath. This means that NO absorption declined by 0.193 after
circulatory arrest and that this represents the portion of NO
absorption that was accounted for by pulmonary blood flow in
endotoxemic rats. Furthermore, 0.473 was absorbed by the unperfused lung tissues while the remaining 0.334 was exhaled in the expired air.
Fig. 6.
A: absorption by lungs of exogenous NO
administered via ventilator intake to rats made endotoxemic by 1 mg/kg
LPS iv (
). Similar absorption measurements made in endotoxemic rats
after circulatory arrest was produced (
). Regression lines were
calculated by method of least squares.
n, No. of rats in each group.
B: identical lung absorption
measurements performed on control rats that were not made endotoxemic
before () and after circulatory arrest (). [NO]e, mixed expired
[NO];
[NO]i, inspired
[NO].
[View Larger Version of this Image (54K GIF file)]
Condition
Fraction of
[NO]i Exhaled
(
NO = [NO] e / [NO]i)
Fraction of [NO]i Absorbed
Total
(1
NO)
Absorbed by Lung Tissue
Absorbed by Blood
Flow
LPS treated (from Fig. 6A)
0.334
0.666
0.473
0.193
Cardiac arrest LPS-treated (from Fig. 6A)
0.527
0.473
0.473
Control (from Fig.
6B)
0.412
0.588
0.440
0.148
Cardiac arrest control (from Fig. 6B)
0.560
0.440
0.440
Coefficients were derived from the 4 lines determined in Fig. 6 by
subtracting the slope of each line from 1.0 because the slopes of these
lines are the fractions of inspired concentration of nitric oxide (NO)
([NO]i) that were not absorbed by the lungs. F
NO, mixed expired
fraction of NO; [NO]e, mixed expired concentration of
NO; LPS, lipopolysaccharide; , change. The arrows indicate that the
preceding value was carried forward to the column or row indicated to
allow NO absorption to be partitioned between tissue and flow.
Circulatory arrest in control rats, which were not producing any
endogenous NO, decreased NO absorption from 0.588 to 0.440. This means
that in normal rats, pulmonary blood flow was responsible for removing
0.558 0.440 = 0.148 of the inspired NO that was absorbed by the
lungs and that 0.440 was absorbed by lung tissues while the remaining
0.412 was exhaled.
The lungs of eight of rats were lavaged to determine both the total number and the relative distribution of leukocytes in the bronchoalveolar airways. The averaged results of bronchoalveolar lavages, performed 3 h after LPS (1 mg/kg iv) or saline injection from four endotoxemic and four control rats, are compared in Table 2. There were no significant differences in either the total number of leukocytes present, or in the relative distributions of macrophages, lymphocytes, and polymorphonuclear leukocytes within the airways of the two groups.
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Finally, Fig. 7 compares the histological
picture of the lungs of an endotoxemic rat, removed 3 h after LPS
treatment, with those removed from a similarly ventilated control rat.
Clearly, there is a marked congestion of the lung vasculature by
leukocytes in the endotoxemic rat, compared with the control animal's
lungs. The major blood cell type present after LPS treatment also
stained positive for neutral esterase and, therefore, appears to be a polymorphonuclear leukocyte. Margination of blood leukocytes is common
after LPS treatment, and leukopenia is an early hallmark of endotoxemia
(29). Neutrophil aggregation within the pulmonary vasculature has been
described frequently in the early stages of lung injury that is caused
by endotoxin (27). Similar histological pictures have been published in
studies of rats (53), rabbits (27), and humans (11). It is also
reported that no changes in the bronchoalveolar lavage profile are
detectable inside the first 6 h after endotoxin is given intravenously
(11).
DISCUSSION
The objective of this study was to investigate whether rats that have been treated with endotoxin exhale gaseous NO and to determine whether the phenomenon might serve any useful purpose in detecting signs of early lung injury. There is every indication that the detection of NO in the exhaled air of endotoxemic animals might prove to be an early biomarker for lung injury induced by endotoxin.
Figure 1 shows that even tiny amounts of LPS for the rat (1 µg/kg iv) can produce detectable levels of NO in the exhaled air. This response is dose related, and levels of alveolar NO in excess 260 ppb are produced by rats that have been injected with 1 mg/kg iv. This dose is still far from one that produces endotoxic shock in rats because it appears that shock-inducing doses of LPS are in the region of 5-20 mg/kg iv (50). The appearance of NO in the expired air (after 60-90 min) is quite rapid, although it takes ~3 h for the response to develop fully. However, the sequelae of endotoxic lung injury in rats, such as capillary leak, pulmonary hypertension, and hypoxemia, are not usually apparent until much later (1, 17). Thus the appearance of NO in exhaled air would seem to be both an early and convenient biomarker of the potential for acute lung injury that is produced by septicemia.
There is now good evidence to believe that the cardiovascular collapse that frequently attends septic shock is the result of a widespread induction of NOS within the systemic vasculature by cytokine products of the acute-phase reaction in response to endotoxins (21, 33). iNOS has been detected in a variety of vascular and other tissues after treatment with LPS, and NO is probably the endothelial-derived relaxing factor that produces profound decreases in total peripheral resistance (30, 43, 50). That a similar activation of NOS in rats is responsible for the appearance of NO in lung air is demonstrated in Fig. 2, which shows the ability of the NOS inhibitors L-NAME and aminoguanidine to suppress the phenomenon. Furthermore, the NO suppression by L-NAME is overcome by injecting an excess (150 mg/kg iv) of L-arginine (results not shown), thus demonstrating the competitive inhibitory nature of L-NAME that has been described previously (23, 32). These results support a role for NOS in the production of the NO that is found in exhaled lung air after LPS treatment.
Although the results shown in Fig. 3, which correlates changes in mean PAP with the appearance of lung NO, are not proof of any causal relationship, it is interesting to speculate whether the decline in mean PAP that occurs at 2 h after endotoxin injection might be related to the appearance of NO in the lung. It is well known that NO has powerful vasodilatory properties. However, because only mean PAP was measured and pulmonary vascular resistance at that time was unknown, no conclusions can be drawn. There were also slight decreases in systemic arterial pressure at the same time, and these could account for resultant decreases in PAP as a result of a decrease in cardiac output. The phenomenon deserves further investigation that is designed to measure changes in pulmonary vascular resistance.
The role of the pulmonary circulation is important to understanding the
mechanisms that produce the NO that is detected in exhaled lung air.
Given the fact that endotoxin is known to induce iNOS activity
throughout the body (33) and that excessive NO production in the
periphery is believed to be a major cause for the hypotension and
cardiovascular collapse that accompany septic shock (30, 43, 50), a
case might be made that massive NO production in the periphery results
in blood-borne NO being carried to and unloaded into the lungs by the
pulmonary circulation, from where it is exhaled. However, the results
illustrated in both Figs. 4 and 5 clearly demonstrate that exhaled NO
has its origin within the lung itself and that the pulmonary blood flow
acts as a sink that removes a portion of the NO generated by the lung in response to endotoxin. The fact that transient reductions in cardiac
output (and thus pulmonary blood flow) lead to gradual increases in the
concentration of NO measured in exhaled air and that the restoration of
lung perfusion rapidly returns the lung [NO]e to control
levels proves that the blood must be carrying NO away from the lung,
rather than unloading it into the lung. The extent to
which the pulmonary circulation acts as a sink to NO production in the
lung in response to endotoxin is illustrated in Fig. 5. When
LPS-induced NO levels in expired air had reached their plateau levels
after 4 h, cardiac arrest and the concomitant cessation of pulmonary
blood flow resulted in an increase in the lung
[NO]e from 170 to 618 ppb (>260%). This means that only a small portion of this NO
actually reaches the alveolar compartment to be exhaled, when the lung
is being perfused, and thus the lung [NO]e is probably only
the tip of the iceberg that is NO generation in the lung in response to
endotoxin. Indeed, the chemical properties of NO are more
conducive to its being kept in solution as
HCO
3, nitrates, nitrites,
nitrosothiols, and peroxynitrites, which are collectively referred to
as reactive nitrogen intermediates (RNIs).
It is well known that NO is a highly reactive and diffusible gas in the lungs, and measurements reported in the literature indicate that it has a membrane diffusion constant (Dm) that is approximately five times greater than that of CO (10, 24). Depending on its residence time in the lungs, most NO that is inhaled will disappear from the alveolar compartment. In humans, it is estimated that >90% of inhaled NO disappears within 5 s (24). Thus, at normal human respiratory rates, virtually all inhaled NO will be removed from the alveolar compartment of the lung. Recently reported studies have shown that the bronchotracheal airways in humans normally have a capacity to generate gaseous NO, which can best be displayed after prolonged breath holding and a slow expiration (20). We have not detected a similar phenomenon in rats during these studies, although tracheostomy and respiratory rates of rats (~1 breath/s) would not be optimal for detecting such NO production. As far as we know, the relative distribution of the gas among various compartments in the lung after its inhalation has not been systematically investigated. However, one might expect because of its high reactivity that it would be taken up by the lung tissues as well as by the circulation. Indeed, it is still not clear just how NO is carried in the blood after it has been absorbed from the lungs by the pulmonary circulation. While hemoglobin has a high affinity for NO, whereupon it is oxidized from the Fe II to Fe III form that is methemoglobin, there is no evidence that this happens during prolonged inhalation of high concentrations of NO (2). During such NO inhalations, measured levels of methemoglobin, which is an Fe III slowly reversible form of hemoglobin, do not exceed those found in the blood normally. Additionally, we have found that even in endotoxemias of 6-h duration, methemoglobin levels do not increase above the levels that are measured in control rats (0.8-1.0%). However, there are reports that both plasma and urinary RNIs are measurably elevated during septicemia (22, 54). Recent studies of NO interactions with hemoglobin (31) have postulated that the NO molecule can be bound by thiol bonds to cysteine residues on the amino termini of the hemoglobin rather than by the heme moiety. Furthermore, the presence or absence of oxygen in hemoglobin is postulated to affect the nature of this form of NO binding and thereby regulate allosterically the loading and unloading of NO by the hemoglobin molecule at different sites within the circulation, depending on the prevailing PO2 levels in the blood.
It was for these reasons that we decided to measure the capacity of the rat lung to absorb inspired NO and to ascertain the effects of endotoxemia and of circulatory arrest on this capacity. The results are summarized in Table 1 and Fig. 6, which plots the [NO]e as a function of [NO]i for each condition, over a range of [NO]i of ~2,000 ppb. Control anesthetized rats that were ventilated at a rate of once per second absorbed 0.588 of the gas (Table 1, Fig. 6B). Because the line passes through the origin, this indicates that comparatively little NO was produced by the control animals, nor was there any concentration-independent removal of NO occurring during the control conditions. The fact that [NO]e is a linear function of the [NO]i means that the amount of NO absorbed by the lungs is a linear function of the amount inspired and, therefore, that the process of NO absorption is first-order kinetics depending on solubility and is an uncatalyzed reaction.
When the measurements were repeated 3 h after the animals had been
treated with LPS, the slope of the line
was decreased
to 0.334. This means that 1 0.334 = 0.666 of the
[NO]i was absorbed by
the lungs. The [NO]e
intercept (i.e., mixed [NO]e) was now offset
upward by 171 ppb and represents the concentration of NO that was
contained in the exhaled air of the rats when they were breathing
NO-free air. This is a reflection of the endogenous NO
production initiated by the endotoxemia. In fact, it is the actual rate
of increase of [NO] in the alveolar compartment per second,
due to the endogenous NO source, because these animals were being
respired once per second. When the animals were circulatory arrested,
two things happened. The
[NO]e intercept was
then offset up to 612 ppb, indicating that the concentration of NO in
expired lung air had risen to 612 ppb, and the fraction of inspired NO present in mixed expired air increased to 0.527, indicating that pulmonary absorption of NO decreased to 1 0.527 = 0.473. Both changes reflect the role that pulmonary blood flow was playing in the
removal of NO from the lungs of endotoxemic rats. Because NO absorption
fell from 0.666 to 0.473 after circulatory arrest, while the
concentration of NO produced by the lung itself rose to 612 ppb in
expired air, we can infer that the pulmonary blood flow was responsible
for the absorption of 0.666 0.473 = 0.193 of the NO that was
being removed by the lungs of the endotoxemic rats when they were being
perfused by blood. Furthermore, 0.473 of the inspired NO was being
absorbed by lung tissues. This absence of blood flow must also have
been responsible for the larger amount of endogenous NO that was
exhaled by the circulatory-arrested animals. However, because of
differences in the fluxes of the NO that is generated endogenously from
within the lung, compared with those of inhaled NO, it is not possible
to quantify the roles of blood flow and lung tissues in a manner
similar to that derived for the inspired exogenous NO gas. However,
because pulmonary blood flow could only have removed 0.193 of any NO
within the alveolar compartment, it is clear that the circulation must
have been previously diverting >50% of the LPS-induced NO and
thereby preventing it from ever entering the alveolar compartment,
because only 27% of the endogenous lung NO (171 ppb) that became
apparent after circulatory arrest (612 ppb) was being exhaled when the blood was flowing through the lungs (Figs. 5 and
6A).
As illustrated in Fig. 6A, where the line of identity for [NO]e = [NO]i intersects the two [NO]e lines, net flux (i.e., production minus absorption) of NO is zero, with or without blood flow. We can then equate the absorption of inhaled NO with the production of endogenous NO. It will be noted that it intersects the endotoxemic [NO]e line at 257 ppb and the circulatory arrested endotoxemic [NO]e line at 1,295 ppb. This means that when the endotoxin-treated animals breathed NO at [NO]i = 257 ppb, the absorption of the gas by the lungs exactly matched delivery of NO from its endogenous source in the lung so that there was no net delivery or absorption of NO in the alveolar compartment, and thus [NO]e = [NO]i. Below this concentration, there was a net delivery of NO into the alveolar air from the endogenous source so that [NO]e > [NO]i. It is most obvious when [NO]i = 0. Above this concentration of 257 ppb there was a net absorption of NO out of the alveolar compartment so that [NO]e < [NO]i. In other words, if we assume that the endogenously produced NO that enters the alveolar compartment is subject to the same absorption as the inspired NO, then at [NO]i = 257 ppb, this lung-generated NO must have been delivered into the alveolar compartment at an effective concentration of 171/0.334 = 512 ppb, where it combined with the originally inspired [NO]i of 257 ppb to yield an effective concentration of 769 ppb. Because 0.666 of the combined NO gases was absorbed by the lungs during the respiratory cycle, the concentration of NO remaining in the alveolar compartment 769 × 0.334 = 257 ppb exactly matched that of the NO in the inspired air; as did the mixed expired air, because the dead space [NO] was also 257 ppb. Furthermore, because this lung-generated NO was being delivered at an effective concentration of 512 ppb (pl/ml) and ventilation was 3 ml/s, then the rate of entry of NO into the alveolar compartment must have been 512 × 3 = 1,536 pl/s or 1.54 nl/s. A similar reasoning would apply to the conditions that existed after cardiac arrest, when the line of identity for [NO]e = [NO]i intersects the arrested [NO]e line at 1,295 ppb. Because of the absence of pulmonary blood flow, the lung-generated NO must now have been delivered into the alveolar compartment at an effective concentration of 612.6/0.527 = 1,162.4 ppb, where it combined with the originally inspired [NO]i of 1,295 ppb to yield an effective concentration of 2,457.4 ppb. Because 0.473 of the combined gases was then absorbed by the lung tissues during the respiratory cycle, the residual concentration of NO in the alveolar compartment 2,457.4 × 0.527 = 1,295 ppb exactly matched that of the [NO]i and [NO]e because of the dead space [NO] was also 1,295 ppb. In this case, by using the same reasoning as above, the rate of entry of lung-generated NO must have been 1,162.4 × 3 = 3,487 pl/s or 3.49 nl/s. This more than doubling of NO entry after circulatory arrest reflects the fact that the absence of lung blood flow no longer prevented entry of endogenous NO into the alveolar compartment by "intercepting" it at its generation site at the vascular endothelium within the lung circulation.
The slopes of
for both the endotoxemic and circulatory-arrested endotoxemic groups
are straight lines over the range of
[NO]i from 0 to 2,000 ppb (Fig. 6A). This means that the
delivery of the lung-generated NO into the alveolar compartment is not
affected by the concentration of NO already in this compartment, at
least over the range of 2,000 ppb NO. It indicates that the lung-generated NO is not entering the alveolar compartment by simple
diffusion but is the result of some concentration-independent mechanism
that may even be the actual process of extrusion of the NO from the
cells that are the source of the gas. It will also be noted that the NO
absorption in endotoxemic rats (0.666) is greater than that determined
for control rats (0.588), even though the LPS-treated lungs are
producing endogenous NO. Because pulmonary blood flow is one factor
that will influence the amount of absorption, this might reflect an
increase in overall blood flow to the lungs produced by the
vasodilatory effects of lung-generated NO on the pulmonary vasculature
itself.
There are two other striking features about these results. First, the circulatory-arrested animals still retained the ability to absorb nearly 50% of the inspired NO (Table 1, Fig. 6, A and B). This means that a large portion of the ability of the lungs to absorb NO resides within the lung tissues themselves, although we are cognizant of the fact that blood resident in the arrested pulmonary vascular bed may continue to act as a sink for NO. However, because the endogenous NO from these animals continues to be generated for >60 min after circulatory arrest (Fig. 4) and because NO has a very high Dm (10, 24), it is difficult to envisage that hemoglobin in resident stagnant blood could act as an appreciable sink for NO absorption for that length of time. A more likely candidate is the lung tissues themselves. It may be that this blood contains enzymes or other factors that promote the fixation of gaseous NO within the lung tissue or lung vasculature, because Spriestersbach et al. (46) have shown that ventilated buffer-perfused excised rabbit lungs unload and exhale all the NO that is presented to them in simple solution, via the perfusing buffer solution. A second striking feature is the ability of the NOS to continue to produce NO in the absence of blood flow to the lungs. The L-arginine-NO radical production pathway is an energy-utilizing process that requires molecular oxygen to oxidize an L-arginine substrate to L-citrulline, with the release of NO (35). The fact that the cells that were producing the NO could survive for >60 min after the cessation of blood flow to the lungs suggests that they reside within the O2 diffusional pathway of the alveolar compartment. Indeed, to support the continued production of NO in the lungs, we had to continue to ventilate the lungs of the dead animals with air. If ventilation was continued with 100% nitrogen instead of air, the [NO]e declined abruptly.
This leads to a consideration of the origins of the NO that is produced during endotoxemia. While we present no direct evidence to implicate any specific cell site in the lungs, there are several factors that, when considered together, seem to point to one particular process and to the involvement of two particular cell types. As outlined in the introduction, the HDR and the acute-phase reactions involving inflammatory responses, mediated by cytokines, are prime candidates for initiating the production of NO in response to endotoxemia. Lung injury that is induced by aggregation and adhesion of neutrophils within the pulmonary vasculature has often been described in response to septicemia (29, 36, 37). Both the bronchoalveolar lavage data and histology reported in the present study would suggest that the alveolar compartment itself is not the site of NO production because there is no evidence of any inflammatory cells within the airways at 3 h. It is more likely that the site of NO production is the endothelial side of the blood-air barrier of the lung rather than the epithelial side. It is known that endotoxin induces the upregulation of iNOS in a wide variety of tissues, including the lung itself (33). Thus it is possible that there might be a direct effect of the LPS on the lung macrophages, airway epithelial, or vascular endothelial cells. That this is not the case is evident from our preliminary communication (49), demonstrating that neutropenia abolishes the ability of LPS to induce gaseous NO in the exhaled air of rats.
A more plausible sequence for the production of NO in response to endotoxin might be as follows. The introduction of LPS into the systemic circulation activates the acute-phase reaction, which initiates the production of adhesion molecules both on the vascular endothelium and on circulating neutrophils. Endotoxin-induced production of the early cytokines appears to be essential in this process. This leads to a margination of the leukocytes and the aggregation and adhesion of neutrophils in affected capillary beds. As was mentioned previously, the pulmonary circulation appears to be particularly susceptible to this phenomenon and significant numbers of neutrophils immigrate into the pulmonary vasculature, where they adhere to capillary endothelia (27). This might account for the transient rise in mean PAP that we documented in Fig. 3. The interaction between the adhering neutrophils and the endothelium cells may be the key factor that leads to the generation of NO. Both cell types are capable of NO production. Neutrophils that have been activated by adhesion and cytokines will produce their characteristic respiratory burst, which includes the release of eicosanoids, oxygen free radicals, proteolytic enzymes, and NO radicals (8, 25, 29, 42). This is the initiation of the inflammatory response that leads eventually to both acute- and late-phase lung injury. On the other hand, vascular endothelial cells are also a rich source of NO, especially when they are perturbed by the adhesion of neutrophils to them. One or possibly both cell types might be the source of the NO that, after a mere 60 min, finds its way into the alveolar compartment and thus appears in exhaled air.
Our observation that during endotoxemia there is a generation of considerable amounts of NO within the lung vasculature, of which >19% is taken up by the reoxygenating pulmonary blood flow, acquires considerable pathophysiological importance, when seen in the context of the findings and hypotheses of Jia et al. (31), with respect to the role of hemoglobin in the transport of NO throughout the body. They have reported that two forms of NO binding to hemoglobin can exist in blood, besides the Fe III metal-bound form of methemoglobin NOHb(Fe III). These are S-nitrosohemoglobin S-NO-Hb(Fe II)O2 (S-NO-HbO2) and nitrosylhemoglobin [Hb(Fe II)NO]. The S-H bonds of cysteine residues are the attachment points of the nitrosyls to the hemoglobin molecule. They have also shown that S-NO-HbO2 only exists in arterial blood, whereas Hb(Fe II)NO is nearly twice as high in venous blood as it is in arterial blood. They propose that the high arterial levels of S-NO-HbO2 are allosterically affected by the reaction of O2 with the heme moiety of hemoglobin and that in low levels of oxygenation, presumably at the periphery where PO2 is decreased, S-NO-HbO2 decomposes to release NO from the erythrocyte. Finally, they propose that the formation of S-NO-HbO2 occurs in the lung by a concomitant formation of oxyhemoglobin and the S-nitrosylation from lung stores of S-nitrosothiols. If this is true, then the endotoxemic rat lung might be a major source of NO, which according to the hypothesis of Jia et al., could export S-NO-HbO2 to the periphery via the erythrocyte, where it could be unloaded and contribute to the massive peripheral vasodilatation that is the hallmark of endotoxic shock. However, in furtherance of this hypothesis, we have found no evidence that NO is unloaded into the lungs by the returning venous blood, but we do present substantial evidence that the arterialized blood passing through the lungs carries off a considerable amount of NO, which never shows up in the form of methemoglobin within the circulation.
A major problem in dealing with the pathology of sepsis is predicting the potential for its occurrence early enough to deal with its sequelae, which can be irreversible and fatal. Gram-negative sepsis is a major risk factor for ARDS, which is associated with the development of multiple-organ failure in patients with sepsis (6, 9, 12, 41). It is also reported that even patients at risk for ARDS who are not septic often have detectable levels of endotoxin in their blood (52). Acute sepsis syndrome is becoming increasingly more common and has very high mortality rates. It has been estimated to affect between 70,000 and 300,000 people each year in this country (6, 7). Establishment of an early marker for identifying, with some degree of certainty, the potential for acute lung injury, ARDS, and sepsis in the clinical population at risk would accrue medical and financial benefits to the patient, the physician, and society in general. We propose that the appearance of gaseous NO in the expired air may be just such an early marker for lung injury.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the generous assistance of Dr. Vahid Mohsenin in performing the bronchoalveolar lavages and their cell counts.
FOOTNOTES
Address for reprint requests: J. T. Stitt, John B. Pierce Foundation Laboratory, 290 Congress Ave., New Haven, CT 06519.
Received 9 April 1996; accepted in final form 10 September 1996.
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